Brock/ Springer Series in Contemporary Bioscience

Genetics and Molecular Biology of Anaerobic Bacteria

Brock/ Springer Series in Contemporary Bioscience

Series Editor: Thomas D. Brock University of Wisconsin-Madison

Tom Fenchel ECOLOGY OF PROTOZOA: The Biology of Free-living Phagotrophic Protists

Johanna Dobereiner and Fabio O. Pedrosa NITROGEN-FIXING BACTERIA IN NONLEGUMINOUS CROP PLANTS

Tsutomu Hattori THE VIABLE COUNT: Quantitative and Environmental Aspects

Roman Saliwanchik PROTECTING BIOTECHNOLOGY INVENTIONS: A ( uide for Scientists

Hans G. Schlegel and Botho Bowien (Editors) AUTOTROPHIC BACTERIA

Barbara Javor HYPERSALINE ENVIRONMENTS: Microbiology and Biogeochemistry

Ulrich Sommer (Editor) PLANKTON ECOLOGY: Succession in Plankton Communities

Stephen R. Rayburn THE FOUNDATIONS OF LABORATORY SAFETY: A Guide for the Biomedical Laboratory

Gordon A. McFeters (Editor) DRINKING WATER MICROBIOLOGY: Progress and Recent Developments

Mary Helen Briscoe A RESEARCHER'S GUIDE TO SCIENTIFIC AND MEDICAL ILLUSTRATIONS

Max M. Tilzer and Colette Serruya (Editors) LARGE LAKES: Ecological Structure and Function

Jurgen Overbeck and Ryszard ]. Chr6st (Editors) AQUATIC MICROBIAL ECOLOGY: Biochemical and Molecular Approaches

John H. Andrews COMPARATIVE ECOLOGY OF MICROORGANISMS AND MACROORGANISMS

(Continued after index)

Madeleine Sebald Editor

Genetics and Molecular Biology of Anaerobic Bacteria

With 186 Figures

Springer-Verlag New York Berlin Heidelberg London Paris

Tokyo Hong Kong Barcelona Budapest

Madeleine Sebald Unite des Anaerobies Institut Pasteur 75724 Paris, France

Cover photograph: Clostridium bifermentans DSM 631. Reproduced from The Prokaryotes, Second Edition, Vol. II. p. 1803. © 1992 Springer-Verlag, New York.

Library of Congress Cataloging-in-Publication Data Genetics and molecular biology of anaerobic bacteria / Madeleine

Sebald, editor. p. cm. - (Brock/Springer series in contemporary bioscience)

Includes bibliographical references and index.

ISBN 978-1-4615-7089-9 ISBN 978-1-4615-7087-5 (eBook) DOl 10.1007/978-1-4615-7087-5

1. Anaerobic bacteria-Genetics. 2. Anaerobic bacteria-Molecular Aspects. I. Sebald, Madeleine. II. Series. QR89.5.G46 1992 589.9'01'5-dc20 91-28037

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© 1993 Springer-Verlag New York, Inc.

Softcover reprint of the hardcover 1st edition 1993

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9 8 7 6 5 4 321

Preface

The field of bacterial genetics has been restricted for many years to Escherichia coli and a few other genera of aerobic or facultatively anaerobic bacteria such as Pseudomonas, Bacillus, and Salmonella. The prevailing view up to recent times has been that anaerobic bacteria are interesting organisms but nothing is known about their genetics. To most microbiologists, anaerobic bacteria appeared as a sort of distant domain, reserved for occasional intrusions by taxonomists and medical microbiologists. By the mid-1970s, knowledge of the genetics and molecular biology of anaerobes began to emerge, and then developed rapidly. This was the result of advances in molecular biology techniques, but also im­portantly because of improvements in basic techniques for culturing anaerobes and for understanding their biochemistry and other areas of in­terest. Investigations in this field were also stimulated by a renewal of interest in their ecology, their role in pathology and in biotransformations, and in the search for alternative renewable sources of energy.

The initial idea for this book came from Thomas D. Brock. When Dr. Brock requested my opinion about two years ago on the feasibility of publishing a book on the genetics of anaerobic bacteria, as a part of the Brock/Springer Series in Contemporary Bioscience, I answered positively but I was apprehen­sive about assuming the role of editor. However, I was soon reassured by the enthusiastic commitment of those I approached to contribute. Eventually, thanks to the caring cooperation of the contributors, the task became relatively easy.

I have tried to include most of the available data on the genetics and molecu­lar biology of both developing and developed topics, and it is my hope that this book will furnish up-to-date information both to those entering and to those already involved in anaerobic microbiology. I also hope it will show that anaerobes are less distant than may be perceived.

Finally, I should like to express my gratitude to Thomas D. Brock, to the contributors, and also to the reviewers. It is a pleasure to acknowledge the assistance of Morris Goldner of the University of Toronto in the editorial pro-cess.

Madeleine Sebald

v

Contents

Preface v Contributors xiii Introduction xix

1 Plasmids, Phages, and Gene Transfer in Methanogenic Bacteria 1 Thomas Leisinger and Leo Meile

2 Methanogen Genes and the Molecular Biology of Methane Biosynthesis 13 John R. Palmer and John N. Reeve

3 Genes for Stable RNAs and Their Expression in Archaea 36 Michael Thomm and Winfried Hausner

4 Molecular Biology of the Acetoclastic Methanogen Methanothrix soehngenii 54 Rik I.L. Eggen and Willem M. de Vos

5 Mutations 64 Madeleine Sebald

6 Conjugative Gene Transfer in Clostridia 98 Michael Young

7 Transformation and Electrotransformation in Clostridia 111 Gilles Reysset

8 Vectors for Use in Clostridium acetobutylicum 120 Nigel P. Minton, Tracy-Jane Swinfield,

vii

viii Contents

John K. Brehm, Sarah M. Whelan, and John D. Oultram

9 Antibiotic Resistance Determinants of Clostridium perfringens 141 Julian I. Rood

10 Genetics and Molecular Biology of Antibiotic Resistance in Clostridium difficile: General and Specific Overview 156 Herbert Hachler and Fritz H. Kayser

11 Genetics and Molecular Biology of Chloramphenicol Acetyltransferase of Clostridium butyricum 174 Walter L. Staudenbauer and Wolfgang Dubbert

12 The Role of Bacteriophages and Plasmids in the Production of Toxins and Other Biologically Active Substances by Clostridium botulinum and Clostridium novyi 179 Mel W. Eklund

13 Molecular Biology of Clostridial ADP-Ribosyltransferases and Their Substrates 195 Klaus Aktories, Gertrud Koch, and Ingo Just

14 Gene Cloning and Organization of the Alpha-Toxin of Clostridium perfringens 211 Richard W. Titball, Helen Yeoman, and Sophie E.C. Hunter

15 Gene Cloning, Organization, and Expression of 6-Toxin of Clostridium perfringens 227 Rodney K. Tweten

16 Molecular Biology of Clostridium perfringens Enterotoxin 235 Per Einar Granum and Gordon S.A.B. Stewart

17 Molecular Genetic Studies of UV-Inducible Bacteriocin Production in Clostridium perfringens 248 Stewart T. Cole and Thierry Garnier

18 Genome Mapping of Clostridium perfringens Type A Strains 255 Stewart T. Cole and Bruno Canard

Contents ix

19 Molecular Biology of the Clostridium difficile Toxins 264 Christoph von Eichel-Streiber

20 Gene Cloning and Expression in Escherichia coli of Clostridial Sialidases 290 Peter Roggentin and Roland Schauer

21 Cloning and Expression of Clostridium acetobutylicum Genes Involved in Carbohydrate Utilization 301 Peter Verhasselt andJos Vanderleyden

22 Cloning and Expression of Clostridium acetobutylicum Genes Involved in Solvent Production 317 George N. Bennett and Daniel J. Petersen

23 Molecular Analysis of Glutamine Synthetase Genes and Enzymes of Clostridium and Bacteroides 344 David R. Woods

24 Phospholipid Biosynthetic Enzymes of Butyric Acid-Producing Clostridia 354 Howard Goldfine

25 The Clostridium pasteurianum Ferredoxin Gene 363 Jesse C. Rabinowitz

26 Organization of the Nitrogen Fixation Genes in Clostridium pasteurianum 373 John L. Johnson, Shu-Zhen Wang, and Jiann-Shin Chen

27 Cloning and Sequencing of the Clos~ridium pasteurianum Genes Encoding Molybdenum-Pterin Binding Proteins 382 Stephen M. Hinton

28 Cloning, Sequencing, and Expressions of Genes Encoding Enzymes of the Autotrophic Acetyl-CoA Pathway in the Acetogen Clostridium thermoaceticum 389 Thomas A. Morton, Chih-Fong Chou, and Lars G. Ljungdahl

x Contents

29 Genes and Proteins Involved in Cellulose Degradation by Mesophilic Clostridia 407 Jean-Pierre Belaich, Anne Belaich, Christian Gaudin, and Chantal Bagnara

30 Genes and Proteins Involved in Cellulose and Xylan Degradation by Clostridium thermocellum 412 Jean-Paul Aubert, Pierre Beguin, and Jacqueline Millet

31 Nucleotide Sequence of the Gene and Primary Structure of the Thermophilic p-Amylase from Clostridium thermosulfurogenes 423 Hideo Yamagata and Shigezo Vdaka

32 The a-Amylase-Pullulanase (apu) Gene from Clostridium thermohydrosulfuricum: Nucleotide Sequence and Expression in Escherichia coli 432 Hannes Melasniemi

33 Molecular Biology of Xylan Utilization by Thermoanaerobes 443 Michael Bagdasarian, Yong-Eok Lee, Chanyong Lee, Menghsiao Meng, and J. Gregory Zeikus

34 Genetics and Molecular Biology of Sulfate-Reducing Bacteria 456 Gerrit Voordouw and Judy D. Wall

35 Gene Transmission, MLS, and Tetracycline Resistance in Bacteroides 474 Francis L. Macrina and C. Jeffrey Smith

36 Transfer of Beta-Lactam Antibiotic Resistance in Bacteroides 490 Kunitomo Watanabe and Kazue Veno

37 Genetics of 5-Nitroimidazole Resistance in Bacteroides 494 Gilles Reysset, Wen-Jin Su, and Madeleine Sebald

38 Genetics of Polysaccharide Utilization Pathways of Colonic Bacteroides Species 505 Abigail A. Salyers, Peter Valentine, and Vivian Hwa

Contents xi

39 Molecular Biology of the Fimbriae of Dichelobacter (Previously Bacteriodes) nodosus 517 John S. Mattick, Matthew Hobbs, Peter T. Cox, and Brian P. Dalrymple

40 Genetic Exchange in Pigmented Bacteroides 546 Donald G. Guiney

41 Porphyromonas gingivalis: Gene Cloning of Determinants of Pathogenicity 552 Barry C. McBride, Umadatt Singh, and Angela Joe

42 Cloning, Structure, and Expression of Genes of the Anaerobic Rumen Bacteria 569 RM. Teather, H.J. Gilbert, and G.P. Hazlewood

43 Antigenic Characterization, Taxonomy, and Genetics of Treponema hyodysenteriae 586 R Sellwood

44 Nucleic Acid Hybridization for Identification and Detection of Gram-Negative Anaerobes 605 Ulf B. Gobel and Klaus Pelz

45 Molecular Biology of Bile Acid 7a-Dehydroxylation in an Intestinal Eubacterium Species 618 Darrell H. Mallonee and Phillip B. Hylemon

46 Cloning and Expression in Escherichia coli of Three Amylase Genes of a Strictly Anaerobic Thermophile, Dictyoglomus thermophilum, and 629 Their Nucleotide Sequences Sueharu Horinouchi and Teruhiko Beppu

47 A Novel Class of Industrially Important Debranching Enzymes: The Thermoanaerobic Amylopullulanases 640 Robert D. Coleman

Appendix: A List of Strict Anaerobes 654 J. Gregory Zeikus, Monique Hermann, Michel Magot, and Madeleine Sebald

Index 675

Contributors

Klaus Aktories Pharmakologisches Institut, Universitatsklinikum, Essen, 0-4300 Essen, Germany

Jean-Paul Aubert Unite de Physiologie Cellulaire, Institut Pasteur, 75724 Paris Cedex IS, France

Michael Bagdasarian Michigan Biotechnology Institute, Lansing, MI 48909, USA

Chantal Bagnara Laboratoire de Chimie Bacterienne, CNRS, 13277 Marseille Cedex 9, France

Pierre Beguin Unite de Physiologie Cellulaire, Institut Pasteur, 75724 Paris Cedex IS, France

Anne Belaich Laboratoire de Chimie Bacterienne, CNRS, 13277 Marseille Cedex 9, France

Jean-Pierre Belaich Laboratoire de Chimie Bacterienne, CNRS, 13277 Marseille Cedex 9, France

George N. Bennett Department of Biochemistry and Cell Biology, Wiess School of Natural Sciences, Rice University, Houston, TX 77251, USA

Teruhiko Beppu Department of Agricultural Chemistry, Faculty of Agri­culture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

John K. Brehm Division of Biotechnology, PHLS Center for Applied Microbiology and Research, Salisbury, Wiltshire, UK

Bruno Canard Laboratoire de Genetique Moleculaire Bacterienne, Institut Pasteur, 75724 Paris Cedex IS, France

Jiann-Shin Chen Department of Anaerobic Microbiology, Virginia Poly­technic Institute and State University, Blacksburg, VA 24061, USA

Chih-Fong Chou VA Medical Center, 154J, Palo Alto, CA 94304, USA

Stewart T. Cole Laboratoire de Genetique Moleculaire Bacterienne, In­stitut Pasteur, 75724 Paris Cedex IS, France

xiii

xiv

Robert D. Coleman Environmental Research Division, Argonne Nation­al Laboratory, Argonne, IL 60439, USA

Peter T. Cox Center for Molecular Biology and Biotechnology, University of Queensland, Brisbane, Queensland 4072, Australia

Brian P. Dalrymple CSRIO Division of Tropical Animal Production, Longpacket Laboratories, Indooroopilly, Queensland 4068, Australia

Willem M. de Vos Bacterial Genetics Group, Department of Microbiol­ogy, Wageningen Agricultural University, Wageningen, The Netherlands

Wolfgang Dubbert Institut fur Mikrobiologie, Technische UniversWit Munchen, D-8000 Munchen 2, Germany

Rik 1.1. Eggen Bacterial Genetics Group, Department of Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands

Christoph von Eichel-Streiber Institut fur Medizinische Mikrobiologie der Johannes Gutenberg Universitat, 6500 Mainz, Germany

Mel W. Eklund Utilization Research Division, Northwest Fisheries Center, NMFS, NOAA, Seattle, WA 98112, USA

Thierry Gamier Laboratoire de Genetique Moleculaire Bacterienne, In­stitut Pasteur, 75724 Paris Cedex 15, France

Christian Gaudin Laboratoire de Chimie Bacterienne, CNRS, 13277 Marseille Cedex 9, France

H.J. Gilbert Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, UK

Ulf B. Gobel Klinikum der Albert-Ludwigs-Universitat Freiburg, Insti­tut fur Medizinische Mikrobiologie und Hygiene, Abteilung Mikrobiolo­gie und Hygiene, D-7800 Freiburg, Germany

Howard Goldfine Department of Microbiology, University of Pennsyl­vania, School of Medicine, Philadelphia, PA 19104-6076, USA

Per Einar Granum Norwegian College of Veterinary Medicine, Depart­ment of Food Hygiene, Oslo 1, Norway

Donald G. Guiney UCSD Medical Center, University of California, San Diego, CA, USA

Herbert Hachler Department of Medical Microbiology, University of ZUrich, CH-8028 Zurich, Switzerland

Winfried Hausner Lehrstuhl fur Mikrobiologie, Universitat Regensburg, D-8400 Regensburg, Germany

G.P. Hazlewood AFRC Institut of Animal Physiology and Genetic Re­search, Cambridge, CB24AT, UK

Monique Hermann Direction Biotechnologie et Environnement, Institut Fram;ais du Petrole, 92506 Rueil Malmaison, France

Stephen M. Hinton Exxon Corporate Research Co. Clinton Township, Annandale, NJ 08801, USA

Contributors

Contributors xv

Matthew Hobbs Center for Molecular Biology and Biotechnology, Uni­versity of Queensland, Brisbane, Queensland 4072, Australia

Sueharu Horinouchi Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Sophie E.e. Hunter Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire SP4 OJQ, UK

Vivian Hwa Department of Microbiology, University of Illinois, Urbana, IL 61801, USA

Phillip B. Hylemon Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Rich­mond, VA 23298, USA

Angela Joe University of British Columbia, Department of Microbiology, Vancouver BC, V6T 1W5, Canada

John 1. Johnson Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA

Ingo Just Pharmakologisches Institut, Universitatsklinikum Essen, D-4300 Essen, Germany

Fritz H. Kayser Department of Medical Microbiology, University of Zurich, CH-8028 Zurich, Switzerland

Gertrud Koch Pharmakologisches Institut, Universitatsklinikum Essen, D-4300 Essen, Germany

Yong-Eok Lee Department of Biochemistry, Michigan State University, East Lansing, MI 48824, USA

Chanyong Lee Department of Biochemistry, Michigan State University, East Lansing, MI 48824, USA

Thomas Leisinger Mikrobiologisches Institut ETH-Zentrum, CH-8092 Zurich, Switzerland

Lars G. Ljungdahl Center for Biological Resource Recovery and Depart­ment of Biochemistry, University of Georgia, Athens GA 30602, USA

Francis 1. Macrina Department of Microbiology and Immunology, Vir­ginia Commonwealth University, Richmond, VA 23298-0678, USA Michel Magot Unite de Microbiologie Industrielle, Sanofi Elf Bio Re­cherches, Labege Innopole, 31328 Labege, France

Darrell H. Mallonee Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Rich­mond, VA 23298, USA

John S. Mattick Centre for Molecular Biology and Biotechnology, Uni­versity of Queensland, Brisbane, Queensland 4072, Australia

Barry e. McBride University of British Columbia, Department of Micro­biology, Vancouver, BC, V6T 1W5, Canada

Leo MeiIe Mikrobiologisches Institut, ETH-Zentrum, CH-8092 Zurich, Switzerland

xvi

Hannes Melasniemi Research Laboratories Alko Ltd., SF-OOlOl Helsinki, Finland

Menghsiao Meng Michigan Biotechnology Institute, Lansing, MI48909, USA

Jacqueline Millet Unite de Physiologie Cellulaire, Institut Pasteur, 75724 Paris Cedex IS, France

Nigel P. Minton Division of Biotechnology, PHLS Center for Applied Microbiology and Research, Salisbury, Wiltshire, UK

Thomas A. Morton Center for Biological Resource Recovery and Depart­ment of Biochemistry, University of Georgia, Athens, GA 30602, USA

John D. Oultram Division of Biotechnology, PHLS Center for Applied Microbiology and Research, Salisbury, Wiltshire, UK

John R. Palmer Department of Microbiology, Ohio State University, Columbus, OH 43210, USA

Klaus Pelz Klinikum der Albert Ludwigs Universitat Freiburg, Institut fur Medizinische Mikrobiologie und Hygiene, Abteilung Mikrobiologie und Hygiene, D-7800 Freiburg, Germany

Daniel J. Petersen Department of Biochemistry and Cell Biology, Wiess School of Natural Sciences, Rice University, Houston, TX 77251, USA

Jesse C. Rabinowitz Department of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720, USA

John N. Reeve Division of Microbiology, Ohio State University, Columbus, OH 44210, USA

Gilles Reysset Unite des Anaerobies, Institut Pasteur, 75724 Paris Cedex IS, France

Peter Roggentin Biochemisches Institut in der Medizinischen Fakultat, Christian-Albrechts-Universitat, D-2300 Kiel, Germany

Julian I. Rood Department of Microbiology, Monash University, Clayton Victoria 3168, Australia

Abigail A. Salyers Department of Microbiology, University of Illinois, Urbana IL 61801, USA

Roland Schauer Biochemisches Institut in der Medizinischen Fakultat, Christian-Albrechts-Universitat, D-2300 Kiel, Germany

Madeleine Sebald Unite des Anaerobies, Institut Pasteur, 75724 Paris Cedex IS, France

R. Sellwood AERC Institute for Animal Health, Compton, Newbury, Berkshire RG16 ONN, UK

Umadatt Singh University of British Columbia, Department of Micro­biology, Vancouver Be, V6T 1W5, Canada

Contributors

Contributors xvii

C. Jeffrey Smith Department of Microbiology and Immunology, East Carolina University, Greenville, NC 27858-4354, USA

Walter 1. Staudenbauer Institut fur Mikrobiologie, Technische Universi­tat Miinchen, D-8000 Miinchen 2, Germany

Gordon S.A.B. Stewart Department of Applied Biochemistry and Food Science, School of Agricultural and Food Science, University of Notting­ham, Sutton Bonington, Loughborough LE12 5RD, UK

Wen-Jin Su Department of Biology, Xiamen University, 361005 Xiamen, Fujian, China

Tracy-Jane Swinfield Division of Biotechnology, PHLS Center for Applied Microbiology and Research, Salisbury, Wiltshire, UK

R.M. Teather Animal Research Centre, Research Branch, Agriculture Canada, Ottawa, Ontario, KIA OC6, Canada

Michael Thomm Institut fUr Biochemie Genetik und Mikrobiologie der Universitat Regensburg, D-8400 Regensburg, Germany

Richard W. Titball Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire SP4 OJQ, UK

Rodney K. Tweten Department of Microbiology and Immunology, Uni­versity of Oklahoma, Health Sciences Center, Oklahoma City, OK 73190, USA

Shigezo Vdaka Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464, Japan

Kazue Veno Institute of Anaerobic Bacteriology, Gifu University School of Medicine, 40 Tsukasa-Machi, Gifu, 500, Japan

Peter Valentine Department of Microbiology, University of Illinois, Urba­na, IL 61801, USA

Jos Vanderleyden F.A. Janssens Laboratory of Genetics, University of Leuven, B-3001 Heverlee, Belgium

Peter Verhasselt F.A. Janssens Laboratory of Genetics, University of Leuven, B-3001 Heverlee, Belgium

Gerrit Voordouw Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

Judy D. Wall University of Missouri, Columbia, Department of Bio­chemistry, Columbia, MO USA

Shu-Zhen Wang Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA

Kunitomo Watanabe Institute of Anaerobic Bacteriology, Gifu University School of Medicine, 40 Tsukasa-Machi, Gifu 500, Japan

Sarah M. Whelan Division of Biotechnology, PHLS Center for Applied Microbiology and Research, Salisbury, Wiltshire, UK

xviii

David R. Woods University of Cape Town, Rondebosch 7700, South Africa

Hideo Yamagata Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464, Japan

Helen Yeoman Chemical Defence Establishment, Porton Down, Salis­bury, Wiltshire SP4 OJQ, UK

Michael Young Department of Biological Sciences, University College of Wales, Aberystwyth, Dyfed SY23 3DA, UK

J. Gregory Zeikus Michigan Biotechnology Institute, Lansing, MI48909, USA

Contributors

Introduction

In comparison to other living organisms, obligate anaerobic microorganisms share two common characters: 1) an extreme sensitivity to molecular oxygen (or its derivatives); and a capacity to produce energy and perform essential biosyntheses without molecular oxygen. This latter property is often called anoxybiontic metabolism and relates to the lack of a complete cytochrome chain and oxygenases. In most anaerobes, the energy required for growth is obtained by fermentation, a redox process in which organic substrates are both donor and terminal electron acceptors. The major exceptions are the auto­trophic methanogenic and acetogenic bacteria, the sulfur-metabolizing archaebacteria, and the sulfate-reducing bacteria which use, respectively, CO2, elemental sulfur, and sulfate for hydrogen oxidation. Energy is trans­formed mostly by enzymatic processes associated with substrate level phos­phorylations (Decker et al., 1970). As discussed by Gottschalk (1986), electron transport phosphorylations occur only in a few anaerobes, the terminal elec­tron acceptor being in all cases more electronegative than oxygen. Strict anaerobes never oxidize organic substrates into CO2 and H2 exclusively (Gott­schalk, 1986).

Anaerobes are unable to grow at the surface of a medium exposed to air (21 % molecular oxygen) and the oxygen is either bactericidal, or bacteriostatic, that is, anaerobes are either oxylabile or oxyduric, respectively (Hungate, 1985). The sensivity to O2 is extremely variable from one group to another, and eval­uation of O2 sensitivity requires that all organisms be cultivated under identi­cal conditions, both for medium composition (including reducing substances) and gaseous environment. Most of the strict, i.e., obligate, anaerobes are procaryotes [domains Archaebacteria (synonym, Archaea) and Eubacteria (synonym, Bacteria)], but there are also some eucaryotes, such as anaerobic fungi and anaerotolerantlanaerobic protozoa (Eucarya). Only the anaerobic procaryotes are considered in this book, however.

There are no geological records of bacteria but, according to current views, the first microorganisms on earth were probably anaerobic (Brock, 1987; Levine, 1988). One may speculate that anaerobes even had a "Golden Age," living in the primordial atmosphere with no molecular oxygen, suffering "just" from overexposure to ultraviolet (UV) irradiation. But, presumably, since about 2.5 x 109 years ago, they have had to face increasing concentra­tions of molecular oxygen, which resulted from photosynthetic activity and

xix

xx

which built up in the atmosphere [0.21%, 2 x 109 years ago; 1.05%, 1 x 109

years ago; 2.1%, 550 x 1()6 years ago; 21%, 400 x 1()6 years ago (Ooud, 1983)]. Anaerobes had to adapt using metabolic, ecological, phylogenetic, and genetic means. They succeeded by developing strategies such as negative aerotaxis, sporulation, adaptation to extreme conditions (temperature, pH, etc.), steno­trophic or eurytrophic metabolic capacities, nutritional interrelationships, attachment to the substrate, and living in close contact with O2 consumers or H2 producers. This resulted in highly adapted, or even bizarre, life styles, like living in a suHur and saline environment at 100°C (Staphylothermus), in the termite hindgut, the bovine rumen, or as endosymbiotic bacteria (endosym­biotic methanogens), of anaerobic protozoa.

One may also speculate that some primordial anaerobes remained as strict anaerobes, while others, after unsuccessful trials to adapt to 0 21 "returned" to an anaerobic metabolism and to an anaerobic mode of life. The anaerobes which belong to the latter category consist of those with generally low or very low levels of enzymes such as catalase and superoxide dismutase, or of those possessing a partial cytochrome chain with a terminal acceptor other than molecular O2•

The extreme diversity of the anaerobic bacteria make them very interesting organisms for a genetic and molecular approach. These ef-forts have recently begun and look very promising from many viewpoints. The chapters of this book reflect this diversity and the interest in complementary approaches such as biotechnology and intraspecific to intergeneric gene exchanges. The latter techniques, first available for clostridia and Bacteroides, are now developed for archaebacteria, sulfate-reducing bacteria, rumen bacteria, and pigmented bac­teria.

The chapters of the book document recent data obtained with techniques as diverse as whole chromosome mapping, performing vectors, genetic ex­changes through phage conversion, transduction, conjugation (including con­jugative plasmids, conjugative transposons, transposonlike structures, and "elements"), protoplast transformation, electrotransformation, gene cloning and sequencing, polymerase chain reaction, and in vitro mutagenesis.

All these approaches are furnishing insight into gene organization and reg­ulation in anaerobes, permitting the characterization and isolation of enzymes from multienzymatic complexes, providing information on structure-function relationships, genetic determination of antibiotic resistance and of antibiotic transfer factors. They also permit manipulation of operons of interest, protein modeling, and the development of diagnostic probes.

The molecular techniques will make it possible to solve complex problems such as mechanisms of pathogenicity and of O2 sensitivity, analysis of prop­erties such as thermophily or hyperthermophily, evolutionary relationship to anaerobes of puzzling structures such as organelles in anaerobic protozoa and the role of anaerobes as a natural reservoir of genes, i.e., antibiotic-resistance genes. Molecular biology is being intensively used in the taxonomy of anaerobes. GC content determinations and DNA-DNA hybridization studies have been fundamental for determining bacterial relationships at the intra­species and interspecies level. A bacterium can no longer be described as a new species unless that sort of information is available. Indeed, the establish­ment of close and remote relationships has been made possible from rRNA sequencing data, particularly from the 165 rRNA sequences, which appear as

Introduction

Introduction xxi

highly conserved throughout evolution. These have resulted in still-tentative but increasingly reliable phylogenetic trees. Molecular taxonomy of anaerobes as a whole has not been treated in this book because it rightly deserves a volume of its own. Indeed, excellent reviews have recently been published on this topic (Cato and Stackebrandt, 1989; Paster et al., 1985; Woese, 1987; Yang et al., 1985; Balows et al., 1991), and valuable information and comments are also given in several chapters of this book.

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Brock, T.D. 1987. Evolutionary relationships of the autotrophic bacteria. p. 499-512, in Schlegel H.G. and Bowien B. (editors), Autotropic bacteria, Springer­Verlag, Science Tech. Madison Wi.

Cato, E.P. and E. Stackbrandt. 1989. Taxonomy and phylogeny. p. 1-26. In Minton N.P. and Clarke D.J. (editors), Clostridia, Plenum Press, New York.

Cloud, P. 1983. The biosphere. Scientific American 249:132-144. Decker, K, K Jungermann and R.K Thauer, 1970. Energy production in anaerobic

organisms. Angewandte Chemie (International edition in English). 9:138-158. Gottschalk, G. 1986. The anaerobic way ofllie of prokaryotes. p. 1415-1424. In Starr

M.P., Stolp, H., Tmper, H.G. Balows, A. and Schlegel, H.G. (editors.) The pro­karyotes, a handbook on habitats, isolation and identification of bacteria, volume 2, Springer-Verlag, Berlin.

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