Proteobacteria: Phylogeny, Metabolic Diversity and Ecological Effects

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MICROBIOLOGY RESEARCH ADVANCES

PROTEOBACTERIA: PHYLOGENY, METABOLIC DIVERSITY AND ECOLOGICAL EFFECTS No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

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MICROBIOLOGY RESEARCH ADVANCES

PROTEOBACTERIA: PHYLOGENY, METABOLIC DIVERSITY AND ECOLOGICAL EFFECTS

MARIA L. SEZENNA EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Proteobacteria : phylogeny, metabolic diversity, and ecological effects / editor, Maria L. Sezenna. p. ; cm. Includes bibliographical references and index. ISBN 978-1-61122-150-3 (eBook) 1. Gram-negative bacteria. I. Sezenna, Maria L. [DNLM: 1. Proteobacteria--classification. 2. Proteobacteria--genetics. 3. Proteobacteria--metabolism. QW 150] QR74.8.P76 2010 614.5'7--dc22 2010034019

Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

vii Phylogeny and Activity of Proteobacteria in Sediments from Lake Furnas Gilberto Martins, Daniel C. Ribeiro, Akihiko Terada, Barth F. Smets, António G. Brito and Regina Nogueira

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Proteobacteria Forming Nitrogen-Fixing Symbiosis with Higher Plants Encarna Velázquez, Paula García-Fraile, Helena Ramírez-Bahena, Álvaro Peixand Raúl Rivas

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Proteobacteria and the Endosymbiotic Origin of Mitochondrion Yu-Juan Zhang and Jian-Fan Wen

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Interpreting Diversity of Proteobacteria Based on 16S rRNA Gene Copy Number Konstantinos Ar. Kormas

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A Harsh Life to IndigenousProteobacteria at the Andean Mountains: Microbial Diversity and Resistance Mechanisms Towards Extreme Conditions Virginia Helena Albarracín, Julián Rafael Dib, Omar Federico Ordoñez, María Eugenia Farías

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vi Chapter 6

Chapter 7

Index

Contents Molecular Structure of a Chromate Resistance Determinant of Pseudomonas sp. Tik3 and its Expression in Different Proteobacteria Strains Sofia Mindlin, Mayya Petrova and Zhosephine Gorlenko Sulfhydryl Glycoconjugates Produced by Filamentous Sheath-Forming Members of -Proteobacteria Minoru Takeda

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143 157

PREFACE Proteobacteria are a major group (phylum) of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter, and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation. This book presents current research from across the globe in the study of proteobacteria, including phylogeny and activity of proteobacteria in sediments from Lake Furnas; proteobacteria forming nitrogen-fixing symbiosis with higher plants; proteobacteria and the endosymbiotic origin of mitochondrion; and interpreting diversity of proteobacteria based on 16S rRNA gene copy number. Chapter 1 - Sediments are sites of intense bacterial activity fostered by the presence of several organic and inorganic electron donors and acceptors that can be metabolised under either aerobic or anaerobic conditions. Among the bacterial groups populating sediments, the Proteobacteria phylum is one of the most abundant. Thus, the aim of the present work was to evaluate the contribution of Proteobacteria to nutrient (N and P) and iron cycling in sediments from the Azorean Lake Furnas (Portugal). The combination of denaturing gradient gel electrophoresis and cloning of the bacterial 16S rRNA gene fragment identified the Proteobacteria phylum as a dominant member of the sediment bacterial community from Lake Furnas. Using quantitative PCR to determine the relative amount of sediment bacteria affiliated to specific groups of Proteobacteria, it was inferred that 4.6% to 7.3% belonged to ammonium-oxidizing bacteria of the Beta-Proteobacteria phylum, Nitrobacterlike nitrite-oxidizing bacteria of the Alpha-Proteobacteriaphylum amounted to 0.3% to 6.0%, Geobacteraceae-like iron reducing bacteria of the DeltaProteobacteria phylum amounted to 1.0% to 2.4%, and Rhodocyclus-like phosphorus accumulating organisms of the Beta-Proteobacteriaphylum

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accounted for 0.2% to 0.5%. Experiments with homogenized sediments in batch conditions indicated that bacteria performing autotrophic nitrification, heterotrophic denitrification, iron-reduction, and biological phosphorus storage/release were active in sediments from Lake Furnas. Lake Furnas is an advanced stage of eutrophication despite the considerable efforts that in the last decades have been made by local authorities towards the reduction of phosphorus inputs to the lake. Conventional remediation measures focused on the amount of P adsorbed into iron oxides that are released to the water column under anoxic conditions. The present work suggested that biological P storage/release by denitrifying bacteria in sediments might as well contribute to the release of phosphorus from sediments. Future measures towards lake restoration should include in addition to the classical procedure an evaluation of the contribution of biological processes in sediments to the eutrophication problem. Chapter 2 - The ability to fix nitrogen is inherent only to prokaryotes and within them several Proteobacteria are able to establish symbiosis with legumes and the non-legume Parasponia in which structures specialized in nitrogen fixation named nodules are formed. Within Proteobacteria, the species able to induce nitrogen-fixing nodules are classified in two different classes, alpha and beta. All ―classical‖ rhizobia are alpha Proteobacteria whereas several species recently described as legume endosymbionts have been classified within the beta Proteobacteria. Although several gamma Proteobacteria have been isolated from nodules, they cannot induce nodule formation in legumes. The ecological relevance of Proteobacteria forming nitrogen fixing endosymbiosis with higher plants is related to that of the legumes which colonise very different habitats being the legume symbiosis the main source of terrestrial fixed nitrogen. In this chapter we revised the current known Proteobacteria forming nitrogen-fixing endosymbiosis with legumes. Chapter 3 - The acquisition of mitochondrion is one of the decisive steps in the evolution of the eukaryotic cell. Today, people almost have believed that mitochondrion had a bacterial endosymbiotic origin. But what kind of bacteria is the endosymbiont? With the accumulation of evidence from the studies of morphology, cell biology, biochemistry, and molecular biology, especially from the recent studies of molecular phylogeny and genomics, proteobacteria, in particular Rickettsiales or their close relatives, are proposed to be the endosymbiont. In this chapter, the authors review the progress of the studies in this field. Chapter 4 - The application of the 16S rRNA gene diversity analysis has revealed the immense microbial diversity of our planet. At the same time, and

Preface

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after of more than two decades of using this methodology along with several important improvements and new techniques, there is still no universal golden rule on how to estimate prokaryotic diversity in a natural sample, as there is in macroecology. A general assumption during studies of prokaryotic diversity is that each found 16S rRNA gene found corresponds to one cell. However, in this paper it is shown that recent genomic data reveal that this is not the case for several bacterial phyla. Since the Proteobacteria, along with the Firmicutes, are the most abundant and diverse bacterial phyla, in this paper the average 16S rRNA gene copy number is presented at the sub-phylum (α-, β-, γ-, δ- and ε-Proteobacteria), order and family level of the Proteobacteria phylum. At the sub-phylum level the average 16S rRNA gene copy number varied between 2.1±1.3 and 5.8±2.8. Since the 16S rRNA gene copy number affects the relative abundance of each proteobacterial species/phylotype found in a clone library, and subsequently the estimation of diversity, the corrected relative abundances of the found proteobacterial phylotypes were estimated in 37 clone libraries from six different natural habitats. It is suggested, that at least in the cases where Proteobacteria consist 50-75% of the clone library, the corrected abundances should be used for diversity estimations. Chapter 5 - High-altitude Andean lake (HAAL) ecosystems of the South American Andes are almost unexplored systems of shallow lakes formed during the Tertiary geological period, distributed in the geographical area called the Puna at altitudes from 3,000 to 6,000 m above sea level, and isolated from direct human activity. They present a broad range of extreme conditions which makes the indigenous microbial communities exceptionally interesting to study physiological mechanisms of adaptation to chemical and physical stresses such as hypersalinity and high levels of UV radiation. Previous work have revealed the outstanding diversity of these environments, being Proteobacteria the most extended and best represented microbial taxa within the extremophilic communities. The aim of this work is to review the microbial diversity of Proteobacteria present at the HAAL and to describe their multiple resistance properties towards the extreme factors that these microbial communities thrived in their natural environments. A special reference to the representatives of the genus Acinetobacter found at the HAAL is also presented. Due to the isolation program held at LIMLA (www.limla.com.ar) during the past four years a one-of-a-kind collection of extremophilic strains from the HAAL was assembled. HAAL microbial diversity was investigated by sampling bacterioplankton, benthonic microorganisms, microbial-mat associated microbes as well as gastrointestinal symbiotic organisms from

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flamingoes living at the lakes. Representatives of Proteobacteria has been profusely isolated from these samples, more exactly from Lakes: Azul, Verde, Negra, Vilama, Aparejos, Chaxas, Salina Grande, Socompa, Dead Man Salar, Tolar Grande, Brava, Diamante, Huaca-Huasi, all of them located above 4000 m, at the Northwest of Argentina. In addition, a more extended coverage of Proteobacteria was detected by non-culture dependent techniques (mainly DGGE), suggesting that much more efforts will be needed to isolate most novel Proteobacteria present at the HAAL. Within Proteobacteria, all the four main groups were represented in our culture collection being the Gammaproteobacteria the class with better coverage. The gammaproteobacteria strains were classified as belonging mainly to Pseudomonas Acinetobacter, Halomonas, Stenotrophomonas, Moraxella, Enterobacter, Serratia, Salinivibrio, Pseudoalteromonas, Aeromonas and Marinobacter. 16S rDNA gene sequence comparison of some isolates with the ones presented at the database indicated an identity lower that 94%, which should point out that these extremophilic communities harbour yet unraveled species. The extreme conditions suffered by these microorganisms at the HAAL made them resistant to factors present as well as not present in their natural environments. Exposure to UV-B radiation during 24 h revealed that most isolates were highly resistant: 33.3% of betaproteobacteria, 44.4% of gammaproteobacteria, 40% of alphaproteobacteria were able to survive through the whole exposition time. In addition, resistance to hipersalinity in most isolates was also observed. Interestingly, antibiotic resistance was also observed in spite of the pristinely and isolation of these lakes. In light of the great adaptability strength of the strains to changing conditions in their original environment, antibiotic resistance may be considered as a consequence of a high frequency of mutational events, which also, may be enhanced by the intense solar irradiation present at the HAAL (UV index in summer: 16- 18). A special reference can be made to the representatives of the genus Acinetobacter isolated from the HAAL. Most of these strains appeared to have multiple resistance profiles to hipersalinity, UV-B irradiation, antibiotics and even arsenic. These ―superbugs‖ can be subjected to further studies as they can be clues to discover new ways of surviving at extreme conditions, a matter that has applications in astrobiology. On the other hand, it will be very interesting to further research on these strains biotechnological potential because as extremophiles they can be source of novel bioactive compounds.

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Chapter 6 - The antibiotic resistant strain Tik3 of Pseudomonas sp. originally isolated from permafrost contains a chromosome-located composite transposon Tn5045. Molecular analysis of Tn5045 structure revealed a chromate resistance transposon as one of its constituent elements. Simultaneously it was shown that the strainTik3 is able to grow in the presence of Cr(VI). The chromate resistance transposon of P. sp. Tik3 has a similar genetic arrangement and is closely related to the transposable element TnOtChr conferring the chromate resistance of Ochrobactrum tritici 5bvl1. Both elements belong to the Tn3 family and contain a group of chrB, chrA, chrC, and chrF genes located between divergently transcribed resolvase (tnpR ) and transposase (tnpA) genes. The transposon Tn5045 was translocated onto the broad-host-range plasmid pRP1.2 and transferred to Escherichia coli. In addition the high-copy number plasmids pGEM-7Zf(-) and pAK1 containing Tn5045 were created. It was found that introduction of the complete operon chrBACF on a high- or low-copy number plasmid into genome of E.coli JF238 didn′t result in a significant increase in chromate resistance. At the same time the increased chromate resistance was detected in Acinetobacter calcoaceticus BD413 cells carrying pAK1::Tn5045. The findings indicate that expression of chromate resistance is regulated differently in different species of Proteobacteria. bacteria form sheath, a tube-like Chapter 7 - Several filamentous extracellular structure, which enfolds a line of cells. The genera Leptothrix and Sphaerotilus are phylogenetically related filamentous -Proteobacteria whose members are known as typical sheath-forming bacteria in various aquatic environments. Despite the extensive studies on taxonomic properties and ecologicalimportance of these bacteria, not much is known about the structural composition of their sheaths. The sheath of both genera is readily degraded with hydrazine, releasing amphoteric heteropolysaccharides, which is made up of pentasaccharide repeating units. In the case of Leptothrix sheath, the pentasaccharide repeating unit is composed of 2-amino-2-deoxy-galacturonic acid, galacturonic acid, galactosamine, glucosamine, whereas the pentasaccharide of Sphaerotilus sheath contains glucuronic acid, galactosamine and glucose. In addition to the sugar moieties, cysteine and glycine are found as the major amino acids in acid hydrolysates of both Sphaerotilus and Leptothrix sheaths. Structural analysis of partial hydrolysates of the sheaths indicated that the glycans are attached to oligopeptides of cysteine and glycine residues through an amide bond involving the C-terminal carboxyl group of the oligopeptides and particular amino groups in the

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pentasaccharides repeating units. The peptideside chains can be spontaneously connected by disulfide bond. The sheaths of the genera Leptothrix and Sphaerotilus are somewhat similar supermolecule constructed by association of sulfhydryl glycoconjugates, which may represent a novel glycoconjugate category.

In: Proteobacteria Editor: Maria L. Sezenna

ISBN: 978-1-61761-198-8 © 2011 Nova Science Publishers, Inc.

Chapter 1

PHYLOGENY AND ACTIVITY OF PROTEOBACTERIA IN SEDIMENTS FROM LAKE FURNAS Gilberto Martinsa, Daniel C. Ribeiroa, Akihiko Teradab1, Barth F. Smetsb, António G. Britoa2 and Regina Nogueiraa a

IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710057 Braga, Portugal. b Department of Environmental Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark.

ABSTRACT Sediments are sites of intense bacterial activity fostered by the presence of several organic and inorganic electron donors and acceptors that can be metabolised under either aerobic or anaerobic conditions. Among the bacterial groups populating sediments, the Proteobacteria phylum is one of the most abundant. Thus, the aim of the present work was to evaluate the contribution of Proteobacteria to nutrient (N and P) 1

Present address: Department of Chemical Engineering, Tokyo University of Agriculture and Technology, Naka-cho, Koganei-shi 184-8588 Tokyo, Japan. 2 Present address: ARH Norte, R. Formosa, 254, 4049-030 Porto, Portugal.

2

Gilberto Martins, Daniel C. Ribeiro, Akihiko Terada et al. and iron cycling in sediments from the Azorean Lake Furnas (Portugal). The combination of denaturing gradient gel electrophoresis and cloning of the bacterial 16S rRNA gene fragment identified the Proteobacteria phylum as a dominant member of the sediment bacterial community from Lake Furnas. Using quantitative PCR to determine the relative amount of sediment bacteria affiliated to specific groups of Proteobacteria, it was inferred that 4.6% to 7.3% belonged to ammonium-oxidizing bacteria of the Beta-Proteobacteria phylum, Nitrobacter-like nitrite-oxidizing bacteria of the Alpha-Proteobacteriaphylum amounted to 0.3% to 6.0%, Geobacteraceae-likeiron reducing bacteria of the Delta-Proteobacteria phylum amounted to1.0% to 2.4%, and Rhodocyclus-like phosphorus accumulating organisms of the Beta-Proteobacteriaphylum accounted for 0.2% to 0.5%. Experiments with homogenized sediments in batch conditions indicated that bacteria performing autotrophic nitrification, heterotrophic denitrification, iron-reduction, and biological phosphorusstorage/release were active in sediments from Lake Furnas. Lake Furnas is an advanced stage of eutrophication despite the considerable efforts that in the last decades have been made by local authorities towards the reduction of phosphorus inputs to the lake. Conventional remediation measures focused on the amount of P adsorbed into iron oxides that are released to the water column under anoxic conditions. The present work suggested that biological P storage/release by denitrifying bacteria in sediments might as well contribute to the release of phosphorus from sediments. Future measures towards lake restoration should include in addition to the classical procedure an evaluation of the contribution of biological processes in sediments to the eutrophication problem.

Keywords:Proteobacteria, sediments,bacterial diversity, activity

NOMENCLATURE amoA - Ammonia monooxygenase gene; anammox - Anaerobic ammonium oxidizing bacteria; AOB - Ammonium oxidizing bacteria; BMFC -BenthicMicrobial Fuel Cell; DGGE -Denaturing gradient gel electrophoresis; DNA- Deoxyribonucleic acid; DNB - Denitrifying bacteria;

Phylogeny and Activity of Proteobacteria in Sediments … dsrA- dissimilatory (bi)sulfite reductase, subunit alpha; Fe - Iron; IRB - Iron reducing bacteria; N - Nitrogen; NH4+- Ammonium; nirK, nirS - Nitrite reductase gene; NO2- - Nitrite; NO3-- Nitrate; NOB - Nitrite oxidizing bacteria; nosZ-Nitrous oxide reductase gene OM - Organic matter; P - Phosphorus; PAO - Polyphosphate accumulating organisms; PCR- Polymerase chain reaction; pHzpc -pH at the point of zero charge; PO43-- Phosphate; qPCR - Quantitative polymerase chain reaction; RFLP - Restrictionfragment length polymorphism; RNA - Ribonucleic acid; rRNA - Ribosomal ribonucleic acid; SRB - Sulphate reducing bacteria; SSCP - Singlestrand conformation polymorphism; TFe - Total iron; TGGE - Temperature gradient gelelectrophoresis TN - Total nitrogen; TP - Total Phosphorus; t-RFLP -terminal-restriction fragment length polymorphism

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Gilberto Martins, Daniel C. Ribeiro, Akihiko Terada et al.

1. INTRODUCTION Sediments are repositories of the overlying water body (for example, ocean, lake, river, or reservoir) composed of organic and inorganic materials (Huettel et al., 2003; Chen and White, 2004). The composition of sediments is influenced byseveral factors namely the chemistry of the overlying water, the level of primary productivity, runoff or rivers, as well as biogeochemical transformations occurring inside sediments (Huettel et al., 2003; Chen and White, 2004; Reitzel et al., 2007). They receive substantial inputs of both allochthonous material (both organic and inorganic) and autochthonous material (mainly organic) from primary productivity (Huettel et al., 2003; Chen and White, 2004).Bacteria in sediments interact with metals and minerals, which are dragged to sediments by settling of materials from runoff, changing their physical and chemical states (Gadd, 2010). Many minerals (calcium carbonates, silicates and iron oxides or sulphides) can also be products of microbial metabolism (Nealson, 1997; Gadd, 2010), as for examplemagnetite that is an end product of Fe(III)-reducing microorganisms in sediments (Lovley, 1991).

1.1. Biogeochemical Profiles in Sediments The composition of sediments in terms of organic matter (OM) (Nelson et al., 2007), total nitrogen (TN) (Haukka et al., 2006; Herrmann et al., 2009) and total phosphorus (TP) (Li et al., 2005; Zeng et al., 2009), as well as in situ physical-chemical parameters, dissolved oxygen and pH(Koretsky et al., 2006), have been reported to drive the dynamics of microbial communities in sedimentary environments. Thus, a better characterization of biogeochemical parameters in sediments combined with sediment microbial communities assessmentmay help to clarify the environmental function of microorganisms in sediments. In general, the determination of biogeochemical profiles in sediments requires sedimentsampling and execution of physical-chemical analyses (Bufflap and Allen, 1995; Laskov et al., 2007). The most common profiles obtained in the solid phase and the respective analytical methods are the OM profile determined by weight loss at ignition (Nelson et al., 2007; Zeng et al., 2009), TN and TP profiles obtained by acid/alkaline or microwave digestion (Andersen, 1976; Johnes and Heathwaite, 1992; Zeng et al., 2009), metals (Tessier et al., 1982; Kostka and Luther, 1994; Koretsky et al., 2006), and

Phylogeny and Activity of Proteobacteria in Sediments …

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individual P species profiles performed by sequential extraction(Psenner and Pucsko,1988; Ribeiro et al., 2008). Profiles in sediment pore-water usually require sampling that can be done either ex situ by squeezing or centrifugation, or in situ using vacuum filtration or dialysis (Bufflap and Allen, 1995). The amount of water sampled by these techniques is relatively small, limiting the number of analyses that can be performed. The use of microsensors shortcuts the necessity of pore water sampling, but its availability might still be limited considering the large list of water parameters to be analysed (Stockdale et al., 2009). The most common elements measured with microsensors are O2, pH, NO3-, NH4+ and H2S (Altmann et al. 2004; Nakamura et al. 2006; Preisler et al., 2007). Microsensors also allow identifying the precise localization of the thin sediment surface layer where aerobic processes take place that might be otherwise overlooked by conventional pore water analysis or mass balance studies (Ahltman et al., 2004). Voltammetric microsensors have also been used to study biogeochemical processes in sediments (Luther et al., 2008; Himmelheber et al., 2008). In a voltammetric work, current is measured while scanning the entire voltage range of the solid-state electrode, which allows the measurement of more than one species at a given time in the same region of space (Brendel and Luther, 1995). To date, O2(aq), H2O2, Fe2+, Mn2+, S2O32-, ΣH2S [H2S + HS- + S2- + S0 + Sx2-], organic-FeIII(aq), and FexSy(aq) species are the commonly elements measured using this technique (Luther et al., 2008; Himmelheber et al., 2008; Himmelheber et al., 2009).

1.2. Biogeochemical Processes Driven by Proteobacteria in Sediments Proteobacteria comprise one of the largest divisions within prokaryotes and account for the vast majority of the known Gram-negative bacteria (Gupta, 2000). Phylogenies based on 16S and 23S rRNA geneshave led to the division of the proteobacterial group into five subdivisions or subclasses that have been arbitrarily designated Alpha, Beta, Gamma, Delta, and Epsilon (Woese, 1987; Gupta, 2000). Nowadays, genetic fingerprinting techniques are routinely used to explore the diversity of microbial communities in the environment (Muyzer et al., 1993; Zak et al., 2006; Moura et al., 2009). These techniques provide a profile of the community diversity based upon the physical separation of unique nucleic acid species (Stahl and Capman, 1994). Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE),both based on differences between taxa in denaturation(chemical for

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DGGE and temperature for TGGE) of a PCR-amplified gene, have frequently been used to characterize the bacterial diversity in lakesediments(Kojima et al., 2006;Nelson et al., 2007; Qu et al., 2008;Martins et al., 2010). Restriction fragment length polymorphism(RFLP) and terminal-RFLP (t-RFLP) are other techniques that profile the microbial community composition based on differences among taxa in the length of restriction fragments of a PCRamplified gene (Zak et al., 2006) and have been used for comparing the community composition and relative abundance of sequences within a targeted microbial group (Polymenakou et al., 2005; Schwarz et al., 2007). Single strand conformation polymorphism (SSCP) is different from the community profiling techniquespreviously described, in that it is based on electrophoretic separation of the original sample RNA, rather than PCR products derived from RNA or DNA. This technique is based on conformational changes in singlestranded DNA, which result from single-point mutations that can be detected as shifts in migration time (Atha et al., 1998) and has been used for profiling microbial communitiesfrom estuarine sediments (MacGregor and Amann, 2006). Another molecular biology tool commonly applied is a metagenomic clone library that is used to screen for functional or taxonomic genes and could result in the discovery of new enzymes, metabolic pathways and organisms with impacts on biogeochemical processes (Zak et al., 2006). As an example, the almost complete assembly of Kuenenia stuttgartiensis genome, an uncultured bacterium, has revealed unique metabolic adaptations associated with anaerobic ammonium-oxidation as well as iron and manganese respiration (Strous et al., 2006). However, in a complex community, it is necessary to analyze an enormous clone library to overcome random sampling of many genomes. The application of the above mentioned techniques suggested that Proteobacteria, Bacteroidetes, Chloroflexi and Actinobacteria are the most common phyla in sediments from different environments such as shallow eutrophic lakes (Tamaki et al., 2005; Zeng et al., 2009), eutrophic reservoirs (Qu et al., 2008), meromictic lakes (Nelson et al., 2007), meso-eutrophic monomictic lakes (Schwarz et al., 2007), freshwater and brackishwater lakes (Kojima et al., 2006) and freshwater suboxic ponds (Briée et al., 2007). Other phyla currently retrieved from freshwater sediments include Acidobacteria, Deferribacteres, Firmicutes and Nitrospirae (Tamaki et al., 2005; Schwarz et al., 2007; Qu et al., 2008; Zeng et al., 2009). The oxidation of organic matter in sediments is coupled to a succession of increasingly less energetically-favourable terminal electron acceptors, namely O2, NO3-, Mn(IV), Fe(III), and SO4-2 resulting in a vertical pattern of redox


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stratification (Canavanet al., 2006; Himmelheber et al., 2009; Thomsen et al., 2004). Oxygen penetration in sediments can extend from millimeters to centimeters (Himmelheber et al., 2008). In the presence of oxygen, the ammonium derived from the ammonification of organic matter or dissimilatory nitrate reduction is oxidized to nitrate via nitrite. The oxidation of ammonium to nitrite is catalysed by ammonium-oxidizing bacteria (AOB), while nitrite- oxidizing bacteria (NOB) oxidize nitrite to nitrate; a two step process called nitrification. This autotrophic process reduces CO2 to cellular carbon. In top sediment layers, nitrifiers have to compete with heterotrophic bacteria, taking up organic carbon, for oxygen (Altmann et al., 2004; Himmelheber et al., 2009).Regarding nitrifiers within Proteobacteria, the majority of known AOB identified by a 16S rRNA or functional gene (ammonia monooxygenase,amoA) approach, have been classified in the Betasubclass of Proteobacteria, genus Nitrosomonas and Nitrosospira (Kowalchuk and Stephen, 2001; Mosier and Francis, 2008). In marine environments, the phylogeny of AOB is also related to members of theGamma-subclass of Proteobacteria, genus Nitrosococcus (Ward and O‘Mullan, 2002; Kowalchuk and Stephen, 2001). Recently, a novel ammonium-oxidizing microorganism, Crenarchaeote, within the domain Archaea has been retrieved in freshwater and marine sediments (Könneke et al., 2005; Francis et al., 2005; Mosier and Francis, 2008). So far, most studies focusing on the diversity of NOB in sediments are based on a 16S rRNA gene-based approach. NOB are phylogenetically affiliated with the genera Nitrobacter (AlphaProteobacteria), Nitrococcus (Gamma-Proteobacteria), Nitrospina (DeltaProteobacteria) and the phylumNitrospira(Nakamura et al., 2006). The nitrate resulting from the activity of nitrifiers diffuses into the nitrate reduction zone and is used as terminal electron acceptor in the oxidation of organic carbon by facultative heterotrophic bacteria, under suboxic conditions; that is oxygen concentrations below 2 mg/L (Fennel et al., 2008). This process called denitrification is done in several steps with the formation of distinct intermediates (NO2-, NO, N2O), eventually resulting in the formation of nitrogen gasas the end product. Denitrifying bacteria (DNB) are widespread among the domain Bacteria (Oakley et al., 2007), with high occurrence within Proteobacteria. Functional genes encoding nitrite reductase (nirS and nirK) and nitrous oxide reductase (nosZ) have been widely used to study the diversity of DNB in sediments(Braker et al., 2000; Smith et al., 2007; Bulow et al., 2008; Magalhães et al., 2008). nirS is normally associated to marine bacteria clones (Pseudomonas sp. and Marinobacter sp.,within GammaProteobacteria, and Thauera, belonging toBeta-Proteobacteria), while nirK is

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related to the genera Alcaligenes (Beta-Proteobacteria) and Hyphomicrobium (Yoshie et al., 2004; Osaka et al., 2006). Until now, nosZ sequences have been classified in the Alpha-subclass of the Proteobacteria (Magalhães et al., 2008). The loss of nitrogen from sedimentsis the result of the combination of nitrification and denitrification processes. An alternative path is the autotrophic nitrogen removal over nitrite mediated by anaerobic ammoniumoxidizing bacteria, designated as anammox bacteria, belonging to the phylumPlanctomycetes. Anammox bacteria are slow-growingmicroorganisms compared to DNB (Oakley et al., 2007) and, in the presence of evenlow concentration of organic matter, may be outcompeted by DNB. Nevertheless, based on experiments using a 15N stable isotope labelling technique, anammox bacteria are reportedly responsible for at least 50 % of N2 losses from sediments (Thamdrup and Dalsgaard, 2002; Dalsgaard et al., 2005), which have previously been entirely attributed to DNB (Ward, 2003). Environmental sequences of anammox bacteria are affiliated to the Candidatus Scalindua clade (Woebken et al., 2008) which primarily contains the Candidatus Scalindua sorokinii from the Black Sea, the first marine anammox bacterium reported (Kuypers et al., 2003). Deeper in the sediment, the remaining organic carbon is oxidized by iron and manganeseoxides in the metal reduction zone(Lovley, 1991, 1995; Nealson, 1997). Fe(III) is present both as amorphous and crystalline forms in sediments, but only the amorphous iron, especially FeOOH, can be reduced either biologically by the activity of iron reducing bacteria (IRB) or chemically by reaction with hydrogen sulphide (Azzoniet al., 2005; Lovley and Phillips, 1987). Most IRB are also capable of reducing manganese oxides (Lovley, 1991). IRB, distributed among the Bacteria and Archaea domains, are phylogenetically and physiologically diverse (Lin et al., 2007; Lovley et al., 2004). Nevertheless, most of the known IRB cluster in the Delta subclass of Proteobacteria(Fredrickson and Gorby, 1996).The first microorganisms found to conserve energy from the complete oxidation of organic compounds to carbon dioxide with Fe(III) serving as the sole electron acceptor belongto the Geobacteraceaefamily in the Delta-subclass of Proteobacteria and are abundant in aquatic sediments (Lovley et al., 1987; Lovley and Phillips, 1988; Stein et al., 2001). Shewanellaspecies in the Gamma-subclass of Proteobacteriaare ubiquitous in the environment and play an important role in Fe(III) and Mn(IV) reduction as well as in the bioremediation of metal and organic contaminants in sediments (Lovley et al., 2004).


9

Dissimilatory Fe(III) reduction has practical applications such as in the bioremediation of subsurface environments contaminated with organic compounds and metals (Lovley, 1995; Petrie et al., 2003) and harvesting of electricity from aquatic sediments using the principle of the microbial fuel cell (Holmes et al., 2004; Rabaey et al., 2005; Martins et al., 2010). The benthic microbial fuel cell (BMFC) consists of an anode embedded in the anoxic sediment and a cathode suspended in the aerobic water column (Reimers et al., 2001). Bacteria in a BMFC mediate the transfer of electrons from carbon sources to the anode thus generating an electric current. The first BMFCs described in literature were associated with marine sediments due to the better ion conductivity between electrodes in saline environments (Tender et al., 2002; Bond et al., 2002). Recently, sediments from rivers and lakes were also used to operate BMFCs (Venkata Mohan et al., 2009). The main application of BMFCs described in literature is long-term power sources for autonomous sensors and communication devices (Reimers et al., 2001; Tender et al., 2002; Holmes et al., 2004; Donovan et al., 2008).Among the electrochemically active microorganisms that can transfer electrons directly from the carbon source to an anode, without the need of electroactive intermediates, the most well known bacteria are Shewanella putrefaciens, a Gamma-Proteobacterium, Geobacter sulfurreducens, Geobacter metallireducens and Desulfuromonas acetoxidans, all Delta-Proteobacteria, and Rhodoferax ferrireducens, a BetaProteobacterium (Du et al., 2007; Logan et al., 2005; Rabaey et al., 2005). As pointed out in several studies, microbial communities associated with BMFC anodes are enriched in Delta-Proteobacteria (Bond et al., 2002; Holmes et al., 2004; Cummings et al., 2003; Reimers et al., 2006). The predominance of these bacteria is dependent on the environment: Desulfuromonas species are more abundant in marine sediments, while in freshwater sediments Geobacter species predominate (Holmes et al., 2004). Phosphorus (P) is present in sediments from eutrophic lakes mainly as organic P and P bound to metal oxides, smaller amounts of labile P, calcium bound P and refractory P are also present. Sediments can act as an internal source of P to the overlying water and several biogeoprocesses have been proposed to explain this phenomenon (Hupfer and Lewandowski, 2008). Processes contributing to the dissolution of phosphate bound to metal oxides are microbial dissimilatory Fe(III) oxide reduction and chemical Fe(III) oxide reduction by hydrogen sulphide. IRB mainly of the Geobacteraceae and Shewanelanaceae families reduce Fe(III) oxides to soluble Fe(II), with the consequent release of phosphate to pore water under anoxic conditions (Azzoni et al., 2005). In sulphate-rich sediments, reduction of iron and

10


manganese oxides is done by hydrogen sulphide resulting from sulphate reduction promoting the dissolution of phosphate to pore water (Nielsen et al., 2010). The pool of non refractory organic P contributes to phosphate dissolution both via organic matter decomposition (Golterman, 2001; Rozan et al., 2002) and biological storage/release of P as polyphosphate (Poly-P) (Hupfer et al., 2008). Decomposition of organic P has been reported to occur by the activity of phosphatases excreted by some DNB, as for example Pseudomonas aeruginosa, Bacillus subtilis, Klebsiella pneumonia, Rhizobium meliloti, Mycobacterium leprae and Agrobacterium tumefaciens (Yiyong et al., 2002; White and Metcalf, 2007). Microorganisms are able to store and release P in organic-rich lakesediments, depending on redox conditions. P accumulating organisms (PAO) store P as Poly-P intracellularly under aerobic conditions (up to 20% of their dry weight) and release intracellular P via enzymatic hydrolysis when conditions turn anaerobic. Well known PAO belong to the genera Rhodocyclus (Beta-Proteobacteria), Acinetobacter (Gamma-Proteobacteria) and Tetrasphaera (Actinobacteria) (Gaechter and Meyer, 1993; Ahn et al., 2001; Tsuneda et al., 2005; Hupfer et al., 2008). It is estimated that Poly-P could represent at least 10% of total P in surficial sediments (Hupfer et al., 2007) and the density of Rhodocyclus-related PAO was reported to be around 1% of total bacteria in sediments (Gloess et al., 2007). In addition, under acidic conditions provided by the oxidation of ammonium to nitrite by the genera Nitrosomonas and Nitrosospira, P dissolution might occur from the pool of calcium bound P (Altmann et al., 2004; Nakamura et al., 2006).

2. CASE STUDY -LAKE FURNAS Lake Furnas is situated in the East side of the island of São Miguel, designated the "The Green Island", which is the largest (759 km2) and most densely populated (140,000 inhabitants) of the archipelago of Azores(Portugal). The lake is in the bottom of a large caldera, located in Furnas‘s volcanic centre (Ribeiro et al., 2008). Lake Furnas is in an advanced state of eutrophication due tothe nutrient load from intensive fertilization and livestock manure that reaches the lakefrom the watershed (Martins et al., 2008).Consequently, algae blooms and the release of phosphorus from sediments during summer are recurrent events in the lake(Santos et al., 2004; Medeiros et al., 2004). To reduce the external input of nutrients, the Regional Government has designated the lakewatershed as ―vulnerable area‖ complying


11

with Nitrates Directive 91/676/EEC and a watershed management plan was approved in 2005.

2.1. Organic Matter, Nutrients, Iron, pH and Oxygen Profiles in Sediments Typical vertical profiles of OM, TP, TN and total iron (TFe), as well as the microprofiles3 of dissolved oxygenand pHin sediments from Lake Furnas are depicted in Figure 1. Profiles of OM, TP and TN were quite homogeneous in depth. Examples of both homogeneous (Zeng et al., 2009) and heterogeneous (Søndergaard et al., 1996; Kelderman et al., 2005; Zeng et al., 2009) sediment profiles have been previously reported. A distinctive profile was obtained for TFe, where the amount of Fe reacheda maximum value below the aerobic layer, at a depth of 1.5 cm from the surface of the sediment. The accumulation of Fe resulting from the reduction of Fe(III) oxides in the absence of oxygen was attributed to the activity of IRB (Koretsky et al., 2006). Regarding the dissolved oxygenpenetration in the sediment, O2 decreased from 7.17 mg/L in the water-air interface to 0.0 mg/L at 0.25 cm below the interface sediment-water. A similar valueof oxygen penetration wasreported for stream sediments (Altmann et al., 2004; Himmelheber et al., 2009). The pH profile in the sediment from Lake Furnas, obtained at the same depth as the oxygen profile, was homogeneous with an average value of 6.42 (Figure 1).

3

Microprofiles of pH and oxygen concentration were obtained ex-situ in the sediment-water interface and in the interstitial water of the sediment from Lake Furnas. Three perspex tubes (10.0 cm depth and 4.1 cm of diameter) open on top were filled with the sediment and immersed in a parallelepiped container filled with 1.7 L lakewater. The water column was open to the air.The ratio of volume of water and area of sediment was 43 m3/m2. The microprofiles were measured with a vertical resolution of 250 m (80 measured points in the sediment). The microsensors, Ox50 (oxygen microsensor) and pH50 (pH microsensor), were operated with a level III microprofiling setup (Unisense), which includes a micromanipulator (MM33-2) with a motor controller (MC232). Data was acquired by computer using an analogical/digital converter (Unisense, ADC216).

Figure 1. Vertical profiles of organic content, nutrients (total P and total N), total Fe, dissolved oxygen and pH profiles in sediments from LakeFurnas; error bars represent standard deviations.


13

The point of zero charge1 (pHZPC) defined as the pH at which the surface charge is zero, based on zeta potential measurements of sediment particles, was pH 5.21 ± 0.05. Thus, the surface charge of sediment particles was positive at pH lower than 5.21 and turned negative at higher pH values. Thisresult suggested that at pH 6.4 the sediment from Lake Furnas was negatively charged, thereby having more affinity to cations (e.g., ammonium and Fe(III) compounds) than anions (e.g., nitrate). In most lakes, the major P input is of organic origin and is the result of lake primary production and catchment (Pettersson, 2001). Particulate organic P that reaches sediments is mineralized and the released phosphate ions are easily adsorbed into inorganic particulates (as for example, Fe and Al oxides, clay minerals with surficial Fe and Al(hydr)oxides, and possibly also Mn oxides) or stored by microorganisms (Gonsiorczyk et al., 1998). A typical P distribution in sediments from two Azorean lakes, Lake Furnas and Lake Fogo, with distinct trophic states is presented in Table 1. As it can be observed, P was mostly present in the sediment from the oligotrophic Lake Fogo as refractory/residual P, while in the eutrophic Lake Furnas organically bound P and metal oxide bound P represented around 66% of total P (Ribeiro et al., 2008; Martins et al., 2008). Table 1. Typical Values for P Chemical Forms in Sediments from two Azorean Lakes: Lake Fogo (Oligotrophic) and Lake Furnas (Eutrophic)

a

P forms

Lake Fogo ( g/g)

Lake Furnas ( g/g)

labile P

2

6

metal bound P

21

189

organic P

16

79a

Ca-bound P

18

44

refractory/residual P

53

86

Inferred based on the average organic content of sediments from Lake Furnas (11%)

1

The batch equilibration technique (Smiciklas et al., 2000) was used to determine pH at the point of zero charge (pHzpc). Approximately 0.1 g of sediment from Lake Furnas was placed into 30 mL of 0.1 M NaCl solution. Initial pH values (pHinitial) of NaCl solutions were adjusted from 2 to 12 by addition of 0.1 M HCl or NaOH. Sediment suspensions were allowed to equilibrate for 24 h in a shaker at room temperature, and the final pH values (pHfinal) were measured.

14


2.2. Phylogeny of Proteobacteria in Sediments The dominant members of the bacterial community inhabiting Lake Furnas sediments was studied by DGGE fingerprinting of a 16S rRNA gene fragment (Figure 2). The PCR-DGGE approach was performed according to Muyzer et al. (1993) and the detailed methodologyis described in Martins et al. (2010). The obtained DGGE profiles of sediment layers collected at several depths were quite similar and the bacterial diversity detected was consistent with previous published studies (Tamaki et al., 2005; Kojima et al., 2006; Nelson et al., 2007, Briée et al., 2007, Schwarz et al., 2007; Qu et al., 2008; Zeng et al., 2009).Of the 33 excised, cloned and sequenced DGGE bands, 24 had a similarity higher than 97% with previously described sequences, 7 had a similarity lower than 94%, suggesting the presence of previously undescribed bacterial diversity(Yang et al., 2001), and only 1 (F49) had a similarity lower than 91%. The phylogenetic analysis revealed that most of the bacterial 16S-rRNA gene sequences retrieved from Lake Furnas sedimentswere assigned to the phylumProteobacteria (11 sequences), the phylum Chloroflexi (5 sequences), and the group Bacteroidetes/Chlorobi (4 sequences). Sequences affiliated to Actinobacteria (1 sequences), Gemmatimonadetes (1 sequence), Cyanobacteria (1 sequence), and Candidate divisions OP1 (1 sequence) and OP11 (1 sequence) were less abundant. Within theProteobacteria phylum, 7 sequences were affiliated to the subclass Alpha-Proteobacteria, 2 sequencesin Delta-Proteobacteria and 2 sequences in Gamma-Proteobacteria. Three sequencesaffiliated to Alpha-Proteobacteria were closely related to Brevundimonas aurantiaca strain 210-31 (band E35, more than 98% similarity) and Sphingomonas sp. (bands E31 and F48, 100% similarity), known as heterotrophic bacteria, while two sequences were closely related to Nitrobacter sp. (band E30, 100% similarity) and Erythrobacter sp. NH89-70 (F53, 100% similarity) that are involved in the cycling of nitrogen. Sequences affiliated to the Delta- (I62 - Geobacter bremensis strain Dfr1, 90% similarity, and I64 - Syntrophus aciditrophicus SB, similarity higher than 92%) and Gamma(B12 -Thiocapsa sp. MTWDM061, 93% similarity, and F54 - Shewanella sp. E505-7, 93% similarity) subclasses of Proteobacteria presented a low similarity to their closest relatives. Members of the Delta- and Gamma-subclasses of Proteobacteria are known to be involved in iron and sulphur cycling (Holmes et al., 2004; Lovley et al., 2004; Martins et al., 2010). The closest relatives of sequences belonging to the phylum Chloroflexi (band A3, B14, G36, I61 and I63) were all obtained from river and sea sediments. The environmental function of the phylumChloroflexi is not yet fully investigated; literature studies reported that they may contribute to the degradation of


15

recalcitrant organic matter (Kindaichi et al.,2004;Miura et al., 2007; Daniel et al., 2009).

Figure 2. DGGE profiles of 16S rRNA gene fragments amplified from DNA extracts from Lake Furnas sediments. Lane F1: 0-1 cm; Lane F2: 1-2 cm; Lane F3: 2.5-3.5 cm; Lane F4: 5-6 cm; Lane F5: 9-10 cm.

The sequences affiliated to the Bacteroidetes/Chlorobi group were retrieved from anoxic environments (bands A15, B13, C21, D25). Bacteroidetes are known as hydrolytic fermentative degraders of polymers in anaerobic habitats, including freshwater sediments (Kirchman, 2002; Schwarz et al., 2007). Less abundant sequences, but present in sediments, were affiliated to Candidate division OP11, commonly related to marine sediments (Harris et al., 2004), Candidate division OP1, whose closest relative retrieved in public data bases was found in a

16


hydrothermal vent biofilm (Stott et al., 2008), and Gemmatimonadetes, retrieved from lakesediments (Qu et al., 2008; Briée et al., 2007) and deep-sea sediments (Li et al., 1999). The presence of Candidate division OP1 in sediments from Lake Furnas might be related to its volcanic origin (Stott et al., 2008; Hugenholtz et al., 1998). The environmental function of these microbial groups remains relatively unknown.

2.3. Quantification of Proteobacteria Involved in the N cycle, Fe Reduction and P Accumulation in Sediments Little is known about the quantitative composition of microbial communities in sediments.Although the efforts made to reveal the diversity of sediment bacterial communities based on traditional cultivation methods (Springet al., 2000; Tamaki et al., 2005), it is widely recognized that only 0.001% to 15% (0.25% in sediments) of the total cell counts in environmental samples can be cultured (Amann et al., 1995, Parkes et al., 2000; Tamaki et al., 2005). The application of traditional culture-dependent methods to identify and quantify specific microbial groups in sediments may fail due to the lack of knowledge about the physiological conditions prevailing in the environment, or to cooperative interspecies growth interactions (Watanabe and Hamamura, 2003). In fact, a comparative study (Tamaki et al., 2005) has shown that with traditional culture-dependent methods it was possible to identify 7 different phyla, while with culture-independent methods (cloning of 16S RNA gene) the number increased to 15. The use of real-time quantitative PCR (qPCR), targeting specific functional or phylogenetic genes, overcame the limitation of traditional quantification methods, and has recently been broadened to the quantification of bacteria in complex environments, such as biofilms (Kindaichi et al., 2006), wastewater treatment plants (Ben-Dov et al., 2007; He et al., 2007), soil (Henry et al., 2004; Stubner, 2004), and sediments (Nakamura et al., 2006; Bedard et al., 2007; Bulow et al., 2008). So far, quantitative studies in sedimentsfocused on the bacterial community responsible for the nitrogen cycle, in particular, nitrifiers (Nakamura et al., 2006) and denitrifiers (Henry et al., 2004; Smith et al., 2007; Bulow et al., 2008).The amount of AOB reported in river sediments, determined by qPCRwith primers targeting 16S rRNA genes, was around106 to 107 cells/cm3(Nakamura et al., 2006). In marine sediments, the amount of AOB Beta-Protobacteria, based onamoA gene copy numbers, ranged from 104 to 107 copies per gram of sediment (Mosier and Francis, 2008). The amount of NOB reported for river


17

sediments,determined by qPCRwith primers targeting Nitrospira-like NOB 16S rRNA genes, ranged from106to 109 cells/cm3(Nakamura et al., 2006). Regarding DNB densities, reported values were around 104 to 108 copies/g sediment (targeting the functional gene nirS) in estuarine sediments (Smith et al., 2007; Bulow et al., 2008). Other bacterial communities quantified in sediments and soil include sulphate reducing bacteria in rice field soils (Stubner, 2004) and marine sediments (Leloup et al., 2007), as well as iron reducing bacteria in freshwater sediments (Cummings et al., 2003;Bedard et al., 2007; Himmelheber et al., 2009). In marine sediments, the amount of SRB, based ondsrA (dissimilatory (bi)sulfite reductase) gene copy numbers was around 105 to 107 cells/g sediment(Leloup et al., 2007), while in freshwater sediments,the amount of SRB was around 107 to 108cells/cm3 (Himmelheber et al., 2009).IRB of the family Geobacteraceae, Anaeromyxobacter spp. and Shewanella spp. represented about 105 to 107 cells/cm3, 106 to 107 cells/cm3 and 104 to 105 cells/cm3 respectively, in freshwater sediments (Bedard et al., 2007; Himmelheber et al., 2009).

Figure 3. Sediment bacterial profiles in Lake Furnas.

18


In sediments from Lake Furnas, the density of Beta-Proteobacteria-like AOB, Nitrobacter-likeNOB,Rhodocyclus-likePAO, and Geobacteraceae-likeIRB, was inferred by qPCR2 (Figure 3) and represented only 9% of total bacteria (Table 2). In average, total bacteria quantified with the EUBAC primer set (Harms et al., 2003) amounted to 8.48 108 ± 8.93 107 cell/g sediment.Bacterial profiles in the first 10 cm of sediment presented an almost homogeneous distribution of bacteria (Figure 3). Similar results have already been reported for stream sediments (Altmann et al., 2004; Nakamura et al., 2006) and anoxic sediments from the Black Sea, where the number of total bacterial cells was constant until a depth of 100 cm (Leloup et al., 2007). Aerobic bacteria (AOB, NOB and PAO) were retrieved both in aerobic and anoxic layers of the sediment.AOBwere the most abundant group (3.59 107±4.84 106cell/g to 5.78 107±3.51 107 cell/g) followed by Nitrobacter-like NOB (2.35 106±4.44 104cell/g to 4.50 107±5.20 106 cell/g), Geobacteraceae-likeIRB (7.46 106±6.33 104cell/g to 2.20 107±7.80 105cell/g), and Rhodocyclus-like PAO (1.27 106±2.73 105cell/g to 3.69 106± 3.31 105cell/g). Bacterial distribution in sediments was certainly influenced by the availability of oxygen in the sediment-water interface (~2.0 mg/L in Lake Furnas) and oxygen penetration into the sediment. Oxygen penetration in sediments from Lake Furnas was less than 1 cm (Figure 1), suggesting the prevalence of anoxic/anaerobic conditions in the sediment. The presence of AOB and NOB in anoxic/anaerobic sediment layers was to be expected because nitrifiers can grow under oxygen limited conditions, as reported in previous studies (Pynaert et al., 2002). In the 2

The amplification of real-time PCR products was done with a Chromo4 real-time PCR detector (MJ/Bio-Rad) using SYBR Green as signal dye. PCR amplification was performed in a 25 L reaction mixture containing 20 L of master mix (iQ™ SYBR® Green Supermix, Bio-Rad 1708882), and 5 L of DNA template (concentrations ranged from 0.10 ng/ L to 1.60 ng/ L). Primers targeting the 16S rRNA were used to quantify total bacteria, 1055f/1392r (Harms et al., 2003), ―Candidatus Accumulibacter‖ as phosphate-accumulating organisms,518f/PAO-846r(He et al.,2007), Geobacteraceae as iron-reducing bacteria, Geo564f Geo840r (Bedard et al., 2007), ammonium-oxidizing bacteria, CTO189fA/B/CTO189fC/RT1r (Kowalchuk et al.,1997; Hermansson and Lindgren, 2001),nitrite- oxidizing bacteria,Nitrobacter spp. FGPS872f/FGPS1269r (Degrange and Bardin, 1995), Nitrospira spp.- Nspra-746r/Nspra-675f (Graham et al., 2007), and anaerobic ammonium oxidizing bacteria,Amx809f/Amx1066r (Tsushima et al., 2007). In the case of denitrifying bacteria, primers targeting both functional genes nirK(nirK876f/nirK1040r) and nirS(nirS4Qf/nirS6Qr) were used(Henri et al., 2004).The purity of amplified products was checked by the appearance of a single melting peak in melting curves, which were obtained by increasing temperature from 62 ºC to 95 ºC at 0.2 ºC/s. Data were analyzed using MJ OpticonMonitor 3.1 (MJ/Bio-Rad) analysis software. Standard curves were generated from a 10-fold dilution series of positive controls (101 to 108 target copies per reaction) included in duplicate in each PCR. The detailed protocol can be found in Martins et al. (in preparation).


19

present work, nitrifying activity was detected in batch assays done with an oxygen concentration as low as 0.1 mg/L (Figure 4c). Nitrifiers deeper in the sediments might have occasionally access to oxygen due to mixing of the sediment layers induced by physical resuspension, formation of gas bubbles and bioturbation (Kemp et al., 1990; Stockdale et al., 2009). Other bacterial groups, not belonging to Proteobacteria, Anammox was the most abundant group and amounted to 107to 108 cells/g sediment. Nitrospira-like nitrite-oxidizing bacteria belonging to the Nitrospira phylum was also present and ranged from 106to 107 cells/g sediment. The amount of DNB quantified with the primer sets targeting the nirSand the nirKgenesamounted to 107 copies/g sediment and 106 copies/g sediment, respectively. Table 2. Average percentage of Proteobacteriaquantified with specific primers for AOB Beta-Proteobacteria, Nitrobacter-like NOB, Geobacteraceaelike IRB, and Rhodocyclus-like PAOin sediments from Lake Furnas Sample depth

AOB (%)

NOB (%)

PAO (%)

IRB (%)

F1 (0 to1 cm)

7.3

1.7

0.5

1.0

F2 (1 to 2 cm)

5.4

0.3

0.4

2.4

F3 (2.5 to 3.5 cm)

5.2

6.0

0.2

1.0

F4 (5 to 6 cm)

5.8

0.5

0.2

1.3

F5 (9 to 10 cm)

4.6

2.6

0.3

1.2

Average

5.7

2.0

0.3

1.4

2.4. Bacterial Activity in Sediments Related to the N cycle, Fe Reduction and P Accumulation Microcosm experiments3 with homogenized sediments were performed to assess bacterial activity in sediments form Lake Furnas. Nitrifying activity was 3

A sediment suspension, prepared by re-suspending 50 g of sediment in 300 mL distilled water, was incubated in the dark, at room temperatureand 150 rpm, for several weeks. Substrates were added periodically to stimulate the activity of specific bacterial groups: i) AOB - CaCO3 (20 mM) and NH4Cl (52 mg/L N), ii) NOB - CaCO3 (20 mM) and NaNO2 (100 mg/L N), iii) DNB - Na-acetate (20 mM) and KNO3 (140 mg/L N), iv) AOB and NOB under residual oxygen concentration CaCO3 (20 mM), NH4Cl (52 mg/L N), and NaNO2 (70 mg/L N), v) PAO - Na-acetate (20 mM), K2HPO4 (20 mg/L P), and KH2PO4 (10 mg/L P), vi) IRB - Na-acetate (20 mM) and FeC6H5O7·H2O (200 mg/L Fe). Anoxic conditions used in DNB and IRB tests were obtained

20


assessed in an aerobic batch assay in the presence of specific substrates and inhibitors (Figure 4a, 4b and 4c).The activity of AOB (Figure 4a) and NOB (Figure 4b) was stimulated by the addition of NH4+ and NO2-, respectively. A decrease in the concentrations of NH4+and NO2- was observed in AOB and NOB assays, respectively, concomitant with an increase in the concentration of NO3-. The simultaneous NH4+ and NO2- oxidationwas assessed in batch assays performed with low oxygen concentrations (below 0.1 mg/L), (Figure 4c). The results showed that the decrease in NH4+ and NO2- concentrations was followed by the nitrificationstoichiometric increase in NO3- concentration, suggesting that AOB and NOB were active in the sediment suspension. To further confirm these results, batch essays with specific inhibitors of AOB and NOB were performed. The addition of N-allythiourea, a specific inhibitor of AOB, suppressed NH4+ oxidation, as well as the addition of chlorate, a specific inhibitor of NOB, inhibited the oxidation of NO2-(results not shown). The activity of DNB (Figure 4d), assessed in anoxic batch assays, was stimulated by the addition of acetate and NO3-. A decrease in the concentration of both acetate and NO3- was observed.Attempts to inactivate biological activity in the sediment byautoclavingand addition of a specific inhibitor for DNB, i.e. hydroquinone, did not prevent the removal of acetate and NO3- completely, but retarded the denitrification rate(results not shown). The addition of acetate and NO3- to batch assays in a cyclic pattern (Figure 5a) resulted in a simultaneous decrease of PO43-and NO3- concentrations, suggesting the activity of denitrifying phosphate-accumulating organisms (Ahn et al., 2001; Tsuneda et al., 2006). It was observed that PO43-was released after NO3depletion in the presence of acetate. In repeated additions of acetate and NO3-, on average, 287 mg/L C, 112 mg/L N, and 59 g/L P were removed in the anoxic phase and in the anaerobic phase (absence of NO3-), 68 g/L P were released.

throughN2 gas sparging during 20 min in the beginning of the test, and after each sampling. To inhibit nitrite and ammonium oxidation activities, 50 mg/L chlorate (Remde and Conrad, 1991), and 10 mg/L N-allythiourea, (Reuschenbach et al., 2003) were used, respectively. Denitrification activity was inhibited by the addition of 50 mg/L hydroquinone (Zhengping et al., 2007). To monitor microbial activity, samples were taken along time, centrifuged at 10000 rpm for 15 min to remove sediment particles, and analyzed. Chemical analyses were performed according to Standard Methods (APHA, 1998).


21

Figure 4. Activity assays of (a) AOB, (b) NOB, (c) AOB and NOB under residual oxygen concentration and (d) DNB in sediments from Lake Furnas.

Iron-reducing activity of sediment bacteria was assessed in anaerobic batch assays in the presence of acetate and soluble Fe(III) (Figure 5b). A decrease in the concentration of soluble Fe(III) was observed.On the contrary, the expected increase in the concentration of Fe(II), resulting from the reduction of Fe(III), was not observed in the same proportion. One possible explanation for this result is that Fe(II)-containing minerals were formed and not accounted by the method used to quantify Fe(II). As suggested in the literature, magnetite might be an

22


endproduct of the activity of Fe(III)-reducing microorganisms in sedimentary environments (Lovley, 1991).

Figure 5. Activity assays of denitrifying PAO (a) and IRB (b) in sediments from Lake Furnas.

Phylogenetic analysis indicated that clones retrieved from Lake Furnas sediments belonging to Delta- and Gamma-subclasses of Proteobacteria were affiliated to iron-reducing Geobacteraceae and Shewanellaceae families, respectively, both well known as electroactive microorganisms (Holmes et al., 2004). Additionally, amplification of sediment DNA with primers directed toward conserved regions of the gene within the Geobacteraceae family (Geo564F, Geo840R) resulted in a discrete PCR product of the expected molecular weight (data not shown). Thus, the electroactivity of Lake Furnas sediments was determined in a microcosm experiment simulating a BMFC and voltage was recorded along time (Figure 6).


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Figure 6. Voltage along time obtained in the benthic microbial fuel cell operated with sediments from Lake Furnas.

The voltage was very low for the first 8 d increasing rapidly afterwards to a maximum value of 53 mV after 12 d, corresponding to a power density of 1 mW/m2. This behaviour might be explained by the formation of an electrochemically active biofilm on the surface of the anode. The open circuit voltage (OCV), representing the maximum voltage that is possible to obtain in optimum conditions, was 387 mV. The power density obtained in the study was considerably lower than power densities reported in literature for BMFCs: typical values are in the range of 0.18 mW/m2 to 49 mW/m2 (Tender et al., 2002;Lowy et al., 2006;He et al., 2007; Schamphelaire et al., 2008). One possible explanation for this result is that fouling of the cathode occurred. After a few days of operation, it was observed the colonization of the cathode by a thick biofilm which probably consumed oxygen and reduced its availability to the cathodic reaction. Tender et al. (2002) reported a decrease in power density in a BMFC due to the coverage of the cathode with sediments and macroalgal detritus. The highest power density reported in literature studies, 49 mW/m2, was obtained in a BMFC operating with a rotating cathode that enhanced oxygen mass transfer to the cathode‘s surface.

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3. CONCLUSION The combination of molecular methods (PCR-DGGE and qPCR) and activity tests suggested that bacteriawithin the Proteobacteria phylum were the most abundant contributed to nitrogen cycling, iron reduction as well as biological phosphorusstorage/release in sediments from the eutrophic Lake Furnas. Nowadays, eutrophication of freshwater reservoirs and lakes is mainly due to inputs of phosphorusfrom sediments to the water column. The present work suggested that the activity of denitrifying bacteriamight also contribute to the release of phosphorus from sediments. In this regards, an evaluation of the sediment microbial compositionand activity is certainly useful for designing requalification strategies towards the good ecological status prescribed by the Water Framework Directive (2000/60/EC). Aprevious study suggested that other polyphosphate-accumulating microorganisms affiliated to Actinobacteria and Gemmatimonadeteswere present in sediments from Lake Furnas. A future study intends to quantify these bacterial groups and to assess their contribution to phosphate release from sediments.

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Chapter 2

PROTEOBACTERIA FORMING NITROGENFIXING SYMBIOSIS WITH HIGHER PLANTS Encarna Velázquez1, Paula García-Fraile1, Helena Ramírez-Bahena2, Álvaro Peix3 and Raúl Rivas1 1

Departamento de Microbiología y Genética. Laboratorio 209. Edificio Departamental de Biología. Doctores de la Reina s/n. Universidad de Salamanca. 37007 Salamanca. Spain. 2 Laboratoire de Ecologie Microbienne. Universite Claude Bernard. Lyon. France. 3 Departamento de Desarrollo Sostenible de Sistemas Agroforestales y Ganaderos. Instituto de Recursos Naturales y Agrobiología. IRNASA-CSIC. Salamanca. Spain.

SUMMARY The ability to fix nitrogen is inherent only to prokaryotes and within them several Proteobacteria are able to establish symbiosis with legumes and the nonlegumeParasponia in which structures specialized in nitrogen fixation named nodules are formed. Within Proteobacteria, the species able to induce nitrogenfixing nodules are classified in two different classes, alpha and beta. All ―classical‖ rhizobia are alpha Proteobacteria whereas several species recently described as legume endosymbionts have been classified within the beta Proteobacteria. Although several gamma Proteobacteria have been isolated from nodules, they cannot induce nodule formation in legumes. The ecological

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Encarna Velázquez, Paula García-Fraile, Helena Ramírez-Bahena et al.

relevance of Proteobacteria forming nitrogen fixing endosymbiosis with higher plants is related to that of the legumes which colonise very different habitats being the legume symbiosis the main source of terrestrial fixed nitrogen. In this chapter we revised the current known Proteobacteria forming nitrogen-fixing endosymbiosis with legumes.

1. THE ALPHA-PROTEOBACTERIA NODULATING LEGUMES AND PARASPONIA Legume-nodulating bacteria were discovered at the end of XIX century and were included in the genus Rhizobium proposed as a single species, Rhizobium leguminosarum (Frank, 1889). This genus was later included in a new family named Rhizobiaceae proposed by Conn (1938). From this date ahead the number of species was slowly increasing until 1988 overall linked to the cross-nodulation concept (Baldwin & Fred, 1929). In this way the species were basically defined on the basis of the host they nodulated, for example R. meliloti was considered as a different species from R. leguminosarum because the strains nodulating Medicago commonly do not nodulate Vicia and viceversa (Jordan& Allen, 1974; Jordan, 1984). Nevertheless, in some cases phenotypic characteristics were also used to define genera and species such as the growth rate that allows the separation of slow growing species into a different genus named Bradyrhizobium (Jordan, 1982). Until 1984 all species nodulating legumes were placed in the family Rhizobiaceae in which only two genera were recognized: Rhizobium and Bradyrhizobium (Jordan, 1984). That year a general revision of the Taxonomy of Prokaryotes was carried out based on the Woese‘s proposal using the 16S rRNA gene sequences as basic criterion for classification (Woese et al., 1984). These authors proposed in this way a new classification of bacteria, placing legumenodulating rhizobia within the alpha subdivision of Proteobacteria. This proposal was accepted slowly by rhizobiologists and the new genus Azorhizobium able to form stem nodules in Sesbania rostrata was still described without sequencing its 16S rRNA gene (Dreyfus et al., 1988). In the same year, a new genus, Sinorhizobium, containing the single speciesSinorhizobiumfredii, was proposed from phenotypic data (Chen et al., 1988). Moreover R. galegae isolated from Galega nodules (Lindström, 1989) and R. huakuii isolated from Astragalus (Chen et al., 1991) were also described supported by phenotypic characteristics.

Proteobacteria Forming Nitrogen-fixing Symbiosis with Higher Plants

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However from 1991 ahead, sequencing of 16S rRNA gene was one of the minimal standards required for description of new rhizobial species (Graham et al., 1991) and the description of genus Sinorhizobium was ratified by partial 16S rRNA gene sequences (Jarvis et al., 1992). Nevertheless, the first description of rhizobial species with 16S rRNA gene sequencing was that of Rhizobium tropici isolated from Leucaena nodules in America (Martínez-Romero et al., 1991). The first attempts to analyse the phylogeny of bacteria nodulating legumes were made in 1993, when two different scientific groups published the phylogenetic analysis of family Rhizobiaceae (Willems & Collins, 1993, Yanagi & Yamasato, 1993). The results of these works confirmed the close phylogenetic relationship of genus Rhizobium with genus Agrobacterium, the high phylogenetic divergence of some species of Rhizobium, and the higher phylogenetic divergence of genera Bradyrhizobium, Azorhizobium and Phyllobacterium. Although no taxonomic decisions were taken in these studies, few years later, the species of genus Rhizobium phylogenetically divergent were 39enegal39red to a new genus named Mesorhizobium (Jarvis et al., 1997) and a new genus with a single species, Allorhizobium undicola,was proposed to include the strains isolated in Senegal from nodules of Neptunia natans (de Lajudie et al., 1998). At the beginning of XXI century some changes have been proposed within the family Rhizobiaceae such as the reclassification of genera Agrobacterium and Allorhizobium into genus Rhizobium proposed by Young et al. (2001). The name of genus Sinorhizobium was changed to Ensifer since after the analysis of the 16S rRNA gene, their species are closely related to a species described before named Ensifer adhaerens (Casida et al., 1982). According to the rules of the Bacteriological Code about nomenclature, the name Ensifer has priority over Sinorhizobium and thus the name Sinorhizobium is no longer valid (Judicial Comission of the International Committee on Systematics of Prokaryotes, 2008). Finally, an important change was the split of the genera from the family Rhizobiaceae into several families and the creation of a new order named Rhizobiales proposed in the Bergey‘s Manual Edition of 2005 (Kuykendall, 2005). The new order Rhizobiales defined by Kuykendall (2005) includes symbiotic, pathogenic and saprophytic bacteria distributed in several families from which the family Rhizobiaceae contains the genera Rhizobium, Agrobacterium, Sinorhizobium and Allorhizobium together with a genus named Ensifer that does not establish any plant interaction. A new family named Phyllobacteriaceae was also proposed (Mergaert and Swings, 2005) and later validated (Validation list No. 107, 2006) that contains the genera Phyllobacterium and Mesorhizobium together with several non-symbiotic genera. The genus Azorhizobium was

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included in the previously existent valid family Hyphomicrobiaceae together with other non-symbiotic genera. Finally, the genus Bradyrhizobium was included in a family named Bradyrhizobiaceae (Garrity et al., 2005). Most of these names were validated in IJSEM (Validation list No. 107) but those of the order Rhizobiales and family Bradyrhizobiaceae are illegitimate being correct names order Hyphomicrobiales and family Nitrobacteriaceae, respectively. Surprisingly, although the Bergey‘s Manual of 2005 recorded the changes in the suprageneric organization of rhizobial species, the reclassification of Young et al. (2001) was not considered and the genera Agrobacterium and Allorhizobium have independent chapters in this Manual. By contrast, genera Sinorhizobium and Ensifer were maintained as separated genera because the resolution of the Judicial Comission was published later. After publication of Bergey‘s Manual, many other species of rhizobia nodulating legumes have been described and currently genus Rhizobium contains 34 species and includes the former genera Agrobacterium and Allorhizobium. The genus Sinorhizobium, currently named Ensifer, contains 13 species, genus Mesorhizobium, 20 species, genus Phyllobacterium, 8 species, genus Azorhizobium, 2 species, the genus Bradyrhizobium 9 species and the genus Shinella contains 4 species. The complete list of valid species of rhizobia is constantly actualized and recorded in the List of Prokaryotic Names with Standing in Nomenclature by Dr. Euzeby (http://www.bacterio.cict.fr). In the Table 1 only are recorded the species able to form nitrogen-fixing symbiosis with legumes. Table 1. Species of “Classical” Rhizobia Belonging to Alpha Proteobacteria Forming Nitrogen-fixing Symbiosis with Higher Plants. Species

Origin host legume

Order Hyphomicrobiales Family Rhizobiaceae Genus Rhizobium R. alkalisoli

Caragana microphylla

R. fabae, R. leguminosarum, R. pisi

Vicia faba, Pisum sativum

R. galegae

Galega orientalis

R. etli, R. gallicum, R. giardinii, R. lusitanum, R. phaseoli, R. tropici

Phaseolus vulgaris

R. hainanense

Desmodium sinuatum

R. huautlense

Sesbania herbacea

Proteobacteria Forming Nitrogen-fixing Symbiosis with Higher Plants R. indigoferae

Indigofera amblyantha, I. carlesii, I. potaninii

raeloessense R.

Astragalus complanatus, A. adsurgens, A. scobwerrimus, A. chrysopterus

R. mesosinicum

Albizia julibrissin, Kummerwia spp., Dalbergia spp.

R. multihospitium

Robinia pseudoacacia, Halimodendron halodendron

R. miluonense

Lespedeza spp.

R. mongolense

Medicago ruthenica

R. tibeticum

Trigonella archiducis-nicolai

R. sullae

Hedysarum coronarium

R. undicola

Neptunia natans

R. yanglingense

Amphicarpaea trisperma

Genus Ensifer (formerly Sinorhizobium) E. arboris, E. kostiense

Acacia 41enegal, Prosopis chilensis

E. saheli, E. terangae

Sesbania spp., Acacia spp.

E. fredii

Glycine max

E. kummerowiae

Kummerowia stipulacea

E. meliloti, E. medicae

Medicago sativa

Genus Shinella Shinella kummerowiae

Kummerowia stipulacea

Family Phyllobacteriaceae Genus Mesorhizobium M. albiziae

Albizia kalkora, A. julibrissin

M. alhagi

Alhagi sparsifolia

M. amorphae

Amorpha fruticosa

M. australianum, M. opportunistum

Biserrula pelecinus

M. caraganae, M. shangrilense

Caragana microphylla, C. intermedia

M. chacoense

Prosopis alba

M. ciceri, M. mediterraneum

Cicer arietinum

M. gobiense, M. tarimense

Glycyrrhiza uralensis, Lotus corniculatus, Oxytropis glabra, Robinia pseudoacacia

M. huakuii, M. septentrionale, M. temperatum

Astragalus adsurgens

M. loti

Lotus corniculatus

M. metallidurans

Anthyllis vulneraria

41

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Encarna Velázquez, Paula García-Fraile, Helena Ramírez-Bahena et al. Table 1 (Continued)

M. plurifarium

Acacia spp.

M. tianshanense

Sophora alopecuroides

Family Nitrobacteraceae Genus Bradyrhizobium B. canariense

Chamaecytisus proliferus

B. elkanii, B. japonicum, B. liaoningenese

Glycine max

B. jicamae, B. pachyrhizi

Pachyrhizus erosus

B. yuanmingense

Lespedeza cuneata

Family Hyphomicrobiaceae Genus Azorhizobium A. caulinodans

Sesbania rostrata

A. dobereinereae

Sesbania virgata

Compiled from Rivas et al. (2009) and Euzéby (2010).

In addition to the validly described species, many other strains belonging to the alpha Proteobacteria have studied and many of them remain unclassified even if their complete genome has been completely sequenced such occurs with the strain NGR234 that is a broad legume range strain able to nodulate several legumes and the non-legume Parasponia (Pueppke & Broughton, 1999).

2. THE NEW ALPHA-PROTEOBACTERIA NODULATING LEGUMES AND PARASPONIA In the 80‘s decade it was already well known that symbiosis determinants are codified on plasmids in the fast growing rhizobia (Zurkowski & Lorkiewic 1979), being most of them autoconjugative and hence easily transferred among strains. This fact evidence that plasmids are not adequate DNA molecules to be used for species description, even if the symbiotic characteristics have been used in rhizobial taxonomy for decades. Moreover, the idea of considering rhizobia as the unique nodulating bacteria was present in the scientific community for more than 100 years. This general belief led to discard all those colonies obtained from nodules that showed non-rhizobial morphology when grown on YMA plates (Vincent, 1970)


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and to preserve the strains whose colonies presented the typical mucoid aspect of rhizobia. The first non-rhizobial bacterium found to be able to nodulate legumes was a Methylobacterium, published in January 2001 (Sy et al., 2001). This genus belongs to alpha Proteobacteria, order Rhizobiales and family Methylobacteriaceae. The species able to originate nodules in Crotalaria was named M. nodulans (Jourand et al., 2004) and harbors the common nodulation nodABC genesand nifH gene encoding structural nitrogenase enzyme (Sy et al., 2001, Jourand et al., 2004). Unidentified strains of methylotrophic bacteria with high nitrogenase activity have been recently isolated from nodules of Crotalaria juncea and Sesbania aculeata in Korea (Madhaiyan et al., 2009). The second non-rhizobial nodulating bacterium found belonging to alpha Proteobacteria and able to nodulate legumes was Devosia neptuniae (Rivas et al., 2003). These strains that were isolated in India from Neptunia natans nodules were previously named Rhizobium neptunii because their colony morphology ressembles that of rhizobial species (Subba-Rao et al., 1995). However, the analysis of their 16S rRNA gene sequences revealed that they belong to genus Devosia within family Hyphomicrobiaceae. This family also includes a classic rhizobial genus, Azorhizobium, isolated from stem nodules of Sesbania rostrata in Senegal (Dreyfus et al., 1988). The strains of D. neptuniae isolated from Neptunia nodules carry nodD and nifH genes closely related to those of R. tropici CIAT899T (Rivas et al., 2002) indicating that they were transferred to D. neptuniae from R. tropici, an American species nodulating Leucaena (MartínezRomero et al., 1991). Later this hypothesis was supported by the finding that R. tropici nodulated Neptunia in America (Zurdo-Piñeiro et al., 2004). Up to date D. neptuniae is the only species of genus Devosia found to be able to nodulate legumes. In successive years other non-rhizobial genera from alpha Proteobacteria were reported as legume endosymbionts (Table 2). In 2002, the nodulation of Aeschynomene indica by Blastobacer denitrificans was reported (van Berkum & Eardly, 2002). This species is able to form effective nodules in the roots of A. indica and reduce the atmospheric nitrogen (van Berkum & Eardly, 2002), which was again confirmed by presence of nifHDK genes by southern hybridization. Genus Blastobacter belongs to the same family as Bradyrhizobium andits reclasiffication into genus Bradyrhizobium has been proposed (van Berkum et al., 2006). However, since this proposal is not yet validated in the official journal of bacterial systematics (IJSEM), it cannot be considered as official. There are other Blastobacter species whose nodulation ability has not been analysed and whose taxonomy should be revised because the type species of this genus, B. henricii

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does is not deposited in culture collections. Several strains named photosynthetic Bradyrhizobium but whose taxonomic status is not correctly established may nodulate stems of Sesbania, a tropical legume also nodulated by Rhizobium (Zurdo-Piñeiro et al., 2004). Apparently these strains do not harbor nodulation genes and constitute a new model for the study of legume symbiosis (Giraud et al., 2007). Table 2. Species Belonging to Alpha Proteobacteria Forming Nodules in Higher Plants Not Included in the “Classical” Rhizobia. Species Family Phyllobacteriaceae Genus Phyllobacterium P. trifolii Family Nitrobacteraceae Genus Blastobacter B. denitrificans Family Hyphomicrobiaceae Genus Devosia D. neptuniae Family Brucellaceae Genus Ochrobactrum O. lupini O. cytisi Family Methylobacteriaceae Genus Methylobacterium M. nodulans

Origin host legume

Trifolium repens

Aeschynomene indica

Neptunia natans

Lupinus honoratus Cytisus scoparius

Crotalaria glaucoides


In spite of the importance of these results as being the first reports on the high diversity of bacteria able to nodulate legumes within the class alpha Proteobacteria, the most relevant finding in this field was the discovery of Ochrobactrum strains within family Brucellaceae able to nodulate legumes. This family contains important human pathogenic species belonging to the genus Brucella and the fact that species close to Ochrobactrum anthropi are capable of nodulate legumes posses the question about the coexistence of virulence and symbiotic determinants in some strains. Although strains of genus Ochrobactrum


45

were initially found in nodules of Acacia mangium, no information on their symbiotic genes was reported (Ngom et al., 2004). A year later, we found that strains from a new species of this genus carrying rhizobia –related symbiotic genes are able to nodulate Lupinus (Trujillo et al., 2005). The strains from O. lupini harbored megaplasmids of 1500, 200 and 150 kbp and the nodulation (nod) and nitrogen fixation (nif) genes were detected in all the sym plasmids using nifH and nodD probes. Later a second new species of Ochrobactrum named O. cytisi carrying symbiotic genes phylogenetically related to those from rhizobial strains was isolated from Cytisusscoparius nodules in Spain (Zurdo-Piñeiro et al., 2007). In the same year a new alpha Proteobacteria nodulating Lupinus in America related to genus Methylobacterium was reported although the strain was not officially asigned to a concrete genus (Andam & Parker, 2007). In 2005 a new species named Phyllobacterium trifolii was isolated from Trifolium pratense nodules (Valverde et al., 2005). Phyllobacterium was originally proposed to accommodate bacteria isolated from leaf nodules of Rubiaceae and Myrsinaceae tropical plants (Knösel et al., 1984). Although the type strain of the species P. trifolii harbors symbiotic plasmids in which the nod and nif genes were located it forms ineffective nodules in the roots of Trifoliumrepens and Lupinus albus (Valverde et al., 2005). Considering that the endosymbionts of many species of legumes remain unstudied probably the number of species able to induce nodules will increase in the future overall within the genera considered as ―classical‖ rhizobia but also in other groups of the alpha Proteobacteria class.

3. THE BETA-PROTEOBACTERIA NODULATING LEGUMES In June of 2001 an impactant paper was published in Nature reporting the nodulation of Mimosa by Burkholderia, a genus belonging to the betaProteobacteria (Moulin et al., 2001). These authors isolated a strain of Burkholderia from nodules of the South African legume, Aspalathuscarnosa, that is also able to form nodules in the roots of Macroptilium atropurpureum, a promiscuous tropical legume, confirming to have the ability of symbiotic nitrogen fixation. The Burkholderia strain carried nodulation (nodABC)genes phylogenetically related to those found in legume symbionts of the class alpha Proteobacteria (―classic‖ rhizobia) supporting the hypothesis of lateral gene transfer in the rhizosphere, crossing the boundary between class alpha Proteobacteria and beta Proteobacteria.

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Therefore, from 2001 ahead the concept of the rhizobia changed and several species of beta Proteobacteria (Table 3) have been reported as legume endosymbiots such as Ralstonia taiwanensis nodulating Mimosa pudica and Mimosa diplotricha (Chen et al., 2003a).In this case the strain was erroneously classified as Ralstonia being later reclassified as Cupriavidus taiwanensis, a beta Proteobacteria belonging to the family Burkholderiaceae from the order Burkholderiales (Vandamme and Coenye, 2004). C. taiwanensis carries ten nodulation genesnodABCIJHASUQ and one regulatory gene nodD on plasmid pRalta (Amadou et al., 2008). Next to nod genes, C. taiwanensis carries 19 genes, presumably arranged in five operons and covering 25kb that are involved in nitrogenase synthesis and functioning (Amadou et al., 2008). Table 3. Species Establishing Legume Symbiosis Belonging to Beta Proteobacteria Species Family Burkholderiaceae Genus Burkholderia B. cepacia complex B. nodosa B. sabiae B. mimosarum B. phymatum B. tuberum Family Burkholderiaceae Genus Cupriavidus C. taiwanensis

Origin host legume

Dalbergia spp. Mimosa bimucronata, M. scabrella Mimosa caesalpiniifolia Mimosa pigra, M. scabrella Machaerium lunatum Asphalatus carnosa

Mimosa pudica, M. diplotricha


In successive years several species of Burkholderia nodulating legumes have been described such as B. mimosarum (Vandamme et al., 2002, Chen et al., 2006), B. phymatum (Vandamme et al., 2002), B. nodosa (Chen et al., 2007) B. tuberum (Elliott et al., 2007a) and B. sabiae (Chen et al., 2008). The species B. mimosarum was isolated from root nodules of M. pigra and M. scabrella from Taiwan, Brazil and Venezuela and the presence of nif and nod genes has been demonstrated (Chen et al., 2005a, b). B. nodosa was isolated from root nodules of M. bimucronata and M. scabrella from Brazil and produced N2-fixing nodules on


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M. pudica, M. diplotricha and M. pigra. Mimosa is also nodulated by B. phymatum (Elliott et al., 2007b). Although Burkholderia mainly nodulate legumes belonging to the subfamily Mimosoideae, the Papilionoideae legumes Cyclopia spp. is nodulate by B. tuberum (Elliott et al., 2007a) and Dalbergialouveli is nodulated by a strain belonging to the Burkholderia cepacia complex (Rasolomampianina et al., 2005). There are more and more works reporting the presence of beta Proteobacteria in nodules of several legumes such as Dalbergia, Caragana, Crotalaria, Mimosa and Prosopis (Rasolomampianina et al., 2005, Barrett and Parker, 2006, Liu et al., 2007, Yan et al., 2007, Benata et al., 2008). It has been reported that beta-Proteobacteria are widespread in legume nodules in tropical countries (Chen et al., 2003b, Chen et al., 2005), in which Burkholderia seems to be the main endosymbiont of legume trees (Barrett and Parker, 2005, Chen et al., 2005). Even some strains of Burkholderiaare more competitive than R. tropici for the nodulation of Mimosa (Elliot et al., 2009). In a recent work Bontemps et al. (2010) have sequenced the ribosomal 16S gene and the genes for nodulation and nitrogen fixation of Burkholderia strains isolated from Mimosa nodules in Brazil. The sequences of symbiotic genes indicated that nodulation is not a new function among Burkholderia species the data obtained suggested a long and stable genetic history of symbiotic abilities for these bacteria. These authors demonstrated that nif genes are widespread in Burkholderia species and other unrelated bacteria but nod genes are restricted to the legume endosymbionts. Therefore the hypothesis of Bontemps et al. (2010) is that Mimosa and Burkholderia have not only an ancient history of coexistence but also that this symbiosis is highly specific. Considering that the Burkholderia strains isolated in Brazil have phylogenetically divergent 16S rRNA and recA genes, several species of this genus could be described in the future. Moreover, a lot of geographical regions and legumes remain still unexplored for which we can expected that diversity of beta-Proteobacteria nodulating legumes is probably much higher than what we currently know.

4. THE NITROGEN-FIXING GAMMA-PROTEOBACTERIA ENDOPHYTIC OF LEGUME NODULES Since the discovery of non-rhizobial alpha and beta Proteobacteria nodulating legumes, the interest to find other legume endosymbionts led to the researchers to deeply analyse the microbiota present in the nodules. The exploration of new

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source plants and the use of alternative isolation and identification methods has produced rapid expansion in the knowledge of endophytic bacteria of legume nodules. In several studies non-pathogenic species of gamma Proteobacteria have been isolated from diverse legumes although no nodulation genes have been detected in these strains up to date (Benhizia et al., 2004, Rasolomampianina et al., 2005; Muresu et al., 2008, Ibáñez et al., 2009). Therefore they cannot induce nodules and probably these strains may colonize root nodules after or during their formation. Although gamma Proteobacteria do not form nodules, nitrogen fixation has previously been demonstrated for representatives of these genera such as Klebsiella that provides fixed nitrogen to non-leguminous plants, such as wheat (Iñíguez et al., 2004). This genus also has a very broad host range and is capable of colonizing the interior of many plants, including legumes such as Medicago species, with fewer than 10 cells in the inoculums (Dong et al., 2003 a and b). This ability has been also observed in Pseudomonas strains colonizing plant roots (Ghiglione et al., 2000). Several genera belonging to the gamma Proteobacteria as Pseudomonas, Escherichia, Leclercia, Pantoea, Klebsiella and Enterobacter have been found associated with nodules in legumes. For example, Pantoea agglomerans and Pseudomonas fluorescens were the most common endophytes in various pea cultivars (Elvira-Recuenco & Van Vuurde, 2000). Pantoea, Enterobacter, Escherichia, Leclercia y Pseudomonas were found in nodules of Hedysarum (Benhizia et al., 2004). Enterobacter asburiae y Serratia marcescens have been found in nodules of Pueraria lobata (Selvakumar et al., 2008). Pseudomonas, Enterobacter and Klebsiella have been recently found in nodules of Arachis hypogaea (Ibáñez et al., 2009). Therfore, although gamma Proteobacteria isolated up to date have not the ability to reproduce nodules many of them are able to fix nitrogen and could contribute to the plant growth since they cohabit with rhizobia in effective and functional nodules being compatible with rhizobial endosymbiosis. The studies of nitrogen legume symbiosis from this point of view will provide a better knowledge of microbial interactions occurring in legume nodules. If the ability to fix nitrogen in legume nodules by gamma Proteobacteria is confirmed, the scientific implications would be significant because the endophytic genera containing nitrogen fixing species could have a great potential in yield improvement of legume and non-legume plants. Moreover the coinoculation of nitrogen fixing endophytes commonly found in nodules with rhizobia could improve the legume and non-legume production. Anyway, the legume symbiosis is still poorly understood and further studies on the legume endosymbionts in different ecosystems and geographical locations


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are necessary. The incredibly fast advances in molecular techniques will surely help with this challenging task of studying the broad diversity of bacteria and their interactions with plants.

ACKNOWLEDGMENTS The authors would like to thank our numerous collaborators and students involved in this research over the years. Funding was provided by Ministerio de Ciencia e Innovación and Junta de Castilla y León from Spain.

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Lupinus albus by strains of Ochrobactrum lupini sp. nov. Applied and Environmental Microbiology, 71, 1318-327. Validation List no. 107. (2006). List of new names and new combinations previously effectively, but not validly, published. International Journal of Systematic and Evolutionary Microbiology, 56, 1-6. Valverde, A., Velázquez, E., Fernández-Santos, F., Vizcaíno, N., Rivas, R., Mateos, P. F., Martínez-Molina, E., Igual, J. M. & Willems, A. (2005). Phyllobacterium trifolii sp. nov., nodulating Trifolium and Lupinus in Spanish soils. International Journal of Systematic and Evolutionary Microbiology, 55, 1985-1989. van Berkum, P. & Eardly, B. D. (2002). The aquatic budding bacterium Blastobacter denitrificans is a nitrogen-fixing symbiont of Aeschynomene indica. Applied Environmental Microbiology, 68, 1132-1136. van Berkum, P., Leibold, J. M. & Eardly, B. D. (2006). Proposal for combining Bradyrhizobium spp. (Aeschynomene indica) with Blastobacter denitrificans and to transfer Blastobacter denitrificans (Hirsch and Muller, 1985) to the genus Bradyrhizobium as Bradyrhizobium denitrificans (comb. nov.). Systematic Applied Microbiology,29, 207-215. Vandamme, P. & Coenye, T. (2004). Taxonomy of the genus Cupriavidus: a tale of lost and found. International Journal of Systematic and Evolutionary Microbiology, 54, 2285-2289. Vandamme, P., Goris, J., Chen, W. M., de Vos, P. & Willems, A. (2002). Burkholderia tuberum sp. nov. and Burkholderia phymatum sp. nov., nodulate the roots of tropical legumes. Systematic and Applied Microbiology, 25, 507512. Vincent, J.M. (1970). The cultivation, isolation and maintenance of rhizobia. In J.M. Vincent (Ed.). A Manual for the Practical Study of Root-Nodule. pp. 113. Oxford: Blackwell Scientific Publications. Willems, A. & Collins, M. D. (1993). Phylogenetic analysis of rhizobia and agrobacteria based on 16S rRNA gene sequences. International Journal of Systematic Bacteriology, 43, 305-313. Woese, C. R., Stackebrandt, E., Weisburg, W. G., Paster, B. J., Madigan, M. T., Fowler, V. J., Hahn, C. M., Blanz, P., Gupta, R., Nealson, K. H. & Fox, G. E. (1984). The phylogeny of purple bacteria: The alpha subdivision. Systematic Applied Microbiology, 5, 315-326. Yan, X. R., Chen, W. F., Fu, J. F., Lu, Y. L., Xue, C. Y., Sui, X. H., Li, Y., Wang, E. T. & Chen, W. X. (2007). Mesorhizobium spp. are the main microsymbionts of Caragana spp. grown in Liaoning Province of China. FEMS Microbiology Letters, 271, 265-273. Yanagi, M. & Yamasato, K. (1993) Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS Microbiology Letters, 107, 115-20.

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Young, J. M., Kuykendall, L. D., Martínez-Romero, E., Kerr, A. & Sawada, H. (2001). A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi,R. undicola and R. vitis. International Journal of Systematic and Evolutionary Microbiology, 51, 89103. Zurdo-Piñeiro, J. L., Rivas, R., Trujillo, M. E., Vizcaíno, N., Carrasco, J. A., Chamber, M., Palomares, A., Mateos, P. F., Martínez-Molina, E. & Velázquez, E. (2007). Ochrobactrum cytisi sp. nov., isolated from nodules of Cytisus scoparius in Spain. International Journal of Systematic and Evolutionary Microbiology 57, 784-788. Zurdo-Piñeiro, J. L., Velázquez, E., Lorite, M. J., Brelles-Mariño, G., Schröder, E. C., Bedmar, E. J., Mateos, P. F. & Martínez-Molina, E. (2004). Identification of fast-growing rhizobia nodulating tropical legumes from Puerto Rico as Rhizobium gallicum and Rhizobium tropici. Systematic and Applied Microbiology, 27, 469-477. Zurkowski, W. & Lorkiewicz, Z. (1979) Plasmid-mediated control of nodulation in Rhizobiumtrifolii. Archives of Microbiology, 123, 195-201.



Chapter 3

PROTEOBACTERIA AND THE ENDOSYMBIOTIC ORIGIN OF MITOCHONDRION 1

Yu-Juan Zhang1, 2and Jian-Fan Wen1§* State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan Province650223, China 2 Graduate School of the Chinese Academy of Sciences, Beijing 100039, China

ABSTRACT The acquisition of mitochondrion is one of the decisive steps in the evolution of the eukaryotic cell.Today, people almost have believed that mitochondrion had a bacterial endosymbiotic origin. But what kind of bacteria is the endosymbiont? With the accumulation of evidence from the studies of morphology, cell biology, biochemistry, and molecular biology, especially from the recent studies of molecular phylogeny and genomics, proteobacteria, in particular Rickettsiales or their close relatives, are proposed to be the endosymbiont.In this paper, we review the progress of the studies in this field.

Keywords: Mitochondrion, Origin, endosymbiotic theory, Proteobacteria, Rickettsiales

*

Corresponding authorEmail addresses:Yu-Juan Zhang: [email protected] and Jian-Fan Wen:[email protected].

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Yu-Juan Zhang and Jian-Fan Wen

1. INTRODUCTION In the biological world, there are two distinctly different cell types, prokaryotic and eukaryotic. Eukaryotic cells, evolving from a prokaryote-like predecessor, are internally more complex than prokaryotic cells. Not only are the genetic materials in eukaryotic cells partitioned into a membrane encircled nucleus, but also other functionally specialized structures (organelles), prominent among which is mitochondrion, are contained. Mitochondrion is a kind of organelle found in virtually all eukaryotic cells. In addition to their role in energy conversion, mitochondria are involved in many metabolisms, such as synthesis of heme groups [1], steroids[2], amino acids, and iron-sulphur (Fe-S) clusters[3]. Prokaryotic cells have no mitochondria. How mitochondrion arose during the evolution of the eukaryotic cell from the prokaryotic cell has been a subject of continuous interest and considerable speculation ever since this issue was first recognized over a hundred years ago. The most popular theory about the origin of mitochondria is the endosymbiotic theory, which had been developing for more than 100 years since its first proposal [4]. The endosymbiotic theory envisions the evolution of the first eukaryotic cell to have resulted from the permanent incorporation of once autonomous, physiologically different prokaryotic cells within a host prokaryotic-type cell. Which kind of bacteria is exactly the ancestor of mitochondrion and how it was transformed from an endosymbiont to a mitochondrionattract widespread concerns. Early simple morphologic and biochemistrystudies can only providethe evidence for the bacteria-origin of mitochondria. With the development of molecular phylogenetic analysis, and genomics, more and more evidence has been accumulated to reveal which kind of bacteria is the endosymbiotic ancestor of mitochondrion, and what had happened in the transformation process from the endosymbiont to the typical mitochondrion. In this paper, we mainly give a brief reviewof the studiesaboutproteobacteria in particular, Rickettsiales or close relatives as the endosymbiotic ancestor of mitochondrionand the process of transition from proteobacteria to mitochondria at genomics and proteomics angles.

Proteobacteria and the Endosymbiotic Origin of Mitochondrion

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2. THE ENDOSYMBIOTIC HYPOTHESIS ABOUT MITOCHONDRION ORIGIN The concept of endosymbiosis and the origin of cell organelles (mitochondria and plastids) have deep historical roots which can be traced back to the late 19th century. In 1883,Andreas Schimper observed the division of chloroplasts in green plants closely resembled that of free-living cyanobacteria, then he tentatively proposed that green plants had arisen from a symbiotic union of two organisms in a footnote [5]. But it was Konstantin Mereschkowski who stated the endosymbiotic theory firstly. His research on lichens led him to coined the term ―symbiogenesis‖—that larger, more complex cells evolved from the symbiotic relationship between less complex ones [6]. Later, Ivan Wallin extended the idea of an endosymbiotic origin to mitochondria in the 1920s. In his 1927 book titled ―Symbionticism and the Origin of Species‖, he described how new species form by the permanent acquisition of symbiotic bacteria. Wallin further posited that mitochondria were once independent bacteria that took up permanent residence inside of existing cells to become what we today know as cell ―organelles‖[7]. However, the above-mentioned theories were initially dismissed or ignored, and fell into great disfavor. More detailed electron microscopic comparisons, combined with the existence of DNA in plastids and mitochondria[8] led to a revival of the endosymbiotic idea in the 1970s. Lynn Margulis advanced the endosymbiotic theory and substantiated it with microbiological evidence in a 1967 paper [4], which was "rejected by about fifteen scientific journals," according to Margulis‘s recall. It was finally accepted by The Journal of Theoretical Biology and is considered today a landmark in modern endosymbiotic theory. She proposed that organelles such as mitochondria living happily inside of nucleated cells evolved from ancient free-living bacteria, and furthermore, these organelles were the source of cytoplasmic DNA genes living outside of the cell‘s nucleus that were confounding geneticists at the time. The paper was initially heavily rejected, as symbiosis theories had been dismissed by mainstream biology at the time. Currently, her endosymbiotic theory is recognized as the key method by which some organelles have arisen and is widely accepted by mainstream scientists.

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3. PROTEOBACTERIA AS THE ENDOSYMBIOTICANCESTOR OF MITOCHONDRIA The evidence that support endosymbiotic theory and proteobacteria as the endosymbiotic ancestor of mitochondria comes from many fields, including: Morphology, Cell Biology, Biochemistry, Molecular biology, Molecular phylogeny, and Genomics. Here below we will review this progress in the field, especially those from molecular phylogeny and genomicsstudies in recent years.

3.1 Evidence from Morphology, Cell Biology, Biochemistry, and Molecular Biology Studies Previous Morphology, Cell Biology and Biochemistry and Molecular Biology studies indicated that mitochondria possess many features similar to those of bacteria. Mitochondria are just about the same size as bacteria. They are surrounded by two membranes, and the innermost of these is like that of a prokaryotic cell membrane and shows differences in composition from the outer membrane, which have similar composition with the endomembrane system in eukaryotic cell; The formation of new mitochondria and new bacteria are acted in a same way. New mitochondria are formed only through a process similar to binary fission, which is usually taken by bacteria; Mitochondria contain their own DNA, which may be remnants of the genome the organelles had when they were independent bacteria, and the mitochondrial DNA is different from that of the cell nucleus but is similar to that of bacteria, in morphylogy and structure. Mitochondrial DNA are made up of single bared circle DNA without interaction with histone, have no 5methylcytosine and can conduct independent replication and transcription in mitochodria; Mitochondria has its own protein synthesis system. The mechanism of protein synthesis in mitochondria are like those found in bacteria. For example, Proteins in mitochondria use N-formylmethionine as the initiating amino acid like those of bacteria, Ribosomes in mitochondria are like those found in bacteria in its size (70s)and structure (have no 5.8SrRNA ). All these lines of evidence indicate mitochondria might have a bacterialorigin, but did not tell which kind of bacteria is the ancestor of mitochondria.


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3.2 Evidence from Molecular Phylogenetic Analysis Phylogenetic analysis is crucial to the establishment of the closest extant relatives of mitochondria, since it helps to make a better choice of a bacterium for detailed molecular analysis and tell us which kind of bacteria is the most likely ancestor of mitochondria. The earliest phylogenetic analysis for this issue is based on the single-gene sequences of chaperonin 60 (Cpn60), which is likely the best tracer of the bacterial origin of organelles. It indicated the sister clade nature of alphaproteobacteria and mitochondria, especially the closest relationship of the genus Rickettsia with mitochondria [9]. Since then the phylogenetic studies based on single gene, such as some protein-encoding genes[10, 11], and ribosomal RNA (rRNA) genes [12] were widely carried out. Their results have consistently shown that the monophyletic cluster of mitochondria emanates from the order Rickettsiales, specifically R. prowazekii. In 1998, sequencing of R. prowazekii genome was finished and provided ample information for phylogenetic analysis. The results based on concatenated sequences of ribosomal proteins and protein sequences of respiratory complexes also strongly supported the closecorrelation of mitochondria and Rickettsiales [13]. However, the specific close relationship between proteobacteria (exactly Rickettsia) and mitochondria is not support by all phylogenetic studies. For example, analysis of Cob and Cox1-3 has revealed that some free-living alphaproteobacteria and R. prowazekii may be equally close relatives of mitochondria [14]. The mitochondrial proteome-wideanalysis of yeastshowed that not all of ca. 50 proteins, which turned out to be of alpha-proteobacterial origin, point to a sisterhood of R. prowazekii and mitochondria. Although some disagreements existed, they do not weaken the proposal that an alpha-proteobacterium, mostly of Rickettsiales or close relatives, is the ancestor of mitochondria.

3.3 Evidence from Genomics Studies Much compelling evidence of proteobacteria-origin of mitochondria comes from the comparison of R. prowazekii and mitochondrial genomes. Among many of the completely sequenced bacterial genomes, R. prowazekiigenome (1,111,523bp) stands out as the ‗most mitochondrial‘ one[13, 15]. It shows striking similarity in the functional profiles of its genes to those of mitochondria:

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no genes required for anaerobic glycolysis are found, while a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratorychain complex exists.In fact, R. prowazekis genome encodes an ATP-generating machinery that is strikingly similar to that of mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from both R. prowazekiiand mitochondria. [13, 16]. Evolutionary analyses of proteins encoded in the R. prowazekii genome have provided the strongest phylogenetic evidence to date for the view that mitochondria descend from alpha-proteobacteria in particular, Rickettsiales or close relatives[16].

4. FROM ENDOSYMBIONT TO MITOCHONDRION Since mitochondria are eukaryotic organelles that originated from the endosymbiosis of an alpha-proteobacterium, then how did the transition from a free-living cell to a specialized organelle happen? Now, it is known that the endosymbiont has experienced a series of changes such as genome reduction, proteome reduction and expansion, protein relocation, and the origin of the mitochondrial import machinery.

4.1 Genome Reduction A critical step in the transition from an autonomous endosymbiont to an organelle was genome reduction. Contemporary mitochondrial genomes generally encode very few proteins. The fresh water protozoan Reclinomonas americana has the largest number of mitochondrial genes known, which encodes 97 proteins and is over 69kb in size [17]. At the other end of the spectrum, Plasmodium falciparum, the causative agent of malaria, contains the smallest reported mitochondrial genome, which contains only five genes and is only 6kb in size [18]. Metazoan mitochondrial genomes are usually 15–20 kb in size with few exceptions, containing the same 37 genes of which 12 or 13 encode proteins [19]. However, the genome ofR. prowazekii, rickettsia-related ancestor of mitochondria, is approximately 1.1 Mb and contains 834 protein-coding genes [13]. Many ancestor alpha-proteobacteria genes were lost from the endosymbiont during transition into the organelle [20], and most of the retained ones were transferred later became active in the nuclear genome[21, 22]. This is consistent


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with a decreased dependence of the endosymbiont in the eukaryotic host after forming an endosymbiosis.

4.2 Proteome Reduction and Expansion Mitochondria have small genomes but may contain thousands of proteins. For example, the A. thaliana mitochondrion contains only 58 protein-encoding genes, but up to about 2,900 proteins encoded by the A. thaliana nuclear genome carrying signal peptides and finally targeting to mitochondria. Some of these mitochondrial proteins are proved to be encoded by genes of alpha-proteobacterial origin, while some other proteins are encoded by genes that origin from the host[23]. Comparative proteomics studies based on a reconstructed ancestral mitochondrial proteome with the proteomes of alpha-proteobacteria and the yeast and human mitochondrial proteomes revealed that only 10–16% of the modern mitochondrial proteome has an origin that can be traced back to the bacterial endosymbiont [21]. YidC/Oxa/Alb3 super family is a group of conserved translocases that are essential for protein insertion into inner membranes of bacteria and mitochondria, and thylakoid membranes of chloroplasts. Eukaryotic Oxa and Alb3 have two separate prokaryotic origins, but the mitochondrial Oxa clade does not group with proteobacterial YidC clade [24]. Other mitochondrial information processing enzymes, such as RNA polymerase, DNA polymerase, and replicative helicases, is not of alpha-proteobacterial origin, but rather were acquired from an ancestor of T-odd phage early in the evolution of the eukaryotic cell, at the time of the mitochondrial endosymbiosis [25-27]. These disagreements might largely due to the different composition of proteome in present mitochondria. Many ancestor alpha-proteobacteria proteins were lost from the endosymbiont during transition into the organelle, and new proteins of diverse origin (like as nucleus) have been recruited to the function of mitochondrion. For the proteins in the latter case, it‘s no surprise for us to find their origins were not alpha-proteobacteria. For those ancestor alpha-proteobacteria proteins which were kept in present mitochondria, they might also lack an observed relationship with alpha-proteobacterial ancestor. In this case, it might due to the inefficiency of some macromolecules to recover true evolutionary relationships spanning prokaryotes and eukaryotes, due to duplications, horizontal gene transfers and rapid sequence evolution among prokaryotes that has erased the ancient phylogenetic signal [28-31]. The low fraction of proto-mitochondrial proteins in modern mitochondria is the result of the combination of a proteome reduction and a proteome expansion

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process [23, 32]. Firstly, some proteins involved in cell envelope synthesis, such as LPS-biosynthesis or lipid synthesis, have been lost from the mitochondrion and moved to other parts of the cell. Secondly, new proteins of diverse origin have been recruited to the function of mitochondrion, such as the ATP/ADP translocase and most of the mitochondrial protein import machinery[33]. Although the ATPproduction system is derived from the bacterial ancestor, the ATP/ADP translocase has a eukaryotic origin [34]. Perhaps the gaining of ADP/ATP translocase is the most radically affected the role of the mitochondrion within the eukaryotic cell, since it provides mitochondria the ability to exchange ATP with the cell‘s cytoplasm[35] and therefore transformed the endosymbiont into an ATP-exporting organelle in the cell[32, 36]. The fraction of alphaproteobacterial–derived proteins is larger in some classes such as coenzyme metabolism or energy production and conversion than in other classes such as translation or protein turnover and chaperones. The (almost) complete renewal of classes such as signal transduction and classes involved in mitochondrial fission and fusion, which is considered as providing the eukaryotic host with effective control of the mitochondria. Although chloroplasts usually have retained a bacterial-type division machinery, most mitochondria use a completely eukaryotic-derived system [37], something that could have facilitated the control of the number and shape of mitochondria in a cell. In the mitochondrial evolution process, the recruitment to new functions of some proteins already present in the endosymbiont also occurred often. One example is Oxa protein, which has homolog YidC in bacteria, but gain both of cotranslational and post-translational transport capabilities in modern mitochondria[38, 39].

4.3 Protein Relocation Actually a substantial portion of reduced proto-mitochondrion proteins in modern mitochondria have not been deleted from the whole cell, instead, they have been retargeted to other organelles in the course of eukaryotic evolution. RNA polymerase is one of these examples. Nuclear-encoded single subunit RNA polymerases, in plastids of tobacco, maize, A. thaliana and spinach, are believed to be derived by duplication and divergence from the gene for mitochondrial transcription[22, 40]. Comparative genomic analysis demonstrated that non-mitochondrial proteins represent more than 50% (68%, 246 proteins, in human; and 57%, 106 proteins, in yeast) of the total set of alpha-proteobacterial derived proteins in the cell [21]. The


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process of retargeting has also affected some classes more than others. For instance, of the 41 yeast proto-mitochondrial–derived proteins whose mutants specifically impair respiration according to a large-scale analysis in yeast [37], 36 (88%) have a mitochondrial localization. This indicates that most of the respiratory metabolism donated by the mitochondrial ancestor has remained inside the mitochondria. In contrast, larger fractions of carbohydrate and nucleotide metabolic pathways that can be traced back to the proto-mitochondrion have been retargeted during evolution. This fraction includes complete pathways or part of them, such as the initial steps from the synthesis of uridine monophosphate (UMP)[41], which is cytosolic in human and yeast; biotin synthesis and fatty-acid beta-oxidation in yeast, which are cytosolic and peroxisomal, respectively, in human; and lipid synthesis, which is cytosolic in human. A large fraction of metabolic enzymes have been retargeted to other compartments of the cell. It means most of what remains of the proto-mitochondrion is a bacterial-derived metabolism that is under the full control of the eukaryotic proteome[21].

4.4 A Crucial Step: The Origin of the Mitochondrial Import Machinery It is known today in the modern mitochondria; most proteins are encoded by nuclear genes, synthesized in the cytoplasm, and subsequently imported into the organelle. Therefore, the emergence of the protein import system was a crucial step in the evolution of mitochondria [32]. Most of the proteins that function in mitochondria carry short N-terminal targeting sequences, recognized by Tom complex (translocase of the outer membrane of mitochondria) or Tim complex (translocase of the inner membrane of mitochondria). Thus they can be translocated to the outermembrane, intermembrance space, inner membrane or matrix of the mitochondria, follow the commands. Every translocation complex is a complicated machinery consisting of dozens of proteins that function as receptor or channel, as well as soluble chaperones that assist in the process. The complexity of the molecular machinery raises the question of how it was created. Comparative sequence analysis and functional complementation experiments suggest: Some components of the import pathways to be directly derived from the eubacterial endosymbiont's own proteins, for example, molecular chaperones such as mHsp70 and Sec61 complexes are derived from the bacterial chaperones DnaK and SecYEG, respectively [42, 43]; Some of the protein translocation apparatus are created de novo during the coevolution of the host and

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endosymbiont, TOM, TIM23, and TIM22 complexes are such instances, which have no obvious protein translocation counterparts in bacteria[42, 43]; A third class of components are lineage-specific and were incorporated into the process of protein import long after mitochondria was established as an organelle and after the divergence of the various eukaryotic lineages[43]. Only three subunits of the TOM complex, Tom40, Tom7, and Tom22, are found commonly in eukaryotes [44]. It has therefore been hypothesized that the small, primitive TOM complex was operational in the mitochondrial outer membrane of the last common ancestor for all eukaryotes [44, 45]. Additional components for the TOM complex, such as Tom6, Tom70, and Tom20, were derived subsequently to increase the efficiency of protein import. The core modules of the translocation machines function independently so that the function or dysfunction of one, during its development in the course of evolution, would not necessarily affect the development of the others.

4.5 Hydrogenosomes and Mitosome – Atypical Mitochondria? Some anaerobic unicellular eukaryotes contain particular organelles that are atypical in terms of organelle biochemistry or morphology, known as hydrogenosomes[46] or mitosomes[47, 48]. Besides some fungi, they are mainly found in some anaerobic protists such as Giardia intestinalis and Trichomonas vaginalis. Recent evidence suggests a common function shared by hydrogenosome and mitosome, such as iron–sulfur cluster assembly [49], which is usually happened in mitochondria. Several genes that encode the proteins participating in the biosynthesis of FeS clusters have been identified in the genomes of protists that harbor hydrogenosome or mitosome, including G. intestinalis, T. vaginalis[50, 51], C. parvum [52], E. histolytica [53] and E. cuniculi [54]. This suggests that the biosynthesis of critical FeS moieties might be a common functional feature of hydrogenosome, mitosome and mitochondria. That whether hydrogenosome and mitosome are primitive or degenerated mitochondria is a controversial question. Very recent data suggests that hydrogenosomes and mitosomes represent highly evolved mitochondria, which were vertical descended from the original mitochondrial endosymbiont [53, 55, 56]. The processes of protein gain, loss, and retargeting have acted in a lineagespecific and selectively manner, resulting in the composition differences of mitochondria proteome in different species[21]. There are three reasons for this proposal: 1) the presence of the limiting double membrane that surrounds these


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mitochondrion-related organelles; 2) the functionally conserved mechanisms of protein import into hydrogenosomes, mitosomes and mitochondria; 3) the independent evolution of these mitochondrion-related organelles in a range of unrelated eukaryotic lineages [57]. However, it‘s still a short time since the discovery of hydrogenomes (4 decades) and mitosomes (5 years). Much remains to be investigated about the true color of hydrogenosome and mitosome, including physiology, metabolic, evolutionary status and so on. Nowadays the endosymbiotic theory is widely accepted by mainstream scientists for explaining the origin of mitochondrion. The evidence gathered together support that proteobacteria, in particular Rickettsiales or close relatives, are most likely ancestor of mitochondria. The process and mechanisms of the transform from endosymbiont to mitochondrion are revealed more and more clearly.

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Chapter 4

INTERPRETING DIVERSITY OF PROTEOBACTERIA BASED ON 16S RRNA GENE COPY NUMBER Konstantinos Ar. Kormas* Department of Ichthyology & Aquatic Environment, School of Agricultural Sciences, University of Thessaly, 384 46 Nea Ionia, Magnisia, Greece

ABSTRACT The application of the 16S rRNA gene diversity analysis has revealed the immense microbial diversity of our planet. At the same time, and after of more than two decades of using this methodology along with several important improvements and new techniques, there is still no universal golden rule on how to estimate prokaryotic diversity in a natural sample, as there is in macroecology. A general assumption during studies of prokaryotic diversity is that each found 16S rRNA gene found corresponds to one cell. However, in this paper it is shown that recent genomic data reveal that this is not the case for several bacterial phyla. Since the Proteobacteria, along with the Firmicutes, are the most abundant and diverse bacterial phyla, in this paper the average 16S rRNA gene copy number is presented at the subphylum (α-, β-, γ-, δ- and ε-Proteobacteria), order and family level of the Proteobacteria phylum. At the sub-phylum level the average 16S rRNA gene copy number varied between 2.1±1.3 and 5.8±2.8. Since the 16S rRNA gene * Correspondence [email protected]

Information:

Tel.:

+30-242-109-3082,

+30-242-109-3157,

E-mail:

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Konstantinos Ar. Kormas copy number affects the relative abundance of each proteobacterial species/phylotype found in a clone library, and subsequently the estimation of diversity, the corrected relative abundances of the found proteobacterial phylotypes were estimated in 37 clone libraries from six different natural habitats. It is suggested, that at least in the cases where Proteobacteria consist 50-75% of the clone library, the corrected abundances should be used for diversity estimations.

The cultivation of microorganisms from the environment was developed for studying purposes and is a fully accepted, well established and continuously improving methodology. However, cultivation is a human innovation and it does not imply that all microorganisms necessarily will have to conform with our cultivation efforts, since in natural conditions the microorganisms rarely -if everencounter cultivation-like conditions. Thus, cultivation-independent approaches (Nocker et al. 2007, Orphan 2009), seem to be more appropriate for the study of the diversity of microorganisms and their communities‘ structure in the environment. The analysis of the 16S rRNA gene diversity, amplified directly from environmental samples, is considered a proxy for the occurrence of prokaryotic species. It is the oldest methodology for the investigation of microbial diversity from practically every natural Earth habitat and has been the most widely used approach over the last two decades (Woese et al. 1990). Microbial, namely prokaryotes and microscopic eukaryotes, taxonomy, biology and ecophysiology has benefited enormously by such studies and the race to unravel as much as possible of the hidden microbial diversity remains a fascinating and rewarding scientific field (Cases & de Lorenzo 2002). In order to assess diversity, it is essential to securely identify the members of the community or population and also their abundances or relative abundances (Bunge 2009). Although in the macroscopic world this is assured by species identification -in most cases based on morphological features- and measuring their abundance, the concept of species in the microbial world is still under debate (Ward 1998, Roselló-Mora & Amann 2001, Konstantinidis et al. 2006). This obstacle has been dealt with the use of phylotypes or operational taxonomic units (OTU) based on 16S rRNA gene sequencing and comparison of similarity (Stackebrandt & Goebel 1994), in most cases with a similarity cut-off limit between 97 and 99% being used. This approach, assumes either that each of the found 16S rRNA phylotypes corresponds to one prokaryotic cell or that it is unknown to how many cells it corresponds and, thus, only semi-quantitative estimations can be inferred. To a degree this approach is justified by the fact that in most studies there are several

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unknown phylotypes that are distantly similar to any of the known (cultivated) species. However, for the cases of high similarity with known species, the ample genomic data that is available might be of help. I use the case of the Proteobacteria as is the most diverse and abundant phylum of the domain Bacteria in nature, along with the Firmicutes, and are found almost everywhere on Earth (Kersters et al. 2006). This paper aims at depicting that the 16S rRNA gene copy numbers for all the available Proteobacteria genomes, varies significantly at different taxonomic levels and shows which taxon provides more safe data for the relative abundance of the 16S rRNA genes in a clone library. The 16S rRNA gene copy number should be taken into account when estimating Proteobacteria diversity from inferred phylogenies as a proxy to corrected relative abundances of the cells that bear them. The only assumption is that for the species with multiple 16S rRNA gene copies, all these copies are identical, although few cases exist which this is not true but recent data suggest that the Proteobacteria are rather on the low range of intra-gene diversity (0.00 – 2.09%, average 0.20%, Pei et al. 2010). These values are very close to the generally accepted threshold of 1.0 – 1.3% difference between two 16S rRNA genes in order to be considered representing two different bacterial species (Stackenbrandt & Ebers 2006). Considering this limitation, this paper aims only to depict that the general rule ―one 16S rRNA for every prokaryotic species‖ which is ubiquitously used and is never stated even as a possible factor of misinterpretation of data in microbial diversity studies, is far from real for the Proteobacteria. It also attempts to reveal trends in the mean number of Proteobacteria taxa so these can be used for the estimation of more realistic values of Proteobacteria diversity. In total, 485 genomes of the phylum Proteobacteria were analysed in this study (Table 1). The 16S rRNA gene copy number for each strain was retrieved from the Microbial Genome Resources (http://www.ncbi.nlm. nih.gov/genomes /MICROBES/microbial_taxtree.html). Only completed genomes as of 01 February 2010 were considered. The δ-Proteobacteria (Emerson et al. 2007) are not discussed here since there is only one cultivated representative from this subphylum (Table 1). A first glance on the distribution of 16S rRNA gene copy number among the available Proteobacteria genomes, shows that four out the five sub-phyla have multiple gene copy numbers. Only in the α-Proteobacteria, 41.2% of the available genomes have one copy of this gene while for the β-, γ- δ- and ε-Proteobacteria this percentage falls gradually, 9.7%, 8.2%, 5.9% and 3.8%, respectively (Figure 1).

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Konstantinos Ar. Kormas

Table 1. Number of Genomes Per Taxon of the Phylum Proteobacteria Used in this Study. Number of Yet-unclassified Taxa in Parentheses. Sub-phylum αβγδεδTotal

Orders 6 7 (1) 17 (5) 4 4 (2) 1 39

Families 19 12 (3) 26 (5) 9 5 (2) 1 72

Genera 49 32 68 18 9 1 177

Species 33 52 115 29 16 1 6

Total genomes 119 72 220 33 26 1 485

The available genomes of the Proteobacteria, which were used in this study, do not cover equally all the taxa of the phylum and, it is most likely that their representation in the database is irrelevant and accidental with their distribution in nature (Lagesen et al. 2010). For example, there are cases where one family includes genomes only of very few of its genera. In addition, the advent of molecular biology technologies and their applications in microbial ecology the last two decades (Amann et al. 1995) have created a rather large number of ―yetunaffiliated‖ taxa, i.e. taxa which either do not include a cultured representative or high-level taxa which include only one or very few cultured representatives. Such limitations, render any effort to apply universal rules regarding the 16S rRNA gene copy number per taxon rather impossible at the time being. Instead it depicts the need for incorporating the 16S rRNA gene copy number in studies of microbial diversity, by applying this approach to the ubiquitous phylum of Proteobacteria, since several of its members are far from having only one 16S rRNA gene copy. The 119 genomes of the α-Proteobacteria represent six orders, 19 families, 49 genera and 33 species of the sub-phylum (Figure 2). The mean copy number of the sub-phylum is 2.1±1.3. Only the Rickettsiales, which includes members with the smallest genomes known to date, and its three families seem to have only one copy of the 16S rRNA gene. The rest of the five orders vary from 1.5±0.7 (Caulobacterales) to 3.6±1.0 (Rhodospirillales). Among these, the Rhizobiales contains the highest number of families, which reflects its wide distribution.


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Figure 1. Distribution of available genomes (01 Feb. 2010) of Proteobacteria according to the copy number of the 16S rRNA gene.

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Figure 2. Variance of the 16S rRNA gene copy number among the α-Proteobacteria orders (top) and families (bottom). The ratio of the number of genera with available genomes in GenBank to the number of genera in Bergey‘s Manual (Staley et al. 2005) is shown above each column; the number of genera not included in Bergey‘s Manual is in parentheses. Standard deviation is indicated in error bars. No / indicates absence from the Bergey‘s Manual.


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Figure 3. Variance of the 16S rRNA gene copy number among the β-Proteobacteria orders (top) and families (bottom). The ratio of the number of genera with available genomes in GenBank to the number of genera in Bergey‘s Manual (Staley et al. 2005) is shown above each column; the number of genera not included in Bergey‘s Manual is in parentheses. Standard deviation is indicated in error bars. No / indicates absence from the Bergey‘s Manual.

In the nine Rhizobiales families, the mean 16S rRNA gene copy number varies significantly from 1.0±0.0 (Aurantimonadaceae) to 5.3±0.9 (Methylobacteraceae). The data from the families Bradyrhizobiaceae (1.6±0.5), Bartonellaceae (2.0±0.0), Brucellaceae (2.5±0.7) and Rhizobiaceae (2.8±0.4) correspond to ≥50% of the known genera of each family and, thus, it is possible to consider their mean 16S rRNA gene copy numbers as representative for each of these families. The number of available genomes within the β-Proteobacteria sub-phylum is smaller (72), representing seven orders, 12 families, 32 genera and 52 species (Table 1). The mean 16S RNA gene copy number of the sub-phylum is

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3.7±1.7, which implies that for this phylum is rather far from realistic to consider it having one copy of the 16S rRNA gene. Six out of the seven subphylum‘s orders include genomes from only one family (Figure 3) and from these families, only for the Nitrosomonadaceae and the Hydrogenophilaceae the available genomes cover ≥50% of the known genera of each family. The Burkholderiales is the dominating order among the β-Proteobacteria available genomes, covering six families, two of which are novel and yet-unclassified. These families show a highly variable mean 16S rRNA gene copy number (1.0±0.0 – 4.5±1.5) but they represent a low number of their known genera, with the exception of the Burkholderiaceae (50%). Thus, at the family level it is not secure yet to assume specific average 16S rRNA copy numbers, as more genomes from more genera of the Burkholderiaceae families are required. The γ-Proteobacteria sub-phylum includes the highest number of available genomes (220; Table 1), obviously due to the wide distribution of its members and their importance for the human health and plants/animals of human interest. The available genomes originate from 17 orders, 26 families, 68 genera and 115 species. As a sub-phylum, it is very unlikely to be considered as a single 16S rRNA gene copy taxon due to its high average value is 5.8±2.8 (Figure 4) which is the highest among all the Proteobacteria sub-phyla. Thirteen orders include single family representatives with available genomes. Only the Moraxellaceae family is well represented (66.7% of the family‘s known genera), whose average number of 16S rRNA gene copies is 5.4±1.0. Each of the four orders of the δ-Proteobacteria sub-phylum with available genomes (33 in total, Table 1) includes at least two families (Figure 5). The available genomes of this sub-phylum point towards a multiple 16S rRNA gene copies group (3.0±1.5). Although most of the families are not satisfactory represented in terms of available genomes of their known genera, the Desulfovibrionaceae (3.5±1.3), the Myxococcaceae (2.4±0.9) and the Syntrophaceae (1.0+2.0) are fairly well represented (≥50%). The ε-Proteobacteria contains the smallest number of available genomes (26, Table 1) but the sub-phylum‘s average 16S rRNA gene copy number is 2.8±0.8. The available genomes refer to the order Campylobacterales and three other orders which are not included in the Bergey‘s Manual (Staley et al. 2005) and two of them are still unclassified (Figure 6). This is due to the increased scientific interest of the last 10 years which resulted in major breakthroughs for this sub-phylum (Campbell 2006). The two families of the Campylobacterales, namely Campylobacteraceae (3.2±0.6) and Helicobacteraceae (2.2±0.8), have available genomes for more of the 2/3 their

Figure 4. Variance of the 16S rRNA gene copy number among the γ-Proteobacteria orders (top) and families (bottom). The ratio of the number of genera with available genomes in GenBank to the number of genera in Bergey‘s Manual (Brenner et al. 2005) is shown above each column; the number of genera not included in Bergey‘s Manual is in parentheses. Standard deviation is indicated in error bars. No / indicates absence from the Bergey‘s Manual.

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Figure 5. Variance of the 16S rRNA gene copy number among the δ-Proteobacteria orders (top) and families (bottom). The ratio of the number of genera with available genomes in GenBank to the number of genera in Bergey‘s Manual (Staley et al. 2005) is shown above each column; the number of genera not included in Bergey‘s Manual is in parentheses. Standard deviation is indicated in error bars. No / indicates absence from the Bergey‘s Manual.

Interpreting Diveristy of Proteobacteria Based on 16S rRNA…

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Figure 6. Variance of the 16S rRNA gene copy number among the ε-Proteobacteria orders (top) and families (bottom). The ratio of the number of genera with available genomes in GenBank to the number of genera in Bergey‘s Manual (Staley et al. 2005) is shown above each column; the number of genera not included in Bergey‘s Manual is in parentheses. Standard deviation is indicated in error bars. No / indicates absence from the Bergey‘s Manual.

known genera, suggesting that the use of a more than one copy of the 16S rRNA gene should be used when phylotypes from any sample are affiliated within these two families. It is indicative for this sub-phylum that only one genome (Helicobacter hepaticus ATCC 51449) was found to have single copy of its 16S rRNA gene. From the above it is evident that the assumption that each found 16S rRNA phylotype in a clone library corresponds to one genome and, subsequently, to one cell, is not valid. Even if one considers the case of a very specialised communityconsisting practically only from members of the α-

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Proteobacteria, which have the lowest average 16S rRNA gene copy number, one could ask whether it is safe or not to assume that each found 16S rRNA phylotype corresponds to one cell, in order to calculate some of the most commonly used diversity indices. The answer is probably negative, based on the available data, since just the lower end of the SD for the sub-phylum (0.8) is practically 1. Table 2. Percentage of Proteobacteria Relative Abundance in Different Clone Libraries. Clone library origin Kazan mud volcano sediments Amsterdam sediments Drinking network

mud water

% Proteobacteria 56.3 – 78.0

Reference Pachiadaki (2010)

et

al.

volcano

15.5 – 66.7

Pachiadaki et al. (in prep.)

distribution

72.4 – 73.6

Kormas et al. (2010a)

19.3 – 44.3

Lymperopou et al. (in prep.)

70.2

Demiri et al. (2009)

Drinking water reservoir Mudshrimp Pestarella tyrrhena gut Norway lobster norvegicus gut

9.9 – 100.0

Meziti et al. (in press)

Terrestrial thermal springs

21.1 – 98.2

Kormas et al. (2009)

Lake water and sediment

30.5 – 37.8

Kormas et al. (2010b)

Nephrops

To further test this issue, the effect of the 16S rRNA gene copy number was investigated in already constructed clone libraries originating from diverse aquatic habitats (Table 2): Twelve clone libraries from the sediments of two active marine mud volcanoes, the Kazan MV (Pachiadaki et al. 2010) and the Amsterdam MV (Pachiadaki, de Lange, Kormas in prep). Two clone libraries from an urban drinking water distribution network (Kormas et al. 2010a).


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Eight clone libraries from a drinking water reservoir (Lymperopoulou, Kormas, Karagouni, in prep.). One clone library from the mudshrimp Pestarella tyrrhena gut (Demiri et al. 2009) and eight clone libraries from the Norway lobster Nephrops norvegicus gut (Meziti et al. in press). Five clone libraries from terrestrial thermal springs (Kormas et al. 2009). Two clone libraries from lake water and sediment bacterial communities (Kormas et al. 2010b). Overall, the relative abundance of all Proteobacteria in the clone libraries from the above studies varied between 9.9% and 100.0%. For each clone library, the Shannon H index was calculated according to the Shannon–Wiener index H and was calculated as follows: H = Σ [pi ln(pi)] where the summation is over all phylotypes i and pi is the proportion of phylotypes relative to the sum of all phylotypes (Shannon & Wiever 1949). H was estimated by assuming the ―one 16S rRNA gene for one cell‖ hypothesis (Hsin) and then by correcting the relative abundance of each Proteobacteria phylotype according to the mean 16S rRNA gene copy number that is valid for the sub-phylum that it belongs (Hcorr), as these were mentioned above. The impact of this difference depends upon the overall abundance of the Proteobacteria in each clone library (Figure 7). When a clone library contains 75%, no clear trend was observed, since in such libraries what is really important is whether they are dominated from one or more sub-phyla. In the former case, ΔH remains zero, while in the latter depends on how many sub-phyla co-exist and which one of is dominating.

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Figure 7. The relationship of the difference between the Shannon-Wiener diversity H calculated with single copy 16S rRNA gene (Hsin) and corrected for each of the Proteobacteria sub-phyla (Hcorr) vs. the relative abundance (%) of Proteobacteria in environmental clone libraries (see details in the text).


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In conclusion, this paper showed that the widely used assumption that each found 16S rRNA gene corresponds to one cell is not always valid based on the available genomic data for the case of the Proteobacteria. In particular, each sub-phylum has different ranges and average 16S rRNA gene copy numbers and this number varies, also, at the order and family level. By applying the average 16S rRNA gene copy number for each phylum, it was shown that in cases of moderate to high (50-75%) of Proteobacteria abundance, the use of this correction results in different diversity estimations, giving a different picture of the bacterial community. More available genomes from the Proteobacteria, but also from other bacterial phyla as well, are needed in order to will assist us in more accurate and pragmatic estimations of bacterial diversity in natural samples.

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Chapter 5

A HARSH LIFE TO INDIGENOUS PROTEOBACTERIA AT THE ANDEANMOUNTAINS: MICROBIAL DIVERSITY AND RESISTANCE MECHANISMS TOWARDS EXTREME CONDITIONS Virginia Helena Albarracín 1,2, Julián Rafael Dib1,3, Omar Federico Ordoñez1 and María Eugenia Farías*1 1

Laboratorio de Investigaciones Microbiológicas de Lagunas Andinas (LIMLA), Planta Piloto de Procesos Industriales Microbiológicos (PROIMI), CCT, CONICET, Tucumán, Argentina 2 Facultad de Ciencias Naturales e Instituto Miguel Lillo, Universidad Nacional de Tucumán, Tucumán, Argentina 3 Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán,Tucumán, Argentina

SUMMARY High-altitude Andean lake (HAAL) ecosystems of the South American Andes are almost unexplored systems of shallow lakes formed * Corresponding author: María Eugenia Farias, LIMLA-PROIMI-CCT, Av. Belgrano y Pasaje Caseros. 4000 Tucumán, Argentina. Tel: +54-381-4344888 Int. 24. Fax: +54-381-4344887 www.limla.com.ar

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V. Helena Albarracín, J. Rafael Dib, O. Federico Ordoñez et al. during the Tertiary geological period, distributed in the geographical area called the Puna at altitudes from 3,000 to 6,000 m above sea level, and isolated from direct human activity. They present a broad range of extreme conditionswhich makes the indigenous microbial communities exceptionally interesting to study physiological mechanisms of adaptation to chemical and physical stresses such as hypersalinity and high levels of UV radiation.Previous work have revealed the outstanding diversity of these environments, being Proteobacteria the most extended and best represented microbial taxa within the extremophilic communities. The aim of this work is to review the microbial diversity of Proteobacteria present at the HAAL and to describe their multiple resistance properties towards the extreme factors that these microbial communities thrived in their natural environments. A special reference to the representatives of the genus Acinetobacter found at the HAAL is also presented. Due to the isolation program held at LIMLA (www.limla.com.ar) during the past four years a one-of-a-kind collection of extremophilic strains from the HAAL was assembled. HAAL microbial diversity was investigated by sampling bacterioplankton, benthonic microorganisms, microbial-mat associated microbes as well as gastrointestinal symbiotic organisms from flamingoes living at the lakes. Representatives of Proteobacteria has been profusely isolated from these samples, more exactly from Lakes: Azul, Verde, Negra, Vilama, Aparejos, Chaxas, Salina Grande, Socompa, Dead Man Salar, Tolar Grande, Brava, Diamante, Huaca-Huasi, all of them located above 4000 m, at the Northwest of Argentina. In addition, a more extended coverage of Proteobacteria was detected by non-culture dependent techniques (mainly DGGE), suggesting that much more efforts will be needed to isolate most novel Proteobacteria present at the HAAL. Within Proteobacteria, all the four main groups were represented in our culture collection being the Gammaproteobacteria the class with better coverage. The gammaproteobacteria strains were classified as belonging mainly to Pseudomonas Acinetobacter, Halomonas, Stenotrophomonas, Moraxella, Enterobacter, Serratia, Salinivibrio, Pseudoalteromonas, Aeromonas and Marinobacter. 16S rDNA gene sequence comparison of some isolates with the ones presented at the database indicated anidentity lower that 94%, which should point out that these extremophilic communities harbour yet unraveled species. The extreme conditions suffered by these microorganisms at the HAAL made them resistant to factors present as well as not present in their natural environments. Exposure to UV-B radiationduring 24 h revealed that most isolates were highly resistant: 33.3% of betaproteobacteria, 44.4% of gammaproteobacteria, 40% of alphaproteobacteria were able to survive through the whole exposition time. In addition, resistance to hipersalinity in most isolates was also observed.

A Harsh Life to Indigenous Proteobacteria at the AndeanMountains: … 93 Interestingly, antibiotic resistance was also observed in spite of the pristinely and isolation of these lakes. In light of the great adaptability strength of the strains to changing conditions in their original environment, antibiotic resistance may be considered as a consequence of a high frequency of mutational events, which also, may be enhanced by the intense solar irradiation present at the HAAL (UV index in summer: 16- 18). A special reference can be made to the representatives of the genus Acinetobacter isolated from the HAAL. Most of these strains appeared to have multiple resistance profiles to hipersalinity, UV-B irradiation, antibiotics and even arsenic. These ―superbugs‖ can be subjected to further studies as they can be clues to discover new ways of surviving at extreme conditions, a matter that has applications in astrobiology. On the other hand, it will be very interesting to further research on these strains biotechnological potential because as extremophiles they can be source of novel bioactive compounds.

Key words: extremophiles, High-AltitudeLakes, Proteobacteria

PUNE-ANDINE HIGH-ALTITUDE LAKES: A DIVERSE SOURCE OF POLI-EXTREMOPHILIC MICROORGANISMS Extreme environments are defined as habitats that experience steady or fluctuating exposure to one or more environmental factors, such as salinity, osmolarity, desiccation, UV radiation, barometric pressure, pH, and temperature. Under these physical conditions human lifecan not be possible. Nevertheless, some microorganisms can colonize these extreme environments and they are called extremophiles; this group includes representatives of all three domains (Bacteria, Archaea, and Eukarya); they are categorized into subgroups according to the specific environmental characteristics of their habitats, i.e. psycrophilic, thermophilic, halophilic, alkalophilic, acidophilic (Seufferheld et al., 2008). Extreme environments have been subject to intensive studies focusing attention on the diversity of organisms and molecular and regulatory mechanisms involved. The products obtainable from extremophiles such as proteins, enzymes (extremozymes) and compatible solutes are of great interest to biotechnology. Examples include biochemicals used for detergent formulations, leather and paper processing, biofuels, bioremediation, UVblocking, and new antibiotics (Sanchez et al., 2009; Bowers et al., 2009). Nevertheless, potentially beneficial biomolecules still remain to be discovered

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V. Helena Albarracín, J. Rafael Dib, O. Federico Ordoñez et al.

from unexplored extreme environments. This field of research has also attracted attention because of its impact on the possible existence of life on other planets (Rothschild and Mancinelli, 2001, Cavicchioli, 2002).

Figure 1. Six extreme lakes were proteobacteria diversity was mainly studied.

Typical examples of extreme environments are the HighAltitudeAndeanLakes (between 3,000 and 6,000 masl) at the northwest of Argentina in the Puna and Andean regions (Fig. 1). Most of these wetlands are completely isolated, experience a wide daily range in temperatures (35 ºC), are slight saline to hypersaline, and are subject to low phosphate availability and to high intensity of solar UV-B radiation (Table 1). Microbial communities living in such aquatic ecosystems are tolerant to large fluctuations in environmental factors in addition to steady-state extreme conditions (Fernandez Zenoff et al., 2006; Zenoff et al., 2006; Dib et al., 2008). These ecosystems have demonstrated to be a great source of microbial diversity and

A Harsh Life to Indigenous Proteobacteria at the AndeanMountains: … 95 interesting strategies that allow microorganisms to survive under severe conditions. Table 1.Characteristics ofHigh altitude Andean lake (HAAL) in Argentina:L. Aparejos, L. Negra, L. Verde, L. Azul, L. Vilama, Salina Grande and L. Socompa.

Wetland Geographic position

L. Aparejos L. Negra L. Verde

L. Azul

L. L. Socom Vilama SalinaGrande pa

Catamarca Catamarca Catamarca Catamarca Jujuy

22º 35´S Global position 27º 34´S 27º40´S 27º 38'S 27º 38´S 66º 68º 23´W 68º 23´W 68º 32' W 68º 32´W 55´W Depth (cm) 10 20 20 100 20 Altitude (masl) 4,200 4,400 4,400 4,400 4,600 pH 6.5 6.8 6.7 7.5 7.1 Arsenic (mg L1 ) 2.5 3 0.8 0.8 11.8 Phosphorus ND**

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