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ADVANCES IN

Applied Microbiology VOLUME 56

This Page Intentionally Left Blank

ADVANCES IN

Applied Microbiology Edited by ALLEN I. LASKIN Somerset, New Jersey

JOAN W. BENNETT New Orleans, Louisiana

GEOFFREY M. GADD Dundee, United Kingdom

VOLUME 56

Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK

This book is printed on acid-free paper. Copyright ß 2004, Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (www.copyright.com), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2004 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2164/2004 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (þ44) 1865 843830, fax: (þ44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’ For all information on all Academic Press publications visit our Web site at www.academicpress.com ISBN: 0-12-002658-9 PRINTED IN THE UNITED STATES OF AMERICA 04 05 06 07 08 9 8 7 6 5 4 3 2 1

CONTENTS Potential and Opportunities for Use of Recombinant Lactic Acid Bacteria in Human Health SEAN HANNIFFY, URSULA WIEDERMANN, ANDREAS REPA, ANNICK MERCENIER, CATHERINE DANIEL, JEAN FIORAMONTI, HELENA TLASKOLOVA, HANA KOZAKOVA, HANS ISRAELSEN, SøREN MADSEN, ASTRID VRANG, PASCAL HOLS, JEAN DELCOUR, PETER BRON, MICHIEL KLEEREBEZEM, AND JERRY WELLS I. Introduction ............................................................................ II. Use of Recombinant LAB to Prevent Infectious Diseases ....................... III. Potential for Immune Modulation of Type I Allergy Using Recombinant LAB ..................................................................... IV. Opportunities for the Treatment of Inflammatory Bowel Diseases Using Recombinant LAB ............................................................. V. LAB as Cell Factories for the Manufacturing of Pharmaceutical Proteins .............................................................. VI. Engineering LAB for Their Safe Use in Humans ................................. VII. Opportunities and Potential Applications of Future Research ................. VIII. Concluding Remarks .................................................................. References ...............................................................................

2 3 14 18 23 28 33 43 45

Novel Aspects of Signaling in Streptomyces Development GILLES P. VAN WEZEL I. II. III. IV. V.

AND

ERIK VIJGENBOOM

Introduction ............................................................................ Aspects of Vegetative Growth and Liquid Cultivation of Streptomycetes .... The Switch to Development ......................................................... Novel Genes in Development ........................................................ Concluding Remarks .................................................................. References ...............................................................................

v

65 67 69 72 83 83

vi

CONTENTS

Polysaccharide Breakdown by Anaerobic Microorganisms Inhabiting the Mammalian Gut HARRY J. FLINT I. II. III. IV. V.

Introduction: Role of Gut Microbial Fermentation in Nutrition ............... Microbial Diversity and Interactions Within Gut Ecosystems .................. Strategies for Polysaccharide Utilization by Gut Anaerobes ................... Applications ........................................................................... Conclusions and Future Prospects ................................................. References ..............................................................................

89 92 94 106 109 110

Lincosamides: Chemical Structure, Biosynthesis, Mechanism of Action, Resistance, and Applications JAROSLAV SPI´ZˇEK, JITKA NOVOTNA´,

AND

TOMA´Sˇ RˇEZANKA

I. Introduction ............................................................................ II. Chemical Structure of Lincosamides and Cultivation of Production Strains .................................................................... III. Lincomycin Biosynthetic Pathway ................................................. IV. Genetic Control of Lincomycin Biosynthesis ..................................... V. Mechanism of Action ................................................................. VI. Resistance Against Lincosamides ................................................... VII. Biological Activity and Applications .............................................. VIII. Gram-Positive Bacteria ............................................................... IX. Gram-Negative Bacteria .............................................................. X. Anaerobic Bacteria .................................................................... XI. Protozoa and Other Organisms ...................................................... XII. Conclusion and Future Prospects ................................................... References ..............................................................................

121 124 130 133 135 137 138 139 141 143 144 145 146

Ribosome Engineering and Secondary Metabolite Production KOZO OCHI, SUSUMU OKAMOTO, YUZURU TOZAWA, TAKASHI INAOKA, TAKESHI HOSAKA, JUN XU, AND KAZUHIKO KUROSAWA I. II. III. IV. V. VI. VII. VIII.

Introduction ............................................................................ General Method for Obtaining Drug-Resistant Mutants ......................... Antibiotic Overproduction by rpsL (Ribosomal Protein S12) Mutations ..... Antibiotic Overproduction by rpoB (RNA Polymerase) Mutations ............ Effect of str and rpoB Mutations in Various Bacteria ............................ Increase of Chemical Tolerance in Pseudomonas ................................ Combined Drug-Resistance Mutations ............................................. Conclusion and Future Prospects ................................................... References ..............................................................................

155 156 157 164 167 171 172 175 179

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CONTENTS

Developments in Microbial Methods for the Treatment of Dye Effluents R. C. KUHAD, N. SOOD, K. K. TRIPATHI, A. SINGH, AND O. P. WARD I. II. III. IV. V.

Introduction ............................................................................ Conventional Methods ................................................................ Microbial Methods .................................................................... Enzymatic Methods ................................................................... Conclusion .............................................................................. References ...............................................................................

185 186 191 205 206 206

Extracellular Glycosyl Hydrolases from Clostridia WOLFGANG H. SCHWARZ, VLADIMIR V. ZVERLOV, AND HUBERT BAHL I. II. III. IV. V.

Introduction ............................................................................ Modular Structure of the Enzymes ................................................. Function of Noncatalytic Modules .................................................. Characterization of Enzyme Systems ............................................... Concluding Remarks .................................................................. References ...............................................................................

215 217 218 225 251 252

Kernel Knowledge: Smut of Corn MARIÁ D. GARCIÁ-PEDRAJAS I. II. III. IV. V.

AND

SCOTT E. GOLD

Introduction ............................................................................ The Fungal Saprophyte ............................................................... The Fungal Pathogen .................................................................. The Host Reaction ..................................................................... Conclusions ............................................................................. References ...............................................................................

263 263 267 282 284 285

Bacterial ACC Deaminase and the Alleviation of Plant Stress BERNARD R. GLICK I. II. III. IV. V. VI.

ACC Deaminase–Containing Bacteria .............................................. Ethylene and Plant Stress ............................................................ Decreasing Plant Stress with ACC Deaminase–Containing Bacteria ........... Modulating Nodulation of Legumes ................................................ Decreasing Stress in ACC Deaminase Transgenic Plants ........................ Conclusions ............................................................................. References ...............................................................................

291 293 295 304 306 307 308

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CONTENTS

Uses of Trichoderma spp. to Alleviate or Remediate Soil and Water Pollution G. E. HARMAN, M. LORITO, AND J. M. LYNCH I. II. III. IV. V. VI.

Introduction ............................................................................ Trichoderma spp. Are Opportunistic Plant Symbionts ......................... Rhizosphere Competence and Co-Metabolism .................................... Root Enhancement by Trichoderma spp. .......................................... Enhanced Extraction and Biodegradation of Toxicants ......................... Conclusions and Future Prospects ................................................. References ..............................................................................

313 314 315 316 316 326 327

Bacteriophage Defense Systems and Strategies for Lactic Acid Bacteria JOSEPH M. STURINO I. II. III. IV. V. VI. VII.

AND

TODD R. KLAENHAMMER

Introduction ............................................................................ Traditional Strategies ................................................................. Molecular Strategies .................................................................. Native Defense Systems .............................................................. Recent Advancements in Genomics ................................................ Engineered Defense Systems ........................................................ Concluding Remarks .................................................................. References ..............................................................................

332 339 342 343 351 353 368 369

Current Issues in Genetic Toxicology Testing for Microbiologists KRISTIEN MORTELMANS AND DOPPALAPUDI S. RUPA I. II. III. IV. V.

Introduction ............................................................................ Genesis of Genetic Toxicology ...................................................... Regulatory Genetic Toxicology Tests ............................................... Regulatory Genetic Toxicology Guidelines ........................................ Concluding Remarks and Outlook .................................................. References ..............................................................................

379 381 382 391 396 397

INDEX ............................................................................................ CONTENTS OF PREVIOUS VOLUMES ...............................................................

403 411

Potential and Opportunities for Use of Recombinant Lactic Acid Bacteria in Human Health SEAN HANNIFFY,* URSULA WIEDERMANN,{ ANDREAS REPA,{ ANNICK MERCENIER,{ CATHERINE DANIEL,{ JEAN FIORAMONTI,} HELENA TLASKOLOVA,k HANA KOZAKOVA,k HANS ISRAELSEN,{ SØREN MADSEN,{ ASTRID VRANG,{ PASCAL HOLS,# JEAN DELCOUR,# PETER BRON,** MICHIEL KLEEREBEZEM,** AND JERRY WELLS*,{{ *Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, United Kingdom {

University of Vienna, Department of Pathophysiology A-1090 Vienna, Austria {

Institut Pasteur de Lille, Department of Microbiology of Ecosystems F59019 Lille, France }

Neurogastroenterology and Nutrition Unit, INRA, F31931 Toulouse 9, France k

Institute of Microbiology, Department of Immunology and Gnotobiology Academy of Sciences of the Czech Republic 142 20 Prague 4, Czech Republic { #

Bioneer A/S, DK-2970 Horsholm, Denmark

Universite´ Catholique de Louvain, Unite´ de Geńe´tique B1348 Louvain-la-Neuve, Belgium

**Wageningen Centre for Food Sciences—NIZO Food Research 6710 BA Ede, The Netherlands {{

Author for correspondence. E-mail: [email protected]

I. Introduction II. Use of Recombinant LAB to Prevent Infectious Diseases A. Recombinant LAB as Vaccine Delivery Vehicles B. Infections of the Respiratory Tract C. Infections of the Gastrointestinal Tract D. Infections of the Urogenital Tract III. Potential for Immune Modulation of Type I Allergy Using Recombinant LAB A. Role of the Indigenous Microflora B. Animal Model of Type I Allergy C. Use of LAB for Prophylaxis and Therapy of Type I Allergy IV. Opportunities for the Treatment of Inflammatory Bowel Diseases Using Recombinant LAB A. Role of LAB in Intestinal Barrier Function

2 3 4 7 9 11 14 15 16 17 18 19

1 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 56 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2164/04 $35.00

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V.

VI.

VII.

VIII.

B. LAB and Intestinal Inflammation C. Recombinant LAB as a Therapeutic Option LAB as Cell Factories for the Manufacturing of Pharmaceutical Proteins A. The Choice of Microbial Host B. The Expression Vector C. Propagation, Fermentation, and Initial Downstream Processing Engineering LAB for Their Safe Use in Humans A. Food-Grade Systems in LAB for Plasmid Maintenance and Chromosomal Insertion B. Biological Containment Systems Opportunities and Potential Applications of Future Research A. Insights from Genome Sequencing and Comparative Genomics B. The Behavior of LAB in the Host C. The Host Response to LAB Concluding Remarks References

20 22 23 23 24 26 28 28 32 33 33 35 39 43 45

I. Introduction Dietary lactic acid bacteria (LAB) are mostly known for their widespread use in the production and preservation of fermented foods and as such have obtained the ‘‘generally regarded as safe’’ (GRAS) status within the food industry (Adams and Marteau, 1995). Some members of this diverse group of bacteria are components of the indigenous gut microflora of both animals and humans and have long been recognized for their health-promoting properties. Indeed, specific strains of LAB, and in particular lactobacilli, have been used as probiotics (Fuller, 1989; Holzapfel et al., 1998; Isolauri et al., 2001) because they are thought to play a crucial role in maintaining a healthy microflora as well as contributing to an expanding list of health-promoting activities (Mercenier et al., 2003). Probiotic LAB have been shown to be beneficial in the treatment of gastrointestinal disorders such as lactose intolerance, travelers’ diarrhea, antibiotic-associated diarrhea, and infections caused by various bacterial and viral pathogens (Heyman, 2000). Clinical trials and animal studies indicate that LAB may also be used to treat the symptoms of atopy and may prevent/reduce the development of allergy (Cross et al., 2001; Kalliomaki and Isolauri, 2003; Kalliomaki et al., 2003; Lodinova-Zadnikova et al., 2003; Tlaskalova-Hogenova et al., 2002). Furthermore, evidence that LAB play a role in controlling intestinal microflora, restoring intestinal

USE OF RECOMBINANT LAB IN HUMAN HEALTH

3

barrier function, and alleviating inflammatory responses has led to their proposed use for therapy and management of immunopathological disorders such as Crohn’s disease, ulcerative colitis, and pouchitis (Campieri and Gionchetti, 1999; Marteau et al., 2003; Shanahan, 2001). Over the last few years, a coordinated effort involving several European laboratories and combining a number of interdisciplinary research strategies has set out to provide experimental evidence for the efficacy of different prototype health products based on the mucosal administration of recombinant LAB (http://www.labdel.eu.com). This consortium also aims to further advance technology for LAB delivery and the safe containment of genetically modified organisms in order to increase the range of potential applications for recombinant LAB and to accelerate their commercial development. In this review, the consortium discusses the potential and future opportunities for the use of recombinant lactic acid bacteria in human health. II. Use of Recombinant LAB to Prevent Infectious Diseases Although vaccines against several major pathogens are in common use, morbidity and mortality from infectious disease remains a considerable burden worldwide. According to World Health Organization (WHO) estimates, infectious diseases caused 14 million deaths in 2001, accounting for 26% of global mortality. This situation is steadily becoming worse because of increasing microbial resistance to antimicrobial drugs worldwide, especially Streptococcus pneumoniae, enterococci, and Gram-negative enteric pathogens (Hakenbeck et al., 1999; Kariuki and Hart, 2001; Threlfall, 2002; Walsh, 2000). Recent evidence is also linking a growing number of infectious agents to an increased risk of cancer, blurring the traditional distinctions between chronic and communicable diseases. In addition, behavioral changes over the last two decades have seen the emergence of new pathogens as well as the re-emergence of ‘‘old’’ infectious diseases thought to be extinct (van Ginkel et al., 2000). Some of the existing vaccines are also not without problems, and more-effective versions need to be developed. In some of the poorest countries, existing vaccines are unaffordable to those most in need, a situation that might be helped by strategies to reduce the cost of vaccination (e.g., through avoidance of the use of syringes and needles). Orally administered vaccines are also an attractive goal in developed countries where combined childhood vaccination schedules generally administered via nonmucosal or percutaneous injections are becoming increasingly more complex to formulate and combine. The main advantage of mucosal immunization (e.g., oral,

4

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nasal, rectal) is the potential to elicit both mucosal and systemic immune responses that would enhance the efficacy of many vaccines. It is not surprising therefore that the development of effective strategies for the delivery of vaccine antigens to the mucosal tissues has received considerable attention over the past decade (Michalek et al., 1994; O’Hagan, 1994; Wells and Pozzi, 1997). The use of recombinant bacteria as carrier systems has received particular attention, most vectors under development being derived from attenuated pathogenic bacteria (Bumann et al., 2000; Gicquel, 1995; Levine et al., 1996; Michalek et al., 1994; Roberts et al., 1994; Sirard et al., 1999; Stahl et al., 1997). In addition to the potential to revert to virulence with associated risk of infection and a public opinion sensitive to the use of recombinant organisms, variation in the immunogenicity of the different attenuated strains has constituted a major problem, and it has been difficult to reach the right balance between the level of attenuation (i.e., lack of disease symptoms) and immunogenicity (i.e., efficacy). An additional concern with the use of attenuated pathogens is that they may still be sufficiently virulent to cause disease in infants, the elderly, or immunocompromised individuals. Therefore the use of noninvasive and nonpathogenic lactic acid bacteria as vehicles for mucosal delivery of vaccine antigens and other therapeutic molecules is an attractive concept. In addition to their application as vaccine vehicles (discussed below), lactic acid bacteria can be used to deliver anti-infectives or antimicrobial products in situ. An example is the use of recombinant Lactobacillus strains expressing anti-idiotypic single-chain Fv antibody to a Streptococcus mutans adhesin (SAI/II) in the prevention of dental caries in a rat model (Kruger et al., 2002). Similarily, recombinant Streptococcus gordonii expressing the microbiocidal molecule H6, which is an anti-idiotypic single chain antibody mimicking a yeast killer toxin, was shown to demonstrate candidacidal activity in a rat model (Oggioni et al., 2001). A. RECOMBINANT LAB

AS

VACCINE DELIVERY VEHICLES

Many lactic acid bacteria are used by the food industry and have a history of safe use or are classified as nonpathogenic. They can therefore be given orally in relatively large doses without risk of potential side effects such as those associated with live attenuated bacterial vectors (Medina and Guzman, 2001). In addition, many LAB strains are acid resistant (Kociubinski et al., 1999) and adhere to mucosal epithelium (Morita et al., 2002), properties that may be beneficial for


5

their use as mucosal delivery vehicles. Expressing vaccine antigens and other therapeutic molecules in recombinant LAB would obviate the need to purify the active component, reducing the overall production and delivery costs. Moreover, these bacteria would ideally be given orally and thus would be amenable to large-scale vaccination programs in populations at risk. In addition to providing protection, LAB-based vaccines could also potentially elicit protective mucosal immune responses against pathogens that are also present as components of the normal microflora in asymptomatic carriers, thus having an impact on carriage rates. While evidence suggests that LAB exhibit low intrinsic immunogenicity when administered by the mucosal route, there is no doubt that specific strains of LAB do possess immunoadjuvant properties and can enhance antigen-specific immune responses when administered in combination with antigen (Link-Amster et al., 1994). It is also clear that the immunoadjuvant properties of LAB vary for different species, which has obvious implications for the selection of specific LAB as vaccine vehicles (Maassen et al., 2000; Repa et al., 2003). In addition, it is important to consider that not all LAB are commensals in humans and that specific strains may be associated with different mucosal sites and environmental niches within the host (Wells and Mercenier, 2003). While selection of an appropriate model species is therefore critical, their inherent diversity can also be seen as an advantage. Their ability to survive at different mucosal surfaces may increase opportunities for use of recombinant LAB as vaccine vehicles against a wider range of diseases. Similarly, differences in the immunomodulatory capacities of different LAB create additional possibilities for tailoring the choice of vehicle to meet the requirements for immunity or to modulate immune outcomes in the treatment of various immunopathological diseases. A theoretical model of the immunomodulatory effects of LAB is shown in Fig. 1. Importantly, the last decade has seen the development of the genetic tools necessary for expression of heterologous proteins in an increasing number of LAB (for reviews see de Vos, 1999a; Langella and Le Loir, 1999; Nouaille et al., 2003; Pouwels et al., 2001; Reuter et al., 2003; Wells and Mercenier, 2003; Wells and Schofield, 1996). Expression systems were first developed in the model LAB strain Lactococcus lactis, a noninvasive, noncolonizing bacterium widely used to produce cheese curds by the fermentation of milk. These included constitutive and inducible promoters that allowed efficient high-level production of antigen under various conditions (de Vos, 1999a; Israelsen et al., 1995a; Kuipers et al., 1997; Wells and Schofield, 1996; Wells et al.,

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FIG. 1. Theoretical model for Th1-enhancing effects of antigen expressing lactic acid bacteria on immune responses based on a previous illustration by Cross et al. (2001). The production of pro-interferon cytokines IL-12 and IL-18 and interferon alpha (INF) by the interaction of certain strains of lactic acid bacteria with antigen presenting cells (APC) (e.g., dendritic cells and macrophages) is supported by in vitro co-culture studies and in vivo animal models. Cell wall components of the Gram-positive lactic acid bacteria may induce pro-Th1 cytokine production through Toll-like receptors (TLRs) and possibly also through other surface receptors. The interaction of LAB with other cell types, including epithelial cells, may also influence the outcome of exposure to LAB and prevent tissue inflammation.

1993a). In more recent times, constitutive and regulated expression systems, some of which have been adapted from Lc. lactis (Kleerebezem et al., 1997; Pavan et al., 2000), have also been used to express heterologous proteins in other LAB, including species of Lactobacillus that have been shown to colonize different niches in both animals and humans (Chang et al., 2003; Grangette et al., 2001, 2002; Hols et al., 1997b; Maassen et al., 1999; Oliveira et al., 2003; Pouwels et al., 1996; Rush et al., 1997; Scheppler et al., 2002). These systems have been further adapted such that the expressed protein can be secreted by LAB or anchored to the bacterial surface (Bernasconi et al., 2002; Dieye et al., 2001; Grangette et al., 2002; Kruger et al., 2002; Oliveira et al., 2003; Reveneau et al., 2002; Ribeiro et al., 2002; Wells et al., 1993b). These improvements have ensured that LAB can be used to effectively deliver antigen to mucosal surfaces either in particulate form or as soluble antigen secreted into the surrounding lumen (Norton et al., 1996). The development of efficient expression systems for LAB has enabled several vaccine antigens derived from a variety of mucosal pathogens to be successfully expressed in different LAB (Mercenier et al., 2000; Seegers, 2002; Thole et al., 2000; Wells and Mercenier, 2003; Wells et al., 1996).


7

Pioneering research carried out by several European laboratories demonstrated that immunization with Lc. lactis and selected species of lactobacilli expressing the tetanus toxin fragment C (TTFC) antigen were capable of eliciting antigen-specific secretory IgA and serum antibodies that were protective against lethal challenge with tetanus toxin (Grangette et al., 2001, 2002; Norton et al., 1997; Robinson et al., 1997; Wells et al., 1993a). In a continuation of this work, it was also shown that Lc. lactis could express and secrete the murine cytokines IL-2 and IL-6, which enhanced antigen-specific immune responses when co-expressed with TTFC (Steidler et al., 1998). These experiments demonstrated that biologically active molecules such as cytokines, enzymes and other molecules could be effectively delivered to mucosal surfaces by using recombinant LAB. This application was further exploited by Steidler et al. (2000), who showed that Lc. lactis strains expressing and secreting murine IL-10 could be used to treat inflammation in two different mouse colitis models. This work has now led to the development of a biologically contained Lc. lactis strain secreting human IL-10 (Steidler et al., 2003), which has been approved by Dutch authorities for use in a small clinical trial as an experimental therapy for use in humans with inflammatory bowel diseases (IBD) (see also Section IV). The aforementioned discoveries demonstrate that there is considerable potential to develop health-based products based on oral delivery of vaccine and other therapeutics when using LAB. Several recent publications have now documented the use of prototype LAB-based vaccines for production and delivery of various molecules targeting a range of mucosal pathogens (Bermudez-Humaran et al., 2002, 2003; Enouf et al., 2001; Gil et al., 2001; Gilbert et al., 2000; Lee et al., 2001; Ribeiro et al., 2002; Xin et al., 2003; Zegers et al., 1999), some of which are discussed in the following sections. B. INFECTIONS OF THE RESPIRATORY TRACT As well as being a reservoir for potentially pathogenic bacteria such as Haemophilus influenzae, Morexella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, S. pneumoniae, and beta-hemolytic streptococci, the respiratory tract is also susceptible to serious infections caused by Bordetella pertussis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, influenza virus, and respiratory syncitial virus (RSV). All these organisms initiate disease at the mucosal surface of the respiratory tract, and thus the efficacy of the host’s response to these infections is dependent on optimal local immune responses at this site. However, vaccines available for diseases caused by many

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of these pathogens have limitations in accessibility or efficacy, highlighting the need for improvements in approaches and products. The need for more-effective control strategies is heightened by reports of increasing antibiotic resistance among isolates from studies on carriage in healthy individuals and from clinical specimens (Metlay and Singer, 2002). Until recently there has been little evidence that probiotic LAB might help prevent respiratory infections despite numerous studies showing their beneficial effects against gastrointestinal infections. However, a number of studies have now shown that the oral application of various LAB and/or their bioactive components can enhance the antimicrobial activity of pulmonary natural killer cells (NK) and macrophages, increase IgAþ cells at the bronchial level, and induce cytokine production by nasal lymphocytes (Hori et al., 2002; Matar et al., 2001; Moineau and Goulet, 1997; Perdigon et al., 1999). Moreover, a number of studies carried out in animals and in humans have shown that LAB may attenuate infections caused by respiratory pathogens. When using animal models, the oral application of different LAB strains increased phagocytic activity in macrophages and enhanced clearance of S. pneumoniae (Cangemi de Gutierrez et al., 2001) and P. aeruginosa (Alvarez et al., 2001). Similarly, LAB were also shown to enhance cellular immunity and reduce influenza virus titers in aged mice (Hori et al., 2001, 2002). These results suggest that selected strains of LAB are capable of preventing respiratory tract infections, perhaps by microbial exclusion and/or by mediating nonspecific immune responses. Interestingly, Cangemi de Gutierrez et al. (2001) also showed that protected mice had increased numbers of lymphocytes in their lamina propria, as well as higher levels of antibodies binding to S. pneumoniae, indicating that a specific immune response may have been elicited. Some studies carried out in humans have further demonstrated the potential of using probiotics to confer protection against respiratory diseases, particularly in young children. In a randomized, double-blind, placebo-controlled study, the consumption of milk containing the probiotic strain Lactobacillus rhamnosus GG modestly reduced the incidence of respiratory infections as well as their severity in young children (1–6 yr) (Hatakka et al., 2001). In a similar study, a live dietary supplement containing Lactobacillus acidophilus and Lactobacillus casei suppressed pneumonia and decreased bronchitis in 6- to 24-month-old children (Rio et al., 2002). These studies have been supported by Gluck et al. (2003), who demonstrated that nasal colonization by pathogenic bacteria (S. aureus, S. pneumoniae, and beta-hemolytic streptococci) was reduced in individuals who ingested milk supplemented with a cocktail of


9

probiotic LAB as compared with individuals fed standard yogurt (Gluck and Gebbers, 2003). While only preliminary, these studies do suggest that oral administration of probiotic LAB may help to reduce the occurrence and severity of respiratory infections in both children and adults. These findings have important implications for the development of recombinant LAB vaccines against respiratory pathogens, because they suggest that LAB themselves may have an effect on immune function that could potentially act as an adjuvant during vaccination. Previous work has already demonstrated that intranasal administration of recombinant Lc. lactis and Lb. plantarum strains expressing TTFC could elicit significant antigen-specific IgA in broncheoalveolar fluids as well as antigen-specific serum antibody and T cell responses in mice (Grangette et al., 2001; Norton et al., 1997). Moreover, these immune responses, which were dose dependent, were sufficient to protect against lethal challenge with injected tetanus toxin. Since then, reports of the construction of LAB strains expressing antigens from a number of respiratory pathogens including S. pneumoniae (Gilbert et al., 2000), Bacillus anthracis (Zegers et al., 1999) and Bordetella pertussis (Lee et al., 1999, 2002) were reported but await proper testing in relevant animal models of disease. More recently, prototype vaccine strains of Lc. lactis and Lb. plantarum expressing pneumococcal PspA antigen were constructed and evaluated in a respiratory challenge model for S. pneumoniae (Hanniffy et al., 2004). PspA, a surface protein and virulence factor found on all isolates of S. pneumoniae (Briles et al., 1998; Crain et al., 1990), is highly immunogenic and is considered a vaccine candidate because it has been shown to confer protection against virulent isolates in different animal models of pneumococcal infection (Bosarge et al., 2001; Briles et al., 1996, 2000). Intranasal administration of LAB expressing PspA have now been shown to elicit mucosal and systemic immune responses that protect against lethal challenge with virulent pneumococcci (Hanniffy et al., 2004). C. INFECTIONS OF THE GASTROINTESTINAL TRACT As constituents of the normal microflora, LAB have long been recognized as playing a role in maintaining oral tolerance and homeostasis in the gut. Additionally, LAB have been used therapeutically to protect against gastrointestinal infections, the most notable example being the use of Lactobacillus GG to treat acute infectious diarrhea in children (Rosenfeldt et al., 2002a,b; Szajewska and Mrukowicz, 2001). The exact

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mechanisms that confer significant clinical benefit following administration of probiotic LAB are uncertain, but various intrinsic properties of the bacteria have been proposed as an explanation for the beneficial effects (Heyman, 2000). In addition to restoring normal intestinal microflora, LAB may help to eliminate enteric pathogens by reinforcing intestinal barrier function, increasing mucosal secretory IgA and humoral immune responses, and boosting specific and nonspecific immunity. In vitro experiments with intestinal epithelial cell lines have demonstrated that LAB probiotic strains can prevent adhesion and invasion by pathogenic bacteria and enhance barrier function in naı¨ve epithelial cells (Lee et al., 2003; Resta-Lenert and Barrett, 2003). Various studies have also shown that LAB can intervene by binding to receptors on gastric and intestinal epithelium typically used by pathogens to gain entry into host cells (Mukai et al., 2002; Neeser et al., 2000). In addition, certain LAB produce relatively large amounts of organic acids (Aiba et al., 1998; Koga et al., 1998; Midolo et al., 1995) and/or bacteriocin-like substances (Kim et al., 2003; Lee et al., 2003; Strus et al., 2001) that have been implicated in the inhibition of Helicobacter pylori, Campylobacter spp., and Clostridium difficile. While the extent to which these components can cause an effect in the gastrointestinal (GI) environment remains questionable, it is becoming increasingly clear that by modulating immune responses, LAB play a vital role in protecting against pathogens. Experiments carried out in animal models have demonstrated the protective capabilities of different LAB against pathogenic Escherichia coli (Ogawa et al., 2001; Shu and Gill, 2001), Salmonella typhimurium (Shu et al., 2000), Helicobacter pylori, and Clostridial species. Shu et al. (2000) demonstrated that compared with control mice, Lb. rhamnosus HN001fed mice exhibited lower morbidity and bacterial translocation rates when challenged with E. coli O157:H7 (Shu et al., 2000), which was associated with significantly higher levels of pathogen-specific IgA and blood leukocyte phagocytic activity in the intestines of these mice. In a separate study, mice administered dietary Bifidobacterium lactis demonstrated increased specific and nonspecific immune responses and reduced intestinal infection by S. typhimurium (Shu and Gill, 2001). These results would suggest that LAB may be capable of enhancing local immune responses that might play an important role in protecting against infectious disease. Using genetic engineering to further enhance probiotic LAB strains holds much promise in the prevention and treatment of infectious diseases of the GI tract. Prototype recombinant Lc. lactis vaccine strains expressing antigen have already been developed against


11

H. pylori (Lee et al., 2001) and rotavirus (Enouf et al., 2001; Gil et al., 2001). Oral administration of recombinant Lc. lactis expressing the H. pylori urease subunit B antigen did stimulate low levels of antigenspecific immune responses in serum, but no protection was observed during challenge with H. pylori (Lee et al., 2001). The influence of different LAB strains and choice of antigen is likely to be critical to any vaccine delivery strategy, particularly when dealing with the GI tract, where persistence and survival of different strains can differ widely (Vesa et al., 2000). Other variables such as interaction of the strain with mucus, intestinal epithelium, and lymphoid cells, as well as the location and amount of antigen expressed in the bacteria, are also likely to influence immune outcomes. Except for recent studies on the effect of antigen quantity on level of antibody response, the impact of many of the aforementioned variables has yet to be assessed experimentally (Wells and Mercenier, 2003). D. INFECTIONS OF THE UROGENITAL TRACT LAB-based vaccine delivery systems may also be appropriate for preventing and treating bacterial vaginosis (BV) as well as specific pathogen-related infections of the urogenital tract (Reid and Bruce, 2001; Reid and Burton, 2002). These include human immunodeficiency virus (HIV), Chlamydia, herpes simplex virus (HSV), papillomavirus, Treponema palladium, and Trichomonas vaginalis, as well as diseases caused by bacterial pathogens such as Neisseria gonorrhoeae, group B Streptococcus, and enteropathogenic E. coli. Lactobacilli are dominant among microflora associated with the urogenital tract of healthy women but are almost completely absent in patients who develop most forms of urinary tract infections. Depletion or disturbance of vaginal Lactobacillus sp. has been associated with the development of bacterial vaginosis as well as increased risk of acquiring HIV and other sexually transmitted diseases (Hillier et al., 1993; Taha et al., 1998; Wiesenfeld et al., 2003). There is evidence that lactobacilli can prevent urinary tract infections by microbial exclusion; by producing bacteriocins, biosurfactants, hydrogen peroxide, and coaggregation molecules; by maintaining a low pH; or a combination of these factors (Reid, 2002). In vitro studies have shown that adhesive lactobacilli can inhibit growth and attachment of uropathogenic bacteria to uroepithelial cells in a strain-specific manner (Boris et al., 1998; McGroarty and Reid, 1988; Osset et al., 2001). Similarly in mice, an endogenous population of Lb. casei in the urinary tract prevented colonization by uropathogenic bacteria in the absence of pathogen-specific immune

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responses (Reid et al., 1985). More recently, a randomized, placebocontrolled trial of 64 healthy women showed that daily consumption of a capsule containing the probiotic strains Lb. rhamnosus GR-1 and Lactobacillus fermentum RC-14 reduced colonization of the vagina by potential pathogenic bacteria and yeast (Reid et al., 2003). While this observed probiotic effect appeared to be mediated predominantly through microbial exclusion and/or production of antagonistic byproducts, an immunological component cannot be discounted. Although further studies are required to determine if LAB can induce protective immune responses in the vagina and bladder, these findings do suggest that daily intake of probiotic LAB could provide a natural, safe, and effective means of stabilizing the continually fluctuating vaginal flora and lower the risk of infection in healthy individuals as well as those prone to urogenital disease. For women, direct application of a LAB-based vaccine by the intravaginal route leading to the induction of local antigen-specific immune responses could provide increased protection against urogenital tract infections, reduce pathogen carriage, and help block sexual transmission of pathogens. Previous findings, however, suggest that the urogenital tract may have a limited ability to mount immune responses to epithelial infections. This is evident from clinical observations that pathogens such as N. gonorrhoeae can be contracted repeatedly in the absence of effective immunity from previous infections (Russell, 2002). In addition, local immunization in order to induce local immune responses would also be effected by hormonal variations that are more pronounced in the urogenital tract than at other mucosal sites and are known to have profound effects on susceptibility to sexually transmitted diseases (Gallichan and Rosenthal, 1996; Gillgrass et al., 2003; Kaushic et al., 2000). While mucosal immunization usually results in higher IgA at the site where immunization occurs, there is evidence that this is not the case in the vagina and that other routes of administration may be more effective. Experiments carried out in mice by Wu et al. (2000) showed that intranasal immunization could induce substantially higher levels of IgG and IgA in vaginal fluids and serum as compared with intravaginal immunization. Such compartmentalization within the common mucosal immune system occurs also in humans, where the route of administration can significantly influence immune responses at remote mucosal surfaces (Kantele et al., 1998). This has been demonstrated by Kutteh et al. (2001), who showed that induction of antibodies (S-IgA) in the female genital tract was best achieved by oral immunization followed by a rectal administration (Kutteh et al., 2001).


13

LAB would offer greater flexibility as vaccine vectors in that they can be administered by whatever route is deemed most suitable for a given clinical situation. The nasal route of administration, for example, may be the best inductive site for a common mucosal immune response and could provide a useful strategy for inducing more-potent, longer-lasting immune responses not just in saliva but in remote secretions (e.g., vaginal fluids) as well as in serum (Wu et al., 2000). Recombinant Lc. lactis and S. gordonii vaccine strains have already been developed against human papillomavirus type 16 (HPV-16) (Bermudez-Humaran et al., 2002, 2003; Cortes-Perez et al., 2003) and HIV (Oggioni et al., 2001; Xin et al., 2003). Similarly, Lactobacillus vaccine strains against Chlamydia psittaci and HIV have also been engineered (Zegers et al., 1999). Xin et al. (2003) also showed that Lc. lactis expressing the envelope protein of HIV protected mice against intraperitoneal challenge by an HIV Env-expressing vaccinia virus. While oral and intranasal administration of a number of these LAB vaccine strains has been effective in inducing antigen-antibody responses in serum, their ability to induce local and remote mucosal responses has not yet been investigated (Cortes-Perez et al., 2003; Xin et al., 2003). Interestingly, vaginal colonization of mice with recombinant strains of S. gordonii, expressing HPV and HIV antigens, has been shown to induce antigen-specific vaginal IgA as well as serum IgG (Medaglini et al., 1998). These strains were also able to induce local and systemic immune responses when repeatedly administered (three inoculations) to the vagina of Cynomologus monkeys (Di Fabio et al., 1998). These findings would indicate that LAB possess adjuvant properties that may be capable of eliciting antigen-specific local as well as systemic immune responses even when applied directly to the vagina. If persistence and colonization of the vagina are essential components of a LAB-based vaccine or therapy, intravaginal immunization would therefore be optimal. Such a strategy would be particularly preferable where the aim is to provide passive immunity in situ. This has recently been addressed by Chang et al. (2003), who engineered Lactobacillus jensenii, a natural human vaginal isolate, to express and secrete the HIV binding protein CD4, which bound the HIV gp120 protein and inhibited HIV-1 entry into target cells in vitro. Lb. jensenii efficiently colonizes the vaginal mucosa of women where it exists as part of a natural ‘‘biofilm’’ composed of bacteria and extracellular matrix materials. It is hoped that resident LAB engineered to express HIV binding proteins that are either surface associated or secreted into the surrounding ‘‘biofilm’’ and mucus layer could effectively neutralize HIV particles by impeding viral access to epithelial cells and prolonging exposure to viral inactivating

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substances that are produced naturally. The potential of this approach has been demonstrated by reports detailing the development of prototype S. gordonii vaccine strains expressing molecules that are microbiocidal against candidiasis (Beninati et al., 2000) and HIV (Giomarelli et al., 2002). Such LAB vaccines, perhaps in tablet form (Bonetti et al., 2003; Maggi et al., 2000; Mastromarino et al., 2002), could be self-administered intermittently to provide ongoing protection against these pathogens. Vaccines such as this could potentially be further improved by combining different LAB strains with different antimicrobial, adhesive, and biochemical characteristics to increase their effectiveness in preventing and treating urogenital diseases. Recombinant LAB vaccines would also be suitable for maternal vaccination strategies aimed at preventing disease in neonates, such as those caused by group B Streptococcus (GBS) and selected strains of E. coli. While antibiotic therapy has proved effective in reducing the incidence of neonatal disease in developed countries, other complications such as premature rupture of the membranes, premature birth, low-birth-weight babies, or stillbirth have been attributed to GBS and other infections of the urogenital tract. A LAB-based vaccine against GBS and other pathogens could reduce the level of colonization in the mother and provide an important first line of defense against the pathogen at mucosal surfaces. In addition, colostrum antibody and transplacentally transferred serum IgG antibodies against GBS could confer immune protection to the newborn. Recombinant strains of Lc. lactis and Lb. plantarum have already been developed that express antigen that has been shown to be protective in an invasive animal model of GBS infection (Seepersaud et al., 2004). These strains are now being tested in animal models for their ability to elicit protective mucosal and systemic responses (Hanniffy and Wells, personal communication). III. Potential for Immune Modulation of Type I Allergy Using Recombinant LAB The prevalence of type I allergy has constantly increased within recent years, leading to the fact that currently up to 25% of the population in industrialized countries suffer from allergic symptoms such as allergic rhinoconjunctivities, allergic asthma, or atopic dermatitis. Besides atopic disposition (Marsh et al., 1994) and allergenic molecules (Scheiner and Kraft, 1995), there are various environmental factors such as air pollutants (von Mutius et al., 1994), changes in nutrition


15

(Bottcher et al., 2000), or ‘‘sterile’’ lifestyles in industrialized countries without bacterial epidemics (Ring, 1997) that may have a causal relationship with the increasing prevalence of the disease. The pathway of IgE production is well described for mice and man and is basically influenced by the reciprocal relationship between Th2 cytokines, mainly IL-4, the switching factor for IgE class, and Th1 cytokines (e.g., interferon gamma [IFN-], which counteracts the activity of IL-4) (Mosmann and Coffman, 1989). In atopic individuals an imbalance favors the production of IL-4, leading to a bias of Th2 responses with an overproduction of IgE against allergenic proteins (Romagnani, 1994). These molecules lead to cross-linking of the allergen-specific IgE bound to the surface of mast cells via specific FC-receptors, thereby triggering the release of anaphylactic mediators, leading to the characteristic allergic symptoms mentioned previously. Until now, specific immunotherapy (SIT) has been the treatment of choice against type I allergy, performed by injecting increasing doses of allergens (Bousquet et al., 1998). Although SIT is effective in the majority of treated patients, there are certain drawbacks, such as frequent injections and long duration of the treatment, leading to poor compliance in patients. Moreover, aluminium salts, which are used as adjuvants in SIT, are potent inducers of Th2 responses, which might reduce the efficacy of the treatment. Thus, there is increasing interest in improving immunotherapy by using Th1-promoting adjuvants (Wheeler and Woroniecki, 2001) and/or administration via a less invasive route, such as mucosal delivery (Morris, 1999). A. ROLE OF THE INDIGENOUS MICROFLORA The indigenous microflora plays an important role in anti-infectious resistance by competitive interaction with pathogenic bacteria but is also important for directly influencing immune responses. This has been demonstrated in animals reared under sterile conditions (germfree animals), in which it has been shown that systemic and local immune responses are more difficult to establish and that, in particular, the induction of oral tolerance is unstable and short-lived (Cebra et al., 1999). Based on these findings, an imbalance of the composition of the indigenous microflora is believed to play a role in the development of inflammatory diseases, such as intestinal bowel disease (Duchmann et al., 1995) and allergies (Rautava and Isolauri, 2002; Wiedermann, 2003). Indeed, differences in the intestinal colonization pattern between children of ‘‘Western lifestyle countries’’ with a high prevalence of allergies and of economically low developed countries

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where allergies are less common has also been reported (Bottcher et al., 2000). The ‘‘hygiene theory’’ proposes that an increasingly hygienic lifestyle has contributed to the increasing prevalence of allergic disease caused by intestinal colonization, with a limited range of microbes (Strachan, 1989). In the last few years, the possible role of specific LAB strains in the prevention of allergic diseases has become more evident. In particular the relationship between the composition of the intestinal flora and the prevalence of allergic diseases has been epidemiologically documented. One study showed that infants from countries with a high prevalence of allergy, such as Sweden and England, have a lower level of intestinal colonization with certain LAB strains than children from countries where allergic diseases are less prevalent, such as Estonia and Nigeria (Bjorksten et al., 1999; Sepp et al., 1997; Simhon et al., 1982). Moreover, it was recently demonstrated that oral administration of a particular LAB strain (Lb. rhamnosus GG) led to reduced atopic dermatitis in children with a positive family history of type I allergy (Kalliomaki et al., 2001), indicating that LAB can directly or indirectly exert an anti-allergic effect. B. ANIMAL MODEL OF TYPE I ALLERGY Among the numerous inhalant allergens, tree pollen of the white birch Betula verrucosa is one of the most important sources responsible for eliciting allergic symptoms. The major allergen of birch pollen is Bet v 1, a 17-kD molecule, to which 95% of birch pollen allergic patients (and 60% exclusively) display IgE binding reactivity. Bet v 1 was the first pollen allergen to be cloned, sequenced, and produced as a recombinant protein in E. coli (Breiteneder et al., 1989) and to have its crystalline structure determined (Gajhede et al., 1996). Recombinant Bet v 1 has also been shown to possess biological properties equivalent to those exhibited by the natural Bet v 1 molecule (Ferreira et al., 1993). Moreover, immune responses to Bet v 1 have been characterized at the B and T cell level in atopic and nonatopic individuals (Ebner et al., 1995). An animal model of allergic sensitization to birch pollen and its major allergen Bet v 1 has been developed in BALB/c mice that have been identified as high IgE responders to this allergen (Bauer et al., 1997; Wiedermann et al., 1998). The established standard sensitization scheme is based on an intraperitoneal injection of recombinant (r) Bet v 1 adsorbed to aluminium hydroxide (Al(OH)3), followed by an aerosol treatment with natural birch pollen extract (BP). This sensitization


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procedure leads to high allergen-specific IgE/IgG1 antibody levels, positive immediate type skin reactions in vivo and Th2-like immune responses in vitro, eosinophilic infiltration within the lungs, as well as broncheoalveolar and airway hyper-responsiveness. Thus the immune responses represent an immunological state comparable to that of human type I allergy/asthma (Wiedermann et al., 1998). Using this model, several mucosal adjuvants have been tested for their capacity to modulate an allergic immune response. In this respect it has been demonstrated that immunomodulation can be achieved by using mucosal adjuvants such as cholera toxin (Wiedermann et al., 1998), cholera toxin subunit B (Wiedermann et al., 1999), or certain bacterial components (CpG-motifs) (Jahn-Schmid et al., 1999). C. USE OF LAB

FOR

PROPHYLAXIS AND THERAPY OF TYPE I ALLERGY

Experiments carried out in mice have shown that IgG1 and IgE antibody levels could be reduced when certain LAB strains were injected or orally applied with a particular antigen/allergen (Matsuzaki et al., 1998; Shida et al., 2002). In light of these studies, experiments were carried out to investigate the capacity of two LAB strains (Lactococcus lactis MG1363 and Lactobacillus plantarum NCIMB8826) to prevent or modulate allergic immune responses. Both LAB strains induced high levels of Th1 cytokines IL-12 and IFN- in naı¨ve murine spleen cell cultures possibly via the mechanisms depicted in Fig. 1. In the murine birch pollen allergy model, intranasal or oral co-application of Lc. lactis or Lb. plantarum with recombinant Bet v 1, prior to or after allergic sensitization, led to increased levels of allergen-specific IgG2a antibodies and in vitro IFN- production, indicating a shift toward Th1 responses. Successful immunomodulation by the mucosal pretreatment was further demonstrated by suppression of allergen-induced basophil degranulation (Repa et al., 2003). These results, based on induction of counter-regulatory Th1 responses, indicated that combined mucosal application of LAB with a specific allergen could provide an effective prophylactic and therapeutic strategy against allergy. The construction of recombinant LAB for local delivery of an allergen could further enhance this protective/immunostimulatory effect and could represent a useful tool for mucosal vaccination against type I allergy. Recently it has been shown that treatment with a recombinant LAB strain expressing the house dust mite allergen Der p1 reduced levels of allergen-specific IL-5 (a Th2 cytokine) in sensitized mice (Kruisselbrink et al., 2001). Similarly, recombinant Lc. lactis and Lb. plantarum strains expressing Bet v 1 have now also been

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constructed and preliminary data indicate that mucosal administration of these strains prior to or after sensitization results in a significant suppression of allergic immune responses (Mercenier and Wiedermann, personal communication).

IV. Opportunities for the Treatment of Inflammatory Bowel Diseases Using Recombinant LAB IBDs, as typified by ulcerative colitis and Crohn’s disease, are characterized by chronic dysregulation of inflammatory immune responses in the gastrointestinal tract. While the pathogenesis of IBD remains unclear, it is thought to involve complex interactions combining host genetic susceptibility, intestinal bacteria, and gut mucosal immune responses (Farrell and Peppercorn, 2002; Rath, 2003). It is now generally accepted that intestinal microflora provide the antigenic stimuli to deregulate mucosal immune responses in genetically susceptible hosts such that they become overly aggressive with reduced tolerance toward the indigenous microflora. The proposed use of probiotics including LAB for therapy and management of IBD has arisen from increasing evidence implicating indigenous bacteria in the pathogenesis of these diseases. Much of this evidence comes from transgenic animal models in which immunopathological disease is induced by the absence of immunologically important molecules but is dependent on the presence of a normal bacterial microflora (Blum and Schiffrin, 2003; Hudcovic et al., 2001; Strober et al., 2002). For example, mice with disrupted IL-2 and IL-10 genes or / T cell receptor mutants that normally develop chronic intestinal inflammation resembling ulcerative colitis in humans remain healthy in germ-free conditions (Strober et al., 2002). These studies confirmed that IBD is likely to result from abnormal immune responses to normal intestinal microflora and demonstrated the importance of regulatory cytokines such as IL-10 in maintaining immune homeostasis at mucosal sites (Blum and Schiffrin, 2003). As a result, there has been increasing interest in using probiotics, particularly those with immunomodulatory capacities including the ability to induce immunoregulatory cytokines such as IL-10 and TGF-. This has led to suggestions that LAB and their associated anti-inflammatory effects may be capable of reinstating mucosal immune homeostasis and may provide an alternative strategy for intervention in IBD patients (Blum and Schiffrin, 2003).


A. ROLE OF LAB

IN INTESTINAL

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BARRIER FUNCTION

One task of the gut is to act as a barrier between the external and internal environments to prevent the entrance of potentially harmful components. This barrier, which can be considered both as physical (paracellular permeability and protective action derived from mucus) and functional (mucosal immune system), is strongly disrupted in inflammatory states. A decrease in mucosal barrier function consistently occurs in experimental colitis as well as in human IBD (Rath, 2003). One of the main actions of probiotics concerns the reinforcement of the intestinal mucosal barrier, an activity which can confer intestinal anti-inflammatory properties to some probiotics (see review, Fioramonti et al., 2003). For example, treatment with Lb. reuteri or Lb. plantarum reduced the level of intestinal permeability in a rat model of methotrexate-induced enterocolitis (Mao et al., 1996). In addition, as well as decreasing intestinal myeloperoxidase levels, the administration of LAB re-established the intestinal microecology and reduced bacterial translocation to extra-intestinal sites. The ability of LAB to enhance barrier function and reduce bacterial translocation may play an important role in preventing subsequent activation of inflammatory responses. Bifidobacterial supplementation has been shown to reduce the incidence of necrotizing enterocolitis in mice by preventing bacterial translocation and subsequent activation of inflammatory mediators such as plasma endotoxin and intestinal phospholipase A2 (Caplan et al., 1999). In another study, Shiba et al. (2003) provided evidence that treatment of Bifidobacterium vulgatus– implanted mice with Bifidobacterium infantis abrogated increases in plasma B cells in the Peyer’s patch, probably by protecting the epithelium layer (including Peyer’s Patch) from invasion by B. vulgatus. LAB may also have a trophic action on colonic mucosa. This has been elegantly shown in rats, where an elemental liquid diet induced an atrophy of colonic mucosa (assessed by the rate of crypt cell production) that was significantly improved by treatment with Lb. casei or Clostridium butyricum (Ichikawa et al., 1999). Strong interactions exist between mucus and colonic bacteria, and some actions of probiotics may involve this protective glycoprotein layer. The inability to degrade mucus by bacteria such as Lactobacillus acidophilus or Bifidobacterium bifidum could itself be seen as protective (Ruseler-van Embden et al., 1995). Indeed, in vitro experiments have shown that adherence by specific strains of Lactobacillus induces mucin gene expression and extracellular secretion of MUC3 by intestinal epithelial cells (Mack et al., 2003). Moreover, there was a direct correlation between

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increased mucin secretion and reduced adherence by enteropathogenic Escherichia coli. Similar interactions between LAB and host epithelial and other host cells may play an important role in protecting against translocation of antigenic stimuli that contribute to the pathogenesis of IBD.

B. LAB

AND INTESTINAL INFLAMMATION

A number of studies have shown that a select number of probiotic strains can reduce experimental colonic inflammation in animals. A study carried out by Fabia et al. (1993) was one of the first to show that treatment with LAB could prevent the development of acetic acid– induced colitis in rats. Intracolonic administration of Lb. reuteri produced normal myeloperoxidase (MPO) activity levels and mucosal permeability and prevented the development of morphologic lesions (Fabia et al., 1993). These pioneering experiments were subsequently reproduced with other bacteria and in other models of experimental colitis. Lb. reuteri, but not Lb. rhamnosus, was shown to be effective in attenuating acetic acid–induced colitis in rats (Holma et al., 2001). Treatment with Bifidobacterium longum in mice (Fujiwara et al., 2003) and C. butyricum in rats (Araki et al., 2000) both reduced the severity of a colitis induced by dextran sulfate sodium (DSS). In addition, Lb. plantarum and Lb. reuteri ameliorated methotrexate-induced entercolitis in rats (Mao et al., 1996). The spontaneous development of colitis in IL-10–deficient mice has also been ameliorated by treatment with Lb. plantarum (Schultz et al., 2002) or Lb. reuteri (Madsen et al., 1999a). In a recent study it has been shown that treatment with a solution of lysed E. coli ameliorated a colitis induced by DSS in mice (Konrad et al., 2003). To date, there is no evidence from animal models that LAB can adversely effect the development of colitis or augment severity of associated symptoms. While the mechanisms remain unclear, evidence from some of these studies indicates that administration of LAB may induce tolerance to bacterial antigens by down-regulating Th1 inflammatory cytokines (Konrad et al., 2003; Schultz et al., 2002). However, it is also possible that LAB may beneficially interact with other commensal bacteria that may play a pathogenic role in IBD. A recent study carried out by Shiba et al. (2003) showed that B. infantis inhibited the growth of B. vulgatus, a putative pathogenic microbe in IBD. In addition, B. infantis also suppressed systemic immune responses to B. vulgatus in a gnotobiotic murine model (Shiba et al., 2003). This study would indicate that LAB-like B. infantis may protect


21

epithelial layers from invasion by other commensal microbes that are believed to play a pathogenic role in IBD. In recent years we have seen probiotics including LAB being used as an experimental therapy against IBD in humans. In a double-blind, placebo-controlled trial, a probiotic preparation (VSL#3), a cocktail of Bifidobacterium, Lactobacillus, and Streptococcus species, was shown to be effective in preventing flare-ups of chronic ileal pouchitis (Gionchetti et al., 2000). The VSL#3 preparation has also been used for maintenance treatment of ulcerative colitis (Venturi et al., 1999) and in preventing postoperative recurrence of Crohn’s disease. More recently, Gionchetti et al. (2003) showed in double-blind, placebo-controlled trial that treatment with VSL#3 was effective in preventing the onset of acute pouchitis in patients with ileal pouch-anal anastomosis. Promising data have been also obtained with E. coli strain Nissle, which was found to have an equivalent to traditional treatment with mesalazine in maintaining remission of ulcerative colitis (Kruis et al., 1997; Rembacken et al., 1999). On a cautionary note, other studies carried out in humans have shown LAB/probiotic therapy to have little or no effect in the treatment of IBD. While feeding Lactobacillus GG to patients with a history of pouchitis and endoscopic inflammation did change the pouch bacterial flora, the treatment proved ineffective as a primary therapy for a clinical or endoscopic response (Kuisma et al., 2003). Similarly, Lactobacillus GG treatment did not prevent endoscopic recurrence 1 year after curative resection for Crohn’s disease nor did it reduce the severity of recurrent lesions (Prantera et al., 2002). While some studies indicate that probiotics do have potential as therapies against IBD, they have provided little insight into the microbiological and immunological mechanisms that underlie these diseases. A recent study showed that ex vivo production of the proinflammatory cytokine tumor necrosis factor alpha (TNF-) by ileal– mucosal explants surgically removed from Crohn’s patients were down-regulated in the presence of Lb. casei and Lactobacillus bulgaricus but not by E. coli and Lactobacillus crispatus (Borruel et al., 2002). This would indicate that LAB interact differently with immunocompetent cells and have different capacities in modulating the production of pro-inflammatory cytokines such as TNF-, which play a key role in the pathogenesis of IBD. More clinical trials are therefore needed to evaluate different LAB strains as therapeutic preparations against the various manifestations of IBD. These studies must also establish the correct placement and dosage of probiotic required for any treatment regimen. Clearly more research is also needed to characterize and establish the mechanisms underlying the interactions of probiotic

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bacteria with the immune system and the role of intestinal microflora in homeostasis. The generated understanding will be of crucial importance for the future screening of lactic acid bacteria affecting immune functions and the development of recombinant strains with enhanced properties. C. RECOMBINANT LAB

AS A

THERAPEUTIC OPTION

While results from experiments carried out in animals are encouraging, the potential of probiotic therapy against IBD in humans remains uncertain. Various studies have now shown that in addition to proven efficacy against experimental colitis in animals, probiotics including LAB also have other beneficial actions on intestinal mucosa. Unfortunately, these studies have yet to establish a link between the two sets of data, and the mechanisms that underlie the anti-inflammatory action of probiotics remain ill-defined. Until more is known about mucosaassociated microflora and the mechanisms that underlie inflammatory diseases, the use of probiotics in therapy of IBD will remain largely empirical. An alternative approach is to genetically engineer LAB to produce and deliver to the intestinal mucosa, molecules that have a therapeutic activity against IBD. Steidler and colleagues (2000) reported the construction of a recombinant Lb. lactis strain genetically engineered to secrete interleukin-10. Intravenous administration of IL10 has previously shown clinical efficacy in the treatment of Crohn’s disease (van Deventer et al., 1997) but can cause side effects that prevent long-term use. In addition, IL-10 is sensitive to acid; therefore intestinal delivery is not an option. In addressing this problem Steidler et al. (2000) showed that Lb. lactis genetically engineered to secrete IL-10 could reduce DSS-induced colitis and prevent spontaneous colitis in IL-10–deficient mice. Unfortunately, only a limited number of molecules or compounds display efficacy against intestinal inflammation when infused in the colonic lumen. Nitric oxide (NO) is one such compound and was shown to reduce inflammation when infused into the colonic lumen of rats (Perner and Rask-Madsen, 1999). Lactobacillus farciminis, which produces NO in vitro, has also been shown to produce NO in the colon and to reduce experimental colitis when given orally to rats (Lamine et al., 2004). Another possibility would be to engineer LAB-secreting antioxidant enzymes such as catalase or superoxide dismutase (SOD), which may prove effective in removing free radicals such as superoxides and hydrogen peroxide produced by leukocytes, which are thought to play a role in amplifying inflammatory responses and subsequent mucosal damage in IBD patients


23

(Babbs, 1992). Indeed, some LAB possess significant antioxidative activity and are intrinsically resistant to oxidative stress, properties that may help some isolates of LAB to serve as defensive components in the intestinal microbial ecosystem (Kullisaar et al., 2002). While use of recombinant LAB in functional foods would not be acceptable, their use as therapeutics may be foreseen where the potential benefits greatly outweigh any potential risks. This is particularly true of IBD for which current treatments are unsatisfactory and the development of new and innovative therapies is urgently needed. The recent approval by Dutch authorities to use genetically engineered Lc. lactis secreting IL-10 as an experimental therapy for humans with IBD gives encouragement for the further development of therapies based on recombinant LAB (Steidler et al., 2003). V. LAB as Cell Factories for the Manufacturing of Pharmaceutical Proteins Several aspects are considered when choosing a host and a gene expression system for the production of proteins for pharmaceutical use (‘‘pharmaceutical proteins’’). These include cost of production, yield, purity, formation of biologically active molecules, and possible contamination by toxic substances either produced by the host cell or present in the growth medium. In addition, commercial production of a pharmaceutical protein and its production process (including applied tools and unit operations) requires approval from the regulatory authorities. These processes must, therefore, follow good manufacturing practices (GMP), ensuring that the production organism and associated manufacturing procedures and materials are well-characterized and documented. When considering LAB for commercial production of pharmaceutical proteins, most issues that need to be addressed are common to all biological production systems and can be divided into four categories relating to (1) the microbial host; (2) the expression vector; (3) propagation, fermentation and initial downstream processing; and (4) quality control of the product. The latter includes testing for the proper activity as well as stability and purity and will not be discussed here, because it is specific to each product. A. THE CHOICE OF MICROBIAL HOST E. coli was the first microorganism to be utilized for the production of pharmaceutical proteins with recombinant DNA technology (Goeddel et al., 1979a,b; Villa-Komaroff et al., 1978), primarily because this bacterium had been extensively studied in laboratories throughout the world.

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E. coli demonstrates several disadvantages from a technological point of view, such as inefficient protein secretion, formation of inclusion bodies, and production of endotoxin. However, because of precedent from its previous use in producing a long list of approved pharmaceutical proteins, E. coli continues to be the preferred bacterial production host for industrial applications. This tendency to revert to a proven and accepted production system seems to have hindered the adoption of LAB as a production host. Although LAB demonstrate several features that make them ideal for producing certain products, pharmaceutical proteins produced by these bacteria have yet to be approved and brought to market. However, it is expected that this bottleneck will be eliminated in the present decade, since several proteins produced in lactic acid bacteria are currently being tested in clinical trials. For regulatory approval of the recombinant product, a historical description of the host strain is required (i.e., description of how the strain was isolated, characterized, and subsequently treated and manipulated in the laboratory prior to use). This should include a risk evaluation describing known and potential application risks and documenting any record of infections and diseases caused by the same species as the selected host strain. Finally, the evaluation should also deal with host strain stability and potential for genetic exchange with other bacteria (e.g., natural competence for DNA uptake). As many LAB are already used in food production and preservation and more recently as probiotics, many of the aforementioned requirements for regulatory approval are already in place for selected strains. Genome sequences are now available for an increasing number of LAB (Klaenhammer et al., 2002), and various postgenomic studies involving DNA arrays (van de Guchte et al., 2002) and 2-D gel electrophoresis (Champomier-Verges et al., 2002; Guillot et al., 2003) have already been carried out with different LAB under different conditions. These approaches can be used to compare production strains with their corresponding source strain, thus contributing to more-robust risk assessment procedures capable of predicting an undesirable effect. Similarly, comparative genomics and other bioinformatic approaches can also be used to identify potential conjugation or mobilization genes, which can be subsequently deleted in order to minimize gene transfer and its associated risks. B. THE EXPRESSION VECTOR The vector for expression of a desired gene can be chromosomally integrated or plasmid borne. In addition to unnecessary and redundant DNA, it is essential that all genes dedicated to gene transfer are


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eliminated. Similarly, if a plasmid is used, the origin of replication should ideally replicate in one or few species only. The entire DNA sequence of the plasmid or any integrated DNA must be determined to trace recombinant DNA during downstream processing as well as in the final product. In addition, the segregational and structural stability of the chromosomal insert or expression plasmid in a production host must be analyzed to determine if it is stable. Usually a selection marker is required for maintenance of the plasmid carrying the desired gene. Previously the regulatory authorities have approved the use of antibiotic selection markers. However, future approvals are expected to include nonantibiotic selection markers only (see Section VI) because of the growing concerns regarding resistant bacteria whose emergence appears to correlate with the extensive use of antibiotics. The ideal promoter for gene expression should fulfil several requirements (Makrides, 1996). While in most cases the activity of the promoter should be as high as possible to produce the greatest amount of protein, high levels of constitutive expression can be lethal to the host cell or result in inhibition of growth, leading to loss of the expression vector or structural instability of the recombinant DNA. An inducible promoter is therefore preferable to coordinate protein production with cell growth in a way that maximizes the ratio of protein yield relative to biomass. Background transcription should be minimal during the growth phase to produce sufficient biomass, at which point induction of the promoter should initiate a burst of gene expression. The last decade has seen the development of elegant and efficient tools for genetic manipulation and gene expression in Lc. lactis, the most extensively studied member of the entire LAB group (reviewed in Section II). For example, the P170 expression system (Fig. 2) utilizes the regulated promoter P170, which is activated in Lc. lactis on transition from exponential to stationary phase (Israelsen et al., 1995b; Madsen et al., 1999b). Consequently, the growth phase is separated from the protein production phase that occurs only when biomass has reached a maximum. The P170 system also utilizes the lactococcal replicon (repB) and the lactococcal hom and thrB genes, thus permiting auxotrophic selection when using a hom-thrB-deficient production strain (Glenting et al., 2002; Madsen et al., 1996). The system has been further adapted to include DNA encoding the SP310mut2* lactococcal secretion signal (Ravn et al., 2000, 2003). The secretion signal directs the preprotein to the translocation apparatus, where it is cleaved off thus releasing the mature protein into the growth medium. Pharmaceutical proteins secreted into the growth medium can be easily purified, thereby reducing downstream processing and potential cost of production.

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FIG. 2. Expression vector pAMJ1223. The P170 promoter, signal peptide SP310mut2*, the auxotrophic selection marker hom-thrB, and the Lc. lactis replicon repB are indicated. The multiple cloning site is located between the signal peptide and the transcription terminator.

DNA microarray and proteomic characterization of global regulatory responses to different environmental stimuli in Lc. lactis (Champomier-Verges et al., 2002; Guillot et al., 2003; van de Guchte et al., 2002) should help the development of new expression systems that are activated under industrial conditions. In addition, Lc. lactis strains are now available that lack extracellular proteases, resulting in reduced degradation of secreted proteins (Madsen, personal communication; Miyoshi et al., 2002). Combining appropriate expression systems with such mutant strains could significantly improve production and secretion of pharmaceutical proteins, making Lc. lactis increasingly attractive as an industrial production host. C. PROPAGATION, FERMENTATION, AND INITIAL DOWNSTREAM PROCESSING The production strain must be propagated into growth medium (propagation medium) that contains no toxic or harmful substances and ensures the stability of the strain. Once the production strain has been characterized with respect to critical parameters (e.g., composition of heterologous DNA, genetic stability, productivity, and byproducts), minimally passaged cultures are used to generate master and working cell banks that are checked at regular intervals.


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LAB produce high amounts of lactic acid with a concomitant pH decrease that inhibits growth of the bacteria. It is therefore important that the fermentation medium is properly buffered or pH controlled to achieve higher cell densities. Alternatively, strains may be developed that either produce low amounts and/or show a high tolerance to lactate. It may also be possible to use a growth medium that produces low amounts of lactate. In any biological production system, the growth medium must be optimized according to product yield and purity. For example, fully synthetic growth media optimized for the P170 controlled expression system has now been developed, comprising commercially available components that meet the standards of United States Pharmacopoeia (USP) or the European Pharmacopoeia (Ph. Eur.). Depending on the protein being produced, the pH of the medium in the fermenter can be adjusted between 4.5 and 6.5. Fermentation is carried out as a fed batch culture in which potassium hydroxide and glucose are added automatically. Following inoculation and determination of exponential growth, the fermentation process requires only limited surveillance, since the P170 system automatically induces gene expression according to the pH and growth phase. As shown in Fig. 3, the protein produced is stable when secreted into the supernatant and can therefore be isolated over an extended time range for subsequent downstream processing. Once the fermentation process is completed, cells are separated from culture supernatant containing

FIG. 3. Data from a fed batch fermentation of a recombinant Lc. lactis strain that secretes the Staphylococcus aureus nuclease. (A) Optical density during the course of fermentation. Samples for analysis of the produced nuclease were taken at the indicated time-points (1–9). In (B), 10 l crude culture supernatant from each time-point was analyzed by SDS-PAGE. The lane numbers correspond to the time-points shown in (A). The gel was stained with Coomassie brilliant blue. Molecular weights (in kilodaltons) are indicated at the right, and the triangle indicates the position of the secreted nuclease.

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the pharmaceutical protein. The supernatant can then be concentrated by dia-filtration in readiness for downstream purification and formulation. A risk evaluation of the P170 Expression System and host strains has already been carried out by the independent Danish Toxicology Centre, which is now being used as a guide by a number of commercial clients to meet with demands from regulatory authorities (Israelsen, personal communication). VI. Engineering LAB for Their Safe Use in Humans The use of genetically modified microbes (GMMs) in humans raises concerns about their survival and proliferation in the environment. Microorganisms have evolved highly efficient systems for horizontal gene transfer such as transformation, conjugation, retromobilization, and transduction to improve their adaptation to changes in their ecological niche. Therefore the transfer of recombinant DNA such as antibiotic resistance markers or other genetic modifications from a well-characterized transgenic microorganism to uncharacterized indigenous species is perceived as a significant risk that must be minimized (Gruzza et al., 1993; Netherwood et al., 1999; Ramos et al., 1995; von Wright and Bruce, 2003). In the context of recombinant live vaccines, for example, a possible scenario could be the transfer of a cloned virulence determinant from a live vaccine strain to a pathogen potentially reinforcing its ability to cause infection. Apart from the transgene of interest, the ideal GMM for use in humans should therefore contain the minimal amount of foreign DNA and must not include an antibiotic resistance marker. Furthermore, the possibilities of transgene horizontal transfer must be minimized, and GMM lethality should be achieved in an unconfined environment. A. FOOD-GRADE SYSTEMS IN LAB FOR PLASMID MAINTENANCE AND CHROMOSOMAL INSERTION A large number of safe and sustainable food-grade systems for genetic modifications of LAB have been developed (initially for Lc. lactis), including endogenous cloning vectors, selection markers for plasmid maintenance, inducible high-level expression systems, and chromosomal insertion strategies (for a review, see de Vos, 1999b; Renault, 2002). The transgene of interest can either be inserted into the chromosome or cloned into a multicopy plasmid, the latter being sometimes necessary to achieve very high levels of expression such as those required for intracellular expression of antigen in live recombinant


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LAB-based vaccines (Grangette et al., 2001). Complete food-grade plasmid-based expression systems have been successfully developed in LAB that minimize the introduction of foreign DNA (self-cloned systems) (de Vos, 1999b). In these systems, all components ideally originate from the cloning host including replicons, the inducible or constitutive expression/secretion cassette, as well as a reliable selection system for plasmid introduction and maintenance. The selection system is of crucial importance to ensure the stability of the transgenic strain during the fermentation processes that precede administration. Nonantibiotic food-grade markers developed for LAB can be grouped in two categories based on the selection method. The first category includes so-called ‘‘dominant markers’’ that do not rely on specific host genes (versatile) and can be readily compared with antibiotic resistance markers. For example, the catabolism of specific sugars (melibiose, sucrose, xylose, starch, and inulin) offers various possibilities for the development of dominant selection markers in Lc. lactis and various lactobacilli (Boucher et al., 2002; Fitzsimons et al., 1994; Hols et al., 1994; Leenhouts et al., 1998; Posno et al., 1991). A second group in this category are genes conferring resistance or immunity (e.g., immunity to bacteriocins [nisI, lafI]) and resistance to cycloserine (alr) and heavy metals (Cdr) (Allison and Klaenhammer, 1996; Bron et al., 2002; Froseth and McKay, 1991; Liu et al., 1996; Takala and Saris, 2002; von Wright et al., 1990). Although dominant markers are convenient, most cannot be used at the industrial scale for safety or economic reasons. Furthermore, their implementation sometimes requires the transfer of numerous genes that can strongly affect plasmid stability or could result in slower growing recombinant strains. The second category includes the complementation markers resulting from specific mutations in the host chromosome, thus permitting a specific plasmid-host combination. Two types of complementation markers can be distinguished in LAB based either on sugar utilization (lactose) or on a specific auxotrophy (pyrimidine, thymine, D-alanine) (Bron et al., 2002; Fu and Xu, 2000; Hashiba et al., 1992; MacCormick et al., 1995; Platteeuw et al., 1996; Ross et al., 1990; Sorensen et al., 2000; Takala et al., 2003). A complementation system for lactose utilization based on the lacF gene has been successfully applied to Lc. lactis on an industrial scale (Kleerebezem, personal communication; Platteeuw et al., 1996). This system, however, uses a growth medium that utilizes lactose as a carbon source, which is not always desirable for implementation in other LAB. Complementation markers based on pyrimidine or thymine auxotrophy have also been described

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that exploit the amber suppressor tRNA gene (supD) and thymidilate synthase gene (thyA), respectively (Fu and Xu, 2000; Sorensen et al., 2000), but again, the requirement for dedicated growth media without these specific compounds represents a major drawback. More recently the use of the alanine racemase gene (alr) as a complementation marker was reported as functional in both Lc. lactis and Lb. plantarum (Bron et al., 2002). Alanine racemase converts L-alanine (L-Ala) into D-alanine (D-Ala), which is essential in cell wall biosynthesis as it is involved in the cross-linking of peptidoglycan. Alanine racemasedeficient strains of both LAB are strictly auxotrophic for D-Ala and undergo cell lysis when starved of D-Ala (Fig. 4) (Bron et al., 2002; Hols et al., 1997a, 1999). Because D-Ala is mainly present in the cell walls of bacteria and concentrations of this compound are extremely low in most growth media, the alr gene offers greater potential as a complementation marker. Significantly, the alr gene can also be used as a dominant marker, since high alanine racemase expression confers cycloserine resistance (Bron et al., 2002). In addition to D-Ala, the peptidoglycan contains other unique compounds (D-glutamate, D-aspartate, meso-diaminopimelate) that are essential and can also be used to supplement growth medium. In the future these compounds and their genes could potentially represent a range of complementation markers that could be used for the development of new food-grade plasmid maintenance or chromosomal integration systems. Although complete food-grade systems based on plasmids have been successfully developed, they often demonstrate intrinsic structural instability and display a high level of potential horizontal gene transfer through mechanisms such as plasmid co-integration and conjugation. To minimize these problems, several food-grade chromosome delivery systems have now been developed for LAB based on homologous recombination (recA-dependent), site-specific recombination (phage-derived systems), retrotransposition (group II intron-derived systems), and transposition (insertion sequence-based systems) (de Vos, 1999b; Frazier et al., 2003; Hols et al., 1994; Maguin et al., 1996; Martin et al., 2000; Romero and Klaenhammer, 1991). With the exception of homologous recombination, all of these delivery systems are based on genetic elements that contribute to genome plasticity and/or horizontal transfer and must be avoided for safe use. Chromosomal integration by homologous recombination can be achieved by a single crossover event (Campbell integration with one homologous region) with a nonreplicating plasmid (suicide vector). Food-grade systems based on this approach have been developed for Lc. lactis by using endogenous plasmids devoid of their repA replication protein (pORIþ plasmids)


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FIG. 4. (A) The effects of D-Ala starvation on viability of wild-type NCIMB8826 (open squares) and the alr deletion mutant MD007 (solid triangles). The zero time-point is set to the beginning of starvation after cell re-suspension in MRS broth not supplemented with D-Ala. The log number of colony forming units/ml CFU ml1 of Lb. plantarum were enumerated over time by dilution and plating on solid medium supplemented with D-Ala. Scanning electron micrographs of the alr mutant MD007 are shown at time zero (B) and after 20 hours (C) in MRS broth lacking D-Ala. The arrows in (C) indicate V-shaped cells that have broken in the middle, resulting in the release of cytoplasmic material.

and food-grade markers of the complementation type (lacF) or the dominant type (scrAB for sucrose utilization) (de Vos, 1999b; Leenhouts et al., 1996, 1998). In some cases, continuous selection for the food-grade marker can result in a chromosomal plasmid amplification of up to 20 copies, thus improving gene dosage (Leenhouts et al., 1998). However, a possible drawback of this system is that replication of the chromosomally integrated plasmid could be reactivated subsequent to horizontal transfer of an incoming plasmid bringing repA in trans. The best strategy for reduced risk of horizontal transfer employs two crossover events whereby the transgene is safely and stably integrated into the chromosome as a single copy and in the absence of additional DNA. This double crossover strategy has recently been used to stably integrate the human interleukin 10 (hIL10) gene into the chromosomal thyA gene of Lc. lactis. Furthermore, the hIL10 gene was translationally fused to the expression signals of the thyA gene to minimize the introduction of foreign DNA. The level of hIL10 expression was reasonably high in comparison to its expression from a

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multicopy plasmid (Steidler et al., 2000, 2003). It is now likely that such a strategy will be used increasingly for the genetic engineering of LAB for other medical applications. B. BIOLOGICAL CONTAINMENT SYSTEMS Biological containment systems can be subdivided into two groups: active systems and passive systems. Active containment is based on the conditional genetic control of either a killing gene (activation) or an essential gene (repression). Various killing genes have been identified in LAB such as those encoded by phage lytic cassettes (lysin or lysin/ holin combination) and DNA restriction systems (de Ruyter et al., 1997; Djordjevic and Klaenhammer, 1996, Djordjevic et al., 1997; Sanders et al., 1997). While a large number of essential genes could potentially be exploited, peptidoglycan biosynthetic genes are particularly attractive, because their inactivation usually results in cell lysis (Curtiss III et al., 1977). Proof of the concept for lysis induction with peptidoglycan hydrolases (lysins) and biosynthetic genes (alr; D-ala-D-ala ligase, ddl) tightly regulated by the nisin-inducible system has already been achieved in the laboratory by using Lc. lactis and Lb. plantarum (Bron et al., 2002; LABDEL project; Prozzi and Fontaine, unpublished data). To be implemented in humans, these active containment systems would require expression signals that are tightly controlled by environmental parameters. These expression signals could either be activated/repressed in the host or subsequent to release of the recombinant strain into the environment. A range of expression cassettes have been isolated from Lb. plantarum that are specifically activated in the GI tract of mice (Bron et al., 2004a; LABDEL project) (see Section VII). These expression systems are now being exploited in Lb. plantarum to specifically induce lysis and consequent release of a biomolecule of interest (therapeutic molecules) into the GI tract via the control of lytic cassettes. A drawback to using active containment systems is that mutations can occur that can inactivate a killing gene/compound or result in constitutive expression of the essential gene (Hols, P., unpublished data; Molina et al., 1998; Szafranski et al., 1997). However, it may be possible to minimize these problems by combining more than one system in a defined recombinant strain (Ronchel and Ramos, 2001). Passive containment systems, by contrast, are robust and very simple and based mainly on complementation of an auxotrophy by supplementing an essential metabolite. This metabolite is ideally not available or occurs in extremely low amounts in the environment. Furthermore, if the compound must be added to growth medium for


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inoculum preparation, it is preferable that it occurs at a low cost to facilitate future industrial applications. Several mutant strains used for plasmid maintenance via complementation markers also offer potential as passive containment systems. These include two auxotrophies that have a high potential for human application since they are bactericidal. Alanine racemase mutants that demonstrate a requirement for D-Ala can be obtained in a large number of bacteria including LAB (Bron et al., 2002; Hols et al., 1997a). D-Ala starvation results in cell death via a lysis process, and experiments are currently underway for containment of S. mutans GMMs designed for anti-caries therapy (Hillman, 2002). The second bactericidal auxotrophy is based on a thyA mutant of Lc. lactis that has recently been evaluated for its efficacy in a pig model (Steidler et al., 2003). Thymine starvation results in activation of the SOS repair system and DNA fragmentation, thus constituting an intrinsic suicide system (Curtiss et al., 1977). Exploiting the thyA gene as a passive containment system by replacing it with a transgene of interest offers additional advantages in terms of biosafety (Steidler et al., 2003). While any reversion of the thyA mutation through homologous recombination with a Lc. lactis thyA gene that might be acquired by horizontal transfer would restore a wild-type phenotype, it should also result in loss of the transgene. Furthermore, thyA mutants of Lc. lactis are severely impaired in phage replication, further reducing the risk of horizontal transfer by phage transduction (Pedersen et al., 2002). This elegant system has recently been applied to the delivery of hIL10 by live Lc. lactis bacteria and has now been approved for use in humans (Steidler et al., 2003).

VII. Opportunities and Potential Applications of Future Research A. INSIGHTS FROM GENOME SEQUENCING AND COMPARATIVE GENOMICS The relatively small genome size of bacteria, combined with the availability of high-throughput sequencing facilities, has stimulated the determination of many bacterial genomes. Over the past decade, the sequences of more than 90 bacterial genomes have become available in the public domain. These efforts have focused primarily on pathogenic bacteria and have included the completion of several genome sequences of food-borne pathogens (Schoolnik, 2002; Wells and Bennik, 2003). However, in more recent times, the genomes of food-grade bacteria have received considerable attention and genome sequences (including partial sequences) are now available for more than 20 LAB as well as a number of related species (Klaenhammer et al., 2002).

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FIG. 5. Phylogenetic tree constructed by using 16 S rDNA sequences of LAB and related bacteria that have been completely or partially analyzed at the genomic level. It should be noted that for a number of species the genome sequence has (partially) been determined for more than one strain of this species. The estimated genome sizes are indicated between brackets. Complete genomic sequences (and thus their exact sizes) are available for the paradigm Gram-positive bacterium Bacillus subtilis (Kunst et al., 1997) and the LAB, Lactococcus lactis subspecies lactis IL1403 (Bolotin et al., 2001) and Lactobacillus plantarum WCFS1 (Kleerebezem et al., 2003).

The first complete LAB genome sequence to be published was that of Lc. lactis subspecies lactis strain IL1403 (Bolotin et al., 2001). More recently, a high-fidelity genome sequence has also been published for Lb. plantarum strain WCFS1 (Kleerebezem et al., 2003). In addition, more LAB genomes are nearing completion (Fig. 5; for a review see Klaenhammer et al., 2002) including draft genome information for a number of LAB that was made available in the public domain in 2002 by the Joint Genome Institute (ftp://ftp.jgi-psf.org/pub/JGI_data/ Microbial/) in collaboration with the lactic acid bacteria genomics consortium. Comparative genomics will be used to determine the evolutionary relationships between different species of LAB and allow analysis of genomic diversity among different strains and closely related species. Studies on pathogenic bacteria have already shown that comparative genome analysis and microarray-based studies of genome composition can indeed reveal genetic factors linked to specific functions or


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plasticity regions associated with lifestyle properties of specific strains (Israel et al., 2001; Pearson et al., 2003; Porwollik et al., 2002; Salama et al., 2000). In the case of LAB, it is already well established that some species are genetically diverse and that strain adaptation is rapid. Moreover, horizontal gene transfer appears to be the rule rather than the exception and is especially important for genes or functions related to mobile genetic elements such as plasmids, transposons, and bacteriophages. Recently, genomic variation within a single LAB species was exemplified by a detailed genome diversity study of Lb. plantarum isolates using DNA microarrays (Molenaar et al., 2003). In addition to high levels of genome diversity, Molenaar et al. (2003) also identified specific genomic regions of relatively high plasticity that may represent lifestyle adaptation islands. These findings show that genomic diversity is a key determinant of bacterial functionality and indicate that specific approaches are required to fully exploit the collective genomic potential of LAB. B. THE BEHAVIOR OF LAB

IN THE

HOST

The human GI tract is colonized by a vast, complex, and dynamic population of commensal microorganisms including food-associated bacteria that contribute to nutrient processing, affect the host’s immune function, and stimulate a variety of other host activities. Molecular approaches based on 16 S rDNA sequencing and profiling of dominant GI tract microbiota have revealed that the majority of commensals belong to unknown, ‘‘novel’’ bacterial species that so far have not been studied under laboratory conditions (Zoetendal et al., 1998). In addition, these studies revealed that while the microbial composition was relatively stable in individual adults, it varies significantly between different individuals (Zoetendal et al., 1998, 2002). Intriguingly, the composition of dominant GI tract microbiota was also shown to be affected by specific host-microbe interactions that appear to be related to the host genotype (Zoetendal et al., 2001). Similar molecular approaches as well as classical cultivation experiments have also shown that several food-grade bacteria are encountered as natural inhabitants of the human GI tract. In particular, certain Lactobacillus species are frequently found among the natural intestinal microbiota and would appear to be relatively numerous in the upper regions of the small intestine (Ahrne et al., 1998; Heilig et al., 2002; Walter et al., 2000, 2001). The increasing availability of genome sequences for several intestinal species of LAB opens new avenues to study their functionality in the gut, including interactions with host cells. For example,

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when ingested, Lb. plantarum WCFS1 was shown to effectively survive passage of the human stomach and to reach the ileum in high numbers as compared with other LAB strains (Vesa et al., 2000). In addition, the bacterium could also be detected in the colon. These characteristics and the availability of its genome sequence (Kleerebezem et al., 2003) render this strain a suitable candidate for the study of bacterial behavior in the GI tract. To date, DNA microarray and proteomic studies carried out in LAB have been confined to in vitro experiments investigating global regulatory responses to different environmental stimuli (ChampomierVerges et al., 2002; Guillot et al., 2003; van de Gucht et al., 2002). Similarly, studies investigating host-LAB interaction have focused primarily on physiological aspects such as intrinsic levels of acid and bile tolerance (Chou and Weimer, 1999; Hyronimus et al., 2000) and the development of complex media to selectively enrich for LAB species that are tolerant to digestive stress (Shah, 2000). For example, a genome-wide genetic screen carried out in Lb. plantarum identified 31 open reading frames (ORFs) whose expression would appear to be induced by bile acids, including genes encoding efflux pumps as well as several membrane and cell wall–associated functions (Bron et al., 2004b,c). While such in vitro experiments might unravel responses by specific microorganisms to certain GI-tract conditions, they will not suffice in portraying bacterial behavior in the GI tract. The full response repertoire will be triggered only in vivo, where all physicochemical conditions are combined with specific host-microbe and microbe-microbe interactions. This notion has led to development of more sophisticated in vivo approaches aimed at identifying bacterial genes that are important during residence in the GI tract. Three main strategies have been developed for the identification of genes that are highly expressed in vivo as compared to laboratory conditions, that is, (recombination-based) in vivo expression technology ((R-)IVET), signature tagged mutagenesis (STM), and selective capture of transcribed sequences (SCOTS). The basic characteristics and relative (dis)advantages of each of these approaches have recently been reviewed (Mahan et al., 2000), but because of their recent application in LAB, (R-)IVET approaches will be explored in somewhat more detail here. To date, four variations of IVET utilizing different reporter genes have evolved that allow the selection of genomic sequences that harbor promoters that are specifically induced in situ, for example, during GI-tract passage (for a review see Angelichio and Camilli, 2002; Mahan et al., 2000). The (R-)IVET approach relies on the generation of transcriptional fusions of random genomic fragments to a promoterless


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reporter gene encoding an enzymatic activity. In the decade following the first report describing IVET technology, this technique has solely been used for the identification of genes that were up-regulated during the infection processes of a number of pathogenic bacteria (Angelichio and Camilli, 2002; Mahan et al., 2000). Recently, however, two reports have described for the first time, the application of (R-)IVET technology to food-grade LAB. An IVET strategy based on in vivo selection of an antibiotic-resistant phenotype was used to identify three in vivo induced (ivi) genes in Lb. reuteri that were expressed at induced levels during colonization of the GI tract of reconstituted Lactobacillus-free mice (Walter et al., 2003). Interestingly, one of these genes encoding a peptide methionine sulfoxide reductase has previously been identified by using IVET in the non-food-associated bacterium S. gordonii during experimental endocarditis (Kili et al., 1999). Although not noticed by the authors, this finding gives a first glimpse into potential overlap that may exist between genetic responses triggered by both pathogenic and non-pathogenic bacteria subsequent to host interaction. A disadvantage of this approach, however, is that it required antibiotic selection pressure during host transit, which was achieved by administering antibiotic to the mice during the course of the experiment. This treatment would have been detrimental to native intestinal microbiota already present in these mice, and it is highly likely that the in vivo conditions encountered by Lb. reuteri would have been significantly changed. More recently, Bron et al. (2004a) have reported the successful application of a second (R-)IVET approach for the identification of ivi genes in Lb. plantarum. In contrast to other IVET systems based on selection of an antibiotic marker, this approach employs the irreversible enzymatic activity of resolvases as a reporter and screening tool. In this system, an antibiotic resistance marker flanked by two resolvase-recognition sites (Fig. 6: loxP-ery-loxP cassette) is integrated into the chromosome of the bacterium of interest. In addition, a promoterless copy of the corresponding resolvase-encoding gene (Fig. 6: cre) is introduced on a plasmid and used as a reporter to trap transcriptional activation (promoter activity) by monitoring changes in the antibiotic resistance phenotype. Since promoter activation leads to irreversible excision of the antibiotic resistance marker, this strategy does not rely on selective pressure during animal experiments. This form of (R-)IVET can therefore be used to monitor induction of bacterial gene expression in a live host possessing an intact unmodified GI tract. Moreover, this strategy is the only IVET approach that functions as a genetic screen as it discards all promoters displaying activity under laboratory

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FIG. 6. Schematic representation describing the (R)-IVET screen performed with Lactobacillus plantarum (Bron et al., 2004a). A loxP-ery-loxP cassette is introduced in the Lactobacillus plantarum chromosome. A library of random Lactobacillus plantarum chromosomal fragments is constructed in pIVET-cre, upstream of a promoterless copy of the cre resolvase gene. Expression of cre leads to excision of the loxP-ery-loxP cassette and erythromycin sensitivity. An erythromycin-resistant sub-library is selected under laboratory conditions and administered to conventional mice. The complete sub-library is recovered from fecal samples by selection for the pIVET-cre encoded chloramphenicol resistance and replicated to select erythromycin-sensitive clones (CmR, EmR). These clones contain pIVET-cre derivatives that harbor a GI tract activated promoter in the Lactobacillus plantarum chromosomal fragment cloned upstream of cre, since loxP-eryloxP excision has occurred during transit through the host intestine. Sequencing of the pIVET-cre inserts is used to identify the corresponding ivi genes of Lactobacillus plantarum (Bron et al., 2004a).

conditions, since the (R-)IVET library is prepared under antibiotic selection pressure prior to administration to the host (Fig. 6). Application of this genetic screen in Lb. plantarum resulted in the identification of 72 genes that were induced during passage of the GI tract in conventional mice (Bron et al., 2004a). A diverse range of functions was predicted for these 72 ivi-genes, including nutrient acquisition, intermediate and/or co-factor biosynthesis, stress response, cell surface proteins, and a number of (conserved) hypothetical proteins. Remarkably, one of the hypothetical proteins identified in this study displays significant homology (32% identity) to the conserved hypothetical protein that was identified by using IVET in Lb. reuteri (Bron et al., 2004a). Moreover, a large number of the functions and pathways identified


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by (R-)IVET in Lb. plantarum have also been described in pathogenic bacteria, where they have also been shown to play an important role during infection. The striking parallels that exist between ivi responses exhibited by pathogens and nonpathogens in vivo may well be explained by the importance of these genes for survival rather than pathogenesis during host residence (Bron et al., 2004a). These studies represent an important step toward elucidating the behavior of food-grade LAB in the complex environment they encounter after ingestion by the consumer. Approaches such as IVET will provide the genetic tools necessary, including the required promoters, that will allow the development of dedicated LAB-based delivery vehicles that will express only desired pharmaceutical proteins in situ. Moreover, these approaches should provide a more geographical and precise insight into the exact locations of ivi-gene activation in the GI tract that may allow the construction of a new generation of site-specific delivery vehicles. Eventually, ivi-promoters could be combined with certain genes (e.g., bacteriophage-derived lytic cassettes) to develop LAB delivery systems that exhibit controlled release of their cellular content (including desirable molecules) at a specific location in the GI tract. IVET and other genomic-based approaches will also provide a better understanding of LAB behavior in the GI tract as well as in other environmental niches. They should facilitate the genetic engineering of mutant strains that can be compared with their wild-type counterparts in appropriate in vitro and in vivo models. Ultimately, it is hoped that these approaches will help define potential probiotic functions and provide a molecular basis to support and explain health benefits that are associated with LAB and related species. C. THE HOST RESPONSE TO LAB The ability of LAB to alter the function of the systemic and immune responses has been the subject of intense research over the last few years. Some LAB have been shown to translocate across the mucosal epithelium of the GI tract, allowing them to come into direct contact with the underlying immune cells and influence immune responses. There is also evidence that some LAB can directly stimulate the immune system at the gut mucosal surface via localized GI tract lymphoid cell loci, increasing lymphocyte populations and cell surface expression in the gut-associated lymphoid tissue (GALT) environment, and facilitate increased immunoglobulin output into the intestinal lumen. It is also becoming increasingly clear that interactions of different LAB with host cells in the mucosa can have different effects on immune

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function. Administration of both viable and/or heat-killed preparations of the probiotic Lb. acidophilus strain UFV-H2b20 has been shown to enhance phagocytic activity in germ-free mice and to improve clearance capacity against circulating E. coli as compared with controls (Neumann et al., 1998). Experiments carried out in mice have shown that oral administration of different LAB increased IgA-producing cells associated with the lamina propria (Vitini et al., 2000). However, this increase did not always correlate with an increase in CD4þ T lymphocytes, suggesting that these bacteria only induced clonal expansion of cells in the lamina propria that were already triggered to produce IgA. In addition, while some LAB did increase macrophages, neutrophils and eosinophils indicative of an inflammatory response, CD8þ T cell populations were diminished or not affected. Other studies carried out in animals and humans have also shown that the administration of various Lactobacilli and Bifidobacteria can significantly increase total IgA as well as antigen or pathogen-specific IgA, thereby contributing to mucosal resistance to gastrointestinal pathogens (Cukrowska et al., 2002; Fang et al., 2000; Fukushima et al., 1998; Herias et al., 1999; Tejada-Simon et al., 1999). In one of these studies, co-administration of Lb. plantarum and an E. coli strain to germ-free rats resulted in significantly increased densities of CD25þ cells in the lamina propria and decreased proliferative spleen cell responses to E. coli (Herias et al., 1999). CD 25þ cells have been shown to be involved in tolerance and down-regulation of immune responses to self and nonself antigens. While the production of IgA may be important in preventing infections by pathogens, it may also be considered anti-inflammatory because it involves mechanisms controlled by Th2 effector cytokines that are more associated with tolerance. These studies have been supported by work carried out by Kirjavainen et al. (1999a) that showed that administration of selected LAB strains could reduce T cell reactivity in mice in a dose-dependent fashion (Kirjavainen et al., 1999a,b). Similarily, in vitro experiments have shown that LAB or their components can inhibit mitogen- and antigen-induced T lymphocyte proliferation and down-regulate both Th1 and Th2 effector cytokines (Chen et al., 2002; Pessi et al., 1999; von der Weid et al., 2001). In addition, LAB have been reported to induce IL-10 production in gut epithelium cells (Haller et al., 2000) and in peripheral blood mononuclear cells isolated from both animals and humans (Miettinen et al., 1996; Vinderola et al., 2004). Interestingly, von der Weid et al. (2001) also identified a Tr1-like population of CD4þ T cells with low proliferative capacity that produced substantial levels of the regulatory cytokines IL-10 and TGF-. These studies would indicate that


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mechanisms controlled by LAB may play an important role in maintaining immune homeostasis and, in particular, maintaining tolerance to the indigenous microbial population. This is supported by experiments in animals that show that select strains of LAB induce IL-10 and suppress Th1-related immunopathologies such as IBD. Conversely, LAB have also been shown to increase lymphocyte proliferation as well as the production of inflammatory cytokines. Experiments carried out by Aattour et al. (2002) in pathogen-free rats showed that oral ingestion of yogurt containing S. thermophilus and Lb. bulgaricus resulted in increased in vitro proliferation of lymphocytes isolated from peripheral blood, spleen, and Peyer’s patches that, for the two latter lymphoid compartments, were accompanied by increased interferon- production. Similarly, Gill et al. (2000) showed that feeding various LAB strains to mice significantly enhanced mitogen-induced proliferation of splenocytes and production of IFN-, increased phagocytic activity of peripheral blood leukocytes and peritoneal macrophages, and enhanced serum antibody responses to antigen administered by the oral and systemic routes. Moreover, numerous studies have also shown that certain LAB or their components are potent inducers of Th1 cytokines (Cleveland et al., 1996; Kato et al., 1999; Maassen et al., 2000) and can decrease Th2 responses (Marshall et al., 1995; Murosaki et al., 1998; Repa et al., 2003; Shida et al., 2002). Similarly, studies carried out in animals and humans have demonstrated that ingestion of dietary LAB can prevent atopy and reduce the development of allergy (Bjorksten et al., 1999; Kalliomaki et al., 2001; Repa et al., 2003; Sepp et al., 1997; Simhon et al., 1982). These studies would indicate that LAB can enhance natural and acquired immunity and may be beneficial in optimizing or enhancing immune responses in healthy and immunocompromised individuals (Gill et al., 2000). In addition, counter-regulatory properties based on their Th1-inducing capacities could provide a useful strategy against Type I allergy (Repa et al., 2003) (see also Section III). It has been demonstrated that different strains of LAB have substantially different capacities to induce IL-12 and TNF- production in dendritic cells (DCs) (Christensen et al., 2002). In addition to exerting less-pronounced effects on IL-6 and IL-10 production, all strains upregulated expression of surface MHC class II and B7-2 indicative of DC activation and maturation. In a separate study, significant functional and phenotypic dichotomy was also observed in DCs exposed either to Lb. rhamnosus or Klebsiella pneumoniae (Braat et al., 2003). Interestingly, compared to K. pneumoniae, Lb. rhamnosus induced lower TNF-, IL-6, and IL-8 production in immature DCs as well as lower IL12 and IL-18 production in mature DCs that were also associated

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with the development of T cells without a typical Th phenotype. Significantly, Christensen et al. (2002) also showed that at least one species of Lactobacillus was capable of inhibiting the activities of other Lactobacillus species with respect to DC activation and production of cytokines. These studies demonstrated that gut microflora, and in particular LAB, can differentially modulate immune responses by stimulating DC activation and function, which in turn may determine whether the DC population may favor a Th1 or Th2 response as well as tolerance. It is now clear that different LAB possess intrinsically different immunoadjuvant capacities that can play a significant role in modulating immune responses. Germ-free animal models will help elucidate the interactions that occur between lactic acid bacteria and the host. This includes their ability to induce innate immunity that has not yet been appropriately compared in germ-free and conventional animals. Like other commensals, lactic acid bacteria will express various commensal associated molecular patterns (CAMPs) that are able to recognize and activate mucosal cells (including epithelial cells) through pattern recognition receptors (Funda et al., 2001; Janeway and Medzhitov, 2002). Recombinant mutants could be compared with wild-type strains to identify specific components, lipoteichoic acid and peptidoglycan for example, which may be responsible for distinct immune responses. The availability of genomic data will accelerate these efforts by facilitating the identification of other key bioactive components such as those displayed on the bacterial surface. Combining post-genomic approaches such as microarrays and proteomics with GF animal models and selective colonization strategies could improve understanding of host-commensal interactions and bring new insights into the mechanisms of mucosal immunity. These efforts can also be combined with ex vivo approaches such as laser microdissection techniques that will allow us to examine individual cellular components as well as general cell populations from different anatomical regions of the immune system. Similarly, these tools can be applied to animal models of human diseases developed under defined gnotobiotic conditions and may help elucidate the etiology of frequent disorders such as those associated with several infectious, inflammatory, autoimmune and neoplastic diseases. These approaches may help to explain why lactic acid bacteria and other commensals do not trigger pathological inflammatory responses in mucosal tissues in normal hosts and may answer intriguing questions regarding their associated benefits.


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VIII. Concluding Remarks The last decade has seen increasing interest in the use of recombinant LAB as mucosal delivery vehicles for vaccines and other therapeutic molecules. It is now clear that sufficient advances in the genetics of LAB have made it possible to construct safe LAB-based recombinant vaccines that are capable of eliciting protection against lethal challenge with toxin or a human pathogen in a relevant disease model. Although multiple doses of LAB vaccines are currently needed to afford immune protection by the intranasal and oral routes of administration, this should not preclude their suitability for use in humans. Multiple doses of the injected DTP and oral polio vaccines (OPV) are already given to infants in the first several months of life and have the potential to be combined with LAB vaccines. Moreover, this technology is relatively inexpensive and could be applied successfully to mass-vaccination programs that are particularly relevant in developing countries. There are also opportunities to enhance the efficacy of LAB vaccines through increased antigen expression or through the combined delivery of multiple immunogens and specific adjuvants. Further insights may be gained through direct comparisons of LAB strains with different persistence and survival characteristics or immunostimulatory properties with different immunization routes or schedules against a selected target disease. Genetic engineering clearly has the potential to further optimize the survival characteristics of selected LAB, define optimal placement and dosage regimes in different clinical settings, and enhance their ability to deliver a pharmaceutical protein. Ultimately, we hope that LAB-based delivery technology will add a new dimension to vaccine development with respect to safety, potential for use in continual vaccination stratagems that encompass both control and eradication of a disease, and increased flexibility and potential use against a wide range of diseases. Opportunities for developing more effective LAB vaccines may also arise from ongoing research on the fate of LAB in the human host and the interaction of LAB with the cells and tissues of the immune system. Genetic engineering may help elucidate the mechanisms by which probiotic strains of LAB exert positive effects on human health and facilitate the identification of the key bioactive components responsible for conferring probiotic traits to these bacteria. These efforts will be further supported by ongoing developments in genomic and postgenomic-based approaches and use of appropriate animal models. As well as providing intriguing answers to questions regarding their therapeutic role in conditions such as allergy and IBD, these studies may help clarify the effect of nutrition and genetic background on the

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development of these diseases to establish the true role of these bacteria in human health. It is foreseen that these approaches will enable the selection of appropriate species that might prove most beneficial under certain conditions and facilitate the identification of new probiotics. In addition, it should be possible to determine optimal probiotic compositions, ensuring that they demonstrate synergistic rather than competing activities. Most importantly, these approaches will help establish proven criteria to substantiate the sometimes ‘‘vague’’ health claims of currently available probiotic preparations. In doing so, these criteria will lead to the possibility of creating custom-designed probiotics that are scientifically proven, increase their range of potential applications, and accelerate their development into health products. Currently, many probiotic strains of LAB are marketed and regulated as conventional foods. The regulatory environment surrounding these products is diverse, but national differences are being harmonized by ongoing European legislation. For example, the 1997 novel foods regulation of the EU parliament governs new food organisms and makes recommendations for assessment of foods containing genetically modified organisms (GMOs). In principle, this framework governs new recombinant strains of probiotic microorganisms and requires information on toxicology, genetic stability, potential for genetic transfer in the host, and the effect of the GMO in humans. Specific health claims for the product would also need to be substantiated by studies in humans resembling what is required by regulatory frameworks governing human pharmaceutical products. The future of recombinant LAB as novel therapies for humans can be foreseen where the potential benefits are significant, particularly where the absence of satisfactory treatments for a particular disease necessitates the development of new and innovative therapies. However, there is an urgent need for clarification to differentiate food supplements from medicines for product applications based on probiotic organisms. As well as ensuring the expansion of both these applications, appropriate legislation would allay fears within the food supplement industry and accelerate the development of health products based on recombinant LAB. It is now clear that biotechnological applications of LAB over the past decade has meant that many food-grade systems for genetic modification are in fact already available, including methods for chromosomal integration and controlled high-level gene expression. Moreover, the recent approval of a recombinant strain of Lc. lactis expressing IL-10 for use in trials with human IBD patients provides further encouragement. We are therefore optimistic that as well as the continued development of prototype health products based on recombinant LAB, the next decade will see more of these reach the marketplace as finished products.


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ACKNOWLEDGMENTS The Partners of the LABDEL consortium are grateful for financial support from The European Framework 5 Programme (EC contract QLK3-CT-QLK3-CT-2000-00340). The authors also wish to thank Andy Carter, Wendy Glennison, Sally M. Hoffer, Maria Marco, Esther van Mullekom, Douwe Molenaar, Willem M. de Vos, Anne Mette Wolff, Astrid Vrang, Renata Stepankova, Deborah Prozzi, Marie Deghorain, Laetitia Fontaine, Vassilia Therodorou, Heimo Breiteneder, and Michael Hisbergues for their contribution to the work described. We are also grateful to Therese Hall for help with coordination of the LABDEL project. Pascal Hols is scientific collaborator at FNRS.

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Novel Aspects of Signaling in Streptomyces Development GILLES P.

VAN

WEZEL*

AND

ERIK VIJGENBOOM

Department of Biochemistry, Leiden Institute of Chemistry 2300RA Leiden, The Netherlands *Author for correspondence. E-mail: [email protected]

I. Introduction II. Aspects of Vegetative Growth and Liquid Cultivation of Streptomycetes III. The Switch to Development A. General Considerations B. Submerged Development C. Towards an Aerial Mycelium D. Influence of Carbon Sources on Developmental Signaling IV. Novel Genes in Development A. Discovery of New Developmental Genes B. The ram and amf Gene Clusters C. Novel Regulators of Sporulation: The SsgA-Like Proteins (SALPs) V. Concluding Remarks References

65 67 69 69 70 70 71 72 72 73 78 83 83

I. Introduction Actinomycetes have an unusually complex life cycle, many aspects of which are globally similar to those observed in some lower eukaryotes, which makes them particularly interesting for the study of bacterial development and evolution (Chater and Losick, 1997). Their ability to produce a large array of biologically active natural products, including the majority of antibiotics, as well as many agents with other medical and agricultural merits, makes these organisms also highly relevant from an industrial perspective. One of the best-characterized genera among the actinomycetes is Streptomyces, the subject of this review, with Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans being the most well-studied species. Pioneering work by David Hopwood 40 to 50 years ago established Streptomyces coelicolor as the model system for the genus (Chater, 1999; Hopwood, 1999). The latter organism has become the paradigm for the study of Streptomyces development (Chater, 1998) and antibiotic production (Bibb, 1996), which was helped by a wealth of mutants and the development of a large genetic toolbox (Kieser et al., 2000). Recently the genome sequences of S. coelicolor and S. avermitilis were completed, taking Streptomyces research into the genomics era (Bentley et al., 65 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 56 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2164/04 $35.00

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2002; Ikeda et al., 2003). The closely related species S. lividans is especially important as an excellent expression host for industrial enzymes. Much more distantly related is the streptomycin producer S. griseus, which is of particular genetic interest for two main reasons: the profound and well-studied effect of a signal molecule, the hormonelike A-factor (2-isocapryloyl-3R-hydroxymethyl--butyrolactone) on its development and antibiotic production (first described in Khoklov et al., 1967; reviewed in Horinouchi, 2002), and its ability to sporulate in submerged cultures (Kendrick and Ensign, 1983; reviewed in Fla¨rdh and van Wezel, 2003). On solid media, a germinating spore will produce one or more hyphae, which will grow and branch to form a vegetative mycelium. Exponential growth is achieved by a combination of tip growth and branching, resulting in a complex mycelial network. At this stage, the vegetative hyphae consist of multi-nucleoid syncytial cells separated by occasional cross-walls (Wildermuth, 1970). Then, as colonies develop, an aerial mycelium is produced, with hydrophobic hyphae breaking through the moist surface, erected into the air. This is the start of the reproductive phase, initiated in response to nutrient depletion and the resulting requirement of mobilization. The substrates required for the production of the aerial hyphae are derived from reuse of material such as nucleic acids, proteins, and storage compounds from the vegetative mycelium. Eventually, sporulation-programmed hyphae are formed, producing chains of mono-nucleoid spores, which are released after a poorly understood maturation process. A typical example of colonies of streptomycetes growing on an agar plate is shown in Fig. 1A (see color insert) and a close-up of sporulating aerial hyphae in Fig. 1B. Mutants that fail to develop an aerial mycelium are called bld (bald, reflecting the ‘‘hairless’’ phenotype), and mutants that fail to produce mature grey-pigmented spores are called whi (white, referring to the production of a nonpigmented fuzzy aerial mycelium). Several reviews have been written on the involvement of bld and whi genes in the control of Streptomyces development and aerial hyphae formation (e.g., Chater, 1998, 2001; Kelemen and Buttner, 1998). In this review we focus on recently discovered genes that play an important role in the two main switches in Streptomyces development, namely the ram gene cluster (for transition from vegetative to aerial growth) and the ssgA-like genes (control of the sporulation process). We also discuss how these and other developmental genes allow streptomycetes to respond to changes in the nutritional state of the microenvironment. Understanding these mechanisms is important for fundamental and applied Streptomyces research.

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FIG. 1. (A) Sporulating colonies of Streptomyces ramocissimus. Clearly visible are aerial hyphae (white outer circle) and spores (grey inner circle); the vegetative mycelium lies below the aerial mycelium and is not visible. The brown pigment secreted by the colonies is melanine. (B) Scanning electron micrograph of sporulating aerial hyphae of Streptomyces coelicolor. Photograph courtesy of Dr. H. K. Koerten (Centre for Electron Microscopy, LUMC, Leiden, The Netherlands). Bar ¼ 10 m.

II. Aspects of Vegetative Growth and Liquid Cultivation of Streptomycetes Models for mycelial growth have been worked out for filamentous fungi, and particularly the penicillin producer Penicillium chrysogenum (Krabben, 1997; Nielsen, 1996; Trinchi, 1971). While at the molecular level the processes are very different in actinomycetes

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(prokaryotes) and filamentous fungi (lower eukaryotes), it appears that at the microscopic level these organisms exhibit similar growth forms and hyphal and mycelial growth kinetics (reviewed in Prosser and Tough, 1991). Therefore several of the kinetic models for growth of streptomycetes may be derived from the better-studied fungi (Bushell, 1988). Exponential growth of the mycelium is achieved by a combination of linear (polar) growth, branching, and—particularly in submerged culture—hyphal breakage (Locci, 1980). The frequency of branching is not constant but apparently is dictated by the growth conditions; nutrient-rich conditions favor branching, to optimally profit from the available nutrients in the habitat (typically the soil), whereas under nutrient-depleted conditions branching is reduced and growth is dictated by tip extension, which favors the formation of socalled ‘‘searching hyphae’’ (Bushell, 1988). Interestingly, branching and cross-wall formation (which often coincide) markedly reduce hyphal strength (Wardell et al., 2002), a phenomenon supported by the observation that ftsZ mutants of S. coelicolor are viable and produce unbranched, long and stable hyphae in the absence of cross-walls (McCormick et al., 1994). The relationship between mycelial morphology and stability is particularly relevant for biotechnological applications, because it determines clump size and therefore indirectly also the efficiency of the production process (Bushell, 1988; Wardell et al., 2002). While development is mostly studied in solid-grown cultures, in the industrial production process large-scale liquid cultures are the reality. Unfortunately, it is difficult to translate morphological principles of one culture type to the other, which is at least in part due to the morphological diversity of liquid-grown mycelium. In batch fermentations, variations as large as three to four orders of magnitude (m to cm scale) occur. Analysis of erythromycin biosynthesis in Saccharopolyspora erythraea showed that production took place only in hyphal fragments with a diameter larger than approximately 90 m (Martin and Bushell, 1996). Mixing problems with larger mycelial clumps also negatively affect the production process, because an oxygen and nutrient gradient exists from the surface of the mycelial clump to its center, affecting growth and production (Bushell, 1988; Huang and Bungay, 1973). Therefore, better understanding of the factors affecting growth and morphology would obviously be of advantage for biotechnological applications. Studies in streptomycetes with fluorescently labeled vancomycin or radiolabelled N-acetyl glucosamine (both of which are incorporated into newly synthesized peptidoglycan) revealed that peptidoglycan


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biosynthetic activity primarily occurs at hyphal tips and at branching sites (Daniel and Errington, 2003; Gray et al., 1990; Young, 2003). Also, the addition of penicillins results in defects particularly at the apical sites of the hyphae, and less at the lateral walls, which may therefore be regarded as a relatively inert murein polymer. Several of the penicillinbinding proteins and other proteins involved in the synthesis and integrity of the cell wall become recruited to cell wall construction sites. The first clear example of a protein associated with apical growth is DivIVA (Fla¨rdh, 2003). Considering that it is involved in driving (the initiation of) linear extension and that its overexpression results in erratic branching, it is conceivable that another important role for DivIVA is to coordinate the initiation of new branching points. The Bacillus subtilis DivIVA homolog plays a direct role in septum-site determination by interacting with the MinCD cell division inhibitor (Edwards and Errington, 1997) and was recently shown to interact with the chromosome segregation machinery to help position the oriC region of the chromosome at the cell pole, in preparation for polar division (Thomaides et al., 2001). Streptomycetes lack a homolog of MinC, and the function of the two MinD homolog is unclear, as minD disruptants have no obvious phenotype (McCormick and van Wezel, unpublished data). The high frequency of co-occurrence of septa and branches (a feature also seen in filamentous fungi) suggests coordination between cell division and branching, and it is perhaps DivIVA that may play a role in this coordinating process, although there is no evidence that DivIVA directly affects cell division (Fla¨rdh, 2003).

III. The Switch to Development A. GENERAL CONSIDERATIONS As a result of their mycelial lifestyle, streptomycetes are sessile microorganisms, and in contrast to other bacteria, the mycelium itself cannot migrate to a more favorable environment, such as by chemotaxis. When deprived of nutrients, the mycelium responds by favoring apical (linear) growth over branching (see Section II) and by the onset of morphological differentiation, resulting in the production of exospores. Under nutrient-limiting conditions, lysis of the vegetative hyphae probably provides the nutrients necessary for the construction of the aerial mycelium (Mendez et al., 1985). During this part of the life cycle, several control mechanisms come into play, such as carbon catabolite repression and stringent response, which constitute important sensors of the nutritional state of the environment and have a

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repressing or activating effect on the onset of sporulation, respectively. These global regulatory processes and their impact on development are discussed elsewhere (Hodgson, 2000; Ingram et al., 1995; Kamionka et al., 2002; Takano and Bibb, 1994; Ueda et al., 1999). B. SUBMERGED DEVELOPMENT Typically, ‘‘development’’ refers to solid-culture differentiation (vegetative mycelium, aerial mycelium, spores) (Chater, 1972). In liquid culture, several streptomycetes, with Streptomyces griseus as the most well-known example, also form submerged spores, produced by sporogenic hyphae at the extremities of liquid-grown vegetative mycelium (recently reviewed in Fla¨rdh and van Wezel, 2003). This process is generally triggered by nutritional shift down, although some streptomycetes also produce submerged spores in nutrient-rich cultures. Comparison of the ultrastructures of submerged spores with surface spores failed to reveal significant structural differences. Perhaps counterintuitively, no differences were found either between the sporogenic hyphae in submerged and solid-grown cultures: both were essentially unbranched and thin-walled (Rueda et al., 2001). The only significant difference was in the sheath, which was thinner and less regularly structured in submerged sporulating hyphae. Some regulatory aspects of submerged sporulation are dealt with in the section on ssgA (Section IV.C). C. TOWARDS AN AERIAL MYCELIUM Early developmental (bld) mutants are not only defective in aerial hyphae formation, but also their antibiotic production is strongly affected (either negatively or positively), which links development and secondary metabolism. Most bld mutants fail to produce antibiotics, although some are antibiotic overproducers. By definition, all nonessential genes that are required for aerial hyphae formation are bld genes, and genes required for any of the developmental stages between the onset of aerial mycelium formation and the production of the spore pigment WhiE are called whi genes (Kelemen et al., 1998). There is evidence that several of the bld gene products are part of a signaling cascade. This was discovered by extracellular complementation experiments, where bld mutants were grown in close proximity to each other, without physical contact (Nodwell et al., 1996; Willey et al., 1991, 1993). A low degree of aerial hyphae formation could be restored by one bld mutant to the other, and typically in a unidirectional


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manner. A detailed overview of the possible function of the bld genes, and their role within the putative signaling cascade, has been provided elsewhere (Kelemen and Buttner, 1998; Nodwell et al., 1999). The best known of the bld mutants is bldA, resulting from mutations in the gene for a leucyl tRNA, necessary for the translation of UUAcontaining transcripts (Leskiw et al., 1991a,b). Approximately 150 genes of the S. coelicolor genome harbor a TTA codon (and are therefore dependent on BldA), many of which are involved in the regulation of development or antibiotic production (Bentley et al., 2002). Interestingly, the failure to obtain bldA deletion mutants of S. coelicolor M145 suggests that at least one bldA-dependent gene is essential for growth in this model strain. Thus, while bldA is an important control point on the way to aerial development, at least in M145 it is also required for the translation of nondevelopmental mRNAs. Recently it was discovered that the activity of several Bld and Whi proteins is not confined to a specific developmental stage. Some of the so-called late bld genes—including bldD, bldM, and bldN—are active not only in a stage temporally related to the switch to aerial mycelium formation but are also required much later in the developmental program. For example, bldM and bldN mutants were originally classified as whiK and whiN, respectively, as several point mutants obtained from classical screens were blocked in later stages of aerial development. Gene disruption and expression studies later showed that a low level of WhiK and WhiN activity was required for the onset of aerial mycelium formation, after which they were renamed bldM and bldN, respectively (Bibb and Buttner, 2003; Bibb et al., 2000). D. INFLUENCE OF CARBON SOURCES ON DEVELOPMENTAL SIGNALING The relationship between the nutritional state of the environment and Streptomyces development is underlined by the medium-dependence of several of the bld mutants, which sporulate on minimal medium agar plates with mannitol but not with glucose (Merrick, 1976; Pope et al., 1996), suggesting a role for glucose repression in Streptomyces, mediated through glucose kinase (Angell et al., 1992). Indeed, glkA mutant derivatives of S. coelicolor bldA mutants do sporulate in the presence of glucose (van Wezel, unpublished data). One bld gene that is of particular interest for the link between carbon source-dependent gene regulation and development is bldB. BldB null mutants have a bald phenotype on all carbon sources and fail to produce aerial hyphae or antibiotics under any condition (Merrick, 1976). Furthermore, bldB mutants are defective in catabolite control

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and do not fall in the hierarchy of extracellular complementation exhibited by other bld mutants (Nodwell et al., 1999; Pope et al., 1996; Willey et al., 1993). Therefore BldB possibly constitutes a key control point in the switch to development. The bldB gene encodes an 11 kDa DNA binding protein that likely functions as a homodimer (Eccleston et al., 2002; Pope et al., 1998). BldB belongs to the family of AbaA-like proteins, with six paralogs on the S. coelicolor genome and—similarly to the SsgA-like proteins (Section IV.C)—no clear homologs outside the actinomycetes. Other connections with glucose metabolism have been reported. Ectopic sporulation was observed in an S. coelicolor mutant with a 7.4 kb deletion around the glkA gene (Kelemen et al., 1995), while an S. griseus das mutant produced ectopic spores at regular intervals in the vegetative hyphae, but only on glucose-containing media, providing another example of a carbon source-dependent (conditional) requirement for a developmental gene (Seo et al., 2002). Recently attention has been directed toward the relationship between high-energy tricarboxylic acid (TCA)-cycle intermediates and development. Interruption of aerobic TCA cycle-based metabolism through mutations in citrate synthase or aconitase resulted in irreversible acidification of the medium during growth on glucose, with obvious defects in morphological differentiation and antibiotic biosynthesis. These effects could at least in part be compensated by buffering of the medium (Viollier et al., 2001a,b). This indicates that the outcome of extracellular complementation experiments, such as for the bld mutants, should be carefully evaluated, as the signal passed on from one Streptomyces strain to another could be the result of an exported (protein) factor, but also of changes in pH or medium composition.

IV. Novel Genes in Development A. DISCOVERY OF NEW DEVELOPMENTAL GENES The quest for novel developmental genes required different strategies and deployment of new experimental approaches. Originally, the discovery of developmental mutants by selecting/screening typically resulted from random mutagenesis experiments with ultraviolet (UV)-irradiated or chemically treated cells. A large collection of bld and whi mutants was obtained, which were classified on the basis of morphological characteristics, helped by scanning electron microscopy (Chater, 1972; Hopwood, 1999; Ryding et al., 1999) or transposon mutagenesis (Gehring et al., 2000). Although these studies were extensive, not all the currently known developmental mutants were thus identified.


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The complete elucidation of the S. coelicolor genome (Bentley et al., 2002), and the concurrent advent of the genomics era to Streptomyces research, allowed a directed search for possible or likely developmental genes on the basis of a presumed homology to developmental genes from Bacillus, or on the basis of development-dependent expression profiles (Donadio et al., 2002; Hesketh and Chater, 2003; Huang et al., 2001). However, several important new classes of developmental genes were recently identified on the basis of physiological criteria such as the acceleration of aerial mycelium formation in S. lividans (ram genes, for rapid aerial mycelium; Ma and Kendall, 1994), restoration of development in the presence of glucose (Seo et al., 2002), or complementation of disturbed submerged sporulation of S. griseus mutants (ssgA-like genes, for sporulation of Streptomyces griseus; Kawamoto and Ensign, 1995b). In the following sections we review the complex data generated by several laboratories on two of these examples, namely the ram/amf gene clusters and the ssgA-like genes. B. THE

RAM AND AMF

GENE CLUSTERS

1. Genetic Organization of the Clusters The ram (in S. coelicolor/S. lividans) and amf (in S. griseus) gene clusters are important for the transition from vegetative to aerial growth, as well as for early stages of aerial growth. Surprisingly, different screens using complementation of certain phenotypes by genomic libraries all resulted in the identification of the same gene clusters: accelerated aerial hyphae development in wild-type S. lividans (Ma and Kendall, 1994); complementation of aerial hyphae development in the A-factor deficient S. griseus strain HH1 (Kudo et al., 1995; Ueda et al., 1998); relief of the dependence on increased copper ion levels for development in S. lividans (Keijser et al., 2000); and complementation of the bldJ mutant (Nguyen et al., 2002). The overall similarity between the ram and amf genes is not very high, but the gene organization is strictly conserved. Both clusters consist of five genes (Fig. 2): ramC/amfT for a serine/threonine (ser/thr) kinase (Hudson et al., 2002), ramS/amfS encoding a small peptide, ramAB/amfAB encoding an integral membrane ABC transporter, and a fifth oppositely oriented gene ramR/amfR, encoding a transcription factor of the two-component regulator family. 2. Transcriptional Control by RamR The ramR gene is expressed from a single promoter that is already active in vegetative mycelium and displays its highest activity after approximately 48 hours of growth on solid media, corresponding with

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FIG. 2. Model for the transcriptional control of the ram cluster by the response regulator RamR, and the function of RamCSAB in the signal transduction. On the basis of sequence analysis, the ABC transporter complex RamAB and the serine/threonine kinase RamC are integrally and C-terminally inserted in the membrane, respectively. In this model, there are two possibilities: (1) RamS is phosphorylated by membrane-bound RamC and subsequently exported by RamAB, and the external RamS then provides a developmental signal (‘‘Signal 2’’) to receiver cells; or (2) it is processed and modified to become SapB, a possibility that is strongly supported by the studies of Kodani et al. (2004). In the latter case it is not yet clear whether all processing and modification functions are provided by RamC or whether other proteins are involved. A model for the primary structure of SapB is presented in the top right corner (based on Kodani et al., 2004). The RamS residues that are modified in SapB are indicated between brackets below the SapB sequence. The developmental signal that triggers RamR expression (‘‘Signal 1’’), and the putative target for RamS signaling, are unknown. Dha, didehydroalanine.

the initiation of aerial hyphae production (Keijser et al., 2002). The same authors showed that ramR transcripts are present throughout aerial growth and even during sporulation, although promoter probing assays with xy/E as a reporter of transcriptional activity suggested a sudden drop in ramR promoter activity upon entry of the aerial growth phase (Nguyen et al., 2002). In all screens, enhanced expression of RamR/AmfR accelerated development, without the need of the other ram/amf genes (Keijser et al., 2000;


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Nguyen et al., 2002; Ueda et al., 1993). The important function of RamR was confirmed by mutational experiments, which showed that S. coelicolor ramR disruption mutants were severely delayed in development, with sparse and significantly delayed aerial mycelium formation. S. lividans ramR mutants were even more delayed in aerial hyphae formation than those of S. coelicolor, in line with the idea that the ram cluster is more important for the development of S. lividans than for the development of S. coelicolor (Keijser et al., 2000). Similarly to most other bld mutants, ram mutants have a conditionally nonsporulating phenotype, with normal development on solid media containing mannitol, and a bld phenotype in the presence of glucose. This once more stresses the important role of carbon catabolite control in the decision to switch to development. Transcriptional analysis showed that expression of ramC is dependent on RamR. The target of RamR was identified in studies by O’Connor et al. (2002) and Nguyen et al. (2002), who showed that RamR has at least one binding site in the ramC promoter region. The ability of recombinant RamR to bind in vitro to the sequence upstream of the ramC promoter demonstrated that phosphorylation of D53, which is essential for the in vivo function of RamR, is not required for its DNA binding activity (Nguyen et al., 2002). Similarly, the corresponding amino acid residue in AmfR, D54, was shown to be critical for restoration by AmfR of sporulation to bld mutant HH1 of S. griseus (Ueda et al., 1993). In S. griseus, expression of amfR is under control of Afactor through the A-factor-dependent activator protein AdpA (Chater and Horinouchi, 2003; Ohnishi et al., 2002; Ueda et al., 1998). The factors controlling ramR expression in S. coelicolor and S. lividans are unknown, but considering the relatively normal phenotype of A-factor null-mutants (Takano et al., 2001), it is unlikely that AdpA also plays an important role in the control of ramR. 3. RamC Is Essential for Development The ramC gene did not show up in the screens as an essential gene for the acceleration of aerial hyphae formation. However, constructed ramC null mutants were bald (O’Connor et al., 2002) or severely delayed in aerial hyphae production (Nguyen et al., 2002), demonstrating the importance of the gene for development. Analysis of the ramC gene product predicted that the protein consists of an N-terminal serine/ threonine protein kinase domain and a C-terminal domain with several membrane spanning regions. In line with this prediction, amino acid residues that are invariant and essential in similar kinases were also essential for RamC function, as shown by complementation

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experiments in which a ramC disruption mutant was transformed with constructs expressing single amino acid substitution mutants of RamC (Hudson et al., 2002). It is attractive to speculate that RamC is associated with the membrane through its C-terminal sequence and functions in close proximity to RamAB (Fig. 2 and Section IV.B.4). Several studies demonstrated that ramC expression occurred at the onset of aerial growth and was dependent on RamR (Keijser et al., 2002; Nguyen et al., 2002; O’Connor et al., 2002). The promoter upstream of ramC also directs transcription of ramS and ramAB, suggesting an operon-like organization. However, the transcription of the ramCSAB gene cluster is complex. The ramAB genes are transcribed much less frequently because of a transcriptional attenuator between ramS and ramA (Keijser et al., 2002). Furthermore, the strong accumulation of ramS transcripts and the low level of ramC transcripts, as detected by Northern hybridization experiments, suggests that after processing of the full-length transcript, the ramC mRNA is rapidly degraded, because there is no promoter in the intergenic region between ramC and ramS (Keijser, 2002). 4. RamS and RamAB What could be the target for the RamC/AmfT kinase activity? The ‘S-peptides’ (RamS/AmfS) are promising candidates for three reasons. The peptides contain several conserved serine and threonine residues as potential phosphorylation sites (Fig. 3). Ueda et al. (2002) showed that the S-peptide has a signaling function and the genes are cotranscribed with the kinase genes. A phosphorylation-dependent function of the S-peptide would fit with a role as signaling molecule. The signal is switched on at a time corresponding to the onset of ramC expression, which in turn is activated by RamR, and switched off again as soon as RamC levels drop. Therefore, the signal presumably does not relate to the half-life of RamS. In a hypothetical model, as depicted in Fig. 2, a cascade of events starting with the induction of ramR

FIG. 3. Alignment of sequences predicted for the three known ‘‘S-peptides.’’ Amino acids shared by at least two proteins are boxed. Residues that form potential target sites for phosphorylation are indicated with asterisks. The accession numbers for the protein sequences are: AAD33774 (RamS S. lividans), BAA33539 (AmfS S. griseus), and BAC75213 (AmfS S. avermitilis). The sequences of S. coelicolor and S. lividans RamS are identical.


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transcription by an unknown signal finally results in the transport of the phosphorylated S-peptide across the membrane. Once outside, the S-peptide will signal the onset of development to neighboring hyphae. Support for this model is provided by several experiments. Disruption of ramS in S. coelicolor resulted in a severe delay of its aerial hyphae development (Nguyen et al., 2002), while an amfS disruption mutant of S. griseus had a bld phenotype (Ueda et al., 2002). Extracellular complementation of the amfS mutant was observed when wild-type S. griseus was grown in close proximity. However, neither amfR disruption mutants nor amfAB disruption mutants were capable of extracellular complementation of the amfS mutant. This is supportive of a model in which the extracellular complementing activity is AmfS, which is not produced by the amfR mutant and not exported by the amfAB mutant (Fig. 2) (Ueda et al., 2002). The observation that the amfAB mutant itself shows normal development suggests that sufficient amounts of AmfS are exported (through other transporters) to provide the developmental signal. However, this contradicts the observation that an amfAB mutant cannot complement an amfS mutant. Therefore, generation of the developmental signal by accumulated AmfS in the cytoplasm, in its native or processed form, is more likely. The role of RamAB/AmfAB as transporters seems to be crucial for RamS/AmfS activity, although this is not supported by the phenotype of the corresponding disruption mutants. The observation that a ramB mutant has a bld phenotype (Ma and Kendall, 1994) could not be reproduced by others. The importance of ramAB is demonstrated in S. lividans by the observation that a triple disruption, ramABR, has a bld phenotype, while the ramR mutant in the end does produce some aerial hyphae (Keijser et al., 2000). Interestingly, a synthetic full-length AmfS peptide was not capable of inducing aerial hyphae formation in the amfS mutant, but a synthetic C-terminal octapeptide did induce aerial growth in the mutant (Ueda et al., 2002). 5. Are RamS and SapB the Same Protein? Another small peptide implicated in the onset of aerial hyphae formation is SapB (Willey et al., 1991), which has been suggested to be identical to RamS (Chater and Horinouchi, 2003). This hypothesis was supported by the observation that SapB levels are significantly higher in strains carrying multiple copies of ramR or ramSABR (Keijser et al., 2002; Nguyen et al., 2002). The N-terminal amino acid sequence of SapB, TG(S/G)RR, is 4/5 identical to residues 22 to 26 of RamS (Keijser et al., 2002; Willey et al., 1991). Assuming that SapB consists of the C-terminal 21 amino acids of RamS, a mass of 2099 Da would be

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expected. However, the reported mass of SapB is 2027 Da (Keijser et al., 2003; Willey et al., 1991, ). In an elegant new study by Kodani et al. (2004), it was shown that the mass of 2027 Da is in agreement with a posttranslational modified peptide consisting of the C-terminal 21 amino acids of RamS, with four out of five serine residues dehydrated resulting in didehydroalanine residues. Two of these residues then react with the two cysteine residues, producing two eight-membered rings with thioether lanthionine bridges (Fig. 2). The latter modifications are suggested to be introduced by the C-terminal domain of RamC that shows significant similarity to lantibiotic modifying enzymes. Whether the N-terminal domain of RamC, having similarity to ser/thr kinases, plays a role in the RamS processing remains to be elucidated. Another question that remains relates to the initial observation that SapB reacts with Schiff’s reagent (Willey et al., 1991), indicating the presence of a sugar residue or another molecule with vicinal hydroxyl groups, which is not explained by the current structure. C. NOVEL REGULATORS OF SPORULATION: THE SSGA-LIKE PROTEINS (SALPS) 1. Occurrence of SALPs Another novel family of developmental regulators first identified in streptomycetes, and later also in other Actinomyces species such as Thermobifida and Streptoverticillium, is that of the SsgA-like proteins (SALPs; Pfam PF04686). The surprising finding that enhanced expression of SsgA directly stimulates sporulation-specific cell division indicates that SsgA is an important control point in the onset of sporulation. This is supported indirectly by phylogenetic evidence, because SALPs are apparently unique to sporulating actinomycetes and are absent from the nonsporulating actinomycetes Corynebacterium glutamicum, Mycobacterium leprae, and Mycobacterium tuberculosis (van Wezel et al., 2000a). The recently completed genome sequences of S. avermitilis and S. coelicolor revealed six and seven ssgA-like genes, respectively (Bentley et al., 2002; Ikeda et al., 2003). The genes encode relatively small (130–140 aa) proteins, which share 30–50% amino acid identity (Keijser et al., 2003; van Wezel et al., 2000a). Homologs of S. coelicolor ssgA (Sco3926), ssgB (Sco1541), ssgD (Sco7622), and ssgE (Sco3158) are found on the S. avermitilis genome (Sav3926, 6810, 1687, and 3605, respectively), with high conservation in these otherwise distantly related species: The ssgB gene products differ in only one amino acid residue. The highest conservation is found in two sections


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of the proteins, corresponding to amino acid residues 13–30 and 40–65 of SsgA. In total, 20 amino acid residues (approximately 15% of the protein) are fully conserved among all 19 SALP proteins identified so far. However, there are no sequences in these proteins that resemble known functional motifs. 2. SsgA Triggers Sporulation-Specific Cell Division Studies on ssgA strongly suggest that it is an activator of sporulationspecific cell division. ssgA was originally identified as a suppressor of the hyper-sporulating mutant SY1 of Streptomyces griseus B2682 and shown to be involved in the regulation of submerged sporulation (Kawamoto and Ensign, 1995a; Kawamoto et al., 1997). The gene is of particular interest for both applied and fundamental aspects of Streptomyces research, as its expression level apparently controls morphology and development. Increased expression of SsgA alters the phenotype of liquid-grown mycelium of S. coelicolor, which normally forms large clumps but produces open mycelial structures (so-called mycelial mats) when expression is increased and shows fragmentation and submerged sporulation at high levels (van Wezel et al., 2000a,b). At these high expression levels, thick and amorphous septa are formed at regular intervals, forming spore-like compartments (Fig. 4). The stimulation by SsgA is apparently specific to sporulation-specific cell division, as ssgA mutants are defective in sporulation but form normal vegetative septa (Jiang and Kendrick, 2000; van Wezel et al., 2000a). Interestingly, ssgA mutants produce viable spores on mannitol-containing media, making it the only known ‘‘conditional’’ whi mutant. Transcriptional analysis showed that ssgA is transcribed from two developmentally regulated promoters in both S. coelicolor and in S. griseus (Traag et al., 2004; Yamazaki et al., 2003). One of these promoters is species-specific, suggesting that ssgA is regulated differently in these two organisms. This is indeed the case. In S. griseus, transcriptional activation of ssgA (further designated ssgAsg to discriminate it from ssgA from S. coelicolor, referred to as ssgAsc) is dependent on the y-butyrolactone A-factor (Horinouchi, 2002; Horinouchi and Beppu, 1995) and involves at least three different regulatory proteins. First, its transcription requires activation by AdpA, a central regulator of the A-factor pathway, which was shown to bind to three distinct sites upstream of ssgA (Yamazaki et al., 2003). The same authors showed that ssgAsg transcription is (probably indirectly) dependent on AdsA, the homolog of the developmental -factor BldN of S. coelicolor. Moreover, ssgAsg is most likely activated by the upstream located ssfR (ssgR in S. coelicolor), which encodes an IcIR-type transcriptional

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FIG. 4. Effect of enhanced expression of SsgA on the morphology of submerged hyphae and on septation of S. coelicolor A3(2). Transmission electron micrographs show


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regulator. Although ssgA is still transcribed in an ssfR mutant, one of its promoters is strongly down-regulated. Interestingly, while transcription of ssgA is fully dependent on A-factor in S. griseus, it most probably is not in S. coelicolor. Here, the upstream located ssgR gene is essential for its activation, and both promoters are silent in an ssgR mutant (Traag et al., 2004). Considering the dominant role of SsgA in triggering Streptomyces cell division, this could explain why A-factor plays such an important role in development of S. griseus but not in S. coelicolor. The apparent requirement for tight control of ssgA transcription may be necessitated by the profound changes in Streptomyces morphology brought about by fluctuations in the ssgA expression level. While transcription of ssgA and ssgR is strongly up-regulated during the onset of sporulation in S. coelicolor, it is not significantly affected in any of the so-called early whi mutants of S. coelicolor (whiA, whiB, whiG, whiH, whiI, and whiJ ) (Traag et al., 2004). This places these genes outside the generally accepted regulatory cascade leading to solid-culture sporulation. This is the first clear example of a sporulation gene that is expressed in a whi-independent manner. The physiological reason for this may be to provide a way to bypass the whi cascade under conditions where aerial hyphae formation is not desired, such as during ectopic or submerged sporulation. It is unclear what morphological changes occur during liquid-culture differentiation, but the fact that it does not require an aerial mycelium implies that there are two routes towards sporulation: one via the traditional whi cascade and one via the whi-independent route. SsgA is essential for submerged sporulation, and if ssgA transcription would be fully dependent on (some of) the whi genes, this process would probably be impossible because it is likely that several of the whi genes are not expressed under submerged conditions. This working hypothesis requires further testing in S. griseus. 3. ssgB Is Essential for Sporulation Expression profiling studies with DNA microarrays by the Cohen laboratory revealed two other SALPs as possibly developmentally regulated, namely ssgB and ssgD (Huang et al., 2001). Of these, ssgB has

S. coelicolor M145 (A–B) and S. coelicolor GSA2 overexpressing SsgA (C–F). (A) image showing vegetative hyphae and cross-walls; (B) Magnification of wild-type vegetative septum; (C) Image showing submerged hypha forming pre-spore-like compartments as the result of the overexpression of SsgA; (D–F) examples of abnormal septa in GSA2. Magnifications: (A) and (C), bar ¼ 1 m; (B, D–F): bar ¼ 0.2 m. Figure reproduced from van Wezel et al., 2000a, with permission from the American Society for Microbiology.

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been subject of more detailed transcriptional and mutational analysis. Like SsgA, SsgB is involved in the regulation of Streptomyces development but acts in an earlier phase of the sporulation process. Detailed studies with confocal fluorescence microscopy and electron microscopy showed that ssgB deletion mutants fail to produce sporulation septa, and genome segregation and condensation were not observed. In contrast to ssgA mutants, the nonsporulating phenotype could not be rescued by growth on mannitol-containing media. These ssgB mutants produce colonies that are larger than those of the parental (wildtype) strain (Keijser et al., 2003), possibly linking SsgB to the process of growth cessation that occurs prior to sporulation-specific cell division (Chater, 1989, 2001; Fla¨rdh et al., 1999). The developmental role of ssgB is underlined by the observation that transcription of ssgB coincides with aerial mycelium formation and depends on the developmental H (Kormanec and Sevcikova, 2002), a factor that itself is developmentally controlled at the transcriptional and post-translational level and plays a role in stress responses (Kelemen et al., 2001; Viollier et al., 2003). However, while sigH mutants are still able to produce spores, ssgB mutants are not (Keijser et al., 2003). This suggests that ssgB is transcribed by at least one other factor, active earlier in the developmental program. Interestingly, the BldD protein is involved in the repression of the sigHp2 promoter, and therefore indirectly of ssgB. BldD is a repressor protein that becomes active at the end of the bld signaling cascade and controls transcription of the developmental factor genes whiG and bldN/whiN. These genes play crucial roles during several stages of aerial mycelium formation. This is a clear example of links that exist between the regulation of the switches to aerial mycelium formation (by the bld genes) and to sporulation (by whi genes). 4. What Is the Function of the SALPs? The mode of action of the SsgA-like proteins is as yet unknown. The relatively highly conserved region corresponding approximately to amino acid residues 20–70 possibly lends a common function to the SALPs, such as interaction with the same protein or protein complex, while the highly variable N- and C-terminal parts are likely to provide functional specificity to the individual SALP proteins. Recent data suggest that they are expressed during distinct phases in the Streptomyces life cycle and may play a role in the coordination of cell division and DNA segregation (Noens, Koerten, and van Wezel, unpublished data). In accordance with this idea, when the amino acid sequence most conserved among SALPs is used in a database screen, the only


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non-SALP hit is MukB, a protein also involved in DNA segregation in bacteria (van Wezel and Vijgenboom, unpublished). Structural and physiological studies are required to elucidate the role of this interesting new family of proteins. V. Concluding Remarks In recent years several new developmental genes have surfaced. The discovery and characterization of genes such as ram/amf and the members of the family of ssgA-like genes have shed new light on the complex morphological development in Streptomyces. Also, the first insights were provided into the relationship between carbon metabolism and development. However, the picture is still far from complete. Important missing links are the signaling molecules, the signal receptors, and the signal transducers, postulated for connecting the individual parts of the developmental machinery, and the exact timing of their expression. Designing new approaches to disclose the identity of many more components of the developmental system is the challenge for the years to come. Helped by the recent elucidation of the complete genome sequences of S. coelicolor and S. avermitilis, and with that of many others soon to follow, significant research efforts employing functional genomics, classical biochemistry, and protein chemistry are needed to identify and characterize the components involved in the morphological development of streptomycetes. ACKNOWLEDGMENTS We are grateful to B. Kraal, B. F. Keijser, and G. Kelemen for very useful comments on the manuscript and to H. K. Koerten for Fig. 1B. We also express our sincere thanks to the many colleagues for the stimulating discussions on the subjects discussed in this review. This work was supported by a grant from the Dutch Royal Academy of Sciences to GPvW.

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Polysaccharide Breakdown by Anaerobic Microorganisms Inhabiting the Mammalian Gut HARRY J. FLINT Microbial Genetics Group Rowett Research Institute Bucksburn, Aberdeen, AB21 9SB, United Kingdom E-mail: [email protected]

I. Introduction: Role of Gut Microbial Fermentation in Nutrition II. Microbial Diversity and Interactions Within Gut Ecosystems A. Diversity, Functional Groups B. Nutritional Interactions and Cross-Feeding III. Strategies for Polysaccharide Utilization by Gut Anaerobes A. Stages in Polysaccharide Utilization B. CFB (Cytophaga–Flavobacterium–Bacteroides) Phylum C. Fibrobacter D. Cellulolytic Ruminococcus Species E. Clostridial Cluster XIVa (C. coccoides/E. rectale) Group F. Bifidobacteria G. Eukaryotes IV. Applications A. Manipulation of Gut Metabolism with Probiotics, Prebiotics, and Enzymes B. Biotechnology V. Conclusions and Future Prospects References

89 92 92 92 94 94 98 101 101 104 105 106 106 106 108 109 110

I. Introduction: Role of Gut Microbial Fermentation in Nutrition A high proportion of the solar energy trapped by plants through photosynthesis is used in the synthesis of polysaccharides. The structural polysaccharides that comprise the plant cell wall are synthesized in vast amounts, approximately 40 Gt of cellulose (Coughlan, 1985) and 30 Gt of hemicellulose globally per annum, together with smaller amounts of energy reserve polysaccharides such as starch and inulin. Herbivorous and omnivorous animals have a variety of strategies for taking advantage of this constantly replenished store of energy. While almost all animals produce digestive amylases, only some invertebrates, such as mollusks and termites (Watanabe et al., 1998; Xu et al., 2001), secrete enzymes capable of degrading structural plant polysaccharides, such as cellulose. Herbivorous mammals therefore rely entirely on the remarkable degradative activities of microorganisms 89 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 56 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2164/04 $35.00

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that colonize their gastrointestinal (GI) tracts for their ability to gain energy from these substrates. The anatomy of the digestive tract in herbivores reflects dramatic evolutionary changes that promote microbial fermentation in either the foregut (reticulo-rumen) or the hindgut (colon and/or cecum) (van Soest, 1984). In ruminants, some 60–70% of the total GI tract volume is given over to this fermentative activity, while in hindgut fermentors, such as the horse, the colon and cecum occupy a similarly large proportion of the total gut volume (Parra, 1978). Depending on the diet, short-chain fatty acids (SCFA) arising from gut microbial fermentation account for 70% of the total energy dietary supply in ruminants and up to 30% in pigs (Bergman, 1990; Fonty and Gouet, 1989). Even in man, where the colon accounts for only 17% of total gut volume, large intestinal fermentation can account for 10% of daily energy supply (Bergman, 1990). Because of its economic importance and accessibility, the rumen has played a key role in developing our understanding of the microbial ecology of anaerobic gut ecosystems. Microbial growth in the rumen provides not only energy sources in the form of SCFA but also microbial protein that can be assimilated by the ruminant further down the GI tract (van Soest, 1984). Optimizing ruminant nutrition and the utilization of poor quality plant material by ruminants for human food production remains an important goal of research. There is also increasing emphasis on reducing pollution caused by nitrogenous waste and methane production by ruminants and on reducing reliance on chemical feed additives (Flint, 1997; Russell and Rychlik, 2001). Whereas rumen microorganisms have access to all dietary carbohydrates, microorganisms in the large intestine can access only those carbohydrates that survive passage through the small intestine. These ‘‘low-’’ or ‘‘non-digestible’’ carbohydrates include the plant structural polysaccharides cellulose, xylan, and pectin and a variety of polysaccharide food additives and oligosaccharides (MacFarlane and Gibson, 1997). In addition, some portion of dietary starch (‘‘resistant starch’’) can escape small intestinal digestion. In man, such dietary carbohydrates have a wide range of claimed health benefits including prevention of colorectal cancer and colitis and lowering of cholesterol (Scheppach et al., 2001; Topping and Clifton, 2001; Wollowski et al., 2000) (Table I). Short-chain fatty acids, especially butyric acid, produced by fermentation provide the major energy sources for the colonic epithelium (Csordas, 1996), and their production rates and molar ratios are influenced by the type and quantity of carbohydrate entering the large intestine (Topping and Clifton, 2001). Other consequences of

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TABLE I SOME EXAMPLES OF DIETARY POLY- AND OLIGO-SACCHARIDES CONSIDERED TO INFLUENCE HUMAN HEALTH THROUGH EFFECTS ON LARGE INTESTINAL METABOLISM

Dietary carbohydrate

Claimed effects on

Reference (examples)

Pectin

Gut transit, polyamine synthesis; cancer protection, antitoxin

Hayashi et al. (2000); Noack et al. (1998); Olano-Martin et al. (2003)

Inulin

Prebiotic (bifidogenic); elevated butyrate; laxative

Kleessen et al. (1997)

Resistant starch

Elevated SCFA, butyrate; fecal bulking; cancer protection

Govers et al. (1999); Wolin et al. (1999)

Barley -glucans

Bile acid excretion

Dongowski et al. (2002)

Psyllium husk, whole cereal flour

Reduced cholesterol, low density lipoprotein

Adam et al. (2001); Anderson et al. (2000)

Fructo-oligosaccharides

Prebiotic (bifidogenic); elevated butyrate

Kleessen et al. (2001)

Lactulose

Laxative, cancer protection

Rowland et al. (1996)

fermentation, such as excessive gas production, can, however, be detrimental. Carbohydrates can also act as prebiotics, selecting for particular groups of bacteria and against others within the intestinal community, resulting potentially in reduced pathogen populations or immune stimulation (Gibson, 1998). Other effects (e.g., sequestration of metabolites, pH, viscosity, and gut transit) do not depend on fermentation but can result in radical alteration of the gut environment. Intestinal fermentation of polysaccharides and oligosaccharides is also a key factor in the nutrition and health of monogastric animals including pigs, poultry, and horses. Understanding the effects of different carbohydrates on gut metabolism requires us to know not only which bacterial groups degrade different substrates but also what strategies they use to compete for energy from the available carbohydrate energy sources. This review attempts to summarize our state of knowledge with particular emphasis on the utilization of dietary polysaccharides by anaerobic gut bacteria, drawing on information from the rumen and large intestinal systems.

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II. Microbial Diversity and Interactions Within Gut Ecosystems A. DIVERSITY, FUNCTIONAL GROUPS The rumen and large intestine are the most densely colonized regions of the mammalian gut, where microbial cell densities can exceed 1011/ml. Rapid depletion of oxygen by the more oxygen-tolerant microorganisms results in communities that are dominated by oxygen-sensitive obligate anaerobes. Thus in the rumen, obligate anaerobes outnumber facultative anaerobes by at least 1000:1 (Hungate, 1966). These are among the most diverse and complex microbial communities found in nature. Analysis of the pig gut microbiota, based on amplified small subunit ribosomal gene sequences, detected 375 bacterial phylotypes (provisional species), of which 309 showed limited resemblance to previously described species (Leser et al., 2002). Similar analyses of the rumen (Tajima et al., 1999; Whitford et al., 1998) and of human fecal (Hayashi et al., 2002; Suau et al., 1999) and human (Hold et al., 2002) and equine (Daly et al., 2001) large intestinal bacteria have produced similar conclusions. The most abundant bacterial groups in all of these anaerobic gut communities appear to be low GþC content Gram-positive bacteria, followed by Gram-negative bacteria belonging to the CFB phylum (Table II). The wealth of available ribosomal sequence data has allowed the development of techniques for microbial detection independent of cultivation that are based on oligonucleotide probes (Fig. 1), quantitative polymerase chain reaction (QPCR), and molecular profiling (e.g., Franks et al., 1998; Tajima et al., 2001; Zoetendal et al., 1998). B. NUTRITIONAL INTERACTIONS AND CROSS-FEEDING The fate of hydrogen is a key factor in anaerobic ecosystems. Methanogenic, acetogenic, sulphate-reducing, and nitrate-reducing microorganisms are all potentially capable of utilizing hydrogen produced by other anaerobes, producing methane, acetate, and hydrogen sulphide and reduced nitrogenous compounds, respectively. Methane formation due to methanogenic Archaea dominates in the rumen. Methanogenesis, acetogenesis, and sulphate reduction all occur in the human large intestine, with their relative contributions apparently depending on the individual, the site within the colon, and the availability of substrates (reviewed by MacFarlane and Gibson, 1997). Reductive acetogenesis was estimated to account for 30% of acetate formation in incubations with human feces (Miller and Wolin, 1996) and is also detectable in

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POLYSACCHARIDE BREAKDOWN BY GUT ANAEROBES TABLE II

APPROXIMATE PROPORTIONS OF DIFFERENT PHYLOGENETIC GROUPS OF BACTERIA IN THE MAMMALIAN GUT, BASED ON 16S rRNA SEQUENCE ANALYSESa Rumenb

Humanc,d

Pige

Horsef

CFB phylum (Bacteroides/Prevotella spp.)

32

26, 31

11

21

Clostridial cluster XIVa (C. coccoides group)

31

46, 44

25

37

Clostridial cluster IV (C. leptum group)

4

15, 20

19

8

Other low G þ C Gram-positives

18

5, 2

33

26

Others

11

7, 3

8

1

a

It should be noted that PCR biases in the construction of 16S rRNA gene libraries may lead to over-representation of some groups and absence of others. b From data of Tajima et al. (1999), cow rumen liquor. c From data of Hold et al. (2002), human colon, three individuals. d From data of Suau et al. (1999), human feces, one individual. e From data of Leser et al. (2002), pig intestine. f From data of Daly et al. (2001), horse intestine.

FIG. 1. Fluorescent in situ recognition of a cellulolytic coccus (Ruminococcus flavefaciens) by using a specific Cy3-labelled 16S rRNA probe. Courtesy of Alan Walker.

the pig large intestine (De Graeve et al., 1994) and in the rumen (Morvan et al., 1994). Hydrogen consumption has the effect of shifting fermentation in hydrogen producers from reduced products such as

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ethanol towards acetate, which can enhance energy metabolism and polysaccharide breakdown by cellulolytic microorganisms (Morvan et al., 1996; Wolin et al., 1997). Hydrogen transfer is only one of many types of nutritional interactions that occur in gut ecosystems. Lactate and succinate do not generally appear as significant fermentation products because of consumption by other species (Fig. 2). Acetate reaches substantial concentrations in most regions of the gut, despite consumption by butyrate producers in the human colon (Barcenilla et al., 2000). A small number of species appear to act as the primary degraders of plant cell wall material in gut ecosystems, but many others benefit through cross-feeding of breakdown products (Fig. 2). Many cellulolytic rumen bacteria, such as Fibrobacter succinogenes and strains of Ruminococcus flavefaciens, release xylo-oligosaccharides that they cannot themselves utilize, and these are efficiently utilized by noncellulolytic bacteria such as Prevotella spp. (Dehority, 1991; Dehority and Scott, 1967; Osborne and Dehority, 1989). In turn, some cellulolytic bacteria depend on other members of the gut microbial community for certain vitamins and precursors for amino acid synthesis (Hungate and Stack, 1982; Scott and Dehority, 1965). While such interactions are best documented for fiber breakdown in the rumen, similar interactions undoubtedly occur in the mammalian large intestine and with other complex substrates. Antagonistic interactions (e.g., bacteriocin formation [Kalmakoff and Teather, 1997]), may also be significant factors in interstrain/species competition.

III. Strategies for Polysaccharide Utilization by Gut Anaerobes A. STAGES IN POLYSACCHARIDE UTILIZATION The main steps involved in the utilization of polysaccharides by individual microorganisms can be summarized as (1) attachment to the substrate; (2) disruption and enzymatic degradation of the substrate; (3) transport of breakdown products into the cell, accompanied by further degradation, metabolism and energy generation. 1. Attachment In the rumen, plant fragments develop a microbiota distinct from that of the lumen that may considered as a biofilm (McAllister et al., 1994). Rather little is known, however, about the initial stages of colonization


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FIG. 2. Nutritional interactions between polysaccharide degrading microorganisms in the rumen. This figure has been modified from Flint (1997), with permission.

of insoluble substrates by obligately anaerobic gut microorganisms. Motility and chemotaxis may assist some bacterial species, fungal zoospores, and protozoa in reaching their substrates. Possible quorum-sensing molecules have been detected in culture fluid from some rumen bacteria (Mitsumori et al., 2003). Initial attachment of cellulolytic bacterial cells is often likely to involve the extensive glycocalyx (Latham et al., 1978; Miron et al., 2001; Roger et al., 1990). In addition, substrate-binding modules in microbial enzymes and

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cell-associated structural proteins mediate specific binding to polysaccharides including cellulose, xylan, and starch. It has been proposed in R. albus that special appendages resembling type IV pili have a role in binding to cellulose (Pegden et al., 1998). In Salmonella typhimurium, biofilm formation appears to involve the synthesis of thin, aggregative fimbriae and of an exopolysaccharide, bacterial cellulose (Romling et al., 2000). 2. Degradation Detailed treatment of the biochemistry and structural biology of the >1,000 individual microbial polysaccharide-degrading enzymes so far studied is beyond the scope of this review, but this information can be accessed in specialist reviews (Bayer et al., 1998; Bourne and Henrissat, 2001; Warren, 1996) and through the excellent CAZY website (http:// afmb.cnrs-mrs.fr/CAZY/index.html). The great majority of such microbial enzymes are multi-modular in their organization. Single polypeptides may consist of one or more catalytic domains, which can differ in their enzymatic specificity (e.g., Flint et al., 1993; Fontes et al., 1995), together with modules responsible for substrate binding and protein: protein interactions, linker regions between domains and modules, and a variety of other domains whose functions have yet to be elucidated. Catalytic domains for glycoside hydrolases that cleave polysaccharide chains have been classified into 91 different families based on their primary amino acid sequences. As far as is known, enzymes belonging to the same family show the same mechanism of hydrolysis (inverting versus retaining) and have similar catalytic sites and threedimensional structures; families with related 3D structures can be further grouped into superfamilies (Henrissat et al., 1995). A variety of substrate specificities, and both exo- and endo-acting enzymes, can be found within the same family. In addition, 13 families of carbohydrate lyase are known, of which seven include enzymes involved in pectin breakdown. Lyases are also involved in the degradation of hostderived polysaccharides such as heparin and chondroitin (Guthrie et al., 1985). Another important group of catalytic domains are those involved in cleaving ester bonds that link substituents such as acetyl groups and phenolic acid residues present in xylans and acetyl and methyl groups in pectins (e.g., Aurilia et al., 2000; Dalrymple et al., 1997; Dongowski et al., 2000; McSweeney et al., 1998). Thirteen different families of carbohydrate esterase are currently described. Similar sequence diversity is encountered among substrate binding modules, with 33 different families so far described. Functionally


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these modules are classified by the size, solubility, composition, and organization of the substrate molecules that they bind (e.g., crystalline cellulose, amorphous cellulose, large or small cello-oligosaccharides) (reviewed in Tomme et al., 1995, 1998). Some binding modules enhance catalytic activity of the enzymes or complexes carrying them against insoluble substrates (e.g., Din et al., 1994). Linkers typically consist of repeated threonines and/or serines and provide flexible regions between different functional domains in large polypeptides as well as sites for glycosylation (Warren, 1996). Dockerin modules are regions of up to 80 amino acids occurring typically at the C-terminus of the polypeptide that contain two inexact repeats of an EF hand-type Ca2þ binding motif. They were initially reported from Clostridium spp. and were shown to interact with cohesin modules present in noncatalytic structural proteins located on the cell surface (Beguin and Lemaire, 1996). Their role is in the assembly of cellulosomes, extremely large (2–6.5 MDa in C. thermocellum) surface–bound complexes of plant cell wall degrading enzymes (Bayer et al., 1998; Beguin and Lemaire 1996). The breakdown of crystalline cellulose may involve processive endocellulases (Gilad et al., 2003) and/or synergy between endo- and one or more exo-acting cellulases (Barr et al., 1996). The degradation of branched polysaccharide substrates, including xylans and pectins, as well as amylopectin starch, relies on synergy between enzymes that cleave the main chain and debranching enzymes (e.g., Biely et al., 1986). Cooperation between many different enzyme specificities is therefore required for the efficient degradation of plant cell walls, in which cellulose fibrils are embedded in a matrix of hemicellulose and pectin. In the cellulosomes of Gram-positive bacteria most of these specificities are concentrated together into high-molecular-weight enzyme complexes (Bayer et al., 1998). 3. Transport Sugars resulting from polysaccharide breakdown can potentially be taken up as monosaccharides, disaccharides, or oligosaccharides by several widespread mechanisms. These include ATP driven, binding protein-dependent systems known as ABC transporters; ion-linked transport (symport, antiport, uniport) via members of the major facilitator superfamily; and PEP-dependent phosphotransferase transport (Saier, 2000). Although first described in Gram-negative bacteria where they involve periplasmic binding proteins, ABC transporters are also abundant in Gram-positive bacteria where binding proteins

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are surface-bound lipoproteins and include systems that can take up relatively large molecules. An ABC transporter for cellobiose/triose has been described in Streptomyces reticuli (Schlosser et al., 1999). The ability to take up oligosaccharides, as the immediate products of polysaccharide breakdown, may be particularly important for survival in the highly competitive environment of the gut, and many anaerobic bacteria show poorer growth on monosaccharides compared with disaccharides or oligosaccharides (e.g., Thurston et al., 1993). Oligosaccharides may be cleaved by hydrolysis before or after transport or by phosphorylysis during transport. A wide variety of di/oligosaccharide phosphorylases is known from various bacteria (Kitaoka and Hayashi, 2002). Cellobiose phosphorylase occurs in many rumen bacteria (Ayers, 1959; Lou et al., 1996), and cellodextrin phosphorylase is reported in Clostridium stercorarium (Reichenbecher et al., 1997). Most of the hexose released by polysaccharide breakdown is metabolized via the EMP pathway in the rumen (Russell and Wallace, 1997) and in the human large intestine (Miller and Wolin, 1996). In bifidobacteria, however, hexoses are metabolized via the fructose6-phosphate shunt, while heterofermentative lactobacilli use the hexose monophosphate shunt pathway (Gottschalk, 1979; MacFarlane and Gibson, 1997). B. CFB (CYTOPHAGA–FLAVOBACTERIUM–BACTEROIDES) PHYLUM The most abundant Gram-negative bacteria in the GI tract belong to the Cytophaga–Flavobacterium–Bacteroides (CFB) phylum. Many species play important roles in polysaccharide breakdown in the gut, although few if any appear to be cellulolytic; the species referred to as ‘‘B. cellulosolvens’’ is in fact related to Gram-positive bacteria. Rumen Prevotella spp. form a phylogenetically distinct subgroup of CFB bacteria that accounts for at least 30% of rumen bacterial diversity (Ramsak et al., 2000; Tajima et al., 1999; Whitford et al., 1998; Wood et al., 1998). Many species are xylanolytic and pectinolytic and utilize these polymers efficiently when in coculture with cellulolytic bacteria (Fondevila and Dehority, 1996; Osborne and Dehority, 1989). Of 188 human colonic Bacteroides strains surveyed by Salyers et al. (1977a,b), most (72%) were able to ferment amylose and amylopectin. Only 25% of strains (mainly B. ovatus and B. eggerthii) fermented xylan, while almost half (including B. ovatus and B. thetaiotaomicron) fermented pectin and polygalacturonate. Although none could ferment pig gastric mucin, some, including B. thetaiotaomicron and B. ovatus


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FIG. 3. Probable location of the major polysaccharide-hydrolyzing activities in different gut groups of anaerobe. Examples are provided by starch utilization in Bacteroides thetaiotaomicron, cell surface enzyme complexes in R. flavefaciens and rumen fungi, and xylanases in Polyplastron multivesiculatum (see text).

strains, could ferment individual host–derived polysaccharides including heparin, chondroitin sulphate, and hyaluronate. In B. thetaiotaomicron, the hydrolytic enzymes responsible for starch breakdown (amylases, pullulanases) are primarily cellassociated and fractionate with periplasmic markers (Anderson and Salyers, 1989) (Fig. 3). Biochemical and subsequent genetic evidence indicated that starch molecules larger than maltohexaose bound to outer membrane proteins, with subsequent transportation and processing by periplasmic enzymes. This ability to sequester large polysaccharide fragments is thought to be important in the ability of B. thetaiotaomicron to compete for nutrients in the large intestine. Four outer membrane proteins encoded by a cluster of starch utilization (sus) genes appear to be involved in starch binding, with susC and susD playing the major roles (Reeves et al., 1997; Shipman et al., 2000). The linked gene susG encodes a low-affinity outer membrane amylase that is thought to hydrolyze starch molecules bound to the cell surface (Shipman et al., 1999). A major starch-degrading enzyme encoded by susA, is a neopullulanase, a periplasmic enzyme able to hydrolyse –(1,4) linkages in amylose, amylopectin, or pullulan (D’Elia and Salyers, 1996). Disruption of susA reduced the growth rate on starch by 30% (D’Elia and Salyers, 1996).

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The complete genome sequence of Bacteroides thetaiotaomicron 5482 (6.26 Mb) is now available. This is one of the largest bacterial genomes and contains at least 172 genes encoding enzymes that are involved in the breakdown of polysaccharides. In addition, there are 71 glycosyltransferase genes that are assumed to be involved in capsular polysaccharide formation (Xu et al., 2003). This gene multiplicity reflects considerable redundancy, including, for example, 31 -galactosidase genes and 8 amylase genes. In addition, the genome contains 163 copies of susC- or susD-related genes, many of them linked to polysaccharide utilization genes, that are presumed to encode outer membrane binding proteins. The abundance of sus genes suggests that the sequestration mechanism described previously for starch may apply quite generally to the utilization of macromolecules in this group of organisms. The apparently remarkable redundancy in polysaccharide utilization genes may simply be required to ensure high enough production of the hydrolytic enzymes, although subtle differences in regulation and specificity may also occur between related enzymes. Curiously, although 11 xylanase genes were apparently identified in its genome, B. thetaiotaomicron does not grow on xylan. These genes, however, belong to glycoside hydrolase family 43 rather than to families 10 or 11, which account for the majority of microbial xylanases. Since many family 43 enzymes function as xylosidases, their real role could be in scavenging oligosaccharides. Xylan degradation clusters identified in the xylan-utilizing species P. bryantii from the rumen (Gasparic et al., 1995a; Miyamoto et al., 2003) and B. ovatus from the human colon (Weaver et al., 1992) in both cases reveal a family 10 xylanase linked to a family 43 enzyme. P. bryantii (formerly P. ruminicola) possesses at least one other family 10 xylanase, with an unusual primary structure (Flint et al., 1997). Xylanase activity appears to be largely cell-associated both in Bacteroides (Hespell and Whitehead, 1990) and in rumen Prevotella bryantii, where assayable xylanase activity is increased fivefold by sonication (Miyazaki et al., 1997). Although P. bryantii possesses a carboxymethylcellulase, its role appears to be in the degradation of mixed link –glucans (Fields et al., 1998) and the cloned enzyme has 1000-fold higher activity against barley –glucan than CM-cellulose (Gasparic et al., 1995b). P. bryantii (formerly P. ruminicola) has the ability to store excess carbon as glycogen, which can account for 60% of cell dry weight (Lou et al., 1997). Under low N conditions, excess carbohydrate can result in severe loss in viability due to the accumulation of methyl glyoxal (Russell, 1998).


101

C. FIBROBACTER Recent estimates of the combined populations of the three major species of cellulolytic bacteria in the rumen (F. succinogenes, R. albus, and R. flavefaciens) by molecular probing are around 4% (Krause et al., 1999), 0.3–3.9% (Weimer et al., 1999), and 4.5% (Michalet-Doreau et al., 2002). In the latter study, R. flavefaciens was found to be the most abundant of the three groups in the ruminant cecum and F. succinogenes in the rumen, while Weimer et al. (1999) reported R. albus as the most abundant in the rumen. These estimates are in broad agreement with previous work based on anaerobic cultivation. A second species F. intestinalis, has been described and as yet undescribed Fibrobacter species appear to be abundant in the cecum of the horse (Lin and Stahl, 1995). F. succinogenes is Gram-negative, but like the CFB group, shows evidence of early evolutionary divergence from proteobacteria (Griffiths and Gupta, 2001). The organization of polysaccharidase enzymes in this species remains unclear, but complete genome sequencing of strain S85 should soon produce the first full picture of the enzyme complement of a cellulolytic gut species. A preliminary report on the F. succinogenes S85 genome indicates that it contains at least 24 endoglucanase and cellodextrinase genes and at least 23 genes concerned with hemicellulose breakdown (Nelson et al., 2002). Sixteen cell surface cellulose binding proteins, including six endoglucanases, have been identified, of which 13 are glycosylated, and it is postulated from the behavior of mutant strains that glycosylation may play an important role in substrate attachment (Miron and Forsberg, 1999). Despite possessing multiple xylanases, F. succinogenes fails to grow on xylan breakdown products and lacks xylose isomerase activity (Matte et al., 1992). This presumably indicates that this bacterium gains a more than sufficient energy supply from cellulose, with xylanases necessary for gaining access to the cellulose in plant cell walls. F. succinogenes is observed to secrete glucose and cellotriose in the presence of excess cellobiose (Russell, 1998; Wells et al., 1995). D. CELLULOLYTIC RUMINOCOCCUS SPECIES As discussed above, two species of ruminococci (R. flavefaciens and R. albus) are among the most abundant cellulose-degrading bacteria in the rumen and may also make a major contribution to plant cell wall breakdown in the large intestine (e.g., of horses [Julliand et al., 1999]

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FIG. 4. (A) A model for the organization of plant cell wall degrading enzymes in the cellulolytic bacterium Ruminococcus flavefaciens (based on Ding et al., 2001, and Rincon et al., 2003). Structural proteins carrying repeat cohesin domains (ScaA and ScaB) together form a scaffold for the assembly of enzyme subunits that include a variety of catalytic domains (CD1,2,3-eg cellulase, xylanase, esterase) and carbohydrate-binding modules (CBMs). Two types of sequence that pair with the cohesins (dockerins) are indicated by different hatchings. A divergent cohesin present in the Sca protein binds a


103

and pigs [Varel and Yen, 1997]). Both species are detectable in human feces (Wang et al., 1997), although their noncellulolytic relatives R. bromii and R. callidus have been isolated more frequently (Moore and Moore, 1995). R. albus, R. flavefaciens. R. bromii, and R. callidus belong to the clostridial cluster IV (Collins et al., 1994), whose members may account for up to 20% of bacteria present in human feces (Franks et al., 1998; Hold et al., 2002; Suau et al., 1999). In R. flavefaciens many polysaccharidases appear to be organized into high-molecular-weight enzyme complexes. Dockerin modules have been found in six of the seven plant cell wall degrading enzymes so far described from R. flavefaciens 17, which include cellulases, mixed link -glucanases, xylanases, and esterases (Aurilia et al., 2000). The xylanase XynA does not contain a dockerin (Zhang and Flint, 1992) and is presumably separate from the complex. A gene cluster has now been identified that encodes three proteins—ScaA, ScaB, and ScaC—that carry repeated cohesin-like domains. The three cohesins in ScaA can interact with one group of enzyme dockerins, while ScaA interacts via its C-terminal dockerin with the seven cohesins found in ScaB (Ding et al., 2001; Rincon et al., 2003) (Fig. 4). The more recently described ScaC has a dockerin that reacts with ScaA and a novel type of cohesin with a distinct binding specificity and may act as a type of adaptor broadening the binding specificity of the complex. Some features of this organization are reminiscent of the cellulosome of Clostridium thermocellum (Bayer et al., 1998), except that the scaffolding protein ScaA has not been found to carry a cellulose-binding module. Mechanisms for binding to cellulose may reside with individual enzymes (Rincon et al., 2001) or with other proteins yet to be identified. A related strain of R. flavefaciens was previously shown to have lost its ability to degrade cotton, but not other forms of cellulose (Stewart et al., 1990), but the molecular basis for this is not yet clear. A distinctive feature of R. flavefaciens, for which the species is named, is the production of a yellow pigment (Kopecny and Hodrova, 1997) during growth on cellulose. Such pigment production is found in other species, such as C. thermocellum (Ljungdahl et al., 1983), and

further set of as yet unidentified proteins. (B) Detection of the scaffoldin protein ScaA by immunogold labelling in cellulose-grown culture of Ruminococcus flavefaciens 17. The protein is seen to be associated with the cell surface, and with substrate particles. This figure is from Rincon et al. (2003), and is reproduced by permission.

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is proposed to play a role in promoting adhesion and/or colonization of the substrate. Evidence also exists for cellulosome-associated enzymes in R. albus F40 (Ohara et al., 2000), and it is also proposed in R. albus that piluslike cell surface appendages are involved in binding the cells to the substrate (Pegden et al., 1998; Rakotoarivivina et al., 2002). Optimal growth and expression of cellulolytic activity in R. albus depends on the availability of phenylacetic and phenylpropionic acids (Stack and Hungate, 1982). R. albus and R. flavefaciens possess cellobiose phosphorylase (Ayers, 1959; Helazcek and White, 1991; Thurston et al., 1993). As noted earlier for Fibrobacter, not all cellulolytic R. flavefaciens strains can grow on the breakdown products of xylans (Dehority and Scott, 1967). A cluster that includes genes for xylose isomerase, -xylosidase genes and components of an oligosaccharide ABC transport system was detected in the xylan-utilizing strain R. flavefaciens 17 but was not detected by DNA hybridization or activity in xylan non utilizing strains of R. flavefaciens (Aurilia et al., 2001). E. CLOSTRIDIAL CLUSTER XIVa (C.

COCCOIDES/E. RECTALE)

GROUP

One of the most numerous groups of anaerobic gut bacteria is the clostridial cluster XIVa, also known as the C. coccoides/E. rectale cluster (Table I). This cluster is represented in the rumen mainly by relatives of Butyrivibrio fibrisolvens, which possess hemicellulase, pectinase, and amylase activities (Dalrymple et al., 1999; Hespell and Cotta, 1995; Stewart et al., 1997). Some strains of B. fibrisolvens are reported to be weakly cellulolytic, and the related cellulolytic species Eubacterium cellulosolvens occurs in the rumen (Stewart et al., 1997). Many cluster XIVa bacteria are butyrate producers, and together with F. prausnitzii, which belongs to cluster IV, they account for the majority of human gut bacteria that are able to produce butyric acid (Barcenilla et al., 2000; Pryde et al., 2002). Butyrate provides the preferred energy source for the colonic epithelium (Csordas, 1996), and its formation can be stimulated by ‘‘low digestible’’ dietary carbohydrates (Kanauchi et al., 1998; McIntyre et al., 1993; Perrin et al., 2001; Pryde et al., 2002). The plant polysaccharide-degrading activities of human fecal representatives of this group have received little attention, however, largely because of a lack of cultured representatives. Abundant butyrate-producing genera found in the human gut, Eubacterium and Roseburia spp., nevertheless, include strains able to utilize xylan, inulin, and starch (Duncan et al., 2002a, 2003).


105

Among nonbutyrate producing species found in cluster XIVa are several Ruminococcus species that are abundant in the human large intestine, notably R. torques, R. obeum, and R. gnavus. R. torques strains are among the few human gut bacteria that have been shown to be able degrade pig gastric mucin (Bayliss and Houston, 1984; Hoskins, 1993; Salyers et al., 1977a,b). Rather little is known about the role of gut bacteria belonging to other clostridial clusters in polysaccharide breakdown, although E. cylindroides (cluster XVI) relatives were stimulated by inulin in a human colonic fermentor simulation (Duncan et al., 2003). F. BIFIDOBACTERIA Although not well represented in 16S rRNA clone libraries (Fig. 1), bifidobacteria can account for 3% or more of human fecal bacteria in adults (Franks et al., 1998) and are particularly abundant in the feces of breastfed infants (Gibson et al., 1995). Bifidobacteria accounted for 70 of 120 colonies of human fecal bacteria able to form clear zones in soluble starch plates (MacFarlane and Englyst, 1986), while in a more recent study only bifidobacterial species and C. butyricum among the human fecal isolates tested were able to form clear zones in high amylose starch (Wang et al., 1999). This suggests a significant role for bifidobacteria in starch breakdown in the human colon, although clear zone formation may not always equate to growth on starch in vivo, and it is not ruled out that other, less readily detected starch-degrading species play a major role. Degradation of xylan by strains of B. adolescentis and B. infantis and of arabinogalactan by B. longum was reported by Salyers et al. (1977). A genome sequence for Bifidobacterium longum (Schell et al., 2002) reveals far fewer polysaccharidase genes than found in B. thetaiotaomicron. Nevertheless, genes concerned with poly/oligosaccharide metabolism still account for 8% of the genome. Despite the absence of pectinases, or amylases, and cellulases, the genome includes around 40 glycoside hydrolases. The genome includes eight high-affinity MalEFG type ABC systems involved with oligosaccharide transport but apparently only one PTS transporter, which it is suggested may reflect selection for rapid uptake of a wide variety of oligosaccharides under gut conditions (Schell et al., 2002). The MalEFG transport system in E. coli takes up maltose and maltodextrins, which then become substrates for amylomaltase, which releases glucose and a larger maltodextrin via a glycosyltransferase reaction. This process is complemented by maltodextrin phosphorylase, which releases

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glucose-1-phosphate from the nonreducing end of maltodextrins (Boos and Shuman, 1998). G. EUKARYOTES Anaerobic chytrid fungi have only relatively recently been recognized as important components of the rumen ecosystem and are now known to occur in the large intestine of other herbivores including horses and camelids (reviewed in Orpin and Joblin, 1997; Trinci et al., 1994). The cellulolytic activity of these fungi can be exceptionally high, as are the specific activities of certain isolated enzymes (Selinger et al., 1996; Wood et al., 1986). There is evidence that their enzymes, which show the type of modular organization characteristic of cellulolytic bacteria (e.g., Dalrymple et al., 1997) may also be organized into cellulosome-like complexes via noncatalytic docking sequences (Fanutti et al., 1995; Steenbakkers et al., 2001). Certain anaerobic protozoa have long been considered to play a role in plant cell wall breakdown in the rumen. Until recently, however, it was unclear whether cellulase or xylanase activity associated with protozoa was due to ingested bacterial or fungal enzymes. Sequences from cDNAs have now resolved this issue at least for Polyplastron multivesiculatum, establishing that it produces its own xylanases (Devillard et al., 1999, 2003). Plant cell wall breakdown by protozoa is likely to differ markedly from that in bacteria and fungi, since protozoa are capable of engulfing large particles and digestion is assumed to occur within food vacuoles (Fig. 3). Since protozoa can account for 50% of the rumen microbial biomass, their contribution to a range of polysaccharide degrading activities may be considerable. IV. Applications A. MANIPULATION OF GUT METABOLISM WITH PROBIOTICS, PREBIOTICS, AND ENZYMES Enzymatic treatment or supplementation of animal feed with polysaccharidases is of considerable commercial importance in pig and poultry production and, increasingly, in ruminant production. In ruminants the main aim is to complement the action of rumen microbes and thus to enhance the degradation of plant fiber (Kung et al., 2002; Wang and McAllister, 2002) (Fig. 5). The potential effects of feed enzymes in monogastric animals are more complex, including alterations in viscosity and passage rates, as well as increasing the release of


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FIG. 5. Alternative approaches for optimizing the breakdown of plant fiber in the rumen. Degradability can potentially be improved by plant breeding, by pretreatment and formulation of feed, or the manipulation of rumen microbial activity.

breakdown products and modifying hind gut fermentation (Bedford and Schultze, 1998; Chesson, 1993). There must be considerable scope for improving the efficacy and formulation of such products through better understanding of interactions with polysaccharide-degrading gut microorganisms. Dietary additives that are used in an attempt to modify gut function or enhance health by acting as substrates to stimulate particular groups of microorganisms are referred to as prebiotics (Gibson and Roberfroid, 1995). Almost all prebiotics are oligosaccharides or polysaccharides, and candidates include inulin, fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xylooligosaccharides, and pectic oligosaccharides (Gibson, 1998). Dietary resistant starch, although mainly present as a normal food component, is increasingly being described as a prebiotic in light of evidence for its beneficial health effects (Topping and Clifton, 2001). The best studied prebiotics are inulin and fructooligosaccharides (FOS). In some cases fecal populations of bifidobacteria have been shown to increase tenfold in humans in response to inulin or FOS in the diet (Gibson et al., 1995; Kleessen et al., 1997; Kruse et al., 1999; Tuohy et al., 2001). It is also clear, however, that inulin and FOS are likely to affect other components of the microbial ecosystem. In rats with an associated human fecal flora, inulin increased members of the E. rectale/C. coccoides group (Kleesen et al., 2001) and stimulation of E. cylindroides, ruminococci and an added Roseburia strain has been demonstrated in an anaerobic fermentor system with mixed human fecal inoculum (Duncan et al., 2003).

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Thus it seems important to understand the effects of prebiotics on the whole microbial community and not just the intended target groups, and to use knowledge of carbohydrate utilization in representative gut microbes to predict responses to novel prebiotics. Lactobacilli and bifidobacteria present in live yogurts are widely used as probiotics and have a range of claimed health benefits (Fooks and Gibson, 2002). The combination of a prebiotic with a probiotic strain able to utilize it has been termed a synbiotic. The ability of bifidobacteria to attach to starch creates the possibility of encapsulation in starch granules as a method of delivery to the hindgut (Crittenden et al., 2001). Commercial interest in probiotics has so far concentrated almost entirely on relatively oxygen-tolerant lactic acid bacteria with a history of food use. There is also considerable potential, however, for exploiting some of the more numerous oxygen-sensitive gut bacteria, as evidenced by successful pilot experiments with Oxalobacter formigenes to degrade oxalate in the human GI tract (Duncan et al., 2002b). Specially selected or modified strains of strict anaerobes have also been introduced into the rumen. Perhaps not surprising given the highly complex nature of the native community, enhancing fiber degradation by introducing particularly active cellulolytic bacterial strains has proved difficult (Krause et al., 2001). The use of added strains to restore rumen conditions altered by intensive feeding regimes (e.g., pH adjustment) so as to favor cellulose breakdown may deserve further attention (Russell and Rychlik, 2001). Perhaps the most promising application, however, has been to degrade plant toxins, such as fluoroacetate (Gregg et al., 1994), offering the potential to extend the range of edible forage. B. BIOTECHNOLOGY Microbial polysaccharidases already have a long history of use and thus a wealth of applications ranging from textile manufacture, washing powders, food and drink production, and animal feed pretreatment to waste recycling and paper making (e.g., Beg et al., 2001; van Bielen and Li, 2002; van den Maarel et al., 2002). While mesophilic enzymes from gut microorganisms have usually not been the first choice, they include some of the highest specific activity enzymes known (Selinger et al., 1996) and represent a hugely diverse reservoir of enzymes that has not yet been fully explored. In addition to single cloned enzymes, the possibility of constructing defined complexes tailored for particular applications (Bayer et al., 1994) is being actively researched in


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nongut Clostridium species (e.g., Fierobe et al., 2001). Not only the catalytic properties of these proteins are of value. Recombinant microbial cellulose binding domains present a range of possibilities ranging from the manipulation of plant growth (Sphigel et al., 1998) to affinity chromatography (Tomme et al., 1998). There is also potential to exploit polysaccharide-degrading gut microorganisms themselves, not only as probiotics (discussed previously) but also in in vitro fermentations. Anaerobic digestors have been tested by using ruminococci to digest cellulosic material (Lynd et al., 2002) to produce potentially valuable chemical feedstocks such as succinic acid (e.g., Golkarn et al., 1997) and for the production of methane (Miller et al., 2000) and hydrogen (Nandi and Sengupta, 1998). V. Conclusions and Future Prospects Recent developments in molecular ecology have emphasized our limited knowledge of diversity and function, particularly among anaerobic bacteria that inhabit the GI tract. If we do not have cultured representatives for, perhaps, 70–90% of microbial colonizers (Daly et al., 2001; Suau et al., 1999), then how can we expect to be able to manipulate the system in any targeted manner, or to explain the selective effects of different substrates and dietary additives? Fortunately, prospects for further progress appear very good. First, it seems likely that most gut anaerobes are not intrinsically unculturable. The extreme situation found in soils and marine environments, where far less than 1% of microbial diversity may be cultured, does not apply to gut microbes, which require a minimum growth rate for survival because of the relatively rapid gut transit. Early anaerobic cultivation methods (Hungate, 1966) appear to have been very successful, and 16S rRNA sequences from cultures obtained in this way should gradually fill in many gaps in the phylogenetic tree (e.g., Ramsak et al., 2000), although the possibility of obligate syntrophs remains. Second, the availability of molecular detection of specific groups by fluorescence in situ hybridization (FISH) and real-time PCR (e.g., Franks et al., 1998; Tajima et al., 2001) and community profiling techniques such as terminal restriction fragment length polymorphism (T-RFLP), denaturing gradient gel electrophoresis (DGGE), and microarrays provides unprecedented power of analysis in vivo. Other techniques such as isotopic labelling (Gray and Head, 2001; Radajewski et al., 2000) can also be used to identify groups that use particular substrates in vivo but have yet to be applied to the gut. Third, genome sequencing of further representative polysaccharide-degrading microorganisms, allied to

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gene expression studies, will help to reveal the transport systems, enzyme systems, and regulatory circuits that allow them to utilize polysaccharides. Ultimately, the ecological niche occupied by a given microbe in the gut should be entirely predictable from its genome sequence, but we have some way to go. Particularly important for functional studies is the ability to do in vivo genetics on a wider range of anaerobic microorganisms, as illustrated by elegant work on hostbacterial interactions in B. thetaiotaomicron (e.g., Bry et al., 1996; Hwa et al., 1992).

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Lincosamides: Chemical Structure, Biosynthesis, Mechanism of Action, Resistance, and Applications JAROSLAV SPI´ZˇEK,* JITKA NOVOTNA´,

AND

ˇ EZANKA TOMA´Sˇ R

Institute of Microbiology Academy of Sciences of the Czech Republic 142 20 Prague 4, Czech Republic *Author for correspondence. E-mail: [email protected]

I. Introduction II. Chemical Structure of Lincosamides and Cultivation of Production Strains III. Lincomycin Biosynthetic Pathway A. Biosynthesis of the Amino Acid Moiety B. Biosynthesis of the Sugar Moiety C. Condensation and Final Modification IV. Genetic Control of Lincomycin Biosynthesis V. Mechanism of Action VI. Resistance Against Lincosamides VII. Biological Activity and Applications VIII. Gram-Positive Bacteria IX. Gram-Negative Bacteria X. Anaerobic Bacteria XI. Protozoa and Other Organisms XII. Conclusion and Future Prospects References

121 124 130 130 130 133 133 135 137 138 139 141 143 144 145 146

I. Introduction Lincosamides constitute a relatively small group of antibiotics with a chemical structure consisting of amino acid and sugar moieties. The naturally occurring members of the group are lincomycin and celesticetin, the latter exhibiting 5% of the biological activity of lincomycin in vivo. Many semi-synthetic derivatives of lincomycin have been prepared. Of these, only the chlorinated derivative clindamycin is highly biologically active and is applied practically. Natural lincosamides are produced by several Streptomyces species, mainly by Streptomyces lincolnensis, S. roseolus, and S. caelestis and by Micromonospora halophytica. Their mechanism of action is via inhibition of protein synthesis in sensitive microorganisms. Lincosamides have an unusual antimicrobial spectrum, being active against only Gram-positive and not Gram-negative aerobic bacteria but widely and potently active against anaerobic bacteria and some protozoa. 121 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 56 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2164/04 $35.00

TABLE I STRUCTURE OF LINCOMYCIN AND RELATED ANTIBIOTICS Name

R1

R2

R3

R4

R5

122

1

Lincomycin A

SCH3

CH3

CH2CH2CH3

OH

H

4

Lincomycin B

SCH3

CH3

CH2CH3

OH

H

5

Lincomycin C

SCH2CH3

CH3

CH2CH2CH3

OH

H

7

Lincomycin D

SCH3

H

CH2CH2CH3

OH

H

6

Lincomycin S

SCH2CH3

CH2CH3

CH2CH2CH3

OH

H

8

Lincomycin K

SCH2CH3

H

CH2CH2CH3

OH

H

10

Lincomycin sulfoxide

CH3

CH2CH2CH3

OH

H

11

1-Demethylthio-1-hydroxylincomycin

CH3

CH2CH2CH3

OH

H

2

Celesticetin A

CH3

H

OCH3

H

14

Celesticetin B

CH3

H

OCH3

H

15

Celesticetin C

CH3

H

OCH3

H

16

Celesticetin D

SCH2CH2OOCCH3

CH3

H

OCH3

H

3

Desalicetin

SCH2CH2OH

CH3

H

OCH3

H

17

N-Demethylcelesticetin

H

H

OCH3

H

OH

SCH2CH2OOCCH2CH(CH3)2

18

Desalicetinsalicylate

9

Clindamycin

12

Clindamycin sulfoxide

13

10 -Demethylclindamycin

SCH3

SCH3

CH3

H

OCH3

H

CH3

CH2CH2CH3

H

Cl

CH3

CH2CH2CH3

H

Cl

H

CH2CH2CH3

H

Cl

123

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The major route of resistance to lincosamides is modification of the 23S rRNA in the 50S ribosomal subunit, similarly to resistance against macrolides and streptogramin B (MLSb resistance). Clindamycin is the main lincosamide antibiotic that is applied in clinical practice. II. Chemical Structure of Lincosamides and Cultivation of Production Strains The chemical structure of lincomycin was investigated by Hoeksema et al. (1964), employing both classical chemical degradation and nuclear magnetic resonance. These studies resulted in the chemical structure of lincomycin (1), (Table I). It consists of an unusual amino acid, viz. trans-N-methyl-4-n-L-proline (propylhygric acid), linked by a peptide bond with the sugar 6-amino-6, 8-dideoxy-1-thioD-erythro--D-galactopyranoside (methylthiolincosamide). Details of the chemical characterization and nuclear magnetic resonance studies of lincomycin and its degradation products were subsequently described (Herr and Slomp, 1967; Magerlein, 1971; Schroeder et al., 1967; Slomp and MacKellar, 1967). The electron-impact mass spectrum and the chemical-ionization mass spectrum of lincomycin were reported later (Horton et al., 1974; Kagan and Grostic, 1972). Lincomycin belongs to a novel class of antibiotics characterized by an alkyl 6-amino-6,8-dideoxy-1-thio-D-erythro--D-galacto-octopyranoside joined with a proline moiety by an amide linkage. The availability of this structure presented an excellent opportunity for chemical and microbiological modification of lincomycin. Before a review of the other naturally occurring members of the lincomycin family of antibiotics, processes used for the cultivation of producing strains (Table II) and methods for separation of lincomycin A will be described. The first patent describing lincomycin production was registered by Upjohn (Bergy et al., 1963). S. lincolnensis var. lincolnensis was cultivated in a 380-liter fermentor at 28 C for 5 days, and 210 liters of cultivation broth were harvested to give approximately 105 g of dried lincomycin A at a purity of 232 biounits/mg. The actinomycete S. vellosus var. vellosus isolated from Arizona soil was used according to Bergy et al. (1981) for the production of lincomycin A without the concomitant production of lincomycin B (see subsequent discussion). The novel actinomycete described in the patent of Argoudelis et al. (1972) produced only lincomycin A. S. espinosus produced 50 g/ml of the desirable lincomycin A during a 4-h cultivation at 28 C in

125

LINCOSAMIDES TABLE II STRAINS PRODUCING LINCOMYCIN GROUP OF ANTIBIOTICS Organisms

Literature

S. lincolnensis 78–11

Peschke et al. (1995), Zhang et al. (1992)

S. lincolnensis NRRL 2936

Bergy et al. (1963), Brahme et al. (1984a), Peschke et al. (1995)

S. lincolnensis RIA 1246

Neusser et al. (1998)

S. lincolnensis UC 5124

Chung and Crose (1990)

S. espinosus NRRL 5729

Peschke et al. (1995)

S. espinosus NRRL 3890


S. espinosus

Argoudelis et al. (1972)

S. espinosus

Reusser and Argoudelis (1974)

S. pseudogriseolus NRRL 3985


S. variabilis var. liniabilis

Argoudelis and Coats (1974)

S. vellosus NRRL 8037

Bergy et al. (1963), Peschke et al. (1995)

shaken flasks by using a classical medium. According to the U.S. patent (Argoudelis and Coats, 1973), S. pseudogriseolus var. linmyceticus cultivated for 2 days at 28 C produces 22 g/ml of lincomycin. Another patent on production of lincomycin A only (Argoudelis and Coats, 1974), without contamination with lincomycin B, was registered by The Upjohn Company. Streptomyces variabilis var. liniabilis produces 26 g/ml of lincomycin A after the 120-h cultivation at 28 C in shaken 500-ml Erlenmeyer flasks. The strain S. lincolnensis used by Zhang (1993) is a phageresistant, lincomycin-overproducing mutant synthesizing approximately 2.5 g of lincomycin A per liter. In the last 5 years, Chinese investigators have become interested in the cultivation of lincomycin-producing strains, especially in the isolation of lincomycin with a simultaneous decrease or complete removal of the undesirable lincomycin B (Dong and Dong, 2001; Li et al., 2001; Wang and Qi, 1999, 2000, 2001). In addition to lincomycin, a very closely related antibiotic was isolated from fermentations of Streptomyces lincolnensis var. lincolnensis. Structural studies indicated this compound to be lincomycin B (i.e., 40 -depropyl-4-ethyllincomycin (2). It had already been

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described in 1965 as a minority component in S. lincolnensis cultures. It exhibits only a 25% antibiotic activity as compared with lincomycin A. As shown in Table I, lincomycin B contains one fewer methylene group in the side chain of propylhygric acid. Several patents (Reusser and Argoudelis, 1974; Visser, 1972; Witz, 1972) were published in which procedures were described to minimize the amount of lincomycin B during the fermentation. The addition of propylproline to the cultivation medium was found to decrease the amount of lincomycin B. It was assumed that propylproline is a likely precursor of propylhygric acid in lincomycin A, whereas ethylproline is a tentative precursor of ethylhygric acid of lincomycin B. Aromatic amino acids, primarily L-tyrosine, L-dihydroxyphenylalanine, and other compounds similar to tyrosine, are by S. lincolnensis incorporated into lincomycin A. It was found that L-tyrosine or L-dihydroxyphenylalanine added to the culture medium induces accumulation of propylproline and also, although to a lesser extent, that of ethylproline. The mechanism by which the addition of propylproline or L-tyrosine and similar compounds influences the biosynthesis of lincomycin has not yet been clarified; however, the results obtained so far indicate that N-demethyllincomycin synthetase, the enzyme catalyzing the linkage between the amino acid and sugar moiety of lincomycin, preferentially utilizes propylproline. Lincomycin A was the first member of its family whose structure had been completely elucidated; however; celesticetin, a related antibiotic, was reported earlier in the culture broth of Streptomyces caelestis, a new actinomycete species. This antibiotic was also isolated by Hoeksema et al. (1955). Employing some of the techniques used in the structural studies on lincomycin, Hoeksema (1968) assigned structure 3 to celesticetin. Desalicetin, the alkaline hydrolysis product of celesticetin, was shown to possess structure 4. Celesticetin and desalicetin proved to be less effective than lincomycin against a number of microorganisms, both in vitro and in vivo (Mason and Lewis, 1964). Celesticetin exhibits a broad antibacterial spectrum, particularly against Gram-positive bacteria. Other new antibiotics, viz. N-demethyl7-O-demethylcelesticetin and N-demethylcelesticetin, were found to be produced in cultures of a strain of S. caelestis. The introduction of various chemicals to the fermentation of S. lincolnensis var. lincolnensis was found to induce the formation of lincomycin-related antibiotics. Thus the addition of DL-ethionine led to the formation of 1-demethylthio-1-ethylthiolincomycin (5) (Argoudelis and Mason, 1965) and 1-demethyl-1-demethylthio-10 -

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ethyl-1-ethylthiolincomycin (6) (Argoudelis et al., 1970); methylthiolincosaminide induced formation of 10 -demethyllincomycin (7); the addition of ethyl thiolinconsaminide yielded 10 -demethyl-1demethylthio-1-ethylthiolincomycin (8). Sulfonamides, sulfanilamide in particular, inhibit N-methylation rather selectively, resulting in the production of 10 -demethyllincomycin (7). Chemical modification (Argoudelis and Stroman, 1984; Birkenmeyer, 1981, 1982a,b; Patt et al., 1984) of lincomycin derivatives consisted mainly in substitution of hydrogen atoms bound to a heteroatom. A number of lincomycin esters, with either organic (from acetate to stearate) or inorganic (phosphoric, carbonic) acids or of lincomycin alkyl derivatives (ethers), were prepared. Substitutions were performed primarily in positions 2, 3, 4, and 7. Salts of lincomycin with inorganic acids (i.e., hydrochlorides and derivatives of sulfamic acid) were also synthesized. Replacement of hydroxyl in the side chain (i.e., C-7 of octose) is another modification of lincomycin. The commercially used derivative with the generic name clindamycin (9) was produced by introduction of chlorine. Additional thio-analogs, of which, for example, the C-1 -anomer exhibits only one tenth of the activity of the natural -anomer, were synthesized. The changed configuration of 1-methyl-4-proline is also very important. It was demonstrated that the derivative containing the D amino acid exhibits only 50% of the antibacterial activity. Although hundreds of lincomycin derivatives were prepared, among them derivatives produced totally by chemical synthesis, clindamycin is the only drug that has been used successfully in clinical practice. Detailed synthetic procedures leading to numbers of lincomycin derivatives and their biological activity were described by Magerlein (1971). A new and stereoselective route to the aminoglycoside components of the antibiotics lincomycin and clindamycin has been described. The key step involves diastereoselective introduction of the amino group at C-6 of D-galactose by (3,3)-sigmatropic rearrangements of the corresponding allylic (Z)-trifluoroacetimidate and (Z)- and (E)-allylic thiocyanates. Epoxidation of the resulting trifluoroacetamide with m-chloroperbenzoic acid led to the epoxide with a high threo-selectivity (Gonda et al., 2000). In another paper (Bowden and Stevens, 2000) a novel synthesis of clindamycin from lincomycin by using N-chlorosuccinimide and triphenylphosphine was reported, resulting in high yields and avoiding the use of tetrachloromethane employed in the currently used manufacturing process. Microbial modification of lincomycin and clindamycin does not lead to the formation of the number and diversity of transformation

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products obtained by chemical modification. However, several types of interesting transformations have been reported. Thus, for instance, lincomycin was glycosylated by using jack bean -mannosidase to produce 7-O--D-mannopyranosyl-lincomycin (Weignerova et al., 2001). The addition of lincomycin to a fermentation of S. lincolnensis var. lincolnensis resulted in the formation of lincomycin sulfoxide (10). This sulfoxide was also prepared by oxidation of lincomycin hydrochloride with hydrogen peroxide (Pospisil et al., 2001). As compared with lincomycin, it exhibits only a 0.01 activity against Sarcina lutea. A lower yield of 1-demethylthio-l-hydroxylincomycin (11) was also isolated from the fermentation broth. Similarly, clindamycin sulfoxide (12) was the major transformation product when clindamycin was added to fermentations of S. armentorus. Trace amounts of sulfoxide were detected in similar fermentations of other Streptomyces species, particularly S. punipalus. Clindamycin sulfoxide is about equally active as lincomycin against Sarcina lutea. In addition to sulfoxide formation, several species of streptomycetes, including those mentioned above, perform a partial 1-demethylation (Argoudelis et al., 1969). S. punipalus was found to demethylate clindamycin (9) to 10 -demethylclindamycin (13), which had previously been prepared by chemical modification. Oxidation of lincomycin with H2O2 in alkaline media leads to N-oxides and the conversion of thiomethyl group to sulfoxides and sulfones. NH4OH favors formation of the S-isomer; both R- and S-isomers of N-oxide form in the presence of NaOH (Pospisil et al., 2004). Argoudelis and Coats (1969) observed that S. rochei grown in a synthetic medium converted lincomycin and lincomycin-related antibiotics to their 3-phosphate esters. Lincomycin 3-phosphate was inactive in vitro against several organisms. In addition to ‘‘classical methods’’ for the analysis of lincomycin and clindamycin, in the last 20 years methods of instrumental analysis based on liquid chromatography (LC) as a separation method and soft ionization techniques of mass spectrometry as an identification method were developed. Reverse-phase (RP) high-performance liquid chromatography (HPLC) for preparation of lincomycin A was patented (Hofstetter, 1982). This process yielded a highly purified lincomycin A with less than 0.5% lincomycin B. Lincomycin salvage mother liquor, containing about 30% lincomycin B, was used as the starting material for preparation of lincomycin B.

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Procedures for the determination of clindamycin in human plasma using HPLC with ultraviolet (UV) detection have been described (Fieger-Bu¨schges et al., 1999; La Follette et al., 1988; Liu et al., 1997). These methods are either not very sensitive or are time-consuming and require laborious sample preparation. More recently, methods using HPLC coupled with MS detection have been published for the quantification of clindamycin in human plasma. Yu et al. (1999) used an LC-MS/MS/ESI method, with acceptable linearity in the 0.05–20 l/ml range and a quantification limit of 0.050 g/ml. Martens-Lobenhoffer and Banditt (2001) used an LC-MS/ APCI method for both human plasma and bone. The method showed good linearity in the 0.1–4.0 g/ml range for plasma with a limit of quantification of 0.1 g/ml. A method for the quantification of clindamycin in animal plasma by using LC-MS/MS/ESI has also been published (Cherlet et al., 2002). Lincomycin was used as the internal standard. Good linearity was observed within the range of 0–10 g/ml. The limit of quantification of the method is 50 g/ml, and the detection limit is 1.3 ng/ml. The method was used for pharmacokinetic studies of clindamycin formulations in dogs. Lincomycin and related antibiotics were analyzed by an MS/MS/CID technique in bovine milk extract. Lincomycin gave a limit of detection of 0.83 pg on-column (Crellin et al., 2003). For a further analysis of lincomycin and spectinomycin, an RP ionpair LC method with a base-deactivated column and pulsed electrochemical detection by means of a gold electrode was used (Szunyog et al., 2002). Capillary electrophoresis was used for a simultaneous determination of five aminoglycoside antibiotics (netilmicin, tobramycin, lincomycin, kanamycin, and amikacin). Under the optimum separation conditions, the aminoglycoside antibiotics were baseline separated within 20 min and the detection limit was below 6.7 M for lincomycin (Yang et al., 2001). HPLC-integrated pulsed amperometric detection was found to be a good primary detection method to complement or replace the absorbance detection method in the case of nonchromophoric sulfurcontaining antibiotics and their sulfur-containing impurities and decomposition products (Hanko et al., 2001). A thin-layer chromatography/densitometric method was developed for the identification and quantitation of oxytetracycline, tiamulin, lincomycin, and spectinomycin in veterinary preparations (Krzek et al., 2000).

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III. Lincomycin Biosynthetic Pathway The lincomycin biosynthetic pathway proceeds via a heterogeneously rooting biphasic pathway, giving rise to propylproline and methylthiolincosamide (Fig. 1). These basic precursors become condensed to N-demethyllincomycin, which is finally methylated to yield lincomycin. A. BIOSYNTHESIS OF THE AMINO ACID MOIETY Tyrosine seems to be the principal precursor of the amino acid moiety. Feeding studies using L-[1-14C]tyrosine and L-[U-14C]tyrosine performed by Witz et al. (1971) suggested that seven of the nine carbons in the propylhygric acid come from tyrosine. The other two carbons, viz. the N-methyl and terminal aliphatic side chain methyl groups, come from S-adenosylmethionine (Argoudelis et al., 1969; Brahme et al., 1984a,b; Witz et al., 1971). Biosynthetic experiments with D-(13C6) glucose suggest that glucose is converted via glycolysis and the hexose monophosphate shunt to phosphoenolpyruvate and erythrose-4-phosphate, respectively, which are in turn converted via the shikimic acid pathway to tyrosine and then to dihydroxyphenylalanine. The pathway probably continues through the 2,3-extradiol cleavage of the aromatic ring of dihydroxyphenylalanine, followed by condensation to form a pyrrolo ring (Brahme, 1984a). A similar biosynthetic route leading from tyrosine through dihydroxyphenylalanine and pyrrolo ring formation lincomycin probably shares with the pyrrolo[1,4]benzodiazepine antibiotics anthramycin, sibiromycin, and tomaymycin (Hurley, 1980; Hurley et al., 1979). The multistep conversion of dihydroxyphenylalanine to the propylproline remains unknown, but it appears to lead to 1,2,3,6-tetradehydro-propylproline, which was shown to accumulate in mutants lacking a reductase that requires lincomycin cosynthetic factor (Kuo et al., 1992) identified as 7,8-didemethyl-8-hydroxy-5deazariboflavin (Kuo et al., 1989). Based on this finding, Kuo and coworkers modified the scheme of Brahme et al. (1984a,b) and postulated the remaining steps leading to propylproline, as shown in Fig. 1. B. BIOSYNTHESIS OF THE SUGAR MOIETY No intermediates of the aminooctose moiety methylthiolincosamide biosynthesis have so far been identified.

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FIG. 1. Hypothetical biosynthetic pathway for lincomycin A. (A) Amino acid branch. (B) Sugar branch. Two alternative routes are proposed for methylthiolincosamide biosynthesis. (B1) From intermediates of the pentose phosphate cycle via formation of a C8 sugar precursor. (B2) From dTDP-glucose via a 6-deoxyhexose pathway and a later extension of the carbon chain. The possible involvement of three of the gene products of the putative methylthio-lincosamide biosynthetic genes is indicated. (C) Condensation and final methylation.

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A likely pathway leading from glucose to methylthiolincosamide was proposed by Brahme et al. (1984b), who studied the metabolic origin of methythiolincosamide by analyzing 13C-13C spin coupling patterns in methythiolincosamide and lincomycin-derived from biosynthetic experiments with D-(13C6)glucose and the specific 13C enrichments in methylthiolincosamide synthesized from specifically labeled substrates. The data obtained by means of both these approaches indicated that the C8 carbon skeleton of methythiolincosamide arises from condensation of a pentose unit (C5) and a C3 unit. The pentose unit could either be derived from glucose through the hexose monophosphate shunt as an intact unit or result from condensation of glyceraldehyde-3-phosphate with a C2 donor such as sedoheptulose7-phosphate via transketolase reaction. The C3 unit probably arises from a suitable donor molecule such as sedoheptulose-7-phosphate and is added via a transaldolase reaction. According to the C3 unit donor origin, the unit could consist either of an intact C3 unit or a C2 unit combined with a C1 unit. The C3 and C5 units are then condensed, giving rise to octose, which is then converted to methylthiolincosamide. The final conversion of the C8-skeleton to methylthiolincosamide was predicted to involve isomerization of the octulose to octose, dephosphorylation and reduction of the C8 carbon, transamination of the precursor 6-ketooctose, and final thiomethylation of the C1 carbon. First attempts to find out the metabolic origin of the sugar moiety were by means of a combination of radioactive labeling and mass spectroscopy. It was determined that the S-methyl group of the methylthiolincosamide subunit is derived from C1 fragments at the oxidation state of methyl groups. Sequencing of the lincomycin gene cluster, and amino acid sequence analysis of the putative protein products, led Peschke et al. (1995) to modify the aforementioned design of the biosynthetic pathway. Eight genes, lmbL through lmbQ, form a subcluster, which probably codes for a set of enzymes involved in sugar metabolism (Fig. 1). Putative proteins LmbO, LmbM, and LmbS show similarity to enzymes involved in the central steps of many NDP-6-deoxyhexose pathways, including sugar-activating pyrophosphorylases (LmbO), NDP-hexose dehydratases (LmbM), and (NDP-) ketosugar (or cyclitol) aminotransferases/ dehydratases (LmbS). The corresponding genes are found mostly in clusters in both Gram-positive and Gram-negative bacteria, and their protein products are involved in the formation of other actinomycete secondary metabolites (Piepersberg, 1994; Pissowotzki et al., 1991; Retzlaff et al., 1993; Stockmann and Piepersberg, 1992) or of extracellular polysaccharides (Thorson et al., 1993).

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The genetic record led Peschke et al. (1995) to the conclusion that methylthiolincosamide biosynthesis involves a nucleotide (probably dTDP) activation step and a series of modification steps on dNTPactivated sugar intermediates. Thus, two totally different pathways, starting from either D-glucose or NDP-activation and modification of a C8 sugar intermediate, both represent routes leading to the production of methylthiolincosamide. The authors assume that the 13C-labelling pattern in methylthiolincosamide published by Brahme et al. (1984b), suggesting that an octulose-phosphate intermediate is formed first could support glucose as a starter unit if there is rapid and efficient equilibrium in the hexose phosphate pool mediated by a hexose monophosphate shunt during the production phase. This type of rearrangement of labelling has been described for other streptomycete products directly derived from glucose (Rinehart et al., 1992). The detection of a gene (lmbR) encoding a transaldolase-like enzyme in the ‘‘sugar subcluster’’ could support an alternative route via a pyranosidic octose intermediate that is NDP-activated and further modified in this form. Finally, a thiomethyl unit at the C1 position of the postulated (NDP-)6-amino-6,8-deoxyoctose intermediate would be added, which could be transferred from 50 -thiomethyladenosine, a side product of polyamine biosynthesis from S-adenosyl-methionine (Piepersberg and Distler, 1997). C. CONDENSATION AND FINAL MODIFICATION Formation of the amide bond between the carboxyl group of the aglycone propylproline and the amino group of the methylthiolincosamide yielding N-demethyllincomycin is apparently the penultimate step in lincomycin biosynthesis. N-demethyllincomycin-synthetase, catalyzing this reaction, is a complex of readily dissociable subunits with nonidentical molecular weights (Hausknecht and Wolf, 1986). The final step in the pathway is N-methylation of N-demethyllincomycin, catalyzed by S-adenosylmethionine: N-demethyllincomycin methyl transferase (Patt and Horvath, 1985). IV. Genetic Control of Lincomycin Biosynthesis Similarly to other antibiotics (Martin and Liras, 1989) produced by actinomycetes, genes coding for lincomycin biosynthesis are clustered together in a single genomic region and closely linked to the corresponding resistance determinants (Chung and Crose, 1990; Wovcha et al., 1986; Zhang et al., 1992).

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FIG. 2. The lincomycin gene cluster. Grey arrows designated A-Z indicate putative biosynthetic genes and black arrows designated rA-rC indicate genes coding for functions that impart lincomycin resistance phenotype.

The 35-kpb-long chromosomal region bearing a lincomycin-production gene cluster from the overproducing industrial strain Streptomyces lincolnensis 78–11 was cloned and sequenced by Peschke et al. (1995). The cluster contains 27 open reading frames with putative biosynthetic or regulatory functions (lmb genes) and three resistance (lmr) genes (Fig. 2). The genes designated lmrA and lmr C flank the cluster and appear to code for proteins probably involved in lincomycin export (Peschke et al., 1995; Zhang et al., 1992). The lmrB gene codes for a protein very similar to several 23S RNA methyltransferases (Zhang et al., 1992). Four other lincomycin producers probably share the overall cluster organization; however, the clusters are embedded in nonhomologous chromosomal surroundings. DNA sequence analysis suggests a complicated transcription pattern with at least 12 possible transcription units. On the basis of experiments using transposon 4560-mediated mutagenesis of the lincomycin production cluster of the strain derived from Streptomyces lincolnensis UC 5124, it is assumed that genes coding for enzymes involved in propylproline synthesis are located close to lmrA gene, whereas those controlling methylthiolincosamide synthesis are located further from it (Chung and Crose, 1990). Thus, biosynthetic genes seem to be organized according to functional pathways within the cluster region; however, the same experiments suggest that genes coding for subunits of the NDL synthetase, the key enzyme catalyzing propylproline and methylthiolincosamide condensation, are located at three widely separated loci (Chung et al., 1997). The functions coded for by only a few of the aforementioned open reading frames were demonstrated experimentally. In addition to resistance genes, only the function of the genes lmbB1 and lmbB2, coding for enzymes converting L-tyrosine and L-dihydroxyphenylalanine, was clarified (Neusser et al., 1998). Gene lmbJ apparently codes for a specific N-demethyllincomycin methyltransferase (Janata et al., 1999). Functions of other proteins coded for by lincomycin cluster genes can only be deduced.

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V. Mechanism of Action Lincosamides belong to antibiotics that block microbial protein synthesis. Protein synthesis includes a large number of steps from activation of amino acid monomers by aminocayl-tRNA synthetases to many steps of chain initiation, elongation, and chain termination of the grown polypeptides on the ribosome. Antibiotics interrupt the timing and specificity of any of these steps, and such disruptions decelerate the growth or are lethal to the microorganism. A molecular mechanism by which clindamycin inhibits ribosomal protein biosynthesis in prokaryotic microorganisms has therefore been unclear. However, recently it was shown that clindamycin’s three-dimensional structure closely resembles the L-Pro-Met and the D-ribosyl ring of adenosine (Fitzhugh, 1998) biomolecules, which occur proximate to one another at the 30 -ends of L-Pro-Met-tRNA and deacylated-tRNA for a brief interval following the formation of a peptide bond between L-Pro-tRNA and L-Met-tRNA. This finding strongly suggests that clindamycin and other lincosamides act as structural analogs of the 30 -ends of L-Pro-Met-tRNA and deacylated-tRNA as they are positioned during an initial phase of pretranslocation in the peptide elongation cycle. Clindamycin thus constitutes a structural analog of an intermediate state in protein biosynthesis. Although the chemical structure of macrolides (e.g., erythromycin), lincosamides (e.g., lincomycin, clindamycin, and celesticetin), and streptogramins is very different, their mechanism of action is similar. Erythromycin binding with the 23S rRNA blocks polypeptide translation, resulting in a release of peptidyl-tRNA intermediates prematurely by blocking the approach to the elongating peptide’s exit tunnel (Schlunzen et al., 2001). Erythromycin also blocks assembly of 50S subunits, probably through its interaction with 23S rRNA. Although the macrolides in general do not directly block the peptide bond-forming step at the peptidyltransferase center of the 50S subunits, it is known that they compete with lincosamide antibiotics that are direct peptidyltransferase inhibitors. A point mutation at A2058 of 23S rRNA induces a triple resistance to macrolides, lincosamides, and streptogramin B. Schlunzen et al. (2001) provided a direct proof with the cocrystal structure of the lincosamide antibiotic clindamycin, in which the 20 - and 30 -hydroxyl groups of the antibiotic sugar moiety form hydrogen bonds with the same exocyclic N6 amino group of A2058. Clindamycin and erythromycin binding show a partial physical overlap. Clindamycin has separately been known to interact with both the A site and the P site of the peptidyltransferase center. In accordance with their mechanism of action, bacteria quite often

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develop cross-resistance to macrolides, lincosamides, and streptogramin B. Lincomycin and clindamycin share a common mechanism in sensitive microorganisms and also exhibit a similar antibacterial spectrum. Nevertheless, slight differences in their antimicrobial activity exist as clindamycin also affects some protozoa—for example, Toxoplasma gondii, Plasmodium falciparum, and Pneumocystis carinii. Administration of 450–600 mg of clindamycin every 6 h and 15–30 mg of primaquine base applied once a day were used successfully for the treatment of infections caused by Pneumocystis carinii (Fishman, 1998), whereas lincomycin did not exhibit any activity. Remington (1990) demonstrated that a combination of clindamycin with primaquine is highly effective in the treatment of pneumonia caused by Pneumocystis carinii, even in AIDS patients. Clindamycin inhibits bacterial protein synthesis and acts specifically on the 50S subunit of the bacterial ribosome, most likely by affecting the process of peptide chain initiation. It may also stimulate dissociation of peptidyl-tRNA from ribosomes (Menninger and Coleman, 1993). Escherichia coli exposed to subminimal inhibitory concentrations of clindamycin shows a decreased adherence to buccal mucosal cells, which may be due to protein synthesis inhibition. Presumably it was suppression of protein synthesis that also enabled Sanders et al. (1983) to show that clindamycin is an effective in vitro inhibitor of the derepression of bacterial -lactamases that would otherwise have been produced by certain nonfastidious Gram-negative bacilli exposed to various -lactam antibiotics. Similarly, Schlievert and Kelly (1984) showed inhibition of toxin production in toxic shock syndrome-producing strains of Staphylococcus aureus by concentrations of clindamycin that do not inhibit bacterial growth. Macrolides and lincosamides are first-choice bacteriostatic antibiotics used in veterinary dermatology. The main antibiotics of these classes are erythromycin, lincomycin, clindamycin, and tylosin. They are well absorbed if administered orally and are able to penetrate well into infected skin. Their spectrum of action comprises bacteria commonly associated with skin infections, including staphylococci. Their main disadvantage is a rapid development of bacterial resistance and occasional gastrointestinal upset (Noli and Boothe, 1999). Macrolides, fluoroquinolones, rifamycins, tetracyclines, trimethoprimsulfamethoxazole, and clindamycin were described as antimicrobial agents of preference for the dermatologist (Epstein et al., 1997). Clindamycin administration results in changes in intestinal microflora. Numbers of enterococcal species increase and those of all anaerobes decrease (Nord and Heimdahl, 1986).

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Some antibiotics also exhibit immunomodulatory effects, clindamycin among them (VanVlem et al., 1996). VI. Resistance Against Lincosamides In general, basic mechanisms of antibiotic resistance include microbial cell impermeability, target site modification, and enzymatic modification or destruction of the antibiotic and its increased efflux. The main type of resistance to lincomycin and clindamycin is the socalled MLSb resistance that renders sensitive microorganisms resistant to macrolides, lincosamides, and streptogramin B (hence MLSb resistance). It is monomethylation or dimethylation of the N6 exocyclic amino group of A2058 by specific ribosome methylation modification enzymes. This type of resistance is associated with genes encoding methyltransferases modifying the common target site of macrolides and lincosamides (i.e., 23S ribosomal RNA—e.g. genes ermA and ermC). A specific gene was also described whose protein product modifies and thus inactivates lincosamide antibiotics (linA). Increased efflux of lincosamides was detected in some microorganisms resistant to them. The occurrence of some genes involved in MLSB resistance in methicillin-resistant strains of Staphylocococcus aureus was investigated, and it was shown that, in the Czech Republic, genes ermC and ermA are the most frequent determinants of MLS resistance (up to 90% cases) and that gene msrA, encoding the protein responsible for the active excretion by the resistant cells of macrolides and streptogramins but not of lincosamides, is less common. Gene linA, whose protein product modifies and thus inactivates lincosamide antibiotics only, is an additional resistance gene that is less frequent (Novotna et al., 2002). Clindamycin and lincomycin share a common or overlapping binding site on the ribosome. As already mentioned, a ribosomal mutation makes the ribosome insensitive to clindamycin. Expression of clindamycin-resistance in Gram-positive cocci may be constitutive or inducible. Staphylococci can also owe their resistance to clindamycin because of enzymatic inactivation of the drug, the enzyme production being specified by small nonconjugative plasmids. The third resistance mechanism to clindamycin involves active efflux of the antibiotic from the periplasmic space. This mainly occurs in Gram-negative bacteria (Leclercq and Courvalin, 1991). Lincomycin resistance in clinical isolates of staphylococci and streptococci has been recognized for several decades. This resistance is plasmid mediated and is encoded on transposons. The resistance

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results from the induction of an enzyme that is normally repressed. The methylated RNA binds lincomycin-type drugs less well than does the nonmethylated RNA. The induction of resistance varies by species, and in most Gram-positive species, erythromycin is a moreeffective inducer of resistance than clindamycin. The plasmids that mediate lincomycin resistance in streptococci and staphylococci are highly similar structurally, indicating that they could have been readily transferred among strains of these species. Inactivation (resistance) of lincosamides by the products of the linA (encoding 3-lincomycin 4-clindamycin O-nucleotidyltransferase) genes of Staphylococcus aureus is one of the resistance mechanism in this bacterium (Matsuoka, 2000). VII. Biological Activity and Applications When usual doses are applied, both lincomycin and clindamycin exhibit bacteriostatic activity (Table III). At higher concentrations that can still be reached in vivo, their effect may be even bactericidal. However, the onset of the bactericidal effect is delayed and is less complete than that of, for example, -lactams. It increases generally in parallel with an increasing concentration of the antibiotic, and even in this respect clindamycin is more effective. On the other hand, the main advantage of lincomycin is that it can be applied at a substantially wider concentration range of clinical therapeutical doses. Macrolides and lincosamides are first-choice bacteriostatic antibiotics used in veterinary dermatology. The main antibiotics of these classes are erythromycin, lincomycin, clindamycin, and tylosin. They are well absorbed if administered orally and are able to penetrate well into infected skin. Their spectrum of action comprises bacteria commonly associated with skin infections, including staphylococci. Their main disadvantage is the rapid development of bacterial resistance and occasional gastrointestinal upset (Noli and Boothe, 1999). Macrolides, fluoroquinolones, rifamycins, tetracyclines, trimethoprim-sulfamethoxazole, and clindamycin have been described as antimicrobial agents of preference for the dermatologist (Epstein et al., 1997). Clindamycin administration results in changes in intestinal microflora. Numbers of enterococcal species increase and those of all anaerobes decrease (Nord and Heimdahl, 1986). Lincomycin was also used as an inhibitor of protein synthesis in experiments concerning photoinactivation in plants and algae (Kato et al., 2002; Sicora et al., 2003).

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LINCOSAMIDES TABLE III EFFECT OF LINCOMYCIN AND CLINDAMYCIN ON SOME COMMON PATHOGENIC BACTERIA MIC (g/ml) Test organism

Lincomycin

Clindamycin

Gram-positive

Generally sensitive

Generally sensitive

Bacillus anthracis

0.25–8.0

0.25–5.0

Staphylococcus aureus

0.2–3.2

0.04–1.6

Staphylococcus epidermidis

0.4–1.8

0.1–0.2

Streptococcus agalactiae

0.1–0.2

0.02–0.1

Streptococcus pneumoniae

0.01–0.8

0.002–0.1

Streptococcus pyogenes

0.04–0.8

0.01–0.2

Streptococcus viridans

0.02–1.0

0.005–0.2

Gram-negative

Generally resistant

Generally resistant

Escherichia coli

1000

64

Haemophilus influenzae

4–16

0.5–16.0

Klebsiella pneumoniae

8

125

Neisseria gonorhoeae

8–64

0.5–4.0

Neisseria meningitis

>32

4

Proteus vulgaris

1000

250

Pseudomonas aeruginosa

>1000

1000

Salmonella schottmuelleri

125

64

The values in Table III can serve for a general orientation only. They were obtained from a number of experimental papers in which different strains were used and the data often varied substantially.

VIII. Gram-Positive Bacteria Clindamycin is active against most of the following bacteria: Staphylococcus aureus, Streptococcus pyogenes, S. pneumoniae, S. viridans and S. bovis, Corynebacterium diphtheriae, Enterococcus durans, Bacillus anthracis, B. cereus, and the Nocardia spp., but unfortunately, it is inactive against Enterococcus faecalis and E. faecium (Gigantelli et al., 1991). In stomatology, clindamycin can be used for the treatment of infections caused by Bacillus melaninogenicus and B. fragilis. Bacterial skin and skin structure infections are caused by aerobic staphylococci and streptococci (Streptococcus pyogenes and Staphylococcus aureus), with aerobic Gram-negative bacilli and anaerobes being involved in more complicated infections. Systemic therapy with lincosamides (clindamycin) has been the cornerstone of the treatment of these infections for many years (Guay, 2003).

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Staphylococcus aureus and Streptococcus pyogenes cause a number of serious infections, such as necrotizing fascitis and toxic shock syndrome, which are associated with the release of bacterial toxins. Animal studies showed that clindamycin is more effective in treating these severe infections than are other drugs (Coyle, 2003). Bacillus anthracis infection can occur in three forms: cutaneous, gastrointestinal, and inhalational, depending on the mode of infection. Anthrax is a zoonotic disease but, unfortunately, the inhalation form can also be used as a biological warfare agent. In addition to other antibiotics, clindamycin was used for treatment (Brook, 2002a). Severe infections caused by Streptococcus pyogenes (group A streptococci) can be treated with clindamycin, acting as an inhibitor of the synthesis of protein M and of extracellular proteins (Bouvet, 1996). Pneumonia caused by Bacillus cereus could be treated with clindamycin (Bastian et al., 1997). Although tests of clindamycin resistance in streptococci are not generally performed in clinical practice, resistance to clindamycin has already been detected in strains of the following species: Streptococcus pyogenes, strains of group B streptococci, and strains of S. pneumoniae. Pneumococci that are multiply resistant to many antibiotics, including clindamycin, were reported from South Africa, but in most areas in the United States they are still clindamycin-sensitive. Staphylococci resistant to clindamycin are more common, and clindamycin sensitivity tests are always performed (Reeves et al., 1991). Type 3 pneumococci produce a capsule composed of cellobiuronic acid units connected in a (1 ! 3) linkage. The genes implicated in the biosynthesis of the type 3 capsule (cap3 genes) were cloned, expressed, and biochemically characterized. The three cap3 genes designated as cap3ABC form an operon. The Cap3A, Cap3B, and Cap3C proteins were biochemically characterized as a UDP-glucose dehydrogenase, the type 3 polysaccharide synthase, and a glucose-1-P uridyltransferase, respectively. The Cap3B protein was expressed in E. coli, and pneumococcal type 3 polysaccharide was synthesized in this heterologous system. When a recombinant plasmid containing cap3B (pLSE3B) was introduced into encapsulated pneumococci of types 1, 2, 5, or 8, the lincomycin-resistant transformants showed a type 3 capsule in addition to that of the recipient type. Unencapsulated (S2) laboratory strains of S. pneumoniae also synthesized a type 3 capsule when transformed with the pLSE3B plasmid (Garcia et al., 1997).

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The increasing prevalence of clindamycin-resistant bacteria (resistant Streptococcus pneumoniae in particular) is leading to new approaches to the management of common respiratory infections in the outpatient setting (Green and Wald, 1996). Clindamycin is an effective therapy for community-acquired, methicillin-resistant Staphylococcus aureus, but there is a risk of development of clindamycin resistance during the treatment of these bacteria (Marcinak and Frank, 2003). Emerging treatments for streptococcal toxic shock syndrome (caused by, for example, Streptococcus pyogenes) include administration of clindamycin and intravenous -globulin (Stevens, 2000). Streptococcus pyogenes, particularly the capsule and protein M, as well as streptococcal toxins, cause severe septic and toxic syndromes. Clindamycin should be used in case of septic shock (Veyssier-Belot et al., 1999). The efficacy of clindamycin and the failure of penicillin to treat a severe group A streptococcal infection and streptococcal toxic shock syndrome were described (Stevens, 1996). An enhanced bactericidal response against -hemolytic streptococci has been found with a combination of penicillin and clindamycin (Seal, 2001). Resistance of enterococci and staphylococci to many antibiotics including clindamycin was described by McManus (1997). Antibiotic resistance of different strains of Bacteroides, Prevotella, and Porphyromonas species and in vitro antimicrobial susceptibility to many antibiotics, including clindamycin, were reviewed by Falagas and Siakavellas (2000). The true fungi and acid-fast and poorly Gram-stainable Mycobacterium tuberculosis are clindamycin-resistant, but clindamycin has some activity against M. leprae. IX. Gram-Negative Bacteria In general, aerobic Gram-negative bacteria are resistant to clindamycin. It was described that in vitro clindamycin is more active against H. influenzae than lincomycin. Campylobacter jejuni is sensitive to clindamycin, but E. coli is much more resistant (128 g/ml) compared with C. jejuni (8 g/ml). Capnocytophaga canimorosus causing bacteremic illness and dog sickness or other animal diseases is clindamycinsensitive. Flavobacteria may also be clindamycin-sensitive (Sheridan et al., 1993).

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Clindamycin has good activity against the Bacteroides fragilis group of anaerobic bacteria (2 g/ml). Unfortunately, the number of clindamycin-resistant strains increases with time, but B. fragilis still remains one of the most sensitive bacteria as compared with B. thetaiotaomicron, B. ovatus, B. vulgatus, and B. distasonis (Tanaka-Bandoh et al., 1995). For instance, in the United States, the resistance is very low, varying to up to 25% of strains. Unfortunately, as usually, it increases proportionally with time. Thus in the B. fragilis group it increased from 4% to 38% in 10 years. A similar situation has also been observed in other parts of the world (Patey et al., 1994; Turgeon et al., 1994). Clindamycin is quite active against other Gram-negative anaerobes such as Prevotella disiens and P. melaninogenica and the Fusobacterium spp. Bacteroides gracilis may be clindamycin-sensitive, but some strains are resistant. Additional Gram-negative bacteria comprising strains of Butyrivibrio, Succinimonas, and Anaerovibrio can be sensitive to clindamycin. Necrotizing fascitis continues to occur due to -hemolytic streptococci but is now also recognized as being due to Vibrio spp. in fishermen and those in contact with warm water in the Gulf of Mexico and Southeast Asia, including Hong Kong. The mechanism of Bacteroides resistance to clindamycin is usually as in Gram-positive bacteria (Jimenez-Diaz et al., 1992; Reig et al., 1992a). The resistance gene in Bacteroides spp. can be located on plasmids or on the chromosome; it can be transferred between species by a plasmid or transposon. Leng et al. (1975) used combinations of clindamycin with gentamicin against Enterobacteriaceae and Pseudomonas aeruginosa and demonstrated synergism. Klastersky and Husson (1977) showed that gentamicin did not interfere with the activity of clindamycin against B. fragilis, and that clindamycin did not influence the activity of gentamicin against E. coli. Adjunctive use of clindamycin, along with mechanical debridement is recommended for the treatment of Actinobacillus actinomycetemcomitans (Gram-negative, facultative anaerobic bacterium)-associated periodontitis as an acceptable therapeutic regimen (Walker and Karpinia, 2002). Laboratory results could be used in clinical practice by comparing the MIC50 of the germs regularly encountered in bone infections (staphylococci, streptococci including enterococci, Gram-negative bacilli, P. aeruginosa, and H. influenzae) with concentrations obtained in the different studies. In these studies it was described that clindamycin has a moderate bone diffusion (Boselli and Allaouchiche, 1999). Mycoplasma and Ureaplasma urealyticum are clindamycin-resistant;

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on the contrary, clindamycin has some activity against Chlamydia trachomatis (Rice et al., 1995) or Coxiella burnetii. X. Anaerobic Bacteria Clostridium tetani and C. perfringens are sensitive to clindamycin, but some C. perfringens strains and strains of C. sporogenes, C. tertium, C. bifermentans, C. novyi, C. ramosum, and C. sordelli may be clindamycin-resistant. A systematic review of studies that investigated the association of clindamycin-like antibiotics with hospital-acquired Clostridium difficile diarrhea was undertaken to summarize the strength of the evidence for this relationship (Thomas et al., 2003). Clostridium difficile is responsible for 300,000 to 3,000,000 cases of diarrhea and colitis in the United States every year. Antibiotics most frequently indicated for this infection are clindamycin, ampicillin, amoxicillin, and cephalosporins (Mylonakis et al., 2001). Clindamycin was an effective drug in the treatment of Gram-positive anaerobic infections (e.g., Clostridium perfringens). Very rapidly, the anti-anaerobic armamentarium was extended with clindamycin, cefoxitin, imipenem, and co-amoxyclavin or piperacillintazobactam. The resistance rate to metronidazole and imipenem remains low, but clindamycin has seen an important decrease in bacterial susceptibility (Bryskier, 2001). Clostridium difficile is now established as a major nosocomial pathogen. C. difficile infection is seen almost exclusively as a complication of antibiotic therapy and is particularly associated with clindamycin and third-generation cephalosporins (Freeman and Wilcox, 1999). Agents with a high potential to induce Clostridium difficile– associated disease include aminopenicillins, cephalosporins, and clindamycin (Job and Jacobs, 1997). Other anaerobic Gram-positive organisms such as Peptococcus, Peptostreptococcus, Eubacterium, Propionibacterium, Bifidobacterium and Lactobacillus spp., Actinomyces israelii or Bifidobacterium, and Eubacterium spp. (Brook and Frazier, 1993) are usually sensitive to clindamycin. Naturally, even here, resistant strains have been described—for example, in Peptostreptococcus spp. (Reig et al., 1992b) or Lactobacillus spp. Vaginal bacterial infections are usually caused by mixed bacterial populations, including Peptostreptococcus sp., Peptococcus sp., B. fragilis, and of the aerobic bacteria by Streptococcus viridans, S. agalactiae, and less by S. pyogenes and other enterobacteria. The mixed

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population can also contain enterococci and staphylococci. With the exception of enterococci, the population is to a considerable extent sensitive to a combination of clindamycin with an aminoglycosidic antibiotic. Clindamycin was also found to inhibit Chlamydia trachomatis. Bacterial vaginosis is an alteration of the vaginal flora, where the normally predominant lactobacilli are replaced by a mixture of organisms including Gardnerella vaginalis and anaerobes and is treated with metronidazole or clindamycin (Priestley and Kinghorn, 1996). In bacterial vaginosis the normal hydrogen peroxide-producing Lactobacillus sp. in the vagina is replaced with high concentrations of characteristic sets of aerobic and anaerobic bacteria. It also occurs in women treated, for example, by orally administered clindamycin (McGregor and French, 2000). Increased doses of clindamycin and lincomycin (at least 8 g per day in adults) have to be administered for the treatment of B. fragilis infections, and even then the effect is not guaranteed. In cases of anaerobic sepsis, usually caused by B. fragilis or Peptostreptococcus sp., the application of clindamycin as the first choice antibiotic is fully justified. In many patients with acne, caused by resistant Propionibacterium acnes, continued treatment with antibiotics such as clindamycin can be inappropriate or ineffective (Cooper, 1998). Clostridium difficile may be clindamycin-sensitive or -resistant, and the proportion of sensitive strains has varied from 10% to 90% in different studies. During outbreaks of diarrhea associated with C. difficile, the strains are usually clindamycin-resistant, and they contain a plasmid, probably located on the chromosome. This codes for transferable macrolide-lincosamide-streptogramin B (MLSB) resistance. This resistance can be transferred from C. difficile to Staphylococcus aureus. In one study, almost all of 161 isolates of C. difficile of serogroups A, F, G, H, and X were susceptible to clindamycin and other antibiotics, but 32 toxigenic isolates of serogroup C were clindamycin resistant. The microbiology, diagnosis, and management of bacteremia caused by anaerobic bacteria (Bacteroides fragilis, Peptostreptococcus sp., Clostridium sp., and Fusobacterium sp.) in children were reviewed by Brook (2002b). XI. Protozoa and Other Organisms Clindamycin has been used as an antimalarial drug (Lell and Kremsner, 2002). It was found effective in animals infected with chloroquine-resistant and chloroquine-sensitive Plasmodium falcipar-

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um. It is also effective against P. vivax but not against the exo-erythrocytic parasites. In P. falciparum clindamycin appears to be quite effective in the treatment of semi-immune subjects, and it enhances quinine activity (Patenotte et al., 1995). Malaria caused by chloroquine-resistant strains can be treated successfully with combinations of clindamycin with a ‘‘classical’’ antimalarial drug (Mordmuller et al., 1998). Clindamycin is effective in experimental toxoplasmosis in mice. In cultured mammalian cells, clindamycin reduces the level of replication of Toxoplasma gondii affecting protein synthesis of free parasites and also impairs the ability of the parasite to infect host cells. Infections caused by Toxoplasma gondii can be treated with clindamycin (Fung and Kirschenbaum, 1996). T. gondii clindamycin-resistant mutants can be selected that usually exhibit cross-resistance to spiromycin and azithromycin (Fichera et al., 1995). The influence of antimicrobial agents on replication and stage conversion of Toxoplasma gondii was described by Gross and Pohl (1996). Molecular genetic tools for the identification and analysis of drug targets in Toxoplasma gondii were reviewed by Roos (1996). Human babesiosis is an important emerging tick-borne disease. Babesia divergens, a parasite of cattle, has been implicated as the most common agent of human babesiosis in Europe and/or Babesia microti in the United States, causing severe disease in splenectomized individuals. Human babesiosis can be treated by clindamycin administered intravenously (Uguen et al., 1997). Current treatment for babesiosis is focused on a regimen of clindamycin and quinine (Kjemtrup and Conrad, 2000). XII. Conclusion and Future Prospects Lincomycin and clindamycin are clinically important antibiotics. According to its worldwide production, the semisynthetic lincosamide derivative clindamycin is one of the 20 most important antibiotic compounds. They are active against most Gram-positive bacteria and against the genera Staphylococcus and Streptococcus in particular. They do not affect Gram-negative bacteria but exhibit a significant antibiotic activity against some anaerobic bacteria. They are used therapeutically, especially in cases where synergistic effects of a mixed anaerobic and aerobic microflora are anticipated (prevention of intraabdominal infection after surgery, stomatological infections, anaerobic sepsis, skin and mucosa infections, and especially infections of bone

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and articular infections). Lincomycin and clindamycin are also useful alternatives to penicillin and its derivatives in the treatment of upper respiratory tract infections in patients with allergy to penicillin. As compared with lincomycin, clindamycin is highly effective in the treatment of toxoplasmosis and pneumocystosis in AIDS patients. Clindamycin and some of its derivatives seem to be promising for the treatment of malaria caused by Plasmodium falciparum, even of strains that developed resistance to chloroquine, sulfonamides, and pyrimethamine. By means of chemical modification of lincomycin, a number of derivatives with improved properties were obtained, whereas biotransformation has so far been less successful. However, with the current knowledge of gene clusters specifying biosynthesis of lincomycin and the related antibiotic celesticetin, one can easily imagine production of new derivatives of lincosamides by genetic engineering, namely by combination of parts of the lincomycin cluster with those of the celesticetin cluster, resulting in production of new derivatives of lincosamides. The knowledge of genomes of more than 100 pathogenic bacteria will make it possible to direct the search for new antibiotics specific for new targets in the pathogens. In addition, the recent completion of the genomes of Streptomyces coelicolor and Streptomyces avermitilis revealed that new secondary metabolites that had not yet been described are coded for by specific genes. Thus, not only hybrid antibiotics but also completely new compounds can still be discovered in the near future. In this respect it appears to the authors that lincosamides are not only useful antibiotics, namely for the treatment of anaerobic and protozoal infections, but that they also may serve as a model for the future development of new derivatives of antibiotics by means of genetic engineering.

ACKNOWLEDGMENTS This work was supported by the Grant Agency of the Czech Republic (grant no. 204/01/ 1004) and by the Institutional Research Concept no. AV 0Z 5020 903. The authors wish to express their thanks to Mrs G. Broucˇkova´ for administrative help. Excellent technical ˇ ezanka (Institute of Chemical Technology and Faculty of Science of assistance of Mr. P. R the Charles University, Prague) is gratefully acknowledged. REFERENCES Argoudelis, A. D., and Coats, J. H. (1969). Microbial transformation of antibiotics. II. Phosphorylation of lincomycin by Streptomyces species. J. Antibiot. 22, 341–343. Argoudelis, A. D., and Coats, J. H. (1973). Process for producing linkomycin. US Patent 3,726,766.

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Ribosome Engineering and Secondary Metabolite Production KOZO OCHI,* SUSUMU OKAMOTO, YUZURU TOZAWA, TAKASHI INAOKA, TAKESHI HOSAKA, JUN XU, AND KAZUHIKO KUROSAWA National Food Research Institute Ibaraki 305-8642, Japan *Author for correspondence. E-mail: [email protected]

I. Introduction II. General Method for Obtaining Drug-Resistant Mutants III. Antibiotic Overproduction by rpsL (Ribosomal Protein S12) Mutations A. Activation of Actinorhodin Production B. Mechanism of Activation C. rpsL Mutations by Site-Directed Mutagenesis IV. Antibiotic Overproduction by rpoB (RNA Polymerase) Mutations V. Effect of str and rpoB Mutations in Various Bacteria A. Antibiotic Overproduction B. Enzyme Overproduction C. Neotrehalosadiamine VI. Increase of Chemical Tolerance in Pseudomonas VII. Combined Drug-Resistance Mutations A. Model Experiment B. Applicability to Industrial Strain VIII. Conclusion and Future Prospects A. Future Prospects References

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I. Introduction Improvement of the productivity of commercially viable microbial strains is an important field in microbiology, especially since wildtype strains isolated from nature usually produce only a low level (1–100 g/ml) of antibiotics. Therefore, a great deal of effort and resources have been committed to improving antibiotic-producing strains to meet commercial requirements. Current methods of improving the productivity of industrial microorganisms range from the classical random approach to using highly rational methods—for example, metabolic engineering. Although classical methods are still effective even without using genomic information or genetic tools to obtain highly productive strains, these methods are always time and resource intensive (Vinci and Byng, 1999; Zhang et al., 2002). One of the current topics is to use microorganisms for bioremediation. Environmental protection efforts have been focused on the development of more effective 155 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 56 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2164/04 $35.00

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processes for the treatment of toxic wastes. Soil bacteria have a wide range of metabolic abilities that make them useful tools for mineralization of toxic compounds (Timmis et al., 1994; Van der Meer et al., 1992). The discovery of microorganisms capable of tolerating, or growing on, high concentrations of organic solvents provides a potentially interesting avenue for development of genetically engineered organisms for treating hazardous wastes. Thus, strain improvement is crucially important to fully exploit the cell’s ability. Since we found a dramatic activation of antibiotic production by a certain ribosomal mutation (a mutation in rpsL gene encoding the ribosomal protein S12) (Shima et al., 1996), we had an idea that bacterial gene expression may be changed dramatically by modulating the ribosomal proteins or rRNA, eventually leading to activation of inactive (silent) genes. Thus, our ultimate aim was to develop ‘‘ribosome engineering’’ for a rational approach to fully elicit the bacterial abilities. In bacteria, the ribosome plays a special role for their own gene expression by synthesis of a bacterial alarmone, ppGpp. Namely, one of the most important adaptation systems for bacteria is the stringent response, which leads to the repression of stable RNA synthesis in response to nutrient limitation (Cashel et al., 1996). The stringent response depends on the transient increase of hyperphosphorylated guanosine nucleotide ppGpp, which is synthesized from GDP and ATP by the relA gene product (ppGpp synthetase) in response to binding of uncharged tRNA to the ribosomal A site. Since bacterial secondary metabolism is often triggered by ppGpp when cells enter into stationary phase, it is important to take the stringent response into consideration in activating or enhancing the bacterial secondary metabolism. In this review, we outline our ribosome engineering and its applicability, especially focusing on strain improvement for antibiotic overproduction in Streptomyces and Bacillus and for enhancement of tolerance to organic chemicals in Pseudomonas. II. General Method for Obtaining Drug-Resistant Mutants One of the most conventional ways to modulate the ribosome is the introduction of mutations conferring resistance to drugs that attack the ribosome. Such drugs include streptomycin, gentamicin, paromomycin, thiostrepton, fusidic acid, kanamycin, chloramphenicol, lincomycin, spectinomycin, and neomycin. The mutants resistant to these drugs frequently possess a point mutation or a deletion mutation within a ribosomal component (ribosomal protein, rRNA, or translation

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factor). An advantage of obtaining the drug-resistant mutants is selectability on the drug-containing plates, even if the incidence of resistant mutants is as low as 10–8–10–10. It is important to use several concentrations of drugs (e.g., 3-, 30-, and 100-fold of MIC) for obtaining a wide variety of mutants. (MIC: minimum inhibitory concentration to suppress cell’s growth). The MIC value varies depending on the species used. It is also recommended to use both the cells and spores grown in plate culture or liquid culture, since mutant forms emerging from spores or cells are often different. Since the frequency of spontaneous mutation is normally 106–108, abundant (107–109) cells or spores may be plated, followed by 1–3 days of incubation (in typical bacteria) or 5–14 days (in Streptomyces) to allow the development of resistant colonies. If the frequency of mutation is very low, more (1010–1011) cells or spores may be spread on a plate. Once mutant colonies develop, it is preferable to carry out single colony isolation before further testing. The mutants resistant to a high level of drugs were stable in their phenotype, whereas mutants resistant to a low level of drugs were unstable in general, as represented by gentamicin-resistant mutants. If a number of resistant colonies develop on the plate, one can select a variety of mutant forms on the basis of colony size, colony morphology, sporulation, pigment formation, etc. To test the ability of each mutant to produce antibiotics (or other metabolites), several kinds of media may be employed because the efficacy of mutation on productivity often depends on the medium used. In this way, one can find overproducing mutants among drug-resistant mutants at a high frequency of 2–40%. To determine the mutated gene, DNA sequence analysis with PCR can be done, focusing on the most probable mutated genes (e.g., ribosomal proteins S12 and L11 genes in streptomycin-resistant and thiostrepton-resistant mutants, respectively).

III. Antibiotic Overproduction by rpsL (Ribosomal Protein S12) Mutations A. ACTIVATION OF ACTINORHODIN PRODUCTION Members of the genus Streptomyces produce a wide variety of secondary metabolites that include about half of the known microbial antibiotics. Advances in understanding the regulation of secondary metabolism in this genus have come from the studies of antibiotic production in Streptomyces coelicolor A3(2) and its close relative Streptomyces lividans 66 (Bibb, 1996; Hopwood et al., 1995; Kieser

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et al., 2000). S. coelicolor produces at least four antibiotics, including the blue-pigmented polyketide antibiotic actinorhodin (Act). S. lividans normally does not produce Act, although the strain has a complete set of Act biosynthetic genes. However, Act production in this organism can be activated by the introduction of certain regulatory genes (Floriano and Bibb, 1996; Horinouchi et al., 1990; MartıńezCosta et al., 1996; Vogtli et al., 1994) or by cultivation under specific conditions (Kim et al., 2001). A strain of S. lividans, TK24, has been found to produce a large amount of Act under normal culture conditions (Shima et al., 1996) (Fig. 1, see color insert). Genetic analyses revealed that a streptomycinresistant mutation, str-6, in TK24 is responsible for activation of Act synthesis and that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu (K88E mutation). It was also shown that introduction of streptomycin-resistant mutations improves Act production in wild-type S. coelicolor (Hesketh and Ochi, 1997) (Fig. 1) and circumvents the detrimental effects on Act

FIG. 1. Activation of antibiotic production by rpsL (encoding ribosomal protein S12) mutations in Streptomyces lividans 66 and Streptomyces coelicolor A3(2). Blue color represents an antibiotic, actinorhodin. K88E means a mutation at lysine-88 altering glutamate.


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production in certain developmental mutants (relA, relC, and brgA) of S. coeliolor (Ochi and Hosoya, 1998; Ochi et al., 1997; Shima et al., 1996). These streptomycin-resistant mutations result in the alteration of the Lys-88 to Glu (K88E) or Arg (K88R) and Arg-86 to His (R86H) in the rpsL gene. In addition to these streptomycin-resistant rpsL mutations, a paromomycin-resistant rpsL mutation (P91S) also can activate Act production in S. coelicolor (Okamoto-Hosoya et al., 2000). These findings indicate that the antibiotic production (secondary metabolism) in streptomycetes is significantly controlled by the translational machinery, that is, the ‘‘ribosome.’’ Much progress has been made in elucidating the organization of antibiotic biosynthesis gene clusters in several Streptomyces species, and a number of pathway-specific regulatory genes have been identified, which are required for the activation of their cognate biosynthetic genes (Wietzorrek and Bibb, 1997). In the Act biosynthetic gene cluster, actII-ORF4 plays such a pathway-specific regulatory role, and the expression level of this gene directly determines the productivity of Act (Arias et al., 1999; Gramajo et al., 1993). Western blot analysis using anti-ActII-ORF4 antibody showed that the expression of ActIIORF4 protein was strongly enhanced in the Act-high-producing rpsL mutant strains (Hu and Ochi, 2001; Okamoto et al., unpublished). Furthermore, RT-PCR experiments revealed that the increase of this regulatory protein can be attributed to the enhanced expression of actII-ORF4 mRNA (Okamoto et al., unpublished). Thus, certain rpsL mutations enhance expression of the actII-ORF4 gene, leading to massive production of Act. In addition to the rpsL mutations that confer a high level of resistance to streptomycin, another type of mutation (ND mutation) conferring a low-level resistance to streptomycin also gave rise to a similar increase in Act production (Hesketh and Ochi, 1997; Shima et al., 1996). The appearance of ND mutants is relatively frequent (105–107), and their ability to produce Act is noticeably higher than that of rpsL mutants. By using proteomic analysis, Okamoto et al. (2003) found that a 46-kDa protein is highly expressed in an S. coelicolor mutant KO-179, a representative strain of such ND mutants. This protein was identified as S-adenosylmethionine (SAM) synthetase, which is a product of the metK gene. These findings imply that SAM synthetase may be involved in the Act-overproducing phenotype observed in these ND mutants. In fact, the introduction of a high-copy-number plasmid containing the metK gene into wild-type strain resulted in an extensive hyperproduction of Act (Fig. 2A, see color insert). Furthermore, addition of SAM to the culture medium activated Act production in wild-type

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FIG. 2. Activation of antibiotic (actinorhodin) production by introducing multi-copy metK gene encoding S-adenosylmethionine synthetase (A), or by directly adding S-adenosylmethionine (B) in S. coelicolor. (Adapted from Okamoto et al., 2003.)

cells (Fig. 2B). Therefore, enhanced Act production in strain KO-179 can be ascribed, at least in part, to the overexpression of SAM synthetase, which leads to an elevation of intracellular SAM level. Consistent with this conclusion, Kim et al. (2003) found that introduction of a multicopy plasmid containing the Streptomyces spectabilis metK gene into S. lividans can induce Act production in this organism. SAM is known to be the methyl donor for the methylation of the various biological substances (DNA, RNA, proteins, and other small molecules). However, the Act biosynthetic pathway does not contain any steps that require SAM as a methyl donor. Interestingly, overexpression of the metK gene stimulated the expression of a pathway-specific regulatory gene


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actII-ORF4 (Okamoto et al., 2003). Furthermore, the addition of SAM also caused overproduction of streptomycin (by Streptomyces griseus) and bicozamycin (by Streptomyces griseoflavus) (Saito et al., 2003a). These findings highlight the significance of SAM as a common intracellular signal molecule for onset of secondary metabolism in widespread Streptomyces species. B. MECHANISM OF ACTIVATION The ribosomal protein S12, a component of the 30S subunit in bacteria, is best characterized with respect to its role in the selection efficiency of cognate tRNAs for an accuracy enhancement (Carter et al., 2000; Kurland et al., 1990). Most of the mutations in S12 protein associated with streptomycin resistance lead to an error restrictive phenotype because of changes in the kinetic properties of the tRNAribosome interaction (Kurland et al., 1996). As already mentioned, antibiotic production by bacteria, including Streptomyces spp., is activated or enhanced by introducing certain mutations into the rpsL gene (encoding the ribosomal protein S12) that confer resistance to streptomycin (Hosoya et al., 1998; Okamoto-Hosoya et al., 2003b; Tamehiro et al., 2003). Recently we found that K88E (which corresponds to position 87 in Escherichia coli S12 protein) rpsL mutant of S. coelicolor A3(2), with an enhanced Act production, exhibits an aberrant protein synthesis activity. To clarify the presence or absence of the causal relationship between this aberrant protein synthesis activity and the observed antibiotic overproduction, we have discovered characteristic properties of the S. coelicolor K88E mutant to synthesize protein in vivo and in vitro (Hosaka, Xu, and Ochi, unpublished; Okamoto-Hosoya et al., 2003a). The results demonstrated that (1) the K88E mutation, like classic S12 mutations (K42N and K42T) in E. coli, confers a restrictive phenotype in addition to resistance to streptomycin; (2) the K88E mutant exhibits a high level of protein synthesis activity in vivo at the late growth phases as examined by measuring the incorporation of labeled leucine; (3) the K88E mutant ribosomes from the latestationary-phase cells have a high capacity for translating both synthetic polynucleotide [poly(U)] and natural mRNA; (4) S150 solution from K88E mutant cells grown to late-stationary-phase supports a higher level of protein synthesis activity in vitro; and (5) the K88E mutant ribosomes are structurally more stable under stress conditions such as amino acid starvation and low concentration of magnesium. We concluded that the increased stability of the 70S complex and the levels of specific translation-associated factor(s) are responsible for the aberrant

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FIG. 3. Outline of mechanism by which mutation in S12 protein activates antibiotic production.

activation of protein synthesis in the S. coelicolor K88E mutant (Fig. 3). This observation is in agreement with our recent findings that the E. coli K87E mutant also shows an aberrant protein synthesis activity at late growth phase (Fig. 4) (Hosaka et al., 2004). Our findings lead to the suggestion that a change from Lys to Glu at position 87 (according to the E. coli numbering) in S12 protein renders cells potentially more active for protein synthesis under the starvation conditions represented by the late growth phase. Such a characteristic would be highly advantageous for the production of proteins from newly transcribed genes (such as those involved in antibiotic production) at the late growth phase. Thus, the aberrant protein synthesis found in the S. coelicolor K88E rpsL mutant could be the cause, at least in major part, of remarkably activated antibiotic production in this mutant strain. C. rpsL MUTATIONS BY SITE-DIRECTED MUTAGENESIS The rpsL mutations found so far in S. coelicolor A3(2) and S. lividans 66 are K43N, K43R, K43T, K88E, K88R, and P91S. Of these, only two mutations (K88E and P91S) effectively activated antibiotic production.


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FIG. 4. In vitro translation of GFP mRNA with ribosomes from E. coli wild-type strain W3110 (s) and K87E mutant (d) grown to late stationary phase (26 h) in LB medium. Upper panel shows fluorographs of GFP that was synthesized in vitro.

It thus appears that certain mutations around the Lys88 region may distinctively affect antibiotic production and that isolation of such mutants could be effective for developing the antibiotic-overproducing strains. However, since most of those mutations are not likely to confer resistance to streptomycin, it would be impossible to identify such mutations by virtue of their ability to resist the drug. To circumvent this difficulty, we used site-directed mutagenesis to create seven novel rpsL mutations (R86L, V87K, K88G, D89R, L90K, G92D, and R94G), and introduced these mutant genes into wild-type S. lividans cells by using a single-copy-number plasmid (Okamoto-Hosoya et al., 2003b). Of these mutations, two (L90K and R94G) activated production of a redpigmented antibiotic (undecylprodigiosin) by S. lividans much more potently than the streptomycin-resistant K88E mutation (Fig. 5, see color insert). Neither the L90K nor the R94G mutation conferred an increase in the level of resistance to streptomycin and paromomycin, indicating nonavailability of these mutant alleles among the resistant isolates. Since

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FIG. 5. Mutations that were constructed by site-directed mutagenesis (upper panel) and effect of a single copy of the mutant rpsL gene on antibiotic (undecylprodigiosin) production. Red color represents undecylprodigiosin. (Adapted from Okamoto-Hosoya et al., 2003b.)

the experimental system chosen in this study to evaluate the new rpsL mutations involves expression of the mutant S12 protein with a plasmid and does not require any special technique for replacing the wild-type rpsL gene on the chromosome with the created mutant genes, this approach could be applicable to a variety of strains (even if no genetic information is available) for improvement of antibiotic productivity. IV. Antibiotic Overproduction by rpoB (RNA Polymerase) Mutations Antibiotic biosynthesis pathways and their genetic regulatory cascades comprise one of the most attractive fields in Streptomyces genetics and are important in considering strain improvement. Onset of the morphological differentiation and the secondary metabolism, including antibiotic production, are thought to be coupled and influenced by a variety of physiological and environmental factors (Chater and Bibb, 1997). Antibiotic production in streptomycetes is generally growth phase–dependent. Thus, the signal molecule for growth rate control, ppGpp, is suggested to play a central role in triggering the onset of antibiotic production in Streptomyces. Namely, the ribosomes play an essential role in adjusting gene expression levels by synthesizing ppGpp in response to nutrient limitation. There is a positive correlation between ppGpp and antibiotic biosynthesis: disruption of the


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FIG. 6. Functional map of E. coli RNA polymerase -subunit and location of rif cluster I within RNA polymerase -subunit in relation to the previously suggested ppGpp-binding site (Chatterji et al., 1998) in E. coli. Positions of the rif mutations found in the present study are designated by arrows. Numbering begins at the start codon of the open reading frame. ML, Mycobacterium leprae; SC, Streptomyces coelicolor A3(2); BS, Bacillus subtilis; EC, Escherichia coli. (Adapted from Ishihama et al., 1990, and Xu et al., 2002.)

ppGpp synthetase gene, relA, or a deletion mutation (designated as relC) in the ribosomal L11 protein gene has been shown to lead to a deficiency in ppGpp accumulation after amino acid depletion (socalled ‘‘relaxed’’ phenotype) accompanied by impairment in antibiotic production (Chakraburtty et al., 1996; Hoyt and Jones, 1999; Jin et al., 2004; Kelly et al., 1991; Ochi, 1987, 1990a,b). The expression level of many genes are regulated by ppGpp, either positively or negatively. Many genetic studies in E. coli suggested that RNA polymerase (RNAP) is the target for ppGpp regulation. Genetic analysis reveals that four major functional domains exist in the RNAP -subunit (Fig. 6). The ppGpp-sensitivity domain is close to another important domain of the RNAP -subunit, the rifampicin (Rif)-binding domain (Ishihama et al., 1990). The crystal structure clearly revealed that Rif-cluster I is involved in the E. coli RNAP active center. Therefore it is reasonable to consider that certain mutations in the Rif-binding domain could affect the activity of RNAP and then may affect the function of the adjacent ppGpp-binding domain. We postulated that the impaired ability to produce antibiotic due to the relA or relC mutation may be circumvented by introducing certain Rif-resistant (rif) mutations into the RNAP -subunit. This hypothesis is based on a notion that the mutated RNAPs may behave like ‘‘stringent’’ RNAP without ppGpp binding. The results from rel mutants of S. coelicolor A3(2) and S. lividans strongly supported this hypothesis (Lai et al., 2002; Xu et al., 2002). The Rif-resistant isolates from the rel mutants regained the ability to produce the colored antibiotic

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actinorhodin, and various types of point mutation were mapped in the so-called Rif-cluster I in the rpoB gene that encodes the RNAP -subunit (Fig. 6). More impressively, gene expression analysis revealed that the restoration of actinorhodin production in the rel rif double mutant strains is accompanied by increased expression of the pathway-specific regulatory gene actII-ORF4, which normally decreased in the rel mutants. Accompanying the restoration of antibiotic production, the rel rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a nutritionally rich medium. Since the dependence of S. coelicolor A3(2) on ppGpp to initiate antibiotic production can apparently be bypassed by certain mutations in the RNAP, the mutant RNAP may function by mimicking the ppGpp-bound form (Fig. 7). This proposal can be supported by the fact that the mutant RNAP behaved like ‘‘stringent’’ RNAP with respect to RNA synthesis, as demonstrated using cells growing in a nutritionally rich medium. To test the feasibility of rif mutation on the breeding of an antibiotic producing strain, Hu et al. (2002) attempted to activate the antibiotic biosynthetic gene cluster in S. lividans. The results demonstrated

FIG. 7. Hypothesis for ppGpp-independent antibiotic (actinorhodin) production. actIIORF4 is the gene encoding a pathway specific regulatory protein ActII-ORF4. rif represents mutated -subunit. relA and relC are mutations that block the synthesis of ppGpp.


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that the biosynthesis of actinorhodin, undecylprodigiosin, and calcium-dependent antibiotic all could be remarkably activated by introducing specific types of rif mutations into the rpoB gene. In most cases, the spontaneously arising rif mutations are mapped in the Rif-cluster I of the -subunit (Jin and Gross, 1988; Severinov et al., 1993, 1994; Singer et al., 1993), and cluster I is only a few angstroms away from the active center of RNAP, as demonstrated in E. coli (Severinov et al., 1995). It is reasonable to consider that the RNAP containing a rif-type -subunit may be structurally similar to a wild-type RNA polymerase modified by ppGpp, because numerous genetic analyses in E. coli have revealed that rif mutations frequently circumvent the ppGpp0 phenotype (Barker et al., 2001a,b). Recent developments in clarification of the ternary structure of RNA polymerase–ppGpp complex by X-ray analysis (Artsimovich et al., 2004) are helpful in assessing the foregoing consideration. V. Effect of str and rpoB Mutations in Various Bacteria A. ANTIBIOTIC OVERPRODUCTION Members of the genera Streptomyces, Bacillus, and Pseudomonas are soil bacteria that produce a high number of agriculturally and medically important antibiotics. The development of rational approaches to improve the production of antibiotics from these organisms is therefore of considerable industrial and economic importance. The impairment in antibiotic production resulting from a relA or relC mutation (that causes a failure to synthesize ppGpp) could be completely restored by introducing mutations conferring resistance to streptomycin (str) (Ochi et al., 1997; Shima et al., 1996). No accompanying restoration of ppGpp synthesis was detected in these relA str or relC str mutants. It is therefore apparent that acquisition of certain str mutations allows antibiotic production to be initiated without the requirement for ppGpp. This offers a possible strategy for improving the antibiotic productivity. Indeed, in addition to actinorhodin production by S. coelicolor and S. lividans, introduction of a str mutation was effective in enhancing antibiotic production by various bacteria. 1. Effect of str Mutation on Streptomyces spp. The effect of an str mutation on antibiotic production in three Streptomyces spp., S. chattanoogensis, S. antibioticus, and S. lavendulae was examined (Table I; Hosoya et al., 1998). When the spores of Streptomyces spp. were spread and incubated on a plate containing 5 or

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OCHI et al. TABLE I ANTIBIOTIC PRODUCTIVITY OF BACTERIAL STREPTOMYCIN-RESISTANT MUTANTS Frequency of str mutants producing increased antibiotic

Microorganism

Antibiotic

Production in parental (¼ wild-type) strain (g/ml)

Streptomyces chattanoogensis

Fredericamycin

10

46%

260

Streptomyces antibioticus

Actinomycin

12

4%

63

Streptomyces lavendulae

Formycin

25

3%

130

Pseudomonas pyrrocinia

Pyrrolnitrin

30%

15

Bacillus cereus

FR900493

7%

550

Bacillus subtilis

Unidentified antibiotic

1.5 76 8a

19%

Highest productivity detected (g/ml)

80a

a unit/ml. Adapted from Hosoya et al., 1998.

30 g of streptomycin per ml, streptomycin resistant (str) mutants developed after 7–14 days at a frequency of 106 to 108. These spontaneous str mutants were characterized from a wide variety of colonies by size, morphology, and pigment formation. In S. chattanoogensis nearly half of the str mutants tested exhibited a significantly increased ability (greater than fivefold) to produce fredericamycin. The highest productivity detected was 26-fold higher than that of the wild-type strain. Similarly, strains producing high levels of actinomycin and formycin could be detected at a relatively high frequency (3%–4%) among str mutants of S. antibioticus and S. lavendulae, respectively. Thus, like actinorhodin production by S. coelicolor A3(2), introduction of mutations conferring resistance to streptomycin was effective for improving the antibiotic productivity of the Streptomyces spp. 2. Effect of str Mutations on Bacillus and Pseudomonas Introduction of the str mutation also improved antibiotic productivity of bacteria such as Bacillus spp. and Pseudomonas spp. (Table I; Hosoya et al., 1998). In B. cereus and P. pyrrocinia, the frequency of antibiotic overproducing strains among str mutants ranged from 7% to 30%. str mutants of B. subtilis developed on GYM agar containing


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either a low (5 g/ml) or a high (400 g/ml) concentration of streptomycin were examined. Antibiotic overproducing strains were detected at a higher frequency among str mutants selected at a high concentration, rather than a low concentration, of streptomycin (19% versus 3%). In contrast to str mutants, none of the mutants resistant to chloramphenicol, tetracycline, lincomycin, or spectinomycin exhibited increased antibiotic production. Unlike the case of S. coelicolor, the str mutations found in B. subtilis all fell into amino acid position K56 (corresponding to K42 in E. coli) of ribosomal protein S12. Mutations K56R, K56T, and K56Q were effective in increasing antibiotic productivity, whereas mutations K56I and K56N were ineffective. B. ENZYME OVERPRODUCTION Introduction of drug-resistant mutations has also been verified to be effective in improving enzyme productivity. Several str mutants of B. subtilis were shown to produce an increased amount (20–30%) of -amylase and protease (Kurosawa and Ochi, unpublished). Jorgensen et al. (personal communication) showed that rpoB mutations are also effective for overproduction (1.5-fold to twofold) of extracellular enzymes such as amylase and protease. Thus these methods may be applicable for overproduction of other enzymes produced by various microorganisms, especially at late growth phase. C. NEOTREHALOSADIAMINE Neotrehalosadiamine (3,30 -diamino-3,30 -dideoxy-,-trehalose; NTD), which is an aminosugar antibiotic produced by Bacillus pumilus and Bacillus circulans, inhibits growth of Staphylococcus aureus and Klebsiella pneumoniae. In contrast, Bacillus subtilis normally does not produce this antibiotic. However, introduction of a certain rifampicinresistant rpoB mutation (rif ) enables cells to activate the dormant ability to produce NTD in B. subtilis (Inaoka et al., 2004). A polycistronic gene, ntdABC, and a monocistronic gene, ntdR, were identified as the NTD biosynthesis operon and a positive regulator for ntdABC, respectively (Fig. 8). Surprisingly, NTD acts as autoinducer for its own biosynthetic process. The mechanism of autoinduction of signaling molecules has been studied extensively in several bacteria in relation to quorumsensing systems that are important for various physiological processes, such as acquisition of competence, sporulation, motility, biofilm formation, bioluminescence, and virulence (Fuqua et al., 2001; Kaiser and Losick, 1993; Kleerebezem and Quadri, 2001). In Gram-positive

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FIG. 8. Scheme of mechanism by which an antibiotic neotrehalosadiamine (NTD) synthesis is regulated via an autoinduction system in Bacillus subtilis. NtdR protein represents a positive regulator for NTD synthesis. In the absence of NTD, NtdR protein cannot function as an activator for ntdABC expression, although NtdR itself can bind to promoter region (upper panel).


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bacteria, most of these signaling molecules are peptides or modified peptides including subtilin and nisin; these antibiotics induce the transcription of their own biosynthesis genes via a two-component signal transduction system. In contrast, NTD acts as an autoinducer for its own biosynthesis operon by directly interacting with NtdR protein (Fig. 8). In our model, the NtdR protein dramatically stimulates ntdABC transcription when the promoter region is occupied by NTD-bound NtdR protein. In line with this notion, NTD-unbound NtdR protein results in a failure to express ntdABC. In addition, this rif mutation was also shown to cause a twofold increase in the activity of the ntdABC promoter recognized by house-keeping sigma factor (A). Therefore this mutant RNAP likely enhances the A-dependent promoters, resulting in a dramatic activation of the NTD biosynthesis pathway by an autoinduction mechanism. On the basis of these findings, together with the results from Streptomyces spp., improvement of RNAP by introduction of a rif mutation could be a useful approach to elicit the bacterial ability. VI. Increase of Chemical Tolerance in Pseudomonas Solvent resistance of bacteria is inheritable and can be generated by random mutagenesis and thus appears to be a useful phenotype for waste processing (Timmis et al., 1994). Therefore development of genetically enhanced microorganisms, through the use of genetic engineering, is important for developing better biological waste processing technologies. The ribosome engineering approach was effective not only in overproduction of useful metabolites but also in improvement of tolerance by bacteria of aromatic compounds, a property that could be useful for bioremediation. Certain str, gen, or rif mutants derived from Pseudomonas putida, which are resistant to streptomycin, gentamicin or rifampicin, respectively, were tolerant to the aromatic compound 4-hydroxybenzoate (4HBA) (Hosokawa et al., 2002). The minimum inhibitory concentration (MIC) of 4HBA for the wild-type strain was 1%, whereas the MIC for mutants was 1.7%. Frequency of 4HBA-tolerant mutants among spontaneous str, gen, and rif mutants was 5–15%, 3–5%, and 3% respectively. These 4HBA-tolerant mutants also tolerated a variety of organic chemicals such as 3-hydroxybenzoate, aliphatic and heterocyclic compounds, chlorobenzoates, as well as the organic solvents toluene and m-xylene. The str mutants had a point mutation in the rpsL gene. Interestingly, str, gen, and rif-phenotypes occurred in spontaneous 4HBA-tolerant mutants that had been selected by successively increasing concentrations (from 0.8% to 5%) of 4HBA, implying that

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breeding approaches by traditional mutagenesis may often involve mutations in the ribosomal component or RNA polymerase. Uptake experiments using [14C]-4HBA revealed that the apparent ability of 4HBA to be taken up by the membrane transport system was reduced twofold to threefold in the mutants compared to the wild-type strain, accounting at least partly for enhanced tolerance to 4HBA (Hosokawa et al., 2002). As the apparent uptake rate is the sum of the actual uptake (influx) and efflux, it is possible that these drug-resistant mutants have acquired a capacity to overproduce wide-specificity efflux pumps by somehow enhancing the expression of the key genes (such as marA, mexAB) that play a role in the efflux system. These findings might help in elucidating the mechanisms of tolerance to toxic organic chemicals, particularly its relationship to transcription and translation machinery. The efficacy of str and rif mutations for elevating the cells’ tolerance to organic solvents such as toluene and xylene suggests a widespread applicability of ribosome engineering in improving the ability of microorganisms to process chemical wastes. VII. Combined Drug-Resistance Mutations A. MODEL EXPERIMENT Introduction of combined drug-resistant mutations was found to be quite effective in increasing the productivity of antibiotics in a hierarchical order (Hu and Ochi, 2001). The increased productivity of actinorhodin by sequential introduction of str, gen, and rif in S. coelicolor A3(2) is shown in Fig. 9A. Mutants with enhanced (1.6-fold to threefold higher) actinorhodin production were detected

FIG. 9. Hierarchical increase of antibiotic production by introducing combined drugresistance mutations in Streptomyces coelicolor wild-type strain 1147 (A) and Streptomyces albus industrial strain SAM-X (B), which produces a high amount (10 mg/ml) of salinomycin. (Adapted from Hu and Ochi, 2001, and Tamehiro et al., 2003.)


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at a high frequency (5–10%) among isolates resistant to streptomycin (str), gentamicin (gen), or rifampin (rif ), which developed spontaneously on agar plates that contained one of the three drugs. Construction of double mutants (str gen and str rif ) by introducing gentamicin or rifampin resistance into an str mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (str gen rif ) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Although analysis of str mutants revealed that a point mutation occurred within the rpsL gene, mutation points in gen mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of Act II-ORF4, a pathway-specific regulatory protein (Hu and Ochi, 2001). The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. Thus, using combined drug-resistant mutations, we can continuously increase the production of an antibiotic in a stepwise manner. Although much progress has been made in improving antibiotic producers (Chater, 1990; Lai et al., 1996; Lee et al., 1999), our method is characterized by the host cell’s amenability (generation of spontaneous drug-resistant mutation) and the method’s applicability to a number of microorganisms. It should also be emphasized that combined resistant mutations (triple mutations) demonstrated no significant impairment in growth or sporulation. Antibiotic production is in general subjected to the suppressive effects caused by an excess of nutrients such as carbon, nitrogen, and phosphate sources. In particular, ammonium and phosphate both appear to be major regulators of antibiotic production, and their control systems may be interrelated in some way. Consistent with this notion, actinorhodin production in wild-type and mutant strains is more or less medium dependent; it is greater in R4 medium (containing less yeast extract and phosphate) than in R3 medium. The triple (str gen rif ) mutants revealed less sensitivity to such suppressive effects. B. APPLICABILITY TO INDUSTRIAL STRAIN Unlike the wild-type strains discussed earlier, improvement of industrial strains is, in general, much more difficult, as productivity has already been raised by various genetic and physiological approaches. Nevertheless, demonstration of the efficacy of drug-resistant mutation in industrial strains is intriguing, because improvement of industrial

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strains is linked directly with economic aspects. Working with a Streptomyces albus strain that had previously been bred to produce industrial amounts (10 mg/ml) of salinomycin, the efficacy of introducing drug-resistant mutations for further strain improvement has been demonstrated (Tamehiro et al., 2003). Mutants with enhanced salinomycin production were detected at a high incidence (7–12%) among spontaneous isolates resistant to streptomycin, gentamicin, or rifampicin. Finally, we demonstrated improvement of the salinomycin productivity of the industrial strain by 2.3-fold by introducing a triple mutation (Fig. 9B). The str mutant was shown to have a point mutation within the rpsL gene (encoding ribosomal protein S12). Likewise, the rif mutant possessed a mutation in the rpoB gene (encoding the RNA polymerase subunit). Combined drug-resistant mutations (triple mutation) caused no impairment of growth and sporulation. Rather, the ability to produce aerial mycelium and spores was, in fact, enhanced. Strikingly, the str mutant with increased salinomycin production exhibited high translation activity at the stationary phase as determined with the in vitro translation assay system (Tamehiro et al., 2003) (Fig. 10). This high-translation activity could be a reason why the str mutant is capable of producing a greater amount of salinomycin. The aberrant protein synthesis ability of the str mutant resulted presumably

FIG. 10. In vitro translation activities of ribosomes prepared from S. albus cells at various growth phase. Translation activities were determined with poly(U)-directed cell-free translation system. (Adapted from Tamehiro et al., 2003.)


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from a more stable ribosome structure, as demonstrated in the presence of a low Mg2þ concentration. Antibiotic production, including salinomycin production, usually commences at the late growth phase (i.e., transition phase or stationary phase). Therefore the enhanced protein synthesis ability at late growth phase would promote the initiation processes (i.e., formation of positive regulatory proteins for antibiotic gene expression) or biosynthetic processes (or both).

VIII. Conclusion and Future Prospects In this review, we demonstrated that a cell’s function can be altered dramatically by modulating the ribosome using a drug-resistance mutation technique. Our approach is characterized by focusing on ribosomal function at late growth phase (i.e., stationary phase). The importance of this phase has been largely overlooked in studies of ribosomes, with a few exceptions (such as a 1990 work by Wada et al.). In summary, our novel breeding approach is based on two different aspects, modulation of the translational apparatus by induction of str and gen mutations, and modulation of the transcriptional apparatus by induction of a rif mutation (Fig. 11, see color insert). Modulation of these two mechanisms may function cooperatively to increase antibiotic productivity. Introduction of mutations conferring resistance to fusidic acid (fus) or thiostrepton (tsp), though not yet published, also causes activation of antibiotic production as well as str mutation. Moreover, these fus and tsp mutations were found to give rise to an aberrant protein synthesis activity, as did the str mutant ribosome (T. Hosaka and K. Ochi, unpublished). Resistance to fusidic acid and thiostrepton is known to come frequently from a mutation in elongation factor G and ribosomal protein L11, respectively. However, no mutations were found within the genes encoding elongation factor G or ribosomal protein L11. It is therefore highly likely that these fus and tsp mutations are located on the genes encoding rRNAs. This is important because it implies the existence of a new way to modulate ribosomal function, in addition to ribosomal protein mutations. In the study of current topics, we have found several important facts (or phenomena), which might be useful in eliciting the cell’s ability. These facts, together with the aforementioned studies, encourage us to construct more elegantly designed and more widely applicable ribosome engineering in the near future.

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FIG. 11. Scheme of ‘‘ribosome engineering’’ to activate cell’s ability.

A. FUTURE PROSPECTS 1. EshA Protein That Affects Developmental Processes Kwak et al. (2001) and our laboratory (Kawamoto et al., 2001; Saito et al., 2003b) independently found a novel 52-kDa protein that is produced during the late growth phase. The disruption of the gene (eshA), which codes for this 52-kDa protein (EshA), was shown to abolish antibiotic production in S. coelicolor A3(2) and S. griseus.


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Conversely, propagation of wild-type eshA with low-copy-number plasmids caused overproduction of antibiotics in both S. griseus and S. coelicolor (Kawamoto et al., 2001; Saito et al., 2003a). Thus it is evident that EshA plays an important role as a positive regulator of antibiotic production. It is also notable that our eshA null mutants show considerable similarity in phenotype to the relA mutant, as characterized by severely impaired ability to produce antibiotics. In fact, eshA null mutant showed a lower level of ppGpp compared to wild-type strain, and the impaired ability to produce antibiotic was completely restored by propagating the relA gene, accompanied by an increase in ppGpp (Saito and Ochi, unpublished). The EshA protein was found to exist as a multimer (20-mers) creating a cubiclike structure with a diameter of 27 nm, possibly forming an icosahedron. EshA may offer a feasible target for strain improvement, because genes homologous to eshA appear to be widely distributed among streptomycetes. 2. ppGpp-GTP Dual Control in B. subtilis Bacilysin is one of the simplest peptide antibiotics produced by B. subtilis. Recently it has been reported that CodY protein regulates the expression of various stationary-phase genes by sensing the intracellular GTP level (Inaoka and Ochi, 2002; Ratnayake-Lecamwasam et al., 2001) (Fig. 12). In fact, sporulation and genetic competence development can be initiated by addition of decoyinine (a GMP synthetase inhibitor) or by inactivation of CodY. Likewise, bacilysin production is apparently controlled by CodY protein, since codY disruption increased the transcription of the bacilysin biosynthesis cluster (Inaoka et al., 2003). However, a codY relA double mutant does not produce bacilysin, indicating that ppGpp plays a pivotal role as a positive regulator even in B. subtilis, and that GTP functions as a negative regulator, producing a synergistic effect on antibiotic production when its level declined (Fig. 12). Thus, unlike antibiotic production in Streptomyces spp., bacilysin production in B. subtilis is controlled by a ‘‘dual regulation’’ system composed of the guanine nucleotides ppGpp and GTP. The codY gene may be a feasible target for activation of secondary metabolism, since many low GþC Gram-positive bacteria contain the CodY homologue. 3. Existence of ppGpp in Plants Plants have a complex signal transduction network activated in response to such stressful conditions as pathogenic infection, wounding, heat shock, drought, and high salinity (Ryan and Moura, 2002;

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FIG. 12. Scheme of a dual regulation system by ppGpp and GTP in bacilysin biosynthesis. ppGpp promotes bacilysin production, while GTP inhibits it via CodY protein.

Xiong et al., 2002). Despite the apparent significance of ppGpp in bacterial gene expression, the importance of ppGpp in plant biology has been largely overlooked. Recently, we unambiguously demonstrated that ppGpp is produced in the chloroplasts of plant cells in response to stressful conditions (Takahashi et al., 2004). Levels of ppGpp increased markedly when plants were subjected to such biotic and abiotic stresses as wounding, heat shock, high salinity, acidity, heavy metal, drought, and ultraviolet irradiation. Abrupt changes from light to dark also caused a substantial elevation in ppGpp levels. Elevation of ppGpp levels was also elicited by treatment with the plant horomones jasmonic acid, abscisic acid and ethylene. In vitro, chloroplast RNA polymerase activity was inhibited in the presence of ppGpp, demonstrating the existence of a bacterial-type stringent response in plants. Given the significance of ppGpp in bacterial physiology, ppGpp apparently plays a critical role in adjusting plant physiology. Our finding is consistent with recent work demonstrating the existence of RelA homologs (At-RSH1 and Cr-RSH) in Arabidopsis and Chlamydomonas (Kasai et al., 2002; van der Biezen et al., 2000). Understanding


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in detail the functions of ppGpp and the signaling cascades it activates is clearly an important challenge that should provide important insight into plant evolution and adaptation to environmental changes, and should also make a contribution in the area of plant breeding. 4. Applicability of Ribosome Engineering to Cell-Free Translation Systems In contrast to the utilization of whole cell, cell-free translation systems have been shown to achieve high-throughput production of a wide variety of foreign-originated proteins. Studies to improve the productivity of protein synthesis in vitro have employed several strategies, such as (1) the development of continuous flow in vitro protein synthesis (Spirin et al., 1988), (2) a source for preparing cell-free extracts (Baranov and Spirin, 1993; Endo et al., 1992), and (3) optimization of translation components, reaction condition, and generation and consumption of an energy source (Kim and Swartz, 2001; Kim et al., 1996; Sawasaki et al., 2002). However, there have been no reports focusing on the ribosome in terms of high-throughput production. Recently, to examine the effects of ribosomal protein S12 mutations on the efficiency of cell-free protein synthesis, we isolated a wide variety of str mutants from E. coli and found that a mutant replacing Lys-42 with Thr (K42T) in the S12 protein shows higher (1.3-fold to twofold) protein production than the wild-type (Chumpolkulwong et al., 2004). Therefore, the method of ribosome engineering may be effective as one of the strategies to enhance protein production in E. coli-based (and even in wheat germ-based) cell-free translation systems.

ACKNOWLEDGMENTS This work was supported by a grant to K. Ochi (for the project ‘‘Construction of Ribosome Engineering’’) from the Organized Research Combination System of Education, Culture, Sports, Science and Technology of Japan. The authors are grateful to S. Kawamoto, Y. Hosoya, H. Hu, K. Matsubara, and K. Hosokawa for their contributions in promoting the project, and to Prof. S. Yokoyama (RIKEN Institute) for valuable discussions and Prof. T. Fukui for encouragement through the project.

REFERENCES Arias, P., Fernańdez-Moreno, M. A., and Malpartida, F. (1999). Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein. J. Bacteriol. 181, 6958–6968.

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Developments in Microbial Methods for the Treatment of Dye Effluents R. C. KUHAD,* N. SOOD,* K. K. TRIPATHI,{ A. SINGH,{,}

AND

O. P. WARD{

*

Department of Microbiology, University of Delhi New Delhi–110 021, India

{

Department of Biotechnology, Ministry of Science and Technology New Delhi–110 003, India {

}

Department of Biology, University of Waterloo Waterloo, Ontario N2L 3G1, Canada


I. Introduction II. Conventional Methods A. Physical B. Chemical III. Microbial Methods A. Biosorption B. Aerobic Biodegradation C. Anaerobic Biodegradation D. Combined Anaerobic/Aerobic Biodegradation IV. Enzymatic Methods V. Conclusion References

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I. Introduction Synthetic chemical dyes are extensively used for textile, paper printing, color photography, cosmetic, pharmaceutical, and leather industries. Dyes are of environmental interest because of their potential of forming toxic aromatic amines. Over the last two decades there has been a tremendous increase in awareness of the toxic and carcinogenic effects of many polluting chemicals that were not considered hazardous in the past (King, 1997). More than 10,000 different dyes and pigments are used industrially with a production of 7 105 tons of these dyes per year (Zollinger, 1987). It is estimated that 2–50% of these dyes are lost into wastewaters, depending on the class of dye used (O’Neill et al., 1999). Since most of these dyes are persistent environmental pollutants, they are not removed from industrial effluents by conventional wastewater treatments (Cripps et al., 1990; Moran et al., 1997; Willmott et al., 1998). Dyes may be toxic and mutagenic, and if they are discharged directly into the environment, they contaminate not only the environment but also traverse through the entire food chain, leading to biomagnification. Through environmental legislation, tougher controls are being applied regarding the requirement to remove dyes from industrial effluents. 185 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 56 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2164/04 $35.00

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Industrial dyes are classified as anionic (direct, acid, and reactive dyes), cationic (basic dyes), and nonionic (disperse dyes) (Dawson, 1981; Mishra and Tripathy, 1993). Anionic and nonionic dyes mostly contain azo or anthraquinone type chromophores. Azo dyes are the most widely used, accounting for more than 60% of the total number of dyes known to be manufactured, and toxic amines in the industrial effluents are the result of reductive cleavage of azo linkages. Anthraquinone dyes are more resistant to degradation because of the fused aromatic structure. High brilliance and intensity of colors make basic dyes highly visible even at low concentration. Chromium-containing metal complex dyes are carcinogenic in nature. More than 90% of about 4,000 dyes tested in a survey had LD50 values >2000 mg/kg with highest toxicities being found among basic and diazo dyes (Shore, 1996). Table I shows examples of some common azo dyes used for microbial dye decolorization studies. Textile industry effluents are characterized as having the high visible color (3000–4500 units), chemical oxygen demand (800–1600 mg/L), alkaline pH range of 9–11, and total solids (6000–7000 mg/L) (Manu and Chaudhari, 2002). A variety of effective physical and chemical treatment methods are available (Nigam et al., 2000; Robinson et al., 2001a). There has been considerable interest in development of biological methods (microbial and enzymatic), because these methods are considered attractive because of their potential low-cost, environmental compatibility, and public acceptability (Dubin and Wright, 1975; Paszczynski and Crawford, 1991). A wide variety of microorganisms, including bacteria, fungi, and algae, are capable of decolorizing a diverse range of dyes (McMullan et al., 2001). Many bacteria are able to degrade dyes both aerobically and anaerobically. Biodegradation of azo dyes by bacteria is often initiated by azoreductase-driven cleavage of azo bonds, followed by aerobic or anaerobic degradation of resulting amines (Stolz, 2001). On the other hand, fungal degradation typically originates from the lignolytic activity to degrade azo dyes aerobically with the aid of lignin peroxidase (Fu and Viraraghavan, 2001). In this chapter, various dye decolorization methods and developments in biological treatment methods for dye are critically reviewed. II. Conventional Methods Physical and chemical methods such as flocculation, electrochemistry, ozonation, bleaching, membrane filtration, irradiation, and adsorption to activated carbon are commonly used for the treatment of

DEVELOPMENTS IN MICROBIAL METHODS

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TABLE I EXAMPLES OF SOME AZO DYES COMMONLY USED IN MICROBIAL DYE DECOLORIZATION STUDIES

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industrial effluents. Most of these methods are highly specific and uneconomical but very effective. Table II shows the merits and demerits of various methods for dye decolorization of industrial effluents. A. PHYSICAL Adsorption is one of the most common physical methods used for dye removal that is economically feasible but is influenced by a number of factors such as surface area of the sorbent, particle size, dye/ sorbent interaction, pH, and temperature (Kumar et al., 1998). Activated carbon is generally very effective for cationic and acid dyes and less effective for dispersed, direct, and reactive dyes (Raghavacharya, 1997). However, the activated carbon adsorption process depends on the type of carbon used, regeneration capacity, and the characteristic of wastewater. A mixture of fly ash and coal can be substituted for activated carbon. Silica gel is another effective adsorbent for removing dyes, but the commercial use is uneconomical because of side reactions such as air binding and fouling with particulate matter. Some naturally occurring material such as peat, wood chips, and agricultural lignocellulosic residues (straws, wood chips, etc.) are potentially economical adsorbents (Nigam et al., 2000; Robinson et al., 2002a,b,c). Dye color removal of up to 90% has been achieved by using steam or chemically pretreated wheat straw, corncobs, and barley husk (Robinson et al., 2002a) in static or continuous packed bed reactor (Robinson et al., 2002b). Widespread availability of agricultural residues offers an economical alternative to activated carbon for dye removal from the industrial effluents. Unlike activated carbon, regeneration is not required when agricultural residues are used, and further potential exists for fermenting dye-adsorbed waste into useful products. Solid state fermentation of the dye-adsorbed residue with white-rot fungi can simultaneously degrade dyes and enrich the nutritional value of the substrate for animal feed or for use of the fermented product used as soil conditioner (Nigam et al., 2000). Membrane filtration can concentrate and separate most of the dyes continuously from the effluent. This approach is typically suitable for low concentrations of dye and water recycling within the plant (Xu and Lebrun, 1999). Although the system is generally resistant to temperature and microbial attack, high capital cost, disposal of concentrated dye, and possibility of clogging are serious disadvantages of membrane technology. Ion exchange is a very effective method for removing both cationic and anionic dyes but less effective for disperse dyes and hence is not widely used (Slokar and Le Marechal, 1997). Usually there is no

TABLE II VARIOUS METHODS FOR DYE DECOLORIZATION OF INDUSTRIAL EFFLUENTS Method Physical

Chemical

Biological

Specific method

Comments

Adsorption

Excellent removal of various dyes; activated carbon is expensive; material loss on regeneration; side reactions with silica gel are undesirable; natural cellulosic material may be cost effective, but specific surface area is comparatively lower

Membrane filtration

Effective in removing all dye types from wastewater, but concentrated dye sludge needs to be disposed of properly

Ion exchange

Both cation and anion dyes can be removed from the effluents; regeneration is possible without loss of material; may not be applicable to all type of dyes; cost is prohibitive

Irradiation

Efficient oxidation of various dyes at lab scale; high volumes of oxygen needed, which makes the system unattractive

Oxidation

Effective decolorization of various kinds of dyes; problems associated with by-product formation; sludge problem may be associated with Fenton’s reagent treatment; ozonation has short 20-min half-life; release of aromatic amines is a concern with NaOCl

Electrochemical

Relatively new method for effective removal; nonhazardous breakdown of products; electricity costs are high

Coagulation

Excellent removal of direct dyes using ferrous sulfate and ferric chloride; poor removal of acid dyes; high volumes of sludge formation; high disposal costs

Biosorption

Microbial biomass to sorb and remove dyes from wastewater is still in the research stage; may not be practical to treat large volumes of industrial effluents; problems associated with disposal of the dye-adsorbed biomass; may be regenerated using chemicals

Biodegradation

Mixed culture consortium in a combined anaerobic/aerobic continuous system for complete removal of dye compounds shows potential; immobilized cell systems appeared to be more practical than free bacterial cells; practical uses of bacterial processes are not well documented; more understanding physiological/genetic information is required

Enzymatic

Laccase and peroxidase preparations offer rapid method for decolorization of dye wastewater; detailed analysis of reaction by-products, scale-up studies and careful economic evaluation are required for commercial applications

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loss of adsorbent on regeneration, but the organic solvents used in this technique are expensive. The irradiation process to treat dye-containing effluent in a dual-tube bubbling reactor requires large volumes of dissolved oxygen in the system, which makes the process economically less attractive (Kumar et al., 1998). Sonophotocatalytic technology, a hybrid technology utilizing photocatalytic and sonochemical processing, has recently been proposed for the treatment of reactive dye-containing wastewater (An et al., 2003). However, more data from scale-up studies are required before realization of a commercial process. B. CHEMICAL Because of the simplicity of the process, chemical oxidation is the most commonly used method for dye decolorization. Chemical oxidation involves the removal of dye resulting from aromatic ring cleavage of the dye molecule. Fenton’s reagent (H2O2/FeSO4) is a suitable method for treating wastewater that is resistant to biological treatment or toxic to the microorganisms. This method can be used for the treatment of both soluble and insoluble dyes (Pak and Chang, 1999). However, flocculation of the reagent and residual dye results in sludge generation containing concentrated impurities, which still requires disposal. The performance is also dependent on the final floc formation and its settling quality. While cationic dyes usually do not coagulate well, acid, direct, mordant, and reactive dyes result in poor quality flocs that do not settle well. Sodium hypochlorite (NaOCl) is effective in azo bond cleavage. However, it is not suitable for disperse dyes. Increased chemical use may have a negative impact in waterways because of the presence of chloride ions and the release of aromatic amines, which are both toxic and carcinogenic. A cyclic polymer of glycoluril and formaldehyde, cucurbituril shows good adsorption capacity for various types of textile dyes, but use of this chemical is cost prohibitive (Karcher et al., 1999). Coagulation with ferrous sulfate and ferric chloride may be another feasible method for removing direct dyes from wastewater. However, poor results with acid dyes and the high disposal cost because of the production of large volumes of flocculated sludge were prohibitive in widespread use of this method (Kumar et al., 1998; Slokar and Le Marechal, 1997). Photochemical methods (UV/H2O2) can be used to degrade organic molecules to CO2 and H2O in a batch or continuous system (Yang et al., 1998). The structure of dye, intensity of UV radiation, and pH affect the


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rate of dye removal. The advantages of this method include the reduction of foul odors and no sludge generation in the process. However, depending on the initial substrate and the extent of treatment, byproducts such as halides, metals, inorganic and organic acids, and aldehydes may be produced. As compared with chlorine (oxidation potential, 1.36) and H2O2 (1.78), ozone (2.08) is a stronger oxidizing agent capable of degrading phenols and chlorinated and aromatic hydrocarbons (Peralto-Zamora et al., 1999). Ozonation leaves dye-containing effluent with no color on toxic byproduct with little or no sludge formation and a low COD. Since the ozone is supplied in its gaseous state, volume of wastewater does not increase. However, because of its short half-life (20 min), the stability of ozone can be strongly affected by the presence of salts, pH, and temperature. Ozonation is a costly process and has been recommended to use in combination with irradiation or membrane filtration. Electrochemical removal of dyes from wastewater is a relatively new process exhibiting efficient color removal and degradation of recalcitrant pollutants (Pelegrini et al., 1999). The method effectively and economically degrades dyes without using chemicals or generating toxic byproducts and sludge build-up. Although a variety of effective physical and chemical treatment methods are commercially available, most of them are either expensive, not adaptable to a wide range of dyes, or do not completely solve the problem of complete decolorization of dye-containing industrial effluents. This has resulted in considerable interest in alternative methods such as microbial, enzymatic, and a combination of physico-chemical and biological methods. III. Microbial Methods Biological methods are currently viewed as effective, specific, less energy intensive, and environmentally benign, since they result in partial or complete bioconversion of organic pollutants to stable nontoxic end products (Baker and Herson, 1994). Both biosorption and biodegradation have been explored as methods of biological treatments of dye-contaminated effluents. A. BIOSORPTION Biosorption includes both adsorption and absorption. The idea of dye removal through absorption originated from the reasonable success achieved in removal of heavy metal contamination from wastewater.

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Both bacterial and fungal cells are reported for their capability of partial or complete removal of industrial dyes by using adsorption process. Not all dyes adsorb to a particular type of biomass. However, with some fungi, adsorption is the only decolorization mechanism, but with white-rot fungi both adsorption and degradation can occur simultaneously or sequentially (Knapp et al., 2001). The potential of 10 actinomycetes isolates to decolorize the polymeric dye polyvinyl amine sulfonate anthrapyridone, Poly R-478, has been studied in our laboratory (Vasdev and Kuhad, 1994). Almost 100% decolorization was observed in 8 d. We also studied the capability of three actinomycetes to decolorize azo dyes (Congo red and Remazol brilliant blue R–RBBR), triphenylmethane dyes (crystal violet and malachite green), the heterocylic dye methylene blue, the polymeric dye Poly R, and xylidine (unpublished results). Decolorization caused by sorption was seen in the case of RBBR, Congo red, and Poly R. In the case of triphenylmethane dyes (0.001%), there was no growth and hence no decolorization was observed, whereas in the case of xylidine no decolorization was observed in spite of good growth. Proteus mirabilis isolated from acclimated sludge from a dyeing wastewater treatment plant rapidly decolorized >95% of a deepred azo dye solution (red RBN) within 20 h of incubation, but a part of decolorization (13–17%) was found to be due to biosorption as confirmed by inactivated bacterial cells (Chen et al., 1999). Although decolorization of dye wastewater by live or dead fungal biomass has been a subject of various studies, only limited information is available on interactions between biomass and molecular structure of dyes (Fu and Viraraghavan, 2001). Dead cells remove dyes through the mechanism of biosorption, which involves physicochemical interactions such as adsorption, deposition, and ion exchange. The extent of dye biosorption depends on the chemical structure of dyes and the functional group of the dye molecules. In the case of Aspergillus niger, different functional groups in the fungal biomass play different roles in biosorption of different dyes (Fu and Viraraghavan, 2002). Electrostatic attractions could be the primary mechanism, and the amino, carboxylic acid, phosphate groups, and lipid fractions could be important binding sites depending on the type of the dye used. Integrity of the cell is also important for the binding capacity. Both Freundlich and Langmuir isotherm models fit well for the biosorption of Reactive brilliant red to Rhizopus oryzae biomass (Gallaghar et al., 1997). The kinetics of activated sludge biomass adsorption for the removal of basic dyes from wastewater follows firstorder processes, controlled by film diffusion (Chu and Chen, 2002).


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The activated sludge biomass process was exothermic in nature, with an activation energy of 3.27 kcal mol1. Yesilada et al. (2002) investigated decolorization of textile dyes by using pellets of the white-rot fungus Funalia trogii. The decolorization activity was significantly affected by dye concentration, amount of pellet, temperature, and agitation of the media. With live cells, the decolorization involved adsorption of the dye compound by the fungal pellet at the initial stage, followed by fungal catabolism (Yesilada et al., 2003). On the other hand, decolorization of media containing the azo reactive dyes Procion and Drimarene with greater than 90% removal under aerobic conditions with Aspergillus foetidus has been reported (Sumathi and Manju, 2000, 2001). Adsorption does not appear to be the principal mechanism of decolorization in white-rot fungi (Knapp et al., 1997). It is likely that degradation occurs after initial adsorption. Prior adsorption to fungal mycelium may serve to bring chromophores into closer contact with the degradative enzymes (Wang and Yu, 1998). The method of using fungal biomass to sorb and remove dyes from wastewater is still in the research stage. Biosorption may not be a practical approach for treating large volumes of dye-contaminated industrial effluents because of the problems associated with disposal of the large volumes of biomass after biosorption of dyes from industrial effluents.

B. AEROBIC BIODEGRADATION Decolorization and degradation of dyes by mixed as well as pure cultures of bacteria and fungi have been studied under aerobic and anaerobic conditions. In most studies, the microbial consortia have been found more effective than pure cultures. In addition to chemical structure, several environmental and nutritional factors such as pH, temperature, amount of oxygen, and co-metabolic carbon sources influence aerobic biodegradation processes. 1. Bacteria Actinomycetes are known to produce extracellular peroxidases that participate in the initial oxidation of lignin to produce various watersoluble polymeric compounds and have also been shown to catalyze hydroxylation, oxidation, and dealkylation reactions against various xenobiotic compounds (Ball et al., 1989; Goszczynski et al., 1994). Species of Streptomyces and Thermomonospora are good examples of actinomycetes capable of effective dye decolorization (Table III). In a

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screening program designed to use actual textile effluents, 83 positive isolates were found (Zhou and Zimmerman, 1993). Pasti-Grigsby and Crawford (1991) investigated the ability of ligninolytic microbes (white-rot fungi and Streptomyces) to mineralize and decolorize textile dyes and found a strong correlation between dye decolorization ability and ligninolytic ability. Decolorization of mono sulphonated monoazo dye derivatives of azo benzene by the Streptomyces spp. was observed with five azo dyes having the common structural pattern of a hydroxy group in the para position relative to the azo linkage and at least one methoxy and/or one alkyl group in an ortho position relative to the hydroxy group. While Streptomyces chromofuscus was unable to mineralize aromatics with sulpho groups and both sulpho and azo groups, it mediated the mineralization of modified dyes containing lignin-like substitution patterns. This work showed

TABLE III BACTERIA AND THEIR MECHANISM OF DYE DECOLORIZATION Bacteria Actinomycetes

Anaerobic

Aerobic

Species

Mechanism of action

Nocardia corallina

Peroxidase

Nocardia globerulla

Peroxidase

Streptomyces badius

Peroxidase

Streptomyces chromofuscus

Peroxidase

Streptomyces viridosporus

Peroxidase

Thermomonospora fusca

Peroxidase

Thermomonospora mesophila

Peroxidase

Clostridium paraputrificum

Anaerobic reduction

Clostridium perfringens

Azoreductase

Proteus vulgaris

Anaerobic reduction

Rhodococcus sp.

Azoreductase

Sphingomonas xenophaga

Anaerobic reduction

Streptococcus faecalis

Anaerobic reduction

Bacillus subtilis

Aerobic biodegradation

Citrobacter sp.

Adsorption and biodegradation

Pseudomonas luteola


Pseudomonas mendocina


Pseudomonas pseudonallei



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that lignocelluloytic bacteria could be used for the biodegradation of anionic azo dyes (Paszczynski et al., 1992). Ball and Cotton (1996) have also studied three well-characterized lignocellulose-degrading actinomycetes, Streptomyces viridosporus, Streptomyces badius, and Thermomonospora mesophila and showed that they decolorized the polymeric dye Poly-R with a maximum decolorization rate of 0.1 unit/day. The potential of different Nocardia species such as N. corallina and N. globerulla for their ability to degrade triphenylmethane dyes has also been demonstrated (Yatome et al., 1991). Several bacterial strains aerobically decolorize azo dyes by reductive mechanisms. Most of these bacteria do not use azo dyes as carbon or energy source and decolorize azo dyes only in the presence of other carbon sources. Govindaswami et al. (1993) reported a Gram-negative rod capable of oxygen-insensitive azo bond cleavage of dyes (such as Acid orange-7 and Acid red-151) during aerobic growth, in glucoseenriched minimal medium that they considered as a potential candidate for incorporation into experimental bioreactors operated for azo dye degradation. Coughlin et al. (1999) isolated a Sphingomonas strain from a wastewater treatment plant that was capable of aerobically degrading a suite of azo dyes by using them as a sole source of carbon and nitrogen. After an analysis of the structures of dyes, they suggested that there were certain positions and types of substituents on the azo dye that determined the degradation of the dye. Their strain decolorized dye with either 1-amino-2-naphthol or 2-amino-1-naphthol in their structure, and the decolorization appeared to be through reductive cleavage of the azo bond. On the other hand, a Proteus mirabilis strain decolorized RRBN by a combination of biodegradative and biosorptive processes. This organism displayed good growth on the contaminant in shake culture, but color removal was best in anoxic static culture (Chen et al., 1999). Sarnaik and Kanekar (1999) described the aerobic mineralization of the triphenyl methane dye methyl violet by a strain of Pseudomonas mendocina MCMB 402. P. mendocina degraded the dye via a number of unidentified metabolites to phenol that then entered the -ketoadipic acid pathway. An et al. (2002) recently reported optimum decolorization of several recalcitrant triphenylmethane and azo dyes by Citrobacter sp. at pH 7–9 and temperature 35–40 C. Color removal by Citrobacter sp. was both by adsorption to cells and enzymatic, as evidenced by the experiments with extracellular culture filtrate. P. luteola cells growing under shaking conditions for 24 h were capable of removing 59–99% of the color of seven azo dyes in static

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conditions (Hu, 2001). There are other reports on the ‘‘aerobic’’ metabolism of azo dyes, where the bacterial strains (e.g., Aeromonas sp., Bacillus subtilis, Proteus mirabilis, P. pseudomallei BNA, P. luteola) were grown aerobically with complex media or sugars, then incubated (often with high cell densities) without shaking in the presence of different azo dyes (Chang and Lin, 2000; Chen et al., 1999; Hayase et al., 2000; Horitsu et al., 1977). However, resting cell cultures presumably become rapidly oxygen depleted, and the reactions observed should therefore be viewed as an anaerobic degradation of azo dyes (Stolz, 2001). To develop novel decolorization processes for practical use, Chang et al. (2001) attempted an immobilized-cell system of P. leuteola with a view to enhance the stability, mechanical strength, and reusability of the biocatalyst. Cell immobilization by entrapment within natural or synthetic matrices is particularly suitable for bacterial dye decolorization since it creates a local anaerobic environment favorable to dye metabolism (Stolz, 2001). P. leuteola cells entrapped in natural and synthetic polymeric matrices efficiently decolorized azo dyes enzymatically. Immobilized cells were less sensitive to dissolved oxygen levels and pH as compared with suspended cells, while the effect of temperature was similar for both suspended and immobilized cells. After four repeated experiments, the decolorization rate of the free cells decreased by nearly 45%, while immobilized cells retained 75–85% of their original activity in different matrices. It is generally recognized that azoreductases play an important role in bacterial dye decolorization. However, only a limited number of studies have attempted molecular characterization of dye decolorization. The genes encoding for azoreductase and other possible proteins involved in decolorization have not been clearly identified. Recently the gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp. KF46F), a strain able to grow with carboxylated azo compound 1-(40 -0 arboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole carbon and energy source (Blu¨mel et al., 2002). The enzyme was heterologously expressed in E. coli. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. While the cell extracts from the recombinant strain demonstrated the turnover of several industrially relevant azo dyes, the whole cells of the recombinant E. coli were unable to take up sulfonated azo dyes and did not show in vivo azoreductase activity. A recombinant E. coli strain NO3 containing genomic DNA fragments from azo-reducing wild-type P. luteola effectively decolorized an azo dye Reactive red 22 at the rate of about 17 mg/g cells/h (Chang


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et al., 2000). In another study from the same group (Chang and Lin, 2001), a 6.3 kb fragment from genome DNA of Rhodococcus sp. containing genes responsible for azo dye decolorization were cloned and expressed in E. coli. The recombinant strain E. coli CY1 decolorized Reactive red 22 at the rate of 8.2 mg/g cells/h with performance of excellent stability during repeated batch operations. Although encouraging laboratory results have been obtained indicating the potential of aerobic bacteria for dye removal, practical uses of bacterial processes for color removal have not been well documented. Immobilized cell systems appeared to be more effective than free bacterial cells. 2. Fungi White-rot fungi are the most widely studied microorganisms for dye decolorization/degradation (Table IV). This group of microorganisms is central to the global carbon cycle as a result of their ability to mineralize the complex polymeric woody plant material lignin. In addition to their natural substrate, white-rot fungi have been found to be capable of mineralizing a diverse range of persistent organic pollutants, which distinguishes them from biodegradative bacteria that tend to be rather substrate specific (Reddy, 1995). The ability of these fungi to degrade such a range of organic compounds results from the relatively nonspecific nature of their ligninolytic enzymes, such as lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase. LiP catalyzes the oxidation of nonphenolic aromatic compounds such as veratryl alcohol, while MnP oxidizes Mnþþ to Mnþþþ, which is able to oxidize many phenolic compounds (Glenn and Gold, 1986). Laccase is a copper-containing enzyme that catalyzes the oxidation of phenolic substrates by coupling the reduction of oxygen to water (Edens et al., 1999). Earlier it was assumed that laccases and peroxidases can only convert a limited type of azo dyes with preferential conversion of dyes carrying a phenolic substituent in paraposition to the azo bond and additional methyl- or methoxy-substituents in 2- or 2,6-position in relation to the hydroxy group (Chivukula and Renganathan, 1995). However, it has been later demonstrated that certain laccases and peroxidase are able to decolorize certain complex azo dyes such as Reactive black 5 (Schliephake et al., 2000). The decolorization of dyes by white-rot fungi was first reported by Glenn and Gold (1983), who developed a method to measure ligninolytic activity of Phanerochaete chrysosporium based on the decolorization of a number of sulphonated polymeric dyes. Subsequently, the decolorization of dyes has also been used to rapidly assess the biodegradative capabilities of diverse white-rot fungi (Chivukula and

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KUHAD et al. TABLE IV FUNGI AND THEIR MECHANISM OF DYE DECOLORIZATION Fungi

White-rot

Other filamentous

Yeast

Species

Mechanism of action

Bjerkandera adusta

Mn-peroxidase

Cyathus bulleri

Laccase

Funalia trogii

Adsorption, biodegradation

Lentinula edodus

Laccase

Phanerochaete chrysosporium

Lignin peroxidase

Phlebia radiata

Peroxidase

Pleurotus ostreatus

Peroxidase

Pycnoporus cinnbarinus

Laccase

Trametes versicolor

Biosorption, ligninase

Trametes hispida

Laccase

Aspergillus foetidus

Biosorption, biodegradation

Aspergillus niger

Adsorption, biodegradation

Aspergillus sojae

Biosorption

Botrytis cineria

Adsorption

Halosarpheia ratnagiriensis

Ligninases

Myrothecum verucaria

Adsorption

Neurospora crassa

Biosorption

Rhizopus arrhizus

Biodegradation

Trichoderma sp.

Biosorption, biodegradation

Candida rugosa

Adsorption

Cryptococcus heveanensis

Adsorption

Dekkera bruxellensis

Adsorption

Klyveromyces maxianus

Adsorption

Klyveromyces waltii

Adsorption

Pichia carsonii

Adsorption

Rhodotorula rubra

Biodegradation

Saccharomyces cerevisiae

Biosorption

Renganathan, 1995; Cripps et al., 1990; Field et al., 1993; Goszczynski et al., 1994; Itoh et al., 1998; Wunch et al., 1997). Wunch et al. (1997) suggested a screening method for selecting fungi capable of removing benzo[a]pyrene based on their ability to decolorize the polymeric dye R-478. For most of the 17 filamentous fungi tested, the disappearance of benzo[a]pyrene was correlated with the ability to decolorize R-478.


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Paszczynski et al. (1991) reported an approach based on modifying the chemical structure of commercial dyes by linking selected substituents into the dyes’ chemical structure to enhance aerobic azo dye transformation by P. chrysosporium. Guaiacyl or syringyl fragments introduced into the dye molecule seem to provide an access point for the fungal ligninolytic enzymes (Martins et al., 2001). The percentage of biological decolorization of Poly-R-478 was higher than 95%. Nearly complete decolorization (95%) of azo dye Orange-II was achieved by immobilized P. chrysosporium in a continuous packed bed reactor for periods longer than 30 days (Mielgo et al., 2001). Other white-rot fungi such as Cyathus, Trametes versicolour, Bjerkandera adusta, Pleurotus, Phlebia, and Thelephora species have also been screened for their dye decolorizing activity (Conneely et al., 1999; Heinfling et al., 1998; Kirby et al., 2000; Pointing et al., 2000; Selvam et al., 2003; Swamy and Ramsay, 1999; Vasdev and Kuhad, 1994). Vasdev et al. (1995) observed effective decolorization of three triphenylmethane dyes by Cyathus bulleri, C. stercorues, and C. straitus. C. Irpex lacteus and Pleurotus ostreatus were selected from 103 wood-rotting fungi for degradation of six different groups of dyes (azo, diazo, anthraquinone based, heterocyclic, triphenylmethane dyes, pthalocyanine) (Novotny et al., 2001). Decolorization of crystal violet and brilliant green by white-rot fungi Coriolus versicolor, Funalia trogii, and P. chrysosporium and one brown-rot fungus, Lacciporus sulphureus, has been reported. Trametes versicolor is capable of efficient decolorization of azo dyes (Toh et al., 2003). Immobilized cultures tend to have a higher level of activity and be more resilient to environmental perturbations than suspension cultures. The decolorization ability of T. versicolor ATCC 20869 was evaluated by using amaranth after immobilization in several natural and synthetic materials such as wheat straw, jute, hemp, maple wood chips, nylon, and polyethylene tetraphthalate fibers (Shin et al., 2002). Jute was found to be the best support material, facilitating good growth of T. versicolor without the loss of jute’s integrity over a 4-week period. Besides white-rot fungi, other filamentous fungi such as A. niger, Trichoderma viride, and A. foetidus have been found to be efficient in decolorizing textile dyes such as scarlet direct red, fast greenish blue, and brilliant direct violet (Fu and Viraraghavan, 2001; Kousar et al., 2000; Sumathi and Manju, 2000). Decolorization of Poly R-478 and Poly S-119 by Penicillium appeared to involve initial adsorption and followed by biodegradation (Zheng et al., 1999). Geotrichum candidum

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Dec 1 was found to decolorize 21 kinds of synthetic dyes, and an extracellular enzyme dye-decolorizing peroxidase (DyP) was responsible for the decolorization of the dyes (Kim and Shoda, 1999). To produce large amounts of DyP with dye-decolorizing activity, Sugano et al. (2000) achieved efficient heterologous expression of DyP from Geotrichum candidum Dec 1 in A. oryzae, and by fusing mature cDNA encoding dyp with the A. oryzae -amylase promotor (amyB). A. oryzae is a safe host with higher growth rate and with the capacity to secrete gram-per-liter quantities of heterologous proteins. Studies on dye decolorization with yeast species are scarce. Biodegradation of crystal violet by oxidative yeast, Rhodotorula rubra (Kwasniewska, 1985), and a number of simple azo dyes by Candida zeylanoides have also been reported (Martins et al., 1999). With live cells, fungal nutrition has been shown to be of significant importance in effective fungal decolorization systems. Influence of various carbon and nitrogen sources, micronutrients, vitamins, and amino acids on fungal decolorization has been investigated by various research groups. Majority of decolorization studies used glucose as the carbon source; however, glycerol, xylose, sucrose, maltose, fructose, cellobiose, starch, ethanol, and xylan have also been used (reviewed by Fu and Viraraghavan, 2001; Knapp et al., 2001; Robinson et al., 2001a). Presence of an added carbon source was found to be essential, particularly in the case of fungal mycelia recycling and reuse. Responses of white-rot fungi P. chrysosporium and Coriolopis gallica were found to be different in N-rich and N-limited artificial textile effluent (Robinson et al., 2001b). Nitrogen supplementation improved enzyme activities and dye decolorization for P. chrysosporium, whereas the additional nitrogen increased enzyme activities for C. gallica but did not improve decolorization. However, in the case of effluent treatment plants, the addition of supplementary carbon or nitrogen sources will depend on the nature of the dye wastewater. Effluents from distilling and paper pulping contain high levels of usable carbohydrates, whereas effluents from dyeing or chemical plants are unlikely to have a sufficient amount of usable carbon sources. Similarly, the presence of usable nitrogen sources in the effluent should be considered for designing medium for industrial effluent treatment. White-rot fungi appear to utilize a wide range of inorganic and organic nitrogen sources. Fungal decolorization offers a promising alternative method to replace or supplement current physicochemical methods. White-rot fungi have the capacity to decolorize a wide range of dyes and colored effluents from the pulp and paper industry, olive milling, cotton


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bleaching, etc., and may be repeatedly used in continuous and fedbatch cultures over a prolonged period. Fungal mycelia can be repeatedly used or stored for several months at 4 C with 100% retention of activity (Zhang et al., 1999). However, there is a further need to understand the mechanism of decolorization by non-white-rot fungi, establish a relationship between dye structure and decolorization, and develop fungal strains that can grow on simple, inexpensive medium and have a high production rate. 3. Bioreactor Processes Decolorization research has been carried out in batch, fed-batch, or semi-continuous and continuous cultures with a range of reactor configurations. The preferred bioreactors were air-lift reactor types, but trickling filters, packed beds, fluidized beds, and stirred tank reactors have also been used in decolorization studies (Bajpai et al., 1993; Zhang et al., 1999). Immobilization on rotating biological contactors (RBC), semi-permeable hollow fiber membrane reactors, and rope has also been proven successful (Marwaha et al., 1998; Yin et al., 1990). Most of the reactors were designed to retain a high biomass in the reactor. White-rot fungi, P. chrysosporium, and T. versicolor have been shown to work effectively as immobilized pellet (Pallerla and Chambers, 1996, 1997). The ability of white-rot fungi to treat effluents from pulp and paper, cotton bleaching, olive mills, and distilleries are now established. However, more scale-up studies with white-rot fungi are required before a commercial process can be realized. C. ANAEROBIC BIODEGRADATION Under anaerobic conditions, many bacteria have been reported to readily decolorize azo dyes by bringing about the reductive cleavage of the azo linkage, which results in dye decolorization and the production of colorless aromatic amines (Chung et al., 1992). The initial step in bacterial azo dye metabolism under anaerobic conditions involves the reductive cleavage of the azo linkage by azoreductases, although the in vivo role of such cytoplasmic enzymes is uncertain (Rafii et al., 1990). It appears that under anaerobic conditions, specific azoreductases are probably only of limited importance for the reduction of azo dyes. This is in contrast to the requirement for true azoreductases under aerobic conditions and readily explains the ubiquitous range of microorganisms that reduce azo compounds under anaerobic conditions. In vitro experiments with a recombinant flavin reductase have

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demonstrated that cytosolic flavin reductases are able to act like azoreductases and may be responsible for unspecific reactions of azo dyes (Russ et al., 2000). Under strict anaerobic conditions, decolorization of dyes can be enhanced in the presence of redox mediators such as benzyl viologen or quinones (Zee et al., 2000). As compared with resting cells, cell extracts show much higher rates of anaerobic reduction of azo dyes, probably because of the low permeability of the cell membranes for highly polar sulfonated azo compounds. Extracellular reduction of azo dyes by microorganisms may also be due to the action of reduced inorganic compounds such as Fe2þ and H2S, which are formed as anaerobic bacterial metabolic reaction end products. The H2S produced by sulfate-reducing bacteria can reduce azo dye Reactive orange 96 (Libra et al., 1997). Decolorization of dyes with pure culture is impractical, as the isolated culture would be dye specific, and their application in largescale wastewater treatment plants with a variety of contaminant dyes is not feasible. Efficient biodegradation of dyes can be accomplished when catabolic activities complement each other in a mixed culture community (Nigam et al., 1996). However, Clostridium paraputrificum was found capable of reducing seven commercially available, structurally related azo dyes (Moir et al., 2001). The rates of reduction of these dyes varied between 24 and 74 nmoles/mg protein/h. Beughmann and Weber (1994) demonstrated that, in anoxic sediment environments, nonionic azo dyes readily undergo biologically mediated reduction to the corresponding amines. Generally, during the anaerobic process, 60–70% reduction in COD can be achieved. The presence of a competitive electron acceptor may be a rate-limiting factor. Bras et al. (2001) have described the behavior of methanogenic and mixed bacterial cultures on color removal of a commercial azo dye, Acid orange 7. Low redox conditions maintained by the methanogenic cultures are supposed to be responsible for color removal (Beydilli et al., 1998). However, Chinwetkitvanich et al. (2000) did not find any relationship between oxidation-reduction potential and color removal. Manu and Chaudhary (2002) investigated anaerobic decolorization of textile wastewater containing azo dyes, Acid orange 7, and Reactive black 3HN. Color removal of >99% and COD removal of 92–95% was achieved, and salts present in textile wastewater inhibited methanogenesis to a limited extent. Recently, Kapdan and Oztekin (2003) reported over 90% decolorization efficiency up to 350 mg/L dyestuff concentration and 200 ml/h feeding rate of textile dyestuff Reactive orange 16 in fed-batch culture


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when using a facultative anaerobic bacterium consortium called PDW. The addition of 3 g/L yeast extract improved the color removal efficiency to around 95%. In anaerobic treatment azo reduction is achieved, but mineralization does not occur. Toxic amines are produced in the environment, and therefore, careful monitoring of the wastewater effluent is required before release into waterways. An advantage of this system could be the simultaneous generation of biogas, which could be recovered to provide heat and power to reduce energy costs. To overcome the problem of the relative recalcitrance of azo dye breakdown products under anaerobic conditions, a sequential or simultaneous two-stage anaerobic/aerobic system could be used. A biofilm-based reactor possibly may be able to completely mineralize contaminant dyes in industrial effluents. D. COMBINED ANAEROBIC/AEROBIC BIODEGRADATION It has been repeatedly suggested that aromatic amines formed during anaerobic cleavage of the azo dyes could be further degraded in an aerobic treatment system. The feasibility of this strategy was first demonstrated for the sulfonated azo dye mordant yellow-3 (Glasser et al., 1992). Haug et al. (1991) showed that under anaerobic conditions, mordant yellow was reduced by the biomass of a bacterial consortium grown aerobically with 6-amino napthalene-2-sulfonic acid. After reaeration, these amines were completely mineralized by the culture. This system was believed to be useful for the treatment of azo dyes containing wastewater because under anaerobic conditions it reduces a wide range of azo dyes and aerobically oxidizes many different amino napthalene sulfonic acids. The anaerobic/aerobic treatment can be carried out either sequentially or simultaneously. Sequential processes may combine the anaerobic and the aerobic step, either alternately in the same reaction vessel or in a continuous system in separate vessels. The simultaneous treatment systems utilize anaerobic zones within basically aerobic bulk phases, such as observed in biofilms, granular sludge, or biomass immobilized in various matrices (Field et al., 1995; Jiang and Bishop, 1994; Kudlich et al., 1996; Tan et al., 1999). In the sequential and simultaneous treatment systems, auxiliary substrates are required that supply the bacteria in the anaerobic zones with a source of carbon and energy and a source of reduction equivalents for the cleavage of the azo bond. Chang and Lin (2000) studied a

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P. luteola strain possessing azoreductase activity to decolorize azo dye, Reactive red-22, with fed-batch processes consisting of an aerobic cell growth stage and an anaerobic fed batch decolorization stage. Tan et al. (1999) studied the degradation of two azo dyes in batch experiments where anaerobic and aerobic conditions were integrated by exposing anaerobic granular sludge to oxygen. The study indicates that aerobic enrichment cultures developed on aromatic amines combined with oxygen tolerant anaerobic granular sludge can potentially be used to completely degrade azo dyes under integrated anaerobic and aerobic conditions. A sequential anaerobic-aerobic treatment process based on mixed cultures of bacteria isolated from textile dye effluent-contaminated soil was used for degradation of sulfonated azo dyes Orange G, Amido black 10B, Direct red 4BS, and Congo red (Rajaguru et al., 2000). In a fixed-bed column using glucose as co-substrate, amines produced by reduction of azo dyes were completely mineralized in a subsequent aerobic treatment. The degradation rates achieved for different dyes ranged from 60.9 mg/d to 571 mg/d. In another anaerobic-aerobic sequencing batch reactor, 60–70% removal of azo-reactive dye by polyphosphate- and glycogen-accumulating organisms was obtained within the first 2 h of the anaerobic stage (Panswad et al., 2001). Different reactor designs have been proposed for effective anaerobic/ aerobic treatment systems for azo dyes, including a system of anaerobic and aerobic rotating biological contactors, anaerobic fixed-film fluidized bed reactors and aerobic activated sludge reactors, a system of anaerobic and aerobic rotating drum reactors, and anaerobic up-flow fix bed columns and aerobic agitated reactors (O’Neill et al., 2000; Rajaguru et al., 2000; Stolz, 2001). However, it is difficult to compare these systems because of differences in the dyes and conditions, the presence of auxiliary carbon sources, and the difficulty of analysis of biological or spontaneous reactions. In continuous anaerobic/aerobic systems, fed with high BOD/COD substrates and low concentrations of dye, a complete decolorization of dyes and significant reduction of BOD and COD can be achieved in the anaerobic stage (Stolz, 2001). Encouraging results have also been obtained in laboratory experiments that demonstrated that the anaerobic breakdown of azo dyes results in products that are significantly more available for subsequent aerobic processes. This observation has formed a basis for a fullscale anaerobic/aerobic treatment plant for the treatment of more than 1000 m3 of dye-containing wastewater per day from the textile processing industry (Krull et al., 2000).


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IV. Enzymatic Methods Oxidative enzymes represent an attractive option for wastewater treatment, have been used for effluents from pulp and paper mills, and have potential application in treatment of dye-contaminated wastewater (Duran and Esposito, 2000; Karam and Nicell, 1997). Laccases (EC 1.10.3.2) are copper-containing glycoproteins that require O2 to oxidize phenols and aromatic amines as well as nonphenolic organic substrates by one-electron abstractions resulting in the formation of H2O and reactive radicals that undergo further depolymerization, repolymerization, demethylation, dehalogenation, or quinone formation (Alexandre and Zhulin, 2000; Thurston, 1994). Decolorization and detoxification of azo, triphenylmethane, and anthraquinonic dyes by laccases from Pyricularia oryzae, Pycnoporous sanguineus, Trametes hirsuta, and Sclerotium rolfsii have been reported (Abadulla et al., 2000; Muralikrishna and Renganathan, 1995; Pointing and Vrijmoed, 2000; Ryan et al., 2003). The broad substrate specificity of laccases can be further extended by addition of redox mediators (Claus et al., 2002). Laccases from the lignin-degrading basidiomycetes T. versicolor and Polyporus pinisitus and the ascomycete Myceliophthora thermophila were found to decolorize synthetic dyes to different extents. The addition of the redox mediator 1-hydroxybenzotriazole further improved the decolorization activity of laccase. In the presence of bentonite or by immobilized system, laccase decolorized both individually and in complex mixture. A commercial laccase formulation containing laccase, a redox mediator, and a nonionic surfactant was used for decolorization of Remazol brilliant blue R (RBBR), an important class of recalcitrant anthraquinone-type dye (Soares et al., 2001). Interestingly, laccase alone did not decolorize RBBR and a small molecular weight redox mediator was necessary for decolorization to occur. Purified laccase has been used to transform novel synthetic disazo dyes (Soares et al., 2002). A laccase isolated and purified from the culture filtrate of edible mushroom Lentinula edodes, was effective in decolorizing various chemically different dyes such as EBBR, bromophenol blue, methyl red, and naphthol blue black without any redox mediator, whereas Reactive orange 16 and red poly(vinylamine) sulfonate anthrapyridone dyes did require some redox mediators (Nagai et al., 2002). A catalase-peroxidase from the alkalothermophilic Bacillus sp. SF effectively treated the textile bleaching effluent both in free and immobilized form (Fruhwirth et al., 2002). An extracellular peroxidase from Streptomyces chromofuscus A11, capable of oxidizing azo dyes, showed

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substrate specificity similar to Mn-peroxidase from P. chrysosporium and horseradish peroxidase (Pasti-Grigsby et al., 1996). Enzymatic approaches to dye decolorization offer a rapid treatment method for dye wastewater. However, scale-up studies and careful economic evaluations are required before this could be applicable at the industrial scale. V. Conclusion Knowledge of physiological and genetic characteristics, biochemical capabilities, and ecology of the relevant microbial species and consortia is an essential prerequisite for successful cleaning up of dyecontaminated water bodies, irrespective of the nature of the dye or the choice of biological treatment system used. Continuous effluent treatment methods using combined anaerobic/aerobic systems for complete removal of dye compounds show some potential. Mixed culture consortia that are capable of surviving in effluents by utilizing the constituents as sources of carbon, energy, and nitrogen would make the process economically feasible. Advances in molecular techniques can help create microbes with improved metabolic capabilities by cloning the gene(s) coding for the decolorizing enzyme(s) into suitable expression systems under strong promoters. Although a robust laccase/ mediator enzyme system is a suitable biocatalyst for rapid treatment of effluents from textile, dye, or printing industries, more studies are required to characterize the nature of reaction products. Moreover, the scale-up of enzyme-based processes needs to be further researched. Moreover, the higher volumes of effluents generated by larger dye plants need to be considered where physical methods of adsorption, extraction, and concentration of dyes and other pollutants may be required before a biological method is feasible (Robinson et al., 2001a). In an integrated system, dye-adsorbed agricultural residues can be further fermented by using white-rot fungi for animal feed or as fertilizer or soil conditioner. ACKNOWLEDGMENTS We thank Manoj Kumar and Jing Ye for their help in preparing the manuscript.

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Extracellular Glycosyl Hydrolases from Clostridia WOLFGANG H. SCHWARZ,* VLADIMIR V. ZVERLOV,{ {,} AND HUBERT BAHL *Technical University of Munich Institute of Microbiology, D-85350 Freising, Germany {

Russian Academy of Science, Institute of Molecular Genetics 123182 Moscow, Russia {

University of Rostock, Institute of Biological Sciences Department of Microbiology, D-18051 Rostock, Germany }


I. Introduction II. Modular Structure of the Enzymes III. Function of Noncatalytic Modules A. Substrate Binding B. SLH Module C. Fibronectin Type III Module IV. Characterization of Enzyme Systems A. Starch Degradation B. Cellulose Degradation C. Xylan and Hemicellulose Degradation V. Concluding Remarks References

215 217 218 219 223 224 225 228 229 247 251 252

I. Introduction Anaerobic bacteria are key players in the fate of rotting biomass. They play a major role in the digestion of biomass by herbivores and insects (such as termites), possibly even as endosymbionts of flagellates common in the intestinal tract of plant-feeding animals, such as the rumen of cattle. The hosts help by mechanical degradation (chewing) and by providing a favorable environment. A part of the natural rotting process of biomass in soil and compost heaps is also performed by the anaerobic bacteria when the easily degradable constituents (e.g., soluble sugars and proteins) of the biomass are already used up. Among the anaerobic bacteria are specialists for the degradation of the insoluble components of biomass that are most difficult to degrade: crystalline starch, hemicellulose, and cellulose. In nature, polysaccharide-degrading bacteria thrive in symbiotic relationships with secondary microorganisms (Ljungdahl and Eriksson, 215 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 56 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2164/04 $35.00

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1985). The enzymes secreted by the primary cellulose degraders break the substrate down into cellodextrins, cellobiose, and glucose, only a part of which is assimilated by the polymer-degrading strains themselves. The rest is utilized by the secondary microbial flora, as are the fermentation products of the anaerobic cellulose degraders: hydrogen, carbon dioxide, alcohols, and short-chain fatty acids. Thus polysaccharide degradation is just the first step in a food chain within a complex ecosystem. Approximately 1.8 1012 metric tons of biomass (dry weight) exist on the continents, which are continuously recycled by enzymatic processes. Polysaccharides from plant material form a major part of the biomass: They are the most important factors in the carbon cycle in nature that regulates the CO2 content of the atmosphere. An estimated 40 GT per year alone of cellulose are produced by land plants—about the same amount is degraded. The natural rotting process is catalyzed by hydrolytic enzymes produced from ubiquitous microorganisms. The energy contained in the resulting sugars drives the build-up of micro- and macrobiotic biomass. But the energy gradient from polysaccharide to CO2 can also be exploited for industrial purposes without increasing the CO2 content in the atmosphere: biomass, through burning or enzymatic hydrolysis, is a CO2-neutral source of environmentally friendly energy for the future. Anaerobic bacteria, among them primarily the clostridia, are an excellent source for hydrolytic enzymes able to hydrolyze polysaccharides in biomass to fermentable sugars. An example of special interest is the utilization of the hydrolytic extracellular enzymes of the solventogenic bacterium Clostridium acetobutylicum for the fermentation of starch to the organic solvents butanol and acetone (Du¨rre, 1998; Gapes, 2000). Although, because of economic reasons, the industrial process at present is not utilized in the Western world, it is still a very attractive alternative to the mineral oil–based production of energy and bulk chemicals, since it runs with renewable substrates, enabling sustainable energy production. Consequently, research on the bacterial solvent production process is going on; for example, a number of new strains degrading a wide range of polysaccharides have been isolated (Du¨rre, 1998; Montoya et al., 2001). Meanwhile, determination of the genomic sequence of C. acetobutylicum made a thorough analysis of its genes possible, and a complete cluster of genes for the expression of a cellulosome was detected (No¨lling et al., 2001). Unfortunately, only few of these genes are expressed, and hydrolysis of cellulosic substrates could not be achieved (Sabathe et al., 2002). Nevertheless, this opens the possibility that related strains may exist that express the whole operon and would then be able to produce solvents directly from cellulose. In

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addition to genetic engineering of producer organisms and new fermentation and product separation technologies, this will help to make the bacterial solvent production economically feasible in the near future. So far none of the strains used for industrial production of acetone and butanol have been able to degrade cellulose as a cheap and available substrate to fermentable sugars. However, only a few of the industrial strains have survived the shutdown of the production facilities. The rest of the valuable strains are permanently lost and cannot be tested. The search for new solventogenic strains capable of efficient lignocellulose hydrolysis is therefore going on, and research on the clostridial extracellular enzymes is an increasingly urgent necessity. This chapter will focus on the most prevalent polysaccharides present in biomass: starch, cellulose, and hemicellulose—the latter two of which are especially difficult substrates to degrade. The unique strategies of the clostridia to cope with these substrate problems are discussed. II. Modular Structure of the Enzymes In contrast to the enzymes isolated from eukaryotic organisms (mostly fungi) and aerobic bacteria, many extracellular enzymes of the anaerobic bacteria have a modular structure—that is, they consist not only of a catalytic module but of a complex arrangement of different modules: one or even more than one catalytic module(s) and in addition, noncatalytic modules. In Fig. 1, a schematic modular structure of a hypothetical clostridial glycosyl hydrolase is depicted. The catalytic module can be accompanied by one, several, or all of the following modules: carbohydrate binding (CBM), immunoglobulin (Ig)-like, dockerin (Doc), fibronectin type III (Fn3), and S-layer homology (SLH). These modules constitute independent folding units that often are covalently connected by flexible linkers such as the so-called PTS boxes (irregular stretches of hydroxy amino acids). As a consequence of the presence of several modules, these enzymes are often quite large, consisting of more than 1,000 amino acids with a molecular mass above

FIG. 1. Schematic representation of the modular structure of a hypothetical clostridial extracellular enzyme. CBM: carbohydrate-binding module; Doc: dockerin module; Fn3: fibronectin type III module; GH: catalytic module of glycosyl hydrolase family; Ig: immunoglobulin-like module; SLH: surface-layer homology module. Numbers indicate the relative position of the modules.

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100 kDa. The order of modules in a given enzyme does not follow strict rules. Noncatalytic modules may appear on the N- or the C-terminal end of the catalytic units. SLH or Doc modules are in most cases located near the C-terminus of the enzymes. In general, the noncatalytic modules may support or even modulate the catalytic activity. Some are stuffer proteins between a catalytic unit and a functionally important noncatalytic module; some are closely connected with a catalytic module and stabilize it against thermal denaturation. Binding modules are known for the substrate (CBM) or for the host cell (SLH). The catalytic modules of these enzymes hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety. Based on amino acid sequence similarities, a classification of glycoside hydrolases into families has been proposed (Henrissat, 1991): The updated list (October 2003) contains 91 families, GH1 to GH91 (Coutinho and Henrissat, 1999a). The catalytic mechanism is a general acid catalysis that requires two critical amino acid residues: a proton donor and nucleophile/base (Davies and Henrissat, 1995). The hydrolysis results in either retention or inversion of the configuration at the anomeric C-atom. The roles of the Doc, Fn3, and SLH modules are summarized in the following section, whereas the function of the Ig module has not yet been successfully addressed. The most complex enzymes are those of the extremely thermophilic cellulose degraders Cellulosiruptor cellulolyticus and the closely related Anaerocellum thermophilum, which contain a so-called multifunctional enzyme system; these are not included in this review (Bayer et al., 2000). They also belong to the order Clostridiales. Many of their cellulases and hemicellulases are composed of more than one catalytic module, connected with binding modules and stuffing peptides. Functionally related and mutually synergistic catalytic components are combined in one polypeptide chain to enhance the effectiveness of enzymatic action. This seems to be an independent evolutionary way towards an enzyme complex that combines all necessary functions in a close spatial arrangement but with more flexibility in structure and composition. The most advanced of these complexes is the cellulosome, which is described now. III. Function of Noncatalytic Modules The functionally most important and best-characterized noncatalytic module in the extracellular enzymes of the clostridia is the CBM. In recent years the SLH module was in the focus of functional analysis,


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whereas only limited knowledge exists on the function of the Fn3 module. Therefore these three modules will be described in this part of the chapter. The function of the Doc module can be found within the description of the cellulosome.

A. SUBSTRATE BINDING The interaction of enzymes with polymeric substrates is severely slowed by the limited diffusion of the enzyme as well as the substrate. This difficulty is greatly overcome by the introduction of binding modules. These are protein modules of about 35 to less than 180 amino acid residues that target the enzyme in a noncatalytic way to suitable areas of the large substrate, a single polysaccharide molecule thread as in soluble or in amorphic parts of insoluble substrates, or a bundle of insoluble substrate molecules as in crystalline cellulose (Linder and Teeri, 1997). This increases the enzyme concentration on the substrate surface and improves substrate interaction (Bolam et al., 1998). The carbohydrate binding modules (CBM) are categorized into families according to sequence homology and the consequent three-dimensional fold (Coutinho and Henrissat, 1999b). A list of the presently known CBMs with links to nucleotide and amino acid sequences and a short compilation of general information on each family is given at the CAZY server (Coutinho and Henrissat, 1999a). Some CBMs have a flat strip of aromatic amino acid residues for binding to the surface of an array of parallel substrate molecules as in crystals; others bind single substrate molecules in a pocket-like structure (reviewed in Bayer et al., 1998). The anchoring is mediated by polar residues such as asparagine or glutamine (Tormo et al., 1996). Some families can be separated into slightly different subfamilies that have the same global fold but differ in their binding abilities. An example is family CBM3, where subfamily CBM3a modules bind tightly to crystalline cellulose, whereas CBM3b modules seem to be more variable, and CBM3c modules modulate the enzymatic activity by feeding a single substrate molecule with a predefined directionality through the active site pocket of the catalytic module, leading to processive cleavage (Sakon et al., 1997). An enzyme with endoglucanase activity is consequently transformed into an exo-glucanase (or a processive endo-glucanase) by the activity of a binding module. This emphasizes the important role of the noncatalytic modules for the enzyme activity, especially for the hydrolysis at different sites on crystalline or amorphic cellulose (Carrard et al., 2000).

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It has been proposed that some CBMs may be degenerated and function as thermostabilizing modules, such as the CBM3c in C. stercorarium cellulase Cel9Z or C. thermocellum cellulase Ce19I (see below). Although it is not clear if such CBMs have lost their binding capacity, they are functionally attached to the catalytic module. This seems to stabilize the structure of the catalytic core and in some cases increases the thermostability up to 30 C (Riedel et al., 1998a). The loss of activity at high temperature on deletion of the CBM may be so drastic that the function of the module was in some cases interpreted as essential for activity through binding. Similar results have been reported for the CBM22 modules that, for example, exert a thermostabilizing effect on xylanase XynA from Thermotoga maritima but at the same time bind to xylan and -1,3-1,4-glucan (Meissner et al., 2000). The authors argue that thermostabilization is a side effect of the close association of the enzyme with its substrate binding module. Despite a very tight binding to the substrate through a CBM, the enzymes seem to diffuse laterally along the substrate molecule (Jervis et al., 1997). This was shown with fungal enzymes in impressive pictures of the crystal surface through atomic force microscopy (Lee et al., 2000). Some dynamic experiments have been performed with the family CBM3a, which is important for the effectiveness of the cellulosome of C. thermocellum. In in vitro experiments, this CBM binds the scaffolding irreversibly to crystalline cellulose and allows the cellulolytic cellulosome components to be effective without a CBM and probably with a greater freedom of movement for activity around the binding site. The binding ability can be investigated primarily by two methods (Tomme et al., 1996). In equilibrium assays, the binding protein is mixed with an insoluble substrate in a suitable buffer; after equilibration, the decrease of protein in the cleared supernatant is estimated. Alternatively, a soluble binding substrate is mixed into the gel matrix of a native polyacrylamide gel; the binding protein is subjected to electrophoresis with these gels, and retardation in comparison with other (nonretarded control) proteins is determined. However, the in vivo function of the CBM can be detected only by constructing deletion mutants of enzymes and thorough characterization of the mode of the enzymatic activity, especially the processivity in the case of the cellulases. The binding specificity is not necessarily identical with the substrate specificity of the associated catalytic module. Especially with the xylanases it is common to find cellulose binding modules. The xylanases present in the cellulosome do not have their own CBMs but


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rely on the cellulose binding capacity of the scaffoldin-linked CBM. This may have to do with the function of these enzymes that serve as supporters in biomass degradation, where xylan is associated with cellulose (see below). In contrast, a CBM6 module was ascribed a role in hemicellulose degradation by binding to insoluble xylan and several soluble polysaccharides (Sun et al., 1998). CBMs are not randomly connected with catalytic modules. A limited number of them play a role in the extracellular enzymes in clostridia, and patterns of module structures emerge that seem to be evolutionarily successful. Examples are the ‘‘themes’’ A to D of the GH9 cellulases depicted by Bayer et al. (2000), where the GH9 cellulases form a repeatedly observed pattern with noncatalytic modules: Theme Theme Theme Theme

A: B: C: D:

GH9 GH9–CBM3c Ig–GH9 CBM4–Ig–GH9

Others are given in the next list, where the preferential binding activity of the CBM families is also indicated. In connection with cellulases: CBM3a (with scaffoldin)—crystalline cellulose CBM3b (often as GH9-CBM3c-CBM3b)—crystalline cellulose CBM3c (often as GH9-CBM3c)—as thermostabilizing module and possibly for substrate feeding (binding amorphous cellulose) CBM4 (often as CBM4-Ig-GH9)—amorphous cellulose or soluble oligosaccharides CBM6—amorphous cellulose, -1,3-glucan, or xylan CBM9 (e.g., GH5-CBM9)—cellulose In connection with xylanases: CBM22—thermostabilizing module, or xylan or -1,3-1,4-glucan CBM4 (also in -glucanase Ct-Lic16A)—amorphous cellulose or soluble oligosaccharides With esterases/glycosidases: CBM6 (with esterase CE1, PL6, PL1, PL11, GH30, GH43, GH39)— amorphous cellulose, -1,3-glucan or xylan In connection with amylases: CBM 20—binding to starch CBM 26—binding to cyclodextrins

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In connection with other enzymes: CBM2 (PL, GH74)—crystalline cellulose, chitin or xylan CBM13 (GH43)—as threefold repeat (xylan binding?) The best-characterized carbohydrate binding module in starch degrading enzymes belongs to the CBM20 family. This module is also known as starch-binding domain: It consists of approximately 100 residues and has granular starch-binding activity. CBM26 has been found in enzymes with different enzymatic activities (e.g., -amylases, amylases, glucoamylases, and especially cyclodextrin glucanotransferases). The presence in the latter enzyme is consistent with the fact that CBM26 strongly interacts with cyclodextrins. In clostridial enzymes this module has been found so far only in a few cases (Table I). Other CBMs with affinity to starch have been identified in not further characterized gene products of C. acetobutylicum (Table I).

TABLE I OCCURRENCE OF CARBOHYDRATE BINDING MODULES IN STARCH DEGRADING ENZYMES FROM CLOSTRIDIAa Module CBM20

Species

Enzyme

Reference

Thermoanaerobacterium thermosulfurigenes

-amylase

Kitamoto et al., 1988

thermosulfurigenes EM1

amylase-pullulanase

Matuschek et al., 1994

cyclodextrin glucanotransferase

Wind et al., 1995

ethanolicus 39E

amylase-pullulanase

Mathupala et al., 1990

saccharolyticum

amylase-pullulanase

Ramesh et al., 1994

thermohydrosulfuricus

amylase-pullulanase

Melasniemi et al., 1990

CBM21

Clostridium acetobutylicum

CAP 0129

No¨lling et al., 2001

CBM25


-amylase


CBM26


CAC 2252


Thermoanaerobacter

CAC 2891

a Several thermophilic Clostridium species have been reclassified as members of the genera Thermoanaerobacterium and Thermoanaerobacter (Lee et al., 1993). Data taken from Coutinho and Henrissat (1999b).


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B. SLH MODULE The function of SLH modules (for surface-layer homology) in the extracellular enzymes of clostridia is not immediately obvious. SLH modules are found in proteins from phylogenetically unrelated bacteria (e.g., Gram-positive and Gram-negative bacteria) and are present in three types of proteins: surface-layer (S-layer) proteins, extracellular enzymes/proteins, and outer membrane proteins (Engelhardt and Peters, 1998). In most cases the SLH module is present in three copies of about 50–60 residues each. A single module is predicted to have the following secondary structure pattern: -helix (HI)—-sheet (S)—loop (LI)—-helix (HII)—loop (LII) (Fig. 2). The overall similarity of SLH modules in proteins from different organisms is low, but they contain at least two highly conserved motifs, a FxDV motif at the N-terminus and an TRAE motif at the beginning of the second -helix. Our data indicate that at least the TRAE motif contributes to the function of SLH modules (unpublished results). In S-layer and outer membrane proteins these modules are generally located at the N-terminus and in enzymes at the C-terminus. Their role as cell-wall targeting modules was initially proposed mostly on the basis of sequence comparison (Fujino et al., 1993; Lupas et al., 1994; Matuschek et al., 1994). Now, because of several in vitro and in vivo studies, there is strong evidence that the SLH modules indeed serve as an anchor to the cell wall for the different protein types (Brechtel et al., 1999; Lemaire et al., 1995; Mesnage et al., 1999; Olabarria et al., 1996; Ries et al., 1997). Although it was initially thought that SLH modules bind to peptidoglycan, it is now clear that the adhesion component in the cell wall is not the peptidoglycan itself but a polymer covalently linked to it (Brechtel and Bahl, 1999; Ilk et al., 1999; Mesnage et al., 1999; Ries et al., 1997; Sa´ra et al., 1996). Complete structural analysis has indicated that these cell-wall associated polymers are teichuronic acids (Ilk et al., 1999). Furthermore, it has been found that they are pyruvylated and that a strong correlation between the existence of SLH modules and genes involved in the addition of pyruvate to the wallassociated polymer exists in different bacteria (Mesnage et al., 2000).

FIG. 2. Consensus sequence and highly conserved motifs of SLH modules. H: -helix; S: -sheet; L: loop.

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FIG. 3. Model of the attachment of extracellular proteins to the cell surface of clostridia. The interaction of SLH modules ( ) in S-layer proteins, enzymes, and functional proteins with polymers ( ) associated with the peptidoglycan (PG) is illustrated. Enzymes can be attached to the cell wall via SLH modules either directly, mediated by a linker protein, or as part of a multienzyme complex. CM: cytoplasmic membrane.

Thus the SLH-mediated anchoring mechanism is one of several, but highly conserved strategy bacteria have developed to display proteins on their surface. In clostridia, SLH modules have been found in S-layer proteins and in several hydrolases (e.g., cellulases, xylanases, amylase-pullulanases) (Fuchs et al., 2003; Matuschek et al., 1996). Figure 3 illustrates how SLH modules mediate the attachment of S-layer proteins, single enzymes, or multienzyme complexes to the cell wall. C. FIBRONECTIN TYPE III MODULE The Fn3 module is one of three types of internal repeats found in the plasma protein fibronectin. Many animal proteins contain the Fn3 module, including extracellular, intracellular, and membranespanning proteins, and adhesion molecules. Surprisingly, Fn3-like modules are also found in bacterial glycosyl hydrolases (Little et al., 1994), including cellulases, pullulanases, and polygalacturonases from clostridia (Matuschek et al., 1996; Zverlov et al., 1998b). In


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Thermoanaerobacterium thermosulfurigenes EM1 Fn3 modules are present in the amylase-pullulanase (AmyB) and the polygalacturonate hydrolase (PglA) (Matuschek et al., 1996). Interestingly, the Fn3 modules of PglA and the exopolygalacturonate hydrolase of Erwinia chrysanthemi (He and Collmer, 1990) share a higher degree of similarity (38% identical residues) than the module of PglA and the two modules of AmyB (27% and 29% identical residues, respectively). On the other hand, the Fn3 modules of AmyB are 64% identical to the corresponding modules in the amylase-pullulanase of Thermoanaerobacter thermohydrosulfuricus E101-69 (Melasniemi et al., 1990). Therefore the Fn3 modules appear to be clustered by protein type and not by organism. Very little information is available on the function of Fn3 modules in extracellular enzymes of bacteria. It has been postulated that they serve as spacers or linkers allowing optimal interaction between the catalytic and substrate-binding modules (Little et al., 1994). In agreement with this suggestion, Watanabe et al. (1994) reported that deletion of the Fn3 module(s) located between the catalytic and substrate-binding modules of a chitinase from Bacillus circulans did not affect binding to chitin but decreased hydrolytic activity of the enzyme to colloidal chitin. Recently the first evidence of a function for Fn3 in a clostridial enzyme during hydrolysis of a polysaccharide was presented. It was shown that the two Fn3 modules of the multi-modular cellobiohydrolase CbhA of Clostridium thermocellum are able to change the surface of cellulose that had been loosened up and crenellated. That promoted hydrolysis by the catalytic domain (Kataeva et al., 2002). IV. Characterization of Enzyme Systems Polysaccharides are difficult substrates for enzymes. They are usually larger than the enzyme itself, and quite often they are not soluble (i.e., they are not hydrated or occur in tight aggregates or even in crystalline form). Moreover, many natural polysaccharides such as hemicelluloses are extremely heterogeneous and contain many different sugar moieties with different linkage types, or they are derivatized. Others may contain only one type of sugar moiety, which, however, as in starch, are linked in different ways (1->4 or 1->6). Others are chemically homogeneous (such as cellulose: -1,4-glucosidic linkages only) but are at least partially crystalline and topologically diverse. Long substrate molecules can be shortened by a statistical hydrolytic cut in one of the many linkages between the building blocks of the substrates. This is the so-called endo-mode of action. The sites for such

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enzymatic attacks may be extremely limited because of occlusion by other molecules, by the viscosity of the polymer solution, or by the tight assembly of many molecules (e.g., by crystal formation). Only freely accessible, hydrated parts of a long molecule can be recognized by enzymes, and it could be postulated that even within the same polymer strand different enzyme types are needed to hydrolyze different topologies. The substrate size poses the problem of diffusion: It is not the substrate that tumbles around until it finds an enzyme pocket, but the enzyme must find its substrate. The diffusion of such large molecules is slow, and hence it is an advantage to stick to the substrate and degrade it successively once a large substrate molecule is found (see the previous section on binding modules). The sequential, processive action is executed by the exo-mode enzymes, which recognize either a reducing or a nonreducing end of the substrate molecule and feed it through the activity pocket, chopping off a monomer (e.g., -glucosidase), a dimer (e.g., -amylase, cellobiohydrolase), or a multimer (several exoglucanases). A synergism between endo-glucanases that produce the open ends, and exo-glycanases and processive enzymes that widen the gap, has been observed (i.e., the sum of the single activities is smaller, as if both types of enzymes act in combination simultaneously). Another synergism exists between the exo-glycanases active from the reducing and the nonreducing end: they can be thought to act from one open cut in a long molecule into both directions, opening a hole (e.g., in the surface of a cellulose crystal). Polysaccharides are degraded by extracellular enzymes (sometimes also called ‘‘depolymerases’’). The resulting monosaccharides or oligosaccharides are either taken up by the cell or degraded extracellularly by secreted glycosidases. Examples are discussed below. Commonly, the potential of a bacterial strain to produce extracellular enzymes is evaluated by assaying the cell free culture supernatant for enzymatic activities. This was done, for example, in an effort to compare the thermophilic polysaccharide hydrolyzing bacteria C. stercorarium and C. thermocellum for enzymes in the culture supernatant (Table II). The cellulolytic activities of these two species were comparable, but C. stercorarium culture supernatant had a higher soluble activity for mixed-linkage glucan and xylan, and especially for glucoside, xyloside, and arabinoside. This can be taken as an indication for a higher activity on hemicellulose in C. stercorarium. The higher cellobiosidase activity of C. thermocellum corroborates its specialization for cellulose as substrate, since the major components of cellulases are cellobiohydrolases that are active on the cellobioside.

227

EXTRACELLULAR CLOSTRIDIUM ENZYMES TABLE II COMPARISON BETWEEN TWO THERMOPHILIC, SACCHAROLYTIC CLOSTRIDIA

Substrate

C. thermocellum (mU/ml)a

Microcrist. cellulose

2

Phosphoric acid swollen cellulose

C. stercorarium (mU/ml)a 2

13

10

140

120

1,3-1,4--glucan (lichenan)

6.500

12.000

Arabino-xylan

3.000

20.000

Carboxymethyl cellulose (CMC)

pNP--glucopyranoside pNP--cellobioside

1,3 12

7 1,7

pNP--xylopyranoside

0,3

2

pNP--arabinofuranoside

1,3

21

a

Activity in cell free culture fluid (grown on cellobiose).

However, the results of such assays have to be interpreted with great care: 1. A great portion of the exo-enzymes in C. thermocellum are located on the cell surface, such as the cellulosome complex and some single enzymes such as the -1,3-glucanase Lic16A (Fuchs et al., 2003); the same is true for one xylanase of C. stercorarium (see below); their activity escapes the assay. 2. Many enzymes are not specific (e.g., -glucosidases are also active on cellobiosides and xylosides). 3. Xylanases of GH10 have high activity on mixed-linkage glucans. Thus, information from the activity of culture supernatants on model substrates alone is not sufficient to estimate the hydrolytic potential of a given bacterium. Nevertheless, C. stercorarium was identified as a thermophilic bacterium with a more general activity on polysaccharides present in biomass, whereas C. thermocellum is known as a specialist for the degradation of cellulose, despite the presence of a number of other enzymatic activities for -1,3-glucan, xylan, mannan, pectin, chitin, and probably other polysaccharides. Valuable information on a complete catabolic pathway of a bacterium comes from fermentation experiments; if a given polymeric substrate can be depolymerized into oligosaccharides and further to monomeric sugars, transported into the cell and metabolized, the complete set of at

228

SCHWARZ et al.

least the hydrolytic enzymes must be present. The presence of the genes alone as shown by gene cloning or genome sequencing is not sufficient; expression and secretion of the proteins must also occur. Furthermore, the presence of a hydrolytic gene (e.g., for an endo-xylanase) is not sufficient to prove xylan degrading activity, even if the enzyme is found extracellularly. To hydrolyze xylan, cellulose, raw starch, and a number of other natural polysaccharides, a network of enzymes is needed, a socalled enzyme system. Examples of such enzyme systems are discussed below. It is obvious from the synergism explained above and from the immobility of most polymeric substrates that a high local concentration of all enzyme components necessary for the substrate degradation is needed for efficient hydrolysis. Two strategies are possible: 1. To increase the concentration of all enzyme components in the medium, or 2. To combine the necessary components in a complex and to add a binding module for the substrate to hold the complex on the substrate once it has found it. Both possibilities are realized by clostridia as is explained with the cellulase enzyme systems below. An intermediate possibility is found with the species Caldicellulosiruptor and Anaerocellum, where large proteins are secreted containing several catalytic and noncatalytic modules in one polypeptide chain. The cellulosome of the clostridia, a protein complex on a scaffolding protein, seems to be the more elegant and flexible solution. A. STARCH DEGRADATION Starch is an abundant polymer in plant biomass and consists of two components: amylose, a linear polymer of -1,4-linked glucose residues, and amylopectin, a branched polymer in which amylose chains are connected via -1,6-linkages. The relative distribution of amylose and amylopectin in a starch molecule and the degree of branching depends on the source of starch. Complete degradation of starch is achieved by endo-acting (-amylase) and exo-acting (-amylase and glucoamylase) enzymes. Enzymes that hydrolyze the -1,6-linkages are named pullulanase or debranching enzymes. Maltose and short oligosaccharides produced during primary hydrolysis are converted to glucose by -glucosidase. Starch is a good substrate for most of the saccharolytic clostridia, and all types of starch hydrolases have been found among them (Mitchell, 2001). Nevertheless, the knowledge


229

about starch-degrading enzymes from clostridia is limited. In contrast to the many Clostridium species able to degrade starch, there are relatively few entries in the database on hydrolases of GH13 (-amylase, pullulanase, cyclodextrin glucanotransferase), GH14 (-amylase), or GH15 (glucoamylase) (Coutinho and Henrissat, 1999b). Furthermore, although starch was one of the preferred substrates for the industrial acetone-butanol fermentation by C. acetobutylicum, very little is known about its starch-degrading enzyme system (Gerischer and Du¨rre, 1988; Paquet et al., 1991). Sequencing of the genome has identified a few genes related to starch degradation in this important organism (Table I; No¨lling et al., 2001). Thermophilic species and their thermostable enzymes have attracted particular interest (Antranikian, 1990). In some of these organisms, which later were reclassified as members of the genera Thermoanaerobacter and Thermoanaerobacterium, a novel type of pullulanase, which hydrolyzes -1,4- and -1,6-linkages, was identified (Spreinat and Antranikian, 1990). Other enzymes that hydrolyze both types of linkages are the glucoamylase and the -glucosidase of C. thermosaccharolyticum and of C. beijerinckii (Albasheri and Mitchell, 1995; Ganghofner et al., 1998; Specka et al., 1991). In addition, synergistic action of pullulanase and -amylase (cyclodextrin glucanotransferase) has been observed (Spreinat and Antranikian, 1992). In Fig. 4, the action of enzymes from clostridia and related bacteria on a starch molecule is illustrated. B. CELLULOSE DEGRADATION Cellulose is a completely insoluble, partially nonhydrated, and crystalline substrate that poses special difficulties for enzymatic hydrolysis. Although it is a chemically homogeneous, unbranched polymer of -1,4-linked glucopyranose residues, it is structurally heterogeneous. Only a very small fraction of the substrate molecules on the surface or in amorphic regions of the crystal are susceptible to immediate enzyme attack. The current understanding of enzymatic cellulose hydrolysis is as follows: an endo-glucanase binds with its attached binding module (CBM) to the surface of a substrate bundle, opens the cellulose molecule at one of a few accessible sites and consequently produces a new reducing and a nonreducing end. The endo-glucanase stays bound near this site and may open other available cellulose chains in reachable distance. Processive glucanases (exo-glucanases) find the open ends and walk successively along the cellulose thread either from the nonreducing or the reducing end. They produce

FIG. 4. Enzymes from clostridia and related bacteria involved in the degradation of starch. Glucose units in the starch molecule with a reducing end are drawn in black; those with a nonreducing end are in grey. Data taken from Coutinho and Henrissat (1999a).


231

cellobiose (cellobiohydrolases) or cellotetraose (processive endo-glucanases), depending on the enzyme type (Reverbel-Leroy et al., 1997), widen the gap, and expose another layer of cellulose chains on the surface of the crystal, which may in turn be attacked by endo-glucanases. The cellodextrins produced are transported into the bacterial cells, where they are hydrolyzed by -glucosidases to glucose or, energetically more favorable, cleaved phosphorolytically by phosphorylases to glucose-1-phosphate. All cellulases cleave a -1,4-glucosidic bond by a hydrolytic reaction (hence ‘‘-1,4-glucanases’’). It is the same type of chemical reaction that takes place, but the mode of attack differs: cellulases may be endoor exo-glucanases (cellobiohydrolases or processive endo-glucanases); exo-glucanases may be active from the reducing or the nonreducing end of the molecule. Only a combination of enzymes with a different mode of action works synergistically and degrades the crystalline substrate effectively (Barr et al., 1996). Some enzymes hydrolyze the glucosidic bond by an inverting mechanism, others by a retaining mechanism (Davies and Henrissat, 1995). However, this difference in the hydrolytic mechanism does not seem to play a role in action modes; neither is the basic fold of an enzyme important, which is reflected in the glycosyl hydrolase family (GH family) to which a catalytic module is assigned (Coutinho and Henrissat, 1999a,b). Some of the GH families contain exo- as well as endo-glucanases (e.g., GH5, GH9). The endo- or exo-mode of a given enzyme is determined by the depth and the accessibility of the active site pocket; the processivity seems to be a function of attached substrate-binding modules and their orientation towards the hydrolytic center of the catalytic module (Barr et al., 1996; Bayer et al., 2000). At least in some enzymes the direction of the processivity could be explained by the way the substrate is bound and released and not by the gross structure of the protein backbone (Parsiegla et al., 2000). An especially high synergism between bacterial cellulases has been described between enzymes of GH48 and GH9 (Riedel et al., 1997), which are present in all cellulase enzyme systems known so far in bacteria. The synergism is higher with higher enzyme concentration, (i.e., the vicinity of proteins of both types is a crucial factor). By placing two enzymes close to each other, the cellulolysis can be optimized. Many clostridia reach this goal by packing enzymes of suitable types together in a cell- or substrate-bound huge enzyme complex, the cellulosome. One species secretes the enzymes separately as a ‘‘soluble’’ enzyme system. Examples for both types are given next. Interestingly, cellulosomes have been found exclusively in clostridia and some closely related Lachnospiraceae, such as Butyrivibrio and

TABLE III BACTERIA PRODUCING THE EXTRACELLULAR PROTEIN COMPLEX, THE CELLULOSOME Taxonom. group Clostridiaceae

Lachnospiraceae

a

Genus Clostridium

Species

Temp.

acetobutylicum

M

Source Sewage

Reference Sabathe et al., 2002

cellulovorans

M

Wood fermenter

Tamaru et al., 2000

cellobioparum

M

Rumen

Lamed et al., 1987

cellulolyticum

M

Compost

Be´laich et al., 1997

josui

M

Compost

Kakiuchi et al., 1998

papyrosolvens

M

Paper mill

Pohlschro¨der et al., 1995

thermocellum

T

Sewage, soil

Lamed et al., 1987

Acetivibrioa

cellulolyticus

M

Sewage

Ding et al., 1999

Bacteroidesa

cellulosolvens

M

Sewage

Lamed et al., 1991

Butyrivibrio

fibrisolvens

M

Rumen

Berger et al., 1990

Ruminococcus

albus

M

Rumen

Ohara et al., 2000

flavefaciens

M

Rumen

Aurilia et al., 2000

succinogenes

M

Rumen

Fields et al., 2000

Unambiguous assignment to the Clostridiaceae due to the 16S rDNA sequence in phylogenetic tree construction by ARB. Temperature: M, mesophilic; T, thermophilic.


233

Ruminococcus strains (Table III). Other species have been sporadically reported but were so far not proven by genetic data. 1. Clostridium stercorarium: A Soluble Cellulase System Only two enzymes that hydrolyze cellulose could be isolated from the culture supernatant of C. stercorarium NCIB 11764, the type strain (Bronnenmeier et al., 1990, 1991). The search for other components in culture supernatants was not successful (Bronnenmeier, personal communication). Extensive screening of genomic libraries in cosmid and bacteriophage Lambda vectors also revealed not more than two genes involved in the production of cellulases: celZ and celY (Schwarz et al., 1989). These genes coded for the cellulases Cel9Z, an endo-glucanase with some exo-glucanase activity, and Cel48Y, an exoglucanase. The gene products were identical to the previously isolated Avicelases I and II, respectively (Bronnenmeier et al., 1997; Jauris et al., 1990). Both enzymes are modular proteins consisting of an N-terminal catalytic module, a CBM3c module, and a binding module of family 3b with high affinity for crystalline cellulose (CBM3b) (Fig. 5). In both enzymes the presence of a cellulose binding module enhances the local concentration of the enzymes on the substrate surface and is necessary for the activity on the solid substrate. The arrangement CBM3c-CBM3b occurs in both enzymes. In addition, both modules in inverse order are located downstream in Cel9Z, resulting in the order CBMc-b-b-c (Jauris et al., 1990). The CBM3c module adjacent to the catalytic module of Cel9Z is essential for the enzymatic activity at elevated temperature and has no experimentally

FIG. 5. Structure of the cellulase cluster in the Clostridium stercorarium genome. The order and approximate size (numbers) of the genes celY and celZ, the direction of transcription (arrows), and the module architecture of the cellulases is indicated.

234

SCHWARZ et al.

detectable binding activity (Riedel et al., 1998a). It may function, however, in feeding the emerging cellulose molecule into the active site pocket and thus determining the orientation of the processive activity of the enzyme, as described previously. No indication for a cellulosomal structure could be found for the cellulolytic activities of C. stercorarium: (1) both isolated enzymes were ‘‘free’’ exo-enzymes and could be purified as single proteins from the culture supernatant; (2) despite the isolation of dozens of glucanase clones, no gene other than celY and celZ was obtained (unpublished observation); and (3) none of the genes for extracellular enzymes cloned so far from C. stercorarium contained the dockerin module, which is typical for all hitherto identified cellulosome components (see below). Nevertheless, the two cellulases, Cel48Y and Cel9Z, constitute a functionally complete enzyme system in which both components are essential for the hydrolysis of crystalline cellulose. The combination of exo- and endo-glucanases is typical for the soluble enzyme systems of the cellulolytic bacteria. Moreover, GH9 and GH48 enzymes are the major components in all bacterial cellulase systems. Cel48Y and Cel9Z show a distinct synergistic interaction in the degradation of microcrystalline cellulose, which is dependent on the ratio of the two enzymes and on the type of the cellulosic substrate (Riedel et al., 1997). The synergism depends on the simultaneous presence of both enzymes and is not expressed by sequential addition of the two activities. To investigate this synergism further, a bifunctional hybrid, Cel48YCel9Z, was constructed with the structure GH48-CBM3c-CBM3b-GH9CMB3c (Riedel et al., 1998b). The large fusion protein (170 kDa) was expressed in E. coli and purified. It exhibited endo- as well as exoglucanase activity, and it retained the thermostability of the parent enzymes. But its cellulolytic activity was threefold to fourfold higher than the sum of the individual enzyme activities, underscoring the effect of packing two catalytic activities physically together. A natural hybrid enzyme, CelA, with a similar structure was identified in the extremely thermophilic, nonclostridial bacterium Anaerocellum thermophilum (Zverlov et al., 1998a). It also consists of GH9 and GH48 modules connected to CBM3 modules. It is able to hydrolyze microcrystalline cellulose. Both catalytic modules showed sequence identities of about 70% to the C. stercorarium cellulases Cel48Y and Cel9Z, respectively, and were active if expressed separately as recombinant proteins. C. stercorarium so far is the only Clostridium species shown to have a soluble cellulase system comparable to that of other bacterial genera,


235

especially the actinomycetes, and does not possess a cellulosome. Other cellulolytic clostridia have not been investigated in such detail (Schwarz, 2003). Whether C. stercorarium, similar to C. acetobutylicum, lost a cellulosome during evolution and became a hemicellulose specialist or if it simply did not acquire the cellulosomal genes can only be determined by genomic sequencing. The reduction or development to the simplest known cellulase systems with only two components is astonishing. This is a singular observation among the bacteria and could be an encouraging model for an industrial cellulase preparation. However, cellulose hydrolysis in C. stercorarium is comparatively slow and incomplete, although it allows growth of the cells on pure cellulose as sole carbon source. C. stercorarium feeds the cello-oligosaccharides into its catabolism by phosphorylation through the cellobiose phosphorylase CepA and cellodextrin phosphorylase CepB, rather than by the energy-wasting hydrolytic -glucosidase action (Reichenbecher et al., 1997). These enzymes can be isolated from the cell extract and seem to be located intracellularly. 2. Clostridium thermocellum Cellulosomes The cell-free culture supernatant of C. thermocellum contains a cocktail of enzymes with high cellulolytic activity on crystalline cellulose (Fig. 6, see color insert). Dependent on the growth phase of the bacterial culture, the majority of this activity is cell bound. Whether

FIG. 6. (Left) Hydrolysis of crystalline cellulose by C. thermocellum. Cellulose powder (MN300) in a thin layer of agar-medium on top of an agar plate is completely degraded around C. thermocellum colonies: the dark background is shining through. (Right) A strip of Whatman No. 1 filter paper is decomposed by a growing culture (left to right: uninoculated, culture after 1 and 2 days).

236

SCHWARZ et al.

enriched from the cell surface or from the culture fluid, there are more than 25 different extracellular enzymes visible in a denaturing electrophoresis gel. Many of these proteins are hydrolytically active on cellulose. However, the complete cellulolytic activity could not be reconstituted from single, isolated components (Bhat et al., 1994; Morag et al., 1996; Wu et al., 1987). Nevertheless, a huge multienzyme complex was isolated, which in intact form was highly active on crystalline cellulose. It was called a cellulosome and could be isolated either from the culture supernatant or from the cells (Lamed et al., 1983). A purification method called ‘‘affinity digestion’’ uses the adsorption of the cellulosomes to cellulose fibers, washing the cellulose-cellulosome complex, followed by the complete hydrolysis of the cellulose. Cellulosomes are then purified by gel filtration chromatography (Morag et al., 1992). This purification scheme makes use of the basic characteristics of the cellulosomes: They not only bind to the substrate but also to the cell surface and thus form a bridge that holds the cell on its much larger substrate. This is energetically favorable because a sufficient enzyme concentration can easily be reached on the cell surface without producing a high amount of extracellular protein. Furthermore, the enzymatic components necessary for optimum synergism stay in close proximity, and the products of hydrolysis are present in high concentration near the cell surface, ready for uptake by the enzyme-producing cells. They are not ‘‘wasted’’ for other competing bacteria (Lynd et al., 2002). The large protein complexes on the outer surface of the bacterial cells could be made visible by electron microscopy, but only after fixation with cationized ferritin (Bayer and Lamed, 1986; Madkour and Mayer, 2003; Mayer et al., 1987). The attachment of the cells to the substrate via the cellulosomes was also observed (Bayer et al., 2000). Isolated, purified cellulosomes of C. thermocellum vary in size depending on the strain (from 2.0 to 6.5 Mda) and may even aggregate to large supercomplexes, called poly-cellulosomes (up to 100 Mda). Approximately 25 genes for cellulosomal genes have been isolated from genomic libraries by random screening for hydrolytic activity (for review see Schwarz, 2001). There is no proof yet for all gene products that they are actually present in the cellulosome. Moreover, a number of components surprisingly have a hydrolytic activity that apparently has nothing to do with cellulose degradation. These components degrade other polysaccharides in biomass—such as mixedlinkage -glucan, pectin, xylan, mannan or chitin—which in natural substrates wrap the cellulose crystals (Blum et al., 2000; Kurokawa et al., 2001; Spinnler et al., 1986; Zverlov et al., 1994, 2002a). This hydrolytic activity is in contrast to the lack of fermentation ability of


237

C. thermocellum for pentoses: evidence is accumulating that the cellodextrins derived from cellulose (not glucose or cellobiose) are the best substrate providing the most energy (Lynd et al., 2002). Hemicellulases seem to have an accessory function in providing access to the preferred substrate. However, it was shown recently that C. thermocellum also ferments -1,3-glucan (Fuchs et al., 2003). Gene cloning, together with immunological investigations, provided clues for the presence of noncatalytic proteins in the cellulosomes that are involved in structure-forming or other functions. Most important was the discovery of a scaffolding protein (CipA, ‘‘cellulosome integrating protein’’), the so-called scaffoldin, which has nine docking sites called cohesins (Fujino et al., 1992; Gerngross et al., 1993). The binding partner on the catalytic cellulosome components is a conserved twofold repeat of 24 amino acid residues, the dockerin (type I) (Tokatlidis et al., 1991). The dockerin sequences of the different cellulosomal genes are well conserved. Slight differences together with the differences in the sequence of the cohesins may lead to preferences of specific cellulosomal components to specific sites. This assumption is not in contrast to experimental results showing that a single cellulosomal component can bind to different cohesins (Fierobe et al., 2001). X-ray analysis of the cohesin structure revealed a flat binding area exposing surface residues for relatively unspecific interaction with the dockerins (Lytle et al., 2000; Mechaly et al., 2000; Shimon et al., 1997). The scaffoldin CipA brings together nine catalytic components in close proximity and thus may stimulate the synergism between the enzymes. However, there are still other modules besides the cohesins in the scaffoldin: a cellulose binding module (CBM3a) on the C-terminus for tight binding to a crystalline cellulose surface and a dockerin module on the N-terminus. This dockerin type II is not as closely related to the dockerins of the catalytic components as they are to each other. Type I and II dockerins bind to their complementary cohesin types but are not cross-reacting (Fierobe et al., 2001). The binding partner for the CipA dockerin was found in other noncatalytic extracellular proteins, SdbA and OlpB (and probably others), which carry S-layer homologous modules, anchoring the proteins in the bacterial cell wall, and cohesins of type II (Leibowitz and Be´guin, 1996; Lemaire et al., 1995). The role of other outer layer proteins, which were identified as reading frames in the genome of C. thermocellum, has still to be elucidated. To date, only one of the dockerin(I)-bearing components of the cellulosome was obviously noncatalytic: CseP with homology to CotH, a spore coat forming structural protein in Bacillus subtilis (Table IV;

238

SCHWARZ et al. TABLE IV LIST OF CELLULOSOMAL COMPONENTS IN THE GENOME OF C. Reading frame/function

Structurea

THERMOCELLUM

Ref.b

Structural component 1. CipA (c) scaffoldin, Cthe1933-1930

2(Coh1)-CBM3a-7(Coh1)X2-Doc2

þ (Fujino et al., 1992; Zverlov et al., in prep.)

GH2 2. Cthe1580

GH2-CBM6-Doc1

GH5 3. CelO cellobiohydrolase, Cthe1674

CBM3b-GH5-Doc1

Zverlov et al., 2002b

4. Cthe1575

GH5-CBM6-Fn3-Doc1

5. CelB endoglucanase, Cthe0374

GH5-Doc1

Grepinet and Be´guin, 1986

6. CelG endoglucanase, Cthe0885

GH5-Doc1

þ (Lemaire and Be´guin, 1993)

7. Cthe0444

GH5-Doc1

GH8 GH8-Doc1

þ (Be´guin et al., 1985; Zverlov et al., in prep.)

CBM4-Ig-GH9-2(Fn3)CBD3b-Doc1

þ (Zverlov et al., 1998b)

10. CelK cellobiohydrolase, Cthe2598

CBM4-Ig-GH9-Doc1

þ (Zverlov et al., 1999)

11. CelD endoglucanase, Cthe0968

Ig-GH9-Doc1

Joliff et al., 1986

12. Cthe1953

GH9-CBM3c-CBM3b-Doc1

8. CelA endoglucanase, Cthe0722 GH9 9. CbhA cellobiohydrolase

13. Cthe0850

GH9-CBM3c-CBM3b-Doc1

14. CelN endoglucanase, Cthe1222

GH9-CBM3c-Doc1

þ (Zverlov et al., in prep.)

15. CelR endoglucanase, Cthe1837

GH9-CBM3c-Doc1


16. CelQ endoglucanase, Cthe0300

GH9-CBM3c-Doc1

þ (Arai et al., 2001)

17. CelF endoglucanase, Cthe0382

GH9-CBM3c-Doc1

Navarro et al., 1991 (continued )

239

EXTRACELLULAR CLOSTRIDIUM ENZYMES TABLE IV (Continued) Structurea

Reading frame/function

Ref.b

Structural component 18. Cthe1308

GH9-CBM3c-Doc1

19. Cthe0727

GH9-Doc1

20. CelT endoglucanase

GH9-Doc1

þ (Kurokawa et al., 2002)

21. XynD xylanase, Cthe0688

CBM22-GH10–Doc1


22. XynC xylanase, C the0626

CBM22-GH10-Doc1

þ (Hayashi et al., 1997)

23. XynA, XynU xylanase, Cthe1161

GH11-CBM4-Doc1-NodB


24. XynB, XynV xylanase

GH11-CBM4-Doc1


25. LicB lichenase

GH16-Doc1

þ (Zverlov et al., 1994a,b)

26. ChiA chitinase

GH18-Doc1

þ (Zverlov et al., 2002a)

27. ManA mannanase, Cthe0533

CBM-GH26-Doc1

þ (Halstead et al., 1999)

28. Cthe2142

GH26-Doc1

29. Cthe1127

GH30-CBM6-Doc1

30. Cthe2333

GH53-Doc1

31. Cthe0269

GH81-Doc1

Xylanases

Other hemicellulases

Putative glycosidases 32. Cthe1665

GH39-2(CBM6)-Doc1

33. Cthe1579

GH43-CBM6-Doc1

34. Cthe0268

GH43-CBM13-Doc1

35. Cthe0484

GH43-2(CBM6)-Doc1

GH48 36. CelS exoglucanase Cthe0939

GH48-Doc1

þ (Wang et al., 1993)

GH74-CBM2-Doc1


Xyloglucanase 37. XghA xyloglucanhydrolase, Cthe2335

(continued )

240

SCHWARZ et al. TABLE IV (Continued) Structurea

Reading frame/function

Ref.b

Structural component Putative carbohydrate esterases 38. Cthe0066

Fn3-CE12-Doc1-CBM6-CE12

39. Cthe1577

CE1-CBM6-Doc1

Putative pectinases 40. Cthe2008

GH28-Doc1

41. Cthe2236

PL1-Doc1-CBM6

42. Cthe1810

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