Envelope
Nucleoid
Ribosomes
Ribosome
0.25 µm
©Dennis Kunkel
mRNA
Flagellum
Model of a bacterial cell (Escherichia coli). Envelope: The cell membrane contains embedded proteins for structure and transport. The cell membrane is supported by the cell wall. In this gram-negative cell, the cell wall is coated by the outer membrane, whose sugar chain extensions protect the cell from attack by the immune system or by predators. Plugged into the membranes is the rotary motor of a flagellum. Cytoplasm: Molecules of nascent messenger RNA (mRNA) extend out of the nucleoid to the region of the cytoplasm rich DNA-binding protein in ribosomes. Ribosomes translate the mRNA to make proteins, which are folded by chaperones. Nucleoid: The chromosomal DNA is wrapped around binding proteins. Replication by DNA polymerase and transcription HU by RNA polymerase occur at the same time within the nucleoid. (PDB codes: ribosome, 1GIX,1GIY; DNA-binding protein, 1P78; RNA polymerase, 1MSW)
E P A 30S
50S
Polypeptide
Flagellar motor
RNA polymerase
DNA
RNA
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A Key to the Icons in Microbiology: An Evolving Science Weblink icons indicate that there is an author-recommended website related to the topic at hand. Animation icons in a figure’s caption indicate that there is a process animation to further illustrate that particular figure. Visit Norton StudySpace (wwnorton.com/studyspace) to access these resources and other review material.
Bacterial Cell Components Outer membrane proteins: Sugar porin (10 nm) Braun lipoprotein (8 nm) Inner membrane proteins: Glycerol porin Secretory complex (Sec) Lipopolysaccharide Envelope
Outer membrane Cell wall Periplasm Inner membrane (cell membrane)
ATP synthase (20 nm diameter in inner membrane; 32 nm total height)
Ribosome
Arabinose-binding protein (3 nm x 3 nm x 6 nm) Acid resistance chaperone (HdeA) (3 nm x 3 nm x 6 nm) Disulfide bond protein (DsbA) (3 nm x 3 nm x 6 nm) Cytoplasmic proteins:
Peptide Cytoplasm
RNA
Periplasmic proteins:
RNA polymerase
Pyruvate kinase (5 nm x 10 nm x 10 nm) Phosphofructokinase (4 nm x 7 nm x 7 nm) Proteasome (12 nm x 12 nm x 15 nm) Chaperonin GroEL (18 nm x 14 nm) Other proteins Transcription and translation complexes: RNA polymerase (10 x 10 x 16 nm)
DNA bridging protein H-NS
DNA
Nucleoid
DNA-binding protein HU
Ribosome (21 x 21 x 21 nm) Nucleoid components: DNA (2.4 nm wide x 3.4 nm/10 bp) DNA-binding protein (3 x 3 x 5 nm) DNA-bridging protein (3 x 3 x 5 nm)
50 nm
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Microbiology An Evolving Science
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Microbiology An Evolving Science
Joan L. Slonczewski Kenyon College
John W. Foster University of South Alabama Appendices and Glossary by
Kathy M. Gillen Kenyon College
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W. W. Norton & Company has been independent since its founding in 1923, when William Warder Norton and Mary D. Herter Norton fi rst published lectures delivered at the People’s Institute, the adult education division of New York City’s Cooper Union. The Nortons soon expanded their program beyond the Institute, publishing books by celebrated academics from America and abroad. By mid-century, the two major pillars of Norton’s publishing program—trade books and college texts—were fi rmly established. In the 1950s, the Norton family transferred control of the company to its employees, and today—with a staff of four hundred and a comparable number of trade, college, and professional titles published each year—W. W. Norton & Company stands as the largest and oldest publishing house owned wholly by its employees. Copyright © 2009 by W. W. Norton & Company, Inc. All rights reserved. Printed in the United States of America. Composition by Precision Graphics Manufacturing by R. R. Donnelley/Willard Illustrations by Precision Graphics Editor: Michael Wright Developmental editors: Carol Pritchard-Martinez and Philippa Solomon Senior project editor: Thomas Foley Copy editor: Janet Greenblatt Production manager: Christopher Granville Photography editor: Trish Marx Marketing manager: Betsy Twitchell Managing editor, college: Marian Johnson Science media editor: April Lange Editorial assistant: Matthew A. Freeman
ISBN: 978-0-393-11337-2 W. W. Norton & Company, Inc., 500 Fifth Avenue, New York, N.Y. 10110 www.wwnorton.com W. W. Norton & Company Ltd., Castle House, 75/76 Wells Street, London W1T 3QT 123456789
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D E D I C AT I O N
We dedicate this book to the memory of our doctoral research mentors. Joan’s doctoral mentor, Bob Macnab, offered an unfailingly rigorous pursuit of bacterial chemotaxis and physiology, and lasting friendship. John was mentored by Al Moat, a gifted microbial physiologist and humorist who instilled in his neophyte students an appreciation for critical thinking and a love for the science of microbiology.
v
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Brief Contents Preface About the Authors
Part 1: The Microbial Cell 1 2 3 4 5 6
Genomes and Chromosomes Transcription, Translation, and Bioinformatics Gene Transfer, Mutations, and Genome Evolution Molecular Regulation Viral Molecular Biology Molecular Techniques and Biotechnology
Part 3: Metabolism and Biochemistry 13 14 15 16
Energetics and Catabolism Respiration, Lithotrophy, and Photolysis Biosynthesis Food and Industrial Microbiology
221 257 303 345 389 431
458 461 505 547 589
626
Origins and Evolution Bacterial Diversity Archaeal Diversity Eukaryotic Diversity Microbial Ecology Microbes and the Global Environment
629 675 721 755 793 831
Part 5: Medicine and Immunology 23 24 25 26 27 28
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218
Part 4: Microbial Diversity and Ecology 17 18 19 20 21 22
vi
2
Microbial Life: Origin and Discovery 5 Observing the Microbial Cell 39 Cell Structure and Function 73 Bacterial Culture, Growth, and Development 115 Environmental Influences and Control of Microbial Growth 149 Virus Structure and Function 181
Part 2: Genes and Genomes 7 8 9 10 11 12
xvii xxviii
Human Microflora and Nonspecific Host Defenses The Adaptive Immune Response Microbial Pathogenesis Microbial Diseases Antimicrobial Chemotherapy Clinical Microbiology and Epidemiology
Appendix 1: Biological Molecules Appendix 2: Introductory Cell Biology: Eukaryotic Cells Answers to Thought Questions Glossary Index
860 863 895 937 979 1029 1063 A-1 A-21 AQ-1 G-1 I-1
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Contents
Preface About the Authors
xvii xxviii
PART 1
The Microbial Cell
2
AN INTERVIEW WITH RITA COLWELL: THE GLOBAL IMPACT OF MICROBIOLOGY CHAPTER 1
Microbial Life: Origin and Discovery 1.1 From Germ to Genome: What Is a Microbe? 1.2 Microbes Shape Human History 1.3 Medical Microbiology Special Topic 1.1 How Did Life Originate? Special Topic 1.2 The Discovery of Viruses 1.4 Microbial Ecology Special Topic 1.3 Microbial Endosymbionts of Animals 1.5 The Microbial Family Tree 1.6 Cell Biology and the DNA Revolution
5 6 11 17 18 22 26 28 29 32
CHAPTER 2
Observing the Microbial Cell 2.1 2.2 2.3 2.4 2.5
Observing Microbes Optics and Properties of Light Bright-Field Microscopy Dark-Field, Phase-Contrast, and Interference Microscopy Fluorescence Microscopy
Special Topic 2.1 Confocal Fluorescence Microscopy 2.6 Electron Microscopy Special Topic 2.2 Three-Dimensional Electron Microscopy Solves the Structure of a Major Agricultural Virus 2.7 Visualizing Molecules
39 40 44 48 55 58 60 62 66 68
vii
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viii
C o n t e n ts
CHAPTER 3
Cell Structure and Function
73
75 78 82 88 Special Topic 3.1 The Unique Cell Envelope of Mycobacteria 92 3.5 The Nucleoid and Gene Expression 98 3.6 Cell Division 101 Special Topic 3.2 Bacteria Have a Cytoskeleton 105 3.7 Specialized Structures 106 3.1 3.2 3.3 3.4
The Bacterial Cell: An Overview How We Study the Parts of Cells The Cell Membrane and Transport The Cell Wall and Outer Layers
Special Topic 3.3 One Swims
Two Kinds of Progeny: One Stays,
108
CHAPTER 4
Bacterial Culture, Growth, and Development 4.1 4.2 4.3 4.4 4.5 4.6
Microbial Nutrition Nutrient Uptake Culturing Bacteria Counting Bacteria The Growth Cycle Biofilms Special Topic 4.1 Resistance
115 116 121 127 131 134 140
Biofilms, Disease, and Antibiotic
4.7 Cell Differentiation
141 142
CHAPTER 5
Environmental Influences and Control of Microbial Growth Environmental Limits on Microbial Growth Microbial Responses to Changes in Temperature Microbial Adaptation to Variations in Pressure Microbial Responses to Changes in Water Activity and Salt Concentration 5.5 Microbial Responses to Changes in pH 5.1 5.2 5.3 5.4
Special Topic 5.1 Signaling Virulence 5.6 Microbial Responses to Oxygen and Other
Electron Acceptors 5.7 Microbial Responses to Nutrient Deprivation and Starvation 5.8 Physical and Chemical Methods of Controlling Microbial Growth 5.9 Biological Control of Microbes
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149 150 152 155 157 158 164 164 168 170 178
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ix
Co n te n ts
CHAPTER 6
Virus Structure and Function 6.1 6.2 6.3 6.4 6.5 6.6 6.7
181
What Is a Virus? Virus Structure Viral Genomes and Classification Bacteriophage Life Cycles Animal and Plant Virus Life Cycles Culturing Viruses Viral Ecology Special Topic 6.1
182 187 191 198 201 208 212 214
West Nile Virus, an Emerging Pathogen
PART 2
Genes and Genomes
218
AN INTERVIEW WITH RICHARD LOSICK: THE THRILL OF DISCOVERY IN MOLECULAR MICROBIOLOGY CHAPTER 7
Genomes and Chromosomes 7.1 DNA: The Genetic Material 7.2 Genome Organization 7.3 DNA Replication Special Topic 7.1 Trapping a Sliding Clamp 7.4 Plasmids and Bacteriophages Special Topic 7.2 Plasmid Partitioning and Addiction 7.5 Eukaryotic Chromosomes: Comparison with Prokaryotes 7.6 DNA Sequence Analysis Special Topic 7.3 The Polymerase Chain Reaction
221 222 223 232 237 243 245 246 248 250
CHAPTER 8
Transcription, Translation, and Bioinformatics 8.1 RNA Polymerases and Sigma Factors 8.2 Transcription Initiation, Elongation, and Termination 8.3 Translation of RNA to Protein Special Topic 8.1 Antibiotics That Affect Transcription Special Topic 8.2 Antibiotics That Affect Translation 8.4 Protein Modification and Folding 8.5 Secretion: Protein Traffic Control 8.6 Protein Degradation: Cleaning House Special Topic 8.3 Ubiquitination: A Ticket to the Proteasome 8.7 Bioinformatics: Mining the Genomes Special Topic 8.4 What Is the Minimal Genome?
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257 258 263 267 268 281 283 285 290 292 293 297
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x
C o n t e n ts
CHAPTER 9
Gene Transfer, Mutations, and Genome Evolution 9.1 The Mosaic Nature of Genomes 9.2 Gene Transfer: Transformation, Conjugation, 9.3 9.4 9.5 9.6 9.7
and Transduction Recombination Mutations DNA Repair Mobile Genetic Elements Genome Evolution Special Topic 9.1
Integrons and Gene Capture
303 304 304 316 320 327 333 336 338
C H A P T E R 10
Molecular Regulation
345
10.1 Regulating Gene Expression 346 10.2 Paradigm of the Lactose Operon 349 10.3 Other Systems of Operon Control 355 Special Topic 10.1 How Do We Study Protein-DNA Binding? 356 10.4 Sigma Factor Regulation 365 10.5 Small Regulatory RNAs 368 10.6 DNA Rearrangements: Phase Variation
by Shifty Pathogens 10.7 Integrated Control Circuits 10.8 Quorum Sensing: Chemical Conversations Special Topic 10.2 The Role of Quorum Sensing in Pathogenesis and in Interspecies Communications 10.9 Genomics and Proteomics: Tools of the Future
370 373 378 380 381
C H A P T E R 11
Viral Molecular Biology 11.1 11.2 11.3 11.4 11.5 11.6
Phage T4: The Classic Molecular Model The Filamentous Phage M13 A (+) Strand RNA Virus: Polio A Segmented (–) Strand RNA Virus: Influenza A Retrovirus: Human Immunodeficiency Virus A DNA Virus: Herpes Simplex Special Topic 11.1
How Did Viruses Originate?
389 391 397 400 406 412 423 424
C H A P T E R 12
Molecular Techniques and Biotechnology 12.1 12.2 12.3 12.4 12.5 12.6
Basic Tools of Biotech: A Research Case Study Genetic Analyses Molecular Analyses “Global” Questions of Cell Physiology Biotechniques of Artificial Evolution Applied Microbial Biotechnology Special Topic 12.1
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DNA Vaccines
431 432 432 436 444 446 450 452
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xi
Co n te n ts
PART 3
Metabolism and Biochemistry
458
AN INTERVIEW WITH CAROLINE HARWOOD: BACTERIAL METABOLISM DEGRADES POLLUTANTS AND PRODUCES HYDROGEN C H A P T E R 13
Energetics and Catabolism 13.1 Energy and Entropy: Building a Cell 13.2 Energy and Entropy in Biochemical Reactions 13.3 Energy Carriers and Electron Transfer Special Topic 13.1 Observing Energy Carriers in Living Cells 13.4 Catabolism: The Microbial Buffet Special Topic 13.2 Swiss Cheese: A Product of Bacterial Catabolism 13.5 Glucose Breakdown and Fermentation 13.6 The Tricarboxylic Acid (TCA) Cycle 13.7 Aromatic Catabolism Special Topic 13.3 Genomic Analysis of Metabolism
461 463 465 469 475 476 480 482 491 496 498
C H A P T E R 14
Respiration, Lithotrophy, and Photolysis 14.1 Electron Transport Systems 14.2 The Proton Motive Force Special Topic 14.1 Testing the Chemiosmotic Hypothesis 14.3 The Respiratory ETS and ATP Synthase 14.4 Anaerobic Respiration Special Topic 14.2 ATP Synthesis at High pH 14.5 Lithotrophy and Methanogenesis 14.6 Phototrophy
505 506 511 512 516 525 526 529 534
C H A P T E R 15
Biosynthesis 15.1 Overview of Biosynthesis 15.2 CO2 Fixation: The Calvin Cycle Special Topic 15.1 The Discovery of 14 C 15.3 CO2 Fixation in Anaerobes and Archaea 15.4 Biosynthesis of Fatty Acids and Polyesters Special Topic 15.2 Polyketide Drugs Are Synthesized by Multienzyme Factories 15.5 Nitrogen Fixation 15.6 Biosynthesis of Amino Acids and Nitrogenous Bases 15.7 Biosynthesis of Tetrapyrroles Special Topic 15.3 Modular Biosynthesis of Vancomycin
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547 548 550 554 560 564 567 570 575 581 584
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xii
C o n t e n ts
C H A P T E R 16
Food and Industrial Microbiology 16.1 Microbes as Food 16.2 Fermented Foods: An Overview 16.3 Acidic and Alkaline Fermented Foods Special Topic 16.1 Chocolate: The Mystery Fermentation 16.4 Ethanolic Fermentation: Bread and Wine Special Topic 16.2 Beer Is Made from Barley and Hops 16.5 Food Spoilage and Preservation 16.6 Industrial Microbiology Special Topic 16.3 Start-Up Companies Take On Tuberculosis
589 590 592 595 600 602 606 608 615 616
PART 4
Microbial Diversity and Ecology
626
AN INTERVIEW WITH KARL STETTER: ADVENTURES IN MICROBIAL DIVERSITY LEAD TO PRODUCTS IN INDUSTRY C H A P T E R 17
Origins and Evolution 17.1 Origins of Life 17.2 Models for Early Life Special Topic 17.1 The RNA World: Clues for Modern Medicine 17.3 Microbial Taxonomy 17.4 Microbial Divergence and Phylogeny Special Topic 17.2 Phylogeny of a Shower Curtain Biofilm 17.5 Horizontal Gene Transfer Special Topic 17.3 Horizontal Transfer in E. coli O157:H7 17.6 Symbiosis and the Origin of Mitochondria
and Chloroplasts
629 631 641 644 647 651 658 661 664 666
C H A P T E R 18
Bacterial Diversity 18.1 Bacterial Diversity at a Glance 18.2 Deep-Branching Thermophiles 18.3 Cyanobacteria: Oxygenic Phototrophs Special Topic 18.1 Cyanobacterial Communities: From Ocean to Animal 18.4 Gram-Positive Firmicutes and Actinobacteria 18.5 Gram-Negative Proteobacteria and Nitrospirae 18.6 Bacteroidetes and Chlorobi 18.7 Spirochetes: Sheathed Spiral Cells
with Internalized Flagella
675 677 685 688 691 692 703 714 715
18.8 Chlamydiae, Planctomycetes, and Verrucomicrobia:
Irregular Cells
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716
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Co n te n ts
xiii
C H A P T E R 19
Archaeal Diversity 19.1 Archaeal Traits and Diversity 19.2 Crenarchaeota: Hyperthermophiles Special Topic 19.1 Research on Deep-Sea Hyperthermophiles 19.3 Crenarchaeota: Mesophiles and Psychrophiles 19.4 Euryarchaeota: Methanogens 19.5 Euryarchaeota: Halophiles Special Topic 19.2 Haloarchaea in the High School Classroom 19.6 Euryarchaeota: Thermophiles and Acidophiles 19.7 Nanoarchaeota and Other Emerging Divisions
721 723 730 733 735 738 744 746 750 753
CHAPTER 20
Eukaryotic Diversity 20.1 Phylogeny of Eukaryotes 20.2 Fungi Special Topic 20.1 Mold after Hurricane Katrina 20.3 Algae 20.4 Amebas and Slime Molds 20.5 Alveolates: Ciliates, Dinoflagellates, and Apicomplexans Special Topic 20.2 A Ciliate Model for Human Aging 20.6 Trypanosomes, Microsporidia, and Excavates
755 756 765 772 774 780 783 785 791
C H A P T E R 21
Microbial Ecology 21.1 21.2 21.3 21.4 21.5 21.6
793
Microbes in Ecosystems Microbial Symbiosis Marine and Aquatic Microbiology Soil and Subsurface Microbiology Microbial Communities within Plants Microbial Communities within Animals
794 798 801 812 820 824
Special Topic 21.1 A Veterinary Experiment: The Fistulated Cow
828
CHAPTER 22
Microbes and the Global Environment 22.1 Biogeochemical Cycles 22.2 The Carbon Cycle Special Topic 22.1 Wetlands: Disappearing Microbial Ecosystems 22.3 The Hydrologic Cycle and Wastewater Treatment 22.4 The Nitrogen Cycle 22.5 Sulfur, Phosphorus, and Metals 22.6 Astrobiology
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831 832 835 838 839 842 847 854
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xiv
C o n t e n ts
PART 5
Medicine and Immunology
860
AN INTERVIEW WITH CLIFFORD W. HOUSTON: AN AQUATIC BACTERIUM CAUSES FATAL WOUND INFECTIONS CHAPTER 23
Human Microflora and Nonspecific Host Defenses 23.1 23.2 23.3 23.4 23.5
Human Microflora: Location and Shifting Composition Risks and Benefits of Harboring Microbial Populations Overview of the Immune System Barbarians at the Gate: Innate Host Defenses Innate Immunity: The Acute Inflammatory Response Special Topic 23.1 Do Defensins Have a Role in Determining Species Specificity for Infection?
23.6 Phagocytosis 23.7 Innate Defenses by Interferon and Natural Killer Cells Special Topic 23.2 Immune Avoidance: Outsmarting the Host’s Innate Immune System 23.8 Complement’s Role in Innate Immunity 23.9 Fever
863 864 871 872 877 880 881 884 886 888 889 891
CHAPTER 24
The Adaptive Immune Response 24.1 Adaptive Immunity 24.2 Factors That Influence Immunogenicity 24.3 Antibody Structure and Diversity Special Topic 24.1 Applications Based on Antigen-Antibody Interactions 24.4 Humoral Immunity: Primary and Secondary
Antibody Responses 24.5 Genetics of Antibody Production 24.6 T Cells, Major Histocompatibility Complex, and Antigen Processing Special Topic 24.2 T Cells That Recognize Self Too Strongly Are Weeded Out in the Thymus 24.7 Complement as Part of Adaptive Immunity 24.8 Failures of Immune System Regulation:
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895 896 898 902 906 908 911 915 918 925
Hypersensitivity and Autoimmunity
926
Special Topic 24.3 Rejection
933
Organ Donation and Transplantation
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xv
Co n te n ts
CHAPTER 25
Microbial Pathogenesis 25.1 Host-Pathogen Interactions 25.2 Virulence Factors and Pathogenicity Islands:
The Tools and Toolkits of Microbial Pathogens 25.3 Virulence Factors: Microbial Attachment 25.4 Toxins: A Way to Subvert Host Cell Function 25.5 Protein Secretion and Pathogenesis Special Topic 25.1 The Bacterial Trojan Horse: Bacteria That Deliver Their Own Receptor 25.6 Finding Virulence Genes Special Topic 25.2 Signature-Tagged Mutagenesis 25.7 Surviving within the Host 25.8 Viral Pathogenesis
937 938 942 944 948 959 963 964 966 969 971
CHAPTER 26
Microbial Diseases 26.1 26.2 26.3 26.4 26.5
Characterizing and Diagnosing Microbial Diseases Skin and Soft-Tissue Infections Respiratory Tract Infections Gastrointestinal Tract Infections Genitourinary Tract Infections Special Topic 26.1 Intracellular Biofilm Pods Are Reservoirs of Infection
26.6 26.7 28.8 26.9
Infections of the Central Nervous System Infections of the Cardiovascular System Systemic Infections Immunization
979 980 982 986 992 997 1000 1007 1014 1017 1023
CHAPTER 27
Antimicrobial Chemotherapy 27.1 27.2 27.3 27.4 27.5 27.6 27.7 27.8
The Golden Age of Antibiotic Discovery Basic Concepts of Antimicrobial Therapy Measuring Drug Susceptibility Mechanisms of Action Antibiotic Biosynthesis The Challenges of Antibiotic Resistance The Future of Drug Discovery Antiviral Agents Special Topic 27.1 Poking Holes with Nanotubes: A New Antibiotic Therapy Special Topic 27.2 Critical Virulence Factors Found in the 1918 Strain of Influenza Virus
27.9 Antifungal Agents
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1029 1030 1032 1034 1037 1046 1047 1052 1054 1055 1057 1059
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xvi
C o n t e n ts
CHAPTER 28
Clinical Microbiology and Epidemiology 28.1 28.2 28.3 28.4 28.5
Principles of Clinical Microbiology Approaches to Pathogen Identification Specimen Collection Biosafety Containment Procedures Principles of Epidemiology Special Topic 28.1 Wired Up
1063 1064 1065 1081 1083 1085
Microbial Pathogen Detection Gets
28.6 Detecting Emerging Microbial Diseases
1092 1092
APPENDIX 1
Biological Molecules A1.1 A1.2 A1.3 A1.4 A1.5 A1.6 A1.7
Elements, Bonding, and Water Common Features of Organic Molecules Proteins Polysaccharides Nucleic Acids Lipids Chemical Principles in Biological Chemistry
A-1 A-2 A-5 A-6 A-10 A-12 A-14 A-16
APPENDIX 2
Introductory Cell Biology: Eukaryotic Cells A2.1 A2.2 A2.3 A2.4 A2.5 A2.6
The Cell Membrane The Nucleus and Mitosis Problems Faced by Large Cells The Endomembrane System The Cytoskeleton Mitochondria and Chloroplasts
Answers to Thought Questions Glossary Index
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A-21 A-22 A-29 A-30 A-32 A-35 A-37 AQ-1 G-1 I-1
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Preface
Among civilization’s greatest achievements are the discovery of microbes and learning how they function. Today, microbiology as a science is evolving rapidly. Emerging species, from Helicobacter pylori to ammonia oxidizers, challenge our vision of where microbes can grow, while emerging technologies, from atomic force microscopy to metagenomic sequencing, expand the frontiers of what we can study. As our understanding of microbes and our ability to study them has evolved, what is taught must also evolve. This textbook was designed to present core topics of microbiology in the context of new challenges and opportunities. Our book gives students and faculty a fresh approach to learning the science of microbiology. A major aim is to balance the coverage of microbial ecology and medical microbiology. We explore the origin of life as a dynamic story of discovery that integrates microfossil data with physiology and molecular biology. This story provides surprising applications in both biotechnology and medicine (Chapter 17, Origins and Evolution). Microbial–host interactions are presented in the context of evolution and ecology, reflecting current discoveries in microbial diversity. For example, Vibrio cholerae, the causative agent of cholera, is discussed as part of a complex ecosystem involving invertebrates as well as human hosts (Part 1, Interview with Rita Colwell). Principles of disease are explained in terms of molecular virulence factors that act upon the host cell, including the horizontal transfer of virulence genes that make a pathogen (Chapter 25, Microbial Pathogenesis). Throughout our book, we present the tools of scientific investigation (emphasizing their strengths and limitations) and the excitement of pursuing questions yet to be answered. We were students when the fi rst exciting reports of gene cloning and the descriptions of molecular machines that compose cells were published. We shared in the excitement surrounding these extraordinary advances, witnessed their impact on the field, and recall how profoundly they inspired us as aspiring scientists. As a result, we believe that conveying the story of scientific advancement and its influence on the way scientists approach research questions, whether classical or modern, is an important motivational and pedagogical tool in presenting fundamental concepts. We present the story of molecular microbiology and microbial ecology in the same spirit as the classical history of Koch and Pasteur, and of Winogradsky and Beijerinck. We drew on all our experience as researchers and educators (and on the input of dozens of colleagues over the past seven years) to create a microbiology text for the twenty-fi rst century.
xvii
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xviii
Pr e f a c e
Major Features Our book targets the science major in biology, microbiology, or biochemistry. We offer several important improvements over other books written for this audience:
00i-xxviii_SFMB_fm.indd xviii
■
Genetics and genomics are presented as the foundation of microbiology. Molecular genetics and genomics are thoroughly integrated with core topics throughout the book. This approach gives students many advantages, including an understanding of how genomes reveal potential metabolic pathways in diverse organisms, and how genomics and metagenomics reveal the character of microbial communities. Molecular structures and chemical diagrams presented throughout the art program clearly illustrate the connections between molecular genetics, physiology, and pathogenesis.
■
Microbial ecology and medical microbiology receive equal emphasis, with particular attention paid to the merging of these fields. Throughout the book, phenomena are presented with examples from both ecology and medicine; for example, when discussing horizontal transfer of “genomic islands” we present symbiosis islands associated with nitrogen fi xation, as well as pathogenicity islands associated with disease (Chapter 9).
■
Current research examples and tools throughout the text enrich students’ understanding of foundational topics. Every chapter presents numerous current research examples within the up-to-date framework of molecular biology, showing how the latest research extends our knowledge of fundamental topics. For example, in the past two decades, advances in microscopy have reshaped our vision of microbial cells. Chapter 2 is devoted to visualization techniques, from an in-depth treatment of the student’s microscope to advanced methods such as atomic force microscopy. Unlike most microbiology textbooks, our text provides size scale information for nearly every micrograph, which is critical when trying to visualize the relationship between different organisms and structures. Examples of current research range from the use of two-hybrid assays to study Salmonella virulence proteins to the spectroscopic measurement of carbon flux from microbial communities.
■
Viruses are presented in molecular detail and in ecological perspective. For example, in marine ecosystems, viruses play key roles in limiting algal populations while selecting for species diversity (Chapter 6). Similarly, a constellation of bacteriophages influences enteric flora. Our coverage of human virology includes the molecular reproductive cycles of herpes, avian influenza, and HIV, including emerging topics such as the role of regulatory proteins in HIV virulence (Chapter 11).
■
Microbial diversity that students can grasp. We present microbial diversity in a manageable framework that enables students to grasp the essentials of the most commonly presented taxa, devoting one chapter each to bacteria, archaea, and the microbial eukaryotes. At the same time, we emphasize the continual discovery of previously unknown forms such as the nanoarchaea and the marine prochlorophytes. Our book is supported by the on-line Microbial Biorealm, an innovative resource on microbial diversity authored by students and their teachers.
■
The physician-scientist’s approach to microbial diseases. Case histories are used to present how a physician-scientist approaches the interplay between the
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Pre face
xix
human immune response and microbial diseases. By taking an organ systems approach, we show how a physician actually interacts with the patient, recognizing that patients complain of symptoms, not a species. Ultimately, we let the student in on the clues used to identify infective microbes. The approach stresses the concepts of infectious disease rather than presenting an exhaustive recitation of diseases and microbes. ■
Scientists pursuing research today are presented alongside the traditional icons. This approach helps students see that microbiology is an extremely dynamic field of science, full of opportunities for them to do important research as undergraduates or as future graduate students. For example, Chapter 1 not only introduces historical figures such as Koch and Pasteur, but also features genome sequencer Claire Fraser-Liggett, postdoctoral researcher Kazem Kashefi growing a hyperthermophile in an autoclave, and undergraduate students studying acid stress in E. coli.
■
Appendices for students in need of review. Our book assumes a sophomorelevel understanding of introductory biology and chemistry. For those in need of review, two appendices summarize the fundamental structure and function of biological molecules and cells.
Organization The topics of this book are arranged so that students can progressively develop an understanding of microbiology from key concepts and research tools. The chapters of Part 1 present key foundational topics: history, visualization, the bacterial cell, microbial growth and control, and virology. Chapter 1 discusses the nature of microbes and the history of their discovery, including the key role of microbial genomes. In Chapter 2, basic tools of visualization, from the student’s microscope to cryo-EM, provide the foundation for understanding how scientists reveal microbial structure. The basic form and function of bacterial cells emerges in Chapter 3, while Chapters 4 and 5 present core concepts of microbial growth in relation to the environment. Chapter 6 introduces virus structure and culture. The six chapters in Part 1 present topics treated in more detail in Parts 2 through 5. The topics of nucleoid structure and virus replication introduced in Chapters 3 and 6 lead into Part 2, where Chapters 7 through 12 present modern genetics and genomics. Chapter 11 presents the life cycles of selected viruses in molecular detail. The topics of cell growth and nutrition introduced in Chapter 4 lead into Part 3 (Chapters 13–16), which presents cell metabolism and biochemistry. Diverse forms of metabolism, such as phototrophy and lithotrophy, are explained on a common basis, the fundamental principles of electron transport and energy conservation. These chapters are written in such a way that they can be presented before the genetics material if so desired. Chapter 16 presents food and industrial microbiology, showing how these fields are founded on microbial metabolism. The principles of environmental responses and growth limits introduced in Chapter 5 lead into Part 4 (Chapters 17–22), which explores microbial ecology and diversity. The roles of microbial communities in local ecosystems and global cycling, introduced in Chapter 4, are presented in greater depth in Chapters 21 and 22. And the chapters of Part 5 (Chapters 23–28) present medical and disease microbiology from an investigative perspective, founded on the principles of genetics, metabolism, and microbial ecology.
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Special Features Throughout our book, special features aid student understanding and stimulate inquiry.
3 5C CH2OPO32– 3ADP
C
O
H
C
OH
H
C
OH
CH2OPO32– 3. Regeneration of ribulose 1,5-bis P
ART PROG RAM
3 CO2 + 3H2O
The art program offers exceptional depth and clarity, using up-to-date graphical methods to enhance understanding. Key processes are shown in both a simplified version and a more complex version. For example, the Calvin-Benson cycle is introduced with a focus on the incorporation of CO2 and formation of energy carriers (Fig. 15.5), followed by a more detailed diagram that includes the chemical structures of all intermediates (Fig. 15.7). Overall, our book provides a greater number of figures and photos than our major competitors.
1. Carboxylation and splitting 6C f 2 3C by rubisco
Ribulose 1,5-bis P
O–
O 6 3C
3 ATP Calvin-Benson Cycle: Overview
C H
C
OH
CH2OPO32–
(Sugar- P intermediates)
3- P glycerate (PGA)
Five G3P become phosphorylated; one G3P enters biosynthesis of glucose.
6 ATP + 6NADPH + 6H+ 5 3C
O
H C
3C
H
C
OH
CH2OPO32– Glyceraldehyde 3- P (6 G3P) Amino acids
2. Reduction of RCOO– to RCOH 6ADP + 6 Pi + 6H2O 6NADP+ CH2OPO32–
3(Ribulose 1,5-bis P ) 5C Glucose
C O
1. Carboxylation H 3(CO2 + H2O)
Simplified view
H 2 3C
Split 6C
C
OH
C
OH
H+
CO2
H
O–
O C
6 ATP
H
C
6(1,3-bis P glycerate) 3C
O C
6NADPH
H
6NADP+ + 6HPO42–
C
C
OH
CH2OPO32–
+
H
OH
C
OH
C
CH2OPO32–
6ADP
CH2OPO32– O C C O– C O
CH2OPO32–
CH2OPO32–
6(3- P glycerate) 3C 2. Reduction
HO
O
O–
OPO32– OH
CH2OPO32– O
6(Glyceraldehyde 3- P ) 3C
H C
H
C
OH
CH2OPO32–
3. Regeneration 3C
3C
3C
3C
3C
3C
6(Glyceraldehyde 3- P ) Dihydroxyacetone 3- P 3C G3- P
3C
Fructose 1,6-bis P
Glucose Dihydroxyacetone 3- P (DHA)
6C
3C Sucrose, starch, cellulose
H2O 2–
HPO4
Fructose 6- P
3C G3- P
6C
Cells
Erythrose 4- P
Xylulose 5- P 5C
4C 3C DHA 3- P Sedoheptulose 1,7-bis P H2 O
7C
HPO42– Sedoheptulose 7- P 7C Xylulose 5- P 5C
Ribulose 5- P
Ribulose 5- P
5C
5C
3C G3- P Ribose 5- P
5C
Ribulose 5- P
5C
3 ATP 3ADP 3(Ribulose 1,5-bis P ) 5C
Expanded view
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Part 1
microbes that can live with or without oxygen. They will grow throughout the tube shown in Figure 5.20. Facultative anaerobes (sometimes called aerotolerant) only use fermentation to provide energy but contain superoxide dismutase and catalase (or peroxidase) to protect them from reactive oxygen species. This allows them to grow in oxygen while retaining a fermentation-based metabolism. Facultative aerobes (such as E. coli) also possess enzymes that destroy toxic oxygen by-products, but have both fermentative and aerobic respiratory potential. Whether a member of this group uses aerobic respiration, anaerobic respiration, or fermentation depends on the availability of oxygen and the amount of carbohydrate present. Microorganisms that possess decreased levels of superoxide dismutase and/or catalase will be microaerophilic, meaning they will grow only at low oxygen concentrations. The fundamental composition of all cells reflects their evolutionary origin as anaerobes. Lipids, nucleic acids, and amino acids are all highly reduced—which is why our bodies are combustible. We never would have evolved that way if molecular oxygen were present from the beginning. Even today, the majority of all microbes are anaerobic, growing buried in the soil, within our anaerobic digestive tract, or within biofi lms on our teeth.
167
Culturing Anaerobes in the Laboratory Many anaerobic bacteria cause horrific human diseases, such as tetanus, botulism, and gangrene. Some of these organisms or their secreted toxins are even potential weapons of terror (for example, Clostridium botulinum). Because of their ability to wreak havoc on humans, culturing these microorganisms was an early goal of microbiologists. Despite the difficulties involved, conditions were eventually contrived in which all, or at least most, of the oxygen could be removed from a culture environment. Three techniques are used today. Special reducing agents (for example, thioglycolate) or enzyme systems (Oxyrase®) that eliminate dissolved oxygen can be added to ordinary liquid media. Anaerobes can then grow beneath the culture surface. A second, very popular, way to culture anaerobes, especially on agar plates, is to use an anaerobe jar (Fig. 5.22A). Agar plates streaked with the organism are placed into a sealed jar with a foil packet that releases H 2 and CO2 gases. A palladium packet hanging from the jar lid catalyzes a reaction between the H2 and O2 in the jar to form H2O and effectively removes O2 from the chamber. The CO2 released is required by some reactions to produce key metabolic intermediates. Some microaerophilic microbes, like the pathogens H. pylori (the major cause of stomach ulcers) and Campylobacter jejuni (a major cause of diarrhea), require low levels of O2 but elevated amounts of CO2. These conditions are obtained by using similar gas-generating packets. For strict anaerobes exquisitely sensitive to oxygen, even more heroic efforts are required to establish an oxygen-free environment. A special anaerobic glove box must be used in which the atmosphere is removed by
THOUGHT QUESTION 5.6 If anaerobes cannot live in oxygen, how do they incorporate oxygen into their cellular components? THOUGHT QUESTION 5.7 How can anaerobes grow in the human mouth when there is so much oxygen there?
A.
■ The M i c r obi a l Cell
xxi
THOUG HT QU ESTIONS
“Thought Questions” throughout the text stimulate students to think critically about their reading. For example, a Thought Question in Chapter 5 (p. 167) asks students to consider how anaerobes incorporate oxygen into their cellular components in spite of their inability to live in oxygen. The question is posed in the context of a discussion of the different levels of oxygen tolerated or required by different types of microbes. Answers to each Thought Question are provided at the back of the book.
B.
Catalyst in lid mediates reaction. H2 + ½O2 f H2O
Airlock
Tracy Grosshans
©Jack Bostrack/Visuals Unlimited
GasPak envelope generates H2 and CO2.
Glove ports
Anaerobic growth technology. A. An anaerobic jar. B. An anaerobic chamber with glove ports.
Figure 5.22
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Part 1
The Microbial Cell AN INTERVIEW WITH Courtesy of Rita Colwell
RITA COLWELL: THE GLOBAL IMPACT OF MICROBIOLOGY
Rita Colwell is Distinguished Professor at the University of Maryland and Johns Hopkins University and served as director of the USA National Science Foundation from 1998 to 2004. Colwell’s decades of research on Vibrio cholerae, the causative agent of cholera, have revealed its natural ecology, its genome sequence, and ways to control it. Colwell originated the concept of viable but nonculturable microorgan-
Rita Colwell, former director of the National Science Foundation.
isms, microbial cells that metabolize but cannot be cultured in the laboratory. She is now chairman of the board of Canon US Life Sciences, Inc., and she represents the American Society for Microbiology at the United Nations Educational, Scientific and Cultural Organization (UNESCO).
Why did you decide to make a career in microbiology?
I was fi rst inspired by the report of my college roommate at Purdue University about a wonderful bacteriology professor, Dr. Dorothy Powelson, probably one of only two women at Purdue who were full professors at the time. I enrolled in Powelson’s course and was truly inspired by this remarkable woman who was so interested in microbiology and made it fascinating for her students. How did you choose to study Vibrio cholerae? What makes this organism interesting?
I chose to study Vibrio cholerae as a result of my having become an “expert” on vibrios through my graduate dissertation on marine microorganisms. Vibrios were the most readily culturable of the marine bacteria and were therefore considered the most dominant. Of course, new information indicates that although vibrios are the dominant bacteria in many estuarine areas, there are other organisms that are very difficult to culture that are important as well.
When I took my fi rst faculty position at Georgetown University, a friend of mine at NIH, Dr. John Feeley, suggested that I study Vibrio cholerae. What makes V. cholerae interesting is that it is a human pathogen of extremely great importance, yet resides naturally in estuaries and coastal areas of the world. What is it like to study this organism?
Vibrio cholerae is naturally occurring (in the environment outside humans) and therefore can never be eradicated; it carries out important functions in the environment, and significant among these is its ability to digest chitin, the structural component of shellfish and many zooplankton. It is at once a “recycling agent” and a public health threat in the form of the massive epidemics of cholera that it causes. You led an international collaboration in Bangladesh training women to avoid cholera by filtering water through sari cloth. How did the sari cloth filtration project come about?
It came about through collaboration with the International Centre for Diarrhoeal Diseases, Bangladesh, located in Dhaka, Bangladesh, and the Mattlab Field Laboratory, which is located in the village area of Mattlab, Bangladesh. Our work had shown that Vibrio cholerae is associated with environmental zooplankton, namely, the copepod. The notion that the copepods are large and could be filtered out and therefore lead to reduced incidence of cholera was a result of my work on the vibrios and the relationships described by my students, notably, Dr. Anwar Huq, who did his thesis on Vibrio cholerae attachment to copepods. Anwar Huq is now an associate professor at the University of Maryland. An important collaborator was Nell Roberts, an outstanding public health microbiologist at Lake Charles, Louisiana, working on public health problems. Nell, Professor Xu (a colleague from Qingdao, China), and I did the critical experiment showing the presence of Vibrio cholerae in water from which blue crabs had been harvested—the cause of an outbreak of cholera in Louisiana back in 1982. We were able to use fluorescent antibody to show the presence of the vibrio on copepods in the water. From there, the idea of sari cloth came about in searching for a very inexpensive fi lter for use by village
I NTE RVI EWS WITH PROM I N E NT SCI E NTISTS
Each Part of the book opens with an interview of a prominent microbiologist working today. In each interview, the authors ask the featured scientist questions about everything from how they fi rst became interested in microbiology to how their thought processes and experiments allowed them to make important discoveries. Interviewees include Karl Stetter, the fi rst person to discover living organisms growing at temperatures above 100°C, and Rita Colwell, past director of the National Science Foundation, who used her understanding of the marine ecology of Vibrio cholerae to help develop public health measures against cholera in developing countries.
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Special Topic 10.2
Part 2
Molecular Regulation
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381
G en e s a n d G e n o me s
The Role of Quorum Sensing in Pathogenesis and in Interspecies Communications
Pseudomonas aeruginosa is a human pathogen that commonly infects patients with cystic fibrosis, a genetic disease of the lung. The organism forms a biofilm over affected areas and interferes with lung function. Key to the destruction of host tissues by P. aeruginosa are virulence factors such as proteases and other degradative enzymes. But these proteins are not made until cell density is fairly high, a point where the organism might have a chance of overwhelming its host. The organism would not want to make the virulence proteins too early and alert the host to launch an immune response. The induction mechanism involves two interconnected quorumsensing systems called Las and Rhl, both comprised of regulatory proteins homologous to LuxR and LuxI of V. fischeri. Many pathogens besides Pseudomonas appear to use chemical signaling to control virulence genes. Genomic analysis has revealed homologs of known quorum-sensing genes in Salmonella, Escherichia, Vibrio cholerae, the plant symbiote Rhizobium, and many other microbes. Some microbial species not only chemically talk among themselves, but appear capable of communicating with other species. V. harveyi, for example, uses two different, but converging, quorum-sensing systems to coordinate control of its luciferase. Both sensing pathways are very different from the V. fischeri system. One utilizes an acyl homoserine lactone (AHL) as an autoinducer (AI-1) to communicate with other V. harveyi cells. The second system involves production of a different autoinducer (AI-2) that contains borate. Because many species appear to produce this second signal molecule, it is thought that mixed populations of microbes use it to “talk” to each other. In the case of V. harveyi, specific membrane sensor kinase proteins are used to sense each autoinducer (Fig. 1). At low cell densities (no autoinducer), both sensor kinases initiate phosphorylation cascades that converge on a shared response regulator, LuxO, to produce phosphorylated LuxO. Phosphorylated LuxO appears to activate a repressor of the lux genes. Thus, at low cell densities, the culture does not display bioluminescence. At high cell density, the autoinducers prevent signal transmission by inhibiting phosphorylation. The cell stops making repressor, which allows another pro-
Autoinducer AI-1
tein, LuxR (not a homolog of the V. fischeri LuxR), to activate the lux operon. The “lights” are turned on. Bonnie Bassler (Fig. 2) and Pete Greenberg (Fig. 3) are two of the leading scientists whose studies revealed the complex elegance of quorum sensing in Vibrio and Pseudomonas species. Other organisms, such as Salmonella, have been shown to activate the AI-2 pathway of V. harveyi, dramatically supporting the concept of cross-species communication. A recent report by Ian Joint and his colleagues has shown that bacteria can even communicate across the prokaryotic-eukaryotic boundary. The green seaweed Enteromorpha (a eukaryote) produces motile zoospores that explore and attach to Vibrio anguillarum bacterial cells in biofilms (Fig. 4). They attach and remain there because the bacterial cells produce acetyl homoserine lactone molecules that the zoospores sense. Part of the evidence for this interkingdom communication involved showing that the zoospores would even attach to biofilms of E. coli carrying the Vibrio genes for the synthesis of acetyl homoserine lactone. The implications of possible inter-
Autoinducer AI-2
Inhibits autophosphorylation
H1
P
P
LuxN D1 (Sensor kinase) LuxLM (synthesizes autoinducer)
H1 LuxQ D1
P
H2 LuxU
LuxS (synthesizes autoinducer)
P
kingdom conversations are staggering. Do our normal flora “speak” to us? Do we “speak” back? For further discussion of molecular communication between prokaryotes and eukaryotes, see Chapter 21.
Courtesy of University of Iowa Medical Photography Unit
C h a p t er 1 0
D2 LuxO
Peter Greenberg, one of the pioneers of cellcell communication research. Peter Greenberg, first at the University of Iowa and now at the University of Washington, has studied quorum sensing in Vibrio species and various other pathogenic bacteria, such as Pseudomonas. Figure 3
Repressor
luxCDABE
LuxR
(+)
Reprinted with permission from AAAS
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Luciferase
The two quorum-sensing systems of V. harveyi. In the absence of autoinducers (AI-1 and AI-2), both sensor kinases trigger converging phosphorylation cascades that end with the phosphorylation of LuxO. Phosphorylated LuxO (LuxO-P) activates a repressor that inhibits expression of the luciferase genes. As autoinducer concentrations increase, they inhibit autophosphorylation of the sensor kinases and the phosphorylation cascade. As a result, repressor levels decrease, which allows the LuxR protein to activate the lux operon.
Figure 1
©Denise Applewhite
xxii
Bonnie Bassler (left) of Princeton University was instrumental in characterizing interspecies communication between bacteria.
Figure 2
Enteromorpha zoospores (red), a type of algae, attach to biofilm-producing bacteria (blue) in response to lactones produced by the bacteria.
Figure 4
SPECIAL TOPICS
Optional “Special Topics” boxes show the process of science and give a human face to the research. Topics are as diverse as scientists discovering “quorum sensing” in pathogenesis (ST 10.2) and undergraduate researchers investigating mycorrhizae in wetland soil (ST 22.1). Whether historical in focus or providing more detail about cutting-edge science, “Special Topics” give students extra background and detail to help them appreciate the dynamic nature of microbiology.
Chapter 3
Cell Structure and Function 3.1 3.2 3.3 3.4 3.5 3.6 3.7
The Bacterial Cell: An Overview How We Study the Parts of Cells The Cell Membrane and Transport The Cell Wall and Outer Layers The Nucleoid and Gene Expression Cell Division Specialized Structures
Microbial cells face extreme challenges from their environment, enduring rapid changes in temperature and salinity, and pathogens face the chemical defenses of their hosts. To meet these challenges, microbes build complex structures, such as a cell envelope with tensile strength comparable to steel. Within the cytoplasm, molecular devices such as the the ribosome build and expand the cell. With just a few thousand genes in its genome, how does a bacterial cell grow and reproduce? Bacteria coordinate their DNA replication through the DNA replisome and the cell fission ring. Other devices, such as
CHAPTE R OPE N E RS
flagellar propellers, enable microbial cells to compete, to communicate, and even to cooperate in building biofilm communities. Discoveries of cell form and function have exciting applications for medicine and biotechnology. The structures of ribosomes and cell envelope materials provide targets for new antibiotics. And devices such as the rotary ATP synthase inspire “nanotechnology,” the design of molecular machines.
The filamentous cyanobacterium Anabaena sp. was engineered to make a cell division protein, FtsZ, fused to green fluorescent protein (GFP). FtsZ-GFP proteins form a ring-like structure around the middle of each cell, where it prepares to divide. Source: Samer
The title page of each chapter presents an intriguing photo related to a recent research article or current application of the chapter topic. For example, Chapter 3 opens with a fluorescence micrograph of Anabaena in which the cell division protein FtsZ fused to “green fluorescent protein” (GFP) fluoresces around the division plane of each cell.
Sakr, et al. 2006. J. Bacteriol. 188.
73 5 µm
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B act e ri al C u l t u re , G ro w t h , an d D e ve l o p m e n t
Dissolution
Planktonic forms
TO SU M MAR I Z E Attachment monolayer
Figure 4.24
Biofilm development.
Microcolonies
Exopolysaccharide (EPS) production
Maturation
Biofilm development in Pseudomonas.
fi rmly attach to the surface. As more and more cells bind to the surface, they can begin to communicate with each other by sending and receiving chemical signals in a process called quorum sensing. These chemical signal molecules are continually made by individual cells. Once the population reaches a certain number (analogous to an organizational qì uorum ”), the chemical signal reaches a specific concentration that the cells can sense. This triggers genetically regulated changes that cause cells to bind tenaciously to the substrate and to each other. Next, the cells form a thick extracellular matrix of polysaccharide polymers and entrapped organic and inorganic materials. These exopolysaccharides (EPSs), such as alginate produced by P. aeruginosa and colanic acid produced by E. coli, increase the antibiotic resistance of residents within the biofi lm. As the biofi lm matures, the amalgam of adherent bacteria and matrix takes on complex three-dimensional forms such as columns and streamers, creating channels through which nutrients flow. Sessile cells in a biofi lm chemically “talk” to each other in order to build microcolonies and keep water channels open. Little is known about how a biofi lm dissolves, although the process is thought to be triggered by starvation. P. aeruginosa produces an alginate lyase that can strip away the EPSs, but the regulatory pathways involved in releasing cells from biofi lms are not clear. It is important to keep in mind that most biofi lms in nature are consortia of several species. Multispecies biofi lms certainly demand interspecies communication, and individual species may perform specialized tasks in the community. Organisms adapted to life in extreme environments also form biofilms. Members of Archaea form biofilms in acid mine drainage (pH 0), where they contribute to the recycling of sulfur, and cyanobacterial biofilms are common in thermal springs. Suspended particles called “marine snow” are found in ocean environments and appear to be floating biofilms comprising many organisms that have
not yet been identified. The particles appear capable of methanogenesis, nitrogen fi xation, and sulfide production, indicating that biofilm architecture can allow anaerobic metabolism to occur in an otherwise aerobic environment.
This feature ensures that students understand the key concepts of each section before they continue with the reading.
Biofi lms TO SU M MAR I Z E: ■
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Biofilms are complex multicellular surface-attached microbial communities. Chemical signals enable bacteria to communicate (quorum sensing) and in some cases to form biofi lms. Biofilm development involves adherence of cells to a substrate, formation of microcolonies, and, ultimately, formation of complex channeled communities that generate new planktonic cells.
4.7
Cell Differentiation
Many bacteria faced with environmental stress undergo complex molecular reprogramming that includes changes in cell structure. Some species, like E. coli, experience relatively simple changes in cell structure, such as the formation of smaller cells or thicker cell surfaces. However, select species undergo elaborate cell differentiation processes. An example is Caulobacter crescentus, whose cells convert from the swimming form to the holdfast form before cell division. Each cell cycle then produces one sessile cell attached to its substrate by a holdfast, while its sister cell swims off in search of another habitat. Other species undergo far more elaborate transformations. The endospore formers generate heat-resistant capsules (spores) that can remain in suspended animation for thousands of years. Yet another group, the actinomycetes, form complex multicellular structures analogous to those of eukaryotes. In this case, cell struc-
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Student Resources
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StudySpace. wwnorton.com/studyspace This student website includes multiple-choice quizzes, process animations, vocabulary flashcards, indices of the Weblink reference sites from the text, and prominent links to Microbial Biorealm.
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Process Animations. Developed specifically for Microbiology: An Evolving Science, these animations bring key figures from the text to life, presenting key microbial processes in a dynamic format. The animations can be enlarged to full-screen view, and include VCR-like controls that make it easy to control the pace of animation.
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Weblink Icons throughout the text point students to the student website, which serves as a portal to websites where they can fi nd more information on a host of topics. Each link was reviewed and approved by the authors to ensure that only high-interest, high-quality sites were selected.
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Microbial Biorealm and Viral Biorealm. A website maintained at Kenyon College provides information on several hundred genera of microbes and viruses, to which interested students have the opportunity to contribute. Pages are monitored and edited by microbiologists at Kenyon.
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Ebook. Same great book at half the price. Microbiology: An Evolving Science is also available as an ebook from nortonebooks.com. With a Norton ebook, students can electronically highlight text, use sticky notes, and work with fully zoomable images from the book.
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Instructor Resources ■
Norton Media Library Instructor’s CD-ROM: • Drawn Art and Photographs. Digital fi les of all drawn art and most photographs are available to adopters of the text. • Process Animations. Developed specifically for Microbiology: An Evolving Science, these animations bring key figures from the text to life, presenting key microbial processes in a dynamic format. The animations can be enlarged to full-screen view and include VCR-like controls that make it easy for instructors to control the pace of animation during lecture. • Editable PowerPoint Lectures for each chapter.
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Norton Resource Library Instructor’s Website. wwnorton.com/instructors Maintained as a service to our adopters, this password-protected instructor website offers book-specific materials for use in class or within WebCT, Blackboard, or course websites. The resources available online are the same as those offered on the Norton Media Library CD-ROM.
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Instructor’s Manual. The manual includes chapter overviews, answers to endof-chapter questions, and a test bank of 2,000 questions. Authored by Kathleen Campbell at Emory University.
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Electronic Test Bank. The Test Bank includes 2,000 questions in ExamView Assessment Suite format.
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Blackboard Learning System Coursepacks. These coursepacks include classroom-ready content.
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Transparencies. A subset of the figures in the text are available as color acetates.
Acknowledgments We are very grateful for the help of many people in developing and completing the book. Our fi rst editor at Norton, John Byram, helped us defi ne the aims and scope of the project. Vanessa Drake-Johnson helped us shape the text, supported us in developing a strong art program, and conceived the title. Mike Wright spared no effort to bring the project to completion and to the attention of our colleagues. Our developmental editors, Philippa Solomon and Carol Pritchard-Martinez, con-
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tributed greatly to the clarity of presentation. Philippa’s strength in chemistry was invaluable in improving our presentation of metabolism. Trish Marx and the photo researchers did a heroic job of tracking down all kinds of images from sources all over the world. Our colleague Kathy Gillen provided exceptional expertise on review topics for the appendices and wrote outstanding review questions for the student website. April Lange’s coordination of electronic media development has resulted in a superb suite of resources for students and instructors alike. We thank Kathleen Campbell for authoring an instructor’s manual that demonstrates a clear understanding of our goals for the book, and Lisa Rand for editing it. Without Thom Foley’s incredible attention to detail, the innumerable moving parts of this book would never have become a fi nished book. Marian Johnson, Norton’s managing editor in the college department, helped coordinate the complex process involved in shaping the manuscript over the years. Chris Granville ably and calmly managed the transformation of manuscript to fi nished product in record time. Matthew Freeman coordinated the transfer of many drafts among many people. Steve Dunn and Betsy Twitchell have been effective advocates for the book in the marketplace. Finally, we thank Roby Harrington, Drake McFeely, and Julia Reidhead for their support of this book over its many years of development. For the quality of our illustrations we thank the many artists at Precision Graphics, who developed attractive and accurate representations and showed immense patience in getting the details right. We especially thank Kirsten Dennison for project management; Karen Hawk for the layout of every page in the book; Kim Brucker and Becky Oles for developing the art style and leading the art team; and Simon Shak for his rendering of the molecular models based on PDB fi les, including some near-impossible structures that we requested. We thank the numerous colleagues over the years who encouraged us in our project, especially the many attendees at the Microbial Stress Gordon Conferences. We greatly appreciate the insightful reviews and discussions of the manuscript provided by our colleagues, and the many researchers who contributed their micrographs and personal photos. We especially thank the American Society for Microbiology journals for providing many valuable resources. Reviewers Bob Bender, Bob Kadner, and Caroline Harwood offered particularly insightful comments on the metabolism and genetics sections, and James Brown offered invaluable assistance in improving the coverage of microbial evolution. Peter Rich was especially thoughtful in providing materials from the archive of Peter Mitchell. We also thank the following reviewers: Laurie A. Achenbach, Southern Illinois University, Carbondale Stephen B. Aley, University of Texas, El Paso Mary E. Allen, Hartwick College Shivanthi Anandan, Drexel University Brandi Baros, Allegheny College Gail Begley, Northeastern University Robert A. Bender, University of Michigan Michael J. Benedik, Texas A&M University George Bennett, Rice University Kathleen Bobbitt, Wagner College James Botsford, New Mexico State University Nancy Boury, Iowa State University of Science and Technology Jay Brewster, Pepperdine University James W. Brown, North Carolina State University Whitney Brown, Kenyon College undergraduate Alyssa Bumbaugh, Pennsylvania State University, Altoona Kathleen Campbell, Emory University Alana Synhoff Canupp, Paxon School for Advanced Studies, Jacksonville, FL Jeffrey Cardon, Cornell College
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Tyrrell Conway, University of Oklahoma Vaughn Cooper, University of New Hampshire Marcia L. Cordts, University of Iowa James B. Courtright, Marquette University James F. Curran, Wake Forest University Paul Dunlap, University of Michigan David Faguy, University of New Mexico Bentley A. Fane, University of Arizona Bruce B. Farnham, Metropolitan State College of Denver Noah Fierer, University of Colorado, Boulder Linda E. Fisher, late of the University of Michigan, Dearborn Robert Gennis, University of Illinois, Urbana-Champaign Charles Hagedorn, Virginia Polytechnic Institute and State University Caroline Harwood, University of Washington Chris Heffelfi nger, Yale University graduate student Joan M. Henson, Montana State University Michael Ibba, Ohio State University Nicholas J. Jacobs, Dartmouth College Douglas I. Johnson, University of Vermont Robert J. Kadner, late of the University of Virginia Judith Kandel, California State University, Fullerton Robert J. Kearns, University of Dayton Madhukar Khetmalas, University of Central Oklahoma Dennis J. Kitz, Southern Illinois University, Edwardsville Janice E. Knepper, Villanova University Jill Kreiling, Brown University Donald LeBlanc, Pfi zer Global Research and Development (retired) Robert Lausch, University of South Alabama Petra Levin, Washington University in St. Louis Elizabeth A. Machunis-Masuoka, University of Virginia Stanley Maloy, San Diego State University John Makemson, Florida International University Scott B. Mulrooney, Michigan State University Spencer Nyholm, Harvard University John E. Oakes, University of South Alabama Oladele Ogunseitan, University of California, Irvine Anna R. Oller, University of Central Missouri Rob U. Onyenwoke, Kenyon College Michael A. Pfaller, University of Iowa Joseph Pogliano, University of California, San Diego Martin Polz, Massachusetts Institute of Technology Robert K. Poole, University of Sheffield Edith Porter, California State University, Los Angeles S. N. Rajagopal, University of Wisconsin, La Crosse James W. Rohrer, University of South Alabama Michelle Rondon, University of Wisconsin-Madison Donna Russo, Drexel University Pratibha Saxena, University of Texas, Austin Herb E. Schellhorn, McMaster University Kurt Schesser, University of Miami Dennis Schneider, University of Texas, Austin Margaret Ann Scuderi, Kenyon College Ann C. Smith Stein, University of Maryland, College Park John F. Stolz, Duquesne University Marc E. Tischler, University of Arizona
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Monica Tischler, Benedictine University Beth Traxler, University of Washington Luc Van Kaer, Vanderbilt University Lorraine Grace Van Waasbergen, The University of Texas, Arlington Costantino Vetriani, Rutgers University Amy Cheng Vollmer, Swarthmore College Andre Walther, Cedar Crest College Robert Weldon, University of Nebraska, Lincoln Christine White-Ziegler, Smith College Jianping Xu, McMaster University Finally, we offer special thanks to our families for their support. Joan’s husband Michael Barich offered unfailing support, and her son Daniel Barich contributed photo research, as well as filling the indispensable role of technical director for the Microbial Biorealm website. John’s wife Zarrintaj (“Zari”) Aliabadi contributed to the text development, especially the sections on medical microbiology and public health.
To the Reader: Thanks! We greatly appreciate your selection of this book as your introduction to the science of microbiology. This is a fi rst edition, and as such can certainly benefit from the input of readers. We welcome your comments, especially if you find text or figures that are in error or unclear. Feel free to contact us at the addresses listed below. Joan L. Slonczewski
[email protected] John W. Foster
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About the Authors
JOAN L . SLONCZEWSKI received her B.A. from Bryn Mawr College and her Ph.D. in Molecular Biophysics and Biochemistry from Yale University, where she studied bacterial motility with Robert M. Macnab. After postdoctoral work at the University of Pennsylvania, she has since taught undergraduate microbiology in the Department of Biology at Kenyon College, where she earned a Silver Medal in the National Professor of the Year program of the Council for the Advancement and Support of Education. She has published numerous research articles with undergraduate coauthors on bacterial pH regulation, and has published five science fiction novels including A Door into Ocean, which earned the John W. Campbell Memorial Award. She serves as At-large Member representing Divisions on the Council Policy Committee of the American Society for Microbiology, and is a member of the Editorial Board of the journal Applied and Environmental Microbiology.
JOHN W. FOSTER received his B.S. from the Philadelphia College of Pharmacy and Science (now the University of the Sciences in Philadelphia), and his Ph.D. from Hahnemann University (now Drexel University School of Medicine), also in Philadelphia, where he worked with Albert G. Moat. After postdoctoral work at Georgetown University, he joined the Marshall University School of Medicine in West Virginia; he is currently teaching in the Department of Microbiology and Immunology at the University of South Alabama College of Medicine in Mobile, Alabama. Dr. Foster has coauthored three editions of the textbook Microbial Physiology and has published over 100 journal articles describing the physiology and genetics of microbial stress responses. He has served as Chair of the Microbial Physiology and Metabolism division of the American Society for Microbiology and is a member of the editorial advisory board of the journal Molecular Microbiology.
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Microbiology An Evolving Science
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Part 1
The Microbial Cell AN INTERVIEW WITH Courtesy of Rita Colwell
RITA COLWELL: THE GLOBAL IMPACT OF MICROBIOLOGY
Rita Colwell is Distinguished Professor at the University of Maryland and Johns Hopkins University and served as director of the USA National Science Foundation from 1998 to 2004. Colwell’s decades of research on Vibrio cholerae, the causative agent of cholera, have revealed its natural ecology, its genome sequence, and ways to control it. Colwell originated the concept of viable but nonculturable microorgan-
Rita Colwell, former director of the National Science Foundation.
isms, microbial cells that metabolize but cannot be cultured in the laboratory. She is now chairman of the board of Canon US Life Sciences, Inc., and she represents the American Society for Microbiology at the United Nations Educational, Scientific and Cultural Organization (UNESCO).
Why did you decide to make a career in microbiology?
I was fi rst inspired by the report of my college roommate at Purdue University about a wonderful bacteriology professor, Dr. Dorothy Powelson, probably one of only two women at Purdue who were full professors at the time. I enrolled in Powelson’s course and was truly inspired by this remarkable woman who was so interested in microbiology and made it fascinating for her students. How did you choose to study Vibrio cholerae? What makes this organism interesting?
I chose to study Vibrio cholerae as a result of my having become an “expert” on vibrios through my graduate dissertation on marine microorganisms. Vibrios were the most readily culturable of the marine bacteria and were therefore considered the most dominant. Of course, new information indicates that although vibrios are the dominant bacteria in many estuarine areas, there are other organisms that are very difficult to culture that are important as well.
When I took my fi rst faculty position at Georgetown University, a friend of mine at NIH, Dr. John Feeley, suggested that I study Vibrio cholerae. What makes V. cholerae interesting is that it is a human pathogen of extremely great importance, yet resides naturally in estuaries and coastal areas of the world. What is it like to study this organism?
Vibrio cholerae is naturally occurring (in the environment outside humans) and therefore can never be eradicated; it carries out important functions in the environment, and significant among these is its ability to digest chitin, the structural component of shellfish and many zooplankton. It is at once a “recycling agent” and a public health threat in the form of the massive epidemics of cholera that it causes. You led an international collaboration in Bangladesh training women to avoid cholera by filtering water through sari cloth. How did the sari cloth filtration project come about?
It came about through collaboration with the International Centre for Diarrhoeal Diseases, Bangladesh, located in Dhaka, Bangladesh, and the Mattlab Field Laboratory, which is located in the village area of Mattlab, Bangladesh. Our work had shown that Vibrio cholerae is associated with environmental zooplankton, namely, the copepod. The notion that the copepods are large and could be filtered out and therefore lead to reduced incidence of cholera was a result of my work on the vibrios and the relationships described by my students, notably, Dr. Anwar Huq, who did his thesis on Vibrio cholerae attachment to copepods. Anwar Huq is now an associate professor at the University of Maryland. An important collaborator was Nell Roberts, an outstanding public health microbiologist at Lake Charles, Louisiana, working on public health problems. Nell, Professor Xu (a colleague from Qingdao, China), and I did the critical experiment showing the presence of Vibrio cholerae in water from which blue crabs had been harvested—the cause of an outbreak of cholera in Louisiana back in 1982. We were able to use fluorescent antibody to show the presence of the vibrio on copepods in the water. From there, the idea of sari cloth came about in searching for a very inexpensive fi lter for use by village
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the president and confirmed by the U.S. Senate. As a microbiologist, I was able to bring a molecular understanding of biology to the NSF, while as an interdisciplinary researcher, I was attuned to the needs of all aspects of science, from astronomy to physics. In the biological sciences, my major impact was in launching the Biocomplexity Initiative, which has been enormously productive and continues to yield new information on biological systems, including those of microbiology.
Vibrio cholerae bacteria (left) colonize copepods such as this one (right).
What do you think are the most exciting areas for students entering microbiology today?
women in Bangladesh. We were able to show that folded sari cloth yielded a 20-micrometer (µm) mesh net. Because plankton are 200 µm or more in size, we could fi lter them out. The hypothesis that I came up with was that by removing the copepods and associated particulates, we could reduce cholera, which proved to be the case.
Microbial diversity and microbial population studies are two emerging areas of huge interest that will lead to a better understanding of microbial evolution and development.
ology laboratory would be negative for their presence. What are the challenges of marine microbiology today? How does marine microbiology impact human health?
What are “viable but nonculturable” organisms?
Courtesy of Rita Colwell
Viable but nonculturable is a state into which gram-negative microorganisms transform under adverse conditions in the environment. In this state, the bacteria are unable to be cultured, even though they remain viable and potentially pathogenic. Hence, they pose a public health risk, since routine tests done in a bacteri-
A Bangladeshi woman filters water through sari cloth. Colwell’s graduate student Anwar Huq compares the filtered and unfiltered water.
The challenges of marine microbiology today are to understand and catalog the extraordinary diversity of marine microorganisms. The world’s oceans function in large part as a result of the activities of marine organisms. Marine microbiology impacts human health because of the many pathogens naturally occurring in the environment. But more than that, marine microorganisms may well be the cycling agent that keeps the blue planet inhabitable for humans. Marine microorganisms actively cycle carbon, nitrogen, phosphorus, and other elements in our oceans and even play a role in the weather by producing dimethyl sulfoxide (DMSO), which is involved in cloud formation and moisture condensation. Why did you move to the National Science Foundation? What difference did you make as a microbiologist heading NSF?
I was asked by the president of the United States to serve as director of the National Science Foundation (NSF). It is a position appointed by
What advice do you have for today’s students?
Develop an expertise as an undergraduate in some area of science, whether it be biology, chemistry, mathematics, physics, or some other area of science or engineering, and be creative and curious about other disciplines. The world of the future will be interdisciplinary and multidisciplinary. How does your family relate to your work?
I have been happily married ever since I graduated from college! We have two daughters. One is a medical doctor (pediatrician). She recently was named an outstanding physician scholar and voted the best physician in her fellowship class by her colleagues. She also worked in Africa on delivery of health care to women in Tanzania for her PhD. We are equally proud of our other daughter, who earned a PhD in evolutionary biology and now works for the U.S. Geological Survey, cataloging rare plants in Yosemite National Park and Forest. 3
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Chapter 1
Microbial Life: Origin and Discovery 1.1 1.2 1.3 1.4 1.5 1.6
From Germ to Genome: What Is a Microbe? Microbes Shape Human History Medical Microbiology Microbial Ecology The Microbial Family Tree Cell Biology and the DNA Revolution
Life on Earth began early in our planet’s history with microscopic organisms, or microbes. Microbial life has since shaped our atmosphere, our geology, and the energy cycles of all ecosystems. A human body contains ten times as many microbes as it does human cells, including numerous tiny bacteria on the skin and in the digestive tract. Throughout history, humans have had a hidden partnership with microbes ranging from food production and preservation to mining for precious minerals. Yet throughout most of our history, humans were unaware that microbes even existed. To study these unseen organisms required a microscope, first developed in the 1600s. In the nineteenth century—the “golden age” of microbiology—microscopes revealed the tiny organisms at work in our bodies and in our ecosystems. The twentieth century saw the rise of microbes as the engines of biotechnology. Microbial discoveries led to recombinant DNA and revealed the secrets of the first sequenced genomes.
Lactobacillus salivarius bacteria grow normally in human skin, where they produce bacteriocins, compounds that protect us from disease-causing bacteria. Their multi-part genome was sequenced by Marcus Claesson and colleagues. (Claesson, et al. 2006. Proceedings of the National Academy of Sciences 103:6718.) Scanning electron micrograph is from Sinead Leahy and D. John, Trinity College, Dublin. Cell length, 1–2 µm.
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Yet before microscopes were developed in the seventeenth century, we humans were unaware of the unseen living organisms that surround us, that float in the air we breathe and the water we drink, and that inhabit our own bodies. Microbes generate the very air we breathe, including nitrogen gas and much of the oxygen and carbon dioxide. They fi x nitrogen into forms used by plants, and they make essential vitamins, such as vitamin B12. Microbes are the primary producers of major food webs, particularly in the oceans; when we eat fi sh, we indirectly consume tons of algae at the base of the food chain. At the same time, virulent pathogens take our lives. Despite all the advances of modern medicine and public health, microbial disease remains the number one cause of human mortality. In the twentieth century, the science of microbiology exploded with discoveries, creating entire new fields such as genetic engineering. The promise—and pitfalls—were dramatized by Michael Crichton’s best-selling science fiction novel and fi lm, The Andromeda Strain (1969; fi lmed in 1971). In The Andromeda Strain, scientists at a top-secret laboratory race to identify a deadly pathogen from outer space—or perhaps from a biowarfare lab (Fig. 1.3A). The fi lm prophetically depicts the computerization of medical research, as well as the emergence of pathogens, such as the human immunodeficiency virus (HIV), that can yet defeat the efforts of advanced science. Today, we discover surprising new kinds of microbes deep underground and in places previously thought uninhabitable, such as the hot springs of Yellowstone National Park (Fig. 1.3B). These microbes shape our biosphere and provide new tools that impact human society. For example, the use of heat-resistant bacterial DNA polymerase (a DNA-replicating enzyme) in a technique called the polymerase chain reaction (PCR) allows us to detect minute amounts of DNA in traces of blood or fossil bone. Microbial technologies led us from the discovery of the double helix to the sequence of the human genome, the total genetic information that defi nes our species. In this chapter, we introduce the concept of a microbe and the question of how microbial life originated. We then survey the history of human discovery of the role microbes play in disease and in our ecosystems. Finally, we address the exciting century of molecular microbiology, in which microbial genetics and genomics have transformed the face of modern biology and medicine. NASA
In 2004, the two Mars Exploration Rovers, Spirit and Opportunity, landed on the planet Mars (Fig. 1.1). The rovers carried scientific instruments to test Martian rocks, to identify minerals and to assess the size and shape of sedimentary particles. The identity of the minerals, as well as their particle structure, could yield clues as to whether the Martian surface had ever been shaped by liquid water. Evidence for water would support the possible existence of living microbes. Why do we care whether microbes exist on Mars? The discovery of life beyond Earth would fundamentally change how we see our place in the universe. The observation of Martian life could yield clues as to the origin of our own biosphere and expand our knowledge of the capabilities of living cells on our own planet. As of this writing, the existence of microbial life on Mars remains unknown, but here on Earth, many terrestrial microbes remain as mysterious as Mars. Barely 0.1% of the microbes in our biosphere can be cultured in the laboratory; even the digestive tract of a newborn infant contains species of bacteria unknown to science. Our “exploration rovers” for microbiology include, for example, new tools of microscopy and the sequencing of microbial DNA. On Earth, the microscope reveals microbes throughout our biosphere, from the superheated black smoker vents at the ocean floor to the subzero ice fields of Antarctica. Bacteria such as Escherichia coli live in our intestinal tract, while algae and cyanobacteria turn ponds green (Fig. 1.2). Protists are the predators of the microscopic world. And viruses such as papillomavirus cause disease, as do many bacteria and protists.
Figure 1.1 Is there microbial life on Mars? On February 9, 2004, the Mars Exploration Rover Spirit (inset) photographed this windswept surface of the planet Mars. Rock samples were tested for distinctive minerals that are formed by the action of water. The presence of liquid water today would increase the chance that microbial life exists on Mars.
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1.1
From Germ to Genome: What Is a Microbe?
From early childhood, we hear that we are surrounded by microscopic organisms, or “germs,” that we cannot see. What are microbes? Our modern concept of a
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Representative microbes. A. Filamentous cyanobacteria produce oxygen for planet Earth (dark-field light micrograph). B. Escherichia coli bacteria colonize the stomata of a lettuce leaf cell (scanning electron microscopy). C. Stentor is a protist, a eukaryotic microbe. Cilia beat food into its mouth. D. Halophilic archaea, a form of life distinct from bacteria and eukaryotes, grow at extremely high salt concentration. E. Mushrooms are multicellular fungi (eukaryotes). They serve the ecosystem as decomposers. F. Papillomavirus causes genital warts, an infectious disease commonly acquired by young adults (model based on electron microscopy).
Figure 1.2
Tony Craddock/Photo Researchers, Inc.
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Universal Studios, 1971
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Microbial discovery: science fiction and science fact. A. In The Andromeda Strain, medical scientists try to feed a baby who was infected by a deadly pathogen from outer space. While the details of the pathogen are imaginary, the film’s approach to identifying the mystery organism captures the spirit of actual investigations of emerging diseases. B. Yellowstone National Park hot springs are surrounded by mats of colorful microbes that grow above 80°C in waters containing sulfuric acid. Bacteria discovered at Yellowstone produce enzymes used in polymerase chain reaction (PCR), a technique of DNA amplification.
Figure 1.3
microbe has deepened through two major research tools: advanced microscopy and the sequencing of genomic DNA. Modern microscopy is covered in Chapter 2, and microbial genetics and genomics are presented in Chapters 6–12.
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A Microbe Is a Microscopic Organism A microbe is commonly defi ned as a living organism that requires a microscope to be seen. Microbial cells range in size from millimeters (mm) down to 0.2 micrometers
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Chapter 1
(µm), and viruses may be tenfold smaller (Table 1.1). Some microbes consist of a single cell, the smallest unit of life, a membrane-enclosed compartment of water solution containing molecules that carry out metabolism. Each microbe contains in its genome the capacity to reproduce its own kind. Our simple defi nition of a microbe, however, leaves us with contradictions. ■
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Super-size microbial cells. Most single-celled organisms require a microscope to render them visible and thus fit the definition of “microbe.” Nevertheless, some species of protists and algae, and even some bacterial cells are large enough to see with the naked eye (Fig. 1.4). The marine sulfur bacterium Thiomargarita namibiensis, called the sulfur pearl of Namibia, grows as large as the head of a fruit fly. Even more surprising, a single-celled plant, the “killer algae” Caulerpa taxifolia, spreads through the coastal waters of California. The single cell covers many acres with its leaf-like cell parts. Microbial communities. Many microbes form complex multicellular assemblages, such as mushrooms, kelps, and biofi lms. In these structures, cells are differentiated into distinct types that complement each other’s function, as in multicellular organisms. And yet, some multicellular worms and arthropods require a microscope to see but are not considered microbes.
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Viruses. A virus consists of a noncellular particle containing genetic material that takes over the metabolism of a cell to generate more virus particles. Although viruses are considered microbes, they are not fully functional cells. Some viruses consist of only a few molecular parts, whereas others, such as the Mimivirus infecting amebas (also spelled amoebae) show the size and complexity of a cell.
NOTE: Each section contains questions to think about. These thought questions may have various answers. Possible responses are posted at the back of the book.
THOUGHT QUESTION 1.1 The minimum size of known microbial cells is about 0.2 µm. Could even smaller cells be discovered? What factors may determine the minimum size of a cell? THOUGHT QUESTION 1.2 If viruses are not functional cells, are they truly “alive”? In practice, our defi nition of a microbe derives from tradition as well as genetic considerations. In this book, we consider microbes to include prokaryotes (cells lacking a nucleus, including bacteria and archaea) as well as certain classes of eukaryotes (cells with a nucleus) that
Table 1.1 Sizes of some microbes. Microbe
Description
Approximate size
Varicella-zoster virus 1 Prochlorococcus Rhizobium Spirogyra Pelomyxa (an ameba)
Virus that causes chicken pox and shingles Photosynthetic marine bacteria Bacteria that fix N2 in symbiosis with leguminous plants Filamentous algae found in aquatic habitat Protists found in solid or aquatic habitat
100 nanometers (nm) = 10–7 meter (m) 500 nm = 5 × 10–7 m 1 micrometer (µm) = 10–6 m 40 µm = 4 × 10–5 m (cell width) 5 millimeters (mm)
Giant microbial cells. A. The largest known bacterium, Thiomargarita namibiensis, a marine sulfur metabolizer, nearly the size of the head of a fruit fly. B. “Killer algae,” Caulerpa taxifolia. All the fronds constitute a single cell, the largest single-celled organism on Earth. Growing off the coast of California.
Figure 1.4 Rachel Woodfield, Merkel & Associates
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Thiomargarita namibiensis Heide N. Shulz, University of Hanover, Germany
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Bacteria Prokaryotes Common ancestor
Archaea Algae and plants Eukarya
Fungi and animals
Eukaryotes
Protists
Figure 1.5 Three domains of life. Analysis of DNA sequence reveals the ancient divergence of three domains of living organisms: Bacteria and Archaea (both prokaryotes) and Eukarya (eukaryotes). The color code shown here is used throughout this book to indicate the three domains.
include simple multicellular forms: algae, fungi, and protists (Fig. 1.5). The bacteria, archaea, and eukaryotes— known as the three domains—diverged from a common ancestral cell. We also discuss viruses and related infectious particles (Chapters 6 and 11). DNA. The sequence of base pairs in DNA encodes all the genetic information of an organism.
Figure 1.6 NOTE: The formal names of the three domains are
Bacteria, Archaea, and Eukarya. Members of these domains are called bacteria (singular, bacterium), archaea (singular, archaeon), and eukaryotes (singular, eukaryote), respectively. The microbiology literature includes alternative spellings for some of these terms, such as “archaean” and “eucaryote.”
Microbial Genomes Are Sequenced Our understanding of microbes has grown tremendously through the study of their genomes. A genome is the total genetic information contained in an organism’s chromosomal DNA (Fig. 1.6). By determining the sequence of genes in a microbe’s genome, we learn a lot about how that microbe grows and associates with other species. For
example, if a microbe’s genome includes genes for nitrogenase, a nitrogen-fi xing enzyme, that microbe probably can fi x nitrogen from the atmosphere into compounds that plants can assimilate into protein. And by comparing DNA sequences, we can measure the degree of relatedness between different species based on the time since they diverged from a common ancestor. Historically, the fi rst genomes to be sequenced were those of viruses. The fi rst genome whose complete DNA sequence was determined was that of a bacteriophage (a virus that infects bacteria), bacteriophage φX174. The DNA sequence of φX174 was determined in 1977 by Fred Sanger (Fig. 1.7A), who shared the 1980 Nobel Prize in Chemistry with Walter Gilbert and Paul Berg for developing the method of DNA sequence analysis. The
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Microbial genome sequencers. A. Fred Sanger, who shared the 1980 Nobel Prize in Chemistry for devising the method of DNA sequence analysis that is the basis of modern genome sequencing. He is reading sequence data from bands of DNA separated by electrophoresis. B. Claire Fraser-Liggett, past president of The Institute for Genomic Research (TIGR), which completed the sequences of H. influenzae and many other microbial genomes.
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Figure 1.7
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Mic r o b ia l L ife : Orig in and Disc ov ery
genome of bacteriophage φX174 includes over 5,000 base pairs, with just ten genes encoding proteins (Fig. 1.8A). Its genome is so compact that several gene sequences actually overlap, sharing the same segment of nucleotides (for example, genes C and K).
The Nobel Prize website presents the lectures and autobiographies of all Nobel Prize winners, including many who were awarded for advances in microbiology.
A. Genome of bacteriophage φX174
Minor spike protein
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DNA synthesis protein
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K DNA maturation protein
Cell lysis protein DNA condensation
B. Genome of Haemophilus influenzae Sma I Not I Sma I Sma I 1800000 1 1700000
Gene classification based on functional categories 100000 Rsr II Sma I 200000
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Translation Transcription Replication Regulatory functions Cell envelope Cellular processes Transport/binding proteins Central intermediary metabolism Amino acid biosynthesis Purines, pyrimidines, nucleosides and nucleotides Energy metabolism Lipid metabolism Secondary metabolite biosynthesis, transport Other categories General function prediction only Function unknown
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The first sequenced genomes. A. The first organism whose genome sequence was determined was bacteriophage φX174, a virus that grows in Escherichia coli (virus diameter, 27 nm). The entire DNA sequence of φX174 contains 5,386 base pairs specifying only ten genes (here labeled A–H and J–K), nine of whose functions have since been determined. Note the highly compact genome, with several overlapping genes. B. The genome of Haemophilus influenzae Rd, a bacterium that causes ear infections and meningitis, was the first DNA sequence completed for a cellular organism (inset, colorized electron micrograph). The genome of H. influenzae contains nearly 2 million base pairs specifying approximately 1,743 genes, which are expressed to make protein and RNA products. The annotated sequence of the genome appears on the website of the National Center for Biotechnology Information. Colored bars indicate gene sequences throughout.
Figure 1.8
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Nearly two decades passed before scientists completed the fi rst genome sequence of a cellular microbe, Haemophilus influenzae, a bacterium that causes ear infections and meningitis in children (Fig. 1.8B). The strain of H. influenzae sequenced has nearly 2 million base pairs, which specify about 1,700 genes. The sequence of H. influenzae was determined by a large team of scientists at The Institute for Genomic Research (TIGR) led by Hamilton Smith and Craig Venter, who devised a special computational strategy for assembling large amounts of sequence data. This strategy was later applied to sequencing the human genome. Led by president Claire Fraser-Liggett, (Fig. 1.7B), TIGR sequenced the genomes of numerous microbes, such as Bacillus anthracis, the bacterium that causes anthrax, and Colwellia psychrerythraea, a cold-loving bacterium growing in Antarctic sea ice. The National Center for Biotechnology Information (NCBI) provides free access to all published genome sequences. The growing availability of sequenced genomes at universities and in industry has generated the new field of comparative genomics, which involves the systematic comparison of all genomic sequences of living species, ranging from microbes to Homo sapiens. Comparative genomics reveals a set of core genes shared by all organisms, further evidence that all life on Earth shares a common ancestry.
1.2
Microbes Shape Human History
Throughout most of human history, we were unaware of the microbial world. Microorganisms have shaped human culture since our earliest civilizations. Yeasts and bacte-
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ria have made foods such as bread and cheese, as well as alcoholic beverages (Fig. 1.9A; also discussed in Chapter 16). “Rock-eating” bacteria known as lithotrophs leached copper and other metals from ores exposed by mining, enabling ancient human miners to obtain these metals. The lithotrophic oxidation of minerals for energy generates strong acid, which accelerates breakdown of the ore. Today, about 20% of the world’s copper, as well as some uranium and zinc, are produced by bacterial leaching. Unfortunately, microbial acidification also consumes the stone of ancient monuments (Fig. 1.9B), a process intensified by airborne acidic pollution. Management of microbial corrosion is an important field of applied microbiology. As humans became aware of microbes, our relationship with the microbial world changed in important ways (Table 1.2, pages 14–15). Early microscopists in the seventeenth and eighteenth centuries formulated key concepts of microbial existence, including their means of reproduction and death. In the nineteenth century, the “golden age” of microbiology, key principles of disease pathology and microbial ecology were established that scientists still use today. This period laid the foundation for modern science, in which genetics and molecular biology provide powerful tools for scientists to manipulate microorganisms for medicine and industry.
Microbial Disease Devastates Human Populations Throughout history, microbial diseases such as tuberculosis and leprosy have profoundly affected human demographics and cultural practices (Fig. 1.10). The bubonic plague, which wiped out a third of Europe’s population in the fourteenth century, was caused by Yersinia pestis, a bacterium spread by rat fleas. Ironically, the plague-induced population decline enabled the social
A.
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Production and destruction by microbes. A. Roquefort cheeses ripening in France. B. Statue undergoing decay from the action of lithotrophic microbes. The process is accelerated by acid rain. Cathedral of Cologne, Germany.
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Figure 1.9
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Mic r o b ia l L ife : Orig in and Disc ov ery A.
The Centers for Disease Control, in Atlanta, is the global center of modern medical statistics and epidemiology.
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Private Collection/Bridgeman Art Library
Bridgeman Art Library
transformation that led to the Renaissance, a period of unprecedented cultural advancement. In the nineteenth century, the bacterium Mycobacterium tuberculosis stalked overcrowded cities, and tuberculosis became so common that the pallid appearance of tubercular patients became a symbol of tragic youth in European literature. Today, societies throughout the world have been profoundly shaped by the epidemic of acquired immunodeficiency syndrome (AIDS), caused by the human immunodeficiency virus (HIV). Historians traditionally emphasize the role of warfare in shaping human destiny, the brilliance of leaders or the advantage of new technology in determining which civilizations rise or fall. Yet the fate of human societies has often been determined by microbes. For example, much of the native population of North America was exterminated by smallpox unwittingly introduced by European invaders. Throughout history, more soldiers have died of microbial infections than of wounds in battle. The significance of disease in warfare was fi rst recognized by the British nurse and statistician Florence Nightingale (1820–1910) (Fig. 1.11). Better known as the founder of professional nursing, Nightingale also founded the science of medical statistics. She used methods invented by French statisticians to demonstrate the high mortality rate due to disease among British soldiers during the Crimean War. Nightingale’s statistics convinced the British government to improve army living conditions and to upgrade the standards of army hospitals. In modern epidemiology, statistical analysis continues to serve as a crucial tool in determining the causes of disease.
Figure 1.10 Microbial disease in history and culture. A. Medieval church procession to ward off the Black Death (bubonic plague). B. The AIDS Memorial Quilt spread before the Washington Monument. Each panel of the quilt memorializes an individual who died of AIDS.
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Figure 1.11 Florence Nightingale, founder of medical statistics. A. Florence Nightingale was the first to use medical statistics to demonstrate the significance of mortality due to disease. B. Nightingale’s polar area chart of mortality data during the Crimean War.
The Florence Nightingale Museum Trust, London
The seventeenth century was a time of growing inquiry and excitement about the “natural magic” of science and patterns of our world, such as the laws of gravitation and motion formulated by Isaac Newton (1642–1727). Robert Boyle (1627–1691) performed the fi rst controlled experiments on the chemical conversion of matter. Physicians attempted new treatments for disease involving the application of “stone and minerals” (that is, the application of chemicals), what today we would call chemotherapy. Minds
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Microscopes Reveal the Microbial World
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were open to at least consider the astounding possibility that our surroundings, indeed our very bodies, were inhabited by tiny living beings.
fi rst microscopist to publish a systematic study of the world as seen under a microscope was Robert Hooke (1635–1703). As Curator of Experiments to the Royal Society of London, Hooke built the fi rst compound microscope—a magnifying instrument containing two or more lenses that multiply their magnification in series. With his microscope, Hooke observed biological materials such as nematode “vinegar eels,” mites, and mold fi laments, illustrations of which he published in Micrographia (1665), the fi rst publication that illustrated objects observed under a microscope (Fig. 1.12). Hooke was the fi rst to observe distinct units of living material, which he called “cells.” Hooke fi rst named the units cells because the shape of hollow cell walls in a slice of cork reminded him of the shape of monks’ cells in a monastery. But his crude lenses achieved at best 30-fold power (30 ×), so he never observed single-celled organisms. Antoni van Leeuwenhoek observes bacteria with a single lens. Hooke’s Micrographia inspired other micros-
copists, including Antoni van Leeuwenhoek (1632–1723), who became the fi rst individual to observe single-celled microbes (Fig. 1.13A). As a young man, Leeuwenhoek lived in the Dutch city of Delft, where he worked as a cloth draper, a profession that introduced him to magnifying glasses. (The magnifying glasses were used to inspect the quality of the cloth, enabling the worker to count the number of threads.) Later in life, he took up
Milton S. Eisenhower Library
Robert Hooke observes the microscopic world. The
Hooke’s Micrographia. An illustration of mold sporangia, drawn by Hooke in 1665, from his observations of objects using a compound microscope.
Figure 1.12
the hobby of grinding ever stronger lenses to see into the world of the unseen. Leeuwenhoek ground lenses stronger than Hooke’s, which he used to build single-lens magnifiers, complete with sample holder and focus adjustment (Fig. 1.13B). First he observed insects, including lice and fleas, then the relatively large single cells of protists and algae, then ultimately bacteria. One day he applied his microscope to observe matter extracted from between his teeth. He wrote, “to my great surprise [I] perceived that the aforesaid matter contained very many small living Animals, which moved themselves very extravagantly.” Over the rest of his life, Leeuwenhoek recorded page after page on the movement of microbes, reporting their size and shape so accurately that in many cases we can determine the species he observed (Fig. 1.13C).
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Lens Sample holder Brian J. Ford
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van Leeuwenhoek microscope (circa late 1600s)
Figure 1.13 Antoni van Leeuwenhoek. A. A portrait of Leeuwenhoek, the first person to observe individual microbes. B. “Microscope” (magnifying glass) used by Leeuwenhoek. C. Spiral bacteria viewed through a replica of Leeuwenhoek’s instrument.
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Table 1.2 Microbes and human history. Date
Microbial discovery
Discoverer(s) Microbes impact human culture without detection
10,000 BC 1,500 BC 50 BC 1546 AD
Food and drink are produced by microbial fermentation. Tuberculosis, polio, leprosy, and smallpox are evident in mummies and tomb art. Copper is recovered from mine water acidified by sulfur-oxidizing bacteria. Syphilis and other diseases are observed to be contagious.
1676 1688 1717
Microbes are observed under a microscope. Spontaneous generation is disproved for maggots. Smallpox is prevented by inoculation of pox material, a rudimentary form of immunization.
1765 1798 1835 1847 1881
Microbe growth in organic material is prevented by boiling in a sealed flask. Cowpox vaccination prevents smallpox. Fungus causes disease in silkworms (first pathogen to be demonstrated in animals). Chlorine as antiseptic wash for doctor’s hands decreases pathogens. Bacterial spores survive boiling, but are killed by cyclic boiling and cooling.
1855 1857 1864 1866 1867 1877 1881 1882 1884 1884 1886 1889 1889 1899
Statistical correlation is shown between sanitation and mortality (Crimean War). Microbial fermentation produces lactic acid or alcohol. Microbes fail to appear spontaneously, even in the presence of oxygen. Microbes are defined as a class distinct from animals and plants. Antisepsis during surgery prevents patient death. Bacteria are a causative agent in developing anthrax. The first artificial vaccine is developed (against anthrax). First pure culture of colonies on solid medium, Mycobacterium tuberculosis. Koch’s postulates are published, based on anthrax and tuberculosis. Gram stain devised to distinguish bacteria from human cells. Intestinal bacteria include Escherichia coli, the future model organism. Bacteria oxidize iron and sulfur (lithotrophy). Bacteria isolated from root nodules are proposed to fix nitrogen. The concept of a virus is proposed to explain tobacco mosaic disease.
Egyptians, Chinese, and others Egyptians Roman metal workers under Julius Caesar Girolamo Fracastoro (Padua)
Early microscopy and the origin of microbes Antoni van Leeuwenhoek (Netherlands) Francesco Redi (Italian) Turkish women taught Lady Montagu, who brought the practice to England Lazzaro Spallanzani (Padua) Edward Jenner (England) Agostino Bassi de Lodi (Italy) Ignaz Semmelweis (Hungary) John Tyndall (Ireland)
“Golden age” of microbiology: principles and methods established Florence Nightingale (England) Louis Pasteur (France) Louis Pasteur (France) Ernst Haeckel (Germany) Robert Lister (England) Robert Koch (Germany) Louis Pasteur (France) Robert Koch (Germany) Robert Koch (Germany) Hans Christian Gram (Netherlands) Theodor Escherich (Austria) Sergei Winogradsky (Russia) Martinus Beijerinck (Netherlands) Martinus Beijerinck (Netherlands)
Cell biology, biochemistry, and genetics 1908 1911 1917
Antibiotic chemicals are synthesized and identified (chemotherapy). Cancer in chickens can be caused by a virus. Bacteriophages are recognized as viruses that infect bacteria.
Paul Ehrlich (USA) Peyton Rous (USA) Frederick Twort (England) and Felix D’Herelle (France) 1924 The ultracentrifuge is invented and used to measure the size of proteins. Theodor Svedberg (Sweden) 1928 Streptococcus pneumoniae bacteria are transformed by a genetic material from dead cells. Frederick Griffith (England) 1929 Penicillin, the first widely successful antibiotic, is made by a fungus. The molecule Alexander Fleming (Scotland), Howard is isolated in 1941. Florey (Australia), and Ernst Chain (Germany) 1933–1945 The transmission electron microscope is invented and used to observe cells. Ernst Ruska and Max Knoll, inventors (Germany); first cells observed by Albert Claude (Belgium), Christian de Duve (Belgium), and George Palade (USA) 1937 The tricarboxylic acid cycle is discovered. Hans Krebs (England) 1938 The microbial “kingdom” is subdivided into eukaryotes and prokaryotes (Monera). Herbert Copeland (USA) 1938 Bacillus thuringiensis spray is produced as the first bacterial insecticide. Insecticide manufacturers (France) 1941 One gene encodes one enzyme in Neurospora. George Beadle and Edward Tatum (USA) 1941 Poliovirus is grown in human tissue culture. John Enders, Thomas Weller, and Frederick Robbins (USA) 1944 The genetic material responsible for transformation of S. pneumoniae is DNA. Oswald Avery, Colin Macleod, and Maclyn McCarty (USA) 1945 Bacteriophage replication mechanism is elucidated. Salvador Luria (Italy) and Max Delbrück (Germany), working in the USA 1946 Bacteria transfer DNA by conjugation. Edward Tatum and Joshua Lederberg (USA) 1946–1956 X-ray diffraction crystal structures are obtained for the first complex biological molecules, Dorothy Hodgkin, J. D. Bernal, penicillin and vitamin B12. and coworkers (England) 1950 Anaerobic culture technique is devised to study anaerobes of the bovine rumen. Robert Hungate (USA) 1950 Bacteria can carry latent bacteriophages (lysogeny). André Lwoff (France) 1951 Transposable elements are discovered in maize and later shown in bacteria, where they Barbara McClintock (USA) play key roles in evolution. 1952 DNA is injected into a cell by a bacteriophage. Martha Chase and Alfred Hershey (USA)
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Table 1.2 Microbes and human history (continued) Date
Microbial discovery
Discoverer(s) Molecular biology and recombinant DNA
1953 1953 1959 1960 1961 1966 1967 1968 1969 1972 1973 1974 1975 1975 1975 1977 1977 1978 1978 1979
The overall structure of DNA is a double helix, based on X-ray diffraction analysis.
Rosalind Franklin and Maurice Wilkins (England) Double-helical DNA consists of antiparallel chains connected by the hydrogen bonding of James Watson (USA) and Francis Crick AT and GC base pairs. (England) Expression of the messenger RNA for the E. coli lac operon is regulated by a repressor Arthur Pardee (England) and François protein. Jacob and Jacques Monod (France) Radioimmunoassay for detection of biomolecules is developed. Rosalyn Yalow and Solomon Bernson (USA) The chemiosmotic hypothesis, which states that biochemical energy is stored in a Peter Mitchell and Jennifer Moyle transmembrane proton gradient, is proposed and tested. (England) The genetic code by which DNA information specifies protein sequence is deciphered. Marshall Nirenberg, H. Gobind Khorana, and others (USA) Bacteria can grow at temperatures above 80° in hot springs at Yellowstone National Park. Thomas Brock (USA) Serial endosymbiosis is proposed to explain the evolution of mitochondria and chloroplasts. Lynn Margulis (USA) Retroviruses contain reverse transcriptase, which copies RNA to make DNA. Howard Temin, David Baltimore, Renato Dulbecco (USA) Inner and outer membranes of gram-negative bacteria (Salmonella) are separated by Mary Osborn (USA) ultracentrifugation. A recombinant DNA molecule is created in vitro (in a test tube). Stanley Cohen, Annie Chang, Robert Helling, and Herbert Boyer (USA) The bacterial flagellum is driven by a rotary motor. Howard Berg, Michael Silverman, and Melvin Simon (USA) mRNA-rRNA base pairing initiates protein synthesis. Joan Steitz and Karen Jakes (USA) and Lynn Dalgarno and John Shine (Australia) The dangers of recombinant DNA are assessed at the Asilomar Conference. Paul Berg, Maxine Singer, and colleagues (USA) Monoclonal antibodies are produced indefinitely in tissue culture by hybridomas, George Kohler and Cesar Milstein (USA) antibody-producing cells fused to cancer cells. A DNA-sequencing method is invented and used to sequence the first genome of a virus. Fred Sanger, Walter Gilbert, and Allan Maxam (USA) Archaea are a third domain of life, the others being eukaryotes and bacteria. Carl Woese (USA) The first protein catalog is compiled for E. coli based on 2D gels. Fred Neidhart, Peter O’Farrell, and colleagues (USA) Biofilms are a major form of existence of microbes. William Costerton and others (Canada) Smallpox is declared eliminated, the culmination of worldwide efforts of immunology, The World Health Organization molecular biology, and public health. Genomics, structural biology, and molecular ecology
1981 Invention of the polymerase chain reaction (PCR) makes available large quantities of DNA. 1981–1986 Self-splicing RNA is discovered in the protist Tetrahymena, evidence that life could have originated as an “RNA world.” 1982 Archaea are discovered with optimal growth above 100°C. 1982 Viable but nonculturable bacteria contribute to ecology and pathology. 1982 1983 1983
Prions, infectious agents consisting solely of protein, are characterized. Human immunodeficiency virus (HIV) is implicated in the development of AIDS. Genes are introduced into plants by using Agrobacterium tumefaciens plasmid vectors.
1984
Acid-resistant Helicobacter pylori are discovered in the stomach, where they lead to gastritis. 1987–2004 Geobacter bacteria that can generate electricity are discovered, and their genomes are sequenced. 1988 Earth’s smallest and most abundant photosynthesizer is Prochlorococcus. 1993 Giant bacterium (Epulopiscium) is identified, large enough to see. 1995 The first genome is sequenced for a cellular organism, Haemophilus influenzae. 2001
The ribosome structure is obtained at near-atomic level by X-ray diffraction.
2008
Over 1,000 genome sequences of bacteria and archaea are publicly available.
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Kary Mullis (USA) Thomas Cech and Sidney Altman (USA) Karl Stetter (Germany) Rita Colwell, Norman Pace, and others (USA) Stanley Prusiner (USA) Luc Montagnier and colleagues (France) Eugene Nester, Mary-Dell Chilton, and colleagues at the Monsanto Company (USA) Barry Marshall and Robin Warren (Australia) Derek Lovley and colleagues (USA) Sallie Chisholm and colleagues (USA) Esther Angert and Norman Pace (USA) Craig Venter, Hamilton Smith, Claire Fraser, and others (USA) Marat Yusupov, Harry Noller, and colleagues (USA) National Center for Biotechnology Information (USA)
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He performed experiments, comparing, for example, the appearance of “small animals” from his teeth before and after drinking hot coffee. The disappearance of microbes from his teeth after drinking a hot beverage suggested that heat killed microbes—a profoundly important principle for the study and control of microbes ever since. Ironically, Leeuwenhoek is believed to have died of a disease contracted from sheep whose bacteria he observed. Historians have often wondered why it took so many centuries for Leeuwenhoek and his successors to determine the link between microbes and disease. Although observers such as Agostino Bassi de Lodi noted isolated cases of microbes associated with pathology (see Table 1.2), the very ubiquity of microbes— most of them actually harmless—may have obscured their more deadly roles. Also, it was hard to distinguish between microbes and the single-celled components of the human body, such as blood cells and sperm. It was not until the nineteenth century that human tissues could be distinguished from microbial cells by the application of differential chemical stains (discussed in Chapter 2). THOUGHT QUESTION 1.3 Why do you think it took so long for humans to connect microbes with infectious disease?
showed that a sealed flask of meat broth sterilized by boiling failed to grow microbes. Spallanzani also noticed that microbes often appeared in pairs. Were these two parental microbes coupling to produce offspring, or did one microbe become two? By long and tenacious observation, Spallanzani watched a single microbe grow in size until it split in two. Thus, he demonstrated cell fission, the process by which cells arise by the splitting of preexisting cells. Even Spallanzani’s experiments, however, did not put the matter to rest. Proponents of spontaneous generation argued that the microbes in the priest’s flask lacked access to oxygen and therefore could not grow. The pursuit of this question was left to future microbiologists, including the famous French microbiologist Louis Pasteur (1822–1895) (Fig. 1.14A). In addressing spontaneous generation and related questions, Pasteur and his contemporaries laid the foundations for modern microbiology. Louis Pasteur reveals the biochemical basis of microbial growth. Pasteur began his scientific career as
a chemist and wrote his doctoral thesis on the struc-
A. Institut Pasteur
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Spontaneous Generation: Do Microbes Have Parents? The observation of microscopic organisms led priests and philosophers to wonder where they came from. In the eighteenth century, scientists and church leaders intensely debated the question of spontaneous generation, the theory that living creatures such as maggots could arise spontaneously, without parental organisms. Chemists of the day tended to support spontaneous generation, as it appeared similar to the changes in matter that could occur upon mixture of chemicals. Christian church leaders, however, supported the biblical view that all organisms have “parents” going back to the fi rst week of creation. The Italian priest Francesco Redi (1626–1697) showed that maggots in decaying meat were the offspring of fl ies. Meat kept in a sealed container, excluding fl ies, did not produce maggots. Thus, Redi’s experiment argued against spontaneous generation for macroscopic organisms. The meat still putrefied, however, producing microbes that seemed to arise “without parents.” To disprove spontaneous generation of microbes, another Italian priest, Lazzaro Spallanzani (1729–1799),
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Open to air
B.
S curve excludes dust and microbes Growth medium
Figure 1.14 Louis Pasteur, founder of medical microbiology and immunology. A. Pasteur’s contributions to the science of microbiology and immunology earned him lasting fame. B. Swan-necked flask. Pasteur showed that in such a flask, after boiling, the contents remain free of microbial growth, despite access to air.
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ture of organic crystals. He discovered the fundamental chemical property of chirality, the fact that some organic molecules exist in two forms that differ only by mirror symmetry. In other words, the two structures are mirror images of one another, like the right and left hands. Pasteur found that when microbes were cultured on a nutrient substance containing both mirror forms, only one mirror form was consumed. He concluded that the metabolic preference for one mirror form was a fundamental property of life. Subsequent research has confi rmed that most biological molecules, such as DNA and proteins, occur in only one of their mirror forms. As a chemist, Pasteur was asked to help with a widespread problem encountered by French manufacturers of wine and beer. The production of alcoholic beverages is now known to occur by fermentation, a process by which microbes gain energy by converting sugars into alcohol. In the time of Pasteur, however, the conversion of grapes or grain to alcohol was believed to be a spontaneous chemical process. No one could explain why some fermentation mixtures produced vinegar (acetic acid) instead of alcohol. Pasteur discovered that fermentation is actually caused by living yeast, a singlecelled fungus. In the absence of oxygen, yeast produces alcohol as a terminal waste product. But when the yeast culture is contaminated with bacteria, the bacteria outgrow the yeast and produce acetic acid instead of alcohol. (Fermentative metabolism is discussed further in Chapter 13.) Pasteur’s work on fermentation led him to test a key claim made by proponents of spontaneous generation. The proponents claimed that Spallanzani’s failure to fi nd spontaneous appearance of microbes was due to lack of oxygen. From his studies of yeast fermentation, Pasteur knew that some microbial species do not require oxygen for growth. So he devised an unsealed flask with a long, bent “swan neck” that admitted air but kept the boiled contents free of microbes (Fig. 1.14B). The famous swannecked flasks remained free of microbial growth for many years; but when a flask was tilted to enable contact of broth with microbe-containing dust, growth occurred immediately. Thus, Pasteur disproved that lack of oxygen was the reason for failure of spontaneous generation in Spallanzani’s flasks. But even Pasteur’s work did not prove that microbial growth requires preexisting microbes. The Irish scientist John Tyndall (1820–1893) attempted the same experiment as Pasteur, but sometimes found the opposite result. Tyndall found that the broth sometimes gave rise to microbes, no matter how long it was sterilized by boiling. The microbes appear because some kinds of organic matter, particularly hay infusion, are contami-
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nated with a heat-resistant form of bacteria called endospores (or spores). The spore form can only be eliminated by repeated cycles of boiling and resting, in which the spores germinate to the growing, vegetative form that is killed at 100°C. It was later discovered that endospores could be killed by boiling under pressure, as in a pressure cooker, which generates higher temperatures than can be obtained at atmospheric pressure. The steam pressure device called the autoclave became a standard method for the sterilization of materials required for the controlled study of microbes. (Microbial control and antisepsis are discussed further in Chapter 5.) While spontaneous generation has been discredited as a continual source of microbes, at some point in the past the fi rst living organisms must have originated from nonliving materials. The origin of life is explored in Special Topic 1.1. TO SU M MAR I Z E: ■
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Microbes affected human civilization for centuries before humans guessed at their existence through their contributions to our environment, food and drink production, and infectious diseases. Robert Hooke and Antoni van Leeuwenhoek were the fi rst to record observations of microbes through simple microscopes. Spontaneous generation is the theory that microbes arise spontaneously, without parental organisms. Lazzaro Spallanzani showed that microbes arise from preexisting microbes and demonstrated that heat sterilization can prevent microbial growth. Louis Pasteur discovered the microbial basis of fermentation. He also showed that providing oxygen does not enable spontaneous generation. John Tyndall showed that repeated cycles of heat were necessary to eliminate spores formed by certain kinds of bacteria. Florence Nightingale quantified statistically the impact of infectious disease on human populations.
1.3
Medical Microbiology
Over the centuries, thoughtful observers, such as Fracastoro and Agostino Bassi (see Table 1.2), noted a connection between microbes and disease. Ultimately, researchers developed the germ theory of disease, the theory that many diseases are caused by microbes. The fi rst to establish a scientific basis for determining that a specific microbe causes a specific disease was
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Mic r o b ia l L ife : Orig in and Disc ov ery
How Did Life Originate?
If all life on Earth shares descent from a microbial ancestor, how did the first microbe arise? The earliest fossil evidence of cells in the geological record appears in sedimentary rock that formed as early as 3.8 billion years ago. Although the nature of the earliest reported fossils remains controversial, it is generally accepted that “microfossils” from over 2 billion years ago were formed by living cells. Moreover, the living cells that formed these microfossils looked remarkably similar to bacterial cells today, forming chains of simple rods or spheres (Fig. 1). The exact composition of the first environment for life is controversial. The components of the first living cells may have formed from spontaneous reactions sparked by ultraviolet absorption or electrical discharge. American chemists Stanley Miller (1930–) and Harold C. Urey (1893–1981) argued that the environment of early Earth contained mainly reduced compounds−compounds that have a strong tendency to donate electrons, such as ferrous iron, methane, and ammonia. More recent evidence has modified this view, but it is agreed that the strong electron acceptor oxygen gas (O2) was absent until the first photosynthetic microbes produced it. Today, all our cells are composed of highly reduced molecules
that are readily oxidized by O2. This seemingly hazardous composition may reflect our cellular origin in the chemically reduced environment of early Earth. In 1953, Miller attempted to simulate the highly reduced conditions of early Earth to test whether ultraviolet absorption or electrical discharge could cause reactions producing the fundamental components of life (Fig. 2A). Miller boiled a solution of water containing hydrogen gas, methane, and ammonia and applied an electrical discharge (comparable to a lightning strike). Astonishingly, the reaction produced a number of amino acids, including glycine, alanine, and aspartic acid. A similar experiment in 1961 by Spanish-American researcher Juan Oró (1923–2004) (Fig. 2B) combined hydrogen cyanide and ammonia under electrical discharge to obtain adenine, a fundamental component of DNA and of the energy carrier adenosine triphosphate (ATP). How could early cells have survived the heat and chemically toxic environment of early Earth? Clues may be found in the survival of archaea that thrive under habitat conditions we consider extreme, such as solutions of boiling sulfuric acid. The specially adapted structures of such microbes may resemble those of the earliest life-forms.
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University of California Museum of Paleontology
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Simulating early Earth’s chemistry. A. Stanley Miller with the apparatus of his early Earth simulation experiment. B. Biochemist Juan Oró demonstrated formation of adenine and other biochemicals from reaction conditions found in comets.
Figure 2
Evidence of ancient microbial life. Microfossils of ancient cyanobacteria from the Bitter Springs Formation, Australia, about 850 million years old.
Figure 1
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Research since Miller’s day has generated as many questions as answers concerning the origin of life. For example: ■
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Were the reduced forms of early carbon and nitrogen, such as methane (CH4) and ammonia (NH4), supplemented by oxidized forms such as CO2, spewed out by volcanoes? If oxidized carbon and nitrogen were available, different kinds of early-life chemistry may have occurred. How did the origin of informational molecules, such as RNA and DNA, coincide with the origin of metabolism? One possibility is that early metabolism was catalyzed by molecules of RNA instead of protein. RNA molecules capable of catalysis, called ribozymes, were discovered in 1982 by Thomas Cech and Sidney Altman, who earned the Nobel Prize in Chemistry in 1989 (Fig. 3). The discovery of ribozymes and thousands of small functional RNAs expressed by genomes led to the theory that early organisms were composed primarily of RNA−the so-called “RNA world.” Geochemical evidence suggests that cells may have originated on Earth as early as 3.8 billion years ago, when Earth was just barely cool enough to allow the existence
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of cells. How could cells have formed so quickly? Could the first cells in fact have come from somewhere else? Most of the molecules that spontaneously formed in Miller’s experiments are also found in meteorites and comets. This observation led Oró to propose that the first chemicals of life could have come from outer space, perhaps carried by comets. But could life itself have an extraterrestrial origin? This controversial concept was proposed by British physicists Fred Hoyle (1915–2001) and Chandra Wickramasinghe. Hoyle and Wickramasinghe argued that features of the infrared spectroscopy of interstellar matter might be explained by the existence of microbes in outer space−microbes that could be brought to Earth by comets or meteors. Alternatively, some scientists propose that the first microbes originated on Mars. Because Mars orbits farther out from the sun than Earth, its surface would have cooled before Earth did; therefore, life might have formed on Mars and traveled to Earth on a meteorite. But the “Mars first” explanation says nothing about how life would have arisen on Mars. Current evidence for the origin and evolution of microbes is discussed in Chapter 17.
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Geoffery Wheeler for Howard Hughes Medical Institute
Cleavage is catalyzed.
Figure 3 Tom Cech, discoverer of catalytic RNA. A. Tom Cech (University of Colorado, Boulder) holding a flask containing Oxytricha nova, microbes that make catalytic RNA, the kind of molecule that in early cells may have served both genetic and catalytic functions. B. Diagram of a catalytic RNA, where horizontal bars represent bases. The RNA catalyzes cleavage of itself.
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sis in Europe, malaria in Africa and the East Indies, and bubonic plague in India.
the German physician Robert Koch (1843–1910) (Fig. 1.15). As a college student, Koch conducted biochemical experiments on his own digestive system. Koch’s curiosity about the natural world led him to develop principles and techniques crucial to modern microbial investigation, including the pure-culture technique and the famous Koch’s postulates for identifying the causative agent of a disease. He applied his methods to numerous lethal diseases around the world, including anthrax and tuberculo-
Museum in the Robert Koch-Institut Berlin
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Growth of Microbes in Pure Culture
Museum in the Robert Koch-Institut Berlin
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Unlike Pasteur, who was a university professor, Koch took up a medical practice in a small Polish-German town. To make space in his home for a laboratory to study anthrax and other deadly diseases, his wife curtained off part of his patients’ examining room. Anthrax interested Koch because its epidemics in sheep and cattle caused economic hardship among local farmers. Today, anthrax is no longer a major problem for agriculture, as its transmission is prevented by effective environmental controls and vaccination. It has, however, gained notoriety as a bioterror agent because anthrax bacteria can survive for long periods in the dormant, desiccated form of an endospore. In 2001, anthrax spores sent through the mail contaminated post offices throughout the northeastern United States, as well as an office building of the United States Senate, causing several deaths (Fig. 1.16). To investigate whether anthrax was a transmissible disease, Koch used blood from an anthrax-infected carcass to inoculate a rabbit. When the rabbit died, he used the rabbit’s blood to inoculate a second rabbit, which then died in turn. The blood of the unfortunate animal had turned black with long, rod-shaped bacilli. Upon introduction of these bacilli into healthy animals, the ani-
Figure 1.15 Robert Koch, founder of the scientific method of microbiology. A. Robert Koch as a university student. B. Koch’s sketch of anthrax bacilli in mouse blood. C. Koch (second from left) during his visit to New Guinea to investigate malaria.
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Museum in the Robert Koch-Institut Berlin
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Figure 1.16 Disinfecting the Hart Senate Office Building, Fall 2001. The building became contaminated with anthrax spores sent through the U.S. mail.
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Cytographics/Visuals Unlimited
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mals became ill with anthrax. Thus, Koch demonstrated an important principle of epidemiology—the chain of infection, or transmission of a disease. In retrospect, his choice of anthrax was fortunate, for the microbes generate disease very quickly, multiply in the blood to an extraordinary concentration, and remain infective outside the body for long periods. Koch and his colleagues then applied their experimental logic and culture methods to a more challenging disease: tuberculosis. In Koch’s day, tuberculosis caused one-seventh of reported deaths in Europe; today, tuberculosis bacteria continue to infect millions of people worldwide. Koch’s approach to anthrax, however, was less applicable to tuberculosis, a disease that develops slowly after many years of dormancy. Furthermore, the causative bacteria, Mycobacterium tuberculosis, are small and difficult to distinguish from human tissue or from different bacteria of similar appearance associated with the human body. How could Koch prove that a particular bacterium caused a particular disease? What was needed was to isolate a pure culture of microorganisms, a culture grown from a single “parental” cell. This had been done by previous researchers using the laborious process of serial dilution of suspended bacteria until a culture tube contained only a single cell. Alternatively, inoculation of a solid surface such as a sliced potato could produce isolated colonies, distinct populations of bacteria, each grown from a single cell. For M. tuberculosis, Koch inoculated serum, which then formed a solid gel after heating. Later, he refi ned the solid-substrate technique by adding gelatin to a defi ned liquid medium, which could then be chilled to form a solid medium in a glass dish. A covered version called the petri dish (also called a petri plate) was invented by a colleague, Richard J. Petri (1852–1921). The petri dish consists of a round dish with vertical walls covered by an inverted dish of slightly larger diameter. Today, the petri dish, generally made of disposable plastic, remains an indispensable part of the microbiological laboratory. Another improvement in solid-substrate culture was the replacement of gelatin with materials that remain solid at higher temperatures, such as the gelling agent agar (a polymer of the sugar galactose). The use of agar was recommended by Angelina Hesse (1850–1934), a microscopist and illustrator, to her husband, Walther Hesse (1846–1911), a young medical colleague of Koch’s (Fig. 1.17). Agar comes from red algae (seaweed) which is used by East Indian birds to build nests; it is the main ingredient in the delicacy “bird’s nest soup.” Dutch colonists used agar to make jellies and preserves, and a Dutch colonist from Java introduced it to Angelina Hesse. The Hesses used agar to develop the fi rst effective growth medium for tuberculosis bacteria. (Pure culture is discussed further in Chapter 4.)
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Figure 1.17 Angelina and Walther Hesse. A. Portrait of the Hesses, who first used agar to make solid plate media for bacterial growth. B. Colonies from a streaked agar plate.
Note that some kinds of microbes cannot be grown in pure culture without other organisms. For example, viruses can be cultured only in the presence of their host cells. The discovery of viruses is explored in Special Topic 1.2.
Koch’s Postulates Link a Pathogen with a Disease For his successful determination of the bacterium responsible for tuberculosis, M. tuberculosis, Koch was awarded the Nobel Prize in Physiology or Medicine in 1905. Koch formulated his famous set of criteria for establishing a causative link between an infectious agent and a disease (Fig. 1.18). These four criteria are known as Koch’s postulates: 1. The microbe is found in all cases of the disease, but is
absent from healthy individuals. 2. The microbe is isolated from the diseased host and
grown in pure culture. 3. When the microbe is introduced into a healthy, sus-
ceptible host (or animal model), the same disease occurs. 4. The same strain of microbe is obtained from the newly diseased host. When cultured, the strain shows the same characteristics as before.
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Mic r o b ia l L ife : Orig in and Disc ov ery
The Discovery of Viruses
The discovery of ever smaller and more elusive microorganisms continues to this day. From the nineteenth century on, researchers were puzzled to find contagious diseases whose agent of transmission could pass through a filter of a pore size that blocked known microbial cells−0.1 µm. One of these researchers was the Dutch plant microbiologist Martinus Beijerinck (1851–1931), who studied tobacco mosaic disease, a condition in which the leaves become mottled and the crop yield is decreased or destroyed altogether. Beijerinck concluded that because the agent of disease passed through a filter that retained bacteria, it could not be a bacterial cell. The filterable agent was ultimately purified by the American scientist Wendell Stanley (1904–1971), who processed 4,000 kilograms (kg) of infected tobacco leaves and crystallized the infective particle. What he had crystallized was the tobacco mosaic virus, the causative agent of tobacco mosaic disease. The crystallization of a virus particle earned Stanley the 1946 Nobel Prize in Chemistry. The fact that an entity capable of biological reproduction could be inert enough to be crystallized amazed scientists and ultimately led to a new, more mechanical view of living organisms. The individual particle of tobacco mosaic virus consists of a helical tube of protein subunits containing its genetic material coiled within (Fig. 1). Stanley thought that the virus was a catalytic protein, but colleagues later determined that it contained RNA as its genetic material. The structure of the coiled RNA was solved through X-ray diffraction crystallography by the British scientist Rosalind Franklin (1920–1958). We now know that all kinds of animals, plants, and microbial cells can be infected by viruses. Toward the end of the twentieth century, even smaller infective particles were discovered consisting of a single mol-
Koch’s postulates continue to be used to determine whether a given strain of microbe causes a disease. Modern examples include Lyme disease, a tick-borne infection that has become widespread in New England and the Mid-Atlantic states, and hantaviral pneumonia, an emerging disease particularly prevalent among Native Americans in the Southwest. Nevertheless, the postulates remain only a guide; individual diseases and pathogens may confound one or more of the criteria. For example, tuberculosis bacteria are now known to cause symptoms in only 10% of the people infected. If Koch had been able to detect these silent bacilli, they would not have fulfi lled his fi rst criterion. In the case of AIDS, the concentration of HIV virus is so low that initially no virus could be
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A. ©Dennis Kunkel/Visuals Unlimited
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RNA
100 nm
B.
Capsid proteins
Tobacco mosaic virus (TMV). A. Particles of tobacco mosaic virus (colorized transmission EM). B. In TMV, a protein capsid surrounds an RNA chromosome.
Figure 1
ecule of RNA (viroids) or of protein (prions). Prions are suspected as a factor in the development of Alzheimer’s disease. (The infectious processes of viruses, viroids, and prions are discussed in Chapters 6, 11, and 26.)
detected in patients with fully active symptoms. It took the invention of the polymerase chain reaction (PCR), a method of producing any number of copies of DNA or RNA sequences, to detect the presence of HIV. Another difficulty with AIDS and many other human diseases is the absence of an animal host that exhibits the same disease. In the case of AIDS, even the chimpanzees, our closest relatives, are not susceptible, although they exhibit a similar disease from a related pathogen, simian immunodeficiency virus (SIV). Experimentation on humans is prohibited, although in rare instances researchers have voluntarily exposed themselves to a proposed pathogen. For example, Australian researcher Barry Marshall ingested Helicobacter pylori to convince
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1. The microbe is found in all cases of disease, but absent from healthy individuals.
No microbe
2. The microbe is isolated from the diseased host and grown in pure culture.
3. When the microbe is introduced into a healthy, susceptible host, the same disease occurs.
4. The same strain of microbe is obtained from the newly diseased host.
Figure 1.18 Koch’s postulates defining the causative agent of a disease.
skeptical colleagues that this organism could colonize the extremely acidic stomach. H. pylori turned out to be the causative agent of gastritis and stomach ulcers, conditions that had long been thought to be caused by stress rather than infection. For the discovery of H. pylori, Marshall and colleague Robin Warren won the 2005 Nobel Prize in Physiology or Medicine. THOUGHT QUESTION 1.4 How could you use Koch’s postulates to demonstrate the causative agent of influenza? What problems would you need to overcome that were not encountered with anthrax?
Immunization Prevents Disease Identifying the cause of a disease is, of course, only the fi rst step to developing an effective therapy and prevent-
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ing further transmission. Early microbiologists achieved some remarkable insights on how to control pathogens (see Table 1.2). The fi rst clue as to how to protect an individual from a deadly disease came from the dreaded smallpox. In the eighteenth century, smallpox infected a large fraction of the European population, killing or disfiguring many people. In Turkey, however, the incidence of smallpox was decreased by the practice of deliberately inoculating children with material from smallpox pustules, which contained naturally attenuated virus. Inoculated children usually developed a mild case of the disease and were protected from smallpox thereafter. The practice of smallpox inoculation was introduced from Turkey to Europe in 1717 by Lady Mary Montagu, a smallpox survivor (Fig. 1.19A). Stationed in Turkey with her husband, the British ambassador, Lady Montagu learned that many elderly women there had perfected the art of inoculation: “The old woman comes with a nut-shell full of the matter of the best sort of small-pox, and asks what vein you please to have opened.” Lady Montagu arranged for the procedure on her own son, then brought the practice back to England where it became widespread. Preventive inoculation with smallpox was dangerous, however, as some infected individuals still contracted serious disease and were contagious. Thus, doctors continued to seek a better method of prevention. In England, milkmaids claimed that they were protected from smallpox after they contracted cowpox, a related but much milder disease. This claim was confi rmed by English physician Edward Jenner (1749–1823), who deliberately infected patients with matter from cowpox lesions (Fig. 1.19B). The practice of cowpox inoculation was called vaccination, after the Latin word vacca for “cow.” To this day, cowpox, or vaccinia virus, remains the basis of the modern smallpox vaccine. Pasteur was aware of vaccination as he studied the course of various diseases in experimental animals. In the spring of 1879, he was studying fowl cholera, a transmissible disease of chickens with a high death rate. He had isolated and cultured the bacteria responsible, but left his work during the summer for a long vacation. No refrigeration was available to preserve cultures, and when he returned to work, the aged bacteria failed to cause disease in his chickens. Pasteur then obtained fresh bacteria from an outbreak of disease elsewhere, as well as some new chickens. But the fresh bacteria also failed to make the original chickens sick (those who had been exposed to the aged bacteria). All of the new chickens, exposed only to the fresh bacteria, contracted the disease. Grasping the clue from his mistake, Pasteur had the insight to recognize that an attenuated (or “weakened”) strain of microbe, altered somehow to eliminate its potency to cause disease, could still confer immunity to the virulent disease-causing form.
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B.
C.
Pasteur was the fi rst to recognize the significance of attenuation and extend the principle to other pathogens. We now know that the molecular components of pathogens generate immunity, the resistance to a specific disease, by stimulating the immune system, an organism’s exceedingly complex cellular mechanisms of defense (see Chapter 23). Understanding the immune system awaited the techniques of molecular biology a century later, but nineteenth-century physicians developed several effective examples of immunization, the stimulation of an immune response by deliberate inoculation with an attenuated pathogen. The way to attenuate a strain varies greatly among pathogens. Heat treatment or aging for various periods often turned out to be the most effective approach. In retrospect, the original success of prophylactic smallpox inoculation was probably due to natural attenuation during the time between acquisition of smallpox matter from a diseased individual and inoculation of the healthy patient. A far more elaborate treatment was required for the most famous disease for which Pasteur devised a vaccine: rabies.
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Corbis
Smallpox vaccination. A. Lady Mary Wortley Montagu, shown in Turkish dress. The artist avoided showing Montagu’s facial disfigurement from smallpox. B. Dr. Edward Jenner, depicted vaccinating 8-year-old James Phipps with cowpox matter from the hand of milkmaid Sarah Nelmes, who had caught the disease from a cow. C. Newspaper cartoon depicting public reaction to cowpox vaccination. Figure 1.19
Bettmann/Corbis
Hulton-Deutsch Collection/Corbis
A.
The rabid dog loomed large in folklore, and the disease was dreaded for its particularly horrible and inevitable course of death. Pasteur’s vaccine for rabies required a highly complex series of heat treatments and repeated inoculations. Its success led to his instant fame (Fig. 1.20). Grateful survivors of rabies founded the Pasteur Institute for medical research, one of the world’s greatest medical research institutions, whose scientists in the twentieth century discovered the virus HIV, responsible for AIDS.
Antiseptics and Antibiotics Control Pathogens Before the work of Koch and Pasteur, many patients died of infections transmitted unwittingly by their own doctors. In 1847, Hungarian physician Ignaz Semmelweis (1818–1865) noticed that the death rate of women in childbirth due to puerperal fever was much higher in his own hospital than in a birthing center run by midwives. He guessed that the doctors in his hospital were transmitting pathogens from cadavers that they had dissected. So he ordered the doctors to wash their hands
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Corbis
A.
Penicillium mold
Mediscan/Visuals Unlimited
B.
Alexander Fleming, discoverer of penicillin. A. Alexander Fleming in his laboratory. B. Fleming’s original plate of bacteria with Penicillium mold inhibiting the growth of bacterial colonies.
Institut Pasteur
Figure 1.21
Figure 1.20 Pasteur cures rabies. Cartoon in French newspaper depicts Pasteur protecting children from rabid dogs.
in chlorine, an antiseptic agent (a chemical that kills microbes). The mortality rate fell; but this revelation displeased other doctors, who refused to accept Semmelweis’s fi ndings. In 1865, the British surgeon Joseph Lister (1827–1912) noted that half his amputee patients died of sepsis. Lister knew from Pasteur that microbial contamination might be the cause. So he began experiments to develop the use of antiseptic agents, most successfully carbolic acid, to treat wounds and surgical instruments. After initial resistance, Lister’s work, with the support of Pasteur and Koch, drew widespread recognition. In the twentieth century, surgeons developed fully aseptic environments for surgery; that is, environments completely free of microbes. Antibiotics. The problem with most antiseptic chemicals
that killed microbes was that if taken internally, they also
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tended to kill the patients. Researchers sought a “magic bullet,” an antibiotic molecule that killed microbes alone, leaving their host unharmed. An important step in the search for antibiotics was the realization that microbes themselves produce antibiotic compounds with highly selective effects. This followed from the famous accidental discovery of penicillin by the English medical researcher Alexander Fleming (1881–1955) (Fig. 1.21A). In 1929, Fleming was culturing Staphylococcus, which infects wounds. He found that one of his plates of Staphylococcus was contaminated with a mold, Penicillium notatum, which he noticed was surrounded by a clear region, free of Staphylococcus colonies (Fig. 1.21B). Following up on this observation, Fleming showed that the mold produced a substance that killed bacteria. We now know this substance as penicillin. In 1941, British biochemists Howard Florey (1898– 1968) and Ernst Chain (1906–1979) purified the antibiotic molecule, which we now know inhibits formation of the bacterial cell wall. Penicillin saved the lives of many Allied troops during World War II, the fi rst war in which an antibiotic became available to soldiers. The second half of the twentieth century saw the discovery of many new and powerful antibiotics. Most of the new antibiotics, however, were produced by
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obscure strains of bacteria and fungi from dwindling ecosystems—a circumstance that focused attention on wilderness preservation worldwide. Furthermore, the widespread and often indiscriminate use of antibiotics has selected for pathogens that are antibiotic resistant. As a result, antibiotics have lost their effectiveness against certain strains of major pathogens. For example, multiple-drug-resistant Mycobacterium tuberculosis is now a serious threat to public health. Fortunately, biotechnology provides new approaches to antibiotic development, including genetic engineering of microbial producers and artificial design of antimicrobial chemicals. This industry has become ever more critical because the indiscriminate use of antibiotics has led to a “molecular arms race” in which our only hope is to succeed faster than the pathogens develop resistance. (Microbial biosynthesis of antibiotics is discussed in Chapter 15, and the medical use of antibiotics is discussed in Chapter 27.) THOUGHT QUESTION 1.5 Why do you think some pathogens generate immunity readily, whereas others evade the immune system? THOUGHT QUESTION 1.6 How do you think microbes protect themselves from the antibiotics they produce?
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Robert Koch devised techniques of pure culture to study a single species of microbe in isolation. A key technique is culture on solid medium using agar, as developed by Angelina and Walther Hesse, in a double-dish container devised by Richard Petri. Koch’s postulates provide a set of criteria to establish a causative link between an infectious agent and a disease. Edward Jenner established the practice of vaccination, inoculation of cowpox to prevent smallpox. Jenner’s discovery was based on earlier observations by Lady Mary Montagu and others that a mild case of smallpox could prevent future cases. Louis Pasteur developed the fi rst vaccines based on attenuated strains, such as the rabies vaccine. Ignaz Semmelweis and Joseph Lister showed that antiseptics could prevent transmission of pathogens from doctor to patient. Alexander Fleming discovered that the Penicillium mold generated a substance that kills bacteria. Howard Florey and Ernst Chain purified the substance, penicillin, the fi rst commercial antibiotic to save human lives.
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1.4
Microbial Ecology
Koch’s growth of microbes in pure culture was a major advance in technology, enabling the systematic study of microbial physiology and biochemistry. In hindsight, this discovery eclipsed the equally important study of microbial ecology. Microbes are responsible for cycling the many minerals essential for all life. Yet barely 0.1% of all microbial species can be cultured in the laboratory—and the remainder make up the majority of Earth’s entire biosphere. Only the outer skin of Earth supports complex multicellular organisms. The depths of Earth’s crust, to at least 2 miles down, as well as the atmosphere 10 miles out into the stratosphere, remain the domain of microbes. So, to a fi rst approximation, Earth’s ecology is microbial ecology.
Microbes Support Natural Ecosystems The fi rst microbiologists to culture microbes in the laboratory selected the kinds of nutrients that feed humans, such as beef broth or potatoes. Some of Koch’s contemporaries, however, suspected that other kinds of microbes living in soil or wetlands existed on more exotic fare. Soil samples were known to oxidize hydrogen gas, and this activity was eliminated by treatment with heat or acid, suggesting microbial origin. Ammonia in sewage was oxidized to nitrate, and this process was eliminated by antibacterial treatment. These fi ndings suggested the existence of microbes that “ate” hydrogen gas or ammonia instead of beef or potatoes, but no one could isolate these microbes in culture. Among the fi rst to study microbes in natural habitats was the Russian scientist Sergei Winogradsky (1856– 1953). Winogradsky waded through marshes to discover microbes with metabolism quite alien from human digestion. For example, Winogradsky discovered that species of the bacterium Beggiatoa oxidize hydrogen sulfide (H2S) to sulfuric acid (H2SO4). Beggiatoa fi xes carbon dioxide into biomass without consuming any organic food. Organisms that feed solely on inorganic minerals are known as chemolithotrophs, or lithotrophs, discussed further in Chapters 4 and 14. The lithotrophs studied by Winogradsky could not be grown on Koch’s plate media containing agar or gelatin. The bacteria that Winogradsky isolated could grow only on inorganic minerals; in fact, some species are actually poisoned by organic food. For example, nitrifiers convert ammonia to nitrate, forming a crucial part of the nitrogen cycle in natural ecosystems. Winogradsky cultured nitrifiers on a totally inorganic solution containing ammonia and silica gel, which supported no other kind of organism. This experiment was an early example of enrichment culture, the use of selective growth
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Geochemical Cycling Depends on Bacteria
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Cyanobacteria
Purple sulfur bacteria Green sulfur bacteria Sulfate reducing bacteria
Joseph Vallino/Marine Biological Laboratory
media that support certain classes of microbial metabolism while excluding others. Instead of isolating pure colonies, Winogradsky built a model wetland ecosystem containing regions of enrichment for microbes of diverse metabolism. This model is called the Winogradsky column (Fig. 1.22). The model consists of a glass tube containing mud mixed with shredded newsprint (an organic carbon source) and calcium salts of sulfate and carbonate. After exposure to light for several weeks, several zones of color develop, full of mineral-metabolizing bacteria. At the top, cyanobacteria conduct photosynthesis, using light energy to split water and produce molecular oxygen. Below, purple sulfur bacteria use photosynthesis to split hydrogen sulfide, producing sulfur. At the bottom, sulfate reducers produce hydrogen sulfide and precipitate iron. The gradient from oxygen-rich conditions at the surface to highly reduced conditions below generates a voltage potential, like a battery cell. We now know that the entire Earth’s surface acts as a battery—for humans, a potential source of renewable energy.
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Figure 1.22 Winogradsky column. A wetland model ecosystem designed by Sergei Winogradsky.
THOUGHT QUESTION 1.7 Why don’t all living organisms fix their own nitrogen?
Microbial endosymbiosis, in many diverse forms, Winogradsky and later microbial ecologists showed that is widespread in all ecosystems. Many interesting cases bacteria perform unique roles in geochemical cycling, involve animal or human hosts (see Special Topic 1.3). the global interconversion of inorganic and organic forms of nitrogen, sulfur, phosphorus, and other minerals. Atmospheric Without these essential convernitrogen (N2) sions (nutrient cycles), no plants or animals could live. Bacteria and archaea fix nitrogen (N2) by reducing it to ammonia (NH3), the form of nitrogen assimilated by plants. This process is called Industrial Animals fixation nitrogen fi xation (Fig. 1.23). consume (commercial Other bacterial species oxidize plants fertilizers) NH4 + in several stages back to nitrogen gas. Soil bacteria Bacterial symbionts of Within plant cells, cerPlant fix N2 to NH4+ legumes fix nitrogen uptake tain bacteria fi x nitrogen as Bacteria endsymbionts, organisms living Bacteria symbiotically inside a larger orBacteria Bacteria ganism. Endosymbiotic bacteNitrate ria known as rhizobia induce the (NO3–) Nitrite Ammonium roots of legumes to form special + (NO2–) (NH ) 4 nodules to facilitate bacterial niBacteria trogen fixation. Rhizobial endosymbiosis was first observed by the Dutch plant microbiologist Figure 1.23 The global nitrogen cycle. All life depends on these oxidative and reductive Martinus Beijerinck (1851–1931). conversions of nitrogen−most of which are performed only by microbes.
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Mic r o b ia l L ife : Orig in and Disc ov ery
Microbial Endosymbionts of Animals
inspired fictional creations, such as the “breathmicrobes” of Joan Slonczewski’s novel A Door into Ocean, which acquire oxygen for human hosts swimming underwater. (Microbial endosymbiosis is discussed further in Section 21.2.)
Fritz Heide/WHOI
Numerous kinds of endosymbiosis have been found in which microbes make essential nutritional contributions to host animals. Ruminant animals, such as cattle, as well as insects such as termites, require digestive bacteria to break down cellulose and other plant polymers. Even humans obtain about 10% of their nutrition from colonic bacteria. A remarkable form of endosymbiosis is seen in marine animal communities such as those inhabiting thermal vents in the ocean floor, where superheated water emerges carrying reduced minerals, such as hydrogen sulfide (Fig. 1). Near these thermal vents, sulfideoxidizing bacteria nourish giant worms and clams. The worms have evolved so as to lose their digestive tracts altogether, while their body fluids carry the hydrogen sulfide (highly toxic to most animals) needed for the bacteria to metabolize. Many invertebrates, such as hydras and corals, harbor endosymbiotic phototrophs that provide products of photosynthesis in return for protection and nutrients. Other kinds of endosymbiosis involve more than nutrition. In the light organs of fish and squid, luminescent bacteria such as Vibrio fischeri respond to host signals, causing light emission. Certain sponges maintain endosymbiotic bacteria that produce antibiotics, preventing infection by pathogens. Similarly, some bacteria that normally inhabit the human skin are believed to protect us from infection. Microbial endosymbiosis has even
Tube worms in a thermal vent ecosystem. Tube worms do not feed themselves but depend on sulfideoxidizing symbionts for nutrition.
Figure 1
Microbes with unusual properties, such as the ability to digest toxic wastes, have valuable applications in industry and bioremediation. For this reason, microbial ecology is a priority for funding by the National Science Foundation (NSF). The NSF program Life in Extreme Environments supports research aimed at documenting
microbes from environments with extreme heat, salinity, acidity, or other factors. One such microbe is a species of Geobacter that reduces rust (iron oxide, Fe2O3 ) to the magnetic mineral magnetite (Fe3O4) while growing at 121°C, a temperature high enough to kill all other known organisms (Fig. 1.24). B.
G. Reguera/UMass, Amherst
A.
Bacterium strain 121
1 µm
Kazem Kashefi/UMass, Amherst
28
An extreme thermophile reduces iron oxide to magnetite. A. Microbiologist Kazem Kashefi (Michigan State University) pulls a live culture of Geobacter out of an autoclave generally used to kill all living organisms at 121°C (250°F). The magnet shows that strain 121 is converting nonmagnetic iron oxide (rust; Fe2O3) to the magnetic mineral magnetite, Fe3O4. B. Strain 121 is a round bacterium with a tuft of rotary flagella (transmission electron micrograph). The black material at right is iron oxide, which feeds the bacteria. Source: Part B reprinted with permission from Kashefi, et al. 2003. Science 301(5635):934. ©2005 AAAS.
Figure 1.24
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Sergei Winogradsky fi rst developed a system of enrichment culture, the Winogradsky column, to grow microbes from natural environments. Chemolithotrophs (or lithotrophs) metabolize inorganic minerals, such as ammonia, instead of the organic nutrients used by the microbes isolated by Koch. Geochemical cycling depends on bacteria and archaea that cycle nitrogen, phosphorus, and other minerals throughout the biosphere. Endosymbionts are microbes that live within multicellular organisms and provide essential functions for their hosts, such as nitrogen fi xation for legume plants. Martinus Beijerinck fi rst demonstrated that nitrogen-fi xing rhizobia grow as endosymbionts within leguminous plants.
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on genetic similarity. For example, two distinct species generally share no more than 95% similarity of DNA sequence.
NOTE: The actual names of microbial species are often changed to reflect new understanding of genetic relationships. For example, the causative agent of bubonic plague has formerly been called Bacterium pestis (1896), Bacillus pestis (1900), and Pasteurella pestis (1923), but is now called Yersinia pestis (1944). The older names, however, still appear in the literature, a point to remember during research. Current and historical nomenclature is compiled at the List of Prokaryotic Names with Standing in Nomenclature.
List of Prokaryotic Names with Standing in Nomenclature
1.5
The Microbial Family Tree
The bewildering diversity of microbial life-forms presented nineteenth-century microbiologists with a seemingly impossible task of classification. So little was known about life under the lens that natural scientists despaired of ever learning how to distinguish microbial species. The famous classifier of species, Swedish botanist Carl von Linné (Carolus Linnaeus, 1707–1778), called the microbial world “chaos.”
Microbes Are a Challenge to Classify Two challenges faced would-be taxonomists with respect to microbes. The fi rst was that the resolution of the light microscope allows little more than visualizing the outward shape of microbial cells, and vastly different kinds of microbes look more or less alike (visualization of cells and molecules is discussed in Chapter 2). This challenge was overcome as advances in biochemistry and microscopy made it possible to distinguish microbes based on their metabolism and cell ultrastructure and ultimately on their DNA sequence. The second challenge was that microbes do not readily fit the classic defi nition of a species—that is, a group of organisms that interbreed. Unlike multicellular eukaryotes, microbes generally reproduce asexually, with only occasional sexual exchange. When they do exchange genes, they may do so with distantly related species (discussed in Chapter 9). Nevertheless, microbiologists devise working defi nitions of microbial species that enable us to usefully describe populations while being flexible enough to accommodate continual revision and change. The most useful defi nitions are based
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Microbes Include Eukaryotes and Prokaryotes In the nineteenth century, taxonomists had no access to DNA information. As they tried to incorporate microbes into the tree of life, they faced a conceptual dilemma in that microbes could not be categorized as either animals or plants, which since ancient times were considered the two “kingdoms” or major categories of life. Taxonomists attempted to apply these categories to microbes—for example, by including algae and fungi with plants. But German naturalist Ernst Haeckel (1834–1919) recognized that microbes differed from both plants and animals in fundamental aspects of their lifestyle, cellular structure, and biochemistry. Haeckel proposed that microscopic organisms constituted a third kind of life—neither animal nor plant— which he called Monera. In the twentieth century, biochemical studies revealed profound distinctions even within the microbial category. In particular, microbes such as protists and algae contain a nucleus bounded by a nuclear membrane, whereas bacteria do not. Herbert Copeland (1902–1968) proposed a system of classification that divided Monera into two groups: the eukaryotic protists (protozoa and algae) and the prokaryotic bacteria. Copeland’s four-kingdom classification (plants, animals, eukaryotic protists, and prokaryotic bacteria) was later modified by Robert Whittaker (1924–1980) to include fungi as a fi fth kingdom of eukaryotic microbes. Whittaker’s system thus generated five kingdoms: bacteria, protists, fungi, and the multicellular plants and animals.
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Eukaryotes Evolved through Endosymbiosis
A.
Lynn Margulis, UMass, Amherst
The five-kingdom system was modified dramatically by Lynn Margulis, at the University of Massachusetts (Fig. 1.25). Margulis tried to explain how it Animalia Plantae Fungi Protoctista Prokaryotes is that eukaryotic cells contain mito- Plants and algae chondria and chloroplasts, membranous organelles that possess their own chromosomes. She proposed that eukaryotes evolved by merging with bacteria Chloroplasts to form composite cells by intracellular B. endosymbiosis, in which one cell internalizes another that grows within it. The endosymbiosis may ultimately generate Protoctista a single organism whose formerly indeProtists and slime molds pendent members are now incapable of independent existence. Margulis proposed that early in the history of life, respiring bacteria Mitochondria similar to E. coli were engulfed by preeukaryotic cells, where they evolved into mitochondria, the eukaryote’s respiraPre-eukaryote tory organelle. Similarly, she proposed that a phototroph related to cyanobacteria was taken up by a eukaryote, giving Cyanobacteria Proteobacteria Bacteria were incorporated by rise to the chloroplasts of phototrophic (phototroph) (respiring) pre-eukaryotes as eukaryotic algae and plants. organelles. Prokaryotes The endosymbiosis theory was highly controversial because it implied Figure 1.25 Lynn Margulis and the serial endosymbiosis theory. A. Fivea polyphyletic, or multiple, ancestry of kingdom scheme, modified by the endosymbiosis theory. B. Lynn Margulis (University living species, inconsistent with the long- of Massachusetts, Amherst) proposed that organelles evolve through endosymbiosis. held assumption that species evolve only by divergence from a common ancesWoese used the sequence of the gene for 16S ribosomal tor (monophyletic). Ultimately, DNA sequence analyRNA (16S rRNA) as a “molecular clock,” a gene whose sis produced compelling evidence of the bacterial origin sequence differences can be used to measure the time of mitochondria and chloroplasts. Both these classes of since the divergence of two species (discussed in Chaporganelles contain circular molecules of DNA, whose ter 17). The divergence of rRNA genes showed that the sequences show unmistakable homology (similarity) to newly discovered prokaryotes were a distinct form of life, those of modern bacteria. DNA sequences and other eviarchaea (Fig. 1.26). The archaea resemble bacteria in their dence established the common ancestry between mitorelatively simple cell structure, in their lack of a nucleus, chondria and respiring bacteria and between chloroplasts and in their ability to grow in a wide range of environand cyanobacteria. ments. Many archaea, however, grow in environments more extreme than any bacterium, such as 110°C at high Archaea Differ from Bacteria pressure. The genetic sequences of archaea differ as much and Eukaryotes from those of bacteria as from those of eukaryotes; in fact, their gene expression machinery is more similar to that of Gene sequence analysis led to another startling advance eukaryotes. in our understanding of the evolution of cells. In 1977, Carl Woese’s discovery replaced the classification scheme Woese, at the University of Illinois, was studying a group of five kingdoms with three equally distinct groups, now of recently discovered prokaryotes that live in seemingly called the three domains: Bacteria, Archaea, and Eukarya hostile environments, such as the boiling sulfur springs (Fig. 1.27). In the three-domain model, the bacterial of Yellowstone, or that exhibit unusual kinds of metaboancestor of mitochondria derives from ancient proteolism, such as production of methane (methanogenesis).
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bacteria (shaded pink), whereas chloroplasts derive from ancient cyanobacteria (shaded green). The three-domain classification is largely supported by the sequences of microbial genomes, although some horizontal transfer of genes occurs both within and between the domains (discussed in Chapter 17).
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TO SU M MAR I Z E: ■
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THOUGHT QUESTION 1.8 What arguments support the classification of Archaea as a third domain of life? What arguments support the classification of archaea and bacteria together, as prokaryotes, distinct from eukaryotes?
Classifying microbes was a challenge historically because of the difficulties in observing distinguishing characteristics of different categories. Ernst Haeckel recognized that microbes constitute a form of life distinct from animals and plants. Herbert Copeland and Robert Whittaker classified prokaryotes as a form of microbial life distinct from eukaryotic microbes such as protists. A.
B.
B.
Carl Woese
James W. Brown, North Carolina State University
A.
16S rRNA sequences reveal three deeply branching domains of life.
Bacteria Green nonsulfur bacteria Gram-positive bacteria
Archaea Crenarchaeota Hyperthermophiles Euryarchaeota Sulfur Halophiles oxidizers Methanogens Last common ancestor
Proteobacteria Root Cyanobacteria
1 µm
Henry Aldrich, University of Florida
Spirochetes Thermotogales
Archaea, newly discovered life-forms. A. Finding archaea in the hot spring Obsidian Pool, at Yellowstone. B. Pyrococcus furiosus, an organism that lives at temperatures above 100°C (transmission electron micrograph).
Figure 1.26
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Entamebas Slime molds Animals Fungi Plants Microsporidia Ciliates Flagellates Trichomonads Diplomonads
Eukarya Carl Woese and the three domains of life. A. Carl Woese (University of Illinois at Urbana-Champaign) proposed that archaea constitute a third domain of life. B. Three domains, a monophyletic tree, based on 16S rRNA sequences. The length of each branch approximates the time of divergence from the last common ancestor. For an up-to-date tree that synthesizes both approaches, see Chapter 17.
Figure 1.27
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Lynn Margulis proposed that eukaryotic organelles such as mitochondria and chloroplasts evolved by endosymbiosis from prokaryotic cells engulfed by proto-eukaryotes. Carl Woese discovered a domain of prokaryotes, Archaea, whose genetic sequences diverge equally from those of bacteria and those of eukaryotes. Many, though not all, archaea grow in extreme environments.
1.6
Cell Biology and the DNA Revolution
During the twentieth century, amid world wars and societal transformations, the field of microbiology exploded with new knowledge (see Table 1.2). More than 99% of what we know about microbes today was discovered since 1900 by scientists too numerous to cite in this book. Advances in biochemistry and microscopy revealed the fundamental structure and function of cell membranes and proteins. The revelation of the structures of DNA and RNA led to the discovery of the genetic programs of model bacteria such as E. coli and the bacteriophage lambda. Beyond microbiology, these advances produced the technology of “recombinant DNA,” or genetic engineering, the creation of molecules that combine DNA sequences from unrelated species. These microbial tools offered unprecedented applications to human medicine and industry.
details never seen before. Ultimately, Ruska built lenses to focus electrons using specially designed electromagnets. Magnetic lenses were used to complete the fi rst electron microscope in 1933 (Fig. 1.28). Early transmission electron microscopes achieved about tenfold greater magnification than the light microscope, revealing details such as the ridged shell of a diatom. Further development steadily increased magnification, to as high as a millionfold. For the fi rst time, cells were seen to be composed of a cytoplasm containing macromolecules and bounded by a phospholipid membrane. By the 1960s, thin sections revealed the entire inner architecture of eukaryotic cells, including intracellular membranes, ribosomes, and organelles such as mitochondria and chloroplasts. Within the smaller cells of bacteria, the DNA-containing nucleoid was revealed, as well as specialized structures, such as photosynthetic organelles called chlorosomes (Fig. 1.29). Subcellular structures, however, raised many questions about cell function that visualization alone could not answer. Biochemists showed how cell function involved numerous chemical transformations mediated by enzymes. A milestone in the study of metabolism was the elucidation by German biochemist Hans Krebs (1900–1981) of the tricarboxylic acid cycle (TCA, or Krebs cycle), by which the products of sugar diges-
Cell Membranes and Macromolecules
Marine Biological Laboratory/WHOI
In 1900, the study of cell structure was still limited by the resolution of the light microscope and by the absence of tools that could take apart cells to isolate their components. Both these limitations were overcome by the invention of powerful instruments. Just as society was being transformed by machines ranging from jet airplanes to vacuum cleaners, the study of microbiology was also being transformed by machines. Two instruments had exceptional impact: The electron microscope revealed the internal structure of cells (Chapter 2), and the ultracentrifuge enabled isolation of subcellular parts (Chapter 3). The electron microscope. In the 1920s, at the Techni-
cal College in Berlin, student Ernst Ruska (1906–1988) was invited to develop an instrument for focusing rays of electrons. Ruska recalled as a child how his father’s microscope could magnify fascinating specimens of plants and animals, but that its resolution was limited by the wavelength of light. He was eager to devise lenses that could focus beams of electrons, with wavelengths far smaller than that of light, to reveal living
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Electron microscopy. An early transmission electron microscope. The tall column contains a series of magnetic lenses.
Figure 1.28
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Nucleoid
Chlorosome
100 µm
ASM, Frigaard et al. 2002. J. Bacteriol.
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Electron microscopy. Transmission electron micrograph of a Chlorobium species, a photosynthetic bacterium. The thin section reveals the nucleoid (containing DNA), the light-harvesting chlorosomes, and envelope membranes.
Figure 1.29
tion are converted to carbon dioxide. The tricarboxylic acid cycle provides energy for many bacteria and for the mitochondria of eukaryotes. But even Krebs understood little of how metabolism is organized within a cell; he and his contemporaries considered the cell a “bag of enzymes.” The full significance of cell structure required experiments on isolated parts of cells. The ultracentrifuge. Whole cells could be separated
from the fluid in which they were suspended by centrifugation, the spinning of samples in a rotor so as to subject the cells to centrifugal forces of a few thousand times that of gravity. Some biochemists proposed that even greater centrifugal forces could separate cell components, even macromolecules such as proteins. The Swedish chemist Theodor Svedberg (1884–1971), at the University of Uppsala, set out to build such a machine: the ultracentrifuge. Svedberg studied the properties of macromolecules such as proteins and polysaccharides. A major challenge was to measure the size of such particles. Svedberg calculated that particle size could be determined based on the rate of the movement of particles in a tube, the subjected to forces of hundreds of thousands times that of gravity. So he designed and built rotors spinning at ever greater rates, rates so high that they required a vacuum to avoid burning up like a space reentry vehicle. Such an advanced centrifuge, used to separate components of cells, is called an ultracentrifuge. Experiments based on electron microscopy and ultracentrifugation revealed how membranes govern energy transduction within bacteria and within organelles such
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as mitochondria and chloroplasts. In the 1960s, English biochemists Peter Mitchell (1920–1992) and Jennifer Moyle (1921–) proposed and tested a revolutionary idea called the chemiosmotic hypothesis. The chemiosmotic hypothesis states that the redox reactions of the electron transport system store energy in the form of a gradient of protons (hydrogen ions) across a membrane, such as the bacterial cell membrane or the inner membrane of mitochondria. The energy stored in the proton gradient in turn drives the synthesis of ATP. The chemiosmotic hypothesis earned Mitchell the Nobel Prize in Chemistry in 1978. Nevertheless, the theory took many years to win acceptance by biologists committed to the “bag of enzymes” model of the cell. Ultimately the role of ion gradients across membranes was recognized as fundamental to all living cells. (Ion gradients in membranes are discussed further in Chapters 3 and 4, and their role in energy transduction is presented in Chapter 14.)
Microbial Genetics Leads the DNA Revolution As the form and function of living cells emerged in the early twentieth century, a largely separate line of research revealed patterns of heredity of cell traits. In eukaryotes, the Mendelian rules of inheritance were rediscovered and connected to the behavior of subcellular structures called chromosomes. Frederick Griffith (1877–1941) showed in 1928 that some unknown substance out of dead bacteria could carry genetic information into living cells, transforming harmless bacteria into a strain capable of killing mice, a process called transformation. Some kind of “genetic material” must be inherited to direct the expression of inherited traits, but no one knew what that material was or how its information was expressed. Then in 1944, Oswald Avery (1877–1955) and colleagues showed that the genetic material responsible for transformation is deoxyribonucleic acid, or DNA. An obscure acidic polymer, DNA was previously thought too uniform in structure to carry information; its precise structure was unknown. As World War II raged among nations, among scientists an epic struggle began: the quest for the structure of DNA. The discovery of the double helix. The tool of choice
for macromolecular structure was X-ray crystallography, a method developed by British physicists in the early 1900s. The field of X-ray analysis included an unusual number of women, including Dorothy Hodgkin (1910–1994), who won a Nobel Prize for the structures of penicillin and vitamin B12. In 1953, crystallographer Rosalind Franklin (1920–1958) joined a laboratory at King’s College to study the structure of DNA (Fig. 1.30A). As a woman and as a
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C.
B.
Photo Researchers, Inc.
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A. Barrington Brown/Photo Researchers, Inc.
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Omikron/Photo Researchers, Inc.
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The DNA double helix. A. Rosalind Franklin discovered that DNA forms a double helix. B. X-ray diffraction pattern of DNA, obtained by Rosalind Franklin. C. James Watson and Francis Crick discovered the complementary pairing between bases of DNA and the antiparallel form of the double helix.
Figure 1.30
Jew who supported relief work in Palestine, Franklin felt socially isolated at the male-dominated Protestant university. Nevertheless, the exceptional quality of her X-ray micrographs impressed her colleagues. The X-shaped pattern in her micrograph (Fig. 1.30B) showed for the fi rst time that the standard B-form DNA was a double helix. Without Franklin’s knowledge, her colleague Maurice Wilkins showed her data to a competitor, James Watson (1928–). The pattern led Watson and Francis Crick (1916–2004) to guess that the four bases of the DNA “alphabet” were paired in the interior of Franklin’s double helix (Fig. 1.30C). They published their model in the journal Nature, while denying that they used Franklin’s data. The discovery of the double helix earned Watson, Crick, and Wilkins the 1962 Nobel Prize in Physiology or Medicine. Franklin died of ovarian cancer before the Nobel Prize was awarded. Before her death, however, she turned her efforts to the structure of ribonucleic acid (RNA). She determined the form of the RNA chromosome within tobacco mosaic virus, the first viral RNA to be characterized. The structure of DNA base pairs led to development of techniques for DNA sequencing, the reading of the sequence of DNA base pairs. Figure 1.31 shows an exam-
ple of DNA sequence data, where each color represents one of the four bases and each peak represents a DNA fragment terminating in that particular base. The order of fragment lengths yields the sequence of bases in one strand. Reading the sequence enabled microbiologists to determine the beginning and endpoint of genes and ultimately entire genomes. The DNA revolution began with bacteria. What
amazed the world about DNA was that such a simple substance, composed of only four types of subunit, is the genetic material that determines all the different organisms on Earth. The promise of this insight was fi rst fulfi lled in bacteria and bacteriophages, whose small genomes and short generation times made key experiments possible. Furthermore, naturally occurring mechanisms of gene exchange in bacteria and viruses provided scientists with the key tools for transferring genes, including those of animals and plants. Consider these examples: ■
Restriction endonucleases led to recombinant DNA. Bacteria make restriction endonucleases, enzymes that cut DNA at positions determined by specific short base sequences. In nature, restriction
C T C T T A G T G G C G C A G C T A A C G C A T T A A G T T G A C C G 300
310
320
330
Figure 1.31 A DNA sequence fluorogram. Sequence obtained by an undergraduate at Kenyon College using an ABI sequencer, from genomic DNA of a microbe isolated from a discarded beverage container. Each colored trace represents the intensity of fluorescence of one of the four bases terminating a chain of DNA.
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Public response. As early as the 1970s, when the DNA revolution was largely limited to bacteria, its implications drew public concern. The use of recombinant DNA to make hybrid organisms—organisms combining DNA from more than one species—seemed “unnatural,” although we now know that interspecies gene transfer occurs ubiquitously in nature. Furthermore, recombinant DNA technology raised the specter of placing deadly genes that produce toxins such as botulin into innocuous human flora such as E. coli. The unknown consequences of recombinant DNA so concerned molecular biologists that they held a conference to assess the dangers and restrict experimentation on recombinant DNA. The conference, led by Paul Berg and Maxine Singer at Asilomar in 1975, was possibly the fi rst time in history that a group of scientists organized and agreed to regulate and restrict their own field.
Asilomar conference on the dangers of recombinant DNA
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Ribosome
Messenger RNA
Courtesy of Gabriel Weiss
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endonucleases protect bacteria from the foreign DNA of viruses. In the test tube, purified restriction endonucleases were used to “cut and paste” DNA from two unrelated organisms, generating recombinant DNA. The construction of artificially recombinant DNA, by “gene cloning,” ultimately made it possible to transfer genes between the genomes of virtually all types of organisms, using processes derived from natural phenomena of bacterial transformation. A thermophilic DNA polymerase was used for polymerase chain reaction (PCR) amplification of DNA. While molecular biologists forged ahead in the laboratory, microbial ecologists discovered new species of bacteria and archaea in increasingly extreme habitats. A hot spring in Yellowstone National Park yielded the bacterium Thermus aquaticus, whose DNA polymerase could survive many rounds of cycling to near-boiling temperature. The Taq polymerase formed the basis of a multibillion-dollar industry of PCR amplification of DNA, with applications ranging from genome sequencing to forensic identification. Gene regulation discovered in bacteria provided models for animals and plants. The information carried by DNA was shown to be expressed by transcription to RNA and then translation of RNA to make proteins. Most of the key discoveries of gene expression were made in bacteria and bacteriophages. The regulation of expression of a bacterial gene was first demonstrated for the lac operon, a set of contiguous genes in E. coli. Transcription of the lac operon was shown to be regulated by a repressor protein binding to DNA. DNA-binding proteins were subsequently demonstrated in all classes of living organisms.
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Figure 1.32 “Protein Jive Sutra.” At Stanford University, in 1971, rock dancers represented the process of protein synthesis in the ribosome. The depiction was immortalized in a film produced by chemist Kent Wilson and directed by Gabriel Weiss, with an introduction by Nobel laureate Paul Berg.
On the positive side, the emerging world of molecular biology drew excitement from students at a time of new ideas and social change. In 1971, the newly discovered process of protein translation by the bacterial ribosome inspired a classic cult film, Protein Synthesis: An Epic on the Cellular Level, directed by Gabriel Weiss and choreographed by Jackie Bennington, America’s 1969 Junior Miss (Fig. 1.32). Introduced by Nobel laureate Paul Berg, the film depicts dancers on the Stanford University football field forming the shape of the ribosome while “messenger RNA” and “transfer RNAs” meet to assemble a “protein.” The dancers sway to “Protein Jive Sutra,” performed by a Haight-Ashbury-inspired rock band. Their excitement echoed that of scientists exploring the extraordinary potential of microbial discovery. THOUGHT QUESTION 1.9 Do you think engineered strains of bacteria should be patentable? What about sequenced genes or genomes?
Microbial Discoveries Transform Medicine and Industry Twentieth-century microbiology transformed the practice of medicine and generated entire new industries of biotechnology and bioremediation. Following the discovery of penicillin, Americans poured millions of dollars of private and public funds into medical research. The March of Dimes campaign for private donations to prevent polio
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Mic r o b ia l L ife : Orig in and Disc ov ery
Table 1.3 Fields of research in microbiology. Field
Subject of study
Experimental microbiology Medical microbiology Epidemiology Immunology Food and industrial microbiology Environmental microbiology Forensic microbiology Astrobiology
Fundamental questions about microbial form and function, genetics, and ecology The mechanism, diagnosis, and treatment of microbial disease Distribution and causes of disease in humans, animals, and plants The immune system and other host defenses against infectious disease Microbial and industrial food products, food contamination, and bioremediation Microbial diversity and microbial processes in natural and artificial environments Analysis of microbial strains as evidence in criminal investigations The origin of life in the universe and the possibility of life outside Earth
Joan Slonczewski, Kenyon College
led to the successful development of a vaccine that has nearly eliminated the disease. With the end of World War II, research on microbes and other aspects of biology drew increasing fi nancial support from U.S. government agencies, such as the National Institutes of Health and the National Science Foundation, as well as from governments of other countries, particularly the European nations and Japan. Further support has come from private foundations, such as the Pasteur Institute, the Wellcome Trust, and the Howard Hughes Medical Institute. Research in microbiology fi nds applications in diverse fields (Table 1.3), all of which recruit microbiologists (Fig. 1.33). Cloned genes in bacteria produce valuable therapeutic proteins, such as insulin for diabetics. Recombinant viruses make safer vaccines. Bacteria and viral recombinant genomes are used to construct transgenic animals and plants. In environmental science, newly discovered microbes provide new ways to bioremediate wastes and control insect pests. On a global level, the management of our planet’s biosphere, with the challenges of pollution and global warming, increasingly depends on our understanding of microbial ecology. American Society for Microbiology
TO SU M MAR I Z E: Microbiologists at work. Students at Kenyon College conduct research on bacterial gene expression.
Figure 1.33 ■
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Genetics of bacteria, bacteriophages, and fungi in the early twentieth century revealed fundamental insights about gene transmission that apply to all organisms. Structure and function of the genetic material, DNA was revealed by a series of experiments in the twentieth century. Molecular microbiology generated key advances, such as the use of restriction enzymes, the cloning of the fi rst recombinant molecules, and the invention of DNA-sequencing technology.
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Genome sequence determination and bioinformatic analysis became the tools that shape the study of biology in the twenty-fi rst century. Microbial discoveries transformed medicine and industry. Biotechnology enables the production of new kinds of pharmaceuticals and industrial products.
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Pa r t 1
Concluding Thoughts The advances in microbial science raise important questions for society. Who shall bear the growing costs of sophisticated research on microbial pathogens? Should genetically modified bacteria be released into the environment for agriculture to increase crop yield? Should deadly viruses be engineered as vectors for gene therapy? This book explores the current explosion of knowledge about microbial cells, genetics, and ecology. We introduce the applications of microbial science to human affairs,
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from medical microbiology to environmental science. Most importantly, we discuss research methods—how scientists make the discoveries that will shape tomorrow’s view of microbiology. In the rest of Part 1, Chapter 2 presents the visualization tools that make possible our increasingly detailed view of the structures of cells (Chapter 3) and viruses (Chapter 6). Chapters 4 and 5 introduce microbial nutrition and growth in diverse habitats, including new information derived from the sequencing of microbial genomes. Throughout, we invite readers to share with us the excitement of discovery in microbiology.
C H A P T E R R E V I EW Review Questions 1. Explain the apparent contradictions in defining micro-
6. Explain how microbes are cultured on liquid and
biology as the study of microscopic organisms or the study of single-celled organisms. What is the genome of an organism? How do genomes of viruses differ from those of cellular microbes? Under what conditions might microbial life have originated? What evidence supports current views of microbial origin? List the ways in which microbes have affected human life throughout history. Summarize the key experiments and insights that shaped the controversy over spontaneous generation. What key questions were raised, and how were they answered?
solid media. Compare and contrast the culture methods of Koch and Winogradsky. How did their different approaches to microbial culture address different questions in microbiology? 7. Explain how a series of observations of disease transmission led to development of immunization to prevent disease. 8. Summarize key historical developments in our view of microbial taxonomy. What attributes of microbes have made them challenging to classify? 9. Explain how various discoveries in “natural” bacterial genetics were used to develop recombinant DNA technology.
2. 3.
4. 5.
Key Terms agar (21) antibiotic (25) antiseptic (25) Archaea (9) archaeon (9) aseptic (25) autoclave (17) Bacteria (9) bacterium (9) chain of infection (21) chemiosmotic hypothesis (33) chemolithotrophs (26) colonies (21) DNA sequencing (34) electron microscope (32)
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endosymbiont (27) enrichment culture (26) Eukarya (9) eukaryote (8) fermentation (17) genome (9) geochemical cycling (27) germ theory of disease (17) immune system (24) immunity (24) immunization (24) Koch’s postulates (21) lithotrophs (26) microbe (7) monophyletic (30)
nitrogen fixation (27) petri dish (21) photosynthesis (27) polyphyletic (30) prokaryotes (8) pure culture (21) recombinant DNA (35) reduced (18) restriction endonuclease (34) spontaneous generation (16) transformation (33) ultracentrifuge (32) vaccination (23) virus (8) Winogradsky column (27)
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Recommended Reading Angert, Esther, Kendall D. Clements, and Norman R. Pace. 1993. The largest bacterium. Nature 362:239–241. Brock, Thomas D. 1999. Robert Koch: A Life in Medicine and Bacteriology. ASM Press, Washington, DC. Brock, Thomas D. 1999. Milestones in Microbiology, 1546 to 1940. ASM Press, Washington, DC. Doudna, Jennifer A., and Thomas R. Cech. 2004. The chemical repertoire of natural ribozymes. Nature 418:222–228. Dubos, Rene. 1998. Pasteur and Modern Science. Translated by Thomas Brock. ASM Press, Washington, DC. Fleishmann, Robert D., Mark D. Adams, Owen White, Rebecca A. Clayton, Ewen F. Kirkness, et al. 1995. Wholegenome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496–512. Hesse, Wolfgang. 1992. Walther and Angelina Hesse—early contributors to bacteriology. ASM News 58:425–428. Joklik, Wolfgang K., Lars G. Ljungdahl, Alison D. O’Brien, Alexander von Graevenitz, and Charles Yanofsky (eds.). 1999. Microbiology: A Centenary Perspective. ASM Press, Washington, DC.
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Margulis, Lynn. 1968. Evolutionary criteria in Thallophytes: A radical alternative. Science 161:1020–1022. Raoult, Didier. 2005. The journey from Rickettsia to Mimivirus. ASM News 71:278–284. Sherman, Irwin W. 2006. The Power of Plagues. American Society for Microbiology Press, Washington, DC. Thomas, Gavin. 2005. Microbes in the air: John Tyndall and the spontaneous generation debate. Microbiology Today (Nov. 5): 164–167. Ward, Naomi, and Claire Fraser. 2005. How genomics has affected the concept of microbiology. Current Opinion in Microbiology 8:564–571. Westall, Frances. 2005. Life on the early earth: A sedimentary view. Science 308:366–367. Woese, Carl R., and George E. Fox. 1977. Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proceedings of the National Academy of Sciences USA 74:5088–5090. Zuniga, Elina I., Bumsuk Hahm, Kurt H. Edelmann, and Michael B. A. Oldstone. 2005. Immunosuppressive viruses and dendritic cells: A multifront war. ASM News 71:285–290.
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Chapter 2
Observing the Microbial Cell 2.1 2.2 2.3 2.4 2.5 2.6 2.7
Observing Microbes Optics and Properties of Light Bright-Field Microscopy Dark-Field, Phase-Contrast, and Interference Microscopy Fluorescence Microscopy Electron Microscopy Visualizing Molecules
Microscopy reveals the vast realm of bacteria and protists invisible to the unaided eye. The microbial world spans a wide range of size—over several orders of magnitude. For different size ranges, we use different instruments, from the simple bright-field microscope to the electron microscope. The microscope enables us to count the number of microbes in the human bloodstream or in dilute natural environments such as the ocean. It shows how microbes swim and respond to signals such as a new food source. Advanced forms of microscopy reveal microbes in remarkable detail. Fluorescence microscopy shows how microbial cells develop and reproduce. Electron microscopy explores the cell’s interior, showing how all the parts of the cell fit together. The electron microscope is used to build models of viruses, and to probe the subcellular structures of membranes, ribosomes, and flagellar motors. Scanning probe microscopy images live microbes with an unprecedented degree of realism.
Atomic-force microscopic image (AFM) of Corynebacterium glutamicum cells, showing the contours of their formidable S-layer. Each cell is 1.0–1.5 µm in length, and about 0.7 µm in diameter. C. glutamicum is a major industrial producer of amino acids and vitamins. Source: Nicole Hansmeier, et al. 2006. Microbiology 152:923–935. Photo from Hansmeier, et al. 2006. SGM Microbiology No. 4.
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O b s e r vin g t h e Mic robial C ell
When the microscope of Antoni van Leeuwenhoek (1632–1723) fi rst revealed the tiny life-forms on his teeth, scientists in England refused to believe it until their own instruments showed the same. Van Leeuwenhoek’s superior lenses were key to his success. Since the time of van Leeuwenhoek, microscopists have devised ever more powerful instruments to search for microbes in unexpected habitats. An example of such a habitat is the human stomach, long believed too acidic to harbor life. In the 1980s, however, Australian scientist Barry Marshall reported finding a new species of bacterium in the stomach. The bacteria, Helicobacter pylori, proved difficult to isolate and culture, and for over a decade, medical researchers refused to believe Marshall’s report. Ultimately, electron microscopy (EM) confirmed the existence of H. pylori in the stomach and helped document its role in gastritis and stomach ulcers. The scanning electron micrograph in Figure 2.1 shows H. pylori colonizing the gastric crypt cells. H. pylori bacteria are comma-shaped rods with a polar tuft of flagella. The contours of cells and flagella are well resolved by the scanning beam of electrons, an achievement far beyond that possible with a light microscope. Today, various kinds of microscopes are used in many settings, from hospitals and veterinary clinics to industrial plants and wastewater treatment facilities. This chapter covers two areas: ■
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Light microscopy. The theory and practice of light microscopy are essential for every student and professional observing microorganisms. Advanced tools for research. Fluorescence microscopy, electron microscopy, and X-ray diffraction crystallography probe ever farther the frontiers of the unseen.
2.1
Observing Microbes
Most microbes are too small to be seen; that is, they are microscopic, requiring the use of a microscope to be seen. But why can’t we see microbes without magnification? The answer is surprisingly complex. In fact, our defi nition of “microscopic” is based not on inherent properties of the organism under study, but on the properties of our eyes. What is “microscopic” actually lies in the eye of the beholder.
Resolution of Objects by Our Eyes The size at which objects become visible depends on the resolution of the observer’s eye. Resolution is the smallest distance by which two objects can be separated and still be distinguished. In the eyes of humans and other animals, resolution is achieved by focusing an image on a retina packed with light-absorbing photoreceptor cells (Fig. 2.2). A group of photoreceptors with their linked neurons forms one unit of detection, comparable to a pixel
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Veronika Burmeister/Visuals Unlimited
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1 µm
Helicobacter pylori within the crypt cells of the stomach lining. Microscopy demonstrated the presence of H. pylori, the causes of stomach ulcers, growing on the lining of the human stomach, a location previously believed too acidic to permit microbial growth. Scanning electron micrograph, colorized to indicate bacteria (green).
Figure 2.1
on a computer screen. The distance between two retinal “pixels” limits resolution. The resolution of the human retina (that is, the length of the smallest object most human eyes can see) is about 150 µm, or one-seventh of a millimeter. In the retinas of eagles, photoreceptors are more closely packed, so an eagle can resolve objects eight times as small or eight times as far away as a human can; hence, the phrase “eagle-eyed” means sharp-sighted. On the other hand, insect compound eyes have one-hundredth the resolution of human eyes because their receptor cells are farther apart than ours. If a science-fictional giant ameba had eyes with a retinal resolution of 2 meters (m), it would perceive humans as we do microbes. NOTE: In this book, we use standard metric units for
size: 1 millimeter (mm) = one thousandth of a meter = 10 –3 m 1 micrometer (µm) = one thousandth of a millimeter = 10 –6 m 1 nanometer (nm) = one thousandth of a micrometer = 10 –9 m 1 picometer (pm) = one thousandth of a nanometer = 10 –12 m Some authors still use the traditional unit angstrom (Å), which equals a tenth of a nanometer, or 10 –10 m.
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Photoreceptors Cone
Eye muscle
Retina
Iris Cornea
Pupil Optic nerve
Lens
Rod
Figure 2.2 Defining the microscopic. We define what is visible and what is microscopic in terms of the human eye. Within the human eye, the lens focuses an image on the retina. Adjacent light receptors are sufficiently close to resolve the image of a human being, even at a distance where its apparent height shrinks to a millimeter.
Resolution Differs from Detection
Microbial Size and Shape
Objects of sizes below the resolution limit may yet be detected; that is, we can observe their presence as a group. Thus, our eyes can detect a large population of microbes, such as a spot of mold on a piece of bread (about a million cells) or a cloudy tube of bacteria in liquid culture (a million cells per milliliter; Fig. 2.3A). Detection, the ability to determine the presence of an object, differs from resolution. When the unaided eye detects the presence of mold or bacteria, it cannot resolve individual cells. To resolve bacterial cells, such as those of the wetland phototroph Rhodospirillum rubrum (Fig. 2.3B), requires magnification. In microscopy, magnification means to increase the apparent size of an image so as to resolve smaller separations between objects and thus increase the information obtained by our eyes.
Different kinds of microbes differ in size, over a range of several orders of magnitude, or powers of ten (Fig. 2.4). Eukaryotic microbes are often sufficiently large (10–100 µm) 2
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Prochlorococcus marinus, 0.4 µm Staphylococcus aureus, 0.9 µm Escherichia coli, 2.5 µm Bacillus megaterium, 4 µm
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Red blood cell, 8 µm Paramecium, 100 µm
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Steffen Klamt, U. Magdeburg
Joan Slonczewski, Kenyon College
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Relative sizes of different cells. Microbial cells come in various sizes, most of which are below the threshold of resolution by the unaided human eye (150 µm). Note that most bacteria are much smaller than the human red blood corpuscle, a small eukaryotic cell.
Figure 2.4
Detecting and resolving bacteria. A. A tube of bacterial culture, Rhodospirillum rubrum. Bacterial cells are detected, though not resolved. B. Bright-field image. Individual cells of R. rubrum are resolved.
Figure 2.3
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that we can resolve their compartmentalized structure under a light microscope. Prokaryotes (bacteria and archaea) are generally smaller (0.4–10 µm); thus, their overall size can be seen, but their internal structures are too small to be resolved. Nevertheless, a few bacterial species, such as Thiomargarita namibiensis, are large enough to be seen without a microscope, while many unculturable marine eukaryotes are as small as the smallest bacteria. Thus, the actual size ranges of eukaryotic and prokaryotic microbes overlap substantially. Many eukaryotes, such as protists and algae, can be resolved under low-power magnification to reveal internal and external structures such as the nucleus, vacuoles, and flagella (Fig. 2.5). Protists show complex shapes and appendages. For example, amebas from an aquatic ecosystem show large nuclei and pseudopods to engulf prey (Fig. 2.5A). Pseudopods can be seen to move through streaming of their cytoplasm. Another protist readily observed by light microscopy is Giardia lamblia, a water contaminant that causes outbreaks of intestinal illness in cities and day-care centers (Fig. 2.5B). In the Giardia cell, we observe two nuclei and multiple flagella. Eukaryotic flagella propel the cell by a whiplike action. Similarly, we can observe the complex internal patterns of chloroplasts in algae and the exceptional variety of crystalline forms in diatoms. For more on microbial eukaryotes, see Chapter 20. Prokaryotic cell structures are generally simpler than those of eukaryotes (see Chapter 3). Figure 2.6 shows representative members of several common cell types, as visualized by light microscopy or by scanning electron microscopy. Note that with light microscopy, the cell shape is barely discernable under the highest power.
C. Spirochetes. Borrelia burgdorferi, causative organism in Lyme disease, among human blood cells (LM).
50 µm
Nucleus
Flagellum
10 µm
Eukaryotic microbial cells. Eukaryotic microbes are large enough that details of internal and external organelles can be seen under a light microscope. A. Amoeba proteus. B. Giardia lamblia.
Figure 2.5
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15 µm
©Dennis Kunkel/ Visuals Unlimited
D. Spirochetes. Leptospira interrogans, causative organism in leptospirosis in animals and humans (SEM).
E. Cocci in pairs (diplococci). Streptococcus pneumoniae, a cause of pneumonia. Methylene blue stain (LM). George Wilder/Visuals Unlimited
Pseudopod
Andrew Syred/Photo Researchers, Inc.
5 µm
2 µm
1 µm
Dennis Kunkel Microscopy
10 µm
B.
Nucleus
B. Rods (bacilli). Lactobacillus acidophilus, gram-positive bacteria (SEM).
A. Filamentous rods (bacilli). Lactobacillus lactis, gram-positive bacteria (LM).
F. Cocci in chains. Anabaena spp., filamentous cyanobacteria. Producers for the marine food chain (SEM). Dennis Kunkel Microscopy
A.
With scanning electron microscopy, the shapes appear clearer, but we still see no subcellular structures. Subcellular structures are best visualized with transmission electron microscopy and fluorescence microscopy (discussed below). Certain shapes of bacteria are common to many taxonomic groups (Fig. 2.6). For example, both bacteria and archaea form similarly shaped rods, or bacilli, and cocci (spheres). Thus, rods and spherical shapes have evolved independently within different taxa. On the other hand, a unique bacterial shape is seen in the spirochetes, which cause diseases such as syphilis and Lyme disease. Spirochetes possess an elaborate spiral structure with internal axial fi laments and flagella as well as an outer sheath (for more on spirochetes, see Section 18.7). Spiral axial fi laments are found only within the spirochete group of closely related species.
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10 µm
Common shapes of bacteria. The shape of most bacterial cells can be discerned with light microscopy (A, C, E), but their subcellular structures and surface details cannot be seen. Surface detail is revealed by scanning electron microscopy (SEM) (B, D, F). These SEM images are colorized to enhance clarity.
Figure 2.6
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0.1 µm = 100 nm
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Human eye Light microscopy Scanning electron microscopy Transmission electron microscopy Atomic force microscopy X-ray crystallography
Microscopy and X-ray crystallography, range of resolution. A. Light microscopy reveals internal structures of a paramecium (a eukaryote). LM, Magnification ×100 at 35 mm size. B. Pseudomonas sp., rod-shaped bacteria, are barely resolved. LM, Magnification ×1200. C. Internal structures of the bacterium Escherichia coli are revealed by transmission electron microscopy (TEM). Magnification ×32,000. D. Individual ribosomes are attached to the messenger RNA molecules that are translated to make peptides (TEM). Magnification ×150,000. E. A ribosome (diameter, 21 nm) cannot be observed directly but can be modeled using X-ray crystallography.
Figure 2.7
NOTE: The genus name Bacillus refers to a specific tax-
onomic group of bacteria, but the term bacillus (plural, bacilli) refers to any rod-shaped bacterium or archaeon.
Microscopy for Different Size Scales To resolve microbes and microbial structures of different sizes requires different kinds of microscopes. Figure 2.7 shows the different techniques used to resolve microbes and structures of various sizes. For example, a paramecium can be resolved under a light microscope, but an individual ribosome requires electron microscopy. ■
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Light microscopy resolves images of individual bacteria based on their absorption of light. The specimen is commonly viewed as a dark object against a light-fi lled field, or background; this is called brightfield microscopy (seen in Figs. 2.7A, B). Advanced techniques, based on special properties of light, include dark-field, phase-contrast, and fluorescence microscopy. Electron microscopy (EM) uses beams of electrons to resolve details several orders of magnitude smaller than those seen under light microscopy.
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In scanning electron microscopy (SEM), the electron beam is scattered from the metal-coated surface of an object, generating an appearance of three-dimensional depth. In transmission electron microscopy (TEM) (Figs. 2.7C, D), the electron beam travels through the object, where the electrons are absorbed by an electron-dense metal stain. Atomic force microscopy (AFM) uses van der Waals forces between a probe and an object to map the three-dimensional topography of a cell. X-ray crystallography detects the interference pattern of X-rays entering the crystal lattice of a molecule. From the interference pattern, researchers build a computational model of the structure of the individual molecule, such as a protein or a nucleic acid, or even a molecular complex such as a ribosome (Fig. 2.7E). Nuclear magnetic resonance (NMR) imaging provides structural information based on the magnetic properties of atomic nuclei.
THOUGHT QUESTION 2.1 You have discovered a new kind of microbe, never observed before. What kind of questions about this microbe might be answered by light microscopy? What questions would be better addressed by electron microscopy?
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Detection is the ability to determine the presence of an object. Resolution is the smallest distance by which two objects can be separated and still be distinguished. Magnification means an increase in the apparent size of an image so as to resolve smaller separations between objects. Eukaryotic microbes may be large enough to resolve subcellular structures under a light microscope, although some eukaryotes are as small as bacteria. Bacteria and archaea are usually too small for subcellular resolution. Their shapes include characteristic forms, such as rods and cocci. Different kinds of microscopy are required to resolve cells and subcellular structures of different sizes.
A. Electrical field Magnetic field
Wavelength (λ)
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Molecular Expressions: Exploring the World of Optics and Microscopy, Florida State University
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TV AM/FM radio waves
Optics and Properties of Light
Light microscopy directly extends the lens system of our own eyes. Light is part of the spectrum of electromagnetic radiation (Fig. 2.8), a form of energy that is propagated as waves associated with electrical and magnetic fields. Regions of the electromagnetic spectrum are defi ned by wavelength, which for visible light is about 400–750 nm. Radiation of longer wavelengths includes infrared and radio waves, whereas shorter wavelengths include ultraviolet rays and X-rays.
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Electromagnetic energy. A. Electromagnetic radiation is composed of electrical and magnetic waves perpendicular to each other. B. The electromagnetic spectrum includes the visual range, used in light microscopy.
Figure 2.8
Light Carries Information All forms of electromagnetic radiation carry information from the objects with which they interact. The information carried by radiation can be used to detect objects; for example, radar (using radio waves) detects a speeding car. All electromagnetic radiation travels through a vacuum at the same speed (c), about 3 × 108 meters per second (m/s). The speed of light (c) is equal to the wavelength (λ) of the radiation multiplied by its frequency (ν), the number of wave cycles per unit time. Frequency is usually measured in hertz (Hz), reciprocal seconds: c = λν Because c is constant, the longer the wavelength λ, the lower the frequency ν. For electromagnetic radiation to resolve an object from neighboring objects or from its surrounding medium, certain conditions must exist:
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Contrast between the object and its surroundings. Contrast is the difference in light and dark. If an object and its surroundings absorb or reflect radiation equally, then the object will be undetectable. Thus, it is hard to observe a cell of transparent cytoplasm floating in water because the aqueous cytoplasm and the extracellular water tend to transmit light similarly, producing little contrast. Wavelength smaller than the object. For an object to be resolved, the wavelength of the radiation must be equal to or smaller than the size of the object. If the wavelength of the radiation is larger than the object, then most of the wave’s energy will simply pass through it, like an ocean wave passing around
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a dock post. Thus, radar, with a wavelength of 1–100 centimeters (cm), cannot resolve microbes, though it easily resolves cars and people. A detector with sufficient resolution for the given wavelength. The human eye has a retina with photoreceptors that absorb radiation within a narrow range of wavelengths, 400–750 nm (0.40– 0.75 µm), which we defi ne as “visible light.” But the distance between our retinal photoreceptors is 150 µm, about 500 times the wavelength of light. Thus, our eyes are unable to access all the information contained in the light that enters. In order for us to exploit the full information capacity of light, the light rays from point sources of light must be spread apart sufficiently to be resolved by our retinal photoreceptors. The spreading of the light rays results in magnification of the image.
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B. Reflection Incident angle θ
Angle of reflection θ′
Energy level raised The object blocks part of the light.
C. Refraction
The wave front of the light bounces off the surface.
D. Scattering
Light Interacts with an Object The physical behavior of light in some ways resembles a beam of particles and in other ways a waveform. The particles of light are called photons. Each photon has an associated wavelength that determines how the photon will interact with a given object. The combined properties of particle and wave enable light to interact with an object in several different ways: absorption, reflection, refraction, and scattering. ■
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Absorption means that the photon’s energy is acquired by the absorbing object (Fig. 2.9A). The energy is converted to a different form, usually heat (infrared radiation), a form of electromagnetic radiation of longer wavelength than light. When a microbial specimen absorbs light, it can be observed as a dark spot against a bright field, as in bright-field microscopy. Some molecules that absorb light of a specific wavelength reemit energy not as heat, but as light with a longer wavelength; this is called fluorescence. Fluorescence is also used in microscopy (discussed later). Reflection means that the wave front redirects from the surface of an object at an angle equal to its incident angle (Fig. 2.9B). Reflection of light waves is analogous to the reflection of water waves. Reflection from a silvered mirror or a glass surface is used in the optics of microscopy. Refraction is the bending of light as it enters a substance that slows its speed (Fig. 2.9C). Such a substance is said to be “refractive” and, by definition, has a higher refractive index than air. Refraction is the key property that enables a lens to magnify an image. Scattering occurs when a portion of the wave front is converted to a spherical wave originating from the object (Fig. 2.9D). If a large number of particles
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The light bends when it enters a substance that changes its speed.
Figure 2.9
A small fraction of the incident light is scattered in all directions. Occurs when the dimensions of the object are close to the wavelength of incident light.
Interaction of light with matter.
simultaneously scatter light, a haze is observed— for example, the haze of bacteria suspended in a culture tube. Special optical arrangements (such as dark-field microscopy, discussed later) can use scattered light to detect (but not resolve) microbial shapes smaller than the wavelength of light.
Refraction Enables Magnification Magnification requires refraction of light through a medium of high refractive index, such as glass. As a wave front of light enters a refractive material, the region of the wave that fi rst reaches the material is slowed, while the rest of the wave continues at its original speed until it also passes into the refractive material (Fig. 2.10). As the entire wave front continues through the refractive material, its path continues at an angle from its original direction. At the opposite face of the refractive material, the wave front resumes its original speed. THOUGHT QUESTION 2.2 Explain what happens to the refracted light wave as it emerges from a piece of glass of even thickness. How does its new speed and direction compare with its original (incident) speed and direction?
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60°
(Glass refractive index greater than for air) 34.5°
Wave front slows in glass, causing a change in direction.
60°
Refraction of light waves. Wave fronts of light shift direction as they enter a substance of higher refractive index, such as glass.
Figure 2.10
In a lens, a parabolic surface focuses impinging light rays. Parallel light rays entering the lens emerge at angles so as to intersect each other at the focal point, equivalent to the focus of the parabola (Fig. 2.12). From the focal point, the light rays continue onward in an expanding wave front. This expansion magnifies the image carried by the wave. The distance from the lens to the focal point (called the focal distance) is determined by the degree of parabolic curvature of the lens and by the refractive index of its material. In Figure 2.12, the object under observation is placed just outside the focal plane, a plane containing the focal point of the lens. All light rays from the object are bent by the lens, converging at the opposite focal point. At the focal point, the light rays continue through until they converge with nonparallel light rays refracted by the lens. The plane of convergence generates a reversed image of the object. The image is expanded, or magnified, by the spreading out of refracted rays. The distances between parts of the image are enlarged, enabling our eyes to resolve fi ner details. FSU Tutorial on Magnification
Light rays entering lens Focus
A wider parabola has a more distant focal point.
THOUGHT QUESTION 2.3 Parabolic lenses are generally “biconvex,” that is, curving outward on both sides. What would happen to parallel light rays that pass through a lens that is concave on both sides? Or a lens that is convex on one side and concave with equal curvature on the other?
Magnification and Resolution
Focus A narrower parabola has a closer focal point.
Figure 2.11 The focus of a parabola. parabola, the more distant the focal point.
The wider the
Refraction magnifies an image when light passes through a refractive material shaped so as to spread its rays, widening the wave front. The shape that accomplishes this widening is that of a parabola. Light rays entering a parabolic surface are focused; that is, they are each bent at an angle such that all rays meet at a certain point, called the focus of the parabola (Fig. 2.11).
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The spreading of light rays does not necessarily increase resolution. For example, an image composed of dots does not gain detail when enlarged on a photocopier, nor does an image composed of pixels gain detail when enlarged on a computer screen. In these cases, resolution fails to increase because the individual details of the image expand in proportion to the expansion of the overall image. Magnification without increase in resolution is called empty magnification. The resolution of detail in microscopy is limited by the degree to which details “expand” with magnification. This expansion of detail is limited by the interference of light rays converging at the focal point. In interference, two wave fronts interact by addition (amplitudes in phase) or subtraction (amplitudes out of phase) (Fig. 2.13A). The result of interference between two waves is a pattern of alternating zones of constructive and destructive interference (brightness and darkness) (Fig. 2.13B). In theory, a perfect lens that focuses all the light from an object should have no interference as its rays
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Object
F
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Focal distance Focal plane
Real image
Focal plane Biconvex lens
Generating an image with a lens. The object is placed within the focal plane of the lens. All light rays from the object are bent by the lens, converging at the opposite focal point. The light rays continue through the focal point, generating an image of the object that is reversed and magnified.
Figure 2.12
A.
B.
Constructive interference (in phase)
Destructive interference (out of phase) D
A1
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D A1
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D = Propagation direction A = Amplitude
Wavelength D A2
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D AR AR
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A1 – A2 = AR Darker
Constructive (additive) and destructive (subtractive) interference of light waves. A. In constructive interference, the peaks of the two wave trains rise together; their amplitudes are additive, creating a wave of greater amplitude. In destructive interference, the peaks of the waves are opposite one another, so their amplitudes cancel, creating a wave of lesser amplitude. B. Two wave fronts approaching at an angle generate an interference pattern in which intensity alternately increases and decreases.
Figure 2.13
converge toward the focal point. In fact, however, the outer edges of the lens introduce imperfection. The converging edges of the wave interfere with each other to create alternating regions of light and dark. At the focal point, the interference forms a central sphere of intensity surrounded by a series of concentric spheres of alternating light and dark (Fig. 2.14A). The viewing plane intersects the concentric spheres to form an Airy disk, a disk containing a bright central peak surrounded by rings
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of light and dark. The Airy disk was originally discovered by the astronomer George Airy (1801–1892), who showed that the stars viewed through a telescope could never appear as true point sources, but only as tiny disks of light surrounded by rings. The same principle applies to objects viewed under a microscope.
FSU Tutorial on Airy Disk
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Airy disk
B. Image plane
Image plane Michael W. Davidson
Airy disk
Interference of converging wave fronts
Intensity
Image plane Lens
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Airy disk
Michael W. Davidson
Image plane
Intensity
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Interference of light waves at the focal point generates an Airy disk. A. The interference of converging spherical wave fronts creates a sphere with a central zone of maximum intensity, surrounded by alternating zones of bright and dark. A plane section through the sphere generates the Airy disk. B. The Airy peak and its surrounding rings.
Figure 2.14
The width of the Airy disk—and hence the limit to resolution of detail—depends on the wavelength of light and the quality of the lens. The shorter the wavelength, the narrower the Airy disk. In addition, the central peak of intensity is sharpest when the specimen is at the focal point, where the specimen is said to be in focus. Suppose an object consists of a collection of point sources of light. Each point source generates an Airy disk of rings surrounding one central peak of intensity. The width of the central peak increases with distance away from the focal point. This width will defi ne the resolution, or separation distance, between any two points of the object (Fig. 2.14B). Full resolution is achieved for two points when there is complete separation of the central peaks of the two Airy disks. This resolution distance determines the degree of detail that can be observed along the edge of an object.
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Contrast between object and background makes it possible to detect the object and resolve its component parts. The wavelength of the radiation must be equal to or smaller than the size of the object if we are to resolve its shape. Absorption means that the energy from light (or other electromagnetic radiation) is acquired by the object. Reflection means that the wave front bounces off the surface of a particle at an angle equal to its incident angle. Refraction is the bending of light as it enters a substance that slows its speed. Scattering occurs when a wave front interacts with an object of smaller dimension than the wavelength. Light scattering enables detection of objects whose detail cannot be resolved.
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Electromagnetic radiation interacts with an object and acquires information we can use to detect the object.
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2.3
Bright-Field Microscopy
In bright-field microscopy, an object such as a bacterial cell is perceived as a dark silhouette blocking the pas-
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Wide Airy disk
Increasing Resolution The optics of a modern bright-field microscope are designed to maximize detail under magnification by a lens. In maximizing the resolution, the following factors need to be considered: ■
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Wavelength. The theoretical maximum magnification can be calculated as the resolution distance of our retina (0.15 mm, or 150,000 nm) divided by the average wavelength of light (550 nm, for green). This gives an approximately 300-fold magnification with increasing resolution. Additional optical factors can bring this magnification closer to 1,000×, although full resolution is rarely reached in practice. Any greater magnification expands only the width of the interference patterns; the image becomes larger but with no greater resolution (empty magnification). Light and contrast. For any given lens system, there is an optimal amount of light that yields the highest contrast between the dark specimen and the light background. High contrast is needed to achieve the maximum resolution at high magnification. Lens quality. All lenses possess inherent aberrations that detract from perfect parabolic curvature. Instead of trying to grind a “perfect” single lens, modern manufacturers construct microscopes with a series of lenses that multiply each other’s magnification and correct for aberrations.
Let us fi rst consider magnification of an image by a single lens. The principles described would hold for any lens. Figure 2.15 shows an objective lens, a lens situated directly above an object or specimen that we wish to observe at high resolution. How can we maximize the resolution of details in the image? An object at the focal point of a lens sits at the tip of an inverted light cone formed by rays of light from the lens converging at the object. The angle of the light cone is determined by the curvature and refractive index of the lens. The lens fi lls an aperture, or hole, for the passage of light; and for a given lens, the light cone is defi ned by an angle θ (theta) projecting from the midline, known as the angle of aperture. As θ increases and the horizontal width of the light cone (sin θ) increases, a wider cone of light passes through the specimen. The wider cone of light rays improves the precision of the image by lessening the proportion of edge interference between wave fronts, thereby narrowing the width of the Airy disk interference pattern. Another way to say this is that as sin θ increases, the resolvable distance decreases. Thus, the greater the angle of aperture of the lens, the better the resolution.
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Objective
θ θ =15°
NA = n sin θ = 1.0 sin 15.0° = 0.25
Specimen
High-power magnification 100ⴛ
θ = 72.1° Objective Specimen
Narrow Airy disk
NA = n sin θ = 1.0 sin 72.1° = 0.95 NA = Numerical aperture n = Refractive index = 1.00 (air) θ = Angle of aperture
Figure 2.15 Resolution and numerical aperture. The resolution depends on numerical aperture (NA), which equals the refractive index (n) of the medium containing the light cone multiplied by the sine of the angle of the light cone (θ). Higher magnification occurs at higher NA.
THOUGHT QUESTION 2.4 In theory, what angle θ would produce the highest resolution? What practical problem would you have in designing a lens to generate this light cone? Resolution also depends on the refractive index of the medium containing the light cone, which is usually air. The refractive index is the ratio of the speed of light in a vacuum to its speed in another medium. For air, the refractive index (n) is taken as 1, whereas lens material has a refractive index greater than 1. The lens bends the light spreading at an angle (θ). The product of the refractive index (n) of the medium times sin θ is the numerical aperture (NA): NA = n sin θ In Figure 2.15, we see the calculation of NA for an objective lens of magnification 10×, and for a lens magnification
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100×. As NA increases, the magnification power of the lens increases, although the increase is not linear. Note that as the lens strength increases and the light cone widens, the lens must come nearer the object. Defects in lens curvature become more of a problem, and focusing becomes more challenging. As θ becomes very wide, too much of the light from the object is lost owing to refraction at the glass-to-air interface. The greater the refractive index of the medium between the object and the objective lens, the more light can be collected and focused. For the highest-power objective lens, generally 100×, a zone of even refractive index is maintained by insertion of immersion oil with a refractive index comparable to that of glass (n = 1.5) (Fig. 2.16). Immersion oil minimizes loss of light rays at the widest angles and makes it possible to reach 100× magnification with minimal distortion.
The Compound Microscope
Objective lens Immersion oil
Refracted light No oil
Glass slide Microscope stage Light
Light
Figure 2.16 Use of immersion oil in microscopy. Immersion oil with a refractive index comparable to that of glass (n = 1.5) prevents light rays from bending away from the objective lens. Thus, more light is collected, NA increases, and resolution improves.
a parfocal system, when an object is focused using one lens, it remains in focus, or nearly so, when another lens is rotated to replace the fi rst.
The manufacture of higher-power lenses is difficult owing to decreasing tolerance for aberration. For this reason, we rarely observe through a single lens. Instead NOTE: Objective lenses can be obtained in several we use a compound microscope, a system of multiple different grades of quality, manufactured with differlenses designed to correct or compensate for aberraent kinds of correction for aberrations. Lenses should tion. A typical arrangement of a compound microscope feature at minimum the following corrections: “plan” is shown in Figure 2.17. In this arrangement, the light correction for field curvature, to generate a field that source is placed at the bottom, shining upward through appears fl at, and “apochromat” correction for spheria series of lenses, including the condenser, objective, and cal and chromatic aberrations. ocular lenses. The condenser consists of one or more lenses that focus parallel light rays from the light source onto a small The image from the objective lens is amplified by a area of the slide. The condensed light rays generate a narsecondary focusing step through the ocular lens. The rower Airy interference pattern and Eye B. thus improve the resolution of the A. objective lens. Between the light source and the condenser sits a diaphragm, a device to Eyepiece vary the diameter of the light column. Ocular lens Lower-power lenses require operation at lower light levels because the excess light makes it impossible to observe Focusing knob the darkening effect of absorbance by the specimen (contrast). Higher-power lenses require more light and thus an open diaphragm. Objective lens Objectives The nosepiece of a compound Specimen on revolving Specimen nosepiece microscope typically holds three or four objective lenses of different Condenser lens Stage magnifying power, such as 4×, 10×, Diaphragm Substage 40×, and 100× (requiring immersion condenser oil). These lenses are arranged so as Light Light source to rotate in turn into the optical column. A high-quality instrument will have the lenses set at different heights Figure 2.17 The anatomy of a compound microscope. A. Light path through a from the slide so as to be parfocal. In compound microscope. B. Cutaway view of a compound microscope.
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ocular lens sits directly beneath the observer’s eye. In the process of magnification, each light ray traces a path toward a position opposite its point of origin, thus creating a mirror-reversed image. This reversal must be kept in mind when exploring a field of cells. Research-grade microscopes include a series of ocular and objective lenses. The magnification factor of the ocular lens is multiplied by the magnification factor of the objective lens to generate the total magnification (power). For example, a 10× ocular times a 100× objective generates 1,000× total magnification. Observing a specimen under a compound microscope requires several steps:
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erates extra rings of light surrounding an object whose dimensions are close to the resolution limit. In Figure 2.18, we see microscopic observation of Rhodospirillum rubrum, photosynthetic bacteria that contribute to wetland productivity. As cells of R. rubrum swim in and out of the focal plane, their appearance changes through optical effects. When a bacterium swims out of the focal plane too close to the lens, resolution declines and the image blurs (Fig. 2.18A). When the bacterium swims within the focal plane, its image appears sharp, with a bright line along its edge. A helical cell like R. rubrum shows well-focused segments alternating with hazier segments that are too close to the lens (Fig. 2.18B). When the cell swims too far past the focal plane, the bright interference lines collapse into the object’s silhouette, which now appears bright or “hollow” (Fig. 2.18C). In fact, the bacterium is not hollow at all; only its image has changed. When the cell extends across several focal planes, different portions appear out of focus (either too near or too far from the lens). In addition, when the end of a cell points toward the observer, light travels through the length of the cell before reaching the observer, so the cell absorbs more light and appears dark (see Fig. 2.18D). The
Position the specimen centrally in the optical column. Only a small area of a slide can be visualized within the field of view of a given lens. The higher the magnification, the smaller the field of view will be seen. Optimize the amount of light. At lower power, too much light will wash out the light absorption of the specimen without contributing to magnification. At higher power, more light needs to be collected by the condenser. To optimize light, the condenser must be set at the correct vertical A. position to focus on the specimen, and the diaphragm must be adjusted to transToo close to lens; image is blurred. mit the amount of light that produces the best contrast. Focus the objective lens. The focusing knob permits adjustment of the focal dis- B. tance between the objective lens and the In focus; image specimen on the slide so as to bring the appears sharp. specimen into the focal plane. Typically, we focus fi rst using a low-power objective, which generates a greater depth of C. field—that is, a region along the optical Too far beyond column over which the object appears in focal point; image appears “empty,” reasonable focus. After focusing under with interference low power, we can rotate a higher-power rings. lens into view, then perform a smaller adjustment of focus. D.
Is the Object in Focus? An object appears in focus (that is, is situated within the focal plane of the lens) when its edge appears sharp and distinct from the background. At higher power, however, recognition of the focal plane is a challenge because of Airy-like interference. The shape of the dark object is actually defi ned by the points of light surrounding its edge. The partial resolution of these points of light gen-
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Steffen Klamt, U. Magdeburg
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Object extends through focal planes.
Rhodospirillum rubrum observed at different levels of focus. A. When a bacterium swims too close to the lens, its image blurs. B. When the bacterium lies within the focal plane, its image appears sharp. If the width of the cell crosses several focal planes, some parts appear sharp, whereas other parts appear blurred. C. When the bacterium lies too far from the lens, its image appears “empty” or “hollow,” surrounded by rings of Airy-like interference. D. When the spirillum extends through the focal plane, different parts show different focal effects (in focus, too near, or too far).
Figure 2.18
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observation and identification of motile bacteria swimming in and out of the focal plane present a challenge even to experienced microscopists. The higher the magnification, the narrower the depth of the focal plane; thus, when observing swimming organisms, there is a tradeoff between magnification and depth of field.
used organic synthesis to invent new coloring agents for women’s clothing. Clothing was made of natural fibers such as cotton or wool, so a substance that dyed clothing would be likely to react with biological specimens. How do stains work? Most stain molecules contain conjugated double bonds or aromatic rings that absorb visible light (Fig. 2.19) and one or more positive charges that bind to negative charges on the bacterial cell envelope (discussed in Section 3.4). Different stains vary with respect to their strength of binding and the degree of binding to different parts of the cell.
THOUGHT QUESTION 2.5 Under starvation conditions, bacteria such as Bacillus thuringiensis, the biological insecticide, repackage their cytoplasm into spores, leaving behind an empty cell wall. Suppose, under a microscope, you observe what appears to be a hollow cell. How can you tell if the cell is indeed hollow or if it is simply out of focus?
Different Kinds of Stains A simple stain adds dark color specifically to cells, but not to the external medium or surrounding tissue (in the case of pathological samples). The most commonly used simple stain is methylene blue, originally used by Robert Koch to stain bacteria. A typical procedure for fi xation and staining is shown in Figure 2.20. First, a drop of culture is fi xed on a slide by treatment with methanol or by incubation on a slide warmer. Either treatment denatures cell proteins, exposing side chains that bind to the glass. The slide is then flooded with methylene blue solution. The positively charged molecule binds to the negatively charged cell envelope. After excess stain is washed off and the slide has been dried, it is observed under highpower magnification using immersion oil. A differential stain stains one kind of cell but not another. The most famous differential stain is the Gram stain, devised in 1884 by the Dutch physician Hans Christian Gram (1853–1938). Gram fi rst used the Gram stain to distinguish pneumococcus (Streptococcus pneumoniae) bacteria from human lung tissue. A similar use of the Gram stain is seen in Figure 2.21A, where S. pneumoniae bacteria appear dark purple against the pink background of human epithelial cells. Not all species of bacteria retain the Gram stain (Fig. 2.21B). Different bacterial species are classified gram-positive or gram-negative, depending on whether they retain the purple stain.
Fixation and Staining Improve Resolution and Contrast The simplest way to observe microbes is to place them in a drop of water on a slide with a coverslip. This is called a wet mount preparation. The advantage of the wet mount is that the organism is viewed in as natural a state as possible, without artifacts resulting from chemical treatment; and live behavior such as swimming can be observed. The disadvantage is that most living cells are transparent and therefore show little contrast with the external medium. With limited contrast, the cells can barely be distinguished from background, and both detection and resolution are minimal. The detection and resolution of cells under a microscope are enhanced by fi xation and staining, procedures that usually kill the cell. Fixation is a process by which cells are made to adhere to a slide in a fi xed position. Cells may be fi xed with methanol or by heat treatment to denature cellular proteins, whose exposed side chains then adhere to the glass. A stain absorbs much of the incident light, usually over a wavelength range that results in a distinctive color. The use of chemical stains was developed in the nineteenth century, when German chemists CH3 N CH3 H3C N –
CH3 H3C
(Cl ) S+
N
N Methylene blue
CH3 N
CH3
C
H3C N+ CH3 CH3
(Cl–)
Crystal violet
Figure 2.19 Chemical structure of stains. Methylene blue and crystal violet are cationic (positively charged) dyes. The positively charged groups react with the bacterial cell envelope, which carries mainly negative charge. Chloride (Cl) is the counter ion.
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Gram Staining Separates Bacteria into Two Classes In the Gram stain procedure (Fig. 2.22), a dye such as crystal violet binds to the bacteria; it also binds to the surface of human cells, but less strongly. After the excess stain is washed off, a mordant, or binding agent, is applied. The mordant used is iodine solution, which contains iodide ions (I– ). The iodide complexes with the positively charged crystal violet molecules trapped inside the cells. The crystal violet–iodide complex is now held more strongly within the cell wall. The thicker the cell wall, the more crystal violet–iodide molecules are held.
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Sandra Richter
Eye of Science/Photo Researchers, Inc.
1. Place a loopful of the culture on a clean slide.
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Gram staining of bacteria (a type of differential staining). A. Gram stain of a sputum specimen from a patient with pneumonia, containing gram-positive Streptococcus pneumoniae (purple cocci). Cell length, 0.5 to 1.0 µm. B. Gram stain of a gingival specimen containing both gram-positive and gram-negative bacteria (purple and pink rods). Cell length, 0.5 to 1.0 µm.
Figure 2.21
4. Fix cells to slide by adding drop of methanol; air-dry.
Gram-positive
Gram-negative 1. Add methanol to fix cells to surface, air-dry.
5. Stain (e.g., with methylene blue, 1 min).
Cells are fixed to slide surface.
Cells are fixed to slide surface.
2. Add crystal violet stain (1 min).
Crystal violet stains cells reversibly.
Crystal violet stains cells reversibly. 6. Wash off stain with water.
3. Add iodine, which binds stain to gram-positive cells (1 min). No change in cells.
Crystal violet stain binds to gram-positive cells.
4. Wash with ethanol (20 sec). 7. Blot off excess water. Stain is removed from gram-negative cells.
No change.
5. Add safranin counterstain (1 min). 8. View under microscope.
Gram-positive cells remain dark purple.
Safranin counterstains gram-negative cells.
The Gram stain procedure. The Gram stain distinguishes between gram-positive cells, with thick cell walls, which retain the crystal violet stain, and gram-negative cells, with thinner cell walls, which lose the crystal violet stain but are counterstained by safranin.
Figure 2.22
Figure 2.20
blue.
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Procedure for simple staining with methylene
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Next, a decolorizer, ethanol, is added for a precise time interval (typically, 20 seconds). The decolorizer removes loosely bound crystal violet–iodide, but grampositive cells retain the stain tightly. The gram-positive cells that retain the stain appear dark purple, while the gram-negative cells are colorless. The decolorizer step is critical because if it lasts too long, the gram-positive cells, too, will release their crystal violet stain. In the final step, a counterstain, safranin, is applied. This process allows the visualization of gram-negative material, which is stained pale pink by the safranin. The Gram stain procedure was originally devised to distinguish bacteria (gram-positive) from human cells (gram-negative). Microscopists soon discovered, however, that many important species, such as the intestinal bacterium Escherichia coli and the nitrogen-fi xing symbiont Rhizobium meliloti, do not retain the Gram stain. It turns out that gram-negative species of bacteria possess a thinner and more porous cell wall than gram-positive species (Fig. 2.23). A gram-negative cell wall has only a single layer of peptidoglycan (sugar chains cross-linked by peptides), whereas a gram-positive cell has multiple layers. The multiple layers of peptidoglycan retain enough stain complex so that the cell appears purple. Among bacteria, the Gram stain distinguishes two groups with distinctive cell wall structures: Proteobacteria, with a thin cell wall plus an outer membrane (gramnegative), and Firmicutes, with a multiple-layered cell wall but no outer membrane (gram-positive). The outer membrane of Proteobacteria (discussed in Chapter 3) often possesses important pathogenic factors, such as the lipopolysaccharide endotoxins that cause toxic shock. During the Gram stain procedure, however, the outer membrane is disrupted by the decolorizer, allowing most of the crystal violet–iodide complex to leak out. Thus, the Gram stain emerged as a key tool for biochemical identification of species, and it remains essential in the clinical laboratory. Note, however, that still other groups of bacteria and archaea have different kinds of cell walls that may stain either positive or negative and are thus not distinguished by the Gram stain. Prokaryotic diversity and identification are discussed further in Chapters 18 and 19.
Other Differential Stains Other differential stains applied to various types of prokaryotes are illustrated in Figure 2.24. These include: ■
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Acid-fast stain. Carbolfuchsin specifically stains mycolic acids of Mycobacterium tuberculosis and M. leprae, the causative agents of tuberculosis and leprosy, respectively (Fig. 2.24A). Spore stain. When samples are boiled with malachite green, the stain binds specifically to the endospore
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Gram-positive
Gram-negative
Peptidoglycan Outer membrane Peptidoglycan Cell membrane
Cell membrane (Inner membrane)
1. Crystal violet is added. +
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2. Iodide complexes with crystal violet.
I–
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– +
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3. Decolorizer releases loosely bound stain.
– +
– +
+
+
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– +
– +
I
+
+
Figure 2.23 Mechanism of the Gram stain. In a gram-positive cell, the crystal violet–iodide complex is retained by multiple layers of peptidoglycan. In a gram-negative cell, the stain leaks out. If the decolorizer is applied for too long, the gram-positive cell will lose its stain as well.
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coat (Fig. 2.24B). It detects spores of Bacillus species such as the insecticide B. thuringiensis and B. anthracis, the causative agent of anthrax, as well as spores of Clostridium botulinum, which produces botulin toxin. Negative stain. Some bacteria synthesize a capsule of extracellular polysaccharide filaments, which protects
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©Lauritz Jensen/Visuals Unlimited
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Differential stains. A. Mycobacterium tuberculosis, acid-fast stain (stained cells are red, 1–2 µm long). LM ×600. B. Clostridium botulinum, endospore stain (stained endospores are blue-green, 2 µm long). LM ×400. C. Gloeocapsa, a colonial blue green alga, whose individual cells are encased in thick sheaths. Staining with India ink makes the sheaths clearly visible. Individual cell size, 3–5 µm.
Figure 2.24
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the cell from predation or from engulfment by white blood cells (discussed in Section 3.4). The capsule is transparent and invisible in suspended cells. However, a suspension of opaque particles such as India ink can be added to darken the surrounding medium. The particles are excluded by the thick polysaccharide capsule, which thus appears clear against the dark background (Fig. 2.24C). This is an example of a negative stain. Antibody stains. Specialized stains utilize monoclonal antibodies to identify precise strains of bacteria or even specific molecular components of cells. The antibody (which binds a specific cell protein) is linked to a reactive enzyme for detection or to a fluorophore (fluorescent molecule) for fluorescence microscopy (see below).
2.4
Dark-Field, Phase-Contrast, and Interference Microscopy
Advanced optical techniques enable us to visualize structures that are difficult or impossible to detect under a bright-field microscope, either because their size is below the limit of resolution of light or because their cytoplasm is transparent. These techniques take advantage of special properties of light waves, including scattering and interference patterns. One application of dark-field illumination is the detection of pathogenic spirochetes, such as Treponema pallidum, the causative organism of syphilis. T. pallidum cells are so narrow (0.1 µm) that their shape cannot be fully resolved by light microscopy. Nevertheless, the spiral form of T. pallidum can be detected by dark-field microscopy (Fig. 2.25).
TO SU M MAR I Z E:
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In bright-field microscopy, resolution depends on: ■ The wavelength of light, which limits resolution to about 200 nm. ■ The magnifying power of a lens, which depends on its numerical aperture (n sin θ). ■ The position of the focal plane, the location where the specimen is “in focus” (that is, where the sharpest image is obtained). A compound microscope achieves magnification and resolution through a series of lenses: the condenser, objective, and ocular lenses. A wet mount specimen is the only way to observe living microbes. Fixation and staining of a specimen kills it but improves contrast and resolution. Differential stains distinguish between different kinds of bacteria with different structural features. The Gram stain differentiates between two major bacterial taxa, Proteobacteria (gram-negative) and Firmicutes (gram-positive). Other bacteria and archaea vary in their Gram stain appearance. Eukaryotes stain negative.
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Dark-Field Observation Detects Unresolved Objects Dark-field optics enables microbes to be visualized as halos of bright light against the darkness, just as stars are
Spirochete cell is too narrow to resolve by light microscopy
©Arthur Siegelman/Visuals Unlimited
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Dark-field observation of bacteria. Treponema pallidum specimen from a patient with syphilis. Note the detection of dust particles. Dark-field LM, ×1,100. Cell length about 10 µm; actual cell width, 0.2 µm.
Figure 2.25
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observed against the night sky. A tiny object whose size is well below the wavelength of light, such as a virus particle, can be detected by light scattering. The wave front of scattered light is spherical, like a wave emitted by a point source (see Fig. 2.9D). Light scattering. The scattered wave has a much smaller amplitude than that of the incident (incoming) wave. Therefore, with ordinary bright-field optics, scattered light is washed out. Detection of scattered light requires a modified condenser arrangement that excludes all light transmitted directly (Fig. 2.26). The condenser contains a “spider light stop,” an opaque disk held by three “spider legs” across an open ring. The ring permits only a hollow cone of light to focus on the object. The incident hollow cone converges at the object, then generates an inverted hollow cone radiating outward. The objective lens is positioned in the central region, where it completely misses the directly transmitted light. For this reason, the field appears dark. However, light scattered by the object radiates outward in a spherical wave. A sector of this spherical wave enters the objective lens and is detected as a halo of light. An intriguing application of dark-field optics is the study of bacterial motility. Motility is important in bacterial diseases such as urethritis, in which the organism needs to swim up through the urethra. The bacterial swimming apparatus consists of helical fi laments called
Transmitted light Scattered light
Light cones
10 µm
Motile bacteria observed under dark-field microscopy. A. Flagellated E. coli observed with low light, which limits scattering. Only cell bodies are detected, no flagella. B. The light intensity is increased. Flagella are detected, although their fine structure is not resolved because their width is below the threshold of resolution by light.
Figure 2.27
flagella, which are rotated by a motor device imbedded in the bacterial cell wall (for flagellar structure, see Section 3.7). The “swimming strokes” of bacteria were first elucidated by Howard Berg (1934–) and Robert Macnab (1940–2003) using dark-field optics to view the helical flagella. The length of flagella is great enough to be resolved with light waves—but their width is not. Thus, dark-field optics is needed to detect and observe the helical flagella (Fig. 2.27). Note, however, that the bacterial cell itself appears “overexposed;” its shape is unresolved owing to the high light intensity.
THOUGHT QUESTION 2.6 Some early observers claimed that the rotary motions observed in bacterial flagella could not be distinguished from whiplike patterns, comparable to the motion of eukaryotic flagella. Can you imagine an experiment to distinguish the two and prove that the flagella rotate? Hint: Bacterial flagella can get “stuck” to the microscope slide or coverslip.
Specimen
Light source
A dark-field condenser system, with a spider light stop. Below the condenser, the spider light stop excludes all but an annular ring of light from the light source. The annular ring converges as a hollow cone of light focused on the specimen. Objects in the specimen scatter light in all directions. The scattered light is collected by the objective lens, but the transmitted light shines outside the range of the objective; thus, in the absence of scattering objects, the field appears dark. Only light scattered by the specimen enters the objective lens.
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10 µm
Videos of swimming bacteria
Spider light stop
Figure 2.26
B.
Objective
Condenser lenses
Light source
A.
R. M. Macnab. 1976. J. Clin. Microbiol. 4(3):258
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Limitations of dark-field microscopy. A disadvantage of dark-field microscopy is that any tiny particle, including specks of dust, can scatter light and interfere with visualization of the specimen. Unless the medium is extremely clear, it can be difficult to distinguish microbes of interest from particulates. Other methods of contrast enhancement, such as phase contrast and fluorescence, avoid this difficulty.
Phase-Contrast Microscopy Phase-contrast microscopy exploits differences in refractive index between the cytoplasm and the surrounding medium or between different organelles. This technique is particularly useful for eukaryotic cells, such as algae and protists,
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P. L. Graumann and R. Losick
The optical system for phase contrast was invented in the 1930s by the Dutch microscopist Frits Zernike (1888–1966), for which he earned the Nobel Prize in Physics. In this system, slight differences in the refractive index of the various cell components are transformed into A. B. differences in the intensity of transmitted light. Zernike’s scheme makes use of the fact that living cells have relatively high contrast owing to their high concentration of solutes. Given the size and refractive index of commonly observed cells, light is retarded by approximately onequarter of a wavelength when it passes through the cell. In other words, after having passed through a cell, light exits 50 µm 2 µm the cell about one-quarter of a wavelength behind the phase of light transmitted directly through the medium. Figure 2.28 Phase and interference effects in microscopy. The Zernike optical system is designed to retard the A. Phase-contrast micrograph of the parasitic protist Entamoeba refracted light by an additional one-quarter of a wavehistolytica. B. Nomarski interference micrograph of Bacillus subtilis. length, so that the light refracted through the cell is slowed The apparent three-dimensional effect is illusory. Note that one by a total of half a wavelength compared with the light bacterium appears as if crossed through another. Source: Part B reprinted by permission from P. L. Graumann and R. Losick. 2001. J Bacteriol. 183(13): transmitted through the medium. When two waves are 4052–4060. out of phase by half a wavelength, they produce destructive interference, canceling each other’s amplitude (see Fig. 2.13). A. The result is a region of darkness B. in the image of the specimen. Phase-contrast 1/2 wavelength As in dark-field microscopy, image Light refracted by the light transmitted through 1/2 in total Phase plate the medium in phase-contrast Phase plate microscopy needs to be separated from the light interacting 1/4 wavelength with the object—in this case, light waves slowed by refraction. Light refracted by Specimen 1/4 by specimen This separation is performed by a Specimen Slide ring-shaped slit, called an “annular ring,” similar in function to the spider light stop. The annular ring stops light from passing Unobstructed light directly through the center of the (phase unaltered Annular ring by specimen) lens system, where the specimen is located, and generates a hollow Light source cone of light, which is focused Light source through the specimen and generates an inverted cone above it (Fig. 2.29). Light passing through the specimen, however, is not Figure 2.29 Phase-contrast optics. A. The specimen retards light by approximately one-quarter of a wavelength. The phase plate contains a central disk of refractive material only retarded; it is also refracted that retards light from the specimen by another quarter wavelength, increasing the phase and thus bent into the central difference to half a wavelength. The light from the specimen and the transmitted light are now region within the inverted cone. fully out of phase, and if they coincide, their waveforms cancel, making the specimen appear Both the refracted light dark. B. In the phase-contrast microscope, the annular ring forms a hollow cone of light. As from the specimen and the outer the light cone passes through the refractive material of the specimen, it is delayed by about cone of transmitted light enter one-quarter of a wavelength, and its path bends inward to the central region. This refracted the phase plate. The phase plate light is surrounded by the hollow cone of unrefracted light. The light refracted by the specimen consists of refractive material enters the lens through the dense central disk of the phase plate, which retards the wave by that is thinner in the region met another quarter wavelength. When the transmitted and refracted light cones rejoin at the focal by the outer (transmitted) light point, they are out of phase; their amplitudes cancel each other, and that region of the image cone. The refracted light passing appears dark against a bright background. Stephen Aley, U of Texas, El Paso
which contain many intracellular compartments. For example, Figure 2.28A shows an image of the protist Entamoeba histolytica, a human parasite, obtained by phase-contrast microscopy. In some cells, the nucleus is clearly visible.
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through the center of the phase plate is retarded by an additional one-quarter wavelength compared with the transmitted light passing through the thinner region on the outside. The overall difference approximates half a wavelength, so that the two waves are out of phase, thus canceling each other’s amplitude. When the light from the inner and outer regions focuses at the ocular lens, the amplitudes of the wave trains cancel and produce a region of darkness. In this system, small differences in refractive index can produce dramatic differences in contrast between the offset phases of light. FSU Phase contrast tutorial
Interference Microscopy Other kinds of optical systems have been devised using light interference to enhance cytoplasmic contrast. Interference microscopy enhances contrast by superimposing the image of the specimen on a second beam of light that generates interference fringes. The interference pattern produces an illusion of shadowing across the specimen. For example, the shapes of the Bacillus subtilis cells illuminated by interference contrast (Fig. 2.28B) are more clearly defi ned than in conventional bright-field microscopy (for comparison see Fig. 2.21). One optical system for producing interference contrast is based on the Wollaston-Nomarski prism. In this system, light is polarized to obtain waves oriented in one direction. The polarized light is then split by the prism into two separate beams, which are out of phase with each other. The two beams recombine to generate interference patterns whose edges are highly sensitive to slight differences in the refractive index of the specimen. TO SU M MAR I Z E: ■
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Dark-field microscopy uses scattered light to detect objects too small to be resolved by light rays. Advantage: Extremely small microbes and thin extracellular structures can be detected. Limitation: The shape of objects is not resolved. Dust particles easily obscure the image of the specimen. Phase-contrast microscopy superimposes refracted light and transmitted light shifted out of phase so as to observe differences in refractive index as patterns of light and dark. Advantage: Live microbes with transparent cytoplasm, especially eukaryotes, can be observed with high contrast. Limitation: Phase contrast is less effective for smaller microbes and for organisms whose cytoplasm has a low refractive index. Interference microscopy superimposes interference bands on an image, accentuating small differences in refractive index.
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Advantage: The shape of cells can be defi ned most clearly. Limitations: Interference microscopy requires complex optical adjustment and is less effective for organisms with low refractive index.
2.5
Fluorescence Microscopy
In fluorescence microscopy, incident light is absorbed by the specimen and reemitted at a lower energy, thus longer wavelength. Fluorescence microscopy offers a powerful way to detect microbes and subcellular structures while avoiding artifacts caused by dust and other nonspecific materials. One use of fluorescence microscopy is to assess microbial populations in highly dilute natural environments (Fig. 2.30). This technique was used by the Oceanic Microbial Observatory at the Bermuda Biological Station for Research to conduct a long-term study assessing the effects on microbial populations of changing water chemistry due to climate changes such as global warming. Populations of microbes, including viruses, bacteria, and protists, are measured using DNA-specific fluorescence. The advantage of this fluorescence technique is that it detects only live organisms whose DNA is intact, distinguishing them from fi ne debris in natural environments.
Fluorescence Requires Excitation and Emission at Different Wavelengths Fluorescence occurs when light of a specific wavelength (the excitation wavelength) is absorbed by an atom (or molecule) capable of promoting an electron to an orbital of higher energy (Fig. 2.31). Because this higher-energy electron state is unstable, the electron decays to an orbital with a slightly lower energy level through the loss of energy as heat. The electron then falls to its original level by emitting a photon of lower energy and longer wavelength, the emission wavelength. The emitted photon has a longer wavelength (less energy) because part of the electron’s energy of absorption was lost as heat. Craig A. Carlson, University of California, Santa Barbara
58
Flagellate
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Figure 2.30 Cell counting using fluorescence microscopy. Bacterioplankton and flagellated protists from the surface waters of the Sargasso Sea near Bermuda are enumerated to investigate their contribution to the oceanic carbon cycle. Live organisms are detected by fluorescence using the DNA-specific fluorophore DAPI.
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3. Electron loses some energy as heat and drops to slightly lower orbital.
2. Electron is raised to orbital of higher energy.
4. Fluorescence emission occurs at longer wavelength.
5. Electron returns to original level.
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Fluorescence can be used to label specific parts of cells. The specificity of the fluorophore can be arranged in several ways:
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Fluorophores Can Label Specific Parts of Cells
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The aromatic groups of DAPI associate exclusively with the base pairs of DNA. Another commonly used DNA-specific fluorophore is acridine orange (Fig. 2.32). These fluorophores provide an extremely sensitive means of detecting diverse microorganisms in the environment, including those whose dimensions are too small to be resolved. The optical system for fluorescence microscopy utilizes fi lters to limit incident light to the wavelength of excitation and the emitted light to the wavelength of emission. The wavelengths of excitation and emission are determined by the specific fluorophore used.
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Fluorescence. Energy gained from UV absorption is released as heat and as a photon of longer wavelength in the visible region. A. Fluorescence on the molecular level. B. Comparison of absorption and emission spectra for a fluorophore.
Figure 2.31
The wavelengths of excitation and emission are determined by the fluorophore, the fluorescent molecule used to stain the specimen. For example, the slides for cell counting in the Bermuda study (see Fig. 2.30) used the DNA-specific stain 4′,6-diamidino-2-phenylindole (DAPI).
Chemical affinity. Certain fluorophores have chemical affi nity for certain classes of biological molecules; for example, the fluorophore acridine specifically binds DNA. Labeled antibodies. Antibodies that specifically bind a cell component are chemically linked to a fluorophore molecule. The use of antibodies linked to fluorophores is known as immunofluorescence. Gene fusion. Genetic recombination can be used to create a hybrid gene expressing a protein that generates fluorescence, such as green fluorescent protein (GFP). DNA hybridization. A short sequence of DNA attached to a fluorophore will hybridize to a specific sequence in the genome, thus labeling one position in the chromosome or nucleoid.
Fluorophore labeling is used to dissect endospore formation in Bacillus species (Fig. 2.33). The cell envelope and DNA origin of replication are labeled by different fluorophores to track movements of DNA during sporulation. Richard Losick and colleagues applied this technique to show how the DNA origin moves toward one pole of the cell, followed by formation of a septum, a new portion of cell envelope, just behind the developing endospore.
S C N NH N CO2H H3C
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Fluorescent molecules (fluorophores) commonly used in microscopy. Because of the abundance of conjugated double bonds, these molecules have closely spaced molecular orbitals that give rise to fluorescence.
Figure 2.32
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DNA origin Septum DNA origin
DNA origin Septum Endospore
Septum DNA origin DNA origin 2 µm
Fluorescence micrograph of Bacillus subtilis cells during sporulation. Red fluorescence arises from membrane stained with the dye FM-464. Green fluorescence arises from green fluorescent protein (GFP) bound to the DNA origin of replication. The yellow color occurs where green fluorescence overlaps red.
Figure 2.33
In the micrograph, the cell envelope is stained red by the fluorescent dye FM-464. The dye FM-464 specifically binds phospholipid membranes. The DNA origin of replication is stained by green fluorescent protein (GFP) fused to a protein that specifically binds the DNA origin of replication. The fluorescence micrographs were taken at two different wavelengths for both excitation and emission to record the two different fluorescent labels. The images were superimposed with two different colors marking envelope and DNA. The result shows how, in sporulation, DNA replication originates not at the midpoint of the cell, as in vegetative growth of most bacteria, but at the poles where the endospore forms.
Special Topic 2.1
Confocal Fluorescence Microscopy
An advanced application of fluorescence is laser scanning confocal microscopy, in which both excitation light and emitted light are focused together. Confocal microscopy is used to produce images of cells at high resolution, with interference effects decreased by laser optics. The images can be “stacked” computationally to model a cell in three dimensions. In Figure 1, we see a confocal image of human tissue culture cells infected by enteroinvasive Escherichia coli. The DNA of bacteria and of the nuclei of HeLa cells (an immortal cancer cell line) are stained blue with DAPI fluorophore. Invading bacteria cause the host to form actin “tails,” which help the bacteria move. Tails are stained green by an actinbinding fluorophore, FITC. In confocal microscopy, a laser beam is focused onto the specimen and scanned across it in two dimensions−that is, in two planes at right angles to each other (Fig. 2). The laser beam excites the fluorophore, causing it to emit light at a longer wavelength. The emitted light passes in reverse direction through the objective, where it encounters a dichroic mirror that allows light transmission at the excitation wavelength but reflects light at the wavelength of emission. The reflected emission rays are then focused and pass through a pinhole, which eliminates all unfocused light. A narrow focused beam, representing one “pixel” of image, enters the photomultiplier tube. Scanning across the specimen yields a two-dimensional pattern of pixels that forms an image.
Bacterium
THOUGHT QUESTION 2.7 What experiment could you devise to determine the actual order of events in DNA movement toward the pole during formation of an endospore?
Actin tail
THOUGHT QUESTION 2.8 Compare and contrast fluorescence microscopy with dark-field microscopy. What similar advantage do they provide, and how do they differ? Fluorescence microscopy has been used to develop advanced optical systems that reconstruct three-dimensional models of cells. An example is confocal fluorescence microscopy (Special Topic 2.1).
HeLa nucleus
Michael S. Donnenberg/University of Maryland
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Human tissue culture cells infected by enteroinvasive Escherichia coli. DNA of bacteria and of HeLa nuclei are stained blue with DAPI fluorophore. Invading bacteria (blue-stained rods, 1–2 µm long) cause the host to form actin “tails,” stained green with an actin-binding FITC fluorophore.
Figure 1
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Fluorescence microscopy involves detection of specific cells or cell parts based on fluorescence by a fluorophore. Cell parts can be labeled by a fluorophore attached to an antibody stain.
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The scanning feature of confocal microscopy is also used to acquire data for high-throughput experiments in which a large number of chemical reactions are arranged in microscopic quantities in an array. An example of an array is a DNA microarray containing probes for all the genes of a genome. Short segments of DNA are arrayed on a microscope slide such that an entire genome of potential protein-encoding genes may be present on a single slide. One use of arrays is to investigate the mode of action of a new antibiotic molecule by testing which of the microbe’s genes are induced or repressed in response to the antibi-
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otic. The DNA array is hybridized to fluorescence-labeled cDNA copies of messenger RNA from bacteria cultured with or without the antibiotic. The confocal laser then scans the slide to detect binding of the genes to cDNA molecules labeled with a fluorophore. This technique makes it possible to measure transcription of all known genes in the genome and reveal those whose expression varies during exposure to an antibiotic. The antibiotic-induced genes indicate the mode of action of the antibiotic within the cell and aid the design of better antibiotics. DNA microarrays are discussed further in Chapter 12.
Fluorescence emission from specimen fluorophores is reflected by mirror.
Only confocal rays (sharing focal point) pass through pinhole.
Dichroic mirror Out of focus In focus
Laser light rays pass through mirror.
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450 510 Wavelength (nm)
Confocal microscopy. In confocal optics, the incident laser beam (blue) passes through a dichroic mirror and reaches the specimen, where its absorption leads to fluorescent emission at a longer wavelength. The fluorescent emission travels back to the dichroic mirror, where it is reflected toward the photomultiplier. Only the confocal rays (those emitted from the focal point) pass through the pinhole and reach the photomultiplier. The laser scans across the specimen, generating a pixilated image. The scanned images can be stacked through a series of focal planes to generate a three-dimensional model.
Figure 2
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Electron Microscopy
All cells are built of macromolecular structures. The tool of choice for observing the shape of these macromolecular structures is electron microscopy (EM). In electron microscopy, beams of electrons are focused to generate images of cell membranes, chromosomes, and ribosomes with a resolution a thousand times that of light microscopy. The modern electron microscope was developed in the 1950s and was popularly known as the centerpiece of any biological research program. In Michael Crichton’s fi lm, The Andromeda Strain, for example, an electron microscope is used to analyze a fictional pathogen from outer space.
The Electron Microscope Focuses Beams of Electrons How does an electron microscope work? Electrons are ejected from a metal subjected to a voltage potential. The electrons travel in a straight line, like photons. Like photons, electrons interact with matter and carry information about their interaction. And like photons, electrons can exhibit the properties of waves. The wavelength associated with an electron is a 100,000 times smaller than that of a photon; for example, an electron accelerated over a voltage of 100 kilovolts (kV) has a wavelength of 0.0037 nm (0.037 Å) compared with 400–750 nm for visible light. The actual resolution of electrons in microscopy is limited, however, not by the wavelength, but by the aberrations of the lensing Optical lens
Magnetic lens
Light source
Electron source
Soft iron pole
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systems used to focus electrons. The magnetic lenses used to focus electrons never achieve the precision required to utilize the full potential resolution of the electron beam. Electrons are focused by means of a magnetic field directed along the line of travel of the beam (Fig. 2.34). As a beam of electrons enters the field, it spirals around the magnetic field lines. The shape of the magnet can be designed to generate field lines that will focus the beam of electrons in a manner analogous to the focusing of photons by a refractive lens. The electron beam, however, forms a spiral because electrons travel around magnetic field lines. Because magnetic lenses generate aberrations, a series of corrective magnetic lenses is generally required to obtain a resolution of about 0.2 nm. This represents a resolution a thousand times greater than the 200-nm resolution of light microscopy. THOUGHT QUESTION 2.9 An electron microscope can be focused at successive powers of magnification, as in a light microscope. At each level, the image rotates at an angle of several degrees. Based on the geometry of the electron beam, as shown in Figure 2.34, why do you think this rotation occurs? Transmission EM and scanning EM. Two major types of
electron microscopy are transmission electron microscopy (TEM) and scanning electron microscopy (SEM). In TEM, electrons are transmitted through the specimen as in light microscopy to reveal internal structure. In SEM, the electron beams scan across the surface of the specimen and are reflected to reveal the contours of its threedimensional surface. The transmission electron microscope closely parallels the design of a bright-field microscope, including a source of electrons (instead of light), a magnetic condenser lens, a specimen, and an objective lens. A projection lens projects the image onto a fluorescent screen and the final images are obtained by a digital camera (chargecoupled device, CCD) (Fig. 2.35). The scanning microscope is arranged somewhat differently than the TEM in that a series of condenser lenses focuses the electron beam onto the surface of the specimen. Reflected electrons are then picked up by a detector (Fig. 2.36). In either case, the overall apparatus required for electron microscopy is complex, resembling the bridge of a Star Trek spaceship.
Copper coils
Image is inverted.
Image is inverted and rotated.
A magnetic lens. The beam of electrons spirals around the magnetic field lines. The U-shaped magnet acts as a lens, focusing the spiraling electrons much as a refractive lens focuses light rays.
Figure 2.34
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Electron Microscopy Requires Specialized Sample Preparation Electron microscopy poses special problems for biological specimens. With the exception of cryo EM (discussed shortly), the entire optical column must be maintained under vacuum to prevent the electrons from colliding with the gas molecules in air. The requirement for a vacuum precludes the viewing of live specimens, which
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B. Transmission electron microscopy
Light microscopy
Electron source (tungsten filament) Zhiping Luo/Texas A&M University
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Objective lens
Transmission electron microscopy. A. The light source is replaced by an electron source consisting of a high-voltage current applied to a tungsten filament, which gives off electrons when heated. The electron beam is focused by a condenser magnet lens onto the specimen. The specimen image is then magnified by an objective magnetic lens. The projector lens, analogous to the ocular lens of a light microscope, focuses the image on the cathode-ray tube (CRT) screen. Note: Because of the greater problem of aberrations in focusing electrons, each magnetic lens shown (condenser, objective, projection) actually represents a series of lenses. B. Using an electron microscope.
Figure 2.35 Projection lens
Image plane Fluorescent screen
Eye
B.
C. Lawrence Migdale/Photo Researchers, Inc.
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Electron gun Vacuum column Electron beam Condensing lenses Monitor
Museum of Science/Boston
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in any case would be quickly destroyed by the electron beam. Moreover, the structure of most specimens lacks sufficient electron density (ability to block electrons) to provide contrast. Thus, the specimen usually requires an electron-dense stain using salts of heavy metals such as gold or uranium.
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Scanning electron microscopy. A. In the scanning electron microscope (SEM), the electron beam is scanned across a specimen coated in gold, which acts as a source of secondary electrons. The incident electron beam ejects secondary electrons toward a detector, generating an image of the surface of the specimen. B. Operating an SEM. C. Loading a specimen into the vacuum column.
Figure 2.36
The specimen can be prepared in one of three ways: ■
Embedded in a polymer for thin sections. A special knife called a microtome cuts slices through the specimen, each slice a fraction of a micrometer thick.
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Sprayed onto a copper grid. The electron beam penetrates straight through the entire object, as if it were transparent. This method is effective for virus particles and for isolated macromolecular complexes. Flash-frozen (for cryo EM). Samples frozen rapidly in refrigerant provide sufficient contrast for detection by a high-intensity electron beam, a recent innovation.
A. SEM of Mycobacterium paratuberculosis
B. TEM of flagellar motors from Salmonella typhimurium
Flagellar motor
For sectioned samples or for samples sprayed onto a grid, the specimen is coated with a heavy metal salt such as 1 µm 50 nm uranyl acetate. The metal salt is deposited around the biological structures, acting as C. TEM of Bacillus anthracis showing envelope and cytoplasm a negative stain. (For comparison, a negative stain used in light microscopy is the Glycoprotein India ink stain, illustrated in Fig. 2.24C.) In Figure 2.37, we see examples of Cell wall SEM and TEM images from gram-positive rod bacteria. The SEM in Figure 2.37A shows an exterior view of the capsules of M. paratuberculosis. The image provides far greater magnification of surface detail than does a bright-field view of rod bacte250 nm ria. In Figure 2.37B, flagellar motors have been isolated or “unplugged” from a bac- Figure 2.37 SEM and TEM images of gram-positive rod-shaped bacteria. terial cell envelope and spread on a grid for TEM. The micrograph reveals details of the motor, including the axle and indirapidly in a refrigerant of high heat capacity (ability to vidual rings. The TEM of B. anthracis in Figure 2.37C absorb heat). The rapid freezing avoids ice crystallizashows a thin section through a bacillus, including cell tion, leaving the water solvent in a glass-like amorphous wall, membranes, and glycoprotein fi laments. The TEM is phase. The specimen retains water content and thus “transparent,” summing electron density throughout the closely resembles its viable form, although it is still ultidepth of the section.
An important limitation of traditional electron microscopy, whether TEM or SEM, is that in most cases it can only be applied to fi xed, stained specimens. The fi xatives and heavy-atom stains can introduce artifacts into the image, especially at fi ner details of resolution. In some cases, different preparation procedures have led to substantially different interpretations of subcellular structure. The development of exceptionally high-strength electron beams now permits low-temperature cryoelectron microscopy (cryo EM). In cryo EM, the specimen is flash-frozen, that is, suspended in water and frozen
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J. Ortiz and W. Baumeister. 2006. Journal of Structural Biology 156:334
THOUGHT QUESTION 2.10 What kind of research questions could you investigate using SEM? What questions would be answered using TEM?
Michio Homma, et al. 1987. Proc. Natl. Acad. Sci. 84
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Cryoelectron microscopy. Cryoelectron tomography of Spiroplasma melliferum. The tomographic slice reveals ribosomes identified using molecular identification software, false-colored yellow.
Figure 2.38
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Microscopy Results Require Careful Interpretation
THOUGHT QUESTION 2.11 What kind of experiments could prove or disprove the interpretations of the images of “nanobacteria” in blood plasma?
The images produced by high-level microscopy can be difficult to interpret. For example, an oval that appears hollow might be interpreted as a cell when in fact it represents a deposit of staining material. A microscopic
Cold Spring Harbor Laboratory Press
Nanobacteria?
Figure 2.39 Artifacts of microscopy. A. Objects attached to the surface of a fibroblast were observed by SEM. The objects (200–300 nm in diameter) were identified as “nanobacteria,” exceptionally small bacteria inhabiting human blood plasma, but they are now believed to be mineral deposits. B. Cultured human cells observed by immunofluorescence microscopy revealed small particles identified as nanobacteria. This observation, however, has not been reproduced by other researchers.
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Emerging Methods of Microscopy
B. Olavi Kajander and Neva Ciftcioglu, University of Kuopio, Finland
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structure that is interpreted incorrectly is termed an artifact. Avoiding artifacts is an important concern in microscopy. Sometimes, published interpretations generate controversy. For example, electron microscopy and immunofluorescence microscopy were used to test for contamination of cultured white blood cells that grew poorly (Fig. 2.39). The SEM images were interpreted to show the presence of tiny prokaryotic cells, 200–300 nm in diameter, attached to cultured fibroblast cells (Fig. 2.39A). These proposed cells were termed “nanobacteria.” Unfortunately, other researchers were unable to confi rm these results and suggested that the objects observed might actually be mineral deposits. Another technique, immunofluorescence microscopy, reportedly showed the invasion of cultured cells by nanobacteria (Fig. 2.39B). The technique required use of fluorescent-tagged antibodies against nanobacteria, but the actual specificity of the antibodies was unclear. The existence of small infective particles in the blood remains a question of interest to medical researchers.
mately destroyed by electron bombardment. The sample does not require staining because the high-intensity electron beams can detect smaller signals than in earlier instruments. Figure 2.38 shows a cryo EM of Spiroplasma melifera. Another innovation in cryo EM is tomography, the acquisition of projected images from different angles of a transparent specimen. Tomography avoids the need to physically slice the sample for thin-section TEM. The images from EM tomography are combined digitally to visualize the entire object. An example is the tomographic image of the spiral bacterium Spiroplasma melliferum, in which individual ribosomes clearly appear (Fig. 2.38). Cryo EM is also used to generate high-resolution models of virus particles and purified structures such as the ribosome (Special Topic 2.2). For these small particles, multiple images can be averaged together by computational analysis. The digitally combined images can achieve high resolution, nearly comparable to that of X-ray crystallography.
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New methods of microscopy are emerging that enable nanoscale observation of cell surfaces, in some cases of living cells suspended in water. A general term for these methods is scanning probe microscopy (SPM). SPM methods differs from light and electron microscopy, in which the sample interacts with a beam of light or electrons. Instead, SPM methods measure a physical interaction, such as the “atomic force” between the sample and a sharp tip. Atomic force microscopy (AFM) measures the van der Waals forces between the electron shells of adjacent atoms of the cell surface and the sharp tip. AFM is particularly useful to study the surfaces of live bacteria. In AFM, an instrument probes the surface of a sample with a sharp tip a couple of micrometers long and often less than 10 nm in diameter (Fig. 2.40A). The tip is located at the free end of a lever that is 100–200 µm long. The lever is deflected by van der Waals forces between the tip and the sample surface. Deflection of the lever is measured by a laser beam reflected off a cantilever attached to the tip as the sample scans across. The measured deflections allow a computer to map the topography of cells in liquid medium with a resolution below 1 nm. In the example in Figure 2.40B, obtained by Mark Martin at Occidental College, AFM reveals the outline of
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Three-Dimensional Electron Microscopy Solves the Structure of a Major Agricultural Virus
Wah Chiu, Baylor College of Medicine
One of the world’s most economically damaging agricultural pathogens is rice dwarf virus. Rice dwarf virus (RDV) is transmitted by leafhopper beetles in China and Southeast Asia, where it systemically infects rice, discoloring the leaves and stunting the plant, devastating the harvest. A retrovirus, RDV has a genome of double-stranded RNA enclosed within an icosahedral capsid of proteins, approximately 70 nm in diameter. Determining the molecular structure of the virus particle may enable us to design antiviral agents.
Three-dimensional cryoelectron microscopy. Dr. Wah Chiu (standing), at Baylor University, succeeded in imaging the structure of rice dwarf virus using threedimensional cryoelectron microscopy.
Figure 1
A.
Wah Chiu (Fig. 1) and colleagues at the National Center for Macromolecular Imaging, at Baylor University, imaged the structure of RDV at high resolution using three-dimensional cryo EM tomography. Cryo EM is especially important for particles that cannot be crystallized for X-ray diffraction analysis, the most common means of molecular visualization. Because the frozen sample remains hydrated, the biological molecules retain the same conformation as in solution. The sample is imaged without heavy-metal stains, and thus higher resolution is obtained (see Fig. 2B). This technique avoids introducing stain artifacts but generates very low contrast. Repeated scans can be summed computationally to obtain an image at higher resolution. In Figure 2A, a cryo EM image reveals the outline of the RDV particles. Each particle was imaged in stereo by rotating the specimen to receive the electron beam at different angles (Fig. 2B). In theory, a three-dimensional picture of the virus could be obtained from a stereo pair of images from one particle. In practice, however, one image pair reveals little information because the ratio of signal to noise is so low, so stereo pairs of images were combined from 4,000 individual RDV particles (see Fig. 2B). A visual model was generated by computation requiring the use of a supercomputer. The computational model of RDV reveals in three dimensions the capsid, a protein shell that encloses the virus’s RNA
B. Electron beam
50 nm
Joanita Jakana, Baylor College of Medicine
Specimen rotates in the beam
RDV particles
Figure 2. Cryoelectron microscopy. A. Cryo EM image of particles of rice dwarf virus (RDV). B. Data from 4,000 stereo pairs of images of individual particles were combined for computational reconstruction of the virus structure.
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Projection images orient, average
Averaged image 2-D transform
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Three-dimensional Fourier transform 3-D inverse transform
Three-dimensional reconstruction
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chromosome (Fig. 3A). The model includes detailed representations of both the outer shell (P8 subunits) and inner shell (P3 subunits). The remarkable resolution of the RDV model provides enough detail to map the alpha helices of a P3 subunit (Fig. 3B), although it does not yet reach the atomic resolution of X-ray diffraction. This structure can be used to model molecular interactions for potential antiviral agents that would inactivate the capsid.
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an E. coli cell invaded by the parasitic bacterium Bdellovibrio bacteriovorus. The cells were observed in water suspension, without stain; and their contours appear without the flattening on a grid required for EM. The infected and uninfected E. coli cells differ in texture. This ability to “catch cells in the act” is an advantage of AFM. A. Laser Photodiode Cantilever/mirror
P3
Alpha helices
Matthew Baker and Wen Jiang, Baylor College of Medicine
B. P3 protein inner shell of RDV
The rice dwarf virus capsid. A. Model of RDV, at resolution 6.8 Å. The outer shell is composed of 396 P8 subunits (bright colors). A cutaway from the outer shell reveals the inner shell (pale color, within black border) composed of 120 P3 subunits. B. The labeled P3 subunit from part A is shown in expanded detail, revealing alpha helices (blue cylinders).
Figure 3
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AFM tip x
Sample z As the tip moves up or down the cantilever/mirror is deflected.
B.
y The sample is moved back and forth in the x-y plane.
Attack phase Bdellovibrio cell
Infected E. coli cell full of Bdellovibrio (note altered texture)
M. Nunez, M. Martin, P. Chan, and E. Spain, Occidental College
P8
Z. Hong Zhou, University of Texas Houston Medical School and Guangying Lu, Peking University
A.
Flagellum still adhering to glass
Remains of lysed E. coli cell (note pili and flagella remaining)
Pili helping cells adhere to glass
Atomic force microscopy enables visualization of untreated cells. A. The atomic force microscope (AFM) has a fine-pointed tip attached to a cantilever that moves over a sample. The tip interacts with the sample surface through atomic force. As the tip is pushed away, or pulled into a depression, the cantilever is deflected. The deflection is measured by a laser light beam focused onto the cantilever and reflected into a photodiode detector. B. Escherichia coli cells under attack by Bdellovibrio bacteriovorus, bacterial predators that invade the periplasmic space of E. coli. The untreated cells allow AFM observation of B. bacteriovorus cells, E. coli cells, pili, and flagella. Each E. coli cell is about 2 µm in length.
Figure 2.40
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Electron microscopy is based on the focusing of electron beams on an object stained with a heavymetal salt that blocks electrons. Much higher resolution is obtained than for light microscopy. Transmission electron microscopy (TEM) involves electron beam penetration of a thin sample. Scanning electron microscopy (SEM) involves scanning of a three-dimensional surface with an electron beam. Cryoelectron microscopy (cryo EM) observes a sample flash-frozen in water solution. Multiple images may be combined digitally to achieve high resolution. Atomic force microscopy, an emerging alternative to EM, uses van der Waals force measurement to observe cells in water solution.
2.7
Visualizing Molecules
To understand the structure of cells, ultimately we need to isolate the cell’s molecules to observe their individual structure and function. The major tool used at present for molecular visualization is X-ray diffraction analysis, or X-ray crystallography. In some cases, cryo EM modeling based on thousands of samples has also reached near atomic resolution. Another emerging alternative to X-ray diffraction for analysis of small molecules and proteins is nuclear magnetic resonance (NMR). The advantage of A.
Crystal
ω
Area detector(s)
NMR is that it presents a dynamic view of molecules in solution, including multiple conformation states. Unlike microscopy, X-ray diffraction does not present a direct view of a sample, but generates computational models. Dramatic as the models are, they can only represent particular aspects of electron clouds and electron density that are fundamentally “unseeable.” That is why molecular structures are represented in different ways that depend on the context—by electron density maps, as models defined by van der Waals radii, or as stick models, for example. Proteins are frequently presented in a cartoon form that shows alpha helix and beta sheet secondary structures.
X-Ray Diffraction Analysis For substances that can be crystallized, X-ray diffraction makes it possible to fix the position of each individual atom in a molecule, because the wavelengths of X-rays are much shorter than the wavelengths of visible light and are of the same magnitude as the dimensions of atoms. X-ray diffraction, like phase microscopy, involves the principle of wave interference (see Fig. 2.13). The interference pattern is generated when a crystal containing many copies of an isolated molecule is bombarded by a beam of X-rays (Fig. 2.41A). The wave fronts associated with the X-rays are diffracted as they pass through the crystal, causing interference patterns. In the crystal, the diffraction pattern is generated by a symmetrical array of many sample molecules (Fig. 2.41B). The larger the number of copies of the molecule in the array, the narrower the interference pattern and the greater the reso-
C.
Focused beam
50-keV electrons
Focusing mirrors (or monochromator)
χ φ
θ
Primary X-ray beam Bruker AXS Inc.
Rotating anode (Cu)
B. Incident waves of wavelength λ
Visualizing molecules by X-ray crystallography. A. Modern apparatus for X-ray crystallography. The X-ray beam is focused onto a crystal, which is rotated over all angles to obtain diffraction patterns. The intensity of the diffracted X-rays is recorded on film or with an electronic detector. B. X-rays are diffracted by rows of identical molecules in a crystal. The diffraction pattern is analyzed to generate a model of the individual molecules. C. Diffraction pattern from a crystal.
Figure 2.41 Array of atoms
a1 a2
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lution of atoms within the molecule. Diffraction patterns obtained from the passage of X-rays through a crystal (Fig. 2.41C) can be analyzed by computation to develop a precise structural model for the molecule, detailing the position of every atom in the structure. The application of X-ray crystallography to complex biological molecules was pioneered by the Irish crystallographer John Bernal (1901–1971) (Fig. 2.42A). Bernal was particularly supportive of women students and colleagues, including Rosalind Franklin (1920–1958) who made important discoveries about DNA and RNA, and Nobel laureate Dorothy Crowfoot Hodgkin (1910–1994). Hodgkin won the 1964 Nobel Prize in Chemistry for solving the crystal structures of penicillin and vitamin B12 (Fig. 2.42B). She later solved one of the fi rst protein structures, that of the hormone insulin. Today, X-ray data undergo digital analysis to generate sophisticated molecular models, such as the one seen in
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Figure 2.43 of anthrax lethal factor, a toxin produced by B. anthracis that kills the infected host cells. The model for anthrax lethal factor was encoded in a Protein Data Bank (PDB) text fi le that specifies coordinates for all atoms of the structure. The Protein Data Bank is a growing world database of solved X-ray structures, freely available on the Internet. Visualization software is used to present the structure as a “ribbon” of amino acid residues, colorcoded for secondary structure. In Figure 2.43, the pinkcolored coils represent alpha helix structures, whereas the blue arrows represent beta sheets (for a review of these secondary structures, see Appendix 1). Protein Data Bank: Research Collaboratory for Structural Bioinformatics Protein and Nucleic Acid Databases Biomolecules at Kenyon College: molecular tutorials by undergraduate students, on anthrax lethal factor and other proteins; as well as instructions to write your own tutorials.
Brikbeck Photo Unit
A.
NOTE: Molecular and cellular biology increasingly rely on visualization in three dimensions. Many of the molecules illustrated in our book are based on structural models deposited in the Protein Data Bank, as indicated by the PDB fi le code. You may view these structures in 3-D by downloading the PDB fi le and viewing with a free plug-in such as Jmol.
C.
Cobalt Corrin ring
Hulton-Deutsch Collection/CORBIS
B.
Adenosyl
Figure 2.42 Pioneering X-ray crystallography. A. John Bernal developed X-ray crystallography to solve the structure of complex biological molecules. B. Dorothy Hodgkin (1910–1994) was awarded the 1964 Nobel Prize in Chemistry for her work in X-ray crystallography. C. Vitamin B12, whose structure was originally solved by Dorothy Hodgkin. The corrin ring structure (see Chapter 15) is built around an atom of cobalt (pink). Carbon atoms are gray; oxygen, red; nitrogen, blue; phosphorus, yellow. Hydrogen atoms are omitted for clarity.
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X-ray crystallography of a protein complex, anthrax lethal factor. The toxin consists of a butterfly-shaped dimer of two peptide chains. This cartoon model is based on X-ray crystallographic data, showing alpha helix (pink coils) and beta sheet (orange arrows). (PDB code: 1J7N)
Figure 2.43
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O b s e r vin g t h e Mic robial C ell
A limitation of X-ray analysis is the unavoidable deterioration of the specimen under bombardment by X-rays. The earliest X-ray diffraction models of molecular complexes such as the ribosome relied heavily on components from thermophiles, microbes that grow at high temperatures. Because these organisms have evolved to grow under higher thermal stress, their macromolecular complexes form more stable crystals than do their homologues in organisms growing at moderate temperatures. X-ray diffraction analysis of crystals from a wide range of sources was made possible by cryocrystallography. In cryocrystallography, as in cryo EM, crystals are frozen rapidly to liquid nitrogen temperature. The frozen crystals have greatly decreased thermal vibrations and diffusion, thus decreasing the radiation damage to the molecules. Models based on cryocrystallography can present multisubunit structures such as the bacterial ribosome complexed with transfer RNAs and messenger RNA. Much of our knowledge of microbial genetics (Chapters 7–12) and metabolism (Chapters 13–16) is based on crystal structures of key macromolecules.
TO SU M MAR I Z E: ■
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X-ray diffraction analysis, or X-ray crystallography, uses X-ray diffraction (interference patterns) from crystallized macromolecules to determine structure at atomic resolution. Cryocrystallography uses frozen crystals with greatly decreased thermal vibrations and diffusion, enabling the determination of structures of large macromolecular complexes, such as the ribosome. Molecular visualization by crystallography can only model the “appearance” of a molecule at atomic resolution. Different models emphasize different structural features and levels of resolution.
Concluding Thoughts The tools of microscopy and molecular visualization described in this chapter have shaped our current understanding of microbial cells—how they grow and divide, organize their DNA and cytoplasm, and interact with other cells. Our current models of cell structure and function are explored in Chapter 3. In Chapter 4, we learn how cells use their structures to obtain energy, reproduce, and develop dormant forms that can remain viable for thousands of years.
CHAPTE R R EVI EW Review Questions 1. What principle defi nes an object as “microscopic”? 2. Explain the difference between detection and
9. Summarize the optical arrangement of a compound
resolution. How do eukaryotic and prokaryotic cells differ in appearance under the light microscope? Explain how electromagnetic radiation carries information and why different kinds of radiation can resolve different kinds of objects. Defi ne how light interacts with an object through absorption, reflection, refraction, and scattering. Explain how refraction enables magnification of an image. Explain how magnification increases resolution and why “empty magnification” fails to increase resolution. Explain how angle of aperture and resolution change with increasing lens magnification.
10. Explain how to focus an object and how to tell when
3. 4.
5. 6. 7.
8.
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microscope. the object is in or out of focus. 11. Explain the relative advantages and limitations of
wet mount and stained preparation for observing microbes. 12. Explain the significance (and limitations) of the Gram stain for bacterial taxonomy. 13. Explain the basis of dark-field, phase-contrast, and fluorescence microscopy. Give examples of applications of these advanced techniques. 14. Explain the difference between transmission and scanning electron microscopy and the different applications of each.
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Key Terms aberration (49) absorption (45) acid-fast stain (54) Airy disk (47) angle of aperture (49) antibody stain (55) artifact (65) atomic force microscopy (AFM) (43, 65) bacillus (plural, bacilli) (42) bright-field microscopy (43) coccus (plural, cocci) (42) compound microscope (50) condenser (50) contrast (44) counterstain (54) cryocrystallography (70) cryoelectron microscopy (cryo EM) (64) dark-field microscopy (45) depth of field (51) detection (41) diaphragm (50) differential stain (52) electron microscopy (43, 62) emission wavelength (58)
empty magnification (46) excitation wavelength (58) fixation (52) flagella (56) fluorescence (45) fluorophore (59) focal plane (46) focal point (46) focus (48) Gram stain (52) gram-negative (54) gram-positive (54) immersion oil (50) interference (46) interference microscopy (58) laser scanning confocal microscopy (60) light microscopy (40, 43) magnification (41) microscope (40) mordant (52) negative stain (54) nuclear magnetic resonance (NMR) (43, 68)
numerical aperture (NA) (49) objective lens (49) ocular lens (50) parfocal (50) reflection (45) refraction (45) refractive index (45) resolution (40) rods (42) scanning electron microscopy (SEM) (43, 62) scattering (45) simple stain (52) spirochete (42) spore stain (54) staining (52) tomography (65) total magnification (51) transmission electron microscopy (TEM) (43, 62) wet mount (52) X-ray crystallography (43, 68) X-ray diffraction analysis (68)
Recommended Reading Chiu, Wah, et al. 2002. Deriving folds of macromolecular complexes through electron cryomicroscopy and bioinformatics approaches. Current Opinion in Structural Biology 12:263. Graumann, Peter L., and Richard Losick. 2001. Coupling of asymmetric division to polar placement of replication origin regions in Bacillus subtilis. Journal of Bacteriology 183:4052–4060. Jiang, Weihang, Juan Chang, Joanita Jakana, et al. 2006. Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging-injection apparatus. Nature 439:612–616. Lucic, Vladan, Friedrich Förster, and Wolfgang Baumeister. 2005. Structural studies by electron tomography: From cells to molecules. Annual Review of Biochemistry 74:833–865.
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Matias, Valério R. F., Ashruf Al-Amoudi, Jacques Dubochet, and Terry J. Beveridge. 2003. Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa. Journal of Bacteriology 185:6112–6118. Murphy, Douglas B. 2001. Fundamentals of Light Microscopy and Electronic Imaging. Wiley-Liss, Hoboken, N.J. Popescu, Aurel, and R. J. Doyle. 1996. The Gram stain after more than a century. Biotechniques in Histochemistry 71:145–151.
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Chapter 3
Cell Structure and Function 3.1 3.2 3.3 3.4 3.5 3.6 3.7
The Bacterial Cell: An Overview How We Study the Parts of Cells The Cell Membrane and Transport The Cell Wall and Outer Layers The Nucleoid and Gene Expression Cell Division Specialized Structures
Microbial cells face extreme challenges from their environment, enduring rapid changes in temperature and salinity, and pathogens face the chemical defenses of their hosts. To meet these challenges, microbes build complex structures, such as a cell envelope with tensile strength comparable to steel. Within the cytoplasm, molecular devices such as the the ribosome build and expand the cell. With just a few thousand genes in its genome, how does a bacterial cell grow and reproduce? Bacteria coordinate their DNA replication through the DNA replisome and the cell fission ring. Other devices, such as flagellar propellers, enable microbial cells to compete, to communicate, and even to cooperate in building biofilm communities. Discoveries of cell form and function have exciting applications for medicine and biotechnology. The structures of ribosomes and cell envelope materials provide targets for new antibiotics. And devices such as the rotary ATP synthase inspire “nanotechnology,” the design of molecular machines.
The filamentous cyanobacterium Anabaena sp. was engineered to make a cell division protein, FtsZ, fused to green fluorescent protein (GFP). FtsZ-GFP proteins form a ring-like structure around the middle of each cell, where it prepares to divide. Source: Samer Sakr, et al. 2006. J. Bacteriol. 188.
73 5 µm
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■ C e ll S tr u c tu r e a nd Func t ion
Chapter 3
The microbial cell has formidable tasks: to obtain nutrients faster than its competitors, to protect itself from toxins and predators, and to reproduce itself. These tasks are accomplished by the cell’s molecular parts. To study the cell requires microscopy as well as isolating cell parts. Chapter 3 presents an overview of the bacterial cell, then explains how such views derive from cell fractionation and genetic analysis. We explore the structures common to most microbial cells, as well as more specialized devices such as light-harvesting complexes and magnetosomes. We dissect the cell’s complex outer layers and see how they interact with the replicat-
Envelope
Nucleoid
ing chromosome to accomplish cell fi ssion. Many of the cell’s structures offer targets for antibiotic design as well as opportunities for biotechnology. Overall, this chapter focuses on prokaryotes, with reference to eukaryotes for comparison (eukaryotic microbes are covered in Chapter 20). Our discussion assumes an elementary knowledge of cell biology (reviewed in Appendices 1 and 2). The Microbial Biorealm online provides a quick, readable reference for microbial species, including those mentioned in this book.
Ribosomes
Ribosome
0.25 µm
©Dennis Kunkel
mRNA
Flagellum
Model of a bacterial cell (Escherichia coli). Envelope: The cell membrane contains embedded proteins for structure and transport. The cell membrane is supported by the cell wall. In this gram-negative cell, the cell wall is coated by the outer membrane, whose sugar chain extensions protect the cell from attack by the immune system or by predators. Plugged into the membranes is the rotary motor of a flagellum. Cytoplasm: Molecules of nascent messenger RNA (mRNA) extend out of the nucleoid to the region of the cytoplasm rich in ribosomes. Ribosomes translate DNA-binding protein the mRNA to make proteins, which are folded by chaperones. Nucleoid: The chromosomal DNA is wrapped around binding proteins. Replication by DNA polymerase and transcription HU by RNA polymerase occur at the same time within the nucleoid. (PDB codes: ribosome, 1GIX,1GIY; DNA-binding protein, 1P78; RNA polymerase, 1MSW)
E P A 30S
50S
Polypeptide
Figure 3.1
Flagellar motor
RNA polymerase
DNA
RNA
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A Model of the Bacterial Cell
The Bacterial Cell: An Overview
Prokaryotic cells show a wide range of form, whose diversity is explored in Chapters 18 and 19. At the same time, most prokaryotes share fundamental traits: ■
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Thick, complex outer envelope. The envelope protects the cell from environmental stress and predators. It also mediates exchange with the environment and communication with other organisms. Compact genome. Prokaryotic genomes are compact, with relatively little noncoding DNA. Small genomes maximize the production of cells from limited resources. Tightly coordinated cell functions. The cell’s subcellular parts work together in a highly coordinate mechanism. Coordinate action enables high reproduction rate.
Here we present a model of the bacterial cell (Fig. 3.1). This model offers an interpretation of how the major components of one bacterial cell fit together. The model represents Escherichia coli, but its general features apply to many kinds of bacteria. Remember that we cannot literally “see” the molecules within a cell, but microscopy and subcellular analysis generate a remarkably detailed view. Within the bacterial cell, the cytoplasm consists of a gel-like network composed of proteins and other macromolecules. The cytoplasm is contained by a cell membrane (or inner membrane, for a gram-negative cell). The membrane is composed of phospholipids, hydrophobic proteins, and other molecules. The cell membrane prevents
Bacterial Cell Components Outer membrane proteins:
In the early twentieth century, the cell was envisioned as a bag of “soup” full of floating ribosomes and enzymes. Modern research shows that in fact the cell’s parts fit together in a structure that is ordered, though flexible.
Sugar porin (10 nm) Braun lipoprotein (8 nm) Inner membrane proteins: Glycerol porin Secretory complex (Sec)
Lipopolysaccharide Envelope
Outer membrane Cell wall Periplasm Inner membrane (cell membrane)
ATP synthase (20 nm diameter in inner membrane; 32 nm total height)
Ribosome
Arabinose-binding protein (3 nm x 3 nm x 6 nm) Acid resistance chaperone (HdeA) (3 nm x 3 nm x 6 nm) Disulfide bond protein (DsbA) (3 nm x 3 nm x 6 nm) Cytoplasmic proteins:
Peptide Cytoplasm
RNA
Periplasmic proteins:
RNA polymerase
Pyruvate kinase (5 nm x 10 nm x 10 nm) Phosphofructokinase (4 nm x 7 nm x 7 nm) Proteasome (12 nm x 12 nm x 15 nm) Chaperonin GroEL (18 nm x 14 nm) Other proteins Transcription and translation complexes: RNA polymerase (10 x 10 x 16 nm)
DNA bridging protein H-NS
DNA
Nucleoid
DNA-binding protein HU
Ribosome (21 x 21 x 21 nm) Nucleoid components: DNA (2.4 nm wide x 3.4 nm/10 bp) DNA-binding protein (3 x 3 x 5 nm) DNA-bridging protein (3 x 3 x 5 nm)
50 nm
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cytoplasmic proteins from leaking out and maintains gradients of ions and nutrients. It is covered by a cell wall, a rigid structure composed of polysaccharides linked covalently by peptides (peptidoglycan). The cell wall forms a single cage-like molecule that surrounds the cell. In gram-negative bacteria such as E. coli, the cell wall consists of a single layer of peptidoglycan, a polymer of sugars and peptides. The cell wall extends within the periplasm, an aqueous layer containg proteins such as sugar transporters. Outside the cell wall lies an outer membrane of phospholipids and lipopolysaccharides (LPSs), a class of lipids attached to long polysaccharides (sugar chains). LPS and other polysaccharides can generate a thick capsule surrounding the cell. The capsule polysaccharides form a slippery mucous layer that inhibits phagocytosis by macrophages (see Section 26.3). The cell wall and outer membrane constitute the envelope. The envelope includes cell surface proteins that enable the bacteria to interact with specific host organisms. For example, E. coli cell surface proteins enable colonization of the human intestinal epithelium, whereas Sinorhizobium cell surface proteins enable colonization of legume plants for nitrogen fi xation. Another common external structure is the flagellum, a helical protein filament whose rotary motor propels the cell in search of a more favorable environment. Within the cell, the cell membrane and envelope provide an attachment point for one or more chromosomes. The chromosome is organized within the cytoplasm as a system of looped coils called the nucleoid. Unlike the round, compact nucleus of eukaryotic cells, the bacterial nucleoid is not bounded by a membrane, and so the coils of DNA can extend throughout the cytoplasm. Loops of DNA from the nucleoid are transcribed by RNA polymerase to form messenger RNA (mRNA) as well as small functional RNA molecules (sRNA). As the mRNA transcripts grow, they bind ribosomes to start synthesizing polypeptide chains. As the polypeptides grow, protein complexes called chaperones help them fold into their functional conformations.
Microbial Eukaryotes How do eukaryotic microbes, such as euglenas and amebas, compare with prokaryotes? Most eukaryotic cells that have been studied are much larger than prokaryotes; some amebas are actually visible to the unaided eye. Yet DNA-based surveys of natural environments, such as the ocean and soil, reveal eukaryotes as small as the smallest bacteria. Thus, eukaryotic cells extend over the full sizerange encompassed by prokaryotes, in addition to reaching much larger sizes. Like bacteria, microbial eukaryotes typically possess a thick outer covering. Fungi possess cell walls of polysaccharide, whereas protists have a pellicle, consisting
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of membranous layers reinforced by protein microtubules. Other kinds of eukaryotic cell surface are reinforced by inorganic materials, such as the silicate shells of diatoms. Eukaryotic microbes contain subcellular organelles composed of membranes that are more extensive and specialized than those of prokaryotes. Examples include the nuclear membrane, golgi, mitochondria, and chloroplasts (reviewed in Appendix 2). These organelles perform some of the physiological functions that are provided by organ systems in multicellular organisms, such as circulation, digestion, and excretion. Mitochondria and chloroplasts are the products of ancestral engulfment of prokaryotic cells, followed by evolution of endosymbiosis, as explained in Chapter 17. Eukaryotic microbial diversity is explored in Chapter 20.
Biochemical Composition of Bacteria The bacterial cell model indicates shape and size, but tells little about chemical composition. Chemistry explains, for example, why wiping a surface with ethanol eliminates microbial life, whereas water has little effect. Water is a universal constituent of cytoplasm, but is excluded by cell membranes. Ethanol, however, dissolves both polar and nonpolar substances; thus, ethanol disintegrates membranes and destroys the secondary structure of proteins. All cells share common chemical components: ■ ■
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Water, the fundamental solvent of life Essential ions, such as potassium, magnesium, and chloride ions Small organic molecules, such as lipids and sugars that are incorporated into cell structures and that provide nutrition by catabolism Macromolecules, such as nucleic acids and proteins, which contain information, catalyze reactions, and mediate transport, among many other functions
Cell composition varies with species, growth phase, and environmental conditions. Table 3.1 summarizes the chemical components of a cell for the model bacterium Escherichia coli during exponential growth. Small molecules. The E. coli cell consists of about 70%
water, the essential solvent required to carry out fundamental metabolic reactions and to stabilize proteins. The water solution contains inorganic ions, predominantly potassium, magnesium, and phosphate. Inorganic ions store energy in the form of transmembrane gradients, and they serve essential roles in enzymes. For example, a magnesium ion is required at the active site of RNA polymerase to help catalyze incorporation of ribonucleotides into RNA.
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Table 3.1 Molecular composition of a bacterial cell, Escherichia coli, during balanced exponential growth.a
Component
Percentage of total weightb
Approximate number of molecules/cell
Number of different kinds
70% 16% 6% 0.7% 3% 1% 1% 1.3% 0.8% 0.1% 0.1%
20,000,000,000 2,400,000 250,000 4,000 25,000,000 1,400,000 2d 50,000,000 1 250,000,000 6,700,000
1 2,000c 200 2,000c 50 1 1 1,000 1 20 2
Water Proteins RNA: rRNA, tRNA, and other small regulatory RNA (sRNA), mRNA Lipids: phospholipids (membrane) lipopolysaccharide (outer membrane) DNA Metabolites and biosynthetic precursors Peptidoglycan (murein sacculus) Inorganic ions Polyamines (mainly putrescine and spermine) a
Values shown are for a hypothetical “average” cell cultured with aeration in glucose medium with minimal salts at 37°C.
b
The total weight of the cell (including water) is about 10–12 gram (g), or 1 picogram (pg).
c
The number of kinds of mRNA and of proteins is difficult to estimate because some genes are transcribed at extremely low levels and because RNA and proteins include kinds that are rapidly degraded. d
In rapidly growing cells, cell fission typically lags approximately one generation behind DNA replication; hence, two identical DNA copies per cell.
Source: Modified from Neidhardt, F., and H. E. Umbarger. 1996. Chemical composition of Escherichia coli, p. 14. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed. ASM Press, Washington, DC.
Frederick C. Neidhardt, U. of Michigan
The cell also contains many kinds of small charged organic molecules, such as phospholipids and enzyme cofactors. A major class of organic cations is the polyamines, molecules with multiple amine groups that are positively charged when the pH is near neutral. Polyamines balance the negative charges of the cell’s DNA, and they stabilize ribosomes during translation. Macromolecules. Many cells show similar content of
water and small molecules, but their specific character is defined by their macromolecules, especially their proteins. Proteins vary among different species; and a given species makes very different proteins, depending on environmental conditions such as temperature, nutrient levels, and entry into a host organism. Individual proteins, encoded by specific genes, are found in very different amounts, from 10 per cell to 10,000 per cell. The total proteins encoded by a genome, capable of expression in the cell, are known collectively as the proteome. Early attempts to define the proteome of a cell were conducted by Fred Neidhardt (Fig. 3.2) and colleagues at the University of Michigan, who created the fi rst protein catalog of E. coli using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE or 2-D gels) (Fig. 3.3). To obtain 2-D gels, cells are lysed by sonication or detergent to release and solubilize as many proteins as possible. The proteins are then subjected to two forms of separation, isoelectric focusing followed by electrophoresis. In isoelectric focusing, proteins are placed in a gel that has a pH gradient, and they migrate along the gradi-
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Proteins of E. coli. Fred Neidhardt and colleagues at the University of Michigan, Ann Arbor, used 2-D gel electrophoresis to create the first protein catalog of E. coli.
Figure 3.2
ent until they reach the point where the number of positively charged residues equals the number of negatively charged residues. At this point, their net charge is zero (that is, the pH equals the protein’s isoelectric point), and the protein stops moving. In electrophoresis, proteins deposited in an electrical field migrate toward a positive electrode. The proteins are coated with a reagent (SDS, or sodium dodecyl sulfate) to confer a uniform negative charge so that their distance of migration depends on
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Molecular weight (kD)
molecular weight (roughly a logarithmic function). The proteins are then stained for detection, and the protein spots can be identified, either by N-terminal protein sequencing or by mass spectrometry. 50 In a 2-D gel of E. coli (Fig. 3.3), about 500 different proteins can be distinguished. The most highly expressed proteins include ribo40 somal proteins and translation factors such as elongation factor Tu (EF-Tu). Outer membrane proteins such as OmpX and ProX also show high concentration. Conversely, many proteins of the inner (cell) membrane are too insoluble 30 in water to appear, and some important regulators are present at levels too low to be seen. Nevertheless, 2-D gels are useful for identifying changes in protein expression that occur 20 under different environmental conditions or when bacteria are invading host cells. Protein synthesis is directed by DNA and RNA. The content of nucleic acids in E. coli is nearly 8% by weight, much higher than in multicellular eukaryotes. For microbes, the high nucleic acid content is advantageous, allowing the cell to maximize reproduction of its chromosome while minimizing resources for proIsoelectric point (pl) 5.0 5.5 6.0 tein-rich cytoplasm. The high level of nucleic acids is actually toxic to human consumers, Figure 3.3 Proteins of E. coli. 2-D gel of proteins of Escherichia coli who lack the enzymes to digest the uric acid grown aerobically in a casein-yeast extract medium. Proteins were identified waste product of digested nucleotides. That is by N-terminal sequence and by mass spectroscopy. DnaK is a cytoplasmic why most kinds of bacteria cannot be eaten as chaperone; EF-Tu is an elongation factor for translation; MalE is an outer a major part of the diet. membrane maltose-binding protein; ProX and OmpX are periplasmic transporters; and AhpC is an antioxidant stress protein. Other kinds of macromolecules are found in the cell wall and outer membrane. The bacterial cell wall consists of peptidoglycan, an organic polymer that constitutes nearly 1% of the cell ■ The biochemical composition of bacteria includes mass, approximately the same mass as that of DNA. This shows the importance (for most species) of maintaining relatively high nucleic acid content, as well as proturgor pressure in dilute environments, where water would teins, phospholipids, and other organic and inorganic otherwise enter by osmosis, causing osmotic shock. constituents. ■ Proteins in the cell vary, depending on the species and environmental conditions. THOUGHT QUESTION 3.1 Which molecules occur in the greatest number in a prokaryotic cell? The smallest number? Why does a cell contain 100 times as many lipid molecules as strands of RNA?
3.2 TO SU M MAR I Z E: ■
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■
Prokaryotic cells are protected by a thick cell envelope. Compact genomes maximize reproductive potential with minimal resources. The model bacterial cell contains a highly ordered cytoplasm in which DNA replication, RNA transcription, and protein synthesis occur coordinately.
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Joan Slonczewski, Kenyon College
78
How We Study the Parts of Cells
How can we determine how all the macromolecules listed in Table 3.1 interact and work together? Electron microscopy largely defi nes how we “see” the cell’s interior as a whole. In Figure 3.1, we clearly saw major parts of B. thuringiensis, such as the endospore and the parasporal crystal. But smaller parts, such as the ribosomes, appear only as small, densely packed particles. Furthermore,
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Subcellular fractionation enables isolation of cell parts such as ribosomes and membranes so that we can study their form and function. Structural analysis by X-ray crystallography and related methods reveals the form of cell components. Genetic analysis dissects the function of cell components based on construction of mutant cells with altered function.
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In this section, we present these methods as applied to a key example of cellular function, that of the bacterial ribosome. Each method has its particular strengths and limitations to reveal form and function.
The ultracentrifuge. Different parts of the cell can be isolated by subcellular fractionation. A key tool of subcellular fractionation is the ultracentrifuge, a device in which solutions containing cell components are rotated in tubes at high speed. The high rotation rate generates centrifugal forces strong enough to separate subcellular particles (Fig. 3.4A). The ultracentrifuge was invented
Isolating Parts of Cells Cellular components, such as ribosomes and flagellar motors, can be isolated readily from cells. The cells must A.
B.
Eric Kaufmann, USDA-ARS-CMAVE
Subcellular fractionation by ultracentrifugation. A. An ultracentrifuge is used to fractionate cell components. B. Salmonella ribosomes (TEM) isolated from the cytoplasm by ultracentrifugation through a linear sucrose density gradient. Polysomes consist of two or more ribosomes attached to mRNA. C. High-speed rotation generates high centrifugal forces, measured in units of gravity (g). The Svedberg unit (S) offers a measure of particle size based on its rate of travel in a tube subjected to high g force. The Svedberg coefficient (number of S units; for example, 30S) is defined in terms of the velocity of the particle in the tube (v), the radius of the rotor (r), and the rotational velocity (ω). The coefficient of S for a given particle depends on its mass (m) and its shape. After centrifugation, fractions are collected from the base of the centrifuge tube. The fractions contain radiolabeled ribosomes. The largest particles (whole ribosomes) sediment near the bottom of the tube, whereas the smaller particles (separate 50S and 30S subunits) appear in upper fractions.
Figure 3.4
C.
100 nm
To centrifuge
From centrifuge
Rotor 30S Sample tube
50S
Axis of rotation
Ribosomes 70S
r
Spins at up to 100,000 g
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Mild detergent lysis. Cells can be lysed with a detergent capable of dissolving membranes but not denaturing proteins. Freezing and thawing. Freezing and then thawing is another way to open cells gently, releasing particles such as ribosomes. Sonication. Cells can be lysed by intense ultrasonic vibrations above the range of human hearing. Enzymes. Enzymes such as lysozyme can break the cell wall, allowing the cell to be lysed by mild osmotic shock.
P. L. Clark and J. King. 2001. J. Bio. Chem. 276
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79
be broken open by techniques that allow subcellular components to remain intact. Here are examples of such techniques:
even higher-resolution images from electron microscopy cannot tell us the chemical composition of ribosomes or how they function in the living cell. To study ribosome function requires isolation and analysis of subcellular parts: ■
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m
v Svedberg unit v S= 2 r
Collect fractions from base of tube
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by the Swedish physicist Theodor Svedberg (1884–1971), who won the 1926 Nobel Prize in Chemistry for the use of ultracentrifugation to separate proteins. Modern ultracentrifuges have titanium rotors that spin in a vacuum to avoid frictional heating, generating forces up to 100,000 times gravity (100 k g). For fractionation, cells are fi rst lysed by one of the methods previously described to obtain a cell lysate, a general term for the contents of a broken cell. The cell lysate is placed in tubes containing a high-density solution, such as sucrose or cesium chloride solution, in which suspended particles sediment slowly. Under a high g force, however, particles sediment at different rates, depending on their size and density. The sedimentation rate is the rate at which particles of a given size and shape travel to the bottom of the tube under centrifugal force. The sedimentation rate is measured by the Svedberg unit, S, which is given by the particle’s rate of sedimentation (v), the radius at which the tube rotates (r), and the rotational velocity (ω): S = v/(ω2r) For a given type of particle in suspension, the sedimentation rate also depends on the particle’s mass and shape. The contribution of particle mass and shape is defi ned as its Svedberg coefficient; for example, the coefficient of the small subunit of the ribosome is 30, for a sedimentation value of 30S. The value of the Svedberg coefficient increases with the average cross-sectional area of the particle. For bacterial ribosomes, ultracentrifugation yields intact ribosomes (70S) as well as separated ribosomal subunits, the large subunit (50S) and the small subunit (30S). Within cells, ribosomes normally exist as a mixture of joined and separate subunits. To isolate the ribosomes, a cell lysate is layered onto a tube of sucrose solution, whose density decreases the sedimentation rate and increases the separation of particles of different size (Fig. 3.4A). The fractions shown in Figure 3.4C were drained sequentially from the base of the tube after centrifugation. The heaviest particle, the 70S ribosome, appears in the fractions nearest the bottom of the tube because it travels fastest under the centrifugal force. THOUGHT QUESTION 3.2 Why does the Svedberg coefficient of the intact ribosome (70S) differ from the sum of the individual subunits of the ribosome (30S and 50S)? The ribosomes can be seen in collected fractions by electron microscopy (Fig. 3.4B). In some cases, two or more ribosomes are connected by a strand of messenger RNA (mRNA). This multiple-ribosome structure, called a polysome, gives our fi rst clue as to the intracellular orga-
073-114_SFMB_ch03.indd 80
nization of the translation apparatus within a bacterial cell (see Fig. 3.1), where a number of ribosomes translate each mRNA at the same time. Furthermore, ribosomes isolated by centrifugation can translate messenger RNA in cell-free systems. Experiments in cell-free systems provide the basis of much of our knowledge of protein synthesis (see Chapter 8). Limitations of subcellular fractionation. Subcellular
fractionation yields clues about internal structure but provides little information about processes that require overall integrity of the cell. For example, the role of the transmembrane electrochemical potential, or proton potential, in ATP synthesis was obscured for many years because biochemists were unable to isolate a cytoplasmic complex responsible for it. Transmembrane ion gradients were in fact observed within membrane vesicles— spheres of membrane isolated by cell disintegration and centrifugation. But it was harder to demonstrate that the entire cell membrane of an intact cell supports a proton potential.
Crystallographic Analysis Reveals Structure Isolated ribosomes can be crystallized for structure analysis by X-ray diffraction crystallography. A crystallographic model of the 30S ribosomal subunit is shown in Figure 3.5A. The 30S subunit consists of ribosomal RNA (rRNA) and 27 different proteins, each of which is encoded by a particular gene. The sequences of all the ribosomal genes, with their predicted protein and RNA products, were fitted with X-ray crystallography data to build a three-dimensional model of the 30S subunit. This model includes three bound transfer RNA (tRNA) molecules, each at a key position for translation: the acceptor site (A) for incoming aminoacyl-tRNA; the peptide site (P) attached to the growing peptide chain; and the exit site (E), where tRNA is released from the ribosome. A similar model was obtained of the 30S ribosomal subunit bound to streptomycin, an antibiotic produced by Streptomyces bacteria (Fig. 3.5B). In this model, the streptomycin molecule contacts a particular ribosomal protein, S12. The contact with S12 distorts the structure of the A site and thus halts protein synthesis. Only the ribosomes of bacteria, not of eukaryotes, have the precise shape to bind streptomycin; thus, this antibiotic kills bacterial pathogens without harming the human patient. Studying ribosome structure enables us to design new antibiotics. Limitations of crystallography. As discussed in Chapter 2, crystallographic analysis applies only to isolated particles and under conditions in which its full function cannot be observed. The technique can solve structures
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A. The ribosome: x-ray diffraction model
B.
X-ray diffraction models relate structure to function. A. The 30S ribosomal subunit, X-ray diffraction model of the 16S RNA (stereo view) showing the acceptor (A) site, the peptide (P) site, and the exit (E) site. To view, place a piece of cardboard vertically between the two images. Position your eyes on opposite sides of the cardboard and force them to focus behind the images. The images will merge and produce a 3-D image. (PDB codes: 1GIX, 1GIY) B. When the ribosome is crystallized with streptomycin, the antibiotic molecule binds to protein S12, near the A site. Streptomycin interferes with the tRNA binding to the A site.
Figure 3.5
Streptomycin
p Promoter for str ybxF operon Ribosomal protein S12
Unknown function
rpsL rpsG Ribosomal protein S7
fus
tuf
Elongation factor G
Elongation factor Tu
Ribosomal protein S12 Mutations in the gene rpsL, encoding ribosomal protein S12, confer resistance to the antibiotic streptomycin.
only for proteins and nucleic acids capable of crystallization. It remains impractical for proteins of flexible, nonrigid structure and for many membrane-soluble proteins.
Genetic analysis of the streptomycin-resistant ribosome. The str operon has genes encoding components of the ribosome. A mutation in rpsL, the gene encoding ribosomal protein S12, alters the protein’s interaction with streptomycin. The mutant gene makes the bacteria resistant to the antibiotic.
Figure 3.6
Genetic Analysis Reveals Function The crystal structure of a macromolecule only yields clues as to function; it does not show the structure in action. The function of cell components can be dissected by genetic analysis. In genetic analysis, mutant strains are selected for loss of a given function, or a strain can be intentionally mutated so as to lose or alter a gene. An example of genetic analysis is the use of ribosome-encoding genes to show how ribosome func-
073-114_SFMB_ch03.indd 81
tion is blocked by the antibiotic streptomycin. The genes specifying each ribosomal protein and RNA (rRNA) are organized in operons of genes under one promoter, such as the str operon (Fig. 3.6). The str operon was named for streptomycin resistance, a phenotype conferred by mutations in the gene rpsL. Mutations conferring streptomycin resistance were known for decades before the structural basis was discovered. These mutations were
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mapped to the gene rpsL encoding the protein S12, whose position in the 30S subunit was shown by X-ray crystallography (see Fig. 3.5B). The S12 protein forms part of the ribosome’s tRNA acceptor (A) site, which receives the incoming aminoacyl-tRNA. Mutation in the gene encoding S12 generates a protein of altered shape, permitting function in the presence of streptomycin. Thus, genetic analysis and crystallography were combined to demonstrate the precise mode of action of an important antibiotic. An exciting extension of genetic analysis is the construction of strains with “reporter genes” fused to genes encoding structures of interest. An example of a reporter gene is green fluorescent protein, which fluoresces at the site of the fused protein. Fluorescent reporter genes enable us to observe the function of proteins within a live cell. In Section 3.6, we will discuss the use of fluorescent reporter genes to observe chromosome replication and cell division. The methods of gene manipulation are discussed in Chapters 8 and 12. THOUGHT QUESTION 3.3 What are the advantages and limitations of biochemical and genetic approaches to cell structure and function? TO SU M MAR I Z E: ■
■
■
Subcellular fractionation isolates cell parts for structural, biochemical, and genetic analysis. X-ray crystallography shows the three-dimensional form of cell components at the atomic level. Genetic analysis shows which genes and proteins are responsible for functions of subcellular complexes such as the ribosome. Mutation of a gene leads to altered function of the cell.
3.3
The Cell Membrane and Transport
The structure that defines the existence of a cell is the cell membrane (Fig. 3.7). The cell membrane consists of a phospholipid bilayer containing lipid-soluble proteins. Overall, the membrane serves two kinds of function: It contains the cytoplasm within the external medium, mediating transport between the two; and it carries many proteins with specific functions, such as biosynthetic enzymes and environmental signal receptors.
Membranes Consist of Phospholipids and Proteins Cell membranes are composed of approximately equal parts phospholipids and proteins. Each phospholipid possesses a charged phosphate-containing “head” that con-
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Extracellular environment
Hopanoid
Phosphatidylglycerol
Transporter protein
Proton-driven ATPase
Cytoplasm
Bacterial cell membrane. The cell membrane consists of a phospholipid bilayer, with hydrophobic fatty acid chains directed inward, away from water. The bilayer contains stiffening agents such as hopanoids, which serve the same function as cholesterol in eukaryotic membranes. Half the volume of the membrane consists of proteins.
Figure 3.7
tacts the water interface, as well as a hydrophobic “tail” packed within the bilayer. A phospholipid consists of glycerol with ester links to two fatty acids and a phosphoryl polar head group (for review, see Appendix 2). The polar head group may have a side chain such as ethanolamine in phosphatidylethanolamine, the major phospholipid of E. coli (Fig. 3.8). Lipid biosynthesis is a key process of cells; for example, a bacterial enzyme for fatty acid biosynthesis, enoyl reductase, is the target of triclosan, a common antibacterial additive in detergents and cosmetics. In the bilayer, all phospholipids face each other tail to tail, keeping their hydrophobic side chains away from the water inside and outside the cell. The two layers of phospholipids in the bilayer are called leaflets. One leaflet of phospholipids faces the cell interior, whereas the other faces the exterior. As a whole, the phospholipid bilayer imparts fluidity and gives the membrane a consistent thickness (about 10 nm). The cell membrane can be thought of as a twodimensional fluid within which float a vast array of hydrophobic proteins and smaller molecules such as hopanoids (see Fig. 3.7). Membrane proteins serve numerous functions that define the capabilities of the cell, including transport, communication with the environment, and structural support. Examples include: ■
Structural support. Some membrane proteins anchor together different layers of the cell envelope (see Section 3.4). Other proteins attached to membranes form part of the cytoskeleton. Still others form the base of structures that extend out from the cell, such as pili,
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CH2 CH2 O
H
O
P
H
H
O
C
C
C
O
O
H
CO
CO
O
H
Glycerol derivative
Phosphatidylethanolamine
Phosphatidylethanolamine. A major bacterial phospholipid consists of glycerol with ester links to two fatty acids and a phosphoethanolamine.
Figure 3.8
■
fi laments, and flagella; these enable adherence and motility. Detection of environmental signals. In Vibrio cholerae, the causative agent of cholera, ToxR is a transmembrane protein whose amino-terminal part reaches into the cytoplasm. When ToxR detects acidity and elevated temperature—signs of the host digestive tract—its amino-terminal domain binds to DNA at a sequence that activates expression of cholera toxin and other virulence factors. Secretion of virulence factors and communication signals. Membrane proteins form secretion complexes to export toxins and cell signals across the envelope. For example, symbiotic nitrogen-fi xing rhizobia require membrane proteins NodI and NodJ to transport nodulation signals out to the host plant roots, where they induce formation of root nodules containing the bacteria.
Proteins embedded within a membrane must have a hydrophobic portion that is soluble within the membrane. Typically, several hydrophobic alpha helices thread back and forth through the membrane. Other regions of peptide extend outside the membrane, containing charged and polar amino acids that interact favorably with water. The combination of hydrophobic and hydrophilic regions effectively locks the protein into the membrane.
Transport across the Cell Membrane Overall, the cell membrane acts as a barrier to sequester water-soluble proteins of the cytoplasm. The membrane’s
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Phosphoryl head group
Fatty acid side chains
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specific components, particularly proteins, determine which substances cross the membrane between the cytoplasm and the outside. Selective transport is essential for cell survival; it means the ability to acquire scarce nutrients while excluding toxins.
H3N+
–
■
Osmosis. Most cells maintain a concentration of total solutes (molecules in solution) that is higher inside the cell than outside. As a result, the internal concentration of water is lower than the concentration outside the cell. Because water can cross the membrane, but its charged solutes cannot, water tends to diffuse across the membrane into the cell, causing expansion of cell volume, in a process called osmosis. The resulting pressure on the cell membrane is called osmotic pressure (reviewed in Appendix 2). Osmotic pressure will cause a cell to burst, or lyse, in the absence of a countering pressure such as that provided by the cell wall. Various solutes cross the membrane by different means. Nonspecific transport, or passive diffusion, requires dissolving in the phospholipid bilayer. Specific transport—for example, of nutrients and toxins—requires specific transport proteins. The presence of specific transporters depends on the microbial species and environmental conditions. Passive diffusion. Small uncharged molecules, such as O2, CO2, and water, easily permeate the membrane. Some molecules, such as ethanol, also disrupt the membrane, an action that can make such molecules toxic to cells. By contrast, strongly polar molecules, such as sugars, generally cannot penetrate the hydrophobic interior of the membrane and require transport mediated by specific proteins. Water molecules permeate the membrane, but their rate of passage is increased by protein channels called aquaporins. Membrane-permeant weak acids and bases. A special case of movement across cell membranes is that of membrane-permeant weak acids and weak bases, which exist in equilibrium between charged and uncharged forms:
Weak acid: HA D H + + A– Weak base: B + H 2O D BH + + OH – Weak acids and weak bases cross the membrane in their uncharged form, HA (weak acid) or B (weak base). On the other side, upon reentering aqueous solution, they dissociate (HA to A– and H+) or reassociate with H+ (B to BH+). In effect, they conduct acid (H+) or base (OH– ) across the membrane, causing acid or alkali stress. A high-proton concentration outside the cell will increase the amount of uncharged weak acid that can freely enter the cell. Thus, if the H+ concentration (acidity) outside the cell is higher than inside, it will drive weak acids into the cell.
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The protonated form of a weak acid (RCOOH) crosses the membrane, _ whereas the deprotonated form (RCOO ) does not.
Membranesoluble
C
Watersoluble
O
O
C
OH O
C
–
O
O CH3 C
+
+ H
O C
OH
CH3
S
+ H+
N O O
Aspirin
NHC Penicillin
R
CH3
H3C
The protonated form of a weak base (RNH3+) does not cross the membrane, whereas the deprotonated form (RNH2) does. +
H
NH2CH3
H3C + H+
N HO CH3
O CHCH2CH2NHCH3 + H+
_
H3C
O
F3C
O
+
N
CH3 H
OH NH2
Membranesoluble
Prozac (fluoxetine)
Watersoluble
OH O HO
O OH Tetracycline
Common drugs are membrane-permeant weak acids and bases. bloodstream. The uncharged form is hydrophobic and penetrates the cell membrane.
Figure 3.9
Many key substances in cellular metabolism are membrane-permeant weak acids and bases, such as acetic acid. Most pharmaceutical drugs—therapeutic agents delivered to our tissues via the bloodstream—are weak acids or bases whose uncharged forms exist at sufficiently low concentration to cross the membrane without disrupting it. Examples of weak acids that deprotonate (acquiring negative charge) at neutral pH include aspirin (acetylsalicylic acid) and penicillin (Fig. 3.9). Examples of weak bases that protonate (acquiring positive charge) at neutral pH include Prozac (fluoxetine) and tetracycline. THOUGHT QUESTION 3.4 Amino acids have acidic and basic groups that can dissociate. Why are they not membrane-permeant weak acids or weak bases? Why do they fail to cross the phospholipid bilayer? Transport proteins. Proteins enable transport that is
highly selective; that is, a given protein will transport only certain nutrients or ions (Fig. 3.10). Molecules that carry net charge, such as inorganic ions (Na+, Cl– ), require specific transport by transport proteins, or transporters. So do organic molecules that carry charge at cytoplasmic pH, such as amino acids. Transporters are highly specific to different ions and organic substances under different environmental conditions. Much of the character and ecological niche of a microbe—the nutrients it can consume, the drugs it can
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O
In its charged form, each drug is soluble in the
Extracellular environment Substrate molecules
Transport driven by gradient of substrate or cotransported molecules. Cytoplasm
Figure 3.10 Membrane transport proteins. Transport proteins enable the uptake of nutrients into the cell. Uptake can be powered by movement down a gradient of substrate or of a cotransported molecule such as sodium ions. Alternatively, transport may be powered by a coupled chemical reaction that spends energy, such as ATP hydrolysis.
resist—depend on its set of transporters encoded by its genome. Transporters evolve in families common to many species; thus, analysis of known genomes predict the transport capabilities in a newly sequenced genome (discussed in Chapter 8). Organisms that live in complex, changing environments express numerous transporters. For exam-
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ple, the genome of Streptomyces coelicolor, a soil-dwelling actinomycete bacterium that produces several antibiotics, shows over 400 different transporters. These include exporters for toxic ions such as arsenate and chromate, uptake of oligosaccharides and peptides, and multipledrug pumps. Transport proteins act by various mechanisms, such as a structural channel, or pore, that allows a nutrient molecule to enter the cell or a toxin to be exported. Transport may be passive or active. In passive transport, molecules accumulate or dissipate along their concentration gradient. Active transport, that is, transport from lower to higher concentration, requires expenditure of energy. The energy for active transport may be obtained by cotransport of another substance down its gradient from higher to lower concentration. For example, many transporters couple uptake of amino acids to uptake of sodium ions. Alternatively, transport may be powered by a coupled chemical reaction that spends energy, such as ATP hydrolysis. Further transport mechanisms are discussed in Chapter 4. A medically important example of active transport is that of drug efflux proteins powered by the hydrogen ion gradient. Efflux proteins pump antibiotics such as tetracycline out of the bacterial cell, enabling harmful bacteria to grow in the presence of antibiotics. Pathogens and cancer cells evolve multiple drug resistance transporters that enable them to survive chemotherapy.
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A.
85
H+
c
a
Cell membrane Fo
␥
b ␦

␣
Cytoplasm F1
⑀
B.
Nanopropeller
Post
F1
␣

Transmembrane Ion Gradients Store Energy Ions such as H+ or Na+ usually exist in very different concentrations inside and outside the cell. The ion gradient (ratio of concentrations) across the cell membrane can store energy obtained from metabolizing food or from photosynthesis. For example, membrane proteins such as cytochrome oxidases use energy from respiration to pump hydrogen ions across the cell membrane, generating a hydrogen ion gradient. The hydrogen ion gradient plus the charge difference (voltage potential) across the membrane form an electrochemical potential. Because this electrochemical potential includes the hydrogen ion gradient, it is called the proton potential. The proton potential, or proton motive force, drives various cell devices, such as the rotary flagella and efflux pumps that expel antibiotics. Most importantly, the proton potential drives the membrane-bound ATP synthase (Fig. 3.11). The role of the proton potential in metabolism is discussed in detail in Chapters 13 and 14. The membrane-bound ATP synthase, also called F1Fo ATP synthase, provides most of the ATP for aerobic respiring cells such as E. coli; and essentially the same complex mediates ATP generation in our own mitochondria. The complex of peptides includes a channel (Fo) for
073-114_SFMB_ch03.indd 85
Figure 3.11 Bacterial membrane ATP synthase. A. The F1Fo complex of ATP synthase is embedded in the cell membrane. The Fo channel/rotor admits hydrogen ions; the H+ current flow across the membrane is driven by the concentration and charge differences between inside and outside the cell. The flow of H+ causes the rotation of F1. Rotation of F1 drives conversion of ADP + Pi to ATP. B. An artificial “biomolecular motor” was built from an ATPase F1 unit attached to a nickel post and a nanopropeller.
hydrogen ions, which drive rotation of the ATPase complex (F1). Rotation of F1 mediates the formation of ATP. The idea that a living organism could contain rotating parts was highly controversial when such parts were first discovered in bacterial flagella (discussed in Section 3.7). Before this discovery, scientists had believed that living body parts could not rotate and that only humans had “invented the wheel.” The discovery of rotary biomolecules has inspired advances in nanotechnology, the engineering of microscopic devices. For example, a “biomolecular motor” was devised using an ATP synthase F1 complex to drive a metal submicroscopic propeller (Fig. 3.11B). In the future, such biomolecular design may be used to create microscopic robots that enter the bloodstream to perform microsurgery.
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Membrane Lipids Differ in Structure and Function Membranes require a certain uniformity to maintain structural integrity and function. Yet individual membrane lipids differ remarkably in structure. Membrane lipids help determine whether an organism grows in a Yellowstone hot spring or in the human lungs, where it may cause pneumonia. Phospholipids vary with respect to their phosphoryl head groups and with respect to their hydrocarbon side chains. Some membrane lipids lack phosphate altogether, substituting other polar groups; and some replace mobile side chains with fused rings. Phosphoryl head groups. At the pH of most biological systems, the phosphoryl head group carries net negative charge, called a phosphatidate (Fig. 3.12A). The negatively charged phosphatidate can contain various organic groups, such as glycerol to form phosphatidylglycerol. A more complex phosphatidate is cardiolipin, or diphosphatidylglycerol, actually a double phospholipid linked by a glycerol (Fig. 3.12B). Cardiolipin increases in concentration in bacteria grown to starvation or stationary phase, possibly because its extended structure stabilizes the membrane.
H H C
H
O–
H
C C
O
P
H
O
OH OH H
C
O
H
O
H C
O C O
H C
O C
H Phosphatidylglycerol
H O–
H H C
O
P
H
O
C O
C O
H C O
C
O
O C H
Other phospholipids have a positively charged head group, such as phosphatidylethanolamine. Phospholipids with positive charge or with mixed charges are concentrated in portions of the membrane that interact with DNA, which has negative charge. Also, some transporter proteins require interaction with positively charged phospholipids. The fatty acid component of phospholipids also varies greatly among bacteria. Certain soil bacteria and pathogens such as Mycobacterium tuberculosis have several hundred different kinds of fatty acids. Fatty acid structures that stiffen the membrane are increased under stress conditions, such as starvation or acidity. Variant fatty acid structures in phospholipids enhance survival under environmental stress. The most common bacterial fatty acids are hydrogenated chains of varying length, typically between 6 and 22 carbons. These chains pack neatly to form a smooth layer. Some fatty acid chains, however, are partly unsaturated (possess one or more carbon-carbon double bonds). If the unsaturated bond is cis, meaning that both alkyl chains are on the same side of the bond, the unsaturated chain has a “kink,” as in oleic acid (Fig. 3.13). The kinked chains do not pack as closely as the straight hydrocarbon chains and thus make the membrane more “fluid.” This is why, at room temperature, unsaturated vegetable oils are fluid, whereas highly saturated butterfat is solid. The enhanced fluidity of a kinked phospholipid improves the function of the membrane at low temperature; hence, bacteria can respond to cold and heat by increasing or decreasing their synthesis of unsaturated phospholipids. Another interesting structural variation is cyclization of part of the chain to create a stiff planar ring with decreased fluidity. The double bond of unsaturated fatty acids can incorporate a carbon from S-adenosyl-LO HO Palmitic acid
H
O
H C OH H
O H C H
O
P O–
O
C
H O
H
C
O C O
H
C
O C
HO Oleic acid (trans) O HO Oleic acid (cis)
H Cardiolipin (diphosphatidylglycerol)
Figure 3.12 Phospholipid head groups. Bacterial membranes contain phospholipids with several kinds of polar head groups. Common examples include phosphatidylglycerol (negatively charged, a phosphatidate), cardiolipin (a double phospholipid joined by a third glycerol), and phosphatidylethanolamine (with a positively charged amine).
073-114_SFMB_ch03.indd 86
HO O Cyclopropane fatty acid
Figure 3.13 Phospholipid side chains. Bacterial lipid side chains include palmitic acid, oleic acid, and cyclopropane fatty acid.
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methionine to form a three-membered ring, becoming a cyclopropane fatty acid (see Fig. 3.13). Bacteria convert unsaturated fatty acids to cyclopropane during starvation and acid stress, conditions under which membranes require stiffening. Cyclopropane conversion is an important factor in the pathogenesis of Mycobacterium tuberculosis (causative agent of tuberculosis) and in the acid resistance of food-borne toxigenic E. coli. Many other structural variants are seen in different species, such as branched chains, hydroxyl and sulfate groups, and polycyclic groups. The less common forms are highly characteristic of particular species and strains. Thus, fatty acid profi les are used to identify certain kinds of pathogens, such as anthrax spores.
HO HO
HO HO
HO HO
OH OH
OH OH
HO HO
OH OH
OH OH
HO HO
Figure 3.14 Hopanoids add strength to membranes. Hopanoids fit between the fatty acid side chains of membranes and limit their motion, thus stiffening the membrane. A hopanoid has five fused rings and an extended hydroxylated tail.
Terpene derivatives stiffen membranes. In addition to
phospholipids, membranes include planar molecules that fi ll gaps between hydrocarbon chains (Fig. 3.14). These stiff, planar molecules reinforce the membrane, much as steel rods reinforce concrete. In eukaryotic membranes, the reinforcing agents are sterols, such as cholesterol. In bacteria, the same function is fi lled by pentacyclic (five-ring) hydrocarbon derivatives called hopanoids, or hopanes. Hopanoids appear in geological sediments, where they indicate ancient bacterial decomposition; they provide useful data for petroleum exploration. Variations in phospholipid side-chain structures reach their extreme in archaea, many of which inhabit the planet’s most extreme environments. All archaeal phospholipids replace the ester link between glycerol and fatty acid
Ester-linked lipids (bacteria and eukaryotes)
OH OH
87
with an ether link, C⫺O⫺C (Fig. 3.15). Ethers are much more stable than esters, which hydrolyze easily in water. Another modification is that archaeal hydrocarbon chains are branched terpenoids, polymeric structures derived from isoprene, in which every fourth carbon extends a methyl branch. The branches strengthen the membrane by limiting movement of the hydrocarbon chains. The most extreme hyperthermophiles, which live beneath the ocean at 110°C, have terpenoid chains linked at the tails, creating a tetraether monolayer. In some
Ether-linked lipids (archaea)
O O
O O
O HO
O Glycerol diester
HO Glycerol diether Diethers condensed here OH O
O
O
O HO Diglycerol tetraether
Figure 3.15 Terpene-derived lipids of archaea. In archaea, the hydrocarbon chains are ether-linked to glycerol, and every fourth carbon has a methyl branch. In some archaea, the tails of the two facing lipids of the bilayer are fused, forming tetraethers; thus, the entire membrane consists of a single layer of molecules (a monolayer).
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Isoprene cyclized to cyclopentane. H2C
O
HC
O
_
O
O
O P O
CH2
OH
O CH
CH2
_
O
CH2
Cyclopentane rings
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Synthesis of terpene-derived lipids. Archaeal lipids are synthesized from isoprene chains. Two isoprene units (five carbons in each) link to form a terpene (ten carbons). Terpenoid multimers, such as the triterpene derivative squalene, generate the lipid chains of archaea, in which most of their double bonds have been hydrogenated. In bacteria, squalene cyclizes to form hopanes and hopanoids, and in eukaryotes, squalene is converted to cholesterol.
Figure 3.16 Isoprene
OH O P
O O
P
O–
O OH Isopentenyl-pyrophosphate
OH O P
O O P
O–
O OH Geranyl-pyrophosphate (terpene) Double length with saturated double bonds Squalene (6 isoprene = tri-terpene)
CH2
OH
O CH O CH2 Branched lipid ether (archaean membranes)
O ■
Squalene epoxide Cyclization
■
H
H
H H
H
HO
H
■
H Cholesterol (eukaryotic membranes)
Hopane (bacterial membranes)
species, the terpenoids cyclize to form cyclopentane rings. These planar rings stiffen the membrane under stress to an even greater extent than the cyclopropyl chains of bacteria. For more on archaeal cells, see Chapter 19. Both cholesterol and hopanoids are synthesized from the same precursor molecules as the unique lipids of archaea (Fig. 3.16). It may be that cholesterol and hopanoids persist in bacteria and eukaryotes as derivatives of lipids once possessed by a common ancestor of bacteria, eukaryotes, and archaea.
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■
Small uncharged molecules, such as oxygen, can penetrate the cell membrane by diffusion. Weak acids and weak bases exist partly in an uncharged form that can diffuse across the membrane and increase or decrease, respectively, the H+ concentration within the cell. Polar molecules and charged molecules require membrane proteins to mediate transport. Such facilitated transport can be active or passive. Active transport requires input of energy from a chemical reaction or from an ion gradient across the membrane. Ion gradients generated by membrane pumps store energy for cell functions. Diverse fatty acids are found in different microbial species and in microbes grown under different environmental conditions. Archaeal membranes have ether-linked terpenoids, which confer increased stability at high temperature and acidity. Some species have cyclized terpenoids, which generate a lipid monolayer.
3.4 TO SU M MAR I Z E: ■
The cell membrane consists of a phospholipid bilayer containing hydrophobic membrane proteins. Membrane proteins serve diverse functions, including transport, cell defense, and cell communication.
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The Cell Wall and Outer Layers
A few prokaryotes, such as the mycoplasmas, have a cell membrane with no outer layers. In most bacteria and archaea, however, the cell envelope includes at least one structural supporting layer outside the cell membrane.
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5 µm
Figure 3.17 The peptidoglycan sacculus. A. Isolated sacculus from Escherichia coli (TEM). B. The structure of the sacculus consists of glycan chains (parallel rings) linked by peptides (arrows). The spaces between links are open, porous to large molecules. C. Sacculus structure resembles the skeleton of a geodesic dome such as that of Epcot Center at Walt Disney World.
J. V. Holtje
A.
C.
The Cell Wall Is a Single Molecule The cell wall confers shape and rigidity to a bacterial cell and helps it withstand the intracellular turgor pressure that can build up as a result of osmotic pressure. The bacterial cell wall, also known as the sacculus, consists of a single interlinked molecule that encloses the entire cell. The sacculus has been isolated from E. coli and visualized by TEM (Fig. 3.17A). In the image shown, the isolated sacculus appears flattened on the sample grid like a deflated balloon. Its geometric structure encloses maximal volume with minimal surface area, analogous to a geodesic dome such as that of Epcot Center. The properties of the cell wall, or sacculus, are largely opposite to those of the membrane: a unimolecular, cage-like structure (Fig. 3.17B), highly porous to ions and small organic molecules. Peptidoglycan structure. Most bacterial cell walls are
composed of peptidoglycan, a polymer of peptide-linked chains of amino sugars. Peptidoglycan is synonymous with murein (“wall molecule”) and consists of parallel polymers of disaccharides cross-linked with peptides of four amino acids, called glycan chains (Fig. 3.18). Peptidoglycan, or murein, is unique to bacteria, although archaea build analogous structures whose overall physical nature is similar. (Archaeal cell wall structures are presented in Chapter 19.) Glycan chains are linked by peptide cross-bridges. The long chains of peptidoglycan consist of repeating units of the disaccharide composed of N-acetylglucosamine (an amino sugar derivative) and N-acetylmuramic acid
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©Kit Kittle/Corbis
The most common structural support is the cell wall. Many species possess additional coverings, such as an outer membrane and capsule.
(glucosamine plus a lactic acid group; see Fig. 3.18). The lactate group of muramic acid forms an amide link with the amino terminus of a short peptide containing four to six amino acid residues. The peptide extension can form cross-bridges connecting parallel strands of glycan. The peptide contains two amino acids in the unusual D mirror form, D-glutamate and D-alanine, a hint that peptidoglycan evolved very early in life’s history, before the bias toward the L form had been established. The third amino acid, m-diaminopimelic acid, has an extra amine group. It is also used by cells as a biosynthetic precursor of lysine. The fourth and sometimes the fi fth amino acids are D-alanine. The second amino group of m-diaminopimelic acid is needed to form a cross-link with the COOH of D-alanine from a neighboring peptide. The cross-link forms by removal of a second D-alanine (fi fth in the chain). The cross-linked peptides of neighboring glycan strands form the cage of the sacculus. The details of peptidoglycan structure vary among bacterial species. Some gram-positive species, such as Staphylococcus aureus (a cause of toxic shock syndrome) have peptides linked by bridges of pentaglycine instead of the D-alanine link to m-diaminopimelic acid. In gramnegative species, the m-diaminopimelic acid is linked to the outer membrane, as discussed shortly.
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N-Acetylglucosamine N-Acetylmuramic acid
H
L-Alanine D-Glutamic
acid m-Diaminopimelic acid D-Alanine D-Alanine M G
C m-Diaminopimelic D-Glutamic D-Alanine acid acid
M
O
O G
G
CH NH
C
C
NH
C
CH
COOH CH3
CH
O
O
OH
O
H
HO
NH C O
N-Acetylglucosamine
CH C
O
O D-Glutamic
acid
H H
CH3
NH
H
H H
CH C
L-Alanine
CH2OH O OH H
CH2OH O H
NH
(CH2)2
NH C
CH3
COOH
C
CH C
CHCOOH H3C
(CH2)3 NH
O
CH3
O
CHCH3
O CH NH
C
CH NH
C
CH3
O
CH2
NH
C
CH3
NH
COOH
CH2
G
CH3
H
L-Alanine
(CH2)2
CH2
COOH
M
M
G
H 3C
H H
O
G
H
NH
H
G
M
O
CH2OH O OH
H
M
M
M
G
H
OH H HO
G
M
CH2OH O
NH
CH
NH C O
Peptide cross-bridge forms with release of D-alanine. Blocked by penicillin.
O m-Diaminopimelic acid
D-Alanine
Cross-bridge formation blocked by vancomycin, which binds D-ala-D-ala and prevents release of terminal D-alanine.
O N-Acetylmuramic acid
Figure 3.18 Peptidoglycan cross-bridge formation. A disaccharide unit of glycan has an attached peptide of four to six amino acids. The amino terminus of the peptide forms an amide bond with the lactate group of muramic acid. On the peptide, the extra amino group of m-diaminopimelic acid can cross-link to the carboxyl terminus of a neighboring peptide. The addition of d-alanine to the peptide is blocked by vancomycin, and the cross-bridge formation by transpeptidase is blocked by penicillin.
Peptidoglycan synthesis as a target for antibiotics.
Synthesis of peptidoglycan requires many genes encoding enzymes to make the special sugars, build the peptides, and seal the cross-bridges. Because peptidoglycan is unique to bacteria, these biosynthetic enzymes make excellent targets for antibiotics (see Fig. 3.18). For example, the transpeptidase that cross links the peptides is the target of penicillin. Vancomycin, a major defense against Clostridium difficile and drug-resistant staphylococci, prevents cross-bridge formation by binding the terminal D-ala-D-ala dipeptide, thus preventing release of the terminal D-alanine. Unfortunately, the widespread use of such antibiotics selects for evolution of resistant strains. One of the most common agents of resistance is the enzyme betalactamase, which cleaves the lactam ring of penicillin, rendering it ineffective as an inhibitor of transpeptidase.
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Strains resistant to vancomycin, on the other hand, contain an altered enzyme that adds lactic acid to the end of the branch peptides in place of the terminal D-alanine. The altered enzyme is no longer blocked by vancomycin. As new forms of drug resistance emerge, researchers continue to seek new antibiotics that target cell wall formation (discussed in Chapter 27).
Gram-Positive and Gram-Negative Bacteria Most bacteria have additional envelope layers that provide structural support and protection from predators and host defenses (Fig. 3.19). Additional molecules are attached to the cell wall and cell membrane, in some cases interpenetrating them. Envelope composition defi nes two major
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Gram-negative
Capsule LPS Porin
LPS
S-layer Teichoic acids
Lipoprotein
Peptidoglycan
Periplasm
Cell membrane
B. Gram-positive (TEM)
C. Gram-negative (TEM)
Capsule
Outer membrane
Peptidoglycan
Peptidoglycan
Inner membrane
Inner membrane
Terry Beveridge
Terry Beveridge
Membrane proteins
Cell envelope: gram-positive and gram-negative. A. The gram-positive cell has a thick cell wall with multiple layers of peptidoglycan, threaded by teichoic acids. The cell wall may be covered by an S-layer. In some gram-positive species, carbohydrate filaments form a capsule. The gram-negative cell has a single layer of peptidoglycan covered by an outer membrane. Some gramnegative species include an S-layer or a capsule (not shown). The cell membrane of gram-negative species is called the inner membrane. B. Gram-positive envelope of Bacillus subtilis (TEM), showing cell membrane, cell wall, and capsule. C. Gram-negative envelope of Pseudomonas aeruginosa (TEM), showing inner membrane, thin cell wall in the periplasm, and outer membrane.
Figure 3.19
categories of bacteria distinguished by the Gram stain (discussed in Chapter 2): ■
■
Gram-positive bacteria have a thick cell wall with multiple layers of peptidoglycan, interpenetrated by teichoic acids. Gram-positive species make up the phylum Firmicutes, such as Bacillus thuringiensis and Streptococcus pyogenes, the cause of “strep throat.” Gram-negative bacteria have a thin cell wall (single layer of peptidoglycan) enclosed by an outer membrane. Gram-negative species make up the phylum Proteobacteria, such as Escherichia coli and nitrogenfi xing rhizobia.
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Outside these two groups, however, many bacterial species do not fit the Gram stain models; consider, for example, the mycobacterial envelope (Special Topic 3.1). Archaeal cell envelopes are highly diverse, and they do not fit the Gram models. THOUGHT QUESTION 3.5 The actual thickness of the cell wall is difficult to determine based solely on electron microscopic observation of the envelope layers. Devise a biochemical experiment that can show the number of layers of peptidoglycan in cells of a given species.
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The Unique Cell Envelope of Mycobacteria
Cell envelopes of exceptional complexity are found in mycobacteria, such as M. tuberculosis and M. leprae, the causative agents of tuberculosis and leprosy, respectively. Some features of mycobacterial cell walls also appear in actinomycetes, a large and diverse family of soil bacteria, many of which produce antibiotics and other industrially useful products.
Figure 1 shows two unusual classes of lipids found in mycobacteria. Mycolic acids contain a hydroxy acid backbone with two hydrocarbon chains, one comparable in length to typical membrane lipids (about 20 carbons), the other about threefold longer. The long chain includes ketones, methoxyl groups, and cyclopropane rings. Only one type of mycolic acid is shown; hundreds of different forms are known. The phenolic glycolipids include a phePhenolic glycolipids nol group, also linked to sugar chains. OH The mycobacterial cell wall includes pepCH3 H tidoglycan linked to chains of galactose, called H HO H OH galactans (Fig. 2). The galactans are attached CH3 O H H to arabinans, polymers of the five-carbon sugar O H H arabinose. The arabinan-galactan polymers are H H known as arabinogalactans. Arabinogalactan O O biosynthesis can be inhibited by ethambutol, a O H3C H major drug against tuberculosis. Mycolic acid Phenol H derivative The ends of the arabinan chains form ester CH3 links to mycolic acids. Mycolic acids provide (CH2)17 the basis for acid-fast staining, in which cells H3C O Ketone retain the dye carbolfuchsin, an important diagnostic test for mycobacteria and actinomycetes (CH2)15 (shown in Chapter 2). In M. tuberculosis and Cyclopropane cis M. leprae, the mycolic acids form a kind of bilayer interleaved with phenolic glycolipids. Long chain with The extreme hydrophobicity of the phenol derivcyclopropanes, atives generates a waxy surface that prevents ketones, and methoxyl HC O phagocytosis by macrophages. branches Overall, the thick, waxy envelope excludes C O HC O many antibiotics and offers exceptional protecC O tion from host defenses, enabling the pathogens of tuberculosis and leprosy to colonize their cis hosts over long periods. However, the mycobacH3CO terial envelope also retards uptake of nutrients. As a result, M. tuberculosis and M. leprae grow extremely slowly and are a challenge to culture in the laboratory. Short chain
Mycobacterial envelope lipids. Mycobacterium tuberculosis possesses unusual envelope components, such as mycolic acids and phenolic glycolipids. These highly hydrophobic molecules form a waxy outer coating that protects cells from host defenses.
Figure 1
HO HOOC
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Phenolic glycolipid
Phospholipid
Capsule
Sugar mycolate
The capsule consists of glycolipids and sugar mycolates.
A section of a gram-positive cell envelope is shown in Figure 3.19. The gram-positive cell wall consists of multiple layers of peptidoglycan, up to 40 in some species. No space appears between the peptidoglycan layers, although the outer band of envelope appears more dense. The peptidoglycan is reinforced by teichoic acids threaded through its multiple layers (Fig. 3.20). Teichoic acids are chains of phosphodiester-linked glycerol or ribitol, with sugars or amino acids linked to the middle OH groups. The negatively charged cross-threads of teichoic acids, as well as the overall thickness of the gram-positive cell wall, help retain the Gram stain. Outside the cell wall, gram-positive cells are often encased in a slippery capsule consisting of loosely bound polysaccharides. The capsule can be visualized under the light microscope by using a negative stain consisting of suspended particles of India ink, which the capsule excludes (shown in Fig. 2.24C).
HO P O
Mycolic acids (attached to arabinan)
CH2
Cell wall core Ethambutol blocks synthesis.
O
H C
O
R
H2C
O
R = D-Ala, D-Lys, or sugar
HO P
Arabinan O
O
CH2 Galactan Peptidoglycan
H C
O
H2C
O
R
HO Cell membrane
P O
O
CH2
Mycobacterial envelope structure. A complex cell wall includes a peptidoglycan layer linked to a chain of galactose polymer (galactan) and arabinose polymer (arabinan). Arabinan forms ester links to mycolic acids, which form an outer bilayer with phenolic glycolipids. Outside the outer bilayer is a capsule of loosely associated phospholipids and phenolic glycolipids.
Figure 2
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H C
O
H2C
O
R
Glycerol teichoic acid
Figure 3.20 Teichoic acids. Teichoic acids in the grampositive cell wall consist of glycerol or ribitol phosphodiester chains. The middle hydroxyl group of each glycerol or ribitol is typically linked to d-alanine, to d-lysine, or to a sugar such as galactose or N-acetylglucosamine.
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The S-layer. An additional protective layer commonly
THOUGHT QUESTION 3.6 Why would laboratory culture conditions select for evolution of cells lacking an S-layer?
found in gram-positive cells, as well as in archaea, is the surface layer, or S-layer. The S-layer is a crystalline layer of thick subunits consisting of protein or glycoprotein (proteins with attached sugars) (Fig. 3.21). S-layers are found in most archaea, as well as in many gram-positive and gram-negative bacteria freshly isolated from natural sources. Each subunit of the S-layer contains a pore large enough to admit a wide range of molecules. The subunits form a smooth layer on the cell wall or outer membrane (see Fig. 3.21). The proteins are arranged in a highly ordered array, either hexagonally or tetragonally. They present a formidable physical barrier to predators or parasites. The S-layer is rigid, but it also flexes and allows substances to pass through it in either direction. If S-layers are common, why were they observed only recently? Bacterial cell structures are generally studied in laboratory cultures. Upon repeated subculturing, however, bacteria lose the genetic ability to synthesize S-layer proteins. Genetic loss of a structure not required in laboratory culture, resulting from accumulation of deleterious mutations, is an example of reductive evolution, also known as degenerative evolution. Loss of a trait occurs in the absence of selective pressure for genes encoding the trait (discussed in Chapter 17). For example, the mycoplasmas are close relatives of gram-positive bacteria, yet they have permanently lost their cell walls as well as the S-layer. Mycoplasmas have no need for cell walls because they are parasites living in host environments, such as the human lung, where they are protected from osmotic shock.
Figure 3.21 The S-layer. The archaeon Thermoproteus tenax has a single tetraether membrane encased by an S-layer (SEM). Note the regular pattern of tiled proteins.
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The Gram-Negative Outer Membrane A gram-negative cell envelope is seen in Fig. 3.19C. The thin layer of peptidoglycan is believed to be a single sheet based on calculations of molecular density. The peptidoglycan is covered by an outer membrane. The gramnegative outer membrane confers defensive abilities and toxigenic properties on many pathogens, such as Salmonella species and enterohemorrhagic E. coli (strains that cause hemorrhaging of the colon). Between the outer and inner (cell) membranes is the periplasm. and lipopolysaccharide (LPS). The inward-facing leaflet of the outer membrane has a phospholipid composition similar to that of the cell membrane (in gram-negative species, it is called the inner membrane or inner cell membrane). The outer membrane’s inward-facing leaflet includes lipoproteins that connect the outer membrane to the peptide bridges of the cell wall. The major lipoprotein is called murein lipoprotein, also known as Braun lipoprotein (Fig. 3.22A). Murein lipoprotein consists of a protein with an N-terminal cysteine triglyceride inserted in the inward-facing leaflet of the outer membrane. The C-terminal lysine forms a peptide bond with the m-diaminopimelic acid of peptidoglycan (murein). The outward-facing leaflet, however, consists of a special kind of phospholipid called lipopolysaccharide (Fig. 3.22B). The LPS lipids have shorter fatty acid chains than those of the inner cell membrane, and some are branched. LPS is of crucial medical importance because it acts as an endotoxin. An endotoxin is a cell component that is harmless so long as the pathogen remains intact, but when released by a lysed cell, endotoxin overstimulates host defenses, inducing potentially lethal endotoxic shock. Thus, antibiotic treatment of an LPS-containing pathogen can kill the cells but can also lead to death of the patient. In LPS the fatty acids are esterified to glucosamine, an amino sugar found also in peptidoglycan. Two glucosamine dimers each condense with two fatty acid chains, which makes four fatty acid chains in all. Like the glycerol of phospholipid, each glucosamine of LPS connects to a phosphate, whose negative charge interacts with water.
Lipoproteins
Paul Messner, et al. 1986. Journal of Bacteriology 166(3):1046
100 nm
THOUGHT QUESTION 3.7 Suppose that one cell out of a million has a mutant gene blocking S-layer synthesis, and suppose that the mutant strain can grow twice as fast as the S-layered parent. How many generations would it take for the mutant strain to constitute 90% of the population?
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Lipoprotein and lipopolysaccharide. A. Murein lipoprotein consists of a protein with an N-terminal cysteine triglyceride inserted in the inwardfacing leaflet of the outer membrane. The protein’s C-terminal lysine forms a peptide bond with the mdiaminopimelic acid of the peptidoglycan (murein) cell wall. B. Lipopolysaccharide (LPS) consists of branched and unbranched short-chain fatty acids linked to a dimer of phosphoglucosamine (also called disaccharide diphosphate). The disaccharide is linked to a core polysaccharide extending out from the cell, which is attached to about 40 repeating units of a distinct polysaccharide known as O antigen.
Figure 3.22
One of the glucosamines is attached to a core polysaccharide, a sugar chain that extends outside the cell. The core polysaccharide consists of about five sugars with side chains such as phosphoethanolamine. It extends to the O polysaccharides, a chain of as many as 200 sugars. The O polysaccharide may extend longer than the cell itself. These polysaccharide chains form a layer that helps bacteria resist phagocytosis by white blood cells.
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A. Gram-negative envelope
LPS
Outer membrane
R1
R2
R3
O
O
NH
CH2
CH
CH2
S
Terminal D-Ala removed
Cys
Murein lipoprotein (aa)65 Lys
D-Ala
NH2
m-A2pm
COOH
Peptidoglycan
NH2
D-Glu L-Ala
GlcNAc
MurNAc
GlcNAc
MurNAc
Proteins of the outer membrane. The outer mem-
brane also contains unique proteins not found in the inner membrane. The various layers of the cell envelope need to maintain distinct identities and properties that distinguish them from each other and from the cytoplasm and periplasm. The composition of the inner and outer membranes and aqueous compartments is characterized by ultracentrifugation. Fractions containing various cell components show that each protein is confi ned to a specific component of the cell (Table 3.2). For example, the proton-translocating ATPase is found
Cytoplasmic membrane
B. Lipopolysaccharide (LPS)
man abe O-polysaccharide repeat 40 units
rha
gal
Table 3.2 Subcellular location of proteins in gram-negative bacteria. Cell fraction
Proteins (examples)
Cytoplasm
Glycolytic enzymes Biosynthesis of amino acids Cytoplasmic chaperones Proton-translocating ATPase Electron transport chain Porins for water and glycerol Sugar taxis receptors Periplasmic chaperones Porins for sugars and peptides Sugar-binding proteins
gln
P O
Inner membrane (cell membrane) Periplasm (periplasmic space) Outer membrane
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NH
Core polysaccharide gln P Glucosamine O phosphate dimer NH
Lipid A Fatty acids
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only in the inner membrane fractions, whereas sugarbinding proteins are only in the outer membrane. THOUGHT QUESTION 3.8 Why would the proteins listed in Table 3.2 be confined to specific cell fractions? Why could a protein not function throughout the cell? Outer membrane proteins of pathogens are important targets for vaccine development. For example, the outer membrane proteins were characterized for Borrelia burgdorferi, the cause of Lyme disease. In the experiment shown in Figure 3.23, fractions of Borrelia outer membrane are separated from whole cells by density gradient centrifugation. The sample is located on a tube of sucrose solution whose concentration (and density) increases with depth in the tube. Within the gradient, suspended fragments of membrane migrate to the position where their density equals that of the solution. The outer membrane fractions collect at a density different from that of fractions containing unbroken cells. The gradient contents are then removed sequentially through an opening at the base of the tube. Once isolated, the outer membrane components are separated by gel electrophoresis to reveal distinctive proteins that may serve as vaccine targets. NOTE: Another type of gradient centrifugation, called equilibrium density gradient centrifugation, uses a cesium chloride solution that forms a gradient when subjected to centrifugal force. Cesium chloride gradients are used most commonly to separate DNA molecules such as plasmids (discussed in Chapter 7).
Outer membranes contain a class of transporters called porins that permit entry of nutrients such as sugars and peptides. Outer membrane porins have a distinctive cylinder of beta sheets, also known as a beta barrel. A typical outer membrane porin exists as a trimer of beta barrels, each of which acts as a pore for nutrients. Figure 3.24 shows a model of the sucrose porin based on a crystal structure. Cells express different outer membrane porins under different environmental conditions. In a dilute environment, cells express porins of large pore size, maximizing the uptake of nutrients. In a rich environment—for example, within a host—cells downregulate expression of large porins and express porins of smaller pore size, selecting only smaller nutrients and avoiding the uptake of toxins. For example, the porin regulation system of gram-negative bacteria enables them to grow in the intestinal region containing bile salts—a hostile environment for grampositive bacteria. Periplasm. The outer membrane is porous to most ions and many small organic molecules, but it prevents passage of proteins and other macromolecules. Thus, the region between the inner and outer membranes of gramnegative cells, including the cell wall, defi nes a separate membrane-bounded compartment of the cell known as the periplasm (see Fig. 3.19). The periplasm contains specific enzymes and nutrient transporters not found within the cytoplasm, such as periplasmic binding protein for sugars, amino acids, or other nutrients. Periplasmic proteins are subjected to fluctuations in pH and salt concentration, because the outer membrane is porous to ions.
Low-density component
Steep sucrose gradient (e.g., 20–70%)
The sample is loaded on top of the sucrose density gradient.
From centrifuge
0.5 µm
Centrifugation
High-density component
Components have migrated to a region in the gradient that matches their own density. 0.5 µm
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J. D. Radolf, et al. 1995. Infection and Immunity 63:2154
To centrifuge
Outer membrane analysis by centrifugation. Borrelia burgdorferi outer membrane vesicles are separated from the cell by equilibrium density gradient ultracentrifugation. The lighter fractions contain outer membrane vesicles, whereas the denser fractions contain unbroken whole cells (inset images, freeze-fracture TEM).
Figure 3.23
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Figure 3.24 Sucrose porin. Outer membrane porin (OMP) for sucrose transport in Salmonella typhimurium (stereo view) based on X-ray crystallography. The porin comprises three beta barrel channels (red, yellow, and blue). Each transports a molecule of sucrose through its channel. To view, place a piece of cardboard vertically between the two images. Position your eyes on opposite sides of the cardboard and force them to focus behind the images. The images will merge and produce a 3-D image. (PDB code: 1A0S)
possess a contractile vacuole to pump water out of the cell, avoiding osmotic shock (Fig. 3.25). The vacuole takes up water from the cytoplasm through an elaborate network of intracellular channels, and then expels the water through a pore.
Eukaryotic Microbes: Protection from Osmotic Shock The problems confronting bacteria, such as adjusting to environmental change, also challenge eukaryotes. A key example is osmotic shock. Eukaryotic microbes possess their own structures to avoid osmotic shock. Algae form cell walls of cellulose fibers, and fungi form walls of chitin. Diatoms form intricate exoskeletons of silicate. Other eukaryotes, however, such as amebas and ciliated protists, possess a flexible outer coating, the pellicle. The pellicle allows great flexibility of shape; it enables uptake of large particles by endocytosis and of larger objects, even entire cells, by phagocytosis. Thus, eukaryotic cells have the nutritional option of engulfi ng prey, an option unavailable to prokaryotes and to eukaryotes with cell walls. Eukaryotic microbes that lack a cell wall A.
THOUGHT QUESTION 3.9 What do you think are the advantages and disadvantages of a contractile vacuole, compared with a cell wall?
TO SU M MAR I Z E: ■
■
The cell wall maintains turgor pressure. The cell wall is porous, but its rigid network of covalent bonds protects the cell from osmotic shock. The gram-positive cell envelope has multiple layers of peptidoglycan, interpenetrated by teichoic acids.
B.
Pore for water expulsion
©Michael Abbey/Visuals Unlimited
Contractile vacuoles
Microtubules for stabilizing position
Ampulla for collecting water
Vacuole filled with water Vacuole after water has been expelled
Protection from osmotic shock in eukaryotic microbes. A. A paramecium, showing two contractile vacuoles (phase-contrast micrograph). The inset images show radiating channels that collect water and expel it from the cytoplasm. B. Diagram of a contractile vacuole.
Figure 3.25
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The S-layer, composed of proteins, is highly porous, but can prevent phagocytosis and phage infection. In archaea, the S-layer serves the structural function of a cell wall. The capsule, composed of polysaccharide and glycoprotein filaments, protects cells from phagocytosis. Either gram-positive or gram-negative cells may possess a capsule. The gram-negative outer membrane regulates nutrient uptake and excludes toxins. The envelope layers include protein pores and transporters of varying selectivity. Eukaryotic microbes are protected from osmotic shock by polysaccharide cell walls or by a contractile vacuole.
3.5
The Nucleoid and Gene Expression
An important function of the cell envelope is to contain and protect the cell’s genome. Figure 3.26 compares the organization of chromosomal material in enteropathogenic E. coli cells with that in a cultured human cell that they have colonized. In this thin-section TEM, each bacterium contains a fi lamentous nucleoid region that extends through the cytoplasm. In contrast, the nucleus of the eukaryotic cell, only a fraction of which
Cell membrane Endoplasmic reticulum Nuclear membrane
is visible in the figure, is many times larger than the entire bacterial cell, and the chromosomes it contains are separated from the cytoplasm by the nuclear membrane.
In Prokaryotes, DNA Is Organized in the Nucleoid All living cells on Earth possess chromosomes consisting of DNA. The genetic functions of microbial DNA are discussed in detail in Chapters 7–12. Here we focus on the physical organization of DNA within the nucleoid of the prokaryotic cell (Fig. 3.27). NOTE: The prokaryotic genome typically consists of a single circular chromosome, but some species have a linear chromosome or multiple chromosomes. In this chapter, we focus on the simple case of a single circular chromosome.
In a bacterial cell, the DNA is organized in loops called domains. All domains connect back to a central point. The central point is the origin of replication (ori), which is attached to the cell envelope at a point on the cell’s “equator,” halfway between the two poles (Fig. 3.28). DNA is replicated by DNA polymerase; the process
Human cell
Nucleoid
Bacterial nucleoid and eukaryotic nucleus. Enteropathogenic Escherichia coli bacteria attaching to the surface of a tissue-cultured human cell (TEM). The human cell has a well-defined nucleus delimited by a nuclear membrane, whereas the E. coli cells have nuclear material more loosely arranged in the nucleoid region. E. coli cells are 1.0–2.0 µm in length.
Figure 3.26
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0.25 µm
©Dennis Kunkel/Visuals Unlimited
R. P. Rabinowitz, et al.
Envelope Cytoplasm E. coli Nucleoid
The nucleoid. The E. coli nucleoid appears as clear regions that exclude the dark-staining ribosomes and contain DNA strands (colorized orange in TEM).
Figure 3.27
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In bacteria, as in eukaryotic cells, the direction of the extra turns opposes the natural twist of the duplex. Turns that oppose the natural twist are called negative superhelical turns. Negative superhelical turns actually unwind the DNA slightly and help expose the base pairs for transcription. In archaea, however, most supercoiling is in the positive direction, tending to wind the DNA more tightly and therefore increase stability of the duplex. The increased stability of positive supercoils is an advantage for archaea growing in extreme environments, such as temperatures above 100°C.
DNA origin
DNA-binding proteins. Prokaryotic DNA is condensed DNA-binding protein
DNA domain
Figure 3.28 Nucleoid organization. The nucleoid forms approximately 50 loops of chromosome called domains, which radiate from the center (shown shaded). Within each domain, the DNA is supercoiled and partly compacted by DNA-binding proteins (shown in green).
is covered in more detail in Chapter 7. At the origin, the DNA double helix begins to unzip, and replication proceeds outward in both directions. Growing cells replicate their DNA continuously, with no resting phase; in fact, the two replicated origins may split again to begin a second round of replication before the fi rst is completed. NOTE: In biology, the word domain is used in several
different ways, each referring to a defi ned portion of a larger entity. ■ DNA domains of the nucleoid are distinct loops of DNA that extend from the origin. ■ Protein domains are distinct functional or structural regions of a protein. ■ Taxonomic domains are genetically distinct classes of organisms, such as bacteria, archaea, and eukaryotes. How does all the cell’s DNA fit neatly into the nucleoid? The DNA is compacted over several levels by supercoiling and by DNA-binding proteins. Supercoiling. The chromosome includes a number of
extra twists, called supercoils or superhelical turns, beyond those inherent in the structure of the DNA duplex (double helix). The supercoiling causes portions of DNA to double back and twist upon itself, which results in compaction. Supercoiling is generated by enzymes such as gyrase and maintained by DNA-binding proteins (shown in green in Fig. 3.28). The enzyme gyrase is a major target for antibiotics such as quinolones, used, for example, to treat bacterial pneumonia and urinary tract infections.
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by winding around various classes of binding proteins (see Fig. 3.28). Some binding proteins, such as Hns and Hu, also function as regulators of gene expression. Binding proteins can respond to the state of the cell; for example, under starvation conditions, when most transcription of RNA ceases, the binding protein Dps is used to organize the DNA into a protected crystalline structure. Such “biocrystallization” by Dps and related proteins may be a key to the extraordinary ability of microbes to remain viable for long periods in stationary phase or as endospores.
Transcription and Translation Are Tightly Coupled The information encoded in DNA is “read” by the processes of transcription and translation to yield gene products. Within the prokaryotic cell, DNA transcription to RNA is coupled tightly to RNA translation to proteins. DNA transcription to RNA. The initial product of a gene
is a single strand of ribonucleic acid (RNA), produced when DNA is transcribed by RNA polymerase (Fig. 3.29). In some cases, the newly made RNA has a function of its own, such as one of the RNA components of a ribosome, the cell’s protein-making machine. For most genes, however, the RNA is a messenger RNA (mRNA)
Rifampin blocks RNA synthesis.
RNA polymerase
Opening to secondary channel for ATP, GTP, CTP, UTP Nascent RNA DNA
DNA transcription to RNA. The DNA information is transcribed into a single strand of RNA.
Figure 3.29
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replicated. This remarkable coordination of replication, transcription, Erythromycin blocks peptide elongation by and translation is a hallmark of the binding 50S subunit. Peptide exit prokaryotic cell; it explains why Transfer RNA channel Chaperone some bacteria can double in as little (GroEL-GroES) as 10 minutes. Tetracycline blocks tRNA As peptide chains are syntheby binding Incoming sized, some kinds bind to chap50S Correctly 30S subunit. amino acid folded protein erones, enzyme complexes that The new peptide needs help from a chaperone 30S help fold the new peptide into its Ribosome to fold properly. functional tertiary structure (see Fig. 3.30). Chaperones were fi rst discovered in E. coli as “heat-shock RNA spontaneously proteins,” expressed at high temforms hairpin loops. perature to protect proteins from denaturation by heat. Since then, Figure 3.30 Translation and protein folding. Messenger RNA (mRNA) is translated chaperones have been discovered into a peptide by the ribosome. Nascent polypeptides are folded into their proper in all organisms, including humans, conformation, assisted by chaperones. where chaperone defects are associated with genetic diseases. that immediately binds to a ribosome for translation to In eukaryotes, note that transcription and translation generate a polypeptide, a linear sequence of amino acid occur only during interphase, when the cell is not underresidues (discussed in Chapter 8). going division. Within the nucleus, nascent (elongating) mRNA is translated by ribosomes, which check the transcript for errors and target faulty transcripts for destrucmRNA translation to protein. A growing bacterial cell tion. The majority of eukaryotic translation, however, typically invests 40% of its energy in translation of mRNA occurs outside the nucleus, in the cytoplasm. by ribosomes. The ribosome, with its large number of protein and RNA components, is probably the most complex subcellular machine to be discovered (Fig. 3.30). Its Compaction Expansion task of converting the four-letter RNA code into 20 amino acids requires the assistance of 27 different transfer RNAs DNA binding Ribosome SRP Protein RNA Origin proteins (tRNAs) and an equal number of aminoacyltransferase enzymes, each encoded by a different gene. Each amino acid is brought by a tRNA to fit into the acceptor site within the ribosome, sandwiched between the 30S and 50S subunits. The amino acid is then transferred onto a peptide chain, where the peptide bond forms. The process of translation is discussed further in Chapter 8. Because of its complexity, the ribosome is targeted by many kinds of antibiotics. For example, tetracyclines block aminoacyl-tRNA from arriving at the 30S subunit. Erythromycin blocks peptide elongation at the 50S subunit. Coupling of transcription and translation. In prokary-
otes, translation is tightly coupled to transcription; the ribosomes bind to mRNA and begin translation even before the mRNA strand is complete. Thus, a growing bacterial cell is full of mRNA strands dotted with ribosomes. These composite mRNA-ribosome structures are known as polysomes. Polysomes can be observed as the mRNA elongates, attached by RNA polymerase to the DNA strand (Fig. 3.31). In rapidly growing bacteria, both transcription and translation occur at top speed while the DNA itself is being
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Polysome
Figure 3.31 Protein synthesis and secretion. Bacterial transcription of DNA to RNA is coordinated with translation of RNA to make proteins. Even before the RNA chain is complete, ribosomes bind to commence translation. Growing peptides destined for the membrane bind the signal recognition particle (SRP) for insertion into the membrane. The DNA is compacted by binding proteins (shown in green), but it is also pulled outward by the nascent chains of RNA (blue) and membraneinserted peptides (orange). Each mRNA is translated by multiple ribosomes, called a polysome (highlighted yellow).
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Inserting proteins in the membrane. Some of the
newly translated proteins are destined for the membrane or for secretion outside the cell. Proteins destined for membrane insertion must be hydrophobic and hence are poorly soluble in the aqueous cytoplasm where translation occurs. How can proteins that are insoluble in water be folded correctly in the cytoplasm? In prokaryotes, membrane and secreted proteins are synthesized in association with the cell membrane (Fig. 3.31). Protein secretion in prokaryotes is managed by several different classes of protein complexes, many of which export toxins and virulence factors, as discussed in Chapter 8. One major secretion system relies on a signal sequence, a short N-terminal sequence rich in hydrophobic residues that heads the coding sequence of a growing polypeptide. As translation begins, the signal sequence binds to a signal recognition particle (SRP), an RNA-protein complex, plus other helper proteins. The SRP complex transfers the polypeptide-ribosome-mRNA complex bearing the signal sequence to the secretory complex within the cell membrane. Translation then continues at the cell membrane, and as it does so, the waterinsoluble protein progressively translocates into or across the membrane. In eukaryotes, membrane proteins are synthesized at the surface of the endoplasmic reticulum (ER). From the ER, eukaryotic proteins are packaged within the Golgi complex for transport to the cell membrane.
3.6
Cell Division
Cell division, or cell fission, requires highly coordinated growth and expansion of all the cell’s parts. Unlike eukaryotes, prokaryotes synthesize RNA and proteins continually while the cell’s DNA undergoes replication. Bacterial DNA replication is coordinated with the expansion of the cell wall and ultimately the separation of the cell into two daughter cells. The DNA replication process is outlined here as it relates to cell division; molecular details are discussed in Chapter 7.
DNA Is Replicated Bidirectionally In prokaryotes, a circular chromosome begins to replicate at its origin, or ori site, a sequence of base pairs on the genetic map. Replication proceeds in both directions (bidirectionally) all around the circle. Some prokaryotes have linear chromosomes, but their replication is poorly understood. At the origin, the DNA double helix begins to unzip, forming two replication forks. At each replication fork, DNA is synthesized by DNA polymerase (Fig. 3.32). The replication fork is propagated by helicase, which unwinds the DNA helix ahead of replication, and pri-
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Leading strand 5′ 3′
5′ 3′ DNA polymerase
Primase RNA primer
Replisome
Helicase
RNA primer
DNA replication
ssDNA-binding proteins 5′ 3′
Figure 3.32 DNA replication. DNA replication proceeds by unzipping the helix and synthesizing a complement for each strand. The replisome consists of two DNA polymerase complexes, one of which synthesizes the leading strand, while the other synthesizes the lagging strand. Further details are discussed in Chapter 7.
mase, which generates RNA primers. The complex of DNA polymerase with its accessory components is called a replisome. The replisome actually includes two DNA polymerase enzymes, one to replicate the “leading strand,” the other for the “lagging strand.” The actual lag time is short compared with the overall time of replication; thus, as the replisome travels along the DNA, it converts one helix into two progeny helices almost simultaneously. NOTE: Each replisome contains two DNA polymerase complexes (for leading and lagging strands), and each dividing nucleoid requires two replisomes (for bidirectional replication), thus four DNA polymerase complexes overall.
Within the cell, replication proceeds outward in both directions around the genome. Thus, bidirectional replication requires two replisomes, one for each replicating fork. A long-standing question has been: Do the two replisomes move oppositely around the DNA, or do they stay in the middle while the DNA helices slide through them?
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Origin of replication
Courtesy of Melanie Berkmen, MIT
A.
Two replisomes
Terminator sequence Origin of replication DNA origin replicates and migrates.
Pol-YFP Ori-CFP
2 µm
The replisome and the DNA origin. A. Melanie Berkmen, working in the laboratory of Alan Grossman, obtains the fluorescence micrographs shown. B. Fluorescence microscopy reveals the DNA origin, labeled blue by a protein fused to cyan fluorescent protein, binding at a sequence near the origin (Ori-CFP). Replisomes are labeled yellow by fusion of a DNA polymerase subunit to yellow fluorescent protein (Pol-YFP) in dividing cells of Bacillus subtilis. The cell envelope is labeled red with the membrane stain FM4-64.
Courtesy of Melanie Berkmen, MIT
B. Terminator sequence DNA replication continues bidirectionally.
Figure 3.33
DNA starts next round.
Septum forms.
To answer this question, fluorescence microscopy is used to observe the process of DNA replication within a growing cell of Bacillus subtilis (Figure 3.33). The DNA origin of replication (ori) and the pair of replisomes are labeled by fluorescence. The origin of replication is labeled blue by a hybrid protein fused to a gene encoding cyan fluorescent protein (CFP). The CFP protein binds to a promoter sequence cloned in B. subtilis near its origin site. The pair of replisomes are labeled yellow by a hybrid protein expressed from a gene encoding a DNA polymerase subunit fused to a gene encoding yellow fluorescent protein (YFP). The replisomes usually locate together near the center of the cell, but sometimes they separate, visible as two yellow spots. The fluorescence data are consistent with a model in which two replisomes are located at the midpoint of the growing cell (Fig. 3.34 ). Each of the two replisomes forms a replicating fork that directs two daughter strands of DNA toward opposite poles. The two copies of the DNA origin of replication (green), attached to the cell envelope, move apart as the cell expands. The termination site (red) remains in the middle of the cell. The two replisomes continue replication at both forks in the middle of the cell. Finally, as the termination site replicates, the two replisomes separate from the DNA. At each new ori site, however, two pairs of new replisomes
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Division into two cells
Replisome movement within a dividing cell. The DNA origin of replication sites (green) move apart in the expanding cell as the pair of replisomes (yellow) stay near the middle, where they replicate around the entire chromosome, completing the terminator sequence last (red). Source: Ivy Lau, et al.
Figure 3.34
2003. Molecular Microbiology 49:731.
have formed. The replication of the new ori sites begins, sometimes even before termination of the previous round of replication. Note that the contents of the cytoplasm must expand coordinately with DNA replication for the cell to generate progeny equivalent to the parent. In a rod-shaped
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Ahmed Touhami, et al. 2004. J. Bacteriol. 186
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Septation in Staphylococcus aureus. A. Furrows appear in the cell envelope, all around the cell equator, as new cell wall grows inward (TEM). B. Two new envelope partitions are complete. C. The two daughter cells peel apart. The facing halves of each cell contain entirely new cell wall.
Figure 3.35
For the cell to divide, DNA replication must be complete. Replication of the DNA termination site triggers growth of the dividing partition of the envelope, called the septum. The septum grows inward from the sides of the cell, at last constricting and sealing off the two daughter cells. This process is called septation. Septation and envelope extention require rapid biosynthesis of all envelope components, including membranes and cell wall. The biosynthetic enzymes required are all of great interest as antibiotic targets. Cell wall biosynthesis poses an interesting theoretical problem: How is it possible to expand the covalent network of the sacculus without breaking links to insert new material, thus weakening the wall? The answer remains unclear. Septation of spherical cells. In spherical cells (cocci), such as Staphylococcus aureus, the process of septation generates most of the new cell envelope to enclose the expanding cytoplasm (Fig. 3.35). Furrows form in the cell envelope, in a ring all around the cell equator, as new cell wall grows inward. The wall material must compose two separable partitions. When the partitions are complete, the two progeny cells peel apart. The facing halves of each cell consist of entirely new cell wall. The spatial orientation of septation has a key role in determining the shape and arrangement of cocci (Fig. 3.36A). When septation always occurs in parallel planes, such as in Streptococcus species, cells form chains. If septation occurs in random orientations, or if cells reassociate loosely after septation, they form compact hexagonal arrays similar to the grape clusters portrayed in classical paintings; hence the Greek-derived term staphylococci.
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A. Eye of Science/Photo Researchers, Inc.
Septation Completes Cell Division
Such clusters are found in colonies of Staphylococcus aureus. If subsequent septation occurs at right angles to the previous division, the cells may form tetrads and even cubical octads called sarcinae. Tetrads and sarcinae are formed by Micrococcus species (Fig. 3.36B).
B.
Kwangshin Kim/Photo Researchers, Inc.
cell, the cell envelope and cell wall must elongate as well to maintain progeny of even girth and length.
Septation orientation determines the arrangement of progeny cells. A. A chain of cocci results from septation in the same plane (Streptococcus, colorized TEM). B. Septation in two planes generates a tetrad (Micrococcus tetragenus, negative stain).
Figure 3.36
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Septation of rod-shaped cells. In rod-shaped cells,
unlike cocci, cell division requires the envelope to elongate before septation, followed by formation of a new polar envelope for each progeny cell. The process of septation involves an intricate series of molecular signals that is at the frontier of current research. Certain mutant strains of E. coli form long filaments instead of dividing normally. This fi lamentation results from a failure to form a septum between cells. The mutant genes responsible for this behavior were called fts for “fila-
Z-ring
mentation temperature sensitive” because the cells divide normally at the permissive temperature but fail to septate at the restrictive temperature, forming long filaments. Some of the fts genes encode proteins directly involved in the formation of the septum. The most dramatic example is the protein FtsZ, which assembles to form the “Z ring,” a constriction ring around the equator (Fig. 3.37). FtsZ is universally found in bacteria and archaea as a key septation protein. FtsZ is also an ancient homolog of tubulin, which is the major component of the mitotic apparatus in eukaryotes. The discovery of FtsZ is interesting because it implies that the processes of mitosis in eukaryotes and cell division in prokaryotes might have evolved from a common process in an ancestral cell. Other bacterial homologs of eukaryotic structural proteins suggest that bacteria possess a kind of cytoskeleton beneath the cell wall (Special Topic 3.2). What signals the cell as to when and where to form the septum? What keeps the cell from septating at positions other than the equator? In fact, defective gene products can lead cells to divide incorrectly with a septum near the pole of the cell. This incorrect septation generates a “minicell,” a small cellular compartment with no DNA. Genes whose defects lead to minicells are called min genes. In E. coli, the protein encoded by minD oscillates in rings from pole to pole (Fig. 3.38). The Min proteins somehow regulate the formation of the septum, ensuring symmetrical cell division. Note, however, that normal division of bacteria is not always symmetrical. For example, Bacillus species undergo an asymmetrical cell division to form an endospore. In this process, one daughter nucleoid forms an inert endospore capable of remaining dormant but viable for many years (discussed in Chapter 4). Other bacteria expand their cells by polar extension; an example is Corynebacterium diphtheriae, the causative agent of diphtheria. Polar extension occurs at variable rates and direction, and thus generates irregularly shaped rods.
Septation and the Z ring. Fluorescence microscopy of E. coli based on FtsZ-GFP, a genetic fusion of FtsZ with green fluorescent protein (GFP). The Z ring of FtsZ subunits forms around the equator of the constricting cell, as the septum grows inward.
Figure 3.37
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B. 30 seconds
Yu-Ling Shih, et al. 2003. PNAS 100:7856
New z-rings
Q. Sun and W. Margolin. 1998. J. Bacteriol.
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Yu-Ling Shih, et al. 2003. PNAS 100:7856
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MinD protein rings regulate cell division. Fluorescence shows MinD-YFP protein rings in an E. coli cell completing division. The rings oscillate from pole to pole (times 0 to 30 s shown).
Figure 3.38
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Bacteria Have a Cytoskeleton
A major distinction of prokaryotes was thought to be the lack of a cytoskeleton, a structural feature prominent in eukaryotes. Bacterial shape was thought to be maintained by the cell wall. In fact, bacterial shape and cell division require cytoskeletal proteins homologous to eukaryotic cytoskeletal components such as tubulin and actin. The first such protein recognized was the Z ring subunit FtsZ, an analog of tubulin. Further components of the bacterial cytoskeleton were revealed by several means: gene defects that confer the loss of cell shape, fluorescent labeling of the corresponding gene products in wild-type cells, and genomic analysis showing bacterial homologues of eukaryotic cytoskeletal components. An example of a “shape loss” mutant is shown in Figure 1. The bacterium Shigella is an enteric pathogen closely related to E. coli that normally grows as a rod, or bacillus (see Fig. 1A). The opposite poles of the rod bind specific pole proteins, which are marked in the micrographs by fluorescentlabeled antibodies. A mutant strain of Shigella was isolated that grows as amorphous spheres (see Fig. 1B). Unlike the rod-shaped cells, the spherical mutants show multiple polemarker proteins at undefined locations. The mutation eliminates expression of mreB, encoding the MreB cytoskeletal protein. Sequence analysis shows a strong relationship between MreB and the eukaryotic filament-forming protein actin. How do MreB proteins maintain the rod shape of the bacterium? Like actin filaments in eukaryotes, MreB proteins form elongated structures beneath the cell membrane. Figure 2A shows how MreB proteins polymerize in a helical arrangement around the cell, thus determining the axis of elongation. Because MreB is required for rod elongation, its gene is absent from bacteria that normally grow as cocci, such as Streptococcus. In Figure 2B, the helical arrangement of MreB is observed by confocal fluorescence microscopy of Caulobacter crescentus, a pond-dwelling bacterium. The MreB proteins are labeled by fluorescent antibodies. Crescent-shaped cells such as C. crescentus usually possess a third kind of shape-determining protein, CreS, in addiA. Parent strain
tion to FtsZ and MreB (see Fig. 2A). CreS is a homologue of the eukaryotic intermediate filament protein. CreS polymerizes along the inner curve of a crescent cell, as seen in Figure 2C. In the micrograph shown, CreS proteins are labeled by recombination of the creS gene with a gene encoding the green fluorescent protein (GFP). The result is a gene fusion expressing the hybrid protein CreS-GFP, which fluoresces green. In the confocal fluorescence micrograph, the CreS-GFP appears blue-green, whereas the cell membrane is labeled red by a membrane-binding fluorophore. The membrane-binding fluorophore reveals the overall crescent outline of the cell and the localization of CreS to the cell’s inner curve. Other bacterial proteins contributing to cell shape and division include the Min proteins, required for equal division of cells, and the Par proteins, which segregate replicated plasmids during cell division. In one group of bacteria, the phylum Verrumicrobia, a tubulin homolog has been discovered that forms tubules in vitro. Such tubules may contribute to the unusual extended shapes of Verrumicrobia. A. Staphylococcus aureus
B.
N. Ausmees, et al. 2003
FtsZ
Escherichia coli
MreB
Caulobacter crescentus
B. mreB mutant
C.
N. Ausmees, et al. 2003
Special Topic 3.2
Spherical mreB mutants of rod-shaped bacteria. A. Rod-shaped Shigella cells show a poleassociated protein (IcsA-GFP) localized to opposite poles. B. Mutant MreB cells form bloated spherical cells with multiple sites localizing IcsA-GFP.
Figure 1
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Trine Nilsen, et al. 2005. J. Bacteriol. 187:6187
5 µm
Trine Nilsen, et al. 2005. J. Bacteriol. 187:6187
Crescentin
Shape-determining proteins. A. Spherical cell size and shape is maintained by FtsZ polymerization to form the Z ring. Elongation of a rod-shaped cell is accomplished by helical polymerization of Mre proteins. Crescent-shaped cells possess a third shape-determining protein, CreS (crescentin), which polymerizes along the inner curve of the crescent. B. MreB localizes in a helical coil within Caulobacter crescentus cells. Images of MreB immunostaining were obtained by confocal fluorescence microscopy. C. Crescentin protein fused to green fluorescent protein GFP (CreS-GFP) localizes to the inner curve of C. crescentus. CreS-GFP colocalizes with the membrane-specific stain FM4-64 (red fluorescence).
Figure 2
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TO SU M MAR I Z E: ■
■
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The nucleoid region contains loops of DNA, supercoiled and bound to DNA-binding proteins. Transcription of DNA occurs in the cytoplasm, often simultaneously with DNA replication. The ribosome translates RNA to make proteins, which are folded by chaperones and in some cases secreted at the cell membrane. DNA is replicated bidirectionally by the replisome. Cell expansion and septation are coordinated with DNA replication. Cell shape is determined by the FtsZ “Z ring” and other cytoskeletal proteins.
3.7
Specialized Structures
We have introduced the major structures required to contain the cell, organize its contents, maintain its DNA, and express its genes. The cell envelope, the nucleoid, and the gene expression and protein translocation complexes are essential for all living prokaryotes. In addition to these fundamental structures, different species have evolved different kinds of specialized devices adapted to diverse metabolic strategies and environments.
Thylakoids and Carboxysomes Conduct Photosynthesis Photosynthetic bacteria, also called phototrophs, need to collect as much light as possible to drive photosynthesis. Light is harvested by protein complexes called phycobilisomes (see Section 14.6). Some of the energy obtained is stored in the form of storage granules.
W. Kili & F. Partensky, Bedford Institute of Oceanography
A.
To maximize their photosynthetic membranes, phototrophs have evolved specialized systems of extensively folded intracellular membrane called thylakoids (Fig. 3.39A). Thylakoids consist of layers of folded sheets (lamellae) or tubes of membranes packed with photosynthetic proteins and electron carriers. Cyanobacteria containing thylakoids structurally resemble chloroplasts, which are believed to have evolved from a common ancestor of modern cyanobacteria. The thylakoids conduct only the “light reaction” of photon absorption and energy storage. The energy obtained is rapidly spent to fi x carbon dioxide, which occurs within carboxysomes. Carboxysomes are polyhedral, protein-covered bodies packed with the enzyme rubisco for CO2 fi xation. Aquatic and marine phototrophs, as well as archaea, often possess gas vesicles to increase buoyancy and keep themselves high in the water column, near the sunlight (Fig. 3.39B). Gas vesicles are specialized vacuoles composed of specific proteins. The vesicles trap and collect gases such as hydrogen or carbon dioxide produced by the cell’s metabolism.
Storage Granules During times of starvation, phototrophs may digest their phycobilisomes for energy and as a source of nitrogen. Alternatively, energy is stored in storage granules composed of glycogen or other polymers, such as polyhydroxybutyrate (PHB) and poly-3-hydroxyalkanoate (PHA). PHB and PHA polymers are of interest as a biodegradable plastic, which bacteria are engineered to produce industrially. Similar storage granules are also produced by nonphototrophic soil bacteria.
B.
Thylakoids Cell envelope
Polyhedral body
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©Tom E. Adams/Visuals Unlimited
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Organelles of phototrophs. A. The marine phototroph Prochlorococcus marinus (TEM). Beneath the envelope lie the photosynthetic double membranes called thylakoids. Carboxysomes are polyhedral, protein-covered bodies packed with rubisco enzyme for CO2 fixation. B. Filaments (chains of cells) of the cyanobacterium Planktothrix. Gas vesicles provide buoyancy, enabling the phototroph to remain at the surface of the water, exposed to light.
Figure 3.39
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The M ic ro b i al Ce l l
motility) is the magnetosome. Magnetosomes are microscopic membrane-bounded crystals of the magnetic mineral magnetite, Fe3O4 (Fig. 3.41). They are found in anaerobic pond-dwelling organisms such as Magnetospirillum gryphiswaldense. The crystals generate a magnetic dipole moment along the length of a bacterium, constraining it to swim along a magnetic field. This magnetic orientation of swimming is called magnetotaxis, the ability to sense and respond to magnetism. Magnetotactic bacteria can be collected by placing a magnet within a jar of pond water; bacteria orienting by the field lines collect nearby. The natural function of magnetosomes appears to be to orient bacterial swimming toward the bottom of the pond. Magnetotactic organisms are anaerobes, which prefer the lower part of the water column, where oxygen concentration is lowest. Because Earth’s magnetic field lines point downward in the northern latitudes, bacteria that are magnetotactic swim “downward” toward magnetic north.
Courtesy of Juergen Wiegel and Manfred Rohde
1 µm
■
External sulfur particles. Sulfur globules dot the surface of Thermoanaerobacter sulfurigignens, an anaerobic thermophilic bacterium that gains energy by reducing thiosulfate (S2O32–) to elemental sulfur (S0).
Figure 3.40
Another type of storage device is sulfur, granules of elemental sulfur produced by purple and green phototrophs through photolysis of hydrogen sulfide (H2S). Instead of disposing of the sulfur, the bacteria store it in granules, either within the cytoplasm (purple phototrophs) or as “globules” attached outside of the cell. Sulfur-reducing bacteria also make extracellular sulfur globules (Fig. 3.40). The sulfur may be usable as an oxidant when reduced substrates become available. Alternatively, the presence of potentially toxic sulfur granules may help cells avoid predation.
THOUGHT QUESTION 3.10 How would a magnetotactic species have to behave if it were in the Southern Hemisphere instead of in the Northern Hemisphere?
Magnetotactic bacteria are being studied for their potential applications in wastewater treatment. Through their anaerobic metabolism, some magnetotactic bacteria accumulate high concentrations of toxic metals from the water. They and the toxic metals they scavenge can then be removed by application of a magnetic field to attract and concentrate the bacteria.
Magnetosomes of Magnetite Direct Motility An unusual structure possessed by magnetotactic species of bacteria (bacteria showing magnetically directed
The Sustainable Energy Research Group, U. of Southampton
Pili and Stalks Enable Attachment
Magnetotactic bacterium. A magnetospirillum is a spiral-shaped magnetotactic bacterium. The spiral cell contains a chain of magnetosomes, magnetite crystals (TEM).
Figure 3.41
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In a favorable habitat, such as a running stream full of fresh nutrients or the epithelial surface of a host, it is advantageous for a cell to adhere to a substrate. Adherence, the ability to attach to a substrate, requires specific adherence structures, such as pili (protein filaments). As bacteria grow and proliferate, however, they face the question of whether to stay where they are or leave their present habitat, where nutrients may be depleted and waste products increased. In rapidly changing environments, cell survival requires motility, the ability to move and relocate. Motility requires structures such as rotary flagella. Some bacteria have it both ways by producing two kinds of progeny, one adherent, the other motile (see Special Topic 3.3). The most common structures that bacteria use to attach to a substrate are pili (singular, pilus), or fi mbriae (singular, fi mbria), straight fi laments of protein monomers called pilin. For example, fi mbriae attach the
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Special Topic 3.3
Two Kinds of Progeny: One Stays, One Swims
What if a single species encounters very different environments throughout its life history, some favorable, others unfavorable? Sometimes, adherence might be advantageous; at other times, motility would be desirable. Bacteria such as E. coli and Salmonella species possess more than one kind of filamentous structure: flagella for motility and pili for attachment once a favorable substrate is reached. An even more dramatic strategy for “having it both ways” is to generate two kinds of daughter cells: one that stays, and one that swims away. This strategy is exemplified by the flagellum-to-stalk transition of the aquatic bacterium Caulobacter crescentus. Caulobacter crescentus exists in two forms, one with a stalk (stalked cell) and one with a single flagellum at one pole (swarmer cell). The swarmer cell swims about freely in an aqueous habitat, such as a pond or a sewage bed. After half an hour of swimming, the swarmer cell undergoes differentiation to shed its flagellum and replace it with a stalk (Fig. 1). Once attached, the stalked cell immediately starts to replicate its DNA. How do the two progeny cells develop different forms? Lucy Shapiro and colleagues at Stanford University showed that the identities of swimmer and stalked cells are determined by the methylation state of their DNA (Fig. 2). The original DNA has methyl groups on its cytosine bases, added during the previous round of DNA replication. These methyl groups allow expression of genes needed for a stalked cell. As the DNA now undergoes a new round of replication, each daugh-
oral pathogen Porphyromonas gingivalis to gum epithelium, where they are associated with periodontal disease (Fig. 3.42). A different kind of pili, the sex pili, serve to attach a “male” donor cell to a “female” recipient cell for transfer of DNA. This process of DNA transfer is called conjugation. The genetic consequences of conjugation are discussed in Chapter 9. Another kind of attachment organelle is a membranebound extension of the cytoplasm called a stalk. The tip of the stalk secretes adhesion factors called holdfast, which fi rmly attach the bacterium in an environment that has proved favorable. The mechanism of stalk and holdfast attachment has been extensively studied in iron-oxidizing bacteria that interfere with mining operations by producing massive biofi lms. An example is Gallionella ferruginea, an iron-oxidizing species that grows a long stalk (Fig. 3.43). The long twisted stalks of adherent Gallionella cells become coated by iron hydroxides, and Gallionella contributes a major part of the process of biomineralization (biological crystallization of minerals) in iron mines.
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ter duplex receives a new unmethylated strand, base-paired with the old strand containing methyl groups. The original two parental strands differ in their methylation patterns; thus, the methylation patterns of the two daughter duplexes allow different gene expression in the two daughter cells, one of which is stalked, the other a swarmer. Once DNA synthesis is complete and the cells have differentiated into different forms, the
Free-swimming Caulobacter crescentus
Stalk Holdfast
Swarmer
Stalked cell
Asymmetrical cell division: A model for development. A swarmer cell of C. crescentus loses its flagellum and grows a stalk. The stalked cell divides to produce a swarmer cell. Photo courtesy of Yves Brun, Indiana University.
Figure 1
Tsute Chen and Margaret J. Duncan, The Forsyth Institute
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Pili: protein filaments for attachment. Porphyromonas gingivalis, a causative agent of gum disease or gingivitis. The P. gingivalis cells show fimbriae along with vesicles budding from the cell’s outer membrane.
Figure 3.42
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B. New DNA becomes methylated, restoring state of stalked cell.
Swarmer methylation pattern
Stalked cell methylation pattern
Bacteria and archaea that are motile generally swim by means of rotary flagella (singular, flagellum). Flagella are helical propellers that drive the cell forward like the motor of a boat. First shown by Howard Berg at the
B. Eleanora I. Robbins, U.S. Geological Survey
A.
Rotary Flagella Enable Motility and Chemotaxis
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DNA segregation into progeny cells. A. Lucy Shapiro and colleagues at Stanford University determined the molecular mechanism of Caulobacter differentiation into stalked and swarmer cells. B. As the stalked cell divides, one daughter helix inherits the strand methylated in the swarmer pattern while the other inherits the stalked cell pattern. After cell division and replication, both new DNA strands are methylated to set the original pattern. The swarmer cell eventually loses its flagellum and reverts to a stalked cell.
Eleanora I. Robbins, U.S. Geological Survey
Gallionella ferruginea: iron-oxidizing stalked bacteria. A. The oval cell of Gallionella ferruginea generates a long twisted stalk encrusted with iron oxides. B. Karen L. Prestegaard, of the University of Maryland, studies iron-oxidizing bacteria attached to iron surfaces, such as this rod in the stream, where the bacteria promote rusting, coating the surface orange.
Figure 3.43
The M ic ro b i al Ce l l
Figure 2
A.
Lucy Shapiro, Stanford University
new DNA becomes methylated, restoring the complementary methylation patterns in each cell. The remaining stalked daughter cell undergoes another round of cell division to produce yet another swarmer cell, as well as a stalked cell. Each swarmer cell eventually converts to a stalked cell and reproduces progeny. By this means, Caulobacter can hedge its bets. Half its progeny always remain at a location that, however favorable, may eventually deteriorate, whereas the other half swims away to find a potentially better environment. Caulobacter represents a simple example of cell differentiation that resembles phenomena seen in more complex developmental systems, such as DNA methylation during cell morphogenesis. Thus, it offers an informative model system for cell differentiation in the development of multicellular organisms.
■
California Institute of Technology, the bacterial flagellar motor was the fi rst rotary device to be discovered in a living organism. Different bacterial species have different numbers and arrangements of flagella. Peritrichous cells, such as E. coli and Salmonella species, have flagella randomly distributed around the cell (Fig. 3.44A). The
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5 µm
flagella rotate together in a bundle behind the swimming cell (Fig. 3.44B) Lophotrichous cells, such as Rhodospirillum rubrum, have flagella attached at one or both ends. In monotrichous (polar) species, such as Pseudomonas aeruginosa, the cell has a single flagellum at one end. Each flagellum is a spiral fi lament of protein monomers called flagellin. The filament is actually rotated by means of a motor driven by the cell’s transmembrane proton current, the same proton potential that drives the membrane-bound ATPase. (Alternatively, the motor is driven by a sodium ion potential, particularly in marine bacteria such as Vibrio cholerae.) The flagellar motor is embedded in the layers of the cell envelope (Fig. 3.45). The motor is observed by electron microscopy, and its parts are defi ned by genetic analysis of mutants with aberrant motility. The motor actually possesses an axle and rotary parts, all composed of specific proteins. Much of the structure and function of the motor were elucidated by Scottish microbiologist Robert Macnab (1940–2003) at Yale University.
Robert Macnab. 1976. J. Clinical Microbiology 4:258
1 µm
Flagellated Salmonella bacteria. A. Salmonella enterica bacterium has multiple flagella (colorized TEM). B. The flagella collect in a bundle behind a swimming cell. Under darkfield microscopy, the cell body appears overexposed, about five times as large as the actual cell.
Figure 3.44
B.
CAMR/A. Barry Dowsett
A.
Flagellar motility benefits the cell by causing dispersal of progeny, decreasing competition. In addition, most flagellated cells have an elaborate sensory system that enables them to swim toward favorable environments (attractant signals, such as nutrients) and away from inferior environments (repellent signals, such as waste products). This sensory system is known as chemotaxis. Chemotaxis requires a way for the cell to move toward attractants and away from repellents. This is accomplished by flagellar rotation either clockwise or counterclockwise relative to the cell (Fig. 3.46A ). When a cell is swimming toward an attractant chemical, the flagella rotate counterclockwise (CCW), enabling the cell to swim smoothly for a long stretch. When the cell veers away from the attractant, receptors send a signal that allows one or more flagella to switch rotation clockwise (CW), against the twist of the helix. This switch in the direction of rotation disrupts the bundle of flagella, causing the cell to tumble briefly, ending up pointed in a random direc-
David DeRosier/Brandeis University
A.
B.
L ring Outer membrane Cell wall
P ring
Inner membrane Rotor C ring 25 nm
Julius Adler, U. of Wisconsin, and May Macnab, Yale Univ.
C.
Figure 3.45 The flagellar motor. A. The basal body, or motor, of the bacterial flagellum (TEM). The image is based on digital reconstruction, in which electron micrographs of purified hook-basal bodies were rotationally averaged. The rings (L, P, and C) correspond to those labeled in the diagram. B. Diagram of the flagellar motor, including major protein components. C. Robert Macnab (University of Wisconsin) identified many components of the motor and chemotaxis signaling.
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B.
A. Counterclockwise (CCW) rotation moves cell toward attractant.
CCW swim toward attractant Attractant Receptors for attractants CW tumble
Random walk Clockwise (CW) rotation stops forward motion so cell tumbles and changes direction.
Swim toward attractant
Figure 3.46 Chemotaxis. A. In peritrichous bacteria, flagella are oriented in a bundle extending behind one pole, while their chemotactic receptors are concentrated at the opposite pole. When the cell veers away from the attractant, the receptors send a signal that allows one or more flagella to switch rotation from counterclockwise (CCW) to clockwise (CW). This switched rotation disrupts the bundle of flagella, causing the cell to tumble briefly before it swims off in a new direction. B. The pattern of movement resulting from alternating swimming and tumbling is a “biased random walk” in which the cell sometimes moves randomly but overall tends to migrate toward the attractant.
tion (Fig. 3.46B ). The cell then swims off in the new direction. The resulting pattern of movement generates a “biased random walk” in which the cell tends to migrate toward the attractant. THOUGHT QUESTION 3.11 Most strains of E. coli and Salmonella commonly used for genetic research actually lack flagella entirely. Why do you think this is the case? How can a researcher maintain a motile strain? Note that bacterial flagella differ completely from the whiplike flagella and cilia of eukaryotes. Eukaryotic flagella are much larger structures containing multiple microtubules enclosed by a membrane. They move with a whiplike motion that forms a flat sine wave, propagated by ATP hydrolysis all along the flagellum. TO SU M MAR I Z E: ■
Phototrophs possess thylakoid membrane organelles packed with photosynthetic apparatus and carboxysomes for carbon dioxide fi xation. Other subcellular structures may include sulfur granules from H 2S photolysis and gas vesicles for buoyancy in the water column.
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■ ■
■
■
■
Storage granules store polymers for energy. Magnetosomes orient the swimming of magnetotactic anaerobic bacteria. Adherence structures enable prokaryotes to remain in an environment with favorable environmental factors. Major adherence structures include pili or fi mbriae (protein fi laments) and the holdfast (a cell extension). Flagellar motility occurs by rotary motion of helical flagella. Chemotaxis involves a biased random walk up a gradient of attractant substance or down a gradient of repellents.
Concluding Thoughts Research techniques of cell fractionation and biochemistry, together with genetic analysis, continue to reveal the structure and function of cells. The intricate mechanisms derived by microbial evolution challenge the inventers of antibiotics as well as designers of molecular machines. As a journalist observed in Science, “When it comes to nanotechnology, physicists, chemists, and materials scientists can’t hold a candle to the simplest bacteria.”
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CHAPTE R R EVI EW Review Questions 1. What are the major features of a bacterial cell, and
5. Compare and contrast the structure of gram-positive
how do they fit together for cell function as a whole? 2. What fundamental traits do most prokaryotes have in common with eukaryotic microbes? What traits are different? 3. Give examples of how our views of ribosome structure and function have emerged from microscopy, cell fractionation, X-ray diffraction crystallography, and genetic analysis. Explain the advantages and limitations of each technique. 4. Outline the structure of the peptidoglycan sacculus, and explain how it expands during growth. Cite two different kinds of experimental data that support our current views of the sacculus.
and gram-negative cell envelopes. Explain the strengths and weaknesses of each kind of envelope. Outline the process of DNA replication, and explain how it is coordinated with cell wall septation. Explain how DNA transcription to RNA is integrated with translation and protein processing and secretion. What are kinds of subcellular structures found in certain cells with different functions, such as magnetotaxis or photosynthesis? Compare and contrast bacterial structures for attachment and motility. Explain the molecular basis of chemotaxis.
6. 7.
8.
9.
Key Terms active transport (85) adherence (107) ATP synthase (85) capsule (76, 93) carboxysome (106) cardiolipin (86) cell membrane (75) cell wall (76) chaperone (100) chemotaxis (110) cholesterol (87) contractile vacuole (97) core polysaccharide (95) cross-bridge (89) diphosphatidylglycerol (86) electrochemical potential (85) electrophoresis (77) endotoxin (94) envelope (76) fimbria (plural, fimbriae) (107) flagellum (plural, flagella) (76, 109) gas vesicle (106) genetic analysis (81) glucosamine (94) glycan (89) helicase (101) holdfast (108) hopane (hopanoid) (87) inner membrane (75, 94) isoelectric focusing (77)
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leaflet (82) lipopolysaccharide (LPS) (76, 94) lysate (80) magnetosome (107) magnetotaxis (107) membrane-permeant weak acid (83) membrane-permeant weak base (83) motility (107) murein (89) murein lipoprotein (94) nucleoid (76) O polysaccharide (95) origin of replication (ori) (98) osmosis (83) osmotic pressure (83) outer membrane (76) passive transport (85) pellicle (76) peptidoglycan (78) periplasm (76) peritrichous (109) permissive temperature (104) phosphatidate (86) phosphatidylethanolamine (82) phosyphatidylglycerol (86) phycobilisome (106) pilin (107) pilus (plural, pili) (107) polyamine (77) polysome (100)
porin (96) proteome (77) reductive evolution (degenerative evolution) (94) replisome (101) restrictive temperature (104) sacculus (89) sedimentation rate (80) septation (103) septum (103) sex pilus (108) signal recognition particle (SRP) (101) S-layer (94) solutes (83) stalk (108) staphylococcus (plural, staphylococci) (103) subcellular fractionation (79) supercoil (99) Svedberg coefficient (80) teichoic acid (93) terpenoid (87) thylakoid (106) transport protein (transporter) (84) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE, 2-D gels) (77) ultracentrifuge (79)
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Recommended Reading Ausmees, Nora, Jeffrey R. Kuhn, and Christine JacobsWagner. 2003. The bacterial cytoskeleton: An intermediate fi lament-like function in cell shape. Cell 115:705–713. Begic, Sanela, and Elizabeth A. Worobec. 2006. Regulation of Serratia marcescens ompF and ompC porin genes in response to osmotic stress, salicylate, temperature and pH. Microbiology 152:485–491. Carter, Andrew P., William M. Clemons, Ditlev E. Brodersen, Robert J. Morgan-Warren, Brian T. Wimberly, and V. Ramakrishnan. 2000. Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407:340–348. Feucht, Andrea, and Jeff Errington. 2005. ftsZ mutations affecting cell division frequency, placement and morphology in Bacillus subtilis. Microbiology 151:2053–2064. Gitai, Zemer, Natalie Dye, and Lucy Shapiro. 2004. An actinlike gene can determine cell polarity in bacteria. Proceedings of the National Academy of Sciences USA 101:8643–8648. Komeili, Arash, Zhuo Li, Dianne K. Newman, and Grant J. Jensen. 2006. Magnetosomes are cell membrane invaginations organized by the actin-like protein MamK. Science 311:242–245. Lemon, Katherine P., and Alan D. Grossman. 1998. Localization of bacterial DNA: Evidence for a factory model of replication. Science 282:1516–1519.
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Nilsen, Trine, Arthur W. Yan, Gregory Gale, and Marcia B. Goldberg. 2005. Presence of multiple sites containing polar material in spherical Escherichia coli cells that lack MreB. Journal of Bacteriology 187:6187–6196. Noji, Hiroyuki, Ryohei Yasuda, Masasuke Yoshida, and Kazuhiko Kinoshita, Jr. 1997. F1F0 Direct observation of the rotation of F1-ATPase. Nature 386:299–302. Parkinson, John S., Peter Ames, and Claudia A. Studdert. 2005. Collaborative signaling by bacterial chemoreceptors. Current Opinion in Microbiology 8:116–121. Ruiz, Natividad, Daniel Kahne, and Thomas J. Silhavy. 2006. Advances in understanding bacterial outer-membrane biogenesis. Nature Reviews Microbiology 4:57–66. Schäffer, Christina, and Paul Messner. 2005. The structure of secondary cell wall polymers: How Gram-positive bacteria stick their cell walls together. Microbiology 151:643–651. Shih, Yu-Ling, Trung Le, and Lawrence Rothfield. 2003. Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles. Proceedings of the National Academy of Sciences USA 100:7865–7870. Sutcliffe, Joyce A. 2005. Improving on nature: Antibiotics that target the ribosome. Current Opinion in Microbiology 8(5):534–542.
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Chapter 4
Bacterial Culture, Growth, and Development 4.1 4.2 4.3 4.4 4.5 4.6 4.7
Microbial Nutrition Nutrient Uptake Culturing Bacteria Counting Bacteria The Growth Cycle Biofilms Cell Differentiation
The adage “To eat well is to live well” is as true for microbes as it is for humans. Microorganisms constantly struggle to survive in natural habitats because they compete for food. Yet a single microbial pathogen can multiply within the course of a day to cause deadly illness, and a few algae can abruptly bloom and cover the entire surface of a lake. In each of these cases, the population explosion results from the sudden availability of food. Over eons, bacteria have evolved ingenious strategies to find, acquire, and metabolize a wide assortment of potential food sources, ranging from glucose to mothballs. This metabolic diversity arose by necessity, from the need to find new sources of food ignored by competitors. The remarkable plasticity of microbial genomes enables adaptation to new food sources. During the course of DNA replication, gene systems involved in the use of one food naturally undergo duplications and mutations, some of which sculpt new biochemical pathways capable of metabolizing novel substrates. This genomic flexibility raises hopes that we can engineer microbial biochemistry to remediate pollution and produce tomorrow’s wonder drugs.
This elaborate bacterial colony is formed by an undomesticated strain of Bacillus subtilis isolated from soil. The complex architecture is the result of a tightly regulated developmental process and provides insight into the organization of bacterial communities. Source: Cover. 2004. Journal of Bacteriology 186(12).
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Microorganisms need sources of carbon in order to grow. Understanding how they use food to increase their cell mass and, ultimately, cell number enables us to control their growth and manipulate them to make useful products. Yeast, for example, consume glucose and break it down to ethanol and carbon dioxide gas. These end products are merely waste to the yeast but are extremely important to humans who enjoy beer. Brewers have learned to control the amount of sugar supplied to yeast growing in fermentation vats to produce just the right amount of alcohol and CO2, which produces beer’s bubbling carbonation. Many a home brewer has learned the hard way that providing too much sugar (in malt) will cause yeast to make too much CO2 gas, turning a homemade fermentor into an unpredictable explosive device. Learning how bacteria grow has also provided a window through which to view the core processes of life. As a result of studying bacterial growth and nutrition, we now know how DNA replicates, how RNA is made, and how proteins are assembled. We have also learned that the availability of nutrients has influenced the evolution of sophisticated microbial processes designed to avoid or survive starvation. For example, bacteria communicate with each other to build elaborate multicellular reproductive structures, such as fruiting bodies, or complex multispecies biofilms, such as those that erode our teeth and rust the surface of ocean liners. In this chapter, we consider the microbial biosphere and the basics of bacterial growth. We’ll consider a variety of questions: How diverse is this biosphere? Is it true that we have identified only about 0.1% of the bacterial species that inhabit Earth? Where do bacteria get their energy? You might be surprised to learn that some bacteria gain energy from light (photosynthesis), whereas others gain energy from oxidizing sulfur. With so many microbes consuming nutrients, how has nature arranged biosystems to avoid depleting Earth of key compounds? To understand the biochemistry behind metabolic diversity, we need to grow microbes. How do we ensure
their growth and measure it? Some bacteria make elaborate, multicellular structures, whereas others form environmentally resistant fortresses called spores. In this chapter, we discuss how microbes obtain energy and nutrients for growth, and how cell populations develop. A more detailed treatment of mechanisms of energy gain and biosynthesis is presented in Chapters 13–15.
4.1
Microbial Nutrition
Bacterial cells, for all their apparent simplicity, are remarkably complex and efficient replication machines. One cell of Escherichia coli, for example, can divide into two cells every 20–30 minutes. At a rate of 30 min per division, one cell could potentially multiply to over 1 × 1014 cells in 24 hours—that is, 100 trillion organisms! Although 100 trillion cells would only weigh about 1 g, after another 24 hours (a total of two days) of replicating every 30 minutes, the mass of cells would explode to 1014 g, or 107 tons. So why are we not buried under mountains of E. coli?
Nutrient Supplies Limit Microbial Growth One factor limiting growth is that microbes commonly encounter environments where essential nutrients are limited. Essential nutrients are those compounds a microbe cannot make itself but must gather from its environment if the cell is to grow and divide. Microbial cells obtain all the essential nutrients for growth from their immediate environment. Consequently, when an environment becomes depleted of one or more essential nutrients, the microorganism will stop growing. How various organisms cope with these periods of starvation until nutrients are restored is a rapidly developing field of microbiology. All microorganisms require a minimum set of macronutrients, nutrients needed in large quantities. Six of these—carbon, nitrogen, phosphorus, hydrogen, oxygen,
Table 4.1 Growth factors and natural habitats of organisms associated with disease. Organism
Diseases
Natural habitats
Growth factors
Shigella Haemophilus
Bloody diarrhea Meningitis, chancroid
Nicotinamide (NAD)a Haemin, NAD
Staphylococcus Abiotrophia Legionella Bordetella Francisella Mycobacterium Streptococcus pyogenes
Boils, osteomyelitis Osteomyelitis Legionnaires’ disease Whooping cough Tularemia Tuberculosis, leprosy
Humans Humans and other animal species, upper respiratory tract Widespread Humans and other animal species Soil, refrigeration cooling towers Humans and other animal species Wild deer; rabbits Humans
Pharyngitis, rheumatic fever
Humans
Glutamate, alanine
Complex requirement Vitamin K, cysteine Cysteine Glutamate, proline, cystine Complex, cysteine Nicotinic acid (NAD),a alanine
a
Both nicotinamide and nicotinic acid are derived from NAD, nicotinamide adenine dinucleotide.
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and sulfur—make up the carbohydrates, lipids, nucleic acids, and proteins of the cell. Four other macronutrients are cations whose roles range from serving as enzyme cofactors (Mg2+, Fe2+, and K+) to acting as regulatory signal molecules (Ca2+). In addition to macronutrients, all cells require very small amounts of certain trace elements, called micronutrients. These include cobalt, copper, manganese, molybdenum, nickel, and zinc, which are ubiquitous contaminants on glassware and in water. As a result, these trace elements are not added to laboratory media unless heroic measures have been taken to fi rst remove the elements from the medium. Micronutrients are required by cells because these elements are frequently essential components of enzymes or can themselves be components of cofactors. Cofactors are small molecules that fit into specific enzymes and aid in the catalytic process. Cobalt, for example, is part of the cofactor vitamin B12. Some organisms, such as the laboratory “workhorse” bacterium E. coli, make all their proteins, nucleic acids, and cell wall and membrane components from this very simple blend of chemical elements and compounds. For many other microbes, this basic set of nutrients is not enough. One example is Borrelia burgdorferi, the causative
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agent of Lyme disease, which requires an extensive mixture of complex organic supplements to grow.
Microbes Evolved to Grow in Different Environments Based in part on the ecological niche it inhabits, an organism may have evolved to require additional growth factors (Table 4.1), specific nutrients that are not required by all cells. For example, why should an organism like Streptococcus pyogenes make glutamic acid or alanine if those amino acids are readily available in its normal environment (such as, the human oral cavity)? Because it never needs to make these compounds, S. pyogenes, as a matter of efficiency, has lost the genes whose protein products synthesize glutamic acid and alanine. A defined minimal medium contains only those compounds needed for an organism to grow (Table 4.2). In the case of S. pyogenes, this would include glutamic acid and alanine in addition to the macro- and micronutrients mentioned earlier. Other organisms have adapted so well to their natural habitat that we still do not know how to grow them in the laboratory. For example, Rickettsia prowazekii, the causative agent of Rocky Mountain
Table 4.2 Composition of commonly used media. Media
Ingredients per liter a
Organisms cultured
Luria Bertani (complex)
Bacto tryptone Bacto yeast extract NaCl pH 7
10 g 5g 10 g
Many gram-negative and gram-positive organisms
M9 medium (defined)
Glucose Na2HPO4 KH2PO4 NH4Cl NaCl MgCl2 CaCl2 pH 7
2.0 g 6.0 g (42 mM) 3.0 g (22 mM) 1.0 g (19 mM) 0.5 g (9 mM) 2.0 mM 0.1 mM
Gram-negative organisms such as E. coli
Azotobacter medium (defined)
Mannitol K2HPO4 MgSO4 • 7H2O FeSO4 • 7H2O
2.0 g 0.5 g 0.2 g 0.1 g
Azotobacter
Sulfur oxidizers (defined)
NH4Cl KH2PO4 MgSO4 • 7H2O CaCl2 Elemental sulfur CO2 pH 3
0.52 g 0.28 g 0.25 g 0.07 g 1.56 g 5%
Thiobacillus thiooxidans
a
Bacto tryptone is a pancreatic digest of casein (bovine milk protein).
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B.
Rickettsia
6 µm
D. J. Silverman, C. L. Wisseman, Jr., and A. Wadell
A.
spotted fever, grows only within the cytoplasm of eukaryotic cells (Fig. 4.1). This obligate intracellular bacterium lost key pathways needed for independent growth because the host cell supplies them. Despite extensive efforts to grow them outside a host cell (axenic growth), cells of R. prowazekii have proved uncooperative in this endeavor. Although it will not grow in a defined medium, we can at least grow Rickettsia in the laboratory using eggs or animal cell tissue culture. But it is estimated that 99.9 % of all the microorganisms on Earth cannot be grown in the laboratory at all. How do we even know that these microbes exist if we cannot grow them? Evidence for their existence has been discovered only recently using newly developed tools of molecular biology. All known microorganisms have a set of genes, associated with ribosomes, whose DNA sequences are highly conserved across the phylogenetic tree. A DNAamplifying procedure called polymerase chain reaction (PCR, described in Section 7.6) can be used to screen for the presence of these genes in soil and water samples. Comparing the DNA sequences of the PCR products with the DNA sequences of similar genes from known organisms reveals that nature harbors many microbes hitherto undiscovered because we cannot grow them in the laboratory. Even though the growth and nutritional requirements of these phantom microbes are unknown, modern genomic techniques can expose their existence. We can gain remarkable insight into the physiology of these “invisible,” nonculturable organisms by comparing their gene sequences, mined by PCR, with known gene sequences from culturable organisms that have well-characterized physiologies.
Microbes Build Biomass through Autotrophy or Heterotrophy Maintaining life on this planet is an amazing process. All of Earth’s life-forms are based on carbon, which they acquire by delicately choreographed processes that recycle key nutrients. The carbon cycle, a critical part of this process, involves two counterbalancing metabolic groups of
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Rickettsia prowazekii growing within eukaryotic cells. A. R. prowazekii growing within the cytoplasm of a chicken embryo fibroblast (SEM). B. Giemsa stain of R. prowazekii growing within cultured human L cells.
Figure 4.1 David Wood, U. of South Alabama College of Medicine
118
organisms, heterotrophs and autotrophs. Their pathways of metabolism are discussed in chapters 13 and 14. Heterotrophs (such as E. coli) rely on other organisms to form the organic compounds, such as glucose, that they use as carbon sources. During heterotrophic metabolism, organic carbon sources are disassembled to generate energy and then reassembled to make cell constituents such as proteins and carbohydrates. This process converts a large amount of the organic carbon source to CO2, which is then released to the atmosphere. Thus, left on their own, heterotrophs would deplete the world of organic carbon sources (converting them to unusable CO2) and starve to death. For life to continue, CO2 must be recycled. Autotrophs assimilate CO2 as a carbon source, reducing it (adding hydrogen atoms; see Chapter 15) to make complex cell constituents made up of C, H, and O (for example, carbohydrates, which have the general formula CH2O). These organic compounds can later be used as carbon sources by heterotrophs. As will be discussed below, autotrophs are subclassified, based on how they obtain energy, into photoautotrophs, organisms that use light for photosynthesis, and chemoautotrophs also known as chemolithotrophs or lithotrophs (literally, rock-eaters), organisms that gain energy by oxidizing inorganic substances such as iron or ammonia. Both photoautotrophs and chemoautotrophs carry out the autotrophic process of CO2 fi xation, which forms part of the carbon cycle between autotrophs and heterotrophs (Fig. 4.2). THOUGHT QUESTION 4.1 In a mixed ecosystem of autotrophs and heterotrophs, what happens when a heterotroph allows the autotroph to grow and begin to make excess organic carbon?
Microbes Obtain Energy through Phototrophy or Chemotrophy Although the macronutrients mentioned earlier provide the essential building blocks (in other words, carbon) to
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B. Autotrophy
A. Heterotrophy
Light absorption
Polysaccharides
CO2 + H2O
Photolysis NADPH or ATP
Mineral oxidation
Glucose (6C) Lipids Peptides Lignin
Glycolysis
Lithotrophy
Glucose
C.
Pyruvate (3C) Acetyl-CoA Acetate (2C) TCA Cycle
Citrate
Wim von Egmond
Fermentation products
CO2 + H2O Oxidative respiration
The carbon cycle. The carbon cycle requires both autotrophs and heterotrophs. A. Heterotrophs gain energy from degrading complex organic compounds, such as polysaccharides, to smaller compounds, such as glucose and pyruvate. The carbon from pyruvate moves through the tricarboxylic acid (TCA) cycle, and CO2 is released. In the absence of a TCA cycle, the carbon can end up as fermentation products, such as ethanol or acetic acid. B. Autotrophs use light energy or energy derived from the oxidation of minerals to capture CO2 and convert it to complex organic molecules. C. Nostoc is an autotroph. These cyanobacteria live in a gelatinous sphere. The spheres shown are colonies (approx. 1 mm diameter) that are just beginning to divide. The individual cyanobacteria are the small cells forming the long strands inside the spheres. Figure 4.2
make proteins and other cell structures, those synthetic processes require an energy source. Depending on the organism, energy can be obtained from chemical reactions triggered by the absorption of light (phototrophy, or photosynthesis) or from oxidation-reduction reactions that transfer electrons from high-energy compounds to make products of lower energy (chemotrophy). Chemotrophic organisms include two classes that use different sources of electron donors: chemoautotrophs and chemoheterotrophs. Chemoautotrophs oxidize inorganic chemicals H2, H2S, NH4, NO2–, and Fe2+ for energy. Chemoheterotrophs oxidize organic compounds such as sugars to obtain energy. NOTE: The following prefi xes for “-trophy” terms help distinguish different forms of energy-yielding metabolism. Carbon source for biomass AutoCO2 is fi xed and assembled into organic molecules. HeteroPreformed organic molecules are acquired from outside and assembled.
Energy source PhotoLight absorption excites electron to high-energy state. Chemo- Chemical electron donors are oxidized. Electron source LithoInorganic molecules donate electrons. Organo- Organic molecules donate electrons.
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In chemotrophy, the amount of energy harvested from oxidizing a compound is directly related to the compound’s reduction state. The more reduced the compound is, the more electrons it has to give up and the higher the potential energy yield. A reduced compound, such as glucose, can donate electrons to a less reduced (more oxidized) compound, such as nicotinamide adenine dinucleotide (NAD), releasing energy (in the form of donated electrons) and becoming oxidized in the process. NAD is a cell molecule critical to energy metabolism and is discussed, along with oxidation-reduction reactions, in Chapter 13. In short, microbes are classified on the basis of their carbon and energy acquisition as follows: ■
■
Autotrophs. Autotrophs build biomass by fi xing CO2 into complex organic molecules. Autotrophs gain energy through one of two general metabolic routes that either use or ignore light: Photoautotrophy generates energy through light absorption by the photolysis (light-activated breakdown) of H 2O or H2S. The energy is used to fi x CO2 into biomass. Chemoautotrophy (or lithotrophy) produces energy from oxidizing inorganic molecules such as iron, sulfur, or nitrogen. This energy is also used to fi x CO2 into biomass. Heterotrophs. Heterotrophs break down organic compounds from other organisms to gain energy and to harvest carbon for building their own biomass. Heterotrophic metabolism can be divided into two classes, also based on whether light is involved.
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Photoheterotrophy produces energy through photolysis of organic compounds. Organic compounds are broken down and used to build biomass. Chemoheterotrophy (or organotrophy) yields energy and carbon for biomass solely from organic compounds. Chemoorganotrophy is commonly called heterotrophy. Note that many species, particularly free-living soil and aquatic bacteria, can utilize more than one of these strategies, depending on environmental conditions. They do this by having multiple gene systems that are expressed under different conditions and whose products carry out different functions. For example, Rhodospirillum rubrum grows by photoheterotrophy when light is available and oxygen is absent; but when oxygen is available, the organism grows by respiration, without absorbing light. The survival and metabolism of any one group of organisms depend on the survival and metabolism of other groups of organisms. Even metazoa (multicellular organisms) rely on microbial metabolism to survive. For example, the cyanobacteria, a type of photosynthetic microorganism that originated 2.5–3.5 billion years ago, produce most of the oxygen we and other metazoa breathe. In fact, cyanobacteria also form the base of the marine food chain. The autotrophic cyanobacteria fi x carbon in the ocean and are eaten by heterotrophic protists. The protists then are devoured by fish that produce the CO2 that is fi xed by the cyanobacteria. The cyanobacteria also depend on the heterotrophic bacteria to consume the molecular oxygen that the cyanobacteria produce, since molecular oxygen is toxic to cyanobacteria.
Energy Is Stored for Later Use Whatever the source, energy once obtained must be converted to a form useful to the cell. This form can be chemical energy, such as that contained in the highenergy phosphate bond in adenosine triphosphate (ATP), or electrochemical energy, where energy is stored in the form of an electrical potential existing between compartments separated by a membrane (see Chapter 14). Energy stored by an electrical potential across the membrane is known as the membrane potential. A membrane potential is generated when chemical energy is used to pump protons (and in some cases Na+) outside of the cell, so that the proton (or Na+) concentration is greater outside the cell than inside. This ion movement produces an electrical gradient across the cell membrane, making the inside of the cell more negatively charged than the outside. The energy stored in the membrane potential can be used directly to move nutrients into the cell via specific transport proteins (see Section 3.3), to drive motors that rotate flagella, and to drive synthesis of ATP.
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The Nitrogen Cycle Depends on Bacteria and Archaea Nitrogen is an essential component of proteins, nucleic acids, and other cellular constituents, and as such is required in large amounts by living organisms. Nitrogen gas (N2) makes up nearly 79% of Earth’s atmosphere, but nitrogen gas is unavailable for use by most organisms because the triple bond between the two nitrogen atoms is highly stable and requires considerable energy to split them. For nitrogen to be used for growth, it must first be “fixed,” or converted to ammonium ions (NH4+). As with the carbon cycle, various groups of organisms collaborate to interconvert nitrogen gas, ammonium ions, and nitrate (NO3–) ions in what is called the nitrogen cycle. One group “fixes” atmospheric nitrogen while other bacteria do the opposite, transforming ammonia to nitrate (nitrification) and then converting nitrate to N2 (denitrification) (Fig. 4.3). For the environmental significance of nitrogen metabolism, see Chapter 22. Nitrogen-fi xing bacteria may be free-living in soil or water or they may form symbiotic associations with plants or other organisms. A symbiont is an organism that lives in intimate association with a second organism. Rhizobium, Sinorhizobium, and Bradyrhizobium species, for example, are nitrogen-fi xing symbionts in leguminous plants such as soybeans, chickpeas, and clover (Fig. 4.4). These plant symbionts take atmospheric nitrogen and convert it to the ammonia that the plant needs to form proteins and other essential compounds. This type of beneficial symbiosis is called mutualism. Although symbionts are the most widely known nitrogen-fi xing bacteria, the majority of nitrogen in soil and marine environments is fi xed by free-living bacteria and archaea. Once fi xed, how does nitrogen get back into the atmosphere? The nitrifying bacteria, such as Pseudomonas, Alcaligenes, and Bacillus, gain energy by oxidizing ammonia to produce nitrate (a form of chemoautotro-
N2
Denitrifiers
Nitrogen fixers
NO3–
NH4+ Nitrifiers
The nitrogen cycle. Dinitrogen gas (N2) is fixed by species of bacteria (nitrogen fixers) that possess the enzyme nitrogenase. Other bacteria oxidize ammonia (NH4+) to generate energy. Still others use oxidized forms of nitrogen, such as NO3–, as an alternative electron acceptor in place of O2. Figure 4.3
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Frank Dazzo, Michigan State University
Rhizobia
Tip of clover root hair 3 µm
Inga Spence/Photo Researchers, Inc.
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fungi require complex organic molecules for growth; their lifestyle involves predation, parasitism, or scavenging the dead. Most protists and fungi need mitochondria, which are the products of an ancient symbiotic partnering with internalized prokaryotes (discussed in Section 17.6). Heterotrophic fungi possess the exceptional ability to digest complex organic compounds such as lignin, a major component of wood and bark. In marine and aquatic systems, protists form a huge part of the food chain, so that eating trout or swordfish means, in effect, consuming trillions of protists. Just like photosynthetic bacteria, eukaryotic algae are photoautotrophs that produce biomass through photosynthesis. In addition to needing chloroplasts for photosynthesis, however, algae require mitochondria for energy production. (Recall that chloroplasts, like mitochondria, evolved from internalized bacteria; see Section 1.7.) Protist algae such as single-celled Euglena are mixotrophic, capable of utilizing photosynthesis or heterotrophic respiration, depending on environmental conditions. TO SU M MAR I Z E: ■
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Rhizobium and a legume. A. Symbiotic Rhizobium cells clustered on a clover root tip (SEM). The rhizobia shown here are clustered on the surface of the root. Soon they will start to invade the root and begin a symbiotic partnership that will benefit both organisms. B. Root nodules. After the rhizobia (approx. 0.9 µm × 3 µm) invade the plant root, symbiosis between plant and microbe produces nodules. Two partly crushed nodules (arrowheads) are shown with pink-colored contents. This color is caused by the presence of the pigment leghaemoglobin, which is found only in the nodules and is not produced by either the bacterium or the plant when grown alone.
Figure 4.4
phy). Denitrifying bacteria are heterotrophs that use the nitrate provided by the nitrifiers to produce a series of nitrogen compounds, ending with gaseous N2. The activity of denitrifying bacteria results in a substantial loss of nitrogen into the atmosphere that roughly balances the amount of nitrogen fi xation that occurs each year. As in the carbon cycle, this, once again, illustrates how nature manages to replenish the planet Earth.
Eukaryotic Microbes Include Consumers and Producers Eukaryotic microbes such as protists and fungi are heterotrophic consumers (discussed in Chapter 20). Protists and
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Microorganisms require certain essential macroand micronutrients to grow. Microbial genomes evolve in response to nutrient availability. Obligate intracellular bacteria lose metabolic pathways provided by their hosts and develop requirements for growth factors supplied by their hosts. In the global carbon cycle, autotrophs use CO2 as a carbon source, either through photosynthesis or through lithotrophy. Autotrophs make complex organic compounds that are consumed by heterotrophs. Nitrogen fi xers, nitrifiers, and denitrifiers contribute to the nitrogen cycle. All the preceding reactions are carried out by prokaryotes (bacteria or archaea), whereas eukaryotes carry out only a limited range of heterotrophic and photosynthetic reactions.
4.2
Nutrient Uptake
Whether a microbe is propelled by flagella toward a favorable habitat or, lacking motility, drifts through its environment courtesy of Brownian movement, it must be able to fi nd nutrients and move them across the membrane into the cytoplasm. The membrane, however, presents a daunting obstacle. Membranes are designed to separate what is outside the cell from what is inside. So, for a cell to gain sustenance from the environment, the membrane must be selectively permeable to nutrients the cell can use. A few compounds, such as oxygen and carbon dioxide,
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can passively diffuse across the membrane, but most cannot. Selective permeability is achieved in three ways: ■
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By the use of substrate-specific carrier proteins (permeases) in the membrane With the aid of dedicated nutrient-binding proteins that patrol the periplasmic space Through the action of membrane-spanning protein channels, or pores, that discriminate between substrates
Microbes must also overcome the problem of low nutrient concentrations in the natural environment (for example, lakes or streams). If the intracellular concentration of nutrients were no greater than the extracellular concentration, the cell would remain starved of most nutrients. To solve this dilemma, most organisms have evolved efficient transport systems that concentrate nutrients inside the cell relative to outside. However, moving molecules against a concentration gradient requires some form of energy. In contrast to environments where nutrients are available but exist at low concentrations (for example, aqueous environments), certain habitats have plenty of nutrients, but those nutrients are locked in a form that cannot be transported into the cell. Starch, a large, complex carbohydrate, is but one example. Many microbes unlock A. Glycerol porin
these nutrient vaults by secreting digestive proteins that break down complex carbohydrates or other molecules into smaller compounds that are easier to transport. The amazing techniques that cells use to extrude these large digestive proteins through the membrane and into their surrounding environment are discussed in Chapter 8.
Facilitated Diffusion: Transport Down a Concentration Gradient Although most transport systems use cellular energy to bring compounds into the cell, a few do not. Facilitated diffusion is a type of system that simply uses the concentration gradient of a compound to move that compound across the membrane from a compartment of higher concentration to a compartment of lower concentration. The best these systems can do is to equalize the internal and external concentrations of a solute; facilitated transport cannot move a molecule against its gradient. The most important facilitated diffusion transporters are those of the aquaporin family that transport water and small polar molecules such as glycerol. Glycerol transport is performed by an integral membrane protein in E. coli called GlpF. The structure of a glycerol channel is shown in Figure 4.5A. This protein channel allows passive uptake B.
Glycerol
Glycerol
1. Glycerol enters the GlpF channel. GlpF
Extracellular fluid
Cell membrane
Cytoplasm 2. A conformational change in GlpF opens the channel to the cytoplasm, and glycerol enters the cell.
Extracellular fluid
Cell membrane
In passive transport, solutes move down the concentration gradient.
Cytoplasm
Figure 4.5 Facilitated diffusion. A. The glycerol transporter of E. coli, viewed from the external side of the membrane, consists of a tetramer of four dimer channels formed by hydrophobic alpha helices alternating back and forth across the cell membrane. Each channel (blue and yellow) contains a glycerol (magenta). (PDB code: 1FX8) B. Facilitated diffusion of glycerol through GlpF. The protein facilitates the movement of the compound from outside the cell, where glycerol is at high concentration, to inside the cell where the concentration of glycerol is low.
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of glycerol, a polar molecule useful to cells for energy and for building phospholipids. The glycerol channel complex is viewed from the outer face of the membrane. The complex is a tetramer of four channels. Each channel transports a molecule of glycerol (magenta). In the membrane, GlpF reversibly assumes two conformations. One form exposes the glycerol-binding site to the external environment, whereas the second form exposes this site to the cytoplasm. When the concentration of glycerol is greater outside than inside the cell, the form with the binding site exposed to the exterior is more likely to fi nd and bind glycerol. After binding glycerol, GlpF changes shape, closing itself to the exterior and opening to the interior (Fig. 4.5B). Bound glycerol is then released into the cytoplasm (influx). Once the cytoplasmic concentration of glycerol equals the exterior concentration, bound glycerol can be released to either compartment. If the internal concentration exceeds the external concentration, a situation that can arise if a cell is moved from a high- to a low-glycerol environment, glycerol will move out of the cell (efflux). However, facilitated diffusion normally promotes glycerol influx, because the cell consumes the compound as it enters the cytoplasm, keeping cytoplasmic concentrations of glycerol low. GlpF homolog
Active Transport by Membrane Transporters Requires Energy Most forms of transport expend energy to take up molecules from outside the cell and concentrate them inside. The ability to import nutrients against their natural gradients is critical in aquatic habitats, where nutrient concentrations are low, and in soil habitats, where competition for nutrients is high. The simplest way to use energy to move molecules across a membrane is to exchange the energy of one chemical gradient for that of another. The most common chemical gradients used are those of ions, particularly the positively charged ions Na + and K+. These ions are kept at different concentrations on either side of the cell membrane. When an ion moves down its concentration gradient (from high to low), energy is released. Some transport proteins harness that free energy and use it to drive transport of a second molecule up, or against, its concentration gradient. This is called coupled transport. The two types of coupled transport systems are symport, where the two molecules travel in the same direction, and antiport, in which the actively transported molecule moves in the direction opposite to the driving ion. An example of a symporter is the lactose permease
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LacY of E. coli, one of the fi rst transport proteins whose function was elucidated. This work was carried out by the pioneering membrane biochemist H. Ronald Kaback. LacY moves lactose inward, powered by a proton that is also moving inward (symport). LacY proton-driven transport is said to be electrogenic because an unequal distribution of charge results (for example, symport of a neutral lactose molecule with H+ results in net movement of positive charge). An example of electroneutral coupled transport, in which there is no net transfer of charge, is that of the Na+/H + antiporter. The Na+/H + antiporter couples the export of Na+ with the import of a proton (antiport). Because molecules of like charge are exchanged, there is no net movement of charge. The mechanism by which Na+/H + antiporters function was elucidated by the Israeli biochemist Etana Padan. Sodium exchange is of universal importance for all organisms and is particularly critical for organisms living in a high-salt habitat (see Section 5.4). THOUGHT QUESTION 4.2 In what situation would antiport and symport be passive rather than active transport? THOUGHT QUESTION 4.3 What kind of transporter, other than an antiporter, could produce electroneutral coupled transport? Symport and antiport transporter proteins function by alternately opening one end or the other of a channel that spans the cell membrane. The channel contains solute-binding sites (Figs. 4.6A and B). When the channel is open to the high-concentration side of the membrane, the driving ion (solute) attaches to binding sites. The transport protein then changes shape to open that site to the low-concentration side of the membrane and the ion leaves. When and where the second (cotransported) solute binds depends on whether the transport protein is an antiporter or a symporter. Movie on antiport With all this ion traffic going on across the membrane, a careful accounting must be kept of how many ions are inside relative to outside. The cell must recirculate ions into and out of the cell to maintain certain gradients if the organism is to survive. Because key ATP-producing systems require an electrochemical gradient across the membrane, it is especially important to keep the interior of the cell negatively charged relative to the exterior. However, because the movement of many compounds is coupled to the import of positive ions, the electrochemical gradient will eventually dissipate, or depolarize, unless positive ions are also exported. Depolarization must be avoided
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B. Antiport
A. Symport
Antiporter opens to the outside, where the concentration of A is less.
Energy is released as one substrate (red) moves down its concentration gradient. This energy moves a second substrate (blue) against its gradient and into the cell.
Substrate A leaves its binding site, and substrate B (blue) then binds to its site.
Outside
Outside
Cell membrane Inside
Inside
Antiporter binds substrate A (red) on the cytoplasmic side of the membrane.
Coupled transport. A. Symport. B. Antiport. Substrate B (blue) is taken up against its gradient because of the energy released by substrate A (orange) traveling down its gradient. C. Ronald Kaback (left) of UCLA elucidated the mechanism of transport by the proton-driven lactose symporter LacY. D. Etana Padan (second from right) of The Hebrew University of Jerusalem dissected the molecular mechanism of the Na+/H+ antiporter NhaA.
Figure 4.6
because a depolarized cell loses membrane integrity and cannot carry out the simple transport functions needed to sustain life. A healthy cell maintains a proper charge balance using the electron transport chain to move protons out of the cell and by exchanging negatively and positively charged ions as needed. In addition to the direct linking of ion transport described for symport and antiport systems, ion circuitry is such that movement of one ion can be linked indirectly to movement of another molecule. For example, proton concentrations are typically greater outside than inside the cell. The inwardly directed proton gradient is called proton motive force. Proton motive force can impel the exit of Na+ through the Na+/H+ antiporter. The resulting Na+ gradient can drive the symport of amino acids into the cell. In this case, Na+ moves back into the cell down its gradient, and the energy released is tied to the import of an amino acid against its gradient.
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Antiporter opens to the inside of the cell. Substrate B is released in exchange for substrate A.
D.
Courtesy of Etana Padan
Courtesy of Ronald Kaback
C.
ABC Transporters Are Powered by ATP As we pointed out in Section 3.3, a major function of proton transport is to create the proton motive force that powers ATP synthesis (for details on proton motive force, see Chapter 14). The energy stored in ATP, whether generated by the proton current or by cytoplasmic means such as fermentation, can drive membrane transport of nutrients. The largest family of energy-driven transport systems is the ATP-binding cassette superfamily, also known as ABC transporters. These transporters are found in bacteria, archaea, and eukaryotes. It is impressive to note that nearly 5% of the E. coli genome is dedicated to producing the components of 70 different varieties of uptake and efflux ABC transporters. The uptake ABC transporters are critical for transporting nutrients such as maltose, histidine, arabinose, and galactose. The efflux ABC transporters are generally used as multidrug efflux pumps that allow microbes to
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survive exposures to hazardous chemicals. Lactococcus, for example, can use one pump, LmrP, to export a broad range of antibiotics, including tetracyclines, streptogramins, quinolones, macrolides, and aminoglycosides, conferring resistance to those drugs (see Section 27.6). An ABC transporter typically consists of two hydrophobic proteins that form a membrane channel and two peripheral cytoplasmic proteins that contain a highly conserved amino acid motif involved with binding ATP. Any conserved amino acid sequence found in a family of proteins is viewed as a “cassette” because the sequence appears to have been inserted into those proteins for specific functions (hence, ATP-binding cassette). The ABC transporter superfamily contains both uptake and efflux transport systems. The uptake systems (but not the efflux systems) possess an additional, extracytoplasmic protein, called a substrate-binding protein (SBP), that initially binds the substrate (also called solute). In gram-negative bacteria, these substrate-binding proteins float in the periplasmic space between the inner and outer membranes. In gram-positive bacteria, which lack an outer membrane, the proteins must be tethered to the cell surface. ABC transport (Fig. 4.7) starts with the SBP snagging the appropriate solute, either as it floats by a gram-positive microbe or as the molecule enters the periplasm of a gramnegative cell. Most substrates nonspecifically enter the periplasm of gram-negative organisms through the outer membrane pores, although some high-molecular-weight substrates, like vitamin B12, require the assistance of a spe-
Solutebinding protein
Solute
1. Solute binds to its cognate periplasmic binding protein, and the complex then binds to the membrane transporter. Periplasm
Cell membrane
Cytoplasm
2. The ATPase activity of one component (yellow) signals the opening of the channel (blue) and movement of the solute into the cell. ATP ADP + P
Figure 4.7
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ATP ADP + P
ABC transporters.
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cific outer membrane protein to move the substrate into the periplasm. Because SBPs have a high affinity for their cognate (matched) solutes, their use increases the efficiency of transport when concentrations of solute are low. Once united with its solute, the SBP binds to the periplasmic face of the channel protein and releases the solute, which now binds to a site on the channel protein. This interaction triggers a structural (or conformational) change in the channel protein that is telegraphed to the nucleotide-binding proteins associated with the cytoplasmic side. On receiving this signal, the nucleotide-binding proteins start hydrolyzing ATP and send a return conformational change through the channel, signaling the channel to open its cytoplasmic side and allow the solute to enter the cell. The three proteins that constitute ABC transporters appear to have arisen from a common ancestral porter and share a considerable amount of amino acid sequence homology. One unusual feature of ABC transporters is that although the membrane channel components and ATP-hydrolyzing components are generally present as distinctly separate polypeptides in uptake transporters, they are fused in efflux systems.
Siderophores Are Secreted to Scavenge Iron Iron, an essential nutrient of most cells, is mostly locked up in nature as Fe(OH)3 , which is insoluble and unavailable for transport. Many bacteria and fungi have solved this transport dilemma by synthesizing and secreting specialized molecules called siderophores (Greek for “iron bearer”) that have a very high affinity for whatever soluble ferric iron is available in the environment. These iron scavenger molecules are produced and sent forth by cells when the intracellular iron concentration is low (Fig. 4.8). In most gram-negative organisms, the siderophore binds iron in the environment, and the siderophore-iron complex then attaches to specific receptors in the outer membrane. At this point, either the iron is released directly and is passed to other transport proteins or the complex is transported across the cytoplasmic membrane by a dedicated ABC transporter. The iron is released intracellularly and reduced to Fe2+ for biosynthetic use. Other gram-negative microorganisms, such as Neisseria gonorrhaeae (the causative agent of gonorrhea), do not use siderophores at all but employ receptors on their surface that bind human iron complexes (for example, transferrin or lactoferrin) and wrest the iron from them.
Group Translocation against a Concentration Gradient The ABC transporters we have just considered increase concentrations of solute inside the cell relative to the outside concentration. They move nutrients “uphill” against
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(PEP), an intermediate in glycolysis, to attach a phosphate to specific sugars during their transport into the cell. Glucose, for example, is conFe3+ verted during transport to glucose 3+ 6-phosphate. The system has a modFe ular design that accommodates dif2a. The complex is transported to the periplasm Enterochelinferent substrates. There are common through an outer membrane protein (FepA) . . . iron complex elements used by all sugars that are transported by the PTS system and elements that are unique to a given carbohydrate (Fig. 4.9 ). Common Outer membrane elements located in the cytoplasm include Enzyme I (PtsI) and a histidine-rich protein called HPr (PtsH). FepA Enzyme I strips the high-energy 2b. . . . and is transported into the cell by an FepB phosphate from PEP and passes it ABC transporter that includes a periplasmic to HPr, which in turn distributes solute-binding protein (FepB) and the integral membrane proteins FepC, G, and D. the phosphate to various substratespecific transport proteins called Enzyme II. A typical Enzyme II comprises three domains (A, B, and C) that may be fused together as a single FepG FepD Cell polypeptide or assembled in a variety membrane of combinations. Regardless of the configuration, the phosphorylated FepC FepC ATPase HPr (HPr-P) transfers its phosphate ATP ATP to the Enzyme IIA domains/proteins, ADP + P ADP + P which relay the phosphate to their 3. Inside the cell, the iron is cognate Enzyme IIB domains/ released and reduced to Fe2+. proteins. Enzyme IIB fi nally delivers the phosphate to the specific 3+ 2+ + sugar that has been transported into Fe Fe the cell by the Enzyme IIC domain embedded in the cytoplasmic memFigure 4.8 Siderophores and iron transport. brane (for example, glucose is transported by Enzyme IIC and converted to glucose 6-phosphate by Enzyme IIB). In Chapter 9, we will see how this physiological sysa concentration gradient. An entirely different system tem impacts the genetic control of many other systems. known as group translocation cleverly accomplishes the same result but without really moving a substance “uphill.” Group translocation alters the substrate during Eukaryotes Transport Nutrients transport by attaching a new group (for example, phosby Endocytosis phate) on it. Because the modified nutrient inside the cell is a different chemical entity than the related compound Like prokaryotic cells, eukaryotic cells possess antiporters outside, the parent solute entering the cell is always movand symporters and use ABC transport systems as multiing down its concentration gradient, regardless of how drug efflux pumps, but they also employ another process, much solute has already been transported. Note that called endocytosis, which often precedes nutrient transthis process uses energy to chemically alter the solute. port across membranes. The lack of a rigid cell surface ABC transporters and group translocation systems both allows areas of the cytoplasmic membrane to invaginate involve active transport, but group translocation systems and pinch off to form membrane-enclosed vesicles called are not ABC transporters. endosomes (see Appendix 2, Fig. A2.8). Pinocytosis is The phosphotransferase system (PTS) is a wella form of endocytosis that pinches off small packets of characterized group translocation system present in medium to spawn small vacuoles (endosomes). Eukarymany bacteria. It uses energy from phosphoenolpyruvate otic microbes such as amebas engulf prey bacteria by a 1. E. coli synthesizes and secretes an iron-binding enterochelin that binds Fe3+.
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Enzyme II
Enzyme II components are modular.
2. Substrates are transformed by phosphorylation during transport.
1. Phosphate from PEP is passed along common elements of the PTS to the modular Enzyme II and ultimately to the substrate.
IIA
IIB
P
P
IIC Mannitol
Common elements of PTS
PEP
EI
HPr ~ P Mannitol 6-P
Pyruvate
EI ~ P
3. Enzyme IIC transports substrate across the cell membrane. Enzyme IIB transfers phosphate to the sugar transported across the cell by IIC.
HPr IIA
IIB
P
P
IIC
Enzyme I Glucose
Glucose 6-P
IIA
IIB
IIC
P P Mannose 6-P Cytoplasm
Mannose
Cell membrane
Extracellular fluid
Group translocation: the phosphotransferase system (PTS) of E. coli. The phosphate group from PEP is ultimately passed to the substrate during transport. The common elements of the PTS are Enzyme I (PtsI) and HPr (histidine-rich protein; PtsH). Each Enzyme II is specific for a given substrate and consists of modular components. Enzyme II for mannitol is one protein with three domains: A, B, and C. Enzyme II for glucose is really two proteins: One protein contains the A domain, and the B and C domains are joined to form the second protein. Mannose Enzyme II is designed with the opposite arrangement. Its A and B domains are fused into one protein, whereas the membrane protein is simply the C domain.
Figure 4.9
form of endocytosis called phagocytosis, producing large vacuoles (phagosomes). Lysosomes, cytoplasmic vacuoles that contain various digestive enzymes, then fuse with phagosomes or endosomes, creating phagolysosomes. As a result of the fusion, digestive enzymes gain access to the endocytosed material, degrading it to a form and size that can be transported across the vacuolar membrane and into the cytoplasm.
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Siderophores are secreted to bind ferric iron and transport it into the cell, where it is reduced to the more useful ferrous form. Siderophore-iron complexes enter cells with the help of ABC transporters. Group translocation systems chemically modify the solute during transport. Eukaryotes use endocytosis, a process in which small areas of the cytoplasmic membrane invaginate, trap extracellular fluid, and pinch off into the cytoplasm.
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Transport systems move nutrients across semipermeable membranes. Facilitated diffusion helps solutes move across a membrane from a region of high concentration to one of lower concentration. Antiporters and symporters are coupled transport systems in which energy released by moving a driving ion (H+ or Na+) from a region of high concentration to one of low concentration is harnessed and used to move a solute against its concentration gradient. ABC transporters use the energy from ATP hydrolysis to move solutes “uphill” against their concentration gradients.
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4.3
Culturing Bacteria
Microbes in nature usually exist in complex, multispecies communities, but for detailed studies, they must be grown separately in pure culture. It is nothing short of amazing—and humbling—that after 120 years of trying to grow microbes in the laboratory, we have succeeded in culturing only 0.1% of the microorganisms around us. Since the time of Koch in the late nineteenth century, microbiologists have used the same fundamental techniques to culture bacteria in the laboratory. It is true that improvements have been made, but the vast majority of the microbial world has yet to be tamed.
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Bacteria Are Grown in Culture Media For those organisms that can be cultured, we have access to a variety of culturing techniques that can be used for different purposes. Bacterial culture media may be either liquid or solid. A liquid, or broth, medium, in which organisms can move about freely, is useful when studying the growth characteristics of a single strain of a single species (that is, a pure culture). Liquid media are also convenient for examining growth kinetics and microbial biochemistry at different phases of growth. Solid media, usually gelled with agar, are useful when trying to separate mixtures of different organisms as they are found in the natural environment or in clinical specimens. University of Manitoba
Pure Cultures Can Be Obtained by Dilution Streaking or by Spread Plate Technique
B.
Yellow-pigmented bacterial colonies
Parm Randhawa, California Seed & Plant Lab, Inc.
A.
20 mm
Wrinkled bacterial colonies Fungus
Solid media are basically liquid media to which a solidifying agent has been added. The most versatile and widely Figure 4.10 Separation and growth of microbes on an agar surface. A. Colonies (1 to 5 mm diameter) of Acidovorax used solidifying agent is agar (for the development of aveane separated on an agar plate. This organism is a plant agar medium, see Section 1.5). Derived from seaweed, pathogen that causes watermelon fruit blotch. B. Mixture of agar forms an unusual gel that liquefies at 100°C but yellow-pigmented bacterial colonies, wrinkled bacterial colonies, does not solidify until cooled to about 40°C. Liquefied and fungus separated by dilution on an agar plate. agar medium poured into shallow, covered petri dishes cools and hardens to provide a large, flat surface on which a mixture of microorganisms can be streaked to separate individual cells. A. B. Each cell will divide and grow to form a distinct, Holding loop visible colony of cells (Fig. 4.10). As shown in Figure 4.11, a drop of liquid culture is collected using an inoculating loop and streaked across the agar plate surface in a pattern called dilution streaking. Organisms fall off the loop as it moves along the agar surface. Toward the end Hold the loop of the streak, few bacteria remain on the loop, flat against the so at that point, individual cells will land and agar and streak across surface. stick to different places on the agar surface. If the medium, whether artificial (for example, laboratory medium) or natural (for example, crab carapace) contains the proper nutrients and Reflame loop Start growth factors, a single cell will multiply into before changing many millions of offspring, forming a microcoldirection of streaking. ony. At fi rst visible only under a microscope, the microcolony grows into a visible droplet called Figure 4.11 Dilution streaking technique. A. A liquid culture is sampled with an inoculating loop and streaked across the plate in three to a colony (Figs. 4.11B and 4.12). A pure culture four areas, with the loop flamed between areas. The result is that dragging of the species, or one strain of a species, can the loop across the agar diminishes the number of organisms clinging to the be obtained by touching a single colony with a loop until only single cells are deposited at a given location. B. Salmonella sterile inoculating loop and inserting that loop enterica culture obtained by dilution streaking. into fresh liquid medium. It is important to note that the “one cell equals one colony” paradigm does not hold for all bacteria. Organisms such as Streptococci or
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John Foster, University of South Alabama
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Bacterial colonies. A. A three-day-old microcolony (biofilm) on the surface of a membrane used to treat municipal wastewater in Fountain Valley, California (SEM). B. Bacterial colony attacking a crab carapace.
John Foster, U. of South Alabama
Douglas Prince, U. of New Hampshire
B.
Figure 4.12
Staphylococci usually do not exist as single cells, but occur as chains or clusters of several cells. Thus, a cluster of 10 Staphyloccocus cells will only form one colony on an agar medium and is called a colony-forming unit (CFU). Another way to isolate pure colonies is the spread plate technique. Starting from a liquid culture of bacteria, a series of tenfold dilutions is made, and a small amount of each dilution is placed directly on the surface of separate agar plates (Fig. 4.13). The sample is spread over the surface of the plate using a heat-sterilized, bent glass rod. The early dilutions, those containing the most bacteria, will produce confluent growth that covers the entire agar surface. The later dilutions, containing fewer and fewer organisms, yield individual colonies. As we shall see later, spread plates not only enable us to isolate pure cultures but also can be used to enumerate the number of viable bacteria in the original growth tube. A viable organism is one that successfully replicates to form a colony. Thus, each colony on an agar plate represents one viable organism present in the original liquid culture.
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Figure 4.13 Tenfold dilutions, plating, and viable counts. A. A culture containing an unknown concentration of cells is serially diluted. One milliliter (ml) of culture is added to 9.0 ml of diluent broth and mixed, and then 1 ml of this 101 dilution is added to another 9.0 ml of diluent (10–2 dilution). These steps are repeated for further dilution, each of which lowers the cell number tenfold. After dilution, 1 ml of each dilution is spread onto an agar plate. B. Plates prepared as in (A) are incubated at 37°C to yield colonies. By multiplying the number of countable colonies (11 colonies on the 10–6 plate) by the reciprocal of the dilution factor you can calculate the number of cells (colony-forming units; CFUs) per milliliter in the original broth tube (11 × 106 = 1.1 × 107 CFUs per ml).
There is some disagreement about the use of the term viable cell. Is an organism that does not replicate but continues to metabolize viable? Consider, for instance, that human beings who have undergone sterilization are incapable of producing offspring, but they are certainly alive. Nevertheless, based on a tradition dating back to the time of Koch, it remains convention within the scientific community that a “viable” cell reproduces to form a colony on a plate. Organisms that appear to metabolize but for some reason cannot replicate have been referred to as
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dormant or “viable but nonculturable.” Increasingly, it seems probable that all viable but nonculturable organisms simply appear that way to us because we have not yet discovered the culture conditions necessary for them to reproduce.
Growth in Complex Media or in Synthetic Media Bacteria can be grown in nutrient-rich but poorly defi ned complex media or in precisely defi ned synthetic media. Recipes for complex media usually contain several poorly defi ned ingredients, such as yeast extract or beef extract, whose exact composition is not known. These additives include a rich variety of amino acids, peptides, nucleosides, vitamins, and some sugars. Some organisms are particularly fastidious, requiring that components of blood be added to a basic complex medium. The complex medium is now called an enriched medium. Complex, or rich, media provide many of the chemical building blocks that a cell would otherwise have to synthesize on its own. For example, instead of making proteins that synthesize tryptophan, all the cell needs is a membrane transport system to harvest prefabricated tryptophan from the medium. Likewise, fastidious organisms that require blood in their media may reclaim the heme released from red blood cells as their own, using it as an enzyme prosthetic group, a group critical to enzyme function (for example, the heme group in cytochromes). All of this saves the scavenging cell a tremendous amount of energy, and as a result, bacteria tend to grow fastest in complex media. An argument can be made that complex media mimics the rich environment that pathogens encounter in an animal host. However, the metabolism of a microbe growing in a complex medium is hard to characterize. How would you know whether E. coli possesses the ability to make tryptophan if the bacterium grows only in complex media? Fortunately, some organisms grow in fully defi ned synthetic media. In preparing a synthetic medium, one starts with water, then adds various salts, carbon, nitrogen, and energy sources in precise amounts. For self-reliant organisms like E. coli or Bacillus subtilis, that is all that is needed. Other organisms, such as Shigella species or mutant strains of E. coli or B. subtilis, require additional ingredients to satisfy requirements imposed by the absence of specific metabolic pathways. THOUGHT QUESTION 4.4 What would be the phenotype (growth characteristic) of a cell that lacks the trp genes (genes required for the synthesis of tryptophan)? What would be the phenotype of a cell missing the lac genes (genes whose products catabolize the carbohydrate lactose)?
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Selective and Differential Media Reveal Differences in Metabolism Microorganisms are remarkably diverse with respect to their metabolic capabilities and resistance to certain toxic agents. These differences are exploited in selective media, which favor the growth of one organism over another, and in differential media, which expose biochemical differences between two species that grow equally well. For example, gram-negative bacteria, with their outer membrane, are much more resistant than gram-positive microbes to detergents like bile salts and certain dyes, such as crystal violet. A solid medium containing bile salts and crystal violet is considered selective because it favors the growth of gramnegative organisms over gram-positive ones. On the other hand, E. coli and S. enterica are both gram-negative, but only E. coli can ferment lactose. Colonies of each can be distinguished on solid media containing lactose, the dye neutral red, and a nonfermentable carbon source like peptone. Both organisms grow in the medium, but E. coli ferments the lactose and produces acidic end products. The lower pH surrounding an E. coli colony causes the dye to enter the cells, turning the colony red. In stark contrast, S. enterica, a major cause of diarrhea, will grow on nonfermentable peptides, but, because it cannot ferment lactose, acidic end products are not produced and the colonies remain white, the natural color of the colony. In this example, growth in differential media easily distinguishes colonies of lactose fermenters from nonfermenters. Several media used in clinical microbiology are both selective and differential. MacConkey medium, for example, selects for growth of gram-negative bacteLactose fermenting (Lac+)
Nonfermenting (Lac–)
John Foster, University of South Alabama
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Figure 4.14 MacConkey medium, a culture medium both selective and differential. Dilution streak of a mixture of Lac+ and Lac– bacteria. Only gram-negative bacteria grow on lactose MacConkey (selective). Only a species capable of fermenting lactose produces pink colonies (differential).
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ria because it contains bile salts and crystal violet, which prevent the growth of gram-positives. The medium also includes lactose, neutral red, and peptones to differentiate lactose fermenters from nonfermenters. Thus, a culture grown in MacConkey medium will consist of gram-negative organisms that can be identified as lactose fermenters (red colonies) or nonfermenters (uncolored colonies; Fig. 4.14). This medium is of particular benefit when diagnosing the etiology (cause) of diarrheal disease because most normal flora are lactose fermenters, whereas two important pathogens, Salmonella and Shigella, are lactose nonfermenters. THOUGHT QUESTION 4.5 The addition of sheep blood to agar produces a very rich medium called blood agar. Do you think blood agar can be considered a selective medium? A differential medium? Hint: Some bacteria can lyse red blood cells. TO SU M MAR I Z E: ■
■ ■
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Microbes in nature usually exist in complex, multispecies communities, but for detailed studies they must be grown separately in pure culture. Bacteria can be cultured on solid or liquid media. Minimal defi ned media contain only those nutrients essential for growth. Complex, or rich, media contain many nutrients. Other media exploit specific differences between organisms and can be defined as selective or differential.
4.4
Counting Bacteria
Counting or quantifying organisms invisible to the naked eye is surprisingly difficult because each of the available techniques measures a different physical or biochemical aspect of growth. Thus, a cell density value (given as
1. Slide with shallow wells and inscribed grid (~400 squares, 0.0025 mm2 each).
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cells per milliliter) derived from one technique will not necessarily agree with the value obtained by a different method.
Direct Counting Enumerates Both Living and Dead Cells Microorganisms can be counted directly using a microscope. A dilution of a bacterial culture is placed on a special microscope slide called a hemocytometer (or, more specifically for bacteria, a Petroff-Hausser counting chamber, Fig. 4.15). Etched on the surface of the slide is a grid of precise dimensions, and placing a coverslip over the grid creates a space of precise volume. The number of organisms counted within that volume is used to calculate the concentration of cells in the original culture. However, “seeing” an organism under the microscope does not mean that the organism is alive. Living and dead cells are indistinguishable by this basic approach. Living cells may be distinguished from dead cells by fluorescence microscopy using fluorescent chemical dyes, as discussed in Chapter 2. For example, propidium iodide, a red dye, intercalates between DNA bases but cannot freely penetrate the energized membranes of living cells. Thus, only dead cells stain red under a fluorescence scope. Another dye, Syto-9, enters both living and dead cells, staining them both green. By combining Syto-9 with propidium iodide, living and dead cells can be distinguished: Living cells will stain green, whereas dead cells appear orange or yellow because both dyes enter, and Syto-9 (green) plus propidium (red) appears yellow (Fig. 4.16). Direct counting without microscopy can be accomplished by a Coulter counter. In the Coulter counter, a microbial culture is forced through a small orifice, through which flows an electrical current. Electrodes placed on both sides measure resistance. Every time
3. Bacterial suspension is added to wells and seeps under the coverslip to fill the shallow space of known volume over the grid.
4. Bacterial cells in each square are counted under a microscope.
0.2 mm apart
Figure 4.15 The Petroff-Hausser chamber for direct microscopic counts. Special slides have a precision grid etched on the surface. The organisms in several squares are counted, and their numbers are averaged. Knowing the dimensions of the grid and the height of the coverslip over the slide enables one to calculate the number of organisms in a milliliter.
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what conditions that gene is expressed and whether all cells in the population express that gene at the same time and to the same extent (Fig. 4.17B). Cheek cell nuclei
Live
Molecular Probes, Inc.
Dead
Live-dead stain. Live and dead bacteria visualized on freshly isolated human cheek epithelial cells using LIVE/DEAD BacLight Bacterial Viability Kit. Dead bacterial cells fluoresce orange or yellow because propidium (red) can enter the cells and intercalate the base pairs of DNA. Live cells fluoresce green because Syto-9 (green) enters the cell. The faint green smears are the outlines of cheek cells.
Figure 4.16
a cell passes through the orifice, electrical resistance increases, and the cell is counted. The Coulter counter, however, works best with larger eukaryotic cells, such as red blood cells; the instrument is not generally sensitive enough to detect individual bacteria. An electronic technique more suited to counting and separating bacterial cells requires an instrument called a fluorescence-activated cell sorter (FACS). In the FACS technique, bacterial cells that synthesize a fluorescent protein (see cyan fluorescent protein, Section 3.6) or that have been labeled with a fluorescent antibody or chemical are passed through a small orifice, as in the Coulter counter, and then past a laser (Fig. 4.17A). Detectors measure light scatter in the forward direction, a measure of particle size, and to the side, which indicates shape or granularity. In addition, the laser activates the fluorophore in the fluorescent antibody, and a detector measures fluorescence intensity. The FACS technique can be used to identify and count different populations of cells within a single culture based on cell size and level of fluorescence (that is, one subpopulation may fluoresce more than or less than another). For example, by placing the green fluorescent protein gene (gfp) under the control of the regulatory DNA sequences of a bacterial gene (creating a gene fusion), researchers can count the cells expressing that gene using FACS analysis; this, in turn, makes it possible to determine under
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Viable Counts Estimate the Number of Cells That Can Form Colonies Viable cells, as noted previously, are those that can replicate and form colonies on a plate. To obtain a viable cell count, dilutions of a liquid culture can be plated directly on an agar surface or added to liquid agar cooled to about 42–45°C. The agar is subsequently poured into an empty Petri plate (pour plate), where the agar cools further and solidifies. Because many bacteria resist short exposures to that temperature, individual cells retain the ability to form colonies on, and in, the pour plate. After colonies form, they are counted, and the original cell number is calculated. For example, if 100 colonies are observed in a pour plate made with 100 microliters (µl) of a 10 –3 dilution of a culture, there are 106 organisms per milliliter of the original culture. Although viable counts are widely used in research, there are problems using this method to measure cell number and determine cultural characteristics. One issue is that colony counting does not reflect cell size or growth stage. Even more problematic is that colony counts usually underestimate the number of living cells in a culture. As noted earlier, metabolically active cells that do not form colonies on agar plates will typically not be counted as alive. Cells damaged for one reason or another, while still alive, may be too compromised to divide. Comparing a viable count with a direct count obtained from a live-dead stain can expose the presence of damaged cells. Organisms such as Streptococci pose another problem. These organisms grow in chains. If individual cells in a chain are not separated prior to plating, each colony seen will have formed from a group of cells. This will cause actual cell numbers to be underestimated and is why the results of this counting technique are reported as colony-forming units (CFUs) rather than cells.
Biochemical Assays Measure Overall Population Size In contrast to methods that visualize individual cells, assays of cell mass, protein content, or metabolic rate measure the overall size of a population of cells. The most straightforward but time-consuming biochemical approach to monitoring population growth is to measure the dry weight of a culture. Cells are collected by centrifugation, washed, dried in an oven, and weighed. Because bacterial cells weigh very little, a large volume of culture must be harvested to obtain measurements, which makes this technique quite insensitive. A more accurate alternative is to measure increases in protein levels, which correlate with increases in cell number. Protein levels are more easily measured and use relatively sensitive assays.
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Optical Density: A Rapid Measure of Population Growth The phenomenon of light scattering was introduced in Chapter 2. We noted that although individual bacteria could not be resolved based on the detection of scattered light by the unaided human eye, the presence of numerous bacteria in a tube of medium could be detected as a cloudy appearance. The decrease in intensity of a light beam due to the scattering of light by a suspension of particles is measured as optical density. Note that only parA. Flow cell body
Sample stream
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Figure 4.17 Fluorescence-activated cell sorter. A. Schematic diagram of a FACS apparatus (bidirectional sorting). B. Separation of GFP-producing E. coli from non-GFP-producing E. coli. The low-level fluorescence in the cells on the left is baseline fluorescence (autofluorescence). The scatterplot displays the same FACS data, showing the size distribution of cells (x-axis) with respect to level of fluorescence (y-axis). The larger cells may be ones that are about to divide.
Fluorescent cells of increasing size Non-GFPproducing cells
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ticles in suspension, such as cells and macromolecules, can scatter light. True solutions, such as a solution of salt or of glucose, are clear even at high concentration. The optical density of light scattered by bacteria is a very useful tool for estimating population size. The method is quick and easy, but because light scattering is a complex function of cell number and volume, optical density provides only an approximate result that must be corrected using a standard curve. Typically, a standard curve is obtained that plots viable counts for cultures versus optical densities as measured by a spectrophotometer. Thereafter, the optical density of a growing culture is measured, and the standard curve is used to estimate cell number at any given point during growth. One inherent problem in estimating cell numbers based on optical density is that a cell’s volume can vary, depending on its growth stage, altering its light-scattering properties. Thus, the cell number estimated by the standard curve may deviate from the true number. Another problem is that dead cells also scatter light. Clearly, using optical density to estimate viable count can be misleading, especially when measuring populations in stationary phase.
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Microorganisms in culture may be counted directly under a microscope, with or without staining, or by using a fluorescence-activated cell sorter. Microorganisms can be counted indirectly, as in viable counts and measurement of dry weight, protein levels, or optical density. A viable bacterial organism is defined as being capable of replicating and forming a colony on a solidmedium surface.
4.5
The Growth Cycle
How do microbes grow? What determines their rate of growth? And when does growth start in a nongrowing population? In nature, the answers to these questions are extremely complex. Most microbes in nature do not exist alone, but in complex communities of microbes and multicellular organisms. Studying the growth of multicellular forms requires considering them as a unit. Yet these same multicellular forms can send off planktonic cells, freeliving organisms that grow and multiply on their own. Nevertheless, all species at one time or another exhibit both rapid growth and nongrowth, as well as many phases in between. For clarity, we present here the principles of rapid growth, while bearing in mind the actual diversity of growth situations in nature. The ultimate goal of any species is to make more of its own kind. This fundamental law ensures survival of the species. A typical bacterium (at least the typical bacterium that can be cultured in the laboratory) grows by increasing in length and mass, which facilitates expansion of its nucleoid as its DNA replicates (see Section 3.3). As DNA replication nears completion, the cell, in response to com-
plex genetic signals, begins to synthesize an equatorial septum that ultimately separates the two daughter cells. In this overall process called binary fission, one parent cell splits into two equal daughter cells (Fig. 4.18A). Note, however, that although a majority of culturable bacteria divide symmetrically in two equal halves, some species divide asymmetrically. For example, the bacterium Caulobacter forms a stalked cell that remains fixed to a solid surface but reproduces by budding from one end to produce small, unstalked motile cells. The marine organism Hyphomicrobium also replicates asymmetrically by budding, releasing a smaller cell from a stalked parent (Fig. 4.18B). Eukaryotic microbes divide by a special form of cell fission involving mitosis, the segregation of pairs of chromosomes within the nucleus (see Section 20.2 and Appendix 2). Some eukaryotes also undergo more complex life cycles involving budding and diverse morphological forms. Nevertheless, a large population of eukaryotic microbes will exhibit the same mathematical functions of growth seen in bacteria; indeed, these same growth patterns are seen in large populations of multicellular organisms, including ourselves.
Unlimited Population Growth Is Exponential The process of reproduction has implications for growth not only of the individual, but also of populations. If we assume that growth occurs without limits, what happens to the population? The unlimited growth of any population obeys a simple law: The growth rate, or rate of increase in population numbers or biomass, is proportional to the population size at a given time. Such a growth rate is called exponential because it generates an exponential curve, a curve whose slope increases continually. Figure 4.18 Symmetrical and asymmetrical cell division. A. Symmetrical cell division, or binary fission, in Lactobacillus sp. (SEM). B. Budding of the marine bacterium Hyphomicrobium (approx. 4 µm long).
B.
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How does binary fission of cells generate an exponential curve? If each cell produces two cells per generation, then the population size at any given time is proportional to 2n, where the exponent n represents the number of generations (replacement of parents by offspring) that have taken place between two time points. Thus, cell number rises exponentially. Many microbes, however, have replication cycles based on numbers other than two. For example, some cyanobacteria form cell aggregates that divide by multiple fission, releasing dozens of daughter cells. The cyanobacterium enlarges without dividing, and then suddenly divides many times without separating. The cell mass breaks open to release hundreds of progeny cells. It is important to note that simple binary fission is not the only kind of reproduction that would generate an exponential curve. Any generation size will yield exponential growth. For example, the imaginary, softballsized, fuzzy creatures called “tribbles” created in an episode of the television series Star Trek supposedly produced 11 offspring per generation and soon overran the starship. The rate of increase of the tribble population is proportional to 11n, where n is the number of tribble generations (Fig. 4.19A). In comparison with bacterial reproduction, which produces only 2 cells per generation, we might expect the growth rate of tribbles to be dramatically faster. In fact, this is the case for the fi rst few generations. But after a few more generations, the bacterial growth curve eventually achieves the same steep slope (that is, rate) as the tribble curve (Fig. 4.19B); the two curves are, in fact, graphical representations of the same kind of exponential function.
A Constant Generation Time Results in Logarithmic Growth A variable not accounted for in Figure 4.19B is the length of time from one generation to the next. In an environment with unlimited resources, bacteria divide at a constant interval called the generation time. The length of that interval varies with respect to many parameters, including the bacterial species, type of medium, temperature, and pH. The generation time for cells in culture is also known as the doubling time, because the population of cells doubles over one generation. For example, one cell of E. coli placed into a complex medium will divide every 20 minutes. After 1 hour of growth (three generations), that one cell will have become eight (1 to 2, 2 to 4, 4 to 8). Because cell number (N) doubles with each division, the increase in cell number over time is exponential, not linear. A linear increase would occur if cell number rose by a fixed amount after every generation (for example, 1 to 2, 2 to 3, 3 to 4). Starting with any number of organisms (N0 ), the number of organisms after n generations will be N0 × 2n. For example, a single cell after three generations (n = 3) will produce 1 cell × 23 = 8 cells
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THOUGHT QUESTION 4.6 A virus such as influenza virus might produce 800 progeny virus particles from one infected host cell. How would you mathematically represent the exponential growth of the virus? What practical factors might limit such growth?
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Reproduction: from one to many. A. Exponential growth of a hypothetical organism (tribbles from the Star Trek series) that produces 11 progeny per generation, compared with an organism that produces 2 offspring per generation. B. Growth curves for a population that increases 11-fold per generation and for a population that increases twofold per generation.
Figure 4.19
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B. Bacterial growth curve
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Thiobacillus thiooxidans grown on sulfur at different starting concentrations.
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Bacterial growth curves. A. Theoretical growth curve of a bacterial suspension measured by optical density at a wavelength of 600 nm. B. Phases of bacterial growth in a typical batch culture. C. Published growth curves of Thiobacillus thiooxidans, an acidophile that oxidizes sulfur to sulfuric acid. Whatever the starting cell density, the culture grows exponentially until it runs out of sulfur; then it enters the stationary phase.
Figure 4.20
Source: C. Yasuhiro Konishi, et al. 1995. Applied and Environmental Biology 61:3617.
The number of generations that an exponential culture undergoes in a given time period can be calculated if the number of cells at the start of the period (N0 ) and the number of cells at the end of the period (Nt) are known. Methods such as viable count are used to make those determinations. To simplify calculations, instead of using base 2 logarithms, the log2 expressions are converted to base 10 through division by a factor of log10 2, which is approximately 0.301. Thus: Nt = N0 × 2n
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In practice, exponential growth occurs only for a short period when all nutrients are in full supply and the concentration of waste products has not become a limiting factor. The rate of exponential growth can be expressed as the mean growth rate constant (k), which is the number of generations (n) per unit time (usually generations per hour). Even if we do not know the generation time, we can calculate k if we know the number of organisms at time zero (N0 ) and the number of organisms after incubation time t (Nt ) as follows: k = n/t = (log10 Nt – log10 N0) ÷ 0.301t Thus, a culture with a generation time of 20 minutes (0.33 hour) will have a mean growth rate constant of 3 generations per hour (k = 3). For a culture doubling every 120 minutes (2 hours), k is 0.5. Note from these examples that the mean generation time (g) in hours is the reciprocal of the mean growth rate constant: g = 1/k The growth rate constant can also be calculated from the slope of log10 N over time, where N is a relative measure of culture density, such as the optical density measured in a spectrophotometer (Fig. 4.20A). The units of N do not matter because we are always looking at ratios of cell numbers relative to an earlier level. For example, we can use the following series of optical density (OD) measurements to measure N:
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OD600
log10 OD600
0 15 30 45 60
0.05 0.08 0.13 0.20 0.33
–1.30 –1.10 –0.89 –0.69 –0.48
If we plot log10 OD600 versus time, we obtain a line with a slope of 0.0136/min. Substituting in equation 4.1, we obtain k, the growth rate constant (doubling time): k = (0.0136/min)(60 min/h) ÷ 0.301 = 2.7 doublings per hour The steeper the slope, the faster the organisms are dividing. THOUGHT QUESTION 4.7 Suppose 1,000 bacteria are inoculated in a tube of minimal salts medium, where they double once an hour; and 10 bacteria are inoculated into rich medium, where they double in 20 minutes. Which tube will have more bacteria after 2 hours? After 4 hours? THOUGHT QUESTION 4.8 An exponentially growing culture has an optical density at 600 nm (OD600) of 0.2 after 30 minutes and an OD600 of 0.8 after 80 minutes. What is the doubling time? THOUGHT QUESTION 4.9 Figure 4.20C shows growth curves for different population densities of Thiobacillus thiooxidans when the concentration of sulfur in the medium is constant. Draw the growth curves you would expect to see if the initial population density was constant but the concentration of sulfur varied. The mathematics of exponential growth is relatively straightforward, but remember that microbes grow differently in pure culture (very rare in nature) than they do in mixed communities, where neighboring cells produce all kinds of substances that may feed or poison other microbes. In mixed communities, the microbes may grow planktonically (floating in liquid), as in the open ocean, or as a biofi lm on solid matter suspended in that ocean. In each instance, the mathematics of exponential growth apply at least until the community reaches a density at which different species begin to compete. THOUGHT QUESTION 4.10 It takes 40 minutes for a typical E. coli cell to completely replicate its chromosome and about 20 minutes to prepare for another round of replication. Yet the organism enjoys a 20-minute generation time growing at 37°C in complex medium. How is this possible?
Stages of Growth in Batch Culture Exponential growth never lasts indefi nitely because nutrient consumption and toxic by-products eventually slow
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the growth rate until it halts altogether. The simplest way to model the effects of changing conditions is to culture bacteria in liquid medium within a closed system, such as a flask or test tube. This is called batch culture. In batch culture, no fresh medium is added during incubation; thus, nutrient concentrations decline and waste products accumulate during growth. These changing conditions profoundly affect bacterial physiology and growth and illustrate the remarkable ability of bacteria to adapt to their environment. As medium conditions deteriorate, alterations occur in membrane composition, cell size, and metabolic pathways, all of which impact generation time. Microbes possess intricate, self-preserving genetic and metabolic mechanisms that slow growth before their cells lose viability. Because many bacteria replicate by binary fission, the plotting of culture growth (as represented by the logarithm of the cell number) versus incubation time allows us to see the effect of changing conditions on generation time and reveals several stages of growth (Fig. 4.20B). Lag phase. Cells transferred from an old culture to fresh
growth media need time to detect their environment, express specific genes, and synthesize components needed to institute rapid growth. As a result, bacteria inoculated into fresh media typically experience a lag period, or lag phase, where cells do not divide. There are several reasons for this. Cells taken from an aged culture may be damaged and require time for repair. Carbon, nitrogen, or energy sources different from those originally used by the seed culture must be sensed, and the appropriate enzyme systems must be synthesized. The length of lag phase varies, depending on the age of the culture, changes in temperature, and the differences between the new and old media (for example, changes in nutrient levels, pH, and salt concentrations). For example, transferring cells from a complex medium to a fresh complex medium results in a very short lag phase, whereas cells grown in a complex medium and then plunged into a minimal defined medium experience a protracted lag phase, during which time they readjust to synthesize all the amino acids, nucleotides, and other metabolites originally supplied by the complex medium. Early log, or exponential, phase. Once cells have retooled their physiology to accommodate the new environment, they begin to grow exponentially and enter what is called exponential, or logarithmic (log) phase. Exponential growth is balanced growth, where all cell components are synthesized at constant rates relative to each other. At this stage, cells are growing and dividing at the maximum rate possible based on the medium and growth conditions provided (such as, temperature, pH, and osmolarity). Cells are largest at this stage of growth. This is the linear part of the growth curve. If cell division were synchronized and all cells divided at the same time, the growth curve during this period would appear as a series of steps with cell
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numbers doubling instantly after every generation time. But batch cultures are not synchronous. Every cell has an equal generation time, but each cell divides at a slightly different moment, making the cell number rise smoothly. Cells enjoying balanced, exponential growth are temporarily thrown into metabolic chaos (unbalanced growth) when their medium is abruptly changed. Nutritional downshift (moving cells from a good carbon source such as glucose to a poorer carbon source such as succinate) or nutritional upshift (moving cells to a better carbon source) casts cells into unbalanced growth. Downshift to a carbon source with a lower energy yield not only means that a different set of enzymes must be made and employed to use the carbon source, but also means that the previous high rate of macromolecular synthesis (such as ribosome synthesis) used to support a fast generation time is now too rapid relative to the lower energy yield. Failure to adjust will lead to increased mistakes in RNA, protein, and DNA synthesis, depletion of key energy stores, and ultimately death. Microbes, however, possess molecular fail-safe systems that immediately dampen rates of macromolecular synthesis until they come into balance with the rest of metabolism. In contrast, nutritional upshift means that the cell will start making more energy (ATP) than it can use at its current rate of macromolecular synthesis. The same fail-safe mechanisms used during downshift will, during upshift, kick macromolecular synthesis into a higher gear, once again establishing balanced growth. Nutritional upshift causes cells to reenter log phase but with a shorter generation time. Late log phase. As cell density (number of cells per milliliter) rises during log phase, the rate of doubling eventually slows, and a new set of growth-phase–dependent genes are expressed. At this point, some species can also begin to detect the presence of others by sending and receiving chemical signals in a process known as quorum sensing (discussed in Chapter 10). Stationary phase. Eventually, cell numbers stop rising
owing to lack of a key nutrient or buildup of waste products. This occurs for bacteria grown in a complex medium when cell density rises above 109 cells per milliliter, but it can occur at lower cell densities if nutrients are limiting. At this point, the growth curve levels off and the culture enters what is called stationary phase. In contrast to bacteria, eukaryotic microorganisms, such as protozoa, enter stationary phase at much lower cell numbers, usually around 106 organisms per milliliter. The reason for this is simply that eukaryotic cells are bigger than bacteria. Bigger cells use more nutrient and run out of it sooner. If they did not change their physiology, microbes would be very vulnerable once entering stationary phase. Because cells in stationary phase are not as metabolically nimble as cells in exponential phase, damage from oxygen radicals and toxic by-products of metabolism would read-
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ily kill them. As an avoidance strategy, some bacteria differentiate into very resistant spores in response to nutrient depletion (see Section 4.7), while other bacteria undergo less dramatic but very effective molecular reprogramming. The microbial model organism E. coli, for example, adjusts to stationary phase by decreasing its size, minimizing the volume of its cytoplasm compared with the volume of its nucleoid. Less nutrient is required to sustain the smaller cell. New stress resistance enzymes are also synthesized to handle oxygen radicals, protect DNA and proteins, and increase cell wall strength through increased peptidoglycan cross-linking. As a result, E. coli cells in stationary phase become more resistant to heat, osmotic pressure, pH changes, and other stresses that they might encounter while waiting for a new supply of nutrients. Death phase. Without reprieve in the way of new nutrients, cells in stationary phase will eventually succumb to toxic chemicals present in the environment. Like the growth rate, the death rate, the rate at which cells die, is logarithmic. The death rate, however, is a negative exponential function. Recall that the increase in cell number during exponential phase is a positive exponential function of time. In death phase, the number of cells that die in a given time period is proportional to the number that existed at the beginning of the time period. Determining microbial death rates is critical to the study of food preservation and to antibiotic development (further discussed in Chapters 5, 16, and 27). Although death curves are basically logarithmic, exact death rates are difficult to defi ne because mutations arise that promote survival, and some cells grow by cannibalizing others. Consequently, the death phase is extremely prolonged. A portion of the cells will often survive for months, years, or even decades. In Figure 4.20C, we see an actual bacterial growth curve from a study of T. thiooxidans, an organism that oxidizes sulfur to sulfuric acid and grows below pH 1. Growth curves are shown for several different starting concentrations of bacteria. In each case, the bacteria grow exponentially for several days, until they have exhausted the sulfur in their growth medium. Their growth then slows until they enter stationary phase.
THOUGHT QUESTION 4.11 What can happen to the growth curve when a culture medium contains two carbon sources, one a preferred carbon source of growth-limiting concentration and a second, nonpreferred source? (See Section 10.3.) THOUGHT QUESTION 4.12 How would you modify the equations describing microbial growth rate to describe the rate of death? THOUGHT QUESTION 4.13 Why are cells in log phase larger than cells in stationary phase?
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Courtesy of Global Medical Instrumentation, Inc.
Fresh media (ingested food) Fresh medium
Culture “flask” (intestines) Culture flask Spent medium, wastes, and excess microbes
Wastes and excess microbes
Chemostats and continuous culture. A. The basic chemostat ensures logarithmic growth by constantly adding and removing equal amounts of culture media. B. Note that the human gastrointestinal tract is engineered much like a chemostat in that new nutrients are always arriving from the throat while equal amounts of bacterial culture exit in fecal waste. C. A modern chemostat.
Figure 4.21
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Longer
Bacterial mass
Generation time
Generation time (min)
In the classic growth curve that develops in closed systems, the exponential phase spans only a few generations. In open systems, however, where fresh medium is continually added to a culture and an equal amount of culture is constantly siphoned off, bacterial populations can be maintained in exponential phase at a constant cell mass for extended periods of time. In this type of growth pattern, known as continuous culture, all cells in a population achieve a steady state, which permits detailed analysis of microbial physiology at different growth rates. The chemostat is a continuous culture system in which the diluting medium contains a limiting amount of an essential nutrient (Fig. 4.21). Increasing the flow rate increases the amount of nutrient available to the microbe. The more nutrient available, the faster a cell’s mass will increase. Because cell division is triggered at a defined cell mass, it follows that the growth rate in a chemostat is directly related to the dilution rate, or flow rate (milliliters per hour divided by the vessel volume). The more nutrient a culture receives as a result of increasing flow rate, the faster those cells can replicate (that is, the shorter the generation time). The complex relationships among dilution rate, cell mass, and generation time in a chemostat are illustrated in Figure 4.22. The curves in this figure represent a typical experimental result, which can vary with organism, limiting nutrient, temperature, and so on. Depending on the experimental conditions, the shapes of the curves may change, but the general relationships will remain similar. Note that at moderate dilution rates (defined differently for each species), an increased flow rate of medium
Steady state
Greater
Nutrient Bacterial mass
Continuous Culture Maintains Constant Cell Mass during Exponential Growth
Nutrient concentration in vessel
Dilution rate (flow rate)
Washout Faster
Relationships between dilution rate, cell mass, and generation time. As the dilution rate increases in a chemostat (meaning more nutrient is fed to the culture), the generation time decreases (the cells divide more quickly) and the cell mass of the culture increases. This continues until the rate of dilution exceeds the division rate, at which point cells are washed from the vessel faster than they can be replaced by division and the cell mass decreases. The y-axis varies, depending on the curve, as labeled in figure.
Figure 4.22
through the system increases division rate (thus, generation time decreases as the cells divide faster). A constant cell mass, or density, is maintained over a range of flow rates because the amount of culture (and cells) removed from the vessel exactly compensates for the increased rate of cell division.
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At faster and faster flow rates, cells are eventually removed more quickly than they can be replenished by division, so cell density (cell mass) decreases in the vessel. This is called washout. Notice, in contrast, that at very low dilution (flow) rates, an increase in rate will actually increase cell density. This is because at extremely low dilution rates, the nutrient is so limiting that cell mass cannot increase to the point necessary for division. If more nutrient becomes available, the system can maintain a higher cell mass and cell density because the cells are able to divide faster than they are removed. With slow flow rates, the removal of fluid is not a significant factor in determining cell density. The turbidostat is essentially a chemostat in which a photoelectric cell constantly monitors the optical density (turbidity) of the culture. The flow of medium through the vessel is then regulated to maintain a constant turbidity and thus cell density. The turbidostat is most suited to high dilution rates, where the numbers of cells can change quickly and overwhelm a less responsive system; the chemostat, which must be adjusted manually, is better suited at low dilution rates. Continuous cultures are used to study large numbers of cells at constant growth rate and cell mass for both research and industrial applications. Most bacteria in nature grow at very slow rates, a situation that can be mimicked in a chemostat. The physiology of these cells is quite different from what is typically observed using batch culture.
Biofilms: A Multicellular Microbe? Biofi lms can be constructed by a single species or by multiple, collaborating species and can form on a range of organic or inorganic surfaces (Fig. 4.23). The gramnegative bacterium Pseudomonas aeruginosa, for example, can form a single-species biofi lm on the lungs of patients with cystic fibrosis or on medical implants (Special Topic 4.1). Distinct stages in biofi lm development include initiation, maturation, maintenance, and dissolution. Bacterial biofi lms form when nutrients are plentiful. The goal is to stay where food is plentiful. Why should a microbe travel off to hunt for food when it is already available? Once nutrients become scarce, however, individuals detach from the community to forage for new sources of nutrient. Biofi lms in nature can take many different forms and serve different functions for different species. In addition, formation of biofi lms can be cued by different environmental signals in different species. Such signals include pH, iron concentration, temperature, oxygen availability, and the presence of certain amino acids.
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The growth cycle of organisms grown in liquid batch culture consists of lag phase, log phase, stationary phase, and death phase. The physiology of a bacterial population changes with growth phase. Continuous culture can be used to sustain a population of bacteria at a specified growth rate and cell density.
4.6
B.
Biofilms
Bacteria are typically thought of as unicellular; but in nature, many, if not most, bacteria form specialized, surface-attached communities called biofilms. Indeed, within aquatic environments, bacteria are found mainly associated with surfaces, a fact that underscores the importance of biofi lms in nature. Biofi lms also play critical roles in microbial pathogenesis and environmental quality, and cost the nation billions of dollars each year in equipment damage, product contamination, and medical infections. For example, pseudomonad or staphylococcal biofi lms can damage ventilators used to assist respiration and can act as direct sources of infection.
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Biofilm
Biofilms. A. A thermophilic microbial mat found attached to a rock in Yellowstone National Park (confocal scanning laser microscope image). The autofluorescence of these cyanobacterial cells of the genus Synechococcus was generated using a 568-nm krypton laser and a 590LP filter. B. Biofilm on a tooth. The biofilm that forms on teeth is called plaque.
Figure 4.23
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Biofilms, Disease, and Antibiotic Resistance
When causing diseases of plants, animals, or humans, bacteria preferentially exist in surface-attached biofilms. Attachment to host tissues and multicellular growth are important in many situations, from simple wound infections caused by Staphylococcus aureus to colonization of the lungs of cystic fibrosis patients by Pseudomonas aeruginosa. One characteristic of bacterial biofilms is a marked increase in antibiotic tolerance. The failure to cure infections that stem from bacterial-pathogen biofilm formation is well documented, but the basis of persistence remains unclear. The literature offers different explanations for increased tolerance of antibiotics by microcolonies, including reduced penetration of drug into the microcolony and an altered stress-resistant physiology by the cells in the interior of the colony. A study of P. aeruginosa biofilms clearly demonstrated the importance of multicellular cooperativity of cells within the biofilm. It appears that quorum sensing induces the drug-
tolerant state. Cells in the biofilm release a chemical signal molecule called an acyl homoserine lactone (AHL). The population of cells respond to these molecules by increasing resistance to certain antimicrobial agents. As shown in Figure 1, cells within a typical biofilm are resistant to the antibiotic tobramycin. The fluorescent stain used in this experiment will stain live cells green and dead cells red. However, simultaneous treatment of P. aeruginosa biofilms with a specific quorum-sensing blocker and the antibiotic tobramycin led to the effective killing of the cells by the antibiotic. The quorumsensing blocker, a novel, synthetically produced brominated furanone, did not reduce the viability of the P. aeruginosa cells. This result is consistent with the furanone specifically interfering with the AHL quorum-sensing system as seen by a microarray analysis. Hence, the shutdown of the AHL quorum-sensing system in the biofilm cells made them sensitive to the antibiotic. B. 10 µg/ml furanone; 100 µg/ml tobramycin Hentzer, et al. 2003. European Molecular Biology Organization
A. No furanone; 100 µg/ml tobramycin
Hentzer, et al. 2003. European Molecular Biology Organization
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Resistance of Pseudomonas aeruginosa biofilms to the antibiotic tobramycin is mediated by cell-cell signaling. Tobramycin sensitivity was tested in the presence and absence of the furanone compound C-30, which specifically inhibits cell-cell signaling. P. aeruginosa biofilms were grown in the absence (left panel) and presence (right panel) of 10 mM C-30. After three days, the biofilms were exposed to 100 µg/ml tobramycin for 24 hours. Bacterial viability was assayed by staining using the LIVE/DEAD BacLight BacterialViability Kit. Red areas are dead bacteria and green areas are live bacteria. Views are top and cross-sectional. Panels show top and side views of the biofilms.
Figure 1
Nevertheless, a common pattern emerges in the formation of many kinds of biofi lms (Fig. 4.24). First, the specific environmental signal induces a genetic program in planktonic cells. The planktonic cells then start to attach to nearby inanimate surfaces by means of flagella, pili, lipopolysaccharides, or other cell
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surface appendages, and begin to coat that surface with an organic monolayer of polysaccharides or glycoproteins to which more planktonic cells can attach. At this point, cells may move along surfaces using a twitching motility that involves the extension and retraction of a specific type of pilus. Ultimately, they stop moving and
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Dissolution
Planktonic forms
Attachment monolayer
Figure 4.24
Biofilm development.
Microcolonies
Maturation
Biofilm development in Pseudomonas.
fi rmly attach to the surface. As more and more cells bind to the surface, they can begin to communicate with each other by sending and receiving chemical signals in a process called quorum sensing. These chemical signal molecules are continually made by individual cells. Once the population reaches a certain number (analogous to an organizational “quorum”), the chemical signal reaches a specific concentration that the cells can sense. This triggers genetically regulated changes that cause cells to bind tenaciously to the substrate and to each other. Next, the cells form a thick extracellular matrix of polysaccharide polymers and entrapped organic and inorganic materials. These exopolysaccharides (EPSs), such as alginate produced by P. aeruginosa and colanic acid produced by E. coli, increase the antibiotic resistance of residents within the biofi lm. As the biofi lm matures, the amalgam of adherent bacteria and matrix takes on complex three-dimensional forms such as columns and streamers, creating channels through which nutrients flow. Sessile cells in a biofi lm chemically “talk” to each other in order to build microcolonies and keep water channels open. Little is known about how a biofi lm dissolves, although the process is thought to be triggered by starvation. P. aeruginosa produces an alginate lyase that can strip away the EPSs, but the regulatory pathways involved in releasing cells from biofi lms are not clear. It is important to keep in mind that most biofi lms in nature are consortia of several species. Multispecies biofi lms certainly demand interspecies communication, and individual species may perform specialized tasks in the community. Organisms adapted to life in extreme environments also form biofilms. Members of Archaea form biofilms in acid mine drainage (pH 0), where they contribute to the recycling of sulfur, and cyanobacterial biofilms are common in thermal springs. Suspended particles called “marine snow” are found in ocean environments and appear to be floating biofilms comprising many organisms that have
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Exopolysaccharide (EPS) production
not yet been identified. The particles appear capable of methanogenesis, nitrogen fi xation, and sulfide production, indicating that biofilm architecture can allow anaerobic metabolism to occur in an otherwise aerobic environment. Biofi lms TO SU M MAR I Z E: ■
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Biofilms are complex multicellular surface-attached microbial communities. Chemical signals enable bacteria to communicate (quorum sensing) and in some cases to form biofi lms. Biofilm development involves adherence of cells to a substrate, formation of microcolonies, and, ultimately, formation of complex channeled communities that generate new planktonic cells.
4.7
Cell Differentiation
Many bacteria faced with environmental stress undergo complex molecular reprogramming that includes changes in cell structure. Some species, like E. coli, experience relatively simple changes in cell structure, such as the formation of smaller cells or thicker cell surfaces. However, select species undergo elaborate cell differentiation processes. An example is Caulobacter crescentus, whose cells convert from the swimming form to the holdfast form before cell division. Each cell cycle then produces one sessile cell attached to its substrate by a holdfast, while its sister cell swims off in search of another habitat. Other species undergo far more elaborate transformations. The endospore formers generate heat-resistant capsules (spores) that can remain in suspended animation for thousands of years. Yet another group, the actinomycetes, form complex multicellular structures analogous to those of eukaryotes. In this case, cell struc-
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ture can change radically; and individual, freewheeling members of a species can relinquish their independence and band together, forming multicellular “organisms.” The actinomycete that produces streptomycin is one example. These differentiation programs illustrate the distinction between the presence of genes in a bacterial genome and their activation. Genes for differentiation are expressed only when the cell needs to survive stress. We will discuss gene activation in more detail in Chapter 10. Eukaryotic microbes also undergo highly complex life cycles. For example, Dictyostelium discoideum is a seemingly unremarkable ameba that grows as separate, independent cells. However, when challenged by adverse conditions such as starvation, the microbe secretes chemical signal molecules that choreograph a massive interaction of individuals to form complex multicellular structures. The developmental cycle of this organism may be compared to other eukaryotic microbes that are human parasites (discussed in Chapter 20). A.
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Endospores Are Bacteria in Suspended Animation Certain gram-positive genera, including important pathogens such as Clostridium tetani (tetanus), Clostridium botulinum (botulism), and Bacillus anthracis (anthrax) have the remarkable ability to develop dormant spores that are heat- and desiccation-resistant. Dessication and heat resistance are properties that make B. anthracis spores a potential bioweapon. Most of our knowledge of bacterial sporulation comes from the gram-positive soil bacterium B. subtilis. When growing in rich media, this microbe undergoes normal vegetative growth and can replicate every 30–60 minutes. However, starvation initiates an elaborate 8-hour genetic program that directs an asymmetrical cell division process and ultimately yields a spore (see Section 10.4). As shown in Figure 4.25 , sporulation can be divided into discrete stages primarily based on morphological
John Foster, U. of South Alabama
B.
Endospore formation. A. Photomicrograph of Bacillus subtilis spores. The cells (approx. 2 µm long) are stained with crystal violet. B. Peter Setlow (right) of the University of Connecticut figured out how proteins regulate the process of differentiation of endospores. C. Stages of endospore formation.
Courtesy of Peter Setlow
Figure 4.25
C. Sporulation
Vegetative growth
Axial filament
Mother cell Germination Stage I. DNA extends into an axial filament.
Spore
Stage II. Septum forms near one pole, separating forespore from mother cell.
Stage VII. Mother cell releases spore.
Forespore
Spore coat
Stage III. Mother cell engulfs the forespore, surrounding it with a second membrane.
Stage VI. Synthesis of dipicolinic acid and incorporation of calcium into spore coat.
Engulfment
Cortex Stage V. Forespore develops a cortex layer of peptidoglycan between original forespore membrane and the membrane from the mother cell.
Stage IV. Chromosomes of mother cell disintegrate. Exosporangium
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appearance. Stage 0 (not shown) represents the point at which the vegetative cell decides to use one of two potential polar division sites to begin septum formation instead of the central division site used for vegetative growth. Stage I involves replicating and stretching the DNA into a long axial fi lament that spans the length of the cell. Ultimately, one of the polar division sites wins out, and in stage II, septation occurs, dividing the cell into two unequal compartments, the forespore, which will ultimately become the spore, and the larger mother cell, from which it is derived. Each compartment contains a chromosome. In stage III of sporulation, the mother cell membrane engulfs the forespore. Next, the mother cell chromosome is destroyed and a thick peptidoglycan layer (cortex) is placed between the two membranes surrounding the forespore protoplast (stage IV). Layers of coat proteins are then deposited on the outer membrane in stage V. Stage VI completes development of spore resistance to heat and chemical insults. This last process includes the synthesis of dipicolinic acid and the uptake of calcium into the core of the spore. Finally, the mother cell, now called a sporangium, releases the mature spore (stage VII). Spores are resistant to many environmental stresses that would kill vegetative cells. The nature of this resistance is due, in part, to desiccation of the spore (they have only 10–30% of a vegetative cell’s water content). But, as discovered by Peter Setlow and colleagues, spores are also packed with small acid-soluble proteins (SASPs) that bind to and protect DNA. The SASP coat protects the spore’s DNA from damage by ultraviolet light and various toxic chemicals. A fully mature spore can exist in soil for at least 50– 100 years and spores have been known to last thousands of years. Once proper nutrient conditions arise, another genetic program, called germination, is triggered to wake the dormant cell, dissolve the spore coat, and release a viable vegetative cell.
Cyanobacteria Differentiate into Nitrogen-Fixing Heterocysts Some of the autotrophic cyanobacteria, such as Anabaena, not only make oxygen through photosynthesis but “fi x” atmospheric nitrogen to make ammonia. This is surprising because nitrogenase, the enzyme required to fi x nitrogen, is very sensitive to oxygen, so one might expect that photosynthesis and nitrogen fi xation would be two mutually exclusive physiological activities. Anabaena have solved this dilemma by developing specialized cells, called heterocysts, that function in nitrogen fi xation (Fig. 4.26). A tightly regulated genetic program converts every tenth photosynthetic cell to a heterocyst, which loses the capacity to fi x CO2 and forms a spe-
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Heterocyst
B. 20 µm
20 µm
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Cyanobacteria and heterocyst formation. A. Light microscope image of cyanobacteria Phanizomenon. B. The cyanobacterial genus Anabaena. The expression of genes in heterocysts is different from their expression in other cells. All cells in the figure contain a cyanobacter gene to which the gene for green fluorescent protein (GFP) has been spliced. Only cells that have formed heterocysts are expressing the fused gene, which makes the cell fluoresce bright green.
Figure 4.26
cialized envelope to limit O2 access. How this organism produces such a precise spacing of heterocysts is currently the subject of intensive research.
Starvation Induces Differentiation into Fruiting Bodies Certain species of bacteria, in the microbial equivalent of barn raising, produce architectural marvels called fruiting bodies. The gram-negative species Myxococcus xanthus uses a gliding motility (involving a type of pilus, not a flagellum) to travel on surfaces as individuals or to move together as a mob (Fig. 4.27). Starvation triggers a developmental cycle in which 100,000 or more individuals aggregate, rising into a mound called a fruiting body. At this point, the system resembles a stage in P. aeruginosa biofi lm formation. However, myxococci within the interior of the fruiting body differentiate into thick-walled, spherical spores that are released into the surroundings. The changes involved in this differentiation process require many cell-cell interactions and a complex genetic program that we do not fully understand.
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Myxococcus swarm erecting a fruiting body. Approximately 100,000 cells begin to aggregate, and over the course of 72 hours they erect a fruiting body.
Figure 4.27
Some Bacteria Differentiate to Form Eukaryotic-like Structures The actinomycetes (see Chapter 19) such as Streptomyces are bacteria that form mycelia and sporangia analogous to the fi lamentous structures of eukaryotic fungi (Fig. 4.28). Several developmental programs tied to nutrient availability are at work in this process (Fig. 4.29). Under favorable nutrient conditions, a germ tube emerges from a germinating spore, grows from its tip (tip extension), and forms branches that grow along the surface of its food source. This type of growth produces an intertwined net-
B.
Aerial hypha Spore-filled hyphae C. Society for General Microbiology, Reading, UK
Kim Findlay and Mark Buttner, John Innes Centre, Norwich, UK
Dennis Kunkel Microscopy, Inc.
A.
work of long multinucleate fi laments (hyphae) collectively called substrate mycelia. After a few days, new genes are activated that cause the hyphae to grow upward, rising above the surface to form aerial mycelia. Compartments at the tips of these aerial hyphae contain 20–30 copies of the genome. Aerial hyphae stop growing as nutrients decline, triggering a developmental program that synthesizes antibiotics and produces spores (arthrospores) that are fundamentally different from endospores. This program lays down multiple septa that subdivide the compartment into single-genome prespores. The shape of the prespore then changes, its cell wall thickens, and deposits
10 µm 1 µm
Mycelia. A. Streptomyces lavendulae substrate mycelia. B. Filamentous colonies of Streptomyces coelicolor, an actinomycete known for producing antibiotics such as streptomycin. C. Streptomyces aerial hyphae. Arrow points to a hyphal spore (approx. 1 µm each).
Figure 4.28
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1. Under favorable conditions one or two germ tubes emerge from a spore and produce a substrate mycelium. Germ tubes
Substrate mycelium
bld genes expressed
18 hrs
30 hrs
10 hrs
2. After 48 to 72 hours, bld genes cause production of aerial hyphae (i.e., aerial mycelia).
Free spore
whi genes expressed
2A. The tips of the hyphae form a spiral compartment containing multiple copies of the genome. Aerial mycelia Mycelia 3. When growth stops, the compartment segments (which requires the whi genes). Each segment changes shape and its wall thickens to become a dessication-resistant spore.
3 days
4 to 10 days
Figure 4.29
Developmental cycle of Streptomyces coelicolor.
are made in the spore that increase resistance to desiccation. These organisms are of tremendous interest, both for their ability to make antibiotics and for their fascinating developmental programs.
THOUGHT QUESTION 4.14 How might Streptomyces and Actinomyces species avoid committing suicide when they make their antibiotics?
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Microbial development involves complex changes in cell forms. Endospore development in Bacillus and Clostridium involves production of dormant, stress-resistant endospores. Heterocyst development enables cyanobacteria to fi x nitrogen anaerobically while maintaining oxygenic photosynthesis. Multicellular fruiting bodies in Myxococcus and Dictyostelium and mycelia in actinomycetes develop in response to starvation, dispersing dormant cells to new environments.
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Concluding Thoughts All systems of microbial development share a common theme. They are all triggered in response to a change in their environment, such as depletion of resources, desiccation, or changes in temperature. Microbial responses to the environment have major implications for the function of ecosystems and for the microbial communities that inhabit the human body for good or ill. In Chapter 5, we survey the mechanisms of some of the major environmental responses of microbes.
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C H A P T E R R E V I EW Review Questions 1. What nutrients do microbes need to grow? 2. Explain the differences between autotrophy, hetero-
6. Under what circumstances would you use a selective
trophy, phototrophy, and chemotrophy. 3. Explain the basics of the carbon and nitrogen cycles. 4. Describe the various mechanisms of transporting nutrients in prokaryotes and eukaryotes. What are facilitated diffusion, coupled transport, ABC transporters, group translocation, and endocytosis? 5. Why is it important to grow bacteria in pure culture?
7. What are the factors that defi ne the growth phases of
medium? A differential medium? bacteria grown in batch culture? 8. Describe the important features of biofi lms. 9. Name three kinds of bacteria that undergo differ-
entiation, and give highlights of the differentiation processes.
Key Terms ABC transporter (124) antiport (123) ATP-binding cassette (124) autotroph (118, 119) axenic growth (118) batch culture (137) binary fission (134) biofilm (140) chemoautotroph/chemoautotrophy (118, 119) chemoheterotroph/chemoheterotrophy (119, 120) chemolithotroph (118) chemostat (139) chemotrophy (119) cofactor (117) colony (128) complex medium (130) confluent (129) continuous culture (139) Coulter counter (131) coupled transport (123) death phase (138) death rate (138) defined minimal medium (117) denitrification (120) differential medium (130) dilution streaking (128) doubling time (135)
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electrogenic (123) electroneutral (123) endosome (126) enriched medium (130) essential nutrient (116) exopolysaccharide (EPS) (142) exponential phase (137) fluorescence-activated cell sorter (FACS) (132) forespore (144) generation time (135) germination (144) gliding motility (144) group translocation (126) growth factor (117) growth rate (134) heterotroph (118, 119) lag phase (137) lithotroph (118) logarithmic (log) phase (137) MacConkey medium (130) macronutrient (116) mean growth rate constant (136) membrane potential (120) microcolony (128) micronutrient (117) mitosis (134) mixotrophic (121) mother cell (144)
mycelium (145) nitrification (120) nitrogen-fixing bacterium (120) optical density (133) permease (122) phagocytosis (127) phagosome (127) phosphotransferase system (PTS) (126) photoautotroph/photoautotrophy (118, 119) photoheterotrophy (120) phototrophy (119) pinocytosis (126) planktonic cells (134) pour plate (132) pure culture (128) quorum sensing (142) selective medium (130) siderophore (125) spread plate (129) stationary phase (138) substrate-binding protein (125) symbiont (120) symport (123) synthetic medium (130) turbidostat (140) twitching motility (141) viable (129) viable but nonculturable (130)
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Recommended Reading Angert, Esther R. 2005. Alternatives to binary fission in bacteria. Nature Reviews Microbiology 3:214–224 . Branda, Steven S., Ashlid Vik, Lisa Friedman, and Robert Kolter. 2005. Biofi lms: the matrix revisited. Trends in Microbiology 13:20–26. Davidson, Amy L., and Jue Chen. 2004. ATP-binding cassette transporters in bacteria. Annual Reviews in Biochemistry 73:241–268. England, Jennifer C., and James W. Gober. 2001. Cell cycle control of cell morphogenesis in Caulobacter. Current Opinion in Microbiology 4:674–680. Finkel, Steven E., and Robert Kolter. 1999. Evolution of microbial diversity during prolonged starvation. Proceedings of National Academy of Sciences USA 96:4023–4027.
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Higgins, Christopher F. 2001. ABC transporters: physiology, structure and mechanism—an overview. Research in Microbiology 152:205–210. Nystrom, Thomas. 2004. Stationary-phase physiology. Annual Review of Microbiology 58:161–181. Piggot, Patrick J., and David W. Hilbert. 2004. Sporulation of Bacillus subtilis. Current Opinion in Microbiology 7:579–586. Skerker, Jeffrey M., and Michael T. Laub, 2004. Cell-cycle progression and the generation of asymmetry in Caulobacter crescentus. Nature Reviews Microbiology 2:325–337.
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Chapter 5
Environmental Influences and Control of Microbial Growth 5.1 5.2 5.3 5.4
5.5 5.6 5.7 5.8 5.9
Environmental Limits on Microbial Growth Microbial Responses to Changes in Temperature Microbial Adaptation to Variations in Pressure Microbial Responses to Changes in Water Activity and Salt Concentration Microbial Responses to Changes in pH Microbial Responses to Oxygen and Other Electron Acceptors Microbial Responses to Nutrient Deprivation and Starvation Physical and Chemical Methods of Controlling Microbial Growth Biological Control of Microbes
Microbes have both the fastest and the slowest growth rates of any known organism. Some hot-springs bacteria can double in as little as 10 minutes, whereas deep-sea sediment microbes may take as long as 100 years. And while the rapid growth of pathogens underlies the rapid spread of disease, Earth’s crust is shaped by microbes that grow very slowly in soil and rock. What determines these differences in growth rate? Nutrition is one factor, but niche-specific physical parameters like temperature, pH, and osmolarity are equally important. A microbe’s physiology is geared to work only within a narrow range of physical parameters. But in nature, the environment can quickly change. Many marine microbes, for instance, can move from deep-sea cold to the searing heat of a thermal vent. How do these organisms survive? Stopgap measures called stress survival responses help, but a more permanent solution is when a species slowly evolves to thrive, not just survive, in extreme environments. How does the biology of these so-called extremophiles permit growth under conditions that seem uninhabitable? In this chapter, we explore the limits of microbial growth and show how this knowledge has helped us control the microbial world.
1 µm
The extremophile Halorubrum lacusprofundi, an Antarctic haloarchaeon that forms biofilms at subzero temperatures in 5 M NaCl. Extremophiles are considered model systems for astrobiology. Source: Shiladitya DasSarma. 2006. Microbe 1:120.
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In 1998, apple and pear growers in Washington and northern Oregon lost crops worth an estimated $68 million owing to outbreaks of fi re blight, a devastating bacterial disease; the causative agent, Erwinia amylovora, destroys apple and pear trees, making them appear as if they were torched by fi re. As with human disease, we try to control microbial plant infections by controlling the growth of microbes. It should not be surprising, then, that antibiotics are often utilized for this purpose. But while many antibiotics are used to protect humans, only two are currently approved for use on plants: streptomycin and oxytetracycline. The fact that resistance to streptomycin has already been demonstrated in Erwinia amylovora underscores the importance of studying all aspects of bacterial growth in the effort to control pathogens of plants, animals, and humans. We begin this chapter by discussing how physical and chemical changes in the environment modify the growth of different groups of microbes. We will also explore how microorganisms adapt to different environments in ways both transient (involving temporary expression of inactive genes) and permanent (modifications of the gene pool). The permanent genetic changes have led to biological diversity. Finally, we will examine the different ways humans try to limit the growth of microorganisms to protect plants, animals, and ourselves. As you proceed through this chapter, you will encounter two recurring themes: that different groups of microbes live in vastly different environments and that microbes can respond in diverse ways when confronted by conditions outside their niche, or comfort zone.
5.1
Environmental Limits on Microbial Growth
With our human frame of reference, we tend to think that “normal” growth conditions are those found at sea level with a temperature between 20°C and 40°C, a nearneutral pH, a salt concentration of 0.9%, and ample nutrients. Any ecological niche outside this window is called extreme and the organisms inhabiting them extremophiles (R. MacElroy fi rst used the term extremophile in 1974). Extremophiles are prokaryotes (bacteria and archaea) that are able to grow in extreme environments. For example, one group of organisms can grow at temperatures above boiling, while another group requires a pH 2 acidic environment to grow. Based on our defi nition of normal conditions, conditions on Earth when life began were certainly extreme. Consequently, the earliest microbes likely grew in these extreme environments. Organisms that grow under conditions that seem “normal” to humans likely evolved from an ancient extremophile that gradually adapted as the environment evolved to that of our present-day Earth.
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Be aware that multiple extremes in the environment can be encountered simultaneously. For instance, in Yellowstone National Park, one can fi nd an extreme acid pool next to an extreme alkali pool, both at extremely high temperatures. Thus, extremophiles typically evolve to survive multiple extreme environments. Extremophiles may provide insight into the workings of extraterrestrial microbes we may one day encounter, since outer space certainly qualifies as an extreme environment. Our experiences with extremophiles should alert us to the dangers of underestimating the precautions necessary in handling extraterrestrial samples. For example, we should not assume that irradiation would be sufficient to sterilize samples from future planetary or interstellar missions. Such treatments do not even kill the extremophile Deinococcus radiodurans found on Earth. Pyrodictium How do we even begin to study organisms that grow in boiling water or sulfuric acid solutions or organisms that we cannot even culture in the laboratory? How do we dissect the molecular response of organisms to changes in an environment such as acid rain or to changes in the human body? Genome sequences present new opportunities for investigating these questions. Bioinformatic analysis allows us to study the biology of organisms that we cannot culture. First, genome sequences from novel organisms are amplified by polymerase chain reaction, or PCR (a technique that multiplies a small sample of DNA; see Section 7.6). PCR amplification can be done directly from the natural environment in which the organisms are found, and the sequences are compared with those of model systems, such as E. coli, whose biochemistry is well known. Genomic comparison quickly reveals whether an organism under study may possess specific metabolic pathways and regulatory responses. Global arrays of DNA and proteins allow us to study the response of an organism to changing environments. Global approaches can reveal in a single experiment the response of all an organism’s genes to a single environmental change, such as change in temperature or pH (Fig. 5.1). DNA microarrays (see Section 10.8) consist of slides with a grid containing DNA probes for every gene in an organism’s genome. They are used to assess which genes are expressed to make RNA in a given organism at a given time or under a given condition. Two- dimensional protein gels (see Section 3.5) achieve two-dimensional separation of proteins based on differences in each protein’s isoelectric point (fi rst dimension) and molecular weight (second dimension). They show which proteins the cell produces when cultured in different environments. Knowing what genes and proteins are expressed in a given situation helps elucidate the molecular strategies that microbes use to grow under different conditions and to defend themselves against environmental stresses.
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B.
Joan Slonczewski, Kenyon College
Alfred Pasieka/Photo Researchers, Inc.
High
Mwt
Acid
pH
Alkaline
Low
Figure 5.1 Response to environmental stress: global analysis of genes and proteins. A. This microarray contains DNA probes (small pieces of DNA sequence) representing each gene in the genome. The DNA probes hybridize to preparations of fluorescent-labeled cDNA (complementary DNA) made from whole-cell RNA of cultures grown under different conditions. Each colored disk represents one gene. Red fluorescence indicates RNA expressed under one condition; green indicates expression under the second condition; yellow indicates expression under both conditions. B. Two-dimensional gels separate the proteins expressed in the cell. Proteins are separated in one dimension by their charge (isoelectric pH point) and in the second dimension by their molecular weights. Gel images are obtained from cultures grown under two different conditions (in this case, Escherichia coli K-12 grown at pH 4.9 versus pH 7.0). Pink spots denote proteins expressed only at pH 4.9; green indicates expression only at pH 7.0. Circled spots were chosen for additional studies.
These techniques of molecular analysis are discussed further in Chapters 8 and 10. We have already mentioned the fundamental physical conditions (temperature, pH, osmolarity) that define an environment and select for (favor) the growth of specific groups of organisms. And within a given microbial community, each species is further localized to a specific niche defined by a narrower range of environmental factors. The reason for this is that every protein and macromolecular structure within a cell is affected profoundly by changes in environmental conditions and through reaction with the by-products of oxygen consumption. For example, a single
enzyme works best under a unique set of temperature, pH, and salt conditions because those conditions allow it to fold into its optimum shape, or conformation. Deviations from these optimal conditions cause the protein to fold a little differently and become less active. While all enzymes within a cell do not boast the same physical optima, these optima must at least be similar and matched to the organism’s environment for the organism to function effectively. As may be apparent from the preceding discussion, there are many different classes of microbes based on their environmental niche. Table 5.1 summarizes these environmental classes.
Table 5.1 Basic environmental classification of microorganisms. Environmental parameter
Classification
Temperature
Hyperthermophile* (growth above 80°C)
pH
Alkaliphile* (growth above pH 9)
Osmolarity
Halophile* (growth in high salt > 2M NaCl) Aerobe (growth only in oxygen)
Oxygen
Pressure
Barophile* (growth at high pressure, greater than 380 atm)
Thermophile* (growth between 50°C and 80°C) Neutralophile (growth between pH 5–8)
Mesophile (growth between 15°C and 45°C) Acidophile* (growth below pH 3)
Psychrophile* (growth below 15°C)
Facultative (growth with or without oxygen)
Microaerophile (growth only in small amounts of oxygen) Barotolerant (grown between 10–495 atm)
Anaerobe (growth only without oxygen)
*considered extremophiles
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Extremophiles inhabit fringe environmental conditions that do not support human life. The environmental niche (such as high salt or acidic pH) inhabited by a particular species is defi ned by the tolerance of that organism’s proteins and other macromolecular structures to the physical conditions within that niche.
rate versus temperature, we get a straight line whose slope (C) can be obtained by substituting in equation 5.1: log (k2/k1) = C/(T1 – T2) The general result of the Arrhenius equation is that growth rate roughly doubles for every 10°C rise in temperature (Fig. 5.2). This is the same relationship observed for any chemical reaction. A.
Microbial Responses to Changes in Temperature
Unlike humans (and mammals in general), microbes cannot control their temperature; thus, bacterial cell temperature matches that of the immediate environment. Because temperature affects the average rate of molecular motion, changes in temperature impact every aspect of microbial physiology, including membrane fluidity, nutrient transport, DNA stability, RNA stability, and enzyme structure and function. Every organism has an optimum temperature at which it grows most quickly, as well as minimum and maximum temperatures that defi ne the limits of growth. These limits are imposed, in part, by the thousands of proteins in a cell, all of which must function within the same temperature range. The fastest growth rate for a species occurs at temperatures where all of the cell’s proteins work most efficiently as a group to produce energy and synthesize cell components. Growth stops when rising temperatures cause critical enzymes or cell structures (such as the cell membrane) to fail. At cold temperatures, growth ceases because enzymatic processes become too sluggish and the cell membrane less fluid. The membrane needs to remain fluid so that it can expand as cells grow larger and so that proteins needed for solute transport can be inserted into the membrane.
Maximum Growth Rate Increases with Temperature
(5.1)
T is the absolute temperature in degrees Kelvin, and C is a second constant that combines the gas constant and the average activation energy of cellular reactions. Over a defi ned temperature range, which differs for each species, growth rate increases (that is, cells divide faster) as temperature increases. If we plot the logarithm of the growth
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Mesophile Psychrophile
10
20
30
40 50 60 80 Temperature °C
80
90
100
At higher temperatures, growth rates fall because enzymes denature.
B.
4.0 3.0 2.0 42° 39° 45° 46°
1.0
37° 36° 33°
At lower temperatures, growth rates fall because of decreases in membrane fluidity and enzymatic activity. 30°
37°
47°
28° 30°
23° 21°
0.5 0.4
23° 19°
0.3
17° 0.2
In general, microbes that grow at higher temperatures can achieve higher rates of growth. Remarkably, the relationship between the maximum growth temperature and the growth rate constant k (number of generations per hour; see Section 4.5) obeys the Arrhenius equation for simple chemical reactions: log k = C/T
Thermophile
0
Growth rate constant, k (hr –1)
5.2
Generations per hour
Extreme thermophile
48°
0.1 3.1
Growth in rich media Growth in minimal media Extrapolated Arrhenius plot 3.2
3.3 1,000/T (°K)
15° 13.5° 3.4
3.5
Relationship between temperature and growth rate. A. Relationship between temperature and growth rates of different groups of microbes. Note that the growth rate increases linearly with temperature and obeys the Arrhenius equation. B. Growth rate constant (k) of the enteric organism Escherichia coli is plotted against the inverse of growth temperature on the Kelvin scale (1,000/T is used to give a convenient scale on the x-axis). This is a more detailed view of a mesophilic growth temperature curve. As temperature rises above or falls below the optimum range, growth rate decreases faster than predicted by the Arrhenius equation.
Figure 5.2
Source: B. Sherrie L. Herendeen, et al. 1979. Journal of Bacteriology 139:185.
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At the upper and lower limits of the growth range, however, the Arrhenius effect breaks down. At high temperatures, critical proteins denature. Lower temperatures decrease membrane fluidity and limit the conformational mobility of enzymes, thereby lowering their activities. As a result, growth fails to occur. The typical growth temperature range spans about 30–40°C, but some organisms have a much narrower range. Within a species, we can identify mutants that are more sensitive to one extreme or the other (heat sensitive or cold sensitive). These mutations often defi ne key molecular components of stress responses, such as the heat-shock proteins (discussed in Chapter 10). Thermodynamic principles limit a cell’s growth to a narrow temperature range. For example, heat increases molecular movement within proteins. Too much or too little movement will interfere with enzymatic reactions. As a result, there is a great biological diversity among microbes as different groups have evolved to grow within very different thermal niches. A species grows within a specific thermal range because its proteins have evolved to tolerate that range. Outside that range, proteins will denature or function too slowly for growth. The upper limit for protists is around 50°C, while some fungi can grow at temperatures as high as 60°C. Prokaryotes, however, have been found to grow at temperatures ranging from below 0°C to above 100°C. Temperatures over 100°C are usually found around thermal vents deep in the ocean, where water temperature can rise to 350°C but the pressure is sufficient to keep water liquid. THOUGHT QUESTION 5.1 Why haven’t cells evolved so that all their enzymes have the same temperature optimum? If they did, wouldn’t they grow even faster?
Microorganisms Are Classified by Growth Temperature Based on their ranges of growth temperature, microorganisms can be classified as mesophiles, psychrophiles, or thermophiles (Fig. 5.2A). Mesophiles include the typical “lab rat” microbes, such as Escherichia coli and Bacillus subtilis. Their growth optima range between 20°C and 40°C, with a minimum of 15°C and a maximum of 45°C. Because they are easy to grow and because human pathogens are mesophiles, much of what we know about protein, membrane, and DNA structure came from studying this group of organisms. Current advances, particularly in obtaining detailed three-dimensional (3-D) views of protein structures, are frequently based on studies of two other classes of organisms whose optimum growth temperature ranges flank
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Transfer RNAs
Ribosome
Figure 5.3 Thermophilic proteins. Special properties of thermophilic proteins facilitate 3-D structure analysis, as shown here. The structure of the Thermus thermophilus 70S ribosome is shown here at 7.8 Å (or 0.78 nm) resolution. Transfer RNAs (green, blue, and yellow) occupy a cavity between two ribosomal subunits. (PDB codes: 1GIX, 1GIY)
that of the mesophiles, namely psychrophiles (on the low temperature side) and thermophiles (on the high temperature side; Fig. 5.3). Because of their more stable folding structures, proteins from the thermophilic extremophiles are generally easier to crystallize than those of mesophiles or psychrophiles, so it is possible to determine their structures by X-ray crystallography (see Section 2.7). Psychrophiles are microbes that grow at temperatures as low as 0°C, but their optimum growth temperature is usually around 15°C. Psychrophiles are prominent flora beneath icebergs in the Arctic and Antarctic (Fig. 5.4). In addition to true psychrophiles, whose growth optima are below 20°C, there are mesophiles that are cold resistant. For instance, the modern practice of refrigeration has selected for cold-resistant, yet mesophilic, pathogens such as Listeria monocytogenes (one cause of food poisoning and septic abortions). Why do these organisms grow so well in the cold? One reason psychrophilic microbes prefer cold is that their proteins are more flexible than those of mesophiles and require less energy (heat) to function. Of course, the downside to the increased flexibility of psychrophilic proteins is that they denature at lower temperatures than their mesophilic counterparts. As a result, psychrophiles grow poorly, if at all, when temperatures rise above 20°C. Another reason psychrophiles favor cold is that their membranes are more fluid at low temperature owing to the high proportion of
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B.
© Gerald & Buff Corsi/Visuals Unlimited
unsaturated fatty acids present; at higher temperatures, their membranes are too flexible and fail to maintain cell integrity. Psychrophilic enzymes are of commercial interest because their ability to carry out reactions at low temperature has potential utility in food processing and bioremediation. Many food products require enzymatic processing. Enzymes help brew beer more quickly, break down lactose in milk, and can remove cholesterol from various foods. Production of foods at lower temperatures would be beneficial, since lower processing temperatures would minimize the growth of typical mesophiles that
1 µm
degrade and spoil food. Another goal is to fi nd organisms that can safely degrade toxic organic contaminants (for example, petroleum) in the cold. Arctic environments are particularly sensitive to pollution because contaminants are slow to degrade in the freezing temperatures. Consequently, the ability to seed Arctic oil spills with psychrophilic organisms armed with petroleumdegrading enzymes could rapidly restore contaminated environments. Thermophiles (Fig. 5.5) are species adapted to growth at high temperature, typically 55°C and higher. Hyperthermophiles grow at temperatures as high as
B.
©Momatiuk-Eastcott/Corbis
A.
Psychrophilic environment and psychrophiles. A. Iceberg, in which psychrophilic organisms like those shown in (B) can be found. These organisms are unclassified. B. Bacteria from South Polar snow (SEM).
Figure 5.4
C.
3 µm
2 µm
B. Boonyaratanakornkit et al., University of California, Berkeley
A.
Douglas G. Capone, U. of Southern California
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Photo by Dudley Foster from RISE expedition, courtesy of William R. Normark, USGS
D.
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Figure 5.5 Thermophilic environments and thermophiles. A. Yellowstone National Park hot spring. B. Thermus aquaticus, a hyperthermophile first isolated at Yellowstone by Thomas Brock. Cell length varies from 3 to 10 µm. C. Thermophile Methanocaldococcus jannaschii grown at 78°C and 30 psi. D. A “smoker,” a hydrothermal vent 2 miles deep in the Pacific Ocean. M. jannaschii was isolated in 1983 in the area of this vent.
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110°C, which occur under extreme pressure (for example, at the ocean floor). These organisms flourish in hot environments such as composts or near thermal vents that penetrate Earth’s crust on the ocean floor and on land (for example, hot springs). The thermophile Thermus aquaticus was the fi rst source of a DNA polymerase used for PCR amplification of DNA. T. aquaticus was discovered in a hot spring at Yellowstone National Park by microbiologist Thomas Brock, a pioneer in the study of thermophilic organisms. Its application to the polymerase chain reaction has revolutionized molecular biology (discussed in Chapter 7). Extreme thermophiles often have specially adapted membranes and protein sequences. The thermal limits of these structures determine the specific high-temperature ranges in which various species can grow. Because enzymes in thermophiles (thermozymes) do not unfold as easily as mesophilic enzymes, they more easily hold their shape at higher temperatures. Thermophilic enzymes are stable, in part, because they contain relatively low amounts of glycine, a small amino acid that contributes to an enzyme’s flexibility. In addition, the amino-termini of proteins in these organisms often are “tied down” by hydrogen bonding to other parts of the protein, making them harder to denature. Like all microbes, thermophiles have chaperone proteins that help refold other proteins as they undergo thermal denaturation. Thermophile genomes are packed with numerous DNA-binding proteins that stabilize DNA. In addition, these organisms possess special enzymes that function to tightly coil DNA in a way that makes it more thermostable and less likely to denature (think of a coiled phone cord that has twisted and bunched up on itself). Special membranes also help give cells additional stability at high temperatures. Unlike the typical lipid bilayers of mesophiles, the membranes of thermophiles manage to “glue” together parts of the two hydrocarbon layers that point toward each other, making them more stable. They do this by incorporating more saturated linear lipids into their membranes. Saturated lipids form straight hydrocarbon tails that align well with neighboring lipids and form a highly organized structure stable to heat. The membranes of mesophiles are composed mostly of unsaturated lipids that bend against each other and align poorly. This property makes the membranes of mesophiles more fluid at lower temperatures.
High Temperatures Induce the Heat Shock Response As insurance against extinction, most microorganisms possess elegant genetic programs that remodel their physiology to one that can temporarily survive inhospitable conditions. Rapid temperature changes experienced
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during growth activate batches of stress response genes, resulting in the heat shock response (discussed in Chapter 10). The protein products of these heat-activated genes include chaperones that maintain protein shape and enzymes that change membrane lipid composition. The heat-shock response, fi rst identified in E. coli by Yamamori and Yura in 1982, has since been documented in all living organisms examined thus far. THOUGHT QUESTION 5.2 If microbes lack a nervous system, how can they sense a temperature change? The appearance of different branches of life in the course of evolution reflects, in some ways, the narrowing of tolerance to heat. Different archaeal species, for example, can grow in extremely hot or extremely cold temperatures and some can grow in the middle range. Bacteria, for the most part, tolerate a temperature range that bridges the archaeal extremes. Eukaryotes are less temperature tolerant than bacteria, with individual species capable of growth between 10°C and 65°C. Archaeal species have been found to grow at greater extremes than bacterial species, and bacteria at greater extremes than eukaryotes. As we will see, this evolutionary relationship holds for other environmental conditions. TO SU M MAR I Z E: ■
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Different species exhibit different optimal growth values of temperature, pH, and osmolarity. The Arrhenius equation applies to growth of microorganisms: Growth rate doubles for every 10°C rise in temperature. Membrane fluidity varies with the composition of lipids in a membrane, which in turn dictates at what temperature an organism can grow. Mesophiles, psychrophiles, and thermophiles are groups of organisms that grow at moderate, low, and high temperatures, respectively. The heat shock response produces a series of protective proteins in organisms exposed to temperatures near the upper edge of their growth range.
5.3
Microbial Adaptation to Variations in Pressure
Living creatures at Earth’s surface (sea level) are subjected to a pressure of 1 atmosphere (atm), which is equal to 0.101 megapascal (Mpa) or 14 pounds per square inch (psi). At the bottom of the ocean, however, thousands of meters deep, hydrostatic pressure averages a crushing 400 atm and can go as high as 1,000 atm (101 Mpa, or 14,000 psi) in ocean trenches (Fig. 5.6). Organisms adapted to
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designed ribosome structures that can withstand pressures even higher than that. Specific applications for barotolerant proteins and pressure-regulated genes have not yet been developed. However, many food processes are carried out at high pressure to minimize bacterial contamination (destructive bacteria will not tolerate the pressure), so it is expected that barophiles will ultimately offer useful biotechnology products, such as enzymes that will carry out food processing at high pressure. Highpressure processing has been used on cheeses, yogurt, luncheon meats, and oysters to kill contaminating bacteria without destroying the flavor or texture of the food.
A. Average height of the land, 870 m B. J. W. Deming & R. Colwell, Applied Environmental Microbiology, 44.
Average depth of the ocean, 3,730 m
Mount Everest, 8,848 m
Mariana Trench, 11,035 m 1.5 µm
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Figure 5.6 Barophilic environments and barophiles. A. Ocean depths. The deepest part of the ocean is at the bottom of the Mariana Trench, a depression in the floor of the western Pacific Ocean, just east of the Mariana Islands. The Mariana Trench is 1,554 miles long and 44 miles wide. Near its southwestern extremity, 210 miles southwest of Guam, lies the deepest point on Earth. This point, referred to as the “Challenger Deep,” plunges to a depth of nearly 7 miles. B. Barophile Shewanella violacea. Length is approx. 1.5 µm.
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THOUGHT QUESTION 5.3 What could be a relatively simple way to grow barophiles in the laboratory?
Barophiles require high pressure to grow, though they die at still higher pressures. Barotolerant organisms grow up to a certain pressure, but die at higher pressures.
Growth rate
grow at overwhelmingly high pressures are called barophiles or piezophiles. From the curves in Figure 5.7, we can see that barophiles actually require elevated pressure to grow, while barotolerant organisms grow well over the range of 1–50 Mpa, but their growth falls off thereafter. Many barophiles are also psychrophilic because the average temperature at the ocean’s floor is 2°C. However, barophilic hyperthermophiles form the basis of thermal vent communities that support symbiotic worms and giant clams (see Chapter 21). How bacteria can survive pressures of 12,000 psi is still a mystery. It is known that increased hydrostatic pressure and cold temperatures similarly reduce membrane fluidity. Because fluidity of the cell membrane is critical to survival, the phospholipids of deep-sea bacteria commonly have high levels of polyunsaturated fatty acids to increase membrane fluidity. It is thought that in addition to these membrane changes, internal structures must also be pressure adapted. For example, ribosomes in the barosensitive organism E. coli dissociate at pressures above 60 Mpa, so barophiles must contain uniquely
Barophiles can grow at pressures up to 1,000 atm but fail to grow at low pressures. Membrane fluidity can be compromised at high pressures and cold temperatures. Specially designed membranes and protein structures are thought to enable the growth of barophiles.
0
20
40
60
80
100
Pressure (Mpa)
Barosensitive organisms die as pressure increases.
Figure 5.7
Relationship between growth rate and
pressure.
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Microbial Responses to Changes in Water Activity and Salt Concentration
Water is critical to life, but environments differ in terms of how much water is actually available to growing organisms; microbes can only use water that is not bound to ions or other solutes in solution. Water availability is measured as water activity (aw), a quantity approximated by concentration. Because interactions with solutes lower water activity, the more solutes there are in a solution, the less water is available for microbes to use for growth. Water activity is typically measured as the ratio of the solution’s vapor pressure relative to that of pure water. A solution is placed in a sealed chamber and the amount of water vapor determined at equilibrium. If the air above the sample is 97% saturated relative to the moisture present over pure water, the relative humidity is 97% and the water activity is 0.97. Most bacteria require water activity to be greater than 0.91 for growth (the water activity of seawater). Fungi can tolerate water activity levels as low as 0.86.
Osmotic Stress Osmolarity is a measure of the number of solute molecules in a solution and is inversely related to aw. The more particles there are in a solution, the greater the osmolarity and the lower the water activity (see Appendix 1). Osmolarity is important for the cell because it is related to water activity and also because a semipermeable membrane surrounds microbial cells, so osmolarity inside the cell can be, and often is, different from the osmolarity outside. The principles of physical chemistry dictate that solute concentrations in two chambers separated by a semipermeable membrane will tend to equilibrate. Equilibrating osmolarity across a semipermeable cell membrane, which does not allow the movement of solutes, requires the movement of water. In hypertonic medium, where the external osmolarity is higher than the internal, water will try to leave the cell in an attempt to equalize osmolarity across the membrane. In contrast, suspension of a cell in a hypotonic medium (one of lower osmolarity than the cell) will cause an influx of water (see Fig. A2.6). This movement of water across cell membranes does not occur primarily by simple diffusion. Special membrane water channels formed by proteins called aquaporins enable water to traverse the membrane much faster than by diffusion and help protect cells against osmotic stress (Fig. 5.8). However, too much water moving in or out of a cell is detrimental. Cells may ultimately explode or implode, depending on the direction in which the water moves. Even bacteria with a rigid cell wall suffer. They may not explode like a human cell, but the forces placed on the cell wall are great.
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H2O
Channel
Aquaporin. Transverse view of the channel. Complementary halves of the channel are formed by adjacent protein monomers. The curvilinear, size-selective (~4 ± 0.5 nm) core of the channel (~18 nm) is primarily lined by hydrophobic residues. (PDB code: 1J4N)
Figure 5.8
Cells Minimize Osmotic Stress across Membranes In addition to moving water, microbes have at least two other mechanisms to minimize osmotic stress across membranes. When stranded in a hypertonic medium (higher osmolarity than the cell), bacteria try to protect their internal water by synthesizing or importing compatible solutes that increase intracellular osmolarity. Compatible solutes are small molecules that do not disrupt normal cell metabolism, even when present at high intracellular concentrations. Increasing intracellular levels of these compounds (for example, proline, glutamic acid, potassium, or betaine) elevates cytoplasmic osmolarity without any detrimental effects, making it unnecessary for water to leave the cell. Cells also contain pressure-sensitive (mechanosensitive) channels that can be used to leak solutes out of the cell. It is believed that these channels are activated by rising internal pressures in cells immersed in a hypotonic medium. When activated, the channels allow solutes to escape, which lowers internal osmolarity. Outside their osmotic comfort range—that is, where the aforementioned housekeeping strategies become ineffective at controlling internal osmolarity—microbes launch a global response in which cellular physiology is transformed to tolerate brief encounters with potentially lethal salt concentrations. Some changes are similar to those provoked by heat shock, such as the increased synthesis of chaperones that protect critical cell proteins from denaturation. Other changes include alterations in outer membrane pore composition (for gram-negative organisms).
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TO SU M MAR I Z E:
Halophiles Require High Salt Concentration
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Some species of archaea have evolved to require high salt (NaCl) concentration. These are called halophiles (Fig. 5.9). In striking contrast to most bacteria, which prefer salt concentrations from 0.1 to 1 M (0.2–5% NaCl), the extremely halophilic archaea can grow at an aw of 0.75 and actually require 2–4 M NaCl (10–20% NaCl) to grow. For comparison, seawater is about 3.5% NaCl. All cells, even halophiles, prefer to keep a relatively low intracellular Na+ concentration. One reason for this is that some solutes are moved into the cell by symport with Na+. To achieve a low internal Na+ concentration, halophilic microbes use special ion pumps to excrete sodium and replace it with other cations, such as potassium, which is a compatible solute. In fact, the proteins and cell components (for example, ribosomes) of halophiles require remarkably high intracellular potassium levels to maintain their structure. Halophilic algae use a different strategy for dealing with hypertonic media. They redirect photosynthesis to make glycerol (a compatible solute) instead of starch. Increasing glycerol production raises internal osmolarity, which stops water from leaving the cell and prevents dehydration. Although halophilic bacteria appear to have lost ecological flexibility by adapting to such an extreme environment, they have gained access to environments that other organisms are not equipped to use. THOUGHT QUESTION 5.4 How might the concept of water availability be used by the food industry to control spoilage?
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5.5
Microbial Responses to Changes in pH
As with salt and temperature, the concentration of hydrogen ions (H +)—actually, hydronium (H 3O +)— also has a direct effect on the cell’s macromolecular structures. Extreme concentrations of either hydronium or hydroxyl ions (OH – ) in a solution will limit growth. In other words, too much acid or base is harmful to cells. Despite this sensitivity to pH extremes, living cells tolerate a greater range in environmental concentration of H + than of virtually any other chemical substance. E. coli, for example, tolerates a pH range of 2–9, a 10,000,000-fold difference. For a brief review of pH, refer to Section A1.7.
B.
Wayne P. Armstrong
A.
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Water activity (aw) is a measure of how much water in a solution is available for a microbe to use. Osmolarity is a measure of the number of solute molecules in a solution and is inversely related to aw. Aquaporins are membrane channel proteins that allow water to move quickly across membranes to equalize internal and external pressures. Compatible solutes are used to minimize pressure differences across the cell membrane. Mechanosensitive channels can leak solutes out of the cell when internal pressure rises. Halophilic organisms grow best at high salt concentration.
0.5 µm
C. Courtesy of S. DasSarma, U. of Maryland Biotechnology Institute
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Halophilic salt flats and halophilic bacteria. A. The halophilic salt flats along Highway 50, east of Fallon, Nevada, are colored pinkish red by astronomical numbers of halophilic bacteria. B. Halobacterium sp. (TEM). Cross section cell width 0.5 to 0.8 µm. C. Shiladitya DasSarma and colleagues at the University of Maryland completed the genome sequence of Halobacterium species NRC-1. They demonstrated novel features of archaeal genetics, including intriguing similarities with molecular regulatory structures in eukaryotes.
Figure 5.9
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Enzymes Show pH Optima, Minima, and Maxima
Growth pH
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[H+] (molarity)
pH
100
0
10–1
1
159
pOH H 3O
+
14 13
The charges on various amino or carboxyl groups Acidophiles 10–2 2 12 within a protein help forge the intramolecular 10–3 3 11 bonds that dictate protein shape and thus pro–4 10 4 10 tein activity. Because H+ concentration affects the 10–5 5 9 protonation of these ionizable groups, altering pH –6 10 6 8 Intracellular can alter the charges on these groups, which in levels –7 Neutralophiles 10 7 7 turn changes protein structure and activity. The compatible –8 10 8 6 with life result is that all enzyme activities exhibit optima, 10–9 9 5 minima, and maxima with regard to pH, much –10 as they do for temperature. As we saw with tem10 10 4 perature, groups of microbes have evolved to 10–11 11 3 inhabit diverse niches, for which pH values can Alkalophiles 10–12 12 2 range from pH 0 to pH 11.5 (Fig. 5.10). However, 10–13 13 1 – species differences in optimum growth pH are not –14 OH 10 14 0 dictated by the pH limits at which critical cell proFigure 5.10 Classification of organisms grouped by optimal teins function. growth pH. Generally speaking, the majority of enzymes, regardless of the pH at which their source organism thrives, tend to operate best between pH 5 and 8.5 (which, if you think about it, is still a range in which the hydrogen ion concentration varies more than 1,000-fold), yet many microbes grow in even more acidic or alkaline environments. pHinitial = 7 pHextracellular = 5 Unlike its temperature, the intracellular pH of a microbe, as well as its osmolarity, is not necessarily the pHfinal = 6 pKa = 5 same as that of its environment. Biological membranes are + – H +A HA HA A– + H+ relatively impermeable to protons, a fact that allows the 99% 1% 50% 50% cell to maintain an internal pH compatible with protein 99 mM 1 mM 1 mM 1 mM function when growing in extremely acidic or alkaline environments. When the difference between the intracellular and extracellular pH (∆pH) is very high, protons can leak through either directly or via proteins that thread the Cell membrane membrane. Excessive influx or efflux of protons can cause problems by altering internal pH. Figure 5.11 Effect of organic acids on internal pH. In Membrane-permeant organic acids, also called this contrived example, the organic acid (HA) has an extracellular weak acids (discussed in Chapter 3), can accelerate the concentration of 2 mM and has a dissociation constant of pH 5. leakage of H+. Unlike H+, the uncharged form of an Because the medium is also pH 5, half of the acid is protonated organic acid (HA) can freely permeate cell membranes (undissociated, or un-ionized) and half is dissociated (ionized). The un-ionized form, because it is uncharged, diffuses across and dissociate intracellularly, releasing a proton that then the membrane to establish equilibrium between the inside and acidifies internal pH (Fig. 5.11). The extent of the pH outside of the cell. However, because the inside of the cell is drop depends on the buffering capacity of the cell’s propH 7 (2 units above the external pH), 99% of the acid will teins. This shuttling of protons can turn a relatively mild dissociate. This lowers the internal HA concentration, so more external pH level (say, pH 6) into a deadly acid stress. A HA enters the cell in search of equilibrium. Because neither the naturally occurring example of organic acid stress is the ionized form of the acid nor the released proton can diffuse out lactic acid produced by lactobacilli during the formation of the cell, both accumulate. At equilibrium, the concentrations of yogurt. The buildup of lactic acid limits the bacterial of HA inside and outside the cell are equal, but for every HA that growth, leaving yogurt with plenty of food value. The enters the cell, ionization of HA has yielded 99 A– molecules and food industry has taken advantage of this phenomenon an equal number of protons, which lower the internal pH to an by preemptively adding citric acid or sorbic acid to cerextent that depends on the buffering capacity of cellular proteins. tain foods. This allows manufacturers to control microbial growth under pH conditions that do not destroy the
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Concentration of hydrogen ions compared to distilled water
Example of solutions at this pH
10,000,000
pH = 0
Battery acid (strong), hydrofluoric acid
1,000,000
pH = 1
Hydrochloric acid secreted by stomach lining
100,000
pH = 2
Lemon juice, gastric acid, vinegar
10,000
pH = 3
Grapefruit, orange juice, soda
1,000
pH = 4
Acid rain, tomato juice
100
pH = 5
Soft drinking water, black coffee
10
pH = 6
Urine, saliva
1
pH = 7
“Pure” water
1/10
pH = 8
Seawater
1/100
pH = 9
Baking soda solution
1/1,000
pH = 10
Great Salt Lake, milk of magnesia
1/10,000
pH = 11
Ammonia solution
1/100,000
pH = 12
Soapy water
1/1,000,000
pH = 13
Bleaches, oven cleaner
1/10,000,000
pH = 14
Liquid drain cleaner
Figure 5.12
pH values of common substances.
8.5 Acid added at a constant rate pHint
7.5 pH
160
6.5
Base added at a constant rate
pHext
5.5
0
40
80
120
Time (min)
Figure 5.13 Maintaining internal pH (pH homeostasis) over a wide range of external pH. Internal pH of Escherichia coli measured following the addition of acid (HCl, indicated by arrow at t = 10 min) to change external pH (circles) and subsequent addition of base (NaOH at t = 52 min). In this experiment, internal pH (pHint) was determined using nuclear magnetic resonance to measure changes in cellular phosphate (closed circles/squares) and methyl phosphate (open circles/ squares). The two phosphate species titrate over different pH ranges. Source: Joan L. Slonczewski, et al. 1981. Proceedings of the National Academy of Sciences USA 78:6271.
flavor or quality of the food. Figure 5.12 illustrates the pH values of various everyday items. Food microbiology is further discussed in Chapter 16.
Neutralophiles, Acidophiles, and Alkaliphiles Grow in Different pH Ranges Cells have evolved to live under different pH conditions not by drastically changing the pH optima of their enzymes but by using novel pH homeostasis strategies that maintain intracellular pH above pH 5 and below pH 8, even when the cell is immersed in pH environments well above or below that range. There are three classes of organisms marked by the pH of their growth range. Neutralophiles generally grow between pH 5 and pH 8, and include most human pathogens. Many neutralophiles, including E. coli and Salmonella enterica, adjust their metabolism to maintain an internal pH slightly above neutrality, which is where their enzymes work best. They maintain this pH even in the presence of moderately acidic or basic external environments (Fig. 5.13). Others allow their internal pH to fluctuate with external
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pH but usually maintain a pH difference (∆pH) of about 0.5 pH units across the membrane at the upper and lower limits of growth pH. The ∆pH value is an important component of the transmembrane proton potential, a source of energy for the cell (see Chapter 13). NOTE: The older term neutrophile used for this group of organisms is also similar to the descriptor for a specific type of white blood cell (neutrophil). To avoid confusion, the term neutrophil should be reserved for the white blood cell and the term neutralophile used to designate microbes with growth optima near neutral pH.
Acidophiles are bacteria and archaea that live in acidic environments. They are often chemoautotrophs (lithotrophs) that oxidize reduced metals and generate strong acids such as sulfuric acid. Consequently, they grow between pH 0 and pH 5. The ability to grow at this pH is due partly to altered membrane lipid profi les (high levels of tetraether lipids) that decrease proton permeability as well as to ill-defi ned proton extrusion mecha-
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Thomas D. Brock, University of Wisconsin, Madison
Sonny X. Li
C. Thomas D. Brock, University of Wisconsin, Madison
A.
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Figure 5.14 Sulfur Caldron acid spring and Sulfolobus acidocaldarius. A. Sulfur Caldron, in the Mud Volcano area of Yellowstone National Park, is one of the most acidic springs in the park. It is rich in sulfur and in Sulfolobus, a bacterium that thrives in hot, acidic waters with temperatures from 60°C to 95°C and a pH of 1–5. B. Thin-section electron micrograph of S. acidocaldarius. Under the electron microscope, the organisms appear as irregular spheres that are often lobed. C. Fluorescent photomicrograph of cells (green) attached to a sulfur crystal. Fimbrial-like appendages (not seen here) have been observed on the cells attached to solid surfaces such as sulfur crystals. Crystal size is approx. 55 mm in real life.
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DSMZ/GBF, Rohde
B.
C.
Wolfgang Kaehler/Corbis
Kazuyoshi Nomachi/Corbis
nisms. Often, an organism that is A. an extremophile with respect to one environmental factor is an extremophile with respect to others. Sulfolobus acidocaldarius, for example, is a thermophile and an acidophile (Fig. 5.14). It uses sulfur as an energy source and grows in acidic hot springs rich in sulfur. Alkaliphiles occupy the opposite end of the pH spectrum, growing best at values ranging from pH 9 to pH 11. They are commonly found in saline soda lakes, which have high salt concentrations and pH values (as high as pH 11). Soda lakes, like Lake Magadi in Africa (Fig. 5.15A), are steeped in carbonates, which explains their extraordinarily alkaline pH. An alkaliphilic organism fi rst identified in Lake Magadi is Halobacter salinarum (also known as a NatronobacteFigure 5.15 A soda lake ecosystem. rium gregoryi), a halophilic archaeon A. Lake Magadi in Kenya. Its pink (Fig. 5.15B). color is due to Spirulina. B. Alkaliphile The cyanobacterium Spirulina is Natronobacterium gregoryi. Cell size another alkaliphile that grows in soda approx. 1 × 3 µm. C. Pink flamingo lakes. Its high concentration of carocolored by ingestion of Spirulina. tene gives the organism a distinctive pink color (note the color of the lake in Fig. 5.15A). Spirulina is also a major food for the famous pink flamingos indigenous to these African lakes and is, in fact, the reason pink flamingos are pink. After the birds ingest these organisms, digestive
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work of the cell (see Section 14.3). They also rely heavily on Na+/H+ antiporters (see Section 4.2) to bring protons into the cell, and this keeps the internal pH well below the extremely alkaline external pH. This is partly why many alkaliphiles are resistant to high salt (NaCl) concentrations; sodium ions are expelled while protons are sucked in. Important aspects of sodium circulation in alkaliphiles are depicted in Figure 5.16. In contrast to proteins within the cytoplasm, enzymes secreted from alkaliphiles are able to work in very alkaline environments. The inclusion of base-resistant enzymes like lipases and cellulases in laundry detergents helps get our “whites whiter and our brights brighter.” Other commercially useful alkaliphilic enzymes produce cyclodextrins from starch. The cyclodextrins are complex cyclic carbohydrates whose structure resembles a hollow truncated cone with a hydrophobic (water-hating) core and hydrophilic (water-loving) exterior (Fig. 5.17). It is a superb vehicle for drug delivery. For example, cyclodextrins have been used in eyedrops to deliver the antibiotic chloramphenicol. The hydrophobic cavity of cyclodextrin can harbor a poorly soluble drug, while the hydrophilic exterior increases its apparent water solubility. The drug goes into solution more easily, permitting smoother absorption into the body.
processes release the carotene pigment to the circulation, which then deposits it in the birds’ feathers, turning them pink (Fig. 5.15C). NOTE: Humans also consume Spirulina as a health food supplement, but do not turn pink because the cyanobacteria are only a small component of the diet.
The internal enzymes of alkaliphiles, like those of acidophiles, exhibit rather ordinary pH optima (around pH 8). The key to the survival of alkaliphiles is the cell surface barrier that sequesters fragile cytoplasmic enzymes away from harsh extracellular pH. Key structural features of the cell wall, such as the presence of acidic polymers and an excess of hexosamines in the peptidoglycan, appear to be essential. The reason for this is not clear. At the membrane, some alkaliphiles also possess a high level of diether lipids, which are more stable than ester-linked phospholipids, that prevent protons from leaking out of the cell (see Section 3.4). Because external protons are in such short supply at alkaline pH, most alkaliphiles use a sodium motive force in addition to a proton motive force to do much of the
Terminal cytochromes pump Na+ instead of H+.
Decarboxylases couple Na+ export with decarboxylation of substrates (e.g., oxalocetate f pyruvate).
Cytochromes
H+
Decarboxylases
Antiport
+
RCOO– + H+
RH + CO2 Na+-driven ATPases export Na+.
ATP
–
Bacterial cytoplasm is negatively charged relative to the exterior.
ADP + Pi
Na+
ADP + Pi ATP Na+ motive force powers motility.
Symport Na+
Flagellar motor Solute
Na+ motive forces drive symport of some substrates.
ATP synthase Na+ Cell membrane Na+
Na+ import through F1Fo ATPase drives ATP synthesis in some organisms.
Na+ circulation in alkaliphiles. Cells of alkaliphiles are designed to use Na+ in place of H+ to do the work of the cell. They require an inwardly directed sodium gradient that can be used to rotate flagella, transport nutrient solutes, and generate energy. The Na+/H+ antiporter is used to keep internal pH lower than exterior pH. Sodium homeostasis inside the cell is maintained by the net effects of efflux and influx mechanisms. Figure 5.16
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pH Homeostasis and Acid Tolerance Mechanisms Enable Cells to Live in Different pH Environments
Hydrophilic hydroxyl group
When cells are placed in pH conditions below their optimum, protons can enter the cell and lower internal pH to lethal levels. Microbes can prevent the unwanted influx of protons by exchanging extracellular K+ for intracellular protons when internal pH becomes too low. At the other extreme, under extremely alkaline conditions, the cells can use the Na+/H+ antiporters mentioned previously (and in Section 4.2) to recruit protons into the cell in exchange for expelling Na+ (Fig. 5.18). Some organisms can also change the pH of the medium using various amino acid decarboxylases and deaminases. Helicobacter pylori, the causative agent of gastric ulcers, employs an exquisitely potent urease to generate massive amounts of ammonia, which neutralizes the acid pH environment. These acid stress and alkaline stress protection systems are usually not made or at least do not become active until the cell encounters an extreme pH. Many, if not all, microbes also possess an emergency global response system referred to as acid tolerance or acid resistance. In a process analogous to the heat-shock response, bacterial physiology undergoes a major molecular reprogramming in response to hydrogen ion stress. The levels of a large number of proteins increase, while the levels of others decrease. Many of the genes and proteins involved in the acid stress response overlap with other stress response systems, including the heat-shock response. These physiological responses include modifications in membrane lipid composition, enhanced pH homeostasis,
Hydrophobic ether group
Figure 5.17 A cyclodextrin. View down the channel in the hydrophobic core lined by C-O-C ether groups, which is surrounded by a hydrophilic exterior covered in -OH hydroxyl groups. The hydrophobic core can harbor drugs that are poorly soluble in water.
THOUGHT QUESTION 5.5 In Chapter 4, we stated that an antiporter couples movement of one ion down its concentration gradient with movement of another molecule uphill, against its gradient. If this is true, how could a Na+/H+ antiporter work to bring protons into a haloalkiliphile growing in high salt at pH 10? Since the Na+ concentration is lower inside the cell than outside and H+ concentration is higher inside than outside (see Fig. 5.16), both ions are moving against their gradients.
Figure 5.18 Proton circulation and pH homeostasis. A typical E. coli cell uses various proton transport strategies to maintain an internal pH near pH 7.8 in the face of different external pH stresses. Proton pumping through cytochromes also establishes a proton gradient, which drives flagellar rotation and solute transport.
In a strongly acidic (pH 2) environment, amino acid decarboxylases drain protons RH from the cell.
Under alkaline stress (pH 9) Na+/H+ antiport systems scavenge protons from the environment.
Cytochromes
–
H+
RCOO
RH + CO2 Under slightly acidic conditions (pH 5), cells use a K+/H+ antiport system K+ to remove internal protons.
–
Na+
+
RCOO + H
pH 7.8 H+
ADP + Pi
ATP
– Symport +
H
Flagellar motor Solute
ATP synthase
Cell membrane H+
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+
H+ E. coli cytoplasm is negatively charged relative to the exterior.
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Special Topic 5.1
Signaling Virulence
One of the burning questions in the study of infectious disease is, “How does a microbe know when it has entered a suitable host?” Most, if not all, pathogens express certain genes only after infection, so they must sense that something in their environment has changed. A change in environment that causes increased virulence is called a virulence signal. One of these virulence signals is pH. On their way to causing disease, many pathogens travel through acidic host compartments. The most obvious are the stomach, which is extremely acidic (pH 1–3), and infected host cell phagosomes and phagolysosomes, which are mildly acidic (pH 4.5–6). Salmonella enterica, a major causative agent of diarrheal disease, provides an intriguing example of virulence that is triggered by low pH. After gaining entrance to the intestine via the mouth and subsequently penetrating intestinal epithelia, this pathogen tricks certain white blood cells, called macrophages, into engulfing the bacterium, placing it within a phagosome (Fig. 1). The microbe then waits for the phagosome (initially pH 6) to partially fuse with a lysosome and form a phagolysosome, after which the pH of the compartment drops to about pH 4.5. Numerous bacterial proteins are then expressed, many of which enable Salmonella to survive in the macrophage. Drugs that prevent phagolysosome acidification, however, render Salmonella extremely susceptible to the various antimicrobial weapons wielded by the macrophage. Salmonella “knows” it
and numerous other changes with unclear purpose. Some pathogens, such as Salmonella, sense a change in external pH as part of the signal indicating the bacterium has entered a host cell environment (Special Topic 5.1). TO SU M MAR I Z E: ■
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Hydrogen ion concentration affects protein structure and function. Thus, enzymes have pH optima, minima, and maxima. Microbes use pH homeostasis mechanisms to keep their internal pH near neutral when in acidic or alkaline media. The addition of weak acids to certain foods undermines bacterial pH homeostasis mechanisms, which prevents food spoilage and kills potential pathogens. Neutralophiles, acidophiles, and alkaliphiles prefer growth under neutral, low, and high pH conditions, respectively. Acid and alkaline stress responses occur when placing a given species under pH conditions that slow its growth. The cell increases the levels of proteins designed to mediate pH homeostasis and protect cell constituents.
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is in a macrophage phagolysosome in part because the pH drops. Recent studies have started to map the complex bacterial regulatory circuits involved in sensing acid pH signals and translating them into defensive actions by the pathogen.
Salmonella Phagosome Brett Finlay, U. of British Columbia
164
Salmonella (1 lm in length) within human epithelial cell phagosomes.
Figure 1
5.6
Microbial Responses to Oxygen and Other Electron Acceptors
Many microorganisms can grow in the presence of molecular oxygen (O2). Some even use oxygen as a terminal electron acceptor in the electron transport chain, a group of membrane proteins (cytochromes) that convert energy trapped in nutrients to a biologically useful form. The use of O2 as the terminal electron acceptor is called aerobic respiration (see Chapter 14).
Oxygen Has Benefits and Risks Electrons pulled from various energy sources (for example, glucose) possess intrinsic energy, an energy that cytochrome proteins can harness. They do this by extracting the energy in incremental stages and using it to move protons out of the cell. This unequal distribution of H+ across the membrane produces a transmembrane electrochemical gradient, a sort of biobattery called proton motive force (discussed in Section 14.2). Once the cell has drained as much energy as possible from an electron,
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Figure 5.19 Role of oxygen as a terminal electron acceptor in respiration. This pumping of protons out of the cell by cytochromes produces more positive charges outside the cell than inside, creating an electrochemical gradient (also called proton motive force). An electron from a high-energy source is passed from one member of the chain to the next. With each transfer of an electron to the next member of the chain, more energy is extracted and converted to proton motive force. At the end of the chain, the electron must be passed to a final or terminal electron acceptor, clearing the path for the next electron. This net process is called respiration.
Terminal electron acceptor ½ O2 H2O
Cytochromes H+ 2. Shuttle molecules like NAD move the electrons to a series of membrane proteins called cytochromes.
2 e–
NAD
Glucose
3. Cytochromes extract energy from the electrons and use that energy to pump H+ out of the cell.
– – 2 e 2 e– 2 e + H+ H
+
Energy source
H+
165
H+
+
NADH + H
Glucose 6-P (reduced)
Acetate (oxidized)
1. Cells remove protons and high-energy electrons from energy sources like glucose. Cell membrane
that electron must be passed to a diffusible, fi nal electron acceptor molecule floating in the medium. This clears the way for another electron to be passed down the cytochrome chain (Fig. 5.19). One such terminal electron acceptor is dissolved oxygen. However, oxygen and its breakdown products are dangerously reactive, a serious problem for all cells. As a result, different species have evolved to either tolerate or avoid oxygen all together. Table 5.2 gives examples of microbes that grow at different levels of oxygen.
Microbes Are Classified by Their Relationship with Oxygen The relationships between microbes and oxygen are varied. Figure 5.20 illustrates a test tube with growth medium. The top of the tube, closest to air, is oxygenated; the bottom of the tube has much lower levels of oxygen. Some microbes grow only at the top of the tube, while others prefer to grow at the bottom, the distributions based on each organism’s relationship with oxygen. A
Table 5.2 Examples of aerobes and anaerobes. Aerobic
Anaerobic
Facultative
Microaerophilic
Neisseria spp. Causative organisms of meningitis, gonorrhea
Azoarcus tolulyticus Degrades toluene
Escherichia coli Normal GI flora; additional pathogenic strains
Helicobacter pylori Causative organism of gastric ulcers
Pseudomonas fluorescens Found in soil and water; degrade pollutants such as TNT and aromatic hydrocarbons
Bacteroides spp. Normal GI flora
Saccharomyces cerevisiae Yeast; used in baking
Lactobacillus Ferments milk to form yogurt
Mycobacterium leprae Causative organism of leprosy
Clostridium spp. Soil microorganisms; causative agents of tetanus and botulism
Bacillus anthracis Causative organism of anthrax
Campylobacter spp. Causative organism of gastroenteritis
Azotobacter Soil microorganisms; fix atmospheric nitrogen
Actinomyces Soil microrganisms; synthesize antibiotics
Vibrio cholerae Causative organism of cholera
Treponema pallidum Causative organism of syphilis
Rhizobium spp. Soil microorganisms; plant symbionts
Desulfovibrio spp. Reduce sulfate
Staphylococcus spp. Found on skin; causative agent of boils
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Micro-aerophilic
High oxygen
Low oxygen
Anaerobes
Facultative
Aerobes
Growth zones
No oxygen
Oxygen-related growth zones in a standing
Figure 5.20
test tube.
strict aerobe is an organism that not only exists in oxygen but also uses oxygen as a terminal electron acceptor. In fact, a strict aerobe can only grow with oxygen present and possesses an aerobic metabolism (aerobic respiration). An aerobe will grow at the top of the tube shown in Figure 5.20. In contrast, a strict anaerobe dies in the least bit of oxygen. Strict anaerobes do not use oxygen as an electron acceptor, but this is not why they die in air. They die because this type of microbe is vulnerable to reactive oxygen molecules (also called reactive oxygen species; ROS) produced by its own metabolism when it is exposed to oxygen. Anaerobes will grow at the bottom of the tube shown in Figure 5.20. Do not confuse the ability of an organism to exist in the presence of oxygen with its ability to use oxygen as
a terminal electron acceptor. Some aerobes can survive in oxygen but do not have the ability to use oxygen. Any organism that possesses NADH dehydrogenase II, aerobe or anaerobe, will, in the presence of oxygen, inadvertently autooxidize the FAD cofactor within the enzyme and produce dangerous amounts of superoxide radicals – ( •O2 ; Fig. 5.21). Superoxide will degrade to hydrogen peroxide (H 2O2), another reactive molecule. Iron, present as a cofactor in several enzymes, can then catalyze a reaction with hydrogen peroxide to produce the highly toxic hydroxyl radical ( •OH). All of these molecules seriously damage DNA, RNA, proteins, and lipids. Consequently, oxygen is actually an extreme environment in which survival requires special talents. Aerobes, but not anaerobes, destroy reactive oxygen species with the aid of enzymes such as superoxide dismutase (to remove superoxide) and peroxidase and catalase (to remove hydrogen peroxide). Aerobes also have resourceful enzyme systems that detect and repair macromolecules damaged by oxidation.
Anaerobes Ferment and Respire without Oxygen Anaerobic microbes fall into several categories. Some anaerobes actually do respire using cytochrome systems, but instead of using oxygen, they rely on alternative terminal electron acceptors like nitrate to conduct anaerobic respiration and produce energy. Anaerobes of another ilk do not possess cytochromes, cannot respire, and so must rely on carbohydrate fermentation for energy (that is, they conduct fermentative metabolism). Fermentation involves the production of ATP energy through substrate-level phosphorylation in a process that does not involve cytochromes. Facultative organisms are
O2 + e–
FAD
These reactions generate ROS.
O2– Superoxide radical union
O2– + H+
superoxide dismutase
H2O2 Hydrogen peroxide Fe2+
(Fenton reaction) catalase 2 H2O2 2 H2O + O2 2 H2O2
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Fe3+ OH– + OH Hydroxyl radical
H2O2
These reactions destroy ROS.
Generation and destruction of reactive oxygen species (ROS). The autoxidation of flavin adenine dinucleotide (FAD) and the Fenton reaction occur spontaneously to produce superoxide and hydroxyl radicals, respectively. The other reactions require enzymes. FAD is a cofactor for a number of enzymes (for example, NADH dehydrogenase II). Catalase and peroxidase do not produce ROS but detoxify hydrogen peroxide.
Figure 5.21
The production of ROS often begins with the autooxidation of FAD.
peroxidase
2 H2O + NAD+
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microbes that can live with or without oxygen. They will grow throughout the tube shown in Figure 5.20. Facultative anaerobes (sometimes called aerotolerant) only use fermentation to provide energy but contain superoxide dismutase and catalase (or peroxidase) to protect them from reactive oxygen species. This allows them to grow in oxygen while retaining a fermentation-based metabolism. Facultative aerobes (such as E. coli) also possess enzymes that destroy toxic oxygen by-products, but have both fermentative and aerobic respiratory potential. Whether a member of this group uses aerobic respiration, anaerobic respiration, or fermentation depends on the availability of oxygen and the amount of carbohydrate present. Microorganisms that possess decreased levels of superoxide dismutase and/or catalase will be microaerophilic, meaning they will grow only at low oxygen concentrations. The fundamental composition of all cells reflects their evolutionary origin as anaerobes. Lipids, nucleic acids, and amino acids are all highly reduced—which is why our bodies are combustible. We never would have evolved that way if molecular oxygen were present from the beginning. Even today, the majority of all microbes are anaerobic, growing buried in the soil, within our anaerobic digestive tract, or within biofi lms on our teeth.
Many anaerobic bacteria cause horrific human diseases, such as tetanus, botulism, and gangrene. Some of these organisms or their secreted toxins are even potential weapons of terror (for example, Clostridium botulinum). Because of their ability to wreak havoc on humans, culturing these microorganisms was an early goal of microbiologists. Despite the difficulties involved, conditions were eventually contrived in which all, or at least most, of the oxygen could be removed from a culture environment. Three techniques are used today. Special reducing agents (for example, thioglycolate) or enzyme systems (Oxyrase®) that eliminate dissolved oxygen can be added to ordinary liquid media. Anaerobes can then grow beneath the culture surface. A second, very popular, way to culture anaerobes, especially on agar plates, is to use an anaerobe jar (Fig. 5.22A). Agar plates streaked with the organism are placed into a sealed jar with a foil packet that releases H2 and CO2 gases. A palladium packet hanging from the jar lid catalyzes a reaction between the H2 and O2 in the jar to form H2O and effectively removes O2 from the chamber. The CO2 released is required by some reactions to produce key metabolic intermediates. Some microaerophilic microbes, like the pathogens H. pylori (the major cause of stomach ulcers) and Campylobacter jejuni (a major cause of diarrhea), require low levels of O2 but elevated amounts of CO2. These conditions are obtained by using similar gas-generating packets. For strict anaerobes exquisitely sensitive to oxygen, even more heroic efforts are required to establish an oxygen-free environment. A special anaerobic glove box must be used in which the atmosphere is removed by
THOUGHT QUESTION 5.7 How can anaerobes grow in the human mouth when there is so much oxygen there?
B.
Catalyst in lid mediates reaction. H2 + ½O2 f H2O
Airlock
Tracy Grosshans
©Jack Bostrack/Visuals Unlimited
GasPak envelope generates H2 and CO2.
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THOUGHT QUESTION 5.6 If anaerobes cannot live in oxygen, how do they incorporate oxygen into their cellular components?
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Anaerobic growth technology. B. An anaerobic chamber with glove ports.
Figure 5.22
A. An anaerobic jar.
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vacuum and replaced with a precise mixture of N2 and CO2 gases (Fig. 5.22B). THOUGHT QUESTION 5.8 What evidence led people to think about looking for anaerobes? Hint: Look up Spallanzani, Pasteur, and spontaneous generation on the Web.
Starvation Slows Cell Metabolism but Activates Key Survival Genes
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Oxygen is a benefit to organisms (aerobes) that can use it as a terminal electron acceptor to extract energy from nutrients. Oxygen is toxic to all cells (anaerobes) that do not have enzymes capable of efficiently destroying the reactive oxygen species. Anaerobic metabolism can either be fermentative or respiratory. Anaerobic respiration requires the organism to possess cytochromes that can use compounds other than oxygen as terminal electron acceptors. Facultative anaerobes grow either in the presence or in the absence of oxygen, but use fermentation as their primary, if not only, means of gathering energy. These microbes also have enzymes that destroy reactive oxygen species, allowing them to grow in oxygen. Facultative aerobes, like facultative anaerobes, grow with or without oxygen and have enzymes that destroy reactive oxygen species. In addition, they possess both the ability for fermentative metabolism and the ability to use oxygen as a terminal electron acceptor.
5.7
Microbial Responses to Nutrient Deprivation and Starvation
It is intuitively obvious that limiting the availability of a carbon source or other essential nutrient will limit
Numerous gene systems are affected when nutrients decline (see Section 10.3 for details). Growth rate slows, and daughter cells become smaller and begin to experience what is termed a “starvation” response, where the microbe senses a dire situation developing but still strives to fi nd new nourishment. The resulting metabolic slowdown generates increased concentrations of critically important small signal molecules such as cyclic AMP and guanosine tetraphosphate (ppGpp) that globally transform gene expression. During this metabolic retooling, transport systems for potential nutrients are produced even if the matching substrates are unavailable. Cells begin to make and store glycogen, presumably as an internal emergency store in case no other nutrient is found. Some organisms growing on nutrient-limited agar plates can even form colonies with intricate geometrical shapes that help the population cope, in some unknown way, with nutrient stress (Fig. 5.23). As a cultural environment progressively worsens, the organism must prepare for famine, and many different stress survival genes become active. The products of these genes afford protection against stressors such as reactive oxygen radicals or temperature and pH extremes. No cell can predict the precise stresses it might encounter while incapacitated, so it is advantageous to be prepared for as many as possible. As described in Chapter 4, some species undergo elaborate developmental processes that ultimately produce dormant spores.
Effects of starvation on colony morphology. A. Starving E. coli colony. B. Pseudomonas dendritiformis C morphotype, grown on hard agar (1.75%) under starvation conditions. The colony consists of branches with chiral twists (colored green), all with the same handedness. The added coloration indicates time of growth (yellow = oldest; red = most recent).
Figure 5.23
B.
John Foster, U. of South Alabama
A.
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growth. Not so obvious are the dramatic molecular events that cascade through a cell undergoing nutrient limitation and ultimately starvation. Optimizing growth rate at suboptimal nutrient levels is an important aim of free-living bacteria, given that intestinal, soil, and marine environments rarely offer nutrient excess.
Eshel Ben-Jacob, Tel Aviv University
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Some Organisms Only Grow in Nutrient-Poor Media Most well-studied organisms thrive on organically rich media (more than 2 g of carbon per liter) but struggle to grow at very low nutrient concentration (1–5 mg of carbon per liter). In natural ecosystems, however, the majority of existing microbes appear to be oligotrophs, organisms with a high rate of growth at extremely low organic substrate concentrations. Oligotrophs are well suited for growth in the nutrient-poor, or oligotrophic, environments found in certain lakes and streams. Some oligotrophs are actually poisoned by concentrated organics or commit suicide in rich media by overproducing excess toxic hydrogen peroxide. Consequently, they require low nutrient levels to survive. In soil, for example, there exist a variety of uncharacterized oligotrophic bacteria that are very sensitive to NaCl and various L-amino acids. How do oligotrophs manage to grow in such impoverished conditions? Some oligotrophic bacteria have thin extensions of their membrane and cell wall called prothecae (stalks) that essentially expand the surface area of the cell and increase capacity to transport nutrients. How other oligotrophs grow remains a mystery.
Microbes Encounter Multiple Stresses in Real Life In keeping with the reductionist approach to science, bacterial stress responses have traditionally been studied in terms of individual stresses. Escherichia coli, for example, increases synthesis of a specific set of proteins when exposed to high temperature and a different set of proteins when exposed to high salt. There are overlapping members of the two sets; that is, several proteins may be highly expressed under both conditions, but each stress response also includes proteins unique to each stress. Environmental situations in the world outside of the laboratory, however, can be quite complex, involving multiple, not just single, stresses. An organism could simultaneously undergo carbon starvation in a high salt, low pH environment. A classic study by Kelly Abshire and Frederich Neidhardt examined this situation using the pathogen Salmonella enterica, a cause of diarrhea. S. enterica invades human macrophage cells and survives in phagocytic vacuoles, where numerous stresses, such as low pH, oxidative stress, and nutrient limitations, are simultaneously imposed on the bacteria. Comparing the proteins synthesized by Salmonella growing in this compartment with the proteins synthesized under single stresses in the laboratory revealed an unexpected response pattern. Although many stress-related proteins were induced in the intracellular environment, no one set of stress-induced proteins was induced in its entirety.
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Furthermore, several bacterial proteins were induced by growth only within the macrophage phagolysosome, suggesting the presence of unknown intracellular stresses. Thus caution is advised when trying to predict cell responses to real-world situations based solely on controlled laboratory studies that alter only single parameters.
Humans Influence Microbial Ecosystems Natural ecosystems are typically low in nutrients (oligotrophic) but teem with diversity, so that numerous species compete for the same limiting nutrients. Maximum diversity in a given ecosystem is maintained, in part, by the different nutrient-gathering profi les of competing microbes. Take a scenario in which microbe A is better than microbe B at gathering phosphate when phosphate levels are low, but microbe B is superior to A at culling limited quantities of nitrogen (Fig. 5.24). Neither organism dominates when both phosphate and nitrogen are in short supply. So even though B can harvest more nitrogen than A, it cannot outgrow A, since the phosphate concentration is low. However, if phosphate is suddenly increased so that it is no longer limiting for either organism, the one better adapted to low nitrogen (microbe B) will outgrow and overwhelm the other. The sudden infusion of large quantities of a formerly limiting nutrient, a process called eutrophication, can lead to a “bloom” of microbes. Organisms initially held in check by the limiting nutrient now exhibit unrestricted growth, consuming other nutrients to a degree that threatens the existence of competing species. Humans have caused nutrient pollution in several ways. Runoff from agricultural fields, urban lawns, and golf courses is one source. Untreated or partially treated domestic sewage is another. Sewage was a primary source of phosphorus eutrophication of lakes in the 1960s and 1970s, when detergents contained large amounts of phosphates. The phosphates acted as water softeners to improve cleaning action, but when washed into lakes, they also proved to be powerful stimulants to algal growth (Fig. 5.25). The resulting algal “blooms” in many lakes led to oxygen depletion and resultant fish kills. Many native fish species disappeared, to be replaced by species more tolerant of the new conditions. TO SU M MAR I Z E: ■
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Starvation is a stress that can elicit a starvation response in many microbes. Enzymes are produced to increase the efficiency of nutrient gathering and to protect cell macromolecules from damage. The starvation response is usually triggered by the accumulation of small signal molecules such as cyclic AMP or ppGpp.
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Maintaining microbial diversity through nutrient limitation. Species A and species B require both nitrogen and phosphate to grow. Top: Neither species dominates because each has a limiting nutrient (N = nitrogen; P = phosphate). Bottom: Following phosphate eutrophication, species B will outgrow species A because while both species have enough phosphate, B is better able to assimilate the limited quantity of nitrogen.
Figure 5.24
Low P Transports phosphate efficiently
Transports phosphate poorly
P
P
P
P
Species B
Species A
N
N
Transports nitrogen poorly Low N
N
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Transports nitrogen efficiently
Neither organism has an advantage. Increase phosphate concentration
High
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Species B can outcompete species A.
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Oligotrophs are organisms that thrive in nutrientpoor conditions. Human activities can cause eutrophication, which damages delicately balanced ecosystems by introducing nutrients that can allow one member of the ecosystem to flourish at the expense of other species.
5.8
Eutrophication
Eutrophication. Algal bloom in an experimental lake resulting from phosphate eutrophication. A divider curtain separates the lake and prevents mixing of water. The bright green color results from cyanobacteria growing on phosphorus added to the near side of the curtain.
Figure 5.25
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Physical and Chemical Methods of Controlling Microbial Growth
Prevention, control, or elimination of microbes that are potentially harmful to humans is one of the primary goals of our health care system. Within the recent past, infectious disease was an imminent and constant threat to most of the human population. The average family in the United States prior to 1900 had four or five children, in part because parents could expect half of them to succumb to deadly infectious diseases. What today would be a simple infected cut in years past held a serious risk of death, and a trip to the surgeon was tantamount
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Sterilization is the process by which all living cells, spores, and viruses are destroyed on an object. Disinfection is the killing, or removal, of diseaseproducing organisms from inanimate surfaces; it does not necessarily result in sterilization. Pathogens are killed, but other microbes may survive. Antisepsis is similar to disinfection, but it applies to removing pathogens from the surface of living tissues, like the skin. Antiseptic chemicals are usually not as toxic as disinfectants, which frequently damage living tissues. Sanitation, closely related to disinfection, consists of reducing the microbial population to safe levels and usually involves cleaning an object as well as disinfection.
Antimicrobials can also be classified on the basis of the specific groups of microbes destroyed, leading to the terms bactericide, algicide, fungicide, and virucide. Furthermore, these agents can be classified either as -static (inhibiting growth) or -cidal (killing cells). For example, antibacterial agents are called bacteriostatic or bactericidal. Chemical substances that kill microbes are germicidal if they kill pathogens (and many nonpathogens). Germicidal agents do not necessarily kill spores. Although these descriptions place emphasis on killing pathogens, it is also important to note that antimicrobial agents can also kill or prevent the growth of nonpathogens. Many public health standards are based on total numbers of microorganisms on an object, regardless of pathogenic potential. For example, to gain public health certification, the restaurants we frequent must demonstrate low numbers of bacteria in their food preparation areas.
Cells Treated with Antimicrobials Die at a Logarithmic Rate Exposure of microbes to lethal chemicals or conditions does not instantly kill all microorganisms. Microbes die according to a negative exponential curve, where cell numbers are reduced in equal fractions at constant intervals. The efficacy of a given agent or condition is measured as decimal reduction time (D-value), which is the length of time it takes that agent (or condition) to
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8 7 Log10 viable organisms
to playing Russian roulette with an unsterilized scalpel. Improvements in sanitation procedures and antiseptics and the advent of antibiotics have, to a large degree, curtailed the incidence and lethal effects of many infectious diseases. Success in this endeavor has played a major role in extending life expectancy and in contributing to the population explosion. A variety of terms are used to describe antimicrobial control measures. The terms convey subtle, yet vitally important, differences in various control strategies and outcomes.
171
6 5 4
100% 10% (90% killed)
3 2
D 100
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Exposure (min)
The death curve and the determination of D-values. Bacteria were exposed to a temperature of 100°C, and survivors were measured by viable count. The D-value is the time required to kill 90% of cells (that is, for the viable cell count to drop by one log10 unit). In this example, the D-value at 100°C (D100) is approximately 1 minute.
Figure 5.26
kill 90% of the population (a drop of 1 log unit, or a drop to 10% of the original value). Figure 5.26 illustrates the exponential death profi le of a bacterial culture heated to 100°C. The D-value is a little over 1 minute. Several factors influence the D-value—that is, the ability of an antimicrobial agent to kill microbes. These include the initial population size (the larger the population, the longer it takes to decrease it to a specific number), population composition (are spores involved?), agent concentration, and duration of exposure. Although the effect of concentration seems intuitively obvious, only over a narrow range is an increase in concentration matched by an increase in death rate. Increases above a certain level might not accelerate killing at all. For example, 70% ethanol is actually better than pure ethanol at killing organisms. This is because some water is needed to help ethanol penetrate cells. The ethanol then dehydrates cell proteins. So, why is death a logarithmic function? Why don’t all cells in a population die instantly when treated with lethal heat or chemicals? The reason is based, in part, on the random probability of the agent causing a lethal “hit” in a given cell. Cells contain thousands of different proteins and thousands of molecules of each. Not all proteins and not all genes in a chromosome are damaged by an agent at the same time. Damage accumulates. Only when enough molecules of an essential protein or a gene encoding that protein are damaged will the cell die. Cells that die fi rst are those that accumulate lethal hits early. Members of the population that die later have, by random chance, absorbed more hits on nonessential proteins or genes, sparing the essential ones.
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Why, if 90% of a population is killed in 1 minute, aren’t the remaining 10% killed in the next minute? It seems logical that all should have perished. Yet after the second minute, 1% of the original population remains alive. This can also be explained by the random hit concept. Although there are fewer viable cells after 1 minute, each has the same random chance of having a lethal hit as when the treatment began. Thus, death rate is an exponential function, much like radioactive decay is an exponential function. A fi nal consideration involves the overall fitness of individual cells. It is mistaken to assume that all cells in a population are identical. For example, at any given time, one cell may express a protein that another cell has just stopped expressing (for example, superoxide dismutase). In that instant, the fi rst cell might contain a bit more of that protein. If the protein is essential or confers a level of stress protection (such as against superoxide), the cell can absorb more punishment before it is dispatched. The presence of lucky individuals expressing the right repertoire of proteins might also explain why death curves commonly level off after a certain point.
Physical Agents That Kill Microbes Physical agents are often used to kill microbes or control their growth. Commonly used physical control measures are temperature extremes, pressure (usually combined with temperature), fi ltration, and irradiation. High temperature and pressure. Even though microbes
were discovered less than 400 years ago, thermal treatment of food products to render them safe has been practiced for over 5,000 years. Moist heat is a much more effective killer than dry heat, thanks to the ability of water to penetrate cells. Many bacteria, for instance, easily withstand 100°C dry heat but not 100°C in boiling water. We humans are not so different, fi nding it easier to endure a temperature of 32°C (90°F) in dry Arizona than in humid Louisiana. While boiling water (100°C) can kill most vegetative (actively growing) organisms, spores are built to withstand this abuse, and thermophiles prefer it. Killing spores and thermophiles usually requires combining high pressure and temperature. At high pressure, the boiling point of water rises to a temperature rarely experienced by microbes living at sea level. Even endospores quickly die under these conditions. This combination of pressure and temperature is the principle behind sterilization using the steam autoclave (Fig. 5.27). Standard conditions for steam sterilization are 121°C (250°F) at 15 psi for 20 minutes, a set of conditions that experience has taught us will kill all spores. These are also the conditions produced in pressure cookers used for home canning of vegetables.
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Safety valve Exhaust valve (to remove steam after sterilization)
Operating valve (controls steam from jacket to chamber) Pressure gauge
Steam to chamber
Steam Steam chamber
Air
Perforated shelf
Steam jacket
Automatic ejector valve is thermostatically controlled and closes on contact with pure steam when air is exhausted To waste Steam supply line
Figure 5.27
Sediment screen Thermometer Pressure regulator for steam supply
Steam autoclave.
Failure to adhere to these heat and pressure parameters can have deadly consequences, even in your own home. For instance, Clostridium botulinum is a sporeforming soil microbe that commonly contaminates fruits and vegetables used in home canning. The improper use of a pressure cooker while canning these goods will allow spores of this pathogen to survive. Once the can or jar is cool, the spores will germinate and begin producing their deadly toxin. All of this happens while the canned goods sit on a shelf waiting to be opened and consumed. Once ingested, the toxin makes its way to the nervous system and paralyzes the victim. Several incidents of this disease, called botulism, occur each year in the United States. (For more on food poisoning, see Chapter 16.) THOUGHT QUESTION 5.9 How would you test the killing efficacy of an autoclave? The food industry uses several parameters to evaluate the efficiency of heat killing. The D-value has already been described. Two additional measures are 12D, the amount of time required to kill 1012 spores (or reduce a population 12 logs); and the z-value, the increase in temperature needed to lower the D-value to 1 /10 its previous value. If, for example, D100 (the D-value at 100°C) and D110 (the D-value at 110°C) for a given organism are 20 minutes and 2 minutes, respectively, then 12D100 equals 240 minutes (that is, 20 minutes × 12), and the z-value
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is 10°C (because a 10°C increase in temperature reduced the D-value to 1 /10, from 20 minutes to 2 minutes). These measurements are extremely important to the canning industry, which must ensure that canned goods do not contain spores of Clostridium botulinum, the previously described anaerobic soil microbe that causes the paralyzing food-borne disease botulism. Because the tastes of certain foods suffer if they are overheated, z-values and 12D-values are used to adjust heating times and temperatures to achieve the same sterilizing result. Take the following example, where D121 is 10 minutes and 12D121 is 120 minutes. Sterilizing food at 121°C for 120 minutes might result in food with a repulsive taste, whereas reducing the temperature and extending the heating time may yield a more palatable product. The D- and z-values are used to adjust conditions for sterilization at a lower temperature. If D121 is 15 minutes and the z-value is known to be 10°C, then reducing temperature by 10°C to 110°C will mean D111 is 150 minutes (10 times D121). As a result, 12D111 is 1,800 minutes. Sterilization may take longer, but food quality is likely to remain high, because the sterilizing temperature is lower. Pasteurization. Originally devised by Louis Pasteur to save products of the French wine industry from devastating bacterial spoilage, pasteurization today involves heating a particular food (such as milk) to a specific temperature long enough to kill Mycobacterium tuberculosis and Coxiella burnetii. These two organisms, the causative agents of tuberculosis and Q fever, respectively, are the most heat-resistant nonspore-forming pathogens known. Many different time and temperature combinations can be used for pasteurization. The LTLT (low-temperature/long-time) process involves bringing milk to a temperature of 63°C (145°F) for 30 minutes. In contrast, the HTST (high-temperature/short-time, also called flash pasteurization) method brings the milk to a temperature of 72°C (161°F) for only 15 seconds. Both processes accomplish the same thing: the destruction of M. tuberculosis and C. burnetii. Cold. Low temperatures have two basic purposes in microbiology: to temper growth and to preserve strains. Bacteria not only grow more slowly in cold, but also die more slowly. Refrigeration temperatures (4–8°C, or 39–43°F) are used for food preservation because most pathogens are mesophilic and grow poorly, if at all, at those temperatures. One exception is the gram-positive bacillus Listeria monocytogenes, which can grow reasonably well in the cold and causes disease when ingested. Long-term storage of bacteria usually requires placing solutions in glycerol at very low temperatures (–70°C). Glycerol prevents the production of razor-sharp ice crys-
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tals that can pierce cells from without or within. This deep-freezing suspends growth altogether and keeps cells from dying. Another technique called lyophilization freeze-dries microbial cultures for long-term storage. In this technique, cultures are quickly frozen at very low temperatures (quick-freezing also limits ice crystal formation) and placed under vacuum, where the resulting sublimation process removes all water from the media and cells, leaving just the cells in the form of a powder. These freeze-dried organisms remain viable for years. Finally, viruses and mammalian cells must be kept at extremely low temperatures (–196°C), submerged in liquid nitrogen. Liquid nitrogen freezes cells so quickly that ice crystals do not have time to form. Filtration. Filtration through micropore fi lters with
pore sizes of 0.2 µm can remove microbial cells, but not viruses, from solutions. Samples from 1 ml to several liters can be drawn through a membrane fi lter by vacuum or can be forced through it using a syringe (Fig. 5.28). Filter sterilization has the advantage of avoiding heat, which can damage the material sterilized. Of course, since these fi lters do not trap viruses, the solutions are not really sterile. Air can also be sterilized by fi ltration. This forms the basis of several personal protective devices. A surgical mask is a crude example, while laminar flow biological safety cabinets are more elaborate (and more effective). These cabinets force air through high-efficiency particulate air (HEPA) fi lters and remove over 99.9% of airborne particulate material 0.3 µm in size or larger. Biosafety cabinets are critical to protect individuals working with highly pathogenic material (Fig. 5.29). Newer technologies have been developed that embed antimicrobial agents or enzymes directly into the fibers of the fi lter
Syringe filter
Bottle top filter Filtration device Unsterile media Sterile membrane filter Connect to vacuum
Syringe with fluid attached here Sterile membrane filter
Sterile solution collected
Sterile solution collected in a sterile container
Figure 5.28
Membrane filtration apparatus.
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Biological safety cabinet. Air is drawn into the chamber and continually passed through the HEPA filter to remove hazardous agents. This prevents the escape of aerosolized infectious agents.
Figure 5.29
B.
A.
HEPA filter
Labconco Corporation
Airflow
(Fig. 5.30). Organisms entangled in these fibers are not just trapped; they are attacked by the antimicrobials and lyse. Irradiation. Public health authorities worldwide are
increasingly concerned about food contaminated with pathogenic microorganisms such as Salmonella species, E. coli O157:H7, L. monocytogenes, and Yersinia enterocolitica. Irradiation, the bombardment of foods with highenergy electromagnetic radiation, has long been a potent, if politically sensitive, strategy for sterilizing food after harvesting. The food consumed by NASA astronauts, for example, has for some time been sterilized by irradiation as a safeguard against food-borne illness in space.
Nikki-Universal Co., Ltd., 2000
Lysed organism
Filter fiber
3 µm
Figure 5.30 Immobilized enzyme filter. The primary function of this enzyme filter is to kill airborne microorganisms caught on the surface of the filter to protect against secondary contamination from microorganisms in air filtration systems. The photo shows lysed bacteria (Bacillus subtilis). The cell walls have been hydrolyzed by enzymatic action, and cell membranes are broken as a result of osmotic pressure pushing outward against the membrane.
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Until recently, public pressure severely limited the use of this technology because of concerns about product safety and exposure of workers. However, the recent use of the U.S. mail service by a bioterrorist to disseminate anthrax spores across the eastern United States has renewed interest in sterilization by irradiation. Foods do not become radioactive when irradiated, but there are concerns that reactive molecules potentially dangerous to humans are produced when high-energy particles are absorbed by the natural chemicals in the food and react to produce toxic substances. Aside from ultraviolvet light, which, owing to its poor penetrating ability, is only useful for surface sterilization, there are three other sources of irradiation: gamma rays, electron beams, and X-rays. Radiation dosage is usually measured in a unit called the gray (Gy), which is the amount of energy transferred to food, microbe, or other substance being irradiated. A single chest X-ray delivers roughly half a milligray (1 mGy = 0.001 Gy). To kill Salmonella, freshly slaughtered chicken can be irradiated at up to 4.5 kilograys (kGy), about 7 million times the energy of a single chest X-ray. When microbes present in food are irradiated, water and other intracellular molecules absorb the energy and create transient reactive chemicals that damage DNA and scramble genetic information. Unless the organism repairs this damage, it will die while trying to replicate. Microbes differ greatly in their sensitivity to irradiation, depending on the size of their DNA, the rate at which they can repair damaged DNA, and other factors. It also matters if the irradiated food is frozen or fresh, as it takes a higher dose of radiation to kill microbes in frozen foods.
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Some Bacteria Are Highly Resistant to Physical Control Measures Deinococcus radiodurans could be nicknamed “Conan the bacterium” and designated as a poster microbe for extremophiles (Fig. 5.31). It was discovered in 1956 in a can of meat that spoiled despite having been sterilized by radiation. The microbe has the greatest ability to survive radiation of any known organism. Based on the amount of radiation it can handle, it has been estimated that it could even survive the amount of radiation released in an atomic blast. The bacterium’s ability to withstand radiation may have evolved as a side effect of developing resistance to extreme drought, since dehydration and radiation produce similar types of DNA damage. Deinococcus
Chemical Agents Supplement Physical Means of Controlling Microbes Disinfection by physical agents is very effective, but numerous situations arise where their use is impractical (kitchen countertops) or plainly impossible (skin). In these instances, chemical agents are the best approach. There are a number of factors that influence the efficacy of a given chemical agent. These include: ■
■
The presence of organic matter. A chemical placed on a dirty surface will bind to the inert organic material present, lowering the agent’s effectiveness against microbes. It sometimes is not possible to clean a surface prior to disinfection (as in a blood spill), but the presence of organic material must be factored in when estimating how long to disinfect a surface or object. The kinds of organisms present. Ideally, the agent should be effective against a broad range of pathogens. Corrosiveness. The disinfectant should not corrode the surface or, in the case of an antiseptic, damage skin.
A.
B.
1 µm
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John R. Battista, Louisiana State University
Deinococcus radiodurans. A. Based on the amount of radiation this organism can survive, the bacterium could withstand radiation equivalent to that from an atomic blast. The nature of the dark inclusion bodies in three of the four cells in the quartet is not known. B. John Battista of Louisiana State University showed that D. radiodurans has exceptional capabilities for repairing radiation-damaged DNA.
Figure 5.31
175
repairing its damaged DNA, although the precise mechanisms remain a mystery. One possible mechanism is that a damaged chromosome in one member of the quartet shown can restore itself using DNA from another member of the quartet. Based on this research, D. radiodurans has been genetically engineered to treat radioactive mercury-contaminated waste from nuclear reactors, a process called bioremediation (discussed in Chapter 22). The genes for mercury conversion were spliced from a strain of E. coli resistant to particularly toxic forms of mercury and inserted into D. radiodurans. The genetically altered superbug was able to withstand the ionizing radiation and transform toxic waste into forms that could be removed safely. Fortunately, there is little need to worry about its being a super pathogen, because the organism does not cause disease and is susceptible to antibiotics.
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Research by microbiologist John Battista has shown that D. radiodurans possesses an unusual capacity for
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John R. Battista, Louisiana State University
The size of the DNA “target” is a major factor in radiation efficacy. Parasites and insect pests, which have large amounts of DNA, are rapidly killed by extremely low doses of radiation, typically with D-values of less than 0.1 kGy (in this instance, the D-value is the dose of radiation needed to kill 90% of the organisms). It takes more radiation to kill bacteria (D-values in the range of 0.3–0.7 kGy) because they have less DNA per cell unit (less target per cell). It takes even more radiation to kill a bacterial spore (D-values on the order of 2.8 kGy) because they contain little water, the source of most ionizing damage to DNA. Viral pathogens have the smallest amount of nucleic acid, making them resistant to irradiation doses approved for foods (viruses have D-values of 10 kGy or higher). Prion particles associated with bovine spongiform encephalopathy (BSE, also known as mad cow disease) do not contain nucleic acid and are only inactivated by irradiation at extremely high doses. Thus, irradiation of food is effective in eliminating parasites and bacteria, but is woefully inadequate for eliminating viruses or prions.
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Stability, odor, and surface tension. The chemical should be stable upon storage, possess a neutral or pleasant odor, and have a low surface tension so it can penetrate cracks and crevices.
The Phenol Coefficient Compares the Effectiveness of Disinfectants Phenol, fi rst introduced by Joseph Lister in 1867 to reduce the incidence of surgical infections, is no longer used as a disinfectant, but its derivatives, such as cresols and orthophenylphenol, are still in use. The household product Lysol is a mixture of phenolics. Phenolics are useful disinfectants because they denature proteins, are effective in the presence of organic material, and remain active on surfaces long after application. Today we know phenolics are toxic and should not be used on living tissues. Nevertheless, based on its potency and its history, phenol is the benchmark against which other disinfectants are measured. The phenol coefficient test involves inoculating a fi xed number of bacteria—for example, Salmonella typhi or Staphylococcus aureus—into dilutions of the test agent. At timed intervals, samples are withdrawn from each dilution and inoculated into fresh broth (which contains no disinfectant). The phenol coefficient is based on the highest dilution (lowest concentration) of a disinfectant that will kill all the bacteria in a test after 10 minutes of exposure, but leaves survivors after only 5 minutes of exposure. This concentration is known as the maximum effective dilution. Dividing the reciprocal of the maximum effective dilution for the test agent (for example, ethyl alcohol) by the reciprocal of the maximum effective dilution for phenol gives the phenol coefficient (Table 5.3). For example, if the maximum effective dilution for agent X is 1 /900 and that of phenol is 1 /90, then the phenol coefficient of X is 900/90 = 10; the higher the coefficient, the higher the efficacy of the disinfectant.
Table 5.3 Phenol coefficients for various disinfectants. Chemical agent Phenol Chloramine Cresols Ethyl alcohol Formalin Hydrogen peroxide Lysol Mercury chloride Tincture of iodine
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Staphylococcus aureus 1.0 133.0 2.3 6.3 0.3 — 5.0 100.0 6.3
Salmonella typhi 1.0 100.0 2.3 6.3 0.7 0.001 3.2 143.0 5.8
Commercial Disinfectants Ethanol, iodine, chlorine, and surfactants (for example, detergents) are all used to reduce or eliminate microbial content from commercial products (Fig. 5.32). The first three are highly reactive compounds that damage proteins, lipids, and DNA. Iodine complexed with an organic carrier forms an iodophor, a compound that is water-soluble, stable, nonstaining, and capable of releasing iodine slowly to avoid skin irritation. Wescodyne and Betadyne (trade names) are iodophors used respectively, for the surgical preparation of skin and for wounds. Chlorine is a disinfectant with universal application. It is recommended for general laboratory and hospital disinfection and kills the HIV virus. Detergents can also be antimicrobial agents. The hydrophobic and hydrophilic ends of detergent molecules (which makes them amphipathic) will emulsify fat into water. Cationic (positively charged) but not anionic (negatively charged) detergents are useful as disinfectants because the cationic detergents contain positive charges that can gain access to the negatively charged bacterial cell and disrupt membranes. Anionic detergents are not antimicrobial but do help in the mechanical removal of bacteria from surfaces. Low-molecular-weight aldehydes such as formaldehyde are highly reactive, combining with and inactivating proteins and nucleic acids. This, too, makes them useful disinfectants. Disposable plasticware like petri dishes, syringes, sutures, and catheters are not amenable to heat sterilization or liquid disinfection. These materials are best sterilized using antimicrobial gases. Ethylene oxide gas (EtO) is a very effective sterilizing agent; it destroys cell proteins, is microbicidal and sporicidal, and rapidly penetrates packing materials, including plastic wraps. Using an instrument resembling an autoclave, EtO at 700 milligrams per liter (mg/L) will sterilize an object after 8 hours at 38°C or 4 hours at 54°C if the relative humidity is kept at 50%. Unfortunately, EtO is explosive. A less hazardous gas sterilant is betapropiolactone. It does not penetrate as well as EtO, but it decomposes after a few hours, which makes it easier to dispose of than EtO.
Antibiotics Selectively Control Bacterial Growth Antibiotics as made in nature are chemical compounds made by one microbe that selectively kill other microbial species. Naturally occurring antibiotics act like tiny molecular land mines. As a defense against competitors, some organisms secrete antibiotic compounds into their surrounding environment, where they remain until encountered by an intruder. If the interloper is susceptible to the antibiotic, the compound will target specific structures or proteins, disrupting their function. The target cell is either rendered helpless (by bacteriostatic antibiot-
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Phenolics
Alcohols
Quaternary ammonium compounds
Aldehydes
(CH2)15CH3
OH H3C
CH2
HO CH2
H
Cl H 3C
Cl Cl Cl Cl Hexachlorophene
C OH
Isopropanol (rubbing alcohol)
H C CH2
Cl–
Cetylpyridinium chloride
O CH3
CH2
CH2
Gases
H2C
C O H Formaldehyde
Ethanol
Phenol OH
OH
H
O Glutaraldehyde
CH3 CH2 N R
C
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+
N
H
Cl
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CH2
O Ethylene oxide
+
Cl–
CH3 R = alkyl, C8H17 to C18H37
CH2 CH2 O
C O
Betapropiolactone
Benzalconium chloride (mixture)
OH CH3
Orthocresol
Figure 5.32
Structures of some common disinfectants and antiseptics.
ics) or made nonviable (by bactericidal compounds). (See Chapter 27 for detailed modes of action.) When purified and administered to patients suffering from an infectious disease, these antibiotics can produce seemingly miraculous recoveries. As we saw in Chapter 1, penicillin, produced by Penicillium notatum, was discovered serendipitously in 1929 by Alexander Fleming. Figure 5.33A shows a threedimensional representation of this molecule, which mimics a part of the microbial cell wall. Because of this mimicry, penicillin binds to biosynthetic proteins involved in peptidoglycan synthesis and prevents cell wall formation. The drug is bactericidal because actively growing cells lyse without the support of the cell wall (Fig. 5.34). Other antibiotics target protein synthesis, DNA replication, cell
membranes, and various enzyme reactions. These interactions are described throughout Parts 1–3 of this text. So how do antibiotic-producing microbes avoid suicide? In some instances, the producing organism lacks the target molecule. Penicillium mold, for instance, lacks peptidoglycan and is immune to penicillin by default. Some bacteria produce antimicrobial compounds that target other members of the same species. In this case, the producing organism can modify its own receptors so that they no longer recognize the compound (as with some bacterial colicins). Another strategy is to modify the antibiotic if it reenters the cell. This is the case with streptomycin produced by Streptomyces griseus. Streptomycin inhibits bacterial protein synthesis and does not discriminate between the protein synthesis machinery of Streptomyces
A.
B.
USPS
Christine L. Case/Skyline College
C.
Penicillin. A. Three-dimensional and flat views of penicillin G molecule produced by the Penicillium mold. To view, place a piece of cardboard vertically between the two images. Position your eyes on opposite sides of the cardboard and force them to focus behind the images. The images will merge and produce a 3-D image. B. U.S. postal stamp showing Penicillium notatum. C. P. notatum culture. The mold excretes penicillin, which inhibits the growth of Staphylococcus aureus.
Figure 5.33
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B.
C.
Effect of ampicillin (a penicillin derivative) on E. coli. Cells were incubated for 1 hour at the antibiotic concentrations shown. Swollen areas of cells in panels B–D reflect weakening cell walls. Cells shown are approx. 2 um long.
Figure 5.34
1.25 µm/ml
2.5 µm/ml
5 µm/ml
10 µm/ml
Art Girard, Anne Klein & A.J. Milici, Pfizer Global Research and Development
A.
D.
and that of others. However, while making enzymes that synthesize and secrete streptomycin, S. griseus simultaneously makes the enzyme streptomycin-6-kinase, which remains locked in the cell. If any secreted streptomycin reenters the cell, this enzyme renders the drug inactive by attaching a phosphate to it. Because many microorganisms have become resistant to commonly used antibiotics, pharmaceutical companies are continually using a variety of drug discovery approaches to search for new antibiotics. Traditional procedures include scouring soil and ocean samples collected from all over the world for new antibiotic-producing organisms and chemically redesigning existing antibiotics so that they can bypass microbial resistance strategies. These procedures are now supplemented by “mining the genomes” of microbes for potential drug targets. The frontiers of chemistry include rational drug design, which relies on computer-based methods for predicting the structure and function of potential new antibiotics.
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The autoclave uses high pressure to achieve temperatures that will sterilize objects. The z-value is a measure of how much more heat is needed to reduce D-value to 1 /10 its original value. Pasteurization is a heating process designed to kill specific pathogens in milk and other food products. Refrigeration is used to prevent microbial growth in foods. Extreme cold (freezing) is used to preserve bacteria. Filtration can remove cells from a solution, but it cannot remove the smallest viruses. Irradiation can kill pathogens in foods without damaging the food itself. Chemical disinfectants are compared to one another based on the phenol coefficient. Antibiotics are compounds produced by one living microorganism that kill other microorganisms.
5.9 TO SU M MAR I Z E: ■ ■ ■
■
■
Sterilization kills all living organisms. Disinfection kills pathogens on inanimate objects. Antisepsis is the removal of potential pathogens from the surfaces of living tissues. Antimicrobial compounds can be bacteriostatic or bactericidal. The D-value is the time (or dose, in the case of irradiation) it takes an antimicrobial treatment to reduce the numbers of organisms to 10% of the original value.
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Biological Control of Microbes
Pitting microbe against microbe is an effective way to prevent disease in humans and animals. One of the hallmarks of a healthy ecosystem is the presence of a diversity of organisms. This is true not only for tropical rain forests and coral reefs, but also for the complex ecosystems of human skin and the intestinal tract. In these environments, the presence of harmless microbial flora can retard the growth of undesired pathogens. The pathogenic fungus Phytophthora cinnamomi, for example, causes root rot in plants but is biologically controlled by fungi belonging to the genus
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Myrothecium. Naturally occurring Staphylococci on human skin produce short-chain fatty acids that retard the growth of pathogenic strains. Another illustration is the human intestine, which is populated by as many as 500 microbial species. Most of these species are nonpathogenic organisms that exist in symbiosis with their human host. Vigorous competition between members of the normal intestinal flora and the production of permeant weak acids by fermentation help control the growth of numerous pathogens. Microbial competition has been widely exploited for agricultural purposes and to improve human health in a process known as probiotics. In general, a probiotic is a food or supplement that contains live microorganisms and improves intestinal microbial balance. Newborn baby chicks, for instance, are fed a microbial cocktail of normal flora designed to quickly colonize the intestinal tract and prevent colonization by Salmonella, a frequent contaminant of factory-farmed chicken. In another example, Lactobacillus and Bifidobacterium have been used to prevent and treat diarrhea in children. Russian Nobel laureate Elie Metchnikoff in 1908 fi rst suggested that a high concentration of lactobacilli in intestinal flora was important for health and longevity in humans. Yogurt is an example of a probiotic containing Lactobacillus acidophilus and a number of other lactobacilli. It is often recommended as a way to restore a normal balance to gut flora (for example, after it has been disturbed by antibiotic treatment) and appears useful in the treatment of inflammatory bowel disease. Phage therapy is another biocontrol method fi rst described in 1907 by Felix d’Herelle at France’s Pasteur Institute, long before antibiotics were discovered. Bacteriophages are viruses that prey on bacteria (discussed in Section 6.1). Each bacterial species is susceptible to a limited number of specific phages. Because the culmination
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of a phage infection is often bacterial lysis, it was considered feasible to treat infectious diseases with a phage targeted to the pathogen. At one time, doctors used phages as medical treatment for illnesses ranging from cholera to typhoid fever. In some cases, a liquid containing the phage was poured into an open wound. In other cases, phages were given orally, introduced via aerosol, or injected. Sometimes the treatments worked; sometimes they did not. When antibiotics came into the mainstream, phage therapy largely faded. Now that strains of bacteria resistant to standard antibiotics are on the rise, the idea of phage therapy has enjoyed renewed interest from the worldwide medical community. Several biotechnology companies have even been formed in the United States to develop bacteriophage-based treatments. TO SU M MAR I Z E: ■
■
■
Biocontrol is the use of one microbe to control the growth of another. Probiotics contain certain microbes that, when ingested, aim to restore balance to intestinal flora. Phage therapy offers a possible alternative to antibiotics in the face of rising antibiotic resistance.
Concluding Thoughts Microbiology as a science was founded on the need to understand and control microbial growth. The initial impetus was to control the diseases of humans as well as the diseases of plants and animals. But as we will see in later chapters, microbiology has developed into a science that has helped us understand the molecular processes of life. Concepts such as biological diversity, food microbiology, microbial disease, and antibiotics will be revisited in later chapters.
C H A P T E R R E V I EW Review Questions 1. Explain the nature of extremophiles and discuss why 2. 3.
4. 5.
these organisms are important. What are the parameters that defi ne any growth environment? List and defi ne the classifications used to describe microbes that grow in different physical growth conditions. What do thermophiles have to do with the PCR reaction? Why is water activity important to microbial growth? What changes water activity?
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6. How do cells protect themselves from osmotic stress? 7. Why do changes in H+ concentration affect cell growth? 8. How do acidophiles and alkaliphiles manage to grow
at the extremes of pH? 9. Because an organism can live in an oxygenated envi-
ronment, does that mean that the organism uses oxygen to grow? Because an organism can live in an anaerobic environment, does that mean it cannot use oxygen as an electron acceptor? Why or why not? 10. What happens when a cell exhausts its available nutrients?
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11. List and briefly explain the various means by which
humans control microbial growth. What is a D-value? What is a phenol coefficient?
12. How do microbes prevent the growth of other
microbes?
Key Terms 12D (172) acidophile (160) aerobic respiration (164) alkaliphile (161) anaerobic respiration (166) antibiotic (176) antisepsis (171) bactericidal (171) bacteriostatic (171) barophile (156) bioremediation (175) decimal reduction time (D-value) (171) disinfection (171) DNA microarray (150) electron transport chain (164) eutrophication (169) extremophile (150) facultative (166)
facultative aerobe (167) facultative anaerobe (167) fermentation (166) fermentative metabolism (166) germicidal (171) halophile (158) heat shock response (155) hyperthermophile (154) laminar flow biological safety cabinet (173) lyophilization (173) membrane-permeant organic acid (159) mesophile (153) microaerophilic (167) neutralophile (160) oligotroph (169) osmolarity (157)
pasteurization (173) phage therapy (179) phenol coefficient test (176) piezophile (156) probiotic (179) psychrophile (153) reactive oxygen species (166) sanitation (171) starvation response (168) steam autoclave (172) sterilization (171) strict aerobe (166) strict anaerobe (166) thermophile (153) two-dimensional protein gels (150) water activity (157) z-value (172)
Recommended Reading Alpuche-Aranda, Celia M., Joel A. Swanson, Wendy P. Loomis, and S. I. Miller. 1992. Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes. Proceedings of the National Academy of Science USA 89:10079–10083. Atomi, Haruyuki. 2005. Recent progress towards the application of hyperthermophiles and their enzymes. Current Opinion in Chemical Biology 9:163–173. Bang, I. S., Jonathan P. Audia, Y. K. Park, and John W. Foster. 2002. Autoinduction of the ompR by acid shock and control of the Salmonella enterica acid tolerance response. Molecular Microbiology 44:1235–1250. Biswas, Biswajit, Sankar Adhya, Paul Washart, et al. 2002. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infection and Immunity 70:204–210. Ferenci, Thomas. 1999. Regulation by nutrient limitation. Current Opinion in Microbiology 2:208–213. Fredrickson, Jim K., H. M. Kostandarithes, S. W. Li, A. E. Plymale, and Mark J. Daly. 2000. Reduction of Fe(III), Cr(VI), U(VI), and Tc(VII) by Deinococcus radiodurans R1. Applied and Environmental Microbiology 66:2006–2011. Horikoshi, Koki. 1998. Barophiles: Deep-sea microorganisms adapted to an extreme environment. Current Opinion in Microbiology 1:291–295. Horikoshi, Koki. 1999. Alkaliphiles, some applications of their products for biotechnology. Microbiology and Molecular Biology Review 63:735–750.
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MacElroy, Robert D. 1974. Some comments on the evolution of extremophiles. Biosystems 6:74–75. Makarova, Kira S., L. Aravind, Yuri I. Wolf, Roman L. Tatusov, Kenneth W. Minton, et al. 2001. Genome of the extremely radiation resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiology and Molecular Biology Reviews 65:44–79. Maurer, Lisa M., Elizabeth Yohannes, Sandra S. BonDurant, Michael Radmacher, and Joan L. Slonczewski. 2005. pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12. Journal of Bacteriology 187:304–319. Peitzman, Steve J. 1969. Felix d’Herelle and bacteriophage therapy. Transactions & Studies of the College of Physicians of Philadelphia 37:115–123. Rathman, M., Michael D. Sjaastad, and Stan Falkow. 1996. Acidification of phagosomes containing Salmonella typhimurium in murine macrophages. Infection and Immunity 64:2765–2773. Rhen, Mikael, and Charles J. Dorman. 2005. Hierarchical gene regulators adapt Salmonella enterica to its host milieus. International Journal of Medical Microbiology 294:487–502. Saxelin, Maija, Soile Tynkkynen, Tiina Mattila-Sandholm, and Willem M. de Vos. 2005. Probiotic and other functional microbes: From markets to mechanisms. Current Opinion in Biotechnology 16:204–211. Thomas, D. N., and G. S. Dieckmann. 2002. Antarctic Sea Ice—a habitat for extremophiles. Science 295:641–643.
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Chapter 6
Virus Structure and Function 6.1 6.2 6.3 6.4 6.5 6.6 6.7
What Is a Virus? Virus Structure Viral Genomes and Classification Bacteriophage Life Cycles Animal and Plant Virus Life Cycles Culturing Viruses Viral Ecology
All kinds of cells, including bacteria, eukaryotes, and archaea, can be infected by particles called viruses. Viruses are the smallest known units of reproduction, some with genomes of less than ten genes. Upon entering a cell, a viral genome subverts the cell’s machinery to reproduce more virus particles, usually killing the host cell. Alternatively, some viral genomes are copied into the host genome, where they replicate silently with the host. Degenerate viral genomes from ancient viral insertions take up a large part of the human genome. Virus structure varies in complexity from single RNA molecules to spore-like packages containing multiple layers of protein and membranes. Some viruses resemble degenerate cells. Viral intracellular life cycles enable them to divert complex host cell processes to the formation of new viruses. How can such particles, barely large enough to be detected by an electron microscope, subvert and consume entire cells and multicellular organisms? Alter-
Tail hub
Tail spikes
natively, how can viruses integrate their genomes into the genome of their host, replicating with their host for future generations?
dsDNA end Coiled dsDNA 0 Capsid
30 nm
The bacteriophage epsilon 15 attacks Salmonella bacteria, pathogens of the human digestive tract. Such bacteriophages help control our enteric bacterial populations, but they also transfer DNA encoding resistance to antibiotics. The structure of the epsilon 15 bacteriophage was solved by cryo-electron microscopy, revealing details of the tail apparatus that inserts the DNA into the host cell. Source: Wen Jiang, et al. 2006. Nature 439:612.
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In February 2003, travelers from Guangdong, China, began succumbing to a mysterious respiratory syndrome from which one in ten died. The disease spread alarmingly through hospitals in Hong Kong, Vietnam, and Toronto, Canada. Its rapid spread caused thousands of travelers worldwide to cancel plans, while residents of affected cities donned face masks (Fig. 6.1A). The disease became known as sudden acute respiratory syndrome (SARS). SARS was caused by a new virus, a member of the coronavirus family, named for their characteristic “corona” of spike proteins (Fig. 6.1B). To fi nd clues for the treatment of SARS, the RNA genome of the new virus was sequenced by Canadian researchers with unprecedented speed. Comparison with known viruses showed SARS virus to be related to coronaviruses infecting pigs and birds (Fig. 6.1C). So the researchers hypothesized that SARS evolved from an animal virus in the food markets of Guangdong. Viruses evolve new strains even faster than cellular pathogens because of their tiny genomes and little or no proofreading of replication. Despite their small size, viruses generate frightening epidemics, from smallpox and polio to AIDS and influenza. Other viruses play crucial roles in the environment, particularly in marine ecosystems, where they cycle carbon and curb toxic blooms of algae. Viral predation selects for much of the species diversity of marine protists and invertebrates.
In research, viruses have provided both tools and model systems for our discovery of the fundamental principles of molecular biology. The fi rst genes mapped, the fi rst regulatory switches defi ned, and the fi rst genomes to be sequenced were all those of viruses. Vectors for gene cloning and gene therapy today continue to be derived from viruses. In this chapter, we introduce major themes of virus structure and function and the fundamental challenges that all viruses face: genome packaging, cell attachment and entry, and the molecular strategies that enable viruses to divert the metabolism of their host cell. Viruses provide key tools and model systems for molecular biology. Our understanding of viruses, particularly bacterial viruses, called bacteriophages, provides a useful background for the molecular biology we will encounter in Part 2 of this book (Chapters 7–12). The molecular biology of viral life cycles is explored further in Chapter 11, and viral disease pathology and epidemiology are discussed in Chapters 25–27.
6.1
What Is a Virus?
A virus is a noncellular particle capable of infecting a host cell, where it reproduces. The virus particle, or virion, consists of an infective nucleic acid (DNA or RNA) con-
CDC/Fred Murphy
B.
A.
C. Coronavirus phylogenetic tree Avian infectious bronchitis virus
Bovine coronavirus
Mouse hepatitis virus
China Photo/Reuters
Porcine gastroenteritis virus SARS Human coronavirus
0.1 substitution/residue
Figure 6.1 Sudden acute respiratory syndrome (SARS): an emerging viral disease. A. In 2003, as SARS first breaks out, a bride and groom in Hubei, China, wear masks to protect themselves from airborne infection. B. A SARS virion (diameter, 78 nm) bristles with spike proteins (TEM). C. The family tree of coronavirus shows that the SARS branch is as distant from a human cold-causing coronavirus as from animal coronaviruses.
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THOUGHT QUESTION 6.1 Which viruses do you know that have a narrow host range, and which have a broad host range?
American Society for Virology International Committee on Taxonomy of Viruses
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Simon. 1969. Virology 38
A.
400 nm
Lowell Georgia/Photo Researchers, Inc.
D.
Courtesy of Claire Moore and Shmuel Rozenblatt, Tel-Aviv University
C.
Capsid
Virus envelope
E.
F.
Norm Thomas/Photo Researchers, Inc.
TMV virions Thomas Shalla, J. Cell. Bio. 21
tained within a protective shell made of protein, called the capsid. The capsid usually has a molecular delivery device that enables transfer of the virion’s genome into the host cell. Viruses that infect bacteria are known as bacteriophages or phages. An example is bacteriophage T2, which infects Escherichia coli (Fig. 6.2A). The T2 and T4 phages have a capsid with a tail that inserts the viral genome into the host cell, where it directs reproduction of progeny virions. Virions are released when the host cell lyses. As cells lyse, their disappearance can be observed as a plaque, a clear spot against a lawn of bacterial cells (Fig. 6.2B). Each plaque arises from a single virion or phage particle that lyses a host cell and spreads progeny to infect adjacent cells. Plaques can be counted as representing individual infective virions from a phage suspension. An example of a virus that infects humans is measles virus. The measles virion has an envelope of membrane that, during infection, fuses with the host cell membrane. After replicating within the infected cell, newly formed measles capsids become enveloped by host cell membrane as they bud out of the host cell (Fig. 6.2C). The spreading virus generates a rash of red spots on the skin of infected patients (Fig. 6.2D) and is occasionally fatal (one in 500 cases). Plants are also infected by viruses, such as tobacco mosaic virus (TMV). Within the plant cell, virions accumulate to high numbers (Fig. 6.2E) and travel through interconnections to neighboring cells. Infection by tobacco mosaic virus results in mottled leaves and stunted growth (Fig. 6.2F). Plant viruses cause major economic losses in agriculture worldwide. Each species of virus infects a particular group of host species, known as the host range. Some viruses can infect only a single species; for example, HIV infects only humans. Close relatives of humans, such as the chimpanzee, are not infected, although they are susceptible to a closely related virus, simian immunodeficiency virus (SIV). On the other hand, the West Nile virus, transmitted by mosquitoes, has a much broader host range, including many species of birds and mammals.
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Figure 6.2 Virus infections. A. Bacteriophage T2 particles form a semicrystalline array within an E. coli cell (TEM). B. Bacteriophage infection forms plaques of lysed cells on a lawn of bacteria. C. Measles virions (diameter, 200–600 nm) bud out of human cells in tissue culture (TEM). D. Child infected with measles shows a rash of red spots. E. Tobacco leaf section is packed with tobacco mosaic virus particles. F. Tobacco leaf infected by tobacco mosaic virus shows mottled appearance.
Viruses Propagate Their Genomic Information Viral propagation exemplifies the central role of information in biological reproduction. The propagation of viruses is mimicked by the spread of “computer viruses,” whose information “infects” computer memory (Fig. 6.3).
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industry; for example, bacteriophages (literally, “bacteria-eaters”) infect cultures of Lactococcus during the production of yogurt and cheese. Plant Infects host computer pathogens such as cauliflower mosaic A GA T virus and rice dwarf virus continue to T C C 01001011 Host computer cause substantial losses in agriculture. 01001011 Host genome 01001011 memory In contrast to our vast arsenal 01001011 Infects host cell of antibiotics (effective against bac01001011 01001011 01001011 01001011 01001011 teria), the number of antiviral drugs 01001011 01001011 01001011 remains depressingly small. Because 01001011 01001011 01001011 A A A A A 01001011 01001011 the machinery of viral growth is T T T T T 01001011 01001011 01001011 01001011 01001011 C C C C C largely that of the host cell, viruses 01001011 01001011 G G G G G 01001011 01001011 present relatively few targets that can A A A A A 01001011 01001011 C C C C C 01001011 be attacked by antiviral drugs with01001011 01001011 T T T T T 01001011 01001011 01001011 out harming the host. However, an 01001011 01001011 01001011 understanding of viral life cycles at the molecular level is now leading to the development of new antiviInfect new computers Infect new cells ral drugs, such as AZT and protease Figure 6.3 Biological viruses and computer viruses. From the standpoint of inhibitors, which combat HIV. information, the behavior of biological viruses and computer viruses are analogous. Despite their lethal potential, viruses have made surprising contributions to medical research. A bacWhen a biological virus infects a host cell, the informateriophage provides a protein that lyses the cell walls of tion in its genome subverts the host cell machinery to anthrax bacteria. Other bacteriophages are used as clonproduce multiple copies of the virus; the multiple copies ing vectors, small genomes into which foreign genes can then escape to infect more host cells. Similarly, when a be inserted and cloned for gene technology. Even lethal computer virus infects a host computer, its program code viruses such as HIV are being developed as vectors for subverts the host to produce multiple copies of the virus, human gene therapy. which then escape to infect more host computers. ComSome viruses introduce copies of their own genomes puter viruses generate epidemics analogous to those of into the host’s genome, a process that can mediate evobiological viruses. The virus’s code can even be designed lution of the host genome. Indeed, studies of molecular to “mutate” in order to foil the “immune system” of antievolution reveal that viral genomes are the ancestral virus software. source of about a tenth of the human genome. Biological virus A GA T T C C
Computer virus 01001011
Viruses Infect All Forms of Life
Viral Genomes
Viruses are ubiquitous, infecting every taxonomic group of organisms, including bacteria, eukaryotes, and archaea. In marine ecosystems, viruses act as major predators and sequester significant amounts of nutrients. For humans, viruses cause many forms of illness, whose influence on our history and culture would be hard to overstate. More people died of influenza in the global epidemic of 1918 than in the battles of World War I. In the past 30 years, the AIDS pandemic caused by HIV has killed 25 million people worldwide and continues to grow. Viruses are part of our daily lives. The most frequent infections of college students are due to respiratory pathogens such as rhinovirus (the common cold) and EpsteinBarr virus (infectious mononucleosis), as well as sexually transmitted viruses such as herpes simplex (HSV) and papilloma (genital warts). Viruses also impact human
The genome of a virus can be small, encoding fewer than ten genes. In cauliflower mosaic virus, for example, the genome encodes only seven genes (Fig. 6.4A), which actually overlap each other in sequence. This overlap in sequence is made possible by the use of different reading frames, start positions for translating codons to amino acids. Many viral genomes, such as that of avian leukosis genome, are encoded by RNA. The RNA genome of avian leukosis virus has protein-encoding genes grouped by functional categories of core capsid, replicative enzymes, and envelope proteins. On the other hand, larger viral genomes, such as that of herpes virus or of bacteriophage T4, have genes dispersed around the chromosome, similar to the genomes of bacteria. The giant Mimivirus, which infects amebas and may cause human pneumonia, is as large as some bacteria
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ORF 7
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ORF 4
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B. Avian leukosis virus genome (RNA) gag pol LTR
Core
Enzymes
Mimivirus infecting an ameba. About 300 nm across, the Mimivirus is larger than some bacteria.
Figure 6.5
env LTR
Envelope
Simple viral genomes. A. Cauliflower mosaic virus has a circular genome of double-stranded DNA, whose strands are interrupted by nicks. The genome encodes seven overlapping genes (open reading frames, ORFs). B. Avian leukosis virus: a single-stranded RNA retrovirus resembling eukaryotic mRNA. Three genes (gag, pol, and env) encode polypeptides that are eventually cleaved to form a total of nine functional products. LTR = long terminal repeat.
Figure 6.4
(Fig. 6.5). Furthermore, Mimivirus has a bacterium-sized genome of 1.2 million base pairs. The Mimivirus virion conducts numerous cellular functions, including DNA repair and protein-folding by chaperones. It probably represents a descendant of a cell that parasitized protist cells until most of its cellular traits were lost through degenerative evolution.
Viroids: Infective Genomes with No Capsid Early in the twentieth century, viruses were believed to be the smallest particles capable of infecting hosts and propagating themselves. Then even smaller virus-like infectious agents were discovered for which the nucleic acid genome is itself the entire infectious particle; there is no protective capsid. Such infectious agents are called viroids. Most viroids are RNA molecules that infect plants. An example is the potato spindle tuber viroid. This viroid consists of a circular, single-stranded molecule of RNA that doubles back on itself to form base pairs interrupted by short unpaired loops (Fig. 6.6A). The RNA folds up into a globular structure that interacts with host
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Didier Raoult, Mediterranean University, Marseille
DNA strands
cell proteins. Most critically, the RNA genome requires a host RNA-dependent RNA polymerase to replicate itself and transcribe its genes. RNA-dependent RNA polymerases occur normally in plant cells, where they contribute to regulation of gene expression. During viroid infection, the RNA-dependent RNA polymerase replicates progeny copies of the viroid, which encodes no products other than itself. Viroids can cause as much host destruction as “true viruses,” and some authors, particularly in plant pathology, classify them as “viruses without capsids.” Some viroids have catalytic ability, comparable to enzymes made of protein. An RNA molecule capable of catalyzing a reaction is called a ribozyme. One class of plant-infecting viroids are the hammerhead ribozymes (Fig. 6.6B). Named for their hammer-shaped tertiary structure, hammerhead ribozymes possess the ability to cleave themselves or other specific RNA molecules. Their ability to cleave very specific RNA sequences has applications in medical research. Hammerhead ribozymes have been engineered to cleave human RNA molecules involved in cancer or in infection by viruses such as HIV. The engineering of ribozymes for medical therapy is an exciting field of biotechnology.
Prions: Infection without Nucleic Acid? A remarkable class of infectious agents is believed to consist of protein only. These agents, known as prions, are believed to be aberrant proteins arising from the host cell. Prions gained notoriety when they were implicated in brain infections such as Creutzfeldt-Jakob disease, popularly known as “mad cow” disease because it may
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A. Potato spindle tuber viroid−circular ssRNA C G G A A C U A A A C U C G U GG U U C C U UGA CG C C A A GG U C C U UGG
GA C
UGU U U
AA AA GG A C U C GG A G G A G C G C U U C A G A G A A GGCG G C U C C U G A GC A G A A A A GA C U CG U U U U U CGC C A A G A GC C A AGUC GGGG C U U C G U U C A U U C U A C A A G C UC U A CAA
GG U U C A C A C C U C C G A U U U G U GG G C
GUC GC C C A C C C U UCC U U U C U U C G G G U U U C C U CG C G C C C G C A G G AC C A C C C C U C G C C C C C U U U GC UG U CGC U U C GG U U U U G GG A A A A G CC G C C C U G A G G A CA G C C G G C G A C C C G U G A G G G G U G G CA G G A A A A A C G G U C A A G C G A G G U C U C A U AA
C A AA
G
C G
C
U G C G C C G G
Figure 6.6 Viroids: infective RNA. A. Potato spindle tuber viroid consists of a circular single-stranded RNA that hybridizes internally. A viroid encodes no genes, but it hijacks the plant cell’s RNA-dependent RNA polymerase to replicate itself. B. A hammerhead ribozyme catalyzes cleavage of itself (at “self-cleavage site”) or of another RNA molecule. (PDB code: 1HMH)
B. Hammerhead ribozyme
form of a normally occurring cell protein that assumes an abnormal conformation or tertiary structure (Fig. 6.7A). The prion form of the protein acts by binding to normally folded proteins of the same class and altering their conformation to that of the prion. The multiplying prion then alters the conformation of other normal subunits, forming harmful aggregates in the cell and ultimately leading to cell death. In the brain, prion-induced cell death leads to tissue deterioration and dementia (Fig. 6.7B). Prion diseases can be initiated by infection with an aberrant protein. More rarely, the cascade of protein misfolding can start with the spontaneous misfolding of an endogenous host protein. The chance of spontaneous unfolding is greatly increased in individuals who inherit certain alleles encoding the protein; thus, spontaneous prion diseases can be inherited genetically. Overall, prion diseases are unique in that they can be transmitted by an infective protein instead of by DNA or RNA; and they propagate conformational change of existing molecules without synthesizing entirely new infective molecules.
Self-cleavage site
Catalytic strand catalyzes RNA cleavage.
be transmitted through defective proteins in beef from diseased cattle. Other diseases believed to be caused by prion transmission include scrapie, a disease of sheep, and kuru, a degenerative brain disease found in a tribe of people who customarily consumed the brains of deceased relatives. In prion-associated diseases, the infective agent is unaffected by treatments that destroy RNA or DNA, such as nucleases or UV irradiation. A prion is an aberrant
A.
Ralph C. Eagle/Photo Researchers, Inc.
Normal conformation
Prion disease. A. The normal conformation of a prion, compared to the abnormal conformation. The abnormal form “recruits” normally folded proteins and changes their conformation into the abnormal form. (PDB code: 1AG2) B. Section of a human brain showing “spongiform” holes typical of Creutzfeldt-Jakob disease.
Figure 6.7
B.
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U A C G G C
Aberrant conformation
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Viruses consist of noncellular particles that infect a host cell and direct its expression apparatus to produce virus particles. A virion consists of a capsid enclosing a nucleic acid genome. Some viruses are further enclosed by a membrane envelope. All classes of organisms are infected by viruses. Usually the hosts are limited to a particular host range of closely related strains or species. Viruses contain infective genomes that take over a cell, reprogramming its cell machinery to make progeny virus particles (virions). Some viral genomes consist of less than 10 genes; others have 100 or 200 genes and may represent degenerate cells. Viroids that infect plants consist of RNA hairpins with no capsid. Prions consist of infectious proteins that induce a cell’s native proteins to fold incorrectly and impair cell function.
6.2
Virus Structure
A packaged structure of a virus achieves two goals: It keeps the viral genome intact, and it enables infection of the appropriate host cell. First, the stable capsid protects the viral genome from degradation and enables it to be transmitted outside the host. Second, in order for the viral genome to reproduce, the virion must either insert its genome into the host cell or disassemble within the host. In the process, the original particle loses its stable structure and its own identity as such, but it generates numerous progeny virions. THOUGHT QUESTION 6.2 What would happen if a virus particle remained intact within a host cell instead of releasing its genome?
Symmetrical Virus Particles Different viral species make different forms of capsids. Virus particles may be symmetrical, in which case the capsid is one of two types, icosahedral or fi lamentous (helical). Each type of capsid exhibits geometrical symmetry. The advantage of symmetry is that it provides a way to form a package out of repeating protein units generated by a small number of genes and encoded by a short chromosomal sequence. The smaller the viral genome, the more genome copies can be synthesized from the host cell’s limited supply of nucleotides. Nevertheless, other viruses, such as smallpox virus, are asymmetrical and may have much larger genomes. Large genomes offer a greater range of functions for viral components.
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Icosahedral viruses. Many viruses package their genome in an icosahedral (20-sided) capsid; examples include poliovirus and the herpes viruses. Icosahedral viral capsids take the form of a polyhedron with 20 identical triangular faces. Bacteriophages often supplement the icosahedral capsid or head coat with an elaborate delivery device. For example, bacteriophage T4 (Fig. 6.8A) has a complex structure consisting of an icosahedral headpiece containing the genome, six jointed “legs” that stabilize the structure on the host cell surface, and a neck piece that channels the nucleic acid into the host cell (Fig. 6.8B). The structure of phage T4 was first observed by microbiologists during the rise of the NASA space program, and its form was compared to that of the “lunar module” that landed on the moon (Fig. 6.8C). Indeed, in the 1960s, the “tailed phages,” such as phage T4 and phage lambda, were to molecular biology what the lunar landings were to space exploration.
T4 Bacteriophage Cell-Puncturing Device, a 3-D interactive tutorial In the capsid, each triangle can be composed of three identical but asymmetrical protein units. An example of an icosahedral capsid is that of the herpes simplex virus (Fig. 6.9A). Each triangular face of the capsid is determined by the same genes encoding the same protein subunits. No matter what the pattern of subunits in the triangle, the structure overall exhibits rotational symmetry characteristic of an icosahedron (Fig. 6.9B): threefold symmetry around the axis through two opposed triangular faces; fivefold symmetry around an axis through opposite points; and twofold symmetry around an axis through opposite edges. Capsid symmetry is important for structure determination and visualization and for the design of antiviral drugs. THOUGHT QUESTION 6.3 Why do viral capsids take the form of an icosahedron instead of some other polyhedron? Virus particles can be observed by standard transmission electron microscopy (TEM), but the details of capsid structure as in Figure 6.9A require visualization by digital reconstruction of cryo EM (discussed in Section 2.6). Recall from Chapter 2 that in cryo EM, the viral samples for TEM are prepared flash-frozen, preventing formation of ice crystals. Flash freezing enables observation without stain. The electron beams penetrate the object; thus, images of individual capsids actually provide a glimpse of the virus’s internal contents. By digitally combining and processing cryo TEM images from a number of capsids, a three-dimensional reconstruction is built for the entire virus particle. In some icosahedral viruses, the capsid is enclosed in an envelope composed of membrane from the host cell in which the virion formed. Figure 6.10A shows how herpes virions capture cell membrane to form their envelopes as
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A. Phage T4
500 nm
©George Chapman/Visuals Unlimited
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Bettmann/Corbis
Genome Capsid (head): 100 nm
Bacteriophage T4 capsid. A. E. coli infected by phage T4 (colorized blue, TEM). B. Phage T4 particle with protein capsid containing packaged double-stranded DNA genome. The capsid is attached to a sheath with tail fibers that facilitate attachment to the surface of the host cell. After attachment, the sheath contracts and the core penetrates the cell surface, injecting the phage genome. C. The structure of phage T4 resembles an Apollo lunar module that landed on the moon.
Figure 6.8 Collar Tail fibers Tail sheath
Endplate
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B.
A.
Capsid proteins
Fivefold axis
DNA Threefold axis Twofold axis
Threefold Depth into virion
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Herpes: Icosahedral capsid symmetry. A. Icosahedral capsid of herpes simplex 1 (HSV-1), envelope removed. Imaging of the capsid structure is based on computational analysis of cryoelectron microscopy (cryo TEM). Images of 146 virus particles were combined digitally to obtain this model of the capsid at 2 nm resolution. B. Icosahedral symmetry includes fivefold, threefold, and twofold axes of rotation. C. The icosahedral capsid contains spooled DNA. Source: Z. H. Zhou, et al. 1999. J. Virology 73:3210
Figure 6.9
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University of Leeds
Envelope
Envelope forms from cell membrane.
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Envelope Tegument Alasdair Steven, NIH
Capsid
National Institute for Arthritis, Musculoskeletal Disease and Skin Disease
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Capsid
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they bud out of the cell. (Other viral species may derive their envelope from intracellular membranes, such as the nuclear membrane or endoplasmic reticulum.) The envelope and capsid contents of herpes virus are shown in Figure 6.10B. The mature envelope (Fig. 6.10C) bristles with glycoprotein spike proteins that plug it onto the capsid. The spike proteins enable the virus to attach and infect the next host cell. Between the envelope and the capsid, additional proteins may be found, called the tegument (Fig. 6.10D). Tegument proteins, also called accessory proteins, serve the virus during the early stages of infection. As the virus enters its host cell, its envelope is dissolved and the tegument proteins are released to start reproduction of new viruses. NOTE: Distinguish the viral envelope (membrane derived from a host cell membrane) from the bacterial cell envelope (protective layers outside the bacterial cell membrane). The bacterial envelope is discussed in Chapter 3.
Filamentous viruses. A second major category of virus
structure is that of filamentous viruses. Filamentous viruses include bacteriophages, such as phage M13 (Fig. 6.11A), as well as animal viruses, such as Ebola virus, which causes a swiftly fatal disease of humans and related primates (Fig. 6.11B). Filamentous phages have applications in human medicine and industry. Phages similar to M13 infect the gram-positive species Propionibacterium freudenreichii, a key fermenting agent for Swiss cheese. Another fi lamentous phage, CTXφ, integrates its sequence into the genome of Vibrio cholerae, where it car-
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Alasdair Steven, NIH
Figure 6.10 Envelope and tegument surround the Herpes capsid. A. TEM of sectioned herpes virions budding off from an infected cell. B. Section showing envelope and tegument proteins surrounding capsid (cryo TEM). C. Reconstruction of Herpes capsid. D. Cutaway reconstruction: spike envelope glycoproteins (yellow), envelope membrane (dark blue), tegument proteins (orange), capsid (light blue).
100 nm
ries the deadly toxin genes required for cholera. On the other hand, an application in nanoscience is the use of fi lamentous phages to nucleate the growth of crystalline “nanowires” for electronic devices. The fi lamentous bacteriophage M13 (Fig. 6.11A) consists of a relatively simple capsid of protein monomers. The monomers are stacked around a coiled genome consisting of a circle of single-stranded DNA. At one end of the fi lament, short tail fibers mediate specific attachment to the host. Attachment occurs at the F pilus of an E. coli bacterium containing an F plasmid that encodes pili. Filamentous viruses show helical symmetry. The pattern of capsid monomers forms a helical tube around the genome, which usually winds helically within the tube. In a helical capsid, the genome is a single-stranded DNA (as in phage M13) or RNA (as in tobacco mosaic virus). Figure 6.12A shows how the RNA strand of tobacco mosaic virus winds in a spiral within a tube of capsid monomers laid down in a spiral array. Such a tube can be imagined as a planar array of subunits that coils around such that each row connects to the row above, generating a spiral (Fig. 6.12B). The length of the helical capsid may extend up to 50 times its width, generating a flexible fi lament. Unlike the icosahedral capsid, which has a fi xed size, the helical capsid can vary in length to accommodate various lengths of nucleic acid. Furthermore, some viruses package several genome segments into separate
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Marcus Drechsler, U. of Bayreuth
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Filamentous viruses. A. The filamentous bacteriophage M13 has a relatively simple helical capsid that surrounds the genome coiled within (TEM). B. Ebola virus filaments.
Figure 6.11
helical capsids. For example, influenza virus packages several different genome segments into separate helical packages of different sizes, contained together within a membrane envelope. Multiple helical packaging enables influenza virus to package different numbers of RNA segments into different virions, thus facilitating rapid evolution of new strains.
stranded DNA genome is stabilized by covalent connection of its two strands at each end (Fig. 6.13B). Instead of a capsid, the DNA is enclosed loosely by a core envelope studded with spike proteins, surrounded by an outer membrane. The core envelope also encloses a large number of accessory proteins needed early in viral infection, such as initiation proteins for transcription of viral genes and RNA-processing enzymes that modify viral mRNA molecules. Asymmetrical viruses usually contain a large number of accessory proteins. The fi rst viruses to be studied, including TMV and poliovirus, had extremely simple
Asymmetrical Virus Particles Some viruses lack a symmetrical capsid. In poxviruses, such as vaccinia and smallpox (Fig. 6.13A), the double-
B.
A.
A planar net of identical subunits a. b.
b. a.
Roll
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J.-Y. Sgro, U. of Wisconsin, Madison
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Figure 6.12 Tobacco mosaic virus: helical symmetry. A. The helical filament of tobacco mosaic virus (TMV) contains a single-stranded RNA genome coiled inside. Image reconstruction is based on X-ray crystallography. B. An array corresponding to helical capsid structure can be simulated by rolling up a planar array into a cylinder, then displacing the array along the vertical axis of the cylinder so that the horizontal elements are displaced by one unit.
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B. Outer membrane A. J. Malkin, et al. Journal of Virology 77:6332
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Core envelope Envelope protein DNA genome Accessory proteins
Figure 6.13 Vaccinia pox virus. A. Vaccinia virions observed in aqueous medium by atomic force microscopy (AFM). B. A pox virion includes an outer membrane and a core envelope membrane containing envelope proteins enclosing the double-stranded DNA genome and accessory proteins. The DNA is stabilized by a hairpin loop at each end.
structures that consisted only of nucleic acid and packaging proteins. Based on these models, it was concluded that a virion consists solely of a packaged genome. Further research, however, revealed that other kinds of viruses contain enzymes and regulatory proteins— encoded by the virus, its host, or both. The proteins may be found either inside the capsid or in the tegument between the capsid and the envelope. Examples include the reverse transcriptase and protease enzymes of HIV and the dozen different enzymes contained by vaccinia poxvirus. Large, asymmetrical viruses contain so many enzymes that they appear to have evolved from degenerate cells. TO SU M MAR I Z E: ■
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The viral capsid is composed of repeated protein subunits, a structure that maximizes the structural capacity while minimizing the number of genes needed for construction. The capsid packages the viral genome and delivers it into the host cell. Icosahedral capsids have regular, icosahedral symmetry. Filamentous (helical) capsids have uniform width, generating a flexible fi lamentous virion. Enveloped viruses consist of a protein capsid and tegument proteins enclosed within membrane derived from the host cell. The envelope includes virus-specific spike proteins. Accessory proteins are contained within the capsid or as tegument components between the capsid and envelope.
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6.3
Viral Genomes and Classification
Living organisms today are classified based on relatedness of their gene sequences. Genetic relatedness can also be used to compare closely related viruses, such as SARS and other coronaviruses. The definition of a virus species, however, is problematic, given the small size and high mutability of viral genomes and the ability of different viruses to recombine their genome segments within an infected host cell. Furthermore, it is not clear whether all viral species are monophyletic, that is, descended from a common ancestor. In fact, it is more likely that different classes of viruses arose from different sources—such as from parasitic cells or from host cell components such as DNA replication enzymes. Viruses are classified based on genome composition, virion structure, and host range.
The International Committee on Taxonomy of Viruses For purposes of study and communication, a working classification system has been devised by the International Committee on Taxonomy of Viruses (ICTV). The ICTV classification system is based on several criteria: ■
Genome composition. The nucleic acid of the viral genome can vary remarkably with respect to physical structure: it may consist of DNA or RNA; it may be single- or double-stranded; it may be linear or circular; and it may be whole or segmented (that is, divided into separate “chromosomes”). Genomes are classified by the Baltimore method (discussed next).
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Capsid symmetry. The protein capsid may be helical or icosahedral, with various levels of symmetry. Envelope. The presence of a host-derived envelope, and the envelope structure if present, are characteristic of related viruses. Size of the virus particle. Related viruses generally share the same size range; for example, enteroviruses such as poliovirus are only 30 nm across (about 1 / 30 the size of bacteria such as E. coli), whereas pox viruses are 200–400 nm, as large as a small bacterium. Host range. Closely related viruses usually infect the same or related hosts. However, viruses with extremely different hosts can show surprising similarities in genetics and structure. For example, both rabies virus and potato yellow dwarf virus are enveloped, bullet-shaped viruses of the rhabdovirus family.
NOTE: In nomenclature, families of viruses are designated by Latin names with the suffi x -viridae: for example, Papovaviridae. Nevertheless, the common forms of such family names are also used, for example, the papovaviruses. Within a family, a virus species is simply capitalized, as in Papillomavirus.
The Baltimore Virus Classification Based on Genome Structure Of the classification criteria just described, many virologists consider genome composition the most fundamental; that is, viruses of the same genome class (such as double-stranded DNA) are more likely to share ancestry with each other than with viruses of a different class of genome (such as RNA). In 1971, David Baltimore proposed that the primary distinction among classes of viruses be the genome composition (RNA or DNA) and the route used to express messenger RNA (mRNA). Baltimore, together with Renato Dulbecco and Howard Temin, were awarded the Nobel Prize in Physiology or Medicine in 1975 for discovering how tumor viruses cause cancer. All cells and viruses need to make messenger RNA (mRNA) to make their fundamental protein components. The production of mRNA from the viral genome is central to a virus’s ability to propagate its kind. Cellular genomes always make mRNA by copying double-stranded DNA. For viruses, however, different kinds of genomes require fundamentally different mechanisms to produce mRNA. The different means of mRNA production generate distinct groups of viruses with shared ancestry. So far, the known mechanisms of replication and mRNA expression defi ne seven fundamental groups of
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viral species (Fig. 6.14). These seven fundamental groups form the basis of the taxonomic survey of viruses in Table 6.1. Viral Biorealm Group I. Double-stranded DNA viruses such as herpes and smallpox make their own DNA polymerase or use that of the host for genome replication. Their genes can be transcribed directly by a standard RNA polymerase, in the same way that a cellular chromosome would be transcribed. The RNA polymerase used can be that of the host cell, or it can be encoded by the viral genome. Group II. Single-stranded DNA viruses require the host DNA polymerase to generate the complementary DNA strand. The double-stranded DNA can then be transcribed by host RNA polymerase. An example is canine parvovirus. Group III. Double-stranded RNA viruses, such as the plant pathogenic reoviruses, require a viral RNAdependent RNA polymerase to generate messenger RNA by transcribing directly from the RNA genome. Since the RNA polymerase is required immediately upon infection, such viruses usually package a viral RNA polymerase with their genome before exiting the host cell. Group IV. (+) sense single-stranded RNA viruses consist of a positive-sense (+) strand, that is, the strand that can serve directly as mRNA to be translated to viral proteins. Replication of the RNA genome, however, requires synthesis of the template (–) strand, that is, the strand complementary to the (+) strand, to form a double-stranded RNA intermediate. Positive-sense (+) RNA viruses are very common. They include the coronaviruses, such as SARS, as well as hepatitis C virus and the flavivirus that causes West Nile encephalitis. Group V. (–) sense single-stranded RNA viruses such as influenza virus have genomes that consist of template or “negative-sense” RNA. Thus, they need to package a viral RNA-dependent RNA polymerase for transcribing (–) RNA to (+) mRNA. The (–) RNA viral genomes are often segmented, that is, they consist of more than one separate linear chromosome—a key factor in the evolution of killer strains of influenza (see Section 11.4). Group VI. Retroviruses, or RNA reversetranscribing viruses such as HIV and feline leukemia virus, have genomes that consist of (+) strand RNA. Instead of RNA polymerase, they package a reverse transcriptase, which transcribes the RNA
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Group II: Single-stranded DNA Requires DNA polymerase to generate a complementary strand.
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Group IV: (+) Single-stranded RNA Requires RNA-dependent RNA polymerase to make a template for mRNA and genome replication.
Group VII: Double-stranded DNA pararetrovirus Requires plant host reverse transcriptase to make dsDNA.
California Institute of Technology
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+ DNA
± DNA
into a double-stranded DNA (for details, see Section 11.5). The double-stranded DNA is then integrated into the host genome, where it directs the expression of the viral genes. Group VII. DNA reverse-transcribing viruses, or pararetroviruses, have a life cycle that requires reverse transcriptase. Animal viruses such as hepatitis B (a hepadnavirus) fi rst copy their double-stranded DNA genomes into RNA, then reverse-transcribe the
■
Group III: Double-stranded RNA Requires RNA-dependent RNA polymerase to make mRNA and genomic RNA.
Figure 6.14 Baltimore classification of viral genomes. A. Seven categories of viral genome composition: I. Double-stranded DNA is transcribed to mRNA. II. Single-stranded DNA generates a double-stranded form within the host cell, which is transcribed to mRNA. III. Double-stranded RNA makes mRNA using RNAdependent RNA polymerase. IV. Single-stranded RNA (+) makes a complementary (–) strand, which is transcribed to mRNA. V. Single-stranded RNA (–) is transcribed to mRNA. VI. Single-stranded RNA (+) is reverse-transcribed to DNA, which is transcribed to mRNA. VII. Double-stranded DNA is transcribed to mRNA, which is reverse-transcribed to regenerate viral genomes for packaging into virions. B. David Baltimore (left), with a graduate student at the California Institute of Technology. Baltimore won the 1975 Nobel Prize in Physiology or Medicine for his work on retroviruses; co-winners were Renato Dulbecco and Howard Temin.
RNA to progeny DNA using a reverse transcriptase packaged in the original virion. In contrast, plant pararetroviruses, such as cauliflower mosaic virus, generate an RNA intermediate that replicates using a reverse transcriptase made by the host cell. Many plant genomes include a gene for reverse transcriptase. Cauliflower mosaic virus is of enormous agricultural significance for its use as a vector to construct pesticide-resistant food crops.
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Table 6.1 Groups of viruses−Baltimore classification. Viruses
Taxonomic group with key traits and examples
Group I. Double-stranded DNA viruses
60 nm
©Gopal Murti/Visuals Unlimited
Replicate using host or viral DNA polymerase.
10 nm
©Tim Baker/Visuals Unlimited
Bacteriophage lambda
Papillomavirus
Nonenveloped bacteriophages Structure includes head, neck, and tail. Myoviridae. Bacteriophage T4 infects Escherichia coli. Siphoviridae. Bacteriophages lambda and Mu infect E. coli. Others infect gram-positive hosts such as Lactobacillus and Streptococcus. Tectiviridae. Infect enteric bacteria. Nonenveloped viruses of animals and protists Adenoviridae. Adenovirus generates tumors in humans. Papillomaviridae. Papillomavirus causes genital warts. Phycodnaviridae. Infect chlorella, algal symbionts of paramecia and hydras. Iridoviridae. Infect insects and amphibians. Regular packaging of capsids in cell confers an iridescent color on the infected cells. Mimiviridae. The largest known viruses, infect Acanthameba. Enveloped viruses of animals Herpesviridae. Herpes simplex I and II cause oral and genital herpes, and varicella-zoster virus causes chickenpox. Poxviridae. Include smallpox and cowpox viruses. Baculoviridae. Baculoviruses infect insects.
50 nm
ICTVdb
Archaeal viruses Fuselloviridae. Fusellovirus STIV infects Sulfolobus sp., growing at pH 2 at 80°C. Fusellovirus His1 infects Haloarcules sp., growing in concentrated salt. Rudiviridae and Lipothrixviridae infect Sulfolobus and Thermoproteus species. Gultaviridae. Rod-shaped virus infect Sulfolobus sp. Ampullaviridae. Bottle-shaped viruses infect Acidianus sp. Haloviridae. Haloviruses infect haloarchea such as Haloferax, Halobacterium, and Haloarcula.
Fusellovirus
Group II. Single-stranded DNA viruses
100 nm
Geminivirus
Robert G. Milne, CNR, Instituto de Fitovirologica Applicata, Torino, Italy
Genome consists of (+) sense DNA; require a host DNA polymerase to generate the complementary strand; nonenveloped. Nonenveloped bacteriophages Inoviridae. Bacteriophage M13 infects E. coli and has a slow-release life cycle. Microviridae. Bacteriophage φX174 infects E. coli. Nonenveloped animal viruses Parvoviridae. Cause various diseases in cats, pigs, and other animals. Circoviridae. Infect pigs and birds. These viruses target the lymphoid tissues and cause immunosuppression. Nonenveloped plant viruses Geminiviridae.Transmitted by aphids to tomato plants and other important crops. Their virions group in “twins,” each member of the pair carrying one DNA circle with part of the genome. Infection requires transmission of both parts.
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Table 6.1 Groups of viruses−Baltimore classification (continued) Viruses
Taxonomic group with key traits and examples
Group III. Double-stranded RNA viruses
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Gary Gaugler/Visuals Unlimited
Require viral RNA-dependent RNA polymerase; usually package the polymerase before exiting host cell.
Rotavirus
Nonsegmented, enveloped bacteriophages Cystoviridae. Infect Pseudomonas species of bacteria. Segmented, nonenveloped viruses of animals and plants Birnaviridae. Infect marine and aquatic fish. Reoviridae. Orthoreoviruses and rotaviruses infect humans and other vertebrates. Cypovirus infects insects. Fijivirus infects plants. Rice dwarf virus (phytoreovirus) is transmitted by leafhopper beetles and causes major economic damage to rice crops worldwide. Varicosaviridae. Infect plants.
Group IV. (+) Sense single-stranded RNA viruses
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©Hans Ackermann/Visuals Unlimited
Require viral RNA-dependent RNA polymerase to generate (–) template for progeny (+) genome; usually nonsegmented.
30 nm
Kenneth Eward/Photo Researchers, Inc.
Bacteriophage MS2
Nonenveloped bacteriophages Leviviridae. Bacteriophages MS2 and Qβ infect E. coli. Nonenveloped animal and plant viruses Bromoviridae. Infect many kinds of plants and are often carried by beetles. Picornaviridae. Poliovirus causes poliomyelitis. Rhinovirus causes the common cold. Apthovirus causes hoof and mouth disease in cattle and other stock. Tobamoviridae. Tobacco mosaic virus infects plants. Potyviridae. Viruses such as plum pox virus infect fruits, peanuts, potatoes, and other plants. Enveloped animal and plant viruses Coronaviridae. Coronaviruses include SARS and animal viruses. Flaviviridae. Flaviviruses infecting humans include West Nile virus, yellow fever virus, and hepatitis C virus. Togaviridae. Include rubella virus and equine encephalitis virus.
Rhinovirus 14
Group V. (–) Sense single-stranded RNA viruses
Sciecne VU/Visuals Unlimited
Require viral RNA-dependent RNA transcriptase.
©Science VU/CDC/ Visuals Unlimited
Influenza virions
Segmented, enveloped viruses Orthomyxoviridae. Influenza virus causes major epidemics among humans and animals. Nonsegmented, enveloped viruses Filoviridae. Ebola virus causes outbreaks among humans and chimpanzees. Rhabdoviridae. Rabies virus infects mammals. Paramyxoviridae. Infect humans and cause measles, mumps, and parainfluenza. Segmented (+/–) strand, enveloped viruses Arenaviridae. Spread by rodents, arenaviruses cause hemorrhagic fever and lymphocytic choriomeningitis. Bunyaviridae. Hantaviruses are spread by rodents and infect humans. Tospoviruses are transmitted by thrips, infecting peanuts, onions, garlic, and other plants.
Ebola virus
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Table 6.1 Groups of viruses−Baltimore classification (continued) Viruses
Taxonomic group with key traits and examples
Group VI. Retroviruses (RNA reverse-transcribing viruses)
100 nm
©Hans Gelderblom/ Visuals Unlimited
Require viral reverse transcriptase to generate DNA copy for integration into host; chromosome package transcriptase before exiting host cell. Retroviridae. Human immunodeficiency virus (HIV) is causing a human pandemic (worldwide epidemic). Feline leukemia virus (FeLV) is endemic among cats.
HIV I
Group VII. Pararetroviruses (DNA reverse-transcribing viruses)
Encyclopedia of Virology. 1995 Academic Press Ltd All Rights Reserved
DNA is transcribed to RNA intermediate; reverse-transcribed to DNA; infect plant cells, which have cytoplasmic reverse transcriptase; or package a viral reverse transcriptase. Nonenveloped plant viruses Caulimoviridae. Transmitted by aphids, cauliflower mosaic virus and related viruses infect cauliflower, broccoli, groundnuts, soybeans, and cassava. The cauliflower mosaic virus promoter sequence is used to construct vectors to put genes into transgenic plants. Badnaviridae. Badnaviruses infect bananas, cocoa plants, citrus, yams, and sugarcane. Enveloped animal viruses Hepadnaviridae. Hepatitis B virus causes widespread disease of the human liver.
Cauliflower mosaic virus
Molecular Evolution of Viruses The phylogeny, or genetic relatedness, of viruses can be determined within families. For example, the herpes family includes double-stranded DNA viruses that cause several human and animal diseases, such as chickenpox, oral and genital herpes infection, and equine herpes respiratory and genital infections. Herpes genomes consist of double-stranded DNA, 120–220 kilobases (kb) encoding about 70–200 genes; an example is that of varicella-zoster virus, the causative agent of chickenpox (Fig. 6.15A). The genome includes two “unique” segments of genes, one long and one short (U L and US), joined by two inverted repeats (IRs). Other herpes genomes share similar structure, though they differ in gene order and IR position. The relatedness of different herpes viruses can be measured by comparing their genome sequences. Comparison is based on orthologous genes, or orthologs. Orthologs are genes of common ancestry in two genomes that share the same function, a topic discussed in Chapter 8. An example is the ribosomal RNA genes whose sequence is used to measure relatedness of cellular organisms. Viruses have no ribosomal RNA, but closely related viruses share other orthologous genes. In pairs of orthologs, the amount of difference in sequence correlates approximately with
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the time following divergence from a common ancestor (a topic discussed in Chapter 17). Sequence comparison places herpes into three classes designated alpha, beta, and gamma (Fig. 6.15B). The alpha class includes human varicella-zoster virus and the oral and genital herpes viruses (HSV-1 and HSV-2), as well as equine herpes virus. The beta class includes cytomegalovirus, a common cause of congenital infections (present at birth), as well as two less known viruses. The gamma class includes Epstein-Barr virus, the cause of infectious mononucleosis, as well as several viruses of animals. The more ancient evolution of viruses, however, is difficult to assess based on DNA or RNA sequence. Unlike cells, viruses do not possess genes common to all species, such as the ribosomal RNA genes used to estimate times of divergence of cellular organisms (discussed in Chapter 17). Furthermore, the genomes of viruses are highly mosaic, that is, derived from multiple sources. Mosaic genomes result from recombination between different viruses coinfecting a host. In some cases, phylogeny is inconsistent with the fundamental chemical composition of the genome. For example, some DNA bacteriophages actually share closer ancestry with RNA bacteriophages than they do with DNA animal viruses. This is because two or more viruses can coinfect a cell and exchange genetic components.
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A. Varicella-Zoster Virus (VZV) Genome (120 kb) Unique long region (UL )
IR
US
■
B.
Alpha herpes viruses
IR
Varicella-zoster (VZV) 100
61 ORFs
Equine herpes (EHV-1) 77
10 ORFs
Figure 6.15 Phylogeny of herpes viral genomes. A. Genome structure of human varicella-zoster virus (VZV), the causative agent of chickenpox. B. Phylogeny of human and animal herpes viruses, based on whole-genome sequence analysis comparing clusters of orthologous groups of genes. Numbers measure genetic divergence.
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Equine herpes (EHV-4) Herpes simplex (HSV-1) Herpes simplex (HSV-2)
Root (common ancestor)
Beta herpes viruses Cytomegalovirus (CMV)
100
Thus, the genomic content of a virus can be influenced by its host range.
Herpes 6 (HHV-6)
73
Herpes 7 (HHV-7)
THOUGHT QUESTION 6.4 How can viruses with different kinds of genomes (RNA versus DNA) combine and exchange genetic information?
90
Epstein-Barr virus (EBV)
As more viral genomes are sequenced, classification methods have been devised to take advantage of sequence information without requiring gene products common to all species. One promising approach is that of proteomics, analysis of the proteome, the proteins encoded by genomes (discussed in Chapter 8). Proteins are identified through biochemical analysis of virus particles and through bioinGram-positive Siphophage hosts formatic analysis of protein sequences encoded (dsDNA) in the genomes. Proteomic analysis is useful for phage λ viruses because their small genomes encode a small number of proteins, which can be readGram-negative ily analyzed. Furthermore, because proteomic hosts analysis is based on numerous gene products, it does not require a single gene (such as rRNA) common to all species. Statistical comparison of all proteins generated by a set of viral speLactobacillus cies reveals underlying degrees of relatedness. An example of viral classification based on proteomic analysis is that of the “proteomic Podophage Gram-positive bacillus tree” of bacteriophages proposed by Rohwer and Edwards (Fig. 6.16). Unlike earlier trees based on a single common gene sequence, the proteomic tree is based on the statistical comparison of Myophage (dsDNA) Gram-negative phage protein sequences predicted by genomic phage T4 enteric DNA of many different species of phages. The proteomic analysis predicts seven major
The bacteriophage proteomic tree. Comparison of all proteins encoded by each genome predicts distinct groups of bacteriophages. Within each group, there are subgroups of phages with shared hosts, since sharing of hosts facilitates genetic recombination and horizontal transfer of genes between different phages. Source: Based on
Figure 6.16
F. Rohwer and R. A. Edwards. 2002. J. Bacteriol. 184:4529.
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Gamma herpes viruses
Saimiriine herpes (HVS-2)
87 82
Equine herpes (EHV-2) Murine herpes (MHV-68)
Lactobacillus or Streptococcus Inophage (ssDNA) phage M13 Plectrophage (ssDNA)
Gramnegative hosts Leviphage (ssRNA)
E. coli
Mycobacteria Gramnegative
Gramnegative E. coli
Siphophage
Gramnegative
Microphage (ssDNA) phage X174
Podophage (dsDNA) phage T7
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evolutionary categories of phage species. These categories appear to group phage species according to host bacteria. For example, phages that infect gram-negative hosts will show more genetic commonality with each other than with phages that infect gram-positive hosts. Shared hosts have a significant impact on phage evolution because coinfecting a host enables phages to exchange genes.
Lambda binding sites
Exterior
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Classification of viruses is based on genome composition, virion structure, and host range. The Baltimore virus classification emphasizes the form of the genome (DNA or RNA, single- or doublestranded) and the route to generate messenger RNA. Proteomic classification includes information from all viral proteins. Statistical analysis reveals common descent of viruses infecting a common host.
Beta barrel within outer membrane
Periplasm
6.4
Bacteriophage Life Cycles
All viruses require a host cell for reproduction. While viruses display a remarkable diversity of reproductive strategies, they all face these same needs for host infection: ■
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■
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Host recognition and attachment. Viruses must contact and adhere to a host cell that can support their particular reproductive strategy. Genome entry. The viral genome must enter the host cell and gain access to the cell’s machinery for gene expression. Assembly of virions. Viral components must be expressed and assembled. Components usually “selfassemble”; that is, the joining of their parts is favored thermodynamically. Exit and transmission. Progeny virions must exit the host cell, then reach new host cells to infect. In the case of multicellular organisms, the virus must eventually reach other multicellular hosts (as discussed in Chapters 27 and 29).
Bacteriophages Attach to Specific Host Cells To commence an infectious life cycle, bacteriophages need to contact and attach to the surface of an appropriate host cell. Contact and attachment is mediated by cell surface receptors, proteins on the host cell surface that are specific to the host species and that bind to a specific viral component. Receptor proteins. The cell surface receptor for a virus
is actually a protein with an important function for the host cell, but the virus has evolved to take advantage of its existence. An extensively studied model system of virusreceptor binding is that of bacteriophage lambda (see Table 6.1, top row). The phage lambda virion attaches
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Figure 6.17 Bacteriophage lambda interacts with its host receptor. Phage lambda enters E. coli by first binding to the maltose porin. The “beta barrel” of maltose porin, shown in blue, is buried in the outer membrane. The phage lambda–binding sites, shown in green, were identified by amino acid substitution mutations that prevent phage binding and confer resistance to lambda on the host cell. (PDB code: 1MAL)
specifically to the maltose porin in the outer membrane of Escherichia coli (Fig. 6.17). Although the protein is often called “lambda receptor protein,” it actually evolved in the host as a way to acquire the sugar maltose to metabolize. Thus, natural selection maintains the maltose porin in E. coli despite the danger of phage infection. The precise domain of the maltose porin that binds to phage lambda was defi ned experimentally by mutations in E. coli that cause amino acid substitution in the protein. Some of the mutant E. coli strains were resistant to phage lambda infection. The mutations that conferred host resistance mapped to the domain of maltose porin that binds the phage capsid.
Phage Genomes Direct Host Cells to Produce Progeny Phages Historically, the life cycles of bacteriophages have provided some of the most fundamental insights in molecular biology. In 1952, Alfred Hershey and Margaret Chase showed that the transmission of DNA by a bacteriophage to a host cell led to production of progeny bacteriophages, thus confi rming that DNA is the hereditary material. In 1950, André Lwoff and Antoinette Gutman showed that a phage genome could integrate itself within a bacterial genome—the fi rst recognition that genes could enter and leave a cell’s genome. Other
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A. Phage T4 DNA insertion Phage T4 attaches to bacterium.
Pa r t 1 Sheath contracts and viral DNA enters bacterium.
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The M ic ro b i al Ce l l
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fundamental concepts of the genetic unit and the basis of gene transcription came from experiments on bacteriophages, as discussed in Chapters 7–9. Most bacteriophages (or phages) insert only their genome into a cell through the cell envelope, thus avoiding the need for the capsid to penetrate the molecular barrier of the cell wall. For example, the phage T4 virion has a neck tube that contracts, bringing the headpiece near the cell surface to insert its DNA (Fig. 6.18A ). After the genome is inserted, the phage capsid remains outside attached to the cell surface and is termed a “ghost.”
B. Phage lambda reproductive cycle
The lytic cycle. In a lytic cycle, when a phage particle injects
Host genome
Phage attaches to host cell and inserts DNA.
Linear dsDNA cyclizes to circular DNA.
Phage particle
its genome into a cell, it immediately proceeds to reproduce as many progeny phage particles as possible. The process of reproduction involves replicating the phage genome as well as expressing phage mRNA to make enzymes and capsid proteins. Some phages, such as T4, digest the host DNA to increase efficiency of phage production. Finally, the host cell lyses, releasing progeny phages. Lysogeny Phage DNA integrates into host genome to form prophage.
Lytic cycle Viral DNase cleaves host cell DNA. Cell synthesizes capsid proteins.
Phage recombines by rejoining the ends of its phosphodiester chain and enters the lytic cycle.
Cell replicates phage DNA. DNA is packaged into capsids. Stress induces excision of phage DNA.
Integrated phage DNA reproduces with host genome.
Phage lyses cell, and progeny phage are released.
Integrated phage DNA replicates with host genome.
Figure 6.18 Bacteriophage reproduction: lysis and lysogeny. A. Phage T4 attaches to the cell surface by its tail fibers, then contracts to inject its DNA. The empty capsid remains outside as a “ghost.” B. Lysis occurs when the phage genome reproduces progeny phage particles, as many as possible, then lyses the cell to release them. In phage lambda, lysogeny can occur when the phage genome integrates into that of the host. The phage genome is replicated along with that of the host cell. The phage DNA, however, can direct its own excision by expressing a site-specific DNA recombinase. This excised phage chromosome then initiates a lytic cycle.
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Phage T4 reproduces entirely by the lytic cycle; this is called a virulent phage. Other phages, such as phage lambda, have the options of reproducing by lysis or by lysogeny (Fig. 6.18B ). Lysis. After phage lambda inserts its DNA, its genes are expressed by the host cell RNA polymerase and ribosomes. “Early genes” are expressed early during the lytic cycle. Other phage-expressed proteins then work together with the cellular enzymes and ribosomes to replicate the phage genome and produce phage capsid proteins. The capsid proteins self-assemble into capsids and package the phage genomes, a process that takes place in defi ned stages, like a factory assembly line. At last, a “late gene” from the phage genome expresses an enzyme that lyses the host cell wall, releasing the mature virions. Lysis is also referred to as a burst, and the number of virus particles released is called the burst size. Lysogeny. A temperate phage, such as phage lambda, can infect and lyse cells like a virulent phage, but it also has an alternative pathway: to integrate its genome into that of the host cell (see Fig. 6.18B). Phage lambda has a linear genome of double-stranded DNA, which circularizes upon entry into the cell. The circularized genome then recombines into that of the host by site-specific recombination of DNA. In site-specific recombination, a recombinase enzyme aligns the phage genome with the host DNA and exchanges the phosphodiester backbone links with those of the host genome. The process of exchange thus integrates the phage genome into that of the host. The integrated phage genome is called a prophage. Integration of the phage genome as a prophage results in lysogeny, a condition in which the phage genome is replicated along with that of the host cell as the host reproduces. Implicit in the term lysogeny, however, is the ability of such a strain to spontaneously generate a lytic burst of phage. For lysis to occur, the prophage (integrated phage genome) directs its own excision from the host genome by an intramolecular process of site-specific recombination. The two ends of the phage genome exchange their phosphodiester backbone linkages so as to come apart from the host molecule. As the phage DNA exits the host genome, it circularizes and initiates a lytic cycle, destroying the host cell and releasing phage particles. The “decision” between lysogeny and lysis is determined by proteins that bind DNA and repress transcription of genes for virus replication (see Section 10.7). Exit from lysogeny into lysis can occur at random, or it can be triggered by environmental stress such as UV light, which damages the cell’s DNA. The regulatory switch of lysogeny responds to environmental cues indicating the likelihood that the host cell will survive and continue to propagate the phage genome. If a cell’s growth is strong, it is more likely that the phage
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DNA will remain inactive, whereas events that threaten host survival will trigger a lytic burst. An analogous phenomenon occurs in animal viral infections such as herpes, in which environmental stress triggers painful outbreaks arising from latent infection, reactivation of a virus that was dormant within cells. The lysogeny of phage lambda provided an early model system for investigating viral persistence in human viral diseases. Viruses transfer host genes. During the exit from lysog-
eny or during latent growth of animal viruses, the virus can acquire host genes and pass them on to other host cells. This process of transferring host genes is known as transduction. A transducing bacteriophage can pick up a bit of host genome and transfer it to a new host cell. In some forms of transduction, the entire phage genome is replaced by host DNA packaged in the phage capsid, resulting in a virus particle that only transfers host DNA. Host DNA transferred by viruses can become permanently incorporated into the infected host genome. The mechanisms of phage-mediated transduction (generalized and specialized) are discussed in Chapter 9. The integration and excision of viral genomes make extraordinary contributions to the evolution of host genomes. Lysogenic prophages often contribute key toxins and virulence factors to pathogens: For example, the CTXφ prophage encodes cholera toxin in V. cholerae; the Shiga toxin prophage confers virulence on enteric pathogens Shigella and E. coli H7:O157; and prophages encode toxins for Corynebacterium diphtheriae (diphtheria) and Clostridium botulinum (botulism). In natural environments, phage-mediated transduction mediates much of the recombination of bacterial genomes. In the laboratory, the ability of phages to transfer genes provided vectors for recombinant DNA technology, the artificial construction of phages to carry genes from animals and plants for gene cloning. When viral transfer of genes was fi rst discovered in the 1960s, it was thought that multicellular eukaryotes had a much lower tolerance for genome change. We have since learned, however, that much of the human genome also shows evidence of gene transfer mediated by ancient viruses, including possible retroviruses similar to HIV (discussed in Chapter 11). Slow release. The slow-release cycle differs from lysis
and lysogeny in that phage particles reproduce without destroying the host cell (Fig. 6.19). Slow release is performed by filamentous phages such as phage M13. In the slow-release life cycle, the single-stranded circular DNA of M13 serves as a template to synthesize a double-stranded intermediate. The double-stranded intermediate slowly generates single-stranded progeny genomes, which are packaged by supercoiling and coating with capsid proteins. The phage particles then extrude through the cell envelope
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Phage attaches to host cell and inserts DNA.
■
Host genome ■
Cell synthesizes duplex.
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Cell replicates circular, single-stranded DNA.
201
Host cell surface receptors mediate attachment of bacteriophages to a cell and confer host specificity. Lytic cycle. A bacteriophage injects its DNA into a host cell, where it utilizes host gene expression machinery to produce progeny virions. Lysogeny. Some bacteriophages can copy their genome into that of the host cell, which then replicates the phage genome along with its own. A lysogenic bacterium can initiate a lytic cycle. Slow release. Some bacteriophages use the host machinery to make progeny that bud from the cell slowly, slowing growth of the host without lysis.
6.5 Phages assemble and exit without lysis.
While phages reproduce and exit cell, the cell reproduces slowly.
Figure 6.19 Bacteriophage life cycle: slow release. In the slow-release life cycle, a filamentous phage produces phage particles without lysing the cell. The host continues to reproduce itself, but more slowly than uninfected cells because much of its resources are being used to make phages.
without lysing the cell. The host cell continues to reproduce, though more slowly than uninfected cells because much of its resources are diverted to virus production. Some phage genomes consist of RNA. RNA phages include the leviviruses such as phage MS2 and Qβ. The RNA phages replicate their genomes through a double-stranded RNA intermediate, analogous to the double-stranded DNA intermediate of the filamentous phage M13; but RNA phages lyse their hosts. RNA phages have very simple genomes composed of only three or four genes. These genes encode a replicase, a coat protein, and a maturation factor. The RNA genomes of these phages are of historical interest as model systems to study ribosomal translation. THOUGHT QUESTION 6.5 What are the relative advantages and disadvantages (to a virus) of the slowrelease strategy, compared with the strategy of a temperate phage, which alternates between lysis and lysogeny?
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Animal and Plant Virus Life Cycles
Animal and plant viruses solve problems similar to those faced by bacteriophages: host attachment, genome entry and gene expression, virion assembly, and virion release. The more complex structure of eukaryotic cells, however, leads to greater complexity and diversity of life cycles than is seen in phages. Viral reproduction may involve intracellular compartments such as the nucleus or secretory system and may depend on tissue and organ development in multicellular organisms. Study of animal virus life cycles reveals potential targets for antiviral drugs, such as protease inhibitors for HIV. NOTE: The virus life cycles in Chapter 6 are simplified. For greater molecular detail, see Chapter 11.
Animal Viruses Bind Receptors and Uncoat Their Genomes Like bacteriophages, animal viruses evolve to bind specific receptor proteins on their host cell. An example of a human virus-receptor interaction is that of rhinovirus (see Table 6.1, Group IV), which causes the common cold. Rhinovirus attaches to ICAM-1, a human glycoprotein needed for intercellular adhesion (Fig. 6.20A). The rhinovirus binds to a domain of ICAM-1 essential for it to bind a lymphocyte protein called integrin. The host receptors play a key role in determining the host range, the group of host species permitting growth. Within a host, receptor molecules can also determine the tropism, or tendency to infect a particular tissue type. Some viruses, such as Ebola virus, exhibit broad tropism, infecting many kinds of host tissues, whereas others, such as papillomavirus, show tropism for only one type, the epithelial tissues. Poliovirus infects only a specific class of human cells that display the immunoglobulin-like receptor protein PVR. Children under age 3 do not yet
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A. Host receptor binding
B. Uncoating at the cell membrane COO
Cytoplasm
–
Measles virus Cell exterior S
ICAM-1 Receptor protein
S S S S
Uncoating of RNA genome
S S S S S
Fibrinogen
Cytoplasm
Cell membrane
S S NH 3+ Lymphocyte integrin Lfa-1–binding site
Rhinovirus-binding site
D. Uncoating at the nuclear membrane
C. Uncoating within endosomes
Adenovirus
Hepatitis C
Cytoplasm Receptor protein
Cytoplasm Receptor protein
Cell membrane Cell membrane
Endosome Lysosome fuses with and acidifies endosome.
Endosome
Viral envelope fuses with endosome membrane. Docking onto nuclear membrane
H+ Uncoating of RNA genome Nucleus Uncoating of dsDNA genome
Nuclear membrane
Receptor binding and genome uncoating. A. Rhinovirus attaches to the intercellular adhesion molecule (ICAM-1), a glycoprotein required by the host cells to bind a lymphocyte integrin, a cell surface matrix protein required for cell-cell adhesion. After binding a specific receptor on the host cell membrane, an animal virus enters the cell, where its genome is uncoated. B. Uncoating at the cell membrane. C. Uncoating within endosomes. D. Uncoating at the nuclear membrane. Source: A. Based on J. Bella, et al. 1998. PNAS 95:4140.
Figure 6.20
202
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express the receptor PVR; thus, they do not experience polio infection of the nervous system. Mice are not normally infected by polio, but when transgenic mice were created to express PVR on their cells, the mice could then be infected. Different strains of a virus may show radically different host tropisms based on their receptor specificity. An example is that of avian influenza strain H5N1. The H5N1 strain infects birds by binding to a glycoprotein (protein with sugar chains) receptor on cell surfaces of the avian respiratory tract. The H5N1 strain requires a receptor protein with a sialic acid sugar chain terminating in galactose linked at the C-3 position (α2, 3), common in the avian respiratory tract. In humans, however, most nasal upper respiratory cells have receptors with galactose linked at the C-6 position (α2, 6). Cells with the C-3 linkage are more common in the human lower respiratory tract. That is why avian influenza H5N1 infection of humans has been relatively rare. However, only a small mutation in the H5N1 envelope protein could enable it to bind to the (α2, 6) receptor more effectively, allowing rapid transmission between humans. THOUGHT QUESTION 6.6 How could humans evolve to resist rhinovirus infection? Is such evolution likely? Why or why not? Genome uncoating. Some animal viruses, such as
poliovirus, attach to the host cell surface and insert their genome into the host cell, as do bacteriophages. In animal virology, this process is called “extracellular uncoating” of the genome. Most animal viruses, however, enter the host cell as intact virions, then undergo intracellular uncoating, a process of virion disassembly in which the genome is released for replication and gene expression. Uncoating of the genome takes place in several different ways. For example, measles virus, a paramyxovirus, enters the cell by binding host receptor proteins, which causes the viral envelope to fuse with the host cell membrane (Fig. 6.20B). The measles RNA genome is then uncoated and released directly into the cytoplasm. Alternatively, a flavivirus such as hepatitis C is taken up by endocytosis (Fig. 6.20C). In endocytosis, the cell membrane forms a vesicle around the virion and engulfs it, forming an endocytic vesicle. The endocytic vesicle fuses with a lysosome, whose acidic environment activates entry of the capsid into the cytoplasm. The capsid then comes apart, uncoating the viral genome. Still other species, such as adenovirus, enter by endocytosis but require transport to the nucleus (Fig. 6.20D). At the nuclear membrane, the virion docks at a nuclear pore and injects its DNA genome into the nucleus. The adenoviral DNA then has access to cellular DNA polymerase and RNA transcriptase.
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Animal Virus Life Cycles The primary factor determining the life cycle of an animal virus is the physical form of the genome. A viral genome made of DNA can utilize some or all of the host replication machinery. If, however, the genome consists of single-stranded RNA, it fi rst needs to synthesize either an RNA-dependent RNA polymerase to generate an RNA template or a reverse transcriptase to generate a DNA template (in the case of retroviruses). DNA virus life cycle. An example of a double-stranded
DNA virus is human papillomavirus (HPV) (see Table 6.1, Group I), the cause of genital warts (Fig. 6.21A). HPV is the most common sexually transmitted disease in the United States and one of the most common worldwide. Certain strains infect the skin, whereas others infect the mucous membranes through genital or anal contact (sexual transmission). Like phage lambda, papillomavirus has an active reproduction cycle and a dormant cycle in which the viral genome integrates into that of the host. Genome integration leads to abnormal cell proliferation causing cauliflower-like warts and can progress to cervical or penile cancer. HPV initially infects basal epithelial cells (Fig. 6.21B). In the basal cells, papilloma virions enter the cytoplasm by receptor binding and membrane fusion. The virion then undergoes uncoating, the disintegration of the protein capsid or coat, releasing its circular double-strand genome (Fig. 6.22). The uncoated viral genome enters the nucleus, where it gains access to host DNA and RNA polymerases. The process of viral reproduction is complicated by the developmental progression of basal cells into keratinocytes and ultimately cells to be shed or sloughed off from the surface (see Fig. 6.21B). Viral replication is largely inhibited until the basal cells start to differentiate into keratinocytes (mature epithelial cells). Host cell differentiation induces the viral DNA to replicate and undergo transcription by host polymerases. The mRNA transcripts then exit the nuclear pores, as do host mRNAs, for translation in the cytoplasm. The translated capsid proteins, however, return to the nucleus for assembly of the virion. Nuclear virion assembly is typical of DNA viruses (with the exception of poxviruses, which replicate entirely in the cytoplasm). As the keratinocytes complete differentiation, they start to come apart and are shed from the surface. The DNA virions are released from the cell during this shedding process. Unfortunately, in the basal cells, HPV has an alternative pathway of integrating its genome into that of the host (analogous to phage lysogeny). Genome integration can disrupt the expression of key host genes, causing transformation to cancer.
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A.
dsDNA virus Receptor protein Epithelial cell Genome is uncoated. Nuclear pore
©Ken Greer/Visuals Unlimited
Host RNA polymerase
Transcription
mRNAs mRNAs
DNA replication
Translation
Host DNA polymerase
B.
Capsid proteins 2. Keratinocytes differentiate; virus replication is activated.
1. HPV infects basal cells, where it remains dormant.
Assembly Nucleus
3. Shedding cells release HPV virions. Only shedding epithelial cells release virions.
HPV integration into host genome transforms cells to cancer.
Human papillomavirus. A. Certain strains of human papillomavirus (HPV) cause warts on the genitals or anus. B. HPV infects basal epithelial cells, where the DNA uncoats but remains dormant. As cells differentiate, viral synthesis is activated. Shedding cells then release virions. An alternative fate of HPV is to integrate in the genome of host basal cells, where it leads to carcinogenesis.
Figure 6.21
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Progeny virions
Papillomavirus life cycle. HPV, a double-stranded DNA virus, enters the cytoplasm, where the protein coat disintegrates. The viral DNA enters the nucleus for replication and transcription by host polymerases. Viral mRNA returns to the cytoplasm for translation of capsid proteins, which return to the nucleus for assembly of virions.
Figure 6.22
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RNA virus life cycle. The picornaviruses include poliovirus as well as rhinovirus, which causes the common cold (see Table 6.1, Group IV). Picornavirus genomes contain (+) strand RNA, allowing viral reproduction to occur entirely in the cytoplasm, without any use of DNA. Picornaviruses bind to a surface receptor, such as ICAM-1 for rhinovirus or the PVR receptor for poliovirus. The (+) strand RNA is uncoated by insertion through the cell membrane into the cytoplasm (Fig. 6.23). The role of endocytosis is debated; poliovirus requires no endocytosis, but rhinovirus genome uncoating may require endocytosis and low-pH induction.
(+) RNA genome
+ RNA genome is inserted into cytoplasm.
Viral (+) RNA is translated to make RNA-dependent RNA polymerase.
+
Viral RNA-dependent RNA polymerase + Replication – Replication
+ Assembly
Progeny virions
Picornavirus life cycle. A picornavirus inserts its (+) strand RNA into the cell. Reproduction occurs entirely in the cytoplasm. A key step is the early translation of a viral gene to make RNA-dependent RNA polymerase. The RNA-dependent RNA polymerase uses the picornavirus RNA template to make (–) strand RNA, which then serves as a template for other viral mRNAs as well as progeny genomic RNA.
Figure 6.23
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After uncoating, a gene in the viral RNA is translated by host ribosomes to make RNA-dependent RNA polymerase. The RNA-dependent RNA polymerase uses the viral RNA template to make (–) strand RNA. The (–) strand RNA then serves as a template for other viral mRNAs as well as progeny genomic RNA. Capsid proteins are translated by host ribosomes, and the capsids self-assemble in the cytoplasm. Virions then assemble at the cell membrane and are released by budding out. Note that other kinds of RNA virus, such as influenza virus, encapsidate a (–) strand genome. In this case, the (–) strand must serve as template to generate mRNA as well as (–) strand progeny genomes. Influenza virus replication includes other interesting molecular complications, discussed in Chapter 11.
Nucleus
Capsid proteins
■
RNA retroviruses. Retroviruses include human immunodeficiency virus (HIV), the causative agent of AIDS, and feline leukemia virus (FeLV), a disease commonly affl icting domestic cats (see Table 6.1, Group VI). A retrovirus uses a reverse transcriptase to make a DNA copy of its RNA genome (Fig. 6.24). Instead of being translated from an early gene, the reverse transcriptase is actually carried within the virion, bound to the RNA genome with a primer in place. The virion contains two copies of the HIV genome, each carrying its own reverse transcriptase. After uncoating in the cytoplasm, the viral RNA is copied into double-stranded DNA. The DNA copy then enters the nucleus, where it integrates by recombining with the host genome. The viral genome then replicates silently in the host cell, generating only a small number of virions without apparent effect on the host. To generate virions, a host RNA polymerase transcribes viral mRNA and viral genomic RNA. The viral mRNA reenters the cytoplasm for translation to produce coat proteins and envelope proteins. The coat proteins are transported by the endoplasmic reticulum (ER) to the cell membrane, where virions selfassemble and bud out. At a certain point, the host cell can suddenly begin to generate large numbers of virions. The cause of accelerated reproduction is poorly understood, although it
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Reverse transcriptase Envelope protein
Virion binds receptor protein.
Uncoating of envelope releases capsid.
Figure 6.24 Retrovirus life cycle. A retrovirus such as human immunodeficiency virus (HIV) uses reverse transcriptase to copy its RNA into double-stranded DNA. The DNA then enters the nucleus to recombine in the host genome, where a host RNA polymerase generates viral mRNA and viral genomic RNA. The viral mRNA enters the cytoplasm for translation. Viral coat proteins are transported by the endoplasmic reticulum to the cell membrane, where virions assemble and bud out.
in Chapter 12). For example, adenovirus and related viruses are being engineered to deliver a corrective gene to the lungs of people with cystic fibrosis.
Receptor protein
Uncoating of capsid releases ss (+) RNA and viral reverse transcriptase.
+ +
THOUGHT QUESTION 6.7 What are the advantages and disadvantages to the virus of replication by the host polymerase, compared with using a polymerase encoded by its own genome?
RT Reverse transcriptase dsDNA copy of genome Translation yields viral proteins.
dsDNA integrates into host genome.
RT Host RNA Pol II
Host RNA polymerase transcribes viral DNA.
Capsid proteins
Plant Virus Life Cycles All kinds of plants are subject to viral infection. Plant viruses pose enormous challenges to agriculture, where the concentrated growth of a single strain (monoculture) provides ideal conditions for a virus to spread.
Plant virus entry to host cells. In contrast to animal viruses and bacteriophages, plant viruses infect cells by mechanisms Assembly Glycoproteins that do not involve specific membrane receptors. This may be because plant cell membranes are covered by thick cell walls impenetrable to virion uptake or genome insertion. Thus, the entry of plant viruses Budding usually requires mechanical transmission, nonspecific access through physical can involve stress conditions such as poor health or pregdamage to tissues, such as abrasions of the leaf surface by nancy. The mechanism of control involves several protein the feeding of an insect. The means of mechanical transregulators encoded by the virus. The full process and mission of plant viruses are limited by the cell wall and by regulation of HIV reproduction is exceedingly complex; the sessile nature of plants. Most plant viruses gain entry further molecular details are discussed in Chapter 11. to cells by one of three routes: Oncogenic viruses. DNA viruses such as HPV and adenovirus can insert their genomes into the chromosome of a host cell in a manner analogous to that of a lysogenic bacteriophage. In some cases, the transfer of host genes to abnormal chromosome locations can generate oncogenes, genes whose expression at inappropriate times causes uncontrolled proliferation of the cell and ultimately cancer. Viruses that carry oncogenes are known as oncogenic viruses. This capacity for gene transfer may be manipulated artificially for gene therapy (discussed
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Contact with damaged tissues. Viruses such as tobacco mosaic virus appear to require nonspecific entry into broken cells. Transmission by an animal vector. Insects and nematodes transmit many kinds of plant viruses. For example, the geminiviruses are inoculated into cells by plant-eating insects such as aphids, beetles, or grasshoppers. Transmission through seed. Some plant viruses enter the seed and infect the next generation.
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Scott Bauer, Agricultural Research Service, USDA
B.
Marina Barba, Istituto Sperimentale per la Patologia Vegetale
Frederick E. Gildow, Pennsylvania State University
C.
A.
Frederick E. Gildow, Pennsylvania State University
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Plum pox is caused by potyvirus. A. Potyvirus, a filamentous (+) strand RNA virus, approximately 800 nm in length (TEM). B. Potyvirus is transmitted by aphids, which suck the plant sap and release potyvirus into the damaged tissues. C. Streaking of flowers caused by potyvirus infection. D. Ring-shaped “pox” appear on the infected fruit and the stone inside.
Figure 6.25
An economically important plant virus is the potyvirus plum pox virus (see Table 6.1, Group IV), a major pathogen of plums, peaches, and other stone fruits. Plum pox virus, a (+) strand RNA virus, is transmitted by aphids (Fig. 6.25). Following infection, the spread of the virus generates streaked leaves and flowers as well as ring-shaped “pox” marks on the surfaces of the fruit and of the stone within.
Cell membrane Nucleus
Plasmodesma Cellulose cell wall
Plant virus transmission through plasmodesmata.
Within a plant, the thick cell walls prevent a lytic burst or budding out of virions. Instead, plant virions spread to uninfected cells by traveling through plasmodesmata (singular, plasmodesma). Plasmodesmata are membrane channels that connect adjacent plant cells (Fig. 6.26). The outer channel connects the cell membranes of the two cells, whereas the inner channel connects the endoplasmic reticulum. Passage through the plasmodesmata requires action by movement proteins whose expression is directed by the viral genome. In some cases, the movement proteins transmit the entire plant virion; in other cases, only the nucleic acid itself is small enough to pass through. The independent movement and transmission of plant viral genomes suggest infective strategies in common with those of viroids, which lack capsids altogether. DNA pararetroviruses. Pararetroviruses possess a DNA
genome that requires transcription to RNA in the cytoplasm, followed by reverse transcription to form DNA genomes for progeny virions (Fig. 6.27). While some pararetroviruses infect humans, such as hepadnavirus, the best-known pararetrovirus is cauliflower mosaic virus, or caulimovirus (see Table 6.1, Group VII). Cauli-
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Cytoplasm Nucleus
Endoplasmic reticulum
Vacuole
Plant cells connected by plasmodesmata. Plasmodesmata offer a route for plant viruses to reach uninfected cells.
Figure 6.26
movirus is an important tool for biotechnology because it has a highly efficient promoter for gene transcription, allowing high-level expression of cloned genes. Vectors derived from caulimoviruses are often used to construct transgenic plants. Caulimovirus is transmitted by secretions from an insect whose bite damages plant tissues, providing access to the cytoplasm. The caulimovirus genome moves from
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the cytoplasm to the cell nucleus through a nuclear pore. Within the nucleus, two promoters on its DNA genome direct transcription to RNA. The two RNA transcripts exit the nucleus for translation by host ribosomes to make viral proteins. A host reverse transcriptase, present in plant cells, generates DNA viral genomes. After virions are assembled in the cytoplasm, movement proteins help transfer them through plasmodesmata into an adjacent cell. A caulimovirus promoter sequence is commonly used in gene transfer vectors for plant biotechnology. The advantage of viral promoters is their highly efficient transcription of a transferred gene, such as one conferring pesticide resistance. In the field, 10% of cruciferous vegetables are typically infected with caulimovirus. Some critics of gene technology are concerned that the prevalence of the caulimovirus promoter in transgenic crops may lead to the evolution of new pararetroviruses. TO SU M MAR I Z E:
dsDNA genome is transmitted through aphid bite.
Plant host cell Nucleus Uncoating of capsid releases genome. RNA transcription
RNA
Virions travel through plasmodesma to uninfected cell. Progeny virions
Host reverse transcriptase RT transcribes RNA to viral DNA.
Assembly of virions
Mature virion with DNA
Capsid proteins Viral gene products are translated.
Movement proteins Capsid proteins
Host cell surface receptors Figure 6.27 Caulimovirus life cycle. The cauliflower mosaic virus, a caulimovirus, mediate animal virus attachuses host RNA polymerase to copy its DNA into RNA and uses host reverse ment to a cell and confer host transcriptase to make DNA copies. The DNA genomes are packaged into progeny specificity and tropism. virions that use virus-encoded movement proteins to travel through plasmodesmata ■ Animal DNA viruses either to an adjacent uninfected cell. inject their genome or enter the host cell by endocytosis. The viral genome requires uncoating for gene expression. tion of virus culture is the need to grow the virus within ■ RNA viruses use an RNA-dependent RNA polya host cell. Therefore, any virus culture system must be a merase to transcribe their messenger RNA. double culture of host cells plus viruses. Culturing viruses ■ Retroviruses use a reverse transcriptase to copy of multicellular animals and plants creates additional their genomic sequence into the chromosome of a complications, as viruses show tropism for particular tishost cell. sues or organs. ■ Plant viruses enter host cells by transmission through a wounded cell surface or an animal vector. Plant viruses Batch Culture of Bacteriophages travel to adjacent cells through plasmodesmata. in Large Populations ■ Pararetroviruses contain DNA genomes but generBatch culture, or culture in liquid medium, enables growth ate an RNA intermediate that requires reverse tranof a large population of viruses for study. Bacteriophages scription to DNA for progeny virions. can be inoculated into a growing culture of bacteria, usually in a culture tube or a flask. The culture fluid is then sampled over time and assayed for phage particles. The growth pat6.6 Culturing Viruses tern usually takes the form of a step curve (Fig. 6.28). To observe one cycle of phage reproduction, phages are To study any living microorganism, we must culture it in added to host cells at a multiplicity of infection (MOI, ratio the laboratory. Culturing viruses provides large numbers of phage to cells) such that every host cell is infected. The of virions and their components for analysis. A complica■
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Burst size =
Rise period
10 phages/ml 200 phages/ml (inoculum)
= 500 phages/ cell infected
Eclipse period 0
0
10
Plaque-forming units/ml
Phages/ml
5
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Cell lysis complete
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Intracellular
108 Extracellular 106
Latent period
104
Eclipse period
102 Rise period
20 Time (min)
30
40
0
4
8 12 16 20 24 28 32 36 40 44 48 52 Hours after viral absorption
Figure 6.28
One-step growth curve of a bacteriophage. After initial infection of a liquid culture of host cells, the titer of virus drops near zero as all virions attach to the host. During the eclipse period, progeny phages are being assembled within the cell. As cells lyse (the rise period), virions are released until they reach the final plateau. The infectious cycle is typically complete within less than an hour.
Figure 6.29
phage particles immediately adsorb to surface receptors of host cells and inject their DNA. As a result, virions are virtually undetectable in the growth medium for a short period after infection; this is called the eclipse period. For some species, it is possible to distinguish between the eclipse period and a latent period, which includes the eclipse period plus the time during which progeny viruses have been formed but remain trapped within the cell. The latent period is particularly significant in animal viruses, where large numbers of virions usually generate progeny through budding out of the host cell (Fig. 6.29).
inoculated virions, assuming that all the original virions infect a cell. The burst size, together with the cell density prior to lysis, determines the concentration of the resultant suspension of virus particles, called a lysate. In the case of bacteriophages, a lysate of phage particles can be extremely stable, remaining infective at room temperature for many years. Eukaryotic viruses tend to be less stable and need to be maintained in culture or deep freeze.
NOTE: The latent period of a lytic virus is the period between initial phage-host contact and the fi rst appearance of progeny phage. This must be distinguished from the latent infection of a virus that maintains its genome within a host cell without reproducing virions.
As cells begin to lyse and liberate progeny viruses, the culture enters the rise period, in which virus particles are appearing in the growth medium. The rise period ends when all the progeny viruses have been liberated from their host cells. If the number of viruses that go on to reinoculate further host cells is small, then the virus concentration at the end point divided by the original concentration of inoculated phage approximates the burst size; that is, the number of viruses produced per infected host cell. The burst size may be estimated by dividing the concentration of progeny virions by the concentration of
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One-step growth curve for a virus. The titer of extracellular virus drops near zero during the latent period, as all virions adsorb to the host. Then progeny virions begin to emerge by budding out from the infected cell. The growth curve of a rapidly replicating animal virus typically takes hours to level off; the “burst” event is not defined as clearly as for phages.
THOUGHT QUESTION 6.8 Why does bacteriophage reproduction give a step curve, whereas cellular reproduction generates an exponential growth curve? Could you design an experiment in which viruses generate an exponential curve? Under what conditions does the growth of cellular microbes give rise to a step curve?
Animal Viruses Are Grown in Tissue Culture In the case of animal and plant viruses, the multicellular nature of the host is an important factor in the pathology and transmission of the pathogen (discussed in Chapters 26 and 27). Animal viruses can be cultured within whole animals by serial inoculation, where virus is transferred from an infected animal to an uninfected one. Culture within animals ensures that the virus strain maintains its original virulence (ability to cause disease). But the process is expensive and laborious, involving large-scale use of animals.
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C h ap t e r 6
5.5 h cells round up
8 h cells detach
24 h cells lyse and clump R. Compans, Emory University, School of Medicine
0 h uninfected
■ Vir u s S tr u c tu r e and Func t ion
Figure 6.30 Poliovirus replication in human tissue culture. Before infection (0 hours), the cultured cells grow in a smooth layer. At 5.5 hours after infection, cells are starting to retract and round up. At 8 hours, infected cells detach from the culture dish. By 24 hours, cells have lysed or in some cases clumped with other cells.
A historic event in 1949 was the fi rst successful growth of a virus in tissue culture. Poliovirus, the causative agent of the devastating childhood disease poliomyelitis, was grown in human cell tissue culture (Fig. 6.30) by John F. Enders, Thomas J. Weller, and Frederick Robbins at the Children’s Hospital in Boston. As heralded that year in Scientific American, “It means the end of the ‘monkey era’ in poliomyelitis research. . . . Tissue-culture methods have provided virologists with a simple in vitro method for testing a multitude of chemical and antibiotic agents.” Since then, tissue culture has remained the most effective way to study the molecular biology of animal and plant viruses and to develop vaccines and antiviral agents. Some viruses can be grown in a tissue culture of cells growing confluently on a surface. The fluid bathing the tissue layer is sampled for virus concentration. As in the case of bacteriophage batch culture, we can defi ne an eclipse period, a latent period before appearance of the fi rst progeny virions in the culture fluid, and a rise period. The intracellular completion of virion assembly within the cell is important for transmission of viral infection because some viruses have the ability to spread their virions from cell to cell without ever existing as such outside. In this way, they can avoid detection by the immune system to which they might be vulnerable. In tissue culture, the time course of animal virus replication is usually much longer (hours or days) than that of bacteriophages (typically less than an hour under optimal conditions). The burst size, however, of animal viruses is typically several orders of magnitude larger than that of phages. The reason for the larger burst size is probably that the volume of the eukaryotic host cell is usually much larger than that of a bacterial host, thus providing a larger supply of materials to build virions. Not all animal viruses exhibit growth that can be represented by a step curve. Some species, called slow viruses, bud off virions relatively slowly, without immediately lysing the host cell. A well-known family of slow viruses is the lentiviruses (literally, “slow viruses”), a group of retroviruses that includes HIV. HIV and related retroviruses are known for their long incubation periods, in some cases many years, during which time extremely low concentrations of virions are produced by infected cells.
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Plaque Isolation and Assay of Bacteriophages For the investigation of cellular microbes, an important tool is the culturing of individual colonies on a solid substrate that prevents dispersal throughout the medium, as described in Chapters 1 and 4. Plate culture of colonies enables us to isolate a population of microbes descended from a common progenitor. But viruses cannot be isolated as “colonies.” The reason is that although viruses can be obtained at incredibly high concentrations, they disperse in suspension. Even on a solid medium, viruses never form a solid visible mass comparable to the mass of cells that constitutes a cellular colony. In viral plate culture, viruses from a single progenitor lyse their surrounding host cells, creating a clear area called a plaque. The plaque assay for lytic bacteriophages was invented early in the twentieth century by the French microbiologist Felix d’Herelle, who was the first to recognize that bacterial cells can be infected by bacteriophages. To perform a plaque assay of bacteriophages, a diluted suspension of bacteriophages is mixed with bacterial cells in soft agar, and the mixture is then poured over a nutrient agar plate (Fig. 6.31). If no bacteriophages are present, the bacteria grow homogeneously as an opaque sheet over the surface (confluent growth). The presence of a phage is detected as a plaque, a clear circle seemingly cut out of the bacterial growth. A plaque is formed when a single virus infects a cell, replicates, and spreads progeny phages to adjacent cells, killing them as well (Fig. 6.32). In contrast to the clear plaques produced by lytic phages (as shown in the figure), temperate (lysogenic) phages make cloudy plaques containing lysogenized viable cells. The prophage in the host genome protects the cells from subsequent infection and lysis by another phage. Plaques offer a convenient way to isolate a recombinant DNA molecule contained in a bacteriophage vector. In Figure 6.32B, the blue plaques result from the phage vector, a derivative of phage M13 that carries a gene encoding the enzyme β-galactosidase. This enzyme converts a colorless compound into a blue dye. When the indicator gene is interrupted by an inserted recombinant gene, the phage produces white plaques, which indicate
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E. coli in rich broth culture
Lambda phage stock 2. Add phage-infected bacteria to molten top agar. Molten top agar
Plaque Isolation and Assay of Animal Viruses
3. Pour immediately onto the agar plate.
Multiplicity of infection MOI = 0.1
50°C H2O bath
4. Rotate to spread evenly. Agar solidifies.
Bottom (broth) agar plate 5. Incubate at 37°C overnight. Bacterial lawn
Soft agar (0.75%) Bottom agar (1.5%) Each plaque contains about 106 phage from one parent.
Plating a phage suspension to count isolated plaques. A suspension of bacteria in rich broth culture is inoculated with a low proportion of phage particles (multiplicity of infection is approximately 0.1). This means that only a few of the bacteria become infected immediately, while the rest continue to grow. Each plaque arises from a single infected bacterium that bursts, its phage particles diffusing to infect neighboring cells.
Figure 6.31
A.
B. Plaques
GNU
For animal viruses, the plaque assay has to be modified because it requires infection of cells in tissue culture. Tissue culture usually involves growth of cells in a monolayer on the surface of a dish containing fluid medium, which would quickly disperse any viruses released by lysed cells. To solve this problem, in 1952 Renato Dulbecco, at the California Institute of Technology, modified the tissue culture procedure for plaque assays (Fig. 6.33A). In Dulbecco’s method, the tissue culture with liquid medium is first inoculated with virus. After sufficient time to allow for viral attachment to cells, the fluid is removed and replaced by a gel medium. The gel retards dispersal of viruses from infected cells, and as the host cells die, plaques can be observed. Figure 6.33B shows a plate culture of adenovirus. Animal viruses that do not kill their host cells require a different kind of assay based on identification of a focus (plural, foci), a group of cells infected by the virus. In a fluorescent-focus assay, the infected cells under the gel overlay are incubated for a sufficient period to allow production of progeny virions. The plasma membranes of the cells are then made permeable by treatment with an organic solvent, and an antiviral antibody is added. Unattached antibodies are then washed away, and a second antibody is added that recognizes the fi rst antibody molecule. The second antibody is conjugated to a fluorophore whose fluorescence reveals
The M ic ro b i al Ce l l
1. Add phages to bacteria.
Blue plaques
Nick Bowlby, Michigan State University
the successful production of recombinant DNA phages. Plaques can be counted and used to calculate the concentration of phage particles, or plaque-forming units (PFUs), in a given suspension of liquid culture. The liquid culture can be analyzed by serial dilution in the same way one would analyze a suspension of bacteria.
■
White plaques
Phage plaques on a lawn of bacteria. A. Phage lambda plaques on a lawn of Escherichia coli K-12. B. Plaques of recombinant phage M13 on E. coli. The original phage expresses β-galactosidase, an enzyme that makes a blue product (blue plaques). White plaques are produced by phage particles whose genome is recombinant (contains a cloned gene interrupting the gene for β-galactosidase).
Figure 6.32
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C h ap t e r 6
■ Vir u s S tr u c tu r e and Func t ion
A.
A. 1. Infect monolayer with virus.
2. Remove liquid medium.
Jane Flint
3. Add gelatin medium.
4. Virus reproduces. Host cells lyse, forming plaques.
Courtesy of Jane Flint, Princeton University
B.
Plate culture of animal viruses. A. Modified plaque assay for animal viruses. The gelled medium retards dispersal of progeny virions from infected cells, restricting new infections to neighbor cells. The result is a visible clearing of cells (a plaque) in the monolayer. B. Plaque assay in which a serial dilution of adenovirus suspension was plated on a monolayer of cells in tissue culture.
100 µm
Focus assays of animal viruses. A. GFP fluorescent-focus assay. B. Transformed-focus (cancer-forming) assay of an oncogenic virus. Cells infected by an oncogenic virus have produced three foci of transformed cells. Transformed cells grow in an uncontrolled manner, which can produce a tumor.
Figure 6.34
TO SU M MAR I Z E: ■
Figure 6.33
foci of cells, each of which has arisen from a single virion in the original inoculum. Another kind of fluorescent-focus assay uses green fluorescent protein (GFP). The viruses are genetically engineered to contain a GFP gene fusion, a labeling technique discussed in Chapter 3. The GFP fluorescence reveals foci of virus-infected cells (Fig. 6.34A). A focus assay can also be used to isolate oncogenic viruses. Oncogenic, or cancer-causing, viruses actually transform their host cells into cancer cells. Cancer cells lose contact inhibition; they grow up in piles instead of remaining in the normal monolayer. These piles of transformed cells, or transformed foci, can easily be visualized and counted. This is known as the transformed-focus assay (Fig. 6.34B).
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H. Kisanuki, et al. 2005. European Journal of Cancer 41:2170
B.
■ ■
■
■
Culturing viruses requires growth in host cells. Bacteriophages may be cultured either in batch culture or as isolated plaques on a bacterial lawn. Batch culture of viruses generates a step curve. Animal viruses are cultured within animals or as plaques in a tissue culture. Fluorescent-focus assays reveal foci of virusinfected cells. Oncogenic viruses are cultured as foci of cancertransformed host cells.
6.7
Viral Ecology
Viruses exist naturally within host organisms in complex ecosystems. Viral persistence in natural ecosystems is significant for human health and for agricultural plants and animals. Persistence in wild populations provides a reservoir of infection that hinders eradication of a pathogen, especially when it is associated with an insect vector (carrier organism) such as ticks or mosquitoes.
Environmental Change Leads to Emergence of Viral Pathogens Changing distribution patterns of insect vectors and animal hosts can generate new epidemics of a pathogen in
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Virus
Packaged capsid
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B.
regions where the virus could not spread before. Such changes in distribution can be brought about by many factors, including global climate change (Special Topic 6.1). Some emerging viruses arise as variants of endemic milder pathogens. Viruses long associated with a host, such as the common cold viruses (rhinoviruses), tend to have evolved a moderate disease state that provides ample opportunities for host transmission. A virus that “jumps” from an animal host, however, may cause a more acute syndrome with higher mortality. The best-known case is the exceptionally virulent emerging strains of influenza, which generally result from intracellular recombination of human strains with strains from pigs or ducks (discussed in Chapter 11). Wild animals consumed for food introduce entirely new species of viruses to human populations. For example, HIV is believed to have emerged from apes whose meat was consumed by humans.
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Viruses infect algae. A. A virus attaches to the surface of a marine phytoplankton, Emiliana huxleyi (SEM). B. Progeny virions assembling within the phytoplankton Pavlova vivescens.
Figure 6.35
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250 nm
Society for General Microbiology, Reading, UK
A.
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in diversity is exemplified in marine phytoplankton, which are highly susceptible to viruses and display an exceptional range of species diversity. In the oceans, viruses are the most numerous and genetically diverse forms of life. The number of bacteriophages and algal viruses can reach 107 (10 million) per milliliter. Figure 6.35 shows examples of marine viruses infecting phytoplankton such as the algae Emiliana huxleyi and Pavlova vivescens. When marine algae overgrow, generating an algal bloom, viruses play a decisive role in controlling the bloom, such as the overgrowth of Emiliana huxleyi (Figure 6.36). Consumer organisms apparently cannot grow fast enough to control such blooms, but Plymouth
Viruses Have Important Roles in Ecosystems
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Limiting host population density. An increase in host population density increases the rate of transmission of viral pathogens. As the host population declines, natural selection results in individuals that are less susceptible. Thus, viruses can limit host density without extinction of the host. An example is the myxovirus introduced into Australia to curb the population of rabbits (which had been previously introduced by British colonists). The myxovirus ultimately maintained the rabbits at a reduced density, less destructive to the ecosystem. Selecting for host diversity. Each viral species has a limited host range and requires a critical population density to sustain the chain of infection. The virus limits its host species to a population density far lower than that sustainable by the available resources, thus preventing the dominance of any one species. In this way, the presence of viruses fosters the evolution of many distinct host species. The role of viruses
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Society for General Microbiology, Reading, UK
Viruses fi ll important niches in all ecosystems. Two important roles of viruses are:
Viral control of marine algal blooms. Phytoplankton bloom of E. huxleyi in the English Channel, satellite image, July 30, 1999. Algal populations are generally controlled by viruses. Source: From the Plymouth Marine Laboratory Remote Sensing Group,
Figure 6.36
based on data from the NERC Dundee Satellite Receiving Station.
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■ Vir u s S tr u c tu r e and Func t ion
Special Topic 6.1
West Nile Virus, an Emerging Pathogen
In the spring of 1999, New Yorkers noticed an unusual number of dead crows and other local birds. Then people began falling ill with an unfamiliar form of encephalitis (brain inflammation) caused by an avian virus never before seen in North America. The virus, a (+) single-stranded RNA virus, comes from the flavivirus family, which includes yellow fever and dengue (breakbone fever). These pathogens are all endemic to warmer environments, such as the Mediterranean and North Africa— hence the name, West Nile virus. Like yellow fever, a disease made infamous by its devastation of the southern United States in the nineteenth century, West Nile virus is transmitted between humans and animals by mosquitoes (Fig. 1). Why the sudden appearance of West Nile virus in the United States? The answer is not known for certain, but some climatologists argue that West Nile and other emerging pathogens arise from unusual weather patterns related to global climate change (Fig. 2). West Nile virus is generally found in species of mosquitoes that fare poorly in the cold of winter. In 1998–1999, however, the unusually warm winter enabled a few mosquitoes carrying the virus to survive until the spring. The spring that followed was drier than usual, forcing birds to spend more time at dwindling water pools, where mosquitoes were concentrated. The July heat wave then increased the rate of viral replication in the mosquitoes, which reached unusual population densities later that summer. As the cycle continued, mosquitoes reinfected birds, which then infected more mosquitoes. Eventually, the cycle included mosquitoes infecting humans. As of this writing, West Nile virus has spread to most of the United States and appears to be endemic to North America. Meanwhile, research continues to determine whether clearer links can be established between climate changes and emerging pathogens.
viruses spread rapidly through the population. By lysing the algae as they grow, viruses return algal carbon and minerals to the surface water before the algae starve to death and their bodies sink to less productive depths. On a global scale, marine viruses play a significant role in the carbon balance. Models of ocean carbon flux typically emphasize the consumption of phytoplankton by grazers and the consumption of grazers by carnivores
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C.
NS2a C
M
E NS1
NS4a NS3
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West Nile virus. A. The mosquito carries West Nile virus. B. West Nile virus particles (50 nm in diameter) forming within an infected cell (TEM). C. Like hepatitis C, West Nile virus is a flavivirus, with a (+) strand RNA of 10,000 nucleotides. The genome encodes ten proteins, three of which are structural proteins (C, M, and E) and seven of which are nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5).
Figure 1
(Fig. 6.37). At each level, some of the carbon fi xed as biomass sinks to the ocean floor, removed from the carbon cycle. But at each level, viral infection and lysis converts the bodies of phytoplankton, grazers, and carnivores into detritus consisting of small organic particles. The organic material is consumed by bacteria, whose rapid respiration releases much CO2. Thus, carbon is diverted from the ocean sink into CO2 returning to the atmo-
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More mosquitoes than usual survived the winter in sewers, damp basements and other sources of still water.
1. Mild winter
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Mosquito population flourished in spring and summer.
2. Dry spring and summer
Lack of rain killed predators of mosquitoes (frogs, lacewings, ladybugs). Many birds congregated at dwindling water sources.
As water in breeding sites evaporated, organic compounds became concentrated, nourishing mosquito larvae.
Mosquito population grew large.
3. July heat wave Once mosquitoes infected with the West Nile virus appeared, the heat caused the virus to proliferate rapidly inside them.
Drenching August rains Downpours formed new breeding sites for mosquitoes, yielding a new crop of the insects.
4. Vicious cycle began.
Infected mosquitoes transmitted the virus to initially uninfected birds.
Infection spread to people. As more and more mosquitoes become infected, they spread the virus to still more birds and ultimately to people.
Initially uninfected mosquitoes picked up the virus from infected birds.
An emerging pathogen. In the changing global climate, an emerging pathogen threatens wildlife and people. West Nile virus, originally found in Africa and the Mediterranean, is now spreading through the Western Hemisphere, probably owing to an increase in warm weather. The spread of the virus involves transfer between mosquitoes and avian, animal, or human hosts.
Figure 2
Source: Adapted from P. Epstein. 2000. Scientific American, August: 36–43.
sphere—a factor that must be considered in models of global warming. TO SU M MAR I Z E: ■
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Environmental change results in new emerging viral pathogens. Host population density is limited by virus infection, and host genetic diversity is increased.
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Marine viruses are the major consumers of phytoplankton. Viral activity substatially impacts the global carbon balance.
Concluding Thoughts In this chapter, we have covered general approaches to studying the structure and function of all kinds of
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■ Vir u s S tr u c tu r e and Func t ion
Marine viruses recycle carbon. Viruses divert the flow of carbon from marine phytoplankton and bacteria away from consumers and into organic particles (detritus). The organic particles are consumed by bacteria that respire CO2.
Figure 6.37
Grazers Carnivores Phytoplankton
Viral shunt Viruses catalyze the movement of nutrients from organisms to organic particles and minerals.
Organic particles
Virus infection returns more carbon as CO2 to atmosphere.
Uninfected cells sink; carbon is removed from cycle.
Heterotrophic bacteria
CO2
viruses. As devastating as viruses can be to their hosts, they also provide engines of genomic change (see Chapter 9) and ecological balance (see Chapter 21). Understanding virus function prepares us to discuss microbial genetics in Chapters 7–10. Chapter 11 then explores in
depth the molecular basis of viral genomes and reproduction. Molecular virology is a field of growing importance for medical and agricultural research, for genetic engineering and gene therapy, and for understanding natural ecosystems.
CHAPTE R R EVI EW Review Questions 1. Compare and contrast the form of icosahedral and fi l-
7. Explain the plate titer procedure for enumerating
amentous (helical) viruses, citing specific examples. How do viral genomes gain entry into cells in bacteria, plants, and animals? Explain the structure and function of the seven Baltimore groups of viral genomes. How do viral genomes interact with host genomes, and what are the consequences for host evolution? Compare and contrast the lytic, lysogenic, and slowrelease life cycles of bacteriophages. What are the strengths and limitations of each? Compare and contrast the life cycles of RNA viruses and DNA viruses in animal hosts. What are the strengths and limitations of each?
viable bacteriophages. How must this procedure be modified to titer animal viruses? Oncogenic viruses? 8. Explain the generation of the step curve of virus proliferation. Why is virus proliferation generally observed as a single step, or generation, in contrast to the life cycles of cellular microbes, outlined in Chapter 4? 9. Explain the key contributions of viruses to natural ecosystems. What may happen in an ecosystem where viruses are absent or fail to cause significant infection?
2. 3. 4. 5.
6.
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Key Terms accessory protein (189) bacteriophage (phage) (183) burst (200) burst size (200, 209) capsid (183) cell surface receptor (198) cloning vector (184) DNA reverse-transcribing virus (193) double-stranded DNA virus (192) double-stranded RNA virus (192) eclipse period (209) endocytosis (203) envelope (187) filamentous virus (189) fluorescent-focus assay (211) focus (211) hammerhead ribozyme (185) host range (183) icosahedral (187) latent period (209) lentivirus (210) lysate (209) lysogeny (200)
mechanical transmission (206) (–) sense single-stranded RNA virus (192) movement protein (207) oncogene (206) oncogenic virus (206) ortholog (196) orthologous gene (196) pararetrovirus (193) plaque (183) plaque assay (210) plaque-forming unit (PFU) (211) plasmodesma (207) (+) sense single-stranded RNA virus (192) prion (185) prophage (200) proteome (197) proteomics (197) reading frame (184) retrovirus (192) reverse transcriptase (192) ribozyme (185)
rise period (209) RNA reverse-transcribing virus (192) RNA-dependent RNA polymerase (192) segmented genome (191) single-stranded DNA virus (192) site-specific recombination (200) slow-release cycle (200) slow virus (210) spike protein (189) tegument (189) temperate phage (200) transduction (200) transform (212) transformed-focus assay (212) tropism (201) uncoating (203) viral envelope (189) virion (182) viroid (185) virulence (209) virulent (200) virus (182)
Recommended Reading Baltimore, David. 1971. Expression of animal virus genomes. Bacteriology Reviews 35:235–241. Edwards, Robert A., and Forest Rohwer. 2005. Viral metagenomics. Nature Reviews Microbiology 3:504–510. Flint, S. Jane, Lynn W. Enquist, Vincent R. Racaniello, and A. M. Skalka. 2003. Principles of Virology: Molecular Biology, Pathogenesis, and Control of Animal Viruses. 2nd ed. ASM Press, Washington, DC. Gromeier, Matthias. 2002. Viruses for treating cancer. ASM News 68:438. Grünewald, Kay, Prashant Desai, Dennis C. Winkler, J. Bernard Heymann, David M. Belnap, et al. 2003. Threedimensional structure of Herpes Simplex Virus from cryoelectron tomography. Science 302:1396–1398. Hull, Roger. 2001. Matthews’ Plant Virology. Academic Press, New York.
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Moineau, Sylvain, et al. 2002. Phages of lactic acid bacteria: From genomics to industrial applications. ASM News 68:388–391. Ptashne, Mark. 2004. Genetic Switch: Phage Lambda Revisited. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Raoult, Didier, Stéphane Audic, Catherine Robert, Chantel Abergel, and Patricia Renesto. 2004. The 1.2-megabase genome sequence of Mimivirus, Science 306:1344–1350. Suttle, Curtis A. 2005. Viruses in the sea. Nature 437:356–361. Suttle, Curtis A. 2007. Marine viruses—major players in the global ecosystem. Nature Reviews Microbiology 5:801–812. Wilson, Willie. 2002. Giant algal viruses: Lubricating the great engines of planetary control. Microbiology Today 29(November):180.
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Part 2
Genes and Genomes AN INTERVIEW WITH Courtesy of Richard Losick
RICHARD LOSICK: THE THRILL OF DISCOVERY IN MOLECULAR MICROBIOLOGY
Richard Losick has taught at Harvard for more than three decades, and his creative teaching was honored when he was named a Howard Hughes Medical Institute (HHMI) Professor. A member of the National Academy of Sciences, Losick has authored more than 200 research articles on bacterial genetics and cell biology. He discovered how Bacillus sporulation is controlled by alternative sigma factors that regulate transcription. To dissect sporulation and the cell division cycle, he uses fluo-
Richard Losick, Harvard College Professor. Losick has made major discoveries on the molecular and cellular biology of Bacillus subtilis.
rescent proteins to visualize individual subcellular parts of bacteria.
Why did you decide to make a career in microbiology? Did you begin research as an undergraduate?
When I was in grade school, I used to read science books and do experiments on my own. I thought that school was boring, and I didn’t realize that science was a subject that I would learn about later in school! When I was an undergraduate at Princeton, I joined the laboratory of a newly arrived professor, Charles Gilvarg, who studied amino acid biosynthesis in bacteria. I fell in love with bacteria from that experience, and I have remained fascinated by bacteria ever since. Why do you study Bacillus subtilis? How is your work relevant to the insecticide B. thuringiensis and the human pathogen B. anthracis?
Bacillus subtilis is extraordinarily rich in its biology. Its ability to metamorphose into a spore is endlessly fascinating. B. subtilis has the capacity to differentiate into specialized cells for DNA uptake (competence), to swim, to swarm on surfaces, to cannibalize sibling cells, and to form architecturally elaborate, multicellular communities. At the same time, its property of genetic competence (gene transfer between
cells) makes it exceptionally facile for molecular genetic manipulation. B. thuringiensis and B. anthracis are less favorable than B. subtilis for genetic manipulation, but the main features of spore formation are very similar in all three organisms. The crystal toxin of B. thuringiensis that has useful insecticidal properties is produced under sporulation control. The remarkable environmental resistance properties of B. subtilis spores are shared with its evil cousin B. anthracis and help us understand the challenge posed by the bioterrorism agent. Your career has spanned a generation of revolutionary developments in genetics and molecular biology, such as the discovery of the sigma factors that regulate transcription of many genes. Would you describe how one of these developments came about?
After winning my PhD from MIT, I came to Harvard to work on a project involving the effect of a virus on bacterial cell membranes. The sigma subunit of E. coli RNA polymerase had just been discovered (by Dick Burgess and Andrew Travers at Harvard and by John Dunn and Ekkehard Bautz at Rutgers), and people were excited about the possible exis-
tence of alternative sigma factors (proteins that regulate large groups of genes). Meanwhile, A. L. Sonenshein was studying how certain bacterial viruses (bacteriophages) get trapped during spore formation by Bacillus subtilis. The phage is virulent but is shut off transcriptionally upon entering a sporulating cell. The phages are unable to grow in cells that have started to sporulate and instead become trapped in what will become the spore. So the poor spore, after waiting for long periods of time to germinate, gets killed upon germination by the enemy within. Putting two and two together, we reasoned that maybe changes in the RNA polymerase during entry into sporulation could help explain the shutdown in viral gene expression. We got so excited about this idea that we began to work on RNA polymerase full time. This eventually led to the discovery that sporulation is governed by a cascade of bacterial sigma factors. Meanwhile, in collaboration with Jan Pero, we found that phage SP01 encodes and produces two alternative sigma factors upon infecting growing cells. These regulatory proteins, the first examples of alternative sigmas, drive the phage program of gene transcription. Since then, alternative sigmas have emerged as a widespread
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Courtesy of Richard Losick
Sporulating cells of Bacillus subtilis with molecular parts labeled by fluorophores. Red fluorescence (FM4-64) labels the cell membrane; blue (DAPI) the DNA; and green (green fluorescent protein) a DNA pump that translocates a chromosome across the division septum from the mother cell (the big cell) into the forespore (the small cell on the upper right). (Cell length, 2–3 µm.)
theme in the control of gene sets in many different kinds of bacteria. What is the significance of sporulation as a developmental process?
Sporulation is an extremely attractive system for understanding cellular differentiation because it is both complicated enough to mimic features of development in higher organisms and accessible enough to be subject to genetic, biochemical, and cytological manipulation. Sporulating cells undergo a true process of cellular differentiation, in which the two cellular compartments of the sporangium communicate with each other in a back-and-forth manner by intricate signaling systems.
cent protein (GFP) as a tool for visualizing the subcellular localization of proteins in cells. Andrew Murray and Andrew Belmont figured out that GFP could also be harnessed to visualize the location of sites on DNA by attaching the fluorescent protein to a protein that binds to specific sequences in DNA. Murray and Belmont used this trick to visualize centromeres in yeast. Andrew Wright and I extended this methodology to visualizing sites on chromosomes in bacteria. Traditional thinking was that origins tended to remain in cell middles, but we found that replication origins moved toward the cell poles during chromosome segregation, and with GFP we could visualize this in living cells. How do students get involved with your research? Do you work with undergraduates as well as graduate students?
Yes, I always have undergraduates as well as graduate students in my lab, and both have contributed to some of our most significant fi ndings. One special undergraduate was Aurelio Teleman, who was both a brilliant student and a gifted investigator. Working with a graduate student, Teleman helped create and exploit the system for visualizing the movement of a specific site in the bacterial chromosome that had been tagged with tandem lactose operon operators. He is now at the European Molecular Biology Laboratory.
As an educator, what innovations have you developed?
I enjoy the challenge of explaining complicated concepts and of making large-class instruction lively and interactive. I run a program for placing freshmen from disadvantaged backgrounds in laboratories of faculty for long-term research mentoring. Such students are often at greatest risk for dropping out of science, but a longterm relationship with a laboratory that begins early during the college years can keep these students (indeed all students) excited about science throughout college and beyond. I believe that a positive experience in experimental work can represent one of the most memorable aspects of undergraduate education. And its benefit is not limited to future scientists. A science major with an associated experience in discovery research is excellent preparation for a wide variety of careers, including medicine, business, law, public policy, journalism, and education. What advice do you have for today’s students?
Join a lab! In science you can learn something that nobody else on Earth ever knew before. There’s a special thrill about learning how living things function—the inner workings of living things. If you learn some new aspect of it, even if it’s a tiny part, you’ve advanced knowledge for the whole of humankind.
In the old days, bacteria were viewed as an amorphous vessel with enzymes floating around inside. Now we understand that bacteria are highly organized, that proteins often have subcellular addresses in bacteria; thus, cytology has become an important aspect of bacterial research. One of the keys to this revolution was the introduction of green fluores-
Photo by Kris Snibbe
How was the technology developed to visualize chromosome dynamics in Bacillus?
Losick teaching undergraduates at Harvard. Harvard Gazette staff photo by Kris Snibbe.
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Chapter 7
Genomes and Chromosomes 7.1 7.2 7.3 7.4 7.5 7.6
DNA: The Genetic Material Genome Organization DNA Replication Plasmids and Bacteriophages Eukaryotic Chromosomes: Comparison with Prokaryotes DNA Sequence Analysis
A genome is all the genetic information that defines a species. We have known that chromosomes consist of DNA for over a hundred years, yet we have only recently been able to sequence complete genomes. Now we are in the postgenomic era in which biological research is driven, in large part, by knowledge of complete genome sequences. Microbial genomes consist of one or more chromosomes made of DNA. Many bacteria have a single, circular chromosome, but some species have multiple chromosomes or even a mixture of linear and circular chromosomes. For example, the genome of Sinorhizobium melliloti, a major contributor to nitrogen fixation, consists of three circular chromosomes. This chapter addresses basic as well as some advanced issues of DNA and genomes. What is DNA? How is it packaged in the cell? Because DNA is essential to life, nature must have devised a remarkable machine to replicate it. But how does this machine work? And do different species use different machines? Microbiology has played a leading role in answering these difficult questions−opening the door to post-genomic studies of all living things.
90°
90°
All cells accurately time the initiation of chromosome replication to cell division. In bacteria, DnaA protein accomplishes this by accumulating during growth and binding to the origin of replication, forming a right-handed helical filament. The figure shows four DnaA tetramers bound to origin DNA. On the left, the initial monomer of is colored grey to distinguish a single subunit of the filament. Gold, green and red mark different domains of subsequent monomers. The image on the right uses different colors to show individual monomers. The torsional strain caused by the filament may destabilize the adjacent DNA to allow assembly of the replication machine. Source: Melissa L. Mott and James M. Berger. 2007. Nature Reviews Microbiology 5:343–354.
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Gopal Murti/Visuals Unlimited
What makes each species unique is its genome, the sum total of its genes. The human genome is also supplemented by the genomes of all the microbes in the digestive tract. Collectively, intestinal microbes contain 100 times more genes than the human genome and provide key metabolic capabilities that humans lack, such as digesting complex plant materials. In the mid-twentieth century, one intestinal bacterium, Escherichia coli, became the focus of efforts to understand genes and genetics. The choice of E. coli was fortunate, as its genetics proved to be especially pliable. In one remarkable experiment, a cell of E. coli was lysed, releasing its chromosome for electron microscopy. What spewed out of this single cell was a strand of DNA 1,500 times longer than E. coli itself. Scientists wondered how this enormous molecule could fit into a single cell, and how an enzyme could duplicate that much DNA (over 4.6 million base pairs) without the whole DNA molecule getting tangled up (Fig. 7.1). And how did it manage to do this in under 20 minutes, the doubling time of the organism? Half a century later, those questions continue to intrigue us. Chapter 7 explores the nature of DNA, the structure of prokaryotic genomes, and the mechanisms used for their replication. We also examine how microbial enzymes that manipulate DNA were used to develop the fundamental techniques of present-day biotechnology, such as DNA restriction analysis, DNA sequencing, and the polymerase chain reaction (PCR). The expression of genes to make RNA and protein is discussed in Chapter 8. The remaining chapters of Part 2 present gene recombination and transmission (Chapter 9), the regulation of gene expres-
Osmotically disrupted bacterial cell with its DNA released.
Figure 7.1
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sion (Chapter 10), the specialized genetic mechanisms of viruses (Chapter 11), and the use of microbial genetic tools for research and biotechnology (Chapter 12). From the advances of the last 50 years, a new molecular and genomic perspective of biology has emerged.
7.1
DNA: The Genetic Material
The ability of plants and animals to transfer genetic traits was known well before we knew that DNA was the carrier of genetic information. Early researchers understood the Mendelian model of gene inheritance and how it dictated the passage of traits from parent to offspring in eukaryotes. This type of gene transfer is known as vertical transmission, as from parent to child. Bacteria, however, appeared to operate differently, because in addition to vertical transmission, they were able to conduct horizontal transmission—the transfer of small pieces of genetic information from one cell into another. The study of horizontal gene transfer in bacteria ultimately led to the discovery of horizontal transfer throughout animals and plants, albeit on a slower time scale because of their longer generation times. Mechanisms of horizontal transmission are discussed in Chapter 9. Our path to understanding DNA began in 1928 with the discovery by Fred Griffith (1881–1941) that some unknown substance from dead cells of a virulent strain of Streptococcus pneumoniae could transfer virulence to living but otherwise harmless strains when both strains were coinjected into mice. This form of horizontal gene transfer, called transformation, led to the discovery in 1944 that genetic information is embedded in the base sequence of deoxyribonucleic acid (DNA) (see Section 1.6). A more contemporary example of transformation is the toxic shock gene that can be moved horizontally among strains of Staphylococcus aureus. As a result of these and many other experiments, we now appreciate that chromosomes are made of contiguous packets of information, called genes. Genes are units of information composed of a sequence of DNA nucleotides of four different types, adenine (A), guanine (G), thymine (T), and cytosine (C). (For review at the level of introductory biology, see Appendix 1.) A structural gene is a string of nucleotides that can be decoded by an enzyme to produce a functional RNA molecule. A structural gene usually produces an RNA molecule that in turn encodes a protein. A DNA control sequence, on the other hand, regulates the expression of a structural gene. DNA control sequences do not encode RNA but regulate the expression (RNA production) of an adjacent structural gene, for example, by binding a repressor protein (discussed in Chapter 8). DNA control sequences are not really genes, since they do not encode RNA or protein. Control sequences include the promoters that signal the start of RNA synthesis from a structural
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Pa r t 2
gene and the binding sites for regulatory proteins that can activate or inactivate that promoter. As noted previously, the entire genetic complement of DNA in a cell that defi nes it as a species is called its genome. Our primary goal in this chapter is to convey what is known about how genomes are maintained and replicated. Information about how computers are used to mine the informational content of DNA sequences and genes is described more fully in Chapters 8 and 10.
Bacterial Genomes Can Be Circular or Linear In the early twentieth century, the chromosomes of bacteria, unlike those of eukaryotes, could not be observed by light microscopy. The reason for this is that bacteria, unlike eukaryotes, do not undergo mitosis, a process in which chromosomes are condensed and thickened about 1,000-fold. Important clues to bacterial chromosome structure were, however, gleaned from painstaking genetic studies. In the 1950s, it was discovered that conjugation, a horizontal gene transfer mechanism requiring cell-tocell contact, could transfer large segments of the bacterial chromosome—not all at once, as in the established Mendelian model of plants and animals, but sequentially over a period of time (it takes 100 minutes to move the entire E. coli chromosome from one cell to another). Thus, even though the bacterial chromosome could not be seen, conjugation allowed genes to be mapped relative to each other based on time of transfer. For example, a donor strain that can synthesize the amino acids alanine and proline can directly transfer the responsible genes to a recipient cell defective in those genes. The transfer process is nonspecific; any gene can be transferred in this way. Completion of transfer also requires recombination, in which the donor DNA fragment replaces the recipient DNA fragment. Successful transfer of the genes for amino acid synthesis enables the formerly defective recipient to form colonies on minimal media lacking either amino acid. But because transfer occurs over time from a fi xed starting point (that is, not all genes are transferred at the same time), it might take one gene 10 minutes of cell contact to be transferred while the second gene, farther away from the starting point of DNA transfer, takes an additional 20 minutes. Knowing that eukaryotic chromosomes are linear, scientists initially expected that bacterial chromosomes would be linear, too. However, the early genetic maps drawn from conjugation experiments just would not fit together in a manner consistent with a linear model— for the simple reason that the bacterial chromosome in E. coli, the organism under study at the time, is circular. We now know that many microbes have circular chromosomes, although some, like the Lyme disease agent Borrelia burgdorferi, have linear chromosomes and others,
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like Agrobacterium tumefaciens, have a mixture of circular and linear chromosomes (Table 7.1). It is instructive to compare genome sizes across the phylogenetic tree, from viruses to humans. The size range is enormous. In general, the simpler the organism, the smaller its genome. At some point, as DNA content is trimmed, the organism loses independence and must parasitize another organism. This begs the question, what constitutes a “minimal” genome? How “lean” can a chromosome become and still support independent growth? TO SU M MAR I Z E: ■
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A genome is all the genetic information that defi nes a species. Prokaryotic genomes are made up of chromosomes and plasmids made of DNA. Prokaryotic chromosomes can be circular or linear, as can plasmids. Functional units of DNA sequences include structural genes and regulatory sequences.
7.2
Genome Organization
New techniques for constructing physical maps of genomes and determining the sequences of whole genomes have revealed tremendous diversity in the size and organization of prokaryotic genomes.
Genomes Vary in Size Bacterial chromosomes range from 580 kilobase pairs (kbp) to 9,400 kbp, and archaeal chromosomes range from 935 kbp to 6,500 kbp. For comparison, eukaryotic chromosomes range from 2,900 kbp (microsporidia) to over 4,000,000 kbp (humans). NOTE: The designation kbp refers to the length of a double-stranded DNA molecule. A 1,000-base-pair double-stranded genome is 1 kbp in size. In contrast, a 1,000-base single-stranded genome (as in some viruses) is referred to as 1 kb.
The smallest cellular genomes identified thus far are those of Mycoplasma. These pathogens rely on their host environment for many products but can still grow outside a host cell. The complete genome of Mycoplasma genitalium consists of only 580 kbp and encodes 480 proteins. M. genitalium lacks the genes required for many biosynthetic functions. These organisms cannot synthesize amino acids or construct cell walls, and they do not have a functional tricarboxylic acid (TCA) cycle. In contrast, free-living bacteria that can grow in soil have larger genomes and dedicate many genes
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Chapter 7
Table 7.1
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Genomes of representative bacteria and archaea.
Species (strain)
Genomic chromosome(s)* (kilobase pairs, kbp)
Plasmid(s)* (kbp)
Total (kbp)
Bacteria Mycobacterium tuberculosis Tuberculosis
4,400
4,400
Mycoplasma genitalium Normal flora, human skin
580
580
Burkholderia cepacia
3,870 + 3,217 + 876
93
8,056
Escherichia coli K-12 (W3110) Model strain for E. coli proteomics
4,600
Anabaena species (PCC 7120) Cyanobacteria: Major photosynthetic producer of carbon source for aquatic ecosystems
6,370
110 + 190 + 410
7,080
911
21 plasmids with sizes between 9–58
>1,250
2,840 + 2,070
214 + 542
5,666
1,660
16 + 58
1,734
3,130 + 288
33 + 33 + 39 + 50 + 155 + 132 + 410
4,270
1,000 kbp
500 kbp
Borrelia burgdorferi Lyme disease Agrobacterium tumefaciens Tumors in plants; genetic engineering vector
4,600
Archaea Methanocaldococcus jannaschii Methanogen from thermal vent Haloarcula marismortui Halophile from volcanic vent
*Colored schematics indicate relative sizes of genomic elements and whether these are circular or linear. Size bars are provided under each column.
to the synthesis or acquisition of amino acids or TCA cycle intermediates. Even different strains of one species, such as Salmonella enterica, may vary considerably in gene distribution (Fig. 7.2). Another feature that distinguishes prokaryotic and eukaryotic genomes is the amount of so-called noncoding DNA. Many, but not all, eukaryotes contain huge amounts of noncoding DNA scattered between genes. In some species (such as humans), over 90% of the total DNA is noncoding. These noncoding regions include
221-256_SFMB_ch07.indd 224
enhancer sequences needed to drive transcription of eukaryotic promoters and DNA expanses that separate enhancers. Enhancer sequences can function at large distances from the gene they regulate. A promoter is the DNA sequence immediately in front of a gene that is needed to activate the gene’s expression. Most of the noncoding spacers appear to be remnants of genes lost over the course of evolution and pieces of defunct viral genomes. Noncoding regions may, however, provide raw material for future evolution.
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The genome of Salmonella enterica serovar Typhimurium LT2. The circular chromosome. Base pairs are indicated around the perimeter. The two outer multicolored circles indicate genes encoded on the separate strands, which means they are transcribed in opposite directions. Color codings represent homologies between eight compared species. Blue indicates genes present only in S. typhimurium LT2. Orange indicates that the gene has a close homolog in all eight genomes compared. Green indicates genes with a close homolog in at least one other Salmonella (S. typhi, S. paratyphi A, S. paratyphi B, S. arizonae, or S. bongori) but not in E. coli K12, E. coli O157:H7, and Klebsiella pneumoniae. Gray indicates other combinations. The black inner circle is the GC content of the LT2 DNA (peaks pointing outward indicate GC-rich areas).
Figure 7.2
■ Genes and Ge n o m e s
225
0 4,500,000 bp
500,000 bp
4,000,000 bp
1,000,000 bp Genes on (+) strand Genes on (–) strand GC ratio
3,500,000 bp
1,500,000 bp
3,000,000 bp
2,000,000 bp 2,500,000 bp
Gene distribution key S. typhimurium-specific Salmonella-specific All eight genomes Others
In contrast to many eukaryotes, prokaryotes tend to have very little noncoding DNA (typically less than 15% of the genome). The completed genome sequence of the bacterium Mycobacterium leprae, however, has a substantial amount (about 50%) of DNA with no known or predicted function, and this noncoding DNA may represent degenerate or inactivated genes. Still, this is less than the percentage of noncoding DNA seen in most eukaryotes.
A. Operon Promoter
Genes Are Organized into Functional Units
B. Regulon
In the simplest case, a gene can stand alone, operating independently of other genes. The RNA produced from a stand-alone gene is said to be monocistronic, coding for one protein. Alternatively, a gene may exist in tandem with other genes in a unit called an operon (Fig. 7.3A). All genes in an operon are situated head to tail on the chromosome and are controlled by a single regulatory sequence located in front of the first gene. The single RNA molecule produced from the operon contains all the information from all the genes in that operon. On a functional level, a collection of genes and operons at multiple positions on the chromosome can hold membership in a regulon when they have a unified biochemical purpose (such as amino acid biosynthesis; Fig. 7.3B). The various mechanisms regulating expression of these functional genetic units are discussed in Chapter 10.
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Promoter Gene 1 Gene 2
Monocistronic RNA
Gene 3
Polycistronic RNA
Transcription start site Operon This gene is transcribed to form the regulatory protein.
Control protein
Operon
Figure 7.3 Gene organization. A. Diagram of a segment of DNA containing a single gene producing a monocistronic message (codes for one protein) and an operon of three genes producing a polycistronic message (from which three different proteins are made, one corresponding to each gene). B. Diagram of a circular bacterial genome, containing genes and operons coordinately controlled by a regulatory protein.
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226
■
Chapter 7
G e n o m e s a n d C hromosomes
DNA Function Depends on Chemical Structure
NOTE: In bacteria, the names of genes are given as
a three-letter abbreviation of the encoded enzyme’s name (for example, the gene dam encodes deoxyadenosine methylase) or the function of related genes (for example, genes designated proA, proB, and proC encode enzymes involved in proline biosynthesis). Bacterial gene names are written in italics with lowercase letters. For example, the genes involved in catabolizing lactose are the lac genes. If several genes are involved in the pathway, a fourth letter, capitalized, is used. Thus, the three genes lacZ, lacY, and lacA are all associated with lactose catabolism. When speaking of a gene product, a nonitalic (roman) font is used, and the fi rst letter is capitalized. Thus, lacZ is the gene, and LacZ is the protein product of that gene.
DNA is composed of four different nucleotides linked by a phosphodiester backbone (Fig. 7.4A). Each nucleotide consists of a nitrogenous base attached through a ring of nitrogen to carbon 1 of 2-deoxyribose in the phosphodiester backbone. The 2-deoxy position that distinguishes DNA from RNA is circled in the figure. A phosphodiester bond (marked) joins adjacent deoxyribose molecules in DNA to form the phosphodiester backbone. Phosphodiester bonds link the 3′ carbon of one ribose to the 5′ carbon of the next ribose. The two backbones are antiparallel so that at either end of the DNA molecule, one strand ends with a 3′ hydroxyl group and the opposite strand ends with a 5′ phosphate. This antiparallel arrangement is necessary so that complementary bases protruding from the two strands can pair properly via hydrogen bonding. Base pairing is not possible if one tries to model DNA strands in a parallel arrangement.
Microbial Genome Database
A. Chemical structure of DNA 5′ end
B. Chemical structure of RNA 5′ end
O –
O
O –
P O
–
O
H
P O O
–
O
H
CH2
2-Deoxyribose
H O
N
–
O
–
O
O
H
O
H CH2 O
N
H O
Phosphodiester bond
–
O
H
H N
H
N H CH2 O
O H2C O
N
O
P O O
N
CH2
5′
4′
H O
N
3′ 2′
O
1′
H
3′ end
–
O
221-256_SFMB_ch07.indd 226
Cytosine (C) O
N
O P H
H
O
O
O–
N
CH2 O
H N
H
H H
N O
O H2C
Cytosine (C)
H
Guanine (G)
Guanine (G)
N
N
H N
H N
OH
P O
O
H
N
H
O
N
3′ O
O O P
–
O–
O
OH O
P O
H
N
O O O P
O–
CH2
H O
N
H
Uracil (U) O
O O–
O
Structures of DNA and RNA.
N
H O
CH2
O P
Figure 7.4
H N
H
O
N N
OH H
P O
O
H
O
Adenine (A) H
O
O H C 2
O
H
O
O
N
N
H
–
N
H
N
N
H
–
O–
O P
H H
O
H
P O O
O
C
N
5′
2′ Hydroxyl
H
N
Ribose
N
N
N
H
N
H CH2 O
O–
O
H
O
H
N
H N
P O O
H N
H
N
H
N
O
O
O P H
P O
H2C
H
H
P O
O
O
O
5′
O
The 2-deoxy position that distinguishes DNA and RNA
N
N
–
H 2′ O 3′
N
N
H
N O
O
H
H
N
P O
H H
C
Adenine (A) O
P O
O
3′ end
Thymine (T)
O
O
OH
3′ end
5′ end
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The nitrogenous bases in DNA are planar heteroaromatic structures stacked perpendicular to the phosphodiester backbone and parallel to each other (see Fig. 7.4A). Purines (bicyclic bases, adenine or guanine) pair with pyrimidines (monocyclic bases, thymine or cytosine). Under physiological conditions of salt (about 0.85%) and pH (pH 7.8), the hydrogen bonding of the bases permits adenine to pair only with thymine (via two hydrogen bonds) and likewise guanine with cytosine (via three hydrogen bonds). These complementary base interactions enable the two phosphodiester backbones to wrap around each other to form the classic double helix, or duplex. The thousands of H-bonds that form between purines and pyrimidines along the interior of a DNA duplex (Fig. 7.5) make the bonding of the two complementary strands of DNA highly specific, so that a duplex is formed only between complementary strands.
O
Sugar Bases Pyrimidine (Cytosine or thymine) Purine (Adenine or guanine)
P
Phosphate group
P CH2
H2C O
C
H H
P G
H
O
P CH2
A
H
T O
P CH2
G
H H
O
T
H H
H2C
O
H2C
P A O
Progressive magnification of the DNA helix. The top half of the molecule illustrates the typical double-helix structure that becomes magnified in the lower half to show individual bases and hydrogen bonding.
Figure 7.5
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THOUGHT QUESTION 7.1 What do you think happens to two single-stranded DNA molecules isolated from different genes when they are mixed together at very high concentrations of salt? At high temperatures (50–90°C), the hydrogen bonds in DNA break, and the duplex falls apart, or denatures, into two single strands. The temperature required to denature a DNA molecule depends on the GC/AT ratio of a sequence. More energy is required to break the three hydrogen bonds of a GC base pair than the two bonds of an AT base pair. Thus, DNA with a high GC content requires a higher denaturing temperature than a similarsized DNA with a lower GC content. The black center ring in Figure 7.2 illustrates how the GC content of bases can change around a single chromosome. When DNA has been heated to the point of strand separation, lowering the temperature permits the two single strands to fi nd each other and reanneal into a stable double helix. The kinetics of DNA denaturation is much faster than that of renaturation, since the latter is a random, hit-or-miss process of complementary sequences fi nding each other. This melting/reannealing property of DNA is exploited in a number of molecular techniques (see the discussion of the polymerase chain reaction in Special Topic 7.3). Note, however, that bacteria and archaea growing at extreme pH or temperature protect their DNA from denaturation through the use of remarkable DNA-binding proteins. The role of DNAbinding proteins in microbial survival is the subject of considerable research. THOUGHT QUESTION 7.2 How do you think the kinetics of denaturation and renaturation are dependent on DNA concentration?
C
P CH2
Although H-bonds govern the specificity of strand pairing, the thermal stability of the helix is predominantly due to the stacking of the hydrophobic base pairs. Stacking creates interactions between these base pairs so that water and ions can interact with the negatively charged, hydrophilic phosphate backbone but not with the interior of the double helix.
P
H
O
227
P
H
O
H2C
■ Genes and G e n o m e s
Ribonucleic Acid (RNA) Differs Slightly from DNA As we learned in Chapter 3, the growing cell continually makes temporary copies of its genes in the form of RNA molecules that direct the synthesis of proteins. DNA and RNA are chemically similar, except that in RNA the sugar ribose replaces deoxyribose and the pyrimidine base uracil replaces thymine (Fig. 7.4B). Functionally,
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Chapter 7
A. Space-filled
Major groove
Minor groove
■
G e n o m e s a n d C hromosomes
groove and the narrow minor groove. The two grooves are generated by the angles at which the paired bases meet each other. These grooves provide DNA-binding proteins access to base sequences buried in the center of the molecule, so that proteins can interact with the bases without the strands being separated (Fig. 7.7).
B. Surface
Major groove
Bacterial Chromosomes Are Compacted into a Nucleoid
Minor groove
Models of DNA. A. Space-filling model of DNA. B. DNA surface modeled using nuclear magnetic resonance. (PDB code: 1K8J)
Figure 7.6
Cro protein
DNA Major groove
Protein recognizing DNA. Cro repressor protein binds DNA within the major groove. (PDB code: 6CRO)
The chromosome of E. coli has over 4.6 million bases in one strand, or over 9 million counting both strands. This is a huge molecule. In fact, this one molecule contributes greatly to the overall negative charge of the cytoplasm. This is because at the normal pH of the cell (pH 7.8), all the phosphates in the backbone (9 million) are unprotonated and negatively charged. You might wonder how the cell handles this macromolecule. In Figure 7.1, we see DNA spewing out of a damaged bacterial cell. Laid out, the chromosome is 1,500 times longer than the cell. It is obvious from this photomicrograph that an intact, healthy cell must compact a huge bundle of DNA into a very small volume. DNA is the second largest molecule in the cell (only peptidoglycan is larger) and comprises a large portion of a bacterial cell’s dry mass, about 3–4%. While packaging 3% of a cell’s dry weight may not seem like a challenge, realize that DNA is further confi ned only to ribosome-free areas of the cell, so the chromosome-packing density reaches about 15 mg/ml. In a test tube, DNA at 15 mg/ml is almost a gel, so how can anything move inside a cell? And how does all this DNA keep from getting hopelessly entangled? As introduced in Chapter 3, cells pack their DNA into a manageable form that still allows ready access to DNAbinding proteins. While bacteria lack a nuclear membrane, they pack their DNA into a series of protein-bound domains collectively called the nucleoid (see Section 3.5). The bacterial nucleoid is distributed throughout the cytoplasm, unlike the compact nucleus of eukaryotes.
Figure 7.7
these two differences prevent enzymes meant to work on DNA, such as DNA polymerases, from acting on RNA. However, uracil can still base-pair with adenine, which means that hybrid RNA-DNA double-stranded molecules (hybridization) can form when base sequences are complementary. In fact, hybridization is a necessary step in the decoding of genes to make proteins. Also, note that DNA in a cell is usually double-stranded, whereas RNA is usually single-stranded. As seen in the space-fi lling model of DNA in Figure 7.6A and the contour map in Figure 7.6B, the B form of the DNA double helix has grooves: the wider major
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DNA Supercoiling Compacts the Chromosome A nucleoid gently released from E. coli appears as 30–100 tightly wound loops (Fig. 7.8A). The boundaries of each loop are defi ned by anchoring proteins called histone-like proteins for their similarity to histones, the DNA-binding proteins of eukaryotes. The double helix within each domain is itself helical, or supercoiled. The easiest way to envision supercoiling is to picture a coiled telephone cord. After much use, a phone cord twists, or supercoils, upon itself. Note that supercoiled phone cords are quite compact, taking up less space than a relaxed cord. Circular DNA works the same way, a property used by the cell to pack its chromosome.
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Bacterial nucleoid. Nucleoid showing domain loops after gentle release from cells. The single-strand nick unwinds (relaxes) only one loop.
■ Genes and Ge n o m e s
229
Figure 7.8
Histone-like anchoring proteins
Remarkably, the supercoiling of one domain of DNA is maintained independently of other loops. The independence of supercoiled domains was demonstrated by introducing one single-strand nick in the phosphodiester backbone of one domain (see Fig. 7.8). You can do this by adding very small amounts of a nuclease (an enzyme that cleaves a nucleic acid). The ends of the broken strand, driven by the energy inherent in the supercoil, rotate about the unbroken complementary strand of the duplex and relax the supercoil. However, supercoils were removed from only the one domain. How is this possible if the chromosome is one circular molecule? The unaffected chromosomal domains remained supercoiled because they were constrained at their bases by anchoring proteins such as Hu and HNS (histone-like proteins) that prevent rotation. How does DNA achieve the supercoiled state? The bacterial cell produces enzymes that can twist DNA into supercoils and relieve supercoils. A single twist introduced into a 300-bp circular DNA molecule creates a single supercoil as shown in Figure 7.9. An enzyme makes a double-stranded break at one point in the circle, passes another part of the DNA through the break, and reseals it. This produces the same result as if one end of the broken circle were twisted one full turn. Twisting in the opposite direction of the helical turn tightens the helix by adding more turns (overwinding). Think of the phone cord again. Look down the length of the cord from one end. If the cord turns left to right (that is, clockwise) down its length, twist it from one end right to left (counterclockwise). This increases the number of twists. The resulting torsional stress of overwinding is relieved when the DNA (or phone cord) subsequently twists upon itself, introducing positive supercoils. In contrast, negative supercoils are formed if one end of a DNA molecule is turned in the same direction as the helix (thereby underwinding the DNA). In terms of the phone cord, if the cord naturally turns clockwise down its length, turn one end clockwise several more times. Torsional stress results in this case, as well, because the maneuver tries to decrease the number of turns in the helix. To maintain the same number of turns, the molecule must supercoil in the opposite direction and form negative supercoils that reduce the torsional strain. The nucleoids of bacteria and most archaea, as well as the nuclear DNA of eukaryotes, are
221-256_SFMB_ch07.indd 229
Nick made in single strand
Supercoiling relaxed in nicked strand
Break
Pass a section of DNA through the break, then reseal.
Figure 7.9 Supercoiling of 300-bp circular DNA. A supercoil can be introduced into a double-stranded circular DNA molecule by (1) cleaving both strands at one site in the molecule, (2) passing an intact part of the molecule between ends of the cut site, and (3) reconnecting the free ends.
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Chapter 7
■
G e n o m e s a n d C hromosomes
kept negatively supercoiled. Because the DNA is underwound, the two strands of negatively supercoiled DNA are easier to separate than positively supercoiled DNA. This is important for transcription enzymes like RNA polymerase that must separate strands of DNA to make RNA. Note, however, that some archaeal species living in acid at high temperature have nucleoids that are positively supercoiled to keep DNA double-stranded in these inhospitable environments (discussed below). Positively supercoiled DNA is harder to denature because it takes excess energy to separate overwound DNA.
Topoisomerases Regulate the Supercoiling of DNA Supercoiling changes the topology of DNA. Topology is a description of how spatial features of an object are connected to each other. Thus, enzymes that change DNA supercoiling are called topoisomerases. To maintain proper DNA supercoiling levels, a cell must delicately balance the activities of two types of topoisomerases. Type I topoisomerases cleave only one strand of a double helix, while type II enzymes cleave both strands. Type I enzymes are generally used to relieve or unwind supercoils, while type II enzymes use energy to introduce them. Figures 7.10 and 7.11 illustrate the mechanisms used by type I and type II topoisomerases, respectively. Type I enzymes are usually single proteins, while type II enzymes have multiple subunits. An example of a type II topoisomerase
is DNA gyrase, whose function is to introduce negative supercoils in DNA (see Fig. 7.11). The active gyrase complex is a tetramer composed of two GyrA and two GyrB proteins. Figure 7.12 shows a three-dimensional representation of DNA gyrase in the midst of generating a supercoil. Enzymes that make or manage bacterial DNA, RNA, and proteins are common targets for antibiotics. For instance, the quinolone antibiotics specifically target bacterial type II topoisomerases. A modern quinolone, ciprofloxacin, was the treatment of choice for anthrax pneumonia during the 2001 anthrax attacks. The patriarchs of this drug family, nalidixic and oxolinic acids, were used to map gyrA and gyrB, the fi rst drug resistance genes identified in E. coli. The modern successors of these drugs, the fluoroquinolones, are among the most widely used antimicrobials in the world. These drugs do not block topoisomerase action but stabilize the complex in which DNA gyrase is covalently attached to DNA (see Fig. 7.11). This forms a physical barrier in front of the DNA replication complex, and the bacterial cell dies. Extreme thermophiles (hyperthermophilic archaea) possess an unusual gyrase called reverse DNA gyrase. In contrast to the DNA gyrase from mesophiles, reverse gyrase introduces positive supercoils into the chromosome. It is proposed that tightening the coil helps protect the chromosome against thermal denaturation. Because the DNA has extra turns, it takes more energy (heat) to separate the strands.
Topoisomerase I cleaves one strand of a double helix, holds on to both ends, and . . .
The helix unwinds in this region, resulting in one less negative supercoil.
Topoisomerase I
1
. . . passes the other, intact strand through the break and religates the strand. 1
2 2 3 3 4 5
4 6 Loops
Circular double-stranded DNA with 5 negative supercoils
4 Loops
4 Negative supercoils
Mechanism of action for type I topoisomerases (Topo I of E. coli). Topoisomerase I relaxes a supercoiled DNA molecule. From left to right: Circular, supercoiled, double-stranded DNA is nicked by topoisomerase I, unwound, and released with one less superturn.
Figure 7.10
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■ Genes and Ge n o m e s
231
DNA duplex cleaved by gyrase
dsDNA
GyrB GyrA
ATP
ATP
ADP ADP
GyrA introduces double-strand break in this section (cylinder) and holds the two ends apart while remaining covalently attached to the DNA.
Courtesy of James Berger
GyrB grabs one section of double-stranded DNA (represented by cylinder).
DNA duplex to pass through break in the duplex above
Three-dimensional representation of DNA gyrase. The 3-D model of gyrase was determined using data from X-ray crystallographic studies (blue and red regions). The gyrase complex is modeled in the process of gripping a broken DNA duplex (shown in green) and transporting a second duplex (the multicolored rosette).
Figure 7.12 GyrA ATPase passes the intact double-stranded section through the double-stranded break.
GyrB rejoins the cleaved DNA and opens at the other end to allow the strand that has passed through to exit.
TO SU M MAR I Z E: ■
■
■
Mechanism of action for type II topoisomerases (DNA gyrase of E. coli). The gyrase enzyme grabs DNA and, in an ATP-dependent process, introduces a double-strand break, passes another part of the double helix through the break, and then reseals the break. The result is the introduction of a negative supercoil.
Figure 7.11
■
■
THOUGHT QUESTION 7.3 DNA gyrase is essential to cell viability. Why, then, are nalidixic acid–resistant cells that contain mutations in gyrA still viable? THOUGHT QUESTION 7.4 Bacterial cells contain many enzymes that can degrade linear DNA. How, then, do linear chromosomes in organisms like Borrelia burgdorferi (the causative agent in Lyme disease) avoid degradation?
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■
■
The smallest genome known for a free-living microbe encodes a possible 480 proteins. Noncoding DNA can constitute a large amount of a eukaryotic genome, while prokaryotes have very little noncoding DNA. DNA is composed of two antiparallel chains of purine and pyrimidine nucleotides in which phosphate links the 5′ carbon of one nucleotide with the 3′ carbon of the next in the chain. This forms a double helix containing a deep major groove and a more shallow minor groove. Hydrogen bonding and interactions between the stacked bases hold together complementary strands of DNA. Supercoiling by topoisomerases compacts DNA into an organized nucleoid. Bacteria, eukaryotes and most archaea possess negatively supercoiled DNA. Archaea living in extreme environments have positively supercoiled genomes. Type I topoisomerases cleave one strand of a DNA molecule and relieve supercoiling; type II enzymes cleave both strands of DNA and use ATP to introduce supercoils.
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232
Chapter 7
Table 7.2
G e n o m e s a n d C hromosomes
Some genes and proteins involved in DNA replication of E. coli.
Protein DnaA DnaB DnaC Pol III (alpha) Primase (DnaG) Gamma and tau subunits Beta (EFI) Single-stranded binding protein Delta (EFIII) Delta prime Psi Chi Theta Epsilon subunit DNA gyrase (subunit beta) DNA Pol II RNA Pol, beta subunit Pol I DNA ligase
7.3
■
Gene
Size
Function
oriC dnaA dnaB dnaC dnaE dnaG dnaX dnaN ssb
245 bp 52,300 Da 52,200 Da 27,800 Da 129,700 Da 65,400 Da 47,500 Da; 71,000 Da 40,400 Da 19,000 Da
holA holB holD holC holE dnaQ (mutD) gyrB
38,500 Da 36,700 Da 15,000 Da 16,500 Da 8,700 Da 27,000 Da 89,800 Da
polB rpoB
120,000 Da 150,000 Da
DNA repair Initiation of replication, also bulk RNA synthesis
polA ligA
103,000 Da 73,400 Da
Gap filling Joins phosphodiester ends
Origin of replication Initiation, binds oriC, DnaB loading Helicase (hexamer), prepriming priming, DNA-dependent rNTPase Loading factor for DnaB DNA Pol III holoenzyme, elongation Priming, RNA primer synthesis, rifampin resistant RNA polymerase Synthesis, part of the gamma complex, promotes dimerization of Pol III Beta clamp, processivity Helix destabilizing Part of gamma complex, clamp loading Part of gamma complex, clamp loading Part of gamma complex, clamp loading Part of gamma complex, clamp loading Pol III dimerization Proofreading, 3′-to-5′ exonuclease Relaxation of supercoils
DNA Replication
Microbial DNA needs to replicate itself as accurately and as quickly as possible so that the organism can grow and compete with other species. Replication efficiency is one reason why bacterial pathogens such as Salmonella can cause disease so quickly after ingestion. In this respect, bacteria differ from multicellular organisms, which need to regulate cell division carefully within their tissues; unregulated growth within tissues leads to cancer. The process of bacterial replication involves an amazing number of proteins and genes coming together in a complex
machine (Table 7.2). Its operation is all the more remarkable considering that some bacteria, such as thermophilic Bacillus species that live in hot springs, can double in less than 15 minutes. The molecular details of bacterial DNA replication are important because they provide targets for new antibiotics, as well as tools for biotechnology such as the polymerase chain reaction (PCR). In addition, the proteins of DNA replication have homologs in the human genome, in which defects are the basis of inherited human diseases such as xeroderma pigmentosum, and can cause a predisposition to certain cancers.
Parental strand 3′
5′
5′
3′
New daughter strands 3′
3′
Replication fork
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Semiconservative replication. A replication bubble with two replication forks. Parental strands are gray, and newly synthesized daughter strands are purple. Replication is called semiconservative because one parental strand is conserved and inherited by each daughter cell genome. It is called bidirectional because it begins at a fixed origin and progresses in opposite directions.
Figure 7.13
Origin
5′
5′
Replication fork
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Pa r t 2
Overview of Bacterial DNA Replication To replicate a molecule containing millions of base pairs poses formidable challenges. How does replication begin and end? How can two identical copies be generated? How is accuracy checked and maintained?
■ Genes and Ge n o m e s
233
Origin, oriC
1. Replication begins at origin.
Semiconservative replication. Replication of cellular
DNA in most cases is semiconservative, meaning that each daughter cell receives one parental strand and one newly synthesized strand (Fig. 7.13). At the replication fork, the advancing DNA synthesis machine separates the parental strands while extending the new, growing strands. The semiconservative mechanism provides a means for each daughter duplex to be checked for accuracy, based on its parent strand. Enzymes that synthesize DNA or RNA can only connect nucleotides together in a 5′-to-3′ direction. That is, every newly made strand begins with a 5′ triphosphate and ends with a 3′ hydroxyl group (see Fig. 7.4A). A polymerase (a chain-lengthening enzyme complex) fastens the 5′ alpha-phosphate of an incoming nucleoside triphosphate to the 3′ hydroxyl end of the growing chain, thus forming a phosphodiester bond. (The alpha-phosphate is the phosphate closest to the sugar base.) This 5′-to-3′ enzymatic constraint produces an interesting mechanistic puzzle. If polymerases can only synthesize DNA in a 5′-to-3′ direction and the two phosphodiester backbones of the double helix are antiparallel, how are both strands of a moving replication fork synthesized simultaneously? One strand presents no problem because it is synthesized in a 5′-to-3′ direction toward the fork, but synthesizing the other new strand in a 5′-to3′ direction would seem to dictate that it move away from the fork (Fig. 7.14). How is this possible? The answer was revealed in Chapter 3 but is discussed in greater detail here.
Terminus, ter
2. Replication bubble forms. Replication forks progress in opposite directions.
3. One strand at each fork is synthesized continuously 5′ to 3′.
Replication bubble
5′ 3′
Fork movement 3′ 5′
4. Second strand at each fork is synthesized discontinuously in Okazaki fragments 5′ to 3′.
Okazaki fragment 3′
5′ Fork 5′ movement 3′
Terminus
THOUGHT QUESTION 7.5 Suppose you have the following capabilities: You can label DNA in a bacterium by growing cells in medium containing either nitrogen 14N or the heavier isotope 15N; you can isolate pure DNA from the organism; and you can subject DNA to centrifugation in a cesium chloride solution, a solution that forms a density gradient when subjected to centrifugal force, thereby separating the light (14N) and heavy (15N) forms of DNA to different locations in the test tube. Given these capabilities, how might you prove that DNA replication is semiconservative? The process of DNA replication is divided into three phases: (1) initiation, which is the melting (unwinding) of the helix and the loading of the DNA polymerase enzyme complex; (2) elongation, which is the sequential addition of deoxyribnucleotides from deoxynucleotide triphosphates, followed by proofreading; and (3) termination,
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5. Replication ends at terminus. 3′
5′
Terminus
Comparing direction of fork movement with direction of DNA synthesis.
Figure 7.14
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in which the DNA duplex is completely duplicated, the negative supercoils are restored, and key sequences of new DNA are methylated!
Replication from a Single Origin Replication begins at a single defined DNA sequence called the origin (oriC). Following initiation, a circular microbial chromosome replicates bidirectionally (see Fig. 7.14, step 1) until it terminates at defined termination (ter) sites located on the opposite side of the molecule. Once the process has begun, the cell is committed to completing a full round of DNA synthesis. As a result, the decision of when to start copying the genome is critical. If it starts too soon, the cell accumulates unneeded chromosome copies; if it starts too late, the dividing cell’s septum “guillotines” the chromosome, killing both daughter cells. Consequently, elaborate fail-safe mechanisms link the initiation of DNA replication with cell mass, generation time, and cellular health, making the timing of initiation remarkably precise. Fundamentals of DNA replication. The basic process
of chromosome replication is outlined in Figure 7.14. After initiation of replication, a replication bubble forms at the origin. The bubble contains two replication forks that move in opposite directions around the chromosome (Fig. 7.14, step 2). DNA polymerases synthesize DNA in a 5′-to-3′ direction. Thus, at each fork, one new DNA strand can be synthesized continuously until the terminus region (Fig. 7.14, step 3). However, because the two DNA strands are antiparallel and the DNA polymerases only synthesize 5′-to-3′, the other daughter strand has to be synthesized discontinuously, in stages—seemingly backward relative to the moving fork (Fig. 7.14, step 4). The fragments of DNA formed on this discontinuously synthesized strand are called Okazaki fragments, after the scientist who discovered them. As we will discuss later, the Okazaki fragments are progressively stitched together to make a continuous, unbroken strand. Ultimately, the two replicating forks meet at the terminus sequence (see Fig. 7.14, step 5) and the two daughter chromosomes separate. Overall, copying of the whole chromosome in E. coli takes about 40 minutes. Chromosome-partitioning processes then move each chromosome to different ends of the cell so that a cell wall can form at midcell. Once the cell wall is complete (this time is variable but generally about 20 minutes), the two daughter cells, with their new chromosomes, can separate. So you would imagine the whole process from the start of replication to cell separation would take about 60 minutes for E. coli. Under optimal growth conditions, however, E. coli cells divide in 20 minutes. How is this possible if the process of replication takes at least 40 minutes? The answer is that a partially replicated chromosome can start new rounds of replication at the two daughter origins even before the
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fi rst round is complete. This overlapping of generations enables the cell to accommodate the 40-minute DNA replication time within a 20-minute generation time. During these rapid cell divisions, the DNA-partitioning mechanisms ensure that the two actively replicating chromosomes move to different ends of the cell, which keeps them from being severed when the cell septum forms. Now let’s examine each step in molecular detail to answer some important questions about mechanism.
Initiation of Replication What determines when replication begins? Initiation is controlled by DNA methylation, and by the binding of a specific initiator protein to the origin sequence. Further molecular events load the elaborate DNA polymerase complex and generate the first RNA primer for the new DNA strand.
DNA Methylation Controls Timing The chromosomal origin of E. coli is a sequence of 245 base pairs designated oriC. It is subject to a critical molecular control mechanism that dictates the precise timing of replication initiation. Initiation of replication at oriC is activated by one protein, DnaA, and inhibited by another, SeqA. Immediately after a cell has divided, the level of active DnaA (DnaA bound to ATP) is low, and the inhibitor SeqA will bind to oriC and prevent ill-timed initiations (before the cell has grown enough to divide again). How does SeqA know to bind just after the origin has replicated? The key is DNA methylation. E. coli uses the enzyme deoxyadenosine methylase (Dam) to attach a methyl group to the adenine residue at position N-6. The methylated adenines appear in all GATC sequences. GATC sequences (the recognition sites of Dam methylase) are scattered throughout the chromosome and occur on both strands. However, just after the origin has replicated, there is a short lag before the newly synthesized strand is methylated. As a result, the origin is temporarily hemimethylated, a situation in which only one of the two complementary strands is methylated. Because SeqA has a high affi nity for hemimethylated origins, this inhibitor will bind most tightly immediately after the origin has been replicated and prevent another initiation event. Eventually, the Dam methylase will methylate the new strand and decrease SeqA binding. The replication initiator protein, DnaA. The onset of
the initiation phase is determined by the concentration of the replication initiator protein, DnaA. DnaA protein recognizes specific 9-bp repeats at oriC. As the cell grows, the level of active DnaA initiator protein rises until it is sufficient to bind to a series of 9-bp repeats at oriC (Fig. 7.15 , step 1). DnaA actually binds as a complex with ATP (DnaA-ATP). This binding initiates the assembly of
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Initiation of replication A
3′ 13-mer
5′
DnaA-ATP 5′ 1. DnaA-ATP proteins bind to the repeated 9-mer sequences within oriC.
3′
9-mer 3′ 5′
2. Binding of DnaA leads to strand separation at the 13-mer repeats.
5′
A A A A
3′
DNA helicase loader (DnaC)
3′
3. DNA helicase (DnaB) and DNA helicase loader (DnaC) associate with the DnaA bound origin.
5′ 5′
A A DNA helicase (DnaB)
A A
3′ 5′
3′
A A
5′
A A
3′
4. DNA helicase loaders open the DNA helicase protein ring and place the ring around the ss (single-stranded) DNA at the origin. Loading of the DNA helicase leads to release of the helicase loader.
DNA primase A 3′
A
5′
A A
3′
5. The DNA helicases each recruit a DNA primase, which synthesizes an RNA primer on each template.
3′ 3′
5′ Sliding clamp DNA polymerase III
3′
6. The clamp loader with DNA polymerase III loads a sliding clamp onto each leading-strand DNA at an RNA primer.
Clamp loader
5′
5′
Sliding clamp
3′
Leading strand
7. DNA polymerase binds to the clamp. Leading-strand synthesis begins and continues to the end of the template. At each lagging strand, a sliding clamp is then loaded.
5′
3′
3′
5′
3′
3′
5′
Clamp loader opens clamps
3′
5′ 3′
Reloaded with new sliding clamps
5′ Leading strand
Initiation of DNA replication. The start of DNA replication is precisely timed and linked to the ratio of DNA to cell mass. In E. coli, the initiator protein DnaA accumulates during growth and then triggers the initiation of replication. It begins with DnaA-ATP complexes binding to 9-mer (9-bp) repeats upstream of the origin. This binding (along with other proteins not shown), first causes the DNA to loop in preparation for melting open by the helicase (DnaB).
Figure 7.15
235
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a membrane-bound replication hyperstructure, a complex assembly of numerous proteins forming a functional unit, at midcell (at the cell equator). The origin sequence (Ori), after moving through the replication complex, cannot trigger another round of replication because of inhibition by SeqA and decreasing levels of unbound DnaA-ATP. Another round of replication can only begin after (1) the origin becomes fully methylated, (2) SeqA dissociates, and (3) the DnaA-ATP concentration rises.
Initiation Requires RNA Polymerases An unexpected feature of DNA replication is that its initiation actually requires RNA polymerases. One RNA polymerase helps separate the strands of the DNA helix at the origin, and a second RNA polymerase produces the primer, or starter, fragments needed to synthesize new DNA. The fi rst of these polymerases (the housekeeping RNA polymerase used to make most of the RNA in the cell; discussed in Chapter 8) transcribes DNA at oriC to produce RNA that helps separate the two DNA strands (Fig. 7.15, step 2, this RNA polymerase is not shown). This separation allows a special DNA helicase (protein DnaB) in association with a DNA helicase loader (DnaC) to bind the two replication forks formed during bidirectional replication (Fig. 7.15, step 3, and see Fig. 7.16). As the lead protein of the replication machine, the helicase (DnaB) uses energy from ATP hydrolysis to unwind the DNA helix as the DNA moves into the DNA polymerase replicating complex. The ringlike DnaB is Beta clamp subunits form a ring that slides along the DNA.
assembled around one DNA strand at each replication fork. After loading DnaB at the origin, DnaC is released (Fig. 7.15, step 4). Coincident with the unwinding of DNA, small single-strand DNA binding proteins (ssb, seen in Fig. 7.17) coat the exposed single-stranded DNA, protecting it from nuclease activities patrolling the cell. DNA-dependent DNA polymerases possess the unique ability to “read” the nucleotide sequence of a DNA template and synthesize a complementary DNA strand. The discovery of this activity earned Arthur Kornberg (1918–) the Nobel Prize in 1959. As remarkable as these enzymes are, no DNA polymerase can start synthesizing DNA unless there is a preexisting DNA or RNA fragment to extend—a primer fragment. The primer fragment possesses a 3′ OH end that can receive incoming deoxy nucleotides. Consequently, once DnaB (helicase) is bound to DNA, the next step in initiation is to make RNA primers at each fork (Fig. 7.15, step 5). In contrast to DNA polymerases, RNA polymerases can synthesize RNA without a primer. A specific RNA polymerase called DNA primase (DnaG) synthesizes short (10–12 nucleotides) RNA primers at the origin, which launches DNA replication. One primase is loaded at each of the two replication forks. Note that primase is different from the RNA polymerase involved in initially separating the DNA strands at the origin.
A Sliding Clamp Tethers DNA Polymerase to DNA At this point, the DNA is almost ready for DNA polymerase. But fi rst, a sliding clamp protein (the beta subunit) must be loaded to keep the DNA polymerase affi xed to the DNA (Fig. 7.15, step 6). Without this clamp, DNA polymerase would frequently “fall off” the DNA molecule (see Special Topic 7.1). A multisubunit complex (called the clamp-loading complex) places the beta clamp, along with an attached pair of DNA polymerase molecules, onto DNA. DNA polymerase (specifically DNA Pol III; discussed later) can then bind to the 3′ OH terminus of the primer RNA molecule and begin to synthesize new DNA (Fig. 7.15, step 7). Figure 7.16 presents the molecular structure of the beta clamp loaded onto DNA.
Elongation: DNA Polymerases Synthesize DNA DNA to be replicated
DNA replication: The sliding clamp of E. coli. The clamp dimer encircles the gray and purple DNA strand. (PDB codes: 1OK7, 1K8J)
Figure 7.16
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Escherichia coli contains five different DNA polymerase proteins designated Pol I through Pol V. All polymerases catalyze synthesis of DNA in the 5′-to-3′ direction. However, only the replication polymerases Pol III and Pol I participate directly in chromosome replication. The other polymerases conduct operations to rescue stalled replication forks and repair DNA damage.
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Special Topic 7.1
Trapping a Sliding Clamp
Michael O’Donnell and colleagues in 1991 performed an elegant experiment that supports the sliding clamp model. O’Donnell hypothesized that if the beta subunit were really a sliding clamp, then once it was loaded onto a small circular DNA molecule, the protein would never come off. It would continually slide around the circle. However, the protein complex would easily slide off a linear DNA molecule. The experiment required detecting differences in molecular weight (or size) between DNA and DNA-protein complexes. Free protein is smaller than the DNA-protein complex formed when the same protein binds to DNA. To measure these size differences, various mixtures of DNA and 3H-labeled clamp proteins were passed through a gel filtration column, a column filled with tiny, hollow gel beads, each of which contains channels that serve as molecular sieves (Fig. 1A). The smaller the molecule, the more easily it can enter the bead channel. Thus, small molecules become temporarily trapped in each bead, taking a long time to move from the top of the gel column to the bottom. Larger molecules, however, are too large to enter the beads. They flow quickly around the beads and exit the column first. Fractions of defined volume are collected over A. Flow
time. Proteins collected in early fractions are larger than those collected in later fractions. Consequently, a DNA–clamp protein complex, being large, will move more quickly through the gel and will appear in the early fractions. Unbound protein will travel more slowly and appear in later fractions. The beta clamp complex was loaded onto a circular DNA molecule and onto the same molecule cut with the restriction enzyme SmaI. The separate mixtures were passed through identical gel filtration columns. Fractions were collected and counted for radioactivity (see Fig. 1B). Note that the protein loaded onto the circular molecule eluted in a much earlier fraction (fraction 12) than did protein loaded onto the linear molecule (fraction 25). The earlier elution indicated that the protein did not slide off the circular molecule and was maintained as a large complex. The late-elution profile of the protein loaded onto the linear molecule indicated that the protein was free to slide off the DNA and failed to maintain a large complex. The bottom panel, however, shows that a protein (for example, eukaryotic EBNA1) bound to the ends of the linear molecule will block exit of the beta clamp. This is evident in the fact that both the circular and linear DNA molecules eluted in the same fractions. B.
Small proteins can enter beads and their movement is retarded.
Beta 100 SmaI 80
Glass column H-Beta dimers (fmol)
Large proteins are excluded from the beads and pass through the column first.
60
3
Polysaccharide gel beads
Circular DNA Linear DNA
40 SmaI
20 0 50
EBNA1 EcoRV
40
EcoRV
30 EBNA1 20 10 0 0
5
10 15 20 25 30 35 40 45 50
Fraction Gel filtration evidence supports the sliding fmol = fempto mole clamp model. A. Principle of gel filtration. Porous beads separate small molecules from large molecules because small molecules are temporarily trapped inside the beads. B. Top: 3H-labeled beta clamp dimers were added to nicked, circular DNA (red line) or linear DNA (black line). After passing through separate gel filtration columns, radioactivity was measured in the material that eluted from the column. The results show that the clamp cannot leave the circular model, so the large protein–DNA complex elutes from the column in an early fraction (fraction 12). The second red peak eluting at fraction 25 is protein that did not load onto DNA. Protein loaded on linear DNA, however, slides off of the molecule and does not form a large complex. Bottom: This is the same experiment, but the DNA contained two EBNA1 binding sites. Clamp protein was loaded onto circular DNA (red line) or the same DNA cut between the sites with EcoRV (linear). EBNA1 protein blocks the ends and prevents the beta clamp from sliding off the linear molecule. Note that the beta clamp mixed with linear DNA (black line) in the bottom panel eluted earlier from the column than in the top panel. Again, the peaks at fraction 28 reflect protein that did not load onto DNA. Source: B. P. Todd
Figure 1
Stukenberg, et al. 1991. Journal of Biological Chemistry 266:11328.
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Elongation of DNA synthesis 1. The leading-strand DNA Pol III enzyme replicates the leading strand. SSBs cover and protect the unreplicated single strand. The DNA helicase remains on the lagging strand, unwinding the dsDNA moving into the replisome complex.
3′ Sliding clamp
5′
Leading-strand DNA polymerase 3′ OH
Sliding clamp
2. Lagging-strand DNA polymerase synthesizes the lagging strand. The lagging strand loops out after passing through the polymerase.
Clamp loader
5′ DNA helicase
Actual strand movement threading through the replisome.
5′ SSB bound to DNA
3′ OH
3′
Lagging-strand DNA polymerase
5′
3′
5′
Okazaki fragments
3′ 5′ 3′ OH 3′
3. After DNA helicase has moved approximately 1,000 bases, a second RNA primer is synthesized on each lagging strand.
5′ RNA primer 3′ 5′
DNA primase
5′
5′
Lagging-strand pol completing 3′ OH previous Okazaki fragment
3′ 5′ 3′ OH 3′
4. When the lagging-strand polymerase bumps into the 5′ end of a previously synthesized fragment, the DNA polymerase is released and the clamp is disengaged.
5′ DNA primase is released 3′
5′
3′ 5′
5′
A. The DNA polymerase dimer acting at a replication fork. Both the leading and lagging strands are synthesized simultaneously in the 5′-to-3′ direction. For clarity, the beta clamp on the lagging strand is shown on the opposite side of Pol III as compared to its position on the leading strand. If the clamp was placed on the left side of polymerase, as it is shown in many models, the lagging strand would have to completely loop around the polymerase and enter the polymerase from the left.
Figure 7.17
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Pa r t 2
DNA polymerase III. The main replication polymerase, Pol III, is a complex, multicomponent enzyme. Because of its complexity, it is often referred to as a “molecular machine.” The DNA synthesis activity of Pol III is held in the alpha subunit of the complex, while other subunits are used for improving fidelity (accuracy of replication) and processivity (a measure of how long the polymerase remains attached to, and replicates, a template). The Pol III epsilon subunit (DnaQ), for example, contains a proofreading activity that prevents mistakes and improves fidelity. Proofreading activities within DNA polymerases scan for mispaired bases that have been inappropriately added to a growing chain. Mispaired bases are detected by their increased mobility. A mispaired base mistakenly linked by a phosphodiester bond to a growing DNA chain is more mobile than the correct base because it does not hydrogen-bond to the template base. This motion halts DNA elongation by Pol III because the base is not properly positioned at the enzyme’s active site. This stalling of Pol III activity triggers an intrinsic 3′-to-5′ exonuclease activity in the epsilon subunit. Exonucleases degrade DNA starting from either the 5′ end or the 3′ end. The exonuclease activity of Pol III cleaves the phosphodiester bond, releasing the improperly paired base from the growing chain. Once the wayward mispaired base has been excised, Pol III activity can proceed.
Both DNA Strands Are Elongated Simultaneously After initiation, each replication fork contains one elongating 5′-to-3′ strand called the leading strand (look back
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at Fig. 7.15, step 7). But how is the opposite strand at each fork replicated? There are no known DNA polymerases capable of synthesizing DNA in the 3′-to-5′ direction, which would seem to be needed if both strands are to be synthesized simultaneously. DNA synthesis of one strand continuing all the way back to the origin is not a solution because it would leave the unreplicated strand at each fork exposed to possible degradation for too long and would double the time needed to complete DNA replication. The cell has solved this dilemma by coordinating the activity of two DNA Pol III enzymes in one complex, one for each strand. The two associated Pol III complexes together are called the replisome. As the dsDNA unwinds at the fork, the problem strand loops out and primase (DnaG) synthesizes a primer. The second Pol III enzyme binds to the primed section of the loop and synthesizes DNA in the 5′-to-3′ direction (imagine the lower template strand in Fig. 7.17A threading from left to right, through the polymerase ring). All the while the second polymerase is moving along in tandem with the fi rst polymerase (on the leading strand) relative to the fork (Fig. 7.17A, step 1). Realize that in actuality, the replisome remains at a fi xed, midcell location in the cell, probably attached to the membrane, and the template DNA threads through it (discussed later). Note that simultaneous extension of the two strands requires that synthesis of the looped strand lag behind the leading strand and that new RNA primers be synthesized periodically by primase (DnaG) every thousand bases or so. Thus, the lagging strand is synthesized discontinuously, in pieces called Okazaki fragments, while the leading strand can be synthesized continuously. As the leading strand moves forward,
3′
3′ 5′
5′ 3′ OH
5′
3′ OH 3′
3′
5′
5′
5′
5′
5′
5. A new clamp is assembled on the newly primed lagging strand. 3′ 5′
Figure 7.17 (continued)
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3′ 5′
6. The DNA polymerase binds to that clamp and begins synthesizing another Okazaki fragment.
B. The DNA polymerase dimer acting at a replication fork.
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advancing the fork, there remains a long stretch of lagging strand complementary to the already replicated leading strand. This lagging strand remains singlestranded but protected by single-stranded DNA binding proteins (SSBs) (Fig. 7.17A, step 2). After about 1,000 bases, DNA primase reenters and synthesizes a new RNA primer in anticipation of lagging strand DNA synthesis (Fig. 7.17A, step 3). At some point, the lagging-strand polymerase bumps into the 5′ end of the previously synthesized fragment. This interaction causes DNA polymerase to disengage from that strand (Fig. 7.17A, step 4), and the clamp loader loads a new clamp near the new RNA primer (Fig. 7.17B, step 5). The DNA polymerase binds to that clamp and begins synthesizing another Okazaki fragment (Fig. 7.17B, step 6). This process repeats every 1,000 bases or so around the chromosome.
5′
Lagging strand OH 3′
THOUGHT QUESTION 7.6 How fast does E. coli DNA polymerase synthesize DNA (in nucleotides per second), given that the genome is 4.6 million base pairs, replication is bidirectional, and the chromosome completes a round of replication in 40 minutes?
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5′
Parental template strand
RNase H cleaves RNA at any point along the hybrid RNA-DNA section.
RNase H
OH 3′
5′
5′
DNA Pol I fills the gap using 3′ OH end of DNA fragment.
Pol I DNA
5′
5′ O–
DNA polymerase I. Discontinuous DNA synthesis
results in a daughter strand containing long stretches of DNA punctuated by tiny patches of RNA primers. This RNA must be replaced with DNA to maintain chromosome integrity. To remove the RNA, cells typically use an RNase enzyme specific for RNA-DNA hybrid molecules (called RNase H). A DNA Pol I enzyme enters after the RNase and synthesizes a DNA patch using the 3′ OH end of the preexisting DNA fragment as a priming site (Fig. 7.18). When DNA Pol I reaches the next fragment, the enzyme removes the 5′ nucleotide and resynthesizes it. This process of replicating DNA increases accuracy and decreases mutations. Once DNA Pol I stops synthesizing, it cannot join the 3′ OH of the last added nucleotide with the 5′ phosphate of the abutting fragment. The resulting nick in the phosphodiester backbone is repaired by DNA ligase, which in E. coli uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond (see Fig. 7.18). DNA ligase from eukaryotes and some other microbes uses ATP rather than NAD in this capacity. A cleavage fragment of DNA Pol I, which can be made in the laboratory, containing DNA polymerase activity is shown with an associated DNA molecule in Figure 7.19. The figure illustrates DNA fi lling a crevice in the protein. The polymerase activity is located in the N terminus of the protein.
RNA primer
DNA ligase
O P
HO
O
O–
NAD AMP + NMN
5′ 5′ O– O
P O
O
DNA ligase repairs the phosphodiester nick.
5′ 5′
RNase H removing the RNA primer. RNase H cleaves the RNA primer (blue). DNA polymerase I uses the preexisting 3′ OH end of the DNA fragment to fill the gap. Finally, DNA ligase repairs the phosphodiester nick using energy derived from the cleavage of NAD.
Figure 7.18
DNA polymerase Klenow fragment
DNA molecule
Active site of DNA Pol I. The DNA molecule fits into a molecular pocket of the polymerase. (PDB code: 1KLN)
Figure 7.19
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near midcell (Fig. 7.20). Cellular DNA stained with a blue fluorescent dye (DAPI) clearly occupied most of the cytoplasmic space.
DNA Replication Generates Supercoils As template DNA is threaded through the replisome, the helicase continually pulls apart the two strands of the DNA helix. This causes the DNA ahead of the fork to twist, introducing positive supercoils. (Try this yourself. Twist two pieces of string together, staple one end of the pair to a piece of cardboard, and then pull the two strands apart from the free end. Notice the supercoiling that takes place beyond, or downstream of, the moving fork.) The increasing torsional stress in the chromosome could stop replication by making strand separation more and more difficult. What prevents the buildup of torsional stress is the DNA gyrase that is located ahead of the fork, removing the positive supercoils as they form. Another topological question, noted earlier, is how DNA polymerase maneuvers through the cell. Although many descriptions of replication give the impression that the polymerase complex travels around the chromosome like a train along a track, DNA is actually fed through a stationary pair of replisomes (each a double Pol III complex) located at the cell membrane. This was shown by Katherine Lemon and Alan Grossman using Bacillus subtilis. The replisomes in this organism were each tagged with green fluorescent protein, and the location of the complex was monitored in replicating cells using fluorescence microscopy. If the replisomes moved like a train on a track, the polymerase-GFP protein would be found at different positions in each cell. Instead, however, in every replicating cell, replisomes were observed as distinct fluorescent foci located at or
Bidirectional replication of a circular bacterial chromosome results in the two membrane-associated replication machines attempting to replicate through the same DNA sequences 180° from the origin—that is, halfway around the chromosome. What tells the polymerases to stop? There are at least two and as many as six terminator sequences (ter) on the chromosome that polymerases enter but rarely, if ever, leave (Fig. 7.21A). One set of terminators deals with the clockwise-replicating polymerase, while the other set halts DNA polymerases replicating counterclockwise relative to the origin. A protein called Tus (terminus utilization substance) binds to these sequences and acts as a counter-helicase when it comes in contact with an advancing helicase (DnaB). The bound Tus protein effectively halts polymerase movement. Six terminator sites ensure that the polymerase complex does not escape and continue replicating DNA. Once DNA polymerases have completed duplicating the chromosome and have been removed at the ter sites, the cell is still faced with what could be called a “knotty” problem. Because of the topology of the chromosome, the two daughter molecules will appear as a catenane of linked rings after replication (Fig. 7.21B). The cell resolves this structure using enzymes called
A.
B.
PolC-GFP DNA
1 µm
Lemon, et al. 1998. Science 282:1516
1 µm
Lemon, et al. 1998. Science 282:1516
PolC-GFP
Terminating Replication
Location of replicative DNA polymerase in living cells. Bacillus subtilis cells containing PolC-GFP (DNA polymerase III tagged with green fluorescent protein) were grown at 30°C in defined minimal medium. Images were captured with a cooled charge-coupled device (CCD) camera. A. PolC-GFP is seen localized as discrete green foci. Membranes were stained orange with vital membrane stain FM4-64. B. Using the same cells, DNA was stained blue with DAPI (blue) and the image overlayed with the one in part A. This image shows that DNA is distributed throughout the cell while PolC-GFP is localized to midcell.
Figure 7.20
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A.
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Figure 7.21 Terminating replication of the chromosome. A. Terminator regions for DNA replication on the E. coli chromosome. Replication forks moving clockwise are trapped by terG, terF, terC, and terB. Counterclockwise moving forks are trapped by terA, terD, and terB. B. Resolution of DNA replication catenanes. On the left is a linked chromosome catenane. The highlighted area is enlarged to the right. XerC and XerD catalyze a breaking and rejoining that passes the chromosomes through each other, resolving the link.
Origin
Counterclockwise fork
Clockwise fork
terG terF terB terC Clockwise fork trap
terB terD terA Counter-
have resolved and move toward the poles, the cell can begin to divide, forming the cell septum.
clockwise fork trap
THOUGHT QUESTION 7.7 Individual cells in a population of E. coli typically initiate replication at different times (asynchronous replication). However, depriving the population of a required amino acid can synchronize reproduction of the population. What happens is that ongoing rounds of DNA synthesis finish, but new rounds do not begin. Replication stops until the amino acid is once again added to the medium, an action that triggers simultaneous initiation in all cells; that is, reproduction of the population becomes synchronized. Why?
B.
Linked catenane
THOUGHT QUESTION 7.8 The antibiotic rifampin inhibits transcription by RNA polymerase, but not by primase (DnaG). What happens to DNA synthesis if rifampin is added to a synchronous culture?
Completed replication
DNA replication in archaea TO SU M MAR I Z E:
XerC
■
Holiday junction ■
XerD
■
Resolution
■
■ ■
XerC and XerD that recognize a specific site (called dif ) on both DNA molecules and catalyze a series of cutting and rejoining steps that essentially pass one molecule through the other. Once the two daughter chromosomes
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■
Replication is semiconservative, with newly synthesized strands lengthening in a 5′-to-3′ direction. It involves initiation, elongation, and termination. Initiation of replication occurs from a fi xed DNA origin attached to the cell membrane. Initiation depends on the mass and size of the growing cell. It is controlled by the accumulation of initiator and repressor proteins and by methylation at the origin. Elongation requires that primase (DnaG) lay down an RNA primer, that DNA polymerase III act as a dimer at each replication fork, and that a sliding clamp keep DNA Pol III attached to the template DNA molecule. The 3′-to-5′ proofreading activity of Pol III corrects accidental errors during polymerization. DNA ligase joins Okazaki fragments. Two replisomes, each containing a pair of DNA Pol III complexes, are fi xed at the membrane, and DNA feeds through them. Termination involves stopping replication forks halfway around the chromosome at ter sites that inhibit helicase (DnaB) activity.
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Pa r t 2
Ringed catenanes formed at the completion of replication are separated by the proteins XerC and XerD.
A.
Plasmid
7.4
Plasmids and Bacteriophages
Two kinds of extragenomic DNA molecules can interact with bacterial genomes: plasmids and the genomes of bacteriophages, viruses that infect bacterial cells. Plasmid-encoded functions can contribute to the physiology of the cell, and in some cases the plasmid or phage DNA itself will integrate into the bacterial genome (see Section 9.2). Eukaryotic genomes and the genomes of eukaryote-specific plasmids and viruses are similarly subject to sharing of proteins and chromosomal interactions, such as insertions. Viruses of eukaryotes and their applications in genetic engineering are discussed in greater detail in Chapters 11 and 12.
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Plasmids are much smaller than chromosomes (Fig. 7.22) and are found in archaea and bacteria as well as in eukaryotic microbes. Plasmids are usually circular, and circular plasmids, like circular chromosomes, are typically negatively supercoiled. Replication of many of these extrachromosomal elements is not tied to chromosome replication. Each plasmid contains its own origin sequence for DNA replication, but only a few of the genes needed for replication. Thus, even when the timing of plasmid replication is not linked to that of the chromosome, many of the proteins used for plasmid replication are actually host enzymes. Which host proteins are used depends on the plasmid. Plasmids can replicate in two different ways. Bidirectional replication, as described in Figure 7.23A, occurs in two directions simultaneously, while rollingcircle replication (Fig. 7.23B) is unidirectional. In rollingcircle replication, a replication initiator (RepA, encoded by a plasmid gene) binds to the origin of replication, nicks one strand, and holds on to one end (5′ PO4) of it while the other end (3′ OH) serves as a primer for host DNA polymerase to replicate the intact, complementary strand. The RepA protein recruits a helicase that unwinds DNA, which becomes coated by single-strand binding proteins. As replication proceeds, the nicked strand progressively peels off until replication is complete. Then the two ends of the nicked single strand are rejoined by the Rep protein and released. A complementary strand is replicated by host enzymes to produce a double-stranded molecule. Although most known plasmids use one or the other strategy, a few can use either bidirectional or unidirection replication, depending on the cell circumstance.
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Figure 7.22 Plasmid map. A. Note the huge difference in size between a circular plasmid DNA molecule and chromosomal DNA after both are gently released from a cell (approx. 1 µm). The arrow points to the circular plasmid. B. Map of plasmid pBR322. This plasmid contains an origin of replication (ori) and genes encoding resistance to ampicillin (amp) and tetracycline (tet). The locations of three unique restriction sites are shown (HindIII, BamHI, and PstI).
If plasmids replicate autonomously, can cells easily lose their plasmids? Plasmids come equipped with selfpreservation genes that help maintain the plasmid in the host (Special Topic 7.2). Plasmids can ensure their inheritance by carrying genes whose functions benefit the host bacterium under certain conditions. For instance, bacterial plasmids can carry genes responsible for antibiotic resistance (discussed in Chapter 28). As long as the antibiotic is present in the environment, any cell that loses the plasmid will be killed or stop growing. Antibiotic resistance plasmids benefit bacteria, but they are a major problem for modern hospitals, where plasmids carrying multiple drug resistance genes are transmitted from harmless bacteria into pathogens. On the other hand, plasmids (such as pBR322) containing drug resistance genes are the workhorses of genetic technology and have benefited society tremendously. Other kinds of host survival genes carried by plasmids include genes providing resistance to toxic metals,
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Plasmid replication: bidirectional versus rolling circle. A. DNA replication begins at a fixed origin (ori ) and proceeds bidirectionally. B. Replication begins at a fixed origin but only moves in one direction. As the polymerase moves, it displaces one strand as it replicates the other. At the end of a round, the polymerase releases the displaced strand as a single-stranded circle that is then replicated.
Figure 7.23
genes encoding toxins that aid pathogenesis, and genes encoding proteins that enable symbiosis. These will be discussed in later chapters. Most of the genes involved in the nitrogen-fi xing symbiosis of Rhizobium, for example, are plasmid-borne. Far from being freeloaders, plasmids often contribute significantly to the physiology of an organism.
In this case, the conjugation mechanism produced by one plasmid will act on the other plasmid. Plasmids released from dead cells can also be taken up intact by some bacteria in a process called transformation. Finally, plasmid transmission in nature can occur by the accidental packaging of plasmids into bacteriophage head coats—in other words, by bacteriophage transduction (discussed in Section 9.2).
Plasmids Are Transmitted between Cells Some plasmid molecules are self-transferable via conjugation, a process that involves cell-to-cell contact to move the plasmid from a donor cell to a recipient (as discussed in Section 9.2). Other plasmids are incapable of conjugation (nontransmissible). A third group can be transferred only if a self-transferable plasmid resides in the same cell.
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Replication Mechanisms of Bacteriophages Bacteriophages show diverse mechanisms of DNA replication, which may differ considerably from those of cells. For instance, many phage genomes replicate unidirectionally rather than bidirectionally, and they may copy
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Plasmid Partitioning and Addiction
ence of an addiction molecule ensures that the plasmid is By virtue of sheer numbers in a cell, high-copy-number plasmids maintained in a population even if that plasmid does not proensure their inheritance into daughter cells through simple difvide any other selective advantage to the cell. fusion (Fig. 1). Low-copy-number plasmids, however, require dedicated partitioning mechanisms to guarantee that each daughter cell receives a A. For low-copy-number plasmids, B. For high-copy-number plasmids, replication is coordinated with random partitioning occurs. copy prior to cell division. Some enterprising plasmids even have chromosome replication. addiction systems designed to kill cells that lose the plasmid. A typical addiction system includes a long-lived toxin and an antidote protein. The antidote is very labile and in Plasmid Plasmid need of constant synthesis if it is to neureplication replication tralize the more stable toxin. Consequently, failure to inherit the plasmid means that the antidote can no longer be made. The antidote that remains will decay, leaving active toxin to kill the errant, plasmid-less cell. Why Cell division Cell division and is this necessary? What happens if a cell and partitioning random segregation division produces a cell with no plasmid? into plasmid into daughter cells daughter cells The cell without the plasmid may be at a competitive advantage for growth because maintaining a plasmid drains energy. Thus, the cells without plasmids could overgrow the plasmid-containing cells, eventually eliminating them from the population. Pres- Figure 1 Partitioning of low-copy-number and high-copy-number plasmids.
only one strand. Often a phage genome encodes specialized polymerase enzymes to conduct its own replication, coordinated with host components (see Section 7.3). Representative phage replication pathways are shown in Figure 7.24. For example, the linear DNA duplex of phage T4 replicates bidirectionally from multiple origins using T4 gene products such as gene product 43, the T4 DNA polymerase. An interesting feature of T4 is that each capsid packs a linear DNA molecule containing redundant sequences at each end. Consequently, the genome is said to be terminally redundant. As indicated in Figure 7.24A, genes at one end of the packaged DNA are identical to genes at the other end. After the T4 DNA has replicated, it must be packaged into new capsids. The packaging apparatus, however, requires a long concatemer of DNA in which multiple genomes are joined end to end. The concatemer is made when the terminally redundant ends of individual copies of T4 DNA recombine to make a single DNA molecule (Fig. 7.24A). From the linear T4 DNA concatemer the individual genomes are packaged into head coats, then cleaved in such a way that 3% of each
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encapsidated DNA is redundant. Because each cleavage occurs farther along the genome, every T4 DNA molecule has a different terminal repeat. The genome is linear, but when the genes in a population of T4 phages are examined as a whole, the terminal repeats make the genome seem circular. For other mechanisms of bacteriophage and viral genome replication, see Chapters 6 and 11.
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Plasmids are autonomously replicating circular or linear DNA molecules that are part of a cell’s genome and can be transferred to other cells. Plasmids replicate by rolling-circle and/or bidirectional mechanisms. Plasmids can be transferred between cells. Phage can replicate by forming linear concatemers that are cut up to fi ll capsid heads. Some phage incorporate single-stranded DNA or RNA into their capsids.
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B. Phage: Lambda Form of genome: Linear DNA, double strand, linear map
A. Phage: T4 Form of genome: Linear DNA, double strand, permuted map ABCDEF
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Genome replication in bacteriophages. A. Phage T4 replicates by the rolling-circle method. Initially, the linear genome circularizes, then replicates as a linear concatemer. From the concatemer, the individual genomes are packaged into head coats, then cleaved. Because the phage head carries 103% of the genome, each encapsidated DNA contains an end-duplicated 3% of its genome. B. Phage lambda replicates its doublestranded DNA in two stages. In the first stage, each strand serves as a template for a complementary strand, replicated in opposite directions (bidirectional rope). In the second stage, rolling-circle replication generates a concatemer, which is precisely cleaved at cos sites.
Genome is cut at cos sites during packaging into a capsid. One phage genome starts at a cos site and ends at a second cos site. cos
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Figure 7.24
cos
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7.5
Eukaryotic Chromosomes: Comparison with Prokaryotes
The chromosomes of eukaryotic microbes such as protists and algae have much in common with those of prokaryotes. Both consist of double-stranded DNA, and both usually replicate by bidirectional replication. Yet important differences are revealed as well—differences in genome structure. Eukaryotic chromosomes are linear, contained within a nucleus, and their replication involves the process of mitosis.
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Eukaryotic Genomes Are Large and Linear Overall, the genomes of eukaryotic nuclei are larger than those of bacteria, sometimes by several orders of magnitude (see Chapter 18). In addition, most eukaryotic chromosomes are linear, whereas most (though not all) bacterial chromosomes are circular. Eukaryotic chromosomes require segregation by mitosis, in order to ensure that each daughter cell receives the correct combination of daughter chromosomes.
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Because their genomes are linear, eukaryotes require a special process to duplicate the chromosome ends, providing a primer for the lagging strand. The primer is provided by a special enzyme called telomerase. At each end of the chromosome (called a telomere) there often is not enough room on the lagging strand to add an appropriate RNA primer. So after each round of replication, the chromosome would be a little shorter and information would eventually be lost. Telomerase is actually a reverse transcriptase, an enzyme that reads RNA as a template to synthesize a complementary DNA molecule. Telomerase uses an intrinsic RNA (an RNA that is part of the enzyme) as a template to add DNA repeat sequences to the end of a lagging strand forming the telomere. The telomere allows for an RNA primer to be synthesized so that the end of the chromosome can be replicated. In this way, genetic information is not lost during division. Telomerase may have evolved from the ancient progenitor cells that contained RNA rather than DNA genomes (see Section 17.2). For cells with RNA genomes to evolve modern chromosomes made of DNA, a reverse transcriptase must have been necessary. The reverse transcriptase may also be the evolutionary source of retroviruses (see Section 11.5). Eukaryotic cells pack their DNA within the confines of a nucleus, using a series of proteins called histones. Histones are rich in arginine and lysine, and therefore, are positively charged, basic proteins that easily bind to the negatively charged DNA. The DNA becomes wrapped around the histones to form units called nucleosomes. For
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protection and condensation, all eukaryotic chromosomes are packaged by histones, proteins that play a regulatory role through methylation and acetylation. Bacteria, too, have DNA-packaging proteins, but they are more diverse and less essential for function (see Sections 3.5 and 7.2). The detailed structure of the genomes of eukaryotes and prokaryotes reveal surprising differences (Fig. 7.25). The genome of the bacterium E. coli is packed with genes (green) encoding proteins or RNA molecules. These genes are separated by very little unused or noncoding DNA (purple) and with only an occasional mobile element, such as an insertion sequence, that can move from one DNA molecule to another. Most prokaryotic genes are organized in coordinately regulated operons such as thrABC, which encodes three enzymes needed to synthesize the amino acid threonine (see Section 10.1). In contrast, 95% of the human genome consists of noncoding sequences composed largely of regulatory sequences and the fossil genomes of ancient viruses. The coding genes are separated by large stretches of noncoding sequences and usually are not situated together in operons. Moreover, coding genes in the human genome are interrupted by introns (DNA within a gene that is not part of the coding sequence for a protein; shown as yellow in the figure) and ancient gene duplications that have decayed into nonfunctional, vestigial pseudogenes (orange). Bacteria also have pseudogenes, but they account for much less of the genome than in eukaryotes. The more rapid replication of bacteria causes pseudogenes to be lost more quickly.
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Genome structure in a prokaryote and in a eukaryote. The genome of the prokaryote Escherichia coli is packed with coding genes (green), with very little unused sequence between them (purple), only an occasional mobile element, such as an insertion sequence (IS). Most genes are organized in operons coordinately regulated by a single regulatory molecule such as thrABC. By contrast, the human genome consists of 95% noncoding sequences consisting largely of regulatory sequences and the decayed genomes of ancient viruses. The coding genes are separated by large stretches and usually are not situated together in operons. Moreover, coding genes are interrupted by introns (yellow). Ancient gene duplications have decayed into pseudogenes (orange). Figure 7.25
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The amount of noncoding DNA present in a eukaryote varies with the complexity of the eukaryotic species (for example, yeasts contain a much lower percentage of noncoding DNA than humans). It is believed that eukaryotes have retained noncoding DNA in their genomes because they rely heavily on it somehow for survival. By themselves, promoters of eukaryotic genes have a very low activity and require enhancer DNA sequences to drive transcriptional activity. Enhancer sequences act at large distances from promoters, and scientists suspect that once enhancers became important, it was necessary to place enough DNA between them to reduce activation of other, unrelated but adjacent promotors.
7.6
DNA Sequence Analysis
We have just described the core concepts of DNA structure, packaging, and replication. This knowledge is crucial to understanding genomics and the fundamentals of genomic analysis. It is now appropriate to discuss the basic techniques used to manipulate DNA. These include isolating genomic DNA from cells, snipping out DNA fragments with surgical precision, splicing them into plasmid vehicles, and reading their nucleotide sequences. These are the techniques that drove the genomic revolution.
DNA Isolation and Purification Archaeal Genomes Combine Features of Bacteria and Eukaryotes Like bacteria, archaea have polygenic operons, and their reproduction is predominantly asexual. Archaea are true prokaryotes in that their cells lack a nuclear membrane. On the other hand, in most species of archaea, the structures of the DNA-packing proteins, RNA polymerase, and ribosomal components more closely resemble those of eukaryotes. Even the DNA polymerase and the origin recognition sequence show greater similarity to those of eukaryotes. Finally, it should be noted that archaeal genomes encode certain unique components such as the metabolic pathway of methanogenesis. (For more on archaea, see Chapter 19.) What experimental data allow us to make such comparisons? Overwhelmingly, we rely on new data from the growing number of microbial genomes sequenced. Comparison of genomes has revolutionized our understanding of evolutionary relationships among microbes. We will now examine the tools of DNA sequence analysis that have made these studies possible. The most important of these tools—restriction mapping, DNA sequencing, and amplification by the polymerase chain reaction—actually harness the molecular apparatus used by bacteria to replicate or protect their own chromosomes. TO SU M MAR I Z E: ■
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Eukaryotic chromosomes are always linear doublestranded DNA molecules that replicate by mitosis. A reverse transcriptase called telomerase is needed to finish replicating the ends of a eukaryotic chromosome. Histones (eukaryotic DNA-packing proteins) play a critical role in forming chromosomes. Introns and pseudogenes are noncoding DNA sequences that make up a large portion of eukaryotic chromosomes. Archaeal chromosomes resemble those of bacteria in size and shape, but archaeal DNA polymerases are more closely related to eukaryotic enzymes.
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The chemical uniformity of DNA means that simple and reliable purification methods can be used to isolate it. A variety of techniques are used to extract DNA from bacterial cells. The cells may be lysed using lysozyme to degrade the peptidoglycan of the cell wall, followed by treatment with detergents to dissolve the cell membranes. The next step is to remove most proteins by precipitating them in a high salt solution. These proteins are removed by centrifugation, and the cleared lysate containing DNA is passed through a column containing a silica resin that specifically binds DNA. The remaining proteins are washed out of the column because they do not stick, and the DNA is eluted with water. The DNA is then concentrated via alcohol precipitation (ethanol or isopropanol). DNA may be precipitated from aqueous solution by adding ethanol and salt because the charge density of the phosphates makes the molecule particularly insoluble in nonaqueous solvents. The precipitated DNA is then redissolved in water or a weak buffer. At this point, the extracted DNA can be examined with a variety of analytical tools. Plasmid or chromosomal DNA molecules released from lysed cells can also be purified using a technique known as equilibrium density gradient centrifugation. (The principles of centrifugation are discussed in Chapter 3.) The density gradient is typically generated using a cesium chloride (CsCl) solution. Under high centrifugal force, the heavy Cs + atoms will increase in concentration towards the end of the tube, decreasing their concentration near the top. This forms a gradient at equilibrium ranging from the least dense at the top of the tube to the densest at the bottom. DNA molecules dissolved in the solution prior to centrifugation will migrate along the gradient according to each molecule’s density. Adding the DNA-binding dye ethidium bromide to the mix will magnify these differences. Because the dye binds in between bases (intercalates), it binds more to linear DNA (such as fragmented chromosomal DNA) than it does to supercoiled molecular plasmids. The different molecules will stop moving down the
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Although the method has largely been supplanted in recent years by PCR techniques (Special Topic 7.3), cloning genes for study typically required cutting the chromosome into distinct pieces, or fragments, that could be selectively “plucked” from the sea of other genes composing the chromosome. Fortunately, bacteria themselves provided a way to produce these small fragments (discussed in Section 9.2). The bacterial proteins involved are called restriction enzymes, which naturally function to clip the “foreign DNA” of invading plasmids and phages at specific points. Different species of bacteria use restriction enzymes that recognize different DNA sequences. The most useful restriction enzymes for molecular biology recognize specific base sequences (usually four to six bases in length) in a DNA molecule and cleave both phosphodiester backbones at sites either near or within the center of the site. The cut may produce blunt ends or staggered ends; in staggered ends, the top strand is cut at one end of the site and the bottom strand cut at the other (Fig. 7.26A). Staggered ends are also called cohesive ends because the protruding strand of one of those ends can base-pair with complementary protruding strands from any DNA fragment cut with the same restriction enzyme, regardless of the source organism. This ability of cohesive ends from different organisms to base-pair forms the basis of recombinant DNA technology. Notice in Figure 7.26A that each recognition sequence, also called a restriction site, is a palindrome in which the top and bottom strands read the same in the 5′-to-3′ direction. This is typical of the sequences recognized by one class of restriction enzymes (there are three general classes). But how do bacteria making these scissor-like DNA enzymes keep from destroying their own DNA? They protect themselves by producing a companion methylating enzyme that recognizes the same DNA sequence recognized by the restriction enzyme and methylates a base in both strands of the
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gradient at different points where their apparent densities match the density of the Cs + gradient. No matter how long you spin, the bands stop at one density point, and they are extremely sharp. In this way, CsCl density gradient centrifugation differs from the sucrose gradient centrifugation, described in Chapter 3, where the gradient is prepared in a test tube before centrifugation. Besides isolating DNA molecules, CsCl density gradient centrifugation can be used to purify DNA/protein complexes and virus particles.
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Bacterial DNA restriction enzymes. A. Target sequences of sample enzymes. The names of these enzymes reflect the genus and species of the source organism. For example, EcoRI comes from Escherichia coli. B. Agarose gel size analysis of EcoRI-restricted DNA fragments. M refers to marker fragments of known size. Smaller fragments move toward the bottom. Figure 7.26
recognition sequence. The restriction enzyme cannot cut a site if either one or both of the strands are methylated. Thus, newly replicated double-stranded molecules are protected, since one strand, the template, remains methylated at all times. Phage DNA that originates from a cell with one type of restriction modification system will not be protected once it infects a cell with a different restriction modification system because the phage will not be methylated in the right places. As such, it faces a greater chance of destruction than of undergoing protective methylation.
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The Polymerase Chain Reaction
A technique that relies on thermostable DNA polymerases isolated from thermophiles has revolutionized many fields, including biological research, medicine, and forensics. The technique is the polymerase chain reaction (PCR), briefly described in Chapter 1. The basic PCR process, outlined in Figure 1, can produce over a millionfold amplification of target DNA. Using PCR, one can produce large quantities of a specific DNA sequence within a few hours. In this technique, specific oligonucleotide primers (usually between 20 and 30 bp) are annealed to known DNA sequences flanking the target gene. A thermostable DNA polymerase uses these primers to replicate the target DNA. Heat-stable DNA polymerases must be used in this process because the reaction mixture must be subjected to repeated cycles of heating to 95°C (to separate DNA strands so that they are available for primer annealing), cooling to 55°C (to allow primer annealing), and heating to 72°C (the optimum reaction temperature for the polymerase). The polymerase Taq, from the thermophile Thermus aquaticus, is often used for this purpose (polymerases from other thermophiles are also used). Because PCR reactions require 25–30 heating and cooling cycles, a machine called a thermocycler is used to reproducibly and rapidly deliver these cycles. This basic PCR technique has been modified to serve many different purposes. Primers can be engineered to con-
tain specific restriction sites that simplify subsequent cloning. Cloning is the process by which DNA from one source is spliced into DNA from another source. Restriction sites are small (4–8 bp) sequences that are recognized and cleaved by endonucleases called restriction enzymes. If the primers used for PCR are highly specific for a gene that is present in only one microorganism, PCR can be used to detect the presence of that organism in a complex environment, such as the presence of the pathogen E. coli O157:H7 in hamburger. PCR has profoundly impacted human society. By making it possible to amplify the tiniest amounts of DNA contaminating a crime scene, PCR has changed our judicial system by providing conclusive evidence in court cases where no evidence would have existed previously. And there may come a day when individual human genomes are sequenced as a standard medical test−with profound ethical and societal implications. The hopes and fears raised by the invention of PCR-based genomic sequencing inspired the science fiction film GATTACA, directed by Andrew Niccol, depicting an imaginary future in which everyone’s destiny is determined by his or her DNA sequence. This knowledge could impact which jobs are available to someone, whether insurance coverage can be withheld, and even whether two individuals can marry.
55°C, 30 seconds primers annealed. 72°C, Taq polymerase Segment to amplify 5′
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Taq polymerase, replicates sequence, 72°C, 60 seconds.
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The polymerase chain reaction. 1. DNA template is heated to 95°C (30 seconds) to separate strands. 2. The reaction is cooled to 55°C for 30 seconds, which allows amplifying DNA primers to anneal. 3. Within seconds, the temperature is raised to 72°C, optimal for Taq polymerase activity. Polymerization is allowed to proceed for about 60 seconds, and then the strands are separated again at 95°C to prepare for another cycle. Each polymerization step increases (amplifies) the target sequence until, ultimately, only the fragment bounded by the primers is amplified. Thus, from a single DNA molecule, potentially 1030 copies of a fragment can be made.
Figure 1
Restriction enzymes from hundreds of bacteria are now commercially available for analyzing DNA. A few examples of such enzymes are shown in Figure 7.26A. Agarose gels can be used to analyze the DNA fragments obtained by treatment with these enzymes. In Figure 7.26B, each lane represents a different, homogeneous
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population of DNA molecules cut with the same restriction enzyme. Because DNA is negatively charged, all DNA molecules travel to the positive pole during electrophoresis. Pore sizes in the agarose are such that they allow small molecules to speed through the gel, while larger molecules take longer to move. Thus, DNA fragments in
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this gel separate on the basis of size—the smaller the fragment is, the farther it travels down the lane. The inserted DNA fragments were of different sizes and so travel different distances in the gel. The sum of the sizes of fragments in each lane yields the size of the original, intact, uncut molecule.
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Cloning: The Birth of Recombinant DNA Technology
The ability to clone genes rests on several seemingly unrelated findings. The discovery of restriction enzymes in the late 1960s was initially considered interesting but esoteric, and its significance was grossly underestimated at the time. Only B. later would the full impact of these enzymes on biological research be realized. The beginning of the recombinant DNA revolution can be traced to 1972. It was known at that time that plasmids were small circular DNA molecules capable of independent replication in bacterial cells. Some of the known plasmids contained a single site for certain restriction enzymes. As already noted, many restriction enzymes generate cohesive ends that can base-pair with the ends of any DNA cut with the same enzyme. The significance of these facts went unrecognized until Stanley Foreign DNA Cohen, Herb Boyer, and Stanley Falkow were relaxing with colleagues in a Waikiki deli after a scientific meeting in 1972 (Figs. 2 3 7.27A and B). In something of a “eureka” moment, the scientists Cleavage by EcoRI endonuclease suddenly realized that it might be possible to use a restriction enzyme (such as EcoRI) to cut a A AT T A AT T piece of DNA from one organism’s chromosome and graft it in T TA A T TA A vitro into a plasmid cut in a single place with the same enzyme (Fig. 7.27C). They also knew that DNA ligase (see earlier discussion) would seal the fragment to the plasmid and form a new artificial Steve Northrup/Time Life Pictures/Getty Images
Steve Northrup/Time Life Pictures/Getty Images
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Figure 7.27 Formation of recombinant DNA olecules. A. Stanley Cohen of Stanford University and B. Herbert Boyer of the University of California, San Francisco, two of the founders of recombinant DNA technology. C. A plasmid vector is cut with a restriction enzyme (EcoRI) that produces cohesive ends. The same enzyme is used to digest foreign DNA, producing a series of fragments all with identical cohesive ends. The cut vector and foreign DNA fragments are mixed. Cohesive ends of the foreign DNA and vector anneal to form a chimeric molecule containing two nicks. DNA ligase is used to seal the nicks and connect the phosphodiester backbones.
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DNA molecule. This gene, as part of the recombinant molecule, could then be introduced into E. coli by transformation (discussed in Section 9.2) and be produced in large numbers through plasmid replication. Three months later, the strategy worked. This seminal work was published by Boyer and Cohen along with students Annie Chang and Robert Helling. Over the next decade, the technique became known as gene cloning, an immensely powerful technology that thrust biology into the genomic era. Cloning strategies One problem with earlier cloning techniques is that E. coli plasmid origins will not function (that is, will not initiate replication) in most other bacterial species, but especially eukaryotes or archaea. This means that cloned genes from these groups of organisms could be studied only in E. coli. The problem with studying these foreign genes in E. coli is that the processing of RNA or proteins naturally carried out in the eukaryote, for instance, will not be possible in E. coli because E. coli does not possess those systems. Nor can function of these eukaryotic proteins be tested in the prokaryotic cell. The solution to these problems is to use a shuttle vector. A shuttle vector is a plasmid that contains a replication origin compatible with E. coli and a second origin that will allow the plasmid to replicate in an unrelated bacterial species, or in a eukaryote or archaeon. In this way, the cloned gene can be genetically manipulated in E. coli using wellcharacterized molecular protocols (that is, creating specific mutations) and then be reintroduced into the source organism to study function. THOUGHT QUESTION 7.9 Knowing that E. coli possesses restriction enzymes that cleave DNA from other species, how is it possible to clone a gene from one organism to another without the cloned gene sequence being degraded? THOUGHT QUESTION 7.10 PCR is a powerful technique, but one can easily contaminate a sample and amplify the wrong DNA−perhaps sending an innocent person to jail. What might you do to minimize this possibility?
DNA Sequencing Based on Sanger Dideoxynucleotides Even with the development of gene cloning, our advance into the genomic age would not have been possible without a rapid way to sequence large pieces of DNA. The most commonly used DNA-sequencing method relies on the Sanger dideoxy strategy. Ingenious in its simplicity, the dideoxy method relies on the fact that the 3′ hydroxyl
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Figure 7.28 Dideoxy nucleotide. DNA nucleotides lack the 2′ OH group on ribose but retain the 3′ OH needed for DNA synthesis. Dideoxy nucleotides lack both the 2′ OH and the 3′ OH. Thus, the 5′ phosphate of a dideoxy nucleoside triphosphate can still form a phosphodiester bond to a growing chain but lacks the 3′ OH acceptor for a new incoming nucleotide.
group on 2′-deoxyribonucleotides (shown in Fig. 7.3A) is absolutely required for a DNA chain to grow. Thus, incorporation of a 2′,3′-dideoxynucleotide (Fig. 7.28) into a growing chain prevents further elongation because without the 3′ OH, extension of the phosphodiester backbone is impossible. The dideoxy method begins with a short (20–30-bp) oligonucleotide DNA primer molecule designed to anneal to the plasmid vector at a site immediately adjacent to the cloned insert to be sequenced. The mixture is first heated to 95°C to separate the DNA strands and then cooled, which allows the primer to bind. DNA polymerase can then build on this primer and synthesize DNA using the cloned insert as a template. A small amount of dideoxyadenosine triphosphate (dideoxy ATP) is included in the DNA synthesis reaction, which already contains all four normal deoxyribonucleotides. Because normal 2′deoxyadenosine is present at high concentration, chain elongation usually proceeds normally. However, the sequencing reaction contains just enough dideoxy ATP to make DNA polymerase occasionally substitute the deadend base for the natural one, at which point the chain stops growing. The result is a population of DNA strands of varying size, each one truncated at a different adenine position. Now imagine using four different dideoxynucleotides corresponding to A, T, C, and G, with each dideoxy base tagged with a different-colored fluorescent dye (Fig. 7.29A ). The result will be a series of different-sized strands tagged at their 3′ end with different colors, depending on the base incorporated. Analyzing the results of this reaction by electrophoresis in a single lane of a polyacrylamide gel will separate the various fragments based on their size. A laser and detector positioned at the bottom of the gel reads the individual fragments as they pass (Fig. 7.29B). Because we know the color of each tagged base, we can use a computer to print a series of colored peaks whose order corresponds to the template DNA sequence (Figs. 7.29C and 7.30). Although the fig-
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Figure 7.29 DNA sequencing using fluorescently tagged dideoxynucleotides to randomly stop chain elongation. The steps consist of synthesizing tagged strands using dideoxynucleotides and then separating the strands. A, B. The reaction products are separated based on size using a polyacrylamide gel apparatus that includes a laser and detector to specifically identify the different tagged fragments. C. The bases are then read and printed out as differently colored peaks.
ure depicts a standard polyacrylamide slab gel, the rapid automated DNA sequencers used today for large-scale projects utilize small capillary tube gels.
Sequencing a genome begins by indiscriminately cloning all the fragments of chromosomal DNA that have been randomly generated, either by restriction digestion (for small fragments) or by sonic breakage (for large pieces). The fragments can either be cloned into conventional plasmid vectors, which may only hold 40–50 kbp of insert DNA, or be cloned into super vectors known as bacterial artificial chromosomes (BACs) that can harbor inserts of 150–1,000 kbp. This random “shotgun” cloning approach must be done in such a way as to ensure overlapping cloned fragments. This is important when finally reassembling the DNA sequences read from various fragments into one contiguous sequence representing the entire chromosome.
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Keith Weller, USDA
Sequencing an Entire Genome
Figure 7.30 Rapid DNA sequencing using an automated DNA sequencer. Cletus Kurtzman inspects a yeast DNA sequence from a previous run while Larry Tjarks loads samples of new sequencing reactions at the National Center for Agricultural Utilization Research. The lanes on the screen represent the sequences of different DNA molecules.
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NOTE: Sonication and some restriction enzymes
generate blunt, rather than cohesive ends. These molecules can still be cloned into linearized plasmids that also contain blunt ends. However, the efficiency of cloning blunt end molecules is much lower than with cohesive ends.
ticularly intriguing recent discovery was finding the entire genome of the bacterium Wolbachia (which infects 20% of the world’s insect population) is embedded within the genome of Drosophila, the common fruit fly. The evolutionary implications of this are under intense investigation. TO SU M MAR I Z E: ■
Next, each clone is sequenced using primers that originate from a sequence in the vector. The vector provides a clear starting point and a known sequence to design a primer. Typically, 500–1,000 bases can be read using the Sanger sequencing technique previously described. New primers are then designed based on the previously read sequence, and the sequencing team continues to “walk” along the fragment until its sequencing is complete. An alternative to “walking” along the chromosome is to break large (greater than 5 kb) cloned fragments into many overlapping, smaller fragments, which are then subcloned into another plasmid vector. The entire sequence of the smaller subclones can usually be determined using the vector primers. It is important to remember that both complementary strands must be sequenced in order to verify a fragment’s sequence and that the region should be sequenced at least two or three times. Once the fragment sequences are verified, overlapping sequences from different fragments are identified using supercomputers and assembled in silico (in a computer) into contigs, which are contiguous sequences spanning several fragments. Ideally, each chromosome of a genome will assemble into a single contig. However, if some gaps remain between contigs, focused sequencing can span them. Due to constantly improving technologies, the length of time it takes to sequence an entire genome has shortened remarkably over the past decade. It originally took many years to sequence the 4.6-million-bp genome of E. coli. Today it would take less than a month. The top DNA sequencing laboratories can sequence nearly 10 million bases per month. How the information from sequences is mined will be discussed more in Chapter 8. Suffice it to say, many important discoveries have resulted from this technology. A par-
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DNA restriction enzymes used for DNA analysis cleave DNA at specific recognition sequences, which are usually 4–6 bp in length and produce either a blunt or a staggered cut. Agarose electrophoresis will separate DNA molecules based on their size. Restriction enzyme digested DNA molecules were fi rst cloned into plasmids in the early 1970s. The most commonly used DNA sequencing technique (Sanger technique) uses nucleotides that lack both the 2′ OH and 3′ OH groups to terminate chain elongation. Entire genomes are sequenced by shotgun cloning of overlapping DNA fragments, each of which is sequenced. Overlapping regions are matched and joined by computer analysis until the entire genome is reconstructed.
Concluding Thoughts We used to think of bacteria as simple, single-celled organisms. While they are single cells, they are far from simple. Complexity is evident in the elegant mechanisms they use to organize and replicate their genomes. The next three chapters will expand on this idea and discuss the way bacteria make proteins, regulate genes, exchange DNA, and, in the process, evolve. Knowing how a bacterial cell replicates and genetically controls the repertoire of available proteins and enzymes provides a unique perspective from which to study the physiology of microbial growth, explored in Part 3 of this textbook. Furthermore, understanding the bacterial genome allows us to explore the physiology of pathogenic as well as nonculturable organisms and gain greater insight into the evolutionary diversity of species that we will discuss in Part 4.
CHAPTE R R EVI EW Review Questions 1. What is the difference between vertical and horizon-
tal gene transmission? 2. Explain the structural types of bacterial genomes. What is a structural gene?
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3. Describe
the four functional levels of gene organization. 4. What are the differences between DNA and RNA?
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5. Explain DNA supercoiling. Why is it important to 6. 7. 8. 9.
microbial genomes? Discuss the mechanisms of topoisomerases. What drug targets a topoisomerase? What are the basic mechanisms involved in DNA replication? How does the bacterial cell regulate the initiation of chromosome replication? What is the gamma complex? Primase? DnaB? DnaC? DNA proofreading?
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10. How is the problem of replicating both strands at a
replication fork solved? 11. What is a catenane? What does it have to do with
DNA replication? 12. Explain the polymerase chain reaction. 13. What is rolling-circle replication? 14. What is DNA restriction and modification? Why is it
important to bacteria? Why is it important to forensic scientists? 15. Explain the basic technique of DNA sequencing.
Key Terms antiparallel (226) catenane (241) cloning (250) conjugation (223) contig (254) denature (227) DNA control sequence (222) DNA ligase (240) enhancer (224) exonuclease (239) histone (247) horizontal transmission (222) hybridization (228) intron (247)
Okazaki fragments (234) operon (225) origin (oriC) (234) palindrome (249) phosphodiester bond (226) plasmid (243) polymerase chain reaction (PCR) (250) primase (236) promoter (224) proofreading (239) pseudogene (247) purine (227) pyrimidine (227) quinolone antibiotic (230)
recombination (223) regulon (225) replication fork (233) restriction enzyme (249) restriction site (250) semiconservative (233) shuttle vector (252) sliding clamp (236) structural gene (222) termination (ter) site (234) topoisomerase (230) vertical transmission (222)
Recommended Reading Actis, Luis, Marcelo E. Tolmasky, and Jorge H. Crosa. 1998. Bacterial plasmids: Replication of extrachromosomal genetic elements encoding resistance to antimicrobial compounds. Frontiers in Bioscience 3:D43–62. Amick, Jean D., and Yves V. Brun. 2001. Anatomy of a bacterial cell cycle. Genome Biology 2:1020.1–1020.4. Boeneman, Kelly, and Elliott Crooke. 2005. Chromosomal replication and the cell membrane. Current Opinion in Microbiology 8:143–148. Cohen, Stanley N., Annie C. Chang, Herbert W. Boyer, and Robert B. Helling. 1973. Construction of biologically functional bacterial plasmids in vitro. Proceedings of the National Academy of Science 70:3240–3244. Dame, Remus T. 2005. The role of nucleoid-associated proteins in the organization and compaction of bacterial chromatin. Molecular Microbiology 56:858–870. Falkow, Stanley. 2001. I’ll have chopped liver please, or how I learned to love the clone. ASM News 67:555.
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Forterre, Patrick, Simon Gribaldo, Daniele Gadelle, and Marie-Claude Serre. 2007. Origin and evolution of DNA topoisomerases. Biochimie 89:427–446. Giovannoni, Stephen J., H. James Tripp, Scott Givan, Mircea Podar, Kevin L. Vergin, et al. 2005. Genome streamlining in a cosmopolitan oceanic bacterium. Science 309:1242–1245. Khodursky, Arkady B., and Nicholas Cozzarelli. 1998. The mechanism of inhibition of topoisomerase IV by quinolone antibacterials. Journal of Biological Chemistry 273:27668–27677. Lemon, Katherine P., and Alan D. Grossman. 1998. Localization of bacterial DNA polymerase: Evidence for a factory model of replication. Science 282:1516–1519. Mott, Melissa L., and James M. Berger. 2007. DNA replication initiation: Mechanisms and regulation in bacteria. Nature Reviews Microbiology 5:343–354. Myllykallio, Hannu, Philippe Lopez, Purificación LópezGarcia, Roland Heilig, William Saurin, et al. 2000. Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon. Science 288:2212–2215.
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Rothfield, L.awrence, Sheryl Justice, and Jorge GarciaLara. 1999. Bacterial cell division. Annual Review of Genetics 33:423–428. Stukenberg, P. Todd, Patricia S. Studwell-Vaughn, and Mike O’Donnell. 1991. Mechanism of the sliding β clamp of DNA polymerase III holoenzyme. Journal of Biological Chemistry 266:11328–11334.
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Thanbichler, Martin, Patrick H. Viollier, and Lucy Shapiro. 2005. The structure and function of the bacterial chromosome. Current Opinion in Genetic Development 115:153–162. Vicente, Miguel, Ana Isabel Rico, Rocio Martinez-Arteaga, and Jesus Mingorance. 2006. Septum enlightenment: Assembly of bacterial division proteins. Journal of Bacteriology 188:19–27.
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Chapter 8
Transcription, Translation, and Bioinformatics 8.1 8.2 8.3 8.4 8.5 8.6 8.7
RNA Polymerases and Sigma Factors Transcription Initiation, Elongation, and Termination Translation of RNA to Protein Protein Modification and Folding Secretion: Protein Traffic Control Protein Degradation: Cleaning House Bioinformatics: Mining the Genomes
The cell accesses the vast store of data in its genome through transcription, which is the reading of a DNA template to make an RNA copy, and translation, the decoding of RNA to assemble protein. The molecular machines that carry out these processes have been studied relentlessly, yet they still hold secrets. After translation, each polypeptide must be properly folded and placed at the correct cellular or extracellular location. How does the cell do this? And what does the cell do with proteins it no longer needs? As we will discuss, elegant chaperone pathways help fold proteins; complex transport systems move proteins out of the cytoplasm; and regulated proteolytic systems properly dispose of unneeded or damaged proteins. What emerges is a picture of remarkable biomolecular integration, homeostatically controlled to maintain balanced growth and ensure survival.
The Escherichia coli DegP protein is a periplasmic protease that degrades misfolded proteins including “sentry” regulators that control certain stress responses. It has been hypothesized that unfolded polypeptides are threaded in an extended conformation into the cage to access the proteolytic sites. The image is a close-up view of the inner cavity of DegP. Source: Ahmad Jomaa, et al. 2007. Journal of Bacteriology 189:706; © 2007, American Society for Microbiology. All Rights Reserved.
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By 1960, it was clear that the genetic code was embedded in DNA, but the code itself remained a mystery, as was the mechanism by which the code produced protein. RNA was a suspected, but unproven, intermediate. Marshall Nirenberg and Heinrich Matthaei, a postdoctoral student of Nirenberg’s at the National Institutes of Health, set out in 1959–1960 to design a cell-free system (a cell lysate of E. coli) to test the hypothesis (Fig. 8.1A). Key to their investigation was the use of synthetic RNA molecules containing simple, known, repeated sequences such as poly-A (consisting only of adenylic acid), poly-U (polyuridylic acid), poly-AAU, and poly-AAAAU. These RNA molecules were tested in a cell-free protein-synthesizing system to see if the repetitive sequence might direct incorporation of a specific amino acid into a protein. Each synthetic polynucleotide was tested in the presence of a radiolabeled amino acid. If the radioactive amino acid was incorporated into a polypeptide, the polypeptide would also be radioactive. On the morning of May 27, 1960, the results of experiment 27Q indicated that the poly-U RNA specified the assembly of radioactive poly-phenylalanine. It was the fi rst break in the genetic code. B.
Courtesy of National Library of Medicine/ National Institute of Health
Courtesy of Maxine Singer
A.
Many scientists contributed to understanding the genetic code. A. Heinrich Matthaei (left) and Marshall Nirenberg (right) were first to crack the genetic code. An early hand-drawn model of what would eventually be known as translation is behind them. B. Maxine Singer, a key contributor to the genetic code experiments, also helped develop guidelines for recombinant DNA research.
Figure 8.1
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Nirenberg reported the result at a conference in Moscow at the height of the Cold War. News that Nirenberg’s poly-U experiment had determined the fi rst “word” of the genetic code was an international media event. In January 1962, the Chicago Sun-Times announced: “No stronger proof of the universality of all life has been developed since Charles Darwin’s The Origin of Species . . . In the far future, the . . . hereditary lineup will be so well known that science may deal with the aberrations of DNA . . . that produce cancer, aging, and other weaknesses of the flesh.” This statement was truly prophetic, for understanding the genetic code was the fi rst step in discovering the basis of many genetic diseases. For his work, Nirenberg, along with Har Gobind Khohara (who developed methods for making synthetic nucleic acids) and Robert Holley (who solved the structure of yeast transfer RNA), won the 1968 Nobel Prize in Physiology or Medicine. Maxine Singer, who also played an important role in deciphering the genetic code by synthesizing the RNA molecules used in the Nirenberg experiments, became a leader in developing guidelines for recombinant DNA research (Fig. 8.1B). Since Nirenberg’s breakthrough, we have learned a tremendous amount about genes and proteins and, in this electronic age, we can use the information in ways never before possible. A new discipline called bioinformatics uses powerful computer technologies to store and analyze gene and protein sequences. Bioinformatic programs can compare a new gene sequence with hundreds of thousands of known genes. These comparisons help predict for any given microbe what genes are present, what proteins are made, and even what food it consumes. Patterns in DNA sequences across species also allow us to pose new questions about microbial life, disease, and evolution. In this chapter, we use E. coli as a paradigm to explore the way microbes interpret the nucleotide sequence of DNA and convert it to proteins. From there we look at what the cell does with those proteins once they are made. Specific proteins are targeted for movement into the periplasm, while other proteins must be inserted into membranes. We also show how damaged proteins are selectively degraded. We conclude with bioinformatics, now an essential tool of the new microbiology. Chapters 9 and 10 then discuss how microbes respond to their environment by controlling the expression of their genes.
8.1
RNA Polymerases and Sigma Factors
To survive and reproduce, every cell needs to form proteins using the information encoded within DNA. Chromosomal DNA is large and cumbersome, so the first step in the process is to make multiple copies of the information in snippets of RNA that can move around the cell and, like
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disposable photocopies, be destroyed once the encoded protein is no longer needed. This copying of the DNA to RNA is called transcription. Much of what we have learned about transcription has come from studying E. coli. However, bioinformatic analyses indicate that the mechanisms and components of transcription in other bacterial species are similar to those characterized in E. coli.
RNA Polymerase Transcribes DNA to RNA An enzyme complex called RNA polymerase, also known as DNA-dependent RNA polymerase, carries out the process of transcription, making RNA copies (called transcripts) of a DNA template. The DNA template strand specifies the base sequence of the new complementary strand of RNA. Transcription involves (1) initiation, which is the binding of RNA polymerase to the beginning of the gene, followed by melting of the helix and synthesis of the fi rst nucleotide of the RNA; (2) elongation, the sequential addition of ribonucleotides from nucleoside triphosphates; and (3) termination, in which
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sequences at the end of the gene trigger release of the polymerase and the completed RNA molecule. RNA polymerase in bacteria consists of a “core polymerase,” which is a protein complex containing the essential components required for elongation of the RNA chain, plus a sigma factor, a protein needed only for initiation of RNA synthesis, not for its elongation. Core polymerase plus sigma factor together are called “holoenzyme.” Core RNA polymerase is a complex of four different subunits: two alpha (α) subunits, one beta (β) subunit, and one beta-prime (β′) subunit for every core enzyme (Fig. 8.2). The beta-prime subunit contains the Mg2+ -containing catalytic site for RNA synthesis, as well as sites for the ribonucleotide and DNA substrates and the RNA products. The three-dimensional structure of RNA polymerase resembles a hand, whose “fi ngers” consist of the beta and beta-prime subunits (Fig. 8.3). DNA fits into a cleft formed by the beta and beta-prime subunits (Fig. 8.2). The alpha subunit assembles the other two subunits into a functional complex and communicates through physical “touch” with
DNA channel
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Figure 8.2 Subunit structure of core RNA polymerase. Two stereo-image views of RNA polymerase. The channel for the DNA template is shown by the yellow line. The molecule is rotated as shown. Subunits are color-coded. Omega is a subunit of unclear function sometimes associated with RNA polymerase. To view the stereo-images, place a piece of cardboard vertically between the two images. Position your eyes on opposite sides of the cardboard and force them to focus behind the images. The images will merge and produce a 3-D image. (PDB code: 1HQM) Source: Robert D. Finn, Elena V. Orlova, Brent Gowen, Martin Buck, and Marin van Heel. 2000. Escherichia coli RNA polymerase core and holoenzyme structures. EMBO J. 19(24):6833–6844.
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Three-dimensional structure of RNA polymerase. RNA polymerase holoenzyme. Note the open channel able to accept DNA. To view the stereo-image, follow the instructions from Figure 8.2. (PDB code: 1L9U)
Figure 8.3
various regulatory proteins that can bind DNA. The resulting protein-protein communications instruct RNA polymerase in what to do after binding DNA. Promoter binding. Where does a gene begin and end?
The helical structure of a DNA sequence appears largely homogeneous compared to proteins, which bend and fold in complex ways. In fact, without sigma factor, the core RNA polymerase binds and releases DNA at random. Yet there must be a mechanism that tells RNA polymerase where a gene starts, because random transcription is extremely wasteful. The proteins responsible for guiding RNA polymerase to genes are the sigma (σ) factors (see Fig. 8.3B). A sigma factor binds RNA polymerase through the alpha subunit and then helps the core enzyme detect a specific DNA sequence, called a promoter, which signals the beginning of a gene. We will discuss how sigma recognizes a promoter later. A single bacterial species can make several different sigma factors, with each sigma factor helping core RNA polymerase find the start of a different subset of genes. However, a single core polymerase complex can bind only one sigma factor at a time. By acting as a sort of “seeingeye” protein, sigma factors help RNA polymerase recognize different classes of promoter sequences. The specific sigma factor used to initiate transcription of a given gene will vary, depending on the gene and on the environmental signals needed to initiate transcription of that gene. However, regardless of which sigma factor is used, core RNA polymerase is required for all gene transcription. Sigma factors regulate major physiological responses.
Individual sigma factors coordinately control genes involved in nitrogen metabolism, flagellar synthesis, heat stress, starvation, sporulation, and many other physiological responses. So far, over 100 sigma factor genes have been sequenced from numerous species. While they all have different amino acid sequences, they also show
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sequence similarities that make them recognizable as sigma factors. The DNA sequence recognized by a given sigma factor can be determined by comparing known promoter sequences of different genes whose expression requires the same sigma. The DNA sequence similarities seen when comparing these different promoters defi nes a consensus sequence likely recognized by the sigma factor (Fig. 8.4A). A consensus sequence consists of the most likely base (or bases) at each position of the predicted promoter. Note that while the promoter is a dsDNA sequence, convention is to present the promoter as the ssDNA sequence of the sense strand, which matches the RNA product sequence. The best consensus sequences are based on a large set of promoters that use the same sigma factor, or other regulator. Some positions in a consensus sequence are highly conserved—that is, the same base is found in that position in every promoter. Other, less conserved positions can be occupied by different bases. Few promoters will actually have the most common base at every position. Even highly efficient promoters usually differ from the consensus at one or two positions. Microbes are guarded by an array of protein sensors that continually sample the internal and external environments. These sensors “look” for chemical deficiencies or dangers and, when triggered, direct the cell to increase synthesis or decrease destruction of the appropriate sigma factor. Accumulation of a specialty sigma factor dislodges other sigma factors from core polymerase and redirects RNA polymerase to the promoters of genes whose products can best solve the problem sensed. Regulation of multiple genes by a single sigma factor enables the cell to coordinately control those genes simply by regulating when a given sigma factor accumulates (Section 10.4). This control is critical to survival in constantly changing environments. THOUGHT QUESTION 8.1 If each sigma factor recognizes a different promoter, how does the cell manage to transcribe genes that respond to multiple stresses, each involving a different sigma factor? Every cell has a “housekeeping” sigma factor that keeps essential genes and pathways operating. In the case of E. coli and other gram-negative rod-shaped bacteria, that factor is sigma-70, so named because it is a 70-kilodalton (kDa) protein (its gene designation is rpoD). Genes recognized by sigma-70 all contain similar promoter consensus sequences consisting of two parts. The DNA base corresponding to the start of the RNA transcript is called nucleotide +1 (+1 nt). Relative to this landmark, the consensus promoter sequences are characteristically centered at –10 and –35 nt before the start of transcription (see Fig. 8.4). Other sigma factors typically recognize different
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A. Strong E. coli promoters tyr tRNA rrn D1 rrn X1 rrn (DXE)2 rrn E1 rrn A1 rrn A2 λ PR λ PL T7 A3 T7 A1 T7 A2 fd VIII
–35 –10 +1 T C T C A A C G T A A C A C T T T A C A G C GG C G • • C G T C A T T T G A T A T G A T G C • G C C C C G C T T C C C G A T A A GGG G A T C A A A A A A A T A C T T G T G C A A A A A A • • T T GGG A T C C C T A T A A T G C G C C T C C G T T G A G A C G A C A A C G A T G C A T T T T T C C G C T T G T C T T C C T G A • • G C C G A C T C C C T A T A A T G C G C C T C C A T C G A C A C GG C GG A T C C T G A A A T T C A GGG T T G A C T C T G A A A • • G A GG A A A G C G T A A T A T A C • G C C A C C T C G C G A C A G T G A G C C T G C A A T T T T T C T A T T G C GG C C T G C G • • G A G A A C T C C C T A T A A T G C G C C T C C A T C G A C A C GG C GG A T T T T T A A A T T T C C T C T T G T C A GG C C GG • • A A T A A C T C C C T A T A A T G C G C C A C C A C T G A C A C GG A A C A A G C A A A A A T A A A T G C T T G A C T C T G T A G • • C GGG A A GG C G T A T T A T G C • A C A C C C C G C G C C G C T G A G A A T A A C A C C G T G C G T G T T G A C T A T T T T A • C C T C T GG C GG T G A T A A T GG • • T T G C A T G T A C T A A GG A GG T T A T C T C T GG C GG T G T T G A C A T A A A T A • C C A C T GG C GG T G A T A C T G A • • G C A C A T C A G C A GG A C G C A C G T G A A A C A A A A C GG T T G A C A A C A T G A • A G T A A A C A C GG T A C G A T G T • A C C A C A T G A A A C G A C A G T G A T A T C A A A A A G A G T A T T G A C T T A A A G T • C T A A C C T A T A GG A T A C T T A • C A G C C A T C G A G A GGG A C A C G A C G A A A A A C A GG T A T T G A C A A C A T G A A G T A A C A T G C A G T A A G A T A C • A A A T C G C T A GG T A A C A C T A G G A T A C A A A T C T C C G T T G T A C T T T G T T • • T C G C G C T T GG T A T A A T C G • C T GGGGG T C A A A G A T G A G T G
B. Consensus sequences of σ70 promoters –35 region –10 region 17 ± 1bp TATAAT T T GACA T C. Sequence of lac promoter –10 region –35 region T A TGT T T T T ACAC A
AA
Down
Up
A
D. Sigma factor
Promoter recognized
Promoter consensus sequence –35 region
–10 region
RpoD σ
Most genes
T T GACA T
TATAAT
RpoH σ32
Heat-shock-induced genes
T C T C N CCC T T G A A
CCCC A T N T A
RpoF σ28
Genes for motility and chemotaxis
RpoS σ38
Stationary phase and stress response genes
70
RpoN σ54
Genes for nitrogen metabolism and other functions
CTAAA T T GACA –24 region C T GGN A
consensus sequences at one or both of these positions (or at nearby locations in some cases); or they shape the overall polymerase complex to bind the promoter. How do sigma factors, or any other DNA-binding protein for that matter, recognize specific DNA sequences when the DNA is a double helix? The phosphodiester backbone is quite uniform, and the bases appear inaccessible owing to base pairing. Recognition of DNA sequences is possible, however, because the alpha-helical portions of DNA-binding proteins recognize base side groups (noncovalent binding) that protrude from the base into the major and minor grooves of DNA. Figure 8.5A illustrates how portions of sigma-70 from E. coli wrap around DNA allowing certain parts of the protein to fit into DNA grooves. A key to understanding how sigma factors recognize different promoters is their structure. Based on amino acid sequence comparisons, sigma factors generally contain four
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–10 and –35 sequences of E. coli promoters. A. Alignment of sigma-70 (σ70)dependent promoters from different genes is used to generate a consensus sequence in panel B (yellow indicates conserved nucleotides, brown denotes transcript start site). B. Consensus sequences of σ70-dependent promoters (red-shaded letters indicate nucleotide positions where different promoters show a high degree of variability). C. Mutations in the lac promoter that affect promoter strength (lac genes encode proteins used to metabolize the carbohydrate lactose). Some mutations can cause decreased transcription (called down mutations), while others cause increased transcription (up mutations). D. Some E. coli promoter sequences recognized by different sigma factors. Figure 8.4
CCG A T A T T C T A T AC T T –12 region T T GC A
highly conserved regions (Figs. 8.5A and B). Part of region 2 of the sigma-70 family recognizes –10 sequences, while region 4 recognizes the –35 sites. A part of region 1 helps separate the DNA strands in preparation for RNA synthesis. The structure of the holoenzyme RNA polymerase complex positioned at a promoter is shown in Figure 8.5C.
THOUGHT QUESTION 8.2 With respect to two different sigma factors with different promoter recognition sequences, predict what would happen to the overall gene expression profile in the cell if one sigma factor were artificially overexpressed? Could there be a detrimental effect on growth? THOUGHT QUESTION 8.3 Why might some genes contain multiple promoters, each one specific for a different sigma factor?
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A. E. coli sigma factor binds to two sequences in a DNA promoter. Region 2 of sigma binds to the –10 region of promoters, while region 4 binds to the –35 promoter region. Cyan marks part of core RNA-P. Purple on DNA denotes the –10 and –35 promoter regions. B. Sigma-70 amino acid homology and domain interactions with different regions of the promoter sequence. Conserved amino acid regions within examples of the sigma-70 family. C. Taq holoenzyme/promoter DNA complex. Double-stranded DNA is shown as atoms. The possible locations of the two alpha subunit C-terminal domains (drawn as gray spheres, labeled I and II) on the DNA UP elements is illustrated. The UP element is a DNA sequence that increases transcription. (PDB codes: 1L9Z, 1K8J) Source: A. Seth A. Darst. 2001. Bacterial RNA
Figure 8.5
A. –10
σ-factor region 2
DNA –35
σ-factor region 4
polymerase. Current Opinion in Structural Biology 11:155–162.
σ-factor region 3 B. Factor
Region –10 Binding 2
1 Ec σ70 Bs σ43 Ec σ32 Bs σ28 Bs σ37 Bs σF Bs σ29 Bs σA Bs σK SP01gp28 SP01gp34 T4gp55
E. coli Bacillus subtilis E. coli
Bacillus subtilis
B. subtilis bacteriophage T4 phage
3
–35 Binding 4
housekeeping housekeeping, major heat shock sporulation stress sporulation sporulation vegetative growth sporulation-specific virus-encoded sigma virus-encoded sigma virus-encoded sigma
N and C termini, and numbers of residues N 1
100
200
300
C 375
C. –10 Extended –10 element element –35 element –30
–10
–20
Transcription start site +1
+10
Transcription downstream
Template 5′
–40 UP
σ2 I II
β1
Nontemplate
Up
st
re
–60 5′
3′
σ2
am
–50
σ3
αI
3′
Mg2+ αII
β2 Sigma factor regions visible in this orientation
β′
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■
■
RNA polymerase holoenzyme consisting of core RNA polymerase and a sigma factor executes transcription of a DNA template strand. Sigma factors help core RNA polymerase locate consensus promoter sequences near the beginning of a gene. The sequences identified and bound by E. coli sigma-70 are located –10 and –35 bp upstream of the transcription start site. Dynamic changes in gene expression result when the relative levels of different sigma factors change.
8.2
Transcription Initiation, Elongation, and Termination
Sigma binding is the fi rst step of transcription initiation. Once the promoter has been activated by binding with its sigma factor, RNA polymerase completes the process of
1. RNA polymerase holoenzyme scans DNA for promoter sequences. RNA polymerase holoenzyme
3′ 5′
β′
α α
“Scanning”
β 5′
σ –35
2a. Binding to the promoter sequence forms the closed complex.
α α
3′ 5′
–10 Promoter
β′
Closed 3′ complex
–10
P Pi
mRNA
α β
Core enzyme
3′
5′
5′
α
Open 3′ complex
β′
Transcription bubble σ 3b. Sigma factor leaves the complex.
Figure 8.6
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Transcription initiation.
263
initiation, in which the closed RNA polymerase complex becomes an open complex. In elongation, the RNA chain is extended, followed by termination, in which the RNA polymerase comes apart from the DNA.
Initiation of Transcription RNA polymerase constantly scans DNA for promoter sequences (Fig. 8.6, step 1). It binds DNA loosely and comes off repeatedly. Once bound to the promoter, RNA polymerase holoenzyme forms a loosely bound, closed complex with DNA, which remains annealed and double-stranded (that is, unmelted) (step 2). To successfully transcribe a gene, this closed complex must become an open complex through the unwinding of one helical turn, which causes DNA to become unpaired in this area (step 3). After promoter unwinding, RNA polymerase in the open complex becomes tightly bound to DNA. Region 1 of the sigma factor is important for DNA unwinding, as is a “rudder” complex in β′ that separates the two stands (shown in Fig. 8.7A). The open complex form of RNA polymerase begins transcription. The first ribonucleoside triphosphate (rNTP) of the new RNA chain is usually a purine. It base-pairs to the DNA template designated position +1, marking the start of the gene. As the enzyme complex moves along the template, subsequent NTPs diffuse through a channel in the polymerase and into position at the DNA template (see Fig. 8.7B). After the fi rst base is in place, each subsequent ribonucleoside triphosphate transfers a nucleoside monophosphate to the growing chain while releasing a pyrophosphate: O RNA 3′ chain
OH
+
–
O
O O
P O
5′
rNTPs (ribonucleotides)
3a. RNA polymerase unwinds DNA and begins transcribing RNA from ribonucleoside triphosphates (rNTPs). 5′
2b. The antibiotic rifamycin will bind to this form of polymerase and prevent formation of the subsequent open complex.
β
σ
–35
3′
■ Genes and Ge n o m e s
–
O
O O
P –
O
P O
nucleoside
O
O RNA 3′ chain
O
P O–
O
nucleoside
3′
3′
OH
–
OH
+
–
O P O–
O O
P
O–
O–
The energy released by cleaving the rNTP triphosphate groups (phosphoric anhydride bond) is used to create the phosphodiester link to the growing polynucleotide chain. As seen in Figure 8.7, the β′ subunit forms part of the pocket, or cleft, in which the DNA template is read. Another part of the β′ cleft is involved in hydrolyzing ribonucleoside triphosphates. Deep at the base of the cleft is the active site of polymerization, defi ned by three evolutionarily conserved aspartate residues that chelate (that is, bond with) two magnesium ions (Mg2+). As we saw for DNA polymerases, these metal ions play a key role in catalyzing the polymerization reaction.
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A.
β′C rudder
DNA enters
Three-dimensional view of transcription by Thermus aquaticus (Taq) RNA polymerase showing DNA, RNA, and the position of the rudder. The process is shown from two different angles. Transcription (polymerase movement on the DNA) is going from right to left in panel A and the opposite in panel B. Color-coded molecular surfaces are as follows: beta subunit, green; beta G flap in red, beta-prime C rudder in gold; alpha and omega, not seen in this view; the DNA template (template strand, purple; nontemplate strand, gray); and the RNA transcript (blue). The directions of the entering downstream DNA and exiting upstream DNA are indicated (black arrows). A. A view perpendicular to the main active site channel, which runs roughly horizontal. Parts of the protein structure are colored. B. In this view, the structure is rotated 90° relative to view (A). With the entering DNA removed for clarity, Mg2+ is seen in the catalytic site; the secondary channel where nucleotides (orange) enter and a part of the RNA strand (blue) are also seen. (PDB codes: 1L9Z, 1K8J) Source: A. Seth A. Darst. 2001. Bacterial RNA polymerase. Current Opinion in Figure 8.7
Nontemplate DNA loops away
DNA exits
–14 RNA exits behind
Structural Biology 11:155–162.
90°
Secondary channel
B.
DNA enters
the template, synthesizing RNA at approximately 45 bases per second. The unwinding of DNA ahead of the moving complex forms a 17-bp transcription bubble. Because of this unwinding, positive supercoils are formed ahead of the advancing bubble. These positive supercoils are removed by enzymes that return the DNA to its normal negative supercoiled state (see Section 7.2). Termination of transcription. We have learned how
DNA exits Mg2+
Secondary channel
Elongation of RNA transcripts. The sigma factor
remains associated with the transcribing complex until about nine bases have been joined; then it dissociates (see Fig. 8.6). The newly liberated sigma factor can then reassociate with an unbound core RNA polymerase to direct another round of promoter binding. Meanwhile, the original RNA polymerase continues to move along
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RNA polymerase recognizes the beginning of a gene and transcribes RNA, but how does it know when to stop? Again, the secret is in the sequence. All bacterial genes use one of two known transcription termination signals. One termination mechanism, called Rho- dependent, relies on a protein called Rho and an ill-defi ned sequence at the 3′ end of the gene that appears to be a strong pause site. Pause sites are areas of DNA that slow or stall RNA polymerase movement. Some pause sites occur within the gene’s open reading frame (ORF), the part of a gene that actually encodes a protein, to allow time for the ribosome to catch up to a transcribing RNA polymerase (discussed in Section 8.3). The transcription termination pause site, however, is located after the ORF, beyond the translation stop codon, because if transcription were to cease before the ribosome reaches the translation stop codon, an incomplete protein would be made. Rho factor binds to an exposed region of RNA after the ORF at GC-rich sequences that lack obvious secondary structure. This is the transcription terminator pause site. Rho monomers assemble as a hexamer around the RNA (Fig. 8.8A). Then, like a raft pulled to shore by winding a rope around a piling, Rho pulls itself to the paused RNA
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A. Rho-dependent termination Translational stop
5′
RNA polymerase Pause site
Rho binds to GC-rich regions.
■ Genes and G e n o m e s
265
poly-A base pairs at the 3′ terminus contain only two hydrogen bonds per pair and are usually weak (that is, easier to melt). The melting of the hybrid molecule releases the transcript and halts transcription. The pause in polymerase movement, and thus transcription, is important to prevent the formation of tighter base pairing downstream of the UA region.
5′
Antibiotic Resistance Helped Reveal Components of RNA Synthesis Machines
Translational stop 5′ RNA wraps around Rho hexamer, pulling Rho toward RNA polymerase. 5′
Contact between Rho and RNA polymerase causes termination.
B. Rho-independent termination 5′
RNA polymerase Movement of polymerase Translational stop GC-rich terminator stem loop
5′
Figure 8.8
Contact between hairpin and RNA polymerase causes termination.
Transcription termination.
polymerase by wrapping downstream RNA around itself via an intrinsic ATPase activity. Once Rho touches the polymerase, an RNA-DNA helicase activity built into Rho appears to unwind the RNA-DNA heteroduplex, which releases the completed RNA molecule and frees the RNA polymerase. The second type of termination event, called Rhoindependent, occurs in the absence of Rho or any other protein cofactors. Rho-independent termination requires a GC-rich region of RNA roughly 20 bp upstream from the 3′ terminus as well as four to eight consecutive uridine residues at the terminus. The GC-rich sequence contains complementary bases and forms an RNA stem, or stem loop, a structure that “grabs” RNA polymerase, causing it to pause (Fig. 8.8B). While the polymerase is paused, the DNA-RNA duplex is weakened because the poly-U,
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How did scientists determine the details of RNA and protein synthesis? The processes were observed in cellfree systems. For example, transcription and translation were observed in the test tube using purified RNA polymerase and ribosomes. In addition, the various protein subunits of RNA polymerase were separated from each other and mixed together again to make an active complex. One particularly clever strategy was based on the genetics of antibiotic resistance. We now know that many antibiotics target specific components of the bacterial transcription and translation machinery. However, organisms, such as E. coli mutate at low frequency to become resistant to a given antibiotic. The resistance mutation often affects the part of the RNA or protein synthesis machine that binds to the antibiotic. The effect is a decreased affi nity of that altered protein for the antibiotic. The subunits that conferred resistance to particular antibiotics were determined in cell-free systems. For example, rifamycin is an antibiotic that inhibits transcription (Special Topic 8.1). To determine which RNA polymerase subunit was targeted by the drug, RNA polymerases from rifamycin-sensitive and -resistant strains were purified, the component parts of the polymerase were separated, and then a chimeric RNA polymerase was reassembled using all but one of the protein subunits from the sensitive strain. The missing subunit was supplied by the resistant strain and the polymerase preparation tested in vitro for an ability to make RNA in the presence of rifamycin. This will happen only when the subunit conveying resistance has been added—in this case, the beta subunit. Subsequent in vitro assays and X-ray crystallography used this information to learn more about the enzymatic mechanism of this subunit.
Different Classes of RNA Have Different Functions Now that we have shown how RNA is synthesized, it is important to note that not all RNA molecules are translated to protein. Six known classes of RNA are made. Each
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Table 8.1 Classes of RNA in E. coli.a RNA class
Function
Number of types
Average size
Approx. half-life
Unusual bases
mRNA (messenger RNA) rRNA (ribosomal RNA)
Encodes protein
Thousands
1,500 nt
3–5 minutes
No
Synthesizes protein
Three
Hours
Yes
Shuttles amino acids
27 typeset (86 genes) 20–30
5S, 120 nt; 16S, 1,542 nt; 23S, 2,905 nt 80 nt
Hours
Yes
intragastrica Complex Simple Particulate Soluble Denatured Native Many Few Effective Ineffective
Differences from self proteins Interactions with host MHC proteins
Low immunogenicity
a
Route of inoculation affects immunogenicity of a given antigen. In general, subcutaneous inoculation gives the strongest immune response while intragastric yields the weakest response.
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C. Varicella
and the only available preventive treatment was to take dried material from lesions of a previous smallpox sufferer, place it on a healthy person, and hope the person survived. Those who survived were protected from subsequent bouts of smallpox but were still susceptible to other diseases. This early observation gave rise to the idea of immunological specificity, which means that an immune response to one antigen is not effective against a different antigen. In other words, the immune response to smallpox will not protect someone against the plague bacillus (Yersinia pestis), which is antigenically different from smallpox. While immunological specificity is important, it is not absolute. As described in Chapter 1, an English country physician named Edward Jenner (see Fig. 1.19) in the late eighteenth century (long before viruses were discovered) learned to protect townsfolk from deadly smallpox disease by inoculating them with scrapings from lesions produced by a tamer disease, cowpox. By this process, Jenner unwittingly transferred the vaccinia virus (which we now know causes cowpox; see Fig. 24.3C) to villagers susceptible to smallpox (caused by the related but more dangerous, variola virus). The resulting immune reaction to vaccinia produced an effective cross-protection against variola and thus prevented smallpox. This illustrates that an immune reaction against one organism or virus may be sufficient to protect against an antigenically related, if not identical, organism. The technique of exposing individuals to “tame” microbes to protect them against pathogens, now generally called vaccination, has been used to protect humans against many microbial and viral pathogens (Table 24.2). Most vaccinations today involve administering crippled (attenuated) strains of the pathogenic microbe or inactivated microbial toxins (for example, diphtheria toxin). Cross-protection, where immunization against one microbe protects against a second, will work only if two
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proteins critical to the pathogenesis of the two different microorganisms share key antigenic determinants. No cross-protection occurs if these determinants differ significantly. A good example is the common cold, which is caused by hundreds of closely related rhinovirus strains (rhinitis, a runny nose, is one of the symptoms of this viral disease). Infection with one strain will not immunize the victim against a second strain. The reason is that the structures of the viral proteins used to attach to the ICAM1 protein on host cells differ dramatically between different strains of rhinovirus (Fig. 24.4A). Antibodies called neutralizing antibodies, which bind to the attachment protein on one strain of rhinovirus, will prevent infection by the same virus strain (Fig. 24.4B) but will not bind a similar but antigenically distinct ICAM-1 receptor protein from a different strain. A key to one lock will not work on a different lock. CDC/PHIL/Corbis
Immunological specificity is the basis of vaccination. A. Photo of a smallpox patient, showing the white pox pustules. B. The smallpox virus, variola major (300 nm long, TEM). C. The vaccinia virus that causes cowpox (360 nm long, electron micrograph). Edward Jenner recognized the similarity between the deadly smallpox and less severe cowpox diseases and used cowpox scrapings to vaccinate humans against smallpox.
Figure 24.3
899
B. Variola major
CDC
A. Smallpox patient
■ M edic ine and I mm u n o l o gy
THOUGHT QUESTION 24.2 How does a neutralizing antibody that recognizes a viral coat protein prevent infection by the associated virus?
Antigens, Immunogens, and Haptens Karl Landsteiner (1868–1943) discovered the ABO blood group system in 1901, for which he received the 1930 Nobel Prize in Physiology or Medicine (Fig. 24.5A). He observed that red blood cells (RBCs) from a type A individual, whose RBCs contained the A antigen, were destroyed (lysed) when transfused into a type B person, whose RBCs contained the B antigen. However, type A RBCs remained intact when introduced into another type A individual, and type B RBCs were undamaged when introduced into a type B individual (Fig. 24.5B). This is the phenomenon of blood group incompatibility, which occurs when specific antibodies in the serum of an individual of one blood type bind to antigens on red blood cells of a different type (that is, foreign RBCs). We now know that type B individuals carry specific anti-A antibodies in their bloodstream, while type A individuals carry specific anti-B antibodies. (Type AB people carry neither antibody, and type O individuals, whose RBCs contain neither A nor B antigen, carry both anti-A and anti-B antibodies.) The discovery by Landsteiner defined the basic concept of immunological specificity and explains why a type A person cannot donate blood to a type B individual, and vice versa. It also explains why a type O person (called a universal donor) can donate to type A, B, AB, or O individuals.
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Table 24.2 Vaccines against viral and bacterial pathogens. Disease
Vaccine
Vaccination recommended for:
Attenuated strain (will still replicate) Inactivated virus (will not replicate) Viral antigen Inactivated virus or antigen Attenuated viruses; MMR combined vaccine
Children 12–18 months Travelers to endemic areas Medical personnel, children 1–18 months Adults over 65 years Children 15–19 months
Attenuated (oral, Sabin) Inactivated (injection, Salk) Inactivated virus Attenuated virus
Children 2–3 years
Viral Chickenpox Hepatitis A Hepatitis B Influenza Measles Mumps Rubella Polio Rabies Yellow fever
Persons in contact with wild animals Military personnel
Bacterial Anthrax Cholera Diphtheria Pertussis Tetanus Haemophilus influenzae type b (meningitis) Lyme disease Meningococcal disease Pneumococcal pneumonia Tuberculosis (Mycobacterium tuberculosis) Typhoid fever Typhus fever
B. anthracis, components of toxin Unencapsulated strain Vibrio cholerae, components Toxoid (inactivated toxin) Acellular Bordetella pertusis Toxoid Bacterial capsular polysaccharide
Children under 5 years
Lipoprotein OspA surface antigen, Borrelia burgdorferi Bacterial capsular polysaccharides, Neisseria meningitidis Bacterial capsular polysaccharides, Streptococcus pneumoniae
Individuals in endemic areas
Attenuated Mycobacterium bovis (BCG vaccine) Killed Salmonella typhi Killed Rickettsia prowazekii
Exposed individuals Individuals in endemic areas Medical personnel in endemic areas and scientists
Antibodies prevent rhinovirus attachment to cell receptors. A. This figure illustrates the complexity of the rhinovirus capsid and shows the attachment of the virus to the cell surface molecule ICAM-1 (intercellular adhesion molecule, shown in reddish brown). (PDB code: 1rhi) B. This figure shows rhinovirus coated with protective (neutralizing) antibodies (green) that block the ICAM-1 receptors on the virus. As a result, the virus fails to attach to and infect the host cell. (PDB code: 1rvf)
Figure 24.4
Agricultural and veterinary personnel, key health care workers Travelers to endemic areas Children 2–3 months
A. Rhinovirus
Military and high-risk individuals Adults over 50 years
B. Antibody-coated rhinovirus
Cell receptor (ICAM-1) Cell
Antibody to virus receptor protein
900
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■ M edic ine and I mm u n o l o gy
901
B.
Karl Landsteiner. The Specificity of Serological Reactions. Dover Publications
Type A
Type B RBCs from a type A person are coated with A antigen. RBCs from a type B person are coated with B antigen.
A antigen
Transfusion
Type A blood contains anti-B antibodies. Type B blood contains anti-A antibodies. Anti-A antibody
Anti-B antibody The anti-A antibodies in a type A person will attack and destroy transfused type B red blood cells.
C.
Carrier-specific antibodies
Protein carrier The small hapten molecule benzene cannot stimulate antibody production in a mouse.
No response
Benzene hapten
Carrier-specific and hapten-specific antibodies
Protein-hapten complex Attaching the hapten to a larger carrier molecule (e.g., BSA protein) will result in production of antibodies to both the carrier and the hapten molecules.
Landsteiner wanted to better understand this idea of immunological specificity. Because at that time nothing was known about antigen or antibody structure, he chose to study very simple molecules that, by themselves, were too small to elicit antibodies. Molecules of molecular weight less than 1,000 are generally not immunogenic (we now know the reason is that they do not bind MHC molecules);
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B antigen
Type A RBC is lysed
Karl Landsteiner, ABO blood groups, and haptens. A. Karl Landsteiner discovered ABO blood groups and haptens. B. Individuals with type A antigens on their red blood cells possesses antibodies to B antigen, while type B individuals have B antigens on RBCs and carry anti-A antibodies. Transfusing RBCs from a type B individual into a type A individual will lead to the anti-B antibodies attacking and destroying the type B red blood cells. C. Basic concept of a hapten.
Figure 24.5
however, Landsteiner discovered that these small molecules would elicit production of specific antibodies if they were covalently attached to a larger carrier protein or other molecule. He called these types of small antigens haptens (derived from the Greek word meaning “to fasten” and the German word for “stuff”). Haptens can be thought of as small incomplete antigens. An example of a hapten is the antibiotic penicillin, a serious cause of immune hypersensitivity reactions in some individuals (see Section 24.8). Landsteiner chose to work with benzene haptens with sulfate bound at different positions of the ring. As illustrated in Figure 24.5C, a protein carrier like bovine serum albumin (BSA) injected into a mouse would elicit antibodies that react against BSA. Because BSA antigen elicits an immune response to itself, it is called an immunogen. In contrast, a mouse injected with the benzenesulfate hapten alone fails to produce antibodies to the hapten. However, when the hapten is attached to BSA and the hapten-BSA complex is injected, the mouse produces antibodies that react to the carrier (BSA) as well as other antibodies that react against the benzene hapten. The reason for this carrier effect will be evident later as we describe how antigens are processed by cells of the immune system. Thus, antigens include immunogens that elicit an immune response by themselves, and haptens that must be attached to an immunogen in order to generate an immune response.
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Th e A d a p tive I mmune Response
TO SU M MAR I Z E: ■
■
■
■
Proteins are better immunogens than nucleic acids and lipids, because proteins have more diverse chemical forms. Antigen-presenting cells are cells, such as phagocytes, that degrade microbial pathogens and present distinct pieces on their cell surface MHC proteins. Immunological specificity means that antibody made to one epitope will not bind to different epitopes (although some weak cross-binding can happen). A hapten is a small compound that must be conjugated to a larger carrier antigen to elicit production of an antibody.
24.3 Antibody Structure and Diversity Antibodies, also called immunoglobulins, are members of the larger immunoglobulin superfamily of proteins. Antibodies are the keys to immunological specificity. They are glycoproteins made by the body in response to an antigen. The immunoglobin superfamily of proteins has in common a 110-amino-acid domain with an internal disulfide bond. The immunoglobulin superfamily includes antibodies and other important binding proteins, such as the major histocompatibility proteins and B-cell receptors described later. Like miniature “smart bombs,” antibody immunoglobulins individually circulate through blood, ignoring all antigens except those for which they were designed. When A. Antigen
an antibody finds its antigenic match, it binds to the antigen and initiates several events designed to destroy the target. Antibodies, in addition to being free-floating, are also strategically situated on the surfaces of B cells, where they enable these lymphocytes to recognize specific antigens. An antibody consists of four polypeptide chains. There are two large heavy chains and two smaller light chains (Fig. 24.6). The four polypeptides combine to form a Y-shaped tetrameric structure held together by disulfide bonds. Two bonds connect the two identical heavy chains to each other. One light chain is then attached near its carboxyl end to the middle of each heavy chain by a single disulfide bond. The antigen-binding sites are formed at the amino-terminal ends of the light and heavy chains. One antibody molecule possesses two identical antigenbinding sites, one on each “arm” of the molecule.
Antibodies Have Constant and Variable Regions There are five classes of antibodies, defi ned by five different types of heavy chains called alpha (α), mu (µ), gamma (γ), delta (δ), and epsilon (ε). The heavy-chain classes are distinguished one from another by regions of highly conserved amino acid sequences, known as constant regions (denoted C, Fig. 24.7). Antibodies containing gamma heavy chains are called IgG; those with alpha, delta, mu, and epsilon heavy chains are called IgA, IgD, IgM, and IgE, respectively. Each antibody class serves a specific purpose in the immune system. B.
+
+
NH3
NH3
F(ab)
F(ab)
Heavy chain +
+
Antigen-binding sites
H3N
S
S S
NH3
S
Light chain
S S S S
–
COO–
OOC
COO–
COO–
Fc
Basic antibody structure. A. Each antibody contains two heavy chains and two smaller, light chains held together by disulfide bonds. The Y-shaped structure contains two antigen-binding sites, one at each arm of the molecule. The two antigen-binding sites are formed by the amino-terminal regions of the heavy and light chain pairs. B. Three-dimensional structure of an antibody. The heavy chains are shown in blue, the light chains in yellow. The F(ab) regions represent the antigen-binding sites. The Fc portion points downward and is used to attach the antibody to different cell surface molecules. (PDB code: 1r70)
Figure 24.6
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In contrast to heavy chains, there are only two classes of light chains: kappa (κ) and lambda (λ). An antibody of any heavy-chain class may contain two kappa chains or two lambda chains, but never one of each. Two-thirds of all antibody molecules carry kappa chains; the rest have lambda chains. The antigen-binding part of an antibody is formed by highly variable amino acid sequences situated at the amino-terminal ends of the light and heavy chains. These variable regions are referred to as the V L region and the V H regions (see Fig. 24.7). The rest of the immunoglobulin chains are composed of the highly conserved constant regions. C H1, C H2, and C H3 denote the three different constant regions in each heavy chain. Each light chain also has a constant region, designated C L. Section 24.5 discusses the genes that code for these regions and the combinatorial possibilities underlying the formation of different antibody molecules. In addition to the two “arms” that bind antigen, every antibody contains a “tail” that serves a different function. Intact antibodies can be dissected into their two functional parts through digestion with the protease papain. This endoprotease cleaves the molecule at the hinge Heavy chain
Antigenbinding site
+
NH3
VH Papain CH1
VL
F(ab)2
κ or λ
S
S
S
S
CL
S S S S COO—
CH2
Pepsin
Complement activation
Fc
Heavy chain
CH3 Macrophage binding —
—
COO
Papain digestion separates individual antigen-binding sites (Fab fragment) from the antibody molecule.
COO
■ M edic ine and I mm u n o l o gy
region, which releases the tail of the antibody (called the Fc region) from the antigen-binding portion of the molecule (called the F(ab) 2 region) (see Fig. 24.7). The c in Fc refers to the ease with which this fragment can be crystallized. The Fc region is not involved in antigen recognition but is important for anchoring antibodies to the surface of certain host cells and for binding components of the complement system.
Isotypes, Allotypes, and Idiotypes Represent Different Levels of Antibody Diversity As noted earlier, antibodies are classified based on variations in amino acid sequences in regions of the light and heavy chains. Certain differences in the constant region give rise to isotypes, which are common to all members of a particular species. IgG, IgM, IgA, IgD, and IgE are antibody isotypes. The IgG isotype is identical among humans but is different from the IgG isotype of monkeys. Within an isotype, there are amino acid differences in the constant region that are shared by some, but not all, members of a species. These are called allo-
Constant and variable regions in antibody structure. It is apparent in this figure that the variable sequences present at the amino termini + NH3 of the heavy and light chains form the antigen binding sites. Functional parts of the antibody can be separated Light following certain protease treatments. Papain chain specifically cleaves the hinge regions of the heavy chains and releases separate Fab arms. Pepsin cleaves in the CH2 constant region and releases the two F(ab) regions as one piece [F(ab)2] from the Fc region. Note that allotypic differences between individuals are generally found in the constant The Fc region is the part region of the light chains (CL), while idiotypic of an antibody that reacts differences between specific antibodies in a with complement or binds single person are found in the variable regions to the surface of cells such as macrophages. that form the antigen-binding sites. Figure 24.7
Heavy chain
μ, γ, α, δ, or ε
VH
Pepsin digest F(ab′)2
Papain digest
CH1
VL
VL
VH
CL
CH1
Fab
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CL
Light chain
S
S
S S
903
S
S S S S S
Pepsin digestion separates the Fc region from the F(ab′)2 portion that contains both antigen-binding sites.
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typic differences. Allotypic differences in an amino acid sequence usually occur in the light chains. For example, John possesses circulating IgG molecules that have allotypic differences from the IgG antibodies circulating in Sherrie. (John and Sherrie have different IgG allotypes.) On another level, alterations in hypervariable regions within a single antibody class in a single person are referred to as idiotypic differences. These differences occur in the antigen-binding sites of the various antibodies of a single individual. Thus, the IgG molecules in Sherrie that bind to epitope A possess idiotypic differences from Sherrie’s IgG molecules that bind epitope B. In sum, the human IgG isotype is composed of different allotypes (constant region differences between individuals), and each allotype is comprised of different idiotypes (variable region differences within an individual). Note that antibodies are proteins and as such are themselves antigens. Thus, the amino acid differences found within a single class of antibody—IgG for example— also represent different epitopes within that class. Isotypic, allotypic, and idiotypic differences define epitopes in antibody molecules that can be detected using immunological techniques such as immunoprecipitation and Western blotting assays described in Special Topic 24.1.
Different Antibody Isotypes Have Different Functions and “Super” Structures All antibody isotypes have the same basic structure. However, each isotype has a unique “super” structure (for example, monomer, dimer), and each is designed to carry out a different task. Some key properties of the five
different immunoglobulin classes are listed in Table 24.3. IgG is the simplest and most abundant antibody in blood and tissue fluids. It is made as a monomer but has four subclasses. Each subclass varies in its amino acid composition and by the number of interchain cross-links. IgG molecules carry out several missions for the immune system. First, they bind and opsonize microbes; that is, they make the microbe more susceptible to phagocytes. Opsonizing IgG antibodies coat the microbe with their Fc portions protruding outward. Phagocytes possess surface Fc receptors that can attach to the Fc region of the antibody to gain a fi rmer “grip” on the microbe, facilitating phagocytosis (discussed in Section 23.6). IgG can also directly neutralize viruses by binding to virus attachment sites and is one of only two antibody types that can activate complement by the classical pathway (to be described in Section 24.7). IgA is secreted across mucosal surfaces and is most commonly found as a dimer (Fig. 24.8A). This explains why IgA can bind four molecules of antigen (each monomer can bind two molecules of antigen). The components of the IgA dimer are linked by disulfide bonds to a protein called the J chain, which joins two IgAs by their Fc regions. A sixth protein, the secretory piece, is wrapped around the IgA dimer during the secretion process. The secreted molecule, now called sIgA (secretory IgA), is found in tears, breast milk, and saliva and on other mucosal surfaces. The molecule sIgA is important for mucosal immunity against pathogens. Circulating IgM is a huge, Ferris wheel–shaped molecule formed from five monomeric immunoglobulins tethered together by the J-chain protein (Fig. 24.8B). It can also be found in monomeric form on the surfaces of B
Table 24.3 Properties of human immunoglobulins. IgG Property
IgG1
IgG2
Mol. wt. (kDa) % Carbohydrate Serum half-life (days) % Total serum Ig Avr. concentration (mg/ml) Ag-binding sites Heavy chain Light chain Produced by fetus Transmitted across placenta Bind complement Opsonizing Bind mast cells
146
146
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IgA IgG3
IgG4
IgA1
165
146
7
21
1
0.5
3
γ3
γ4
α1
160 7–11 6 15–20%
3 21
20 70%
9
3
γ1
γ2
IgA2
IgD
IgE
970 9–12 10 5–10%
184 9–11 3 0.2%
188 12 2 0.002%
0.03 2 δ κλ ?
5 × 10–5 2 ε κλ +/–
No No No No
No No No Yes
κλ +/–
κλ +/–
1.5 5–10 µ κλ +++
Yes Yes Yes No
No No No No
No Yes No No
2
0.5
IgM
2–4 α2
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cells, where it forms part of the B-cell receptor. IgM is the fi rst antibody isotype detected during the early stages of an immune response. Unlike the smaller IgG immunoglobulins, IgM is so large that it cannot cross the placenta (see Table 24.3). Two other antibody isotypes are present at very low levels in the blood. IgD is a monomer that can neither bind complement nor cross the placenta. These molecules are, however, abundantly found on the surface of B cells.
A. IgA
905
Attached to the cell surface by their Fc regions, IgD, along with monomeric IgM, can bind antigen and signal B cells to differentiate and make antibody. IgE is also present in trace amounts in the blood, but is found more prominently bound to the surface of mast cells and basophils, where it has potent biological activity. Mast cells and basophils contain granules loaded with inflammatory mediators. The primary role of IgE is to amplify the body’s response to invaders. Once secreted into serum, IgE
IgA is secreted as a dimer held together by the secretory piece and J chain and can bind four identical antigens.
VL J chain
CL S S
VH1
S S
CH2 S S S S S S
CH3
S S S S
CH1
S S
Secretory piece
VH
B. IgM IgM forms a pentamer held together by disulfide bonds and the J chain. IgM can bind ten antigens.
CH1
S
S
S
VL
S
CL S S
CH2 CH3
S
S S
S
S
S S
S
S S
S S
S
S
S
S
CH4
S
S
S
S
J chain links adjacent IgMs
Carbohydrate
S S S
S S
S
S
S
S
S
S
S S
Structures of IgA and IgM.
S
Figure 24.8
The antibodies are made as multimers of two (IgA) or five (IgM) immunoglobulin
molecules.
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Special Topic 24.1
Th e A d a p tive I mmune Response
Applications Based on Antigen-Antibody Interactions
A typical antibody molecule will bind only one type of antigen but has two antigen-binding sites. Because of the ability to bind to more than one antigen molecule, antibodies can cross-link antigens in solution, ultimately forming complexes too large to remain soluble (Fig. 1). The phenomenon, called immunoprecipitation, is normally only observed in vitro, where the concentration of antigen and antibody can be manipulated experimentally. Immunoprecipitation occurs only with appropriate ratios of antigen and antibody molecules. Too many antigen molecules (antigen excess; Fig. 1A) or too few antigen molecules (antibody excess; Fig. 1B) result in complexes too small to immunoprecipitate. Large complexes are formed only at an appropriate antigen:antibody ratio called equivalence (Fig. 1C). Equivalence is the point where the number of antigenic sites is roughly equal to the number of antigenbinding sites. Immunoprecipitation is the basis for many experimental immunological techniques. For example, the concentration of an antigen can be determined in vitro, or antibodies can be used to identify and remove specific antigens from a complex mixture because the specific antigen-antibody complex falls out of solution (Figs. 2A and B). Figure 2B shows how specific proteins can be isolated from a complex cell extract using immunoprecipitation. Antibody is added to a complex mix of different antigens. The antibody, however, can only bind to its specific antigen. Beads coated with a molecule known as protein A (derived from Staphylococcus aureus)
A. Antigen excess
No complex is possible because there are more antigens than antigen-binding sites.
B. Antibody excess
No complex is possible because there are more antigen-binding sites than antigens.
are added to specifically purify that antigen-antibody complex. Protein A binds to the Fc portion of an IgG antibody molecule. Consequently, all the antigen-antibody complexes will become bound to the bead. Because the bead is heavy, centrifugation will remove the desired antigen from the complex mixture. Another application, called radial immunodiffusion, allows the concentration of an antigen in a solution to be determined (Fig. 2C). This technique involves visualizing a ring of precipitation in an agarose gel impregnated with antibody. Antigen placed within a well will diffuse outward until reaching a zone of equivalence with the embedded antibody. At this point, antigen-antibody complexes precipitate and form a ring a certain distance from the well. Antigen concentration progressively decreases the farther the antigen diffuses away from the well. Consequently, the higher the concentration of antigen that is originally present in the well, the farther it will have to diffuse before the zone of equivalence is reached and a ring of precipitation forms. The concentration of antigen in an unknown solution is determined by comparing the radius of immunoprecipitation formed against a standard curve in which known concentrations of antigen are plotted against the radius of the ring of precipitation. Another important research technique involving antibodies is the Western blot, which is used to detect the presence of a specific protein in cell extracts. As described in Section 12.1, proteins are separated using SDS PAGE
C. Equivalence
Complexes are possible when antigen numbers equal the antigen-binding sites.
Figure 1 Basis of immunoprecipitation. Only when the number of epitopes and antigen-binding sites are roughly equivalent will a large complex form and fall out of solution.
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electrophoresis. The proteins are transferred (that is, blotted) from the gel onto a nitrocellulose or other membrane. The membrane is then probed with an antibody directed against a specific protein. Antibody sticks to the protein in quesA. Precipitating aggregate Antibody A
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907
tion, and because that antibody is labeled in some way (for example, a radioactive or fluorescent probe has been added), the protein band can be visualized by autoradiography or phosphoimaging.
B. Antigenic determinants (epitopes)
Antigen
Cells
Incubate with Met; all proteins labelled.
Wash; lyse cells.
35
Antibody A Antibody B
Antigen-combining site
Add antigen-specific antibody to lysate.
C. Zone of equivalence
Protein to isolate
A B
Lysed cell extract Remove antibody-antigen complex with protein A on beads. ©2006 EDVOTEK, Inc.
C
Protein A–covered bead Protein A
Elute protein; analyze by SDS:PAGE.
200 98
Visualize by autoradiography.
68 45 31
Applications based on antigen-antibody behavior. A. Precipitating aggregate. Antibodies to different antigenic determinants residing on antigen molecules can cross-link the antigens to make a huge, insoluble complex that precipitates. B. Purifying proteins. Removing specific proteins from a complex cell extract using immunoprecipitation. In the experiment, antibody to a specific cellular protein (shown as yellow box) is added to a lysed cell extract. Protein A beads are then added. Protein A will bind to the Fc region of the antigen-antibody complex. Centrifugation then is used to pull down the beads and the protein with it. The protein is eluted from the antibody and analyzed by SDS PAGE (Protein A remains covalently attached to the bead). C. Radial immunodiffusion. The agarose plate shown is embedded with antibodies specific for a certain antigen. Different concentrations of the antigen are placed in each well. The antigen diffuses into the agarose. A ring of precipitation occurs when the concentration of the diffusing antigen reaches a zone of equivalence with the antibody in the plate. The more antigen placed in the well, the larger the diameter of the precipitation ring. Well A was loaded with more antigen than wells B or C.
Figure 2
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B. Mast cell (TEM)
5 μm
Laura Hale, Duke University
A. Mast cell (SEM)
C. Hayfever
1 μm
Mark Clarke
Chapter 24
Laura Hale, Duke University
908
Mast cells. A. Scanning EM of a mast cell. B. Electron transmission EM showing granules (arrow). C. Hayfever is the result of degranulation of IgE-coated mast cells, which release histamine and other pharmacological mediators.
Figure 24.9
attaches to mast cells (Figs. 24.9A and B), again by way of its Fc region, and, like a Venus flytrap, waits until its matched antigen binds to its antigen-binding site. When surface IgE molecules of two mast cells are cross-linked by antigen, a signal is sent internally that triggers degranulation (see hypersensitivity, Section 24.8). The subsequent release of histamine and other pharmacological mediators from these granules helps orchestrate the acute inflammation that takes place during early host responses to microbial infection (that is, while the antibody response is gearing up). The system is also responsible for severe allergic hypersensitivities, such as anaphylaxis, and milder forms like hay fever (see Fig. 24.9C).
TO SU M MAR I Z E: ■
■
■
■
■
Antibodies, or immunoglobulins, are members of the immunoglobulin superfamily. Antibodies are Y-shaped molecules that contain two heavy chains and two light chains. There are five classes (isotypes) of heavy chains. Each antibody isotype is defined by the structure of the heavy chain. Each antibody molecule contains two antigenbinding sites. Each binding site is formed by the hypervariable ends of a heavy and light chain pair. The Fc portion of an antibody can bind to specific receptors on host cells. This binding is antigen independent.
THOUGHT QUESTION 24.3 There are immune disorders in which an individual overproduces a specific class of antibody, for example hypergammaglobulinemia. How could the radial immunodiffusion technique be used to identify what class of antibody is in excess?
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24.4 Humoral Immunity: Primary and Secondary Antibody Responses After a lag period of several days following primary immunization or natural infection, antibodies begin to appear in the serum (the fluid that remains after blood clots). During the lag period, a series of molecular and cellular events occur that cause a distinct subset of B cells to proliferate and differentiate into antibody-secreting plasma cells and memory B cells. This process is known as the primary antibody response, the events of which will be discussed later. A secondary exposure to the antigen, which can take place months or years after the initial encounter, will trigger a rapid, almost instantaneous increase in the production of antibodies and is called the secondary antibody response (Fig. 24.10). This quick response occurs thanks to the memory B cells formed during the primary response. Once stimulated, memory B cells rapidly differentiate into plasma cells and secrete antibody. The net result of the primary antibody response is the early synthesis and secretion of IgM molecules specifically directed against the antigen, also called the immunogen. Later during the primary response, a process known as isotype switching (class switching) occurs, by which the predominant antibody type produced becomes IgG rather than IgM (discussed shortly). Antibodies made during this primary phase, while specific for the immunogen, are actually not of the highest affi nity. Mechanisms to increase antibody affi nity occur later. As the immunogen is cleared from the body, the levels of both IgG and IgM decline because the plasma cells that produced them die. Plasma cells have a life span of only 100 days. However, the immune system has been primed to respond more aggressively to the immunogen should it encounter it again. This is because the memory B cells produced as a result of the primary response react to anti-
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Serum titer of antibodies
Primary response
Booster dose Primary vaccination IgM
1
2
3
4
5
6
7
Time (weeks)
Figure 24.10 Primary versus secondary antibody response. Primary vaccination or infection leads to the early synthesis of IgM followed by IgG. Reinfection or a second, booster dose of a vaccine results in a more rapid antibody response comprised mainly of IgG due to memory B cells formed during the primary response.
gen more quickly and take less time to make antibody than naive B cells that had no prior exposure to the antigen. B cells are maintained in the body because, unlike plasma cells, they continue to divide. If a person encounters the same antigen at a later time, memory B cells quickly proliferate and differentiate into plasma cells with no lag phase. Thus, memory B cells quickly initiate the secondary antibody response (or anamnestic response, from the Greek anamnesis, meaning “remembrance”). During the secondary response, copious amounts of IgG antibody are secreted from plasma cells made from memory B cells that have undergone isotype switching. These antibodies have a higher specificity for the antigen than the antibodies produced during the primary response. Small amounts of IgM are also produced from the few memory cells that did not undergo isotype switching during the primary response. (Most memory B cells undergo isotype switching and produce IgG rather than IgM, but a few do not.) The speedy production of antibody during the secondary response is the basis of immunization. Pathogens can do considerable harm during the lag phase of the primary response. To avoid this, an innocuous version of a pathogen, or a harmless piece of it, can be injected into a person to trigger the primary response without producing disease (or, at worst, producing only a mild form of the illness). Immunization thus primes the immune system to respond efficiently and without delay upon encountering the real pathogen. Table 24.2 lists a variety of viral and bacterial diseases for which immunizations are available. THOUGHT QUESTION 24.4 The mother of a newborn was found to be infected with rubella, a viral disease. Infection of the fetus could lead to serious consequences for the newborn. How could you determine if the newborn was infected while in utero?
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B-Cell Differentiation into Plasma Cells Occurs by Clonal Selection
Secondary response IgG
0
■ M edic ine and I mm u n o l o gy
As mentioned earlier, each B cell circulating throughout the body or ensconced in a lymphoid organ is programmed to synthesize antibody that reacts with a single epitope. In a process called clonal selection, an invading antigen will inadvertently select which B-cell clone will proliferate to large numbers and differentiate into antibody-producing plasma cells or memory B cells. In this way, large amounts of antibody specific for the antigen are made. The mechanism of clonal selection begins with the antigen binding to a matching B cell (a B cell preprogrammed to bind to that antigen) (Fig. 24.11). Mature naive B cells (those that have not previously encountered antigen) can produce only IgM and IgD, both of which have identical antigen specificities. These two antibody classes are displayed like tiny satellite dishes on the B-cell surface, anchored by their Fc regions through hydrophobic transmembrane segments. These surface antibodies are the keys to stimulating the proliferation and differentiation of B cells into antibodyproducing plasma cells or memory B cells. Upon binding to its corresponding antigen via these surface antibodies, the B cell is said to become activated, whereby it multiplies and differentiates into a plasma cell that ultimately synthesizes only one antibody isotype (for example, IgG1 or IgA2). Clonal selection has begun. (Remember that some of the activated B cells become memory B cells and do not become antibody-secreting plasma cells.) Each membrane-bound antibody on the B cell is associated with two other membrane proteins called Igα and Igβ (these are not immunoglobulins but are designated Ig because they associate with the surface antibody). The complex is called the B-cell receptor (Fig. 24.12). Each B cell may have upwards of 50,000 B-cell receptors. When B cells bind antigen, they often, but not always, differentiate into plasma cells (some become memory cells). During this transformation process, antibody heavy-chain isotype switching (class switching) occurs. Class, or isotype, switching changes the class of antibodies produced. After binding to the antigen, the surface B-cell receptors begin to cluster in a process called capping. Capping activates Igα and Igβ to initiate a phosphorylation signal cascade directed into the nucleus (Fig. 24.13). The transcription factors at the end of the cascade stimulate transcription of genes that contribute to cellular activation and DNA recombination events involved in heavy-chain class switching. In addition, B-cell receptors trigger endocytosis of a bound antigen. Once inside the B cell, the antigen is degraded, processed, and repositioned on the B-cell membrane, a step that will ultimately enable T cells to directly bind and communicate with the B cell, giving the B cell “permission” to become an antibody-secreting plasma cell. How these antigens are placed on the B-cell membrane will be discussed in Section 24.6.
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C h ap t e r 2 4
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Th e A d a p t ive I mmune Response
Antigen binding to the right B cell B cells with different antibody receptors
Clonal expansion
1C
Memory B cell 4C 2C
4C clone
3C 4C antibody release
4C Antigen binds to the B cell coated with matching antibody.
4C plasma cell
Figure 24.11 Clonal selection theory. The B-cell population is composed of individuals that have specificity for different antigens. When a B cell contacts its cognate antigen, an intracellular signal is generated, leading to proliferation and differentiation of that clone (clonal expansion). Plasma cells and memory B cells result. Antigen-binding sites
S
S
S
Monomeric IgM
S
S S S S
Carbohydrates Outside Igβ Inside
Igα
Igα
Igβ Membrane
(the capping process), a step essential for triggering differentiation (see Fig. 24.13). Proteins, however, which are the largest group of antigens, do not contain multiple repeating units. Proteins possess many small, discrete, single epitopes, making cross-linking of B-cell receptors difficult. B-cell responses to these types of antigens require help from specific T cells, and this constitutes the second route to B-cell activation. Thus, B cells usually require multiple signals to initiate a primary response. How T cells help foster B-cell activation will be discussed in Section 24.6. TO SU M MAR I Z E: ■
Figure 24.12 B-cell receptor. The B-cell receptor is formed as a complex between a monomeric IgM and Igα and Igβ in the membrane. Igα and Igβ are not immunoglobulins.
■
There are two routes by which antigens can stimulate B cells to differentiate into plasma cells. In one, called the T-cell-independent route, antigens that possess multiple repeating epitopes can directly cross-link B-cell receptors
■
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The primary antibody response to an antigen begins when B cells differentiate into antibodyproducing plasma cells and memory B cells. IgM antibodies are generally the fi rst class of antibodies made during the primary response. Isotype switching occurs during the primary response when a subclass of B cells switches during differentiation from making IgM to making other antibody isotypes. The secondary antibody response occurs during subsequent exposures to an antigen and arises because memory B cells are activated. IgG is the predominant antibody made.
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Mature B cell
B-cell receptors
■ M edic ine and I mm u n o l o gy
911
cassettes), the random introduction of somatic mutations, and the generation of different codons during antibody gene splicing. In humans, the process of generating antibody diversity takes place constantly over a lifetime.
DNA Rearrangements in Gene Splicing and Hypermutation Generate Antibody Diversity
Antigen
Binding antigen causes the surface B-cell receptors to cluster (capping) and activate Igα and Igβ.
Activation
Capping and activation of the B cell. The capping process initiates a signal cascade that activates differentiation and proliferation of the B cell.
Figure 24.13
■
■
Clonal selection is the rapid proliferation of a subset of B cells during the primary or secondary antibody response. A B-cell receptor consists of a membrane-bound antibody in association with the Igα and Igβ proteins. Binding of antigen to the B-cell receptor triggers B-cell proliferation and differentiation.
24.5 Genetics of Antibody Production It is estimated that each human can synthesize 1011 different antibodies. Given that each B cell displays antibodies to only one antigenic determinant, it follows that there are 1011 different B cells in the body. We have learned, however, that each person possesses only about 1,000 genes or gene segments involved in antibody formation. How are 1011 different antibodies made from only 103 genes? Susumu Tonegawa was awarded the 1987 Nobel Prize in Physiology or Medicine for discovering that antibody genes can move and rearrange themselves within the genome of a differentiating cell. Three steps are involved: rearrangement of antibody gene segments (or
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The fi rst step in making a specific antibody occurs during the formation of a B cell from a progenitor stem cell in bone marrow. Immunoglobulin genes in a bone marrow stem cell consist of many gene segments that can rearrange in many possible combinations. During differentiation of a stem cell into a mature B cell, DNA segments are deleted in a process called gene splicing, decreasing the number of gene segments in the mature B-cell DNA. The process starts at the 5′ end of an immunoglobulin gene cluster, which corresponds to the variable (V) end of the ultimate peptide (Fig. 24.14). The 5′ end encodes the Nterminal end of the protein that ultimately binds antigen (that is, the antigen-binding site). In both the heavy- and light-chain genes, there are a number of tandem gene cassettes encoding potential variable regions separated by recombination signal sequences (RSS). These sequences allow recombination to bring two widely separated gene segments together. There are approximately 135 V gene segments for the heavy and light chains. The light-chain variable-region gene cluster lies upstream of a cluster of J (joint or joining)-region genes, which are ultimately used to join the variable region to the light-chain constant regions (C), whose genes reside farther downstream in the DNA. The arrangement of the heavy-chain genes is slightly more complex. In this case, the heavy-chain V cluster is followed by a D (diversity) cluster, then the J region. NOTE: Do not confuse the J-region gene segments used to make heavy- and light-chain proteins with the J-chain protein that holds together IgM and IgA multimers. They are completely different and unrelated. The J-region gene segments do not encode the J chain.
During the formation of each mature B cell, recombination between the various recombination signal sequences deletes all but one heavy-chain variable region (see Fig. 24.14). As a result of the deletion, a single V segment is placed next to one D region. The structure of the particular peptide encoded by the selected V region begins to limit antigen recognition properties. At the same time, all but one D segment and one J segment are deleted. The result is the VDJ portion of the heavy chain. A similar sequence of events occurs for the light chains, only the product is VJ. Table 24.4 illustrates the amount of
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RSS region Heavy-chain variable-region genes
Joining genes
Diversity genes
Heavy-chain constant-region genes
1. Germ-line DNA V1
5′
V2
D1 D2 D3-4
V3–4
J1
J2
J3
J4
C
Recombination and deletion 2. B-cell DNA
D2
V1
5′
J3
B-cell DNA recombines to delete all but one V, D, and J cassette.
C
Transcription V1
J3
D2
C
3. B-cell mRNA
4. Heavy-chain peptide
Translation mRNA is spliced and translated.
V 5. Light-chain peptide
D J
+
V
C (μ or δ)
J C
V
Assembly of heavy and light chains
J C
V J
S
S
D
C S S S S
6. Antibody
C terminus
C terminus S
S
Heavy chain
Light chain
Variable region
Constant region
Figure 24.14 Formation of the VDJ regions of heavy chains. Note that only a small subset of the V, D, and J genes listed in Table 24.4 are actually shown in this model.
Table 24.4 Antibody diversity attributed to combinatorial joining in the human germ line. Number of Chain type
V regions
D regions
J regions
λ light chains κ light chains Heavy chains Number of possible antibodies
30 0 4 40 0 5 65 27 6 7,800 × 200 = 1.56 × 106 7,800 × 120 = 0.94 × 106 1.56 × 106 + 0.94 × 106 = 2.5 × 106 combinations
Number of combinations 30 × 4 = 120 40 × 5 = 200 1,000 × 30 × 4 = 120,000 65 × 27 × 6 = 7,800
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antibody diversity in humans that can be achieved simply by this combinatorial rejoining (a total of about 2.5 × 106 antigens can be recognized). Where does the rest of the diversity arise? In addition to recombination, the V regions of the germ lines are susceptible to high levels of somatic mutation, resulting in the hypervariable regions. Hypermutation happens every time a memory B cell is exposed to the antigen: The memory B cells divide and the hypervariable regions mutate. Additional diversity comes from the junctions of VJ and VDJ, where recombinational splicing can occur between different nucleotides. Each gene splice event can generate additional codons, so the resulting peptides will differ by one or more amino acids. The interactions between the light- and heavy-chain hypervariable regions in an antibody form the antigen-binding sites. In sum, a combination of gene splicing and random mutations creates the remarkable level of antibody diversity we each possess. As noted earlier, the human body is capable of responding to 1011 antigens; yet there are only 10 8 estimated antigens in nature. This apparent overkill suggests that the immune system is well prepared to cope with any possible antigen it could encounter. Unfortunately for humans, enterprising microbes, such as the trypanosomes that cause sleeping sickness, can stay one step ahead of the immune system by changing the structure of key surface antigens. Changing the antigenic structure of a protein renders useless those antibodies made to the previous structure. It isn’t hard to understand why multicellular organisms, like humans, need the capacity to make any one of billions of different antibodies quickly. Pathogens can undergo many generations of growth within the single life span of a human host. So humans have to generate recombinant clones of cells quickly to overcome rapidly dividing pathogens.
Isotype Class Switching Is the Result of Gene Splicing The fi rst stage in isotype switching involves the transition of the immature B cell in bone marrow to a mature B cell, which ends up in circulation or in lymph nodes. The immature B cell (a B cell that has not yet “seen” the antigen but has already assembled its immunoglobulin VDJ-binding site) produces only monomeric IgM as part of the B-cell receptor on its cell surface. The immature B cell ultimately differentiates into a mature B cell (a naive B cell), which then makes both IgM and IgD B-cell receptors (the naive B cell is referred to as IgM+ IgD +). This type of immunoglobulin class switching does not involve recombination at the DNA level, but occurs through the splicing of RNA. For instance, the immunoglobulin mRNA transcript in these cells includes both the mu- and delta-region sequences (the sequences that
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encode the IgM and IgD isotypes, respectively). Unprocessed, the transcript will yield IgM. However, the part of the transcript containing the mu constant region can be spliced out, in which case the processed transcript will make IgD. The primary immune response is launched when a mature (naive) B cell receptor fi nds its matched antigen. As mentioned, in the early stages of the response, plasma cells produced from these B cells secrete only IgM, and an antigen-activated mature B cell will make IgM and IgD (IgM+ IgD +). If this activated B cell also receives appropriate signals from a certain type of T cell known as a helper T cell (T H cell), further immunoglobulin isotype switching will occur; the switched B cell may then make either IgG, IgA, or IgE. The type of switch is influenced by small peptides called cytokines that are secreted by helper T cells. How does the switch occur? Notice in Figure 24.15 that the constant-region gene segments defi ning different antibody heavy-chain classes are arranged in tandem after a VDJ region. The mechanism by which a B cell switches to make IgG (C regions gamma 1–4), IgE (C-epsilon), or IgA (C-alpha 1 and 2) is very similar to VDJ formation. Each constant segment, except delta, contains a repeating DNA base sequence called a switch region. Recombination between these switch regions will delete the intervening DNA between the VDJ region and one of the constant regions. The primary RNA transcript produced after this recombination event has all of its introns spliced out before translation. Because the VDJ region is the same regardless of which C H gene is selected, whatever antibody is produced will have the same antigenic specificity as the original IgM. However, the heavy-chain switch selection process is not random. The type of cytokine present at the time of the switch will influence which C H gene is selected. Note that any heavy-chain peptide can combine with any light chain. However, a single, mature B cell can only make one type of heavy chain and one type of light chain. THOUGHT QUESTION 24.5 Why does the delta region have no switch region? THOUGHT QUESTION 24.6 Why do individuals with type A blood have anti-B and not anti-A antibodies?
Making Memory B Cells As illustrated in Figure 24.11, an antigen-activated mature B cell may become a memory B cell. These are B cells that have already undergone class switching (that is, they are committed to making IgG) but are
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Immunoglobulin germ-line genes for heavy chain
Switch region (SR) μ Variableregion genes
Constant region genes SRγ
Cμ (lgM)
Deleted
C Cδ γ
VDJ
Cα SRμ
Assembled IgA heavy-chain gene
Cα1–2 (lgA)
Cε
Looping out; deletion of intervening DNA
SRα Cε (lgE)
Cγ1–4 (lgG)
Cδ (lgD)
Cμ
VDJ
SRε
SRα
Cα
VDJ SRμ
SRα
Transcription VDJ Cα
mRNA for α (IgA) heavy chain
Translation IgA heavy chain
Figure 24.15 Heavy-chain class switching. As a B cell becomes activated, a switch in antibody isotype will occur. The switch involves recombination between isotype cassettes that brings one heavy-chain constant region (Cα in the example, for IgA) in tandem with a VDJ sequence. RSS = recombination signal sequences.
very long-lived. Normally, chromosome ends (called telomeres) in every cell progressively shorten with each round of DNA replication. When they become too short, the cell dies. Memory B-cell production is associated with an increase in telomerase activity. Recall that telomerase is a reverse transcriptase that shares common ancestry with the reverse transcriptase of the virus HIV. Telomerase activity maintains the length of the telomeres and prolongs the life of the cell. Memory B cells hypermutate the VJ regions of antibody genes (as already described). These mutations increase the affi nity of the antibody produced during the secondary response. Hypermutation helps fi ne-tune the immune response, making it even more specific toward a given microbe. Note, too, that as an immune response progresses, antigen becomes scarce. This means that only B cells with the highest affi nity antibodies as part of
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their B cell receptors will remain activated. The process is known as affinity maturation. THOUGHT QUESTION 24.7 Why do immunizations lose their effectiveness over time? TO SU M MAR I Z E: ■
■
■
Each B cell is programmed to make primarily a single antibody isotype that specifically binds one epitope. Antigens bound to the surface IgM or IgD antibodies on mature B cells are internalized, degraded, and redisplayed on MHC molecules at the B-cell surface. B cells differentiate into antibody-secreting plasma cells after a helper T cell interacts with the MHCantigen complex.
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■
■
During the primary antibody response, IgM is the predominant class of antibody secreted. Memory B cells rapidly proliferate and differentiate into antibody-secreting plasma cells upon a second encounter with an antigen/epitope. Most antibody made during this secondary response is IgG. Diversity of idiotype occurs via a complex series of splicing events between adjacent DNA cassettes as well as mutational events in DNA sequences encoding the hypervariable regions of heavy and light chains.
24.6 T Cells, Major Histocompatibility Complex, and Antigen Processing The following section describes how antigens are recognized by T cells and how T cells then influence both humoral and cellular immunities. In the process, we will expand on how B cells become activated once they bind antigen, and will begin to explain why a host usually does not react against itself.
The T Cell Links Humoral and Cell-Mediated Immunity Recall that there are two general types of immune response: humoral (antibody-based) immunity and cellmediated immunity. Different types of infection tilt the immune response toward one or the other. B cells are clearly linked to the humoral immune system because they ultimately become plasma cells that make antibodies. T cells, on the other hand, serve as a nexus, or link, between humoral and cell-mediated responses. They play integral roles in both antibody production and cellmediated immunity. Although derived from the same progenitor stem cell as B cells, T cells develop in the thymus (rather than in the bone marrow, where B cells develop) and contain surface antigens different from those of B cells. In general, T cells can be divided into two broad groups differentiated by the type of cellular differentiation (CD) proteins present on their cell surfaces (Table 24.5).
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One of the two major types of T cells has already been mentioned: the so-called helper T cells (T H cells). The second major type of T cells are known as cytotoxic T cells (TC cells). Helper T cells display the surface antigen CD4, while cytotoxic T cells display CD8. Helper T cells come in three types: T H1 cells, which assist in the activation of cytotoxic T cells; T H 2 cells, which stimulate B-cell differentiation into plasma cells; and T H0 cells, which are really undifferentiated precursors of T H1 and T H 2 cells. Cytotoxic T (TC ) cells are the “enforcers” of the cell-mediated immune response. They destroy the membranes of host cells infected with viruses or bacteria. The ratio between the number of T H1 cells and T H 2 cells produced during an infection affects whether the immune response will lean more toward humoral (antibody, T H 2) or cell-mediated (T H1) immune mechanisms. As will be described, different cytokines produced during infection influence whether an activated T H0 cell develops into a T H1 or T H 2 cell. Why are T H and TC cells different? The reasons are many but it all starts with their relative abilities to recognize two different protein complexes that are present on antigen-presenting cells. These complexes are called the major histocompatibility proteins.
The Major Histocompatibility Complex (MHC) Is Critical to the Immune System The factors affecting immune responsiveness are many, but the major histocompatibility complex (MHC) proteins encoded by a set of genes on a single chromosome, play a particularly important role. MHC proteins differ between species and among individuals within a species. They help determine whether a given antigen is recognized as coming from the host (a self antigen) or from another source (a foreign antigen), in a phenomenon called histocompatibility. Thus, the name major histocompatibility complex. There are two classes of MHC molecules found on cell surfaces. Both classes belong to the immunoglobulin family of proteins, but they are not immunoglobulins. Class I MHC receptors (Fig. 24.16A) are found on all nucleated cells. Class II MHC molecules (Fig. 24.16B), on the other hand, have a more limited distribution, principally on antigen-presenting cells.
Table 24.5 Major classes of T cells. T-cell type
Coreceptor
MHC restriction
Cytokines produced
Major function
Helper T cell TH1 TH2 Cytotoxic T cell
CD4 CD4 CD8
Class II Class II Class I
IFN-γ, IL-2, TNF-β IL-4, IL-5, IL-6 IFN-γ, TNF
Activating cytotoxic T cells B-cell helper Killing virus-infected and cancer cells
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A. Major histocompatibility proteins Class I MHC
Peptidebinding area
S S
α1
β1
α2
S S
α1
S S
α2
Class II MHC
β2
S S
S S
α3
β2
S S
Microglobulin
Cell membrane Cytoplasmic tail
Cytoplasmic tails
B. MHC binding to antigen α chain
C. MHC-antigen binding
Antigen MHC
They are always expressed by the antigen-presenting dendritic cells and B cells, for example, but can also be made by macrophages under the influence of cytokines such as interferon-gamma (IFNγ). The salient feature of MHC molecules is that they bind antigen, presenting it on the cell surface. The antigen-binding clefts of different MHC molecules differ markedly and have distinct binding affi nities for different antigenic peptides. MHC molecules are critical to the immune system because T cells only recognize antigens associated with MHC proteins; they do not recognize free-floating antigens. (B cells, on the other hand, do recognize freefloating antigens.) Once a T cell recognizes an antigenpresenting cell with a foreign antigen attached to an MHC molecule, the T cell becomes activated and in turn activates the immune system. It is important to note that not all potentially antigenic peptides can be recognized in one animal. An animal will not respond to an antigen if its antigen-presenting cells lack the MHC molecule needed to bind that antigen. This is the basis of tolerance to the self antigens present on an animal’s own tissues. Tolerance means your immune system does not launch an immune response to your own antigens. How tolerance to self-antigens develops is described in Special Topic 24.2.
+
NH2
+
NH3
Antigen Processing and Presentation Occur by Two Paths –
OOC
β chain Extracellular space Plasma membrane
–
Cytosol
OOC
Figure 24.16 Major histocompatibility proteins. A. MHC Class I molecules are composed of a 45-kDa chain and a small peptide called β2-microglobulin (12 kDa). MHC Class II molecules contain an alpha chain (30-34 kDa) and a beta chain (26–29 kDa). The peptide-binding regions of both classes show variability in amino acid sequence that yield different shapes and grooves. Peptide antigens nestle in the grooves and are held there awaiting interaction with T-cell receptors. CD8 T cells recognize antigen peptides associated with class I molecules, while CD4 T cells recognize peptides bound to class II molecules. B. Antigen binding to an MHC. C. Top view of antigen (red) nestled in the MHC peptide-binding site. (PDB code: 1bii)
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How are foreign antigens placed, or presented, on host cell surfaces? In the initial stages of an immune response, microbes either infect (as with viruses) or are engulfed by (as with bacteria) antigen-presenting cells. Inside the APCs, foreign proteins are degraded into smaller pieces (for example, converted to peptides). These smaller pieces are placed within MHC-binding clefts and transported back to the cell surface. Whether an antigen peptide binds to class I or class II molecules generally depends on how the antigen initially entered the cell (Fig. 24.17). Antigens synthesized within the cytoplasm of an APC (called endogenous antigens), as would occur during infections with viruses and intracellular bacteria, will attach to class I MHC molecules on the endoplasmic reticulum and are moved to the cell surface. Antigens produced outside of the APC (called exogenous antigens), as are most bacterial antigens, will enter the cell via phagocytosis and attach to class II MHC molecules transported to the acidic phagosome or lysosome (Fig. 24.17, right side). The MHC class II–peptide complex is then carried to the cell surface. Once the antigen is presented on the APC cell surface, T cells can interact with it via the T-cell receptors. Interactions between antigen-presenting cells and naive T cells occur within the lymph nodes, the spleen, or Peyer’s patches in the gut. So the APCs must make their way to those locations. How T cells then distinguish between MHC I and MHC II presentation is explained later.
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MHC II
1. Foreign proteins made in the cytoplasm (e.g., viruses) are degraded by the proteasome (LMP) to peptides.
1. MHC II assembly starts in ER with α and β chains associating with the invariant chain used for all MHC II molecules. This chain resides in the groove that will bind antigen.
Nucleus TAP LMP
MHC I
MHC II
Virus protein
Rough endoplasmic reticulum
Peptides 2. Peptides (microbial antigens) are translocated into the ER by TAP protein.
3. Peptides are loaded onto MHC I.
Invariant chain 2. MHC II moves through the Golgi and into late endosomal compartments. Golgi complex
3. The invariant chain is degraded to open the peptide-binding site. Digested invariant chain 4. Secretory vesicles with peptide–MHC I complexes fuse with membrane.
Endocytic compartments
Enzyme
Peptide antigens Exogenous antigen
4. The microbe is phagocytized into the endosome and degraded by proteases. The peptide antigens are placed on MHC II. The complex is exported to the cell surface.
Figure 24.17 Processing and presentation of antigens by antigen-presenting cells on class I and class II MHC proteins. Microbial proteins made in the host cytoplasm (upper left) are degraded and peptides are placed on MHC class I molecules in the ER. Microbial proteins made outside the cell (lower center) are endocytosed, degraded in the endosome, and placed on MHC class II molecules. TAP = transporter of antigen peptides; LMP = low-molecular-mass polypeptide component of the proteasome.
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T Cells That Recognize Self Too Strongly Are Weeded Out in the Thymus
Greek mythology tells of Narcissus, a young man punished by the gods for scorning the women who fell in love with him. One day Narcissus saw his own beautiful reflection in a pool of water. He unwittingly fell in love with his own reflection, fell into the pool, and drowned. Narcissus’s inability to identify his own image is tantamount to the inherent danger posed by an immune system. To avoid disaster, immune systems must be able to distinguish what is self (meaning antigens present in our own tissues) from what is not self. Chapter 23 discusses how innate immunity accomplishes this task, in part through the use of pattern recognition proteins such as the Toll-like receptors. Equally important are the ways adaptive immune mechanisms avoid recognizing self. At the outset of development, the immune system is fully capable of reacting against self. One protective mechanism the body uses to avoid attacking itself is to delete any T cells that react against self antigens. In humans, this occurs throughout life as new T cells are made. In this process, T cells undergo a two-stage selection or “education” process in the thymus to recognize self versus nonself. This education process involves the use of self antigens, not foreign antigens. T cells bearing T-cell receptors (TCRs) that weakly recognize self MHC proteins displayed on thymus epithelial cells are allowed to survive (positive selection). These T cells leave the thymus to seed secondary lymphoid organs such as the spleen. T cells in the thymus that recognize self MHC peptides too strongly, however, are killed, or deleted from the population (negative selection). Almost 95% of T cells entering the thymus die during these positive and negative selection processes.
T-Cell Receptors Bind Antigens on Antigen-Presenting Cells (APCs) T-cell receptors (TCRs) are antigen-binding molecules (not immunoglobulins) present on the surfaces of T cells (Fig. 24.18). These receptors do not bind soluble antigen. A TCR, in general, will only bind to antigens attached to MHC surface proteins that are present on antigen-presenting cells. However, the TCRs of cytotoxic T cells can also bind viral antigens present on any virus-infected cells, whether or not that cell is normally considered an APC. The T-cell receptor is composed of several transmembrane proteins. The part used to recognize antigen is composed of two molecules, alpha and beta. Much like the immunoglobulins, the alpha and beta proteins of the TCR are formed from gene clusters that undergo
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The positive selection process is needed because T cells must be able to recognize self MHC to “see” the associated antigen. Antigen bound to MHC actually increases the affinity to the matched TCR. If our T-cell repertoire included cells that bound self MHC too tightly, then our T cells would constantly react to our own MHC molecules, regardless of what antigen peptides were attached. In contrast to negative selection for B cells, which occurs in bone marrow, T-cell education is limited to the thymus. But, if the thymus only expresses thymus antigens, how can T cells that respond to antigens expressed on other host cells (for example, heart cells) be removed? The answer is a special gene activator in thymus cells that allows them to synthesize all human proteins in small amounts. This expression is necessary to complete T-cell education within the thymus. You might ask how someone who has had their thymus removed (a treatment for myasthenia gravis) can live if the organ is critical for T-cell maturation and for deleting selfreactive T cells. Actually, within a few years after birth, the thymus begins to lose its utility such that very little function remains in adults. Research has shown that some T-cell maturation can occur extrathymically in secondary lymphoid tissues, although not as efficiently as in the thymus. This is at least part of the reason why children and adults with thymectomies are able to live relatively normal lives. The situation is more serious with newborns who have had their thymuses removed. Newborns have not had sufficient time to populate their secondary extrathymic lymphoid organs with T cells.
gene rearrangements. The diversity of TCR antigen receptors comes from random recombinations of various V, D, and J segments (analogous to, but different from, the immunoglobulin genes) and to variability in the precise joining of segments. There is no hypermutation, however. The TCR alpha and beta proteins are found in a complex with four other peptides; together these form the CD3 complex. When stimulated, these ancillary CD3 complex proteins recruit and activate intracellular protein kinases and launch a phosphorylation cascade that triggers proliferation of the T cell.
Activation of TH0 Helper Cells Figure 24.19 summarizes the steps that lead to T-cell activation in the lymph node and highlights how acti-
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TCR
N
S S
S S
Recognition
α
CD3
S S
CD3 S S
ε
β
δ
γ
ε
S S
+ –
–
+ –
+
C
–
Cell membrane
C
Signaling
ζ Figure 24.18 The T-cell receptor (TCR) and CD3 complex. The T-cell receptor proteins are associated with CD3 proteins at the cell surface. Antigen binds to the alpha and beta subunits. The positive and negative charges holding the complex together come from amino acids in the peptide sequences. Once bound to antigen, the complex transduces a signal into the cell. This signal triggers T-cell proliferation.
vated T cells influence the two arms of adaptive immunity: humoral and cellular. We will refer to Figure 24.19 often within the next sections. Two molecular signals are required to activate T cells. The fi rst step, regardless of the T-cell type, involves the cross-linking of T-cell receptors by antigen-MHC proteins on antigen-presenting cells (Fig. 24.19 , step 1). In the case of T H0 cells, this involves class II MHC complexes. But this fi rst step alone is not enough to activate a T cell. A second signal is required by T H cells, which involves the binding of a CD28 molecule present on the T-cell surface to a molecule called B7 protein on the APC cell surface (Fig. 24.19, step 2). Different cytokines produced during infection will then convert activated T H0 cells to T H1 or T H2 cells (Fig. 24.19, steps 3 and 4). Activation of cytotoxic T cells (TC cells) will be examined later.
B-Cell Activation Revisited As with T cells, B cells need two signals to become activated. The fi rst signal, described earlier, occurs when antigen cross-links B-cell receptors on B-cell membranes (in the process called capping). But to become activated
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during a primary immune response, B cells usually require helper T cells. But not just any helper T cell can stimulate a B cell; specificity is involved. How does a B cell gain specific T-cell help? T cells (the T H 2 type) know which B cells to help via their T-cell receptors. As part of the B-cell capping mechanism, some antigen bound by B-cell receptors becomes internalized, to be processed and presented back on the B cell’s surface MHC receptor (see Fig. 24.17, step 4). T H 2 cells that are activated by dendritic cells presenting antigen A, for instance, can use their T-cell receptor to bind to antigen A presented on the matched B cells. This contact allows CD40 on the B cell to bind CD154 on the T cell, an interaction that triggers the second intracellular signal needed for B-cell activation (Fig. 24.19, step 5). During the secondary response, B cells still need help to become plasma cells but do not need direct contact with helper T cells. Memory B cells that have antigen bound to their B-cell receptors can respond to the soluble IL-4 and IL-6 cytokines secreted by activated helper T cells without having direct contact with the T H cell. IL4 stimulates B-cell proliferation, while IL-6 directs differentiation into antibody-secreting plasma cells (Fig. 24.19, step 6).
Production of Cytotoxic T cells As with helper T cells, the fi rst signal needed to activate cytotoxic T cells is the binding of the TCR to an antigen-MHC complex (in this case, MHC class I) on an antigen-presenting cell (see Fig. 24.19, step 1). Figure 24.20 shows a closer look at the interaction between an antigen-presenting cell and a CD8 cytotoxic T cell. This interaction occurs in a lymph node. As with the production of helper T cells, this interaction alone will not activate the cytotoxic T cell. The second signal needed here is actually a cytokine called IL-2 produced by the T H1 class of helper T cells (Fig. 24.19, step 7). The fi rst signal (the TCR–antigen–MHC I complex) initiates the synthesis of IL-2 receptors on the TC cell surface. Upon subsequent binding of IL-2, the TC cell gains cytotoxic activity, after which it can leave the lymph node, travel to the site of infection, and kill any cell bearing the same peptide–MHC class I complex (for example, cells infected with the same virus that triggered TC production) (Fig. 24.21). The B7-CD28 interaction that was needed to activate helper T cells is not required for TC cell activation but can help.
Superantigens Do Not Require Processing to Activate T Cells Normal antigens require processing by antigenpresenting cells. Each peptide produced by antigen processing is ultimately recognized in the context of
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1. The first T-cell activation signal is the linking of TCR to antigen bound to MHC class I (for TC cells) or class II (for TH cells) receptors on APC surfaces. The CD8 molecules that help define TC cells selectively bind MHC class I, while the CD4 molecules on TH cells selectively bind to MHC class II.
Humoral immunity favored Extracellular pathogens TH0
Signal 1
Cell-mediated immunity favored
MHC ΙΙ
2. The second signal needed to activate TH0 cells is the binding of CD28 on the T-cell surface to a B7 protein on the APC cell surface, which stimulates cytokines to convert activated TH0 cells to TH1 or TH2 cells.
CD28
Signal 1 CD4
B7 Signal 2
MHC Ι
Intracellular pathogens
CD8
TCR Antigen 3. Whether activated TH0 cells develop into TH1 or TH2 depends on what cytokines are present after infection. Mast cells produce IL-4, which promotes TH0 differentiation into TH2 cells.
Activated TH
IL-4 IL-5 IL-6
IL-12
TH2
IL-2
IFNγ
IL-4 IL-10 CD154
Processed antigen
Macrophage
TH1 cell
TH2 cell
Signal 2
CD40
B cell 6. Activated memory B cells respond to IL-4 and IL-6 cytokines secreted by activated T cells. IL-4 stimulates B-cell proliferation, while IL-6 directs differentiation into antibody-secreting plasma cells.
Viruses and bacteria
IL-4
4. Cytokines such as IFN-γ produced by TH1 cells inhibit TH2 development, while IL-4 and IL-10 secreted by TH2 cells inhibit TH1 differentiation.
Signal 2
7. Once TH1 cells are produced, they secrete cytokine IL-2, which is the second signal needed to activate TC cells. Activated TC cells secrete molecules such as perforin and granzymes that destroy infected target cells.
Parasites and allergens Mast cells
TC
APC
5. Activated TH cells (the TH2 subset) link via TCR to B cells presenting MHC II molecules bound to matched antigen. This contact allows binding between CD40 on the B cell to CD154 on the T cell, which triggers the second signal needed for B-cell activation.
Plasma cell Signal 1 Cross-link B-cell receptor
Activates macrophages containing intracellular bacteria
8. TH1 cells can also activate macrophages harboring intracellular bacteria. To activate the macrophage, the TH1 cell TCR binds to MHC II–antigen complex and secretes IFN-γ.
Summary of the activation of humoral and cell-mediated pathways. Intracellular pathogens generally activate cell-mediated immunity by stimulating cytotoxic T cells. Extracellular pathogens tend to activate humoral immunity. The balance between cell-mediated and humoral immune responses to a given infection is regulated by the balance between the production of TH1 (cell-mediated) versus TH2 (antibody) helper cells. This balance is influenced by whether the foreign antigen was made by intracellular pathogens (TH1-favored) or extracellular pathogens (TH2-favored), and whether antigen presentation (APC) occurred through a macrophage (TH1-favored) or a mast cell (TH2-favored). TH1 cells will encourage activation of cytotoxic T cells (cell-mediated immunity; best for killing intracellular pathogens), while TH2 promotes antibody production (humoral immunity, best for attacking extracellular pathogens). See text for more details. Figure 24.19
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self MHC molecules by a specific cognate T-cell receptor. However, when an antigen is introduced into a host, there are only a few T cells with the proper TCR needed to recognize that antigen—in the range of 1–100 cells in a million. Proliferation of the T cells through antigen-dependent activation increases that number and increases the immune response to that antigen. Some proteins, called superantigens, stimulate T cells much more rapidly by bypassing the normal route of antigen processing. In fact, recognition as an antigen is not even involved. Certain microbial toxins, such as staphylococcal toxic shock syndrome toxin (TSST), are actually superantigens. These proteins, as illustrated in Figure 24.22, simultaneously bind to the outside of T-cell recep-
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tors on T cells and to the MHC molecules on antigenpresenting cells (for example, macrophages). This joining of the T cell and macrophage activates more T cells than a typical immune reaction, and stimulates release of massive amounts of inflammatory cytokines from both cell types. The effect of superantigens can be devastating because cytokines like tumor necrosis factor can overwhelm the host immune system regulatory network and cause severe damage to tissues and organs. The result is disease and sometimes death. Jim Henson, the creator of Kermit the frog and other Muppet characters, died in 1990 from complications of pneumonia caused by a potent superantigen produced by Streptococcus pyogenes. This example is but one of many ways that overreaction of the immune system causes morbidity (disease) and mortality (death), more so than the direct effects of the pathogen involved.
Infected cell Nucleus Viral mRNA
Viral DNA 1. Virus produces peptides that are processed to the surface with MHC I.
2. CD8 on the T cell recognizes MHC I proteins and facilitates docking of the T-cell receptor.
The CD4 and CD8 Surface Proteins Help T Cells Differentiate between MHC I and MHC II Surface Proteins One question not yet addressed is how do the TCRs on TC and T H0 cells “know” to respectively bind MHC class I and class II antigen complexes? The answer is based on the different clusters of cellular differentiation (CD) molecules displayed on TC and T H0 cell surfaces. The surface CD proteins CD8 and CD4 help T cells distinguish between MHC class I and class II molecules on antigen-presenting cells. The CD8 molecules on TC cells selectively bind MHC class I while CD4 molecules on T H cells selectively bind to MHC class II (see Fig. 24.19, step 1). Antigens presented on class I MHC molecules generally arise from intracellular pathogens such as viruses and some bacteria, situations that will require cell-mediated immunity for resolution. In contrast, antigen peptides presented on class II MHC molecules originate from extracellular infections and are only recognized by CD4 T H cells. Once activated, cytotoxic CD8 TC cells secrete molecules such as perforin and granzymes that destroy infected target cells (similar to natural killer cells; discussed in Section 23.7). Perforin forms a pore in target cells that is used to deliver toxic enzymes (granzymes).
Viral peptides
CD8
MHC I
Peptide 3. This T cell is turned into a killer T cell that will destroy the virus-infected target cell.
T-cell receptor
Cytotoxic T cell (CD8+)
Presentation of a viral antigen to a T cell. CD8 protein on a T cell directs the interaction between a T-cell receptor and a viral antigen bound to a class I MHC protein on an antigen-presenting cell.
Figure 24.20
Cytotoxic T-cell action. A. APC A. An antigen-presenting cell presenting viral peptide on MHC I surface molecules interacts with and activates a cytotoxic T cell in a lymph node. B. The activated TC cell then leaves the lymph node and migrates to the site of infection where it Cytotoxic can recognize the viral peptide presented T cell on the MHC class I receptor on an infected cell. This interaction authorizes the cytotoxic T cell to kill the infected cell.
B.
Figure 24.21
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Viral peptide
TH1 cell
IL2
Activated TC cell
Infected cell
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A.
Cytoplasm
Antigen-presenting cell
After processing, antigens bind within MHC and TCR molecules. Antigen CHO
α2
β2
α1
β1
αV αC
βV βC
MHC II
T-cell receptor
α2
β2
α1
β1
αV αC
βV βC
Exterior Superantigen Superantigens bind immediately to outside of MHC and TCR molecules.
T lymphocyte
Superantigens. A. The difference between presentation of antigen and superantigen. Antigen presentation requires the antigen to bind within the binding pockets of MHC and TCR molecules. Superantigens do not require processing. They can bind directly to outer aspects of the TCR and MHC proteins, linking and activating the two cell types. B. Staphylococcal toxic shock syndrome toxin is one example of a potent superantigen. Alpha helices are shown as red ribbons, while sections of the beta sheet are shown as blue ribbons. (PDB code: 2qil)
B.
Figure 24.22
MHC binding
TCR binding
Because they directly attack host cells, CD8 TC cells are the major “enforcers” of the cellular immune system, along with macrophages and NK cells. Why does the presentation of antigens on MHC I molecules activate cytotoxic T cells rather than B cells and antibody production? A major reason is that intracellular pathogens (for example, viruses) hide inside host cells, where they are protected from antibody. Consequently, these pathogens are best killed when the harboring host cell is also sacrificed (via cellular immunity). Because class I MHC molecules are found on all nucleated cells, any infected cell can potentially activate TC cells, converting them to cytotoxic T cells that ultimately kill the infected cell (TC cells are not actually cytotoxic until they are activated). The infected host cell is sacrificed for the good of the whole animal, human or otherwise. Whereas antigens presented on class I MHC molecules elicit only cell-mediated immunity, antigens presented on class II molecules can actually stimulate either arm of the immune system. In contrast to CD8 TC cells, CD4 helper T cells do not directly kill host cells. Instead, activated CD4 T H cells release various cytokines called lymphokines that, among other things, attract additional
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white cells to the area. Table 24.6 lists a fraction of the many different cytokines produced by various cell types and their influence over the immune system. Activated T H1 cells secrete cytokines that stimulate the cytotoxic TC cells of the cellular immune system (see Fig. 24.19, step 7), whereas activated T H2 cells directly interact with the class II MHC–antigen peptide complexes on B cells, as described earlier, and stimulate those cells to differentiate and produce antibody (see Fig. 24.19, step 5). AIDS (acquired immunodeficiency syndrome) provides a vivid and tragic illustration of the importance of CD4 T cells in immunity. The human immunodeficiency virus (HIV) binds to CD4 molecules and thus is able to invade and infect CD4 + T cells. As the disease progresses, the number of CD4 T cells declines below its normal level of about 1,000 per microliter. When the number of CD4 T cells drops below 400 per microliter, the ability of the patient to mount an immune response declines dangerously. Not only does the patient become hypersusceptible to infections by pathogens, but the individual becomes susceptible to infection by commensal organisms as well. Most patients actually die from these opportunistic infections.
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Table 24.6 Select cytokines that modulate the immune response. Cytokine
Sample sources
General functions
IL-1
Many cell types, including endothelial cells, fibroblasts, neuronal cells, epithelial cells, macrophages
IL-2 IL-3
TH1 cells T cells, mast cells, keratinocytes
IL-4
TH2 cells, mast cells
IL-5
TH2 cells
IL-6
IL-10
TH2 cells, macrophages, fibroblasts, endothelial cells, hepatocytes, neuronal cells Monocytes, endothelial cells, T cells, keratinocytes, neutrophils TH2 cells, B-cells, macrophages, keratinocytes
IFN-α/β IFN-γ TNF-α TNF-β
T cells, B cells, macrophages, fibroblasts TH2 cells, cytotoxic T cells, NK cells T cells, macrophages, NK cells T cells, B cells
Affects differentiation and activity of cells in inflammatory response; central nervous system; acts as endogenous pyrogen Stimulates T-cell and B-cell proliferation Stimulates production of macrophages, neutrophils, mast cells, others. Promotes differentiation of CD4 and T cells into TH2 helper T cells; promotes proliferation of B cells Acts as a chemoattractant for eosinophils; activates B cells and eosinophils Stimulates T-cell and B-cell growth; stimulates production of acute-phase proteins Chemoattracts PMNs; promotes migration of PMNs through endothelium Inhibits production of IFN-γ, IL-1, TNF-α, IL-6 by macrophages Promotes antiviral activity Activates T cells, NK cells, macrophages Exerts wide variety of immunomodulatory effects Exerts wide variety of immunomodulatory effects
IL-8
The MHC Restriction Rule Limits T-Cell Reactivity As noted previously, T cells can only recognize antigens complexed to an MHC molecule. Actually, it must be a self, MHC molecule. The MHC molecules of each person are unique. Thus, T cells from one individual normally do not recognize peptides placed on antigen-presenting cells from another individual, a property known as MHC restriction. CD4 TH cells are class II MHC restricted, while CD8 TC cells are class I restricted. Rolf Zinkernagel (University of Zurich) and Peter Doherty (St. Jude Children’s Hospital, Memphis) discovered this phenomenon in 1976 with a simple yet profoundly insightful experiment. They demonstrated that mouse cytotoxic T cells would kill only virus-infected target cells that possessed the same MHC surface protein. If the infected target cell came from a different strain of mouse containing a different MHC protein, the infected target cell was spared. Thus, there are two components to T-cell recognition: self and nonself. The self component comes from the MHC receptor, while the nonself component comes from the pathogen. This concept won Zinkernagel and Doherty the 1996 Nobel Prize in Physiology or Medicine.
Cytokines Tip the Balance between Cell-Mediated Immunity and Antibody Production We have already discussed how cytokines made from T H1 cells help activate cells involved with cell-mediated
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immunity (the CD8 TC cells, macrophages, and natural killer cells) and that other cytokines from T H2 cells trigger B cells to class-switch their immunoglobulin. But what determines whether T H1 or T H2 cells predominate during an infection? Part of the answer is found in the contingent of cytokines generated by macrophages or mast cells during different types of infection. Infections by viruses and bacteria favor the production of T H1 cells, whereas allergens and parasites favor differentiation to T H2. Early in an infection, bacteria and viruses stimulate cells of the innate immune system (macrophages and natural killer cells) to produce cytokines that tilt development of T H0 into T H1 (one such cytokine is IL-12). During interactions with an allergen, on the other hand, mast cells produce IL-4, which promotes T H0 differentiation into T H2 cells (see Fig. 24.19, step 3). There is also cross-inhibition of T H1 and T H 2 development. Cytokines such as interferon-gamma (IFN-γ) produced by T H1 cells inhibit T H 2 development, while interleukins IL-4 and IL-10 secreted by T H 2 cells inhibit T H1 differentiation (Fig. 24.19, step 4). As a result, once the immune system starts to go down one pathway, the other pathway is held back. But this is not an all-or-none response. For instance, a viral infection will also result in T H 2 cells that stimulate antibody production. The tilt toward T H1 or T H 2 predominance occurs because the body realizes which pathway, cell mediated (T H1) or humoral (T H 2), will more effectively clear a particular infection.
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Microbial Evasion of Adaptive Immunity Efficient as our immune system is, numerous viral and bacterial pathogens have developed effective means for avoiding the adaptive immune response. Many viruses produce proteins that downregulate production of class I MHC on infected cell surfaces. This will limit antigen presentation, since MHC I is needed for that process. On the other hand, losing MHC I should expose the infected host cell to natural killer cells because NK cells attack peers that lack MHC I (discussed in Section 23.7). To surmount this obstacle, it appears that human cytomegalovirus places a decoy MHC I-like molecule on the surface of infected cells. These decoys are thought to bind inhibitory receptors on NK surfaces that block NK cell cytotoxicity. Bacteria, like viruses, are masters of illusion when it comes to the immune system. A major cause of gastric ulcers, Helicobacter pylori expresses proteins from a cluster of pathogenicity genes that trigger apoptosis of T cells. (Apoptosis is a programmed cell death.) Other bacteria have evolved mechanisms that interfere with signal transduction pathways controlling the expression of cytokines. For example, YopP from Yersinia enterocolitica, one cause of gastroenteritis, inhibits a specific signal transduction pathway needed to produce TNF, IL-1, and IL-8. Thus, Yersinia avoids the detrimental effects of those pro-inflammatory cytokines. Another method of immune avoidance is employed by various mycobacteria, some of which cause tuberculosis and leprosy (Fig. 24.23). These bacteria induce the production of anti-inflammatory cytokines, which dampen the immune response. Mycobacterium-infected macrophages produce IL-6, which inhibits T-cell activation, and IL-10, which downregulates the production of MHC II molecules needed for specific activation of T cells. Fortunately for humans, in most cases the immune system catches on to these tricks and through redundant humoral and cellular mechanisms manages to resolve these infections.
also requires two signals. One activation pathway involves interferon-gamma (IFN-γ) produced by nearby infected or damaged cells, followed by binding of the macrophage to lipopolysaccharide (LPS) or other microbial components. Once activated, the macrophage becomes highly aggressive in terms of phagocytosis and increases production of numerous antimicrobial reactive oxygen intermediates. As noted earlier, some microorganisms such as mycobacteria, the causative agents of tuberculosis and leprosy, are intracellular pathogens that grow primarily in the phagolysosomes of macrophages. There they are shielded from the effects of both antibodies and cytotoxic T cells. These pathogens live in the usually hostile environment of the phagocyte either by inhibiting the fusion of lysosomes to the phagosomes in which they grow or by preventing the acidification of the vesicles needed to activate lysosomal proteases. However, macrophages can rid themselves of these pathogens if the macrophage is activated by a T H1 cell (see Fig. 24.19, step 8). Macrophages, even unactivated, are able to process some of the intracellular bacteria and place antigen from them on their class II MHC molecules. T H1 cells then activate these macrophages though binding of their TCR molecules to the macrophage MHC II–antigen complex and by secreting interferon-gamma. The activated macrophages can then kill any bacteria that may be growing within them. Because activated macrophages are such effective assassins of microbial pathogens, why are macrophages not always kept in an active state? A major reason is that once activated, macrophages also damage nearby host tissue through the release of reactive oxygen radicals and proteases. Thus, effective killing of microbial pathogens comes at the expense of host tissue damage. TO SU M MAR I Z E: ■
Activated TH1 Cells Can Also Activate Macrophages Macrophages, too, must be activated to become highly effective killers of microbes. Activation of macrophages
■
The major histocompatibility complex consists of membrane proteins with variable regions that can bind antigens. Class I MHC molecules are on all nucleated cells, while antigen-presenting cells contain both class I and class II MHC molecules. Antigen-presenting cells (APCs) such as dendritic cells present antigens synthesized during an intra-
10 μm
Courtesy of E. Hoffman and S. Kuznetsov, Univ. of Rostock
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Macrophage
Mycobacterium bovis growing within cultured macrophages (cell line J774). Left panel is a differential interference contrast image of an infected macrophage. Right image is the same cell viewed by fluorescence microscopy. M. bovis is carrying green fluorescent protein. The macrophage was stained for F-actin (red) and nucleus (blue).
Figure 24.23
M. bovis
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cellular infection on their surface class I MHC molecules, but place antigens from engulfed microbes or allergens on their class II MHC molecules. Activation of a TH0 cell requires two signals: TCR/ CD4 binding to an MHC II–antigen complex on an antigen-presenting cell, and CD20-B7 interaction. TH0 cell differentiation to T H1 or T H2 cells is influenced by different cytokine “cocktails” secreted by macrophages and NK cells during an infection. Activation of a B cell into an antibody-producing plasma cell usually requires two signals: antigen binding to BCR and binding to a T H2 cell activated by the same antigen. Activation of cytotoxic T cells requires two signals: TCR/CD8 molecules that recognize MHC I–antigen complexes and cytokines such as IL-2 made from activated T H1 cells. The activated cytotoxic T cells in turn destroy infected host cells. Superantigens stimulate T cells by directly linking TCR on a T cell with MHC on an APC without undergoing APC processing and surface presentation.
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Antigen lgG
C1
1. C1 complex binds FC region.
2. C1 complex cleaves C4 and C2, whose fragments rejoin to make C3 convertase.
C4
C2
3. C3 convertase
Enzyme 4. C3 convertase cleaves C3 to C3a + C3b. C3b joins with C3 convertase to make C5 convertase.
THOUGHT QUESTION 24.8 Transplant rejection is a major consideration when transplanting most tissues because host TC cells can recognize allotypic MHC on donor cells. So, why are corneas easily transplanted from a donor to just about any other person? THOUGHT QUESTION 24.9 Why does attaching a hapten to a carrier protein allow production of antihapten antibodies?
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C3
C3 is shared by the alternate pathway.
C3a
C3b
5. C5 convertase
The alternate pathway is identical from here.
C5
6. C5 convertase cleaves C5 to C5a + C5b.
C5a C5b
24.7 Complement as Part of Adaptive Immunity Chapter 23 describes how complement can attack invading microbes before a specific immune response is launched. In what is called the alternative pathway (see Section 23.8), C3b binding to LPS sets off a cascade of reactions resulting in the formation of a membrane attack complex (MAC) pore composed of C5b, C6, C7, C8, and C9 proteins (see Fig. 23.29). In addition to the alternative pathway, antibodies made during the humoral response to a pathogen offer another route to activate complement called the classical complement pathway. Called “classical” because it was the fi rst complement pathway to be discovered, it requires a few additional proteins before reaching C3, the linchpin factor connecting the two pathways. The classical cascade begins with the binding of the complement protein C1 complex to the Fc region of an antibody that is part of an antigen-antibody complex, as you would find when antibody binds to a bacterial or viral pathogen (Fig. 24.24). The bound C1 complex then cleaves
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C7 7. C5b leads to formation of the membrane attack complex.
C8
C5b C6 C9
Figure 24.24
Classical complement cascade.
two other complement factors, C2 and C4, not used in the alternative pathway. Two fragments, one each for C2 and C4, unite to form another protease called C3 convertase. This enzyme cleaves C3 into C3a and C3b. C3b, which in the alternative pathway is stabilized by interacting with LPS, combines in the classical pathway with the C3 convertase to make a C5 convertase (note that this C5 convertase is different from the C5 convertase formed in the alternative pathway; see Fig. 23.31). The subsequent steps leading to formation of a membrane attack complex are the same as in the alternative pathway. As before, C5b
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binds to a target membrane and is joined by C6, C7, and C8. Multiple C9 proteins then assemble around the membrane attack complex and form a pore that compromises the integrity of the target cell. One might wonder why we need complement with all the other immune responses at our disposal, but the need becomes evident in individuals with complement deficiencies. These patients are extremely susceptible to recurrent septicemic (blood) infections by organisms such as Neisseria gonorrhoeae (the cause of gonorrhea) that normally do not survive forays into the bloodstream because they are killed by complement. Why does reinfecting Neisseria not quickly succumb to all the other defenses, such as antibodies and cytotoxic T cells? Because key antigens on the bacterial surface undergo phase variations (see Sections 10.6 and 27.7), many antibodies made to cells of N. gonorrhoeae participating in the initial infection are useless during a second infection.
mation but are important for amplifying the immune reaction. These peptides act as chemoattractants to lure more inflammatory cells into the area. C3b, which does participate in MAC formation, also can act as an opsonin when it is bound to a bacterial cell. An opsonin is a protein factor that can facilitate phagocytosis. Because phagocytes contain C3b receptors on their surfaces, it is easier for them to grab and engulf cells coated with C3b. THOUGHT QUESTION 24.10 Do bone marrow transplants in a patient with severe combined immunodeficiency require immunosuppressive chemothereapy? THOUGHT QUESTION 24.11 How can a stem cell be differentiated from a B cell at the level of DNA? TO SU M MAR I Z E: ■
Regulation of Complement Activation
The classical pathway for complement activation begins with an interaction between the Fc portion of an antibody bound to an antigen and C1 factor in blood. (The alternative pathway does not need antibody but begins when C3b binds to LPS on a bacterium.) The Fc-C1 complex reacts with C2 and C4, leading to production of C3b and a novel C5 convertase specific to the classical pathway. After C5 convertase cleaves C5, the classical pathway is the same as the alternative pathway, resulting in the formation of a membrane attack complex (MAC). CD59 and/or factor H prevent inappropriate MAC formation in host cells.
How do normal body cells prevent self-destruction following complement activation? The sequential assembly of the membrane attack complex provides several places where regulatory factors can intervene. One such factor is the host cell surface protein CD59. This protein will bind any C5b-C8 complex trying to form in the membrane and prevent C9 from polymerizing. Thus, no pore is formed and the host cell is spared. (Complement will not normally attack normal or infected host cells, since both contain CD59.) Another regulatory mechanism hinges on a normal serum protein called factor H. This protein prevents the inadvertent activation of complement in the absence of infection. In the absence of factor H, the uncontrolled activation of complement could damage host cells despite the presence CD59. To short-circuit the cascade, factor H binds to C3bBb, displaces Bb from the complex, and acts as a cofactor for factor I protease that then cleaves C3b. Without C3b, the complement cascade stops. Some bacteria, such as some strains of Neisseria gonorrhoeae, have learned to protect themselves from complement by binding factor H. Normal flora can also assist host cells in resisting complement. The intestinal microbe Bacteroides thetaiotamicron protects host cells from complement-mediated cytotoxicity by upregulating a decay-accelerating factor present in host cell membranes. Decay-accelerating factor stimulates decay of complement factors and prevents their deposition at the cell surface.
Sometimes the immune system overreacts to certain foreign antigens and causes more damage than the antigen (microbe) alone might cause. Furthermore, some foreign antigens possess structures similar in shape to host structures and can trick the immune system into reacting against self. These immune miscues are called allergic hypersensitivity reactions, and the antigen causing the reaction is called an allergen. There are four types of hypersensitivity reactions (Table 24.7). The fi rst three types of hypersensitivity are antibody mediated; the fourth is cell mediated.
Other Roles for Complement Fragments
Case History: Type I Hypersensitivity
The C3a, C4a, and C5a cleavage fragments generated by the complement cascade do not participate in MAC for-
A bee stings a 9-year-old boy walking with his mother at the zoo. Within minutes the boy begins sweating and itching. His
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Table 24.7 Summary of hypersensitivity reactions. Type
Description
Time of onset
Mechanism
Manifestations
I
IgE-mediated hypersensitivity
2–30 min
II
Antibody-mediated cytotoxic hypersensitivity
5–8h
III
Immune complex–mediated hypersensitivity
2–8h
Systemic anaphylaxis, local anaphylaxis, hay fever, asthma, eczema Blood transfusion reactions, hemolytic disease of the newborn, autoimmune hemolytic anaemia Systemic reactions, disseminated rash, arthritis, glomerulonephritis
IV
Cell-mediated hypersensitivity
24–72h
Ag induces cross-linking of IgE bound to mast cells with release of vasoactive mediators Ab directed against cell surface antigens mediates cell destruction via ADCC or complement Ag-Ab complexes deposited at various sites induce mast cell degranulation via Fc receptor; PMN degranulation damages tissue (Arthus reaction, localized) Memory TH1 cells release cytokines that recruit and activate macrophages
Contact dermatitis, tubercular lesions
Ag = antigen; Ab = antibody; ADCC = antibody-dependent cell cytotoxicity; PMN = polymorphonuclear leukocyte
chest then starts to tighten and he has tremendous difficulty breathing. Terrified, he looks to his equally frightened mother for help. This is a classic and severe example of type I (immediate) hypersensitivity, sometimes called anaphylaxis. (Anaphylaxis is defi ned as the contraction of smooth muscle and dilation of capillaries due to release of pharmacologically active substances.) Type I hypersensitivity occurs within minutes of a second exposure to an allergen when the allergen reacts with IgE-coated mast cells. Recall that the primary role of IgE is to amplify the body’s response to invaders by causing mast cells to release inflammatory mediators (see Section 24.3). On initial exposure, an allergen elicits the production of IgE antibodies specific to the allergen (bee venom in this example). The Fc portions of these antibodies bind to Fc receptors on the surfaces of mast cells. IgE-coated mast cells are then said to be sensitized. Most allergen molecules contain several identical antigenic sites. Thus, a single allergen particle during a second exposure can bind to adjacent surface IgE molecules. The result is a bridge between adjoining binding sites (Fig. 24.25). This cross-linked complex inhibits the mast cell enzyme adenylate cyclase, so that intracellular levels of cyclic adenosine monophosphate (cAMP) fall. The lower levels of intracellular cAMP cause the mast cell granules to quickly migrate to the cell surface and release their contents in a process called degranulation. Understanding this process is clinically important because anaphylaxis inhibitors such as epinephrine (also known as adrenaline) stimulate adenylate cyclase activity, stopping the anaphylaxis cascade in its tracks.
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Mast cell degranulation releases chemicals with potent pharmacological activities. The most important of these is histamine, which binds to histamine receptors (H1 receptors) present on most body cells. Antihistamines have a structure similar to histamine and work by binding H1 receptors to prevent histamine from binding. When histamine binds H1, it activates an enzyme (phospholipase A) that releases from the cell membrane a 20-carbon fatty acid called arachidonic acid. Arachidonic acid is then converted to leukotrienes by 5-lipooxygenase A and to prostaglandins by cyclooxygenase. Another important chemical released during degranulation is tumor necrosis factor. All of these mediators (leukotrienes, prostaglandins, and tumor necrosis factor) cause the smooth muscles nearby to contract. This constricts small blood vessels and expands capillary pores. Fluid is then forced from the circulation into the tissues. The immediate consequence is swelling (edema) in the joints and around the eyes and a rash (similar to hives), with burning and itching of the skin due to nerve involvement. In the clinical example involving the bee sting, the contraction of lung smooth muscles led to breathing difficulties. What we described was a severe type I allergic reaction caused by a bee sting. Type I hypersensitivity reactions do not usually involve the whole body. Most type I reactions are more localized and cause what is called atopic (“out of place”) disease. Hay fever, or allergic rhinitis, is a common manifestation of atopic disease that can be caused by the inhalation of dust mite feces (Fig. 24.26), animal skin or hair (dander), and certain types of grass or weed pollens. This disease affects the eyes, nose, and upper respiratory tract. Atopic asthma, another form of type I hypersensitivity resulting from inhaled
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Ragweed pollen 1. Allergy-prone person first encounters allergen (e.g., ragweed).
B cell
IgE 2. Large amount of ragweed IgE is made.
Feces Dust mite
Plasma cell
Fc receptor
Figure 24.26 Inhaled allergen. Dust mite (200 to 600 micrometers) and dust mite feces (colorized SEM).
Mast cell
4. On second encounter, cross-linking of surface-Ig on IgE-primed mast cells lowers cAMP levels, which causes release of chemicals such as histamine.
Chemicals
Epinephrine can prevent degranulation by increasing cAMP.
5. Person suffers sneezing, runny nose, watery eyes, and itching.
Symptoms
Figure 24.25
Events leading to type I hypersensitivity
reactions.
allergens, affects the lower respiratory track and is characterized by wheezing and difficulty breathing. The administration of antihistamines is an effective treatment of allergic rhinitis, but the more important chemical mediators of asthma are the leukotrienes produced dur-
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3. Anti-ragweed IgE attaches to mast cells.
ing what is called late-phase anaphylaxis. In late-phase anaphylaxis, mast cells release chemotactic factors that call in eosinophils. Eosinophils entering the affected area produce large amounts of leukotrienes that, like histamine, cause vasoconstriction and inflammation. At this point, antihistamines have little effect. Effective treatment includes inhaled steroids to minimize inflammation and bronchodilators (for example, albuterol) to widen bronchioles and facilitate breathing. A more recently developed medication, SINGULAIR™, works by blocking leukotriene receptors in the lung. During severe asthma attacks, injected epinephrine is critical. Epinephrine will open airways to ease breathing through direct hormonal action. People sensitized to allergens are not necessarily doomed to suffer with the allergy their entire life. A clinical treatment called desensitization can sometimes be used to prevent anaphylaxis. Desensitization involves injecting small doses of allergen over a period of months. This process is thought to produce IgG molecules that circulate, bind, and neutralize allergens before they contact sensitized mast cells. Desensitization has been useful in cases of asthma, food allergies, and bee stings, although results are variable and by no means guaranteed. There is a relatively new treatment for allergic asthma, omalizumab (Xolair), that can actually prevent sensitization. Omalizumab is a monoclonal antibody (an antibody preparation of one antibody type that only binds one epitope), administered by injection that selectively binds IgE. It prevents IgE from binding to Fc receptors on mast cells and, thus, prevents sensitization.
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Why don’t all antigens generate type I hypersensitivity? The answer is that most antigens do not elicit high levels of IgE antibodies. It is unclear what makes certain antigens capable of this and what makes different individuals prone to different allergies.
Case History: Type II Hypersensitivity A hospitalized male patient with blood group B accidentally receives a transfusion with type A blood. Within hours, the individual experiences chills and lowered blood pressure and has blood in his urine, all symptoms of ABO blood group incompatibility.
Swiss Society of Neonatology
This is a form of type II hypersensitivity. Type II hypersensitivity starts with antibody binding to cell surface antigens (donor blood cells, in this case). This differentiates it from type III hypersensitivity (considered next), which involves antibody binding to soluble antigens. The classic type II hypersensitivity reaction is illustrated by blood group incompatibilities resulting from transfusions. Destruction of the foreign red blood cells can occur by two routes. In the fi rst route, antibody-dependent cell-mediated cytotoxicity is triggered when NK cells or macrophage surface Fc receptors bind to the Fc regions of antibodies bound to foreign red blood cell antigens. The second route involves complement activation, in which complement (C1) binds to the Fc regions of circulating antibodies affi xed to foreign red blood cells. The complement cascade then creates a membrane attack complex (MAC) that destroys the RBC. Another form of type II hypersensitivity is Rh incompatibility disease of the fetus and newborn (Fig. 24.27). Similar to ABO antigens, Rh antigens are chemical groups on red blood cells. Incompatibility between mother (Rh–) and fetus (Rh+) can lead to a hypersensitivity reaction in the fetus, especially in a second pregnancy. During birth of an Rh+ child to an Rh– mother, fetal red blood cells enter the mother’s bloodstream. The Rh– mother’s immune system will make anti-Rh+ IgG antibodies that can pass through the placenta. This is a problem during a subsequent preg-
Type II hypersensitivity. Targeting foreign red blood cells in Rh disease of the newborn.
Figure 24.27
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nancy because these antibodies will attack an Rh+ fetus. Rh disease can be prevented if the mother is passively given anti-Rh+ immunoglobulin by intramuscular injection within a day after birth. This antibody binds the fetal Rh+ antigen and prevents the mother’s immune system from making Rh+ antibody and the attendant memory B cells. Passive immunity only lasts four to six weeks, long enough to clear the fetal antigens from the mother’s bloodstream.
Case History: Type III Hypersensitivity A 16-year-old girl is treated for acne with minocycline (a tetracycline derivative). Two weeks later she develops a fever and a urticarial rash (Fig. 24.28B) and experiences muscle and joint pain. (Urticaria are hives, an itchy rash caused by tiny amounts of fluid that leak from blood vessels just under the skin surface.) Her symptoms resolve after five days of antiinflammatory steroid treatment. This is an example of immune complex disease, also called type III hypersensitivity. In contrast to type II hypersensitivity, type III hypersensitivity is initiated by IgG antibody binding to soluble antigen, which in this case is minocycline. Penicillin sensitivity, usually associated with type I anaphylaxis, can also involve a type III hypersensitivity reaction. Normally, antigen-antibody complexes are readily cleared by macrophage engulfment, but an excessive amount of complexes may overwhelm the system and circulate in the blood, free to bind complement (C1 factor, classical pathway). The binding of C1 triggers a cascade in which complement fragments recruit and activate polymorphonuclear leukocytes (PMNs). The complement-activated PMNs release lysosomal proteases and reactive oxygen species that damage host cells in the area. A more recent model for type III hypersensitivity is shown in Figure 24.28A. In this model, type III hypersensitivity is very similar to type I hypersensitivity in that mast cells are involved. The difference is that the immune complexes involve IgG molecules that are not prebound to mast cells. Once made, the soluble IgG-antigen complexes bind to Fc receptors on mast cells, causing degranulation and PMN recruitment. The PMNs use their Fc receptors to bind the complexes formed or deposited near tissues. The PMNs then release enzymes that damage cells in the area. Tissue deposition is important for disease because PMNs can more easily engage the deposited complex than if it is floating in blood, and the factors released will maintain a higher concentration in the local area. Because antigen-antibody complex deposition occurs throughout the body, the result is still a systemic disease. Systemic type III hypersensitivity will resolve once the immune complexes are cleared. In the case history described, steroid treatment was administered to minimize inflammation as the complexes were cleared. In addition to systemic effects, there are also localized type III reactions. In 1903, a Frenchman named Maurice Arthus (1862–1945) discovered a phenomenon now
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A. IgG immune complex
1. The Fc region of an antigen-antibody complex binds to mast cells.
Case History: Type IV Hypersensitivity Mast cell
FcγRIII
2. Chemotactic chemicals released attract PMNs to the area.
Vessel PMN recruitment
3. PMN Fc receptors bind to antigen-antibody compexes. PMNs release damaging enzymes.
FcγRIII PMN
Fluid serum proteins
Granule contents damage tissue
emedicine.com
B.
Figure 24.28 Type III hypersensitivity. A. Development of hypersensitivity. B. Urticarial rash caused by serum sickness, a form of type III hypersensitivity that can follow the administration of serum. The rash is similar to that formed after the administration of nonprotein drugs (for example, minocycline).
named for him—the Arthus reaction. Every few weeks, he would inject the same set of rabbits with horse serum. With each succeeding injection, he noted an increasingly severe reaction at the injection site. On early rounds only redness was noted, but after the fi fth round, the lesions
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became necrotic and slow to heal. This is a localized type III reaction, where antigen-antibody complexes form at the vessel walls.
The patient is a 19-year-old woman from Argentina. As a child she received the BCG vaccine for tuberculosis. Upon entering university in the United States, she is required to take a skin test for tuberculosis. She tries to refuse but is told it is a requirement and allows the nurse to apply the test to her arm. Three days later, the test site has a large red lesion and the skin is starting to slough (peel off). Type IV hypersensitivity, also known as delayedtype hypersensitivity (DTH), is the only class of hypersensitivity to be triggered by antigen-specific T cells. Because T cells have to react and proliferate to cause a response, it generally takes 24–48 hours before a reaction is noticed. This delay distinguishes type IV hypersensitivity from the more rapid antibody-mediated allergic reactions (types I–III). The woman in the case study was initially sensitized with BCG (an attenuated strain of M. bovis, a close relative of M. tuberculosis) as a result of childhood vaccination. Because the organism is intracellular, the vaccination produced a cell-mediated immunity, complete with preactivated memory T cells (similar to memory B cells). When she was reinoculated by the skin test, the memory T cells activated and elicited a localized reaction at the site of injection. There are two stages to the development of type IV sensitivity. The fi rst stage, sensitization, involves processing and presentation of antigen on cutaneous dendritic cells (called Langerhans cells). These APCs travel to the lymph nodes, where T H0 cells can react to them as described earlier (see Figure 24.19), generating activated T cells and a subset of memory T cells. On second exposure, two routes leading to delayed-type hypersensitivity (DTH) are possible. In the fi rst pathway, memory T H1 cells, upon binding antigen that is complexed to class II MHC receptors (this happens at the site of infection), will release IFN-gamma, TNF-beta, and IL-2 (Fig. 24.29A). These cytokines recruit macrophages and PMNs to the site and will activate macrophages and natural killer cells to release a number of other inflammatory mediators that will damage innocent, uninfected bystander host cells. In the second pathway, memory TC cells can recognize antigen on class I MHC receptors, become activated and directly kill the host cell presenting the antigen (Fig. 24.29B). A hallmark of type IV hypersensitivity is that transferring white blood cells, not serum, can transfer the sensitivity to a naive animal. This is because T cells, not serum antibody, are responsible for the reaction. Forms of delayed-type hypersensitivity reactions include contact dermatitis (such as poison ivy) and
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OH Antigen
OH
An unsaturated pentadecylcatechol
Cellular infiltrate
Dendritic cell
Swelling Blood vessel
3. PMN cells attack the tissues in the area of the antigen and cause damage. Ken Greer/Visuals Unlimited
1. T cells react to antigen on dendritic cells.
PMN T H1 Macrophage Recruitment
T cell
2. T cells secrete cytokines that “call in” macrophages and PMNs from the circulation.
Cytokine secretion
Contact dermatitis. Pentadecylcatechols are chemicals present on the surface of poison ivy leaves. They are haptens that bind to proteins in dermal cells, where they can activate T cells. The result of the ensuing delayed-type hypersensitivity is contact dermatitis.
Figure 24.30
B. Cytotoxic T cells recognize allergen-altered epithelial cells.
TC
TC
MHC I
T-cell receptor
Antigen-altered epithelial cells
Autoimmunity Occurs When the Immune System Is Tricked into Recognizing Self as Nonself
Cell lysis/necrosis apoptosis Granzymes and cytokines trigger apoptosis.
Mechanisms of damage in delayed-type hypersensitivity (type IV). A. TH1 cells react to antigen on dendritic cells. B. Antigen-sensitized cytotoxic T cells recognize allergen haptens attached to proteins of other cells. This recognition triggers granzyme release and cytokine production that triggers apoptosis of the target cell.
Figure 24.29
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allograft rejection. The antigens involved with contact dermatitis are usually small haptens that have to bind and modify normal host proteins to become antigenic (Fig. 24.30). In this case, the hapten-modified protein is processed, and fragments containing the bound hapten are presented on the surfaces of antigen-presenting cells.
The ability to distinguish between self antigens and foreign antigens is crucial to our survival; without this ability, our immune system would constantly attack us from within. Normally, the body develops tolerance to self; occasionally, however, an individual loses immune tolerance against some self antigens and mounts an abnormal immune attack against his or her own tissues. The attack can involve antibodies or T cells and is called an autoimmune response. Autoimmune responses may or may not be associated with pathological changes (autoimmune disease). Almost 30% of the population will have an autoimmune antibody by age 65, but many will not exhibit disease. The mechanisms that lead to autoimmune disease are essentially hypersensitivity reactions. An autoimmune response results in autoimmune disease when an autoantibody or autoimmune lymphocyte causes damage to tissue components. Tissue damage can ensue when antibody to self activates antibodydependent cell cytotoxicity (see Section 24.5). In this
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scenario, autoantibodies affi xed to host cells can bind to NK cell Fc receptors and cause the NK cell to kill the tissue cell. How might autoimmune antibodies be formed? One proposed mechanism starts with the occasional escape of self-reacting B cells from the negative selection process. The negative selection process normally kills autoreactive B cells. Self-reacting B cells that escape this process are usually not a problem because the specific helper T cells needed to activate them were deleted from the T-cell population. However, the self-reacting B cell can still be activated. The B cell can, of course, take up and process a self antigen (or a foreign antigen that mimics a self antigen). The problem comes if the antigen also contains a nonself epitope. This nonself epitope will also be presented on a B-cell receptor. When this happens, T cells specific to the nonself epitope will recognize the nonself peptide and activate that B cell. However, the B cell is programmed to make antibody to the self antigen, not the nonself antigen. Those antibodies will begin to attack the host antigen. A good example of this is the M protein (pili) of Streptococcus pyogenes, a microbe that causes “strep throat” and the autoimmune disease rheumatic fever. M pili contain an epitope that resembles cardiac antigen, but the cardiaclike epitope is flanked by epitopes that are not related to the human host. The cardiac-like epitope can bind surface antibody on an escaped self-reacting B cell and be taken up. Because the nonself epitope and the heart-like epitope are contained within the same protein, the nonself epitope will “piggyback” its way into the B cell. The self epitope is processed and placed on the B-cell surface MHC. When this happens, T cells specific to the nonself epitope
will recognize the non-self peptide and activate the B cell. Because the B cell is programmed to make antibody to the cardiac antigen, those self-reacting antibodies are made, begin to bind to cardiac tissue, and trigger autoimmune disease. In the case of S. pyogenes, cardiac tissue is damaged and rheumatic fever results. This similarity between the epitope in M protein and cardiac tissue is an example of antigenic mimicry. Another type of autoimmune reaction involves cytotoxic T cells. Type 1 diabetes, also known as juvenile diabetes, is an autoimmune disease that usually occurs during childhood, when T cells attack and destroy the insulin-producing islet cells in the pancreas. (In contrast to type 1 diabetics, type 2 diabetics continue to produce insulin, but their cells no longer respond to it.) A theoretical series of events has been proposed that involves molecular mimicry and starts with a viral infection of some sort. In this unproven model, an infecting virus (one study suggests Coxsackie virus), possesses an antigen with a shape similar to the glutamic acid decarboxylase in pancreatic beta islet cells. The cytotoxic T cells produced in response to the viral infection will also attack the beta cells, destroying them and the ability to make insulin. Other autoimmune diseases involve the production of autoantibodies that bind and block certain cell receptors. Graves’ disease (hyperthyroidism), for example, occurs when an autoantibody is made that binds to the thyroid-stimulating hormone (TSH) receptor. This mimics TSH binding and continually stimulates the production of T3 and T4, which generally increases metabolic rate. The thyroid is not destroyed but in fact enlarges to form a goiter. Examples of other important autoimmune diseases are listed in Table 24.8.
Table 24.8 Examples of autoimmune diseases. Autoimmune disease
Autoantigen
Type II hypersensitivity−mediated by antibody to cell surface antigens Acute rheumatic fever Streptococcal M protein, cardiomyocytes Autoimmune hemolytic anemia Rh blood group Goodpasture’s syndrome Graves’ disease
Basement membrane collagen Thyroid-stimulating hormone receptor
Myasthenia gravis
Acetylcholine receptor
Pathology Myocarditis, scarring of heart valves Destruction of red blood cells by complement, phagocytosis Pulmonary hemorrhage, glomerulonephritis Ab stimulates T3, T4 production, hyperthyroidism Interrupts electrical transmission, progressive muscular weakness
Type III hypersensitivity−mediated by antibody complexes with small soluble antigens (immune complex) Systemic lupus erythematosus DNA, histones, ribosomes Arthritis, vasculitis, glomerulonephritis Type IV hypersensitivity−mediated by antigen specific T cells Type I diabetes Pancreatic β cell antigen Multiple sclerosis Myelin protein
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β cell destruction Demyelination of axons
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Organ Donation and Transplantation Rejection
It is now possible to donate organs from one person to another because we can minimize the risk of the recipient’s immune system rejecting the donated tissue. As a result of their “education” process, T cells that survive the selection process by and large do not have TCRs that “see” isogenic MHC proteins on other cells of the body. However, about 5% of the surviving T cells can recognize and bind to allotypic MHC proteins on cells from other individuals (donors). This is because the allotypic MHC resembles a self MHC-foreign antigen complex. For this reason, transplanting organs from a donor with one type of MHC protein into a recipient with a different type of MHC (called an allograft) typically ends badly, with the recipient rejecting the graft. The allotypic MHC proteins of the donor play a major role in this transplantation rejection process. To understand transplant rejection, it is important to remember that during normal antigen presentation, the T-cell receptor actually recognizes part of the presenting MHC molecule (self) as well as the peptide (antigen) presented. Consequently, allograft rejection is initiated in one of two ways. TCR on a recipient’s T cells can directly bind the allotypic MHC (that is, the nonself MHC) on donor target cells that are presenting peptides (Fig. 1A). Alternatively, T cells from the recipient can indirectly recognize pieces of the donor allotypic MHC presented on the recipient’s host cell through normal antigen-processing pathways (Fig. 1B). In the direct route, it does not matter whether the donor’s antigen-presenting cells are presenting self peptides (from the recipient) or foreign peptides (from the donor). The allotypic differences in MHC are sufficient to trigger a response. The indirect route, however, requires that foreign donor peptides be presented on recipient APCs. Once activated by one of these routes, cytotoxic T cells will destroy all the donor cells containing the allotypic MHC proteins. The tissue is rejected. Transplant rejection due to allotypic differences in MHC proteins might appear to contradict the MHC restriction rule for T-cell responses that was noted earlier. By this rule preactivated T cells are restricted to act only on other host cells, that is, cells with compatible MHC proteins. Transplant rejection, however, is the result of T-cell action on foreign cells with incompatible MHC proteins and so appears to violate the MHC restriction rule. This paradox can be understood if we look at the time scales involved. In a typical laboratory experiment, when virus-infected cells of one MHC allotype are mixed with already activated T cells of a different MHC allotype, the in vitro cytotoxic reaction is monitored only for an hour. In this
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time period, activated cytotoxic T cells will not kill virus-infected cells of a different allotype but could kill virus-infected cells of the same isotype MHC. In transplant rejection, however, an unactivated T cell initially recognizes the donor cell allotypic MHC protein as nonself. The subsequent in vivo response to nonself will take days before rejection occurs. There are probably a few CD8 TC cells in the in vitro experiment that could recognize the allotypic MHC antigen, but because they are so few in number, activation would take a long time. MHC type matching is an important consideration for a successful transplant. Then how, you may ask, can we successfully carry out blood transfusions, which do not require MHC matching? Because human red blood cells, which are not nucleated, do not contain MHC proteins, tissue rejection via MHC incompatibility does not occur. However, the A or B antigens present on donor red blood cells can be recognized as foreign. A. Direct route Foreign Self or MHC foreign antigen Recipient T cell TCR
APC graft cell
CD8 B. Indirect route Self MHC Foreign antigen Recipient T cell TCR
APC recipient
CD8
Transplantation rejection. A. In the direct route to tissue rejection, T cells from the recipient recognize the foreign MHC on a donor (graft) cell and become activated. B. The indirect route occurs when pieces of foreign donor MHC (allo-MHC) are presented on the surface of recipient APCs. Recipient TC cells then respond and become activated.
Figure 1
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Allergens cause the host immune system to overrespond or react against self. Type I hypersensitivity involves IgE antibodies bound to mast cells by the antibody Fc region. Binding of antigen to the bound IgE causes mast cell degranulation. Can occur within minutes of an exposure. Type II hypersensitivity (for example, Rh incompatibility) involves antibodies binding to cell surface antigens. This can trigger cell-mediated cytotoxicity or activate the complement cascade. Can occur within hours of an exposure. Type III hypersensitivity is an immune complex disease involving antibody complexes with small, soluble antigens. The complexes can activate complement or bind to mast cells. The result is the recruitment of PMNs that damage host cells in the area. May take weeks to form. Type IV hypersensitivity (delayed-type hypersensitivity) involves antigen-specific T cells. T H1 cells release cytokines that activate macrophages and NK cells. TC cells can directly kill cells that present the antigen. Reaction seen within a few days of exposure. Autoimmune disease is caused by the presence of lymphocytes that can react to self.
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Autoreactive B cells (B cells that make antibody directed against self epitopes) are activated when they present a nonself epitope on their surface MHC. T cells that recognize the nonself epitope activate the B cell, which then secretes antibody against the self epitope. Cytotoxic T cells can produce autoimmune disease by killing host cells that make a self protein that closely resembles a foreign antigen.
Concluding Thoughts Immunity is a remarkable feat of nature. The realization that any one person’s immune system can recognize and respond to virtually any molecular structure and yet remain selectively “blind” to his or her own antigens is hard to comprehend. But even more remarkable is that pathogenic microbes have managed ways to outmaneuver the interconnected redundancies and safeguards of the immune system. In some cases, the pathogen evolves to become less harmful and coexists with the host; indeed, pathogens in nature are far outnumbered by closely related strains that are harmless or even beneficial to their hosts. In other cases, however, evolution generates a never-ending arms race between pathogen and host. The next chapter describes some of the microbial strategies that contribute to the success of a pathogen.
CHAPTE R R EVI EW End of Chapter Questions 1. Defi ne antigen, epitope, hapten, and antigenic deter2. 3. 4. 5. 6. 7. 8. 9.
minant. What is the basic difference between humoral immunity and cellular immunity? Why are proteins better immunogens than nucleic acids? What makes IgA antibody different from IgG? What is immunoprecipitation and radial immunodiffusion? Explain isotypic, allotypic, and idiotypic differences in antibodies. How is IgE involved in allergic hypersensitivity? Discuss differences in the primary and secondary antibody responses. Outline the basic steps that turn a B cell into a plasma cell.
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10. What is isotype switching, and how is antibody
diversity achieved? 11. What signals are needed to activate helper T cells? 12. What signals activate cytotoxic T cells? 13. Discuss the differences between transplant rejections
of nucleated cells and rejection of blood cells. 14. How do superantigens activate T cells? 15. Discuss the differences between the alternative and
classical pathways of complement activation. 16. How does the host prevent membrane attack com-
plexes from being formed in host cells? 17 . Describe the differences between hypersensitivity
types I, II, III, and IV. 18. How does a B cell programmed to make an antibody
against self become activated in the absence of specific T-cell help?
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Key Terms activated (909) adaptive immune response (896) adenylate cyclase (927) allergen (926) allograft (933) allotype (904) anaphylaxis (927) antibody (897) antigen (897) antigenic determinant (897) antigen-presenting cell (APC) (898) atopic (927) autoimmune response (931) B-cell receptor (909) B-cell tolerance (898) capping (909) cell-mediated immunity (897) classical complement pathway (925) clonal (897) clonal selection (909) constant region (902) cytotoxic T cell (TC cell) (915) cytokine (913) decay-accelerating factor (926) desensitization (928) edema (927) epitope (897) equivalence (906)
F(ab)2 region (903) factor H (926) Fc region (903) gene splicing (911) granzyme (921) hapten (901) heavy chain (902) helper T cell (TH cell) (913) humoral immunity (897) idiotype (904) immunogen (897) immunogenicity (898) immunoglobulin (902) IgA (904) IgD (905) IgE (905) IgM (904) immunological specificity (899) immunoprecipitation (906) isotype (903) isotype switching (class switching) (908) late-phase anaphylaxis (928) light chain (902) major histocompatibility complex (MHC) (898) memory B cell (908) MHC restriction (923)
negative selection (918) opsonize (904) perforin (921) plasma cell (908) positive selection (918) primary antibody response (908) radial immunodiffusion (906) recombination signal sequence (RSS) (911) secondary antibody response (908) serum (908) sIgA (904) superantigen (921) switch region (913) threshold dose (898) tumor necrosis factor (927) type I (immediate) hypersensitivity (927) type II hypersensitivity (929) type III hypersensitivity (929) type IV (delayed-type) hypersensitivity (DTH) (930) vaccination (899) variable regions (903) Western blot (906)
Recommended Reading Hornef, Mathias W., Mary Jo Wick, Mikael Rhen, and Staffan Normark. 2002. Bacterial strategies for overcoming host innate and adaptive immune responses. Nature Immunology 3:1033–1040. Kaufmann, Stefan H., and Ulrich E. Schaible. 2005. Antigen presentation and recognition in bacterial infections. Current Opinion in Immunology 17:79–87 Manz, Rudolf A., Anja E. Hauser, Falk Hiepe, and Andreas Radbruch. 2005. Maintenance of serum antibody levels. Annual Review of Immunology 23:367–386 Medzhitov, Ruslan, and Charles A. Janeway, Jr. 2002. Decoding the patterns of self and nonself by the innate immune system. Science 296:298–300.
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Müller-Alouf, Heidi, Christophe Carnoy, Michel Simonet, and Joseph E. Alouf. 2001. Superantigen bacterial toxins: State of the art. Toxicon 39:1691–1701 Nestle, Frank O., Arpad Farkas, and Curdin Conrad. 2005. Dendritic-cell-based therapeutic vaccination against cancer. Current Opinion in Immunology 17:163–169. Orange, Jordan S., Marlys S. Fassett, Louise A. Koopman, Jonathan E. Boyson, and Jack L. Strominger. 2002. Viral evasion of natural killer cells. Nature Immunology 3:1006–1011. Robinson, Harriet L., and Rama Rao Amara. 2005. T-cell vaccines for microbial infections. Nature Medicine 11:s25–s32. Savino, Wilson. 2006. The thymus is a common target organ in infectious diseases. PLoS Pathogens 2:472–483
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Chapter 25
Microbial Pathogenesis 25.1 25.2
25.3 25.4 25.5 25.6 25.7 25.8
Host-Pathogen Interactions Virulence Factors and Pathogenicity Islands: The Tools and Toolkits of Microbial Pathogens Virulence Factors: Microbial Attachment Toxins: A Way to Subvert Host Cell Function Protein Secretion and Pathogenesis Finding Virulence Genes Surviving within the Host Viral Pathogenesis
Mammals have developed elaborate physical, chemical, and immunological defenses that protect against disease-causing microbes, or pathogens. Yet every fortress has its weakness. Pathogenic microbes have identified those weaknesses and exploit them to gain entry. The result is disease. What makes a pathogen different from a commensal organism? The answer varies, depending on the pathogen. Some bacterial pathogens can avoid being phagocytosed by host cells, whereas others actively encourage it. Why do some microbes prefer to live intracellularly? Even more mysterious are those bacterial pathogens that develop a latent stage in the host, where they are undetectable, but still emerge and cause disease. Pathogens also differ widely with respect to their effects on a host. Some pathogens efficiently slay their hosts. Others persist for many years without killing. Which is the more successful pathogen? The fundamental question of microbial pathogenesis is how an organism too small to be seen with the naked eye can kill a human that is 1 million times larger. This chapter will explore the varied methods that different bacterial and viral pathogens use to accomplish this feat.
Pseudocolored transmission electron micrograph (magnification ×4,000) showing a Vero cell infected for 8 days with Coxiella burnetii, an obligate intracellular bacterium and the agent of human Q fever. C. burnetii multiplies within a large vacuole, called a parasitophorous vacuole (green). The organism directs development of this vacuole, a process presumably aided by the potent antiapoptotic properties of the organism, which keeps the host cell from killing itself. Source: Elizabeth Fischer of the Research Technologies Branch, Rocky Mountain Laboratories Microscopy Unit. From Daniel E. Voth, et al. 2007. Infection and Immunity 75:4263.
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A 23-year-old Hispanic mother brought her 3-yearold daughter into the emergency room. The child was lethargic, had a fever of 40°C (104°F), and was having difficulty breathing. The mother explained that the family arrived in the United States from El Salvador the previous week. The attending physician noted an extreme swelling of the child’s cervical lymph nodes, giving the girl a thick, “bull-neck” appearance. She also noticed the beginnings of a membranous growth at the back of the child’s throat that was beginning to obstruct the trachea. It was grayish in color and bled when scraped. When asked, the distraught mother admitted that the child had not received any vaccinations before arriving in New Mexico. Suspecting the nature of the child’s illness, the physician granted immediate admission to the hospital and ordered administration of penicillin and a specific antitoxin. The results of a throat swab sent to the microbiology lab confi rmed the physician’s suspicion. The root of the child’s disease was Corynebacterium diphtheriae, which causes diphtheria. Corynebacterium diphtheriae is a deadly pathogen able to kill humans by attacking the respiratory and cardiac systems. When untreated, death often comes by suffocation when the gray membrane completely covers the trachea. This pathogen is a gram-positive, nonspore-forming rod identical in appearance to many common commensal throat microbes, such as the more docile C. striatum. So, two gram-positive rods, identical in appearance, are both found in the human throat. Why is one a killer and the other not? The child in the case history most likely came in contact with the pathogen before leaving El Salvador, where vaccinations in some areas are difficult to obtain. The result was diphtheria. The question we want to ask in this chapter is not how this could have been prevented, but what genetic distinctions separate closely related disease-causing pathogens from innocuous nonpathogens. In this instance, the difference between friend and foe is a bacteriophage genome embedded in the genome of the organism. This phage carries the gene for diphtheria toxin, whose properties will be described later. Pathogens, such as C. diphtheriae, that kill their hosts or only transiently reside there must also be prepared for life outside the host. These microbes possess an alternate physiology that allows survival in nonhost environments such as a lake or soil. However, some pathogens are not as versatile and die when separated from their host. They must be passed directly from person to person. If the host dies before transmission, the pathogen dies with it. Thus, to kill or not to kill is an important question each pathogen must address. This chapter will discuss the various relationships that occur between pathogens and their hosts and the factors that contribute to pathogenesis, the processes by which microbes cause disease in a host. The degree of harm that is caused depends on the mechanisms the pathogen has at its disposal.
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25.1 Host-Pathogen Interactions Parasites, in the broadest sense, include bacteria, viruses, fungi, and protozoa that colonize and harm their hosts. However, the term pathogen is typically used to refer to bacterial, viral, and fungal agents of disease. Diseasecausing protozoans and worms, on the other hand, are normally called parasites. Pathogens and parasites infect their animal and plant hosts in a variety of ways and enter into different host pathogen relationships, depending on the site of colonization. For example, organisms that live on the surface of a host are called ectoparasites. The fungus Trichophyton rubrum, one cause of athlete’s foot, is an ectoparasite (Fig. 25.1). Wuchereria bancrofti, the worm parasite that causes elephantiasis, is an endoparasite because it lives inside the body (Fig. 25.2). Before examining the mechanisms microbes use to cause disease, it is helpful to know the terminology of pathogenesis. An infection occurs when a pathogen or parasite enters or begins to grow on a host. Be aware that A.
Microconidia © 2007 doctorfungus.org
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An ectoparasite. A. Athlete’s foot is caused by the fungus Tricophyton rubrum. B. Colony morphology and microscopic, branching conidia (blowup) of T. rubrum. Conidia are described in Chapter 20.
Figure 25.1
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W. Peters. A Colour Atlas of Arthropods in Clinical Medicine. 1992; Elsevier.
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An endoparasite. The disease filariasis, commonly known as “elephantiasis” for obvious reasons, is caused by the worm Wuchereria bancrofti (blowup), which enters the lymphatics and blocks lymphatic circulation. Adult worms are threadlike and measure 4–10 cm in length. The young microfilaria (shown) are approximately 0.5 mm in length. Though not a problem in the United States, it is found throughout middle Africa, Asia, and New Zealand.
Figure 25.2
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severe that disease is (virulence). Pathogenicity, overall, is shaped by the genetic makeup of the pathogen. In other words, an organism is more—or less—pathogenic depending upon the tools at its disposal (such as toxins), and their effectiveness. Virulence is a measure of the degree or severity of disease. For instance, Ebola virus and the closely related Marbury virus have case fatality rates near 70%. This means that they are highly virulent (Fig. 25.4). On the other hand, rhinovirus, the cause of the common cold, is very effective at causing disease but never kills its victims. So it is highly infective but has a low virulence. Both organisms are pathogenic, but with one you live and the other you die. One way to measure virulence is to determine how many bacteria or virions are required to kill 50% of an experimental group of animal hosts. This is called the LD50 (lethal dose 50%). An organism with a low LD50, in which very few organisms are required to kill 50% of the hosts, is more virulent than one with a high LD50 (Fig. 25.5). For organisms that colonize but do not kill the host, the infectious dose needed to colonize 50% of the experimental hosts (ID50 ) can be measured. Infectious dose is measured by determining how many microbes are required to cause disease symptoms in half of an experimental group of hosts. Although one might be able to measure the infectious dose, rather than lethal dose, for a lethal pathogen, it is not typically done. Because it gives a clear end point, LD50 is much easier to use when trying to determine the effectiveness of a given treatment (an antibiotic, for example) or quantitate the role of a given gene in pathogenesis.
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CDC/Lois Norman
the term infection does not necessarily imply overt disease. Any potential pathogen growing in or on a host is said to cause an infection, but that infection may be only transient because immune defenses kill the pathogen before noticeable disease results. Indeed, most infections go unnoticed. For example, every time you have your teeth cleaned by a dentist your gums bleed and your oral flora transiently enter the bloodstream, but you rarely suffer any consequences. Primary pathogens, also called frank pathogens, are disease-causing microbes possessing the means to breach the defenses of a healthy host. For example, Shigella flexneri, the cause of bacillary dysentery, is a frank pathogen. When ingested, it can survive the natural barrier of an acidic (pH 2) stomach, enter the intestine, and THOUGHT QUESTION 25.1 Is a microbe with an begin to replicate. Opportunistic pathogens, on the LD50 of 5 × 104 more or less virulent than a microbe other hand, only cause disease in a compromised host. with an LD50 of 5 × 107? Pneumocystis jiroveci (previously P. carinii) is an opportunistic pathogen that causes life-threatening infections in AIDS patients, whose A. B. immune systems have been eroded (Fig. 25.3A). Some microbes even enter into a latent state during infection, where the organism cannot be found by culture. Herpes virus, for instance, can enter the peripheral nerves and remain dormant for years, then suddenly emerge to cause cold sores (Fig. 25.3B). The bacterium Rickettsia prowazekii causes epidemic typhus, but it can also enter a latent phase and months or years later cause a disease relapse called recrudescent typhus. The term pathogenicity refers to an Figure 25.3 Opportunistic and latent infections. A. Pneumocystis jiroveci cysts organism’s ability to cause disease. It is (5 to 10 µm diameter) in broncheoalveolar material. Notice how the fungi look like defined in terms of how easily an organ- crushed Ping-Pong balls. B. Cold sore produced by a reactivated herpes virus hiding ism causes disease (infectivity) and how latent in nerve cells.
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ing yellow fever from infected to uninfected individuals (Fig. 25.7) in what is called horizontal transmission. The mosquito can also bequeath this particular virus to its offspring via infected eggs in a form of vertical transmission called transovarial transmission. Although yellow fever is not a problem in the United States today, West Nile virus, a flavivirus closely related to yellow fever, currently claims several victims each year in this country. Because insects are instrumental in transmitting pathoFigure 25.4 Highly virulent Ebola virus. A. Ebola virus (approx. 1 µm long, TEM). gens, killing insect vectors is an B. The body of a victim of Marburg virus is placed in a coffin for safe burial in Angola. important way to halt the spread Marburg and Ebola cause hemorrhagic infections in which patients bleed from the mouth, nose, eyes, and other orifices. They have a 70–80% mortality rate. of disease. Interventions include spraying insecticide in a community during egg-hatching season or using other microbes as assassins “trained” to kill the 100 vector. For example, Bacillus thuringiensis will kill many Agent 1 types of insects that carry infectious agents. More recently, 75 an insect virus called baculovirus has been developed that Agent 2 LD50 = 400 kills the Culex mosquito vectors carrying the West Nile virus. The advantage of these vector-targeting microbes is 50 that they do not kill other insects or animals, as do many chemical insecticides. LD50 = 600 25 Another critical factor in an infection cycle is the reservoir of infection. A reservoir is an animal, bird, or insect that normally harbors the pathogen. In the case of yellow fever, the mosquito is not only the vector, but the 0 100 200 300 400 500 600 700 800 900 1,000 reservoir as well, because the insect can pass the virus to Dose (organisms administered per animal) future generations of mosquitoes through vertical transmission. The virus causing eastern equine encephalitis Figure 25.5 Measurement of virulence. Each LD50 measurement requires infecting small groups of animals with (EEE), however, uses birds as a reservoir. The microbe is increasing numbers of infectious agent and viewing how many normally a bird pathogen and is transmitted from bird to animals die. The number of microbes that kill half the animals is bird via a mosquito vector. The virus does not persist in called the LD50 dose. In this example, agent 1 is more virulent the insect, but transmission by the insect vector keeps the than agent 2. virus alive by passing it to new avian hosts. Humans or horses entering geographic areas harboring the disease (called endemic areas) can also be bitten by the mosInfection Cycles Can Be Direct or Indirect quito. When this happens, they become accidental hosts Somehow, pathogens must pass from one person or animal and contract disease. The virus does not replicate to high to another if a disease is to spread. The route an organtiters in mammals, which means that horses and humans ism takes to accomplish this is called the infection cycle. are poor reservoirs for the virus. However, the virus does A cycle of infection can be simple or complex (Fig. 25.6). replicate to high numbers in the avian host. Reservoirs Organisms that spread directly from person to person, such are critically important for the survival of a pathogen and as the rhinovirus or Shigella, have simple infection cycles. as a source of infection. If the eastern equine encephalitis Inanimate objects through which pathogens can be relayed virus had to rely on humans to survive, the virus would to hosts are called fomites. More complex cycles often cease to exist because of limited replication potential involve vectors, usually insects, as intermediaries. Vectors and limited access to mosquitoes. It is important to note serve to carry infectious agents from one animal to another. that the reservoir of a given pathogen might not exhibit A mosquito vector, for example, transfers the virus causdisease. Percent mortality
CDC
B.
F. A. Murphy, University of Texas Medical Branch, Galveston
A.
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Microbes can be transmitted indirectly from one person to another by inanimate objects, collectively called fomites, or by an insect vector.
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Infection cycles. Infectious agents can be transmitted horizontally from one member of a species to another by a variety of means: fomites, aerosolization, direct contact, or insect vector. Vertical transmission is passage from parent to offspring during birth, while accidental transmission happens when a host that is not part of the normal infectious cycle unintentionally encounters that cycle.
Figure 25.6 Fomites Microbes can be transmitted by direct contact or by aerosolization (e.g., sneeze). Direct transmission
Some microbes are ordinarily transmitted from animal to animal by insect vectors. Humans can accidentally be infected by the insect.
Accidental Insect Transmission of an infectious agent from an insect to its offspring is called vertical transmission.
Vertical transmission Insect
Insect offspring
Reservoir Courtesy of Historical Collections & Services, Claude Moore Health Sciences Library, U. of Virginia
A.
Insect vector and yellow fever. A. Walter Reed, a member of the University of Virginia Medical School class of 1869, proved in 1901 that the mosquito Aedes aegypti was the vector of transmission for yellow fever, a disease named for the jaundice produced by liver damage. B. Aedes aegypti. Yellow fever is caused by a flavivirus (inset, TEM) carried by this mosquito. The virus varies in size from 50 to 90 nm. Yellow fever remains endemic in the northern part of South America and in central Africa.
CDC/Science Source/Photo Researchers, Inc.
Figure 25.7
CDC
B.
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Even a simple infectious cycle can become more complex. For example, rhinovirus can be spread person-toperson by a sneeze (airborne) or through the sharing of inanimate objects (fomites) such as contaminated utensils (fork, pen), towels, cloth handkerchiefs, and doorknobs. Handshaking is also an efficient means of transferring some pathogens. Imagine that one person in a city of 100,000 people has a cold and sneezes on her hands. Then, without washing her hands, she goes through the day shaking hands with 10 people, and each of those people shake hands with another 10 people per day, and so on. If there were no repeat handshakes, and if none of the contacts washed their hands, it would take only four days to spread the virus throughout the population. In this example, eventually the entire populace of the city would come in contact with the virus, but not everyone would actually contract disease. Additional factors influence whether the virus successfully replicates in a given individual.
Portals of Entry How do infectious agents gain access to the body? Each organism is adapted to enter the body in different ways. Food-borne pathogens (for example, Salmonella, E. coli, Shigella, and rotavirus) are ingested by mouth and ultimately colonize the intestine. They have an oral portal of entry. Airborne organisms, in contrast, infect through the respiratory tract. Some microbes enter through the conjunctiva of the eye, others through the mucosal surfaces of the genital and urinary tracts. Agents that are only transmitted by mosquitoes or other insects enter their human hosts via the parenteral route, meaning injection into the bloodstream. Wounds and needle punctures can also serve as portals of entry for many microbes. For instance, shared needle use between drug addicts has been an important factor in the spread of HIV. TO SU M MAR I Z E: ■
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Infection with a microbe does not always lead to disease. Primary pathogens have mechanisms that help the organism circumvent host defenses. Opportunistic pathogens cause disease only in a compromised host. Pathogenicity refers to the mechanisms a pathogen uses to produce disease and how efficient the organism is at causing disease, whereas virulence is a measure of disease severity. Diseases can be spread by direct or indirect contact between infected and uninfected persons/animals or by insect vectors. Pathogens use portals of entry best suited to their mechanisms of pathogenesis.
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25.2 Virulence Factors and Pathogenicity Islands: The Tools and Toolkits of Microbial Pathogens Pathogens can be distinguished from their avirulent counterparts by the presence of virulence factors that help establish the organism in the host and that alter host functions to cause disease. Virulence factors, which are encoded by virulence genes, include toxins, attachment proteins, capsules, and other devices used to avoid host innate and adaptive immune systems. All of these factors enhance the disease-producing capability, or pathogenicity, of the pathogen. Extensive sequencing efforts have allowed us to compare genomes of many pathogens and expose some “footprints” of their evolution. For example, in bacterial pathogens, most chromosomes are dotted with clusters of pathogenicity genes that encode virulence functions. These gene clusters, called pathogenicity islands, can be considered the toolboxes of pathogens (originally discussed in Section 9.7). Many virulence genes reside in pathogenicity islands, although many others do not. Some virulence genes reside on plasmids (for example, the genes for the diarrhea-producing labile toxin of certain E. coli strains) or in phage genomes (such as the genes encoding the diphtheria toxin of Corynebacterium diphtheriae). Many of the genes in pathogenicity islands were originally inherited through horizontal transmission from other organisms by, for example, conjugation or transduction (discussed in Section 9.2). But what do the pathogenicity genes do? Some genes encode molecular “grappling hooks,” such as pili that attach to host cells. Once attached, microbes can secrete toxins that injure the host cell. Other bacteria wall themselves off to prevent damage by host inflammatory responses. Some bacterial pathogens are even capable of what could be called “host cell reprogramming.” These organisms inject proteins directly into the host cell to disrupt normal signaling pathways. This reprogramming causes the target cell to either engulf the bacterium, commit suicide (undergo apoptosis), or provide an even more intimate attachment platform at the cell surface.
Pathogenicity Islands Are Different from the Rest of the Genome How do new pathogens evolve? DNA sequencing efforts suggest that gene swapping followed by divergent evolution is a major force in the development of emerging pathogens. Horizontal gene transfers move whole blocks of DNA (more than 10 kbp) from one organism to another, placing the blocks directly in the chromosome in what is called a genomic island. If the island increases
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the “fitness” of a microorganism (pathogen) that interacts with a host, it is called a pathogenicity island. Genomic islands generally reveal themselves by several anomalies that they possess with respect to the rest of the host genome: ■
They are often linked to a tRNA gene and generally have a GC/AT ratio very different from the rest of the chromosome (Fig. 25.8). For example, a plot of GC content along the length of a chromosome may reveal that most of the genome has a 50% GC content. But somewhere in the middle, a 50-kbp region sticks out on the graph, showing a content of 40%. This probably reflects the GC content of the microbe that donated the island. The reason tRNA genes are often
A.
Genomic pathogenicity island tRNA
int
abc def ghi
IS IS
DR
Core
DR
B. 60 C+G %
■
Pathogenicity island
50 40 10
20
50
60 kbp
Model pathogenicity island. A. Schematic model of a pathogenicity island. The DNA block is linked to a tRNA gene and flanked by direct repeats (DR) that may be “footprints” of a transposon or viral-mediated transfer. The integrase gene (int) and insertion sequences (IS) may also be remnants of transposition. B. The guanine + cytosine (GC) content of the island is different from that of the core genome.
Figure 25.8
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the targets for insertion of pathogenicity islands is not known. One hypothesis is that the conserved secondary structure of tRNA genes provides a structural motif that facilitates integration by an integrase. They are typically flanked by genes with homology to phage or plasmid genes. This is thought to reflect the transfer vector used to move the island from one organism to another. They carry gene clusters with specific functions, such as protein export systems that secrete toxins (for example, type III secretion systems that inject toxic proteins directly into target host cells; see Section 25.5). These are the operons that contribute to the fitness of the organism in pathogenic circumstances.
Table 25.1 lists several pathogenicity islands and their functions. It provides examples of pathogenicity islands present in different bacteria, names a key function of the products of the island, and gives the disease caused. Various pathogenicity island functions will be described as the chapter proceeds.
Shigella and Escherichia Are Examples of Evolution through Horizontal Transmission Figure 25.9 shows a schematic comparison of the circular Shigella flexneri genome with those of E. coli K-12 (a commonly used avirulent lab strain) and E. coli O157:H7 (a virulent strain of enterohemorrhagic E. coli). All three gram-negative rods are closely related but differ greatly in terms of pathogenic potential and mechanisms. S. flexneri and E. coli O157:H7 cause bloody diarrhea, while E. coli K-12 has a commensal origin. DNA sequence analysis has revealed chromosome regions that are common among these organisms, as well as genomic islands specific to individual species and strains (Fig. 25.9). The core genes needed for sustaining growth (that is, for transcription, translation, replication, and so on) are common to all
Table 25.1 Examples of pathogenicity islands. Pathogenicity island
Function
Organism
Disease
HPI (high pathogenicity island) VPI (Vibrio pathogenicity island) PAI III (pathogenicity island III) SPI-1 and SPI-2 (Salmonella pathogenicity islands) SHI-1 and SHI-2 (Shigella islands) YSA (Yersinia secretion apparatus) Cag PI (cytotoxin-associated gene) icm/dot (intracellular multiplication)
Iron uptake Toxin production Encodes adhesins
Yersinia spp. Vibrio cholerae Uropathogenic E. coli
Plague, enterocolitis Cholera Urinary tract infection
Type III secretion Type III secretion Type III secretion Type IV secretion Type IV secretion
Salmonella enterica Shigella flexneri Yersinia spp. Helicobacter pylori Legionella pneumophila
Gastroenteritis Bloody diarrhea Plague, enterocolitis Gastric ulcers, gastric cancer Legionnaires’ disease
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2
1
K-12 Shigella
Terminus Shigella flexneri 2a 4,607,203 bp
Comparison of the Shigella flexneri 2a chromosome with those of E. coli K-12 and O157:H7 (EDL933). Segments of the three genomes between 1 MB and 2 MB are shown. Gray line indicates DNA sequences shared among the organisms (O157, top arc; K-12, second arc; Shigella, third arc). Colored boxes depict genomic islands (including pathogenicity islands) present in each organism. The bottom arc illustrates the GC content of the Shigella genome. Each data point along the graph indicates GC content relative to AT content as averaged over a sliding 10 kbp window. Note how major differences in Shigella GC content shown above or below the center line (indicating 50% GC content) often correlate with the genomic islands depicted in the third arc. Arrows indicate some islands with obvious correlation to GC content.
Figure 25.9
three organisms, whereas other genes may be present in only one. These unique genes and islands are thought to be the result of horizontal gene transfers originating from widely different genera. Within the unique DNA segments of S. flexneri and E. coli O157:H7 are genes encoding host cell attachment, toxin secretion, and the toxins themselves—all of which are absent from K-12. The degree of gene shuffling that was required to separate these otherwise similar bacteria is remarkable. The following sections describe some of the specific tools pathogens use to undermine the integrity of the body. From attachment, to toxins, to intracellular invasion, the infection process is like a chess match, with each side, human and microbe, trying to outmaneuver the other. TO SU M MAR I Z E: ■
■
■
Pathogenicity islands are DNA sequences within a species that are acquired by horizontal gene transfer from a different species. Virulence genes encode genes whose products enhance the disease-causing ability of the organism. Many virulence genes can be found within pathogenicity islands, but some are located outside of an obvious genomic island or reside in plasmids. Pathogenicity islands contain distinct features, such as GC content and the remnants of phage or plasmids, that mark them as being different from the rest of the genome.
hitchhiking with a vector. Once at the site, attachment mechanisms are needed to stay there. The human body has many ways to exclude pathogens. The lungs use a mucociliary elevator (see Fig. 23.5) to rid themselves of foreign bodies, the intestine uses peristaltic action to ensure that its contents are constantly flowing, and the bladder uses contraction to propel urine through the urethra with tremendous force. How do bacteria ever manage to stay around long enough to cause problems? Like a person grasping a telephone pole during a hurricane, successful pathogens moving through the body manage to grab onto host cells and tenaciously hold on. Thus, the fi rst step toward infection is attachment, also called adhesion. An adhesin is the general term for any microbial factor that promotes attachment. Viruses attach to the host through their capsid or envelope proteins, which bind to specific host cell receptors discussed in Chapters 6 and 11. Bacteria have a variety of similar strategies. They can use hairlike appendages called pili (also called fi mbriae), whose tips contain receptors for mammalian cell surface structures. Or they can use a variety of adherence proteins or other molecules (adhesins) that are not part of a pilus. Sometimes they use both. Table 25.2 summarizes bacterial attachment strategies.
Types of Pili
25.3 Virulence Factors: Microbial Attachment Regardless of the disease, pathogens must reach a colonization site either through their own motility or by
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Different pili from different bacterial species have been classified historically based on phenotype. In some cases the phenotype classes have turned out to be inconsistent with sequence-based homologies. We will consider three groups in this chapter.
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Table 25.2 Specific attachments of bacteria to cell or tissue surfaces. Bacterium
Adhesin
Host receptor
Attachment site
Disease
Streptococcus pyogenes Streptococcus mutans Streptococcus salivarius Streptococcus pneumoniae Staphylococcus aureus Neisseria gonorrhoeae Enterotoxigenic E. coli Uropathogenic E. coli Uropathogenic E. coli Bordetella pertussis Vibrio cholerae
Protein F
Amino terminus of fibronectin
Pharyngeal epithelium
Sore throat
Glucan
Salivary glycoprotein
Pellicle of tooth
Dental caries
Lipoteichoic acid
Unknown
Buccal epithelium of tongue
None
Cell-bound protein
N-acetylhexosamine galactose disaccharide Amino terminus of fibronectin
Mucosal epithelium
Pneumonia
Mucosal epithelium
Various
Urethral/cervical epithelium
Gonorrhea
Treponema pallidum Mycoplasma Chlamydia
Cell-bound protein N-methylphenylalanine pili Type I fimbriae (pili) Type I fimbriae (pili) P pili (pyelonephritisassociated pili) Pili (“filamentous hemagglutinin”) N-methylphenylalanine pili Peptide in outer membrane Membrane protein Unknown
Glucosamine galactose carbohydrate Species-specific carbohydrate(s) Complex carbohydrate
Intestinal epithelium
Diarrhea
Urethral epithelium
Urethritis
P-blood group
Upper urinary tract
Pyelonephritis
Galactose on sulfated glycolipids Fucose and mannose carbohydrate Surface protein (fibronectin)
Respiratory epithelium Intestinal epithelium
Whooping cough Cholera
Mucosal epithelium
Syphilis
Sialic acid Sialic acid
Respiratory epithelium Conjunctival or urethral epithelium
Pneumonia Conjunctivitis or urethritis
Type I pili are a group that, in general, adhere to mannose residues on host cell surfaces. Because adding free mannose will inhibit attachment of most type I pili, this binding is called mannose sensitive. Another group of pili is called mannose resistant because the pili do not bind to mannose residues. There are at least two types of mannose-resistant pili. Members of one type, sometimes called type III pili, bind to red blood cells treated with tannic acid. The other more commonly studied type is called type IV pili. Unlike type I pili, which simply stick out from the cell surface, type IV pili are more dynamic and are assembled on the cell surface through a very different pathway. There are other types of attachment pili that do not fall neatly into one of these three groups. For clarity, it is important to note that the primary classification of pili is now based largely on protein sequence information (deduced from DNA sequence) and may contradict earlier phenotype-based schemes. For instance, the pyelonephritis adhesion pili (Pap) of the uropathogenic E. coli are mannose resistant, since they bind to a digalactoside on
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host surfaces (called the P blood group antigen). However, Pap is very similar to type I pili based on amino acid sequence homology.
Pilus Assembly How bacteria assemble pili on their cell surfaces is an engineering marvel. The shafts of pili are cylindrical structures composed of identical pilin protein subunits. Several different proteins adorn the tip, including one at the very apex that binds to host receptors (Fig. 25.10A). In addition to these structural components, numerous other proteins work together as a machine to assemble the structure. Genes encoding a given pilin protein and the associated assembly apparatus are typically arranged on the chromosome as an operon (Fig. 25.10B). In Figure 25.11, the assembly of a type I pilus is shown, using uropathogenic E. coli Pap pili as a model. The mechanism is representative of other type I pili; only the names of the proteins will differ for each system. Protein components are secreted into the periplasm by the SecA-
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C. Hal Jones, et al. 1995. PNAS USA 92:2081
A. FimH adhesion tip (TEM)
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Inner membrane
Figure 25.10 Attachment pili and encoding operon. A. Highresolution micrograph showing a type I pilus (TEM). The FimH adhesin at the tip is the protein that binds to the cell receptor. B. Genetic organization of the type I gene cluster, which includes genes involved in pilus assembly. The genes are designated fim A-H.
Outer membrane
Subunits synthesized PapD General secretion, SecAY, SecB
PapC G
FG
E F
B. Type I pilus gene cluster
G
Tip pilus components B E
A
Major pilus subunit Regulation
I
C
D
Periplasmic Outer chaperone membrane usher
F
G
E
H
Adapters/ Mannoseinitiators/ binding terminators adhesin
A
H
dependent general secretory system (discussed in Section 8.4). Once in the periplasm, the subunits are chaperoned by PapD to the membrane site of assembly, which is marked by the presence of the usher protein PapC. PapC proteins form channels in the outer membrane large enough to accommodate individual pilus subunits and, like an usher in a theater, direct the subunits to their proper places. Chaperoning of the pilin building blocks by PapD is necessary to prevent pilin subunits from inadvertently assembling in the periplasm. As illustrated in Figure 25.11, appropriate assembly of pili at the usher site starts with the tip protein, PapG, which will ultimately bind to carbohydrates on host membranes after the pilus is complete. After PapG, the ushers add PapF and PapE, forcing PapG farther away from the surface. Then a series of identical PapA pilin subunits are strung together to form the shaft. The PapA subunits assemble by swapping domains, essentially linking themselves together like pieces of a jigsaw puzzle. Interestingly, shear forces generated by fast-moving urine in the urethra may actually tighten bacterial adhesion to target cells. The tip protein, FimH, which is analogous to PapG but for a different type I pilus, contains two domains, the pilin domain (pale yellow) and the lectin domain (blue), as shown in Figure 25.12. The pilin domain integrates FimH into the tip of the pilus. The lectin domain binds the host receptor. Scientists have described a mechanism whereby a shear force, such as might be experienced by uropathogenic E. coli bound to bladder cells during urination, extends the interdomain
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A
A
PapH = cap
A
AE
AE
E
E
E
F G
F G
F G
Assembly of type I pili. The figure illustrates pyelonephritis-associated pilus (Pap) assembly but is representative of other type I pili. Only the names of the proteins will differ. Proteins are secreted by the Sec system to the periplasm, where they are chaperoned by PapD to the site of assembly. PapC, also called the usher, assembles the individual proteins in the proper order. Assembly starts with the tip protein, PapG, marked at the far right, which ultimately binds to carbohydrates on host membranes. The subunits fit together like pieces of a jigsaw puzzle. The arrow at the head of the elongating pilus indicates direction of pilus growth.
Figure 25.11
linker between the FimH lectin and pilin domains and strengthens the hold of the lectin domain on the mannose receptor. It is not clear whether shear ends up changing the existing site from a low-affi nity to a high-affi nity binding site or if the conformational shift exposes a second, hidden, mannose-binding site. Regardless, it becomes harder to pry E. coli from its target host cell. Thus, fluid forces in the human body, as occur with salivating, swallowing, sneezing, urinating, and weeping, may in some cases strengthen bacterial adhesion instead of detaching and flushing away the infectious agents.
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Figure 25.12 Structure of type I pili tip protein. FimH contains two domains, the pilin domain (yellow) and the lectin domain (blue). The pilin domain integrates FimH into the tip of the pilus. The lectin domain binds the host receptor. When shear is created, the receptor-binding site on the lectin domain is pulled in one direction (yellow arrows), and residue T158 (orange arrow) is pulled in the opposite direction. (PDB codes: 2co4, 1tr7)
Pilin domain
Interchain linker Lectin domain
C To pilin domain, fimbria, and rest of bacteria
N To receptor and host cell
1. Pilin PilA is made as a preprotein and inserted into inner membrane.
A.
Pre-PilA
2. PilD is a peptidase that removes a leader sequence from PilA preproteins prior to pilus assembly. NTP
3. PilT and PilF are NTP-binding proteins that provide energy for retraction and assembly.
PilD
PilF
Fiber formation PilT
Surface localization PilQ
NTP PilA Disassembly/ degradation 4. The secretin PilQ is required for the type IV pilus to cross the outer membrane.
Inner membrane
PilC1 and Y1
Other pili with an important role in pathogenesis are the type IV pili. Type IV pili are found in a broad spectrum of gram-negative bacteria and share amino acid homology in their major pilin structure (Fig. 25.13). Species with type IV pili include V. cholerae, Pseudomonas aeruginosa (Fig. 25.13B), certain pathogenic strains of E. coli, Neisseria meningitidis, and N. gonorrhoeae. All type IV pili use similar secretion and assembly machinery involving at least a dozen proteins. One major difference between the assembly of type IV and type I pili is that type IV pilus proteins are never free in the periplasm; they are transported directly from the cytoplasm through a channel in the outer membrane (Fig. 25.13A). Thus, type IV pilus assembly is SecA independent. As described here, the blueprints for this mechanism were adapted and modified through evolution to secrete other proteins via what is generally called type II protein secretion (discussed in Section 25.5).
Figure 25.13 Type IV pili. A. Model of pilus assembly and disassembly. In this example, PilA is the pilin protein, and PilC1 and Y1 form the attachment tip. Diameter of the filament is approximately 6 nm. Note that assembly/disassembly requires hydrolysis of nucleoside triphosphate and takes place at the inner membrane, not in the periplasm. B. Photographic evidence of type IV pilus extension and retraction in cells of P. aeruginosa. Filament b retracts, then filament d extends at 6 seconds and retracts. Filament c attaches briefly at its distal tip (note straightening at 24 seconds), then begins to retract. Time in seconds. Fluorescent microscopy. C. Type IV pili are essential for the interaction of Neisseria meningitidis with brain endothelial cells. Type IV pili are green in this SEM. Diplococcal cells are approx. 1.6 µm in diameter. Sources: A. Bardy, Ng, and Jarrel. 2003 Microbiol. 149:295–304. B. J. M. Skerker and H. C. Berg. 2001. PNAS 98:6901–6904.
Outer membrane B. Extension and contraction of Type IV pili
C. Type IV pili of Neisseria meningitidis
t=0
8 μm
d 6
12
18
24
36
b a 104
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116
122
128
134
c
Courtesy of Xavier Nassif
b a
Jeffrey M. Skerker and Howard C. Berg. 2001. PNAS 98: 6901
c
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Type IV pili can actually make cells move because the assembly process involves the reiterative elongation and retraction of the pili. The process, called “twitching motility,” occurs when the pilus elongates, attaches to a surface, then depolymerizes from the base, which shortens the pilus and pulls the cell forward. This mechanism is akin to using a grappling hook to scale a building. The gliding motility of the slime mold Myxococcus xanthas is also due to type IV pili. The type IV pili of Neisseria meningitidis, shown in Figure 25.13C, are essential for crossing the blood-brain barrier and causing bacterial meningitis. Bacteria also carry afi mbriate adhesins (proteins that aid in attachment but do not form pili) that mediate binding to host tissues (Fig. 25.14). Some examples include Bordetella pertactin (which binds to integrin), Streptococcus protein F (binds to fibronectin), Streptococcus M protein (binds to fibronectin and complement regulatory factor H), and intimin of enteropathogenic E. coli (binds to Tir; discussed in Section 25.5). Fimbriae (pili) often mediate the initial binding between bacterium and host, after which a more intimate attachment is created by an afi mbriate attachment protein. In the case of Neisseria gonorrhoeae, once the type IV pilus has attached to the surface of the mucosal epithelial cell, the fi lamentous pilus contracts, pulling the bacterium down onto the host cell membrane. Tight secondary interactions are then mediated by the neisserial Opa membrane proteins, another example of an afimbriate adhesin (Opa is named because of the opacity it adds to colony appearance).
TO SU M MAR I Z E: ■
■
■
Bacteria use pili and nonpilus adhesins to attach to host cells. Type I pili produce a static attachment to the host cell, whereas type IV pili continually assemble and disassemble. Nonpilus adhesins are bacterial surface proteins, or other molecules, that can tighten interactions between bacteria and target cells.
25.4 Toxins: A Way to Subvert Host Cell Function Following attachment, many microbes secrete protein toxins (called exotoxins) that kill host cells and unlock their nutrients (because dead host cells ultimately lyse). Bacterial pathogens have developed an impressive array of toxins that take advantage of different key host proteins, or structures. All gram-negative bacteria also possess a nonprotein yet toxic compound called endotoxin that can hyper-activate host immune systems to harmful levels.
Microbial Exotoxins Have Many Modes of Action Microbial exotoxins fall into five broad categories based on their mechanisms of action (Table 25.3). Three of these classes are illustrated in Figure 25.15.
B. B. pertussis colonizing the trachea
A. M protein Maria Fazio and Vincent A. Fischetti, Rockefeller University
Cilia
M protein fibrils
Nonpilus adhesins. A. M protein surface fibrils on Streptococcus pyogenes (TEM). Cell size 0.5 to 1 µm diameter. B. Colonization of tracheal epithelial cells by Bordetella pertussis (SEM). This organism uses a surface protein called pertactin as well as a pilus called filamentous hemagglutinin (FHA) to bind bronchial cells. Bacteria Nonciliated cells 2 μm
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Figure 25.14
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■
■
■
Cell membrane disruption. Members of the fi rst class, exemplified by the alpha (α) toxin of Staphylococcus aureus, disrupt the cell membrane and cause leakage of cell constituents (Fig. 25.15A). Protein synthesis disruption. A second class, exemplified by diphtheria and Shiga toxins, targets eukaryotic ribosomes and destroys protein synthesis (Fig. 25.15B). Second messenger pathway disruption. The third broad mechanism of action involves the toxin subverting host cell secondary messenger pathways. Cholera toxin and E. coli ST (stable toxin), for instance, cause runaway synthesis of cAMP (described later) and cGMP (Fig. 25.15C), respectively, in target cells. Elevated cAMP or cGMP levels, in turn, trigger critical changes in ion transport and fluid movement. Superantigens. The fourth class includes toxins that act as superantigens and activate the immune system without being processed by antigen-presenting cells (discussed in Section 24.6). The pyrogenic toxins of Staphylococcus aureus (toxic shock syndrome toxin) and Streptococcus pyogenes are examples of superantigenic toxins. Proteases. The fi fth class of toxins consists of proteases. One example is tetanus toxin, a protease that cleaves host components involved in nerve signal transmission.
This section focuses on the key concepts of microbial toxin activity. Section 25.5 discusses how bacteria secrete these toxins. A. Damage cellular membranes/matrices α-Toxin
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A common structural theme among many, but not all, bacterial toxins is that they have two subunits, usually called A and B. These two-subunit complexes are termed AB toxins. The actual toxic activity in AB toxins resides within the A subunit. The role of the B subunit is limited to binding host cell receptors. Thus, the B subunit for each toxin delivers the A subunit to the host cell. Many AB toxins have five B subunits arranged as a ring, in the center of which is nestled a single A subunit (Fig. 25.16A). A major AB toxin subclass comprises toxins that have an ADP-ribosyltransferase enzymatic activity. These enzyme toxins transfer the ADP-ribose group from an NAD molecule to a target protein (Fig. 25.16B). The ADPribosylated protein has an altered function. Sometimes the function is destroyed (for example, protein synthesis is destroyed by diphtheria toxin); other times, the protein is locked into an active form insensitive to regulatory feedback control (for example, cAMP synthesis continues unchecked in the presence of cholera toxin). Mechanisms of selected toxins representing the various classes are described in this section. Alpha toxin. The hemolytic alpha toxin is produced by Staphylococcus aureus, an organism that causes boils and blood infections. Alpha toxin forms a transmembrane, oligomeric (seven member) beta barrel pore in target cell membranes. It is easy to see how the resulting leakage of cell constituents and influx of fluid cause the target cell to burst. To form the pore, hydrophobic areas of each monomer face the lipids of the membrane and hydrophilic residues face the channel interior.
B. Inhibit protein synthesis
C. Activate secondary messenger pathways
Shiga toxin
A1
949
Stable toxin
Na+
B A2 Cap and rim
Cl–
Receptor-mediated endocytosis via Gb3
H2O
Gb3
Stem A1 GTP
cGMP
mRNA Ribosome
Amino terminal of nascent peptide
+ NH3
Three classes of microbial toxins. These classes are defined by mode of action. A. Pore-forming toxins assemble in target membranes and cause leakage of compounds into and out of cells. B. Shiga toxin attaches to ganglioside Gb3, enters the cell, and cleaves 28S rRNA in eukaryotic ribosomes to stop translation. C. Enterotoxigenic E. coli heat-stable toxin affects cGMP production. The result is altered electrolyte transport: inhibition of Na+ uptake and stimulation of Cl– transport. Water follows the resulting electrolyte imbalance and leaves the cell. Figure 25.15
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Table 25.3 Characteristics of bacterial toxins.a Toxin
Organism
Mode of action
Host target
Disease
Toxin implicated in diseaseb
Pore former
Glycophorin
Diarrhea
(Yes)
Pore former
Cholesterol
Gas gangrenec
Unknown
Pore former Pore former
Plasma membrane Cholesterol
(Yes) Yes
Pore former
Plasma membrane
UTIs Food-borne systemic illness, meningitis Abcessesc
Pore former
Cholesterol
Pneumoniac
(Yes)
Pore former
Cholesterol
Strep throat Scarlet fever
Unknown
ADP-ribosyltransferase
Elongation factor 2
Diphtheria
Yes
N-glycosidase
28S rRNA
HC and HUS
Yes
ADP-ribosyltransferase
Elongation factor 2
Pneumoniac
(Yes)
Deamidase ADP-ribosyltransferase Stimulates guanylate cyclase ST-like? Adenylate cyclase
Rho G proteins G proteins Guanylate cyclase receptor Unknown ATP
UTIs Diarrhea Diarrhea
Unknown Yes Yes
Diarrhea Anthrax
Unknown Yes
Deamidase
Rho G proteins
Rhinitis
(Yes)
ADP-ribosyltransferase
G protein(s)
Yes
ADP-ribosyltransferase
Monomeric G-actin
Pertussis (whooping cough) Botulism
Unknown
ADP-ribosyltransferase Glucosyltransferase Glucosyltransferase ADP-ribosyltransferase
Rho G protein Rho G protein(s) Rho G protein(s) G protein(s)
Botulism Diarrhea/PC Diarrhea/PC Cholera
Unknown (Yes) Unknown Yes
Superantigen Superantigen (and serine protease?) Superantigen
TCR and MHC II TCR and MHC II
Yes Yes
Superantigens
TCR and MHC II
Food poisoningc Scalded skin syndromec Toxic shock syndromec Toxic shock syndrome Scarlet fever
Damage membranes Aerolysin Perfringolysin O Hemolysind Listeriolysin O Alpha toxin Pneumolysin Streptolysin O
Aeromonas hydrophila Clostridium perfringens Escherichia coli Listeria monocytogenes Staphyloccocus aureus Streptococcus pneumoniae Streptococcus pyogenes
(Yes)
Inhibit protein synthesis Diphtheria toxin Shiga toxins Exotoxin A
Corynebacterium diphtheriae E. coli/Shigella dysenteriae Pseudomonas aeruginosa
Activate second messenger pathways CNF LT ST d
E. coli E. coli E. coli
EAST Edema factor
E. coli Bacillus anthracis Bordetella pertussis B. pertussis
Dermonecrotic toxin Pertussis toxin C2 toxin C3 toxin Toxin A Toxin B Cholera toxin
Clostridium botulinum C. botulinum Clostridium difficile C. difficile Vibrio cholerae
Activate immune response Enterotoxins Exfoliative toxins
S. aureus S. aureus
Toxic shock toxin S. aureus Pyrogenic exotoxins
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S. pyogenes
TCR and MHC II
Yes Yes Yes
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Table 25.3 Characteristics of bacterial toxinsa (continued) Toxin
Organism
Mode of action
Host target
Disease
Toxin implicated in diseaseb
B. anthracis C. botulinum
Metalloprotease Zinc metalloprotease
Anthrax Botulism
Yes Yes
Clostridium tetani
Zinc metalloprotease
MAPKK1/MAPKK2 VAMP/synaptobrevin SNAP-25 syntaxin VAMP/synaptobrevin
Tetanus
Yes
Protease Lethal factor Neurotoxins A–G Tetanus toxin a
Abbreviations: CNF, cytotoxic necrotizing factor; LT, heat-labile toxin; ST, heat-stable toxin; CLDT, cytolethal distending toxin; EAST, enteroaggregative E. coli heat-stable toxin; TCR, T-cell receptor; MHC II, major histocompatibility complex class II; MAPKK, mitogen-activated protein kinase kinase; VAMP, vesicle-associated membrane protein; SNAP-25, synaptosomal-associated protein; UTI, urinary tract infection; HC, hemorrhagic colitis; HUS, hemolytic uremic syndrome; PC, antibioticassociated pseudomembranous colitis. b
Yes, strong causal relationship between toxin and disease; (yes), role in pathogenesis has been shown in animal model or appropriate cell culture.
c
Other diseases are also associated with the organism.
d
Toxin is also produced by other genera of bacteria.
Source: C. K. Schmidt, K. C. Meysick, and A. O’Brien. 1999. Bacterial toxins: Friends or foes? Emerging Infectious Diseases 5:224–234.
A completed pore and a cutaway view exposing the channel are illustrated in Figure 25.17A and B. Diagnostic microbiology laboratories visualize hemolysins (proteins that lyse red blood cells) such as alpha toxin by inoculating bacteria onto agar plates containing sheep red blood cells (Fig. 25.17C). The clear, yellow zones around the S. aureus colonies growing on blood agar indicate that the microbe secretes a hemolysin. Cholera and E. coli labile toxins. Vibrio cholerae (Fig. 25.18A) produces a severe diarrheal disease called cholera that generally affl icts malnourished people populating poor countries like Bangladesh, or countries in which access to clean water has been disrupted by war or natural disasters. This microbe produces a gastrointestinal enterotoxin nearly identical to one produced by
Figure 25.16 AB toxins. A. A typical AB toxin consists of an A subunit and a pentameric B subunit joined noncovalently. B. Many AB toxins are ADPribosyltransferase enzymes that modify protein structure and function.
A.
some strains of E. coli associated with what is known as “traveler’s diarrhea.” Enterotoxins specifically affect the intestine. The E. coli enterotoxin is called labile toxin (LT) because it is easily destroyed by heat. Cholera toxin (CT) and labile toxin are both AB toxins with identical modes of action, which is to increase the level of cAMP made inside the host cell. After the bacteria attach to the cells lining the intestinal villa (Figs. 25.18C and D), they secrete these AB toxins, the structure of which is shown in Figure 25.18B. Both toxins have five B subunits arranged as a ring around a single A subunit. The B subunits bind to ganglioside GM1 on eukaryotic cell membranes and deliver the A subunit to the target cell (Fig. 25.19A , steps 1 to 4). The A subunit possesses the toxic part of the molecule, an ADPribosyltransferase, which must be activated by the host.
B. CONH2 N Ribose
A subunit
B subunit pentamer
ADP-ribosyltransferase
+ P
Target protein (active)
CONH2
+ P
N H
Ribose
Ribose N
N N
N
NH2 NAD
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Ribose P
P
N
Target protein
N
Nicotinamide
N N NH2
ADP-ribosyl protein (inactive)
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B. Cross section of alpha hemolysin
C. Hemolysis by S. aureus
MicrobeLibrary.org
A. Alpha hemolysin
Figure 25.17 Alpha hemolysin of Staphylococcus aureus. A. Three-dimensional figure of the pore complex comprising seven monomeric proteins. (PDB code: 7ahl) B. Cross section showing the channel. C. A blood agar plate inoculated with S. aureus. The alpha toxin is secreted by the organism and diffuses away from the producing colony. It forms pores in the red blood cells embedded in the agar, causing them to lyse. This is visible as a clear area surrounding each colony.
The binding of CT or LT to GM1 triggers endocytosis and the formation of a toxin-containing vacuole. The phagosome is then transported to the endoplasmic reticulum. During this time, the A subunit is cleaved by a host protease into two fragments called A1 and A2, which are still held together by a disulfide bond. The reducing environment at the cell surface reduces that bond and frees the A1 peptide containing active ADP-ribosylase
C. Brush border of intestine
B. Cholera toxin
© 2006 Dennis Kunkel Microscopy, Inc.
A subunit
1 μm
Gopal Murti/Photo Researchers
B5 subunit
1 μm
D. V. cholerae attachment GM1-OS
Intestinal cell surface
Figure 25.18 Pathogenesis of cholera. A. Vibrio cholerae (SEM).Note the slight curve of each cell and the presence of a single polar flagellum. B. Three-dimensional structure of cholera toxin binding ganglioside GM1 on the intestinal cell surface. (PDB code: 1s5f) C. Brush border of intestine (transmission EM). V. cholerae binds to the fingerlike villi on the apical surface. D. View of V. cholerae binding the surface of a host cell (SEM).
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V. cholerae
1 μm
Luz Blanco
A. Vibrio cholerae (SEM)
into the endoplasmic reticulum, which exports the toxin into the cytoplasm. The mission of A1 peptide is to modify (ADPribosylate) a membrane-associated GTPase (called a G protein) that binds to adenylate cyclase and controls its activity (Fig. 25.19A, step 5). To understand how the toxin produces diarrhea, it is helpful to observe what happens in the absence of toxin (Fig. 25.19B).
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A. A1 subunit B subunit
A2 subunit
Adenylate cyclase 1. The 5B:1A toxin complex binds the ganglioside GM1 on host membrane lipid rafts.
ATP
GTPase
GM1
Cl–
ADPR NAD
2. The toxin is endocytosed.
cAMP
NAm
5. The A1 peptide attaches an ADP ribose to an amino acid within the host G protein that regulates adenylate cyclase. 3. The phagosome containing CT is taken to the endoplasmic reticulum.
K+ H2O Na+ HCO3– 6. Cyclic AMP levels rise and activate ion transport systems, causing an electrolyte imbalance. Water from the cell follows the ions, causing diarrhea.
Nucleus
4. The A1 subunit is removed from the B subunits and exported into the cytoplasm.
Endoplasmic reticulum
B. 1. G-factor complex (α, β, γ) bound to hormone receptor. When bound to hormone, GTP exchanges for GDP on Gα.
Hormone
Hormone receptor
α β γ GTP
Figure 25.19 Cholera toxin mode of action. A. Delivery of cholera toxin into target cells and deregulation of adenylate cylase activity. B. Normal regulation of human cell adenylate cyclase. Human cells have two G-factor complexes, Gs and Gi, that stimulate and inhibit adenylate cyclase, respectively.
β γ GDP
α
4. Gα factor with GDP returns to receptor complex to await stimulation.
α GTP
GDP 2. Gα-GTP disengages from receptor, interacts with adenylate cyclase, and either stimulates (if Gsα) or inhibits (if Giα) cAMP production.
Pi 3. An intrinsic GTPase activity within Gα inactivates the G factor.
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GTP hydrolysis
Adenylate cyclase ATP α GTP
cAMP
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The intestinal epithelium has both absorptive and secretory functions. Transport is characterized by a net absorption of NaCl, short-chain fatty acids (SCFA), and water, allowing extrusion of a feces with very little water and salt content. In addition, the epithelium secretes mucus, bicarbonate, and KCl. A major player in the secretion of Cl– is the cystic fibrosis transmembrane conductance regulator (CFTR). Chloride export via CFTR is activated indirectly by cAMP. (CFTR is so named because a genetically based defect in the CFTR transporter/regulator manifests as the lung disease cystic fibrosis.) Human cells have two types of G-factor complexes called Gs and Gi that stimulate and inhibit adenylate cyclase, respectively. The G-factor complexes share beta and gamma subunits but have distinct alpha subunits (Gs-alpha and Gi-alpha). The complexes are normally bound to specific membrane hormone receptors (Fig. 29.19B). Stimulation of the hormone receptor allows GTP to bind the associated G-alpha subunit (replacing a bound GDP). Subsequently, the G-alpha–GTP protein leaves the receptor complex and binds adenylate cyclase, either stimulating cAMP production (if Gs-alpha) or inhibiting it (if Gi-alpha). Normally, an intrinsic GTPase activity within the G-alpha protein degrades GTP to GDP. G-alpha protein bound to GDP is inactive and cannot control adenylate cyclase. G-factor-associated GTP turnover controls cAMP levels and thus CFTR activity in normal cells. In the disease state (Fig. 25.19A, step 5), G-alpha protein is ADP-ribosylated by cholera or labile toxin. Modified G-alpha protein can still activate adenylate cyclase, but it has a defective GTPase. Because the altered G factor cannot degrade GTP, the protein continually stimulates cAMP production. This increases the levels of cAMP tremendously. Elevated levels of cAMP stimulate a host enzyme called protein kinase A that activates various ion transport channels (Fig. 25.19, step 6). One of these channels is the cystic fibrosis transmembrane conductance regulator that controls chloride transport (discussed in Section 24.4). As a result, chloride, sodium, and other ions leave the cell and, in an attempt to equilibrate osmolarity, water leaves as well. Because the affected cells line the intestine, the escaping water enters the intestinal lumen, leading to watery stools, or diarrhea. How is diarrhea a benefit to the microorganism? A major benefit to the organism is to propagate the species. The more diarrhea that is produced and expelled, the greater the number of organisms are made and disseminated throughout the environment. This increases the chance that another host will ingest the organism, ensuring survival of the species. Diarrhea can also benefit a pathogen by decreasing competition with other organ-
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isms as they are swept away. In fact, the vast majority of organisms found in the diarrheal fluids of cholera patients are V. cholerae bacteria. Different pathogens have discovered alternative ways to alter host cAMP levels. The gram-negative pathogen Bordetella pertussis causes a childhood respiratory infection called whooping cough, so named for the whooping sound made as a child tries to take a breath after a fit of coughing. This microbe also secretes an ADP-ribosylating toxin, but one that modifies Gi-alpha (the inhibitory factor that normally downregulates adenylate cyclase activity). ADP-ribosylation in this instance prevents that inhibition, with the result (again) of runaway cAMP synthesis. Pertussis toxin, however, is structurally different from cholera toxin. In addition, the organism makes a “stealth” adenylate cyclase that is secreted from the bacterium but remains inactive until it enters host cells, where it becomes active by binding the calcium-binding protein calmodulin. The resulting increase in cAMP levels causes inappropriate triggering of certain host cell signaling pathways. THOUGHT QUESTION 25.2 Antibodies to which subunit of cholera toxin will best protect a person from the toxin’s effects? Diphtheria toxin. Other exotoxins can affect host protein
synthesis. The microbe Corynebacterium diphtheriae produces a potent toxin that causes the disease diphtheria by inhibiting protein synthesis in eukaryotic cells (Fig. 25.20A). The organism remains in the pharynx while the toxin spreads throughout the body. Symptoms of diphtheria include the formation of an airway-obstructing pseudomembrane that forms over the trachea (Fig. 25.20B) and enlarged lymph nodes that give the patient a bullneck appearance (Fig. 25.20C). The pseudomembrane consists of fibrin, bacteria, and inflammatory cells; it adheres fi rmly to underlying tissue. The toxin, not growth of the organism, is solely responsible for these symptoms. The gene encoding the toxin actually resides in the genome of a bacteriophage that lysogenizes C. diphtheriae (lysogeny is discussed in Section 10.7). The phage is called Beta phage and is a prime example of how a phage genome may, over generations, degenerate to become a pathogenicity island, losing its former identity as a phage genome. Diphtheria toxin is also an ADP-ribosylase and another example of an AB toxin. But unlike cholera toxin, whose A and B subunits are synthesized as separate peptides from separate genes, diphtheria toxin is synthesized as a single polypeptide. Figure 25.21A illustrates the monomeric and natural dimeric forms of diphtheria toxin. There are three domains within the peptide. A
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CDC/P. B. Smith
©Princess Margaret Hospital, Hong Kong
B.
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A.
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Pathogenesis of diphtheria. A. Methylene blue stain of Corynebacterium diphtheriae (size 1 to 8 µm long). B. Pseudomembrane formed across trachea. C. Bull-neck appearance due to enlarged cervical lymph nodes.
Figure 25.20
central receptor-binding domain (the B subunit of the toxin) binds to the target cell receptor on the membrane. There is also a C-terminal transmembrane domain that allows the protein to embed in the host membrane. The third domain, located at the amino terminus, carries the ADP-ribosylase activity and is the A subunit of the peptide toxin. As with cholera toxin, diphtheria toxin must be cleaved to become active. As illustrated in Figure 25.22, the receptor-binding domain (B) binds the host membrane, and the toxin (A + B) then enters the cell by endocytosis. Once the toxin enters the cell, its peptide backbone
A.
Catalytic domains
Dimer
is cleaved, but the halves are held together by disulfide bonds. When the inside of the endosome vesicle acidifies (by H+ -pumping ATPases), the disulfide bonds are reduced (cleaved), allowing the transmembrane domain of subunit A to embed in the host endosome membrane and then to pass through the vesicle membrane. The transmembrane domain then facilitates passage of the A peptide (that is, the catalytic domain) through the vesicle membrane. Once inside the cytoplasm, the catalytic domain (A subunit) ADP-ribosylates an important protein synthesis factor called elongation factor 2 (EF2). This effectively halts protein synthesis and kills the cell.
B. Monomer
DNA NH3+
NH3+
C
R COO–
COO–
Receptorbinding domain T
Adenine Guanine
Receptorbinding domains Transmembrane domains
Adenine Guanine
Thymine Cytosine
Diphtheria toxin and the diphtheria toxin repressor. A. View of diphtheria toxin showing swapped domains of a dimer and a monomer of the toxin. To form a dimer, the receptor-binding domains of two monomers swap to produce a stable structure. (PDB code: 1SGK) B. Diphtheria toxin repressor (DtxR) bound to the dtx promoter DNA. When bound to iron, the repressor binds to the dtx gene in the beta phage to prevent toxin synthesis. (PDB code: 1C0W)
Figure 25.21
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Diphtheria toxin A B
Toxin receptor
1. Diphtheria toxin’s receptor-binding domain (B) binds host membrane.
2. Membrane-bound toxin (A + B) enters by endocytosis.
Membrane Receptormediated endocytosis
A B
3. Catalytic subunit A is cleaved but held to the B subunit by disulfide bonds. Endosome vesicle acidifies; the disulfide bonds are reduced.
A B A
4. The transmembrane domain facilitates passage of the catalytic A peptide through the vesicle membrane. NAD
Why is diphtheria toxin made? Iron availability appears to be a key factor. The body holds its iron very tightly in proteins such as lactoferrin and ferritin. So to an invading microbe, the body seems to be a very iron-poor environment. This makes it difficult for the pathogen to grow. Diphtheria toxin offers the organism a way to rob the host’s iron stores. Diphtheria toxin genes (dtx) are controlled by a repressor (DtxR) that binds iron (see Fig. 25.21B). When iron is plentiful, the repressor binds as a dimer to the promoter region of the dtx gene and prevents transcription. However, when free iron is scarce, as is the case in the body, there is not enough iron to activate the repressor and the dtx gene is expressed. As a result, diphtheria toxin is made, target cells are killed, and iron is released for bacterial use. This quest for iron is another common theme used by many pathogens to control pathogenic mechanisms. THOUGHT QUESTION 25.3 Would patients with iron overload (excess free iron in the blood) be more susceptible to infection?
EF2 EF2
ADPR 5. The catalytic A domain ADP-ribosylates elongation factor 2 (EF2). This halts protein synthesis and kills the cell.
Ribosome
mRNA Nascent peptide Elongation ceases
Diphtheria toxin mode of action. ADP-ribose, ADPR.
Figure 25.22
Today, thanks to intense vaccination efforts using inactivated toxin (called a toxoid), the disease diphtheria is rarely seen in this country. However, scientists are now learning to modify diphtheria toxin to serve new therapeutic purposes. Some experimental anticancer therapies have used gene-splicing techniques to replace the receptor-binding domain of diphtheria toxin with protein domains that bind unique receptors on cancer cells. The catalytic (toxic) and transmembrane domains of the toxin are left intact, but the new receptor domain targets the toxin to cancer cells. The toxin then enters and kills the cancer cell without harming innocent (noncancerous) bystander cells. Though showing promise, recombinant diphtheria toxin has not yet been proven effective in clinical trials.
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Shiga toxin: Shigella and E. coli O157:H7. S. flexneri
and E. coli O157:H7 (also known as enterohemorrhagic E. coli) cause food-borne diseases whose symptoms include bloody diarrhea. These organisms produce an important toxin known as Shiga toxin (or Shiga-like toxin). The toxin has five B subunits for binding and one A subunit imbued with toxic activity. The A subunit, upon entry, destroys protein synthesis by cleaving 28S rRNA in eukaryotic ribosomes. Strains that produce high levels of this toxin are associated with acute kidney failure known as hemolytic uremic syndrome. E. coli O157:H7 is a recently emerged pathogen. The organism can colonize cattle intestines without causing bovine disease; as a result, undetected bacteria can easily contaminate meat products following slaughter. The organism is sometimes referred to as the “Jack-in-the Box” microbe, a reference to the fi rst large U.S. outbreak associated with fast-food hamburgers at a Washington state Jack-in-the-Box restaurant in 1993. Both E. coli and Shigella have remarkable acid resistance mechanisms that rival that of the gastric pathogen Helicobacter pylori. This acid resistance makes these pathogens infectious at a very low infectious dose. THOUGHT QUESTION 25.4 How might you experimentally determine if a pathogen secretes an exotoxin? Anthrax. A century ago, anthrax (caused by Bacillus
anthracis; Fig. 25.23A) was mainly a disease of cattle
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C.
A.
1. Protective antigen subunit (PA) is made as a single peptide.
PA Cytoplasm
2. PA binds to a host cell surface, where a human protease cleaves off the orange part shown in part B.
Membrane
Arthur Friedlander (from NIAID)
Anthrax toxin receptor 3. Seven PA fragments autoassemble in the membrane to form a pore.
EF or LF
4. The other two components of anthrax toxin, EF and LF, bind to the ring and are carried into the cell by endocytosis.
B. Single PA protein Heptamer
5. EF and LF are expelled through the PA pore into the cytoplasm.
Bacillus anthracis and anthrax toxin. A. Bacillus anthracis (SEM). Approx. 2 µm in length. Splenic tissue from a monkey. Spores are not visible. B. Single subunit and heptamer of protective antigen. (PDB code: 1TZO) C. Mechanism of toxin entry.
Figure 25.23
and sheep. Humans only acquired the disease accidentally. Today we fear the deliberate shipment of B. anthracis through the mail or its dispersion from the air ducts of heavily populated buildings. What makes this grampositive, spore-forming microbe so dangerous? In large part, its lethality is due to the secretion of a plasmidencoded tripartite toxin. The core subunit of the toxin is called protective antigen (PA) because immunity to this protein protects hosts from disease. Protective antigen is made as a single peptide but then binds to the host cell surface, where a human protease cleaves off a fragment (Fig. 25.23B). The remaining part of PA can autoassemble in the membrane to form a heptameric (sevenmembered) pore. The other two components of anthrax toxin, edema factor (EF) and lethal factor (LF), bind to separate rings and are carried into the cell (see Fig.
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25.23C). The complex is endocytosed, and the two proteins carried in are passed through the pore into the host cytoplasm. Edema factor and lethal factor are the toxic parts of anthrax toxin. Both are enzymes that attack the signaling functions of the cell. Edema factor is an adenylate cyclase that remains inactive until entering the cytoplasm, where it binds calmodulin. This binding activates adenylate cyclase, resulting in a huge production of cAMP, and inactivates calmodulin from its normal function in the cell. Lethal factor is actually a protease that cleaves several host protein kinase kinases, each of which is part of a critical regulatory cascade affecting cell growth and proliferation. A protein kinase kinase is an enzyme that phosphorylates, and thereby activates, another protein kinase
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that can then phosphorylate one or more subsequent target proteins. One consequence of subverting these phosphorylation cascades is a failure to produce signals that recruit immune cells to fight the infection. We have examined only a few of the many toxins employed by pathogens. Some of the others, including tetanus and botulism toxins, will be described in the next chapter. What should be apparent from our brief sampling is the evolutionary ingenuity that pathogens have used to try to tame the human host. THOUGHT QUESTION 25.5 Internet problem: What other toxins are related to the cholera enterotoxin A subunit? B subunit? Anthrax tutorial
Identifying New Protein Toxins How does one identify and characterize the toxin of a new pathogen? In part that depends on whether there is an animal model for the disease (that is, whether the organism will infect a laboratory animal like the guinea pig or mouse and cause disease similar to that of humans). If there is a suitable animal model, one can grow the pathogen in laboratory media and harvest cellfree growth media into which the suspected exotoxin was secreted. The supernatant can be injected directly into a small number of mice, and effects on the health of the animals can be monitored over time. It is important to also use control mice that have received only fresh
A. LPS membrane
laboratory media (no toxin). If an effect is noted in the test mice, the exotoxin-containing supernatant can be treated with proteinase. If the toxin is protein, the treatment will destroy toxic activity. Subsequently, a variety of protein purification techniques, such as ion-exchange and molecular sieve chromatography, can be used to purify the protein. One can also utilize tissue culture cells to test for the presence of a cell-free toxin in growth media. In using this method, one thing to keep in mind is that the effect may be tissue specific: The toxin may work on one tissue but not another. Another concern is whether the laboratory medium used to grow the bacterial cells is sufficient to elicit toxin production. A high-iron medium, for example, will prevent synthesis of diphtheria toxin. Thus, it is important to mimic the host environment as closely as possible when coaxing a pathogen to make exotoxin in vitro.
Endotoxin (LPS) Is Made Only by Gram-Negative Bacteria Another important virulence factor common to all gramnegative microorganisms is endotoxin present in the outer membrane (discussed in Chapter 3). Not to be confused with secreted exotoxins, endotoxin is an embedded part of the bacterial cell surface and an important contributor to disease. Endotoxin, otherwise called lipopolysaccharide (LPS), is composed of lipid A, core glycolipid, and a repeating polysaccharide chain (Fig. 25.24). LPS molecules form the outer leaflet of the gram-negative outer membrane. As bacteria die, they release endotoxin. Endotoxin can then bind macrophages or B cells and trigger
B. Gram-negative bacterial endotoxin (lipopolysaccharide, LPS)
Water
Lipid A n O-specific oligosaccharide subunit O-specific polysaccharide chain
LPS
Water
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(inner)
Core oligosaccharide Core glycolipid
Endotoxin. A. Model of a lipopolysaccharide membrane of Pseudomonas aeruginosa, consisting of 16 lipopolysaccharide molecules (red) and 48 ethylamine phospholipid molecules (white). B. Basic structure of endotoxin, showing the repeating O antigen side chain that faces out from the microbe and the membrane proximal core glycolipid and lipid A (contains endotoxic activity).
Figure 25.24 Pacific Northwest National Laboratory
Membrane
(outer)
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the release of TNF-alpha, interferon, IL-1, and other cytokines. The release of these active agents causes a variety of symptoms, such as: ■
■ ■
■ ■
A.
Fever Activation of clotting factors, leading to disseminated intravascular coagulation Activation of the alternate complement pathway Vasodilation, leading to hypotension (low blood pressure) Shock due to hypotension Death when other symptoms are severe
The lipid A moiety of LPS possesses endotoxic activity (Fig. 25.24).
B.
Mediscan/Visuals Unlimited
NOTE: Lipopolysaccharides are also the outer membrane structures called O antigens that are used to classify different strains of E. coli as well as other gram-negative organisms (for example, E. coli O157 versus E. coli O111).
The role of endotoxins can be seen in infections with the gram-negative diplococcus Neisseria meningitidis (Fig. 25.25A), a major cause of bacterial meningitis. N. meningitidis has, as part of its pathogenesis, a septicemic phase where the organism can replicate to high numbers in the bloodstream. The large amount of endotoxin present causes a massive depletion of clotting factors that leads to internal bleeding, most prominently displayed to a physician as small pinpoint hemorrhages called petechiae on the patient’s hands and feet. (Fig. 25.25B). Capillary bleeding near the surface of the skin causes petechiae. One danger of treating massive gram-negative sepsis with antibiotics is that the enormous release of endotoxin from dead bacteria could well kill the patient. Untreated gram-negative sepsis is, however, almost always fatal, so its treatment, albeit risky, is imperative.
Effect of Neisseria meningitidis endotoxin. A. Neisseria meningitidis (cell 0.8 to 1 µm diameter, SEM). B. Petechial rash caused by N. meningitidis.
Figure 25.25
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There are five categories of protein exotoxins based on mode of action. These include toxins that cause membrane disruption, inhibition of protein synthesis, or alteration of host cell signal molecule synthesis, as well as superantigens and target-specific proteases. AB subunit toxins are common. The B subunit promotes penetration through host cell membranes, and the A subunit has toxic activity. S. aureus alpha-toxin forms pores in host cell membranes. Cholera toxin, E. coli labile toxin, and pertussis toxin are AB toxins that alter host cAMP production by adding ADP-ribose groups to different G-factor proteins.
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Diphtheria toxin is an AB toxin that adds an ADPribosyl group to eukaryotic elongation factor 2—a modification that stops protein synthesis. Shiga toxin is an AB toxin that cleaves host cell 28S rRNA in host cell ribosomes. Anthrax toxin is a three-part AB toxin with one B subunit (protective antigen) and two different A subunits that affect cAMP levels (edema factor) and cleave host protein kinases (lethal factor). Lipopolysaccharide, known as endotoxin, is an integral component of gram-negative outer membranes and an important virulence factor that triggers massive release of cytokines from host cells. The indiscriminate release of cytokines can trigger fever, shock, and death.
25.5 Protein Secretion and Pathogenesis A recurring theme among bacterial pathogens is the secretion of proteins that destroy, cripple, or subvert host target cells. The bacterial toxins described in Section 25.4 are secreted into the surrounding environment, where they float randomly until chance intervenes and they hit a membrane-binding site. However, many pathogens attach
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to a tissue and inject bacterial proteins directly into the host cell cytoplasm. The proteins may not kill the cell but redirect host signaling pathways in ways that benefit the microbe. Protein secretion pathways were introduced in Section 8.5, focusing on ATP-binding cassette (ABC) proteins as a model. Additional secretion models are described here in their critical role of delivering pathogenicity proteins such as toxins. A particularly interesting aspect of these secretory systems is that many of them evolved from, and bear structural resemblance to, other more innocuous systems. Other molecular processes that are evolutionarily related to secretion include: ■
■
■
Type IV pilus biogenesis (homologous to type II protein secretion) Flagellar synthesis (homologous to type III protein secretion) Conjugation (homologous to type IV protein secretion)
Outer membrane
D
Polymerization C
N
Periplasm Depolymerization
G M Secdependent export
L
Inner membrane
E
Table 25.4 gives examples of virulence proteins associated with these export systems.
Cytoplasm ATP
Type II Secretion Systems Resemble Pilus Assembly
ADP + Pi
Type II secretion offers a clear example of how Figure 25.26 Type II secretion. C, D, E, G, L, M, and N are protein components of the secretion system. Source: Modified from Moat, Foster, and Spector. nature has modified the blueprints of one system 2002. Microbial Physiology, 4th ed. to do a very different task. DNA sequence analysis has revealed that the genes used for type IV pilus periplasm, where they then encounter the appropribiogenesis (see Section 25.3) were duplicated at some ate type II secretion system. Because of this periplaspoint during evolution and redesigned to serve as a promic “layover,” the proteins are folded before secretion. tein secretion mechanism. Type IV pili have the unusual ability to extend and retract from the outer membrane, Type II secretion systems use the pilus-like structure a property that produces the gliding motility of Myxoas a piston to ram the folded proteins through an outer coccus (see Section 4.7) and the twitching motility of membrane pore structure and into the surrounding void Neisseria and Pseudomonas. As you might guess, assem(Fig. 25.26). Piston action occurs via cyclic assembly and bly/disassembly of these appendages is quite complex. disassembly of pilus-like proteins, driving the pilus-like Type II protein secretion mechanisms mirror this comstructure through an outer membrane pore and then plexity. Proteins to be secreted fi rst make their way, via retracting. Cholera toxin is a well-known example of a the Sec-dependent general secretion pathway, to the protein expelled by a type II secretion mechanism.
Table 25.4 Secretion systems for bacterial toxins. Secretion type
Features
Examples
I II Autotransporters
E. coli alpha-hemolysin, Bordetella pertussis adenyl cyclase Pseudomonas aeruginosa exotoxin A, elastase Gonococcal and Haemophilus influenzae IgA proteases
III
SecA dependent SecA dependent, similar to type IV pili SecA dependent to periplasm, self-transport through outer membrane SecA independent, syringe
IV
Related to conjugational DNA transfers
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Yersinia Yop proteins, Salmonella Sip proteins, EPEC EspA proteins, TirA B. pertussis toxin, Helicobacter CagA
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Jorge E. Galàn and Alan Collmer. Science 284:1322
C. PrgI
Outer membrane InvG PrgH
Periplasm
PrgK
In the last decade of the previous century, it was discovered that the etiological agents of Black Death and various forms of diarrhea (caused by species of Yersinia, Salmonella, and Shigella) could somehow take their bacterial virulence proteins made in the cytoplasm and drive them directly into the eukaryotic cell cytoplasm without the protein ever getting into the extracellular environment. Direct delivery is a good idea because it eliminates the dilution that happens when a toxin is secreted into media. Another advantage of this strategy is that it avoids the need to tailor the toxin to fit a preexisting host receptor. What kind of molecular machine can directly deliver cytoplasmic bacterial proteins into target cells? Research has shown that some microbes use tiny molecular syringes embedded in their membranes to inject proteins directly into the host cytoplasm; this mechanism of delivery is called type III secretion. Figure 25.27 shows electron micrographs of actual type III secretion needles and a model of the complex. Genes encoding type III systems are actually related to flagellar genes, whose products export the flagellin proteins through the center of a growing flagellum (discussed in Chapter 3). It appears that flagella genes were evolutionarily reengineered to act more like molecular syringes (see Fig. 25.27C). The bacterial virulence proteins secreted by type III systems subvert normal host cell signaling pathways and cause dramatic rearrangements of host membranes that often lead to engulfment of the microbe (Fig. 25.28). The genes encoding type III systems are usually located within pathogenicity islands inherited from other microbial sources.
Inner membrane SpaO
SpaP, Q, R, S InvA OrgB InvC
Cytoplasm
ATP ADP + Pi
The needle complex of the S. typhimurium type III secretion system. A. Unlike other secretion systems, the type III mechanism injects proteins directly from the bacterial cytoplasm into the host cytoplasm. The proteins in these systems are related to flagellar assembly proteins. Shown here are TEMs of osmotically shocked S. typhimurium with needle complexes visible in the bacterial envelope (arrows). B. Purified needle complexes (electron micrograph). C. A schematic representation of the S. typhimurium needle complex and its putative components.
Figure 25.27
Source: Modified from Moat, Foster, and Spector. 2002. Microbial Physiology, 4th ed.
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B.
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Shigella flexneri
Roger Wepf, Philippe Sansonetti, and Ariel Blocker, EMBL, Heidelberg
150 nm
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Figure 25.28 Shigella invades a host cell ruffle produced as a result of type III secretion. Shigella flexneri entering a HeLa cell (scanning transmission microscopy). The bacterium (small diameter approx. 1 µm) interacts with the host cell surface and injects (via its type III secretion apparatus) its invasin proteins, which act to choreograph a local actin-rich membrane ruffle at the host cell. The ruffle engulfs the bacterium and eventually disassembles, internalizing the bacterium.
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pathogenicity islands that are absent from related, but harmless, E. coli strains. Only two will be discussed here. Salmonella pathogenicity island 1 (SPI-1) encodes a type III protein secretion system that delivers a cocktail of at least 13 different protein toxins (called effector proteins) directly into the cytosol of host epithelial cells in the gut (Fig. 25.29). Inside epithelial cells, these effector proteins interfere with signal transduction cascades and modulate the host response. One mission of these effectors is to induce cytoskeletal rearrangements that cause ruffl ing of the eukaryotic membrane around the microbe (see Fig. 25.28). The membrane ruffl ing starts the process of engulfment. Salmonella induces this response as a way to avoid the normal endocytic process. Once Salmonella enters an epithelial cell or a macrophage, it fi nds itself in a vacuole. In the normal course of events, an enzyme-packed lysosome would then fuse with the phagosome and release its contents in an effort to kill the invader. Salmonella, however, possesses a second pathogenicity island called SPI-2 that subverts this host response. SPI-2 uses another type III secretion system to inject proteins that alter vesicle trafficking, thereby reducing phagosome-lysosome fusion so that the intracellular bacteria are spared. The pathogenesis of Salmonella is even more complex than just outlined. There are 12 recognized pathogenicity islands present in the genome.
Many bacterial pathogens use this type of secretion system, including plant pathogens such as Pseudomonas syringae (the cause of blight, a disease of many plants in which leaves or stems develop brown spots). Secretion is normally triggered by cell-cell contact between host and bacterium. The use of type III secretion will be examined during our discussion of Salmonella and E. coli pathogenesis (Special Topic 25.1). Because of their importance to virulence, type III secretion systems are the subject of intensive research designed to exploit them as potential drug targets. Salmonella pathogenesis. The enteric pathogen S. enterica uses type III secretion systems to invade the eukaryotic host cell and become an intracellular parasite. Following ingestion of contaminated food or water, this bacterium attaches to and invades M cells that are interspersed along the intestinal wall. M cells are specialized intestinal epithelial cells (see Figure 23.17) that sample the intestinal flora and transfer pathogens across the epithelial barrier for recognition by the immune system. Salmonella subverts the normal function of M cells and causes an inflammatory response that leads to diarrhea. During its evolutionary journey toward becoming a pathogen, Salmonella has acquired at least five and as many as ten
Salmonella Membrane ruffling
Type III secretion
Actin Effector proteins
Cl– H2O
Cell death Cdc42 Gene expression
Cytokines
JNK Host cell
Schematic overview of Salmonella pathogenesis. Effector proteins injected by Salmonella into a host M cell affects the activity of host proteins that trigger cell death (apoptosis), influence gene expression (Cdc42 and JNK), influence electrolyte movements, and induce actin rearrangement. Actin rearrangement produces the ruffling of host membrane to engulf the organism.
Figure 25.29
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The Bacterial Trojan Horse: Bacteria That Deliver Their Own Receptor
E. coli type III secretion and cell-cell interaction. A. EspA filaments form a bridge between enteropathogenic E. coli (EPEC) and an epithelial cell during the early stages of attaching and effacing lesion formation (SEM). These filaments are part of the type III secretion apparatus, functioning as a molecular syringe to inject proteins from pathogenic E. coli into the host cell. B. Model of cytoskeletal components within the EPEC pedestal. EPEC injects Tir protein into the host cell, where it moves to the membrane and acts as a receptor for intimin. Tir also communicates through phosphorylation with other host proteins to cause a change in actin cytoskeleton, which leads to pedestal formation. C. Pedestal formation (SEM).
Figure 1
A.
0.25 μm
B.
Knutton, et al. 1998. The EMBO Journal 17(8):2166
Many pathogens rely on key host surface structures such as gangliosides to recognize and attach to the correct host cell. But the host species can evolve to become resistant to infection when the gene encoding the receptor (or receptor synthesis) mutates. The mutation could lead to complete loss of the protein or a change in its shape to prevent recognition or alter its function. An example is the T-cell surface protein CCR5, which acts as a receptor for HIV virus (see Section 11.5). Individuals with a genetic defect that eliminates CCR5 are immune to HIV infection, so absent methods for preventing and curing HIV infection, humans would evolve a level of resistance to HIV. Some microbes circumvent possible loss of a host receptor by not relying on the natural array of host receptors in the first place. Instead, these bacterial pathogens use a type III secretion system to insert their own receptors into target cells (Fig. 1A). One such group of enterprising pathogens is enteropathogenic E. coli (EPEC). The adhesion molecule on the bacterial cell is called intimin. Intimin is a 94-kDa integral outer membrane bacterial protein needed for intimate adherence to host cells. However, there is no natural receptor for intimin on host membranes. As discovered by Brett Finley and his colleagues in Vancouver, British Columbia, EPEC must deliver its own intimin receptor, a 57-kDa bacterial protein called Tir (for translocated intimin receptor) that the bacteria inject into the host cell. The genes that encode intimin, Tir, and the secretion apparatus are all part of an EPEC pathogenicity island. Once injected and placed in the host membrane, Tir binds intimin on the bacterial surface (Fig. 1B). The result is the more intimate adherence between the bacterium and host cell surface required for infection to proceed. In addition, host protein kinases phosphorylate Tir at tyrosine residue 474. Phosphorylated Tir directly triggers a remarkable reorganization of host cellular cytoskeletal components (actin, alpha-actinin, ezrin, talin, and myosin light chain) such that a membrane “pedestal” is formed, raising the microbe up (Fig. 1C). The result of this attachment is the characteristic attaching and effacing (A/E) lesion, characterized by destruction of the microvilli and pedestal formation.
Pedestal EPEC
Tir Intimin α-Actinin
Y P NcK
Host factor
Host factors that control actin filamentation
N-WASP
Arp2/3 Actin
C.
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Type IV Secretion Resembles Conjugation Systems As Chapter 9 describes, many bacteria can transfer DNA from donor to recipient cells via a cell-cell contact system known as conjugation (discussed in Section 9.2). The conjugation systems of some pathogens have, through evolution, been modified into new systems that transport proteins, or proteins plus DNA, directly into target cells. Agrobacterium tumefaciens, for example, uses its Vir system to transfer the tumor-producing Ti plasmid and some effector proteins into plant cells. The result is a plant cancer called crown gall disease. The bacterium responsible for whooping cough in humans, Bordetella pertussis, also uses a type IV secretion system to export pertussis toxin, but simply exports the toxin without injecting it into the host (Fig. 25.30). Type IV systems also differ with respect to whether the protein is taken directly from the cytoplasm, like CagA from Helicobacter, or from the periplasm, as with pertussis toxin. In the latter case, the SecA-dependent general secretory system fi rst delivers the toxin to the periplasmic space.
Outer membrane
Pertussis toxin Periplasm
Inner membrane
Sec-dependent export
ATP Cytoplasm ADP + Pi
Pi + ADP
ATP
Type IV secretion of pertussis toxin. Evolutionarily related to conjugation systems, this type IV system in Bordetella pertussis takes pertussis toxin from the periplasm (moved there by SecA-dependent transport) across the outer membrane.
Figure 25.30
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THOUGHT QUESTION 25.6 Protein and DNA have very different structures. Why would a protein secretion system be derived from a DNA-pumping system?
TO SU M MAR I Z E: ■
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Many pathogens use specific protein secretion pathways to deliver toxins. Type II secretion systems use a pilus-like extraction/retraction mechanism to push proteins out of the cell. Type III secretion uses a molecular syringe to inject proteins from the bacterial cytoplasm into the host cytoplasm. Type IV secretion utilizes an entourage of proteins that resemble conjugation machinery to secrete proteins from either the cytoplasm or periplasm.
25.6 Finding Virulence Genes All pathogens must enter a host; fi nd their unique niche; avoid, circumvent, or subvert normal host defenses; multiply; and eventually be transmitted to a new susceptible host. Although certain common pathogenic tactics have come to be appreciated, each microbe has a unique “pathogenic signature” that permits survival and leads to its freedom to multiply. When an emerging pathogen causes disease, how do we figure out its mechanisms of virulence? Virulence constitutes a measurable phenotype of an organism. Genes encoding virulence factors are expressed just like those for other traits (for example, expression of the lac operon confers the ability to metabolize lactose; discussed in Section 10.2). For metabolic or biosynthetic pathways, identifying the requisite genes is relatively straightforward. For instance, mutations in genes for amino acid biosynthesis produce a clear phenotype that can be observed on an agar plate. If the pathway is defective, the amino acid is not made and must be added to the medium or the mutant will not grow. Because virulence is a consequence of growth in a host, there is no way to identify a phenotype for virulence gene mutants in vitro. Virulence mutants fail to survive or fail to cause disease in animals. So how are these genes identified? If the genome has been sequenced and a gene bears similarity to a known virulence gene present in another organism, that gene can be selectively mutated and the resulting mutant strain tested for virulence, usually by measuring LD50. But what of genes not assigned an annotated function? They, too, could encode virulence factors. And what of genes that are expressed only during an infection? How do we find those?
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In Vivo Expression Technologies Can Identify Virulence Genes Bacterial genes that are exclusively expressed by the pathogen only during growth within an animal host can now be discovered using what is called in vivo expression technology (IVET). The first step in IVET is to cripple a pathogen so that it cannot grow in the host animal unless a “rescue” gene that produces the missing factor is expressed. Next, the rescue gene is placed without its promoter into a plasmid to make what is called a promoter trap plasmid. The promoter of the plasmid-borne rescue gene is then replaced with random promoters cloned from the microbial genome. If one of the cloned promoters drives expression of the bacterial rescue gene after the organism is injected into the animal, the organism will flourish and kill the host. The trick is to find the promoters that express in vivo but that do not express, or that very poorly express, in laboratory media. Promoters that fit these criteria respond only to unknown in vivo conditions within the host and can be used to determine what those in vivo conditions are. The IVET procedure was first described in 1995 by J. Mekalanos and colleagues at Harvard University using a promoter trap plasmid in which random fragments of Salmonella DNA were inserted (that is, cloned) upstream of a promoterless cat-lacZ fusion in the plasmid (Fig. 25.31). The cat gene encodes chloramphenicol acetyltransferase, an enzyme that inactivates chloramphenicol, making the strain resistant to the drug. The collection of random clones was then injected into mice that were immediately injected with chloramphenicol. When a promoter allowed in vivo expression of the cat gene, the microbe became resistant to the drug, grew in the animal, and traveled to the spleen. The researchers knew that some of the in vivo expressed promoters would also be expressed in vitro (for example, promoters that drive genes encoding ribosome proteins). However, the goal was to identify promoters only expressed in the host, a characteristic that would indicate the presence of a downstream gene dedicated to virulence. So, the next task was to identify which of the in vivo expressed promoters was not expressed in vitro. To do this, the bacteria extracted from the spleen were plated on artificial media containing X-gal (an indicator of betagalactosidase enzyme made from the lacZ gene). A colony that was blue indicated that the promoter was expressed in vitro. A white colony indicated that the promoter was not expressed in vitro, but since the strain grew in the mouse, that same promoter must be exclusively expressed in vivo. DNA sequence analysis of the insert identified the promoter and, based on the promoter’s location on the bacterial genome, led to the conclusion that downstream genes were expressed specifically in vivo. This approach has now been used to identify potential virulence genes in several pathogens.
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1. The cat gene encodes chloramphenicol acetyltransferase, an enzyme that inactivates chloramphenicol. cat Cloned promoter fragment
lacZ Transform
Plasmid origin
2. DNA fragments from Salmonella are cloned in front of the promoterless cat-lacZ cassette, and the plasmids are transformed into a Salmonella strain.
Salmonella
3. Clone library is injected into mice.
4. Mice are immediately injected with chloramphenicol.
Wait two days; selects for in vivo Cm resistance 5. Bacteria extracted from the spleen are plated on X-Gal media to screen for the absence of lactose fermentation (white colonies).
Figure 25.31 In vivo expression technology. A library of promoter trap plasmids containing randomly cloned fragments of Salmonella DNA is transferred into Salmonella by transformation. The cloned pool is fed or injected into mice that have themselves been injected with chloramphenicol. Plasmids containing promoters expressed in vivo will yield Cm-resistant bacteria that can survive for two days and travel to the spleen. Bacteria recovered from the spleen are tested for an inability of the promoter fragment to drive lacZ expression in vitro. Lactosefermenting colonies will turn blue when grown on X-Gal medium.
You could ask why these virulence or pathogenicity genes are needed for in vivo growth of pathogens if commensal microbes, which also grow in vivo, do not need them. The reason is that commensal microbes exist in distinct niches within or on the body. Pathogens have mechanisms to subvert or bypass host defenses to gain entrance and grow in privileged sites normally unassailable by commensal bacteria. Finding these genes is the goal of IVET and other gene selection technologies (Special Topic 25.2).
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Special Topic 25.2
Signature-Tagged Mutagenesis
An especially elegant approach to identifying virulence genes is signature-tagged mutagenesis. Signature-tagged mutagenesis is a negative selection procedure that will allow the researcher to find mutants that do not replicate in
1. Mutants created. KmR
Tn5
DNA tag (or bar code)
hosts. The process, as applied to Salmonella, starts with a pool of mutants (Fig. 1, step 1). Each member of the pool is mutated (randomly) in a different gene and each mutant gene is tagged with a different oligonucleotide (like a bar code)
Signature-tagged allele replacement (STAR)
4. Samples of the different mutants from one plate are pooled and injected into a mouse or other host animal. Strains recovered from the spleen are grown and pooled; then their DNA is extracted (output pool).
DNA tag (or bar code)
2. Each mutant gene is tagged with a different oligonucleotide.
Put into infection model Recover and plate out
3. Individual mutants are placed in separate wells of a microtiter plate.
5. Tags from input and output pools are amplified and used to probe colony blots of the original mutants.
Microtiter plate
Replicate mutants onto filters Amplify and hybridize input pool of tags.
Output
Input
Amplify and hybridize output pool of tags.
6. Comparison of hybridization of input and output pools will identify mutants unable to survive the virulence screen.
Signature-tagged mutagenesis. DNA on the filter from mutants unable to survive in mouse will not hybridize to anything amplified from the output pool.
Figure 1
Using Modern Tools to Define Pathogenesis Helicobacter pylori and gastric ulcers. Helicobacter pylori is a gram-negative microbe, transmitted orally, that causes a variety of stomach diseases, including ulcers and cancer (Fig. 25.32). H. pylori has a genome size of 1.6 megabases (Mb) encoding 1,590 genes. The organism, however, is extremely difficult to grow in vitro and does
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not easily exchange genetic material. Because of this, classical approaches for studying its pathogenicity that rely on mutagenesis, clonal selection, and gene transfer are nearly impossible. However, cutting-edge gene array technology, which can identify the repertoire of genes expressed under any condition (see Section 10.9), and signature-tagged mutagenesis have successfully identified numerous genes involved in the virulence of Helicobacter. A basic scheme of
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treated to separate DNA strands and submerged into a solution containing the pooled radioactive PCR tag fragments made from the bacteria that survived in the mice and made it to the spleen (see step 4). Hybridization is spotted when a colony spot becomes radioactive. The conclusion is that representative bacteria from that colony survived in vivo. In contrast, a colony on the nitrocellulose membrane that does not hybridize to any of the probes in the mix (and thus is not radioactive) represents an attenuated strain that did not make it through the mouse selection (step 6). This strain contains a mutation (with a linked unique tag) within a virulence gene needed for in vivo survival of the bacterium. The gene can then be identified through various DNAsequencing strategies. The SPI-2 pathogenicity island of Salmonella enterica was discovered using this technique (Fig. 2).
(step 2). Each individual mutant with its unique sequence tag is placed in a separate well in a 96-well microtiter plate (step 3). Samples of the 96 different mutants from one plate are pooled and injected into a mouse or other host animal. Strains with mutations in pathogenicity genes will not grow in the mouse and will not be recovered from the spleen. These defective virulence mutants are identified using what is called a colony blot. For the colony blot, strains recovered from the spleen are grown and pooled, and then their DNA is extracted (step 4). The various tagged genes in this pool of DNA are then amplified and radiolabeled using PCR (all of the unique tag sequences are flanked by common sequences to which PCR primers are made). The individual clones originally arrayed in the microtiter dish are blotted onto a nitrocellulose membrane (step 5). This colony blot membrane is
tRNAval
■
SPI-2
31.0′
30.5′
ydhE ssaU T S R Q P O N Function Secreted protein Specific chaperone Transcription regulator Secretion apparatus
V
ssrA sseGF E D C B A sscB A M L K JIHG E D C B
B 319 242 70 ORF
245 ttrRS
32 pykF
ttrBCA 408 48 ORF
Tetrathionate reductase Genes with unknown function Homologue of E. coli protein
The Salmonella pathogenicity island SPI-2 was identified using signature-tagged mutagenesis. The island is located between 30.5 and 31 centisomes. ORFs are indicated by colored block arrows pointing in the direction of transcription. Gene designations are positioned below each ORF. The functional classes of SPI-2 genes are represented by different colors.
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pathogenesis has been suggested as a result (Fig. 25.33). As with most microbial diseases the fi rst step is colonization. Following ingestion of the pathogen, H. pylori flagella propel the organism toward the mucosa (step 1). As it approaches the mucosa, the pathogen produces intracellular and perhaps extracellular urease, which converts urea to CO2 and ammonia, locally reducing the acidic pH of the stomach (step 2). Two other enzymes, collagenase and mucinase, soften the mucus lining, which allows the
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bacteria to reach the stomach’s epithelial lining (step 3). The epithelial lining is much less acidic than the lumen, permitting the organism to grow and divide. Once at the epithelium, Helicobacter produces various adhesins, such as BapA or HpaA, to bind host cells (step 4). Once the organism has adhered, tissue damage occurs following the release of vacuolating cytotoxin (VacA) and neutrophil-activating protein (NAP), which activates neutrophils and mast cells that further damage local tissue (step 5).
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Helicobacter pathogenesis. A. H. pylori (SEM). Note the tuft of flagella at one pole. Cell length approx. 2 µm. B. Helicobacter attached to gastric mucosa (SEM). C. Helicobacter (red) binding to human gastric epithelial cells (colorized SEM). Intimate binding facilitates translocation of the bacterial effector protein CagA into the gastric cell via type IV secretion.
Figure 25.32
1. H. pylori flagella propel the organism toward the mucosa.
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Figure 25.33
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Summary of steps involved in Helicobacter colonization.
VacA forms a hexameric pore in the host membrane and induces apoptosis (programmed cell death). Another protein, CagA, is injected into host epithelial cells, where it is phosphorylated; CagA then interacts with host signaling proteins and activates host signal transduction pathways that can stimulate growth, possibly leading to cancer (step 6). As you can see, genomics has been a powerful tool in the study of Helicobacter. From DNA sequence alone, a fairly detailed picture of Helicobacter physiology and pathogenesis was proposed based on homologies to known enzymes from other organisms (see Fig. 8.42). Several aspects of these models have been confirmed experimentally. Whipple’s disease. It is difficult to characterize a pathogen if you cannot even grow it in culture. The microorganism Tropheryma whipplei causes a gastrointestinal illness called Whipple’s disease, whose symptoms include diarrhea, intestinal bleeding, abdominal pain, loss of appetite, weight loss, fatigue, and weakness. Though identified, the causative agent had never been grown outside of fibroblast cells. This made it nearly impossible to study its physiology. Once the com-
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plete genome sequence of two strains of T. whipplei became available, however, Didier Raoult and his colleagues at the Marseille School of Medicine discovered how to feed this fastidious bacterium. The genome (less than 1 Mb) contained a great deal of functional information. Computer modeling studies used this information to discover that the organism lacks the machinery to make several amino acids. With this knowledge, the investigators were able to design a cell-free culture medium that supported the growth of three different strains of T. whipplei as well as a new strain isolated from a heart valve. Similar applications of modern genomic techniques should lead to other successes in growing previously unculturable intracellular pathogens. TO SU M MAR I Z E: ■
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In vivo expression technology (IVET) is a positive selection tool to identify genes required for a pathogen to grow in vivo. Signature-tagged mutagenesis is a technique that uses negative selection to identify genes required for in vivo growth. Genomic tools were used to predict mechanisms of Helicobacter pylori pathogenesis and the growth requirements for the Whipple’s disease microbe.
25.7 Surviving within the Host Once inside a host, a successful pathogen must avoid detection and destruction for as long as possible. Many products of the virulence factor arsenal help the microbe escape or resist innate immune mechanisms. Others are dedicated to stealth, that is, hiding from the immune system.
Intracellular Pathogens In an effort to escape both nonspecific and humoral immune mechanisms (see Chapter 24), many pathogens, called intracellular pathogens, seek refuge by invading host cells. Antibodies and phagocytic cells will not penetrate live host cells, so hiding there provides temporary safe harbor for the pathogen. Some bacteria dedicate their entire lifestyle to intracellular parasitism. Rickettsia, for example, for reasons unknown, will not grow outside a living eukaryotic cell. Other microbes, such as Salmonella and Shigella, are considered facultative intracellular pathogens. Facultative intracellular pathogens can live either inside host cells or free. We have discussed how intracellular parasites get into cells, but how do they withstand intracellular attempts to kill them? Once inside the phagosome, intracellular pathogens have three options to avoid being killed by the phagolysosome. They can escape the phagosome, prevent phagosome-lysosome fusion, or survive the result.
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from the phagosome. The gram-negative bacillus Shigella dysenteriae and the gram-positive bacillus Listeria monocytogenes, both of which cause food-borne gastrointestinal disease, use hemolysins to break out of the phagosome vacuole before fusion. In this way, they completely avoid lysozymal enzymes. Once free in the cytoplasm, they are thought to enjoy unrestricted growth. Yet even in the cytoplasm, these microbes have found a way to redirect host cell function to their own ends. A fascinating aspect of escaping the phagosome involves motility. Shigella and Listeria are both nonmotile at 37°C in vitro; however, they both move around inside the host cell, even though they have no flagella. How do they move? These species are equipped with a special device at one end of the cell that mediates host cell actin polymerization. The polymerizing actin, called a “rocket tail,” propels the organism forward through the cell (Fig. 25.34A) until it reaches a membrane. The membrane is then pushed into an adjacent cell, where the organism once again ends up in a vacuole. This strategy allows the microbe to spread from cell to cell without ever encountering the extracellular environment, where it would be vulnerable to attack.
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lular pathogens avoid the hazard of lysosomal enzymes by preventing lysosomal fusion with the phagosome. Salmonella, Mycobacterium, Legionella, and Chlamydia are good examples. For example, Legionella pneumophila grows inside alveolar macrophage phagosomes and produces the potentially fatal Legionnaires’ disease, so named for the veterans group that suffered the first recognized outbreak in 1976. The organism, once inside a phagosome, secretes proteins through the vesicle membrane and into the cytoplasm. These bacterial proteins interfere with the cell-signaling pathways that cause phagosome-lysosome fusion. The result is that L. pneumophila can grow in a friendlier vesicle. Thrive under stress. In what could be called the “grin and bear it” strategy, some intracellular pathogens prefer the harsh environment of the phagolysosome. Coxiella burnetii, for example, grows well in the very acidic phagolysosome environment (Fig. 25.34B). This obligate intracellular organism causes a flu-like illness called Q fever (Query fever). The symptoms of Q fever include sore throat, muscle aches, headache, and high fever. It has a mortality rate of about 1%, so most people recover to good health. The organism allows phagosome-lysosome fusion because the acidic environment that results is needed for it to survive and grow.
THOUGHT QUESTION 25.7 How can one determine if a bacterium is an intracellular parasite? Why some bacteria are obligate intracellular pathogens is unclear. One intracellular bacterium, Rickettsia prowazekii, a cause of epidemic typhus, appears to be an “energy parasite” that can transport ATP from the host
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Intracellular pathogens: Shigella motility and Coxiella development. A. Intracellular Shigella flexneri (fluorescence microscopy), fluorescently stained red (1 µm in length), are propelled through the host cytoplasm by actin tails stained green. B. TEM image showing a typical vacuole in J774A.1 mouse macrophage cells infected with C. burnetii at 2 hours and 6 hours postinfection. The organism, which prefers to live in the acidified vacuole, undergoes a form of differentiation that changes its shape and alters its interactions with the host cell.
Figure 25.34
cytoplasm and exchange it for spent ADP in the bacterium’s cytoplasm. But this does not explain its obligate intracellular status, since giving rickettsia ATP outside a host does not allow the bacterium to grow. Other factors remain to be discovered. THOUGHT QUESTION 25.8 Why would killing a host be a bad strategy for a pathogen?
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This topic was fi rst discussed in the chapters on host defense (Chapter 23) and immunology (Chapter 24). Many bacteria, such as Streptococcus pneumoniae and Neisseria meningitidis, produce a thick polysaccharide capsule that envelopes the cell. Capsules help organisms resist phagocytosis in several ways. Recall that phagocytes must recognize bacterial cell surface structures or surface-bound C3b complement factor to begin phagocytosis (see Section 23.6). Capsules cover bacterial cell wall components and mannose-containing carbohydrates that phagocytes normally use for attachment. The uniformity and slippery nature of capsule composition makes it difficult for phagocytes to lock onto the bacterial cell. But what about complement factor C3b, which can bind to the bacterial cell? Phagocytes have surface C3b receptors that latch onto C3b molecules. Capsules will envelop any C3b complement factor that binds to the bacterial surface, thereby hiding it from the phagocyte. Fortunately, immune defense mechanisms can circumvent this avoidance strategy by producing opsonizing antibodies (IgG) against the capsule itself (see Section 24.3). The Fc regions of antibodies that bind to the capsule stick out away from the bacterium, where they can be recognized by the Fc receptors on phagocyte membranes. Binding of the Fc region to the phagocyte’s Fc receptor can then trigger phagocytosis. Pathogens can also use proteins on the cell surface to avoid phagocytosis. Staphylococcus aureus has a cell wall protein called protein A that binds to the Fc region of antibodies, hiding the bacteria from phagocytes. This works in two ways. Protein A can bind to the Fc region of an antibody before the antibody binding site ever fi nds its bacterial target. Thus, the business end of the antibody— that is, the part with antigen-binding sites—is pointing away from the microbe. However, even if the antibody fi nds its antigen target on one bacterium, protein A from a second bacterium can bind to that Fc region, once again blocking phagocyte recognition. Other microbes can trigger apoptosis in target host cells. Proteins made by the pathogen enter the host cell and trigger this programmed cell death. If a macrophage is targeted for self-destruction, it cannot destroy the microbe. Another immune avoidance strategy used by microorganisms is to shift their antigenic structure. Genes encoding flagella, pili, and other surface proteins often use, as part of their regulatory features, site-specific inversions to express alternative proteins (for example, Salmonella phase variation) or slipped-strand mispairing to add or remove amino acids from a sequence (see Section 10.6). Many bacteria and viruses also use mimicry to confuse the immune system (discussed in Section 24.6). In some cases, microbial proteins are made that look like cytokines.
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These factors manipulate the balance of T helper cells and send immunity down the wrong path for combating the microbe. All of these strategies are designed to buy the microbe more time to overwhelm the host. Chapter 26 presents additional mechanisms bacteria use to survive in vivo.
Regulating Virulence: How Do Pathogenic Bacteria Know Where They Are? Many pathogens can grow either outside or inside a host. To accommodate the needs imposed by these different environments, microbes will employ alternate physiologies appropriate to each. Why make a type III secretion system if there are no host cells around? So how do microbial pathogens know whether they are in a host or in a pond? And what bacterial genes are exclusively expressed while in a host? The same types of regulatory mechanisms that sense environmental conditions in a pond are used by the microbe to intuit its whereabouts in a host. That is, various sensing systems act in concert to recognize any specific environmental niche. Two-component signal transduction systems, discussed in Section 10.1, are used to monitor magnesium concentrations, which are characteristically low in a host cell vacuole. Other regulators measure pH, which will be acidic in the same vacuole. There are many other examples. The point is that there is no one in vivo sensor system. The various regulators collaborate to trigger the expression of virulence genes. As one example, the concentration of free iron, which is typically very low in the host, is an important signal used to induce the synthesis of virulence proteins whose purpose is to liberate iron bound up in host proteins. However, regulators sensitive to other in vivo signals must also be activated to achieve a successful infection. Cell-cell communication is also important during infections. Pseudomonas aeruginosa, for example, has at least two quorum-sensing systems that detect secreted autoinducers. As the number of bacteria in a given space increases, so, too, does the concentration of the chemical autoinducer. When the autoinducer reaches a critical concentration, it diffuses back into the bacterium or binds to a surface receptor and triggers the expression of bacterial target genes. Genes included in the P. aeruginosa quorumsensing regulons encode Pseudomonas exotoxin A and other secreted proteins, such as elastase, phospholipase, and alkaline protease. Why would a pathogen employ quorum sensing to regulate virulence factors? One reason may be to prevent alerting the host that it is under attack before enough microbes can accumulate through replication. Tripping the host’s alarms too early would make eliminating infection easy. However, releasing toxins and proteases once a large number of bacteria have amassed could overwhelm the host.
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Knowledge of quorum sensing has also provided an opportunity for treating disease. We know that bacteria can communicate with each other by secreting, and sensing, autoinducer chemicals. We also know that these molecules will accumulate in the growth environment and trigger collaborative responses, such as biofi lm formation, among members of the population. A new chemical has been developed that interferes with quorum sensing and could serve as a new type of antibiotic. The compound, N-(2-oxocyclohexyl)-3-oxododecanamide, is an analog of the homoserine lactone autoinducers used by P. aeruginosa. The compound can bind, but not activate, the regulatory proteins LasR and RhlR. When tested in vitro, this compound reduced biofi lm formation and decreased production of virulence factors. TO SU M MAR I Z E: ■
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Intracellular bacterial pathogens attempt to avoid the immune system by growing inside host cells. Different mechanisms are used to avoid intracellular death. Hemolysins are used by certain pathogens to escape from the phagosome and grow in the host cytoplasm. Actin tails are used by some microbes to move within and between host cells. Inhibiting phagosome-lysosome fusion is one way pathogens can survive in phagosomes. Specialized physiologies enable some organisms to survive in the normally hostile environment of fused phagolysosomes. Extracellularly, pathogens evade the immune system by hiding in capsules, by changing their surface proteins, by triggering apoptosis, or by using molecular mimicry. Two-component signal transduction systems can regulate virulence gene expression in response to the environment. Quorum sensing may prevent pathogens from releasing toxic compounds too early during infection.
25.8 Viral Pathogenesis The pathogenic strategies of viruses are at once similar to and yet distinctly different from those of bacteria. On their own, viruses are inert objects, lacking motility and unable to replicate. Like tiny molecular mines, they float in the environment until bumping into a suitable target cell. There is no need to transport food, expel waste, or generate energy, because the host does all that. But each virus must still attach to a host cell and subvert its biochemistry, directing it to make more virus. Because of the differences between bacteria and viruses, important insights into pathogenicity can be derived by comparing infection strategies used by different viruses.
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The Common Cold Versus Influenza You have probably wondered why you repeatedly get colds if the immune system is so effective. Why are you not protected from recurring infections after having one cold? The common cold is caused by over 100 known serotypes of rhinovirus, a small (30 nm), nonenveloped single-strand RNA virus. Serotypes of a given organism differ from one another in the antigenic makeup of a specific protein. The host receptor for these viruses is ICAM-1 (see Fig. 24.3), which is also important for the extravasation of leukocytes through blood vessels (see Section 23.5). Many of the differences among the rhinovirus strains can be traced to the structure of one of the capsid proteins, VP1, which is thought to bind ICAM-1. Because of these differences, neutralizing antibodies to one strain of rhinovirus will not neutralize a different strain. Thus, we repeatedly contract a cold because we are infected with a different strain. Unlike many viruses, rhinoviruses prefer to replicate at 33°C. As a result, they grow in the cooler regions of the respiratory tract, such as nasal passages. The reason these viruses cause a runny nose is that virus-infected cells release bradykinin and histamine, both of which affect fluid loss from local blood vessels (discussed in Section 24.5). Certain strains of coronavirus also cause common cold symptoms. It is significant to note, however, that a recent variant of coronavirus is also responsible for the newer, more deadly disease known as severe acute respiratory syndrome (SARS). THOUGHT QUESTION 25.9 Why do rhinovirus infections fail to progress beyond the nasopharynx? Influenza infection proceeds very differently from rhinovirus infections, even though the two are often confused by the public. Influenza virus fi rst establishes a local upper respiratory tract infection, where it targets and kills mucus-secreting, ciliated epithelial cells, destroying this primary defense. If the virus spreads down to the lower respiratory tract, it can cause shedding of bronchial and alveolar epithelium down to the basement membrane. As a result, oxygen and CO2 exchange is compromised and breathing becomes difficult. Systemic symptoms, such as muscle aches, are due to the release of interferon and lymphokines in response to the infection. Because natural defenses are compromised, persons infected with flu are very susceptible to superinfection with bacterial pathogens such as Haemophilus influenzae. In fact, these bacterial infections are so prevalent in persons infected with influenza virus that Haemophilus influenzae was originally suspected as the causative agent of flu. What makes flu so dangerous is that every 10 to 15 years the virus undergoes a major genetic alteration
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called an antigenic shift that results in a new pandemic strain. The change takes place in the gene encoding hemagglutinin and produces a change in the protein’s antigenic form. (Hemagglutinin binds to and agglutinates red blood cells. It is used by the virus for attachment to host cells.) As a result of the change in form, antibodies that recognized the old hemagglutinin will not recognize and neutralize the new one. Antigenic shift occurs when two different strains of influenza infect the same cell, usually in animals like pigs or chickens. Recall that influenza has a segmented RNA genome. Thus, as the two viruses disgorge their eight nucleic acid segments, recombinations can occur between them, generating a chimeric, and thus new, form of the hemagglutinin gene (see Section 11.4). This new influenza virus can then jump to humans, and a new pandemic begins. During the time between major shifts, minor changes in antigen structure also occur. Because they are minor, the process is called antigenic drift. Antigenic shifts and drifts are the reasons why a new vaccine (the flu shot) is required each year. These shots are highly recommended for the elderly, who are very susceptible to the disease, and are a good idea for everyone else. An estimated 21 million people worldwide died of the flu in the pandemic of 1918 (Fig. 25.35). No one then knew what caused the disease. Influenza was originally thought to be of bacterial etiology, and not until 1933 was it established that a virus was at fault. Many flu epidemics have taken place since then, but none have been so deadly. Though different from influenza, what was so frightening about the SARS outbreak that began in 2003 is that it had a fatality rate of greater that 10%, making it more deadly than the 1918 influenza strain, which had a fatality rate of 2–4%. The 1918 influenza virus was thought lost, and with it the opportunity to learn why it was so deadly. Such knowledge could prove important as we face the threat of another deadly strain, which, as of this writing, is the avian flu. However, we now have a new opportunity to learn about this deadly strain. In February 1997, Ann Reid, Jeffery Taubenberger, and their colleagues at the Armed Forces Institute of Pathology reported the partial sequences of five influenza genes recovered from the preserved lung tissue of a U.S. soldier who died from influenza in 1918. Additional sequences of this virus were obtained from an Alaskan influenza victim who was buried in the permafrost in November 1918. The freezing helped preserve the integrity, if not the viability, of the flu genome. In 2005, a group of scientists at the CDC, using these sequences, actually regenerated a live version of the 1918 pandemic virus, which is kept under tight security and containment. Its resurrection is expected to reveal secrets of its pathogenesis that may help us combat the inevitable emergence of a new, equally deadly flu virus.
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Flu epidemic of 1918. A. Influenza unit, U.S. Army hospital in Luxembourg. Notice the mouth covering designed to prevent spread of the disease. B. A Kansas emergency hospital trying to cope with the 1918 flu. C. Life expectancy in the United States in the twentieth century. Notice the dramatic dip in 1918, the result of deaths from influenza.
Figure 25.35
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Acquired immunodeficiency syndrome (AIDS) became an enormously important disease in the late twentieth century and continues to grip us today. There are between 30 to 40 million cases worldwide. As a result of infection by the retrovirus HIV (human immunodeficiency virus), the patient becomes severely immunocompromised and susceptible to a wide variety of infectious diseases. Death in patients with AIDS is usually the result of these secondary infections. Chapter 11 discusses the detailed molecular biology of HIV; here we will deal with the pathogenesis of the disease. In contrast to rhinovirus, which exclusively binds to one cell receptor, ICAM-1, HIV binds multiple cell receptors via its glycoprotein 120 (gp120, also called the spike protein; see Section 11.5 and Fig. 25.36A). For HIV, the basic receptor unit on host cells is CD4 (meaning T cells are the usual targets), in combination with a chemokine receptor CCR5 (see Fig 11.29A). By binding to the chemokine receptor, HIV can block chemokine binding and disconnect communication between the target cell and the immune system. You might still expect that the infection would be stopped because another aspect of the viral infection should, on its own, trigger apoptosis of the infected cell. Viral protein gp120 cross-links CD4 surface molecules on T cells, which, coupled with antigen binding to the T-cell receptor, should trigger apoptosis. The ensuing programmed cell death would simultaneously kill the infecting virus. However, the virus makes a protein (negative factor, NEF) that hinders apoptosis. NEF is one of several HIV proteins that affect pathogenesis (Table 25.5). As a result, the virus can replicate and bud from these lymphocytes (Fig. 25.36B). Despite the presence of NEF, however, the lymphocytes eventually do die. Because CD4 + T cells are the primary target of HIV, the levels of these T cells decline dramatically in an infected patient and cause an immunodeficiency, particularly in immune regulation. Monitoring T-cell numbers, therefore, is a critical diagnostic tool. Comparing HIV infection and AIDS, the disease it causes, underscores the difference between infection and disease. Detection of HIV in an individual does not equate with that person having overt disease. The individual can go for years without developing symptoms. But even if infected individuals are not showing signs of disease, they are fully able to transmit the virus to another person sexually, through blood transfusion, or through the use of shared hypodermic needles. As with many viral diseases, AIDS begins with flulike symptoms—fever, headache, fatigue, sore throat, and sometimes a rash. Consequently, symptomology is never a reliable way to diagnose HIV infection. The detection of antibodies is a good initial screen to gauge whether a person has been exposed to the virus. Once exposure has been confi rmed by a serum test (a change in test result
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from negative to positive is called seroconversion), it is important to use PCR techniques to measure actual HIV viral load in a patient’s blood. The higher the viral load the faster the disease will progress. Thus, for proper disease management, it is important to know viral load in addition to monitoring T-cell numbers. The symptoms of AIDS itself begin to manifest once T-cell numbers fall to around 300 per microliter of blood (the normal range is 500–1,500). It can often take four or
five years (or more) before a patient starts to experience this decline. With this immunocompromised state come secondary infections such as shingles (herpes zoster virus), Pneumocystis pneumonia, tuberculosis, and oral thrush (a fungal infection of the mouth caused by the yeast Candida albicans). Late-stage AIDS is defi ned by CD4 + counts below 200, at which point a malignancy called Kaposi’s sarcoma can develop (Fig. 25.36C). Kaposi’s sarcoma is a malignancy originating in endothelial or lymphatic cells, and tumors can arise anywhere—gastrointestinal tract, mouth, lungs, skin, or brain. An important cause of AIDS symptoms is the HIV Tat protein (trans activator of the HIV promoter). A transactivator is a protein that can diffuse through the cytoplasm to bind and regulate a target gene sequence. The Tat protein is needed for viral replication. In addition, it is released from infected monocytes and enters other cells, where it alters host regulatory cascades and gene expression. The result is an increase in the production of nuclear factor kappa beta (NF κβ), which helps further disassemble the
Table 25.5 HIV proteins that affect pathogenesis. Proteins
Role in pathogenesis
TAT (transcriptional trans-activator)
Secreted from infected cells; accelerates HIV gene transcription by host polymerase, has chemokine-like properties, acts as growth factor for endothelial cells, alters gene expression in target cells Induces G2 cell cycle arrest and nuclear import of the preintegration complex Downregulates cell surface CD4 and MHC class I (important for virus release); enhances virion infectivity by inhibiting apoptosis. Counteracts a host cellular protein that naturally inhibits HIV replication Enhances HIV release from infected cells and CD4 degradation by targeting CD4 to the proteasome
VPR (viral protein R) NEF (negative factor) VIF (virion infectivity factor) VPU (viral protein U)
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C. Nondisease state: Normally, p53 neutralizes EF2, a protein that is required to activate genes encoding essential cell division proteins. EF2 stimulates cell division. p53
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immune system, and activation of endothelial cell growth, which can lead to Kaposi’s sarcoma. Some believe that a vaccine using inactivated Tat will be useful in preventing AIDS. Other HIV proteins important to the pathogenesis of HIV and AIDS are presented in Table 25.5. The relative incidence of AIDS by race since 1986 is examined in Figure 25.36D. Since 1990, the overall incidence, approximately 40,000 annual cases, has declined in the United States, mainly as a result of improvements in antiviral therapies. However, the decrease is also due to a drop in the numbers of white males affl icted. The
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Human papillomavirus. A. Human papillomavirus (40–50 nm diameter, TEM). B. View of hand warts. C. Part of the strategy HPV uses to deregulate cell division in infected cells. Protein p53 normally helps limit cell division by inactivating EF2. Virus E6 protein, however, removes p53, so the cell will begin to divide uncontrollably and become cancerous.
Figure 25.37
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numbers for women, African Americans, and Hispanic minorities continue to rise.
Human Papillomavirus Human papillomavirus (HPV) is a very common virus that causes warts, an abnormal growth of skin tissue (Figs. 25.37A and B). HPV can produce warts on the feet, hands, vocal cords, mouth, and genital organs. Over 60 types of HPV have been identified so far. Each type infects different parts of the body. Some strains of HPV have also been associated with cervical and penile cancer. Genital HPV is one of the most common sexually transmitted diseases among college students. How does this virus cause excessive growth of tissues? Once inside host cells, HPV synthesizes a protein called E6 (Fig. 25.37C). E6 binds to a host protein, p53, that normally prevents cells from completing a cell cycle if their DNA is not properly replicated. Protein p53 inhibits cell division by binding to a transcription factor called EF2, preventing it from activating two oncogenes c-myc and c-fos. Transcription of c-myc and c-fos is needed for mitosis, so blocking their transcription prevents cell division and controls growth. The HPV protein E6 targets p53 for destruction by the cell proteosome (discussed in Section 8.6). With p53 thus removed, c-myc and c-fos proteins are made continually. As a result, cell division proceeds unabated to produce warts or, in the worst-case scenario, cervical cancer. After the initial infection has resolved, the virus may remain latent in tissues and can reactivate if immune system function is impaired. An effective HPV vaccine that will protect against cervical cancer has been developed, although guidelines for its use remain controversial. The problem is that it must be administered before a girl becomes sexually active (and potentially exposed) in order to work. The FDA and CDC have recommended its use in women and girls between ages 9 and 26. TO SU M MAR I Z E: ■
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(for example, capsid). Because of these differences, neutralizing antibodies made during a previous infection are ineffective against the new strain. Viral infection can increase a patient’s susceptibility to other, less virulent microbes. Antigenic shifts in a viral antigen (as in the hemagglutinin of influenza) can lead to a new pandemic of the disease. Animals coinfected with two viruses can serve as incubators for antigenic shifts or the evolution of new viruses. Binding to host cells can involve a single host receptor (as in rhinovirus) or multiple receptors (as in HIV). Virus-infected cells can secrete proteins that enter uninfected cells and disrupt signaling pathways.
Concluding Thoughts This chapter has only scratched the surface of all that is known about microbial pathogenesis. It should be clear
at this point that attachment, immune avoidance, and subversion of host signaling pathways are common goals of most successful pathogens, be they bacterial, viral, or parasitic. But even with all we know, there remains much to learn. The remaining chapters deal with the basic principles used to diagnose disease and eradicate offending pathogens, but many pathogens remain hard to detect and difficult, if not impossible, to kill. So the more we know about the mechanisms microbes use to cause disease, the better we will be at developing effective countermeasures. As you will learn in the coming chapters, new pathogens are constantly emerging. Over the last few decades, we have seen the development of HIV, SARS, avian flu, hantavirus, West Nile virus, E. coli O157:H7, and the re-emergence of flesh-eating streptococci, to name but a few. Can we ever stop pathogens from emerging? Probably not. For every countermeasure we develop, nature designs a counter-countermeasure. Our hope is that continuing research into the molecular basis of pathogenesis and antimicrobial pharmacology will keep us one step ahead.
CHAPTE R R EVI EW Review Questions 1. Describe the differences between infection versus dis-
11. Explain the mechanisms of secretion carried out by
ease; pathogenicity versus virulence; LD50 versus ID50. What is meant by direct versus indirect routes of infection? What are the characteristics of a good reservoir for an infectious agent? Name the various portals of entry for infectious agents and name a disease associated with each. Describe the basic features of a pathogenicity island. Explain various ways bacteria can attach to host cell surfaces. Describe the basic steps by which pili are assembled on the bacterial cell surface. How do type I and type IV pili differ? Explain the five broad categories of toxin mode of action. What is ADP-ribosylation and how does it contribute to pathogenesis? Explain the differences between exotoxins and endotoxins.
type II and type III protein secretion systems. What are the paralogous origins of these systems? Describe the key features of Salmonella pathogenesis. How can genomic approaches help identify pathogens in an infection? What different mechanisms do intracellular pathogens use to survive within the infected host cell? Describe different molecular strategies that microbes use to avoid the immune system. How do bacteria determine whether or not they are in a host environment? What is antigenic shift? How is attachment of influenza virus different from HIV? Why does HIV produce an immune deficiency while influenza does not? Explain the basics of how human papilloma virus can trigger cancer.
2. 3. 4. 5. 6. 7.
8. 9. 10.
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12. 13. 14. 15. 16. 17. 18.
19.
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Key Terms AB toxin (949) adhesin (944) ADP-ribosyltransferase (949) antigenic shift (972) ectoparasite (938) edema factor (EF) (957) endemic area (940) endoparasite (938) endotoxin (948) exotoxin (948) facultative intracellular pathogen (969) fimbria (944) fomite (940) frank pathogen (939) genomic island (942) horizontal transmission (940) in vivo expression technology (IVET) (965) infection (938)
infection cycle (940) infectious dose (939) intimin (963) intracellular pathogen (969) Kaposi’s sarcoma (974) labile toxin (LT) (951) latent state (939) LD50 (939) lethal factor (LF) (957) opportunistic pathogen (939) parasite (938) pathogen (938) pathogenesis (938) pathogenicity (939) pathogenicity island (942) petechia (959) pilus (944) primary pathogen (939) protective antigen (PA) (957)
protein A (970) receptor-binding domain (955) reservoir (940) Salmonella pathogenicity island 1 (SPI-1) (962) SPI-2 (962) transmembrane domain (955) type I pilus (945) type II secretion (960) type III pilus (945) type III secretion (961) type IV pilus (945) vector (940) vertical transmission (transovarial transmission) (940) virulence (939) virulence factor (942)
Recommended Reading Aktories, Klaus, and Joseph T. Barbieri. 2005. Bacterial cytotoxins: Targeting eukaryotic switches. Nature Reviews in Microbiology 3:397–410. Autret, Nicolas, and Alain Charbit. 2005. Lessons from signature-tagged mutagenesis on the infectious mechanisms of pathogenic bacteria. FEMS Microbiology Reviews 27:703–717. Burrows, Lori L. 2005. Weapons of mass retraction. Molecular Microbiology 57:878–888. Campellone, Kenneth G., Andrew Giese, Donald J. Tipper, and John M. Leong. 2002. A tyrosine-phosphorylated 12-amino-acid sequence of enteropathogenic Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to actin pedestals. Molecular Microbiology 43:1227–1241. Dean, Paul, Marc Maresca, and Brendan Kenny. 2005. EPEC’s weapons of mass subversion. Current Opinion in Microbiology 8:28–34. Dietrich, Guido, Sebastian Kurz, Claudia Hübner, Christian Aepinus, Stephanie Theiss, et al. 2003. Transcriptome analysis of Neisseria meningitidis during infection. Journal of Bacteriology 185:155–164. Dobrindt, Ulrich, Franziska Agerer, Kai Michaelis, Andreas Janka, Carmen Buchrieser, et al. 2003. Analysis of genome plasticity in pathogenic and commensal Escherichia coli isolates by use of DNA arrays. Journal of Bacteriology 185:1831–1840. Dobrindt, Ulrich, Bianca Hochhut, Ute Hentschel, and Jörg Hacker. 2004. Genomic islands in pathogenic and environmental microorganisms. Nature Reviews Microbiology 2:414–424.
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Groisman, Eduardo, and Josep Casadesús. 2005. The origin and evolution of human pathogens. Molecular Microbiology 56:1–7. Hamon, Mélanie, Hélène Bierne, and Pascale Cossart. 2006. Listeria monocytogenes a multifaceted model. Nature Reviews in Microbiology 4:423–434. Hensel, Michael, Jill E. Shea, Colin Gleeson, Michael D. Jones, Emma Dalton, and David W. Holden. 1995. Simultaneous identification of bacterial virulence genes by negative selection. Science 269:400–403. Hensel, Michael. 2004. Evolution of pathogenicity islands of Salmonella enterica. International Journal of Medical Microbiology 294:95–102. Jin, Qi, Zhenghong Yuan, Jianguo Xu, Yu Wang, Yan Shen, et al. 2002. Genome sequence of Shigella flexneri 2a: Insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157. Nucleic Acid Research 30:4432–4441. Liu, Tie F., Kimberley A. Cohen, Jason G. Ramage, Mark C. Willingham, Andrew M. Thorburn, and Arthur E. Frankel. 2003. A diphtheria toxin-epidermal growth factor fusion protein is cytotoxic to human glioblastoma multiforme cells. Cancer Research 15:1834–1837. Mahan, Michael J., John W. Tobias, James M. Slauch, Philip C. Hanna, R. John Collier, and John J. Mekalanos. 1995. Antibiotic based selection for bacterial genes that are specifically induced during infection of a host. Proceedings of the National Academy of Science 92:669–673.
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Mota, Luís Jaime, Isabel Sorg, and Guy R. Cornelis. 2005. Type III secretion: The bacteria-eukaryotic cell express. FEMS Microbiology Letters 252:1–10. Pallen, Mark J., and Brendan W. Wren. 2007. Bacterial pathogenomics. Nature 449:835–842. Renesto, Patricia, Nicolas Crapoulet, Hiroyuki Ogata, Bernard La Scola, Guy Vestris, et al. 2003. Genome-based design of a cell-free culture medium for Tropheryma whipplei. Lancet 362:447–449. Saenz, Henri L., and Christoph Dehi. 2005. Signature-tagged mutagenesis: Technical advances in a negative selection method for virulence gene identification. Current Opinion in Microbiology 8:612–619. Schilling, Joel D., Matthew A. Mulvey, and Scott J. Hultgren. 2001. Structure and function of Escherichia coli type 1 pili: New insight into the pathogenesis of urinary tract infections. Journal of Infectious Diseases 183:S36–S40. Smith, Kristina M., Yigong Bu, and Hiroaki Suga. 2003. Induction and inhibition of Pseudomonas aeruginosa quorum
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sensing by synthetic autoinducer analogs. Chemistry & Biology 10:81–89. Taubenberger, Jeffrey K., Ann H. Reid, and Thomas G. Fanning. 2005. Capturing a killer flu. Scientific American 292:48–57. Taubenberger, Jeffrey K., Ann H. Reid, A. E. Krafft, K. E. Bijwaard, and Thomas G. Fanning. 1997. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science 275(5307):1793–1796. Thomas, Wendy E., Elena Trintchina, Manu Forero, Viola Vogel, and Evgeni V. Sokurenko. 2002. Bacterial adhesion to target cells enhanced by sheer force. Cell 109:913–923. Thompson, Lucinda J., and Hilde de Reuse. 2002. Genomics of Helicobacter pylori. Helicobacter 7:1–17. Wiles, Siouxsie, William P. Hanage, Gad Frankel, and Brian Robertson. 2006. Modelling infectious disease—time to think outside the box? Nature Reviews in Microbiology 4:307–312.
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Chapter 26
Microbial Diseases
26.1 26.2 26.3 26.4 26.5 26.6 26.7 26.8 26.9
Characterizing and Diagnosing Microbial Diseases Skin and Soft-Tissue Infections Respiratory Tract Infections Gastrointestinal Tract Infections Genitourinary Tract Infections Infections of the Central Nervous System Infections of the Cardiovascular System Systemic Infections Immunization
In the American Civil War, twice as many people died from infections of their battle wounds as from the wounds themselves. Clearly, developments in medicine had not kept pace with innovations in rifle accuracy. Today, antibiotics can quell most wound infections. Yet millions of people still die as a result of dangerous microbes that spread by food, air, body fluids, and even insects. How do clinicians recognize these many different types of infections? What are the key symptoms or laboratory tests that help determine if a person suffers from a viral disease or bacterial disease? Microbial diseases are with us daily and continue to be one of the main contributors to global mortality and morbidity. The emergence of new pathogens, the increasing development of drug resistance, and the threat of bioterrorism have combined to create an increased demand for investigations into disease mechanisms and the body’s ability to combat the organisms responsible. Along with those basic studies, effective diagnostic algorithms are needed to quickly identify infectious diseases and prevent their spread. This chapter explores the general classes and causes of microbial diseases and introduces the art of diagnosis.
Scanning electron micrograph of primary cultured rabbit tracheal epithelial cells following 5 minutes of coincubation with virulent wild-type Bordetella bronchiseptica, a close relative of B. pertussis, the cause of human whooping cough. In this model for early host-pathogen interactions, wild-type B. bronchiseptica preferentially adheres to cilia and is rarely seen bound to aciliated cells or aciliated portions of ciliated cells. This adherence pattern impedes the ciliary elevator that normally removes bacteria from the lung. B. bronchiseptica is a common respiratory pathogen of cats and dogs but can cause whooping cough in immuno-compromised humans. Bacteria (approx. 1 µm in length) have been pseudocolored pink for easier visualization.
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A visibly ill 16-year-old girl was taken to her pediatrician, where she complained of diarrhea, high fever (39.9°C; 102°F), and vomiting. She told the physician that she was healthy until two days prior. Upon physical exam, the doctor noticed that the girl had a much lower than normal blood pressure (76/48 mmHg), a rapid heart rate of 120 beats per minute, and an erythematous (red) rash on her trunk. Because of her deteriorating condition, the patient was admitted to the pediatric intensive care unit at the local hospital, and cultures were obtained. She was immediately given intravenous fluids and IV antibiotics, yet she died within a week. Patient history taken upon admission revealed that the girl had started her menstrual period four days before becoming ill, providing an important clue as to the cause of the disease. This tragedy was part of a larger story that emerged in the late 1970s and early 1980s, when women started dying from this dangerous, new (emerging) disease. Now known as toxic shock syndrome, scientists and physicians learned that it was caused by certain strains of Staphylococcus aureus that produced a superantigen type of toxin (toxic shock syndrome toxin, or TSST; see Section 24.6). Why was the disease not seen in previous decades? The answer, surprisingly, turned out to be the use of one brand of superabsorbent tampons (since removed from the market). The tampons produced a rich growth environment for S. aureus. So if the patient were colonized by a TSST-producing strain, huge amounts of toxin would be released to circulate in the bloodstream. Today we recognize that toxic shock syndrome is a possible consequence of any S. aureus infection and can occur in both men and women. We now have tools to diagnose it quickly. In Chapter 25, we discussed the arsenal of weapons that microbes use to cause disease (including TSST). But what of the diseases themselves? Microbial diseases are often presented from the microbe’s point of view—for example, where in the body does Staphylococcus aureus cause disease? Another approach, which we take in this chapter, is to look at infections from the vantage point of the infected organ. We might ask, for example, what organisms cause vaginal infections versus lung infections, and how are they differentially diagnosed? Are the patient’s complaints consistent with gastrointestinal tract disease or a lung infection? This approach is more attuned to the practices of health care workers and the foundation by which a physician interacts with a patient. An individual who goes to a clinician complaining of a fever, cough, and chest pain does not typically report having encountered a particular bacterium. The physician must determine from the patient that the disease is localized to the chest and then intuit that it may be a respiratory tract infection. Appropriate samples are then collected and sent to a clinical microbiology laboratory, where data are collected to confirm or dispute the presence of a specific etiologic agent such as Streptococcus pneumoniae or other lung pathogen.
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In this chapter, microbial diseases are classified as respiratory, gastrointestinal, genitourinary, nervous system, skin and soft-tissue, bone and joint, cardiovascular, and generalized (disseminated) infections. We do not present an exhaustive compendium of microbial illnesses but a representative sampling of infections that illustrate key aspects of microbial disease. The material is arranged and presented so as to integrate your knowledge of microbiology and immunology within the framework of the practice of medicine and the study of infectious disease.
26.1 Characterizing and Diagnosing Microbial Diseases Although this chapter will classify representative microbial diseases by organ system, there are other very instructive ways to group and study microbial infections. These include the organism and portal-of-entry approaches. Each has clear benefits and pitfalls. The organism approach is useful when examining the different ways a given species can cause disease. For instance, E. coli can cause urogenital tract infections, gastrointestinal infections, and meningitis. How do the various strains causing these diseases differ from one another? Are virulence factors present in the uropathogenic strain that are absent or different in the diarrhea-producing strain? (Virulence factors are discussed in Chapter 25; see Sections 25.2 and 25.3.) Pathogens are also commonly classified by their route of infection (or portal of entry; see Chapter 23). In this approach, pathogens are classified as food-borne, airborne, blood-borne, or sexually transmitted. The port-of-entry approach has its merits, but it sometimes seems at odds with the disease itself. Salmonella typhi, for example, is categorized as a food-borne pathogen. Food-borne pathogens typically have a fecal-oral route of infection and cause pronounced gastrointestinal disease. Yet S. typhi does not actually cause gastrointestinal disease until very late in the disease process. After ingestion, it quickly enters the bloodstream from the intestine to produce a serious septicemia (blood infection) called enteric (typhoid) fever. At this stage, the organism cannot be found in feces. (Much later the organism can reenter the intestine via the gall bladder and cause mild gastrointestinal symptoms.) The obvious value of knowing the route of infection is that society can design effective measures to avoid disease. Hand washing and proper food preparation, for instance, will effectively prevent the septicemia of typhoid fever. But clearly, all organisms entering via the gastrointestinal tract do not cause gastrointestinal disease. The organ systems approach has its drawbacks as well, given that organisms that initially affect one organ system can disseminate to infect other organ systems. Neisseria gonorrhoeae, for example, initially infects the genitourinary tract, but can disseminate via the bloodstream, causing a
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Patient Histories Provide Important Diagnostic Clues A problem often faced by clinicians is that many infectious diseases display similar symptoms, making diagnosis difficult. Vibrio cholerae and enterotoxigenic E. coli, for instance, both produce diarrheal diseases characterized by cramps, lethargy, and liters of watery stool each day. Cholera, however, is not commonly seen in the United States. Nevertheless, a clinician might suspect cholera if a patient recently traveled to or arrived from an endemic area (a specific geographical locale such as India or Bangladesh) where the disease is regularly observed. This travel information is not gained by examining the patient but comes from taking a “patient history.” Knowledge of the patient’s hobbies can be equally important for diagnosing a disease. This is another critical part of taking a patient’s history. For example, a person who is suffering from enlarged glands, fever, and headaches and who was recently rabbit hunting may have been exposed to the gram-negative rod Francisella tularensis, an intracellular pathogen that infects various wild animals and is the cause of tularemia, an illness also known as “rabbit fever” (Fig. 26.1A). A hunter with these symptoms could have accidentally infected a cut while cleaning
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the kill. Tularemia is an example of a zoonotic disease, an infection that normally affects animals but that can be transmitted to humans. How can knowing a person’s occupation be important? Consider the case of a man with acute pneumonia. The clinician discovers that the man is a sheep farmer. Knowing this, Coxiella burnetii, a pathogenic bacterial species that infects sheep and is shed in large quantities in placental materials, would be considered one of the possible sources of infection. The organism grows intracellularly only (it is an obligate intracellular pathogen). It infects sheep, cattle, and goats but does not usually cause clinical disease in these animals. C. burnetii is secreted in body fluids and shed in high numbers in the animal’s amniotic fluids and placenta, heavily contaminating the soil. When the contaminated soil dries, the microbes become aerosolized whenever the dirt is disturbed. Humans (such as farmers) that inhale the dried particles can develop the lung infection called Q fever, another type of zoonotic disease (Fig. 26.1B). One more example of an “occupational hazard” is illustrated by a woman with severe respiratory disease who works in a leather factory, handling animal hides. Knowing her occupation, a clinician would investigate whether she might have contracted anthrax (woolsorter’s disease) from inhaling spores present on the animal hide. Because patient histories are so helpful in diagnosing infectious diseases, we will present several case histories in the following sections and segue into discussions of various microbes that can infect each organ system. Key aspects of infectious diseases will be revealed as we proceed. It should be stressed that further insight into pathogenesis and new innovations in diagnosis will come from the remarkable number of microbial genomes whose sequences have been completed as well as from those that are still under way. A sampling of the pathogens whose genomes have already been sequenced is presented in Table 26.1. Many of the ORFs identified have no known function and
A.
B.
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Examples of bacteria that cause zoonotic diseases. A. Francisella tularensis (colorized SEM), the cause of tularemia. Tularemia is a highly infectious disease, usually spread via a tick vector but also through cuts. B. Coxiella burnetii (colorized TEM), the cause of Q fever. This irregularlyshaped organism undergoes developmental stages that range in size from 0.2 µm to 1 µm.
Figure 26.1
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blood infection. Yersinia enterocolitica, a cause of gastroenteritis, can disseminate from the intestine and cause abscesses elsewhere in the body. Yet we favor the organ systems approach because it is illuminating to know how microbial diseases present themselves to physicians treating patients. The physician’s examination usually begins not by taxonomic evaluation of the agent or knowledge of the portal of entry, but with symptoms emanating from the organ system that has been affected. You will benefit, however, from studying infectious disease from various perspectives, and you should keep in mind factors such as the portal of entry and the taxonomy of the infectious organism.
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Table 26.1 Examples of pathogens whose genomes have been sequenced.a Pathogen
Disease
Escherichia coli O157:H7 Salmonella typhi Yersinia pestis Haemophilus influenzae Haemophilus ducreyi Vibrio cholerae Vibrio vulnificus Pseudomonas aeruginosa Coxiella burnetii Neisseria meningitidis Bordetella pertussis Helicobacter pylori Campylobacter jejuni Rickettsia prowazekii Brucella suis Bacillus anthracis Staphylococcus aureus Streptococcus pyogenes Listeria monocytogenes Clostridium tetani Clostridium perfringens Mycoplasma pneumoniae Mycobacterium tuberculosis Mycobacterium leprae Chlamydia trachomatis Borrelia burgdorferi Treponema pallidum Leptospira interrogans
Diarrhea, hemolytic uremic syndrome (HUS) Typhoid fever Plague Meningitis, otitis media Chancroid (sexually transmitted) Cholera (severe diarrhea) Septicemia, wound infections Wound infections, septicemia, respiratory infections Q fever (respiratory) Meningitis, septicemia Whooping cough (respiratory) Stomach ulcers, gastric cancer Diarrhea Epidemic typhus Flu-like disease, brucellosis Anthrax (respiratory or wound) Boils, toxic shock syndrome, wound infections, osteomyelitis (bone) Strep throat, wound infections Flu-like, diarrhea, stillbirths Tetanus Wound infections Respiratory Tuberculosis (respiratory) Leprosy Eye infection, urethritis (sexually transmitted) Lyme disease Syphilis (sexually transmitted) Septicemia
a
To view genomes of pathogens, access the “Kyoto Encyclopedia of Genes and Genomes” site on the Internet or the “Genome” site at Entrez PubMed.
may eventually explain differences in host and tissue specificity and reveal new toxins or other pharmacologically active molecules produced by virulent microbes. TO SU M MAR I Z E: ■
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Pathogens can be classified as food-borne, airborne, blood-borne, or sexually transmitted. Patient histories are vital in diagnosing microbial diseases. Zoonotic diseases are animal diseases accidentally transmitted to humans.
26.2 Skin and Soft-Tissue Infections Skin infections range from simple boils to severe, complicated so-called “flesh-eating” diseases (Table 26.2). Recall that the integrity of the skin as well as the presence
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of normal skin flora prevent infection. However, even minor insults to the skin (such as a paper cut) can result in infections, most of which are caused by the gram-positive pathogen Staphylococcus aureus. Healthy individuals develop infections of the skin only rarely; however, people with underlying immunosuppressive diseases such as diabetes are at much higher risk.
Boils Staphylococcus aureus is a common cause of the painful skin infections called boils, or carbuncles (Fig. 26.2). This gram-positive organism, often a normal inhabitant of the nares (nostrils), can infect a cut or gain access to the dermis via a hair follicle. It possesses a number of enzymes that contribute to disease, including coagulase, which helps coat the organism with fibrin, thereby walling off the infection from the immune system and antibiotics. As a result, boils generally require surgical drainage as well as antibiotic therapy. As noted earlier, some strains of S.
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Figure 26.2
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aureus also produce toxic shock syndrome toxin, a superantigen that can lead to serious systemic symptoms (see Chapters 24 and 25). A particularily dangerous strain of S. aureus called methicillin-resistant S. aureus (MRSA) has recently emerged. S. aureus infections are commonly treated with penicillin-like drugs, such as methicillin. MRSA has developed resistance to methicillin and many other penicillin-like drugs through a mutation that alters one of the proteins (called a penicillin-binding protein; PBP) involved in cell wall synthesis. Because methicillin is normally used as a first line of defense against staphylococcal infections, treatment failure of MRSA can be life-threatening, and alternative drugs, such as vancomycin, need to be used. MRSA first appeared as agents of nosocomial infections—infections that occur after a patient enters a hospital. However, these antibioticresistant organisms are no longer contained only in the hospital. Individuals who have not been in a hospital are being infected with MRSA in what are called communityacquired infections. This is occuring at an epidemic rate in the United States (an incidence of approximately 20 per 100,000 population). Seventy percent of Staphylococcal skin infections are now caused by MRSA. Thus, a physician can no longer assume that a patient walking into the office with a staphylococcal infection will respond to methicillin. The doctor must assume that it may be MRSA. As a result, treatment regimens around the country are being forced to change. Today, vancomycin and linezolid are the antibiotics typically used to treat staph infections. Antibiotics are discussed in Chapter 27. Other staphylococcal diseases are caused by toxinproducing strains in which the organism remains localized but the toxin disseminates. We have already mentioned TSST, but there are other toxins. For example, some strains of Staphylococcus aureus produce a toxin called exfoliative toxin that causes a blistering disease in children called staphylococcal scalded skin syndrome (Fig. 26.2C). Exfoliative toxin, like TSST, is a superanti-
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© 2007 Interactive Medical Media LLC
Staphylococcus aureus. A. S. aureus (colorized SEM). B. Exfoliative toxin from some strains of S. aureus cause scalded skin syndrome.
gen, but it also cleaves a skin cell adhesion molecule that when inactivated results in blisters. Table 26.2 presents other common infections of the skin and soft tissues.
Case History: Necrotizing Fasciitis by “Flesh-Eating” Bacteria One weekend in June, Cassi was camping with her three children. She suffered a minor cut on her finger, which she bandaged properly. She also injured the left side of her body while playing sports with her kids. Not thinking much of either of her minor injuries, she went to bed. Two days later, Cassi was extremely ill. Her symptoms included vomiting, diarrhea, and a fever. She was also in severe pain where she had injured her side, and the area had begun to bruise (the skin was not broken). By the next day, she could barely get out of bed, and by the end of the night, she was breathing with difficulty and could not see. Her side began to leak fluid and blood. Cassi was admitted to the hospital in shock, with no detectable blood pressure. An infectious disease specialist diagnosed the problem as necrotizing fasciitis, and she was rushed into surgery. In an effort to save her life, about 7% of her body surface was removed. Because the large wound infection in her side would need to resolve before a skin graft could be performed to repair it, the hole in Cassi’s body was left wide open (Fig. 26.3A). After nearly three months and several operations, Cassi recovered. What kind of organism can cause this type of devastating disease? The disease necrotizing fasciitis, also known as flesh-eating disease, is rare and is often caused by the gram-positive coccus Streptococcus pyogenes, a microbe normally associated with sore throat infections (pharyngitis). Although sometimes described as a recently emerging infectious disease, necrotizing fasciitis was first discovered in 1783, in France. Its incidence may have risen recently owing to the increased use of anti-inflammatory drugs, which increase a person’s susceptibility to infection. In this case history, Cassi probably had this organism on her skin
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Table 26.2 Common infectious diseases of the skin. Disease
Symptoms
Etiological agent
Virulence factors
Boils Peeling skin on infants, systemic toxin Skin lesions on the face, mostly children Sore throat, fever, rash Skin lesions, usually facial, that spread to cause systemic infection Rapidly progressive cellulitis
Staphylococcus aureus (G+ cocci); fibrin wall around abscess renders it poorly accessible to antibiotics
Coagulase, protein A, TSST, leucocidin, exfoliative toxin
Streptococcus pyogenes (G+ cocci)
M-protein pili, C5a peptidase, hemolysin, pyrogenic toxins, others
Bacterial Folliculitis Scalded skin syndrome Impetigo Scarlet fever Erysipelas
Necrotizing fasciitis
Viral Rubellaa Measlesa Chickenpoxa Shingles Smallpox Warts
Discolored, pimply rash; mild disease unless congenital Severe disease, fever, conjunctivitis, cough, rash Generalized discolored lesions Pain and skin lesions, usually on trunk in adults Raised, crusted skin rash, highly contagious Rapid growth of skin cells
Rubella virus [ssRNA(+)] Rubeola virus [ssRNA(–)] Varicella-zoster (dsDNA)
Variola major (dsDNA) Papillomaviruses (dsDNA)
Fungal Dermatomycosis Sporotrichosis
Blastomycosis
Candidiasis
Aspergillosis Zygomycosis a
Dry, scaly lesions like athlete’s foot Granulomatous, pus-filled lesions; can disseminate to lungs or other organs Granulomatous, pus-filled lesions; can disseminate to lungs or other organs Patchy inflammation of mouth (thrush) or vagina; can disseminate in immunocompromised patients Infects wounds, burns, cornea, external ear Mainly affects diabetic patients; can rapidly disseminate
Dermatophytes Sporothrix schenckii
Blastomyces dermatitidis
BAD1 adherence
Candida albicans
Proteinase, phospholipase, Ssn6/Tup1 regulators, others
Aspergillus spp.
PacC/fos1 regulators, gliotoxin
Mucor and Rhizopus spp.
Vaccine is available against this agent.
when the injury to her side occurred. The injured area probably suffered an invisible microabrasion, providing a good growth environment for the organism, leading to the secretion of potent toxins and death of surrounding tissues. Rapid, aggressive antibiotic treatment is required in these extreme cases, even before the clinical microbiology lab has had time to identify the organism. Therapy can
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include several antibiotics, such as clindamycin and metronidazole, which act against anaerobes and gram-positive cocci, and gentamicin, a drug particularly effective against gram-negative microbes. (Chapter 27 further discusses these and other antibiotics.) Often, antibiotic treatment of patients with necrotizing fasciitis is ineffective because of insufficient blood supply to the affected tissues.
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Courtesy Cassi Moore
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THOUGHT QUESTION 26.1 Why would treatment of an infection sometimes require multiple antibiotics? Antibiotic treatment
Is There an Immunological Predilection to Necrotizing Faciitis? S. pyogenes is best known for causing sore throats and for the immunological sequelae that can develop, such as rheumatic fever and glomerulonephritis. Sequelae are secondary diseases that develop after resolution of a primary infection. They are often the result of a cross-reactivity between bacterial and host antigens. The immune system, activated by the bacterial antigen, begins to attack the cross-reacting self antigens and damages tissues (see Section 24.8). Why some patients infected with S. pyogenes develop necrotizing faciitis while others do not may be due more to the immunological status of the patient than to the virulence arsenal of the microbe, but its arsenal is vast nonetheless. The source of many established putative virulence factor genes in S. pyogenes are the many prophages present in its genome. Prophages constitute approximately 10% of the organism’s genome. One study found that a soluble factor produced by human pharyngeal cells can facilitate activation of at least some of these phages and cause horizontal transfer of the associated virulence factors between strains of this pathogen.
Courtesy Cassi Moore
Flesh-eating Streptococcus pyogenes. A. Flesh removed from a patient in an effort to stop the spread of necrotizing fasciitis. B. Gangrene of the fingers caused by the same infection in the same patient.
Figure 26.3
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with a sore throat. Then a skin rash develops that typically starts on the face and spreads down the body. The virus replicates in the lymph nodes and spreads to the bloodstream (viremia), where it can infect endothelial cells of the blood vessels. The rash occurs when T cells begin to interact with these infected cells. Although skin rash is the main symptom of measles, infection can also cause respiratory symptoms and complications, including pneumonia, bronchitis, croup, and even a fatal encephalitis in immunocompromised patients. In the United States, measles has been almost completely eliminated by the measles, mumps, and rubella (MMR) multivalent vaccine (one exception is the AIDS patient, where measles can be fatal). Worldwide, however, measles is still a serious problem in places where vaccinations are not routine. Rubella virus, a togavirus, causes a maculopapular rash known as German measles, or three-day measles. The rash is similar but less red than that of measles (Fig. 26.4). German measles is an infection that primarily affects the skin and lymph nodes and is usually transmitted from
Several viruses can produce skin rashes, although their route of infection is usually through the respiratory tract. Measles, for example (see Section 6.1), is a highly contagious viral infection caused by a paramyxovirus, whose hallmark symptom is skin rash (Fig. 6.2). Also known as rubeola, the fi rst signs are fever, cough, runny nose, and red eyes occurring 9–12 days after exposure. A few days later, spots in the mouth appear (Koplik’s spots) along
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Peter Rowe, MD/DermAtlas
Viral Diseases Causing Skin Rashes
Figure 26.4
German measles.
Skin rash caused by rubella
virus.
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person to person by aerosolization of respiratory secretions. It is not dangerous in adults or children; the virus can, however, cross the placenta in a pregnant woman and infect her fetus. If the virus crosses the placenta within the fi rst trimester, the result is congenital rubella syndrome, which can cause death or serious congenital defects in the developing fetus. Other viruses affecting the skin, such as chickenpox, the related disease shingles, and smallpox, are discussed in Chapters 6 and 11 and included in Table 26.2.
the mucociliary elevator (described in Section 23.1), as will growth of the bacterium itself. Because a compromised mucociliary elevator makes it difficult to expel the microbe, the patient’s susceptibility to secondary bacterial infection increases. Hence, cold sufferers are advised to drink plenty of fluids, which help decrease the viscosity of mucus and consequently improve mucociliary elevator function.
TO SU M MAR I Z E:
In March, an 80-year-old resident of a New Jersey nursing home had a fever accompanied by a productive cough with brown sputum (mucous secretions of the lung that can be coughed up). He reported to the attending physician that he had pain on the right side of his chest and suffered from night sweats. Blood tests revealed that his white blood cell (WBC) count was 14,000/µl with a makeup of 77% segmented forms (polymorphonuclear leukocytes, PMNs) and 20% bands (immature PMNs). The chest radiograph revealed a right upper lobe infiltrate with cavity formation (Fig. 26.5A). From this information, the clinician made a diagnosis of pneumonia. Microscopic examination of the patient’s sputum revealed gram-positive cocci in pairs and short chains surrounded by a capsule (Fig. 26.5B). Bacteriological culture of his sputum and blood yielded Streptococcus pneumoniae.
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S. aureus and S. pyogenes are common bacterial causes of skin infections. The organisms usually infect through broken skin. Methicillin-resistant S. aureus (MRSA) has become an important cause of community-acquired staphylococcal infections. Necrotizing fasciitis is usually caused by S. pyogenes but can be the result of other infections. Infections of the skin can disseminate via the bloodstream to other sites in the body. Rubeola and Rubella viruses infect through the respiratory tract, but their main manifestation is the production of similar maculopapular skin rashes.
26.3 Respiratory Tract Infections Lung and upper respiratory tract infections are among the most common diseases of humans. Many different bacteria, viruses, and fungi are well adapted to grow in the lung. Successful lung pathogens come equipped with appropriate attachment mechanisms and countermeasures to avoid various lung defenses (such as alveolar macrophages). Although many microbes can cause lung infection, most respiratory diseases are of viral origin, and most viral infections (such as the common cold) do not spread beyond the lung. Fortunately, viral diseases by and large are self-limiting and typically resolve within two weeks; however, the damage caused by a primary viral infection can lead to secondary infections by bacteria. Bacterial infections of the lung, whether of primary or secondary etiology, require intervention. Today this means antibiotic therapy. Before the advent of antibiotics, the only recourse was to insert a tube into the patient’s back to drain fluid accumulating in the pleural cavity around the lung (a pathological process known as pleural effusion). Unless released, the pressure on the lung will collapse the alveoli and make breathing difficult. Bacterial infections of the lung that arise secondarily to viral disease occur, in part, because the patient dehydrates. The resulting increase in mucus viscosity hampers
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Case History: Bacterial Pneumonia
Note that pneumonia is a disease, not a specific infection. Many different microbes can cause pneumonia (Table 26.3). The pneumococcus S. pneumoniae accounts for about 25% of community-acquired cases of pneumonia, but pneumococcal pneumonia occurs mostly among the elderly and immunocompromised, including smokers, diabetics, and alcoholics. A breakdown of pneumonia cases by causative organism is shown in Figure 26.5C. The noses and throats of 30–70% of a given population can contain S. pneumoniae. The microbe can be spread from person to person by sneezing, coughing, or other close personal contact. Pneumococcal pneumonia may begin suddenly, with a severe shaking chill usually followed by high fever, cough, shortness of breath, rapid breathing, and chest pains. After being aspirated into the lung, the microbe will grow in the nutrient-rich edema fluid of the alveolar spaces. Neutrophils and alveolar macrophages then arrive to try to stop the infection. They are called into the area from the circulation by chemoattractant chemokines released by damaged alveolar cells. The thick polysaccharide capsule of the pneumococcus, however, makes phagocytosis very difficult. In an otherwise healthy adult, pneumococcal pneumonia usually involves one lobe of the lungs; thus, it is sometimes called lobar pneumonia. The infi ltration of PMNs and fluid lead to the typical radiological fi ndings of diffuse cloudy areas. In contrast, infants, young children,
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Courtesy Yale University School of Medicine
A. Lobar pneumonia
RUL infiltrate RML fissure
Bacteria PMN
LeBeau/CMSP
B. Streptococcus pneumoniae
C. Relative incidence of pneumonia Streptococcus pneumoniae Chlamydophila pneumoniae Viral Mycoplasma pneumoniae Legionella Haemophilus influenzae Gram-neg enteric bacteria Chlamydophila psittacii Coxiella burnetii Staph. aureus Moraxella catarrhalis Other
Pneumonia caused by Streptococcus pneumoniae. A. X-ray view of a patient with lobar pneumonia. Infiltration in the right upper lobe is caused by S. pneumoniae. The sharp lower border represents the upper boundary of the middle lobe fissure (arrow). B. Micrograph of S. pneumoniae. Sputum sample showing numerous PMNs and extracellular diplococci in pairs and short chains. Bacteria range from 0.5 µm to 1.2 µm in diameter. C. Relative incidence of pneumonia caused by various microorganisms.
Figure 26.5
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and elderly people more commonly develop an infection in other parts of the lungs, such as around the air vessels (bronchi), causing bronchopneumonia. The white cell count in the case history is telling. The patient had an elevated WBC count (normal is 5,000 to 10,000/µl) and elevated band cells (normal is 0–8%). These increases are indicative of a bacterial, not viral, infection. Neutrophils (PMNs), the front line combatants against infection, rise in response to bacterial infections and are first released from bone marrow as immature band cells, whose presence is a sure sign of bacterial infection. Several outbreaks of pneumococcal pneumonia have occurred over recent years in nursing homes, where numerous residents have been affected. This underscores the importance of elderly people receiving the pneumococcal polysaccharide vaccine (PPV) as a hedge against infection. While there are over 80 antigenic types of pneumococcal capsular polysaccharide, the injected vaccine contains only the 23 types that are most often associated with disease. A vaccine that is formulated to respond to multiple antigens is termed a multivalent vaccine. The immune response that results will protect vaccinated individuals against infection by those antigenic types. The vaccine is recommended for individuals over 65 as well as for those who are immunocompromised. The patient in this case history failed to receive the vaccine. In addition to causing serious infections of the lungs (pneumonia), S. pneumoniae can invade the bloodstream (bacteremia) and the covering of the brain (meningitis). The death rates for these infections are about one out of every 20 who get pneumococcal pneumonia, about two out of 10 who get bacteremia, and three out of 10 who get meningitis. Individuals with special health problems, such as liver disease, AIDS (caused by HIV), or organ transplants, are even more likely to die from the disease because of their compromised immune systems. An emerging infectious disease problem throughout the United States and the world is the increasing resistance of S. pneumoniae to antibiotics. At least 30% of the strains isolated are already resistant to penicillin, the former drug of choice for treating the disease. Chapter 27 discusses why antibiotic resistance is on the rise for this and other microbes.
Case History: Disseminated Disease from a Fungal Lung Infection A 35-year-old male boxer named Tyrrell, recently admitted to a Maryland hospital, was in good health until six months ago, when he developed a chronic cough that produced bloodtinged white sputum. He also experienced flu-like symptoms, a decrease in appetite, and weight loss. One month prior to admission, he became so short of breath that he could no longer continue boxing. At that time, an X-ray taken in the emergency
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room showed right upper lobe infiltrate, indicating pneumonia (Fig. 26.6A). A tuberculosis skin test was negative. He was given a prescription for the antibiotic azithromycin (a commonly used macrolide antibiotic to treat bacterial infections of the respiratory tract) and discharged. Despite antibiotic treatment, the cough never diminished and he developed several painless subcutaneous nodules. The largest nodule was on his left leg, contained pus, caused pain, and eventually hindered walking (Fig. 26.6B). This prompted his current admission to the hospital. The patient’s history revealed that he installed home insulation for a living and had not traveled outside the area for the past year. He complained of fevers, chills, night sweats, and a 10-kg (22-lb) weight loss. His right tibia was tender to the touch, indicating bone involvement. He denied having prior pneumonia, sinus infection, arthritis, hematuria (blood in the urine), numbness, or muscle weakness. He had no history of intravenous drug use and had been in a monogamous relationship for four years. His white cell count was 10,500/ mm3 with a normal differential of PMNs and band cells. In this case, the expression of frankly purulent material (pus) from the left leg nodule suggested that this was an infectious process. The infection in this patient probably started in the lung (clued by the cough), after which the organism disseminated throughout the body via the bloodstream. Fungus is a probable cause, given the chronic nature of the patient’s symptoms. Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, also causes a chronic lung infection and might have been suspected except for the negative TB skin test. The tuberculin skin test involves injecting a small amount of mycobacterial antigen called PPD (purified protein derivative) under the skin of the lower arm. A person who has been infected with M. tuberculosis will exhibit a localized delayed-type hypersensitivity reaction at the site of injection, although this does not equate to currently active disease. The most likely fungal causes of infection in this case history are the endemic mycoses, such as histoplasmosis, blastomycosis, and coccidiomycosis. This patient had never traveled to the western United States, where coccidioidomycosis is endemic. This ruled out exposure to Coccidioides. Histoplasmosis most commonly presents as a flu-like pulmonary illness, with erythema nodosum (tender bumps on skin) and arthritis (swollen joints) or arthralgias (joint pain), none of which the patient had. Blastomycosis can disseminate to the lung, skin, bone, and genitourinary tract, which is consistent with the pattern of organ involvement seen in this patient. Cryptococcus, an encapsulated yeast, is not the likely cause, since it typically requires an immunocompromised host to cause disease, not the case in this instance. (Cryptococcus, which causes cryptococcosis, is an opportunistic pathogen that commonly infects AIDS patients.) The most prevalent clinical form of cryptococcosis is meningoencephalitis, although
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A. Pneumonia infiltrate © 1998–2007. The Johns Hopkins University on Behalf of its Division of Rheumatology. All Rights Reserved
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B. Leg lesion © 1998–2007. The Johns Hopkins University on Behalf of its Division of Rheumatology. All Rights Reserved
Chapter 26
C. Colony of Blastomyces dermatitidis
Image Courtesy of Creighton University Department of Pathology
988
Pneumonia and metastatic disease caused by Blastomyces dermatitidis. A. Diffuse infiltrate in the right lung (arrow). B. Metastatic leg lesion at tibia. C. Fungal colony of Blastomyces dermatitidis.
Figure 26.6
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disease can also involve the skin, lungs, prostate gland, urinary tract, eyes, myocardium, bones, and joints. Amphotericin B, an antifungal agent, was fi nally given to this patient. His fever lowered almost immediately, and the skin nodules diminished. After two weeks, a fungus was found in the cultures of the nodule biopsy, bronchoalveolar lavage (washes), and urine (Fig. 26.6C). This fungus was identified by a DNA probe as Blastomyces dermatitidis, confi rming the diagnosis of blastomycosis. Blastomyces dermatitidis is a dimorphic fungus that resides in the soil of the Ohio and Mississippi River valleys and the southeastern United States. The portal of entry is the respiratory tract, and infection is usually associated with occupational and recreational activities in wooded areas along waterways, where there is moist soil with a high content of organic matter and spores. The incubation period ranges from 21 to 106 days. This patient most likely inhaled conidia (fungal spores) from the soil while crawling underneath houses installing insulation. The physician learned that he used only a T-shirt to cover his mouth and nose, not an effective method of keeping spores from entering the respiratory tract. He should have worn a respirator. Several critical features of this case help differentiate it from the preceding case of pneumococcal pneumonia. First, the initial macrolide antibiotic, azithromycin, should have killed most bacterial sources of infection. Second, the X-ray finding of diffuse infiltrate is more indicative of fungal lung infection than bacterial infection, which in a patient of this age would likely be confined to one lobe. The patient was young and in good health prior to the infection, making it unlikely to be pneumococcal pneumonia. The blood count was also a clue. Fungal infections do not usually cause an increase in WBCs or an increase in band cells. Finally, the metastatic lesions (infectious lesions that develop at a secondary site away from the initial site of infection) on the leg were in no way consistent with S. pneumoniae. They arise when the organism moves through the bloodstream from the primary site of infection to another body site, where it can begin to grow. Many infectious diseases start out as a localized infection but end up disseminating throughout the body to cause metastatic lesions.
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developed world. In 1985, however, owing primarily to the newly recognized HIV epidemic and a growing indigent population, TB resurfaced, especially in inner-city hospitals. In 1991, highly virulent multidrug-resistant (MDR) strains of M. tuberculosis were reported. These strains not only produced fulminant (rapid onset) and fatal disease among patients infected with HIV (the time between TB exposure to death is 2–7 months), but also proved highly infectious. Tuberculin skin test conversion rates of up to 50% are reported in exposed health care workers. A positive tuberculin skin test is seen as a delayed-type hypersensitivity reaction to M. tuberculosis proteins [called partially purified derivative (PPD)] injected under the skin. More information on mycobacteria can be found in Section 18.4. M. tuberculosis primarily causes a respiratory infection, but can disseminate through the bloodstream to produce abscesses in many different organ systems. Disseminated disease is called miliary TB because the size of the infected nodules, called tubercles, approximates the size of millet seeds. The organisms are spread from person to person (no animal reservoir) through aerosolization of respiratory secretions. Once in the lung, the bacteria are phagocytized by macrophages and survive ensconced within modified phagolysosomes. A delayedtype hypersensitivity response results, and small hard tubercles form. Over time, the tubercles develop into caseous lesions that have a cheese-like consistency and can calcify into the hardened Ghon complexes seen on typical X-ray fi ndings (Fig. 26.7).
CDC
NOTE: The term “metastasis” means to spread disease from one organ to another noncontiguous organ. Only microbial infections and malignant cancer cells can metastasize.
Tuberculosis as a Reemerging Disease Until recently, tuberculosis, caused by the acid-fast bacillus Mycobacterium tuberculosis, was considered of passing historical significance to physicians practicing in the
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Calcified Ghon complex of tuberculosis. The arrow points to a complex in a patient’s right upper lobe. Note the difference in appearance compared to Figure 26.6A.
Figure 26.7
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Table 26.3 Microbes causing respiratory tract diseases. Disease
Symptoms
Etiologic agent
Inhalation anthrax Diphtheria
Fever, muscle aches, hypotension, respiratory failure Tracheal pseudomembrane
Bacillus anthracis (G+ rod) Corynebacterium diphtheriae (G+ rod)
Whooping cough
Bordetella pertussis (G– coccobacilli)
Tuberculosis
Fever, runny nose, sneezing, violent cough followed by inhalation “whoop” Fever, chills, cough, bloody sputum, fatigue, weight loss
Pneumonia
Infects cystic fibrosis patients; fever, chills, cough, chest pain
Pseudomonas aeruginosa (G– rod)
Pneumonia
Fever, chills, cough, chest pain
Psittacosis
Fever, sore throat, chest pain, runny nose
Streptococcus pneumoniae (G+ diplococcus) Chlamydophila psittaci
Pneumonia
Chlamydophila pneumoniae
“Walking pneumonia”
Fever, sore throat, chest pain, runny nose (also associated with cardiovascular disease) Fever, sore throat, nonproductive cough, chills
Legionnaires’ disease (legionellosis)
Fever, chest pain, chills, cough, muscle pain, vomiting, diarrhea
Legionella pneumophila (G– rod)
CMV disease
Fever, chills, cough, chest pain
Cytomegalovirus (CMV); dsDNA
RSV disease
Fever, chills, cough, chest pain
Respiratory syncytial virus; ssRNA (–)
Influenza
Fever, chills, cough, chest pain, sore throat, muscle pain Sore throat, fever, runny nose
Influenza and parainfluenza viruses; ssRNA (–), segmented Adenovirus; dsDNA
Fever, chills, cough, chest pain
SARS virus; ssRNA (+)
Aspergillosis Histoplasmosis
Lung, sinuses, fever, chills, breathing difficulty Flu-like; pulmonary infiltrate
Aspergillus spp. Histoplasma capsulatum
Coccidioidomycosis Blastomycosis Pneumocystosis
Flu-like; pulmonary infiltrate Chest pain, cough, skin lesion, pulmonary infiltrate Infects AIDS patients; cough, fever, weight loss
Coccidioides immitis Blastomyces dermatitidis Pneumocystis jiroveci (formerly carinii )
Bacterial
Mycobacterium tuberculosis (acid-fast bacillus)
Mycoplasma pneumoniae (wall-less microbe)
Viral
Severe acute respiratory syndrome
Fungal
a
Bacillus Calmette-Guérin (a weakened strain of the bovine tuberculosis strain).
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Virulence properties
Source
Treatment
Vaccine
Peptide capsule, PA, LF, and EF toxins Diphtheria toxin
Airborne Airborne; localizes to nasopharynx Respiratory droplets Human
Ciprofloxacin Penicillin
+, Military +
Erythromycin
+ + BCGa
Multivalent from capsule antigens
Tracheal cytotoxin, adenylate cyclase toxin, filamentous hemagglutinin (adhesin) Cord factor, wax D Intracellular growth
Exotoxin A, phospholipase C, exopolysaccharide, others Capsule, pneumolysin
Water, soil
Combination therapy (rifampin, isoniazid, ethambutol, pyrazinamide) Quinolones, aminoglycosides
Inhalation
Macrolides, quinolones, ceftriaxone
Obligate intracellular growth; prevents phagolysosome fusion Intracellular, obligate; prevents phagolysosome fusion Adhesin tip
Bird droppings, dust; inhalation Person-to-person
Tetracycline, erythromycin
Intracellular growth, hemolysin, cytotoxin, protease
Inhalation, person-toperson Inhalation
Saliva, tears, breast milk Respiratory droplets Respiratory droplets Respiratory droplets Respiratory droplets
Inhalation Inhalation; bird, chicken, bat droppings Inhalation Inhalation Inhalation
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Quinolone Erythromycin and other macrolides
Erythromycin
Gangcyclovir
Experimental
Treat symptoms; ribovarin Relenza
+
Treat symptoms
+, Military
Amphotericin B, voriconazole Amphotericin B, itraconazole
Amphotericin B, fluconazole Amphotericin B, itraconazole Trimethoprim sulfamethoxazole
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After a period of 4–12 weeks, patients with disease exhibit a productive cough generating sputum and experience fever, night sweats, and weight loss. They also become tuberculin test positive owing to delayed-type hypersensitivity. However, it is important to note that a positive tuberculin skin test does not signify active disease, only that the person was infected at one time. The bacterium may have been killed by the immune system without having caused disease. Treatment of active disease is aggressive and involves a four-drug regimen including isoniazid, rifampin, pyrazinamide, and ethambutol given over a course of several months. MDR strains are treated with a nine-drug regimen.
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Viral Diseases of the Lung Numerous viruses can cause lung infections (see Table 26.3). Influenza, rhinovirus, and SARS are discussed in Chapters 6, 11, and 25. An important viral lung infection not discussed elsewhere is respiratory syncytial disease, caused by respiratory syncytial virus (RSV). A negative-sense, single-strand RNA, enveloped virus, RSV is the most common cause of bronchiolitis and pneumonia among infants and children under 1 year of age. Illness begins most frequently with fever, runny nose, cough, and sometimes wheezing. RSV is spread from respiratory secretions through close contact with infected persons or by contact with contaminated surfaces or objects. Infection can occur when the virus contacts mucous membranes of the eyes, mouth, or nose and possibly through the inhalation of droplets generated by a sneeze or cough. Unlike rubella or rubeola, which infect the respiratory tract and disseminate though the body, RSV remains localized in the lung. The majority of children hospitalized for RSV infection are under 6 months of age. RSV can cause repeated infections throughout life, usually associated with moderateto-severe cold-like symptoms. Severe lower respiratory tract disease may occur at any age, especially among the elderly or people with compromised cardiac, pulmonary, or immune systems. As yet, a vaccine to control this disease is not available. Table 26.3 presents many other bacterial, fungal, and viral microbes that can cause respiratory tract infection. Be aware that very different diseases can produce similar symptoms. For instance, people constantly confuse influenza (the flu) with the common cold. Symptomatically, they may start out similarly, but there are telling differences. Influenza is characterized by fever, myalgia (muscle aches), pharyngitis (sore throat), and headache. A runny nose is not one of the symptoms. The common cold, however, manifests as a runny nose, nasal congestion, sneezing, and throat irritation. No myalgia. The observant clinician will note the difference.
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The mucociliary elevator is a primary defense mechanism used by the lung to avoid infection. Pneumonia is a disease that can be caused by many microorganisms. An elevated white cell count in blood is an indicator of bacterial infection. Pneumococcal vaccine should be administered to the elderly because they are often immunocompromised. Fungal agents commonly cause long-term, chronic infections. Tuberculosis is an ancient yet reemerging bacterial disease with an increasing mortality rate caused by the development of multidrug-resistant strains, the susceptibility of HIV patients, and an increasing indigent population. Localized infections in the lung can disseminate via the bloodstream to form metastatic lesions at other body sites. Respiratory syncytial virus is one of several viruses that can cause lung disease but rarely disseminates.
26.4 Gastrointestinal Tract Infections Nearly everyone has experienced diarrhea—a condition characterized by frequent loose bowel movements accompanied by abdominal cramps. Hundreds of millions of cases occur each year in the United States. The loose stools usually result from inflammation (called gastroenteritis) due to viral growth, bacterial growth, or toxin production, causing large amounts of water and electrolytes to leave the intestinal cells and enter the intestinal lumen. As a result, the patient not only suffers diarrhea, but can become dangerously dehydrated. As with respiratory tract infections, most diarrheal disease is viral in origin, with rotavirus being the primary culprit. Among the bacteria, the gram-negative curved bacillus Campylobacter is the most frequent cause of self-limiting diarrheal disease. The main symptoms of gastroenteritis, whether bacterial or viral, are watery diarrhea and vomiting. A more severe form of gastroenteritis is called dysentery, whose symptoms include abdominal pain, a persistent desire to empty the bowels, and diarrhea with passage of blood or mucus. Bacterial causes of dysentery include several Shigella species and some strains of E. coli. An ameba, Entamoeba histolytica, can also cause a form of the disease called amebic dysentery. Features of bacterial dysentery are discussed in more detail later in this section. Remarkably, Staphylococcus aureus causes gastrointestinal disease without ever producing infection. Everyone has heard of the local church picnic where scores of people
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became violently ill within hours of eating unrefrigerated potato salad. S. aureus is the usual cause of these disasters. Not all strains of S. aureus cause food poisoning, but certain strains can secrete enterotoxins into tainted foods such as pies, turkey dressing, or potato salad. After ingestion, the toxin travels to the intestine, where it enters the bloodstream and stimulates nerves leading to the vomit center in the brain. Because the toxin is preformed, symptoms occur quickly after ingestion. Within 2–6 hours, the poisoned patient will begin vomiting and may also experience diarrhea. The disease, though violent, is not lifethreatening and usually resolves spontaneously within 24–48 hours. In contrast, diarrhea caused by infectious agents, such as Salmonella enterica, that must fi rst grow in the victim do not occur until 12–24 hours after ingestion, sometimes longer. A clinician noting quick onset of symptoms in a patient will immediately suspect staphylococcal food poisoning. Obviously, antibiotic treatment is not needed for staph food poisoning but may be indicated for other gastrointestinal infections.
Antibiotics Are Often Inappropriate When Treating Gastroenteritis Although antibiotic treatment of infectious gastroenteritis seems intuitive, it is rarely used. Most gastrointestinal infections are viral, and so antibiotics are ineffective. Likewise, gastroenteritis caused by bacteria usually resolves spontaneously without antibiotic treatment. Of course, severe systemic disease stemming from gastroenteritis can develop, often in the young or elderly. Diseases such as typhoid fever (Salmonella typhi) or bacillary dysentery (Shigella dysenteriae) will respond well to antibiotics. In some cases, antibiotic treatment can actually trigger gastrointestinal disease. For example, the antibiotic clindamycin can kill most normal intestinal flora except the naturally resistant gram-positive anaerobe Clostridium difficile, the causative agent of pseudomembranous enterocolitis. Unrestrained by microbial competition, C. difficile will grow in the intestine and produce specific toxins that can damage intestinal cells. The organism’s growth leads to inflammation and the formation of pseudomembrane structures along the intestinal wall (refer to Fig. 23.8). Because they block the intestinal mucosa, pseudomembranes cause malabsorption of nutrients and water, which results in diarrhea. As the pseudomembrane enlarges, it begins to slough off and pass into the stool. Diagnosis of this disease involves PCR identification of the organism or immunological identification of the toxin in fecal samples.
Case History: Diarrhea and Dysentery In April, a 6-year-old girl from Montgomery County, Pennsylvania, arrived at the ER with bloody diarrhea, a temperature of
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39°C, abdominal cramping, and vomiting. Hospital admission was five days after a kindergarten field trip to the local dairy farm. The child’s health history was otherwise unremarkable. At the time of hospital admission, the parents were questioned as to the child’s activities during the trip. They confirmed that the child purchased a snack while at the farm. Upon laboratory analysis, a fecal smear was positive for leukocytes, and isolation of organisms confirmed the presence of gram-negative rods that produced Shiga toxins 1 and 2. Subsequent testing of the isolate by pulsedfield gel electrophoresis was indistinguishable from E. coli O157: H7. By this time, the child developed further problems. Symptoms included puffy face and hands as well as neurological abnormalities. Initial examination suggested renal failure, and laboratory analyses supported this diagnosis with thrombocytopenia (reduced blood platelet count) and hemolytic uremic syndrome (HUS; renal failure). The child was treated by fluid and electrolyte replacement (intravenous). Antibiotics were not administered. In this case history, the presence of leukocytes in a fecal smear is a sign that the intestinal pathogen has invaded the epithelial mucosa of the intestine. Breaching this barrier sends out a chemical call to neutrophils, which then enter the area. Shigella dysenteriae, Salmonella enterica, and enteroinvasive E. coli (EIEC) are invasive; because they actually enter enterocytes, they are considered intracellular pathogens. Enterohemorrhagic E. coli (EHEC), which also produces leukocytes and blood in stools, is not an intracellular parasite (it is not invasive), but causes damaging attachment and effacing lesions, described in Section 25.2, that causes destruction of the mucosal epithelium. The resulting inflammation, in conjunction with damage caused by Shiga toxin, leads to blood and white cells in the stool. E. coli O157:H7, the etiological agent in the case history, is a common serotype of EHEC. There are at least six different classes of pathogenic E. coli that differ based on their repertoire of pathogenicity islands, plasmids, and virulence factors. They include EIEC and EHEC, already mentioned, enterotoxigenic E. coli (ETEC) and uropathogenic E. coli, (UPEC), both described in Chapter 25, as well as enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC). All but UPEC cause gastrointestinal disease. To tell them apart, each group has telltale O and H antigens that can be identified using serology. NOTE: “O antigen” is part of the bacterium’s LPS, while “H antigen” is flagellar protein. Thus, O157:H7 denotes the specific version of LPS (O157) and flagellar protein (H7) found on E. coli O157:H7. Other pathogenic strains of E. coli have different O and H antigens.
Shiga toxin. Shigella and EHEC, the agent in the preced-
ing case history, both produce toxins called Shiga toxin 1 and 2 that are encoded by genes of bacteriophage genomes
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embedded in the bacterial chromosome. These toxins inhibit host protein synthesis and, in the process, damage endothelial cells in the kidney and brain. Endothelial damage triggers the formation of platelet-fibrin microthrombi (clots) that occlude blood vessels in the various organs, leading to two major syndromes—hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TPP). HUS occurs when the microthrombi are limited to the kidney. The microclots clog the tiny blood vessels in this organ and cause decreased urine output, ultimately leading to kidney failure and death. In TPP, the clots occur throughout the circulation. This will cause reddish skin hemorrhages called petichiae and purpura. Neurological symptoms (for example, confusion, severe headaches, possibly coma) then arise from microhemorrhages in the brain. The hemorrhaging occurs because platelets needed for normal clotting have been removed from the circulation as they form the microthrombi. The decreased number of platelets is called thrombocytopenia. The toxins, which are absorbed through the intestine and disseminated via the bloodstream, have five B subunits used to bind to target cell membranes and one A subunit imbued with toxic activity (see Section 25.4). The A subunit, upon entry, destroys protein synthesis by cleaving an adenine from 28S rRNA in eukaryotic ribosomes. Enterohemorrhagic E. coli (EHEC). E. coli O157:H7 is a
recently emerged pathogen that can colonize cattle intestines without causing disease and as a result can contaminate meat products following slaughter. The organism is sometimes referred to as the “Jack-in-the Box” microbe, a reference to the fi rst documented large-scale U.S. outbreak in 1993, linked to fast-food hamburgers purchased at a Washington state Jack-in-the-Box restaurant. As many as 600 people were sickened in that outbreak, and 3 children died. Though 1993 marked the fi rst large-scale outbreak in the United States, the fi rst report of the disease in this country occurred 11 years earlier, in 1982. E. coli O157:H7 rarely affects the health of the reservoir animal. But when an infected steer is slaughtered, fecal contamination of the carcass can happen, despite manufacturers’ considerable efforts to prevent it. Grinding the tainted meat into hamburger will distribute the microbe throughout. As a result, cooking burgers to 160°C is essential to kill any existing EHEC. Also be aware that cross-contamination between foods is possible. Using the same cutting board to prepare meat and salad is a great way to contaminate the salad, which will not be cooked. Despite its common association with hamburger, vegetarians are not safe from this organism. During heavy rains, waste from a cattle farm can easily wash into nearby vegetable fields unless precautions are taken. If the cattle waste contains E. coli O157:H7, the crops are contaminated. One such outbreak occurred in 2006, when spinach from certain areas of California became contami-
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nated with this pathogen, prompting a nationwide recall of bagged spinach and a month without spinach salad. Early on, the remarkably low infectious dose of E. coli O157:H7 mystified researchers. However, we have since learned that E. coli has an impressive level of acid resistance that rivals that of the gastric pathogen Helicobacter pylori. Acid resistance mechanisms permit survival of E. coli in the acidic stomach and enable a mere 10–100 individual organisms to cause disease. As already noted, EHEC strains produce two toxins that are identical to the Shiga toxins produced by Shigella species. The toxins cleave host ribosomal RNA, thereby halting translation. As discussed earlier, one consequence of Shiga toxin is HUS. The development of HUS, as in the case history described, is a common consequence of E. coli O157:H7 infection. Unfortunately, HUS can only be treated with supportive care, such as blood transfusions and dialysis throughout the critical period until kidney function resumes. Antibiotic treatment can increase the release of Shiga toxins from the organisms and actually trigger HUS. Thus, antimicrobial therapy is not recommended. In contrast to the case just described, many gastrointestinal infections do not produce fecal leukocytes or blood in the stool. Diarrheal diseases caused by V. cholerae (cholera) or enterotoxigenic E. coli (ETEC), which produces a cholera-like disease, do not involve invasion of the intestinal lining by the microbe and yield copious amounts of watery diarrhea. In these two toxin-driven diseases, the bacteria attach to cells lining the intestine and secrete toxins that are imported into the target cells (see Section 25.4). Epidemiology of EHEC. Epidemiology is the study of factors and mechanisms involved in the spread of disease. The father of epidemiology was the British physician John Snow, who, even before there was a clear connection between bacteria and disease, managed to trace the source of a cholera epidemic in London in 1854 to a single well in the city. He found that the addresses of all the cholera patients clustered around just one of the city’s numerous water pumps that had, as it turns out, been contaminated with human feces. Sealing the well ended the epidemic. The U.S. Centers for Disease Control (CDC) used the same basic strategy to identify the risk factors associated with the case study presented earlier. Fifty-one infected patients and 92 controls (children who visited the farm but did not become ill) were interviewed. Infected patients were more likely than controls to have had contact with cattle, an important reservoir for E. coli O157:H7. All 216 cattle on the farm were sampled by rectal swab, and 13% yielded E. coli O157:H7 with a DNA restriction pattern indistinguishable from that isolated from the patients. This finding indicated that the cattle were the source of infection. Activities that promoted hand-mouth contact, such as nail-biting and purchasing food from an outdoor
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concession were more common among the children that contracted disease (fecal-oral route of infection). Furthermore, separate areas were not established for eating and interactions with farm animals. Visitors could touch cattle, calves, sheep, goats, llamas, chickens, and a pig while eating and drinking. Hand-washing facilities were unsupervised and lacked soap, and disposable hand towels were out of the children’s reach. All of these situations provided opportunity for infection. Type III secretion and diarrhea. Type III secretion was fi rst described in Section 25.5, where we discussed the pathogenesis of Salmonella enterica, but these secretion systems are present in numerous gram-negative pathogens, such as EHEC in our case history. Recall that type III protein secretion systems directly inject proteins from the cytoplasm of a bacterial pathogen into the cytoplasm of a target eukaryotic host cell. The system delivers proteins across three membranes—two for the gram-negative bacterial pathogen and one for the target cell. In addition to stimulating bacterial entry into host cells, bacterial proteins injected by type III transport systems cause host cells to secrete pro-inflammatory cytokines. The cytokines then “call in” inflammatory cells and alter ion transport through the epithelial membrane. Excessive export of ions such as chloride causes water to leave the cell in an attempt to equilibrate the internal and external ionic concentrations. The water entering the intestine results in diarrhea. We are just beginning to understand how the type III–translocated effector proteins induce these host cellular responses and how this leads to intestinal inflammation during an infection.
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Although most gastrointestinal disease is intestinal in locale, specialized microbes can also target the stomach, with its harsh acidic environment.
Case History: Ulcers−It’s Not What You Eat Gary is a 34-year-old accountant who immigrated to Nebraska from Poland seven years ago. Since his teenage years, he has been bothered periodically by episodes of epigastric pain (pain around the stomach), nausea, and heartburn. Antacids usually alleviated the symptoms. Over the years, he received several courses of treatment with Tagamet or Pepcid to reduce acid secretion and provide relief. Recently, an upper-GI endoscopy was performed, in which a long, thin tube tipped with a camera and light source was inserted into Gary’s mouth and into his stomach. The view through the endoscope showed some reddened areas in the antrum (bottom part) of the stomach. The endoscope was also equipped with a small clawlike structure that obtained a small tissue sample from the lining of Gary’s stomach. A urease test performed on the antral biopsy turned positive in 20 minutes. Histological examination of the biopsy confirmed moderate chronic active gastritis (inflammation of the stomach lining) and revealed the presence of numerous spiral-shaped organisms. Cultures of the antral biopsy were positive for H. pylori.
Painful and sometimes life-threatening gastric ulcers were for many years blamed on spicy foods and stress. These factors were believed to cause increased acid production that ate away at the stomach lining, even though the gastric mucosa is normally well protected from stomach acid, which can fall as low as pH 1.5. This argued against the model but was ignored. In the 1980s, based on their discovery of odd, helical-shaped bacteria present in the biopsies of gastric B. ulcers, Australians Robin Warren H. pylori and Barry Marshall (a medical intern at the Royal Perth Hospital at the time; Fig. 26.8A) proposed that bacteria, not pepperoni, caused ulcers (Fig. 26.8B). Their hypothesis was viewed with skepticism and declared as heresy by the established medical community. Faced with disbelief bordering on ridicule, the young intern drank a vial of the helical organisms and waited. A week later he began vomiting and suffered other painful symptoms of gastritis. Barry Marshall Figure 26.8 A bacterial cause of could not have been happier. He had gastric ulcers. A. Australian physician proved his point. We now know that Barry Marshall was so sure he was right this curly-shaped microbe causes the about the cause of stomach ulcers, he vast majority of stomach ulcers. swallowed bacteria to prove his point. The discovery of Helicobacter B. View of H. pylori in stomach crypts pylori and its association with gas(colorized SEM). Bacteria are approximately tric ulcer disease led to an upheaval 2 µm in length.
Courtesy of Barry Marshall
CNRI/Photo Researchers, Inc.
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in gastroenterology. Prior to this discovery, treatment focused on suppressing acid production, which did not provide long-term relief. Within one year after acidsuppressive therapy, up to 80% of patients suffer a relapse of their ulcer. Therapy now includes antimicrobial treatment to kill the bacteria and acid suppression therapy to prevent further inflammation while the ulcer heals. Warren and Marshall, who recovered from his gastritis, received the 2005 Nobel Prize in Physiology or Medicine for their groundbreaking work. The exact mechanism by which the organism causes gastric ulcers is not known, although a variety of virulence factors have been described (see Section 25.6). The urease enzyme of H. pylori is a characteristic virulence feature. Urease converts urea, produced by the gastric lining, to ammonia and carbon dioxide. The ammonia neutralizes acid in and around the organism and allows the microbe to survive in the stomach. In an animal model, the urease enzyme of H. pylori was found to be important for bacterial colonization in the stomach. Treatment with urease inhibitors such as acetohydroxamic acid, however, does not lead to eradication of H. pylori. This failure is likely due to protection afforded the organism once it reaches the mucous layer that blankets the gastric epithelium (see Fig. 26.8B). The pH of this environment is closer to neutral, so urease may no longer be needed. Tools useful for diagnosing H. pylori include histology, rapid urease testing, and serology (for example, enzyme-linked immunosorbent assay (ELISA) to detect antibody to the CagA antigen). Enzyme-linked immunosorbent assay (ELISA). ELISA is a common immunological tool to detect the presence, in serum, of antibodies to a specific organism—an indication of infection. The assay is performed by coating wells in a plastic dish with an antigen (for example, Helicobacter CagA). Serum from the patient is then added to the well. If antibodies to CagA are present, they will bind to the CagA antigen. Unbound antibody is removed by washing, and a secondary antibody that binds human IgG is added to the well. These anti-antibodies have an enzyme linked to them. A sandwich is formed as follows: [plastic dish]–[CagA protein]–[anti-CagA antibody]–[anti-IgG antibody]–[enzyme]. When the appropriate enzyme substrate is added to the well, the enzyme acts on it to produce light or a chromogenic product that can be detected. The more anti-CagA antibody present in the serum, the more light or product is produced by the linked enzyme. Applications of ELISA and further discussion of the methodology involved are described in Section 28.2. H. pylori infection, cancer, and bad breath. Problems
caused by H. pylori are not limited to gastric ulcers. The microbe has also been associated with gastric cancer. The evidence is not yet conclusive, but many individuals with
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gastric cancer are also colonized by H. pylori. The most compelling experimental proof is that gerbils infected with this organism develop gastric cancer. In addition, when the CagA protein of H. pylori is injected into gastric epithelial cells, it is phosphorylated on a tyrosine residue and activates a regulatory cascade that causes the gastric cell to proliferate (Fig. 25.33). The connection between H. pylori and gastric cancer is sobering when you consider that H. pylori can be detected in about two-thirds of the world’s population, especially in impoverished countries. There is also the unusual case of a patient with a 60year history of halitosis (extreme bad breath) that was resistant to all standard therapies but was cured by a triple-drug therapy regimen used for H. pylori. This case is an illustration of how microorganisms can cooperate to cause disease. H. pylori did not cause the smell. It appears that the patient did not produce much stomach acid. As a result, the urease produced by H. pylori easily neutralized the remaining stomach acid, allowing anaerobes ingested with food to start putrefying the stomach contents, leading to the foul-smelling breath. Other examples of disease caused by microbial cooperation will be described later.
Rotavirus Is the Single Greatest Cause of Gastroenteritis Many people wrongly think that most cases of diarrhea are caused by a bacterial agent. Actually, a virus, rotavirus, causes more intestinal disease than any bacterial species. It is highly infectious; it is endemic around the globe; and it affects all age-groups, although children between 6 and 24 months are most severely affected. Spread by the fecal-oral route, it is estimated that by age 3, all children have had a rotavirus infection. The incubation period is approximately two days, after which the victim suffers frequent watery, dark green, explosive diarrhea. All of this may be accompanied by nausea, vomiting, and abdominal cramping. Severe dehydration and electrolyte loss due to the diarrhea will cause death unless supportive measures, such as fluid replacement, are undertaken. There is no cure, but most patients recover if rehydrated properly. Few deaths from rotavirus occur in the United States, but each year more than 600,000 children worldwide die from this viral diarrhea, which is why efforts to develop a safe, effective vaccine are so important. Success in this endeavor seems close at hand.
Protists (Protozoa) Are Another Major Cause of Diarrheal Diseases Most students of biology are familiar with protozoa (more properly called protists) such as paramecia and amebas, but many are surprised to learn that some species of protists cause serious human diseases. For instance,
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A.
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B.
Entamoeba histolytica and Cryptosporidium parvum cause the diarrheal diseases amebic dysentery and cryptosporidiosis, respectively. In 2005, the CDC tallied 2,640 cases of cryptosporidiosis, a reportable disease in the United States. Two other amebas, Nagleria and Acanthamoeba, cause amebic menigoencephalitis. Because some species of Acanthamoeba can infect the eye, soft contact wearers should take precautions to prevent contamination of their lenses. The flagellated protozoan Giardia lamblia is a major cause of diarrhea throughout the world. In the United States alone, G. lamblia was responsible for over 11,000 reported cases of giardiasis diarrhea in 2005 and likely caused thousands more that were not reported. Giardia lamblia enters a human or other host as a cyst present in drinking water contaminated by feces (Fig. 26.9A). Aside from humans, G. lamblia can be found in various rodents, deer, cattle, and even household pets. It is very infectious. Ingestion of as few as 25 cysts can lead to disease. Following ingestion, the hard, outer coating of the cyst is dissolved by the action of digestive juices to produce a trophozoite, which attaches itself to the wall of the small intestines and reproduces (Fig. 26.9B). Offspring quickly encyst and are excreted out of the host’s body. Asymptomatic carriers of G. lamblia are common—it has been estimated that anywhere from 1 to 30% of children in U.S. day-care centers are carriers. Disease usually manifests as greasy stools alternating between a watery diarrhea, loose stools, and constipation. However, some patients will experience explosive diarrhea. Diagnosis is usually done by observing the cysts or trophozoite forms of the protozoan in feces. Metronidazole is a drug often used to cure the disease. As for prevention, proper treatment of community water supplies is essential. We have examined only a handful of the microbes that cause gastrointestinal infection. Others are presented in Table 26.4.
Oliver Meckes/Nicole Ottawa/Photo Researchers
Giardia lamblia cyst
Fecal flora
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Giardia lamblia. This protist is a major cause of diarrhea in the world. A. Cysts (7 to 14 µm) present in fecal matter (colorized SEM). B. Trophozoite form (colorized SEM, 5 to 15 µm in length).
Figure 26.9
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Diarrhea leads to dehydration, for which fluid replacement is a critical treatment. Antibiotic treatment is usually not recommended. Staphylococcal food poisoning is not an infection. It is a toxigenic disease. Antibiotic treatments can sometimes cause gastrointestinal disease (for example, pseudomembranous enterocolitis by Clostridium difficile). Bacteria that invade intestinal epithelial mucosal cells lead to the presence of blood and white blood cells in fecal contents. This occurs with intracellular pathogens such as Shigella, Salmonella, and EIEC. Bacteria that do not invade intestinal cells usually produce watery diarrhea. EHEC is an exception because the attachment and effacing lesions it produces result in bloody stools. Bacterial toxins produced by bacterial enteric pathogens can cause systemic symptoms. John Snow founded the science of epidemiology while studying a cholera outbreak in London. The bacterium H. pylori, a common cause of gastric ulcers, lives in the stomach and is highly acid resistant. Rotavirus is the single greatest cause of diarrhea worldwide. Giardia lamblia is a major protozoan cause of diarrhea worldwide.
26.5 Genitourinary Tract Infections Although the genital and urinary tracts are different organ systems, their close association in the body has led them to be grouped together when discussing infections.
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Table 26.4 Microbes causing diseases of the gastrointestinal tract. Disease
Symptoms
Etiologic agent
Symptoms within 4 h of ingestion; nausea, vomiting, diarrhea Symptoms begin quickly; flaccid paralysis
Staphylococcus. aureus (G+)
Virulence factors
Source
Treatment
Enterotoxin
Preformed toxin in foods
Supportive
Clostridium botulinum (G+, anaerobe) Salmonella enterica [gram negative (G–)]
Neurotoxin
Preformed toxin in food
Antiserum
Type III secretion, intracellular growth
Chickens, other animals; fecal-oral
Oral rehydration; antibiotics if severe
Salmonella typhi (G–)
Type III secretion, intracellular growth, PhoPQ regulators, Vi antigen capsule Labile and stable toxins Intimin, Tir, type III secretion, Shiga toxin
Human carriers (gall bladder reservoir), food, water
Quinolones
Humans; food, water Contaminated foods (hamburger) and crops Human fecal-oral route
Oral rehydration
Bacterial Staphylococcal food poisoning Botulism
Salmonellosis
Traveler’s diarrhea
Symptoms after 18 h; abdominal pain, diarrhea; invade intestinal M cells Headache, fever, chills, abdominal pain, rash (rose spots), hypotension, diarrhea in late stages Watery diarrhea
Gastroenteritis
Bloody diarrhea, HUS
Shigellosis
Bloody diarrhea, HUS
Cholera
Watery diarrhea
Gastroenteritis
Diarrhea, blood in stool
Gastroenteritis
Watery diarrhea, nausea
Pseudomembranous enterocolitis
Fever, abdominal pain, diarrhea, pseudomembrane in colon Fever, muscle pain, watery diarrhea, blood in stool, headache Abdominal pain, bleeding, heartburn
Typhoid fever
Gastroenteritis
Gastric ulcers
Enterotoxigenic Escherichia coli Enterohemorrhagic E. coli
Shigella spp. (G–)
Shiga toxin, type III secretion, intracellular growth, actin-based motility, escape phagosome Vibrio cholerae (G–) Cholera toxin, TCP pili, ToxR regulator V. parahaemolyticus Enterotoxin (G–) Clostridium Alpha toxin perfringens (G+) C. difficile (G+) Cytotoxin, antibiotic resistance Campylobacter jejuni (G–) Helicobacter pylori (G–)
Oral rehydration; antibiotics if severe Oral rehydration; antibiotics if severe
Human wastecontaminated water Raw seafood
Oral rehydration, antibiotics
Soil, food
Self-limiting
Animals, normal flora
Vancomycin
Self-limiting
Cytotoxin, Poultry, enterotoxin, unpasteurized adhesin milk Adhesin, urease ?? CagA, vacuolating toxin
Erythromycin
??
Fecal-oral route
Oral rehydration
??
Fecal-oral
Oral rehydration
Triple drug (omeprazole, clarithromycin, metronidazole)
Viral Stomach “flu”
Nausea, vomiting, diarrhea (Most common cause)
Noroviruses (Norwalk virus) Rotavirus
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Urinary Tract Infections The urinary tract includes the kidneys, ureters, urinary bladder, and urethra. Infections anywhere along this route are called urinary tract infections (UTIs). Urinary tract infections are the second most common type of bacterial infection in humans, ranking in frequency just behind respiratory infections such as bronchitis or pneumonia. Along with numerous office visits, bladder infections and other urinary tract infections result in 100,000 hospital admissions and $1.6 billion in medical expenses each year. Most sufferers are women. One in five women will get a urinary tract infection at some point in her life, and 20– 40% of those infected will develop recurrent infections. Urine, as produced in the kidneys and stored in the bladder, is normally sterile. Microorganisms must be introduced into the bladder to cause infection. Active infection of the urinary tract occurs in one of three basic ways: ■
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Descending infection from the kidney. Descending infection occurs when an infected kidney sheds bacteria that descend via the ureters into the bladder. Kidney infections arise when microorganisms are deposited in the kidneys from the bloodstream. Ascending infection to the kidney. This is when an established infection in the bladder ascends along the ureter to infect the kidney. Infection from the urethra to the bladder. Bacteria residing along the superficial urogenital membranes of the urethra can ascend to the bladder. This is more common in women than men. Microorganisms can also be introduced into the bladder by means of mechanical devices such as catheters or cystoscopes that are passed through the urethra into the bladder.
THOUGHT QUESTION 26.2 Why do you think most urinary tract infections occur in women? Urine is bacteriostatic to most of the commensal organisms inhabiting the perineum and vagina, such as Lactobacillus, Corynebacterium, diphtheroids, and Staphylococcus epidermidis. In contrast, many gram-negative organisms thrive in urine. As a result, most urinary tract infections are caused by facultative gram-negative rods from the GI tract. The most common etiological agents of UTI are: ■
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Certain serotypes of E. coli that comprise the uropathogenic E. coli (75% of all UTI) Klebsiella, Proteus, Pseudomonas, Enterobacter (20%) S. aureus, Enterococcus, Chlamydia, fungi, Staphylococcus saprophiticus, other (5%)
Case History: Classic Urinary Tract Infection Lashandra is 24 years old and has been experiencing back pain, increased frequency of urination, and dysuria (painful urination)
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over the past three days. She consulted her general practitioner, who requested that a mid-stream specimen of urine be examined. This was the first time Lashandra had ever suffered from persisting dysuria. Upon microscopic examination, the urine was found to contain more than 50 leukocytes per µl and 35 red blood cells per µl. No epithelial squamous cells were seen. The urine culture plated on agar medium yielded more than 105 colonies per milliliter (meaning more than 105 organisms per milliliter in the urine) of a facultative anaerobic gram-negative bacillus capable of fermenting lactose. The fi rst question to ask in this case is whether the patient had a significant UTI. The purpose of the midstream urine collection is to provide laboratory data to make this determination. Even though urine in the bladder is normally sterile, urine becomes contaminated with normal flora that have adhered to the urethral wall. In a mid-stream collection, the patient urinates briefly, stops to position a collection jar, and resumes urinating to collect a midstream sample. This minimizes the number of organisms in the sample by washing away organisms clinging to the urethra before actually collecting the sample. Nevertheless, the collected sample will still contain low numbers of organisms representing normal flora of the urethra. A diagnosis of UTI is made when the number of bacteria in the sample becomes greater than 105/ml. (However, that number can be as low as 1,000/ml in symptomatic women.) The patient in the case history is symptomatic and has sufficient numbers of bacteria in her urine to indicate a UTI. The laboratory found the organism to be a gramnegative bacillus that ferments lactose, suggesting E. coli as the likely culprit. Given that the normal habitat of E. coli is the gastrointestinal tract and this is the fi rst UTI suffered by the patient, the infection is likely the result of an inadvertent introduction of the microbe into the urethra. The organism makes its way up the urethra and into the bladder. If the patient had a recurring UTI, it could result from organisms entering the bladder from a descending route along the ureter from an infected kidney (also see Special Topic 26.1). Gram-negative rods not only thrive in urine, but may be adapted to cause urinary tract infections through specialized pili. These pili have terminal receptors for glycolipids and glycoproteins present on urinary tract epithelial cells (Fig. 26.10A). Uropathogenic strains of E. coli, for example, typically have P-type pili, with a terminal receptor for the P antigen (a rather appropriate name for a bladderspecific virulence factor). The P antigen is a blood group marker found on the surface of cells lining the perineum and urinary tract; it is expressed by approximately 75% of the population. These individuals are particularly susceptible to UTIs. The P antigen can also be shed and found in vaginal and prostate secretions. When this happens, the secreted P antigens can be protective by acting as a decoy.
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Special Topic 26.1
Intracellular Biofilm Pods Are Reservoirs of Infection
Courtesy of Scott Hultgren
Gregory G. Anderson, et al. 2003. Science 301:105
Gregory G. Anderson, et al. 2003. Science 301:105
Gregory G. Anderson, et al. 2003. Science 301:105
Doctors used to think that women suffering from recurrent bladder infections were repeatedly infected as a result of sexual activity or poor hygiene, a presumption many women with chronic infections found frustratA. ing and offensive. New evidence indicates that bacteria may actually hide inside bladder cells, forming a reservoir for reinfection. Specialized colonies of E. coli have been found living in the surface layer of cells lining the bladders of infected mice (Fig. 1A). Though the mouse served as a model system, the bacteria used were 50 µm 5 µm 1 µm originally isolated from humans. These strains of E. coli cause about 80% of all B. urinary tract infections. The bacteria use pili to latch onto proteins coating the bladder cells. Those proteins form a protective substance, known as uroplakin, that strengthens the epithelial cells and shields them from toxins that may build up in the urine (Fig. 1B). The bacteria can slip C. D. under the protective barrier and penetrate superficial cells lining the bladder. Once inside the cell, the microbes begin to multiply, and the epithelial cell fills with bacteria also coated with uroplakin, taking on the appearance of a pod (Fig. 1C). The pod-like E. coli biofilms we 20 µm describe here are notoriously resistant to antibiotic treatment and attacks by the immune system. Living inside host cells and surrounded by uroplakin, the E. coli in the bladder pods have devised a clever mechanism for surviving. Bacteria living on the edges of the biofilm may break free, leading to subsequent rounds of infection. Understanding the cycle of infection and pod for20 µm mation may help researchers design drugs to prevent new urinary tract infections or Figure 1 Bladder pods of uropathogenic E. coli. Scott Hultgren’s laboratory stop established ones. (Washington University, St. Louis) has discovered that intracellular bacterial communities Although pods have so far been extend like pods into the bladder lumen. A. Increasing magnifications of large found only in mice, researchers are trackintracellular communities of uropathogenic E. coli (UTI89) inside pods on the surface of ing women with persistent infections to see a mouse bladder infected for 24 hours (SEM). B. Confocal photos of a whole-mounted whether pods are also present in humans. bladder infected with UTI89 expressing green fluorescent protein from the plasmid Biofilms hiding inside cells elsewhere in the pcomGFP. Uroplakin on the surface of the two pods is revealed by treatment with body also may act as reservoirs for other antibody to uroplakin (primary antibody) and tetramethyl rhodamine isothiocyanate– hard-to-treat chronic or recurring infections, labeled secondary antibody (red). The series starts on the left with the lumenal surface and progresses toward the right in sections that move downward through the epithelium. such as ear infections. Optical section thickness, 1 µm. C. Hematoxylin- and eosion-stained sections of UTI89Source: G. G. Anderson, J. J. Patermo, J. D. infected mouse bladders show a bacterial factory 6 hours after inoculation (top panel) Schilling, R. Roth, J. Heuser, and S. J. Hultgren. and a pod 24 hours after inoculation (bottom panel). Bacteria in the pod were densely 2003. Intracellular bacterial biofilm-like pods in packed and shorter and completely filled the host cell. D. Scott Hultgren. urinary tract infections. Science 301:105–107.
1000
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A.
2 µm
B.
PAI II 102 kb hlyll/prf
PAI III 76,8 kb sfa/iro
PAI I 75,8 kb hlyl
Courtesy of Hilde Merkert, University of Wuerzburg
Uropathogenic E. coli
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comial infections). In these cases, the causal organism is less likely to be E. coli and more likely to be another gramnegative bacterium or Staphylococcus. Many UTIs resolve spontaneously, but others can progress to destroy the kidney or, via gram-negative septicemia, the host. As a result, antibiotic therapy is recommended. In older patients, UTIs frequently show atypical symptoms, including delirium, which disappears when the UTI is treated. THOUGHT QUESTION 26.3 Urine samples collected from six hospital patients were placed on a table at the nurses’ station awaiting pickup from the microbiology lab. Several hours later, a courier retrieved the samples and transported them to the lab. The next day, the lab reported that four of the six patients had UTI. Would you consider these results reliable? Would you start treatment based on these results? What makes uropathogenic E. coli different from other E. coli? This is a question still under investigation, but genomic analysis has exposed five pathogenicity islands unique to these strains (Fig. 26.10B). The functions of these pathogenicity islands are still under investigation.
0/100 min
Sexually Transmitted Diseases leuX selC 97 thrW 82 5.6 E. coli 536 chromosome pheV 64
PAI V >40 kb kps
asnT 44
PAI IV 31 kb ybt
Uropathogenic E. coli. A. Bladder cell with adherent uropathogenic E. coli (SEM). Bacteria are approx. 1 µm in length. B. Distribution of pathogenicity islands (PAI) in uropathogenic E. coli. The location of each insert is given in map units within the circle representing the genome. The 0–100 map units are called centisomes. Each centisome is approximately 44 kb of DNA. O is arbitrarily placed at the thr (threonine) gene. The origin of replication on this map is near 82 centisomes. A chromosomal gene flanking the insert is also provided. The size of each island is provided above the insert. A key virulence gene for each island is listed. Figure 26.10
They bind to the bacterial receptor, preventing binding of the organism to the surface epithelium. Individuals most susceptible to UTIs are those who express P antigen on their cells but lack P antigen in their secretions. Urinary tract infections are among those most frequently acquired during a hospital stay (so-called noso-
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Sexually transmitted diseases (STDs) are defined as infections transmitted primarily through sexual contact, which may include genital, oral-genital, or anal-genital contact. The organisms or viruses involved are generally very susceptible to drying and require direct physical contact with mucous membranes for transmission. Because sex can take many forms in addition to intercourse, these microbes can initiate disease in the urogenital tract, rectum, or oral cavities. Examples of common sexually transmitted diseases are given in Table 26.5.
Case History: Secondary Syphilis A pregnant 18-year-old woman came to the county urgentcare clinic with a low-grade fever, malaise, and headache. She was sent home with a diagnosis of influenza. She again sought treatment seven days later after she discovered a macular rash (flat, red) developing on her trunk, arms, palms of her hands, and soles of her feet. Further questioning of the patient when serology results were known revealed that one year ago, she had a painless ulcer on her vagina that healed spontaneously. The vaginal ulcer, the long latent period, and secondary development of rash on the hands and feet described in the case history are classic symptoms of syphilis. Syphilis was recognized as a disease as early as the sixteenth century, but the organism responsible, the spirochete Treponema pallidum, was not discovered until 1905 (Fig. 26.11A). The illness has several stages. The disease
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Table 26.5 Microbes causing sexually transmitted diseases. Disease
Symptoms
Etiologic agent
Virulence factors
Treatment
Gonorrhea
Purulent discharge, burning urination; can lead to sterility
Type IV pili, phase variation
Ceftriaxone
Syphilis
1oChancre, 2oJoint pain, rash 3oGummata, aneurism, CNS damage Watery or mucoid urethral discharge, burning urination
Neisseria gonorrhoeae (G–) Treponema pallidum (spirochete) Chlamydia trachomatis
Motility
Penicillin
Intracellular growth; prevents phagolysosome fusion Cytotoxin
Azithromycin
Metronidazole
?
Erythromycin
gp120, rev and nef proteins, tat protein Cell fusion protein, complementbinding protein, latency E6, E7 proteins
Azidothymidine, AZT, protease inhibitors, Zidovudine Acyclovir, iododeoxyuridine
Trichomoniasis
Vaginal itching, painful urination, strawberry cervix
Chancroid
Painful genital lesion
Acquired immunodeficiency syndrome (AIDS) Genital herpes
Fever, diarrhea, cough, night sweats, fatigue, opportunistic infections
Genital warts
Trichomonas vaginalis (protozoan) Haemophilus ducreyi (G–) HIV
Painful ulcer on external genitals, painful urination
Herpes simplex 2
Warts on external genitals
Human papillomavirus
Vaccine now available
CDC/Susan Lindsley
B. has often been called the great A. imitator because its symptoms in the second stage, as exhibited in the case history, can mimic many other diseases. The incubation stage can last from two to six weeks after transmission, during which time the organism multiplies and spreads throughout the body. Primary syphilis is an inflammatory reaction at the site of infection called a chancre. About a centimeter in diameter, the chancre is painless and hard, and it contains spirochetes. Patients are usu- Figure 26.11 Syphilis. A. Treponema pallidum (darkfield microscopy). Organisms are ally too embarrassed to seek 10–25 µm long. B. Rash of secondary syphilis. medical attention, and because it is painless, hope that it will “go away.” It does go away many different diseases, which contributes to the “great after several weeks, and without scarring. The disease imitator” label. The patient remains contagious in this has now entered the primary latent stage. Over the next stage. Some patients eventually progress over years to five years, symptoms may be absent, but at any time, as tertiary syphilis, in which a great number of symptoms described in the case history, the infected person can can develop, mostly cardiovascular and nervous systems. develop the rash typical of secondary syphilis (Fig. The patient can develop dementia and eventually dies 26.11B). The rash can be similar to rashes produced by from the disease.
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Nongonococcal urethritis
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The presence of the organism in tissues can be detected with fluorescent antibody, but the initial screen is usually serological (that is, patient serum is tested for antibodies). Antibiotics are useful for eradicating the organism, but there is no vaccine, and cure does not confer immunity. The disease is particularly dangerous in pregnant women. The treponeme can cross the placental barrier and infect the fetus to cause congenital syphilis. At birth, infected newborns will have notched teeth (visible on X-rays), perforated palates, and other congenital defects. Women should be screened for syphilis as part of their prenatal testing to prevent these congenital infections. Columbus and the New World theory of syphilis. An outbreak of syphilis that spread throughout Europe soon after Columbus and his crew returned from the Americas (1493) led to the theory that Columbus brought the treponeme to Europe from the New World. However, the theory that Columbus brought syphilis to Europe is most likely wrong. Native Americans buried before Columbus arrived in America showed no signs of syphilis. Furthermore, in recent years, pre-Columbian skeletons—such as those unearthed at the Hull friary in England—have been found with distinctive signs of syphilis. While some historians disagree, the progenitor organism was probably brought to Europe by African slaves long before the discovery of America, and it was in Europe that it developed into venereal syphilis. Thus, the crew of the Columbus voyages may actually have brought the disease to America along with smallpox and measles. The Tuskegee experiment. Unfortunately, much of what we know of syphilis is the result of the infamous Tuskegee experiment conducted in the 1930s in Alabama. The study was entitled “Untreated Syphilis in the Negro Male.” Through dubious means and deception, a group of African-American males were enlisted in a study that promised treatment but whose real purpose was to observe how the disease progressed without treatment. Today, such experiments are barred thanks to strict institutional review board (IRB) oversights in which human subjects must sign informed consent forms. An interesting treatise on the Tuskegee experiment can be found on the Web at the National Center for Case Study Teaching in America (Search for Bad Blood).
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The chlamydiae are unusual gram-negative organisms with a unique developmental cycle. They are obligate intracellular pathogens that start as a small, nonreplicating infectious elementary body that enters target eukaryotic cells. Once inside vacuoles, they begin to enlarge into replicating reticulate bodies (Fig. 26.12). As the vacuole fills, the reticulate bodies divide to become new nonreplicating elementary bodies. Chlamydia trachomatis and Chlamydophila pneumoniae can both cause STDs, as well as other diseases, such as trachoma of the eye or pneumonia. People most at risk of developing genitourinary tract infections with chlamydia are young, sexually active men and women; anybody who has recently changed sexual partners; and anybody who has recently had another sexually transmitted disease. The astute clinician knows that when one STD is discovered, others may also be present. A recent report indicates that Chlamydia can bind to sperm in a process called hitchhiking. It is thought that this interaction helps the organism spread to females. Left untreated, chlamydia can cause serious health problems. In women, the organism can produce pelvic 1. Elementary bodies bind and enter eukaryotic cell by phagocytosis.
EBs
2. Elementary body differentiates into reticulate body.
RBs
3. Reticulate bodies replicate.
4. Reticulate bodies differentiate to elementary bodies and form inclusions.
Attachment 5. Elementary bodies are released.
Chlamydial Infections Are Often Silent Chlamydia is the most frequently reported sexually transmitted infectious disease in the United States, according to the Centers for Disease Control and Prevention, but many people are completely unaware that they are infected. Three-fourths of infected women, for instance, have no symptoms.
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Figure 26.12
Replication cycle of Chlamydia.
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inflammatory disease. Pelvic inflammatory disease is a damaging infection of the uterus and fallopian tubes that can be caused by several different microbial species. The damage produced can lead to infertility, tubal pregnancies, and chronic pelvic pain. Men left untreated can suffer urethral and testicular infections and a serious form of arthritis.
Case History: Gonorrhea A 22-year-old mechanic saw his family doctor for treatment of painful urination and urethral discharge. His medical history was unremarkable. The patient was sexually active, with three regular and several “one time–good time” partners. Physical examination was unremarkable except for prevalent urethral discharge. The discharge was Gram stained and sent for culture. The Gram stain revealed many pus cells, some of which contained numerous phagocytosed gram-negative diplococci (Fig. 26.13A). Blood was drawn for syphilis serology, which proved negative. The patient was given an intramuscular injection of ceftriaxone (250 mg), and oral tetracycline (500 mg, four times a day) was prescribed for seven days. The bacteriology lab was able to recover the bacteria seen in the Gram-stained smear of the urethral discharge. The organism produced characteristic colonies on chocolate agar (agar plates containing heat-lysed red blood cells that turn the medium chocolate brown; Fig. 26.13B). The case was subsequently reported to the state public health department. Upon his return visit, the patient’s symptoms had resolved, and a repeat culture was negative. The patient confirmed that all three of his regular sexual contacts were seen at the STD clinic. The disease here is classic gonorrhea caused by Neisseria gonorrhoeae. A characteristic distinguishing Neisseria infections from Chlamydia infections is that bacterial cells A.
are seen in gonorrheal discharges but not in chlamydial discharges. Gonorrhea has been a problem for centuries and remains epidemic in this country today. Symptoms generally occur 2–7 days after infection but can take as long as 30 days to develop. Most infected men exhibit symptoms; only about 10–15% of men do not. Symptoms include painful urination, yellowish white discharge from the penis, and in some cases swelling of the testicles and penis. The Greek physician Galen (AD 129–ca199) originally mistook the discharge for semen. This led to the name gonorrhea, which means “flow of seed.” In contrast to men, most infected women (80%) do not exhibit symptoms and constitute the major reservoir of the organism. If they are asymptomatic, they have no reason to seek treatment and thus can spread the disease. When symptoms are present, they are usually mild. A symptomatic woman will experience a painful burning sensation when urinating and will notice vaginal discharge that is yellow or occasionally bloody. She may also complain of cramps or pain in her lower abdomen, sometimes with fever or nausea. As the infection spreads throughout the reproductive organs (uterus and fallopian tubes), pelvic inflammatory disease occurs (see earlier discussion of chlamydia). It is important to note that there is no serological test or vaccine for gonorrhea because the organism frequently changes the structure of its surface antigens. Although N. gonorrhoeae is generally serum sensitive owing to its sensitivity to complement, certain serumresistant strains can make their way to the bloodstream and carry infection throughout the body. As a result, both sexes can develop purulent arthritis (joint fluid containing pus), endocarditis, or meningitis. An infected mother can also infect her newborn during parturition (birth), leading to a serious eye infection called ophthalmia neonatorum. Because of this risk and because most infected
B.
C. N. gonorrhoeae
John W. Foster
A. M. Siegelman/Visuals Unlimited
N. gonorrhoeae
from NATURE IMMUNOLOGY cover (March 2002). Photo Courtesy of Ian C. Boulton and Gray-Owen
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CD4+ T cell
Neisseria gonorrhoeae. A. Within pus-filled exudates, the gram-negative diplococci are found intracellularly inside PMNs. The intracellular bacteria (approx. 0.6 to 1 µm in diameter) in this case are no longer viable, having been killed by the antimicrobial mechanisms of the white cell. B. Colonies of N. gonorrhoeae growing on chocolate agar (agar plates containing heat-lysed red blood cells that turn the medium chocolate brown). C. N. gonorrhoeae binding to CD4+ T cells (colorized SEM), inhibiting T-cell activation and proliferation, which may explain the ease of reinfection.
Figure 26.13
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THOUGHT QUESTION 26.4 Considering that N. gonorrhoeae is exquisitely sensitive to ceftriaxone, why do you suppose the patient was also treated with tetracycline? And why can one person be infected repeatedly with N. gonorrhoeae?
develop (the average time between infection and AIDS is 11 years), nearly all HIV-infected individuals eventually become ill and die from the disease. The fi rst stage of the disease, previously known as AIDS-related complex (ARC), can include fever, headache, a macular rash, weight loss, and the appearance of antibodies to HIV in serum. These early, relatively mild symptoms can manifest within a few months of infection, resolve within a few weeks, and then may recur. The debilitated immune system leads to secondary infections by the yeast Candida albicans (candidiasis) and related species (Fig. 26.14B). After several years, the disease can progress to the second stage, AIDS, marked by a significant depletion of the CD4+ T cell population. In 1993, the CDC revised its A. 40
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30 25
Male-to-male sexual contact Injection-drug use Heterosexual contact
20 15 10 5 0
1983
1987
1991
1995
1999
2003
Year of diagnosis B.
C.
Raphael Ojoh/Thatchers.org
Discovered in 1981, HIV caused the greatest pandemic of the late twentieth century and remains a serious problem today, especially in Africa, where it is estimated that 10–30% of the population are infected with HIV (prevalence in the United States is less than 1%). HIV has claimed the lives of more than 22 million people worldwide, over half a million in the United States alone. The molecular biology and virulence of HIV are discussed in Chapters 11 and 25. This section focuses on the disease that HIV causes—acquired immunodeficiency syndrome (AIDS). HIV, a lentivirus in the retroviral family, is a prominent example of viruses that can be transmitted either sexually (vaginally, orally, anally, homosexually, or heterosexually) or through direct contact with body fluids, such as occurs with blood transfusion or the sharing of hypodermic needles by intravenous drug users. HIV is not transmitted by kissing, tears, or mosquito bite. It can, however, be transferred from mother to fetus through the placenta (transplacental transfer). Figure 26.14A illustrates the incidence of AIDS cases by year. The drop-off beginning in 1990 corresponds to the development of more effective treatment regimens. Starting in 1999, however, the trend began climbing upward once again. The proportion of AIDS cases by sex and ethnicity is described in Chapter 25 (see Fig. 25.37). Having entered the bloodstream, HIV infects CD4+ T cells and replicates very rapidly, producing a billion particles per day. The subsequent decrease in CD4+ T cells leads to the disease symptoms collectively called AIDS. Though the symptoms of AIDS may take years to
Number (thousands)
35
Human Immunodeficency Virus (HIV) Causes Sexually Transmitted and Blood-Borne Disease
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12 µm
© F. C. Skvara/Visuals Unlimited
women are asymptomatic, all newborns receive antimicrobial eyedrops at birth. Because adults engage in a variety of sexual practices, N. gonorrhoeae can also infect the anus or the pharynx, where it can develop into a mild sore throat. These infections generally remain unrecognized until a sex partner presents with a more typical form of genitourinary gonorrhea. Because no lasting immunity is built up, reinfection with N. gonorrhoeae is possible. This is due in part to the phase variation in various surface antigens that takes place and because the organism can apparently bind to CD4+ T cells, inhibiting their activation and proliferation to become memory T cells (Fig. 26.13C).
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Figure 26.14 Acquired immunodeficiency syndrome. A. Number of AIDS cases in the United States by major transmission category and year. In 1993, government agencies developed a specific definition of HIV infection that is used for public health surveillance only (vertical dotted line). It is estimated that 250,000–300,000 HIV-infected individuals are unaware of their HIV infection. B. Oral candidiasis. The white patches are caused by secondary infection by the yeast Candida albicans. C. Pneumocystis jiroveci infection of the lung. Note the cuplike appearance of the fungus, almost like crushed Ping-Pong balls. Organisms range from 2 to 6 µm in diameter.
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definition of AIDS to include all persons with a CD4+ cell count of less than 200/µl. Once the CD4+ T cell population falls below 400 cells/µl, opportunistic infections begin to arise and disease processes begin. Opportunistic infections include pneumonia by Mycobacterium avium– intracellulare or Pneumocystis jiroveci (Fig. 26.14C), cryptococcal meningitis, Histoplasma capsulatum infection, and tuberculosis. The third stage of AIDS includes changes in mental cognition, muscular action, and reflexes. Cardiovascular disease and brain tumors are also common. The neurological changes appear coincident to inflammation and demyelination of neurons. The fourth stage of disease is marked by the appearance of various cancers triggered when the depressed immune system cannot detect and destroy cancers initiated by secondary agents. For instance, Kaposi’s sarcoma (Fig. 25.37C) is a common cancer seen in AIDS patients caused by human herpes virus type 8 (HHV8). Diagnosis of AIDS involves detecting anti-HIV antibodies and determining CD4+ cell count in a patient. Assay for HIV to determine viral load is done via quantitative PCR to detect HIV-specific genes such as gag, nef, or pol. Remember, a person who is HIV-positive does not necessarily have AIDS; it may take years to develop. A vaccine is not yet available to prevent AIDS, in part because the envelope proteins of the virus (see Fig. 11.29) typically change their antigenic shape. However, progression of the disease can be controlled by antimicrobials that inhibit two HIV enzymes critical to the replication of the virus—reverse transcriptase and protease; this is discussed further in Chapter 27.
The Protozoan Trichomonas vaginalis Causes a Common Vaginal Infection Trichomonas vaginalis is a flagellated protozoan (Fig. 26.15) that causes an unpleasant sexually transmitted vaginal disease called trichomoniasis. Approximately 2–3 million infections occur each year in the United States. It can occur in men or women; however, men are usually asymptomatic. Even among infected women, 25–50% are considered asymptomatic carriers. There is no cyst in the life cycle of T. vaginalis, so transmission is via the trophozoite stage only (discussed in Chapter 20). The female patient with trichomoniasis may complain of vaginal itching and/or burning and a musty vaginal odor. An abnormal vaginal discharge also may be present. Males will complain of painful urination (dysuria), urethral or testicular pain, and lower abdominal pain. Owing to colonization by lactobacilli (which produce large amouts of acidic lactic acid), the normal, healthy vagina has a pH of less than 4.5. However, since Trichomonas vaginalis feeds on bacteria, the pH of the vagina rises as the numbers of lactobacilli decrease. Definitive diagnosis
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Figure 26.15 Trichomonas vaginalis (SEM), a protist that causes a common sexually transmitted disease (size 7 lm ë 10 lm).
requires demonstrating the flagellated protozoan in secretions by microscopy. PMNs, which are the primary host defense against the organism, are also usually present. As with giardiasis, this disease is treated with metronidazole. THOUGHT QUESTION 26.5 Human immunodeficiency virus was discussed in this section and in Chapter 25. Like the plague, it is a blood-borne disease. Why, then, do fleas and mosquitoes fail to transmit HIV? TO SU M MAR I Z E: ■
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Urinary tract diseases can result from ascending (to the kidney) or descending (from the kidney) routes of infection. The most common route leading to bladder infection, however, is through the urethra. E. coli is the most common cause of UTI. Syphilis, gonorrhea, and chlamydia are the most common sexually transmitted diseases. A patient with one sexually transmitted disease often has another sexually transmitted disease. Complement sensitivity prevents dissemination by N. gonorrhoeae. In contrast, N. meningitidis, a cause of meningitis, frequently disseminates in the bloodstream because it is complement resistant. HIV depletion of CD4+ T cells results in lethal secondary infections and cancers. Trichomonas vaginalis is a flagellated protozoan that causes a sexually transmitted vaginal disease. The reservoir for this organism is the male urethra and female vagina.
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26.6 Infections of the Central Nervous System
gel electrophoresis and were found to be indistinguishable (that is, they had identical DNA restriction patterns).
Microbes cannot gain easy access to the brain in large measure because of the blood-brain barrier, a fi lter mechanism that allows only selected substances into the brain. The blood-brain barrier works to our advantage when harmful substances, such as bacteria, are prohibited from entering. However, it works to our disadvantage when substances we want to enter the brain, such as antibiotics, are kept out. The barrier is not a single structure but is a function of the way blood vessels, especially capillaries, are organized in the brain. Furthermore, the endothelial cells in those vessels have tight junctions that do not allow most compounds or microbes to cross. And yet, brain infections do occur.
Meningitis is an inflammation of the meninges—the membrane that surrounds the brain and spinal cord. Meningitis can be either bacterial or viral in origin. Sinus and ear infections can extend directly to the meninges, while septicemic spread requires passage through the blood-brain barrier. Viral meningitis is serious but rarely fatal in people with a normal immune system. The symptoms generally persist for seven to ten days and then completely resolve. Bacterial meningitis is usually caused by Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus influenzae. Symptoms of bacterial meningitis can include sudden onset of fever, headache, neck pain or stiffness, painful sensitivity to strong light (photophobia), vomiting (often without abdominal complaints), and irritability. Prompt medical attention is extremely important because the disease can quickly progress to convulsions and death. The meningococcus Neisseria meningitidis (Fig. 26.16B) can colonize the human oropharynx, where it causes mild, if any, disease. At any given time, 10–20% of the healthy population can be colonized and asymptomatic. The organism spreads directly by person-to-person contact or indirectly via droplet nuclei from sneezing or fomites. The problem arises when this organism enters the bloodstream. Unlike the gonococcus, N. meningitidis is very resistant to complement owing to its production of a polysaccharide capsule. The capsule allows the microbe to produce a transient blood infection (bacteremia) and reach the blood-brain barrier. N. meningitidis uses type IV pili to adhere to these endothelial cells and becomes intracellular. Then, utilizing the process of transcytosis, in which a cell moves from one side of a host cell to another, the microbe moves to the brain side of the capillary, where it exits. Once in the cerebral spinal fluid, microbes can multiply almost at will. Figures 26.16C and D shows the remarkable damage
In April 2001, a 4-month-old infant from Saudi Arabia was hospitalized with fever, tender neck, and purplish spots (purpuric spots) on her trunk (Fig. 26.16A). Suspecting meningitis, the clinician took a cerebral spinal fluid (CSF) sample and examined it by Gram stain. The smear revealed gram-negative diplococci inside PMNs. The CSF was turbid with 900 leukocytes per µl, and Neisseria meningitidis was confirmed by culture. The child was treated with cefotaxime and made a full recovery. Her father, the person who brought her in, was clinically well. However, meningococcus was isolated from his oropharynx, as well as from the throat of the patient’s 2-year-old brother. Isolates from the patient, her father, and her brother were positive by agglutination with meningococcus A, C, Y, W135 polyvalent reagent. The father’s vaccination certificate confirmed that he had received a quadrivalent meningococcal vaccine. All three isolates were sent to the WHO (World Health Organization) Collaborating Center, which confirmed meningococcus serogroup W135. The three isolates were examined by pulsed-field
N. meningitidis
D.
University of Wisconsin, Madison Brain Collection
Purpuric spot
C.
Manfred Kage/Peter Arnold
B. John Radcliffe Hospital/Photo Researchers
A.
CDC/Edwin P. Ewing, Jr.
Case History: Meningitis
Bacterial meningitis. Meningococcal disease is dreaded by parents and medical practitioners alike for its rapid onset and the difficulty of obtaining a timely and accurate diagnosis. A. Purpuric spots produced by local intravascular coagulation due to Neisseria meningitidis endotoxin. The rash in meningitis typically has petechial (small) and purpuric (large) components. B. N. meningitidis (SEM, approx. 1 µm diameter). C. Normal brain. D. Autopsy specimen of meningitis due to Streptococcus pneumoniae. Note the greening of the brain, compared with the pink normal brain.
Figure 26.16
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that N. meningitidis and other microbes, such as S. pneumoniae, cause in the brain. Several antigenic types of capsules, called type-specific capsules, are produced by different strains of pathogenic N. meningitidis: types A, B, C, W135, and Y. Types A, C, Y, and W135 are usually associated with epidemic infections seen among people kept in close proximity, such as college students or military personnel. Type B meningococcus is typically involved in sporadic infections. Antibodies to these capsular antigens are used to classify the capsular types of the organisms causing an outbreak. Knowing the capsular type of organism involved in each case helps determine whether the disease cases are related and where the infection may have started. Meningococcal meningitis is highly communicable. As a result, close contacts, such as the parents or siblings of any patient with meningococcal disease, should receive antimicrobial prophylaxis within 24 hours of diagnosis; a single dose of ciprofloxacin (a quinolone antibiotic, discussed in Section 27.4) can be given to adults, and two days of rifampicin given to children. Highly susceptible populations can be immunized with a vaccine containing the four capsular structures. Generally, large numbers of people housed together are susceptible to this disease. For instance, it is a significant problem in college dormitories, prompting several colleges to request that incoming students be vaccinated. THOUGHT QUESTION 26.6 Normal cerebral spinal fluid is usually low in protein and high in glucose. The protein and glucose content does not change much during a viral meningitis, but bacterial infection leads to greatly elevated protein and lowered glucose levels. What could account for this?
Case History: Botulism−It Is What You Eat In June, a 47-year-old resident of Oklahoma was admitted to the hospital with rapid onset of progressive dizziness, blurred vision, slurred speech, difficulty swallowing, and nausea. Findings on examination included drooping eyelids, facial paralysis, and impaired gag reflex. He developed breathing difficulties and required mechanical ventilation. The patient reported that during the 24 hours before onset of symptoms, he had eaten home-canned green beans and a stew containing roast beef and potatoes. Analysis of the patient’s stool detected botulinum type A toxin, but no Clostridium botulinum organisms were found. The patient was hospitalized for 49 days, including 42 days on mechanical ventilation, before being discharged.
Bacterial Neurotoxins That Cause Paralysis Imagine a disease that causes complete loss of muscle function. Using secreted exotoxins, two microbes cause
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lethal paralytic diseases. In one instance, the victim suffers a flaccid paralysis in which the muscles go limp, as in the case history just given, causing paralysis and respiratory difficulty. Voluntary muscles fail to respond to the mind’s will because botulinum toxin interferes with neural transmission. The disease (called botulism) is typically food-borne and is caused by an anaerobic, gram-positive, spore-forming bacillus named Clostridium botulinum. In striking contrast to botulism is tetanus, a very painful disease in which muscles continuously and involuntarily contract (called tetany or spastic paralysis). Tetanus is caused by tetanospasmin, a potent exotoxin made by another anaerobic, gram-positive spore-forming bacillus called Clostridium tetani (Fig. 26.17A). Tetanospasmin interferes with neural transmission, but in contrast to botulism toxin, it causes excessive nerve signaling to muscles, forcing the victim’s back to arch grotesquely while the arms flex and legs extend. The patient remains locked this way until death. Spasms can be strong enough to fracture the patient’s vertebrae. Figure 26.17B shows the result of injecting a mouse’s hind leg with just a tiny amount of tetanus toxin. In both botulism and tetanus, death can result from asphyxiation. Botulism is typically caused by ingesting preformed toxin, although infected wounds or germination of ingested spores can also occasionally produce disease. The microbe germinates in the food and produces toxin. Because the organism is an anaerobe, an anaerobic environment must be present. Home-canning processes are designed to remove oxygen in the canned food as well as sterilize the food, but if the sterilization process is not complete, surviving spores germinate and produce toxin. The toxin is susceptible to heat, but will remain active in improperly cooked food. After ingestion, the toxin is absorbed from the intestine. As in the case history given here, it is not unusual for the organism to be absent from stool samples. This is why serological identification of the toxin is important. Note that a rare form of botulism, called infant botulism or “floppy head syndrome,” can occur when infants are fed honey. Honey can harbor C. botulinum spores that can germinate in the gastrointestinal tract after which the growing vegetative cells will secrete toxin. THOUGHT QUESTION 26.7 Knowing the symptoms of tetanus, what kind of therapy would you use to treat the disease?
Toxin structure. Botulism and tetanus toxins share 30–40% identity and have similar structures and nearly identical modes of action. Botulinum and tetanus toxins are each composed of two peptides—a large, or heavy, fragment analogous to a B subunit of AB toxins and a small, or light, fragment analogous to an A subunit. Both
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A. Clostridium tetani
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B. Spastic paralysis due to tetanus toxin
Tetany
A. M. Siegelman/Visuals Unlimited
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Courtesy American Society for Microbiology
Spores
D. Tetanus toxin structure Binding domain
Catalytic domain
C. Basic structure of tetanus and botulinum toxins 1. The binding domain binds to receptor molecules (gangliosides) of the nerve cell.
COO– Ganglioside binding domain Heavy chain Translocation domain Proteolytic S cleavage S Zn
Light chain Catalytic domain
3. The protease toxin disrupts release of neurotransmitter.
2. The translocation domain makes a pore for passage of the toxin.
NH2
Translocation domain
Figure 26.17 Tetanus and botulinum toxins. A. Photomicrograph of Clostridium tetani (cell length 4–8 µm). B. Mouse injected with tetanus toxin in left hind leg. C. Schematic diagram of tetanus and botulinum toxins. D. Three-dimensional representation of the tetanus neurotoxin with the domains marked. (PDB code: 3BTA)
toxins are initially made as single peptides (about 150 kDa) that are cleaved after secretion to form two fragments (heavy and light chains) that remain tethered by a disulfide bond (Fig. 26.17C). Each heavy chain includes the binding domain, which binds to receptor molecules (gangliosides) on the nerve cell membrane, and a translocation domain that makes a pore in the nerve cell through which the toxin passes. The light chains (catalytic domains) are proteases that disrupt the movement of exocytic vesicles containing neurotransmitters needed for contraction or relaxation. Figure 26.17D shows a threedimensional rendition of tetanus toxin with the three domains marked. Mechanism of action. Both toxins bind to target mem-
branes and are brought into the cell by endocytosis. The
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low pH that forms in the endosome reduces the disulfide bonds holding the two halves of the toxin together and causes the heavy chain to assemble as a channel through the membrane. That channel allows release of the proteolytic light chain into the cytoplasm. The toxic subunit then cleaves key host proteins such as synaptobrevin (VAMP), involved in the exocytosis of vesicles containing neurotransmitters (Fig. 26.18). With such a high degree of mechanistic similarity, why do tetanus and botulism toxins have such drastically different effects? The answer is based on where each toxin acts in the nervous system. Both toxins enter at peripheral nerve ends where nerve meets muscle. The disease botulism results from the ingestion of preformed toxin in poorly prepared, contaminated foods. Whether the organism is also ingested is immaterial. Once botulism toxin
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A. Motor neuron
Acetylcholine
Signal
Signal
Synapse Muscle
Neuromuscular junction
B. Neuron cell VAMP (synaptobrevin) Synaptic vesicle Syntaxin Ca2+ Proteins that mediate fusion of vesicle membrane to cell membrane
SNAP-25 Plasma membrane
Synapse Muscle cell
C.
enters a peripheral nerve, it will immediately cleave peptides, such as VAMP, associated with the exocytic release of the neurotransmitter acetylcholine (Fig. 26.18). Without acetylcholine to activate nerve transmission, muscles will not contract and the patient is paralyzed. Although botulism disease is now rare, the toxin has gained renewed interest in recent years. It is considered a select biological toxic agent of potential use to bioterrorists, but that is not where most interest lies. Because it can safely relax muscles in a localized area if injected in small doses, botulism toxin, or Botox, is used cosmetically by plastic surgeons to reduce facial wrinkles in their patients. Tetanus, in contrast to botulism, is not a food-borne disease. C. tetani spores are introduced into the body by trauma (such as by stepping on a dirty, rusty nail). Necrotic tissue then provides the anaerobic environment required for germination. Growing, vegetative cells release tetanospasmin, which enters the peripheral nerve cells at the site of injury. But rather than cleaving targets here, the toxin travels up axons in the direction opposite to nerve signal transmission until it reaches the spinal column, where it becomes fi xed at the presynaptic inhibitory motor neuron. There the toxin also cleaves proteins like VAMP, but the function of vesicles in these nerves is to release inhibitory neurotransmitters that dampen nerve impulses. Tetanus toxin blocks release of the inhibitory neurotansmitters GABA and glycine into the synaptic cleft, leaving nerve impulses unchecked. As a result, impulses come too frequently and produce the generalized muscle spasms illustrates the characteristic of tetanus. Figure 26.19 mechanism of action of tetanus toxin.
D. Toxin binding Endocytosis
THOUGHT QUESTION 26.8 How do the actions of tetanus toxin and botulinum toxin actually help the bacteria colonize or obtain nutrients? THOUGHT QUESTION 26.9 If C. botulinum is an anaerobe, how might botulism toxin get into foods?
Case History: Eastern Equine Encephalitis Mechanism of action of botulism toxin. A. The neuromuscular junction. Inset shows vesicles filled with neurotransmitters. B. A series of proteins within the nerve are needed to allow synaptic vesicles to bind to the nerve endings. Fusion of the membranes releases acetylcholine into the neuromuscular junction. Botulinum toxin types A and E cut SNAP-25. Botulinum toxins B, D, F, and G cut VAMP. Botulinum toxin C1 cuts syntaxin and SNAP-25. C, D. Toxin binds via the heavy chain and is endocytosed into the nerve terminal. Once the toxin cleaves its target, the nerve terminal is no longer able to release acetylcholine. Figure 26.18
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In August, Mr. C brought his 21-year-old son, Rich, to a New Jersey emergency room. Rich appeared dazed and had trouble responding to simple commands. When questioned about his son’s activities over the past few months, Mr. C told the physician that Rich planned to enter veterinary school in the fall and had spent the month of July relaxing and sunning himself on New Jersey beaches. Aside from the shore, his favorite locale was a pond in a wooded area near a horse farm. On the afternoon prior to admission, Rich became lethargic and tired. He returned home and went to bed. That evening, his father woke him for supper but Rich was confused and had no appetite. By 11 p.m., Rich had a fever of 40.7°C and could not respond to questions. A few hours later, when his father had
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Spinal cord
Inhibitory neuron
Excitory neuron
GABA
ACH
Tetanus toxin
Nerve signal
Axon
C. tetani
Necrosis
Tetanospasmin
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contract the disease in the same way. Rich contracted it inadvertently while visiting the pond. The disease, an encephalitis, is often fatal (35% mortality) but fortunately rare. One reason human disease is rare is that the species of mosquito that usually transmits the virus between marsh birds does not prey on humans. But sometimes a human-specific mosquito bites an infected bird and then transmits EEE virus to humans. Another reason human disease is rare is that the virus generally does not fare well in the body. Many persons infected with EEE virus have no apparent illness because the immune system thwarts viral replication. However, as already noted, mortality is high in those individuals that do develop disease. An interesting point in the case history is that diagnosis relied on detecting an increase in antibody titer to the virus. Typically, at the point that disease symptoms first appear, the body has not had time to generate large amounts of specific antibodies. After a week or so, often when the patient is nearing recovery (convalescence), antibody titers have risen manyfold. The rule of thumb is that a greater than fourfold rise in antibody titer between acute disease and convalescence (or in this case death) indicates that the patient has had the disease. While this knowledge could not help the patient in this case, it was valuable in terms of public health and prevention strategies. Several bacterial, fungal, and viral causes of encephalitis and meningitis are given in Table 26.6.
Prion Diseases Are Caused by Proteins Periphery Neuromuscular junction
Figure 26.19 Retrograde movement of tetanus toxin to an inhibitory neuron. Tetanus toxin enters the nervous system at the neuromuscular junction and travels retrogradely up the axons until reaching an inhibitory neuron located in the central nervous system. There it cleaves VAMP protein (Fig. 26.18B) associated with exocytosis of vesicles containing inhibitory neurotransmitters. GABA = gamma-aminobutyric acid; ACH = acetylcholine.
trouble rousing him, he brought Rich to the ER. Over the next week, Rich’s condition worsened to the point where his limbs were paralyzed. Two weeks later, he died. Serum samples taken upon entering the hospital and a few days before he died showed a sixfold rise in antibody titer to eastern equine encephalitis (EEE) virus. Brain autopsy showed many small foci of necrosis in both the gray and white matter. The EEE virus, a member of the family Togaviridae, is transmitted from bird to bird by mosquitoes. Horses
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An unusual infectious agent called the prion has been implicated as the cause of a series of relatively rare but invariably fatal brain diseases (Table 26.7). Prions are infectious agents that do not have a nucleic acid genome. It seems that a protein alone can mediate an infection. The prion has been defined as a small proteinaceous infectious particle that resists inactivation by procedures that modify nucleic acids. The discovery that proteins alone can transmit an infectious disease has come as a surprise to the scientific community. Diseases caused by prions are especially worrying, since prions resist destruction by many chemical agents and remain active after heating at extremely high temperatures. There have even been documented cases in which sterilized surgical instruments, originally used on a prion-infected person, still held infectious agent and transmitted the disease to a subsequent surgery patient. How can a nonliving entity without nucleic acid be an infectious agent? Prions associated with human brain disease are thought to be aberrantly folded forms of a normal brain protein. The theory is that when a prion is introduced into the body and manages to enter to the brain, it will cause normally folded forms of the protein to refold incorrectly. The improperly folded proteins fit together like Lego blocks to produce damaging aggregated structures within brain cells.
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Table 26.6 Microbes causing meningitis/encephalitis. Type of meningitis
Etiologic agent
Bacterial (septic)
Streptococcus pneumoniae (G+) Neisseria meningitidis (G–) Haemophilus influenzae type B (G–) Other G– bacilli Group B streptococci (G+)
Listeria monocytogenes (G+) Mycobacterium tuberculosis
Staphylococcus aureus (G+) Staphylococcus epidermidis (G+) Aseptica
Treatment
Vaccine
Capsule, pneumolysin
Lung
Ampicillin
Multivalent, capsule
Capsule IgA protease, endotoxin Polyribitol capsule IgA protease Endotoxin
Throat
Cephalosporin (3rd generation), ceftriaxone Cephalosporin (3rd generation), ceftriaxone
Multivalent, capsule
Sialic acid capsule, streptolysin, inhibition of alternate complement path Intracellular growth, PrfA regulator, actin-based motility Cord factor, wax D, intracellular growth Coagulase, protein A, TSST, leucocidin Biofilm, slime
Fungi (e.g., Coccidioides, Cryptococcus) Amebas (e.g., Naegleria) T. pallidum Mycoplasmas Leptospira
Viral
Typical initial infection or source
Virulence factors
Viruses (90% caused by enteroviruses) EEE virus
Eastern equine encephalitis West Nile West Nile virus disease
Adhesin tip Burrowing motility
Ear infection
Type b polysaccharide
Septicemia Neonate infected during parturition
Ampicillin
Mild GI disease of mother
Ampicillin plus gentamicin
Lung
Combination therapy BCG (rifampin, isoniazid, ethambutol, pyrazinamide) Methicillin, vancomycin
Septicemia
Complication of surgical procedure
Vancomycin, methicillin
Sinusitis, direct spread to meninges Swimming in contaminated waters Syphilis Respiratory Septicemia, water contaminated with animal urine
Ketoconezole, fluconazole
Capsular
Amphotericin B
Erythromycin Erythromycin
Self-limiting
?
Mosquito bite
None; often fatal
?
Mosquito bite
Supportive therapyb
a
Aseptic meningitis refers to an inability to isolate bacterial sources by ordinary means.
b
Supportive therapy means hospitalization, intravenous fluids, airway management, respiratory support, prevention of secondary infections.
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Table 26.7 Prion diseases. Disease
Susceptible animal
Incubation period
Disease characteristics
Creutzfeldt-Jakob [sporadic, familial, new variant (vCJD)] Kuru Gerstmann-Straussler-Scheinker syndrome Fatal familial insomnia
Human Human Human Human
Months (vCJD) to years Months to years Months to years Months to years
Mad cow disease Wasting disease
Cattle Deer
5 years Months to years
Spongiform encephalopathy (degenerative brain disease) Spongiform encephalopathy Genetic neurodegenerative disease Genetic neurodegenerative disease with untreatable insomnia Spongiform encephalopathy Spongiform encephalopathy
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N. meningitidis is resistant to serum complement because it produces a type-specific capsule. This allows the organism to reach and then cross the blood-brain barrier. A vaccine for N. meningitidis is available, but none exists for N. gonorrhoeae. Tetanospasmin causes spastic paralysis. Botulinum toxin causes flaccid paralysis.
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ISM/Phototake
Some investigators suggest that living, infectious agents, such as Spiroplasma (a spiral-shaped genus whose members lack cell walls), can somehow precipitate a conformational change in the normal brain protein, converting it to an infectious prion protein. This proposal is not, however, universally accepted and has yet to be proved. Prion diseases are often called spongiform encephalopathies because the postmortem appearance of the brain includes large, spongy vacuoles in the cortex and cerebellum. These are visible in a brain sample from a victim of one of these diseases, called Creutzfeldt-Jakob disease (CJD) (Figs. 26.20A and B). Most mammalian species appear to develop these diseases. Since 1996, mounting evidence points to a causal relationship between outbreaks in Europe of a disease in cattle called bovine spongiform encephalopathy (BSE, or “mad cow disease”) and a disease in humans called variant Creutzfeldt-Jakob disease (vCJD). Both disorders are invariably fatal brain disorders. They have unusually long incubation periods (measured in years) and are caused by an unconventional transmissible agent. As of this writing, only four cases of BSE have been detected in the United States. Due to aggressive surveillance efforts in the United States and Canada (where only nine cases have been found), it is unlikely, but not impossible, that BSE will be a food-borne hazard to humans in this country. The CDC monitors the trends and current incidence of typical and variant CJD in the United States (Fig. 26.20C).
B. Carolina Biological Supply Company/ Phototake
A.
C.
CJD in the county Unspecified location of CJD in the state
Spongiform encephalopathies. A. Normal brain section. B. Section of brain taken from a CJD victim. Note the “Swiss-cheese” appearance, indicating brain damage. C. Locations of CJD (not vCJD) cases in the USA.
Figure 26.20
■
■
Serological diagnosis of an infectious disease can be done if a fourfold rise in specific pathogen antibody titer occurs between the acute and convalescent stages. Spongiform encephalopathies are believed to be caused by nonliving proteins called prions.
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26.7 Infections of the Cardiovascular System Infections of the cardiovascular system include septicemia, endocarditis (inflammation of the heart’s inner lining), pericarditis (inflammation of the heart’s outer lining) and, possibly, atherosclerosis (the deposition of fatty substances along the inner lining of arteries). These are all life-threatening diseases. Septicemia is, by strict defi nition, the presence of bacteria or viruses in the blood. The presence of viruses is a condition called viremia, while bacteria in the circulation is more specifically called bacteremia. In practice, however, the terms septicemia and bacteremia are often used interchangeably. Septicemia can develop from a local infection situated anywhere in the body, although blood factors such as complement can nonspecifically kill many types of bacteria that enter the blood. Nevertheless, gram-positives, gram-negatives, aerobes, and anaerobes can all produce septicemia under the right conditions. Endocarditis can be either viral or bacterial in origin. It can be a consequence of many bacterial diseases, such as brucellosis, gonorrhea, psittacosis, staphylococcal and streptococcal infections, candidiasis, and Q fever. Among the many viral causes are Coxsackie virus, ECHO virus, Epstein-Barr, and HIV. Bacterial infections of the heart are always serious. Viral infections, although common, are rarely life-threatening in healthy individuals and are usually asymptomatic.
A.
Vegetation
B.
Vegetation
Case History: Bacterial Endocarditis Elizabeth is 38 years old and has a history of mitral valve prolapse (a common congenital condition in which a heart valve does not close properly). She was recently admitted to the hospital complaining of fatigue, intermittent fevers for five weeks and headaches for three weeks, symptoms the physician recognized as possible indications of endocarditis. Elizabeth reported having a dental procedure a few weeks prior to the onset of symptoms. A sample of her blood placed in a liquid bacteriological media grew gram-positive cocci, which turned out to be Streptococcus mutans, a member of the viridans streptococci. As a result, the diagnosis of bacterial endocarditis was confirmed. The patient began a one-month course of intravenous penicillin G and gentamicin therapy and eventually recovered to normal health. Endocarditis (inflammation of the heart) is traditionally classified as acute or subacute, based on the pathogenic organism involved and the speed of clinical presentation. Subacute bacterial endocarditis (SBE) has a slow onset with vague symptoms. It is usually caused by a bacterial infection of a heart valve (Fig. 26.21). Subacute bacterial endocarditis infections are usually (but not
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Courtesy of Raymond Ho, Dept. of Pathology, The University of Hong Kong
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Views of bacterial endocarditis. A. Whole-heart view, showing vegetations on the mitral valve. B. Close-up of mitral valve endocarditis, showing vegetation (arrow).
Figure 26.21
always) caused by a viridans streptococcus from the oral flora (for example, Streptococcus mutans, a common cause of dental caries). Viridans streptococci is a general term used for normal flora streptococci whose colonies produce green alpha hemolysis on blood agar (viridans from the Greek viridis, to be green). Most patients who develop infective endocarditis have mitral valve prolapse (90%), although this is frequently not the case when patients are intravenous drug abusers or have hospital-acquired (nosocomial) infections. As in the case history presented here, bacterial endocarditis often begins at the dentist’s office. Following a dental procedure (such as tooth restoration), oral flora can transiently enter the bloodstream and circulate.
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S. mutans, which is not normally a serious health problem, can become lodged onto damaged heart valves, grow as a biofi lm, and secrete a thick glycocalyx coating that encases the microbes and forms a vegetation on the valve, damaging it further. If untreated, the condition can be fatal within six weeks to a year. When more virulent organisms, such as Staphylococcus aureus, gain access to cardiac tissue, a rapidly progressive (acute) and highly destructive infection ensues. Symptoms of acute endocarditis include fever, pronounced valvular regurgitation (back flow of blood through the valve), and abscess formation. Most patients with subacute bacterial endocarditis present with a fever that lasts several weeks. They also complain of nonspecific symptoms, such as cough, shortness of breath, joint pain, diarrhea, and abdominal or flank pain. Endocarditis is suspected in any patient who has a heart murmur and an unexplained fever for at least one week. It should also be considered in an intravenous drug abuser with a fever, even in the absence of a murmur. In either case, a defi nitive diagnosis requires blood cultures that grow bacteria. Blood cultures involve taking samples of a patient’s blood from two different locations (such as two different arms). Growth of the same organism in cultures taken from two body sites rules out inadvertent contamination with skin flora, which would likely yield growth only in one culture. The blood is then added to liquid culture medium and incubated at 37°C. Incubation should be done aerobically and anaerobically. Curing endocarditis is difficult because the microbes are usually ensconced in a nearly impenetrable glycocalyx. Consequently, eradicating microorganisms from the vegetations almost always requires hospitalization, where high doses of intravenous antibiotic therapy can be administered and monitored. Antibiotic therapy usually continues for at least a month, and in extreme cases, surgery may be necessary to repair or replace the damaged heart valve. Although at increased risk, patients with heart valve prolapse should not shy away from the dentist. Prophylactic treatment with penicillin or erythromycin taken an hour before a procedure will kill any oral flora that enter the bloodstream and prevent the development of endocarditis. Viruses such as adenovirus and some enteroviruses can also cause endocarditis as well as a condition known as myocarditis, an inflammation of the heart muscle. THOUGHT QUESTION 26.10 A patient presenting with high fever and in an extremely weakened state is suspected of having a septicemia. Two sets of blood cultures are taken from different arms. One bottle from each set grows Staphylococcus aureus, yet the laboratory report states that the results are inconclusive. New blood cultures are ordered. Why might this be?
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Atherosclerosis and Coronary Artery Disease Atherosclerosis can occur in most blood vessels. Coronary artery disease is the result of atherosclerotic deposits in the arteries of the heart, which can produce a heart attack if blood flow is occluded. The causes of atherosclerosis have been studied and debated for many years. It now appears that microbes may have an important role in the development of this disease. Chlamydophila pneumoniae, for example, is suspected of involvement. Serological studies indicate that a large number of patients with coronary heart disease have antibodies to this organism. Although it is still not clear if Chlamydophila truly causes atherosclerosis, one possible model is presented in Figure 26.22. Infection of alveolar macrophages leads to circulating “activated” macrophages that generate inflammatory cytokines and factors that result, among other things, in lowered high-density cholesterol (the “good” cholesterol) and increased clotting—all of which can damage endothelial cells and form plaques. In support of an infection model, some studies indicate that antibiotic treatment will reduce the number of recurrent heart attacks in patients with acute coronary artery disease.
Malarial Parasites Feed on Blood Malaria is the most devastating infectious disease known. Each year 300–500 million people develop malaria worldwide, and 1–3 million of these people, mostly children, die. Fortunately, the disease is relatively rare in the United States; only around 1,000 cases occur annually and almost all are acquired as a result of international travel to endemic areas (Fig. 26.23A). Once again, this illustrates the diagnostic importance of knowing a patient’s history. The disease is caused by four species of Plasmodium; P. falciparum (the most deadly), P. malariae, P. vivax, and P. ovale. The life cycle of Plasmodium, discussed in detail in Chapter 20, is complex and involves two cycles—an asexual erythrocytic cycle in the human and a sexual cycle in the mosquito (discussed in Chapter 20; Fig. 20.37). In the erythrocytic cycle, the organisms enter the bloodstream through the bite of an infected female Anopheles mosquito (the mosquito injects a small amount of saliva containing Plasmodium). The haploid sporozoites travel immediately to the liver, where they undergo asexual fission to produce merozoites. Released from the liver, the merozoites attach to and penetrate red blood cells, where Plasmodium consumes hemoglobin and enlarges into a trophozoite. The protist nucleus divides, so that the cell, now called a schizont, contains up to 20 or so nuclei. The schizont then divides to make the smaller, haploid merozoites (Fig. 26.23B). The glutted red blood cell eventually lyses, releasing merozoites that can infect new red blood cells (Fig. 20.36).
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1. C. pneumoniae infects alveolar macrophages in lung. Chlamydophila pneumoniae elementary bodies “Activated” circulating monocyte 2. Activated macrophage circulates and releases cytokines.
Cytokines
C reactive protein Triglycerides Fibrinogen High-density lipoprotein cholesterol
Tissue factor
Clotting cascade and thrombin Atheromatous plaque
Damage to endothelial cells
3. Cytokines damage endothelial cells. Infected macrophages may become resident, lipid-laden, “foam” macrophages in tissue.
Endothelial cells Smooth muscle cells Foam cells C. pneumoniae (tissue macrophages)
Model outlining a possible role for Chlamydophila pneumoniae in cardiovascular disease. Upward and downward arrows indicate increases or decreases in factors. Atheromatous plaques consist of fatty deposits of cholesterol that narrow blood vessels.
Figure 26.22
B.
Areas where malaria is prevalent
Dennis Kunkel/Visuals Unlimited
A.
Malaria is a major disease worldwide. A. Endemic areas of the world where malaria is prevalent. B. Plasmodium falciparum (TEM). Schizont after completion of division. A residual body of the organism (yellow-green) is left over after division. The erythrocyte has lysed and only a ghost cell remains; no cytoplasm is seen surrounding the merozoites just being released. Free merozoites are seen outside the membrane.
Figure 26.23
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Sudden, synchronized release of the merozoites and red cell debris triggers the telltale symptoms of malaria— violent, shaking chills followed by high fever and sweating. The erythrocytic cycle, and thus the symptoms, repeats every 48–72 hours. After several cycles, the patient goes into remission lasting several weeks to months, after which there is a relapse. Much of today’s research focuses on why malarial relapse happens. Why does the immune system fail to eliminate the parasite after the first episode? When Plasmodium invades the red blood cells, it lines the blood cells with a protein, PfEMP1, that causes them to stick to the sides of blood vessels. This removes the parasite from circulation, but the protein cannot protect the parasite from patrolling macrophages, which eventually detect the invader and recruit other immune cells to fight it. So, during a malarial infection, a small percentage of each generation of parasites switches to a different version of PfEMP1 that the body has never seen before. In its new disguise, P. falciparum can invade more red blood cells and cause another wave of fever, headaches, nausea, and chills. These sticky surface proteins are the antigens that the body’s immune system recognizes and attacks. Once the immune system fights off one version of falciparum malaria, the parasite alters which gene is expressed. The resulting antigenic variation blinds the immune system, allowing a new wave of illness. The body now has to repeat the recognition and attack responses all over again. The parasite has 60 cloaking genes, called var, that can be turned on and off individually, changing the organisms’ antigenic structure, like a criminal repeatedly changing his disguise to elude police. In April 2005, Australian scientists Alan Cowman, Brendan Crabb, and colleagues (Walter and Eliza Hall of Institute of Medical Research) showed that var genes are regulated by chromosome packaging, which unwraps one gene to be expressed at a time and literally packs away the inactive genes. DNA can be encased so securely by some proteins that other proteins cannot access the nucleic acid for transcription, a process known as epigenetic silencing. Becoming immune to all the types of malaria can take upwards of five years and requires constant exposure; otherwise the immunity is lost. Many children do not live long enough to gain immunity to malaria in all its forms. Diagnosis involves microscopic demonstration of the protist within erythrocytes (Wright stain) or through serology to identify antimalarial antibodies. Treatment regimens include chloroquine or mefloquine, which kill the organisms in their erythrocytic asexual stages, and primaquine, effective in the exoerythrocytic stages. The chloroquine family of drugs acts by interfering with the detoxification of heme generated from hemoglobin digestion. Malaria parasites accumulate the hemoglobin released from red blood cells in plasmodial lysosomes, where digestion occurs. The parasites use the amino acids from hemoglobin to grow but
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find free heme toxic. To prevent eating themselves to death from accumulating too much heme, the organism detoxifies heme via polymerization, which produces a black pigment. Many antimalarial drugs, such as chloroquine, prevent polymerization by binding to the heme. As a result, the increased iron (from heme) level kills the parasite. Chloroquine is given prophylactically to persons traveling to endemic areas. Unfortunately, Plasmodium has been developing resistance to these drugs, forcing development of new ones. The antigenic shape-shifting carried out by this parasite has so far stymied development of an effective vaccine. TO SU M MAR I Z E: ■
■ ■
■
■
Blood cultures are useful in diagnosing septicemia and endocarditis. Endocarditis can have acute or subacute onsets. Subacute bacterial endocarditis is usually an endogenous infection caused by S. mutans. Chlamydophila pneumoniae infection may contribute to atherosclerosis. Malaria, caused by Plasmodium species, manifests as repeated episodes of chills, fever, and sweating owing to the organism’s ability to alter the antigenic appearance of its surface proteins and evade the immune response.
26.8 Systemic Infections Many pathogenic bacteria can produce a septicemia as a way to disseminate throughout the body and infect other organs. These organisms cause what are considered systemic infections.
Case History: The Plague A 25-year-old New Mexico rancher was admitted to an El Paso hospital because of a two-day history of headache, chills, and fever (40°C). The day before admission, he began vomiting. The day of admission, an orange-sized, painful swelling in the right groin area was noted (Fig. 26.24A). A lymph node aspirate and a smear of peripheral blood were reported to contain gram-negative rods that exhibited bipolar staining (Fig. 26.24B). The patient’s white blood cell count was 24,700/µl (normal is 4,300–10,800/µl), and platelet count was 72,000/µl (normal is 130,000–400,000/µl). In the two weeks prior to becoming ill, the patient had trapped, killed, and skinned two prairie dogs, four coyotes, and one bobcat. The patient had cut his left hand shortly before skinning a prairie dog. PCR and typical biochemical testing of a gram-negative rod isolated from blood cultures identified the organism as Yersinia pestis, the organism that causes plague. The patient received an antibiotic cocktail of gentamicin and tetracycline. He eventually recovered after six weeks in intensive care.
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ervoir. Upon returning to the city, the rat flea passed the organism on to other rats, which then died in droves. The rat fleas, deprived of their normal meal, were forced to feed on city dwellers, passing the disease on to them. Individuals bitten by an infected flea or accidentally infected through a cut while skinning an infected animal fi rst exhibit the symptoms of bubonic plague. Bubonic plague emerges as the organism moves from the site of infection to the lymph nodes, producing characteristically enlarged nodes called buboes (see Fig. 26.24A). From the lymph nodes, the pathogen can enter the bloodstream, causing septicemic plague. In this phase, the patient can go into shock from the massive amount of endotoxin in the bloodstream. Neither bubonic nor septicemic plague is passed from person to person. As the organism courses through the bloodstream, however, it will invade the lungs and produce pneumonic plague, which can be easily transmitted from person to person through aerosol droplets generated by coughing (Fig. 26.24D). Pneumonic plague is the most dangerous form of the disease because it can kill A. B. quickly and spread rapidly through a population. Pneumonic plague is so virulent that an untreated patient can die within 24–48 hours. Identification of the organism is usually achieved postmortem. The organism has numerous virulence factors, including so-called V and W cell surface lipoprotein antigens that inhibit phagocytosis. Another factor, the F1 protein surNotice the bipolar staining face antigen, is partly responsible for Y. pestis survival in the gut of the flea. 4 µm It blocks flea digestion, making the flea feel “starved” even after a blood C. D. meal. Therefore, the flea jumps from host to host in a futile effort to feel full. This curious effect on the insect Infection in vector is another unique aspect of lung how plague spreads so quickly. Y. pestis also uses type III secretion systems to inject virulence proteins (YopB and YopD) into host cell membranes. Unlike Salmonella, which uses type III–secreted proteins to gain entrance into host cells, Y. pestis is not primarily an intracellular pathogen. Injection of the Yop proteins disrupts the actin cytoskeleton and so helps the organism evade phagocytosis. By evading phagocyFigure 26.24 The plague. A. Classic bubo (swollen lymph node) of bubonic plague. tosis, the organism avoids triggering B. Yersinia pestis, bipolar staining (length 1 to 3 µm). C. Prairie dogs are often hosts to an inflammatory response and profleas that carry plague bacilli. D. X-ray of pneumonic plague, showing bilateral pulmonary duces massive tissue colonization. infection.
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CDC
Science Source/Photo Researchers
Craig K. Lorenz/Photo Researchers
CDC
Plague is caused by the bacterium Yersinia pestis, which can infect both humans and animals. During the Middle Ages, the disease, known as the Black Death, decimated over a third of the population of Europe. Such was the horror it evoked that invading armies would actually catapult dead plague victims into embattled fortresses. This was probably the fi rst use of biowarfare. Contrary to popular belief, Y. pestis is present in the United States. The organism is endemic in 17 western states. It is normally transmitted from animal to animal, typically rodents like rats and even prairie dogs (Fig. 26.24C) by the bite of infected fleas. Figure 26.25 illustrates the various infective cycles of the plague bacillus. Humans are not typically part of the natural infectious cycle. However, in the absence of an animal host, the flea can take a blood meal from humans and thereby transmit the disease to them. During the Middle Ages, urban rats venturing back and forth to the countryside became infected by the fleas of wild rodents that served as a res-
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Plague has disappeared from Europe; the last major outbreak occurred in 1772. The reason for its disappearance is not known but is thought to be the result of human interventions. Although it wasn’t until the nineteenth century that doctors understood how germs could cause disease, Europeans recognized by the sixteenth century that plague was contagious and could be carried from one area to another. Beginning in the late seventeenth century, governments created a medical boundary, or cordon sanitaire, between Europe and the areas to the east from which epidemics came. Ships traveling west from the Ottoman Empire were forced to wait in quarantine before passengers and cargo could be unloaded. Those who attempted to evade medical quarantine were shot.
Case History: Lyme Disease Brad, a 9-year-old from Connecticut, developed a fever and a large (8-cm) reddish rash with a clear center (Erythema migrans) on his trunk (Fig. 26.26A). He also had some left facial nerve palsy. Brad had returned a week previously from a Boy Scout camping trip to the local woods, where he did a lot of hiking. When asked by his physician, Brad admitted finding a tick on his stomach while in the woods, but thought little of it. The doctor ordered serological tests for Borrelia burgdorferi
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(the organism responsible for Lyme disease), Rickettsia rickettsii (which produces Rocky Mountain spotted fever), and Ehrlichia equi (which causes ehrlichiosis). The ELISA test for B. burgdorferi came back positive, confirming a diagnosis of Lyme disease. The boy was given a three-week regimen of doxycycline (a tetracycline derivative), which led to resolution of the rash and palsy. Lyme arthritis was first reported in Lyme, Connecticut, in the 1970s, but the causative organism, B. burgdorferi, was not identified until 1982. Since then, Lyme disease (aka borreliosis) has become the most common vector-borne illness in the United States and is considered an emerging infectious disease. The main endemic areas are the northeastern coastal area from Massachusetts to Maryland, Wisconsin and Minnesota, and northern California and Oregon. Lyme disease is also common to parts of Europe, such as Sweden, Germany, Austria, Switzerland, and Russia. Person to person
Direct human-tohuman transmission
Pathways Usual Occasional Rare or theoretical
■ M edic ine and I mm u n o l o gy
May progress to
Secondary plague pneumonia
Bubonic plague Pneumonic plague epidemic
Direct contact
Sylvatic cycle between fleas and rodents
Direct contact
Wild rodent
Urban cycle between fleas and rodents Infective flea
Direct contact Infective flea
Domestic rodent
Infective flea Contaminated soil
Direct contact
Direct contact
Direct contact Infective flea Wild rodent Sylvatic cycle
Urban cycle
Domestic rodent
Figure 26.25 The cycles of plague. The sylvatic cycle occurs in the wild, where fleas transmit the organism between rodents. An accidental interaction with urban rats can trigger a similar urban cycle. Humans can be infected through contact with infected fleas coming from either cycle. This starts bubonic plague symptoms that can progress to pneumonic plague. Pneumonic plague is highly infectious, which can cause epidemic spread of the disease.
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A.
B.
CDC
5 µm
Courtesy Jeffrey Nelson
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D. Maintenance host
Life cycle
Incidental hosts
C.
David M. Phillips/Photo Researchers
Egg
Larva
Nymph
Figure 26.26 Lyme disease. A. Erythema migrans rash. B. Borrelia burgdorferi (dark-field microscopy), the agent of Lyme disease (cell length 5 to 30 µm). C. Ixodes vector (SEM). D. Host associations of Ixodes scapularis. Tick stages are drawn actual size. Ticks engorged with blood will be larger.
B. burgdorferi is a spirochete (Fig. 26.26B) transmitted to humans by ixodid ticks (hard ticks; Figs. 26.26 C and D). In the northeast and central United States, where most cases occur, the deer tick Ixodes scapularis transmits the spirochete, usually during the summer months. In the western United States, I. pacificus is the tick vector. During its nymphal stage (the stage after taking its fi rst blood meal), I. scapularis is the size of a poppy seed. Its bite is painless, so it is easily overlooked. Infection takes place when the tick feeds because the spirochete is regurgitated into the host. However, the organism grows in the tick’s digestive tract and takes about two days to
979-1028_SFMB_ch26.indd 1020
Adult
make its way to the tick’s salivary gland, so if the tick is removed before that time, the patient will not be infected. Once it is transferred to the human, the microbe can travel rapidly via the bloodstream to any area in the body, but prefers to grow in skin, nerve tissue, synovium (joint lining), and the conduction system of the heart. Lyme disease has three stages. Three general stages of Lyme disease are recognized. Stage 1 infections occur 3–30 days after the initial exposure. Approximately 75% of patients experience an erythema migrans rash, usually at the site of the tick bite, that varies in appearance but
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Hepatitis Viruses Target the Liver Hepatitis is a general term meaning inflammation of the liver. Hepatitis is caused by several viruses, includ-
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ing hepatitis A, B, and C viruses. Although these viruses are members of very different families, they all target the liver. We include them under the section on systemic infections because their infectious routes take them to the bloodstream before arriving at the liver. Hepatitis A virus (HAV) is a single-stranded RNA picornavirus (Fig. 26.27A) that causes an acute infection spread person-to-person by the fecal-oral route, but can also result from eating undercooked shellfish collected from contaminated waters. The organism replicates in the intestinal endothelium and is disseminated via the bloodstream to the liver. After replicating in hepatocytes, the progeny enter the bile and are released into the small intestine, explaining why stools are so infectious. Though the virus has an early viremic stage after leaving the intestine, it is rarely transmitted by transfusion because the viremic stage is transient, ending after development of liver symptoms. In contrast, hepatitis B and C viruses produce persistent viremia and are readily transmitted by transfusion. Many people who are infected with HAV are asymptomatic or exhibit very mild symptoms that include nausea, vomiting, diarrhea, low-grade fever, and fatigue. As the virus attacks the liver, the patient may become jaundiced (from accumulation of bilirubin in the skin), and their urine will turn dark brown. There is no specific treatment, but the disease usually lasts for only a few months and then resolves without establishing a carrier state. Disease can, however, be prevented if immunoglobulin is given to someone who has had contact with an infected individual. There is an inactivated hepatitis A vaccine available called HepA vaccine, which is administered after 1 year of age. For those not vaccinated, frequent hand washing is important for preventing the spread of the disease because it interrupts the fecal-oral cycle.
B.
120 nm
160 nm
Dennis Kunkel/Visuals Unlimited
A.
Gopal Murti/Visuals Unlimited
is classically erythematous with central clearing (“bullseye” rash). This stage is often associated with constitutional symptoms such as fever, myalgias, arthralgias, and headache. Stage 2 occurs weeks to months after the initial infection. In this stage, the patient can be quite ill with malaise, myalgias (muscle pain), arthralgias (joint pain), or neurological or cardiac involvement. Common neurological manifestations include Bell’s palsy (facial paralysis), inflammation of spinal nerve roots, and chronic meningitis. The most common cardiac manifestation is an irregular heart rhythm. Stage 3 borreliosis occurs months to years later and can involve the synovium, nervous system, and skin, though skin involvement is more common in Europe than in North America. Arthritis occurs in the majority of previously untreated patients; it is usually intermittent, involves the large joints, particularly the knee, and lasts from weeks to months in any given joint. Joint fluid analysis typically shows a WBC count of 10,000 to 30,000/µl. Late neurological involvement may include peripheral neuropathy and encephalopathy, manifested by memory, mood, and sleep disturbances. Treatment with antibiotics is recommended for all stages of Lyme disease, but is most effective in the early stages. Treatment for early Lyme disease (stage 1) is a course of doxycycline for 14–28 days. Lyme arthritis is typically slow to respond to antibiotic therapy. Despite antimicrobial drug treatment, patients with persistent active arthritis and persistently positive PCR tests may have incomplete microbial eradication and may be the most likely to benefit from repeated treatment with injected antibiotics. Curiously, 25% of people infected with B. burgdorferi never experience erythema migrans, and many infected individuals are also unsure of tick bites. Hence, patients with Lyme disease may present with arthritis as their fi rst complaint. Although this makes diagnosis extremely difficult, knowing that the patient lives or recently traveled to an endemic area can provide a critical clue. Many other bacteria can cause septicemia and systemic illness (Table 26.8). Gram-negative organisms like E. coli, S. typhi, and Francisella and gram-positive microbes like S. aureus, Enterococcus, and Bacillus anthracis can grow in the bloodstream if they can gain entrance. Even anaerobes that are normal inhabitants of the intestine (for example, Bacteroides fragilis) can be lethal if they escape the intestine and enter the blood, as might happen following surgery. This is one reason surgical patients are given massive doses of antibiotics immediately before and after surgery.
■
Structures of hepatitis A and hepatitis B viruses. Both viruses are icosahedral in shape. A. Hepatitis A (TEM), spread by fecal-oral route, is a single-stranded RNA virus in the Picornaviridae family (30 nm in diameter). B. Hepatitis B (TEM) is an enveloped, double-stranded DNA virus in the Hepadnaviridae family (40 nm in diameter).
Figure 26.27
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Table 26.8 Microbes causing systemic disease. Etiologic agent
Virulence properties
Borrelia burgdorferi (spirochete)
Disease
Symptoms
Lyme disease
Stage 1: rash Stage 2: chills, headache, malaise, systemic involvement Stage 3: neurological changes Fever, weakness, sweats, splenomegaly, osteomyelitis, endocarditis, others Fever, photophobia, headache, abdominal pain, skin rash, liver involvement, jaundice, Chills, fever, headache, muscle pain, splenomegaly, coma
Fever, chills, headache, muscle pain, rash, bacteremia Septicemia, chills, fever, hypotension, rash (rose spots)
Francisella tularensis (G– rod) Salmonella typhi (G– rod)
Typhoid fever
Septicemia, chills, fever, hypotension
Vibriosis
Serious with immunocompromised patients; fever, chills, multi-organ damage, death Buboes (swollen lymph glands), high fever, chills, headache, cough, pneumonia, septicemia
Salmonella cholerasuis (G–) rod) Vibrio vulnificus (G– curved rod)
Brucellosis
Leptospirosis
Epidemic typhus
Tularemia
Typhoid fever
Bubonic plague
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Source
Treatment
Vaccine
Antigenic variation, OspE (binds complement)
Deer tick
Penicillin, tetracycline
+
Brucella abortis (G– rod)
Intracellular, growth in monocytes
Doxycycline
Leptospira interrogans (spirochete)
Burrowing motility
Animal products, unpasteurized milk Urine of infected animals
Rickettsia prowazekii (G– rod)
Obligate intracellular growth, escapes phagosome Intracellular
Yersinia pestis (G– rod)
Type III secretion, intracellular growth, PhoPQ regulators, Vi antigen capsule Intracellular growth, invasin Cytolysin, capsule
Erythromycin, penicillin
Human louse, flying squirrel flea
Tetracycline, chloramphenicol
Rabbits, rodents, insect vectors Gallbladder of human carrier
Gentamicin, streptomycin C. profloxacin, ceftriaxone
Animals, poultry
Ceftriaxone
Seawater, raw oysters
Tetracycline plus aminoglycoside
Intracellular Rodents, rodent growth, fleas, human type III respiratory secretion aerosol, of YOPs potential (Yersinia bioterrorism outer agent proteins), phospholipase D, toxin
Vi antigen
Streptomycin or tetracycline
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In contrast to HAV, hepatitis B virus is a partially double-stranded circular DNA virus (family Hepadnaviridae) that causes diseases of varying severity. These include acute and chronic hepatitis; cirrhosis; and hepatocarcinoma. The virus also wears a membrane envelope donned when progeny viruses are released from infected cells. The virion coat protein, a surface antigen, is called HBsAg. The virus makes an excess amount of HBsAg, so it is sometimes extended as a tubular tail on one side of the virus particle and is often found in the blood of infected individuals in the form of noninfectious fi lamentous and spherical particles (Fig. 26.27B). The presence of HBsAg in blood in an indicator of HBV infection. HBV is primarily transferred via blood transfusions, contaminated needles shared by IV drug users, and any human body fluid (saliva, semen, sweat, breast milk, tears, urine, feces). It can be transferred transplacentally to a fetus and can be sexually transmitted. Infection by HBV has two stages: a short-term acute phase and a long-term chronic phase that, if it extends beyond six months, may never resolve. Symptoms resemble the flu but with jaundice and brown urine. Liver damage caused by HBV infection is in large part due to an efficient cell-mediated immune response. Cytotoxic T cells and natural killer cells cause immune lysis of infected liver cells. Over the long term, chronic hepatitis will lead to a scarred and hardened liver (cirrhosis), the only recourse being a liver transplant. Fortunately, about 90% of those infected are able to fight off infection and never proceed to the chronic stage. A HepB vaccine (made from recombinant HBsAg) is available. Its administration is recommended after birth followed by booster shots administered by 2 months and 18 months of age. Hepatitis C virus (HCV) causes another form of hepatitis. HCV is a single-stranded linear DNA virus with a lipid coat; it is a member of the Flaviviridae family. It is transmitted by blood transfusions and is, in fact, responsible for 90% of transfusion-related cases of hepatitis. It can also be transmitted by needle sticks and, less frequently, by sex. Over 100 million people worldwide are infected with HCV. Most (80%) HCV-infected individuals do not exhibit symptoms, and in those that do, symptoms may not appear for 10–20 years. At least 75% of patients that exhibit symptoms ultimately progress to chronic hepatitis requiring a liver transplant. Fortunately, infection can be detected using an ELISA assay. Liver biopsies of HCV patients are used to determine the extent of liver damage, which in turn helps establish the stage of disease. Prevention of HBC or HCV infection for health care personnel includes avoiding inadvertent needle sticks and, if one should occur, the administration of immunoglobulin within seven days. Chronic hepatitis can be treated with interferon but only with modest success. Though vaccines have been developed for HAV and HBV, no vaccine is yet available for HCV. It is important to note that since hepatitis viruses can be spread via contaminated
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blood products, all blood donations collected by the Red Cross and other agencies are tested for the presence of these viruses, as well as for HIV. Thus, the blood supply is safe. TO SU M MAR I Z E: ■
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■
■
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Septicemia is caused by many gram-positive and gram-negative bacterial pathogens. It can start with the bite of an infected insect, introduction via a wound, escape from an abscess, or by the pathogen penetrating mucosal epithelium (as through the intestine or vagina); it can lead to disseminated, systemic disease. Plague has sylvatic and urban infection cycles involving transmission between fleas and rats. Y. pestis-infected flea bites lead to bubonic plague. Bubonic plague can progress to septicemic and pneumonic stages. Aerosolized respiratory secretions will directly spread Y. pestis pneumonic plague from person to person (no insect vector). Lyme disease is caused by the spirochete Borrelia burgdorferi, which is transmitted from animal reservoirs to humans by the bite of Ixodes ticks. Three stages of Lyme disease: stage 1, a bull’s-eye rash (erythema migrans); stage 2, joint, muscle, and nerve pain; stage 3, arthritis with WBCS in the joint fluid. Hepatitis is caused by several unrelated viruses; among them, HAV, HBV, and HCV account for most disease. HAV is transmitted by the fecal-oral route, does not establish chronic infection, and can be prevented by a vaccine. HBV and HCV can be transmitted by blood products (such as transfusions) and shared hypodermic needles and can lead to chronic hepatitis. Vaccine for HBV, but not HCV, is available.
26.9 Immunization In our discussion of various infections caused by bacteria and viruses, we often noted the availability of vaccines that prevent disease. In Chapter 24, we also listed many of the available vaccines and their basic makeup (see Table 24.2). As noted in that table, some vaccines utilized killed organisms (examples are the hepatitis A vaccine or the Salk inactivated polio vaccine), while others contained attenuated microbes (BCG for tuberculosis or the Sabin live polio vaccine) or consist of purified components of an infectious agent (Streptococcus pneumoniae and Haemophilus influenzae b capsular antigens). Some vaccines are injected in combination as polyvalent vaccines to control multiple diseases (measles, mumps, and rubella), while others are given individually but may change from year to year (influenza viral capsid proteins).
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Table 26.9 Recommended childhood and adolescent immunization schedule, by vaccine and age−United States, 2007. Age r Birth
Vaccine Hepatitis B
a
1 month
HepB (dose1) b
Diphtheria, tetanus, pertussis
c
Inactivated poliovirus Measles, mumps, rubella Varicella
4 months
6 months
12 months
HepB (dose 2)
Rotavirus
Haemophilus influenzae type b
2 months
HepB
Rota (dose1)
Rota (dose 2)
DTaP (dose1)
DTaP (dose 2) DTaP (dose 3)
Hib (dose1)
Hib (dose 2)
IPV (dose1)
IPV (dose 2)
Rota (dose 3) Hibc (dose 3)
IPV (dose 3)
d
MMR (dose 1)
e
Varicella (dose 1)
Meningococcal
f
Pneumococcalg Influenza
PCV (dose 1)
PCV (dose 2)
PCV (dose 3)
h
Hepatitis A
Hib (dose 4)
PCV
Influenza (yearly) i
HepA series
This schedule indicates the recommended ages for routine administration of currently licensed childhood vaccines, as of December 1, 2005. Indicates age groups that warrant special effort to administer those vaccines not previously administered. Range of recommended ages
Catch-up immunization
Assessment at age 11–12 years
a
Hepatitis B vaccine (HepB). At birth: All newborns should receive monovalent HepB soon after birth and before hospital discharge.
b
Diphtheria and tetanus toxoids and acellular pertussis vaccine (DTaP).
c
Haemophilus influenzae type b conjugate vaccine (Hib).
d
Measles, mumps, and rubella vaccine (MMR).
e
Varicella vaccine.
f Meningococcal vaccine (MCV4). Meningococcal conjugate vaccine (MCV4) should be administered to all children at age 11–12 years as well as to unvaccinated adolescents at high school entry (age 15 years). Vaccine contains four types of capsule. g
Pneumococcal vaccine. The heptavalent pneumococcal conjugate vaccine (PCV) is recommended for all children 2–23 months and for certain children aged 24–59 months. The final dose in the series should be administered at age ≥12 months. Pneumococcal polysaccharide vaccine (PPV) is recommended in addition to PCV for certain high-risk groups. PPV contains 23-capsule antigens.
h Influenza vaccine. Influenza vaccine is recommended annually for children aged ≥6 months with certain risk factors (including, but not limited to, asthma, cardiac disease, sickle-cell disease, human immunodeficiency virus infection, diabetes, and conditions that can compromise respiratory function or handling of respiratory secretions or that can increase the risk of aspiration), health-care workers, and other persons (including household members) in close contact with persons in groups at high risk (see MMWR 2005; 54[No. RR-8]). i
Hepatitis A vaccine (HepA). HepA is recommended for all children at age 1 year (i.e., 12–23 months).
Source: http://www.immunize.org/cdc/child-schedule.pdf
Contrary to what you may think, multiple vaccines can be given simultaneously and safely. Most vaccines are administered during childhood, when the diseases can be most devastating. Table 26.9 provides the current immunization schedule recommended for children and adolescents by the Centers for Disease Control and Prevention. As you will notice, all of these vaccines are given in multiple doses, called booster doses. The exception to this rule is the influenza vaccine, which is given in a single dose but changes every year. The reason for multiple doses, as noted in Chapter 24, is that secondary exposure to an antigen provides a more robust and long-lasting immunity. However, most vaccines are not administered until 2 months of age because maternal antibody crossing
979-1028_SFMB_ch26.indd 1024
through the placenta to the fetus (or to a newborn through breast milk) will persist for a short time in the newborn, temporarily protecting the baby from disease and possibly dampening the response to an antigen administered during that time. You might wonder whether all members of a community must be vaccinated against a given microbe in order to lower the risk of disease for every individual in that community. It depends on the disease. For infections spread by person-to-person contact, the risk of disease to an unvaccinated person can actually be lowered dramatically if only around two-thirds of the community are vaccinated. This concept, called community (or herd) immunity, works because it interrupts the transmission
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Pa r t 5
15 months
18 months
24 months
4–6 years
11–12 years
M edic ine and I mm u n o l o gy
13–14 years
15 years
1025
16–18 years
HepB series
DTaP (dose 4)
DTaP
Tdap
Tdap
IPV MMR (dose 2)
MMR Varicella (dose 2)
Vaccines within broken line are for selected populations
MCV4
MCV4
MCV4 (dose 1) PCV
MCV4 PPV
Influenza (yearly) HepA series (dose 2)
of the disease. If one individual contracts the disease, the chance that he or she will come into contact with another unvaccinated person and transmit the disease is much reduced. Thus, the risk of disease to any one unvaccinated person is lessened as a result of community immunity. Herd immunity works well for diseases such as diphtheria, whooping cough (pertussis), measles, and mumps. However, herd immunity will not lower the risk of an unvaccinated person contracting tetanus, which is not spread by person-to-person contact. Clostridium tetani, the agent whose toxin causes tetanus, is a ubiquitous soil organism transmitted through punctured skin. The risk of tetanus for an unvaccinated person doesn’t change even if every other person in the community is vaccinated against tetanus. It is important to note that the vast majority of people who receive vaccines suffer no, or only mild, reactions, such as fever or soreness at the injection site. Very rarely do more serious side effects, such as allergic reactions, occur. Vaccines are extremely safe. You may fi nd some unsettling, erroneous information on the Internet purporting a link between vaccinations and other diseases, such as diabetes or autism, but no well-controlled scientific study supports these claims. Clearly, the risks, including death, associated with these preventable infectious diseases are far greater than the minimal risk associated with being vaccinated against them. As proof of this point, the undervaccination of children in the United States in the 1980s and 1990s has led to an increase in cases of whooping cough (25,616 in 2005), caused by Bordetella pertussis. Nevertheless, the successful vaccination programs car-
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ried out in the United States have come close to eradicating many diseases once feared, such as polio, rubella, and diphtheria, and have dramatically reduced the morbidity and mortality of many others. The one caveat to this is that the current risk of infection for some diseases (because they are so rare) is lower than the risk of an adverse reaction to immunization. However, failing to vaccinate, as just mentioned, will result in a population susceptible to the microbe and a resurgence of serious disease. TO SU M MAR I Z E: ■
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Vaccines can be made from live attenuated organisms, killed organisms, or purified microbe components. Herd immunity can help protect unimmunized persons from diseases transmitted from person to person. Serious side effects very rarely result from immunizations.
Concluding Thoughts This chapter described key concepts of infectious disease using just a small sample of disease-causing pathogens. Many more pathogens can be explored in further studies. The principal goal with this and the previous chapter was to illustrate how microbial metabolism can undermine the physiology of a human host. The next chapter will describe how humans fight back using pharmacology to sabotage the physiology of the infecting microbes.
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CHAPTE R R EVI EW Review Questions 1. How are pathogens classified as to their portal of
14. Would you suspect Salmonella infection in a cluster
entry? Discuss some common skin infections. What causes boils? What are the symptoms of necrotizing fasciitis? What is the difference between primary and secondary infections? How do pneumococci avoid engulfment by phagocytes? What causes the clouding seen in X-rays of infected lungs? What are the key features of the pneumococcal vaccine? Name the common fungal causes of lung disease. What is a metastatic lesion? Why is diarrhea watery? What is the most common microbial cause of diarrhea? List common bacterial agents that cause diarrhea.
of nauseated patients rushed to the hospital directly from a church picnic? Why or why not? What is the significance of finding leukocytes in stool? What is one reservoir of E. coli O157:H7? How is a diagnosis of a UTI made? What is an important virulence determinant of uropathogenic E. coli? What is the most common sexually transmitted disease? How is gonorrhea different in men and women? Why will N. gonorrhoeae not usually disseminate in the bloodstream, while N. meningitidis will? Name the major causes of bacterial meningitis. What are the two routes of infection? If tetanus and botulism toxins have the same mode of action, why do they cause opposite effects on muscles? What are some virulence factors of Y. pestis?
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Key Terms bacteremia (1014) blood-brain barrier (1007) bubonic plague (1018) chancre (1002) chlamydia (1003) coagulase (982) congenital syphilis (1003) dysentery (992) epidemiology (994) exfoliative toxin (983) influenza (992)
meningitis (1007) metastatic lesion (989) multivalent vaccine (987) necrotizing fasciitis (983) pneumonic plague (1018) primary syphilis (1002) prion (1011) respiratory syncytial virus (RSV) (992) rotavirus (996) rubella virus (985) secondary syphilis (1002)
septicemia (1014) septicemic plague (1018) sequela (985) sexually transmitted disease (STD) (1001) spongiform encephalopathy (1013) tertiary syphilis (1002) tetanospasmin (1008) transcytosis (1007) viremia (1014) zoonotic disease (981)
Recommended Reading Broudy, Thomas, and Vincent Fischetti. 2003. In vivo lysogenic conversion of Tox– Streptococcus pyogenes to Tox+ with lysogenic streptococci or free phage. Infection and Immunity 71:3782–3786. Clark, Ian A., Alison C. Budd, Lisa M. Alleva, and William B. Cowden. 2006. Human malarial disease: A consequence of inflammatory cytokine release. Malaria Journal 5:85. Duerr, Ann, Judith N. Wasserheit, and Lawrence Corey. 2006. HIV vaccines: New frontiers in vaccine development. Clinical Infectious Diseases 43:500–511.
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Eiden, Martin, Anne Buschmann, Leila Kupfer, and Martin H. Groschup. 2006. Synthetic prions. Journal of Veterinary Medicine 53:251–256. Eugene, Emmanuel, Isabelle Hoffmann, Celine Pujol, Pierre-Oliver Couraud, Sandrine Bourdoulous, and Xavier Nassif. 2002. Microvilli-like structures are associated with the internalization of virulent capsulated Neisseria meningitidis into vascular endothelial cells. Journal of Cell Science 115:1234–1241. Kim, Kwang Sik. 2006. Microbial translocation of the bloodbrain barrier. International Journal for Parasitology 36:607–614.
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Perrin, Agnès, Stéphane Bonacorsi, Ettiene Carbonnelle, Driss Talibi, Philippe Dessen, et al. 2002. Comparative genomics identifies the genetic island that distinguish Neisseria meningitidis, the agent of cerebrospinal meningitis, from other Neisseria species. Infection and Immunity 70:7063–7072. Petersen, Andreas M., and Karen A. Krogfelt. 2003. Helicobacter pylori: an invading microorganism? A review. FEMS Immunology and Medical Microbiology 36:117–126.
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Russell, David G. 2007. Who puts the tubercle in tuberculosis? Nature Reviews in Microbiology 5:39–47. Trevejo, Rosalie T., Margaret C. Barr, and Robert Ashley Robinson. 2005. Important emerging bacterial zoonotic infections affecting the immunocompromised. Veterinary Research 36:493–506.
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Chapter 27
Antimicrobial Chemotherapy 27.1 27.2 27.3 27.4 27.5 27.6 27.7 27.8 27.9
The Golden Age of Antibiotic Discovery Basic Concepts of Antimicrobial Therapy Measuring Drug Susceptibility Mechanisms of Action Antibiotic Biosynthesis The Challenges of Antibiotic Resistance The Future of Drug Discovery Antiviral Agents Antifungal Agents
The discovery of antibiotics less than 80 years ago has played a major role in increasing life expectancy throughout the world. Prior to 1918, the average life expectancy in the United States was 45–50 years. That number is now between 70 and 75 years, thanks in part to antibiotics. Yet we face the possibility that antibiotics will become useless. Over several decades, antibiotics have been used indiscriminately to treat patients and are often included in animal feed—not to treat animals but to grow larger ones. These abuses, combined with the ability of bacteria to become antibiotic resistant, have led experts to predict an impending crisis where the human race is vulnerable to infectious diseases we once thought had been conquered. Important questions about chemotherapeutic agents will be addressed in this chapter, including: Why do antimicrobials inhibit the growth of bacteria, but not humans or animals? What is antibiotic resistance? How do clinicians know which antibiotic to use to treat an infection? We will also discuss what makes a good antibiotic target, and how new antibiotics are discovered.
The genome of Streptomyces avermitilis, shown here as an SEM, may yield new antibiotics through unrecognized pathways of secondary metabolism. S. avermitilis produces avermectins, a group of antiparasitic agents used in human and veterinary medicine. The genome was found to contain 9,025,608 bases that encode at least 7,574 potential open reading frames (ORFs). Thirty gene clusters related to secondary metabolite biosynthesis were identified, corresponding to 6.6% of the genome. It is thought that some of these pathways may yield other useful antibiotics. Source: Cover of Nature Biotechnology. May 2003. 21(5).
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■ A n t im ic r o b ial C hemot herapy
27.1 The Golden Age of Antibiotic Discovery Antibiotics (from the Greek, meaning “against life”) are compounds produced by one species of microbe that can kill or inhibit the growth of other microbes. While
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A.
B. H N O O
S N
CH3 CH3
COO–
Penicillin G D.
C. © Hulton-Deutsch Collection/Corbis
E.
©1998 Australian Broadcasting Corporation
Antibiotics undeniably have been a benefit to modern society. We live longer and contribute more to society than in the past, in large measure because of antibiotics. Infections we consider minor today often killed their victims just 50 or 60 years ago. But there has been a downside. Consider the case of a 56-year-old man with diabetes who entered the hospital for a heart transplant. The operation went well, but one week after the surgery, he developed a severe chest wound infection notable for exuded pus. Treatment with methicillin, a penicillin derivative commonly used to treat infections, failed and the patient became comatose. The diagnostic microbiology laboratory ultimately identified the agent as a methicillin-resistant Staphylococcus aureus (MRSA), prompting the surgeon to immediately place the patient on intravenous vancomycin for six weeks. Vancomycin is an antibiotic, structurally different from methicillin, that can usually kill methicillin-resistant bacteria. Fortunately, this patient recovered; too often, they do not. Where did the infection come from? Surprising as it may seem, this drug-resistant pathogen was a resident of the hospital itself. To find the source, nasal swabs were taken of all hospital personnel and screened for the presence of S. aureus. The results revealed that several members of the surgery team actually harbored this organism as part of their normal flora. But which individual was the actual source of the patient’s infection? In what could be described as an example of forensic microbiology, each strain was subjected to pulsed-field gel electrophoresis, and the resulting genomic restriction patterns were compared with that of the isolate from the infected patient. A match pointed to the source. The unwitting culprit turned out to be the perfusionist who manipulated the tubing used for cardiopulmonary bypass. Hospital-acquired, or nosocomial, infections are not unusual. As many as 5–10% of all patients admitted to acutecare hospitals acquire nosocomial infections. This leads to over 80,000 deaths each year. The deaths are due, in part, to the poor health of the patient and in part to the antibioticresistant nature of bacteria lurking in hospitals. In fact, as much as 60–70% of staph infections that develop in a hospital setting are the result of methicillin-resistant S. aureus. We begin this chapter with a discussion of the golden age of antibiotic discovery (1940–1960) and move on to describe the basic concepts of antibiotics and their use and misuse. We will also delve into the ways genomic and proteomic approaches broaden our ability to search for new antibiotics and identify new antibiotic targets—all part of our attempt to stay one step ahead of evolving, antibiotic-resistant pathogens.
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Figure 27.1 The dawn of antibiotics. A. Alexander Fleming’s photo of the dish with bacteria and penicillin mold. B. The chemical structure of penicillin G. C. Alexander Fleming at work in his laboratory. D. Howard Florey. E. Pictures taken in 1942, shortly after the introduction of penicillin, show the improvement in a child suffering from an infection four days (panel 3) and nine days (panel 5) after treatment. Panels 5 and 6 show her fully recovered.
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B. NH2 O © Underwood & Underwood/Corbis
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C.
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we think of antibiotics as being a recent biotechnological development, their use has historical precedent. Ancient remedies called for cloths soaked with organic material to be placed on wounds to allow them to heal faster. This organic material likely contained “natural antibiotics” that killed bacteria and prevented further infection. The medicinal properties of molds were also recognized for centuries. Historical accounts refer to the ancient Chinese successfully treating boils with warm soil and molds scraped from cheeses, and in England, a paste of moldy bread was a home remedy for wound infections up until the beginning of the twentieth century. The modern antibiotic revolution began with the discovery of penicillin in 1928 by Sir Alexander Fleming (1881–1955). His discovery was actually a rediscovery, and was arguably one of the greatest examples of serendipity in science. Although Fleming generally receives the credit for discovering penicillin, a French medical student, Ernest Duchesne (1875–1912), originally discovered the antibiotic properties of Penicillium in 1896. Duchesne observed that Arab stable boys at the nearby army hospital kept their saddles in a dark and damp room to encourage mold to grow on them. When asked why, they told him the mold helped heal saddle sores on the horses. Intrigued, Duchesne prepared a solution from the mold and injected it into diseased guinea pigs. All recovered. Although he submitted his work as a dissertation to the Pasteur Institute, it was ignored because of his young age and because he was unknown. Penicillium was forgotten in the scientific community until Fleming rediscovered it one day in the late 1920s. Petri dishes were glass in those days and could be rewashed and sterilized. Fleming was preparing to wash a pile of old Petri dishes he’d used to grow the pathogen S. aureus. He opened and examined each dish before tossing it into a cleaning solution. He noticed that one dish had grown contaminating mold, which in and of itself was not unusual in old plates, but all around the mold the staph bacteria had failed to grow (Fig. 27.1A). He took a sample of the mold and found it was from the penicillium family, later identified as Penicillium notatum. The mold appeared to have synthesized a chemical, now known as penicillin (Fig. 27.1B), that diffused through the agar, killing cells of S. aureus before they could form colonies. Fleming (Fig. 27.1C) presented his fi ndings in 1929, but they raised little interest, since penicillin appeared to be unstable and would not remain active in the body long enough to kill pathogens. As World War II began, an Oxford professor named Howard Florey (Fig. 27.1D), together with his colleague Ernst Chain, rediscovered Fleming’s work, thought it held promise, and set about purifying penicillin. To their amazement, when the purified penicillin was injected into mice infected with staphylococci or streptococci, the majority of mice survived. Subsequent human trials also proved successful (Fig. 27.1E), and penicillin gained wide
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NH NH H2N O HO O CHO
H OH N OH
NH NH2
H3C OH O HO HO
O OH
Streptomycin
NHCH3
Figure 27.2 The discoverers of sulfanilamide and streptomycin. A. Gerhard Domagk discovered sulfanilamide. B. Chemical structure of sulfanilamide, an analog of paraaminobenzoic acid (PABA), a precursor of the vitamin folic acid, necessary for growth. Sulfonilamide inhibits one of the enzymes that converts PABA into folic acid. C. Selman Waksman discovered streptomycin in 1944. D. Chemical structure of streptomycin.
use, saving countless lives during the war. As a fitting tribute to serendipity, Fleming, Florey, and Chain received the 1945 Nobel Prize in Physiology or Medicine for their work. Duchesne’s work, however, went unrecognized by the scientific community for decades. The next landmark discovery in antibiotics was made by Gerhard Domagk (1895–1964), a German physician at the Institute of Experimental Pathology and Bacteriology who investigated antimicrobial compounds in the 1930s (Fig. 27.2A). In 1935, Domagk’s 6-year old child was affl icted with a serious streptococcal infection induced by an innocent pinprick to the fi nger. The infection spread to the lymph nodes under her arm and became so severe that lancing and draining the pus 14 times did little to help.
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The only remaining alternative was to amputate the arm. Unfortunately, even this drastic measure would probably not save her life. Frustrated, Gerhard Domagk took what would appear to be drastic measures. He administered a dose of an innocuous red dye he was investigating that, on agar plates (the usual medium for testing antibiotics), had shown absolutely no ability to inhibit the growth of streptococcus. Nevertheless, Domagk’s daughter recovered completely. How did Domagk conceive of such a therapy when the conventional method of screening for antibiotic activity on agar plates indicated that this compound (Prontosil) was useless? The answer lay in his prior use of laboratory animals to study the drug. When administered to mice, Prontosil was very effective at preventing infection. Had he not used live animals, Domagk would never have discovered that Prontosil was metabolized by the body into another compound, sulfanilamide, clearly lethal to the streptococcus. This fi nding led to an entire class of drugs, the sulfa drugs, which saved hundreds of thousands of lives, including that of his own child. Sulfanilamide is an analog of para-aminobenzoic acid (PABA), a precursor of folic acid, a vitamin necessary for nucleic acid synthesis (Fig. 27.2B). The drug stops bacterial growth by inhibiting the conversion of PABA to folic acid. Folic acid is not synthesized by humans, but is a dietary supplement. Furthermore, bacteria do not transport folic acid, which explains why the sulfa drugs can inhibit bacterial growth in humans without affecting human cells. In a dark turn, the pressing need for effective antibiotics during World War II dictated a change in the in vivo screening methods used by Domagk’s German employer. Animal testing was abandoned in favor of human testing, but the humans were not volunteers. They were concentration camp prisoners intentionally infected with bacterial diseases, such as gangrene, and then treated with new chemical compounds. Domagk’s possible participation in these experiments, even if unwilling, was a source of controversy that haunted him long after the war ended. Yet his contributions to medicine continued as he also developed the fi rst effective chemotherapy for tuberculosis via the thiosemicarbazones and isoniazid, still used today. During the same period of history, Selman Waksman (1888–1973) at Rutgers University began screening 10,000 strains of soil bacteria and fungi for an ability to inhibit growth or kill bacteria (Fig. 27.2C). In 1944, this Herculean effort paid off with the discovery of streptomycin, an antibiotic produced by the actinomycete Streptomyces griseus (Fig. 27.2D). Waksman’s discovery of streptomycin triggered the antibiotic gold rush that is still under way. TO SU M MAR I Z E: ■
The importance of antibiotics in treating disease was recognized in the early 1940s.
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Some antimicrobial agents are initially inactive until converted by the body to an active agent. Florey and Chain purified penicillin and capitalized on Fleming’s discovery of penicillin.
27.2 Basic Concepts of Antimicrobial Therapy Antibiotics comprise the vast majority of chemotherapeutic agents used to treat microbial diseases. As already noted, the term antibiotic originally referred to any compound produced by one species of microbe that could kill or inhibit the growth of other microbes. Today, the term is also used for synthetic chemotherapeutic agents, such as sulfonamides, that are clinically useful but chemically synthesized. There are a large number of natural and synthetic compounds that affect microbial growth. However, their utility in a clinical setting is dictated by certain key characteristics.
Antibiotics Exhibit Selective Toxicity As early as 1904, the German physician Paul Ehrlich (1854–1915) realized that a successful antimicrobial compound would be a “magic bullet” that selectively kills or inhibits the pathogen but not the host. This seemingly obvious premise was innovative at the time. Ehrlich made several discoveries based on this concept, the most celebrated of which was the arsenical compound known as salvarsan. Salvarsan proved to be quite active in killing the syphilis agent, Treponema pallidum. Syphilis, a sexually transmitted disease, had been untreatable and the source of considerable long-term suffering. Ehrlich’s “magic bullet” concept is now known as selective toxicity. Selective toxicity is possible because key aspects of a microbe’s physiology are different from those of eukaryotes. For example, suitable bacterial antibiotic targets include peptidoglycan, which eukaryotic cells lack, and ribosomes, which are structurally distinct between the two domains of life. Thus, chemicals like penicillin, which prevents peptidoglycan synthesis, and tetracycline, which binds to bacterial 30S ribosomal subunits, inhibit bacterial growth but are essentially invisible to host cells, since they do not interact with them at low doses. While their intended targets are bacterial cells, some antibiotics, particularly at high doses, can interact with elements of eukaryotic cells and cause side effects that harm the patient. For example, chloramphenicol, a drug that targets bacterial 50S ribosomal subunits, can interfere with the development of blood cells in bone marrow, a phenomenon that may result in aplastic anemia (failure to produce red blood cells). The toxicity of an antibiotic can also depend on the age of the patient. Tetracycline, for instance, can cause defects in human bone growth plates
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and should not be administered to children. Problems can even arise if the drug does not directly impact mammalian physiology. For example, many people develop an extreme allergic sensitivity toward penicillin, a situation in which the treatment of an infection may end up being worse than the infection itself. Physicians must be aware of these allergies and use alternative antibiotics to avoid harming their patients. As a student of microbiology, it is important that you distinguish between drug susceptibility and drug sensitivity. A microbe is susceptible to the drug’s action, but a human can develop an allergic sensitivity to the drug.
Antimicrobials Have a Limited Spectrum of Activity No one antimicrobial drug affects all microbes. As a result, antimicrobial drugs are classified based on the type of organisms they affect. Thus, we have antifungal, antibacterial, antiprotozoan, and antiviral agents. The term antibiotic is usually reserved for antimicrobial compounds that affect bacteria. Even within a group, one agent might have a very narrow spectrum of activity, meaning it only affects a few species, while another antibiotic will inhibit many species. For instance, penicillin has a relatively narrow spectrum of activity, primarily killing gram-positive bacteria. However, ampicillin is penicillin with an added amino group that allows the drug to more easily penetrate the gram-negative outer membrane. As a result of this chemically engineered modification, ampicillin kills gram-positive and gramnegative organisms and is described as having a spectrum of activity broader than penicillin. There are also
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antimicrobials that exhibit extremely narrow activities. One example is isoniazid, which is clinically useful only against Mycobacterium tuberculosis, the agent of tuberculosis. Table 27.1 presents the spectrum of activity of select antibiotics.
Antibiotics Are Classified as Bacteriostatic or Bactericidal Nonscientists typically believe that all antibiotics kill their intended targets. This is a misconception. Many drugs simply prevent the growth of the organism and let the body’s immune system dispatch the intruding microbe. Thus, antimicrobials are also classified on the basis of whether or not they kill the microbe. An antibiotic is bactericidal if it kills the target microbe; it is bacteriostatic if it merely prevents bacterial growth.
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Antimicrobial agents may be produced naturally or artificially. Selective toxicity refers to the ability of an antibiotic to attack a unique component of microbial physiology that is missing or distinctly different from eukaryotic physiology. Antibiotic side effects on mammalian physiology can limit the clinical usefulness of an agent. Antibiotic spectrum of activity refers to the range of microbes that a given drug affects. Bactericidal antibiotics kill microbes; bacteriostatic antibiotics inhibit their growth.
Table 27.1 Antibacterial activity (spectrum) of antimicrobial agents. Aerobic bacteria Spectrum
Anaerobic bacteria
Gram (+)
Gram (–)
Gram (+)
Gram (–)
Broad
+
+
+
+
Intermediate
+
+
+
±
+
± + ± +
+
±
+
±
+ + +
+
Narrow + + + + +
+
+
Examples Cefoxitin, chloramphenicol, imipenem, tetracyclines Carbenicillin, ticarcillin, ceftiofur, amoxicillin/ clavulanic acid, cephalosporins Ampicillin, amoxicillin Aztreonam, polymyxin Benzyl penicillin G Aminoglycosides, spectinomycin, sulfonamides, trimethoprim Fluoroquinolones Lincosamides, macrolides, pleuromutilins Bacitracin, vancomycin Nitroimidazoles
± variable activity, depending on species/strain
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MIC of ampicillin needed to stop the growth of S. aureus will be different from that needed to inhibit Shigella dysenteriae. The reasons that a drug may be more effective against one organism than another include the ease with which the drug penetrates the cell and the affi nity of the drug for its molecular target. So how do we measure MIC? As shown in Figure 27.3, an antibiotic is serially diluted along a row of test tubes containing nutrient broth. After dilution, the organism to be tested is inoculated at low, constant density into each tube, and the tubes are usually incubated overnight. Growth of the organism is seen as turbidity. Note that in Figure 27.3 the tubes with the highest concentration of drug were clear, indicating no growth. The tube containing the MIC is the tube with the lowest concentration of drug that shows no growth. However, the MIC does not tell whether a drug is bacteriostatic or bactericidal.
27.3 Measuring Drug Susceptibility One critical decision a clinician must make when treating an infection is what antibiotic to prescribe for the patient. There are several factors to consider, including: ■
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The relative effectiveness of different antibiotics on the organism causing the infection. This includes learning whether the organism isolated from a specific patient has developed resistance to the drug. The average attainable tissue levels of each drug. An antibiotic may appear to work on an agar plate, but the concentration at which it affects bacterial growth may be too high to be safe in the patient. In fact, an important aspect of designing new antibiotics is to enhance the pharmacological activity of an existing drug—for example, modifying it so that the body does not break it down or quickly secrete it in urine.
THOUGHT QUESTION 27.1 The drug tobramycin is added to a concentration of 1,000 µg/ml in a tube of broth from which serial twofold dilutions were made. Including the initial tube (tube 1), there are a total of ten tubes. Twenty-four hours after all the tubes are inoculated with Listeria monocytogenes, turbidity is observed in tubes 6–10. What is the MIC?
Minimal Inhibitory Concentration Reflects Antibiotic Efficacy The in vitro effectiveness of an agent is determined by measuring how little of it is needed to stop growth. This is classically measured in terms of an antibiotic’s minimal inhibitory concentration (MIC), defi ned as the lowest concentration of the drug that will prevent the growth of an organism. But the MIC for any one drug will differ among different bacterial species. For example, the
0.125
0.25
µg/ml 0.5 1.0
2.0
4.0
8.0
John W. Foster
0.06
THOUGHT QUESTION 27.2 What additional test performed on an MIC series of tubes will tell you whether a drug is bacteriostatic or bactericidal?
Figure 27.3 Determining minimal inhibitory concentration. In this series of tubes, tetracycline was diluted serially starting at 8 µg/ml (far right tube). Each tube was then inoculated with an equal number of bacteria. Turbidity indicates that the antibiotic concentration was not sufficient to inhibit growth. The MIC in this example is 1.0 µg/ml.
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MIC determinations are very useful for estimating a single drug’s effectiveness against a single bacterial pathogen isolated from a patient but not very practical when trying to screen 20 or more different drugs. Dilutions take time—time that the technician, not to mention the patient, may not have. The time required to evaluate antibiotic effectiveness can be reduced by using a strip test (like the Etest® shown in Fig. 27.4) that avoids the need for dilutions. The strip, containing a gradient of antibiotic, is placed over a fresh lawn of bacteria spread on an agar plate. While the bacteria are trying to grow, the drug diffuses out of the disk and into the media. Drug emanating from the more concentrated areas of the strip will travel faster and farther through the agar than will drug from the less concentrated areas of the strip. Thus, the higher concentrations will kill or inhibit the growth of cells farther from the strip than the lower concentration areas. The result is a zone of inhibition where the antibiotic has stopped bacterial growth. The MIC is the point at which the elliptical zone of inhibition intersects with the strip.
Kirby-Bauer Disk Susceptibility Test Although the strip test eliminates the time and effort needed to make dilutions, it would take 20 or more plates to test an equal number of antibiotics for just one bacterial
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Numbers reflect the relative concentrations of antibiotic that are present at various points within the zone of inhibition. The concentrations along the periphery of the clear zone are equal, and reflect the MIC, which in this case is 0.047 µg/ml.
An MIC strip test. The Etest® (AB Biodisk) is a commercially prepared strip that creates a gradient of antibiotic concentration (µg/ml) when placed on an agar plate. The MIC corresponds to the point where bacterial growth crosses the numbered strip.
John W. Foster
A.
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isolate. Clinical labs can receive up to 100 or more isolates in one day, so individual MIC determinations are not practical. A simplified agar diffusion test, however, which can test 12 antibiotics on one plate, makes evaluating antibiotic susceptibility a manageable task. Named for its inventors, the Kirby-Bauer assay uses a series of round fi lter paper disks impregnated with different antibiotics. A dispenser (Fig. 27.5) delivers up to 12 disks simultaneously to the surface of an agar plate covered by a bacterial lawn. Each disk is marked to indicate the drug used. During incubation, the drugs diffuse away from the disks into the surrounding agar and inhibit growth of the lawn to different distances. The zones of inhibition vary in width, depending on which antibiotic is used, the concentration of drug in the disk, and the susceptibility of the organism to the drug. The diameter of the zone correlates to the MIC of the antibiotic against the organism tested. Correlations between MIC values and Kirby-Bauer zone sizes are made empirically. Every disk containing a given antibiotic is impregnated with a standard concentration of drug, and every antibiotic has a quantifiable MIC that will differ when tested against different bacterial species and strains. The outermost ring of the no-growth zone in a Kirby-Bauer disk test must, by
Figure 27.5 The Kirby-Bauer disk susceptibility test. A. Device used to deliver up to 12 disks to the surface of a Mueller-Hinton plate. The device is placed over the plate, and the plunger is depressed to deliver the disks. B. Disks impregnated with different antibiotics are placed on a freshly laid lawn of bacteria and incubated overnight. The clear zones around certain disks indicate growth inhibition. Shown are the results with normal Staphylococcus aureus. C. Methicillin-resistant Staphylococcus aureus (MRSA). Note the lack of inhibition by the oxacillin disk. This strain is resistant to both methicillin and oxacillin. D. Streptococcus pneumoniae. The brownish tint of the blood agar plates outside the zones of bacterial inhibition is caused by a hemolysin secreted by the lawn of pneumococci. Penicillin (P); oxacillin (OX); erythromycin (E); clindamycin (CC); rifampin (RA); vancomycin (VA); tetracycline (TE); chloramphenicol (C); cephazolin (CZ); sulbactam-ampicillin (SAM); norfloxacin (NDR); sulfa-trimethoprim (SXT). D.
John W. Foster
C.
John W. Foster
B.
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Figure 27.4
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definition, contain the minimal concentration of drug needed to prevent growth on agar. Thus, if species A and B have MIC values for penicillin of 4 µg/ml and 40 µg/ml, respectively, then species A will exhibit a proportionally larger zone of inhibition than species B in the disk test. A graph plotting MIC on one axis and zone diameter on the other provides the correlation. After incubating agar plates in the Kirby-Bauer test, the diameters of the zones of inhibition around each disk are measured, and the results are compared with a table listing whether a zone size is wide enough (meaning the MIC is low enough) to be clinically useful. Table 27.2 shows susceptibility data for S. aureus. The concentration of antibiotic used and the zone size that is considered clinically significant are correlated with the average attainable tissue level for each antibiotic. For the antibiotic to remain effective in vivo, it is important that the tissue concentration of the drug remain above the MIC; otherwise, invading bacteria will not be affected. Correlating antibiotic MIC with tissue level. The average attainable tissue level for a drug depends on how quickly the antibiotic is cleared from the body via secretion by the kidney or destruction in the liver. It also depends on when side effects of the drug start to appear. As shown in Figure 27.6, as long as the concentration of the drug in tissue or blood remains higher than the MIC, the drug will be effective. The concentration can be kept at sufficient levels either by administering a higher dose, which runs the risk of side effects, or by giving a second dose at a time when the levels from the first dose have declined. This is why patients are told to take doses of some antibiotics four times a day and other antibiotics only once a day. To ensure reproducibility, the Kirby-Bauer test was standardized a half century ago. Reproducibility means that results from a laboratory in California will match those in Alabama, Ohio, or any other state. The following are standardizations used to make the test reproducible and easier. ■
Size of the agar plate. The plates used (150 mm) are larger than standard agar plates (100 mm) to accommodate more disks and to maintain sufficient dis-
20 18
Peak
16 Concentration (µg/ml)
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14 12 10 8 6 MIC 4 2 0 0
5
Time above MIC
10
15 Hours
20
25
30
Figure 27.6 Correlation between MIC and serum or tissue level of an antibiotic. This graph illustrates the serum level of ampicillin over time. The important consideration here is how long the serum level of the antibiotic remains higher than the MIC. Once the concentration falls below the MIC, owing to destruction in the liver or clearance through the kidneys and secretion, the infectious agent fails to be controlled by the drug−in this case about 7–8 hours after the initial dose. To maintain a serum level higher than the MIC, a second dose would be taken. The shaded area of the curve represents time above MIC.
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tance between disks so that zones of inhibition do not overlap. Depth of the media. Antibiotics diffuse out of impregnated disks in not two, but three dimensions. Because diffusion cannot occur very far downward in a thinly poured agar, the drug is forced to move more laterally. Thus, the zone of inhibition measured from a thinly poured agar plate will be larger than the zone from a thick agar plate. Media composition. Media composition can also affect results. For example, most common laboratory media contain para-aminobenzoic acid (PABA), which is used by the cell to make folic acid, a vitamin needed for purine and pyrimidine synthesis.
Table 27.2 Susceptibility results for Staphylococcus aureus. Zone of inhibition diameter (mm) Antibiotic Ampicillin Chloramphenicol Erythromycin Gentamicin Streptomycin Tetracycline
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Resistant 14 >18
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Sulfonamides are analogs of PABA that competitively inhibit one of the enzymes needed to convert PABA to folic acid. Media containing PABA, such as standard nutrient agar, will flood the bacterial cell with PABA and limit the ability of the sulfa drugs to compete for the enzyme. Thus, even though the drug might be effective in vivo, on the agar plate there would be no zone of inhibition. The standardized media used for the Kirby-Bauer test is called Mueller-Hinton agar, which contains no PABA. The number of organisms spread on the agar plate. The number of organisms placed on an agar surface is inversely proportional to the size of the zone of inhibition. The more organisms there are, the smaller the zone. The reason for this is that there is a time lag between dropping a disk on a plate and the diffusion of the antibiotic. The more organisms there are on a plate, the less time it takes for them to form visible growth; so by the time the antibiotic gets to them, visible growth has already formed. As a result, a standard optical density solution of each organism is prepared, and a cotton swab is used to spread the entire agar surface. Size of the disks. A standard diameter of 6 mm means that all antibiotics start diffusing into agar at the same point. Concentrations of antibiotics in the disks. The higher the concentration of antibiotic in a disk, the faster the drug can diffuse through the plate and kill bacteria (or at least inhibit growth) before replicating cells are able to form visible growth. To avoid differences between labs, the concentration of each given drug impregnating a disk has been standardized. Incubation temperature. Incubation temperature will not affect growth and diffusion equally. To avoid differences, a temperature of 37°C is standard.
THOUGHT QUESTION 27.3 You are testing whether a new antibiotic will be a good treatment choice for a patient with a staph infection. The Kirby-Bauer test using the organism from the patient shows a zone of inhibition of 15 mm around the disk containing this drug. Clearly, the organism is susceptible. But you conclude from other studies that the drug would not be effective in the patient. What would make you draw this conclusion?
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The spectrum of an antibiotic and the susceptibility of the infectious agent are critical points of information required before prescribing antibiotic therapy. Minimal inhibitory concentration (MIC) of a drug, when correlated with average attainable tissue levels of the antibiotic, can predict the effectiveness of an antibiotic in treating disease. MIC is measured using tube dilution techniques but can be approximated using the Kirby-Bauer disk diffusion technique.
27.4 Mechanisms of Action As noted earlier, selective toxicity of an antibiotic depends on enzymes or structures unique to the bacterial target cell. The following aspects of a microbe’s physiology are classic targets: ■ ■ ■ ■ ■ ■
Cell wall Cell membrane DNA synthesis RNA synthesis Protein synthesis Metabolism
Table 27.3 summarizes the general targets of common antibiotics. Chapters 3, 7, and 8 describe these cellular components and provide the tools needed to understand how antibiotics work.
Cell Wall Antibiotics Bacterial cell walls are an obvious structure that could be the basis for selective toxicity because peptidoglycan does not exist in mammalian cells; thus, antibiotics that target the synthesis of these structures should selectively kill bacteria. The following case history illustrates the use of two-cell-wall targeting antibiotics and also reveals how bacteria can evolve to escape destruction.
Case History: Meningitis A 3-year-old child was brought to the emergency room crying, with a stiff neck and high fever. Gram stain of cerebral spinal fluid revealed gram-positive cocci, generally in pairs. The diagnosis is
Table 27.3 Mechanisms of action of antimicrobial agents. Target
Antibiotic examples
Cell wall synthesis Protein synthesis Cell membrane Nucleic acid function Intermediary metabolism
Penicillins, cephalosporins, bacitracin, vancomycin Chloramphenicol, tetracyclines, aminoglycosides, macrolides, lincosamides Polymyxin, amphotericin, imidazoles vs. fungi Nitroimidazoles, nitrofurans, quinolones, rifampin; some antiviral compounds, especially antimetabolites Sulfonamides, trimethoprim
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meningitis. The physician immediately prescribed intravenous ampicillin. Unfortunately, the child’s condition worsened, so antibiotic treatment was changed to a third-generation cephalosporin (which will cross the blood-brain barrier). The patient began to improve within hours and was released after two days. A report from the clinical microbiology laboratory identified the organism as Streptococcus pneumoniae. Both of the antibiotics used in this case kill bacteria by targeting cell wall synthesis. Synthesis of peptidoglycan is a complex process but is represented simply in Figure 27.7. Basically, sugar molecules called N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) are made by the cell and linked together by a transglycosylase enzyme into long chains assembled at the cell wall. N-Acetylmuramic acid contains a short side chain of amino acids that is assembled enzymatically, not by a ribosome. Rigidity of the macromolecular structure, essential for maintaining cell shape, is achieved by cross-linking the side chains from 1
adjacent strands. The enzyme transpeptidase (D-alanylcarboxypeptidase/transpeptidase) catalyzes the cross-link. Figure 27.7 also indicates where several antibiotics target various stages of this assembly process. D-alanine
Peptidoglycan is assembled outside the cell membrane. Before we can explain how cell wall antibiotics
work, it is important to know how the cell wall is made. Synthesis of peptidoglycan starts in the cytoplasm with a uridine diphosphate (UDP)-NAM molecule. The amino acids L-alanine, D-glutamic acid, and L-lysine (or diaminopimelic acid in gram-negative organisms) are individually and sequentially added to NAM (Fig. 27.7, step 1); and then a dipeptide of D-alanine is attached (step 2). Next, the NAM-pentapeptide is transferred to a membranesituated, 55-carbon, lipid molecule called bactoprenol (step 3) and UMP is released. Another sugar molecule, NAG, is then linked to NAM, once again through a UDP intermediate (step 4). All of this takes place on the cytoplasmic
UDP–NAM
L-Alanine L-ala
D-Glutamic D-ala
Cycloserine
L-Lysine
2 D-ala–D-ala
Cycloserine inhibits formation of the dipeptide.
acid
(diaminopimelic acid in gram-negative bacteria)
UDP Ala Glut Lysine D-ala D-ala
Cytoplasm P 3 P
Membrane
UDP 4
UMP P
NAG UDP P
P
P
NAM
NAG
5 C55 (Bactoprenol)
Bacitracin Bactitracin inhibits incorporation of disaccharide units.
P
P P
8
NAG
P
transglycosylase
NAM
P
P
6
Vancomycin
PBPs (transpeptidases) (transglycosylases) Penicillin
Pentaglycine
7
Vancomycin and penicillin inhibit peptide cross-linking. transpeptidase
Figure 27.7 Peptidoglycan synthesis and targets of antibiotics. A series of small-molecular-weight compounds are joined to form a disaccharide unit that will be added to preexisting chains of this unit. Steps in the synthesis are described in the text. Red lines indicate inhibition. Cycloserine inhibits ligation of the two d-alanines (step 2). Bacitracin inhibits the linking of the disaccharide units, while vancomycin and the beta-lactams, such as penicillin, inhibit the peptide cross-linking of peptidoglycan side chains.
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side of the membrane. Bactoprenol carrying NAM-NAG then moves to the outer side of the cytoplasmic membrane (step 5) where transpeptidases and transglycosylases (two so-called penicillin-binding proteins) bind to the D-alaD-ala part of the pentapeptide. Transglycosylase attaches the new disaccharide unit to an existing peptidoglycan chain (step 6). Transpeptidase then links two peptide side chains with a pentaglycine cross-link (in Staphylococcus aureus). The pentaglycine connects L-lysine on one side chain and the penultimate D-ala on the other side chain (step 7). The terminal D-ala is removed in the process. Other bacteria do not use a pentaglycine cross-link but directly form a peptide bond between L-lysine (or DAP) and the penultimate D-ala. Cross-linking, which strengthens the cell wall, can take place between either the same strand or adjacent strands. In the last part of the cycle, one of the phosphates on the now liberated bactoprenol is removed and the lipid moves back to the cytoplasmic side of the membrane, ready to pick up and taxi another unit of peptidoglycan to the growing chain (step 8). Penicillin and other beta-lactam antibiotics target penicillin-binding proteins. Penicillin is an antibiotic
derived from cysteine and valine, which combine to form A.
β-Lactam ring
H N
R
S
O
N
CH3 CH3
COO Basic structure
H2N
=
SH CH
CH2
COO–
–
Cysteine
CH
+ H2N
CH3
CH3 CH COO– Valine
B. R groups R=H 6-Aminopenicillanic acid O R=
CH
C –
COO Carbenicillin
O R=
CH2
C
Penicillin G O R=
CH
C
O
NH2 Ampicillin
Figure 27.8 The structure of penicillins. A. Penicillanic acid (R = H) is derived from cysteine and valine. B. The R group marked in (A) can be any one of a number of different groups, some of which are shown here. Modifying this group changes the pharmacological properties and antimicrobial spectrum.
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1039
the beta-lactam ring structure shown in Figure 27.8A. Different R groups can be added to the basic ring structure to change the antimicrobial spectrum and stability of the derivative penicillin (Fig. 27.8B). Penicillin itself chemically resembles the D-ala-D-ala piece of peptidoglycan. This molecular mimicry allows the drug to bind transpeptidase and transglycosylase (which is why they are called penicillin-binding proteins), preventing their activities and halting synthesis of the chain. The consequence is a disaster for bacteria that are trying to grow larger and larger. Eventually, the growing cell bursts for lack of cell wall restraint. Penicillin, then, is a bactericidal drug (unless the treated organism is suspended in an isotonic solution). Penicillin is most effective against gram-positive organisms because the drug has difficulty passing through the gram-negative outer membrane. Ampicillin, which was used in the case history, is a modified version of penicillin that more easily penetrates this membrane and is more effective than penicillin against gram-negative microbes. Thus, ampicillin has a broader spectrum of activity than penicillin. As noted earlier, antibiotic resistance is a growing problem throughout the world. Bacteria develop resistance to penicillin in two basic ways. The first is through inheritance of a gene encoding one of the beta-lactamase enzymes, which cleave the critical ring structure of this class of antibiotics. Beta-lactamase is transported out of the cell and into the surrounding media (for gram-positives) or the periplasm (for gram-negatives), where it can destroy penicillin before the drug even gets to the cell. Bacteria that produce beta-lactamase are still susceptible to certain modified penicillins and cephalosporins engineered to be poor substrates for the enzyme. Methicillin, for example, works well against beta-lactamase-producing microbes. The second way a microbe can become resistant is through mutations in genes encoding key penicillinbinding proteins. Resistance occurs when the mutated gene produces an altered protein that no longer binds to the antibiotic. Methicillin-resistant bacteria use this strategy. Hospitals take special interest in methicillinresistant S. aureus (MRSA) because very few drugs can kill it. One of the few remaining antibiotics effective against MRSA is vancomycin. Unfortunately, resistance to this drug is also developing. The penicillin-resistant S. pneumoniae in the preceding case history actually had an altered penicillin-binding protein. No beta-lactamase producing S. pneumoniae has ever been found. Cephalosporins are another type of beta-lactam antibiotic originally discovered in nature but modified in the laboratory to fight microbes that are naturally resistant to penicillins (especially Pseudomonas aeruginosa). Over the years, the basic structure of cephalosporin has undergone a series of modifications to improve its effectiveness against penicillin-resistant pathogens. Each modification is increasingly more complex and produces what is referred to as a new “generation” of cephalosporins.
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C h ap t e r 27
There are currently four generations of this semisynthetic antibiotic (Fig. 27.9). Unfortunately, the microbial world constantly adapts and eventually becomes resistant to new antibiotics. In the case of the cephalosporins, new beta-lactamases evolve that can attack the sterically buried beta-lactam rings in these molecules. It is also important to note that since the core feature of these drugs is the beta-lactam ring, persons who are sensitive to penicillins may also suffer a hypersensitivity reaction to cephalosporins. Treatment note: The infecting strain of S. pneumoniae in the preceeding case history turned out to be resistant to ampicillin. If the patient had been an adult, a fluoroquinolone (discussed in Chapter 7) might have been the best secondary drug of choice because its target, a type II topoisomerase, is unrelated to cell wall synthesis. However, quinolones are not recommended for children, as in this case, due to potential side-effects. Other beta-lactam A. Cephalexin
S O H2N
O
N N
OH O
H
B. Cefoxitin
COO– O
S
O N
C
N O H
H
OCONH2 S
CH3 C. Ceftriaxone
NH2 N
S
O
H
H
N H3C
O
N
H3C
S
N
O
N
S
HO
N
OH
N
O
O
D. Cefepime
S
H2N N
C
O
H
C
N
S
N
N
OCH3 O –
OOC
CH2
+
N
H 3C
Figure 27.9 Cephalosporin generations. Representative examples. A. First generation: cephalexin (Keflex). B. Second generation: cefoxitin. C. Third generation: ceftriaxone. D. Fourth generation: cefepime. Note that with each successive generation, the side groups become more complex. Highlighted areas indicate the core structure of each of the cephalosporins, with beta-lactam rings.
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antibiotics, such as the third-generation cephalosporins, may still work on penicillin-resistant S. pneumoniae since the modified antibiotic often can still bind the altered PBP. Nevertheless, vancomycin or an oxazolidinone are the best last choices, since cephalosporin-resistant strains of S. pneumoniae are now appearing. NOTE: Archaea peptidoglycan contains talosaminuronic acid instead of muramic acid and lacks the Damino acids found in bacterial peptidoglycan. Because of this, archaea are insensitive to penicillins, which interfere with bacterial transpeptidases.
Cell wall antibiotics target other steps in peptidoglycan synthesis. Another antibiotic that affects cell wall
synthesis is bacitracin, a large polypeptide molecule produced by Bacillus subtilis and Bacillus licheniformis (Fig. 27.10A). The antibiotic inhibits cell wall synthesis by binding to the bactoprenol lipid carrier molecule that normally transports monomeric units of peptidoglycan across the cell membrane and to the growing chain (see Fig. 27.7). Bacitracin binds to and inhibits dephosphorylation of the carrier, thereby preventing the carrier from accepting a new unit of UDP-NAM. Resistance to bacitracin can develop if the organism can rapidly recycle the phosphorylated lipid carrier molecule through dephosphorylation or if the organism possesses an efficient drug export system (discussed in Section 27.6). Normally, bacitracin is used only topically because of serious side effects, such as kidney damage, that can occur if it is ingested. Cycloserine (made by Streptomyces garyphalus) is one of several antimicrobials used to treat tuberculosis (Fig. 27.10B). Relative to bacitracin, it acts at an even earlier step in peptidoglycan synthesis. Cycloserine inhibits the two enzymes that make the D-ala-D-ala dipeptide. As a result, the complete pentapeptide sidechain on N-acetylmuramic acid cannot be made (see Fig. 27.7). Without these alanines, cross-linking cannot occur and peptidoglycan integrity is compromised. Vancomycin, a very large and complex glycopeptide produced by Amycolatopsis orientalis (Fig. 27.10C), binds to the D-ala-D-ala terminal end of the disaccharide unit and prevents the action of transglycosylase and transpeptidases (see Fig. 27.7). The mechanism of resistance is very different for vancomycin and penicillin. This difference makes vancomycin particularly useful against penicillinresistant bacteria. To prevent development and spread of vancomycin-resistant bacteria, this antibiotic is typically used only as a drug of last resort. Resistance can develop when products from a cluster of van genes collaborate to make D-lactate and incorporate it into the ester D-ala-Dlactate to which vancomycin cannot bind. Another enzyme in the van gene cluster prevents the accumulation of D-alaD-ala; as a result, the lactate version is incorporated into peptidoglycan. Peptidoglycan containing D-ala-D-lactate
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B. Cycloserine
A. Bacitracin
CH3
CH3 CH3 N
NH2
H3C
O
O NH
OH N H
O
O
H N
H3C
H3C
S
H3C
N H
H N
N H
NH O
H N
NH2 CH3
NH
HO H3C
O
O
O
O
HN
O
HO
CH3 O
CH3
O O HN – OOC H
O NH O
OH O O
O Cl
HN
O N
C. Vancomycin
O H 2N
1041
M edic ine and I mm u n o l o gy
HO
N H H H
O
O
Cl H N
O
OH OH
OH
N OH H O
H N H O NH2
O N H
H N
Me Me
Me
OH OH
OH
Figure 27.10 Other antibiotics that affect peptidoglycan synthesis. A. Bacitracin is produced by Bacillus subtilis. It is generally used only topically to prevent infection. B. Cycloserine, an analog of d-alanine, is one of several drugs used to treat tuberculosis. C. Vancomycin is a cyclic polypeptide made by Amycolatopsis orientalis, previously classified as a streptomycete. These antibiotics, especially bacitracin and vancomycin, are synthesized by exceedingly complex biochemical pathways in the producing organisms.
functions just fi ne, but since vancomycin cannot bind the D-lactate form, the organism is resistant to the antibiotic. It is important to note that antibiotics targeting cell wall biosynthesis generally only kill growing cells. These drugs do not affect static or stationary phase cells because in this state the cell has no need for new peptidoglycan.
Gramicidin channel
Gramicidin
THOUGHT QUESTION 27.4 When treating a patient for an infection, why would combining a drug such as erythromycin with a penicillin be counterproductive?
Lipid bilayer
Drugs That Affect Bacterial Membrane Integrity Poking holes in a bacterial cytoplasmic membrane is an effective way to kill bacteria. There are a few compounds useful in this regard, among them a group called the peptide antibiotics, of which gramicidin is an example. Gramicidin, produced by Bacillus brevis, is a cyclic peptide composed of 15 alternating D- and L-amino acids. It inserts into the membrane as a dimer, forming a cation channel that disrupts membrane polarity (Fig. 27.11). Polymyxin (from Bacillus polymyxa), another polypeptide antibiotic, has a positively charged polypeptide ring that binds to the outer (lipid A) and inner membranes of bacteria, both of which are negatively charged. Its major lethal effect seems to be to destroy the inner membrane, much like a detergent. These antibiotics are used only topically to treat or prevent infection. Because they can also form channels across human cell membranes, they are never ingested. Polymyxin has been fused to some bandage materials used to treat burn patients who are particularly susceptible to gram-negative infections (e.g., P. aeruginosa).
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Figure 27.11 Gramicidin is a peptide antibiotic that affects membrane integrity. Gramicidin forms a cation channel through cell membranes through which H+, Na+, or K+ can freely pass. (PDB code: 1grm)
Drugs That Affect DNA Synthesis and Integrity Bacteria generally make and maintain their DNA using enzymes that closely resemble those of mammals. Thus, you might consider it impossible to selectively target bacterial DNA synthesis. As you will see, this is not the case.
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Case History: Pneumonia Due to a Gram-Negative Anaerobe
Because humans lack that pathway, sulfa drugs are selectively toxic toward bacteria.
A 23-year-old woman arrived at the emergency room by ambulance with fever, chills, and severe muscle aches. She developed a nonproductive cough, had difficulty breathing, had pleuritic chest pain, and became hypotensive (low blood pressure). An X-ray showed lower lobe infiltrate in the lungs, and the clinical laboratory reported the presence of the gram-negative anaerobe Fusobacterium necrophorum in blood cultures. The patient was diagnosed with pneumonia and treated with metronidazole, a DNA-damaging agent specific for anaerobes. She fully recovered.
Quinolones. Another group of drugs inhibit DNA syn-
thesis by targeting microbial topoisomerases such as DNA gyrase and topoisomerase IV (discussed in Section 7.2). Because these enzymes are structurally distinct from their mammalian counterparts, drugs can be designed to selectively interact with them without interfering with mammalian DNA metabolism. In 1963, one such drug, nalidixic acid, was discovered as a by-product of the synthesis of chloroquine, an antimalarial drug. Nalidixic acid, which targets DNA gyrase, has a very narrow antimicrobial spectrum, covering only a few gram-negative organisms. However, various chemical modifications, such as adding fluorine and amine groups, have increased its antimicrobial spectrum and its half-life in the bloodstream. The result is the class of drugs known as the quinolones. (The mode of action of quinolones and fluoroquinolones was discussed in Section 7.2.)
There are several classes of drugs, including sulfa drugs, quinolones, and metronidazole, that selectively affect the synthesis or integrity of DNA in microorganisms. Sulfa drugs. The sulfa drugs, originally discovered
by Domagk, belong to a group of drugs known as antimetabolites because they interfere with the synthesis of metabolic intermediates. Ultimately, the sulfa drugs act to inhibit the synthesis of nucleic acids. Drugs such as sulfamethoxazole work at the metabolic level to prevent the synthesis of tetrahydrofolic acid (THF), an important cofactor in the synthesis of nucleic acid precursors (Fig. 27.12). All organisms use THF to synthesize nucleic acids, so why are the sulfa drugs selectively toxic to bacteria? The selectivity occurs because mammalians do not synthesize a precursor of THF called folic acid. Higher mammals generally rely on bacteria and green leafy vegetables as sources of folic acid. Bacteria make folic acid from the combination of PABA, glutamic acid, and pteridine. Sulfamethoxazole, a structural analog of PABA, competes for one of the enzymes in the bacterial folic acid pathway and inhibits both folic acid and THF production (Fig. 27.12C). A. COO–
H2N
Para-aminobenzoic acid (PABA)
H2 N
SO2NH2
Sulfanilamide (SFA)
Metronidazole. Also known as Flagyl, metronidazole
is an example of a drug that is harmless until activated, known as a prodrug. Metronidazole is activated after receiving an electron (is reduced) from the microbial protein cofactors flavodoxin and ferredoxin, found in microaerophilic and anaerobic bacteria such as Bacteroides (Fig. 27.13). Once activated, the compound begins nicking DNA at random, thus killing the cell. Because the etiological agent in our case history was an anaerobe, metronidazole was an effective therapy. Metronidazole is also effective against protozoans such as Giardia and Entamoeba. Aerobic microbes, although in possession of ferredoxin, are incapable of reducing metronidazole, presumably because oxygen is reduced in preference to metronidazole.
C. Normal folic acid formation
P
PABA
G P PABA G
B. Pteridine (P)
Folic
H N
Synthesis
Enzyme
COO– Glutamic acid (G) PABA
acid
Folic acid formation blocked
Folic acid
P
SFA
G
P
P SFA G
SFA
G
No synthesis
Enzyme
Figure 27.12 Mode of action of sulfanilamides. A. The structures of PABA and sulfanilamide are very similar. B. PABA, pteridine, and glutamic acid combine to make the vitamin folic acid. C. Normal synthesis of folic acid requires that all three components engage the active site of the biosynthetic enzyme. The sulfa drugs replace PABA at the active site. The sulfur group, however, will not form a peptide bond with glutamic acid, and the size of sulfanilamide sterically hinders binding of pteridine so folic acid cannot be made.
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O2N
CH3
N
CH2CH2OH
Single e– transfers
N
Nitrose free radical form of metronidazole
–
ON
N
CH3
CH2CH2OH
Figure 27.13 Activation of metronidazole. Single-electron transfers are made by ferredoxin and flavodoxin from anaerobes. Ferredoxin and flavodoxin are reducing agents capable of reducing very low redox potential compounds.
THOUGHT QUESTION 27.5 The enzyme DNA gyrase, a target of the quinolone antibiotics, is an essential protein in DNA replication. The quinolones bind to and inactivate this protein. Research has proved that quinolone-resistant mutants contain mutations in the gene encoding DNA gyrase. If the resistant mutants contain a mutant DNA gyrase and DNA gyrase is essential for growth, why are these mutations not lethal?
CH3 CH3 OH
O
CH3
OH OH
Antibiotics that inhibit transcription, such as rifampicin and actinomycin D (Fig. 27.14), were described in Special Topic 8.1. These drugs are considered bactericidal in action and are most active against growing bacteria. The tricyclic ring of actinomycin D binds DNA from any source. As a result, it is not selectively toxic and not used to treat infections. Rifampin (also called rifampicin), on the other hand, does exhibit selective toxicity and is often prescribed for treatment of tuberculosis or meningococcal meningitis. Curiously, because rifampin is reddish orange, it turns bodily secretions, including breast milk, orange. The astute physician will warn the patient of this highly visible but harmless side effect to avoid unnecessary anxiety when the patient’s urine changes color.
Protein Synthesis Inhibitors
NH N
O O
O
CH3
N
N
CH3
OH
Rifampin
RNA polymerase residues D. Actinomycin D and DNA
C. Actinomycin D Pro D-Val
Sar
Me-Val
Pro
O Thr
Sar
Me-Val
DNA
O D-Val Thr O
O N
NH2 O
O CH3
CH3
Actinomycin D Antibiotics that inhibit transcription. A. Rifampin. B. Rifampin-binding site on the RNA polymerase beta subunit. (PDB code: 1i6v) C. Actinomycin D. D. Actinomycin D (yellow and red) interacting with DNA. Covalent intercalation of actinomycin interferes with DNA synthesis and transcription. (PDB code: 1DSC)
Figure 27.14
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1043
The differences between prokaryotic and eukaryotic ribosomes account for the selective toxicity of antibiotics that specifically inhibit bacterial protein synthesis. How various antibiotics inhibit protein synthesis was discussed in Section 8.3. We recommend reviewing Chapter 8 for details. As a brief review, protein synthesis inhibitors can be classified into several groups based on structure and function (Fig. 27.15). Note that most of these antibiotics work by binding and B. Rifampin and RNA polymerase interfering with the function of bacterial rRNA, which differs from eukaryotic rRNA. Also recall that protein synthesis inhibitors are, by and large, bacteriostatic.
A. Rifampin HO O H3C O H3C H3C O H3C H3C
M edic ine and I mm u n o l o gy
RNA Synthesis Inhibitors
N
Prodrug form of metronidazole
■
Case History: Erysipelas in a Penicillin-Sensitive Patient Sixteen-year-old Jamal arrived at the emergency room after two days of fever, malaise, chills, and neck stiffness. His most notable symptom was a painful, red, rapidly spreading rash covering the right side of his face. The rash covered his entire cheek, which was swollen, and extended into his scalp. About seven days earlier, Jamal had a severe sore throat. However, since it subsided in two days, he was not clinically evaluated. Throat cultures taken on admission revealed group A Streptococcus pyogenes, suggesting that the rash was a case of erysipelas caused by this organism. Although penicillin would be the drug of choice, Jamal is known to be allergic to this antibiotic.
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A. Gentamicin H2N Cyclohexane ring O HO Amino sugar O
OH
O
O
C. Erythromycin D O
CONH2
HO O OH
H3C Lactone ring HO H3C H3C
NH2
HO H3C
R1 O H NH2 HN R2
B. Doxycycline CH3 OH N(CH3)2 H H OH
CH3
O
HNCH3
E. Chloramphenicol
O
OH CH3
F. Linezolide O
CH3 N H
CH3 N HO O CH3 CH3
H3CO
D. Clindamycin
H H N O HO
H Cl O2N
CH3
H OH O
CH3
O
CH3
OH
H3C
OH CH3 O
OH H3C O
H
NH
C
C
C
O CHCl2
N
O
O
H
OH CH2OH SCH3
F
N
O H
C N H
CH3
OH
Figure 27.15 Protein synthesis inhibitors. A. The aminoglycoside gentamicin. B. The tetracycline doxycycline. C. The macrolide antibiotic erythromycin. D. The lincosamide antibiotic clindamycin. E. Chloramphenicol. F. Linezolide.
When a patient is known to be immunologically sensitive to the usual drug of choice, a structurally distinct drug is best. Often, that drug will be one that inhibits protein synthesis; in this case, the drug chosen was the macrolide azithromycin. Drugs that inhibit protein synthesis can be subdivided into different groups based on their structures and on what part of the translation machine is targeted.
Drugs That Affect the 30S Ribosomal Subunit The classification of antibiotics affecting protein synthesis is initially based on the bacterial ribosomal subunit targeted. Thus, one class of antibiotics interferes with 30S subunit function, and the other scrambles 50S subunit activities. Aminoglycosides. There is considerable variation in
structure among different aminoglycosides, but all contain a cyclohexane ring and amino sugars (Fig. 27.15A). The aminoglycosides are unusual among protein synthesis inhibitors in that they are bactericidal rather than bacteriostatic. Most of them bind 16S rRNA and cause translational misreading of mRNA, which is why these drugs are bactericidal. The resulting synthesis of jumbled peptides wreaks havoc with physiology and kills the cell. Streptomycin and gentamicin (see Fig. 27.15A) are two widely used drugs in this class. Ototoxicity (hearing damage) is a major, but uncommon, side effect of these antibiotics (approximately 0.5–3% of patients treated with gentamicin suffer from this toxicity). Hearing is generally affected at frequencies above 4,000 Hz.
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Tetracyclines. Tetracycline antibiotics are characterized by a structure with four fused cyclic rings; hence the name. Figure 27.15B shows one frequently used example, called doxycycline. Tetracyclines are bacteriostatic and work by binding to and distorting the ribosomal A site that accepts incoming charged tRNA molecules. Doxycycline is used to treat early stages of Lyme disease (Borrelia burgdorferi), acne (Corynebacterium acne), and other infections. An important adverse side effect of tetracyclines is that they can interfere with bone development in a fetus or young child. Tetracycline use by pregnant mothers will also cause yellow discoloration of the infant’s teeth. As a result, this drug is not recommended for pregnant women or nursing mothers.
Drugs That Affect the 50S Subunit Five classes of drugs subvert translation by binding to the 50S ribosomal subunit. Most of these drugs were discussed in Chapter 8 and will be recapped here only briefly. ■
Macrolides, all of which contain a 16-member lactone ring (Fig. 27.15C), inhibit translocation of the growing peptide (bacteriostatic action). Commonly prescribed examples are erythromycin and azithromycin. Azithromycin was the antibiotic used to treat the S. pyogenes infection in our case history, although other drugs could have been used. Because it is structurally dissimilar to any of the beta-lactam antibiotics, such as penicillin, it can be used safely in patients who are penicillin sensitive.
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■
■
■
Lincosamides (Fig. 27.15D), such as clindamycin, are similar to macrolides in function but have a different structure. Chloramphenicol (Fig. 27.15E) inhibits peptidyl transferase activity (bacteriostatic). Bone marrow depression leading to aplastic anemia is the most common serious side effect and limits its clinical use. Oxazolidinones (Fig. 27.15F) are a recently discovered class of antibiotics effective against many antibiotic-resistant microbes. In fact, this is the fi rst new class of antibiotics discovered in over 35 years. Oxazolidinones such as linezolid bind to the 23S rRNA in the 50S subunit of the prokaryotic ribosome and prevent formation of the protein synthesis 70S initiation complex. This is a novel mode of action; other protein synthesis inhibitors either block polypeptide extension or cause misreading of mRNA. Linezolid binds to the 50S subunit near where chloramphenicol binds, but does not inhibit peptidyl transferase. Resistance is limited because most bacterial genomes have multiple operons encoding 23S rRNA. Usually more than one of these genes must mutate to confer high-level resistance. The more unmutated 23S rRNA genes there are, the more ribosomes susceptible to the antibiotic will be present. Oxazolidinones are useful primarily against gram-positive bacteria. Gram-negative bacteria are intrinsically resistant because of multidrug efflux pumps and decreased permeability due to the outer membrane. Streptogramins (Fig. 27.16), produced by some Streptomyces species, fall into two groups, A and B. Streptogramins belonging to group A have a large nonpeptide ring. Streptogramin B members are cyclic peptides. The two groups differ in their modes of action, although both inhibit bacterial protein synthesis by binding to the peptidyl transferase site. Group A streptogramins bound to the peptidyl transferase site distort the ribosome to prevent binding of tRNA to the ribosome A site. In contrast, group B streptogramins are thought to narrow the peptide exit channel, preventing exit of the peptide and thereby blocking translocation.
Natural streptogramins are produced as a mixture of A and B, the combination of which is more potent than either individual compound alone (an example of synergy). In tribute to this synergistic action, the drug combination is marketed under the name Synercid. Synergy between the two drugs occurs because the A type streptogramin alters the binding site for the B type drug, increasing its affi nity. Bacteria can develop resistance either through ribosomal modification (the modification in 23S rRNA is the same one that provides resistance to macrolides), via the production of inactivating enzymes, or by active efflux of the antibiotic.
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M edic ine and I mm u n o l o gy
A. Streptogramin A O H3C H3C
OH
N H
O
O
CH O
H 3C
O N
N
O
CH3 B. Streptogramin B OH N
CO NH
H3C
CH
CH
Peptide bond CO
NH
CH3 CH2 CH
H2C CO
CH2 CH2
N
CH CO
O N OC
CH NH CO
CH
N
H2C
CH2
OC
CH2
Figure 27.16 The streptogramins. A. Streptogramin A is a large nonpeptide ring structure. B. Streptogramin B is a cyclic peptide.
CO
CH3
CH CH2
N H3C
CH3
TO SU M MAR I Z E: ■
■
■
■
■
■
■
Antibiotic specificity for bacteria can be achieved by targeting a process that occurs only in bacteria, not host cells; by targeting small structural differences between components of a process shared by bacteria and hosts; or by exploiting a physiological condition such as anaerobiosis present only in certain bacteria. Antibiotic targets include cell wall synthesis, cell membrane integrity, DNA synthesis, RNA synthesis, protein synthesis, and metabolism. Antibiotics targeting the cell wall bind to the transglycosidases, transpeptidases, and lipid carrier proteins involved with peptidoglycan synthesis and cross-linking. Antibiotics interfering with DNA include the antimetabolite sulfa drugs that inhibit nucleotide synthesis, quinolones that inhibit DNA topoisomerases, and a drug, metronidazole, that, when activated, randomly nicks the phosphodiester backbone. Inhibitors of RNA synthesis target RNA polymerase (rifampin) or bind DNA and inhibit polymerase movement (actinomycin D). Aminoglycosides and tetracyclines bind the 30S subunit of the prokaryotic ribosome. A variety of antibiotics bind the 50S ribosomal subunit and inhibit translocation (macrolides, lincosamides), peptidyl transferase (chloramphenicol), formation of 70S complex (oxazolidinones), or peptide exit through the ribosome exit channel (streptogramins).
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Core biosynthetic pathway for penicillin and the cephalosporins. A. Penicillin and cephalosporins share a biochemical pathway. The pcbAB, pcbC, and penDE genes encode ACV synthetase, IPN synthase, and IPN acyltransferase, respectively. CefD is required to make penicillin N, a precursor of the cephalosporins, and CefE is a synthase that makes the first cephalosporin structure in the biosynthetic sequence.
Figure 27.17
A.
H
H H3N+ –
OOC
[CH2]3
COO–
+
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+
H3N+
H [CH2]3
–
OOC
CO
H3N
H
L-Cysteine
3 ATP 3 AMP + 3 P Pi
27.5 Antibiotic Biosynthesis
H N
O
H
S
C
CH2
C
NH2
C
+
CH2 COO–
L-2-Aminohexandioate L-2-Aminoadipate
pcbAB
Antibiotics are considered secondary metabolites because they often have no apparent use in the producing organism. This most likely means a purpose has yet to be identified. Although the production of antibiotics is thought to help one microbe compete with another in nature, growth inhibition may not have been the original purpose of secondary metabolite production. The relative complexity of the biosynthetic pathways suggests a more immediate purpose—for example, cell-cell signaling— that evolved into cross-species inhibition. Antibiotic biosynthetic pathways are sometimes relatively simple (as in penicillin) and sometimes quite complex. Figure 27.17A outlines the relatively straightforward biosynthetic pathway used to make penicillin and the related cephalosporins, all of which contain a beta-lactam ring. Note that the ring results from the joining of two amino acids, cysteine and valine. Several different types of fungi can produce these drugs. All use similar pathways and evolutionarily related genes (Fig. 27.17B). Polyketide antibiotics such as erythromycin (Fig. 27.15D) are synthesized in a modular fashion by modular polyketide synthase enzymes (discussed previously in Special Topic 15.2). The synthesis strategy is reminiscent of the way fatty acids are made. Malonyl-ACP units containing different R groups are successively condensed by modular subunits of the polyketide synthase complex until elongation is terminated by the thioesterase. The repeating forms of polyketides enable these longchain molecules to be synthesized by a small number of enzymes. Curiously, the peptide antibiotics such as bacitracin or the gramacidins are not synthesized using ribosomes. Complex biosynthetic pathways are involved that do not rely on mRNA. Nonribosomal synthesis of peptide antibiotics is discussed in Section 15.6 and Special Topic 15.3. One theory of why certain microbes make antibiotics is that they prevent the growth of competing organisms, but how do microbes that make antibiotics avoid committing suicide? Fungi that make penicillin do not face any consequence for having done so because the organism does not contain peptidoglycan. Actinomycetes that produce compounds such as streptomycin or chloramphenicol, however, could be susceptible to their own secondary metabolite. Ribosomes isolated from Streptomyces griseus, for example, are fully sensitive to the streptomycin pro-
H
S
H H3N+
H H
C
CH3 CH3
COO– L-Valine
The dotted lines show where the bonds will be formed.
CH3
C
CH3
C
COO– H (ACV) N-[(5S)-5-amino-5-carboxypentanoyl]L-cysteinyl-D-valine O2
pcbC
2 H2O H3N+
H [CH2]3
–
OOC
CO
H N
O
H
H
C
C
C
N
S
CH3 penDE
C
Penicillin G
CH3
C
COO–
H Isopenicillin N cefD
H3N+
H
–
OOC
[CH2]3
CO
H N
O
H
H
C
C
C
N
S
CH3
C
CH3
C
COO–
H Penicillin N
O2 + 2-oxoglutarate
cefE
H2O + CO2 + succinate + H
H3N –
OOC
[CH2]3
CO
H N
O
H
H
C
C
CH2
C
N
C
S
C
CH3
Other cephalosporins
–
COO Deacetoxycephalosporin C
duced by this organism. S. griseus avoids killing itself in two ways. First, the organism synthesizes an inactive precursor of streptomycin, 6-phosphorylstreptomycin, which is secreted from the cell and, once outside the mycelium, becomes activated by a specific phosphatase. In addition, this streptomycete has an enzyme that inactivates any streptomycin that may leak back into the mycelium. Other organisms protect themselves by methylating key residues on their rRNA to prevent drug binding or set up permeability barriers that thwart reentry of the antibiotic.
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B.
ACV synthase
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IPN synthase IPN acyltransferase
P. chrysogenum chromosome I
pcbAB
pcbC
A. nidulans chromosome VI
pcbAB
pcbC
A. chrysogenum chromosome VI
pcbAB
pcbC
penD
penD
Late genes N. lactamdurans
S. clavuligerus
pbp
cefE
cmcT
cefD
cefE
cefD
cefF
lat
pcbAB
pcbC
lat
pcbAB
pcbC
bla
B. Penicillin gene clusters in various fungi that synthesize penicillins and cephalosporins. The arrows indicate the orientation of the genes. Note that the pcbAB-pcbC promoter region is transcribed bidirectionally. Genes involved in cephalosporin synthesis are labeled cef. The bla gene encodes a beta-lactamase that protects the producing organism Nocardia lactamdurans and appears to serve as an ancestral homolog to bla genes found in clinical pathogens.
Figure 27.17 (continued)
THOUGHT QUESTION 27.6 Why might a combination therapy of an aminoglycoside antibiotic and cephalosporin be synergistic? THOUGHT QUESTION 27.7 Could genomics ever predict the drug resistance phenotype of a microbe? If so, how? TO SU M MAR I Z E: ■
■
■
Antibiotics are synthesized as secondary metabolites. Microbes may make antibiotics to eliminate competitors in the environment. Antibiotic producers prevent self-destruction by various antibiotic resistance mechanisms.
27.6 The Challenges of Antibiotic Resistance In this section, we discuss the various ways microbes can become resistant to antibiotics and explore the evolutionary road to resistance. In addition, we address how resistance moves between species and how we can deal with the spread of resistance throughout the world.
Case History: Multiple-Drug-Resistant Pneumonia A 14-year-old boy with fever (39°C), chills, and left-sided pleuritic chest pain was referred to a hospital emergency department by his general practitioner. A chest X-ray showed left
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lower lobe pneumonia. The boy reported that he was allergic to amoxicillin and cephalosporins (as a child he had developed a rash to these agents) and had been taking daily doxycycline (tetracycline) for the previous three months to treat mild acne. He was admitted to the hospital and treated with intravenous erythromycin because of his reported beta-lactam allergies, but he continued to feel sick. The day after admission, both sputum and blood cultures grew Streptococcus pneumoniae. After 48 hours, antibiotic susceptibility results indicated that the microbe was resistant to penicillin, erythromycin, and tetracycline. Armed with this information, the clinician immediately changed antibiotic treatment to vancomycin. The boy’s fever resolved over the next 12 hours, and he made a slow but full recovery over the next week. Unfortunately the scenario presented in this case is far too common and has become an extremely serious concern. Figure 27.18 illustrates the rapid rise of penicillin resistance among S. pneumoniae in the world. Another instance of emerging antibiotic resistance is unfolding in Europe and the Far East. The non-Enterobacteriaceae gram-negative rod Acinetobacter baumanii is increasingly seen as a dangerous cause of nosocomial infections. It commonly colonizes hospitalized patients, particularly those in intensive care units. Before 1998, there were almost no cases of multidrug-resistant A. baumanii. The rate is now as high as 8%. The organism is resistant to drugs as diverse as ciprofloxacin (a quinolone), amikacin (an aminoglycoside), penicillins, third-generation cephalosporins, tetracycline, and chloramphenicol. Imipenem, a relatively new class of beta-lactam drugs, is currently useful, but resistance to it is also likely to develop.
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80
Spain
60
40
20
Spain
% of S. pneumoniae strains showing penicillin resistance
100
Hungary Iceland Sweden Norway Finland United Kingdom Ireland Luxembourg The Netherlands Germany Austria Belgium France Italy Greece Crete Portugal Spain Hungary Poland Slovenia Czech Republic Croatia USA Brazil Ivory Coast South Africa Saudi Arabia Kuwait Vietnam Sri Lanka Taiwan China Korea Japan
1048
0 1940: Introduction of penicillin
1967: 1979 1990s First report of a penicillin-resistance strain
2000s
Figure 27.18 The rise of penicillin-resistant Streptococcus pneumoniae throughout the world. Numbers reflect the number of penicillin-resistant strains among clinical isolates (strains of disease-causing bacteria isolated from patients).
There are four basic forms of antibiotic resistance. The resistant organism can: ■
■
■
■
Modify the target so that it no longer binds the antibiotic. Mutations in key penicillin-binding proteins and ribosomal proteins, for instance, can confer resistance to methicillin and streptomycin, respectively. These mutations occur spontaneously and are not typically transferred between organisms. Destroy the antibiotic before it gets into the cell. One example is the enzyme beta-lactamase (or penicillinase), which is made exclusively to destroy penicillins. The sites of ring cleavage and structure of the enzyme are illustrated in Figure 27.19. Add modifying groups that inactivate the antibiotic. For instance, there are three classes of enzymes that modify and inactivate aminoglycoside antibiotics. The results of these types of enzyme modifications are illustrated for kanamycin in Figure 27.20. Pump the antibiotic out of the cell using specific (for example, tetracycline export) and nonspecific transport proteins. This strategy works because the pumps bail drugs out of the cell faster than the drugs can get in. Some are single-component pumps present in the cytoplasmic membrane of gram-negative and gram-positive bacteria (for example, NorA in S. aureus, PmrA in S. pneumoniae, and the TetA and B proteins in gram-negatives). Other drug efflux pumps are multicomponent systems present in gram-negative bacteria only (discussed shortly). Efflux in either case is usually energized by proton motive force.
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A.
Enzyme-Ser
H
H
R
CONH
C
C
OH
+
C
N
O
S
CH3 C
C COO
CH3 –
β-Lactam B.
Penicillin
Figure 27.19 Destroying penicillin. A. Beta-lactamase (or penicillinase) cleaves the beta-lactam ring of penicillins and cephalosporins. There are two types of penicillinases, based on where the enzyme attacks the ring. In either type, a serine hydroxyl group launches a nucleophilic attack on the ring. B. Structure of a beta-lactamase and location of the penicillinbinding site. (PDB code: 1XX2)
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■
Pa r t 5
A.
HO
6′
HO
6′
HO
AAC (6′)-li H2N
HO H2N
O
NH2
HO
+
HO
NH3 O
O
NH2
H3N
Aminoglycoside phosphotransferase (APH) catalyzes ATP-dependent phosphorylation (yellow) of a hydroxyl group. OH + NH3
HO + O NH3
HO +
OH O
+
HO
NH3 O
O
HO +
H3N
OH O
HO O + NH3
Kanamycin
APH
P
–
HO
ANT
OH
+
H3N
O
OH + NH3
HO O NH3 +
Kanamycin (3′)-phosphate
Aminoglycoside adenylyltransferase (ANT) catalyzes ATP-dependent adenylation (yellow) of a hydroxyl group. OH + NH3
HO O
O
HO
O
O–
HO
+
+
ATP
THOUGHT QUESTION 27.9 Mutations in the ribosomal protein S12 (encoded by rpsL) confer resistance to streptomycin. Given a cell containing both rpsL+ and rpsLR genes, would the cell be streptomycin resistant or sensitive? (Recall that genes encoding ribosome proteins for the small subunit are designated rps. A + indicates the wild-type allele, while R indicates a gene whose product is resistant to a certain drug.) A particularily dangerous type of drug resistance is mediated by what are called multidrug resistance (MDR) efflux pumps (Fig. 27.21). A single pump in
O
O
HO P Pi
HO
NH3
AMP
THOUGHT QUESTION 27.8 Fusaric acid is a cation chelator that normally does not penetrate the E. coli membrane, which means E. coli is typically resistant to this compound. Curiously, cells that develop resistance to tetracycline become sensitive to fusaric acid. Resistance to tetracycline is usually the result of an integral membrane efflux pump that pumps tetracycline out of the cell. What might explain the development of fusaric acid sensitivity?
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+
NH3
ADP
ATP
C.
HO
O O
Kanamycin
HO
NH2
HO
B.
HO
O
HO
HO
HO
H N O
O
NH2 CoASH AcCoA
1049
Figure 27.20 Aminoglycoside-inactivating enzymes. Different enzymes can inactivate aminoglycoside antibiotics.
Aminoglycoside acetyltransferase (AAC) catalyzes acetyl-CoAdependent acetylation of an amino group.
NH2 O
M edic ine and I mm u n o l o gy
H3 N
OH O
OH + NH3
HO O + NH3
4′-Adenylyl kanamycin
Multi-drug exporter Outer membrane channel
Outer membrane Accessory protein
Periplasm
Transporter Cytoplasmic membrane
Figure 27.21 Basic structure of a multidrug efflux pump in gram-negative bacteria. These efflux systems have promiscuous binding sites that can bind and pump a wide range of drugs out of the bacterial cell.
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this class can export many different kinds of antibiotics with little regard for structure. MDR pumps of gramnegative microbes are similar to the ABC export systems described in Section 4.2. They include three proteins: an inner membrane pump protein (fueled by proton motive force, a distinction from true ABC exporters), an outer membrane channel connected to the pump protein, and an accessory protein that may link the other two proteins. For instance, the ArcB transporter, shown in Figure 27.22, almost indiscriminately binds antibiotics in a large central cavity (a promiscuous binding site) and uses proton motive force to move those compounds through a pore and out of a funnel that connects to an outer membrane channel, TolC. Antibiotic efflux pumps are now believed to contribute significantly to bacterial antibiotic resistance because of the very broad variety of substrates they recognize and because of their expression in important pathogens. Strains of the pathogen M. tuberculosis, for instance, have developed multiple drug resistance phenotypes due in part to MDR pumps. Approximately 2 million people die from tubercu-
losis annually, mostly in developing nations. What is even more alarming is that an increasing number of M. tuberculosis strains isolated from patients exhibit multidrug resistance. Although most of the antibiotic resistance in most M. tuberculosis multidrug resistant strains is due to the accumulation of independent mutations in several genes, MDR pumps are thought to increase the level of resistance. Chemists typically try to tweak the structure of an antibiotic to overcome a specific type of resistance mechanism; but the MDR pumps act on an exceptionally wide range of antibiotics, almost without regard to structure.
How Does Drug Resistance Develop?
As discussed in earlier chapters, nature has engineered a certain degree of flexibility in the way genomes are replicated and passed from one generation to the next. DNA repair pathways involving lesion bypass polymerases (for example, UmuDC; see Section 9.5) are thought to play a large role in randomized adaptive and evolutionary processes. For instance, at some point during evolution, gene duplication and mutaAntibiotic—Out tional reshaping generated a gene product able to cleave the beta-lactam ring, creating an organism resistant to penicillin. However, antibiotic resistance in all species does not occur de novo through Funnel gene duplication and/or mutation. Why reinvent the wheel—or, in this case, drug resistance? Gene transfer mechanisms such as conjugation, described in Chapter 9, can move antibiotic resistance genes from one organism to another and from Pore one species to another. In fact, several drug resistance genes found in pathogenic bacteria actually had their start in the chromosomes of the drug-producing organisms and were passed on through Central cavity gene swapping. For instance, Streptomyces clavuligerus produces a beta-lactamase Antibiotic (encoded by the bla gene) that protects this organism from the penicillin it proMembrane duces. Transfer of a drug resistance gene is particularly evident if the gene has been incorporated into a plasmid and that plasmid is found in a new species. An interesting case study of antibiotic resistance is provided by the grampositive bacterium Enterococcus faecalis, Antibiotic—In a natural inhabitant of the mammalian gastrointestinal tract that can cause Figure 27.22 Structure of the E. coli ArcB multidrug resistance efflux life-threatening disease if granted access pump. The structure is trimeric and driven by proton motive force. Dotted lines to other body sites (as in subacute bacoutline the central cavity, pore, and efflux funnel through which the drug is pumped. terial endocarditis; see Section 26.7). Connection to TolC in the outer membrane is not shown. (PDB code: 2GIF)
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E. faecalis is naturally resistant to numerous antibiotics, which makes disease treatment particularly challenging. Vancomycin is one of the last lines of defense for treating serious E. faecalis infections. Unfortunately, increasing numbers of vancomycin-resistant strains have arisen in recent years. The completed genome sequence of one vancomycin-resistant strain illustrates the reason. The organism has an incredible propensity for incorporating mobile genetic elements that encode drug resistance. About a quarter of the genome consists of mobile or exogenously acquired DNA. These include 7 probable phages, 38 insertion elements, numerous transposons, and integrated plasmid genes. One such mobile element encodes vancomycin resistance in the sequenced strain. Recently, multidrug resistance in various microbes (for example, Salmonella enterica) has also been attributed to the presence of integrons. Integrons are gene expression elements that account for rapid transmission of drug resistance because of their mobility and ability to collect resistance gene cassettes (see Special Topic 9.2). We should note that the development of antibiotic resistance is not without consequence to the bacterium. Altering one aspect of an organism’s physiology for the better may weaken another area. Bacteria may become resistant to a certain antibiotic, but that resistance comes at a price. For example, the altered DNA gyrase that affords resistance to quinolones may not function as well as the “normal” gyrase. Thus, in a situation where both resistant and susceptible organisms cohabit the same environment, the wild-type (sensitive) strain may grow faster and eventually overwhelm the mutant strain— unless fluoroquinolone is present, of course.
How Did We Get into This Mess? Consider the following case: A young mother brings her 4-year-old child to the physician. The child is screaming because he has an extremely painful sore throat. Simply looking at the throat is not diagnostic. The raw tissue could mean the child is suffering from a bacterial infection, in which case antibiotics are needed. Alternatively, a virus could be the cause, a situation where antibiotics do nothing but pacify the parent. More often than not, the clinician will prescribe an antibiotic without ever knowing the cause of disease. The problem is this: the more an antibiotic is used, the more opportunities there are to select for an antibiotic-resistant organism. The presence of drug does not cause resistance but will kill off or inhibit the growth of competing bacteria that are sensitive, thereby allowing the resistant organism to grow to high numbers. When should antimicrobials be administered? Certainly, in life-threatening situations where time is of the essence, antibiotics should be administered even before
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knowing the cause of the infection. On the other hand, the most prudent course to take when a patient has a simple infection is to confi rm a bacterial etiology, and then prescribe. An exception to this may be in an elderly or otherwise immunocompromised individual, who may be more susceptible to secondary bacterial infections that can occur subsequent to viral disease. Another proposed source of antibiotic resistance is the widespread practice of adding antibiotics to animal feed. No one quite knows why, but feeding animals antibiotics in their food makes for larger, and therefore more profitable, animals. Some estimates suggest that 70% of all antibiotics used in the United States is fed to healthy livestock. The consequence of this is that the animals may serve as incubators for the development of antibiotic resistance. Even if the resistance develops in nonpathogens, the antibiotic genes produced can be transferred to pathogens. Efforts have been under way since the 1960s to curb this practice.
Fighting Drug Resistance Several strategies are being used to stay one step ahead of drug-resistant pathogens. In some instances, dummy target compounds that inactivate resistance enzymes have been developed. Clavulanic acid, for example, is a compound sometimes used in combination with penicillins such as amoxicillin. Clavulanic acid, a beta-lactam compound with no antimicrobial effect, competitively binds to beta-lactamases secreted from penicillin-resistant bacteria. Because the enzyme releases bound clavulanic acid very slowly, the amoxicillin remains free to enter and kill the bacterium. Another strategy is to alter the structure of the antibiotic in a way that sterically hinders the access of modifying enzymes. Figure 27.23 illustrates how adding a side chain to gentamicin, converting it to amikacin, blocks the activity of various aminoglycoside-modifying enzymes. Of course, we are now seeing resistance to amikacin develop (this resistance is ribosome based, involving mutational alteration of the S12 protein or 16S rRNA). Linking antibiotics is another strategy currently used to limit resistance. Recent advances have been made in linking a quinolone to an oxazolidinone to form a hybrid antibiotic with dual modes of action (Fig. 27.24). Because it has two modes of action, this hybrid antibiotic may limit the development of antibiotic resistance. Here’s why: The rate of spontaneous resistance to a given antibiotic is roughly 1 out of 107 cells. For spontaneous resistance to develop to two antibiotics, that probability rises to 1 out of 1014, making it very unlikely that an organism can become doubly drug resistant. However, multidrug resistance efflux pumps, integron cassettes, and plasmids carrying multiple antibiotic resistance genes can overwhelm that approach.
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C h ap t e r 27
TO SU M MAR I Z E:
A. Gentamicin
■
These sites all open to attack from aminoglycoside modifying enzymes.
■
AAC(2′)
H3C H3CHN
HO 3′′
HO
O 1
3
ANT(2′′) APH(2′′)
4′ 3′
O
OH 4 O NH2
6
H2N
2′
H2N
O
2′′
6′
N
R1
R2
■
AAC(6′) ■
Gentamicin R1 R2 C1 CH3 CH3 C1a H H C2 CH3 H
AAC(3)
■
B. Amikacin AAC(2′) H3C HO
H2N
3′′
H2NCH2CH2
OH
6
HO
ANT(2′′) APH(2′′)
H2N
O
2′′
CH
O
1
C
NH
2′
O
HO
4′
3′
6′
4
O 3
■
APH(3′) ANT(4′)
O NH2
Still open to enzymatic attack
OH NH2
27.7 The Future of Drug Discovery
AAC(6′)
AAC(3)
OH This side chain protects amikacin from attack by AAC(3,2′), APH(3′,2′′), and ANT(2′′) by steric hindrance.
Figure 27.23 Fighting drug resistance. A. Sites where gentamicin is vulnerable to enzymatic inactivation. AAC = aminoglycoside acetyltransferase; ANT = aminoglycoside adenylyltransferase; APH = aminoglycoside phosphotransferase. Inset shows the R groups for different gentamicin compounds C1, C1a , and C2 . B. Gentamicin can be chemically modified at the highlighted sites to prevent loss of activity due to enzyme action. The side groups block access to enzyme active sites by steric hindrance (that is, the added group prevents the active site from interacting with its target structure), but does not inactivate the antibiotic.
O O O
O
N
F
F O
N
O OH
N
N
Quinolone
Figure 27.24 Quinolone-oxazolidinone hybrid. Physically combining two different antibiotics may help reduce the emergence of drug-resistant bacteria.
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Is humankind doomed to a future where antibiotics will no longer work? The general consensus is no. Through prudent use of current antibiotics and innovative approaches for finding new ones, humans should continue to effectively control evolving bacterial pathogens. How does one screen for new drugs that target specific proteins? Certainly the classic approach in which microbes, plants, and even animals collected from around the world are screened for their abilities to make new antibiotics is still valid and remains the most fruitful source of new drugs. However, the ability to search for novel bacterial drug targets or validate theoretical targets has been revolutionized by genome sequence analysis and associated genetic techniques. Powerful computational and bioinformatic methods are required in the initial identification and selection of molecular targets, followed by a series of postgenomic approaches to validate and characterize the targets, devise screens for effective inhibitor molecules, and pursue structurebased drug design to handle the inescapable emergence of future resistance. The modern drug discovery process can be outlined as follows: 1. Identify new targets (such as unique essential
enzymes) based on genomics.
NH Oxazolidinone
Antibiotic resistance is a growing problem worldwide. Mechanisms of antibiotic resistance include modifying the antibiotic, destroying the antibiotic, altering the target to reduce affi nity, and pumping the antibiotic out of the cell. Multidrug resistance pumps use promiscuous binding sites to bind antibiotics of diverse structure. Antibiotic resistance can arise spontaneously through mutation, can be inherited by gene exchange mechanisms, or can arise de novo through gene duplication and mutational reengineering. Indiscriminate use of antibiotics has significantly contributed to the rise in antibiotic resistance. Measures to counter antibiotic resistance include chemically altering the antibiotic, using combination antibiotic therapy, and adding a chemical decoy.
2. Find or design compounds that inhibit the target in
vitro and show that the inhibitory compound has actual antibacterial activity. 3. Show that the target within the bacterial cell is the same as the in vitro target.
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4. Optimize the MICs against susceptible species by
altering the compound’s structure. 5. Examine the compound’s spectrum of activity (grampositive, gram-negative, aerobe, anaerobe, etc.) and the rate at which antibiotic resistance develops in pathogens. 6. Determine the new drug’s toxicity to animals and humans, and its pharmacological properties (for instance, how long does it stay at therapeutic levels in a patient). The functions of genes that constitute potential new drug targets fall into three broad categories: those required only for growth of bacteria in laboratory media (in vitro expressed genes), those required only for bacterial infection in vivo (the in vivo induced genes, discussed in Section 25.6), and those required for bacterial growth both in vitro and in vivo (housekeeping genes). Products of novel and essential genes falling into the second and third categories constitute new potential drug targets. Take, for example, the case of S. pneumoniae. Twenty years ago, S. pneumoniae was uniformly sensitive to penicillin. Today, a growing number of strains have become resistant (as illustrated in the multiple-drug-resistant pneumonia case history presented earlier; also see Fig. 27.18). The discovery of proteins within this microbe that could potentially be targeted by new antibiotics would offer hope of future treatments. Recently, a computerassisted strategy has helped identify several potential drug targets in this pathogen. The search consisted of scanning the pneumococcal genome for sequence motifs commonly found in cell-surface-exposed or virulencerelated proteins of other bacteria. One such motif, called the choline-binding domain (CBD), is used by a variety of bacteria to interact with host cells. These domains consist of repeats of 2–10 amino acids. The rationale used to fi nd potential drug targets in the pneumococcus was to look for pneumococcal proteins that contain these domains. It was already known that various streptococcal species had one such protein, CbpA, which functions as a surface adhesin and plays an important role in nasopharyngeal colonization. By performing genome-wide searches with the C-terminal choline-binding region of cbpA, Khoosheh Gosink and his collaborators at St. Jude Children’s Hospital (Memphis, TN) identified six genes predicted to encode CBD-containing proteins ranging from 20 to 80 kDa (CbpD, E, F, G, I, and J). They constructed mutations in each gene and tested the mutants for pathogenicity. Mutants defective in CbpG adhered poorly to nasopharyngeal cells and were less virulent in a mouse infection model. Therefore, CbpG appears to play an important role in invasion and infection of the mucosa and the bloodstream. Its structural similarity to proteases suggests that it may be an excellent
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candidate for target-based drug development because numerous chemical inhibitors of proteases already exist. Another advance in drug design utilizes a combination of genomic and proteomic approaches. The basis of this technique is to develop fluorescent molecules that bind only to active proteins of a given family of proteins. The probes can be created by rational design in which knowledge of the active site structure allows chemists to engineer chemicals that interact with that site. A true inhibitor of that protein and thus, a potential antibiotic, will prevent binding of the tagged probe, an event that can be viewed after directly blotting cell extracts on a membrane fi lter, much like a Western blot. In the absence of inhibitor, the probe will bind the spot, which will fluoresce. However, the presence of an inhibitor that binds well to the active site will prevent binding of the probe and the spot will not fluoresce. Alternatively, if the probe binds to more than one protein, the same test can be performed using gel electrophoresis separation techniques. Proteins are separated on a nondenaturing gel so that the protein in question will retain its shape and its ability to bind probe. Tagged probe, with or without a potential inhibitor, is added to duplicate gels or a blot of those gels. The protein band will fluoresce in the absence of inhibitor or in the presence of a poor inhibitor. The band will not light up if an effective inhibitor is present. The inhibitors are often made using more or less random combinatorial chemistry techniques, in which random variations of a molecule are created and tested. A library of these related compounds, which could number in the thousands, is robotically screened for inhibitory activity. Genomic and combinatorial chemistries are certainly the cutting edge of antibiotic discovery. However, the old brute force screening of natural products was recently used in a novel way to discover a promising new class of antibiotic. Merck scientists screened 250,000 natural product extracts for an ability to specifically inhibit bacterial fatty acid biosynthesis. Fatty acid biosynthesis is an attractive target for antibiotic development because the process and proteins involved are different from those of eukaryotes. The strategy specifically targeted the protein FabF because it is an essential component of fatty acid synthesis and is conserved among key pathogens such as S. aureus. Scientists engineered a strain of S. aureus to contain a gene expressing an antisense RNA to fabF mRNA. When the antisense RNA was induced, it bound to fabF mRNA and prevented efficient translation. As a result, the level of FabF protein in the cell decreased. The strain could still grow but would be exquisitely sensitive to any compound that targeted the remaining FabF protein. Fewer molecules of FabF present per cell means that fewer molecules of an active ingredient in a natural product are needed to stop fatty acid synthesis. Thus, zones of inhibition on an
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agar plate will be wider than they would be if cells produced normal amounts of FabF. This novel targeted screening method led to the discovery of the antibiotic platensimycin, made by Streptomyces platensis, an organism isolated from South African soil. Platensimycin was subsequently shown to bind FabF and exhibit bacteriostatic, broad-spectrum activity, acting on gram-positive and gram-negative bacteria. It is only the third entirely new antibiotic developed in the last four decades. The novel chemical structure of platensimycin and its unique mode of action provide a great opportunity to develop a new class of critically needed antibiotics—a class that selectively targets fatty acid synthesis. More information on fatty acid biosynthesis and inhibitors can be found in Chapter 15. Another type of rationale for developing new antimicrobial compounds is presented in Special Topic 27.1. TO SU M MAR I Z E: ■
■
■
Potential targets for rational antimicrobial drug design include proteins expressed only in vivo or proteins expressed both in vivo and in vitro. Candidate antimicrobial compounds can be designed to interact at the active site of a known enzyme and inhibit its activity. Combinatorial chemistry is used to make random combinations of compounds that can be tested for enzyme inhibitory activity and antimicrobial activity.
27.8 Antiviral Agents A father pleading with a physician to give his child antibiotics when the infant is suffering with a cold is an all too common dilemma faced by the general practitioner,
but there is nothing of substance the physician can do. The common cold is caused by the rhinovirus, and no antibiotic designed for bacteria can touch it. So why are there so few antiviral agents in the clinician’s arsenal? The reason is that applying the principle of selective toxicity is much harder to achieve for viruses than it is for bacteria. Viruses routinely usurp host cell functions to make copies of themselves. Thus, a drug that hurts the virus is likely to also harm the patient. Nevertheless, there are several useful antiviral agents in which selective viral targets have been found and exploited. Some of these agents are listed in Table 27.4. Select examples are discussed in this section. Note that all of the molecular mechanisms of viruses presented in Chapter 11 are studied as potential drug targets.
Antiviral Agents That Prevent Virus Uncoating or Release Membrane-coated viruses are vulnerable at two stages: when the virus is invading the host cell and after viral propagation, when the progeny viruses release from the host cell. The flu virus presents a good example of both.
Case History: Antiviral Treatment of Infant Influenza A 9-month-old infant arrived at the Johns Hopkins Hospital with an acute onset of fever, cough, regurgitation from his gastrostomy feeding tube, and dehydration. This illness occurred following a series of chronic problems, including respiratory syncytial virus bronchiolitis (infection and inflammation of the bronchioles) and neonatal group B streptococcal sepsis. [Neonatal sepsis is often caused by Lancefield group B Streptococcus agalactiae described in Chapter 28.] Physical
Table 27.4 Examples of antiviral agents. Virus
Agent
Mechanism of action
Result
Influenza
Amantadine Zanamivir Acyclovir Famciclovir
Prevents viral uncoating Prevents viral release Halts DNA synthesis Halts DNA synthesis
Gancyclovir Foscarnet
Inhibits viral M2 protein Neuraminidase inhibitor Guanosine analog Prodrug of penciclovir a guanosine analog Similar to acyclovir Analog of inorganic phosphate
Ribavirin
RNA virus mutagen
Zidovudine (AZT) Nevirapine Nelfinavir
Nucleoside analog, resembles thymine Binds to allosteric site Protease inhibitor
Herpes simplex virus and varicella-zoster virus (shingles) Cytomegalovirus
Respiratory synticial virus and chronic hepatitis C HIV
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Binds and inhibits virus-specific DNA polymerase Causes catastrophic replication errors Inhibits reverse transcriptase Inhibits reverse transcriptase Prevents viral maturation
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Special Topic 27.1
Poking Holes with Nanotubes: A New Antibiotic Therapy
Reza Ghadiri and his colleagues at the Scripps Research Institute have designed tiny peptide molecules that will selfassemble within bacterial membranes to form lethal nanotubes that puncture the cell surface (Fig. 1A). The component parts of the tube are six- and eight-residue cyclic D, L alpha peptides. Built with alternating right- and left-handed amino acids joined end to end, the peptides expose their amino acid side chains outside of the ring. (In nature, the great majority of peptides contain only left-handed L-amino acids, but synthetic peptides can use both right- and left-handed forms.) This alternating D and L configuration allows adoption of a planar ring structure and stacking of rings to form a tube. Ghadiri and his colleagues were able to design the amphipathic peptides to insert themselves selectively into bacterial cell membranes (differences in the phospholipid content of mammalian and bacterial membranes enables selective toxicity). Once the peptides enter the charged environment of the bacterial
membrane, they become sticky and form hollow, antiparallel, hydrogen-bonded stacks (Fig. 1B). These nanotubes effectively poke holes in the membrane and disrupt the normal electrical potential and ion gradients that bacteria use to maintain homeostasis, generate energy, and carry out chemical reactions necessary for survival. By forming nanotubes and poking holes in the cells, cyclic peptides disrupt these gradients and kill the cells. This is not just a theoretical whimsy. The authors have found these peptides to be highly effective in curing methicillin-resistant S. aureus infections in mice. Questions of selectivity and toxicity have yet to be addressed, so it will be years before the use of nanotubes in humans can be realized. Sources: Fernandez-Lopez, S., H.-S. Kim, E. C. Choi, M. Delgado, J. R. Granja, et al. 2001. Antibacterial agents based on the cyclic D, L-α-peptide architecture. Nature 412:452–455. Dartois, V., J. Sanchez-Quesada, E. Cabezas, E. Chi, C. Dubbelde, et al. 2005. Systemic antibacterial activity of novel synthetic cyclic peptides. Antimicrobial Agents and Chemotherapy 49:3302–3310.
A. O
R
H N
R O D
HN
H H N
R
O D
O
O L
R
Self-assembly R
D
HN O
N H
L
R
NH
O
R
O
N H
D N H
N
OH
NH
R
N
O
L
L
R
O H
O
N
H H O N N
H
O
O H
OH
O
R
N H
b
O
O
H H O N N O
H
O R
N
H
H N
R
O
O
H H O
N
H
N
N N
N N
O
N
N N
O
O
N
OH
O
OH H
H
H N
N
O
R R
a
N
H
OH
O
N N
N
R
O OH H
H O
N N H H O
H
O
N N O
R
N
H
c
B.
Antibiotic nanotubes. A. The diagram on the left illustrates the cyclic nature of peptides that form nanotubes. R groups define different amino acids shown as single letter designations inside the ring. The cyclic peptides possess outward facing side chains that allow them to stack via hydrogen bonds (red dotted lines) within a membrane structure to form penetrating nanotubes. The lower figure shows a bacterial membrane under nanotube attack. (Cyclic peptides are depicted as ring structures. There are three possible sequence-dependent modes of membrane permeation; a, intramolecular pore; b, barrel stave; and c, carpet-like). Nanotubes operating through the carpet-like mode of action would have a greater potential for membrane discrimination because of its polyvalent display of surfaceexposed hydrophilic side chains for potential interactions with various membrane constituents. B. A top view down a nanotube.
Figure 1
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C h ap t e r 27
exam revealed fever, a severe cough resulting in respiratory distress, a rapid heart rate, and moderate dehydration. Nasopharyngeal aspirate was positive for influenza A antigen. The patient was treated with amantadine when influenza was diagnosed. He gradually improved and was discharged home four days after admission. An unusually severe form of influenza spread across the United States in 2003. Most states reported a higher than normal number of influenza-related deaths of children and young adults. Several factors, however, helped keep the outbreak from becoming an epidemic of larger proportions. First, the administration of flu vaccine afforded the population what is called herd immunity (discussed in Section 26.9). Herd immunity occurs even when only a fraction of a population is immunized. This fraction of individuals will not become infected and so cannot spread the disease to others. At the very least, this slows progression of infection throughout the population. (It is impossible to immunize all humans against any given disease. However, it is estimated that immunizing twothirds of a population can eliminate the disease by cutting off transmission. Even this, however, is rarely achieved.) The second factor that prevented an influenza pandemic was the availability of antiviral agents that can limit the disease course. As a result of studying the molecular biology of influenza, scientists discovered two selective targets. Influenza virus (200 nm) is encased in a membrane envelope donned when the virion buds from an infected cell. As described in Section 11.4, the envelope contains the viral proteins neuraminidase and hemagglutinin. Spikes of hemagglutinin bind to glycoprotein receptors on the host cell and trigger receptor-mediated endocytosis. The virus ends up inside the resulting endosome. After the endosome is formed, proton pumps in the membrane acidify endosomal contents. The drop in pH changes the structure of hemagglutinin on the viral membrane so it can now bind to receptors on the endocytic membrane. The result is fusion of the two membranes and release of the virion into the cytoplasm. For this change in A. Amantadine H H H N+
B. Zanamivir HO HO HN
hemagglutinin structure to occur, it is essential that the interior of the enveloped virion also be acidified, and this is facilitated by the formation of a channel by the virusencoded M2 envelope protein. Amantadine (Fig. 27.25A), a drug that has been used to treat some strains of the flu virus, is a specific inhibitor of the influenza M2 protein; however, it is useful against the influenza type A viral strains only. It is also effective against the avian H5N1 strain that has occasionally infected humans and is feared as the source of a future influenza pandemic. Unfortunately, amantadine-resistant strains are circulating in Asia due in part to the widespread use of amantadine by Chinese poultry farmers. The second target of the influenza virus is the envelope protein neuraminidase. The newer antiflu drugs, such as zanamivir (Relenza), are neuraminidase inhibitors that act against types A and B influenza strains (Figs. 27.25B and C). Neuraminidase on the viral envelope allows virus particles to leave the cell in which they were made. Neuraminidase inhibitors prevent this release and cause the virus particles to aggregate at the cell surface, reducing the number of virus particles released. The contributions of different NA and HA genes on the severity of influenza are discussed in Special Topic 27.2. Amantadine and the neuraminidase inhibitors, when used within 48 hours of disease onset, decrease shedding and reduce the duration of influenza symptoms by approximately one day. However, flu symptoms generally last only from three to ten days. While this does not sound like a substantial benefit, in the elderly, shortening the course of the flu can minimize damage to the lungs, which in turn reduces the chance of developing lifethreatening secondary bacterial infections such as pneumonia and bronchitis.
Antiviral DNA Synthesis Inhibitors Most antiviral agents work by inhibiting viral DNA synthesis. These drugs chemically resemble normal DNA nucleosides, molecules containing deoxyribose and analogs of
C. OH H O COO–
O HN HN
NH2
Figure 27.25 Inhibitors of influenza proteins. A. Amantadine inhibits the M2 protein. B. Neuraminidase inhibitor zanamivir. C. Neuraminidase without (left) and with (right) bound inhibitor. (PDB: 2HTQ)
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Special Topic 27.2
■ M edic ine and I mmu n o l o gy
Critical Virulence Factors Found in the 1918 Strain of Influenza Virus
As Chapter 26 describes, the influenza virus that swept the globe from 1918 to 1919 took the lives of 20–40 million people, making it a killer greater than the first World War. One might wonder whether today’s drugs could prevent a similar disaster should that virus strain reemerge after all these years. This question may now have been answered. A team of scientists working in a fortresslike biohazard research facility has used forensic molecular biology techniques to partially re-create that deadly virus. Their initial goal was to determine why the 1918 virus was so lethal compared with typical influenza strains, but they also wanted to learn if the antivirals on hand today could avert a 1918-like disaster. Christopher F. Basler, of the Mount Sinai School of Medicine in New York, and his colleagues incorporated several genes from the 1918 flu into an influenza strain that was adapted to kill mice. The viral genes introduced included those that encode hemagglutinin (HA), neuraminidase (NA), and protein M2. The team actually expected that those genes would reduce the virulence of the mouse-adapted virus. This
adenine, guanine, cytosine, or thymine. Viral enzymes then add phosphate groups to these nucleoside analogs to form DNA nucleotide analogs. The DNA nucleotide analogs are then inserted into the growing viral DNA strand in place of a normal nucleotide. Once inserted, however, new nucleotides cannot attach to the nucleoside analogs, and DNA synthesis stops (Fig. 27.26). These DNA chainterminating analogs are selectively toxic because viral polymerases are more prone to incorporate nucleotide analogs into their nucleic acid than are the more selective host cell polymerases. Antiviral DNA synthesis inhibitors work on DNA viruses or retroviruses, but not on viruses such as influenza with its RNA genome.
was because flu viruses isolated from people rarely prove lethal to rodents, and genes from human-adapted strains typically weaken the rodent-adapted viruses. Not so for the HA and NA genes of the 1918 flu. The engineered virus containing both of the 1918 HA and NA genes efficiently killed mice, but the presence of only one or the other of the two genes lowered virulence. This suggests that the specific combination of HA and NA may underlie the 1918 flu disaster. Even more remarkable was that treating the recombinant virus-infected mice with an NA inhibitor prevented 90% of the mice from dying. Furthermore, all mice infected with a recombinant flu strain carrying the 1918 gene for M2 survived when treated with M2 inhibitors, even if treatment began 6 hours after infection. The conclusion is that HA and M2 are critical components of influenza virulence. Thus, the currently available flu drugs targeting HA and M2 may help prevent a new pandemic of the 1918 influenza strain or a similar flu, such as the H5N1 avian flu.
course of antiretroviral therapy (AZT) will be helpful in preventing the transmission of the virus to her child. The husband had an HIV viral load of 10,000 copies per milliliter and was started on protease inhibitor treatment. O
O CH3
HN O
O
N –
O
HO H
O
H
H
O H
H H
H OH H
Deoxyribonucleotide containing thymine O
N HO
O
N
O
O
H
Zidovudine
A married couple come to the community clinic for prenatal care. He is 20 years old. She is 19 and reportedly two months pregnant with her first child. She denies intravenous (IV) drug use or a history of other sexual partners and has no history of sexually transmitted disease; however, a routine prenatal HIV antibody screen is reported as positive for HIV-1. Careful questioning of the patient and her husband elicits from him a history of IV drug use five years earlier. An HIV antibody screen for him is also positive. The laboratory results indicate that the wife may not yet require therapy (she has a low viral load—that is, less than 1,000 copies per milliliter of blood— and a high CD4 T-cell count), but since she is pregnant, a short
P O–
H N3
CH3
HN
Case History: Treatment of HIV
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1057
N
O N
NH N
N
NH2
N
NH2
O
HO Acyclovir
NH
H H
H H
OH H Deoxyguanosine
Figure 27.26 Antiviral inhibitors that prevent DNA synthesis. Zidovudine (AZT) and acyclovir are analogs of thymine and guanine nucleotides, respectively. Because the analogs have no 3′ OH to which another nucleotide can add, chain elongation ceases.
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Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors Human immunodeficiency virus (HIV) is an RNA retrovirus that uses a reverse transcriptase to make DNA that then integrates into host nuclear DNA to form a provirus (discussed in Section 11.5). The antiretroviral drug zidovudine (abbreviated ZDV or AZT), which was used in the case history, is a nucleoside analog recognized by reverse transcriptase. Once incorporated into a replicating HIV DNA molecule, the DNA chain-terminating property of AZT prevents further DNA synthesis. Because HIV transmission from the mother to the neonate can occur at delivery or by breast-feeding, treatment of the mother and the child are important steps to prevent transmission. There are also nonnucleoside reverse transcriptase inhibitors. For example, the drug delavirdine binds directly to reverse transcriptase and allosterically inactivates the enzyme. Because HIV can mutate rapidly and become resistant to single-drug therapies, treatment usually involves administering combinations of three or more antiretroviral drugs.
Protease Inhibitors To make optimum use of its limited provirus DNA sequence, HIV makes long nonfunctional polypeptide chains that are proteolytically cleaved to make the actual proteins and enzymes used to replicate and produce new virions. For example, the gag and pol genes reside next to each other in the HIV genome and are transcribed as a single mRNA molecule (discussed in Chapter 11). The Gag and Pol open reading frames overlap but are offset from one another by one base. This mRNA produces two polyproteins called Gag and Gag-Pol, the latter being the result of a shift in reading frames that takes place during translation. Once made, both polyproteins are cleaved by HIV protease. The Gag protein is proteolytically cleaved to make different capsid components (p17, p24, and p15, which is further cleaved to make nucleocapsid protein p7), while Gag-Pol is cleaved to make reverse transcriptase and integrase (Fig. 27.27A). Protease inhibitors such as Viracept® and Lopinavir® are a powerful new class of drugs that block the HIV protease (Fig. 27.27B). When the protease is inactivated, even though new virus particles are made, the polyproteins remain uncleaved and the virus cannot mature. Because immature HIV particles cannot infect other cells, progress of the disease stalls. It is important to note that protease inhibitors do not cure AIDS; they can only decrease the number of infectious copies of HIV. Antiviral therapy with protease inhibitors is recommended for patients with symptoms of AIDS and for
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A. p15
p24 gag polyprotein Protease Protease
p17
Protease
Protease B.
Inhibitor
Figure 27.27 HIV protease inhibitor. A. Representation of HIV protease cleavage of a single gag polyprotein into multiple, smaller proteins. B. The protease enzyme is shown as a ribbon structure, while the protease inhibitor BEA 369 is shown as a stick model. (PDB code: 1EBY)
asymptomatic patients with viral loads above 30,000 copies per milliliter. Treatment should be considered even for patients with viral loads above 5,000 copies per milliliter, as for the husband in the case history presented earlier. TO SU M MAR I Z E: ■
■
■
■
Fewer antiviral agents are available when compared to antibacterial agents because it is harder to identify viral targets that provide selective toxicity. Preventing viral attachment to, or release from, host cells are mechanisms of action for antiviral agents such as amantadine and Zanamivir used to treat influenza virus. Inhibiting DNA synthesis is the mode of action for most antiviral agents, although they only work for DNA viruses and retroviruses. HIV treatments include reverse transcriptase inhibitors that prevent synthesis of DNA and protease inhibitors that prevent the maturation of viral polyproteins into active forms.
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Pa r t 5
27.9 Antifungal Agents
A. Clotrimazole
Fungal infections are much more difficult to treat than bacterial infections in part because fungal physiology is more similar to that of humans than is bacterial physiology. The other reason is that fungi have an efficient drug detoxification system that modifies and inactivates many antibiotics. Thus, to have a fungistatic effect, repeated applications of antifungal agents are necessary to keep the level of unmodified drug above MIC levels.
1059
M edic ine and I mm u n o l o gy
B. Griseofulvin H3C O
O O
CH3 O
Cl N
O
O
H3C
CH3
Cl
Core imidazole N ring
C. Nystatin
Case History: Blastomycosis
H3C
A 37-year-old male presented to the emergency department of a Florida hospital with persistent fever, malaise, and a painful right arm mass. He denied trauma to the arm. White blood cell count was elevated at 27,000/µl, and chest X-ray revealed a left lung infiltrate. Bronchoscopy revealed granulomatous inflammation containing a single yeast-like mass. Incision and drainage was performed on the arm mass and cultures obtained. Serum cryptococcal antigen tests were negative, as were tests for Bartonella henselae and Toxoplasma. Cultures from the right arm grew out a fungal form similar to that identified from the bronchoscopy specimens. A tentative diagnosis of Blastomyces dermatitides was confirmed using PCR. The patient was placed on amphotericin B, and his fevers and leukocytosis subsequently subsided. His medication was changed to fluconazole for a recommended duration of six months.
H3C
1029-1062_SFMB_ch27.indd 1059
OH O
OH
OH CH3
OH HO
OH
HO O
–
OOC
OH
O O OH D. Amphotericin B H3C HO
Superficial mycoses (fungal infections), such as athlete’s foot, and systemic mycoses, such as blastomycosis, require very different treatments. Imidazole-containing drugs (clotrimazole, miconazole) are often used topically in creams for superficial mycoses (Fig. 27.28A). Others, such as itraconazole, are administered orally. Superficial mycoses include infections of the skin, hair, and nails, as well as Candida infections of moist skin and mucous membranes (for example, vaginal yeast infections). The imidazole-containing drugs appear to disrupt the fungal membrane by inhibiting sterol synthesis. More chronic dermatophytic infections typically require another antifungal agent called griseofulvin, produced by a Penicillium species (Fig. 27.28B). Griseofulvin disrupts the mitotic spindle and derails cell division (called metaphase arrest). This does not kill the fungus, but as the hair, skin, or nails grow and are replaced, the fungus is shed. Vaginal yeast infections caused by Candida are often treated with nystatin, a polyene antifungal agent synthesized by Streptomyces that forms membrane pores (Fig. 27.28C). The name nystatin, by the way, came about because two of the people who discovered it worked for the New York State Public Health Department. The serious, sometimes fatal consequences of systemic mycoses require more aggressive therapy. The
O
NH2
CH3 OH
OH
O O CH3
OH OH
OH
OH OH O
OH COO–
H3C H3C NH2 O
O OH
OH
Figure 27.28 Examples of antifungal agents. A. Clotrimazole belongs to the group of imidazole antifungals, which are so named because they all contain an imidazole ring. B. Griseofulvin is produced by Penicillium griseofulvum. C. Nystatin is a polyene macrolide produced by Streptomyces noursei. D. Amphotericin B is a polyene produced by Streptomyces nodosus.
drugs used in these instances include amphotericin B (produced by Streptomyces; see Fig. 27.28D) and fluconazole. Amphotericin B binds to the sterols in fungal membranes and destroys membrane integrity. It has a high affi nity for ergosterol, which is prevalent in fungal, but not mammalian, membranes. Fluconazole, on the other hand, inhibits the synthesis of ergosterol. Thus, fungal cells grown in the presence of fluconazole make defective membranes. Typically, curing systemic fungal infections
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■ A n t im ic r o b ial C hemot herapy
Table 27.5 The major antifungal agents and their common uses. Clinical application Systemic mycoses Drug
Coccidioidomycosis
Histoplasmosis
Blastomycosis
Paracoccidioidomycosis
+ – –
+ – –
+ – –
+ – –
– – +
– – +
– – +
– – +
+ +
+ ?b
+ ?
+ ?
–
–
–
Polyenes Amphotericin B Nystatin Pimaricin Imidazoles Clotrimazole Miconazole Ketoconazole Triazoles Itraconazole Fluconazole Antimetabolite 5-Fluorocytosinec a.
mc, Mucocutaneous but not systemic candidiasis.
b.
Insufficient data.
c.
Used only in combination with amphotericin B.
requires long-term treatment to prevent disease relapse. Table 27.5 lists a number of other commonly used antifungal agents. TO SU M MAR I Z E: ■
■
■ ■ ■
Fungal infections are difficult to treat because of similarities in human and fungal physiologies. Imidazole-containing antifungal agents inhibit sterol synthesis. Griseofulvin inhibits mitotic spindle formation. Nystatin produces membrane pores. Amphotericin B binds to membranes and destroys membrane integrity.
Concluding Thoughts Antibiotics have done much to improve the health and well-being of people and animals around the globe. Although they have successfully kept at bay infectious diseases dreaded for centuries (such as the plague and tuberculosis), a crisis of antibiotic resistance looms because of the irresponsible use of antimicrobial agents. The fact that very few new, clinically useful antimicrobials have been discovered over the last quarter century should give us pause. Redoubling efforts to find new drugs combined with the responsible use of existing antibiotics are required to maintain our advantage over constantly evolving pathogens.
CHAPTE R R EVI EW Review Questions 1. What is selective toxicity? Provide examples. 2. Explain the difference between antibiotic susceptibil-
5. What is the Kirby-Bauer test? Does it tell whether a
ity and antibiotic sensitivity. 3. What does the term spectrum of antibiotic activity mean? 4. Provide examples of bacteriostatic and bactericidal antibiotics.
6. Give examples of drugs that target cell wall synthe-
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drug is bacteriostatic or bactericidal? sis; RNA synthesis; protein synthesis; DNA replication. What are their modes of action? 7. What is the mechanism by which peptide antibiotics are synthesized by the producing organism?
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■
M edic ine and I mm u n o l o gy
1061
Clinical application Opportunistic mycoses Aspergillosis
Candidiasis
Cryptococcosis
Dermatophytosis
+ – –
+ mca –
+ – –
– – –
– – –
mc mc +
– – –
+ + +
+ –
mc +
+ +
+ +
Sporotrichosis Sporotrichosis
+
+
+
–
Phaeohyphomycosis
8. How do antibiotic-producing microorganisms pre9. 10. 11. 12.
vent suicide? Why is antibiotic resistance a growing problem? What are the four basic mechanisms of antibiotic resistance? Explain the basic concept of an MDR efflux pump. Discuss the current concepts of the origin of antibiotic resistance.
Other
Mycotic keratitis
13. What are some mechanisms used to combat the
development of drug resistance? 14. Why are there few antiviral agents available to treat
disease? 15. What is herd immunity? 16. How does amantadine inhibit influenza? 17. Discuss the general modes of action of antifungal
agents.
Key Terms amphotericin B (1059) antibiotic (1030) bacitracin (1040) bactericidal (1033) bacteriostatic (1033) chloramphenicol (1045) cycloserine (1040) gramicidin (1041) griseofulvin (1059) Kirby-Bauer assay (1035) lincosamide (1045)
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macrolide (1044) minimal inhibitory concentration (MIC) (1034) Mueller-Hinton agar (1037) multidrug resistance (MDR) efflux pump (1049) mycosis (1059) neuraminidase inhibitor (1056) nosocomial (1030) nystatin (1059) oxazolidinone (1045)
penicillin-binding protein (1039) quinolones (1042) secondary metabolite (1046) selective toxicity (1032) spectrum of activity (1033) streptogramin (1045) transglycosylase (1038) transpeptidase (1038) vancomycin (1040) zone of inhibition (1034)
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Recommended Reading Cohen, Mitchell L. 2000. Changing patterns of infectious disease. Nature 406:762–767. Dibner, Julia J., and James D. Richards. 2005. Antibiotic growth promoters in agriculture: History and mode of action. Poultry Science 84:634–643. Fernandez-Lopez, Sara, Hui-Sun Kim, Ellen C. Choi, Mercedes Delgado, Juan R. Granja, et al. 2001. Antibacterial agents based on the cyclic D,L-α-peptide architecture. Nature 412:452–455. Fernandez-Tornero, Carlos, Rubens Lopez, Ernesto Garcia, Guillermo Gimenez-Gallego, and Antonio Romero. 2001. A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA. Nature Structural Biology 8:1020–1024. Freiberg, Christoph, and Heike Brötz-Oesterhelt. 2005. Functional genomics in antibacterial drug discovery. Drug Discovery Today 10:927–935. Gosink, Khoosheh K., Elizabeth R. Mann, Chris Guglielmo, Elaine I. Tuomanen, and H. Robert Masure. 2000. Role of novel choline binding proteins in virulence of Streptococcus pneumoniae. Infection and Immunity 68:5690–5695. Hubschwerlen, Christian, Jean-Luc Specklin, Daniel K. Baeschlin, Yves Borer, Sascha Haefeli, et al. 2003. Structure–activity relationship in the oxazolidinone–quinolone hybrid series: Influence of the central spacer on the antibacterial activity and the mode of action. Bioorganic & Medicinal Chemistry Letters 13:4229–4233. Johnson, Kirk W., Denene Lofland, and Heinz E. Moser. 2005. PDF inhibitors: An emerging class of antibacterial drugs. Current Drug Targets—Infectious Disorders 5:39–52.
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Kloss, Patricia, Liqun Xiong, Dean L. Shinabarger, and Alexander S. Mankin. 1999. Resistance mutations in 23S rRNA identify the site of action of the protein synthesis inhibitor linezolid in the ribosomal peptidyl transferase center. Journal of Molecular Biology 294:93–101. Marquez, Beatrice. 2005. Bacterial efflux systems and efflux pumps inhibitors. Biochimie 87:1137–1147. Paulsen, I. T., L. Banerjee, G. S. Myers, K. E. Nelson, R. Seghadri, et al. 2003. Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science 299:2071–2074. Shah, P. M. 2005. The need for new therapeutic agents: What is the pipeline? Clinical Microbiology and Infection 11 (Suppl 3):36–42. Swaney, Steve M., Hiroyuki Aoki, M. Clelia Ganoza, and Dean L Shinabarger. 1998. The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrobial Agents and Chemotherapy 42:3251–3255. Turnidge, John, and David J. Paterson. 2007. Setting and revising antibacterial susceptibility breakpoints. Clinical Microbiology Reviews 20:391–408. Walsh, Christopher. 2003. Antibiotics: Actions, Origins, Resistance. ASM Press, Washington, DC. Wang, Jun, Stephen M. Soisson, Katherine Young, Wesley Shoop, Srinivas Kodalil, et al. 2006. Platensimycin is a selective FabF inhibitor with potent antibiotic properties. Nature 441:358–361.
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Chapter 28
Clinical Microbiology and Epidemiology 28.1 28.2 28.3 28.4 28.5 28.6
Principles of Clinical Microbiology Approaches to Pathogen Identification Specimen Collection Biosafety Containment Procedures Principles of Epidemiology Detecting Emerging Microbial Diseases
Throughout history, infectious diseases have killed more people than all of our wars combined. Our present success in controlling the spread of disease is due in large part to worldwide surveillance agencies that are equipped to detect outbreaks quickly, before major epidemics develop. These agencies rely on smaller clinical microbiology laboratories scattered throughout the world. These laboratories help clinicians diagnose infectious diseases. This chapter discusses the principles of clinical microbiology and epidemiology that are used to identify, treat, and contain outbreaks. How do clinical laboratories know what organisms to suspect in a given case? And what tests will unequivocally identify the right pathogen? How are the real pathogens found among the normal flora? Finding the source of an outbreak is also critical. How do epidemiologists identify “patient zero” and recognize and contain emerging diseases? Last but not least, we’ll consider bioterrorism. Clinical scientists and epidemiologists are trained to detect bioterrorist attacks using the same principles they employ to detect naturally occuring infectious diseases.
Community-acquired methicillin-resistant S. aureus (in blue) destroys a neutrophil attempting to phagocytose it, thus breaching the body’s first line of defense. Recent outbreaks of community-acquired MRSA have become a serious health problem. Source: Jovanka Voyich, et al. 2006. The Journal of Infectious Diseases 194:1761. Photo from David W. Dorward/Rocky Mountain Laboratories/NIAID.
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Wilfrid Saintilus lay immobile on a straw mat when fi rst seen by Dr. Paul Farmer in 1996. The 33-year-old peasant farmer had been sent home without hope by health care workers in a small clinic near his village in Haiti. He was pronounced “paralyzed from the waist down”; it was said that “nothing more could be done.” Farmer, however, quickly realized that Wilfrid’s problem was not paralysis but a long-term Salmonella infection, very likely contracted from an unclean drinking water supply—a problem for 80% of Haiti’s population. Farmer realized that, while normally a gastrointestinal pathogen, Salmonella can occasionally disseminate via the bloodstream and infect bones, causing a painful disease called osteomyelitis. The microbe had infected Wilfrid’s hip, causing pain so severe he could not move. His leg muscles had atrophied over time, and he now weighed under 100 pounds. Contrary to prediction, Wilfrid did not die. Dr. Farmer’s diagnostic skills and antibiotics saved his life. Thousands of stories like this, most with more tragic outcomes, underscore the integral roles that clinical microbiology and epidemiology have in health care. In Wilfrid’s case, a simple laboratory test would have revealed a fully treatable Salmonella infection and prevented his enormous physical and emotional pain. As in Chapters 24–27, we use case histories in this chapter to introduce various strategies and methodologies used by modern medicine to diagnose infectious diseases. The approach is, by necessity, selective. Not all techniques, nor all diseases, can be described. Our goal is not to catalog the many kinds of infectious diseases but to demonstrate general principles and problem-solving approaches used in identifying disease-causing microbes. It will become apparent in the process that modern tools of clinical microbiology are indispensable in the diagnosis and treatment of infectious disease.
28.1 Principles of Clinical Microbiology As in any good detective mystery, the fi rst step in investigating an infectious disease is to identify the most likely suspects. This can be accomplished, in part, from observing the disease symptoms in a patient and knowing what organisms typically produce those symptoms. It also helps if the physician is aware of a similar disease outbreak under way in the community. Beyond these clues, the etiological agent must be identified through biochemical, molecular, serological, or antigen detection strategies.
Why Take the Time to Identify an Infectious Agent? This is the fi rst question many students ask when contemplating the effort and expense required to identify the
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genus and species of an organism causing an infection. Why not simply treat the patient with an antibiotic and be done with it? While this approach sounds appealing, there are several compelling reasons for identifying an infectious agent. Many bacteria are resistant to certain antibiotics.
As discussed in Chapter 27, antibiotic resistance is an increasingly serious global problem. Characterizing a microbe’s antibiotic resistance profi le is thus part of any microbial identification process. Understanding which antibiotics are effective in treating an infectious agent will help the physician avoid prescribing an inappropriate drug. Furthermore, knowing which drugs are ineffective enables health organizations to track the spread of antibiotic-resistant strains. For example, before 1970, most strains of Neisseria gonorrheae were susceptible to penicillin. Today, most are penicillin resistant, due in part to the widespread use of penicillin to treat gonorrhea during and after the Vietnam War, when U.S. soldiers fi rst contracted penicillinase-producing N. gonorrhoeae (PPNG). There are strain-specific disease complications. Many diseases have serious complications that are common to a given organism or strain of organism. For example, children whose sore throats are caused by certain strains of Streptococcus pyogenes can develop serious complications affecting the heart and kidney long after the infection has resolved. These complications are the immunological consequence of bacterial and host antigen crossreactivity; they are called sequelae because they occur after the infection itself is over. Life-threatening sequelae such as rheumatic fever and acute glomerular nephritis caused by certain strains of S. pyogenes produce severe damage to the heart and kidney, respectively. Knowing early on that S. pyogenes has caused a child’s sore throat allows the physician to prescribe antibiotics that will quickly eradicate the infection and prevent development of the sequelae. NOTE: Penicillin or a penicillin-like antibiotic is the usual treatment for S. pyogenes infection. Contrary to what you might expect, S. pyogenes has not developed any penicillin-resistant strains.
Tracking the spread of a disease can lead to its source. Consider a situation in which ten infants scat-
tered throughout a city develop bloody diarrhea. The clinical laboratory identifies the same strain of Shigella sonnei, a gram-negative bacillus, as the cause in each case. Finding the same strain in all cases suggests they probably originated from the same source. Shigellosis is transferred from person to person by what is called the fecal-oral route. Carriers of Shigella will shed this
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organism in their feces. Inadequate hand washing after defecation will leave bacteria on their hands, which can then transfer the pathogen to foods or utensils or to another person by touching. Uninfected persons can become infected after eating the contaminated food or placing their fingers in their mouth. Public health officials, armed with the knowledge that all the patients had the same strain of Shigella, then question the parents and learn that all of the children attend the same day-care center. By testing the other children and workers in that center, officials can stop the infection, called shigellosis, from spreading. This investigative process, called epidemiology, is covered more completely later in this chapter. TO SU M MAR I Z E:
Identifying a pathogen enables us to: ■ ■ ■
Use appropriate antibiotics, if necessary. Anticipate possible sequelae. Track the spread of the disease.
28.2 Approaches to Pathogen Identification Pathogens can be identified on the basis of a variety of observations, including the patient’s symptoms, fi nding organisms in stained clinical specimens, biochemical clues, and serology (the presence in a patient’s serum of antibodies reactive against a specific microbe).
Bacterial Pathogen Identification Requires Knowledge of Microbial Physiology and Genetics Thousands of bacterial species are capable of causing disease. How can a laboratory quickly, sometimes within hours, identify which one causes a given infection? The solution, in part, comes from knowing the biochemical and enzymatic features of these bacteria. Because no two species have the same biochemical “signature,” the clinical lab can look for reactions, or combinations of reactions, that are unique to a given species. The same is now true at the genetic level. In the current genomics-dominated era, DNA sequences of many pathogens have been completely elucidated. This has enabled design of polymerase chain reaction (PCR) primers that rapidly detect species-specific genes or even genes that are unique to highly pathogenic strains within a species. One example of such a gene is the attaching and effacing locus (eae) that is present in enterohemorrhagic, but not commensal, strains of E. coli. Finding E. coli in a fecal stool sample is not unusual. Finding E. coli with the eae gene, however, indicates that it is a pathogen.
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Case History: Medical Detective Work A 38-year-old woman with no significant previous medical history came to the emergency room complaining of a mild sore throat persisting for three days. Her symptoms included arthralgia (joint pain), myalgia (muscle pain), and low-grade fever. The day before, she had a severe headache with neck stiffness, nausea, and vomiting. She was not taking any medications, had no known drug allergies, and did not smoke. She lived with her husband and two children, all of whom were well. Cerebral spinal fluid (CSF) was collected from a spinal tap. The CSF appeared cloudy (it should be clear) and contained 871 white blood cells per microliliter (normal is 0–10/µl); the glucose level was 1 mg/dl (normal is 50–80 mg/dl); and the total protein level was 417 mg/dl (normal is under 45 mg/dl). Gram stain of a CSF smear revealed gram-negative rods. The CSF sample was sent to the diagnostic laboratory for microbial identification. As discussed in Thought Question 26.6, low glucose and elevated protein levels in CSF are indicators of bacterial infection, not viral. The increase in white blood cells revealed that the woman’s immune system was trying to fight the disease. The presence of gram-negative rods in the CSF smear confi rmed a diagnosis of bacterial meningitis, since CSF should be sterile. Now it was up to the clinical laboratory to determine the etiological agent.
Clinical Laboratories Use Problem-Solving Algorithms to Identify Bacteria How does a clinical microbiology laboratory handle incoming specimens such as the one considered here? What are the fi rst steps toward identifying the etiological agent of a disease? Over the years, clinical microbiologists have developed algorithms (step-by-step problemsolving procedures) that expose the most likely cause of a given infectious disease. For instance, there are only a limited number of microbes known to cause meningitis. The microbiologist poses a series of binary “yes” or “no” questions about the clinical specimen in the form of biochemical or serological tests. Typical questions in this case might include: “Is an organism seen in the CSF of a patient with symptoms of meningitis?” “Is the organism gram-positive or gram-negative?” “Does it stain acidfast?” Answers to a fi rst round of questions will then dictate the next series of tests to be used. Because speed is of the essence in deciding how to treat the patient, a series of tests is not always carried out sequentially. To save time, a slew of tests are carried out simultaneously, but the results are interpreted sequentially based on the algorithm. In our case history, for instance, consider the most common causes of bacterial meningitis: Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, and Escherichia coli. The CSF sample was
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Gram-stained by a microbiologist and simultaneously plated onto three media—chocolate agar, blood agar, and Hektoen agar. Chocolate agar is an extremely rich medium that looks brown owing to the presence of heatlysed red blood cells (Figs. 28.1A and B). Because it is so nutrient-rich, all four organisms will grow on chocolate agar. However, nutritionally fastidious organisms such as N. meningitidis and H. influenzae will not grow well, if at all, on ordinary blood agar because these bacteria cannot lyse red blood cells and release required nutrients. Less fastidious organisms, such as S. pneumoniae and E. coli, will grow on blood agar, but only E. coli can grow on Hektoen agar (Figs. 28.1C–E), which is a selective and differential medium for enteric gram-negative rods. Differential and selective media are described in Section 4.3. In our case history, the Gram stain of the CSF revealed gram-negative rods, which ruled out Neisseria
(a gram-negative diplococcus) and Streptococcus pneumonia (a gram-positive diplococcus). The organism in CSF did grow on blood agar, which eliminated Haemophilus influenzae (a gram-negative, nonenteric rod) as a candidate. It also grew on Hektoen, where it produced orange lactose-fermenting colonies. Thus, the organism was a gram-negative, enteric rod and likely E. coli. Additional biochemical tests confi rming the identity of the organism had to be carried out, but this simple example shows how simultanteous tests can be interpreted. Identifying gram-negative bacteria. The gram-negative bacterium in this case was subjected to a battery of biochemical tests packaged as 20 separate chambers in a patented analytical profile index (API 20E) strip that can be used for pathogen identification (Fig. 28.2). The strip
A. Chocolate agar
C. Hektoen agar
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E. S. enterica colonies on Hektoen
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D. E. coli colonies on Hektoen
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B. Colonies of N. gonorrhoeae
Chocolate agar and Hektoen agar; two widely used clinical media. A. Uninoculated chocolate agar. Its color is due to gently lysed red blood cells that provide a rich source of nutrients for fastidious bacteria. B. Chocolate agar inoculated with a species of Neisseria. This organism will not grow well on typical blood agar because important nutrients remain locked within intact red blood cells. C. Uninoculated Hektoen agar, which contains lactose, peptone, bile salts, thiosulfate, an iron salt, and the pH indicators bromthymol blue and acid fuchsin. The bile salts prevent growth of gram-positive microbes. D. Hektoen agar inoculated with Escherichia coli. This organism ferments lactose to produce acidic fermentation products that give the medium an orange color owing to the pH indicators acid fuchsin and bromphenol blue. E. Hektoen agar inoculated with Salmonella enterica. This organism does not ferment lactose but instead grows on the peptone amino acids. The amines produced are alkaline and produce a more intense blue color with bromthymol blue. Salmonella species also produce hydrogen sulfide gas from the thiosulfate. Hydrogen sulfide reacts with the medium’s iron salt to produce an insoluble, black iron sulfide precipitate visible in the center of the colonies.
Figure 28.1
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example, in the indole chamber (ninth well from the left in Fig. 28.2B), a red reaction is positive and indicates that the organism can produce indole from tryptophan. A colorless chamber would be a negative result. Similar B. E. coli results after 24 hours API strips are available for the identification of gram-positive bacteria and yeasts. The results of the API chambers can be interpreted in two ways. First, as a dichotomous key, a stepwise interpretation can be C. P. mirabilis results after 24 hours done by lab personnel starting with a key reaction, such as lactose fermentation. The reaction is read as positive or negative based on the color. Then, following a printed flowchart, the technician would go to the next key Figure 28.2 API 20E strip technology for the biochemical reaction, indole production, and read it as posidentification of Enterobacteriaceae. A. Uninoculated API strip. Each itive or negative. If the organism was a lactose well contains a different medium that tests for a specific biochemical fermentor and the indole test was positive, capability. The color of the media after 24-hour incubation indicates a positive the choices have been narrowed to E. coli or or negative reaction (see Table 28.1). B. API results for E. coli. Plus (+) and Klebsiella species. Another reaction is read to minus (–) indicate positive and negative reactions, respectively. distinguish between the remaining choices. C. API results for Proteus mirabilis. The process continues until a single species is identified. A simplified example is shown in Figure 28.3 using a limited number of enteric gramrequires overnight incubation and tests whether the negative species. In reality, many more reactions than the organism can ferment a series of carbon sources. It also 11 shown have to be used to make a defi nitive identificaprovides evidence of specific end products produced as tion because of species differences with respect to a single a result of fermentation. The results appear as differentreaction. For example, Figure 28.3 shows that K. pneucolored reactions in each chamber and are scored as posimoniae and K. oxytoca exhibit opposite indole reactions, tive or negative, depending on the color (Table 28.1). For Courtesy of A. Philippon, Faculté de Médecine René Descartes
A. Uninoculated strip
Table 28.1 Reading the API 20. Tests
Substrate
Reaction tested
Negative results
Positive results
ONPG ADH LDC ODC CIT H2S URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA
ONPG Arginine Lysine Ornithine Citrate Na thiosulfate Urea Tryptophan Tryptophan Na pyruvate Charcoal gelatin Glucose Mannitol Inositol Sorbitol Rhamnose Sucrose Melibiose Amygdalin Arabinose
Beta-galactosidase Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Citrate utilization H2S production Urea hydrolysis Deaminase Indole production Acetoin production Gelatinase Fermentation/oxidation Fermentation/oxidation Fermentation/oxidation Fermentation/oxidation Fermentation/oxidation Fermentation/oxidation Fermentation/oxidation Fermentation/oxidation Fermentation/oxidation
Colorless Yellow Yellow Yellow Pale green/yellow Colorless/gray Yellow Yellow Yellow Colorless No diffusion of black Blue/blue-green Blue/blue-green Blue/blue-green Blue/blue-green Blue/blue-green Blue/blue-green Blue/blue-green Blue/blue-green Blue/blue-green
Yellow Red/orange Red/orange Red/orange Blue-green/blue Black deposit Red/orange Brown-red Red (2 min) Pink/red (10 min) Black diffuse Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow
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Gram-negative rod Glucose Neg
Lactose (ONPG)
Pos
Pseudomonas spp.; others
Neg
Indole
Pos Neg
H 2S Neg
Neg
Neg
Neg
Pos
LDC
Citrate Neg
Pos
Pos
ADH Pos
Salmonella Proteus enterica mirabilis
Neg
ODC
Shigella flexneri
Neg
Yersinia pestis Providencia stuartii
Neg
Pos
Citrobacter freundii
Sorbitol Neg
Pos
H 2S Urease
Pos
VP Neg
Pos
Citrate
Pos
Pantoea Pos
Enterobacter cloacae Pos
Urease Neg
Kluyvera Pos
Klebsiella oxytoca
Escherichia coli
Yersinia enterocolitica
Klebsiella pneumoniae
Serratia marsescens
Figure 28.3 Simplified biochemical algorithm to identify gram-negative rods. The diagram presents a dichotomous key using a limited number of biochemical reactions and selected organisms to illustrate how species identifications can be made using biochemistry. Abbreviations and reactions: Lactose fermentation to produce acid, ONPG (orthonitrophenyl beta-d-galactoside, cleaved by betagalactosidase); sorbitol fermentation to produce acid, indole production from tryptophan; VP (Voges-Proskauer test) indicates production of acetoin or 2,3-butanediol; LDC (lysine decarboxylase) cleaves lysine to produce CO2 and cadaverine; ODC (ornithine decarboxylase) cleaves ornithine to make CO2 and putrescine; ADH (arginine dihydrolase) is the stepwise degradation of arginine to citrulline and ornithine; citrate utilization as a carbon source; H2S is the production of hydrogen sulfide gas; urease produces CO2 and ammonia from urea. (Note: “Neg” for Pseudomonas in terms of glucose indicates an inability to ferment glucose. Pseudomonas can still use glucose as a carbon source.)
even though they are of the same genus. Even within a given species, only a certain percentage of strains might be positive for a given reaction. The inherent danger in using a dichotomous key is that one anomolous result can lead to an incorrect identification. In a second approach, the API strips can be used to generate a seven-digit identification number that points to the identity of the bacterium. To generate this number, individual reactions are given numerical values based on whether they give a positive or negative result, and then the number values from three reactions are added to produce a single digit. Because there are 21 reactions (the 21st reaction is an oxidase test performed on colonies), the end result will be a seven-digit number. The example in Figure 28.4A shows how this is done. Once the number is generated, it is compared to a database of numbers generated by computer. The database was compiled by taking all possible test results and applying them to all species of Enterobacteriaceae. Each species has a known probability of being positive for a given reaction. As discussed in Section 17.3, taking each spe-
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cies and multiplying the probabilities for the results generated by the test organism will generate a probability score. The test organism is identified as the species with the highest score. This probability approach is considerably more accurate than the dichotomous key. Most clinical laboratories in the United States and Europe now use automated identification systems that are even more sophisticated than API. These include BioMerieux’s VITEC, Dade Behring’s WalkAway, and Becton, Dickinson and Company Phoenix systems (Fig. 28.4B). All of them use cards or plates with 30 or more biochemical or enzymatic reactions. As with the API chambers, the results appear as colored reactions that can be read by computer. The BD Phoenix system, for example, will convert the results into a 10-digit code that is used to identify genus and species. Identifying nonenteric gram-negative bacteria. The
procedures we have outlined will accurately identify members of Enterobacteriaceae, but pathogenic gramnegative bacilli can also be found in other phylogenetic
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A. ONPG ADH LDC 1 2 4
+
–
2
+
+
H2S 4
+
7
URE TDA 1 2
+
+
IND 4
VP 1
–
–
GEL GLU 2 4
3
+ 6
+
MAN INO SOR 1 2 4
–
– 0
–
RHA SAC MEL 1 2 4
–
– 0
–
AMY ARA 1 2
–
–
OX 4
–
0
Figure 28.4 Generating an identification number from the API 20E strip. A. Results from Figure 28.2C were used to generate an identification number. The 20 reactions in the API plus an oxidase reaction performed separately are divided into seven reaction triplets. Each reaction within a triplet is given a value (1, 2, or 4). If the reaction is positive, that value is added to the value of any other positive reaction in that group, and the resulting number is placed in the oval. The result is a sevendigit number that will be unique to a given genus and species of enteric microorganism. Note that because of biochemical diversity within different strains of a species, several different numbers may be generated; but because of the way the test is designed, the numbers will still be unique to the species. B. Automated microbiology system. The BD Phoenix™ system uses plates with numerous reaction wells and a computerized plate reader to automatically identify pathogenic bacteria. The numbers and their evaluation can be done automatically using this type of instrument.
Courtesy and © Becton, Dickinson and Company
B.
families. One possibility in the preceding case history is that the gram-negative bacillus seen in CSF smears would not grow on blood agar or on the other selective media, but would grow as small, glistening colonies on chocolate agar. This result would implicate the gramnegative rod Haemophilus influenzae. Meningitis caused by H. influenzae was a major problem prior to 1988, before the introduction of vaccinations with H. influenzae type b capsule material. Growth of H. influenzae requires hemin (X factor) and NAD (V factor), so confi rming the idenA.
tity H. influenzae involves growing the organism on agar medium containing hemin and NAD. This can be done by placing small fi lter paper disks containing these compounds on a nutrient agar surface (not blood agar) that has been covered with the organism. H. influenzae will only grow around a strip containing both X and V factors. Alternatively, X and V factors can be incorporated into Mueller-Hinton agar, as shown in Fig. 28.5A. The organism grows on chocolate medium because the lysed red blood cells release these factors. Although XV growth B.
V factor
X and V factor
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X factor
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–
ODC CIT 1 2
Figure 28.5 Haemophilus influenzae growth factors and Neisseria meningitidis oxidase reaction. A. H. influenzae will grow on an agar plate (here, Mueller-Hinton agar) only when the medium has been fortified with both X factor (hemin) and V factor (NAD), but not either one alone. B. Oxidase positive reaction for Neisseria meningitidis. Oxidase reagent was dropped onto colonies of N. meningitidis grown on chocolate agar. The test is called the cytochrome oxidase test, but it really tests for cytochrome c.
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phenotype is still used for identification, fluorescent antibody staining is more specific (discussed shortly). A completely different identification scheme would have been used in the preceding meningitis case if the laboratory had discovered the organism to be a gramnegative diplococcus (rather than a gram-negative rod). A gram-negative diplococcus would suggest Neisseria meningitidis. N. gonorrhoeae is also possible, but less likely because the gonococcus lacks the protective capsule meningococci used to survive in the bloodstream. Without this capsule, N. gonorrhoeae is not as resistant to serum complement and cannot disseminate to the meninges. The fi rst test to determine if the organism is a species of Neisseria is the cytochrome oxidase test (Fig. 28.5B). In this test, a few drops of the colorless reagent N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride are applied to the suspect colonies. The reaction, which only takes place if the organism possesses both cytochrome oxidase and cytochrome c, turns the p-phenylenediamine reagent (and the colony) a deep purple/black. While many bacteria possess cytochrome oxidase, Neisseria is one of only a few genera that also contain cytochrome c in their membranes. Oxidase-positive organisms use cytochrome oxidase to oxidize cytochrome c, which then oxidizes p-phenylenediamine. Other oxidase-positive bacteria include Pseudomonas, Haemophilus, Bordetella, Brucella, and Campylobacter species—all of them gram-negative rods. None of the Enterobacteriaceae, however, are oxidase-positive because they lack cytochrome c. An oxidase-positive, gram-negative diplococcus is very likely a member of Neisseria. Differentiation between species of Neisseria is based on their ability to grow on certain carbohydrates; or it can be determined using immunofluorescent antibody staining tests that test for the presence of different capsule antigens in N. meningitidis. Identifying gram-positive pyogenic cocci. Recall the case of the woman with necrotizing fasciitis (see Section 26.2). How did the laboratory determine that the etiological agent was Streptococcus pyogenes? A sample algorithm, or flowchart (Fig. 28.6), shows how it was done. The physician sends a cotton swab containing a sample from a lesion to the clinical laboratory. The laboratory technician streaks the material onto several media: (1) blood agar (which will grow both gram-positive and gram-negative organisms), (2) blood agar containing the inhibitors colistin and naladixic acid (called a CNA plate, this agar will grow only gram-positives), and (3) MacConkey agar (which will grow only gram-negative organisms (see Section 4.3). The suspect organism in this case grows on the CNA and blood plates. Because they grow in the presence of the gram-negative inhibitory compounds in CNA, one would immediately sus-
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pect the organism to be gram-positive, an assumption borne out by the Gram stain. NOTE: Though the skin is normally populated by many different microorganisms (normal flora), samples from an infected lesion are overwhelmingly populated by the etiological agent. This occurs because the pathogenic microbe outgrows normal flora. Using selective media to isolate the infectious agent will further simplify diagnosis by reducing growth of any normal flora that may still be present.
The algorithm tells the laboratory technician that since the organism is a gram-positive coccus, the next step is to test for catalase production. Catalase, which converts hydrogen peroxide (H 2O2) to O2 and H 2O, clearly distinguishes staphylococci from streptococci (remember, Gram stain morphology alone is insufficiently reliable to make that distinction). The catalase test is performed by mixing a colony with a drop of H 2O2 on a glass slide. Effusive bubbling due to the release of oxygen indicates catalase activity (see Fig. 28.6). Staphylococci are catalase-positive, while the streptococci are catalase-negative. Note that many other organisms possess catalase activity, including the gram-negative rod E. coli. However, based on the algorithm, E. coli would not be considered, since it does not grow on CNA agar and is not gram-positive. NOTE: When performing a catalase test from colonies grown on blood agar, be sure not to transfer any of the agar, since red blood cells also contain catalase.
Having established that the organism is catalasenegative, the technician examines the blood plate for evidence of hemolysis. Three types of colonies are possible: nonhemolytic, alpha-hemolytic, and beta-hemolytic. Nonhemolytic streptococci do not produce any lytic zone. Alpha-hemolytic strains produce large amounts of hydrogen peroxide that oxidize the heme iron within intact red blood cells to produce a green product. As a result, alphahemolytic streptococci produce a green zone around their colonies called alpha hemolysis—even though the red blood cells remain intact. (For example, S. mutans, a cause of dental caries and subacute bacterial endocarditis, is alpha-hemolytic.) Still other streptococci produce a completely clear zone of true hemolysis surrounding their colony. This is called beta hemolysis. Complete hemolysis of red blood cells occurs owing to the export of enzymes, called hemolysins, that lyse red cell membranes. The flowchart indicates that the organism from the case history was beta-hemolytic. The fi nal relevant test in this flowchart involves susceptibility to the antibiotic bacitracin, which identifies the
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CNA agar Blood agar MacConkey agar
Growth Growth No growth
© Michael Abbey/Visuals Unlimited
Lesion
1071
Gram stain (gram-positive cocci)
Neg
Pos
Hemolysis on blood agar
Coagulase
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Catalase
β
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None
Staphylococcus aureus Novobiocin Resistant
Bacitracin
Optochin
Neg Courtesy of Hardy Diagnostics
Pos α
S. saprophiticus
Susceptible
S. epidermidis
Algorithm for identifying gram-positive pathogenic cocci. The red arrows follow the identification of S. pyogenes. The bacitracin and optochin results are designated “positive” if the organism is susceptible and “negative” if the organism is resistant to the agent.
Figure 28.6 Pos
Neg Neg
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Pos
S. pneumoniae
S. pyogenes
most pathogenic group of beta-hemolytic streptococci. The beta-hemolytic streptococci are subdivided into many different groups, known as the Lancefield groups, based on differences in the composition of their carbohydrate peptidoglycans. These cell wall differences are distinguished from each other immunologically and divide the streptococci into Lancefield groups A through U. Rebecca Lancefield, for whom the classification scheme is named, was the fi rst to use immunoprecipitation to group the streptococci (Fig. 28.7). The vast majority of streptococcal diseases are caused by group A beta-hemolytic strepto-
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cocci (also called GAS), defi ned as the species Streptococcus pyogenes. Unfortunately, the Lancefield classification procedure is somewhat time-consuming and not readily amenable as a rapid identification method. However, the group A beta-hemolytic streptococci are uniformly susceptible to the antibiotic bacitracin. Thus, a simple antibiotic disk susceptibility test can be used to indicate the group A beta-hemolytic streptococci (that is, S. pyogenes). But beware; many bacteria are bacitracin sensitive, so like the catalase test, the bacitracin test must be used in conjunction with an algorithm to be useful for identification. The technician must follow the appropriate algorithm before assigning importance to this or any other test result. It is irrelevant, for instance, if an alphahemolytic organism is bacitracin susceptible. Some of these may exist, but they are not associated with disease.
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Marine Biological Laboratory
THOUGHT QUESTION 28.1 Use Figure 28.6 to identify the organism from the following case. A sample was taken from a boil located on the arm of a 62-year-old man. Bacteriological examination revealed the presence of gram-positive cocci that were also catalase-positive, coagulase-positive, and novobiocin resistant.
Rebecca Lancefield. In 1918, Dr. Lancefield joined the Rockefeller Institute for Medical Research in New York City, where she studied the hemolytic streptococci, known then as Streptococcus haemolyticus. She was the first to use serum precipitation methods to classify S. haemolyticus into groups according to differences in cell wall carbohydrate antigens. The basic technique is still used today and is known as the Lancefield classification scheme in her honor.
Figure 28.7
The organism in our case of necrotizing fasciitis, however, was beta-hemolytic, so bacitracin susceptibility indicated that the organism was S. pyogenes. The other tests named in Figure 28.6 are equally important for the identification of gram-positive infectious agents. For example, Streptococcus pneumoniae is an important cause of pneumonia. Like S. pyogenes, S. pneumoniae is a catalase-negative, gram-positive coccus; but unlike S. pyogenes, it is alpha-hemolytic. Optochin susceptibility is a property closely associated with S. pneumoniae while other alpha-hemolytic strains of streptococci are resistant to this compound. Thus, an optochin susceptibility disk test is a useful tool for identifying S. pneumoniae. Coagulase is a key reaction used to distinguish the pathogen Staphylococcus aureus, which causes boils and bone infections, from other staphylococci, such as the normal skin species S. epidermidis. To conduct a coagulase test, a tube of plasma is inoculated with the suspect organism. If the organism is S. aureus, it will secrete the enzyme coagulase. Coagulase will convert fibrinogen to fibrin and produce a clotted, or coagulated, tube of plasma. Coagulase-negative staphylococci can still be medically important, however. S. saprophiticus, for instance, is an important cause of urinary tract infections. It can be distinguished from S. epidermidis based on resistance to novobiocin.
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Pathogen Identifications Based on Molecular Genetics Many diagnostic laboratories have implemented DNAbased methods to detect and type bacteria and viruses. Molecular detection methods for bacteria are often more rapid than the traditional culture-based methods described earlier. DNA detection (for example, PCR) takes only a few hours, while culture-based identification takes days to weeks. DNA/RNA detection methods are especially useful for viruses, which otherwise require elaborate electron microscopy to view morphology or serology to detect an increased presence of antiviral antibodies. The problem with serology is that by the time these antibodies become detectable in blood, the patient is already recovering from the disease. It is also important to note that molecular detection techniques may provide the only means of identifying newly recognized, or emerging, pathogens. The polymerase chain reaction (PCR) is the most widely used molecular method in the clinical laboratory’s diagnostic arsenal. DNA primers that bind to unique genes in a pathogen’s genome can be used to specifically amplify DNA or RNA present in a clinical specimen. Successful amplification is visualized as an appropriately sized fragment in agarose gels following electrophoresis. Why is PCR needed to detect the presence of these nucleic acids? Without the PCR amplification steps, clinical samples usually provide too little nucleic acid from infecting microorganisms to be detected. For example, Mycobacterium tuberculosis, the cause of tuberculosis, can take weeks to grow on standard bacteriological media. Detecting the presence of its DNA in the specimen, however, would allow a very rapid diagnosis. Unfortunately, the amount of bacterial DNA present in the sample is infi nitesimal, too little to detect by standard techniques such as Southern blot. By amplifying M. tuberculosis nucleic acid by PCR, however, one copy of DNA will become billions of copies. PCR is very quick. Preparing a clinical specimen for PCR by extracting the DNA or RNA usually takes less than an hour. PCR itself is completed in 2–3 hours. Detection of the PCR-amplified DNA by DNA gel electrophoresis takes an additional 1–2 hours. So, what might take two to three days (or sometimes weeks) using biochemical algorithms may take less than a day by molecular strat-
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egies. Most clinical laboratories are now equipped with thermocyclers (the instrument used to precisely and rapidly cycle temperature for PCR reactions) and can carry out this type of identification protocol for organisms for which specific primers are available. Table 28.2 lists several instances in which DNA detection tests are useful. A specific example presented in Figure 28.8 illustrates the use of PCR to type different strains of the anaerobic pathogen Clostridium botulinum, the cause of food-borne botulism. These organisms are not typed serologically, as is the case for S. pneumoniae. C. botulinum is divided into different types based on what neurotoxin genes they possess. In this example, multiplex PCR was used to simultaneously search for these toxin genes. Multiplex PCR uses multiple sets of primers, one pair for each gene, combined in a single tube with a specimen. Care must be taken to be sure that the primers chosen make different-sized products, do not interfere
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with each other, and do not produce artifactual products that can confuse interpretation. Multiplex PCR can help identify sets of specific genes present in a single species or can screen for the presence of multiple pathogens in a clinical sample. In the latter, primer sets are designed to amplify genes unique to each pathogen.
Case History: Outbreak of HospitalAcquired Wound Infections in New Delhi From August through December, 45 patients in the pediatric surgery unit of a New Delhi hospital developed postoperative wound infections. Of these, 42 were outpatients, while 3 were inpatients who had undergone major surgery. The diseases ranged from chronic ear infections to bacteremia associated with the use of hemodialysis equipment. Thirty-two clinical samples of pus and wound exudates were tested for acid-fast bacilli by the Ziehl-Neelsen method (Fig. 28.9A).
Table 28.2 Some DNA-based detection tests. Test
Intended use
Specimen for PCR
Transport conditions
Detection of HIV proviral DNA
Diagnosis of HIV infection in newborns; to resolve indeterminate serologic results Diagnosis of whooping cough (pertussis)
>2 ml whole blood in EDTA tube. EDTA prevents coagulation by chelating (binding) ions Nasopharyngeal swab
Room temperature, within 24 hours after collection
Diagnosis/surveillance of Lyme disease
Tick
Detection of Bordetella pertussis Detection of Borrelia burgdorferi
Room temperature, within 24 hours after collection
Detection of Rickettsia rickettsii
Diagnosis/surveillance of Rocky Mountain spotted fever
Detection of Ehrlichia chaffeensis
Diagnosis/surveillance of human monocytic ehrlichiosis
Detection of Norwalk and other small round-structured viruses Typing of Mycobacterium tuberculosis
Diagnosis of viral gastroenteritis; investigation of food-borne and waterborne outbreaks
>1 ml diarrheal stool in sterile container
Wrap in moist tissue in small plastic bag 4°C (cold packs); within 24 hours after collection Wrap in moist tissue in small plastic bag Blood: room temperature Skin lesion biopsy within 24 hours after collection; skin: 4°C Wrap in moist tissue in small plastic bag Room temperature, within 24 hours after collection 4°C (cold packs) within 72 hours after collection
Determine relatedness of M. tuberculosis isolates/ investigation of suspected outbreaks Determine relatedness of isolates/investigation of suspected nosocomial or food-borne outbreaks
Sputum, urine
Room temperature
Food, feces
4°C (cold packs), within 24 hours after collection (if Campylobacter suspected, transport at room temperature)
Typing of various gram-negative and gram-positive bacteria
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Skin biopsy; >1 ml spinal fluid; or urine Tick >2 ml whole blood in EDTA tube Skin biopsy Tick >2 ml whole blood in EDTA tube
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School of Veterinary Medicine, UC Davis
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7
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–
(bp) 1030 515 300 150
+
Multiplex PCR identification of Clostridium botulinum. C. botulinum cells are not typed serologically based on surface antigens, but on the basis of what toxin genes a strain possesses. Typing isolates can be done by a single PCR reaction that uses primer pairs specific for each of the four major toxin genes: Type A, B, E, and F. Because multiple products are sought in a single reaction, this is called multiplex PCR. Each lane in the agarose gel was loaded with multiplex products from different isolates of C. botulinum and subjected to electrophoresis. The slower-moving fragments (toward top of gel) are larger than those moving farther down the gel toward the positive pole. Lane 1, DNA size markers; lane 2, type A (cntA); lane 3, type B (cntB); lane 4, type E (cntE); lane 5, type F (cntF); lane 6, type A, B, and F; lane 7, type B, E, and F; lane 8, type A, B, E, and F.
Figure 28.8
The same smear samples were cultured on LowensteinJensen slants (Fig. 28.9B) and examined for growth over a course of several weeks. Biochemical tests indicated that the organism was a mycobacterium in each case. The slowgrowing organism, Mycobacterium abscessus, was identified by PCR. Based on the DNA fingerprint, the source of the outbreak was traced to the tap water in the operating room and to a defective autoclaving process (the result of a leaking vacuum pump and faulty pressure gauge in the autoclave). This case history points out an underlying problem with some biochemical identifications. It took weeks to grow enough organisms to perform these biochemical tests. An alternative PCR test that probed for mycobacterium-specific genes encoding 16S rDNA could have been performed directly on the clinical specimen and would have confi rmed the diagnosis immediately, much sooner than by biochemical methods. The PCR test that was done was not performed until after the cells were grown in the laboratory. M. tuberculosis can be presumptively diagnosed in the clinical laboratory by the acid-fast stain, a technique fi rst described in 1882 (called the Ziehl-Neelsen stain) that is used to find the tubercle bacillus in a patient’s sputum (Fig. 28.9A). The acid-fast stain enables a technician to visualize bacteria such as Mycobacterium species that are not stained by the Gram stain. Mycobacteria have a very
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waxy outer coat composed of mycolic acid that resists penetration by most dyes, an obstacle the acid-fast stain was designed to overcome. The original Ziehl-Neelsen acid-fast stain used phenol and heat to drive carbol fuchsin (a red dye) into mycobacterial cells on glass slides. Destaining with an acid alcohol solution removes the stain from all cell types except mycobacteria. The slide is subsequently counterstained with methylene blue, after which the mycobacteria will be seen as curved, red rods (called acid-fast bacilli), while everything else will appear blue. A more modern version of the acid-fast stain uses the fluorochrome auramine O to stain the mycolic acid. This dye also resists removal by an acid alcohol wash, so when observed under a fluorescence microscope, the mycobacteria will fluoresce bright yellow. Although the acid-fast stain is very useful, sometimes organisms cannot be found in a sputum sample; and even if they are found, confi rmatory tests are needed for a definitive diagnosis. Relying on growth of the organism means that diagnosis may not be clear for several weeks. For our case history, a rapid DNA-based method would have made the job of determining species much easier. This test probes a 383-bp DNA sequence located at the end of a heat-shock gene that is highly conserved among all mycobacterial species. As a result, the sequence can be amplified by PCR regardless of the strain. However, fragments amplified from different species will have somewhat different DNA sequences. The alterations in DNA sequence can be exposed by restriction digestion. Sequences from different species will produce unique digestion fragment patterns that can then be used for identification. The clinical laboratory compared the restriction patterns produced from mycobacteria isolated from different areas throughout the hospital in the case history. The results allowed the infection control staff to trace the source of all the infections to the tap water in the operating room. In addition to PCR, restriction fragment length polymorphisms (RFLPs), a form of DNA fi ngerprinting, can be used to track a given strain during the course of an epidemic. This technique relies on small differences in sequence that occur between strains of a single species. Because of these small differences in sequence, the number and location of restriction enzymes sites in the chromosome will differ. In the example in Figure 28.9, DNA from a strain of Mycobacterium tuberculosis is cut with restriction enzymes, and the digested fragments are separated by gel electrophoresis (Figs. 28.9C and D). The gel is then probed with a radioactively labeled fragment of insertion sequence IS6110 (a fluorescent probe can also be used). IS6110 is a repetitive DNA sequence present at multiple sites in the chromosome. The radioactive probe can hybridize only to DNA fragments that contain IS6110 sequences. Strains of
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Courtesy of Claudio Basilico, M.D./New York University
C. Genetic fingerprint 1. Restriction enzymes cleave chromosomal DNA at restriction sites.
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B. M. tuberculosis colonies on Lowenstein-Jensen agar
CDC
A. Acid-fast stain
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IS6110, a repetitive DNA sequence
Restriction sites
2006. Journal of Clinical Microbiology 43:4473
D. 2. Some DNA fragments contain IS6110 (yellow).
3. Gel electrophoresis separates fragments by size. 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 9 101010
4. Fragments containing IS6110 hybridize to a specific radioactive probe.
Morphology, growth and DNA fingerprinting of Mycobacterium tuberculosis. A. Acid-fast stain of M. tuberculosis. B. Lowenstein-Jensen medium enables growth of mycobacterial species, some of which grow extremely slowly. The colonies have a “bread-crumb-like” appearance. C. Genetic fingerprinting of Mycobacterium tuberculosis isolates. Fragments containing IS6110 are marked yellow. D. Fragments containing IS6110 hybridize to the specific radioactive probe. A characteristic banding pattern (fingerprint) appears for each isolate. Isolates with similar banding patterns are assigned the same number in this example.
Figure 28.9
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M. tuberculosis obtained from different outbreaks would likely display different hybridization patterns. Strains isolated from the same outbreak would look very similar, if not identical.
Conversion of mRNA to cDNA by reverse transcription
Case History: West Nile Virus
5′
A 55-year-old man was admitted to a local hospital complaining of headache, high fever, and neck stiffness. The man appeared confused and disoriented. He also complained of muscle weakness. History indicated he had received several mosquito bites approximately two weeks previously. A blood specimen was sent to the laboratory. The report the following day indicated that the patient was suffering from West Nile virus.
3′
West Nile virus is primarily an infection of birds and culicine mosquitoes (a group of mosquitoes that can transmit human diseases), with humans and horses serving as incidental, dead-end hosts. Replication of virus in this bird-mosquito-bird cycle begins when adult mosquitoes emerge in early spring and continues until fall. Among humans, the incidence of disease peaks in late summer and early fall. Birds provide an efficient means of geographical spread of the virus. As a result, over the past several years, the virus has spread throughout much of the United States. Isolating a disease-causing virus is extremely challenging. Most laboratories are not equipped for the special tissue culture techniques required to grow viruses. Consequently, most viral infections, including human West Nile virus infections, are usually diagnosed by measuring the antibody response of the patient. For instance, the presence of West Nile virus–specific IgM in cerebrospinal fluid is a good indicator of current West Nile virus infection, but it is indirect and not conclusive. Real-time PCR is a molecular test that can quickly reveal the presence of the virus itself. Real-time quantitative PCR (RTQ-PCR, or qPCR) is used routinely for the high-throughput diagnosis of viral pathogens such as West Nile virus. Because West Nile virus (Flaviviridae family) contains single-stranded RNA, its RNA must fi rst be converted to DNA using reverse transcriptase before PCR can be attempted. The quantitative advantage of RTQ-PCR is that you can estimate the number of virus particles present in the sample based on the number of viral RNA molecules there. The basic technique is as follows: RNA is fi rst extracted from the sample. A subsequence of viral RNA is then converted to cDNA using a single primer and reverse transcriptase (Fig. 28.10). The more virus particles there are in the sample, the more viral RNA will be present and the more cDNA product is made. The cDNA is then amplified by PCR using two specific primers and Taq polymerase (see Section 12.3; Fig. 12.15). The trick
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5′
3′ RT
3′
AAAAA TTTTT
5′ 3′
RT
A AAAA TTTTT
5′
1. Oligo dT primer is bound to mRNA.
2. Reverse transcriptase (RT) copies first cDNA strand.
RT 5′ A AAAA TTTTT
3′
5′
5′
3′
3′
5′
3. Reverse transcriptase digests and displaces mRNA and copies second strand of cDNA.
4. The result is double-stranded cDNA.
Figure 28.10 Reverse transcriptase synthesis of DNA. Reverse transcriptase (RT) uses mRNA as a template to make DNA. The DNA can then be amplified using standard PCR methods. Molecules of eukaryotic and viral mRNA usually contain poly-A tails at their 3′ ends. An oligonucleotide poly-T primer added to the reaction tube will anneal to the poly-A tail and allow RT to synthesize the first strand of a complementary DNA (cDNA). A second primer specific to the viral gene is then used to prime RT synthesis of the opposite strand (second-strand synthesis), using the first DNA strand as template. Subsequent amplification by PCR requires the addition of a thermostable DNA polymerase (Taq) that can withstand the denaturing and DNA synthesis temperatures required for amplifying the cDNA.
for quantitation is in the method used to detect amplification. In one method, a third, fluorescent oligonucleotide (called the probe) is added to the PCR reaction (see Fig. 12.15A). The probe contains a fluorescent dye at the 3′ end and a chemical dye at the 5′ end that quenches (that is, absorbs) energy emitted from the fluorescent dye. As long as the two chemicals are kept in close proximity by the intact probe, no light is emitted. The probe is designed to anneal to a sequence in between the binding sites of the two other primers (modifications on the ends of the probe prevent it from being used as a primer). So in a successful amplification, Taq polymerase will synthesize DNA from the two outside primers and degrade the probe oligonucleotide as it passes through that area. This cleavage separates the dye from the quencher, and the dye begins to fluoresce. The greater the amount of cDNA there was to begin with, the fewer cycles it takes to register a fluorescence increase over background (Fig. 28.11). THOUGHT QUESTION 28.2 Why does finding IgM to West Nile virus indicate current infection? Why wouldn’t finding IgG do the same?
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10,000 1,000 100 10 0 –1,000
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Cycle
Figure 28.11 Results of real-time PCR. A. The exponential increase in PCR products after each cycle of hybridization and polymerization. The switch to yellow indicates the point where the increase in product plateaus because the primers have been exhausted. B. Increase in relative fluorescence units (RFU) as the fluorescent dye is released from the dual-labeled probe during real-time PCR. The blue curve representing PCR product is flat for the first 22 cycles because the amount of DNA made, and therefore level of fluorescent dye released, remains below background level (red line). The more starting DNA there is, the sooner RFU values will increase over background (that is, fewer cycles are needed to see the increase over background). The slope eventually decreases because the fluorescent probe has become limiting.
Identifications Based on Serology Case History: Ebola Between October and November 2000, 62 residents of a small village north of Gulu, a town in Uganda, became ill with high fever, diarrhea, headache, vomiting, and gastrointestinal bleeding from the rectum. As many as 36 patients died. By January, 425 cases were reported, of whom 224 died. The presumptive diagnosis was Ebola. Laboratory confirmation tests included viral antigen detection and antibody ELISA tests. Laboratoryconfirmed Ebola patients were defined as patients who were either positive for Ebola virus antigen or Ebola IgG antibody. Once identified, rigorous quarantine mechanisms were implemented to limit spread of the disease to other villages. What are these tests? The ELISA test detects antibodies indirectly or antigens directly. The enzyme-linked immunosorbent
assay (ELISA) can detect antigens or antibodies present in nanogram and picogram quantities. One form of ELISA detects serum antibodies. It is carried out in a 96-well microtiter plate, which allows multiple patient serum samples to be tested simultaneously. An anti-
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B. Amount of DNA 1 2 4 8 16 32 64 128 256 512 1,024 2,048 4,096 8,192 16,384 32,768 65,536 131,072 262,144 524,288 1,048,576 2,097,152 4,194,304 8,388,608 16,777,216 33,554,432 67,108,864 134,217,728 268,435,456 536,870,912 1,073,741,824 1,400,000,000 1,500,000,000 1,550,000,000 1,580,000,000
PCR base line subtracted RFU
A. Cycle number 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
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gen from the virus (Ebola in this case) is attached (or adsorbed) to the plastic of the wells (Fig. 28.12). Albumin or powdered milk is used to block the remaining sites on the plastic that could result in false positives. Patient serum is then added. Ebola-specific antibodies present in the serum will react with the antigen attached to the microtiter plate. The antigen-antibody complex is then reacted with goat-antihuman IgG to which an enzyme has been attached, or conjugated (for example, horseradish peroxidase). This forms an antibody “sandwich” that attaches the enzyme to the well. The chromogenic substrate for the enzyme is added next (for example, tetramethylbenzidine). If enzyme-conjugated antibody has bound to any human IgG, the enzyme will convert the substrate to a colored product (blue for tetramethybenzidine). Enzyme activity can be measured with an ELISA plate reader. The amount of colored product formed, detected as absorbance with a spectrophotometer, will be an indication of the amount of antiEbola antibody present in the patient sample. THOUGHT QUESTION 28.3 Why does adding albumin or powdered milk prevent false positives in ELISA?
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Enzyme-Linked Immunosorbent Assay (ELISA)
Antigen capture 4. If Ebola antigen is present, the conjugated antibody will be captured by the complex. The addition of substrate will lead to production of a colored product. Substrate
Product 3. Enzyme-conjugated anti-Ebola antibody is then added.
Albumin ELISA plate
2. Ebola antigen from patient serum will be captured by antibody on plate.
Albumin Ebola antigen Plastic of microtiter plate
Figure 28.13 Antigen capture ELISA. ELISA technique to capture Ebola antigens circulating in patient serum.
Rinse off excess and add patient serum
Human anti-Ebola antibody from patient serum binds to Ebola antigen.
Wash off unbound serum and add conjugated antibody
Rabbit antihuman IgG antibody with attached (conjugated) enzyme.
Wash off unbound conjugated antibody and add substrate
Rate of conversion of substrate to colored product is propotional to the amount of anti-Ebola antibody that was present in the patient’s serum.
Enzyme-linked immunosorbent assay (ELISA). ELISA to detect anti-Ebola antibodies circulating in patient serum. The 96-well plate can be used to make dilutions of a single patient’s serum to more precisely determine the amount of anti-Ebola antibody, or it can be used to test samples from multiple patients. Figure 28.12
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1. Anti-Ebola monoclonal antibody is attached to the plate surface.
Antigen capture is another ELISA technique, but in this instance, anti-Ebola antibody, not viral antigen, is absorbed to the wells of a microtiter plate (Fig. 28.13). Patient serum is then added to the wells. If the serum contains Ebola antigen, the antigen will be captured by the antibody in the well. Then a second, enzyme-conjugated antibody against the Ebola antigen is added. The more antigen present in the serum, the more enzyme-linked antibody will affi x to the well. Addition of the appropriate chromogenic substrate will produce a colored product that can be measured. Antibody against Ebola may be easier to detect than viral antigen because antibodies will be present at higher levels than the virus itself. But because there is a delay between the time when the virus is fi rst present in serum and when the body manages to make antibody, a speedier diagnosis can be made by directly detecting viral antigen. While bacterial infections are commonly diagnosed by growing the infecting organism on artificial medium in the clinical laboratory, viral diseases are usually diagnosed by immunological means. Viruses are more difficult to grow than bacteria and do not exhibit the biochemical diversity so useful for identifying different bacterial species. In addition to immunological tests, viral diseases can be diagnosed using molecular approaches, such as PCR, to identify viral DNA or RNA sequences. THOUGHT QUESTION 28.4 Specific antibodies against an infectious agent can persist for years in the bloodstream. So how is it possible that antibody titers can be used to diagnose diseases such as infectious mononucleosis?
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cent antibody stain of pleural fluid from a patient with Legionnaires’ disease.
A.
Other Microbes B.
Space does not permit a complete listing of the various diseases and the methods used to identify the etiological agents, but Table 28.3 presents some additional examples. In general, bacteria that are easily cultured are grown in the laboratory, after which biochemical tests are performed. Bacterial, viral, and fungal species that are difficult to grow are typically identified using immunological techniques. These tests either identify antigen from the microbe in infected tissues or measure a rise in antibody titer. As noted Figure 28.14 Fluorescent antibody stain. A. S. pneumoniae capsule (cells approx. 0.8 µm, fluorescence microscopy). The capsule is the green halo. The earlier, DNA-based methodologies center of the halo is the cell. B. Legionella pneumophila (approx. 1 µm in length) are gaining increasing acceptance. from a respiratory tract specimen. Eukaryotic microbial parasites such as Plasmodium species (the cause of malaria), Giardia lamblia (which Fluorescent Antibody Staining causes giardiasis, a diarrheal disease), and Entamoeba histolytica (the cause of amebic dysentery) can be idenChapter 27 presents a case history involving an 80-yeartified via their telltale morphologies under the microold nursing home resident who contracted pneumonia scope, making biochemical tests unnecessary. caused by Streptococcus pneumoniae. The laboratory diag© G. J. Delisle & L. Tomalty. Queens University, Kingston, Ontario, Canada. Licensed for use by ASM Microbe Library
CDC/M. S. Mitchell
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nosis was probably made using the biochemical algorithm previously described. However, there are over 80 serological types of S. pneumoniae, each one containing a different capsular antigen. How can the lab identify which antigenic type has caused the infection? One way is to stain the organism with antibodies. Figure 28.14A illustrates the result of staining a smear of the isolated streptococcus with fluorescently tagged antibodies directed against a specific antigenic type of capsule. Viewed under a fluorescence microscope, the organism is “painted” green when the right antibody binds to the capsule. In the case of pneumonia, this knowledge probably will not help in the treatment of the individual patient, but its broader value is in determining if a single type of organism is responsible for an outbreak of pneumonia, which in turn is of epidemiological value for identifying the source of the bacterium. On the other hand, fluorescent antibody staining techniques are critically important for rapidly identifying organisms that are difficult to grow. Infected tissues can be subjected to direct fluorescent antibody staining. Figure 28.14B, for example, shows a direct fluores-
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TO SU M MAR I Z E: ■
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Selective media are used to inhibit growth of one group of organisms while permitting the growth of others (such as gram-positive bacteria versus gramnegative bacteria). This technique is often used to prevent growth of normal flora while permitting the growth of pathogens. Differential media exploit the unique biochemical properties of a pathogen to distinguish it from similar-looking nonpathogens. Identification of bacterial species can be accomplished with biochemical analyses, molecular techniques (for example, PCR), and/or immunological methods (for example, ELISA). Viral diseases are often diagnosed using immunological tests, such as ELISA, that measure the presence of antibody or antigen, or by real-time quantitative PCR. Fluorescent antibody staining can rapidly identify organisms or antigens present in tissues.
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Table 28.3 Identification procedures for selected diseases. Agent
Disease
Identification
Corynebacterium diphtheriae
Diphtheria
Bordetella pertussis
Pertussis (whooping cough)
Legionella pneumophila
Campylobacter spp.
Legionellosis, Legionnaires’ disease, Pontiac fever Campylobacteriosis
Leptospira interrogans
Leptospirosis
Listeria monocytogenes
Listeriosis
Chlamydia trachomatis Treponema pallidum Francisella tularensis
Chlamydial genital infections Syphilis Tularemia
Yersinia pestis
Plague
Material from nose and throat cultured on a special medium; in vivo or in vitro tests for toxin Smears of nasopharyngeal secretions stained with fluorescent antibody; ELISA for toxin in respiratory secretions; culture on special media Culture on special medium is preferred method; antigen detection by ELISA; Legionella nucleic acid can be identified in clinical material using PCR and nucleic acid probes Isolate bacteria on selective media incubated at 42°C in atmosphere of nitrogen containing 5% oxygen and 10% carbon dioxide; then biochemical testing performed Serological tests early in the illness and after 2–3 weeks to detect rise in antibody titer Culture of blood and spinal fluid; selective and enrichment cultures performed on food samples to grow potential pathogens; DNA probe for rapid identification of colonies Identification of C. trachomatis antigen in urine or pus using monoclonal antibody; nucleic acid probes Direct fluorescent antibody staining; serological tests Cultures using cysteine-containing media; fluorescent antibody stain of pus; detection of rise in antibody titer Identification of capsular antigen using fluorescent antibody or ELISA
Viruses Rhinovirus Influenza
Common cold Flu
Hantavirus
Hantavirus pulmonary syndrome
Herpes simplex
Mumps
Different strains cause cold sores, ocular lesions, and genital lesions Mumps
Rotavirus HIV
Diarrhea AIDS
Strain identification requires use of specific antibodies Tests comparing influenzal antibody levels in blood samples taken during acute and convalescent stages of illness Antigen detection in tissues using electron microscopy or monoclonal antibody; ELISA and Western blot tests for IgG and IgM antibodies in victim’s blood Identifying the viral antigen in clinical material using fluorescent antibody or DNA probes
Rise in antibody titer, or presence of IgM antibody to mumps virus in victim’s blood Electron microscopy or ELISA of diarrheal stool for virus Detection of antibody to HIV-1 in patient’s blood
Fungi Coccidioides immitis
Histoplasma capsulatum
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Coccidioidomycosis, infection of lung; can disseminate to almost any tissue Histoplasmosis, intracellular infection of lung; sometimes disseminates
Observation of large, thick-walled, round spherules from clinical specimens; PCR identification
Stained material from pus, sputum, tissue, etc., examined for intracellular H. capsulatum yeast phase; blood tests for antibody to the organism
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lem began about one week earlier with ill-defined pain in the same area. He had a white blood cell count of 24,900 with 87% granulocytes. An abdominal computed tomography scan revealed an abscess adjacent to his rectum. A needle aspiration drained 20 mL of yellowish, foul-smelling fluid from the abscess. Aerobic cultures of this specimen plated on blood and MacConkey agars were negative. Why didn’t the infectious agent grow?
28.3 Specimen Collection Physicians must collect, and the laboratory must process, a wide variety of clinical specimens. Types of specimens range from simple cotton swabs of sore throats, in which the swab is placed into a liquid transport medium before being sent to the laboratory, to urine and fecal samples that are transported directly. Tables 28.2 and 28.3 outline some of these samples and the techniques used to collect them.
The problem in this instance is related to specimen collection and processing. Internal abscesses located near the gastrointestinal tract are often anaerobic infections, in this case caused by the gram-negative rod Bacteroides fragilis, a strict anaerobe (Fig. 28.15A).
Case History: Abdominal Abscess A 4-year-old boy was admitted to the hospital for evaluation and treatment of persistent pain in the rectal area. His prob-
CDC/Don Stalons
A.
Figure 28.15 Anaerobic infection. A. Gram stain of Bacteroides fragilis (1.5 to 4 µm in length). B. Vacutainer anaerobic specimen collector. Plunging the inner tube to the bottom will activate a built-in oxygen elimination system. The anaerobic indicator changes color when anaerobiosis has been achieved.
B. 1. Remove tube from package
2. Remove plunger with attached swab. Collect sample. Plastic foil package Plunger
3. Reinsert swab and press plunger through stopper so that inner tube drops to bottom of outer tube.
4. Mix by swirling. Transport to laboratory.
Stopper
Swab Inner tube
Platinum catalyst
Anaerobic indicator
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Intestinal microbes, the majority of which are anaerobic, can sometimes escape the intestine if the organ is damaged in some way. The specimen in this instance should have been collected under anaerobic conditions by aspiration into a nitrogen-fi lled tube prior to transport to the clinical laboratory. Alternatively, a swab of the abcess material can be inserted into a special transport tube that has a built-in oxygen elimination system (Fig. 28.15B). Because it was collected, transported, and handled in air, many anaerobic microbes were probably killed by the oxygen. Because B. fragilis has a stress response system that permits survival of this anaerobe for one or two days in oxygen, some of the bacteria may have survived transport. The laboratory still had a chance to fi nd the organism, which raises the second problem in the case. Once the lab received the specimen, they cultured it only under aerobic conditions. The laboratory should have also incubated a series of plates anaerobically (see Section 5.6; Figure 5.22). This case, therefore, illustrates the importance of both proper specimen collection and proper processing. Some body sites should not contain any microorganisms when collected from a healthy individual. These include blood, cerebral spinal fluid, and urine from the
A. Urinary catheter Bladder
B. Throat swab Will & Deni McIntyre/Photo Researchers
Figure 28.16 Specimen collection. A. Urinary catheter showing placement in the urethra. B. Throat swab. C. Sputum collection. A TB patient has coughed up sputum and is spitting it into a sterile container. (The patient is sitting in a special sputum collection booth that prevents the spread of tubercle bacilli. The booth is decontaminated between uses.) D. Lumbar puncture to obtain cerebral spinal fluid.
bladder. Because these sites are sterile, specimens can be plated onto nonselective agar media as well as selective media. Nonselective media, such as blood or chocolate agars, can be used because any organism found in these specimens is considered significant. Urine collection can be problematic, however. When collected from a catheterized patient, urine should be sterile (Fig. 28.16A). Catheterization involves passing a thin, sterile tubing through the urethra and directly into the bladder. (Catheterization is primarily used to assist urination by immobilized patients, but it also provides a convenient way to collect urine for bacteriological examination.) Collections made from the tube should be sterile unless an infection is present. Unfortunately, the simple process of inserting the catheter through the non-sterile urethra can sometimes introduce organisms into the bladder and precipitate an infection. Also, the urine should be collected from the catheter, never from the collection bag. Urine may sit for hours in the collection bag, so organisms initially present at low numbers have time to replicate to high numbers even if the patient does not have a urinary tract infection (UTI). When a catheter is not in place, urine is most commonly collected by what is called a midstream clean-
Urethra
D. Lumbar puncture Catheter
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Tribune photo by Nancy Stone
Courtesy of the CDC
C. Sputum collection
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catch technique, which is performed by the patient. In this procedure, the external genitals are fi rst cleaned with a sterile wipe containing an antiseptic. The patient then partially urinates to wash as many organisms as possible out of the urethra and then collects 5–15 ml of the midstream urine in a sterile cup. This urine sample will not usually be sterile because of urethral contamination, but the number of bacteria will be low. The clinical laboratory determines how many organisms per milliliter are present in the midstream catch and informs the physician as to whether an infection is present. Finding more than 100,000 organisms per milliliter of urine from a midstream clean catch is considered indicative of an infection even in an asymptomatic patient; 10,000 or fewer is considered normal. If the patient actually has symptoms of a UTI, however, then any count greater than 1,000 CFUs/ml of a single species is considered significant and should be treated. Identifying pathogens present at sites that contain normal flora is more challenging. A stool or fecal sample, for instance, is normally teaming with microbial flora. These specimens are typically plated onto selective media (for example, MacConkey, Hektoen, CNA agar) to eliminate or decrease the number of normal flora that might contaminate the specimen. The following represent techniques used to collect specimens from sterile and nonsterile body sites:
Collections from normally sterile sites: Cerebral spinal Lumbar punctures (spinal taps; fluid Fig. 28.16D); testing for meningitis Urine samples Midstream clean catch or from catheters placed in the bladder; testing for urinary tract infections Blood samples Generally taken by syringe from two body sites and placed in liquid media for aerobic and anaerobic culture. The same organism isolated from blood samples taken from the two sites is considered the likely etiological agent.
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THOUGHT QUESTION 28.5 Two blood cultures, one from each arm, were taken from a patient with high fever. One culture grew Staphylococcus epidermidis, but the other blood culture was negative (no organisms grew out). Is the patient suffering from septicemia caused by S. epidermidis? THOUGHT QUESTION 28.6 A 30-year-old woman with abdominal pain went to her physician. After the examination, the physician asked the patient to collect a midstream urine sample that they would send to the lab across town for analysis. The woman complied and handed the collection cup to the nurse. The nurse placed the cup on a table at the nurse’s station. Three hours later, the courier service picked up the specimen and transported it to the laboratory. The next day, the report came back “greater than 200,000 CFUs/ml; multiple colony types; sample unsuitable for analysis.” Why was this determination made?
TO SU M MAR I Z E: ■
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Collections from sites with normal flora: Swabs Throat swabs (Fig. 28.16B), for example, should be placed in specialized liquid nutrient transport medium Sputum Deep lung secretions expectorated for oral collection (Fig. 28.16C) Stool samples Cup or rectal swab; for identifying diarrhea-causing microbes Abscesses Needle aspirations
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Special collection precautions must be taken when collecting specimens from abscesses of suspected anaerobic etiology. Common specimens include blood, pus, urine, sputum, throat, stool, and CSF. Specimens from sites containing normal flora must be handled differently than specimens taken from normally sterile body sites.
28.4 Biosafety Containment Procedures Case History: Fatal Meningitis On July 15, an Alabama microbiologist was taken to the emergency room with acute onset of generalized malaise, fever, and diffuse myalgias. She was given a prescription for oral antibiotics and released. On July 16, she became tachycardic and hypotensive and returned to the hospital. She died 3 hours later. Blood cultures were positive for N. meningitidis serogroup C. Three days before the onset of symptoms, the patient had prepared a Gram stain from the blood culture of a patient subsequently shown to have meningococcal disease; the microbiologist also handled agar plates containing cerebrospinal fluid (CSF) cultures from the same patient. Coworkers reported that in the laboratory, aspirating fluids from blood culture bottles was typically performed at the open laboratory bench. No biosafety cabinets, eye protection, or masks were used for this procedure. Testing at CDC indicated that the isolates from both patients were indistinguishable. The laboratory at the hospital infrequently processed isolates of N. meningitidis and had not processed another meningococcal isolate during the previous four years.
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Medical and laboratory personnel are exposed to extremely dangerous pathogens on a daily basis. The microbiologist in this case did not take appropriate measures to protect herself and ended up with a laboratoryacquired infection leading to meningitis. The CDC has
published a series of regulations designed to protect workers at risk of infection by human pathogens. Infectious agents are ranked by the severity of disease and ease of transmission. Based on this ranking, four levels of containment are employed (Table 28.4).
Table 28.4 Biological safety levels and select agents. Containment level Level 1
Level 2
Level 3
Level 4
Class of disease agent
Category I Agents not known to cause disease
Category III Agents may cause disease by inhalation route
Category IV Dangerous and exotic pathogens with high risk of aerosol transmission; only six such labs in the United States
Recommended safety measures
Basic sterile technique; no mouth pipetting
Category II Agents of moderate potential hazard; also required if personnel may have potential contact with human blood or tissues Level 1 procedures plus limited access to lab; biohazard safety cabinets used; hepatitis vaccination recommended
Representative organisms in class
Bacillus subtilis E. coli K12 Saccharomyces
Level 2 procedures plus ventilation providing directional airflow into room, exhaust air directed outdoors; restricted access to lab (no unauthorized persons) Bacillus anthracis (anthrax) Brucella spp. (brucellosis) California encephalitis Coxiella burnetii (Q fever) EEE (Eastern equine encephalitis) Japanese encephalitis virus La Crosse encephalitis LCM (lymphocytic choriomeningitis) Mycobacterium tuberculosis Rabies Rickettsia prowazekii (typhus fever) Rift Valley fever SARS (severe acute respiratory syndrome) Variola major (smallpox) and other poxviruses VEE (Venezuelan equine encephalitis) West Nile virus Yellow fever
Level 3 procedures plus one-piece positivepressure suits; lab completely isolated from other areas present in the same building or is in a separate building Ebola Guanarito virus Hantavirus Junin virus Kyasanur Forest virus Lassa fever Machupo virus Marburg virus Tick-borne encephalitis viruses
Bordetella pertussis Burkholderia mallei (glanders) Campylobacter jejuni Chlamydia spp. Clostridium spp. Corynebacterium diphtheriae Cryptococcus neoformans Cryptosporidium parvum Dengue Diarrheagenic E. coli Entamoeba histolytica Francisella tularensis (tularemia) Giardia lamblia Haemophilus influenzae Helicobacter pylori Hepatitis Legionella pneumophila Listeria monocytogenes Mycoplasma pneumoniae Neisseria spp. Pathogenic Vibrios Salmonella Shigella spp. Staphylococcus aureus Toxoplasma Yersinia pestis Yersinia enterocolitica
Organisms in blue are on the list of CDC select agents that are considered possible agents of bioterrorism.
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Kevin Karem/CDC
Category I organisms have little to no pathogenic potential and require the lowest level of containment (biosafety level 1). Standard sterile techniques and laboratory practices are sufficient. Category II agents have greater pathogenic potential but are not generally transmitted by the respiratory route. They require more rigorous containment procedures, such as limiting laboratory access when experiments are in progress and the use of biological laminar flow cabinets if aerosolization is possible (biosafety level 2). Category III pathogens can be transmitted via the respiratory route and present a high risk of infection. To safely handle these organisms, level 2 procedures are supplemented with a lab design that ensures that ventilation air flows only into the room (negative pressure) and that exhaust air vents directly to the outside. Negative pressure will keep any organism that may aerosolize from escaping into hallways. In addition, access to the lab is strictly regulated and includes double-door air locks at the entrance (biosafety level 3). By law, extremely dangerous pathogens such as the Ebola virus may only be studied at a biosafety level 4 containment facility. Practices here dictate that lab personnel wear positive-pressure lab suits connected to a separate air supply (Fig. 28.17). The positive pressure ensures that if the suit is penetrated, organisms will be blown away from the breach and not sucked into the suit. As reasonable as these regulations may seem, they were not always in effect. Prior to 1970, scientists had an almost cavalier approach
Figure 28.17 Biosafety level 4 containment. Dr. Kevin Karem at the CDC performs viral plaque assays to determine the neutralization potential of serum from smallpox vaccination trials. He is protected by a positive pressure suit working in a biosafety level 4 laboratory. The airflow into his suit is so loud he must wear earplugs to protect his hearing.
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toward handling pathogens. For instance, culture material was routinely transferred from one vessel to another by mouth pipetting (essentially using a glass or plastic pipette as a straw). This is now forbidden for obvious reasons. TO SU M MAR I Z E: ■
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Various levels of protective measures are used when handling potentially infectious biological materials. Category I agents are generally not pathogenic and require the lowest level of containment. Category II agents are pathogenic but not typically transmitted via the respiratory tract. Laminar flow hoods are required. Category III agents are virulent and transmitted by respiratory route. They require laboratories with special ventilation and air-lock doors. Category IV agents are highly virulent and require the use of positive-pressure suits.
28.5 Principles of Epidemiology Case History: Inhalation Anthrax On October 16, a 56-year-old African-American U.S. Postal Service worker became ill with a low-grade fever, chills, sore throat, headache, and malaise. This was followed by minimal dry cough, chest heaviness, shortness of breath, night sweats, nausea, and vomiting. On October 19, he arrived at a local hospital, where he presented with a normal body temperature and normal blood pressure. He was not in acute distress but had decreased breath sounds and rhonchi (dry sounds in lungs due to congestion). No skin lesions were observed, and he did not smoke. Total WBC count was normal, but there was a left shift in the differential—that is, more polymorphonuclear lymphocytes (PMNs; see Section 26.3). A chest X-ray showed bilateral pleural effusions (accumulation of fluids in the lung) and a small right lower lobe air space opacity. Within 11 hours, blood cultures taken upon admission grew Bacillus anthracis. Ciprofloxacin, rifampin, and clindamycin antibiotic treatments were initiated, and the patient recovered. His job at the post office was to sort mail. From October 4 to November 2, 2001, the Centers for Disease Control and Prevention (CDC) and various state and local public health authorities reported 10 confirmed cases of inhalational anthrax and 12 confirmed or suspected cases of cutaneous anthrax in persons who worked in the District of Columbia, Florida, New Jersey, and New York. Many of them were postal workers. It was clear that a biological attack was in progress. The word epidemiology is derived from the Greek, meaning “that which befalls man.” In scientific parlance, epidemiology examines the distribution and determinants of disease frequency in human populations. Put more simply, epidemiologists determine the source of a disease outbreak
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and the factors that influence how many individuals will succumb to the disease. Epidemiological principles are also used to determine the effectiveness of therapeutic measures and to identify new syndromes, such as SARS (severe acute respiratory syndrome) and Lyme disease. Some of the basic concepts of epidemiology were already covered in Chapter 25 when we discussed infection cycles. Now we will explore how those principles are used to track disease. Epidemiological early-warning systems require an extensive organization that coordinates information from many sources. In the United States, that duty falls to the Centers for Disease Control and Prevention (CDC). On the world stage, it is the World Health Organization (WHO). Any disease considered highly dangerous or infectious is fi rst reported to local public heath centers, usually within 48 hours of diagnosis. The local centers forward that information to their state agencies, which then report to the CDC in Atlanta. This is how authorities in 2001 quickly recognized that an outbreak of anthrax was under way. The terms endemic and epidemic are often used when referring to disease outbreaks. A disease is endemic if it is always present in a population at a low frequency. For example, Lyme disease, caused by the spirochete Borrelia burgdorferi, is endemic to the Northeastern United States because the organism has found a reservoir in deer and ticks. Recall that a reservoir is an animal, bird, or insect that harbors the infectious agent and is indigenous to a geographical area. Humans become infected only when they come in contact with the reservoir. Thus, the disease incidence is low but relatively constant. An epidemic disease, on the other hand, is when large numbers of individuals in a population become infected over a short time. This is due, in part, to rapid and direct human-to-human transmission. Figure 28.18A illustrates the difference in the frequency of cases observed between endemic and epidemic disease. An endemic disease can become epidemic if the population of the reservoir increases, which allows for more frequent human contact, or if the infectious agent evolves to spread directly from person to person, bypassing the need for a reservoir. This is the concern with the H5N1 avian flu virus, which is endemic in animals and birds in Asia (see Sections 11.4 and 25.8). A pandemic is an epidemic that occurs over a wide geographical area, usually the world. The bubonic plague pandemic in the fourteenth century and the AIDS pandemic in the late twentieth and early twenty-first centuries are good examples.
Finding Patient Zero When trying to contain the spread of an epidemic, it is vital to track down the first case of the disease (known as the index case or patient zero) and then identify everyone who has had contact with the individual so that they can be treated or separated from the general population (that is,
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A. Endemic versus epidemic
Endemic
Epidemic
Time B.
AP Photo/Jean-Marc Bouju
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Figure 28.18 The difference between endemic and epidemic disease. A. An endemic disease is continuously present at a low frequency in a population. If the disease frequency suddenly rises, this constitutes an epidemic. B. A health care worker stands outside a quarantined area housing Ebola patients in the Ivory Coast. Epidemics can be minimized if infected persons are kept segregated from the general population (quarantined) to avoid spread of the infectious agent.
quarantined). When a new disease arises, the epidemiological search for the index case starts only after a number of patients have been diagnosed and a new disease syndrome declared. This is what happened with AIDS in the 1970s. Identifying an index case within a specific community is easier if the disease syndrome is already recognized, as was the case with the 2003 SARS outbreak in Singapore. According to the World Health Organization, a suspected case of SARS is defi ned as an individual who
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has a fever greater that 38°C, who exhibits lower respiratory tract symptoms, and who has traveled to an area of documented disease or has had contact with a person affl icted with SARS. The index case in Singapore was a 23-year-old woman who had stayed on the ninth floor of a hotel in Hong Kong while on vacation. A physician from southern China who stayed on the same floor of the hotel during this period is believed to have been the source of her infection, as well as that of the index patients who precipitated subsequent outbreaks in Vietnam and Canada. During the last week of February, the woman, who had returned to Singapore, developed fever, headache, and a dry cough. She was admitted to Tan Tock Seng Hospital, Singapore, on March 1 with a low white blood cell count and patchy consolidation in the lobes of the right lung. Tests for the usual microbial suspects (Legionella, Chlamydia, Mycoplasma) were negative. Electron microscopy of nasopharyngeal aspirations showed virus particles with widely spaced club-like projections (Fig. 28.19A). At the time of her admission to the hospital, the clinical features and highly infectious nature of SARS were not known. Thus, for the fi rst six days of hospitalization, the patient was in a general ward, without barrier infection control measures. During this period, the index patient
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infected at least 20 other individuals, including hospital staff, nearby patients, and visitors. Within weeks, the WHO named the disease in China SARS and issued travel alerts (discussed further in Section 28.6). These alerts allowed Singapore health officials to rapidly identify the index patient and her contacts. As a result, they were able to limit spread of the illness. When all was said and done, SARS killed fewer than 1,000 victims worldwide, although thousands more became ill and recovered (Fig. 28.19B). Today, SARS remains a threat but one of lesser concern. Methicillin-resistant Staphylococcus aureus (MRSA) and H5N1 avian flu are considered more pressing dangers.
Identifying Disease Trends Depends on Physician Notification of Health Organizations How do epidemiologists first recognize that an epidemic is under way, and then identify the agent and its source? Certain diseases, because of their severity and transmissibility, are called reportable or notifiable diseases (Table 28.5). Physicians are required to report instances of these diseases to a central health organization, such as the CDC in the United States and the WHO. As a result, the incidences of certain diseases within a population can be tracked and upsurges noted. An emerging disease not on the list of notifiable diseases can be detected as a cluster of patients with unusual symptoms or combinations of symptoms. This is possible because diseases of unknown etiology are also reported to health authorities. A new disease could manifest with common symptoms (for example, the cough and fever of SARS) that cannot be linked to a known disease agent by clinical tests. An upsurge in cases, either of a reportable disease or an emerging disease, will set off institutional “alarms” that initiate epidemiological efforts to determine the source and cause of the outbreak.
Courtesy of CDC/Dr. Fred Murphy
A.
B.
Anat Givon/AP Photo
John Snow (1813–1858), the Father of Epidemiology
Figure 28.19 Severe acute respiratory syndrome (SARS). A. The coronavirus SARS (TEM). B. Citizens of China, including the military, donned surgical masks in 2003 to slow the spread of SARS.
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The fi rst case in which the source of a disease outbreak was methodically investigated took place in the midnineteenth century. During a serious outbreak of cholera in London in 1854, John Snow (Fig. 28.20A) used a map to plot the locations of all the diarrheal cases he learned about. The source of the infection was unknown, but Snow thought that if the cases clustered geographically, he might gain a clue as to the source. Water in that part of London was pumped from separate wells located in the various neighborhoods. Snow’s map revealed a close association between the density of cholera cases and a single well located on Broad Street (Fig. 28.20B). Simply removing the pump handle of the Broad Street well put an
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Table 28.5 Notifiable infectious diseases. Bacterial Anthrax Botulism Brucellosis Campylobacter infection Chlamydia infection Cholera Diphtheria Ehrlichiosis E. coli O157:H7 infection Gonorrhea Haemophilus influenzae, invasive disease
Hansen’s disease (leprosy) Legionellosis Leptospirosis Listeriosis Lyme disease Meningitis, infectious Pertussis Plague Psittacosis Q fever
Rocky Mountain spotted fever Salmonellosis (nontyphoid fever types) Shigellosis Staphylococcal enterotoxin Streptococcal invasive disease Streptococcus pneumoniae, invasive disease Syphilis Tuberculosis Tularemia Typhoid fever Vancomycin-resistant Staphylococcus aureus (VRSA)
Viral Dengue fever Hantavirus infection Hepatitis, viral Human immunodeficiency virus infection Measles
Mumps Varicella (chickenpox), fatal cases only (all types) Poliomyelitis Yellow fever Rabies Rubella and congenital rubella syndrome Smallpox
Fungal Coccidioidomycosis
Parasitic Amebiasis Cryptosporidiosis Cyclosporiasis
Giardiasis Trichinosis Microsporidiosis
Malaria
A.
Early epidemiology. A. John Snow. B. The map of London that Snow used to pinpoint the source of the cholera outbreak in 1854. The Broad Street well is found within the red circle. Each black bar represents a death from cholera.
B.
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The John Snow Archive and Research Companion
The John Snow Archive and Research Companion
Figure 28.20
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THOUGHT QUESTION 28.7 Methicillin, a betalactam antibiotic, is very useful in treating staphylococcal infections. The development of methicillin-resistant strains of Staphylococcus aureus (MRSA) is a very serious development because there are few antibiotics that can kill these strains. Imagine a large metropolitan hospital in which there have been eight serious nosocomial infections with MRSA and you are responsible for determining the source of infection so it can be removed. How would you accomplish this using common bacteriological and molecular techniques?
Genomic Strategies Help Identify Nonculturable Pathogens Microbiologists have successfully developed many strategies to identify the causes of infectious diseases. Robert Koch in the late 19th century devised a set of postulates that, when followed, can identify the agent of a new disease (discussed in Section 1.3). One important feature of Koch’s postulates is that the suspected organism be grown in pure culture. However, there are bacterial diseases for which the agent could not be cultured. How might they be identified?
Case History: Whipple’s Disease In April, a 50-year-old man had an abrupt onset of watery diarrhea with a stool frequency of up to ten times every 24 hours. This was the latest episode in a six-year history of illness beginning with recurring fevers, flu-like symptoms, profuse night sweats, and painful joint swelling. The current bout of diarrhea was associated with gripping lower abdominal pain, especially after meals. No blood or mucus was found in the stools. Blood and stool cultures tested negative for known infectious agents. Serology was also negative for syphilis, brucellosis, toxoplasmosis, and leptospirosis. His weight fell rapidly from 83 kg to 73 kg within four weeks of onset. A flexible sigmoidoscopy showed only diffuse mild erythema in the bowel. However, the appearance of the small bowel was consistent with malabsorption, a disease in which nutrients are poorly absorbed by the intestine. A duodenal biopsy to look for the organisms of Whipple’s disease showed large macrophages in the lamina propria (a layer of loose connective tissue beneath the epithelium of an organ). Bacterial rods characteristic of Whipple’s disease were seen with electron microscopy. PCR analysis of tissue samples confirmed the diagnosis.
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First diagnosed in 1907 by George Whipple, the symptoms of this disease are malabsorption, weight loss, arthralgia (joint pain), fevers, and abdominal pain. Any organ system can be affected, including the heart, lungs, skin, joints, and central nervous system. The cause of Whipple’s disease went undiscovered for 85 years but was suspected to be of bacterial etiology even though an organism was never successfully cultured. The agent was fi nally identified in 1992 not by culturing, but by blindly amplifying 16S rDNA sequences from biopsy tissues. All bacterial 16S rRNA genes have some sequence regions that are highly homologous across species and other sequences that are unique to a species. Tissues from numerous patients diagnosed with Whipple’s disease were subjected to PCR analysis using the common 16S rDNA primers. If a bacterial agent were present, it was predicted that PCR should successfully amplify a DNA fragment corresponding to the agent’s 16S rRNA gene. All tissues produced such a fragment, indicating that there were bacteria in the tissues. DNA sequence analysis of these fragments indicated that the organism was similar to actinomycetes but was unlike any of the known species. The organism is actually a gram-positive soil-dwelling actinomycete that has been named Tropheryma whipplei in honor of the physician who fi rst recognized the disease. Because of its bacterial etiology, Whipple’s disease can be treated with antibiotics, usually trimethoprim sulfamethoxazole (Bactrim, Septra, Cotrim). Figure 28.21 shows an in situ hybridization for T. whipplei RNA in a tissue biopsy.
D. N. Fredricks & D. A. Relman. 2001. J Infect Dis 183:1229
end to the epidemic, proving that the well water was the source of infection. This approach succeeded brilliantly despite the fact that the infectious agent that causes cholera, Vibrio cholerae, was not recognized until 1905, over 50 years later. Identifying clusters of patients affl icted with a given disease is still used to identify potential sources of infectious disease outbreaks.
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T. whipplei RNA (blue) Host nuclei Host cytoskeleton 50 µm
Figure 28.21 Whipple’s disease. Fluorescent in situ hybridization of a small intestinal biopsy in a case of Whipple’s disease (confocal laser scanning microscopy). In this test, a fluorescently tagged DNA probe that specifically hybridizes to T. whipplei RNA is added to the tissue. Other fluorescent probes are used to visualize host nuclei and cytoskeleton. T. whipplei rRNA is blue, nuclei of human cells are green and the intracellular cytoskeletal protein vimentin is red. Magnification approximately 200×.
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This story reveals that Koch’s postulates (see Figure 1.18) must sometimes be modified when identifying the cause of a new disease. In this instance, the organism could not be cultured in pure form in the laboratory but was found, via molecular techniques, in all instances of the disease. Note that more recent studies have successfully cultured T. whipplei in vitro. The medium used was formulated based on nutritional requirements deduced from knowing the DNA sequence of the T. whipplei genome (discussed in Section 25.6). THOUGHT QUESTION 28.8 What are some reasons why some diseases spread quickly through a population while others take a long time?
Molecular Approaches Can Be Used for Disease Surveillance A worldwide pandemic of pulmonary tuberculosis currently affects over 2 billion people. Many of the Mycobacterium tuberculosis infections are caused by multidrug-resistant strains that are difficult, if not impossible, to kill with existing antibiotics (see Section 26.3). This problem is especially serious among refugee populations attempting to flee war-torn countries. As a result, it is important to screen these refugees as they enter neighboring countries with low incidences of tuberculosis. Although chest X-rays are mandatory in many cases, a positive image will be obtained only if the disease is at a relatively advanced stage. Actively infected individuals who have not developed the characteristic lung tubercles seen on X-ray will not be identified. Unfortunately, the acid-fast staining of sputum samples (discussed in Section 28.2) also fails to detect individuals at an early stage of infection. Studies have shown, however, that PCR techniques are much more sensitive for detecting these individuals. As time progresses, PCR surveillance strategies will be used more often to track the worldwide ebb and flow of microbial diseases. PCR and restriction fragment length polymorphism (RFLP) strategies are already used for epidemiological purposes to type (that is, determine relatedness of) different microbial isolates by generating a complex DNA profi le that is specific for a particular strain (Section 28.2).
Bioterrorism “A wide-scale bioterrorism attack would create mass panic and overwhelm most existing state and local systems within a few days,” said Michael T. Osterholm, director of the Center for Infectious Disease Research and Policy at the University of Minnesota. “We know this from simulation exercises.” These words were spoken in October 2001.
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Less than a month after the September 11 attack on the World Trade Center, some unknown person(s) sent weapons-grade anthrax spores through the U.S. mail. Thankfully, only 5 persons died and a mere 25 became ill. But even though the efficiency of the attack was poor, the impact was enormous. Over 10,000 people took a two-month course of antibiotics after possible exposure, and mail deliveries throughout the country were affected. The simple act of opening an envelope suddenly became a risky endeavor. As a result of this attack and other events, the CDC and the National Institutes of Health (NIH) have assembled a list of select agents (marked in blue in Table 28.4) that could potentially be used as bioweapons. A bioweapon is considered to be any infectious agent or toxin that has high virulence and/or mortality rate. Microorganisms considered bioweapons can be used to conduct biowarfare, in which the intent is to infl ict massive casualties, or bioterrorism, in which there may be a few casualties but widespread psychological trauma. Although the list of select agents is recent, biowarfare is not new. In the Middle Ages, victims of the Black Death (plague caused by Yersinia pestis) were flung over castle walls using catapults; during the French and Indian War, in the eighteenth century, Jeffery Amherst distributed smallpox-infected blankets to Native Americans; and during World War II, the Japanese Imperial Army experimented with infectious disease weapons using Chinese prisoners as guinea pigs. Even the United States has participated through the development of weapons-grade anthrax spores. It was originally thought that the strain of B. anthracis used in the 2001 anthrax attack was the same as the strain developed by the military. We now know that the strain used in the attack had a more ordinary pedigree. The fi rst documented act of bioterrorism in the United States occurred in 1984, when followers of the cult leader Bhagwan Shri Rajneesh tried to control a local election in Dalles, Oregon, by infecting salad bars with Salmonella. Over 900 people became ill. Rajneesh was sentenced to 20 years in prison. How effective are bioweapons? The method by which a biological agent is dispersed plays a large role in its effectiveness as a weapon. Only a few people became ill during the 2001 anthrax attack not only because of the epidemiological surveillance, but because anthrax is inherently difficult to disperse. Thousands of spores must be inhaled to contract disease, which means that effective dispersal of the spores is critical for the use of anthrax as a weapon. Once spores hit the ground, the threat of infection is limited. Weapons-grade spores are very fi nely ground so that they stay airborne longer. But as we saw, the letter-borne dispersal system was not very effective in terms of generating large numbers of victims. Nevertheless, the potential threat of weapons-grade anthrax
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Dealing with bioterrorism. A. Members of a hazardous materials team near Capitol Hill during the anthrax attacks in 2001. B. An Illinois man suffering from smallpox, 1912.
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ons (see Table 28.4). Research with organisms considered to be select agents is tightly regulated. Because Yersinia pestis, for instance, is a select agent, laboratory personnel working with it must now possess security clearance with the Department of Justice (even though the organism itself can be handled under biosafety level 2 conditions). The laboratory must also register with the CDC to legally possess this pathogen and access to the lab and the organism must be tightly controlled. Much has improved since Michael Osterholm offered his dire assessment of a wide-scale bioterrorism attack. Education and surveillance procedures have been bolstered, and new detection technologies are being developed (Special Topic 28.1). We will probably never be fully protected from attack, biological or otherwise, but recent efforts have improved the situation. Bioterrorism Preparedness Act
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John Snow founded the discipline of epidemiology. Epidemiology is a field that examines factors that determine the distribution and source of disease. Endemic, epidemic, and pandemic are terms for different frequencies of disease in different geographical areas. Finding patient zero is important for containing the spread of disease. Molecular approaches using PCR and nucleic acid hybridization are used to identify nonculturable pathogens and to track disease movements. Bioweapons, when they have been used, typically kill few people but create great fear. The CDC has assembled a list of select agents with bioweapon potential.
A.
B.
Kenneth Lambert/AP
Figure 28.22
M edic ine and I mm u n o l o gy
Illinois Department of Public Health
on the battlefield led the U.S. military in 2006 to resume vaccinating all soldiers serving in Iraq, Afghanistan, and South Korea. An effective bioweapon would be one that capitalizes on person-to-person transmission. In an easily transmitted disease, one infected person could disseminate disease to scores of others within one or two days. So in terms of generating massive numbers of deaths, anthrax was a poor choice. But the goal of most terrorists is not to kill large numbers of people, but to terrorize them. In that regard, the anthrax attack succeeded. The most effective bioweapon in terms of infl icting death (biowarfare) would have a low infectious dose, be easily transmitted between people, and be one to which a large percentage of the population is susceptible. Smallpox fits these criteria and would be the bioweapon of choice (Fig. 28.22). Fortunately, however, smallpox has been eradicated (almost) from the face of the Earth and is not easily obtained. Two laboratories still harbor the virus—one in the United States and one in Russia. It is believed that the virus has been destroyed in all other laboratories. Since smallpox is the perfect biowarfare agent, it is imperative that the last two smallpox repositories remain secure. While the good news is that smallpox disease has been eradicated, the bad news is that no individual born after 1970 has been vaccinated. As a result, anyone under 35 years of age is susceptible to smallpox. Even those of us who received the smallpox vaccination over 35 years ago are at risk, since our protective antibody titers have diminished. A terrorist attack with smallpox would cause terrible numbers of deaths. The vaccine does, however, exist. Were a smallpox attack to be launched, the vaccine would be rapidly administered to limit the spread of disease. Nevertheless, the economic and psychological impact of a smallpox epidemic would be devastating. The CDC has assembled a list of so-called “select agents” that are considered possible bioterrorist weap-
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Special Topic 28.1
Microbial Pathogen Detection Gets Wired Up
The emergence of pathogenic infectious diseases resulting from both natural and intentional acts is a growing global concern. Acts of bioterrorism, particularly, represent a formidable challenge that dictates the need for rapid and efficient means of pathogen detection. The ideal detection system would integrate sample processing and pathogen characterization into a single automated device that would eliminate labori-
Target probe (pathogen DNA)
Capture probe
ous, time-consuming sample processing and costly detection. One promising detection system involves a hybridizationbased bioelectronic DNA detection chip that is in the testing phases. The basic design, illustrated in Figure 1, requires two hybridization events to generate a detection signal. First, a DNA capture probe is affixed to a gold electrode chip. The capture probe is complementary to one part of a gene that is specific to a given pathogen. Next, a solution containing unknown DNA is injected into the chip. If the target gene is present, the capture probe will hybridize to a single-stranded part of the gene’s DNA and “capture” it. The actual detection signal is then generated following a second hybridization. The second probe used is complementary to the downstream region of the same captured gene. This probe is not affixed to the gold support but contains a ferrocene label at one end. If the target DNA is captured, the second probe will anneal
Signal probe 5′
Fe
Electronic detection of pathogens. Schematic representation of the eSensor DNA Detection System. A self-assembled monolayer is generated on a gold electrode. For electrochemical detection to occur across the monolayer, two hybridization events must occur: the first between the capture probe (shown in purple perpendicular to the electrode) and the target (for example, HPV DNA, shown in gray), and the second between an adjacent region of the target HPV DNA and a second, ferrocene-labeled signal probe (shown in purple parallel to the monolayer).
Figure 1
Fe
5′
Fe
Ferrocene labels
3′
Self-assembled monolayer
Gold electrode
28.6 Detecting Emerging Microbial Diseases Case History: SARS A 48-year-old man was hospitalized in a Dutchess County, New York, hospital with a 101°F fever, headache, and body aches. He also had difficulty breathing. He had just returned from a business trip to China.
SARS: An Epidemiological Success Story Within seven weeks in early 2003, epidemiologists armed with global technologies and rapid DNA-sequencing techniques tracked, named, identified, completely sequenced and contained a newly emerging disease with a scary death rate: severe acute respiratory syndrome (SARS). This was a remarkable feat, especially when we consider that after
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the first cases of AIDS appeared in 1981, it took over three years just to identify the virus, and it took seven years to track down the Lyme disease spirochete, Borrelia burgdorferi, after Lyme disease was first recognized in 1975. We now know that SARS fi rst developed in Asia, then began to spread by jet to other countries, such as Canada. While the death rate seemed modest at about 5%, it was higher than the 4% reached during the 1918 flu epidemic that killed 25–40 million people worldwide. SARS clearly posed a formidable threat. Unprecedented cooperation between the WHO, the CDC, and numerous other health organizations around the world played a major role in tracking and containing the disease. Patient information, case histories, possible treatment regimens flooded into a secure WHO website. The most exciting posting was the 30,000-bp sequence of the SARS genome, taken from one-millionth of a gram of genetic
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and bring the ferrocene molecules in contact with the gold electrode, generating an electrochemical signal. These signals are then detected by a scanning AC voltammetry technique (voltammetry is an electroanalytical method whereby information about an analyte is obtained by measuring current as potential is varied). As shown in Figure 2, Suzanne Vernon and colleagues at the Centers for Disease Control (CDC) have used this approach to detect the presence of human papillomavirus (HPV, a cause of genital warts) in a dramatic test of the system.
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Of course, an ideal system to use in the field would also include an automated DNA extraction technology, a development that is yet to come. Nevertheless, the elegance of this design promises to be invaluable for pathogen detection under a variety of circumstances. Source: S. D. Vernon, D. H. Farkas, E. R. Unger, V. Chan, D. L. Miller, et al. 2003. Bioelectronic DNA detection of human papillomaviruses using eSensor™: A model system for detection of multiple pathogens. BMC Infectious Diseases 3:12.
HPV-negative sample 1.0E-7
8.0E-8
8.0E-8
Signal output (nA)
Signal output (nA)
HPV-positive sample 1.0E-7
6.0E-8 4.0E-8 2.0E-8 4.6E-11 –100
0
100
200 300 400 Potential (mV)
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500
600
6.0E-8 4.0E-8 2.0E-8 4.6E-11 –100
0
100
200 300 400 Potential (mV)
500
600
Figure 2 Bioelectronic signal output. The eSensor bioelectronic representative signal output. The peaks in the first panel reveal the electrochemical signal generated when the ferrocene from the DNA probe comes in contact with the gold electrode. The second panel represents a negative control sample.
material isolated from a Toronto patient. The following 2003 timeline illustrates the remarkable speed with which all this took place: February 14—China first reports 305 cases of atypical pneumonia to WHO. March 15—WHO issues emergency travel alert, names illness SARS. March 17—Leading epidemiologists join WHO in conference call, agree to unprecedented cooperation. April 4—U.S. President George Bush authorizes quarantine of SARS patients. April 12—Canadian scientists in Vancouver post the completed genome on the Internet. April 16—WHO announces that SARS is caused by a pathogen never before seen in humans, a new coronavirus.
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Technology Helps New Infectious Agents Emerge and Spread Despite all we know of microbes, despite the many ways we have to combat microbial diseases, our species, for all its cleverness, still lives at the mercy of the microbe. Lyme disease, MRSA, SARS, Ebola, E. coli O157:H7, HIV, “flesheating” streptococci, hantavirus—all of these and many other new diseases have emerged over the last 30 years. Worse yet, forgotten scourges, such as tuberculosis, have reappeared. Yet in the 1970s, medical science was claiming victory over infectious disease. What happened? Part of the equation has been progress itself. Travel by jet, the use of blood banks, and suburban sprawl have all opened new avenues of infection. People unwittingly infected by a new disease in Asia or Africa can, traveling by jet, bring the pathogen to any other country in the world within hours. The person may not even show
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symptoms until days or weeks after the trip. This means that diseases can spread faster and farther than ever before. In addition, newly emerged blood-borne pathogens can spread by transfusion. This was a major problem with HIV before an accurate blood test was developed to screen all donated blood. Although human encroachments into the tropical rain forests have often been blamed for the emergence of new pathogens, one need go no farther than the Connecticut woodlands to find such developments. Borrelia burgdorferi, the spirochete that causes Lyme disease, lives on deer and white-footed mice and is passed between these hosts by the deer tick (Fig. 26.26). This infectious cycle has been going on for years. Humans have crossed paths with these animals long before the disease erupted in our communities. Why have we suddenly become susceptible? The answer appears to be suburban development. In the wild, foxes and bobcats hunt the mice that carry the Lyme agent. These predators disappear as developers clear land and build roads and houses, leaving the infected mice and ticks to proliferate. Humans in these developed areas are more likely to be bitten by an infected tick and contract the disease than in prior decades. Luckily, many diseases that successfully leap from animals to humans find the new host to be a dead end, unable to spread the disease to others. There are numerous examples in which technology and progress have had the unintended consequence of breeding disease. Here are just a few:
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Mad cow disease: Modern farming practices (North America and Europe) of feeding livestock the remains of other animals helped spread transmissible spongiform encephalopathies similar to Creutzfeldt-Jakob disease associated with prions. Because the prion is infectious, the brain matter from one case of mad cow disease could end up infecting hundreds of other cattle, which in turn increases the chance that the disease could spread to humans. Lyme disease: Suburban development in the northeastern United States destroys predators of the mice that carry Borrelia burgdorferi. Hepatitis C: Transfusions and transplants spread this blood-borne disease. Influenza: Live poultry markets in Asia serve as breeding grounds for avian flu viruses that can jump to humans. Enterohemorrhagic E. coli (for example, E. coli O157:H7): Modern meat-processing plants can accidentally grind trace amounts of these acid-resistant, fecal organisms into beef while making hamburgers.
Natural environmental events can also trigger upsurges in the incidence of unusual diseases. A good example of this involves the hantavirus (also known as Sin Nombre virus). An unprecedented outbreak of hantavirus pulmonary disease occurred in the Four Corners
Multidrug-resistant tuberculosis Cryptosporidiosis Antibiotic-resistant “Staph” bacteria
Variant Creutzfeldt-Jakob disease (vCJD) Diphtheria H7N7 bird flu
E. coli 0157:H7 Cyclosporiasis
Hepatitis C
Cholera
Dengue fever Hantavirus pulmonary syndrome
Cholera
Yellow fever
Rift Valley fever HIV (AIDS virus)
Lassa fever Marburg virus
Ebola hemorrhagic fever
Cholera Drug-resistant malaria
E. coli 0157:H7 H5N1 bird flu Drug-resistant malaria Nipah virus
Hendra virus
Human monkeypox Plague
Antibiotic-resistant “Staph” bacteria
Typhoid fever
Lyme disease West Nile fever
Whitewater Arroyo virus
Severe acute respiratory syndrome (SARS)
Enterovirus 71
Locations of some emerging and reemerging infectious diseases. The examples given represent extreme increases in the reported cases. Many of these diseases, such as HIV and cholera, are widespread but show alarming increases in the areas indicated.
Figure 28.23
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area of Arizona, New Mexico, Colorado, and Utah in 1993 when rain lead to greater than normal increases in plant and animal numbers. The resulting tenfold increase in deer mice, which carries the virus, made it more likely that infected mice and humans would come in contact.
Detecting Emerging and Reemerging Pathogens A world map showing the general locations of emerging and reemerging diseases is shown in Figure 28.23. A reemerging disease is one thought to be under control but whose incidence has risen. For example, the incidence rate of tuberculosis dropped sharply in the 1950s, but the number of recent cases has increased just as dramatically. Tuberculosis is a reemerging disease. The trigger for its reemergence was the AIDS pandemic beginning in 1980. Immunocompromised AIDS patients are highly susceptible to infection by many organisms, including Mycobacterium tuberculosis. Mycobacterium tuberculosis is also reemerging among non-AIDS patients owing to the development of drugresistant strains. Drug resistance in M. tuberculosis developed largely because of noncompliance on the part of patients to complete their full courses of antibiotic treatment. Treatment usually involves three or more antibiotics to reduce the risk that resistance to any one drug will develop. Many patients failed to take all three drugs simultaneously, allowing the organism to develop resistance to one drug at a time until it became resistant to all of them. These highly drug-resistant strains are almost impossible to kill. The link between noncompliance and the development of drug resistance is the primary reason why tuberculosis patients are now required to take the multiple antibiotics prescribed in the presence of the physician or a member of the medical staff. As described in Section 28.5, highly infectious diseases are identified using aggressive epidemiological surveillance. Communications between local, national, and world health organizations are critical and helped expose the rise in tuberculosis. To see for yourself how emerging diseases are monitored, search the Internet for
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1095
a website titled ProMed-mail. There you will fi nd daily reports posted from around the world that describe new outbreaks of infectious diseases. For example, a posting on June 21, 2006, reports that “11 students at a Franklin elementary school (Boston) have confirmed cases of salmonella, and state health officials are investigating whether owl pellets used in a class project were the source of the bacterial infection.” The source was eventually confirmed to be the owl pellets. Another posting from the Congo in Africa notes reports of 100 cases of suspected pneumonic plague, including 19 deaths in the Ituri district, Oriental province. The site also posts reports of undiagnosed illnesses that may be the initial signs of emerging diseases. For instance, on June 23, 2006, there is a report of an undiagnosed die-off of wild birds in Zambia, which may be due to avian flu. By tracking these reports, trends of disease spread can be determined. THOUGHT QUESTION 28.9. Using the “Maps of Outbreaks” provided on the ProMed-mail website, identify the countries considered to have anthrax epidemics.
TO SU M MAR I Z E: ■
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Emerging diseases can spread quickly around the world as a result of air travel. Modern technology and urban growth have provided opportunities for new diseases to emerge.
Concluding Thoughts This chapter has examined the basic principles used to collect, detect, and track pathogenic microorganisms. As you can see, the task of controlling the spread of disease is daunting and ever changing because microorganisms continue to evolve. Known pathogens change, eluding eradication efforts by the immune system and antibiotics, while new pathogens keep emerging. The world is depending on the next generation of microbiologists to face the growing threat of these evolving microbes.
C H A P T E R R E V I EW Review Questions 1. Why is it important to identify the genus and species
4. If a colony on a nutrient agar plate is catalase-positive,
of a pathogen? 2. What is an API strip, and what is its use in clinical microbiology? 3. Describe three examples of selective media.
does this mean it is made up of gram-positive microorganisms? Why or why not? 5. Describe the types of hemolysis visualized on blood agar.
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6. What is the clinical significance of a group A, beta7. 8. 9. 10. 11. 12.
hemolytic streptococcus? How does one distinguish S. aureus from S. epidermidis? Why are PCR identification tests preferable to biochemical approaches? How is RTQ-PCR performed? Describe an ELISA. Name some sterile and nonsterile body sites. List seven common types of clinical specimens collected for bacteriological examination.
13. Describe key features of the four levels of biological
containment. 14. How is a pandemic different from an epidemic? 15. How can genomics help identify nonculturable
pathogens? 16. List and provide short descriptions of four emerging
diseases. 17. Name four select agents and the diseases they cause.
Key Terms bioweapon (1090) emerging disease (1095) endemic (1086) enzyme-linked immunosorbent assay (ELISA) (1077)
epidemic (1086) index case (1086) multiple PCR (1073) pandemic (1086) patient zero (1086)
quarantined (1086) reemerging disease (1095) reportable (notifiable) disease (1087) reservoir (1086) sequela (1064)
Recommended Reading Baker, Edward L., Margaret A. Potter, Deborah L. Jones, Shawna L. Mercer, Joan P. Cioffi, et al. 2005. The public health infrastructure and our nation’s health. Annual Review of Public Health 26:303–318. Bengis, R. G., F. A. Leighton, J. R. Fischer, M. Artois, T. Morner, and C. M. Tate. 2004. The role of wildlife in emerging and re-emerging zoonoses. Reviews in Science and Technology 23:497–511. Espy, Mark, James Uhl, Lynne Sloan, Seanne Buckwalter, Mary Jones, et al. 2006. Real time PCR in clinical microbiology: Application for routine laboratory testing. Clinical Microbiology Reviews 19:165–256. Fournier, Pierre-Edouard, Michel Drancourt, and Didier Raoult. 2007. Bacterial genome sequencing and its use in infectious diseases. Lancet Infectious Diseases 7:711–723. Gaydos, Charlotte A., Mellisa Theodore, Nicholas Dalesio, Billie J. Wood, and Thomas C. Quinn. 2004. Comparison of three nucleic acid amplification tests for detection of Chlamydia trachomatis in urine specimens. Journal of Clinical Microbiology 42:2041–3045. Kuiken, Thijs, Ron Fouchier, Guus Rimmelzwaan, and Albert Osterhaus. 2003. Emerging viral infections in a rapidly changing world. Current Opinion in Biotechnology 14:241–246. Madoff, Lawrence C., and John P. Woodall. 2005. The Internet and global monitoring of emerging diseases: Lessons from
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the fi rst 10 years of ProMed-mail. Archives of Medical Research 36:724–730. Molinari, J. A. 2003. Diagnostic modalities for infectious diseases. Dental Clinics of North America 47:605–621. Pejcic, Bobby, Roland De Marco, and Gordon Parkinson. 2006. The role of biosensors in the detection of emerging infectious diseases. Analyst 131:1079–1090. Raman, Lakshmy Anantha, Noman Siddiqi, Mohammed Shamim, Monorama Deb, Geeta Mehta, and Seyed Ehtesham Hasnain. 2000. Molecular characterization of Mycobacterium abscessus strains from a hospital outbreak. Emerging Infectious Diseases 6:561–562. Relman, David A., Thomas M. Schmidt, Richard P. MacDermott, and Stanley Falkow. 1992. Identification of the uncultured bacillus of Whipple’s disease. New England Journal of Medicine 327:283–301. Shi, Pei-Yong, Elizabeth Kauffman, Ping Ren, Andy Felton, Jennifer Tai, et al. 2001. High throughput detection of West Nile Virus RNA. Journal of Clinical Microbiology 39:1264–1271. Shvartzman, Pesach, and Yussuf Nasri. 2004. Urine culture collected from gel-based diapers: Developing a novel experimental laboratory method. The Journal of the American Board of Family Practice 17:91–95.
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Appendix 1
Biological Molecules
A1.1 A1.2 A1.3 A1.4 A1.5 A1.6 A1.7
Elements, Bonding, and Water Common Features of Organic Molecules Proteins Polysaccharides Nucleic Acids Lipids Chemical Principles in Biological Chemistry
This appendix reviews information typically covered in an introductory biology course. We first cover the chemical bonding principles needed to understand biological molecules, with an emphasis on the special properties of water. We then discuss organic molecules, paying particular attention to four important classes of organic biomolecules—proteins, polysaccharides, nucleic acids, and lipids. Finally, we explore common chemical principles, such as concentrations, thermodynamics, equilibrium, pH, and oxidation-reduction reactions.
A computer model showing the structure of the enzyme RNA polymerase II. The molecule comprises 12 subunits. This enzyme synthesizes a complementary mRNA strand from a strand of DNA during a process called transcription. It recognizes a start sign on the DNA strand and then moves along the strand building the mRNA until it reaches a stop sign. mRNA is the intermediary between DNA and its protein product. Source: Mark J. Winter/Photo Researchers, Inc.
A-1
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Bio lo g ic a l M o lec ules
Living cells are remarkably complex machines, able to integrate and respond to multiple stimuli, to catalyze reactions, and to replicate themselves. Yet despite all the various tasks that cells perform, 98% of the mass of living organisms consists of only six elements: hydrogen (H), oxygen (O), nitrogen (N), phosphorus (P), sulfur (S), and carbon (C); and 90% of the mass is accounted for by just C, H, and O. Of the compounds formed from these elements, the most abundant in cells is water. The remainder of the cell consists, for the most part, of just four different kinds of organic (carbon-based) macromolecules: proteins, nucleic acids, carbohydrates, and lipids. In one respect, cells can be thought of as compartments that orchestrate chemical reactions between these organic molecules. It is clear that to understand life, we must understand the properties of water and organic molecules and of their building blocks, the chemical elements.
A1.1 Elements, Bonding, and Water Cells consist mostly of water and organic molecules. These cellular components are formed from atoms of various elements. An atom consists of a positively charged nucleus that contains protons and neutrons, surrounded by negatively charged electrons. The hydrogen nucleus consists of a single proton. Protons, neutrons, and electrons differ in their mass and charge as summarized in Table A1.1. The elements can be organized into a periodic table as in Figure A1.1, indicating each element’s atomic number (the number of protons) and atomic mass (the mass in grams of 1 mole of the element). The defi ning characteristic of an element is the atomic number. For example, all carbon atoms have six protons in their nucleus. To maintain neutrality, atoms have negatively charged electrons equal in number to the positively charged protons. The atomic mass is found by adding together the number of protons and neutrons. The atomic mass is an average that takes into account the relative abundance of each isotope. Isotopes are atoms of an element that differ in the number of neutrons. For example, the most abun-
Table A1.1 The mass and charge of atomic particles. Particle Proton Neutron Electron
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Mass (atomic mass unit) 1 1 0.0005
Charge (electronic charge unit) +1 0 –1
dant isotope of carbon is carbon-12 (with six neutrons), but there are naturally occurring isotopes of carbon-13 (seven neutrons) and carbon-14 (eight neutrons). Because carbon-13 and carbon-14 are rare, the average atomic mass is close to but not exactly 12. While some isotopes are stable, others decay at a known rate and give off radioactivity. Carbon-14 has a half-life (the amount of time it takes for half of the sample to decay) of 5,700 years and is used in radiocarbon dating to determine the age of organic material. Carbon-14 and shorter-lived isotopes such as tritium (hydrogen with a mass of 3—one proton and two neutrons) are used by scientists as tracers to follow specific atoms in metabolic pathways.
Bonds Hold Elements Together to Form Molecules Atoms combine by sharing electrons to form molecules. For example, two atoms of oxygen combine to form molecular oxygen, O2. Molecules may also contain more than one kind of element; an example is water, H 2O. The symbols O2 and H2O are examples of molecular formulas, a shorthand notation indicating the number and type of atoms present in a molecule. Each column (group) in the periodic table contains elements of similar reactivity as a result of the similarity in their electronic configurations, particularly of electrons in the outermost shell. The shell closest to the nucleus can hold a maximum of two electrons and the next shell a maximum of eight electrons. Figure A1.2A shows all the electrons for hydrogen, carbon, nitrogen, and oxygen. Each unpaired electron is capable of participating in a bond. If we examine Figure A1.2A, it is clear that H can form one bond, C four bonds, N three bonds, and O two bonds. For example, carbon has four unpaired electrons in its outermost shell; each can form a bond with an unpaired electron from another atom. The two atoms involved in this bond “share” the two electrons. This sharing of electrons is termed a covalent bond. Covalent bonds are very strong and difficult to break. In methane (CH4), carbon forms four covalent bonds with four hydrogen atoms (Fig. A1.2B). Methane is stable because both carbon and hydrogen have filled their outer shells. Atoms can also share more than one pair of electrons with another atom, forming double or triple bonds. In carbon dioxide, CO2, each oxygen shares two pairs of electrons with carbon; and in diatomic nitrogen, N2, the nitrogen atoms each share three pairs of electrons (see Fig. A1.2B). The bonding in molecules can be represented in a shorthand representation by a structural formula, in which covalent bonds are shown as a line between two atoms (Fig. A1.2C). Another way atoms can obtain full outer shells is by gaining or losing electrons. A complete transfer of electrons can occur between two atoms that have a large
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Appe n dix 1
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A-3
B iolog ic al Mo l e cu l e s
Main-group elements
Main-group elements
1 1A
18 8A
Atomic number
1
1
2
3
H
2
1.00794
2 2A
3
4
Chemical symbol
Li
Be
6.941 11
9.01218 12
Na
Mg
Transitional elements
22.9898 24.3050
4
5
6
7
Metals Nonmetals Metalloids Noble gases
Atomic mass (average of all isotopes)
3 3B
4 4B
5 5B
6 6B
7 7B
8
9 8B
10
11 1B
12 2B
He
13 3A
14 4A
15 5A
16 6A
17 7A
5
6
7
8
9
4.00260 10
N
O
F
Ne
B
C
10.811 13
12.011 14
Al
Si
26.9815 28.0855
14.0067 15.9994 18.9984 20.1797 15
16
17
18
P
S
Cl
Ar 39.948
30.9738
32.066
35.4527
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
K
Ca
Sc
Ti
V
Cr
Mn
Fe
Co
Ni
Cu
Zn
Ga
Ge
As
Se
Br
Kr
39.0983
40.078
44.9559
38
39
47.88 40
55.847 44
58.9332 45
58.693 46
63.546 47
65.39 48
69.723
37
49
72.61 50
74.9216 51
78.96 52
79.904 53
83.80 54
Ag
Cd
In
Sn
50.9415 51.9961 54.9381 43 42 41
Rb
Sr
Y
Zr
Nb
Mo
Tc
Ru
Rh
Pd
85.4678
87.62
88.9059
91.224
92.9064
95.94
(98)
101.07
102.906
106.42
107.868 112.411 114.818 118.710 81 80 82 79
Sb
Te
I
Xe
121.76
127.60 84
126.904 85
131.29 86
55
56
57
72
73
74
75
76
77
78
Cs
Ba
*La
Hf
Ta
W
Re
Os
Ir
Pt
Au
Hg
Tl
Pb
Bi
Po
At
Ra
132.905
137.327
138.906
178.49
180.948
183.84
186.207
190.23
192.22
195.08
196.967
200.59
204.383
207.2
208.980
(209)
(210)
(222)
83
87
88
89
104
105
106
107
108
109
110
111
112
114
116
Fr
Ra
†Ac
Rf
Db
Sg
Bh
Hs
Mt
**
**
**
**
**
(223)
226.025
227.028
(261)
(262)
(263)
(262)
(265)
(266)
(269)
(272)
(277)
*Lanthanide series
58
59
60
61
62
63
64
65
66
67
68
69
70
71
Ce
Pr
Nd
Pm
Sm
Eu
Gd
Tb
Dy
Ho
Er
Tm
Yb
Lu
144.24 92
(145) 93
150.36 94
151.965 95
157.25 96
158.925 97
162.50 98
164.930 99
167.26 100
168.934 101
173.04 102
174.967 103
140.115 140.908 90 91
†Actinide series
Th
Pa
U
232.038 231.036 238.029
Np
Pu
Am
Cm
Bk
Cf
Es
Fm
Md
No
Lr
237.048
(244)
(243)
(247)
(247)
(251)
(252)
(257)
(258)
(259)
(260)
**Not yet named Symbol Ac Al Am Sb Ar As At Ba Bk Be Bi Bh B Br Cd Ca Cf C Ce Cs Cl Cr
Name Actinium Aluminum Americium Antimony Argon Arsenic Astatine Barium Berkelium Beryllium Bismuth Bohrium Boron Bromine Cadmium Calcium Californium Carbon Cerium Cesium Chlorine Chromium
Figure A1.1
Symbol Co Cu Cm Db Dy Es Er Eu Fm F Fr Gd Ga Ge Au Hf Hs He Ho H In I
Name Cobalt Copper Curium Dubnium Dysprosium Einsteinium Erbium Europium Fermium Fluorine Francium Gadolinium Gallium Germanium Gold Hafnium Hassium Helium Holmium Hydrogen Indium Iodine
Symbol Ir Fe Kr La Lr Pb Li Lu Mg Mn Mt Md Hg Mo Nd Ne Np Ni Nb N No Os
Symbol O Pd P Pt Pu Po K Pr Pm Pa Ra Rn Re Rh Rb Ru Rf Sm Sc Sg Se Si
Name Oxygen Palladium Phosphorus Platinum Plutonium Polonium Potassium Praseodymium Promethium Protactinium Radium Radon Rhenium Rhodium Rubidium Ruthenium Rutherfordium Samarium Scandium Seaborgium Selenium Silicon
Symbol Ag Na Sr S Ta Tc Te Tb Tl Th Tm Sn Ti W U V Xe Yb Y Zn Zr
Name Silver Sodium Strontium Sulfur Tantalum Technetium Tellurium Terbium Thallium Thorium Thulium Tin Titanium Tungsten Uranium Vanadium Xenon Ytterbium Yttrium Zinc Zirconium
Periodic table of the elements. The atomic number (number of protons) and atomic mass are shown for each element.
difference in electronegativity, a measure of the affi nity of an atom for electrons. A large electronegativity indicates a strong attraction for electrons. Of the elements listed in Table A1.2, oxygen has the greatest attraction for electrons and sodium the weakest. If two elements
SFMB_app01.indd A-3
Name Iridium Iron Krypton Lanthanum Lawrencium Lead Lithium Lutetium Magnesium Maganese Meitnerium Mendelevium Mercury Molybdenum Neodymium Neon Neptunium Nickel Niobium Nitrogen Nobelium Osmium
with greatly different electronegativities come into close contact, one element can “steal” an electron from the other. For example, sodium (Na) and chlorine (Cl) interact to form table salt, NaCl. The electronegative Cl strips an electron away from Na, and both Cl– and Na+ now
1/17/08 12:01:44 PM
A-4
Appendix 1
■
Bio lo g ic a l M o lec ules
A.
A. H
C
N
O
B.
Loss of electron Na+
Na Cation formation
B.
Na+
Gain of electron
H
Cl–
Cl H
Cl–
C
H
O
C
O
N
Anion formation
N
Formation of ions and ionic crystals. A. The loss of an electron from an atom of sodium to form the cation Na+ and the gain of an electron by a chlorine atom to form the chloride anion Cl–. B. Oppositely charged anions and cations—in this case, Cl– and Na+—are attracted to one another and form crystals of table salt (sodium chloride). Figure A1.3
H
C.
H H
C
O C O
H
N N
H
Covalent bonding of hydrogen, carbon, nitrogen, and oxygen. A. The electrons present in hydrogen (H), carbon (C), nitrogen (N), and oxygen (O). B. Electron sharing in the four single bonds present in methane, CH4; the two double bonds present in carbon dioxide, CO2; and the triple bond present in diatomic nitrogen, N2. C. Structural formulas for methane, carbon dioxide, and diatomic nitrogen. Each line represents a covalent bond. Lone pairs of electrons in the outermost shell are represented by dots. Figure A1.2
Table A1.2 Electronegativities of some common elements. Element
Electronegativity
Oxygen Chlorine Nitrogen Sulfur Carbon Hydrogen Sodium
3.44 3.16 3.04 2.58 2.55 2.10 0.93
Water Is the Solvent of Life Living organisms consist mostly of water, and water has many unique properties that render it particularly suitable for sustaining life. To appreciate these properties, we must understand the forces that hold water together: polar covalent bonds and hydrogen bonds. For molecules such as H2 and O2, there is no charge distribution within the molecule because both atoms in the molecule have the same electronegativity. Therefore, the electrons in the covalent bond are shared equally and form nonpolar covalent bonds. In contrast, the shared electrons in H2O spend more of their time around the highly electronegative oxygen than in the vicinity of the less electronegative hydrogen (Fig. A1.4A). A bond with unequal electron sharing is a polar covalent bond, so called because the molecule has partial positive and negative poles. Polar covalent bonds occur in individual water
A.
B. δ– O
have full outer shells (Fig. A1.3A). Both Cl– and Na+ are charged atoms called ions, in which the number of electrons and protons are unequal. Anions are negatively charged ions, and cations are positively charged ions. Anions and cations can form ionic crystals held together by ionic bonds, electrostatic attractions between anions and cations (Fig. A1.3B). In the absence of water, ionic crystals maintain their integrity, but they are destabilized in the presence of water. To understand why, we need to understand the structure of water.
SFMB_app01.indd A-4
Electrons
H
H
δ+
δ+
Hydrogen bond
δ–
δ+ Polar covalent bond
Polar covalent and hydrogen bonds in water. A. The polar covalent bonds in an individual water molecule. Displacement of electrons toward oxygen causes oxygen to have a partial negative charge and the hydrogen atoms to have a partial positive charge. Hence, water is polar. B. A hydrogen bond between two water molecules.
Figure A1.4
1/17/08 12:01:44 PM
Appe n dix 1
■
A-5
B iolog ic al Mo l e cu l e s
Table A1.3 Bond types and strengths. Type of bond
Description
Bond strength in water (kJ/mol)a
Covalent Ionic Hydrogen
Sharing of electrons Electrostatic attraction between anion and cation Electrostatic attraction between a hydrogen bound to a nitrogen or oxygen and a second nitrogen or oxygen Electrostatic attraction between temporary, shifting electron clouds
210–418 12.5 4
van der Waals
0.42 (per atom)
a
Bond strengths are given in water, which is similar to the environment in the aqueous cytoplasm. Anhydrous bond strengths differ from those listed.
molecules, creating a partial charge separation within the molecule. Water molecules experience a strong electrostatic attraction for one another as a result of the charge separation present in individual molecules. This electrostatic attraction, known as a hydrogen bond, occurs between a hydrogen bound to an oxygen or nitrogen and a second oxygen or nitrogen, either in the same or a different molecule. Hydrogen bonds are short-lived and constantly break and re-form in liquid water. Figure A1.4B shows the polar covalent and hydrogen bonds present in water. The hydrogen bonds in water contribute to its unique properties that enable it to support life. Water is a liquid over a large temperature range because hydrogen bonds cause water molecules to associate, favoring the liquid state over the gas. The hydrogen bonds that keep water a liquid over a wide range of temperatures also endow water with a high specific heat, the amount of energy needed to raise the temperature of 1 gram (g) of a substance by 1ºC. The high specific heat of water moderates the temperature of all aqueous environments, oceans and cells alike. The polar nature of water is also responsible for its properties as a solvent. Compounds that are ionic or polar themselves tend to dissolve in water and are termed hydrophilic. For example, the ionic bonds in NaCl are very strong in the absence of water, but are easily dissolved in the presence of water. This is because the polar water can surround and interact with the sodium and chloride ions and shield them from each other (Fig. A1.5A). Water also dissolves polar compounds. In this case, water does not break the polar covalent bonds; rather, individual, intact polar molecules are surrounded by water owing to electrostatic interactions. In contrast, compounds that are mostly nonpolar do not dissolve in water and are termed hydrophobic. Nonpolar molecules have no partial charges to attract water. Because water hydrogen-bonds with other water molecules, it tends to exclude nonpolar compounds, forcing them together (Fig. A1.5B). The aggregated nonpolar compounds can be further stabilized by van der Waals forces, weak temporary
SFMB_app01.indd A-5
A. δ
δ
δ– δ–
δ+
δ+
Na+
δ+
δ+
δ– –
δ+
δ+
–
Cl–
δ+
δ+ δ+
δ+ δ
+
δ
+
B.
Interactions between water and solutes. A. Water surrounds and interacts with individual ions or polar molecules, causing them to dissolve in water. B. Nonpolar molecules do not dissolve in water. Instead, to minimize the disruption of hydrogen bonding among water molecules, nonpolar molecules aggregate in water.
Figure A1.5
electrostatic attractions between molecules caused by random movements of their electron clouds. Table A1.3 lists the bonds we have discussed and indicates the strength of the bonds in water.
A1.2 Common Features of Organic Molecules Now that we have discussed water, let’s take a look at some of the organic molecules found in cells. Organic molecules are those that contain a carbon-carbon bond.
1/17/08 12:01:45 PM
A-6
Appendix 1
■
Bio lo g ic a l M o lec ules
Condensation H
Monomer
OH + H
Monomer
OH Hydrolysis
H2O + H
Monomer
Monomer
OH
Figure A1.6 Condensation and hydrolysis reactions. Monomers can be covalently bonded to form polymers through a condensation reaction that liberates a water molecule. Polymers can be broken apart into monomers through hydrolysis (water-splitting) reactions.
The major macromolecular components of cells are proteins, polysaccharides, nucleic acids, and lipids. Although the macromolecules differ from each other in their structures and cellular functions, they all share some common features: ■
■
■
As polymers, the macromolecules found in cells are composed of smaller units called monomers. The monomers are joined to one another by a common reaction, called a condensation, that involves splitting out a molecule of water for each monomer unit added. Conversely, the polymers can be broken apart into monomers by hydrolysis, the addition of a molecule of water, as depicted in Figure A1.6. The three-dimensional shape, or structure, of a molecule is critical for proper function. The structure is determined by which atoms are present and how they are bonded together. Structural formulas like those in Figure A1.2C represent the sequence of bonds in a molecule; however, this does not adequately convey information about the three-dimensional shape of the molecule. It is particularly important to understand the shape of carbon-containing molecules because carbon is the backbone for most cellular macromolecules. The bonding orbitals in the second shell of carbon are arranged so that they point to the vertices of a tetrahedron; methane, the simplest hydrocarbon, actually has the shape of tetrahedron, with the hydrogen atoms at the vertices (Fig. A1.7A). Figure A1.7B and C show two different models used to depict the three-dimensional shape of molecules. The space-filling model shows the volume filled by the outer shell of the atoms. The stick model shows the length and orientation of interatomic bonds between the nuclei of each pair of atoms. There are inherent difficulties in presenting three-dimensional representations, such as the models just described, on a two-dimensional page. Images on a computer screen in conjunction with special glasses can give a sense of the actual three-dimensional structures of molecules. Viewers can use the mouse to rotate the molecule to see it from any angle. Often the viewer can choose whether to view the molecule as a space-fi lling or stick representation. An important feature of any organic molecule is the number and type of functional groups present. Functional groups are small groups of atoms with characteristic bonding, shape, and reactivity. A few of the more common ones are shown in Table A1.4. Know-
SFMB_app01.indd A-6
A.
B.
C.
Molecular models of methane. A. A molecule of methane showing the tetrahedral arrangement of the electrons in the second (valence) shell of carbon. B. A space-filling model of methane. C. A stereo-image view of a stick model of methane. To view the stereo-image, place a piece of cardboard vertically between the two images. Position your eyes on opposite sides of the cardboard and force them to focus behind the images. The images will merge and produce a 3-D image.
Figure A1.7
ing which functional groups are present in a molecule allows us to infer something about the structure and reactivity of the molecule. Information about the functional groups present in a molecule is often indicated by a molecule’s name; for example, amino acids contain an amino group and a carboxyl (carboxylic acid) group. We will discuss different classes of molecules individually, but in living cells, molecules of different types are frequently found in combination. For example, sugars decorate some proteins (glycoproteins) and lipids (glycolipids), and the ribosome is a complex of proteins and ribonucleic acids. Let us now examine the structure of the fundamental biological macromolecules in detail.
A1.3 Proteins Cells express thousands of different proteins and may contain more than 2 million protein molecules. Proteins perform many functions, such as catalyzing reactions,
1/17/08 12:01:46 PM
■
Appe n dix 1
A-7
B iolog ic al Mo l e cu l e s
Table A1.4 Common functional groups. Functional group
General structure
Example
O
Aldehyde
R
C
H
H
H
O
C
C
Comments Can react with alcohols H R
H
Acetylaldehyde H
Alkane
R
C
H
H
H
H
H
C
C
H
H
OH
O C
+ HO
R′
R
H
C
OR′
H
Nonpolar, tends to make molecules containing it hydrophobic; nonreactive
H
Ethane H
H
Amino
R
H
N
H
C
H
Acts as a base by accepting a proton; found in amino acids
N H
H
R NH2 + H+
Methylamine O
Carboxyl
R
H
C
O H
H
C
O
OH
H
CH3 C
Acetic acid
Ester
R
C
R1 O
O R
P
O–
Acetate
O
O
NH3+
Acts as an acid by donating a proton; the ionized form name ends in ate (i.e., acetate)
O
C
R
O
R2
+ H+
Proton
Common linkage found in lipids
O–
Phosphodiester Hydroxyl
R
O
H
H
H
H
C
C
H
H
Polar, makes compounds more soluble through hydrogen bonding; found in alcohols and sugars
OH
Ethanol CH2OH
O
Ketone
R
C
Found in many intermediates of metabolism
C O
R
H C
OH
H
Dihydroxyacetone O
H C
O
Phosphate
R
O
O–
P O
–
H
C
OH O
H
C
O
H
O–
P –
O
Glyceraldehyde-3-phosphate
serving as receptors and transporters, providing structure, and aiding movement. Proteins can carry out such diverse functions because they can fold into a variety of three-dimensional structures.
The Building Blocks of Proteins Are Amino Acids Although different proteins can have vastly different structures, all proteins are composed of the same
SFMB_app01.indd A-7
When more than one phosphate is linked together, a high-energy bond forms due to repulsion of the negative oxygens
building blocks: amino acids. All 20 of the amino acids commonly occurring in nature have the same general structure. Each amino acid contains a central carbon atom (the alpha carbon) covalently bound to four different moieties—a hydrogen atom, an amino group, a carboxyl group, and a side chain (R, residue) that is unique for each amino acid (Fig. A1.8A). The alpha carbon is an example of a chiral carbon, a carbon with four different groups attached to it. Chiral carbons exist in two different forms called optical isomers, or enantiomers.
1/17/08 12:01:47 PM
A-8
■
Appendix 1
Bio lo g ic a l M o lec ules
A. Ionized form
B.
H H3N+
C O–
R Alpha carbon
Asymmetrical carbon atoms
O
C
Fit to template is impossible for isomer.
Molecule fits template.
Side Carboxyl chain group
Molecule
Mirror image
Figure A1.8 Amino acid structure and chiral carbons. A. All amino acids contain a central carbon (the alpha-carbon) bonded to an amino group, a carboxyl group, a hydrogen, and a variable group or side chain, designated R. At cellular pH, the amino and carboxyl groups are ionized. Because the alpha-carbon is bound to four different molecules, it is a chiral carbon. B. Chiral molecules exist in two different forms that are mirror images of each other. The two forms cannot be superimposed, and only the correct isomer can interact with a template (for example, an enzyme).
Optical isomers have the same molecular formula and the same order of bonds within the molecules, but a different arrangement of their atoms in space. Optical isomers are actually mirror images of each other, like your left and right hands, and the two forms cannot be super-
imposed (Fig. A1.8B). Each of the amino acid isomers is designated L or D, based on the configuration at the alpha carbon. The amino acids used to make proteins all have the L configuration at the alpha carbon, and all of these amino acids can be derived by substituting the different
B. Polar amino acids
A. Charged amino acids Basic amino acids H3N+ C
Acidic amino acids H
H
H COO–
H3N+ C
COO–
H3 N + C
COO–
CH2
CH2
CH2
CH2
CH2
C
NH
CH2
CH2 C H
N+ H
CH2 N H3
C
H
H
C
COO– H3N+ C
COO–
Arginine (Arg or R)
H COO– H3N+ C
COO–
CH2
CH2
CH2
CH2
C
CH2
OH
CH3
Serine (Ser or S)
Threonine (Thr or T)
H2N
O
H C
OH
C H2N
Glutamate (Glu or E)
Asparagine (Asn or N)
NH2
Lysine (Lys or K)
H COO– H3N+ C
CH2 COO–
N H2
H COO– H3N+ C
COO–
Aspartate (Asp or D)
+
H H3N+ C
CH2 CH
NH
+
H3 N +
O
Glutamine (Gln or Q)
Histidine (His or H)
C. Hydrophobic amino acids H H3N+ C
H COO–
CH3
H3N+ C
H COO–
H3N+ C
CH H3C CH3
H
C
H COO– CH3
CH2 CH3
H3 C
H
H
H3 N + C
COO–
H3 N + C
COO–
CH2
CH2
CH
CH2
CH3
H3 N + C
H COO–
CH2
Valine (Val or V)
Isoleucine (Ile or I)
Leucine (Leu or L)
H COO–
CH2
Methionine (Met or M)
H3 N + C
COO–
CH2 C
S
CH NH
OH
CH3
Alanine (Ala or A)
H3 N + C
Phenylalanine (Phe or F)
Tyrosine (Tyr or Y)
Tryptophan (Trp or W)
D. Special amino acids H H3N+ C
H
H COO–
CH2
H3N+ C
COO–
H
SH
Cysteine (Cys or C)
Figure A1.9
SFMB_app01.indd A-8
Glycine (Gly or G)
C
COO–
H2N+
CH2
H2C
CH2
Proline (Pro or P)
Twenty common amino acids.
The grouping of amino acids is based on their side chains, highlighted in yellow.
1/17/08 12:01:48 PM
■
Appe n dix 1
R groups for one another. Some unusual D-amino acids, with the opposite configuration at the alpha carbon, are found in bacterial cell walls. The side chains of amino acids can be grouped according to their hydrophobicity, charge, or presence of specific functional groups. Figure A1.9 depicts the 20 common amino acids, along with their three-letter abbreviations and single-letter codes. Several amino acids have unusual features: Glycine does not contain a chiral carbon and therefore does not have optical isomers; proline has a ring structure that interrupts the regular geometry of the polypeptide chain; and the side chain of cysteine is capable of forming inter- and intramolecular disulfide (–S–S–) bonds.
A.
There Are Four Levels of Protein Organization
B.
The first level of organization, the primary structure, is simply the linear sequence of amino acids. This is genetically determined; the DNA code specifies the amino acid sequence. During protein synthesis, amino acids are connected together by a condensation reaction to form a covalent peptide bond. The peptide bond forms between the carboxyl group of one amino acid and the amino group of a second amino acid, as shown in Figure A1.10A. The portion of the amino acid incorporated into the peptide after condensation is called an amino acid residue. Note that the peptide chain has an amino terminus (N-terminus) and a carboxyl terminus (C-terminus); numbering of the amino acid residues starts at the amino terminus (Fig. A1.10B). The bonds on either side of the alpha carbon can rotate, but there is no rotation around the peptide bond, so the backbone of a protein does not rotate freely. The secondary structures of proteins are regular patterns that repeat over short regions of the polypeptide chain. Figure A1.11A depicts two common secondary structures, the alpha helix and the beta sheet. The alpha helices are flexible, coiled structures formed by a series of hydrogen bonds between an oxygen in a carboxyl group and a hydrogen in an amino group five amino acids down the chain. The beta-sheets are more rigid than the alpha helices. Beta sheets form as a result of extensive hydrogen bonding between regions of the protein lying next to each other. Both alpha helix and beta sheet secondary structures are stabilized by hydrogen bonding between the oxygen and hydrogen atoms of the peptide bonds in the main chain; the side chains do not directly participate. Proline, however, acts as a helix breaker because its ring structure does not allow the proper rotation around the alpha carbon that is necessary to form an alpha helix. The tertiary structure of a protein is its unique three-dimensional shape. At the tertiary level of organization, regions distant in the primary structure may be brought close together. The nature and order of the side chains (the primary structure) determines how a protein folds into its fi nal tertiary structure. The large number
SFMB_app01.indd A-9
H
H
+
C
H N H
H
H
O
N C
C H
OH
R1
A-9
B iolog ic al Mo l e cu l e s
O C O–
R2 H2O
Peptide linkage H
H
O
H
C
C
N C
O
+
H N H
R1
H
C O–
R2
N-terminus
C-terminus
Amino terminus +
H3N
H
O
C
C
CH2
Carboxyl terminus H N
C
C
H
CH2 O
H
H
O
N
C
C
CH2 COO
OH
H C
N
C
H
CH2 O
–
H
H
O
N
C
C
O–
CH3
CH2 S CH3
1
2
3 Residue number
4
5
Figure A1.10 The peptide bond and primary protein structure. A. Formation of a peptide bond. Amino acids are joined by a condensation reaction between the carboxyl group of one amino acid and the amino group of another to form a covalent linkage called a peptide bond. B. The primary structure of a pentapeptide, a chain of five amino acids. This pentapeptide has the sequence SFDMA. A protein can be formed of hundreds or thousands of amino acids.
of amino acid side chains is responsible for the diversity of protein structures and hence functions. As proteins emerge from the ribosome, they are bound by chaperone proteins that help them fold into their functional shape, known as the native conformation. In soluble proteins, amino acids with hydrophobic side chains tend to cluster inside the protein to minimize reactions with water, while polar amino acids are exposed on the surface (Fig. A1.11B). Tertiary structure is stabilized by hydrogen bonding between side chains and between side chains and the main chain. Ionic interactions between acidic and basic side chains also contribute to tertiary structure. The one covalent interaction found stabilizing tertiary structure is disulfide bonding between two cysteine residues. The interactions that contribute to protein tertiary structure are shown in Figure A1.11C, and a ribbon representation of the three-dimensional structure of a protein is illustrated in Figure A1.11D. Some proteins form stable, functional complexes with other proteins. These multisubunit proteins exhibit the fourth level of protein organization, quaternary structure (Fig. A1.11E). The forces that hold interacting polypeptide
1/17/08 12:01:48 PM
A-10
■
Appendix 1
Bio lo g ic a l M olec ules
B.
A. R
H
H
R O
H
N
O C N H H R C C O
N H
R
O N
O C
H
H
C
H
C
C
N
C
C
O HH
N
H C
N
C
C H
O C
H
H
H
H
H N
N C R O H N C C H
R C N R C O H H H C C N C R R C O H O N H C C H R C N R C O H
α-helix
R
Unfolded polypeptide
H N
R
C O
R
H N O
H
C.
C C
Hydrogen bond between peptide groups C O
R
N
C H
R H C
CH2
β-pleated sheet
OH
CH3
O
H3C
CH2CH
Hydrogen bond between side chain and peptide group
C O R
H
CH3
H N
C O H N
C N H
R
H
Folded conformation in aqueous environment
R
C O
C
Polar side chains
C C
H
O C N H C H
H
N H O
O C N H C H
H
C
R
Hydrophobic core region contains nonpolar side chains.
O
C R
C
Nonpolar side chains
O C R C
H N C
H
N
R
Hydrogen bonds can form to polar side chains on the outside of the molecule.
H
C
R
Hydrogen bond between two side chains
–
O
CCCH2
Ionic bond
H3C
Hydrophobic interaction O C
(CH2)4
CHCH2
+ NH3
Disulfide bond CH2
S
S
CH2
H CH2
D.
N CH2C
OH H
E.
α-helix β-sheet
Turns No specific secondary structure
chains together are the same noncovalent interactions and disulfide bonds responsible for protein tertiary structure. It is important to note that proteins are not static structures. The individually weak hydrogen bonds, ionic interactions, and van der Waals forces that contribute to protein structure are constantly being broken and reformed. Because these noncovalent bonds do not break all at once, the large number of such bonds present act collectively to maintain protein integrity. Some proteins are dynamic entities that may permute through a number of semistable states. Such conformational changes may
SFMB_app01.indd A-10
Figure A1.11 Secondary, tertiary, and quaternary structures of proteins. A. Secondary structures of proteins: alpha helix and beta sheet. B. Tertiary structure of a protein. C. Types of bonding that maintain protein tertiary structure. D. A ribbon representation of the tertiary structure of protein G of Streptococcus species. (PDB code: 2nmq) E. Quaternary structure of the multisubunit enzyme phosphofructokinase (a key enzyme in glycolysis). Each subunit is shown in a different color. (PDB code: 3pfk)
be stochastic, random fluctuations due to the intrinsic thermal (kinetic) energy of the protein. Alternatively, the conformational changes may be influenced by external factors. For many proteins, alterations in conformation are critical for protein function.
A1.4 Polysaccharides Polysaccharides are complex carbohydrates, composed of monosaccharide (simple-sugar) monomers. Carbohy-
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Appe n dix 1
A.
H
O 1
C
H 2C
H
O 1
OH
HO
3 CH2OH
C 2
C H
O
CH2OH
3 CH2OH
D-Glyceraldehyde
L–Glyceraldehyde
(An aldose)
(An aldose)
B.
H
O H 2C HO designation is determined by orientation at this carbon. D
3
C
Dihydroxyacetone (A ketose) H
O C 1
C 1
1
OH
HO 2 C
H
H
HO
H
3
C
HO
CH2OH
C 2
O
C
H
3
H 4C
OH
H 4C
OH
H 4C
OH
H 5C
OH
H 5C
OH
H 5C
OH
6 CH2OH
D–Glucose
6 CH2OH
6 CH2OH
D–Mannose
D–Fructose
Figure A1.12 Structural formulas for some monosaccharides. A. The three-carbon sugars dihydroxyacetone and glyceraldehyde. The aldehyde and ketone functional groups are shown in yellow. Glyceraldehyde contains a chiral carbon and has two optical isomers. B. The hexose sugars glucose, mannose, and fructose. The numbering of the carbons starts at the carbon of the aldehyde group or at the free carbon closest to the ketone group. The d designation is determined from the orientation of the hydroxyl group on a chiral carbon farthest from the ketone or aldehyde functional group (highlighted).
drates are so named because their molecular formulas are multiples of CH2O (hydrated carbon). Carbohydrates are a preferred energy source for many cells and also serve to store energy. They also play a structural role, as in cell walls. Moreover, when attached to proteins or lipids, they can serve an informational role.
Monosaccharides Are Aldehydes or Ketones with Multiple Hydroxyl Groups
Two Monosaccharides Condense to Form a Disaccharide
Monosaccharides differ from each other in the number of carbons present, in whether they have an aldehyde or a ketone group, and in the orientation of the hydroxyl H
1C
Disaccharides form when two monosaccharides undergo a condensation reaction to form a covalent glycosidic bond. The glycosidic bond often occurs between the C-1 carbon of one monosaccharide and the C-4 carbon of another
O
H
2C
OH
HO
3C
H
H
4
C
OH
H
C 5
OH
H
6C
OH
H Straight-chain form
6 4
HO
4
5 3
2
6
6
CH2OH OH
HO
1
H
OH O
Intermediate form
A-11
groups. The smallest sugars, glyceraldehyde and dihydroxyacetone, have three carbons (Fig. A1.12A). These two sugars have the same molecular formula, C3H6O3 , but since their atoms are arranged differently, they are structural isomers of each other. In addition, because glyceraldehyde contains a chiral carbon at C-2, it also has an optical isomer. In contrast to proteins, where the L-amino acids predominate, it is the D-sugars that are of biological importance. The six-carbon sugars (hexoses) are particularly important in the cell. Hexose sugars include glucose, mannose, and fructose (Fig. A1.12B). Hexoses all have the molecular formula C6H12O6 and are structural isomers of each other. While fructose is a keto sugar (ketose), mannose and glucose are both aldehydes (aldoses). The difference between mannose and glucose lies in the orientation of the hydroxyl group on one of the chiral carbons, C-2. The carbons in sugars are numbered starting with the carbon of the aldehyde group or the free carbon closest to the ketone group so the aldehyde group is always C-1. In sugars of five or more carbons, one of the hydroxyl groups can react with the aldehyde or ketone group, forming a ring structure. The ring form predominates because it is more energetically favorable than the straight-chain sugar. In the formation of the cyclic form of glucose, two products can be formed (Fig. A1.13). Depending on how the bond between C-1 and C-2 was oriented during the formation of the ring, the cyclic form obtained will be alpha-glucose or beta-glucose. Both forms interconvert rapidly in aqueous solution, and both forms are found as components of biological structures. The hydroxyl groups of sugars can react with other functional groups to form modified sugars. Examples include fructose 1,6-bisphosphate, an intermediate in glycolysis, and N-acetylglucosamine, a component of the peptidoglycan cell wall (Fig. A1.14).
CH2OH
C
■ B iolog ic al M ol e cu l e s
HO
CH2OH O HO 5 3
2
4 1
OH OH α-Glucose
H
+
HO
CH2OH O HO 5 3
2
1
OH
OH H β-Glucose
Figure A1.13 Straight-chain and cyclic forms of glucose. The straight-chain form of glucose can cyclize to form two different ring structures, alpha-glucose and beta-glucose. In this process, a new chiral carbon is formed at C-1, the anomeric carbon.
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A-12
Appendix 1
■
Bio lo g ic a l M olec ules
A. Sugar phosphate O –
B.
Phosphate group
CH2OH
O
O P
O
O–
CH2 O CH2 O P O– H HO O– H OH OH H Fructose
Fructose 1,6-bisphosphate
O OH
HO HO N
H3C
H
O N-acetylglucosamine
Figure A1.14 Modified sugars. A. Phosphorylated sugars, such as fructose 1,6-bisphosphate, are intermediates in glycolysis. B. N-acetylglucosamine is a component of bacterial cell walls.
monosaccharide (Fig. A1.15). The structure of the disaccharide varies, depending on which monosaccharides are involved and whether the C-1 carbon participating in the bond is in the alpha or beta form. Oligosaccharides consist of a few monosaccharides linked together, while polysaccharides are much longer chains of monosaccharides. Polysaccharides are unbranched if all the linkages are 1,4. In branched polysaccharides, additional sugars are attached to other hydroxyl groups (for example, at C-6).
A1.5 Nucleic Acids Cells contain two kinds of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the hereditary material of the cell; it encodes the information to make proteins. Three different types of RNA play key roles in protein synthesis: Messenger RNA (mRNA) is transcribed from DNA and is the template for protein synthesis; transfer RNAs (tRNAs) bind amino acids and deliver them to the ribosome; and ribosomal RNA (rRNA) is a catalytic component of the ribosome.
A Chain of Nucleotides Forms the Primary Structure of Nucleic Acids
RNA, 2-deoxyribose in DNA), a nitrogenous base, and a phosphate group (Fig. A1.16A). The nitrogenous base is attached to the C-1 of the sugar. Nitrogenous bases come in two structural classes: purines (adenine, A; and guanine, G), and pyrimidines (cytosine, C; thymine, T, found only in DNA; and uracil, U, found only in RNA). The phosphate group is esterified to the 5′-hydroxyl group of the sugar. (The prime indicates that the numbering refers to the sugar portion of the nucleotide; the base is numbered without primes.) A nucleoside is similar to a nucleotide, except that it has only two components—a sugar and a base (that is, it lacks a phosphate group). The names of the nucleosides are listed in Table A1.5. Nucleotides are formed when a phosphate group forms a covalent ester bond to the C-5 carbon of the sugar portion of a nucleoside. The nomenclature for nucleotides uses the abbreviation for the nitrogenous base and adds the abbreviation MP, DP, or TP to designate the phosphate group as a monophosphate, diphosphate, or triphosphate, respectively (Fig. A1.16B). A small “d” in front of the nucleotide indicates that the sugar is 2-deoxyribose. For example, adenosine diphosphate is ADP, and deoxycytosine triphosphate is dCTP. In addition to being the precursors for nucleic acid synthesis, the ribose nucleotides have cellular functions of their own. For example, ATP is an energy carrier in the cell, and GTP and cyclic adenosine monophosphate (cAMP) are signaling molecules.
Table A1.5 Bases and nucleosides. Base (abbreviation)a
Nucleoside (base plus ribose or deoxyribose)
Adenine (A) Guanine (G) Cytosine (C) Thymine (T) Uracil (U)
Adenosine Guanosine Cytidine Thymidine Uridine
a
Nucleic acids are polymers of nucleotides. Nucleotides have three components—a pentose sugar (ribose in
HO
CH2OH O HO OH OH α-D-Glucose
+
HO
CH2OH O HO
A,T,G, C, and U are abbreviations for the nucleotide bases; however, they are also used when referring to the nucleotides in a DNA or RNA strand to indicate which base is present in the primary structure.
HO
OH OH
β-D-Glucose
H2 O
CH2OH O HO
α-1,4-Glycosidic linkage α OH O
CH2OH O HO
α-D-Glucose
OH OH
β-D-Glucose Maltose
Figure A1.15 Formation of the disaccharide maltose. Maltose forms by a condensation reaction between two molecules of d-glucose, so that the C-1 of one molecule of glucose is linked by an oxygen atom to the C-4 of a second molecule of glucose. The hydroxyl groups of the second glucose can be either alpha or beta (the beta form is shown). The covalent glycosidic bond is called an α-1,4-glycosidic linkage because the oxygen on the C-1 carbon of the glycosidic bond is in the α position.
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■ B iolog ic al M ol e cu l e s
Appe n dix 1
A. Nucleotide
–
O P
5
O
4
O–
3
Phosphate group
H
HOCH2
OH
O
1
H
H C
3
H C
4C
C H
H C
OH OH
Ribose
C.
H C
–
CH 2
O
P
C H
O
N
N
N
H
–
N
O
H
N
H
H
H
Deoxyribose
Cytosine (both DNA and RNA)
Thymine (DNA only)
Uracil (RNA only)
O
O
P
CH 2
Deoxyribose
F.
H O
O
P
CH 2
H
–
O
H O
O
P
CH 2
O
O
–
O
3′ end
CH2
OH
CH2
H
O –
O
H
N
Oxygen
N
O
O
–
O
O
P O
N
CH2 4′
H O
N
3′ 2′
O
1′
H
H2C
H
H
5′
Phosphorus
N
O
N
N
N
H
H
N H
N
O–
O
H H
N
H N
N
O
O P H
O
0.34 nm
H
H H
O
H
O–
O P
H H
C
N H CH2 O
O H
H
P O O
O H C 2
N
N
O
A
N O
H
C C
N
H
O
H H
N
N
O–
ction
Hydrogen
ction
3′
3.4 nm
Major groove
ire -3′ d 5′-to
Carbon in sugar-phosphate “backbone”
H
N
H
N H O
O
O P
O
P O
H2C
H
ire -3′ d 5′-to
O
O
Minor groove
A T C G TA
N
H
O
Bases
5′
N
O
O
H
–
T
H O
H 2′ O 3′
N
N
H N
H
E.
2 nm
H OH OH Ribose
H
H
N
P O N
Phosphodiester bond O
H C H
Adenine (A) O A
–
3′ linkage 5′ linkage
D.
H
O
O
G C A T C G
H C
3′ end
Thymine (T) 5′ end
T
–
O
T
H
ATP (Adenosine triphosphate)
O
O
A G T
O
C H
AMP (Adenosine monophosphate) ADP (Adenosine diphosphate)
Base
C
5′ end
5′
O Alpha
Adenosine
H
O
G C
CH2
O –
H
N
OH H
H
O
3′
–
H
N O
H
O P
O O Gamma Beta
O CH3
N
O P –
O H
H
O P
H
N N Adenine
H
O
–
C G A C
Phosphate groups O O O
H
H 2N
N
N N
Adenine Guanine Pyrimidines
N
2
G
O
N
N
NH2
1
H 3
2
H
NH2
O
H
OH
O
H
N
N
5
4C
O
NH2
Nitrogenous base N O The highlighted 1 nitrogens on 2 the bases link 5-carbon to the sugar. sugar
5
HOCH2
B. ATP
Purines
O
A-13
N O
O H C 2
Cytosine (C)
H
O O P
O–
Guanine (G) 5′ end
3′ end
Figure A1.16 Nucleotide and DNA structure. A. A nucleotide consists of a five-carbon sugar (ribose or deoxyribose), a phosphate group, and a nitrogenous base. B. A nucleotide may be a monophosphate, diphosphate, or triphosphate. The phosphate attached to the sugar is designated alpha, the beta phosphate is in the middle, and the gamma phosphate is distal to sugar. C. The primary structure of DNA. The orientation of the strand is determined by the presence of a free phosphate on the 5′ end and a free hydroxyl on the 3′ end. The sequence of a nucleic acid is read from the 5′ end to the 3′ end, so for this portion of the DNA strand, the primary structure is GCTA. D, E, F. The secondary structure of DNA. D. A ribbon structure of the DNA double helix showing the purinepyrimidine base pairs. E. A space-filling model of DNA. Note that the base pairs stack on top of each other like the rungs of a ladder. F. Hydrogen bonding between the base pairs. Two hydrogen bonds form between A and T, three hydrogen bonds between G and C. Note the antiparallel orientation of the two strands.
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A-14
Appendix 1
■
Bio lo g ic a l M olec ules
Amino acid attached Ser to 3′ end C AC 3′
5′
tRNA Hydrogen bond Anticodon mRNA U GU C A
A
The nucleotide triphosphate monomers (collectively designated NTPs or dNTPs) form nucleic acids through reactions catalyzed by nucleic acid polymerase enzymes. The phosphate at the C-5 position forms a diester linkage with the hydroxyl group at C-3, releasing pyrophosphate. At one end of the nucleic acid is a free phosphate group; at the other end is a free C-3 hydroxyl group. The terminus with a free phosphate is called the 5′ end, and the terminus with the free C-3 hydroxyl is called the 3′ end (Fig. A1.16C). Repeating sugar-phosphate linkages form the “backbone” of the molecule, while the information content of nucleic acids resides in the sequence of bases attached to the backbone. This linear sequence of nucleotides, from the 5′ end to the 3′ end, is the primary structure of the nucleic acid.
5′
The Secondary Structure of DNA Is the Famous Double Helix Two strands of DNA, held together by hydrogen bonding between a purine and a pyrimidine on opposite strands, twist around each other to form a helix (Figs. A1.16 D and E). Two hydrogen bonds form between adenine and thymine, and three hydrogen bonds form between cytosine and guanine (Fig. A1.16F). The sugar-phosphate backbone of DNA is on the outside of the helix. The base pairs are inside the helix, but portions of them are accessible to proteins such as transcription factors at two grooves in the helix—a wide major groove and a narrower minor groove. The double-stranded structure of DNA is critical to its function as the hereditary material. Each strand of DNA encodes the information to make a new double helix based on strict base-pairing rules.
The Secondary Structure of RNA Is Diverse In contrast to the invariant double-helix structure of DNA, RNA molecules have a variety of secondary structures that allow RNA molecules to perform a number of different functions in the cell. Secondary structures are possible because RNA is single-stranded and capable of complementary base pairing. Base pairing in RNA is similar to the base pairing exhibited by DNA, except that in RNA, uracil replaces thymine. Base pairing is used in making mRNA from a DNA template and also in protein synthesis, where a tRNA charged with an amino acid matches its anticodon with the codon on the mRNA (Fig. A1.17). Because RNAs are single-stranded, they can undergo intramolecular as well as intermolecular base pairing. Base pairing within a single RNA molecule can lead to interesting three-dimensional shapes that may allow the RNA to function as a catalyst. The activity of catalytic RNAs (ribozymes) may be enhanced by the reactive hydroxyl group at the C-2 of the ribose sugar, which is lacking in DNA.
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3′
Codon
Figure A1.17 RNA secondary structure. A tRNA folds up into a secondary structure stabilized by the hydrogen bonds shown in the figure. The tRNA forms complementary base pairs with an mRNA.
A1.6 Lipids Lipids are a structurally diverse class of molecules that have diverse functions. They are major structural components of cell membranes, they store energy, and they act as cellular signals. The common structural feature of lipid molecules is that they contain a substantial number of nonpolar C–H and C–C covalent bonds. Because lipids are nonpolar they are hydrophobic and do not dissolve in water.
Lipids Can Be Hydrophobic or Amphipathic An example of a hydrophobic lipid is isoprene (Fig. A1.18), which functions as a building block for more complex cellular lipids such as squalene. Other lipid
HO H2C H2C
C C
H
CH3 CH2
Isoprene
H2C H2C
C
O
Carboxyl group
CH2 CH2
Hydrocarbon chain
CH3
Fatty acid
Figure A1.18 Lipid building blocks. Isoprene is used to make squalene and cholesterol. Fatty acids are components of triglycerides and phospholipids.
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Appe n dix 1
■ B iolog ic al M ol e cu l e s
building blocks include the fatty acids (Fig. A1.18). In contrast to isoprene, the fatty acids have both a hydrophobic portion (the hydrocarbon tail) and a hydrophilic component (the carboxylic acid). Molecules with both hydrophobic and hydrophilic portions are described as amphipathic.
A. Saturated fatty acid OH
Fatty Acids May Be Saturated or Unsaturated
H2C
H2C H2C H2C
CH2 CH2 CH2 CH2
HC CH2
CH2
HC
CH2
HC CH2
CH3
CH2 CH2 Linoleic acid
CH2 CH3
Figure A1.19 Saturated and unsaturated fatty acids. A. Palmitic acid is a saturated fatty acid; there are no double bonds between the carbon atoms. B. Linoleic acid is an example of a polyunsaturated fatty acid (it has more than one double bond). Note that the double bonds create kinks in the hydrocarbon tail.
Triglycerides are a compact energy source for cells (Fig. A1.20). Phospholipids, key components of the cell membrane, contain fatty acids attached to two of the hydroxyl groups of glycerol and a phosphate covalently attached to the third hydroxyl. The structure of phospholipids and how they function in cell membranes is fully explored in Appendix A2.1.
CH
CH2
OH
OH
OH
OH
CH2
CH2
HC
CH2
C
CH2
H2C
+ 3 fatty acid molecules O
CH2
H2C
Palmitic acid
The three hydroxyl groups on glycerol can undergo a condensation reaction with the carboxylic acid portion of fatty acids to form triesters, known as triglycerides.
Glycerol (an alcohol)
CH2
H2C
H2C
Glycerol Is an Important Building Block of Many Lipids
Formation of a triglyceride. Condensation reactions form covalent ester linkages between the carboxyl groups of three fatty acids and the three hydroxyl groups of glycerol.
O C
CH2
H2C
The cell contains many different fatty acids, which differ in the number of carbons (usually even) and their saturation (Fig. A1.19). In saturated fatty acids, the carboncarbon bonds are all single bonds, and the carbons are bonded to the maximum number of hydrogen atoms. Unsaturated fatty acids contain one or more double bonds between adjacent carbons in the hydrocarbon tail. Monounsaturated fatty acids have one double bond; polyunsaturated fatty acids have multiple double bonds. Saturated fatty acids can pack together in an arrangement where they can be stabilized by van der Waals forces between adjacent hydrocarbon chains. Thus, saturated fatty acids (including animal fats such as lard) tend to be solids at room temperature. In unsaturated fatty acids, the double bond causes a kink in the chain that prevents tight packing, so unsaturated fatty acids, such as vegetable oils, tend to remain fluid at room temperatures.
Figure A1.20
B. Unsaturated fatty acid OH
O C H2C
A-15
OH O
C CH2
OH O
C
O 3 H2O
CH2
CH
CH2
O
O
O
C CH2
O
C CH2
O
C
Ester linkage
CH2
CH2
Triglyceride
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A-16
Appendix 1
■
Bio lo g ic a l M olec ules
A1.7 Chemical Principles in Biological Chemistry To Express Very Large or Very Small Quantities, We Use Scientific Notation Scientists often express very large numbers (such as the number of microorganisms in a liter of seawater) or very small numbers (such as the diameter of a bacterium) with the help of exponents. For example, a million, or 1,000,000, is often written as 1 × 106. The positive exponent 6 indicates how many decimal places to the right of the number 1 our number is. One-millionth, or 0.000001, is written as 1 × 10 –6 to indicate six decimal places to the left of the number 1. Special prefi xes can be used to modify the magnitude of units. These are listed in Table A1.6. For example, 1 × 10 –6 is designated by the symbol µ (which stands for “micro”), so a length of 0.2 micrometer (µm) is equivalent to 2 × 10 –7 meter (m). When scientists work with a set of numbers that range over many orders of magnitude, they often use a logarithmic scale. For example, the pH scale is based on base 10 logarithms. Base 10, or common, logarithms are abbreviated log, as opposed to natural logarithms, abbreviated ln. Base 10 logarithms are defi ned as log 10x = x. For example the log of 1 × 10 –4 = –4. The log of 10 = 1, and the log of 1 = 0.
Molarity Is a Unit Commonly Used to Measure Concentration Scientists often measure concentrations. Concentration refers to how much of something is present in a given volume. A frequent way to report concentration is in units of molarity. Molarity is defi ned as the number of moles of substance per liter of solution (usually water). One mole is Avogadro’s number (6.02 × 1023 ) of molecules. If you wanted to make a 1-molar solution of NaCl, it would be impossible to know when you had added 6.02 × 1023 mol-
ecules to a liter of water. It’s more convenient to weigh out a mole of NaCl on a balance. The weight of a mole of a substance is Avogadro’s number times the weight of a molecule. For example, NaCl has a mass of 58.5 atomic mass units (amu), 23 from sodium and 35.5 from chlorine (see Fig. A1.1). Each amu is equal to 1.66 × 10 –24 grams (g), so one molecule of NaCl weighs 9.71 × 10 –23 g. A mole of NaCl then weighs 9.7 × 10 –23 g times 6.021 × 1023 (Avogadro’s number) = 58.5 g. (Note that this weight is the same as just adding together the atomic masses of Na and Cl and expressing it in grams.) So to make a 1molar solution of NaCl, you would take 58.5 g of NaCl and add water up to a liter.
The Change in Free Energy Determines Whether a Reaction Proceeds Spontaneously Reactions in organisms, such as the synthesis of proteins from amino acids by condensation, can occur only if they are energetically favorable. The study of energy and matter changes is called thermodynamics. All systems (including living systems, such as cells) must obey the laws of thermodynamics. The first law of thermodynamics states that energy is neither created nor destroyed. In a closed system (a system that is not exchanging energy with its environment), the total amount of energy remains constant. Although the amount of energy remains constant, energy can be converted from one form to another. The second law of thermodynamics states that in energy transformations, some energy becomes unavailable to do work and is lost as disorder, or entropy. In other words, entropy tends to increase. The laws of thermodynamics are summarized in Figure A1.21. The total energy in a system, called enthalpy, can be expressed in the following equation, where H stands for
Energy transformation
Table A1.6 Common numerical prefixes. Prefix
Symbol
Factor
kilo deci centi milli micro nano pico femto
k d c m µ n p f
103 10–1 10–2 10–3 10–6 10–9 10–12 10–15
SFMB_app01.indd A-16
Energy before
Usable energy after (free energy) Unusable energy after (entropy)
The first and second laws of thermodynamics. The first law states that in an energy transformation, the total amount of energy remains constant. Both sides have the same amount of energy (the balance reads zero). The second law states that in an energy transformation, the amount of usable energy, or free energy, decreases and the amount of unusable energy, or entropy, increases.
Figure A1.21
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Appe n dix 1
enthalpy, G stands for free energy, S stands for entropy, and T stands for temperature:
■ B iolog ic al Mo l e cu l e s
A-17
may result in a negative change in free energy, especially at high temperatures.
H=G+T⋅S Because scientists are interested in the amount of free energy—that is, the amount of energy available, or free, to do work—the energy equation is usually written in terms of G: ∆G = ∆H – T ⋅ ∆S The triangle is the Greek letter delta, which signifies “change in.” Here, a change in energy is defi ned as the energy of the products minus the energy of the reactants. Thus, ∆G means Gproducts – Greactants , and ∆S indicates Sproducts – Sreactants.
Systems Tend Toward the Most Stable, Lowest Free Energy State Reactions are favored when the free energy of the products is less than the free energy of the reactants, that is, when ∆G is negative. Reactions with a negative ∆G are termed spontaneous, or exergonic, because they are energetically favorable and can occur without an input of energy. Reactions with a positive ∆G are endergonic. They are not energetically favorable, and energy needs to be added for them to proceed. Reactions with a ∆G of zero are at equilibrium, and neither products nor reactants are favored. The sign of ∆G is determined by changes in both enthalpy and entropy. For example, the following reaction, the oxidation, or “burning,” of methane to release carbon dioxide and water, is spontaneous and has a negative change in free energy: CH4 + O2 D CO2 + H2O + energy The bonds in methane and oxygen have more energy than the bonds in CO2 and H2O. Because the products CO2 and H2O have less energy than the reactants, the change in enthalpy for this reaction, ∆H (Hproducts – Hreactants), is negative and the reaction is said to be exothermic. A reaction does not need to be exothermic to be spontaneous. Consider an ice cube placed at room temperature. The ice cube will spontaneously melt even though this reaction is endothermic, absorbing heat from the environment. Ice melting is spontaneous because of the increase in entropy in going from a solid to a liquid. Remember that entropy, S, is a measure of the disorder of a system. Gases are more disordered than liquids, and liquids are more disordered than solids. Thus, the change in entropy, ∆S (Sproducts – Sreactants ), from solid water (ice) to liquid water is positive because the product, liquid water, has more entropy than ice. Positive changes in entropy
SFMB_app01.indd A-17
There is a Relationship between the Standard Free Energy Change and Chemical Equilibrium To compare changes in free energy among different reactions, a standard change in free energy, ∆G° (the circle is pronounced naught), is defi ned as the change in free energy when the concentrations of all reactants and products are at 1 molar. ∆G° is a constant and depends on the nature of the products and reactants. Another constant, ∆G°′ refers to the standard change in free energy at pH 7. A standard free energy change can be related to an equilibrium constant, Keq, that indicates whether products or reactants will be favored at equilibrium. Reactions between biomolecules can be written as chemical equations. For example, in the following reaction, reactants A and B form products C and D: kf A+BDC+D kr The double arrow indicates that the reaction is reversible; A and B can be forming C and D at the same time that C and D are reverting to A and B. What determines whether the forward or reverse reaction will predominate? The forward and reverse rates depend on rate constants kf and kr, respectively. The rate constants depend on the nature of the interacting molecules and are an indication of how easily they will react. The forward and reverse rates also depend on concentrations of reactants and products: Rate forward = [A][B]kf Rate reverse = [C][D]kr If the forward and reverse reactions are allowed to proceed, eventually equilibrium will be reached. All chemical reactions have a preferred state called equilibrium, where there is no net change in the reaction. Equilibrium is not static; rather, at equilibrium, the rate of the forward reaction equals the rate of the reverse reaction: [A][B]kf = [C][D]kr For every reaction, there is an equilibrium constant, Keq, that indicates the relative ratios of products and reactants at equilibrium: Keq = kf/kr = [C][D]/[A][B] A Keq of 1 means that neither products nor reactants are favored; a Keq greater than 1 indicates that products are
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A-18
Appendix 1
■
Bio lo g ic a l M olec ules
favored at equilibrium; and a Keq less than 1 means that reactants predominate. Keq and ∆G° are related as follows where R is the gas constant (8.3 × 10 –3 kJoule/mole ⋅ degree) and T is the temperature in kelvins: ∆G°= – RT ln Keq A value of 298 K is often used (this is roughly 25°C). The relationship between Keq and ∆G° is usually shown with the base 10 logarithm instead of the natural log: ∆G° = – 2.303 RT log Keq This equation defi nes a relationship between ∆G° and Keq that is discussed in Chapter 13. When Keq is 1 (neither products nor reactants favored), ∆G° (defi ned as the change in free energy when concentrations of all substances are at 1 molar) is zero because the reaction is at equilibrium at that point. A Keq of less than 1 (reactants favored) corresponds to a positive ∆G° because at 1-molar concentrations of reactants and products, the reaction will move to the left. A Keq of more than 1 (products favored) corresponds to a negative ∆G° because at 1-molar concentrations, the reaction will move to the right.
Free Energy in Cells and the Law of Mass Action Although the standard changes in free energy are useful for comparing the energetics and equilibria of different reactions, they do not reflect what is happening in the cell where concentrations of substances are probably not 1 molar. While Keq and ∆G° are constants and unique for a particular reaction, ∆G depends on the actual concentrations of products and reactants as shown in the equation relating ∆G to ∆G°:
left to reestablish equal energy on both sides. This law of mass action is the tendency of a reaction to reestablish equilibrium after perturbations in the concentrations of products or reactants. The law of mass action means that a reaction with a positive ∆G° can be made spontaneous and driven to the right by keeping the concentration of reactants high or the concentration of products low. The cell employs this strategy in metabolic pathways, where the product of one reaction is constantly removed by a subsequent reaction.
The Rate at Which a Reaction Proceeds Depends on the Activation Energy It is important to realize that although a reaction with a large positive Keq and negative ∆G° is spontaneous, it may be slow. The value of ∆G° says nothing about the rate of a reaction. Everyday examples of spontaneous but slow reactions are the aging process in humans and the rusting of metal. A reaction will be slow, even though it is spontaneous, if it must pass through an unstable, highenergy transition state on the way to forming products. The activation energy (Ea) is the energy needed to reach this transition state (Fig. A1.22). For reactions with low activation energies, random collisions between reactants may provide enough energy to boost them up and over the Ea. For reactions with high activation energies, collisions between molecules may not provide enough energy for them to reach the transition state. Enzymes are biological catalysts that can speed up reaction rates by stabilizing transition states and lowering the activation energy. Most enzymes are proteins that specifically bind reactants and provide an environment
∆G = ∆G° + 2.303 RT log ([C][D]/[A][B])
SFMB_app01.indd A-18
Uncatalyzed reaction Ea
Free energy
In this equation, the concentrations of products and reactants are not their equilibrium concentrations, but the actual concentrations present at a given point in time. Because the concentrations of products and reactants will change as a reaction proceeds, the value of ∆G will change over time. At equilibrium, where the total energy on each side of the reaction is equivalent, ∆G is zero. For an exergonic reaction at equilibrium, the total energy of reactants and products is equivalent because the amount of products is greater than the amount of reactants. If, at equilibrium, reactants (A, B) are added or products (C, D) are removed, ∆G will become negative. A negative ∆G causes the reaction to move to the right to reestablish equilibrium. As A and B convert to C and D, ∆G becomes less and less negative until it reaches zero again at equilibrium. If, at equilibrium, products are added or reactants are removed, ∆G will become positive and the reaction will proceed to the
Activation energy (Ea) in the absence of an enzyme.
In the presence of an enzyme, Ea decreases and the reaction rate increases.
Ea Reactants ∆G°
Catalyzed reaction
∆G° is negative; therefore, the reaction is spontaneous. ∆G° is unchanged by adding an enzyme.
Products Course of reaction
Figure A1.22
Enzymes, activation energies, and reaction
rates.
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■ B iolog ic al M ol e cu l e s
Appe n dix 1
that facilitates product formation. Although enzymes increase reaction rates, they do not change the ∆G° or Keq of a reaction. Figure A1.22 illustrates how the difference in free energy between products and reactants is unchanged even in the presence of an enzyme that lowers the activation energy.
Many Biological Processes Are Sensitive to Changes in pH Many biological processes occur only within a narrow range of hydrogen ion concentrations. Hydrogen ions (H+) are also referred to as protons because they have lost their single electron and consist of only a proton. Hydrogen ion concentration is reported using a pH (power of hydrogen) scale, where pH is the negative logarithm of the hydrogen ion concentration: pH = –log [H+]. In pure water, which is considered neutral, [H+] is 1 × 10 –7 molar (M), a pH of 7. Because the pH scale is logarithmic, every pH unit corresponds to a tenfold change in hydrogen ion concentration. A solution with a pH of 6 has a hydrogen ion concentration of 1 × 10 –6 M, ten times the hydrogen ion concentration at pH 7. Hydrogen ion concentration [H+] multiplied by hydroxide ion concentration [OH– ] always equals 1 × 10 –14. Hence, the concentrations of H+ and OH– are reciprocally related (if one goes up, the other goes down). If the pH is less than 7, a solution is acidic (more H+, less OH– ). If the pH is greater than 7, the solution is basic, or alkaline (less H+, more OH– ). Acids (for example, carboxyl groups) donate protons, and bases (for example, amino groups) accept protons (Fig. A1.23). At intracellular pH (near 7), the carboxylic acid and amino group of amino acids are both ionized (charged), the carboxyl group carrying a negative charge and the amino group a positive charge. The ionization of these groups can change if the pH changes. At lower pH (more acidic solution) protons move back onto the ionized carboxylic acid. For example, the side chain of an acidic amino acid such as glutamate (see Fig. A1.9) is ionized at normal cell pH but may regain a proton and become
glutamic acid at a lower pH. At higher pH values (lower proton concentrations), amino groups lose protons to become uncharged. Disturbances in the ionization state of carboxyl or amino groups on the side chains of amino acid residues can disrupt ionic bonds between these groups and lead to protein denaturation. This effect of pH on protein tertiary structure is one reason why cells can tolerate only a narrow range of intracellular pH.
Oxidation-reduction (Redox) Reactions Are an Important Way of Transferring Energy in Biological Systems Biomolecules may undergo an important class of chemical reactions termed redox reactions. Redox reactions involve the transfer of electrons from one molecule to another or from one atom to another. The molecule that gains electrons becomes reduced, and the molecule that loses electrons becomes oxidized (Fig. A1.24). (A useful mnemonic device to remember this is “LEO the lion says GER.” LEO: lose electrons oxidation; GER: gain electrons reduction.) Redox reactions are always coupled. If one atom loses electrons, another atom must gain electrons. A. e– A
B
e– Reduced compound A (reducing agent)
Oxidized compound B (oxidizing agent)
e– A
e–
Oxidized compound A
H CONH2
+
+ H + 2e
CONH2 N
R
O
O CH3 C
OH
Acid
Base
O–
NAD + R Proton
+
H
O
H
CH3N+H3
Acid
Base
Figure A1.23
SFMB_app01.indd A-19
+
Organic acids and bases.
NADH H
+
C OH
The amino group of an organic base accepts a proton from water, leaving OH– in solution. CH3 NH2
R NAD+
C.
H+
+
OH–
H
–
N The carboxyl group of a carboxylic acid dissociates, generating H+.
B
Reduced compound B
B. H
+
CH3 C
A-19
R′
NADH + R
C
R′ + H+
O
Figure A1.24 Oxidation-reduction reactions. A. A reducing agent A donates electrons to reduce compound B. Because A loses electrons, it becomes oxidized itself in the process. B is the oxidizing agent. B. NADH is a common biological reducing agent. C. NAD+ is reduced to NADH as an alcohol is oxidized to an aldehyde.
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A-20
Appendix 1
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Bio lo g ic a l M olec ules
Carbon has been oxidized since electrons are moved farther away from the carbon atoms.
C
Figure A1.25 The oxidation of methane. Bonding electrons were shared equally by carbon and hydrogen in methane, but are closer to oxygen in carbon dioxide and water. Carbon and hydrogen have been oxidized; oxygen has been reduced.
Oxidation
H H
Oxygen has been reduced since electrons have moved closer to oxygen atoms.
H
+
O
O
H Methane
2 Oxygen
O
C
O
+
Reduction Carbon dioxide
H
O
H
Energy
2 Water
Oxygen, with its large electronegativity, usually gains electrons and becomes reduced in redox reactions. Molecules that become reduced are called oxidizing agents because they cause something else to become oxidized. In contrast, reducing agents can donate electrons, reduce other molecules, and become oxidized themselves in the process. Molecules with reduced carbons contain more energy than their oxidized counterparts. Common reducing agents in the cell are NADH, NADPH, and FADH 2. These are all high-energy molecules that can donate electrons. In addition to achieving a complete transfer of electrons, redox reactions can also occur if electrons are
SFMB_app01.indd A-20
+
shifted toward or away from an atom. For example, in the burning (oxidation) of methane, electrons have moved away from the carbon and toward the oxygen (Fig. A1.25). Oxygen usually acts as an oxidizing agent; thus, we expect it to get reduced (gain electrons). By default, then, methane is oxidized; indeed, while the bonding electrons were shared fairly equally between C and H in methane, they are now close to the O in both carbon dioxide and water and farther away from the C and H. Thus, methane has been oxidized and oxygen has been reduced. This reaction releases energy because CO2 and H 2O are the most stable lowest-energy forms available when carbon, hydrogen, and oxygen are combined.
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Appendix 2
Introductory Cell Biology: Eukaryotic Cells A2.1 A2.2 A2.3 A2.4 A2.5 A2.6
The Cell Membrane The Nucleus and Mitosis Problems Faced by Large Cells The Endomembrane System The Cytoskeleton Mitochondria and Chloroplasts
This appendix presents a review of cell biology principles generally covered in an introductory-level biology class. Cell biology encompasses fundamental principles of cell structure and function. Because all cells are enclosed by a cell membrane and need to regulate the transport of materials across the membrane, we cover this topic first. We then go on to discuss structures unique to eukaryotic cells. All eukaryotic cells contain a nucleus, an organelle that houses the DNA. We describe the structure of the nucleus and the process of mitosis−a process that ensures the accurate separation of replicated chromosomes during cell division. We then explore the challenges encountered by eukaryotic cells owing to their large size compared to most prokaryotic cells. The endomembrane system, a network of internal membranes is discussed next, followed by a discussion of the cytoskeleton, a collection of proteins that form the basis of cell architecture and are responsible for cell movement. Finally, we discuss mitochondria and chloroplasts, the energy-producing organelles of eukaryotic cells.
This eukaryotic cell (lymphocyte white blood cell) shows numerous major organelles such as a large nucleus (orange) and multiple mitochondria (blue). (TEM ×20,550) Source: ©Gopal Murti/Visuals Unlimited
A-21
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Appendix 2
■
I n tr o d u c to r y C ell B iolog y : E ukary ot ic C ells
The cell is the basic unit of life. All organisms consist of either a single cell or a collection of cells. All cells can potentially perform a common set of tasks, including replication, catalysis, and regulation. The structures that enable these functions are found in all cells and include the cell membrane, DNA, and ribosomes (Fig. A2.1). Although the detailed organization of these structures differs in cells from the three domains (Bacteria, Archaea, and Eukarya; see Table A2.1), the fundamental structure and function of the cell membrane, DNA, and ribosomes are the same in all cells. This appendix describes the cell structures that are found across all three domains of life, then focuses on those that are unique to the eukaryotic domain, specifically organelles and the cytoskeleton.
A2.1 The Cell Membrane All cells are enclosed by a cell membrane (sometimes called the plasma membrane or cytoplasmic membrane) that creates an internal environment distinct from the external environment. The aqueous fluid inside the membrane is called cytoplasm (or cytosol). Major functions of the cell membrane include the regulated transport of substances into and out of the cell and the reception of signals from the external environment. Membranes are also critical for energy production. As shown in Figure A2.2, membranes consist mainly of lipids and proteins, but also contain some carbohydrates found in hybrid structures such as glycolipids and glycoproteins (sugars joined to lipids or to proteins, respectively).
Although the membrane contains more lipid molecules than protein molecules, proteins may contribute most of the mass. The number and nature of proteins within a membrane depend on the membrane under consideration. For example, the inner membrane of the mitochondrion contains a rich array of proteins involved in energy production Membrane proteins can be classified based on how they interact with membranes (Fig. A2.2A). Transmembrane proteins (integral proteins) span the bilayer. Transmembrane proteins are amphipathic meaning they have both hydrophobic and hydrophilic portions; the hydrophilic portions face the cytoplasm and the extracellular environment, and the hydrophobic components span the membrane. The transmembrane domains are often alpha-helices containing 15–20 amino acid residues (Fig. A2.2B). The portions of the protein that are intracellular, in the membrane, and extracellular depend on the protein and can be quite different for different proteins. Peripheral membrane proteins are associated with the cell membrane but are not directly inserted into the bilayer. Some peripheral membrane proteins are retained at membranes through noncovalent interactions with transmembrane proteins, others by noncovalent interactions with the lipids of the membrane.
Lipids Are Responsible for Forming Membranes The predominant lipids in membranes are phospholipids. Phospholipids consist of a core of glycerol, to which two fatty acids and a modified phosphate group are attached
Table A2.1 Comparison of cell structures in the three domains. Feature
Bacteria
Archaea
Eukarya
Genome
Usually circular DNA Usually one chromosome May have plasmids Usually lacks introns Nucleoid region in cytoplasm
Usually circular DNA Usually one chromosome May have plasmids
Linear DNA Multiple chromosomes, in pairs Plasmids rare May have introns Contained within membrane-bound nucleus Glycerol bonded to straight-chain fatty acids via ester linkages
Location of DNA Cell membrane
Cell wall
Glycerol bonded to straight-chain fatty acids via ester linkages
Usually present, composed of peptidoglycan Internal membranes Uncommon May have mesosomes or energy-transducing lamellae Ribosomes Sensitive to chloramphenicol and streptomycin
SFMB_app02.indd A-22
Nucleoid region in cytoplasm Glycerol bonded to branched fatty acids via ether linkages Usually present, composed of pseudopeptidoglycan Uncommon
Not sensitive to chloramphenicol and streptomycin
If present composed of cellulose (algae) or chitin (fungi) Extensive membranous organelles
Not sensitive to chloramphenicol and streptomycin
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Appe n dix 2
■ I nt roduc t ory C ell B iolog y : E ukary oti c Ce l l s
A-23
A. Prokaryotic cell Flagellum
Ribosomes
Nucleoid Cytoplasm Chromosome Cell membrane Cell wall
On average, prokaryotes are about 10 times smaller than eukaryotic cells in diameter and about 1,000 times smaller than eukaryotic cells in volume. B. Generalized plant cell
Cell wall Cell membrane (Plasma membrane)
Peroxisome Chloroplast
Smooth endoplasmic reticulum Rough endoplasmic reticulum Nuclear envelope Nucleolus Chromatin Golgi complex Cytoskeletal element Mitochondrion Central vacuole Ribosomes
C. Generalized animal cell
Cell membrane (Plasma membrane)
Smooth endoplasmic reticulum
Cytoskeletal element
Rough endoplasmic reticulum Golgi complex
Nuclear envelope Nucleolus
Mitochondrion
Chromatin Ribosomes Peroxisome Centrioles
Lysosome
Figure A2.1 The prokaryotic cell and the eukaryotic cell. A. The prokaryotic cell typically contains a single compartment, and its DNA is organized in the nucleoid region. B, C. Eukaryotic cells are typically much larger than prokaryotic cells and contain organelles.
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A-24
Appendix 2
■
I n tr o d u c to r y C ell B iolog y : E ukary ot ic C ells
B.
A. Cholesterol Carbohydrate attached to protein Glycoprotein
Extracellular fluid
Glycolipid Phospholipid bilayer
Transmembrane protein spans the membrane.
Peripheral protein associates with the membrane but does not span it.
Cytoplasm
Cytoskeletal proteins
The cell membrane. A. A cutaway view of the cell membrane. B. Ribbon diagram from X-ray crystallographic data of the E. coli EmrD protein, a multi-drug transporter. Red portions are transmembrane alpha helices and yellow portions are intracellular and extracellular loops. (PDB code: 2gfp) Source: Yin, et al. 2006. Science 312:741.
A2.2
via ester linkages (Fig. A2.3A). Unlike eukaryotes and bacteria, archaea have phospholipids with ether linkages. Phospholipids are amphipathic; the fatty acid hydrocarbon tails are hydrophobic, and the phosphate head group is hydrophilic. Amphipathic lipids are most stable in water when the hydrophilic portions interact with water and the hydrophobic portions cluster together away from water. One way phospholipids can achieve stability is by forming a bilayer. Indeed, the cell membrane is a phospholipid bilayer, two layers of phospholipids whose hydrocarbon fatty acid tails face the interior of the bilayer and whose charged phospholipid head groups face the aqueous cytoplasm and extracellular environment (Fig. A2.3B). The phospholipid layer in contact with the cytoplasm is called the inner leaflet, and the layer in contact with the environment is called the outer leaflet. Cells contain a number of different phospholipids that differ in how the phosphate head group is modified (Fig. A2.3C). Phospholipids can also differ in the length and saturation of the fatty acids chains (see Section A1.6 for fatty acid structures), and, as we shall see, these structural variations have functional consequences. In addition to phospholipids, eukaryotic membranes contain a variable amount of the steroid cholesterol (Fig. A2.3D). Like phospholipids, cholesterol is amphipathic, with a hydrophilic hydroxyl group and hydrophobic hydrocarbon rings and tail. In membranes, cholesterol is oriented so that the hydrophilic hydroxyl group interacts with the phosphate head groups of phospholipids, while the hydrophobic rings and tail of cholesterol interact with the phospholipid hydrocarbon tails. The amount of cho-
SFMB_app02.indd A-24
lesterol present in membranes varies among cells and also among organelles within a cell. Cholesterol contributes to membrane integrity by providing mechanical stability to the phospholipid bilayer. Bacterial membranes do not contain cholesterol but do contain hopanoids that serve similar roles.
Movement of Membrane Lipids and Proteins Membranes are not static structures; rather, many membrane components (a mosaic of various lipids and proteins) can undergo rapid movement. The fluid mosaic model of membranes states that membrane components are free to diffuse in the plane of the membrane. Recent evidence indicates that certain membrane proteins may be restricted to specific regions of the membrane by interactions with cytoskeletal proteins and that these membrane regions may be enriched with specific lipids (for discussion of cytoskeletal proteins, see Section A2.5). Although many phospholipids and membrane proteins can move laterally within a leaflet, they do not “flip-flop” from one leaflet of the bilayer to the other (Fig. A2.3E). Flip-flop of phospholipids is rare owing to the highly unfavorable interactions of charged head groups moving through the hydrophobic interior of the membrane. Thus, the inner and outer leaflets of the membrane may be made up of different phospholipids. It is thought that such phospholipid asymmetry may be important for the correct functioning of membrane proteins—that is, that proteins may work best when surrounded by particular phospholipids.
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■ I nt roduc t ory C ell B iolog y : E ukary oti c Ce l l s
Appe n dix 2
A. Phospholipid
B. Phospholipid bilayer
O– O
–
O P
HO P
O
H 2C O
H
CH2
CH
H C
O
Aqueous extracellular environment
O– Polar, hydrophilic “head”
OH Phosphate
O
H
A-25
H
C C
Outer leaflet
H
OH OH OH Glycerol
C OC O CH2 CH2
OH O
OH
Nonpolar, hydrophobic fatty acid “tails”
O
Inner leaflet
Polar, hydrophilic “head”
Fatty acids
C. Selected phospholipids
D. Cholesterol structures
+
NH3
NH3 H
CH2
O
O
O
O–
O
O CH2
CH
O
O
C O C
Fatty acid
Fatty acid
O
O
CH2
O
Phosphatidylethanolamine Phosphatidylserine E. Phospholipid motions
O–
O CH2
CH
O
O
C O C –
P
CH2
O
OH
Polar head group CH3 CH3 CH3 CH CH2
Nonpolar hydrocarbon tail
CH2 CH2 CH CH3 CH3
Phosphatidylcholine F. Membrane fluidity Fluid
Lateral diffusion
Rigid planar steroid ring structure
Fatty acid
O
C O C
CH2
O–
P
Fatty acid
P
Fatty acid
O
CH2
CH2
Fatty acid
CH
COO–
C
CH2
O CH2
CH3 H3C + CH3 N
+
CH2
O
Aqueous cytoplasmic environment
Viscous
Flip-flop (rarely occurs)
Flexion
Rotation
Unsaturated, kinked fatty acids, loosely packed
Saturated, straight fatty acids, closely packed
Figure A2.3 Phospholipids, cholesterol, the lipid bilayer, and membrane fluidity. A. Generic (saturated) phospholipid. B. Orientation of phospholipids in the bilayer. C. Some phospholipids present in cell membranes. D. Structural formula and schematic drawing of cholesterol. E. Motions of phospholipids in membrane bilayers. F. The ratio of saturated and unsaturated fatty acids in the phospholipids affects membrane fluidity.
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A-26
Appendix 2
■
I n tr o d u c to r y C ell B iolog y : E ukary ot ic C ells
Glycolipids and glycoproteins also contribute to membrane asymmetry because the carbohydrate moieties always face the extracellular environment. Membrane fluidity refers to the movement of membrane phospholipids within the plane of the membrane, and this fluidity is important for proper membrane function. For example, transport across the membrane is affected by membrane fluidity. Decreased fluidity is associated with decreased transport rates. Because a drop in temperature decreases fluidity, cold temperatures may slow transport processes across the membrane. The composition of the membrane, especially the types of phospholipids present, can have a dramatic effect on membrane fluidity. For example, saturated fatty acids decrease membrane fluidity because the linear hydrocarbon tails pack together well. In contrast, unsaturated fatty acids have kinks in the hydrocarbon chains that limit packing and increase fluidity (Fig. A2.3F). The length of the fatty acid chains also affects fluidity. Phospholipids with longer hydrocarbon chains have increased hydrophobic interactions with neighboring lipids and thus decreased membrane fluidity. Organisms can alter membrane fluidity in response to temperature stress by changing the length and degree of saturation of fatty acids present in membrane phospholipids. For example, as environmental temperatures drop, both eukaryotes and prokaryotes maintain membrane fluidity by replacing long-chain fatty acids with shorter chains and increasing the percentage of unsaturated fatty acids in their membranes. Cholesterol also influences membrane fluidity. The effects of cholesterol on membrane fluidity are complicated and depend on factors such as the ratio of saturated to unsaturated fatty acids in the membrane. Cholesterol may prevent packing of saturated fatty acids, thus
increasing fluidity. In membranes with unsaturated fatty acids, cholesterol may fi ll in the spaces between adjacent phospholipids, stabilizing them and decreasing fluidity. In this case, cholesterol can decrease the permeability of the membrane to hydrophobic substances by packing in between the hydrocarbon chains and preventing substances from slipping through.
Membranes Are Semipermeable: Some Substances Pass through Them Easily; Others Do Not The major functions of membranes (such as containing cytoplasmic components, regulating what substances enter and leave cells and organelles, and producing energy) depend on the semipermeable nature of membranes. Semipermeable (also called selectively permeable) membranes are permeable to some substances but not to others. In general, the cell membrane is permeable to hydrophobic molecules and impermeable to charged molecules (Fig. A2.4). Diffusion across the membrane also depends on the size of the molecule. The membrane is freely permeable to small nonpolar molecules such as O2. Larger nonpolar molecules can also diffuse across the membrane, albeit more slowly. Molecules that are polar but small (such as ethanol and water) can also diffuse across the membrane. The membrane is impermeable to large polar molecule such as glucose and to charged molecules, regardless of their size. The impermeability of the membrane to charged substances such as ions is important for energy production at membranes because the proton motive force depends on the ability of the membrane to separate compartments of different ion concentrations.
Selective permeability of cell membranes.
Figure A2.4
Freely permeable
Hydrophobic molecules O2, CO2, N2
SFMB_app02.indd A-26
Slightly permeable
Small, uncharged polar molecules H2O, glycerol
Impermeable
Impermeable
Large, uncharged polar molecules Glucose, sucrose
Ions H+, Na+, HCO3– Ca2+, Cl–,Mg2+, K+
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Appe n dix 2
■ I nt roduc t ory C ell B iolog y : E ukary oti c Ce l l s
Transport Proteins Move Many Substances across Cell Membranes Although ions and large polar molecules such as glucose cannot diffuse directly across the cell membrane, they do need to enter cells. In both eukaryotes and prokaryotes, movement of molecules across the cell membrane is accomplished through specific transmembrane proteins such as channels and transporters. Channels can also increase the diffusion of molecules that move across the membrane too slowly on their own to supply the cell’s needs. For example, aquaporins can increase the rate of water movement across the membrane. There are many different types of transporters, each differing in its energy requirements and in the types of molecules that it transfers across the membrane.
■
Diffusion Moves Substances across Cell Membranes and within Cells
■
Diffusion is the net movement of molecules from an area of high concentration to one of low concentration. It is a spontaneous process because it is accompanied by an increase in entropy (positive ∆S) that results in a negative free energy change (∆G). The process requires no energy input and is brought about by the random, thermal movement of molecules. Many factors can influence the rate of diffusion of a molecule across a membrane. Diffusion rates can be determined with an artificial membrane system as depicted in Figure A2.5. Molecules are added to the left side of the beaker, and samples from the right side are analyzed at various time points for the presence of the test molecule. Factors that influence diffusion of molecules across a membrane include: ■
■
Temperature. Increased temperatures mean faster motion. The faster the molecules are moving, the faster they will arrive at the membrane and cross it. Solubility of the molecules in the membrane. To cross the membrane, the molecules must penetrate
A.
■
■
A-27
it. Hydrophobic molecules will dissolve in the membrane and cross it; charged molecules will not. Surface area of the membrane. To cross the membrane, molecules must fi rst encounter it. The chances of this happening are increased with an increase in membrane surface area. Concentration gradient of the dissolved molecules. A larger concentration gradient speeds up diffusion because the more molecules there are, the more will encounter the membrane and cross. Thickness of the membrane. Diffusion rates are inversely proportional to the square of the distance the solute must travel across the membrane. The thinner the membrane, the faster the molecules can get across. Mass of the molecule. Friction between a molecule and its medium is a source of resistance that slows down motion. Larger molecules with more mass experience more resistance and cross the membrane more slowly. These factors can be expressed as follows: Diffusion rate ∝ temperature × surface area × concentration gradient mass × distance2
Conditions for the diffusion of gases and other substances across the cell membrane will be most favorable when the surface area of the membrane is large, the concentration gradient across the membrane is high, and the membrane is thin.
Transport of Water Across the Cell Membrane Must Be Tightly Controlled Osmosis is the diffusion of water across a selectively permeable membrane from regions of high water concentration (low solute) to regions of low water concentration
B.
Water
Water
Hydrophobic solute
Lipid bilayer Solute diffuses from left to right according to the laws of diffusion.
Figure A2.5
SFMB_app02.indd A-27
Diffusion across a phospholipid bilayer.
A. An artificial bilayer system. B. Diffusion across an artificial membrane.
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A.
B. Higher solute concentration (lower water concentration)
Isotonic solution
Hypertonic solution
Hypotonic solution
Decreases
Increases
Lipid bilayer
Lipid bilayer Solute molecules do not cross the membrane.
Cell volume
No change
Osmosis
Water moves toward the compartment with lower water concentration.
If too much water enters, cell lysis may occur.
Osmosis and water balance. A. Osmosis. B. Movement of water across the cell membrane, and shrinkage or expansion of the membrane in isotonic, hypertonic, and hypotonic environments. Arrows indicate net water movement.
Figure A2.6
(high solute) (Fig. A2.6A). Cells must maintain osmotic balance with the surrounding environment. The direction of water movement depends on the concentration of dissolved solutes in the cell relative to the cell’s environment. When a cell is in an isotonic environment (equal concentrations of dissolved solutes inside the cell and out), there is osmotic balance, and water will enter and exit the cell at equal rates (that is, there is no net movement of water). In a hypertonic environment (higher concentration of solutes outside the cell), there is a net loss of water from a cell. The cell shrinks, and the concentration of cell contents increases. In a hypotonic environment (lower concentration of solutes outside the cell), there is a net uptake of water by the cell; the cell swells, and the cell components are diluted (Fig. A2.6B). If enough water enters, the cell is destroyed by lysis—a rupturing of the cell membrane and dispersal of cell contents. Both hypertonic and hypotonic environments can cause other problems for cells. Proteins have specific salt requirements, and intracellular environments that have salt concentrations that are either higher or lower than normal for a cell can cause denaturation of proteins, with potentially fatal results for the cell. Thus, transport of water across the cell membrane must be tightly controlled, and cells need mechanisms that allow them to live in environments that are not isotonic. Most cells live in environments that are hypotonic. To deal with this challenge, most prokaryotes and many
SFMB_app02.indd A-28
eukaryotes have a cell wall external to the cell membrane. As water enters by osmosis and pushes against the cell wall (turgor pressure), the wall resists the tension and pushes back with an equal but opposite force known as wall pressure. The wall pressure is an inward pressure exerted by the cell wall against the cell membrane (Fig. A2.7). When turgor pressure and wall pressure are equal in magnitude, the cell is at equilibrium with respect to
Stiff cell wall pushes back with equal and opposite force.
Wall pressure When turgor pressure equals wall pressure, the cellular water and environmental water are at equilibrium.
Outside of cell
Cell wall Cell membrane Turgor pressure
Inside of cell
Expanding volume of cell pushes membrane out.
Figure A2.7
Turgor pressure and wall pressure.
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Appe n dix 2
water movement. Organisms that lack a cell wall employ other strategies to deal with hypotonic environments. For example, some freshwater protists that lack a cell wall expel excess water through a contractile vacuole. In contrast to freshwater microbes, ocean-dwelling organisms face the problem of water loss. Many of these organisms accumulate solutes known as osmolytes to ensure that they are isotonic to the external environment, thus preventing water loss.
Eukaryotes Can Move Substances across the Cell Membrane by Endocytosis and Exocytosis All cells use diffusion and transport proteins to move molecules into or out of the cell, but some eukaryotes can, in addition, use endocytosis and exocytosis to achieve this end. In endocytosis, parts of the cell membrane bud into the cytoplasm and eventually separate from it to form endosomes (Fig. A2.8). Endosomes are a type of vesicle, a small membranous sphere found within a cell. The interior of these endosomes contains extracellular material. Phagocytosis (cell eating) is a form of endocytosis in which large extracellular particles are brought into the cell. Pinocytosis (cell drinking) is endocytosis of the extracellular fluid. Endocytosis is a controlled, energy-requiring process that relies on many proteins, including cytoskeletal proteins. Exocytosis is the reverse of endocytosis. In exocytosis, intracellular vesicles fuse with the cell membrane, and the contents of the vesicles are released to the extracellular environment. Cells can use exocytosis to release wastes.
Endocytosis
Vesicle (endosome)
Exocytosis
Secretory vesicle
Cell membrane
Figure A2.8
SFMB_app02.indd A-29
Endocytosis and exocytosis.
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A2.2 The Nucleus and Mitosis All cells need to synthesize proteins, and all cells contain DNA, the genetic material that encodes the information needed to specify protein primary structure. The central dogma of molecular biology holds that DNA is transcribed into messenger RNA (mRNA), and mRNA is translated into protein on ribosomes. Although this central dogma of molecular biology holds for all cells, details in the structure of DNA and ribosomes vary among the three domains of life (see Table A2.1). For example, ribosomes always function in protein synthesis, but ribosomes from organisms in different domains differ in their sedimentation rates and their sensitivity to various antibiotics. DNA always encodes the information needed for protein synthesis, but whereas bacterial chromosomes are usually circular, eukaryotic chromosomes are typically linear. Another difference between DNA in prokaryotes and DNA in eukaryotes is the location of DNA within the cell. Prokaryotic DNA is found in an area of the cytoplasm known as the nucleoid, while eukaryotic DNA is enclosed by a membrane-bound nucleus.
Eukaryotic DNA Is Housed in the Membrane-bound Nucleus Eukaryotes derive their name (eukaryote means “true kernel”) from the fact that they possess a nucleus, and, indeed, the nucleus is often the most prominent feature of eukaryotic cells viewed under a microscope (Fig. A2.9A). The nucleus is an organelle, an intracellular membranebound compartment with a specific function. The nucleus contains chromatin, a complex of DNA and proteins. The nuclear membrane (envelope) consists of two concentric phospholipid membranes. The outer nuclear membrane is continuous with the membrane of the endoplasmic reticulum (ER), and the space between the two nuclear membranes is continuous with the lumen (inside) of the ER (Fig. A2.9B). Nuclei contain a region called the nucleolus, where ribosome assembly begins. At the nucleolus, multiple rRNA (ribosomal RNA) genes are transcribed, and the resulting rRNA combines with ribosomal proteins imported into the nucleus from the cytoplasm to form the ribosomal subunits. The ribosomal subunits then need to exit the nucleus. The nuclear membrane contains nuclear pore complexes (NPCs) that allow for transport of material into and out of the nucleus. Metabolites and small proteins can diffuse through the NPCs, but larger proteins and organelles cannot enter by diffusion. Large proteins that need to enter the nucleus are actively transported in through the NPCs. These selectively imported proteins contain a nuclear localization signal, a sequence of amino acids that acts like a zip code to direct them into the nucleus. In addition to their role in protein import, NPCs also function in exporting mRNAs out of the nucleus.
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A.
B. The nuclear envelope is a double membrane. Outer membrane Inner membrane Nucleolus
Nucleus
Figure A2.9
The nucleus.
©Gopal Murti/Visuals Unlimited
At some sites, the nuclear envelope is continuous with the endoplasmic reticulum. Chromatin Nuclear envelope
Pores in nuclear envelope (NPCs)
A. An electron micrograph of a eukaryotic yeast cell showing the prominent nucleus. B. Diagram of a
nucleus.
Eukaryotic Cells Replicate by Mitosis Cells need to ensure an accurate replication and division of their DNA. Prokaryotes replicate by fission, as described in Chapter 3. Eukaryotic cells replicate their nuclear DNA and divide by mitosis. Mitosis is a series of steps that segregates duplicated chromosomes and ensures that each daughter cell receives a copy of the genetic material. When the cell is not undergoing mitosis, it is in interphase (Fig. A2.10A). During interphase, the individual chromosomes are long and thin and not visible by a light microscope. Interphase can be divided into three phases: G1, S, and G2. Cells that are not committed to dividing are in G1, the fi rst gap phase. If cell division is to occur, then the chromosomes are replicated during S phase (S for “synthesis”). The duplicated chromosomes, called sister chromatids, remain attached to each other at the centromere (Fig. A2.10B). After chromosome replication, a second gap phase, G2, occurs, after which mitosis can proceed. Mitosis is divided into a number of steps: prophase, metaphase, anaphase, and telophase (Fig. A2.10C). During prophase, the chromosomes condense and become visible by light microscopy. The nuclear membrane may break down. The mitotic spindle, responsible for separating the sister chromatids to opposite poles of the cell, begins to form. The mitotic spindle is a network of microtubules (see Section A2.5) that originate from centrosomes. There are two centrosomes, and these migrate to opposite sides of the cell. Each centrosome contains two centrioles, and each centriole contains nine sets of microtubules in a radial formation (see Section A2.5). The free ends of the microtubules establish connections with the sister chromatids at a structure called the kinetochore.
SFMB_app02.indd A-30
At metaphase, the spindle apparatus is complete and each sister chromatid is connected to a microtubule. The chromosomes are arranged along an imaginary plane in the middle of the cell. In the next phase of mitosis, anaphase, the microtubules shorten and pull the sister chromatids apart, separating the replicated chromosomes. At the end of anaphase, each set of chromosomes is located on opposite sides of the cell. During, telophase, the final step in mitosis, the nuclear membrane re-forms around the chromosomes and the chromosomes become long and thin again. Cytokinesis also occurs during telophase and partitions the original cell into two daughter cells by the formation of a cell membrane between them. The preceding description of mitosis forms a synopsis of this process as it occurs in eukaryotic cells from multicellular organisms, where it has been particularly well studied. However, some unicellular eukaryotes exhibit differences from the canonical mitotic phases. In yeast, for example, the nuclear envelope does not break down. Despite these differences, the end result, the separation of duplicated chromosomes into two daughter cells, is the same.
A2.3 Problems Faced by Large Cells Most eukaryotic cells range in size from 10 to 100 micrometers (µm) in diameter, about ten times as large as the typical prokaryotic cell (1–10 µm diameter). Cells face two major challenges as a result of increased cell size. The fi rst problem is that as cells increase in size, their volume (the cytoplasm) increases faster than their surface area (the cell
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Appe n dix 2
A.
B. Unreplicated chromosome
Division M
A-31
Replicated chromosome
Chromosome
Mitosis
G2
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Second gap Centromere
S
First gap
DNA synthesis
G1
Interphase
Sister chromatids
Number of chromosomes: 4
Number of chromosomes: 4
C. Prior to Mitosis Chromosomes replicate, forming sister chromatids Centrosomes
Mitosis Early mitotic spindle
Kinetochore
Centrioles
Chromosomes replicate in parent cell.
Prophase: Chromosomes condense, and mitotic spindle begins to form. Nuclear envelope breaks down.
Metaphase: Chromosomes migrate to middle of cell.
Anaphase: Sister chromatids separate. Chromosomes are pulled to opposite poles of the cell.
Telophase: The nuclear envelope re-forms. Cytokinesis: The cell divides.
Figure A2.10 The cell cycle and mitosis. A. Stages of the eukaryotic cell cycle. G1, S, and G2 make up interphase (in gray). B. Duplication of the chromosomes during S phase. C. The phases of mitosis. See text for details.
membrane) (Fig. A2.11). Cells are fi lled with metabolically active cytoplasm that requires nutrients and energy and produces wastes. Energy production, nutrient import, and waste disposal are events that take place at the cell membrane. As cells increase in size, the cell membrane area may not be able to keep up with the demands placed on it by a proportionally larger cytoplasm. The eukaryotic cell’s answer to this problem is the endomembrane system, an extensive network of internal membranes that effectively increases the membrane surface area without an increase in cell volume. The second problem associated with an increase in size is related to diffusion. The amount of time it takes a molecule to diffuse a given distance is proportional to the distance squared. For example, if it takes a particular molecule 1 second to diffuse 1 µm, then it takes that same molecule 100 seconds to diffuse 10 µm. Many biochemical reactions depend on the partners in the reaction fi nding each other by diffusion (that is, their rate is diffusion controlled), so longer diffusion times result in slower reactions. Furthermore, signals received at the cell membrane
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r
r
r = 1µm Surface area (4πr2) = 12.6µm2 Volume (4⁄3πr3) = 4.2µm3 Surface = 3.0 Volume
r = 2µm Surface area = 50.3µm2 Volume = 33.5µm3 Surface = 1.5 Volume
Figure A2.11 Cell volume increases faster than surface area. Because the surface area increases by the radius squared and the volume increases by the radius cubed, the volume increases faster than the surface area.
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need to be communicated throughout the cell. In a large cell, the amount of time it takes for a molecule to traverse the cell by diffusion may be too slow for the cell to rapidly adjust to signals it receives from the environment or from other sites within the cell. To deal with this problem, eukaryotic cells possess a cytoskeleton, a group of proteins that, among other things, maintains cell shape and moves molecules around the cell, relieving the cell from the need to rely on diffusion for transport. As we shall see in the next two sections, the endomembrane system and the cytoskeleton have other advantages for the eukaryotic cell.
■
■
Organelles can provide different environments that allow disparate reactions to occur simultaneously. For example, proteins are being synthesized by ribosomes in the neutral pH cytoplasm, while at the same time proteins are being hydrolyzed within the acidic organelles known as lysosomes. Compartmentalization protects cytoplasmic components from harmful substances. For example, hydrogen peroxide (H2O2), a product of cellular oxidation reactions, is produced and converted to water within peroxisomes. Localizing the reaction within the peroxisome keeps the toxic peroxide away from other cell components, such as proteins and DNA, that are sensitive to oxidative stress.
A2.4 The Endomembrane System The endomembrane system is a series of compartments found inside eukaryotic cells and bounded by membranes that are separate from the cell membrane. Organelles of the endomembrane system include the endoplasmic reticulum (ER), the Golgi complex (also called the Golgi apparatus), lysosomes, and peroxisomes. Different organelles contain unique subsets of proteins that contribute to their function. To a large extent, the function of the ER and the Golgi complex is to direct to their proper cellular location proteins destined for lysosomes, for the cell membrane, or for secretion from the cell.
Organelles Provide Many Advantages to the Eukaryotic Cell ■
■
Endomembranes increase the membrane surface area without increasing the cell volume. Separating cellular contents in small, enclosed compartments increases the concentrations of enzymes and their substrates, allowing reactions to proceed faster (Fig. A2.12).
Some Eukaryotic Cells Use Lysosomes to Digest Organic Matter Lysosomes are membrane-bound organelles that help eukaryotic cells obtain nourishment from macromolecular nutrients. Lysosomes contain many hydrolytic enzymes (for example, proteases, nucleases, and lipases) and have an acidic pH of around 5. Lysosomes are formed when vesicles containing hydrolytic enzymes and proton pumps bud off from the Golgi complex (Fig. A2.13A). Lysosomes then fuse with vesicles containing the material to be digested. Often this material comes from outside the cell via phagocytosis (Figs. A2.13A and B). Phagocytosis and lysosomal digestion help the eukaryotic cell because they effectively increase the membrane surface area over which nutrients can be absorbed. Bacteria lack these processes; to obtain nutrition from large molecules in their environment, bacteria must secrete digestive enzymes. The extracellularly digested materials are subsequently transported across the bacterial cell membrane through specific transporters (see Section 4.2). In contrast, lysosomes allow for intracellular digestion, and digested material crosses the lysosomal membrane into the cytoplasm. Any waste products left in the lysosome can leave the cell via exocytosis.
The Area Inside the Endoplasmic Reticulum Is Separate from the Cytoplasm
Organelle
Figure A2.12 Advantages of organelles. Solutes (red dots) are concentrated in organelles, reactive molecules are separated from the cytoplasm, and membrane surface area is increased without an increase in cell volume.
SFMB_app02.indd A-32
The endoplasmic reticulum is continuous with the outer nuclear membrane, and the lumen (interior) of the ER is continuous with the space between the two nuclear membranes (Fig. A2.14A). As well as being continuous with the space between the nuclear membranes, the lumen of the ER is spatially equivalent to the interior spaces of other endomembrane components and to the outside of the cell. This means that material in the ER does not need to cross a membrane to enter these other spaces, and
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Appe n dix 2
A.
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A-33
B. Food particle is taken in by phagocytosis.
Extracellular environment Cell membrane
Products of digestion Phagosome Food particles digested
Cytoplasm
Paramecia
Primary lysosome Golgi complex
0.25 mm
Eric Grave/Photo Researchers
Ameba
Figure A2.13 Lysosomes. A. Lysosomes contain hydrolytic enzymes to digest material brought into the cell by phagocytosis. B. Amobas engulfing paramecia (light microscopy). The paramecia will be digested with the aid of lysosomes.
mixing can occur via vesicle fusion. For example, material contained within the lumen of the ER can mix with the contents of the Golgi complex or with the extracellular milieu by fusion of vesicles from the ER with Golgi membrane or the plasma membrane. These topologically equivalent areas, indicated by a common color in Figure A2.14A, are completely separated from the cytoplasm by endomembranes, so that the ER can be used to sequester substances that must be held at low concentrations in the cytoplasm, for example, calcium ions.
Smooth ER and Rough ER Have Different Structures and Functions There are two morphologically and functionally distinct types of endoplasmic reticulum—smooth ER and rough ER, as shown in the micrograph of Figure A2.14C. The smooth ER is the site of lipid synthesis and some detoxification of noxious compounds. The rough ER is the site where transmembrane proteins, secreted proteins, and resident proteins of the ER, Golgi, or lysosomes are
A.
B. Golgi
ow Fl
rial ate m of
Vesicle
trans Golgi cisternae C. Endoplasmic reticulum cis Golgi cisternae
Lumen of ER Smooth ER Rough ER Nucleus
Ribosome
Figure A2.14 The endoplasmic reticulum and Golgi complex. A. The relationship of the ER to other cellular membranes and the flow of material through vesicles from the rough ER to the cell membrane. B. Electron micrograph of Golgi cisternae. C. Electron micrograph showing rough and smooth ER.
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Rough ER
Robert Bolender & Donald Fawcett/ Visuals Unlimited
Cytoplasm
SPL/Photo Researchers
Cell membrane
Smooth ER
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translated. The rough ER appears rough because its cytoplasmic surface is studded with ribosomes. These ribosomes are located on the rough ER because the protein being synthesized by the ribosome has a signal sequence on its amino terminus (the fi rst part of the protein translated from the mRNA). A signal sequence is a specific sequence of amino acids that directs proteins to a specific cellular location. The signal sequence that directs proteins to the ER recognizes a receptor (the signal recognition particle) on the rough ER membranes (Fig. A2.15A), so the protein-ribosome complex is directed to the surface of the ER membrane. Note that the ribosomes attached to the rough ER are identical to cytoplasmic ribosomes and only attach to the ER transiently because of the type of protein they are translating, proteins that contain the correct signal sequence.
A.
Membrane of endoplasmic reticulum
Cytoplasm
Signal sequence
1. Signal sequence is synthesized by ribosome.
Ribosome Receptor
RNA
2. Signal sequence interacts with receptor protein in ER membrane.
3. Protein enters ER. Signal sequence is removed.
The Translation of Proteins across Rough Endoplasmic Reticulum After the ribosome docks with the signal recognition particle, the protein is threaded through the ER membrane as it is translated. Secreted proteins and proteins destined for the lumen of an organelle are threaded completely through the ER membrane and end up in the lumen of the ER (see Fig. A2.15A). In contrast, transmembrane proteins are not threaded completely through, and part of the protein spans the membrane (Fig. A2.15B). The membrane-spanning regions of transmembrane proteins usually contain a continuous stretch of about 20 hydrophobic amino acids that form an alpha helix with the hydrophobic side chains facing out toward the hydrophobic hydrocarbons of the membrane. As the nascent polypeptide chains are threaded across the ER membrane, the unfolded proteins are bound by chaperonins. Chaperonins (also known as heat-shock proteins) prevent partially folded proteins from clumping together and help proteins attain their correct tertiary structure. In the ER, proteins may be modified. The signal sequence that directed the proteins to the ER is usually cleaved off. The environment inside the ER allows disulfide bonds to form between cysteine residues of some proteins. Other ER proteins have oligosaccharide groups covalently attached, a posttranslational modification known as glycosylation. The enzymes that attach the sugars are found only in the ER lumen. In transmembrane proteins of the cell membrane, sugars always face the extracellular environment because the extracellular environment is topologically equivalent to the lumen of the ER (see Fig. A2.14A). Resident ER proteins, those that will stay and function in the ER, are retained inside the ER because they contain a sequence of amino acids that acts as an ER retention signal.
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Interior of rough endoplasmic reticulum (lumen)
B. Cleavage site Ribosome
H2N 1. Translocation
Signal sequence
H2N 2. Translocation stops.
Stop-transfer peptide
H2N Mature transmembrane protein
COOH
H2N
NH2
3. Cleavage of start transfer peptide.
COOH
Figure A2.15 Insertion of proteins into the ER. A. Proteins are targeted to the ER by a signal sequence on the growing polypeptide chain. Secreted proteins and proteins destined for the lumen of an organelle end up in the lumen of the ER. B. Transmembrane proteins are not completely inserted through the ER membrane, and a portion of the protein remains within the membrane.
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these proteins serve as a cell skeleton and impart specific shapes to eukaryotic cells. However, cytoskeletal proteins are multifunctional and are also involved in whole-cell movements and movements of substances within the cell.
The Golgi Complex Directs the Transport of Proteins Proteins not retained in the ER pass on to the Golgi complex by way of vesicles. The Golgi complex consists of separate membrane stacks (cisternae) that each contain unique enzymes. The cis face of the Golgi complex is nearest to the ER, and the trans face is farthest from the ER, closest to the cell membrane (see Figs. A2.14A and B). As proteins pass through the cisternae, the carbohydrates on them may be trimmed and modified. These modified carbohydrates can serve as address tags to target proteins to particular organelles. For example, proteins tagged with mannose-6-phosphate (mannose phosphorylated on its number 6 carbon) are selectively sent to lysosomes; that is, vesicles enriched in proteins containing mannose6-phosphate bud off from the Golgi and are directed to lysosomes. Proteins not targeted to lysosomes or marked for retention in the Golgi complex, may be sent to the cell membrane. Vesicles leaving the Golgi complex may fuse with the cell membrane, releasing their contents to the extracellular environment (see Fig. A2.14A). Transmembrane proteins in these vesicles can then become part of the cell membrane. Regions of transmembrane proteins that were inside vesicles will face the extracellular environment, and cytoplasmic portions will remain cytoplasmic.
Intermediate Filaments Are Formed of Various Proteins Intermediate filaments (Fig. A2.16A) consist of various fibrous proteins that have a diameter of about 10 nm. Intermediate fi laments often form a meshwork under the cell membrane and, in cells that lack a cell wall, help impart and maintain cell shape. Intermediate fi laments also strengthen the cell by resisting tension placed on the cell membrane. The proteins that make up intermediate fi laments vary with cell type. Intermediate fi laments are fairly stable and are not thought to undergo acute changes in length the way microfi laments and microtubules do.
Microfilaments Are Polymers of the Protein Actin Microfilaments, also known as actin fi laments, have a diameter of 7 nm (Fig. A2.16B). They are formed when individual actin monomers (globular actin, or G-actin) polymerize, in a process fueled by ATP hydrolysis, to form chains of fi lamentous actin (F-actin). Two F-actin chains twist around each other to form microfi laments that have a plus end and a minus end. Microfi laments are dynamic structures, growing and shrinking in a controlled manner. New monomer units add to the plus end and dissociate from the minus end. Whether actin will polymerize
A2.5 The Cytoskeleton Eukaryotic cells contain proteins called intermediate filaments, microfilaments, and microtubules that are collectively termed the cytoskeleton. As their name implies,
A. Intermediate filament
A-35
B. Microfilament
C. Microtubule
8–12 nm
7 nm 25 nm Actin monomer β α Tubulin dimer
Fibrous subunit
alpha-Tubulin monomer beta-Tubulin monomer
D. Kinesin “walks” along a microtubule track. Transport vesicle
ATP
ADP + Pi
ATP
ADP + Pi
Kinesin Microtubule
–
End
+
End
Figure A2.16 Cytoskeletal proteins. A. Intermediate filaments are ropelike assemblages of various proteins. B. Microfilaments consist of two strands of actin polymers twisted together. C. Microtubules are polymers of tubulin dimers. D. Fueled by the hydrolysis of ATP, the motor protein kinesin can move vesicles or organelles toward the plus end of microtubules.
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or depolymerize depends on a number of factors, including the concentration of G-actin. The critical concentration is a measure of the ability of actin to polymerize. At G-actin concentrations below the critical concentration, F-actin will depolymerize, and at concentrations greater than the critical concentration, G-actin will polymerize. The plus end of microfi laments has a critical concentration less than that of the minus end, so actin is preferentially added on to the plus end and removed from the minus end. Some microfi laments play a structural role in the cell and work in conjunction with intermediate filaments to maintain cell shape. These structural microfi laments have protein caps at both ends to prevent changes in microfi lament length. Other microfi laments have functions that require dynamic changes in length. For example, the pseudopod movement of an ameba depends on the polymerization of actin at the leading edge of growth. The plus end of the microfi lament is located underneath the cell membrane of the extending pseudopod, and polymerization is enhanced by the actin-binding protein profi lin. Microfi laments are also responsible for cytoplasmic streaming, a mixing of the cytoplasm that aids diffusion. The protein myosin works with microfilaments to generate the forces needed for cell streaming, pseudopod formation, and cytokinesis, the separation of daughter cells after nuclear division.
Microtubules Are Polymers of Tubulin Microtubules have a larger diameter (25 nm) than microfi laments and intermediate fi laments (Fig. A2.16C). The hollow microtubule structure consists of 13 tubulin dimers; one alpha-tubulin protein plus one beta-tubulin protein forms one tubulin dimer. Like microfilaments, microtubules have plus (faster-growing) and minus (slower-growing) ends and are dynamic structures that can polymerize and depolymerize. Polymerization is an energy-requiring process, and the necessary energy is obtained by coupling polymerization to GTP hydrolysis. Microtubules aid movement of substances within the cell and are also involved in powering whole-cell movement by cilia and flagella. Traffic of proteins through the endomembrane system (see Section A2.4) relies on the controlled movement of vesicles from one cellular compartment to the next. Microtubules provide tracks that can move vesicles from one organelle to the next in an efficient, directed fashion. Working with microtubules to accomplish this are motor proteins. Motor proteins such as kinesin and dynein can capture cargo (for example, vesicles or organelles) and walk them along microtubule tracks in an ATPdependent process (Fig. A2.16D). Kinesin moves cargo toward the plus end of microtubules, while dynein moves
SFMB_app02.indd A-36
cargo toward the minus end. In addition to moving vesicles, microtubules are responsible for the segregation of duplicated chromosomes during mitosis.
Eukaryotic Cilia and Flagella Cilia and flagella are thin extensions of the cell membrane that can move in a whiplike fashion, driven by interactions between microtubules and the motor protein dynein. Flagella (singular, flagellum) are relatively long, and cells usually have only one or two of them; cilia (singular, cilium) are shorter and more numerous. Both flagella and cilia can move cells through space. Cilia may also aid in food capture—for example, by sweeping extracellular fluid into the gullet of a paramecium. Eukaryotic cilia and flagella differ from bacterial cilia and flagella; bacterial flagella depend on the proton motive force to rotate a motor that causes flagellar movement, whereas eukaryotic flagella rely on ATP hydrolysis by dynein to move the flagella in a whiplike fashion. The study of protists has played a key role in determining the structure and function of flagella and cilia. Much information about flagella structure has come from studies of a Chlamydomonas species, a unicellular alga that has two long flagella (Fig. A2.17A). Dynein was fi rst discovered in the cilia of the unicellular eukaryote Tetrahymena (Fig. A2.17B). Flagella and cilia have the same structure and mechanism of action, so for convenience we will restrict the following discussion to flagella. A cross section through a flagellum reveals a central bundle of microtubules called the axoneme (Fig. A2.17C). The axoneme originates at a microtubule-organizing center called the basal body, which is similar to the centriole (see Section A2.2). The minus ends of the microtubules are at the basal body; the plus ends at the tip of the flagellum are capped to prevent changes in length. As shown in the electron micrograph in Figure A2.17D, the axoneme has a characteristic arrangement of two central microtubules and nine microtubule doublets around the periphery. The microtubule doublets are connected to each other and to the central microtubules through protein cross-links called bridges and spokes. (Fig. A2.17E). The motor protein dynein connects adjacent microtubule doublets. If the cell membrane is removed from the surface of a flagellum and the protein cross-links (but not dynein) are dissolved, the microtubule doublets are observed to lengthen in the presence of ATP. This is due to the action of dynein, which slides the microtubule doublets past each other (Fig. A2.17F). In intact flagella, the tension imparted as dynein tries to slide microtubules past each other is translated into a bending motion. Controlled cycles of dynein activation and inactivation on opposite sides of the axoneme lead to the whiplike motion of flagella.
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Appe n dix 2
2 µm
D.
E.
Microtubule doublet
Basal body Flagellum
Microtubules
Dynein Bridge Spoke
Axoneme
Microtubule doublet – End
Central pair
Outer doublet
Microtubules
Cell membrane
F. Cell membrane
Courtesy of David R. Mitchell
75 nm
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C. Biomedia Museum, U. of Paisley
B. Tetrahymena Dartmouth Electron Microscope Facility
A. Chlamydomonas
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Direction of movement – End
+ End Dynein + End
The force generated by dynein propels the adjacent microtubule doublet in the direction of its minus end. In intact flagella, this force is translated into a whiplike motion.
Figure A2.17 Flagella and cilia. A. The protist Chlamydomonas has two long flagella (fluorescence micrograph). B. The protist Tetrahymena has numerous cilia (scanning electron micrograph). C. Structure of flagella and cilia. D. Cross section of axoneme (TEM). E. Cross section of axoneme indicating bridges and spokes formed by cross-linking proteins. F. Force generated by dynein on a microtubule doublet.
A2.6 Mitochondria and Chloroplasts Mitochondria and chloroplasts are organelles involved in cellular energy production. Mitochondria perform oxidative respiration and are found in nearly all eukaryotes. Chloroplasts perform photosynthesis and are found only in photosynthetic eukaryotes, such as green algae. Both organelles are thought to have become part of eukaryotic cells through a process of endosymbiosis. The endosymbiosis theory states that mitochondria and chloroplasts were once free-living prokaryotes that became ingested, but not digested, by a larger (possibly eukaryotic) cell (Fig. A2.18A). A symbiotic relationship developed, with the larger eukaryotic cell providing protection to the prokaryote and the prokaryote providing energy to the eukaryote. As we shall see, the structure of mitochondria and chloroplasts strongly supports the endosymbiosis theory.
Mitochondria Produce ATP by Oxidative Respiration Mitochondria are the powerhouses of the eukaryotic cell. A cell may contain tens or hundreds of mitochondria, depending on its energy needs. Mitochondria have two membranes, an outer membrane and an inner membrane (Fig. A2.18B). The inner membrane has numerous infoldings called cristae that increase its surface
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area. It is thought that as a result of the endocytosis of the prokaryote, the inner mitochondrial membrane is derived from the prokaryote and the outer membrane is derived from the larger eukaryote. Supporting this idea is the fact that the inner membrane has structural characteristics of a prokaryotic cell membrane, while the outer membrane is similar to the host eukaryotic membrane. For example, 20% of the phospholipids in the inner membrane are cardiolipin, a phospholipid largely absent from eukaryotic membranes. Mitochondria contain two distinct compartments: the intermembrane space between the two membranes and the matrix inside the inner membrane. Different stages of oxidative respiration occur in specific compartments. As predicted by the endosymbiosis theory, the topology of these processes is similar in prokaryotes and mitochondria (Table A2.2). For example, in prokaryotes, the citric acid cycle occurs in the cytoplasm; in mitochondria, the citric acid cycle takes place inside the matrix, the metabolic equivalent of the prokaryotic cytoplasm. The fact that mitochondria contain their own DNA and ribosomes lends further support to the endosymbiosis theory, since these cell features would be a necessary part of any free-living organism. Both the DNA and ribosomes of mitochondria show similarities with the DNA and ribosomes of prokaryotes. For example, like prokaryotic DNA, mitochondrial DNA is circular, and mitochondrial ribosomes are sensitive to antibiotics that disrupt
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Appendix 2
■
I n tr o d u c to r y C ell B iolog y : E ukary ot ic C ells
A. Membrane of larger cell
B.
C. Outer membrane
Outer membrane Inner membrane
Inner membrane Intermembrane space
Endosymbiosis theory, mitochondria, and chloroplasts. theory. B. Structures in a mitochondrion. C. Chloroplast structure.
Figure A2.18
Granum Thylakoids Stroma
Matrix Cristae
Double membrane
George Chapman/ Visuals Unlimited
Don W. Fawcett/ Visuals Unlimited
Membrane of smaller cell
A. Origin of organelles according to endosymbiosis
Table A2.2 Location of oxidative respiration components in prokaryotes and mitochondria. Feature
Prokaryotes
Mitochondria
Electron transport chain (ETC) ATP synthase Citric acid cycle Proton motive force
Cell membrane Cell membrane Cytoplasm Protons diffuse from lower-pH extracellular environment into the cytoplasm across the cell membrane.
Inner membrane Inner membrane Matrix Protons diffuse from the lower-pH intermembrane space into the matrix across the inner membrane.
prokaryotic ribosomes. Although nuclear DNA encodes some mitochrondrial proteins, phylogenetic analysis has shown that these nuclear genes are related to prokaryotic genes. Furthermore, the generation of new mitochondria is not tied to the replication of the cell, and the division of mitochondria within the cell is similar to the fission seen in prokaryotic cells.
Chloroplasts Allow Eukaryotes to Perform Photosynthesis Chloroplasts are organelles found only in photosynthetic eukaryotes. In the light reactions of photosynthesis, chloroplasts convert light energy from the sun to ATP and reduced NADPH. In the subsequent light-independent reactions, the ATP and NADPH are used to reduce CO2 to sugar. Like mitochondria, chloroplasts are probably the result of endosymbiosis. Bacteria related to cyanobacteria are thought to be the prokaryotic partners that gave
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rise to chloroplasts. Like eukaryotic chloroplasts, cyanobacteria contain chlorophylls a and b and perform aerobic photosynthesis. In addition, cyanobacteria contain extensive internal membranes called thylakoids that contain chlorophyll and participate in the light reactions of photosynthesis. Chloroplasts have three membranes whose topology can be understood in light of the endosymbiosis theory (Fig. A2.18C). The outer membrane appears to be derived from the host eukaryotic cell; the inner membrane is equivalent to the bacterial cell membrane; and the thylakoid membrane is derived from the prokaryotic thylakoid membranes. The region inside the inner membrane is called the stroma and is equivalent to the bacterial cytoplasm. The thylakoid membrane is packed with the chlorophyll pigments that give chloroplasts their green color. ATP and NADPH are produced in the stroma and used there in the light-independent reactions. As would be expected as a result of endosymbiosis, chloroplasts, like mitochondria, contain their own circular DNA and their own ribosomes.
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Answers to Thought Questions
Chapter 1 1.1 The minimum
size of known microbial cells is about 0.2 µm. Could even smaller cells be discovered? What factors may determine the minimum size of a cell? ANSWER: The smallest cells known, about 0.2 µm in length, are cell wall-less bacteria called mycoplasmas; for example, Mycoplasma pneumoniae, a causative agent of pneumonia. Bacteria might be discovered that are smaller than 0.2 µm, but it is hard to see how their cell components such as ribosomes (about a tenth this size) could fit inside such a small cell. The volume required for DNA and the apparatus of transcription and translation probably sets the lower limit on cell size.
1.2 If viruses are not functional cells, are they truly “alive”? ANSWER: A traditional definition of a life form includes an entity with the capability for metabolism and homeostasis (maintaining internal conditions of its cytoplasm) as well as reproduction and response to its environment. Viruses reproduce themselves indefinitely, and respond to the environment of the host cell, but lack metabolism or homeostasis outside their host cell. Nevertheless, viruses such as herpesviruses contain numerous metabolic enzymes that participate in the metabolism of their host. Certain large viruses such as the mimivirus appear to have evolved from cells. Some microbiologists argue that viruses should be considered “alive” if reproduction is the main criterion, and if the viral “environment” is considered the inside of the host cell. 1.3
Why do you think it took so long for humans to connect microbes with infectious disease? ANSWER: For most of human history we were unaware of the existence of microbes. Even after microscopy had revealed their existence, the incredible diversity of the microbial world and the difficulties in isolating and characterizing microbial organisms made it difficult to discern the specific effects of microbes. All healthy people contain microbes; and most disease-causing microbes are indistinguishable from normal flora by light microscopy. Not all microbial diseases are directly transmittable from human to
human; they may require complex cycles with intermediate hosts, such as the fleas and rats that carry bubonic plague.
1.4 How could you use Koch’s postulates to demonstrate the causative agent of influenza? What problems would you need to overcome that were not encountered with anthrax? ANSWER: In order to use Koch’s postulates to demonstrate the causative agent of influenza, an animal model host would be needed. Secretions from diseased patients could be applied to different animal species, such as monkeys and mice, in order to find an animal that shows signs of the disease. To determine the causative agent of disease, the patient’s secretions could be filtered in order to separate bacteria and viruses. Only the filtrate would cause disease, as it contains viruses (relevant to Koch’s postulates 1 and 3). Viruses, however, are more difficult to isolate in pure culture than bacteria (postulate 2), a problem Koch did not address. Furthermore, some viruses, such as HIV (human immunodeficiency virus) have no animal model; they grow only in human cells. Today, viruses are usually isolated in a tissue culture. Once isolated, the virus could be used to inoculate a new host animal (if an animal model exists) or a tissue culture, and determine whether infection results (postulates 3 and 4). Another problem Koch did not address was the detection of infectious agents too small to be observed under a microscope. Today, antibody reactions are used to determine whether an individual has been exposed to a putative pathogen. An antibody test could be used to determine whether healthy and diseased individuals had been exposed to the isolated virus. 1.5 Why do you think some pathogens generate immunity readily, whereas others evade the immune system? ANSWER: Some pathogens (microbes that cause disease) have external coat proteins that strongly stimulate the immune system and induce production of strong antibodies. Other pathogens have evolved to avoid the immune system by changing the identity of their external proteins. Immunity also varies greatly with the host’s status. The very young and very old generally have weaker immune systems than people in the prime of their lives. Some pathogens, such as HIV, will directly attack the immune system, limiting the immune response to the pathogen. AQ-1
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A n sw e r s t o Th o u g h t Q u e st io ns
1.6
How do you think microbes protect themselves from the antibiotics they produce? ANSWER: Microbes protect themselves from the antibiotics they produce by producing their own resistance factors. As discussed in later chapters, microbes may synthesize pumps to pump the antibiotics out; or they make altered versions of the target macromolecule, such as the ribosome subunit; or they make enzymes to cleave the antimicrobial substance.
1.7 Why don’t all living organisms fix their own nitrogen? ANSWER: Nitrogen fixation requires a tremendous amount of energy, about thirty molecules of ATP per dinitrogen molecule converted to ammonia (discussed in Chapter 15). In a community containing adequate nitrogen sources, organisms that lose the nitrogen fixation pathway would make more efficient use of their energy reserves than those that spend energy to fix nitrogen from the atmosphere. Another consideration is that nitrogenase is an oxygen-sensitive enzyme, whereas plants, animals, and fungi are aerobes. In order to fix nitrogen, aerobic organisms need to develop complex mechanisms to exclude oxygen from nitrogenase. 1.8 What arguments support the classification of Archaea as a third domain of life? What arguments support the classification of archaea and bacteria together, as prokaryotes, distinct from eukaryotes? ANSWER: The sequence of 16S rRNA (small-subunit rRNA) and other fundamental genes differs as much between archaea and bacteria as it does between archaea and eukaryotes. The composition of archaeal cell walls and phospholipids is completely distinct from those of bacteria and eukaryotes. Some aspects of gene expression, such as the RNA polymerase complex, are more similar between archaea and eukaryotes than between archaea and bacteria. On the other hand, archaeal and bacterial cells are prokaryotic; they both lack nuclei and complex membranous organelles. Archaeal metabolism and lifestyles are more similar to those of bacteria than eukaryotes. Some archaea and bacteria sharing the same environment, such as high-temperature springs, have undergone horizontal transfer of genes encoding traits such as heat-stable membrane lipids. 1.9 Do you think engineered strains of bacteria should be patentable? What about sequenced genes or genomes? ANSWER: Microbes—as well as multicellular organisms, such as transgenic mice—have been patented, and the patents have stood up in court. DNA sequence per se is not patentable, but specific plans for use of DNA sequence can include the sequence as part of the patent. The reason for granting these patents is to encourage medical research by companies that need to earn a profit. The disadvantage of patents is that they restrict information flow and under-
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cut competition. Furthermore, religious and philosophical arguments have been made that patenting live organisms cheapens life. Current laws attempt to reach a balance among these concerns.
Chapter 2 2.1 You have discovered a new kind of microbe, never observed before. What kind of questions about this microbe might be answered by light microscopy? What questions would be better addressed by electron microscopy? ANSWER: Light microscopy could answer questions such as: What is the overall shape of this cell? Does it form individual cells or chains? Is the organism motile? Only light microscopy can visualize an organism alive. Electron microscopy can answer questions about internal and external subcellular structures. For example, does a bacterial cell possess external filamentous structures such as flagella or pili? If the dimensions of the unknown microbe are smaller than the lower limits of a light microscope’s resolution, EM may be the only way to observe the organism. Viruses are often characterized by shape, and this shape is observed by electron microscopy.
2.2 Explain what happens to the refracted light wave as it emerges from a piece of glass of even thickness. How does its new speed and direction compare with its original (incident) speed and direction? ANSWER: The part of the wave front that emerges first travels faster than the portion still in the glass, resulting in the wave front bending toward the surface of the glass. Ultimately, the wave travels in the same direction and with the same speed as before it entered the glass. The path of the emerging light ray is parallel to the path of the light ray entering the glass and is shifted over by an amount dependent on the thickness of the glass. This refraction will alter the path of the beam of light and decrease the amount reaching the lens of the microscope. Immersion oil has the same refractive index as glass and will limit the amount of light lost in this way. 2.3 Parabolic lenses are generally “biconvex,” that is, curving outward on both sides. What would happen to parallel light rays that pass through a lens that is concave on both sides? Or a lens that is convex on one side and concave with equal curvature on the other? ANSWER: When light passes through a lens that is concave (curving inward) on both sides, the light rays diverge within the lens material, then diverge again more steeply on their way out. If the lens is concave on one side and convex with equal curvature on the opposite side, then the light rays will diverge within the lens but will emerge parallel, although slightly farther apart than when they entered.
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Answers t o Thoug ht Que s ti o n s
2.4 In theory, what angle θ would produce the highest resolution? What practical problem would you have in designing a lens to generate this light cone? ANSWER: In theory, an angle theta (θ) of 90 degrees would produce the highest resolution. However, a 90-degree angle of theta generates a cone of 180 degrees, which would require the object to sit in the same position as the objective lens; in other words, to have a focal distance of zero. In practice, the cone of light needs to be somewhat less than 180 degrees, in order to allow room for the object and to avoid substantial aberrations (light-distorting properties) in the lens material. 2.5
Under starvation conditions, bacteria such as Bacillus thuringiensis, the biological insecticide, repackage their cytoplasm into spores, leaving behind an empty cell wall. Suppose, under a microscope, you observe what appears to be a hollow cell. How can you tell if the cell is indeed hollow or if it is simply out of focus? ANSWER: You could tell whether the cell is out of focus or actually hollow by rotating the fine-focus knob to move the objective up and down while observing the specimen carefully. If the hollow shape appears to be the sharpest image possible, it is probably a hollow cell. If the hollow shape turns momentarily into a sharp, dark cell, it was probably out of focus before. You could also use a confocal microscope to visualize the center of the hollow cell.
2.6 Some early observers claimed that the rotary motions observed in bacterial flagella could not be distinguished from whiplike patterns, comparable to the motion of eukaryotic flagella. Can you imagine an experiment to distinguish the two and prove that the flagella rotate? Hint: Bacterial flagella can get “stuck” to the microscope slide or coverslip. ANSWER: To prove that flagella rotate, you can “tether” the bacteria to the microscope slide by getting one of its flagella stuck to the slide. A simple way to tether bacteria is by using a slide coated with anti-flagellin antibody. When the flagellum is stuck to the slide, its motor continues to rotate; thus, the entire cell now rotates. The rotation of the cell body can easily be seen by video microscopy. If the flagella moved in a whiplike fashion, the tethered cell would move back and forth, not rotate.
2.7 What experiment could you devise to determine the actual order of events in DNA movement toward the pole during formation of an endospore? ANSWER: One way to track the movement of DNA during sporulation would be to stain the DNA with a dye such as propidium iodide at various stages of sporulation. Another way to determine the order of events in sporulation could be to observe mutant strains of bacteria that contain defects at different points in the sporulation process. (Sporulation is discussed further in Chapter 4.)
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2.8 Compare and contrast fluorescence microscopy with dark-field microscopy. What similar advantage do they provide, and how do they differ? ANSWER: Both dark-field and fluorescence enable detection (but not resolution) of objects whose dimensions are below the wavelength of light. Dark-field technique is based on light scattering, which detects all small objects without discrimination. Fluorescence, however, provides a means to label specific parts of cells, such as cell membrane or DNA, or particular species of microbes, using fluorescent antibody tags. 2.9 An electron microscope can be focused at successive powers of magnification, as in a light microscope. At each level, the image rotates at an angle of several degrees. Based on the geometry of the electron beam, as shown in Figure 2.34, why do you think this rotation occurs? The image rotates because the electron beam is not straight, as for photons, but travels in a spiral through the magnetic field lines. As magnification increases, the spiral expands, and it reaches the image plane at a slightly different angle than before.
ANSWER:
2.10 What kind of research questions could you investigate using SEM? What questions would be answered using TEM? ANSWER: SEM could be used to examine the surface of cells: Do the cells possess a smooth surface, or does their surface contain protein complexes or bulges that serve special functions? How do pathogens attach to the surface of cells? TEM can be used to determine the intracellular structure of attachment sites as well as of internal organelles. TEM can also visualize the shape of macromolecular complexes such as flagellar motors or ribosomes. 2.11 What kind of experiments could prove or disprove the interpretations of the images of “nanobacteria” in blood plasma? ANSWER: Attempt to observe the proposed “nanobacteria” in the presence of a general antibacterial agent such as sodium azide. If the particles still appear despite the sodium azide, they cannot be alive and are probably inorganic in nature. Another approach is to use PCR amplification of 16S ribosomal RNA sequences to establish the existence of a novel isolate. So far, the PCR sequences obtained from “nanobacteria” in blood plasma have been identified as those of an environmental microbe that commonly contaminates PCR samples. Chapter 3 3.1 Which molecules occur in the greatest number in a prokaryotic cell? The smallest number? Why does a cell contain 100 times as many lipid molecules as strands of RNA?
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AQ-4
A n sw e r s t o Th o u g h t Q u e st io ns
ANSWER: Inorganic ions occur in the largest number in a prokaryotic cell (250 million/cell). They are also the smallest in size. DNA molecules are found in the smallest number (one large molecule, branched during replication). A prokaryotic cell contains a hundred times as many lipid molecules as strands of RNA because lipids are small structural molecules, highly packed. RNA molecules are long macromolecules that are either packed into complexes (such as ribosomal RNA), or are temporary information carriers (messenger RNA), present only as needed to make proteins.
3.2 Why does the Svedberg coefficient of the intact ribosome (70S) differ from the sum of the individual subunits of the ribosome (30S and 50S)? ANSWER: The Svedberg coefficient is a nonlinear function. A particle’s mass, density, and shape will determine its S value. It depends on the frictional forces retarding the particle’s movement, which in turn are based on the average cross-sectional area of the particle. The sum of the crosssectional areas of the two separate subunits is greater than the cross-sectional area of the complex; thus, the sum of their S units (30S + 50S) is greater than that of the intact complex (70S). 3.3
What are the advantages and limitations of biochemical and genetic approaches to cell structure and function? ANSWER: Biochemistry is useful to separate particles and identify their functions in isolation. In some cases, particles can also be combined together to study the function of a complex such as the cell-free translation system. On the other hand, biochemistry may miss aspects of function that are observed only in the intact cell. Genetics reveals the functions of specific genes in the phenotype of a living cell. On its own, however, genetics reveals little about the spatial relationships of gene products working together. Together, genetics and biochemistry reveal a more complete view of how cellular structures function.
3.4 Amino acids have acidic and basic groups that can dissociate. Why are they not membrane-permeant weak acids or weak bases? Why do they fail to cross the phospholipid bilayer? ANSWER: At neutral pH, amino acids each have both a positively charged amine and a negatively charged carboxylate, that is, they can act as either a weak acid or a weak base. Charged ions, no matter what their size, will not freely pass a plasma membrane. If either charged group becomes neutralized by acid or base, the other group remains charged, so the molecule as a whole will never cross the membrane. 3.5 The actual thickness of the cell wall is difficult to determine based solely on electron microscopic observation of the envelope layers. Devise a biochemical experi-
SFMB_answers.indd AQ-4
ment that can show the number of layers of peptidoglycan in cells of a given species. ANSWER: To demonstrate the number of layers of peptidoglycan in a cell wall, determine the percentage by weight of peptidoglycan in the cell sample. Estimate the surface area of cells based on scanning EM. Given the known thickness of a layer of peptidoglycan, calculate how many layers would be needed to yield the amount of peptidoglycan present in the cell.
3.6 Why would laboratory culture conditions select for evolution of cells lacking an S-layer? ANSWER: Degeneration of protective traits is a common problem when conducting research on microbes that can produce thirty generations overnight. Their rapid reproductive rate gives ample opportunity for spontaneous mutations to accumulate over an experimental time scale. In the case of the S-layer, in a laboratory test tube free of predators or viruses, mutant bacteria that fail to produce the thick protein layer would save energy compared to S-layer synthesizers, and would therefore grow faster. Such mutants would quickly take over a rapidly growing population. 3.7 Suppose that one cell out of a million has a mutant gene blocking S-layer synthesis, and suppose that the mutant strain can grow twice as fast as the S-layered parent. How many generations would it take for the mutant strain to constitute 90% of the population? ANSWER: After each generation, the parent strain increases two-fold, whereas the mutant numbers increase by a factor of four. The mutant fraction is equal to (4N)/[4N + (106 × 2N)], where N is the number of mutant cells after a given generation. Plotting this out on an Excel spreadsheet shows that by the twenty-third generation, the mutant fraction approximates 90%. 3.8 Why would the proteins listed in Table 3.2 be confined to specific cell fractions? Why could a protein not function throughout the cell? ANSWER: Each protein has evolved a specific function optimized for a specific part of the cell. For example, waterconducting porins are found solely in the inner membrane (cell membrane), which is otherwise impermeable to water. The outer membrane, which is water-permeable, is the sole location for specific porins transporting small peptides and sugars. The sugars then need to be taken across the inner membrane by transport proteins that evolved to function best in this location. Similarly, different chaperones (proteins that aid peptide folding) have evolved to function best in the cytoplasmic environment or in the periplasmic environment, membrane-enclosed regions that differ substantially in pH and ion concentrations. In a different chemical environment of the cell, the protein denatures and loses its functional structure.
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Answers t o Thoug ht Que s ti o n s
3.9
What do you think are the advantages and disadvantages of a contractile vacuole, compared with a cell wall? ANSWER: The disadvantage of a contractile vacuole is that it requires continual input of energy to bail out the water. On the other hand, the contractile vacuole permits the existence of a cell that is flexible enough to engulf other cells as prey. A cell wall does not require energy (other than the initial energy to synthesize it). A cell wall is inflexible and does not allow another cell to be engulfed as prey.
3.10 How would a magnetotactic species have to behave if it were in the Southern Hemisphere instead of in the Northern Hemisphere? In the Northern Hemisphere, the field lines for magnetic north point downward; in the Southern Hemisphere, the opposite is true. Thus, if downward direction is the aim of magnetotaxis, bacteria existing in the two hemispheres would have to respond oppositely to the magnetic field; in the Southern Hemisphere, anaerobic magnetobacteria swim toward magnetic south. Near the equator, the proportions of north-seeking and south-seeking bacteria are roughly equal.
ANSWER:
3.11 Most strains of E. coli and Salmonella commonly used for genetic research actually lack flagella entirely. Why do you think this is the case? How can a researcher maintain a motile strain? ANSWER: The motility apparatus requires fifty different genes generating different protein components. Cells that acquire mutations eliminating expression of the motility apparatus gain an energy advantage over cells that continue to invest energy in motors. In a natural environment, the nonmotile cells lose out in competition for nutrients despite their energetic advantage; but in the laboratory, cells are cultured in isotropic environments such as a shaking test tube, where motility confers no advantage. These culture conditions lead to evolutionary degeneration of motility, as they do for the S-layer (see Thought Question 3.7). In order to maintain a motile strain, bacteria are cultured on a soft agar medium containing an attractant nutrient. As cells consume the attractant, they generate a gradient and chemotaxis leads them to swim outward. By subculturing only bacteria from the leading edge of swimming cells, one can maintain a motile strain.
Chapter 4 4.1 In a mixed
ecosystem of autotrophs and heterotrophs, what happens when a heterotroph allows the autotroph to grow and begin to make excess organic carbon? ANSWER: At first the growth of the heterotroph might outpace the growth of the autotroph, using the carbon sources faster
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than the autotroph can make them. As the organic carbon sources diminish through consumption, growth of the heterotroph decreases, but the CO2 formed by the heterotroph will allow the autotroph to grow and make more organic carbon. Ultimately, the ecosystem comes into balance.
4.2 In what situation would antiport and symport be passive rather than active transport? ANSWER: Antiport and symport would be passive when both molecules are moving down their concentration gradients. A symport or an antiport system can do work to move a molecule from a low concentration to a high (against a concentration gradient) as long as the co-transported molecule is moving from high to low concentration. When this happens, it is a form of active transport. If a symport or antiport moves both molecules with the concentration gradient (from high concentration to low), it is passive transport, assuming the molecules are moving no faster than the rate of diffusion. 4.3 What kind of transporter, other than an antiporter, could produce electroneutral coupled transport? ANSWER: Electroneutral coupled transport can occur by symport if molecules of opposite charge are symported; for example Na+ flux together with Cl–. 4.4 What would be the phenotype (growth characteristic) of a cell that lacks the trp genes (genes required for the synthesis of tryptophan)? What would be the phenotype of a cell missing the lac genes (genes whose products catabolize the carbohydrate lactose)? ANSWER: The difference lies in the function of the two pathways. The trp operon is a biosynthetic operon. Errors in the biosynthetic pathway will lead to a failure to produce tryptophan. Therefore, a trp auxotrophic mutant will only grow on defined medium if tryptophan is added. The lactose operon involves the catabolism of a carbon source, lactose. If any of these genes are damaged, the cells are no longer able to use lactose as a carbon source. A lac mutant will not grow on defined medium with lactose as the sole carbon source. 4.5 The addition of sheep blood to agar produces a very rich medium called blood agar. Do you think blood agar can be considered a selective medium? A differential medium? Hint: Some bacteria can lyse red blood cells. ANSWER: Blood agar can be considered differential, because different species growing on blood have different abilities to lyse the red blood cells in the agar. Some do not lyse, others completely lyse red blood cells (secreted hemolysin produces complete clearing around a colony), while still others only partially lyse the blood (the secreted hemolysin produces a greening around the colony). It will therefore differentiate between hemolytic and non-hemolytic bacteria. The medium is very rich and does not prevent the growth of any organism so is not considered selective.
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AQ-6
A n sw e r s t o Th o u g h t Q u e st io ns
4.6
A virus such as influenza virus might produce 800 progeny virus particles from one infected host cell. How would you mathematically represent the exponential growth of the virus? What practical factors might limit such growth? ANSWER: In theory, the growth rate of the virus would be proportional to 800n. In practice, however, all the virus particles would never find 800 different host cells to infect. Furthermore, it turns out that only a small proportion of the influenza virus progeny are viable (see Chapter 11).
4.7 Suppose 1,000 bacteria are inoculated in a tube of minimal salts medium, where they double once an hour; and 10 bacteria are inoculated into rich medium, where they double in 20 minutes. Which tube will have more bacteria after 2 hours? After 4 hours? After 2 hours, the minimal medium will contain 1,000 × 22 = 4,000 bacteria, whereas the rich medium will contain only 10 × 2(2 h)(3/h) = 640 bacteria. After 4 hours, the minimal medium will contain 1.6 × 105 bacteria, whereas the rich medium will have surpassed this count, reaching nearly 4.1 × 105. ANSWER:
4.8 An exponentially growing culture has an optical density at 600 nm (OD600) of 0.2 after 30 minutes and an OD600 of 0.8 after 80 minutes. What is the doubling time? ANSWER: Because the OD600 has increased from 0.2 to 0.8, there are four times as many cells after 80 minutes as after 30 minutes, and the culture has doubled twice. Two doublings occurred in 50 minutes; therefore the doubling time is 25 minutes. 4.9 Figure 4.20C shows growth curves for different population densities of Thiobacillus thiooxidans when the concentration of sulfur in the medium is constant. Draw the growth curves you would expect to see if the initial population density was constant but the concentration of sulfur varied. ANSWER:
Cell number
1 mM 0.5 mM 0.1 mM
Time (days)
4.10 It takes 40 minutes for a typical E. coli cell to completely replicate its chromosome and about 20 minutes to prepare for another round of replication. Yet the organism enjoys a 20-minute generation time growing at 37°C in complex medium. How is this possible? ANSWER: After the DNA is replicated about halfway around the chromosome, each daughter half-chromosome initi-
SFMB_answers.indd AQ-6
ates a second round of replication, so the time needed to divide from one cell to two is effectively halved. Most cells in a log-phase culture in rich medium actually have four copies of the DNA origin of replication, each with a separate attachment site on the cell envelope, the future midpoint of a cell two generations ahead (see Chapter 3).
4.11 What can happen to the growth curve when a culture medium contains two carbon sources, one a preferred carbon source of growth-limiting concentration and a second, nonpreferred source? (see Section 10.3) ANSWER: There are two possibilities. If the enzyme systems needed to utilize both carbon sources are always made, the growth curve will look normal because both will be used simultaneously. Usually the enzyme system for the nonpreferred carbon source is not produced until the preferred source is used up. In this case, a second lag phase will interrupt the exponential phase. This is called a diauxic growth curve and is commonly seen when cells are grown on both glucose and lactose. Lactose is the nonpreferred carbon source and is used second. The second lag phase marks the exhaustion of one nutrient and the gearing up by the cell to use the other (see Chapter 10). 4.12 How would you modify the equations describing microbial growth rate to describe the rate of death? ANSWER: The death rate applies to a period of declining cell numbers. Therefore, the logarithm of the cell number ratio of N1 to N0 will be a negative number, and this factor will need to be preceded by a negative sign to convert it to a positive “halving time,” or half-life of the culture. 4.13 Why are cells in log phase larger than cells in stationary phase? ANSWER: Cells strive to maintain a certain DNA/mass ratio. In so doing, they balance the amount of biochemical processes needed to sustain viability. If the cell mass becomes large relative to the number of copies of a given, critical gene, the amount of enzyme produced may not be sufficient to keep the cell alive and growing. In addition, the DNA/mass ratio serves as a signal to trigger cell division. Thus, when a cell divides faster than it replicates its chromosome, it must start a second round of replication before it finishes the first. This type of replication ensures that at least one chromosome duplication will be complete at the time of division. Because fast-growing cells contain more than one chromosome, they will increase in size to maintain the desired DNA/mass ratio. If the ratio were not maintained, cell division would not occur when needed. 4.14 How might Streptomyces and Actinomyces species avoid committing suicide when they make their antibiotics? ANSWER: Bacteria that produce antibiotics need to make defenses against the antibiotic within their own cytoplasm.
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For example, their genes can express an altered form of the target molecule, such as a ribosomal subunit; or they can make pumps to pump the antibiotic out of the cell.
Chapter 5 5.1 Why haven’t cells evolved so that all their enzymes have the same temperature optimum? If they did, wouldn’t they grow even faster? ANSWER: An enzyme’s function is not determined by temperature alone. There are other physicochemical constraints based on the variety and complexity of functions different enzymes must carry out. The thousands of different enzyme molecules must work in a coordinated fashion to support the basic functions of life. By having some enzymes work below or above their optimum temperatures, the rate of the reactions they catalyze will be altered. A population will evolve based on the entire organism’s ability to reproduce, not the speed at which each individual chemical reaction is carried out. The primary goal of a microbe is not just to grow fast but also to survive. Growing too fast could deplete food sources and produce toxic by-products too quickly.
5.2 If microbes lack a nervous system, how can they sense a temperature change? ANSWER: Most bacteria respond to outside stimuli, such as heat, by altering their gene expression. They sense heat by monitoring the level of misfolded proteins, a consequence of too high a temperature. The mechanism does not perceive heat per se, but recognizes the deleterious effects of moving outside the optimum growth temperature range so that the cell can launch an emergency response. The same mechanisms can sense other environmental stresses that misfold proteins, such as acid stress. See Chapter 10. 5.3 What could be a relatively simple way to grow barophiles in the laboratory? ANSWER: High pressures can be maintained using a syringe. Scientists would place medium in a stainless steel syringe and maintain pressure with a calibrated vise. 5.4 How might the concept of water availability be used by the food industry to control spoilage? ANSWER: Food preservation traditionally includes water exclusion by salt, as seen in hams, back bacon, salted fish, etc., or by high concentration of sugar, as in canned fruit or jellies. The lower aw prevents microbial growth. Dehydrating foods will also prevent microbial growth. 5.5 In Chapter 4, we stated that an antiporter couples movement of one ion down its concentration gradient with movement of another molecule uphill, against its gradient. If this is true, how could a Na+/H+ antiporter work to bring
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protons into a haloalkaliphile growing in high salt at pH 10? Since the Na+ concentration is lower inside the cell than outside and H+ concentration is higher inside than outside (see Fig. 5.16), both ions are moving against their gradients. ANSWER: In this situation the cell has to expend some energy to make the antiporter work. The energy involved is rooted in the charge difference between the inside of the cell (negative charge) and the outside of the cell (positive charge, called delta psi, ∆ψ. See Chapter 13). The antiporter in this case must also exchange a different number of NA+ and H+ ions, maintaining an electrical charge difference across the membrane. For example, export 2NA+ and import 1H+.
5.6 If anaerobes cannot live in oxygen, how do they incorporate oxygen into their cellular components? ANSWER: They incorporate oxygen from their carbon sources (e.g., CO2 and carbohydrates such as glucose), all of which contain oxygen. This form of oxygen will not damage the cells. 5.7 How can anaerobes grow in the human mouth when there is so much oxygen there? ANSWER: A synergistic relationship occurs between facultatives and anaerobes within a tooth biofilm. The facultatives consume oxygen within the biofilm microenvironment, which allows the anaerobes to grow underneath them. 5.8 What evidence led people to think about looking for anaerobes? Hint: Look up Spallanzani, Pasteur, and spontaneous generation on the web. ANSWER: The priest Lazzaro Spallanzani (1729–1799), during his quest to disprove spontaneous generation, said, “Every beast on Earth needs air to live, and I am going to show just how animal these little animals are by putting them in a vacuum and watching them die.”He dipped a glass tube into a culture, sealed one end and attached the other to a vacuum. He was astonished to find that the microbes lived for weeks. He then wrote: “How wonderful this is. For we have always believed there is no living being that can live without the advantages air offers it. “ Fifty years later, Pasteur observed that air could kill some organisms. After looking at a drop of liquid from a fermentation culture, he wrote, “There is something new here—in the middle of the drop they are lively, going every which way, but on the edge they were stiff as pokers.” 5.9 How would you test the killing efficacy of an autoclave? ANSWER: Construct a death curve by measuring survival of a known quantity of spores (e.g., Bacillus stearothermophilus) after autoclaving for various lengths of time. Spores should be used because they are more resistant to heat than any vegetative cell. Typically, autoclaves are regularly checked with spore strips that change color once the endosopores are no longer viable.
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A n sw e r s t o Th o u g h t Q u e st io ns
Chapter 6 6.1 Which viruses
do you know that have a narrow host range, and which have a broad host range? ANSWER: Examples of viruses with a narrow host range include poliovirus (poliomyelitis), which infects only humans and chimpanzees; smallpox, which infects only humans; and feline T-cell leukemia virus, which infects only cats. Examples of viruses with a broad host range include rabies, which infects numerous species of mammals, and influenza strains, which show preference for particular species but can jump between various mammals and birds.
6.2
What would happen if a virus particle remained intact within a host cell instead of releasing its genome? ANSWER: The virus would be unable to reproduce, because DNA polymerases could not reach its genome for reproduction and RNA polymerase could not transcribe its genes to make gene products.
6.3 Why do viral capsids take the form of an icosahedron instead of some other polyhedron? ANSWER: The icosahedron is a polyhedron of 20 triangular faces, the largest number possible. Thus, the icosahedron turns out to be the largest and most economical form to enclose space based on a small repeating unit. Natural selection probably favored viruses that could build the largest capsid based on the smallest amount of genetic information. 6.4 How can viruses with different kinds of genomes (RNA versus DNA) combine and exchange genetic information? ANSWER: DNA viruses require messenger RNA intermediates to express their proteins. It is possible that a DNA virus could acquire the ability to package its RNA transcript in a capsid, rather than its DNA. Alternatively, RNA retroviruses form DNA intermediates within their host cell; these DNA intermediates might recombine with the DNA genome of another virus. 6.5 What are the relative advantages and disadvantages (to a virus) of the slow-release strategy, compared with the strategy of a temperate phage, which alternates between lysis and lysogeny? ANSWER: A disadvantage of slow release is that the phages can never reproduce progeny phage as rapidly as in a lytic burst. The drain on resources of the host cell infected by a slow-release virus causes it to grow more slowly compared with uninfected cells; in contrast, a lysogenized cell suffers little or no reproductive deficit compared with uninfected cells. An advantage of reproduction by slow release is the continual release of phage, while avoiding the possibility of releasing all particles into an environment where no other host cell exists. 6.6 How could humans evolve to resist rhinovirus infection? Is such evolution likely? Why or why not?
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Resistance to all rhinovirus infections might evolve through a mutation in the host gene encoding ICAM-1. The mutation would have to prevent rhinovirus binding without impairing the protein’s ability to bind integrin. Such evolution is unlikely because of the importance of integrin binding and because rhinovirus infection is rarely fatal; thus, there is little selection pressure to evolve inherited resistance. Note, however, that the immune system rapidly generates immunity to particular strains of rhinovirus. Over a lifetime, most individuals acquire immunity to many rhinovirus strains but remain susceptible to others.
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6.7 What are the advantages and disadvantages to the virus of replication by the host polymerase, compared with using a polymerase encoded by its own genome? ANSWER: An advantage of using the host polymerase is energetic; the virus avoids the energetic cost of manufacturing a polymerase to package with each virion. This is an advantage to the virus, since its reproductive potential is limited by the energy resources of its host cell. Furthermore, because DNA and RNA polymerases are so central to cell function, the host species is unlikely to evolve a mutant form of the polymerase that resists the virus. On the other hand, the advantage of the virus making its own polymerase is that the viral polymerase can evolve to better meet the needs of its own replication, such as high speed and low accuracy to generate frequent variants. 6.8 Why does bacteriophage reproduction give a step curve, whereas cellular reproduction generates an exponential growth curve? Could you design an experiment in which viruses generate an exponential curve? Under what conditions does the growth of cellular microbes give rise to a step curve? ANSWER: Lytic viruses appear to make a step curve because the number of progeny per infected cell is 100 or more, released simultaneously. After two or three generations the cell cycles would fall out of synchrony, and the curve would smooth out, but the later cell cycles are rarely observed in practice because by then the supply of host cells is exhausted. If, however, an extremely low ratio of viruses to host cells is provided, the growth of virus particles will eventually generate an exponential curve. By contrast, the growth of cellular microbes is rarely observed during the first few doublings. By the time we measure the population, the cells are all undergoing different stages of division, and the population growth overall generates a smooth exponential curve. If, however, we observe the growth of a synchronized population of cells, we see a step curve of cell division two. Chapter 7 7.1 What do you think happens to two single-stranded DNA molecules isolated from different genes when they are mixed together at very high concentrations of salt?
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ANSWER: In high salt conditions, the stacking of hydrophobic bases is so strongly favored that two single strands of DNA will form a duplex no matter what the sequence of base pairs.
7.2 Do you think the kinetics of denaturation and renaturation are dependent on DNA concentration? ANSWER: The speed of denaturation does not depend on DNA concentration, but the speed of renaturation does. The higher the concentration of ssDNA, the more likely it is that complementary sequences will find each other and the faster the duplex can re-form. 7.3
DNA gyrase is essential to cell viability. Why, then, are nalidixic acid-resistant cells that contain mutations in gyrA still viable? ANSWER: The gyrA mutations alter only the nalidixic acid binding site on GyrA, not its gyrase activity. In other words, active DNA gyrase is still made, but the drug cannot bind to it.
7.4 Bacterial cells contain many enzymes that can degrade DNA. How, then, do linear chromosomes in organisms like Borrelia burgdorferi (the causative agent of Lyme disease) avoid degradation? ANSWER: DNA-digesting exonucleases act on free 5’ or 3’ ends. The Borrelia linear chromosomes possess covalently closed hairpin ends called telomeres and do not possess free 5’ or 3’ groups. 7.5 Suppose you have the following capabilities: You can label DNA in a bacterium by growing cells in medium containing either nitrogen 14N or the heavier isotope 15N; you can isolate pure DNA from the organism; you can subject DNA to centrifugation in a cesium chloride solution, a solution that forms a density gradient when subjected to centrifugal force, thereby separating the light (14N) and heavy (15N) forms of DNA to different locations in the test tube. Given these capabilities, how might you prove that DNA replication is semiconservative? 15 ANSWER: Grow bacteria in medium containing heavy N, so that all the DNA made by the cells will be in the heavy form. Transfer the cells to medium containing only 14N and allow the cells to divide for one generation. Extract the DNA and centrifuge the preparation in cesium chloride. If replication is semiconservative, a hybrid DNA band will be seen at a position located between and equidistant from where heavy DNA and light DNA would be located. The hybrid band will be composed of one heavy strand and one light strand, which makes it of intermediate weight (density). This, in fact, was how Meseleson and Stahl proved semiconservative replication in 1958, five years after the discovery that DNA was double-stranded. 7.6 How fast does E. coli DNA polymerase synthesize DNA (in nucleotides per second), given that the genome is 4.6
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million base pairs, replication is bidirectional, and the chromosome completes a round of replication in 40 minutes? ANSWER: It takes E. coli 40 minutes (2,400 seconds) to complete the replication of its 4,639,221-bp chromosome. Since Pol III works as a dimer to synthesize both strands simultaneously, we will only consider one strand in the calculation. The rate is 4,639,221 nt per 2,400 seconds = 1,933 nt per second. But there are two replication forks, so each polymerase dimer only synthesizes one-half of the chromosome, which means that each polymerase still synthesizes a remarkable 800–1000 nt per second.
7.7 Individual cells in a population of E. coli typically initiate replication at different times (asynchronous replication). However, depriving the population of a required amino acid can synchronize reproduction of the population. What happens is that ongoing rounds of DNA synthesis finish, but new rounds do not begin. Replication stops until the amino acid is once again added to the medium, an action that triggers simultaneous initiation in all cells; that is, reproduction of the population becomes synchronized. Why? Initiation requires synthesis of the initiator protein DnaA. Consequently, depriving the population of an amino acid prevents protein synthesis, which precludes synthesis of DnaA. Because DnaA is not required to complete already initiated rounds of replication, all rounds already started are completed, but reinitiation cannot occur. Adding the amino acid once again will allow all cells to simultaneously make DnaA so that initiation is triggered in all cells at the same time.
ANSWER:
7.8 The antibiotic rifampin inhibits transcription by RNA polymerase, but not by primase (DnaG). What happens to DNA synthesis if rifampin is added to a synchronous culture? Initiation of DNA synthesis requires primer transcription at the origin by RNA polymerase, an enzyme sensitive to rifampin. Primase (DnaG), which synthesizes RNA primers in the lagging strand throughout DNA synthesis, is resistant to rifampin. So adding rifampin to a synchronized culture will prevent new rounds of DNA replication but will not affect already initiated rounds.
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7.9 Knowing that E. coli possesses restriction enzymes that cleave DNA from other species, how is it possible to clone a gene from one organism to another without the cloned gene sequence being degraded? ANSWER: The best way to overcome the restriction barrier is to use a mutant strain of E. coli in which its restriction system has been inactivated. DNA entering the cell will not be degraded and, if the modification system is still in place, will be modified. Once modified, that DNA can be safely transferred to other E. coli strains that still have their restriction enzymes.
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A n sw e r s t o Th o u g h t Q u e st i ons
7.10
PCR is a powerful technique, but one can easily contaminate a sample and amplify the wrong DNA— perhaps sending an innocent person to jail. What might you do to minimize this possibility? ANSWER: Use sterile, filtered micropipet tips. The filters prevent contamination of the micropipet and subsequent reactions. Also, perform a control PCR reaction without adding a DNA sample. If the water, buffers, or tubes are contaminated with a given DNA, a PCR product will appear even when DNA template has not been added deliberately to the reaction.
Chapter 8 8.1 If each sigma factor recognizes a different promoter, how does the cell manage to transcribe genes that respond to multiple stresses, each involving a different sigma factor? ANSWER: In these situations, a given gene will have multiple promoters. Each promoter will be recognized by a different sigma factor and will begin transcription at different distances from the ORF start codon.
8.2 With respect to two different sigma factors with different promoter recognition sequences, predict what would happen to the overall gene expression profile in the cell if one sigma factor were artificially overexpressed. Could there be a detrimental effect on growth? ANSWER: Since sigma factors compete for the same site on core polymerase, overexpressing one sigma factor could displace the other sigma factor from the RNA polymerase population and compromise expression of those target genes. If those genes were important to survival, the cell could die. 8.3 Why might some genes contain multiple promoters, each one specific for a different sigma factor? ANSWER: The gene might need to be expressed under multiple conditions at different levels. If a given condition increases expression of an alternate sigma factor, the target gene will need a promoter that the new sigma can recognize. As the need disappears and the sigma factor diminishes in concentration, a promoter that uses the housekeeping sigma will be needed. For example, the gene for DnaK heat shock protein has promoters for RpoH (sigma-32) and sigma-70, the housekeeping sigma factor. The level of protein needed during normal growth is supplied by sigma-70. Upon encountering heat stress, the RpoH sigma factor level increases and mediates an increase in DnaK production. 8.4 How might the redundancy of the genetic code be used to establish evolutionary relationships? ANSWER: The codon preferences of different microorganisms are based in part on their GC content. Thus, an organism with an AT-rich genome will preferentially use codons
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for a given amino acid that have As and Ts over those with Gs and Cs. Evolutionarity, finding a long AT-rich region encoding mRNA with AT bias within a chromosome that is otherwise GC-rich, suggests that the AT-rich region was horizontally inherited from some illicit DNA exchange with another species. (See Chapter 9.)
8.5 Synthesis of ribosomal RNAs and ribosomal proteins cause a major energy drain on the cell. How might the cell regulate synthesis of these molecules when growth rate slows as a result of amino acid limitation? ANSWER: As energy levels fall, fewer amino acids are made, which means that fewer charged tRNA molecules are assembled. This will cause ribosomes to pause during translation until finding the right charged tRNA. This pause causes synthesis of a signal molecule called guanosine tetraphosphate (ppGpp), which interacts with RNA polymerase and selectively stops transcription of the rRNA genes. This also stops new ribosome synthesis. As cells divide, the ribosomes will dilute to a lower steady-state level. Since less rRNA is made, fewer partners for ribosomal proteins are available and unassembled ribosomal proteins accumulate. These proteins bind sequences in their own mRNA molecules that are similar to the target sequences on rRNA. By binding to their mRNA, these ribosomal proteins can prevent their own translation. (See Chapter 10.) 8.6 While working as a member of a pharmaceutical company’s drug discovery team, you have found that a soil microbe snatched from the jungles of South America produces an antibiotic that will kill even the most deadly, drug-resistant form of Enterococcus faecalis, which is an important cause of heart valve vegetations in bacterial endocarditis.Your experiments indicate that the compound stops protein synthesis. How could you more precisely determine the antibiotic’s mode of action? ANSWER: One way is to take a culture of sensitive bacteria and isolate resistant mutants (bacteria that are not killed by the antibiotic), purify their ribosomes, and separate the 30S and 50S ribosomal subunits. Cross-mix subunits from sensitive and resistant cells (for example, mix 30S subunits from sensitive cells with 50S subunits from resistant cells). Then measure protein synthesis by the hybrid ribosomes with and without the drug. If resistance is due to an altered ribosomal protein or RNA, then the subunit mix containing the altered component will make protein regardless of whether the drug is present. Once identified, the responsible ribosomal subunits from resistant and sensitive cells can be broken down further into their component parts, reconstituted in hybrid form, and again tested for an ability to make protein in the presence of drug. This reductive approach will likely, but not always, uncover the target ribosomal protein or rRNA.
8.7 How might one gene code for two proteins with different amino acid sequences?
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By having two different translation start sites in different reading frames. While this is not a common occurrence, it happens.
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8.8 Why involve RNA in protein synthesis? Why not translate directly from DNA? ANSWER: Because transcription enables the cell to amplify the gene sequence information into multiple copies of RNA. Amplification means that more ribosomes can be engaged in translating the same protein, causing the concentration of the protein to rise more quickly than if only a single gene were used. The transcriptional process also presents an opportunity to regulate production of a protein. 8.9 Codon 45 of a 90-codon gene was changed into a translation stop codon. This produced a shortened, truncated protein. What kind of mutant cell could produce a full-length protein from the gene without removing the stop codon? ANSWER: A mutant in which a tRNA gene has been altered by mutation so that the anticondon of the tRNA “sees” the stop codon as an amino acid codon. This mutated tRNA molecule will transfer its amino acid to the peptide chain. The attached amino acid can be used to bridge the gap caused by the stop condon, and a full-length protein is made. These modified tRNAs are called suppressor tRNAs because they suppress the mutant phenotype. 8.10 An ORF 1,200 bp in length could encode a protein of what size and molecular weight? ANSWER: There are three bases per codon, so the ORF can encode 400 amino acids. The average molecular weight of an amino acid is 110 Da. Therefore, the hypothetical protein will be approximately 44,000 Da (44 kDa). Chapter 9 9.1 Transfer of an F factor from an F+ cell to an F– cell
converts the recipient to F+. Why does transfer of an Hfr not do the same? ANSWER: The last piece of an Hfr to transfer is the F factor and oriT. It is a rare event that an entire chromosome will transfer from one cell to another, so most Hfr transfers do not result in transfer of oriT and so cannot initiate conjugation.
9.2 Would it be easier to demonstrate generalized transduction using a temperate phage (which generates a lysogen) or a virulent phage? ANSWER: Both can be used, but more care must be taken with a virulent phage. The reason is that a lytic phage can kill a potential recipient through superinfection. Superinfection occurs when a normal phage particle and a transducing particle infect the same cell. The normal lytic phage will lyse the cell before a recombinant can be formed. Generalized trans-
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duction usually occurs more easily with a temperate phage (like P22 or P1) because a superinfecting temperate phage will usually just lysogenize the cell and not kill it. Therefore, the host DNA delivered by the transducing particle has time to recombine into the recipient genome. Lytic phage, like the variant of the lysogenic P1 phage called P1 vir, commonly used for E. coli transduction, can be used in the lab if care is taken to avoid superinfection. (The vir designation stands for virulent because the mutant P1 phage lost the ability to lysogenize.) If citrate is added moments after mixing P1 vir transducing lysate with a recipient, the ions needed for phage absorption are chelated, thereby limiting superinfection. In nature, where dilution of phage may be very great, there is less chance of superinfection and death. In this situation, transduction can easily be performed by lytic phage.
9.3 How do you think phage DNA containing restriction sites evade the restriction-modification screening systems of its host? ANSWER: Phage DNA will survive because sometimes the modification enzyme gets to the foreign DNA before the restriction enzyme. Once methylated, the phage DNA will be shielded from restriction and the methylated molecule will replicate unchallenged. If, on the other hand, a foreign DNA fragment (not necessarily a phage) has been modified and the conditions are right, it might recombine into the host chromosome and convey a new character to the strain.
9.4 Type I restriction-modification system genes are often designated hsdR, hsdM, and hsdS, which code, respectively, for the restriction (HsdR), modification (HsdM), and sequence recognition (HsdS) proteins. Can a plasmid grown in a wild-type strain be used to transform a strain defective in hsdR? An hsdS mutant? Can a plasmid grown in an hsdM mutant strain be transformed into an hsdR defective strain? ANSWER: A plasmid grown in a wild-type strain will be methylated, but methylated or not, it can survive in a restrictionless hsdR mutant or an hsdS mutant that lacks the recognition protein needed by both the restriction and modification subunits. Since an hsdM mutant lacks the modification protein, it cannot protect its genes from restriction enzymes and would commit suicide, so this last experiment cannot be done.
In a transductional cross between an A+B+C+ genotype donor and an A– B– C – genotype recipient, 100 A+ recombinants were selected. Of those 100, 15% were also B+, while 75% were C+. Is gene B or C closer to gene A? ANSWER: Gene C, because it was cotransduced with A at the highest frequency.
9.5
9.6 Calculate the mutation rate for a gene in which the number of mutations increases from 0 to 50 as cell number increases from 101 to 108.
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A n sw e r s t o Th o u g h t Q u e st i ons
One calculation that quickly estimates mutation rate is m [ln2/Nt ] where m = number of mutation in the culture and Nt = the number of cells in the culture. Because bacterial cultures grow logarithmically, ln2/Nt estimates the number of generations that went into making the culture. Thus: ANSWER:
50[ln2/(108 – 101)] = 50[0.69/(108 – 101)] = 50[0.69/approx. 108] = 50/1.44(108) = mutation rate mutation rate approx. equals 3.47 × 10–7
9.7 It has been reported that hypermutable bacterial strains are overrepresented in clinical isolates. Out of 500 isolates of Haemophilus influenzae, for example, 2–3% were mutator strains having mutation rates 100–1,000 times higher than lab reference strains. Why might mutator strains be beneficial to pathogens?
ANSWER: The mutator strains may speed microbial evolution, which could help the microbe outwit the immune system or escape the effects of administered antibiotics.
9.8 Diagram how a composite transposon like Tn10 can generate inversions or deletions in target DNA during transposition. Hint: This occurs when the transposon tries to jump from one site to another in the same chromosome using the ends of the inverted repeats closest to the tetracycline resistance gene (see Fig. 9.35). If you draw it right, you will see that in the end, the tetracycline gene is lost. ANSWER: From the diagram below you can see that inversions or deletions occur when the inner rather than outer ends of the inverted repeats are the targets of the transposase. The tetracycline resistance gene is lost, and the chromosome will contain a deletion or inversion depending on the orientation of the chromosome (looped or unlooped) at the time of transposase action.
9.9 The heat-shock gene dnaK in E. coli encodes a chaperone (Hsp70), homologs of which are found in all three domains of life. However, there is no other phylogenetic DIAGRAM FOR ANSWER TO THOUGHT QUESTION 9.8 evidence that a dnaK-like gene Deletion Inversion was present in the most recent z a b c d z a b c d tet tet common ancestor of all organisms. For instance, some speTarget site Target site cies of archaea do not encode a dnaK homolog, while other a z a z tet tet archaeal species do. In fact, molecular phylogenies indicate that the archaeal homologs are d quite closely related to those b of bacteria. It seems that the c d c Target site b most recent common ancestor of all dnaK (or hsp70) genes Target site existed more recently than the common ancestor of all organtet z isms. That being the case how z a tet do you suppose hsp70 genes d a arose in archaea? b ANSWER: The gene is thought to have been moved by some type of horizontal gene transfer mechanism from domain Bacteria to some members of domain Archaea.
d
c
c b z d
d
z
a
b
Chapter 10 10.1 If the gene lacZ has
a c
c
b
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c
b
a
d
a nonsense mutation in its open reading frame, will lacY be translated?
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LacY mRNA will still be translated because each gene in this polycistronic mRNA (lacZYA) has its own ribosome binding site and the lacY ribosome binding site is functional. A transitional stop codon in an upstream gene does not usually affect transcription of the downstream RNA.
ANSWER:
10.2 Null mutations completely eliminate the function of a mutated gene. Predict the effect of the following null mutations on the induction of β-galactosidase by lactose and predict whether the lacZ gene is expressed at high or low levels: lacI, lacO, lacP, crp, cya (the gene encoding adenylate cyclase). Also, what effect will those mutations have on catabolite repression? ANSWER: Loss of LacI repressor will lead to constitutive expression of lacZ but will not affect catabolite repression. A lacO mutant will not bind LacI repressor, so the phenotype will mimic that of a lacI mutation. A lacP mutation will prevent expression of lacZYA because RNA polymerase will not bind. Mutations in crp or cya will prevent catabolite repression, but lacZYA induction by lactose will be normal. However, without the cAMP-CRP complex, expression can never achieve maximal levels. 10.3 Predict what will happen to the expression of lacZ under the following partial diploid conditions (see Section 9.1). The genotypes of these strains are presented as follows: chromosomal genes/plasmid gene. (a) lacI lacO+P+Z+Y+A+/plasmid lacI+; (b) lacO lacI+P+Z+Y+A+/ plasmid lacO+; (c) crp lac I+O+P+ Z+Y+A+/plasmid crp+. + ANSWER: (a) The lacI gene on the plasmid will produce LacI repressor protein that can diffuse through the cytoplasm, bind chromosomal lacO, and repress the lacZYA operon. Because the complementing gene is on a different DNA molecule than the mutation, the gene is said to work in trans. (b) Because the lacO gene does not produce a diffusible product (e.g., protein or RNA), the plasmid lacO+ cannot complement a lacO mutation in trans, the strain will not make β-galactosidase. Thus, the lacO gene only functions in cis, that is, when it resides next to the gene it regulates. (c) The crp gene produces a diffusible protein product, so it can function in trans and complement a crp mutation. The strain will make β-galactosidase to the highest level, in the presence of inducer lactose. 10.4 Researchers often use isopropyl-β-Dthiogalactopyranoside (IPTG) rather than lactose to induce the lacZYA operon. IPTG structure resembles lactose, which is why it can interact with the LacI repressor, but it is not degraded by β-galactosidase. Why do you think the use of IPTG is preferred in these studies? ANSWER: There are at least two reasons. First, the level of IPTG inducer will not change, but the level of lactose inducer will continually decrease as it is consumed. This can affect the kinetics of induction. Second, the act of degrading lactose produces glucose and galactose. Glucose, as the
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preferred carbon source, will catabolite repress the lacZYA operon, once again affecting the kinetics of induction.
10.5 Predict the phenotype of a spoIIAA mutant that completely lacks this anti-anti-sigma factor (see Fig. 10.16). ANSWER: The spoIIAA mutant will have no way to efficiently remove anti-sigma F factor from sigma F. As a result, sigma F will almost never be active, even in the forespore. The cell can get as far as asymmetrical cell division to make what would be the forespore but since sigma F and antisigma F equally distribute in both compartments, sigma F will not be activated. 10.6
What is the phenotype of a fljA mutant? A fliC
mutant? A fljA mutant lacks the repressor needed to turn off FliC. Thus, a cell will switch back and forth from making H2 flagellin to making both H1 and H2 flagellin. A fliC mutant, however, will switch from being motile to nonmotile. In one orientation, the invertible element will allow H2 flagellin to be made, but in the opposite orientation, no flagellin will be made, at which point the cell will not be motile.
ANSWER:
10.7 The plaques of normal phage lambda appear as clear rings with cloudy centers of lysogenized cells. What do you think the plaque will look like in a cI mutant? A cII mutant? A cro mutant? ANSWER: Both the cI and cII mutant plaques will be completely clear (no cloudy center) because without CI, the virus can only go down the lysis pathway. A cro mutant may not even make a plaque, since Cro is needed to stop CI transcription and allow lysis. 10.8 Predict why UV irradiation can activate lambda prophage. Hint: What other host system is activated by UV? The RecA coprotease activated by the ssDNA molecules resulting from excessive UV irradiation not only stimulates autocleavage of the LexA repressor that controls the SOS response but also facilitates autocleavage of the lambda CI repressor. This allows transcription from lambda promoters PL and PR and sends the integrated prophage down the lytic path.
ANSWER:
10.9 Predict the phenotype of a glnB mutant. Will it be a glutamine auxotroph or not? What about a glnD or a glnE mutant? A glnB mutant will not dephosphorylate NtrB or remove AMP from GlnA. The cell will continue to make GlnA and fail to inactivate it. Thus, the cell will overproduce glutamine. A glnD mutant, however, will not add UMP to GlnB, so GlnB will not direct GlnE to remove AMP from GlnA (remember, GlnA without AMP is active). As a result, GlnA will remain inactive and the cell will likely require glutamine. The same will occur with a glnE mutant. ANSWER:
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10.10 Genes encoding luciferase can be used as “reporters.” What do you think would happen if the promoter for an SOS response gene were fused to the luciferase open reading frame? ANSWER: It could serve as a real-time biosensor for an environmental stress response. You could fuse the luciferase gene to the recA promoter and examine a real-time increase in fluorescence after ultraviolet irradiation by inserting the whole culture into a spectrofluorometer. The opening figure in Chapter 9 illustrates this strategy, using green fluorescent protein rather than luciferase. 10.11 What do you think would happen if a culture were coinoculated with a V. fischeri luxI mutant and a luxA mutant, neither of which produces light? The luxI mutant would glow. The luxA mutant would still make autoinducer as it grew. This autoinducer would accumulate in the culture medium, then diffuse and enter the luxI mutant cells, where it would trigger induction of the lux operon and production of luciferase.
ANSWER:
10.12 Why might the mass of a protein excised from a gel fail to match any of the ORF molecular weights predicted by the genome sequence? ANSWER: The protein might be altered by phosphorylation, acetylation, removal of a leader sequence, or otherwise proteolytically processed. Identification might still be made if certain unmodified fragments of the protein match up with predicted fragments. Chapter 11 11.1 Why would phage T4 production require a tenfold higher rate of DNA replication than does the host cell? Why would the phage substitute all of its cytosine with an unusual base that requires greater energy to synthesize? ANSWER: The phage T4 genome needs to replicate itself and generate progeny as fast as possible, in order to use as much of the host cell’s resources as possible before the cell deteriorates. By replacing cytosine with a modified base, the phage can avoid degradation of its chromosome by its own endonucleases (which cleave host DNA) or by those of the host (which are made to cleave phage DNA).
11.2 The phage T4 chromosome is a linear piece of DNA; yet the genomic map of T4 is circular. Why? ANSWER: Each headpiece contains just enough space to package slightly more than the length of a genome. The head-filling mechanism of DNA packaging means that successive genomes on a concatemer each get cut at different positions within the genome sequence. Thus, any map of gene order based on recombination linkages between genes, or based on the sequencing of a population of genome molecules, will generate a circular map.
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11.3 Which numbered genes from Figure 11.8 might be responsible for each of the defective phage populations presented in Figure 11.9D? ANSWER: Mutant 1 may have a defect in a gene whose product is needed for tail fiber attachment, such as gene 63. Mutant 2 may be defective for tail fiber assembly (genes 34 or 57). Mutant 3 could be defective for sheath assembly around the internal tube (gene 18). 11.4 Compare and contrast the reproductive strategies of phages T4 and M13. ANSWER: Both species of phage aim to maximize production of progeny and sustain an unbroken chain of infection. Both recognize and attach to proteins of the cell envelope that are essential for host cell structure, and both are specific for E. coli; T4 attaches to OmpC, whereas M13 attaches to the F pilus. T4 injects its DNA only, discarding its capsid, whereas M13 injects its DNA and recycles its coat proteins into progeny phage. T4 immediately destroys its host genome and reshapes its host’s biosynthetic apparatus to accelerate production of phage, whereas M13 moderates its rate of production so that the host will continue to grow and acquire nutrients, perhaps generating more phage in the long run. Most of T4 assembly occurs in the cytoplasm, whereas most of M13 assembly occurs in the cell membrane. T4 produces an enzyme that lyses the host to liberate all phage progeny, whereas M13 exits through a capped pore that maintains the integrity of the host, enabling gradual sustained production of phage. 11.5 Why does influenza virus have to provide its polymerase ready-made, whereas poliovirus does not? ANSWER: The influenza viral chromosome is (–) strand RNA. When it enters the host cytoplasm, it cannot be translated by ribosomes, and no host polymerase makes RNA from RNA. Therefore, the virion must provide an RNAdependent RNA polymerase to generate a (+) strand RNA for translation to proteins. Poliovirus, however, injects (+) strand RNA, which can immediately be translated by host ribosomes to generate RNA-dependent RNA polymerase. Explain why the expression (8!/88) approximates the proportion of infective particles of influenza virus. What assumption might be changed to make the proportion greater or less?
11.6
ANSWER: An infective particle can start by packaging any one of the eight chromosomes. Once the first is chosen, seven possibilities remain for the second, six for the third, and so forth. Thus there are 8! ways to obtain a perfect set out of 88 total ways to pick eight segments at random. This expression assumes, however, that exactly eight segments are packaged in every virion. If the average number of segments packaged is less than eight, the proportion of infectives falls off. If the number of segments is usually more than eight, the proportion of virions containing at least one of each segment increases.
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11.7
How do attachment and entry of HIV resemble attachment and entry of influenza virus? How do attachment and entry differ between these two viruses? ANSWER: Attachment and entry of HIV requires the envelope spike proteins to bind receptors in the host plasma membrane, just as the influenza envelope protein hemagglutinin binds the sialic acid protein in the host membrane. In both cases, initial receptor binding signals major rearrangement of a viral envelope protein so as to insert a fusion peptide into the host membrane. The processes differ between these two virus species, in that influenza virus induces formation of an endocytic vesicle, whose acidification triggers membrane fusion and release of the core contents into the host cytoplasm. HIV virions, however, do not induce endocytosis and do not require acidification to induce membrane fusion and release of the core into the cytoplasm.
11.8
Compare and contrast the priming of chromosome replication in HIV with the priming mechanisms of poliovirus and influenza virus. ANSWER: Poliovirus uses a viral protein (Vpg) to provide the 3’ OH needed to prime synthesis of RNA. In influenza virus, RNA synthesis is primed by “capped” fragments of host mRNA. Instead of host mRNA, HIV utilizes tRNA for primers.
11.9 Compare and contrast the fate of the HSV chromosome with that of the chromosome of HIV. ANSWER: HSV-1 contains a DNA chromosome, which is transported to the nuclear membrane within an intact capsid. HIV contains twin RNA chromosomes, which are released in the cytoplasm upon dissolution of the capsid. The RNA chromosomes are copied to double-stranded DNA for transport into the nucleus. In HSV-1, the DNA chromosome circularizes and generates concatemeric duplicates by rolling-circle replication. In HIV, the replicated DNA circularizes but immediately integrates into the host chromosome. In both cases, the viral DNA can persist for decades in latent infection.
Chapter 12 12.1 In this postgenomic era, how might you generally design a strategy to construct a strain of E. coli that cannot synthesize histidine? ANSWER: The genomic sequence of E. coli is known. We also know which genes encode the steps of histidine biosynthesis. The strategy today would be to engineer a plasmid containing DNA sequences (about 200 bp) that flank the histidine biosynthesis gene you want to delete, but in place of the target gene, you would instead insert an antibiotic resistance gene (e.g., kanamycin resistance). The plasmid used would
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be a “suicide” plasmid that cannot replicate in the strain you wish to mutate but which can replicate in other permissive strains (strains that produce a specific protein the plasmid needs to replicate). The permissive strain allows you to make a lot of the plasmid. The recombinant plasmid is then moved into the nonpermissive target strain by transformation, and the transformation mixture is plated on nutrient agar medium containing the antibiotic. Since the plasmid cannot replicate in this strain, the only way the target strain can become resistant to the antibiotic is if the chromosomal sequences flanking the drug marker in the plasmid undergo homologous recombination with the same DNA sequences on the actual chromosome. This double recombination event “surgically” removes the target gene from the chromosome and replaces it with the antibiotic resistance marker. In our example, the newly constructed mutant can grow in the presence of antibiotic, but only if histidine is present.
12.2 You have monitored expression of a gene fusion where lacZ is fused to the glutamate decarboxylase gene, gadA. You find that there is a 50-fold increase in expression of gadA (as measured for -galactosidase) when the cells carrying this fusion are grown in media at pH 5.5 as compared to cells grown at pH 8. What additional experiment must you do to tell if the control is transcriptional or translational? ANSWER: You must compare these results with those obtained with an operon fusion to the same gene. If both fusions show an equal 50-fold increase, then the gene is controlled at the transcriptional level. If the gene fusion shows a 50-fold increase in induction but the transcriptional fusion shows no induction, then regulation occurs postranscriptionally, most likely at the translational level.
12.3 What would you conclude if the Northern blot of the gad genes showed no difference in mRNA levels in cells grown at pH 7.7 and pH 5.5 but the Western blot showed more protein at pH 5.5 than at pH 7.7? Regulatory control would likely be at the posttranscriptional level, either through increasing mRNA stability, translational efficiency, or protein stability.
ANSWER:
12.4 Another regulator of glutamate decarboxylase (Gad) production does not affect the production of gad mRNA but is required to accumulate Gad protein. What two regulatory mechanisms might account for this phenotype? ANSWER: Control of translation or control of protein degradation. 12.5 How could you use two-hybrid analysis to determine if three proteins can interact? You suspect that proteins A and C can simultaneously bind to protein B and that this interaction then allows A to interact with C in the complex. ANSWER: Place three plasmids in the cell. One makes protein A fused to the Gal4 DNA-binding domain, the second plasmid makes protein C fused to the Gal4 activator domain,
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and the third plasmid simply makes protein B. If the yeast cell only contains the first two plasmids, no interaction will occur and activation of lacZ will not take place. If all three plasmids are in the same yeast cell, protein B will be the center of a sandwich, linking the A and C fusion proteins. The fusion proteins can then interact and activate lacZ.
12.6 How would you have to modify real-time PCR to quantitate the level of a specific mRNA? ANSWER: You would have to first convert the mRNA into cDNA using the enzyme reverse transcriptase. An oligonucleotide primer hybridizing to the to the 3’ end of the mRNA is acted on by reverse transcriptase to synthesize DNA (called complementary DNA, or cDNA). Normal real-time PCR techniques can then quantitate the cDNA. 12.7
You have just employed phage display technology to select for a protein that tightly binds and blocks the eukaryotic cell receptor targeted by anthrax toxin. When this receptor is blocked, the toxin cannot get into the target cell. You suspect that the protein may be a useful treatment for anthrax. How would you recover the gene following phage display and then express and purify the protein product? ANSWER: The gene can be cut out of the phage genome using restriction enzymes or amplified by PCR using known phage DNA sequences that flank the gene. The fragment can then be cloned into a His6 tag expression vector. This requires that the sequence of the gene be known so that the DNA encoding the His6 tag can be placed in-frame with the open reading frame of the receptor-blocking protein. The plasmid containing the His6-tagged protein gene is then induced to overexpress the protein in E. coli. The vector will contain an inducible promoter (e.g., lacP) to drive expression of the gene. The His6-tagged protein can then be purified by pouring cell extracts over a nickel column, as described in the text.
12.8 Would insect resistance to an insecticidal protein be a concern when developing a transgenic plant? Why? How would you design a transgenic plant to limit the possibility of insects developing resistance? ANSWER: Insects have, in fact, developed resistance to single insecticidal proteins. The most common resistance mechanism involves a change in the membrane receptors in the midgut to which activated Bt toxins bind. Resistance can be due to a reduced number of Bt toxin receptors or to a reduced affinity of the receptor for the toxin. Some insects, such as the spruce budworm, can inactivate specific toxins by precipitating them with a protein complex present in the midgut. While developing a transgenic plant, several steps could be taken to limit the development of resistance in an insect population. The insecticidal gene could be fused to a promoter that is only expressed at a time when the plant is most susceptible to attack. Alternatively, the gene could be fused to a promoter that is only expressed in a tissue of the
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plant that is most vulnerable to attack. This would limit the time during which the insects can develop resistance. For instance, cotton plants attacked by bollworms could produce toxin only in young boll tissues, the most important part of the plant. In addition to specifically protecting the critical plant tissue, this strategy would only affect one generation of bollworms, avoiding the constant selection pressure that hastens evolution of resistance. Another technique would be to engineer two different insecticidal proteins into the plant genome that will not exhibit cross-resistance. In other words, even if an insect develops resistance to one toxin, it will still remain susceptible to the second.
Chapter 13 13.1 Why can’t
photosynthesis be driven by solar microwaves or by X-rays? ANSWER: The energy of microwaves is a hundred times lower than that of visible light and near infrared. Microwaves have too little energy to form or break chemical bonds. On the other hand, photons of higher energy such as X-rays break molecular bonds indiscriminately, in a manner that enzymes cannot control.
13.2 Methanogens are a small proportion of the microbial community in soil, and the ∆G of methanogenesis is small. Yet methanogens produce large volumes of methane, large enough to contribute significantly as a greenhouse gas to global warming. Why would methanogens produce a relatively large quantity of waste product? ANSWER: For methanogenesis, the ∆G per reaction cycle is small. Thus, the organism must run numerous cycles in order to store sufficient energy to build biomass. 13.3 The soil bacterium Geobacter can metabolize acetic acid by the following reaction: CH3COOH + 2H2O → 2CO2 + 4H2 Yet the ∆G°′ is +95 kJ/mol. What could happen in the soil enabling Geobacter to grow? ANSWER: Geobacter must grow in the presence of different species of bacteria that oxidize the H2 produced by Geobacter. Removal of H2 drives forward the acetate catabolism. The sum of the acetate reaction plus the hydrogen oxidation yields a negative ∆G°′. This joint metabolism of two species is called syntrophy. The rate of reaction in this syntrophy is further enhanced at high temperature, which magnifies the term –∆RT ln K.
13.4 After ATP has been dephosphorylated to ADP, how else might ADP provide energy for the cell? ANSWER: Within the cell, some enzymes can hydrolyze ADP to AMP, breaking the second high-energy phosphodiester bond.
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13.5
Linking an amino acid to its cognate tRNA is driven by ATP hydrolysis to AMP plus pyrophosphate. Why release PPi instead of Pi? ANSWER: The formation of aminoacyl-tRNA must be irreversible until the ribosome is ready to release the tRNA. The pyrophosphate from ATP is immediately cleaved to 2Pi , preventing the reversal of aminoacyl-tRNA formation.
13.6 In the microbial community of the bovine rumen, the actual ∆G value has been calculated for glucose fermentation to acetate: C6H12O6 + 2H2O → 2C2H3O2– + 2H+ + 4H2 + 2CO2 ∆G = –318 kJ/mol If the actual ∆G for ATP formation is 44 kJ/mol and each glucose fermentation yields four molecules of ATP, what is the thermodynamic efficiency of energy gain? Where does the lost energy go? ANSWER: The energy efficiency is (4 × 44 kJ/mol)/ (318 kJ/mol) × 100 = 55%. The remaining energy is dissipated as heat. (Kohn and Boston, 2000.)
13.7 What would happen to the cell if pyruvate kinase catalyzed PEP conversion to pyruvate but failed to couple this reaction to ATP production? ANSWER: If PEP conversion to pyruvate occurred without ATP production, then the energy liberated by this reaction would be lost as heat. No energy would be channeled into cell function and growth. 13.8 Some bacteria make an enzyme, dihydroxyacetone kinase, that phosphorylates dihydroxyacetone to dihydroxyacetone phosphate. Why would this enzyme be useful? ANSWER: Bacteria can obtain dihydroxyacetone from their environment, using a transporter protein, then phosphorylate the substrate and direct it into glycolysis. Some bacteria can grow on dihydroxyacetone as a sole carbon source.
before it splits in two. The phosphorylated end yields glyceraldehyde 3-phosphate, which enters the EMP pathway to generate ATP, ending up as pyruvate. The unphosphorylated three-carbon unit yields pyruvate directly, with no ATP.
13.11 How does the structure of coenzyme A resemble that of NADH? How does it differ? ANSWER: Coenzyme A and NADH each consist of ADP esterified to a nitrogenous compound. The linked compound for coenzyme A, however, is not nicotinamide (as in NADH) but a linear molecule terminating in a thiol (SH). 13.12 Compare the reactions catalyzed by pyruvate dehydrogenase and pyruvate formate lyase (see Section 13.4). What conditions favor each reaction, and why? ANSWER: The pyruvate dehydrogenase complex (PDC) is favored in the presence of oxygen because the electrons transferred to NADH can enter the electron transport chain, eventually combining with oxygen to release energy. In the absence of oxygen, pyruvate formate lyase is favored to yield fermentation products that can be excreted from the cell without reducing more energy carriers. At high pH, formate and acetate production is especially favorable because the extra acid counteracts alkalinity. Suppose a cell is pulse-labeled with 14C-acetate (fed the C label briefly, then “chased” with unlabeled acetate). Can you predict what will happen to the level of radioactivity observed in isolated TCA intermediates? Plot a curve showing your predicted level of radioactivity, as a function of number of rounds of the cycle. ANSWER: The amount of radioactivity measured in TCA intermediates will rise steeply as labeled acetate is incorporated, then will decrease by half with each succeeding cycle, as the order of the carbons is randomized by succinate.
13.13
14
14
C in TCA intermediates
13.9
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C label in TCA intermediates
13.10 Explain why the ED pathway generates only one ATP, whereas the EMP pathway generates two. ANSWER: The EMP pathway primes the six-carbon sugar with two phosphoryl groups. The sugar then splits into two three-carbon units (glyceraldehyde 3-phosphate), each of which generates two ATPs for one of the original ATPs. By contrast, the ED pathway only phosphorylates the sugar once
1
14
In the EMP pathway, how are the two water molecules generated? ANSWER: Each water molecule is removed from 2-phosphoglycerate, converting R⫺CH2OH to R⫽CH2. The water molecule accounts for the water equivalent from incorporation of Pi during oxidation by NAD+. The Pi ultimately is used to convert one ADP to ATP, a reaction that includes water formation.
0.8
0.6
0.4
0.2 0 0
2 4 Rounds of TCA cycle
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13.14 How might the enzymes that catalyze acenaphthene catabolism be discovered? ANSWER: A genomic library of Pseudomonas strain A2279 might be used to transform another species that lacks the ability to degrade acenaphthene. If a transformant capable of degradation is found, the genes can be mapped and the deduced amino acid sequences compared with known enzymes to provide clues as to their mode of action. Other genes in the same operons might be found to catalyze other steps in the catabolic pathways. Chapter 14 14.1 Pseudomonas fluorescens, a soil bacterium at neu-
tral pH, oxidizes NADH with nitrate (NO3–) at the neutral pH. What is the value of E°′? ANSWER: To calculate the reduction potential E°′: NADH + H+ + NO3– → NAD+ + NO2– + H2O E°′ = 320 mV + 420 mV = 740 mV
14.2 Could a bacterium obtain energy from succinate as an electron donor with nitrate (NO3–) as an electron acceptor? ANSWER: For nitrate reduction: Succinate + NO3– → fumarate + NO2– + H2O E°′ = –33 mV + 420 mV = 387 mV Although succinate is a relatively poor electron donor, nitrate is a strong electron acceptor. This reaction should provide energy for bacterial metabolism. What do you think happens to ∆ψ as the cell’s external pH increases or decreases? What happens to ∆p in bacteria growing at a pH two or three units above their internal pH? ANSWER: As external pH changes, ∆pH increases or decreases, affecting the magnitude of ∆p. Some bacteria, such a Bacillus species, regulate their electrical potential with counter ions in order to maintain overall ∆p at about 200 mV. Other species, such as E. coli, seem to tolerate a range of ∆p from near zero to 250 mV. Alkaliphiles growing at high pH appear to spend much of their ∆p to maintain the inverted ∆pH. How they stay energized is not understood.
14.3
14.4 Suppose that de-energized cells (∆p = 0) with an internal pH 7.6 are placed in a solution at pH 6. What do you predict will happen to the cell’s flagella? What does this demonstrate about the function of ∆p? ANSWER: The flagella will rotate, driven solely by the ∆pH component of ∆p. This result is consistent with the hypothesis that the trans-membrane potential ∆p drives flagellar rotation.
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14.5 What is the advantage of the oxidoreductase transferring electrons to a pool of mobile quinones, which then reduce the terminal reductase (cytochrome complex)? Why does each oxidoreductase not interact directly with a cytochrome complex? ANSWER: The mobile quinone pool serves to connect diverse electron donors with diverse electron acceptors. If each oxidoreductase had to interact specifically with a different terminal oxidase, the pathways of electron transport would be limited; for example, NADH might only donate electrons to O2, whereas succinate might only donate electrons to nitrate. Instead, all potential electron donors can be coupled with all potential acceptors. 14.6 Why are most electron transport proteins fixed within the cell membrane? What would happen if they “got loose” in aqueous solution? ANSWER: If the electron transport proteins came away from the membrane into water solution, they could carry their energized electrons back into the cytoplasm or lose them outside the cell. In either case, they could no longer convert the electron energy to a proton gradient. 14.7 Why would bacteria evolve dehydrogenases that fail to pump protons even when sufficient donor potential exists? ANSWER: Dehydrogenases that fail to transport protons despite sufficient reduction potential are needed for cases in which reoxidation of the donor is required but the proton gradient is already high enough to drive generation of ATP. Too high a transmembrane voltage can disrupt the membrane. 14.8 Experiments suggest that NADH dehydrogenase 2 is more often coupled with cytochrome bo, and dehydrogenase 1 more often with cytochrome bd. Why might this be the case? ANSWER: The non-proton-pumping oxidoreductase NDH2 may be used to moderate the amount of proton gradient generated with abundant oxygen. The proton-pumping NDH-1 may be useful under low-oxygen conditions, where the cell is forced to use the high-affinity oxidase (cyt b/d), which fails to pump the extra 2H+.
14.9 How might the bacterial cell compensate for environmental conditions in which ∆p falls below 100 mV, a suboptimal level for ATP synthesis? To generate sufficient ATP when ∆p is low, the bacteria could synthesize a greater number of F1Fo complexes for the membrane. The overall rate of ATP synthesis would thus be increased. ANSWER:
14.10 Would E. coli be able to grow in the presence of an uncoupler that eliminates the proton potential supporting ATP synthesis?
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ANSWER: Yes, E. coli can grow with the proton gradient eliminated, but only with a rich supply of nutrients for substrate phosphorylation to generate ATP (for example, from glycolysis). Also, the external pH and salt levels must be maintained close to those of the cytoplasm, to minimize the need for ion transport.
14.11 The proposed scheme for uranium removal requires injection of acetate under highly anoxic conditions, with less than 1 part per million (ppm) dissolved oxygen. Why must the acetate be anoxic? ANSWER: Oxygen is the strongest terminal electron acceptor. If O2 is present, bacteria will use it preferentially (instead of U6+) to oxidize the acetate to CO2. 14.12 Hydrogen gas is so light that it rapidly escapes from Earth. Where does all the hydrogen come from to be used for hydrogenotrophy and methanogenesis? ANSWER: Hydrogen is produced in substantial quantities as a by-product of fermentation. It may seem surprising that organisms would readily excrete quantities of energy-rich H2, but in the absence of a good electron acceptor (or the enzymes to utilize electron acceptors), hydrogen may be just another waste product. Hydrogen gas trapped underground supports large communitites of methanogens and hydrogenotrophs. The human colonic bacteria generate so much hydrogen that all parts of the body show traces of hydrogen gas. 14.13 Suppose you discover bacteria that require a high concentration of Fe2+ for photosynthesis. Can you hypothesize what the role of Fe2+ may be? How would you test your hypothesis? ANSWER: The organism uses reduced iron as an electron donor for its photosystem (Fe2+ → Fe3+). To test this, grow the organism on a defined concentration of Fe2+. Measure the amount of iron oxidized and the amount of carbon fixed into biomass; if the Fe2+ is an electron donor for the photosystem, the two numbers should show a linear correlation. Chapter 15 15.1 Propose a
simple experiment to reveal the key intermediate to receive CO2. 14 ANSWER: Add a very short pulse of [ C] CO2 to the cells. The pulse must contain sufficient CO2 to begin fixation, but insufficient to continue beyond one “turn” of the cycle. In this case, the radiolabel will accumulate in the intermediate that needs to assimilate CO2 for the next round. This kind of experiment ultimately identified the key intermediate, ribulose 1,5-bisphosphate.
15.2 Speculate why rubisco catalyzes a competing reaction with oxygen. Why might researchers be unsuccessful in attempting to engineer rubisco without this reaction?
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The oxygenation reaction might have an essential function in regulation of metabolism. For example, it might help prevent excessive reduction of cell components or fixation of too much carbon to be used in biosynthesis. Given the universal existence of the oxygenation reaction in bacterial and chloroplast rubiscos, it seems unlikely that oxygenation serves no purpose. For this reason, the attempt to engineer rubisco without oxygenation may not succeed.
ANSWER:
15.3 Why does ribulose 1,5-bisphosphate have to contain two phosphoryl groups, whereas the other intermediates of the Calvin cycle contain only one? ANSWER: Only ribulose 1,5-bisphosphate needs to split into two molecules (3-phosphoglycerate). Each of the two products needs to have its own phosphate as a tag for the enzymes to recognize it within the cycle. 15.4 Which catabolic pathway (see Chapter 13) includes some of the same sugar-phosphate intermediates as those of the Calvin cycle? What might this suggest about the evolution of the two pathways? The pentose phosphate pathway includes ribulose 5-phosphate, erythrose 4-phosphate, and sedoheptulose 7-phosphate in a similar series of carbon exchanges. Perhaps these pathways evolved from a common amphibolic pathway of sugar consumption and biosynthesis. Alternatively, the one pathway evolved earlier, then the sugar intermediates were available for evolution of the second pathway. ANSWER:
15.5 For a given species, uniform thickness of a cell membrane requires uniform chain length of its fatty acids. How do you think chain length may be regulated? ANSWER: In E. coli, the chain length of a growing fatty acid appears to be limited by beta-ketoacyl-ACP synthase, which binds only precursor acyl-ACPs shorter than 18 carbons. 15.6 Suggest two reasons why transamination is advantageous to cells. ANSWER: Transamination enables cells to store amine groups in nontoxic form, readily available for biosynthesis. The availability of multiple enzymes of transamination from different amino acids enables cells to quickly recycle existing resources into the amino acids most needed by the cell in a given environment. For example, if a sudden supply of glutamine appears, cells can immediately distribute its amines into all twenty amino acids. 15.7 Which energy carriers (and how many) are needed to make arginine from 2-oxoglutarate? (See Fig. 15.24.) ANSWER: Arginine biosynthesis requires three ATP molecules and three NADPH molecules (including two for converting two molecules of 2-oxoglutarate to glutamate). An additional ATP is spent converting acetate to acetyl-CoA.
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15.8
Why are purines and pyrimidines not synthesized separately from the sugar phosphate? ANSWER: Purines and pyrimidines are highly hydrophobic, insoluble in the cytoplasm. The ribose phosphate component solubilizes the molecule, enabling synthesis to occur in the cytoplasm, where needed to make RNA and DNA.
15.9
Why are the ribosyl nucleotides synthesized first, then converted to deoxyribonucleotides as necessary? What does this suggest about the evolution of nucleic acids? ANSWER: Ribonucleic acid is believed to be the original chromosomal material of cells. Cells evolved to synthesize RNA first; then later, as DNA was used, pathways evolved to synthesize it by modification of RNA, which the cell already had the ability to make.
Chapter 16 16.1 Why do the lipid components of food experience relatively little breakdown during fermentation? ANSWER: Lipids are highly reduced molecules, largely hydrocarbon with relatively low oxidizing potential. Thus, lipids cannot undergo as many intramolecular redox reactions as do sugars, which readily generate energy through anaerobic fermentation.
16.2 Why does oxygen allow excessive breakdown of food, compared with anaerobic processes? ANSWER: Oxygen functions as the terminal electron acceptor for the complete breakdown of all kinds of organic molecules to water and CO2.
The fermentation can be controlled by introducing a crude starter culture obtained from a previous batch of the food product or from a natural source of a particular microbe; for example, rice straw is a source of Bacillus natto for natto production. The fermentation type can be manipulated by the addition of factors, such as brine, that retard growth of all but a few strains. In pidan, for example, the high concentration of sodium hydroxide limits bacterial growth to alkali-tolerant strains of Bacillus.
ANSWER:
16.6 Compare and contrast the role of fermenting organisms in the production of cheese and bread. ANSWER: In cheese production, fermentation causes major biochemical changes in the food, such as the buildup of acids and the breakdown of proteins to smaller peptides and amino acids. Minor by-products, such as methanethiols and esters accumulate to levels that confer flavors. In yeast bread, by contrast, the only significant product of fermentation is the carbon dioxide that leavens the dough. The small amount of ethanol produced evaporates during cooking. A form of bread in which extended fermentation does generate flavor is injera, for which the dough ferments for three days. 16.7 Compare and contrast the role of lowconcentration by-products in the production of cheese and beer. ANSWER: In both cheese and beer, minor by-products such as esters contribute flavor. In both cases, oxidation leads to off-flavors. In cheese, however, the exclusion of oxygen usually prevents off-flavors. In beer, the yeast requires a low level of oxygen; thus, significant amounts of acetaldehyde and diacetyl are produced and must be eliminated by a secondary fermentation.
16.3 In an outbreak of listeriosis from unpasteurized cheese, only the refrigerated cheeses were found to cause disease. Why would this be the case? ANSWER: In the cheeses kept at room temperature, other naturally occurring bacteria outgrew the pathogenic Listeria, whereas in the refrigerator, only the Listeria could grow. (Note, however, that many other potential pathogens, such as Salmonella, are inhibited by refrigeration.)
16.8 Why would bacteria convert trimethylamine oxide (TMAO) to trimethylamine? Would this kind of spoilage be prevented by exclusion of oxygen?
16.4 Cow’s milk contains 4% lipid (butterfat). What happens to the lipid during cheese production? ANSWER: Lipids undergo little catabolism, because the fermentation conditions are anaerobic. During coagulation, lipid droplets become trapped in the network of denatured protein and are largely retained in the bulk of the cheese. “Low-fat” cheeses are made from skim milk, which eliminates the lipids before fermentation.
16.9 Is it possible for physical or chemical preservation methods to completely eliminate microbes from food? Explain. ANSWER: Preservation methods either slow microbial growth or induce microbial death. Microbial death follows a negative exponential curve, as discussed in Chapter 5. In theory, the exponential curve never reaches zero, so total exclusion of microbes is impossible. In practice, there is a high probability of totally eliminating microbes if the treatment time extends several “half-lives” beyond the time at which microbial concentration declines to less than one per total volume.
16.5 In traditional fermented foods, without pure starter cultures, what determines the kind of fermentation that occurs?
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TMAO acts as a terminal electron acceptor—that is, an alternative to oxygen for anaerobic respiration, as discussed in Chapter 14. Exclusion of oxygen inhibits only aerobic bacteria; TMAO respirers continue to grow and can spoil the fish.
ANSWER:
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16.10
Why would different industrial strains or species be used to express different kinds of cloned products? ANSWER: Different industrial strains have biochemical systems that favor different products. Some fungi naturally possess the highly complex pathways to generate antibiotics, as well as regulatory timing to turn on these pathways after the culture has grown to high population density. On the other hand, bacteria such as B. subtilis are the most genetically tractable and predictable in their growth cycles and the easiest to manipulate to express recombinant products such as human genes.
16.11 Why would an herbicide resistance gene be desirable in an agricultural plant? What long-term problems might be caused by microbial transfer of herbicide resistance genes into plant genomes? Introduction of an herbicide resistance gene allows application of higher amounts of herbicide to crops in order to control growth of weeds. But the higher concentrations of herbicide may also have greater side effects on animals and on human consumers of the crop. In the long run, the herbicide resistance gene is likely to escape into weed plants through natural genetic transfer mechanisms. Thus, eventually the weeds may require still higher concentrations of herbicide. While the costs versus benefits of new gene modifications remain poorly understood, it must be recognized that all modern crops today are the product of many generations of genetic manipulation.
ANSWER:
Chapter 17 17.1 What would have happened to life on Earth if the sun were a different stellar class, substantially hotter or colder than it is now? ANSWER: If the sun were hotter, too much ultraviolet and gamma radiation would reach the Earth, breaking chemical bonds of living organisms so rapidly that life could not be sustained. If the sun were colder, too little radiation with sufficient energy would be available to drive photosynthesis. In either case, life as we know it could not have evolved on Earth.
17.2 Outline the strengths and limitations of each model of the origin of living cells. Which aspects of living cells does each model explain? ANSWER: The three models are complementary in that each explains aspects of modern cells not addressed by the others. The prebiotic soup model accounts for the major classes of compounds used by cells, such as nucleosides, TCA cycle intermediates, amino acids, and fatty acids. It also suggests the origin of membranes as soap bubble-like micelles. It does not, however, account for the evolution of metabolic pathways and replication of genetic informa-
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tion. The metabolist model accounts for the prevalence of major cellular reactions such as carbon fixation and the TCA cycle. It does not explain the evolution of membranes and genetic material. The RNA world accounts for the central role of RNA in living cells; of all molecular classes, RNA and ribonucleotides probably serve the widest range of functions as information carriers, agents of catalysis, and genetic regulators. The origin of membranes is not addressed.
17.3 Suppose living organisms were to be found on Mars. How might such a find shed light on the origin and evolution of life on Earth? ANSWER: If life on Mars were to show a completely different basis than that of Earth—for example, based on silicon polymers instead of carbon—such a find would support the view that life originated independently on each planet, rather than traveling from one planet to the other; or that both planets were seeded from somewhere else. If life on Mars is based on similar macromolecules, perhaps even showing the same genetic code, this would support the view that life arose on Mars first; or that both planets were seeded from the same source. 17.4 Based on Figure 17.18, what would be the identification of a straight, nonsheathed gram-negative bacterium that has sulfur granules, is motile, and is 1.0 µm wide? What would happen if you happened to assign the bacterium a width of 0.9 µm? ANSWER: The bacterium would key out as Beggioatoa sp. If the cell width were measured as 0.9 µm, however, the identification would proceed down a completely different, wrong track, at the first step of the key. This is one disadvantage of the dichotomous key. 17.5 In Figure 17.19B, were any test results atypical for Salmonella enterica? What would happen if another atypical result had been obtained? What would happen if the actual isolate had not been one of those in the database? ANSWER: The negative result for dulcitol fermentation is atypical. If you multiply the probabilities, for most of the tests a second atypical result will decrease the score for S. enterica by about an order of magnitude but will still leave it at the top of the list. Thus the probabilistic indicator is relatively tolerant of atypical results for species within the database. On the other hand, if an organism outside the database is isolated it cannot be correctly identified.
17.6
What kind of DNA sequence changes have no effect on gene function? ANSWER: Base substitutions that do not change the amino acid specified by the codon have no effect on gene function. For example, CUA → CUG still encodes leucine. Additionally, it turns out that a majority of the amino acids in any given protein can be replaced by an amino acid of
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similar form (for example leucine → valine) without significantly affecting function of the gene.
17.7 Solve the three alignment problems shown in Figure 17.22. How could errors or ambiguities in alignment lead to mistakes in interpreting the molecular clock? ANSWERS:
A. Add the new sequence from organism F to this alignment: Seq A
CCCCAGCUUCGGCUGGGGGAGG
Seq B
CCUUAGCGAAAGCU–AAGGAGG
Seq C
CCUCAGCGUGAGCU–GAGGAGG
Seq D
CCCAAGCUUU–GCUAUGGGAGG
Seq E
CC––AGCUUUGGCU–––GGAGG
Seq F
CC–AAGCGAGAGCU–UGG–AGG
B. Make an alignment of these RNAs: RNA 1 – G G G G G U U C G C C C C C A A – RNA 2 – G G G A G U U U G C U C C C A A – RNA 3 – G A G G G U U C G C C C U C A U – RNA 4 – G G G A G G A A A C U C C C A C A RNA 5 – G A G G G G U G A C U C U C A C A RNA 6 G G A G G G U U U – C C C U C A U –
C. Which group of Archaea does ‘Isolate X’ most likely belong to based on the RNA shown? ANSWER:
Position Number
Isolate X
Crenarchaea
Euryarchaea
Korarchaea
4 5 8 11 15 18 19 20 21 22
C A G G C U G A A G
(C or U) U A C R (A or G) U A A A G
C U G G C U G A A G
U A C G U C Y (C or U) U Y (C or U) A
Isolate X shows 9/10 key positions in common with the Euryarchaea, as compared with 5/10 Crenarchaea and 2/10 Korarchaea. Thus Isolate X most likely belongs to the Euryarchaean clade. Errors in alignment could lead to overestimate of the time of divergence between organisms. For example, if position 15 were aligned by counting bases in Isolate X without skipping the loop extension, base 15 could be given as G, which would lead to only 8/10 positions in common with Euryarchaea.
17.8 What are the major sources of error in constructing phylogenetic trees?
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Phylogenetic trees are affected by variability in the number of substitutions, or rate of mutation in different strains. The tree is distorted by errors in sequence alignment, and by systematic errors due to failure of the fundamental assumptions of the molecular clock. These assumptions include the constant rate of mutation for all branches; constant generation time; and true orthology of the gene chosen (that is, the encoded product has the same function and hence the same degree of selection pressure in all taxa under consideration).
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17.9 What are the limits of evidence for horizontal gene transfer in ancestral genomes? What alternative interpretation might be offered? ANSWER: Horizontal gene transfer is inferred based on the appearance of genes in clade A that are absent from other members of the clade but present in clade B. The degree of similarity between genes in the two clades, however, must be high enough to exclude the possibility that the genes in question were retained from a common ancestor of the two clades, but lost from other members of clade A. This possibility is difficult to exclude in the case of deep-branching clades, where all genes have had a long time to diverge. For example, the large number of archaean genes present in deep-branching thermophilic bacteria such as Thermatoga may include some inherited from the last common ancestor. 17.10
Besides mitochondria and chloroplasts, what other kinds of entities within cells might have evolved from endosymbionts? ANSWER: Some of the large“megaplasmids”found in bacteria and protists are as large as genomic chromosomes and contain numerous housekeeping genes. These megaplasmids may have originated as endosymbiotic cells that lost all their membranes through reductive evolution. Similarly, some of the giant viruses such as Mimivirus and smallpox, as well as phages such as T4, possess a wide spectrum of housekeeping genes. These viruses may have originated as cellular parasites that underwent reductive evolution.
Chapter 18 18.1 Which taxonomic
groups in Table 18.1 stain gram-positive and which gram-negative? Which group contains both gram-positive and gram-negative species? For which groups is the Gram stain undefined, and why? ANSWER: See Table 18.1. Most Firmicutes and Actinobacteria stain gram-positive, as do the Cyanobacteria. These bacteria have relatively thick cell walls that retain the stain. The Proteobacteria, Nitrospira, and Bacteroides/Chlorobi groups stain gram-negative. The Thermus/Deinococcus group includes both gram-positive and gram-negative staining members. For Chlamydia and Planctomyces, the
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Gram stain is irrelevant because they lack the cell wall that retains the stain. For Spirochetes, many species are too narrow to observe the stain under light microscopy.
18.2 Which groups of species share common structure and physiology within the group? Which groups show extreme structural and physiological diversity? ANSWER: Cyanobacteria all share oxygenic photosynthesis through thylakoid membranes. Their overall cell structure and organization, however, takes diverse forms. Spirochetes all share the common structure of sheathed flexible spiral with internal flagella; most share anaerobic or facultative heterotrophy. Chlamydia and Planctomyces groups of species each share general structural features. Other groups, particularly Firmicutes and Actinomycetes, show considerable diversity of form and physiology. The Proteobacteria display more extreme diversity of metabolism than any other division. 18.3 What taxonomic questions are raised by the apparent high rate of gene transfer between archaea and thermophilic bacteria? ANSWER: If gene transfer results in a species containing a quarter of its genes from organisms outside its domain, such a mosaic genome raises questions of how to define the species and the domain. How can a species be defined if its genome contains large portions from distantly related sources? Other interesting questions relate to the means of gene transfer. How do such distantly related organisms as bacteria and archaea maintain a compatible mechanism of gene transfer? 18.4 What are the relative advantages and disadvantages of propagation by hormogonia, as compared with akinetes? ANSWER: Hormogonia are motile, and thus capable of active chemotaxis toward a more favorable environment. On the other hand, hormogonia have active metabolism that requires nutrition; if the environment lacks nutrients, the hormogonia will die. Akinete cells can persist until environmental conditions improve, but they cannot actively seek out a new location. 18.5 What are the relative advantages and disadvantages of the different strategies for maintaining separation of nitrogen fixation and photosynthesis? ANSWER: Temporal separation has the advantage that all cells possess both capacities for nitrogen fixation and photosynthesis. On the other hand, it eliminates the ability of a chain of cells to conduct both processes simultaneously—the benefit of heterocysts. Heterocysts face the problem of operating in close proximity to photosynthetic cells generating toxic oxygen; this problem may be solved by associated respiring bacteria. Globular clusters of cells can bury their nitrogen fixers within the cluster;
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this arrangement effectively excludes oxygen, but it may lack flexibility during environmental change. Endosymbiotic nitrogen fixation within a respiring eukaryote is probably the most effective strategy of all, as the host provides oxygen-removing proteins such as leghemoglobin. Endosymbiosis, however, requires the presence of an appropriate host organism.
18.6 Why would Streptomyces produce antibiotics targeting other bacteria? ANSWER: Streptomyces species may produce antibiotics to curb the growth of bacterial competitors with smaller genomes and faster rates of reproduction. The lysed cells release nutrients that feed growing mycelia of Streptomyces. 18.7 Why might genes for the proteorhodopsin retinal photopump be more likely to transfer horizontally than the bacteriochlorophyll-based photosystems PS I and PS II? ANSWER: Proteorhodopsin requires only the one gene encoding the pump, plus one or two genes to produce retinal. This involves a relatively small amount of sequence to transfer, and the encoded products generate proton potential on their own, without requiring interaction with recipient enzymes. By contrast, PSI and PSII each involve multiple electron carriers that must function together and interact with the recipient electron transport chain.
18.8 Why do you think it took many years of study to realize that Escherichia coli and other Proteobacteria can grow as a biofilm? ANSWER: E. coli and its relatives grow exceptionally well in liquid culture. Liquid culture is attractive because it enables quantitative measurement of defined aliquots of microbial population. However, repeated subculturing in liquid medium selects for planktonic (non-biofilm) cells. Eventually, the biofilm-forming property may be lost if mutants outgrow the original genotype in liquid medium. 18.9 Compare and contrast the formation of cyanobacterial akinetes; firmicute endospores; actinomycete arthrospores; and myxococcal myxospores. ANSWER: An endospore forms as the daughter product (forespore) of a single cell. Within the same cell, endospore development is supported by the motherspore, which disintegrates after release of the endospore. Endospores have tough coatings of calcium picolinate; they are heatresistant. By contrast, arthrospores and myxospores are less durable and are not heat-resistant, although they can persist in the environment for an extended period. Arthrospores form through binary fission of actinomycete filaments. Myxospores are formed by a multicellular fruiting body. In all three cases, spore formation can be induced by depletion of nutrients; and the spore-producing entity is left behind to die.
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A n sw e r s t o Th o u g h t Q u e st i ons
Chapter 19 19.1 If two deeply diverging clades each show a wide range of growth temperature, what does this suggest about the evolution of thermophily or psychrophily? ANSWER: The two clades diverged before temperature adaptation occurred, and adaptations to high or low temperature must have evolved independently in the two clades.
19.2
What might be the advantages of flagellar motility for a hyperthermophile living in a thermal spring or in a black smoker vent? What would be the advantages of growth in a biofilm? ANSWER: Flagellar motility enables isolated cells to detect a new nutrient source, or an approximate temperature range, and approach it through chemotaxis. Growth in a biofilm attached to a substrate prevents the microbes from floating away from the nutrient source, or from being carried away in the flow from the vent.
19.3 What problem with cell biochemistry is faced by acidophiles that conduct heterotrophic metabolism? ANSWER: Heterotrophic metabolism generates fermentation products such as acetate and lactate, which act as permeant acids. Permeant acids become protonated outside the cell, at low pH; the protonated forms then permeate the membrane, returning into the cell. Given the high transmembrane pH difference maintained by Sulfolobus, one would expect even small traces of fermentation acids to cross the membrane in the protonated form, then dissociate and accumulate to toxic levels of organic acids. It is unknown how Sulfolobus solves this problem. 19.4
What conclusions might be drawn if viruses of mesophilic archaea are found to have RNA genomes? What if they all have DNA genomes only? If mesophilic viruses show RNA genomes, then it is likely that only double-stranded DNA is sufficiently stable for viruses to persist in the environment of hyperthermophiles. If only double-stranded DNA viruses are found throughout the archaea, this would suggest that all archaeal viruses evolved from viruses infecting a common ancestral cell that was a thermophile. The latter hypothesis would require supporting evidence from archaeal cell physiology and phylogeny.
ANSWER:
19.5 What do the multiple metal requirements suggest about the evolution of methanogens? ANSWER: The requirement for so many different metals may suggest that methanogens evolved in habitats such as geothermal vents where superheated water carries up high concentrations of dissolved metal ions. 19.6 Compare and contrast the metabolic options available for Pyrococcus and for the crenarchaeote Sulfolobus.
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ANSWER: Sulfolobus catabolizes sugars and amino acids aerobically, using O2 as terminal electron acceptor. P. abyssi catabolizes sugars and amino acids anaerobically, using S0 as terminal electron acceptor. Sulfolobus oxidizes sulfur lithotrophically, from S2– to S0, SO32–, and ultimately SO42–. P. abyssi, however, reduces sulfur lithotrophically with H2 to form H2S.
19.7 Compare and contrast sulfur metabolism in Pyrococcus and in Ferroplasma. 0 ANSWER: Pyrococcus species reduce S with hydrogens from – organic substrates, forming HS and H2S. Ferroplasma species oxidize sulfur in the form of FeS2 (using oxidant Fe3+) forming sulfuric acid. The result is extreme acidification of their environment.
Chapter 20 20.1 Why would yeasts remain unicellular? What are the relative advantages and limitations of hyphae? ANSWER: Yeast grow in environments with sufficient dissolved nutrients to absorb from the medium. The advantage of forming hyphae is that they enable penetration of other organisms, and hence provide access to nutrients. On the other hand, hyphae formation limits the rate of dispersal of progeny cells. Since yeasts grow in environments where dissolved nutrients can be absorbed from the medium, they do not need to produce hyphae and can proliferate more rapidly than mycelial fungi.
20.2 Why would some fungi have lost their sexual life cycle? What are the advantages and limitations of sexual reproduction? ANSWER: The sexual life cycle involves significant genetic and metabolic costs to the organism. Reductive evolution leading to loss of sexual reproduction might eliminate an energy drain and perhaps enable greater proliferation with fewer resources. On the other hand, the sexual life cycle provides a valuable means of generating diversity through genetic recombination, so that the population may respond to environmental change. Asexual fungi, like prokaryotes, must rely on mutation and gene transfer by viruses and mobile sequence elements to generate genetic diversity. 20.3 What are the advantages and limitations of motile gametes, as compared to non-motile spores? ANSWER: Motile gametes have the advantage of rapid dispersal on their own and the potential for chemotaxis toward a food source or toward a gamete of the opposite mating type. On the other hand, motility uses up energy that could alternatively be invested in production of a greater number of non-motile gametes. Motile gametes
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are especially useful in a watery habitat, but are little use in a terrestrial habitat, where air currents or animal hosts must be used for dispersal.
20.4
Compare the life cycle of an oomycete (Figure 20.17C) with that of a chytridiomycete (Figure 20.11C). How are they similar, and how do they differ? ANSWER: Both chytridiomycetes and oomycetes undergo alternation of generations. Each possesses the alternative route of an asexual diploid cycle between mycelium, motile zoospores and cysts. At the cellular and molecular level, however, the two kinds of organism differ radically. The chytridiomycete flagellum differs from the oomycete paired flagella, one of which has brush-like side hairs. The oomycete gametes have highly specialized forms that fuse by a process completely different from that of chytridiomycete zoospore fusion.
20.5
How are coralline algae able to grow at greater depths than coral? Corals generally grow symbiotically with green algae, which contain chlorophyll but lack accessory photopigments. Corallines are red algae, possessing both chlorophyll and phycoerytherins. The latter absorb blue and green light that are not absorbed by green algae, and thus coralline algae penetrate deeper in the water column.
ANSWER:
20.6 What might happen when an ameba phagocytoses algae? ANSWER: If light is available, the algae may be retained as endosymbionts providing energy through photosynthesis. For example, Chlorachnion possesses obligate chloroplastbearing endosymbionts descended from green algae. Alternatively, the ameba may digest all but the algal chloroplast, which persists for some time providing photosynthetic products. 20.7
What kind of habitats would favor a flagellated
ameba? ANSWER: A dilute watery habitat would favor flagella, which allow more rapid propulsion than pseudopods. Pseudopod motility requires a solid substrate.
20.8 Compare and contrast the processes of conjugation in ciliates and in bacteria. ANSWER: Conjugation in ciliates is a completely different process from conjugation in bacteria, although the purpose (genetic exchange) is similar. In ciliates, two cells form a bridge allowing cytoplasm to flow directly between them, whereas in bacteria, a donor cell attaches to another (by pili in some cases), then a protein complex transfers DNA across both cell envelopes, without direct cytoplasmic contact. In bacterial conjugation, DNA is transferred unidirectionally from the donor cell to the recipient, whereas in ciliates there is reciprocal exchange of DNA. A donor bac-
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terium generally transfers only part of its genome, whereas ciliates exchange entire copies of theirs.
20.9 For ciliates, what are the advantages and limitations of conjugation, as compared with gamete production? ANSWER: The process of conjugation avoids the necessity of dissolving the intricate cell structure of the ciliate in order to form gametes that fuse or fertilize each other. On the other hand, conjugation requires two diploid organisms to find each other and make contact for several hours, during which time feeding is suspended and the pair is vulnerable to predation.
Chapter 21 21.1 From Chapters
13–15, give examples of microbial metabolism that fit patterns of assimilation and dissimilation. ANSWER: Microbes can assimilate carbon either by reducing carbon dioxide or by oxidizing methane. Dissimilation of organic carbon occurs by fermentation and respiration. Nitrogen is assimilated by N2 fixation, and by incorporation of NH4+ into glutamine and glutamate. Nitrogen is dissimilated by deamination of amino acids, and by lithotrophic oxidation.
21.2 Does “viable but nonculturable” mean that growth is permanently shut down? ANSWER: Nonculturable bacteria might be induced to grow again, perhaps when the correct growth conditions are discovered. Some “unculturable” isolates have been made to grow by new methods after decades of attempts. Alternatively, some nonculturable cells may come from multicellular assemblages such as biofilms in which reproductive growth is limited to a portion of the cells. In this case, some of the population is permanently prevented from growth, but other cells continue to grow. 21.3 How do viruses select for increased diversity of microbial plankton? Why would this be so? ANSWER: Since viruses tend to infect only a narrow host range, their existence favors the evolution of a large number of different species with highly dispersed populations. Highly dispersed populations minimize the chance of viral transmission from one host to another. 21.4 Design an experiment to test the hypothesis that the presence of mycorrhizae enhances plant growth in nature. ANSWER: Such an experiment requires a control based on the natural environment, where various unknown factors may be very different than in the laboratory. One possibility is to compare the growth of seedlings in natural soil
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versus sterilized natural soil. However, this experiment would not prove that fungi are the cause of enhanced growth in unsterile soil. The sterilization procedure (usually involving heat and pressure) could break down key nutrients in the soil. A follow-up experiment might be to grow the plants in the presence of a fungus inhibitor in sterilized and unsterilized soil.
21.5 High levels of nitrate or ammonium ion corepress the nod genes through NodR. Why? ANSWER: Nitrate and ammonia are the main forms of nitrogen assimilated by plants. If they are abundant in the soil, the plant does not need rhizobial symbionts; therefore, development of rhizobia-legume symbiosis is inhibited. 21.6
One unanswered question is, how do symbiotic rhizobia reproduce? Why do bacteroids develop if they cannot proliferate? ANSWER: Various answers have been proposed. Not all of the invading bacteria become bacteroids; some continue to undergo cell division, particularly within senescing tissues of the plant. These bacteria benefit from plant growth, which is sustained by the bacteroids whose genes they share. Alternatively, the entire plant-bacteroid system may benefit rhizobia that grow just outside the plant, in the rhizosphere.
21.7 Compare and contrast the processes of plant infection by rhizobia and by fungal haustoria. ANSWER: Both rhizobial bacteria and fungal haustoria penetrate the volume of a plant cell, but they keep the plant cell membrane intact, its invagination always surrounding the invading cell. Rhizobia establish a complex, highly regulated exchange of nutrients with the host, receiving catabolites and oxygen in exchange for ammonium and cycling the components of amino acids. By contrast, haustoria establish one-way removal of nutrients such as sucrose, while providing no nutrients in return. Fungal pathogens weaken the structure of the host plant and decrease or halt its growth. 21.8 Why does ruminant fermentation leave food value for the animal host? How is the animal able to obtain nourishment from waste products that the microbes could not use? ANSWER: The rumen interior is anaerobic. In the absence of oxygen as a terminal electron acceptor, microbes are forced to generate waste products in which the electrons are put back onto the electron donors (fermentation; see Chapter 13). When the short-chain fatty acid wastes enter the animal’s bloodstream, the blood is full of oxygen, which enables complete digestion to CO2 and water. 21.9 How do you think cattle feed might be altered or supplemented to decrease methane production?
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ANSWER: Several methods have been proposed to limit methanogenesis. One is to feed cattle the antibiotic monensin, an inhibitor of sodium transport, which is required for methanogenesis. Another approach is to feed cattle an electron acceptor for H2, such as fumarate, which bacteria use to generate short-chain acids instead of methane. These approaches have been used with only partial success—not surprising given the complexity of the system.
Chapter 22 22.1 Why is oxidation state critical for the acquisition, usability, and potential toxicity of cycled compounds? Cite examples based on your study of microbial metabolism. ANSWER: Many examples can be cited. In the case of carbon, CO2 can be fixed by many species with substantial input from photosynthesis or hydrogen donors. The reduced form methane, however, can be assimilated only by methanotrophic bacteria, usually with oxygen as electron acceptor. Nitrogen gas can be assimilated only by nitrogen-fixing bacteria and archaea, whereas NH4+ can be assimilated by many plants and microbes. But NH4+ can also be oxidized by lithotrophs to NO3–, a substance potentially toxic to humans.
22.2 What would happen if wastewater treatment lacked microbial predators? Why would the result be harmful? ANSWER: Without predators, too many planktonic bacteria would remain in the wastewater after sedimentation of the sludge. The bacteria could be killed by chlorination, but the treated water would have significant BOD because the bacterial remains provide an organic carbon source for respirers. 22.3 How many kinds of biomolecules can you recall that contain nitrogen? What are the usual oxidation states for nitrogen? ANSWER: Amino acids, nucleotide bases, polyamines for DNA stabilization, peptidoglycan (both amino sugar and peptide chains), and the heme derivatives of cytochromes, chlorophyll, and vitamin B12 all include nitrogen (as do many other blochemicals). The oxidation states of nitrogen in living organisms are nearly always reduced, either R⫺NH2, R⫽NH, or R⫺N⫽R. An exception is the neurotransmitter NO (nitric oxide). 22.4 In the laboratory, which kind of bacteria would likely grow on artificial medium including NH2OH as the energy source: Nitrosomonas or Nitrobacter? ANSWER: Nitrosomonas is more likely to utilize NH2OH, since it performs the intermediate oxidation of NH2OH during nitrification of ammonia.
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22.5 The nitrogen cycle has to be linked with the carbon cycle, since both contribute to biomass. How might the carbon cycle of an ecosystem be affected by increased input of nitrogen? ANSWER: One hypothesis is that the injection of nitrogen into an ecosystem accelerates growth of producers (phytoplankton in the ocean or trees in a forest) and therefore facilitates net removal of CO2 from the atmosphere. Overall, however, the additional fixed carbon may end up dissipated by consumers and decomposers. 22.6
Compare and contrast the cycling of nitrogen and sulfur. How are the cycles similar? How are they different? Both nitrogen and sulfur cycling involve interconversion between different oxidation states. Most of these interconversion reactions are performed solely by microbes, many of them solely by bacteria. Examples include nitrification of ammonia and denitrification to N2, as well as sulfide oxidation and photolysis. In both cases, oxidation produces strong acids (HNO3, H2SO4). The major sources and sinks differ; nitrogen is obtained primarily from the atmosphere as N2, whereas sulfur is at high levels in the ocean and soil. Sulfur is rarely limiting, whereas nitrogen frequently is. Sulfur participates extensively in phototrophy; nitrogen shows little involvement in phototrophy; phototrophy based on nitrate reduction has been observed. ANSWER:
22.7 Compare and contrast the cycling of nitrogen and phosphorus. How are the cycles similar? How are they different? ANSWER: Nitrogen and phosphorus are both limiting nutrients in many ecosystems—marine, aquatic, and terrestrial. Addition of either element into an aquatic system may cause algal bloom and eutrophication. On the other hand, the two elements differ in their major sources: the atmosphere for nitrogen, and crustal rock for phosphate. Within biomass, nitrogen exists almost entirely in reduced form, whereas phophorus is entirely oxidized. Phosphorus cycles through the biosphere mainly as inorganic or organic phosphates, whereas nitrogen cycles through a broad range of oxidation states, from NH3 to NO3–. 22.8
Do you think that iron fertilization would succeed in maintaining lowered atmospheric CO2? What consequences might you predict for widespread iron fertilization? ANSWER: The algal bloom could occur in a population of toxin-producing algae, which would cause massive die-off of fish. Alternatively, if iron fertilization were widespread, the increased population density of algae would lead to increased viral predation, which would mineralize the algae, returning their carbon to the atmosphere. Other scenarios are possible, but it appears unlikely that iron fertilization would lead to a sustained decrease in atmospheric CO2.
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Chapter 23 23.1 How can an anaerobic microorganism grow on skin or in the mouth, both of which are exposed to air? ANSWER: Facultative organisms living in proximity to the anaerobes will deplete oxygen in the environment, especially around nooks and crannies (e.g., between teeth and gums, gingival pockets) that would ordinarily prevent anaerobes from growing. These small spaces have limited access to oxygen.
23.2 What can a person with mitral valve prolapse do to prevent formation of subacute bacterial endocarditis when visiting the dentist? ANSWER: Take antibiotics prophylactically. A high dose of antibiotic (usually amoxicillin or acephalosporin) taken 1 hour before the procedure will produce a high enough blood level to kill bacteria. A patient that is hypersensitive to β-lactam antibiotics such as amoxicillin will be prescribed an alternate antibiotic such as azithromycin.
23.3 Why do many gram-positive microbes that grow on the skin, such as S. epidermidis, grow poorly or not at all in the gut? ANSWER: Bile salts present in the intestine (not on the skin) easily gain access to and destroy cytoplasmic membranes of gram-positive organisms (unless the organism possesses bile hydrolases). Gram-negative microbes have an extra protection in the form of an outer membrane and so can survive better in the intestine. 23.4 How might normal flora escape the intestine and cause disease at other body sites? ANSWER: Normal flora can escape through intestinal perforations resulting from gunshot or knife wounds, surgery, or cancer. 23.5
Why can the colon be considered a fermenter? ANSWER: The contents are continually flowing through the intestinal tube, with food containing substrates for fermentation ingested at one end and waste containing fermentation products removed from the other.
23.6
Why do defensins have to be so small? ANSWER: They need to be small so they can get through the outer membrane of gram-negative organisms and the thick peptidoglycan maze of gram-positive organisms.
23.7 If NK cells can attack infected host cells coated with antibody, why won’t neutrophils? ANSWER: Actually, neutrophils (PMNs) can attack infected cells coated with antibody—but the killing mechanism is different than ADCC. Human neutrophils do not make perforin nor the other ADCC-related compounds, called granzymes, used by NK cells to kill target cells. In addition to that difference,
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NK cells possess a type of Fc receptor not found on neutrophils, which means the intracellular signaling pathways are different between NK cells and neutrophils. Neutrophils can, however, be activated when their Fc receptors bind antibody. Activated neutrophils make reactive oxygen products and can release a variety of peptides including defensins, cathelicidin, and myeloperoxidase, which can all damage target cells.
23.8 If increased fever limits bacterial growth, why would bacteria make pyrogenic toxins? ANSWER: The pyrogenic toxins have other effects that compromise and damage the host. The toxins can induce cytokines that damage local host cells or confuse the immune system. This can provide the pathogen with nutrients and help it hide from the immune system longer. Pyrogenic toxins include lipopolysaccharide and protein toxins such as toxic shock syndrome toxin (see Chapter 25). Chapter 24 24.1 Two different stretches of amino acids in a single protein form a three-dimensional antigenic determinant. Will the specific immune response to that three-dimensional antigen also respond to one of the amino acid stretches alone? ANSWER: Most likely no. It is the three-dimensional shape formed by the two stretches that is recognized as an antigen. A denatured protein that contains both amino acid stretches will not possess the three-dimensional shape of the antigen. On the other hand, there can be other specific immune responses that involve different subsets of lymphocytes that individually recognize each of the amino acid stretches that form the three-dimentional antigenic determinant. As an analogy, take a computer image of a friend’s face and shuffle the facial features. Turn the nose upside down, exchange the eyes with the mouth, and lower the ears. Since you were programmed to respond to the original facial configuration, you likely would not recognize the rearranged face as a whole. But you might find that the nose looks familiar.
24.2 How does a neutralizing antibody that recognizes a viral coat protein prevent infection by the associated virus? ANSWER: Neutralizing antibodies usually bind attachment proteins on the virus and sterically prevent them from binding to host cell receptors (see Fig. 24.4). Some antibodies to enveloped viruses might trigger the complement cascade (see later in the chapter), destroying the membrane. 24.3 There are immune disorders in which an individual overproduces a specific class of antibody, for example hypergammaglobulinemia. How could the radial immunodiffusion technique be used to identify what class of antibody is in excess?
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ANSWER: Because they are proteins, immunoglobulin antibodies are also antigens. Therefore, antibodies can be made that react to epitopes in the heavy chains. Antibodies to human IgG can be raised in rabbits, for example. Radial immunodiffusion using agarose impregnated with rabbit anti-human IgG antibodies can be used to measure the concentration of human IgG in serum loaded into a well. The larger the diameter of the resulting ring, the more IgG there is in the serum sample. A patient that has an unusually high amount of IgG antibody relative to other antibody isotypes, as compared to a normal healthy person, would be diagnosed with IgG hypergammaglobulinemia.
24.4 The mother of a newborn was found to be infected with rubella, a viral disease. Infection of the fetus could lead to serious consequences for the newborn. How could you determine if the newborn was infected while in utero? ANSWER: Since maternal IgM antibodies cannot cross the placenta, finding IgM antibodies to rubella antigens in the newborn’s circulation indicates that the fetus was infected and initiated its own immune response. If the newborn has only IgG antibodies to rubella (no IgM antibodies), this indicates that the child was not infected and that maternal IgG crossed the placenta. 24.5
Why does the delta region have no switch region? ANSWER: Because B cells at the early stages have both IgM and IgD surface antibodies. Recombination at the DNA level is not involved because alternative RNA-splicing events after transcription determine whether an IgM or IgD molecule is made.
24.6 Why do individuals with type A blood have antiB and not anti-A antibodies? ANSWER: Because the B-cell population that would react to type A antigen was deleted during B-cell maturation. 24.7 Why do immunizations lose their effectiveness over time? ANSWER: Because memory B cells eventually die. Without some exposure to antigen, those memory cells will not be replaced. 24.8 Transplant rejection is a major consideration when transplanting most tissues because host TC cells can recognize allotypic MHC on donor cells. So why are corneas easily transplanted from a donor to just about any other person? ANSWER: The cornea is not normally vascularized. So even though corneal cells express MHC proteins, circulating host T cells do not have an opportunity to interact with them. The cornea will not be rejected. This is called an immune privileged site.
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24.9
Why does attaching a hapten to a carrier protein allow production of anti-hapten antibodies? ANSWER: B cells with anti-hapten surface antibody (as part of the B-cell receptor) can take up hapten but cannot present the hapten to a helper T cell. The same B cell can also take up the hapten bound to a carrier molecule, and because the carrier molecule is larger than the hapten, the B cell will present the carrier epitope to the helper T cell. The helper T cell stimulates the B cell, which was already programmed to make anti-hapten antibody, to differentiate into plasma and memory B cells.
24.10 Do bone marrow transplants in a patient with severe combined immunodeficiency require immunosuppressive chemothereapy? ANSWER: No. Since the patient has no T cells to recognize foreign antigens, the transplant is not rejected.
24.11 How can a stem cell be differentiated from a B cell at the level of DNA? ANSWER: Gene splicing will have taken place in the B cell but not the stem cell. Thus, the B cell will have fewer cassettes for each of the V, D, and J regions, while the stem cell will have all of them. PCR techniques can be used to view those differences. Chapter 25 25.1 Is a microbe with an LD50 of 5 × 104 more or less
virulent than a microbe with an LD50 of 5 × 107? ANSWER: Because it takes fewer cells to cause disease, the microbe with the smaller LD50 (5 × 104) is the more virulent.
25.2 Antibodies to which subunit of cholera toxin will best protect a person from the toxin’s effects? ANSWER: Antibodies to the B subunit will be more protective. Inactivating the B subunit will prevent binding of the toxin to cell membranes. The A subunit active site is typically sequestered in these toxins and inaccessible to antibody. Furthermore, once the A subunit has entered a host cell, antibodies cannot enter and neutralize it. 25.3 Would patients with iron overload (excess free iron in the blood) be more susceptible to infection? ANSWER: Withholding iron from potential pathogens is a host defense strategy because when iron is plentiful, the microbe does not have to expend energy to get it and so can readily grow. On the other hand, low iron can also be a signal to express various virulence genes, so for some organisms, high iron might hinder infection. 25.4 How might you experimentally determine if a pathogen secretes an exotoxin?
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The microbe can be grown in liquid culture and the cells removed either by centrifugation or filtration. If the organism makes an exotoxin, it may well be present in the cell-free supernatant. The presence of a toxin can be determined by injecting the supernatant into an animal model (e.g., mice) and examining the result (death or altered function; see tetanus toxin below). Alternatively, the supernatant can be administered to a layer of tissue culture cells and the health of the monolayer noted.
ANSWER:
25.5 Internet problem: What other toxins are related to the cholera enterotoxin A subunit? B subunit? ANSWER: Go to Pub-med (http://www.ncbi.nlm.nih.gov/ PubMed/) In the search pop-down window mark “Protein.” Then type “cholera enterotoxin” or “cholera B subunit.” Click on the appropriate result. Highlight and copy the amino acid sequence at the bottom of the document. Then, back at the NCBI home page, click on “Blast”, select “Protein-Protein Blast,” paste the B subunit sequence into the Search box and click “Blast.”After clicking “Format” on the next page, all the homology results will be displayed. Items with E-values having large negative numbers represent the most significant homologies. Note that the toxin of Citrobacter freundii, another gram-negative rod, possesses a B subunit similar to that of cholera, but not a similar A subunit (you must perform a second “Blast” for this). Scroll down the page to find the actual alignments between the query protein and homolog. 25.6 Protein and DNA have very different structures. Why would a protein secretion system be derived from a DNA-pumping system? ANSWER: Conjugation systems actually move DNA that is attached to a pilot protein at the 5’ end. A pilot protein is made by the conjugation system, then binds to the 5’ end of the DNA to be transferred, and “pilots” the DNA through the conjugation pore. So a modified conjugation system that moves protein only is not as much of a leap as might initially be thought. 25.7 How can one determine if a bacterium is an intracellular parasite? ANSWER: Microscopic examination to see if bacteria are found within cultured mammalian cells is usually not satisfactory. The difficulty lies in determining if the organism is inside the host cell or just bound to its surface—or, if it is inside, whether it is a live or dead bacterium. One commonly used approach is to add to infected cell monolayers an antibiotic that can kill the microbe but will not penetrate the mammalian cells. The protein synthesis inhibitor gentamicin is typically used. A bacterium that invades a host cell will gain sanctuary from gentamicin and grow intracellularly. Extracellular bacteria and bacteria attached to the outside of the host cell are killed. Counting viable colony-forming units of bacteria released from the mammalian cells by gentle detergent treatments at various
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times will reveal if the organism grew intracellularly. Of course, this will only work if the microorganism is not an obligate intracellular parasite.
ples gave the organisms time to replicate and increase their numbers. Consequently, the lab results should be viewed with suspicion.
25.8 Why would killing a host be a bad strategy for a pathogen? ANSWER: The goal of any microbe is to maintain its species. If a microbe does not have an opportunity to easily spread to a new host, killing its host would be tantamount to suicide.
26.4 Considering that N. gonorrhoeae is exquisitely sensitive to ceftriaxone, why do you suppose the patient was also treated with tetracycline? And why can one person be infected repeatedly with N. gonorrhoeae? ANSWER: Because STDs often travel in pairs and because the initial symptomolgy is similar, the clinician will want to “cover” the patient for possible chlamydia infection. Chlamydiae are not susceptible to ceftriaxone. The large, single dose of ceftriaxone (as opposed to smaller multiple injections given over days) was given because patients with gonorrhea are typically poorly compliant and fail to return for subsequent injections. A person who experiences gonorrhea is not protected from reinfection because the organism’s surface antigens undergo phase variations (see Section 10.6) and may downregulate the immune system (see Fig 26.13C).
25.9 Why do rhinovirus infections fail to progress beyond the nasopharynx? ANSWER: For one thing, rhinoviruses are susceptible to acid pH (pH 3.0), so they are unable to replicate in the gastrointestinal tract. They also grow best at 33°C, which may help explain their predilection for the cooler environs of the nasal mucosa.
Chapter 26 26.1 Why would treatment of an infection sometimes require multiple antibiotics? ANSWER: Sometimes treatment is initiated before knowing the infecting microbe or its susceptibility to different drugs. Without knowing the antibiotic susceptibility of the infecting agent, a physician will use multiple antibiotics to ensure that one will kill or inhibit the growth of the microbe. Combinations of antibiotics are also used because different antibiotics are effective against different sets of microbes (e.g., gram-positive vs. gram-negative, or aerobe vs. anaerobe).
26.2 Why do you think most urinary tract infections occur in women? ANSWER: The major reason is anatomy. Most bladder UTIs come from access through the urethra. Since the urethra in men is longer than in women, it usually takes a catheter to introduce bacteria into a male bladder. The trip for the infecting organism in women is much shorter. However, in people older than 50, UTIs become more common in both men and women, with less difference between the sexes. The reason is not clear.
26.3 Urine samples collected from six hospital patients were placed on a table at the nurse’s station awaiting pickup from the microbiology lab. Several hours later, a courier retrieved the samples and transported them to the lab. The next day, the lab reported that four of the six patients had UTI. Would you consider these results reliable? Should you start treatment based on these results? ANSWER: Because urine is good growth media for many bacteria, the delay of several hours in picking up the sam-
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26.5 Human immunodeficiency virus was discussed in this section and in Chapter 25. Like the plague, it is a blood-borne disease. Why, then, do fleas and mosquitoes fail to transmit HIV? ANSWER: When insect vectors take a blood meal, they typically defecate or regurgitate simultaneously. So theoretically, they could serve as a vector for HIV. Pathogens such as the West Nile virus that are transmitted by insect vectors actually grow in their insect hosts; however, HIV does not. Because it is unusually fragile, HIV will die quickly. There have been no cases of HIV transmitted by insect vectors. 26.6 Normal cerebral spinal fluid is usually low in protein and high in glucose. The protein and glucose content does not change much during a viral meningitis, but bacterial infection leads to greatly elevated protein and lowered glucose levels. What could account for this? There are several explanations. Bacteria and infiltrating PMNs will consume glucose and alterations in the blood-brain barrier can lead to decreased transport of glucose into the spinal fluid. As a result of these factors, glucose levels plummet. Growth of the bacteria and infiltration of PMNs accounts for the increase in CSF protein levels. Viruses are very small and consist mostly of nucleic acids, so even at high numbers, they will not significantly add to CSF protein content. Since viruses do not grow in CSF directly, they will not consume glucose. In addition, viral meningitis does not cause a great infiltration of PMNs into the CSF, another reason glucose levels remain high and protein levels remain low.
ANSWER:
26.7 Knowing the symptoms of tetanus, what kind of therapy would you use to treat the disease? ANSWER: At the first sign of muscle spasm, antitoxin should be given. If tetany is severe, muscle relaxants can relieve spasms.
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26.8
How do the actions of tetanus toxin and botulinum toxin actually help the bacteria colonize or obtain nutrients? ANSWER: This is a difficult question to answer. Few scientists have speculated. Recall that the toxins are encoded by genes in resident bacteriophages that became part of the clostridial genome through horizontal transfer from some other source. Since the organisms normally reside in soil, the actual function of these toxins may have something to do with survival in that habitat. The toxin’s effects on humans may simply be an unfortunate accident. However, with tetanus toxin, there may be benefit in that muscle spasms could limit oxygen delivery to infected tissues, enabling a more anaerobic environment for growth. Cell death may also release iron or other nutrients useful to C. tetani.
26.9 If C. botulinum is an anaerobe, how might botulism toxin get into foods? ANSWER: The most common way botulism toxin gets into food today is via home canning. The canning process involves heating food in jars to very high temperatures. The heat destroys microorganisms and drives out oxygen; both processes help to preserve foods. If the jars are not heated to sterilization conditions, spores of C. botulinum will survive. When the jars are cooled for storage at room temperature, the spores germinate; the organism then grows in the anaerobic medium and releases the toxin. When the food is eaten, the toxin is eaten, too. 26.10 A patient presenting with high fever and in an extremely weakened state is suspected of having a septicemia. Two sets of blood cultures are taken from different arms. One bottle from each set grows Staphylococcus aureus yet the laboratory report states that the results are inconclusive. New blood cultures are ordered. Why might this be? There must have been something different about the two strains of staphylococci grown in the separate bottles. For example, they may have different antibiotic susceptibility patterns when tested against a battery of antibiotics. One strain may be sensitive to penicillin while the other strain is resistant. Since the expectation is that a single strain initiated the infection, both isolates should exhibit the same drug susceptibility pattern. The laboratory suspects contamination from separate sources. ANSWER:
Chapter 27 27.1 The drug tobramycin is added to a concentration
of 1,000 µg/ml in a tube of broth from which serial twofold dilutions were made. Including the initial tube (tube 1), there are a total of ten tubes. Twenty-four hours after all the tubes are inoculated with Listeria monocytogenes, turbidity is observed in tubes 6–10. What is the MIC?
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62.5 µg/ml, the concentration in tube 5, the last tube with no growth. (Relative to tube 1, tube 5 has been diluted 24 (16-fold dilution: 1,000/500/250/125/62.5.) ANSWER:
27.2
What additional test performed on an MIC series of tubes will tell you whether a drug is bacteriostatic or bactericidal? ANSWER: Streaking a portion of the broth from the dilution tubes showing no growth. If the drug is bacteriostatic, colonies will form on the agar plate because during streaking, the bacteria are removed from the presence of the drug. If the antibiotic is bactericidal, no colonies will form because the organisms are dead before plating. This method determines the minimum bactericidal concentration (MBC) of an antibiotic. MBC would be defined as the lowest dilution that did not yield viable cells.
27.3
You are testing whether a new antibiotic will be a good treatment choice for a patient with a staph infection. The Kirby-Bauer test using the organism from the patient shows a zone of inhibition of 15 mm around the disk containing this drug. Clearly, the organism is susceptible. But you conclude that the drug would not be effective in the patient. What would make you draw this conclusion? ANSWER: If the average attainable tissue level of the drug is below the MIC, the drug will not be effective.
27.4
When treating a patient for an infection, why would combining a drug such as erythromycin with a penicillin be counterproductive? ANSWER: Erythromycin, a bacteriostatic drug, will stop growth, which indirectly stops cell wall synthesis and renders the microbe insensitive to penicillin.
27.5
The enzyme DNA gyrase, a target of the quinolone antibiotics, is an essential protein in DNA replication. The quinolones bind to and inactivate this protein. Research has proved that quinolone-resistant mutants contain mutations in the gene encoding DNA gyrase. If the resistant mutants contain a mutant DNA gyrase and DNA gyrase is essential for growth, why are these mutations not lethal? ANSWER: The mutations cause changes in DNA gyrase that have little to no effect on function but that do prevent the drug from binding. Thus, the mutant organism will continue to twist its DNA and grow well with or without the drug.
27.6
Why might a combination therapy of an aminoglycoside antibiotic and cephalosporin be synergistic? ANSWER: The two drugs given together could act synergistically because the cephalosporin can weaken the cell wall and allow the aminoglycoside easier access to the cell interior, where it can attack ribosomes. This is especially useful in organisms that have some resistance to both drugs.
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27.7
Could genomics ever predict the drug resistance phenotype of a microbe? If so, how? ANSWER: Yes. If the organism’s genome possesses genes whose deduced protein sequences harbors significant similarity to antibiotic resistance proteins from other organisms, one can predict a similar drug resistance. Definitive proof of drug resistance requires actual in vitro testing.
28.2 Why does finding IgM to West Nile virus indicate current infection? Why wouldn’t finding IgG do the same? ANSWER: Upon infection with any organism, IgM antibodies are the first to rise. After a short time the levels of IgM decline as IgG levels rise. IgG, however, can remain in serum for years, making it a poor prognosticator of current infection.
27.8
28.3 Why does adding albumin or powdered milk prevent false positives in ELISA? ANSWER: Antibodies are proteins. They can stick to plastic just as easily as the antigen being tested. Without blocking all the possible binding sites on plastic with albumin, any antibody from the patient’s serum could stick to the plastic instead of to the antigen and react with the secondary enzyme-conjugated antihuman antibody.
Fusaric acid is a cation chelator that normally does not penetrate the E. coli membrane, which means E. coli is typically resistant to this compound. Curiously, cells that develop resistance to tetracycline become sensitive to fusaric acid. Resistance to tetracycline is usually the result of an integral membrane efflux pump that pumps tetracycline out of the cell. What might explain the development of fusaric acid sensitivity?
Fusaric acid is imported by the tetracycline efflux pump. This phenomenon can be used to isolate mutants with deletions of transposons encoding tetracycline resistance. Transposons, such as Tn10, which carries tetracycline resistance, can spontaneously delete at around a frequency of 10–6, but finding one out of a million tetracyclinesusceptible cells is impossible without a positive selection. Fusaric acid provides that positive selection, since the cell with the Tn10 deletion will be resistant to fusaric acid. ANSWER:
27.9
Mutations in the ribosomal protein S12 (encoded by rpsL) confer resistance to streptomycin. Given a cell containing both rpsL+ and rpsLR genes, would the cell be streptomycin resistant or sensitive? (Recall that genes encoding ribosome proteins for the small subunit are designated rps. A + indicates the wild-type allele, while R indicates a gene whose product is resistant to a certain drug. ANSWER: This merodiploid cell would contain two sets of ribosomes, one set, containing normal S12, would be sensitive to streptomycin; another set, containing the resistant S12, would be resistant. Because streptomycin causes mistranslation of mRNA on sensitive ribosomes, inappropriate proteins that can kill the cell would still be synthesized. Thus, the cell would remain sensitive to streptomycin. Note, however, that the recessive nature of antibiotic resistance seen in this case is not the norm. Resistance is a dominant trait in a majority of cases.
Chapter 28 28.1 Use Figure
28.6 to identify the organism from the following case. A sample was taken from a boil located on the arm of a 62-year-old man. Bacteriological examination revealed the presence of gram-positive cocci that were also catalase-positive, coagulase-positive, and novobiocin resistant. ANSWER: Staphylococcus aureus. The novobiocin test is irrelevant in this situation.
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28.4 Specific antibodies against an infectious agent can persist for years in the bloodstream. So how is it possible that antibody titers can be used to diagnose diseases such as infectious mononucleosis? ANSWER: During the course of a disease, the body’s immune system will increase the amount of antibody made specifically against the infectious agent. Thus, one compares the antibody titer in a blood sample taken from a patient in the active, or acute, phase of disease with the antibody titer several weeks later, when the patient is in the recovery, or convalescent, stage. Seeing a greater than fourfold rise in a specific antibody titer (for example, in mononucleosis) indicates that the patient’s immune system was responding to the specific agent. Remember, simply finding IgG against an organism or virus in serum only indicates that the patient was exposed to that microbe at some time in the past. 28.5 Two blood cultures, one from each arm, were taken from a patient with high fever. One culture grew Staphylococcus epidermidis, but the other blood culture was negative (no organisms grew out). Is the patient suffering from septicemia caused by S. epidermidis? ANSWER: Probably not. S. epidermidis is a common inhabitant of the skin and could easily have contaminated the needle when blood was taken from the patient. The fact that only one of the two cultures grew this organism supports this conclusion. If the patient had really been infected with S. epidermidis, both blood cultures would have grown this organism. 28.6 A 30-year-old woman with abdominal pain went to her physician. After the examination, the physician asked the patient to collect a midstream urine sample that they would send to the lab across town for analysis. The woman complied and handed the collection cup to the nurse. The nurse placed the cup on a table at the nurse’s station. Three hours later, the courier service picked up the
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specimen and transported it to the laboratory. The next day, the report came back “greater than 200,000 CFUs/ml; multiple colony types; sample unsuitable for analysis.” Why was this determination made? ANSWER: Although the CFU number is high enough to consider relevant, UTIs are typically caused by a single organism. The fact that the lab found many different colony types suggests a problem with specimen collection. In this case, not refrigerating the sample allowed the small number of urethral contaminants to overgrow the specimen.
28.7 Methicillin, a beta-lactam antibiotic, is very useful in treating staphylococcal infections. The development of methicillin-resistant strains of Staphylococcus aureus (MRSA) is a very serious development because there are few antibiotics that can kill these strains. Imagine a large metropolitan hospital in which there have been eight serious nosocomial infections with MRSA and you are responsible for determining the source of infection so it can be removed. How would you accomplish this using common bacteriological and molecular techniques? ANSWER: Samples from all the affected patients and from the hospital staff would be screened for the presence of S. aureus resistant to methicillin. Each strain would then be analyzed for restriction fragment length polymorphisms (RFLP). Strains from all the patients will likely have identical restriction patterns if they came from the same source. The source is then identified by determining which staff member possesses MRSA with the same pattern. The source may also be inanimate, such as surgical equipment or ventilation apparatus. A connection between patients
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and specific staff members or instruments would also have to be demonstrated.
28.8 What are some reasons why some diseases spread quickly through a population while others take a long time? ANSWER: There are several factors. One is mode of transmission; airborne diseases can spread more quickly than food-borne disease, for instance. Sexually transmitted diseases spread slower still. Herd immunity is another factor. Herd immunity is based on the number of individuals within a population that are resistant to a disease. Someone immune to the disease cannot pass it on. The more immune people there are in a population (or herd), the slower the spread of the epidemic to susceptible people. 28.9 Using the “Maps of Outbreaks” provided on the ProMed-mail website, identify the countries considered to have anthrax epidemics. ANSWER: The latest map available at the time this was written was drawn from reported occurrences in 2003. At that time, 14 countries had epidemic occurrences of anthrax (Turkey, Niger, Chad, Guinea, Sierra Leone, Liberia, Ivory Coast, Ghana, Togo, Ethiopia, Zambia, Zimbabwe, Myanmar, and Commonwealth of Independent States). Many others, such as Mexico, are endemic. The United States has sporadic cases of anthrax. Note that the ProMed-mail website as of 2008 has an interactive map that shows the locations of specific disease outbreaks that have taken place during the past 30 days across the globe.
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Glossary
A bacterial toxin containing two protein subunits, A and B. Subunit A is the toxic protein and subunit B binds host cell receptors.
adenylate cyclase An enzyme that converts ATP into cyclic
ABC transporter
An ATP powered transport system that contains an ATP binding cassette.
adhesin Any cell surface factor that promotes attachment of an organism to a substrate.
aberration An imperfection in a lens.
ADP-ribosyltransferase A bacterial toxin that enzymatically transfers the ADP-ribose group from NAD+ to target proteins, altering the target protein’s structure and function.
AB toxin
abiotic Produced without organisms; occurring in the absence
of life. absorption
In optics, the capacity of a material to absorb light.
acceptor end The amino acid attachment site at the 3’ end of
adenosine monophosphate, cAMP. adherence The ability of an organism to attach to a substrate.
adsorb The attachment of virions to a host cell. aerated (A) horizon The layer of soil below the organic hori-
tRNAs.
zon, containing decomposed organic particles and minerals.
acceptor site (A site) The region of the ribosome that binds an
aerial mycelium A hypha that extends above the surface and
incoming charged tRNA.
produces spores at its tip.
accessory protein A protein found in the viral capsid or tegument that is needed early in the viral life cycle.
aerobic respiration The use of oxygen as the terminal electron acceptor in an electron transport chain. A proton gradient is generated and used to drive ATP synthesis.
acid-fast stain A diagnostic stain for mycobacteria, which retain the dye fuchsin due to the presence of mycolic acids in the cell wall. acidophile An organism that grows fastest in acid (generally
defined as below pH 5).
agar A polymer of galactose that is used as a gelling agent.
A bright central point surrounded by rings of light and dark caused by the pattern of interference of spherical wavefronts converging at the focal point.
Airy disk
acquired immunodeficiency syndrome (AIDS) A disease caused by HIV that leads to the destruction of T cells and the inability to fight off opportunistic infections.
akinete A specialized spore cell formed by some filamentous
A phylum of high GC content gram-positive
bacteria.
algal bloom An overgrowth of algae on the water surface, caused by an increase in a limiting nutrient.
activated sludge Organic material concentrated from waste-
alkaline fermentation
Actinobacteria
water, containing microbes that digest the material to inorganic compounds. activation energy The energy needed for reactants to reach the
transition state between reactants and products.
cyanobacteria. alga
A microbial eukaryote that contains chloroplasts.
Bacterial fermentation in conjunction with proteolysis and amino acid catabolism that generates ammonia in amounts that raise pH.
alkaliphile An organism with optimal growth in alkali (generally defined as above pH 9).
activator A protein that increases gene transcription.
allergen An antigen that causes an allergic hypersensitivity
active transport An energy-requiring process that moves
reaction.
molecules across a membrane against their electro-chemical gradient. acyl carrier protein (ACP)
allograft The transplantation of tissue from a donor with one type of major histocompatibility complex (MHC) protein into a recipient with a different type of MHC.
adaptive immune response See adaptive immunity.
allosteric site A regulatory site on a biological molecule distinct from the ligand/substrate binding site.
adaptive immunity Immune responses activated by a specific
allotype An amino acid sequence in the antibody constant
A protein that can carry an acetyl group for anabolic pathways such as fatty acid synthesis.
antigen and mediated by B cells and T cells.
region that is shared by some, but not all, members of a species.
adenosine triphosphate (ATP) A ribonucleotide with three
alternation of generations A life cycle that alternates between
phosphates and the base adenine. It has many functions in the cell including precursor for RNA synthesis and energy carrier.
a haploid cell population and a diploid cell population. alveolar macrophage Macrophages, located in the lung alve-
oli, that phagocytose foreign material.
G-1
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Alveolata (alveolate) A group of ciliated or flagellated protists with complex cortical structure. ameba (or amoeba) A protist that moves via pseudopods. amensalism An interaction between species that harms one partner but not the other. amino acid The monomer unit of proteins. Each amino acid
anti-attenuator stem loop An mRNA secondary structure whose formation prevents assembly of a downstream transcriptional termination stem loop. The anti-attenuator stem permits transcription of the downstream structural genes. antibiotic A molecule that can kill or inhibit the growth of
selected microorganisms. antibody A host defense protein produced by B cells in response
contains a central carbon covalently bound by a hydrogen, an amino group, a carboxylic acid group and a side chain. An exception is proline, in which the side chain is cyclized with the central carbon.
to a specific antigenic determinant. Antibodies bind to their corresponding antigenic determinant.
aminoacyl-tRNA transferase
An enzyme that attaches a specific amino acid to the correct tRNA thereby charging the tRNA.
visualize cell components recognized by the antibody with high specificity.
aminoglycosides Bacteriocidal protein synthesis inhibitors
antibody-dependent cell-mediated cytotoxicity (ADCC) The
used as antibiotics. ammonification
The generation of ammonia from organic
antibody stain The attachment of a stain to an antibody to
process by which natural killer cells destroy viral protein expressing antibody-coated host cells.
nitrogen.
anticodon Three nucleotides in the middle loop of a tRNA that
A taxonomic class of unshelled amebas that move via lobed pseudopods. Also known as Lobosea.
antigen A compound, recognized as foreign by the cell, that
Amoebozoa
amphibolic Metabolic pathways that are reversible and can be
used for both catabolism and anabolism. amphipathic A molecule with both hydrophilic and hydropho-
bic portions. amphotericin B An anti-fungal drug that binds the fungal spe-
cific sterol ergosterol and destroys membrane integrity. anabolism The building up of complex biomolecules from
smaller precursors. anaerobic photosystem I A protein complex that harvests
light from a chlorophyll, splits an electron from a small molecule such as H2S or H2O, and stores energy in the form of NADPH. anaerobic photosystem II A protein complex that splits an
electron from bacteriochlorophyll and stores energy in the form of a proton potential. anaerobic respiration The use of a molecule other than oxygen as the final electron acceptor of an electron transport chain.
base pair with a codon in mRNA. elicits an adaptive immune response. antigenic determinant A small segment of an antigen that is capable of eliciting an immune response. An antigen can have many different antigenic determinants. antigenic shift A genetic change in a pathogen’s surface protein that prevents recognition by antibodies and host immune cells produced in response to the previous version of the protein. antigen-presenting cell (APC) An immune cell that can process
antigens into antigenic determinants and display those determinants on the cell surface for recognition by other immune cells. antimicrobial agent A chemical substance that can kill
microbes or slow their growth. antiparallel The orientation of the two strands in opposite
directions. Commonly refers to a nucleic acid double helix with one strand in the 5’ to 3’ orientation and the other strand in the 3’ to 5’ orientation.
anammox reaction
antiport A transport protein in which the molecules being transported move in opposite directions across the membrane.
anaphylaxis A severe hypersensitivity reaction caused by
antisense RNA A non-coding RNA that binds to a complementary sequence of protein-coding RNA and (usually) prevents its translation.
The anaerobic oxidation of ammonium to nitrogen gas (using nitrate as electron acceptor); yields energy. chemically induced contraction of smooth muscles and dilation of capillaries.
anaplerotic reaction Metabolic reactions, occurring in all
organisms, that fix small amounts of CO2 to regenerate TCA cycle intermediates. angle of aperture The width of a light cone (theta, θ) that
antisepsis The removal of pathogens from living tissues. antiseptic A chemical that kills microbes. anti-sigma factor A protein that inhibits a specific sigma fac-
tor, preventing transcription initiation.
projects from the midline of a lens. Greater angles of aperture increase resolution.
AP endonuclease An enzyme that cleaves the DNA backbone
anion A negatively charged ion.
AP site A position in DNA where there is no base attached to
annotation Deciphering genome sequences, including identi-
fication of genes and prediction of gene function. antenna complex A complex of chlorophylls and accessory
at regions missing a nitrogenous base. the sugar of the backbone. apical complex A specialized structure that facilitates entry of
Apicomplexan parasites into host cells.
pigments in the photosynthetic membrane that collects photons and funnels them to a reaction center.
Apicomplexa A taxonomic group of parasitic alveolates that possess an apical complex used for entry into a host cell.
anthropogenic Caused by humans.
apicoplast An organelle unique to Apicomplexans, derived from genetic reduction of a chloroplast, that no longer performs photosynthesis but provides an essential function in fatty acid metabolism.
anti-anti-sigma factor A protein that inhibits an anti-sigma factor, allowing the target sigma factor to participate in initiating transcription.
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apurinic site A DNA site missing a purine base due to hydroly-
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autoimmune response A pathology caused by lymphocytes
sis of the bond linking the base to the sugar.
that can react to self antigens.
aquaporin A membrane-embedded channel that increases the
autoinducer A secreted molecule that induces quorum sensing
rate of water diffusion across the membrane.
behavior in bacteria.
The structure formed by penetrating hyphae of endomycorrhizae.
archaea
autoradiography The visualization of a radioactive probe by exposing the probed material to X-ray film, followed by photographic development of the film.
Archaea One of the three domains of life, consisting of organ-
autotroph An organism that can reduce carbon dioxide to produce organic carbon for biosynthesis.
arbuscules
Prokaryotic organisms that are members of the domain Archaea. isms with a last common ancestor not shared with members of Bacteria or Eukarya. Organisms are prokaryotic (lacking nuclei, unlike eukaryotes) and possess ether-linked phospholipid membranes (unlike bacteria). The second eon (major time period) of Earth’s existence, from 3.8 to 2.5 gigayears (Gyr, 109 years) before the present. The earliest geological evidence for life dates to this eon.
Archaean eon
archaeon
See pl. archaea.
aromatic A ring-shaped organic molecule with π electrons
delocalized equally around the ring. A small vegetative cell, produced by mature mycelia, that gets dispersed. arthrospore
artifact A structure viewed through a microscope that is incorrectly interpreted. Ascomycota (ascomycete) A taxonomic group of fungi whose
mycelia form paired nuclei. Haploid ascospores are produced in pods called asci. ascospore The spore produced by an Ascomycete fungus.
axenic growth The ability of an organism to grow in the absence of any other species, as, for example, in a pure culture. azidothymidine (AZT) A nucleotide analog that inhibits reverse transcriptase and was the first drug clinically used to fight HIV infections. B cell An adaptive immune cell that can give rise to antibodyproducing cells. bacillus pl. bacilli A bacterium with a linear, rod-like shape. bacitracin A topical antibiotic that affects cell wall synthesis. bacteremia
A bacterial infection of the blood.
bacteria sing. bacterium Prokaryotic organisms that are mem-
bers of the domain Bacteria One of the three domains of life, consisting of organ-
isms with a last common ancestor not shared with members of Archaea or Eukarya. Organisms are prokaryotic (lacking nuclei, unlike eukaryotes) and possess ester-linked phospholipid membranes (unlike archaea). bactericidal An agent that kills bacterial cells.
cete fungi.
bacteriochlorophyll The chlorophylls of anaerobic phototrophs; they absorb photons most strongly in the far red end of the light spectrum.
aseptic An environment that is free of microbes.
bacteriophage (phage) A virus that infects bacteria.
assembly The packaging of a viral genome into the capsid to assimilation An organism’s acquisition of an element, such as
bacteriorhodopsin (BR) An archaeal membrane-embedded protein that contains retinal and acts as a light-driven proton pump; homologous to the bacterial proteorhodopsin.
ascus pl. asci A spore-containing pod produced by Ascomy-
form a complete virion. carbon from CO2, to build into body parts.
bacteriostatic An agent that inhibits the growth of bacterial
assimilatory nitrate reduction The uptake of nitrate by plants
cells.
and bacteria for use in biosynthetic pathways.
bacterium
atomic force microscopy (AFM) A technique that maps the
bacteroid Cell-wall-less, undividing, differentiated rhizobial
three dimensional topography of a object using van der Waals forces between the object and a probe. atomic mass
The mass (in grams) of one mole of an element.
See pl. bacteria.
cell within a plant cell. The bacteroid provides fixed nitrogen for the plant. Bacteroidetes A phylum of gram-negative bacteria; nearly all
atomic number The number of protons in an atom, it is unique
their members are obligate anaerobes.
for each element.
banded iron formation A geological formation consisting of layers of oxidized iron (Fe3+) which indicates formation under oxygen-rich conditions.
A protein complex that synthesizes ATP from ADP and inorganic phosphate using energy derived from the transmembrane proton potential. It is located in the prokaryotic cell membrane and in the mitochondrial inner membrane. ATP synthase
attach
See adsorb.
attenuator stem loop An intramolecular mRNA structure con-
sisting of a base-paired stem connected by a single-strand loop. The stem loop structure causes transcription to terminate. Its formation requires efficient translation of a leader peptide sequence. An appliance that uses pressurized steam to sterilize materials by raising the temperature above the boiling point of water at standard pressure. autoclave
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barophile See piezophile. An organism that requires high
pressure to grow. base excision repair A DNA repair mechanism that cleaves
damaged bases off the sugar-phosphate backbone. After endonuclease activity at the AP site, a new correct DNA strand is synthesized complementary to the undamaged strand. Basidiomycota (basidiomycete)
A clade of fungi that form
mushrooms. basidiospore A haploid spore formed by a basidiomycete
through meiosis of a basidium.
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basidium A diploid cell, formed by the fusion of paired nuclei,
bright-field microscopy A type of light microscopy in which
that lines the gills of mushrooms (Basidiomycota). basophile A white blood cell, stained by basic dyes, that
the specimen absorbs light and appears dark against a light background.
secretes compounds that aid innate immunity.
bubonic plague A disease caused by the bacterium Yersinia pes-
The growth of bacteria in a closed system without inputs of nutrients.
budding A form of reproduction in which mitosis of the mother
B-cell receptor A B-cell membrane protein complex con-
cell generates daughter cells of unequal size.
taining an antibody in association with the Igα and Igβ immunoglobulins.
burst size The number of virus particles released from a lysed
B-cell tolerance The exposure of B cells to a high antigen dose, preventing future antibody production against that antigen.
calorimetry A technique to measure the amount of heat released or absorbed during a reaction.
The region of the Earth’s crust where the soil layer
Calvin cycle (Calvin-Benson cycle, Calvin-Benson-Bassham cycle, CBB cycle) The metabolic pathway of carbon fixation in
benthic organism Organisms that live on the ocean floor or
which the CO2 condensing step is catalyzed by rubisco. Found in chloroplasts and in many bacteria.
batch culture
bedrock
ends. within the sediment.
tis; characterized by swollen lymph nodes that often turn black.
host cell.
porin protein.
candidate species A newly described microbial isolate that may become accepted as an official species.
binary fission The process of replication in which one cell
capsid
divides to form two daughter cells of equal size. bioburden The microorganisms that normally inhabit a par-
capsule A slippery outer layer composed of polysaccharides that surrounds the cell envelope of some bacteria.
ticular body ecosystem.
carbon dioxide fixation
beta barrel A cylinder of beta-sheets, found for example in a
biofilm
A community of microbes growing on a solid surface.
A protein shell that surrounds a virion’s nucleic acid.
The enzymatic covalent incorporation of inorganic carbon dioxide (CO2) into an organic compound.
biogeochemical cycles
The recycling of elements needed for life (such as carbon or nitrogen) through the biotic and abiotic components of the biosphere.
carbon monoxide reductase pathway The carbon fixation
biogeochemistry
The metabolic interactions of microbial communities with the abiotic (mineral) components of their ecosystems.
carbon sink An ecosystem that removes carbon dioxide from
bioinformatics
A discipline at the intersection of biology and computing that analyzes gene and protein sequence data.
carboxysome A protein-bounded compartment containing rubisco to fix CO2 .
biological oxygen demand (BOD) The amount of oxygen
cardiolipin (diphosphatidylglycerol) A double phospholipid
process in methanogenic archaea, so called because the key enzyme can fix CO as well as CO2. the atmosphere, for example by fixation into biomass or by dissolving into marine water.
removed from an environment by aerobic respiration.
linked by glycerol.
biological signature See biosignature.
carotenoids Accessory photosynthetic pigments that absorb photons in the green end of the spectrum.
biomass The mass found in bodies of living organisms. bioprospecting The search for organisms with potential com-
mercial applications. bioremediation The use of microbes to detoxify environmental
contaminants. biosignature (biological signature) A chemical indicator of
life. biosphere The area containing the sum total of all life on
Earth. biosynthesis See anabolism. The building up of complex bio-
molecules from smaller precursors. biotic Referring to processes caused by living organisms.
An oceanic thermal vent containing high concentrations of minerals such as iron sulfide.
black smoker
borreliosis A tick-borne disease caused by Borrelia burgdorferi,
which may involve skin lesions and arthritis; also known as Lyme disease. botulism A food-borne disease caused by a Clostridium botuli-
num toxin, involving muscle paralysis. bradykinin A cell signaling molecule that promotes extravasa-
tion, activates mast cells, and stimulates pain perception.
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catabolism The cellular breakdown of large molecules into smaller molecules, releasing energy. catabolite repression The inhibition of transcription of an operon encoding catabolic proteins in the presence of a more favorable catabolite, such as glucose. catalytic RNA An RNA capable of enzymatic reactions. Also known as a ribozyme. catenane Linked rings of DNA found immediately after repli-
cation of circular chromosomes. cathelicidin An antimicrobial peptide that is synthesized as an inactive precursor and activated extracellularly. cation
A positively charged ion.
cell membrane (plasma membrane, cytoplasmic membrane)
The phospholipid bilayer that encloses the cytoplasm. A transmembrane protein that senses a specific extracellular signal and may be the docking site for a specific virus.
cell surface receptor
cell wall A rigid structure external to the cell membrane. The
molecular composition depends on organism; composed of peptidoglycan in bacteria.
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cell-mediated immunity A type of adaptive immunity, employing mainly T cell lymphocytes. cellular slime mold A slime mold in which the individual cells retain their own cell membranes.
G-5
A microbial opisthokont closely related to
choanoflagellate
animals and fungi. cholesterol
A sterol lipid found in eukaryotic cell membranes.
cellulolytic bacteria Bacteria that catabolyze cellulose; found
Chromosomal DNA complexed with proteins. Usually refers to a eukaryotic chromosome.
in the rumen of cattle fed a high cellulose diet.
chromophore
Cercozoa A taxonomic group of shelled amebas that have thin,
chrysophyte
filamentous pseudopods.
chromatin
A light-absorbing redox cofactor.
A clade of flagellated heterokont protists possessing chloroplasts as secondary endosymbionts; also known as “golden algae.”
chain of infection The serial passage of a pathogenic organism from an infected individual to an uninfected individual, thus transmitting disease.
Chydridomycota (chytridiomycete)
chancre A painless, hard lesion due to an inflammatory reaction at the site of infection with Treponema pallidum, the causative agent of syphilis.
ciliate An Alveolate that has paired cilia.
A protein that helps other proteins fold into their correct tertiary structure. chaperone
Curd that has been cut and piled in order to remove the liquid whey.
cheddared curd
A solid or semi-solid food product prepared by coagulating milk proteins. Its production commonly involves microbial fermentation.
cheese
chemoautotroph An organism that oxidizes inorganic compounds to yield energy and reduce carbon dioxide.
The oxidation of inorganic compounds to yield energy used to reduce carbon dioxide. chemoautotrophy
An organism that oxidizes organic compounds to yield energy. chemoheterotroph
The oxidation of organic compounds to yield energy without the use of light. chemoheterotrophy
chemokine An attractant for white blood cells that is produced by damaged tissues.
A human T-cell membrane protein that binds chemokine hormones, but is also used by HIV for attachment and infection.
chemokine receptor (CCR)
chemolithotroph (lithotroph, chemoautotroph)
See chemo-
A taxonomic group of fungi that produce flagellated zoospores. Includes ruminal endosymbionts and parasites of amphibians.
cistron A functional unit of RNA, containing the information from a single gene. clade
See monophyletic.
class switching
See isotype switching.
classical complement pathway An antibody-mediated path-
way for complement activation. classification The recognition of different forms of life and their placement into different categories. clonal A population of genetically identical cells, all descendants of a single cell. clonal selection The rapid proliferation of a subset of B cells
during the primary or secondary antibody responses. cloning vector A small genome that can carry specific genes
for cloning. cloning The insertion of DNA into a plasmid where it can be replicated.
The inducible expression and insertion of CO2 and bicarbonate (HCO3–) transporters into carboxysomes to enhance CO2 levels near rubisco. CO2-concentrating mechanism (CCM)
coastal shelf Shallow regions of the ocean less than 200 meters
deep.
autotroph.
coccus pl. cocci A spherically shaped microbial cell.
chemostat
A continuous culture system in which the introduced media contains a limiting nutrient.
codon
chemotaxis
The ability of organisms to move towards or away from specific chemicals.
coenzyme A (CoA) A non-protein cellular organic molecule that can carry acetyl groups and participates in metabolism.
Metabolism that yields energy from oxidationreduction reactions without using light energy.
coevolution The evolution of two species in response to one
Chlamydia A phylum of intracellular parasitic bacteria that lose
cofactor A metallic ion or a coenzyme required by an enzyme to perform normal catalysis.
chemotrophy
most of their cell envelope during intracellular growth and generate multiple spore-like structures that escape to infect the next host. Chlorobi A phylum of gram-negative bacteria. They are obli-
gate anaerobes, “green sulfur” phototrophs that photolyze sulfides or H2. A magnesium-containing pigment that captures light energy at the start of photosynthesis. chlorophyll
An organelle of endosymbiotic origin that conducts oxygenic photosynthesis; found in algae and plant cells.
chloroplast
A membranous photosynthetic organelle found in bacterial groups such as Chloroflexus.
chlorosome
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A set of three nucleotides that encodes a particular amino acid.
another.
cointegrate A DNA molecule formed by a single site recom-
bination event joining two participating circular DNA molecules. colony A visible cluster of microbes on a plate, all derived from a single founding microbe. commensal organism An organism that benefits from, but nei-
ther helps nor harms the host. In medical usage, some commensals benefit the host. Commensal organisms are normally found at various nonsterile host body sites. commensalism An interaction between two different species that only benefits one partner.
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competence factor A species-specific secreted bacterial pro-
contrast Differential absorption or reflection of electromagnetic radiation between an object and background that allows the object to be distinguished from the background.
tein that induces competence for transformation.
coral bleaching
compatible solute A small non-toxic molecule that can accumu-
late in a cell to prevent cell water loss in hypertonic environments.
competent A cell that is able to take up DNA from the
The death or expulsion of coral algal symbionts. One cause is an increase in temperature.
environment.
coralline alga An algal species that calcifies its fronds into
complement Innate immunity proteins in the blood that form
hardened shapes similar to corals.
holes in bacterial membranes, killing the bacteria.
core particle A viral capsid enclosing its nucleic acid genome.
complementary DNA (cDNA) A DNA synthesized comple-
core polysaccharide
mentary to an RNA template via reverse transcriptase. complex medium A nutrient-rich growth solution including
undefined chemical components such as beef broth.
A sugar chain that attaches to the glucosamine of lipopolysaccharides and extends outside the cell.
coreceptor A cell surface receptor needed for viral entry along with a primary receptor.
transposon A transposon containing a gene for the transposase enzyme which is needed for replicative transposition.
corepressor A small molecule that must bind to a repressor to
composite transposon A transposon containing genes in
the outer covering of an alveolate.
addition to those of transposition, such as antibiotic resistance or catabolic functions.
cotransduced Genes that are transferred together to a recipient cell during transduction.
compound microscope A microscope with multiple lenses to compensate for lens aberration and increase magnification.
Coulter counter A device to count cells based on increasing
compromised host An animal with a weakened immune
counterstain A secondary stain used to visualize cells that do
complex
system.
allow the repressor to bind operator DNA. cortical alveolus One of the vesicles that forms a network in
electrical resistance as cells pass through a small hole. not retain the first stain.
condensation A chemical reaction that joins two molecules
and produces a water molecule. condenser A lens that focuses parallel light rays from the light source onto a small area of the specimen to improve the resolution of the objective lens.
coupled transport The movement of a substance against its electrochemical gradient (from lower to higher concentration, or from opposite charge to like charge) using the energy provided by the simultaneous movement of a different chemical down its electrochemical gradient.
conditional lethal mutation A mutation that leads to death under one growth condition but permits growth under a second condition.
C-reactive protein A peptide that stimulates the complement
A lawn of organisms that have completely covered
a surface.
Crenarchaeota One of the two major divisions of Archaea, containing sulfur thermophiles and marine mesophiles.
congenital syphilis Syphilis contracted in utero.
cross-bridge An attachment that links parallel molecules such
conjugation Horizontal gene transmission involving cell-cell
as the peptide link between glycan chains in peptidoglycan.
contact. In bacteria, pili draw together the donor and recipient cell envelopes, and a protein complex transmits DNA across. In ciliated eukaryotes, a conjugation bridge forms between two cells connecting their cytoplasm, through which micronuclei are exchanged.
cryocrystallography X-ray crystallography on crystals that are
conjugative transposon A transposon that can be transfered from one cell to another via conjugation.
cryptogamic crust A low-growing ground cover consisting of an algal-fungal symbiont, similar to lichens.
consensus sequence A sequence of nucleotides or amino acids with a common function at many nucleic acid or protein positions. Consists of the base pair or amino acid most frequently found at each position in the sequence.
curd Coagulated milk proteins produced by the combined action of lactic acid-producing bacteria and stomach enzymes of certain mammals such as cattle.
constant region The region of an antibody that defines the
ing chlorophylls a and b. They are closely related to chloroplasts.
confluent
class of a heavy chain or a light chain. Organisms that acquire nutrients from producers, either directly or indirectly. consumers
cascade, induced by cytokines in the liver. Elevated levels in the blood may be associated with heart disease.
flash-cooled in liquid nitrogen. Electron microscopy in which the sample is cooled rapidly in a cryoprotectant medium that prevents freezing. The sample does not need to be stained.
cryoelectron microscopy (cryo-EM)
Cyanobacteria A phylum of photoautotrophic bacteria containcyclic photophosphorylation A photosynthetic process in
which chlorophyll serves as both the initial electron donor and the final electron acceptor. ATP is produced via the proton potential from an electron transport system, but no NADPH is generated.
contig Overlapping fragments of cloned DNA that are contiguous along a chromosome.
cycloserine A polypeptide antibiotic that inhibits peptidogly-
continuous culture A culture system in which new medium is
can synthesis.
continually added to replace old medium. contractile vacuole An organelle in eukaryotic microbes that
cystic fibrosis transmembrane conductance regulator (CFTR) A chloride channel found in respiratory epithelia.
pumps water out of the cell.
Mutations in the CFTR gene lead to cystic fibrosis.
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A membrane protein that donates and receives
depth of field A region of the optical column over which a specimen appears in reasonable focus.
cytokine A small, secreted host protein that binds to receptors on various endothelial and immune system cells, regulating the cells responses.
derepression An increase in gene expression caused by the
cytochrome
electrons.
decrease in concentration of a corepressor.
cytoplasm (cytosol) The aqueous solution contained by the cell
desensitization A clinical treatment to reduce allergic reactions by exposing patients to small doses of the allergen.
membrane in all cells and outside the nucleus (in eukaryotes).
detection The ability to determine the presence of an object.
cytoskeleton A collection of filamentous proteins that impart
detritus Discarded biomass that can be consumed by decomposers.
structure to and aid movement of cells; in a eukaryote, these include intermediate filaments, microtubules and microfilaments. cytotoxic T cell (TC cell) T cells that express CD 8 on their
cell surface and can secrete toxic proteins such as perforin and granzymes. dark-field microscopy The detection of microbes too small to
be resolved by light rays by observing the light they scatter. dead zone An anoxic region of an ocean, devoid of most fish
and invertebrates.
diaphragm A device in a microscope to vary the diameter of the light column, changing the amount of light admitted.
A taxonomic group of protists (Bacillariophyta) known for intricate, silica-containing bipartite shells.
diatom
diauxic growth A biphasic cell growth curve caused by the depletion of the favored carbon source and a metabolic switch to the second carbon source.
phase, in which cells die faster than they replicate.
dichotomous key A tool for identifying organisms, in which a series of yes/no decisions successively narrows down the possible categories of species.
death rate The rate at which cells die; exponential during the
differential medium A growth medium that can distinguish
death phase The period of cell culture following stationary
death phase.
between various bacteria based on metabolic differences.
decay-accelerating factor A host cell membrane protein that
stimulates decay of complement factors and prevents their deposition at the cell surface.
differential stain A stain that differentiates among objects
by staining only particular types of cells or specific sub-cellular structures.
decimal reduction time (D-value) The length of time it takes for a treatment to kill 90% of a microbial population, and hence a measure of the efficacy of the treatment.
diffusion The energy independent net movement of a sub-
A chemical that removes loosely bound stain from
dilution streaking A method of spreading of bacteria on a plate
decolorizer
a specimen. decomposer
in order to obtain colonies arising from an individual bacterium. Organism that consumes dead biomass.
defensins Small, positively charged peptides, produced by ani-
mal tissues, that destroy the cell membranes of invading microbes. A solution of known compounds for organismal growth that contains only the minimal components required for growth.
defined minimal medium
degradosome
stance from a region of high concentration to a region of lower concentration.
complex A
multi-enzyme
complex
that
degrades RNA. degranulate The release of antimicrobial granule contents
by the fusion of granule membranes to cytoplasmic or vacuolar membranes. degron A degradation signal contained within a protein. dehalorespiration The reduction of halogenated organic mol-
Dinoflagellata (dinoflagellate) A taxonomic group of tertiary endosymbiont alveolates with two flagella, one of which is wrapped distinctively around the cell equator.
An enzyme that coordinately oxygenates two adjacent ring carbons.
dioxygenase
diphosphatidylglycerol See cardiolipin. diplococcus The paired cocci of Neisseria species. direct repeat Two identical sequences in a DNA molecule,
aligned in the same direction. Discicristata (discicristate) A protist taxonomic group whose
members have mitochondria with distinctive disc-shaped cristae. disinfection The removal of pathogenic organisms from inani-
ecules by H2.
mate surfaces.
deletion The loss of nucleotides from a DNA sequence.
dissemination The movement of virions from the initial site of
denature The loss of secondary and tertiary structure in a
infection to other regions of the body.
protein or nucleic acid due to high temperature or chemical treatment.
dissimilation
dendritic cell An antigen-presenting white blood cell that pri-
marily takes up small soluble antigens from its surroundings. Energy-yielding metabolism that involves the reduction of nitrate (NO3–) to nitrite (NO2–), diatomic nitrogen, N2, and in some cases ammonia (NH3). denitrification
A heterotrophic bacterium that uses nitrate (NO3–) as the final electron acceptor in an electron transport chain and produces nitrogen gas, N2, or ammonia.
denitrifying bacterium
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An organism’s catabolism or oxidation of nutrients to inorganic minerals that are released into the environment.
dissimilatory denitrification Metabolic reduction of nitrate or nitrite to yield energy; anaerobic respiration of nitrate or nitrite. dissimilatory metal reduction A type of anaerobic respiration
that uses metal cations as terminal electron acceptors. dissimilatory nitrate reduction The reduction of nitrate and
nitrite through a series of decreased oxidation states back to atmospheric nitrogen and/or ammonia.
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dissimilatory nitrate reduction to ammonia (DNRA) A metabolic pathway in which nitrate serves as an electron acceptor and hydrogen gas (H2) as the electron donor forming water and ammonia.
electrogenic A transport system that results in a net movement of charged molecules across a membrane.
D-loop formation A triplex DNA molecule that forms as an
electron donor A reduced molecule (e.g., NADH) that can
intermediary structure during generalized recombination. DNA control sequence A region of DNA, such as the promoter
region, that controls the expression of structural genes but is not itself transcribed to RNA. An enzyme cells use to create a covalent bond at a nick in the phosphodiester backbone. Also used in molecular biology laboratories to join pieces of DNA.
DNA ligase
DNA microarray (or microchip) A microchip containing short
DNA sequences corresponding to all the open reading frames in an organism affixed to specific locations. It can be used to measure the amount of specific mRNA molecules transcribed in cells. A labeled DNA sequence that hybridizes only to a particular complementary DNA sequence.
DNA probe
DNA reverse-transcribing virus A virus with a double-
stranded DNA genome that generates an RNA intermediate and thus requires reverse transcriptase to generate progeny DNA genomes. DNA sequencing A technique to determine the order of bases
in a DNA sample. A technique in which fragments of similar genes are combined to generate new genes with potentially new or improved functions. DNA shuffling
domain 1. One of the three major taxonomic groups of living
organisms. 2. A region of a protein with a particular structure and function. dormant cell A vegetative cell that has entered a physiological
state where it remains metabolically active but fails to replicate or form colonies. Also called the viable-but-nonculturable state, it is distinct from spore forms such as endospores. doubling time The generation time of bacteria in culture. The amount of time it takes for the population to double. downstream processing The recovery and purification of the
commercial product produced by industrial microbes. dry weight The weight of a population of cells after the water
has been removed. early gene A viral gene expressed early in the infectious cycle. eclipse period The time after viral genome injection into host cell but before complete virions are formed. ecosystems Communities of species plus their environment
(habitat). ectomycorrhizae Mycorrhizae that colonize the surface of plant
roots. Their mycelia do not penetrate the root cells. ectoparasite A harmful organism that colonizes the surface of
a host. edema Tissue swelling due to fluid accumulation. edema factor (EF) A component of anthrax toxin with adenyl-
ate cyclase activity. A type of potential energy formed by the combined concentration gradient of a molecule and the electrical potential across a membrane.
electrochemical potential
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electron acceptor An oxidized molecule (e.g., NAD+) that can
accept electrons. donate electrons. electron microscope A microscope that obtains high resolu-
tion and magnification by focusing electron beams on samples using magnetic lenses. electron transport chain See electron transport system.
A collection of membrane proteins that converts the energy of redox reactions into a proton potential.
electron transport system (ETS) (electron transport chain)
electronegativity The affinity of an atom for electrons. The
greater the electronegativity, the stronger the attraction for electrons. electroneutral A transport system that does not result in any net change in charge across the membrane. electrophoresis A technique to separate charged proteins and
nucleic acids based on how rapidly they migrate in an electric field through a gel. electrophoretic mobility shift assay (EMSA) A technique to observe DNA-protein interactions based on the ability of a bound protein to slow the voltage-driven migration of DNA through a gel. electroporation A laboratory technique that temporarily makes the cell membrane more leaky to allow the uptake of DNA. elementary body The endospore-like form of Chlamydia
transmitted outside host cells. eluviated horizon The soil layer below the aerated horizon that periodically experiences water saturation from rain.
A glycolytic pathway in which glucose-6-phosphate isomerizes to fructose-6phosphate, ultimately yielding 2 pyruvate, 2 ATP, and 2 NADH.
Embden-Myerhof-Parnas (EMP) pathway
emission wavelength The wavelength of light emitted by a fluorescent molecule. It is of a lower energy and longer wavelength than the excitation wavelength. empty magnification Magnification without an increase in
resolution. endemic A disease that is always present in a population, although the frequency of infection may be low. endemic area The geographical region where an pathogenic
organism or virus is found, usually colonizing or infecting animals indigenous to the area. endergonic A reaction that requires an input of energy to
proceed. endocarditis An inflammation of the heart’s inner lining. endocytosis The budding in of the cell membrane to form a
vesicle that contains extracellular material. endogenous retrovirus A retroelement that contains gag, env,
and pol genes. endoliths Bacteria that grow within the crystals of solid rock. endomycorrhizae Mycorrhizae in which fungal hyphae penetrate plant root cells. endoparasite A parasite that lives inside the host.
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endosome A vesicle formed from the pinching in of the cell
During centrifugation, cell fractions migrate to locations in the centrifuge tube that match their density.
membrane.
equivalence The antigen:antibody ratio that leads to immuno-
endospore
A durable, inert, heat-resistant spore that can remain viable for thousands of years.
precipitation of large, insoluble complexes. error-prone repair Low accuracy DNA repair mechanisms that
endosymbiont An organism that lives as a symbiont inside
allow mutations.
another organism.
error-proof repair DNA repair mechanisms that minimize the
endophyte An endosymbiont of vascular plants.
endosymbiosis An intimate association between different
formation of mutations.
species in which one partner population grows within the body of another organism.
erythema migrans A bulls’ eye rash characteristic of borreliosis
(Lyme disease).
endosymbiosis theory The theory that mitochondria and
essential nutrient A compound that an organism cannot
chloroplasts were originally free-living prokaryotes that formed an internal symbiosis with early eukaryotes.
synthesize and must acquire from the environment in order to survive.
Lipopolysaccharides in the outer membrane of gram negative bacteria that become toxic to the host after the bacterial cell has lysed.
ethanolic fermentation A fermentation reaction yielding 2 ethanol and 2 CO2 as products.
energy The ability to do work.
through an oxygen, C—O—C. Found in archaeal phospholipids between the glycerol and the fatty acids.
endotoxin
energy carrier Molecules in the cell, such as ATP and NADH,
that serve as energy currency. They are produced during catabolic reactions, and can be used to drive energy-requiring reactions. enhancer A non-coding DNA region in eukaryotes that can lead to activation of transcription when bound by the appropriate transcription factor. Its location on the chromosome can be far removed from the regulated gene. enriched medium A growth solution for fastidious bacteria, consisting of complex media plus additional components. enrichment culture The use of selective growth media to allow only certain microbes to grow.
ether link A covalent attachment of two organic molecule
Eukarya One of the three domains of life, consisting of organ-
isms with a last common ancestor not shared with members of Archaea or Bacteria. Cells possess nuclei, unlike cells of bacteria and archaea. A cell that contains a nucleus and is a member of the domain Eukarya.
eukaryote pl. eukaryotes
Eumycota True fungi, a taxonomic group of opisthokont eukaryotes with chitinous cell walls; the group most closely related to animals. euphotic (photic) zone The region of the ocean that receives
enterotoxins
Proteins that damage the intestine of the host and cause diarrhea, produced by some gram-negative pathogens.
light capable of supporting photosynthesis. Also known as the photic region.
enthalpy A measure of the heat energy in a system.
Euryarchaeota One of the two major divisions of Archaea, containing methanogens, halophiles, and extreme acidophiles.
Entner-Doudoroff (ED) pathway A glycolytic pathway in which glucose-6-phosphate is initially oxidized to 6-phospho-gluconate, and ultimately yields 1 pyruvate, 1 ATP, 1 NADH and 1 NADPH. entropy A measure of the disorder in a system. envelope Structures external to the cell membrane such as a
cell wall or outer membrane. enzyme A biological catalyst; a protein or RNA that can speed
up the progress of a reaction without itself being changed. eosinophil A white blood cell that stains with the acidic dye
eosin, and secretes compounds that facilitate innate immunity. epidemic A disease outbreak in which large numbers of indi-
viduals in a population become infected over a short time. epidermis The outer protective cell layer in most multicellular
animals. Warm upper layer of a freshwater lake, above the thermocline, a region where temperature drops steeply.
epilimnion
episome A DNA element that can exist as part of the chromo-
some or independently, as a plasmid. epitope See antigenic determinant. equilibrium A dynamic state in which there is no net change
in a reaction. equilibrium density gradient centrifugation A technique to
separate cell components based on their differential densities.
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A lake in which overgrowth of heterotrophic microbes has eliminated oxygen, leading to a decrease in animal life.
eutrophic lake
eutrophication A sudden increase of a formerly limiting nutrient in an aquatic environment, leading to overgrowth of algae and grazing bacteria and subsequent oxygen depletion.
A protist group that shows extensive evolutionary reduction and lacks mitochondria.
Excavata
excitation wavelength The wavelength of light that must be absorbed by a molecule in order for the molecule to fluoresce. exergonic A spontaneous reaction that releases free energy. exit site (E site) The region of the ribosome that holds the
uncharged, exiting tRNA. Fusion of vesicles with the cell membrane to release vesicle contents extracellularly.
exocytosis
exon An expressed or protein-coding portion of a eukaryotic
gene. exonuclease An enzyme that cleaves DNA from the end.
A thick extracellular matrix of polysaccharides and entrapped materials that forms around the microbes in a biofilm.
exopolysaccharide (EPS)
exotoxin Protein toxin, secreted by bacteria, that kills or damages host cells.
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exponential A mathematical function that is raised to a power. It gives a curve whose slope continually increases. exponential phase A phase in bacterial cell culture when bac-
teria are growing at their maximal possible rate given the conditions, usually exponentially. Same as log phase. extravasation The movement of immune cells out of blood
vessels and into surrounding infected tissue. extremophile An organism that only grows in an extreme environment; that is, an environment including one or more conditions that are “extreme” relative to the conditions for human life. F – cell The DNA recipient cell in conjugation
A filamentous structure for motility. In prokaryotes, a helical protein filament attached to a rotary motor; in eukaryotes, an undulating cell membrane-enclosed complex of microtubules and ATP driven motor proteins.
flagellum pl. flagella
flavin adenine dinucleotide (FADH2, FAD+)
An energy carrier in the cell that can donate (FADH2) or accept (FAD+) electrons. flavonoids Signaling molecules released from legumes to attract nitrogen-fixing rhizobia.
Biofilms formed on microbial filaments and the soluble organic content of wastewater.
flocs
F(ab)2 region The amino-terminus variable “arms” of an anti-
fluid mosaic model A model of the cell membrane in which proteins are free to diffuse laterally within the membrane.
body that bind to a specific antigen.
fluorescence
F + cell The DNA donor cell that transmits the fertility factor F+
The emission of light from a molecule that absorbed light of a shorter, higher energy wavelength.
to an F – cell during conjugation.
fluorescence resonance energy transfer (FRET)
factor H A normal serum protein that prevents the inadvertent
activation of complement in the absence of infection. facultative An organism that can grow in the presence or absence of a given environmental factor, such as oxygen. facultative aerobe (facultative anaerobe) An organism that
can grow either in the presence or absence of oxygen. facultative anaerobe See facultative aerobe. facultative intracellular pathogen A pathogen that can live
either inside host cells or outside host cells. fatty acid synthase complex A collection of all the enzymes
and binding proteins necessary for fatty acid synthesis. The region of an antibody that binds to specific receptors on host cells in an antigen-independent manner. It is found in the carboxy-terminal “tail” region of the antibody.
Fc region
feline leukemia virus (FeLV)
A retrovirus that is a major cat
pathogen. femtoplankton Marine or aquatic viruses. fermentation The production of ATP via substrate level phos-
phorylation, using organic compounds as both electron donors and electron acceptors. fermentative metabolism See fermentation. fermented food Food products that are biochemically modi-
fied by microbial growth. An iron- and sulfur-containing protein that transfers electrons in electron transport systems. ferredoxin
fertility factor (F factor) A specific plasmid (transferred by an
F+ donor cell) that contains the genes needed for pilus formation and DNA export. field The background observed in microscopy as opposed to
the specimen of interest. filamentous virus A viral structure type consisting of a helical
capsid surrounding a single stranded nucleic acid. filopodium A needle-shaped pseudopod. fimbria pl. fimbriae See pilus. Firmicutes A phylum of low GC content gram-positive
bacteria. fixation The adherence of cells to a slide by a chemical or heat
treatment. flagellate An alveolate that has paired flagella.
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The detectable transfer of fluorescent energy from one molecule to another. Since the participating molecules must be near each other, FRET can be used to monitor protein-protein interactions in cells and is also used in real time PCR.
fluorescence-activated cell sorter (FACS) A device that can count cells and sort them based on differences in fluorescence. fluorescent-focus assay An assay to detect viruses that do not kill host cells, based on intracellular detection of viruses using anti-viral antibodies or green fluorescent protein modified viruses. fluorophore A fluorescent molecule used to stain specimens for fluorescent microscopy. focal plane A plane that contains the focal point for a given
lens. focal point The position at which light rays that pass through
a lens intersect. focus
A group of cells infected by a virus.
folate (folic acid) A heteroaromatic cofactor, that is required
by some enzymes. An inanimate object on which pathogens can be transmitted from one host to another. fomite
food contamination See food poisoning. food irradiation The exposure of food to ionizing radiation,
sterilizing the food for long-term storage. food poisoning The presence of human disease-causing
microbial pathogens or toxins in food. food spoilage Microbial changes that render a food unfit or
unpalatable for consumption. food web A network of interactions in which organisms obtain or provide nutrients for each other, for example by predation or by mutualism. foraminiferan An ameba with a calcium carbonate shell and a
helical arrangement of chambers. forespore In sporulation of gram-positive bacteria, the smaller
cell compartment formed through asymmetrical cell division; it develops into the endospore. fossil fuels Ancient organismal remains that have been converted to hydrocarbons through microbial digestion followed by reduction under high pressure underground; extracted and burned by humans for energy.
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F-prime (F) factor or F plasmid A fertility factor plasmid that
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genus name A taxonomic rank consisting of closely related
contains some chromosomal DNA.
species.
frameshift mutation A gene mutation involving the insertion
geochemical cycling
or deletion of nucleotides that cause a shift in the codon reading frame.
geomicrobiology See biogeochemistry.
frank pathogen See primary pathogen.
geosmin A molecule released from decaying Streptomyces cells;
freeze-drying The removal of water from food, by freezing
under vacuum, to limit microbial growth.
it causes the characteristic odor of soil and can affect the taste of drinking water.
fruiting body A multicellular fungal or bacterial reproductive
germ theory of disease The theory that many diseases are
The global interconversion of various inorganic and organic forms of elements.
structure.
caused by microbes.
frustule The silica bipartite shell produced by diatoms.
germicidal A substance that kills cells but not spores.
functional group A cluster of covalently bound atoms that
germination The activation of a dormant spore to generate a
behaves with specific properties and functions as a unit.
vegetative cell.
fungus pl. fungi A heterotrophic opisthokont eukaryote with
Gibbs free energy change (DG) In a chemical reaction, a mea-
chitinous cell walls. Mainly includes Eumycota, but traditionally may refer to fungus-like protists such as the oomycetes.
sure of how much energy available to do work is released or required as the reaction proceeds.
fusion peptide A portion of a viral envelope protein that
gliding motility The movement of cells individually or as a col-
changes shape to facilitate envelope fusion with the host cell membrane.
global warming The overall rise in temperature of our bio-
gain-of-function mutation A mutation that enhances the
sphere over the past hundred years.
activity or allows new activity of a gene product. Contrast with loss-of-function mutation.
glucosamine A glucose modified with an amine group.
gamogony The differentiation of parasitic haploid cells into
male and female gametes. gas chromatography
A technique to separate and quantify
gasses in a sample. gas vesicle An organelle that traps gasses to increase buoy-
ancy of aquatic microbes. GC content The proportion of an organism’s genome consisting of guanine-cytosine base pairs. gene fusion A technique to measure control of gene transcrip-
tion or translation by inserting a reporter gene into a target gene. The reporter relies on both the promoter and the ribosome binding site of the target gene. Also known as reporter fusion. gene silencing The inhibition of transcription from an mRNA
by a complementary small interfering RNA. Also known as RNA interference or RNAi. gene splicing The cutting and pasting of DNA to produce new
gene variants. generalized
recombination Recombination between two
DNA molecules that share long regions of DNA homology. generalized transduction A phage-mediated gene transfer pro-
cess in which any donor gene can be transferred to a recipient cell. generation time The species-specific time period for doubling
of a population (for example, by bacterial cell division) in an given environment, assuming no depletion of resources. genetic analysis Determination of the function of cell RNAs
and proteins based on the phenotype of cells in which the gene encoding the RNA or protein is mutated. genome The complete genetic content of an organism. The
sequence of all the nucleotides in a haploid set of chromosomes. genomic island A region of DNA sequence whose properties indicate that it has been transferred from another genome. Usually comprises a set of genes with shared function, such as pathogenicity or symbiosis support.
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lective over surfaces using pili.
An enzyme that condenses NH4+ with 2-oxoglutarate to form glutamate. The condensation requires reduction by NADPH. glutamate dehydrogenase (GDH)
glutamate synthase (GOGAT) An enzyme that converts 2-
oxoglutarate plus glutamine into two molecules of glutamate. An enzyme that condenses NH4+ with glutamate to form glutamine.
glutamine synthetase (GS)
glycan See polysaccharide. glycolysis The catabolic pathway of glucose oxidation to pyru-
vate, generating ATP and NADH. An alternative to the tricarboxylic acid cycle, induced under low glucose conditions. glyoxalate bypass
gnotobiotic animal An animal that is germ-free or colonized by a known set of microbes. Golgi complex A series of membrane stacks that modifies proteins and helps sort them to the correct eukaryotic cell compartment. Gram stain A differential stain that distinguishes between cells that possess a thick cell wall and retain a positively-charged stain (gram-positive) from cells with a thin cell wall and outer membrane that fail to retain the stain (gram-negative). gramicidin A peptide antibiotic that disrupts membrane
integrity. gram-negative Cells that do not retain the Gram stain. gram-negative bacteria Bacteria that fail to retain the Gram stain because they have a thin cell wall. gram-positive Cells that do retain the Gram stain and appear dark purple after staining. gram-positive bacteria Bacteria that retain the Gram stain by virtue of their thick cell wall reinforced by teichoic acids. granuloma A thick lesion formed around a site of infection. granzyme A cytotoxic T cell-secreted enzyme that damages
target cells.
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The first level of consumers that feed directly on
producers.
hepatitis An inflammation of the liver, caused by infection or by exposure to a toxic substance.
green alga (chlorophyte) Microbial eukaryotes containing chloroplasts; primary endosymbionts, closely related to plants (Viridiplantae).
herd immunity The slowing of disease spread due to some population members being immune and thus unable to infect other members.
greenhouse effect The trapping of solar radiation heat in the atmosphere by CO2; a cause of global warming.
heteroaromatic Aromatic rings containing non-carbon atoms
griseofulvin An
heterocyst A specialized nitrogen-fixing cell in filamen-
grazers
anti-fungal antibiotic that inhibits cell
division.
such as the nitrogenous bases found in nucleotides. tous cyanobacteria that maintains a reduced environment and excludes O2.
group translocation A form of active transport in which the transported molecule is modified after it enters the cell, thus keeping a favorable inward concentration gradient for the unmodified extracellular molecule.
Heterokonta (heterokont) A taxonomic group of protists whose flagellated members possess two flagella of unequal length.
growth factor A compound needed for the growth of only cer-
heterolactic fermentation A fermentation reaction in which
tain cells.
the products are lactic acid, ethanol and CO2.
growth rate The rate of increase in population number or
heterotroph An organism that relies on external sources of
biomass.
organic carbon compounds for biosynthesis.
gut-associated lymphoid tissue (GALT) Lymphatic tissues
Hfr A high frequency recombination bacterial strain, caused by
such as tonsils and adenoids that are found in conjunction with the gastrointestinal tract and contain immune cells.
high-performance liquid chromatography (HPLC) A tech-
Haber process
Industrial nitrogen fixation, in which dinitrogen is hydrogenated by methane (natural gas) under extreme heat and pressure to form ammonia.
nique for the high-resolution separation of chemical products through columns packed with beads of various physical properties.
habitat The environment where a particular species grows.
histone A protein that helps compact eukaryotic chromosomes
the presence of a chromosomally integrated F factor.
Hadean eon The first period of Earth’s existence, from 4.5 to
in nucleosomes.
3.8 gigayears (109) ago.
holdfast Adhesion factors secreted by the tip of a stalk to firmly
half-life The amount of time it takes for one half of a radioac-
attach an organism to a substrate.
tive sample to decay.
Holliday structure (junction) A cross-like configuration of recombining DNA molecules that forms during generalized recombination.
haloarchaeon
An archaeal species that inhabits high salt
environments. Halobacteriales An order of Euryarchaeota that contains
homologous Genes that derived from a common ancestral
haloarchaea.
gene.
halophile An organism that requires a high extracellular
hopane (hopanoid) Five-ringed hydrocarbon lipids found in
sodium chloride concentration for optimal growth.
bacterial cell membranes.
halorhodopsin (HR) An archaeal light-driven chloride pump.
horizontal gene transfer The passage of genes from one cell
hapten A small compound that must be conjugated to a larger
into another mature cell.
carrier antigen in order to elicit production of an antibody that binds to it.
horizontal transfer See horizontal gene transfer.
haustorium A bulbous hyphal extension of a fungal plant
organism into another, non-progeny organism.
pathogen into the host cell.
hormogonium A short motile chain of three to five cells produced by filamentous bacteria to disseminate their cells.
heat-shock protein A chaperone protein that is induced by high temperature stress. heat-shock response A coordinated response of cells to higher than normal temperatures. It includes changes in the membrane and expression of heat shock genes.
horizontal transmission The transfer of a pathogen from one
host range The species that can be infected by a given
pathogen. human immunodeficiency virus (HIV) A human-specific retrovirus that is the causative agent of AIDS.
heavy chain The larger protein that comprises an antibody. An antibody contains two identical heavy chain and two light chain proteins.
humic material Phenolic molecules, derived from lignin, that
helicase A protein that unwinds the DNA helix.
antibodies.
helper T cell (TH cell) A T lymphocyte that secretes cytokines
humus Organic breakdown products of lignin that are found
that modulate B-cell class switching.
in the soil.
heme An organic molecule containing a ring of conjugated
hybridization The annealing of a nucleic acid strand with another nucleic acid strand containing a complementary sequence of bases. The binding of one nucleic acid strand with a complementary strand.
double bonds surrounding an iron atom. It is involved in redox reactions and oxygen binding.
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are resistant to degradation and hence very stable in soil. humoral immunity A type of adaptive immunity mediated by
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hydric soil Soil that undergoes periods of anoxic water
saturation. hydrogen bond An electrostatic attraction between a hydrogen
bound to an oxygen or nitrogen and a second, nearby oxygen or nitrogen. hydrogenosome Found in some eukaryotes, an organelle that
ferments carbohydrates in a pathway generating H2; may have evolved from mitochondria.
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immunization The stimulation of an immune response by deliberate inoculation with a weakened pathogen, in hopes of providing immunity to disease caused by the pathogen. immunogen See antigen. immunogenicity A measure of the effectiveness of an antigen in eliciting an immune response. immunoglobulin A member of a family of proteins that contain
hydrogenotrophy The use of molecular hydrogen (H2) as an
a 110-amino-acid domain with an internal disulfide bond. Members include antibodies and major histocompatibility proteins.
electron donor for a variety of electron acceptors.
immunological specificity
hydrological cycle The cyclic exchange of water between
atmospheric water vapor and the Earth’s bodies of liquid water. hydrolysis
The cleaving of a bond by the addition of a water
The ability of antibodies produced in response to a particular epitope to bind that epitope almost exclusively. Antibodies made to one epitope bind only weakly, if at all, to other epitopes.
molecule.
immunomodulin A protein made by normal bacterial flora that
hydrophilic Soluble in water. Includes ionic and polar
influences the host immune response by modifying the secretion of host proteins, such as a cytokine.
molecules. hydrophobic Non-polar; insoluble in water. cycle A carbon fixation pathway that condenses hydrated CO2 and acetyl-CoA to form 3-hydroxypropionate. 3-hydroxypropionate
hyperthermophile An organism with optimum growth at
extremely high temperatures (generally considered as above 80°). hypertonic An environment that has more solutes than another
environment separated by a membrane. Water will tend to flow towards the hypertonic solution. hypha Thread-like filaments forming the mycelium of a
fungus. hypolimnion The lower, colder region of a body of freshwater,
below the epilimnion. hypotonic An environment that has fewer solutes than another
environment separated by a membrane. hypoxia
A state of lower than normal oxygen concentration.
immunoprecipitation The antibody mediated cross-linking of antigens to form large, insoluble complexes. Immunoprecipitation is used in research labs and is normally only seen in vitro.
A laboratory technique to uncover bacterial genes that are only expressed during growth within a host.
in vivo expression technology (IVET)
incubation stage The time after infection when an organism multiplies but the patient remains asymptomatic. index case Also known as patient zero, the first case of an
infectious disease and an important piece of data for helping contain the spread of disease. indigenous flora Microbes found naturally in a particular location, often in association with a food substrate. inducer A molecule that binds to a repressor and prevents repressor binding to the operator sequence. inducer exclusion The ability of glucose to cause metabolic
The recognition of the class of a microbe isolated
changes that prevent the cellular uptake of less favorable carbon sources that could cause unnecessary induction.
idiotype Amino acid differences in hypervariable regions (N-
induction Increased transcription of target genes due to an inducer binding to a repressor and preventing repressor-operator binding.
identification
in pure culture. terminus of heavy and light chains) within a single antibody class in an individual that allow recognition of different antigens. IgA An antibody class containing the alpha heavy chain. It can be
secreted and found in tears, saliva, breast milk, etc. IgD An antibody isotype that contains the delta heavy chain
and is found on B cell membranes. IgE An antibody isotype that contains the epsilon heavy chain
and is involved in degranulation of mast cells. IgM The first antibody isotype detected during the early stages
of an immune response. It contains the Mu heavy chain and is found as a pentamer in serum. immersion oil An oil with a refractive index similar to glass
that minimizes light ray loss at wide angles, thereby minimizing wavefront interference and maximizing resolution. immune avoidance The changing of cell surface proteins by
pathogens to prevent antibody detection and prolong infection. immune system An organism’s cellular defense system against
pathogens. immunity The resistance to a specific disease.
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industrial fermenter The equipment used to grow microbes on
an industrial scale. industrial microbiology The commercial exploitation of microbes. industrial strain A microbial strain whose characteristics are
optimized for industrial use. infection The growth of a pathogen or parasite in or on a host. infection cycle The route a pathogen takes as it passes from one host into another. infection thread A column of rhizobial cells that projects down
a tube into a plant root cell during the early stages of the rhizobia-legume symbiosis. infectious dose (ID50) The number of microbes required to
cause disease symptoms in half of an experimental group of hosts. informational gene A DNA sequence that encodes a product
essential for transcription or translation. A technique based on infrared spectrometry that can identify small molecules in a sample; it can be used to measure levels of gasses in the upper atmosphere.
infrared chemical analysis
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initiation factors Proteins required for initiation of protein
isotonic A state in which two solutions separated by a semi-
synthesis in bacteria. injera A highly fermented Ethiopian flat bread using the grain
permeable membrane are in osmotic balance. A cell in an isotonic environment will neither gain nor lose water.
teff.
isotope
innate immunity See non-adaptive immunity. inner leaflet The layer of the cell membrane phospholipid bilayer that faces the cytoplasm. inner membrane In gram-negative bacteria, the membrane in
contact with the cytoplasm, equivalent to the cell membrane. insertion The addition of nucleotides into the middle of a DNA
sequence. insertion sequence (IS) A simple transposable element that
consists of a transposase gene flanked by short, inverted repeat sequences that are the target of transposase. integrase An enzyme that catalyzes the integration, via a dou-
ble cross-over, of one DNA molecule into another at a specific sequence. integron A large transposon that can contain many different antibiotic resistance genes. interference The interaction of two wave fronts. Interference can be additive (amplitudes in phase, constructive) or subtractive (amplitudes out of phase, destructive). interference microscopy Observation of an object using con-
trast enhanced by superimposing interference bands upon an image to accentuate small differences in refractive indexes. interferon A host-secreted immuno-modulatory protein that
inhibits viral replication. interleukin 1 (IL-1) A cytokine release by macrophages. intermediate filament A eukaryotic cytoskeletal protein that is
composed of various proteins depending on the cell type.
An atom of an element with a specific number of neutrons. For example carbon-12, carbon-13, and carbon-14 are all isotopes of carbon.
isotope ratio The ratio of amounts of two different isotopes of an element. The isotope ratio may serve as a biosignature if the ratio between certain isotopes of a given element is altered by biological activity. isotype A species-specific antibody class, defined by the
structure of the heavy chain. IgG, IgA, and IgE are examples of isotypes. A change in the predominant antibody isotype produced by a cell. isotype switching (class switching)
joules (J) The standard SI unit for energy. Kaposi’s sarcoma A malignancy originating in endothelial or lymphatic cells that is often found in late stage AIDS patients.
A heterokont protist, also known as “brown algae,” that grows as multicellular sheets at the water’s surface.
kelp
kimchi
A popular Korean food based on brine-fermented
cabbage. Kirby-Bauer test Method for determining antibiotic susceptibility. Antibiotic-impregnated disks are placed on an agar plate whose surface has been confluently inoculated with a test organism. The antibiotic diffuses away from the disk and inhibits growth of susceptible bacteria. The width of the inhibitory zone is proportional to the susceptibility of the organism. knockout mutation A mutation that completely eliminates the activity of the gene product.
an E. coli-produced receptor injected into host cells.
Four criteria that should be met for a microbe to be designated the causative agent of an infectious disease.
intracellular parasite A parasite that lives within a host cell.
Korarchaeota
intron In eukaryotic genes, an intervening sequence that does
labile toxin (LT)
intimin A pathogenic E. coli adhesion protein that binds tightly to
not code for protein and is spliced out of the mRNA prior to translation. inversion A flipping of a DNA fragment within a chromosome.
Inversion may allow or repress the transcription of a particular gene. inverted repeat A DNA sequence that is found in an identical
Koch’s postulates
A deeply branching division of Archaea.
An E. coli enterotoxin, destroyed by heat, that increases cellular cAMP concentrations. lactate fermentation A fermentation reaction that generates
lactic acid from reduction of pyruvic acid. lactic acid fermentation See lactate fermentation. lag phase A phase of slow growth or no growth right after bac-
teria are inoculated into new media.
but inverted form at two sites on the same double helix (e.g., 5’ ATCGATCGnnnnnnCGATCGAT 3’).
lamellar pseudopod A pseudopod with a sheet-like morphology.
ionophore A small organic molecule that binds to an ion and
laminar flow biological safety cabinet An air filtration appli-
solubilizes it in the membrane, allowing the ion to move down its electrochemical gradient.
ance that removes pathogenic microbes from within the cabinet. Langerhans cell Specialized, phagocytic dendritic cells that are
irradiation The exposure of a substance to high-energy elec-
the predominant cell type in skin-associated lymphatic tissue.
tromagnetic radiation, usually for the purpose of sterilization.
laser scanning confocal microscopy A type of fluorescence
isoelectric focusing A technique that separates proteins based
on their charge.
microscopy in which the excitation and emitted light are focused together, producing high resolution images.
isoelectric point The pH at which there is no net charge on an amino acid or a protein.
late gene A gene that is expressed late in the viral infectious cycle, such as genes for capsid proteins or lysozyme.
isolate A microbe that has been obtained from a specific location and grown in pure culture.
latent period The time in the viral life cycle when progeny virions have formed but are still within the host cell.
isoprenoid Condensed isoprene chains, found in archaeal
latent state When a pathogenic agent is dormant in the host
membrane lipids.
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and can not be cultured.
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late-phase anaphylaxis Anaphylaxis caused by leukotrienes
released by eosinophils recruited by mast cells. lateral transfer See horizontal gene transfer.
The tendency of a reaction at equilibrium to return to equilibrium after the concentrations of products or reactants is altered. law of mass action
leaching
The process of metal dissolution from ores.
leader sequence A short DNA sequence preceding a structural gene. In amino acid operons it contains multiple codons for the amino acid synthesized by the downstream structural genes. The leader sequence and translating ribosome help determine whether the structural genes are transcribed.
One of the two lipid layers in a phospholipid bilayer. The inner leaflet of the cell membrane faces the cytoplasm.
leaflet
leaven The generation of air spaces in bread due to carbon
dioxide production by yeast fermentation. leghemoglobin An iron-bearing plant protein that sequesters oxygen to maintain an anoxic environment for nitrogenase within cells containing bacteroids.
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lymph node A secondary lymphatic organ, formed by the convergence of lymphatic vessels, that traps foreign particles from local tissue and presents them to resident immune cells. lyophilization A method to freeze-dry microbes or food for
long term storage. lysate The contents of broken cells; may include virus
particles. lyse
To break open cells by disruption of their cell membrane.
lysis The rupture of the cell by a break in the cell membrane. lysogen A bacterial cell that harbors a complete, yet quiescent,
phage genome. Depending on the phage, the phage genome may be integrated into the bacterial chromosome or exist as an autonomous plasmid in the cell. lysogeny A viral life cycle in which the viral genome integrates
into and replicates with the host genome, but retains the ability to initiate host cell lysis. lysosome An acidic eukaryotic organelle that aids digestion of molecules. Not found in plant cells.
lentivirus A member of a family of retroviruses that propagate
lytic A viral life cycle in which the virus produces new virions and lyses the cell, releasing virions.
slowly. An example is HIV.
M cell A phagocytic innate immune cell (microfold cell) found
lethal dose (LD50)
between intestinal epithelial cells.
lethal factor (LF) A component of anthrax toxin that cleaves
A differential, selective medium that selects for gram-negative bacteria and can differentiate between lactose fermenters and non-fermenters.
A measure of virulence, it is the number of bacteria or virions required to kill 50% of an experimental group of hosts.
host protein kinases. An organism formed by a mutualistic relationship between algae and fungi. lichen
MacConkey medium
macronucleus A form of nucleus found in ciliates, derived from gene amplification and rearrangement of micronuclear DNA, that contains actively transcribed genes.
The smaller of the two different proteins comprising an antibody. Each antibody contains two heavy chains and two light chains.
macronutrient A nutrient an organism needs in large
light microscopy Observation of a microscopic object based on light absorption and transmission.
ing cell of the immune system. magnetosome An organelle containing the mineral magnetite
lignin A complex aromatic organic compound that forms the
that allows microbes to sense a magnetic field.
key structural support for trees and woody stems.
magnetotaxis The ability to direct motility along magnetic
light chain
quantities. macrophage A mono-nuclear, phagocytotic, antigen present-
limiting nutrient A nutrient that is in short supply and limits
field lines.
growth.
An increase in the apparent size of a viewed object as an optical image.
lipopolysaccharide (LPS) Structurally unique phospholipids
found in the outer leaflet of the outer membrane in gram-negative bacteria. Many are endotoxins.
magnification
Transmembrane cell proteins important for recognizing self and for presenting foreign antigens to the adaptive immune system.
major histocompatibility complex (MHC)
(chemolithotroph) An autotrophic organism that gains energy by oxidizing inorganic compounds. See chemoautotroph.
malaria A disease caused by the apicomplexan Plasmodium falciparum, transmitted by mosquitoes.
lithotrophy (chemolithotrophy) Energy-yielding metabolism
malolactic fermentation Fermentation of
lithotroph
that uses an inorganic electron donor; usually includes fixation of CO2 into biomass.
L-malate (a side product of glucose fermentation) by Oenococcus oeni bacteria, an important process in wine making.
littoral zone
The upper water layer of aquatic habitats that light can penetrate.
marine snow Microbial biofilms on inorganic particles suspended in marine water.
logarithmic (log) phase See exponential phase. long terminal repeat (LTR)
A repeated nucleic acid sequence at the 5’ and 3’ ends of a provirus.
mast cell White cells that secrete proteins that aid innate immunity. Mast cells reside in connective tissues and mucosa and do not circulate in the bloodstream.
loss-of-function mutation A mutation that eliminates or decreases the function of the gene product.
matrix protein A protein found in some viruses that is located between the capsid and the membrane envelope.
lumen The interior of an intracellular membrane-bound compartment.
mean generation time The reciprocal of the mean growth rate
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constant; the mean time period for doubling of a population.
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mean growth rate constant The number of organismal gen-
erations per unit time, k. mechanical transmission
methylotrophy The ability of an organism to oxidize single-
carbon compounds such as methanol, methylamine, or methane. A nonspecific mode of viral entry
metranidazole An antibiotic activated by anaerobic bacteria
into damaged tissue.
that kills cells by nicking the DNA.
membrane attack complex (MAC) A cell-destroying pore produced in the membrane of invading bacteria by the host cell complement cascade.
MHC restriction The ability of T lymphocytes to only recognize
membrane potential Energy stored as an electrical voltage dif-
ference across a membrane.
centration lower than that of the atmosphere, and can not grow in high oxygen environments.
membrane-permeant organic acid See membrane-permeant
microbe
antigens complexed to self-MHC molecules. microaerophilic An organism that requires oxygen at a con-
weak acid.
An organism or virus too small to be seen with the unaided human eye.
membrane-permeant weak acid An acid that exists in charged
microcolony A small colony of bacteria only visible with the
and uncharged forms such as acetic acid. The uncharged form can cross the membrane.
microfilament A eukaryotic cytoskeletal protein composed of
membrane-permeant weak base A base that exists in charged and uncharged forms such as methylamine. The uncharged form can dissolve in the membrane. memory B cell A long-lived type of lymphocyte preprogrammed to produce a specific antibody. After encountering its activating antigen, memory B cells differentiate into antibodyproducing plasma cells.
A partial diploid strain with chromosomal and Fprime factor copies of particular genes. merodiploid
merozoite The form of Plasmodium falciparum, the causative
agent for malaria, which invades red blood cells. mesocosm A small controlled model ecosystem. mesophile An organism with optimal growth between 20°C
aid of a microscope. polymerized actin. microfossil A microscopic fossil in which calcium carbonate deposits have filled in the form of ancient microbial cells. micronucleus A form of nucleus found in ciliates that contains
a diploid set of chromosomes and undergoes meiosis for sexual exchange by conjugation. micronutrient A nutrient that an organism needs in small quantity, typically a vitamin or a mineral. microplankton Plankton consisting of cells 20–200 µm in
diameter. microscope A tool that increases the magnification of specimens to enable viewing at higher resolution.
and 40°C.
microtubule A eukaryotic cytoskeletal protein composed of polymerized tubulin.
messenger RNA (mRNA) An RNA molecule that encodes a
minicell A small spherical cell fragment lacking DNA, gener-
protein. metabolist model A model of early life in which the cen-
tral components of intermediary metabolism arose from selfsustaining chemical reactions based on inorganic chemicals. metastatic lesions Lesions of infection, or of cancerous cells, that
develop at secondary sites away from the initial site of infection.
ated by improper septation near a pole. The lowest concentration of a drug that will prevent the growth of an organism.
minimal inhibitory concentration (MIC)
miso A Japanese condiment, made from ground soy and rice, salted and fermented by the mold Aspergillus oryzae.
tissues.
missense mutation A point mutation that alters the sequence of a single codon, leading to a single amino acid substitution in a protein.
methane gas hydrate A crystalline material in which meth-
mitochondrion A eukaryotic organelle that produces ATP
metazoan A multicellular animal with cells organized into
ane molecules are surrounded by a cage of water molecules. This molecular configuration is found in the deep ocean. methanogen An organism that uses hydrogen to reduce CO2 and other single-carbon compounds to methane, yielding energy. methanogenesis An energy-yielding metabolic process that
produces methane. It is unique to archaea. methanotroph An organism that oxidizes methane to yield
through the use of an electron transport chain to produce a proton motive force. O2 is the final electron acceptor to produce H2O. mitosis The orderly replication and segregation of eukaryotic chromosomes, usually prior to cell division. mitosporic fungus A species of fungus that generates spores
by mitosis and lacks a known sexual cycle. mixotrophic An organism that can switch among metabolic
energy.
strategies, such as heterotrophy and phototrophy, depending on the environmental conditions.
methanotrophy The metabolic oxidation of methane to yield
mixotrophs
energy. methyl mismatch repair A DNA repair system that fixes misincorporation of a nucleotide after DNA synthesis. The unmethylated daughter strand is corrected to complement the methylated parent strand. methylene blue A cationic dye commonly used as a simple
stain for bacteria.
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Organisms capable of both photosynthetic and heterotrophic metabolism.
modulon A group of genes, operons and regulons that is co-
ordinately activated in response to a particular stimulus. molarity A unit of concentration measured as the number of moles of solute per liter of solution.
The use of DNA or RNA sequence information to measure the time of divergence among different species.
molecular clock
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molecular formula A notation that indicates the number and
mycorrhizae A symbiotic relationship between plant roots and
type of atoms in a molecule. For example H2O is the molecular formula for water.
fungi.
molecular mimicry A structural similarity between two differ-
myxospore Durable spherical cells produced by the fruiting body of Myxobacteria.
ent molecules.
Nanoarchaeota
monocistronic The RNA produced from a single gene.
A deeply branching division of Archaea; includes thermophilic cells of extremely small size. Plankton consisting of cells 2–20 µm in
monocyte A white cell with a single nucleus that can differen-
nanoplankton
tiate into macrophages or dendritic cells.
diameter.
monophyletic A group of organisms that includes an ancestral
nasopharynx The area leading from the nose to the oral cavity.
species and all of its descendents.
native conformation The fully folded, functional form of a
monophyletic group See monophyletic.
protein.
monosaccharide
The monomer unit of sugars. Monosaccharides have a molecular formula of CH2O.
natto A soybean product, similar to tempeh, produced by alka-
mordant A chemical binding agent that causes specimens to
necrotizing fasciitis A severe skin infection, also know as
retain stains better.
flesh-eating disease, usually caused by the gram-positive coccus Streptococcus pyogenes.
mother cell The larger cell that forms during the asymmetric
cell division leading to spore formation. The mother cell will engulf the forespore.
line fermentation.
motherspore See mother cell.
negative selection The destruction of T cells bearing T-cell receptors (TCRs) that bind strongly to self-MHC proteins displayed on thymus epithelial cells.
motility The ability of a microbe to generate self-directed
negative-sense
movement. mucociliary elevator The ciliated mucus lining of the trachea,
(–)
strand
(see
also
template
strand)
Single-stranded RNA whose sequence is complementary to that of mRNA. Also the DNA strand that is complementary to an mRNA.
bronchi, and bronchioles that sweeps foreign particles up and away from the lungs.
negative stain A stain that colors the background and leaves
Mueller-Hinton agar A specialized, standardized, para-amino benzoic acid-free media used for the Kirby-Bauer test.
neuraminidase inhibitors Anti-influenza drugs that target
multidrug resistance (MDR) efflux pump A transmembrane
the specimen unstained. neuraminidase on the viral envelope and decrease the number of virus particles released.
protein pump that can export many different kinds of antibiotics with little regard for structure.
neutralophile An organism with an optimal growth range in
multiplex PCR A polymerase chain reaction that uses multiple pairs of oligonucleotide primers to amplify several different DNA sequences simultaneously.
neutrophil An innate immune system white blood cell that can
murein See peptidoglycan. murein lipoprotein The major lipoprotein that connects the outer membrane of gram-negative bacteria to the peptidoglycan cell wall. mutagen A chemical that damages DNA and leads to
mutations. mutation A heritable change in the DNA sequence. mutation frequency The fraction of mutant cells (defective in a
given gene) within the total cell population. mutation rate The number of mutations introduced into DNA
per generation (cell doubling). mutator strain A strain of cells with a high mutation rate, usu-
ally due to a mutation in a DNA repair enzyme. mutualism A symbiotic relationship in which both partners
benefit. mycelia See mycelium. mycelium pl. mycelia A fungal hypha that projects into the
air (aerial mycelium) or into the growth substrate (substrate mycelium). mycolic acid One of a diverse class of sugar-linked fatty acids
found in the cell envelopes of mycobacteria. mycology
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The study of fungi.
environments between pH 5 and 8. phagocytose and kill microbes. A description of an organism’s environmental requirements for existence and its relations with other members of the ecosystem.
niche
nicotinamide adenine dinucleotide (NADH, NAD+)
An energy carrier in the cell that can donate (NADH) or accept (NAD+) electrons. nitrification The oxidation of reduced nitrogen compounds to nitrite or nitrate. nitrifier An organism that converts reduced nitrogen compounds to nitrite or nitrate. nitrifying bacterium A bacterium that performs nitrification,
gaining energy by oxidizing ammonium (NH4+) to nitrate (NO3–). nitrogen fixation The ability of some prokaryotes to reduce
inorganic diatomic nitrogen gas (N2) to two molecules of ammonium ion (2 NH4+). nitrogenase The enzyme that catalyzes the conversion of dini-
trogen (N2) to two molecules of ammonium ion (NH4+). nitrogen-fixing bacterium A bacterium that can reduce diatomic nitrogen gas, N2, into ammonium ion (NH4+). Nitrospirae A phylum of gram-negative bacteria, many of
which are lithotrophs, oxidizing nitrite to nitrate. Signaling molecules released by rhizobial cells and sensed by legumes that help initiate the rhizobia-legume symbiosis.
Nod factors
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nomenclature The naming of different taxonomic groups of
organisms. non-adaptive
immunity
(innate
immunity) Non-specific
mechanisms for protecting against pathogens.
oncogene A gene that through mutation or inappropriate expression can lead to cancer. oncogenic virus A virus that causes cancer.
trons in the bond are shared equally by the two atoms.
A taxonomic group of heterkont protists whose life cycle resembles that of fungi; they were formerly classified as fungi.
non-ribosomal peptide antibiotic Secondary metabolite with
open reading frame (ORF)
antimicrobial activity synthesized by modular enzymes and not by ribosomes.
encode a protein.
nonpolar covalent bond A covalent bond in which the elec-
nonsense mutation A mutation that changes an amino acid
Oomycetes
A DNA sequence predicted to
operational gene A DNA sequence that encodes a product
codon into a premature stop codon.
not essential for transcription or translation but involved in cell functions such as metabolism, stress response, or pathogenicity.
nonsense suppressor An altered (mutant) tRNA molecule
operator A DNA sequence that binds a repressor protein, pre-
whose anticodon will bind to a nonsense codon (premature stop codon) in a mutant mRNA and allow translation to proceed.
venting transcription of a gene downstream. (Less common, refers to a sequence binding an activator protein.)
nori A Japanese food obtained from the red algae Porphyra sp.
operon A collection of genes that are in tandem on a chromosome and are transcribed into a single RNA.
normal flora The resident population of harmless organisms normally present at nonsterile body sites (e.g. the intestine). Northern blot A technique to detect specific RNA sequences. Sample RNA is subjected to gel electrophoresis, transferred to a blot, and probed with a labeled cDNA that will hybridize to target RNA sequences. nosocomial Hospital-acquired. N-terminal rule The tendency of the N-terminal amino acid of a protein to influence protein stability.
A technique that provides structural information based on the absorption and emission of electromagnetic radiation resulting from changes in the spin state of atomic nuclei.
nuclear magnetic resonance (NMR)
nucleocapsid protein (NP)
A protein that coats a viral
operon fusion A technique to monitor transcriptional regulation by fusing a reporter gene containing its own ribosome binding site to the 3’ end of an operon. Unlike the “gene fusion” technique, only the promoter of the target gene directs expression of the reporter. opine A specialized amino acid used as a carbon and nitrogen source by the plant pathogen Agrobacterium tumefasciens.
The eukaryotic clade that contains fungi, multicellular animals, and some protist-like organisms.
Opisthokonta
opportunistic pathogen A microbe that normally is not patho-
genic but can cause infection or disease in an immune compromised host organism. opsonization The coating of pathogens with antibodies that
genome.
aid pathogen phagocytosis by innate immune cells.
nucleoid The looped coils of a bacterial chromosome.
opsonize The binding of IgG antibodies to microbes to enhance
nucleolus A region inside the nucleus where ribosome assem-
bly begins. nucleotide The monomer unit of nucleic acids, consisting of a
five-carbon sugar, a phosphate and a nitrogenous base. A DNA repair mechanism that cuts out damaged DNA. New correct DNA is synthesized by DNA polymerase I.
nucleotide excision repair (NER)
nucleus A eukaryotic organelle that contains the DNA. numerical aperture (NA) The product of the refractive index of the medium and sin θ. As NA increases the magnification increases.
microbial phagocytosis by host immune cells. optical density A measure of how many particles are suspended in a solution based on light scattering by the suspended particles. optical isomer Also known as a stereoisomer, a molecule that
has a mirror image. Molecules that contain a chiral carbon can have optical isomers. oral groove
A ciliate structure for food uptake.
organelle A membrane-bound compartment within eukary-
otic cells that serves a specific function. organic horizon The top, surface layer of the soil.
O polysaccharide
A sugar chain that connects to the core polysaccharide of lipopolysaccharides.
organic molecule A molecule that contains a carbon-carbon
objective lens In a compound microscope, the lens closest to
organotrophy Metabolism of organic compounds to yield
bond.
the specimen that generates the initial magnification.
usable energy.
ocular lens In a compound microscope, the lens situated clos-
origin (oriC) The region of a bacterial chromosome where
est to the observer’s eye. It is also called the eyepiece.
DNA replication initiates.
Short fragments of DNA that are synthesized on the lagging strand during DNA synthesis.
origin of replication (ori) A DNA sequence at which DNA
Okazaki fragments
oligotroph An organism that can only grow in environments containing extremely low concentrations of organic nutrients. oligotrophic lake A lake having low concentration of organic nutrients, the opposite of eutrophic.
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replication initiates. In a bacterial chromosome this site is also attached to the cell envelope. oropharynx The area between the soft palate and the upper edge of the epiglottis. ortholog See orthologous gene.
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orthologous Genes present in more than one species that
pasteurization The heating of food at a temperature and time
derived from a common ancestral gene and encode the same function.
combination that will kill spores of Mycobacterium tuberculosis and Coxiella burnetii.
orthologous gene A gene present in more than one species
pathogen A bacterial, viral, or fungal agent of disease.
that derived from a common ancestral gene and encodes the same function.
pathogenesis The processes through which microbes cause
osmolarity A measure of the number of solute molecules in
pathogenicity The ability of a microorganism to cause disease.
solution. osmosis The diffusion of water from regions of high water
concentration (low solute) to regions of low water concentration (high solute) across a semi-permeable membrane. osmotic pressure Pressure exerted by the osmotic flow of
water through a semipermeable membrane. outer leaflet The layer of the cell membrane phospholipid
bilayer that faces away from the cytoplasm. outer membrane A membrane external to the cell wall in
gram-negative bacteria. A large increase in the oxygen consumption of immune cells during phagocytosis of pathogens as the immune cells produce oxygen radicals to kill the pathogen. oxidative burst
oxidative phosphorylation An electron transport chain that uses diatomic oxygen as a final electron acceptor and generates a proton gradient across a membrane for the production of ATP via ATP synthase.
An oxidized compound is one that has lost electrons. Oxidation is the loss of electrons.
oxidized, oxidation
An electron transport system protein that accepts electrons from one molecule (oxidizing that molecule), and donates electrons to a second molecule, thereby reducing the second molecule. oxidoreductase
An ATP-producing photosynthetic pathway consisting of photosystems I and II. Water serves as the initial electron donor (generating O2) and NADP+ is the final electron acceptor, generating NADPH. oxygenic Z pathway
palindrome A DNA sequence in which the top and bottom
disease in a host. pathogenicity island A type of genomic island, a stretch of
DNA that contains virulence factors and may have been transferred from another genome. pelagic
Referring to the open ocean.
pellicle A thick, flexible cell covering found in protists. penicillin An antibiotic, produced by the Penicillium mold, that
blocks cross-bridge formation during peptidoglycan synthesis. penicillin-binding proteins Bacterial proteins, involved in cell wall synthesis, that are targets of the penicillin antibiotic. pentose phosphate shunt (PPS) An alternate glycolytic path-
way in which glucose-6-phosphate is first oxidized, then decarboxylated to ribulose-5-phosphate, ultimately generating 1 ATP and 2 NADPH. peptide bond The covalent bond that links two amino acid
monomers. peptidoglycan (murein) A polymer of peptide-linked chains of
amino sugars; a major component of the bacterial cell wall. peptidyltransferase The rRNA enzymatic ability to form pep-
tide bonds. The ribosomal site that contains the growing protein attached to a tRNA.
peptidyl-tRNA site (P site)
perforin A cytotoxic protein, secreted by T cells, that forms pores in target cell membranes. peripheral membrane protein A protein that is associated with
a membrane but does not transverse the phospholipid bilayer. permease A substrate-specific carrier protein in the membrane.
pandemic An epidemic that occurs over a wide geographic
permissive temperature A temperature at which a temperature-sensitive mutation in a gene is masked, permitting growth of the organism.
area.
peroxisome
strands have the same sequence in the 5’ to 3’ direction.
panspermia The hypothesis that life forms originated else-
A eukaryotic organelle that converts hydrogen peroxide to water.
where and “seeded” life on Earth.
petechiae
A technique to separate compounds based on their differential migration in a solvent that wicks up through paper. Compounds are separated based relative solubility in the solvent.
petri dish A round dish with vertical walls covered by an
paper chromatography
paralogous Genes that arise by gene duplication within a spe-
cies and evolve to carry out different functions. parasite Bacteria, viruses, fungi, or protozoan (protist) that col-
onizes and harm its host; the term usually refers to protozoa. parasitism A symbiotic relationship in which one member
benefits and the other is harmed. parfocal In a microscope with multiple objective lenses, having the objective lenses set at different heights that maintain focus when switching among lenses. passive transport Net movement of molecules across a mem-
brane without energy expenditure by the cell.
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Pinpoint capillary hemorrhages due to the absence of clotting factors; may indicate the presence of endotoxin.
inverted dish of slightly larger diameter. The smaller dish can be filled with a substrate for growing microbes. phage display A technique in which a phage particle contains recombinant coat proteins expressed by genes encoding a coat protein fused to the protein of interest, such as a vaccine antigen. phagocytosis A form of endocytosis in which large extracellular particles are brought into the cell. phagosome A large intracellular vesicle that forms as a result of phagocytosis. phase variation A gene regulatory mechanism that changes the amino acid sequence of a protein from one antigenic type to another. One mechanism involves site specific recombination that flips a DNA sequence in a chromosome.
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phase-contrast microscopy Observation of a microscopic
object based on the differences in the refractive index between cell components and the surrounding medium. Contrast is generated as the difference between refracted light and transmitted light shifted out of phase. A test of the ability of a disinfectant to kill bacteria; the higher the coefficient the more effective the disinfectant.
phenol coefficient test
phenol red broth test A clinical test for particular bacterial
fermentation pathways, indicating the presence or absence of particular species, based on fermentative acids changing a pH indicator. phosphatidate A negatively charged phosphate head group of
a phospholipid. phosphatidylethanolamine A type of phospholipid with a
positively charged ethanolamine attached to the phosphate group. phosphatidylglycerol A type of phospholipid with a glycerol
phycobilisome A protein complex that captures light in photosynthetic bacteria. phycoerythrin A photopigment that give red algae their char-
acteristic color. phylogenetic tree A diagram depicting estimates of the relative
amounts of evolutionary divergence among different species. phylogeny A measurement of genetic relatedness. The classifi-
cation of animals based on their genetic relatedness. phylum The taxonomic rank one level below domain; a group
of organisms sharing a common ancestor that diverged early from other groups. phytoestrogens See flavonoids. phytoplankton Phototrophic marine bacteria, algae, and pro-
tists, the primary producers in pelagic food webs. picoplankton Plankton consisting of cells 0.3–2 µm in
diameter. picornavirus A class of icosahedral RNA viruses such as
attached to the phosphate group.
poliovirus.
phosphodiester bond The bond that covalently attaches to
piezophile See barophile. An organism that requires high
adjacent nucleotides in a nucleic acid.
pressure to grow.
phospholipid The major component of membranes. A typical phospholipid is composed of a core of glycerol to which two fatty acids and a modified phosphate group are attached.
pilin The protein monomer that polymerizes to form a pilus.
phospholipid bilayer Two layers of phospholipids; the hydrocarbon fatty acids tails face the interior of the bilayer, and the charged phosphate groups face the cytoplasm and extracellular environment. phosphorylation The enzyme-catalyzed addition of a phos-
phoryl group onto a molecule.
pilus pl. pili (fimbria, pl. fimbriae) A straight protein filament composed of a tube of protein monomers that extend from the bacterial cell envelope. pinocytosis A form of endocytosis in which only extracellular fluid and small molecules are brought into the cell. Planctomycetes A phylum of free-living bacteria that have
stalked cells and reproduce by budding. Their nucleoid is surrounded by a membrane.
phosphotransferase system (PTS) A group translocation system that uses phosphoenol pyruvate to transfer phosphoryl groups onto the incoming molecule.
plankton
photoautotroph Organisms
plaque A cell-free zone on a lawn of bacterial cells caused by
that perform photosynthesis, using light energy to reduce carbon dioxide.
photoautotrophy The reduction of carbon dioxide using light
Organisms that float in water.
planktonic cells Isolated cells, growing individually in a liquid
without connections to other cells. viral lysis.
as an energy source.
plaque assay An assay to determine the presence of bacteriophages based on their ability to form plaques.
photoheterolithotroph An organism that can use heterotrophy, lithotrophy, or photosynthesis for energy production, depending on environmental conditions.
plaque-forming unit (PFU) A measure of the concentration of phage particles in liquid culture.
photoheterotrophy The production of energy by the photoly-
sis of organic compounds. photolysis The first energy-yielding phase of photosynthesis,
plasma cell A short-lived antibody-producing cell. plasmid An extrachromosmal genetic element that may be present in some cells.
the light-driven separation of an electron from a molecule coupled to an electron transport system.
plasmodesma pl. plasmodesmata A membrane channel in
photoreactivation A light-induced, photolyase-catalyzed repair
plasmodial slime mold A slime mold in which the mass of aggregating cells becomes a multinucleate single cell.
of pyrimidine dimers. photosynthesis The metabolic ability to absorb and convert
solar energy into chemical energy for biosynthesis; a precise definition includes CO2 fixation.
plants that connects adjacent plant cells.
plasmodium The giant multinucleate cell formed by plasmodial slime molds. pneumonic plague A highly virulent and contagious Yersinia
photosystem I and II See anaerobic photosystem I and II.
pestis lung infection.
phototroph A general term for organisms that can obtain metabolic energy through light absorption, with or without CO2 fixation.
point mutation A change in a single nucleotide within a nucleic
phototrophy Obtaining energy from chemical reactions trig-
poliovirus.
acid sequence. poliomyelitis (polio) The paralytic disease caused by the
gered by the absorption of light.
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poliovirus A picornavirus that is the causative agent of poliomyelitis.
primary syphilis The initial inflammatory reaction (chancre) at the site of infection with Treponema pallidum.
poliovirus receptor (Pvr) The cell surface receptor unique to
primase
humans and simians through which poliovirus gains entry to cells.
An RNA polymerase that synthesizes short RNA primers complementary to a DNA template to launch DNA replication.
polyamine A positively charged molecule containing multiple amine groups.
RNA transcript.
polycistronic An RNA produced from an operon containing
primer extension A technique to determine the 5’ end of an
several genes and hence containing several functional sequences (usually encoding different proteins).
prion An infectious agent that causes propagation of misfolded host proteins; usually consists of a defective version of the host protein.
polymerase chain reaction (PCR)
A method to amplify DNA in vitro using many cycles of DNA denaturation, primer annealing, and DNA polymerization with a heat-stable polymerase.
probabilistic indicator A means of quickly identifying microbes in the clinical setting, based on a battery of biochemical tests performed simultaneously on an isolated strain.
polyphyletic An organism or group of organisms that have multiple evolutionary origins.
probiotic A food or nutritional supplement that contains live
polysaccharide
A polymer of sugars.
microorganisms and aims to improve health by promoting beneficial bacteria.
polysome
A cell structure consisting of multiple ribosomes performing translation on the same mRNA molecule.
prokaryote pl. prokaryotes Organism whose cell or cells lack a nucleus; includes both bacteria and archaea.
porin A transmembrane protein complex that allows movement of specific molecules across the cell membrane or the outer membrane.
promoter A non-coding DNA regulatory region immediately upstream of a structural gene that is needed for transcription initiation.
positive selection Growth of a population under a condition
promoter trap plasmid A laboratory technique to discover
that favors a particular genotype and prevents growth of other genotypes. In immunology: the survival of T cells bearing T-cell receptors (TCRs) that don’t recognize self-MHC proteins displayed on thymus epithelial cells.
promoters that only drive gene expression under particular circumstances. proofreading An enzymatic activity of some nucleic acid poly-
merases that attempts to correct mispaired bases.
positive-sense (+) strand A molecule of of DNA that has the
prophage A phage genome integrated into a host genome.
same sequence as mRNA (except for T replacing U). In virology: a molecule of RNA (mRNA) that can be directly translated into viral proteins.
propionic acid fermentation The fermentation of lactic acid to propionic acid by Propionibacterium species; used in the production of Swiss cheese.
pour plate Also known as a Petri dish, a round, shallow-sided container into which molten agar is poured and subsequently cooled.
prosthetic group A non-protein component of an enzyme required for catalytic ability.
prebiotic soup A model for the origin of life based on the abiotic
formation of fundamental biomolecules and cell structures such as membranes out of a“soup”of nutrients present on early Earth. predator A consumer that feeds on grazers. preliminary mRNA transcript (pre-mRNA) A eukaryotic mes-
senger RNA prior to intron removal.
protease inhibitor A molecule that inhibits a protease enzyme; some are used as anti-HIV drugs to block the virally encoded protease needed to complete HIV assembly. protective antigen (PA) The core subunit of anthrax toxin, so
called because immunity to this protein protects against disease. protein A A Staphylococcus aureus cell-wall protein that binds
primary antibody response The production of antibodies
to the Fc region of antibodies, hiding the S. aureus cells from phagocytes.
upon first exposure to a particular antigen. B cells become activated and differentiate into plasma cells and memory B cells.
Proteobacteria A large, metabolically and morphologically diverse, phylum of gram-negative bacteria.
primary endosymbiont A lineage of organisms derived from a single endosymbiotic event.
proteome All the proteins expressed in a cell at a given time.
the chancre of primary syphilis and the rash of secondary syphilis.
The “complete proteome” includes all the proteins the cell can express under any condition. The “expressed proteome” represents the set of proteins made under a given condition.
primary pathogen A disease-causing microbe that can breach
proteomics The biological field of proteome analysis.
the defenses of a healthy host. Same as a frank pathogen.
proteorhodopsin A bacterial membrane protein that contains retinal and acts as a light-driven proton pump; homologous to the archaeal protein bacteriorhodopsin.
primary latent stage The period of syphilis infection between
primary producer An organism that produces biomass (reduced
carbon) from inorganic carbon sources such as CO2. primary recovery The initial isolation of commercial product from industrial microbes.
protist
primary structure The first level of organization of polymers, consisting of the linear sequence of monomers, for example the sequence of amino acids in a protein or nucleotides in a nucleic acid.
proton potential The potential energy of the concentration
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A motile single-celled eukaryote.
proton motive force See proton potential.
gradient of protons (hydrogen ions, H+) plus the charge difference across a membrane.
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provirus A viral genome that is integrated into the host cell
receptor-binding domain A region of a protein responsible for
genome.
binding to a receptor.
pseudogene A gene that is no longer functional.
recombinant DNA DNA that has been combined with other
pseudomurein See pseudopeptidoglycan.
DNA to create novel DNA sequences.
pseudopeptidoglycan A peptidoglycan-like molecule composed
recombination The replacement of host DNA with donor DNA.
of sugars and peptides that is found in some archaeal cell walls.
recombination signal sequence (RSS) A DNA region downstream of antibody heavy and light chain genes that allows recombination between widely separated gene segments.
pseudopod A locomotory extension of cytoplasm bounded by the cell membrane.
An organism with optimal growth at temperatures below 20°C. psychrophile
pure culture A culture containing only a single strain or spe-
cies of microorganism. A large number of microorganisms all descended from a single individual cell. purine A nitrogenous base with fused rings found in nucleo-
recombinational repair A DNA repair mechanism that relies on recombination between an undamaged chromosome and a gap that occurred during replication of damaged DNA. red alga See Rhodophyta. redox couple The oxidized and reduced states of a compound. For
example, NAD+ and NADH form a redox couple.
putrefaction Food spoilage due to the decomposition of pro-
reduced, reduction A reduced compound is one that has gained electrons. Reduction is the gain of electrons.
teins and amino acids.
reduction potential See voltage potential.
pyrimidine A single-ring nitrogenous base found in nucleo-
reductive acetyl-CoA pathway A carbon assimilation pathway in which two CO2 molecules are condensed and reduced by 2 H2 to form an acetyl group.
tides, such as adenine and guanine.
tides such as cytosine, thymine, or uracil. pyrogen Any substance that induces fever. pyruvate dehydrogenase complex (PDC) The multi-subunit enzyme that couples the oxidative decarboxylation of pyruvate to acetyl-CoA and NADH production.
reductive evolution The loss or mutation of DNA encoding
quarantine The separation of infectious individuals from the
reductive (reverse) TCA cycle
general population to limit the spread of infection. quaternary structure The highest level of protein structure, in
unselected traits. reductive pentose phosphate cycle See Calvin cycle.
A CO2 fixation pathway that generates acetyl-CoA through reversal of TCA cycle reactions. It requires ATP and NADPH.
which more than one polypeptide chain interact and function together.
reflection Deflection of an incident light ray by an object, at an
quinol A reduced electron carrier that can diffuse laterally within membranes.
refraction The bending and slowing of light as it passes
quinolones A group of antibiotic drugs that inhibit DNA syn-
refractive index The degree to which a substance causes the
thesis by targeting bacterial topoisomerases such as DNA gyrase. quinone An oxidized electron carrier that can diffuse laterally
refraction of light, a ratio of the speed of light in a vacuum to its speed in another medium.
within membranes.
regolith Loose rock fragments covering bedrock; commonly
angle equal to the incident angle. through a substance.
quinone pool The oxidized quinones and reduced quinols that
refers to the surface layer of Mars or of the moon.
diffuse freely within the phospholipid membrane and are able to transfer electrons between many different redox enzymes.
regulatory protein (regulator) A protein that can bind DNA
quorum sensing The ability of bacteria to sense the presence of
other bacteria via secreted chemical signals called autoinducers.
and modulate transcription in response to a metabolite. regulon A group of genes and operons that is co-ordinately regulated and shares a common biochemical function.
radial immunodiffusion A laboratory technique to determine the concentration of an antigen in solution.
release factor A molecule that enters a ribosome A site containing
Radiolaria A taxonomic group of amebas with a silicate shell
replication fork During DNA synthesis the region of the chro-
an mRNA stop codon and initiates protein cleavage from the tRNA.
penetrated by filamentous pseudopods.
mosome that is being unwound.
rancidity Food spoilage due to the oxidation of fats; may or
replisome A complex of DNA polymerase and other accessory
may not involve microbial activity.
molecules that performs DNA replication.
rank A level of taxonomic hierarchy, such as phylum, class,
reporter gene A gene, such as lacZ (β-galactosidase) or gfp
order or family. reaction center The chlorophyll molecule that donates its
(green fluorescent protein), whose protein product can be easily quantified; commonly used in a gene fusion.
excited electron to an electron transport system.
repression The down-regulation of gene transcription.
reading frame The position in a nucleic acid sequence from
which triplet codons encode amino acids.
repressor A regulatory protein that can bind to a specific DNA sequence and inhibit transcription of genes.
real-time PCR A technique using fluorescence to detect the
reservoir 1. An organism that maintains a virus in an area by
products of PCR amplification as the reaction progresses, in order to quantify the amount of DNA in a sample.
serving as a high titer host. 2. A part of the biosphere containing significant amounts of an element needed for life.
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resolution The smallest distance that two objects can be separated and still be distinguished as separate objects. respiration The oxidation of reduced electron donors through
a series of membrane-embedded electron carriers to a final electron acceptor. The energy derived from the redox reactions is stored as an electrochemical gradient across the membrane, which may be harnessed to produce ATP. response regulator A cytoplasmic protein that is phosphor-
ylated by a sensor kinase and modulates gene transcription depending on its phosphorylation state. restriction endonuclease A bacterial enzyme that cleaves dou-
A regulatory RNA sequence (upstream of an operon) that forms a stem-loop structure stabilized by the operon’s product and modulates transcription and translation. riboswitch
ribozyme A catalytic RNA molecule. ripening The aging of cheese. rise period During viral culture, the time when cells lyse and viral progeny enter the media. RNA reverse-transcribing virus See retrovirus. RNA world A model of early life in which RNA performed all the informational and catalytic roles of today’s DNA and proteins.
ble-stranded DNA within a specific short sequence, usually a palindrome. Often called a restriction enzyme.
RNA-dependent RNA polymerase An enzyme that produces
restriction enzyme See restriction endonuclease.
rod
restriction site A DNA sequence recognized and cleaved by a restriction enzyme. restrictive temperature A temperature at which a tempera-
ture-sensitive mutation in a gene leads to the mutant phenotype, which generally includes failure to grow. reticulate body The metabolically and reproductively active form of Chlamydia. reticuloendothelial system A collection of cells that can phagocytize and sequester extracellular material. retinal A vitamin A related cofactor in opsin proteins; it under-
goes a conformational change in response to photon absorption. retroelements Non-virion-producing,
ancient
retroviral
genomes found in host genomes. retrotransposon Retroelements that contain only partial ret-
an RNA complementary to a template RNA strand. 1. A bacteria with a linear shape (also referred to as a bacillus). 2. A photoreceptor cell.
rooted tree A phylogenetic tree showing the relative distances between different species, including the earliest time of divergence from a common ancestor. Rous sarcoma virus (RSV) A retrovirus that was the first virus
shown to cause cancer. rubisco, ribulose 1,5-bisphosphate carbon dioxide reductase/oxidase The enzyme that catalyzes the carbon fixation
step in the Calvin cycle. The first chamber of the digestive tract of ruminant animals such as cattle; the main site for microbial digestion of feed.
rumen
sacculus The single covalent molecule that comprises the bac-
terial cell wall. saprophytes Fungal decomposers.
A cubical octad cluster of cells formed by septation at right angles to the previous cell division.
roviral sequences but may contain reverse transcriptase to allow further movement into the host genome.
sarcina pl. sarcinae
retrovirus A virus containing a positive single-stranded RNA genome; uses reverse transcriptase to generate a double-stranded DNA.
sargassum weed Unrooted kelp forests that float in marine
water.
stranded DNA molecule from a single-stranded RNA template.
Electron microscopy in which the electron beams scan across the specimen’s surface to reveal the three-dimensional topology of the specimen.
reversion test A way to screen the mutagenic potential of
scattering Interaction of light with an object resulting in prop-
reverse transcriptase An enzyme that produces a double-
compounds by their ability to fix a gene that is necessary for the biosynthesis of a needed metabolite. rhinovirus
A picornavirus that causes the common cold.
scanning electron microscopy (SEM)
agation of spherical light waves at relatively low intensity. schizogony Mitotic reproduction of parasitic cells to achieve a large population within a host tissue.
rhizobia Bacterial species of the order Rhizobiales that form highly specific mutualistic associations with plants in which the bacteria fix nitrogen for the plant.
secondary antibody response A memory B cell-mediated
rhizoplane The surface of plant roots.
secondary endosymbiont An organism evolved through engulfment of a primary endosymbiont.
rhizosphere The soil environment surrounding plant roots. Rho-dependent A bacterial transcription termination mecha-
nism that requires Rho protein. Rhodophyta (rhodophyte) A taxonomic group of unicellular
primary algae containing red pigment; also known as “red algae.” Rho-independent A bacterial transcription termination mecha-
nism that requires a GC-rich region near the transcript terminus. ribosomal RNA (rRNA) RNA molecules that include the scaffolding and catalytic components of ribosomes. ribosome-binding site See Shine-Dalgarno sequence. A
stretch of nucleotides upstream of the start codon in an mRNA that binds to a specific location of the ribosome.
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rapid increase in the production of antibodies in response to a repeat exposure to a particular antigen.
secondary metabolite See secondary product. secondary product Biosynthetic products that are not essential
nutrients but enhance nutrient uptake or inhibit competing species (e.g. antibiotics). secondary structure The second level of organization of poly-
mers, consisting of regular patterns that repeat, such as the double helix in DNA or the beta-sheet in proteins. secondary syphilis A rash that may appear at some point after the primary latent stage of syphilis. sedimentation rate The rate at which particles of a given size and shape travel to the bottom of a tube under centrifugal force. The rate depends on the particle’s mass and cross-sectional area.
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segmented genome A viral genome that consists of more than
one nucleic acid molecule.
A eukaryotic DNA element that can lead to decreased transcription when bound by an appropriate transcription factor.
selective medium A medium that allows the growth of certain
silent mutation A mutation that does not change the amino
species or strains of organisms but not others.
acid sequence encoded by an open reading frame. The changed codon encodes the same amino acid as the original codon.
silencer
The ability of a drug, at a given dose, to harm the pathogen and not the host.
simple stain A stain that makes an object more opaque, increas-
semiconservative The mode of DNA replication whereby
ing its contrast with the external medium or surrounding tissue.
each new double helix contains one old, parental strand and one newly synthesized daughter strand.
single-celled protein Edible microbes of high food value such as Spirulina or some yeasts.
semipermeable membrane (selectively permeable membrane) A membrane that is permeable to some substances
siphonous alga A form of alga consiting of a single cell
selective toxicity
but impermeable to other substances. sensor kinase A transmembrane protein that phosphorylates itself in response to an extracellular signal, and transfers the phosphoryl group to a receiver protein.
that forms extended siphon-like tubes and fronds without cell partitions.
septation The formation of a septum, a new section of cell wall
site-specific recombination Recombination between DNA molecules that do not share long regions of homology but do contain short regions of homology specifically recognized by the recombination enzyme.
and envelope to separate two prokaryotic daughter cells.
skin-associated lymphoid tissue (SALT)
septicemia An infection of the bloodstream. septicemic plague Infection of the bloodstream by Yersinia
pestis. septum A plate of cell wall and envelope that forms to separate two daughter cells. sequelae A serious, harmful immunological consequence of bacterial and host antigen cross-reactivity that occur after the infection itself is over. An example is rheumatic fever.
Immune cells, such as dendritic cells, located under the skin that help eliminate bacteria that have breached the skin surface.
S-layer A crystalline protein surface layer replacing or external to the cell wall in many species of archaea and bacteria. sliding clamp A protein that keeps DNA polymerase affixed to DNA during replication. slime mold An organism in which unicellular amebas can
aggregate into a fruiting body.
serovar A strain of a microbial species that expresses envelope proteins with an antigenicity distinct from other strains.
sludge The solid products of wastewater treatment.
serum The non-cell, liquid component of the blood.
that modulate translation.
sex pilus A pilus specialized for DNA transfer between
small subunit rRNA (in bacteria, 16S rRNA) A ribosomal RNA
bacteria. Shine-Dalgarno sequence See ribosome-binding site. In bacteria, a stretch of nucleotides upstream of the start codon in an mRNA that hybridizes to the 16S rRNA of the ribosome, correctly positioning the mRNA for translation. shuttle vector A plasmid with origins of replication for recog-
nized by both bacteria and eukaryotes. side effects Drug effects that harm the patient. siderophore A high-affinity iron binding protein used to scavenge iron from the environment and deliver it to the siderophore-producing organism. sigma factor A protein needed to bind RNA polymerase for the initiation of transcription in bacteria.
“small RNA” (smRNA)
Non-protein coding regulatory RNAs
found in the small subunit of the ribosome. Its gene is often sequenced for phylogenetic comparisons. soil A complex mixture of decaying organic and mineral matter
that covers the terrestrial portions of the planet. solute Any dissolved molecule. sorbitol MacConkey agar A media that can detect whether a bacterial strain can ferment sorbitol. Used clinically to test for E. coli O157:H7. SOS response A coordinated cellular response to extensive DNA damage. It includes error-prone repair. sourdough An undefined yeast population, derived from a
previous batch of dough, that is used in bread production. Southern blot A technique to detect specific DNA sequences.
A receptor that recognizes the signal sequence of peptides undergoing translation. The complex attaches to the cell membrane of prokaryotes (or the rough endoplasmic reticulum of eukaryotes), where it docks the protein-ribosome complex to the membrane for protein membrane insertion or secretion.
Sample DNA segments are separated by gel electrophoresis, transferred to a blot, and probed with a labeled DNA that will hybridize to complementary DNA sequences.
signal sequence A specific amino acid sequence on the aminoterminus of proteins that directs them to the endoplasmic reticulum (of a eukaryote) or the cell membrane (of a prokaryote).
species A single, specific type of organism, designated by a genus and species name.
signal recognition particle (SRP)
signature-tagged mutagenesis A technique to discover
pathogenicity genes by creating randomly mutated genes, each tagged with a different oligonucleotide. Mutant strains unable to infect an organism contain mutations in pathogenicity genes, which can be isolated.
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specialized transduction (restricted transduction) Transduc-
tion in which the phage can transfer only a specific, limited number of donor genes to the recipient cell.
A viral glycoprotein that connects the membrane to the capsid or the matrix and may be involved in viral binding to host cell receptors.
spike protein
spirochete A bacterium with a tight, flexible spiral shape; a species of the phylum Spirochetes (Spirochaetes).
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A phylum of bacteria with a unique morphology, a flexible, extended spiral that twists via intracellular flagella.
stromatolite A mass of sedimentary layers of limestone pro-
spongiform encephalopathies Brain-wasting diseases caused
which covalent bonds are shown as a line between atoms.
by prions.
structural gene A string of nucleotides that encodes a functional RNA molecule.
Spirochetes (Spirochaetes)
spontaneous generation The theory, much debated in the
duced by a marine microbial community over many years. structural formula A representation of molecular structure in
nineteenth century, that under current Earth conditions life can arise spontaneously from non-living matter.
structural isomer A molecule with the same molecular formula
sporangiospore A haploid spore that can germinate to form a
subcellular fractionation A procedure to separate cell compo-
haploid mycelium. sporangium A fungal organ that disburses non-motile spores
as a different molecule but a different arrangement of atoms. nents; often includes ultracentrifugation. substrate level phosphorylation Formation of ATP by the enzy-
via ballistic expulsion.
matic transfer of phosphate from a substrate molecule onto ADP.
spore stain A type of differential stain that is specific for the
substrate mycelium A long multinucleate filament (hypha) emanating from the germinating spore of some species that grows along a food source.
endospore coat of various bacteria, typically a firmicute species. sporogony The development of a parasite’s zygote into a spore-like form transmissible to the next host. spread plate A method to grow separate bacterial colonies by
plating serial dilutions of a liquid culture.
substrate-binding protein An extra-cytoplasmic protein that binds specific substrates and delivers them to their cognate uptake ABC transporters.
staining The process of treating microscopic specimens with a stain
sugar acid Sugars containing a carboxylic acid group; carbox-
to enhance their detection or to visualize specific cell components.
ylate sugars
stalk An extension of the cytoplasm and envelope that attaches a microbe to a substrate.
sulfa drugs Antibiotics that that inhibit nucleotide synthesis.
stalked ciliate
A ciliate that adheres to a substrate and uses its cilia to obtain prey.
stored inside the cell. May refer to extracellular sulfur deposits, also called sulfur globules.
standard Gibbs free energy change (DG°) The free energy change under standard conditions of 1 atmosphere pressure, 298°K and 1 M concentrations of all products and reactants.
superantigen Molecules that directly stimulate T cells without undergoing antigen presenting cell processing and surface presentation.
standard reduction potential (E°) A standard value of E, at
supercoil An extra twist or turn found in DNA, either positive
standard temperature and pressure and assuming initial 1 M concentrations of all reactants and products.
(increases DNA winding) or negative (decreases DNA winding). superoxide radical
A hexagonal arrangement of cells formed by septation in random orientations.
Svedberg coefficient A measure of particle size based on
staphylococcus pl. staphylococci
sulfur granule Elemental sulfur generated by metabolism and
A highly reactive oxygen species, O2–, that is toxic to cell components.
acid of a protein.
the particle’s sedimentation rate in a tube subjected to a high g force.
starter culture A mixture of fermenting microbes added to a
swarming A behavior in which some microbial cells differenti-
start codon A codon (usually AUG) that signals the first amino
food substrate to generate a fermented product.
ate into large swarmer cells and swim together as a unit.
stationary phase A period of no net increase in replication that sterilization The destruction of all cells, spores and viruses on
A repeating DNA sequence in antibody constant segment genes that serves as a recombination site during isotype switching.
an object.
symbiogenesis An evolutionary process by which two or more
One of three codons (UAA, UAG, UGA) that do not encode an amino acid and trigger the end of translation.
symbiont An organism that lives in a close association with
follows the exponential growth phase.
stop codon
generalized recombination, the enzyme-catalyzed pairing of single stranded DNA with its complementary strand in the DNA duplex.
strand
invasion During
switch region
species become intimately associated. another organism. symbiosis The intimate association of two unrelated species.
strict aerobe An organism that performs aerobic respiration
Bacteroids sequestered within a sac of plantderived membrane.
symbiosome
and can only grow in the presence of oxygen.
symport A transport protein where the molecules being trans-
strict anaerobe An organism that cannot grow in the presence
ported move in the same direction across the membrane.
of oxygen.
synergism
stringent response A cellular response to idle ribosomes
Cooperation between species in which both species benefit but can grow independently. The cooperation is less intimate than symbiosis.
(often indicating low carbon and energy stores) that includes a decrease in rRNA and tRNA production.
synthetic medium A bacterial growth solution that contains
stroma The compartment contained by the inner chloroplast
defined, known components.
membrane where the light-independent reactions of photosynthesis occur (CO2 fixation).
syntrophy Metabolic
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cooperation between two different
species.
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tailed phage A phage such as T4 that contains a genome delivery device called the tail.
thylakoid
tandem repeat A stretch of directly repeating DNA sequence
Ti plasmid A plasmid found in tumorigenic strains of A. tumefasciens that can be used as a vector to introduce DNA into plant cells.
(direct repeats) without any intervening DNA. taxon pl. taxa A category of organisms grouped together based on genetic relatedness. taxonomy The description of distinct life forms and their orga-
nization into different categories. tegument The contents of a virion between the capsid and the
envelope. teichoic acid Chains of phosphodiester-linked glycerol or ribitol that thread through and reinforce the cell wall in gram-positive bacteria. telomerase A eukaryotic enzyme with reverse transcriptase activity that is required to replicate the ends of linear chromosomes to avoid chromosome shortening. telomere The end of a linear chromosome, composed of a
repeated DNA sequence. tempeh A mold-fermented soy product, popular as a food in
parts of Asia. temperate phage Phage capable of lysogeny. template (–) strand A DNA strand (or an RNA strand in some
viruses) that is used as a template for the synthesis of mRNA. terminal electron acceptor The final electron acceptor at the end of an electron transport system.
A sequence of DNA that halts replication of DNA by DNA polymerase.
termination (Ter) site
terpenoid A branched lipid derived from isoprene that is found
in hydrocarbon chains of archaeal membranes. terraforming The idea of transforming the environment of a
planet to make it suitable for life from Earth. tertiary structure The third level of polymer structure, the unique three-dimensional shape of a polymer. tertiary syphilis A final stage of syphilis, manifested by cardio-
vascular and nervous system symptoms. tetanospasmin The tetanus-causing potent exotoxin produced
A chlorophyll-containing membrane folded within a phototrophic bacterium or a chloroplast.
tmRNA A molecule resembling both tRNA and mRNA that
rescues ribosomes stalled on damaged mRNAs lacking a stop codon. tobacco mosaic virus An RNA virus that is the causative agent of tobacco mosaic disease.
A member of a eukaryotic transmembrane glycoprotein family that recognizes a particular pathogenassociated molecular pattern (PAMP) present on pathogenic microorganisms.
Toll-like receptor (TLR)
tomography The acquisition of projected images of a transparent specimen from different angles that are digitally combined to visualize the entire specimen. topoisomerase A enzyme that can change the supercoiling of
DNA. total magnification The magnification of the ocular lens multiplied by the magnification of the objective lens. transamination The transfer of an ammonium ion between
two metabolites. transcript An RNA copy of a DNA template. transcription The synthesis of RNA complementary to a DNA
template. transcriptional attenuation A transcriptional regulatory mechanism in which translation of a leader peptide affects transcription of downstream structural genes. transcriptome The set of transcribed genes in a cell at given time.
The “complete trancriptome” includes all the possible RNA transcription products from a given genome. The “expressed transcriptome”represent the set of RNAs present during a given condition. transcytosis The movement of a cell or substance from one side of a polarized cell to the other side, using an intracellular route.
by Clostridium tetani.
transduction The transfer of host genes between bacterial cells via a phage head coat.
tetracyclines A class of antibiotics with four fused cyclic rings, that inhibits translation.
transfer RNA (tRNA) An RNA that carries an amino acid to
tetraether A molecule containing four ether links. An example
is found in archaeal membranes, when two lipid side chains form ether linkages with a pair of side chains from the other side of the bilayer. An essential primary product that contains four pyrroles (five-membered rings containing nitrogen), each with one or two double bonds. A precursor for many important cell cofactors such as chlorophylls and vitamin B12.
tetrapyrrole
thermocline A region of the ocean where temperature
decreases steeply with depth, and water density increases. thermophile An organism adapted for optimal growth at high temperatures, usually 55°C or higher.
An order of crenarchaeote containing hyperthermophilic organisms.
Thermoproteales
threshold dose The concentration of antigen needed to elicit
adequate antibody production.
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the ribosome. The anticodon on the tRNA matches the codon on the mRNA. transform The conversion of cultured cells into cancer cells. transformation The internalization of free DNA from the envi-
ronment into bacterial cells. transformed-focus assay The detection of oncogenic viruses
based on their ability transform cells, generating foci of unrestricted cell growth. transglycosylase An enzyme that links N-acetyl glucosamine
and N-acetyl muramic acid into chains during bacterial cell wall synthesis. transition mutation A type of point mutation in which a purine is replaced by a different purine or a pyrimidine is replaced by a different pyrimidine. translation The ribosomal synthesis of proteins based on trip-
let codons present in mRNA.
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translational control A regulatory mechanism that modulates
type I (immediate) hypersensitivity An IgE mediated allergic
protein production by influencing the translation of mRNA. translocasome A bacterial cell membrane protein complex
reaction that causes degranulation of mast cells within minutes of exposure to the antigen.
that imports external DNA during transformation.
type I pilus A pilus that adheres to mannose residues on host
translocation The energy-dependent movement of the ribosome to the next triplet codon along an mRNA.
type I protein secretion An ATP-binding cassette transport
cell surfaces.
membrane.
system that moves molecules directly into or out of bacterial cells.
transmembrane protein A protein with a membrane-spanning
type II hypersensitivity An immune condition in which antibodies
transmembrane domain A region of a protein that spans a
region.
bind to cell surface antigens, triggering cell-mediated cytotoxicity or activation of the complement cascade.
A type of electron microscopy in which electron beams are transmitted through a thin specimen to reveal internal structure.
type II secretion
transovarial transmission The passage of genes or pathogens from parent to offspring via the egg cell.
type III hypersensitivity An immune over-reaction triggered
transmission electron microscopy (TEM)
transpeptidase An enzyme that cross-links the side-chains
A bacterial protein secretion system that uses a type-IV pilus-like extraction/retraction mechanism to push proteins out of the cell.
by IgG antibody binding to soluble antigens.
from adjacent peptidoglycan strands during bacterial cell wall synthesis.
type III pilus A bacterial pilus that does not bind to mannose but binds to tannic acid.
transport protein (transporter) A membrane protein that
type III secretion A bacterial protein secretion system that uses a molecular syringe to inject bacterial proteins into the host cytoplasm.
moves specific molecules across a membrane. transposable element A segment of DNA that can move from
one DNA region to another. transposase A transposable element-encoded enzyme that
catalyzes the transfer of the transposable element from one DNA region to another. transposition The process of moving a transposable element
from one DNA region to another. transposon A transposable DNA element that contains genes
in addition to those required for transposition. transversion A point mutation in which a purine is replaced by a pyrimidine or vice versa. tricarboxylic acid (TCA) cycle A metabolic cycle that catabolizes the acetyl group from acetyl-CoA to 2 CO2 with the concomitant production of NADH, FADH2, and ATP. trophic level A level of the food web representing the con-
sumption of biomass of organisms from a lower trophic level. tropism The tissue types infected by a specific virus in a given
host. trypanosome A parasitic discicristate protist that has a cortical
skeleton of microtubules culminating in a long flagellum. tumor necrosis factor (TNF) A cytokine released by several cell
type IV (delayed-type) hypersensitivity (DTH) An immune response that develops 24 to 72 hours after exposure to an antigen that the immune system recognizes as foreign. DTH is triggered by antigen-specific T cells. The response is delayed because the T cells need time to proliferate after being activated by the allergen. type IV pilus A dynamic pilus that can repeatedly assemble and disassemble; it mediates twitching motility. ubiquitin (Ub) A small eukaryotic protein that can be attached to other proteins to target them for degradation via the proteasome. ultracentrifuge A machine that exposes samples to high centrifugal forces and can be used to separate subcellular components. uncoupler A molecule that makes a membrane permeable
to protons, dissipating the proton motive force and uncoupling electron transport from ATP synthesis. unrooted tree A phylogenetic tree showing only the relative distances between different species, without indicating which of these diverged earliest from the common ancestor. upstream processing The culturing of industrial microbes to produce large quantities of desired product. vaccination Exposure of an individual to a weakened version
types (e.g., macrophages) in response to cell damage.
of a microbe to provoke immunity and prevent development of disease upon re-exposure.
turbidostat A continuous culture device that can measure opti-
van der Waals force
cal density and through changing culture flow rates, maintain a specific cell density.
vancomycin
A type of bacterial movement on solid surfaces where a specific pilus extends and retracts. twitching motility
two-component signal transduction system A message relay
system composed of a sensor kinase protein and a response regulator protein that regulates gene expression in response to a signal (usually an extracellular signal). two-dimensional polyacrylamide gel electrophoresis (2-D PAGE, 2-D gels) A technique to separate proteins based on
differences in charge and molecular weight.
SFMB_glossary.indd G-27
Weak, temporary electrostatic attraction between molecules caused by shifting electron clouds.
A glycopeptide antibiotic that inhibits bacterial cell wall synthesis in a mechanism distinct from penicillin inhibition.
variable region The amino-terminal regions of antibody light
and heavy chains that confer specificity to antigen-binding and define the antibody idiotype. vasoactive factor A cell signaling molecule that increases cap-
illary permeability. vector An organism (e.g., insect) that can carry infectious agents
from one animal to another. In molecular biology, a molecule of DNA into which exogenous DNA can be inserted to be cloned.
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vegetative cell A metabolically active, replicating bacterial cell. vegetative mycelium A branched filament produced by vegetative cells that expands into the substrate.
A phylum of free-living aquatic bacteria with wart-like protruding structures containing actin. Verrucomicrobia
vertical transfer See vertical transmission. vertical transmission The passage of genes from parent to
offspring. vesicle A small membrane-bound sphere found within a cell.
A mutualistic association between plant roots and certain fungi, involving hyphal penetration of plant root cells. vesicular-arbuscular mycorrhiza (VAM)
A technique to detect specific proteins. Proteins are subjected to gel electrophoresis, transferred to a blot, and probed with enzyme-linked or fluorescently-tagged antibodies that specifically bind the protein of interest.
Western blot
wet mount A technique to view living microbes with a microscope by placement of the microbes in water on a slide under a coverslip. wetlands Regions of land that undergo seasonal fluctuations in water level and aeration. whey The liquid portion of milk after proteins have precipitated out of solution, usually during cheese production. Winogradsky column A tube containing a stratified environ-
a colony on an agar plate.
ment that causes specific microbes to grow at particular levels; a type of enrichment culture for the growth of microbes from wetland environments.
viable but nonculturable (VBNC) An organism that is meta-
X-ray crystallography
viable An organism that can replicate, for instance by forming
bolically active but can not replicate to form a colony on a plate by current means of culture. Also called a dormant cell.
A technique to determine the positions of atoms (atomic coordinates) within a molecule or molecular complex, based upon the diffraction of X-rays by the molecule.
viral envelope A host-derived membrane that surrounds a
X-ray diffraction analysis
virus capsid. viremia The presence of large numbers of virions in the
bloodstream. A taxonomic group of eukaryotes that includes the land plants and primary endosymbiont algae.
Viridiplantae
virion A virus particle viroid An infectious naked nucleic acid. virulence A measure of the severity of a disease caused by a
pathogenic agent.
See X-ray crystallography.
yeast A unicellular fungus. yeast two-hybrid system An in vivo technique to determine protein-protein interactions in which DNA sequences encoding proteins of interest of are fused separately to the DNA-binding and transactivation domains of a yeast transcription factor. The recombinant yeast is then tested for expression of a reporter gene. yogurt A semi-solid food produced through acidification of milk by lactic acid-producing bacteria.
See dead zone.
virulence factor A trait of a a pathogen that enhances the
zone of hypoxia
pathogen’s disease-producing capability.
zone of inhibition A region of no bacterial growth on an agar
virus An acellular particle containing a genome that can repli-
cate only inside a cell.
plate due to the diffusion of a test antibiotic. Correlates to the minimal inhibitory concentration.
virus shedding The release of virions from the host organism
zoonotic disease An infection that normally affects animals
into the environment.
but can be transmitted to humans.
voltage potential difference (E°) The difference in electrical
zooplankton Heterotrophic marine microbes.
potential between the oxidized and reduced forms of a molecule; the tendency of a molecule to accept electrons.
zoospore A flagellated reproductive cell produced by chytridiomycete fungi.
wastewater treatment A series of wastewater transformations designed to lower biological oxygen demand and eliminate human pathogens before water is returned to local rivers.
endosymbiont.
water activity A measure of the water that is not bound to sol-
utes and is available for use by organisms. water table The layer of soil that is permanently saturated with
water.
SFMB_glossary.indd G-28
zooxanthella
A phototrophic dinoflagellate that is a coral
Zygomycota (zygomycete) A clade of fungi forming nonmo-
tile haploid gametes that grow toward each other, fusing to form the zygospore. zygospore In zygomycetes, the diploid structure formed by the fusion of two gamete-bearing hyphae.
1/17/08 12:38:16 PM
Index
Page numbers followed by a t denote tables; page numbers followed by an f refer to figures; page numbers set in boldface type refer to defi nitions or major discussions.
A ABC transporter, 124–125, 289, 289f, 960, 1050 Abdominal abscess, 1081–1082 Aberration, lens, 49 Abiotic, 641, 832 Abiotrophia, 116t ABO blood group system, 899, 901f, 929 Abscess, abdominal, 1081–1082 Abshire, Kelly, 169 Absorption, 45, 45f AB toxin, 949, 951, 951f, 954 Acanthamoeba, 997 Acceptor end, 270, 270f Acceptor site (A site), 274, 275f Accessory protein, 189, 416, 416t Ace Lake, Antarctica, 722, 722f Acenaphthene, 500, 501f Acetaldehyde, in beer production, 604, 605f, 607 Acetate acetyl-CoA conversion to, 493 as fermentation product, 488f, 489 metabolism of, 466 methanogenesis from, 743 Acetic acid, 84 Acetobacter, in cocoa fermentation, 601 Acetohydroxamic acid, 996 Acetone, as fermentation product, 490 Acetylcholine, 1010, 1010f Acetyl-CoA, 489 activation of, 564 in fermentation pathways, 489, 489f
reductive TCA cycle, 560, 561f TCA cycle and, 491, 491f, 493, 494f Acetyl-CoA carboxylase, 564, 565, 565f Acetyl-CoA pathway, reductive, 561–562, 562f Acetyl phosphate, 491f, 493 Acid, A-19, A-19f Acid-fast stain, 54, 55f, 699, 1074, 1075f Acid fermentation of dairy products, 594t, 595–597, 595f–598f cheese production, 596, 597f cheese varieties, 596, 596f curd formation, 595–596 examples, 594t flavor generation in cheese, 597, 598f of fish, 594t of meat, 594t of vegetables, 594t, 598–599 cabbage fermentation, 599 examples, 594t soy fermentation, 598–599, 599f Acidianus brierleyi, 562 Acidophile, 160–161 archaeal, 721f, 751, 752f defi nition, 151t environmental conditions for growth, 797t Sulfolobus acidocaldarius, 161, 161f, 530f, 734 Acidovorax aveane, 128f Acid resistance in E. coli, 432–433, 434f Acids, use in food preservation, 614 Acid stress response, 163 Acineta, 786f Acinetobacter A. baumanii, 1047 A. calcoaceticus, 306 Acne, 866, 866f ACP (acyl carrier protein), 564
Acquired immunodeficiency syndrome (AIDS), 413–414 antiviral agents, 1057–1058, 1057f, 1058f CD4 T cell count, 922 description, 973–975, 1005–1006, 1005f diagnosis, 1006 HIV virus detection, 22 impact of, 12 incidence, 974f stages, 1005–1006 symptoms, 1005 Acridine orange, 59, 59f Actin, 969, A-35–A-36, A-35f Actinobacteria description, 683 representative groups, 680t Actinomyces, 700, 866 Actinomycetales, 680t, 684 Actinomycetes, 684 acid-fast stain, 699 animal and plant associations, 700, 700f cell differentiation, 142–143, 145–146, 145f, 146f composting and, 462f examples, 680t GC content, 699 irregularly shaped, 702, 702f Micrococcaceae, 703, 703f nonmycelial, 700–702, 701f, 702f Streptomyces, 699–700, 699f Actinomycetes bovis, 883 Actinomycin D, 269, 269f, 1043, 1043f Actinomycosis, 700 Actinophrys sol, 761t Activation B cell, 909, 910, 911f, 919, 920f defi nition, 909 macrophage, 924 T cell, 918–919, 920f, 921 Activation energy (Ea), 473, 473f, A-18–A-19, A-18f Activator, 46, 347, 347f, 348
Active transport, 85, 123–124 Acute phase reactant protein, 890 Acyclovir, 426, 1054t, 1057f Acyl carrier protein (ACP), 564 Acyl homoserine lactone (AHL), 141 Adaptive immune response, 896 Adaptive immunity, 895–934 antibody structure and diversity, 902–908, 902f–903f, 904t, 905f–908f anticapsular antibodies, 885 antigen-presenting cells (APCs), 898, 918 cell-mediated immunity, 897, 915, 920f, 923 cells involved in, 873–876, 873f–875f complement, 925–926, 925f defi nition, 873 genetics of antibody production, 911, 912f, 912t, 913–915, 914f humoral immunity, 897, 908–911, 909f–911f, 915, 920f immunogenicity, factors influencing, 898–899, 898t, 899f–901f, 900t, 901–902 innate immunity compared to, 873 major histocompatibility complex (MHC), 915–916, 916f, 917f, 923 microbial evasion, 924, 924f overview, 895–898, 897f T-cells, 915–916, 915t, 918–919, 919f–922f, 921–925 types, 897 ADCC (antibody-dependent cell-mediated cytotoxicity), 888 Adenine, 18, 227, 323, 329, A-12, A-12t, A-13f
I-1
SFMB_index.indd I-1
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I-2
Ind ex
Adeno-associated virus, 454, 454f Adenosine diphosphate (ADP), 469f, 470, 473–474, 474f Adenosine triphosphate (ATP) ADP phosphorylation to, 469f, 470, 473–474, 474f as energy currency, 461, 469 energy transfer, 470 hydrolysis of, 470 measuring concentration with NMR, 475, 475f produced in Entner-Doudoroff pathway, 482, 482f, 485–486, 486f glucose catabolism, 470–471 glycolysis, 482, 482f, 483f–484f, 484–485 pentose phosphate shunt, 482, 482f, 486–487, 487f TCA cycle, 493, 494f, 495 structure, A-13f synthesis bacteriorhodopsin and, 534 chemiosmotic hypothesis, 511–513, 511f, 513f at high pH, 526–527, 526f use in ABC transporters, 124–125, 125f Calvin cycle, 552–553, 553f nitrogen fi xation, 571 reductive acetyl-CoA pathway, 561, 562f reverse TCA cycle, 560, 561f Adenosine triphosphate (ATP) synthase, 85, 85f, 511, 523, 523f, 524f, 526–527, 527f, 534, 538 Adenoviridae, 194t Adenovirus, 423t entry and uncoating, 202f, 203 as gene therapy vector, 454–455, 455f heart diseases, 1015 Adenylate cyclase, 353, 353f, 927 Adherence, 107 Adhesin, 944, 945t, 948, 948f ADP (adenosine diphosphate), 469f, 470, 473–474, 474f ADP-ribosyltransferase, 949, 951f A/E (attaching and effacing) lesion, 963, 963f
SFMB_index.indd I-2
Aeras Global TB Vaccine Foundation, 616 Aerated horizon, 812, 812f Aerial mycelia, 700 Aerobe defi nition, 151t examples, 165t facultative, 167 strict, 166 Aerobic respiration, 164, 165f, 167 Aeromonadales, 681t Aeromonas A. hydrophila, 861, 861f infection, 860, 860f, 861 Aeropyrum A. pernix, 731, 731t horizontal gene transfer, 661 Aerotolerant anaerobe, 167 AFM (atomic force microscopy), 39, 43, 43f, 65, 65, 67, 67f African sleeping sickness, 791 Agar, 21 Agaricus bisporus, 591, 591f Agarose gel electrophoresis, 249f, 250–251 Aggregation substance, 310 Agrobacterium tumefaciens chromosome structure, 223 crown gall disease, 310–311, 311f disease, 706, 707f genome size, 224t in plant engineering, 621, 622f Ti plasmid, 310–311 Vir system, 964 AHL (acyl homoserine lactone), 141 AhpC protein, 78f AIDS. See Acquired immunodeficiency syndrome (AIDS) AIDS Memorial Quilt, 12f Airy, George, 47 Airy disk, 47, 48f, 49, 49f Akinetes, 689, 690t Akkermans, Antoon, 676 ALA (5-aminolevulinic acid), 582, 582f Alcaligenes A. faecalis, 490, 490f nitrification, 120 phenol red broth test, 490, 490f Alcanivorax borkumensis, 431f Alcohol, structure of, 177t Alcohol dehydrogenase, 604 Alcoholic fermentation beer, 604–605, 605f, 606–607, 607f examples, 594t wine, 605–607, 605f, 608f
Aldehyde as disinfectant, 176 functional group, A-7t monosaccharides as, A-11 structure, 177t Aleuria, 771f Algae bloom, 169, 170f, 811 brown, 779, 780f cell wall, 97 coralline, 778, 778f cryptophyte, 763, 763f defi nition, 763 edible, 591, 591f golden, 779 green, 761t, 763, 763f, 774–776, 775f–777f colonial, 776, 777f fi lamentous, 776, 776f sheet-forming, 776, 777f siphonous, 776 unicellular, 774–776, 775f halophilic, 158 lichen, 798–799 microfossils, 636, 636f mixotrophs, 852 phylogeny, 761t red, 761t, 763, 763f, 778, 778f symbiosis endosymbiosis, 666–667, 668f, 763, 763f, 778–779, 824, 825f lichen, 666 secondary endosymbiosis, 671–672, 671f viruses of, 213–214, 213f Algal bloom, 169, 170f, 811 Alginate, 142 Algorithm, for bacterial identification, 1065–1072 Alicyclobacillus, 462f Alkaline fermentation, 599–602 description, 593 examples, 594t natto, 600–601, 602f pidan, 602, 602f vegetables, 601–602 Alkaline stress response, 163 Alkaliphile, 161–162 ATP synthesis in, 526, 526f defi nition, 151t environmental conditions for growth, 797t soda lake ecosystem, 161, 161f sodium circulation, 162f Alkane (functional group), A-7t Allergen defi nition, 926 inhaled, 928f type I hypersensitivity reaction, 927–929
Allergy. See Hypersensitivity Allograft, 933 Allolactose, 351, 351f Allomyces, 760t Allosteric regulation, 485 Allosteric site, 474 Allotype, antibody, 903–904 Alpha-amanitin, 771, 771f Alpha helix, A-9, A-10f Alpha hemolysis, 1070, 1071f Alpha-ketoglutarate, 493, 494f, 496 Alpharetrovirus, 413t Alpha toxin, of Staphylococcus aureus, 949, 951, 952f Alternation of generations, 767 Altman, Sydney, 19, 644, 644f Alu sequence, 422 ALV (avian leukosis virus), 184, 185f Alveolar macrophage, 878, 1015 Alveolata (alveolates), 761t–762t, 764, 764f, 783–790, 783f–790f Alvin (submersible vessel), 802, 802f Alzheimer’s disease, 22 Amanita (amanita), 590, 771, 771f Amanitin, 591 Amantadine, 1054t, 1056, 1056f Ameba description, 763 diversity, 761t, 781 foraminiferans, 783, 783f genetics, 781 meningitis/encephalitis, 1012t Mimivirus infection, 184, 185f movement, 780f, 781, 781f overview, 780 radiolarians, 783, 783f reproduction, 781 structure, 42, 42f Amebic dysentery, 997 Amensalism, 799t, 801 American Society for Microbiology, 36 Ames, Bruce, 325 Ames test, 325–326, 326f Amherst, Jeffery, 1090 Amikacin, 1051, 1052f Amino (functional group), A-7t Amino acid, A-7–A-9, A-8f catabolism flavor generation from, 597, 598f overview, 479, 479f metabolist model of early life and, 642, 642f in meteorites, 854
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In d ex
in Murchison meteorite, 576f synthesis, 575–580 arginine biosynthesis, 577, 578f aromatic amino acids, 577–578, 579f assimilation of NH4 +, 576–577, 576f diversity and complexity of, 575 major pathways, 575–576, 575f for nonribosomal peptide antibiotics, 579–580, 584–585, 584f–585f substrates for biosynthesis of, 548, 549f transamination, 577 Aminoacyltransferase, 100 Aminoacyl-tRNA transferase, 271, 272f Aminoglycoside inactivation, 1048, 1049f mechanism of action, 1044 5-aminolevulinic acid (ALA), 582, 582f Ammonia, 843 dissimilatory nitrate reduction to, 847 nitrification, 845 in nitrogen cycle, 120, 120f oxidation, 708 produced in nitrogen fi xation, 570–571, 570f, 844 Ammonia-oxidizing crenarchaeotes, 737 Ammonification, 845 Ammonium, 843 assimilation for amino acid synthesis, 576–577, 576f from nitrogen fi xation, 844 oxidation, 529 Amoeba proteus, 42f Amoebozoa, 761t, 763 Amphibolic pathways, 485 Amphipathic, 641, A-15, A-22, A-24 Amphotericin B, 989, 1059, 1059f, 1060t–1061t Ampicillin effect on E. coli, 178f spectrum of activity, 1033, 1039 structure, 1039f Amplicon, 446 Ampullaviridae, 194t Amycolatopsis orientalis, 1040, 1041f Anabaena A. spiroides, 573 akinetes, 689 appearance, 42f, 73f, 679t
SFMB_index.indd I-3
genome, 688 genome size, 224t heterocyst, 144, 144f, 573 Anabolism, 462, 548. See also Biosynthesis Anaerobe abdominal abscess, 1081 carbon cycling, 836, 836f carbon dioxide fi xation in, 560–564, 561f–563f culturing in laboratory, 167–168, 167f defi nition, 151t examples, 165t facultative, 167 specimen collection, 1081f strict, 166, 167 Anaerobe jar, 167, 167f Anaerobic benzoate catabolism, 499–500, 500f Anaerobic dechlorinators, 697–698, 698f Anaerobic glove box, 167–168 Anaerobic respiration, 525–529 alternative electron acceptors and donors, 525, 525f description, 166, 525 dissimilatory metal reduction, 528–529 by facultative anaerobes, 167 in lake water column, 528f nitrate reduction, 525–527 sulfur reduction, 528 Anaerobiosis, N2 fi xation and, 572, 574 Analytical profi le index (API), 1066–1068, 1067f, 1067t, 1069f Anammox reaction, 529 Anamnestic response, 909 Anaphase, A-30, A-31f Anaphylaxis description, 927 late-phase, 928 Anaplerosis, 495 Anaplerotic reactions, 560 The Andromeda Strain (fi lm), 6, 7f, 62 And the Band Played On (fi lm), 414, 414f Anfi nsen, Christian, 283 Angert, Esther, 695, 695f Angle of aperture, 49 Animal gnotobiotic, 872 microbial communities within, 824–829 digestive communities, 826–828, 826f–828f sponge communities, 825, 825f zooxanthellae, 824, 825f
phylogeny, 760t, 758 virus life cycle DNA virus, 203, 204f oncogenic viruses, 206 receptor binding, 201, 202f, 203 RNA retrovirus, 205–206, 206f RNA virus, 205, 205f uncoating of genome, 202f, 203 Anion, A-4 Annotation of the DNA sequence, 293–294 Ant, leaf-cutter, 666, 667f Antenna complex, 537–538, 538f Anthracnose fungus, 823f, 824 Anthrax bioterrorism, 20, 20f, 1090–1091, 1091f case history, 1085 as occupational hazard, 981 Robert Koch and, 20–21, 20f Anthrax lethal factor, 69, 69f Anthrax toxin, 956–958, 957f Anti-anti-sigma factor, 365 Anti-attenuator stem loop, 362, 363f Antibiotic. See also specific antibiotics bacteriocidal, 1033 bacteriostatic, 1033 biosynthesis, 1046–1047, 1046f–1047f control of bacterial growth, 176–178, 177f, 178f defi nition, 25, 176, 1030 development, 26 discovery of, 25–26, 1029, 1030–1032, 1030f drug discovery, 178 drug susceptibility, measuring, 1034–1037 Kirby-Bauer disk susceptibility test, 1034–1037, 1035f minimal inhibitory concentration (MIC), 1034, 1034f, 1035f, 1036, 1036f future discovery, 1052–1054 for gastroenteritis, 993 halocins, 750 hybrid, 1051 livestock use, 1051 mechanisms of action, 1037–1045, 1037t cell membrane, 1041, 1041f cell wall, 1037–1041, 1038f–1041f DNA synthesis, 1041–1042, 1042f, 1043f
I-3
protein synthesis inhibitors, 1043–1045, 1044f, 1045f RNA synthesis, 1043, 1043f nanotubes, 1055, 1055f nonribosomal peptide, 579–580, 584–585, 584f–585f overview, 1029–1030 production as secondary products, 566 resistance, 26, 1047–1052 biofi lms and, 141, 141f, 142 causes of, 1051 characterizing microbes by resistance profi le, 1064 development of, 1050–1051 fighting, 1051, 1052f integrons and, 338–339 mechanisms of, 1048–1050, 1048f–1050f plasmids, 243 use in RNA synthesis research, 265 selective toxicity, 1030 spectrum of activity, 1033, 1033t target transcription, 268–269, 268f–269f translation, 281, 281f use in plants, 150 Antibody. See also Humoral immunity anticapsular, 885, 970 autoimmune, 931–932 defi nition, 897 diversity generation, 911, 912t, 913 ELISA detection, 1077–1078, 1078f fluorescent antibody staining, 1079, 1079f genetics of production, 911, 912f, 912t, 913–915, 914f hypersensitivity reactions type I, 926–929, 928f type II, 929, 929f type III, 929–930, 930f immunofluorescence, 59 isotypes, 903–905, 904t, 905f, 908 IgA, 904, 904t, 905f IgD, 904t, 905 IgE, 904t, 905, 908 IgG, 903–905, 904t IgM, 904–905, 904t, 905f isotype (class) switching, 908, 909, 913, 914f
1/18/08 5:15:14 PM
I-4
Ind ex
Antibody (continued) labeled, 59 monoclonal, 928 opsonization, 885, 887, 970 primary antibody response, 908–909, 909f production by plasma cells, 908–909 properties, 904t removal by efflux proteins, 85 secondary antibody response, 908–909, 909f structure, 902–903, 902f, 903f, 905f in Western blot analysis, 437–438, 437f Antibody-dependent cell-mediated cytotoxicity (ADCC), 888 Antibody stain, 55 Anticapsular antibody, 885, 970 Anticodon, 270, 270f Antifungal agents, 1059–1060, 1059f, 1060t–1061t Antigen applications based on antigen-antibody interactions, 906–907 immunoprecipitation, 906, 906f, 907f radial immunodiffusion, 906, 907f Western blot, 906–907 B-cell response to, 909–910, 910f, 911f binding to MHC, 916, 916f defi nition, 873, 897 denatured, 897f epitopes, 897, 897f immunogenicity, factors influencing, 898–899, 898t, 899f, 901, 901f phase variations, 926 presentation, 898, 916, 917f, 919, 921–922, 921f superantigen, 919, 921, 922f, 949 threshold dose, 898 Antigen capture ELISA, 1078, 1078f Antigenic determinant, 897, 899 Antigenic shift, 972 Antigen-presenting cell (APC), 876, 895f, 898, 916, 917f, 918, 919, 920f, 921 Antihistamine, 927, 928
SFMB_index.indd I-4
Antimicrobial, 1029–1061. See also Antibiotic antifungal agents, 1059–1060, 1059f, 1060t–1061t antiviral agents, 1054, 1054t, 1056–1058, 1056f–1058f biosynthesis, 1046–1047, 1046f–1047f discovery, 1029, 1030–1033, 1030f future discovery, 1052–1054 mechanisms of action, 1037–1045, 1037t, 1038f–1045f resistance, 1047–1052, 1048f–1050f, 1052f spectrum of activity, 1033, 1033t susceptibility, measuring, 1034–1037, 1034f–1036f, 1036t Antimicrobial peptides, natural, 879–880, 879t Antiparallel, arrangement of DNA, 226 Antiport, 123–124, 124f Antisense RNA, 368, 1053 Antisepsis, 171 Antiseptic agent, 25 Anti-sigma factor, 365, 367–368 Antiviral agent DNA synthesis inhibitors, 1056–1057 examples, 1054t for HIV, 1057–1058, 1057f, 1058f for influenza, 1054, 1056, 1056f targets of, 184 Ants, leaf-cutter, 700 APC (antigen-presenting cell), 876, 895f, 898, 916, 917f, 918, 919, 920f, 921 AP endonuclease, 329 Aphotic zone, 801, 801f Aphthovirus, 400t, 401 API (analytical profi le index), 1066–1068, 1067f, 1067t, 1069f Apical complex, 788 Apicomplexan, 764, 788–790, 788f–790f Apicoplast, 789, 790 Apoptosis immune avoidance and, 970 of T cells, 924 AP site, 329 Apurinic site, 323–324, 324f Aquaporin, 122, 157, 157f
Aquatic microbiology Alpha Proteobacteria, 706 freshwater, 809–811, 809f–811f marine, 81–809, 801f, 804f–805f, 806t, 807f–808f Aquifex A. aeolicus, 685 A. pyrophilus, 685 appearance, 679t “archaeal” traits, 678 autotrophy, 685 genome, 685 Aquificales, 678, 679t, 685 Arabinans, 92, 93f AraC, 355–357, 359–361, 359f, 360f ara operon, 359–360, 359f Arbuscule, 817, 817f ArcB transporter, 1050, 1050f Archaea (Domain), 9, 721–753 bacteria and eukaryotes compared, 657, 660–661, 660t, 722t, A-22t biofi lms, 142 Crenarchaeota ammonia-oxidizing, 737 hyperthermophiles, 730–735, 730f–735f, 731t mesophiles, 735–736 phylogeny, 726, 727f, 728t psychrophiles, 736–737, 737f discovery of, 30 Euryarchaeota acidophiles, 751, 752f halophiles, 744–748, 744f–746f, 745t, 748f–749f, 750 methanogens, 738–744, 739f–743f, 739t phylogeny, 727, 727f, 728t–729t thermophiles, 750–751 gene regulation, 725–726, 726f genome, 248 genome size, 224t genomic analysis, 304 haloarchaea, 744–750 applications, 751 classroom use of, 746, 746f examples, 745t habitat, 721, 744, 744f, 747 metabolic pathways, 749f phototrophy, 721, 724, 727, 747–748, 748f phylogeny, 727, 727f, 728t pigmentation, 744, 744f, 746
structure and physiology, 744–745, 745f, 747 hyperacidophile, 721f, 729t, 751, 752f hyperthermophiles barophiles, 731–733, 732t, 733f Desulfurococcales, 730–733, 731f, 731t Euryarchaeota, 750–751, 750f examples, 731t habitats, 721, 730, 730f Korarchaeota, 753 phylogeny, 726, 728t, 729t reverse gyrase, 724 sulfolobales, 734–735, 734f, 735f initiation factors, 276 introns, 364 Korarchaeota, 727, 727f, 729t, 753 lack of pathogens, 661, 723 low-∆G energetics, 468 mesophiles, 735–736 metabolism, 722t, 724, 725f methanogens examples, 739t habitat, 721, 723, 740–742, 741f histones, 726 metabolism, 724, 738, 742–743, 742f, 743f phylogeny, 727, 729t structure, 738–740, 739f, 740f Nanoarchaeota, 727, 727f, 729t, 753, 753f overview, 721–723 phylogeny Crenarchaeota, 726, 727f, 728t Euryarchaeota, 727, 727f, 728t–729t Korarchaeota, 727, 727f, 729t Nanarchaeota, 727, 727f, 729t positive supercoils, 99, 230 proteasome, 291, 291f psychrophiles, 721, 722, 722f, 726, 728t, 736–737, 737f secretion system, 289 S-layer, 94, 94f structure cell envelope, 722t, 723–724, 723f nucleic acid, 722t, 724–725 terpene-derived membrane lipids, 87–88, 87f, 88f thermophiles, 722, 726, 728t, 729t, 735, 750–751
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In d ex
Archaeal viruses, 194t, 734–735 Archaean eon, 633, 635, 641, 642, 643 Archaeoglobus A. fulgidus appearance, 750f horizontal gene transfer, 661 metabolism, 751, 752f metabolism, 727, 751, 752f Archaeon, 9 Archaeosine, 725 Arenaviridae, 195t argA gene, 322 Arginine biosynthesis, 577, 578f Aromatic amino acids, biosynthesis of, 577–578, 579f Aromatic catabolism, 458, 479–481, 496–501 benzoate catabolism, 497, 497f, 499–500, 500f polynuclear aromatic hydrocarbons (PAHs), 500, 501f Aromatic ring, 471 Arrhenius equation, 152–153, 152f Arsenic, microbial metabolism of, 852t Arthritis, Lyme, 1019, 1021 Arthrobacter A. globiformis, 702f appearance, 702, 702f bioremediation by, 702 Arthrospore, 690t, 692, 700 Arthus, Maurice, 929 Arthus reaction, 930 Artifact, 65, 65f Ascomycota (ascomycetes), 760t, 769–771, 770f, 771f Ascospore, 769, 770f Ascus, 767, 769, 770, 770f Aseptic, 25 Asilomar conference, 35 Asimov, Isaac, 854 A site (acceptor site), 274, 275f Aspergillosis, 984t, 990t–991t, 1061t Aspergillus A. niger, 619 A. orgyae, 599 A. versicolor, 760t cheese spoilage, 609 conidiophores, 770 disease, 984t, 990t–991t, 1061t Hurricane Katrinaassociated mold, 772 industrial strains, 619 reproduction, 767 soybean fermentation, 599 Aspirin, 84, 84f
SFMB_index.indd I-5
Assembly, viral, 394 bacteriophage M13, 399f, 400 HIV, 420 influenza virus, 408–409, 411 poliovirus, 403f, 405 T4, 394, 395–397, 396f Assimilation, 795 Assimilatory nitrate reduction, 845 Asthma, 927–928 Astrobiology, 854–857, 855f, 857f -ate (suffi x), 482 Athalassic lake, 747 Atherosclerosis, 1015, 1016f Athlete’s foot, 938, 938f Atmosphere of Earth, 633 of Mars, 855 Atom, A-2 Atomic force microscopy (AFM), 39, 43, 43f, 65, 65, 67, 67f Atomic mass, A-2, A-3f Atomic number, A-2, A-3f Atopic disease, 927–928 ATP. See Adenosine triphosphate (ATP) ATP-binding cassette, 124, 125, 289, 960 ATP citrate lyase, 560, 561f ATP synthase, 85, 85f, 511, 523, 523f, 524f, 526–527, 527f, 534, 538 Attaching and effacing (A/E) lesion, 963, 963f Attenuation pathogen, 23–24 transcriptional, 361–362, 363f translational control, 348–349 Attenuator stem loop, 362, 363f att site, 312, 313f, 314, 320, 338f, 339 Autoaggregation, in Pseudomonas, 345f Autoclave, 17, 172, 172f Autoimmune response, 931–932, 932t chronic inflammation, 883–884 Autoinducer, 378, 379, 379f, 380 Autoradiography, 436 Autotroph/autotrophy, 118, 119, 119f Avery, Oswald, 33, 304 Avian influenza, 203, 389f, 408f, 409, 1086 Avian leukosis virus (ALV), 184, 185f, 413t
Avogadro’s number, A-16 Axenic growth, 118 Axoneme, A-36, A-37f Azam, Farooq, 803 Azidothymidine (AZT), 413, 418 Azithromycin, 1044 Azoarcus evansii, benzoate catabolism by, 500, 500f Azotobacter growth media for, 117t nitrogen fi xation, 570, 572 Azotobacter medium, 117t AZT, 184
B BAC (bacterial artificial chromosome), 253 Bacillales, 680t, 692–694, 693f, 694f Bacillaraceae, 778 Bacillariophyta, 779 Bacillary dysentery, 939, 993 Bacille Calmette-Guérin (BCG), 616–617 Bacilli, 42, 42f, 43 Bacillus alkaline fermentation, 593, 600–602, 602f alkalithermophilic, 694 antibiotic production, 1040 asymmetrical cell division, 104 B. alkalophilus, 694 B. anthracis anthrax, 1085, 1090–1091 anthrax lethal factor, 69, 69f anthrax toxin, 956–958, 957f Bacillus subtilis compared, 218 as bioweapon, 1090–1091, 1091f endospores, 143 genome sequencing, 11 respiratory tract infection, 990t–991t scanning electron micrograph, 957f spore stain, 54 transmission electron microscopy (TEM), 64, 64f B. brevis, 1041 B. cereus, 449–450, 450f B. halodurans, 694 B. licheniformis, 1040 B. megaterium ATP synthase, 527 size, 41f B. natto, 600–601, 602f B. polymyxa, 1041 B. pseudofirmus ATP synthase, 527, 527f
I-5
B. subtilis antibiotic production, 1040, 1041 appearance, 680t colony morphology, 115f DNA polymerase location, 241, 241f DNA replication, 102, 102f endospore formation, 143, 143f genome, 693 glutamine synthase gene, 297 growth requirements, 130 industrial strains, 619 interference microscopy, 57f, 58 proteome, 385, 385f sigma factors, 218–219 sporulation, 59–60, 60f, 218–219, 219f, 693f B. thermophilus, 694 B. thuringiensis Bacillus subtilis compared, 218 insecticidal gene/protein, 311, 450–451, 451f, 451t, 694, 694f pathogenicity, 940 spore stain, 54 chromosome dynamics, visualizing, 219 composting and, 462 description, 692–694 doubling time, 232 endospore formation, 692 metallo-β-lactamase, 449–450, 450f nitrification, 120 nitrite reduction, 526 skin flora, 864 sporulation, 218–219 thermophilic, 694 variation in strains, 663 Bacitracin, 1040 action of, 1038f biosynthesis, 1046 structure, 1041f test for beta-hemolytic streptococci, 1071–1072, 1071f Bacteremia, 867, 987, 1014 Bacteria (Domain), 9 Archaea and Eukarya compared, 657, 660–661, 660t, 677–678, A-22t cardiovascular system infections, 1014–1015, 1014f, 1016f central nervous system infections, 1007–1008, 1007f, 1012t
1/18/08 5:15:15 PM
I-6
Ind ex
Bacteria (Domain) (continued) common traits of, 677–678, 677f diversity, 675–718 bacteroidetes and chlorobi, 682t, 684, 714–715, 714f, 715f cyanobacteria, 679t, 683, 688–692, 688f–691f gram-negative proteobacteria and nitrospirae, 680t–682t, 684, 703–714, 704f–705f, 707f–713f gram-positive fi rmicutes and actinobacteria, 680t, 683–684, 692–703, 692f–703f irregularly shaped bacteria, 683t, 684, 716–718, 717f, 718f lithotrophic reactions, 705t overview, 675–677 phototrophic bacteria, 687t phylogeny, 678, 678f representative groups of, 679t–683t spirochetes, 682t, 684, 715–716, 715f, 716f spore types, 690t thermophiles, 678, 679t, 683, 685–688, 685f, 686f unclassified and unculturable bacteria, 684–685 DNA replication, 232–243 edible, 592 gene naming conventions, 226 genome organization, 223–231 identification procedures, 1080t notifiable diseases, 1088t respiratory tract infections, 986–987, 987f, 990t–991t skin infections, 984t succession in digestive tract of newborn baby, 676, 676f Bacterial artificial chromosome (BAC), 253 Bacteriochlorophyll, 536–537, 537f, 540, 541f, 542f, 686, 704, 705 Bacteriocidal, 171, 1033 Bacteriophage as cloning vectors, 184 CTX φ, 189, 200
SFMB_index.indd I-6
culturing batch culture, 208–209, 209f plaque isolation and assay, 210–211, 211f defi nition, 183 DNA replication mechanisms, 244–245, 246f epsilon 15, 181f fd, 448 fi lamentous structure, 189, 190f φX174, 9–10, 10f icosahedral structure, 187, 188f lambda genome replication, 246f host receptor interaction, 198, 198f, 199f life cycle, 198–200, 199f lysis/lysogeny decision, 373–375, 374f plaques, 373f specialized transduction, 312, 313f, 314 structure, 194t life cycle attachment, 198 genome entry, 198–199 lysogeny, 199f, 200 lytic cycle, 199–200, 199f needs for host infection, 198 slow-release, 200–201, 201f transduction, 200 M13 adsorption, 398, 399f assembly, 399f, 400 DNA delivery, 398 export, 399f genome map, 398f nanowires, 397 phage display, 448, 449f plaques, 210, 211f replication, 398–400, 399f slow-release cycle, 200–201 structure, 189, 190f, 397–398, 397f, 398f MS2, 195t, 201 P22, 311, 312f plaques, 373f transposable elements, 333 phage display, 448–450, 449f, 450f phage therapy, 179 phylogeny, 196 proteomic tree, 197, 197f Qβ, 201 RNA, 195t, 201 T4 adsorption to host, 391–392, 393f
assembly, 394, 395–397, 396f DNA delivery, 392–393, 393f early genes, 393–394 evolution of, 395 genome map, 392f genome replication, 245, 246f genome size, 184 injector device of, 392–393, 393f late genes, 394 lysis and phage release, 394 lytic cycle, 199–200 mutations, 395–397, 396f replicative cycle, 393–395, 394f, 395f structure, 183, 183f, 188f, 391, 391f transduction, 200, 311–314, 312f–314f Bacteriorhodopsin, 513, 534, 535, 535f, 638–639, 638f, 724, 747–748, 748f, 750 Bacterioruberin, 744, 744f Bacteriostatic, 171, 1033 Bacterium, 9 Bacteroid, 820, 822 Bacteroidales, 682t Bacteroides appearance, 682t B. fragilis, 714, 714f abdominal abscess, 1081–1082 disease from, 871 gram stain, 1081f stress response system, 1082 B. thetaiotaomicron, 478, 714, 742 benefits to host, 869 decay-accelerating factor, upregulation of, 926 farming of intestinal mucus, 485 fermentation by, 684 gas gangrene, 872f intestinal flora, 869 metronidazole activation, 1042 Bacteroidetes, 682t, 684, 714–715, 869 Bacteroids, 706, 707f Bactoprenol, 1038, 1039 Baculoviridae, 194t Baculovirus, 621–623, 623f, 624f, 940 Badnaviridae, 196t Baeocytes, 689 Bait protein, 441, 443 Baldauf, Sandra, 758 Baltimore, David, 192, 193f
Baltimore virus classification system, 192–193, 193f, 194t–196t Banded iron formation (BIF), 639, 639f, 640f Bangia, 636f Barns, Susan, 655, 753 Barophile, 151t, 156, 156f, 730, 797t, 808 Barophilic hyperthermophile, 731–733, 732t, 733f Barry, Clifford, 617 Base, A-19, A-19f Base, nucleic acid description, A-12, A-12t tautomeric shifts, 323, 323f Base analog, 322t Base excision repair (BER), 327t, 328–329, 330f Basic Local Alignment Search Tool (BLAST), 297 Basidia, 772–773, 773f Basidiomycota (basidiomycetes), 760t, 771–773, 771f, 773f Basidiospore, 773, 773f Basler, Christopher F., 1057 Basophil, 874 Bassham, James, 550 Bassler, Bonnie, 381, 381f Batch culture bacteriophage, 208–209, 209f defi nition, 137 growth stages, 136f, 137–138 Batrochochytrium dendrobatidis, 768, 768f BATS (Bermuda Atlantic Time-Series), 804, 805f Battista, John, 175, 175f Bautz, Ekkehard, 218 B cell activation, 909–910, 911f, 919, 920f capping, 909, 911f, 919 clonal selection, 909–910, 910f description, 876 development, 876 maturation, 913 memory, 908, 909, 913–914 plasma cells, 908–909 selection in bone marrow, 918 self-reacting, 932 B cell receptor, 909, 910, 910f B-cell tolerance, 898 BCG (bacille CalmetteGuérin), 616–617 BCG vaccine, 930 Bdellovibrio, 712–713, 713f B. bacteriovorus, 67, 67f Bdellovibrionales, 682t
1/18/08 5:15:16 PM
In d ex
Beadle, George, 769 Bedrock, 812, 812f Beer, 116, 604–605, 605f, 606–607, 607f Bee sting, 927–928 Beggiatoa commensalism, 799t, 800 nitrogen fi xation, 570 sulfur oxidation, 709 Winogradsky’s work on, 26 Beijerinck, Martinus, 22, 27, 570 Béjà, Oded, 534 Belmont, Andrew, 219 Bennington, Jackie, 35 Benson, Andrew, 550 Benthic organism, 801 Benthos, 801, 801f, 808, 810 Benzalconium chloride, 177t Benzene hapten, 901, 901f Benzene ring, stability of, 496 Benzoate catabolism aerobic, 497, 497f anaerobic, 499–500, 500f Benzoic acid, 614 BER (base excision repair), 327t, 328–329, 330f Berg, Howard, 56, 109 Berg, Paul, 9, 35, 35f Bergey’s Manual of Determinative Bacteriology, 649 Berkmen, Melanie, 102f Bermuda Atlantic Time-Series (BATS), 804, 805f Bernal, John, 69, 69f β-galactosidase, 350, 350f, 351f, 434–435 in bacteriophage plaque assay, 210, 211f Beta hemolysis, 1070–1071, 1071f Beta-lactamase, 90, 449–450, 1039, 1048, 1048f, 1050, 1051 Betapropiolactone, 177t Betaretrovirus, 413t Beta sheet, A-9, A-10f Bfp (bundle-forming pilus) gene, 664 Bicarbonate (HCO3 – ) transporters, 558–559 BIF (banded iron formation), 639, 639f, 640f Bifidobacteriales, 680t Bifidobacterium, 179, 676, 870, 871f Bile salts, 869 Bill and Melinda Gates Foundation, 616 Binary fission, 134 Bioburden, 864, 866t Biochemical composition of bacteria, 75f, 76–78, 77t
SFMB_index.indd I-7
Biochemical oxygen demand (BOD), 802, 839–840, 839f, 840f Biocomplexity Initiative, 3 Biodegradation, of petroleum waste, 499 Bioelectric DNA detection chip, 1092–1093 Bioethics, 432 Biofi lm anaerobic, 851, 852f antibiotic tolerance and, 141, 141f, 142 bladder pods, 1000, 1000f cyanobacterial, 691, 691f defi nition, 140 in endocarditis, 1015 Escherichia coli, 1000 examples, 140f formation, 140–142, 142f Gamma Proteobacteria, 710 hyperthermophiles, 733, 734f phylogeny of a shower curtain biofi lm, 658, 658f, 659f pseudomonad, 711, 711f Pseudomonas aeruginosa, 380 role of type IV pili in, 444, 445f Biogeochemical cycles, 831–854 carbon cycle, 835–838 aerobic, 835–836, 836f anaerobic, 836, 836f global carbon balance, 837, 837f wetlands and, 838, 838f defi nition, 833 hydrologic cycle, 839–842 biochemical oxygen demand, 839–840, 839f, 840f wastewater treatment, 840–842, 841f, 842f iron, 850–853, 851f metal metabolism, 822t, 853–854 nitrogen cycle, 842–847, 843f–846f denitrification, 846, 846f dissimilatory nitrate reduction, 847 nitrification, 845–846, 845f nitrogen fi xation, 844–845, 845f nitrogen sources, 843 nitrogen triangle, 843–844, 844f overview, 832–835 phosphate, 849–850, 850f sources and sinks of elements, 833–834, 833t sulfur, 847–849, 848f, 849f
Biogeochemistry, 832 Bioinformatics, 258, 293–298 annotation, 293–294 defi nition, 294 genomic predictions, 298, 299f homologs, 297 ORF identification, 294–295, 295f orthologs, 297, 297f paralogs, 297, 297f programs and web sites, 297–298 sequence alignment, 296 unculturable organisms, 150 Biological control of microbes, 178–179 Biological molecules, A-1–A-20. See also specific classes of molecules bonds, A-2–A-5, A-4f, A-5f lipids, A-14–A15, A14f–A15f nucleic acids, A-12, A-12t, A-13f, A-14, A-14f organic, features of, A-5–A-6, A-6f polysaccharides, A-10–A-12, A-11f, A-12f proteins, A-6–A-10 amino acids, A-7–A-9, A-8f functions, A-6–A-7 structure, A-9–A-10, A-9f, A-10f Biological safety cabinet, 173, 174f Bioluminescence, 378–381, 378f–381f Biomass, 796, 804 Biomineralization, 108 Biomolecular motor, 85, 85f Biopanning, 448–449 Bioprospecting, 617–619 Bioremediation, 175 dehalorespiration, 532, 532f Biosafety containment procedures, 1083–1085, 1084t, 1085f Biosignature (biological signature), 635t, 637, 856 Crenarchaeota, 730, 730f cyanobacterial hopanoids, 638 isotope ratio, 637–638, 637f, 646, 646f, 856 of life on Mars, 855–856 Biosphere, 633 Biosynthesis, 547–586 amino acids, 575–580 arginine biosynthesis, 577, 578f
I-7
aromatic amino acids, 577–578, 579f assimilation of NH4 +, 576–577, 576f diversity and complexity of, 575 major pathways, 575–576, 575f for nonribosomal peptide antibiotics, 579–580, 584–585, 584f–585f transamination, 577 carbon dioxide fi xation, 550–564 Calvin cycle, 550–560, 550f, 551t, 552f–553f, 557f 3-hydroxypropionate cycle, 551t, 562, 563f, 564 reductive acetyl-CoA pathway, 551t, 561–562, 562f reductive (reverse) TCA cycle, 551t, 560, 561f defi nition, 548 energy costs of, 548, 550 fatty acid, 564–566, 565f nitrogen fi xation, 570–575 anaerobiosis and, 572, 573f, 574 assimilation of nitrogen forms, 570, 570f early discoveries of, 570–571, 570f mechanism, 571–572, 572f, 573f regulation of, 574–575, 574f overview, 548–550 polyesters, 566, 566f polyketides, 567–569, 567f–569f purines and pyrimidines, 578–579, 580f regulation, 550 substrates for, 548, 549f TCA cycle intermediates as substrates for, 495–496 tetrapyrroles, 581–583, 586 Biotechnology, 431–456 applied microbial, 450–455 Bacillus thuringiensis, insecticidal proteins, 450–451, 451f gene therapy, 454–455, 454f, 455f vaccines, 452–454, 453f, 454t artificial evolution techniques, 446–450 DNA shuffl ing, 447–448, 448f phage display, 448–450, 449f, 450f
1/18/08 5:15:16 PM
I-8
Ind ex
Biotechnology (continued) case study (acid resistance in E. coli), 432–433 genetic analyses, 432–435, 435f global analyses, 444–446 history of, 431 molecular analyses, 436–443 scope of, 431, 432 tools DNA mobility shift, 438, 438f DNA protection analysis, 441, 442f fluorescence resonance energy transfer (FRET), 446 fluorescently tagged cells, 444–445, 445f multiplex PCR, 445–446, 446f Northern blots, 436–437, 436f primer extension analysis, 440–441, 440f protein-protein interaction maps, 444, 444f protein purification, 438–440, 439f real-time PCR, 446, 447f Southern blots, 436, 437 two-hybrid analysis, 441–443, 443f Western blots, 437–438, 437f Bioterrorism, 20, 20f, 1090–1091, 1091f Biotic, 832 Bioweapons, 1090 Birnaviridae, 195t 1,3-bisphosphoglycerate, 484f, 485 Black Death, 1018. See also Plague Black smoker, 731, 732f, 733, 733f, 802, 802f Bladder infection, 999–1001, 1000f, 1001f BLAST (Basic Local Alignment Search Tool), 297 Blastochloris, 704, 810f Blastomyces dermatitidis, 984t, 988, 988f, 989 Blastomycosis, 984t, 990t–991t, 1059, 1060t Blood agar, hemolysis on, 1070–1071, 1071f Blood-brain barrier, 1007 Bloom, 169, 170f Blue baby syndrome, 846
SFMB_index.indd I-8
BLV (bovine leukemia virus), 413t BOD (biochemical oxygen demand), 802, 839–840, 839f, 840f Boil, 982–983 Bond, A-2–A-5 covalent, A-2, A-4, A-4f hydrogen, A-4f, A-5 ionic, A-4, A-4f nonpolar, A-4 polar, A-4, A-4f types and strengths, A-5t Bordetella adherence to cilia, 979f B. bronchiseptica, 979f B. pertussis attachment mechanism, 945t DNA-based detection test, 1073t genome reduction, 340 identification procedures, 1080t pertactin, 948f respiratory tract infection, 990t–991t toxin, 954 tracheal colonization, 948f type IV secretion system, 964, 964f whooping cough, 1025 growth factors and natural habitat, 116t pertactin, 948, 948f Borrelia B. burgdorferi, 715f, 716 appearance, 42f, 682t, 1020f DNA-based detection test, 1073t genome size, 224t growth requirements, 117 linear chromosome, 223 Lyme disease, 1019–1021, 1020f, 1022t, 1086, 1094 outer membrane proteins, 96, 96f reservoir, 1086 B. recurrentis, 715f, 716 Botox, 1010 Botstein, David, 333, 333f Botulism, 998t case history, 1008 infant, 1008 toxin action of, 613, 613f, 1009–1010, 1010f cosmetic use, 694, 694f, 1010 structure, 1008–1009, 1009f Bovine leukemia virus (BLV), 413t
Bovine serum albumin (BSA), as protein carrier, 901, 901f Bovine spongiform encephalopathy (BSE), 1013 Boyer, Herb, 251, 251f Boyle, Robert, 12 Bradykinin, 883, 884f Bradyrhizobium nitrogen fi xation, 120, 706 symbiosis genes, 340 Braun lipoprotein, 94 Brazier, Martin, 637 Bread dough, leavened, 594t production, 602, 603–604, 603f, 604f spoilage, 610t, 611 Bregoff, Herta, 571 Bright-field microscopy, 43, 48–55 Brochothrix thermosphacta, 609 Brock, Thomas, 154f, 155, 685, 731 Bromoviridae, 195t Bronchopneumonia, 987 Brown, James W., 655f Brown algae, 591, 779, 780f Brucella (brucellosis), 706, 1022t B. abortis, 1022t Brugya malayi, 668, 668f BSA (bovine serum albumin), as protein carrier, 901, 901f BSE (bovine spongiform encephalopathy), 1013 Bt gene, 694 Bubble boy, 896, 896f Bubo, 1018, 1018f Bubonic plague, 11, 12f, 1018, 1018f, 1019f, 1086. See also Plague Budding, 766, 767f Buick, Roger, 646, 646f Bunyaviridae, 195t Burgess, Dick, 218 Burkholderia aromatic catabolism, 499 B. cepacia, 224t, 709 Burkholderiales, 681t Burst, 200 Burst size, 200, 209, 210 Butanol, as fermentation product, 488f, 489, 490 Button mushroom, 591 Butyrate, as fermentation product, 488f, 489, 490
C C-1027, 567, 567f, 568, 569 Cabbage, fermentation of, 599, 599f
Cactus beer, 485 Cadaverine, 479, 609 cadBA operon, 479, 479f CagA protein, 968, 968f, 996 Caldisphaera, 735 Caldisphaerales, 735 Calibration, phylogenetic tree, 656 Calmodulin, 957 Calorimetry, 466 Calvin, Melvin, 550, 774 Calvin cycle categories of organisms performing, 550–551 detail view of, 557f discovery of, 551–552, 552f intermediates, 551–552 overview of, 551, 552–553, 553f phases of, 553–554 regulation, 558–560 ribulose 1,5-bisphosphate regeneration, 554–556, 558 rubisco mechanism of action, 555–556, 556f Cambray, Guillaume, 338f CAMP (cyclic AMP), 353, 353f CAMP-CRP, activation of transcription by, 354, 354f Campi, Carlo, 738 CAMP receptor protein (CRP), 353, 354, 354f Campylobacter C. jejuni gastrointestinal tract infections, 998t as microaerophilic, 167 diarrhea from, 992 food-borne illness, 612t identification procedures, 1080t Campylobacterales, 682t Cancer AIDS-related, 1006 chemotherapy, artificial ribozymes for, 645 gastric, 996 human papillomavirus and, 203 oncogenic viruses, 206, 212, 212f recognition by natural killer cells, 887–888 Candida antifungal agents for, 1059, 1061t C. albicans budding, 767f candidiasis, 984t, 1005, 1005t genetic code variations, 267 intestinal flora, 869
1/18/08 5:15:17 PM
In d ex
oral infection, 1005, 1005f single-celled protein, 592 thrush, 975 vaginal flora, 766 C. utilis industrial strains, 619 in cocoa fermentation, 601 skin flora, 864 use in injera bread production, 604 Candidate species, 649, 685 Candidiasis, 984t, 1005, 1005f Candor Chasma, Mars, 855, 855f Canning, 172–173, 614 Cannulae, 732, 733f Capping, 909, 911f, 919 Capsid, 183, 187, 188f, 189, 189f, 192 Cap snatching, 411 Capsule, 76, 93 anticapsular antibodies, 885, 970 immune avoidance and, 970 Neisseria meningitidis, 970, 1008 Streptococcus pneumonia, 885, 886f, 970, 986 Carbamoyl phosphate, 577, 578f Carbenicillin, 1039f Carbohydrates catabolism, 477–479, 478f distinguishing sugars by NMR, 477f polysaccharide, 477 hydrolysis of, 478 structure, A-10–A-12, A-11f, A-12f sugar aldose and ketose, A-11 disaccharides, A-11–A-12, A-12f isomers, A-11 modified, A-11, A-12f monosaccharides, A-11, A-11f Carbolfuchsin, 54 Carbon aerobic and anaerobic metabolism, 797t assimilation, 795 chiral, A-7, A-8f crustal, 835 dissimilation, 795 fi xation in carbon cycle, 835, 837 formation in stars, 631, 632f isotopes, A-2 14 C, discovery of, 551, 554–555, 554f–555f isotope labeling, 551 isotope ratio, 637–638, 637f measuring environmental, 834, 835f
SFMB_index.indd I-9
oxidation states, 834, 834t rate of cycling, 833t reservoirs, 833–834, 833t, 835 Carbonation, 116 Carbon cycle, 835–838 aerobic, 835–836, 836f anaerobic, 836, 836f description, 118, 119f global carbon balance, 837, 837f hydrologic cycle interaction, 839, 839f wetlands and, 838, 838f Carbon dioxide atmospheric, 633 as greenhouse gas, 832 history of levels, 832f measuring, 834, 835t from ethanolic fermentation, 602–603, 603f fi xation, 118, 119, 119f, 550, 550–564 Calvin cycle, 550–560, 550f, 551t, 552f–553f, 557f carbon monoxide reductase pathway, 724 3-hydroxypropionate cycle, 551t, 562, 563f, 564 metabolist model of early life and, 642, 642f in methanogenesis, 724 pathways, table of, 551t reductive acetyl-CoA pathway, 551t, 561–562, 562f reductive (reverse) TCA cycle, 551t, 560, 561f global flux, 837, 837f methanogenesis from, 743, 743f sources and sinks, 835 use in food preservation, 614 Carbon monoxide dehydrogenase, 562, 562f Carbon monoxide reductase pathway, 724 Carbon sink, 837 Carboxydothermus, 692 Carboxyl (functional group), A-7t Carboxysome, 106, 106f, 558, 558f, 559f, 688 Carbuncle, 982 Cardiolipin, 86, 86f Cardiovascular system infections, 1014–1017 Chlamydophila pneumoniae, 1015, 1016f
endocarditis, 1014–1015, 1014f malaria, 1015, 1016f, 1017 Cardiovirus, 400t Carlson, Craig, 804 Carotenoids, 536–537, 537f Casein, 595–596 Catabolism aromatic, 496–501 benzoate catabolism, 497, 497f, 499–500, 500f polynuclear aromatic hydrocarbons (PAHs), 500, 501f carbon sources for, 476f, 478f classes of, 476–477 coupling with biosynthesis, 468 defi nition, 461, 462 fermentation, 487–491 diagnostic applications, 489–490, 490, 490f food and industrial applications, 489–490 pathways, 488f, 489 glucose, 482–487, 482f Entner-Doudoroff pathway, 482, 485–486, 485f, 486f glycolysis (EmbdenMeyerhof-Parnas), 482, 483–485, 483f, 484f pentose phosphate shunt, 482, 486–487, 487f substrates for, 477–481 amino acids, 479 aromatic compounds, 479 carbohydrate, 477–479, 477f, 478f lipids, 479 tricarboxylic acid (TCA) cycle, 491–496 acetyl-CoA entry, 491, 491f, 493 description, 491, 493, 494f intermediates, 493, 495–496 oxidative phosphorylation and, 495, 496f regulation of, 495 variations of, 491 Catabolite activator protein (CAP), 353. See also cAMP receptor protein (CRP) Catabolite repression, 351, 352f, 353, 353f, 479 Catalase, 166–167, 166f, 1070 Catalytic RNA description, 266 properties, 266t Catechol, 497, 497f Catechol dioxygenase, 497
I-9
Catenane, 241–242, 242f cat gene, 965, 965f Cathelicidins, 879t, 880 Cation, A-4 Caulerpa taxifolia, 8, 8f, 776, 777f Cauliflower mosaic virus genome, 184, 185f, 193 life cycle, 207–208, 208f structure, 196t Caulimoviridae, 196t Caulobacter budding, 134 C. crescentus, 706 cell differentiation, 142 flagellum-to-stalk transition, 108–109 proteomics, 385 shape-determining proteins, 105, 105f Caulobacteriales, 680t CBB (Calvin-BensonBassham) cycle. See Calvin cycle CCD (charge-coupled device), 62 CCM (CO2-concentrating mechanism), 558–559, 559f CCR (chemokine receptor), 417 CCR5 gene, 417–418 CCR5 protein, 390, 390f CD3, 918, 919f CD40, 919 CD154, 919 CDC (Centers for Disease Control and Prevention), 12, 1086 CD4 cell count in AIDS patient, 1006 cytokines, 922 HIV infection of, 417, 922, 973–974, 1005–1006 MHC proteins and, 921–923 N. gonorrhoeae binding to, 1004f CD8 cell, 919, 920f, 921–922, 933 CD47 glycoprotein, 884 CD molecule, 921 cDNA (complementary DNA), 383, 440f, 441 CD4 receptor, 417, 417f, 422 Cech, Thomas, 19, 19f, 642, 644, 644f Cefepime, 1040f Cefoxitin, 1040f Ceftriaxone, 1040f Cell biology, 32–33 cell membrane, A-22–A-29, A-24f–A-29f cell size, A-30–A-32, A-31f chloroplasts, A-37, A-38, A-38f
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I-10
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Cell biology (continued) cytoskeleton, A-35–A-36, A-35f endomembrane system, A-32–A-35, A32f–A-34f flagella and cilia, A-36, A-37f mitochondria, A-37–A-38, A-38f, A-38t mitosis, A-30f, A-31f nucleus, A-29, A-30f Cell-cell communication, 971 Cell counting, using fluorescence microscopy, 58, 58f Cell cycle, A-30, A-31f Cell differentiation, 142–146 actinomycetes, 142–143, 145–146, 145f, 146f in Caulobacter crescentus, 109, 142 cyanobacteria, 144, 144f endospore formation, 143–144, 143f fruiting bodies, 144, 145f heterocyst formation, 144, 144f Cell division asymmetrical, 104, 134, 134f binary fission, 134 in ciliates, 784 DNA replication, 101–102, 101f, 102f mitosis, 134 of Pyrodictium abyssi, 732f septation, 103–104 rod-shaped cell, 104, 104f spherical cell, 103, 103f symmetrical, 134, 134f Cell envelope. See Envelope Cell-free system, 80, 258, 265 Cell function, coordination of, 75 Cell-mediated immunity. See also Adaptive immunity activation, summary of, 920f cytokine modulation of, 923 defi nition, 897 Cell membrane, A-22–A-29 as antibiotic target, 1041 archaeal, 87–88, 87f, 88f description, 75–76, A-22 electron microscope visualization of, 32 fluidity, A-24, A-25f, A-26 in barophiles, 156 in psychrophiles, 153–154 ion gradients, 85 lipids, 82, 82f, 83f, 86–88, 86f–88f, A-22, A-24, A-25f
SFMB_index.indd I-10
proteins, 82–83, 82f, A-22, A-24 selective permeability, 121–122 semipermeable, 157, A-26, A-26f structure, 74f, 82–83, 82f, A-22, A-24, A-24f transport across, 83–85, 84f diffusion, A-27, A-27f endocytosis, A-29, A-29f exocytosis, A-29, A-29f osmosis, A-27–A-29, A-28f transport proteins, A-27 Cells. See also specific cell types of innate and adaptive immune systems, 873–876, 873f–875f origin of, 19 Cell structure. See also specific structures bacterial biochemical composition, 75f, 76–78, 77t cell membrane, 75–76, 82–88 cell wall and other layers, 76, 88–98 envelope, 74f–75f, 75, 76 eukaryotes compared, 76 fundamental traits, 75 model, 74f–75f, 75–76 nucleoid, 74f–75f, 76, 98–99, 98f–99f specialized structures, 106–111 study of cell parts, 78–82 eukaryotic, 32–33, 76 cell membrane, A-22–A-29, A-24f–A-29f cell size, A-30–A-32, A-31f chloroplasts, A-37, A-38, A-38f cytoskeleton, A-35–A-36, A-35f endomembrane system, A-32–A-35, A32f–A-34f flagella and cilia, A-36, A-37f mitochondria, A-37–A-38, A-38f, A-38t mitosis, A-30f, A-31f nucleus, A-29, A-30f Cell surface receptor, 198 Cellular slime mold, 761t, 781, 782, 782f Cellulose breakdown of, 462 catabolism, 478 structure, 476f Cell wall algal, 97
antibiotics targeting, 1037–1041, 1038f–1041f archaeal, 724 description, 76 fungi, 76, 765 gram-negative bacteria, 76, 91, 91f gram-positive bacteria, 91, 91f, 93, 93f mycobacterial, 92, 93f oomycete, 773 peptidoglycan, 89–90, 89f, 90f turgor pressure and wall pressure, A-28, A-28f Cenarchaeum symbiosum, 736, 736f Centers for Disease Control and Prevention (CDC), 12, 1086 Central nervous system infections, 1007–1013 botulism, 1008–1010, 1009f, 1010f eastern equine encephalitis (EEE), 1010–1011 meningitis, 1007–1008, 1007f prion diseases, 1011, 1013, 1013f, 1013t table of etiologic agents, 1012t tetanus, 1008–1010, 1009f, 1011f Cephalexin, 1040f Cephalosporin biosynthesis, 1046, 1046f–1047f generations, 1040, 1040f mechanism of action, 1039–1040 Ceratium C. furca, 762t C. tripos, 762t Cercozoa, 761t, 763–764 Cerebral spinal fluid (CSF), 1065–1066 Cesium chloride gradient, 96 Cetylpyridinium chloride, 177t c-fos, 975, 975f CFP (cyan fluorescent protein), 102, 102f, 444, 445f CFU (colony-forming unit), 129, 132 Chagas’ disease, 791 Chain, Ernst, 25, 1031 Chain of infection, 21 Chan, Russel, 333 Chancre, 1002 Chang, Annie, 252 Chaos carolinense, 761t
Chaperone (chaperonin), 100, 283, A-34 cyclophilia A, 416 GroEL and GroES, 285, 285f role of, 283, 285, 292, 365–366 sigma factors and, 365–366 of thermophiles, 155 Charge-coupled device (CCD), 62 Charophyta, 760t Chase, Margaret, 198 Cheddared, 596, 597f Cheese curd formation, 595–596 defi nition, 595 flavor generation, 597, 598f production, 11, 11f, 596, 597f spoilage, 609, 610t Swiss cheese production, 480–481, 480f–481f varieties, 596, 596f Chemiosmotic hypothesis, 33, 511, 511f–513f, 512–513 Chemoautotroph, 118, 119, 463t Chemoheterotroph, 119 Chemokine description, 883 in inflammation, 883 Chemokine receptor (CCR), 417 Chemolithotroph, 26, 118, 507 Chemostat, 139–140, 139f Chemotaxis defi nition, 110 in inflammation, 883 movement described, 110–111, 111f Chemotherapy, 1029–1061. See also specific drugs Chemotrophy, 119 Chesapeake PERL, Inc., 623 Chickenpox, 426, 984t, 986 Chiral carbon, A-7, A-8f Chirality, 17 Chi site, 317, 318f Chisolm, Sallie, 793f Chitin, 765 Chiu, Wah, 66, 66f Chlamydia (disease), 1003–1004 Chlamydia (genus) attachment mechanism, 945t C. trachomatis, 717, 717f, 1003 appearance, 683t identification procedures, 1080t developmental cycle, 716, 717f replication cycle of, 1003f Chlamydiae, 684, 716–717
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Chlamydiales, 683t Chlamydomonas, 775–776, 775f C. reinhardtii, 774–775, 775f flagella, A-36, A-37f Chlamydophila C. pneumoniae, 717, 1003 in atherosclerosis, 1015, 1016f respiratory tract infection, 990t–991t C. psittaci, 990t–991t developmental cycle, 716 Chloramine, 176t Chloramphenicol, 1045 action of, 281 side effects, 1032 structure, 281f, 1044f Chlorarachinophyceae, 761t Chlorella carbon fi xation, 551–552, 552f, 554, 555f endosymbiosis by, 666–667, 668f, 670 photosynthesis, 774 Chloride pump, halorhodopsin, 747–748, 748f Chlorine anaerobic dechlorinators, 697–698, 698f as disinfectant, 176 Chlorobenzoate, 499 Chlorobi, 682t, 684, 687t, 714–715 Chlorobiales, 682t Chlorobium, 714–715, 810 C. tepidum, 560, 561f, 715f electron micrograph, 33f photosystem I, 540 reductive (reverse) TCA cycle, 560, 561f Chloroflexales, 679t Chloroflexi appearance, 686f description, 683, 686 phototrophy, 686, 687t Chloroflexus appearance, 686f C. aurantiacus, 686, 686t appearance, 679t 3-hydroxypropionate cycle, 562, 563f chlorosomes, 686 Chlorophyll antenna complex, 537–538, 538f biosynthesis, 581–582 of cyanobacteria, 688 description, 536 light absorption, 536–537, 537f reaction center, 538, 539 structure, 537f, 581f Chlorophyta, 761t, 774–776, 775f–777f
SFMB_index.indd I-11
Chloroplast, 763, A-37 endosymbiosis theory, 30, 30f, 67f, 670, A-38, A-38f evolution, 763, 763f genome, 670–671, 671f origin of, 757 photosynthesis and, A-38 Chloroquine, 1017 Chlorosome, 32, 33f, 686 Choanoflagellates, 760t, 758, 759f Chocolate agar, 1066, 1066f Chocolate production, 589f, 600–601, 601f Cholera, 998t diagnostic cues, 981 epidemiology, 1087, 1088f, 1089 pathogenesis of, 952f sari cloth fi ltration project, 2–3, 3f, 794 stomach acidity and, 868 Cholera toxin, 951–952, 952f, 953f, 954 Cholesterol, 87, 88, 88f, A-24, A-25f, A-26 Choline-binding domain (CBD), 1053 Chopra, Ashok, 860 Chromatiales, 681t Chromatin, A-29 Chromatium, 709, 710f Chromium, microbial metabolism of, 852t Chromophore, 536 Chromosome compaction into nucleoid, 228 E. coli, 222, 222f, 223 eukaryotic, 246–248 nucleoid, 76 structure, 223 supercoiling, 228–230, 229f–231f, 241 walking, 254 Chroococcales, 679t Chroococcus, 689 Chrysophytes, 763, 779 Chymosin, 596 Chytridiomycota, 760t, 763, 768, 768f, 827 Cilia Bordetella bronchiseptica adherence to, 979f description, 783–784, 784f, A-36 mucociliary elevator, 868, 868f structure, A-37f Ciliate, 764 Ciliophora (ciliates), 783–787 cell structure, 784, 784f examples, 761t genetics, 784
model for human aging, 785, 785f reproduction, 784, 786, 786f stalked, 786f, 787 Circoviridae, 194t CI repressor, 374–376, 374f, 375f Cistron, 273 Citrate, 491, 493, 494f Citromicrobium, 705f, 706 Civil War, American, 979 CJD (Creutzfeldt-Jakob disease), 185–186, 186f, 1013, 1013f, 1013t, 1094 Clade, 651 Cladina evansii, 799f Cladosporium, 772, 772f Claragen, 616 Classical complement pathway, 925, 925f Classification challenges of, 29 Copeland’s four-kingdom system, 29 description, 647 disease, 980–981 Lancefield, 1071, 1072f of microorganisms by environmental parameter, 151t of organisms by relationship with oxygen, 165–168, 165t, 166f taxonomic hierarchy of, 647, 648t virus Group I (double-stranded DNA viruses), 192, 193f, 194t Group II (single-stranded DNA viruses), 192, 193f, 194t Group III (doublestranded RNA viruses), 192, 193f, 195t Group IV ((+) sense single-stranded RNA viruses), 192, 193f, 195t Group V ((–) sense single-stranded RNA viruses), 192, 193f, 195t Group VI (RNA reversetranscribing viruses), 192–193, 193f, 196t Group VII (DNA reversetranscribing viruses), 193, 193f, 196t phylogeny and, 196–198, 197f proteomic analysis, 197–198, 197f
I-11
Whittaker’s five-kingdom system, 29 Woese’s three-domain system, 30–31, 31f Claverie, Jean-Michel, 425 Clavulanic acid, 1051 Clindamycin, 1045 for necrotizing fasciitis, 984 structure, 1044f triggering of enterocolitis, 993 Clinical microbiology, 1063–1095 biosafety containment procedures, 1083–1085, 1084t, 1085f emerging disease detection, 1092–1095, 1092f–1094f epidemiology, 1085–1091, 1086f–1089f, 1088t, 1091f pathogen identification, 1065–1080 algorithm use, 1065–1072 electronic, 1092–1093, 1092f–1093f fluorescent antibody staining, 1079, 1079f gram-negative bacteria, 1066–1068, 1067f–1069f, 1067t gram-positive pyogenic cocci, 1070–1072, 1071f, 1072f by molecular genetics, 1072–1076, 1073t, 1074f–1077f nonenteric gramnegative bacteria, 1068–1070, 1069f overview, 1065 for selected diseases, 1088t selective and differential media, 1066, 1066f by serology, 1077–1078, 1078f principles of, 1064–1065 specimen collection, 1081–1083, 1081f, 1082f Clonal response, 897 Clonal selection, 909–910, 910f Cloning defi nition, 250 history of, 251–252 procedure, 251f shotgun, 253 Cloning vector, 184 Clostridiales, 680t, 694–696, 694f–696f
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I-12
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Clostridium C. acetobutylicum, 694 fermentation by, 490 C. botulinum, 694–695, 694f bioterrorism, 167 botulism, 998t, 1008–1010, 1009f, 1010f endospores, 143 endospore stain, 54, 55f food-borne illness, 172–173, 611, 612t food spoilage, 172–173 gastrointestinal tract infections, 998t multiplex PCR, 1073, 1074f prophage in, 200 toxin, 613, 613f, 694, 694f, 1008–1009, 1009–1010, 1009f, 1010, 1010f C. difficile, 695, 870f gastrointestinal tract infections, 998t pseudomembranous enterocolitis, 993 C. pasteurianum nitrogenase, 571 C. perfringens gastrointestinal tract infections, 998t C. tetani, 694 appearance of, 1009f endospores, 143 tetanus, 1008–1010, 1009f, 1011f, 1025 vaccine in transgenic plants, 454t C. thermoaceticum carbon dioxide assimilation, 555 reductive acetyl-CoA pathway, 561 endospore formation, 692, 694–695, 694f fermentation by, 489 intestinal flora, 869 nitrogen fi xation, 845 Clotrimazole, 1059f, 1060t–1061t ClpPC protease, 367 ClpP protein, 291 Clp protease, 291 ClpXP protease, 283 CLUSTALW program, 296f Clusters of cellular differentiation (CD) molecule, 921 CmpABCD, 559 c-myc, 975, 975f CNA agar, 1070 CNO cycle, 631, 632f CoA. See Coenzyme A (CoA) Coagulase, 982, 1072 Coastal shelf, 801
SFMB_index.indd I-12
Cocci description, 42, 42f septation in, 103, 103f Coccidioides, 988 identification procedures, 1080t meningitis/encephalitis, 1012t Coccidioidomycosis, 990t–991t, 1060t Cockayne syndrome, 332 Cocoa beans, fermentation of, 589f, 599, 600–601, 600f–601f CO2-concentrating mechanism (CCM), 558–559, 559f Codon, 267, 268–269 Coenzyme A (CoA), 489 discovery of, 489 role in fermentation, 488f, 489 structure of, 489f Coenzyme B12 biosynthesis, 581, 582, 583 structure, 581f Coenzyme M, 742 Coevolution, 666 667f Cofactor, 117 as electron carrier, 510 electron transport system, 516–518, 517f F420, 742f, 743 for methanogenesis, 742 Cohen, Stanley, 251, 251f Cohen-Bazire, Germaine, 535, 536, 536f Cohesive ends, 249 Coinfection, 409 Cointegrate, 316 Colanic acid, 142 Cold, growth control and, 173 Cold sores, 423, 939f Colibase, 297 Colibri, 297 Colicin E3, 276, 276f Colistin, 1070 Colony defi nition, 21, 128 microcolony, 128, 129f Colony-forming unit (CFU), 129, 132 Columbus, 1003 Colwell, Rita, 2–3, 2f, 794, 802 Colwellia psychrerythraea, 11 Commensalism, 799t, 800–801 Commensal organism, 864, 871–872. See also Microflora Commercial products from microbes, 615–621, 618t Common cold, 899, 972 Communication, cell-cell, 971
Community (herd) immunity, 1024–1025 Compatible solute, 157 Competence factor, 305f, 306 Competent, 306 Complement, 873 in adaptive immunity, 925–926 alternative pathway, 889, 890f classical pathway, 889, 925, 925f C-reactive protein, 890, 891f fragments, roles of, 926 in innate immunity, 889–891 lectin pathway, 889 membrane attack complex, 889–890, 925–926, 925f regulation of activation, 926 Complementary DNA (cDNA), 383, 440f, 441 Complex media, 130 Complex transposon, 335, 336f Composite transposon, 335, 336f Composting, 462, 462f Compound microscope, 50–51, 50f Compromised host, 871 Computer virus, 183–184, 184f Concentration gradient, energy and entropy in, 467–468, 467f Condensation, A-6, A-6f, A-15, A-15f Condenser, 50 Conditional lethal mutations, 396, 397 Confluent growth, 129 Confocal fluorescence microscopy, 60–61, 61f Conformation, native, A-9 Congenital syphilis, 1003 Conidiophore, 770, 772f Conjugation, 223, 776, 786 chromosome transfer, 223 ciliate, 786, 786f green algae, 776, 776f plasmid, 244, 306–311 Conjugative transposon, 335–336 Conjunctiva, 866 Consensus sequence, promoter, 260 Constant region, antibody, 902–903, 903f Consumer, 464, 796 Contact dermatitis, 930–931, 931f
Containment procedures, biosafety, 1083–1085, 1084t, 1085f Contig, 254 Continuous culture, 139–140, 139f Contractile vacuole, 97, 97f, 776, 784 Contrast, 44, 49, 52 Convergent evolution, 756 Conway, Tyrrell, 485, 485f Copeland, Herbert, 29 Copepod, 2–3, 3f, 794, 794f Copper leaching, 531, 531f oxidation, 530, 531 Coral bleaching, 788, 824 endosymbionts, 788 zooxanthellae in, 824, 825f Coralline algae, 778, 778f Cordon sanitaire, 1019 Coreceptor, 398 Core particle, HIV, 415 Core polysaccharide, 95, 95f Corepressor, 347, 347f, 355 Corn, transgenic, 451 Coronary artery disease, 1015, 1016f Coronaviridae, 195t Coronavirus genome, 192 pathogenesis, 972 severe acute respiratory syndrome (SARS), 1086–1087, 1087f Corrosion anaerobic, 530, 531f of iron, 851, 852f Cortical alveoli, 764 Corynebacterium C. diphtheriae, 702, 702f beta phage, 954 diphtheria, 938, 942 diphtheria toxin, 954–956, 955f, 956f identification procedures, 1080t methylene blue stain of, 955f polar extension, 104 prophage in, 200 pseudomembrane, 954, 955f respiratory tract infection, 990t–991t C. glutamicum, 39f C. striatum, 938 irregular shape, 702 mycolic acids, 700 cos site, 312, 313f Cotransduced, 319 Cotranslational export, 286f Coulter counter, 131–132 Counterstain, 54
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Counting bacteria biochemical assays, 132 direct counting, 131–132, 32f dry weight, 132 fluorescence-activated cell sorter (FACS), 132, 133f optical density, 133 viable counts, 132 Coupled transcription and translation in prokaryotes, 282, 282f Coupled transport, 123–124, 124f Covalent bond, A-2, A-4, A-4f, A-5t Cowman, Alan, 1017 Cowpox, 23, 24f, 899 COX (cyclooxygenase), inhibitors of, 883 Coxiella burnetii appearance of, 981f pasteurization and, 173 phagosome, survival in, 969, 970f Q fever, 707, 937f, 969, 981 Coxsackie virus, 400t, 401, 932 Crabb, Brendan, 1017 C-reactive protein, 890, 891f Crenarchaeol, 730, 730f Crenarchaeota ammonia-oxidizing, 737 biosignature, 730, 730f hyperthermophiles, 730–735, 730f–735f, 731t meaning of name, 730 mesophiles, 735–736 phylogeny, 726, 726, 727f, 728t psychrophiles, 736–737, 737f Creosote, 500, 501f Cresol, 176, 176t CreS protein, 105, 105f Creutzfeldt-Jakob disease (CJD), 185–186, 186f, 1013, 1013f, 1013t, 1094 Crichton, Michael (author), 6, 62 Crick, Francis, 34, 34f, 641 Crimean War, 12, 12f Crinipellis perniciosa, 610 Crohn’s disease, 884, 885f Cro repressor protein, 228f, 374–375, 374f Cross-bridges, 89, 90f Crossover, 316–317, 318f Crown gall, 310, 706, 707f, 822, 823f, 964 CRP (cAMP receptor protein), 353, 354, 354f, 356f
SFMB_index.indd I-13
Crust cryptogamic, 818, 819f, 820 Earth’s, 632–633, 633f, 835 Cryocrystallography, 70 Cryoelectron microscopy (cryo-EM), 64–65, 64f, 66, 66f Cryptococcus (cryptococcosis), 988 antifungal agents for, 1061t meningitis/encephalitis, 1012t Cryptogamic crust, 778, 818, 819f, 820 Cryptophyte algae, 763, 763f Cryptosporidium parvum, 997 Crystal violet, 52, 53f, 54f CsCl density gradient centrifugation, 248–249 CSF (cerebral spinal fluid), 1065–1066 CTX φ bacteriophage, 189, 200 Culture batch bacteriophage, 208–209, 209f growth stages, 136f, 137–138 continuous, 139–140, 139f enrichment, 26–27 pure, 21, 128–129 starter, 596, 597f tissue culture, for growing animal viruses, 209–210, 210f Culturing viruses, 208–212, 209f–212f Curd, 595 Cyan fluorescent protein (CFP), 102, 102f, 444, 445f Cyanobacteria. See also specific species appearance, 7f carbon dioxide fi xation, 550, 550f cell structure, 688–689, 688f chlorophylls, 536 chloroplast origin and, 30, 30f, 670 colonial, 689, 690f communities, 691, 691f description, 683 eutrophication of lakes, 170f evolution of, 639 fi lamentous, 689, 689f gas vesicles, 106f heterocyst, 144, 144f hopanoids as biosignatures, 637f, 638 lichen, 798–799 microfossils, 18f, 636–637, 636f
nitrogen fi xation, 571, 573f, 688, 689, 799–800 oxygen production, 120, 832 photosynthesis, 27, 535–536, 688, 691 phototrophy, 687t, 688–692 representative groups, 679t Spirulina, 161–162, 161f, 592, 592f, 679t, 688 stromatolite, 629f, 630, 630f, 635, 635t symbiotic, 691, 691f thylakoids, 106 in Winogradsky column, 27, 27f Cyanocobalamin. See vitamin B12 Cyclic AMP (cAMP) catabolite repression and, 353 control via phosphotransferase system, 353, 353f Cyclic photophosphorylation, 540 Cyclodextrin, 162, 163f Cyclooxygenase (COX), inhibitors of, 883 Cyclopentane ring, 723f, 724 Cyclophilin A, 415f, 416 Cyclopropane fatty acid, 86f, 87 Cycloserine, 1038f, 1040, 1041f Cyclotron, 554, 554f Cymopolia, 776, 777f C. barbata, 761t Cyst, of Giardia lamblia, 997, 997f Cystic fibrosis, 454, 878, 879f Cystic fibrosis transmembrane conductance regulator (CFTR), 878, 879f, 954 Cystoviridae, 195t Cytochrome, 510, 510f, 516, 519–520, 540 Cytochrome bc, 542f Cytochrome bc oxidase, 522f Cytochrome bd quinol oxidase, 521–522, 521f Cytochrome bf, 541 Cytochrome bo quinol oxidase complex, 519f, 520, 520f, 521 Cytochrome c, 517f, 1070 Cytochrome oxidase test, 1069f, 1070 Cytokine, 913 C-reactive protein stimulation by, 890 creating new by DNA shuffl ing, 447 defi nition, 872 extravasation, role in, 882–883, 882f, 883f
I-13
helper T-cell secretion of, 913 immune response modulation, 923, 923t interferon, 886–887, 887t Toll-like receptor and, 878 type III secretion and, 995 Cytokinesis, A-30, A-31f Cytomegalovirus, 196, 990t–991t Cytoplasm (cytosol), 74f–75f, 75, A-22 Cytoplasmic membrane, A-22. See also Cell membrane Cytosine, 227, A-12, A-12t, A-13f deamination of, 323, 323f, 329 tautomeric shift, 323, 323f Cytoskeleton, A-35 bacterial, 105, 105f intermediate fi laments, A-35, A-35f microfi laments, A-35–A-36, A-35f microtubules, A-35f, A-36 Cytotoxic T cell (TC cell), 915, 915t, 919, 920f, 921–922, 921f
D Dairy products acid fermentation, 595–597, 595f–598f cheese production, 596, 597f cheese varieties, 596, 596f curd formation, 595–596 examples, 594t flavor generation in cheese, 597, 598f spoilage, 609, 610t Dalgarno, Lynn, 275 Dali, Salvador, 655, 656f Dam (deoxyadenosine methylase), 234 DAPI (4′,6-diamidino-2phenylindole), 58f, 59, 59f, 60, 241, 241f Dark-field microscopy, 45, 55–56, 55f, 56f Dark reaction, 536 Darwin, Erasmus, 630 DasSarma, Priya, 746, 746f DasSarma, Shiladitya, 158f, 745, 746 Dead Sea, 744, 744f, 747 Dead zone, 840, 840f, 846 Deamination, 479 Death curve, 171f Death phase, 138 Death rate, 138 Decarboxylation, 479 Decay-accelerating factor, 926
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I-14
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Dechlorinators, anaerobic, 697–698, 698f Decimal reduction time (D-value), 171, 171f, 172–173, 175 Decolorizer, 53f, 54, 54f Decomposer, 796 Decomposition, of lignin to humus, 813, 815, 815f Deep-branching thermophiles, 678, 679t, 683, 685–688, 685f, 686f Defensin, 879–880, 879t, 880f, 881, 881f Defi ned minimal medium, 117 Deforestation, 837 Degenerative evolution, 652 DegP protein, 257f Degranulation, 880 Degron, 291 Dehalobacter restrictus, 698, 698f Dehalococcoides, 532f Dehalorespiration, 532, 532f Dehydration, 613 Dehydrogenation, 507 Deinococcales, 679t, 683 Deinococcus appearance, 679t D. radiodurans, 150, 175, 175f DNA repair, 320 genome, 320 resistance of, 683, 686 description, 686 Delayed-type hypersensitivity (DTH), 930–931, 931f, 932t Deletion, 321, 321f, 322 De Lodi, Agostino Bassi, 16 DeLong, Edward, 735, 736, 736f Deltaretrovirus, 413t DeMontigny, Bree, 741, 741f Denaturation, DNA, 227 Denaturing gradient gel electrophoresis (DGGE), 676 Dendritic cell, 875, 930, 931f antigen-presenting cell (APC), 876, 895f appearance, 875f development, 874f Dengue (breakbone fever), 214 Denitrification, 120, 120f, 121, 570, 846, 846f Denitrifying bacteria, 120 Deoxyadenosine methylase (Dam), 234, 327 Deoxyribonuclease (DNase), 356–357, 358f, 441, 442f, 447
SFMB_index.indd I-14
Deoxyribonucleic acid. See DNA Deoxyribose, 226 Depolarization, 123–124 Depth of field, 51 Derepression, 347, 347f Dermatitis, contact, 930–931, 931f Dermatomycosis, 984t Dermatophytosis, 1061t Desensitization, 928 Desulfobacterales, 682t Desulfobacterium biofi lm, 851 D. autotrophicum, 561 reductive acetyl-CoA pathway, 561 Desulfovibrio biofi lm, 851 D. desulfuricans, 852f, 854, 854f sulfur reduction, 847 uranium reduction, 854, 854f Desulfurococcales, 730–733, 731f, 731t Desulfurococcus, 734 D. mobilis, 730–731, 731f, 731t Desulfuromonadales, 682t Detection, 41 Detergent lysis, 79 Detergents, 162, 176 Detritus, 797 DGGE (denaturing gradient gel electrophoresis), 676 D’Herelle, Felix, 179, 210 Diabetes, 932 Diacetyl, 604, 605f, 607 Diagnosis, of disease, 981–982 4′,6-diamidino-2-phenylindole (DAPI), 58f, 59, 59f, 60, 241, 241f Diaminopimelic acid, 89, 90f, 94, 95f Diaphragm, 50 Diarrhea, 992–995 Diatom, 97, 755f, 762t, 764, 778, 779, 779f Diauxic growth, 351, 352f Dichotomous key, 649, 650f, 1068 Dictyostelium discoideum, 143, 782, 782f Dideoxy nucleotide, 252, 252f Didnium, 784, 784f Differential media, 130, 1066, 1066f Differential stain, 52, 54–55, 55f Differentiation. See Cell differentiation Diffusion, A-27, A-27f entropy and, 467 facilitated, 122–123, 122f passive, 83
Digestive communities, 826–828, 826f–828f Digestive vacuole, 784, 784f Dihydroxyacetone, A-11f Dihydroxyacetone kinase, 483 Dihydroxyacetone phosphate, 483, 484f Dilution streaking, 128, 128f Dimethylmercury, 853, 853f Dimethyl sulfide, 848 Dimethyl sulfoxide (DMSO), 3 DinB protein, 331 2,4-dinitrophenol (DNP), 515 Dinoflagellate, 762t, 763, 787–788, 787f, 824, 825f Dioxygenase, 497 Diphosphatidylglycerol, 86, 86f Diphtheria, 938, 955f Diphtheria, tetanus, pertussis vaccine, 1024t–1025t Diphtheria toxin, 938, 942, 954–956, 955f, 956f Dipicolinic acid, 144 Diplococci, 709 Diplomonadida (diplomonads), 762t, 764 Direct counting, 131–132, 132f Direct repeat, 370 Disaccharide, A-11–A-12, A-12f Discicristata, 762t, 764 Disease, 979–1025 cardiovascular system, 1014–1017 central nervous system, 1007–1013 classification schemes, 980–981 diagnosis, 981–982 emerging, 1094f, 1095 gastrointestinal tract, 992–997 genitourinary tract, 997–1006 germ theory of, 17 human history and, 11–12, 12f immunization for prevention of, 23–24, 1023–1025, 1024t–1025t Koch’s postulates, 21–23 notifiable (reportable), 1087, 1088t overview, 979–980 reemerging, 1094f, 1095 respiratory tract, 986–992 skin and soft tissue, 982–986 systemic, 1017–1023 zoonotic, 981, 981f Disinfection, 171, 175
Disorder. See Entropy Dissemination, 406 Dissimilation, 795 Dissimilatory denitrification, 525 Dissimilatory metal reduction, 528–529 Dissimilatory nitrogen reduction, 846, 847 Divergence archaeal, 727f molecular clocks, 652–653, 652f mutation, 652 natural selection, 652 phylogenetic trees and, 653–656 of three domains of life, 656–657, 660–661 Diversity bacterial, 675–718 ecosystem, 169, 170f eukaryotic, 755–791 Division. See Cell division D-loop formation, 317, 318f DMSO (dimethyl sulfoxide), 3 DNA antiviral DNA synthesis inhibitors, 1056–1057, 1057f base pairing, 226–227, 227f bioelectric DNA detection chip, 1092–1093, 1092f compaction into nucleoid, 228 complementary DNA (cDNA), 383, 440f, 441 denaturation, 227 double helix, grooves of, 228, 228f drugs targeting, 1041–1042, 1042f, 1043f evolution of, 425, 644 GC/AT ratio, 227 as genetic material, 222–223 isolation and purification, 248–249 methylation (See DNA methylation) noncoding, 224–225, 247–248 nucleotides, A-12 organization with nucleoid, 98–99, 99f origin of, 19 repair (See DNA repair) replication (See DNA replication) revolution, 33–35 sequencing, 9–11, 9f, 10f, 34, 34f, 252–254, 253f
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In d ex
structure, 226–227, 226f–228f, A-13f, A-14 discovery of, 33–34, 34f supercoiling, 99, 99f, 228–230, 229f–231f, 241 transformation by, 33 DnaA protein, 221f, 234, 235f, 236 DNA-binding protein, 99, 99f, 155 DnaB protein, 235f, 236, 241 DNA control sequence, 222–223 DnaC protein, 235f, 236 DNA-dependent RNA polymerase. See RNA polymerase DNA fi ngerprinting, 1074, 1075f, 1076 DNA footprinting, 357, 358f DnaG protein, 236, 239 DNA gyrase, 230, 231f, 241, 1042 DNA helicase, 235f, 236, 238f, 327–328 DNA helicase loader, 235f, 236 DNA hybridization, 59, 437 DnaJ protein, 365–366, 366f DnaK protein, 285, 285f, 365–366, 366f DNA ligase, 240 in DNA repair, 328, 329, 329f, 330f in DNA replication, 240, 240f use in gene cloning, 251, 251f DNA methylase, 315f DNA methylation in Caulobacter crescentus, 108–109 control of replication by, 234 for protection against restriction endonuclease, 315, 315f restriction enzymes and, 249 spontaneous, 324 DNA microarray, 150, 151f, 382–383, 382f DNA microchip, 382–383, 382f DNA mobility shifts, 438, 438f DNA polymerase, 240, 240f action of, 98 bacteriophage T4, 394f, 395 direction of synthesis, 234 in DNA repair, 328, 328f, 329, 329f, 330, 330f in DNA shuffl ing technique, 447, 448f in F factor replication, 307–308
SFMB_index.indd I-15
location in living cells, 241, 241t in PCR procedure, 155 polymerase I, 328, 328f, 329, 329f, 330, 330f polymerase III, 238f–239f, 239–240, 307–308 polymerase IV, 331 polymerase V, 331, 332f primer requirement of, 236 proofreading activity, 238, 323 removal, 241 replisome, 101–102, 101f, 239, 241 sliding clamp and, 235f, 236, 236f, 237 Taq polymerase, 35, 250, 250f, 446, 447f, 750–751, 1076 thermostable, 250, 250f vent, 750–751 DNA protection analysis, 441, 442f DNA-protein binding, 356–357 DnaQ protein, 238 DNA repair, 327–333 in Deinococcus radiodurans, 320 error-prone repair, 327, 331 error-proof repair, 327–330 base excision repair, 327t, 328–329, 330f mismatch repair, 327–328, 327t, 328f nucleotide excision repair, 327t, 328, 329f photoreactivation, 327t, 328 recombinational repair, 327t, 329–330, 331f RecA, 320 SOS repair, 331, 332f types, table of, 327t DNA replication, 232–243 bacteriophage, 244–245, 246f bidirectionality of, 101 catenanes, 241–242, 242f cell division, 134 direction of, 233, 233f, 234 elongation, 236, 238–240, 238f–239f, 240f F factor, 307–308 fundamentals of, 234 genes and proteins involved, 232t initiation of, 234, 235f, 236 Okazaki fragments, 234, 239–240 origin of, 98–99, 99f, 101, 102, 102f, 234 phases of, 233–234 plasmids, 243, 244f rolling-circle, 308, 311
semiconservative nature of, 232f, 233 sliding clamp, 235f, 236, 236f, 237 supercoil generation, 241 of telomeres, 247 termination, 241–242, 242f DNA reverse-transcribing viruses, 193, 193f, 196t DNase (deoxyribonuclease), 356–357, 358f, 441, 442f, 447 DNA sequencer, 253f DNA sequencing, 34, 34f, 252–254, 253f DNA shuffl ing, 447–448, 448f DNA transcription. See Transcription DNA vaccine, 452, 453f, 616 DNA viruses examples, 423, 423r life cycle, 203, 204f replication, 423 DNP (2,4-dinitrophenol), 515 Dobzhansky, Theodor, 336 Doherty, Peter, 923 Domagk, Gerhard, 1031–1032, 1031f Domains, DNA, 98, 99, 99f Domains of life, 9, 9f, 30–31, 31f comparison of, 660t divergence, 656–657, 660–661 phylogenetic tree, 656, 657t Doolittle, Ford, 661, 662 A Door into Ocean (novel), 28 Double helix, DNA, 33–34, 34f, A-14 Double-stranded DNA viruses, 192, 193f, 194t Double-stranded RNA viruses, 192, 193f, 195t Doubling time, 135 Doudna, Jennifer, 644–645, 645f Downstream processing, 619, 620f Doxycycline, 1021, 1044, 1044f Dps protein, 99 Drosophila Wolbachia endosymbiosis, 667–668 Wolbachia genome in, 254 Drug efflux pumps, 516, 516f, 1048–1050, 1049f–1050f Drug resistance, 1047–1052, 1048f–1050f, 1052f Drug susceptibility, measuring, 1034–1037 Kirby-Bauer disk susceptibility test, 1034–1037, 1035f
I-15
minimal inhibitory concentration (MIC), 1034, 1034f, 1035f, 1036, 1036f Dry weight, 132 DsRed, 623, 624f DTH (delayed-type hypersensitivity), 930–931, 931f, 932t dtx gene, 956 Duchesne, Ernest, 1031 Dulbecco, Renato, 192 Dunn, John, 218 Dust mite, 928 Dutch elm disease, 823f, 824 D-value (decimal reduction time), 171, 171f, 172–173, 175 Dynein, A-36, A-37f Dysentery, 992
E EAEC (enteroaggregative Escherichia coli), 993 eae gene, 1065 Early genes, phage T4, 393–394 Earth atmosphere, 633 elemental composition of, 632–633, 633f Eastern equine encephalitis (EEE), 940, 1010–1011, 1012t Ebenau-Jehle, Christa, 500 Ebola case history, 1077 ELISA detection, 1077–1078, 1078f structure, 189, 190f, 195t tropism, 201 virulence, 939, 940f Echovirus, 400t Eclipse period, 209, 209f Ecological niche, 663 Ecology, microbial, 793–829 communities within animals, 824–829 digestive communities, 826–828, 826f–828f sponge communities, 825, 825f zooxanthellae, 824, 825f communities within plants, 820–824 plant pathogens, 822, 823f, 824, 824f rhizobia and legumes, 820–822, 820f–823f ecosystems, microbes in, 794–798 abiotic factors, 797–798 aerobic and anaerobic metabolism, 797, 797t
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I-16
In d ex
Ecology, microbial (continued) ecosystems, microbes in (continued) food web, 795–797, 796f role of microbes, 795 freshwater communities, 809–811, 809f–811f marine, 801–809 food webs, 806, 807f habitats, 801–803, 801f ocean floor, 808–809, 808f plankton, 803–804, 803t, 804f–805f, 806 overview, 793–794, 794f soil and subsurface, 812–820 decomposition, 813, 815, 815f dry land, 818, 819f, 820 food web, 813, 814f mycorrhizae, 815–817, 816f, 817f overview, 812–813, 812f, 813f rhizoplane and rhizosphere, 815, 816f wetlands, 817–818, 818f symbiosis, 798–801 amensalism, 799t, 801 commensalism, 799t, 800–801 mutualism, 798–800, 798f–800f, 799t parasitism, 799t, 801 synergism, 799t, 800 Ecology, virus, 212–215, 213f–216f emergence of pathogens, 212–213, 214, 214f, 215f roles in ecosystem, 213–215, 213f, 216f EcoRI, 314f, 315 Ecosystem defi nition, 794 human influences on, 169 Ectomycorrhizae, 816, 816f Ectoparasite, 938, 938f Edema, 927 Edema factor (EF), 957 Edgar, Robert, 396, 397 ED (Entner-Doudoroff) pathway, 478, 482, 485–486, 485f, 486f, 724, 725f Edwards, Katrina, 530 EEE (eastern equine encephalitis), 940, 1010–1011, 1012t EF (edema factor), 957 EF-1α, 758, 759f Efflux protein, 85 Efflux pump, 124–125, 516, 516f, 1048–1050, 1049f–1050f
SFMB_index.indd I-16
EF-G, 279, 279f, 281 EF-Tu, 78, 78f, 277–279, 278f EF-Tu-GTP, 277, 279, 279f Eggs, alkaline fermentation of, 602, 602f EHEC (enterohemorrhagic Escherichia coli), 993–995, 1065 gastrointestinal tract infections, 998t Shiga toxin, 956 spread of, 1094 Ehrlich, Paul, 1032 Ehrlichia chaffeensis, 1073t EIAV (equine infectious anemia virus), 413t EIEC (enteroinvasive Escherichia coli), 993 Electrical potential, 514–515, 514f–515f Electric current, 505–506 Electrochemical potential, 85 Electrogenic, 123 Electromagnetic spectrum, 44, 44f Electron, A-2–A-3, A-2t Electron acceptor, 469, 506, 797 in anaerobic respiration, 525, 525f, 528f concentration of, 508–509 in lithotrophy, 507 reduction potential and, 507–509, 508t in respiration, 507 in soil, 818 terminal, 472 Electron donor, 469, 506 in anaerobic respiration, 525, 525f concentration of, 508–509 hydrogenotrophy, 532 in lithotrophy, 507 reduction potential and, 507–509, 508t in respiration, 507 Electronegativity, A-3, A-4t Electroneutral, 123 Electron microscope, 32 cell structure visualization, 32, 33f development of, 32, 32f Electron microscopy, 43, 62, 62–67 cryoelectron microscopy (cryo-EM), 64–65, 64f mechanism, 62, 62f sample preparation, 62–64 scanning electron microscopy (SEM), 40, 40f, 42, 42f, 43, 43f, 62, 63f, 64, 64f
transmission electron microscopy (TEM), 43, 43f, 62, 62, 63f, 64, 64f Electron transfer electron transport system (ETS), 471–472 FADH 2, 472–473 NADH, 471–472, 472f Electron transport chain, 164, 506 Electron transport system (ETS), 471–472, 506–507 anaerobic respiration, 525–529 electron donors and acceptors, 506, 507 energy storage, 507–509 environmental modulation, 521–522 lithotrophy, 507, 529–534 location in membranes, 509–511, 509f photolysis, 507, 535–543 proton motive force (PMF), 511–516, 511f respiratory, 507, 516–525 TCA cycle and, 495 uncouplers, 515, 515f Electrophoresis defi nition, 77 of proteins, 77–78 two-dimensional polyacrylamide gel, 77, 78, 78f Electrophoretic mobility shift assay (EMSA), 356, 357f, 438, 438f Electroporation, 305 Element, A-2–A-4, A-4t Elementary body, 690t, 717, 717f, 1003, 1003f Elements composition of Earth, 632–633, 633f composition of gramnegative bacteria, 833t formation within stars, 631–632, 632f sources and sinks, 833–834, 833t Elephantiasis, 668, 668f, 938, 939f ELISA. See Enzyme-linked immunosorbent assay (ELISA) Elongation factor 1α (EF-1α), 758, 759f Elongation factor Tu (EF-Tu), 78, 78f, 277–279, 278f Eluviated horizon, 812, 812f Embden-Meyerhof-Parnas (EMP) pathway, 482, 483–485, 483f, 484f archaeal modification of, 724, 725f
ATP generation, 484–485 description, 483 energy changes, 483f phosphorylation and splitting of glucose, 483 in Pyrococcus, 751 regulation of, 485 steps in, 484f Emerging disease, 1095 epidemiology, 1094f, 1095 viral, 212–213, 214, 214f, 215f Emiliana huxleyi, 213, 213f Emission wavelength, 58 EMP. See Embden-MeyerhofParnas (EMP) pathway Empty magnification, 46 EMSA (electrophoretic mobility shift assay), 356, 357f, 438, 438f Encephalitis eastern equine (EEE), 1010–1011 table of etiologic agents, 1012t Encephalitozoon intestinalis, 791 Encephalomyocardiovirus, 400t Endemic, 1086, 1086f Endemic area, 940 Endergonic reaction, A-17 Enders, John F., 210 Endocarditis, 867, 868f, 1014–1015, 1014f Endocytosis, 203, A-29, A-29f influenza entry by, 409, 411 poliovirus entry by, 402 virus entry by, 203 Endogenous retroviruses, 422, 422f Endolith, 797t, 813, 813f, 855 Endolithic microbes, 632, 633f, 836 Endomembrane system, A-32–A-35 advantages of, A-32 endoplasmic reticulum, A-32–A-34, A-33f, A-34f Golgi complex, A-33, A-33f, A-35 lysosomes, A-32, A-32f Endomycorrhizae, 816–817, 817f Endoparasite, 938, 939f Endopeptidase, 291 Endophyte, 700, 700f Endoplasmic reticulum (ER), 101 protein translation, A-34, A-34f rough, A-33–A-34, A-33f smooth, A-33, A-33f structure, A-32–A-34, A-33f
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In d ex
Endosome, 126, 202f, 402, A-29 Endospore Bacillus, 693–694, 693f, 694f characteristics, 690t Clostridium, 694–695, 694f, 695f description, 683 destruction of, 17 fluorescence microscopy of sporulation, 59–60, 60f formation, 143–144, 143f germination, 144 Endosymbiont, 666 algae, 763, 778–779, 787, 824, 825f Alpha Proteobacteria, 706, 707f of animals, 28, 28f of invertebrates, 667–668, 668f, 669f, 670 nitrogen fi xation by, 27, 706, 707f secondary, 755f Endosymbiosis, 666, 757 coral, 788 dinoflagellate, 787, 788 eukaryotic evolution, 30, 757, 758 microbial, 666–667, 668f mitochondria and chloroplasts, origin of, 670–672, 670f, 671f primary, 758, 763f secondary, 671–672, 671f, 758, 763, 763f, 778–779 tertiary, 672, 787 Endosymbiosis theory, A-37, A-38f Endothermic reaction, A-17 Endotoxin, 94, 948, 958–959, 958f, 959f Energy, 461–475 acquisition by living organisms, 463f, 463t activation, 473, 473f, A-18–A-19, A-18f (See also Free energy change) carriers, 469–475, 469f, 472f change in, A-16–A-17, A-16f chemotrophy, 118–120 in concentration gradients, 467–468, 467f costs of biosynthesis, 548, 550 defi nition, 463 entropy and, 463–468 Gibbs free energy change, 464–469 laws of thermodynamics, A-16–A-17, A-16f lithotrophy, 463t
SFMB_index.indd I-17
organotrophy, 462, 463t phototrophy, 118–120 reduction potential, 507–509, 508t solar, 464, 464f storage in electron transport system, 507–509 in ion gradients, 85 as membrane potential, 120 transformation in photosynthesis, 539f use to build order, 464 Energy carriers. See also specific carriers ATP, 469–471, 469f defi nition, 469 FADH 2, 472–473 NADH, 469, 471–473, 472f NADPH, 472–473 observing in living cells, 475 Enhancer, 224, 248, 364 Enoyl reductase, 82, 567, 568f Enriched medium, 130 Enrichment culture, 26–27 Entamoeba E. hartmanni, 869 E. histolytica, 781, 997 phase-contrast microscopy, 57, 57f metronidazole for, 1042 Enteric (typhoid) fever, 980 Enteroaggregative Escherichia coli (EAEC), 993 Enterobacter, 710 Enterobacterales, 681t Enterobacteriaceae, 709–711, 710f API strip for identification of, 1066–1068, 1067f, 1067t, 1069f intestinal flora, 869 Enterochelin, 368f Enterococcus, 697 E. faecalis, 697 antibiotic resistance, 1050–1051 ATP synthase, 523 conjugation, 310 Tn916, 336 urethal flora, 871 Enterohemorrhagic Escherichia coli (EHEC), 993–995, 1065 gastrointestinal tract infections, 998t Shiga toxin, 956 spread of, 1094 Enteroinvasive Escherichia coli (EIEC), 993 Enteromorpha, 381, 381f
Enteropathogenic Escherichia coli (EPEC), 98, 98f, 993 attaching and effacing lesion, 963, 963f intimin, 948, 963 Enterotoxigenic Escherichia coli (ETEC), 993, 994 attachment mechanism, 945t gastrointestinal tract infections, 998t heat-stable toxin, 949f vaccine in transgenic plants, 454t Enterotoxin, 872 Enterovirus, 400t, 1015 Enthalpy, A-16–A-17 defi nition, 464 in microbial metabolism, 465–466 Entner-Doudoroff (ED) pathway, 478, 482, 485–486, 485f, 486f, 724, 725f Entophysalis, 636f Entropy, 463, 464, A-16–A-17, A-16f in biochemical reactions, 465–467 in concentration gradients, 467–468, 467f Gibbs free energy change, 464–465 in microbial metabolism, 465–466 Envelope archaeal, 722t, 723–724, 723f bacterial defi nition, 76 functions of, 75 gram-negative bacteria, 91, 91f, 94–96, 95f–97f gram-positive bacteria, 91, 91f, 93–94, 93f, 94f model of, 74f–75f mycobacterial, 92, 92f, 93f peptidoglycan, 89–90, 89f, 90f reductive (degenerative), 94 S-layer, 94, 94f viral, 187, 189, 189f, 192 Env gene, HIV, 416, 420 Environmental management, 853 Environmental niche. See Niche Environmental sample, 685 Environmental Sample Processor, 733, 733f
I-17
Environment influence on growth classification by environmental parameter, 151 nutrient deprivation/ starvation, 168–170, 168f, 170f osmolarity, 151t, 157–158, 157f, 158f overview, 150–151 oxygen, 151t, 164–168, 165f–167f, 165t pH, 151t, 158–164, 159f–164f pressure, 151t, 155–156, 156f temperature, 151t, 152–155, 152f–154f Enzyme allosteric regulation, 485 allosteric site, 474 as biological catalyst, A-18–A-19 cofactors, 117 defi nition, 473 fi lter, 173–174, 174f industrial, 626, 627 industrial production, 616, 617 lowering of activation energy, 473, 473f in lysosomes, A-32, A-33f modification of, 283 modular, 567, 568f pH optima, minima, and maxima, 159 psychrophilic, 154 reaction catalysis by, 473–475 in thermophiles (thermazymes), 155 in vitro evolution, 449–450, 450f Enzyme-linked immunosorbent assay (ELISA) antigen capture, 1078, 1078f description, 1077 Ebola antibody detection, 1077, 1078f for Helicobacter pylori, 996 toxin assay, 860 Eosinophil, 874, 874f Epcot Center, 89, 89f EPEC (enteropathogenic Escherichia coli), 98, 98f, 993 attaching and effacing lesion, 963, 963f intimin, 948, 963 Epidemic, 1086, 1086f Epidemic typhus, 1022t Epidemiology, 994 bioterrorism, 1090–1091, 1091f
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I-18
In d ex
Epidemiology (continued) case histories, 1085, 1089, 1092 disease spread, 1093–1095, 1094f emerging disease detection, 1092–1095, 1092f–1094f father of, 1087, 1088f, 1089 molecular detection and surveillance, 1089–1090, 1092–1093 notifiable diseases, 1087, 1088t patient zero, 1086–1087 principles of, 1085–1091 terminology, 1086 Epidermis, 864 Epifluorescence microscopy, for enumeration of marine plankton, 804, 805f Epigenetic silencing, 1017 Epilimnion, 809–810, 811 Epinephrine, 927, 928 Episome, 308 Epitope, 897, 897f, 904 EPS (exopolysaccharide), 142, 142f Epsilon 15 bacteriophage, 181f Epsilonretrovirus, 413t Epstein, Richard, 396, 397 Epstein-Barr virus, 196, 423t Epulopiscium fishelsonii, 695–696, 696f, 806 Equilibrium, A-17 Equilibrium density gradient centrifugation, 96, 96f Equine infectious anemia virus (EIAV), 413t Equine rhinotracheitis B virus, 400t Equivalence (antigen: antibody ratio), 906, 906f ER. See Endoplasmic reticulum (ER) Erbovirus, 400t Ergosterol, 765 Erithromicrobium ramosus, 706 Error-prone PCR, 449, 450 Error-prone repair, 327, 331 Error-proof repair, 327, 327–330, 328f–331f Erwinia, 610, 711 E. amylovora, 150 Erysipelas, 984t, 1043 Erythromycin, 1044 action of, 281 biosynthesis, 1046 prophylactic use, 1015 structure, 281f, 567f, 1044f synthesis, 566, 567, 568f
SFMB_index.indd I-18
Erythrose 4-phosphate, 487, 487f, 548 Escherichia, nitrogen fi xation by, 845 Escherichia coli ABC transporters, 124–125 acid fitness island, 340 acid resistance, 432–443, 434f, 956 alternative electron donors and acceptors, 525 ampicillin effect on, 178f API results for, 1067f appearance, 7f, 43f, 681t arginine biosynthesis in, 578f aromatic amino acid biosynthesis, 579f atomic force microscopy, 67, 67f ATP synthase, 523f bacteriophage lambda attachment, 198, 198f bacteriophage M13, 211f, 398 biofi lms, 1000 cadBA operon, 479, 479f catabolite repression in, 351, 352f, 353 cell division, 134 cell wall, 76 chaperones, 100 chromosome compaction, 228 chromosome structure, 222, 222f classes of RNA in, 266t conjugation, 306–309, 306f–310f cytochrome bo quinol oxidase, 519f, 520, 520f Dam methylase, 327 DegP protein, 257f diseases, 710 DNA gyrase, 230, 231f DNA polymerases, 236 DNA replication DnaA protein, 235f genes and proteins involved in, 232t origin of replication, 234 time required for, 234 electron transport system location, 509 enteroaggregative E. coli (EAEC), 993 enterohemorrhagic E. coli (EHEC), 993–995, 1065 gastrointestinal tract infections, 998t Shiga toxin, 956 spread of, 1094 enteroinvasive E. coli (EIEC), 60, 60f, 993
enteropathogenic E. coli (EPEC), 98, 98f, 993 attaching and effacing lesion, 963, 963f intimin, 948, 963 enterotoxigenic E. coli (ETEC), 993, 994 attachment mechanism, 945t gastrointestinal tract infections, 998t heat-stable toxin, 949f vaccine in transgenic plants, 454t evolution through horizontal transmission, 943–944, 944f eye flora, 866 fatty acid composition and temperature, 566 fermentation by, 489 GC content, 337 genome evolution, 336–337 genomic analysis, 304 sequencing, 254 size, 224t structure, 247, 247f glutamine synthase gene, 297 glycerol transporter, 122–123, 122f growth in mucus, 870 growth requirements, 117, 130 heat-shock response of, 155, 365–366, 366f Hektoen agar, 1066f hemolysin transport, 290 horizontal gene transfer, 661, 664, 664f, 665f human homolog genes, 331–332 immune avoidance, 888 infectious dose, 432 interspecies mating, 310 intestinal flora, 869 lactose fermentation, 130 lactose operon, 349–355 lactose permease, 123 model of cell structure, 74f–75f, 75–76 as model organism, 32, 709 molecular composition, 76, 77t multiplex analysis, 446f mutator strain, 325f nucleoid, 98, 98f O157:H7, 710, 943–944, 944f case history, 993–995 in cattle, 994 food-borne illness, 612t horizontal gene transfer in, 664, 664f, 665f
host colonization, 611, 611f infectious dose, 994 rumen acidity, 827 Shiga toxin, 200, 613, 956 sorbitol fermentation test, 490, 490f survival curve in eggplant salad, 614, 614f overlapping generations, 234 phage T4 adsorption, 391 phenol red broth test, 490, 490f phosphatidylethanolamine, 82 phosphotransferase system (PTS) of, 127f pH range, 158, 160, 160f pili Pap (type I), 945–946, 946f type IV, 947 promoters, 261f proteins of, 77, 77f, 78, 78f proteome, 385f proton circulation, 163f RecA-mediated recombination, 317, 317f, 318f replication rate, 116 ribosomes, number of, 272 in rumen, 827, 828, 828f sacculus, 89, 89f septation, 104, 104f shape-determining proteins, 105f shuttle vector, 252 siderophores, 126f sigma factors, 260–261, 261f, 262f signal recognition particle, 286 size, 41f specialized transduction, 312, 313f, 314 starvation and colony morphology, 168f stress responses, 169 surface proteins, 76 tmRNA, 283, 284f topoisomerase I, 230f toxins labile toxin, 951–952, 954 stable toxin, 949, 949f type III secretion, 963, 963f, 995 ultraviolet light, response to, 303f urinary tract infections, 999–1001, 1000f, 1001f uropathogenic E. coli (UPEC), 945–946, 945t, 993, 999–1001, 1000f, 1001f vitamin B12 production, 870
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Escovopsis, 666, 667f ESensor DNA Detection System, 1092f–1093f Essential nutrients, 116 Ester functional group, A-7t use in food preservation, 614 ETEC (enterotoxigenic Escherichia coli), 993, 994 attachment mechanism, 945t gastrointestinal tract infections, 998t heat-stable toxin, 949f vaccine in transgenic plants, 454t Etest, 1034, 1034f Ethambutol, 617, 617f, 992 Ethanol as decolorizer, 53f, 54 detoxification by liver alcohol dehydrogenase, 604 for DNA precipitation, 248 as fermentation product, 488f, 489, 490 phenol coefficients, 176t structure, 177t Ethanolic fermentation, 471, 489, 490 bread production, 602, 603–604, 603f, 604f description, 593 examples, 594t of fruit, 605–607, 605f, 608f of grain, 604–605, 605f, 606–607, 607f overview, 593, 602–603 Ether link, 87, 87f, 724 Ethylene oxide, 176, 177t ETS. See Electron transport system (ETS) Eugenol, 614 Euglena E. gracilis, 762t, 791 mixotrophy, 121 Euglenida, 762t, 791 Euglenozoa, 762t, 764 Euglyphida, 761t Eukarya (Domain), 9 archaea and bacteria compared, 657, 660–661, 660t Bacteria and Archaea compared, A-22t cell size, 41–42, 42f defi nition, 8, 9 diversity, 755–791 algae, 761t, 763, 774–780, 775f–780f alveolates, 761t–762t, 764, 783–790, 783f–790f
SFMB_index.indd I-19
amebas, 761t, 763–764, 780–781, 780f, 781f, 783, 783f animals, 760t, 758 excavates, 762t, 791 fungi, 760t, 758, 763, 765–774, 765f–773f heterokonts, 762t, 764 microsporidia, 760t, 791 newly discovered species, 764 opisthokonts, 760t, 758, 759f overview, 755–756, 756f phylogeny, 756–757, 757f, 758 plants, 760t representative groups of, 760t–762t slime molds, 763, 781–782, 782f trypanosomes, 762t, 790f, 791 water molds, 773, 774f gene regulation, 364 genomes, 246–248, 247f heterotrophy, 121 historical overview, 756–757, 757f, 758 in kingdom Monera, 29 microbial transfer of genes into, 310–311, 311f noncoding DNA, 225 nutrient transport by endocytosis, 126–127 osmotic shock, protection from, 97, 97f phase variation in, 373 proteasome, 291, 291f serial endosymbiosis theory, 30, 30f Eukaryotic cell, A-21–A-38 Bacteria and Archaea compared, A-22t cell membrane, A-22–A-29, A-24f–A-29f chloroplasts, A-37, A-38, A-38f cytoskeleton, A-35–A-36, A-35f endomembrane system, A-32–A-35, A32f–A-34f flagella and cilia, A-36, A-37f mitochondria, A-37–A-38, A-38f, A-38t mitosis, A-30f, A-31f nucleus, A-29, A-30f overview, A-21–A-22, A-21f size, A-30–A-32, A-31f structure, A-23f Euphotic zone, 801, 801f Europa, 856–857, 857f Euryarchaeota acidophiles, 751, 752f
halophiles, 744–748, 744f–746f, 745t, 748f–749f, 750 methanogens, 738–744, 739f–743f, 739t phylogeny, 726, 727, 727f, 728t–729t thermophiles, 750–751 Eutrophication, 169, 170f, 811 Eutrophic lake, 809f, 811 Everglades, 842, 842f Evolution. See also Origins of life artificial techniques, 446–450 DNA shuffl ing, 447–448, 448f phage display, 448–450, 449f, 450f chloroplast, 763, 763f coevolution, 666, 667f convergent, 756 directed, 448–450 of DNA, 644 genome, 336–341 core gene pool, 337, 337t divergent, 340 E. coli, 336–337 flexible gene pool, 337, 337t gene duplication, 340, 340f genome reduction, 340 genomic islands, 337, 339, 339f horizontal gene transfer, 337, 339–340 horizontal gene transfer, 661–664, 662f–665f mesorhizobial, 304 molecular clock, 652–653, 652f natural selection, 652 random mutation and, 652–653 reductive, 424–425, 424f, 425f, 496, 652, 668, 669f, 670, 756 Scopes trial, 630, 631f symbiosis, 666–672 viral, 196–198, 197f, 424–425, 424f in vitro, 447–450, 448f Excavata (excavates), 762t, 764, 791 Excitation wavelength, 58 Exergonic reaction, A-17 Exfoliative toxin, 983, 983f Exit site (E site), 274, 275f Exocytosis, A-29, A-29f Exonuclease, 238, 295, 295f Exopolysaccharide (EPS), 142, 142f Exothermic reaction, A-17 Exotoxin AB toxin, 949, 951, 951f, 954
I-19
alpha toxin, 949, 951, 952f anthrax toxin, 956–958, 957f botulism, 613, 613f, 694, 694f, 1008–1009, 1009–1010, 1009f, 1010, 1010f characteristics of, 950t–951t cholera toxin, 951–952, 952f, 953f, 954 diphtheria, 954–956, 955f, 956f E. coli labile toxin, 951–952, 954 modes of action, 948–958, 949f Shiga toxin, 949f, 956 tetanus, 1008–1010, 1009f, 1011f toxic shock syndrome toxin (TSST), 921, 980, 983 ExPASy (Expert Protein analysis System), 297 Exponential growth, 134, 134–139, 135f Exponential phase, 137 Extracellular environment, sensing of, 347–348, 348f Extraterrestrial microorganisms, 861 Extravasation, 880, 882–883, 882f, 883f Extreme hyperthermophile, terpene-derived lipids of, 87–88 Extreme thermophile, 155. See also Hyperthermophile Geobacter, 28, 28f reverse DNA gyrase, 230 Extremophile, 797–798, 797t. See also specific microbes; specific types acidophile, 161, 161f antarctic haloarchaea, 149f Bacillus spp., 694 defi nition, 150 Eye, microflora of, 865f, 866
F F(ab) 2 region, 902f, 903, 903f FabF protein, 1053–1054 Facilitated diffusion, 122–123, 122f FACS (fluorescence-activated cell sorter), 132, 133f Factor H, 926 Factor IX gene, 455 Facultative, 151t, 165t, 166–167, 166f Facultative aerobe, 167 Facultative anaerobe, 167 Facultative intracellular pathogen, 969 Facultative phototrophy, 544
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FAD (flavin adenine dinucleotide), 166, 166f FADH 2, 472–473, 472f, 493, 494f, 495, 560 Fairy ring mushrooms, 756, 757f, 773 Falkow, Stanley, 251 Famiciclovir, 1054t Farmer, Paul, 1064 Fatal familial insomnia, 1013t Fatty acid, 82, 83f, 86, 86f, 87 from ruminal digestion, 826 saturated, A-15, A-15f synthesis mechanism, 564, 565f regulation, 565–566 unsaturation, 564 unsaturated, A-15, A-15f Fatty acid synthase complex, 564 F+ cell, 307, 307f F – cell, 307, 307f Fc region, antibody, 902f, 903, 903f Fd bacteriophage, 448 Feces, bacteria in, 869, 869f Feeley, John, 2 Feigon, Juli, 785 Feline leukemia virus (FeLV), 205, 413, 413t Femtoplankton, 803, 803t Fennessy, Siobhan, 838, 838f Fenton reaction, 166f Fe protein, 571, 572f Fermentation, 17, 166, 167, 463t, 487–491 acidic, 593, 594t, 595–599, 595f–599f alkaline, 593, 594t, 599–602, 602f of cocoa, 589f, 599, 600–601, 600f–601f description, 476 diagnostic applications, 489–490, 490, 490f ethanolic, 471, 489, 490, 593, 594t, 602–608, 603f–608f food and industrial applications, 489–490 Gamma Proteobacteria, 710 heterolactic, 489, 593, 599 history of, 590, 590f lactic acid, 489, 593 malolactic, 606–607 mixed acid, 489 Pasteur’s work on, 17 pathways, 488f, 489 propionic acid, 593 ruminal, 826–827 Swiss cheese production, 480–481, 480f Fermentation system, 619–621, 619f, 620f Fermentative metabolism, 166
SFMB_index.indd I-20
Fermented foods acidic fermentation, 593, 594t, 595–599, 595f–599f alkaline fermentation, 593, 594t, 599–602, 602f description, 592–593 ethanolic fermentation, 593, 594t, 602–608, 603f–608f heterolactic fermentation, 593 lactic acid fermentation, 593 overview, 592–595, 593f propionic acid fermentation, 593 purposes of, 593 table of, 593, 594t traditional, 593 Ferredoxin, 540, 541f, 560, 1042, 1043f Ferretti, Joseph J., 860 Ferroplasma, 727, 751 F. acidarmanus, 530, 751 Fertility factor (F factor), 307–309, 307f, 308f, 310f Fertilizer nitrogen, 843 phosphate, 850 Fever, 891 F1Fo ATP synthase, 523, 523f, 524f, 534 Fibrobacter flavefaciens, 827 Filamentous phages. See also specific bacteriophages nanowires, 397 structure, 397–398, 397f Filamentous virus, 189–190, 190f Filariasis, 668, 668f Filopodia, 707, 781 Filoviridae, 195t Filtration, 173–174, 173f, 174f Fimbriae, 107, 108f, 944. See also Pilus FimH protein, 946, 947f Finley, Brett, 963 Fire blight, 150 Firmicutes description, 683 endospore-forming rods Bacillales, 692–694, 693f, 694f Clostridiales, 694–696, 694f–696f envelope, 692f intestinal flora, 869 non-endospore-forming anaerobic dechlorinators, 697–698, 698f Lactobacillales, 696 Listeria, 696, 697f Mollicutes, 698–699, 698f staphylococci and streptococci, 696–697, 697f
phototrophy, 687t representative groups, 680t Fish, spoilage of, 609, 610t Fis protein, 370 Fistula, 827, 828, 828f FITC fluorophore, 60, 60f Fitness islands, 340 Fixation, 52, 550. See also Carbon dioxide, fi xation; Nitrogen fi xation Flagella chemotaxis and, 110–111, 111f dark-field microscopy, 56, 56f defi nition, 56, 76, 109 eukaryotic, 42, 758, 764, 764f, A-36, A-37f flagellum-to-stalk transition, 108–109 number and arrangement, 109–110 periplasmic, 715, 715f phase variation, 320 rotary motor, 109, 110, 110f structure, 110 unpaired, 758 Flagellate, 764 Flagellin, 110, 370–371, 371f, 961 Flamingo, pink, 161–162, 161f Flammulina velutipes, 591 Flash pasteurization, 173 Flavin adenine dinucleotide (FAD), 166, 166f Flavin mononucleotide (FMN), 517, 517f, 518, 518f Flaviviridae, 195t, 1023 Flavivirus, 214 genome, 192 yellow fever, 940, 941f Flavobacteriales, 682t Flavobacterium, use in wastewater treatment, 841 Flavodoxin, 1042, 1043f Flavonoid, 820, 821 Fleming, Alexander, 25, 25f, 177, 431, 1030f, 1031 Flesh-eating bacteria, 983–985, 985f FlgM anti-sigma factor, 365 Flocs, 841, 841f Floppy head syndrome, 1008 Florey, Howard, 25, 1030f, 1031 Flu. See Influenza Fluconazole, 1059, 1060t–1061t Fluid mosaic model, A-24 Fluorescence, 45, 58–60, 59f Fluorescence-activated cell sorter (FACS), 132, 133f
Fluorescence microscopy, 58–60, 58f, 59f, 60f, 131, 132f Fluorescence resonance energy transfer (FRET), 446 Fluorescent antibody staining, 1079, 1079f Fluorescent-focus assay, 211–212, 212f 5-fluorocytosine, 1060t–1061t Fluorophore, 59, 59f F-met-leu-phe peptide, 883 FMN (flavin mononucleotide), 517, 517f, 518, 518f Focal plane, 46, 47f, 51–52 Focal point, 46, 47, 47f, 49 Focus, 48, 51–52, 211 Folate, 562 Folding, protein, 283, 285 Folic acid, 1032, 1042, 1042f Folliculitis, 984t Fomite, 940, 941f, 942 Food-borne pathogen, 611, 611f, 612t, 613, 613f Food contamination, 609 Food irradiation, 614 Food microbiology, 589–615 fermented foods acidic fermentation, 593, 594t, 595–599, 595f–599f alkaline fermentation, 593, 594t, 599–602, 602f ethanolic fermentation, 490, 593, 594t, 602–608, 603f–608f heterolactic fermentation, 593 lactic acid fermentation, 593 overview, 592–595, 593f pathways, 488f propionic acid fermentation, 593 purposes of, 593 table of, 593, 594t traditional, 593 food-borne pathogens, 611, 611f, 612t, 613, 613f food contamination (food poisoning), 609 food preservation, 613–615, 614f microbes as food, 590–592 edible algae, 591, 591f edible bacteria and yeasts, 591–592, 592f edible fungi, 590–591, 591f microbial growth control irradiation, 174–175 pasteurization, 173 refrigeration, 173
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steam sterilization, 172–173 spoilage, 154, 608–611 classes of food change, 608 dairy products, 609, 610t defi nition, 608–609 meat and poultry, 609, 610t plant foods, 610–611, 610t seafood, 609, 610t Food poisoning description, 609 by Staphylococcus aureus, 992–993, 998t Food preservation, 613–615, 614f Food spoilage, 154, 608–611 classes of food change, 608 dairy products, 609, 610t defi nition, 608–609 meat and poultry, 609, 610t plant foods, 610–611, 610t seafood, 609, 610t Food web, 796, 796f planktonic, 806, 807f soil, 813, 814f Foot-and-mouth disease virus, 400t, 401, 454t Foraminiferans, 761t, 764, 783, 783f Forespore, 144, 693, 695, 695f Formaldehyde as disinfectant, 176 in northern blot procedure, 436 structure, 177t Formalin, 176t Formate, 488f, 489 Forterré, Patrick, 425, 644, 751 Foscarnet, 1054t Fossil, 18, 18f microfossils, 635–637, 635t, 636f, 637f stromatolite, 629f, 630, 630f, 635, 635t Fossil fuels, 837, 848–849 Fowl cholera, 23 F pilus bacteriophage attachment to, 189 phage M13 attachment to, 398, 399f F-prime (F′) plasmid or F′ factor, 309, 310f Fractionation, subcellular, 79–80, 79f Frameshift mutation, 321f, 322 Francisella F. tularensis, 981, 981f, 1022t identification procedures, 1080t growth factors and natural habitat, 116t Frank, A. B., 815–816 Frankia, 700, 700f
SFMB_index.indd I-21
Franklin, Rosalind, 22, 33–34, 34f, 69 Frank pathogen, 939 Fraser-Liggett, Claire, 9f, 499 Free energy change equilibrium and, A-17–A-18 reaction spontaneity, A-16–A-17 Freeze-drying, 173, 613 Freezing, 613–614 Freshwater microbial communities, 809–811, 809f–811f FRET (fluorescence resonance energy transfer), 446 Fromont-Racine, Micheline, 444 FRT sequence, 455, 455f Fructose, A-11f Fructose 1,6-bisphosphate, 474, 483, 484f, 485, A-12f Fructose 6-phosphate, 482, 483, 483f, 484f, 485, 487, 487f Fruit alcoholic fermentation of, 605–607, 605f, 608f spoilage, 610, 610t Fruiting body, 144, 144f fungal, 590 myxobacteria, 712, 712f slime mold, 763 Frustule, 779 Fts genes, 104 FtsH protease, 375 FtsY protein, 286f FtsZ protein, 73f, 104, 104f, 105, 105f Fuel cell, 505, 506 Fumarate, 493, 494f Fumarate reductase, 560, 561f Functional group, A-6, A-7t Fungi. See also specific fungi absorptive nutrition, 765–766 anthracnose fungus, 823f, 824 antifungal agents, 1059–1060, 1059f, 1060t–1061t Ascomycota, 769–771, 770f, 771f Basidiomycetes, 771–773, 771f, 773f cell wall, 76 Chytridiomycota, 768, 768f decomposers, 813, 815 defi nition, 758 edible, 590–591, 591f heterotrophy, 121 historical view of, 756–757, 757f, 758 hyphae, 765–766, 765f, 766f
identification procedures, 1080t imperfect, 767 lichen, 798–799 meningitis/ encephalitis, 1012t mitosporic, 767 mutualism and, 666, 667f mycorrhizae, 813, 815–817, 816f, 817f notifiable diseases, 1088t overview, 765 parasitic, 813 phylogeny, 763 plant pathogens, 823f, 824 respiratory tract infections, 987–989, 988f, 990t–991t skin infections, 984t unicellular, 766–767, 767f Zygomycota, 769, 769f Fur (ferric uptake regulator), 356f, 368–369, 368f Fuselloviridae, 194t Fuselloviruses, 194t, 734–735 Fusion peptide, 409 Fusobacterium, 867, 867f
G GABA (gamma-aminobutyric acid), 433, 434f, 1010, 1011f gadA gene, 435, 436f, 437, 438, 440f, 441 gadB gene, 436f, 437, 441 GadB protein, 439f, 440 gadC gene, 436f, 437 GadE protein, 438, 438f, 440, 441, 442f GadW protein, 438 GadX protein, 441, 442f gag gene, HIV, 416 Gain-of-function mutation, 321 Galactan, 92, 93f Galen, 1004 Gallionella G. ferruginea, 108, 109f iron oxidation, 509 Gallo, Robert, 413, 414, 414f, 416 GALT (gut-associated lymphoid tissue), 877, 878, 878f GAL4 transcription factor, 442–443, 443f Galvani, Luigi, 506 Gamma-aminobutyric acid (GABA), 433, 434f, 1010, 1011f Gammaretrovirus, 413t Gamogony, 789, 789f, 790 Gamonts, 790 Gancyclovir, 1054t Gas gangrene, 872f Gastric ulcer, 995–996
I-21
Gastroenteritis, 992–993, 996, 998t Gastrointestinal illness, foodborne, 611 Gastrointestinal tract infection, 992–997 antibiotic use, 993 enterohemorrhagic E. coli (EHEC), 993–995 Helicobacter pylori, 995–996, 995f overview, 992–993 protozoal, 996–997, 997f rotavirus, 996 table of common causes, 998t Gas vesicle, 106, 106f, 688, 746, 747, 750 Gates Foundation, 616 GATTACA (movie), 250 GC content, 337 defi nition, 683 gram-positive bacteria, 683, 684 of halophiles, 745 pathogenicity island, 943, 943f GC to AT transition, 323 Gel shift, 438, 438f Geminiviridae, 194t Geminivirus, 194t Gemmata obscuriglobus, 718f Gene composition of, 222 housekeeping, 663 informational, 662, 663 naming conventions, 226 operational, 662 organization, 225, 225f pseudogenes, 247 reporter, 82 rescue, 965 Gene arrays, 381–383 Gene cloning, 252 Gene delivery, 454–455, 454f, 455f Gene duplication, 340, 340f Gene expression, regulating, 346–349 arabinose operon, 359–360, 359f AraC regulator, 355–357, 359–361, 359f, 360f attenuation, 361–362, 363f in eukaryotes, 364 genomic and proteomic tools, 381–386 history of research, 349 integrated control circuits, 349, 373–378 lactose operon, 349–355 levels of control, 348–349 DNA sequence alteration, 348 integrated control circuits, 349, 373–378
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Gene expression, regulating (continued) levels of control (continued) posttranslational control, 349 transcriptional, 348 translational, 348–349, 364 phase variation, 370–373 eukaryotes, 373 gene inversion, 370–371, 371f slipped-strand mispairing, 371–372, 372f quorum sensing, 378–381 regulatory proteins, 346–347, 347f sensing extracellular environment, 347–348, 348f sigma factor, 365–367, 366f, 367f small regulatory RNAs, 368–370, 368f, 369f stringent control, 362–364, 364f Gene expression, starvation response and, 168 Gene fusion, 132, 434–435, 435f Gene gun, 452 Gene inversion, 370 Gene mapping by conjugation, 308f, 309 Genencor International, 590 Generalized recombination, 316, 316–320, 318f, 319f Generalized transduction, 311 Generation time, 135 Gene regulation archaeal, 725–726, 726f operons, 35 Gene splicing, 911, 912f, 913 Gene therapy, 184, 416, 422, 454–455, 454f, 455f, 896, 896f Genetically modified organisms (GMOs), 454 Genetic analysis, 81–82, 81f Genetic code deciphering of, 258, 258f description, 267–269, 267f metabolist model of early life and, 642, 642f redundancy, 271 Genetic drift, 652 Genetic engineering, 32 Genetics of antibody production, 911, 912f, 912t, 913–915, 914f pathogen identification based on, 1072–1076, 1073t, 1074f–1077f
SFMB_index.indd I-22
Gene transfer conjugation, 306–311, 306f–308f, 310f horizontal, 222, 661–664, 662f–664f from microbes into eukaryotes, 310–311, 311f prevention by restrictionmodification systems, 315–316, 315f, 315t transduction, 311–314, 312f–314f transformation, 304–306, 305f vertical, 222 Genital herpes, 423, 426, 426f Genital warts, 203, 204f Genitourinary tract infection, 997–1006 sexually transmitted diseases (STDs), 1001–1006, 1002f–1006f urinary tract infection, 999–1001, 1000f, 1001f microflora, 865f, 866t, 870–871 Genome archaeal, 248 bacterial, 223 bioinformatics and, 293–298 circular, 223 compact, 75 defi nition, 9, 221, 223, 303 eukaryotic, 246–248, 247f evolution, 336–341 core gene pool, 337, 337t divergent, 340 E. coli, 336–337 flexible gene pool, 337, 337t gene duplication, 340, 340f genome reduction, 340 genomic islands, 337, 339, 339f horizontal gene transfer, 337, 339–340 flexibility of microbial, 115 map, bacteriophage T4, 392f minimal, 297 organization compaction into nucleoid, 228 DNA structure, 226–227, 226f–228f functional units, 225 gene distribution, 224, 225f noncoding DNA, 224–225, 247–248 size, 223–224, 224t supercoiling, 228–230, 229f–231f
reduction, 340, 668, 670 replication bacteriophage, 244–245, 246f DNA, 232–243 plasmid, 243–244, 244f segmented, 406–409, 407f sequence analysis, 248–254 sequencing, 9–11, 9f, 10f size, 223–224, 224t viral, 184–185, 185f Genome scanning, 568–569, 569f Genomic analysis gene arrays, 381–383, 382f of metabolism, 498f, 499 Genomic islands, 337, 339, 339f, 662, 664, 942–943, 944f Gentamicin, 1044 modification, 1051, 1052f for necrotizing fasciitis, 984 structure, 1044f Genus name, 647 Geobacter, 28, 28f cytochromes, 516 G. metallireducens, 712 electricity generation by, 506, 506f uranium reduction, 528–529 Geochemical cycling, 27 Geological evidence for early life, 633, 634f, 635–638, 635t, 636f–637f Archaean eon, 633, 635 biosignatures, 635t, 637, 637f Hadean eon, 633 isotope ratios, 635t, 637–638, 646, 646f microfossils, 635–637, 635t, 636f, 637f stomatolites, 629f, 630, 630f, 635, 635ft Geomicrobiology, 832 Geosmin, 699 German measles, 985–986, 985f Germicidal, 171 Germination, 144 Germ theory of disease, 17 Gerstmann-StrausslerScheinker syndrome, 1013t Geyser, 730 GFP. See Green fluorescent protein (GFP) Gfp gene, 444 Ghadiri, Reza, 1055 Ghon complex, 989, 989f Giardia lamblia, 764, 790f, 791, 997, 997f metronidazole for, 1042 structure, 42, 42f
Gibberellin, 606, 607f Gibbs free energy change (∆G) components, 464–465 concentration and, 466–467, 467t defi nition, 464 factors that determine, 466 negative, 465 standard, 466 Gibbs-Helmholtz equation, 465 Gibson, Jane, 458 Gilbert, Walter, 9 Gilvarg, Charles, 218 Giovannoni, Stephen, 804 Glaucocystophyta, 670 Gliding motility, 144, 960 glnA gene, 376–377, 377f GlnB protein, 376–377, 377f Global warming, 831–832 Globigerinella aequilateralis, 783f Gloeocapsa, 689, 690f Glomales, 769 Glomerulonephritis, 985 GlpF protein, 122–123, 122f gltB gene, 297 Gluconate, 485 Gluconeogenesis, 560 Glucosamine, 94, 95 Glucose ATP produced by catabolism, 470–471 catabolism, 482–487, 482f in archaea, 724, 725f Entner-Doudoroff pathway, 482, 485–486, 485f, 486f glycolysis (EmbdenMeyerhof-Parnas), 482, 483–485, 483f, 484f pentose phosphate shunt, 482, 486–487, 487f complete oxidation, 495, 496f lac operon repression, 351, 352f, 353, 353f, 355 straight-chain and cyclic forms of, A-11, A-11f structural formula, A-11f Glucose 6-phosphate, 482, 483, 483f, 484f, 485, 486, 486f, 487f Glutamate in arginine biosynthesis, 577, 578f synthesis, 575f, 576, 577 Glutamate decarboxylase, 433–434, 434f, 436f Glutamate dehydrogenase, 576–577, 576f Glutamate synthase, 576–577, 576f, 685 Glutamine synthesis, 575f, 576, 577
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Glutamine synthetase, 376–377, 377f, 576–577, 576f Glutaraldehyde, 177t Glycan chain, 89 Glyceraldehyde, A-11f Glyceraldehyde 3-phosphate (G3P) Calvin cycle and, 552–553, 553f, 556, 557f, 558 in Embden-MeyerhofParnas (EMP) pathway, 483, 484f in Entner-Doudoroff pathway, 486, 486f in pentose phosphate pathway, 487 Glycerol, A-15 in archaeal membranes, 723–724, 723f bacterial storage in, 173 as compatible solute, 158 as fermentation product, 490 in phospholipids, 82, 83f transport, 122–123, 122f Glycerol 3-phosphate, as biosynthesis substrate, 548, 549f Glycine, in vitamin B12 synthesis, 548, 548f Glycocalyx, 1015 Glycolysis, 467 description, 482, 483–485, 483f, 484f reverse, 560 standard free energy change of reactions, 467 Glycosidic bond, A-11, A-12f Glycosylase, 329 Glyoxalate, 563f, 564 Glyoxylate, 495 Glyoxylate bypass, 495 GMOs (genetically modified organisms), 454 Gnotobiotic animal, 872 Golden age of microbiology, 11 Golden algae, 779 Gold reduction, 529 Golgi complex, A-33, A-33f, A-35 Gonorrhea, 709, 1004–1005, 1064 Gordon, Jeffrey, 742 Gosink, Khoosheh, 1053 Gottesman, Susan, 369, 369f Gouda cheese, 596, 597f Gp120, HIV, 973 Grain alcoholic fermentation of, 604–605, 605f, 606–607, 607f spoilage, 610, 610t Gram, Hans Christian, 52
SFMB_index.indd I-23
Gramicidin, 1041, 1041f, 1046 Gram-negative, 54 Gram-negative bacteria cell envelope, 91, 91f, 94–96, 95f–97f elemental composition of, 833t envelope, 692f identification of pathogens enteric, 1066–1068, 1067f–1069f, 1067t nonenteric, 1068–1070, 1069f Nitrospirae, 684, 714 outer membrane, 94–96, 95f–97f Proteobacteria, 684, 703–714, 704f–705f, 707f–713f representative groups, 680t–682t subcellular location of proteins in, 95t transformation, 306 Gram-positive, 54 Gram-positive bacteria Actinobacteria, 683, 684, 699–703, 699f–703f cell envelope, 91, 91f, 93–94, 93f, 94f envelope, 692f Firmicutes, 683, 692–699, 692f–698f identifying pyogenic cocci, 1070–1072, 1071f, 1072f representative groups, 680t transformation, 305–306, 305f Gram stain, 52, 53f, 54, 54f Granulobacter bethesdensis, 706 Granuloma, 883, 884, 885f Granzyme, 921 Grapes, fermentation of, 605–607, 605f, 608f Graves’ disease, 932 Gray (Gy), 174 Grazer, 796 Great Irish Famine, 773 Great Salt Lake, 744, 746, 747 Green algae (chlorophytes), 763, 763f, 774–776, 775f–777f colonial, 776, 777f fi lamentous, 776, 776f sheet-forming, 776, 777f siphonous, 776 unicellular, 774–776, 775f Greenberg, Pete, 381, 381f Green chemistry, 617 Green fluorescent protein (GFP) FACS analysis, 132 in focus assay of animal viruses, 212, 212f
fusion proteins, 59, 105, 105f reporter gene, 82, 434 tracking cells with, 444–445, 445f visualizing subcellular events, 219 Greenhouse effect, 633, 835 Greenhouse gases, 831–832 Green nonsulfur bacteria, 686 Green sulfur bacteria photosystem I, 541f reductive TCA cycle, 560 Griffith, Frederick, 33, 222, 304 Griseofulvin, 1059, 1059f GroEL, 285, 285f GroES, 285, 285f Grossman, Alan, 241 Ground beef, bacterial growth in, 613, 614f Group B streptococci, 1012t Group translocation, 125–126, 126, 127f Growth axenic, 118 batch culture, 136f, 137–138 confluent, 129 continuous culture, 139–140, 139f culturing bacteria, 127–131 environmental influences classification by environmental parameter, 151 nutrient deprivation/ starvation, 168–170, 168f, 170f osmolarity, 151t, 157–158, 157f, 158f overview, 150–151 oxygen, 151t, 164–168, 165f–167f, 165t pH, 151t, 158–164, 159f–164f pressure, 151t, 155–156, 156f temperature, 151t, 152–155, 152f–154f essential nutrients, 116 exponential, 134–135, 135f limitation by nutrient supply, 116–117 macronutrients, 116–117 micronutrients, 117 nonculturable organisms, 118, 130 pure culture, 128–129 Growth control, 170–179 biological control, 178–179 chemical agents, 175–178 antibiotics, 176–178, 177f, 178f commercial disinfectants, 176, 177f
I-23
influences on efficacy, 175–176 phenol coefficients, 176, 176t death as logarithmic function, 171–172, 171f physiologic agents, 172–175 cold, 173 fi ltration, 173–174, 173f, 174f high temperature and pressure, 172–173, 172f pasteurization, 173 resistance of bacteria to, 175 terminology, 171 Growth curve, 136f, 137–138 Growth cycle, 134–140 Growth factor, 116t, 117 Growth rate defi nition, 134 pressure effect on, 156f temperature influence on, 152–153, 152f variation in, 149 GrpE, 365–366, 366f GTP (guanidine triphosphate), 470 Guanidine triphosphate (GTP), 470 Guanine, 227, 323, 324, A-12, A-12t, A-13f Guanosine tetraphosphate (ppGpp), 363, 364f Guillardia theta, 671f, 672 Gultaviridae, 194t Gustafson, Dan, 838, 838f Gut-associated lymphoid tissue (GALT), 877, 878, 878f Gutman, Antoinette, 198 Gymnodium, 787f Gyr (gigayear), 631 GyrA protein, 230, 231f Gyrase, 99 reverse, 230, 724 GyrB protein, 230, 231f
H Haber, Fritz, 843 Haber process, 571 Hadean eon, 633 Haeckel, Ernst, 29, 756 Haemophilus growth factors and natural habitat, 116t H. influenzae genome sequence, 10f, 11 growth factors, 1069, 1069f meningitis, 1007, 1012t, 1069 pinkeye, 866
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I-24
In d ex
Haemophilus (continued) H. influenzae (continued) protein interaction map, 444f superinfection, 972 type b vaccine, 1024t–1025t transformation, 306 Half-life, A-2 Halitosis, 996 Hall, Eliza, 1017 Hall, Walter, 1017 Haloarchaea, 638–639, 638f, 744, 744–750 applications, 751 classroom use of, 746, 746f examples, 745t habitat, 721, 744, 744f, 747 metabolic pathways, 749f phototrophy, 721, 724, 727, 747–748, 748f phylogeny, 727, 727f, 728t pigmentation, 744, 744f, 746 structure and physiology, 744–745, 745f, 747 Haloarcula, 747 H. marismortui, 224t H. quadrata, 745t H. valismortis, 745t Halobacteriales, 744 Halobacterium appearance, 158f bacteriorhodopsin, 513, 724 classroom use, 745, 746, 746f genome, 748 genome sequence, 158f glucose metabolism, 724, 725f H. halobium, 724 H. salinarum, 534, 534, 535, 535f, 745t habitat, 747 NRC-1, 727, 745, 746, 746f genome sequence, 748 metabolism, 748, 749f Halobacter salinarum, 161 Halocin, 750 Halococcus, 747 appearance, 728t H. morrhuae, 745t Haloferax H. dombrowskii, 745f H. volcanii characteristics, 745t electron transport system, 510, 510f Halophile, 158, 158f archaea, 7f defi nition, 151t environmental conditions for growth, 797t Haloquadra walsbyi, 747 Halorhodopsin, 724, 747–748, 748f Halorubrum lacusprofundi, 149f, 745t, 747
SFMB_index.indd I-24
Halothiobacillus neapolitanus, 558f Haloviridae, 194t Hammerhead ribozyme, 185, 186f Hanseniaspora, 606 Hantavirus, 22, 1094–1095 H antigen, 993 Hapten, 901, 901f, 931 Hartwell, Leland, 766 Harwood, Caroline, 458–459, 458f, 544, 703 Hassid, Zev, 555f Haustorium, 824, 824f Hayfever, 908, 908f Heat-shock protein (HSP), 100, 285 Heat-shock response, 155, 163 Heavy chain, antibody, 902, 902f, 903, 911, 912f, 913, 914f Hektoen agar, 1066, 1066f Helicase, 101, 241 Helicobacter pylori, 22–23 adhesins, 967, 968f appearance, 713f description, 713–714 electron microscopy, 40, 40f evasion of adaptive immunity, 924 gastric cancer and, 996 gastrointestinal tract infections, 998t genome annotation, 298, 299f genome size, 966 halitosis, 996 as microaerophilic, 167 neutrophil-activating protein, 967, 968f pathogenesis, 966–968, 968f protein interaction maps, 444 reductive evolution, 424 scanning electron micrograph, 995f stomach colonization, 868, 869f ulcers, 995–996, 998t urease of, 163 vacuolating cytotoxin, 967–968, 968f variation in strains, 663 Heliobacteraceae, 695 Heliozoan, 764 Helium, formation in stars, 631, 632f Helix-turn-helix motif, 359, 360f, 361 Helling, Robert, 252 Helothrix, 686 Helper T cell (T H cell), 913, 915t, 918–919, 920f, 921–924 Hemagglutinin, 406, 407f, 408f, 409, 972, 1056, 1057
Heme, 516, 517, 520, 1017 Heme b, 517f, 581f Hemin, as growth factor for Haemophilus influenzae, 1069, 1069f Hemocytometer, 131 Hemolysin alpha of Staphylococcus aureus, 949, 951, 952f secretion of, 289, 289f, 290 Hemolysis, 1070–1071, 1071f Hemolytic uremic syndrome (HUS), 956, 994 Hemophilia, 455 Henson, Jim, 921 Hepadnaviridae, 196t, 1021f, 1023 Hepadnavirus, 207 HEPA (high-efficiency particulate air) fi lter, 173, 174f Hepatitis, defi nition, 1021 Hepatitis A vaccine, 1021, 1024t–1025t Hepatitis A virus (HAV), 400t, 1021, 1021f Hepatitis B vaccine, 1023, 1024t–1025t Hepatitis B virus (HBV) disease, 1021f edible vaccine, 453 electron micrography, 1021f genome, 193 vaccine in transgenic plants, 454t Hepatitis C virus (HCV), 1023, 1094 entry and uncoating, 202f, 203 genome, 192 internal ribosome entry site (IRES), 644–645, 645f Herd immunity, 1024–1025, 1056 Herpes simplex virus (HSV) attachment, 427 disease, 423, 426, 426f DNA vaccine for HSV-2, 452, 453f entry, 427 genome, 426f latent infection, 426 replication, 427, 428f structure, 187, 188f, 189f, 426–427, 426f Herpesviridae, 194t Herpes virus. See also specific viruses genome, 192, 196, 197f genome size, 184 latent state, 939, 939f phylogeny, 196, 197f Hershey, Alfred, 198 Hesse, Angelina, 21, 21f
Hesse, Walther, 21, 21f Heteroaromatic ring, 471, 472f Heterocyst, 144, 144f, 572, 573f, 574, 689 Heterokont, 762t, 764 Heterolactic fermentation, 489, 593, 599 Heterotroph/heterotrophy, 118, 119, 119f, 121, 462 Hexachlorophene, 177t Hexose sugar, A-11 Hfr cell, 308f, 309 HHV8 (human herpes virus type 8), 1006 High-efficiency particulate air (HEPA) fi lter, 173, 174f High-energy bond, 470 High-performance liquid chromatography (HPLC), 552 Hill, Russell, 825 Hin recombinase, 370, 371f hisG gene, 325–326, 326f His-tagged proteins, 439–440, 439f Histamine, 883, 884f, 927 Histone, 247, 726, 726f Histoplasma capsulatum (histoplasmosis), 988, 990t–991t, 1006, 1060t, 1080t History, microbes and human, 11–17, 12f, 13f, 14t–15t, 16f Hitchhiking, 1003 HIV. See Human immunodeficiency virus (HIV) HIV-1 integrase, 336, 336f Hly ABC transporter, 289, 289f Hns protein, 99, 229 Hodgkin, Dorothy Crowfoot, 33, 69, 69f Holdfast, 108 Holley, Robert, 258 Holliday, Robin, 317 Holliday junction, 317, 318f, 319f Homeostasis, of Earth, 832 Homolog, 656 Homologous gene, 297 Honey, 1008 Hooke, Robert, 13, 13f Hopanes, 87 Hopanoids, 87, 87f, 88, 88f, 638 Horizon, 812, 812f Horizontal gene transfer conjugation, 223 defi nition, 222, 305, 648 in E. coli O157:H7, 664, 664f, 665f evidence for, 337
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In d ex
genome evolution, 337, 339–340 genomic evidence of, 661 in hyperthermophiles, 627 overview, 661 transformation, 222 vertical transfer distinguished from, 661, 662–663, 662f, 663f Horizontal transmission, 222, 940, 941f Hormogonia, 689 Horseradish peroxidase, 438 Horta, 530, 530f, 531 Hospital-acquired infection, 1030 Host colonization defi nition, 611 by food-borne pathogens, 611, 611f Host-pathogen interactions, 938–942 infection cycles, 940, 941f, 942 portals of entry, 942 terminology, 938–939 Host range, virus, 183, 192 Hot spring, 730, 730f Housekeeping gene, 663 Housekeeping protein, 345 Houston, Clifford, 860–861, 860f Hoyle, Fred, 19 HPLC (high-performance liquid chromatography), 552 hpr gene, 322 HPr protein, 126, 127f HPV. See Human papillomavirus (HPV) HSP (heat-shock protein), 100, 285 HSV. See Herpes simplex virus HTLV (human T-cell leukemia virus), 413 HTST (high-temperature/ short-time), 173 Human homologs of bacterial repair genes, 331–332 noncoding DNA, 247 Human herpes virus type 8 (HHV8), 1006 Human immunodeficiency virus (HIV), 413, 413t accessory proteins, 416, 416t AIDS, 413–414, 973–975, 974f, 1005–1006, 1005f antiviral drugs for, 184, 1057–1058, 1057f, 1058f
SFMB_index.indd I-25
assembly, 420 attachment, 417, 417f case history, 1057 detection by PCR, 22 discovery, 413, 414 entry, 417–418 evolution of, 413 exit, 422 for gene delivery, 455 genome, 415f, 416, 418, 418f host range, 183 integration into host genome, 419f, 420 invasion of CD4 T cells, 922 life cycle, 205–206, 206f mutation rate, 414 pathogenesis, 973–975, 974t prevalence, 1005 regulation of replication, 414–415 replication, 420, 421f, 422 resistance to, 390, 390f reverse transcriptase, 418, 419f, 420 structure, 196t, 414f, 415, 415f, 974f transmission, 1005 Human papillomavirus (HPV), 423t bioelectric DNA detection chip, 1092–1093, 1092f–1093f edible vaccine, 453 life cycle, 203, 204f pathogenesis, 975, 975f vaccine, 975 Human T-cell leukemia virus (HTLV), 413, 413t Humic material (humus), 528, 815, 815f Humoral immunity. See also Adaptive immunity activation, summary of, 920f clonal selection, 909–910, 910f cytokine modulation of, 923 defi nition, 897 isotypic (class) switching, 908, 909, 913, 914f overview, 897 primary antibody response, 908–909, 909f secondary antibody response, 908–909, 909f Hungate, Robert, 827 Hunsen, Mo, 477f Hunt, Tim, 766 Hu protein, 99, 229 Huq, Anwar, 2, 794 Hurricane Katrina, 771, 772 HUS (hemolytic uremic syndrome), 956, 994 HveA, 427
Hybridization, 228, 436 in Northern blots, 436 RNA-DNA, 228 in Southern blots, 437 Hydric soil, 818 Hydrogen fusion to form helium, 631, 632f oxidation, 465 production by Rhodopseudomonas palustris, 459, 459f Hydrogen bond base pairing in DNA, 226–227, 226f, 227f description, A-5, A-5t Hydrogen ion, pH and, 158, 159f, 160f Hydrogenophilales, 681t Hydrogenosomes, 768 Hydrogenotrophy, 465, 532, 532f, 532t, 685, 848 Hydrogen peroxide, 169 in catalase test, 1070 phenol coefficients, 176t Hydrogen sulfide (H 2S) formation, 847, 848, 849 oxidation, 847–848 oxidation of, 808–809 Hydrologic cycle, 839–842 biochemical oxygen demand, 839–840, 839f, 840f defi nition, 839 wastewater treatment, 840–842, 841f, 842f Hydrolysis, 470, A-6, A-6f of ATP, 470 polysaccharide, 478 Hydronium ion, 158 Hydrophilic, A-5 Hydrophobic, A-5 Hydrothermal vent, 154f, 155, 802, 802f Hydroxyl (functional group), A-7t Hydroxyl ion, pH and, 158, 159f Hydroxyl radical, 166 5-hydroxymethylcytosine, 394, 394f 3-hydroxypropionate cycle, 562, 563f Hyperacidophile, 721f, 729t, 751, 752f Hypermutation, 913, 914 Hypersensitivity reactions overview, 926, 927t type I, 926–929, 928f type II, 929, 929f, 932t type III, 929–930, 930f, 932t type IV, 930–931, 931f, 932t Hyperthermophile, 153–154 archaeal, 726, 727, 728t, 729t barophiles, 731–733, 732t, 733f
I-25
biofi lms, 733, 734f deep-branching thermophiles, 678, 679t, 683, 685–688, 685f, 686f defi nition, 151t Desulfurococcales, 730–733, 731f, 731t discovery, 626–627, 627f environmental conditions for growth, 797t Euryarchaeota, 750–751, 750f examples, 731t genomes, 626–627 Geobacter, 28, 28f growth rate, 798 habitat, 721 habitats, 721, 730, 730f Korarchaeota, 753 phylogeny, 726, 728t, 729t reverse gyrase, 230, 724 sulfolobales, 734–735, 734f, 735f symbiotic, 627, 627f Hyperthermus butylicus, 728t Hyperthyroidism, 932 Hypertonic environment, A-28, A-28f Hyphae fungal, 763, 765–766, 765f, 766f Streptomyces, 145, 145f Hyphomicrobium, 134, 134f Hypolimnion, 811 Hypotonic environment, A-28, A-28f Hypoxanthine, 329 Hypoxia, 840, 846, 846f
I ICAM-1 (intercellular adhesion molecule), 201, 202f, 205, 882, 883f, 899, 900f, 972 Iceberg, psychrophiles and, 153, 154f Icosahedral capsid, 187 Icosahedral viruses, 187, 188f, 189, 189f ICSP (International Committee on Systematics of Prokaryotes), 649 ICTV (International Committee on Taxonomy of Viruses), 191 ID50, 939 Identification description, 647 dichotomous key, 649, 650f DNA methods, 649 probabilistic indicator, 649 Idiotype, antibody, 904 IgA, 904, 904t, 905f
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I-26
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IgD, 904t, 905 IgE, 904t, 905, 908, 927–929, 928f IgG, 903, 904–905, 904t, 908–909, 909f IgM, 904–905, 904t, 905f, 908–909, 909f Ignicoccus, 627, 627f, 727, 729t, 753 I. islandicus, 731, 731f, 731t IL-1, 882, 923t IL-2, 919, 923t IL-3, 923t IL-4, 919, 923, 923t IL-5, 923t IL-6, 919, 923t IL-8, 923t IL-10, 923t IL-12, 923 Immersion oil, 50, 50f Immobilized pH gradient (IPG strip), 383, 384f Immune avoidance, 370, 888, 970–971, 1017 Immune system adaptive, 895–934 antibody structure and diversity, 902–908, 902f–903f, 904t, 905f–908f antigen-presenting cells (APCs), 898, 918 cell-mediated immunity, 897, 915, 920f, 923 cells involved, 873–876, 873f–875f complement, 925–926, 925f genetics of antibody production, 911, 912f, 912t, 913–915, 914f humoral immunity, 897, 908–911, 909f–911f, 915, 920f immunogenicity, factors influencing, 898–899, 898t, 899f–901f, 900t, 901–902 major histocompatibility complex (MHC), 915–916, 916f, 917f, 923 nonadaptive (innate) compared to, 873 overview, 895–898, 897f T-cells, 915–916, 915t, 918–919, 919f–922f, 921–925 autoimmunity, 931–932, 932t cytokine modulation of response, 923, 923t defi nition, 24, 873
SFMB_index.indd I-26
hypersensitivity reactions overview, 926, 927t type I, 926–929, 928f type II, 929, 929f, 932t type III, 929–930, 930f, 932t type IV, 930–931, 931f, 932t innate adaptive compared to, 873 avoidance of, 888, 888f cells involved, 873–876, 873f–875f chemical barriers, 878–880, 879t, 880f, 881, 881f complement, 889–891, 890f, 891f fever, 891 inflammatory response, 880, 882–884, 882f–885f interferon, 886–887, 887t natural killer cells, 887–889, 889f phagocytosis, 884–886, 886f, 887f physical barriers, 877–878, 878f, 879f overview, 872–877 cells involved, 873–876, 873f–875f lymphoid organs, 876–877, 876f types, 873 transplantation rejection, 933 Immunity defi nition, 24 herd, 1024–1025, 1056 Immunization. See also Vaccination defi nition, 24 description, 1023–1025 schedule, 1024t–1025t Immunofluorescence, 59 Immunogen, 897 Immunogenicity defi nition, 898 factors influencing, 898–899, 898t, 899f–901f, 900t, 901–902 Immunoglobulin, 902. See also Antibody Immunological specificity, 899, 899f, 901, 902 Immunomodulin, 872 Immunoprecipitation, 906, 906f, 907f Impetigo, 984t Inactivated poliovirus vaccine, 1024t–1025t Index case, 1086–1087 India ink, 55, 55f, 93
Indigenous flora, 593 Inducer, 347 Inducer exclusion, 355 Induction, 347, 347f, 349 Industrial fermenter, 619, 619f, 620f Industrial microbiology, 615–624. See also Food microbiology bioprospecting, 617–619 defi nition, 615 fermentation systems, 619–621, 619f, 620f goal of commercial success, 615 molecular products, 615–619, 618t production in animal or plant systems, 621–624 Agrobacterium tumefaciens in plant engineering, 621, 622f baculovirus in insects, 621–623, 623f, 624f Industrial strain, 618 Infant, bacterial succession in digestive tract of, 676, 676f Infection cardiovascular system, 1014–1017 central nervous system, 1007–1013 chemical barriers to, 878–880, 879t, 880f, 881, 881f defi nition, 938–939 gastrointestinal tract, 992–997 genitourinary tract, 997–1006 nosocomial, 1030 physical barriers to, 877–878, 878f, 879f respiratory tract, 986–992 skin and soft tissue, 982–986 systemic, 1017–1023 Infection cycle, 940, 941f, 942 Infection thread, 706, 707f, 821, 821f Infectious dose, 939 Inflammatory response basic, 882f chronic, 883–884, 885f extravasation, 880, 882–883, 882f, 883f summary of, 884f vasoactive factors, 882–883 Influenza, 990t–991t, 992 antigenic shift, 972 assembly, 408–409, 411, 412f attachment, 409
avian, 203, 389f, 408f, 409, 1086 cap snatching, 411 case history, 1054, 1056 coinfection, 409 disease, 406, 409 emerging strains, 213 entry, 409, 410f envelope synthesis, 411, 412f epidemic of 1918, 972, 973f, 1057 evolution, 192 genome, 192, 205, 406, 407, 407f pathogenesis, 972 reassortment, 408f, 409 receptor, 409, 409f replicative cycle, 409, 411, 412f spread of, 1094 structure, 190, 195t, 406–407, 406f, 407f treatment, 1054, 1054t, 1056, 1056f tropism, 203 vaccine, 972, 1024, 1024t–1025t virulence, 1057 Informational gene, 662, 663 Initiation factors, 276–277, 277f Injector device, of phage T4, 392–393, 393f Injera, 604 Innate immunity adaptive immunity compared to, 873 avoidance of, 888, 888f cells involved, 873–876, 873f–875f chemical barriers, 878–880, 879t, 880f, 881, 881f complement, 889–891, 890f, 891f defi nition, 873 fever, 891 inflammatory response, 880, 882–884, 882f–885f interferon, 886–887, 887t natural killer cells, 887–889, 889f phagocytosis, 884–886, 886f, 887f physical barriers, 877–878, 878f, 879f lungs, 878 mucous membranes, 877–878, 878f skin, 877 Inner leaflet, A-24 Inner membrane, 75, 94 Inositol hexaphosphate, 598 Inositol 1-phosphate synthase, 685
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In d ex
Inoviridae, 194t Insect baculovirus in, 621–623, 623f, 624f vector, 940 Insecticidal proteins, of Bacillus thuringiensis, 450–451, 451f Insertion, 321, 321f, 322 Insertion sequence (IS), 333, 334, 335 The Institute for Genomic Research (TIGR), 9f, 11 Integrase, 312, 313f, 320, 339 Integrated control circuit, 349, 373–378 nitrogen regulation, 376–377, 377f phage lambda, 373–376, 373f–375f Integrin, 882 Integron, 336, 338–339, 338f Intercellular adhesion molecule (ICAM-1), 201, 202f, 205, 882, 883f, 899, 900f, 972 Interference, 46, 47, 47f, 48f Interference microscopy, 57f, 58 Interferon, 447–448, 886–887, 887t, 916, 923, 923t Interleukin IL-1, 882, 923t IL-2, 919, 923t IL-3, 923t IL-4, 919, 923, 923t IL-5, 923t IL-6, 919, 923t IL-8, 923t IL-10, 923t IL-12, 923 Intermediate fi lament, A-35, A-35f Internal ribosome entry site (IRES), 644–645, 645f International Committee on Systematics of Prokaryotes (ICSP), 649 International Committee on Taxonomy of Viruses (ICTV), 191 International Journal of Systematic and Evolutionary Microbiology, 649 Intestine, microflora of, 865f, 866t, 869–870, 869f, 870f Intimin, 948, 963 Intracellular pathogen, 969–970, 970f Intron, 247, 295, 295f, 364, 725 Inversion, 321, 321f, 322
SFMB_index.indd I-27
Inverted repeat, 333, 334f, 335, 356, 370 In vitro evolution, 447–448, 448f In vivo expression technology (IVET), 965, 965f Iodine as disinfectant, 176 as mordant, 52, 53f, 54f Iodophor, 176 Ion gradient chemiosmotic hypothesis and, 33 energy stored in, 85 Ionic bond, A-4, A-4f, A-5t Ionizing radiation, 614 IPG (immobilized pH gradient) strip, 383, 384f IRES (internal ribosome entry site), 644–645, 645f Iridoviridae, 194t Irish potato famine, 432, 610 Iron aerobic and anaerobic metabolism, 797t banded iron formations, 639, 639f, 640f corrosion of, 531–532, 531f, 851, 852f Earth’s content of, 632, 633f electricity from ironreducing bacteria, 506, 506f formation in stars, 631, 632f oxidation, 530, 708–709, 709f phototrophy, 639, 640f reduction, 528, 530–531 transport by siderophores, 125, 126f Iron cycle, 850–853, 851f Iron sulfur cluster, 517, 517f, 518, 518f Irradiation, 174–175, 614 IS (insertion sequence), 333, 334, 335 Ishiwatari, Shigetane, 694 Isocitrate, 493, 494f, 495 Isoelectric focusing, 77, 383, 384, 384f Isoelectric point, 150, 383 Isoflavonoid, 845 Isolate, 649 Isomer optical, A-8 structural, A-11 Isoniazid, 992, 1033 Isoprene, A-14–A-15, A-14f Isoprenoid, 723f, 724 Isopropanol as fermentation product, 488f, 489 structure, 177t Isotonic environment, A-28, A-28f
I-27
Isotope, 551, A-2 Isotope ratio, 635t, 637–638, 637f, 646, 646f, 834, 856 Isotypes, antibody, 903–905, 904t, 905f Isotype switching (class switching), 908, 909, 913, 914f Itraconazole, 1060t–1061t Ivermectin, 567f IVET (in vivo expression technology), 965, 965f Ivors, Kelly, 591f Ixodes, 1020, 1020f
Knockout mutation, 322 Koch, Robert, 20–21, 20f, 25, 1089 Koch’s postulates, 21–23, 1089, 1090 Koplik’s spots, 985 Korarchaeota, 727, 727f, 729t, 753 Kornberg, Arthur, 236 Kovach, Janet, 746f Krebs, Hans, 32, 489, 491 Krulwich, Terry, 526–527, 526f Kurtzman, Cletus, 253f Kuru, 186, 1013t Kustu, Sydney, 574, 574f Kyoto Protocol, 832
J
L
Jacob, François, 349, 349f Jagendorf, André, 512–513 Jannasch, Holger, 802, 802f Jenner, Edward, 23, 24f, 452, 899 Joint, Ian, 381 Joint genome institute, 298 Jumping genes, 333
Labile toxin (LT), 951, 952, 954 LacI (lactose catabolism regulator), 356f Lac operon. See Lactose operon Lactate, as fermentation product, 488f, 489 Lactate fermentation, 480f, 481, 489 Lactic acid, 472 Lactic acid bacteria, 696 Lactic acid fermentation, 593 Lactobacillales, 680t, 696 Lactobacillus, 696 in cocoa fermentation, 601 in Emmentaler cheese, 595f evolution, 692 in kimchi production, 599 L. acidophilus appearance, 42f probiotic, 871f as probiotic, 179, 696 vaginal microflora, 871 L. helveticus, 481, 595f L. lactis, 42f L. salivarius, 5f lactic acid fermentation, 593 milk fermentation, 595 oral flora, 866 probiotic, 870, 871f as probiotic, 179 vaccine delivery, 872 Lactococcus, 696 bacteriophage infection, 184 efflux pump, 125 vaccine delivery, 872 Lactose fermentation, 130–131, 130f transport and catabolism, 351f Lactose operon, 35, 349–355 activation by cAMP-CRP, 354 components of, 349–350, 350f discovery of, 349
K Kaback, H. Ronald, 123, 124f Kahng, Hyung-Yeel, 499 Kamen, Martin, 551, 554–555, 554f, 571, 774 Kanamycin, 1048, 1049f Kaposi’s sarcoma, 974, 974f, 975, 1006 Karem, Kevin, 1085f Kashefi, Kazem, 28f Kay, Mark, 454 KEGG (Kyoto Encyclopedia of Genes and Genomes), 297 KEGG Pathway Database, 491 Keilin, David, 510, 510f, 511 Kelley, Scott, 658 Kelp, 591, 764, 779, 780f Keratin, 877 Keratinocyte, 877 Ketoconazole, 1060t–1061t Ketone (functional group), A-7t Ketosynthase, 567, 568f Khohara, Har Gobind, 258 Kimchi, 599, 599f Kinesin, A-35f, A-36 Kirby-Bauer assay, 1035–1037, 1035f Klebsiella, 710 eye flora, 866 K. pneumoniae, 572, 574, 574f nitrogen fi xation, 845 nitrogen regulation, 572, 574, 574f Kleckner, Nancy, 333, 333f Kloeckera in cocoa fermentation, 601 grape fermentation, 606
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Lactose operon (continued) glucose repression of, 351, 352f, 353f inducer exclusion, 355 induction of, 350f, 351 Lactose permease, 123, 124f, 350, 350f, 351f LacY protein, 123, 124f LacZ gene in lactose operon, 349–355 recombination and, 319 as reporter gene, 326, 434 Lagging strand, 101, 101f, 238f–239f, 240 Lag phase, 137 Lake, James, 662 Lake Magadi, Kenya, 161, 161f Lambda. See Bacteriophage, lambda Lamellar pseudopod, 781 Laminar flow biological safety cabinets, 173 Laminari, 591 Lancefield, Rebecca, 1071, 1072f Lancefield classification, 1071, 1072f Landfi lls, methanogenesis in, 740, 741f Landsteiner, Karl, 899, 901, 901f Langerhans cell, 877, 930 Laser scanning confocal microscopy, 60–61, 61f Late genes, phage T4, 394 Latent period, 209, 209f Latent state, 939 Late-phase anaphylaxis, 928 Lateral gene transfer, 648 LAT protein, 427 Law of mass action, A-18 LD50 (lethal dose 50%), 939, 940f Leaching, 531, 531f Leader sequence, 362 Leading strand, 101, 101f, 238f–239f, 239–240 Leaf-cutter ant, 666, 667f Leaflet, 82 Leavened bread dough, 594t Leavening, 602, 603, 604 Lectin, 598, 889 LEE (locus of enterocyte effacement), 664, 664f, 665f Leeuwenhoek, Antoni van, 13, 13f, 40 Leghemoglobin, 706, 707f Legionella growth factors and natural habitat, 116t immune avoidance, 888 L. pneumophila, 711–712, 711f within amebas, 780
SFMB_index.indd I-28
fluorescent antibody staining, 1079f growth in macrophages, 969 identification procedures, 1080t parasitism, 799t respiratory tract infection, 990t–991t in water sources, 658 Legionellales, 681t, 711 Legumes, nitrogen fi xation for, 706, 707f, 820–822, 820f–823f, 845, 845f Leguminous plants, 120, 121f Leishmania (leishmaniasis), 762t, 790f, 791 Lemon, Katherine, 241 Lens aberrations, 49 magnetic, 62, 62 objective, 49, 50, 51, 56 ocular, 50–51 Lentinula edodes, 591 Lentivirus, 210, 413, 413t, 420, 422 LepB, 287, 287f Leprosy, 701, 702f Leptospira (leptospirosis), 716 L. interrogans, 1022t appearance, 42f identification procedures, 1080t meningitis/encephalitis, 1012t Leptospirillum, 714 Leptothrix, 636, 636f Lethal factor (LF), 957 Leuconostoc heterolactic fermentation, 593 L. mesenteroides in kimchi production, 599 in sauerkraut production, 599 Leukemia virus, as gene vector, 896 Leukotriene, in type I hypersensitivity, 927, 928 Leviviridae, 195t Leviviruses, 201 LexA protein, 331, 332f LF (lethal factor), 957 Li, Changsheng, 740 Lichen, 666, 760t, 778, 798–799, 798f, 799f, 818, 819f Liebl, Wolfgang, 721f Life, origin of. See Origins of life Ligand, regulator binding to, 346–347
Light information carried by, 44–45 interaction with object, 45, 45f photons, 45 speed of, 44 wavelength, 44–45 Light chain, antibody, 902, 902f, 903, 911, 913 Light microscopy, 40, 42, 42f, 43, 43f, 44–55 Light reaction, 536 Light scattering, 56 Lignin decomposition of, 458, 479, 528, 815, 815f defi nition, 815 re-cycling by aromatic catabolism, 496 structure, 476f, 815f Limiting nutrient, 811 Lincosamides, 1045 LINE (long interspersed nuclear repeat), 422 Linezolid, 983, 1044f, 1045 Linné, Carl von (Carolus Linnaeus), 29 Lipid amphipathic, A-15 archaeal, 723–724, 723f catabolism of, 479 hydrophobic, A-14–A-15 structure, 476f, A-14–A-15, A14f–A-15f substrates for biosynthesis of, 548, 549f Lipid A, 95f, 958–959 Lipmann, Fritz, 489, 491 Lipopolysaccharide (LPS), 76, 94–95, 95f, 684 complement activation by, 889, 890f, 925 endotoxin, 948, 958–959, 958f, 959f Toll-like receptor binding to, 877–878 Lipoprotein, 94, 95f Liposome, 513 Lipothrixviridae, 194t Liquid nitrogen, 173 Lister, Joseph, 25, 176, 696 Listeria, 696, 697f immune avoidance, 888 interferon response to, 887 L. monocytogenes cold-resistance, 153, 173 food-borne illness, 611, 611f, 612t identification procedures, 1080t meningitis, 1012t pathogenesis, 696, 697f movement inside host cell, 969
origin of name, 696 phagocytosis, 885 List of prokaryotic names with standing in nomenclature, 647 Lithosphere, 632, 633f Lithotroph/lithotrophy, 11, 11f, 463t as assimilatory metabolism, 797 Beta Proteobacteria, 708–709 in carbon cycle, 836, 836f carbon dioxide fi xation, 550 defi nition, 26, 118, 507 Gamma Proteobacteria, 709 hydrogenotrophy, 532, 532f, 532t iron cycle and, 850, 851f metal oxidation, 530–531, 531f nitrogen oxidation, 529 in ocean floor habitat, 808 Proteobacteria, 704, 705t sulfur oxidation, 530–531, 530f, 531f Littoral zone, 810 Live-dead stain, 131, 132f Lobaria pulminaria, 799, 799f Lobosea, 761t Locus of enterocyte effacement (LEE), 664, 664f, 665f Logarithm, A-16 Logarithmic (log) phase, 137 Long interspersed nuclear repeat (LINE), 422 Long terminal repeat (LTR), 420 Lon protease, 331 Lon protein, 291 Lophotrichous, 110 Losick, Richard, 218–219, 218f, 367 Loss-of-function mutation, 321 Lovley, Derek, 468, 506, 506f Lowenstein-Jensen agar, 1074, 1075f LPS. See Lipopolysaccharide (LPS) LTLT (low-temperature/longtime) pasteurization, 173 LTR (long terminal repeat), 420 Luciferase, 378, 379f Lumbar puncture, 1082f Lumen, thylakoid, 538 Luminol, 438 Lung, physical barriers to infection, 878 Lung infection. See Respiratory tract, infection Luria Bertani medium, 117t
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Lwoff, André, 198, 349, 349f Lyme disease, 715f, 716, 1019–1021, 1020f, 1022t emergence and spread of, 1094 as endemic disease, 1086 Koch’s postulates and, 22 Lymph node, 876, 876f Lymphocyte. See also B cell; T cell appearance, 874f in lymphoid organs, 876 transmission electron micrograph, A-21f Lymphoid organ, 876–877, 876f Lyophilization, 173, 613 Lysate, 80, 209 Lysis, A-28 by detergent, 79 osmotic pressure and, 83 Lysogen, 373–374 Lysogeny, 199f, 200, 373, 373–374 Lysol, 176t Lysosome description, A-32 function, A-32, A-32f phagosome-lysosome fusion, 875f, 885 Lysozyme, 79, 394, 724 Lytic cycle, 199–200, 199f, 373
M M13. See Bacteriophage, M13 MAC. See Membrane attack complex (MAC) MacCarty, Maclyn, 304 MacConkey medium, 130–131, 130f, 1070 MacElroy, R., 150 MacLeod, Colin, 304 Macnab, Robert, 56, 110, 110f Macrocystis, 591 Macrolide, 1044 Macromolecule, 33, 76, 77–78 Macronucleus, 784, 785, 786, 786f Macronutrient, 116–117 Macrophage activation, 924 alveolar, 878, 1015 antigen-presenting cells, 876 in chronic inflammation, 883, 885f description, 875 growth of bacteria within, 969, 970f Mycobacterium-infected, 924, 924f phagocytosis by, 875, 875f release of inflammatory mediators, 882
SFMB_index.indd I-29
Salmonella survival in, 164 scanning electron micrograph, 863f, 875t Mad cow disease, 185–186, 1013, 1013t, 1094 Maedi-Visna virus, 413t Magnesium, ATP and, 469–470, 469f Magnetic resonance imaging (MRI), 475 Magnetite, 28, 28f, 107, 107f Magnetosome, 107, 107 Magnetospirillum gryphiswaldense, 107 Magnetotaxis, 107 Magnification defi nition, 41 empty, 46 refraction and, 45–46 resolution, 46–48 total, 51 Major histocompatibility complex (MHC), 887, 898 allotypic, 933 antigen binding to, 898 class I, 915–916, 916f, 917f, 921–922 class II, 888, 915–916, 916f, 917f, 921–922 restriction rule, 923 role in immune system, 915–916, 917f T cell differentiation between classes, 921–922 in transplantation rejection, 933, 933f Major histocompatibility complex (MHC) restriction, 923 Malachite green, 54 Malaria description, 1015, 1017 endemic areas, 1016f immunity, 1017 Malate, 493, 494f, 495 MalE protein, 78f Malolactic fermentation, 606–607 Malonyl-CoA, 564, 565f Malting of barley, 606, 606f, 607f Maltose, A-12f Maltose-binding protein (MBP), 287, 440 Maltose porin, 198, 198f Malyl-CoA, 562, 563f, 564 Manganese aerobic and anaerobic metabolism, 797t microbial metabolism, 852t reduction of, 528 Mannose, A-11f Mantle, Earth’s, 632, 633f
Mapping, of marine phytoplankton, 804, 805f, 806 Mapping gene position by conjugation, 308f, 309 Marburg virus, 939, 940f March of Dimes, 35–36, 401 Margulis, Lynn, 30, 30f, 670, 670f, 756 Marine crenarchaeotes, 735–736, 736f Marine microbiology, 801–809 current challenges of, 3 food webs, 806, 807f habitats, 801–803, 801f ocean floor, 808–809, 808f plankton, 803–804, 803t, 804f–805f, 806 Marine snow, 803, 804f Marine viruses, 213–215, 213f, 216f Mars, 855–856, 855f Mars Exploration Rovers, 6, 6f Marshall, Barry, 22–23, 40, 713, 995, 995f, 996 Martin, Mark, 65 Mash, 606, 607f Mass action, law of, A-18 Massion, Gene, 733f Mass number, 551 Mast cell bradykinin binding, 883, 884f description, 874–875 electron micrograph, 908f histamine release, 883, 884f IgE and, 908, 927–928 type I hypersensitivity, 927 Matrix proteins, 406, 407f Matthaei, Heinrich, 258, 258f Maximum likelihood, 653, 654 Maximum parsimony, 653 Mazel, Didier, 338f MBP (maltose-binding protein), 287, 440 McClintock, Barbara, 333 M cell, 878, 878f McInerney, Michael, 468 m-diaminopimelic acid, 89, 90f, 94, 95f Mean generation time, 136 Mean growth rate constant, 136 Measles, 183, 183f, 202f, 203, 984t, 985 Measles, mumps, and rubella (MMR) multivalent vaccine, 985, 1024t–1025t Meat, spoilage of, 609, 610t Mechanical transmission, of viruses, 206 Mechanosensitive channel, 157
I-29
Media complex, 130 composition of common, 117t defi ned minimal, 117 differential, 130 enriched, 130 MacConkey, 130–131, 130f nutrient-poor, 169 selective, 130 synthetic, 130 Medical microbiology, 17–26 Mefloquine, 1017 Megasphaera elsdenii, 827 Mekalanos, J., 965 Membrane. See also Cell Membrane evolutionary origin of, 641–642 of thermophiles, 155 Membrane attack complex (MAC), 889, 925–926, 925f Membrane-permeant weak acid, 83–84, 84f, 159, 159f Membrane-permeant weak base, 83–84, 84f Membrane potential, 120 Membrane protein, 82–83, 82f, 84–85, 84f Memory B cell, 908, 909, 913–914 Memory T cell, 930 Meningitis case history, 1007, 1037–1038, 1083 defi nition, 1007 Haemophilus influenzae, 1069 meningococcal, 1007–1008, 1007f Meningococcal meningitis, 1007–1008, 1007f Meningococcal vaccine, 1024t–1025t 7-mercaptoheptanoylthreonine phosphate, 742f Mercury contamination, 853 conversions in the environment, 853f microbial metabolism, 852t Mercury chloride, 176t Merismopedia, 679t, 689, 690f Merodiploid, 309 Merozoite, 788f, 789, 789f, 790, 1015, 1017 Mesocosm, 834 Mesophile, 153 archaeal, 726, 727, 728t defi nition, 151t Mesorhizobia loti, 304, 340 Messenger RNA (mRNA). See also Translation coupled transcription and translation, 282, 282f
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Messenger RNA (mRNA) (continued) description, 266 Northern blots and, 436–437, 436f properties, 266t ribosome-binding site, 275 stability, 266, 348 translation, 100, 100f translation initiation sequences, 348 Metabacterium polyspora, 695, 695f Metabolic activity, as biosignature, 856 Metabolism archaeal, 724, 725f of fi rst cells, 638–639, 638f genomic analysis, 498f, 499 vectorial, 512 Metabolist model, 641, 642, 642f Metagenome, 479, 707 Metagenomic analysis, of marine microbial DNA, 806 Metal oxidation, 530–531, 531f Metal reduction, dissimilatory, 528–529 Metals, microbial metabolism of, 852t, 853–854 Metaphase, A-30, A-31f Metastatic lesion, 989 Metazoa, 760t, 758 Metchnikoff, Elie, 179 Meteor bombardment, of early Earth, 633 Meteorites amino acids in, 854 panspermia hypothesis and, 646 Methane covalent bonds, A-2, A-4f as greenhouse gas, 810, 818 methanotrophs, 706 molecular models of, A-6f oxidation, 533, A-20, A-20f Methane gas hydrates, 742, 742f Methanobacteriales, 738 Methanobacterium M. thermoautotrophicum, 738 characteristics, 739t structure, 739, 739f structure, 739 Methanobrevibacter in human digestion, 741, 742 M. ruminantium, 738, 739t M. smithii, 741, 742 Methanocaldococcus. See Methanococcus Methanococcus cell wall, 740 M. jannaschii, 154f
SFMB_index.indd I-30
characteristics, 739t classification, 647 genome size, 224t genomic analysis, 304 initiation factors, 276 reductive acetyl-CoA pathway, 562 structure, 738 M. vaniellii, 739t Methanoculleus M. nigri, 729t M. olentangii, 739t Methanofuran, 742f, 743 Methanogen, 532, 738 associated with periodontal disease, 723 cell wall, 724 of early Earth, 643 examples, 739t habitat, 721, 723, 740–742, 741f histones, 726 metabolism, 724, 738, 742–743, 742f, 743f phylogeny, 727, 729t reductive acetyl-CoA pathway, 561 ruminant, 827, 827f structure, 738–740, 739f, 740f Methanogenesis, 463t, 532, 724 biochemistry cofactors, 742f, 743 methanogenesis from acetate, 743 methanogenesis from carbon dioxide, 743, 743f bovine, 741, 741f by early microbes, 639 greenhouse effect and, 643 in human digestive tract, 741–742 pathways, 533, 533f, 738 reverse, 751 in soil and landfi lls, 740 in wetland soil, 818 Methanohalophilus zhilinae, 739t Methanomicrobium, 740 Methanopyrus, 738 M. kandleri, 739t Methanosaeta, 740, 740f M. concilii, 739t Methanosarcina cell wall, 739–740 M. barkeri characteristics, 739t methanogenesis, 466 structure, 738, 739f M. mazei, 729t M. thermophila, 291 Methanosphaera stadtmanae, 741
Methanospirillum hungatei, 739, 739t Methanothermus fervidus, 738 characteristics, 739t histones in, 726, 726f structure, 738, 739f Methanotroph, 706 Methanotrophy, 533 Methemoglobinemia, 846 Methicillin, 1039 Methicillin-resistant Staphylococcus aureus (MRSA), 697, 983, 1030, 1039 Methionine, fermentation of, 481, 481f Methionine deformylase, 283 Methylbacterium, 658 Methylene blue, 52, 53f 2-methylhopane, 637f, 638 Methylmercury, 853, 853f Methyl mismatch repair, 327–328, 327t, 328f Methylotrophy, 706 Metronidazole activation of, 1042, 1043f Giardia lamblia, 997 mechanism of action, 1042 for necrotizing fasciitis, 984 for trichomoniasis, 1006 MHC. See Major histocompatibility complex (MHC) MIC (minimal inhibitory concentration), 1034, 1034f, 1035f, 1036, 1036f Micelle, 641 Miconazole, 1060t–1061t Microaerophile, 151t, 165t, 167 Microarray, DNA, 150, 151f, 382–383, 382f Microbe classification, 29 defi nition, 7 genome sequencing, 9–11, 9f, 10f human history and, 11–17, 12f, 13f, 14t–15t, 16f shapes, 42, 42f size, 7–8, 8f, 8t, 41–42, 41f Microbial Biorealm, 677 Microbial communities, 8 Microbial ecology, 26–29 Microbiology fields of, 36t golden age, 11 Microchip, DNA, 382–383, 382–383, 382f Micrococcaceae, 703 Micrococcus M. luteus, 680t, 703, 703f M. tetragenus, 103, 103f Microcolony, 128, 129f Microfi lament, A-35–A-36, A-35f
Microflora location and composition, 864–871 eye, 865f, 866 genitourinary tract, 865f, 866t, 870–871 intestine, 865f, 866t, 869–870, 869f, 870f nasal cavity, 865f, 866–867, 867f oral cavity, 865f, 866–867, 866t, 867f overview, 864, 865f respiratory tract, 867–868, 868f skin, 864, 865f, 866, 866f, 866t stomach, 865f, 868, 869f risks and benefits of, 871–872, 872f Microfossil, 18, 18f, 635–637, 635t, 636f, 637f, 856 Micrographia (Hooke), 13, 13f Micronucleus, 784, 785, 786, 786f Micronutrient, 117, 833 Microplankton, 803, 803t Microscope, 40 history of early, 13, 13f, 16 Hooke’s, 13 Leeuwenhoek’s, 13, 13f, 40 Microscopy artifacts, 65, 65f atomic force (AFM), 39f, 43, 43f, 65, 67, 67f bright-field, 43, 48–55 confocal, 60–61, 61f dark-field, 45, 55–56, 55f, 56f electron, 62–67 cryoelectron microscopy, 64–65, 66, 66f scanning electron microscopy (SEM), 40, 40f, 42, 42f, 43, 43f, 62, 63f, 64, 64f transmission electron microscopy (TEM), 43, 43f, 62, 63f, 64, 64f of eukaryotic cells, 42, 42f fluorescence, 58–60, 58f, 59f, 60f interference, 57f, 58 light, 40, 42, 42f, 43, 43f, 44–55 magnification, 41, 45–46 phase-contrast, 56–58, 57f of prokaryotic cells, 42, 42f resolution, 43f scanning probe (SPM), 65 types, 43, 43f Microsporidia, 760t, 758, 759f, 791 Microtome, 63
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Microtubule, 764, 764f, A-35f, A-36 Microviridae, 194t Milk fermentation, 595 spoilage, 609, 610t Miller, Stanley, 18, 18f, 19, 641 Minimal inhibitory concentration (MIC), 1034, 1034f, 1035f, 1036, 1036f Mimiviridae, 194t Mimivirus, 8, 184–185, 185f, 424–425 Mine drainage, 850, 851f Mineral deposits, as biosignature, 856 min gene, 104, 104f Min protein, 105 Minus (–) sense singlestranded RNA viruses, 192 Mismatch repair, 327–328, 327t, 328f Miso, 599 Missense mutation, 321 Mitchell, Peter, 33, 468, 511, 512, 512f, 513 Mite, dust, 928 Mitochondria ATP production, A-37 description, A-37 electron transport system, 509–510, 509f, 522–523, 522f endosymbiosis theory, 30, 30f, 670, 670f, A-37, A-38f gene similarities to rickettsia, 298 genome, 670–671, 671f origin of, 757 as rickettsial descendents, 707 Mitosis, 134, A-30, A-31f Mitosporic fungi, 767 Mixed-acid fermentation pathway, 488f, 489 Mixotricha, 826 M. paradoxa, 800, 800f Mixotroph, 806, 807f, 852 Mixotrophy, 121, 778 MLVs (mouse leukemia viruses), 448 M9 medium, 117t MMLV (Moloney murine leukemia virus), 413t MMTV (mouse mammary tumor virus), 413t Mobile genetic element, 333–336 Mobile symbiosis island, 340 Mobility shifts, DNA, 438, 438f438 Moist heat, 172 Molarity, A-16
SFMB_index.indd I-31
Mold, 772, 772f Mold-ripened cheese, 596 Molecular clock, 30, 652–653, 652f Molecular detection methods, 1072–1076 DNA-based, 1073t DNA fi ngerprinting, 1074, 1075f, 1076 polymerase chain reaction (PCR), 1072–1074, 1073t, 1074f, 1076, 1077f Molecular formula, A-2 Molecule. See also Biological molecules bonding, A-2–A-5, A-4f, A-5t organic, A-5–A-6 visualizing, 68–70 Mollicutes, 680t, 698–699, 698f Moloney murine leukemia virus (MMLV), 413t Monera (kingdom), 29 Monocyte, 874f, 875 Monod, Jacques, 349, 349f Monophyletic, 30 Monophyletic group, 651–652 Monosaccharide, A-11, A-11f Monosodium glutamate (MSG), 591, 599 Monotrichous (polar), 110 Montagnier, Luc, 413, 414, 414f Montagu, Lady Mary, 23, 24f Monterey Bay Aquarium Research Institute, 733, 733f Morchella hortensis, 769, 770f Mordant, 52 Morel, 769, 770f Morowitz, Harold, 642, 642f Mother cell, 144 Motherspore, 693 Motility gliding, 144, 960 twitching, 141, 948, 960 Motor protein, A-35f, A-36 Mouse leukemia viruses (MLVs), 448 Mouse mammary tumor virus (MMTV), 413t Movement protein, 207 Moyle, Jennifer, 33, 512, 512f MreB proteins, 105, 105f MRI (magnetic resonance imaging), 475 mRNA. See Messenger RNA (mRNA) MRSA (methicillin-resistant Staphylococcus aureus), 697, 983, 1030, 1039 MS2 bacteriophage, 195t, 201
MSG (monosodium glutamate), 591 Mucociliary elevator, 868, 868f, 944 Mucor, 769, 769f, 984t Mucous membrane, as barrier to infection, 877–878, 878f Mucus, intestinal, 870 Mueller-Hinton agar, 1037 Multidrug resistance (MDR) efflux pumps, 1049–1050, 1049f, 1050f Multiple sequence alignment, 296f, 297 Multiplex polymerase chain reaction (PCR), 445–446, 446f, 1073, 1074t Multiplicity of infection, 374–375 Multivalent vaccine, 987 Murchison meteorite, 576f Murein, 89 Murein lipoprotein, 94, 95f Murray, Alison, 736, 737f Murray, Andrew, 219 Mushroom, 771–773 appearance, 7f edible, 590–591, 591f fairy ring, 756, 757f, 773 fruiting body, 771f life cycle, 773f Mutagen defi nition, 323 identification of, 325–326, 326f table of, 322t Mutagenesis, signaturetagged, 966–967, 966f–967f Mutation Ames test as mutagenicity screen, 325–326, 326f causes of, 322–324, 322t, 323f, 324f conditional lethal, 396, 397 defi nition, 320 divergence and, 652 frameshift, 321f, 322 frequency, 324 gain-of-function, 321 genetic analysis, 81–82, 81f genotype and phenotype effects of, 322 hypermutation of immunoglobulin genes, 913, 914 insertion, 321, 321f, 322 knockout, 322 loss-of-function, 321 missense, 321 nonsense, 321, 321f, 396–397
I-31
point, 321, 321f random, 652–653 rate, 324–325 silent, 321 spontaneous, 323 suppressor, 397 temperature-sensitive, 396 transition, 321 transposon mutagenesis, 432–433, 433f transversion, 321 Mutation frequency, 324 Mutation rate, 324–325 molecular clocks and, 652–653 Mutator strain, 328 Mut proteins, 327–328, 328f mutS gene, 332 Mutualism, 666, 668, 691, 700, 798–800, 798f–800f, 799t Mycelia, 145, 145f, 700 Mycelium, 765 Mycena, 813, 815f Mycetozoa, 761t Mycobacterium, 700–702 growth factors and natural habitat, 116t growth within macrophages, 924, 924f immune avoidance, 924, 924f M. avium-intracellulare, 1006 M. bovis, 924f, 930 M. leprae acid-fast stain, 54 cell wall, 92 genome, 701, 702f leprosy, 701, 702f noncoding DNA, 225 M. marinum, 884, 885f M. paratuberculosis, 64, 64f M. smegmatis, 702 M. tuberculosis acid-fast stain, 54, 55f, 1074, 1075f antibiotics against, 616–617, 617f appearance, 700f BCG vaccine, 616 cell wall, 92, 701f chronic inflammation from, 883–884, 885f diagnostic test, 617 disease surveillance, 1090 envelope lipids, 92f fatty acids, 86, 87 genome, 701 genome size, 224t Ghon complex, 989, 989f human history and, 12 on Lowenstein-Jenen agar, 1075f meningitis, 1012t
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In d ex
Mycobacterium (continued) M. tuberculosis (continued) molecular detection methods, 1072, 1073t, 1074, 1075f multiple-drug-resistant (MDR), 26, 989, 992, 1050 mycolic acids, 564, 700 pasteurization and, 173 reemergence, 1095 respiratory tract infection, 990t–991t Robert Koch and, 21 treatment, 1033 tuberculosis, 988, 989, 1095 M. ulcerans, 701 phagocytosis, 885, 886f Mycolic acid, 92, 92f, 93f, 700 Mycology, 756 Mycoplasma appearance, 698f attachment mechanism, 945t description, 698–699 genome, 699 genome size, 223 M. genitalium appearance, 680t genome size, 223, 224t, 297 scanning electron micrograph, 297f M. mobile, 699 M. mycoides, 294, 294f M. penetrans, 699 M. pneumoniae respiratory tract infection, 990t–991t meningitis/encephalitis, 1012t reductive evolution, 424 Mycorrhizae, 813, 815–817, 816f, 817f vesicular-arbuscular, 769, 817 in wetlands, 838 Mycoses, 1059 Myeloperoxidase, 886 Myocarditis, 1015 Myoviridae, 194t Myr (megayear), 631 Myrothecium, 179 Myxobacteria, 712, 712f Myxococcales, 682t Myxococcus appearance, 682t gliding motility, 960 M. xanthus, 712, 712f fruiting body formation, 144, 145f type IV pili, 948 Myxosarcina, 689, 690f Myxospore, 690t, 712, 712f Myxovirus, 213
SFMB_index.indd I-32
N N-acetylglucosamine (NAG), 89, 90f, 1038, A-12f N-acetylmuramic acid (NAM), 89, 90f, 1038 Nacy, Carol, 616, 616f NAD, 119, 1069, 1069f NADH, 471–472, 472f generated in EntnerDoudoroff pathway, 482, 486, 486f generated in glycolysis, 482, 482f, 483, 483f, 484, 484f generated in TCA cycle, 493, 494f, 495 measuring concentration with NMR, 475, 475f strength as electron donor, 508 NADH dehydrogenase, 519, 521, 521f, 522, 522f, 524 NADH dehydrogenase (NADH:quinone oxidoreductase, NDH-1), 518–520, 518f, 521f NADH dehydrogenase II, 166 NADPH, 472–473 generated in Entner-Doudoroff pathway, 482, 486, 486f pentose phosphate shunt, 482f, 486–487, 487f use in benzoate catabolism, 500, 500f Calvin cycle, 552–553, 553f 3-hydroxypropionate cycle, 562, 563f NADPH oxidase, 886 Naegleria, 997, 1012t Na+/H+ antiporter, 123–124, 124f, 162, 163, 163f Nalidixic acid, 1042, 1070 Nanarchaeota, 727, 727f, 729t Nanoarchaeum, 627f, 727 N. equitans, 627f, 729t, 753, 753f Nanobacterium, 65, 65f Nanoplankton, 803, 803t Nanotechnology, 85, 85f Nanotube, 1055, 1055f Nanowires, 189, 397 NAP (neutrophil-activating protein), 967, 968f Naphthalene, 461f Naqvi, Syed Wajih, 846 Narcissus, 918 Nasal cavity, microflora of, 865f, 866–867, 867f Nasopharynx, 867, 867f
National Center for Biotechnology Information (NCBI), 11 National Center for Biotechnology Information (NCBI) Taxonomy Database, 677 National Institutes of Health, 36 National Science Foundation (NSF), 28, 36 Native conformation, A-9 Natronobacterium, 747 N. gregoryi, 161, 161f, 745t Natronococcus, 727 N. occultus, 745t Natronomonas pharaonis, 745t Natto, 593, 600–601, 602f Natural killer cell, 887–889, 889f, 924 Natural selection, 652 NdhF4 transporter, 558–559, 559f Nebula RCW-49, 857, 857f Necrotizing fasciitis, 983–985, 984t, 985f Nectin, 427 Nef protein, HIV, 416t, 422 Negative factor (NEF), 973, 974t Negative selection, 918 Negative stain, 54–55, 55f, 64, 93 Neidhardt, Fred, 77, 77f, 169 Neisseria, 709 horizontal gene transfer, 661 N. gonorrhoeae, 709 antigen phase variations, 926 appearance of, 681t, 1004f attachment mechanism, 945t chocolate agar, 1066f complement and, 926 dissemination of, 980–981 gonorrhea, 1004–1005 immune response to, 873 iron transport, 125 nitrate reduction, 527 Opa adhesin, 948 penicillinase-producing (PPNG), 1064 penicillin resistance, 1064 phase variation, 371–372, 1005 pili, 947 transformation, 305 N. lactamica, 663 N. meningitidis, 709 capsule, 970, 1008 distinguished from N. lactamica, 663
endotoxin, 959, 959f meningitis, 1007–1008, 1007f, 1012t oxidase reaction, 1069f, 1070 pathogenesis, 959 petechial rash, 959, 959f pili, 947, 947f transcytosis, 1007 vaccine, 1008 N. sicca, 709 oral flora, 866 oxidase test, 1070 transformation, 305, 306 twitching motility, 960 Neisseriales, 681t Nelfi navir, 1054t Nematodes endosymbionts of, 668, 668f soil, 813, 814f Neocallomastix, 768, 768f NER (nucleotide excision repair), 327t, 328, 329f Nervous system infections. See Central nervous system infections Neuraminidase, 406, 407f, 408f, 409, 1056, 1057 Neuraminidase inhibitors, 1056, 1056f Neurospora, 767, 769 Neuston, 801, 801f Neutralophile, 151t, 160 Neutron, A-2, A-2t Neutrophil alpha defensins in, 879 appearance, 874f in bacterial pneumonia, 986–987 description, 874 extravasation, 880, 882–883, 882f, 883f Neutrophil-activating protein (NAP), 967, 968f Nevirapine, 1054t Newborn, bacterial succession in digestive tract of, 676, 676f Newman, Dianne, 639, 640f Newton, Isaac, 12 NF κβ (nuclear factor kappa beta), 974 N-formylmethionine (f-Met), 283 Ng, Wailap, 748 Niccol, Andrew, 250 Niche, 151, 794–795 Nicotinamide adenine dinucleotide. See NAD; NADH nif genes, 574, 574f Nightingale, Florence, 12, 12f Nikkomycins, 765 Nirenberg, Marshall, 258, 258f, 267
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Nitrate, 843 in drinking water, 845f, 846 in nitrogen cycle, 120–121, 120f production, 845 as terminal electron acceptor, 472 Nitrate reductase, 526 Nitrate reduction assimilatory, 845 dissimilatory, 525–527, 846, 847 Nitric oxide (NO), 570, 886 Nitric oxide synthetase, 886 Nitrification, 120, 120f, 570, 845–846, 845f Nitrifier, 26, 529, 708, 708f, 845 Nitrifying bacteria, 120 Nitrite, 843 blue baby syndrome, 846 for food preservation, 615 production, 845 Nitrite reductase, 526 Nitrobacter, 681t N. winogradskyi, 844f in nitrification process, 845 Nitrogen aerobic and anaerobic metabolism, 797t ammonia-oxidizing crenarchaeotes, 737 from anthropogenic sources, 832 assimilation, 570, 570f atmospheric, 633, 843 eutrophication by, 811 formation in stars, 631, 632f gas, 120–121, 120f global reservoirs, 833t, 834 isotope, 834 liquid, 173 oxidation, 526–527, 529, 834, 834t, 842 rate of cycling, 833t regulation, 376–377, 377f stability, 570 Nitrogenase, 144, 459, 571, 572, 572f, 573f, 844 Nitrogen cycle, 27f, 120–121, 120f, 842–847, 843f–846f denitrification, 846, 846f dissimilatory nitrate rreduction, 847 nitrification, 845–846, 845f nitrogen fi xation, 844–845, 845f nitrogen sources, 843 nitrogen triangle, 843–844, 844f Nitrogen fi xation, 27, 570–575, 844–845, 845f Alpha Proteobacteria, 706, 707f
SFMB_index.indd I-33
anaerobiosis and, 572, 573f, 574 assimilation of nitrogen forms, 570, 570f by cyanobacteria, 688, 689 early discoveries of, 570–571, 570f energy and oxygen regulation during, 823f by Frankia, 700 Haber process, 571 heterocysts, 144, 144f mechanism, 571–572, 572f, 573f mutualism, 666 in nitrogen cycle, 120, 844–845, 845f regulation of, 574–575, 574f by rhizobia, 820–822, 820f–823f by Rhodopseudomonas palustris, 459f Nitrogen-fi xing bacteria, 120, 120f Nitrogenous bases, in DNA, 226–227 Nitrogen triangle, 843–844, 844f Nitrosamines, 846 Nitrosococcus, 708 Nitrosolobus, 708 Nitrosomonadales, 681t Nitrosomonas, 708 N. europa, 708f in nitrification process, 845 Nitrosopumilales, 737 Nitrosopumilus maritimus, 737 Nitrosovibrio, 708 Nitrospira, 714 appearance, 682t in nitrification process, 845 Nitrospirae, 682t, 684, 714 Nitrous oxide, 846, 846f NK (natural killer) cell, 887–889, 889f, 924 Nobel Prize, 10 Nocardia, 702 N. lactamdurans, 1047f in wastewater treatment, 841, 841f Nod genes, 820f, 821 Nomenclature, 29, 647 Nonadaptive immunity, 873. See also Innate immunity Nonculturable organism, 118. See also Viable but nonculturable (VBNC) Nonpolar covalent bond, A-4 Nonribosomal peptide antibiotics (NRPS), 579–580, 584–585, 584f–585f
Nonsense mutation, 321, 396–397 Nonsense suppressor, 397 Nori, 591, 591f, 778, 778f Normal flora, 869 Norovirus, 611, 612t, 998t Northern blot, 436–437, 436f Norwalk-like virus, 611, 612t Norwalk virus DNA-based detection test, 1073t vaccine in transgenic plants, 454t Nosocomial infection, 1030 Nostoc, 119f, 688, 688f, 689, 689f Nostocales, 679t Notifiable diseases, 1087, 1088t Novobiocin, 1072 Novozymes, 617 NPC (nuclear pore complex), A-29 NRPS (nonribosomal peptide antibiotics), 579–580, 584–585, 584f–585f NSF (National Science Foundation), 28, 36 N-terminal rule, 291 NtrB, 376–377, 377f NtrC, 376–377, 377f, 574, 574f, 577 Nuclear factor kappa beta (NF κβ), 974 Nuclear magnetic resonance (NMR) spectroscopy, 43, 68 distinguishing sugars by, 477f for observation of energy carriers in living cells, 475, 475f Nuclear pore complex (NPC), A-29 Nucleic acid. See also DNA; RNA content of E. coli, 78 structure, A-12, A-12t, A-13f, A-14, A-14f Nucleocapsid proteins, 407, 407f Nucleoid, 74f–75f, 76, 98–99, 98f–99f, 228, 229f Nucleolus, A-29 Nucleomorph, 671, 671f, 763 Nucleoside, A-12, A-12t Nucleoside analog, 1056–1057, 1057f Nucleosome, 247 Nucleotide, 226, A-12, A-13f, A-14 Nucleotide, dideoxy, 252, 252f Nucleotide excision repair (NER), 327t, 328, 329f
I-33
Nucleus, A-29, A-30f Numerical aperture, 49–50, 49f Numerical prefi xes, A-16, A-16t Nurse, Paul, 766 Nutrient deprivation, microbial response to, 168–170, 168f, 169f essential, 116 growth factor, 116t, 117 macronutrient, 116–117 micronutrient, 117 pollution, 169, 170f Nutrient uptake, 121–127 Nutrition, 116–121 autotrophy, 118, 119, 119f carbon cycle, 118, 119f chemotrophy, 119–120 heterotrophy, 118, 119, 119f, 121 mixotrophy, 121 nitrogen cycle, 120–121, 120f phototrophy, 119–120 Nystatin, 1059, 1059f, 1060t–1061t
O O antigen, 959, 993 Objective lens, 49, 50, 51, 56 Ocean. See also Marine microbiology barophiles and, 155–156, 156f thermophiles, 154f, 155 Ocean floor habitat, 808–809 Oceanospirilles, 681t Ocular lens, 50–51 O’Donnell, Michael, 237 Oenococcus oeni, 606, 608f Okazaki fragments, 234, 239–240 Oleic acid, 86f Oligosaccharide, A-12 Oligotroph, 169 aquatic and soil, 706 environmental conditions for growth, 797t Oligotrophic lake, 809, 809f Omalizumab, 928 OmpX protein, 78, 78f Oncogene, 206 Oncogenic viruses, 206, 212, 212f O-nitrophenyl galactoside (ONPG), 434 Oomycota (oomycetes), 762t, 763, 773, 774f Opa gene, 371–372 Opa membrane protein, 948 Oparin, Aleksandr, 641 Open reading frame (ORF) annotation of DNA sequence, 294
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Open reading frame (ORF) (continued) defi nition, 264 identifying by computer analysis, 294–295, 295f Operational gene, 662 Operator defi nition, 46 lactose operon, 350f, 351 lambda, 375 trp, 361, 361f Operon archaeal, 725 for aromatic catabolism, 499, 499f cadBA, 479, 479f coordinate regulation, 348 defi nition, 225, 349 lactose, 35, 349–355 str, 81–82, 81f tryptophan, 361–362, 361f, 363f vir, 621, 622f Operon fusion, 434, 435f Ophiostoma novo-ulmi, 823f, 824 Opine, 621, 622f Opisthokonts, 760t, 758, 759f O polysaccharide, 95, 95f Opportunistic pathogen, 871 Aeromonas as, 861 in AIDS, 1006 defi nition, 939 Opsonization, 885, 887f, 904 Optical density, 133, 136–137 Optical isomer, A-8 Optimum temperature, 152 Optochin susceptibility, 1071f, 1072 Oral cavity, microflora of, 865f, 866–867, 866t, 867f Oral groove, 784, 784f ORF. See Open reading frame (ORF) ORFfi nder program, 294 Organ donation, 933 Organelle, A-29, A-32, A-32f Organic acid, effect on internal pH, 159–160, 159f Organic horizon, 812, 812f Organic molecule, A-5–A-6 Organic respiration, 463t Organotroph, 462 Organotrophy, 462, 463t oriC, 234 Origin of replication (ori), 98–99, 99f, 101, 102, 102f, 234 F factor, 307 oriC, 234 oriT, 307, 307f, 308, 309 oriV, 307
SFMB_index.indd I-34
Origins of life, 18–19, 629–647 conditions required for, 631 elements of life, 631–633 elemental composition of Earth, 632–633, 633f formation within stars, 631–632, 632f geological evidence for early life, 633, 634f, 635–638, 635t, 636f–637f Archaean eon, 633, 635 biosignatures, 635t, 637, 637f Hadean eon, 633 isotope ratios, 635t, 637–638, 646, 646f microfossils, 635–637, 635t, 636f, 637f stomatolites, 629f, 630, 630f, 635, 635ft metabolism of fi rst cells, 635t, 637–638, 638–639, 638f, 646, 646f models for early life, 641–647 metabolist model, 641, 642, 642f panspermia, 646 prebiotic soup, 641–642, 641f RNA world, 641, 642–643, 643f, 644–645, 644f, 645f unresolved questions, 643, 646 oxygenic photosynthesis, 639 timeline for, 640t oriT, 307, 307f, 308, 309 oriV, 307 Ornithine, 577 Ornston, L. Nicholas, 458 Oró, Juan, 18, 18f, 19, 641 Oropharynx, 867, 867f Orthocresol, 177t Orthologous gene (ortholog), 196, 297, 297f, 663 Orthomyxoviridae, 195t Orthomyxovirus, 406 Orthophenylphenol, 176 Oscillatoria, 689, 689f, 691 carbon dioxide fi xation, 550f O. spongeliae, 691f Oscillatoriales, 679t Osmolarity, 157 Osmosis, 83, A-27–A-29, A-28f Osmotic pressure, 83 Osmotic shock, 78, 97, 97f Osmotic stress, 157 Osteomyelitis, 1064 Osterholm, Michael T., 1090, 1091 Ostreococcus tauri, 764
Ototoxicity, of aminoglycosides, 1044 Outer leaflet, A-24 Outer membrane, 76, 94–96, 95f–97f Outgroup, 656 Oxaloacetate, 491, 493, 494f, 495, 496 amino acid biosynthesis, 575, 575f, 577 as biosynthesis substrate, 548, 549f Oxazolidinone, 1045, 1052f Oxidase test, 1069f, 1070 Oxidation-reduction (redox) reaction, A-19–A-20, A-19f–A-20f of early life-forms, 638 electron transport system and, 471–472 reduction potential, 507–509, 508t Oxidative burst, 886 Oxidative phosphorylation, 495 Oxidative respiration, A-37–A-38, A-38t Oxidoreductase, 510, 518, 522, 524, 525, 525f, 528, 529, 540 Oxidoreductase protein complex, 518–520, 518f, 519f 8-oxo-7-dehydrodeoxyguanosine, 324, 324f 2-oxoglutarate, 493, 494f, 496 in amino acid synthesis, 575f, 577 as biosynthesis substrate, 548, 549f in reverse TCA cycle, 560, 561f 2-oxoglutarate:ferrodoxin oxidoreductase, 560, 561f Oxygen benefits and risks, 164–165 biochemical oxygen demand (BOD), 802, 839–840, 839f, 840f classification of organisms by relationship with, 165–168, 165t, 166f aerobes, 165t, 166, 166f, 167 anaerobes, 165t, 166–168, 166f facultative, 165t, 166f, 167 microaerophile, 165t, 166f, 167 in Earth’s atmosphere, 633 as electron acceptor, 508, 509, 521, 797 formation in stars, 631, 632f as oxidizing agent, A-20
production by cyanobacteria, 120 reactive oxygen species, 166, 166f as terminal electron acceptor, 164–165, 165f, 472 Oxygenic Z pathway, 540–542, 543f Oxymonadida, 762t Oxytricha fallax, 785, 785f
P p53 protein, 975 PABA (para-aminobenzoic acid), 1031f, 1032, 1036–1037, 1042, 1042f Pace, Norman R., 655, 656f, 658, 753 Packaging proteins, M13, 400 pac sequence, 311 PAD1, 310 Padan, Etana, 123, 124f PAH (polynuclear aromatic hydrocarbon), 500, 501f Palindrome, 249 Palmaria palmata, 761t Palmitic acid, 86f PAMP (pathogen-associated molecular pattern), 877 Pandemic, 1086 Paneth cells, 879–880, 880f, 881 Panspermia, 646 Pap (pyelonephritis adhesion pilus), 945 Paper chromatography description, 551 intermediates of carbon dioxide fi xation, 551–552, 552f Papillomaviridae, 194t Papillomavirus, 423t, 984t appearance, 7f genome, 192 life cycle, 203, 204f structure, 194t tropism, 201 Papovaviridae, 192 Papovavirus, 423t Para-aminobenzoic acid (PABA), 1031f, 1032, 1036–1037, 1042, 1042f Parabasalidea, 762t Paracoccidioidomycosis, 1060t Paralogous genes (paralogs), 297, 297f, 656, 663 Paralysis flaccid, 1008 spastic, 1008, 1009f Paramecium appearance, 43f
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cell structure, 784, 784f contractile vacuole, 97f cortex, 764f endosymbiotic algae within, 666–667, 668f, 670 P. bursaria, 666–667, 668f, 670 size, 41f Paramyxoviridae, 195t Paraphysomonas, 762t Pararetroviruses, 193, 193f, 196t, 207–208, 208f Parasite defi nition, 938 ectoparasites, 938, 938f endoparasites, 938, 939f notifiable diseases, 1088t Parasitism, 666, 799t, 801 Parasporal body, 451, 451f Parfocal, 50 Par protein, 105 Partially purified derivative (PPD), 988, 989, 992 Parvoviridae, 194t Passive diffusion, 83 Passive transport, 85 Pasteur, Louis, 16–17, 16f, 23–24, 25, 25f, 173, 476 Pasteurization, 173, 614 Pathogen classification schemes, 980–981 defi nition, 938 frank, 939 genome sequencing of, 981–982, 982t identification, 1065–1080 algorithm use, 1065–1072 electronic, 1092–1093, 1092f–1093f fluorescent antibody staining, 1079, 1079f gram-negative bacteria, 1066–1068, 1067f–1069f, 1067t gram-positive pyogenic cocci, 1070–1072, 1071f, 1072f by molecular genetics, 1072–1076, 1073t, 1074f–1077f nonenteric gram-negative bacteria, 1068–1070, 1069f overview, 1065 for selected diseases, 1088t selective and differential media, 1066, 1066f by serology, 1077–1078, 1078f intracellular, 969–970, 970f opportunistic, 861, 939 primary, 939
SFMB_index.indd I-35
Pathogen-associated molecular pattern (PAMP), 877 Pathogenesis, 937–976 defi nition, 938 host-pathogen interactions, 938–942 infection cycles, 940, 941f, 942 portals of entry, 942 terminology, 938–939 pathogenicity islands, 942–944, 943f, 944f protein secretion, 959–964 type III secretion system, 960t, 961–963, 961f–963f type II secretion system, 960, 960f, 960t type I secretion system, 960t type IV secretion system, 960t, 964, 964t survival within host, 969–971, 970f extracellular immune avoidance, 970–971 intracellular pathogens, 969–970, 970f virulence, 971 toxins, 948–959 endotoxin, 948, 958–959, 958f, 959f exotoxin, 948–958, 949f, 950t–951t, 951f–953f, 955f–957f identifying new, 958 viral, 971–976 cold viruses, 972 human immunodeficiency virus, 973–975, 974f human papillomavirus, 975, 975f influenza, 972, 973f virulence factors, 942, 944–948, 945t, 946f–948f virulence genes, identifying, 964–969, 965f–967f Pathogenicity, 939 Pathogenicity islands, 339–340, 339f, 611, 613, 613f, 664, 942–944, 943f, 943t, 961, 962, 963 Patient zero, 1086–1087 Pause site, transcription termination, 264–265 Pavlova vivescens, 213, 213f P22 bacteriophage, 311, 312f, 333, 373f PBP (penicillin-binding protein), 983, 1039 pBR322, 243f
PCR. See Polymerase chain reaction (PCR) Pearson, Ann, 737 Pectin, 476f Pediococcus, 696 Pelagibacter, 704 P. ubique, 647, 707 Pelagic habitat, 801, 801f Pellicle, 76, 97 Pelomyxa, 8t Pelvic inflammatory disease (PID), 1003–1004 Penicillin action of, 90 allergy to, 1033 beta-lactamase action on, 1048, 1048f biosynthesis, 1046, 1046f–1047f discovery of, 25, 25f, 177, 1031 as hapten, 901 hypersensitivity reaction, 929 mechanism of action, 1038f, 1039–1040 prophylactic use, 1015 resistance, 1039 selective toxicity, 1032 spectrum of activity, 1033 structure, 177, 177f, 1030f, 1039f as weak acid, 84, 84f Penicillin-binding protein (PBP), 983, 1039 Penicillium antibiotic production, 1059 asexual reproduction, 767 in cheese production, 596, 596f, 597, 609 conidiophores, 770 Hurricane Katrinaassociated mold, 772 P. griseofulvum, 1059f P. notatum, 25, 177, 177f, 1031 P. roqueforti, 596f penicillin discovery, 1031 Pentadecylcatechol, 931f Pentaglycine, 1039 Pentose phosphate shunt (PPS), 482, 486–487, 487f PEP (phosphoenolpyruvate), 126, 127f, 473–475, 473f, 483, 484f, 485 Pepsin, 596 Peptidase, 291 Peptide bond, A-9, A-9f Peptides, phage display of, 448–449, 449f Peptidoglycan, 76, 78 as antibiotic target, 1038f, 1039–1041 in mycobacteria, 92, 93f sacculus, 89f
I-35
structure, 89, 90f, 677, 677f synthesis as a target for antibiotics, 90, 1038–1039, 1038f, 1040–1041 variant forms, 677, 677f Peptidyl-transferase, 274 Peptidyl-tRNA site (P site), 274, 275f Peptostreptococcus intestinal flora, 869 P. anaerobius, 827 Perforin, 888, 889, 889f, 921 Periodic table of the elements, A-2, A-3f Periodontal disease, 867f Peripheral membrane protein, A-22, A-24 Periplasm, 76, 91f, 94, 96 Peritrichous, 109 Permease, 122 Permissive temperature, 104 Pero, Jan, 218 Peroxidase, 166–167, 166f Peroxisome, A-32 Persistence of Memory (painting), 655, 656f Pertactin, 948, 948f Pertussis toxin, 954, 964, 964f PEST sequence, 292 PET (positron-emission tomography), 555 Petechia, 959 Peterson, Johnny, 860 Petichiae, 994 Petri, Richard J., 21 Petri dish, 21 Petroff-Hausser counting chamber, 131, 131f131 Peyer’s patches, 876f, 877, 878, 878f PfEMP1 protein, 1017 PFU (plaque-forming unit), 211 pH classification of organisms by optimal pH, 151t, 159f, 160–162 acidophiles, 160–161, 161f alkaliphiles, 161–162, 161f, 162f neutralophiles, 160 enzyme optima, minima, and maxima, 159 homeostasis, 163–164 microbial response to changes in, 158–164 organic acids effect on internal, 159–160, 159f sensitivity of biological processes to, A-19 values of common substances, 160f as virulence signal, 164
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PHA (polyhydroxyalkanoate), 106, 431, 566f Phaeophyceae, 778–779 Phage. See Bacteriophage Phage display, 398, 448–450, 449f, 450f Phage therapy, 179 Phagocytosis, 97, 127, A-29 images of, 886f immune avoidance, 970 inhibition by Yersinia pestis, 1018 killing mechanisms, 885–886 by macrophages, 874, 875f by neutrophils, 874 oxidative burst, 886 recognition of foreign cells and particles, 884–885 Phagolysosome, 127, 164, 169, 969 Phagosome, 127, 874, 875f, 885–886, 952, 969 Phagosome-lysosome fusion, inhibition of, 969 Phanizomenon, 144f Phase-contrast microscopy, 56–58, 57f Phase variation, 320, 348, 370 in eukaryotes, 373 immune avoidance, 370 Neisseria gonorrhoeae, 1005 slipped-strand mispairing, 371–372, 372f PHB (polyhydroxybutyrate), 106, 709 pH difference, transmembrane, 514–515, 514f, 515f Phenol, 176, 176t, 177t Phenol coefficient test, 176, 176t Phenolic glycolipid, 92, 92f, 93f Phenolics, 176 structure, 177t in wine production, 607 Phenol red broth test, 490, 490f Phenylalanine, biosynthesis of, 577, 578f 2-phenylethylamine, 601, 601f Pheromone, 310 PhoQ, 348 Phosphate eutrophication, 169, 170f, 811 for food preservation, 615 functional group, A-7t Phosphate cycle, 849–850, 850f Phosphatidate, 86 Phosphatidylethanolamine, 82, 83f Phosphatidylglycerol, 86, 86f
SFMB_index.indd I-36
Phosphoanhydride bond, 470 Phosphodiester bond, 226, 226f, 233 Phosphoenolpyruvate (PEP), 126, 127f, 473–475, 473f, 483, 484f, 485 Phosphofructokinase, 485, A-10f 6-phosphogluconate, 482, 483f, 485, 486, 486f, 487f Phosphoglucosamine, 95f Phosphoglucose isomerase, 485 3-phosphoglycerate (PGA) in Calvin cycle, 552–553, 553f, 556, 556f as carbon fi xation intermediates, 552, 552f rubisco and, 556, 556f 2-phosphoglycolate, 556 Phospholipid, 82, 82f, 83f, 86–87, 86f, A-22, A-24, A-26. A-25f Phospholipid bilayer, A-24, A-25f, A-27f 5-phosphoribosyl-1pyrophosphate (PRPP), 579, 580f Phosphorylation, 470, 495 Phosphorylation relay system, 347–348, 348f Phosphotransferase system (PTS), 126, 127f, 353, 353f, 470 Photic zone, 801 Photoautotroph/ photoautotrophy, 118, 119, 463t Photoferrotrophy, 639, 709, 710f Photoheterotrophy, 463t, 477, 534 Chloroflexi, 686, 687t Proteobacteria, 704–706, 705f retinal-based, 747–748, 748f by Rhodopseudomonas palustris, 459f Photolysis, 535 Photon, 45 Photoreactivation, 327t, 328 Photoreceptor, 40, 41f, 45 Photoswitch, 750 Photosynthesis anaerobic, 639 bacteriorhodopsin, 638–639, 638f in carbon cycle, 835, 836f carboxysomes, 106, 106f cyanobacteria, 27 defi nition, 535 by early microbes, 638–639, 638f energy transformation, 539f
nitrogen-based, 846 purple sulfur bacteria, 27 thylakoids, 106, 106f Photosynthetic membrane, 538, 539f Photosystem I, 540, 541, 541f Photosystem II, 540, 541, 541f, 686 Phototaxis, 748 Phototroph/phototrophy, 534–543 in archaea, 724 bacteriorhodopsin, 534–535, 535f carboxysomes, 106, 106f Chloroflexi, 686, 687t cyanobacteria, 687t, 688–692 defi nition, 119, 507, 535 endosymbiotic, 28 facultative, 544 Gamma Proteobacteria, 709, 710f gas vesicles, 106, 106f iron, 639, 640f, 709, 710f photolysis, 534–543 antenna complex, 537–538, 538f defi nition, 534 electron transport system, 538–539 light absorption by chlorophylls, 536–537, 537f overview, 536 oxygenic, 540–542, 543f photosystem I, 540–541, 541f photosystem II, 540–541, 542f reaction center, 538, 539 phycobilisomes of, 106 taxa, key features of, 686, 687t thylakoids, 106, 106f Phycobilisome, 106 Phycodnaviridae, 194t Phycoerythrin, 688, 778 Phylloquinone, 540, 541f Phylogenetic tree, 653, 655–656 archaeal, 727f bacteria, 678f calibration, 656 of eucaryotes, 757f limitations of, 655 maximum likelihood, 653, 654 maximum parsimony, 653 root and unrooted, 655, 656f of sphingomonad bacteria, 659f for three domains of life, 656, 657f
Phylogeny, 651–661 of archaea, 726–727, 727f, 728t–729t bacteria, 678, 678f clades, 651 defi nition, 646 divergence, 652 eukaryotes, 756–757, 757f, 758 gene transfer and, 661–663, 662f, 663f monophyletic group, 651–652 shower curtain biofi lm, 658, 658f, 659f species identification, 649 three domains of life, 656–657, 660–661, 660t virus, 196–198, 197f Phylotype, 869 Phylum, 678 Physarum polycephalum, 782 Phytate, 598 Phytoestrogen, 820 Phytophthora, 773 P. cinnamomi, 178 P. infestans, 432, 773, 774f P. ramosum, 774f Phytoplankton, 804f defi nition, 774, 806 mapping marine, 804, 805f, 806 satellite image of, 831f viruses of, 213, 213f Picoplankton, 803, 803t Picornaviridae, 195t Picornavirus, 400. See also specific viruses examples, 400–401, 400t hepatitis A virus, 1021, 1021f life cycle, 205, 205f Picrophilus P. oshimae, 729t P. torridus, 721f PID (pelvic inflammatory disease), 1003–1004 Pidan, 602, 602f Piezophile, 156 Pilin, 107, 946, 947f Pilus for adherence, 107–108, 108f assembly, 945–948, 946f, 947f bacteriophage M13 attachment to, 398 defi nition, 107, 944 motility and, 948, 960 M protein of Streptococcus pyogenes, 932 sex, 108 types, 944–945 type I, 945, 946, 946f, 947f type III, 945
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type IV, 945, 947–948, 947f urinary tract infection pathogens, 999, 1000 use in electron transfer, 506, 506f Pimaricin, 1060t–1061t Pinkeye, 866 Pinocytosis, 126, A-29 Piptoporus, 771 Pisolithus tinctorius, 816 Plague bubonic, 11, 12f, 1018, 1018f, 1019f, 1086 case history, 1017 cycles of, 1019f disappearance from Europe, 1019 pneumonic, 1018, 1018f, 1019f septicemic, 1018 Planctomyces, 731 P. bekefii, 717, 718f Planctomycetales (planctomycetes), 683t, 684, 717, 718f Planets, life on other, 854–857, 855f, 857f Plankton counting and identifying, 803–806 biomass, 804 epifluorescence microscopy, 804, 805f incorporation of radiolabeled substrates, 804 mapping marine plankton, 804, 805f, 806 metagenomic analysis of DNA, 806 description, 803 food web, 806, 807f size categories, 803, 803t Planktonic cells, 134 Planktos Foundation, 852 Planktothrix, 106f Plant Agrobacterium tumefaciens use in engineering, 621, 622f cell structure, A-23f edible vaccines, 452–454, 454t microbial communities within, 820–824 plant pathogens, 822, 823f, 824, 824f rhizobia and legumes, 820–822, 820f–823f mycorrhizae, 813, 815–817, 816f, 817f phylogeny, 760t single-celled, 8
SFMB_index.indd I-37
virus, 183, 183f entry into host, 206–207, 207f pararetroviruses, 207–208, 208f transmission through plasmodesmata, 207, 207f Plantensimycin, 1054 Plant pathogens, 610 Plaque, 140f, 183, 183f, 373f Plaque assay of animal viruses, 211–212, 212f bacteriophage, 210–211, 211f invention, 210 Plaque-forming unit (PFU), 211 Plasma cell, 908, 909 Plasma membrane, A-22. See also Cell membrane Plasmid, 243 copy number, 245 genes on, 243–244 inheritance, 243, 245 mobilizable, 309 partitioning, 245, 245f purification, 248 replication, 243, 244f structure, 243, 243f transferable, 306–311 transmission between cells, 244 Plasmodesmata, 207, 207f Plasmodial slime mold, 761t, 781, 782 Plasmodium, 782 Plasmodium, 1015, 1017 P. falciparum, 764, 788f, 789–790, 789f, 790, 1015, 1016f, 1017 P. malariae, 1015 P. ovale, 1015 P. vivax, 1015 Plastid defi nition, 763 kleptoplasty, 763 Plastocyanin, 541 Platensimycin, 547f Pleurocapsa, 689, 690f Pleurocapsales, 679t Pleurotus, 591 Plum pox virus, 207, 207f Plus (+) sense single-stranded RNA viruses, 192, 193f, 195t PMN. See Polymorphonuclear leukocyte (PMN) Pneumococcal pneumonia, 986–987, 987f Pneumococcal vaccine, 987, 1024t–1025t Pneumocystis jiroveci, 939, 939f, 1005f, 1006 Pneumocystosis, 990t–991t
Pneumonia bacterial, 986–987, 987f, 990t–991t case history, 1042, 1047 fungal, 987–989, 988f multiple-drug-resistant, 1047 relative incidence of, 987 Pneumonic plague, 1018, 1018f, 1019f Point mutation, 321, 321f Polar covalent bond, A-4 Polaromonas vacuolata, 461f pol gene, HIV, 416 Poliomyelitis (polio), 35–36, 400, 401, 401f Polio virus, 400 assembly, 403f, 405 attachment, 401–402, 402f disease, 400, 401, 401f dissemination, 405–406, 405f entry, 402–403 exit, 405 gene expression, 403, 405 genome, 401, 403f PVR receptor, 201, 203, 205 replication, 404f, 405 replication in human tissue culture, 210, 210f structure, 190–191, 401, 402f tropism, 201, 203 Poliovirus receptor (PVR), 401–402, 402f Polyamine, 77 Polychlorinated aromatics, 476f Polychlorinated biphenyls, 480 Polycistronic RNA, 273 Polyester, synthesis, 566, 566f Polyglutamate, 601, 602f Polyhedral bodies, 688 Polyhydroxyalkanoate (PHA), 106, 431, 566f Polyhydroxybutyrate (PHB), 106, 709 Polyketides, production of, 566, 567–569, 567f–569f Polymerase chain reaction (PCR), 250 epidemiological use, 1089, 1090 error-prone, 449, 450 examples of use, 1073t multiplex, 445–446, 446f, 1073, 1074t for Mycobacterium tuberculosis detection, 1072, 1074 real-time, 446, 447f, 1076, 1077f
I-37
reverse transcriptase PCR (RT-PCR), 559 Taq polymerase, 35 Polymixin B, 880f Polymorphonuclear leukocyte (PMN) description, 874 development of, 874f in type III hypersensitivity reaction, 929, 930f types, 874 Polymyxin, 1041 Polynuclear aromatic hydrocarbon (PAH), 500, 501f Polyoxins, 765 Polyphyletic, 30 Polypurine tract, 420 Polysaccharide, 477 hydrolysis of, 478 structure, A-10–A-12, A-11f, A-12f Polysome, 79f, 80 Polyvinylidene fluoride (PVDF), 437 Population, 794 Porin, 96, 97f, 684 Porphyra, 778, 778f Porphyromonas gingivalis, 108, 108f, 489 Portal of entry, 942 Portobello mushroom, 591, 771 Positive selection, 918 Positive-sense (+) strand, 192 Positron-emission tomography (PET), 555 Posttranslational control, 349 Potato blight disease, 432 Potato scab disease, 700 Potato spindle tuber viroid, 186f Potato virus X, 444, 445f Potyviridae, 195t Potyvirus, 207, 207f Poultry, spoilage of, 609, 610t Pour plate, 132 Powelson, Dorothy, 2 Poxviridae, 194t Poxviruses, 423t life cycle, 203 structure, 190, 191f PPD (purified protein derivative), 988, 989, 992 PPS (pentose phosphate shunt), 482, 486–487, 487f Prebiotic soup model, 641–642, 641f Predator, 796 Prefi x, numerical, A-16, A-16t Preliminary mRNA transcript (pre-mRNA), 95
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I-38
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Pressure, microbial adaptation to variations in, 151t, 155–156, 156f Prestegaard, Karen L., 109f Prevotella, 827, 867, 867f Prey protein, 441, 443 Prievr, Daniel, 751 Primary antibody response, 908, 909, 909f Primary endosymbionts, 758 Primary pathogen, 939 Primary producer, 795 Primary recovery, 620f, 621 Primary structure, protein, A-9, A-9f Primary syphilis, 1002 Primase, 101, 235f, 236, 239 Primate T-lymphotrophic virus (PTLV-1), 413, 413t Primer in DNA replication, 235f, 236, 240, 240f in PCR, 250, 250f Primer extension, 440f, 441 Prion, 22, 185–186, 186f, 1011, 1013, 1013f, 1013t Probabilistic indicator, 649–650, 651f Probiotic, 179, 696, 870, 871f Prochlorales, 679t Prochlorococcus, 688, 688f, 689, 793f P. marinus, 41f, 106f, 536, 688 in planktonic food web, 806 size, 8t Prokaryote cell structure, A-23f defi nition, 8 noncoding DNA, 225 proteasome, 291, 291f size, 42 Promoter araBAD, 359f, 360 caulimovirus, 207, 208 consensus sequence, 260, 261, 261f CRP activation of, 354, 354f defi nition, 224, 260 lambda, 374–375, 374f RNA polymerase binding, 260 sigma factor recognition, 260–261, 261f, 262f sRNA gene, 369 Prontosil, 1032 Prophage, 200, 312, 373, 985 Prophase, A-30, A-31f Propidium, 131, 132f Propionate, as fermentation product, 488f, 489 Propionibacteria, 680t Propionibacterium P. acnes acne, 864, 866 catabolism by, 476
SFMB_index.indd I-38
P. freudenreichii, 189, 480f, 481, 548 propionic acid fermentation, 593, 597 vitamin B12 synthesis, 548 Propionic acid, 614 Propionic acid fermentation, 481, 593 Prosthecobacter P. dejongeii, 718 P. fusiformis, 683t Protease, 291 HIV, 420 toxins, 949, 951t Protease inhibitor, 184, 422, 1058, 1058f Proteasome, 291, 291f Protective antigen, 957 Protein chaperone, A-9 degradation, 290–293, 291f degradation signals, 291 fold-or-destroy triage system, 292, 293f proteasomes, 291, 291f, 292f ubiquitination and, 292, 292f drug efflux, 85 E. coli proteins, 77, 77f, 78, 78f electrophoresis, 77–78, 78f folding, 100–101, 100f, 283, 285 membrane, 84–85, 84f movement of proteins, A-24, A-26 peripheral, A-22, A-24f transmembrane, A-22, A-24f transport, A-27 modification, 283 mRNA translation, 100, 100f outer membrane, 95–96, 96f, 97f posttranslational control of gene expression, 349 secretion, 285–290 export out of cytoplasm, 286 export through outer membrane, 288t, 289–290 export to cell membrane, 286–287 export to periplasm, 287–289 Sec-dependent pathway, 287–289, 287f type I secretion, 289–290, 289f, 290f secretion and pathogenesis, 959–964, 960f–964f, 960t secretion in prokaryotes, 101
structure primary, A-9, A-9f quaternary, A-9–A-10, A-10f secondary, A-9, A-9f tertiary, A-9, A-9f tagging, 439–440, 439f translation at endoplasmic reticulum, A-34 transport, 84–85, 84f transport of, A-35 Western blot analysis, 437–438, 437f Protein A, 970 Protein Data Bank (PDB), 69 Protein-DNA interaction DNA protection assay and, 441, 442f methods of study, 356–357 Protein G, of Streptococcus, A-10f “Protein Jive Sutra,” 35, 35f Protein kinase, 887 Protein kinase kinase, 957 Protein-protein interaction map, 444, 444f Protein purification, 438–440, 439f Protein synthesis inhibitors aminoglycosides, 1044 case history, 1043 chloramphenicol, 1045 lincosamides, 1045 macrolides, 1044 oxazolidinones, 1045 streptogramins, 1045, 1045f structure, 1044f, 1045f tetracyclines, 1044 Proteobacteria Alpha, 704–707 Agrobacterium, 706, 707f endosymbionts, 706, 707f methylotrophy, 706 oligotrophs, aquatic and soil, 706 photoheterotrophy, 704–706 rickettsias, 706, 708f Beta, 708–709 lithotrophs, 708–709 pathogens, 709 Delta, 712–713 bdellovibrio, 712–713, 713f myxobacteria, 712, 712f description, 684 envelope, 692f Epsilon, 713–714 Gamma, 709–712 aerobic rods, 711–712, 711f Chromatium, 709, 710f Enterobacteriaceae, 709–711, 710f sulfur lithotrophs, 709
metabolism, 703–704, 704f lithotrophy, 704, 705t photoheterotrophy, 704–706 phototrophy, 687t representative groups, 680t–682t Alpha, 680t–681t Beta, 681t Delta, 682t Epsilon, 682t Gamma, 681t Proteolysis sigma factor control by, 365–366 tag, 283 Proteome, 77, 197 Bacillus subtilis, 385f defi nition, 383 Escherichia coli, 385f Proteomics bacteriophage tree, 197f defi nition, 197 two-dimensional analysis, 383–386, 384f–386f viral, 197–198 Proteorhodopsin, 534, 704, 724, 748 Proteus eye flora, 866 P. mirabilis, 710, 1067f P. vulgaris, 710 Prothecae, 169 Protist, 756, 758 heterotrophy, 121 historical view of, 756–757, 757f, 758 pellicle of, 76, 97 phylogeny, 758, 763–764 Proto-cell, 641 Proton, A-2, A-2t Proton motive force, 124, 162, 164, 165f ATP synthesis, 511–513, 511f–513f description, 511 electrical potential, 514–515, 514f, 515f pH difference, 514–515, 514f, 515f Proton potential, 85 Proton pump, bacteriorhodopsin, 747–748, 748f Proton-translocating ATPase, 95, 95t Protozoa diarrheal diseases, 996–997, 997f historical view of, 756 Provirus, 420 ProX, 78, 78f Prozac (fluoxetine), 84, 84f PRPP (5-phosphoribosyl-1pyrophosphate), 579, 580f
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Pseudogene, 247 Pseudomembrane, 954, 955f Pseudomembranous enterocolitis, 870, 870f, 993, 998t Pseudomonadaceae, 711 Pseudomonadales, 681t Pseudomonas acenaphthene catabolism, 500, 501f appearance, 43f benzoate catabolism, 497 camphor catabolism, 462 nitrification, 120 nitrogen fi xation, 845 P. aeruginosa autoaggregation, 345f biofi lm, 140, 141, 141f, 142, 142f, 144, 711 cryo-EM of, 64f, 65 in cystic fibrosis, 878, 879f cytochrome c, 516, 517f disease, 711 flagellum, 110 fluorescently tagged cells, 445f lipopolysaccharide (LPS), 958f penicillin resistance, 1039 pili, 947, 947f quorum sensing, 380, 971 respiratory tract infection, 990t–991t P. dendritiformis, 168f P. fluorescens biofi lm, 711f disease, 711 P. syringae, 962 as psychrotroph, 609 twitching motility, 960 type III secretion, 962 in wastewater treatment, 841 Pseudomurein, 724 Pseudopeptidoglycan, 678, 724 Pseudopod, 763, 764, 780, 780f, 781, 781f Pseudouridine, 271 P site (peptidyl-tRNA site), 274, 275f Psychrophile, 153–154, 154f, 728t of Ace Lake, Antarctica, 722, 722f crenarchaeotes, 736–737 defi nition, 151t environmental conditions for growth, 797t habitat, 721 marine methanogens, 742 origin of life, 643 phylogeny, 726 Psychrotroph food spoilage, 609 Listeria as, 611
SFMB_index.indd I-39
Ptashne, Mark, 375, 375f PTLV-1 (primate Tlymphotrophic virus), 413 PTS (phosphotransferase system), 126, 127f, 353, 353f, 470 Pulque, 485 PUMA2, 297 Pure culture, 21, 128 dilution streaking, 128–129 spread plate technique, 129 Purine, 227, 323–324 biosynthesis, 578–579, 580f transitions and transversions, 321 Puromycin, 281 Purple bacteria carbon dioxide fi xation, 550, 550f phototrophy, 536–537, 538 Purple membrane, 534, 535f Purple nonsulfur bacteria, 704 Purple sulfur bacteria, 704 photosynthesis, 27 in Winogradsky column, 27, 27f Purpura, 994, 1007f Putrefaction, 609 Putrescine, 479, 609 Pyelonephritis adhesion pilus (Pap), 945 Pyrazinamide, 992 Pyrenoid, 775 Pyrimidine, 227 biosynthesis, 578–579, 580f dimer, 324, 325f transitions and transversions, 321 Pyrite, 850 Pyrobaculum P. islandicum, 735 reductive TCA cycle, 560 Pyrococcus habitat, 750 horizontal gene transfer, 661 P. abyssi, 751 P. furiosus, 31f DNA polymerase, 751 Embden-MeyerhofParnas (EMP) pathway variation, 724, 725f P. horikoshii, 750f P. woesii, 750 Pyrodictium, 732, 848 P. abyssi, 726, 731–732, 731t, 732f, 733 P. brockii, 532, 731–732, 731t, 732f, 733 P. occultum, 462, 626, 627f, 655, 731–732, 732f, 733 Pyrogen, 891 Pyrophosphate, 470
Pyrrole, 581, 582 Pyrsonympha, 762t Pyruvate decarboxylation, 491–493, 491f, 492f fermentation, 488f, 489 from glucose catabolism, 482–486, 482f–484f, 486f–487f phosphoenolpyruvate conversion to, 473–474 as terminal electron acceptor, 472 Pyruvate dehydrogenase complex (PDC), 493, 492f, 499 Pyruvate kinase, 474–475, 474f, 489
Q Qβ bacteriophage, 201 Q fever, 707, 937f, 969, 981 Quarantine, 1086 Quartz, 632, 633f, 639f Quaternary ammonium compounds, 177t Quaternary structure, protein, A-9, A-10f Quencher dye, 446 Queuosine, 725 Quinine, 789 Quinol, 517, 518, 519, 519f, 520, 528, 540 Quinolone antibiotics, 230, 1042, 1052f Quinone, 517, 518, 520, 528, 540 Quinone pool, 519, 519 Quorum sensing, 141, 142, 378, 971 examples, 379t in Pseudomonas aeruginosa, 380, 971 in Vibrio, 378–381, 378f–381f visual demonstration, 378f
R Rabbits, myxovirus in, 213 Rabies virus vaccination, 24, 25f vaccine in transgenic plants, 454t Racker, Efrem, 513 Radial immunodiffusion, 906, 907f Radiation electromagnetic, 44 irradiation, 174–175, 614 solar, 464, 464f Radiocarbon dating, A-2 Radioisotope 14 C, 551, 554–555, 554f–555f
I-39
incorporation of radiolabeled substrates, 804 labeling with, 551 tracer, 495 Radiolarians, 761t, 783, 783f Ragsdale, Steven, 741, 741f Ralstonia, 465 Rancidity, 609 Rank, 647 Raoult, Didier, 969 Rash, urticarial, 929, 930f RDV (rice dwarf virus), 66–67, 66f, 67f Reaction activation energy, A-18–A-19, A-18f endergonic, A-17 endothermic, A-17 exergonic, A-17 exothermic, A-17 law of mass action, A-18 oxidation-reduction (redox), A-19–A-20, A19f–A-20f spontaneous, A-16–A-17 Reaction center, 538, 539, 541 Reactive oxygen species (ROS), 166, 166f, 324, 324f, 886 Reading frame, 184, 185f open reading frame (ORF) annotation of DNA sequence, 294 defi nition, 264 identifying by computer analysis, 294–295, 295f recognition by ribosome, 275–276 Real-time PCR, 446, 447f, 1076, 1077f RecA-GFP fusion, 303f RecA protein, 316–318, 317f, 318f, 330, 331, 332f RecBCD complex, 317, 318f Receptor-binding domain, 955 Recombinant DNA, 32, 35 Recombinase, Flp, 455, 455f Recombination advantages of, 316, 319 defi nition, 223 generalized, 316–320, 318f, 319f in horizontal gene transfer, 223 site-specific, 316, 320 Recombinational repair, 327t, 329, 330, 331f Recombination signal sequence (RSS), 911 Red algae, 636f, 761t, 763, 763f, 778, 778f
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I-40
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Red blood cells scanning electron micrograph, 873f size, 41f Redi, Francesco, 16 Redox couple, 507 Redox reaction. See Oxidationreduction (redox) reaction Redox state, 473 Red tide, 787f, 788 Reduced compounds, of early Earth, 18, 19 Reduction potential, 507–509, 508t Reductive acetyl-CoA pathway, 561–562, 562f Reductive (degenerative) evolution, 424–425, 424f, 425f, 496, 652, 668, 669f, 670, 756 Reductive pentose phosphate cycle, 550. See also Calvin cycle Reductive (reverse) TCA cycle, 551t, 560, 561f Reed, Walter, 941f Reemerging disease, 1094f, 1095 Reeve, John, 726, 726f Reflection, 45, 45f Refraction, 45, 45f, 46, 46f Refractive index, 45, 49–50 Refrigeration, 173, 609, 613 Regulator, 346 Regulatory binding site, 356 Regulatory protein defi nition, 346 general concepts of transcriptional control by, 346–347, 347f Regulon, 225, 225f, 365, 368 Reid, Ann, 972 RelA, 363 Release factor, 280, 280f Rennet, 596 Reoviridae, 195t Reovirus, 192 RepA protein, 243 Replication. See DNA replication Replication fork, 101, 102, 233 Replisome, 101–102, 101f, 102f, 239, 241 Reportable diseases, 1087, 1088t Reporter fusion, 434–435, 435f Reporter gene, 82, 434 Repression of anabolic (biosynthetic) pathways, 355 defi nition, 346
SFMB_index.indd I-40
Repressor CI, 371–376, 374f, 375f Cro, 374–375, 374f general concepts, 346–347, 347f Lac, 351, 351f, 352f role of, 348 tryptophan, 361, 361f Rescue gene, 965 Reservoir, 940, 1086 Resolution description, 40 detection distinguished from, 41, 41f electron microscope, 62 increasing, 49 magnification and, 46–48 numerical aperture and, 49–50, 49f range, 43f staining for improvement, 52 Resolvase, 335, 336f Respiration aerobic, 164, 165f, 167 anaerobic, 166, 167, 525–529 alternative electron acceptors and donors, 525, 525f dissimilatory metal reduction, 528–529 in lake water column, 528f nitrate reduction, 525–527 sulfur reduction, 528 as catabolism, 476 description, 507 electron transport system, 507, 516–525 ATP synthesis, 523, 523f, 524f cofactors, 516–518, 517f oxidoreductase protein complexes, 518–520, 518f, 519f pathways, 520–523, 520f–522f sodium pumps, 524 mitochondrial, 509–510, 522–523, 522f sulfate, 646, 646f Respiratory syncytial virus (RSV), 990t–991t, 992 Respiratory tract infection, 986–992 bacterial pneumonia, 986–987, 987f, 990t–991t fungal, 987–989, 988f, 990t–991t overview, 986 table of common, 990t–991t
tuberculosis, 989, 989f, 990t–991t, 992 viral, 990t–991t, 992 microflora, 867–868, 868f Response regulator, 346, 348 Restricted transduction, 311. See also Specialized transduction Restriction endonuclease (restriction enzyme), 34–35, 249, 315 action of, 249 agarose gel analysis of DNA fragments created by, 249f, 250–251 DNA methylation and, 249 recognition sequence, 249, 249f, 250 role in cell, 314f, 315 structure of, 314f types I, II, and III, 315–316 Restriction fragment length polymorphisms (RFLPs), 1074, 1090 Restriction-modification system, 315–316, 315t Restriction site, 250 Restrictive temperature, 104 Reticulate body, 717, 717f, 1003, 1003f Reticuloendothelial system, 875 Retina, resolution of human, 40, 41f Retinal, 534, 535f Retortamonadida, 762t Retroelements, 422, 422f Retrotransposons, 422 Retroviridae, 196t Retrovirus description, 411, 413 DNA shuffl ing experiments, 447, 448 endogenous, 422, 422f evolution, 425 examples, 413t genome, 192–193, 193f, 196t life cycle, 205–206, 206f Reverse gyrase, 230, 724 Reverse transcriptase, 418 activities of, 418, 419f, 420 in DNA microarray analysis, 382f, 383 HIV, 416, 418, 419f, 420 in pararetroviruses, 193, 208, 208f in real-time PCR technique, 1076, 1076f in retroviruses, 192, 205, 206f, 413, 418 telomerase, 425 Reverse transcriptase PCR (RT-PCR), 559 Reversion test, 325 Rev protein, HIV, 416t
R factor, 309 RFLPs (restriction fragment length polymorphisms), 1074, 1090 Rhabdoviridae, 195t Rheumatic fever, 932, 985 Rheumatoid arthritis, 883 Rh incompatibility disease, 929, 929f Rhinitis, allergic, 928 Rhinovirus, 400t, 401, 899, 900f infection cycle, 942 host receptor for, 201, 202f, 205 pathogenesis, 972 structure, 195t Rhizobia endosymbiosis, 27 nitrogen fi xation by, 820–822, 820f–823f, 845, 845f Rhizobiales, 681t Rhizobium in infection thread, 821f membrane proteins, 83 nitrate utilization, 570 nitrogen fi xation, 120, 570, 706, 707f, 820 plasmid genes, 244 size, 8t symbiosis, 120, 121f Rhizoplane, 815, 816f Rhizopus, 769, 769f, 984t R. oligosporus, 598–599, 599f use in tempeh production, 598–599, 599f Rhizosphere, 310, 813, 815, 816f Rho-dependent transcription termination, 264–265, 265f Rhodobacter bacteriochlorophylls, 536 carbon dioxide fi xation, 550 photolysis in, 538 R. sphaeroides, 505f appearance, 704, 705 carbon fi xation, 559 photosynthetic membranes, 550f Rhodobacterales, 681t Rhodococcus, 702 amplification plot, 447f benzoate catabolism, 497 Rhodocyclales, 681t Rhodomicrobium, 704 R. acidophila, 705f Rhodophyta, 761t, 778 Rhodopseudomonas, 738, 810, 848 antenna complex, 538f R. palustris, 458–459, 458f, 544, 639, 703, 795 R. viridis, 538f
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Rhodospirales, 681t Rhodospirillum bacteriochlorophylls, 536 carbon dioxide fi xation, 550 photolysis in, 538 R. rubrum, 810 appearance, 681t detection versus resolution, 41f flagella, 110 metabolism, 705 microscopic observation, 51, 51f nitrogen fi xation, 571 photoheterotrophy, 120 photosystem II, 540 phototrophy, 536 respiration in, 120 shape, 704 tetrapyrrole biosynthesis, 582 Rho-independent transcription termination, 265, 265f Rhopaloeides odorabile, 825, 825f Rho protein, 264–265 RhyB, 368f, 369 Ribavirin, 1054t Ribonucleic acid. See RNA Ribose, 227 Ribose 5-phosphate, 578–579, 580f Ribosomal RNA (rRNA) description, 266 processing, 273, 273f properties, 266t secondary structure, 274f Ribosomal RNA (rRNA) gene, 272–273, 273f molecular clock and, 30, 652 phylogeny and, 653, 655, 655f, 656f, 657, 657f, 658, 659f, 660 Ribosome, 644–645, 645f. See also Translation antibiotics targeting, 100, 1044–1045 appearance of, 43f barophilic, 156 catalytic RNA, 642–643 isolation by ultracentrifugation, 79f, 80 mRNA translation, 100 polysome, 79f, 80, 100, 100f as ribozyme, 274–275 streptomycin-resistant, 80, 81f stringent control and, 362–364, 364f structure, 74f, 271–274, 272f, 273f thermophilic, 153f translocation, 278
SFMB_index.indd I-41
unsticking stuck, 282–283 X-ray diffraction crystallography, 80, 81f Ribosome-binding site, 275, 369 Ribosome recycling factor (RRF), 280, 280f Riboswitch, 644 description, 583 regulation of coenzyme B12 synthesis, 583, 583f Ribotyping, 869 Ribozyme, 185, 186f, 274, 642, 644, 645, 645f discovery of, 19, 19f ribosome as, 274–275 viroid, 425 Ribulose 1,5-bisphosphate, 552, 553, 553f, 555, 556, 556f, 557f, 558 Ribulose 1,5-bisphosphate carbon dioxide reductase/oxidase. See Rubisco Ribulose 5-phosphate, 482, 483f, 486, 487, 487f Rice dwarf virus (RDV), 66–67, 66f, 67f Rich, Peter, 513 Rickettsia (genus), 681t DNA-based detection test, 1073t R. prowazekii, 1022t as energy parasite, 969–970 gene similarities to mitochondria, 298 growth requirements, 117–118 intracellular growth, 118, 118f latent state, 939 R. rickettsii, 706, 1073t Rickettsia (group) appearance, 708f description, 706–707 genome degeneration, 510 mitochondria as descendents of, 707 Rifampin mechanism of action, 1043, 1043f structure, 1043f for tuberculosis, 992 Rifamycin, 265, 268, 268f Ripening, 596 Rise period, 209, 209f RNA antibiotics targeting, 1043 antisense, 368, 1053 catalytic, 19, 19f, 642–643 classes of, 265–266, 266t messenger (See Messenger RNA) nucleotides, A-12
origin of, 19 ribosomal (See Ribosomal RNA) ribozyme, 185 small RNA (smRNA) description, 266 identification of, 369–370, 369f properties, 266t regulation by, 368–369, 368f stability, 266 structure, 226f, 227–228, A-14 synthesis (See Transcription) tmRNA description, 266 function of, 283, 284f properties, 266t structure, 284f transcription, 99–100, 99f transfer (See Transfer RNA) viroids, 185, 186f RNA-dependent RNA polymerase, 185, 192, 205 RNA polymerase, A-1f action of, 99 antibiotic resistance, 265 archaeal, 725, 726f CRP interactions with, 354, 354f primase, 236 promoter binding, 260 role in DNA replication, 236 sigma factor, 259, 260–261, 261f, 262f structure of core polymerase, 259–260, 259f structure of holoenzyme, 260f transcription elongation, 264 transcription initiation, 263, 263f transcription termination, 264–265, 265f RNA reverse-transcribing viruses, 192–193, 193f, 196t RNase control of gene expression by, 348 RNase H, 240, 240f RNA virus life cycle, 205, 205f minus (–) strand, 406–411, 406f–410f, 412f plus (+) strand, 400–406, 401f–405f RNA world, 19, 425, 641, 642–643, 643f, 644–645, 644f, 645f Robbins, Frederick, 210
I-41
Roberts, Nell, 2 Rocky Mountain Spotted Fever, 706, 708f Rod, 42, 42f Rolling-circle replication bacteriophage M13, 399 bacteriophage T4, 394, 395f herpes simplex virus, 427 Ronson, Clive, 304 Roosevelt, Franklin, 401, 401f Root nodules, 121f Roquefort cheese, 11f ROS (reactive oxygen species), 166, 166f, 324, 324f, 886 Roseiflexus, 686 Rosing, Minik, 637f, 638 Rossman, Michael, 392, 393f Rotary biomolecules, 85, 85f Rotavirus, 996 gastroenteritis, 996, 998t structure, 195t vaccine, 1024t–1025t Rous sarcoma virus (RSV), 413, 413t rpoD gene, 260 rpoH gene, 365, 366f rpsL gene, 81–82, 81f RRF (ribosome recycling factor), 280, 280f rRNA. See Ribosomal RNA (rRNA) RSS (recombination signal sequence), 911 RSV (Rous sarcoma virus), 413, 413t RSV (respiratory syncytial virus), 990t–991t, 992 Rubella, 984t, 985–986 Ruben, Samuel, 554, 555f Rubeola virus, 984t Rubisco, 553 description, 554–555 mechanism of, 555–556, 556f radioisotope preference, 637 Ruby, Edward, 378f Rudiviridae, 194t Rumen, 741, 741f, 826, 826f, 828, 828f Ruminant endosymbionts of, 28 microbial communities in, 826–828, 826f–828f Ruminococcus, 478 R. albus, 827 Ruska, Ernst, 32 Russell, James, 827, 828, 828f RuvAB, 317, 318f, 319f RuvC, 317, 318f
S Saccharomyces, 592 in cocoa fermentation, 601 genetic code variations, 267
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I-42
In d ex
Saccharomyces (continued) S. cerevisiae beer production, 606 budding, 603f, 767f ethanolic fermentation by, 602 grape fermentation, 606 life cycle, 767f as protein source, 592 reproduction, 767f secretion systems, 288 use in food microbiology, 766 use in research, 602, 603f Sacculus, 89, 89f, 724 Sadoff, Jerald, 616 Safranin, 53f, 54 Saintilus, Wilfrid, 1064 Salmonella anti-sigma factor, 365 bacteriophage epsilon 15, 181f bacteriophage P22, 333 as bioweapon, 1090 DNA vaccine delivery by, 452 as facultative intracellular pathogen, 969 food-borne illness, 612t immune avoidance, 888, 888f interspecies mating, 310 irradiation to control, 173 magnesium sensing by PhoQ, 348 mixed acid fermentation, 489 multiplex analysis, 446f nitrogen fi xation, 845 osteomyelitis, 1064 pathogenesis, 962, 962f pathogenicity island, 611, 613, 613f, 967f in phagosome, 164, 164f phase variation, 970 P22 phage, 311 replication efficiency, 232 ribosomes, 79f S. cholerasuis, 1022t S. enterica, 287 bacteriophage P22, 373f defensins and host range of infection, 881, 881f flagella, 110f gastrointestinal tract infections, 998t gene distribution, 224, 225f Hektoen agar, 1066f horizontal gene transfer, 661 lactose nonfermenter, 130–131 multidrug resistance, 1051
SFMB_index.indd I-42
pathogenicity islands, 337, 339 phase variation, 320, 370–371, 371f probabilistic indicator, 649–650, 651f serovars, 648 stress response, 169 use in Ames test, 325–326, 326f virulence signaling, 164 S. typhi, 648, 1022t antibiotic therapy for, 993 gastrointestinal tract infections, 998t phenol coefficients tests, 176, 176t route of infection, 980 S. typhimurium defensins and host range of infection, 881, 881f sucrose porin, 97 transmission electron microscopy (TEM), 64f type III secretion system, 961f signature-tagged mutagenesis, 966–967, 967f in vivo expression technology, 965, 965f Salmonella pathogenicity island 1 (SPI-1), 962 Salmonellosis, 998t SALT (skin-associated lymphoid tissue), 877 Salt, halophile requirement for, 158 Saltern, 744, 744f, 747 Salt-tolerant organism, 801 Salvarsan, 1032 Sample preparation, electron microscope, 62–64 Samuel, Buck, 742 Sanger, Fred, 9, 9f Sanger dideoxy strategy of DNA sequencing, 252–254, 253f Sanitation, 171 Saprophytes, 813 Sarcinae, 703 SAR11 cluster, 647 SAR86 cluster, 681t Sargassum natana, 780f Sargassum weeds, 779 SARS. See Severe acute respiratory syndrome (SARS) SASPs (small acid-soluble proteins), 144 Sauerkraut, 599 SBP (substrate-binding protein), 125
SbtA, 559 Scalded skin syndrome, 983, 983f, 984t Scanning electron microscopy (SEM), 40, 40f, 42, 42f, 43, 43f, 62, 63f, 64, 64f Scanning probe microscopy (SPM), 65 Scarlet fever, 984t Scattering, 45, 45f Schizogony, 789, 789f, 790 Schizont, 1015, 1016f Schlesner, Heinz, 675f Schopf, William, 637 Schwalm, Christina K., 746f Schwan, Rosane, 600–601, 600f SCID (severe combined immunodeficiency), 896, 896f Scientific notation, A-16 Scopes, John, 630 Scopes trial, 630, 631f Scrapie, 186 SDS (sodium dodecyl sulfate), 77 Sea anemones, zooxanthellae in, 824 Seafood, spoilage of, 609, 610t Sea lettuce, 776 Seaweed, 591 SecA, 287, 287f, 947, 960 SecB, 287, 287f Sec-dependent general secretion pathway, 287–289, 287f Secondary antibody response, 908, 909, 909f Secondary endosymbionts, 758 Secondary metabolite (secondary product), 550, 566, 567–569, 1046 Secondary structure DNA, A-14 protein, A-9, A-10f RNA, A-14, A-14f Secondary syphilis, 1002, 1002f SecYEG translocon, 286, 287 Sedimentation rate, 80 Sedohepulose 7-phosphate, 487, 487f Segmented genome, 191, 406, 407–409, 407f Selectin, 882 Selectively permeable membrane, 121–122, A-26, A-26f Selective media, 130, 1066, 1066f Selective toxicity, 1032 Selenium, 509, 509f, 852t Seliberia stellata, 706
Selifonov, Sergey, 500 SEM (scanning electron microscopy), 40, 40f, 42, 42f, 43, 43f, 62, 63f, 64, 64f Semiconservative replication, 232f, 233 Semipermeable membrane, A-26, A-26f Semmelweis, Ignaz, 24–25 Sensor kinase, 347–348, 348f Sensor system, in vivo, 971 Septation defi nition, 103 rod-shaped cell, 104, 104f spherical cell, 103, 103f Septicemia, 1014 Septicemic plague, 1018 Septum, 103 SeqA protein, 234, 236 Sequelae, 985, 1064 Sequella, Inc., 616–617 Sequence alignment, DNA, 296, 296f Sequence motif, 297 Sequencer, DNA, 253f Serial endosymbiosis theory, 30, 30f Serology, identifications based on, 1077–1078, 1078f Serovar, 648 Serratia marcescens, 611 Serum, 908 Serum sickness, 930f Setlow, Peter, 143f, 144 Severe acute respiratory syndrome (SARS) case study, 1092 disease, 972, 990t–991t epidemiology, 1086–1087, 1087f, 1092 genome, 192 outbreak, 182, 182f Severe combined immunodeficiency (SCID), 896, 896f Sex pilus, 108, 306–307, 306f Sexually transmitted disease (STD) Chlamydia, 1003–1004, 1003f defi nition, 1001 gonorrhea, 1004–1005, 1004f human immunodeficiency virus, 1005–1006, 1005f syphilis, 1001–1003, 1002f table of common, 1002t trichomoniasis, 1006, 1006f Shapes of bacteria, 42, 42f Shapiro, Lucy, 108, 109f Shen, Ben, 568 Shewanella violacea, 156f Shiga toxin, 613, 949f, 956, 993–994
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Shiga toxin prophage, 200 Shigella acid resistance, 956 as facultative intracellular pathogen, 969 food-borne illness, 612t gastrointestinal tract infections, 998t genome reduction, 340 growth factors and natural habitat, 116t growth requirements, 130 lactose nonfermenter, 131 movement inside host cell, 969, 970f S. dysenteriae, 993 S. flexneri evolution through horizontal transmission, 943–944, 944f as frank pathogen, 939 invasion of cell, 961f Shiga toxin, 956 S. sonnei, 1064–1065 shape loss mutant, 105, 105f Shiga toxin, 200, 956, 993 transmission of, 1064–1065 Shigellosis, 998t Shilts, Randy, 414 Shine, John, 275 Shine-Dalgarno sequence, 275 Shingles, 426, 984t, 986 Short interspersed nuclear repeat (SINE), 422 Shotgun cloning, 253 Shower curtain biofi lm, 658, 658f, 659f Shulman, Robert, 475 Shuttle vector, 252 Sialic acid, in influenza receptor, 409, 409f Siderophore, 125, 126f, 850 SIgA, 904 Sigma-54, 574 Sigma-70, 260, 261, 262f Sigma F, 366–368 Sigma factor Bacillus subtilis, 218–219 description, 259 housekeeping, 260 promoter recognition, 260–261, 261f, 262f regulation, 365–368 anti-anti-sigma factor, 365 anti-sigma factor, 365 heat-shock response, 365, 366f proteolytic degradation, 365–366 in sporulation, 366–368, 367f of synthesis, 365
SFMB_index.indd I-43
regulation of physiological responses by, 260–261 Sigma G, 366 Sigma H, 365–366 Sigma K, 366 Sigma S, 365 Signal peptidase, 287 Signal recognition particle (SRP), 100f, 101, 286, A-34 Signal sequence, 286, A-34, A-34f Signature-tagged mutagenesis, 966–967, 966f–967f Silencer, 364 Silent mutation, 321 Silicon dioxide, 632, 633f, 639, 639f Simian immunodeficiency virus (SIV), 22, 183, 413, 413t Simian virus 40 (SV40), 423t Simple stain, 52, 53f SINE (short interspersed nuclear repeat), 422 Singer, Maxine, 35, 258, 258f Single-celled protein, 592, 592f Single-stranded DNA binding protein (SSB), 238f, 240 Single-stranded DNA viruses, 192, 193f, 194t Sin Nombre virus, 1094–1095 Sinorhizobium, 76 nitrogen fi xation, 120, 572, 706, 799 S. melliloti, 221 Siphonous algae, 776 Siphoviridae, 194t Site-specific recombination, 200, 316, 320 SIV (simian immunodeficiency virus), 22 Size metric units for, 40 of microbes, 41–42, 41f resolution and, 40 Skin acne, 866, 866f anatomy, 866f infections, 982–986 boils, 982–983 fungal, 984t necrotizing fasciitis, 983–985, 984t, 985f staphylococcal, 982–983, 983f, 984t table of common, 984t viral, 984t, 985–986, 986f microflora, 864, 865f, 866, 866f, 866t
physical barriers to infection, 877 Skin-associated lymphoid tissue (SALT), 877 S-layer, 94, 94f archaeal, 724, 730, 734, 735f Deinococcus, 686 gram-positive bacteria, 683, 692f Sleeping sickness, 790f, 791 Sliding clamp, 235f, 236, 236f, 237 Slime mold, 781–782 cellular, 761t, 781, 782, 782f historical view of, 756–757 phylogeny, 763 plasmodial, 761t, 781, 782 Slipped-strand mispairing, 371–372, 372f Slonczewski, Joan, 28 Slow-release cycle, 200–201, 201f Slow viruses, 210 Sludge, 841 Small acid-soluble proteins (SASPs), 144 Smallpox, 984t, 986 as bioweapon, 1090, 1091 genome, 192 immune response, 898–899 lesions, 1091f preventative inoculation with, 23 structure, 190 vaccination, 23, 24f, 899, 1091f Small RNA (smRNA) description, 266 identification of, 369–370, 369f properties, 266t regulation by, 368–369, 368f Small-subunit rRNA, 653, 654f Smith, Gale, 622 Smith, Hamilton, 11 Snow, John, 994, 1087, 1088f, 1089 Soap scum biofi lm, 658, 658f SOD (superoxide dismutase), 166, 167 Soda lakes, 161, 161f, 747 Sodium, circulation in alkaliphiles, 162, 162f Sodium dodecyl sulfate (SDS) polyacrylamide gel, 383 Sodium/hydrogen (Na+/H+) antiporter, 123–124, 124f, 162, 163, 163f Sodium motive force, 162, 524 Soft-tissue infections, 982–986 Soil description, 812
I-43
methanogenesis in, 740, 741f nitrogen fi xation in, 845 Soil microbiology, 812–820 Alpha Proteobacteria, 706 decomposition, 813, 815, 815f dry land, 818, 819f, 820 food web, 813, 814f mycorrhizae, 815–817, 816f, 817f overview, 812–813, 812f, 813f rhizoplane and rhizosphere, 815, 816f wetlands, 817–818, 818f Soil profi le, 812f Solar bacteria, 695 Solar radiation, 464, 464f Solfataras, 750 Solute compatible, 157 defi nition, 83 interactions between water and, A-5, A-5f Sonenshein, A. L., 218 Sonication, 79 Sorangium cellulosum, 712 Sorbic acid, 614 Sorbitol MacConkey agar, 490, 490f SOS response, 303f, 331, 332f Southern, Edwin M., 436 Southern blot, 436, 437 Soybean, 845, 845f Soy fermentation, 598–599, 599f Soylent Green (fi lm), 592, 592f Soy sauce, 599 Space-fi lling model, A-6 Spallanzani, Lazzaro, 16 Specialized transduction, 311, 312, 313f, 314 Species, 647 candidate, 649, 685 defi nitions of, 648–649 molecular defi nition, 663 naming, 649 taxonomic rank, 647 Species name, 647 Specimen collection, 1081–1083, 1081f, 1082f Spectrum of activity, 1033, 1033t Speed of light, 44 Spermatozoan, 760t, 758 Sphingomonadales, 681t Sphingomonas, 658, 659f SPI-2, 962 Spike protein description, 189 herpes simplex virus, 189f, 427 HIV, 415, 415f, 416, 417, 417f Spirillum serpens, 713f
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Spirochaeta, 716 Spirochaetales, 682t Spirochete cell structure, 715–716, 716f description, 684 diversity, 716 examples, 682t structure, 42, 42f Spirogyra, 8t, 776, 776f Spiroplasma, 699, 1013 S. melliferum, 64f, 65 Spirulina, 161–162, 161f, 592, 592f, 679t, 688 SPM (scanning probe microscopy), 65 Spoilage. See Food spoilage Sponge choanocytes, 758, 759f microbial communities in, 825, 825f symbiotic cyanobacteria, 691, 691f tube, 759f Spongiform encephalopathy, 1013, 1013f Spontaneous generation, 16, 17, 630 Sporangiospore, 769, 769f Sporangium, 144, 769 Spore stain, 54, 55f Sporogony, 789, 789f Sporothrix schenckii, 984t Sporotrichosis, 984t Sporozoite, 790, 1015 Sporulation Bacillus, 693, 693f Bacillus subtilis, 218–219, 219f sigma factors involved, 366–368, 367f Spread plate technique, 129 Sputum collection, 1082f SQ109, 617, 617f Squalene, 88f, A-14 SRP (signal recognition particle), 100f, 101 SSB (single-stranded DNA binding protein), 238f, 240 SspB protein, 283, 284f Stable isotope ratio, 834 Stahl, David, 737 Stain acid-fast, 54, 55f antibody, 55 chemical structure, 52f differential, 52, 54–55, 55f Gram, 52, 53f, 54, 54f live-dead, 131, 132f negative, 54–55, 55f, 64, 93 simple, 52, 53f spore stain, 54, 55f Staining, 52 Stainzyme, 617, 618
SFMB_index.indd I-44
Stalk, 108 caulobacter, 108–109, 108f–109f, 134 Hyphomicrobium, 134, 134f Stalked ciliates, 786f, 787 Standard Gibbs free energy change, 466 Standard reduction potential, 507–509, 508t Stanier, Roger, 535–536, 536f Stanley, Wendell, 22 Staphylococci (group) origin of term, 103 septation, 103 Staphylococcus Alexander Fleming and, 25 antibiotic resistance, 697 appearance, 697f biological control by, 179 colony-forming unit (CFU), 129 diseases, 696 growth factors and natural habitat, 116t S. aureus alpha toxin, 949, 951, 952f attachment mechanism, 945t coagulase test, 865f, 1072 dilution streaking technique, 128f diseases, 696 endocarditis, 1015 food poisoning, 612t, 992–993, 998t inflammatory response to, 880, 882f Kirby-Bauer disc susceptibility test, 1035f, 1036t meningitis, 1012t methicillin-resistant (MRSA), 697, 983, 1030, 1063f penicillin effect on, 1031 peptidoglycan structure, 89, 677 phenol coefficients tests, 176, 176t protein A, 970 scalded skin syndrome, 983, 983f scanning electron micrograph, 983f septation in, 103, 103f shape-determining proteins, 105f size, 41f skin infections, 982–983, 983f, 984t throat flora, 867 toxic shock, 222, 949, 980, 983 S. epidermidis amensalism, 799t eye flora, 866
meningitis, 1012t as normal fora, 865f S. saprophiticus distinguished from, 1072 skin flora, 696, 864 throat flora, 867 urethal flora, 871 S. oralis, 867 S. saprophiticus, 1072 urinary tract infection, 1001 Starch, structure of, 476f Starch bloat, 827 Starfish stinkhorn, 771, 771f Stars, element formation in, 631, 632f Start codon, 268 Starter culture, 596, 597f Star Trek, 530, 530f Starvation response, 168 Stationary phase, 138 STD. See Sexually transmitted disease (STD) Steam autoclave, 172, 172f Stedman, Ken, 734 Steitz, Joan, 275–276, 276f Stem cell bone marrow, 873, 874f gene replacement therapy, 896 Stem loop, 265 Stentor, 7f, 786f, 787 Stephenson, Marjorie, 512 Sterilization defi nition, 171 ethylene oxide, 176 fi lter, 173 steam, 172–173 Sterol, 87 Stetter, Karl, 626–627, 626f 685 Stick model, A-6 Stomach microflora, 865f, 868, 869f Stonewort, 760t Stop codon, 268, 322 Storage granule, 106–107, 107f Storz, Gisela, 369, 369f Strand invasion, 317, 318f Strep throat, 697, 872 Streptococci (group) GAS (group A streptococci), 1071 hemolysis, 1070–1071, 1071f Lancefield classification, 1071, 1072f Streptococcus appearance, 697f colony-forming units (CFUs), 132 description, 697 evolution, 692 in kimchi production, 599 milk fermentation, 595 oral flora, 866 protein F, 948
S. haemolyticus, 1072f S. mutans attachment mechanism, 867, 945t endocarditis, 1014–1015 S. pneumoniae appearance, 42f attachment mechanism, 945t capsule, 885, 886f, 970, 986 competence factor, 306 fluorescent antibody staining, 1079, 1079f Gram stain, 52, 53f Griffiths’ experiments with, 222, 304 Kirby-Bauer disc susceptibility test, 1035f meningitis, 1007, 1007f, 1008, 1012t optochin susceptibility, 1071f, 1072 penicillin-resistant, 1039, 1040, 1047, 1048f pinkeye, 866 pneumonia, 697, 986–987, 987f, 990t–991t respiratory tract infection, 990t–991t S. pyogenes attachment mechanism, 945t genome, 985 growth factors and natural habitat, 116t growth medium for, 117 identification of, 1070–1072, 1071f M protein, 932, 948, 948f necrotizing fasciitis, 983–985, 984t, 985f pneumonia, 921 pyrogenic toxins, 949 sequelae, 1064 strep throat, 697, 872 vaccine, 872 S. salivarius attachment mechanism, 867, 945t in cheese production, 481 vaccine delivery, 872 S. thermophilus, in Emmentaler cheese, 595f septation in, 103, 103f transformation in, 304, 305f, 306 Streptogramins, 1045, 1045f Streptomyces antibiotic production, 80, 268–269, 281, 513, 547f, 566, 567, 567f, 666, 667f, 700, 1059 appearance, 680t, 699f
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arthrospores, 692, 700 description, 699–700 differentiation, 145–146, 145f, 146f electron micrograph, 268f metabolic pathways, 738 mutualism, 700 S. avermitilis, 567, 1029f S. clavuligerus, 1050 S. coelicolor antibiotic production by, 550 appearance, 699, 699f classification, 647 colonies, 145f developmental cycle, 146f genome, 700 small-subunit ribosomal RNA, 654f transporters, 85 S. erythrens, 281 S. erythreus, 567 S. fulvissimus, 513 S. garyphalus, 1040 S. globisporus, 567 S. griseus antibiotic production, 1032 chromosome, 699f hyphae, 814f odor, 814f streptomycin inactivation, 1046 streptomycin production, 177 S. lavendulae, 145f S. mediterranei, 268 S. nodosus, 1059f S. noursei, 1059f S. orientalis, 584 S. platensis, 1054 S. scabies, 700 soil odor and, 813, 814f Streptomycin action of, 281 discovery of, 1031f, 1032 production of, 177–178, 584, 1046 ribosome interaction with, 80, 81–82, 81f structure, 281f synthesis of, 550 Stress response, 149, 163, 169 Strict aerobe, 166 Strict anaerobe, 166, 167 Stringent response, 362–364, 364f, 565 Stroma, thylakoid, 538 Stromatolite, 629f, 630, 630f, 635, 635t Str operon, 81–82, 81f Structural formula, A-2 Structural gene, 222 Structural isomer, A-11 Subacute bacterial endocarditis, 1014, 1015
SFMB_index.indd I-45
Subcellular fractionation, 79–80, 79f Subcellular organelle, 76 Substrate-binding protein (SBP), 125 Substrate oxidoreductase, 518 Succinate, as fermentation product, 488f, 489 Succinate dehydrogenase, 522, 522f, 523 Succinomonas, 478 Succinyl-CoA, 493, 494f in reverse TCA cycle, 560, 561f in vitamin B12 synthesis, 548, 548f Succinyl-CoA synthetase, 493, 494f Sucrose porin, 96, 97f Suctorians, 786f, 787 Sudden oak death, 773, 774f Sugar aldose and ketose, A-11 disaccharides, A-11–A-12, A-12f isomers, A-11 modified, A-11, A-12f monosaccharides, A-11, A-11f Sugar acid, 482 SulA gene, 331, 332f Sulfa drug, 1042 Sulfamethoxazole, 1042 Sulfanilamide discovery of, 1031f, 1032 mode of action, 1042, 1042f Prontosil metabolized to, 1032 structure, 1031f Sulfated polygalactans, 778 Sulfate-reducing bacteria early forms of, 638 evolution of, 646, 646f marine, 528, 808 in Winogradsky column, 27, 27f Sulfate reductase, 646 Sulfide, in wine production, 607 Sulfide-oxidizing bacteria, 802 Sulfite, for food preservation, 615 Sulfolobales, 734–735 Sulfolobus appearance, 728t conjugation, 306 fuselloviruses, 734–735 glucose metabolism, 724, 725f hydrogen sulfide oxidation, 530, 530f 3-hydroxypropionate cycle, 562 iron oxidation, 467 metabolism, 734 RNA polymerase, 725, 726f
S. acidocaldarius, 161, 161f, 530f, 734 S. metallicus, 562 S. solfataricus, 731t, 734 structure, 734, 734f Sulfur aerobic and anaerobic metabolism, 797t global reservoirs, 833t, 834 oxidation, 530–531, 530f, 531f, 834, 834t rate of cycling, 833t Sulfur cycle, 847–849, 848f, 849f Sulfur granule, 107, 107f Sulfurospirillum barnesii, 509, 509f Sulfur-oxidizing bacteria, 117t, 708–709, 808 Sulfur triangle, 847, 848f Sullivan, John, 304 Summers, Max, 622 Superantigen, 919, 921, 922f, 949 Supercoil, 99, 99f, 228 Supercoiling negative, 229–230 positive, 230 process, 228–230 regulation by topoisomerases, 230, 230f, 231f Supernova, 631, 632f Superoxide dismutase (SOD), 166, 167, 287 Superoxide radical, 166 SV40 (simian virus 40), 423t Svedberg, Theodor, 33, 80 Svedberg coefficient, 79f, 80 Svedberg unit (S), 79f, 80, 271 Swan-necked flask, 16f, 17 Swarmer cell, 108–109, 108f Swarming, 710–711, 710f Swiss cheese, 480–481, 480f–481f, 866 Switch region, 913 Symbiodinium, in corals and sea anemones, 824, 825f Symbiogenesis, 671–672 Symbiont defi nition, 120 digestive methanogenic, 741, 741f hyperthermophilic, 753, 753f Symbiosis, 798–801 amensalism, 799t, 801 coevolution, 666, 667f commensalism, 799t, 800–801 cyanobacteria, 691, 691f defi nition, 666, 798 endosymbiosis, 666–670, 668f, 669f
I-45
hyperthermophiles, 627, 627f mutualism, 666, 668, 691, 798–800, 798f–800f, 799t nitrogen fi xation, 845, 845f parasitism, 799t, 801 synergism, 799t, 800 Symport, 123–124, 124f Synaptobrevin (VAMP), 1009–1010, 1010f, 1011f Synechococcus biofi lm, 140f carboxysomes, 558f Chloroflexus combined with, 686f genome, 688 niche, 795 in planktonic food web, 806 S. elongatus, 536f structure, 689 Synechocystis, 559f, 691 Synergism, 799t, 800 Synthetic media, 130 Syntrophy, 738, 800, 826 Syphilis case history, 1001–1003 congenital, 1003 New World theory of, 1003 primary, 1002 secondary, 1002, 1002f tertiary, 1002 treatment, 1032 Tuskegee experiment, 1003 Systemic infections, 1017–1023 hepatitis, 1021, 1021f, 1023 Lyme disease, 1019–1021, 1020f, 1022t plague, 1017–1019, 1018f, 1019f, 1022t table of diseases, 1022t Syto-9, 131, 132f
T T4. See Bacteriophage, T4 Tagging protein, 439–440, 439f Tailed phage, 391 Talosaminuronic acid, 1040 Tamiflu (oseltamivir), 406 Tandem repeat, 370 Taq polymerase, 35, 250, 250f, 446, 447f, 750–751, 1076 TAT (twin arginine translocase), 288f, 289 TATA-binding protein (TBP), 364, 725–726, 726f Tat protein, HIV, 416, 416t, 974, 974t, 975 Tatum, Edward, 769 Taubenberger, Jeffery, 972 Tautomeric shifts, 323, 323f
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Taxis chemotaxis defi nition, 110 in inflammation, 883 movement described, 110–111, 111f magnetotaxis, 107 phototaxis, 748 Taxon, 647 Taxonomy classification, 647, 648t defi nition, 647 identification, 649–650, 650f, 651f nomenclature, 647 nongenetic categories for medicine and ecology, 648 species defi nition, 648–649 Tay-Sachs disease, 454 TBGp3 protein, 444, 445f TBP (TATA-binding protein), 364, 725–726, 726f TCA cycle. See Tricarboxylic acid (TCA) cycle TC cell. See Cytotoxic T cell (TC cell) T cell activation, 895f, 897, 918–919, 920f, 921 antigen presentation, 919, 921–922, 921f apoptosis, 924 in autoimmune disease, 932 classes of, 915t cytotoxic (TC ), 915, 915t, 919, 920f, 921–922, 932 development, 876 education, 918, 933 helper (T H ), 913, 915, 915t, 918–919, 920f, 921–924 HIV infection of, 973–974 linking humoral and cellmediated immunity, 915 maturation, 918 memory, 930 selection process in thymus, 918, 933 in transplantation rejection, 933, 933f in type IV hypersensitivity, 930–931, 931f T-cell receptor (TCR), 918, 919f T-DNA, 621, 622f Tectiviridae, 194t Teeth, biofi lm on, 140f Teff, 604 Tegument, 189, 189f, 426–427 Teichoic acid, 93, 93f, 890 Teleman, Aurelio, 219 Tellurite, 475 Tellurium, 706
SFMB_index.indd I-46
Telomerase, 247, 425, 914 Telomere, 247, 699f, 700, 785, 914 Telophase, A-30, A-31f TEM (transmission electron microscopy), 43, 43f, 62, 63f, 64, 64f Temin, Howard, 192 Tempeh, 593, 598–599, 599f Temperate phage, 200 Temperature classification by growth temperature, 151t, 153–155, 154f Earth current, 633 early, 643 effect on metabolism, 797–798 fever, 891 greenhouse effect, 633, 835 heat shock response, 155 influence on growth, 152–155, 152f Temperature-sensitive mutants, 396 Template strand, 192, 259 Terminal electron acceptor, 472 Terminal oxidase, 519–520, 521, 521f, 522f, 528 Termination (ter) site, 234, 241, 242f Termites, 741 Terpenoid, 87–88, 87f, 88f Terrafi rnubg, 856 Tertiary structure, A-9, A-10f Tertiary syphilis, 1002 Tests, 783, 783f Tetanospasmin, 1008 Tetanus, 1008–1010, 1009f, 1011f, 1025 Tetanus toxin action of, 1009–1010, 1009f, 1011f structure, 1008–1009, 1009f Tetracycline action of, 281 mechanism of action, 1044 resistance transposons, 333, 336, 336f selective toxicity, 1032 structure, 281f toxicity, 1032–1033 as weak acid, 84, 84f Tetraether, 723f, 724 Tetrahydrofolate (THF), 562, 562f, 1042 Tetrahydromethanopterin, 742f Tetrahymena, 642, A-36, A-37f Tetrapyrrole biosynthesis of, 581–583, 586 description, 581
in reductive acetyl-CoA pathway, 562, 562f TFIIB (transcription factor B), 725–726, 726f Thalassic lake, 747 Thalassiosira pseudonna, 755f T H cell. See Helper T cell (T H cell) Thermales, 679t Thermal vent, 28, 28f, 154f, 155, 731, 732f, 733, 733f, 808–809, 808f Thermoanaerobacter sulfurigignens, 107f Thermocline, 802 Thermococcales, 727, 750–751 Thermococcus DNA polymerase, 751 habitat, 750 T. litoralis, 751 Thermocrinis ruber, 685, 685f Thermodynamics energy and entropy, relationship between, 464 Gibbs energy, 464–468 laws of, A-16–A-17, A-16f Thermophile, 153, 154–155, 154f, 730, 730f archaeal, 722, 726, 728t, 729t, 735, 750–754 (See also hyperthermophile) deep-branching, 678, 679t, 683, 685–688, 685f, 686f defi nition, 151t environmental conditions for growth, 797t habitats for, 730, 730f origin of life, 643 Thermoplasma, 656, 657f glucose metabolism, 724, 725f T. acidophilum flagella, 751 genome sequence, 751 Thermoplasmatales, 751 Thermoproteales, 735 Thermoproteus, 560, 735 Thermosphaera aggregans, 731t, 733, 734f Thermotogales, 678, 679t, 685 Thermotoga maritima genome, 685 horizontal gene transfer, 661 isolation of, 685 Thermozyme, 155 Thermus composting and, 462 description, 686 T. acidophilum proteasome, 291 T. aquaticus appearance, 154f
discovery, 154f, 155 DNA polymerase from, 35, 678, 750 Taq polymerase, 250, 250f T. thermophilus ribosome, 153 Thermus aquaticus (Taq) RNA polymerase, 262f, 264f Thiesen, Ulrike, 658 Thiobacillus copper oxidation, 709f sulfur oxidation, 708–709, 847 T. ferrooxidans carbon dioxide fi xation, 550f iron oxidation, 531, 531f, 709, 709f sulfuric acid production, 850 sulfur oxidation, 849 use in gold recovery, 531 T. thiooxidans growth curves, 136f, 138 growth media for, 117t Thiocapsa, 846 Thioesterase, 567, 568f Thiogalactoside transacetylase, 350, 350F Thioglycolate, 167 Thiomargarita namibiensis, 8, 8f, 42 Thiomicrospira, 808 Thiothrix, 650f Thiotrichales, 681t Thiovulum, 682t Thomas, George, 785, 785f Thrasher, Adrian, 896, 896f Threobromine, 601, 601f Threshold dose, antigen, 898 Thrombocytopenia, 994 Thrombotic thrombocytopenic purpura (TPP), 994 Thylakoid, 106, 106f, 538, 539f, 688 Thymidine, uptake of radiolabeled, 804, 805f Thymidine glycol, 324, 324f Thymine, 227, A-12, A-12t, A-13f Thymus, 918 Thyroid-stimulating hormone (TSH), 932 TIGR (The Institute for Genomic Research), 9f, 11 Tincture of iodine, 176t Ti plasmid (tumor-inducing plasmid), 310–311, 621, 622f, 964 Tir (translocated intimin receptor), 963
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Tisdale, John, 838, 838f Tissue culture, for growing animal viruses, 209–210, 210f Tjarks, Larry, 253f TLR (Toll-like receptor), 877–878, 884–886 TMAO (trimethylamine oxide), 609 tmRNA description, 266 function of, 283, 284f properties, 266t structure, 284f TMV. See Tobacco mosaic virus (TMV) Tn3, 335, 336f Tn5, 336, 336f Tn10, 333, 335, 336f Tn916, 335–336 TNF-α (tumor necrosis factor alpha), 882, 923t TNF-β (tumor necrosis factor beta), 923t TNT (trinitrotoluene), 462 Tobacco mosaic virus (TMV) chromosome, 34 infection with, 183, 183f Rosalind Franklin and, 22, 34 structure, 22, 22f, 190–191, 190f Tobamoviridae, 195t Tobramycin, 141, 141f Togaviridae, 195t TolA protein, 398, 399f TolC protein, 290, 290f, 1050, 1050f Toll-like receptor (TLR), 877–878, 884–886 Toluene catabolism, 497, 499 Tomography, 65 Tonicity, A-28, A-28f Tooth decay, 867 Topoisomerase, 230, 230f, 231f, 1042 TorA, 289 Total magnification, 51 Toxic shock syndrome, 980 Toxic shock syndrome toxin (TSST), 921, 980, 983 Toxin assays for, 860 endotoxin, 948, 958–959, 958f, 959f exotoxin AB toxin, 949, 951, 951f, 954 alpha toxin, 949, 951, 952f anthrax toxin, 956–958, 957f botulism, 613, 613f, 694, 694f, 1008–1009, 1009–1010, 1009f, 1010, 1010f
SFMB_index.indd I-47
characteristics of, 950t–951t cholera toxin, 951–952, 952f, 953f, 954 diphtheria, 954–956, 955f, 956f E. coli labile toxin, 951–952, 954 modes of action, 948–958, 949f Shiga toxin, 949f, 956 tetanus, 1008–1010, 1009f, 1011f toxic shock syndrome toxin (TSST), 921, 980, 983 Toxoid, 956 Toxoplasma gondii, 612t, 788, 788f TPMP (triphenylmethylphosphonium), 514–515, 515f TPP (thrombotic thrombocytopenic purpura), 994 Tracer isotopes, 495 Trachoma, 1003 Traditional fermented food, 593 Transamination, 577 Transcript, 259 Transcription as antibiotic target, 268, 268f control of gene expression, 348 coupled with translation, 282, 282f defi nition, 259 description, 99–100, 99f elongation, 264 initiation, 263, 263f RNA polymerase promoter binding, 260 sigma factor, 259, 260–261, 261f, 262f structure, 259–260, 259f, 260f, 262f termination, 264–265, 265f translation coupled to, 100 Transcriptional attenuation, 362, 363f Transcriptional fusions, 434, 435f Transcription factor B (TFIIB), 725–726, 726f Transcription factors archaeal, 725–726, 726f eukaryotic, 364 Transcriptome, 383 Transcytosis, 1007 Transducing particles, 312, 314 Transduction, 200 generalized, 311–312, 312f of plasmids, 244, 311
specialized, 311, 312, 313f, 314 Transfer RNA (tRNA), 266 acceptor end, 270 amino acid attachment to, 271, 272f anticodon, 270 archaeal base modifications, 725 modified bases, 270–271, 271f number of, 100 processing, 273f properties, 266t ribosome binding sites for, 274, 275f stability, 271 structure, 270–271, 270f Transfer RNA (tRNA) genes, pathogenicity islands and, 339 Transform, 212 Transformation, 33, 222, 304 Avery’s experiment, 33 competence, 306 electroporation, 305 gram-negative organisms, 306 gram-positive organisms, 305–306 Griffith’s experiment, 33 natural, 305 of plasmids, 244 in Streptococcus, 304, 305f Transformed-focus assay, 212, 212f Transfusion reaction, 929 Transgenic plants, vaccine production in, 452–454, 454t Transglycosylase, 1038, 1039, 1040 Transition, 321 Translation, 267–283 antibiotics targeting, 281, 281f in cell-free system, 80 coupled with transcription, 282, 282f defi nition, 99, 267 description, 100, 100f elongation, 277–280, 278f, 279f eukaryotic, 100 genetic code, 267–269, 267f initiation, 276–277, 277f of proteins across rough endoplasmic reticulum, A-34, A-34f reading frame recognition, 275–276 ribosome assembly, 272–273, 273f ribosome structure, 271–274, 272f, 273f
I-47
termination, 280, 280f transcription coupled to, 100 tRNA binding, 274–275f tRNA structure, 270–271, 270f, 271f Translational control, 364 description, 348–349 in eukaryotes, 364 stringent control, 362–364, 364f Translational fusions, 434, 435f Translation initiation sequence, 348 Translation rescue molecule, 283 Translocasome, 305–306 Translocation, 278 Transmembrane domain, 955 Transmembrane protein, A-22, A-24f Transmission horizontal, 940, 941f transovarial, 940 vertical, 940, 941f Transmission electron microscopy (TEM), 43, 43f, 62, 62, 63f, 64, 64f Transovarial transmission, 940 Transpeptidase, 90, 1038, 1039, 1040 Transphosphorylation, 348 Transplantation rejection, 933, 933f Transport. See also Nutrient uptake ABS transporters, 124–125, 125f across the cell membrane, 83–85, 84f active, 123–124 antiport, 123–124, 124f coupled, 123–124, 124f endocytosis, 126–127 facilitated diffusion, 122–123, 122f group translocation, 125–126, 127f of siderophores, 125, 126f symport, 123–124, 124f Transport protein (transporter), 84–85, 84f, 468, A-27 Transposable elements Barbara McClintock and, 333 defi nition, 333 naming convention, 333 Transposase, 333, 334, 334f, 335f, 336, 336f, 455 Transposition defi nition, 334 discovery of, 333
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Transposition (continued) nonreplicative, 334, 334f, 335f replicative, 334, 334f, 335f Transposome complex, 334, 335f Transposon complex, 335, 336f composite, 335, 336f conjugative, 335–336 defi nition, 335 drug resistance, 338 mutagenesis, 432–433, 433f retrotransposon, 422 Transversion, 321 Traveler’s diarrhea, 998t Travers, Andrew, 218 Trench, Mariana, 156f Treponema normal flora, 716 T. pallidum appearance, 715f, 716 attachment mechanism, 945t dark-field microscopy, 55, 55f genomic analysis of metabolism, 498f, 499 identification procedures, 1080t meningitis/encephalitis, 1012t reductive evolution, 496 syphilis, 1001–1003, 1002f, 1032 Tribbles, 135, 135f Tricarboxylic acid (TCA) cycle, 478, 491–496 acetyl-CoA entry, 491, 491f, 493 biosynthesis substrates from, 548, 549f description, 491, 493, 494f elucidation by Krebs, 32–33 heterotrophy and, 119f intermediates, 493, 495–496 metabolist model of early life and, 642, 642f oxidative phosphorylation and, 495, 496f reductive (reverse), 551t, 560, 561f regulation of, 495 variations of, 491 Trichodesmium, 691, 691f Trichomonas T. hominis, 869 T. vaginalis, 1006, 1006f Trichoplusia ni, 621, 623, 624f Triclosan, 82 Tricophyton rubrum, 938, 938f Trigger factor, 286 Triglycerides, A-15, A-15f
SFMB_index.indd I-48
Trimethylamine oxide (TMAO), 609 Trinitrotoluene (TNT), 462 Triphenylmethylphosphonium (TPMP), 514–515, 515f tRNA. See Transfer RNA (tRNA) Tropheryma whipplei, 968–969, 1089–1090, 1089f Trophic levels, 796 Tropism, 201 trp operon, 355, 361–362, 361f, 363f, 577 TrpR (tryptophan synthesis regulator), 356f Truffle, 591, 769, 815 Trypanosoma T. brucei, 790f, 791 T. cruzi, 791 Trypanosomes, 762t, 790f, 791 Tryptophan, biosynthesis of, 577, 578f TSH (thyroid-stimulating hormone), 932 TSST (toxic shock syndrome toxin), 921, 980, 983 Tuber aestivum, 769 Tuberculin skin test, 988, 989, 992 Tuberculosis antibiotics against, 616–617, 617f, 700 BCG vaccine, 616 diagnostic test, 617 Ghon complex of, 989, 989f as reemerging disease, 1095 Robert Koch and, 21 Tube worm, 28, 28f, 808f, 809 Tularemia, 981, 981f, 1022t Tumor necrosis factor, 927 Tumor necrosis factor alpha (TNF-α), 882, 923t Tumor necrosis factor beta (TNF-β), 923t Tumpey, Terrence, 408f Tungsten, 751 Turbidostat, 140 Turgor pressure, 89, A-28, A-28f Tus (terminus utilization substance), 241 Tuskegee experiment, 1003 12D, 172–173 Twin arginine translocase (TAT), 288f, 289 Twitching motility, 141, 960 Two-component signal transduction systems, 348, 348f, 376–377, 377f, 971 Two-dimensional gel isoelectrical focusing, 383–384, 384f
protein identification, 385, 386f Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE or 2-D gels), 77, 78, 78f Two-dimensional protein gel, 150, 151f Two-hybrid analysis, 441–443, 443f Tye, Bik-Kwoon, 333 Tyndall, John, 17 Type I (immediate) hypersensitivity, 926–929, 927t, 928f Type II hypersensitivity, 929, 929f, 932t Type III hypersensitivity, 929–930, 930f, 932f Type IV hypersensitivity, 930–931, 931f, 932t Type I pilus, 945, 946, 946f, 947f Type III pilus, 945 Type IV pilus, 945 description, 947–948, 947f Neisseria meningitidis, 947, 947f, 948, 1007 role in biofi lms, 444, 445f Type I secretion, 289–290, 289f, 290f Type II secretion, 960, 960f, 960t Type III secretion, 960t, 961–963, 961f–963f, 995, 1018 Type IV secretion, 964 Typhoid fever, 980, 993, 998t, 1022t Tyrosine, biosynthesis of, 577, 578f
U Ubiquinone, 517, 517f, 518 Ubiquitin, 292, 292f Ulcer, 995–996, 998t Ultracentrifugation, 95 Ultracentrifuge, 32, 33, 79–80, 79f Ultraviolet (UV) light, DNA damage from, 322t, 324, 325f Ulva, 776, 777f UmuDC, 331, 332f Unclassified organism, 684–685 Uncoating, 202f, 203 Uncoupler, 515, 515f Unculturable organism, 150, 684–685 Unrooted tree, 655 Unsaturation, of fatty acids, 564
UPEC (uropathogenic Escherichia coli), 945–946, 945t, 993, 999–1001, 1000f, 1001f Upstream processing, 619, 620f Uracil, 227–228, 329, A-12, A-12t, A-13f Uranium contamination, 854, 854f as energy source for endoliths, 813 microbial metabolism, 852t reduction, 528–529 Urate oxidase, 592 Urease, 163, 996 Urey, Harold C., 18, 641 Uribe, Ernest, 512 Urinary tract infection (UTI), 871 description, 999–1001, 1000f, 1001f specimen collection, 1082–1083, 1082f Urine, 999 Urine collection, 1082–1083, 1082f Uropathogenic Escherichia coli (UPEC), 945–946, 945t, 993, 999–1001, 1000f, 1001f Uroplakin, 1000, 1000f Uroporphyrinogen III, 582, 582f Urticaria, 929, 930f UV (ultraviolet) light, DNA damage from, 322t, 324, 325f UvrABC exonuclease, 328, 329f
V Vaccination, 23, 24f, 899. See also Immunization Vaccine. See also specific diseases DNA, 452, 453f, 616 edible, 452–454, 454t hepatitis A virus, 1021, 1024t–1025t hepatitis B virus, 1023, 1024t–1025t influenza, 972, 1024, 1024t–1025t outer membrane proteins as targets, 96 pneumococcal, 987, 1024t–1025t rabies, 24, 25f safety, 1025 schedule, immunization, 1024t–1025t smallpox, 23, 24f, 899, 1091, 1091f table of, 900t types, 1023
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Vaccinia virus, 190, 191f, 423t, 425f, 899 Vacuolating cytotoxin (VacA), 967–968, 968f Vaginal microflora, 871 Valinomycin, 513, 513f VAM (vesicular-arbuscular mycorrhizae), 769, 817 VAMP (synaptobrevin), 1009–1010, 1010f, 1011f Vampirella, 813 Vanadium, microbial metabolism of, 852t Vancomycin, 1040–1041 action of, 90, 1038f biosynthesis of, 584–585 resistance, 1039, 1051 for staphylococcal infections, 983 structure, 584f, 1041f Van der Waals forces, 65, A-5, A-5t Van Niel, Cornelis B., 795 Variable region, antibody, 903, 903f Variant Creutzfeldt-Jakob disease (vCJD), 1013, 1013t Varicella, 426, 1024t–1025t Varicella-zoster virus (VZV), 423t, 984t genome, 196, 197f latent infection, 426 size, 8t Varicosaviridae, 195t Variola virus, 423t, 899, 984t Vasoactive factors, 882, 883 VBNC (viable but nonculturable), 2, 3, 130, 802–803 VCAM-1 (vascular adhesion molecule), 882, 883f vCJD (variant CreutzfeldtJakob disease), 1013, 1013t Vector, 940, 941f Vectorial metabolism, 512 Vegetables acid fermentation, 598–599 cabbage fermentation, 599 examples, 594t soy fermentation, 598–599, 599f alkaline fermentation, 601–602 spoilage, 610, 610t Vegetative cells of Bacillus, 693 defi nition, 693 Vegetative mycelia, 700 Venter, Craig, 11, 707, 806, 850 Vernon, Suzanne, 1093 Verrucomicrobia, 684, 718
SFMB_index.indd I-49
Verrucomicrobium spinosum, 675f Verrumicrobia, 105 Verrumicrobiales, 683t Vertical transfer, 661, 662–663, 662f, 663f Vertical transmission, 222, 940, 941f Vesicle, A-29 Vesicular-arbuscular mycorrhizae (VAM), 769, 817 Vetter, David Philip, 896, 896f Viable, 129 Viable but nonculturable (VBNC), 2, 3, 130, 802–803 Viable count, 129f, 132 Vibrio V. anguillarum, 381 V. cholerae attachment, 945t, 952f bacteriophages of, 189, 200 chitin breakdown by, 2 cholera, 2–3, 794, 868, 981, 1087, 1088f, 1089 cholera toxin, 951–952, 952f, 953f, 954 copepod association, 2–3, 3f, 794, 794f flagella motor, 110 gastrointestinal tract infections, 998t glutamine synthase gene, 297 integron, 339 multiplex analysis, 446f pili, 947 salt tolerance, 801 sari cloth fi ltration project, 2–3, 3f, 794 scanning electron micrograph, 952f sodium pump, 524 ToxR protein, 83 vaccine in transgenic plants, 454t V. fischeri bioluminescence, 28, 346 quorum sensing, 378–380, 378f, 379f V. harveyi, 380–381, 380f V. parahemolyticus gastrointestinal tract infections, 998t multiplex analysis, 446f V. vulnificus food-borne illness, 612t multiplex analysis, 446f systemic disease, 864, 1022t Vibrionales, 681t Vibriosis, 1022t Vif protein, HIV, 416t
Viral envelope, 187, 189, 189f Viral replication bacteriophage M13, 398–400, 399f bacteriophage T4, 393–395, 394f, 395f destructive, 420 herpes simplex virus, 427, 428f HIV, 420, 421f, 422 influenza A, 409, 411, 412f poliovirus, 404f, 405 rolling-circle, 394, 395f, 399, 427 Viremia, 406, 1014 Viridans streptococcus, 1014 Viridiplantae, 760t–761t Virion, 182–183 Virion host shuttle factor (Vhs), 427 Viroid, 22, 185, 186f, 425 vir operon, 621, 622f Virulence defi nition, 209, 939 measurement of, 939, 940f regulating, 971 of virus in tissue culture, 209 Virulence factor attachment, 944–948, 945t, 946f–948f description, 942 Virulence gene, 942 fi nding, 964–969, 965f–967f horizontal gene transfer, 664, 665f Virulence signals, 164 Virulent phage, 200 Virus, 8, 182. See also Bacteriophage; specific viruses animal DNA virus, 203, 204f oncogenic viruses, 206 receptor binding, 201, 202f, 203 RNA retrovirus, 205–206, 206f RNA virus, 205, 205f uncoating of genome, 202f, 203 antiviral agents, 1054, 1054t, 1056–1058, 1056f–1058f archaeal, 734–735.194t cardiovascular system infections, 1014, 1015 central nervous system infections, 1010–1011, 1012t classification (See Virus classification) computer, 183–184, 184f
I-49
culturing animal viruses in tissue culture, 209–210, 210f animal virus plaque isolation and assay, 211–212, 212f bacteriophage batch culture, 208–209, 209f bacteriophage plaque isolation and assay, 210–211, 211f discovery of, 22 DNA evolution and, 644 ecology, 212–215, 213f–216f emergence of pathogens, 212–213, 214, 214f, 215f roles in ecosystem, 213–215, 213f, 216f envelope, 192 evasion of adaptive immunity, 924 evolution, 196–198, 197f in freshwater communities, 810 gastrointestinal tract infection, 996, 998t genome classification based on, 191, 192–193, 193f, 194t–196t, 196–198 overlapping reading frames, 184, 185f sequencing, 9 size, 184–185, 185f uncoating, 202f, 203 host range, 183, 192 identification procedures, 1080t infection mechanisms, 427, 429 life cycle animal virus, 201–206, 202f, 204f, 206f bacteriophage, 198–201, 198f–199f, 201f plant virus, 206–208, 207f marine, 213–215, 213f, 216f molecular biology, 389–429 herpes simplex, 423, 423t, 426–427, 426f, 428f human immunodeficiency virus (HIV), 390, 390f, 411, 413–423, 413t, 414f–415f, 417f–419f, 421f infection mechanisms, 427, 429 influenza, 406–411, 406f–410f, 412f phage M13, 397–400, 397f–399f
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I-50
In d ex
Virus (continued) molecular biology (continued) phage T4, 391–397, 391f–396f polio, 400–406, 400t, 401f–405f nomenclature, 192 notifiable diseases, 1088t oncogenic, 206, 212, 212f origin of, 424–425, 424f, 425f overview, 181–182 pathogenesis, 971–976 cold viruses, 972 human immunodeficiency virus, 973–975, 974f human papillomavirus, 975, 975f influenza, 972, 973f of plankton, 806 plant, 183, 183f entry into host, 206–207, 207f pararetroviruses, 207–208, 208f transmission through plasmodesmata, 207, 207f plaque, 183, 183f propagation, 183–184, 184f respiratory tract infections, 990t–991t, 992 size, 192 skin infections, 984t, 985–986, 986f structure, 181, 182–183, 187–191 asymmetrical particles, 190–191, 191f envelope, 187, 189, 189f symmetrical particles, 187–190 fi lamentous viruses, 189–190, 190f icosahedral viruses, 187, 188f, 189, 189f tropism, 201, 203 ubiquity of, 184 Virus classification Group I (double-stranded DNA viruses), 192, 193f, 194t Group II (single-stranded DNA viruses), 192, 193f, 194t Group III (double-stranded RNA viruses), 192, 193f, 195t Group IV ((+) sense singlestranded RNA viruses), 192, 193f, 195t
SFMB_index.indd I-50
Group V ((–) sense singlestranded RNA viruses), 192, 193f, 195t Group VI (RNA reversetranscribing viruses), 192–193, 193f, 196t Group VII (DNA reversetranscribing viruses), 193, 193f, 196t phylogeny and, 196–198, 197f proteomic analysis, 197–198, 197f Virus shedding, 406 Vitamin B12, 69, 69f, 548, 548f Volta, Alessandro, 738 Volvox, 776, 777f Von Stockar, Urs, 468 Von Tappeiner, Hermann, 738 Vorticella, 761t, 787 Vpr protein, HIV, 416t, 420 Vpu protein, HIV, 416t
W Wächterhäuser, G., 642 Waksman, Selman, 1031f, 1032 Walleye dermal sarcoma virus (WDSV), 413t Wall pressure, A-28, A-28f Warfare, significance of disease in, 12 Warren, Robin, 23, 713, 995, 996 Warts, 975, 975f, 984t Waste treatment, methanogenesis and, 740, 740f Wastewater, dichotomous key for bacterial identification, 649, 650f Wastewater treatment, 708, 708f, 840–842, 840f, 842f Wasting disease, 1013t Water cellular, 76 hydrogen bond, A-4f, A-5 of Mars, 855, 855f membrane channels, 157, 157f polar bonding in, A-4–A-5, A-4f solute interactions with, A-5, A-5f as solvent of life, A-4–A-5 Water activity, 157 Water molds, 762t, 773, 774f Water table, 812, 812f Watson, James, 34, 34f, 304
Wavelength, 44–45 electron, 62 emission, 58 excitation, 58 resolution and, 49 WDSV (walleye dermal sarcoma virus), 413t Weak acid, membranepermeant, 83–84, 84f Weak base, membranepermeant, 83–84, 84f Web biology, 298 Weiss, Gabriel, 35, 35f Weitzmann, Chaim, 490 Weller, Thomas J., 210 Western blot, 437–438, 437f, 906–907 West Nile virus, 1012t case history, 1076 as emerging pathogen, 214, 214f, 215f genome, 192, 214f host range, 183 PCR detection, 1076 vectors, 940 Wetland, 838, 838f defi nition, 818 soil, 817–818, 818f Wetland restoration, 842, 842f Wet mount, 52 Whey, 595 Whipple, George, 1089 Whipple’s disease, 968–969, 1089–1090, 1089f White blood cells development of, 873–874, 874f scanning electron micrograph, 873f types of, 874–875, 874f Whittaker, Robert, 29 Whooping cough, 954, 1025 Wickramasinghe, Chandra, 19 Wilkins, Maurice, 34 Wilson, Kent, 35f Wine, 605–607, 605f, 608f Winogradsky, Sergei, 26–27, 709 Winogradsky column, 27, 27f Witches’-broom fungus, 610 Woese, Carl, 30, 31f, 633, 652, 656, 657f Wolbachia in Drosophila genome, 254 endosymbiosis, 667–668, 668f genome reduction, 668, 670
metabolic pathways, 669f, 670 Wollaston-Nomarski prism, 58 Wong-Staal, Flossie, 415f, 416 Woolsorter’s disease, 981 World Health Organization (WHO), 1086–1087, 1092–1093 Wort, 606, 606f Wright, Andrew, 219 WspR protein, 345f Wuchereria bancrofti, 938, 939f
X Xanthophyceae, 779 Xenobiotic, 476 XerC protein, 242, 242f XerD protein, 242, 242f Xeroderma pigmentosa, 332 Xerophile, 797t X-Gal, 435 X-ray crystallography, 33–34, 34f, 43, 43f, 68–70, 68f, 69f, 80–81, 81f X-ray diffraction analysis, 68–70 XylS activator, 361
Y Yanofsky, Charles, 362, 363f, 577 Yeast, 766–767 alternation of generations, 767, 767f budding, 766–767, 767f edible, 591–592 fermentation, 17, 116, 490, 593, 594t, 602–608, 603f–608f Yeast two-hybrid system, 442–443, 443f Yellow fever, 214, 940, 941f Yellow fluorescent protein (YFP), 102, 102f, 444, 445f Yellowstone National Park acidic springs, 161f Archaea from, 31f, 722 bioprospecting, 618 Chloroflexus, 686f extremophiles, 150 hot springs, 6, 7f, 30, 31f, 35, 154f hyperthermophiles/ thermophiles, 35, 140f, 154f, 155, 685, 686f, 730, 734 phylogenetic tree for thermophiles isolated from, 655, 656f
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In d ex
Yersinia Y. enterocolitica dissemination, 981 evasion of adaptive immunity, 924 Y. pestis appearance of, 1018f as bioweapon, 1090, 1091 identification procedures, 1080t name changes, 29 plague, 11, 1017–1019, 1018f, 1019f, 1022t
SFMB_index.indd I-51
sodium pump, 524 type III secretion, 1018 virulence factors, 1018 YFP (yellow fluorescent protein), 102, 102f, 444, 445f Yogurt, 159, 179, 595 Young, Neil, 852
Z Zanamivir, 1054t, 1056, 1056f
Zernike, Frits, 57 Zidovudine (AZT), 1054t, 1057f, 1058 Ziehl-Neelsen stain, 1074 Zinkernagel, Rolf, 923 Zinser, Eric, 793f Zone of inhibition, 1034 Zooglia, in wastewater treatment, 841 Zoonotic disease, 981, 981f Zoospore, 760t, 758, 759, 768, 768f
I-51
Zooxanthellae, 762t, 788, 824, 825f Z pathway of photolysis, 540 Z ring, 104, 104f, 105, 105f Z-value, 172–173 Zygomycosis, 984t Zygomycota (zygomycetes), 760t, 769, 769f Zygospore, 769, 769f Zymomonas, 485
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Genomes of representative bacteria and archaea.
Species (strain)
Genomic chromosome(s)* (kilobase pairs, kbp)
Plasmid(s)* (kbp)
Total (kbp)
Bacteria Mycobacterium tuberculosis Tuberculosis
4,400
4,400
Mycoplasma genitalium Normal flora, human skin
580
580
Burkholderia cepacia
3,870 + 3,217 + 876
93
8,056
Escherichia coli K-12 (W3110) Model strain for E. coli proteomics
4,600
Anabaena species (PCC 7120) Cyanobacteria: Major photosynthetic producer of carbon source for aquatic ecosystems
6,370
110 + 190 + 410
7,080
911
21 plasmids with sizes between 9–58
>1,250
2,840 + 2,070
214 + 542
5,666
1,660
16 + 58
1,734
3,130 + 288
33 + 33 + 39 + 50 + 155 + 132 + 410
4,270
1,000 kbp
500 kbp
Borrelia burgdorferi Lyme disease Agrobacterium tumefaciens Tumors in plants; genetic engineering vector
4,600
Archaea Methanocaldococcus jannaschii Methanogen from thermal vent Haloarcula marismortui Halophile from volcanic vent
*Colored schematics indicate relative sizes of genomic elements and whether these are circular or linear. Size bars are provided under each column.
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Three domains of life. Characteristic
Traits of living organisms All cells on Earth resemble each other
Chromosomal material RNA transcription Translation Protein Cell structure
Double-stranded DNA Common ancestral RNA polymerase Common ancestral rRNAs and elongation factors Common ancestral functional domains Aqueous cell compartment bounded by a membrane
Comparison of domains Bacteria
Archaea
Eukaryotes
Archaea resemble bacteria Cell volume DNA chromosome DNA organization Gene organization Metabolism Multicellularity
1–100 µm3 (usually) Circular (usually) Nucleoid Multigene operons Denitrification, N2 fixation, lithotrophy, respiration, and fermentation Simple
1–106 µm3 Linear Nucleus with membrane Single genes Respiration and fermentation Simple or complex
Archaea resemble eukaryotes Intron splicing RNA polymerase Transcription factors Ribosome sensitivity to chloramphenicol, kanamycin, and streptomycin Translation initiator Cell wall
Introns are rare Bacterial Bacterial
Introns are common Eukaryotic form Eukaryotic form
Sensitive Formylmethionine Peptidoglycan
Resistant Methionine (except mitochondria use formylmethionine) Pseudopeptidoglycan or other polymer; or protein S-layer
Bacteria resemble eukaryotes and differ from archaea Methanogenesis Thermophilic growth Photosynthesis Chlorophyll light absorption Membrane lipids (major) Pathogens infecting animals or plants
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No Up to 90ºC Many species; bacteriochlorophyll Proteorhodopsin derived from archea Red and blue Ester-linked fatty acids
Yes Up to 120ºC Haloarchaea only; bacteriorhodopsin Green (central range of solar spectrum) Ether-linked isoprenoid
No Up to 70ºC Many species; chlorophyll (bacterial origin) Red and blue (chloroplasts of bacterial origin) Ester-linked fatty acids
Many pathogens
No pathogens
Many pathogens
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