Microbial sulfur metabolism

Christiane Dahl • Cornelius G. Friedrich (Editors)

Microbial Sulfur Metabolism

Christiane Dahl Cornelius G. Friedrich (Editors)

Microbial Sulfur Metabolism With 65 Figures, 11 in Color and 27 T ables

Professor Dr. Cornelius G. Friedrich Chair Technical Microbiology Department of Biochemical and Chemical Engineering University of Dortmund D-44221 Dortmund Germany

Dr. Christiane Dahl Institute for Microbiology & Biotechnology Rheinische Friedrich-WilhelmsUni versität Bonn Meckenheimer Allee 168 D-53115 Bonn Germany

Library of Congress Control Number: 2007929727

ISBN-13 978-3-540-72679-1

Springer-Verlag Berlin Heidelberg New York

This work is subject to cop yright. All rights are reserv ed, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the pro visions of the German Cop yright Law of September 9, 1965, in its current v ersion, and permission for use must al ways be obtained from Springer . Violations are liable to prosecution under the German Cop yright Law. Springer-Verlag is a part of Springer Science+Business Media springer.com © Springer-Verlag Berlin Heidelberg 2008 The use of general descripti ve names, re gistered names, trademarks, etc. in this publication does not imply, e ven in the absence of a specif ic statement, that such names are e xempt from the rele vant protective laws and regulations and therefore free for general use. Editor: Dr. Christina Eckey, Heidelberg Desk Editor: Anette Lindqvist, Heidelberg Production: SPi Typesetting: SPi Cover Design: Design & Production, Heidelber g Printed on acid-free paper

149/3152-HM

543210

Preface

Sulfur is an essential element for the living cell. Sulfur occurs in oxidation states of +2 to −6, is highly reacti ve and is used by prokaryotes not only to b uild up cell constituents b ut also for ener gy transformation. Basic research has re vealed in recent years an increasing rate of information on prokaryotic reactions, proteins and genes involved in sulfur transformations. Ne w insights are emer ging concerning enzyme systems involved in sulfur metabolism, genomics and proteomics of sulfurmetabolizing bacteria and archaea, and the ecology of prokaryotes oxidizing and reducing sulfur compounds. Furthermore, ne w methods ha ve been de veloped and are being applied to study microbial sulfur metabolism. To summarize the f ast-moving de velopments of recent years, to e xchange knowledge and to discuss future developments and research needs, the “International Symposium on Microbial Sulfur Metabolism” was held in Münster, Germany, from 29 June to 2 July 2006. This symposium brought together 85 scientists from 16 countries and w as felt to be timely after a pre vious meeting on bacterial sulfur metabolism which took place in London in 1982. The symposium in Münster focused on prokaryotic sulfur ener gy metabolism, the biochemistry of the enzymes in volved, the molecular genetics of such enzyme systems as well as on the ecosystems harboring sulfur -metabolizing prokaryotes. Pathways of sulfur metabolism present in dif ferent physiological groups were compiled. A collection of in vited lectures presented the state of the art re garding biochemistry, ecology, proteomics, genomics and e volution of chemotrophic and phototrophic sulfur -oxidizing bacteria, anaerobic sulf ate-reducing bacteria and hyperthermophilic sulfur-metabolizing archaea. The symposium setting and time schedule encouraged informal discussion and e xchange between young and established scientists. This proceedings volume presents the essence of the symposium represented by 23 invited lectures which introduce and report cutting-edge research in the v arious fields. The ef forts of the authors ha ve created a book which compiles the state of the art on sulfur metabolism in phototrophic and chemotrophic bacteria and archaea. The book is or ganized according to the se ven major topics of the symposium: (1) sulf ate-reducing bacteria, (2) genomics/proteomics of sulfur metabolizing prokaryotes, (3) biochemistry of sulfur -compound oxidation, (4) metabolism of or ganosulfur compounds, (5) dissimilatory sulfur metabolism in archaea, (6) ecology of sulfur bacteria and (7) specific methods and applied aspects. v

vi

Preface

To each topic leading e xperts in the f ield contrib ute se veral chapters to yield a detailed picture of the current state of the art. Preparing the symposium, we were not a ware of all the f ascinating research in these fields. Also, we had limited space and could not include the contents of fascinating lectures which were selected from submitted abstracts. Also, some topics are not co vered by this v olume, lik e sulfur acti vation and assimilation pathw ays in prokaryotes and higher eukaryotes, transport of sulfur compounds, and biosynthesis of sulfur -containing cell constituents. The reader is, ho wever, referred to the volume Sulfur Metabolism in Phototrophic Organisms (R. Hell, C. Dahl, D. Knaf f and T. Leustek, 2008, eds, Springer, New York, in press). The organizers of this symposium thankfully acknowledge the substantial financial support from the Federation of the European Microbiological Societies (Brussels), the Deutsche Forschungsgemeinschaft (Bonn), the Fonds der Chemischen Industrie (Frankfurt), the Vereinigung für allgemeine und angewandte Mikrobiologie (Frankfurt) and the Gesellschaft für Biochemie und Molekularbiologie (Frankfurt). Their support enabled a scientifically lively meeting, and the attending community decided on a follo w-up meeting which will be or ganized by and Inês Pereira and Christiane Dahl will take place in Portugal in 2009. Bonn, Dortmund, March 2007

Christiane Dahl and Cornelius Friedrich

Contributors

Adam P. Arkin, Howard Hughes Medical Institute, Department of Bioengineering, University of California, Berk eley, CA 94720, USA, Physical Biosciences Division, E.O. La wrence Berk eley National Laboratory , Berk eley, CA 94720, USA, and Virtual Institute of Microbial Stress and Survival, Berkeley, CA 94710, USA Nurgul C. Balci, Biochemistry Department, Uni versity of Missouri, Columbia, MO 65211, USA Frank Bardischewsky, Lehrstuhl für Technische Mikrobiologie, F achbereich Biound Chemieingenieurwesen, Universität Dortmund, 44221 Dortmund, Germany Simón Beard, Laboratory of Molecular Microbiology and Biotechnology , Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile Anke Behr ens, F achbereich Biologie, Uni versität K onstanz, 78457 K onstanz, Germany Donald A. Bryant, Department of Biochemistry and Molecular Biology Pennsylvania State University, University Park, PA 16802, USA

, The

Thomas Büchert, Fachbereich Biologie, Uni versität Konstanz, 78457 K onstanz, Germany Donald E. Canf ield, Nordic Center for Earth Ev olution and Institute of Biology , University of Southern Denmark, 5230 Campusv ej, 5000 Odense M, Denmark Leong-Keat Chan, College of Marine and Earth Studies and Dela Biotechnology Institute, University of Delaware, Newark, DE 19711, USA

ware

An Chi, Laboratory of Molecular Microbiology and Biotechnology, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile Alasdair M. Cook, Department of Biology, Universität Konstanz, 78457 Konstanz, Germany Christiane Dahl, Institut für Mikrobiologie & Biotechnologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Meck enheimer Allee 168, 53115 Bonn, Germany vii

viii

Contributors

Karin Denger, Department of Biology, The University, 78457 Constance, Germany Lina De Smet, Laboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry, Microbiology and Zoophysiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Stephan Duller , Department of Microbial Ecology Althanstrasse 14, 1090 Vienna, Austria

, Uni versity of V ienna,

Ulrich Ermler, Max-Planck-Institut für Biophysik, 60438 Frankfurt, German y Giovanni Esposito, Subdepartment of En vironmental T echnology, Agricultural University of W ageningen, Biotechnion, Bomenwe g 2, P .O. Box 8129, 6700 EV Wageningen, The Netherlands Jörg Fischer , Lehrstuhl für T echnische Mikrobiologie, F achbereich Bio- und Chemieingenieurwesen, Universität Dortmund, 44221 Dortmund, Germany Bettina Franz, Institut für Mikrobiologie und Biotechnologie, Rheinische FriedrichWilhelms-Universität Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany, and Fachbereich Oecotrophologie, Mikrobiologie und Lebensmittelhygiene, Hochschule Niederrhein, Rheydter Straße 277, 41065 Mönchengladbach, German y Cornelius G. Friedrich, Lehrstuhl für T echnische Mikrobiologie, F achbereich Bio- und Chemieingenieurwesen, Uni versität Dortmund, 44221 Dortmund, Germany Niels-Ulrik Frigaard, Copenhagen Biocenter, Department of Molecular Biology , University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark Günter Fritz, Fachbereich Biologie, Uni versität K onstanz, 78457 Constance, Germany Frauke Grimm, Institut für Mikrobiologie und Biotechnologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Meck enheimer Allee 168, 53115 Bonn, Germany Thomas E. Hanson, College of Marine and Earth Studies and Dela Biotechnology Institute, University of Delaware, Newark, DE 19711, USA Petra Hellwig, Laboratoire d’Électrochemie, Institut Chimie, Uni Pasteur, 4 Rue Blaise Pascal, 67000 Strasbourg, France

ware

versité Lous

Joost Hoek, Nordic Center for Earth Evolution and Institute of Biology, University of Southern Denmark,5230 Campusvej, 5000 Odense M, Denmark Michael Hügler , Biology Department, W oods Hole Oceanographic Institution, Woods Hole, MA 02543, USA, and Leibniz Institute of Marine Sciences, 24105 Kiel, Germany Donald F. Hunt, Department of Chemistry, University of Virginia, Charlottesville, VA 22904-4319, USA

Contributors

ix

Carlos A. J erez, Laboratory of Molecular Microbiology and Biotechnology , Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile Eric F. Johnson, Virginia Bioinformatics Institute , V irginia Polytechnic Institute and State University, Washington Street, MC 0477, Blacksburg, VA 24061, USA Ulrike Kappler, School of Molecular & Microbial Sciences, Centre for Metals in Biology, The University of Queensland, Brisbane, QLD 4072, Australia Arnulf Kletzin, Institute of Microbiology and Genetics, Darmstadt Uni versity of Technology, Schnittspahnstrasse 10, 64287 Darmstadt, German y Peter M.H. Kr oneck, Fachbereich Biologie, Uni versität K onstanz, 78457 Constance, Germany Piet N.L. Lens, Subdepartment of En vironmental T echnology, Agricultural University of W ageningen, Biotechnion, Bomenwe g 2, P .O. Box 8129, 6700 EV Wageningen, The Netherlands Shuang-Jiang Liu, State K ey Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People’ s Republic of China Alexander Loy , Department of Microbial Ecology Althanstrasse 14, 1090 Vienna, Austria

, Uni versity of V ienna,

William Martin, Institut für Botanik III, Heinrich Heine Uni versität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany Roel J .W. Meulepas, Subdepartment of En vironmental Technology, Agricultural University of W ageningen, Biotechnion, Bomenwe g 2, P .O. Box 8129, 6700 EV Wageningen, The Netherlands Rachael Mor gan-Kiss, Colle ge of Marine and Earth Studies and Dela Biotechnology Institute, University of Delaware, Newark, DE 19711, USA

ware

Biswarup Mukhopadh yay, Virginia Bioinformatics Institute, Department of Biochemistry and Department of Biology , Virginia Polytechnic Institute and State University, Washington Street, MC 0477, Blacksburg, VA 24061, USA Grazyna Orawski, Lehrstuhl für Technische Mikrobiologie, Fachbereich Bio- und Chemieingenieurwesen, Universität Dortmund, 44221 Dortmund, Germany Inês A. Cardoso Pereira, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República – EAN, 2784–505 Oeiras, Portugal Alexander Prange, Fachbereich Oecotrophologie, Mikrobiologie und Lebensmittelhygiene, Hochschule Niederrhein – Niederrhein Uni versity of Applied Sciences, Rheydter Straße 277, 41065 Mönchengladbach, Germany, and Center for Adv anced Microstructures and De vices, Louisiana State Uni versity, 6980 Jefferson Highway, Baton Rouge, LA 70806, USA

x

Contributors

Armin Quentmeier, Lehrstuhl für T echnische Mikrobiologie, F achbereich Biound Chemieingenieurwesen, Universität Dortmund, 44221 Dortmund, Germany Barbara Rapp-Giles, Biochemistry Department, University of Missouri, Columbia, MO 65211, USA Dagmar Rother, Lehrstuhl für T echnische Mikrobiologie, F achbereich Bio- und Chemieingenieurwesen, Universität Dortmund, 44221 Dortmund, Germany Ricardo Sampaio, Subdepartment of En vironmental T echnology, Agricultural University of W ageningen, Biotechnion, Bomenwe g, 2, P .O. Box 8129, 6700 EV Wageningen, The Netherlands Savvas Sa vvides, Laboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry, Microbiology and Zoophysiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Alexander Schiffer, Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany Jeffrey Shabanowitz, Department of Chemistry, University of Virginia, Charlottesville, VA 22904-4319, USA Stefan M. Sie vert, Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA Theo H.M. Smits, Department of Biology, Universität Konstanz, 78457 Konstanz, Germany Dimitry Y. Sorokin, Winogradsky Institute of Microbiology , Russian Academy of Sciences, Prospect 60-let Octyabrya 7/2, 117811 Mosco w, Russia, and Department of Biotechnology , Delft Uni versity of T echnology, Julianalaan 67, 2628 BC Delft, The Netherlands Jan Stout, Laboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry , Microbiology and Zoophysiology , Ghent Uni versity, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Craig D . Taylor, Biology Department, W oods Hole Oceanographic Institution, Woods Hole, MA 02543, USA Ursula Theissen, Institut für Botanik III, Heinrich Heine Uni versität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany Hans G. T rüper, Institut für Mikrobiologie & Biotechnologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Meck enheimer Allee 168, 53115 Bonn, Germany Lissette Valenzuela, Laboratory of Molecular Microbiology and Biotechnology , Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile

Contributors

xi

Marcus V allero, Subdepartment of En vironmental T echnology, Agricultural University of W ageningen, Biotechnion, Bomenwe g 2, P .O. Box 8129, 6700 EV Wageningen, The Netherlands Jozef Van Beeumen, Laboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry, Microbiology and Zoophysiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Bjorn Vergauwen, Laboratory of Protein Biochemistry and Protein Engineering, Department of Biochemistry, Microbiology and Zoophysiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Gerrit V oordouw, Department of Biological Sciences, Uni Calgary, AB T2N 1N4, Canada Michael W agner, Department of Microbial Ecology Althanstrasse 14, 1090 Vienna, Austria

versity of Calgary ,

, Uni versity of V ienna,

Judy D. Wall, Biochemistry Department, Uni versity of Missouri, Columbia, MO 65211, USA, and Virtual Institute of Microbial Stress and Survi val, Berkeley, CA 94710, USA Carl O. Wirsen, Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

1

2

2.2

3

Genetics and Genomics of Sulfate Respiration in Desulfovibrio. . . . Judy D. Wall, Adam P. Arkin, Nurgul C. Balci, Barbara Rapp-Giles

1

1.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Approach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Sulfate Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Lactate Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Hydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Transmembrane Electron-Conducting Complexes . . . . . . . . . . . . . 1.7 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 5 6 7 8 9 11

Living on Sulfate: Three-Dimensional Structure and Spectroscopy of Adenosine 5¢-Phosphosulfate Reductase and Dissimilatory Sulfite Reductase . . . . . . . . . . . . . . . . . . . . . . . . . . Günter Fritz, Alexander Schiffer, Anke Behrens, Thomas Büchert, Ulrich Ermler, Peter M.H. Kroneck

13

2.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adenosine 5′-Phosphosulfate Reductase . . . . . . . . . . . . . . . . . . . . 2.2.1 Molecular Properties of APSR . . . . . . . . . . . . . . . . . . . . . . 2.2.2 Three-Dimensional Structure of APSR . . . . . . . . . . . . . . . 2.2.3 Reaction Mechanism of APSR. . . . . . . . . . . . . . . . . . . . . . 2.3 Dissimilatory SIR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13 14 15 17 17 20 21

Respiratory Membrane Complexes of Desulfovibrio . . . . . . . . . . . . . Inês A. Cardoso Pereira

24

3.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Membrane Complexes Conserved in Sulfate Reducers . . . . . . . . .

24 25

xiii

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4

Contents

3.2.1 The Qmo Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2 The Dsr Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Membrane Complexes Found Only in Desulfovibrio spp. . . . . . . . 3.3.1 The Hmc and 9Hc Complexes . . . . . . . . . . . . . . . . . . . . . . 3.3.2 The Tmc Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

26 29 30 31 31 32 33

Biochemical and Evolutionary Aspects of Eukaryotes That Inhabit Sulfidic Environments . . . . . . . . . . . . . . . . . . . . . . . . . . Ursula Theissen, William Martin

36

4.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Animals in Sulfidic Environments . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Sulfide-Oxidizing Enzymes in Eukaryotes. . . . . . . . . . . . . . . . . . . 4.4 The Possible Functions of SQR-Related Genes in Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Sulfide and Eukaryotic Evolution. . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Evolution and Ecology of Microbes Dissimilating Sulfur Compounds: Insights from Siroheme Sulfite Reductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alexander Loy, Stephan Duller, Michael Wagner

5.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Evolution of Dissimilatory Sulfite Reductases. . . . . . . . . . . . . . . . 5.2.1 Sulfate/Sulfite-Reducing Microorganisms . . . . . . . . . . . . . 5.2.2 DsrAB-Containing Syntrophs: Former Sulfate/Sulfite-Reducing Microorganisms? . . . . . . 5.2.3 Sulfur-Oxidizing Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4 The Root and Major Branches of the DsrAB T ree . . . . . . 5.2.5 Other Non-DsrAB Dissimilatory Sulfite Reductases . . . . . 5.3 Molecular Insights into the Ecology of DsrAB-Emplo ying Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1 PCR-Based Surveys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2 Metagenomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

36 37 38 39 40 42 43

46 46 47 47 50 51 52 52 53 53 54 55 56

Genomic and Evolutionary Perspectives on Sulfur Metabolism in Green Sulfur Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . Niels-Ulrik Frigaard, Donald A. Bryant

60

6.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

60

Contents

xv

6.1.1 6.1.2

Green Sulfur Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genome Sequencing Projects of Green Sulfur Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Compounds Oxidized by Green Sulfur Bacteria . . . . . . . . . . . . . . 6.3 Enzymes Involved in Sulfur-Compound Oxidation . . . . . . . . . . . . 6.3.1 Overview of the Putative Sulfur Compound Oxidation Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2 Dissimilatory Sulfite Reductase . . . . . . . . . . . . . . . . . . . . . 6.3.3 Sulfide:Quinone Reductase. . . . . . . . . . . . . . . . . . . . . . . . . 6.3.4 Flavocytochrome c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.5 Sulfite Oxidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.6 Thiosulfate Oxidation by the Sox System . . . . . . . . . . . . . 6.3.7 A Novel Complex: SoyYZ . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Assimilatory Sulfur Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Possible Phage-Mediated Lateral Gene Transfer . . . . . . . . . . . . . . 6.6 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Differential-Expression Proteomics for the Study of Sulfur Metabolism in the Chemolithoautotr ophic Acidithiobacillus ferrooxidans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lissette Valenzuela, An Chi, Simón Beard, Jeffrey Shabanowitz, Donald F. Hunt, Carlos A. Jerez

7.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Sulfur Metabolism in A. ferrooxidans . . . . . . . . . . . . . . . . . . . . . . 7.3 Proteomics of A. ferrooxidans Grown in Sulfur Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Thiosulfate Sulfur Transferases from A. ferrooxidans . . . . . . . . . . 7.5 Other Proteins Involved in Sulfur Metabolism. . . . . . . . . . . . . . . . 7.6 High-Throughput Proteomics of Periplasmic Proteins Induced by Growth of A. ferrooxidans on Sulfur Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Sulfur and Light? History and “Thiology” of the Phototrophic Sulfur Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . Hans G. Trüper 8.1 8.2 8.3 8.4

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discovery of Sulfur-Oxidizing Microorganisms . . . . . . . . . . . . . . Identification of Conspicuous Inclusions as Sulfur . . . . . . . . . . . . Enrichment Cultures – First Taxonomy – and the Question of Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . 8.5 Pure Cultures of Phototrophic Sulfur Bacteria at Last! . . . . . . . . .

61 62 63 65 66 66 67 69 69 70 71 71 72 72 73

77

77 78 78 79 82 83 85 85 87 88 88 89 91 92

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Contents

8.6 The Age of Enzymology and Isotope Labeling . . . . . . . . . . . . . . . 8.7 Advent of Molecular Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8 Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

93 95 96 97

Thiosulfate and Sulfur Oxidation in Pur ple Sulfur Bacteria . . . . . . Frauke Grimm, Bettina Franz, Christiane Dahl

101

9.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Oxidation of Thiosulfate in A. vinosum . . . . . . . . . . . . . . . . . . . . . 9.2.1 sox Genes in A. vinosum. . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2 Sox Proteins in A. vinosum. . . . . . . . . . . . . . . . . . . . . . . . . 9.2.3 Inactivation and Complementation of sox Genes in A. vinosum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Oxidation of Stored Sulfur in A. vinosum . . . . . . . . . . . . . . . . . . . 9.3.1 The dsr Operon and Proteins Encoded Therein . . . . . . . . . 9.3.2 Distribution of dsr Genes in Organisms with Dissimilatory Sulfur Metabolism and Phylogenetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.3 Model of the Sulfur Oxidation P athway in A. vinosum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

101 103 104 105

9

10

Sulfur Oxidation in Chlorobium tepidum (syn. Chlorobaculum tepidum): Genetic and Proteomic Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leong-Keat Chan, Rachael Morgan-Kiss, Thomas E. Hanson 10.1

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2 Sulfur-Compound Dynamics in C. tepidum Batch Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Genetic Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.1 Organization of Genes Encoding Putative Sulfur Oxidation Functions. . . . . . . . . . . . . . . . . . . . . . . 10.2.2 Mutations Affecting Sulfur Oxidation Have Secondary Effects on Light Harvesting . . . . . . . . 10.2.3 Additional Genetic Techniques Are Needed . . . . . . . . . 10.3 Proteomic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.1 Why Proteomics? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3.2 Proteomic Analysis of Subcellular Fractions . . . . . . . . . 10.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

106 107 107 108 111 113 113

117 117 117 118 120 120 120 121 122 122 122 124 125

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11

xvii

Structural Insights into Component SoxY of the Thiosulfate-Oxidizing Multienzyme System of Chlorobaculum thiosulfatiphilum . . . . . . . . . . . . . . . . . . . . . . . . . . . Jan Stout, Lina De Smet, Bjorn Vergauwen, Savvas Savvides, Jozef Van Beeumen 11.1 11.2

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SoxY Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.1 Overall Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.2 SoxY Monomer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.3 SoxY Dimer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.4 SoxY Tetramer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.5 Location of the Disulfide Bridges and the Potential Sulfur Binding Site . . . . . . . . . . . . . . . 11.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Redox Control of Chemotrophic Sulfur Oxidation of Paracoccus pantotrophus. . . . . . . . . . . . . . . . . . . . . . . . . Cornelius G. Friedrich, Armin Quentmeier, Frank Bardischewsky, Dagmar Rother, Grazyna Orawski, Petra Hellwig, Jörg Fischer The Sulfur-Oxidizing Enzyme System of Paracoccus pantotrophus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Abundance of the sox Genes in Bacteria . . . . . . . . . . . . . . . . . . . 12.3 The Physiological Function of the Flavoprotein SoxF. . . . . . . . . 12.4 The Periplasmic Partners of SoxV for Transfer of Electrons. . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

127

127 128 128 130 130 131 132 133 136 139

12.1

13

Bacterial Sulfite-Oxidizing Enzymes – Enzymes for Chemolithotrophs Only? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ulrike Kappler 13.1 13.2 13.3 13.4 13.5

Introduction – Sulfite in the Environment and in Cell Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sulfite-Oxidizing Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and Function of Sulfite-Oxidizing Enzymes . . . . . . . . Phylogeny of Sulfite-Oxidizing Enzymes . . . . . . . . . . . . . . . . . . Diversity of Enzymes Within the Sulfite Oxidase Family . . . . . . 13.5.1 Group 1 – SOE Like Enzymes Originating from Pathogenic Microorganisms . . . . . . . . . . . . . . . . . . . . . . 13.5.1.1 Group 1A Enzymes: YedY and Related Proteins . . . . . . . . . . . . . . . . . . . 13.5.1.2 Group 1B – 30-kDa Mo-Domain Proteins . .

139 142 144 146 148 151

151 152 153 154 157 157 158 159

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13.5.2 Group 2: “Classic” Sulfite-Oxidizing Enzymes and Nitrate Reductases . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.2.1 Group 2A: Sulfite Oxidases and Plant Nitrate Reductases . . . . . . . . . . . . . 13.5.2.2 Group 2B: SoxCD-Like Enzymes – “Sulfur Dehydrogenases” . . . . . . . . . . . . . . . 13.5.2.3 Group 2C: SorAB-Like Sulfite Dehydrogenases . . . . . . . . . . . . . . . . . 13.5.2.4 Other Sulfite-Oxidizing Enzymes in Group 2 . . . . . . . . . . . . . . . . . . . 13.5.3 Group 3: Sulfite-Oxidizing Enzymes – Enzymes from Archaea, Phototrophic and Soil Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.6 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

15

161 161 162 163 164 164 165 166

Sulfonates and Organotrophic Sulfite Metabolism . . . . . . . . . . . . . . Alasdair M. Cook, Theo H.M. Smits, Karin Deng er

170

14.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Biosynthesis of Organosulfonates . . . . . . . . . . . . . . . . . . . . . . . . 14.3 Dissimilation of Organosulfonates . . . . . . . . . . . . . . . . . . . . . . . . 14.4 The Detoxification or Fate of Sulfite . . . . . . . . . . . . . . . . . . . . . . 14.5 Sulfite Dehydrogenases in Sulfonate Metabolism . . . . . . . . . . . . 14.6 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

170 173 175 176 178 180 181

Oxidation of Sulfur and Inorganic Sulfur Compounds in Acidianus ambivalens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arnulf Kletzin

184

15.1 15.2

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sulfur and Sulfur Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3 A. ambivalens and A. tengchongensis SORs . . . . . . . . . . . . . . . . 15.3.1 SOR 3D Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3.2 SOR Subunit and Active-Site Structure . . . . . . . . . . . . . 15.3.3 SOR Reaction Mechanism . . . . . . . . . . . . . . . . . . . . . . . 15.4 Oxidation of Soluble Sulfur Compounds in Acidianus . . . . . . . . 15.4.1 Sulfite:Acceptor Oxidoreductase . . . . . . . . . . . . . . . . . . 15.4.2 Thiosulfate:Quinone Oxidoreductase . . . . . . . . . . . . . . . 15.4.3 Tetrathionate Hydrolase . . . . . . . . . . . . . . . . . . . . . . . . . 15.4.4 Sulfide:Quinone Oxidoreductase . . . . . . . . . . . . . . . . . . 15.5 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

184 186 188 190 194 194 196 196 197 197 198 198 199

Contents

16

A Novel Coenzyme F 420 Dependent Sulfite Reductase and a Small Sulfite Reductase in Methanogenic Archaea . . . . . . . . . Eric F. Johnson, Biswarup Mukhopadhyay 16.1 16.2

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Incompatibility of Methanogenesis and Sulfate Reduction, Sulfite As the Key Determinant . . . . . . . . . . . . . . . . . . . . . . . . 16.3 Inevitable Exposure of a Methanogen to Sulf ite in Hydrothermal Vents and on Early Earth. . . . . . . . . . . . . . . . 16.4 Use of Sulfite As a Sulfur Source by Methanocaldococcus jannaschii and Other Methanogens . . . . 16.5 Expression of a Novel Coenzyme F 420 Dependent Sulfite Reductase in Methanocaldococcus jannasc hii During Growth on Sulfite . . . . . . . . . . . . . . . . . . . . 16.6 Fsr, Combining Structural Components of Two Different Dissimilatory Metabolic Machineries to Bring About a Sulfite Reduction Function . . . . . . . . . . . . . . . . . . . . . 16.7 Purified Fsr Exhibits Properties Predicted from the Primary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.8 Fsr, a Sulfite Detoxification Tool and an Assimilatory Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.9 Homologs of Fsr in Other Organisms. . . . . . . . . . . . . . . . . . . . 16.10 Small Sulfite Reductases in Methanogens . . . . . . . . . . . . . . . . 16.11 Conclusion and Hypotheses . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Archaeal and Bacterial Sulfur Oxygenase-Reductases: Genetic Diversity and Physiological Function . . . . . . . . . . . . . . . . . . Shuang-Jiang Liu

17.1 17.2 17.2.1 17.2.2 17.2.3 17.2.4 17.2.5 17.3 17.3.1 17.3.2 17.4

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diversity of Archaeal SORs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SORAb from A. brierleyi . . . . . . . . . . . . . . . . . . . . . . . . . SORAa from A. ambivalens . . . . . . . . . . . . . . . . . . . . . . . SORAt from A. tengchongensis . . . . . . . . . . . . . . . . . . . . SORSt from S. tokodaii . . . . . . . . . . . . . . . . . . . . . . . . . . SORSm from S. metallicus . . . . . . . . . . . . . . . . . . . . . . . . Efforts To Identify Bacterial SORs. . . . . . . . . . . . . . . . . . . . . . . . SORAqa from A. aeolicus . . . . . . . . . . . . . . . . . . . . . . . . . SORAct from Acidithiobacillus sp. strain SM-1 . . . . . . . SOR Links Elemental Sulfur Oxidation to ATP Synthesis via Sulfite:Acceptor Oxidoreductase and Thiosulfate:Acceptor Oxidoreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.5 Physiological Regulation of SOR Activity in Archaea . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xix

202 202 206 206 207 207 209 210 211 212 212 213 214 217 217 219 219 220 220 220 221 221 221 221 222 223 223

xx

18

19

Contents

Diversity of Halophilic Sulfur-Oxidizing Bacteria in Hypersaline Habitats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dimitry Y. Sorokin

225

18.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2 Description of Habitats Investigated. . . . . . . . . . . . . . . . . . . . . . 18.3 Enrichment Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.4 Moderately Halophilic Aerobic SOB . . . . . . . . . . . . . . . . . . . . . 18.5 Extremely Halophilic Aerobic SOB . . . . . . . . . . . . . . . . . . . . . . 18.6 Moderately Halophilic Thiodenitrifyers . . . . . . . . . . . . . . . . . . . 18.7 Extremely Halophilic Denitrifying SOB . . . . . . . . . . . . . . . . . . 18.8 Oxidation of Thiocyanate at High Salt. . . . . . . . . . . . . . . . . . . . 18.9 Fatty Acids in the Membrane Lipids . . . . . . . . . . . . . . . . . . . . . 18.10 Conclusions and Future Perspectives . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

225 226 227 228 232 232 233 234 234 235 236

Sulfur Oxidation at Deep-Sea Hydrothermal Vents. . . . . . . . . . . . . . Stefan M. Sievert, Michael Hügler, Craig D. Taylor, Carl O. Wirsen

238

19.1 19.2

238 239 239 241 241 241 246

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of Sulfur-Oxidizing Bacteria . . . . . . . . . . . . . . . . . . . . . . . 19.2.1 Symbiotic Sulfur-Oxidizing Bacteria . . . . . . . . . . . . . . . 19.2.2 Free-Living Sulfur-Oxidizing Bacteria. . . . . . . . . . . . . . 19.2.2.1 Gammaproteobacteria . . . . . . . . . . . . . . . . . . 19.2.2.2 Epsilonproteobacteria . . . . . . . . . . . . . . . . . . 19.2.2.3 Aquificaceae. . . . . . . . . . . . . . . . . . . . . . . . . . 19.2.2.4 Carbon Metabolism in Sulfur-Oxidizing Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3 Sulfur Oxidation Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3.1 Types of Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3.2 Endosymbionts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3.3 Free-Living Sulfur-Oxidizing Bacteria. . . . . . . . . . . . . . 19.3.3.1 Beggiatoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3.3.2 Thiomicrospira crunogena and Epsilonproteobacteria. . . . . . . . . . . . . . . 19.3.3.3 Oxidation of H2 by Sulfur-Oxidizing Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4 “Snowblower” Vents As Signs for Sulfide Oxidation in the Subseafloor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4.1 The Subseafloor Biosphere. . . . . . . . . . . . . . . . . . . . . . . 19.4.2 Filamentous-Sulfur Formation in the Laboratory. . . . . . 19.4.3 “Snowblowers” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4.4 Diversity of Filamentous-Sulfur-Forming Bacteria . . . . 19.5 Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

246 247 247 248 248 248 249 250 251 251 251 252 252 253 254

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20

xxi

Speciation Analysis of Microbiologically Produced Sulfur by X-ray Absorption Near Edge Structure Spectroscopy . . . Alexander Prange 20.1 20.2

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XAS: X-ray Absorption Near-Edge Structure and Extended X-ray Absorption Fine Structure . . . . . . . . . . . . . . 20.2.1 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.2 Advantages of XANES Spectroscopy . . . . . . . . . . . . . . 20.2.3 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2.4 Quantitative Analysis of XANES Spectra . . . . . . . . . . . 20.3 Sulfur K-Edge XANES Spectroscopy and Speciation of Microbiologically Produced Sulfur . . . . . . . . . . . . . . . . . . . . . 20.3.1 Speciation of Sulfur in Sulfur Glob ules of Phototrophic and Chemotrophic Sulfur Bacteria . . . . 20.3.2 Speciation of “Elemental Sulfur” Taken Up by A. vinosum. . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Controls on Isotope Fractionation During Dissimilatory Sulfate Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joost Hoek, Donald E. Canfield 21.1 21.2

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sulfur Isotope Fractionation During Dissimilatory Sulfate Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.1 Pure Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2.2 Natural Populations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3 Stepwise Reduction of Sulfate and Sulfur Isotope Fractionation Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.4 Multiple Sulfur Isotopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.5 Conclusions and Future Research. . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Bioprocess Engineering of Sulfate Reduction for Environmental Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Piet N.L. Lens, Roel J.W. Meulepas, Ricardo Sampaio, Marcus Vallero, Giovanni Esposito 22.1 22.2 22.3

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sulfate Reduction in Methanogenic Wastewater Treatment. . . . . Sulfate-Reducing Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3.1 High-Rate Sulfate-Reducing Bioreactors . . . . . . . . . . . . 22.3.1.1 Inocula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3.1.2 Electron Donor. . . . . . . . . . . . . . . . . . . . . . . . 22.3.2 Passive Sulfate-Reducing Systems . . . . . . . . . . . . . . . . .

259 259 260 260 264 264 265 266 267 269 270 273 273 275 275 276 277 279 282 283 285

285 286 288 288 288 289 291

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22.4

Sulfate Reduction for Metal Recovery/Reuse . . . . . . . . . . . . . . . 22.4.1 Metal Sulfide Precipitation . . . . . . . . . . . . . . . . . . . . . . . 22.4.2 Biogenic Sulfide for Metal Sulfide Precipitation . . . . . . 22.4.3 Selective Metal Precipitation . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

292 292 292 293 293

Impact of Nitrate on the Sulfur Cycle in Oil Fields . . . . . . . . . . . . . . Gerrit Voordouw

296

23.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2 The Oil Field Sulfur Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.3 Effect of Nitrate Injection on SRB Physiology . . . . . . . . . . . . . . 23.4 Mechanism of Souring Control . . . . . . . . . . . . . . . . . . . . . . . . . . 23.5 Prospects for Nitrate Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

296 297 298 300 301 301

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

303

23

Chapter 1

Genetics and Genomics of Sulfate Respiration in Desulfovibrio Judy D. Wall, Adam P. Arkin, Nurgul C. Balci, Barbara Rapp-Giles

Abstract Bacteria that have evolved to use sulfate as a terminal electron acceptor must commit to spending ener gy for sulf ate activation before there is a return on the investment allowing net energy gain. How sulfate is used and how electron flow is controlled have provided challenging topics for research for many years. Having the complete genome sequences of se veral of these bacteria is a monumental step in the elucidation of these questions. This information has pro vided the tools for determining the quantity of transcripts for genes under def ined growth conditions, not just the relative changes in transcripts in two growth conditions. A comparison of the hybridization signal of messenger RNA with that of genomic DNA with oligonucleotide microarrays of all open reading frames re veals the dif ferences in steady-state levels of transcripts for each gene. Gro wth of Desulfovibrio vulgaris Hildenborough on defined medium with lactate as a carbon and reductant source and with sulfate as the electron acceptor has been e xamined by this procedure for levels of gene e xpression. Relative functional importance w as inferred from the le vels of gene transcription, in spite of the recognized limitations of this interpretation. Not surprisingly, genes encoding established functions for sulfate reduction were highly expressed. However, the high molecular mass c-type cytochrome genes thought to encode a most important transmembrane electron conduit for sulfate reduction were expressed at quite low levels.

1.1 Intr oduction Sulfate-reducing bacteria (SRB) are Gram-ne gative deltaproteobacteria, ubiquitously present in soils, that are able to obtain energy by the dissimilatory reduction of sulf ate. These bacteria are considered anaerobes, although their genome sequences have revealed multiple genes putati vely encoding enzymes that reduce oxygen or detoxify its products (Klenk et al. 1997; Heidelber g et al. 2004; Rab us et al. 2004). The e ver-evident and of fensive end product of sulf ate reduction, sulfide, has brought much attention to the activities of the SRB in the environment. The black precipitates in sediments and shorelines, discoloration of paper during the milling processes (Postgate 1984) and the corrosion of ferrous metals (Hamilton 1 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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2003) are a fe w of the less desirable ef fects. On the other hand, the lo w redox potentials achieved by these bacteria also provide them with the capacity of reducing a number of toxic metals, thereby changing their solubilities, and offering a potential method for remediation of metal-contaminated en vironments (Lovley et al. 1991; Gorby and Lovley 1992). Members of the genus Desulfovibrio are perhaps the most easily and rapidly cultured of the SRB and, therefore, have been the subject of the most intensive biochemical and molecular research (Postgate 1984; Peck 1993; V oordouw 1993). Still, there are gaps in understanding ener gy generation by these anaerobes. F or example, what is the role of hydrogen, formate, carbon monoxide, or ethanol during the respiration of sulfate with lactate or pyruvate as an electron donor? The genome sequences available for a few of the SRB are offering us the boundaries, the “parts list,” for our inquiries into these questions. Of course, it does not help that all the parts are not definitively labeled. Hydrogen metabolism has played a prominent role in the metabolism of man y anaerobes and the SRB are no exception. Hydrogen can support sulfate respiration, is produced during fermentati ve gro wth, and is apparently also in volved in the metabolism of a number of or ganic acids. A hydrogen transient is observ ed upon inoculation of Desulfovibrio strains into medium containing organic acids and sulfate (Hatchikian et al. 1976; Tsuji and Y agi 1980). A contro versial role for this production and consumption of hydrogen was proposed by Odom and Peck (1981a) to be an obligate chemiosmotic vectorial electron transfer for energy supplementation, called “hydrogen c ycling.” In this model, the oxidation of or ganic substrates generates protons and electrons that are substrates for c ytoplasmically located hydrogenase(s). Hydrogen produced in the cytoplasm then diffuses across the cytoplasmic membrane, where the periplasmic hydrogenases oxidize the hydrogen, recapturing the electrons for transfer back to the c ytoplasm for sulf ate reduction and liberating the protons to contrib ute to the proton moti ve force. Alternati ve explanations for this burst of hydrogen have been offered, such as a necessary redox adjustment of electron transport components (Tsuji and Yagi 1980) or the need for fermentative A TP production to initiate sulf ate activation by A TP sulfurylase (Lupton et al. 1984). The complete genome sequence of several SRB allo ws a closer examination of this model.

1.2 A pproach As a part of a collaborative effort to understand how the SRB reduce toxic metals and how environmental stresses impact this ability, a number of experiments have been undertaken to e xamine the changes in transcription and protein e xpression during stress in Desulfovibrio vulgaris Hildenborough (V irtual Institute for Microbial Stress and Survi val 2002). T o e xamine the dif ferentially e xpressed genes, microarray analysis of transcripts of putati ve open reading frames (ORFs) have been used (Li et al. 2005). To normalize the data for comparison of expression

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levels across stresses, the transcript hybridization to microarrays for all e xperiments has been compared with genomic DNA hybridization. As a result, a sizeable data set has been obtained that pro vides the e xpression level of all genes in the microarray. Experiments for stress analyses were performed with defined medium containing sodium lactate (60 mM) as an electron donor and sodium sulf ate (50 mM) as an electron acceptor (LS4D medium; Mukhopadhyay et al. 2006). D. vulgaris was grown from freezer stocks to an optical density at 600 nm of 0.3 (approximately 1 × 108 cells per milliliter) and the stress w as imposed. Table 1.1 lists the various treatments for which data were collected. Triplicates for the control and the treated cultures were sampled at the initiation of the treatment and at specif ied intervals, usually not e xceeding 4 h, following the treatment. The a verage of the triplicates was a single data point. In all, 173 data points comparing transcript and genomic DNA hybridization to ORF probes have been analyzed. From these data, the relati ve abundance of transcripts present for a given gene in an exponentially growing culture of D. vulgaris respiring sulf ate with lactate at 30°C w as determined. Figure 1.1 illustrates the distribution of log 2 of the hydridization signal for transcripts di vided by that of genomic DN A for tw o dif ferent genes. Data points that were not signif icantly above the experimental noise were not included in the average calculation. It should be pointed out that at least 50% of the data points were from untreated control cultures. In addition, an y gi ven treatment or stress resulted in the dif ferential expression of only a fe w hundred genes out of about 3,600 ORFs in the genome. Thus, for re gulated genes, the a verage expression would not e xpect to be biased by the v arious stresses, b ut regulation would be e vident in an increased standard deviation of the average. In the following discussion, expression levels of genes involved in various aspects of metabolism are presented. However, to obtain a reference for the meaning of the data, Table 1.2 provides expression levels for comparison genes (operon predictions

B

Number of Observations

Number of Observations

A

Log2 RNA/ Genomic DNA

Log2 RNA/ Genomic DNA

Fig. 1.1 Histogram of transcripts of a hisD, D VU0796, a verage log 2(RNA/genomic DN A) = –13.6 ± 0.7 (173 total observ ations) and b sat, D VU1295, a verage log 2(RNA/genomic DNA) =−9.2 ± 0.8 (171 total observ ations). Cultures were gro wn as described (T able 1.1) for RNA preparation and hybridization to microarrays (Mukhopadhyay et al. 2006)

Table 1.1 Treatments examined for transcriptional responses in Desulfovibrio vulgaris Hildenborough Treatment Concentration or condition Comparison culture Cold Heat Oxygen Alkaline pHa Acid pHb Nitritec Nitrate Sodiumd Potassium Chromate Stationary phase

8°C 50°C 0.1% pH 10 pH 5.5 2.5 mM 105 mM 250 mM 250 mM 0.45 µM 0.8 OD600

30°C 37°C No O2 pH 7 pH 7 No NO2− No NO3− No added Na + No added K + No CrO42− Mid exponential phase 0.3 OD 600

pH was adjusted by addition of KOH. pH was adjusted by addition of H 2SO4. c Growth occurred after cells reduced the nitrite concentration belo w 0.5 mM. d Total concentration of sodium in the treatment w as about 462 mM; other components were present as sodium salts. a

b

Table 1.2 Expression levels of D. vulgaris Hildenborough reference genes during e xponential growth phase of cells respiring sulf ate with lactate as an electron donor Putative gene name Average log expb ± SD Operon DVU numbersa Ribosomal proteins

Tryptophan biosynthesis

High molecular mass cytochrome c

DVU1302 DVU1303 DVU1304 DVU1305 DVU1306 DVU1307 DVU1308 DVU0465 DVU0466 DVU0467 DVU0468 DVU0469 DVU0470 DVU0471 DVU0529 DVU0530 DVU0531 DVU0532 DVU0533 DVU0534 DVU0535 DVU0536

rpsJ rplC rplD rplW rplB rpsS rplV trpE trpG trpD trpC trpF-1 trpB-2 trpA rrf2 rrf1 hmcF hmcE hmcD hmcC hmcB hmcA

−10.1 ± 0.8 −10.8 ± 0.8 −10.4 ± 0.8 −11.5 ± 0.8 −11.1 ± 0.8 −11.0 ± 0.8 −11.2 ± 0.7 −14.6 ± 0.9 −15.0 ± 0.9 −14.2 ± 0.9 −14.4 ± 1.0 −14.3 ± 1.1 −13.7 ± 1.1 −13.7 ± 0.9 −15.1 ± 1.4 −15.0 ± 1.4 −15.6 ± 1.5 −15.2 ± 1.4 −15.6 ± 1.4 −14.7 ± 1.2 −14.9 ± 1.5 −15.1 ± 1.8

c

SD standard deviation. a DVU numbers from TIGR annotation (Heidelber g et al. 2004). b Average log e xp is the a verage log 2 of the RN A to genomic DN A signal from whole genome transcript microarrays from cultures treated as in T able 1.1. In calculating a verage expressions, fewer than 10% of the 173 data points a vailable for each gene were eliminated because of poor signal-to-noise ratio unless otherwise indicated. c For DVU0535, 27 data points were belo w the cutoff criterion.

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were as described by Price et al. 2005). Ribosomal protein genes are expected to be rather highly e xpressed during e xponential growth and the log 2 of the ratio of the messenger RNA to DNA (a large negative number because of the greater quantity of DNA used for hybridization) was in the range of −10.9. That for the tryptophan operon was −14.3, an operon that must function in medium lacking tryptophan, yet, because large quantities of this amino acid are not needed, w ould not be e xpected to be highly expressed. Finally the operon for the high molecular mass cytochrome c was expressed at a still lo wer level, −15.2. The latter w as an une xpectedly low value for transcription of this operon thought to encode an important conduit for electrons for sulfate reduction.

1.3 Sulfate

Metabolism

The enzymology of sulfate reduction by Desulfovibrio strains is rather mature (Peck and LeGall 1982; Peck 1993) and enzymes involved were reported to beconstitutively present in sulfate-respiring cells (Odom and Peck 1981b). Four cytoplasmic enzymes are sufficient for conversion of sulfate to sulfide in an eight electron reduction pathway. Annotated in the D. vulgaris genome, ATP sulfurylase (DVU1295, sat) activates the sulfate-generating adenosine 5′-phosphosulfate (APS) in preparation for the first twoelectron reduction. Inorganic pyrophosphate is released which is clea ved by an inorganic pyrophosphatase (DVU1636, ppaC) to “pull” the reaction. APS is then reduced by APS reductase, a tw o-subunit enzyme (D VU0846/0847, apsBA). Sulf ite is the reduced product that becomes the substrate for the six-electron reduction by sulf ite reductase, also known as desulfoviridin in the Desulfovibrio strains (DVU0402–0404, dsrABD; DVU2776, dsrC). In vitro, this enzyme is capable of producing sulf ide as the final end product of sulfate reduction. Table 1.3 shows the remarkably high level of expression of the genes for sulfate respiration that exceeds that of ribosomal protein genes. Of the candidate ORFs for

Table 1.3 Expression levels of putative genes coding for enzymes of sulf ate reduction in D. vulgaris Hildenborough Putative gene Average log exp Protein function DVU number name ± SD Sulfate adenylyltransferase Adenosine 5′-phosphosulfate reductase Sulfite reductase

Inorganic pyrophosphatase a

DVU1295 DVU0846 DVU0847 DVU0402 DVU0403 DVU0404 DVU2776 DVU1636

sat apsB apsA dsrA dvsB dsrD dsrC ppaC

−9.2 ± 0.8 −8.8 ± 0.7 −8.9 ± 0.8 −9.3 ± 0.9 −9.5 ± 0.7 −8.8 ± 1.0 −10.3 ± 1.1 a −11.1 ± 1.0

Of the 173 data points a vailable, 50 were below the cutoff for the signal-to-noise ratio.

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putative sulfate permeases (DVU0053/0279/0746/0747/1999) none was found to be expressed at these high levels nor did one have higher expression than others. Thus, the data did not point to a gene responsible for transport of this substrate, a function that remains to be specifically identified. The role of other possible intermediates during sulfite reduction such as thiosulfate or trithionate has recei ved significant attention through the years (Drak e and Akagi 1978; Peck and LeGall 1982; Postgate 1984). Recent deletion analysis in D. gigas proposes these polysulfides as intermediates in sulfite reduction (Broco et al. 2005). Although genes predicted to encode thiosulf ate reductases have been annotated in the genomes available, enzymes designed to handle other polysulfides have not become evident.

1.4 Lactate

Oxidation

Electron donors preferred by Desulfovibrio tend to be strain-specif ic, although most incomplete oxidizers gro w readily with or ganic acids such as lactate and pyruvate and most grow with hydrogen or formate. The enzymology of the oxidation of these substrates is much less well deciphered than that for sulfate reduction. Multiple genes have been annotated as lactate permeases and dehydrogenases in the Desulfovibrio genomes. Genes annotated as glycolate dehydrogenase may actually function as lactate dehydrogenases since the structures of substrates and products are quite similar (see EC 1.99.14 in BREND A 1987). Ho wever, it is clear from biochemical experiments that the primary lactate dehydrogenase is a membranebound flavoprotein that is extremely unstable in air (Hansen 1994). Multiple annotations for genes encoding p yruvate ferredoxin oxidoreductase and for formate acetyltransferase also occur in the genome databases. Ho wever, conserved genes ha ve been annotated for phosphate acetyltransferase and acetyl kinase. Those two genes appear in a region of the genome encoding two conserved operons that together possibly contain information for complete metabolism of lactate, i.e., in D. vulgaris a putati ve lactate permease (D VU3026), two genes annotated as glycolate oxidase and an FeS-cluster-binding protein for electron handling (DVU3027/3028) that could be a lactate dehydrogenase, p yruvate ferredoxin oxidoreductase (D VU3025), the phosphate acetyltransferase (D VU3029) and the acetyl kinase (D VU3030). The proximity of genes for the potentially complete metabolism of lactate suggests common re gulation. Table 1.4 shows the expression levels for the genes in these tw o operons of D. vulgaris growing with lactate as electron and carbon sources. The transcripts for these genes are almost as abundant as those for ribosomal proteins. Transcripts for other annotated lactate dehydrogenases or pyruvate ferredoxin oxidoreductases are detected at lo wer le vels (data not sho wn). The interpretation of this dif ferential expression awaits mutagenesis studies, although it is tempting to speculate that the region encoding an apparently full complement of enzymes for lactate oxidation to acetate might be essential for lactate gro wth of the cells.

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Table 1.4 Expression levels of the gene re gion for lactate oxidation to acetate in D. vulgaris Hildenborough DVU number Putative gene name Average log exp ± SD

D

DVU3025 DVU3026 DVU3027 DVU3028 VU3029 DVU3030 DVU3031 DVU3032 DVU3033

por lldP glcD glpC pta ackA COG-Pta NA NA

−10.8 ± 0.9 −12.9 ± 1.0 −11.7 ± 1.0 −12.2 ± 1.0 −11.8 ± 1.0 −11.9 ± 1.0 −11.8 ± 0.9 −12.1 ± 1.0 −12.2 ± 1.0

Arrows indicate operon arrangement and transcription direction of genes. NA not annotated with gene name.

1.5 Hydr ogenases For the “hydrogen c ycle” to function, it is necessary to ha ve hydrogenases located on either side of the c ytoplasmic membrane. Early biochemical analyses were ambiguous in establishing the e xact position of the kno wn hydrogenases of various Desulfovibrio strains because of the dif ficulty in achieving clean cell fractionations (Odom and Peck 1981b). Ho wever, with the sequence of the encoding genes available, it became clear that the biochemically identif ied Fe-only , NiFe and NiFeSe hydro genases of D. vulgaris were periplasmically located (Peck 1993; Voordouw 2000). Only when the genome sequence of D. vulgaris was completed were candidates for c ytoplasmically located hydrogenases con vincingly re vealed. The operon for carbon monoxide dehydrogenase (CODH) biosynthesis and that for an Ech-like hydrogenase, a membrane-bound NiFe hydrogenase that has similarity to NADH–quinone oxidoreductases (comple x I), were annotated. T able 1.5 sho ws the expression levels of these tw o operons. Clearly the CODH operon (D VU2286– 2293) is more highly expressed under the culturing conditions chosen than the catalytic sub unit of CODH (D VU2098/2099) or the Ech operon (D VU0429–0434). Curiously these operons are not conserv ed in the genomes of the other SRB sequenced to date. Thus, the critical nature of the c ytoplasmic hydrogenases in the pathways of sulfate respiration have yet to be established. Interestingly the genome sequences ha ve also shown that the periplasmic hydrogenases are more redundant than pre viously thought. At least four isozymes ha ve been identified by sequence analysis, adding one to the number biochemically determined. All appeared to be e xpressed at reasonable le vels when D. vulgaris was cultured on lactate plus sulfate: Fe-only hydrogenase (DVU1769/1770) at log2 of −13.8 ± 1.4; NiFe isozyme-1 (DVU1921/1922), −13.1 ± 1.7; NiFe isozyme-2 (DVU2525/2526), −14.4 ± 1.0; and NiFeSe hydrogenase (D VU1917/1918), −11.5 ± 1.5. The observ ation that

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J.D. Wall et al. Table 1.5 Expression levels of putati ve cytoplasmic hydrogenase complexes in D. vulgaris Hildenborough DVU number Putative gene name Average log exp ± SD CODH hydrogenase operon DVU2286 DVU2287 DVU2288 DVU2289 DVU2290 DVU2291 DVU2292 DVU2293 DVU2098 DVU2099

cooM cooK cooL cooX cooU cooH hypA cooF cooS cooC-2

−12.4 ± 0.7 −12.1 ± 0.5 −12.0 ± 0.5 −11.9 ± 0.5 −11.6 ± 0.7 −11.8 ± 0.6 −10.8 ± 0.6 −11.0 ± 0.8 −13.1 ± 1.2 −15.2 ± 0.8 a

echF echE echD echC echB echA

−15.0 ± 1.1 ND −14.8 ± 0.9 −14.5 ± 0.8 −15.0 ± 1.0 −14.0 ± 0.8

Ech hydrogenase operon DVU0429 DVU0430 DVU0431 DVU0432 DVU0433 DVU0434

Arrows indicate operon arrangement and transcription direction of genes. ND insufficient useable data for calculation. a Fifty-four of 173 data points were not signif icant.

transcripts for the NiFeSe enzyme were most ab undant w as une xpected since the biochemical and mutational data indicated that the Fe-only hydrogenase accounts for most of the hydrogenase activity of the periplasm (Pohorelic et al. 2002).

1.6

Transmembrane Electron-Conducting Complexes

To complete the circuit of electrons in the “hydrogen cycling” model, there must be mechanisms that conduct the electrons across the c ytoplasmic membrane from the periplasmically oxidized hydrogen to sulf ate. In addition, gro wth on hydrogen or formate w ould also require a transmembrane conduit for the electrons generated from the periplasmic oxidation of these substrates. The high molecular mass c ytochrome c comple x (Table 1.2) that has a he xadecaheme c ytochrome f acing the periplasm was the first such transmembrane complex (TMC) described (Rossi et al. 1993). It w as proposed to function for electron transfer from periplasmically oxidized hydrogen, re gardless of the origin of the hydrogen, to the c ytoplasm for sulfate reduction (Voordouw 2000). Deletions of this operon, however, demonstrate that this TMC is not essential for this activity (Dolla et al. 2000) and the expression levels of the genes were une xpectedly low (Table 1.2).

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Much progress has been achieved in the molecular analysis of additional TMCs of the SRB and sulfur -oxidizing bacteria (Matias et al. 2005; Pereira et al. 2006; Table 1.6). A three-subunit conserved complex, Qmo (DVU0848–0850), is encoded promoter distal in the same operon as the genes for APS reductase. The location of these genes suggests a possible role in pro viding electrons for APS reduction. A six-gene operon (DVU1286–1291) coding for another apparent TMC suggested to provide electrons to DsrAB, the bisulfite reductase, has also been identified. This operon includes a type II tetraheme cytochrome c3, DsrJ. Recently, the isolation and characterization of the Tmc comple x (DVU0263–0266) was reported. Although a role in electron transfer from periplasmic oxidations or from reduced menaquinones to sulf ate w ould seem lik ely for this comple x, no e xperimental e vidence supported this possibility (Pereira et al. 2006). An additional complex with components sharing sequence similarity with heterodisulfide reductase (DVU2399–2405) and another with similarity to sodium-translocating N ADH:quinone oxidoreductase complex (DVU2791–2798, rnf) have been annotated b ut do not yet ha ve functions assigned. Table 1.6 shows that each of these putative TMCs appears to be expressed at levels similar to those of ribosomal protein genes. The Rnf complex genes were somewhat less ab undantly transcribed b ut were still more ab undant than Hmc genes. The need for different conduits to supply electrons to APS reductase and to bisulf ite reductase w as proposed man y years ago (Peck 1993). Thus, tw o conduits w ould be predicted. Why the multiplicity of TMCs, all of which appear to be synthesized? The information pro vided by genome sequences has served to emphasize that our models for ener gy generation in the SRB are still inadequate.

1.7 Conclusions For the first time, we now have a glimpse at the relative transcription of all putative ORFs in the genome of a sulf ate-reducing bacterium, D. vulgaris Hildenborough. Certainly this information is limited by differential stability of transcripts, stability of the proteins encoded, and enzyme acti vity regulation. However, a few surprises have been observed. As predicted, genes for the sulf ate-reducing enzymes were highly e xpressed. The apparent redundanc y for other steps in metabolism of substrates, i.e., periplasmic hydrogenases and TMCs, was affirmed by robust expression of the genes for the redundant systems. In contrast, c ytoplasmic hydrogenases (CODH complex and Ech) were eightfold dif ferent in e xpression le vels. The much greater transcription le vels of the CODH comple x might suggest a more important role under these experimental conditions. Finally, the low level of expression of the Hmc complex might suggest that it may be supplementary to other TMCs or that it may be more important during growth on other substrates or in other cellular growth phases.

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J.D. Wall et al. Table 1.6 Expression levels of genes coding for putative transmembrane complexes in D. vulgaris Hildenborough DVU number Putative gene name Average log exp ± SD Type II c3 transmembrane complex DVU0258 DVU0259 DVU0260 DVU0261 DVU0262 DVU0263 DVU0264 DVU0265 DVU0266

COG-BaeS divK mtrA COG-UspA NA tmcA tmcB tmcC tmcD

−14.1 ± 1.0 −9.7 ± 1.0 −11.2 ± 1.0 −11.6 ± 1.0 −12.2 ± 0.9 −12.0 ± 0.9 −11.4 ± 1.0 −12.2 ± 1.0 −11.8 ± 1.0

qmoA qmoB qmoC NA

−11.0 ± 0.7 −12.0 ± 0.7 −12.8 ± 0.7 −12.1 ± 0.7

dsrP dsrO dsrJ dsrK dsrM NA

−11.6 ± 0.9 −12.2 ± 0.9 −12.2 ± 0.9 −12.6 ± 0.9 −12.3 ± 0.9 −13.2 ± 0.7

NA NA NA hdrA hdrB hdrC eutG

−11.8 ± 1.0 −11.5 ± 1.0 −12.1 ± 0.7 −12.0 ± 0.9 −11.5 ± 0.9 −11.2 ± 1.0 −9.1 ± 1.4

dhcA rnfC rnfD rnfG rnfE rnfA rnfB apbE

−12.6 ± 1.0 −13.4 ± 1.0 −14.1 ± 1.0 −13.7 ± 1.1 −14.7 ± 0.9 −14.4 ± 1.0 −13.6 ± 0.9 −13.3 ± 1.0

Qmo transmembrane complex DVU0848 DVU0849 DVU0850 DVU0851 Dsr transmembrane complex DVU1286 DVU1287 DVU1288 DVU1289 DVU1290 DVU1291 Heterodisulfide reductase DVU2399 DVU2400 DVU2401 DVU2402 DVU2403 DVU2404 DVU2405 Rnf transmembrane complex DVU2791 DVU2792 DVU2793 DVU2794 DVU2795 DVU2796 DVU2797 DVU2798

Arrows indicate operon arrangement and transcription direction of genes. NA not annotated with gene name.

1 Genetics and Genomics of Sulfate Respiration in Desulfovibrio 11 Acknowledgements. This w ork w as part of the V irtual Institute for Microbial Stress and Survival (http://vimss.lbl.go v) supported by the US Department of Ener gy, Office of Science, Office of Biological and En vironmental Research, Genomics Program:GTL through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Ener gy; the DOE Ener gy Biosciences Program, Of fice of Basic Ener gy Sciences, grant number DE-FG02-87ER13713, and a Uni versity of Missouri Life Sciences Post Doctoral Fellowship to N.C.B.

References BRENDA (1987) Cologne Uni versity Bioinformatics Center . http://www .brenda.uni-koeln.de/ index.php4 Broco M, Rousset M, Oliveira S, Rodrigues-Pousada C (2005) Deletion of flavoredoxin gene in Desulfovibrio gigas re veals its participation in thiosulf ate reduction. FEBS Lett 579:4803–4807 Dolla A, Pohorelic BK, V oordouw JK, V oordouw G (2000) Deletion of the hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough hampers h ydrogen metabolism and low-redox-potential niche establishment. Arch Microbiol 174:143–151 Drake HL, Akagi JM (1978) The dissimilatory reduction of bisulfite by Desulfovibrio vulgaris. J Bacteriol 136:916–923 Gorby Y A, Lo vley DR (1992) Enzymatic uranium precipitation. En viron Sci T echnol 26:205–207 Hamilton WA (2003) Microbially influenced corrosion as a model system for the study of metal microbe interactions: a unifying electron transfer h ypothesis. Biofouling 19:65–76 Hansen TA (1994) NAD(P)-independent lactate dehydrogenase from sulfate-reducing prokaryotes. Methods Enzymol 243:21–23 Hatchikian EC, Chaigneau M, Le Gall J (1976) Analysis of g as production by gro wing cultures of three species of sulf ate reducing bacteria. In: Schle gel HG, Gottschalk G, Pfennig N (eds) Microbial production and utilization of g ases. Goltze, Göttingen, pp 109–118 Heidelberg JF, Seshadri R, Haveman SA, Hemme CL, Paulsen IT, Kolonay JF, Eisen JA, Ward N, Methe B, Brinkac LM, Daugherty SC, Deboy RT, Dodson RJ, Durkin AS, Madupu R, Nelson WC, Sullivan SA, Fouts D, Haft DH, Selengut J, Peterson JD, Davidsen TM, Zafar N, Zhou L, Radune D, Dimitro v G, Hance M, T ran K, Khouri H, Gill J, Utterback TR, Feldblyum TV , Wall JD, Voordouw G, Fraser CM (2004) The genomic sequence of the anaerobic, sulf atereducing bacterium Desulfovibrio vulgaris Hildenborough. Nat Biotechnol 22:554–559 Klenk HP, Clayton RA, Tomb JF, White O, Nelson KE, Ketchum KA, Dodson RJ, Gwinn M, Hickey EK, Peterson JD, Richardson DL, K erlavage AR, Graham DE, K yrpides NC, Fleischmann RD, Quackenbush J, Lee NH, Sutton GG, Gill S, Kirkness EF, Dougherty BA, McKenney K, Adams MD, Loftus B, Peterson S, Reich CI, McNeil LK, Badger JH, Glodek A, Zhou L, Ov erbeek R, Gocayne JD, Weidman JF, McDonald L, Utterback T , Cotton MD, Spriggs T , Artiach P, Kaine BP, Sykes SM, Sadow PW, D’Andrea KP, Bowman C, Fujii C, Garland SA, Mason TM, Olsen GJ, Fraser CM, Smith HO, Woese CR, Venter JC (1997) The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus. Nature 390:364–370 Li X, Zhili H, Zhou J (2005) Selection of optimal oligonucleotide probes for microarrays using multiple criteria, global alignment and parameter estimation. Nucleic Acids Res 33:6114–6123 Lovley DR, Phillips EJP , Gorby Y A, Landa E (1991) Microbial reduction of uranium. Nature 350:413–416 Lupton FS, Conrad R, Zeikus JG (1984) Ph ysiological function of h ydrogen metabolism during growth of sulfidogenic bacteria on organic substrates. J Bacteriol 159:843–849 Matias PM, Pereira IA C, Soares CM, Carrondo MA (2005) Sulphate respiration from h ydrogen in Desulfovibrio bacteria: a structural biology o verview. Prog Biophys Mol Biol 89:292–329

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Mukhopadhyay A, He Z, Alm EJ, Arkin AP , Baidoo E, Bor glin SC, Chen W, Hazen TC, He Q, Holman H-Y, Huang K, Huang R, Jo yner DC, Katz N, Keller M, Oeller P, Redding A, Sun J, Wall J, W ei J, Y ang Z, Y en H-C, Zhou J, K easling JD (2006) Salt stress in Desulfovibrio vulgaris Hildenborough: an integrated genomics approach. J Bacteriol 188:4068–4078 Odom JM, Peck HD Jr (1981a) Hydrogen cycling as a general mechanism for energy coupling in the sulfate reducing bacteria, Desulfovibrio sp. FEMS Microbiol Lett 12:47–50 Odom JM, Peck HD Jr (1981b) Localization of dehydrogenases, reductases, and electron transfer components in the sulfate-reducing bacterium Desulfovibrio gigas. J Bacteriol 147:161–169 Peck Jr HD (1993) Bioenergetic strategies of the sulfate-reducing bacteria. In: Odom JM, Singleton R Jr (eds) The sulfate-reducing bacteria: contemporary perspectives. Springer, New York, pp 41–76 Peck HD Jr, LeGall J (1982) Biochemistry of dissimilatory sulphate reduction. Philos Trans R Soc Lond Ser B 298:443–466 Pereira PM, T eixeira M, Xa vier AV, Louro R O, Pereira A C (2006) The TMC comple x from Desulfovibrio vulgaris Hildenborough is in volved in transmembrane electron transfer from periplasmic hydrogen oxidation. Biochemistry 45:10359–10367 Pohorelic BK, Voordouw JK, Lojou E, Dolla A, Harder J, Voordouw G (2002) Effects of deletion of genes encoding Fe-only hydrogenase of Desulfovibrio vulgaris Hildenborough on hydrogen and lactate metabolism. J Bacteriol 184:679–686 Postgate JR (1984) The sulphate reducing bacteria, 2nd edn. Cambridge Uni versity Press, Cambridge Price MN, Huang KH, Alm EJ, Arkin AP (2005) A novel method for accurate operon predictions in all sequenced prokaryotes. Nucleic Acids Res 33:880–892 Rabus R, Ruepp A, Frick ey T, Rattei T, Fartmann B, Stark M, Bauer M, Zibat A, Lombardot T , Becker I, Amann J, Gellner K, Teeling H, Leuschner WD, Glockner FO, Lupas AN, Amann R, Klenk HP (2004) The genome Desulfotalea psychrophila, a sulf ate-reducing bacterium from permanently cold artic sediments. Environ Microbiol 6:887–902 Rossi M, Pollock WBR, Reij MW , K eon RG, Fu R, V oordouw G (1993) The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex. J Bacteriol 175:4699–4711 Tsuji K, Yagi T (1980) Signif icance of h ydrogen burst from gro wing cultures of Desulfo vibrio vulgaris Miyazaki and the role of hydrogenase and cytochrome c3 in energy production system. Arch Microbiol 125:35–42 Virtual Institute for Microbial Stress and Survival (2002) Publications. http://vimss.lbl.gov/findings/ publications.php Voordouw G (1993) Molecular biology of the sulfate-reducing bacteria. In: Odom JM, Singleton R Jr (eds) The sulf ate-reducing bacteria: contemporary perspecti ves. Springer , Ne w Y ork, pp 88–130 Voordouw G (2000) A uni versal system for the transport of redox proteins: early roots and latest developments. Biophys Chem 86:131–40

Chapter 2

Living on Sulfate: Three-Dimensional Structure and Spectroscopy of Adenosine 5′-Phosphosulfate Reductase and Dissimilatory Sulfite Reductase Günter Fritz, Alexander Schiffer, Anke Behrens, Thomas Büchert, Ulrich Ermler, Peter M.H. Kroneck

Abstract The reduction of sulfate to sulfide and the reverse reaction are widespread biological processes. Hereby , microor ganisms play a central role. Plants also reduce sulfate for the purpose of biosynthesis, and both plants and animals convert reduced sulfur compounds to sulfate. Sulfate respiration is used for energy conservation by strictly anaerobic bacteria and archaea. The redox equi valents generated by the oxidation of or ganic compounds are transferred to sulf ate as the terminal electron acceptor. There are three key enzymes localized in the cytoplasm or at the cytoplasmic aspect of the inner membrane: ATP sulfurylase (ATPS), adenosine 5′-phosphosulfate reductase (APSR), and dissimilatory sulf ite reductase (SIR). Sulfate (S6+) cannot be directly reduced by dihydrogen or organic acids, it has to be activated to adenosine 5 ′-phosphosulfate (APS) catalyzed by A TPS. The enzyme APSR (cofactors flavin adenine dinucleotide, [4Fe4S]) catalyzes the conversion of APS to sulf ite (S 4+) and AMP, followed by the comple x multicomponent enzyme SIR (cofactors siroheme, [4Fe4S]) which catalyzes the reduction of sulf ite (S 4+) to sulfide (S 2−). In this contrib ution we present the three-dimensional structures of APSR from Archaeoglobus fulgidus and of catalytically rele vant reaction intermediates. In addition, we discuss spectroscopic and structural data of SIR purif ied from this organism.

2.1 Intr oduction The biogeochemical cycles of the basic elements of life, such as nitrogen, oxygen, and sulfur , ha ve attracted the interest of man y researchers o ver the past fe w decades. Of similar importance, the biochemistry of the transition metals has been extensively studied because of their functions as cof actors, or as part of cof actors in enzymes, and as structural elements in proteins. Man y processes strictly depend on transition metal ions and their ability to catalyze multielectron redox and hydrolytic transformations (Kroneck 2005). Sulfur can e xist in the biosphere in se veral oxidation states, such as S 6+ in sulf ate, S 4+ in sulf ite, S 0 in elemental sulfur , or 13 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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S2− in hydrogen sulfide (Amend and Shock 2001). Interconversions of these species constitute their biogeochemical c ycles which are sustained by comple x biological processes, with bacteria playing a prominent role. Numerous studies suggest that the ability to reduce sulf ate was developed early during prokaryotic e volution. As life may have originated in hot environments (Achenbach-Richter et al. 1987), the occurrences of sulfate-reducing prokaryotes among hyperthermophilic archaea and deep-branching thermophilic bacteria indicate an early origin of this process (Dahl and Trüper 2001). Isotopic data suggest that dissimilatory sulf ate reduction began 2.8 billion to 3.1 billion years ago (Schidlo wski 1983) but acquired global significance only after sulfate concentrations had considerably increased in the Precambrian oceans approximately 2.35 billion years ago (Cameron 1982). Dissimilatory sulfate reduction operates under strictly anaerobic conditions and represents an important element within the biogeochemical sulfur cycle (Peck 1959; LeGall and Fauque 1988; Hansen 1994). The sulfur-oxidizing pathway proceeds in the opposite direction, starting from sulfide, elemental sulfur, or thiosulfate. Sulfatereducing bacteria, such as Desulfovibrio sp., use sulf ate as the terminal electron acceptor, and hydrogen sulf ide is formed as a f inal product. The y deri ve ener gy from the dissimilatory reduction of sulfate by dihydrogen or organic substrates. The electron transport chain catalyzing this reaction involves periplasmic hydrogenases, several multi-heme cytochromes, such as cytochrome c3 and nonaheme c, and other both membrane-bound and c ytoplasmic redox enzymes (Matias et al. 2005; Pires et al. 2006). In this chapter we will focus on tw o enzymes involved in sulf ate respiration: (1) adenosine 5 ′-phosphosulfate reductase (APSR), which con verts adenosine 5′-phosphosulfate (APS) to sulf ite and AMP , and (2) sulf ite reductase (SIR), which reduces sulfite to hydrogen sulf ide. These multimetal enzymes ha ve been purified to homogeneity under the e xclusion of dioxygen and characterized biochemically and spectroscopically. The structural information on both APSR and SIR from the thermophilic archeon Archaeoglobus fulgidus (Stetter et al. 1987) was obtained by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. Furthermore, catalytically competent intermediates could be trapped and characterized.

2.2 Adenosine

5′-Phosphosulfate Reductase

Sulfate cannot oxidize H2 or organic acids in view of its negative redox potential of −516 mV; thus, it has to be acti vated to APS at e xpense of ATP (via ATP sulfurylase), which shifts the standard redox potential (APS/AMP+HSO 3−) to −60 mV (Thauer et al. 1977). Note that the formation of APS is ender gonic and probably driven by the subsequent clea vage of p yrophosphate. All dissimilatory APSRs isolated so f ar contain fla vin adenine dinucleotide (F AD) and FeS clusters; the y catalyze the two-electron reduction of APS to sulf ite (Lampreia et al. 1994):

2 Three-Dimensional Structure and Spectroscopy of Adenosine

APS + 2e −
AMP + HSO3 −

(

15

)

E 0′ APS AMP + HSO3 − = −60 mV

Hydrolytic cleavage of the S–O–P moiety in APS yields approximately 80 kJ mol−1 (Lipmann 1958), which is among the highest v alues reported so f ar for an X–O–P bond in a biological molecule. This ener gy is utilized by APSR in the reducti ve transformation of APS to sulfite and AMP.

2.2.1

Molecular Properties of APSR

The molecular parameters of APSR from sulf ate-reducing bacteria ha ve been a matter of debate. Originally , an α2β-subunit composition with one F AD and tw o [4Fe4S] prosthetic groups (α approximately 70 kDa, β approximately 20 kDa) was proposed (Lampreia et al. 1994), or an α2β2 complex with one single iron–sulfur cluster/αβ heteromer (Verhagen et al. 1994). This FeS cluster w as thought to consist of more than four iron atoms. Analysis of the genes encoding theα-subunit and the β-subunit of APSR from the sulf ate-reducing hyperthermophilic A. fulgidus revealed a putati ve FAD-binding domain on the α-subunit. On the β-subunit, the arrangement of seven cysteine residues suggested the presence of a ([3Fe4S][4Fe4S]) cluster similar to 7Fe ferredoxins (Speich et al. 1994). Recently, a purification procedure w as de veloped which led to acti ve samples of APSR from four dif ferent microorganisms, Desulfovibrio desulfuricans , D. vulgaris , Thiobacillus denitrif icans, and A. fulgidus. Their UV–vis spectra were practically identical, and the analytical data indicated the presence of one F AD and tw o [4Fe4S] centers per αβ heterodimer as depicted in a structural model (Fritz et al. 2000). Purif ication under the strict exclusion of dioxygen led to a 10% increase in acti vity of APSR, and the absence of dioxygen was crucial for the crystallization of the enzyme. APSR purified under an inert atmosphere (N 2/H2 95%/5%) did not sho w any EPR signal at g=2.01 characteristic for a [3Fe4S] center . In these preparations the enzyme w as in a partially reduced state according to UV–vis and EPR spectra (Büchert et al. 1999). Dithionite reduction of APSR resulted in a rhombic EPR spectrum with gz, gy and gz at 2.08, 1.94 and 1.90. One single [4Fe4S] cluster (center I) became reduced (0.9–1.0 spin per mole); there were no EPR signals originating from the second FeS center. The reduction potentials of center I in APSR of D. desulfuricans and of A. fulgidus were determined by an EPR-monitored titration to be −59 ± 12 and −57 ± 5 mV (pH 7.0), which are close to literature v alues (Lampreia et al. 1994; Verhagen et al. 1994). Note that these v alues are rather high for [4Fe4S] clusters, which usually have values ranging between −200 and −500 mV (Beinert et al. 1997). Such low potentials were found for the potentials of FeS center II of APSR from D. desulfuricans and A. fulgidus, −540 ± 15 and −520 ± 10 mV, respectively. The lineshape and the parameters of the EPR spectra of FeS center I in all four APS reductases were independent of the ionic strength of the buffer, indicating that FeS center I is shielded from the solv ent. At pH 8, reduction of APSRs with

16

G. Fritz et al.

dithionite ga ve EPR signals characteristic for magnetically interacting [4Fe4S] centers, including the so-called g≈4 signal due to dipolar interaction of tw o S=1/2 systems (Fritz et al. 2002a). An increase in ionic strength clearly influenced the lineshape of these signals. Since the ionic strength did not influence the EPR spectra of FeS center I, it w as concluded that FeS center II must be af fected because of partial exposure to the solv ent. Consequentially, FeS center I w as assigned to the [4Fe4S] cluster in close proximity to F AD (Sect. 2.2.2). Upon addition of both sulfite and AMP to APSR, EPR signals from FeS center I and a distinct flavin radical were detected in agreement with the absorption maximum observ ed at 380 nm which is typical for the fla vosemiquinone radical anion. The assignment of the high-potential FeS center I to the b uried [4Fe4S] cluster and the lo w-potential center II to the surf ace-exposed [4Fe4S] recei ved strong support from features in the crystal structure of APSR from A. fulgidus (Fritz et al. 2002b). A different number of backbone amides interacted with the sulfur atoms of the tw o [4Fe4S] sites. Local dipoles formed by the amide groups stabilized the additional ne gative char ge upon reduction of FeS center I; thus, the reduction potential was shifted to a more positi ve value. Fewer interactions are observ ed for FeS cluster II at the surf ace, leading to a lower reduction potential. Upon addition of sulfite to APSR of all four organisms, a slight increase around 320 nm was observed indicative for the formation of a sulfite adduct in the N5 position of the isoalloxazine ring of the F AD moiety. Addition of AMP to the APS reductase–sulf ite complex of the sulfate-reducing organisms in the absence of dioxygen led to the disappearance of the band at 320 nm and caused a slight increase around 380 nm as well as a decrease around 448 nm. The data are consistent with the decay of the fla vin N5-sulfite adduct and reduction of FAD and reduction of one [4Fe4S] cluster; part of the fla vin was only one-electron-reduced as indicated by the formation of a stable anionic flavin radical according to EPR spectroscopy. Only the enzyme purified from T. denitrificans did not form a flavin radical under these conditions. After addition of sulfite and AMP to APS reductase under exclusion of dioxygen, the FAD and one iron–sulfur cluster became reduced immediately. The physiological electron donor/acceptor of APS reductase in sulfate reduction and sulfur oxidation still remains unknown. As electrons generated in the periplasm must be transferred to APS reductase in the cytoplasm, there must exist an electron donor shuttling electrons from a transmembrane electron translocating comple x to APS reductase (see Chap. 3 by Pereira). Preliminary e xperiments with crude extracts from D. desulfuricans indicated that a low molecular mass component (less than 1 kDa), perhaps a thiol, might be in volved. Thus, the reactivity of APSR with different thiols was investigated, including glutathione, l-cysteine, 2-mercaptoethanol, dithiothreitol, 2-mercaptoethylamine and coenzyme M. Among these thiols, dithiothreitol w orked best. 2-Mercaptoethylamine and coenzyme M were much less active; glutathione, l-cysteine and 2-mercaptoethanol remained without ef fect under our experimental conditions. With 10 mM dithiothreitol, the degree of reduction of APSR from D. desulfuricans was comparable to that achie ved with sulfite and AMP. Ho wever, the specif ic acti vity of APSR, with dithiothreitol as the

2 Three-Dimensional Structure and Spectroscopy of Adenosine

17

electron donor and APS as the electron acceptor , w as much lo wer compared with the standard acti vity assay, with reduced methylviologen and APS (Büchert et al. 1999).

2.2.2

Three-Dimensional Structure of APSR

Recently, the X-ray structures of APSR from A. fulgidus in several enzymatic states have been solved, including the structure of the FAD–sulfite adduct which allowed a structure-based reaction mechanism to be de veloped (Fritz et al. 2002b; Schif fer et al. 2006). The enzyme consists of the α-subunit (75 kDa) and the β-subunit (20 kDa) arranged as an α2β2 heterotetramer (Fig. 2.1). The α-subunit harbors the FAD prosthetic group and can be di vided into three domains. This architecture classifies APSR as member of the fumarate reductase family (Lancaster 2003). The β-subunit consists of a bacterial ferredoxin-type se gment with tw o [4Fe4S] clusters, a three-stranded antiparallel β-sheet and a tail with a length of 50 Å (Fig. 2.2). The global part of the β-subunit is embedded into a broad cleft of the α-subunit, while its long tail wraps around the α-subunit (Fig. 2.1). The reaction of APSR consists of an electron transfer step and the reducti ve cleavage of the ester moiety of APS. T wo electrons are transferred to the [4Fe4S] centers and from there via the conserv ed Trp B48 to the isoalloxazine ring via its si side; the hydrolytic reaction occurs at the re side of FAD within a 17-Å-long channel (Fritz et al. 2002b; Schif fer et al. 2006; Fig. 2.1).

2.2.3

Reaction Mechanism of APSR

The reaction catalyzed by APSR comprises a nucleophilic attack of the N5 atom of reduced FAD on the sulfur of APS presumably via an FAD–APS intermediate (Massey et al. 1969). This intermediate decays to AMP and the F AD– sulfite adduct, which is subsequently clea ved and sulf ite is f inally liberated (Figs. 2.3, 2.4). This mode of action w as originally postulated by Michaels et a l. (1970) and has now been experimentally corroborated on the basis of structures of APSR in catalytically relevant states (Fig. 2.4). In the oxidized state (FADox) the isoalloxazine moiety of the F AD cofactor exhibits a similarly bent confor mation as observ ed in the structure of the reduced enzyme. In the APS-bound state (FADox–APS) the substrate APS is embedded into a 17-Å-long substrate channel in such a way that the isoalloxazine ring is pushed towards the channel bottom, thereby producing a compressed enzyme–substrate comple x. A clamp formed by residues Arg A317 and Leu A278 to f ix the adenine ring, as well as the curved APS conformation appear to be key factors to hold APS in a strained conformation. This energy-rich state becomes relaxed during the attack of APS on the reduced FAD. A relaxed FAD–sulfite adduct is observed in the structure

18

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Fig. 2.1 Left: The adenosine 5 ′-phosphosulfate reductase (APSR) αβ heterodimer from Archaeoglobus fulgidus. The α-subunit, which harbors the flavin adenine dinucleotide (FAD; yellow), is shown in blue; the β-subunit, with the two [4Fe4S] clusters, is shown in red. The substrate-binding channel is illustrated by approximately 35 tightly bound w ater molecules ( green), indicating a strong electrostatic f ield favorable for binding char ged groups (Fritz et al. 2002b; Schif fer et al. 2006). Right: Active-site channel and electron transfer pathw ay

Fig. 2.2 Alignment of the Fe–S-binding domain of the APSR β-subunit (red) from A. fulgidus with the ferredoxin ( green) from Clostridium acidiurici ; the β-subunit has an elong ated loop (magenta) that presumably represents the docking site for the ph ysiological electron donor . Electron transfer over a distance of about 30 Å proceeds from the protein surf ace to FAD via the two [4Fe4S] clusters and conserved TrpB48 to the C8 methyl group of FAD (Fritz et al. 2002b)

2 Three-Dimensional Structure and Spectroscopy of Adenosine

19

Fig. 2.3 The FAD–sulfite adduct of APSR from A. fulgidus. The three sulfite oxygens are hydrogen-bonded; His A398 and Arg A265 appear to be key residues for substrate binding and catalysis (Schiffer 2004)

A

CH3

-

N N H

N N H

W 234 NH

R 265

NH

B

O

O

N 74

HN

APS

NH

O

O

H 398

HN

+

NH2

O

CH3

O

E 141

R 265

H2N

NH

S O

+

NH2

H2N

N

O

NH

R 265

NH

+

NH2

O

N 74

HN

O

H2 O O

H 398 HN

O

N

O

E 141

N N H

W 234

N 74

HN

O

O

N

O

O

NH

OH 2

H 398

HN

CH 3 N

O

H2O

H 2N

N H

OH 2

C

CH3

N

OH 2

O

R

2e

W 234

O

P

O

-

F

O

N

-

O

N

-

H N N

W 234

R 265

H O

+

O

H 2N

O

E 141

N 74

HN

O

H2O

OH 2 O R

O

O

-

P

-

NH

S O-

O

NH2

NH

N

N

O

H 398

O

HN

E 141

N

3

HSO

E

CH3

W 234

N NH

S

O O

OH 2

O O

NH H2 N

+

NH2

HN

D

O

CH3

+

O

AMP E 141

O O

H3 O

H2 O O

+

R 265

NH

NH2 NH2

O

-

NH

O

+

O

O O

NH N

O O

H2 O

O

P

N 74

HN

S

O

OH 2

O O

N

NH

-

N

N

W 234

N 74

HN

H 398 N

NH

OH 2

H3O

R 265

-

N

N

E 141

H 398

R

Fig. 2.4 Reaction cycle of APSR. A represents reduced APSR, B APSR–adenosine 5′-phosphosulfate (APS), D APSR–AMP, E reduced APSR–sulf ite and F oxidized APSR; B does not e xactly represent the APSR–APS state, as the latter contains F AD in the oxidized state; the postulated short-lived state C was modeled (Schiffer et al. 2006)

20

G. Fritz et al.

of the FAD–sulfite state. Finally, a FAD–sulfite–AMP1 state could be trapped with AMP within v an der Waals distance. This structure documents ho w adjacent negative charges can be stabilized by the protein, which is crucial for the back reaction to form APS from AMP and sulf ite (Fritz et al. 2002b; Schif fer et al. 2006).

2.3 Dissimilatory

SIR

SIRs are key enzymes for both biosynthetic assimilation of sulfur and dissimilation of oxyanions, such as sulf ate, for energy conservation (LeGall and F auque 1988). Found throughout the three major kingdoms of li ving or ganisms, man y of these enzymes employ a siroheme that is e xchange-coupled with an iron–sulfur cluster (Belinsky 1996; Crane et al. 1995). SIRs catalyze the six-electron reduction of sulfite to sulfide (Thauer et al. 1977): HSO3 − + 6e − + 6H + → HS− + 3H2 O E 0′ ( HSO3 − HS− ) = −116 mV. SIR has been described as α2β2γmδn multimers with α approximately 50 kDa, β approximately 45 kDa, γ approximately 11 kDa, δ approximately 8 kDa, and a total molecular mass of approximately 200 kDa (Steuber and Kroneck 1998). The enzyme has been isolated from se veral microorganisms (Lee et al. 1973; Moura et al. 1988; Lui et al. 1994; Steuber et al. 1995; Dahl and T rüper 2001). The γ-subunit from Pyrobaculum aerophilium reveals a novel fold consisting of a β-hairpin and an orthogonal helix bundle. A flexible seven-residue C-terminal arm with a C-terminal c ysteine is suggested to be in volved in interaction with the α2β2 tetramer (Cort et al. 2001). In the X-ray structure of the A. fulgidus γ-subunit, however, this highly conserved C-terminal arm adopts a well-defined confirmation and the C-terminal c ysteine might be a constituent of a redox-acti ve disulfide bond (Mander et al. 2005). The δ-subunit from D. vulgaris contains a winged helix motif, suggesting its participation in DNA-binding (Mizuno et al. 2003). The proposed binding of sulf ate or sulf ite (Karkhof f-Schweizer et al. 1995) was meanwhile ruled out (Hittel and Voordouw 2000). Active SIR from A. fulgidus was recently purified under the strict e xclusion of dioxygen (Schiffer 2004). The enzyme consisted of an α-subunit (51 kDa) and a β-subunit (45 kDa) arranged as an α2β2 heterotetramer. Its specific activity (approximately 50 nmol sulf ite min −1 mg −1) w as lo wer than v alues obtained for crude extracts of A. fulgidus with reduced methylviologen (Dahl et al. 1994; Dahl and Trüper 2001); note that those initial rates were also found by Schiffer (2004) for the crude extract of A. fulgidus. The enzyme from A. fulgidus exhibited a complicated set of EPR resonances at low and high magnetic field (Schiffer 2004; Fritz et al. 2005) The interpretation of the high-spin EPR signals and the assignment of the indi vidual resonances to the

2 Three-Dimensional Structure and Spectroscopy of Adenosine

21

metal sites of dissimilatory SIR represents a major challenge. In the oxidized state there were two types of high-spin signals, with spin S = 5/2 and S = 9/2 (Pierik and Hagen 1991). The signals with spin S = 5/2 were present in assimilatory as well as dissimilatory SIRs (Pierik and Hagen 1991; Wolfe et al. 1994), whereas the S = 9/2 signals were only observ ed in se veral dissimilatory SIRs, including the enzymes from D. vulgaris and A. fulgidus. The spin S = 5/2 signal results from the coupled high-spin siroheme center. As shown by Mössbauer spectroscop y, the siroheme is in the high-spin state and is strongly e xchange coupled to the [4Fe4S] 2+ cluster (Christner et al. 1981). There are e xchange and hyperfine interactions between the heme iron and the iron–sulfur cluster (Belinsk y 1996). The iron content of dissimilatory SIR has been a matter of controversy, with ten to 24 Fe/α2β2γnδm (Steuber and Kroneck 1998). F or the A. fulgidus enzyme 22–24 non-heme Fe/α2β2 were reported, indicative for the presence of six [4Fe4S] clusters (Dahl et al. 1994). Recently , SIR from A. fulgidus was crystallized in the absence of dioxygen (Schiffer 2004). The green-brown crystals diffracted well below 2.5 Å and were suitable for X-ray structure analysis, which is currently in progress. Acknowledgements. Financial support from the Deutsche F orschungsgemeinschaft (P.M.H.K.) and the Max-Planck-Gesellschaft (U.E.) is gratefully ackno wledged.

References Achenbach-Richter L, Gupta R, Stetter , K O, W oese CR (1987) W ere the original eubacteria thermophiles? Syst Appl Microbiol 9:34–39 Amend JP, Shock EL (2001) Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and Bacteria. FEMS Microbiol Re v 25:175–243 Beinert H, Holm RH, Münck E (1997) Iron-sulfur clusters: nature’ s modular , multipurpose structures. Science 277:653–659 Belinsky MI (1996) Exchange model of the {[Fe 4S4]-Fe} active site of sulf ite reductase. Chem Phys 201:343–356 Büchert T, Fritz G, Kroneck, PMH (1999) T owards the natural electron donor of adenosine-5 ′phosphosulfate (APS) reductase from Desulfovibrio desulfuricans Esse x. In: Ghisla S, Kroneck PMH, Macheroux P, Sund H (eds) Fla vins and flavoproteins. Rudolf Weber Agency for Scientific Publications, Berlin, pp 803–806 Cameron EM (1982) Sulf ate and sulf ate reduction in the early Precambrian oceans. Nature 296:145–148 Christner JA, Münck E, K ent TA, Janick PA, Salerno JC, Sie gel L M (1984) Exchange coupling between siroheme and [4Fe-4S] cluster in E. coli sulf ite reductase. Mössbauer studies and coupling models for a 2-electron reduced enzyme state and comple xes with sulf ide. J Am Chem Soc 106:6786–6794 Cort JR, Santhana Mariappan SV , Kim C-Y, Park M S, Peat T S, W aldo G S, T erwilliger T C, Kennedy MA (2001) Solution structure of Pyrobaculum aer ophilium DsrC, an archaeal homologue of the g amma sub unit of dissimilatory sulf ite reductase. Eur J Biochem 268: 5842–5850 Crane BR, Siegel LM, Getzoff ED (1995) Sulf ite reductase at 1.6 Å: e volution and catalysis for reduction of inorganic anions. Science 270:59–67

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Dahl C, T rüper HG (2001) Sulf ite reductase and APS reductase from Archaeoglobus fulgidus . Methods Enzymol 331:472–441 Dahl C, Speich N, T rüper HG (1994) Enzymology and molecular biology of sulf ate reduction in extremely thermophilic archaeon Archaeoglobus fulgidus. Methods Enzymol 243:331–352 Fritz G, Büchert T , Huber H, Stetter K O, Kroneck PMH (2000) reductases from archaea and bacteria are 1:1 alphabeta-heterodimeric iron-sulfur flavoenzymes. High similarity of molecular properties emphasizes their central role in sulfur metabolism. FEBS Lett 473:63–66 Fritz G, Büchert T , Kroneck PMH (2002a) The function of the [4Fe-4S] clusters and F AD in bacterial and archaeal adenosine 5 ′-phosphosulfate reductases. Evidence for fla vin-catalyzed reduction of adenosine 5′-phosphosulfate. J Biol Chem 277:26066–26073 Fritz G, Roth A, Schif fer A, Büchert T , Bourenk ov G, Bartunik HD, Huber H, Stetter K O, Kroneck PMH, Ermler U (2002b) Crystal structure of the adenosine 5´-phosphosulfate reductase from the h yperthermophilic Archaeon Archaeoglobus fulgidus at 1.6 Å resolution. Proc Natl Acad Sci USA 99:1836–1841 Fritz G, Einsle O, Rudolf M, Schiffer M, Kroneck PMH (2005) Key bacterial multi-centered metal enzymes involved in nitrate and sulfate respiration. J Mol Microbiol Biotechnol 10:223–233 Hansen T A (1994) Metabolism of sulf ate-reducing prokaryotes. Antonie V an Leeuwenhoek 66:165–185 Hittel DS, Voordouw G (2000) Overexpression, purification and immunodetection of DsrD from Desulfovibrio vugaris (Hildenborough). Antonie Van Leeuwenhoek 77:13–22 Karkhoff-Schweizer RR, Huber D P, Voordouw G (1995) Conservation of the genes for dissimilatory sulf ite reductase from Desulfovibrio vulgaris and Archaeoglobus fulgidus allo ws their detection by PCR. Appl Environ Microbiol 61:290–296 Kroneck PMH (2005) The biogeochemical c ycles of the elements and the e volution of life. In: Sigel A, Sigel H, Sigel RKO (eds) Metal ions in biological systems, vol. 43. Taylor & Francis, Baton Rouge, pp 1–7 Lampreia J, Pereira AS, Moura JJG (1994) Adenosine 5 ′-phosphosulfate reductase from sulf atereducing bacteria. Methods Enzymol. 243:241–260 Lancaster CRD (2003) Wolinella succinogenes quinol:fumarate reductase and its comparison to E. coli succinate:quinone reductase. FEBS Lett 555:21–28 Lee J-P, LeGall J, Peck HD (1973) Isolation of assimilatory- and dissimilatory-type sulfite reductases from Desulfovibrio vulgaris. J Bacteriol 115:529–542 LeGall J, F auque G (1988) Dissimilatory reduction of sulfur compounds. In: Zehnder AJB (ed) Biology of anaerobic microorganisms. Wiley, New York, pp 587–639 Lipmann F (1958) Biological sulf ate activation and transfer: studies on a mechanism of group activation and its role in biosynthesis are described. Science 128:575–580 Lui S M, Soriano A, Cowan JA (1994) Electronic properties of the dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough): comparitative studies of optical spectra and relative reduction potentials for the [Fe 4S4]-sirohaem prostetic centers. Biochem J 304:441–447 Mander GJ, Weiss MS, Hedderich R, Kahnt J, Ermler U, W arkentin E (2005) X-ray structure of the γ-subunit of a dissimilatory sulf ite reductase: f ixed and fle xible C-terminal arms. FEBS Lett 579:4600–4604 Massey V, Müller F, Feldberg R, Schuman M, Sullivan PA, Howell LG, Mayhew SG, Matthews RG, Foust GP (1969) The reactivity of flavoproteins with sulfite. Possible relevance to the problem of oxygen reactivity. J Biol Chem.244: 3999–4006 Matias PM, Pereira IA C, Soares CM, Carrondo MA (2005) Sulphate respiration from h ydrogen in Desulfovibrio bacteria: a structural biology o verview. Prog Biophys Mol Biol 89:292–329 Michaels GB, Da vidson JT, Peck HD Jr (1970) A fla vin-sulfite adduct as an intermediate in the reaction catalyzed by aden ylyl sulf ate reductase from Desulfovibrio vulgaris . Biochem Biophys Res Commun 39:321–328 Mizuno N, Voordouw G, Miki K, Sarai A, Higuchi Y (2003) Crystal structure of dissimilatory sulfite reductase D (DsrD) protein – possible interaction with B- and Z-DN A by Its wingedhelix motif. Structure 11:1133–1140

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Moura I, LeGall J, Lino AR, Peck HD, F auque G, Xa vier AV, DerVartanian D V, Moura JJG, Huynh BH (1988) Characterisation of tw o dissimilatory sulf ite reductases from the sulf atereducing bacteria. Mössbauer and EPR studies. J Am Chem Soc 110:1075–1082 Peck HD Jr (1959) The A TP-dependent reduction of sulf ate with h ydrogen in e xtracts of Desulfovibrio desulfuricans. Proc Natl Acad Sci USA 45:701–708 Pierik AJ, Hagen WR (1991) S = 9/2 EPR signals are evidence against coupling between the siroheme and the Fe/S cluster prosthetic groups in Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase. Eur J Biochem 195:505–516 Pires RH, Venceslau SS, Morais F, Texeira M, Xavier AV, Pereira IAC (2006) Characterization of the Desulfovibrio desulfuricans A TCC 27774 DsrMKJOP comple x – a membrane-bound redox complex involved in the sulfate respiratory pathway. Biochemistry 45:249–262 Schidlowski M, Hayes JM, Kaplan IR (1983) Isotopic inferences of ancient biochemistries: carbon, sulfur, hydrogen, and nitrogen In: Schopf JW (ed) Earth’s earliest biosphere, its origin and evolution. Princeton University Press, Princeton, pp 149–186 Schiffer A (2004) Structural and functional in vestigations on multi-site metallo enzymes of the biological sulfur cycle. Dissertation, Universität Konstanz Schiffer A, Fritz G, Kroneck PMH, Ermler U (2006) Reaction mechanism of the iron-sulfur flavoenzyme adenosine-5′-phosphosulfate reductase based on the structural characterization of different enzymatic states. Biochemistry 45:2960–2967 Speich N, Dahl C, Heisig P,Klein A, Lottspeich, F, Stetter KO, Trüper HG (1994) Adenylylsulphate reductase from the sulphate-reducing archaeon Archaeoglobus fulgidus: cloning and characterization of the genes and comparison of the enzyme with other iron-sulphur fla voproteins. Microbiology 140:1273–1284 Stetter KO, Lauerer G, Thomm M, Neuner A (1987) Isolation of e xtreme thermophilic sulf ate reducers: Evidence for a novel branch of archaebacteria. Science 236:822–824 Steuber J, Kroneck PMH (1998) Desulfo viridin, the dissimilatory sulf ite reductase from Desulfovibrio desulfuricans (Essex): new structural and functional aspects of the membranous enzyme. Inorg Chim Acta 275–276:52–57 Steuber J, Arendsen AF, Hagen WR, Kroneck PMH (1995) Molecular properties of the dissimilatory sulfite reductase from Desulfovibrio desulfuricans (Essex) and comparison with the enzyme from Desulfovibrio vulgaris (Hildenborough). Eur J Biochem 233:873–879 Thauer RK, Jungermann K, Deck er K (1977) Ener gy conserv ation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180 Verhagen MFJM, K ooter IM, W olbert RBG, Hagen WR (1994) On the iron-sulfur cluster of adenosine phosphosulf ate reductase from Desulfovibrio vulgaris (Hildenborough). Eur J Biochem 221:831–837 Wolfe BM, Lui SM, Cowan JA (1994) Desulfoviridin, a multimeric-dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). Eur J Biochem 223:79–89

Chapter 3

Respiratory Membrane Complexes of Desulfovibrio Inês A. Cardoso Pereira

Abstract Despite many years of research the process of sulfate respiration is still not fully understood. The mechanisms and components associated with ener gy conservation have not been clearly identified, and the electron donors to the cytoplasmic adenosine 5′-phosphosulfate (APS) and sulfite reductases are not known. Recently, considerable progress has been achie ved through genome analysis and other biochemical and genetic studies. This re view presents our current kno wledge of transmembrane redox complexes of Desulfovibrio spp. that are proposed to play a role in the respiratory electron transfer chain. T wo of these complexes, Qmo and Dsr , are apparently conserv ed in all sulf ate reducers, pointing to an essential role in sulfate respiration, most likely as electron donors to the APS and sulfite reductases, respecti vely. In contrast, the Hmc, 9Hc and Tmc comple xes are only present in Desulfovibrio or ganisms, suggesting a role in alternati ve pathways. The presence of the latter comple xes correlates with the lar ge pool of periplasmic cytochromes c found in Desulfovibrio spp., which act as electron donors to the comple xes upon periplasmic oxidation of hydrogen or formate. Future studies are required to establish the e xact function of all the comple xes discussed, namely, their electron donors and acceptors and their in volvement in energy-conserving mechanisms.

3.1 Intr oduction It has long been recognized that dissimilatory sulfate reduction is associated with oxidative phosphorylation, and is a true respiratory process (Peck 1960). However, despite many years of research into sulf ate-reducing bacteria (SRB) it has still not been clearly established ho w the electron transport chain is associated with the generation of a proton-moti ve force. The terminal reductases (APS reductase and sulfite reductase) are c ytoplasmic and so are not directly in volved in proton translocation. A typical complex I or bc1 complex is not present. Several important intervenients in the electron transport chain ha ve not been identif ied, such as the electron donors to APS and sulfite reductases, or the electron acceptor of the lactate dehydrogenase. It is also not clear what is the role of important 24 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

3 Respiratory Membrane Complexes of Desulfovibrio 25

electron carriers such as NAD(P)H (Kremer and Hansen 1989) and menaquinone (Collins and Widdel 1986). Several bioenergetic mechanisms have been proposed to explain energy conservation in SRB. Odom and Peck (1981) proposed a mechanism of hydrogen cycling for Desulfovibrio vulgaris growing in lactate/sulf ate. In this mechanism electrons from lactate oxidation are transferred to a cytoplasmic hydrogenase that produces H2. This diffuses to the periplasm, where its reoxidation generates electrons that are shuttled across the membrane for the c ytoplasmic reduction of sulf ate, lea ving protons in the periplasm that generate a pH gradient. This mechanism seems not to be applicable to all SRB since genome analysis shows that a cytoplasmic hydrogenase is absent in se veral organisms. In addition, H 2 formation from lactate oxidation to pyruvate is energetically very unfavorable, suggesting that other mechanisms are operative. More recent e vidence indicates that c ycling of other reduced inter mediates, like CO or formate, may also function in Desulfovibrio (Voordouw 2002; Heidelberg et al. 2004). Genome analysis points to the e xistence of differences in energy metabolism between dif ferent SRB, e.g., D. vulgaris v ersus Desulfotalea psychrophila (Pereira et al. 2007). The former has a much higher number of periplasmic cytochromes, hydrogenases and formate dehydrogenases, suggesting that in D. vulgaris c ycling of reduced intermediates may play a more important role than in Dt. psychrophila. Chemiosmotic processes in which energy conservation is achieved by a membrane-bound electron transport chain that transfers protons to the periplasm ha ve also been proposed (W ood 1978; Lupton et al. 1984), and are most probably operati ve since electron-transport-dri ven proton translocation has been demonstrated for several Desulfovibrio spp. (Fitz and Cypionka 1991). Whatever the mechanisms operating, membrane-associated electron transport is a requirement. These membrane processes most lik ely involve menaquinone and may contribute to ener gy conserv ation through standard mechanisms lik e redox loops (Jormakka et al. 2003). In recent years, considerable progress has been achie ved in our understanding of membrane-bound redox proteins in SRB, through genetic, genomic and biochemical studies (Matias et al. 2005; Pereira et al. 2007). These studies revealed the presence of several transmembrane redox complexes, which are probably involved in the electron transfer chain. These comple xes are unique to sulfur metabolizing organisms and contain several novel and interesting proteins, but further studies are required to establish their precise physiological function.

3.2

Membrane Complexes Conserved in Sulfate Reducers

Analysis of the membrane electron transport comple xes present in the complete genomes of four sulf ate-reducers [ D. vulgaris Hildenborough (Heidelber g et al. 2004), D. desulfuricans G20 (Joint Genome Initiati ve 1997), Dt. psyc hrophila (Rabus et al. 2004) and Archaeoglobus fulgidus (Klenk et al. 1997)] re veals that only two such complexes are conserved among the four organisms, suggesting they may be the only ones essential for sulf ate reduction. The y are the QmoABC

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complex, which was isolated from D. desulfuricans ATCC 27774 (Pires et al. 2003), and the DsrMKJOP complex, first isolated from A. fulgidus (Mander et al. 2002) and more recently also from D. desulfuricans ATCC 27774 (Pires et al. 2006). Indirect evidence suggests that QmoABC is involved in electron transfer to the APS reductase and DsrMKJOP is in volved in electron transfer to the sulf ite reductase (Pires et al. 2003, 2006; Haveman et al. 2004; Dahl et al. 2005; Mussmann et al. 2005). A striking point re garding the two complexes is that both contain sub units that are related to sub units of heterodisulf ide reductases (Hdr) of methanogens (Fig. 3.1), which catalyze the reduction of the heterodisulf ide of tw o thiol coenzymes (CoMSH and CoBSH). The heterodisulf ide CoM–S–S–CoB is formed in the last step of methanogenesis and acts as the terminal electron acceptor in the respiratory chain of these or ganisms (Hedderich et al. 1999). Its reduction is link ed to energy conservation by generation of a proton-moti ve force. In hydrogenotrophic methanogens like Methanothermobacter marburgensis the Hdr is soluble and composed of three subunits, HdrA, HdrB and HdrC. HdrA is a flavo-FeS protein, HdrC is also an FeS protein, and HdrB contains tw o five-cysteine motifs that are proposed to bind also FeS cluster(s) (Hedderich et al. 2005). In methylotrophic methanogens like Methanosarcina sp. the Hdr is membrane-bound and composed of only tw o subunits, HdrD and HdrE. HdrD is a homologue of a hypothetical fusion of the HdrBC subunits. HdrE is an inte gral membrane subunit containing two heme b groups. The catalytic subunits of both Hdrs are HdrB and HdrD, which contain a catalytic FeS cluster that forms a paramagnetic [4Fe4S] 3+ center upon oxidation in the presence of HS–CoM or HS–CoB (Hedderich et al. 2005). The tw o types of Hdrs have different electron donors. The membrane-bound HdrED enzyme receives electrons from the membrane cofactor methanophenazine via the cytochrome b HdrE subunit, whereas the soluble HdrABC enzyme forms a comple x with the F420-non-reducing hydrogenase that catalyzes reduction of the heterodisulfide by H2 (Stojanowic et al. 2003).

3.2.1

The Qmo Complex

The Qmo comple x was isolated from the membranes of D. desulfuricans ATCC 27774 (Pires et al. 2003). It is composed of three subunits and contains two hemes b, tw o fla vin adenine dinucleotide groups and se veral iron–sulfur centers. The genes encoding these proteins form a putati ve operon and were named qmoABC for “quinone-interacting membrane-bound oxidoreductase. ” Homologous genes are found in the genomes of the sulf ate reducers D. vulgaris Hildenborough, D. desulfuricans G20, Dt. psychrophila, Desulfotomaculum reducens MI-1 (Joint Genome Initiati ve 1997) and A. fulgidus . Interestingly , the qmo genes are also present in sulfur -oxidizing bacteria lik e the phototrophic Chlorobium tepidum (Eisen et al. 2002) and Chlorobium chlorochromatii (Joint Genome Initiative 1997), and the chemotrophic Thiobacillus denitrificans (Beller et al. 2006; Fig. 3.2). In several of these genomes the qmo genes are found adjacent to the APS reductase

3 Respiratory Membrane Complexes of Desulfovibrio 27

Fig. 3.1 The proteins discussed in the text, as deduced from sequence data. Related subunits are in similar shades of gray. The putative catalytic [4Fe4S] center in HdrB, HdrD, DsrK, HmcF and TmcB is depicted in light gray; 4C represents a conserved four-cysteine motif

genes ( apsAB), pro viding e vidence of a physiological relationship between these proteins. In some cases the ATP sulfurylase gene (sat) is also present in the same locus. QmoA and QmoB are predicted to be cytoplasmic flavo-FeS proteins that are both related to HdrA (Fig. 3.1), b ut QmoA is smaller and sho ws sequence similarity only to an N-terminal segment of HdrA and QmoB (Pires et al. 2003). The QmoB protein probably resulted from a gene fusion since the N-terminus re gion is similar to HdrA and the C-terminus re gion is similar to the δ-subunit of the F 420-non-reducing hydrogenases (MvhD). The QmoC protein seems to be also the result of a gene fusion as the N-terminus region is hydrophilic and shows similarity to HdrC, including the binding sites for two [4Fe4S] clusters, whereas the C-terminus is hydrophobic and includes six transmembrane helices. This membrane-bound domain is similar to HdrE, and the UV–vis spectrum of the QmoABC comple x supports the presence of tw o hemes b. Thus, QmoC belongs to the f amily of membrane sub units of respiratory comple xes that bind tw o hemes b on opposite sides of the bilayer and that are responsible for electron transfer with the membrane quinones in a process that may be associated with generation of a proton gradient (Berks et al. 1995). Ho wever, QmoC is a unique case within this family, since it is the first example, to our knowledge, of a protein that contains an additional hydrophilic domain with iron–sulfur centers. The sequence of QmoC strongly suggests that menaquinone is the electron donor to the QmoABC complex, and the tw o hemes of QmoC are reduced by menadiol, a menaquinol analogue. Interestingly, in Dm. reducens and T. denitrificans the qmoC gene is not found, and instead two genes related to hdrC and hdrB are present (Fig. 3.2).

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Fig. 3.2 Representation of the qmo and dsr gene arrangement in several of the organisms discussed. * the dsrS gene is not found in Thiobacillus denitrificans

The exact function of the QmoABC comple x remains to be established; ho wever, the present evidence points to its involvement in the sulfate respiratory chain, and more specifically as a probable electron donor to the APS reductase (Pires et al. 2003; Haveman et al. 2004; Mussmann et al. 2005). Ho wever, no in vitro electron transfer could be observ ed between the isolated QmoABC comple x and APS reductase (Pires et al. 2003). This may ha ve been due to experimental problems or may indicate that an additional redox partner is in volved. The macroscopic redox potentials of the two hemes b in the Qmo complex were determined to be −20 and +75 mV. These potentials are in a suitable range to be in volved in electron transfer from menaquinol (−70 mV) to APS (E0′ APS/SO32− = − 60 mV). Thus, the QmoABC complex provides a link between the menaquinone pool and the cytoplasmic reduction of sulfate. If oxidation of menaquinol by QmoC occurs at the heme closest to the positive side of the membrane, protons may be released to the periplasm and electrons transferred to the negative side of the membrane to QmoAB, with subsequent reduction of APS. Hence, electron transfer through QmoABC may lead to formation of a proton gradient through a redox-loop mechanism, b ut this has to be verified experimentally.

3 Respiratory Membrane Complexes of Desulfovibrio 29

3.2.2

The Dsr Complex

The DsrMKJOP comple x was isolated from A. fulgidus (Mander et al. 2002) and also from D. desulfuricans ATCC 27774 (Pires et al. 2006). Sequence analysis reveals that DsrM is a membrane c ytochrome b lik e HdrE and the C-terminal domain of QmoC (Fig. 3.1). DsrM is predicted to contain six transmembrane helices and has four conserv ed histidines, which are lik ely candidates to bind tw o hemes b. DsrK is predicted to be a c ytoplasmic iron–sulfur protein that is related to the catalytic subunit HdrD. DsrK contains only one of the five-cysteine motifs of HdrD, which are probably in volved in binding the [4Fe4S] catalytic center for disulfide reduction. The DsrJ protein contains three heme c binding sites, and an N-terminal signal peptide for e xport to the periplasm. This peptide is not clea ved off in the corresponding protein of the A. fulgidus complex, and probably serves as a membrane anchor for the periplasmic c ytochrome. The DsrJ sequence sho ws no homology to other c ytochromes in the databases and so corresponds to a no vel family of cytochromes c. There are not enough histidines in DsrJ for all the hemes to have bishistidine ligation that is usually found in multiheme c ytochromes. The Dsr O protein is a periplasmic FeS protein that belongs to the family of ferredoxinlike subunits found in several respiratory enzymes. Dsr O includes a typical signal peptide for translocation to the periplasm. DsrP is an inte gral membrane protein predicted to contain ten transmembrane helices. It is related to the membrane subunit of Escherichia coli hydrogenase-2 (HybB), which acts as a menaquinone reductase, and to a whole f amily of membrane subunits of respiratory enzymes. The operon coding for the Dsr complex is present in the genomes of all sulfatereducing organisms sequenced to date. In tw o bacteria that reduce sulf ite, but not sulfate, Moorella thermoacetica and Desulfitobacterium hafniense, the Dsr complex is encoded in the same locus as the dsrAB genes coding for the two subunits of the dissimilatory sulfite reductase (Fig. 3.2). Strikingly , both the sulf ite reductase and the Dsr comple x are also found in or ganisms that oxidize reduced sulfur compounds like the phototrophs Allochromatium vinosum and C. tepidum, or the chemotroph T. denitrif icans (Sander et al. 2006). In these or ganisms the dsrAB and dsrMKJOP are also part of the same gene cluster that includes other conserved dsr genes like dsrC and dsrN . It w as in A. vinosum that the dsrMK genes were f irst identified as belonging to the same gene cluster as dsrAB (and were thus named also dsr for “dissimilatory sulf ite reductase”), and these genes were sho wn to be obligatory for sulfur oxidation (Pott and Dahl 1998). Spectroscopic characterization of the D. desulfuricans Dsr complex confirms the presence of a [4Fe4S]3+ cluster (Pires et al. 2006), with similar characteristics to the one reported in A. fulgidus, and analogous to that observed in Hdrs where it acts as the catalytic site (Hedderich et al. 2005). On the basis of sequence and spectroscopic data, the hemes b in DsrM are proposed to be bishistidine-ligated, whereas the three hemes c in DsrJ have each different coordination with one bishi stidine, one histidine/methionine and another a v ery unusual histidine/c ysteine coordination (Pires et al. 2006). There are v ery few precedents for cysteine coordination in

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hemes c, which include the SoxAX c ytochrome (Bamford et al. 2002) that is involved in thiosulf ate oxidation (Friedrich et al. 2005), the triheme PufC c ytochrome of the photosynthetic reaction center (Alric et al. 2004), and possibly a new green c ytochrome from Halochromatium salexigens (Van Driessche et al. 2006). The histidine/cysteine coordinated heme cannot be fully reduced. T reatment with DMNH2 led only to approximately40% reduction of the hemes. There is considerable e vidence to indicate that the Dsr comple x is part of the same metabolic pathway as the sulfite reductase: all prokaryotic genomes that contain a DsrAB dissimilatory sulfite reductase contain also a DsrMKJOP complex; in A. vinosum the DsrKJO proteins associate with the DsrABC proteins (Dahl et al. 2005), and the genes for the sulf ite reductase and Dsr comple x are coordinately regulated by sulf ide (Pott and Dahl 1998; Dahl et al. 2005). In this or ganism the proteins encoded by the dsr genes were shown to be essential for oxidation of intracellular stored sulfur (Pott and Dahl 1998; Dahl et al. 2005; Sander et al. 2006). However, the precise physiological role of the Dsr comple x has still not been established. Sequence analysis suggests that electron transfer may occur in the periplasm, in the membrane and in the c ytoplasm. The unique nature of DsrJ prevents any hints as to its physiological function. It is not an electron acceptor for the hydrogenase/type I c ytochrome c3 (TpI c3) couple, and its heme coordination is suggestive of a specialized role, possibly catalytic. The c ytoplasmic DsrK protein is most probably a catalytic sub unit, gi ven its similarity to the catalytic sub unit HdrD of Hdrs. This suggests that DsrK may be in volved in catalyzing a thiol/ disulfide type of redox chemistry.

3.3

Membrane Complexes Found Only in Desulfovibrio spp.

Desulfovibrio spp. are characterized by a high content of periplasmic ormembraneassociated cytochromes c (Matias et al. 2005; Pereira and Xa vier 2005). Of these, the tetraheme TpI c3 is v ery ab undant, suggesting an important role in ener gy metabolism. It is thus some what surprising that neither Dt. psychrophila nor A. fulgidus have a TpIc3 (and indeed have very few cytochromes c), showing that it is not essential for sulf ate reduction. In Desulfovibrio spp. the pool of periplasmic cytochromes c act as electron acceptors for periplasmic hydrogenases and formate dehydrogenases (Heidelberg et al. 2004; Elantak et al. 2005; Matias et al. 2005). In these or ganisms the tw o classes of proteins lack the c ytochrome b membrane subunit typically present in such enzymes (Pereira et al. 2007). Dt. psychrophila and A. fulgidus have hydrogenases and formate dehydrogenases with a cytochrome b membrane sub unit, which transfers electrons to the menaquinone pool. In Desulfovibrio or ganisms the electrons resulting from periplasmic hydrogen and formate oxidation are lik ely to be transferred from the pool of c ytochromes c3 to one of several transmembrane complexes found only in Desulfovibrio spp., which include a cytochrome c subunit also belonging to the cytochrome c3 family (Matias et al. 2005; Fig. 3.1). The c ytochrome c sub units of three of these comple xes,

3 Respiratory Membrane Complexes of Desulfovibrio 31

HmcA, 9HcA and TmcA, ha ve been isolated and characterized, and recently the Tmc complex was also isolated. It is still not clear whether these three comple xes transfer electrons to the menaquinone pool and/or directly to the c ytoplasm for reduction of sulf ate. The sub units of the Hmc, 9Hc and Tmc comple xes ha ve a strong sequence similarity between them.

3.3.1

The Hmc and 9Hc Complexes

The first transmembrane comple x to be recognized in Desulfovibrio spp. w as the Hmc complex of D. vulgaris (Rossi et al. 1993). Sequence analysis indicates that this complex has a sub unit composition strikingly similar to the Dsr comple x in terms of the type of sub units present: a c ytoplasmic FeS protein related to HdrD, two inte gral membrane proteins, a periplasmic ferredoxin-lik e protein and a periplasmic cytochrome c (Fig. 3.1). This suggests that both comple xes ha ve related functions, but the actual sequence identity between subunits is very low. The multiheme cytochrome c subunit is the most dissimilar since it is a large, 65-kDa, sixteen-heme cytochrome in Hmc and a small three-heme cytochrome of 15 kDa in Dsr. The HmcA cytochrome is a poor electron acceptor for the periplasmic hydrogenases, b ut its reduction rate increases signif icantly in the presence of TpI c3 (Matias et al. 2005). Se veral studies indicate that the Hmc comple x accepts electrons from periplasmic hydrogen oxidation and a mutant deleted in the hmc operon grew at a slo wer rate than the wild type with hydrogen and sulf ate (Dolla et al. 2000). Since deletion of the hmc operon does not pre vent gro wth on hydrogen, there are probably other proteins that can fulf ill the same role, and a lik ely candidate is the Tmc complex described in Sect. 3.3.2. In D. desulfuricans no HmcA has been detected, b ut a nine-heme c ytochrome (9HcA) that is structurally v ery similar to the C-terminal domain of HmcA is present (Matias et al. 2005). This c ytochrome is part of a transmembrane redox complex (9Hc) that lacks the heme b and c ytoplasmic FeS sub units of Hmc, b ut includes two membrane subunits (9HcC and 9HcD) and a periplasmic FeS sub unit (9HcB) (Saraiva et al. 2001; Fig. 3.1). The 9HcA cytochrome is a much better electron acceptor for the hydrogenases than HmcA, b ut its reduction is most probably also mediated by TpIc3 (Matias et al. 2005).

3.3.2

The Tmc Complex

The TmcABCD complex is the first one of this f amily to have been isolated (Pereira et al. 2006). Its cytochrome c subunit (TmcA) had previously been characterized in several Desulfovibrio spp. and w as named type II c ytochrome c3 (TpIIc3) since it has se veral features that distinguish it from TpIc3 (Matias et al. 2005). The Tmc complex is encoded in a ten-gene operon present in D. vulgaris Hildenborough and D. desulfuricans G20.

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Four of the genes encode re gulatory proteins and one gene encodes a hypothetical protein. The other four genes, tmcABCD, encode the functional proteins of the complex (Fig. 3.1). The tmcB gene codes for a c ytoplasmic FeS protein, homologous to HmcF, and of the same f amily as DsrK and HrdD, and includes a binding site for a putatively catalytic [4Fe4S] center. The tmcC gene encodes a membrane cytochrome b homologous to HmcE, and of the same f amily as DsrM and HdrE. The tmcD gene encodes a tryptophan-rich protein that sho ws no similarity to an y proteins in the databases. The electron paramagnetic resonance spectrum of the oxidized comple x confirms the presence of the TmcB FeS center with similar characteristics to that of the D. desulfuricans DsrK and assigned to a [4Fe4S] 3+ center. TpIIc3 (TmcA) is ef ficiently reduced by the pair hydrogenase/ TpIc3 (Matias et al. 2005). Reduction of the Tmc comple x with H 2 and hydrogenase/ TpIc3 led to almost complete reduction of all the redox centers. This supports the prediction that the Tmc complex is a transmembrane conduit for electrons resulting fromperiplasmic hydrogen oxidation. The hemes of the Tmc comple x were not reduced with the menaquinol analogue DMNH2. Conversely, the reduced Tmc complex could transfer electrons to DMN, b ut the reduction rate w as similar using only TpII c3, suggesting the process may be nonphysiological. These results do not support, b ut also cannot discard, the involvement of the menaquinone pool in electron transfer through Tmc.

3.4 Conclusions We have, nowadays, a lot more pieces of the puzzle of sulf ate respiration, but the whole picture is still far from complete. It seems quite certain that the Qmo and Dsr complexes will be involved in the electron transfer pathw ays to the APS reductase and sulfite reductase, respectively, but the precise mechanism of interaction is not clear and there may be other molecules in volved. Another important conclusion is that menaquinol is most lik ely the electron donor to the Qmo comple x, which finally assigns a role for the membrane quinone pool in sulfate respiration. It seems very plausible that oxidation of menaquinol and electron transfer to APS reductase by Qmo may lead to a proton gradient across the membrane, but this has to be verified experimentally. Reduction of menaquinone is likely to occur at least in the first step of lactate oxidation. The similar sub unit architecture of the Dsr , Hmc and Tmc comple xes suggests their functions may be related. In particular, the similarity between the subunits DsrK, HmcF and TmcB points to a common (or similar) electron acceptor on the c ytoplasmic side. This electron acceptor may be a disulfide-containing species that is reduced to a thiol, which in turn could be an electron donor for the DsrAB sulf ite reductase. This thiol/disulfide could either be a small molecular weight compound, or a thiol group of a protein. One v ery likely candidate for this latter case is the DsrC protein that has two strictly conserved cysteines at the C-terminal in all or ganisms that have a dissimilatory sulfite reductase (Cort et al. 2001; Mander et al. 2005). DsrC is present in all organisms containing a dissimilatory sulf ite reductase, irrespective of whether

3 Respiratory Membrane Complexes of Desulfovibrio 33

they are sulfur-compound reducers or oxidizers (Sander et al. 2006). In Desulfovibrio spp. the DsrAB sulf ite reductase forms a stable comple x with DsrC (Pierik et al. 1992). DsrC is not cotranscribed with DsrAB and is actually one of the most highly expressed proteins in D. vulgaris Hildenborough (Haveman et al. 2003). The similarity between Dsr, Hmc and Tmc complexes does not extend, however, to the periplasmic cytochrome subunit. As described above, the role of the Hmc and Tmc complexes is most likely as receptors for electrons resulting from periplasmic H2, and possibly also formate, oxidation. A proton gradient will be associated with oxidation of these two compounds as protons are left in the periplasm and electrons are transferred through the membrane for reduction of sulf ate. For now the role of DsrJ remains as an open question, as does the possibility of electron transfer between the menaquinone pool and the Dsr , Hmc or Tmc complexes. In conclusion, despite considerable progress in our understanding of membrane proteins in SRB, many important questions remain, like the precise role of the membrane complexes, the mechanism(s) of proton translocation and the electron donors to the terminal reductases. Future research should aim to answer these questions before we can fully understand the bioener getics of sulf ate respiration. Acknowledgements. I w ould lik e to thank all my colleagues whose names appear in the references and in particular Miguel Teixeira and António Xavier, who introduced me to the study of Desulfovibrio, for many enlightening discussions and for their support over the years. Our work was funded by Fundação para a Ciência e T ecnologia, MCES, Portugal.

References Alric J, Tsukatani Y, Yoshida M, Matsuura K, Shimada K, Hienerw adel R, Schoepp-Cothenet B, Nitschke W, Nagashima KV, Vermeglio A (2004) Structural and functional characterization of the unusual triheme cytochrome bound to the reaction center of Rhodovulum sulfidophilum. J Biol Chem 279:26090–26097 Bamford VA, Bruno S, Rasmussen T, Appia-Ayme C, Cheesman MR, Berks BC, Hemmings AM (2002) Structural basis for the oxidation of thiosulf ate by a sulfur c ycle enzyme. EMBO J 21:5599–5610 Beller HR, Chain PSG, Letain TE, Chakicherla A, Larimer FW , Richardson PM, Coleman MA, Wood AP, Kelly DP (2006) The genome sequence of the oblig ately chemolithoautotrophic, facultatively anaerobic bacterium Thiobacillus denitfificans. J Bacteriol 188:1473–1488 Berks BC, Page MD, Richardson DJ, Reilly A, Ca vill A, Outen F, Ferguson SJ (1995) Sequence analysis of subunits of the membrane-bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron-carrying arm of a redox loop. Mol Microbiol 15:319–331 Collins MD, W iddel F (1986) Respiratory quinones of sulf ate-reducing and sulfur -reducing bacteria – a systematic investigation. Syst Appl Microbiol 8:8–18 Cort JR, Mariappan SVS, Kim CY, Park MS, Peat TS, Waldo GS, Terwilliger TC, Kennedy MA (2001) Solution structure of Pyrobaculum aerophilum DsrC, an archaeal homologue of the gamma subunit of dissimilatory sulfite reductase. Eur J Biochem 268:5842–5850 Dahl C, Engels S, Pott-Sperling AS, Schulte A, Sander J, Lubbe Y , Deuster O, Brune DC (2005) Novel genes of the dsr gene cluster and e vidence for close interaction of Dsr proteins during

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sulfur oxidation in the phototrophic sulfur bacterium Allochromatium vinosum . J Bacteriol 187:1392–1404 Dolla A, Pohorelic BKJ, V oordouw JK, V oordouw G (2000) Deletion of the hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough hampers h ydrogen metabolism and low-redox-potential niche establishment. Arch Microbiol 174:143–151 Eisen JA, Nelson KE, Paulsen IT, Heidelberg JF, Wu M, Dodson RJ, Deboy R, Gwinn ML, Nelson WC, Haft DH, Hickey EK, Peterson JD, Durkin AS, Kolonay JL, Yang F, Holt I, Umayam LA, Mason T, Brenner M, Shea TP, Parksey D, Nierman WC, Feldblyum TV, Hansen CL, Craven MB, Radune D, V amathevan J, Khouri H, White O, Gruber TM, K etchum KA, V enter JC, Tettelin H, Bryant D A, Fraser CM (2002) The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, green-sulfur bacterium. Proc Natl Acad Sci 99:9509–9514 Elantak L, Dolla A, Durand MC, Bianco P , Guerlesquin F (2005) Role of the tetrahemic sub unit in Desulfovibrio vulgaris Hildenborough formate deh ydrogenase. Biochemistry 44: 14828–14834 Fitz RM, Cypionka H (1991) Generation of a proton gradient in Desulfovibrio vulgaris . Arch Microbiol 155:444–448 Friedrich CG, Bardische wsky F, Rother D, Quentmeier A, Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Microbiol 8:253–259 Haveman SA, Brunelle V , Voordouw JK, V oordouw G, Heidelber g JF, Rab us R (2003) Gene expression analysis of ener gy metabolism mutants of Desulfovibrio vulgaris Hildenborough indicates an important role for alcohol deh ydrogenase. J Bacteriol 185:4345–4353 Haveman SA, Greene EA, Stilwell CP, Voordouw JK, Voordouw G (2004) Physiological and gene expression analysis of inhibition of Desulfovibrio vulgaris Hildenborough by nitrite. J Bacteriol 186:7944–7950 Hedderich R, Klimmek O, Kroger A, Dirmeier R, K eller M, Stetter K O (1999) Anaerobic respiration with elemental sulfur and with disulf ides. FEMS Microbiol Rev 22:353–381 Hedderich R, Hamann N, Bennati M (2005) Heterodisulf ide reductase from methanogenic archaea: a new catalytic role for an iron-sulfur cluster. Biol Chem 386:961–970 Heidelberg JF, Seshadri R, Haveman SA, Hemme CL, Paulsen IT, Kolonay JF, Eisen JA, Ward N, Methe B, Brinkac LM, Daugherty SC, Debo y RT, Dodson RJ, Durkin AS, Madupu R, Nelson WC, Sullivan SA, F outs D, Haft DH, Selengut J, Peterson JD, Da vidsen TM, Zaf ar N, Zhou LW, Radune D, Dimitrov G, Hance M, Tran K, Khouri H, Gill J, Utterback TR, Feldblyum TV, Wall JD, Voordouw G, Fraser CM (2004) The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. Nat Biotechnol 22:554–559 Jormakka M, Byrne B, Iwata S (2003) Protonmotive force generation by a redox loop mechanism. FEBS Lett 545:25–30 Joint Genome Initiative (1997) http://www.jgi.doe.gov. Cited 1 Oct 2006 Klenk HP, Clayton RA, T omb JF, White O, Nelson KE, K etchum KA, Dodson RJ, Gwinn M, Hickey EK, Peterson JD, Richardson DL, K erlavage AR, Graham DE, K yrpides NC, Fleischmann RD, Quack enbush J, Lee NH, Sutton GG, Gill S, Kirkness EF , Dougherty B A, McKenney K, Adams MD, Loftus B, Venter JC et al. (1997) The complete genome sequence of the h yperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus . Nature 390:364–370 Kremer DR, Hansen TA (1989) Demonstration of HOQNO and antimycin-a sensitive coupling of NADH oxidation and Aps and sulf ite reduction in a Marine Desulfovibrio Strain. FEMS Microbiol Lett 58:43–47 Lupton FS, Conrad R, Zeikus JG (1984) Ph ysiological-function of h ydrogen metabolism during growth of sulfidogenic bacteria on organic substrates. J Bacteriol 159:843–849 Mander GJ, Duin EC, Linder D, Stetter KO, Hedderich R (2002) Purification and characterization of a membrane-bound enzyme comple x from the sulf ate-reducing archaeon Archaeoglobus fulgidus related to heterodisulf ide reductase from methanogenic archaea. Eur J Biochem 269:1895–1904

3 Respiratory Membrane Complexes of Desulfovibrio 35 Mander GJ, Weiss MS, Hedderich R, Kahnt J, Ermler U, W arkentin E (2005) X-ray structure of the g amma-submit of a dissimilatory sulf ite reductase: f ixed and fle xible C-terminal arms. FEBS Lett 579:4600–4604 Matias PM, Pereira IA, Soares CM, Carrondo MA (2005) Sulphate respiration from h ydrogen in Desulfovibrio bacteria: a structural biology o verview. Prog Biophys Mol Biol 89:292–329 Mussmann M, Richter M, Lombardot T, Meyerdierks A, Kuever J, Kube M, Glockner FO, Amann R (2005) Clustered genes related to sulf ate respiration in uncultured prokaryotes support the theory of their concomitant horizontal transfer. J Bacteriol 187:7126–7137 Odom JM, Peck HD Jr (1981) Hydrogen c ycling as a general mechanism for ener gy coupling in the sulfate-reducing bacteria, Desulfovibrio sp. FEMS Microbiol Lett 12:47–50 Peck HD (1960) Evidence for oxidati ve phosphorylation during the reduction of sulf ate with hydrogen by Desulfovibrio desulfuricans. J Biol Chem 235:2734–2738 Pereira IA C, Xa vier AV (2005) Multi-heme c c ytochromes and enzymes. In: King RB (ed) Encyclopedia of inorganic chemistry, vol 5, 2nd edn. Wiley, New York, pp 3360–3376 Pereira IAC, Haveman SA, Voordouw G (2007) Biochemical, genetic and genomic characterization of anaerobic electron transport pathw ays in sulphate-reducing delta-proteobacteria. In: Barton LL, Hamilton W A (eds) Sulphate-reducing bacteria: en vironmental and engineered systems. Cambridge University Press, Cambridge (in press) Pereira PM, T eixeira M, Xa vier AV, Louro R O, Pereira IA (2006) The Tmc comple x from Desulfovibrio vulgaris Hildenborough is in volved in transmembrane electron transfer from periplasmic hydrogen oxidation. Biochemistry 45:10359–10367 Pierik AJ, Duyvis MG, v an Helvoort JM, W olbert RB, Hagen WR (1992) The third sub unit of desulfoviridin-type dissimilatory sulfite reductases. Eur J Biochem 205:111–115 Pires RH, Lourenco AI, Morais F, Teixeira M, Xavier AV, Saraiva LM, Pereira IA (2003) A novel membrane-bound respiratory comple x from Desulfovibrio desulfuricans A TCC 27774. Biochim Biophys Acta 1605:67–82 Pires RH, Venceslau SS, Morais F , Teixeira M, Xa vier AV, Pereira IAC (2006) Characterization of the Desulfovibrio desulfuricans ATCC 27774 DsrMKJOP comple x – a membrane-bound redox complex involved in sulfate respiration. Biochemistry 45:249–262 Pott AS, Dahl C (1998) Sirohaem sulfite reductase and other proteins encoded by genes at the dsr locus of Chromatium vinosum are involved in the oxidation of intracellular sulfur. Microbiology 144:1881–1894 Rabus R, Ruepp A, Frick ey T, Rattei T, Fartmann B, Stark M, Bauer M, Zibat A, Lombardot T , Becker I, Amann J, Gellner K, T eeling H, Leuschner WD, Glockner FO, Lupas AN, Amann R, Klenk HP (2004) The genome of Desulfotalea psyc hrophila, a sulf ate-reducing bacterium from permanently cold Arctic sediments. Environ Microbiol 6:887–902 Rossi M, Pollock WB, Reij MW , K eon RG, Fu R, V oordouw G (1993) The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex. J Bacteriol 175:4699–4711 Sander J, Engels-Schwarzlose S, Dahl C (2006) Importance of the DsrMKJOP complex for sulfur oxidation in Allochromatium vinosum and phylogenetic analysis of related complexes in other prokaryotes. Arch Microbiol 186:357–366 Saraiva LM, da Costa PN, Conte C, Xavier AV, LeGall J (2001) In the facultative sulphate/nitrate reducer Desulfovibrio desulfuricans ATCC 27774, the nine-haem c ytochrome c is part of a membrane-bound redox complex mainly expressed in sulphate-grown cells. Biochim Biophys Acta 1520:63–70 Stojanowic A, Mander GJ, Duin EC, Hedderich R (2003) Ph ysiological role of the F-420-nonreducing h ydrogenase (Mvh) from Methanothermobacter marb urgensis. Arch Microbiol 180:194–203 Van Driessche G, De vreese B, Fitch JC, Me yer TE, Cusano vich MA, V an Beeumen JJ (2006) GHP, a ne w c-type green heme protein from Halochromatium sale xigens and other proteobacteria. FEBS J 273:2801–2811 Wood PM (1978) Chemiosmotic model for sulf ate respiration. FEBS Lett 95:12–18

Chapter 4

Biochemical and Evolutionary Aspects of Eukaryotes That Inhabit Sulfidic Environments Ursula Theissen, William Martin

Abstract Various eukaryotes inhabit environments that harbor high concentrations of sulfide, which is a potent inhibitor of complex IV in the mitochondrial respiratory chain. They must therefore posses means by which the y can detoxify sulf ide, or u se alternative electron routes that circumv ent oxygen as the terminal acceptor , or both. The biochemical mechanisms through which eukaryotes deal with sulf ide are beginning to come into focus, with sulf ide:quinone oxidoreductase and the ener gy metabolism germane to anaerobic mitochondria standing in the fore ground. This chapter briefly covers current progress in understanding the biochemistry of sulf ide detoxification and utilization by eukaryotes. In light of ne wer views of ocean geochemistry (“Canf ield” oceans), both the anaerobic biochemistry of mitochondria and their capacity to deal with sulf ide are most easily interpreted as e volutionary holdovers from the anoxic and sulfidic phase of ocean history between about 2.3 billion and about 0.6 billion years ago.

4.1 Intr oduction For prokaryotes, it is well known that sulfide is a rich and widely used energy source. Many bacteria can survi ve with sulf ide as their only electron source (Kelly et al. 1997; Brune 1995). In eubacteria, sulf ide is usually oxidized by the enzymes flavocytochrome c (van Beeumen et al. 1991) and sulfide:quinone oxidoreductase (SQR). Bacterial SQR has been characterized in detail (Reinartz et al. 1998; Griesbeck et al. 2002) and an enzymatic mechanism has been proposed (Griesbeck et al. 2002). The enzyme catalyzes the transfer of electrons from sulfide to quinones as their entry point into the photosynthetic or respiratory membrane. But prokaryotes are not the only inhabitants of sulf idic en vironments; man y eukaryotes inhabit sulfidic environments as well. Traditionally, sulfide is viewed as an environmental toxin for eukaryotes, rather than as an ener gy source, because sulfide is a potent toxin that has long been kno wn to inhibit comple x IV of the 36 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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mitochondrial respiratory chain (Nicholls 1975). The purpose of this chapter is to point out some e xamples of animals that inhabit sulf ide-rich en vironments, to summarize the means with which the y deal with sulf ide, and to consider some of the evolutionary implications of sulfide metabolism in eukaryotes. The physiology of animals that inhabit sulfidic environments has been reviewed by Grieshaber and Völkel (1998).

4.2

Animals in Sulfidic Environments

Animals can be confronted with sulf ide from biological or geological sources. Some animals, like the giant tubeworm Riftia pachyptila, live at deep-sea hydrothermal vents, the exhalate of which can contain more than 0.3 mM sulfide (van Dover 2000). A far more common, and less spectacular, kind of sulfidic environment inhabited by animals is marine sediment. The sulf ide in marine sediments is generally of biogenic origin, stemming mainly from sulfate-reducing prokaryotes (Trüper 1984). V arious in vertebrates, for e xample, the lugw orm Arenicola marina or the ribbed mussel Geukensia demissa , inhabit such sulf idic marine sediments, where sulf ide concentrations can reach 3 µM to 2 mM (Fenchel and Riedl 1970; Völkel and Grieshaber 1992; Völkel et al. 1995), in some cases up to 8 mM (Lee et al. 1996). Animals that li ve in sulf idic en vironments must possess strate gies to a void sulfide-poisoning and/or or to utilize sulfide outright. Several evolutionary strategies are apparent. Man y species of mussels ha ve opted for what might be the simplest solution to short-term sulf ide exposure: they simply close their shells. Animals lacking protecti ve shells, lik e w orms, must f ind dif ferent solutions, because biological membranes are permeable for sulf ide (Beerman 1924). The oligochaete Tubificoides benedii precipitates FeS in the outer mucus layer, which leads to the black color of the animals (Dubilier et al. 1995). Other animals harb or ectosymbiotic bacteria on their body surf ace that af ford protection by oxidizi ng sulfide as an energy source. This has been described for various worms, including the priapulid Halicryptus spinulosus (Oeschger and Schmaljohann 1988; Oeschger and Janssen 1991). Animals that must endure high concentrations or long-term e xposure to sulfide require biochemical means of dealing with sulf ide. The giant gutless tube worm Riftia pachyptila at hydrothermal vents hosts sulfide-oxidizing bacterial symbionts (Dubilier et al. 2001). The animals ha ve not only to tolerate sulf ide, but they also have to transport sulf ide and oxygen to their symbionts, chemoautotrophs that supply the w orms’s reduced carbon, thereby functionally replacing the digesti ve tract. Many such worms have specialized blood proteins suited to this lifestyle. For Riftia, two different extracellular hemoglobins ha ve been described that bind not only oxygen, but sulfide in addition, simultaneously and reversibly (Arp et al. 1985). The sulf ide binding occurs via conserv ed c ysteine residues and disulf ide groups (Zal et al. 1998; Bailly et al. 2002).

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Other in vertebrates lik e the lugw orm Arenicola marina and the mussel Geukensia demissa inhabit sulf idic environments (marine sediments) b ut they do not host intracellular endosymbionts. Both species possess their o wn biochemical means of sulf ide oxidation, the main product of which is thiosulf ate. This has been shown for Arenicola (Völkel and Grieshaber 1992) andGeukensia (Doeller et al. 2001) as well as for the bi valve Solemya r eidi (O’Brien and Vetter 1990). In the case of the ribbed mussel Geukensia demissa , sulfide oxidation tak es place in the gill mitochondria and is directly linked to ATP synthesis: sulfide-supported oxygen consumption that matches the ener gy demand of ciliary beating (Doeller et al. 2001). These mitochondria are thus chemolithoheterotrophic, since the electrons for A TP synthesis via the respiratory chain stem from an inor ganic donor. For the lugworm, Völkel and Grieshaber (1994) showed that the oxidation of sulfide to thiosulfate takes place in mitochondria. The y proposed a model for the mitochondrial respiratory chain in volving an SQR similar to the bacterial enzyme (Völk el and Grieshaber 1996, 1997). If sulf ide concentrations do not exceed 30 µM, at which concentration mitochondrial c ytochrome c oxidase is inhibited, the electrons from sulf ide are thought to be transferred to ubiquinone by the SQR-lik e enzyme and then used for oxygen-dependent A TP production (Völkel and Grieshaber 1997).

4.3

Sulfide-Oxidizing Enzymes in Eukaryotes

Beyond the well-documented circumstances that sulfide is consumed and thiosulfate is produced, and that mitochondria are in volved, comparatively little is kno wn about the molecular details or biochemical mechanisms by which eukaryotes oxidize environmental sulfide. Vande Weghe and Ow (1999) identif ied a gene from the fission yeast Schizosaccharomyces pombe in a screen for heavy-metal tolerance, hmt2. The S. pombe HMT2 gene product showed sequence similarity to the biochemically characterized SQR from the alphaproteobacterium Rhodobacter capsulatus (Schütz et al. 1997) and furthermorecatalyzed electron transfer from sulf ide to quinone, suggesting that HMT2 is, functionally , an SQR enzyme. But the af finities of the S. pombe HMT2 protein for both substrates were e xtremely low (in the millimolar range), raising doubts about the in vivo function of the S. pombe protein (Griesbeck et al. 2002). Vande Weghe and Ow (1999) reported that SQR homologues occur in a few animal genomes. A closer look into the databases re vealed SQR homologs in virtually all sequenced genomes from animals, and man y unicellular eukaryotes as well (Theissen et al. 2003). The widespread occurrence of SQR-related genes in eukaryotes poses tw o questions: What are the products of these eukaryotic genes actually doing? Does eukaryotic sulfide metabolism make any sense in an evolutionary context?

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4.4 The Possible Functions of SQR-Related Genes in Eukaryotes On the one hand, the occurrence of SQR homologues in eukaryotes such as humans or fruit flies, neither of which inhabit sulf idic en vironments, w ould appear as a bit of a puzzle. But on the other hand, it places some older biochemical findings in a new light. In 1972, rat mitochondria were reported to oxidize sulfide (Curtis et al. 1972; Bartholome w et al. 1980). Fish mitochondria ha ve also been shown to oxidize sulf ide (Bagarinao and V etter 1990). More recently , isolated chicken mitochondria were sho wn to oxidize sulf ide, and the sulf ide oxidation was coupled with A TP synthesis (Y ong and Searc y 2001), similar to the case mentioned earlier for Geukensia (Doeller et al. 2001). Are SQR homologues doing the job? As a BLAST search with the S. pombe SQR sequence (accession number NP_ 596067) will quickly reveal, SQR homologues occur in genomes of all theeukaryotic lineages in question. W e have isolated a complementary DN A encoding an SQR homologue from Arenicola marina (Theissen 2006) and e xpressed it in bak er’s yeast, which lacks an SQR homologue in the genome sequence. The Arenicola enzyme expressed in yeast catalyzes the electron transferfrom sulfide to ubiquinone, and with considerably better substrate af finities than reported for the S. pombe protein, but it does not produce thiosulf ate directly; rather, additional enzymes appear to be necessary in the pathway that leads to the formation of thiosulfate from sulfide in the lugworm (Theissen 2006). Reports of enzymatic activity for eukaryotic SQR homologues other than the initial report on S. pombe (Vande Weghe and Ow 1999) are apparently lacking in the literature so f ar. Sulfide has been discussed as an atypical neuromodulator , in addition to the gases NO and CO (Baranano et al. 2001); hence, one possible role for SQR in eukaryotes that do not inhabit sulf idic environments might involve the modulation of physiological responses. Endogenous sulfide production has been described not only for marine in vertebrates lik e Arenicola marina and the mussel Tapes philippinarum (Julian et al. 2002) that deal with high sulf ide environmental concentrations daily, but also for various mammals, which do not (Goodwin et al. 1989; W arenycia et al. 1989; Savage and Gould 1990). Starting from l-cysteine, eukaryotes can synthesize endogenous sulfide in at least four dif ferent w ays (re viewed in Kamoun 2004). In mitochondria, c ysteine aminotransferase (EC 2.6.1.3) and 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) can be in volved in sulf ide production (re viewed in Kamoun 2004). Cysteine aminotransferase catalyzes the reaction of l-cysteine with a ketoacid (e.g., α-ketoglutarate) with formation of 3-mercaptop yruvate and an amino acid (e.g., l-glutamate). 3-Mercaptopyruvate is desulfurated by 3-mercaptop yruvate sulfurtransferase, resulting in formation of sulf ide and p yruvate (Julian et al. 2002). In the c ytosol, sulfide can be generated by c ystathione β-synthase (CBS; EC 4.2.1.22). Alongside endogenoussulfide production in mammals, considerable amounts of sulfide can be

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produced by anaerobic sulfate-reducing bacteria in the human colon, posing a challenge to cells of the intestinal epithelium (MacF arlane et al. 1992). Such findings suggest that even such animals that are not exposed to environmental sulfide nonetheless require biochemical means of dealing with sulf ide, albeit at lower concentrations than those e xperienced by sulf ide-exposed marine in vertebrates. F ailure to deal with endogenous sulf ide can ha ve dire consequences in humans. For example, overproduction of sulf ide owing to enhanced CBS acti vity can e xacerbate cogniti ve ef fects in Do wn-syndrome patients (Chadef aux et al. 1985; Kamoun 2001) and insuf ficient detoxif ication of sulf ide produced in the human colon can lead to inflammatory diseases and might af fect the frequency of colon cancer (Pitcher et al. 2000). Whether mammalian SQR plays a signif icant physiological role in sulfide metabolism, or not, remains to be sho wn.

4.5

Sulfide and Eukaryotic Evolution

Thoughts about eukaryotic e volution are traditionally couched in the conte xt of oxygen, and v ery rarely ha ve an ything to do with sulf ide whatsoe ver. Ho wever, that view needs to change for reasons that we will briefly outline here. F or several decades, the popular opinion about eukaryote e volution has been that the earliest eukaryotes were anaerobic, fermenting amoebae, that the origin of mitochondria corresponded to the origin of the aerobic, respiring lifestyle among eukaryotes, and that ATP yield from glucose was the prime advantage conferred by themitochondrial endosymbiont. This notion is as old as all modern formulations of endosymbiotic theory. Margulis (p. 229 in Sagan 1967), for example, wrote: “The anaerobic breakdown of glucose to pyruvate along the Embden–Meyerhof pathway occurred in the soluble cytoplasm under the direction of the host genome.…The greater amounts of energy available after the incorporation of the mitochondrion resulted in lar ge cells with amoebiod and c yclotic mo vement.” That vie w meshed well with the geological view that was emerging at about the same time, namely , that the origin of eukaryotes (and their mitochondria) corresponded temporally and causally to the global rise in atmospheric oxygen le vels about tw o billion years ago. Quoting Margulis again (p. 225 in Sagan 1967): “The subsequent e volution of aerobic metabolism in prokaryotes to form aerobic bacteria (protoflagella and protomitochondria) presumably occurred during the transition to the oxidizing atmosphere. ” Seen from today’s standpoint, quite a few things have changed since 1967, both from the biological and the geological perspecti ve. (Curiously , ho wever, the popular opinion about the conte xt of mitochondrial origin has remained lar gely unchanged.) From the biological perspecti ve it is no w clear that eukaryotic anaer obes are not restricted to any kind of “early branching” lineages. Rather, anaerobic eukaryotes occur across many independent lineages spanning the breadth and depth of eukaryote evolution (Embley and Martin 2006). It is furthermore clear that those eukaryotic lineages that were once thought to be the most primiti ve and were also thought to lack mitochondria ha ve mitochondria after all, b ut anaerobic forms of

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the organelle called hydrogenosomes and mitosomes (Müller 1993, 2003; T ovar et al. 1999, 2003; v an der Giezen and T ovar 2005; v an der Giezen et al. 2005; Embley and Martin 2006; Martin and Müller 2007). From the geological perspecti ve, things ha ve changed since 1967 as well, perhaps even more dramatically . The vie w that the appearance of oxygen in the atmosphere at about two billion years before the present corresponded to some sort of oxygen catastrophe for the entire planet is no longer current among geologists studying the history of oxygen on Earth. Instead, a newer model is now current that is often designated as “intermediate oxidation state” or “Canfield ocean” (Canfield 1998; Canfield et al. 2000; Anbar and Knoll 2002; Shen et al. 2003; Poulton et al. 2004; Arnold et al. 2004; Brocks et al. 2005). Summarized briefly, this newer view of Canfield oceans suggests that during the time from the appearance of oxygen in the atmosphere at about 2.3 billion years ago up until about 0.6 billion years ago marine sulfate reduction was globally widespread in the oceans, leading to anoxic and highly sulf idic w ater belo w the photic zone. The most recent e vidence to support this vie w comes from geological f indings that suggest the appearance of the Ediacara fauna (the earliest metazoan fossils) to correspond with the completion of ocean oxygenation and the end of anoxic and sulf idic (Canfield) oceans about 550 million years ago (Fike et al. 2006; Canfield et al. 2007). That newer view of oxygen history on Earth (Canf ield oceans) has major and far-reaching consequences for our understanding of early eukaryote e volution, although the community of biologists is not awakening to this realization as rapidly as it probably should. Ho w might Canf ield oceans af fect the vie ws of biologists concerning the course of e volution during the last about tw o billion years? Three main points are of importance. First, and perhaps foremost in the conte xt of this v olume, the biochemistry of sulfur metabolism w ould move to center stage for understanding ecosystems and their inhabitants during the last two billion years of evolution. Put another way, the global signif icance and chemical impact of sulf ate reducers (and other microbes that depend on redox reactions in volving sulfur) and their main end product – sulfide – in marine en vironments could be seen on a le vel comparable to that traditionally attached to oxygen production by c yanobacteria. Biologists ha ve always made a big fuss about the difference between anaerobic and aerobic habitats in evolution; if we trust the geologists (as we probably should) it would appear that the difference between sulfidic and non-sulfidic habitats during Earth history might be just as big. Atmospheric oxygen is one thing, b ut the brunt of e volution during the time from 2.3 billion to 0.6 billion years ago was going on in the oceans, not in the atmosphere. Biologists w ould probably do well to let the message of Canf ield oceans sink into their thinking about biochemistry and e volution during that time. Second, with e vidence continuing to pour in about Canf ield oceans (Canf ield 1998; Canfield et al. 2000; Anbar and Knoll 2002; Shen et al. 2003; Poulton et al. 2004; Arnold et al. 2004; Brocks et al. 2005), geologists are telling us in no uncer tain terms that the oceans were anoxic and sulfidic during the time from 2.3 billion to 0.6 billion years ago, the time during which the major eukaryotic lineages were emerging and di versifying. Hence, the widespread occurrence of SQR among

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eukaryotes should hardly be surprising, because eukaryotes were “born and raised” in sulfidic marine environments (Theissen et al. 2003; Martin et al. 2003; Emble y and Martin 2006). Eukaryotic sulf ide metabolism (also among eukaryotes that do not inhabit sulf idic en vironments today) is thus easily seen as a holdo ver from anaerobic and sulfidic times and is easily understood in that conte xt. Third, the widespread occurrence of the anaerobic lifestyle among eukaryotes would no longer need to be seen as a secondary adaptation, but as a direct holdover from the not-too-distant anaerobic (and/or microaerophilic) past of the eukaryotic lineage. Indeed, biologists still tend to equate the concept of “possessing mitochondria” with “oxygen. ” But mitochondria no w appear to be ubiquitous among all eukaryotes, including the anaerobic forms (Emble y and Martin 2006; T ovar et al. 2006), which do not require oxygen. The mitochondria of eukaryotic anaerobes fall into basically three types. The f irst type are those that possess quinones and produce ATP via anaerobic respirations such as the succinate-producing mitochondria of many worms (van Hellemond et al. 1995; T ielens et al. 2002; T ielens and v an Hellemond 2007), and the denitrifying mitochondria of some benthic forams (Risgaard-Petersen et al. 2006) or the succinate-producing mitochondria of man y marine invertebrates (Grieshaber and Völk el 1998). Notably, eukaryotes that produce succinate as a major end product v ery often contain rhodoquinone as well because it is needed for the fumarate reductase reaction (T ielens et al. 2002; v an Hellemond et al. 2003). The second type are hydrogenosomes, which produce ATP (and molecular hydrogen) b ut lack c ytochromes (Müller 1993, 2003; Martin and Müller 1998; Emble y and Martin 2006; Müller and Martin 2007). The third type are mitosomes, which apparently do not produce A TP at all b ut still fulf ill some important biochemical functions for the cell (v an der Giezen et al. 2005; v an der Giezen and T ovar 2005). Man y biologists w ould still lik e to vie w eukaryotes as ancestrally oxygen-dependent organisms, but in light of Canf ield oceans, it w ould seem far more reasonable to vie w the ability to produce A TP without the help of molecular oxygen as an attrib ute that w as present in the eukaryote common ancestor.

4.6 Conclusion Various eukaryotes inhabit sulfidic environments. Some eukaryotes can use sulfide as an electron donor for ATP synthesis in their mitochondrial respiratory chain. The enzyme that oxidizes sulfide in eukaryotes, SQR, is a mitochondrial enzyme, but in contrast to the situation in prokaryotes, the nature of the oxidized sulfur product in the eukaryotic SQR reaction is not yet kno wn. Geologists are telling us that the oceans were anaerobic and sulfidic during the time from about 2.3 billion to about 0.6 billion years ago. Eukaryotes arose and underwent their early di versification in a global ecological setting dominated by anaerobic and sulf idic environments. It is therefore not surprising to see the biochemical traces of that anaerobic and sulf idic past preserved in the biochemistry of modern eukaryotic groups.

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References Anbar AD, Knoll AH (2002) Proterozoic ocean chemistry and e volution: a bioinor ganic bridge. Science 297:1137–1142 Arnold GL, Anbar AD, Barling J, L yons TW (2004) Molybdenum isotope e vidence for widespread anoxia in mid-Proterozoic oceans. Science 304:87–90 Arp AJ, Childress JJ, Fisher CR (1985) Blood g as transport in Riftia pachyptila. Biol Soc Wash Bull 6:289–300 Bagarinao T , V etter RD (1990) Oxidati ve detoxif ication of sulf ide by mitochondria of the California killifish Fundulus parvipinnis and the speckled sanddab Citharichthys stigmaeus. J Comp Physiol 160B:519–527 Bailly X, Jolli vet D, Vanin S, Deutsch J, Zal F , Lallier F, Toulmond A (2002) Ev olution of the sulfide-binding function within the globin multigenic f amily of the deep-sea h ydrothermal vent tubeworm Riftia pachyptila. Mol Biol Evol 19:1421–1433 Baranano DE, Ferris CD, Snyder SH (2001) Atypical neural messengers. Trends Neurosci 24:99–106 Bartholomew TC, Po well GM, Dodgson KS, Curtis CG (1980) Oxidation of sodium sulf ide by rat-liver, lungs and kidney. Biochem Pharmacol 29:2431–2437 Beerman H (1924) Some physiological actions of hydrogen sulfide. J Exp Zool 41:33–43 Brocks JJ, Lo ve GD, Summons RE, Knoll AH, Log an GA, Bo wden SA (2005) Biomark er evidence for green and purple sulphur bacteria in a stratif ied P alaeoproterozoic sea. Nature 437:866–870 Brune DC (1995) Sulfur compounds as photosynthetic electron donors. In: Blank enship RE, Madigan MT, Bauer CE (eds) Anoxygenic photosynthetic bacteria. Kluwer , Dordrecht, pp 847–870 Canfield DE (1998) A new model for Proterozoic ocean chemistry. Nature 396:450–453 Canfield DE, Habicht KS, Thamdrup B (2000) The Archean sulfur c ycle and the early history of atmospheric oxygen. Science 288:658–661 Canfield DE, Poulton SW , Narbonne GM (2007) Late-Neoproterozoic deep-ocean oxygenation and the rise of animal life. Science 315:92–95. doi:10.1126/science.1135013 Chadefaux B, Rethore MO, Raoul O, Ceballos I, Gilgenkrantz S, Allard D (1985) Cystathionine beta synthase gene dosage effect in trisomy 21. Biochem Bioph ys Res Commun 128:40–44 Curtis CG, Bartholomew TC, Rose FA, Dodgson KS (1972) Detoxification of sodium 35S sulfide in rat. Biochem Pharmacol 21:2313–2321 Doeller JE, Grieshaber MK, Kraus D W (2001) Chemolithoheterotrophy in a metazoan tissue: thiosulfate production matches A TP demand in ciliated mussel gills. J Exp Biol 204: 3755–3764 Dubilier N, Giere O, Grieshaber MK (1995) Morphological and ecoph ysiological adaptations of the marine oligochaete Tubificoides benedii to sulfidic sediments. Am Zool 35:163–173 Dubilier N, Mülders N, Ferdelman T , de Beer D, Pernthaler A, Klein M, W agner M, Erséus C, Thierman F , Krie ger J, Giere O, Amann R (2001) Endosymbiotic sulphate-reducing and sulphide-oxidizing bacteria in an oligochaete w orm. Nature 411:298–302 Embley TM, Martin W (2006) Eukaryotic e volution, changes and challenges. Nature 440:623–630 Fenchel TM, Riedl (1970) The sulf ide system: a ne w biotic community underneath the oxidized layer of marine sand bottoms. Mar Biol 7:255–268 Fike DA, Grotzinger JP, Pratt LM, Summons RE (2006) Oxidation of the Ediacaran ocean. Nature 444:744–747 Goodwin LR, Francom D, Diek en FP, Taylor JD, W arenycia NW, Reiffenstein RJ, Do wling G (1989) Determination of sulfide in brain tissue by gas dialysis/ion chromatography: post-mortem studies and two case reports. J Anal Toxicol 13:105–109 Griesbeck C, Schütz M, Schödl T , Bathe S, Nausch L, Mederer N, V ielreicher M, Hauska G (2002) Mechanism of sulf ide-quinone reductase in vestigated using site-directed mutagenesis and sulfur analysis. Biochemistry 41:11552–11565 Grieshaber MK, Völkel S (1998) Animal adaptations for tolerance and e xploitation of poisonous sulfide. Annu Rev Physiol 60:30–53

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Julian D, Statile JL, W ohlgemuth SE, Arp AJ (2002) Enzymatic h ydrogen sulfide production in marine invertebrate tissues. Comp Biochem Physiol A 133:105–115 Kamoun P (2001) Mental retardation in Do wn syndrome: a h ydrogen sulf ide hypothesis. Med Hypotheses 57:389–392 Kamoun P (2004) Endogenous production of h ydrogen sulf ide in mammals. Amino Acids 26:243–254 Kelly DP, Shergill JK, Lu WP, Wood AP (1997) Oxidati ve metabolism of inor ganic sulfur compounds by bacteria. Antonie Van Leeuwenhoek 71:95–107 Lee RW, Kraus D, Doeller JE (1996) Sulf ide-stimulation of oxygen consumption rate and c ytochrome reduction in gills of the estuarine mussel Geukensia demissa. Biol Bull 191:421–430 MacFarlane GT, Gibson GR, Cummings JH (1992) Comparison of fermentation reactions in different regions of the human colon. J Appl Bacteriol 72:57–64 Martin W, Müller M (1998) The h ydrogen hypothesis for the first eukaryote. Nature 392:37–41 Martin W, Müller M (eds) (2007) Origin of mitochondria and h ydrogenosomes. Springer, Heidelberg Martin W, Rotte C, Hoffmeister M, Theissen U, Gelius-Dietrich G, Ahr S, Henze K (2003) Early cell evolution, eukaryotes, anoxia, sulfide, oxygen, fungi first (?), and a tree of genomes revisited. IUBMB Life 55:193–204 Müller M (1993) The hydrogenosome. J Gen Microbiol 139:2879–2889 Müller M (2003) Ener gy metabolism. P art I: anaerobic protozoa. In: Marr J (ed) Molecular medical parasitology. Academic, London, pp 125–139 Nicholls P (1975) The ef fect of sulf ide on c ytochrome aa3. Isosteric and allosteric shifts of the reduced?α-peak. Biochim Biophys Acta 396:24–35 O’Brien J, Vetter RD (1990) Production of thiosulfate during sulphide oxidation by mitochondria of the symbiont-containing bivalve Solemya reidi. J Exp Biol 149:133–148 Oeschger R, Janssen HH (1991) Histological studies on Halicryptus spinulosus (Priapulida) with regard to environmental hydrogen sulfide resistance. Hydrobiologia 222:1–12 Oeschger R, Schmaljohann R (1988) Association of various types of epibacteria with Halicryptus spinulosus (Priapulida). Mar Ecol Prog Ser 48:285–293 Pitcher MCL, Beatty ER, Cummings JH (2000) The contribution of sulphate reducing bacteria and 5-aminosalicylic acid to faecal sulphide in patients with ulcerative colitis. Gut 46:64–72 Poulton SW, Fralick PW , Canfield DE (2004) The transition to a sulphidic ocean ∼1.84 billion years ago. Nature 431:173–177 Reinartz M, Tschäpe T, Brüser T, Trüper HG, Dahl C (1998) Sulfide oxidation in the phototrophic bacterium Chromatium vinosum. Arch Microbiol 170:59–68 Risgaard-Petersen N, Langezaal AM, Ingv ardsen S, Schmid MC, Jetten MS, Op den Camp HJ, Derksen JW, Pina-Ochoa E, Eriksson SP , Nielsen LP , Re vsbech NP, Cedhagen T , v an der Zwaan GJ (2006) Evidence for complete denitrif ication in a benthic foraminifer . Nature 443:93–96 Sagan L (1967) On the origin of mitosing cells. J Theor Biol 14:225–274 Savage JC, Gould DH (1990) Determination of sulfides in brain tissue and rumen fluid by ion-interaction reversed-phase high-performance liquid chromatography. J Chromatogr 526:540–545 Schütz M, Shahak Y , Padan E, Hauska G (1997) Sulf ide quinone reductase from Rhodobacter capsulatus. J Biol Chem 272:9890–9894 Shen Y, Knoll AH, Walter MR (2003) Evidence for low sulphate and anoxia in a mid-Proterozoic marine basin. Nature 423:632–635 Theissen U (2006) Die Sulf id:Chinon Oxidoreduktase des W attwurms Arenicola marina : Funktion, Mechanismus und Evolution. Dissertation, University of Düsseldorf Theissen U, Hof fmeister M, Grieshaber M, Martin W (2003) Single eubacterial origin of eukaryotic Sulf ide:quinone oxidoreductase, a mitochondrial enzyme conserv ed from the early evolution of eukaryotes during anoxic and sulf idic times. Mol Biol Evol 20:1564–1574 Tielens A GM, v an Hellemond JJ (2007) Anaerobic mitochondria: properties and origins. In: Martin W, Müller M (eds) Origin of mitochondria and hydrogenosomes. Springer, Heidelberg, pp 85–103

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Tielens AGM, Rotte C, van Hellemond JJ, Martin W (2002) Mitochondria as we don’t know them. Trends Biochem Sci 27:564–572 Tovar J, Fischer A, Clark CG (1999) The mitosome, a no vel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica. Mol Microbiol 32:1013–1021 Tovar J, León-Avila G, Sánchez LB, Sutak R, Tachezy J, van der Giezen M, Hernández M, Müller M, Lucocq JM (2003) Mitochondrial remnant or ganelles of Giardia function in iron-sulphur protein maturation. Nature 426:172–176 Trüper HG (1984) Microor ganisms and the sulfur c ycle: In: Müller A, Krebs B (eds) Sulfur: Its significance for chemistry , for the geo-, bio-, and cosmosphere and technology . Studies in inorganic chemistry. Elsevier, Amsterdam, pp 351–365 van der Giezen M, Tovar J (2005) Degenerate mitochondria. EMBO Rep 6:525–530 van der Giezen M, T ovar J, Clark CG (2005) Mitochondrion-deri ved organelles in protists and fungi. Int Rev Cytol 244:175–225 van Dover CL (2000) The ecology of deep-sea h ydrothermal vents. Princeton Uni versity Press, Princeton van Hellemond JJ, Klockie wicz M, Gaasenbeek CPH, Roos MH, T ielens A GM (1995) Rhodoquinon and comple x II of the electron transport chain in anaerobically functioning eukaryotes. J Biol Chem 270:31065–31070 van Hellemond JJ, van der Klei A, van Weelden SW, Tielens AG (2003) Biochemical and evolutionary aspects of anaerobically functioning mitochondria. Philos T rans R Soc Lond B Biol Sci 358:205–213 (not cited in the te xt!) van Beeumen JJ, Demol H, Samyn B, Bartsch RG, Me yer TE, Dolata MM, Cusano vich MA (1991) Covalent structure of the diheme c ytochrome subunit and amino-terminal sequence of the fla voprotein sub unit of fla vocytochrome c from Chromatium vinosum . J Biol Chem 266:12921–12931 Vande Weghe JG, Ow DW (1999) A fission yeast gene for mitochondrial sulfide oxidation. J Biol Chem 274:13250–13257 Völkel S, Grieshaber MK (1992) Mechanisms of sulfide tolerance in the peanut worm Sipunculus nudus (Sipunculida) and in the lugw orm Arenicola marina (Polychaeta). J Comp Ph ysiol B 162:469–477 Völkel S, Grieshaber MK (1994) Oxygen-dependent sulf ide detoxif ication in the lugw orm Arenicola marina. Mar Biol 118:137–147 Völkel S, Grieshaber MK (1996) Mitochondrial sulf ide oxidation in Arenicola marina: Evidence for alternative electron pathways. Eur J Biochem 235:231–237 Völkel S, Grieshaber MK (1997) Sulphide oxidation and oxidati ve phosphorylation in the mitochondria of the lugworm Arenicola marina. J Exp Biol 200:83–92 Völkel S, Hauschild K, Grieshaber MK (1995) Sulf ide stress and tolerance in the lugw orm Arenicola marina during low tide. Mar Ecol Prog Ser 122:205–215 Warenycia MW, Goodwin LR, Benishin CG, Reif fenstein RJ, Francom DM, Taylor JD, Dicken FP (1989) Acute hydrogen sulfide poisoning: demonstration of selecti ve uptake of sulfide by the brainstem by measurement of brain sulf ide levels. Biochem Pharmacol 38:973–981 Yong R, Searcy DG (2001) Sulfide oxidation coupled to ATP synthesis in chicken liver mitochondria. Comp Biochem Physiol B 129:129–137 Zal F, Leize E, Lallier FH, Toulmond A, Van Dorsselaer A, Childress JJ (1998) S-sulfohemoglobin and disulfide exchange: the mechanisms of sulfide binding by Riftia pachyptila hemoglobins. Proc Nat Acad Sci 95:8997–9002

Chapter 5

Evolution and Ecology of Microbes Dissimilating Sulfur Compounds: Insights from Siroheme Sulfite Reductases Alexander Loy, Stephan Duller, Michael Wagner

Abstract Sulfur microorganisms have been thri ving on Earth since the da wn of life and are still of central importance for the functioning of modern ecosystems. Here, we summarize the current perception of the e volution of dissimilatory siroheme sulfite reductases (DSRs), antique key enzymes in the energy metabolism of sulfur microbes. We further gi ve recent e xamples of the di versity and ecology of uncultured sulfur-dissimilating microorganisms; unprecedented insights that were only made possible by e xploiting DSR-encoding genes as molecular mark ers in environmental surveys.

5.1 Intr oduction Some of the f irst microor ganisms under the anoxic, reduced atmosphere of the primordial Earth gained ener gy for gro wth and maintenance of cellular processes by dissimilating sulfur compounds (Canfield and Raiswell 1999; Huston and Logan 2004). Today, phylogenetically distinct bacteria and archaea still ha ve the unifying ability to emplo y sulfur compounds as either electron donors or acceptors for energy-generating redox reactions. In the environment they are thus pivotal for the biogeochemical cycling of sulfur, but also of carbon, as sulfur oxidation/reduction in these microbes is coupled to carbon dioxide assimilation or heterotrophic breakdown of organic matter. A presumably ancient group of enzymes, that could ha ve played a fundamental role in mediating biological conversions of sulfur compounds from the very first appearance of these microorganisms on Earth, are sulfite reductases (SRs), catalyzing the six-electron reduction of sulf ite to sulfide. The electron transfer is mediated by a metallocof actor that is composed of a metalloporphyrin, the so-called siroheme, bound to an iron–sulfur [Fe 4S4] cluster in the acti ve redox centers of the protein (Crane and Getzof f 1996; Crane et al. 1995; Sie gel et al. 1978). Apart from the e volutionarily related ammonia-forming assimilatory nitrite reductases, SRs are the only proteins kno wn to contain siroheme, a reduced iron tetrahydroporphyrin of the isobacteriochlorin class, or siroamide, an amidated siroheme variant, as a prosthetic group (Matthe ws et al. 1995). Because siroheme is vital for the catalytic acti vity of the enzyme, a typical siroheme–[Fe 4S4] binding 46 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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motif of four cysteines Cys-X5–Cys–Xn-Cys-X3–Cys is thus highly conserved in all SRs. Two main categories of SRs can be distinguished on the basis of their phylogeny: cellular function and structure (Crane and Getzof f 1996). Assimilatory SRs produce sulfide for incorporation in biomass and are present in man y microorganisms, algae, fungi, and plants. In contrast, dissimilatory SRs (DSRs) are restricted to specialized groups of bacteria and archaea, allowing them to gain energy by reducing/ oxidizing sulfur compounds. While DSRs are essential in the energy metabolism of all sulf ate/sulfite-reducing microor ganisms (SRMs) that anaerobically respire sulfite and/or sulf ate and thus catalyze a unique step in the reducti ve part of the sulfur cycle (Rabus et al. 2000), some b ut not all phototrophic and chemotrophic microorganisms that oxidize reduced sulfur species also possess DSRs.

5.2

Evolution of Dissimilatory Sulfite Reductases

5.2.1 Sulfate/Sulfite-Reducing

Microorganisms

A typical sulf ate/sulfite-reducing pathw ay, in which a dissimilatory (bi)sulf ite reductase catalyzes the energy-yielding final step, is common to all SRMs in vestigated so f ar. This DSR consists of a heterotetramer core with an α2β2 quaternary protein structure. The α- and β-subunits are encoded by the adjoining genes dsrA and dsrB, respectively, which most lik ely arose by duplication of an ancient dsr gene and are thus paralogous (Dahl et al. 1993, Karkhof f-Schweizer et al. 1995, Molitor et al. 1998; Fig. 5.1). Initial phylogenetic analysis of only a fe w described SRMs sho wed that the tree based on their DsrAB sequences w as largely congruent with the 16S ribosomal RNA (rRNA) based tree, indicating that in the course of SRM e volution, dsrAB was mainly inherited via v ertical transmission from the parent or ganism to its progeny (Wagner et al. 1998). Ho wever, already with this limited dataset, inconsistencies in the branching pattern and phylogenetic distances in DsrAB and 16S rRNA gene-based trees were indicative of possible lateral gene transfer (LGT) e vents of dsrAB (Larsen et al. 1999; W agner et al. 1998). In order to investigate this further, subsequent studies have significantly extended the dsrAB dataset by including sequences from several representatives of all known major lineages of SRMs, namely , the bacterial phyla Proteobacteria, Firmicutes, Thermodesulfobacteria, and Nitrospira, and the archaeal phyla Crenarchaeota and Euryarchaeota (Friedrich 2002; Klein et al. 2001; Molitor et al. 1998; Zverlov et al. 2005). Phylogenetic data, such as characteristic insertions/deletions within dsrAB sequences, collectively confirmed the initial view that vertical transmission w as the main e volutionary process responsible for the distribution of dsrAB among today’ s microor ganisms and that a fe w def ined SRM groups most lik ely recei ved their dsrAB by LGT . Those SRMs with an unusual (i.e., laterally acquired) dsrAB include members of the genus

Fig. 5.1 Phylogeny of assimilatory and dissimilatory siroheme sulf ite reductases. The unrooted trees are based on a short, highly conserv ed sequence stretch surrounding the siroheme-binding site. It is noteworthy that the resolution of the trees is limited because only 89 amino acid positions were considered for treeing. a The schematic maximum-lik elihood tree (ProtML-Molphy) sho ws the affiliation of ungrouped sequences and was edited with iTOL (http://itol.embl.de/). b Polytomic nodes in the consensus tree connect branches for which a relati ve order could not be determined unambiguously by applying maximum-parsimon y and maximum-lik elihood (ProtML-Molphy , ProML-Phylip) treeing methods. P arsimony bootstrap values (100 resamplings) are sho wn for all nodes. Dashed lines indicate enzymes for which a dissimilatory function remains to be pro ven. SOBs sulfur-compound-oxidizing bacteria, SRMs sulfate/sulfite-reducing microorganisms

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Thermodesulfobacterium and some lo w-G+C Gram-positi ve bacteria of the phylum Firmicutes, namely, Moorella thermoacetica, Ammonifex degensii, and Desulfotomaculum subcluster Ib (including Sporotomaculum hydr oxybenzoicum), Ic, Id, and Ie bacteria. The dsrAB donor(s) for these bacteria probably originated from the Deltaproteobacteria; Desulfobacterium anilini and the related SRM strain mXyS1 are v ertical descendants of a common deltaproteobacterial ancestor that might ha ve functioned as a dsrAB donor for Firmicutes with a laterally acquired dsrAB (Zverlov et al. 2005; Fig. 5.2). Another postulated case in volved an ancient horizontal transfer of dsrAB e ven across the boundaries of the domains of life; members of the euryarchaeotal genus Archaeoglobus contain bacterial v ersions of dsrAB. The only other archaea known to carry dsrAB are members of the sulfite-reducing, crenarchaeotal genus Pyrobaculum (Fitz-Gibbon et al. 2002; Molitor et al. 1998), representing the deepest branch in the DsrAB tree (Fig. 5.2). The observation of dsrAB LGTs among major lineages of SRMs ine vitably evokes tw o important questions. What are the mechanisms underlying these LGTs and what is the competiti ve advantage conferred by the acquisition of a xenologous dsrAB? Although definite answers to these questions are not known, some hypothetical ones are shortly discussed. An initial theory that a whole gene set for sulfate reduction could be tightly clustered in the genomes of SRMs and thus mobilized as a metabolic island (Klein et al. 2001) w as rapidly rejected because of the following findings. Although apsA (encoding the α-subunit of the adenosine 5´-phosphosulfate reductase, another key enzyme in the dissimilatory sulfate reduction pathway) was also subject to a fe w LGTs among SRMs, apsA and dsrAB LGT patterns did not match (Friedrich 2002). The notion that genes with key functions in sulf ate reduction, such as aps and dsr, are not physically linked and thus not cotransferred w as confirmed by sequencing the genomes of Archaeoglobus fulgidus (Klenk et al. 1997), Desulfovibrio vulgaris (Heidelberg et al. 2004), and Desulfotalea psyc hrophila (Rabus et al. 2004). Ho wever, the “metabolic island” theory was recently resurrected by the unexpected discovery of a whole cluster of genes for sulfate reduction on genomic fragments from yet uncultured and unidentif ied marine microor ganisms (Mussmann et al. 2005). This sho wed that LGT of dsrAB might be mediated by (a combination of) different processes; e.g., the x enologous displacement (K oonin et al. 2001) of preexisting dsrAB with a foreign v ersion from another phylogenetically distant SRM and/or by acquisition of a complete gene set for sulfate reduction by a nonSRM, the latter resulting in an ob vious metabolic adv antage for the recipient. The mechanistic bases of dsrAB LGT, i.e., if conjugation, transformation, or transduction play a role, are unkno wn. Ho wever, gi ven that SRMs can be infected by phages (Rapp and W all 1987; Walker et al. 2006), it is concei vable that transduction is/was mediating the evolution of sulfate/sulfite respiration, in analogy to cyanophages that are important vectors for reshuffling and exchange of photosynthetic genes in and among Prochlorococcus and Synechococcus species (Lindell et al. 2004; Zeidner et al. 2005).

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Fig. 5.2 Phylogeny of DsrAB sulf ite reductase f amilies. The consensus tree w as constructed on the basis of 367 amino acid alignment positions using the neighbor-joining method with the Kimura model of amino acid substitution. Polytomic nodes connect branches for which a relative order could not be determined unambiguously by applying distance-matrix, maximum-parsimon y, and maximum-likelihood treeing methods. Parsimony bootstrap values (100 resamplings) are indicated for highly supported (v alues greater than 75%) branches. Selected en vironmental dsrAB clones from Loy et al. (2004), Mussmann et al. (2005), Sabehi et al. (2005), and V enter et al. (2004) are shown in bold. The bar indicates estimated sequence di vergence. LA-dsrAB microorganisms with a laterally acquired dsrAB (Klein et al. 2001; Zv erlov et al. 2005)

5.2.2 DsrAB-Containing Syntrophs: Former Sulfate/Sulfite-Reducing Microorganisms? An une xpected peculiarity of some b ut not all dsrAB-containing and dsrABexpressing bacteria such as Pelotomaculum and Sporotomaculum is that the y are not able to grow with sulfite and/or sulfate as electron acceptors although the y are

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closely affiliated with SRMs (Brauman et al. 1998; Imachi et al. 2006). It is possible that some of these dsrAB-carrying non-SRMs use organosulfonates as electron acceptors for anaerobic respiration instead, e.g., Bilophila wadsworthia degrades taurine to sulf ite, the actual substrate for its DSR (Cook et al. 1998). Ho wever, a whole range of organosulfonates did not support growth of the spore-forming, lowG+C bacteria of the genus Pelotomaculum (Imachi et al. 2006). It thus remains a mystery why some bacteria are endued with acti vely expressed dsrAB genes, b ut cannot utilize sulf ate, sulf ite, and/or or ganosulfonates for anaerobic respiration. One might speculate that these microbes were formerly SRMs b ut have lost this trait owing to the necessity to cope with a lo w-sulfate/sulfite, methanogenic environment (Imachi et al. 2006). Hence, the presence ofdsrAB in these bacteria, which often live in close association with hydrogen-consuming microor ganisms for the syntrophic oxidation of substrates, w ould be a genetic remnant and thus indicati ve of an ancient sulf ate/sulfite-respiring potential. This theory recei ves some support from physiological data on other syntrophs such as members of the deltaproteobacterial genus Syntrophobacter, which are also frequently encountered in methanogenic environments (Lo y et al. 2004; Lueders et al. 2004), b ut still ha ve retained their sulfate-reducing capability (Harmsen et al. 1998; W allrabenstein et al. 1994). Syntrophic dsrAB-containing non-SRMs, syntrophic SRMs, and “authentic” SRMs are phylogenetically intermingled, indicating an e volutionary connection between the lifestyles of SRMs and syntrophs. An alternati ve explanation is that the actual substrate for the DSR in syntrophic bacteria has not yet been identif ied. Genomic and metagenomic analyses of Pelotomaculum species are under w ay and might provide some answers to this riddle.

5.2.3 Sulfur-Oxidizing

Bacteria

Apart from microorganisms that do or did gain energy by reducing sulfate/sulfite, it has long been kno wn that also some phototrophic and chemotrophic sulfur compound-oxidizing bacteria (SOBs) contain a re versely operating DsrAB-type DSR (rDSR; Schedel and T rüper 1979). Ho wever, we only recently be gan to understand the actual physiological function of this enzyme and how widespread it is among SOBs; rDSRs occur in members of the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria and of the phylum Chlorobi (green sulfur bacteria) (Dahl et al. 1999, Sabehi et al. 2005; Fig. 5.2). On the basis of mutagenesis studies of Allochromatium vinosum, it is evident that rDSR in these SOBs is essential for the oxidation,and thus mobilization, of intracellularly stored sulfur or polysulf ides (obligate intermediates during oxidation of sulf ide and thiosulfate). Direct oxidation of e xternal sulfide is not mediated by DsrAB but via alternati ve pathw ays (Dahl et al. 2005). Currently a vailable phenotypic and genotypic data suggest that the presence of rDSR is associated with the capability of forming intracellular sulfur globules and lack of soxCD, genes encoding a sulfur dehydrogenase (Friedrich et al. 2005).

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The Root and Major Branches of the DsrAB Tree

The DsrAB tree is subdi vided into three distinct, well-supported branches that represent three different DsrAB protein families (Molitor et al. 1998): (1) bacterial DsrAB from microbes with the (former) capability to reduce sulfite, sulfate, and/or organosulfonates (including the archaeon Archaeoglobus with a laterally acquired bacterial dsrAB), (2) bacterial DsrAB from SOBs, and (3) the archaeal DsrAB from sulfite-reducing Pyrobaculum species (Fig. 5.2). Re vealing whether DsrAB in Pyrobaculum is truly of archaeal nature will require the discovery and phylogenetic analysis of additional sequences from this branch. Candidate archaeal species that showed faint growth with sulfate and/or sulfite as the terminal electron acceptor but that have thus far been overlooked as potential SRMs have been proposed (Dhillon et al. 2005). The genome of one of these species, Caldivirga maquiling ensis, is currently being sequenced. As mentioned already, dsrA and dsrB arose by a gene duplication event and thus allow one to determine the root of the DsrAB tree by so-called paralogous rooting (Klein et al. 2001; Fig. 5.1). Pyrobaculum occupies the deepest position in the nearly bilaterally symmetrical DsrA and DsrB branches, indicating that, ifdsrAB in Pyrobaculum are archaeal, the duplication of an ancestral dsr gene preceded the diversification of the domains Archaea and Bacteria and that the ancestral DsrAB functioned in the reducti ve direction (Molitor et al. 1998). The latter f inding is supported by biogeochemical data suggesting that the rise of sulfite respiration took place early in Earth’s genesis, possibly even before the evolution of sulfate respiration (Skyring and Donnelly 1982). In the further course of e volution, the bacterial DsrAB version presumably underwent a functional split, leading to maintenance of the ancestral, sulfite-reducing enzyme type in SRMs and the f irst appearance of a new reversely operating DSR in SOBs.

5.2.5

Other Non-DsrAB Dissimilatory Sulfite Reductases

Besides DsrAB, tw o further siroheme SRs, AsrABC and Fsr , are presumably involved in dissimilatory processes. AsrABC is best studied in Salmonella enterica serovar Typhimurium and is encoded by a functional operon consisting of three genes asrA, asrB, and asrC (Clark and Barrett 1987; Huang and Barrett 1990, 1991). The deduced amino acid sequences of asrA and asrC contain conserved cysteine residues that are characteristic for [Fe 4S4]–ferredoxin binding domains (Huang and Barrett 1991). An additional siroheme–[Fe 4S4]-binding motif is only present in the γ-subunit AsrC, which is homologous to DsrA and DsrB (Dhillon et al. 2005). In S. enterica serovar Typhimurium, asr genes are part of a larger set of genes, which act in concert to facilitate a very specific metabolism, i.e., B 12-dependent anaerobic growth by oxidizing ethanolamine or 1,2-propanediol with tetrathionate as an electron acceptor (Price-Carter et al. 2001). Interestingly , this assemblage of man y genes, including genes for three sulfur compound reducing enzyme systems ( ttr, phs, and asr),

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1,2-propanediol catabolism (pdu), and de novo synthesis of B12 (cbi), is absent in the genomes of closely related Escherichia coli strains. Thus, B 12-dependent anaerobic degradation of small molecules, dri ven by reduction of sulfur compounds, is a characteristic trait of Salmonella species. It has been suggested that this metabolism evolved during the divergence of Salmonella from E. coli by a combination of acquisition of no vel genes and loss of ancestral genes. Because tetrathionate can be reduced by many other enteric bacteria (Barrett and Clark 1987), it seems lik ely that ttr, phs, and asr genes are ancestral and hence were evolutionary eradicated from the genomes of E. coli strains (Price-Carter et al. 2001). Clearly , additional studies are necessary to shed further light on this hypothesis. For example, the distribution of asr genes among different microbial taxa is not well kno wn. A BLASTp search against all 623 microbial genome sequences (3 October 2006) re vealed that asrABC genes are present in the gammaproteobacterial genera Salmonella and Photobacterium and in the lo w-G+C Gram-positive bacteria Clostridium (Harrison et al. 1984; Laishle y et al. 1984), Thermoanaerobacterium, and Moorella (Fig. 5.1). A ne w type of SR, Fsr , w as recently disco vered in the methanogenic archaeon Methanocaldococcus jannaschii (Johnson and Mukhopadhyay 2005). Although sulf ite can be inhibitory to methanogens (Balderston and P ayne 1976), other methanogens such as M. jannaschii, a strictly hydrogenotrophic, thermophilic microorganism, not only tolerate but even grow with sulfite as the sole source of sulfur (Daniels et al. 1986; Rothe and Thomm 2000). Fsr is an unusual, chimeric protein, with the N-terminal half being an H 2F420 dehydrogenase and the C-terminal half being a siroheme SR, which might ha ve been generated by fusion of a laterally acquired DSR gene and a fqoF or fpoF gene, coding for an H 2F420 dehydrogenase sub unit (Johnson and Mukhopadhyay 2005). The physiological role of Fsr appears to be detoxif ication of sulf ite rather than sulf ite-reduction-based ener gy production, as M. jannasc hii could thus far not been grown with acetate (as the sole carbon source), hydrogen, and sulfite. In the phylogenetic tree, the siroheme-binding sequence range of Fsr forms a monophyletic group with AsrC and other SRs (e.g., from Moorella and Clostridium), whose functions are unkno wn. Ho wever, with the e xception of the DsrA and DsrB branches (see abo ve), the direction of e volution, i.e., the root of the tree, cannot be inferred from Fig. 5.1.

5.3 Molecular Insights into the Ecology of DsrAB-Employing Microorganisms 5.3.1 PCR-Based

Surveys

The fortunate state that SRMs and SOBs are clearly separated in the “bacterial” part of the DsrAB tree (Fig. 5.2) and that the course of DsrAB evolution within these two functional guilds largely paralleled their 16S rRN A evolution makes dsrAB an ideal

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molecular mark er for determinati ve and ecological studies. Specif ic primer sets were developed (and are continuously updated upon the availability of new complete dsrAB sequences) together with protocols for amplification, cloning, and comparative sequence analysis of a lar ge dsrAB fragment from almost all SRMs (e xcluding Pyrobaculum species) (Wagner et al. 2005). T wo fascinating discoveries were made after application of these primer sets for culti vation-independent recovery of dsrAB sequences from a wide v ariety of ecosystems and geographic re gions (Dhillon et al. 2003; Leloup et al. 2007; Lo y et al. 2004). Firstly , man y en vironmental dsrAB sequences are not affiliated with known SRMs but occupy basal positions in the SRM branch of the DsrAB tree (Fig. 5.2); hence, these no vel dsrAB variants might derive from microbes that either are members of recognized major taxa not yet kno wn to contain SRMs or represent yet unknown microbial classes or phyla. Secondly, dsrAB richness in many habitats is dominated by these no vel sequence types. This observation, and the possibility that these environmental surveys underestimated the number and diversity of yet uncultured SRMs (because the primers are based on only a fe w complete dsrAB sequences deriving mainly from cultured SRMs), indicates that these previously unrecognized SRMs are of signif icant ecological importance. Although the current set of culti vated SRMs already is an assemblage from di verse microbial phyla, we now understand that they only constitute the “tip of the iceberg” of the natural SRM diversity. However, only the dsrAB sequences are known from these novel SRMs. Even the DsrAB-based phylogeny may not reflect the phylogeny of the SRMs carrying these novel dsrAB owing to the blurring effect of possible LGT events. Our knowledge of the distribution of rDSR among described SOBs, a prerequisite for sound analysis and interpretation of en vironmentally retrieved dsrAB sequences from this functional guild, is scant. An initial step to wards closing this gap in our knowledge was made by de veloping specific dsrAB-targeted primer sets for SOBs (Duller and Loy, unpublished data). SOBs from dif ferent taxonomic groups are cur rently being screened for the presence of dsrAB. Furthermore, the applicability of these primers for environmental surveys of dsrAB-carrying SOBs is also being tested with samples that contain microbial communities of v arying complexity. With the e xception of dsrAB from Pyrobaculum, virtually nothing is kno wn about the phylogenetic breadth or en vironmental di versity of dsrAB sequences belonging to the “archaeal” DsrAB f amily. Ho wever, it is note worthy that Pyrobaculum aerophilum has two different dsrAB copies in its genome (Fig. 5.2). The presence of multiple copies of a functional gene in one strain is not uncommon and might e xtend an or ganism’s ability to cope with v arying en vironmental conditions, given that the different gene versions code for functional enzymes with slightly different characteristics (Tchawa Yimga et al. 2003).

5.3.2 Metagenomics Recent studies emphasize the potential of metagenomics (DeLong 2002; Handelsman 2004) for en vironmental analysis of uncultured sulfur or ganisms without the need for PCR or culti vation. F osmid libraries containing se veral

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thousand clones with insert sizes ranging from 32 to 44 kb were created from sediment samples from an intertidal sand flat in the W adden Sea and from the deep ocean at the Hydrate Ridge (Mussmann et al. 2005). Detailed sequence analysis of three selected fosmids (1) supported earlier theories about possible mechanisms of gene flo w among SRMs, o wing to the presence of genomic islands for sulf ate reduction (see abo ve) and (2) enabled e xtensions of the current model of sulfur-based energy metabolism. In another study , bacterial artif icial chromosome (B AC) libraries with insert sizes averaging 80 kb were established from surf ace waters of the Mediterranean Sea and the Red Sea in order to better comprehend the genetic variability and physiological capabilities of proteorhodopsin-containing microor ganisms (Sabehi et al. 2005). These microbes gain ener gy with help of a light-dri ven proton pump, the membrane-spanning proteorhodopsin, and are one of the most ab undant microbial guilds on Earth. Surprisingly , one of the 11 proteorhodopsin gene-carrying B AC clones that were completely sequenced also contained a whole “re verse” DSR operon with high sequence similarity to, and identical arrangement of, dsr genes as in A. vinosum . Additionally, the dsrAB sequence from this Mediterranean B AC clone clustered tightly with nine dsrAB sequences from a lar ge, shotgun-librarybased environmental sequencing project of the Sar gasso Sea (V enter et al. 2004; Fig. 5.2), suggesting ubiquity of the respecti ve microbes in the photic zones of the oceans. Some anoxygenic phototrophs gain energy by complete oxidation of dimethyl sulfide to sulfate (Jonkers et al. 1999) and possess a “re verse” DSR (Dahl et al. 1999). In contrast to hydrogen sulfide, which is spontaneously oxidized under oxic conditions, dimethyl sulf ide is the most important v olatile biogenic sulfur compound in ocean w aters and thus is mainly responsible for the transfer of marine-derived sulfur to the air (Lo velock et al. 1972). Once released into the atmosphere, dimethyl sulf ide is chemically oxidized to acidic aerosol sulf ates, which serv e as condensation nuclei for cloud formation and thus increase the absorption and scattering of incoming sunlight (cloud albedo) o ver the remote oceans. However, up to about 90% of the dimethyl sulf ide is biologically oxidized by marine microor ganisms before it can dif fuse into the atmosphere (Kiene and Bates 1990). It has been speculated that these novel proteorhodopsin- and “reverse”DSR-exploiting microbes are part of this important microbial community that regulates the sea-to-air flux of dimethyl sulf ide (Sabehi et al. 2005).

5.4 Conclusions Genetic, biochemical, and phylogenetic studies ha ve raised our a wareness of novel aspects of functional properties of DSRs and the evolutionary flow of their coding genes among di verse microbial lineages. This kno wledge gain w as extended by ecological in vestigations that sho wed an unforeseen di versity of DSR genes in the environment. However, revealing the identities and (eco) physiologies of these previously “hidden” microbes with novel DSR gene variants remains a challenge. In this respect, the genetically modifiable SOBs A. vinosum (Pott and

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Dahl 1998) and Chlorobaculum (Chlorobium) tepidum (Frigaard and Bryant 2001) could become v aluable model systems for heterologous e xpression analysis in order to pro ve the functions of en vironmental dsrAB and for re vealing the theoretical phylogenetic and functional boundaries of lateral dsrAB transfers among different microbial taxa. Acknowledgements. We acknowledge support from the Fonds zur Förderung der wissenschaftlichen Forschung (project P18836-B17) to A.L. and the bmb+f (project 01 LC 0021A-TP2 in the framework of the BIOLOG II program) to M.W . Kasper Kjeldsen and Mik e Taylor are acknowledged for v aluable comments on the manuscript. We are indebted to Da ve Stahl and Michael Friedrich for long-term collaboration on the ecology/e volution of SRMs and we thank Michael Klein, Vladimir Zverlov, Natuschka Lee, Doris Steger, Stephanie Füreder, Ivan Barisic, Sebastian Lücker, and Christian Baran yi, who ha ve contributed in man y ways to our w ork on microbes of the sulfur cycle.

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Siegel LM, Murph y MJ, Kamin H (1978) Siroheme: methods of isolation and characterization. Methods Enzymol 52:436–447 Skyring GW, Donnelly TH (1982) Precambrian sulfur isotopes and a possible role for sulf ite in the evolution of biological sulfate reduction. Precambrian Res 17:41–61 Tchawa Yimga M, Dunfield PF, Ricke P, Heyer J, Liesack W (2003) Wide distribution of a novel pmoA-like gene copy among type II methanotrophs, and its expression in Methylocystis strain SC2. Appl Environ Microbiol 69:5593–5602 Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, Wu D, Paulsen I, Nelson KE, Nelson W, Fouts DE, Le vy S, Knap AH, Lomas MW , Nealson K, White O, Peterson J, Hoffman J, P arsons R, Baden-T illson H, Pf annkoch C, Rogers YH, Smith HO (2004) Environmental genome shotgun sequencing of the Sar gasso Sea. Science 304:66–74 Wagner M, Roger AJ, Flax JL, Brusseau GA, Stahl DA (1998) Phylogeny of dissimilatory sulfite reductases supports an early origin of sulf ate respiration. J Bacteriol 180:2975–2982 Wagner M, Lo y A, Klein M, Lee N, Ramsing NB, Stahl D A, Friedrich MW (2005) Functional marker genes for identif ication of sulf ate-reducing prokaryotes. Methods Enzymol 397:469–489 Walker CB, Stolyar SS, Pinel N, Y en H-CB, He Z., Zhou J, W all JD, Stahl DA (2006) Recovery of temperate Desulfovibrio vulgaris bacteriophage using a novel host strain. Environ Microbiol 8:1950–1959 Wallrabenstein C, Hauschild E, Schink B (1994) Pure culture and c ytological properties of Syntrophobacter wolinii. FEMS Microbiol Lett 123:249–254 Zeidner G, Bielawski JP, Shmoish M, Scanlan DJ, Sabehi G, Beja O (2005) Potential photosynthesis gene recombination between Prochlorococcus and Synechococcus via viral intermediates. Environ Microbiol 7:1505–1513 Zverlov V, Klein M, Lücker S, Friedrich MW, Kellermann J, Stahl DA, Loy A, Wagner M (2005) Lateral gene transfer of dissimilatory (bi)sulf ite reductase re visited. J Bacteriol 187:2203–2208

Chapter 6

Genomic and Evolutionary Perspectives on Sulfur Metabolism in Green Sulfur Bacteria Niels-Ulrik Frigaard, Donald A. Bryant

Abstract Green sulfur bacteria (GSB) are anaerobic photoautotrophs that oxidize sulfide, elemental sulfur, thiosulfate, ferrous iron, and hydrogen for growth. We present here an analysis of the distrib ution and e volution of enzymes involved in oxidation of sulfur compounds in GSB based on genome sequence data from 12 strains. Sulf ide:quinone reductase (SQR) is found in all strains. Chlorobium ferrooxidans, which cannot gro w on sulfide but grows on Fe 2+, has apparently lost all genes in volved in oxidation of sulfur compounds other than sqr. Instead, this or ganism possesses genes in volved in assimilatory sulf ate reduction, a trait that is unusual in GSB. The dissimilatory sulf ite reductase (Dsr) enzyme system, which appears to be in volved in elemental sulfur utilization, is found in all sulf ide-utilizing strains e xcept Chloroherpeton thalassium. The absence of Dsr enzymes in this early di verging GSB, in combination with phylogenetic analyses, suggests that the Dsr system in GSB could be a recent acquisition, which w as obtained by lateral gene transfer in part from sulf ideoxidizing bacteria and in part from sulfate-reducing bacteria. All thiosulfate-utilizing GSB strains ha ve an identical sox gene cluster. The soxCD genes, which are found in certain other thiosulf ate-utilizing organisms like Paracoccus pantotrophus, are absent from GSB. Fla vocytochrome c, adenosine 5 ′-phosphosulfate reductase, ATP-sulfurylase, the Qmo complex, and other enzymes related to the utilization of sulfur compounds are found in some, b ut not all sulfide-utilizing strains. Ev en though dif ferent GSB strains superf icially e xhibit a similar sulfur oxidation phenotype, this may be caused by dif ferent combinations of enzymes. Thus, genome analyses have revealed that GSB have greater diversity in sulfur metabolism than previously suspected.

6.1 Intr oduction Inorganic sulfur metabolism in prokaryotic or ganisms is a comple x topic o wing to the complex chemistry of sulfur and the multitude of enzymes that ha ve evolved to catalyze its chemistry . Nonetheless, the ability to use inor ganic sulfur compounds for gro wth is widespread among v ery dif ferent prokaryotes of both archaeal and 60 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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bacterial affiliation. The phototrophic sulfur bacteria oxidize reduced inorganic sulfur compounds for photosynthetic CO2 fixation and growth under anaerobic conditions. Despite decades of research on the enzymology and genetics of sulfur -compound oxidation in these bacteria, much remains to be learned about the biochemistry and evolution of this essential part of their metabolism. Recently , genome sequence information has become available for 12 strains of phototrophic green sulfur bacteria (GSB). This information holds the k ey to substantial advances in understanding the inorganic sulfur metabolism of these bacteria, which constitute an interesting model not only for bacterial oxidation of sulfur compounds b ut also for the e volution of a complex metabolic network in different strains of a closely related group of bacteria. Based on genome sequence information, this chapter discusses the distrib ution and possible functions of kno wn and putative enzymes metabolizing sulfur compounds as well as the evolution of these enzymes and the metabolic networks that they constitute in GSB. Other recent publications on this subject are also a vailable (Hanson and Tabita 2001, 2003; Frigaard and Bryant 2008).

6.1.1

Green Sulfur Bacteria

Phototrophic sulfur bacteria thrive either in planktonic or benthic forms in aquatic, anoxic en vironments where sulf ide and light coincide. In these en vironments, sulfide is often produced by sulf ate-reducing bacteria, with which phototrophic sulfur bacteria sometimes form more or less stable multicellular aggre gates, but the sulfide may also be of geological or anthropogenic origin. The y are di vided into the GSB and the purple sulfur bacteria (PSB), both of which ha ve been studied in pure cultures for about 100 years. The GSB, which owe their name and color to their pigmentation by bacteriochlorophyll c, d, and e, comprise the f amily Chlorobiaceae and represent the only cultivated members of the phylum Chlorobi (Garrity and Holt 2001). The PSB owe their name and color to their pigmentation by bacteriochlorophyll a and b and various carotenoids, and all are members of the class Gammaproteobacteria, which is a major subdi vision of the highly di verse phylum Proteobacteria (Imhoff et al. 2005). Sulf ide and thiosulfate can also support photosynthetic growth of some purple nonsulfur bacteria, which belong to the class Alphaproteobacteria, b ut these compounds are usually not the preferred substrates for growth of these bacteria (Imhof f et al. 2005). The ecology of GSB and that of PSB are to some e xtent similar (v an Gemerden and Mas 1995) and their oxidati ve sulfur metabolism probably shares man y characteristics (Brune 1989, 1995). Ho wever, most other aspects of their physiology and e volution are rather different. For example, GSB are generally much less physiologically versatile than PSB. GSB are commonly found in sulfide-rich freshwater and estuarine environments and are rare in marine en vironments (Ov ermann 2000; Garrity and Holt 2001). They typically occur in the w ater column, in sediments, or within microbial mats, and may occasionally be found as dense accumulations in planktonic forms in

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stratified lak es or in benthic forms as microbial mats in sulf ide-rich springs. Recently characterized unusual habitats include the anoxic zone 100 m below the surface of the Black Sea (Ov ermann et al. 1992; Mansk e et al. 2005), deep-sea hydrothermal v ents in the P acific Ocean (Beatty et al. 2005), and the microbial mats of Octopus and Mushroom Springs in Yellowstone National Park (Ward et al. 1998). GSB are obligately anaerobic and obligately photoautotrophic (Ov ermann 2000; Garrity and Holt 2001). All characterized GSB strains use the reductive (also called reverse) tricarboxylic acid cycle for CO2 fixation. In addition, most GSB can assimilate a small number of simple, or ganic compounds such as acetate, b ut only in the presence of CO 2 and a photosynthetic electron donor . Most strains use electrons derived from oxidation of sulfide, but some strains can also oxidize elemental sulfur, thiosulf ate, H 2, and Fe 2+ (Sect. 6.2). All GSB characterized to date ha ve unique light-harvesting organelles known as chlorosomes, which allow highly efficient capture of light ener gy (Frigaard and Bryant 2006). This ability pro vides a substantial competitive advantage over PSB at low light intensities.

6.1.2

Genome Sequencing Projects of Green Sulfur Bacteria

Twelve strains of GSB ha ve been selected for genome sequencing (Fig. 6.1). The genome of one of the best characterized strains, Chlorobaculum tepidum TLS (previously known as Chlorobium tepidum TLS), was sequenced and annotated in 2002 by The Institute for Genome Research (Eisen et al. 2002). Other strains are cur rently at various stages of genome sequencing and annotation at the Joint Genome Institute and in the laboratories of Donald A. Bryant, Stephan C. Schuster (both of The Pennsylv ania State Uni versity, USA), and Jör g Ov ermann (LudwigMaximilians-Universität, Germany). Currently, genome sequence data are publicly available for ten GSB strains and can be accessed and analyzed on the Web sites of the Joint Genome Institute (2007a) and the National Center for Biotechnology Information (2007). These ten genomes were sequenced by a traditional shotgun cloning approach. The draft genomes of Chlorobaculum parvum NCIMB 8327d (= DSMZ 263T; pre viously kno wn as Chlorobium vibrioforme subsp. thiosulfatophilum; the “d” indicates that this strain contains bacteriochlorophyll d) and Chloroherpeton thalassium ATCC 35110T have recently been determined by p yrosequencing (D.A. Bryant and S.C. Schuster , unpublished results), and PCR-based methods are currently being used for gap closure. The information in this chapter is based on the genome sequence data a vailable for the ten GSB strains currently present in the NCBI GenBank and has been supplemented with information from the two unfinished sequences. Recent re views on the physiological and metabolic inferences derived from genome sequence data of GSB are a vailable (Frigaard et al. 2003, 2006; Frigaard and Bryant 2004; 2008). At present, the only a vailable genome sequence information for PSB is that for the halophilic Halorhodospira halophila SL1 (Joint Genome Institute (2007b)).

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Fig. 6.1 Neighbor-joining phylogenetic tree of the 16S ribosomal RN A gene of selected strains of green sulfur bacteria (GSB). Common strain designations are sho wn. Asterisks indicate the demonstrated ability to grow on thiosulfate. Strains for which genome sequence data are available are mark ed in bold. Sequence accession numbers are either from the JGI data base or from GenBank. The tree is based on 1,115 nucleotide positions and was made with MEGA version 3.1 (Kumar et al. 2004). Bootstrap v alues in percent are shown for 1,000 replications. Except for the position of Chlorobium chlorochromatii CaD3, whose position is not resolv ed, minimum-evolution and maximum-parsimony analyses support the topology of this tree

6.2

Compounds Oxidized by Green Sulfur Bacteria

In addition to being obligately phototrophic, GSB are obligately lithoautotrophic, which means they can only grow by oxidizing inorganic compounds and reducing CO 2 in the presence of light. Inorganic compounds known to be oxidized by GSB include sulf ide (S2−), elemental sulfur (S 0), polysulf ides (S n2−), thiosulf ate (S 2O32−), tetrathionate (S4O62−), hydrogen (H 2), and ferrous iron (Fe 2+) (Brune 1995; Heising et al. 1999; Garrity and Holt 2001). Most strains can oxidize sulfide and H2, while oxidation of thiosulfate and Fe2+ is less commonly encountered in cultivated strains. GSB are not known to oxidize sulfite (SO32−) for growth, although it probably is an intracellular intermediate in the oxidation of other sulfur compounds (Sect. 6.3.5). Some of the sulfur compounds utilized by the genome-sequenced strains are sho wn in Table 6.1. GSB have a high af finity for sulf ide, and this is usually the preferred substrate even if other sulfur substrates are available. Sulfide is usually initially only incompletely oxidized to elemental sulfur , which is deposited e xtracellularly as highly refractive sulfur globules. These sulfur globules are usually but not always oxidized completely to sulfate when the sulfide has been consumed. Several strains of GSB are capable of gro wth on thiosulfate (see the overview in Imhof f 2003). Such strains are often preferred for laboratory w ork because

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thiosulfate can conveniently be included in high concentrations in liquid or solid growth media and does not inhibit gro wth. Some of these strains ha ve been shown to photochemically disproportionate elemental sulfur into sulfide and thiosulfate in the absence of CO2 (strains DSMZ 249, 255, 257, and 263) – an ability not observed in strains that cannot utilize thiosulf ate (Trüper et al. 1988, Brune 1989). Two thiosulfate-utilizing strains of GSB have also been reported to utilize tetrathionate (Brune et al. 1989). Table 6.1 Phenotypes and genotypes of genome-sequenced green sulfur bacteria b Electron donora Genotype Strain S

2−

S0

S2O32−

sqr dsr fcc soy

sox

apr sat qmo PSRLC3

Chlorobaculum + + + ++ +− + − − − − parvum DSMZ 263 Chlorobaculum + + + ++ +− + + + + − tepidum TLS Chlorobium + − − ++ +− + + + + + chlorochromatii CaD3 Chlorobium + + + ++ ++ + + + + − clathratiforme DSMZ 5477 Chlorobium − − − +− −− − − − − − ferrooxidans DSMZ 13031 Chlorobium + + − ++ ++ − − − − + limicola DSMZ 245 Chlorobium + + − ++ −− − − − − + luteolum DSMZ 273 Chlorobium + + − ++ ++ − − − − + phaeobacteroides DSMZ 266 Chlorobium + + + ++ +− + − − − + phaeovibrioides DSMZ 265 Chloroherpeton + + − +− ++ − − − − + thalassium ATCC 35110 Prosthecochloris + ND ND ++ +− − + + + − sp. BS1 Prosthecochloris + + − + ++ + − − −− + aestuarii DSMZ 271 ND not determined. a Garrity and Holt (2001), Heising et al. (1999), Vogl et al. (2006). b The following abbreviations designate more than one gene: apr, aprBA; dsr, dsrNCABLEFHTMKJOP; fcc, fccAB; sox, soxJXYZAKBW; soy, soyYZ; qmo, qmoABC.

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“Chlorobium ferrooxidans” DSMZ 13031, a GSB strain that oxidizes Fe 2+, has been characterized (Heising et al. 1999). This strain also oxidizes H 2 but appears to have lost the ability to oxidize sulfur compounds because it does not grow on sulfide, elemental sulfur, or thiosulf ate. This phenotype is lar gely confirmed by the absence of many genes related to oxidation of sulfur compounds in its genome (T able 6.1). It is not known how common or important this mode of “photoferrotrophy” is in nature, but photosynthetic growth with Fe2+ as an electron donor has also been demonstrated in some purple bacteria (Widdel et al. 1993; Ehrenreich and Widdel 1994).

6.3

Enzymes Involved in Sulfur-Compound Oxidation

Several enzymes potentially involved in sulfur metabolism can readily be identified in the genome sequences by sequence homology with enzymes for whichfunctional information is available (Table 6.1). This information, combined with biochemical and physiological information about the strains, can be used to produce a putati ve scheme for the metabolic reactions in GSB (Fig. 6.2). The enzymes and the metabolic capabilities they may confer are discussed in the follo wing sections.

Fig. 6.2 Overview of the proposed pathways in the oxidative sulfur metabolism of GSB. Not all GSB strains have all pathways shown here. See text for details. The electron carriers menaquinone (MK) and c ytochrome c (CycA) are reoxidized by the photosynthetic processes that f ix CO 2. (Derived from information in Eisen et al. 2002 and Dahl 2008)

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6.3.1 Overview of the Putative Sulfur Compound Oxidation Enzymes Sulfide:quinone reductase (SQR) and fla vocytochrome c probably constitute tw o alternative pathw ays of sulf ide oxidation (Sects. 6.3.3, 6.3.4). The dissimilatory sulfite reductase (Dsr) system probably is in volved in the oxidation of sulfur globules (Sect. 6.3.2). A putative thiol (R–SH) is shown in Fig. 6.2 that may be involved in the oxidation of the sulfur glob ules (Brune 1989, 1995; Hanson and T abita 2001). The Sox system is in volved in oxidation of thiosulf ate and can account for this activity observed in all strains capable of oxidizing thiosulf ate (Sect. 6.3.6). The adenosine 5 ′-phosphosulfate (APS) reductase (Apr), sulf ate aden ylyltransferase (Sat), and quinone-interacting membrane-bound oxidoreductase (Qmo) complex probably constitute a sulf ite-oxidizing system (Sect. 6.3.5). A putati ve alternative sulfite-oxidizing system has been identif ied in the GSB strains that do not have the Apr, Sat, and Qmo enzymes (Sect. 6.3.5). Homologs of polysulfide reductase, and heterodisulfide reductase are also found in GSB (Frigaard and Bryant 2008). Ho wever, functions cannot easily be assigned to these proteins in GSB because (1) the y are too distantly related to characterized enzymes, (2) they are not distributed among the GSB strains in a manner that obviously correlates with known physiological traits, and (3) the sulfur compound and hydrogen oxidation properties of the GSB strains can be accounted for by other enzymes.

6.3.2 Dissimilatory

Sulfite Reductase

The well-studied PSB, Allochromatium vinosum, contains a gene cluster with high sequence similarity to the dissimilatory sulf ite reductase dsr gene cluster of sulfate-reducing bacteria (Dahl et al. 2005; Dahl 2008). The dsr gene cluster in A. vinosum, dsrABEFHCMKLJOPNRS, is essential for the oxidation of intracellular sulfur glob ules, and thus it is assumed that the Dsr enzyme system in this organism functions in the oxidati ve direction to produce sulf ite (Pott and Dahl 1998; Dahl et al. 2005; Sander et al. 2006). GSB contain a v ery similar dsr cluster, dsrNCABLEFHTMKJOP, the only dif ference being the absence of dsrRS and the presence of dsrT. This cluster is present in all GSB, e xcept Chl. ferrooxidans and Chp. thalassium, and it most likely encodes the same function as in A. vinosum. The absence of dsr genes in Chl. ferrooxidans is consistent with the observation that this bacterium is incapable of growth on elemental sulfur and sulfide. Because this strain appears to have ancestors that are sulfur- and sulfide-oxidizing GSB that contain the dsr genes (Fig. 6.1), it seems highly lik ely that Chl. ferrooxidans has lost the dsr genes as a consequence of adapting to gro wth on Fe 2+. The absence of dsr genes in Chp. thalassium is especially interesting for tw o reasons: firstly, this or ganism is a v ery early diverging GSB, and, secondly , this

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organism gro ws poorly on elemental sulfur . Lik e other GSB, Chp. thalassium grows well on sulf ide and forms e xtracellular sulfur glob ules as an oxidation product (Gibson et al. 1984); ho wever, this elemental sulfur is only v ery slowly oxidized, and this behavior could be due to the absence of the Dsr system. It is at present unclear what might constitute an alternati ve sulfur -oxidizing system in Chp. thalassium . Such a system might someho w in volve the rib ulose-1,5bisphosphate carboxylase/oxygenase (Rubisco) lik e protein (RLP), which is present in all GSB, including Chp. thalassium, and which has been sho wn to be involved in growth on elemental sulfur in Cba. tepidum TLS (Hanson and Tabita 2001, 2003). It is an interesting possibility that it might have been the acquisition of the Dsr -dependent system, which seems to be in volved in ef ficient and complete oxidation of elemental sulfur , that led to the relati vely recent, e xplosive radiation of the lineages of GSB that are not closely related to Chloroherpeton (Fig. 6.1). Phylogenetic analyses of the DsrA protein and other Dsr proteins in GSB show that these proteins constitute a monophyletic group (Fig. 6.3a). Thus, the DsrA phylogeny is congruent with the 16S ribosomal RNA phylogeny at least at the phylum level. However, the dsr genes have experienced lateral gene transfer (LGT) within the GSB phylum; for e xample, DsrA from Prosthecochloris aestuarii DSMZ 271 is located within the Chlorobium/Chlorobaculum cluster (Fig. 6.3a). On the basis of further phylogenetic analyses, the c ytoplasmic DsrAB sulfite reductase and other c ytoplasmic Dsr proteins in GSB are most closely related to the Dsr proteins from other sulf ide-oxidizing prokaryotes (Sander et al. 2006). This is in contrast to the subunits of the membrane-bound DsrMKJOP complex, which are most closely related to the DsrMKJOP proteins from sulfate-reducing prokaryotes. In addition, the DsrT protein (unkno wn function) is only found in GSB and sulf ate-reducing prokaryotes and not in other sulf ideoxidizers. This suggests that the Dsr system in GSB has an intriguing chimeric nature.

6.3.3 Sulfide:Quinone

Reductase

SQR catalyzes the oxidation of sulfide with a membrane-bound isoprenoid quinone as the electron acceptor . This enzyme occurs in both chemotrophic and phototrophic prokaryotes (Griesbeck et al. 2000; Theissen et al. 2003). Membrane-bound SQR activity has been demonstrated biochemically in GSB, and this enzyme presumably feeds electrons into the photosynthetic electron transfer chain (Shahak et al. 1992). The genome sequences of all 12 GSB strains, including Chl. ferrooxidans and Chp. thalassium, encode either one or tw o homologs of the biochemically characterized SQRs from Rhodobacter capsulatus (CAA66112) and Oscillatoria limnetica (AAF72962) (Fig. 6.3b). The SQR homologs of GSB are flavoproteins with predicted masses of about 53 kDa, and each contains all three conserv ed cysteine residues that are essential for sulfide oxidation in R. capsulatus SQR (Griesbeck et al. 2002).

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Fig. 6.3 Neighbor-joining phylogenetic tree of a DsrA and b sulfide:quinone reductase (SQR) proteins from GSB and other or ganisms. Bootstrap values in percent are sho wn for 1,000 replicates. Nodes with less than 50% support in neighbor-joining and minimum-evolution analyses are collapsed

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It is interesting that Chl. ferrooxidans contains an SQR (ZP_01385816), because this or ganism cannot gro w on sulf ide as the sole electron donor (Heising et al. 1999). This organism may benefit from SQR activity as a supplement to its energy metabolism. Alternati vely, it could also use SQR as a protecti ve mechanism to remove sulfide, which prevents growth when it is present in high concentrations.

6.3.4 Flav ocytochrome c Flavocytochrome c is a periplasmic enzyme consisting of a lar ge sulfide-binding FccB flavoprotein subunit and a small FccA c ytochrome c subunit (Brune 1995). Except for Chl. ferr ooxidans and Chlorobium luteolum DSMZ 273, an fccABencoded flavocytochrome c is found in all GSB strains for which genome sequence data are a vailable. The fla vocytochrome c of GSB consists of a 10-kDa FccA cytochrome c553 subunit, which binds a single heme, and an approximately 47-kDa sulfide-binding FccB fla voprotein sub unit. FccAB is constituti vely e xpressed in Chlorobium limicola DSMZ 249 (Verté et al. 2002). Although flavocytochrome c from various organisms has been shown to oxidize sulfide and reduce cytochrome c in vitro, the exact function and significance of this protein in vi vo is still not clear . While man y sulfide-utilizing organisms produce flavocytochrome c, some sulf ide-utilizing GSB and PSB do not, which clearly demonstrates that fla vocytochrome c is not essential for sulf ide oxidation (Brune 1995). Additionally, a mutant of the purple sulfur bacterium A. vinosum DSMZ 180, in which fla vocytochrome c has been eliminated genetically , exhibits sulfide and thiosulf ate oxidation rates similar to the wild type (Reinartz et al. 1998). If indeed the FccAB fla vocytochrome c oxidizes sulfide in vivo, both GSB and PSB apparently have alternative sulfide-oxidizing enzyme systems, possibly SQR (Sect. 6.3.3) and the Dsr system (Sect. 6.3.2), that may be quantitati vely more important. However, it is also possible that fla vocytochrome c is adv antageous under certain growth conditions and that such conditions ha ve not yet been identified.

6.3.5 Sulfite

Oxidation

Although GSB cannot gro w on sulf ite as sole sulfur source and electron donor , sulfite appears to be the product of the Dsr enzyme system (Sect. 6.3.2, Fig. 6.2). The only kno wn dissimilatory sulf ite oxidation enzyme that has homologs with high sequence similarity in GSB is the Apr-type APS reductase (also called adenosine 5 ′-phosphosulfate reductase). (A CysH-type APS reductase is also found in two GSB strains, but the gene encoding this enzyme is part of an assimilatory sulfate reduction gene cluster; see Sect. 6.4.) The aprAB genes, encoding the Apr enzyme, are only found in four GSB strains (T able 6.1). In each of the four strains these genes occur in a cluster with the sat gene encoding an A TP sulfurylase and

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the qmoABC genes encoding a heterodisulfide-reductase-like quinone oxidoreductase known as the Qmo complex that has its substrate-binding site in the c ytoplasm (Pires et al. 2003). In combination, the products of this sat–aprBA–qmoABC gene cluster might constitute an enzyme system (Sat–Apr–Qmo) that in principle could oxidize sulfite to sulf ate, with APS as an intermediate, and reduce a membranebound quinone to quinol (Fig. 6.2). A similar aprBA–qmoABC operon with high sequence similarity occurs in some sulfate-reducing Desulfovibrio-like strains. It is therefore possible that the sat–aprB A–qmoABC gene cluster in GSB is deri ved from sulfate-reducing organisms. How sulfite is oxidized in GSB that lack the putative Sat–Apr–Qmo system is not clear. However, with the sole e xception of Cba. parvum DSMZ 263 (Table 6.1), an unusual homolog of polysulfide reductase, denoted polysulfide-reductase-like complex 3 (PSRLC3), is present in all Dsr -containing GSB that lack the Sat–Apr–Qmo system (Frigaard and Bryant 2008). PSRLC3 is a membrane-bound molybdopterinbinding enzyme that has a quinone oxidoreductase domain in the membrane and, unlike other common polysulf ide reductases, its substrate-binding site in the c ytoplasm. Man y kno wn and putati ve sulf ite oxidoreductases are molybdopterinbinding, oxotransferase enzymes and the PSRLC3 complex may constitute such an enzyme in GSB. Phylogenetic analyses sho w that the PSRLC3 comple x in Chp. thalassium is basal to all other PSRLC3 comple xes in GSB. This suggests that (1) the PSRLC3 complex was present in an early ancestor of GSB and (2) GSB lacking a PSRLC3 complex may have lost it by gene elimination, perhaps as a consequence of acquiring the Sat–Apr–Qmo system.

6.3.6

Thiosulfate Oxidation by the Sox System

In the chemolithoautotrophic alphaproteobacterium Paracoccus pantotrophus, seven sox genes (soxXYZABCD) constitute a complete thiosulfate-oxidizing enzyme system (Friedrich et al. 2001, 2005). In this system, thiosulfate is bound to the SoxYZ carrier by an oxidation reaction catalyzed by SoxAX and further processed by the hydrolase SoxB and the oxidase SoxCD to regenerate the SoxYZ carrier and liberate two sulfate molecules per thiosulf ate molecule. In GSB the sox cluster , orf1015–soxXYZA– orf1020–soxBW, is conserved in the genomes of five strains (Table 6.1). This includes all four thiosulfate-utilizing strains and one strain (CaD3) that has not been reported to grow on thiosulfate. Because of the organizational conservation and the congruent phylogeny of the genes in this cluster , the genes CT1015 and CT1020 are lik ely involved in the Sox system. Thus, these two genes are now denoted as soxJ and soxK, respectively. The soxCD genes, which are essential components of the Sox system in P. pantotrophus, do not occur in the genome sequences of GSB. Instead, owing to the conservation of the soxJ/orf1015 and soxK/orf1020 genes in the GSB sox gene cluster, the process(es) in GSB that re generates the SoxYZ comple x probably in volves the SoxJ and SoxK proteins. No other easily identif iable thiosulf ate oxidation enzymes (such as rhodaneses) are encoded in the GSB genome sequences in a

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manner that obviously matches the pattern of thiosulfate utilization. Thus, thiosulfate utilization in GSB can almost certainly be attrib uted to the Sox system. A similar SoxCD-independent, thiosulfate-oxidizing Sox system is present in the PSBA. vinosum (see Chap. 9 by Grimm et al.). SoxY (J. van Beeumen, personal communication) and the SoxYZ complex (B.C. Berks, personal communication) from GSB ha ve recently been crystallized and their structures determined.

6.3.7

A Novel Complex: SoyYZ

The heterodimeric SoxYZ comple x carries sulfur substrates on a conserv ed cysteine residue in the SoxY subunit (Sect. 6.3.6; Quentmeier and Friedrich 2001). The soxYZ gene cluster has been duplicated in five GSB and is here denoted soyYZ (Table 6.1). A signal sequence at the amino termini of the SoyY sequences suggests that, like SoxYZ, So yYZ is a periplasmic comple x. Neither SoxZ nor So yZ has a signal sequence, and both are probably transferred across the c ytoplasmic membrane as part of comple xes with SoxY or So yY, respectively. In all GSB that ha ve soyYZ, these genes are located immediately upstream of the fccAB genes in an apparent operon; therefore, it is attracti ve to propose that So yY and So yZ form a complex in the periplasm that carries a sulfur substrate and that this complex reacts with the periplasmic FccAB flavocytochrome c. However, not all GSB that encode fccAB also encode soyYZ. The presence of soyYZ does not correlate with thiosulfate utilization and the substrate SoyYZ may carry is unclear. In most or ganisms of all taxonomic af filiations that ha ve SoxY, the sulfur substrate-carrying cysteine residue of SoxY is located at the C-terminus within the motif GGC(G1–2)–COOH. SoyY differs from all known SoxY proteins by having a C-terminus in which the putati ve sulfur-substrate-binding cysteine is the terminal residue. The proximity of the C-terminal carboxyl group and the thiol group of the substrate-carrying cysteine residue in So yY is likely to affect the chemistry at this site in a manner that does not occur in SoxY . If this is the case, this might e xplain the evolution of this particular motif in SoyY. In GSB the conserved motif in SoyY is VXAQAC–COOH. The soyY gene has only been found in one or ganism other than GSB: the anaerobic, sulf ide-oxidizing, chemoautotrophic Alkalilimnicola ehrlichei MLHE-1, which based on ribosomal RNA phylogeny is closely related to PSB of the Ectothiorhodospiraceae family.

6.4 Assimilatory

Sulfur Metabolism

It is often stated in the literature that GSB cannot perform assimilatory sulf ate reduction (Lippert and Pfennig 1969). Nevertheless, a recently isolated strain, Chl. ferrooxidans DSMZ 13031, grows with sulfate as the sole sulfur source and cannot utilize sulf ide, thiosulf ate, or elemental sulfur for gro wth (Heising et al. 1999).

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In agreement with this observation, the Chl. ferrooxidans genome encodes a single gene cluster that includes the assimilatory sulfate reduction genes cysIHDNCG and the sulfate permease genes cysPTWA, which are transcribed in opposite directions. These assimilatory sulfate reduction genes share a high degree of sequence similarity with those from the clostridiaClostridium thermocellum and Desulfitobacterium hafniense. However, sequence analyses sho w that the APS reductase encoded by cysH in Chl. ferrooxidans is related to the plant-type enzyme that uses APS and not 3′-phosphoadenosine 5 ′-phosphosulfate (P APS) as a substrate. An identical cys gene cluster is observ ed in Chl. luteolum DSMZ 273, b ut not in an y other GSB genome. This raises the possibility that Chl. luteolum DSMZ 273 also is capable of assimilatory sulf ate reduction and gro wth in the absence of reduced sulfur compounds using alternative electron donors.

6.5

Possible Phage-Mediated Lateral Gene Transfer

Mobile genetics elements are poorly characterized in GSB. T ransposases and other insertion elements are found in the genome sequences, and a few plasmids have also been identif ied (Méndez-Alv arez et al. 1994). Ho wever, no phage has yet been characterized that infects GSB, b ut there is no reason to belie ve that such a phage does not e xist. In f act, the presence of RN A-directed DNA polymerases and integrases in se veral GSB genomes in dif ferent genetic clusters is a strong indication that viral infections of GSB do occur . Phage can potentially cause lateral e xchange of host genes between successi ve hosts and are thus interesting from an e volutionary point of view. One such example may be found in an 11,000-bp island in Chl. phaeovibrioides DSMZ 265 (Frigaard and Bryant 2008). This island contains the Chlorobium-type sox cluster with eight genes, in addition to a transposase, an integrase, and an RNA-directed DNA polymerase. Genes that are unrelated to sulfur metabolism surround this “thiosulf ate utilization island. ” It is possible that this island is a remnant structure deri ved from an RN A viral genome. The sox cluster could have been transferred into the viral genome by a transposase in a previous host and then integrated laterally into the genome of strain DSMZ 265.

6.6 Conclusions On the basis of genome sequence analyses, some conclusions can be made about the sulfur compound oxidation enzymes in GSB. SQR (encoded by sqr) is the only known sulfur-oxidizing enzyme that is found in all GSB strains. Most strains utilizing sulfide or elemental sulfur contain the dissimilatory sulf ite reductase dsrNCABLEFHTMKJOP genes. Although Chp. thalassium appears to ha ve a (probably less ef ficient) alternati ve enzyme system for elemental sulfur oxidation, the dsr genes appear to be involved in elemental sulfur utilization in all other GSB strains.

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All thiosulfate-utilizing strains have an identical sox gene cluster (soxJXYZAKBW). The soxCD genes found in certain other thiosulfate-utilizing organisms like P. pantotrophus are absent from GSB. A putative complex denoted SoyYZ, related to the thiosulfate-binding SoxYZ comple x, could be in volved in the processing of an as yet unidentif ied sulfur compound in GSB. Genes encoding fla vocytochrome c (fccAB), APS reductase ( aprAB), A TP sulfurylase ( sat), and a heterodisulf ide reductase homolog ( qmoABC) were found in some, b ut not all strains. Gi ven the patchy distribution of these and other enzymes among the strains, it seems lik ely that different enzymes perform some sulfur-oxidation activities, such as sulfite oxidation, in dif ferent strains of GSB. The Fe 2+-oxidizing Chl. ferrooxidans, which cannot grow on sulfide, has no genes obviously involved in sulfur utilization other than sqr, but contains a full complement of genes in volved in assimilatory sulf ate reduction (cysIHDNCG), a trait that is not widely distrib uted among the GSB. Analyses indicate that, although the phylogenies of some enzymes (e.g., the DsrA protein; Fig. 6.3a) are congruent with the or ganismal phylogeny at the phylum level, the phylogenies of other enzymes are not (e.g., the SQR protein; Fig. 6.3b). Some enzyme systems that are only present in some strains e xhibit a phylogeny incongruent with the cellular core phylogeny (e.g., the Sox and the Sat–Apr– Qmo systems), and thus these systems appear to result from LGT rather than gene elimination. In the case of the sox gene cluster, evidence for phage-mediated LGT was identified. As a f inal point, although the GSB are closely related, with the e xception of Chp. thalassium , genomic analyses clearly sho w that gene elimination and LGT substantially influence the distribution of sulfur-metabolism genes both within the Chlorobi and among prokaryotes from other phyla. These observ ations illustrate the dynamic structures of prokaryotic genomes and in addition demonstrate that even organisms that superficially appear to be v ery closely related on the basis of their cellular core machinery ne vertheless can ha ve une xpected dif ferences in physiology and life style. Acknowledgements. N.-U.F gratefully acknowledges support from the Danish Natural Science Research Council (grant 21–04–0463). D.A.B. gratefully ackno wledges support for genomics studies of GSB from the United States Department of Ener gy (grant DE-FG02–94ER20137) and the National Science Foundation (grant MCB-0523100).

References Beatty JT, Ov ermann J, Lince MT , Mansk e AK, Lang AS, Blank enship RE, V an Do ver CL, Martinson TA, Plumle y FG (2005) An oblig ately photosynthetic bacterial anaerobe from a deep-sea hydrothermal vent. Proc Natl Acad Sci USA 102:9306–9310 Brune DC (1989) Sulfur oxidation by phototrophic bacteria. Biochim Bioph ys Acta 975:189–221 Brune DC (1995) Sulfur compounds as photosynthetic electron donors. In: Blank enship RE, Madigan MT , Bauer CE (eds) Anoxygenic photosynthetic bacteria. Kluwer , Dordrecht, pp 847–870

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Dahl C (2008) Inor ganic sulfur compounds as electron donors in purple sulfur bacteria. In: Govindjee (series ed) Adv ances in photosynthesis and respiration, v ol 27, Hell R, Dahl C, Knaff DB, Leustek T (eds) Sulfur metabolism in phototrophic organisms. Springer, New York (in press) Dahl C, Engels S, Pott-Sperling AS, Schulte A, Sander J, Lübbe Y , Deuster O, Brune DC (2005) Novel genes of the dsr gene cluster and e vidence for close interaction of Dsr proteins during sulfur oxidation in the phototrophic sulfur bacterium Allochromatium vinosum . J Bacteriol 187:1392–1404 Eisen JA, Nelson KE, Paulsen IT, Heidelberg JF, Wu M, Dodson RJ, Deboy R, Gwinn ML, Nelson WC, Haft DH, Hickey EK, Peterson JD, Durkin AS, Kolonay JL, Yang F, Holt I, Umayam LA, Mason T, Brenner M, Shea TP, Parksey D, Nierman WC, Feldblyum TV, Hansen CL, Craven MB, Radune D, V amathevan J, Khouri H, White O, Gruber TM, K etchum KA, V enter JC, Tettelin H, Bryant D A, Fraser CM (2002) The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, green-sulfur bacterium. Proc Natl Acad Sci USA 99:9509–9514 Ehrenreich A, Widdel F (1994) Anaerobic oxidation of ferrous iron by purple bacteria, a new type of phototrophic metabolism. Appl Environ Microbiol 60:4517–4526 Friedrich CG, Rother D, Bardischewsky F, Quentmeier A, Fischer J (2001) Oxidation of reduced inorganic sulfur compounds by bacteria: emergence of a common mechanism? Appl Environm Microbiol 67:2873–2882 Friedrich CG, Bardische wsky F, Rother D, Quentmeier A, Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Microbiol 8:253–259 Frigaard N-U, Bryant DA (2004) Seeing green bacteria in a ne w light: genomics-enabled studies of the photosynthetic apparatus in green sulfur bacteria and f ilamentous anoxygenic phototrophic bacteria. Arch Microbiol 182: 265–276 Frigaard N-U, Bryant D A (2006) Chlorosomes: Antenna or ganelles in photosynthetic green bacteria. In: Shively JM (ed) Complex intracellular structures in prokaryotes. Springer, Berlin, pp 79–114 Frigaard N-U, Bryant D A (2008) Genomics insights into the sulfur metabolism of phototrophic green sulfur bacteria. In: Govindjee (series ed) Advances in photosynthesis and respiration, vol 27, Hell R, Dahl C, Knaf f D, Leustek T (eds) Sulfur metabolism in phototrophic or ganisms. Springer, New York (in press) Frigaard N-U, Gomez Maqueo Che w A, Li H, Maresca J A, Bryant DA (2003) Chlorobium tepidum: Insights into the structure, ph ysiology, and metabolism of a green sulfur bacterium derived from the complete genome sequence. Photosynth Res 78:93–117 Frigaard N-U, Gomez Maqueo Chew A, Maresca JA, Bryant DA (2006) Bacteriochlorophyll biosynthesis in green bacteria. In: Grimm B, Porra R, Rüdiger W , Scheer H (eds) Adv ances in photosynthesis and respiration, vol 25. Springer, Dordrecht, pp 201–221 Garrity GM, Holt JG (2001) Ph ylum BXI. Chlorobi phy. nov. In: Boone DR, Castenholz R W (eds) Bergey’s manual of systematic bacteriology, vol 1, 2nd edn. Springer, New York, pp 601–623 Gibson J, Pfennig N, W aterbury JB (1984) Chloroherpeton thalassium gen. no v. et spec. no v., a non-filamentous, flexing and gliding green sulfur bacterium. Arch Microbiol 138:96–101 Griesbeck C, Hauska G, Schütz M (2000) Biological sulfide oxidation: Sulfide-quinone reductase (SQR), the primary reaction. In: Pandalai SG (ed) Recent research developments in microbiology, vol 4. Research Signpost, Trivadrum, pp 179–203 Griesbeck C, Schütz M, Schödl T , Bathe S, Nausch L, Mederer N, V ielreicher M, Hauska G (2002) Mechanism of sulf ide-quinone reductase in vestigated using site-directed mutagenesis and sulfur analysis. Biochemistry 41:11552–11565 Hanson TE, Tabita FR (2001) A ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress. Proc Natl Acad Sci USA 98:4397–4402 Hanson TE, Tabita FR (2003) Insights into the stress response and sulfur metabolism re vealed by proteome analysis of a Chlorobium tepidum mutant lacking the Rubisco-lik e protein. Photosynth Res 78:231–248

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Heising S, Richter L, Ludwig W , Schink B (1999) Chlorobium ferr ooxidans sp. no v., a phototrophic green sulfur bacterium that oxidizes ferrous iron in coculture with a “Geospirillum” sp. strain. Arch Microbiol 172:116–124 Imhoff JF (2003) Phylogenetic taxonomy of the f amily Chlorobiaceae on the basis of 16S rRN A and fmo (Fenna-Matthe ws-Olson protein) gene sequences. Intl J Syst Ev ol Microbiol 53:941–951 Imhoff JF, Hiraishi A, Süling J (2005) Anoxygenic phototrophic purple bacteria. In: Brenner DJ, Krieg NR, Staley JT (eds) Bergey’s manual of systematic bacteriology, vol 2, part A, 2nd edn. Springer, New York, pp 119–132 Joint Genome Institute (2007a) Integrated microbial genomes. http://img.jgi.doe.gov. Cited 15 Jan 2007 Joint Genome Institute (2007b) Microbial genomics. http://genome.jgi-psf.or g/mic_cur1.html. Cited 15 Jan 2007 Kumar S, T amura K, Nei M (2004) MEGA3: inte grated softw are for molecular e volutionary genetics analysis and sequence alignment. Brief Bioinformatics 5:150–163 Lippert KD, Pfennig N (1969) Die V erwertung von molekularem Wasserstoff durch Chlorobium thiosulfatophilum. Arch Microbiol 65:29–47 Manske AK, Glaeser J, K uypers MAM, Overmann J (2005) Ph ysiology and phylogeny of green sulfur bacteria forming a monospecif ic phototrophic assemblage at a depth of 100 meters in the Black Sea. Appl Environ Microbiol 71:8049–8060 Méndez-Alvarez S, Pavón V, Esteve I, Guerrero R, Gaju N (1994) Transformation of Chlorobium limicola by a plasmid that confers the ability to utilize thiosulf ate. J Bacteriol 176:7395–7397 National Center for Biotechnology Information (2007) Genomic biology . http://www.ncbi.nlm. nih.gov/Genomes. Cited 15 Jan 2007 Overmann J (2000) The family Chlorobiaceae. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The prokaryotes: an evolving electronic resource for the microbiological community, 3rd edn, release 3.1. Springer, New York Overmann J, Cypionka H, Pfennig N (1992) An e xtremely low-light-adapted phototrophic sulfur bacterium from the Black Sea. Limnol Oceanogr 37:150–155 Pires RH, Lourenço AI, Morais F , T eixeira M, Xa vier AV, Sarai va LM, Pereira IA C (2003) A no vel membrane-bound respiratory comple x from Desulfovibrio desulfuricans A TCC 27774. Biochim Biophys Acta 1605:67–82 Pott AS, Dahl C (1998) Sirohaem sulfite reductase and other proteins encoded by genes at the dsr locus of Chromatium vinosum are involved in the oxidation of intracellular sulfur. Microbiology 144:1881–1894 Quentmeier A, Friedrich CG (2001) The cysteine residue of the SoxY protein as the active site of protein-bound sulfur oxidation of Paracoccus pantotrophus GB17. FEBS Lett 503:168–172 Reinartz M, Tschäpe J, Brüser T, Trüper HG, Dahl C (1998) Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum. Arch Microbiol 170:59–68 Sander J, Engels-Schwarzlose S, Dahl C (2006) Importance of the DsrMKJOP complex for sulfur oxidation in Allochromatium vinosum and phylogenetic analysis of related complexes in other prokaryotes. Arch Microbiol 186:357–366 Shahak Y, Arieli B, P adan E, Hauska G (1992) Sulf ide quinone reductase (SQR) acti vity in Chlorobium. FEBS Lett 299:127–130 Theissen U, Hoffmeister M, Grieshaber M, Martin W (2003) Single eubacterial origin of eukaryotic sulfide:quinone oxidoreductase, a mitochondrial enzyme conserv ed from the early e volution of eukaryotes during anoxic and sulf idic times. Molec Biol Evol 20:1564–1574 Trüper HG, Lorenz C, Schedel M, Steinmetz M (1988) Metabolism of thiosulf ate in Chlorobium. In: Olson JM, Ormerod JG, Amesz J, Stack ebrandt E, Trüper HG (eds) Green photosynthetic bacteria. Plenum, New York, pp 189–200 van Gemerden H, Mas J (1995) Ecology of phototrophic sulfur bacteria. In: Blankenship RE, Madigan MT, Bauer CE (eds) Anoxygenic photosynthetic bacteria. Kluwer , Dordrecht, pp 49–85

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Verté F , K ostanjevecki V, De Smet L, Me yer TE, Cusano vich MA, V an Beeumen JJ (2002) Identification of a thiosulf ate utilization gene cluster from the green phototrophic bacterium Chlorobium limicola. Biochemistry 41:2932–2945 Vogl K, Glaeser J, Pf annes KR, Wanner G, Ov ermann J (2006) Chlorobium chlorochromatii sp. nov., a symbiotic green sulfur bacterium isolated from the phototrophic consortium “Chlorochromatium aggregatum”. Arch Microbiol 185:363–372 Ward DM, Ferris MJ, Nold SC, Bateson MM (1998) A natural vie w of microbial biodi versity within hot spring cyanobacterial mat communities. Microbiol Mol Biol Re v 62:1353–1370 Widdel F, Schnell S, Heising S, Ehrenreich A, Assmus B, Schink B (1993) Ferrous iron oxidation by anoxygenic phototrophic bacteria. Nature 362:834–836

Chapter 7

Differential-Expression Proteomics for the Study of Sulfur Metabolism in the Chemolithoautotrophic Acidithiobacillus ferrooxidans Lissette Valenzuela, An Chi, Simón Beard, J effrey Shabanowitz, Donald F. Hunt, Carlos A. Jerez Abstract Acidithiobacillus ferr ooxidans obtains its ener gy from the oxidation of ferrous iron, elemental sulfur , or partially oxidized sulfur compounds. The ability of this microorganism to solubilize metal sulf ides is successfully applied in biomining operations. Genomic, metagenomic, and high-throughput proteomic studies of the global regulatory responses that biomining microor ganisms use to adapt to their changing environment are just be ginning to emerge. To further study some of the components involved in sulfur metabolism, differential expression proteomics of total periplasmic proteins was done by high-resolution LTQ FT ion trap mass spectrometry. Of 216 proteins found in the periplasm, several of them changed their le vels of synthesis during growth of A. ferrooxidans ATCC 23270 in thiosulf ate, elemental sulfur and ferrous iron. Thirty-four percent of them corresponded to unkno wn proteins. F orty-one proteins were exclusively present in sulfur-grown cells and 14 in thiosulfate-grown cells. The putative genes coding for these proteins were localized in the a vailable genomic sequence of A. ferrooxidans ATCC 23270. The genomic conte xt around se veral of these genes suggests their in volvement in sulfur metabolism and possibly in sulfur oxidation and formation of FeS clusters. Man y of the periplasmic proteins changing their expression during growth in sulfur compounds may ha ve important roles yet to be described in the sulfur metabolism of this acidophilic microorganism. This knowledge will eventually help to improve mineral bioleaching processes.

7.1 Intr oduction Acidithiobacillus ferrooxidans is a chemolithoauthotrophic bacterium that obtains its energy from the oxidation of ferrous iron, elemental sulfur or partially oxidized sulfur compounds (Olson et al. 2003; Harrison 1984; Lundgren 1980; Suzuki 2001; Rawlings 2002). Effective tools for the study of A. ferrooxidans genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary le vel. An ef ficient and reproducible technique for DN A transfer is still missing (V alenzuela et al. 2006; Rawlings 2005). 77 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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A. ferrooxidans was the first biomining microorganism to have its genome entirely sequenced and the annotation of all its genes has recently been made a vailable (J. Craig Ventner Institute 2007). This information has been v ery useful to man y researchers to look for the genome-wide candidate genes for important metabolic pathways and several important physiological functions and to predict for the functions of many new genes. The main focus of research has been the ener gy metabolism which is directly responsible of bioleaching. Some researchers used “chromosome w alking” to f ind genes in volved in sulfur and iron metabolisms (Valenzuela et al. 2006; Ra wlings 2005). Genomic, metagenomic, and highthroughput proteomic studies of the global re gulatory responses that biomining microorganisms use to adapt to their changing en vironment are just be ginning to emerge (Valenzuela et al. 2006). In this chapter, we will concentrate specifically on proteomic analysis of A. ferrooxidans to better understand its sulfur metabolism. This knowledge together with that obtained in other bioleaching microor ganisms will allow future improvements in industrial bioleaching processes.

7.2

Sulfur Metabolism in A. ferrooxidans

The aerobic oxidation of elemental sulfur by A. ferrooxidans and other microorganisms is carried out by a sulfur dioxygenase (Rohwerder et al. 2003; Rohwerder and Sand 2003; Silv er and Lundgren 1968a; Müller et al. 2004; Sugio et al. 1987). Recently, thiosulfate has been postulated as a key compound in the oxidation of the sulfur moiety of p yrite (Schippers and Sand 1999). Iron(III) ions are exclusively the oxidizing agents for the dissolution. Thiosulf ate would be consequently degraded in a cyclic process to sulfate, with elemental sulfur being a side product. This explains why only Fe(II) ion oxidizing bacteria are capable of oxidizing these metal sulfides (Schippers and Sand 1999). All reactions comprising this oxidation ha ve been sho wn to occur chemically (Sand et al. 1995, 2001). However, sulfur -compound-oxidizing enzymes such as the tetrathionate hydrolase of A. ferrooxidans, A. thiooxidans , or the former Thiobacillus acidophilus (now renamed Acidiphilium acidophilum) may also be involved in the process (De Jong et al. 1997; K elly et al. 1997; Suzuki 1999; Friedrich et al. 2001). In addition, enzymes for thiosulfate or sulfite oxidation of A. ferrooxidans or A. thiooxidans may successfully compete with the chemical reactions with iron(III) ions as an oxidizing agent (Schippers and Sand 1999).

7.3 Pr oteomics of A. ferrooxidans Grown in Sulfur Compounds Two-dimensional polyacrylamide gel electrophoresis (2D P AGE) in combination with mass spectrometry is currently the most widely used technology for comparative bacterial proteomic analysis (Gygi et al. 2000). A set of A. ferrooxidans ATCC

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19859 proteins dif ferentially expressed when gro wn in metal sulf ides, thiosulfate, elemental sulfur, and ferrous iron media were characterized by using 2D P AGE (Ramirez et al. 2004). N-terminal amino acid sequencing and tandem mass spectrometry analysis of these proteins allo wed the identif ication and localization of their corresponding genes in the a vailable genomic sequence of A. ferrooxidans ATCC 23270. The genomic conte xt around se veral of these genes suggests their involvement in the ener gy metabolism of A. ferrooxidans. Two groups of proteins could be distinguished. Proteins highly upregulated by growth on sulfur compounds but downregulated by growth on ferrous iron are particularly interesting, such as a 44-kDa outer -membrane protein, an e xported 21-kDa putati ve thiosulf ate sulfur transferase protein, a 33-kDa putati ve thiosulf ate/sulfate binding protein, and a 45-kDa putati ve capsule polysaccharide e xport protein (WcbC). Polysaccharides may play a role in the adherence capability of bacteria to solid surf aces. In this regard, it is kno wn that most leaching bacteria gro w attached to the surf ace of the solid substrates such as elemental sulfur and metal sulf ides. On the other hand, A. ferrooxidans proteins that are downregulated when growing on sulfur but upregulated when growing on ferrous iron were also analyzed by 2D PAGE (Ramirez et al. 2004). These include rustic yanin, a cytochrome c552, a putative phosphate binding protein (PstS), the small and large subunits of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco), and a 30-kDa putati ve CbbQ protein, amongst others. These results suggested a separation of the iron and sulfur utilization pathways. Rusticyanin in addition of being highly e xpressed on ferrous iron was also newly synthesized as determined by metabolic labeling, although at lower le vels during gro wth on sulfur compounds and iron-free metal sulf ides (Ramirez et al. 2004). These results were in agreement with those of Yarzabal et al. (2004). The capacity of A. ferrooxidans to oxidize thiosulfate and tetrathionate was found to be inhibited by the presence of ferrous iron (Das et al. 1993). Ho wever, during the gro wth of A. ferr ooxidans on iron-containing metal sulf ides, such as pyrite and chalcopyrite, we found elevated expression of proteins involved in both ferrous iron and sulfur compound utilization, indicating that the tw o ener gygenerating pathw ays are simultaneously induced depending on the type and the concentration of the available oxidizable substrates (Ramirez et al. 2004). In agreement with these results, it was previously suggested that A. ferrooxidans can simultaneously utilize both ferrous iron and elemental sulfur as ener gy sources (Espejo and Romero 1987).

7.4

Thiosulfate Sulfur Transferases from A. ferrooxidans

Rhodanese acti vity has been pre viously reported in A. ferr ooxidans (Tabita et al. 1969). This enzyme is a thiosulf ate:cyanide sulfur transferase (TST), which breaks the S–S bond present in thiosulfate, generating sulfur and sulfite. Other enzymes may also participate in the mechanism proposed by Schippers and Sand (1999), such as the thiosulfate-oxidizing enzyme of A. ferrooxidans (Silver and Lundgren 1968b).

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Fig 7.1 Possible cellular localization and in vitro thiosulf ate:cyanide sulfur transferase activities of rhodanese-lik e proteins from Acidithiobacillus ferr ooxidans. The genes coding for proteins P14, P15, P16, P16.2, and P21 were cloned and expressed in Escherichia coli and the thiosulfate: cyanide sulfur transferase activities were determined. Activities were in the low (+) to high (+++) ranges or were absent (−). The presence of signal peptide in P21 is also indicated ( tilde)

Recently, new rhodanese-like proteins were identif ied, the expression of which is re gulated depending on the gro wth substrate and is probably related to sulfur metabolism and/or oxidation (Acosta et al. 2005). Eight nucleotide sequences containing a single rhodanese domain are present in the genome of A. ferrooxidans ATCC 23270 (Fig. 7.1): p11, p14, p14.3, p15, p16, p16.2, p21, and p28 (Acosta et al. 2005). Amino acid sequence comparisons of all eight proteins allo wed us to identify the potential catalytic cysteine residues and other highly conserved rhodanese family features. The genomic conte xts of some of the rhodanese-lik e genes suggested their implication in sulfur oxidation and metabolism, formation of FeS clusters and detoxification mechanisms. Several of the putati ve rhodanese genes were successfully isolated, cloned, and o verexpressed in Escherichia coli and their TST and 3-mercaptop yruvate:cyanide sulfur transferase (MST) acti vities were determined. On the basis of their sulfur transferase acti vities and structural comparisons of catalytic sites and electrostatic potentials between homologymodeled A. ferrooxidans rhodaneses and the reported crystal structures of E. coli GlpE (TST) and SseA (MST) proteins, tw o of the rhodanese-lik e proteins, P15 and P16.2, could clearly be def ined as TSTs, and P14 and P16 could possibly correspond to MSTs (Acosta et al. 2005). Ne vertheless, several of the eight A. ferrooxidans rhodanese-like proteins may ha ve some dif ferent functional acti vities yet to be discovered. The comparison of messenger RN A (mRNA) abundance of some of the genes involved in sulfur metabolism in A. ferr ooxidans gro wn on dif ferent oxidizable substrates was started by performing a preliminary pilot DN A macroarray formed

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Fig. 7.2 Analysis of cotranscription of modA2 and doxDA2 genes of A. ferrooxidans by reversetranscription (RT) PCR. A Map of the positions of the genes in the genomic conte xt of p21. Arrows indicate locations of primers used for the R T and PCR reactions. B Agarose gel electrophoresis of RT-PCR products. The primers used in each reaction are numbered. The R T reaction was carried out on 3 µg of total RNA obtained from thiosulfate-grown cells of A. ferrooxidans. RT reactions with (+ lanes) and without ( − lanes) the Moloney murine leukemia virus reverse transcriptase enzyme were carried out in order to e xclude amplification due to genomic DN A contamination. A control with genomic DNA was also included (C). Sizes of DNA markers are shown on the left of each gel. Expected sizes (in base pairs) for the corresponding R T-PCR products are given at the bottom of the gels

with 70 dif ferent genes (Acosta et al. 2005). As already mentioned, the gene p21 codes for a putati ve thiosulf ate sulfur transferase protein and all putati ve genes upstream of it ha ve been found to form a cluster (Ramirez et al. 2002, 2004). The y were all highly expressed in cells grown on sulfur compared with the le vels seen on ferrous iron (Acosta et al. 2005). This clearly supports our pre vious proposal based on proteomic analysis that the rhodanese-like gene p21 forms part of a group of genes related with sulfur oxidation (Ramirez et al. 2002). In addition, the DNA macroarray results obtained for p21 were v alidated by our pre vious reverse-transcription PCR studies, indicating the induced e xpression of p21 by gro wth on sulfur compounds both at the transcriptional (Acosta et al. 2005) and at the translational levels (Ramirez et al. 2002, 2004). Unlike cytoplasmic rhodaneses, P21 was located in the periphery of A. ferrooxidans cells (Fig. 7.1) and w as regulated depending on the oxidizable substrate. If P21 and some of the proteins coded by its adjacent genes (Fig. 7.2a) are involved in thiosulfate metabolism, one should e xpect an increased e xpression of these proteins when the cells are grown on p yrite, thiosulfate, or sulfur, as we ha ve observed by proteomics (Ramirez et al. 2002). However, we could not detect an in vitro TST activity for purified P21 (Ramirez et al. 2004). Protein P21 may not be a periplasmic rhodanese enzyme but rather part of a possible comple x in charge of thiosulf ate oxidation.

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This putative complex could be dif ferent from the Sox model proposed for sulfur oxidation in man y bacteria (Friedrich 1998) since we did not f ind an y sox-like genes in the genome of A. ferrooxidans (Ramirez et al. 2004).

7.5

Other Proteins Involved in Sulfur Metabolism

Genes modA1 and modA2 form a part of the genomic conte xt of p21 (Fig. 7.2a). Proteins ModA1 and ModA2 may be a part of the A TP-binding cassette (ABC) superfamily of transporters (Self et al. 2001). These transport systems are in volved in both uptake and efflux and have different substrate specificities. Structural modeling of ModA proteins with crystal structures of kno wn similar proteins strongly suggests a conserv ed functional mechanism for the transport of thiosulf ate/sulfate or molybdate in A. ferrooxidans. Previously, we found that modA1 is cotranscribed with p21 and a putati ve thiosulf ate:quinone oxidoreductase (TQR) ( doxDA1) (Ramirez et al. 2004). modA1 is upregulated both in sulfur and thiosulfate, whereas the mRNA of ModA2 is e xpressed only in cells gro wn on thiosulf ate and it is not cotranscribed with doxDA2 (Fig. 7.2b). By e xpression proteomics (Fig. 7.3) it is clear that ModA2 is synthesized in much higher amounts in cells grown on thiosulfate. On the other hand, this protein appears entirely repressed in cells gro wn on sulfur or iron. The almost absent expression of ModA1 and ModA2 on ferrous iron suggests that these putati ve

Fig. 7.3 ModA2 is synthesized in higher le vels in thiosulf ate-grown A. ferrooxidans. A Total proteins from A. ferrooxidans ATCC 23270 gro wn in thiosulf ate were separated by 2D sodium dodecyl sulf atepolyacrylamide gel electrophoresis (P AGE). A rectangular section containing protein ModA2 is indicated. B The sections where ModA2 migrates in 2D P AGE are compared for the total proteins of A. ferrooxidans grown in thiosulf ate (T), ferrous iron ( Fe), or elemental sulfur (S). The proteins were separated by 2D nonequilibrium pH gel electrophoresis with a pH gradient between 3.0 (right side of the g el) and 10.0 (left side of the g el). Spots were detected by silver staining and analyzed by Delta 2D version 3.3 software. The positions of spots corresponding to ModA2 are indicated by circles. Molecular mass standards (in kilodaltons) are seen on the left of the g els. Numbers on the right of the sections indicate the relati ve intensity v olumes of the circled spots with respect to all detected spots in the corresponding gels

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Table 7.1 Real-time PCR quantitation of the levels of gene e xpression of doxDA1 and doxDA2 in cells grown in different oxidizable substrates Ratio doxDA1 doxDA2 Sulfur/Fe(II) 2.5 3.8 Thiosulfate/Fe(II) 6.7 3.3 Pyrite/Fe(II) 10.8 6.7 Unpublished results. The experiments were performed twice with both independent total RN A and complementary DN A preparations. V alues sho wn are the average of these experiments.

transporters may function more likely as periplasmic thiosulfate or molybdate binding proteins in this acidophile. A. ferrooxidans harbors duplicated doxDA genes (Fig. 7.2a) that are homologous to the genes encoding TQR in A. ambivalens, an enzyme oxidizing thiosulfate with tetrathionate as a product and ferricyanide or decylubiquinone as electron acceptors (Müller et al. 2004). As pointed out v ery recently by Friedrich et al. (2005), this gene duplication points to a yet undemonstrated significance in thiosulfate metabolism in A. ferrooxidans. doxDA1 and doxDA2 are in the same genomic conte xt of p21 (Fig. 7.2) and as seen in T able 7.1, their e xpression determined by real-time PCR is enhanced se veral fold in cells gro wn on elemental sulfur or on thiosulf ate compared with the expression levels of cells grown on ferrous iron. Similar behavior was seen by DNA macroarray analysis in the case of doxDA1 (Acosta et al. 2005). The upre gulated e xpression of the doxDA genes may be of particular rele vance during the degradation of the mineral pyrite, where much higher levels of transcription are seen (T able 7.1). In agreement with these results, thiosulf ate is an important intermediate during p yrite oxidation (Schippers and Sand 1999). These results support the signif icance of the tw o doxDA genes in thiosulf ate metabolism in A. ferrooxidans and are in f avor of the idea that acidophilic bacteria oxidize sulfur compounds by a system different from the Sox enzyme system.

7.6 High-Throughput Proteomics of Periplasmic Proteins Induced by Growth of A. ferrooxidans on Sulfur Compounds Several of the proteins involved in sulfur and iron oxidation have been described as forming part of the periplasm of A. ferrooxidans; therefore, to further study some of the components involved in sulfur metabolism, differential proteomic analysis of total periplasmic proteins w as performed using high-resolution L TQ FT ion trap mass spectrometry.

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We identified 216 proteins in the periplasm of A. ferrooxidans ATCC 23270, several of them changing their levels of synthesis when growing the bacterium on thiosulfate, elemental sulfur, and ferrous iron media (Fig. 7.4). Thirty-four percent of them corresponded to unkno wn proteins. F orty-one proteins were e xclusively present in sulfur-grown cells and 14 in thiosulfate-grown cells (unpublished results). The putati ve genes coding for all the proteins were localized in the a vailable genomic sequence of A. ferrooxidans ATCC 23270. The genomic conte xt around several of these genes suggests their in volvement in sulfur metabolism and possibly in sulfur oxidation and formation of FeS clusters. Some of the periplasmic proteins were upre gulated by growth on sulfur compounds, for e xample, the exported P 21, the ModA1 and ModA2 putative transporters, and a sulfide:quinone oxidoreductase.

Fig. 7.4 Distribution of periplasmic proteins from cells of A. ferrooxidans ATCC 23270 grown in different oxidizable substrates

Fig. 7.5 A speculative working model for sulfur oxidation in A. ferrooxidans. Proteins resulting from our studies are shaded. (Data tak en and adapted from Ra wlings 2005 and Rohwerder and Sand 2003)

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A summary of our findings and those from other researchers are put together in a speculati ve w orking model for sulfur -compound oxidation in A. ferr ooxidans (Fig. 7.5). Many of the proteins changing their e xpression during growth in sulfur compounds may have important roles yet to be described in the sulfur metabolism of this acidophilic microorganism.

7.7 Conclusions ●

●

●

●

Proteomics is a po werful tool for the study of dif ferential e xpression of microorganisms such as A. ferrooxidans. Acidophilic chemolithoautotrophs such as A. ferrooxidans do not possess a Sox system, and therefore should oxidize thiosulf ate by other means. During the oxidation of a metal sulf ide such as p yrite, the two doxDA genes of A. ferr ooxidans are upre gulated, in agreement with the proposed thiosulf ate mechanism for pyrite oxidation. A. ferr ooxidans has a thiosulf ate dehydrogenase acti vity. It remains to be demonstrated whether this activity belongs to the DoxDA proteins.

References Acosta M, Beard S, Ponce J, V era M, Mobarec JC, Jerez CA (2005) Identif ication of putati ve sulfurtransferase genes in the e xtremophilic Acidithiobacillus ferr ooxidans A TCC 23270 genome: structural and functional characterization of the proteins. OMICS 9:13–28 Das A, Mishra AK, Ro y P (1993) Inhibition of thiosulf ate and tetrathionate oxidation by ferrous iron in Thiobacillus ferrooxidans. FEMS Microbiol Lett 112:67–72 De Jong GAH, Hazeu W , Bos P , K uenen G (1997) Polythionate de gradation by tetrathionate hydrolase of Thiobacillus ferrooxidans. Microbiology 143:499–504 Espejo R T, Romero P (1987) Gro wth of Thiobacillus ferr ooxidans on elemental sulfur . Appl Environ Microbiol 1907–1912 Friedrich CG (1998) Ph ysiology and genetics of sulfur -oxidizing bacteria. Adv Microb Ph ysiol 39:235–289 Friedrich CG, Rother D, Bardischewsky F, Quentmeier A, Fischer J (2001) Oxidation of reduced inorganic sulfur compounds by bacteria: emer gence of a common mechanism? Appl En viron Microbiol 67:2873–2882 Friedrich CG, Bardische wsky F, Rother D, Quentmeier A, Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Microbiol 8:253–259 Gygi SP, Corthals GL, Zhang Y , Rochon Y, Aebersold R (2000) Ev aluation of tw o-dimensional gel electrophoresis-based proteome analysis technology . Proc Natl Acad Sci USA 97: 9390–9395 Harrison AP (1984) The acidophilic Thiobacilli and other acidophilic bacteria that share their habitat. Annu Rev Microbiol 38:265–92 J. Craig Ventner Institute (2007) The new JCVI. http://www.tigr.org. Cited 16 Jan 2007 Kelly DP , Sher gill JK, Lu W -P, W ood AP (1997) Oxidati ve metabolism of inor ganic sulfur compounds by bacteria. Antonie Van Leeuwenhoek 71: 95–107 Lundgren DG (1980) Ore leaching by bacteria. Annu Re v Microbiol 34:263–283

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Müller FH, Bandeiras TM, Urich T , Teixeira M, Gomes CM, Kletzin A (2004) Coupling of the pathway of sulphur oxidation to dioxygen reduction: characterization of a no vel membranebound thiosulphate:quinone oxidoreductase. Mol Microbiol 53:1147–1160 Olson GJ, Brierle y JA, Brierley CL (2003) Bioleaching re view part B: progress in bioleaching: applications of microbial processes by the minerals industries. Appl Microbiol Biotechnol 63:249–257 Ramirez P, Toledo H, Guiliani N, Jerez CA (2002) An exported rhodanese-like protein is induced during growth of Acidithiobacillus ferrooxidans in metal sulf ides and dif ferent sulfur compounds. Appl Environ Microbiol 68:1837–1845 Ramirez P, Guiliani N, V alenzuela L, Beard S, Jerez CA (2004) Dif ferential protein e xpression during growth of Acidithiobacillus ferrooxidans on ferrous iron, sulfur compounds, or metal sulfides. Appl Environ Microbiol 70:4491–4498 Rawlings DE (2002) Heavy metal mining using microbes. Annu Re v Microbiol 56:65–91 Rawlings DE (2005) Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microb Cell F act 4:13 Rohwerder T, Sand W (2003) The sulfane sulfur of persulfides is the actual substrate of the sulfuroxidizing enzymes from Acidithiobacillus and Acidiphilium spp. Microbiology149:1699–1709 Rohwerder T , Gehrk e T , Kinzler K, Sand W (2003) Bioleaching re view part A: progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation. Appl Microbiol Biotechnol 63:239–248 Sand W, Gehrke T, Hallmann R, Schippers A (1995) Sulfur chemistry, biofilm, and the (in)direct attack mechanism – a critical e valuation of bacterial leaching. Appl Microbiol Biotechnol 43:961–966 Sand W, Gehrke T, Jozsa PG, Schippers A (2001) (Bio)chemistry of bacterial leaching-direct vs. indirect bioleaching. Hydrometallurgy 59:159–175 Schippers A, Sand W (1999) Bacterial leaching of metal sulfides proceeds by two indirect mechanisms via thiosulfate or via polysulfides and sulfur. Appl Environ Microbiol 65:319–321 Self WT, Grunden AM, Hasona A, Shanmug am, KT (2001) Molybdate transport. Res Microbiol 152:311–321 Silver M, Lundgren DG (1968a) Sulfur -oxidizing enzyme of Ferrobacillus ferr ooxidans (Thiobacillus ferrooxidans). Can J Biochem 46:457–461 Silver M, Lundgren DG (1968b) The thiosulf ate-oxidizing enzyme of Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans). Can J Biochem 46:1215–1220 Sugio T, Mizunashi W, Inagaki K, Tano T (1987) Purification and some properties of sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans. J. Bacteriol 169:4916–4922 Suzuki I (1999) Oxidation of inorganic sulfur compounds: chemical and enzymatic reactions. Can J Microbiol 45:97–105 Suzuki I (2001) Microbial leaching of metals from sulf ide minerals. Biotechnol Adv 19: 119–132 Tabita R, Silv er M, Lundgren DG (1969) The rhodanese enzyme of Ferrobacillus ferrooxidans (Thiobacillus ferrooxidans). Can J Biochem 47:1141–1145 Valenzuela L, Chi A, Beard S, Orell A, Guiliani N, Shabano witz J, Hunt DF , Jerez CA (2006) Genomics, metagenomics and proteomics in biomining microor ganisms. Biotechnol Adv 24:197–211 Yarzabal A, Appia-Ayme C, Ratouchniak J, Bonnefo y V (2004) Re gulation of the e xpression of the Acidithiobacillus ferr ooxidans rus operon encoding tw o c ytochromes c, a c ytochrome oxidase and rusticyanin. Microbiology 150:2113–2123

Chapter 8

Sulfur and Light? History and “Thiology” of the Phototrophic Sulfur Bacteria Hans G. Trüper

Abstract This chapter describes how our present knowledge of sulfur metabolism of phototrophic sulfur bacteria accumulated through se veral major steps of experimental progress. Among these are the following: discovery of microbial cells with conspicuous inclusions (“purple” bacteria and colorless ones); verification that such inclusions consist of sulfur; detection of phototactic behavior in purple bacteria; enrichment cultures (“Winogradsky columns”), consequent detection of sulfide requirement and of the liquid stage of sulfur inclusions, “glob ules”; identification of the pigments as bacteriochlorin (no w chlorophylls) and bacterioerythrin (no w carotenoids); discovery of Chlorobium, a green bacterium that deposits sulfur globules outside its cells; disco very of photosynthesis in purple non-sulfur bacteria; postulation of a photosynthetic metabolism in purple sulfur bacteria by combination of photosynthesis and chemosynthesis; e vidence that the red (“purple”) and green sulfur bacteria perform anaerobic photosynthesis (carbon dioxide f ixation) dependent on the oxidation of reduced sulfur compounds; disco very of Ectothiorhodospira, a halophilic phototrophic purple sulfur bacterium that deposits sulfur globules outside its cells; systematic de velopment of enrichment and pure culture media and techniques for the Chromatiaceae and Chlorobiaceae; the f irst specific studies on sulfur metabolism in purple bacteria on whole cells and crude extracts; purification of adenosine 5′-phosphosulfate reductase and reverse siroheme sulfite reductase; f inding and purif ication of sulf ide quinone reductase; disco very and characterization of the dsr gene cluster in Allochromatium vinosum; isolation and gene sequence of the sulfur glob ule encoating periplasmic proteins in Chromatiaceae; determination of the inner structure of the sulfur globules by X-ray absorption near -edge spectroscop y in phototrophic and chemotrophic sulfur bacteria; characterization of sulfite oxidoreductase; characterization of the thiosulfate-oxidizing (Sox) multienzyme complex in Allochromatium vinosum.

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8.1 Intr oduction The chemical element sulfur (L. sulfur or sulfur, Gr. thion) as a material that naturally occurs in v olcanic areas, in calcite and gypsum deposits and at certain beaches has been known to humans since prehistoric times and – as it b urns with an unusual blue flame and a stinging smell – has been ascribed magic po wers as well as connections to de vilish underground spirits and hell. Only since 1870 has it been known that considerable amounts of elemental sulfur may occur in li ving beings as well.

8.2

Discovery of Sulfur-Oxidizing Microorganisms

Since 1786 (Müller 1786, in Denmark) se veral microorganisms (then considered either animals or plants, animalcula or infusoria) have been described to contain intracellular conspicuous light-refringent “granules” of unkno wn composition. Christian Gottfried Ehrenber g (1838) described the disco very of the f irst purple sulfur bacteria “Monas okenii” and “Ophidomonas jenensis” in 1836 with the following words (here translated into English): In 1836, 18 September, the day of the constitutional opening of the 14th meeting of the German Association of Naturalists (in Jena), founded by (Ludwig) Oken, during an excursion that I had undertaken with Professor Weisse I found — somewhat below the church in Ziegenhain – this beautifully red monad in considerable amounts in a small basin of the village creek. It formed handwide red patches, and between its le gions in considerable numbers Ophidomonas jenensis occurred, a ne w genus of armored monads, together with Euglena viridis and Spirogyra. During favorable development this form can easily cause a v ery intensive blood color in the stagnant water. The animalcules collected on 18 September in a little bottle, which I demonstrated to the zoological section of the naturalists convention in Jena, still e xisted in small numbers on 11 December in Berlin, and while I am writing this I ha ve them right beside me together with Ophidomonas alive under the microscope. After Ehrenberg’s description of the two organisms was published in 1838, in the following years further forms were described. The Italian botanist T revisan (1842) described the lar ge f ilamentous Beggiatoa with similar conspicuous inclusions occurring in the sulfur springs of the Euganean Hills west of Padua. The Swiss botanist Maximilian Perty (1852) introduced the genus name Chromatium and described the species Chromatium vinosum, C. weissei, C. violascens and C. erubescens, however, without making attempts to isolate them or to study their nutritional physiology. He just called them “pigment bacteria. ” The British biologist Ray Lankester (1873) described the reddish microbial mats on the mud surf ace in ponds as Bacterium rubescens and for the f irst time classified them as Bacteria; he called the characteristic red pigment “bacteriopurpurin. ” The Danish botanist

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Eugen Warming (1875) consequently coined the term “purple bacteria” for these organisms and added further ne w species. The f irst scientist who started to experiment be yond the predominantly simple descriptions w as Theodor W . Engelmann (1883), who disco vered and described in 1883 the light-e xcitation reactions of purple bacteria. W orking with Bacterium photometricum (probably Allochromatium vinosum) crude cultures, he found that these bacteria assemble in the light in the so-called Engelmann’s light trap, thus finding a behavior similar to that of green algae, where he had found a correlation between phototaxis and photosynthesis before (Dre ws 2005). At that time to biologists oxygen production in the light meant photosynthesis. Although he believed in it, Engelmann was not able to pro ve oxygen production in purple bacteria in a con vincing manner.

8.3

Identification of Conspicuous Inclusions as Sulfur

It had also been recognized that some of the microbes containing the conspicuous inclusions were purple or red in color , while others were not (T able 8.1). The Swiss botanists Cramer and Meyer-Ahrens in 1870 (Müller 1870) and Ferdinand Cohn (1872, 1875), the German botanist founder of bacterial systematics, in 1872 and in 1875 were the first who unequivocally proved that such cellular inclusions of Beggiatoa and of the colored microbes, respecti vely, consisted of elemental sulfur. Cramer and Me yer-Ahrens spok e of “granules” and considered their strong refraction as a sign for their solid state, while Cohn spoke of granules and crystals. Cohn considered the occurrence of elemental sulfur as “a singular phenomenon in the plant w orld” and although studies of en vironmental samples in the laboratory by himself as well as by Ser gei N. W inogradsky (1887) sho wed that these organisms depended on sulfide in their aquatic medium both hypotheses brought forward for this dependence could not really be pro ven: on one hand it was believed that these organisms reduced sulfate under production of hydrogen sulfide and elemental sulfur; on the other it was thought that they oxidized hydrogen sulfide to elemental sulfur. Winogradsky was the first to realize the nonsolid nature of the sulfur inclusions and called them “globules.” He observed that only in dead cells the sulfur transformed into crystals. Much later (Winogradsky 1949) he wrote that it w as easy to pro ve that the inclusions in Beggiatoa cells are not in a solid stage: “ En ef fect il est facile de demontr er qu’elles sont de consistance sirupeuse en c hauffent des f ilaments bourrés de soufr e a 70° dans un peu d’eau toute les inclusions qui r emplissent la cellule des f ilaments ne tardent pas alor s à confluer en formant une seule gr osse goutte par cellule .” As a consequence one should not speak of “granules” b ut of “droplets” or better “glob ules” of sulfur . Actually it is the global shape that causes the strong light refringence, as one can easily observe by comparing them with microscopically small air or gas b ubbles. Winogradsky considered the metabolism of purple sulfur bacteria as a type of chemolithotrophy like in Beggiatoa, where he had observed the oxidation of

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H.G. Trüper Table 8.1 Genera of conspicuous or prominent colored and colorless sulfur bacteria. (List incomplete after 1900. Data from Buchanan and Gibbons 1974) 1786 “Animalcula infusoria” O.F. Müller = Monas (muelleri) 1875 Warming = Thiovulum muelleri (=majus) 1913 Hinze (CSB) 1833 Micraloa Kützing = Lamprocystis 1886 Schroeter (PSB) 1838 Ophidomonas Ehrenberg = Thiospirillum 1888 Winogradsky (PSB) 1838 Monas (okenii) Ehrenberg = Chromatium 1852 Perty (PSB) 1842 Beggiatoa Trevisan = Beggiatoa (CSB) 1865 Beggiatoa (nivea) Rabenhorst = Thiothrix 1888 Winogradsky (CSB) 1887 Spirillum (rubrum) Esmarch = Rhodospirillum 1907 Molisch (PB) 1888 Thiocystis Winogradsky (PSB) 1888 Amoebobacter Winogradsky (PSB) 1888 Thiodictyon Winogradsky (PSB) 1888 Thiothece Winogradsky (PSB) 1888 Thiopedia Winogradsky (PSB) 1888 Thiocapsa Winogradsky (PSB) 1888 Rhabdochromatium Winogradsky (PSB) 1888 Thiopolycoccus Winogradsky (PSB) 1888 Thiosarcina Winogradsky (PSB) 1893 Achromatium (oxaliferum) Schewiakoff (CSB) 1902 (Thiobacillus thioparus) Nathanson; 1904 Thiobacillus Beijerinck (CSB) 1905 Thiospirillum winogradskyi Omelianski = Thiospira 1914 Visloukh (CSB) 1906 Chlorobium Nadson (green PSB) 1907 Thioploca Lauterborn (CSB) 1912 Bacterium (bovista) Molisch = Thiobacterium 1924 Janke (CSB) 1915 Achromatium (mobile) Lauterborn = Macromonas 1924 Utermöhl, Koppe 1936 Ectothiorhodospira Pelsh (PSB) 1999 Thiomargarita Schulz et al. (CSB) Entries gi ven as year of description, name of or ganism, name of author and subsequent nomenclaturalchanges. CSB chemolithotrophic sulfur bacterium, PSB phototrophic sulfur bacterium, PB phototrophic non-sulfur bacterium.

hydrogen sulfide to sulfur and sulfate in the dark under consumption of molecular oxygen. He realized that in purple bacteria this process apparently occurred under anaerobic conditions, and concluded that the necessary oxygen w ould apparently be pro vided by a light-dependent splitting of w ater. In principle, however, he considered the metabolism of colorless bacteria and that of purple sulfur bacteria to be identical. By 1904 it had become clear through the w ork of Nadson (1903) and Arcichowskij (1904) that the pigments of the purple bacteria consisted of two different types, a green component, then called bacteriochlorin (today called chlorophylls) and a red component, then called bacterioerythrin (today called carotenoids). Still these days, lar ge-cell bacteria with conspicuous sulfur glob ule inclusions may be newly found, as the disco very of the rather huge bacterium Thiomargarita by Schulz et al. (1999) showed.

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8.4 Enrichment Cultures – First Taxonomy – and the Question of Photosynthesis An important step forw ard w as the introduction of enrichment cultures in glass cylinders; these were only much later called “W inogradsky columns” after their inventor. These allo wed the maintenance of enrichment cultures of colored sulfur bacteria in the laboratory over longer periods of time. After Erwin v on Esmarch (1887) had established the f irst pure culture of a “Spirillum rubrum ,” Hans Molisch performed a lar ge study on purple bacteria, included this or ganism in his studies and renamed it Rhodospirillum. Molisch applied varieties of Winogradsky colums to enrich so-called non-sulfur purple bacteria as well. He discovered, isolated and described a larger number of new genera and species of purple bacteria, ho wever only of purple non-sulfur bacteria (that he called Athiorhodaceae), which – as he found out – assimilate organic substances in the light. He had detected a new type of photosynthesis which we now call photoorganoheterotrophy, and ascribed the two pigments roles similar to those of the chlorophyll and the carotenoids in carbon dioxide assimilation of green plants (Molisch 1907). But Molisch never studied purple sulfur bacteria! Summarizing his studies on sulfur bacteria, W inogradsky (1887) produced a paper of 73 pages reporting numerous – from our present view – simple little experiments and observations, which were helpful to de velop a first view on these organisms. In this paper he also proposed a first taxonomic scheme for the sulfur bacteria (Sulfobacteria), which he simply subdivided into colorless and red sulfobacteria, the former group represented by the tw o genera Beggiatoa and Thiothrix, the latter by the 11 generaThiocystis, Lamprocystis, Amoebobacter, Thiopolycoccus, Thiodictyon, Thiothece, Thiocapsa, Thiopedia, Chromatium, Rhabdochromatium and Thiospirillum. The differentiating criteria were, as was the tradition of botanists and zoologists, only morphological characters. Johannes Buder (1919) confirmed Engelmann’s theories that the purple bacteria perform a photosynthetic metabolism. He ar gued as follo ws. The dependence of these or ganisms upon or ganic substances or upon hydrogen sulf ide cannot be denied. Living under anaerobic conditions, they are forced to produce the required oxygen themselves. The possession of photosynthesis pigments thus makes possible that an oxidation of organic substances is also possible under anaerobic conditions. Buder made clear that Engelmann, W inogradsky and Molisch had been working with different organisms. He realized that purple bacteria represented a ne w type of metabolism: They assimilate carbon dioxide or organic compounds anaerobically in the light. He thus united Engelmann’ s photosynthesis and W inogradsky’s chemosynthesis. The def inite proof for the phototrophic nature of the purple and green sulfur bacteria, however, as well as the similarities with and the dif ferences from plant photosynthesis came in 1931 from the Dutch American microbiologist Cornelis B. van Niel, w orking in P acific Grove, CA, USA. Using numerous illuminated cultures in closed bottles under the absence of oxygen, v an Niel in long-term

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experiments established the stoichiometric relationship between hydrogen sulf ide oxidation, sulfur and sulfate formation, disappearance of carbonate from the medium and increase in cellular carbon. He realized the surprising similarity with the material turnover in green plant photosynthesis and established the generalized stoichiometric photosynthesis equation: CO2 + 2H2 A ⎯light ⎯⎯ → CH2 O + H2 O + 2 A For the phototrophic sulfur bacteria the reducing agent H2A is hydrogen sulfide (or sulfur or thiosulfate) and for the photosynthesis of plants it is water, with the oxidation products molecular sulfur (and/or sulfate) and molecular oxygen, respectively. Using water labeled with the oxygen isotope 18O, Ruben et al. (1941) pro ved that in plant photosynthesis the O2 formed is indeed derived from water. The similarities between the purple sulfur and non-sulfur bacteria were further strengthened by the finding that species of both groups were able to use molecular hydrogen as the H2A of the general photosynthesis equation (Roelofsen 1934; Gaf fron 1935).

8.5

Pure Cultures of Phototrophic Sulfur Bacteria at Last!

Until the 1960s most of the research on anaerobic phototrophic bacteria aimed at elucidating the mechanisms of photosynthesis, carbon dioxide f ixation, pigment synthesis, phototaxis, dark metabolism and diversity of metabolic physiology were done with the more easily culti vable purple non-sulfur bacteria Rhodospirillum rubrum, Rhodobacter capsulatus and Rhodobacter sphaeroides, or with more or less alle ged pure cultures of Allochromatium (then Chromatium) vinosum. Since 1953 also the green sulfur bacteria (genus Chlorobium) had been cultivated in pure cultures and studied thoroughly by Helge Larsen (1953). The great breakthrough came when Norbert Pfennig in the early 1960s f irst improved Winogradsky columns (by quantif ication of ingredients in the mud, and preincubation in the dark), from which he – step by step – deduced an optimal growth medium for red and green phototrophic sulfur bacteria, “Pfennig’s medium” (consisting of three or four separately sterilized solutions), and introduced the technique of “feeding” with neutralized sulfide (this was necessary because sulfide tolerance turned out to be limited e ven in these sulf ide-requiring bacteria), scre w-capped bottles (the “Pfennig bottle”) instead of the glass-stoppered ones of v an Niel, which easily got contaminated, agar shake dilution series, capillary isolation of single large cells, regular routine controls for contaminating sulf ate-reducing and heterotrophic anaerobic bacteria and man y other tricks. The lar ge-cell purple sulfur bacteria described by Ehrenberg, Perty, Cohn and W inogradsky became cultivable and Pfennig’s medium also turned out to be optimal for the green sulfur bacteria. So Pfennig isolated one after the other of these “old literature bacteria,” and proved, how careful the “old scientists” had percei ved and studied these or ganisms (Pfennig and T rüper 1989). Besides the “feeding technique” the secret of his medium w as that he based it on a

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delicate bicarbonate b uffering system and that the lar ge-cell species as well as man y green bacteria depended upon the availability of vitamin B12, which in the Winogradsky columns had been provided by the anaerobic prokaryotes present in the mud (Schle gel and Pfennig 1961; Pfennig 1961, 1962; Trüper 1970). Practically all the follo wing important research on phototrophic sulfur bacteria owes homage to Pfennig. By in venting the methods for handling these f astidious bacteria he practically opened a “barn door” for us, like Robert Hungate and Ralph Wolfe did for the strict anaerobic rumen bacteria and methanogens, Roger Stanier for the Cyanobacteria, Roger Whittenb ury for the methylotrophs, Karl Stetter for the hyperthermophiles and Fritz Widdel for the sulfate reducers. I am personally glad that I was an eyewitness and coworker during these fruitful years of Pfennig in Göttingen. After that I spent most of my research life until retirement working – together with man y of my students – to elucidate the sulfur metabolism of purple (including Ectothiorhodospira) and green phototrophic sulfur bacteria even by taking detours or side interests such as assimilatory pathw ays in yeasts, sulfur and non-sulfur purple bacteria as well as dissimilatory sulfur metabolism in the anaerobic Thiobacillus denitrif icans and in sulf ate reducers lik e Desulfovibrio and extreme thermophilic Archaeoglobus and Pyrobaculum species. Often the choice of another model or ganism helped to overcome a dead end in our research on phototrophic sulfur bacteria. In those years the progresses in sulfur metabolism research – no matter whether on oxidative or reductive pathways – were due to rapid developments in enzymology, protein chemistry and radioacti ve labeling techniques besides more reliable analytical chemistry of sulfur compounds.

8.6

The Age of Enzymology and Isotope Labeling

The book The Biochemistry of Inorganic Compounds of Sulfur by Roy and Trudinger (1970) presented the state of the art at that time. The bibliography in that book reveals that until it appeared, there existed several highly active groups working on dissimilatory sulfur metabolism in thiobacilli ( sensu lato) or sulf ate-reducing bacteria, while only a few fighters had taken on the phototrophic sulfur bacteria. Some leading laboratories on sulfur metabolism in thiobacilli then were those of M.I.H. Aleem, J.P . Aubert, W .P. Hempfling, D.P . K elly, H. Lees, M. Okizumi, W . Ostro wski, H.D. Peck, S.C. Rittenber g, R.L. Stark ey, I. Suzuki, P .A. Trudinger, W.W. Umbreit and W. Vishniac and on dissimilatory sulfur metabolism in sulfate reducers were those of J.B. Adams, J.M. Akagi, L.L. Campbell, C. Furusaka, M. Ishimoto, J. Le Gall, H.D. Peck and J.R. Postgate. The fe w fighters on phototrophic sulfur bacteria came from only three laboratories: Arnold Smith from June Lascelles’ laboratory , Thiele and I from Pfennig’s laboratory and Harry D. Peck, who had a big laboratory behind him and great experience in the two other fields mentioned above. I entered this field with a minor part of my doctoral thesis, repeating the long-term stoichiometry e xperiments of v an Niel (1931) with a pure culture of Chromatium okenii in the form of short-term ones in a special vessel using radio actively labeled

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carbon and sulfur compounds. Besides e xactly conf irming the stoichiometric turnover relationships with this technique, I found that light-dependent anaerobic oxidation of sulfide by Chromatium okenii depended upon the presence of car bon dioxide, indicating that a close coupling e xisted between sulfide oxidation and photosynthetic carbon dioxide f ixation (T rüper 1964a, b; T rüper and Schlegel 1964). We further pro ved by radioacti ve labeling that in the light thiosulf ate in Thiocapsa roseopersicina and Allochromatium (then Chromatium) vinosum is split into the sulfane group, forming intracellular sulfur globules, and the sulfone group that appears in the medium as sulfate (Trüper and Pfennig 1966), confirming results of Arnold Smith and June Lascelles with Allochromatium vinosum (Smith 1965, 1966; Smith and Lascelles 1966). Smith (1965) further isolated an enzyme from that organism which catalyzes the oxidation of thiosulf ate to tetrathionate. After a f irst hint gi ven by Peck (1961), Thiele (1966, 1968) in Göttingen and Peck (1966) in Athens, GA, USA, independently reported the f irst finding of an adenosine 5′-phosphosulfate (APS) reductase activity in Chromatium and Thiocapsa strains. Thiele quit the f ield of sulfur research soon after 1966, and I took the chance to work in Peck’s laboratory for a few weeks, during which we found APS reductase activities in several green and purple phototrophic sulfur bacteria, but not, however, in an y of the non-sulfur purple bacteria tested. It also became clear that this enzyme acti vity was originally membrane-bound and leached of f at dif ferent degrees in different organisms. It was practically impossible to get it into the clear supernatant from Allochromatium vinosum strain D, while it easily leached of f in Thiocapsa roseopersicina (Trüper and Peck 1970). The latter enzyme w as purified (Trüper and Rogers 1971); the corresponding one from Chlorobium limicola followed (Kirchhoff and Trüper 1974). We were f acing an oxidati ve chain from sulf ide to sulf ate with at least sulfur , sulfite and APS as intermediates. In addition, thiosulfate as a substrate was either split into sulfur and (end-product) sulfate or oxidized to tetrathionate. All electrons set free during these oxidations w ould go into photosynthesis, as the stoichiometry studies had proven before. Our aims then were clear b ut far away, and the w ay to get there w as foggy. In those years cells were def ined as “bags full of enzymes, ” although it w as hard to imagine that all these different proteins in addition to all intermediates, educts and products, harmless as well as poisonous ones, should coe xist in one compartment. We spoke of soluble enzymes when they came out in the supernatant after ultracentrifugation and of particle-, membrane- or chromatophore-bound enzymes, when they resided in the pellet. The former could be tested by combined optical enzyme tests, the latter only with complicated substrate or product determinations including radioactive labeling methods. As we were concerned with oxidation reactions we had to f ind suitable electron acceptors for testing before we could f ind the genuine ones. As soon as the outer membrane and the periplasmic space of Gram-negative bacteria had been disco vered and the concept of the “periplasm” had been de veloped (Mitchell 1961) and became known, we suddenly had to deal with two compartments

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within which our reactions could tak e place in vi vo. In addition we had to answer the topological questions at which side of the cellular membrane such enzymes were situated that we had localized in the insoluble fraction. This meant that the ultrastructure of the bacteria that are capable of storing sulfur glob ules inside their cells became very important. Several studies proposed that the so-called chromatophores in Chromatiaceae that obviously carried the photosynthetic pigments are in vaginations of the cellular membrane, thus forming a lar ge interconnected intracellular vesicular system, the so-called intrac ytoplasmic membrane system, the inside of which topologically was part of the periplasm (Remsen 1978). Working with cell extracts under normal, i.e., aerobic laboratory conditions, brought up the question whether we w ould not measure competing reactions with oxygen. As sulfide and sulf ite are strong reductants the y would easily interfere with man y metabolic redox reactions in the cell besides being undoubtedly toxic to metal-containing and other enzymes. This meant that we also had to look for possible or ganic carrier molecules that would mask such aggressi ve features. Another important question w as that of the exact chemical nature of the conspicuous sulfur globules. How could waterinsoluble elemental sulfur participate in biochemical reactions? The first leap forward from that situation was the purification of a reverse sirohemecontaining sulfite reductase from both Thiobacillus denitrificans and Allochromatium vinosum in 1979 by Michael Schedel (Schedel et al 1979; Schedel and T rüper 1979). Besides that we w orked intensively on c cytochromes, flavocytochromes, iron–sulfur proteins, ATP sulfurylases and ADP sulfurylases. In 1986 Dan Brune (Brune and Trüper 1986) found the first evidence for the possible participation of sulf ide quinone reductase as a possible f irst step in sulf ide oxidation. This line w as successfully pursued later by Hauska and Shahak (Shahak et al. 1999).

8.7

Advent of Molecular Genetics

As soon as we had learned the basic methods of molecular biology , Christiane Dahl and her group brought Schedel’ s results to a ne w high by disco vering the dsr gene cluster (Pott and Dahl 1998, Dahl et al. 2005; Chap. 9 by Grimm et al.). K obchai Pattaragulwanit in our laboratory developed a new method to use gene technology in Allochromatium vinosum and thus came up with the then sensational f inding that the sulfur glob ules in Chromatiaceae are contained in en velopes consisting of tw o to three types of proteins, the sulfur globule proteins (SGP), which are free of sulfhydryl groups. In their genes they revealed, however, typical leader sequences characterizing them as periplasmic proteins (P attaragulwanit et al. 1998). Thus, the sulfur glob ules are stored periplasmically in the intracytoplasmic membrane vesicles! With cinematographic techniques we proved with fixed living cells that the intracellular sulfur globules do not originate at the cellular membrane to be moved to the inner part of the cell – as postulated by Remsen (1978) – b ut that the y are formed at an y place in the cell (Herrmann 1984; Herrmann and T rüper, unpublished data), which is further proof for the continuity of the intrac ytoplasmic membrane system.

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After a long period of cooperation with the inor ganic chemist Ralf Steudel, Berlin, on the nature of the sulfur in the glob ules (Steudel 1989), finally Alexander Prange, for the first time employing X-ray absorption near-edge spectroscopy (at the cyclotron of the University of Bonn Physics Department), succeeded in determining the status of this sulfur in li ving cells of phototrophic as well as chemolithotrophic sulfur bacteria (Prange et al. 2002; Chap. 20 by Prange). Ulrike Kappler w orking with the enzyme sulf ite oxidoreductase (sulf ite dehydrogenase) as the alternative to the APS pathway had to switch fromAllochromatium vinosum to Starkeya (formerly Thiobacillus) novella before she succeeded (Kappler et al. 2000, 2001; Chap. 13 by Kappler). On the basis of path-breaking studies by Don K elly’s group on aerobic thiobacilli (Lu and Kelly 1983a–c, 1988), Cornelius Friedrich and his co workers studied the periplasmic thiosulfate-oxidizing multienzyme (Sox) system in Paracoccus panthotrophus (Friedrich et al. 2001, 2005, Chap. 12), a system that also e xists in Allochromatium vinosum and Thiocapsa sp., as w as found by Dahl’ s group (Hensen et al. 2006). In Chromatiaceae this multienzyme system is probably not in volved in sulf ide oxidation. I leave the e xplanation of the present status of the art in this f ield to the ne xt generation. I am v ery happy and thankful that we ha ve come so f ar through many frustrating but also many highly exciting periods of w ork. As far as the w ork was done in our laboratory I thank my ingenious diploma and doctoral students, postdocs and coworkers, and those who will continue. I apologize for not ha ving been able to mention the merits of all the other colleagues who had and ha ve their share in the progress of sulfur metabolism research. To do that I would have needed about some 20 lecture hours.

8.8 Further

Reading

For further reading I recommend the proceedings of international scientific symposia, workshops and meetings on microbial sulfur metabolism and related f ields of the last 30 years: 1974: 1979: 1979: 1979: 1980: 1982: 1983:

“Meeting on the sulfur cycle,” Wageningen, The Netherlands (van Egeraat and Huntjens 1975) “Symposium on biology of sulfur ,” London, UK (Ciba F oundation 1980) “Low molecular weight sulfur containing natural products, ” Rome, Italy (Cavallini et al. 1980) “SCOPE/UNEP w orkshop on the global biogeochemical sulfur c ycle,” Pushchino, Russia (Ivanov and Freney 1983) “Biology of inorganic nitrogen and sulfur,” Bochum, Germany (Bothe and Trebst 1981) “Sulfur bacteria,” London, UK (Postgate and Kelly 1982) “Sulfur, its significance for chemistry, for the geo-, bio- and cosmosphere and technology,” Bielefeld, Germany (Müller and Krebs 1984)

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1984: 1988: 1995: 1996: 1998:

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“Evolution of the global biogeochemical sulfur c ycle,” Tallinn, Estonia (Brimblecombe and Lein 1989) “The nitrogen and sulfur c ycles,” Southampton, UK (Cole and Fer guson 1988) “First international symposium on DMSP and related sulfonium compounds,” Mobile, AL, USA (Kiene et al. 1996) “Processes and structures in marine methane and sulf ide biotopes, ” Winterscheid, Germany (Grieshaber and Fischer 1996) “The biological sulfur c ycle: en vironmental science and technology ,” Wageningen, The Netherlands (Lens and Hulshof f Pol 1998)

Of the meetings listed above, only that of the Royal Society in London in 1982 had the scope of the International Symposium in Münster in 2006. Acknowledgements. I am personally glad and proud that I ha ve been an e yewitness and co worker during the fruitful years of Norbert Pfennig in Göttingen in the so-called Sulfur Department. Since then, the f ascination of phototrophic bacteria has ne ver left my mind. W ithout the marv elous book by Schlegel (1999) on the history of microbiology I w ould not have been able to write this chapter. I thank him wholeheartedly!

References Arcichowskij V (1904) Zur Frage über das Bakteriopurpurin. Bull Jard Bot St Petersbour g 4:97 Bothe H, Trebst A (eds) (1981) Biology of inor ganic nitrogen and sulfur. Springer, Berlin Brimblecombe P, Lein A Y (1989) SCOPE 39: Ev olution of the global biogeochemical sulphur cycle. Wiley, Chichester Brune DC, T rüper HG (1986) Nonc yclic electron transport in chromatophores from photolithotrophically grown Rhodobacter sulfidophilus. Arch Microbiol 145:295–301 Buchanan RE, Gibbons NE (eds) (1974) Bergey’s manual of determinative bacteriology, 8th edn. Williams and Wilkins, Baltimore Buder J (1919) Zur Bakteriologie des Bakteriopurpurins und der Purpurbakterien. Jahrb Wiss Bot 58:525–628 Cavallini D, Gaull DG, Zappia V (eds) (1980) Natural sulfur compounds. Plenum, Ne w York Ciba F oundation (1980) Ciba F oundation symposium 72, ne w series. Excerpta Medica, Amsterdam Cohn F (1872) Untersuchungen über Bakterien. Beitr Biol Pflanzen 1 2:127–224 Cohn F (1875) Untersuchungen über Bakterien. Beitr Biol Pflanzen 1 3:141–207 Cole JA, Ferguson SJ (eds) (1988) 42nd symposium of the SGM. Cambridge Uni versity Press, Cambridge Dahl C, Engels S, Pott-Sperling A, Schulte A,Sander J, Lübbe Y , Deuster O, Brune DC (2005) Novel genes of the dsr gene cluster and e vidence for close interaction of dsr proteins during sulfur oxidation in the phototrophic sulphur bacterium Allochromatium vinosum. J Bacteriol 187:1392–1404 Drews G (2005) Contrib utions of Theodor W ilhelm Engelmann on phototaxis, chemotaxis and photosynthesis. Photosynth Res 83:25–34 Ehrenberg CG (1838) Die Infusionsthierchen als v ollkommene Or ganismen, ein Blick in das tiefere organische Leben der Natur. Voss, Leipzig Engelmann TW (1883) Bacterium photometricum. Pflüger’s Arch Ges Physiol 30:95–124 Friedrich CG, Bardische wsky F, Rother D, Quentmeier A, Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Microbiol 8:253–259

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Friedrich CG, Rother D, Bardischewsky F, Quentmeier A, Fischer J (2001) Oxidation of reduced inorganic sulphur compounds by bacteria: emergence of a common mechanism? Appl Environ Microbiol 67:2873–2882 Gaffron H (1935) Über den Stoffwechsel der Purpurbakterien. Biochem Z 275:301–319 Grieshaber MK, Fischer U (eds) (1996) Abstracts and program of the workshop on processes and structures in marine methane and sulf ide biotopes. Shaker, Aachen Hensen D, Sperling D, T rüper HG, Brune DC, Dahl C (2006) Thiosulf ate oxidation in the phototrophic sulfur bacterium Allochromatium vinosum. Mol Microbiol 62:794–810 Herrmann R (1984) Photographische Dokumentation der Bildung v on Schwefeleinschlüssen in Zellen von Chromatien. Diploma thesis, Fachhochschule Köln, Cologne Ivanov MI, Frene y JR (eds) (1983) SCOPE 19: The biogeochemical sulphur c ycle. W iley, Chichester Kappler U, Bennett B, Rethmeier J, Schw arz G, Deutzmann R, McEw an A G, Dahl C (2000) Sulfite:cytochrome c oxidoreductase from Thiobacillus novellus – purif ication, characterization, and molecular biology of a heterodimeric member of the sulf ite oxidase f amily. J Biol Chem 275:13202–13212 Kappler U, Friedrich CG, T rüper HG, Dahl C (2001) Evidence for tw o pathways of thiosulf ate oxidation in Starkeya novella (formerly Thiobacillus novellus). Arch Microbiol 175:102–111 Kiene RP, Visscher PT, Keller MD, Kirst GO (eds) (1996) Biological and en vironmental chemistry of DMSP and related sulfonium compounds. Plenum, Ne w York Kirchhoff J, T rüper HG (1974) Adenosine 5 ′-phosphosulfate reductase of Chlorobium limicola. Arch Microbiol 100:115–120 Lankester ER (1873) On a peach-coloured bacterium – Bacterium rubescens . Q J Microsc Sci 13:408–425 Larsen H (1954) On the microbiology and biochemistry of the photosynthetic green sulfur bacteria. K Nor Vidensk Selsk Skr 1 Lens PNL, Hulshoff Pol LW (eds) (1998) Biodegradation 9:157–318 Lu WP, Kelly DP (1983a) Thiosulphate oxidation, electron transport and phosphorylation in a cell-free system from Thiobacillus A2. J Gen Microbiol 129:1661–1671 Lu WP, Kelly DP (1983b) Partial purification and resolutution of a thiosulphate-oxidizing system from Thiobacillus A2. J Gen Microbiol 129:1673–1681 Lu WP, Kelly DP (1983c) Purification and some properties of tw o principal enzymes of the thiosulphate-oxidizing multi-enzyme system from Thiobacillus A2. J Gen Microbiol 129:3549–3564 Lu WP, Kelly DP (1988) Cellular location and partial purif ication of the “thiosulphate-oxidizing enzyme” and “trithionate h ydrolase” from Thiobacillus tepidarius . J Gen Microbiol 134:877–885 Mitchell P (1961) Approaches to the analysis of specif ic membrane transport. In: Goodwin TW , Lundberg O (eds) Biological structure and function, v ol 2. Academic, New York, pp 581–603 Molisch H (1907) Die Purpurbakterien nach neuen Untersuchungen. Fischer , Jena Müller A, Krebs B (eds) (1984) Studies in inor ganic chemistry 5. Elsevier, Amsterdam Müller C (1870) Chemisch-Ph ysikalische Beschreibung der Thermen v on Baden in der Schweiz (Canton Aargau). Zehnder, Baden Müller OF (1786) Animalcula infusoria fluviatila et marina. Ha vniae, Copenhagen Nadson GA (1903) Observations sur les bactèries pourprèes. Bull Jard Bot St Petersbour g 3:109 Pattaragulwanit K, Brune DC, Trüper HG, Dahl C (1998) Molecular genetic evidence for extracytoplasmic localization of sulphur glob ules in Chromatium vinosum . Arch Microbiol 169:434–444 Peck HD (1961) Enzymatic basis for assimilatory and dissimilatory sulf ate reduction. J Bacteriol 82:933–939 Peck HD (1966) Some evolutionary aspects of inorganic sulfur metabolism. In: Lecture series on theoretical and applied aspects of modern microbiology . Uni versity of Maryland, Colle ge Park, pp 1–22

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Perty M (1852) Zur Kenntnis kleinster Lebensformen. Jent and Reinert, Bern Pfennig N (1961) Eine v ollsynthetische Nährlösung zur selekti ven Anreicherung einiger Schwefelpurpurbakterien. Naturwissenschaften 48:136 Pfennig N (1962) Über die K ultur von Chromatium okenii. Vortr Gesamtgeb Bot 1:84–85 Pfennig N, Trüper HG (1989) Section 18. Anoxygenic phototrophic bacteria. In: Staley JT, Bryant MP, Pfennig N, Holt JG (eds) Bergey’s manual of systematic bacteriology, vol 3. Williams and Wilkins, Baltimore, pp 1635–1709 Postgate JR, Kelly DP (eds) (1982) Philos Trans R Soc Lond Ser B 298:431–602 Pott AS, Dahl C (1998) Sirohaem sulfite reductase and other proteins encoded by genes at the dsr locus of Chromatium vinosum are in volved in the oxidation of intracellular sulphur . Microbiology 144:1881–1894 Prange A, Chauvistré R, Modrow H, Hormes J, Trüper HG, Dahl C (2002) Quantitative speciation of sulphur in bacterial sulphur glob ules: X-ray absorption spectroscop y reveals at least three different species of sulphur. Microbiology 148:267–276 Remsen CC (1978) Comparati ve subcellular architecture of photosynthetic bacteria. In Clayton RK, Sistrom WR (eds) The photosynthetic bacteria. Plenum, Ne w York, pp 31–60 Roelofsen PA (1934) On the metabolism of the purple sulfur bacteria. Proc K Ned Acad W et 37:660–668 Roy AB, Trudinger PA (1970) The biochemistry of inor ganic compounds of sulphur. Cambridge University Press, Cambridge Ruben S, Randall M, Kamen M, Hyde JL (1941) Hea vy oxygen (O 18) as a tracer in the study of photosynthesis. J Am Chem Soc 63:877–878 Schedel M, Trüper HG (1979) Purification of Thiobacillus denitrificans siroheme sulfite reductase and investigation of some of its molecular and catalytic properties. Biochim Bioph ys Acta 568:454–467 Schedel M, Vanselow M, Trüper HG (1979) Siroheme sulfite reductase isolated from Chromatium vinosum. Arch Microbiol 121:29–36 Schlegel HG (1999) Geschichte der Mikrobiologie. Leopoldina, Halle Schlegel HG, Pfennig N (1961) Die Anreicherungskultur einiger Schwefelpurpurbakterien. Arch Mikrobiol 38:1–39 Schulz HN, Brinkhoff T, Ferdelman TG, Henández Mariné M, Teske A, Jørgensen BB (1999). Dense population of a giant sulfur bacterium in Namibian shelf sediments. Science 284:493–495 Shahak J, Schütz M, Bronstein M, Griesbeck C, Hauska G, P adan E (1999) Sulfide-dependent anoxygenic photosynthesis in prokaryotes: sulf ide-quinone reductase (SQR), the initial step. In: Peschek GA, Löffelhardt W, Schmetterer G (eds) The phototrophic prokaryotes, Kluwer/Plenum, New York, pp 217–228 Smith A (1965) The discriminative oxidation of the sulphur atoms of thiosulphate by a photosynthetic sulphur bacterium – Chromatium strain D. Biochem J 94:27P Smith A (1966) The role of tetrathionate in the oxidation of thiosulphate byChromatium sp. strain D. J Gen Microbiol 42:371–380 Smith A, Lascelles J (1966) Thiosulphate metabolism and rhodanese in Chromatium sp. strain D. J Gen Microbiol 42:257–270 Steudel R (1989) On the nature of the elemental sulfur (S°) produced by sulfur-oxidizing bacteria – a model for S° glob ules. In: Schle gel HG, Bowien B (eds) Biology of autotrophic bacteria Science Technology, Madison, pp 289–303 Thiele HH (1966) W achstumsphysiologische Untersuchungen an Thiorhodaceae: Wasserstoffdonatoren und Sulfatreduktion. Doctoral thesis, University of Göttingen Thiele HH (1968) Sulphur metabolism in Thiorhodaceae. V . Enzymes of sulphur metabolism in Thiocapsa floridana and Chromatium species. Antonie V an Leeuwenhoek J Microbiol Serol 34:350–361 Trevisan FS (1842) Prospetto della flora eug anea. Coi tipi del seminario, P adua, pp 56–57 Trüper HG (1964a) CO 2-Fixierung und Intermediärstof fwechsel bei Chromatium ok enii Perty. Arch Mikrobiol 49:23–50

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Trüper HG (1964b) Sulphur metabolism in Thiorhodaceae. II. Stoichiometric relationship of CO 2 fixation to oxidation of h ydrogen sulphide and intracellular sulphur in Chromatium ok enii. Antonie Van Leeuwenhoek J Microbiol Serol 30:385–394 Trüper HG (1970) Culture and isolation of phototrophic sulfur bacteria from the marine en vironment. Helgol Wiss Meeresunters 20:6–16 Trüper HG, Peck HD (1970) F ormation of adenosine 5 ′-phosphosulfate in phototrophic bacteria. Arch Mikrobiol 73:125–142 Trüper HG, Pfennig N (1966) Sulphur metabolism in Thiorhodaceae. III. Storage and turno ver of thiosulphate sulphur in Thiocapsa floridana and Chromatium species. Antonie V an Leeuwenhoek J Microbiol Serol 32:261–276 Trüper HG, Rogers LA (1971) Purification and properties of adenosine 5′-phosphosulfate reductase from the phototrophic sulfur bacterium Thiocapsa r oseopersicina. J Bacteriol 108:1112–1121 Trüper HG, Schlegel HG (1964) Sulphur metabolism in Thiorhodaceae. I. Quantitati ve measurements on growing cells of Chromatium okenii. Antonie Van Leeuwenhoek J Microbiol Serol 30:225–238 van Egeraat AWSM, Huntjens JLM (eds) (1975) Plant Soil 43:1–228 van Niel CB (1931) On the morphology and ph ysiologyof the purple and green sulphur bacteria. Arch Mikrobiol 3:1–112 von Esmarch E (1887) Über die Reinkultur eines Spirillum. Zbl Bakteriol I 1:225–230 Warming E (1875) Om nogle v ed Danmarks k yster le vende bakterier . V idensk Medd Dansk Naturhist Foren Khobenhavn 306–324 Winogradsky SN (1887) Über Schwefelbacterien. Bot Ztg 45:489–507, 513–523, 529–539, 545–559, 569–576, 585–594, 606–610 Winogradsky SN (1949) Microbiologie du sol. Problèmes et méthodes. Cinquantes ans de recherches. Masson, Paris

Chapter 9

Thiosulfate and Sulfur Oxidation in Pur ple Sulfur Bacteria Frauke Grimm, Bettina Franz, Christiane Dahl

Frauke Grimm and Bettina Franz contributed equally to this work.

Abstract In chemotrophic and phototrophic sulfur oxidizers that do not form sulfur deposits a periplasmic thiosulf ate-oxidizing multienzyme complex (Sox complex) has been described to be responsible for formation of sulfate from thiosulfate. In the anoxygenic phototrophic sulfur bacterium Allochromatium vinosum intracellular sulfur globules are an obligate intermediate during the oxidation of thiosulf ate to sulfate. Despite this fundamental difference A. vinosum possesses five sox genes in two independent loci (soxBXA and soxYZ) encoding proteins related to components of the Sox complex from Paracoccus pantotrophus. Three sox-encoded proteins were purified from A. vinosum: the heterodimeric c-type c ytochrome SoxXA, the monomeric SoxB and the heterodimeric thiosulfate-binding protein SoxYZ. Gene inactivation and complementation studies proved that these proteins are essential for thiosulf ate oxidation to sulf ate. The intermediary formation of sulfur globules in A. vinosum appears to be related to the lack of soxCD genes, the products of which are proposed to oxidize SoxY -bound sulfane sulfur. In their absence the latter is instead transferred to gro wing sulfur globules. The oxidation of the stored sulfur is completely dependent on the proteins encoded in the dsr operon. The dissimilatory sulf ite reductase (DsrAB) interacts with membranebound as well as soluble Dsr proteins. From membranes the protein is copurif ied with the transmembrane electron-transporting comple x DsrMKJOP. Furthermore, the soluble cytoplasmic proteins DsrC and DsrEFH are found in the same fraction, indicating an interaction of DsrC and DsrEFH with the reverse sulfite reductase. From the soluble fraction DsrAB is copurif ied with DsrL, a homodimeric iron–sulfur fla voprotein with NADH:acceptor oxidoreductase acti vity. The observ ed interactions of Dsr proteins serve as a basis for an impro ved model of sulfur oxidation in purple sulfur bacteria.

9.1 Intr oduction Thiosulfate (S2O32−) plays an important role in the natural sulfur cycle, especially in freshw ater sediments (Jør gensen 1990; Sorokin et al. 1999; Podgorsek and Imhoff 1999). It is a rather stable and environmentally abundant sulfur compound 101 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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of intermediate oxidation state. Generally, there are three different ways of bacterial utilization of thiosulfate: oxidation with tetrathionate or sulf ate as the end product, reduction to products like hydrogen sulfide, and disproportionation to sulfur and sulfite or hydrogen sulf ide and sulf ate (Jørgensen 1990). In this re view, we concentrate on the oxidation of thiosulf ate. In man y or ganisms lik e some Pseudomonas and Halomonas species (Sorokin et al. 1999; Podgorsek and Imhoff 1999), tetrathionate is the end product of thiosulf ate oxidation. It is formed by oxidative condensation of two thiosulfate anions catalyzed by thiosulfate dehydrogenase (EC 1.8.2.2; thiosulf ate:acceptor oxidoreductase). More widespread than the formation of tetrathionate is the complete oxidation of thiosulfate to sulf ate. Two different pathways appear to e xist: In numerous f acultatively chemolithotrophic or photolithotrophic or ganisms like P. pantotrophus or Rhodovulum sulfidophilum both sulfur atoms of thiosulfate are oxidized to sulfate without the appearance of sulfur deposits as intermediates (Appia-A yme et al. 2001; Friedrich et al. 2001, 2005), whereas in phototrophic purple sulfur bacteria and many chemotrophic sulfur oxidizers lik e magnetotactic bacteria, Beggiatoa sp. or Thiothrix the formation of conspicuous globules of polymeric, water-insoluble sulfur appears to be an important step during thiosulf ate oxidation (Nelson and Castenholz 1981; Dahl 1999; Ho warth et al. 1999; Dahl and Prange 2006; Hensen et al. 2006; W illiams et al. 2006). T able 9.1 sho ws selected or ganisms forming sulfur globules from thiosulfate. The oxidation of sulfur deposits is one of the least understood steps of sulfur metabolism. The immense di versity of sulfur -forming prokaryotes is reflected by the f acts that the site of sulfur deposition (intracellular or e xtracellular) as well as the chemical nature of the deposited sulfur can vary (Table 9.1). Universal biochemical mechanisms may therefore not exist (Brune 1995a; Prange et al. 2002; Dahl et al. 2002; Friedrich et al. 2005; Dahl and Prange 2006). One enzyme for which an in volvement in the degradation of stored sulfur w as suggested is dissimilatory sulf ite reductase. In the chemolithotrophic sulfur oxidizer Thiobacillus denitrificans and the photolithotrophic sulfur oxidizer A. vinosum (formerly Chromatium vinosum; Imhoff et al. 1998) this enzyme is assumed to be operating in “re verse,” performing the six-electron oxidation from sulfide to sulfite (Schedel et al. 1979; Schedel and T rüper 1979). The genetically accessible Gammaproteobacterium A. vinosum , an anoxygenic purple sulfur bacterium of the f amily Chromatiaceae, utilizes reduced sulfur compounds like sulfide, thiosulfate and sulfur as electron donors for reducti ve carbon dioxide f ixation during photolithoautotrophic gro wth (Brune 1995a). A. vinosum employs tw o dif ferent pathw ays for the oxidation of thiosulf ate: oxidation to tetrathionate or complete oxidation to sulf ate (Hensen et al. 2006). The formation of intracellular sulfur globules from sulfide and thiosulfate is obligatory en route to sulfate (Pott and Dahl 1998). The glob ules are located in the periplasm (Pattaragulwanit et al. 1998) and are surrounded by an envelope consisting of three different proteins, SgpA, SgpB and SgpC (Brune 1995a). The sulfur inside is present as sulfur chains probably carrying so f ar unidentified organic residues at one or at both ends (Prange et al. 2002). The reverse sulfite reductase is encoded by

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Table 9.1 Selected organisms forming sulfur globules from thiosulfate Organism/group Comments Systematic affiliation References Chemotrophic sulfur bacteria Magnetotactic coccus Intracellular and MC-1 extracellular Extracellular Thermothrix Intracellular, Beggiatoa sp. periplasmic, cyclo-octasulfur Intracellular, Thiothrix periplasmic Extracellular, some Thioalkalivibrio strains intracellular in periplasm Phototrophic bacteria Extracellular Chlorobaculum parvumDSM 263 a Intracellular, Chromatiaceae periplasmic, organic polysulfanes a

Alphaproteobacteria

(Williams et al. 2006)

Betaproteobacteria Gammaproteobacteria

(Odintsova et al. 1996) (Nelson and Castenholz 1981; Prange et al. 2002) (Odintsova et al. 1993; Howarth et al. 1999) (Sorokin et al. 2001)

Gammaproteobacteria Gammaproteobacteria

Chlorobi Gammaproteobacteria

(Steinmetz and Fischer 1982) (Smith and Lascelles 1966; Brune 1989; Prange et al. 2002)

Formerly Chlorobium vibrioforme subsp. thiosulfatophilum (Imhoff 2003).

the first two genes of a lar ge cluster dsrABEFHCMKLJOPNRS, which is essential for the oxidation of sulfur deposited in sulfur glob ules to the f inal product sulf ate (Pott and Dahl 1998; Dahl et al. 2005). In this review we concentrate on A. vinosum and summarize the current kno wledge of the pathw ays of thiosulf ate and intracellular sulfur oxidation to sulf ate. Since the second product of thiosulf ate oxidation, tetrathionate, is not further utilized by A. vinosum , this pathw ay is not described in detail and the interested reader is referred to the data presented by Hensen et al. (2006).

9.2

Oxidation of Thiosulfate in A. vinosum

A wealth of biochemical and molecular genetic information on thiosulf ate oxidation is available about organisms that do not form sulfur deposits during thiosulfate utilization. The groups of Don Kelly and Cornelius Friedrich found and characterized a periplasmic thiosulfate-oxidizing multienzyme comple x (Sox) in P. versutus (Lu et al. 1985) and P. pantotrophus (Rother et al. 2001; Friedrich et al. 2001). In P. pantotrophus the Sox complex is essential for thiosulf ate oxidation in vi vo and catalyzes reduction of

104

F. Grimm et al.

cytochrome c coupled to the oxidation of thiosulf ate, sulfide, sulfite and elemental sulfur in vitro. The proposed mechanism for thiosulf ate oxidation requires four different proteins: SoxB, SoxXA, SoxYZ and SoxCD (Friedrich et al. 2001). The heterodimeric SoxYZ has been identif ied as the substrate-binding molecule of the complex (Quentmeier and Friedrich 2001). SoxXA is a heterodimeric heme enzyme that is reduced while oxidatively coupling the sulfur compound to SoxYZ. The monomeric, manganese-containing SoxB has been proposed to act as a sulf ate thiol esterase or sulfate thiol hydrolase and is responsible for hydrolytic clea vage of a sulf ate group from the bound sulfur substrate. SoxCD oxidizes the remaining sulfane sulfur, acting as a sulfur dehydrogenase. Further action of SoxB releases a second sulf ate molecule and thereby restores SoxYZ. Further details about properties and regulation of the system are given in Chap. 12 by Friedrich et al. Much less is kno wn about thiosulf ate oxidation involving the intermediate deposition of sulfur either inside or outside the cells. As mentioned in Sect. 9.1, sulfur globules are formed as an obligatory intermediate during the oxidation of thiosulf ate to sulf ate in A. vinosum (Pott and Dahl 1998). Studies with radioacti vely labeled thiosulf ate in purple sulfur bacteria demonstrated that the sulfane and the sulfone sulfur atoms of thiosulfate are oxidized by dif ferent pathw ays (Smith and Lascelles 1966; T rüper and Pfennig 1966). Only the sulf ane sulfur accumulates in sulfur glob ules before further oxidation, whereas the sulfone sulfur is rapidly con verted into sulfate. Thus, the initial step of thiosulfate oxidation is a clea vage of the molecule. In the past, the detection of thiosulfate-reducing enzyme acti vities (i.e., rhodaneses and thiosulf ate reductases) in phototrophic and chemotrophic sulfur bacteria led to the assumption that thiosulf ate would be cleaved into sulfate and sulfide in the presence of suitable thiol acceptors like glutathione and dihydrolipoic acid, and that the H 2S formed during the proposed reaction would be immediately oxidized to stored sulfur (Brune 1989, 1995a; Dahl 1999). However, genetic proof for this assumption w as missing and it w as recognized quite early that rhodanese as well as thiosulfate reductase occur in a wide range of organisms not able to metabolize thiosulf ate (Brune 1989). During the past se veral years clusters of sox genes were identif ied in thiosulf ate-oxidizing green sulfur bacteria (Petri et al. 2001; Eisen et al. 2002; Verté et al. 2002; Frigaard and Bryant 2008; see also Chap. 6 by Frigaard and Bryant). Therefore, an essential role of rhodanese or thiosulfate reductase during the initial steps of thiosulf ate oxidation in sulfur -storing bacteria appeared increasingly unlikely. Now, sox genes have also been identified in A. vinosum, proteins essential for thiosulfate oxidation have been purified and mutational analysis has shown that the earlier models of thiosulfate oxidation via intermediate sulfur formation have to be completely revised (Hensen et al. 2006).

9.2.1

sox Genes in A. vinosum

Nucleotide sequence analysis revealed that, unlike the situation in P. pantotrophus, the genes soxXAB and soxYZ are located in tw o independent gene re gions in A. vinosum and genes coding for SoxCD are not present in the organism (Hensen et al. 2006).

9 Thiosulfate and Sulfur Oxidation in Purple Sulfur Bacteria

105

The genes soxB and soxXA are transcribed divergently. The two sequenced DNA fragments include ten further open reading frames. Especially notable is the rhd gene, the product of which contains a conserv ed domain typical for rhodaneses, enzymes responsible for sulfur group transfer that are found in all three domains of life. Since the sulfane sulfur is transferred to the sulfur globules, a rhodanese could well be part of a thiosulf ate oxidizing pathw ay (Hensen et al. 2006).

9.2.2

Sox Proteins in A. vinosum

Three periplasmic proteins SoxXA, SoxB and SoxYZ were identified in and purified from A. vinosum (Hensen et al. 2006). Except SoxZ, all are predicted to be synthesized as precursors carrying signal peptides. A Sec-dependent transport is postulated for SoxXA. The protein w as purified as a heterodimer (SoxX 11 kDa, SoxA 29 kDa). Covalently bound heme is present in both sub units. A. vinosum SoxA is predicted to bind one heme lik e the protein from Starkeya no vella (Kappler et al. 2004), while two heme binding sites are present in P. pantotrophus and R. sulfidophilum SoxA (Friedrich et al. 2000; Bamford et al. 2002). Since the structural analysis of R. sulfidophilum SoxXA revealed that the additional aminoterminal SoxA heme is at too great a distance from the other hemes to allow efficient electron tunneling (Bamford et al. 2002), it is not clear whether the different heme contents of SoxA cause different functions. Although SoxXA was purified under aerobic, nonreducing conditions, the UV–vis spectrum was that of a typical reduced c550-type c ytochrome. All other SoxXA proteins described so f ar ha ve been isolated in the oxidized state (Friedrich et al. 2000; Cheesman et al. 2001; Kappler et al. 2004). SoxA is expressed at a low constitutive level in the absence of thiosulfate and its formation is strongly increased in the presence of thiosulfate (Hensen et al. 2006). SoxB w as isolated as a monomeric protein (62 kDa) from A. vinosum and processing and transport by the T at pathw ay w as e xperimentally v erified (Hensen et al. 2006). This implies transport as a mature, folded protein probably containing a cof actor. SoxB of P. pantotr ophus contains tw o manganese atoms per monomer (Friedrich et al. 2000) and this is probably also the case in A. vinosum. SoxYZ was purified as a heterodimer (SoxY 12.7 kDa, SoxZ 11.2 kDa) (Hensen et al. 2006). Experimental e vidence w as obtained for a co valent attachment of thiosulfate to a strictly conserv ed cysteine at the carboxy terminus of SoxY . This is in accordance with the suggestion of Quentmeier and Friedrich (2001), who proposed SoxY as the substrate-binding molecule in the Sox comple x of P. pantotrophus. For SoxY a T at-dependent transport is predicted, and SoxZ is v ery lik ely cotransported with SoxY, as has also been proposed for P. pantotrophus SoxZ (Friedrich et al. 2001).

106

F. Grimm et al.

9.2.3 Inactivation and Complementation of sox Genes in A. vinosum On the basis of different mutants, the importance of the sox genes and the encoded proteins for thiosulf ate oxidation in A. vinosum was determined. The A. vinosum mutants DsoxX, DsoxB, DsoxBX and DsoxY entirely lacked sulfate production from thiosulfate. In contrast, the inacti vation of ORF9/ rhd had no detectable ef fect on thiosulfate utilization (Hensen et al. 2006). Therefore, the products of the latter genes do not seem to play a vital role in the oxidation of thiosulfate to sulfate under the experimental conditions chosen, while the proteins SoxXABYZ are absolutely essential. The A. vinosum DsoxX and DsoxY mutants were complemented in trans. Thiosulfate oxidation w as completely restored to the wild-type phenotype in the complemented mutant DsoxX and sulfate was again the major product. In the complemented DsoxY mutant the thiosulfate oxidation rate was still significantly lower than in the wild type, b ut the principal capability to oxidize thiosulf ate to sulf ate was clearly reestablished. In summary , the complementation e xperiments verified that the observed lack of sulf ate formation from thiosulf ate was indeed caused by inactivation of sox genes (Hensen et al. 2006). On the basis of these results and the model suggested by Friedrich et al. (2001) for non-sulfur-storing bacteria, a model for thiosulf ate oxidation in sulfur -storing organisms is proposed (Fig. 9.1): the initial oxidation and co valent binding of

S

SgpC

Periplasm

SgpA

+ 2 H + 2 e-

Flavocytochrome c

2 HS¯

SoxX

SgpA

“SO”

SgpB SgpA

SoxZ

SgpC

SgpA SgpC

S2O32-

SoxY SgpB

SgpB

¯SSnS¯

-

SoxA

SgpC SgpC SgpB

2 e-

SgpB

SgpA

O -O S OS

-

S S

S

SoxY

SoxY SoxZ

SoxZ

¯SS¯

SO42-

SoxB

H2O

+ 2 H + 2 e-

Sulfide: quinone oxidoreductase

QH2 Q +

2H

Cytoplasm

Fig. 9.1 Model for the oxidation of sulf ide and thiosulf ate to intracellularly stored sulfur in Allochromatium vinosum. A sulfur globule is represented with its envelope consisting of the three proteins SgpA, SgpB and SgpC (Brune 1995b; P attaragulwanit et al. 1998).

9 Thiosulfate and Sulfur Oxidation in Purple Sulfur Bacteria

107

thiosulfate to SoxYZ is catalyzed by SoxXA and sulf ate is then h ydrolytically released by SoxB. Owing to the lack of the “sulfur dehydrogenase” SoxCD, the sulfane sulfur atom still hook ed up to SoxY cannot be directly further oxidized in organisms like A. vinosum . Probably, the sulfur is instead transferred to gro wing sulfur globules. Such a suggestion is feasible as the sulfur glob ules in A. vinosum and in many if not all other or ganisms forming intracellular sulfur deposits reside in the bacterial periplasm (Pattaragulwanit et al. 1998; Dahl and Prange 2006) and therefore in the same cellular compartment as the Sox proteins. How the transfer of SoxY-bound sulfur to the sulfur globules is achieved is currently unclear as the lack of the potential sulfur transferase encoded by the rhd gene did not lead to a detectable phenotype. Possibly, other sulfur transferases present in the cells function as a backup system (Hensen et al. 2006).

9.3

Oxidation of Stored Sulfur in A. vinosum

In A. vinosum the oxidation of thiosulf ate and that of sulf ide merge at the le vel of stored sulfur. During sulf ide oxidation, sulfur stored in sulfur glob ules is the f irst macroscopically and microscopically observ able product (Dahl and Prange 2006). The mechanism by which the periplasmically stored sulfur is made available to the cytoplasmic sulfite reductase is unclear . In sulf ate-reducing bacteria dissimilatory sulfite reductase catalyzes the six-electron reduction of sulf ite to sulf ide. It has therefore been proposed that the stored sulfur has to be reducti vely activated to the oxidation state of sulf ide in A. vinosum in order to serv e as a substrate for sulf ite reductase operating in reverse (Schedel et al. 1979). The importance of the dsr gene region for the oxidation of stored sulfur has been shown by interposon mutagenesis (Pott and Dahl 1998; Dahl et al. 2005).

9.3.1

The dsr Operon and Proteins Encoded Therein

The re verse dissimilatory sulf ite reductase (DsrAB) of A. vinosum is encoded together with 13 other proteins in the dsr operon, dsrABEFHCMKLJOPNRS (Pott and Dahl 1998; Dahl et al. 2005). The dsrAB gene products form the c ytoplasmic α2β2-structured sulfite reductase, which is closely related to the dissimilatory sulfite reductases of sulfate-reducing prokaryotes (Hipp et al. 1997). The prosthetic group of DsrAB is siroamide–[Fe4S4], with siroamide being an amidated form of the classic siroheme. The dsrN encoded protein resembles cobyrinic acid a,c-diamide synthases and catalyzes the glutamine-dependent amidation of siroheme. A DdsrN mutant showed a reduced sulfur oxidation rate. A. vinosum is apparently able to incorporate siroheme instead of siroamide into sulf ite reductase, thereby retaining some function of the enzyme (Lübbe et al. 2006). The dsrEFH genes are located adjacent to dsrAB. The products of these three genes sho w significant similarity to

108

F. Grimm et al.

each other and form a single tight 75-kDa comple x with an α2β2γ2 structure (Dahl et al. 2005). DsrC is a small soluble c ytoplasmic protein with a highly conserv ed C-terminus including tw o conserved cysteine residues. Proteins closely related to DsrEFH and DsrC have recently been shown to act as parts of a sulfur relay system involved in thiouridine biosynthesis at transfer RN A wobble positions inEscherichia coli (Numata et al. 2006; Ik euchi et al. 2006). The dsrM-encoded protein is predicted to be a membrane-bound b-type cytochrome and shows similarities to a subunit of heterodisulf ide reductases from methanogenic archaea (Sander et al. 2006). The cytoplasmic iron–sulfur protein DsrK exhibits relevant similarity to the catalytic subunit of heterodisulfide reductases. DsrP is another inte gral membrane protein. The periplasmic proteins DsrJ and DsrO are a triheme c-type cytochrome and an iron–sulfur protein, respectively. DsrKJO were copurified from membranes, pointing at the presence of a transmembrane electron-transporting complex consisting of DsrMKJOP (Dahl et al. 2005). Indi vidual in frame deletions of the dsrMKJOP genes led to the complete inability of the mutants to oxidize stored sulfur (Sander et al. 2006). DsrL is a c ytoplasmic iron–sulfur fla voprotein with N ADH: acceptor oxidoreductase acti vity (Y. Lübbe and C. Dahl, unpublished data). In frame deletion of dsrL completely abolished the oxidation of stored sulfur (Lübbe et al. 2006). DsrR and DsrS are soluble cytoplasmic proteins of unknown function. The dsr genes, with the e xception of the constituti vely e xpressed dsrC, are expressed and the encoded proteins are formed at a lo w basic le vel e ven in the absence of sulfur compounds. An increased production of all Dsr proteins is induced by sulfide and/or stored sulfur (Dahl et al. 2005).

9.3.2 Distribution of dsr Genes in Organisms with Dissimilatory Sulfur Metabolism and Phylogenetic Analysis Dissimilatory sulfite reductase and other Dsr proteins occur in sulf ate-reducing prokaryotes, where sulf ite reductase catalyzes the reduction of sulf ite to sulf ide as the f inal step of sulf ate reduction, as well as in sulfur -oxidizing prokaryotes, in which the sulf ite reductase w orks in the re verse direction (Hipp et al. 1997; Dahl et al. 2005; Sander et al. 2006). When the occurrences of the v arious dsr genes in sulfur -oxidizing and sulf ate-reducing prokaryotes are compared, it becomes apparent that certain genes, dsrABCNMKJOP, represent a core unit, whereas other dsr genes are specific for either sulfur-oxidizing or sulfate-reducing prokaryotes (Table 9.2). The gene dsrD appears to be typical for sulf ate/sulfitereducing prokaryotes, whereas the genes dsrEFH and dsrL appear to be restricted to the sulfur oxidizers. Phylogenetic analysis of Dsr proteins yielded two separate clusters consisting of proteins from sulfate reducers, on the one hand, and of proteins from sulfur oxidizers, on the other (Sander et al. 2006). Astonishingly, the DsrMKJOP proteins of the members of the green sulfur bacteria (phylum Chlorobi) do not cluster with the proteins of other sulfur oxidizers b ut af filiate with the sulf ate/sulfite-reducing

Chlorobium limicola Chlorobium clathratiforme Prostecochloris aestuarii Prostecochloris vibrioformis SULFATE/SULFITE REDUCERS Deltaproteobacteria Desulfovibrio vulgaris Desulfovibrio desulfuricans

Betaproteobacteria Thiobacillus denitrificans Gammaproteobacteria Allochromatium vinosum Alkalilimnicola ehrlichii Halorhodospira halophila Chlorobi Chlorobaculum tepidum Chlorobium phaeobacteroides

SULFUR OXIDIZERS Alphaproteobacteria Magnetospirillum magnetotacticum Magnetococcus sp.

+

+ + +

+ +

+ + + +

+ +

+ + +

+ +

+ + + +

+ +

+

+

+

+

+

+ +

− − − −

− −

− − −

−

−

−

− −

+ + + +

+ +

+ + +

+

+

+

− −

+ + + +

+ +

+ + +

+

+

+

+ +

+ + + +

+ +

+ + +

+

+

+

+ +

+ + + +

+ +

+ + +

+

+

+

− −

− − − −

− −

+ − −

+

−

?

− −

− − − −

− −

+ − −

+

−

−

+ +

+ + + +

+ +

− − −

−

−

−

(continued)

NC_002937 NC_007519, AJ249777, CP000112

NC_002932 NZ_AAIB01000016 (DSM266), NZ_AAIB01000004 (DSM 266), NZ_AAIC01000113 (BS1), NZ_AAIC01000057 (BS1) NZ_AAHJ01000040 NZ_AAIK01000042 NZ_AAIJ01000019, NZ_AAIJ01000014 NZ_AAJD01000006

U84760 NZ_AALK01000002 NZ_AAOQ01000001

NZ_AAFH01000005, NC_007404

NZ_AAAP01003833, NZ_ AAAP01003703, NZ_AAAP01003586 NZ_AAAN02000064, NZ_AAA02000091, NZ_AAAN03000009

Table 9.2 Occurrence of dsr genes in selected sulfur-oxidizing and sulfate/sulfite-reducing prokaryotes dsr genes Organisms GeneBank accession numbers AB C D EFH L N MKJOP R S T

9 Thiosulfate and Sulfur Oxidation in Purple Sulfur Bacteria 109

Desulfitobacterium hafniense Euryarchaeota Archaeoglobus fulgidus

Organisms Desufotalea psychrophila Syntrophobacter fumaroxidans Clostridia Moorella thermoacetica

Table 9.2 ( continued)

+

+

+

+

+

+

+

+

−

D + +

dsr genes AB C + + + +

−

−

−

EFH − −

−

−

−

L − −

+

+

+

N + +

+

+

+

−

−

−

MKJOP R + − + −

−

−

−

S − −

+

+

+

T + +

NC_000917

NZ_AADT03000031, NZ_ AADT02000020, NC_AADT03000011 NZ_AAAW03000053

GeneBank accession numbers CR522870 NZ_AAJF01000004, NZ_AAJF01000055

110 F. Grimm et al.

9 Thiosulfate and Sulfur Oxidation in Purple Sulfur Bacteria

111

prokaryotes. This phenomenon suggests a horizontal gene transfer , which is also supported by the presence of dsrT (or ORF9; Mussmann et al. 2005) in the green sulfur bacteria, a gene otherwise only found in sulfate/sulfite-reducing prokaryotes (Sander et al. 2006).

9.3.3

Model of the Sulfur Oxidation Pathway in A. vinosum

The periplasmic flavin adenine dinucleotide containing fla vocytochrome c and the membrane-bound sulfide:quinone oxidoreductase (SQR) have long been suspected to be responsible for sulf ide oxidation in A. vinosum (Brune 1995a; Fig. 9.1). Flavocytochrome c deficient mutants of A. vinosum (Reinartz et al. 1998) sho wed no impact on sulfide oxidation rates, indicating SQR to be the main sulfide-oxidizing enzyme in this organism. The primary in vitro product of the SQR reaction is soluble polysulfide (Griesbeck et al. 2002). Polysulf ides were also detected as the primary product of sulfide oxidation by whole cells of A. vinosum (Prange et al. 2004). The initial product of sulf ide oxidation released from the enzyme is probably disulfide. Polysulfide anions of different chain lengths are in equilibrium with each other (Griesbeck et al. 2002). Longer -chained polysulf ides are spontaneously formed from the initial disulfide by disproportionation. It is currently unknown how polysulfides are converted into sulfur globules containing organic polysulfanes. In A. vinosum, sulf ide and thiosulf ate oxidation mer ge at the le vel of stored sulfur (Fig. 9.1), which is an obligate intermediate during the oxidation of both compounds (Pott and Dahl 1998; Prange et al. 2004; Hensen et al. 2006). As outlined already , the only gene re gion known so f ar to be essential for the oxidation of stored sulfur is the dsr operon. Since the proteins encoded at the dsr locus are either c ytoplasmic or membrane-bound and cannot act directly on the extracytoplasmic sulfur glob ules (Fig. 9.2), it is proposed that the sulfur is reductively activated, transported to and further oxidized in the cytoplasm (Pott and Dahl 1998; Dahl et al. 2005). DsrL e xhibits NADH:acceptor oxidoreductase acti vity (Y. Lübbe and C. Dahl, unpublished data) . Interestingly , the protein carries a thioredoxin motif CysXXCys immediately preceding the carboxy-terminal iron– sulfur cluster binding sites. This indicates a potential disulf ide reductase acti vity which we could not yet pro ve experimentally. Still, the possibility exists that DsrL uses NADH as electron donor for reduction of a disulfidic or persulfidic compound. Thus, it is possible that DsrL is in volved in the reducti ve release of sulf ide from a carrier molecule – probably an organic perthiol – that may transport sulfur from the periplasmic sulfur globules to the cytoplasm, where it is further metabolized by Dsr proteins (Dahl et al. 2005). Glutathione amide is a lik ely candidate for carrying sulfur from the periplasm to the cytoplasm. This derivative of glutathione has been found to be lar gely converted into its perthiolic form when A. vinosum is gro wn photoautotrophically on sulf ide (Bartsch et al. 1996). Recently , transporters ha ve been characterized in E. coli mediating e xport (Pittman et al. 2005) and import (Suzuki et al. 2005) of glutathione. Shuttlingof glutathione amide between cytoplasm

112

F. Grimm et al. SgpC

SgpA

SgpB

SgpB

Periplasm

SgpC

SgpA

SgpA

“SO”

SgpC SgpB

Sulfite: acceptor oxidoreductase

SgpB

HSO3-

SgpC

SgpA

SgpC SgpB

SgpA

2 e2 H2O

DsrO

RSSH RSH

4 [FeS]

DsrJ

APS reductase

3 Heme c

Heme bL

QH2

Q

DsrM

?

DsrP

AprM Q

HemebH QH2 DsrK

RSSH NADH

RSH

HS SH HS SH DsrC DsrC

2 [Fe4S4]

+ NAD

HSDsrL 1 [FeS] FAD 2 [Fe4S4]

DsrF DsrF

HSO3-

APS

Sat

ATP sulfurylase

DsrA DsrA siroamide- siroamide-

SH

DsrH DsrH

SO42ATP PPi

AMP 3 x 2 e−+ 6 H+

+

2H

AprA

DsrC DsrC

2[Fe4S4]

DsrE DsrE HS

AprB 2[Fe4S4]

FAD

HS S S SH

3 H2O

DsrL 1 [FeS] FAD 2 [Fe4S4] SH

SO42-

SH

DsrB [Fe4S4]

2 [Fe4S4]

DsrB [Fe4S4]

Sulfite reductase

Cytoplasm

Fig. 9.2 Model for the oxidation of intracellularly stored sulfur to sulf ate in A. vinosum and involvement of the proteins of the dsr locus. The scheme is based on sequence analysis of the encoding genes and on biochemical information where available. The potential presence of sulfite: acceptor oxidoreductase is inferred from the f act that adenosine 5 ′-phosphosulfate (APS) reductase is not essential (Dahl 1996). Ho wever, neither the protein nor the corresponding genes could yet be proven in A. vinosum.

and periplasm in purple sulfur bacteria lik e A. vinosum therefore also appears feasible. DsrL, being an essential protein for sulfur oxidation, is copurif ied with the sulfite reductase (Y. Lübbe and C. Dahl, unpublished data). Sulf ide released from the perthiol could therefore be directly passed to dsrAB-encoded sulfite reductase, thereby reducing losses caused by e vaporation of gaseous H 2S. The DsrMKJOP membrane complex is copurified with sulfite reductase, DsrEFH and DsrC (Dahl et al. 2005). This is in accordance with the suggestion that DsrMKJOP from dissimilatory sulfate reducers transfer electrons to sulf ite reductase (Pires et al. 2006; see also Chap. 3 by Pereira). Taken together these observations indicate that sulfite reductase specifically interacts with the soluble protein DsrL on one hand and with membrane-bound Dsr proteins and DsrEFHC on the other hand. Electrons released from the oxidation of sulf ide by sulf ite reductase may be fed into photosynthetic electron transport via DsrC and DsrMKJOP , which w ould be analogous to the pathway postulated for sulf ate reducers, operating in the re verse direction. DsrM could operate as a quinone reductase, DsrP as a quinol oxidase and f inally the c-type cytochrome DsrJ would be reduced (Dahl et al. 2005). From here, electrons could be transferred to high-potential iron protein (HiPIP), the primary electron

9 Thiosulfate and Sulfur Oxidation in Purple Sulfur Bacteria

113

donor to the photosynthetic reaction center (V ermeglio et al. 2002). The function of DsrEFH remains unclear , b ut as it occurs e xclusively in sulfur oxidizers and shows some interaction with DsrC, it may be important for the pathw ay to operate in the sulf ide oxidizing direction. In the f inal step, sulf ite is oxidized to sulf ate, either directly by a postulated sulfite:acceptor oxidoreductase or via the nonessential enzymes adenosine 5′-phosphosulfate reductase and A TP sulfurylase (Dahl 1996; Fig. 9.2).

9.4 Conclusions In the purple sulfur bacterium A. vinosum, Sox and Dsr proteins ha ve been established to be absolutely essential for the oxidation of thiosulf ate and stored sulfur , respectively. Clusters of sox and dsr genes ha ve also been identif ied in the only distantly related green sulfur bacteria as well as in other sulfur-storing phototrophic and chemotrophic sulfur oxidizers. This suggests that the mechanisms of thiosulfate oxidation via sulfur deposition and of the oxidation of deposited sulfur are evolutionary highly conserved and that studies in A. vinosum can contribute to the elucidation of sulfur oxidation pathways in other sulfur-storing bacteria. Acknowledgements. We thank Bir gitt Hüttig for e xcellent technical assistance and ackno wledge f inancial support from the Deutsche F orschungsgemeinschaft (grants Da 351/3-3 and 351/3-4 and Da 351/4-1 and 351/4-2). W e also thank Hans G. T rüper for ongoing interest and support.

References Appia-Ayme C, Little PJ, Matsumoto Y, Leech AP, Berks BC (2001) Cytochrome complex essential for photosynthetic oxidation of both thiosulf ate and sulfide in Rhodovulum sulfidophilum. J Bacteriol 183:6107–6118 Bamford VA, Bruno S, Rasmussen T, Appia-Ayme C, Cheesman MR, Berks BC, Hemmings AM (2002) Structural basis for the oxidation of thiosulf ate by a sulfur c ycle enzyme. EMBO J 21:5599–5610 Bartsch RG, Ne wton GL, Sherrill C, F ahey RC (1996) Glutathione amide and its perthiol in anaerobic sulfur bacteria. J Bacteriol 178:4742–4746 Brune DC (1989) Sulfur oxidation by phototrophic bacteria. Biochim Biophys Acta 975:189–221 Brune DC (1995a) Sulfur compounds as photosynthetic electron donors. In: Blankenship RE, Madigan MT, Bauer CE (eds) Anoxygenic photosynthetic bacteria. Kluwer , Dordrecht, pp 847–870 Brune DC (1995b) Isolation and characterization of sulfur glob ule proteins from Chromatium vinosum and Thiocapsa roseopersicina. Arch Microbiol 163:391–399 Cheesman MR, Little PJ, Berks BC (2001) No vel heme ligation in a c-type cytochrome involved in thiosulf ate oxidation: EPR and MCD of SoxAX from Rhodovulum sulf idophilum. Biochemistry 40:10562–10569 Dahl C (1996) Insertional gene inactivation in a phototrophic sulphur bacterium: APS-reductasedeficient mutants of Chromatium vinosum. Microbiology 142:3363–3372

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Dahl C (1999) Deposition and oxidation of polymeric sulfur in prokaryotes. In: Steinbüchel A (ed) Biochemical principles and mechanisms of biosynthesis and biode gradation of polymers. Wiley-VCH, Weinheim, pp 27–34 Dahl C, Prange A (2006) Bacterial sulfur glob ules: occurrence, structure and metabolism. In: Shively JM (ed) Inclusions in prokaryotes. Springer , Heidelberg, pp 21–51 Dahl C, Prange A, Steudel R (2002) Natural polymeric sulfur compounds. In: Steinbüchel A (ed) Miscellaneous biopolymers and biode gradation of synthetic polymers, v ol 9. W iley-VCH, Weinheim, pp 35–62 Dahl C, Engels S, Pott-Sperling AS, Schulte A, Sander J, Lübbe Y , Deuster O, Brune DC (2005) Novel genes of the dsr gene cluster and e vidence for close interaction of Dsr proteins during sulfur oxidation in the phototrophic sulfur bacterium Allochromatium vinosum . J Bacteriol 187:1392–1404 Eisen JA, Nelson KE, Paulsen IT, Heidelberg JF, Wu M, Dodson RJ, Deboy R, Gwinn ML, Nelson WC, Haft DH, Hickey EK, Peterson JD, Durkin AS, Kolonay JL, Yang F, Holt I, Umayam LA, Mason T, Brenner M, Shea TP, Parksey D, Nierman WC, Feldblyum TV, Hansen CL, Craven MB, Radune D, V amathevan J, Khouri H, White O, Gruber TM, K etchum KA, V enter JC, Tettelin H, Bryant D A, Fraser CM (2002) The complete genome sequence of Chlorobium tepidum TLS a photosynthetic, anaerobic, green-sulfur bacterium. Proc Natl Acad Sci USA 99:9509–9514 Friedrich CG, Quentmeier A, Bardische wsky F, Rother D, Kraft R, K ostka S, Prinz H (2000) Novel genes coding for lithotrophic sulfur oxidation of Paracoccus pantotr ophus GB17. J Bacteriol 182:4677–4687 Friedrich CG, Rother D, Bardischewsky F, Quentmeier A, Fischer J (2001) Oxidation of reduced inorganic sulfur compounds by bacteria: emer gence of a common mechanism? Appl En viron Microbiol 67:2873–2882 Friedrich CG, Bardische wsky F, Rother D, Quentmeier A, Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Microbiol 8:253–259 Frigaard NU, Bryant D A (2008) Genomic insights into the sulfur metabolism of phototrophic green sulfur bacteria. In: Go vindjee (series ed) Adv ances in photosynthesis and respiration, vol. 27, Hell R, Dahl C, Knaff DB, Leustek T (eds) Sulfur metabolism in phototrophic or ganisms. Springer, New York (in press) Griesbeck C, Schütz M, Schödl T , Bathe S, Nausch L, Mederer N, V ielreicher M, Hauska G (2002) Mechanism of sulf ide-quinone oxidoreductase in vestigated using site-directed mutagenesis and sulfur analysis. Biochemistry 41:11552–11565 Hensen D, Sperling D, T rüper HG, Brune DC, Dahl C (2006) Thiosulf ate oxidation in the phototrophic sulfur bacterium Allochromatium vinosum. Mol Microbiol 62:794–810 Hipp WM, Pott AS, Thum-Schmitz N, Faath I, Dahl C, Trüper HG (1997) Towards the phylogeny of APS reductases and sirohaem sulf ite reductases in sulf ate-reducing and sulfur -oxidizing prokaryotes. Microbiology 143:2891–2902 Howarth R, Unz RF, Seviour EM, Seviour RJ, Blackall LL, Pickup RW, Jones JG, Yaguchi J, Head IM (1999) Ph ylogenetic relationships of f ilamentous sulfur bacteria ( Thiothrix spp. and Eikelboom type 021N bacteria) isolated from w astewater-treatment plants and description of Thiothrix eikelboomii sp. no v., Thiothrix unzii sp. no v., Thiothrix fructosivorans sp. no v. and Thiothrix defluvii sp. nov. Int J Syst Bacteriol 49:1817–1827 Ikeuchi Y, Shigi N, Kato J, Nishimura A, Suzuki T (2006) Mechanistic insights into sulfur relay by multiple sulfur mediators in volved in thiouridine biosynthesis at tRN A wobble positions. Mol Cell 21:97–108 Imhoff JF (2003) Phylogenetic taxonomy of the family Chlorobiaceae on the basis of 16S rRNA and fmo (Fenna-Matthews-Olson protein) gene sequences. Int J Syst Ev ol Microbiol 53:941–951 Imhoff JF, Süling J, Petri R (1998) Ph ylogenetic relationships among the Chromatiaceae, their taxonomic reclassification and description of the ne w genera Allochromatium, Halochromatium, Isochromatium, Marichromatium, Thiococcus, Thiohalocapsa, and Thermochromatium. Int J Syst Bacteriol 48:1129–1143 Jørgensen BB (1990) The sulfur c ycle of freshw ater sediments: role of thiosulf ate. Limnol Oceanogr 35:1329–1342

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Kappler U, Aguey-Zinsou K-F, Hanson GR, Bernhardt PV, McEwan AG (2004) Cytochrome c551 from Starkeya novella: characterization, spectroscopic properties, and ph ylogeny of a diheme protein of the SoxAX family. J Biol Chem 279:6252–6260 Lu W-P, Sw oboda EP, K elly DP (1985) Properties of the thiosulf ate-oxidizing multi-enzyme system from Thiobacillus versutus. Biochim Biophys Acta 828:116–122 Lübbe YJ, Youn H-S, Timkovich R, Dahl C (2006) Siro(haem)amide in Allochromatium vinosum and relevance of DsrL and DsrN, a homolog of cobyrinic acid a,c diamide synthase for sulfur oxidation. FEMS Microbiol Lett 261:194–202 Mussmann M, Richter M, Lombardot T, Meyerdierks A, Kuever J, Kube M, Glöckner FO, Amann R (2005) Clustered genes related to sulf ate respiration in uncultured prokaryotes support the theory of their concomittant horizontal transfer. J Bacteriol 187:7126–7137 Nelson DC, Castenholz RW (1981) Use of reduced sulfur compounds by Beggiatoa sp. J Bacteriol 147:140–154 Numata T, Fukai S, Ikeuchi Y, Suzuki T, Nureki O (2006) Structural basis for sulfur relay to RNA mediated by heterohexameric TusBCD complex. Structure 14:357–366 Odintsova EV, Wood AP, K elly DP (1993) Chemolithoautotrophic gro wth of Thiotrix r amosa. Arch Microbiol 160:152–157 Odintsova EV, Jannasch H, Mamone JA, Langworthy TA (1996) Thermothrix azorensis sp. nov., an oblig ately chemolithoautotrophic, sulfur -oxidizing, thermophilic bacterium. Int J Syst Bacteriol 46:422–428. Pattaragulwanit K, Brune DC, Trüper HG, Dahl C (1998) Molecular genetic evidence for extracytoplasmic localization of sulfur glob ules in Chromatium vinosum . Arch Microbiol 169:434–444 Petri R, Podgorsek L, Imhof f JF (2001) Ph ylogeny and distrib ution of the soxB gene among thiosulfate-oxidizing bacteria. FEMS Microbiol Lett 197:171–178 Pires RH, Venceslau SS, Morais F , Teixeira M, Xa vier AV, Pereira IAC (2006) Characterization of the Desulfovibrio desulfuricans ATCC 27774 DsrMKJOP comple x – a membrane-bound redox complex involved in the sulfate respiratory pathway. Biochemistry 45:249–262 Pittman MS, Robinson HC, Poole RK (2005) A bacterial glutathione transporter (Escherichia coli CydDC) exports reductant to the periplasm. J Biol Chem 280:32254–32261 Podgorsek L, Imhoff JF (1999) Tetrathionate production by sulfur oxidizing bacteria and the role of tetrathionate in the sulfur c ycle of Baltic Sea sediments. Aquat Microb Ecol 17:255–265 Pott AS, Dahl C (1998) Sirohaem-sulfite reductase and other proteins encoded in the dsr locus of Chromatium vinosum are in volved in the oxidation of intracellular sulfur . Microbiology 144:1881–1894 Prange A, Chauvistre R, Modrow H, Hormes J, Trüper HG, Dahl C (2002) Quantitative speciation of sulfur in bacterial sulfur glob ules: X-ray absorption spectroscopy reveals at least three different speciations of sulfur. Microbiology 148:267–276 Prange A, Engelhardt H, T rüper HG, Dahl C (2004) The role of the sulfur glob ule proteins of Allochromatium vinosum: mutagenesis of the sulfur globule protein genes and expression studies by real-time RT PCR. Arch Microbiol 182:165–174 Quentmeier A, Friedrich CG (2001) The cysteine residue of the SoxY protein as the active site of protein-bound sulfur oxidation of Paracoccus pantotrophus GB17. FEBS Lett 503:168–172 Reinartz M, Tschäpe T, Brüser T, Trüper HG, Dahl C (1998) Sulfide oxidation in the phototrophic bacterium Chromatium vinosum. Arch Microbiol 170:59–68 Rother D, Heinrich HJ, Quentmeier A, Bardischewsky F, Friedrich CG (2001) Novel genes of the sox gene cluster, mutagenesis of the flavoprotein SoxF, and evidence for a general sulfur-oxidizing system in Paracoccus pantotrophus GB17. J Bacteriol 183:4499–4508 Sander J, Engels-Schwarzlose S, Dahl C (2006) Importance of the DsrMKJOP complex for sulfur oxidation in Allochromatium vinosum and phylogenetic analysis of related complexes in other prokaryotes. Arch Microbiol 186:357–366 Schedel M, Trüper HG (1979) Purification of Thiobacillus denitrificans siroheme sulfite reductase and in vestigation of some molecular and catalytic properties. Biochim Bioph ys Acta 568:454–467

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Schedel M, Vanselow M, T rüper HG (1979) Siroheme sulf ite reductase from Chromatium vinosum. Purif ication and in vestigation of some of its molecular and catalytic properties. Arch Microbiol 121:29–36 Smith AJ, Lascelles J (1966) Thiosulphate metabolism and rhodanese in Chromatium sp. strain D. J Gen Microbiol 42:357–370 Sorokin DY, Lysenko AM, Mityushina LL, T ourova TP, Jones BE, Raine y FA, Robertson LA, Kuenen GJ (2001)Thioalkalimicrobium aerophilum gen. nov., sp. nov. and Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus gen. nov., sp. nov., Thioalkalivibrio nitratis sp. nov. and Thioalkalivibrio denitrificans sp. nov., novel obligately alkaliphilic and obligately chemolithoautotrophic sulfur -oxidizing bacteria from soda lak es. Int J Syst Ev ol Microbiol 51:565–580 Sorokin DY, Teske A, Robertson LA, K uenen JG (1999) Anaerobic oxidation of thiosulf ate to tetrathionate by oblig ately heterotrophic bacteria, belonging to the Pseudomonas stutzeri group. FEMS Microbiol Ecol 30:113–123 Steinmetz MA, Fischer U (1982) Cytochromes of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum. Purif ication, characterization and sulfur metabolism. Arch Microbiol 19:26 Suzuki H, Koyanagi T, Izuka S, Onishi A, K umagai H (2005) The yliA, -B, -C, and - D genes of Escherichia coli K-12 encode a no vel glutathione importer with an A TP-binding cassette. J Bacteriol 187:5861–5867 Trüper HG, Pfennig N (1966) Sulphur metabolism in Thiorhodaceae. III. Storage and turno ver of thiosulphate sulphur in Thiocapsa floridana and Chromatium species. Antonie V an Leeuwenhoek Int J Gen Mol Microbiol 32:261–276 Vermeglio A, Li J, Schoepp-Cothenet B, Pratt N, Knaff DB (2002) The role of high-potential iron protein and cytochrome c(8) as alternative electron donors to the reaction center ofChromatium vinosum. Biochemistry 41:8868–8875 Verté F , K ostanjevecki V, de Smet L, Me yer TE, Cusano vich MA, v an Beeumen JJ (2002) Identification of a thiosulf ate utilization gene cluster from the green phototrophic bacterium Chlorobium limicola. Biochemistry 41:2932–2945 Williams TJ, Zhang CL, Scott JH, Bazylinski D A (2006) Evidence for autotrophy via the reverse tricarboxylic acid c ycle in the marine magnetotactic coccus strain MC-1. Appl En viron Microbiol 72:1322–1329

Chapter 10

Sulfur Oxidation in Chlorobium tepidum (syn. Chlorobaculum tepidum): Genetic and Proteomic Analyses Leong-Keat Chan, Rachael Morgan-Kiss, Thomas E. Hanson

Abstract Chlorobium tepidum (syn. Chlorobaculum tepidum ) has become the model system of choice for understanding the unique biological attrib utes of the green sulfur bacteria, the Chlorobiaceae. This chapter describes how genome sequence enabled genetic and proteomic approaches are being applied to under stand pathways of anaerobic sulfur oxidation in C. tepidum. Reduced sulfur compounds are the sole source of e xogenous reductant that C. tepidum utilizes to drive all anabolic pathw ays necessary for cellular gro wth, including carbon and nitrogen fixation. The stoichiometries of sulfur -compound conversions in batch cultures confirm that sulf ide oxidation occurs via e xtracellular elemental sulfur. No intermediate is apparent for the oxidation of thiosulfate to sulfate, but thiosulfate oxidation appears to be stimulated when cells are gro wn autotrophically . Mutation of predicted sulfur oxidation genes leads to pleiotropic phenotypes that appear to affect the organization of photopigments in cells, suggesting that sulfur oxidation and light harv esting are tightly inte grated processes in C. tepidum. In concert with genetic approaches, proteomics coupled with subcellular fractionation is being used to identify proteins that are potentially involved in the oxidation of extracellular elemental sulfur. Observations on the ne xt generation of genetic techniques to augment those that currently e xist in C. tepidum and to e xtend proteomic observations are presented throughout.

10.1 Intr oduction 10.1.1 Bac kground Anaerobic sulfur oxidation is an important, but poorly understood aspect of the global sulfur cycle. This chapter will detail our recent efforts at identifying the relevant genes encoding enzymes of anaerobic sulfur oxidation in the green sulfur bacterium Chlorobium tepidum (syn. Chlorobaculum tepidum; Imhoff 2003). To this end, we have tak en tw o complementary approaches to this goal: genome-directed genetic analysis of predicted sulfur oxidation genes and proteomic analyses to identify 117 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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proteins with appropriate subcellular localization and properties to participate in the oxidation of elemental sulfur, which is accumulated extracellularly. C. tepidum oxidizes reduced sulfur compounds to provide all necessary reducing equivalents during growth. This includes ATP and proton moti ve force generation via photosynthetic electron transport (Brune 1995), dinitrogen fixation via nitrogenase in the absence of combined nitrogen (W ahlund and Madigan 1993), carbon dioxide fixation via the reducti ve tricarboxylic acid c ycle (Buchanan and Arnon 1990), and all other anabolic processes requiring NADH, NADPH, ferredoxin (Fd), and other redox mediators. Reduced Fd is the initial product of photosynthetic electron transport and is utilized directly for carbon dioxide and nitrogen f ixation. Reduced Fd can also be converted to NADPH via a recently described Fd:NADP+ oxidoreductase (Seo and Sakurai 2002). Light harv esting for photosynthetic electron transport is accomplished via the unique antenna structure of the chlorosome and ener gy transfer is accomplished by a type I reaction center (Frigaard and Bryant 2004). Much is kno wn about ho w C. tepidum generates intracellular redox mediators downstream of the reaction center. Much less is kno wn about the input side of the photosynthetic electron transport chain, where more limited information has been gleaned from biochemical studies of other green sulfur bacteria. C. tepidum is an obvious model system to pursue this question as it is genetically amenable (Frigaard and Bryant 2001; Hanson and T abita 2001) and the genome has been sequenced and annotated (Eisen et al. 2002).

10.1.2 Sulfur-Compound Dynamics in C. tepidum Batch Cultures C. tepidum is capable of using multiple forms of reduced sulfur with dif fering redox potentials to feed into the photosynthetic electron transport chain. In a typical batch culture medium, both sulf ide and thiosulf ate are pro vided to the cells (Wahlund et al. 1991). Under these conditions, sulfide is oxidized first and elemental sulfur accumulates as e xtracellular sulfur glob ules. Elemental sulfur accumulation is stoichiometric relati ve to the sulf ide consumed, with small amounts of thiosulf ate produced in some e xperiments (L.K. Chan, R.M. Morgan-Kiss, T.S. Weber, and T .E. Hanson, unpublished results). It has been reported that sulf ide is required for the gro wth of C. tepidum (Wahlund et al. 1991); ho wever, recent e xperiments in our laboratory and others ha ve sho wn that this is not the case and that C. tepidum can be gro wn with thiosulf ate and elemental sulfur as electron donors in the absence of e xogenously pro vided sulfide (data not shown). The oxidation of elemental sulfur commences only after sulf ide has been depleted to undetectable levels in the medium. This poses an interesting challenge to C. tepidum and other or ganisms that use elemental sulfur as an electron donor . Elemental sulfur is sparingly soluble (less than 5 µg l −1) and so to e xtract reducing

10 Sulfur Oxidation in Chlorobium tepidum (syn. Chlorobaculum tepidum) 119 Table 10.1 Thiosulfate consumption and sulfate production in triplicate batch cultures of Chlorobium tepidum WT2321 under autotrophic and mixotrophic conditions Culture

S2O32− consumed (mM)

SO42− produceda (mM)

SO42−/S2O32−

Mixotrophic

2.0

4.7

2.4

Autotrophic

9.0

18.9

2.1

Values were corrected for sulfate produced from elemental sulfur by subtracting the maxim al amount of elemental sulfur observed from the total sulfate produced. a

equivalents from this material C. tepidum must ha ve a specif ic mechanism for accessing and mobilizing it. The problem faced by C. tepidum conceptually resembles the problem of organisms that utilize insoluble materials as electron acceptors under anaerobic conditions. These or ganisms usually either directly attach to the substrate to f acilitate reduction or utilize e xtracellular redox mediators to reduce surfaces at a distance (Lies et al. 2005). In both cases, it appears that outer membrane associated cytochromes are important for deli vering reducing equivalents to the cell surface. Conceptually, it seems reasonable that a similar mechanism w orking in re verse could participate in the oxidation of e xtracellular elemental sulfur (Sect. 10.3). Thiosulfate oxidation commences after the onset of elemental sulfur oxidation. The e xtent of thiosulf ate oxidation appears to be controlled by the demand for reducing equivalents. This is ob vious when comparing mixotrophic (carbon dioxide and acetate as the carbon source) and autotrophic (carbon dioxide as the sole carbon source) gro wth. As sho wn in T able 10.1, mixotrophic batch cultures consumed 4.5-fold less thiosulf ate than autotrophic cultures. Biomass yields under autotrophic and mixotrophic conditions are essentially identical (data not sho wn), suggesting that C. tepidum is able to integrate signals for redox demand and adjust its sulfur-oxidizing capability appropriately. Sulfate is the final product of anaerobic sulfur oxidation produced by C. tepidum. It begins to accumulate when elemental sulfur oxidation commences, but no sooner. This observation, along with the 1:1 stoichiometry of elemental sulfur production from sulfide (L.K. Chan, R.M. Mor gan-Kiss, T.S. Weber, and T.E. Hanson, unpublished results), suggests that sulfide cannot be oxidized to sulfate without elemental sulfur as an intermediate. Sulf ate is produced proportionately to the amount of elemental sulfur and thiosulfate oxidized. The stoichiometry of sulfate production conforms to the expected value of 2:1, though this is somewhat elevated in mixotrophic cultures (T able 10.1), perhaps suggesting that cultures with lo wer demand for reducing equivalents store some form of sulfur as a hedge against lean times. The stoichiometry described above further suggests that C. tepidum fully oxidizes thiosulfate without any significant accumulation of side products. This is noteworthy as C. tepidum, like all other green sulfur bacteria and some purple sulfur bacteria, lacks genes encoding the SoxCD sulfur dehydrogenase that is required for the complete oxidation of thiosulf ate in the Paracoccus pantotrophus sulfur -oxidizing system studied by Friedrich et al. (2001).

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10.2 Genetic

Analyses

10.2.1 Organization of Genes Encoding Putative Sulfur Oxidation Functions Examination of the complete, annotated genome sequence of C. tepidum led to the observation that a number of the genes encoding presumptive sulfur oxidation enzymes are tightly clustered on the genome. The three largest clusters are outlined in Table 10.2. Together, these three gene clusters contain 52 kilobases of DN A encoding 68 proteins, which correspond to 2.4% of the total genome and 3.0% of the total number of protein coding genes in C. tepidum. As noted in Chap. 6 by Frigaard and Bryant, these genes are generally well conserved in other green sulfur bacterial genomes.

10.2.2 Mutations Affecting Sulfur Oxidation Have Secondary Effects on Light Harvesting The tight clustering of man y sulfur oxidation genes led us to use an in vitro transposition mutagenesis approach (Hayes 2003) to isolate insertion mutations in defined regions of the C. tepidum genome. We have used a transposon, TnOGm, derived from the plasposon pTnModOGm to generate mutant strains in a number of the C. tepidum sulfur islands, including those listed in T able 10.2. Interestingly, many of the isolated mutants display altered coloration relati ve to the wild-type parental strain that apparently results from shifts in the major in vi vo absorption band associated with bacteriochlorophyll c in the chlorosome (Table 10.3). Most of these strains display moderate to severe defects in growth and sulfur oxidation. One particular mutant in Sulfur Island-I, C5, carries a TnOGm insertion that has replaced approximately 5 kb of DNA encoding two subunits of the Hdr/Qmo complex, an Sqr homolog and se veral hypothetical proteins (Chan et al. 2007). This strain displays a blueshifted λmax for chlorosomal bacteriochlorophyll c at 750 nm relative to the 755 nm maximum of the wild type. In this strain, as in all TnOGm strains containing

Table 10.2 C. tepidum genomic sulfur islands Name

Genes

SI-I

CT0841CT0877 CT2238CT2252 CT1009CT1027

SI-II SI-Sox Totals

No. of base pairs

Putative sulfur oxidation

Hypothetical

Gene Products

26,636

18

18

12,505

11

3

13,567

8

10

Dsr, ApsBA, Sat, Hdr/Qmo, Sqr Dsr, siroheme biosynthesis Sox complex (no SoxCD)

52,708

37

31

10 Sulfur Oxidation in Chlorobium tepidum (syn. Chlorobaculum tepidum) 121 Table 10.3 Shifts in the major chlorosome absorption peak in TnOGm insertions in Sulfur Island I

C. tepidum strains carrying

TnOGm site

Genes

No. of strains

In vivo λmaxa (nm)

MeOH λmaxa (nm)

None (WT2321) SI-I-2

– CT0854-CT0869

SI-I-3

CT0869-CT0877

– 2 2 5 7 9

755 750 760 747 750 752

669 669 669 669 669 669

a Measurements are the means of three independent cultures. The standard de triplicates was 2 nm or less.

viation in all

similar shifts examined to date, the absorption maximum difference disappears when pigments are e xtracted into methanol (T able 10.3), indicating that the shift is due to the arrangement of the bacteriochlorophyll molecules in the chlorosome rather than the bulk properties of the molecules themselv es. Strain C5 w as also found to display many other properties suggesting that this strain is fundamentally compromised in the way that it deals with incident light ener gy, including increases in baseplate bacteriochlorophyll a fluorescence yield, which may indicate poor ener gy transfer efficiency between the chlorosome and reaction center (W ang et al. 1990; Melo et al. 2000). None of the genes deleted in this mutant are obvious candidates for chlorosome structural proteins, so it seems that the ef fect of this mutation is likely indirect. Strain C5 is also se verely compromised for gro wth (Chan et al. 2007) and sulfur oxidation (L.K. Chan, R.M. Mor gan-Kiss, T.S. Weber, and T.E. Hanson, unpublished results); therefore, we propose that the observ ed alterations in light harv esting and antenna function are a secondary result of defects in sulfur oxidation pathways. Similar effects were seen in a strain of C. tepidum lacking the rib ulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) like protein encoded by CT1772 that was also defective in thiosulfate oxidation (Hanson and Tabita 2001). The major difference between these two strains is that the Rubisco-like protein mutant had a decrease in the level of bacteriochlorophyll per protein, while strain C5 displays no such defect. These results suggest that C. tepidum regulates the function of its antenna apparatus in response to the availability of reductant, as has been reported by others w orking with chlorosomes in Chloroflexus aurantiacus and C. tepidum (Wang et al. 1990; Melo et al. 2000). Details regarding the nature of such a signal and the mechanism by which it is transmitted are currently unclear, but the fact that mutants compromised in sulfur oxidation af fect this regulation indicates a sulfur oxidation intermediate may be in volved.

10.2.3

Additional Genetic Techniques Are Needed

While the ability to mak e targeted gene disruptions in C. tepidum has been and will continue to be v aluable for understanding the biology of this or ganism, additional techniques will be required to enable more sophisticated and subtle e xperimental

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manipulations of the genome. T wo primary areas are vie wed as lik ely to yield the greatest benefit. The first is epitope tagging where a gene is modif ied in place on the C. tepidum chromosome to produce a v ariant protein that can be recognized by tagspecific antibodies. Methods for appending a hexahistidine tag to specific gene products by manipulating the genes in place on the chromosome were recently reported for Escherichia coli (Morgan-Kiss and Cronan 2004). This allo ws the detection and quantification of protein e xpression without the need for generating a specif ic antibody for each protein of interest. In addition, the hexahistidine tag allows purification of the tagged protein by chromatographic methods (Morgan-Kiss and Cronan 2004). This enables the detection and analysis of protein complexes and can greatly facilitate biochemical analysis of the gene product. The second is to de velop a chromosomal e xpression system with a re gulated promoter. Currently, no plasmids e xist for the complementation of mutants in C. tepidum . Failing the de velopment of these systems, one can en vision complementation of mutants by ectopic copies of genes under a re gulated promoter. This is one route to the e xpression of site-directed mutants in genes of interest in a null mutant background to enable a more detailed understanding of structure–function relationships in the original physiological background. We are currently attempting to adapt protocols for both techniques for use in C. tepidum.

10.3 Pr oteomic Analysis 10.3.1 Why

Proteomics?

As noted in T able 10.2, a lar ge number of genes, almost 50% of the total, in the Sulfur Islands of C. tepidum encode hypothetical proteins with unknown functions relative to sulfur oxidation. Thus, their biological functions ha ve yet to be disco vered despite prior in vestigations into the biochemistry of sulfur oxidation. While the genetic approach described in Sect. 10.2 will yield some information as to what genes are important, understanding what proteins are e xpressed during gro wth on particular sulfur compounds and what their location is in the cell (or outside of it) can provide a second line of e vidence for the involvement of particular gene products in the oxidation of particular sulfur compounds. This will be particularly true if some sulfur oxidation genes are essential to C. tepidum’s viability.

10.3.2

Proteomic Analysis of Subcellular Fractions

Specific hypotheses amenable to proteomic analyses can be posed re garding particularly problematic aspects of anaerobic sulfur oxidation lik e the oxidation of extracellular elemental sulfur. One such hypothesis that can be directly tested more easily by proteomics than by current genetic techniques in C. tepidum is whether or

10 Sulfur Oxidation in Chlorobium tepidum (syn. Chlorobaculum tepidum) 123 Table 10.4 Proteins identified from inner- and outer-membrane fractions of C. tepidum No. of MS/MS peptides a CT no.

Annotation

Total membrane

Outer membrane

CT2144 CT1499 CT0893 CT1804 CT1353 CT1447 CT0254 CT0641 CT1157 CT0638 CT2033

Outer surface protein FmoA, Bchla binding protein Hypothetical protein Hypothetical protein OmpA family protein Serine protease OmpH, outer-surface protein PscD, reaction center protein Hypothetical protein Peptidoglycan-associated lipoprotein ATP synthase F1, α subunit

12 21 10 30 5 7 7 2 3 5 8

16 16 15 12 9 9 4 0 0 0 0

Liquid chromatography–tandem mass spectrometry (MS/MS) was performed on proteolytically digested bands from 1D sodium dodec yl sulfate polyacrylamide gel electrophoresis gels of the indicated fractions.

a

not C. tepidum contains electron transfer proteins associated with the outer membrane or cell surf ace. This w ould provide a clear mechanism for transferring reducing equi valents either directly to elemental sulfur to liberate sulf ide and polysulfides, or to a redox-shuttling compound. Outer -membrane proteins ha ve been implicated in sulfur oxidation in Thiobacillus ferroxidans and in mediating the reduction of e xtracellular electron acceptors in Shewanella oneidensis MR-1 (Buonfiglio et al. 1993; Lies et al. 2005). A method previously used to enrich outer-membrane proteins from the Gram-negative marine bacterium Hyphomonas jannaschiana (Shen et al. 1989) was applied to C. tepidumand was found to reliably provide fractionation of chlorosome-depleted membranes. This method relies on the selecti ve solubilization of inner -membrane proteins by nonionic detergents. Proteins have been identified from C. tepidum total (inner and outer) and outer-membrane fractions (Nonidet P-40 insoluble fraction) by standard tandem mass spectrometry methods. The results indicate that the fractionation protocol has specifically enriched outer -membrane proteins from a total membrane preparation (Table 10.4). This is clearly seen when the distrib ution of known inner-membrane proteins lik e the F1 A TPase α subunit and PscD reaction center sub unit are e xamined. Peptides for these proteins were found solely in the total membrane fraction, and not in the outer -membrane fraction. The enrichment of predicted outer -surface and outer membrane proteins lik e CT2033 and OmpA w as observed in terms of the number of peptides detected in the outer-membrane fraction. The exception to this rule is the FmoA protein, which was found to be abundant in both fractions. The FmoA protein is a peripherally membrane associated protein in C. tepidum that mediates the association between the inner membrane and the chlorosome (Imhoff 2003). This result indicates that the “outer-membrane” fraction prepared by selecti ve detergents probably enriches both outer -membrane proteins and peripheral-membrane proteins together . The localization of CT1087, sulf ide:

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quinone oxidoreductase that is likely a peripheral-membrane protein (Shahak et al. 1992), to this outer-membrane fraction also supports this notion (L.K. Chan, R.M. Morgan-Kiss, T.S. Weber, and T.E. Hanson, unpublished results). The outer-membrane and peripheral-membrane fraction has been e xamined by heme staining after electrophoresis and three hemoproteins were re vealed (Chan et al. 2007). These are currently being identif ied (R.M. Morgan-Kiss, L.K. Chan, and T.E. Hanson, unpublished results). Given the caveat regarding the composition of the outer -membrane fraction, these hemoproteins may be either peripheralmembrane proteins or outer-membrane proteins. The C. tepidum genome encodes 26 potential hemoproteins that contain a consensus CXXCH heme attachment motif. Fifteen of these are associated with functional annotations and represent expected hemoproteins of the Dsr comple x, Sox system, and others. The remaining 11 potential hemoproteins are not functionally annotated. If an y of these are found in the outer-membrane fraction, then the next obvious step will be to determine their subcellular localization by emplo ying the classic microbial genetic technique of alkaline phospatase translational fusions. These fusions are only active when the y are transported to the cell e xterior and can be used in concert with β-galactosidase fusion proteins to assess protein topology (Haardt and Bremer 1996). Construction of these fusions in the C. tepidum chromosome will further develop the capabilities for genetics in this system by demonstrating the use of translational reporter fusions.

10.4 Conclusions The combination of both genetic and proteomic techniques to address hypotheses proposed on the basis of genomic sequences is a hallmark of the postgenomics era. Both approaches ha ve their strengths and weaknesses and ha ve only recently been applied to C. tepidum . The de velopment and application of impro ved genetic techniques and higher -throughput proteomic techniques will continue to impro ve the ability to perform more sophisticated and subtle e xperiments in these f ascinating organisms. With the accumulation of additional green sulfur bacterial genomes (see Chap. 6 by Frigaard and Bryant), the ne xt large challenge in the field will be to extend the techniques to these additional strains so that unique genetic features of each can be assessed in their proper biological context. In addition, the extension of theunderstanding developed through these studies in laboratory cultures must be extended to environmental populations to truly understand how the green sulfur bacteria function in situ. Acknowledgements. The authors would like to thank Joy Lawani, Jessica Martin, Egle Burbaite, Tim Weber, and Michele Madorma for excellent technical assistance over the course of the project and Ann O’Brien for protein identif ication. This project was supported by grants from the National Science Foundation (MCB-0447649 to T.E.H. and Delaware EPSCoR grant EPS-0447610 through the Delaware Biotechnology Institute), and utilized common instrumentation f acilities provided in part by the National Institutes of Health (P20-RR116472-04 from the IDeA Netw orks of Biomedical Research Excellence program of the National Center for Research Resources).

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References Brune DC (1995) Sulfur compounds as photosynthetic electron donors. In Bauer CE (ed) Anoxygenic photosynthetic bacteria. Kluwer, Amsterdam, pp 847–870 Buchanan BB, Arnon DI (1990) A re verse Krebs c ycle in photosynthesis: consensus at last. Photosynth Res 24:47–53 Buonfiglio V, Polidoro M, Flora L, Citro G, Valenti P, Orsi N (1993) Identification of two outer membraneproteins involved in the oxidation of sulphur compounds in Thiobacillus ferrooxidans. International Symposium on Adv ances on Bioh ydrometallurgy: Microbiol Appl 11:43–50 Chan LK, Morgan-Kiss R, Hanson TE (2007) Genetic and proteomic studies of sulfur oxidation in Chlorobium tepidum. In: Hell R, Dahl C, Leustek T , Knaff D (eds) Sulfur in phototrophic organisms. Springer, New York (in press) Eisen JA, Nelson KE, Paulsen IT, Heidelberg JF, Wu M, Dodson RJ, Deboy R, Gwinn ML, Nelson WC, Haft DH, Hickey EK, Peterson JD, Durkin AS, Kolonay JL, Yang F, Holt I, Umayam LA, Mason T, Brenner M, Shea TP, Parksey D, Nierman WC, Feldblyum TV, Hansen CL, Craven MB, Radune D, V amathevan J, Khouri H, White O, Gruber TM, K etchum KA, V enter JC, Tettelin H, Bryant D A, Fraser CM (2002) The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, green-sulfur bacterium. Proc Natl Aacad Sci USA 99:9509–9514 Friedrich CG, Rother D, Bardischewsky F, Quentmeier A, Fischer J (2001) Oxidation of reduced inorganic sulfur compounds by bacteria: emer gence of a common mechanism? Appl En viron Microbiol 67:2873–2882 Frigaard NU, Bryant D A (2001) Chromosomal gene inacti vation in the green sulfur bacterium Chlorobium tepidum by natural transformation. Appl Environ Microbiol 67:2538–2544 Frigaard NU, Bryant DA (2004) Seeing green bacteria in a ne w light: Genomics-enabled studies of the photosynthetic apparatus in green sulfur bacteria and f ilamentous anoxygenic phototrophic bacteria. Arch Microbiol 182:265–276 Haardt M, Bremer E (1996) Use of phoA and lacZ fusions to study the membrane topology of ProW, a component of the osmore gulated ProU transport system of Escherichia coli . J Bacteriol 178:5370–5381 Hanson TE, Tabita FR (2001) A ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress. Proc Natl Acad Sci USA 98:4397–4402 Hayes F (2003) T ransposon-based strategies for microbial functional genomics and proteomics. Annu Rev Genet 37:3–29 Imhoff JF (2003) Ph ylogenetic taxonomy of the f amily Chlorobiaceae on the basis of 16s rRN A and fmo (Fenna-Matthe ws-Olson protein) gene sequences. Int J Syst Ev ol Microbiol 53:941–951 Lies DP, Hernandez ME, Kappler A, Mielk e RE, Gralnick J A, Newman DK (2005) Shewanella oneidensis MR-1 uses o verlapping pathw ays for iron reduction at a distance and by direct contact under conditions relevant for biofilms. Appl Environ Microbiol 71:4414–4426 Melo TB, Frig aard NU, Matsuura K, Razi Naqvi K (2000) Electronic ener gy transfer in volving carotenoid pigments in chlorosomes of tw o green bacteria: Chlorobium tepidum and Chloroflexus aurantiacus. Spectrochim Acta A Mol Biomol Spectrosc 56:2001–2010 Morgan-Kiss RM, Cronan JE (2004) The Escherichia coli fadK (ydiD) gene encodes an anerobically regulated short chain acyl-CoA synthetase. J Biol Chem 279:37324–37333 Seo D, Sakurai H (2002) Purif ication and characterization of ferredoxin-N AD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum. Biochim Biophys Acta 1597:123–132 Shahak Y, Arieli B, P adan E, Hauska G (1992) Sulf ide quinone reductase (SQR) acti vity in Chlorobium. FEBS Lett 299:127–130 Shen N, Dag asan L, Sledjeski D, W einer RM (1989) Major outer membrane proteins unique to reproductive cells of Hyphomonas jannaschiana. J Bacteriol 171:2226–2228

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Wahlund TM, Madigan MT (1993) Nitrogen f ixation by the thermophilic green sulfur bacterium Chlorobium tepidum. J Bacteriol 175:474–478 Wahlund TM, Woese CR, Castenholz RW, Madigan MT (1991) A thermophilic green sulfur bacterium from Ne w Zealand hot springs, Chlorobium tepidum sp. no v. Archi v Microbiol 156:81–90 Wang J, Brune DC, Blank enship RE (1990) Ef fects of oxidants and reductants on the ef ficiency of excitation transfer in green photosynthetic bacteria. Biochim Bioph ys Acta 1015:457–463

Chapter 11

Structural Insights into Component SoxY of the Thiosulfate-Oxidizing Multienzyme System of Chlorobaculum thiosulfatiphilum Jan Stout, Lina De Smet, Bjor n Vergauwen, Savvas Savvides, Jozef Van Beeumen

Abstract We discuss the crystal structure of component SoxY of the SoxYZ complex that is known to play a key role in the sulfur-oxidizing multienzyme system of the green sulfur bacterium Chlorobaculum thiosulfatiphilum. The protein appears to be structurally similar to a monomeric immunoglob ulin-like protein that oligomerizes into a tetramer via conserv ed contact re gions between the monomers. The tetramer is a dimer of dimers and e xhibits one lar ge hydrophobic contact region in each dimer, and two small hydrophilic interface patches between the dimers. At the tetramer interf ace patch, tw o conserv ed redox-acti ve C-terminal cysteines form an intersub unit disulfide bridge. Depending on the redox state of the cysteines, the tetramer is in equilibrium with the dimers, each one of which is a candidate to covalently bind a thiosulfate molecule by means of a thiol–disulfide exchange reaction with the interprotein disulfide bonds. The significant conservation level of the interfaces, the specific interactions between the subunits in the tetramer, and the dimer–tetramer equilibrium suggest that these SoxY oligomers are biologically relevant. A possible role for these protomers in the mechanism of the Soxsystem is proposed.

11.1 Intr oduction The oxidation of thiosulf ate proceeds in Eubacteria and Archaea mainly via tw o distinct pathw ays: (1) the “tetrathionate” pathw ay, which is mainly restricted to acidophilic thiobacilli and oxidizes thiosulf ate to sulf ate via tetrathionate as an intermediate (Kelly et al. 1997), and (2) the ubiquitous periplasmic sulfur oxidizing (Sox) pathway found in neutrophilic, respiratory, and phototrophic Proteobacteria and in green sulfur bacteria (Friedrich et al. 2001, Friedrich 2005). The best characterized Sox systems to date are those of Paracoccus versutus (Lu and Kelly 1983; Lu et al. 1985; Lu 1986) and Paracoccus pantotr ophus (Friedrich et al. 2000; Rother et al. 2001), where the combination of four components of the latter organism, SoxYZ, SoxAX, SoxB, and SoxCD, results in an acti ve system exhibiting a full thiosulfate oxidizing activity and generates eight electrons and two sulfate molecules as end products (Friedrich et al. 2000). Although the in volvement of 127 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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these components, and, more recently, of other Sox components such as SoxW and SoxV (Bardische wsky and Friedrich 2001; Appia-A yme and Berks 2002; Bardischewsky et al. 2006a), SoxR and SoxS (Rother et al. 2005), SoxT (Lahiri et al. 2006), and SoxF (Bardische wsky et al. 2006b), in the Sox system has been established, their specific role, with the e xception of that of SoxYZ, remains hard to determine. SoxYZ has the capacity to bind reduced sulfur substrates via a thioether or a thioester bond at a conserved C-terminal cysteine of the SoxY subunit (Quentmeier and Friedrich 2001), and it is thought to present this acti vated sulfur substrate molecule to the other oxidizing Sox enzymes. Although this widely accepted vie w is supported by clear e vidence (Quentmeier and Friedrich 2001), it remains unknown, ho wever, ho w this sulfur substrate binding step e xactly occurs. In the reaction model proposed by Friedrich et al. (2001), the SoxAX c ytochrome c is proposed to mediate the binding, and an appropriate reaction mechanism based on the crystal structure of SoxAX for this binding event was postulated by Bamford et al. (2002). In this model, SoxAX transfers a sulfur substrate molecule, covalently bound to a conserv ed cysteine residue that also functions as the sixth axial ligand of a heme prosthetic group, to the sulfhydryl group of a strictly conserv ed cysteine residue located in a highly conserved C-terminal sequence motif of SoxY. An alternative mechanism was also proposed in which SoxY binds the sulfur substrate via an intersubunit disulfide bridge formed with a second SoxY molecule (Quentmeier et al. 2003). This hypothesis w as based on the observ ation that SoxYZ e xhibits redox activity by means of its conserv ed cysteine residue (Quentmeier et al. 2003) and is further supported by recent results indicating that SoxYZ, ha ving SoxY disulfide-bridged subunits and SoxY–persulf ide subunits, increases the thiosulf ateoxidizing activity of the system (A. Quentmeier, P Jannig, and C.G. Friedrich, personal communication). We here present the crystal structure of a “standalone” tetrameric SoxY from the green sulfur bacterium Chlorobium limicola f. sp. thiosulfatophilum DSM 249T , recently renamed Chlorobaculum thiosulfatiphilum (Imhof f 2003). The structure re veals specif ic and well-conserv ed contact interf aces between the subunits which are linked via an intersubunit disulfide bond, offering a first detailed picture of the structural basis of its redox acti vity.

11.2 SoxY

Structure

11.2.1 Ov erall Structure The SoxY crystal structure reveals an αβ-protein consisting of an N-terminal α-helix and a β-sandwich domain (Fig. 11.1). The two SoxY subunits present in the asymmetric unit interact e xtensively with each other and form an e xtended β-sandwich structure with six and eight β-strands in the upper and the lower layer, respectively (Fig. 11.1d, e). On their turn, two of these SoxY dimers interact with each other via

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Fig. 11.1 a The SoxY tetramer of Chlorobaculum thiosulfatiphilum viewed from the side, showing dimer A and dimer A′, which are related via a crystallographic twofold axis (dashed line with filled ellipsoid on top ). Tetramerization occurs by means of tw o small interf ace patches at the distal ends of the oligomer and is indicated by two dashed ellipsoids . b Sodium dodec yl sulfate polyacrylamide gel electrophoresis of crystallized SoxY (lanes 1 and 2) and purified SoxY (lanes 3 and 4). Lanes 1 and 3 are protein samples boiled at 95°C in Laemmli b uffer. Lanes 2 and 4 are protein samples treated with b-mercaptoethanol before boiling. c SoxY monomer sho wing the secondary structure elements. The N-terminal α-helix (S1–F20) is connected via a loop to a β-sandwich domain (I31–G122) ha ving seven antiparallel β-strands: β-strands a (I31–K34), b (A43–T51), c (N58–T63), c´ (M70–L77), e (P82–M90), f (E94–A102), and g (K105–T116). The nomenclature of the β-strands was taken from Bork et al. (1994). The red β-strands (a, b, and e) form β-sheet I. The blue β-strands (c, c c´, f, and g) constitute β-sheet II. d, e The SoxY dimer orientated with both β-sheets in the plane of and orthogonal to the page, respecti vely. The tw o monomers are shown in different colors

a tw ofold crystallographic axis and assemble into a tetramer (Fig. 11.1a). This agrees well with analytical gel f iltration e xperiments sho wing that recombinant SoxY is present in solution as a 52-kDa tetramer (Stout et al. 2006). Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis re vealed that the tetramer is composed of dimer sub units which are co valently link ed via a disulfide bridge (Fig. 11.1b), a structural property that w as also demonstrated for SoxY of P. pantotrophus (Quentmeier et al. 2003). Interestingly, when solubilized, crystalline SoxY migrates in a SDS polyacrylamide gel like untreated recombinant SoxY does, i.e., as dimer sub units link ed via a disulf ide bridge. This is quite

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remarkable since the thiol-reducing reagent dithiothreitol (DTT) is absolutely required for successful crystallization (Stout et al. 2006) and is e xpected to reduce solvent-exposed disulfide bonds at millimolar concentrations (10 mM), even under the acidic crystal growth conditions (pH 4.0). Being the only cysteine residue in the mature SoxY, C120 has logically been assigned to be the site of co valent linkage. Consequently, a total of tw o disulf ide bridges are formed in the tetramer . Unfortunately, no detailed structure for the disulf ide bridge re gion could be determined, owing to the v ery poor quality of the electron densities of the C120 residues. This is an indication for a high structural fle xibility of this re gion which in all likelihood is correlated with the high glycine content of the GGCG(G) motif. Nevertheless, the molecular assembly of the tetramer mak es it feasible to deduce the position of the disulfide bridges, as will be discussed later.

11.2.2 So xY Monomer The 13-kDa SoxY monomer consists of an N-terminal α-helix (S1–F20) pack ed against an immunoglob ulin (Ig) lik e domain (I31–I117). Both structural elements are connected by a loop (G21–T30) co vering one side of the β-sandwich domain (Fig. 11.1c). Variations are known to exist in the 3D topology of Ig domains, on the basis of which distinct Ig sets have even been defined (Williams and Barclay 1988; Bork et al. 1994; Harpaz and Chothia 1994). The Ig-lik e fold of SoxY is an s-type Ig domain on the basis of the classif ication scheme of Bork et al. (1994).

11.2.3 So xY Dimer The two monomers within the crystal asymmetric unit use one edge of their Ig-like domains to form a β-sandwich dimer constituted of two extended β-sheets: β-sheet I consists of strands a, b, and e of sub units A and B, and β-sheet II is composed of strands c, c´, f, and g of both sub units. Both β-sheets in SoxY exhibit a continuous hydrogen-bonding network (Fig. 11.1d, e). This is in contrast to canonical extended β-sandwiches which have one continuous (sheet II) and one discontinuous (sheet I) β-sheet, separated in the middle by w ater molecules (Richardson and Richardson 2002). In the dimer interf ace of SoxY , however, a total of 12 direct intersub unit hydrogen bonds are present between the main chains of β-strands c′ and e and their equivalent β-strands of the opposite monomer. The structural and physicochemical characteristics of the SoxY dimer interf ace are similar to those of other stable protein–protein interf aces in kno wn protein complexes. The dimer interf ace b uries a surf ace of 1,522 Å 2, which agrees well with canonical protein–protein interf aces (1,200–2,000 Å 2) (Lo Conte et al. 1999; Bahadur et al. 2004), and is mainly hydrophobic (70.5%). It has a substantial fraction (40%) of completely b uried interf ace atoms (Stout et al. 2007), a v alue in

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accordance with the observ ed values corresponding to completely shielded inter face atoms (34–36%) (Lo Conte et al. 1999; Bahadur et al. 2003). The burial of the hydrophobic surface is assumed to be an important feature of stable protein–protein interfaces (Young et al. 1994; Jones and Thornton 1996; Tsai et al. 1997). In the case of SoxY, the major part of the hydrophobic residues is located at the centre of the dimer interface. These residues are flanked at the bottom and the top by the two layers of intersubunit hydrogen bonds (12 in total) that are part of the β-ladder network of the two extended β-sheets described above. Hydrogen bonds, being formed by complementary electrostatic, polar, or charged-atom groups of both proteins, are thought to impose some degree of specificity in protein–protein binding events (Xu et al. 1997a, b). Several studies reported that interface residues are, on average, more conserved than solvent-exposed surface residues (Valdar and Thornton 2001a; Caf frey et al. 2004; Mintseris and Weng 2005). In the case of SoxY , bootstrap analyses demonstrated that the “core” of the dimer interf ace, which consists of ten residues that have at least 95% of their total surf aces b uried at the interf ace, is signif icantly conserved (Stout et al. 2007).

11.2.4 So xY Tetramer In contrast to the dimer interf ace, contacts that stabilize the tetramer occur at the distal ends of the tetramer by virtue of tw o small interface patches, separated by a solvent-filled space between the two SoxY dimers (Fig. 11.1a). The buried surface area per tetramer interf ace patch is 630 Å 2 and the tetramer interf ace is less polar (nonpolar area fraction of 57.1 %) than the dimer interface (Stout et al. 2007). Two strongly conserv ed protein re gions of both SoxY sub units mak e up the tetramer interface: the a–b loop connecting β-strands a and b (P36–G42) and the be ginning of β-strand b (A43–P46), R111–I117 of β-strand g, and the equi valent regions of the symmetry-related subunit. The center of the interface is composed of the strictly conserved peptide sequences E37–E40, exhibiting a β-like conformation, of the a–b loop of both subunits. These short peptides are orientated in an antiparallel way but do not interact via direct intersub unit backbone hydrogen bonds. Instead, six ordered w ater molecules and tw o ordered chloride ions intercalate between the peptides and set up a hydrogen-bonding netw ork with both sub units. The chloride ions appear to be an artif act of crystal cryo-cooling at 100 K, as they are replaced by water molecules in a room-temperature crystal structure. Owing to the high level of hydration, and in contrast to the dimer interf ace, only a small fraction (6.7%) of the tetramer interface atoms are fully buried. Both sides of one tetramer interface patch are defined by two strictly conserved salt bridges (3.8 Å) between E40 and K114 of the symmetry-related subunit (Fig. 11.2). A second glutamate residue, E37, of the symmetry-related sub unit with respect to E40, is positioned with its carboxyl group between the carboxyl group of E37, and NZ of K114 and is within hydrogen-bonding distance of OE2 of E40 (2.8 Å). This

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Fig. 11.2 The tetramer interf ace patch, made up by the sub units A and A ′, with the tw o conserved salt bridges (dashed lines) between E40–K114´ and E40´–K114 defining both sides of the patch. E37´ and E37 are within hydrogen-bonding distance of E40´ and E40´, respectively. K114, K114´, and the differently colored K92, K92´, R89, and R89´ are potential candidates to interact with a modified, sulfur substrate bound cysteine

type of electrostatic interaction, where a third char ged residue interacts with a pair of salt bridge forming residues, occurs frequently at protein–protein interfaces (Xu et al. 1997a). By analogy to the dimer interf ace, a bootstrap procedure to assess the a verage conservation of the tetramer interf ace revealed that about 0.001% of randomly picked sets of surface residues (leaving out the dimer interface residues) have at least the same (or higher) le vel of conserv ation, indicating that the tetramer interface patches are highly conserv ed regions of the protein surf ace (Stout et al. 2007).

11.2.5 Location of the Disulfide Bridges and the P otential Sulfur Binding Site Although the reacti ve C-terminal c ysteines could not be modeled, the position of the disulfide bridges can be deduced on the basis of the quaternary structureof SoxY which brings tw o symmetry-related C120 residues in close proximity to one another to allow formation of a disulfide bridge. The last modeled residues of both β-strands g, I117 and I117´, protrude from the tetramer interf ace into the solv ent (Fig. 11.2). It is therefore lik ely that the intersub unit disulfide bond between the C120 residues is also e xposed to the solv ent, residing at the top of each tetramer interface patch. Several residues which are located on, or in the vicinity of, the tetramer interf ace patch may electrostatically stabilize the resulting adduct after sulfur substratebinding. The conserved residues K114 and K114´, which are also in volved in salt-bridge formation as described in Sect. 11.2.4, and the conserv ed residues R89 and R89´ are

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good candidates to interact with the ne gatively char ged S-thiocysteinesulfonate (Fig. 11.2). The nonconserv ed residues K92 and K92´ are also potential candidates for such an interaction.

11.3 Discussion To date, most biochemical data ha ve been generated using the SoxYZ heterodimer (Friedrich et al. 2000; Rother et al. 2001; Quentmeier and Friedrich 2001; Quentmeier et al. 2003), which is generally believed to be a stable, obligate complex that is only active in the form of a heterodimer. A combination of structural and biochemical results, however, suggests that the individual proteins, apart from forming a SoxYZ comple x, may also e xist on their o wn. First, a recently determined SoxZ structure, showing an apparent dimer , indicates that also this protein can be stable without being associated with another protein (pdb 1v8h, unpublished data). Second, the SoxY structure discussed here e xhibits specific intersubunit interactions, which argues against the notion that SoxYZ is an obligate comple x in which both components depend on each other for their folding and structural inte grity. A f irst strong ar gument supporting the biological rele vance of the SoxY oligomers is the statistically signif icant conserv ation le vels of the dimer and tetramer interf aces indicated by bootstrap analyses. These are in agreement with several studies which state that protein–protein interf aces are more conserved than solvent-exposed protein surfaces (Valdar and Thornton 2001a; Caffrey et al. 2004; Mintseris and Weng 2005) and biologically irrelevant crystal contact points (Valdar and Thornton 2001b). The high conserv ation level of the tetramer interf ace can be explained by the f act that these residues are in the vicinity of the sulfur substrate binding cysteine, and therefore can also be in volved in transient interactions with other components of the Sox system. Hence, the e volutionary pressure on these residues may be higher than that on other surf ace residues since their combination is likely to be optimal for interactions with the dif ferent Sox enzymes. In addition, a number of biochemical data and structural and physicochemical analyses of the interf aces convincingly support the rele vance of SoxY oligomers. Analytical gel filtration demonstrated that reduced SoxY is eluted as a dimer, indicating that the dimer on its o wn constitutes a stable protomer (Stout et al. 2007). Furthermore, three major properties of the dimer interf ace strongly ar gue for the SoxY dimer as a specif ic protein–protein comple x. Firstly, a considerable surf ace area (1,522 Å 2) is b uried at this dimer interf ace, which, secondly , has a distinct central core of hydrophobic residues. Several studies (Miller 1989; Tsai et al. 1997; Bahadur et al. 2003) have emphasized the presence of such a hydrophobic center in medium and large protein–protein interfaces, strengthening the basic paradigm that hydrophobicity is a major stabilizing factor in protein–protein association (Chothia and Janin 1975). Thirdly, the considerable number of hydrogen bonds (12 in total) argues for a biologically rele vant protein–protein interface. These hydrogen bonds not only make a considerable energetic contribution to protein–protein binding, but also enforce binding specif icity due to the electrostatic complementarity of the

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hydrogen-bond donor and acceptor groups (Xu et al. 1997a, b). All hydrogen bonds at the dimer interf ace are part of the hydrogen-bond ladder pattern across the tw o extended β-sheets. This arrangement of tw o ro ws of hydrogen-bond donor and acceptor groups is specif ic for β-sandwiches and lik ely restricts potential binding partners to proteins having a similar arrangement of donor and acceptor groups. In contrast to the e xtended dimer interf ace, the tw o dimers within the tetramer interact with each other via two small interface patches at the top and the bottom of the tetramer. These contact interf aces can be interpreted as biologically irrele vant crystal contacts on the basis of their limited b uried surf ace area (Bahadur e t al. 2004), and their high le vel of hydration (Rodier et al. 2005). This interpretation, however, w ould be in contrast to their high conserv ation le vels. In f act, a small interface for tetramerization can mak e it easier for dimer–tetramer transitions to occur. Such transitions were observ ed for P. pantotrophus SoxYZ (Quentmeier et al. 2003) and for C. thiosulfatiphilum SoxY (Stout et al. 2007) to be dependent on the oxidation state of the C-terminal c ysteine and the molecule adhered to this residue. It has been stated that a considerable number of proteins can change their activity by forming weak transient oligomers (Nooren and Thornton 2003). It w as reasoned that these proteins can easily stabilize or weak en their complexes by creating or breaking a limited number of interactions at small interf aces, thus making a dynamic response to a change in en vironment or to a co valent modif ication possible. In the case of SoxY, the interface reveals a number of hydrogen bonds and four conserved salt bridges which were sho wn by the gel f iltration experiments to be of less importance for the inte grity of the tetramer than the disulf ide bridges (Stout et al. 2007). Indeed, treatment of SoxY with dif ferent reducing agents such as DTT, glutathione, sulf ide, and sulf ite, where the last three sulfur compounds remain covalently linked to the C-terminal c ysteine, resulted in the dissociation of the SoxY tetramer into dimers. An e xception to this beha vior, ho wever, w as observed for a SoxY–thiosulfate adduct which remains a tetramer after the covalent addition of this substrate. These observations are suggestive for the possibility that the chemical nature of the adhered molecule plays a k ey role in the stabilization of the tetramer interface. The second feature, the creation of an intersub unit disulfide bond, can also be e xplained by the specif ic electrostatic properties of the tetramer interface. Such an event can only take place when two dimers approach each other and interact in a way that eventually leads to the closure of this bond. The formation of the conserved intersubunit salt bridges at the tetramer interf ace may well be of major importance in this event. Salt bridges have been shown to play an important role in the rate of association in protein–protein interactions (V ijayakumar et al. 1998; Selzer et al. 2000; Selzer and Schreiber 1999, 2001) and it is lik ely that the constitutent residues orientate and enhance tw o SoxY dimers for tetramerization. However, from an energetic point of view, the contribution of the salt bridges is not sufficient to make a permanent tetrameric assembly . This is based on the observ ation that reduced SoxY is present as dimers in solution, and the SoxY tetramer will thus redissociate into dimers if the intersubunit disulfide bonds are not formed. This dissociation must proceed rapidly because of the limited b uried surface area of the tetramer and its high hydration level.

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Fig. 11.3 One tetramer interface region of SoxY viewed from the top. The proposed β-turns are drawn as black curved lines. The yellow S–S motif represents the proposed disulf ide bridge

Although we could not model the C-terminal re gion, GGCGG, and the concomitant disulfide bond, we can propose a potential conformation for these residues. Double glycine motifs, on a regular basis, occur in β-turns as the second and third residues of this structural element (Chou and F asman 1979). W e therefore suggest that the I117–C120 sequence has a β-turn conformation. On the basis of these assumptions, we propose that the β-turns reside at the top of the tetramer interface re gions, each forming a “hook”-lik e structure, in such a w ay that their fourth residues, C120 residues, are in suf ficiently close proximity to form a disulfide bridge. According to crystallographic rules, this should be symmetrical and centered on a crystallographic tw ofold axis (Fig. 11.3). As mentioned abo ve, these bridges should in principle be accessible for other compounds which act on this bond. Although biochemical studies have so far not revealed a role for SoxY and SoxZ homoprotomers, the SoxY and SoxZ structures are suggesti ve that an e xchange from SoxYZ to the individual constituents may be part of the reaction mechanism. A generally accepted issue is that SoxZ, lacking a signal peptide, needs SoxY for translocation to the periplasm (Friedrich et al. 2000), which implies that SoxYZ heterodimers need to be formed in the cytoplasm. Both proteins can be transported to the periplasm by the T at mechanism, a system capable of translocating folded proteins (Berks et al. 2003). In the periplasm, these heterodimers get in volved in thiosulfate oxidation, which may occur via two possible scenarios. In one scenario, SoxYZ e xchanges sub units before sulfur substrate binding. The SoxY dimers, being redox-acti ve, then assemble into SoxY tetramers with closure of the intersubunit disulfide bridges, an e vent that is lik ely mediated via a thiol oxidoreductase system. Both SoxY protomers comprise the acti ve sulfur substrate binding species on which the other sulfur-oxidizing Sox enzymes act. The drawback in this model is that it disregards SoxZ, which is believed to coordinate the sulfur substrate molecules (Quentmeier and Friedrich 2001). The second scenario does tak e SoxZ into account. SoxZ would mediate the interaction between the heterodimer and the

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proteins responsible for disulf ide bond closure and sulfur substrate addition, respectively, meanwhile protecting the disulf ide bridge and the co valently linked sulfur substrate. An exchange from SoxYZ heterodimers to SoxY homodimers and tetramers would then happen after substrate binding, when SoxY of fers its bound sulfur molecules to the other Sox enzymes. In both scenarios, ho wever, one SoxY protomer of fers tw o co valently bound sulfur molecules instead of one at each encounter with another component of the sulfur-oxidizing system, which should in principle be a more ef ficient way of presenting sulfur substrate molecules to the Sox system. Future research needs to address the role of homo-oligomeric SoxY and SoxZ proteins and, in particular , to in vestigate the role of the intersub unit disulf ide bridges between SoxY monomers.

References Appia-Ayme C, Berks BC (2002) SoxV , an orthologue of the CcdA disulf ide transporter , is involved in thiosulf ate oxidation in Rhodovulum sulf idophilum and reduces the periplasmic thioredoxin SoxW. Biochem Biophys Res Commun 296:737–741 Bahadur RP, Chakrabarti P, Rodier F, Janin, J (2003) Dissecting subunit interfaces in homodimeric proteins. Proteins 53:708–719 Bahadur RP, Chakrabarti P , Rodier F , Janin J (2004) A dissection of specif ic and non-specif ic protein-protein interfaces. J Mol Biol 336:943–955 Bamford VA, Bruno S, Rasmussen T, Appia-Ayme C, Cheesman MR, Berks BC, Hemmings AM (2002) Structural basis for the oxidation of thiosulf ate by a sulfur c ycle enzyme. EMBO J 21:5599–5610 Bardischewsky F, Friedrich CG (2001) The shxVW locus is essential for oxidation of inor ganic sulfur and molecular hydrogen by Paracoccus pantotrophus GB17: a novel function for lithotrophy. FEMS Microbiol Lett 202:215–220 Bardischewsky F , Quentmeier A, Friedrich, CG (2006a) The fla voprotein SoxF functions in chemotrophic thiosulfate oxidation of Paracoccus pantotrophus in vi vo and in vitro. FEMS Microbiol Lett 258:121–126 Bardischewsky F, Fischer J, Holler B, Friedrich CG (2006b) SoxV transfers electrons to the periplasm of Paracoccus pantotrophus – an essential reaction for chemotrophic sulfur oxidation. Microbiology 152:465–472 Berks BC, Palmer T, Sargent F (2003) The Tat protein translocation pathway and its role in microbial physiology. Adv Microb Physiol 47:187–254 Bork P, Holm L, Sander C (1994) The immunoglob ulin fold. Structural classif ication, sequence patterns and common core. J Mol Biol 242:309–320 Caffrey DR, Somaroo S, Hughes JD, Mintseris J, Huang ES (2004) Are protein-protein interfaces more conserved in sequence than the rest of the protein surf ace? Protein Sci 13:190–202 Chothia C, Janin J (1975) Principles of protein-protein recognition. Nature 256:705–708 Chou PY, Fasman GD (1979) Prediction of beta-turns. Bioph ys J 26:367–383 Friedrich CG, Quentmeier A, Bardische wsky F, Rother D, Kraft R, K ostka S, Prinz H (2000) Novel genes coding for lithotrophic sulfur oxidation of Paracoccus pantotrophus GB17. J Bacteriol 182:4677–4687 Friedrich CG, Rother D, Bardischewsky F, Quentmeier A, Fischer J (2001) Oxidation of reduced inorganic sulfur compounds by bacteria: emer gence of a common mechanism? Appl En viron Microbiol 67:2873–2882 Friedrich CG, Bardische wsky F, Rother D, Quentmeier A, Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Microbiol 8:253–259

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Harpaz Y, Chothia C (1994) Man y of the immunoglob ulin superfamily domains in cell adhesion molecules and surface receptors belong to a new structural set which is close to that containing variable domains. J Mol Biol 238:528–539 Imhoff JF (2003) Phylogenetic taxonomy of the f amily Chlorobiaceae on the basis of 16S rRN A and fmo (Fenna-Matthe ws-Olson protein) gene sequences. Int J Syst Ev ol Microbiol 53:941–951 Jones S, Thornton JM (1996) Principles of protein-protein interactions. Proc Natl Acad Sci USA 93:13–20 Kelly DP, Shergill JK, Lu WP, Wood AP (1997) Oxidati ve metabolism of inor ganic sulfur compounds by bacteria. Antonie Van Leeuwenhoek 71:95–107 Lahiri C, Mandal S, Ghosh W, Dam B, Roy P (2006) A novel gene cluster soxSRT is essential for the chemolithotrophic oxidation of thiosulf ate and tetrathionate by Pseudaminobacter salicylatoxidans KCT001. Curr Microbiol 52:267–273 Lo Conte L, Chothia C, Janin J (1999) The atomic structure of protein-protein recognition sites. J Mol Biol 285:2177–2198 Lu WP (1986) A periplasmic location for the thiosulf ate-oxidizing multi-enzyme system from Thiobacillus versutus. FEMS Microbiol Lett 34:313–317 Lu WP, Kelly DP (1983) Purification and some properties of two principal enzymes of the thiosulfateoxidizing multi-enzyme system from Thiobacillus A2. J Gen Microbiol 129:3549–3564 Lu WP, Sw oboda BEP, K elly DP (1985) Properties of the thiosulf ate-oxidizing multi-enzyme system from Thiobacillus versutus. Biochim Biophys Acta 828:116–122 Miller S (1989) The structure of interf aces between sub units of dimeric and tetrameric proteins. Protein Eng 3:77–83 Mintseris J, Weng Z (2005) Structure, function, and e volution of transient and oblig ate proteinprotein interactions. Proc Natl Acad Sci USA 102:10930–10935 Nooren IM, Thornton JM (2003) Structural characterisation and functional signif icance of transient protein-protein interactions. J Mol Biol 325:991–1018 Quentmeier A, Friedrich CG (2001) The cysteine residue of the SoxY protein as the active site of protein-bound sulfur oxidation of Paracoccus pantotrophus GB17. FEBS Lett 503:168–172 Quentmeier A, Hellwig P, Bardischewsky F, Grelle G, Kraft R, Friedrich CG (2003) Sulfur oxidation in Paracoccus pantotrophus: interaction of the sulfur -binding protein SoxYZ with the dimanganese SoxB protein. Biochem Biophys Res Commun 312:1011–1018 Richardson JS, Richardson DC (2002) Natural β-sheet proteins use negative design to avoid edgeto-edge aggregation. Proc Natl Acad Sci USA 99:2754–2759 Rodier F , Bahadur RP , Chakrabarti P , Janin J (2005) Hydration of protein-protein interf aces. Proteins 60:36–45 Rother D, Henrich HJ, Quentmeier A, Bardischewsky F, Friedrich CG (2001) Novel genes of the sox gene cluster, mutagenesis of the flavoprotein SoxF, and evidence for a general sulfur-oxidizing system in Paracoccus pantotrophus GB17. J Bacteriol 183:4499–4508 Rother D, Ora wski G, Bardische wsky F , Friedrich CG (2005) SoxRS-mediated re gulation of chemotrophic sulfur oxidation in Paracoccus pantotrophus. Microbiology 151:1707–1716 Selzer T, Schreiber G (1999) Predicting the rate enhancement of protein comple x formation from the electrostatic energy of interaction. J Mol Biol 287:409–419 Selzer T, Schreiber G (2001) Ne w insights into the mechanism of protein-protein association. Proteins 45:190–198 Selzer T, Albeck S, Schreiber G (2000) Rational design of f aster associating and tighter binding protein complexes. Nat Struct Biol 7:537–541 Stout J, De Smet L, P anjikar S, Weiss MS, Savvides SN, Van Beeumen J (2006) Crystallization, preliminary crystallographic analysis and phasing of the thiosulfate binding protein SoxY from Chlorobium limicola f. thiosulfatophilum. Acta Crystallogr Sect F 62: 1093–1096 Stout J, Van Driessche G, Savvides SN, Van Beeumen J (2007) X-ray crystallographic analysis of the sulfur carrier protein SoxY from Chlorobium limicola f. thiosulfatophilum reveals a tetrameric structure. Protein Sci 16:589–601 Tsai CJ, Lin SL, Wolfson HJ, Nussinov R (1997) Studies of protein-protein interfaces: a statistical analysis of the hydrophobic effect. Protein Sci 6:53–64

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Valdar WSJ, Thornton JM (2001a) Protein-protein interfaces: analysis of amino acid conservation in homodimers. Proteins 42:108–124 Valdar WSJ, Thornton JM (2001b) Conserv ation helps to identify biologically rele vant crystal contacts. J Mol Biol 313:399–416 Vijayakumar M, W ong KY, Schreiber G, Fersht AR, Szabo A, Zhou HX (1998) Electrostatic enhancement of dif fusion-controlled protein-protein association: comparison of theory and experiment on barnase and barstar. J Mol Biol 278:1015–1024 Williams AF , Barclay AN (1988) The immunoglob ulin superf amily domains for cell surf ace recognition. Annu Rev Immunol 6:381–405 Xu D, Tsai CJ, Nussino v R (1997a) Hydrogen bonds and salt bridges across protein-protein interfaces. Protein Eng 10:999–1012 Xu D, Lin SL, Nussinov R (1997b) Protein binding versus protein folding: the role of hydrophilic bridges in protein associations. J Mol Biol 265:68–84 Young L, Jernig an RL, Co vell DG (1994) A role for surf ace h ydrophobicity in protein-protein recognition. Protein Sci 3:717–729

Chapter 12

Redox Control of Chemotrophic Sulfur Oxidation of Paracoccus pantotrophus Cornelius G. Friedrich, Armin Quentmeier, Frank Bardischewsky, Dagmar Rother, Grazyna Orawski, Petra Hellwig, Jörg Fischer

Abstract The reaction c ycle of the reconstituted Sox enzyme system of Paracoccus pantotr ophus requires the periplasmic proteins SoxYZ, SoxXA, SoxB, and SoxCD. The heme enzyme SoxXA covalently binds the sulfur substrate to the thiol of the single c ysteine residue of SoxY located at its carboxy-terminal end. Bound sulfur is then oxidized to sulf ate by a series of reactions. These involve sulfur dehydrogenase SoxCD which oxidizes the protein-bound sulf ane sulfur to sulfone in a unique six-electron transfer . Bound sulfone is then hydrolyzed off by the sulfate thiohydrolase SoxB to regenerate SoxYZ. The flavoprotein SoxF enhances the rate of sulfur oxidation in vivo as evident from mutant analysis and we have specified its action in vitro. SoxYZ unlik e the other Sox proteins is inactivated upon reduction. When the Sox system is reconstituted with inactivated SoxYZ, the thiosulf ate-oxidizing activity is drastically decreased. SoxF re verses this inactivation and may mediate a conformational change of SoxYZ possibly by a transient interprotein disulfide. The membrane protein SoxV and the thioredoxin SoxS are essential for chemotrophic growth as evident from homogenote mutants defective in these proteins. Evidence is presented that both proteins transfer reductant from the c ytoplasm to the periplasm and that SoxYZ is the f inal target of this transfer to balance the redox state of the Sox enzyme system or reduce a SoxY..Y interprotein disulfide.

12.1 The Sulfur-Oxidizing Enzyme System of Paracoccus pantotrophus Paracoccus pantotrophus is a facultatively aerobic alphaproteobacterium which grows heterotrophically with v arious carbon sources and chemoautotrophically with thiosulfate under aerobic conditions (Robertson and K uenen 1983; Ludwig et al. 1993, Rainey et al. 1999). The sulfur-oxidizing (Sox) enzyme system ofP. pantotrophus is encoded by the sox gene cluster , which comprises 15 genes or ganized in three transcriptional units soxRS, soxVW, and soxXYZA-H. The soxR gene encodes a DNA-binding repressor protein of the ArsR f amily (Rother et al. 2005). Seven sox 139 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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genes encode polypeptides which form four periplasmic proteins, designated according to the gene nomenclature. These proteins reconstitute the Sox enzyme system in vitro and oxidize hydrogen sulf ide, sulfur , thiosulf ate, and sulf ite with horse c ytochrome c as the final electron acceptor (Eq. 12.1) (reviewed in Friedrich et al. 2005): −

S − SO3 − + 5H2 O + 8Cytc3+ → 2SO 4 2 − + 8Cytc 2 + + 10H + .

(12.1)

The SoxYZ complex is the central protein and interacts with SoxXA, SoxCD, and SoxB. Sulfur is oxidized when co valently bound to the thiol of the in variant Cys138 of SoxY (Quentmeier and Friedrich 2001; Fig. 12.1). The heme enzyme SoxXA, a complex of the monoheme c-type cytochrome SoxX and the diheme c-type cytochrome SoxA, is proposed to link the sulfur substrate to the thiol of Cys138 of SoxY (Bamford et al. 2002; Dambe et al. 2005). The molybdoprotein cytochrome comple x SoxCD catalyzes a unique six-electron transfer and oxidizes the outer (sulfane) sulfur atom of cysteinepersulfide of SoxY to the sulfone oxidation state to yield l-cysteine-S-sulfate and acts as sulfur dehydrogenase. The dimanganese SoxB protein is a paralog of the zinc-containing 5 ′ nucleotidases and is proposed to hydrolyze of f sulf ate from the l-cysteine-S-sulfate to

Fig. 12.1 Model of the reaction c ycle of thiosulf ate oxidation by the Sox enzyme system of Paracoccus pantotrophus and reactivation of SoxYZ by the flavoprotein SoxF. The capital letters indicate the respecti ve Sox proteins, the central protein SoxYZ in its acti ve form is indicated in boldface, and the inactive form is indicated in fine type. The SoxY–Y interprotein disulfide of the heterotetrameric SoxY–Y(Z) 2 represents a hypothetical intermediate in the transition of the inactive to active form of SoxYZ catalyzed by SoxF. TCEP tris(2-carboxyethyl)phosphine

12 Redox Control of Chemotrophic Sulfur Oxidation of Paracoccus pantotrophus 141

regenerate SoxYZ acting as sulfate thioesterase (Friedrich et al. 2001; Quentmeier and Friedrich 2001; Rother et al. 2001; Bardische wsky et al. 2005). Therefore, the sulfur-binding protein SoxYZ is the central protein of the Sox enzyme system and reacts with SoxXA, SoxB, and SoxCD (Fig. 12.1). Moreo ver, SoxYZ is redox-active although it does not contain a cof actor or metal (Quentmeier et al. 2003), and reduction in vitro by the non-sulfur reductant tris(2-carboxyethyl) phosphine (TCEP) inactivates SoxYZ (Quentmeier and Friedrich 2001). Genetic inactivation of SoxB or SoxC disables P. pantotrophus from gro wing with and oxidizing thiosulfate and hydrogen sulfide to sulfate (Wodara et al. 1997; Rother et al. 2001). The fla voprotein SoxF is not part of the reconstituted Sox enzyme system and its inacti vation does not disable P. pantotr ophus from gro wing chemotrophically with thiosulf ate (Rother et al. 2001) b ut decreases the rate of thiosulfate oxidation (Bardischewsky et al. 2006b). SoxF is a periplasmic monomer of 42,797 Da, contains co valently bound fla vin adenine dinucleotide (F AD), and is closely related to the fla voprotein sub units of fla vocytochromes c of phototrophic and chemotrophic sulfur -oxidizing bacteria. SoxF lik e flavocytochrome c (FCSD) has sulfide dehydrogenase (SDH) activity in vitro with horse cytochrome c as an electron acceptor to yield tw o electrons per mole of hydrogen sulfide (Quentmeier et al. 2004). The SDH activity of FCSDs of various sulfur-oxidizing bacteria was taken as evidence for its significance in sulfur energy metabolism (K usai and Y amanaka 1973; K ostanjevecki et al. 2000; reviewed in Cusanovich et al. 1991) while genetic evidence did not support this view (Reinartz et al. 1998). The soxVW genes comprise a transcriptional unit, and soxV encodes the membrane protein SoxV with six channel-forming transmembrane helices and two c ysteine residues f acing each other on the inner side and able to form a protein disulfide for transport of reductant. In this respect SoxV is homologous to CcdA of P. pantotr ophus and other bacteria. CcdA is essential for c ytochrome c biogenesis (Bardische wsky and Friedrich 2001). The soxW gene encodes a periplasmic thioredoxin of 166 amino acids after maturation. The Sox enzyme system of P. pantotrophus requires the function of SoxV. Although c-type cytochromes are essential for chemotrophic sulfur oxidation and SoxV transfers electrons to the periplasm it does not reduce apoc ytochromes and is not involved in cytochrome c maturation. Instead, a thioredoxin is the proposed electron acceptor as the nonessential thioredoxin SoxW is reduced by SoxV (Bardischewsky et al. 2006a). The crucial periplasmic electron acceptor of SoxV has not been reported so f ar. Here we summarize the current kno wledge of the Sox system and present unpublished results demonstrating that this enzyme system is subject to a subtile redox control. We present preliminary results on the central protein, SoxYZ, and its inactivation upon reduction and sho w that the fla voprotein SoxF reacti vates SoxYZ. Evidence is presented that the redox partner of SoxV is the thioredoxin SoxS, which in turn may interact with SoxYZ.

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Abundance of the sox Genes in Bacteria

The increasing availability of partial and complete microbial genome sequences has enabled the detection of sox genes among the Bacteria, while in Archaea these genes are not present. Besides the P. pantotrophus sox gene cluster, mostly incomplete sox clusters were reported from se ven bacterial strains in 2001 (Friedrich et al. 2001) and from 17 strains in 2005 (Friedrich et al. 2005). T o date (scan closed August 31, 2006) a total of 38 sox gene clusters are kno wn, most of which are derived from genomic sequences of chemotrophic and phototrophic bacteria of different genera (Fig. 12.2). Among the bacteria listed in Fig. 12.2 se veral strains are unable to gro w chemotrophically with thiosulf ate like, e.g., Ralstonia eutropha. Transfer of the P. pantotrophus sox structural genes to R. eutropha does not add this physiological trait (F. Bardischewsky, unpublished data). For other strains like, e.g., Bradyrhizobium japonicum, Nitrobacter hamburgensis, Polaromonas sp., orMethylobium petrophilum, chemoautotrophic growth with inorganic sulfur compounds is unknown. On the other hand enzymes involved in sulfur oxidation ha ve been described from chemotrophic and phototrophic bacteria, the respective genes of which were not identified (reviewed in Brune 1989; Kelly et al. 1997; Friedrich 1998). R. eutropha, Polaromonas sp., and Dechloromonas aromatica (Fig. 12.2), lik e the other strains listed in Fig. 12.2, harbor complete sets of sox genes soxYZ, soxXA, soxB, and soxCD, the products of which are required for thiosulfate oxidation in vitro. These strains, ho wever, lack soxVW and soxRS required for sulfur oxidation to sulf ate in vi vo by P. pantotrophus. Among the strains lacking these genes are the green phototrophic bacteria Chlorobaculum tepidum (formely Chlorobium tepidum; Imhoff 2003), Chlorobium limicola, and Chlorobium clathratiforme (formerly Pelodictyon phaeoclathratiforme; Imhoff 2003) which gro w well photoautotrophically with thiosulf ate. The Chlorobiaceae miss the soxCD genes, which are also missing in the kno wn sequences of the phototrophic purple sulfur bacterium Allochromatium vinosum (formerly Chromatium vinosum; Imhoff et al. 1998). SoxCD is essential for gro wth of the chemotroph P. p antotrophus (Wodara et al. 1997). Instead of the soxCD genes, green sulfur bacteria and A. vinosum harbor the dsr operon which is indispensable for oxidation of stored sulfur (Dahl

Fig. 12.2 Map of the sox gene cluster of P. pantotrophus and sox gene homologs of other bacteria. Capital letters designate the sox genes of P. pantotrophus. Open reading frames (ORFs) predicting homologous proteins are indicated by the same color. Pink/violet arrows without frame indicate genes encoding sulfite dehydrogenases and their cytochromes (see chapter 13). Bright yellow arrows as for Rod. cap. indicate sulf ide-quinone oxidoreductase genes. ORFs not encoding Sox homologous proteins are gi ven in white. Par.pan., P. pantotrophus GB17; Par.den., P. denitrificans 1222; Rhd.sph., Rhodobacter sphaeroides; Rhv.sul., Rhodovulum sulfidophilum; Rhp.pal., Rhodopseudomonas palustris; Sul.NAS, Sulfitobacter sp. NAS-14.1; Sul.EE, Sulfitobacter sp. EE-36, Rho.bac., rhodobacterales bacterium; Ros.nub., Roseovarius nubinhibens; Ros.217, Roseovarius sp. 217; pom., Sil. Silicibacter pomeroyi; Bra.sp, Bradyrhizobium sp.; Bra.jap., Bradyrhizobium japonicum ; Sta.nov., Starkeya novella; Psb.sal., Pseudaminobacter salicylatoxidans KCT001; Met.ext., Methylobacterium

12 Redox Control of Chemotrophic Sulfur Oxidation of Paracoccus pantotrophus 143

Fig. 12.2 (continued) extorquens; Tms.den., Thiomicrospira denitrificans; Tms.cru., Thiomicrospira crunogena; Mel.pet., Methylobium petroleophilum; Ane.deh., Anaeromyxobacter dehalogenans; Pol. sp, Polaromonas sp.; Dec.aro., Dechloromonas aromatica; Ral.eut., Ralstonia eutropha, Ral.sol., Ralstonia solanacearum; Ral.met., Ralstonia metallidurans; Thm.the., Thermus thermophilus; Chl. tep., Chlorobaculum tepidum; Chl.lim., Chlorobium limicola, Chl.chl., Chlorobium chlorochromatii CaD3; Pel.pha., Pelodictyon phaeoclathr atiforme; Nit.ham., Nitrobacter hamburgensis; Tms.den., Thiomicrospira denitrificans; All.vin., Allochromatium vinosum, Aqu.aeo., Aquifex aeolicus; Mgc. MC1, Magnetococcus MC-1;Mgs.mag., Magnetospirillum magnetotacticum; Alk.ehr., Alkalilimnicola ehrlichii; Mec.cap., Methylococcus capsulatus; Rod.cap., Rhodobacter capsulatus

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et al. 2005). The function of dsrAB encoding dissimilatory sulf ite reductase and the interaction of other Dsr proteins is specif ied in this volume and suggests their function in oxidation of inorganic sulfur to sulfate in an alternative route in which elemental sulfur is an obligate intermediate (see Chap. 9 by Grimm et al.). Therefore, other enzymes are proposed in other bacteria which oxidize sulfur compounds to sulf ate. These enzymes should be functional equi valents to sulfur dehydrogenase SoxCD, the membrane protein SoxV , and the thioredoxins SoxW and SoxS. In the genomes of most bacteria harboring the sox structural genes tw o other genes are located either within the sox gene cluster or separate from it. One gene is homologous to soxF which encodes the flavoprotein SoxF in P. pantotrophus. The other gene (soxE) encodes a small c-type cytochrome (Fig. 12.2) which, ho wever, is not closely related to those of other sources. In A. vinosum these genes, designated fccAB, encode the fla vocytochrome c complex which e xhibits SDH acti vity in vitro. The function of FCSD, which is located in the periplasm in most strains but which is membrane-bound in others (V isser et al. 1997), has been a matter of debate. In vitro, sulfur is the product of FCSD. The periplasmic location of the enzyme in A. vinosum is in agreement with the periplasmic storage of sulfur in purple sulfur bacteria (Pattaragulwanit et al. 1998).

12.3

The Physiological Function of the Flavoprotein SoxF

This section is devoted to the physiological function of SoxF in P. pantotrophus. The in vi vo function of SoxF dif fers from the SDH acti vity determined in vitro (Quentmeier et al. 2004). Sulf ide is not a free intermediate of thiosulf ate oxidation as e vident from the mechanism of sulfur oxidation by the Sox enzyme system (Friedrich et al. 2001, 2005; Rother et al. 2001). Also, homologs of the soxF gene are missing in some sulfur -oxidizing bacteria lik e, e.g., in the genome of Thiomicrospira denitrif icans (Fig. 12.2; see also Chap. 19 by Sie vert et al.). Therefore, SDH activity is probably not functionally important in vi vo. Also, SDH yields only tw o electrons, while from sulf ide the Sox enzyme systems yields four electrons in vitro and eight electrons in vi vo. Strain GB soxF∆ carries a deletion in the soxF gene which eliminates 90 amino acids around the F AD-binding site and which causes complete de gradation and inacti vation of the SoxF protein. The specific growth rate of strain GBsoxF∆ with thiosulfate and carbon dioxide is about half of that of the wild type. Whole cells of strain GBsoxF∆, however, oxidize thiosulfate and hydrogen sulf ide to sulf ate as e vident from the requirement of about 2 mol of oxygen per mole of thiosulfate or sodium sulfide (Bardischewsky et al. 2006b). This result again suggests a role different from SDH for SoxF in sulfur metabolism. In the 44–65% ammonium sulf ate precipitate of cell-free e xtracts of strain GBsoxF∆ the specif ic thiosulf ate-oxidation rate is about 50% of that of the same fraction of the wild type. Addition of purif ied SoxF to enzyme assays containing the ammonium sulf ate fraction from strain GB soxF∆ increases the acti vity to the

12 Redox Control of Chemotrophic Sulfur Oxidation of Paracoccus pantotrophus 145

wild-type level. However, SoxF is unable to metabolize thiosulfate and sulfide is not an intermediate of thiosulf ate oxidation by the Sox enzyme system; therefore, SoxF is proposed to enhance the acti vity of some component of the Sox enzyme system. However, SoxF added to the Sox enzyme system reconstituted from homogeneous Sox proteins does not af fect the thiosulf ate-oxidizing acti vity (Rother et al. 2001; Bardischewsky et al. 2006b). Consequently , the action of SoxF must relate to some condition given in cell-free extracts but not in the reconstituted Sox enzyme system. Proteins of cell-free extracts are considered to be reduced. The key protein of the Sox enzyme system, SoxYZ, is sensiti ve to reduction by the non-sulfur reductant TCEP. Reduction of SoxYZ does not only inhibit SoxYZ as is plausible from shifting the equilibrium of the reaction (Eq. 12.2) to the left side, b ut it also inactivates SoxYZ (Quentmeier and Friedrich 2001): SoxZY − S− + − S − SO3 − + 2Cytc3+ → SoxZY − S − S − SO3 − + 2Cytc 2 + . (12.2) Preincubation of SoxYZ with 1 mM TCEP prior to reconstitution of the Sox enzyme system selecti vely inacti vates SoxYZ as e vident from the 50–90% decrease in thiosulfate-oxidizing activity of the system. Reduction of the other Sox proteins prior to reconstitution of the Sox enzyme system does not af fect its thiosulfate-oxidizing activity (A. Quentmeier , unpublished data). Changes in the redox state of redox-acti ve proteins are often link ed to conformational changes upon change in the char ge of metals, heme iron ligation, or protein disulf ide formation as is kno wn from c ytochromes, thioredoxins, fla vodoxin, photosystem I and II, or re gulatory proteins (Takano and Dickerson 1980; Swenson et al. 1999; Arnesano et al. 2003; Lee et al. 2004; Range et al. 2006; re viewed in Ritz and Beckwith 2001). Infrared spectroscop y of SoxYZ suggests a conformational change upon various treatments of the protein (P. Hellwig, unpublished data). Addition of SoxF of P. pantotrophus to the Sox enzyme system reconstituted with TCEP-inactivated SoxYZ reactivated the thiosulf ate-oxidizing activity of the whole system with time (A. Quentmeier , unpublished data). When SoxF w as included in the assay cytochrome c was reduced with progressively increasing rate. This progressive increase suggested an action of SoxF which was linked to the reaction cycle of the reconstituted Sox enzyme system. As the inacti vated SoxYZ was the bottleneck of the o verall reaction, v ery likely this protein is the tar get of the reactivating activity of SoxF. This SoxYZ-reacti vating reaction represents a no vel reaction for a fla voprotein which we consider physiologically signif icant (C.G. Friedrich, A. Quentmeier , D. Rother , F. Bardische wsky, and P . Hell wig, unpublished results). How does the SDH activity of SoxF relate to this function? SoxF transfers electrons to horse c ytochrome c and oxidizes thiols as is e vident from its SDH acti vity. SoxYZ does not contain a redox center or metal and the single cysteine of SoxY is, therefore, proposed to donate one thiol for formation of an interprotein disulf ide to form a SoxY ..Y homodimer (“ ..” stands for protein disulfide). Dimers of the subunits of SoxYZ covalently linked by protein disulfides have been identif ied by sodium dodec yl sulfate gel electrophoresis (Quentmeier et al. 2003). Interprotein disulfides like SoxY..Z and SoxZ..Z are not considered to

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be crucial for the Sox enzyme system since the single c ysteine of P. pantotrophus SoxZ is not present in SoxZ of several other sources (J. Fischer, unpublished data). The current model of sulfur oxidation by the Sox enzyme system proposes the free thiol of SoxY–Cys138 as a hook for the co valent linkage of the sulfur substrate. Therefore, the SoxY ..Y homodimer is considered to be a transient intermediate for a conformational rearrangement of SoxY to yield its acti ve form (Fig. 12.1). This assumption, ho wever, implies the re-reduction of the interprotein disulf ide of SoxY ..Y to two SoxY and poses the question from where the electrons for re-reduction of SoxY ..Y originate.

12.4 The Periplasmic Partners of SoxV for Transfer of Electrons The reversible formation of protein disulfide bonds is an important means for transport of electrons, protein biogenesis, protein stability , and enzyme catalysis (reviewed in Fabianek et al. 2000; Ritz and Beckwith 2001). The membrane protein SoxV is a paralog of CcdA, which is essential for re-reduction of apoc ytochromes, and the functional dif ference is e vident from the phylogenetic tree of the related proteins (Bardischewsky et al. 2006a). The function of SoxV in transfer of reductant to the periplasm is essential as is e vident from disruption of the soxV gene by an ΩKmr cassette. Since soxVW comprise a transcriptional unit, the inserted Ω-cassette in soxV acts in a polar manner on soxW e xpression, leading to a SoxW-negative phenotype. Strain GBΩV is unable to grow chemotrophically with thiosulfate and the in vi vo thiosulf ate-dependent oxygen uptak e rate is only less than 10% of that of the wild type. Genetic complementation of the soxV gene restored the ability for chemotrophic gro wth, while complementation of the soxW gene did not (Bardische wsky et al. 2006b). The thiosulf ate-oxidizing ability of strain GBΩV could also be restored by chemical complementation with dithiothreitol (DTT; Fig. 12.3). Such chemical complementation has similarly been described for CcdA in Escherichia coli (Sambongi and Ferguson 1994) and is in accordance with the direction of transfer of electrons by SoxV to the periplasm. This poses the question of the periplasmic redox partner of SoxV. The electron donor to reduce SoxV is presumably a c ytoplasmic thioredoxin as the thioredoxin is reduced by SoxV. SoxW, however, is not essential for thiosulfate oxidation in vi vo (Bardischewsky et al. 2006a). Recently , the periplasmic thioredoxin SoxS w as identif ied to be essential for thiosulf ate oxidation as is e vident from a homogenote mutant strain GBΩS in which the soxS gene is disrupted by the Ω-Kmr interposon. This mutant forms about 10% of the specific thiosulfate-oxidizing activity as compared with the wild type. In this mutant, trans complementation of soxS restores the wild-type phenotype (G. Ora wski, unpublished data). Also, simi larly as in the mutant GBΩV (Fig. 12.3) the mutation in strain GBΩS can be complemented chemically by inclusion of 1 mM DTT to the mineral medium to yield almost the wild-type level of thiosulfate-oxidizing activity (F. Bardischewsky, unpublished d ata).

12 Redox Control of Chemotrophic Sulfur Oxidation of Paracoccus pantotrophus 147

Fig. 12.3 Chemical complementation of thiosulfate-oxidizing ability of the homogenote mutant P. pantotr ophus GB ΩV. Strain GB ΩV w as culti vated mixotrophically with thiosulf ate. In the stationary growth phase the culture was split and further aerated at 30°C by shaking. One culture was untreated (open diamonds); the other was treated with 1 mM dithiothreitol final concentration (closed diamonds). The thiosulfate-dependent oxygen uptake rate of whole cells w as determined at the time intervals indicated. (From Bardischewsky et al. 2006a, with permission)

Fig. 12.4 Model of the route of reductants from the cytoplasm for redox conditioning of SoxYZ. The letters indicate the Sox proteins

The chemical complementation of strain GB ΩS transposes the pre vious question one step further to: Which is the redox partner of the thioredoxin SoxS? The reduced and the oxidized state of SoxY can be deduced from the availability of the free sulfhydryl of Cys138 and its reactivity with the sulfhydryl reactant disodium 4-acetamido-4 ′-maleimidstilbene-2,2′-disulfonic acid (AMS; cited in Bardischewsky et al. 2006a). In freshly prepared cell-free extracts of the wild type,

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SoxY appears predominantly in the reduced state since AMS binds to SoxY and increases the molecular mass by about 500 Da. Ho wever, SoxY of strain GB ΩS cannot be trapped by AMS, suggesting the sulfhydryl of Cys138 SoxY is either oxidized or present in a conformation in which the sulfhydryl is inaccessible for chemical modification by AMS (F. Bardischewsky, unpublished data). With this set of experiments we have obtained evidence for the route of electrons from the c ytoplasm via SoxV to SoxS. SoxYZ possibly requires a conformation which allows access to the sulfhydryl of Cys138 to which the sulfur substrates and SoxXA bind. SoxS donates electrons and is essential for chemotrophic gro wth. Therefore, we suggest SoxYZ as a redox partner of the thioredoxin SoxS to enable its transition to the active form (Fig. 12.4). It is not yet clear whether an enzymatic step is required to catalyze the conformational change which is lik ely to be link ed to the activation of SoxYZ. Our current w ork aims at answering this question.

References Arnesano F, Banci L, Bertini I, Mang ani S, Thompsett AR (2003) A redox switch in CopC: an intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites. Proc Natl Acad Sci USA 100:3814–3819 Bamford VA, Bruno S, Rasmussen T, Appia-Ayme C, Cheesman MR, Berks BC, Hemmings AM (2002) Structural basis for the oxidation of thiosulf ate by a sulfur c ycle enzyme. EMBO J 21:5599–5610 Bardischewsky F, Friedrich CG (2001) Identification of ccdA in Paracoccus pantotrophus GB17: disruption of ccdA causes complete def iciency in c-type c ytochromes. J Bacteriol 183:257–263 Bardischewsky F, Quentmeier A, Rother D, Hell wig P, K ostka S, Friedrich CG (2005) Sulfur dehydrogenase of Paracoccus pantotrophus: the heme-2 domain of the molybdoprotein c ytochrome c complex is dispensable for catalytic activity. Biochemistry 44:7024–7034 Bardischewsky F, Fischer J, Höller B, Friedrich CG (2006a) SoxV transfers electrons to the periplasm of Paracoccus pantotrophus – an essential reaction for chemotrophic sulfur oxidation. Microbiology 152:465–472 Bardischewsky F , Quentmeier A, Friedrich CG (2006b) The fla voprotein SoxF functions in chemotrophic thiosulfate oxidation of Paracoccus pantotrophus in vi vo and in vitro. FEMS Microbiol Lett 258:121–126 Brune D (1989) Sulfur oxidation by phototrophic bacteria. Biochim Bioph ys Acta 975:189–221 Cusanovich MA, Meyer TE, Bartsch RG (1991) Flavocytochrome c. In: Müller F (ed) Chemistry and biochemistry of flavoenzymes. CRC, Boca Raton, pp 377–399 Dahl C, Engels S, Pott-Sperling AS, Schulte A, Sander A, Lübbe Y, Deuster O, Brune DC (2005) Novel genes of the dsr gene cluster and e vidence for close interaction of Dsr proteins during sulfur oxidation in the phototrophic sulfur bacterium Allochromatium vinosum . J Bacteriol 187:1392–1404 Dambe T, Quentmeier A, Rother D, Friedrich CG, Scheidig AJ (2005) Structure of the c ytochrome complex SoxXA of Paracoccus pantotrophus, a heme enzyme initiating chemotrophic sulfur oxidation. J Struct Biol 152:229–234 Fabianek RA, Henneck e H, Thön y-Meyer L (2000) Periplasmic protein thiol:disulf ide oxidoreductases of Escherichia coli. FEMS Microbiol Rev 24:303–316 Friedrich CG (1998) Physiology and genetics of sulfur-oxidizing bacteria. Adv Microbial Physiol 39:235–289

12 Redox Control of Chemotrophic Sulfur Oxidation of Paracoccus pantotrophus 149 Friedrich CG, Rother D, Bardischewsky F, Quentmeier A, Fischer J (2001) Oxidation of reduced inorganic sulfur compounds by bacteria: emer gence of a common mechanism? Appl En viron Microbiol 67:2873–2882 Friedrich CG, Bardische wsky F, Rother D, Quentmeier A, Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Microbiol 8:253–259 Imhoff JF (2003) Phylogenetic taxonomy of the f amily Chlorobiaceae on the basis of 16S rRN A and fmo (Fenna-Matthe ws-Olson protein) gene sequences. Int J Syst Ev ol Microbiol 53:941–951 Imhoff JF, Süling J, Petri R (1998) Ph ylogenetic relationships among the Chromatiaceae, their taxonomic reclassification and description of the ne w genera Allochromatium, Halochromatium, Isochromatium, Marichromatium, Thiococcus, Thiohalocapsa and Thermochromatium. Int J Syst Bacteriol 48:1129–1143 Kelly DP, Shergill LK, Lu W-P, Wood AP (1997) Oxidative metabolism of inorganic sulfur compounds by bacteria. Antonie Van Leeuwenhoek 71:95–107 Kostanjevecki V , Brige A, Me yer TE, Cusano vich MA, Guisez Y , v an Beeumen JJ (2000) A membrane-bound fla vocytochrome c-sulfide dehydrogenase from the phototrophic purple sulfur bacterium Ectothiorhodospira vacuolata. J Bacteriol 182:3097–103 Kusai A, Y amanaka T (1973) Cytochrome c (553, Chlorobium thiosulfatophilum ) is a sulf idecytochrome c reductase. FEBS Lett 34:235–237 Lee C, Lee SM, Mukhopadhyay P, Kim SJ, Lee SC, Ahn W-S, Yu M-H, Storz G, Ryu SE (2004) Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nat Struct Mol Biol 11:1179–1185 Ludwig W, Mittenhuber G, Friedrich CG (1993) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Int J Syst Bacteriol 43:363–367 Pattaragulwanit K, Brune DC, Trüper HG, Dahl C (1998) Molecular genetic e vidence for cytoplasmic localization of sulfur globules in Chromatium vinosum. Arch Microbiol 169:434–444 Quentmeier A, Friedrich CG (2001) The c ysteine residue of the SoxY protein as the acti ve site of protein-bound sulfur oxidation of Paracoccus pantotr ophus GB17. FEBS Lett 503:168–172 Quentmeier A, Hellwig P, Bardischewsky F, Grelle G, Kraft R, Friedrich CG (2003) Sulfur oxidation in Paracoccus pantotrophus: interaction of the sulfur -binding protein SoxYZ with the dimanganese SoxB protein. Biochem Biophys Res Comm 312:1011–1018 Quentmeier A, Hellwig P, Bardischewsky F, Wichmann R, Friedrich CG (2004) Sulfide dehydrogenase activity of the monomeric flavoprotein SoxF of Paracoccus pantotrophus. Biochemistry 43:14696–14703 Rainey FA, Kelly DP, Stackebrandt E, Burghardt J, Hiraishi A, Katayama Y, Wood AP (1999) A re-evaluation of the taxonomy of Paracoccus denitrificans and a proposal for the combination Paracoccus pantotrophus comb. nov. Int J Syst Bacteriol 49:645–651 Range K, Ayala I, York D, Barry BA (2006) Normal modes of redox-active tyrosine: conformation dependence and comparison to experiment. J Phys Chem 110:10970–10981 Reinartz M, Tschäpe J, Brüser T, Trüper HG, Dahl C (1998) Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum. Arch Microbiol 170:59–68 Ritz D, Beckwith J (2001) Roles of thiol-redox pathw ays in bacteria. Annu Re v Microbiol 55:21–48 Robertson LA, K uenen JG (1983) Thiosphaera pantotropha gen. no v. sp. no v.: a f acultatively anaerobic, facultatively autotrophic sulfur bacterium. J Gen Microbiol 129:2847–2855 Rother D, Henrich H-J, Quentmeier A, Bardischewsky F, Friedrich CG (2001) Novel genes of the sox gene cluster, mutagenesis of the flavoprotein SoxF, and evidence for a general sulfur-oxidizing system in Paracoccus pantotrophus GB17. J Bacteriol 183:4499–4508 Rother D, Ora wski G, Bardische wsky F, Friedrich CG (2005) SoxRS mediated re gulation of chemotrophic sulfur oxidation in Paracoccus pantotrophus. Microbiology 151:1707–1716 Sambongi Y, Fer guson SJ (1994) Specif ic thiol compounds complement def iciency in c-type cytochrome biogenesis in Escherichia coli carrying a mutation in a membrane-bound disulphide isomerise-like protein. FEBS Lett 353:235–238

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Swenson RP, Kasim M, Bradle y LH, Druhan LJ (1999) Role of conformational dynamics and associated electrostatic and hydrogen bonding interactions in the regulation of redox potentials in the Clostridium beijerinckii flavodoxin. In: Ghisla S, Kroneck P, Macheroux P, Sund H (eds) Flavins and flavoproteins. Agency for Scientific Publications, Berlin, pp183–186 Takano T, Dickerson RE (1980) Redox conformation changes in ref ined Tuna cytochrome. Proc Natl Acad Sci 77:6371–6375 Visser JM, de Jong GAH, Robertson LA, Kuenen JG (1997) A novel membrane-bound flavocytochrome c sulfide dehydrogenase from the colorless sulfur bacterium Thiobacillus sp. W5. Arch Microbiol 167:295–301 Wodara C, Bardische wsky F, Friedrich CG (1997) Cloning and characterization of sulf ite dehydrogenase, tw o c-type c ytochromes, and a fla voprotein of Paracoccus denitrif icans GB17: essential role of sulf ite deh ydrogenase in lithotrophic sulfur oxidation. J Bacteriol 179:5014–5023

Chapter 13

Bacterial Sulfite-Oxidizing Enzymes – Enzymes for Chemolithotrophs Only? Ulrike Kappler

Abstract All known sulfite-oxidizing enzymes that ha ve been studied in molecul ar detail belong to the sulf ite oxidase f amily of molybdoenzymes. The f irst bacterial enzymes in this family were only characterized in 2000, b ut by now it has become clear that bacterial enzymes originating from man y dif ferent types of bacteria may actually be the most ab undant proteins in this enzyme f amily. This chapter provides an overview of sulfite oxidase like bacterial enzymes as well as an analysis of their phylogeny.

13.1 Introduction – Sulfite in the Environment and in Cell Metabolism Sulfites form naturally during the decomposition of reduced sulfur compounds such as thiosulf ate, polythionates and sulfonates (Ro y and T rudinger 1970), and in the absence of oxygen, sulfites can persist in the environment (Hayes et al. 2006; Sorokin 1995). Another natural process, namely, sulfur dioxide becoming dissolved in water, can also lead to the formation of hydrogen sulf ite (HSO 3−) and sulf ite (SO 32–). The sulfite anion is a relati vely strong nucleophile and can therefore be used as a reducing agent (SO 42–/HSO32– E°′ = −516 mV; Thauer et al. 1977). As a result of their reactivity, sulfites and so-called sulfiting agents (sulfur dioxide, bisulfites, metabisulfite) are a major class of industrial chemicals. The y are used in applications such as leather tanning, paper milling, photography and, probably most importantly, the food industry. Use of sulf ites in food as a conditioning or preserv ation agent is v ery common, and residual sulf ites in food can cause se vere allergic reactions in some humans following ingestion (Lester 1995; McEvily et al. 1992). In all living cells, sulfur-containing compounds play a major role in the form of coenzymes, amino acids and redox-active molecules. In order to be able to synthesize these compounds, most cells reduce sulf ate to the le vel of sulfur/sulf ide and then incorporate it into the biomass. In this energy-consuming process, sulfate undergoes a two-step activation to 3′-phosphoadenosine 5′-phosphosulfate, from which sulfite is then released and reduced to the desired state via the reaction of a sulfite reductase (Carroll et al. 2005; Kappler and Dahl 2001). 151 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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However, sulfites are also commonly formed as a result of metabolic processes that break do wn sulfonates, sulfur -containing amino acids or reduced inor ganic sulfur compounds. In vertebrates, sulfite generally arises during amino acid breakdown (Griffith 1987). It is then oxidized by a sulfite oxidase (SO) to sulfate, which can be easily e xcreted (Rajagopalan 1980). The formation of free sulf ite within a cell can lead to irre versible damage via disruption of protein disulf ide bonds and damage to bases present in the DN A, and therefore an ef ficient mechanism for removing sulfite from cells by detoxification, export and/or binding of the compound in another form has to be present. Accumulating sulf ite can also cause systemic effects such as damage to the central nervous system and increased oxidative stress (Chamulitrat 1999; Zhang et al. 2004). Ho wever, there may be other , re gulatory roles for sulf ite in the human body as well: ele vated sulfite levels are associated with inflammatory conditions and host defence (Mitsuhashi et al. 1998, et al. 2005; Ratthe et al. 2002). Plants generally encounter sulf ite after e xposure to atmospheric sulfur dioxide or as a result of amino acid breakdo wn. However, the physiological role of the peroxisomal plant SO, which uses oxygen as its preferred electron acceptor, is still being investigated (Mendel and Bittner 2006). In microbial cells sulfite can arise from a variety of reactions, such as dissimilatory sulfur compound oxidation, amino acid degradation, sulfur assimilation, or following sulfonate breakdo wn. Microbial cells can also become e xposed to sulf ite present in the environment, e.g. in the anaerobic regions of the Black Sea (Sorokin 1995). In as far as it has been in vestigated to date, the common strate gy for sulfite detoxification in Bacteria and Archaea seems to in volve an oxidation of sulf ite to sulfate, which can proceed either via a direct formation of sulf ate from sulf ite, or via the indirect APS reductase pathw ay (re viewed in Kappler and Dahl 2001). Despite the fact that the direct oxidation pathway does not allow for a conservation of energy via substrate-level phosphorylation, it appears to be the more common of the two (Kappler and Dahl 2001). The enzymes catalysing the direct oxidation of sulfite to sulfate will be the focus of the remainder of this chapter .

13.2 Sulf ite-Oxidizing Enzymes At present two types of enzymes catalysing the direct oxidation of sulfite to sulfate are recognized: SOs (EC 1.8.3.1) and sulf ite dehydrogenases (SDH; EC 1.8.2.1). Both enzymes are metalloproteins that possess a molybdenum-containing redox centre. Additional redox-acti ve centres (e.g. haem groups) may also be present. The main dif ference between the tw o types of enzymes lies in their ability to transfer electrons to oxygen: SOs transfer electrons to oxygen, ferric yanide and sometimes cytochrome c, while SDHs use either or both of the latter tw o electron acceptors, but do not transfer electrons to oxygen. T ypical SOs are the plant SO (known electron acceptors oxygen, ferric yanide) (Eilers et al. 2001; Hemann et al. 2005) and the well-studied v ertebrate SOs that can use all three electron acceptors

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listed abo ve b ut appear to use a c ytochrome c as their natural electron acceptor (Enemark and Cosper 2002; Rajagopalan 1980). SDHs ha ve been reported in man y bacteria, including the soil bacterium Starkeya no vella (Kappler et al. 2000), Sulfitobacter species (Pukall et al. 1999; Sorokin 1995), v arious “Thiobacilli”, alkanesulfonate-de grading and photosynthetic bacteria (Cook et al. 2006; re viewed in Kappler and Dahl 2001). While for some bacterial SDHs a monohaem cytochrome c has been established as the natural electron acceptor (Kappler et al. 2000; Y amanaka et al. 1981), in man y cases this acceptor is not known. In fact, bacterial enzymes can be divided into two groups on the basis of their preference for either ferric yanide or cytochrome c as an electron acceptor (Kappler and Dahl 2001). The preference for either of these electron acceptors might be an important indicator of the function and/or localization of the respecti ve sulf ite-oxidizing enzymes (SOEs): Enzymes that use a cytochrome c as their natural electron acceptor clearly have to be located in an extracellular compartment or, if they were membrane proteins, they would have to possess a periplasmic/extracellular domain that transfers electrons to cytochrome c. A preference for ferricyanide as an artificial electron acceptor (reported, e.g., for the SOEs from Thiobacillus acidophilus and Comamonas acido vorans; Kappler and Dahl 2001) is also found in the SO from Arabidopsis thaliana that uses oxygen as its preferred electron acceptor (Mendel and Bittner 2006). Although an extracellular location cannot be e xcluded for ferric yanide-dependent SOEs, such a preference might be indicative of SOEs with an intracellular or membrane location which have been sho wn to e xist in some chemolithotrophic and alkanesulfonate-de grading bacteria (Cook et al. 2006; Kappler and Dahl 2001). It should also be noted that while the plant and animal SOs and the so far characterized bacterial enzymes are all soluble proteins, both soluble and membrane-bound bacterial SOEs have been reported (reviewed in Kappler and Dahl 2001).

13.3

Structure and Function of Sulfite-Oxidizing Enzymes

All SOs and SDHs characterized to date belong to the SO family of molybdoenzymes. This enzyme f amily comprises the kno wn SOEs and enzymes related to these as well as the assimilatory nitrate reductases found in plants (Hille 1996). In all enzymes of the SO f amily, a molybdenum atom chelated by the dithiolene groups of a single p yranopterin cofactor (also kno wn as molybdenum cof actor or Moco) forms the active site (Hille 1996). During catalysis, the Mo centre c ycles between the Mo(VI) and Mo(IV) states (Enemark et al. 2006; Enemark and Cosper 2002; Hille 1996), and in the oxidized Mo(VI) state the SOE molybdenum site has been shown to contain two oxo and three sulfur ligands (Fig. 13.1), one of which is from a conserv ed c ysteine residue. The geometry of the molybdenum site is square pyramidal (Kisker et al. 1997a) and the equatorial oxo ligand (Fig. 13.1) is directly involved in the oxidation–reduction reaction catalysed by the SOEs. The spectroscopic

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Fig. 13.1 The sulfite oxidase active site

and catalytic properties of SOEs ha ve been the subject of man y publications and review articles and the reader is referred to these (Enemark et al. 2006; Enemark and Cosper 2002; Hille 1996, 2005). Although the SOEs from v ertebrates, plants and bacteria all belong to the same enzyme family there are significant structural differences between them: while both the v ertebrate and the plant SOs are homodimers containing tw o Mo sites per enzyme molecule (Kisker et al. 1997a; Schrader et al. 2003), the bacterial enzyme from Starkeya novella is a heterodimer with only one Mo site per enzyme molecule (Kappler et al. 2000; Kappler and Baile y 2005). Both the chick en SO and the bacterial SDH contain a haem group, but while the chicken SO contains a haem b, the bacterial SDH contains a haem c located on a second sub unit that forms a per manent complex with the Mo-containing subunit of the enzyme. Crystal structures are a vailable for several enzymes of the SO f amily (Fischer et al. 2005; Kappler and Baile y 2005; Kisk er et al. 1997a; Loschi et al. 2004; Schrader et al. 2003). The structure of the chick en SO w as the f irst to be solv ed and re vealed a threedomain architecture: In this SO a mobile haem b binding domain is connected via a flexible linker region to a central molybdenum-binding and a C-terminal dimerization domain which mediates formation of the SO homodimer (Kisk er et al. 1997a). Both the plant SO and the bacterial SDH were shown to also contain two of these domains, namely the molybdenum-binding and the dimerization domain. In contrast, the YedY protein (Loschi et al. 2004) lacks both the dimerization and the haem b domain. Although both the chicken SO and the bacterial SDH contain haem groups, only the structure of the bacterial enzyme has allo wed insights into intramolecular electron transfer in these SOEs (Kappler and Baile y 2005). Homology modelling suggests that the electron-transfer competent conformation of the chick en SO is very similar to that seen in the bacterial SorAB SDH, which highlights the functional similarity of these two structurally different SOEs.

13.4

Phylogeny of Sulfite-Oxidizing Enzymes

Although SOE activities were first described in bacteria more than 40 years ago and biochemical e vidence for SOEs e xists for man y bacterial species (re viewed in Kappler and Dahl 2001), it remained unclear for a long time whether these bacterial

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enzymes belonged to the SO f amily or whether the y formed a separate class of enzymes. While we can still only speculate on whether the latter may be true of some enzymes still awaiting discovery, it is now clear that many bacterial enzymes exist that are molybdoproteins and belong of the SO f amily. The first bacterial SO-like/SDH proteins to be characterized on both the protein and the genetic le vel were the SoxCD sulfur dehydrogenase from Paracoccus pantotrophus (Quentmeier et al. 2000; W odara et al. 1997), which is part of a multienzyme complex and requires the other complex components for activity, and the SorAB SDH from Starkeya novella (Kappler et al. 2000), which is a true SDH and functions independently of other proteins. Since then SDHs ha ve been characterized from Deinococcus r adiodurans, Acidiphilium acidophilum and Campylobacter jejuni (D’Errico et al. 2006; deJong et al. 2000; Myers and K elly 2005). An unusual enzyme, the Y edY protein from Escherichia coli, which is clearly related to the SO f amily but is of unknown function, w as also characterized and crystallized (Brokx et al. 2005; Loschi et al. 2004). However, with the increasing number of completed genome sequences there is more and more evidence that genes encoding enzymes belonging to the SO f amily are actually widespread in bacteria. W e ha ve recently repeated our phylogenetic analysis of the SO family reported in Kappler and Dahl (2001) 6 years ago: The number of SOE-related gene sequences in GenBank has no w increased to o ver 500 (from around 23), and ne w sequences appear at high frequencies. All sequences considered in the analysis contain the conserv ed cysteine residue that serv es as a ligand to the Mo atom in this enzyme af mily; only residues present in all aligned sequences were used for the determination of the phylogenetic relationships (Kappler and Dahl 2001). A number of entries in the conserved protein domains database (CDD) (MarchlerBauer et al. 2005) refer to the Moco-dimerization and dif ferent Moco-binding domains (Table 13.1). All putati ve proteins considered here contain at least one of these signature sequences. On the basis of our analysis, three groups of SOEs can no w be clearly distinguished (Fig. 13.2), and they have been designated as group 1 – “pathogen enzymes”, group 2 – “classic SOEs and nitrate reductases” and group 3 –“enzymes from Archaea, phototrophic and soil bacteria”. The groups have been arbitrarily named on the basis of either enzyme function or the pro venance of sequences. The sequences identified originate from many different bacterial phyla, including theProteobacteria, the green non-sulfur bacteria, the high-GC Gram-positi ve Bacteria, Bacilli, Actinomycetes, Myxobacteria and Plantomycetes as well as sequences from both Euryarchaeota and Crenarchaeota. Only protein sequences of group 2 SOEs have the two-domain architecture seen in several crystal structures with a Mo-binding domain and a dimerization domain found in tandem on one polypeptide (Kappler and Bailey 2005; Kisker et al. 1997a; Schrader et al. 2003). Both group 1 and group 3 sequences lack the dimerization domain, and the Mo-binding domain of group 3 is further reduced by the loss of parts of the N-terminal sequence.

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Table 13.1 Sulfite oxidase family related entries in the CDD database (Marchler -Bauer et al. 2005) CDD numbering NAME CDD28617 CDD28613 CDD28614 CDD28616 CDD28609 CDD28615 CDD28612 CDD28611 CDD28610 CDD22936 CDD23450 CDD11749

Bact_SorA_Moco SO_family_Moco_dimer eukary_SO_Mico Bact_SoxC_Moco SO_family Moco eukary_NR_Moco arch_bact_SO family bact_SO_family_Moco YedY_like_Moco pfam00174 Oxidoreductases_molybdopterin-binding pfam03404 Mo-co_dimer COG2041 Sulfite oxidase and related enzymes

Fig. 13.2 The phylogeny of the sulf ite oxidase enzyme f amily. The three main subf amilies are indicated by the boxes. Shading is used to accentuate the dif ferent major groupings within the subfamilies. The relative position of sequences of enzymes that are discussed in the text are highlighted in bold and labelled . The ph ylogenetic tree w as generated using the neighbour -joining method. SDH sulfite dehydrogenase

The average deduced molecular mass of the Mo-binding proteins in the dif ferent groups is between 30 and 35 kDa for group 1, 40 and 60 kDa for group 2 (SOEs only) and 20 and 25 kDa for group 3. Interestingly, with the exception of the E. coli YedY protein (a group 1 enzyme), all of the well-characterized SOEs are found in group 2, which therefore has been designated as containing the “classic SOEs and nitrate reductases”. To the best of my kno wledge no protein belonging to group 3 has been characterized in any detail so far.

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Diversity of Enzymes Within the Sulfite Oxidase Family

13.5.1 Group 1 – SOE-Like Enzymes Originating from Pathogenic Microorganisms SOE-like enzymes in group 1 mainly originate from known bacterial pathogens such as Pseudomonas aeruginosa , pathogenic and nonpathogenic E. coli strains, Salmonella, Yersinia, Shewanella, Burkholderia and Ralstonia species. Exceptions to this are sequences from or ganisms related to the Roseobacter lineage, Rhodobacter species and Thiobacillus denitrificans, which are also found in this group. On the basis of the structure of the Mo-binding domain, the group 1 SOEs can be di vided into tw o subgroups, group 1A, which contains Y edY-like enzymes (average molecular mass of Mo domain around 35 kDa, 300–330 amino acids), and group 1B, which contains SOEs with a Moco domain of around 30 kDa (approximately 240–270 amino acid chain length). W ithin group 1A, sequence homologies of around 50% identity (approximately 66–68% similarity) are found. W ithin group 1B, identity v alues are around 40% (approximately 60% similarity), while between the two groups identities fall to around 24% (approximately 40% similarity). In an analysis of group 1 sequences for the presence of signal peptides (Signal P and TatP; Bendtsen et al. 2005; Nielsen et al. 1999) that might indicate an e xtracellular localization most proteins returned ambiguous results or were predicted to lack a signal peptide. There were dif ferences e ven in the predicted location of closely related sequences such as Y edY from E. coli (periplasmic) and from Yersinia species (no signal peptide predicted). It w ould then seem that v arious cellular locations can be assumed for the proteins in this group of SOEs, none of which have been predicted to be membrane bound. In contrast, the genetic context of the group 1 SOE genes is very conserved: all genes encoding group 1 SOEs are associated with genes encoding conserved, membrane-bound proteins, which, lik e the group 1 SOEs themselv es, fall into two categories. Genes encoding group 1A proteins occur together with genes encoding proteins with six transmembrane helices and belonging to the UPF0191 cate gory of conserv ed proteins (Marchler -Bauer et al. 2005). It is likely that all of these proteins bind haem b similar to YedZ, which is a representative of this group and has been described as the second sub unit of YedY (Brokx et al. 2005). In contrast, genes encoding group 1B SOEs are found in association with genes encoding proteins of the COG4117 “thiosulf ate reductase cytochrome b subunit” type (Marchler-Bauer et al. 2005) that contain four conserved transmembrane helices. It w ould then appear that all group 1 SOEs interact with a second sub unit which in all cases is a membrane-bound, haem b binding protein which should allow for transfer of electrons to or from the quinone-pool (see also later and Fig. 13.4).

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Group 1A Enzymes: YedY and Related Proteins

The YedY protein from E. coli is the only group 1 enzyme that has been characterized to date, and its crystal structure clearly sho wed that it is a member of the SO family (Loschi et al. 2004). There are several interesting differences between YedY and other SO/SDH proteins: A crucial and conserv ed residue in the other SO enzymes is Arg55 (SorAB SDH numbering), and in Y edY this residue is replaced by an asparagine residue (Kappler and Baile y 2005; Kisk er et al. 1997a; Loschi et al. 2004). In addition, instead of a positi vely charged substrate-binding pock et similar to the one of the group 2 SOEs (Kappler and Baile y 2005; Kisk er et al. 1997a), the Y edY substrate-binding pocket is mainly hydrophobic with se veral tyrosine (Tyr47, Tyr231) and tryptophan (Trp223, Trp246) residues. A single charged residue, Glu104, occupies a position close to the Mo centre that is v ery similar to that of a catalytically important aspartate residue in bacterial dimethyl sulfoxide and N-oxide reductases (Loschi et al. 2004). Our analyses sho wed that both residues that are close to the Mo active site and those lining the substrate-binding pock et are conserved in the majority of Y edY-like group 1A protein sequences. The sequence around the Mo-binding Cys102 (YedY numbering) is also highly conserved (Fig. 13.3). The in vivo function of YedY is unclear at present: YedY lacks sulfite-oxidizing activity, and may function as a reductase in vivo, as some enzymatic acti vity has been obtained with sulfoxides and N-oxides (Loschi et al. 2004). Y edY interacts with the haem b binding membrane protein YedZ (Em7 = − 8 mV). YedZ was shown to interact weakly with wild-type Y edY and strongly with a Y edY C102S v ariant (Brokx et al. 2005). YedZ was also shown to interact with menadiol, which indicates

Fig. 13.3 Conserved amino acid residues around the conserv ed, Mo-binding c ysteine. Strictly conserved residues ( bold), conserv ed residues ( normal type ), conserv ed acti ve-site residues (boxes), conserved cysteine residue (shaded box)

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that the YedYZ-catalysed reaction may be linked to the quinone pool in vivo (Brokx et al. 2005). Within group 1A, there e xist a number of subgroups, and one of these contains enzymes from bacteria of the Roseobacter lineage such as Silicibacter pomeroyii, Roseobacter and Sulfitobacter species (Wagner-Doebler and Biebl 2006). During the oxidation of organosulfonates by these bacteria sulfite is produced and oxidized by an as yet uncharacterized SDH with a preference for ferric yanide as an electron acceptor (data from Silicibacter pomeroyii; Denger et al. 2006). The only SOE-like protein identified in the Silicibacter genome in addition to this group 1 protein is related to the SoxC sulfur dehydrogenase which is involved in thiosulfate oxidation via a multienzyme comple x (thiosulfate-oxidizing multienzyme system, T OMES) and has neither sulfite-oxidizing capacity in the absence of other T OMES proteins nor an ability to transfer electrons to ferric yanide (Quentmeier et al. 2000). It will therefore be interesting to determine whether the Roseobacter lineage group of YedY-like proteins has a role in the de gradation of or ganosulfonates or whether a novel type of SDH is involved in the process. This may be the case if, as so far predicted, this ferric yanide-linked SDH is located in the c ytoplasm: both the Y edYlike and the SoxC-lik e SOEs found in Silicibacter pomeroyi are predicted to be periplasmic proteins (Fig. 13.4). 13.5.1.2

Group 1B – 30-kDa Mo-Domain Pr oteins

The group 1B enzymes dif fer from the group 1A enzymes not only in their association with a dif ferent type of membrane sub unit, b ut also in the much lower degree of sequence conserv ation found around the Mo-binding c ysteine residue (Fig. 13.3). In addition, se veral of the acti ve-site/substrate binding pocket residues conserved in group 1A SOEs are not conserved in group 1B SOEs, and this includes the asparagine residue which in Y edY occupies a position similar to that of the crucial Arg55 (SorAB numbering) found in group 2 SOEs. The substrate-binding pocket appears to be less hydrophobic than that of YedY, which could be an indication of a dif ferent substrate spectrum/reaction catalysed: only T rp223 is strictly conserv ed; Tyr231 is present in some cases. It will, however, be necessary to determine the crystal structure of a group 1B enzyme t o determine whether these observations are meaningful. Most of the group 1B SOEs (i.e. proteins from Pseudomonas, Burkholderia and Rhodopseudomonas) appear to be located in the c ytoplasm, while a few may have an N-terminal transmembrane helix. In contrast, for another subgroup containing sequences from cyanobacterial and myxococcal species the cellular localization is ambiguous, with most sequences returning either clearly ambiguous prediction results or being predicted to contain a signal peptide (Fig. 13.4). The group 1B enzyme originating from Thiobacillus denitrificans is of particular interest, as a membrane-bound SOE that transferred electrons to ferric yanide has been purified from this organism (Aminuddin and Nicholas 1974). Experiments with crude extracts or membrane fractions sho wed that sulf ite oxidation in Thiobacillus

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Fig. 13.4 The most frequently encountered enzyme conformations in the three dif ferent groups of sulf ite-oxidizing enzymes. Top: Group 1A and group 1B enzymes sho wn in tw o possible conformations, with the Mo-binding sub unit either in the c ytoplasm or in the periplasm. Bottom: Group 2 and group 3 enzymes. Group 2 enzymes are sho wn with the Moco-dimer domain. CSO chicken liver sulfite oxidase, HSO human sulfite oxidase, PSO plant sulfite oxidase, SorAB SDH SorAB sulfite dehydrogenase, SoxCD S°DH Sox CD sulfur dehydrogenase, TMH transmembrane helices, c c-type cytochrome, b b-type cytochrome

denitrificans was inhibited by v arious inhibitors of the electron-transfer chain, indicating that the reaction was coupled to a reduction of the quinone pool. It is possible that the enzyme described by Aminuddin and Nicholas is identical to the group 1B enzyme considered here.

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It is also interesting to note that for some group 1B SOEs originating from Mycobacterium avium, Rubrobacter xylanophilus and a Nocardioides sp., a gene fusion between the genes encoding the Mo-binding and the membrane sub unit has occurred.

13.5.2 Group 2: “Classic” Sulfite-Oxidizing Enzymes and Nitrate Reductases A large body of literature is available for the sulfite-oxidizing and nitrate-reducing proteins that fall into this group. Especially the v ertebrate SOs have been covered extensively, and the reader is referred to se veral e xcellent re views on the topic (Enemark et al. 2006; Enemark and Cosper 2002; Kisk er et al. 1997b; Mendel 2005; Mendel and Bittner 2006). Three subgroups, group 2A – “SOs and plant nitrate reductases”, group 2B – “SoxCD sulfur dehydrogenases” and group 2C – “SorAB SDHs” are easily distinguishable within the group 2 SOEs. There is some v ariation in the a verage predicted molecular masses of the SOEs in the three groups: “SOs” tend to have a molecular mass of around 55–60 kDa, the nitrate reductases about 95– 100 kDa, SoxC-lik e proteins around 45 kDa and SorA-lik e proteins about 42 kDa. All group 2 SOEs show the unique combination of a “Moco-dimer domain” and a Moco-binding domain (Fig. 13.4). In se veral cases, fusions of other domains to the central Mo and dimerization domains ha ve occurred. This is most ob vious for the plant assimilatory nitrate reductases and the v ertebrate SOs: in the former a C-terminal fusion with a c ytochrome b domain (pf am 00173), a fla vin adenine dinucleotide binding domain (pf am 00970) and an NAD-binding domain (pfam 00175) has occurred, while in the case of the SOs an N-terminal fusion with a cytochrome b encoding gene has occurred. Most of the group 2 SOEs occur in e xtracytoplasmic compartments of the cell (Fig. 13.4). There seems to be a lesser degree of conservation in the amino acids surrounding the Mo-binding c ysteine residue in this group (Fig. 13.3), and those residues that are conserv ed dif fer mark edly from those conserv ed in the tw o other groups of SOEs. As the major groupings contain proteins that are comparatively well studied and have already been described in the introductory sections, the y will be dealt with here only briefly. 13.5.2.1

Group 2A: Sulfite Oxidases and Plant Nitrate Reductases

The “SOs” subgroup contains sequences from most forms of life, including mammals, birds, amphibians, insects, plants and fungi. Although all characterized SOs from higher animals are soluble proteins located in the mitochondrial

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intermembrane space, the SO-related genes from Xenopus laevis encode a protein with an N-terminal membrane domain (amino acids 59–78) that precedes the haem b and the Moco-dimer domains. There are even some bacterial sequences located in this group, which appear to originate from Mycobacterium bo vis, Mycobacterium tuber culosis, Oceanicola granulosus and Streptomyces nodosus . All four of these sequences lack signal peptides as well as haem b domains. The tw o mycobacterial genes are associated with genes encoding MmpL-type membrane proteins. MmpL proteins are a f amily of lipid transporters, some of which ha ve been sho wn to be in volved in the virulence and pathogenesis of mycobacteria (Domenech et al. 2005). It remains to be established whether these are true SOEs and what function they might have in their respective source organisms. 13.5.2.2

Group 2B: SoxCD-Like Enzymes – “Sulfur Dehydrogenases”

This group contains protein sequences that are related to the SoxC sub unit of the SoxCD protein that w as first described in Paracoccus species (K elly et al. 1997; Wodara et al. 1997). SoxCD is a periplasmic (SoxCD)2 heterotetramer (Quentmeier et al. 2000) in which SoxC contains the Mo redox centre, while SoxD is a c-type cytochrome containing either one or tw o haem groups. SoxCD is part of a T OMES (Friedrich et al. 2005; K elly et al. 1997), and enhances the reaction rate of the T OMES comple x when assayed together with other complex components (Quentmeier et al. 2000). The e xact nature of the reaction catalysed by the SoxCD protein is unkno wn, and in isolated form it has a lo w affinity for sulf ite. SoxCD has been proposed to oxidize a sulf ane sulfur atom bound to a conserv ed cysteine present in one of the other T OMES components to a sulfone group (Bardische wsky et al. 2005). A crystal structure for SoxCD is, at present, not available. Genes encoding SoxC-like SOEs usually occur in T OMESencoding sox gene clusters (Friedrich et al. 2005) and this is borne out by our analyses which show that this is true for most of the group 2B sequences. The majority of genes encoding SoxC-lik e SOEs are associated with genes encoding SoxD c ytochrome subunits. These SoxD proteins e xist in tw o different forms that bind one or tw o haem groups respecti vely (Appia-Ayme et al. 2001; Kappler et al. 2001; Quentmeier et al. 2000). The function of the monohaem or dihaem forms is unclear as it has recently been sho wn that a single haem group is sufficient for SoxCD function within the TOMES (Bardischewsky et al. 2005). The two SoxD forms differ in their domain structure: both monohaem and dihaem SoxD proteins contain an N-terminal COG3258(c ytochrome c)/2863(cytochrome c553)-like domain, which is follo wed by a C-terminal COG3474(c ytochrome c556 and cytochrome c′) domain in dihaem SoxD proteins. Consequently , monohaem SoxD proteins have about 180–200 amino acids, while the dihaem forms can ha ve up to about 400 residues. In addition, the SoxD proteins that ha ve been studied so f ar contain a conserved CxxxC motif, which is similar to a motif found in metal-binding redox proteins such as PrrC and Sco (McEw an et al. 2002) and could indicate

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the presence of another redox centre in SoxCD proteins. This CxxxC motif, ho wever, is absent from a group of monohaem SoxD proteins that are associated with SoxC-related protein sequences fromBurkholderia, Nitrobacter andBradyrhizobium species (e.g. accession nos. ZP_00457665, ABA06238, NP_772761). This group of sequences is also conspicuous for another reason: they are not associated with a sox gene cluster. It therefore seems possible that they represent a novel type of SoxCDrelated enzyme which may function in a dif ferent metabolic conte xt from the SoxCD “sulfur dehydrogenases” that are part of a T OMES. 13.5.2.3

Group 2C: SorAB-Like Sulfite Dehydrogenases

The SorAB protein from Starkeya novella was the first true bacterial SOE to be characterized in sufficient detail to enable its classif ication as a member of the SO f amily (Kappler et al. 2000). It is a true SDH in that it does not transfer electrons to oxygen, and it was also the first bacterial SOE in group 2 for which a crystal structure was solved (Kappler and Bailey 2005). SorAB is a periplasmic heterodimer of a large Moco-dimer domain (40.2 kDa) and a small cytochrome c sub unit (8.8 kDa) The protein as well as some SorAB v ariants containing site-directed mutations have been studied in detail (Doonan et al. 2006; Kappler et al. 2006; Raitsimring et al. 2005). Proteins related to the SorAB SDH are found in organisms such as Xanthobacter, Campylobacter, Ralstonia, Rhizobia, Nitrobacter, Kineococcus and Brevibacterium. Most of the genes encoding SorA-related proteins are associated with a gene encodi ng a monohaem c ytochrome c; however, in some cases such as Sulfitobacter EE-26 and N AS-14, the sorA-like genes are associated with genes encoding dihaem cytochromes. There is a high degree of diversity between the haem proteins associated with the SorA-lik e SOEs, and sequence homology between these “SorB-lik e” proteins is usually limited to phylogenetically closely related sequences. So far all characterized members of this enzyme family are true SDHs with a periplasmic location and a haem c binding second subunit. One example of such a protein is the recently characterized Campylobacter jejuni SDH (Myers and Kelly 2005) that cross-reacts with anti- Starkeya novella SDH antibodies. T he Campylobacter SDH has been suggested to be in volved in survival of Campylobacter under microaerobic conditions in the environment or to serve as a mechanism for detoxif ication of sulfite (Myers and Kelly 2005). Again, within the group of SorA-related protein sequences there are e xceptions from the “rule”, and a whole subgroup of sequences originating from “high-GC Gram-positive” bacteria (e.g. Kineococcus, Mycobacterium or Streptomyces) and some euryarchaeota (Haloarcula, Natronomonas) are not located in the vicinity of a gene encoding a haem c binding sub unit. The sequences contained in this group also share another characteristic: on average they contain more than 500 amino acids, and ha ve been predicted to contain three to f ive transmembrane domains using the TMHMM program (Moller et al. 2001). These membrane domains appear to be e xclusively located in the N-terminal re gion of these proteins (amino acids

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1–200), while the Moco-dimer domains are located between amino acid 250 and the C-terminus and are predicted to reside on the extracytoplasmic face of the membrane, a characteristic shared by most other group 2 SOEs (Fig. 13.4). It would then seem that this group of enzymes represents another no vel type of SOE-fusion protein. The N-terminal part of these membrane-bound SOEs contains no conserv ed domains, and although there are almost al ways f ive transmembrane domains present, there is no signif icant de gree of conserv ation between these N-terminal membrane domains. 13.5.2.4

Other Sulfite-Oxidizing Enzymes in Group 2

A small number of group 2 enzymes do not appear to belong directly to an y of the three major subgroups. These are SOE-related enzymes from various microorganisms, including Arthrobacter (ZP_00410553), Roseovarious (ZP_00961287), Sinorhizobium (AAK65805) and Deinococccus r adiodurans (AAF12408). This last enzyme has recently been shown to be a ferricyanide-dependent, molybdenumcontaining SDH by D’Errico et al. (2006). The enzyme was constitutively expressed in Deinococccus, but its exact function remains some what unclear and it has been suggested to be involved in intracellular dissimilatory sulfite oxidation. In summary it appears as if most of the group 2 SOEs are soluble, mainly extracytoplasmic proteins and are often associated with haem groups, although there are enzymes (e.g. the plant SOs) that lack additional redox centres. This analysis of the group has also uncovered some novel enzyme groups such as the bacterial enzymes in group 2A, the “SoxCD”-lik e proteins that are not part of a T OMES and the haemless, membrane-bound SorA-related enzymes.

13.5.3 Group 3: Sulfite-Oxidizing Enzymes – Enzymes from Archaea, Phototrophic and Soil Bacteria The third group of SOEs is the one that contains the most reduced Mo-binding domain: in addition to the absence of the dimerization domain, which leads to a reduction of the Mo-binding domain to around 30 kDa in the group 1 SOEs, the domain has been further reduced to an average molecular mass of 22–25kDa by the loss of some N-terminal parts of the sequence. In addition, to the best of my knowledge, none of these enzymes ha ve ever been studied, so only some general observ ations can be made. The putative protein sequences in this group originate from a v ariety of archaeal species (Fig. 13.2) belonging to bothCrenarchaeota and Euryarchaeota (Sulfolobus sp., Ferroplasma, Pyr obaculum, Ar chaeoglobus, Halobacterium ) as well as bacterial species belonging to the high-GC Gram-positi ve Bacteria, Firmicutes (bacilli), the Thermus/Deinococccus group, cyanobacteria and several a-Proteobacteria. In addition individual sequences from a green non-sulfur bacterium, a Planctomycete, a Solibacter and an Acidobacterium are also present.

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Protein sequence identities in the archaeal group are between 30 and 40%, while the sequences from bacteria have about 35–47% identity. Between these groups the amino acid identity levels fall to 25–33%. Despite this, there is some conserv ation of residues surrounding the conserv ed, Mo-binding c ysteine between the tw o groups (Fig. 13.3) and the conserved residues found in group 3 are most similar to those found in the group 1 SOEs. The vast majority of enzymes in this group lack an e xport signal (Signal P or TatP programmes, Bendtsen et al. 2005; Nielsen et al. 1999), and transmembrane helices appear to be absent from all of the group 3 enzymes, which suggest that group 3 SOEs are cytoplasmic enzymes (Fig. 13.4). The genes encoding the group 3 proteins are not found in a conserv ed genetic environment. The only e xception is a major group of α-Proteobacterial sequences that originate from Rhodopseudomonas, Bradyrhizobium and Xanthobacter species. The genes encoding these proteins appear to occur together with genes encoding an OsmC-like protein (COG1765, also COG 2945); ho wever, the gene encoding this OsmC-like protein may not be another sub unit for these group 3 SOEs, b ut may simply be conserved because all the sequences found in that cluster are from closely related bacterial species.

13.6 Conclusions The combination of genome sequencing, adv ances in techniques for the study of proteins and the isolation of man y ne w bacterial species from sulfur -containing habitats has led to great adv ances in the study of bacterial and archaeal sulfur metabolism in recent years, as the phylogenetic analysis of the bacterial SOEs presented here clearly indicates. There are still many challenges, and analysis of the genetic data alone may help with the creation of a systematic overview of an enzyme family such as the SO family, but only studies of the in vivo function of the enzymes in question can pro vide clues as to their true function and ho w they are integrated into general metabolism. It is clear from our analyses though that SOEs occur in many bacterial species, only some of which are known sulfur chemolithotrophs. Microbial sulfur oxidation occurs over nearly the entire pH scale, and notably most of the sequences represented in the analysis presented here of the SO enzyme family originate from neutrophilic microbes. It is interesting that despite several publications re garding SOEs from Acidothiobacillus ferr ooxidans (reviewed in Kappler and Dahl 2001), no SOE-related enzyme w as detected in the Acidithiobacillus genome. By the same token, only reports of the presence of sulfite-oxidizing activities are available for the recently disco vered alkali and haloalkaliphilic sulfur-oxidizing bacteria (Sorokin et al. 2000), and it will be interesting to inspect the genome sequences of these or ganisms once they become available. There is also the open question as to why some bacteria appear to contain a large number of genes encoding SOE-related enzymes belonging to se veral of the three

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groups described above. This is the case forRhizobia, Burkholderia, Campylobacter, Ralstonia, Streptomyces and others. In conjunction with our analysis of the phylogen y of the SOEs and the genetic context in which the dif ferent enzymes are found, this apparent redundanc y of genes encoding enzymes from the same f amily also suggests that the number of different metabolic functions and possibly also reactions that can be carried out by the bacterial enzymes of the SO family is much greater than recognized at present. As most of the SOEs that ha ve been studied to date are soluble proteins, it will be especially interesting to in vestigate the properties of the membrane-bound SOEs found in some “high-GC Gram-positi ve” bacteria, and to unco ver the role of the archaeal enzymes from both the Crenarchaeota and the Euryarchaeota. An e xciting possibility w ould be that these enzymes might contain tungsten rather than molybdenum at their active site, which would make them the first tungsten-containing enzymes in this metalloprotein family. Note: A more detailed representation of the dif ferent branches of the SOE tree could not be included in this article o wing to space restraints. Acknowledgement. U.K. thanks the University of Queensland for a grant and a fello wship.

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D’Errico G, Di Salle A, La Cara F, Rossi M, Cannio R (2006) Identif ication and characterization of a novel bacterial sulfite oxidase with no heme binding domain from Deinococcus radiodurans. J Bacteriol 188:694–701 Domenech P, Reed MB, Barry CE (2005) Contribution of the Mycobacterium tuberculosis MmpL protein family to virulence and drug resistance. Infect Immun 73:3492–3501 Doonan CJ, Kappler U, Geor ge GN (2006) Structure of the acti ve site of sulf ite dehydrogenase from Starkeya novella. Inorg Chem 45:7488–7492 Eilers T, Schwarz G, Brinkmann H, W itt C, Richter T , Nieder J, K och B, Hille R, Haensch R, Mendel RR (2001) Identif ication and biochemical characterization of Arabidopsis thaliana sulfite oxidase – a new player in plant sulfur metabolism. J Biol Chem 276:46989–46994 Enemark JH, Cosper MM (2002) Molybdenum enzymes and sulfur metabolism. Met Ions Biol Syst 39:621–654 Enemark JH, Astashkin AV, Raitsimring AM (2006) In vestigation of the coordination structures of the molybdenum(V) sites of sulfite oxidizing enzymes by pulsed EPR spectroscopy. Dalton Trans 3501–3514 Fischer K, Barbier GG, Hecht HJ, Mendel RR, Campbell WH, Schwarz G (2005) Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site. Plant Cell 17:1167–1179 Friedrich CG, Bardische wsky F, Rother D, Quentmeier A, Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Microbiol 8:253–259 Griffith OW (1987) Mammalian sulfur amino acids metabolism: an o verview. Methods Enzymol 143:366–376 Hayes MK, Taylor GT, Astor Y, Scranton MI (2006) Vertical distributions of thiosulfate and sulfite in the Cariaco Basin. Limnol Oceanogr 51:280–287 Hemann C, Hood BL, Fulton M, Hansch R, Schw arz G, Mendel RR, Kirk ML, Hille R (2005) Spectroscopic and kinetic studies of Arabidopsis thaliana sulfite oxidase: nature of the redox-active orbital and electronic structure contrib utions to catalysis. J Am Chem Soc 127:16567–16577 Hille R (1996) The mononuclear molybdenum enzymes. Chem Re v 96:2757–2816 Hille R (2005) Molybdenum-containing hydroxylases. Arch Biochem Biophys 433:107–116 Kappler U, Bailey S (2005) Molecular basis of intramolecular electron transfer in sulfite-oxidizing enzymes is re vealed by high resolution structure of a heterodimeric comple x of the catalytic molybdopterin subunit and a c-type cytochrome subunit. J Biol Chem 280:24999–25007 Kappler U, Dahl C (2001) Enzymology and molecular biology of prokaryotic sulf ite oxidation. FEMS Microbiol Lett 203:1–9 Kappler U, Bennett B, Rethmeier J, Schw arz G, Deutzmann R, McEw an A G, Dahl C (2000) Sulfite: cytochrome c oxidoreductase from Thiobacillus novellus – purification, characterization and molecular biology of a heterodimeric member of the sulf ite oxidase f amily. J Biol Chem 275:13202–13212 Kappler U, Friedrich CG, T rüper HG, Dahl C (2001) Evidence for tw o pathways of thiosulf ate oxidation in Starkeya novella (formerly Thiobacillus novellus). Arch Microbiol 175:102–111 Kappler U, Bailey S, Feng CJ, Hone ychurch MJ, Hanson GR, Bernhardt PV , Tollin G, Enemark JH (2006) Kinetic and structural evidence for the importance of Tyr236 for the integrity of the Mo active site in a bacterial sulf ite dehydrogenase. Biochemistry 45:9696–9705 Kelly DP, Shergill JK, Lu WP, Wood AP (1997) Oxidative metabolism of inorganic sulfur compounds by bacteria. Antonie Van Leeuwenhoek 71:95–107 Kisker C, Schindelin H, Pacheco A, Wehbi WA, Garrett RM, Rajagopalan KV, Enemark JH, Rees DC (1997a) Molecular basis of sulfite oxidase deficiency from the structure of sulfite oxidase. Cell 91:973–983 Kisker C, Schindelin H, Rees DC (1997b) Molybdenum-cof actor-containing enzymes: Structure and mechanism. Annu Rev Biochem 66:233–267 Lester MR (1995) Sulf ite sensiti vity – signif icance in human health. J Am Coll Nutr 14: 229–232

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Loschi L, Brokx SJ, Hills TL, Zhang G, Bertero MG, Lo vering AL, Weiner JH, Strynadka NCJ (2004) Structural and biochemical identif ication of a no vel bacterial oxidoreductase. J Biol Chem 279:50391–50400 Marchler-Bauer A, Anderson J, Cherukuri P, DeWeese-Scott C, Geer L, Gwadz M, He S, Hurwitz D, Jackson J, K e Z, Lanczycki C, Liebert C, Liu C, Lu F , Marchler G, Mullokando v M, Shoemaker B, Simonyan V, Song J, Thiessen P, Yamashita R, Yin J, Zhang D, Bryant S (2005) CDD: a Conserv ed Domain Database for protein classif ication. Nucleic Acids Res 33:D192–196 McEvily AJ, Iyeng ar R, Otwell WS (1992) Inhibition of enzymatic bro wning in foods and beverages. Crit Rev Food Sci Nutr 32:253–273 McEwan AG, Lewin A, Davy SL, Boetzel R, Leech A, Walker D, Wood T, Moore GR (2002) PrrC from Rhodobacter sphaeroides, a homologue of eukaryotic Sco proteins, is a copper -binding protein and may have a thiol- disulfide oxidoreductase activity. FEBS Lett 518:10–16 Mendel RR (2005) Molybdenum: biological activity and metabolism. Dalton Trans 3404–3409 Mendel RR, Bittner F (2006) Cell biology of molybdenum. Biochim Bioph ys Acta 1763: 621–635 Mitsuhashi H, Nojima Y , Tanaka T, Ueki K, Maeza wa A, Y ano S, Naruse T (1998) Sulf ite is released by human neutrophils in response to stimulation with lipopolysaccharide. J Leuk oc Biol 64:595–599 Mitsuhashi H, Yamashita S, Ik euchi H, K uroiwa T, Kaneko Y, Hiromura K, Ueki K, Nojima Y (2005) Oxidati ve stress-dependent con version of h ydrogen sulf ide to sulf ite by acti vated neutrophils. Shock 24:529–534 Moller S, Croning MDR, Apweiler R (2001) Ev aluation of methods for the prediction of membrane spanning regions. Bioinformatics 17:646–633 Myers JD, K elly DJ (2005) A sulphite respiration system in the chemoheterotrophic human pathogen Campylobacter jejuni. Microbiology 151:233–242 Nielsen H, Brunak S, V onHeijne G (1999) Machine learning approaches to the prediction of signal peptides and other protein sorting signals. Protein Eng 12:3–9 Pukall R, Buntefuß D, Frühling A, Rohde M, Kroppenstedt RM, Bur ghardt J, Lebaron P, Bernard L, Stackebrandt E (1999) Sulfitobacter mediterraneus sp. nov., a new sulfite-oxidizing member of the alphaproteobacteria. Int J Syst Ev ol Microbiol 49:513–519 Quentmeier A, Kraft R, K ostka S, Klockenkamper R, Friedrich CG (2000) Characterization of a new type of sulf ite deh ydrogenase from Paracoccus pantotr ophus GB17. Arch Microbiol 173:117–125 Raitsimring AM, Kappler U, Feng CJ, Astashkin A V, Enemark JH (2005) Pulsed EPR studies of a bacterial sulfite-oxidizing enzyme with pH invariant hyperfine interactions from exchangeable protons. Inorg Chem 44:7283–7285 Rajagopalan KV (1980) Sulfite oxidase (sulfite: ferricytochrome c oxidoreductase). In: Coughlan MP (ed) Molybdenum and molybdenum-containing enzymes. Per gamon, Oxford, pp 243–272 Ratthe C, Pelletier M, Roberge CJ, Girard D (2002) Activation of human neutrophils by the pollutant sodium sulfite: effect on c ytokine production, chemotaxis, and cell surf ace expression of cell adhesion molecules. Clin Immunol 105:169–175 Roy AB, Trudinger PA (1970) The chemistry of some sulfur compounds. In: Ro y AB, Trudinger PA (eds) The biochemistry of inor ganic sulfur compounds. Cambridge Uni versity Press, London, pp 7–42 Schrader N, Fischer K, Theis K, Mendel RR, Schw arz G, Kisk er C (2003) The crystal structure of plant sulfite oxidase provides insights into sulfite oxidation in plants and animals. Structure 11:1251–1263 Sorokin DY (1995) Sulfitobacter pontiacus gen. no v., sp. no v. – a ne w heterotrophic bacterium from the black sea specialized on sulf ite oxidation. Microbiology 64:295–305 Sorokin DY, Kuenen GJ, Jetten MSM (2000) Denitrif ication at extremely high pH v alues by the alkaliphilic, oblig ately chemolithoautotrophic, sulfur -oxidizing bacterium Thioalkalivibrio denitrificans strain ALJD. Arch Microbiol 175:94–101

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Thauer RK, Jungermann K, Deck er K (1977) Ener gy conserv ation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180 Wagner-Doebler I, Biebl H (2006) En vironmental biology of the marine Roseobacter linea ge. Annu Rev Microbiol 60:255–280 Wodara C, Bardische wsky F, Friedrich CG (1997) Cloning and characterization of sulf ite dehydrogenase, tw o c-type c ytochromes, and a fla voprotein of Paracoccus denitrif icans GB17: Essential role of sulf ite deh ydrogenase in lithotrophic sulfur oxidation. J Bacteriol 179:5014–5023 Yamanaka T, Yoshioka T, Kimura K (1981) Purif ication of sulphite c ytochrome c reductase of Thiobacillus no vellus and reconstitution of its sulphite oxidase system with the purif ied constituents. Plant Cell Physiol 22:613–622 Zhang X, Vincent AS, Halliwell B, Wong KP (2004) A mechanism of sulfite neurotoxicity – direct inhibition of glutamate dehydrogenase. J Biol Chem 279:43035–43045

Chapter 14

Sulfonates and Organotrophic Sulfite Metabolism Alasdair M. Cook, Theo H.M. Smits, Karin Denger

Abstract One is used to considering sulf ite oxidation as part of a lithotrophic process (e.g. SorAB or Sox system), much of which in volves neutral or ionic inorganic sulfur species on the outer surf ace of the c ytoplasmic membrane. In contrast, the processes referred to in this chapter in volve or ganic compounds, which (1) include a highly stable sulfonate substituent (C−SO3−), (2) are involved in the organotrophic growth of the organism and (3) much of whose metabolism takes place in the cytoplasm. Many phenomena are associated with this life-style. The sulfonate may be a natural product, e.g. taurine or sulfoquino vose, whose synthesis can in volve sulf ite, or a x enobiotic laundry deter gent, b ut it is ef fectively al ways a char ged species, so an uptak e system is essential. T wo kno wn systems are mentioned, A TP binding cassette transporters and tripartite A TPindependent periplasmic transporters. Annual dissimilation of me gatonnes of organosulfonates essentially al ways in volves intracellular sulf ite generated by diverse enzymic cleavages in bacteria, archaea and possibly eukarya. The fate of this sulfite in anaerobes is often sulf ide. Aerobes occasionally e xcrete sulf ite directly; more frequently, the sulfite is oxidized to sulfate. Many aerobic bacteria excrete only sulfate, but many others excrete also some sulfite, which is rapidly oxidized to sulfate, even under anoxic conditions. The nature and location of these sulf ite dehydrogenases are still unclear , b ut periplasmic SorAB is apparently used by some bacteria. In contrast to the cytochrome c coupled SorAB, there is at least one widespread, uncharacterized sulfite dehydrogenase, which is assayed with ferric yanide as the e lectron acceptor. Dissimilation of a sulfonate releases sulfite (sulfate) in about 500-fold excess of the sulfur requirement for gro wth, so e xporters, tw o classes of which ha ve been detected, are essential to prevent the cell from suffering osmotic stress.

14.1 Intr oduction The sulfur c ycle has man y f acets of dif ferent magnitudes, and this conference (ISMSM) e xamined processes in volving the major lithotrophic mass flux es and placed emphasis on membrane-associated processes. Here, the focus is mo ved to organotrophy with biosynthesis, biotransformation and dissimilation of a group of 170 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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Fig. 14.1 Representative aliphatic organosulfonates from the atmosphere, v ertebrates, spiders, bacteria, archaea, plants and algae. The arrows indicate some degradative routes in the literature (Cook and Denger 2002; Cook et al. 2006). Natural sulfonates are ob viously ubiquitous, and the C−SO 3− bond is not degraded by, e.g., mammals, which excrete organosulfonates (Huxtable 1992). The widespread utilization of organosulfonates is by microbes, whereby up till no w largely bacteria were meant (Cook and Denger 2002; Cook et al. 1999, 2006): utilization by archaea, suggested by sequence data (Rein et al. 2005), has been supported by the f irst experimental data (J. van der Oost and T.H.M Smits, unpublished data), and utilization by a dinoflagellate is suspected (Mayer et al. 2006)

organic compounds, namely or ganosulfonates, which contain sulfur in oxidation state +5 (V airavamurthy et al. 1994; see also Chap. 20 by Prange). These are ancient compounds, some of which ha ve only recently been disco vered, e.g. methanesulfonate (Fig. 14.1) with its signif icance in the sulfur c ycle (Baker et al. 1991). Another sulfonate, discovered before organic chemistry became a synthetic science, is taurine, 2-aminoethanesulfonate (Fig. 14.1): it w as initially called ‘Gallen-Asparagin’ (T iedemann and Gmelin 1827), and the name ‘T aurin’ w as attributed to Gmelin (Demarçay 1838). A century later, den Dooren de Jong (1926) included taurine in some of his studies on microbial nutrition. Man y natural organosulfonates of increasing complexity, and sometimes at enormous concentrations, are cited in reviews (Huxtable 1992; Jacobson and Smith 1968; Yancey et al. 2002)

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Fig. 14.2 A natural arylsulfonate and three commercially available arylsulfonates. The natural product (Hickford et al. 2004) is juxtaposed with one known desulfonation (during ring cleavage) (Junker et al. 1994a): a typical desulfonation prior to ring clea vage (Junker et al. 1994b) and desulfonation subsequent to ring cleavage (Feigel and Knackmuss 1993; Schleheck et al. 2004) are illustrated

and research papers (Abraham et al. 2004; Suzuki et al. 2002; Vollrath et al. 1990) (Fig. 14.1), which lead us to conclude that large quantities of natural sulfonates are being c ycled in the food webs in marine and terrestrial en vironments. Natural aliphatic sulfonates are widely kno wn, b ut natural arylsulfonates are also being found (Budzikiewicz et al. 1998; Hickford et al. 2004; Ov enden and Capon 1999), one of which is shown in Fig. 14.2. The degradation of these natural arylsulfonates is presumably the background to the degradation of anthropogenic arylsulfonates. Mankind now uses megatonnes of

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sulfonated surfactants annually, especially linear alk ylbenzenesulfonate (Knepper and Berna 2003; Fig. 14.2). Dyestuffs often contain sulfonated moieties (Fig. 14.2). It is normal to formulate cationic pharmaceuticals with sulfonates, both aromatic and aliphatic (O’Neil 2001): recently , a major aid in assisting patients to abstain from alcohol, and that is about 4% of the population, is a sulfonate, which is dosed at 2 g day –1 (Cook et al. 2006). Candidate drugs (sulfonates) for strok e and Alzheimer’s disease are also dosed at high le vels (Cook et al. 2006). Kondo’s group made the f irst attempts to elucidate de gradative pathw ays for taurine and isethionate (K ondo and Ishimoto 1972), with the consequence that sulfite was recognized as a de gradative intermediate. About the same time, biodegradable sulfonated surfactants were introduced in commerce, and Cain recognized that the de gradation of model arylsulfonates in volved sulf ite as an intermediate (Johnston et al. 1975): it no w appears that all enzymic desulfonation reactions studied involve the release of sulf ite (Cook and Denger 2002; Cook et al. 1999, 2006; Lie et al. 1998). Biosynthesis of natural sulfonates can also in volve sulfite (see later). The amounts of sulfite in these organotrophic processes do not compete with those in lithotrophic metabolism, b ut these several megatonnes of sulfite, the biodiversity in the metabolic pathw ays and the physiological problems generated (and solved) in these pathways are the backgrounds to this re view.

14.2 Biosynthesis

of Organosulfonates

A major part of our understanding of the biosynthesis of non-carbohydrate, aliphatic sulfonates comes from White (1984, 1986), who set out to characterize the biosynthesis of coenzyme M in methanogenic archaea, and of a sulfolipid in bacteria (capnine; Fig. 14.1). The tw o turned out to be related (Fig. 14.3), and the later discovery that coenzyme M is in volved in the clea vage of epoxides in aerobic bacteria in volved in alkene de gradation (Coleman and Spain 2003) broadens the rele vance of White’ s central pathway. White’s pathway is involved not only in the generation of sulfolactate for bacterial endospore formation, and l-cysteate for capnine-like lipids (Graham et al. 2002), b ut, presumably, also for taurine and taurolipids (Fig. 14.3). The k ey to the sulfonation reaction is a Michael addition of sulf ite to the double bond of phosphoenolpyruvate by phosphosulfolactate synthase (EC 4.4.1.19; ComA) (Fig. 14.3). The synthesis of sulfoquino vose (Fig. 14.1) is also an addition of sulf ite to an activated compound, in this case UDP-glucose (Sanda et al. 2001). Half the sulfur in plants is apparently present as sulfoquino vose, and the corresponding sulfolipid is found in photosynthetic algae, protists and bacteria, so this reaction also consumes considerable amounts of sulfite in the biosphere. The mammalian synthesis of taurine in volves an oxygenation of c ysteine to cysteine sulf inate, a decarboxylation and an unkno wn oxidation to taurine (Stipanuk 2004; Fig. 14.4). The important fact here is that the taurine is excreted. Mammals cannot clea ve the C−SO 3− bond (Huxtable 1992). Dietary c ysteate is apparently excreted as sulfolactate and sulfop yruvate (Weinstein and Griffith 1988).

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Fig. 14.3 The biosynthesis of coenzyme M in methanogens ( bold arrows) and our interpretation (normal arrows) of the generalized pathway to supply different microorganisms with sulfolactate (spore-formers), l-cysteate for sulfolipids (Cytophagales), taurine for sulfolipids (marine bacteria and some algae) and coenzyme M for the aerobes which also use the cof actor in biodegradation

Fig. 14.4 Synthesis of taurine in mammals and spiders, and e xcretion of sulfonates. Taurine has many functions in mammals (Huxtable 1992), b ut after being functional, the compound is excreted, largely in urine. l-Cysteate in mammals is dietary, and transamination and excretion are indicated (Weinstein and Griffith 1988). Large amounts of sulfonates are involved in the function of spiders’ webs (Vollrath et al. 1990)

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Another major source of e xtracellular sulfonates is spiders’ webs (V ollrath et al. 1990; Fig. 14.4). So not only do man y or ganisms produce sulfonates (Fig. 1 4.3), which will be released when herbi vores graze, b ut man y or ganisms e xcrete sulfonates (Fig. 14.4) directly into the en vironment, where biotransformation or dissimilation can take place. Recent research (Domin y et al. 2006) sho ws that the oxygenation reaction (EC 1.13.11.20) found in mammals is also present in some bacteria. Apparently this can function as a second pathw ay to supply sulfolactate for spore formation.

14.3 Dissimilation

of Organosulfonates

We ha ve seen that the sulfonates are costly to generate. Either a cell in vests a high-energy bond to obtain a sulfonate, or it risks oxidati ve stress by involving an oxygenase. The carbon–sulfonate bond is a strong bond, about as strong as a carbon–carbon bond, and the first organic chemists were astonished at theresistance of taurine to strong acid or alkali. The consequence for the biodegradation of organosufonates is that a v ery stable bond must be brok en. We believe that the natural organosulfonates are phylogenetically ancient entities (Huxtable 1992; K elly and Murrell 1999), and that the biodi versity we see in desulfonation mechanisms reflects the long exposure of microbes to organosulfonates. The di versity seen in the desulfonation of arylsulfonates will serv e as an introduction. Note that we are talking about dissimilation: M. K ertesz, T. Tralau and A. Schmalenber ger (personal communication) introduced a dif ferent set of desulfonative enzymes involved in the assimilation of sulfonate sulfur . Some arylsulfonates are desulfonated concomitantly with activation of the ring by multicomponent dioxygenases, as for 4-toluenesulfonate (Fig. 14.2). One case is kno wn in which desulfonation is concomitant with the simpler dioxygenation in volved in ring cleavage (Fig. 14.2). And in the third e xample, (di)oxygenations generate the molecule which can be subtly manipulated and desulfonated by hydrolysis, as in the case of linear alk ylbenzenesulfonate (Fig. 14.2). In each case, sulf ite is the stoichiometric product of the enzyme reaction. When we consider the aliphatic sulfonates (Fig. 14.5), we again see sulfite as the stoichiometric product of desulfonation. Methylsulfonate monooxygenase is another multicomponent oxygenase (K elly and Murrell 1999), and we presume it to be archetypal for man y similar reactions (Cook et al. 2006). Suitably placed substituents on sulfonates allow less spectacular desulfonations, as can be seen for sulfolactate and l-cysteate (Fig. 14.5) (Cook et al. 2006). The reaction we kno w best, inasmuch as we have sketches of complete pathways (see later), is sulfoacetaldehyde acetyltransferase (Xsc) (Fig. 14.5). As in all the desulfonation reactions in Fig. 14.2, the enzymes are soluble and in the c ytoplasm. Now that desulfonations have been introduced, it is relevant to draw attention to, e.g., l-cysteate sulfo-lyase (Fig. 14.5) in a dif ferent manner. The substrate car ries three char ged moieties, and the three products carry one each. None of these

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Fig. 14.5 Some desulfonation reactions. Methanesulfonate monooxygenase is a multicomponent system (MsmABCD), which generates formaldeh yde and sulfite from the substrate. Sulfolactate sulfo-lyase has tw o subunits (SuyAB) and tightly bound Fe(II). l-Cysteate sulfo-lyase (CuyA) represents a third desulfonation mechanism with cof actor p yridoxal 5 ′-phosphate ( PLP). The sulfoacetaldehyde acetyltransferase (Xsc) reaction in volves thiamin diphosphate ( ThDP) as a cofactor

compounds will pass through a protein-free bimolecular lipid leaflet. The cell needs to keep its carbon source in the cell for ener gy conservation and growth, so the pyruvate disappears. This potentially leaves the cell with problems, because the nitrogen supply is in about fourfold excess and the sulfur supply is in about 500-fold excess. Not only that, this sulfur source, sulfite, is considered to be toxic. Exploding may be one answer to toxin at high osmotic pressure, b ut it seems a bit e xtreme, and considering the amount of desulfonation in extant microorganisms (Figs. 14.2, 14.5), it is obviously not the response that cells ha ve developed.

14.4

The Detoxification or Fate of Sulfite

We would like to introduce the critical situation gradually , with the desulfonation reaction that we kno w from pathw ays whose genes are found in man y genomes (Brüggemann et al. 2004; Cook and Denger 2002, 2006; Cook et al. 2006; Denger et al. 2006a; Gorzynska et al. 2006; Rein et al. 2005). Our hypothesis for the dissimilation of taurine in anaerobic Desulfotalea psychrophila is given in Fig. 14.6.

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Fig. 14.6 Hypothetical pathw ay of taurine de gradation in Desulfotalea psyc hrophila LSv54. Given that the organism grows with taurine (R. Rabus, unpublished data), we deduced the pathway from the genome sequence (Rab us et al. 2004) and our e xperience with related pathw ays in other organisms (Denger et al. 2006a; Gorzynska et al. 2006). Ack acetate kinase, Ald alanine dehydrogenase, Tpa taurine:p yruvate aminotransferase, DsrAB dissimilatory sulf ite reductase, Pta phosphotransacetylase

Gene candidates for a tripartite ATP-independent periplasmic transporter, TauKLM, are present, as are genes to encode a taurine:p yruvate aminotransferase and for the oxidative release by alanine dehydrogenase of the ammonium ion, which presumably is e xported by the AmtB f acilitator. So here we ha ve presumably resolv ed the problem of accumulation of the ammonium ion. Desulfonation by Xsc is presumably follo wed by ener gy conserv ation in volving dissimilatory sulf ite reductase (see Chap. 2 by Fritz et al. and Chap. 3 by Pereira), so there is nothing unusual about a sulfate reducer letting HS– (H2S?) diffuse out of the cell. The cell has a secondary source of ATP, via acetate kinase, which will lead to the disposal of acetate; again, this is nothing unusual in an anaerobe. Another of our isolates, Desulfonispora thiosulfatig enes, e xcretes thiosulf ate in place of sulf ide (Denger et al. 1999). Y et another isolate, Desulfovibrio sp. strain GRZCYSA, generates sulf ate and sulf ide (Laue et al. 1997). Disposal of these oxyanions is more difficult to explain, but we have no further data on these processes in anaerobes, so let us consider the corresponding process in aerobes. The DOE Joint Genomic Institute has recently reannotated the genome sequence of Burkholderia xeno vorans LB400 and incorporated our suggestions about taurine metabolism (Brüggemann et al. 2004; Rein et al. 2005; Ruf f et al. 2003), while V. Sauvé and B. Berks (personal communication) and C. Dahl (personal communication; see Chap. 9 by Grimm et al.) recommended locating c ytochrome c in the periplasm or the cytoplasmic membrane. This allo ws the development of the follo wing scheme (Fig. 14.7). The inducible pathw ay presumably consists of, in the carbon-rele vant aspect, an ATP binding cassette transporter, a cytochrome c coupled, membrane-bound taurine dehydrogenase (TauXY), the desulfonation by Xsc, phosphate acetyltransferase (phosphotransacetylase; Pta) and further metabolism of acetylcoenzyme A. The inorganic aspects involve ammonia and sulfite. We presume that the AmtB facilitator is present to

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Fig. 14.7 Degradative pathw ay for taurine in Burkholderia xeno vorans LB400. The genome sequence, experimental data on taurine dehydrogenase (TDH), Xsc and Pta (Ruff et al. 2003), with support for sulf ite dehydrogenase (SorAB) from a dif ferent strain (T able 14.1), and other data (Denger et al. 2006a; Gorzynska et al. 2006; Rein et al. 2005) form the basis for this f igure

excrete ammonia. We assume that there is at least one sulf ite dehydrogenase present, SorAB (see later), and the or ganism presumably uses the gene product of orfX, in the xsc-pta-orfX cluster (Brüggemann et al. 2004), to excrete sulfite to the periplasm, where most of the oxyanion is oxidized immediately. Some transient sulfite is detected outside the cell (Table 14.1), which presumably indicates a f aster excretion of sulfite via OrfX than oxidation via SorAB. The overall picture is thus quite comple x. There is the desulfonati ve pathway itself, including the sulfite exporter, but apparently with an independent cytochrome c for taurine dehydrogenase, an independent AmtB and an independent sulf ite dehydrogenase. The glyoxylate pathway is also needed. So our intelligent microbes have a sophisticated strate gy to deal with the comple xities of metabolizing this apparently simple molecule, taurine. There is considerable biodi versity in this small pathw ay (Figs. 14.6, 14.7). The diversity continues in the unresolved details of the sulfite dehydrogenases.

14.5

Sulfite Dehydrogenases in Sulfonate Metabolism

Lectures at this conference dealt with three major sulfite dehydrogenases, periplasmic SorAB (see Chap. 13 by Kappler), an aspect of the periplasmic Sox system (see Chap. 12 by Friedrich et al.; V. Sauvé and B. Berks, personal communication) and the intracellular , indirect pathw ay via adenosyl phosphosulf ate (see Chap. 2 by

No data None detected Inducible Paracoccus pantotrophus NKNCYSAa (+) Paracoccus denitrificans NKNISb (+) No data None detected None detected Paracoccus versutus N-MTc (+) No data None detected Inducible Silicibacter pomeroyi DSS-3d One None None detected Inducible Rhodobacter sphaeroides 2.4.1e None None None detected None detected Burkholderia sp. strain ICD f No data No data Inducible No data Burkholderia xenovorans LB400g None 2? No data No data Comamomas sp. strain SFCD1 h No data No data Inducible No data Delftia acidovorans NATi No data No data None detected Inducible Alcaligenes faecalis MT-1c No data No data None detected Inducible The columns referring to genes contain information deri ved or inferred from genome sequences. The three right-hand columns refe physiological data. a Rein et al. (2005). b Brüggemann et al. (2004). c Weinitschke et al. (2006). d Gorzynska et al. (2006). e Denger et al. (2006a). f King et al. (1997). g Unpublished data (from JGI and from S. Weinitschke, K. Denger and S.M. Cook). h King and Quinn (1997). i Mayer et al. (2006).

No Yes Yes No No Yes Yes Yes No Yes r to biochemical and

Table 14.1 The nature of the sulf ite dehydrogenases in volved in aerobic gro wth of bacteria utilizing taurine. Paracoccus spp. can usually e xpress the sox genes, so the parentheses indicate that this property has not been conf irmed in these strains Cytochrome-c-coupled Ferricyanide-coupled Sulfite excreted Organism soxCD sorAB sulfite dehydrogenase sulfite dehydrogenase during growth

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Fritz et al. and Chap. 3 by Pereira) in anoxygenic phototrophs. W e have not found the indirect pathway in aerobes (Denger et al. 2006a), which is unsurprising, given the sensitivity of the enzymes to oxygen (see Chap. 2 by Fritz et al.). W e suspect that the Sox system is seldom involved, because no organism with the genes on its genome seems to express a cytochrome c coupled sulfite dehydrogenase (Table 14.1). We suspect that SorAB is sometimes in volved (Fig. 14.7), because Quinn’ s group found cytochrome c coupled sulfite dehydrogenase in their Burkholderia sp. strain ICD (King et al. 1997; Ruff et al. 2003), which could correspond with the presence of candidate sorAB genes in B. xenovorans LB400 (Table 14.1). Quinn’s group (King and Quinn 1997) also found candidate SorAB in Comamonas sp. strain SFCD1. However, the option to oxidize sulf ite in the periplasm seems to be only one possibility, and some or ganisms use dif ferent options with dif ferent substrates. Silicibacter pomeroyi DSS-3 utilizes taurine, induces a sulf ite dehydrogenase and excretes sulfate via an unkno wn exporter; no sulf ite is observ ed. However, when the organism utilizes l-cysteate with induction of sulfite dehydrogenase, it excretes sulfite almost quantitati vely, apparently via CuyZ, a paralogue of T auZ, the presumed sulf ate e xporter in man y Alphaproteobacteria (Denger et al. 2006a, b; Gorzynska et al. 2006; Rein et al. 2005). This sulf ite dehydrogenase is ar guably cytoplasmic, because a periplasmic enzyme w ould not allow sulfite to accumulate to significant amounts extracellularly. We suspect that this sulf ite dehydrogenase in S. pomeroyi DSS-3 represents a major group of unkno wn sulfite dehydrogenases, which is found in man y of our isolates (Table 14.1). The enzyme w as discovered by Reichenbecher et al. (1999) in a strain of Delftia acidovorans, and only in D. acidovorans have we been able to elute active enzyme from a chromatography column (K. Denger, unpublished data). We hope to be able to characterize this enzyme in the near future. Some organisms have no detectable sulf ite dehydrogenase (Table 14.1), one of which (Paracoccus denitrificans NKNIS) leaks some sulf ite during sulfate formation and one of which ( Rhodobacter sphaer oides 2.4.1) does not. W e are, thus, uncertain whether yet more sulfite dehydrogenases await discovery, or whether the assay conditions used were unsuitable.

14.6 Conclusions Organosulfonates are widespread in Nature. Lar ge amounts of sulf ite are used to generate these compounds. Some sulfonates are generated via enzymic or atmospheric oxygenation of sulfhydryl groups. These or ganosulfonates are sometimes functional as synthesized, b ut lar ger compounds (e.g. lipids, surfactants and even nucleic acids) are also generated. All these natural compounds are subject to biode gradation in food webs, and there are indications that not only bacteria, but also archaea and protists can cleave

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the stable carbon–sulfonate bond. There is a broad range of enzymes in volved in desulfonation and desulfonative pathways. The product of desulfonation is al ways sulfite. This sulf ite has man y possible fates, depending on the type of metabolism in the host or ganism. Similar degradative pathways are found in strict anaerobes, f acultative anaerobes (where they function under both oxic and anoxic conditions) and strict aerobes. Under aerobic condit ions, the diversity of sulfite dehydrogenases is incompletely understood. Cells ha ve de veloped f airly comple x sets of responses to the multiplicity of charged compounds generated during the de gradation of these char ged organosulfonates. We think that we can describe these phenomena, b ut our understanding at the molecular level is still very limited. Acknowledgements. We are grateful to R. Rabus for growing D. psychrophila and to the DOE Joint Genomic Institute for making sequence data a vailable on their Web site, especially for B. xenovorans LB400 and R. sphaeroides 2.4.1. Man y undergraduate students in our advanced teaching laboratory also contributed to the data. The research in our laboratory w as funded by the Uni versity of Constance, the DFG, the European Union (SUITE), ECOSOL and CLER, and the LBS Stiftung Umwelt und Wohnen.

References Abraham W-R, Strömpl C, V ancanneyt M, Bennasar A, Swings J, Lünsdorf H, Smit J, Moore ERB (2004) Woodsholea maritima gen. nov., sp. nov., a marine bacterium with a low diversity of polar lipids. Int J Syst Ev ol Microbiol 54:1227–1234 Baker SC, Kelly DP, Murrell JC (1991) Microbial degradation of methanesulphonic acid: a missing link in the biogeochemical sulphur c ycle. Nature 350:627–628 Brüggemann C, Denger K, Cook AM, Ruff J (2004) Enzymes and genes of taurine and isethionate dissimilation in Paracoccus denitrificans. Microbiology 150:805–816 Budzikiewicz H, Fuchs R, Taraz K, Marek-Kozaczuk M, Skorupska A (1998) Dihydropyoverdin7-sulfonic acids – unusual bacterial metabolites. Nat Prod Lett 12:125–130 Coleman NV, Spain JC (2003) Distrib ution of the coenzyme M pathw ay of epoxide metabolism among ethene- and vinyl chloride-degrading Mycobacterium strains. Appl Environ Microbiol 69:6041–6046 Cook AM, Denger K (2002) Dissimilation of the C 2 sulfonates. Arch Microbiol 179:1–6 Cook AM, Denger K (2006) Metabolism of taurine in microor ganisms: a primer in molecular diversity? Adv Exp Med Biol 583:3–13 Cook AM, Laue H, Junker F (1999) Microbial desulfonation. FEMS Microbiol Re v 22:399–419 -sulfonates. Arch Microbiol Cook AM, Denger K, Smits THM (2006) Dissimilation of C 3 185:83–90 Demarçay H (1838) Ueber die Natur der Galle. Ann Pharm 27:270–291 den Dooren de Jong LE (1926) Bijdrage tot de k ennis van het mineralisatieproces. Nijgh & v an Ditmar, Rotterdam Denger K, Stackebrandt E, Cook AM (1999) Desulfonispora thiosulfatigenes gen. nov., sp. nov., a widespread, taurine-fermenting, thiosulf ate-producing, anaerobic bacterium. Int J Syst Bacteriol 49:1599–1603

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Denger K, Smits THM, Cook AM (2006a) Genome-enabled analysis of the utilization of taurine as sole source of carbon or nitrogen by Rhodobacter sphaeroides 2.4.1. Microbiology 152:3197–3206 Denger K, Smits THM, Cook AM (2006b) L-Cysteate sulfo-lyase, a widespread, p yridoxal 5′-phosphate-coupled desulfonati ve enzyme purif ied from Silicibacter pomer oyi DSS-3 T. Biochem J 394:657–664 Dominy JE Jr, Simmons CR, Karplus P A, Gehring AM, Stipanuk MH (2006) Identif ication and characterization of bacterial c ysteine dioxygenases: a ne w route of c ysteine de gradation in eubacteria. J Bacteriol 188:5561–5569 Feigel BJ, Knackmuss H-J (1993) Syntrophic interactions during de gradation of 4-aminobenzenesulfonic acid by a two species bacterial culture. Arch Microbiol 159:124–130 Gorzynska AK, Denger K, Cook AM, Smits THM (2006) Inducible transcription of genes involved in taurine uptake and dissimilation by Silicibacter pomeroyi DSS-3T. Arch Microbiol 185:402–406 Graham DE, Xu H, White RH (2002) Identif ication of coenzyme M biosynthetic phosphosulfolactate synthase: a new family of sulfonate biosynthesizing enzymes. J Biol Chem 277:13421–13429 Hickford SJH, Küpper FC, Zhang G, Carrano CJ, Blunt JW, Butler A (2004) Petrobactin sulfonate, a new siderophore produced by the marine bacterium Marinobacter hydrocarbonoclasticus. J Nat Prod 2004:1897–1899 Huxtable RJ (1992) Physiological actions of taurine. Physiol Rev 72:101–163 Jacobson JG, Smith LH (1968) Biochemistry and ph ysiology of taurine and taurine deri vatives. Physiol Rev 48:424–511 Johnston JB, Murray K, Cain RB (1975) Microbial metabolism of aryl sulphonates. A reassessment of colorimetric methods for the determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzene sulphonates. Antonie Van Leeuwenhoek 41:493–511 Junker F, Field JA, Bangerter F, Ramsteiner K, Kohler H-P, Joannou CL, Mason JR, Leisinger T, Cook AM (1994a) Oxygenation and spontaneous deamination of 2-aminobenzenesulphonic acid in Alcaligenes sp. strain O-1 with subsequent meta ring clea vage and spontaneous desulphonation to 2-hydroxymuconic acid. Biochem J 300:429–436 Junker F, Leisinger T, Cook AM (1994b) 3-Sulphocatechol 2,3-dioxygenase and other dioxygenases (EC 1.13.11.2 and EC 1.14.12.-) in the de gradative pathw ays of 2-aminobenzenesulphonic, benzenesulphonic and 4-toluenesulphonic acids in Alcaligenes sp. strain O-1. Microbiology 140:1713–1722 Kelly DP, Murrell JC (1999) Microbial metabolism of methanesulfonic acid. Arch Microbiol 172:341–348 King JE, Jaouhari R, Quinn JP (1997) The roleof sulfoacetaldehyde sulfo-lyase in the mineralization of isethionate by an environmental Acinetobacter isolate. Microbiology 143:2339–2343 King JE, Quinn JP (1997) Metabolism of sulfoacetate by en vironmental Aureobacterium sp. and Comamonas acidovorans isolates. Microbiology 143:3907–3912 Knepper TP, Berna JL (2003) Surf actants: properties, production, and en vironmental aspects. In: Knepper TP, Barceló D, de Voogt P (eds) Analysis and f ate of surfactants in the aquatic environment. Elsevier, Amsterdam, pp 1–50 Kondo H, Ishimoto M (1972) Enzymatic formation of sulfite and acetate from sulfoacetaldehyde, a degradation product of taurine. J Biochem 72:487–489 Laue H, Denger K, Cook AM (1997) Fermentation of c ysteate by a sulf ate-reducing bacterium. Arch Microbiol 168:210–214 Lie TL, Leadbetter JR, Leadbetter ER (1998) Metabolism of sulfonic acids and other organosulfur compounds by sulfate-reducing bacteria. Geomicrobiol J 15:135–149 Mayer J, Denger K, Smits THM, Holleme yer K, Groth U, Cook AM (2006) N-Acetyltaurine dissimilated via taurine by Delftia acidovorans NAT. Arch Microbiol 186:61–67 O’Neil MJ (2001) International nonproprietary names (INN) for radicals and groups proposed for pharmaceutical substances by the W orld Health Or ganization. In: The Merck inde x. Merck, Whitehorse Station Ovenden SPB, Capon RJ (1999) Echinosulfonic acids A-C and echinosulfone A: novel bromoindole sulfonic acids and a sulfone from a southern Australian marine sponge, Echinodictyum. J Nat Prod 62:1246–1249

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Rabus R, Ruepp A, Frick ey T, Rattei T, Fartmann B, Stark M, Bauer M, Zibat A, Lombardot T , Becker I, Amann J, Gellner K, T eeling H, Leuschner WD, Glöckner F-O, Lupas AN, Amann R, Klenk H-P (2004) The genome of Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic sediments. Environ Microbiol 6:887–902 Reichenbecher W , K elly DP , Murrell JC (1999) Desulfonation of propanesulfonic acid by Comamonas acidovorans strain P53: evidence for an alkanesulfonate sulfonatase and an atypical sulfite dehydrogenase. Arch Microbiol 172:387–392 Rein U, Gueta R, Denger K, Ruff J, Hollemeyer K, Cook AM (2005) Dissimilation of cysteate via 3-sulfolactate sulfo-lyase and a sulf ate e xporter in Paracoccus pantotr ophus NKNCYSA. Microbiology 151:737–747 Ruff J, Denger K, Cook AM (2003) Sulphoacetaldeh yde acetyltransferase yields acetyl phosphate: purif ication from Alcaligenes defr agrans and gene clusters in taurine de gradation. Biochem J 369:275–285 Sanda S, Leustek T , Theisen MJ, Gara vito RM, Benning C (2001) Recombinant Arabidopsis SQD1 converts UDP-glucose and sulf ite to the sulfolipid head group precursor UDPsulfoquinovose in vitro. J Biol Chem 276:3941–3946. Schleheck D, Knepper TP, Fischer K, Cook AM (2004) Mineralization of individual congeners of linear alkylbenzenesulfonate (LAS) by def ined pairs of heterotrophic bacteria. Appl En viron Microbiol 70:4053–4063 Stipanuk MH (2004) Sulfur amino acid metabolism: pathw ays for production and remo val of homocysteine and cysteine. Annu Rev Nutr 24:539–577 Suzuki T, Wada T, Saigo K, Watanabe K (2002) Taurine as a constituent of mitochondrial tRNAs: new insights into the functions of taurine and human mitochondrial diseases. EMBO J 21:6581–6589 Tiedemann F, Gmelin L (1827) Einige neue Bestandtheile der Galle des Ochsen. Ann Phys Chem 9:326–337 Vairavamurthy A, Zhou W , Eglinton T , Manowitz B (1994) Sulfonates: a ne w class of or ganic sulfur compounds in marine sediments. Geochim Cosmochim Acta 58:4681–4687 Vollrath F, Fairbrother WJ, Williams RJP, Tillinghast EK, Bernstein DT, Gallagher KS, Townley MA (1990) Compounds in the droplets of the orb spider’ s viscid spiral. Nature 345:526–528 Weinitschke S, Denger K, Smits TMH, Hollemeyer K, Cook AM (2006) The sulfonated osmolyte N-methyltaurine is dissimilated by Alcaligenes faecalis and by Paracoccus ver sutus with release of methylamine. Microbiology 152:1179–1186 Weinstein CL, Griffith OW (1988) Cysteinesulfonate andβ-sulfopyruvate metabolism. Partitioning between decarboxylation, transamination, and reduction pathw ays. J Biol Chem 263:3735–3743 White RH (1984) Biosynthesis of the sulfonolipid 2-amino-3-hydroxy-15-methylhexadecane1-sulfonic acid in the gliding bacterium Cytophaga johnsonae. J Bacteriol 159:42–46 White RH (1986) Intermediates in the biosynthesis of coenzyme M (2-mercaptoethanesulfonic acid). Biochemistry 25:5304–5308 Yancey PH, Blake WR, Conley J (2002) Unusual organic osmolytes in deep-sea animals: adaptations to h ydrostatic pressure and other perturbants. Comp Biochem Ph ysiol A Mol Inte gr Physiol 133:667–676

Chapter 15

Oxidation of Sulfur and Inorganic Sulfur Compounds in Acidianus ambivalens Arnulf Kletzin

Abstract Mechanisms of archaeal sulfur and inor ganic sulfur comp ound oxidation were almost exclusively studied in Acidianus species, extremely thermophilic and acidophilic (pH opt 2–3), coccoid microor ganisms living in acidic v olcanic environments (solf ataras) w orldwide. The y utilize H 2, H 2S, S 0, polythionates, and metal sulf des as the most important sources of metabolic ener gy for CO 2 f xation during aerobic growth. The sulfur oxidation pathways include a soluble sulfur oxygenase reductase (SOR), membrane-bound thiosulf ate and sulf te oxidoreductases, a soluble tetrathionate hydrolase, and an oxidati ve adenosine 5 ′-phosphosulfate reductase pathw ay. Here, the current kno wledge of the biochemistry of these enzymes is discussed with a special focus on the implications of the recently published 3D structure of the SOR.

15.1 Intr oduction Oxidation and reduction of elemental sulfur (S 0) and inor ganic sulfur compounds (ISCs) for ener gy conserv ation is a common property of (hyper -) thermophilic Archaea. This is not surprising gi ven the ab undance of ISCs in v olcanic environments. Most culti vated isolates are anaerobes and thri ve by reduction of S 0 with inorganic gases or or ganic nutrients as electron donors (Schönheit and Schäfer 1995; Kletzin 2007). Other Archaea found predominantly in acidic hydrothermal environments oxidize S 0 and ISCs aerobically to sulfuric acid (Huber and Prangishvili 2005; Kletzin 2006). Most of these isolates belong to the Sulfolobales order within the Crenarchaeota kingdom: they comprise the strictly aerobic genera Sulfolobus and Metallosphaera, the strictly anaerobic Stygiolobus, and the facultatively anaerobic Acidianus and Sulfurisphaera (the sixth genus, Sulfurococcus, is probably lost) (Huber and Prangishvili 2005). Sulfolobales inhabit solf ataras, which are small, steam-heated pools of boiling surf ace water or mud, named after the Solfatara caldera near Naples, Italy (Fig. 15.1). Optimal gro wth conditions are pH 2–3 and 65–92°C in the laboratory , whereas the in situ temperatures are typically at the ambient boiling point. Members of Sulfolobales also contrib ute to bioleaching of base and precious metals and to the formation of acidic drainage 184 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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Fig. 15.1 Solfataric fumarole and hot spring at Caldeira V elha, São Miguel, Açores, Portug al. The dark color is the result of plant debris dropping into the solf atara from the surrounding area. The fumarole w as the source of Stygiolobus acoricus, an obligatory anaerobic Crenarchaeote of the Sulfolobales order (Segerer et al. 1991). (Photo, Arnulf Kletzin)

downstream of mines and self-heating slug heaps (Huber and Prangishvili 2005). In contrast, bacterial sulfur oxidizers are physiologically and phylogenetically diverse and include anaerobic, phototrophic as well as aerobic, chemolithoautotrophic or mixotrophic bacteria. Several members of the Sulfolobales developed into archaeal model or ganisms, especially Sulfolobus acidocaldarius , S. solfataricus , S. tok odaii, and Acidianus ambivalens. S. solfataricus and S. acidocaldarius gro w on v arious or ganic substrates. S. acidocaldarius, the first hyperthermophile to be isolated, w as originally described as a f acultatively autotrophic sulfur oxidizer (Brock et al. 1972). However, the strain presently available in culture collections (DSM 639) is not able to do so an ymore (Norris and Johnson 1998). It is assumed that the original cultures, which were isolated by successi ve rounds of serial dilution, had not been strictly pure but consisted of a mixture of microscopically indistinguishable heterotrophic and autotrophic strains. T oday’s type strains were probably gro wn from single colonies after plating techniques for hyperthermophiles had been improved. Acidianus species and especially A. brierleyi are metabolically more v ersatile. They gain ener gy by autotrophic sulfur or hydrogen oxidation with air or S 0 as electron acceptors (Huber et al. 1992). A. brierleyi has been shown to grow by aerobic chemolithotrophic sulfur and hydrogen oxidation, by oxidation of pyritic metal ores, or by anaerobic sulfur reduction (Huber and Prangishvili 2005). It also grows heterotrophically on organic substrates with or without sulfur and e ven by anaero-

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bic molybdate respiration (Brierle y and Brierle y 1982). The biochemistry of archaeal sulfur metabolism w as most thoroughly studied in A. ambivalens and in the obligatorly anaerobic heterotrophic euryarchaeote Pyrococcus furiosus (Kletzin 2006). A. ambivalens has the adv antage that it is f acultativly anaerobic and can grow both by hydrogen and sulfur oxidation with sulfur and oxygen as electron acceptors, respectively. A. ambivalens is also part of the aerobic bioleaching community used for biohydrometallurgical metal recovery from low-grade ores. A more general o verview of the biochemistry of dissimilatory sulfur oxidation and reduction in Archaea was given recently in Kletzin (2006). This contrib ution will summarize adv ances of the physiology and especially the biochemistry of sulfur and ISC oxidation in Acidianus species in more detail, focusing on A. ambivalens and especially on its sulfur oxygenase reductase (SOR).

15.2

Sulfur and Sulfur Oxidation

Sulfur is the 14th most abundant element in the earth’s crust. The bulk of the sulfur deposits are found as sulfidic metal ores or as sulfate sediments (Middelburg 2000). A significant amount of ISCs gets into the circulation owing to volcanic activity. S0 and ISCs are pre valent in hydrothermal e xhalations and can amount up to 10% of the dry volume. Sulfur is an element with a complex inorganic chemistry. S0 is almost insoluble in water (5 µg l −1 at 25°C, solubilities at higher temperatures are unkno wn) (Boulegue 1978). H 2S, polysulf ides, metal sulf ides (MeS and MeS 2), S 0, and the sulfur oxyanions sulfite, thiosulfate, polythionates, and sulfate are the biologically relevant sulfur species (Roy and Trudinger 1970). Sulfur compounds ha ve the tendency to form homoatomic chains and rings reactly with each other easily . Thus, man y ISCs will react rapidly at ele vated temperatures to form the thermodynamically most stable product under the given conditions (Steudel 2000). The oxidation of S 0 to sulfuric acid proceeds in several steps and involves intermediates like sulfite, thiosulfate, tetrathionate, and e ven sulfide. Several pathways are distinguished depending on the or ganisms, the environment, and the pH of the medium (re viewed in T akakuwa 1992; K elly et al. 1997; Friedrich et al. 2005; Kletzin 2006). The Sox comple x is currently the best-understood ISC-oxidizing enzyme system. It is found in the periplasm of chemolithotrophic aerobic or phototrophic anaerobic members of the Bacteria growing at more or less neutral pH. Its composition is modular: at least eight polypeptides collaborate to oxidize most ISCs in an oxygen-independent way with cytochrome c as an electron acceptor and without formation of free intermediates (Friedrich et al. 2001, 2005). Sox complexes or genes thereof are neither found in Archaea nor in acidophilic S0 or ISC-oxidizing Bacteria. These microor ganisms, re gardless of whether the y are mesophiles or (hyper-) thermophiles, possess an array of different enzymes. A coherent model of ISC oxidation in acidophiles comparable to that of the Sox comple x is lacking. S0 is oxidized by a cytoplasmic SOR, a remarkable enzyme that catalyzes an

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oxygen-dependent sulfur disproportionation reaction (Emmel et al. 1986; Kletzin 1989; He et al. 2000; Fig. 15.2). In contrast, mesophilic and acidophilic bioleaching Bacteria like Acidithiobacillus thiooxidans possess a periplasmic sulfur oxygenase instead (Rohwerder and Sand 2003). Molecular details are a vailable only for the

Fig. 15.2 Hypothetical model of S 0 oxidation in Acidianus ambivalens and of the reaction mechanism of the sulfur oxygenase reductase (SOR). a Enzymes, enzyme locations and activities, and possible nonenzymic reactions (not stoichiometric) in Acidianus ambivalens. b Hypothetical reaction mechanism of the SOR. CM cytoplasmic membrane, SAOR sulfite:acceptor oxidoreductase, SQR sulfide:quinone oxidoreductase, TQO thiosulfate:quinone oxidoreductase, CQ caldariella quinone, TTH tetrathionate h ydrolase; APS adenosine 5 ′-phosphosulfate, APSR adenosine 5′-phosphosulfate reductase, APAT adenosine 5 ′-phosphosulfate:phosphate aden ylyltransferase, AK adenylate kinase, straight arrows enzyme reactions, dotted arrows nonenzymic reactions

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SORs from se veral hyperthermophiles b ut not for the Acidithiobacillus sulfur oxygenases. The oxidation products of the Acidianus SOR are utilized by other oxidoreductases, including sulfide:quinone oxidoreductase (SQR), tetrathionate-forming thiosulfate oxidoreductase (membrane-bound), sulf ite oxidoreductase (membranebound), and tetrathionate hydrolases (TTH; soluble) (Fig. 15.2a). To unra vel the pathw ays and mechanisms of sulfur oxidation in acidophilic Archaea and to fill in some of these gaps, we purif ied and characterized several of the “dissimilatory sulfur enzymes” from A. ambivalens in the last fe w years (Fig. 15.2a) and established the role of A. ambivalens as the model or ganism for sulfur oxidation in thermoacidophilic Archaea.

15.3

A. ambivalens and A. tengchongensis SORs

The initial enzyme in the archaeal S0 oxidation pathway is unique in several aspects. The SOR catalyzes an oxygen-dependent sulfur disproportionation reaction to sulfite, thiosulfate, and hydrogen sulf ide in a 1:1 stoichiometry of the oxidized and reduced products. SOR acti vity is measured under aerobic conditions using f inely dispersed sulfur in a deter gent-containing reaction b uffer. The enzyme does not require external cofactors for activity (Kletzin 1989; He et al. 2000) (Eq. 15.1): 4S0 + O2 + 4H 2 O → 2HSO3 − + 2H 2 S + 2H + ( sum ) . (15.1) Thiosulfate formation is probably the result of nonenzymic sulf with excess S0 (Eq. 15.2):

ite condensation

pH ≥ 6 ⎯⎯⎯ ⎯⎯ → S2 O3 − + H + ( thiosulfate formation ) . (15.2) S0 + 2HSO3 − ← ⎯ pH ≤ 4

It is not kno wn whether thiosulf ate is a primary product of the SOR or whether it is al ways formed nonenzymically . The ratio between the observ ed thiosulfate and sulfite production is pH-dependent and temperature-dependent; the thiosulfate fraction will increase with temperature and/or pH (Kletzin 1989, and unpublished data). This observ ation suggests a nonenzymic thiosulf ate formation and ar gues against a reaction mechanism that w ould include direct thiosulfate formation by the enzyme. The SOR reaction (Eq. 15.1) can be formally di vided into two partial reactions: an oxygenase and a disproportionation reaction (Eqs. 15.3, 15.4): S0 + O2 + H2 O → HSO3 − + H + ( oxygenase ) (15.3) and 4S0 + O2 + 4H 2 O → 2HSO3 − + 2H 2 S + 2H + ( sum ) .

(15.4)

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Equation 15.4 is identical to the hydroxyl-catalyzed sulfur disproportionation that occurs at alkaline pH and at ele vated temperatures (Roy and Trudinger 1970; Kletzin 1989). I n contrast, the glutathione-dependent sulfur oxygenase of mesophilic Acidithiobacillus species does not ha ve a reductase or disproportionase activity. KM and Kcat values of the A. ambivalens SOR are not very favorable (23 mM and 2.2 s−1, respectively; Urich et al. 2004); however, they are understandable given the complex 3D structure (Sect. 15.3.1) and the poor solubility of the substrate. The SOR activity is inhibited by thiol-binding reagents, pointing to the involvement of cysteines in catalysis (Kletzin 1989; Urich et al. 2004; Chen et al. 2005). Three conserved cysteine residues are present in the v arious SOR sequences (Urich et al. 2004; Fig. 15.3). Site-directed mutagenesis sho wed that only one of these (Cys31 in A. ambivalens numbering) is indispensable and cannot be replaced by alanine or serine, while mutagenesis of the other tw o cysteines resulted in reduced acti vities (Chen et al. 2005; Urich et al. 2005). Cys101/Cys104 double mutants retained up to 30% of wild type acti vity, thereby conf irming that these residues are not essential (Fig. 15.6b; Urich et al. 2005). Electron paramagnetic resonance spectroscopy and redox titration showed that the isolated A. ambivalens SOR contains a mononuclear non-heme iron center in the highspin Fe3+ state. The center has an uncommonly low reduction potential (E0′ = −268 mV, protein as isolated). The signal disappeared upon reduction with dithionate or incubation of the SOR with S 0 at elevated temperature (Urich et al. 2004). It w as intriguing to find that the reduction potential was more than 300 mV lower than usually found for this type of iron center and that it w as low enough to explain the S 0 reducing activity of the enzyme [E0′ (H2S/S0) = −270 mV; Thauer et al. 1977]. A SOR characterized from the related strain A. tengchongensis was highly similar (88% identity; Fig. 15.3). A third enzyme with similar properties w as also described from a phylogenetically uncharacterized “ S. brierle yi” isolate (the isolate w as most probably an Acidianus species); however, a reductase acti vity was not reported (Table 15.1). Other sor genes were identified in the genomes of the crenarchaeote S. tokodaii, of the Euryarchaeota Ferroplasma acidarmanus and Picrophilus torridus, and of the hyperthermophilic bacterium Aquifex aeolicus. They shared 35–69% identical residues with the tw o Acidianus enzymes (Fig. 15.3). Additional sor genes were identif ied in community studies of a commercial gold bioleaching plant recently (Chen et al. 2007). One of the deduced SOR sequences w as identical to that of Ferroplasma acidarmanus (SORFA), another (SOR SB) w as only approximately 50% identical to the archaeal sequences and belonged to a no vel mesophilic Acidithiobacillus SM-1 strain isolated from the bioreactor at an enrichment temperature of 45°C. The third sequence, SOR SA, was similar b ut not identical to the SM-1 sequence (75% identity). When the SOR SB gene was expressed in Escherichia coli, the resulting protein sho wed the typical SOR reaction at an optimal temperature of 75–80°C. It w as speculated that the bacterium acquired the sor gene from a hyperthermophile recently (Chen et al. 2007). The SORs constitute a unique protein family without similarities to other families (Pfam 07682; Wellcome Trust Sanger Institute 2006; Urich et al. 2004). Interestingly,

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Fig. 15.3 Multiple alignment of SOR sequences available in public databases with details from the Acidianus ambivalens 3D structure. A1–A9 and B1–B8 α-helices and β-sheets, respectively; #, conserv ed c ysteine residues; +, iron-coordinating residues; † gate-k eeping phen ylalanine residues from the pore at the enzyme’ s fourfold symmetry axis; ‡, methionine residues at the pore to the acti ve-site pocket. Accession numbers as follo ws: Acidianus ambivalens, P29082; Acidianus tengchongensis, AAK58572; Sulfolobus tokodaii, NP_377053; Ferroplasma acidarmanus, ZP_00608922; Picrophilus torridus , YP_023579; Acidithiobacillus strain SM-1, DQ480733 (Chen et al. 2007); Aquifex aeolicus, AAC06723; uncultured bacterium with SOR-SA, DQ480731, and DQ480732 (Chen et al. 2007). An in-frame stop codon was found immediately upstream of the Ferroplasma acidarmanus Faci1674 open reading frame (X, position 49). It was treated as an unknown residue, because of the similarity of the deduced amino acid sequences of the upstream re gion to the other SOR sequences (Urich et al. 2004). It is not kno wn whether this represents a pseudogene or whether Ferroplasma acidarmanus produces acti ve SOR under suitable conditions. Similar , a reading frame shift at position 96 of the SOR SA sequence was corrected for the purpose of the alignment (Chen et al. 2007). (Extended from Urich et al. 2004, 2006)

sor genes were missing in the S. solfataricus and S. acidocaldarius genomes, which were originally described as f acultative chemolithoautotrophic, sulfur -dependent aerobes (Brock et al. 1972; Zillig et al. 1980). The observ ation is in accordance with other reports that both strains cannot gro w chemolithotrophically on S 0 anymore.

15.3.1

SOR 3D Structure

Electron microscope preparations of A. tengc hongensis cells treated with immunogold-labeled anti-SOR antisera sho wed that the enzyme is most lik ely associated with the cytoplasmic membrane (Chen et al. 2005). Hollow globular

Source W

ild-type SOR

Wild-type sulfur oxygenase

Recombinant SOR (Escherichia coli) (

Acidithiobacillus Acidianus tengchongensis

b 35,000 35,172b 34,491 Subunit molecular mass 35,187b 36,311 c c Holoenzyme molecular mass 844,488 871,464 550,000 NR pHopt/pH range 7–7.4/4–8 NR 6.5–7.5 /NR 5/3.5–9 Topt/Tmax 85°C/108°C NR 65°C/>80°C 70°C/>90°C Specific oxygenase activity at 10.6 2.8 0.9 753f 3.76 optimal temperatured (U mg −1) 29.7 Specific reductase activity at 2.6 0.66 NR 45.2f NR optimal temperaturee (U mg −1) 3.3 Reference (Kletzin 1989) (Urich et al. 2004) (Emmel et al. 1986) (He et al. 2000) NR not reported. a Including ten amino acid C-terminal Streptag. b From a sequence without N-terminal methionine. c From sequence and X-ray crystallography without N-terminal methionine. d 1 U was defined as 1 µmol of sulfite plus thiosulfate formed per minute assuming that thiosulf ate is formed nonenzymically. e 1 U was defined as 1 µmol of H 2S per minute. f Wild-type and recombinant enzymes.

Recombinant SOR (Escherichia coli)a

Table 15.1 Properties of the sulfur oxygenase reductases ( SORs) and sulfur oxygenase Acidianus ambivalens “Sulfolobus brierleyi”

sp. strain SM-1

(Chen et al. 2007)

NR 7.5/NR 75–80°C/NR

Recombinant SOR Escherichia coli)

15 Oxidation of Sulfur and Inorganic Sulfur Compounds 191

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Fig 15.4 Structural model of the SOR holoenzyme and holoenzyme assembly vie wed from the noncrystallographic fourfold symmetry axis. a Secondary structure model of the SOR holoenzyme. Orange α-helices, green β-sheets, gray coils, blue spheres iron atoms. b Molecular surface representation of the holoenzyme. Gray carbon, blue nitrogen, red oxygen c Cross section of the holoenzyme showing the interior lar ge cavity and the sub units. The trapezoid denotes the dimer shown in d and in Fig. 15.5b; the arrows denote the entrances to the active-site pockets. d Model of holoenzyme assembly via homodimers. The figure was prepared with PyMOL (DeLano 2002). (After Urich et al. 2006)

particles of 15.5 nm in diameter appeared in electron microscope pictures of the purified A. ambivalens SOR (Kletzin 1989; Urich et al. 2004). X-ray crystallographic analysis to 1.7-Å resolution showed that the SOR subunits assemble to a spherical homoicosatetramer (i.e., 24 subunits) with 432 point group symmetry and an e xternal diameter of 150 Å (Fig. 15.4a). The sub units surround an empty cavity with a diameter of 71–107 Å (Urich et al. 2006; Fig. 15.4c). The resulting molecular mass of 844 kDa for the nati ve SOR (871 kDa for the recombinant enzyme; Table 15.1) was higher than anticipated from biochemical analyses (550–730 kDa; Urich et al. 2004, 2006). Narro w pores at the fourfold symmetry axes provide an entrance to the cavity (Figs. 15.4c, 15.5a). Two rings of four phenylalanine side chains each close the pores in the present 3D model (Fig. 15.5a). The SOR thus provides an enclosed reaction and/or storage compartment

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Fig. 15.5 Structural details of the SOR. a Model of the pore at the crystallographic fourfold axis of the SOR vie wed from the side; one of the four sub units that form the pore was remo ved for a better vie w of the interior (Urich et al. 2006). The tw o rings of four phenylalanine residues each are highlighted in a ball-and-stick model ( F132 and F140). b Model of a SOR homodimer in surf ace ( upper part ) and secondary structure representat ion (lower part) (Urich et al. 2006). The iron atom is shown as a sphere; the arrows denote the entrance pore to the acti ve-site pock et. Two neighboring methionine residues contrib ute significantly to pore formation (Met296/Met297, yellow in the upper part and sticks in the lower part of the model). c Coordination of the iron ( dashed gr ay lines ) and potential hydrogen-bonding netw ork ( dashed blue lines ) around the iron site; red spher es ordered water molecules; gi ven are all O–N or H 2O–O distances belo w 3.1 Å. The f igure w as prepared with PyMOL (DeLano 2002)

physically separated from the c ytoplasm (Fig. 15.4c). Re versible denaturation experiments had suggested that the holoenzyme assembles via homodimeric building blocks (Urich et al. 2004). The 3D structure sho wed that each subunit contacts five neighboring subunits directly; however, one of these contacts has a significantly higher intersub unit contact area compared with the r est (16% vs. 8% or less; Urich et al. 2006; Fig. 15.4d), thus supporting the conclusions drawn from biochemical work. The 24 Fe sites are well separated from each other (minimal distance 38 Å). They are not e xpected to interact during catalysis. The y reside inside a pock et in the interior of each subunit and are accessible through narrow entrance pores from the internal cavity only (Urich et al. 2006; Figs. 15.4c, 15.6a); thus, sulfur

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and the reaction products both ha ve to pass tw o bottlenecks restricting access and exit, respectively. These constrictions could contrib ute to the high KM and the lo w K cat v alues observ ed. The narro w pores also suggest that the actual substrate might be a linear sulfur species (e.g., polysulf ides) and not the circul ar α-S8 ring.

15.3.2

SOR Subunit and Active-Site Structure

Each subunit consists of a β-barrel core surrounded by α-helices (Urich et al. 2006; Fig. 15.5b). The spacious acti ve-site pocket (18 Å × 18 Å × 6 Å) is located outside the barrel and is lined by at least 21 amino acid residues, including the three conserved cysteines. Residue Cys31 showed additional electron density, which proved to be a persulfide modification (Css; Fig. 15.6). The iron, located at the f ar end of the pock et (Fig. 15.6a), is coordinated in a structural motif known as “2-His 1-carboxylate facial triad”. Two histidine ligands, a bidentate glutamate, and tw o water molecules complete the octahedral geometry (Costas et al. 2004; Urich et al. 2006; Figs. 15.5c, 15.6a, b). Mutation of any of the three iron ligands to alanine resulted in the loss of acti vity and iron-binding capabilities, whereas replacement of the glutamate by aspartate resulted in some residual activity and concomitantly low iron occupancy (approximately 1%; Urich et al. 2005; Fig. 15.6b). It was concluded from the structural and the mutational analysis that the iron site and Css31 constitute the core of the acti ve site, whereas the roles of the remaining c ysteines are less well def ined. The minimal iron–c ysteine distance is 7.8 Å (Cys101), whereas the distance to the Css31 is 8.9 Å (Fig. 15.6b). We concluded therefore that sulfur is co valently bound to the persulf ide moiety of Css31 and that the linear enzyme-bound polysulf ide chain aligned to the iron site is the f inal substrate of the reaction. An interesting ef fect w as observ ed when Cys101 was mutated to serine: the activity and iron content of the enzyme dropped to almost zero, showing that distant mutations can trigger effects on iron incorporation into the enzyme. The low reduction potential of the iron site is probably the result of a surroundi ng network of hydrogen bonds (Fig. 15.5c). Glu87 seems to be crucial for enzyme activity since mutagenesis drastically alters not only the specif ic acti vity of the enzyme but also the stoichiometry of the reaction products (K. Se yfarth and A. Kletzin, unpublished results).

15.3.3

SOR Reaction Mechanism

Some conclusions regarding the reaction mechanism could be deri ved from properties of other oxygenases with a mononuclear non-heme iron atom, from the structure, and from the mutagenesis experiments. The oxygenase reaction (Eq. 15.3) requires

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Fig. 15.6 The active site of the SOR and ef fect of mutations (Urich et al. 2006). a Surface representation of the acti ve-site pock et with the iron ( magenta), the coordinating histidine and glutamic acid residues, and the c ysteine persulf ide ( sticks); arrow pock et entrance pore. b Secondary structure representation of a sub unit with iron ( magenta) and with important residues. Dots Fe-coordinating w ater lig ands; dashed lines Fe–S and S–S distances. Mutations as follows: † zero acti vity; ↓ reduced acti vity; ↓↓ strongly reduced acti vity; ⇑ increased acti vity. The figure was prepared with PyMOL (DeLano 2002)

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the iron to be in the +2 state for dioxygen binding (Fe 3+ is unable to acti vate O 2) (Costas et al. 2004). Upon reduction, the octahedral coordination sphere of the iron should change to f ive ligands in a yet unkno wn geometry. Dioxygen binding usually results in a reacti ve and short-li ved Fe 4+-peroxo intermediate poised to attack the substrate (Fig. 15.2b). It is expected that the abundance of free electron pairs in the sulfur chain of the SOR w ould immediately refill the electron gap of the Fe 4+. In contrast, the disproportionation reaction (Eq. 15.4) requires the action of a strong nucleophile (e.g., OH−) without the need for the presence of dioxygen. The ordered water molecule (W at127; Fig. 15.2c) is a good candidate for the missing nucleophile. The core acti ve site of the SOR is thus composed of the iron site and the modified Css31. Substrate entry has to proceed through the hydrophobic channels along the fourfold axes of the sphere and through the pore of the active site (Figs. 15.5a, 15.6a). The presence of a persulf ide suggests that S 0 is covalently bound to Css31, a process that is equally possible with sulfur and polysulf ides as substrates (Fig. 15.2b, reactions 1a and 1b). The linear polysulf ide chain aligns to the iron site, thereby replacing the remaining water ligand(s). A mixed reaction follows starting with a hydrolytic release of hydrogen sulfide from the chain, thereby forming a sulfino intermediate that is a strong reductant in itself. A polarized w ater molecule or hydroxyl ion might pro vide the nucleophile required for this attack. The following sequence of events is less obvious. Oxygen could bind either to the iron or to the sulf ino group and get reduced to the peroxide state. The peroxide is a strong oxidant that could attack the sulfur chain and release sulfite. The scheme outlined here predicts that the enzyme is not necessarily a dioxygenase b ut rather a monooxygenase. Pre vious w ork by Emmel et al. (1986) showed moderate 18O incorporation from 18O2 into sulfite, supporting the monooxygenase hypothesis.

15.4

Oxidation of Soluble Sulfur Compounds in Acidianus

15.4.1 Sulfite:Acceptor

Oxidoreductase

Sulfite:acceptor oxidoreductase (SA OR) and other sulf ite-oxidizing enzymes are known from many (micro-) organisms (Kappler and Dahl 2001). Two pathways are important for Acidianus: 1. SAOR activity as part of the Sox comple x is found in man y bacteria. SA ORs feed electrons typically via c-type c ytochromes into the respiratory chain (Friedrich et al. 2001, 2005). Fe w of these enzymes are membrane-bound (Kappler and Dahl 2001). 2. An alternative sulfite oxidation pathway coupled to substrate-level phosphorylation was identified in Thiobacillus denitrificans and in A. ambivalens. It involves the indirect sulf ite oxidation via an adenosine 5 ′-phosphosulfate (APS) reductase

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(Fig. 15.2a) and an adenosine 5 ′-phosphosulfate:phosphate adenylyltransferase (APAT) (Brüser et al. 2000). The acti vities of a SA OR (membrane fraction), APS reductase, AP AT, and adenylate kinase (soluble fraction) were demonstrated in A. ambivalens (Zimmermann et al. 1999; Fig. 15.2a) sho wing that both sulf ite oxidation pathw ays are realized. ATP sulfurylase acti vity was not found. The enzymes ha ve not yet been purif ied and molecular details are not known.

15.4.2 Thiosulfate:Quinone

Oxidoreductase

Periplasmic or soluble tetrathionate-forming thiosulf ate oxidoreductases or dehydrogenases were found in se veral Bacteria (Visser et al. 1997; Nakamura et al. 2001). The proteins v ary considerably in subunit composition, molecular mass, and cofactor content. Some contain c-type hemes. Gene or protein sequences are not kno wn b ut the proteins do not appear to be similar to thiosulf ate-oxidizing moieties of the Sox complex. In contrast, a membrane-bound tetrathionate-forming thiosulf ate:quinone oxidoreductase (TQO) w as isolated from aerobically gro wn A. ambivalens cells (Müller et al. 2004). It reduced ferric yanide and decyl ubiquinone and used methylene blue as an electron donor during the re verse reaction. The protein contained bound caldariella quinone. Optimal acti vity was observed at 85°C and pH 5. The 102-kDa glycosylated holoenzyme consists of 28- and 16-kDa subunits, suggesting an α2β2 stoichiometry. Oxygen electrode measurements sho wed an electron transport from thiosulf ate to molecular oxygen via the terminal heme copper quinol: oxygen oxidoreductase. The TQO sub units were identical to DoxA and DoxD, originally described as parts of the A. ambivalens terminal oxidase (Müller et al. 2004). Both enzymes were copurif ied in previous work (Purschke et al. 1997) and might form a supercomplex in the membrane. The recently isolated thiosulf ate-oxidizing haloarchaeon Natronorubrum sp. HG 1 (Sorokin et al. 2005) contained a membrane-associated tetrathionate synthase or a thiosulf ate:acceptor oxidoreductase, whose acti vity depended specifically on ele vated concentrations of Cl −. Its function is similar to the TQO described here but the protein has not been purified yet and the sequence is not known.

15.4.3 T etrathionate Hydrolase The fate of the tetrathionate formed by the TQO is not well understood. W e had proposed that a thiosulf ate/tetrathionate c ycle might e xist because tetrathionate is unstable in the presence of strong reductants lik e H 2S and sulf ite and is reduced to thiosulfate in vitro at high temperatures (Xu et al. 1998, 2000; Kletzin et al. 2004;

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Fig. 15.2a). Such a cycle would feed electrons indirectly from the S0 disproportionation into the quinone pool. W e found, ho wever, a tetrathionate hydrolase (TTH) acti vity in tetrathionate-gro wn cells of A. ambivalens recently (F . Müller and A. Kletzin, unpublished data). The enzyme produced sulf ate, thiosulf ate, and elemental sulfur from tetrathionate but neither H 2S nor sulfite and higher polythionates. A TTH w as also purif ied from Acidithiobacillus ferr ooxidans (Kanao et al. 2006). The homodimeric and membrane-bound protein produced S0, sulfate, and thiosulfate from tetrathionate at an acidic pH optimum. N-terminal sequencing allowed the identification of the gene and the deduced amino acid sequence from the genome, showing that the protein belongs to a superfamily of pyrroloquinoline quinone-containing enzymes. BLAST searches showed that the most similar homologues were not present in other bacterial genomes but in S. tokodaii (three probably paralogous genes) and A. ambivalens (two paralogs; Kanao et al. 2006; A. Kletzin, unpublished observ ation). These genes might e ventually encode the protein(s) with TTH acti vity. There were no homologues in S. acidocaldarius and S. solfataricus, suggesting that this protein and its genes are restricted to the “true” sulfur oxidizers.

15.4.4 Sulfide:Quinone

Oxidoreductase

Sulfide:quinone oxidoreductases (SQRs) catalyzing the oxidation of hydrogen sulfide with quinones as electron acceptors are widely distrib uted in the microbial world. The y are type II f amily fla voproteins similar to N ADH:quinone oxidoreductases (NADH-OR). These enzymes typically consist of a single subunit and are unable to pump protons during N ADH oxidation (Gomes et al. 2001). Multiple homologues of SQRs/NADH-OR genes are found in archaeal genomes, including those of members of the Sulfolobales. The only type II N ADH-OR known from Archaea w as purified and characterized from A. ambivalens . Sequence comparisons showed that the enzyme contains all the cysteine residues conserved in SQRs, but it remains to be demonstrated that the enzyme has an SQR acti vity.

15.5 Conclusions The recent advances in the biochemistry and structure resolution of the SOR allo w for the design of experiments to unravel the reaction mechanism of the enzyme and eventually also the pathw ays of its assembly . Extensive mutagenesis e xperiments will provide derivatives that can be analyzed with v arious spectroscopic methods. Therefore there is hope that we will be able to get a handle on the “dif ficult” inorganic sulfur biochemistry using the SOR as a model system. Regardless of the recent adv ances in structure resolution and mutational analysis, a puzzling question remains unanswered, namely, why a cytoplasmic and not a periplasmic or membrane-bound enzyme is used for the initial step of sulfur

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oxidation (Rohwerder and Sand 2003; Friedrich et al. 2005). One could hypothesize that the closed sphere allows the utilization of the high reactivity of the S0 disproportionation at ele vated temperature and near -neutral pH, so little acti vation energy is required. The highly reactive products of the reaction are another problem that could be solved elegantly. These could be liberated in a more controlled fashion posing less danger for damage to other proteins. The scheme of S0 oxidation pathways and of electron transport in A. ambivalens presented in Fig. 15.2a is outlined from the presently kno wn enzymes and enzyme activities; however, it is tentative and many gaps are open. Sulfur is oxidized by the SOR; the products hydrogen sulf ide, thiosulfate, and sulfite are oxidized by membrane-bound oxidoreductases. In addition, there is the APS oxidation pathw ay. Unsolved questions include how the sulfur gets into the cell, how sulfur gets inside the SOR, and how the products get out. Some of these gaps, however, will be hopefully closed in the near future. Acknowledgements. I wish to thank Felicitas Pfeifer for her support. This w ork was supported by grants from the Deutsche F orschungsgemeinschaft (Kl885/3-1, Kl885/3-2, and Kl885/3-3).

References Boulegue J (1978) Solublity of elemental sulfur in w ater at 298 K. Phosphorus Sulfur 5:127–128 Brierley CL, Brierle y J A (1982) Anaerobic reduction of molybdenum by Sulfolobus species. Zentralbl Bakteriol Hyg I Abt Orig C 3:289–294 Brock TD, Brock KM, Belly R T, Weiss RL (1972) Sulfolobus: A ne w genus of sulfur -oxidizing bacteria living at low pH and high temperature. Arch Microbiol 84:54–68 Brüser T, Selmer T , Dahl C (2000) “ ADP sulfurylase” from Thiobacillus denitrif icans is an adenosine 5 ′-phosphosulfate:phosphate aden ylyltransferase and belongs to a ne w f amily of nucleotidyltransferases. J Biol Chem 275:1691–1698 Chen ZW, Jiang CY, She Q, Liu SJ, Zhou PJ (2005) Key role of cysteine residues in catalysis and subcellular localization of sulfur oxygenase-reductase of Acidianus tengc hongensis. Appl Environ Microbiol 71:621–628 Chen ZW, Liu YY , Wu JF, She Q, Jiang CY , Liu SJ (2007) No vel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates. Appl Microbiol Biotechnol 74:688–698 Costas M, Mehn MP, Jensen MP, Que L Jr (2004) Dioxygen acti vation at mononuclear nonheme iron active sites: enzymes, models, and intermediates. Chem Re v 104:939–986 DeLano WL (2002) The PyMOL molecular graphics system, version 0.97. DeLano Scientific, San Carlos http://www.pymol.org Emmel T, Sand W, König WA, Bock E (1986) Evidence for the e xistence of a sulfur oxygenase in Sulfolobus brierleyi. J Gen Microbiol 132:3415–3420 Friedrich CG, Rother D, Bardischewsky F, Quentmeier A, Fischer J (2001) Oxidation of reduced inorganic sulfur compounds by bacteria: emer gence of a common mechanism? Appl En viron Microbiol 67:2873–2882 Friedrich CG, Bardische wsky F, Rother D, Quentmeier A, Fischer J (2005) Prokaryotic sulfur oxidation. Curr Opin Microbiol 8:253–259 Gomes CM, Baucleines TM, T eixeira H (2001) A ne w type-II N ADH dehydrogenase from the archaeon Acidianus ambivalens: characterization and in vitro reconstitution of the respiratory chain. Bioenerg Biomember 33:1–8

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He Z, Li Y , Zhou P, Liu S (2000) Cloning and heterologous e xpression of a sulfur oxygenase/ reductase gene from the thermoacidophilic archaeon Acidianus sp. S5 in Escherichia coli . FEMS Microbiol Lett 193:217–221 Huber G, Drobner E, Huber H, Stetter K O (1992) Gro wth by aerobic oxidation of molecular hydrogen in archaea – a metabolic property so f ar unkno wn for this domain. Syst Appl Microbiol 15:502–504 Huber H, Prangishvili D (2005) The order Sulfolobales. In: Dw orkin M, Falkow S, Rosenberg E, Schleifer K-H, Stack ebrandt E (eds) The prokaryotes: an e volving electronic resource for the microbiological community, 3rd edn, release 3.19. Springer, New York Kanao T, Kamimura K, Sugio T (2006) Biochemical and genetic characterization of tetrathionate hydrolase from iron-oxidizing bacterium Acidithiobacillus ferrooxidans. In: Querellou J, Lamy C (eds) Extremophiles 2006. International Society for Extremophiles, Brest Kappler U, Dahl C (2001) Enzymology and molecular biology of prokaryotic sulf ite oxidation. FEMS Microbiol Lett 203:1–9 Kelly DP, Shergill JK, Lu WP, Wood AP (1997) Oxidati ve metabolism of inor ganic sulfur compounds by bacteria. Antonie Van Leeuwenhoek 71:95–107 Kletzin A (1989) Coupled enzymatic production of sulfite, thiosulfate, and hydrogen sulfide from sulfur: purif ication and properties of a sulfur oxygenase reductase from the f acultatively anaerobic archaebacterium Desulfurolobus ambivalens. J Bacteriol 171:1638–1643 Kletzin A (2006) Metabolism of inor ganic sulfur compounds in Archaea. In: Garrett RA, Klenk H-P (eds) Archaea. Ev olution, ph ysiology, and molecular biology . Blackwell, Oxford, pp 261–274 Kletzin A (2007) General characteristics and important model or ganisms. In: Cavicchioli R (ed) Archaea. Molecular and Cellular Biology. ASM-Press, Washington, pp 14–92 Kletzin A, Urich T , Müller F , Bandeiras TM, Gomes CM (2004) Dissimilatory oxidation and reduction of elemental sulfur in thermophilic archaea. J Bioener g Biomembr 36:77–91 Middelburg JJ (2000) The geochemical sulfur c ycle. In: Lens PNL, Hulshof f PL (eds) Environmental technologies to treat sulfur pollution. IWA, London, pp 33–46 Müller FH, Bandeiras TM, Urich T , Teixeira M, Gomes CM, Kletzin A (2004) Coupling of the pathway of sulphur oxidation to dioxygen reduction: characterization of a no vel membranebound thiosulphate:quinone oxidoreductase. Mol Microbiol 53:1147–1160 Nakamura K, Nakamura M, Y oshikawa H, Amano Y (2001) Purif ication and properties of thiosulfate dehydrogenase from Acidithiobacillus thiooxidans JCM7814. Biosci Biotechnol Biochem 65:102–108 Norris PR, Johnson DB (1998) Acidophilic microor ganisms. In: Horik oshi K, Grant WD (eds) Extremophiles: microbial life in extreme environments. Wiley, New York, pp 133–154 Purschke WG, Schmidt CL, Petersen A, Schäfer G (1997) The terminal quinol oxidase of the hyperthermophilic archaeon Acidianus ambivalens exhibits a novel subunit structure and gene organization. J Bacteriol 179:1344–1353 Rohwerder T, Sand W (2003) The sulfane sulfur of persulfides is the actual substrate of the sulfuroxidizing enzymes from Acidithiobacillus and Acidiphilium spp. Microbiology 149:1699–1710 Roy AB, Trudinger PA (1970) The chemistry of some sulphur compounds. In: The biochemistry of inorganic compounds of sulphur. Cambridge University Press, Cambridge, pp 7–29 Schönheit P, Schäfer T (1995) Metabolism of h yperthermophiles. World J Microbiol Biotechnol 11:26–57 Segerer AH, T rincone A, Gahrtz M, Stetter K O (1991) Stygiolobus azoricus gen. no v., sp. no v. represents a novel genus of anaerobic, extremely thermoacidophilic archaebacteria of the order Sulfolobales. Int J Syst Bacteriol 41:495–501 Sorokin DY, Tourova TP, Muyzer G (2005) Oxidation of thiosulfate to tetrathionate by an haloarchaeon isolated from hypersaline habitat. Extremophiles 9:501–504 Steudel R (2000) The chemical sulfur c ycle. In: Lens PNL, Hulshof f PL (eds) En vironmental technologies to treat sulfur pollution. IWA, London, pp 1–32

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Takakuwa S (1992) Biochemical aspects of microbial oxidation of sulfur compounds. In: Oae S (ed) Organic sulfur chemistry: Biochemical aspects. CRC, Boca Raton, pp 1–43 Thauer RK, Jungermann K, Deck er K (1977) Ener gy conserv ation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180 Urich T, Bandeiras TM, Leal SS, Rachel R, Albrecht T , Zimmermann P, Scholz C, T eixeira M, Gomes CM, Kletzin, A (2004) The sulphur oxygenase reductase from Acidianus ambivalens is a multimeric protein containing a lo w-potential mononuclear non-haem iron centre. Biochem J 381:137–146 Urich T, Kroke A, Bauer C, Se yfarth K, Reuff M, Kletzin A (2005) Identif ication of core acti ve site residues of the sulfur oxygenase reductase from Acidianus ambivalens by site-directed mutagenesis. FEMS Microbiol Lett 248:171–176 Urich T, Gomes CM, Kletzin A, Frazão C (2006) X-ray structure of a self-compartmentalizing sulfur cycle metalloenzyme. Science 311:996–1000 Visser JM, de Jong GAH, Robertson LA, K uenen JG (1997) Purif ication and characterization of a periplasmic thiosulfate dehydrogenase from the obligately autotrophic Thiobacillus sp. W5. Arch Microbiol 166:372–378 Wellcome Trust Sanger Institute (2006) Pfam. http://www.sanger.ac.uk/pfam Xu Y, Schoonen MAA, Nordstrom DK, Cunningham KM, Ball JW (1998) Sulfur geochemistry of hydrothermal waters in Yellowstone National Park: I. The origin of thiosulfate in hot spring waters. Geochim Cosmochim Acta 62:3729–3743 Xu Y, Schoonen MAA, Nordstrom DK, Cunningham KM, Ball JW (2000) Sulfur geochemistry of hydrothermal w aters in Y ellowstone National P ark, Wyoming, USA. II. F ormation and decomposition of thiosulf ate and polythionate in Cinder Pool. J V olcanol Geotherm Res 97:407–423 Zillig W, Stetter KO, Wunderl S, Schulz W, Priess H, Scholz I (1980) TheSulfolobus-“Caldariella” group: Taxonomy on the basis of the structure of DN A-dependent RNA polymerases. Arch Microbiol 125:259–269 Zimmermann P, Laska S, Kletzin A (1999) T wo modes of sulf ite oxidation in the e xtremely thermophilic and acidophilic archaeon Acidianus ambivalens. Arch Microbiol 172:76–82

Chapter 16

A Novel Coenzyme F 420 Dependent Sulfite Reductase and a Small Sulfite Reductase in Methanogenic Archaea Eric F. Johnson, Biswarup Mukhopadhyay

Abstract Recently a no vel, highly acti ve, coenzyme F 420 dependent sulf ite reductase (Fsr) has been discovered in Methanocaldococcus jannaschii. Three other extremophilic methanogens and an uncultured archaeon from a consor tium performing anaerobic oxidation of methane (A OM) carry Fsr homologs. Methanogens require sulf ide and most are sensiti ve to sulf ite. Since Fsr is induced by sulf ite, reduces sulf ite to sulf ide with H 2F420, and seems to be associated with the membrane, it is a sulf ite detoxif ication and assimilation enzyme. The N-terminal half of Fsr is a homolog of H F dehydrogenase 2 420 (FqoF/FpoF). FqoF/FpoF is the electron input unit of a membrane-bound electron transport system of late-e volving methylotrophic methanogens and Archaeoglobus fulgidus , a sulf ate reducing archaeon emplo ying the partial reverse methanogenesis pathw ay. The C-terminal half (Fsr -C) represents a dissimilatory sulfite reductase sub unit (DsrA). While only four methanogens carry Fsr , e very methanogen carries a small putati ve sulf ite reductase with sequence features of Fsr -C. These observ ations lead to follo wing hypotheses. At one time methanogenesis and sulfate reduction involving a sulfite reductase, two of the oldest energy-conserving respiratory metabolisms of Earth, e xisted in one organism that performed sulfate reduction driven AOM. Fsr gave rise to FqoF/FpoF and DsrA, or from a small sulf ite reductase of methanogens DsrA and Fsr (a fusion with FqoF/FpoF) e volved.

16.1 Intr oduction Hydrogenotrophic methanogenesis and dissimilatory sulf ate reduction (Eqs. 16. 1, 16.2; Fig. 16.1A), tw o of the oldest ener gy-conserving respiratory systems on Earth, developed at least 2.7 billion to 3.2 billion and 3.7 billion years ago, respectively (Leigh 2002; Teske et al. 2003). The recent disco very of a no vel and highly active sulf ite reductase in Methanocaldococcus jannasc hii (Johnson and Mukhopadhyay 2005) and the presence of putati ve small sulf ite reductases in methanogens raise the question whether at one time these two ancient metabolisms 202 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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Fig. 16.1 Methanogenesis and sulf ate reduction ( A), energy metabolism of Archaeoglobus fulgidus (B) and anaerobic methane oxidation ( C). The site of inhibition of methanogenesis by sulfite is shown. Pyruvate and lactate in B are e xamples of ener gy substrates for A. fulgidus; the meth yl group of lactate or p yruvate enters the re verse methanogenesis pathw ay as methyl- tetrahydromethanopterin (Möller-Zinkhan et al. 1989). The dotted line in c indicates the in volvement of tw o or ganisms. Methyl (− CH3), methylcoenzyme; methylreductase, methylcoenzyme M reductase.

existed in a single or ganism. This question is also rele vant to anaerobic oxidation of methane (AOM) (Boetius et al. 2000; Orphan et al. 2002; Eq. 16.3; Fig. 16.1C). 4H 2 + SO 4 2 − + H + → HS− + 4H 2 O, ∆ G 0′ = −152.2 kJ mol −1 SO 4 2 −

( Thauer et al. 1977 )

(16.1)

4H 2 + CO2 → CH 4 + 2H 2 O, ∆ G ′ = −131 kJ mol CH 4 −1

0

( Thauer et al. 1977 )

(16.2)

CH 4 + SO 4 + H → CO 2 + HS + 2H 2 O, ∆ G ′ = −21 kJ mol CH 4 2−

+

−

−1

0

( Shima and Thauer 2005)

(16.3)

HSO3 + 6e + 6H → HS + 3H 2 O, ∆ E ′ = −116 mV −

−

+

( Thauer et al. 1977 )

0

(16.4)

Sulfite (SO 3 ) is an obligatory intermediate in the eight-electron reduction of sulfate to sulfide (Fig. 16.1A). This strong nucleophile is toxic to cells of all types owing to its reacti vity toward proteins and sulfhydryl groups (W edzicha 1992). Therefore, it must be reduced rapidly to sulf ide. Consequently, sulfite reductase, 2−

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which catalyzes the six-electron reduction of sulf ite (SO 32−) or bisulfite (HSO 3–) (Eq. 16.4, Fig. 16.2A–D), is a k ey enzyme in the reduction of sulf ate. Two types of sulf ite reductases are kno wn. Assimilatory sulf ite reductases (Asr) generate sulfide for the synthesis of c ysteine, which in turn pro vides sulfur for all sulfur containing compounds in a cell (Crane and Getzoff 1996). Asr is used by certain bacteria, fungi and plants. Dissimilatory sulf ite reductases (Dsr) participate in dissimilatory sulf ate reduction, an ener gy-conserving process in a group of anaerobic bacteria and archaea. In this metabolism, electrons deri ved from the oxidation of complex materials such as carbohydrates and hydrocarbons or simple compounds such as acetate and H 2 are transferred to sulfate (Widdel 1988). Asr contains siroheme, an iron octacarboxylic tetrahydroporphyrin of the isobacteriochlorin type (LeGall and Fauque 1988). Dsr contains siroamide, a siroheme with one of the acetate chains amidated (Matthe ws et al. 1995; Lubbe et al. 2006; Fig. 16.2). The Asr of Escherichia coli (Sir) is composed of two units: an octameric flavoprotein component (SirFP) made up of 66-kDa flavin mononucleotide (FMN) containing- and flavin adenine dinucleotide (FAD) containing subunits and a tetrameric heme-protein component (SirHP) composed of siroheme-containing 64-kDa subunits (Crane and Getzof f 1996; Fig. 16.2A). SirFP deri ves electrons from NADPH, a hydride donor or tw o-electron-restricted electron carrier , and then via protein-bound FMN and F AD (tw o-electron/one-electron switch) it passes these electrons to the [4Fe-4S]–siroheme groups of SirHP . SirHP reduces sulf ite to sulfide. In the Asr of Arabidopsis thaliana, a monomeric SirHP-type unit reduces sulfite with the electrons provided by a NADPH:ferredoxin reductase orphotosystem I via a ferredoxin (Nakayama et al. 2000; Fig. 16.2B). The Dsr enzymes are tetramers of DsrA and DsrB sub units (Fig. 16.2C), which sho w lo w primary sequence similarities to the E. coli and plant enzymes (Johnson and Mukhopadhyay 2005; Fig. 16.2A). DsrA carries siroheme (Crane and Getzof f 1996; Fig. 16.2C). DsrA and DsrB share substantial sequence similarities with each other and are believed to ha ve originated from a gene duplication e vent (Dhillon et al. 2005). DsrB lacks a conserv ed cysteine residue of the consensus siroheme-binding site, and it probably does not carry the siroheme cof actor (Dahl et al. 1993; Crane and Getzoff 1996). The electron-donating units for dissimilatory sulf ite reductases are

Fig. 16.2 Assimilatory and dissimilatory sulfite reductases (A–D) and A. fulgidus H2F420:quinone oxidoreductase or Fqo comple x (E). Most dissimilatory sulf ite reductases are α2β2 proteins, b ut α2β2γ2 structures have also been observed, where the function of the γ-subunit is unknown (Crane and Getzoff 1996). The quaternary structure for Methanocaldococcus jannaschii coenzyme F 420 dependent sulfite reductase (Fsr) is not kno wn. The question mark in d indicates that it is not known whether Fsr contains bound flavin. e Fqo and Fpo complexes (Deppenmeier 2004). These are similar to respiratory complex I of Escherichia coli and mitochondria. Sir or SR, sulfite reductase; SirFP and SirHP, flavoprotein and hemoprotein subunits of sulfite reductase, Fd, ferredoxin; PS I, photosystem I; Cyt, cytochrome; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; MQ, menaquinone; FqoF or FpoF, H 2F420 dehydrogenase sub unit. (Modif ied from Johnson and Mukhopadhyay 2005)

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yet to be clearly identified (Crane and Getzoff 1996). For Desulfovibrio desulfuricans Dsr, a membrane-bound comple x (DsrMKJOP) is a strong candidate for this role, and homologs for the subunits of this complex are present in every sulfate-reducing organism for which the genome sequence has been determined (Pires et al. 2006). During anaerobic growth, Salmonella enterica expresses a small ssulfite reductase, which has been named anaerobic sulf ate reductase or Asr (Huang and Barrett 1991). Since this enzyme serv es a dissimilatory and not an assimilatory function (Huang and Barrett 1991), we rename the enzyme SalDsr. The siroheme-containing subunit of this enzyme (SalDsrC) is a homolog of DsrA (Dhillon et al. 2005; Johnson and Mukhopadhyay 2005).

16.2 Incompatibility of Methanogenesis and Sulfate Reduction, Sulfite as the Key Determinant If an or ganism has to perform both methanogenesis and dissimilatory sulf ate reduction, it has to deal with the apparent incompatibility of these tw o metabolisms (Fig. 16.1A). As mentioned in Sect. 16.1, sulfite, an intermediate in sulfate reduction, is toxic to all types of cells. Methanogens ha ve an additional target for sulfite. Sulfite inhibits methanogenesis (Balderston and P ayne 1976), the only means of ener gy production for a methanogen (Wolfe 1992). This inhibition is most lik ely due to the f act that sulf ite reacts with and inacti vates methylcoenzyme M reductase (Beck er and Ragsdale 1998; Mahlert et al. 2002; Fig. 16.1A). A similar incompatibility is expected in sulf ate-dependent A OM (Eq. 16.3), which combines re verse methanogenesis (reverse of Eq. 16.2, in volving methylcoenzyme M reductase) with dissimilatory sulfate reduction (Hinrichs et al. 1999; Boetius et al. 2000; Shima and Thauer 2005; Eq. 16.1; Fig. 16.1C). Perhaps for this reason A OM is accomplished through a combined action of at least two organisms, one of which is an archaeon catalyzing methane oxidation and the other is a sulf ate-reducing bacterium (Hinrichs et al. 1999; Boetius et al. 2000; Fig. 16.1C). This antagonism is also reflected in the metabolic properties and genomic potential of Archaeoglobus fulgidus. This sulfate-reducing archaeon carries most elements of the methanogenesis pathway except methylcoenzyme M reductase and associated genes (Klenk et al. 1997; Fig. 16.1B). Ho wever, it should be noted that these concepts need to bejustified by the Ki value of methylcoenzyme M reductase for sulfite, which is yet to be determined.

16.3 Inevitable Exposure of a Methanogen to Sulf ite in Hydrothermal Vents and on Early Earth The submarine hydrothermal v ent methanogens recei ve nutrition from the v ent fluid (Jannasch 1989; McCollom and Shock 1997), which is rich in nutrients for autotrophic growth. However, the temperature of the v ent fluid (300–350°C) is detrimental to all

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known life forms (Jannasch 1989). Cold sea water that permeates through the v ent wall brings the temperature of the v ent fluid do wn to a le vel where h yperthermohilic methanogens can grow (McCollom and Shock 1997). This process also brings oxygen into the vent. Sulfide, which is present at a high level in the vent fluid (5–7mM; Jannasch 1989), reacts with this oxygen and helps to establish anaerobic conditions that a strict anaerobe, such as a methanogen, needs. On the other hand, this reaction between sulfide and a low level of oxygen has the potential of producing sulfite, an incomplete oxidation product of sulfide. Therefore, a deep-sea hydrothermal vent methanogen must be able to tolerate sulfite. It has been suggested that the development of a fully oxic atmosphere on sulfide-containing early Earth follo wed a protracted oxygenation period (Shen et al. 2003; Kah et al. 2004; Poulton et al. 2004). This early oxygenation event presented a situation similar to that described above for the hydrothermal vents and consequently caused sulfite production and selection of methanogens with a sulf ite detoxification ability.

16.4 Use of Sulfite As a Sulfur Source by Methanocaldococcus jannaschii and Other Methanogens Methanocaldococcus jannasc hii, a deeply-rooted, hyperthermophilic, strictly hydrogenotrophic, methanogenic archaeon, is an inhabitant of the deep-sea hydrothermal vents (Jones et al. 1983; Boone et al. 1993). It tolerates sulf ite up to a level of 40 mM, where observ able growth and methane formation occur (E.F . Johnson and B. Mukhopadhyay, unpublished data). With 20 mM sulfite, growth andmethanogenesis is slow, but the f inal cell density is comparable to that obtained with the optimal sulf ite levels of 0.5–2 mM. Methanothermococcus thermolithotrophicus, Methanothermobacter thermautotrophicus, and Methanothermobacter marburgensis, which are thermophiles, can use sulfite as their sole sulfur source (Daniels et al. 1986); a concentration of 1 mM for sulfite is optimal and higher levels are inhibitory . A complete inhibition of gro wth occurs at 1.25 mM or above for Methanothermococcus the rmolitotrophicus and at 4 mM or abo ve for Methanothermobacter thermautotr ophicus and Methanothermobacter marburgensis. Methanosar cina acetivor ans and Methanococcus maripaludis cannot grow in the presence of 0.5 mM sulfite (E.F. Johnson and B. Mukhopadhyay, unpublished data). Methanopyrus kandleri , Methanocaldococcus igneus , Methanococcus vannielli and Methanoplanus limicola can tolerate sulfite (in the presence of sulfide) but cannot use this oxyanion as their sole sulfur source (Rothe and Thomm 2000).

16.5 Expression of a Novel Coenzyme F 420 Dependent Sulfite Reductase in Methanocaldococcus jannaschii During Growth on Sulfite When grown with sulfite as the sole sulfur source, Methanocaldococcus jannaschii expresses a 70-kDa polypeptide in a growth-phase-independent manner (Fig. 16.3B). This polypeptide corresponds to open reading frame (ORF) MJ0870, and it is not

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Fig. 16.3 Sodium dodec yl sulf ate polyacrylamide gel elecrophoresis of Methanocaldococcus jannaschii cell lysates. Cells were harv ested from cultures grown with sulfide (S 2−) (A) or sulfite (SO32−) ( B) at indicated ages (in hours). A lysate w as prepared by boiling cells in a solution containing 62.5 mM tris(hydroxymethyl)aminomethane hydrochloride buffer pH 6.8, 10% glycerol, 5% 2-mercaptoethanol. The gels were stained with Coomassie blue. The overexpressed band in the SO32− lane is MJ0870 or Fsr. a, b and g represent α-, β- and γ-subunits of methylcoenzyme M reductase. M molecular mass standards

detectable in cells gro wn with sulfide (Fig. 16.3A). Although originally annotated as the β-subunit of a coenzyme F420 reducing hydrogenase (FrhB) (Bult et al. 1996), MJ0870 encodes a coenzyme F420 dependent sulfite reductase (Fsr), a novel enzyme (Sect. 16.6). Coenzyme F 420 is an 8-hydroxy-5-deazariboflavin derivative, which is found in the methanogens, certain sulf ate-reducing archaea and actinomycetes (DiMarco et al. 1990; Purw antini et al. 1997). Similar to the nicotinamide coenzymes, F 420 is a h ydride carrier and is restricted to tw o-electron transfer reactions (DiMarco et al. 1990), and H 2F420 (reduced F 420) is a more potent reductant than NAD(P)H (Eqs. 16.5, 16.6). F420 + 2e − + 2H + → H 2 F420 , ∆ E 0′ = −350 mV

( DiMarco et al. 1990 ) NAD ( P ) + 2e − + 2H + → NAD ( P ) H + H + , ( Thauer et al. 1977 )

(16.5) ∆ E ′ = −320 mV 0

(16.6)

HSO3 − + 3H 2 F420 → HS− + 3H 2 O + 3F420 , ∆ G 0′ = −135 kJ mol −1 HSO3 −

( Johnson and Mukhopadhyay 2005)

(16.7)

The reduction of sulf ite to sulf ide with H 2F420 is e xergonic (Eq. 16.7). Extracts of Methanocaldococcus jannaschii cells grown with sulfite as the sulfur source oxidize H2F420 with sulfite with a specif ic activity of 1.3–1.7 µmol min −1 mg −1 protein, but cells gro wn with sulf ide lack this acti vity (Johnson and Mukhopadhyay 2005). Methanocaldococcus jannaschii has the potential of e xpressing two F 420-reducing hydrogenases (Bult et al. 1996), which w ould supply H 2F420 for the Fsr reaction.

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16.6 Fsr, Combining Structural Components of Two Different Dissimilatory Metabolic Machineries to Bring About a Sulf ite Reduction Function The 620-residue MJ0870 polypeptide has tw o distinct domains (Fig. 16.2D): 1. Fsr-N (residues 1–311): A primary structure analysis clearly identif ies this part of Fsr as a homolog of H F dehydrogenase (FqoF/FpoF) (Johnson and 2 420 Mukhopadhyay 2005). All specialized sequence features of FqoF/FpoF are present in Fsr -N. For example, in both sequence and location, C 15C18C21C25P26 and C 42H47C50C54P55 of MJ0870 correspond to tw o ferredoxin-type [ Fe 4–S4] motifs of FqoF and FpoF. A phylogenetic analysis also identifies Fsr-N as an FqoF homolog (Fig. 16.4A). FqoF is the electron-funneling unit of a membranebased energy transduction system, called the H2F420:quinone oxidoreductase (Fqo) complex, found in A. fulgidus (Deppenmeier 2004; Fig. 16.2E). A. fulgidus generates H 2F420 from the oxidation of methyl groups from substrates such as lactate and pyruvate. This oxidation occurs via a partial re verse methanogenesis pathway (Möller-Zinkhan et al. 1989; Fig. 16.1B). FqoF then oxidizes H 2F420 and introduces the electrons derived from this oxidation into the membrane-resident Fqo complex (Deppenmeier 2004; Fig. 16.2E). The Fqo comple x is similar to

Fig. 16.4 Phylogenetic tree for the N-terminal and C-terminal halv es of Methanocaldococcus jannaschii Fsr and its homologs. A, N-terminal half of Fsr or MJ0870 (residues 1–311);B, C-terminal half of Fsr or MJ0870 (residues 325–620). The proteins are identif ied by NCBI’ s open reading frame or accession numbers. Values near the branches are bootstrap confidence levels. Bar number of substitutions per site. − N, N-terminal half; −C, C-terminal half; FrhB and FruB, β-subunits of F420-dependent h ydrogenases; FdhB, β-subunit of F 420-dependent formate deh ydrogenase; DsrA and DsrB, subunits of dissimilatory sulfite reductase; SalDsrC, hemoprotein subunit of Salmonella enterica anaerobic sulf ite reductase; RCIX2197 and RCIX2692, putati ve assimilatory sulf ite reductases of an uncultured methanogenic archaeon from rice rhizosphere.

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the bacterial and mitochondrial respiratory N ADH dehydrogenase comple x or complex I (Deppenmeier 2004). Methanogens belonging to the genus Methanosarcina, which are phylogenetically closely related to A. fulgidus (Woese et al. 1990), carry out partial re verse methanogenesis and possess an Fqo-type system called H2F420:phenazine oxidoreductase (Fpo). In this complex, FpoF is the H 2F420 dehydrogenase (Deppenmeier 2004). It seems that the Fqo/ Fpo system is specif ically associated with the methyl group oxidation and reverse methanogenesis pathw ay. Also, the genomes of Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus lack readily identif iable FqoF/FpoF homologs; these hydrogenotrophs are more deeply rooted compared with A. fulgidus and Methanosar cina (W oese et al. 1990). These observations led to the assumption that the H 2F420 dehydrogenase comple x and FpoF/FqoF are absent in strictly hydrogenotrohic methanogens (Baumer et al. 1998). W e no w kno w that an FpoF/FqoF homolog is indeed present in Methanocaldococcus jannaschii and certain strictly hydrogenotrophic methanogens as Fsr -N (Fig. 16.4A). Hence, Fsr -N is e xpected to retrie ve electrons from H 2F420 (Fig. 16.2D). 2. Fsr-C (residues 325–620): This part of Fsr is a dissimilatory sulfite reductase homolog (Johnson and Mukhopadhyay 2005). A vailable data on sulf ite reductases (Crane and Getzof f 1996) suggest that in MJ0870 Ar g355 and Arg423 are in volved in binding sulf ite and C 428C434C468C472 represents the siroheme–[Fe4-S4]-binding element. Phylogenetically, Fsr-C is most similar to SalDsrC (Fig. 16.4B); therefore, Fsr -C is lik ely to house siroheme and carry out sulfite reduction. In combination, Fsr -N and Fsr -C create an enzyme that w ould retrieve electrons from H2F420, perhaps via bound flavin, and transfer these electrons to the siroheme via Fe4–S4 clusters for use in the reduction of sulfite (Fig. 16.2D); hence, functionally Fsr is similar to E. coli Sir (Fig. 16.2A). One of the unique properties of Fsr is that the electron harvesting and the sulfite reduction units are physically link ed on a single sub unit (Fig. 16.2D); in other sulf ite reductases these are represented by two polypeptides (Fig. 2A–C).

16.7 Purified Fsr Exhibits Properties Predicted from the Primary Structure Purified Fsr contains siroheme and converts sulfite to sulfide with H 2F420 as the reductant with an electron transfer rate of 23–32 µmol min−1 mg−1 (Johnson and Mukhopadhyay 2005). The apparent Km values for sulfite and H2F420 are 12 and 21 µM, respectively. Therefore, Fsr is a highly acti ve enzyme with high af finities for its substrates. Fsr oxidizes H 2F420 with methylviologen (an artif icial one-electron carrier) and reduces sulfite to sulfide with reduced methylviologen,

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which suggest the e xistence of one-electron paths between the retrie val of reducing equivalents from H 2F 420 and reduction of sulf ite; such acti vities are found in dissimilatory sulf ite reductases as well (Lee et al. 1973; Dahl et al. 1993). The Fsr partial reactions transfer electrons at a rate of 90–110 µmol min−1 mg −1, which is 3–5 times f aster than that of the complete H 2F420-utilizing reaction (Johnson and Mukhopadhyay 2005). The transfer of electrons between two functional units might represent a rate-limiting step in the complete reaction. Fsr cannot utilize NADH or NADPH in place of H2F 420 or thiosulfate and sulf ate in place of sulf ite. In the Fsr reaction, about 70% of the reducing equi valents pro vided by H 2F 420 are reco vered as sulf ide. This gap could be due to either an error in the measurement of sulfide or the production of partially reduced compounds. Production of partially reduced compounds such as trithionate and thiosulf ate has been observ ed with certain sulf ite reductases (LeGall and F auque 1988). F or Fsr, the production of thiosulf ate as an intermediate is unlik ely because the enzyme is unable to reduce thiosulfate.

16.8 Fsr, a Sulfite Detoxification Tool and an Assimilatory Enzyme In cells receiving sulfite, Fsr and methylcoenzyme M reductase subunits are expressed at comparable levels (Fig. 16.3b); the latter is a catabolic enzyme and represents up to 30% of the cellular protein in a methanogen (Rouviere and W olfe 1987; Thomas et al. 1987). From the data presented in Sect. 16.7, it can be calculated that the extracts of Methanocaldococcus jannaschii grown with sulfite reduce this oxyanion with H 2F420 at a rate of 2.7–3.7 µmol min −1 mg −1 protein. With reduced methylviologen as the electron donor, this rate would be 8.1–18.5 µmol min−1 mg−1 protein. With A. fulgidus , where sulf ite reductase is an ener gy-metabolism enzyme, cell e xtract sulfite reductase acti vity as measured with methylviologen is 0.07 µmol min −1 mg −1 protein (Dahl et al. 1994). This comparison shows that Methanocaldococcus jannaschii Fsr behaves like a catabolic enzyme. Upon centrifugation at 160,000g, about 26% of the cell extract Fsr activity is found in the pellet fraction and 65% in the denser section of the supernatant (E.F . Johnson and B. Mukhopadhyay , unpublished preliminary data), suggesting a loose association of Fsr with the membrane. These observations lead to the hypothesis that in response to an e xposure to sulf ite, Methanocaldococcus jannaschii expresses Fsr at a high cellular le vel and places this highly active enzyme near the membrane. This arrangement allo ws the or ganism to convert sulfite to sulf ide before it enters the cell and thereby to protect its methylcoenzyme M reductase from inacti vation. Since Methanocaldococcus jannasc hii requires sulfide for growth (Jones et al. 1983), this detoxif ication process also yields an essential nutrient and serv es an assimilatory purpose. It should be noted that the appearance of Fsr activity in the 160,000 g pellet fraction of cell extracts could be due

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to an association of the enzyme with other soluble components of the cell, which could create a rather lar ge complex. If that were the case, coenzyme F 420 reducing hydrogenase, which generates H 2F420, might belong to such a comple x.

16.9

Homologs of Fsr in Other Or ganisms

Fsr homologs have been found in three methanogens and an uncultured archaeon (ORFs MTH280, MK0779, Mbur_0619 and GZ27A8_52; Fig. 16.4). Since MTH280 is closely related to MJ0870 (Fig. 16.4) and its host,Methanothermobacter thermautotrophicus, can use sulfite as a sulfur source (Daniels et al. 1986), this ORF is lik ely to represent an Fsr enzyme. Phylogenetically, MK0779 is a bit distant from both MJ0870 and MTH280 (Fig. 16.4). Also Methanopyrus kandleri cannot use sulf ite as a sole sulfur source (Rothe and Thomm 2000), but has the genomic potential for catalyzing nitrate to ammonia (Slesarev et al. 2002). Therefore, MK0779 probably represents an F 420-dependent nitrite reductase. When Fsr w as discovered, only three Fsr homologs (MJ0870, MTH280 and MK0799) were known, and these belong to deeply rooted, thermophilic, strictly hydrogenotrophic hosts (Johnson and Mukhopadhyay 2005). The identif ication of Mb ur_0619 and GZ27A8_52, which form a new homolog group (Fig. 16.4), extends the host range of this novel enzyme to certain late-e volving and psychrophilic archaea that perform re verse methanogenesis and li ve in a methane-rich en vironment. Methanococcoides b urtonii, which carries Mbur_0619, is an obligately methylotrophic methanogen belonging to the Methanosarcinales (Franzmann et al. 1992). It w as isolated from a methane-saturated environment with permanent temperatures of 1–2°C. The source of GZ27A8_52 is an uncultured anaerobic-methanotrophic archaeon (Hallam et al. 2004). This or ganism is peripherally related to the Methanosarcinales and a member of a cold seeps consortium that performs reverse methanogenesis and sulfate-reduction-driven AOM. It is not known whether these microorganisms tolerate sulfite and/or use it as a sulfur source.

16.10

Small Sulfite Reductases in Methanogens

A small siroheme sulf ite reductase (sub unit size, 23 kDa) has been isolated from Methanosarcina barkeri (Moura et al. 1986). The physiological electron donor and the in vivo role for this enzyme are not known. From a BLAST search of the respective genome using Fsr-C as the query we found that every methanogen carries at least one ORF with the potential of encoding a small (22.4–37.2 kDa) siroheme sulf ite reductase (Fig. 16.4b). These ORFs are related to Fsr -C (Fig. 16.4b), b ut are not linked to an Fsr -N unit. The pre viously isolated Methanosarcina bark eri sulf ite reductase most likely belongs to this group. Since many methanogens are sensitive to sulfite (Sects. 16.2, 16.4), it is unlik ely that the small sulf ite reductases confer an ability to tolerate or utilize externally supplied sulfite as their sulfur source. The possible roles for these ORFs are discussed in the follo wing section.

16 A Novel Coenzyme F420 Dependent Sulfite Reductase

16.11 Conclusion

213

and Hypotheses

It is possible that in addition to its assimilatory and detoxif ication roles, Fsr allows a nonmethanogenic mode of energy generation via H 2-dependent sulfite or nitrite reduction in certain methanogens. This system could be a remnant or a precursor of an ancient sulf ate reduction pathw ay. Fsr could also be the ancestor of FqoF/FpoF, the electron input unit of the ener gy-transduction systems in late-e volving archaea, and the sub units of the dissimilatory sulf ite reductases. If Fsr is simply a detoxif ication enzyme, what w as its role before the appearance of oxygen on Earth? One possible early role of this enzyme is in the synthesis of coenzyme M, where sulf ite is the proposed precursor of a sulfonate group (Graham et al. 2002); coenzyme M is essential for methanogenesis. Methanogens lack sulf ate reduction enzymes and require sulfide for growth. One way to generate the needed sulf ite would be to oxidize sulfide. In the highly reducing, anaerobic en vironment of early Earth this conversion was definitely an endergonic process (Eq. 16.8): HS− + 3H 2 O → HSO3 − + 3H 2 , ∆ G 0′ = +171.7 kJ mol −1 HSO3 −

( Thauer et al. 1977 )

(16.8)

It was probably dri ven by a membrane-associated Fsr that utilized a re verse electron transport system. Also, a membrane-resident Fsr w ould ha ve prepared a methanogen for dealing with the toxicity of sulfite during the early oxygenation of Earth. However, a limited distribution of Fsr genes and a wide distribution of DsrAB genes (Stahl et al. 2002) question the hypothesized ancestral nature of Fsr. On the other hand, it has to be considered that Fsr thus far has been found in or ganisms that live in extreme habitats, which have not been extensively explored. It is also equally possible that the need for sulf ite in coenzyme M biosynthesis w as met by the small sulf ite reductase that is found in methanogens, and this enzyme in combination with FqoF ga ve rise to Fsr . This hypothesis is reasonable because, unlik e Fsr , the small sulf ite reductases are present in e very methanogen and therefore must play a vital role. These proteins are also closely related to Fsr -C (Fig. 16.4b). In all, sulfite reduction is probably an ancient process in the methanogens. The existence of Fsr homologs in methane-oxidizing archaea and methyl group oxidizing methanogens raises the possibility that some of these or ganisms may carry both the methanogenesis/re verse methanogenesis and sulf ate reduction machineries. It no w seems more lik ely that sulf ate reduction and methanogenesis at one time e xisted in one organism. Acknowledgement. We thank Endang Purw antini for discussions and help in phylogenetic analysis, Christiane Dahl for a review of the manuscript and helpful suggestions, and Dwi Susanti, Jason Rodriguez and Carol V olker for comments. This w ork w as supported by N ASA Astrobiology: Exobiology and Evolutionary Biology grant NNG05GP24G to B.M.

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References Balderston WL, P ayne WJ (1976) Inhibition of methanogenesis in salt marsh sediments and whole-cell suspensions of methanogenic bacteria by nitrogen oxides. Appl Environ Microbiol 32:264–269 Baumer S, Murakami E, Brodersen J, Gottschalk G, Ragsdale SW , Deppenmeier U (1998) Methanosarcina species. The F 420H2:heterodisulfide oxidoreductase system from 2-Hydroxyphenazine mediates electron transfer from F 420H2 dehydrogenase to heterodisulfide reductase. FEBS Lett 428:295–298 Becker DF, Ragsdale SW (1998) Acti vation of methyl-SCoM reductase to high specif ic activity after treatment of whole cells with sodium sulf ide. Biochemistry 37:2639–2647 Boetius A, Ra venschlag K, Schubert CJ, Rick ert D, W iddel F, Gieseke A, Amann R, Jor gensen BB, W itte U, Pf annkuche O (2000) A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407:623–626 Boone DR, Whitman WB, Rouviére P (1993) Microbiology , di versity and taxonomy of methanogens. In: Ferry JG (ed) Methanogenesis: ecology, physiology, biochemistry and genetics. Chapman and Hall, New York, pp 35–80 Bult CJ, White O, Olsen GJ, Zhou L, Fleischmann RD, Sutton GG, Blak e JA, FitzGerald LM, Clayton RA, Gocayne JD, K erlavage AR, Dougherty B A, Tomb JF, Adams MD, Reich CI, Overbeek R, Kirkness EF, Weinstock KG, Merrick JM, Glodek A, Scott JL, Geoghagen NSM, Weidman JF, Fuhrmann JL, Nguyen D, Utterback TR, K elley JM, Peterson JD, Sado w PW, Hanna MC, Cotton MD, Roberts KM, Hurst MA, Kaine BP , Borodo vsky M, Klenk H-P , Frasher CM, Smith HO, W oese CR, V enter JC. (1996) Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 273:1058–1073 Crane BR, Getzof f ED (1996) The relationship between structure and function for the sulf ite reductases. Curr Opin Struct Biol 6:744–756 Dahl C, Kredich NM, Deutzmann R, T ruper HG (1993) Dissimilatory sulphite reductase from Archaeoglobus fulgidus: physico-chemical properties of the enzyme and cloning, sequencing and analysis of the reductase genes. J Gen Microbiol 139(Pt 8):1817–1828 Dahl C, Speich N, T ruper HG (1994) Enzymology and molecular biology of sulf ate reduction in extremely thermophilic archaeon Archaeoglobus fulgidus. Methods Enzymol 243:331–349 Daniels L, Belay N, Rajagopal BS (1986) Assimilatory reduction of sulf ate and sulfite by methanogenic bacteria. Appl Environ Microbiol 51:703–709 Deppenmeier U (2004) The membrane-bound electron transport system of Methanosarcina species. J Bioenerg Biomembr 36:55–64 Dhillon A, Gosw ami S, Rile y M, T eske A, Sogin M (2005) Domain e volution and functional diversification of sulfite reductases. Astrobiology 5:18–29 DiMarco AA, Bobik T A, Wolfe RS (1990) Unusual coenzymes of methanogenesis. Annu Re v Biochem 59:355–394 Franzmann PD, Springer N, Ludwig W, Conway de Macario E, Rohde M (1992) A methanogenic archaeon from Ace Lake, Antarctica: Methanococcoides burtonii sp. nov. Syst Appl Microbiol 15:573–581 Graham DE, Xu H, White RH (2002) Identif ication of coenzyme M biosynthetic phosphosulfolactate synthase: a ne w f amily of sulfonate-biosynthesizing enzymes. J Biol Chem 277: 13421–13429 Hallam SJ, Putnam N, Preston CM, Detter JC, Rokhsar D, Richardson PM, DeLong EF (2004) Reverse methanogenesis: testing the h ypothesis with en vironmental genomics. Science 305:1457–1462 Hinrichs KU, Hayes JM, Sylv a SP , Bre wer PG, DeLong EF (1999) Methane-consuming archaebacteria in marine sediments. Nature 398:802–805 Huang CJ, Barrett EL (1991) Sequence analysis and expression of the Salmonella typhimurium asr operon encoding production of h ydrogen sulf ide from sulf ite. J Bacteriol 173:1544–1553

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Jannasch HW (1989) Chemosynthetically sustained ecosystems in the deep sea. In: Schle gel HG, Bowien B (eds) Autotrophic bacteria. Springer, New York, pp 147–166 Johnson EF, Mukhopadhyay B (2005) A ne w type of sulf ite reductase, a no vel coenzyme F 420dependent enzyme, from the methanarchaeon Methanocaldococcus jannaschii. J Biol Chem 280:38776–38786 Jones WJ, Leigh JA, Mayer F, Woese CR, Wolfe RS (1983) Methanococcus jannaschii sp. nov., an extreme thermophilic methanogen from a submarine h ydrothermal vent. Arch Microbiol 136:254–261 Kah LC, L yons TW, Frank TD (2004) Lo w marine sulphate and protracted oxygenation of the Proterozoic biosphere. Nature 431:834–838 Klenk HP, Clayton RA, T omb JF, White O, Nelson KE, K etchum KA, Dodson RJ, Gwinn M, Hickey EK, Peterson JD, Richardson DL, K erlavage AR, Graham DE, K yrpides NC, Fleischmann RD, Quack enbush J, Lee NH, Sutton GG, Gill S, Kirkness EF , Dougherty B A, McKenney K, Adams MD, Loftus B, Peterson S, Reich CI, McNeil LK, Badger JH, Glodek A, Zhou L, Ov erbeek R, Gocayne JD, W eidman JF, McDonald L, Utterback T , Cotton MD, Spriggs T, Artiach P, Kaine BP, Sykes SM, Sado w PW, D’Andrea KP, Bowman C, Fujii C, Garland SA, Mason TM, Olsen GJ, Fraser CM, Smith HO, W oese CR, Venter JC (1997) The complete genome sequence of the h yperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus. Nature 390:364–370 Lee JP, LeGall J, Peck HD, Jr . (1973) Isolation of assimilatory- and dissimilatory-type sulf ite reductases from Desulfovibrio vulgaris. J Bacteriol 115:529–542 LeGall J, F auque G (1988) Dissimilatory reduction of sulfur compounds. In: Zenhder AJB (ed) Biology of anaerobic microorganisms. Wiley, New York, pp 587–693 Leigh JA (2002) Evolution of energy metabolism. In: Staley JT, Reysenbach AL (eds) Biodiversity of microbial life: foundation of earth biosphere. W iley, New York, pp 103–120 Lubbe YJ, Youn HS, Timkovich R, Dahl C (2006) Siro(haem)amide in Allochromatium vinosum and rele vance of DsrL and DsrN, a homolog of cobyrinic acid a,c-diamide synthase, for sulphur oxidation. FEMS Microbiol Lett 261:194–202 Mahlert F, Bauer C, Jaun B, Thauer RK, Duin EC (2002) The nick el enzyme meth yl-coenzyme M reductase from methanogenic archaea: In vitro induction of the nickel-based MCR-ox EPR signals from MCR-red2. J Biol Inor g Chem 7:500–513 Matthews JC, Timkovich R, Liu MY, Le Gall J (1995) Siroamide: a prosthetic group isolated from sulfite reductases in the genus Desulfovibrio. Biochemistry 34:5248–5251 McCollom TM, Shock EL (1997) Geochemical constraints on chemolithoautotrophic metabolism by microor ganisms in seafloor h ydrothermal systems. Geochim Cosmochim Acta 61: 4375–4391 Möller-Zinkhan D, Börner G, Thauer RK (1989) Function of methanofuran, tetrahydromethanopterin, and coenzyme F 420 in Archaeoglobus fulgidus. Arch Microbiol 152:362–368 Moura I, Lino AR, Moura JJ, Xavier AV, Fauque G, Peck HD Jr, LeGall J (1986) Low-spin sulfite reductases: a ne w homologous group of non-heme iron-siroheme proteins in anaerobic bacteria. Biochem Biophys Res Commun 141:1032–1041 Nakayama M, Akashi T , Hase T (2000) Plant sulf ite reductase: molecular structure, catalytic function and interaction with ferredoxin. J Inor g Biochem 82:27–32 Orphan VJ, House CH, Hinrichs KU, McK eegan KD, DeLong EF (2002) Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments. Proc Natl Acad Sci USA 99:7663–7668 Pires RH, Venceslau SS, Morais F, Teixeira M, Xavier AV, Pereira IA (2006) Characterization of the Desulfo vibrio desulfuricans A TCC 27774 DsrMKJOP comple x – a membrane-bound redox complex involved in the sulfate respiratory pathway. Biochemistry 45:249–262 Poulton SW, Fralick PW, Canfield DE (2004) The transition to a sulphidic ocean approximately 1.84 billion years ago. Nature 431:173–177 -dependent glucose-6-phosphate Purwantini E, Gillis TP , Daniels L (1997) Presence of F 420 dehydrogenase in Mycobacterium and Nocardia species, b ut absence from Streptomyces and Corynebacterium species and methanogenic Archaea. FEMS Microbiol Lett 146:129–134

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Rothe O, Thomm M (2000) A simplified method for the cultivation of extreme anaerobic Archaea based on the use of sodium sulf ite as reducing agent. Extremophiles 4:247–252 Rouviere PE, Wolfe RS (1987) Use of sub units of the meth ylreductase protein for taxonomy of methanogenic bacteria. Arch Microbiol 148:253–259 Shen Y, Knoll AH, Walter MR (2003) Evidence for low sulphate and anoxia in a mid-Proterozoic marine basin. Nature 423:632–635 Shima S, Thauer RK (2005) Methyl-coenzyme M reductase and the anaerobic oxidation of methane in methanotrophic Archaea. Curr Opin Microbiol 8:643–648 Slesarev AI, Mezhe vaya KV, Makaro va KS, Polushin NN, Shcherbinina O V, Shakho va VV, Belova GI, Aravind L, Natale DA, Rogozin IB, Tatusov RL, Wolf YI, Stetter KO, Malykh AG, Koonin EV, Kozyavkin SA (2002) The complete genome of h yperthermophile Methanopyrus kandleri A V19 and monophyly of archaeal methanogens. Proc Natl Acad Sci USA 99:4644–4649 Stahl DA, Fishbain S, Klein M, Baker BJ, Wagner M (2002) Origins and diversification of sulfaterespiring microorganisms. Antonie Van Leeuwenhoek 81:189–195 Teske A, Dhillon A, Sogin ML (2003) Genomic markers of ancient anaerobic microbial pathways: sulfate reduction, methanogenesis, and methane oxidation. Biol Bull 204:186–191 Thauer RK, Jungermann K, Deck er K (1977) Ener gy conserv ation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180 Thomas I, Dubourguier H-C, Presiner G, Debeire P, Albagnac G (1987) Purification of component C from Methanosarcia mazei and immunolocalization in Methanosarcinaeae. Arch Micorbiol 148:193–201 Wedzicha BL (1992) Chemistry of sulphiting agents in food. F ood Addit Contam 9:449–459 Widdel F (1988) Microbiology and ecology of sulfate- and sulfur-reducing bacteria. In: Zehnder A (ed) Biology of anaerobic microorganisms. Wiley, New York, pp 469–585 Woese CR, Kandler O, Wheelis ML (1990) T owards a natural system of or ganisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 87:4576–4579 Wolfe RS (1992) Biochemistry of methanogenesis. Biochem Soc Symp 58:41–49

Chapter 17

Archaeal and Bacterial Sulfur Oxygenase-Reductases: Genetic Diversity and Physiological Function Shuang-Jiang Liu

Abstract Many chemolithotrophs oxidize elemental sulfur for energy transformation under acidothermophilic conditions. Research has revealed that some of these acidothermophilic sulfur oxidizers adopt sulfur oxygenase-reductase (SOR) for catalysis of the initial reactions in volved in such elemental sulfur oxidation. Archaeal SORs were f irstly purified from Acidianus brierleryi and Acidianus ambivalens, and were subsequently characterized at molecular le vels. Acidianus tenc hongensis represents an e xample of an acidothermophilic archaeon from hot springs in China. Oxidation of elemental sulfur by this archaeon is initiated by SOR, and this SOR gene w as cloned, e xpressed in Escherichia coli . Furthermore, archaeal SOR gene from Sulfolobus tok odaii was identified from genome data, and when it w as cloned in E. coli, functional SOR was synthesized. More recently, bacterial SORs have been identified from a microbial community in a bioleaching reactor by a metagenomic method. Bacterial SORs ha ve been identif ied also from Acidithiobacillus s pecies and Aquifex aeolicus.

17.1 Intr oduction Sulfur oxygenase-reductase (SOR) catalyzes the con version of elemental sulfur into sulfite and sulfide: 4S 0 + O 2 + 4H 2O → 2Η 2SO3 + 2H2S. Under the conditions for enzymatic catalysis, a s pontaneous reaction of sulf ite and elemental sulfur occurs and thiosulfate is produced. Thus, the total reaction catalyzed by SOR is generally expressed as follows: 5S0 + O2 + 4H 2 O → H 2 SO3 + H 2 S2 O3 + H 2 S.

(17.1)

The f irst SOR w as e videnced and purif ied from Acidianus brierle yi (formerly Sulfolobus brierleyi; Emmel et al. 1986). Although the reductase acti vity was not reported, the properties of this so-called sulfur oxygenase are quite comparable to those of the later -described SORs from Acidianus ambivalens (Kletzin 1989) and Acidianus tengchongensis (He et al. 2000; Sun et al. 2003). 217 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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Fig. 17.1 Phylogenetic relationships among archaeal and bacterial sulfur oxygenase-reductase (SOR) genes. Two genes, sorPt and sorFa, have not been functionally identified, and their hosts do not grow on sulfur as an ener gy source. The gene sorFa further carries a mutation and has been recognized as a pseudogene that has lost its function. sorAt (AF267286) from Acidianus tengchongensis; sorAb from Acidianus brierle yi (unf inished genome sequence); sorAa (X56616) from Acidianus ambivalens ; sorSt (B A000023) from Sulfolobus tok odaii; sorPt (AE017261) from Picrophilus torridus ; sorFa from Ferroplasma acidarmanus ; sorAqa (AE000657) from Aquifex aeolicus; sorAct (DQ480734) from Acidithiobacillus strain SM-1; sorSA (DQ480732) from metagenomic DNAs of bioleaching bioreactors. Bar one base difference per 1,000 bases

At present, knowledge of SORs is mainly deri ved from investigations on sulfur metabolisms in the archaeal species, namely, A. ambivalens and A. tengchongensis. But SORs from bacterial species have been reported recently from Aquifex aeolicus and Acidithiobacillus sp. (Chen et al. 2007; N. Pelletier, M. Guiral, G. Leroy, M.-T. Guidici-Orticoni and C. Aubert, unpublished data). These ne w disco veries will certainly provide more information on both the SOR di versity and its function in sulfur metabolism in the future. The phylogenetic relationship of all the currently known SORs is sho wn in Fig. 17.1. The nine SORs are phylogenetically grouped

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into three subclusters: a lar ge subcluster of six SORs from archaeal strains, a subcluster of two SORs from a bioleaching reactor and Acidithiobacillus sp., and a subcluster of a single SOR from A. aeolicus (Fig. 17.1). This chapter focuses on the SOR di versity, physiology and potential application in bioleaching processes. The biochemistry of SOR is described in Chap. 15 by Kletzin.

17.2

Diversity of Archaeal SORs

The archaeal SORs represent the majority of the currently kno wn SORs (Fig. 17.1). So f ar five SORs have been identified: SOR At from A. tengchongensis (He et al. 2000), SOR Ab from A. brierleyi (Emmel et al. 1986; Sun et al. 2003), SORAa from A. ambivalens (Kletzin 1989), SOR St from Sulfolobus tok odaii (unpublished data) and SOR Sm from Sulfolobus metallicus . The genes coding for SORAb and SORSt were identified according to high identities of amimo acid residue sequences from genome projects, and recently SOR Ab and SOR St have been conf irmed to be acti ve by cloning and e xpression in Escherichia coli. S. tok odaii is unable to gro w with elemental sulfur as the sole ener gy source; thus, the physiological function of SOR St is still unkno wn. Evidence shows that an SOR occurs in S. metallicus (S. Bathe, PE Caldwell and PR Norris, unpublished data), but this SOR has not been characterized at molecular levels. T wo other SOR-lik e genes ha ve been detected in the genomes of Picrophilus torridus and Ferroplasma sp.; ho wever, it is not clear if these SOR-like genes encode active SOR enzymes.

17.2.1 SOR

Ab

from A. brierleyi

A sulfur oxygenase was purified from A. brierleyi (formerly S. brierleyi; Emmel et al. 1986). This enzyme catalyzed the oxidization of elemental sulfur to sulfite, and efforts to determine other products were apparently not made by the researchers. The holoenzyme has a molecular mass of 560,000 kDa, and is composed of a homosub unit of 35 kDa. Its optimal pH and temperature for activity were determined to be 7.0 and 65°C, respectively, and had an apparent Km for sulfur of 0.05 M. SOR Ab was firstly termed as a sulfur oxygenase (Emmel et al. 1986), owing to no observation of sulfide production (indicating the reductase activity of SOR). Evidence that SORAb is similar to the SORs was obtained from western blotting tests, which showed that A. brierleyi cells grown with elemental sulfur contained a protein that immunologically reacted with an antibody specific to the SOR from A. tengchongensis (Sun et al. 2003). Later, an SOR identical at the protein le vel to the SOR from A. tengchongensis was discovered in the genome of A. brierleyi (There is only one nucleotide dif ference, and this difference does not result in amino acid change; unpublished data).

220

17.2.2 SOR

S.-J. Liu Aa

from A. ambivalens

SORAa w as purif ied from A. ambivalens (formerly Desulfurolobus ambivalens ; Kletzin 1989) and subsequently the SOR gene w as cloned and sequenced (Kletzin 1992). This was the first SOR that was characterized at genetic level and was the first reported observ ation where elemental sulfur w as simultaneously oxidized and reduced (an disproportional reaction) during enzymatic catalysis. Similar to SOR Ab from A. brierleyi, this holoenzyme of SORAa has molecular mass of 550,000kDa, and is composed of a homosub unit of 40 kDa (35.6 kDa calculated from the sequence). The optimal pH and temperature for acti vity were 7.4 and 85°C, respecti vely. When observed under the electron microscope, SOR Aa had a hollo w globular morphology, with a diameter of 15.6nm. SORAa has recently been crystallized and its structure was solved at 1.7-Å resolution (Urich et al. 2006, see also Chap. 15 by Kletzin).

17.2.3 SOR

At

from A. tengchongensis

By application of a pair of primers that targeted the conserved motif K-V-C-M-V-Y and the C-terminus W -R-E-Y-L-N, an 840-bp DN A fragment w as amplif ied from the thermophilic sulfur-oxidizing A. tengchongensis (He et al. 2000, 2004). This DNA fragment was used to probe the SOR gene from genomic DN As of A. tengchongensis, and a 3.7-kb EcoRI fragment was obtained. Sequence analysis of this 3.7-kb DNA fragment revealed an open reading frame (ORF) that sho wed 88% identity to SOR Aa. This ORF was cloned in E. coli, and recombinant E. coli cells massively synthesized a protein with SOR activity (He et al. 2000). This work enabled a procedure to be developed for purification of large amounts of SOR for further biochemical and structural studies on SOR. The purified recombinant SOR At has a holoenzyme molecular mass of 550 kDa, and is composed of a homosub unit of 35 kDa. The optimal pH and temperature for acti vity were determined to be 5.0 and 70°C, respecti vely, which are lo wer compared with the v alues for SORAa. The lower temperature for SOR At is apparently related to the optimal gro wth temperature of A. tengchongensis (70°C). By application of site-directed mutagenesis, all three cysteine residues were identif ied to be necessary for enzymatic acti vity, and the importance of these cysteine residues has been conf irmed by crystal structures of SOR At (unpublished data) and SOR Aa (Urich et al. 2006).

17.2.4 SOR

St

from S. tokodaii

Data-mining of the S. tok odaii genome with SOR At as a probe re vealed an ORF homologous to those of SORs. This ORF w as cloned by PCR technique and expressed in E. coli cells with the same method as for SOR At. Enzymatic assays indicated the recombinant SOR St catalyzed oxidation of elemental sulfur to sulf ite and thiosulfite (unpublished data). SORSt showed 68.1 and 64.9% sequence identities

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to SORAa and SORAt/SORAb, respectively, and is the only elemental sulfur oxidizing enzyme found in Sulfolobus species. S. tokodaii is not able to autotrophically grow with elemental sulfur as the sole energy source, but coupling of sulfur oxidation and CO2 fixation was observed in e xtended cultivation and in the presence of or ganic compounds such as alanine.

17.2.5 SOR

Sm

from S. metallicus

Very recently, a new SOR gene was identified in S. metallicus by using a subtractive hybridization approach (S. Bathe, PE Caldwell and PR Norris, unpublished data). Although details are not available, it is expected that this SORSm will have catalytic properties similar to those of other SORs.

17.3

Efforts To Identify Bacterial SORs

Although man y chemolithotrophic bacteria oxidize elemental sulfur for ener gy production, they metabolize elemental sulfur via various enzyme(s) such as the Sox system. Involvement of enzymes similar to SOR had been indicated in a pre vious study (Tano and Imai 1968), b ut this observation was not confirmed until recently when functional SORs originating from bacteria were identif ied.

17.3.1 SOR

Aqa

from A. aeolicus

Data-mining of the A. aeolicus genome discovered two putative sulfur metabolism systems: the thiosulf ate-oxidizing multienzyme system and the SOR system. SORAqa w as cloned and e xpressed in E. coli . Biochemical characterization of SORAqa revealed this enzyme is similar to the archaeal SORs with respect to holoenzyme composition, catalytic properties, etc. Some unique features were reported, such as a more compact structure at 80°C than at 20°C and in volvement o f aromatic residues in maintaining a stable structure (N. Pelletier , M. Guiral, G. Leroy, M.-T. Guidici-Orticoni and C. Aubert, unpublished data).

17.3.2 SOR

Act

from Acidithiobacillus sp. strain SM-1

Novel putati ve bacterial SOR-lik e genes that are v ery phylogenetically dif ferent from the SORAqa gene were identified with metagenomic methods from a microbial community for preoxidation of gold concentrates. One of the putati ve genes w as

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cloned from the metagenome of the microbial community , and this gene, namely , sorSB, w as later located in Acidithiobacillus sp. strain SM-1 (Chen et al. 2007). SORAct (previously SORSB) from Acidithiobacillus sp. strain SM-1 was synthesized with recombinant E. coli , and SOR acti vity w as detected. Further biochemical properties and the physiological role of SOR Act in elemental sulfur oxidation with Acidithiobacillus sp. strain SM-1 are currently under in vestigation.

17.4 SOR Links Elemental Sulfur Oxidation to ATP Synthesis via Sulfite:Acceptor Oxidoreductase and Thiosulfate:Acceptor Oxidoreductase SOR simultaneously oxidizes and reduces elemental sulfur . As can be seen from Eq. 17.1, neither the oxidation nor the reduction of elemental sulfur by SOR is directly coupled to ATP generation or to electron transportation across the cytoplasmic membrane. The linkage between sulfur oxidation and A TP generation w as not understood for man y years until a sulf ite:acceptor oxidoreductase (SA OR) and a thiosulfate:acceptor oxidoreductase (T AOR) were disco vered in sulfur -oxidizing

So

SO42−

2e

2H+

CW

Electron carriers

TAOR

?

SAOR

?

CM

SOR S4O62− S2O62− +

H2S

So

SO32− SO42− 2H+ + 2e

ATP Synthase

H2O

+ 1/2 O2 CO2 Fixation

ATP

2H+ ADP + Pi

So

Fig. 17.2 Coupling sulfur oxidation and ATP generation in A. tengchongensis and A. ambivalens. By application of immunogold electron microscop y technique, the SOR moieties were located at both the c ytoplasmic membrane and the periplasmic membrane. Enzymatic acti vities of SOR, sulfite:acceptor oxidoreductase ( SAOR) and thiosulf ate:acceptor oxidoreductase ( TAOR) were simultaneously determined in the membrane fraction of elemental sulfur (S 0) grown A. tengchongensis cells (Chen et al. 2005). The T AOR was also purif ied from the membrane fraction of S 0grown A. ambivalens cells (Müller et al. 2004). These findings suggest that functional coupling of the three activities possibly happens at the periplasmic membrane CW cell wall, CM cytoplasmic membrane, Pi inorganic phosphate

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A. tengchongensis (Chen et al. 2005) and A. ambivalens (Müller et al. 2004). It is now known that the major physiological function of SOR is to supply substrates (sulfite and thiosulf ite) that will be further oxidized by SA OR and T AOR. The oxidations of sulf ite to sulf ate by SA OR and of thiosulf ate to tetrathionate by TAOR are coupled with electron transportation across the c ytoplasmic membrane and subsequently with ATP generation (Fig. 17.2). A recently characterized TAOR from A. ambivalens uses quinone as the electron acceptor (Müller et al. 2004).

17.5

Physiological Regulation of SOR Activity in Archaea

To date, knowledge of regulation of SOR is very limited. Regulation of SOR activity via posttranslational modif ication has not been reported. Kletzin (1992) reported that transcription of the SOR gene under aerobic conditions w as much higher that under anerobic conditions for A. ambivalens. S. metallicus showed upregulation of SOR gene transcription when gro wn on sulfur and, to a lesser e xtent, on p yrite (S. Bathe, P.E. Caldwell and P.R. Norris, unpublished data). Acknowledgements. The author ackno wledges Z.-W . Chen for his kind assistance in the preparation of the f igures. The research w as supported by the National Natural Science Foundation of China (30621005) and the Ministry of Science and Technology (973 project no. 2004CB719600).

References Chen ZW, Jiang CY, She Q, Zhou PJ, Liu SJ (2005) Key role of cysteine residues in catalysis and subcellular localization of sulfur oxygenase reductase of Acidianus tengc hongensis. Appl Environ Microbiol 71:621–628 Chen, ZW, Liu YY, Wu JF, She Q, Jiang CY, Liu SJ (2007) Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates. Appl Microbiol Biotechnol 74:688–698 Emmel T, Sand W, Koenig WA, Bock E (1986). Evidence for the existence of a sulfur oxygenase in Sulfolobus brierleyi. J Gen Microbiol 132:315–3420 He ZG, Li Y, Zhou P, Liu SJ (2000) Cloning and heterologous e xpression of a sulfur oxygenase/ reductase from the thermoacidophilic archaeon, Acidianus sp. S5 in Escherichia coli. FEMS Microbiol Lett 193:217–221 He ZG, Zhong H, Li Y (2004) Acidianus tengchongensis sp. no v., a ne w species of acidother mophilic archaeon isolated from an acidothermal spring. Curr Microbiol. 48:159–63 Kletzin A (1989) Coupled enzymatic production of sulfite, thiosulfate, and hydrogen sulfide from sulfur: purif ication and properties of a sulfur oxygenase reductase from the f acultatively anaerobic archaebacterium Desulfurolobus ambivalens. J Bacteriol 171:1638–1643 Kletzin A (1992) Molecular characterization of the sor gene, which encodes the sulfur oxygenase/ reductase of the thermoacidophilic Archaeum Desulfurolobus ambivalens . J Bacteriol 174:5854–5859 Müller FH, Bandeiras TM, Urich T , Teixeira M, Gomes CM, Kletzin A (2004) Coupling of the pathway of sulphur oxidation dioxygen reduction: characterization of novel membrane-bound thiosulphate:quinone oxidoreductase. Mol Microbiol 53:1147–1160

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Sun CW, Chen ZW, He ZG, Zhou PJ, Liu SJ (2003) Purif ication and properties of the sulfur oxygenase/reductase from the acidothermophilic archaeon,Acidianus strain S5. Extremophiles 7:131–134 Tano T, Imai K (1968) Ph ysiological studies on thiobacilli. P art II. The metabolism of colloidal sulfur by the cell-free enzyme system of Thiobacillus thiooxidans . Agric Biol Chem 32:51–54 Urich T, Gomes CM, Kletzin A, Frazão C (2006) X-ray structure of a self-compartmentalizing sulfur cycle metalloenzyme. Science 311:996–999

Chapter 18

Diversity of Halophilic Sulfur-Oxidizing Bacteria in Hypersaline Habitats Dimitry Y. Sorokin

Abstract The culturable di versity of halophilic obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) in v arious aquatic hypersaline habitats, such as inland chloride–sulfate lakes, sea solar saltern and deep-sea salt brines, w as found to be une xpectedly high. Six dif ferent groups of halophilic SOB belonging to the Gammaproteobacteria were found. T wo groups of moderately halophilic strictly aerobic SOB dominated at 2 M NaCl, including representati ves of the genus Halothiobacillus (at fully aerobic conditions) and the genus Thiomicrospira (at microoxic conditions). In a fe w cases, halothiobacilli also dominated at 4 M NaCl. Under denitrifying conditions at 2 M NaCl, moderately halophilic and facultatively anaerobic SOB capable of complete denitrif ication of nitrate were found. They are a member of a new genus, “Thiohalomonas”, with closest relatives among marine thiodenitrifyers. At moderate salinity and with thioc yanate as a substrate, a pure culture of moderately halophilic SOB capable of gro wth with thioc yanate and thiosulfate up to 4 M NaCl was obtained, and these SOB are a member of a new genus “Thiohalophilus” distantly related to the genus Thiomicrospira. Two groups of extremely halophilic SOB growing between 2 and 4 M NaCl with an optimum at 3 M NaCl dominated in enrichments at 4 M NaCl. The group of obligately aerobic extreme halophiles, members of a ne w genus “ Thiohalospira”, are related to the Ectothiorhodospiraceae, and f acultatively anaerobic nitrate-reducing e xtreme halophiles, members of a new genus “Thiohalorhabdus”, are distantly related to the genus Acidithiobacillus.

18.1 Intr oduction The di versity of halophilic sulfur -oxidizing bacteria (SOB) able to de velop optimally in NaCl brines remains largely unexplored. Apart from a single moderately halophilic species Halothiobacillus halophilus , disco vered 15 years ago in an Australian hypersaline lak e (Wood and K elly 1991; K elly et al. 1998; K elly and Wood 2000), nothing is kno wn about such chemolithoautotrophic bacteria. Our

225 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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recent research on natronophilic SOB inhabiting saline and highly alkaline soda lakes pro vided e vidence for the widespread potential of chemolithoautotrophic SOB to grow at very high concentrations of sodium carbonate/sodium bicarbonate (Sorokin and K uenen 2005a, b; Sorokin et al. 2005a). This prompted us to start similar research on the di versity of SOB in hypersaline chloride–sulf ate habitats with neutral pH. Extremely halophilic heterotrophic haloarchaea gro wing optimally at 3–4 M NaCl were traditionally re garded as dominating prokaryotes in h ypersaline habitats, such as sea salterns and hypersaline lak es (Oren 2002). Recently , ho wever, evidence started to emer ge indicating the importance of bacterial components in the extremely halophilic prokaryotic communities (Antón et al. 2002; Sorokin et al. 2006a). Among the chemolithotrophic bacteria, SOB ha ve a good chance to adapt to extreme conditions, such as high salt, o wing to a v ery high ener gy yield from complete oxidation of sulf ide/thiosulfate to sulf ate (Oren 1999). Ho wever, so f ar, no culturable SOB phenotypes, equal to haloarchaea with re gard to their salt response, are kno wn. Since functional genes of sulfur -oxidation pathways are not conserved and only recently started to become a subject for molecular analysis (Friedrich et al. 2001, 2005), the culture-independent approach is not yet a vailable for di versity analysis of SOB. Therefore, traditional methods of enrichment and isolation in pure culture remain the main option for biodi versity studies of SOB. Hypersaline aquatic habitats are di vided into marine-dependent (thalassic), which include sea solar salterns, hypersaline lagoons and deep-sea brines, and inland (athalassic) lak es formed either by e vaporative concentration of incoming diluted solutions (primary evaporates) or by dissolution of ancient salt depositions (secondary evaporates). The sea solar salterns ha ve been studied most e xtensively, being relatively easy to access and having an advantage for investigators in offering a whole range of salinity gradients within a short distance (various stages of evaporation). Less is kno wn about microbial communities in hypersaline lak es, located mostly in remote areas with an e vaporative climate. The principal dif ference between these tw o is the much higher magnesium content in the thalassic brines and, usually, the higher sulfate content in inland lakes. In our search for halophilic SOB we mainly focused on inland hypersaline lak es, but also used samples from a sea saltern and from a deep-sea salt brine, formed during dissolution of ancient salt deposits, for a comparison.

18.2

Description of Habitats Investigated

Hypersaline aquatic habitats in six dif ferent regions were e xamined in this study , including four sites of hypersaline inland lak es, a sea solar saltern and a deep-sea salt brine. The main area of study w as in the K ulunda Steppe, southwest Siberia, located along the northeast Kazakhstan border . It harbours numerous salt lak es, ranging from shallo w ponds to v ery large water bodies (Issachenk o 1951) with a

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Table 18.1 Characteristics of the hypersaline habitats investigated Mineral composition

Region

Type

Name

Inland lakes 26 hypersaline Kulunda Steppe lakesa (southwest Siberia) Northeast Mongolia Lake Dzun-Davst and Lake Dolon-Davst South Russia Lake Baskunchak Crimea peninsula Lake Marfinskoe (Ukraine) and Lake Kayashskoe Slovenia (Adriatic Sea saltern Sečovljec coast) East Medi-terranean Deep-sea Urania Basind brine

pH

Total salts (g l−1)

Na+ (M)

Na+/ Cl− Mg2+ (M)

7.5–8.5 100–380 0.9–5.5 14b

7.5–8.1 220–320

ND

ND

Cl/ SO42−

0.65–4.4 4.5b

ND

ND

6.2 8.0

360 115–145

6.5

320

4.1

5.2

5.2

18.6

6.8

220

3.10

11.1

3.30

34.7

a For enrichment purposes sediments were combined into eight groups: group 1, salinity 10–15%, north-central; group 2, salinity 16–22%, north-central; group 3, salinity 22–28%, south; groups 4–8, five individual samples from lakes with salinity 30–38%. b Average data. c Data of Gunde-Cimerman (2000). d Data of M. Yakimov (personal communication).

total salt content from 10 to 38% (w/v), a pH range from 7.5 to 8.5, and with Na +, Mg2+, Cl − and SO 42− as the dominant ions in the brines (T able 18.1). Other lak e provinces, in northeast Mongolia, south Russia (Lak e Baskunchak is the biggest salt lak e in Russia and an important source of cooking salt) and in the Crimea peninsula were studied only briefly (Table 18.1). In addition, a sample from a final evaporation pond in a Sečovlje Adriatic Sea saltern (Gunde-Cimerman et al. 2000) and a sample of deep-sea brine from the eastern Mediterranean Urania Basin (Sass et al. 2001; van der Wielen et al. 2005) were included in the analysis.

18.3 Enrichment

Strategy

In general, two basic mineral media were used to enrich and isolate moderate and extreme halophiles, with 2 and 4 M NaCl, respecti vely. Commonly , thiosulf ate (10–20 mM) w as used as the ener gy source and, in some cases, also sulf ide, tetrathionate (5 mM) or thioc yanate (10 mM). NaHCO 3 served as a carbon source

228

D.Y. Sorokin Sediments from hypersaline lakes, sea saltern, deep-sea brines

Enrichments at 2 M NaCl

Fully aerobic, colonies

Halothiobacillus

Microaerophilic

Thiomicrospira halophila

Enrichments at 4 M NaCl

Anaerobic denitrifying

S2O32−/ NO3−

Thiohalomonas

Aerobic, microaerophilic

NCS−/ NO3−

Thiohalospira

Anaerobic denitrifying

Thiohalorhabdus

Thiohalophilus

Fig. 18.1 General scheme sho wing culturable di versity of halophilic sulfur -oxidizing bacteria (SOB) from h ypersaline habitats. Halothiobacillus spp. and Thiomicrospira halophila , aerobic moderate halophiles; “Thiohalospira”, aerobic extreme halophiles; “Thiohalomonas”, thiodenitrifying moderate halophiles; “ Thiohalorhabdus”, thiodenitrifying e xtreme halophiles; “Thiohalophilus”, f acultatively anaerobic and thioc yanate-utilizing moderate halophile. Dashed lines indicate occasional selections

and additional alkaline b uffer (pH 7–8). T o prevent loss of CO 2 and e vaporation, aerobic culti vation w as performed in closed bottles with 10% liquid v olume at static conditions. Microaerophilic (2% oxygen in the gas phase) and denitrifying cultures were gro wn in 100-ml serum bottles with b utyl rubber stoppers and with 10 ml (aerobic) to 80 ml(anaerobic) of the medium. With sulfide as a substrate, the gradient cultivation technique (Nelson and Jannasch 1993) w as emplo yed. Solid medium containing 2–3 M NaCl was prepared by mixing complete liquid medium containing 4 M NaCl and 30–40 mM thiosulfate with 4–6% (w/v) agarose at different ratios at 50°C. The plates were incubated in closed jars at 0–20% O 2/5% CO2 (v/v) in the gas phase. Various types of enrichments of SOB from hypersaline habitats and their general results are represented in Fig. 18.1.

18.4

Moderately Halophilic Aerobic SOB

The aerobic enrichments at 2 M NaCl usually de veloped quite rapidly , oxidizing 20 mM thiosulf ate within 1 week. In the lak e sediments, direct serial dilutions indicated the presence of 10 5–107 viable cells in 1 cm3. In fully aerated cultures, short motile rods forming large sulfur-containing colonies dominated and could be

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Fig. 18.2 Typical cell morphology of halophilic SOB from h ypersaline habitats. a Moderately halophilic aerobic Halothiobacillus sp. HL 1, thin section, bar 0.5 µm; b moderately halophilic aerobic Thiomicrospira halophila HL 5,bar 0.5µm; c extremely halophilic aerobic “Thiohalospira” HL 4, bar 1 µm; moderately halophilic denitrifying “ Thiohalomonas” HLD 1; e, e xtremely halophilic denitrifying “Thiohalorhabdus” HLD 8; f moderately halophilic, f acultatively anaerobic and thiocyanate-utilizing “Thiohalophilus” HRhD 2, thin section, bar 0.5 µm

easily obtained in pure culture. In static cultures with a high liquid-to-gas ratio and with sulf ide as a substrate in gradient enrichments, a highly motile small vibrio became the dominant morphotype. It also could produce tin y sulfur colonies on thiosulfate plates after prolonged incubation. After isolation in pure culture, the vibrio strains could easily gro w at fully aerated culture. Ov erall, four strains with rod-shaped cells (Fig. 18.2a) and three strains with vibrio cells (Fig. 18.2b) ha ve

7

3 10

1 20 7

Type

1

2 4

6 3 5

SL SL, ST SL, ST

SL SL, ST

SL, MB

Habitat

Thiomicrospira “Thiohalomonas”

Halothiobacillus

Affiliation

Denitrification

0.5–3.5 (1.5) 1.0–3.0 (1.5– 2.0) 1.0–4.0 (2.5) 2.0–5.0 (3.0) 2.0–4.5 (3.0)

− +

0.5–4.0 (1.0– − 1.5)

Salt range (optimum) (M NaCl) Sulfur − −

−

56.1–57.1 58.0–60.0

64.0–67.7

1 1

2

0.10 5.6 0.03–0.04 2.0–2.5 0.052 4.2

0.25 3.5 0.03–0.04 4.0–4.9

0.20–0.35 4.0–4.5

Y (mg protein mmol−1)

Growth kinetics

GeneCNS− S intermediate oxidation G+C (mol%) speciesa µ (h–1)

Genetic properties

+ + 58.2 1 “Thiohalophilus” − Tetrathionate − 65.8–67.0 3 “Thiohalospira” + − 65.0–65.8 1 “Thiohalorhabdus” SL inland lakes, MB deep-sea brines, ST sea salterns, m specific growth rate at optimal salinity, Y specific growth yield. a On the basis of DNA–DNA hybridization.

Number of isolates

Table 18.2 Types of culturable halophilic sulfur-oxidizing bacteria (SOB) in hypersaline habitats: summary

230 Dimitry Y. Sorokin

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231

been isolated in pure culture from the Siberian and Mongolian lak es (Table 18.2). The rod-shaped isolates were identif ied as members of the genus Halothiobacillus and contained at least two different gene-species (DNA–DNA hybridization below species level). The vibrio strains were genetically almost identical to each other and represent a ne w species within the genus Thiomicrospira, Thiomicrospira halophila (Sorokin et al. 2006c; Fig. 18.3).

Fig. 18.3 Phylogenetic position of representati ve strains of halophilic SOB from h ypersaline habitats within the Gammaproteobacteria based on 16S ribosomal RN A gene sequence analysis. Tree topography and evolutionary distances are given by the neighbour-joining method with Jukes and Cantor distances. Numbers at the nodes indicate the percentage of bootstrap v alues for the clade in 1,000 replications. Only values above 90% are shown

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With deep-sea brines from Urania Basin, only the rod-shaped phenotype w as present in the enrichments at 2 M NaCl. The isolate from the Urania Basin, strain HL-U1, clustered with Halothiobacillus hydr othermalis according to the 16S ribosomal RN A (RN A) gene analysis (Fig. 18.3). T wo more Halothiobacillus species, strain HL 20 and strain HL 27, were isolated from the aerobic enrichments at 4 M NaCl, when a specialized group of extremely halophilic SOB “Thiohalospira” (Sect. 18.5) were either at low number or completely absent. Strain HL 20dominated an enrichment culture with tetrathionate as a substrate inoculated with a sediment sample from the Mongolian lak es, while strain HL 27 w as one of the dominant organisms in the enrichment culture from the Crimean lak es with thiosulfate. All these isolates were moderately halophilic with an optimum around 1 M NaCl.

18.5

Extremely Halophilic Aerobic SOB

Enrichment cultures at 4 M NaCl were much slo wer than at 2 M NaCl, the f irst indication of thiosulf ate consumption usually appearing only after 10 days of incubation. Despite this, positi ve results were obtained for most of the samples studied, except for those from the deep-sea brine of Urania Basin. This indicated the uni versal presence of SOB populations able to de velop at saturating salt concentrations. Moreover, they were as abundant in the lake sediments as moderate halophiles (10 3–107 cm−3). The dominant phenotype observ ed at 4 M NaCl in most cases was a thin motile spirillum (Fig. 18.2c). Since it did not form colonies, the pure culture isolation w as achie ved in se veral rounds of dilution to e xtinction. Overall, 20 strains of this phenotype were obtained from salt lak es and a saltern using medium with 4 M NaCl, and with thiosulf ate, sulf ide or tetrathionate as substrates. The group included at least three dif ferent gene species, from Siberian and Mongolian lakes and from a Slo venian saltern. On the basis of the 16S rRN A gene sequence analysis, it represents a ne w lineage in the Gammaproteobacteria with the pro visional name “ Thiohalospira”, clustering with the members of the family Ectothiorhodospiraceae (Fig. 18.3). All strains are e xtreme halophilies not known before among the SOB (Table 18.2). Another specific property of this group was production of lar ge amounts of tetrathionate as an intermediate of thiosulf ate oxidation (up to 80% conversion), which was finally oxidized to sulfate.

18.6

Moderately Halophilic Thiodenitrifyers

Anaerobic enrichments at 2 M NaCl with thiosulf ate as the electron donor and nitrate the as electron acceptor were positive in nine enrichments with various lake sediments and from a saltern (Table 18.2). Nitrite and N2O were observed as major nitrogen intermediates and elemental sulfur as an occasional intermediate during oxidation of thiosulf ate to sulf ate. Six pure cultures were obtained from the

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233

Kulunda lakes, and a single strain each from the Mongolian lak es, Lake Baskunchak, the Crimean lak es and the Slo venian saltern. All these isolates were f acultatively anaerobic, denitrifying, moderately halophilic SOB with long, nonmotile, rod-shaped cells (Fig. 18.2d). Despite the f act that nitrite and N 2O were produced as intermediates during anaerobic growth with nitrate and that washed cells, grown with nitrate, could reduce both intermediates in the presence of thiosulfate, anaerobic growth occurred only with nitrate. Growth was also observed under microoxic conditions at O2 concentrations below 5% (v/v) in the gas phase. This SOB group harbours moderate halophiles with a relatively narrow salt range for growth (Table 18.2). According to the results of DNA–DNA hybridization and sequencing of the 16S rRNA gene, all HLD strains consisted a single gene species and formed a ne w lineage within the Gammaproteobacteria, with the closest relatives among a cluster of marine yet not described thiodenitrifyers (Nercessian et al. 2005; S. Sie vert and G. Muyzer, unpublished data; Fig. 18.3). The pro visional name for this genus is “Thiohalomonas”.

18.7

Extremely Halophilic Denitrifying SOB

Positive anaerobic enrichments with thiosulf ate and nitrate at 4 M NaCl were obtained from the four lak e samples and from the saltern. Despite e xtremely slow development (3–5 mM thiosulf ate consumed within 1 month), all positi ve enrichments resulted in the isolation of a pure culture of the dominant SOB morphotype with long, flexible, nonmotile, rod-shaped cells (Fig. 18.2e). A similar phenotype w as also found in tw o aerobic enrichments at 4 M NaCl. Strain HL 19 w as dominant in an mixotrophic enrichment (acetate/thiosulf ate). During the first stage of this enrichment, heterotrophic haloarchaea utilized acetate and concomitantly oxidized thiosulf ate to tetrathionate (Sorokin et al. 2005b). When all acetate had been utilized, a mixture of e xtremely halophilic SOB started to develop using tetrathionate as the energy source. One of the dominantphenotypes (strain HL 19) w as separated by using dilution series with tetrathionate as a substrate. It w as similar in morphology and its ability to gro w anaerobically with nitrate to HLD strains isolated at 4 M NaCl from denitrifying enrichments. Another similar strain, HL 28, was isolated from an aerobic enrichment culture at 4 M NaCl from one of the Crimean lak es, where it w as de veloping in a mixture with the “Thiohalospira” (Sect. 18.5). The group shared se veral common physiological properties. In contrast to the moderately halophilic denitrifying HLD strains (see abo ve), they grew well under microoxic conditions (2–5% v/v O 2) and some of them e ven in fully aerated cultures. Tetrathionate was a major intermediate of aerobic thiosulfate oxidation to sulfate in this group, similar to the aerobic e xtreme halophiles from the “Thiohalospira” group. Under anaerobic conditions, with either thiosulf ate or tetrathionate as substrates, nitrate was only reduced to nitrite, and sulfuraccumulated as an intermediate. Washed cells, grown with nitrate, however, very slowly reduced

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nitrite and, more acti vely, N 2O in the presence of thiosulf ate as an electron donor . These bacteria represent a second group of e xtremely halophilic SOB found in hypersaline habitats (Table 18.2). All these isolates were related at the species level and formed a new deep lineage within the Gammaproteobacteria (new genus “Thiohalorhabdus”) distantly related to the genus Acidithiobacillus (Fig. 18.3).

18.8

Oxidation of Thiocyanate at High Salt

Thiocyanate (N≡C–S−) is a difficult substrate for SOB and almost nothing is known about its utilization at high salt. Despite some growth and thiocyanate consumption being observed in aerobic enrichments at 2 M NaCl, no pure cultures were obtained because of the presence of high numbers of heterotrophs. Under anaerobic conditions with thiocyanate as an electron donor and nitrate as an electron acceptor at 2 M NaCl, a stable binary culture w as selected which e ventually resulted in the isolation of strain HRhD 2 capable of aerobic gro wth with thiocyanate as the only substrate (Fig. 18.2f). The f inal products of thioc yanate metabolism were sulf ate and ammonium. With both thiosulfate and thiocyanate it could grow within a broad salt range from 1.0 to 4.0 M NaCl (T able 18.2). The bacterium w as able to gro w anaerobically with thiosulfate using nitrite (but not nitrate) as the electron acceptor at low concentrations (below 2 mM) with N2O as an intermediate of denitrification. COS was detected as an intermediate of thioc yanate metabolism, which indicated the “COS pathway” (Kelly and Baker 1990) for the primary thiocyanate degradation in strain HRhD 2. On the other hand, the presence of high c yanase activity in the cells, grown with thiocyanate, cannot be rationally explained at this moment. Phylogenetic analysis of strain HRhD 2 placed it in a ne w lineage within the Gammaproteobacteria distantly related to the genus Thiomicrospira, for which a provisional name “Thiohalophilus” is suggested (Fig. 18.3).

18.9

Fatty Acids in the Membrane Lipids

Since it is the cell membrane which is essential in the “salt out” strate gy used by halophilic Proteobacteria in their adaptation to li ve in brines, the composition of the membrane lipids is an essential property w orth investigating. A comparison of the fatty acid composition in the type strains of four new genera of halophilic SOB described in preceding sections pro vided interesting data (Table 18.3). First of all, palmitic acid (16:0) w as the dominant species in all halophilic SOB. Secondly , hexadecenic acid (16:1w7) was another dominant species in moderately halophilic genera Thiohalomonas and Thiohalophilus, but not in extremely halophilic genera. The latter, represented by the genera Thiohalospira and Thiohalorhabdus, despite their dif ferent phylogenetic position, had quite a similar f atty acid composition

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235

Table 18.3 Comparison of dominant fatty acid composition of the polar lipids in halophilic SOB Percentage of the total Moderate halophiles

Extreme halophiles

Fatty acid

Species

Thiohalomonas Thiohalophilus Thiohalospira Thiohalorhabdus

Hexadecenic acid

16:1w7

27.27

22.00

16:1w5 16:0 10Me16

2.97 25.41 1.26

0.25 33.70

10-Methylhexadecanic acid 11-Methyl-heptadecenic acid Isoheptadecenic acid Cyclopropane– heptadecanic acid Octadecenic acid

11Me17:1 2.53 i17:1w5

10.8

7.37

31.9 44.5

29.36 43.43

44.5

43.43

32.41

17cyc

8.38

0.15

18:1w9

5.10

0.15

18:1w7 Octadecanic 18:0 acid Cyclopropane– 19cyc nonadecanic acid

11.84 0.49

1.11 0.21

0.31 5.8 3.3

3.35 3.43

3.26

with an e xtremely high content of methylated C16 and C17 species, which can be considered as a specif ic feature of these ne w SOB lineages. Thiohalophilus also had a v ery specif ic molecular mark er – isoheptadecenic acid (i17:1w5), which was c ompletely absent in the other genera. Comparison with the other extremely halophilic (Halovibrio-Halospina) and natronophilic (Thioalkalivibrio) Gammaproteobacteria indicated that only the presence of signif icant amount of 16:0 is a common trait among all these e xtremophiles.

18.10

Conclusions and Future Perspectives

Unexpectedly high culturable di versity of halophilic SOB w as detected in hypersaline habitats. Two moderately halophilic aerobic groups belong to the kno wn genera in the Gammaproteobacteria, while e xtremely halophilic aerobes, moderately and extremely halophilic thiodenitrifyers and moderately halophilic thioc yanate-utilizing SOB all represent new lineages within the Gammaproteobacteria.

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Very similar halophilic SOB species were found in inland (athalassic) salt lak es and solar salterns (thalassic) in Europe, dif ferent climatically and in the chemical composition of its brines. Perhaps, total e xtreme NaCl content and v ery specific metabolism are more important than the other parameters. This is conf irmed by a very high requirement both for Na + and Cl − in all groups of halophilic SOB found in hypersaline habitats, both thalassic and athalassic. On the other hand, all of them could grow at very low Mg content and without an y added Ca (data not shown). The most interesting ne w SOB disco vered in hypersaline habitats are the tw o groups of extreme halophiles – a previously unknown ecotype of SOB. High viable cell numbers in the sediments indicate that they may represent one of the dominant bacterial populations there. Both groups have the so-called tetrathionate pathway of thiosulfate oxidation to sulfate, which is common in SOB living in extreme habitats, such as members of Acidithiobacillus, Thermothiobacillus and Halothiobacillus (Kelly and Wood 2000). Despite harsh conditions, their gro wth yield seems to be within the usual range (K elly et al. 1997), implying that these bacteria may possess special adjustments in their bioener getic mechanisms, which w ould be most interesting to study. The array of ne w halophilic SOB from hypersaline en vironments, available in culture, of fers interesting prospects for future research on their physiology and biochemistry. Especially interesting topics might be the mechanisms of salt tolerance in chemolithoautotrophic SOB, the biochemistry and genetics of their sulfur-oxidizing and denitrif ication pathways and the biochemistry of thioc yanate metabolism in halophiles. A preliminary description of the new groups has recently been published elsewhere (Sorokin et al. 2006b). Acknowledgements. This work was supported by an NWO-RFBR grant (047.011.2004.010) by RFBR grant 07-04-00153 and by the Program on Molecular and Cell Biology RAS. The work was done in collaboration with G. Muyzer , T.P. Tourova and A.M. L ysenko (genetic and ph ylogenetic analysis). We are grateful to M. Yakimov and L. Gerasimenko for the possibility to w ork with their samples.

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Kelly DP, Baker SC (1990) The or ganosulfur cycle: aerobic and anaerobic processes leading to turnover of C 1-sulfur compounds. FEMS Microbiol Rev 87:241–246 Kelly DP , W ood AP (2000) Reclassif ication of some species of Thiobacillus to the ne wly designatedgenera Acidithiobacillus gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov. Int J Syst Evol Microbiol 50:511–516 Kelly DP , Sher gill JK, Lu W -P, W ood AP (1997) Oxidati ve metabolism of inor ganic sulfur compounds by bacteria. Antonie Van Leeuwenhoek 71:95–107 Kelly DP , Stack ebrandt E, Bur ghardt J, W ood AP (1998) Conf irmation that Thiobacillus halophilusand Thiobacillus hydrothermalis are distinct species within the γ-subclass of the Proteobacteria. Arch Microbiol 170: 138–140 Nelson DC, Jannasch HW (1983) Chemolithoautotrophic growth of a marine Beggiatoa in sulfidegradient cultures. Arch Microbiol 136:262–269 Nercessian O, Fouquet Y, Pierre C, Prieur D, Jeanthon C (2005) Diversity of Bacteria and Archaea associated with a carbonate-rich metalliferous sediment sample from the Rainbo w vent field on the Mid-Atlantic Ridge. Environ Microbiol 7:698–714 Oren A (1999) Bioenergetic aspects of halophilism. Microbiol Mol Biol Re v 63:34–348 Oren A (2002) Halophilic microorganisms and their environments. Kluwer, Dordrecht Sass A, Sass H, Coolen MJ, Cypionka H, Ov ermann J (2001) Microbial communities in the chemocline of a hypersaline deep-sea basin (Urania Basin, Mediterranean Sea). Appl Environ Microbiol 67:5392–5402 Sorokin DY, Kuenen J G (2005a) Haloalkaliphilic sulfur -oxidizing bacteria in soda lak es. FEMS Microbiol Rev 29:685–702 Sorokin DY, Kuenen J G (2005b) Alkaliphilic chemolithotrophs from soda lakes. FEMS Microbiol Ecol 52:287–295 Sorokin D Y, Banciu H, Robertson LA, K uenen JG (2005a) Haloalkaliphilic sulfur -oxidizing bacteria. In: Dw orkin, F alkow S, Rosenber g E, Schleifer K-H, Stack ebrandt E (eds) The prokaryotes: an evolving electronic resource for the microbiological community. Release 3.20. http://141.150.157.117:8080/prokWIP/index.htm Sorokin DY, Tourova TP, Muyzer G. (2005b) Oxidation of thiosulfate to tetrathionate by a haloarchaeon from hypersaline habitat. Extremophiles 9:501–504 Sorokin DY, Tourova TP, Galinski EA, Belloch C, T indall BJ (2006a) Extremely halophilic denitrifying bacteria from h ypersaline inland lak es Halovibrio denitrif icans sp. no v. and Halospina denitrif icans gen. no v., sp. no v., and e vidence that the genus name Halovibrio (Fendrich 1989) with the type species H. variabilis should be associated with DSM 3050. Int J Syst Evol Microbiol 56:379–388 Sorokin DY, Tourova TP, Lysenko AM, Muyzer G (2006b) Di versity of culturable halophilic sulphur-oxidizing bacteria in hypersaline habitat. Microbiology 152:3013–3023 Sorokin D Y, T ourova TP , K olganova TV , Spiridono va EM, Ber g IA,, Muyzer G (2006c). Thiomicrospira halophila sp. no v., a no vel, moderately halophilic, oblig ately chemolithoautotrophic sulfur -oxidizing bacterium from h ypersaline lak es. Int J Syst Ev ol Microbiol 56:2375–2380 van der Wielen PWJJ, Bolhuis H, Borin S, Daf fonchio D, Corselli C, Giuliano L, D’Auria G, de Lange GJ, Huebner A, Varnavas SV, Thomson J, Tamburini C, Marty D, McGenity TJ, Timmis KN (2005) The enigma of prokaryotic life in deep h ypersaline anoxic basins. Science 307:121–123 Wood AP, Kelly DP (1991) Isolation and characterisation of Thiobacillus halophilus sp. no v., a sulphur-oxidizing autotrophic eubacterium from a W estern Australian hypersaline lake. Arch Microbiol 156:277–280

Chapter 19

Sulfur Oxidation at Deep-Sea Hydrothermal Vents Stefan M. Sievert, Michael Hügler, Craig D. Taylor, Carl O. Wirsen

Abstract Microbial oxidation of geothermally produced reduced sulfur compounds is at the nexus of the biogeochemical carbon and sulfur c ycles at deep-sea hydrothermal vents. A vailable information indicates that microbial symbionts and freeliving gammaproteobacteria of the genera Thiomicrospira, Halothiobacillus, and Beggiatoa are important sulfur-oxidizers above the seafloor at these systems. In addition, bacteria belonging to the Epsilonproteobacteria have been identified as a major component of microbial communities at deep-sea vents. We have previously identified a novel sulfuroxidizing epsilonproteobacterium, Candidatus Arcobacter sulf idicus, which produces sulfur in f ilamentous form that is morphologically and chemically similar to material observed before and after submarine v olcanic eruptions. In the meantime, man y autotrophic epsilonproteobacteria ha ve been isolated and characterized from deep-sea vents, providing further evidence that these organisms play an important role in sulfur and carbon cycling in these environments. These kinds of bacteria may form an important component of a subseafloor biosphere, a currently poorly def ined, yet potentially critical component of deep-sea hydrothermal v ents. Man y autotrophic bacteria and archaea occurring at deep-sea hydrothermal vents, including epsilonproteobacteria, use the reductive tricarboxylic acid c ycle for autotrophic carbon fixation, questioning the paradigm of the Calvin–Benson–Bassham c ycle being at the base of the food web of these ecosystems. In the future, inte grated geochemical and biological studies are needed to further adv ance our understanding of chemoautotrophic sulfur oxidation at deep-sea vents, which will be greatlyfacilitated by having the genomes of representative sulfur-oxidizing bacteria available.

19.1 Intr oduction At deep-sea hydrothermal vents, microorganisms mediate the transfer of energy from the geothermal source to the higher trophic le vels. In particular the microbial oxidation of reduced sulfur compounds through chemolithotrophic processes, principally involving H 2S, has been identif ied to be at the ne xus of the biogeochemical carbon and sulfur c ycles of these systems, where H 2S is primarily produced via sea water– rock interactions within the high-temperature zone (about 400°C) near the sheeted 238 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

19 Sulfur Oxidation at Deep-Sea Hydrothermal Vents

239

dike–gabbro interface (Jannasch and Mottl 1985; Jannasch 1995; b ut see Karl 1995 for a v ery w orthwhile critical look at this aspect; Fig. 19.1). On the basis of thermodynamic modeling, the oxidation of the H 2S contained in the hydrothermal fluids upon contact with oxygenated sea water represents the major ener gy source available at deep-sea v ents (McCollom and Shock 1997), supporting a physiological grouping of microorganisms collectively referred to as the colorless sulfur -oxidizing bacteria. These or ganisms are characterized by their ability to oxidize H 2S or other partially oxidized sulfur compounds for the mixotrophic or autotrophic incorporation of CO 2 into cellular material by using either oxygen or nitrate as electron acceptors. At deep-sea hydrothermal v ents, sulfur-oxidizing bacteria either e xist as free-li ving forms in the mixing zone between oxygenated sea water and reduced hydrothermal fluids, either above or below the seafloor, or in a symbiotic relationship with v arious invertebrates (Jannasch and Mottl 1985; T able 19.1). Thermodynamic calculations suggest that sulfide oxidation is most favorable at relatively low temperatures (below 20°C) owing to an increasing a vailability of oxygen (McCollom and Shock 1997). This suggests that organisms employing this metabolic strategy, in particular at higher temperatures, are able to utilize v ery low oxygen concentrations, to use alternati ve electron acceptors, such as nitrate, or to employ energy-efficient metabolic pathways, S to e.g., for carbon f ixation. While the importance of microbial oxidation of H 2 support chemoautotrophic production abo ve the seafloor at deep-sea hydrothermal vents is well established, much less is known about the composition and extent of the microbial communities in the subseafloor portions of these systems and the global impact of their activities (Wilcock et al. 2004).

19.2

Types of Sulfur-Oxidizing Bacteria

19.2.1 Symbiotic

Sulfur-Oxidizing Bacteria

Although sulfur -oxidizing bacteria li ving in symbiosis with in vertebrates ha ve never been cultured, se veral studies ha ve in vestigated their phylogen y and physiology (reviewed in Nelson and Fisher 1995; Stewart et al. 2005). In particular, recent genomic and proteomic in vestigations ha ve signif icantly adv anced our understanding of these symbioses (Mark ert et al. 2007; Ca vanaugh 2006; Newton et al. 2007). T o date most sulfur -oxidizing endosymbionts ha ve been sho wn to belong to the γ subdivision of the Proteobacteria (Gammaproteobacteria) (Stewart et al. 2005), although recently members of the ε subdivision of the Proteobacteria (Epsilonproteobacteria) have also been identified as endosymbionts (Suzuki et al. 2005a; Urakawa et al. 2005). Many deep-sea hydrothermal vent invertebrates have also been found to live in a stable association with an epibiotic community, which, on the basis of present information, are mostly dominated by Epsilonproteobacteria (Campbell et al. 2006). Although the metabolism of these epibionts has not been determined in each case, sulfur oxidation appears to be common.

Fig. 19.1 A mid-ocean ridge h ydrothermal vent site and potential microbial habitats in the subseafloor . Seawater cycles through the seafl oor where it is geothermally altered. Hot, reducing fluid containing millimolar concentrations of H2S ascend to the seafloor either exiting undiluted

240 S.M. Sievert et al.

19 Sulfur Oxidation at Deep-Sea Hydrothermal Vents

19.2.2

Free-Living Sulfur-Oxidizing Bacteria

19.2.2.1

Gammaproteobacteria

241

In addition to the symbionts, a vailable information indicates that free-li ving bacteria belonging to the genera Thiomicrospira, Halothiobacillus, and Beggiatoa within the Gammaproteobacteria are important sulfur -oxidizers abo ve the seafloor at deep-sea hydrothermal v ents (Brinkhof f et al. 2005; Durand et al. 1993; T eske and Nelson 2006). Up to no w no pure cultures are a vailable for Beggiatoa spp. found at hydrothermal v ents. However, physiological and in situ studies ha ve contributed to a better understanding of these or ganisms, which build e xtensive, up to se veral centimeter thick mats at hydrothermal v ents in Guaymas Basin (T eske and Nelson 2006). Thiomicrospira spp. ha ve been frequently isolated or detected from a v ariety of deep-sea hydrothermal v ents (Brinkhoff et al. 2005), whereas Halothiobacillus hydrothermalis has been isolated from a deep-sea hydrothermal v ent site in the Fiji Basin (Durand et al. 1993). Recently, the complete genome of Thiomicrospira crunogena strain XCL-2 was published, re vealing man y adaptations that ha ve allo wed this or ganism to live at the dynamic oxic–anoxic interface at deep-sea vents (Scott et al. 2006). As an example, only a lo w oxygen adapted cbb3-type cytochrome oxidase could be identified, indicating that the general habitat of this organism is characterized by microaerobic conditions. 19.2.2.2

Epsilonproteobacteria

Until recently Thiomicrospira spp. and H. hydrothermalis represented the only pure cultures of mesophilic, obligately chemolithoautotrophic sulfur -oxidizing bacteria from deep-sea v ents. Ho wever, in recent years Epsilonproteobacteria ha ve been increasingly recognized as important members of the microbialcommunities at deep-sea vents, ranging from black smok er chimney walls and associations with in vertebrates (epibionts, and endosymbionts) to the shallo w subsurf ace (Campbell et al. 2006). These bacteria appear to be predominantly chemolithoautotrophic, and frequently isolates have been obtained that are able to generate ener gy by oxidizing reduced sulfur compounds (Takai et al. 2003, 2006; Inagaki et al. 2003; Nakaga wa et al. 2005). In fact, organisms related to Sulfurimonas autotrophica might be the most pre valent free-living sulfur-oxidizers at deep-sea vents (Inagaki et al. 2003). In the meantime, the Fig. 19.1 (Continued) through black smokers or mixing in v arying proportions with sea water in the subseafloor before being dischar ged from the seafloor at dif fuse-flow v ent sites. The latter creates a range of physicochemical conditions and energy sources that can be exploited by different types of microbes li ving in the subseafloor. The stylized cell depicts a chemolithoautotrophic sulfur-oxidizing bacterium that can use oxygen or nitrate as an electron acceptor . The growth of these organisms in the subseafloor is primarily e xpected to occur at temperatures between 4 and 50°C. (Compiled from Jannasch and Mottl 1985, Jannasch 1995, Huber et al. 2003, T ivey 2004, and Wirsen 2004)

Mussel periostracum, Galapagos Rift Hydrothermal fumarole, TOTO caldera, Mariana Arc, Western Pacific Active hydrothermal vent, North Fiji Basin Guaymas Basin

East Pacific

γ

γ

Thiomicrospira crunogena strain MA-3

Thiomicrospira crunogena strain L-12 Thiomicrospira thermophila strain I78

γ

γ

γ

Halothiobacillus hydrothermalis

Beggiatoa spp.

Riftia pachyptila ES

γ

γ

Vestimentiferan tube worm casing, 21°N EPR Vestimentiferan tube worm casing, Galapagos Rift Polymetal sulfide rock, TAG site – MAR

Thiomicrospira crunogena strain TH-55 Thiomicrospira crunogena strain XCL-2

γ

Mesophile

Mesophile

11–45

15–55

10–35

4–41

ND

4–38.5

ND

ND

6.0–9.0

5.0–8.0

5.5–8.5

5.5–8.5

ND

5.0–8.5

ND

ND

0.6

0.7

0.2

0.8

0.45

0.8

ND

ND

67.4

43.8

44.4

44.6

43.1

44.2

S2−

S0, S2−, S2O32−, S4O62− 2− S

S0, S2−, S2O32−, OC

S0, S2−, S2O32−

S0, S2−, S2O32−

S0, S2−, S2O32−

S0, S2−, S2O32−

O2, NO32−

O2, NO32−

O2

O2

O2

O2

O2

O2

APS

APS

soxB detected

APSR and TSO activity

ND

ND

Sox, SQR

ND

Durand et al. (1993)

Ruby and Jannasch (1982) Takai et al. (2004, 2005)

Wirsen et al. (1998)

Jannasch (et al. 1985) Scott et al. (2006)

Nelson et al. Rubisco activity (1989) Nelson and CBB, rTCA Fisher (1995), Markert et al. (2007)

ND

CBB

CBB

ND

CBB

ND

Table 19.1 Free-living and endosymbiotic sulfur-oxidizing bacteria isolated from or identif ied at deep-sea hydrothermal vent sites and some of their characteristics Growth Sulfur oxi- Carbon Phylotemperature pH dation path- fixation Electron Electron genetic Isolation/observa(°C) pathway Organisma acceptors way affiliation tion siteb range m (h−1) G+C donors References

Water sample, Galapagos vent

Beggiatoa mat, Galapagos vent

α

α

Strain AG33

Strain NF18

15–42

15–42

15–42

ND

ND

ND

ND

Galapagos vent

ND

ND

ND

ND

Western Pacific

γ

ND

ND

Indian Ocean Ridge ND

Western Pacific

γ

Alviniconcha sp. type 1 ES Ifremeria nautilei ES

Scaly foot gastropod γ ES α Strain TB66

Western Pacific

γ

Psychrophile ND

East Pacific

ND

Mesophile

East Pacific

Alviniconcha hessleri ES

Calyptogena mag- γ nifica ES Bathymodiolus ther- γ mophilus ES

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

S2O32−, OC

O2, NO32−

O2, NO32−

O2

S2O32−, OC S2O32−, OC

ND

ND

ND

ND

ND

ND

ND

O2

S2−, S2O32−, OC ND

O2

S2−, S2O32−

ND

ND

ND

ND

ND

ND

ATPS activity

ATPS activity

APS

ND

(continued)

Teske et al. (2000)

Newton et al. (2006) Rubisco Nelson and activity Fisher (1995) Rubisco Stein et al. activity (1988), Suzuki et al. (2005b) Suzuki et al. Calvinc (2006) Desbruyeres Rubisco activity et al. (1994), Urakawa et al. (2005) ND Goffredi et al. (2004) ND Teske et al. (2000) ND Teske et al. (2000)

CBB

Sediments, Mid10–40 Okinawa Trough hydrothermal field

ε

Sulfurimonas autotrophica strain OK10

5.4–8.6

5.0–9.0

Paralvinella nest, 4–35 Iheya North field, MidOkinawa Trough

Gas bubbling 10–40 sediment, Iheya North field, Mid-Okinawa Trough

Sulfurovum lithotrophicum strain 42BKT

4.5–9.0

ND

Sulfurimonas par- ε alvinellae strain GO25

ND

9°N EPR, 13°N EPR

ε

Arcobacter spp.

Growth temperature pH (°C) range

Organisma

Isolation/observation siteb

Phylogenetic affiliation

Table 19.1 ( continued)

0.46

0.04

0.5

ND

m (h−1)

48.0

37.6

35.2

ND

G+C

O2, NO32−

O2, NO32-

s0, S2O32

O2

O2d

Electron acceptors

S0, S2O32−, H2

S0, S2−, S2O32−

S2−d

Electron donors rTCAd

Sor activity Sox?

rTCA

SOR activ- rTCA ity, Sox?e

SOR activ- rTCA ity, Sox?e

ND

Inagaki et al., 2004

Takai et al. (2005, 2006)

Takai et al. (2005), Inagaki et al. (2003)

Taylor and Wirsen (1997), Taylor et al. (1999), Wirsen et al. (2002), Moussard et al. (2006)

Sulfur oxi- Carbon fixdation path- ation pathway way References

Q vent, 9°N EPR

AF

Persephonella marina strain EX-H1

4.7–7.5

4.7–7.5

55–75

55–80

ND

ND

ND

ND

0.14

0.09

ND

ND

ND

37.4

38.5

ND

ND

ND

O2, NO32−, ND S0

S0, S2O32−, H2

ND

O2, NO32−

S0, S2O32−, H2

ND

ND

ND

ND

ND

ND

ND

ND

ND

rTCA

ND

ND

rTCAc

rTCA

Götz et al. (2002), Hügler et al. (2007)

Götz et al. (2002)

Urakawa et al. (2005)

Suzuki et al. (2006)

Suzuki et al. (2005a)

ES endosymbiont, γ Gammaproteobacteria, α Alphaproteobacteria, ε Epsilonproteobacteria, AF Aquificales, EPR East Pacific Rise, APS adenosine 5′-phosphosulfate pathway, ATPS ATP sulfurylase, APSR adenosine 5′-phosphosulfate reductase, sox Sox pathway, SOR sulfite:acceptor oxidoreductase, SQR sulfide:quinone oxdidoreductase, TSO thiosulfate oxidase, Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase, rTCA reductive tricarboxylic acid cycle, CBB Calvin–Benson–Bassham cycle, ND no data, OC organic carbon. a Only one representative of vestimentiferans, vesicomyid clams, and mytilids is listed. No epibionts are listed, e ven though sulfur oxidation is a likely metabolism. b For symbionts, the geographical area of occurrence of the host is gi ven. c Isotopic evidence. d Based on data obtained with Candidatus Arcobacter sulfidicus. e Based on the genome of Sulfurimonas denitrificans.

Rebecca’s Roost, Guaymas Basin

AF

Manus Basin, Southwestern Pacific

Persephonella guaymasenis strain EX-H2

Alviniconcha sp. ES ε

Western Pacific

ε

Alviniconcha sp. type 2 ES

ND

Indian Ocean Ridge ND

ε

Alviniconcha aff. hessleri ES

246

S.M. Sievert et al.

genome of the sulfur-oxidizing bacterium Sulfurimonas denitrificans (formerly known as Tms. denitrificans; Timmer-Ten Hoor 1975) has been sequenced, representing not only the first autotrophic, but also the first free-living epsilonproteobacterium for which a genome sequence is a vailable. Although it w as isolated from marine sulf idic sediments, it appears to share quite a few physiological characteristics with S. autotrophica and S. paralvinellae, two autotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents that are its closest cultured relati ves (Inagaki et al. 2003; T akai et al. 2006). The genome of S. denitrificans has a size of about 2.2 Mb with a coding density of approximately 94% (Sie vert 2006). As has been observ ed for Tms. crunogena (Scott et al. 2006), man y features of the genome of S. denitrificans indicate that it has been streamlined for obligate autotrophy (Sie vert 2006). Its genome further re veals a more versatile metabolism than previously thought, possibly explaining the success of this and closely related species in v arious environments (Campbell et al. 2006). The sensitivity of S. denitrificans towards O2 is well known (Timmer-Ten Hoor 1975), and on the basis of the genome it actually appears that this bacterium is an obligate denitrifier that can also gro w at low O2 concentrations. Interestingly, S. denitrificans appears to rely on a periplasmic nitrate reductase rather than a c ytoplasmic membrane-bound one that operates in all kno wn organisms producing N 2 from nitrate (Richardson and Watmough 1999). In the future, it will be interesting to compare this genome with the genomes of other free-li ving epsilonproteobacteria that are currently being sequenced or have been sequenced (Campbell et al. 2006). 19.2.2.3

Aquificaceae

In addition to Gammaproteobacteria and Epsilonproteobacteria, chemolithoautotrophic bacteria of the genus Persephonella within the Aquificaceae have been isolated from deep-sea hydrothermal v ents that are capable of oxidizing reduced sulfur compounds, as well as H 2, under microaerobic conditions and with nitrate at temperatures up to 80°C (Götz et al. 2002). Interestingly , Persephonella marina strain EX-H1, the genome of which has been sequenced, can also gro w under anaerobic conditions by using S 0 as an electron-acceptor . This indicates that environmental conditions might be highly dynamic, necessitating the need for metabolicversatility. 19.2.2.4

Carbon Metabolism in Sulfur-Oxidizing Bacteria

Until recently the Calvin–Benson–Bassham (Calvin) c ycle w as basically the only autotrophic carbon-fixation pathway known too in colorless sulfur -oxidizing bacteria (Table 19.1). However, all autotrophic Epsilonproteobacteria and Aquificae studied to date use the reductive tricarboxylic acid (rTCA) cycle for converting inorganic carbon into biomass (Hügler et al. 2005, 2007; T akai et al. 2005; Suzuki et al. 2005a). Since these organisms appear to constitute an important component of microbial communities at deep-sea hydrothermal vents, carbon fixation through the rTCA cycle might be

19 Sulfur Oxidation at Deep-Sea Hydrothermal Vents

247

more important in these habitats than previously thought (Campbell et al. 2006; Hügler et al. 2007). Recently, evidence has also been presented that the gammaproteobacterial endosymbiont of Riftia pachyptila uses the rTCA cycle for autotrophic carbon fixation (Markert et al. 2007). The fact that the rTCA cycle requires significantly less ATP and fewer reducing equi valents to synthesize a three-carbon unit compared to the Calvin cycle could be of rele vance in a potentially ener gy limiting en vironment, e.g., for organisms gro wing under microaerobic conditions. Based on the simultaneous presence of reduced sulfur and or ganic compounds the argument has also been made that the predominant sulfur-oxidizers at deep-sea hydrothermal vents might actually be mixotrophs rather than obligate autotrophs (Karl 1995); ho wever, to date this hypothesis has not been rigorously tested. It might be e xpected that obligate chemolithoautotrophs are the dominant types in the subseafloor portion of deep-sea v ents, whereas facultative autotrophs or mixotrophs might fare better in dense animal patches where or ganic matter concentrations are lik ely to be higher (Karl 1995; Jannasch 1995). Clearly, more work in this area is needed, e.g., measuring the concentration and composition of dissolved organic matter at various locations within a given vent field in parallel with quantifying the different metabolic types.

19.3 19.3.1

Sulfur Oxidation Pathways Types of Pathways

Significant adv ances ha ve been made in our understanding of sulfur oxidation pathways in a variety of sulfur-oxidizing bacteria over the last few years, mainly by the pioneering genetic studies on the phototrophic sulfur -oxidizing bacterium Allochromatium vinosum and facultatively autotrophic sulfur-oxidizing bacteria, in particular Paracoccus pantotrophus GB17 (see Chap. 9 by Grimm et al. and Chap. 12 by Friedrich et al. for further details). These studies coupled with the sequencing of bacterial genomes have revealed that neutrophilic sulfur-oxidizing bacteria basically use tw o types of sulfur oxidation pathw ays. One in volves a multienzyme complex catalyzing the complete oxidation of reduced sulfur compounds to sulfate (Sox pathway; Kelly et al. 1997; Friedrich et al. 2001), and the other has sulfite and elemental sulfur as important intermediates (Kappler and Dahl 2001; Pott and Dahl 1998; Shahak et al. 1999). The biochemistry and the genetic basis of these pathways are described in more detail elsewhere in this volume, and only a few aspects of relevance to this chapter will be discussed here. The pathw ay in volving sulf ite and sulfur as intermediates proceeds from the transformation of sulf ide to polysulf ide, most lik ely via sulf ide:quinone reductase (Shahak et al. 1999). Sulfur glob ules are formed and again remobilized using dissimilatory siroheme sulf ite reductase to produce sulf ite (Pott and Dahl 1998). Sulfite is either oxidized completely to sulf ate by sulf ite:acceptor oxidoreductase (SOR), which is encoded by sorAB, or via adenosine 5 ′-phosphosulfate (APS)

248

S.M. Sievert et al.

using the enzymes APS reductase (APSR) and A TP sulfurylase (A TPS) in a reaction akin to a re versal of dissimilatory sulf ate reduction (APS pathw ay; Kappler and Dahl 2001). The Sox multienzyme comple x is capable of oxidizing sulfide, sulfite, sulfur, and thiosulfate to sulfate (Friedrich et al. 2001). It has been demonstrated that four proteins are required for a fully functional complex in vitro: SoxB, SoxXA, SoxYZ, and SoxCD (Friedrich et al. 2001). SoxCD has homologies to SorAB, but in contrast has been shown to act as a sulfur dehydrogenase (Friedrich et al. 2001). It has recently been shown that organisms that lack soxCD, but do have soxB, soxXA, and soxYZ, use the Sox system to oxidize thiosulfate to sulfur, which is either stored inside the cell or excreted (Hensen et al. 2006). It might well be that organisms missing the Sox system completely might not be able to use thiosulf ate at all. Acidophilic sulfur-oxidizing bacteria and thermoacidophilic sulfur-oxidizing archaea use dif ferent sulfur oxidation pathw ays that will not be further discussed here (see Friedrich et al. 2001).

19.3.2 Endosymbionts Until recently, the sulfur oxidation pathway of sulfur-oxidizing (endo) symbionts has remained incompletely characterized (Nelson and Fisher 1995). A major adv ance in our understanding of sulfur oxidation in endosymbiotic Gammaproteobacteria came from a recent proteomic study of the endosymbiont of Riftia pac hyptila (Mark ert et al. 2007). Besides APSR and ATPS, dissimilatory siroheme sulfite reductase could be identified and a model based on these data has been proposed that is similar to that of Chlorobaculum tepidum (formerly Chlorobium tepidum ) (Eisen et al. 2002), except that no sox genes have been found. Possibly , this could e xplain why thiosulfate does not appear to be used by the symbiont. A similar pathw ay might also be used by the symbiont of Calyptogena magnifica (Newton et al. 2007). Interestingly , this symbiont contains sox genes ( soxZYXAB) lik e Chlorobaculum tepidum and Allochromatium vinosum (Eisen et al. 2002; Hensen et al. 2006), in line with its capability to use thiosulf ate and to store sulfur glob ules inside the cell (Nelson and Fisher 1995).

19.3.3

Free-Living Sulfur-Oxidizing Bacteria

19.3.3.1

Beggiatoa

Previous work on Beggiatoa has sho wn that the obligately chemolithoautotrophic marine strain MS-81-1c uses the APS pathway involving APSR and ATPS or SOR to oxidize sulfite (Hagen and Nelson 1997). At present, it is not known how sulfide is oxidized to sulf ite, but it is lik ely that it proceeds similarly as in pho totrophic sulfur bacteria which are also kno wn to store elemental sulfur inside their cells.

19 Sulfur Oxidation at Deep-Sea Hydrothermal Vents

249

It is e xpected that Beggiatoa found at hydrothermal v ents use the same pathw ay, but no direct evidence has been yet obtained. However, the genome of a hydrothermal vent Beggiatoa is currently being sequenced as part of the marine microbial genome sequencing project of the Gordon and Betty Moore Foundation (2007) and may lead to confirmation of this hypothesis.

19.3.3.2

Thiomicrospira crunogena and Epsilonproteobacteria

Until now there were also no indications of which sulfur oxidation pathw ay might be used by Tms. crunogena, H. hydrothermalis, or sulfur -oxidizing Epsilonproteobacteria. New information from recently sequenced genomes, however, points to the importance of the Sox system in free-living sulfur-oxidizing bacteria at deep-sea hydrothermal vents. This sulfur oxidation pathway appears to be operating in Tms. crunogena XCL-2 and S. denitrificans (Scott et al. 2006; Sievert 2006), the latter of which might be representati ve for other Epsilonproteobacteria. Details of the Sox system are described in great detail in Chap. 12 by Friedrich et al. and we wish only to illustrate the main dif ferences from f acultative autotrophic sulfur -oxidizers in which this pathw ay has been studied (Friedrich et al. 2001). First of all, the arrangementof sox genes in Tms. crunogena XCL-2 and S. denitrificans is different. The sox genes do not occur in one cluster, as in the model organism P. pantotrophus GB17 (Friedrich et al. 2001), but in different parts of the genome (Fig. 19.2). In this regard, dif ferences also e xist between Tms. cruno gena and S. denitrif icans. S. denitrificans has basically tw o clusters, one containing soxZYXAB and another one containing soxZYCD (Sie vert 2006). In Tms. cruno gena one cluster contains soxZYXA; soxCD and soxB are located elsewhere (Scott et al. 2006). At present the reasons for this dif ference in arrangement are not kno wn. Interestingly , both S. denitrificans and Tms. crunogena have soxCD, although their other sox genes are more closely related to those of C. tepidum, A. vinosum, and Thiobacillus denitrificans, which do not ha ve soxCD and produce sulfur glob ules (Hensen et al. 2006). Tms. crunogena also forms sulfur from thiosulfate (Javor et al. 1991) and appears to be the first sulfur-oxidizing bacterium to do so e ven though it has soxCD. Possibly soxCD is re gulated and turned on and of f depending on en vironmental conditions. Elemental sulfur formation by S. denitrificans has not been reported. Recently, sulfur oxidation enzymes were also measured in some other autotrophic epsilonproteobacteria, including Sulfurimonas autotrophica and Sulfurimonas paralvinellae (Takai et al. 2005). In this case, SOR activity was detected using an assay that would not be expected to measure such activity were these organisms to use the Sox system (C.G. Friedrich, personal communication). This indicates that other Sulfurimonas autotrophica and Sulfurimonas paralvinellae might either not use the Sox system or use a modified version of it. In this regard it is interesting to note, that the soxC sequence identities of S. denitrif icans and Tms. cruno gena to the soxC sequences of those or ganisms that have the complete sox gene set are signif icantly lower than when soxC sequences from or ganisms with a complete sox gene set are

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compared among themselv es (44% compared with more than 63%) (Scott et al. 2006; Sievert 2006). Thus, tak en together with the f act that the genes are not in a cluster with the other sox genes (Fig. 19.2), the possibility e xists that SoxCD in S. denitrificans and Tms. cruno gena is re gulated and functions dif ferently, possibly exhibiting sulf ite dehydrogenase acti vity. It is also w orth mentioning that APSR activity has been found in Tms. thermophila (Takai et al. 2005), although the gene coding for this enzyme has not been detected in the genome of Tms. cruno gena XCL-2 (Scott et al. 2006). Possibly , dif ferent pathw ays e xist in dif ferent Thiomicrospira species. A soxB has been detected in H. hydrothermalis, indicating that it might also use the Sox pathw ay for sulfur oxidation (Petri et al. 2001). 19.3.3.3 Oxidation

of H2 by Sulfur-Oxidizing Bacteria

The genome of S. denitrificans revealed the presence of a hydrogenase, indicating that it might also be able to use H 2 as an electron donor . This w as confirmed in subsequent growth experiments (S. Sievert, unpublished data), making S. denitrificans physiologically similar to S. paralvinellae (Takai et al. 2006). It appears that the ability to use both H 2 and reduced sulfur compounds as electron donors might be more widespread at deep-sea v ents than pre viously thought, representing a v ery useful strategy for li ving in these dynamic en vironments (Nakagawa et al. 2005, Takai et al. 2006). A SoxX

SoxY

SoxZ

SoxZ

SoxY

SoxA

SoxD

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SoxC

B SoxX

SoxY

SoxZ

SoxC

SoxA

SoxD

SoxB C SoxX

SoxY

SoxZ

SoxA

SoxB

SoxC

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Fig 19.2 Gene arrangement of soxXYZABCD inSulfurimonas denitrificans (a) and Thiomicrospira crunogena XCL-2 ( b), as opposed to that in the f acultative autotrophic sulfur -oxidizing Paracoccus pantotrophus GB17, which contains a complete sox gene cluster (c)

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19.4 “Snowblower” Vents As Signs for Sulfide Oxidation in the Subseafloor 19.4.1

The Subseafloor Biosphere

The discovery of microbial populations beneath the deep ocean floor at hydrother mal vents has far-reaching implications, ranging from speculation on the origins of life to the biogeochemistry of the oceans (Summit and Baross 2001). Though the biota residing in the subseafloor ecosystem potentially has a strong influence on a variety of biogeochemical processes, it is a relatively poorly defined component of hydrothermal systems (Wilcock et al. 2004). W e have yet to determine the nature and extent of the microbiology of this ecosystem, its contrib ution to subseafloor primary production, and its influence on the geosphere. Consequently , little is known about the importance of sulfur oxidation in the interior and subsurf ace portion of hydrothermal systems, which is in stark contrast to our knowledge of the importance of this process in supporting dense animal communities abo ve the seafloor. Potentially, hydrothermal fluids flo wing through cracks and pores within the oceanic crust provide rich environments for subseafloor biological communities (Fig. 19.1). Indeed, sampling of diffuse-flow vents immediately after volcanic eruptions or diking events and during subsequent monitoring points to the e xistence of a subseafloor biosphere that might contrib ute considerably to the primary productivity of these hydrothermal systems (Holland et al. 2004).

19.4.2

Filamentous-Sulfur Formation in the Laboratory

By simulating the conditions that are lik ely to occur in the upper microaerobic subseafloor ecosystem at these v ent sites, i.e., active mixing of hydrothermal fluid containing H 2S and oxygenated deep-sea-bottom w ater resulting in a lo w O 2/high H2S environment (Fig. 19.1), a new type of H 2S-oxidizing bacterium was enriched from coastal sulf idic sediments (T aylor and W irsen 1997). This highly motile vibrioid bacterium is unique among prokaryotes in that it excretes sulfur in filamentous form (f ilaments 0.5–2.0- µm thick by 20–500- µm long) as a product of its metabolism (Taylor and Wirsen 1997; Sievert et al. 2007). This bacterium belongs to the genus Arcobacter within the Epsilonproteobacteria and has been pro visionally named Candidatus Arcobacter sulfidicus (CAS) (Wirsen et al. 2002). The fixation of CO2 occurs via the rTCA cycle at rates equivalent to or higher than those of other sulfur-oxidizing autotrophs utilizing the Calvin c ycle (e.g., Tms. crunogena) (Wirsen et al. 2002; Hügler et al. 2005). Ov erall, CAS seems to be well adapted to the conditions supposedly pre vailing in the upper subseafloor portion of deep-sea hydrothermal vents: the organism is microaerophilic, tolerates high sulfide concentrations, is able to f ix N 2, and the entangling f ilamentous sulfur it produces forms mats that permit retention in high fluid flo w environments.

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19.4.3 “Sno wblowers” The metabolic capability of forming filamentous sulfur is not limited to the coastal strain, as it has no w been documented to occur in other sulf idic en vironments (Sievert et al. 2007). Filamentous sulfur produced by the coastal strain of CAS was first compared with and found to be morphologically and chemically similar to white flocculent material collected from extensive discharges, so-called blizzard or snowblower v ents, observ ed during and after a v olcanic eruption at the 9°N deep-sea hydrothermal vent site on the East Pacific Rise (EPR) in 1991 (Nelson et al. 1991; Haymon et al. 1993; Taylor and Wirsen 1997). In this case the filamentous material accumulated in mats of up to 5-cm thickness (Nelson et al. 1991; Haymon et al. 1993). The process of microbial iflamentous-sulfur formation was documented to occur in situ on a colonization de vice deployed at M v ent (Titanium Ring for Alvinella Colonization, TRA C, at 9°N EPR) as well as in shipboard e xperiments using inocula collected from these sites (Taylor et al. 1999).

19.4.4

Diversity of Filamentous-Sulfur-Forming Bacteria

We could further confirm that the formation of filamentous sulfur at 9°N EPR is also mediated by Arcobacter spp. 16S ribosomal RNA sequences closely related, but not identical to CAS were retrieved from the TRAC device (M vent TRAC clones) and from shipboard reactors (shipboard reactor clones) (Fig. 19.3). It is interesting, however, that the sequences from the shipboard reactors and the coastal laboratory strain CAS form one cluster that is distinct from the cluster formed by the sequences obtained from TRAC (Fig. 19.3). This might be related to temperature, as all reactor enrichments and CAS cultures were incubated between 20 and 25°C, whereas the M vent TRAC enrichments were e xposed to temperatures of 40–50°C (T aylor et al. 1999). The sequences from the in situ incubation device might have originated from filamentous-sulfur-producing organisms that grow at higher temperatures, and thus did not ultimately become established in the shipboard reactors. Recently , the formation of filamentous-sulfur mats by Arcobacter spp. has also been reported from the 13°N deep-sea hydrothermal v ent site on the EPR (Moussard et al. 2006). The sequences obtained in this study form a cluster with the M v ent TRAC clones (Fig. 19.3, L50-sequences) indicating the e xistence of h ydrothermal v ent-specific filamentous-sulfur-forming Arcobacter populations. Furthermore, arcobacter sequences have also been detected after an eruptive event in the outflow of a diffuseflow v ent at Axial V olcano, Juan de Fuca Ridge. (Huber et al. 2003), indicating the presence of these bacteria in the subseafloor at yet another geographic area (Fig. 19.3, Mark er 33 sequences). All of this indicates that f ilamentous-sulfur formation may be an important process at hydrothermal v ents, extending into the shallow subsurf ace biosphere, dri ven by inor ganic nutrients alone (i.e., H 2S and CO2, N 2), and thus contrib uting to overall organic matter production at deep-sea hydrothermal vent sites (Fig. 19.1).

19 Sulfur Oxidation at Deep-Sea Hydrothermal Vents

0.05

Clone L50-WB6 M vent TRAC clone a7 M vent TRAC clone a1 M vent TRAC clone a9 M vent TRAC clone a2 Clone L50-WB53 M vent TRAC clone a6 Marker 33-FL74B00 Vestimentiferan symbiont clone (D83061) M vent TRAC clone a11 M vent TRAC clone a3 M vent TRAC clone a10 Marker 33-FL88B00 Marker 33-FL70B00 Marker 33-PA62B98 M vent TRAC clone a8 Deep-sea hydrothermal vent chimney clone CHA3-437 Japan Trench deep-sea desiment clone JTB129 Shipboard reactor clone CB2C3 Shipboard reactor clone CB2E4 Shipboard reactor clone CB2B10 Shipboard reactor clone CB2D1 Candidatus Arcobacter sulfidicus Marker 33-PA 28B00 Marker 33-FL 76B00 Marker 33-FL 58B00 Arcobacter sp. strain Solar Lake Black Sea sediment clone B4b1 Arcobacter cryaerophilus Arcobacter skirrowi Activated sludge clone T31 Arcobacter butzlerii Arcobacter nitrofigilis Oilfield sulfur oxidizer strain FWKO-B Geospirillum barnesii Sulfurospirillum arcachonense Campylobacter jejuni Vent cap clone VC2.1 Bac31 Alvinella pompejana epibiont clone APG44b Alvinella pompejana epibiont clone APB13b Rimicaris exoculata epibiont Pele's vent clone PVB_OTU_3 Sulfurimonas denitrificans Helicobacter pylori Wolinella succinogenes Riftia pachyptila endosymbiont Thiomicrospira crunogena Calyptogena magnifica endosymbiont

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Arcobacter

Fig. 19.3 16S ribosomal RN A based neighbor -joining distance tree depicting the ph ylogenetic relationship of the f ilamentous sulfur -producing microbe, Candidatus Arcobacter sulf idicus and sequences obtained from ship-board reactors and an in situ incubation at M vent at 9°N East Pacific Rise (EPR) (M vent TRAC clones), as well as sequences obtained from a f ilamentous-sulfur mat at 13°N EPR (clones L50-WB6 and L50-WB53) and clones obtained from a dif fuse-flow hydrothermal vent habitat (Marker 33) at Axial Volcano, Juan de Fuca Ridge, during a time series after an eruption (Huber et al. 2003) to select cultured and en vironmental proteobacterial sequences. The tree w as constructed with sequences containing at least 1,300 bp by using the phylogenetic softw are ARB (Technische Universität München 2007). The partial Mark er 33 sequences where inserted into the tree by applying parsimon y criteria without allo wing for changes in the o verall tree topology. The scale bar represents 0.05 estimated changes per nucleotide

19.5 Conclusions

and Outlook

Research conducted at deep-sea hydrothermal v ents o ver the last decade has revealed that sulfur oxidation is mediated by a di verse group of or ganisms. Traditionally, Gammaproteobacteria, either free-li ving or in a symbiotic association, were seen as the main sulfur -oxidizers at these systems. Only recently ha ve we be gun to appreciate the importance of Epsilonproteobacteria for autotrophic carbon production in general and in particular for sulfur oxidation (Campbell et al. 2006). It might well be that or ganisms related to Sulfurimonas spp. might be the predominant sulfur-oxidizers at deep-sea vents (Inagaki et al. 2003). The Sox pathway emerges as a potentially v ery important sulfur oxidation pathw ay at deep-sea hydrothermal v ents. Man y autotrophic prokaryotes occurring at deep-sea v ents appear to use alternati ve CO 2-fixation pathw ays, in particular the rTCA c ycle, questioning the paradigm of the Calvin cycle being at the base of the food chain at deep-sea hydrothermal v ents. Future w ork on this subject promises to be a v ery fruitful area of research.

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Along these lines, it will be important to (1) quantify epsilonproteobacteria and other autotrophic or ganisms, (2) assess whether these or ganisms are acti ve in their environment by analyzing RN A or intact polar lipids (Martinez et al. 2006; Sturt et al. 2004), and (3) implement studies that couple the identity of microorganisms more directly with their function, e.g., autotrophic carbon f ixation, and the measurements ving these of CO 2-fixation rates, to come to a better understanding of what is dri unique ecosystems and to arri ve at overall productivity estimates. The methodology to address the latter has been de veloped (e.g., Gray and Head 2001; Orphan et al. 2001), but so far has largely not been applied to deep-sea hydrothermal vents, mainly because conducting these studies at vents represents a great challenge, particularly in light of the physical, chemical, and biological heterogeneity of these systems. An important task for the future will also be to better define the contribution of the subseafloor biosphere to the overall productivity of these ecosystems. It w as pointed out soon after the disco very of the deep-sea hydrothermal v ents that “bacteria grow most abundantly in the shallo w crust where upwelling hot, reducing hydrothermal fluid mixes with downwelling cold, oxygenated sea water. The predominant production of biomass, however, is the result of symbiotic associations between chemolithoautotrophic bacteria and certain invertebrates” (Jannasch and Mottl 1985). While the validity of the f irst part of the statement has been pro ven in subsequent studies, we presently do not ha ve enough information to assess whether most of the production occurs above or below the seafloor. Clearly, more research in this area is w arranted. Finally, the genomes of representati ves of the four dominant groups of sulfuroxidizing bacteria at deep-sea v ents are available, i.e., endosymbionts, as well as freeliving bacteria belonging to the Gammaproteobacteria, the Epsilonproteobacteria, or Aquificales. Coupled with metagenomic studies (Xu 2006), these genomes already ha ve and will continue to pro vide unique opportunities to adv ance our understanding of this important process at deep-sea hydrothermal v ents. Acknowledgements. Preparation of this manuscript w as supported by National Science Foundation grant OCE-0452333 and a fello wship from the Hanse-W issenschaftskolleg (http:// www.h-w-k.de) to S.M.S. Research conducted in our laboratories (S.M.S., C.D.T ., C.O.W.) on filamentous-sulfur formation and alternative carbon-fixation pathways was supported by National Science F oundation grants OCE-0452333, IBN-0131557, and IBN-9630054, as well as N ASA Astrobiology Institute grant NN A04CC04A. M.H. w as supported through a WHOI postdoctoral scholarship. Thanks are due to Stephen J. Molyneaux for e xcellent technical support and for help in preparing this manuscript, as well as to Jack Cook for preparing Fig. 19.1.

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Ruby EG, Jannasch HW (1982) Ph ysiological characteristics of Thiomicrospira sp. strain L-12 isolated from deep-sea hydrothermal vents. J Bacteriol 149:161–165 Shahak Y, Schütz M, Bronstein M, Hauska G, P adan E (1999) Sulf ide-dependent anoxygenic photosynthesis in prokaryotes: sulf ide:quinone reductase (SQR), the intial step. In: Peshek GA, Löffelhardt W, Schmetterer C (eds) The phototrophic prokaryotes. Kluwer/Plenum, Ne w York, pp 211–228 Scott KM, Sievert SM, Abril FN, Ball LA, Barrett CJ, Blake RA, Boller AJ, Chain PSG, Clark JA, Davis CR, Detter C, Do KF, Dobrinski KP, Faza BI, Fitzpatrick KA, Freyermuth SK, Harmer TL, Hauser LJ, Hügler M, K erfeld CA, Klotz MG, K ong MW, Land M, Lapidus A, Larimer FW, Longo DL, Lucas S, Malfatti SA, Massey SE, Martin DD, McCuddin Z, Meyer F, Moore JL, Ocampo LH Jr , P aul JH, P aulsen IT, Reep DK, Ren Q, Ross RL, Sato PY , Thomas P , Tinkham LE, Zeruth GT (2006) The genome of deep-sea v ent chemolithoautotroph Thiomicrospira crunogena XCL-2. PLoS Biol 4:e383. doi:10.1371/journal.pbio.0040383 Sievert SM (2006) The genome of the sulfur -oxidizing bacterium Thiomicrospira denitrificans: a model for epsilonproteobacterial autotrophs at v ents and other redox interfaces. In: American Society for Microbiology, General Meeting, Orlando Sievert SM, Wieringa EBA, Wirsen CO, Taylor CD (2007) Growth and mechanism of filamentoussulfur formation by Candidatus Arcobacter sulf idicus in opposing oxygen-sulf ide gradients. Environ Microbiol 9:271–276 Stein JL, Cary SC, Hessler RR, Ohta S,etter V RD, Childress JJ, Felbeck H (1988) Chemoautotrophic symbiosis in a hydrothermal vent gastropod. Biol Bull 174:373–378 Stewart FJ, Newton ILG, Cavanaugh CM (2005) Chemosynthetic endosymbioses: adaptations to oxic-anoxic interfaces. Trends Microbiol 13:439–448 Sturt HF, Summons RE, Smith K, Elvert M, Hinrichs KU (2004) Intact polar membrane lipids in prokaryotes and sediments deciphered by high-performance liquid chromatograph y/electrospray ionization multistage mass spectrometry – ne w biomark ers for biogeochemistry and microbial ecology. Rapid Commun Mass Spectrom 18:617–628 Summit M, Baross JA (2001) A novel microbial habitat in the mid-ocean ridge subseafloor . Proc Nat Acad Sci USA 98:2158–2163 Suzuki Y, Sasaki T, Suzuki M, Nogi Y, Miwa T, Takai K, Nealson KH, Horikoshi K (2005a) Novel chemoautotrophic endosymbiosis between a member of the Epsilonproteobacteria and the hydrothermal-vent gastropod Alviniconcha aff. hessleri (Gastropoda: Provannidae) from the Indian Ocean. Appl Environ Microbiol 71:5440–5450 Suzuki Y, Sasaki T, Suzuki M, Tsuchida S, Nealson KH, Horik oshi K (2005b) Molecular ph ylogenetic and isotopic evidence of two lineages of chemoautotrophic endosymbionts distinct at the subdivision level harbored in one host-animal type: the genus Alviniconcha (Gastropoda: Provannidae). FEMS Microbiol Lett 249:105–112 Suzuki Y, K ojima S, Sasaki T , Suzuki M, Utsumi T , W atanabe H, Uraka wa H, Tsuchida S, Nunoura T, Hirayama H, Takai K, Nealson KH, Horik oshi K (2006) Host-symbiont relationships in hydrothermal vent gastropods of the genus Alviniconcha from the southwest P acific. Appl Environ Microbiol 72:1388–1393 Takai K, Inag aki F, Nakagawa S, Hirayama H, Nunoura T , Sak o Y, Nealson KH, Horik oshi K (2003) Isolation and ph ylogenetic diversity of members of pre viously uncultivated epsilonproteobacteria in deep-sea hydrothermal fields. FEMS Microbiol Lett 218:167–174 Takai K, Hirayama H, Nakag awa T, Suzuki Y, Nealson KH, Horik oshi K (2004) Thiomicrospira thermophila sp. no v., a no vel microaerobic, thermotolerant, sulfur -oxidizing chemolithomixotroph isolated from a deep-sea h ydrothermal fumarole in the T OTO caldera, Mariana Arc, western Pacific. Int J Syst Evol Microbiol 54:2325–2333 Takai K, Campbell BJ, Cary SC, Suzuki M, Oida H, Nunoura T , Hirayama H, Nakag awa S, Suzuki Y, Inagaki F, Horikoshi K (2005) Enzymatic and genetic characterization of carbon and ener gy metabolisms by deep-sea h ydrothermal chemolithoautotrophic isolates of Epsilonproteobacteria. Appl Environ Microbiol 71:7310–7320

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Takai K, Suzuki M, Nakagawa S, Miyazaki M, Suzuki Y, Inagaki F, Horikoshi K (2006) Sulfurimonas paralvinellae sp. no v., a no vel mesophilic, h ydrogen- and sulfur -oxidizing chemolithoautotroph within the Epsilonproteobacteria isolated from a deep-sea h ydrothermal v ent polychaete nest, reclassification of Thiomicrospira denitrif icans as Sulfurimonas denitrif icans comb . no v. and emended description of the genus Sulfurimonas. Int J Syst Evol Microbiol 56:1725–1733 Taylor CD, Wirsen CO (1997) Microbiology and ecology of filamentous sulfur formation. Science 277:1483–1485 Taylor CD, Wirsen CO, Gaill F (1999) Rapid microbial production of f ilamentous sulfur mats at hydrothermal vents. Appl Environ Microbiol 65:2253–2255 Technische Universität München (2007) The ARB project. http://www.arb-home.de Teske A, Nelson D (2006) The genera Beggiatoa and Thioploca. In: Dw orkin M, F alkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) Proteobacteria: gamma subclass. The prokaryotes. A handbook on the biology of bacteria, v ol 6, 3rd edn. Springer, New York, pp 784–812 Teske A, Brinkhof f T , Muyzer G, Moser DP , Rethmeier J, Jannasch HW (2000) Di versity of thiosulfate-oxidizing bacteria form marine sediments and h ydrothermal v ents. Appl En viron Microbiol 66:3125–3133 Timmer-Ten Hoor A (1975) A ne w type of thiosulphate oxidizing, nitrate reducing microor ganisms: Thiomocrospira denitrificans sp. nov. Neth J Sea Res 9:344–350 Tivey MK (2004) The remarkable di versity of seafloor vents. Oceanus 42:60–65 Urakawa H, Dubilier N, Fujiwara Y, Cunningham DE, Kojima S, Stahl DA (2005) Hydrothermal vent gastropods from the same family (Provannidae) harbor epsilon and gammaproteobacterial endosymbionts. Environ Microbiol 7:750–754 Wilcock WSD, DeLong EF, Kelley DS, Baross JA, Cary SC (eds) (2004) The subseafloor biosphere at mid-ocean ridges. Geophysics monographs 144. American Geological Union, Washington Wirsen (2004) Is life thriving deep beneath the seafloor? Oceanus 42:72–77 Wirsen CO, Brinkhof f T , K uever J, Muyzer G, Molyneaux S, Jannasch HW (1998) A ne w Thiomicrospira strain from the Mid-Atlantic Ridge compared to kno wn hydrothermal v ent isolates. Appl Environ Microbiol 64:4057–4059 Wirsen CO, Sievert SM, Cavanaugh CM, Molyneaux SJ, Ahmad A, Taylor LT, DeLong EF, Taylor CD (2002) Characterization of an autotrophic sulf ide-oxidizing marine Arcobacter sp. that produces filamentous sulfur. Appl Environ Microbiol 68:316–325 Xu J (2006) Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances. Mol Ecol 15:1713–1731

Chapter 20

Speciation Analysis of Microbiologically Produced Sulfur by X-ray Absorption Near Edge Structure Spectroscopy Alexander Prange

Abstract The first part of this chapter presents a basic and brief introduction to X-ray absorption spectroscop y with special re gard to its application in microbiology and its great adv antages as an in situ method for speciation analysis. The second part summarizes X-ray absorption near edge structure spectroscopic investigations of microbiologically produced sulfur. Two examples are presented in more detail, the speciation of sulfur in sulfur glob ules of phototrophic and chemotrophic sulfur bacteria and the speciation of elemental sulfur tak en up by Allochromatium vinosum.

20.1 Intr oduction Over the last three decades X-ray absorption spectroscop y (XAS) has de veloped as an incisi ve probe of the local structure around selected atomic species in solids, liquids, and g ases. F oremost among its strengths are its applicability to amorphous materials and its “tunability”, which means the ability to probe the environments of dif ferent elements in a sample by selecting a suitable incident X-ray energy. The amount of information a vailable from a single XAS spectrum is relatively small compared with that a vailable from X-ray dif fraction; however, the information obtained from a well-chosen experiment can be particularly incisive and in some cases inaccessible by any other technique. The purpose of this chapter is to provide (for nonphysicists) a basic and brief introduction to XAS as it is been traditionally practiced. It is written from the point of view of a user of this technique and focuses on the information which is of interest and should be mentioned when “just thinking about using XAS to in vestigate (micro-)biological samples” as a nonphysicist (the author is a microbiologist with special interest in biophysical methods and using XAS). Furthermore, an o verview and some e xamples of the

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application of XAS for investigating sulfur speciation analysis1 in microbiological systems are given.

20.2 XAS: X-ray Absorption Near-Edge Structure and Extended X-ray Absorption Fine Structure An XAS spectrum is typically di vided into tw o re gions: the X-ray absorption near-edge structure (XANES) and e xtended X-ray absorption f ine structure (EXAFS). Although both re gions have in principle the same physical origin, the distinction is con venient for the interpretation of the results obtained by this spectroscopy. The XANES re gion is strongly sensiti ve to formal oxidation state (effective charge), electronegativity of neighboring atoms, and coordination chemistry (e.g., octahedral, tetrahedral coordination) of an absorbing atom (f ine structure about 10 eV below and up to about 50–100 eV above the absorption edge), while the EXAFS re gion can be used to determine the radial distances, coordination number, and neighbors species of an absorbing atom (fine structure from about 100 to 1,000 eV above the absorption edge). More detailed insight into XAS (including technical details) can be found in the excellent books by Teo (1986), Stoehr (1996), and Koningsberger and Prins (1988). F or more information on the application of XAS in biology and applied sciences and an elementary introduction to XAS, the reader is referred to Behrends (1992a, b), Prange and Modro w (2002), and Prange et al. (2007). For the latest developments and an overview of scientific questions of current interest, the conference proceedings of the X-ray absorption f ine structure conferences (XAFS 12 2005; XAFS 13 2007) are recommended.

20.2.1 Experimental Synchrotron light with a continuous spectrum from infrared light with ener gy of approximately 0.03 keV to hard X-rays with ener gy of approximately 500 keV or less is obtained when a relati vistic electron is accelerated vertically to its direction of mo vement. In synchrotron radiation (light) sources of dif ferent generations (in Germany ANKA, Karlsruhe, BESSY , Berlin, DEL TA, Dortmund, ELSA, Bonn, HASYLAB, Hamburg; worldwide about 50 storage rings dedicated to basic and applied research using synchrotron radiation are in operation; lightsources.or g 2006), highly relativistic electrons are “stored” (in high v acuum) to travel along a 1 The term “speciation” has often been used in the literature in dif ferent ways. In this chapter, the terms “speciation” and “speciation analysis” and “chemical species, ” respecti vely, are used according to IUP AC def initions. Speciation analysis: analytical acti vities of identifying and/or measuring the quantities of one or more indi vidual chemical species in a sample. Chemical species: specific form of an element def ined as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure (Templeton et al. 2000).

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circular path for many hours at about 99.9999985% of the speed of light (tra veling approximately 300,000 km s −1) and emitting synchrotron radiation tangentially . In simple XAS e xperiments, e.g., for in vestigating sulfur speciation at the sulfur K-edge, polychromatic X-rays are produced by a synchrotron radiation source and then monochromatized by dif fraction from a double-crystal monochromator (Lemonnier et al. 1978). A schematic sk etch of an XAS e xperiment (transmission and fluorescence modes, see later) is displayed in Fig. 20.1; in Fig. 20.2 an authentic XASexperiment

Fig. 20.1 Experimental setup for X-ray absorption spectroscopy measurements A in transmission mode and B in fluorescence mode. In transmission mode, fluorescence photons are also present, but they are not drawn for reasons of clarity. Furthermore, in fluorescence mode, the beam is also transmitted through the sample, b ut is also not dra wn, nor are the free electrons that occur when the beam hits the sample (electron yield mode)

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Fig. 20.2 Experimental setup of the beamline BN3 for X-ray absorption near edge structure (XANES) spectroscopy measurements in transmission mode: A sample holder, B chamber for the sample, C manual valve, D first ionization chamber (measuring I0), E second ionization chamber (measuring I1), F first electrometer (displays I0), G second electrometer (displays I1), H wall of lead cuboids (radiation protection), and I barometer (measuring the pressure inside the chambers). The monochromator (not shown) is located behind the w all of lead cuboids

(beamline BN3, synchrotron laboratory at ELSA; Physikalisches Institut Uni versität Bonn 2007) is shown with a transmission mode setup. Only the X-ray photons hitting the first crystal that are of the correct wavelength will be redirected to the second crystal, whereas the others are absorbed [Si(111) or InSb(111) crystals are usually used for probing sulfur]. The parallel second crystal is used as a mirror mainly to restore the beam to its original direction. The monochromatic X-rays then pass through the sample, which should be prepared in such a w ay that it absorbs approximately half of the incident X-rays. An X-ray absorption spectrum can be measured in three dif ferent modes: in transmission mode, in fluorescence mode, or in electron yield mode. In transmiss ion mode, the intensity of the X-ray beam is measured before ( I0) (incident beam) and after passing through the sample ( I1) (transmitted beam) using (gas) f illed ionization chambers (detectors), measuring the “missing photons”. In this case, the number of X-ray photons absorbed by core electrons to create a photoelectron (and a “ hole” in the shell) is determined. The XAS spectrum then sho ws the v ariation of log ( I0/I1) versus the energy (electronvolts). The detection limit is matrix (sample) and elem ent concentration dependent and can be estimated with 1% of tar get element within a surrounding matrix, e ven for lo w-Z elements lik e sulfur. In fluorescence mode, the fluorescence ( If) from the sample matrix, which is as a secondary ef fect of the

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Fig. 20.3 Sulfur K-edge XANES spectra of sulfur globules of Allochromatium vinosum measured in transmission mode ( black line ) and in fluorescence mode ( gray line ) at the DCM beamline, Center for Adv anced Microstructures and De vices, Baton Rouge, LA, USA (Prange 2002). A. vinosum was grown photoorganoheterotrophically on malate, then sulfide solution was added, and formation of sulfur globules started. Spectra were recorded 2 h after sulfide addition and are nor malized at 2,510 eV

absorption of X-rays, is determined using a fluorescence detector . In this case, the number of fluorescence photons emitted from the sample, when an electron in the upper le vel f ills the “hole” in the core le vel, is determined. The resulting XAS spectrum shows the variation of If/I0 versus the energy (Stoehr 1996). The fluorescence mode is typically used when v ery small concentrations of a tar get element are present and in favorable cases measurements can be performed even below the parts per million range. Both modes, transmission and fluorescence, provide – as one can expect – comparable results, when the samples are prepared correctly (Sect. 20.2.3). As an e xample, Fig. 20.3 illustrates sulfur K-edge XANES spectra of the purple sulfur bacterium Allochromatium vinosum (with sulfur globules) measured in both modes (for details on XANES spectroscop y of bacterial sulfur glob ules, see Sects. 20.3.1, 20.3.2; Prange et al. 1999, 2002a, b; Franz et al. 2007) The third possibility to measure an XAS spectrum, the electron yield mode (detection of electrons created when the synchrotron light hits an element), has – to the best of author’ s kno wledge – ne ver been used to probe microbiologically produced sulfur. Therefore, this mode is not discussed in this chapter (for an elementary introduction to the electron yield mode, see Prange et al. 2007).

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Advantages of XANES Spectroscopy

For speciation analysis of an element of interest in a (micro-)biological sample, XANES spectroscopy is the technique of first choice. The main advantages, from a “biological point of view” are as follows. Nearly all elements of interest (Z ≥ 6) can be in vestigated separately , independent of the other surrounding elements within a matrix. Only a relatively small amount of sample is necessary, normally a few hundred milligrams is adequate. The method is (almost) nondestructive (as an approximation, starting from 10 23 particles, the number of particles in 1 mole, in a sample and from approximately 10 10 photons, which can hit the sample within 1 s, and a measurement duration of 10 min, the fraction of “hit” atoms can be estimated to be only 10 −10). As X-rays ha ve a high penetration strength, measuring a sample in a v acuum is normally not necessary. Besides solid materials it is possible to in vestigate noncrystalline materials lik e liquids and gases, and measurements with high spatial resolution can be performed as well as measurements of specifically resolved (e.g., chemical) reactions. First results, e.g., propositions on v alencies, can be easily and directly obtained when using the “fingerprint approach,” i.e., the comparison of the XANES spectrum of the sample with those of suitable reference compounds. Considering all these statements, it becomes clear that in situ measurements are possible, which is the most important advantage (for more information, see Prange and Modro w 2002).

20.2.3 Sample

Preparation

The preparation of samples in general and of biological samples in particular is a crucial step. Especially homogeneity , thickness, and concentration of the tar get element play a k ey role. A “perfect sample” is thin and homogeneous, has a lo w, but not too lo w concentration of the tar get element and is easy to handle, i.e., a powder. In general, for all types of samples and the mode of measurement, the preparation has to be optimized to avoid effects which lead to distorted spectra and thus false results. All three modes of measuring a XANES spectrum imply pitfalls, which should be avoided: for transmission mode these are pinhole and/or thickness effects, for fluorescence mode self-absorption ef fects, and for electron yield mode accumulation of char ge on the sample. Therefore, only a fe w general remarks are given related to preparation of samples for XANES measurements in transmission and fluorescence modes. Measuring samples in transmission mode, one has to avoid possible pinhole and/or thickness ef fects (Parrat et al. 1957; Stern and Kim 1981). Especially when measuring po wder samples the risk of artif acts is high at soft X-ray energies (e.g., for sulfur) owing to pinhole effects. Therefore, in addition to requiring the right thickness (the X-rays must be able to pass through the sample!), the sample must be uniformly and homogeneously prepared, and free of pinholes. If these conditions can be met, which is sometimes not so easy ,

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measurements in transmission mode are simple to perform and yield excellent data. For samples with v ery low concentration of the tar get element (parts per million level and lower), fluorescence mode is preferred. However, the key problem related to this mode is self-absorption. Detailed considerations concerning the measurement modes were recently published (Geor ge et al. 2002; Prange et al. 2002c) for the case of measuring sulfur K-edge XANES spectra of bacterial sulfur glob ules and elemental sulfur, and the reader is referred to the detailed information presented on the advantages and disadvantages of the different modes by these authors.

20.2.4

Quantitative Analysis of XANES Spectra

The interpretation of XANES is complicated by the f act that there is not a simple analytically exact description of XANES; however, there is much chemical information, notably formal v alency and coordination en vironment, a vailable from XANES measurements. F or e xample, the chemical shifts of the so-called white line (f irst strong maximum in a XANES spectrum) can be used as a “ruler” to determine the valency of sulfur in an unknown compound, just by using the energy position of the white line (Sect. 3 in Prange et al. 2007; Fig. 20.4). XANES analysis is often based on linear combinations of known spectra from “reference compounds” (examples of XANES spectra of dif ferent reference compounds are sho wn in Fig. 20.4), which can provide ratios of valency states and/or phases, the so-called quantitative analysis. The fact that the local en vironment of the absorbing atoms is probed implies that XANES spectra are additive, i.e., the spectrum of a mixture of substances A and B can be composed pf the separately measured spectra of A and B, respecti vely. This

Fig. 20.4 Sulfur K-edge XANES spectra of dif ferent reference compounds: a cyclo-octasulfur, b polymeric sulfur, c methionine sulfone, d cysteic acid, and e zinc sulfate. Spectra are normalized at 2,510 eV. The typical energy positions of the white lines for dif ferent sulfur species are given

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additivity is the basis for the “quantitati ve analysis” of XANES spectra, which means the decomposition of a sum spectrum into the components of which it is composed. To achie ve this decomposition, a “quality function” def ined by the difference between experimental data and a linear combination of spectra contained in a basis set can be minimized (Modro w et al. 2001; Prange et al. 2002a, 2003). More sophisticated linear algebra techniques such as f actor analysis can also be (and are) applied to XANES spectra. Ho wever, ab initio calculations of all spectral features of a spectrum of a real sample are still difficult to perform and are not always reliable. This situation is improving, but at this point a fully quantitative treatment of XANES using ab initio calculations is rarely a vailable (Rehr and Ankudinov 2001).

20.3 Sulfur K-Edge XANES Spectroscopy and Speciation of Microbiologically Produced Sulfur Although XANES spectroscop y has become a more or less routine technique in physics, chemistry, environmental sciences, and geology, only some studies – apart from EXAFS investigations on metalloproteins originating from microor ganisms – have been performed in the f ield of microbiology in general and only v ery few studies of sulfur speciation (and of other lo w-Z elements) in particular. For more information on sulfur K-edge XANES spectroscopy, a detailed section is given in the article by Prange et al. (2007) highlighting the possibilities and limits when in vestigating dif ferent sulfur compounds. In general, when re garding the interests of a microbiologist w orking with sulfur bacteria, XANES spectroscop y can determine in situ the v alency and v alencies, respecti vely, of sulfur (and of course other elements) within a sample (Fig. 20.5). It can distinguish between different modif ications of sulfur with one formal v alency, e.g., sulfur chains of different lengths (up to four sulfur atoms) (Chauvistré et al. 1997) and between

Fig. 20.5 Sulfur K-edge XANES spectrum of “sulf ide feeding solution,” prepared according to Siefert and Pfennig (1984). The spectrum is normalized at 2,510 eV

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polymeric sulfur and sulfur rings of dif ferent ring sizes (Prange et al. 1999; Franz et al. 2007). It also yields information on/determines atoms bound to the sulfur atom in the second and the third coordination shell as well as the bond itself (Chauvistré et al. 1997; Prange et al. 2007). Furthermore, it can – if one has the “correct” or at least v ery similar reference samples – determine the quantitati ve speciation (Sect. 20.2.4). Before presenting our current knowledge of speciation of microbiologically produced sulfur obtained by XANES spectroscop y, the sulfur K-edge XANES spectrum of a typical “feeding sulfide solution” for sulfur bacteria (Siefert and Pfennig 1984) shown in Fig. 20.5 should be considered. This e xample illustrates the great potential of XANES spectroscopy to elucidate the sulfur speciation, which might be of great interest for microbiologists working with and feeding sulfur bacteria in the laboratory. The spectrum reveals at least five dominant sulfur species (sulfide, S0/disulfide, sulfoxide, sulfone, sulfonate; cf., Fig. 20.4) present in the solution instead of only one species, namely, sulfide, which might be e xpected in such a solution (Prange 2002). However, it has to be k ept in mind that this is qualitati ve information. F or relative percentages of single sulfur species contributing to the spectrum, a quantitative analysis (Sect. 20.2.4) must be performed. Table 20.1 summarizes the studies performed so f ar on sulfur bacteria and microbiologically produced sulfur by using XANES spectroscop y; two examples are discussed in the following sections.

20.3.1 Speciation of Sulfur in Sulfur Glob ules of Phototrophic and Chemotrophic Sulfur Bacteria The most detailed studies using sulfur K-edge XANES spectroscop y ha ve been performed on sulfur in the sulfur globules of the purple sulfur bacterium A. vinosum (Table 20.1). Furthermore, the sulfur in globules or granules of some other phototrophic and chemotrophic sulfur bacteria has been in vestigated in detail (Prange et al. 2002a; Table 20.1). A detailed description of investigations of bacterial sulfur globules, including a historical outline from the early be ginning in the nineteenth century, is given in Dahl and Prange (2006). XANES spectroscopy revealed at least three different sulfur speciations in bacterial sulfur globules, reflecting the different ecological and physiological properties of dif ferent metabolic groups of bacteria: cyclo-octasulfur dominates in the sulfur glob ules of Beggiatoa alba and Thiomargarita namibiensis. In the chemotrophic sulfur bacterium Acidithiobacillus ferrooxidans (gro wn at pH 2) sulfur occurs predominantly as polythionates. In sulfur globules of purple and green sulfur bacteria, the stored sulfur mainly consists of sulfur chains, most probably terminated by an organic group at one or both ends (mono-organylsulfanes/bisorganylsulfanes) (Prange et al. 2002a). Here, the in vestigation of sulfur glob ules in intact cells of A. vinosum v ersus isolated sulfur glob ules is briefly presented, as this is an e xcellent e xample to

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Table 20.1 Overview of investigations characterizing microbiologically produced sulfur by X-ray absorption near edge structure spectroscopy Microbiologically Microorganism produced sulfur References Allochromatium vinosum DSMZ 180T

Thiocapsa roseopersicina DSMZ 219, Marichromatium purpuratum DSMZ 1591 T Amoebobacter purpureus Chlorobaculum parvuma DSMZ 263 Chlorobaculum tepidumb Chloroflexus aurantiacus

Halorhodospira halophila DSMZ 244T, Halorhodospira abdelmalekii DSMZ 2110 Beggiatoa alba DSMZ 1416 Marine Beggiatoa Thiomargarita namibiensis Acidithiobacillus ferrooxidans DSMZ 584 “Candidatus Acrobacter sulfidicus”

Speciation of sulfur in intercellulary stored sulfur globules Elemental sulfur uptake Speciation of sulfur in intercellulary stored sulfur globules Speciation of sulfur in extracellulary stored sulfur globules Speciation of sulfur in extracellulary stored sulfur globules Speciation of sulfur in extracellulary stored sulfur globules Speciation of sulfur stored intracellularly Speciation of sulfur stored intracellularly Speciation of sulfur stored intracellularly Sulfur filaments

Acidianus ambivalens

Sulfur filaments

Epsilonproteobacteria

Sulfur oxidation states in bacterial filaments (micro-XANES) Sulfur speciation in filaments (natural samples) Sulfur filaments

Epsilonproteobacteria and Gammaproteobacteria Thermoanaerobacter sulfurigignens Phomopsis viticola DSMZ 1789 Oscillatoria limnetica

S0 formed by this fungus Intracellularly deposited sulfur granules

XANES X-ray absorption near-edge structure. Formerly Chlorobium tepidum. b Formerly Chlorobium vibrioforme (f. thiosulfatophilum). a

Prange et al. (1999, 2002a–c), Pickering et al. (2001), George et al. (2002), Dahl and Prange (2006) Franz et al. (2007) Prange et al. (1999, 2002a) Pickering et al. (2001) Prange et al. (1999, 2002a) Pickering et al. (2001) Pickering et al. (2001) Prange et al. (1999, 2002a) Prange et al. (2002a) Pickering et al. (2001) Prange et al. (2002a) Prange et al. (2002a) Prange (2002), A. Prange and S.M. Sievert (unpublished data) A. Prange, A. Kletzin, H. Lichtenberg and J. Hormes (unpublished data) Lopez-Garcia et al. (2003) Engel et al. (2007) Prange (2002), Lee et al. (2007a, b) A. Prange (unpublished data) A. Prange and J. Rethmeier (unpublished data)

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clearly point out the necessity to use a nondestructi ve and in situ method lik e XANES spectroscopy. Measurements of isolated (isolated under aerobic conditions) sulfur globules from anaerobically gro wn A. vinosum showed completely dif ferent spectra from those of intact cells (also prepared under aerobic conditions) (Prange et al. 2002a). Quantitati ve analysis of these spectra with suitable reference compounds (Sect. 20.2.4) sho wed that sulfur is predominantly present as cyclo-octasulfur and minor as sulfate. This is in contrast to the chain sulfur structure for sulfur globules of intact cells of A. vinosum (about 80% sulfur chains; about 20% C–S–H/C–S–S–C). During extraction of the globules from the cells, the integrity of the cells w as destro yed and, therefore, the sulfur w as directly e xposed to oxygen from the air, probably leading to changes in the chemical speciation.

20.3.2 Speciation of “Elemental Sulfur” Taken Up by A. vinosum Elemental sulfur (S 0) has the formal v alency of zero; ho wever, elemental sulfur tends to catenate and to form chains with v arious lengths (S ∞ or S µ) and ring sizes (Sn) (Steudel 2000; Steudel and Eckert 2003). All sulfur allotropes are hydrophobic, not wetted by w ater, and they hardly dissolve in water. They can be investigated by XANES spectroscop y and distinguished according to dif ferences in the spectral features (Prange et al. 1999). The thermodynamically most stable form o f elemental sulfur at ambient temperature and pressure is c yclic, orthorhombic asulfur (a-S8) (cyclo-octasulfur or S8 rings) (Roy and Trudinger 1970). At 20°C pure a-S8 has a green-yello w color, turning to white after cooling to −80°C (Steudel 1996). In contrast, the customary commercial typical elemental sulfur (“flo wers of sulfur”) remains yello w after cooling to −80°C (Steudel 1996). It mainly consists of S8 rings, some polymeric sulfur chains, and traces of S 7 rings which are responsible for the yello w color (Steudel and Holz 1988). Elemental sulfur sublimed at ambient pressure (flo wers of sulfur) al ways contains some polymeric sulfur (Steudel and Eckert 2003). Polymeric sulfur, which is frequently used in the rubber industry for vulcanization of natural and synthetic rubbers, consists of chainlik e macromolecules. The bonding ener gy between S–S bonds in polymeric sulfur , however, is relatively weak (2.4 kJ mol −1 weaker than in cyclo-octasulfur) (Steudel et al. 1985; Steudel 1996; Steudel and Eck ert 2003); therefore, chainlik e sulfur (polymeric sulfur), might be the more easily accessible species of elemental sulfur for microorganisms. This hypothesis gains some support from the recent study of Urich et al. (2006), who in vestigated the influence of dif ferent sulfur species on enzyme functions in the sulfur oxygenase-reductase from Aquifex aeolicus . Theoretical considerations on the basis of the crystal structure of this enzyme led to the hypothesis that linear sulfur b ut not c yclic sulfur species can serv e as a substrate for this enzyme (see Chap. 15 by Kletzin). Following this hypothesis, Franz et al. (2007) investigated by XANES spectroscopy whether only one sulfur species is used or at least preferred when A. vinosum takes up elemental sulfur (flowers of sulfur) and forms globules. A. vinosum took up only

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a part of the elemental sulfur added to the medium, formed sulfur glob ules, and oxidized them to sulfate. Some sulfur remained as sulfur platelets in the medium. The sulfur used and oxidized by A. vinosum was quantified via the sulf ate formed and compared with the contents of polymeric sulfur and cyclo-octasulfur, respectively, determined by XANES spectroscop y. It w as shown that A. vinosum uses only the polymeric sulfur (sulfur chain) fraction of elemental sulfur and is probably unable to take up and form sulfur globules from S8 rings. Probably, the speciation of elemental sulfur plays a key role in bacterial sulfur oxidation in a more general w ay and Franz et al. (2007) hypothesize that sulfur chains are also the “microbiologically preferred form” when elemental sulfur is tak en up by other microorganisms. Acknowledgements. The results presented were partly g ained in collaboration with dif ferent colleagues, who are gratefully ackno wledged: J. Hormes and H. Modro w and the SyLi group (Institute of Physics, University of Bonn); the X-ray spectroscopy group of the Center for Advanced Microstructures and De vices (Louisiana State Uni versity, Baton Rouge); C. Dahl, B. Franz, and H.G. T rüper (Institute for Microbiology & Biotechnology , Uni versity of Bonn). J. Hormes is thanked for many helpful discussions and for critical reading of the manuscript.

References Behrens P (1992a) X-ray absorption spectroscop y in chemistry . II. X-ray absorption near edge structure. Trends Anal Chem 11:237–244 Behrens P (1992b) X-ray absorption spectroscopy in chemistry. I. Extended X-ray absorption fine structure. Trends Anal Chem 11:218–222 Chauvistré R, Hormes J, Hartmann E, Etzenbach N, Hosch R, Hahn J (1997) Sulfur K-shell photoabsorption spectroscopy of the sulfanes R-Sn-R, n=2–4. Chem Phys 223:293–302 Dahl C, Prange A (2006) Bacterial sulfur glob ules: occurrence, structure and metabolism. In: Shi vely M (ed) Inclusions in prokaryotes. Microbiology monographs. Springer , Berlin, pp 21–51 Engel AS, Lichtenberg H, Prange A, Hormes J (2007) Speciation of sulfur from naturally-occuring, filamentous microbial mats from sulfidic cave springs using X-ray absorption near edge spectroscopy. FEMS Microbiol Lett 269:54–62 Franz B, Lichtenber g H, Hormes J, Modro w H, Dahl C, Prange A (2007) Utilization of solid ‘elemental’ sulfur by the phototrophic purple sulfur bacterium Allochromatium vinosum: a sulfur K-edge X-ray absorption spectroscop y study. Microbiology 153:1268–1274 George GN, Pick ering IJ, Yu EY, Prince RC (2002) X-ray absorption spectroscop y of bacterial sulfur globules. Microbiology 148:2267–2268 Koningsberger DC, Prins R (eds) (1988) X-ray absorption: principles, applications, techniques of EXAFS, SEXAFS and XANES. Wiley, New York Lee Y -J, Dashti M, Prange A, Raine y F A, Rohde M, Whitman WB, W iegel J (2007a) Thermoanaerobacter sulfurigignens sp. nov., a novel anaerobic thermophilic bacterium reducing 1 M thiosulf ate to elemental sulfur and tolerating 90 mM sulf ite. Int J Syst Ev ol Microbiol 57:1429–1434 Lee Y-L, Prange, A, Lichtenber g H, Rohde M, Dashti M, W iegel J (2007) In situ speciation of sulfur glob ules from thiosulf ate reduction by Thermoanaerobacter sulfur gignens and Thermoanaerobacterium thermosulfurigenes. J Bacteriol ( in Press) Lemonnier M, Collet O, Depaute x C, Esteva JM, Raoux D (1978) High v acuum two crystal soft X-ray monochromator. Nucl Instrum Methods Phy Res Sect A 152:109–111

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lightsources.org (2006) lightsources.org home. http://www.lightsources.org Lopez-Garcia P, Duperron S, Philippot P, Foriel J, Susini J, Moreira D (2003) Bacterial di versity in hydrothermal sediment and epsilonbacterial dominance in e xperimental microcolonizers at the Mid-Atlantic Ridge. Environ Microbiol 5:961–976 Modrow H, Visel F, Zimmer R, Hormes J (2001) Monitoring thermal oxidation of sulfur crosslinks in SBR-elastomers by quantitati ve analysis of sulfur K-edge XANES-spectra. Rubber Chem Technol 74:281–294 Parrat LG, Hemstead CF, Jossem EL (1957) “Thickness effect” in absorption spectra near absorption edges. Phys Rev 105:3781–3787 Pickering IJ, George GN, Yu EY, Brune DC, Tuschak C, Overmann J, Beatty JT, Prince RC (2001) Analysis of sulfur biochemistry of sulfur bacteria using X-ray absorption spectroscop y. Biochemistry 40:8138–8145 Prange A (2002) Molekular genetische und ph ysikalisch-chemische Untersuchungen an den Schwefelkugeln photo- und chemotropher Schwefelbakterien. Verlag dissertation.de, Berlin Prange A, Modro w H (2002) X-ray absorption spectroscop y and its application in biological, agricultural and environmental research. Rev Environ Sci Biotechnol 1:259–276 Prange A, Arzber ger I, Engemann C, Modro w H, Schumann O, T rüper HG, Steudel R, Dahl C, Hormes J (1999) In situ analysis of sulfur in the sulfur globules of phototrophic sulfur bacteria by X-ray absorption near edge spectroscop y. Biochim Biophys Acta 1428:446–454 Prange A, Chauvistré R, Modro w H, Hormes J, T rüper HG, Dahl C (2002a) Quantitati ve speciation of sulfur in bacterial sulfur glob ules: X-ray absorption spectroscopy reveals at least three different species of sulfur. Microbiology 148:267–276 Prange A, Dahl C, Trüper HG, Behnke M, Hahn J, Modrow H, Hormes J (2002b) Investigation of S-H bonds in biologically important compounds by sulfur K-edge X-ray absorption spectroscopy. Eur Phys J D 20:589–596 Prange A, Dahl C, T rüper HG, Chauvistré R, Modro w H, Hormes J (2002c) X-ray absorption spectroscopy of bacterial sulfur globules: a detailed reply. Microbiology 148:2268–2270 Prange A, Birzele B, Krämer J, Modro w H, Chauvistré R, Hormes J, Köhler P (2003) Characterization of sulfur speciation in low molecular weight subunits of glutenin after reoxidation with potassium iodate and potassium bromate at dif ferent pH v alues using X-ray absorption near-edge structure (XANES) spectroscopy. J Agric Food Chem 51:7431–7438 Prange A, Hormes J, Modrow H (2007) X-ray absorption spectroscopy as a tool for the detection and identif ication of sulfur compounds in photototrophic or ganisms. In: Hell R, Dahl C, Leustek T, Knaff D (eds) Sulfur metabolism in phototrophic or ganisms. Advances in photosynthesis and respiration. Springer, Berlin (in press) Physikalisches Institut Universität Bonn (2007) Webseite der Bonner Synchrotronstrahlungsgrup pe. http://syli04.physik.uni-bonn.de/ Rehr JJ, Ankudinov AL (2001) Progress and challenges in the theory and interpretation of X-ray spectra. J Synchrotron Radiat 8:61–65 Roy AB, T rudinger PA (1970) The biochemistry of inor ganic compounds of sulfur . Cambridge University Press, London Siefert E, Pfennig N (1984) Convenient method to prepare neutral sulf ide solution for cultivation of phototrophic sulfur bacteria. Arch Microbiol 139:100–101 Stern EA, Kim K (1981) Thickness effect on the extended-X-ray-absorption-fine-structure amplitude. Phys Rev B 23:1228–1232 Steudel R (1996) Das gelbe Element und seine erstaunliche Vielseitigkeit. Chemie in Unserer Zeit 30:226–234 Steudel R (2000) The chemical sulfur c ycle In: Lens P , Hulshof Pol L (eds) En vironmental technologies to treat sulfur pollution. IWA, London, pp 1–31 Steudel R, Holz B (1988) Detection of reacti ve sulfur molecules (S 6, S 7, S 9, S m) in commercial sulfur, in sulfur minerals, and in sulfur metals slo wly cooled to 20°C. Z Naturforsch B 43:581–589 Steudel R, Eckert B (2003) Solid sulfur allotropes. In Steudel R (ed) Elemental sulfur and sulfur-rich compounds I. Topics in current chemistry 230. Springer, Berlin, pp 1–79

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Steudel R, Strauss R, K och L (1985) Quantitati ve HPLC-Analyse und Thermodynamik der Schwefelschmelze. Angew Chem 97:58–59 Stoehr J (1996) NEXAFS spectroscop y. Springer series in surf ace sciences, v ol 25. Springer , Berlin Templeton DM, Ariese F, Cornelis R, Danielsson L-G, Muntau H, v an Leeuwen HP, Łobi΄nski R (2000) Guidelines for terms related to chemical speciation and fractionation of elements. Definitions, structural aspects, and methodological approaches. IUP AC recommendations 2000. Pure Appl Chem 72:1453–1470 Teo BK (1986) EXAFS: basic principles and data analysis. Inor ganic chemistry concepts, v ol 9. Springer, Berlin Urich T, Gomes CM, Kletzin A, Frazão C (2006) X-ray structure of a self-compartimentalizing sulfur cycle metalloenzyme. Science 311:996–1000 XAFS 12 (2005). Proceedings of the 12th international X-ray absorption fine structure conference (XAFS 12), Malmö, Sweden, June 23–27, 2003. Ph ys Scr T115 XAFS 13 (2007). Conference proceedings of the 13th international conference on X-ray absorption fine structure, Stanford, CA, USA, July 9–14, 2006. Am Inst Ph ys Proc Ser (in press)

Chapter 21 Controls on Isotope Fractionation During Dissimilatory Sulfate Reduction Joost Hoek, Donald E. Canfield

Abstract Sulfur isotopes are fractionated during dissimilatory sulf ate reduction; therefore, sulfur isotope studies ha ve been useful in elucidating the role of sulf ate reduction in the sulfur cycle, and in understanding the early evolution of the sulfur metabolism on Earth. Sulfur isotope fractionation during sulf ate reduction occurs when sulfate is transported into the cell and is reduced to sulf ide through a series of reversible reactions operating with dif ferent efficiencies and distinct fractionation factors. The magnitude of fractionation depends on the relationship between: (1) the exchange of sulfate across the cell membrane and (2) the exchange of sulfur between the different internal sulfur pools. Sulf ate exchange and sulfate reduction rates are controlled by different environmental factors that include temperature, and electron donor and sulf ate concentration. Recent considerations of the minor isotopes of sulfur , 33S and 36S, pro vide ne w insights into the controls on biological fractionation of sulfur isotopes. This approach has pro vided a mechanism for distinguishing the relati ve contributions of dif ferent sulfur metabolisms to the sulfur isotope record in modern and ancient environments. While recent experimental and modeling work has improved our understanding of the factors controlling fractionation during sulfate reduction, it has also highlighted several unanswered questions. For example, precise fractionation factors for individual enzymatic reduction steps remain unknown. Furthermore, the reversibility of sulfate transport and the reversibility of sulfite reduction in vivo have never been tested. These factors play critical roles in controlling isotope fractionation during sulf ate reduction and af fect the predictive success of any isotope fractionation model.

21.1 Intr oduction There are four stable isotopes of sulfur , including the major isotopes 32S and 34S, with natural abundances of 95.04 and 4.20%, respecti vely, and the minor isotopes 33 S and 36S, with natural ab undances of 0.749 and 0.0156%, respecti vely. The relative abundances of sulfur isotopes in nature deviate from these values as a result of biological and inor ganic processes that in volve the transformation of sulfur compounds. These isotope fractionation processes have traditionally been grouped 273 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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into se veral mechanistic cate gories that include equilibrium and kinetic processes. The basis for equilibrium fractionation is related to mass-dependent differences in bond ener gies between light and hea vy isotopes. F or tw o chemical species at equilibrium, fractionations result from the minimization of free energy associated with isotope e xchange reactions (Bigeleisen and Mayer 1947; Ure y 1947). Kinetic fractionations on the other hand comprise a much lar ger group of reactions that are characterized by unidirectional reactions and transport. Kinetic fractionations result from mass-dependent dif ferences in vibrational ener gies of the transition state, those of the reactants, and also the reaction path and its relation to the potential ener gy surface that describes the different states (Bigeleisen and Wolfsberg 1958). They have also been described in terms of the relationship between v elocity and kinetic ener gy for isotopically substituted species (Mook 2000). Although fractionations resulting from metabolic (biological) processes are typically considered to result from kinetic fractionations, Johnston et al. (2005a) make a distinction between fractionations resulting from reactions that are intrinsic to indi vidual chemical and physical processes, and multistep metabolic and biological processes, which may include multiple equilibrium and kinetic fractionation effects. The isotopic composition of sulfur in a sample is al ways expressed relative to the major isotope, 32S, with the following d notation:

{(

d 34 S = ⎡⎢ ⎣

3x

S

32

)

S

sample

(

3x

S

32

)

S

standard

}

⎤ − 1 × 1000, ⎥⎦

(21.1)

where 3xS is 33S, 34S, or 36S, and “standard” refers to a reference sample (Cañon Diablo Troilite), which has the well-constrained natural isotope ab undances, mentioned abo ve. Fractionations between tw o sulfur pools are e xpressed e xactly in terms of ε, with units of per mil: e A − B = 1000 × (a A − B − 1) ,

(21.2)

where α(A–B) is the fractionation factor between two different sulfur pools A and B. It has long been observ ed that or ganisms metabolizing sulfur compounds, particularly during dissimilatory sulfate reduction, fractionate sulfur isotopes (Thode et al. 1951). On the basis of these observations, the isotopic composition of sulfur compounds in nature has been used to elucidate the role of microbial metabol isms in the c ycling of sulfur , both in modern en vironments and in ancient en vironments preserved in the geologic record. Because the c ycling of sulfur is in volved in atmospheric oxygen re gulation, sulfur isotopes ha ve also been used to decipher the history of atmospheric oxygen and, consequently , the oxidation state of Earth’s surface environments (Canfield and Teske 1996). This chapter e xpands on previous reviews of the biogeochemistry of sulfur isotopes by Canfield (2001a) and Brüchert (2004) by focusing on recent xe perimental and modeling work, particularly with the inclusion of the minor sulfur isotopes, which has contrib uted significant additional insights into our understanding of the mechanisms of sulfur isotope

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fractionation during dissimilatory sulfate reduction and sulfur compound disproportionation. A consideration of additional research steps that should be taken, as revealed by the recent research ef forts, will be discussed.

21.2 Sulfur Isotope Fractionation During Dissimilatory Sulfate Reduction 21.2.1 Pure

Cultures

The sequential reduction of sulfate to sulfide during dissimilatory sulfate reduction leads to a fractionation of sulfur isotopes. The fractionation of 34S during dissimilatory sulfate reduction by pure cultures has been e xtensively studied, especially for mesophilic Desulfovibrio species (Thode et al. 1951; Harrison and Thode 1958; Kaplan and Rittenberg 1964; Kemp and Thode 1968; Smejkal et al. 1971; Chambers et al. 1975; McCready 1975; Böttcher et al. 1999; Bolliger et al. 2001; Detmers et al. 2001; Habicht et al. 2005; Johnston et al. 2005a; Canfield et al. 2006). Results from these pure-culture studies show wide-ranging fractionations of 34S from 3 to 46‰, with an average around 18‰. While some of this v ariability results from inherent differences between organisms (Bolliger et al. 2001; Brüchert et al. 2001; Detmers et al. 2001; Kleik emper et al. 2004), en vironmental variables such as temperature, electron donor type and concentration, and sulf ate concentration e xert significant control on the magnitude of sulfur isotope fractionation for indi vidual species of sulfate-reducing microorganisms (Harrison and Thode 1958; Kaplan and Rittenberg 1964; Kemp and Thode 1968; Chambers et al. 1975; Habicht et al. 2005; Canf ield et al. 2006). Research documenting the influence of dif ferent environmental variables on fractionation has sho wn that for indi vidual species of sulf ate-reducing microorganisms, the extent of fractionation depends on factors that influence cell specific rates of sulf ate reduction (e xpressed in moles per cell per unit time). Growth temperature, for example, has a significant impact on cell-specific rates of sulfate reduction. It is well known that cell-specific rates of sulfate reduction decrease when sulf ate reducers are gro wn at temperatures belo w their optimal growth temperature. It has generally been observ ed that when an or ganic electron donor is supplied during fractionation e xperiments, the magnitude of fractionation increases with decreasing cell-specif ic sulf ate reduction rates that result from lo wer growth temperatures (Harrison and Thode 1958; Kaplan and Rittenberg 1964; Kemp and Thode 1968; Chambers et al. 1975). Dif ferent temperature-controlled fractionation patterns, ho wever, ha ve also been observ ed. Canfield et al. (2006) conducted fractionation e xperiments using a temperature gradient block where they grew Desulfovibrio desulfuricans on lactate, over the complete range of growth temperatures. They observed a positive trend between sulfate reduction rates and fractionation. Furthermore, the highest fractionations

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were observed at the highest and lowest growth temperatures. Hoek et al. (2006) performed similar temperature-gradient e xperiments with a chemolithoautotrophic and thermophilic sulf ate-reducing bacterium. The y obtained similar fractionation patterns, with the highest fractionations occurring at the lowest and the highest growth temperatures. Cell-specific sulf ate reduction rates are also controlled by electron donor concentration. It is generally observed that fractionations decrease withincreasing sulfate reduction rates resulting from increasing concentrations of both or ganic substrates and hydrogen (Kaplan and Rittenber g 1964; Hoek et al. 2006). Interestingly, when H 2 is used as an electron donor , fractionations are signif icantly reduced when compared with fractionations produced during sulf ate reduction with or ganic compounds. The reasons for reduced fractionations with H2 are unclear , b ut Kaplan and Rittenber g (1964) suggest that with H 2 the reduction of sulfate to sulfite (through adenosine 5′-phosphosulfate, APS) is ratelimiting, allowing only limited expression of the fractionation during subsequent enzymatic reductions downstream from this step. It is important to note, however, that most experiments with H2 as an electron donor have been conducted in batch culture with H 2-saturated headspace. Hoek et al. (2006) measured fractionation during H 2-limited sulf ate reduction. The y found that fractionations increased from approximately 3 to 37‰ when H 2 supply was changed from nonlimiting to limiting gro wth conditions. Their results highlight the importance of electron donor concentrations in controlling the magnitude of isotope fractionations. In addition to lo wer fractionations with gro wth on H 2, suppressed fractionations have also been observed under low sulfate concentrations (below about 200 µM) (Harrison and Thode 1958; Habicht et al. 2002).

21.2.2 Natural

Populations

The direct determination of isotope fractionation during sulf ate reduction for natural populations of sulf ate-reducing bacteria has been e xplored for microbial mats and marine sediments (Habicht and Canfield 1996, 1997, 2001; Canfield et al. 2000; Canfield 2001b). In these experiments, natural populations were incubated at in situ temperatures and amended with a v ariety of organic substrates and a range of sulf ate concentrations. These studies measured maximum fractionations (approximately 45‰) within the same range as is typically observed in pure-culture studies. The lo w fractionations frequently observ ed in pure cultures were not measured in natural populations metabolizing under in situ conditions, and this is attributed to the generally lo wer specif ic rates of sulf ate reduction for natural populations. Canf ield (2001b) correlated the e xtent of fractionation of sulfur isotopes by natural populations of sulf ate-reducing microor ganisms with the concentration of or ganic substrate and the specif ic sulf ate reduction rates, with excess or ganic substrate producing higher specif ic rates of sulf ate reduction and reduced fractionations.

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277

21.3 Stepwise Reduction of Sulfate and Sulfur Isotope Fractionation Models The fractionation of sulfur isotopes during dissimilatory sulf ate reduction results from a series of sequential biochemical reactions that operate at dif ferent efficiencies and with different fractionation factors. The biosynthetic pathw ays and associated fractionation processes of dissimilatory sulf ate reduction ha ve been e xtensively studied (Harrison and Thode 1958; Peck 1961; Kaplan and Rittenberg 1964; Kemp and Thode 1968; Rees 1973). On the basis of the observ ed isotope fractionation trends summarized above, Rees (1973) developed a kinetic model that describes the principal steps in the sulfate reduction process as 1 ATP ↓ 2 e ↓3 ↓4 ⎯⎯ ⎯ → ⎡SO 4 2 − ( in ) ← ⎯⎯⎯ ⎯⎯⎯ → APS ← ⎯⎯⎯ ⎯⎯ → SO32 − ⎤ ⎯e⎯⎯ → H 2 S. (21.3) SO 4 2 − ( out ) ← ⎯ ⎯ ⎣⎢ ⎦⎥ −

−

In this reaction netw ork, sulf ate is acti vely tak en up by the cell together with sodium ions or protons to preserv e char ge balance (step 1). This occurs via membrane-bound transport proteins, and is reversible (Cypionka 1995), allowing exchange of sulfate in and out of the cell. A small isotope fractionation of −3 to 0‰ (eSO4(out)−SO4(in)) is thought to be associated with this step. Once sulfate enters the cell, it is activated with ATP by ATP sulfurylase to form APS (step 2), which is reduced to sulfite (step 3) by APS reductase. Steps 2 and 3 are both considered reversible. No fractionation is e xpected with the acti vation of sulf ate, but a 22– 25‰ isotopic fractionation ( eSO4−SO3) is assigned to APS reduction to sulf ite (Harrison and Thode 1957, 1958). The f inal reduction of sulf ite to hydrogen sulfide along step 4 occurs by the dissimilatory sulfite reductase. Although sulfite reductase enzymes catalyze the oxidation of sulfide to sulfite in oxidative metabolisms (Dahl and T rüper 1994), the re versibility of sulf ite reduction (step 4) in vivo has never been demonstrated (Canfield 2001a). A 25‰ isotope fractionation (eSO −H S) has been ascribed to this step (K emp and Thode 1968; Rees 1973). 3 2 According to the model proposed by Rees (1973), the o verall isotope fractionation e xpressed during sulf ate reduction depends greatly on which steps limit the sulfate reduction process. If sulf ate e xchange across the cell membrane is ratelimiting, then most, if not all, the sulfate entering the cell will be reduced and only minimal fractionation will be expressed. Conversely, fractionation will bemaximized when isotope exchange between the reversible steps is maximized. In most isotope fractionation models, this is best achie ved when the microbial metabolism is suppressed. In this case, all the fractionations associated with the indi vidual steps will be preserv ed and e xpressed in sulf ide that lea ves the cell. Based on these principles, Canfield et al. (2006) constructed a quantitati ve model that can be used to interpret all the observ ed fractionation patterns summarized in Sect. 21.2. The model builds on the reaction network for sulfate reduction originally developed by Rees (1973), and formalized by F arquhar et al. (2003): SO 4 2 − ( out )

1,j1 ,a1 2 ,j 2 ,a 2

⎡ 2− ⎢SO 4 ( in ) ⎣

4a 5a

APS

4 b ,j 4 ,a 4 5 b ,j 5 ,a 5

⎤ 3,j ,a SO32 − ⎥ 3 3 H 2 S. (21.4) ⎦

278

J. Hoek, D.E. Canfield

The numbers designate different steps, ϕ represents mass flow, and a is the fractionation factor associated with each step. Canf ield et al. (2006) used the same fractionation values as those used by Rees (1973). Branching points within the network control mass balance where material flo w has tw o possible paths. The f irst branching point is defined for the transport of sulfate across the cell membrane and a second branching point is def ined for the e xtent to which sulf ite formation is re versible. The mass flow of sulfur at each branch point is described by a set of flux terms, f3 and f5. For the first branch point, f3 = j3/(j3+j2) describes the fraction of sulfur leaving the cell as sulfide. For the second branch point, f5 = j5/(j5 + j3) describes the fraction of sulf ite that is further reduced to sulf ide. Using these terms and isotope mass balance, Canfield et al. (2006) developed a set of equations that describe the influence off3 and f5, and the isotopic composition of internal and e xternal sulfate, on the isotopic composition of sulfide resulting from sulfate reduction. Depending on the e xact relationship between the e xtent to which (1) sulf ate is exchanged across the cell membrane and (2) sulfur exchanges between the internal sulfur pools, Canfield et al. (2006) were able to reproduce all observed fractionation patterns. Although f3 and f5 are not unique for a given fractionation value, generally speaking, there is a much greater range of possible f5 than f3 for any given fractionation value (Hoek et al. 2006; Fig. 21.1). This implies that the e xchange of sulf ate in and out of the cell v aries less, and e xerts a greater influence on the e xtent of fractionation than the e xchange of internal sulfur reservoirs below fractionations of about 20‰ (eSO4(out)−H2S). Brunner and Bernasconi (2005) recently proposed an alternati ve to the “Rees network” summarized above. Their model differs from that developed by Rees (1973) 1 0.9

0‰

0.8 0.7 0.6

10 ‰

f3 0.5 0.4 20 ‰

0.3 0.2

30 ‰

0.1

40 ‰

0 0

0.1

0.2

0.3

0.4

0.5 f5

0.6

0.7

0.8

0.9

1

Fig. 21.1 The range of possible f3 and f5 v alues for gi ven fractionation v alues. Selected (εSO (out)−H S) values of each line are shown. There is only a single possible f3,f5 pair for the extreme 4 2 fractionation values of −3‰ (f3,f5=1,0) and 47‰ (f3,f5=0,0). (From Hoek et al. 2006)

21 Controls on Isotope Fractionation During Dissimilatory Sulfate Reduction

279

in that it incorporates a possible trithionate pathw ay in the reduction of sulf ite, and that sulfite reduction is considered reversible. Additionally, they propose much larger fractionation factors (εSO3−H2S = 58 ‰) during the sulf ite reduction step. The implications of this model are that maximum fractionations of more than 70‰, as may occur in nature, are possible during dissimilatory sulfate reduction. Owing to the complexity of this netw ork, however, it is dif ficult to rigorously test this model using only 34S isotope analyses. This alternative model can, however, be evaluated by incorporating the minor isotopes 33S and 36S in addition to 34S (see below).

21.4 Multiple

Sulfur Isotopes

The minor isotopes of sulfur , 33S and to a lesser de gree 36S, have recently been included in isotope fractionation studies of dissimilatory sulf ate reduction and sulfur-compound disproportionation (Farquhar et al. 2003; Johnston et al. 0205a, b). Only limited w ork has been done with minor sulfu r isotopes owing to their lo w natural abundance making analyses technically dif ficult. Results of these studies provide additional insights into the controls on biological fractionation of sulfur isotopes. Mass-dependent fractionations result from mass dif ferences between the different sulfur isotopes, with 33S fractionating close to half as much (0.515) as 34S and 36 S fractionating about twice as much (1.91) as 34S, compared with 32S. For 33S this mass-dependent relationship is reflected as the well-constrained fractionation array of δ33S∼0.515δ34S that is observed in the geologic record. It is generally recognized, however, that this linear relationship is an approximation deri ved from the po wer law of the isotope fractionation f actors (α):

(

a 33 32 = a 34 32

)

0.515

(21.5)

.

Because of the dependence of isotope fractionations on the natural logarithm of the fractionation f actors, Mook (2000) def ined a f actor to describe mass-dependent fractionations: q = ln

(

33

a AB

) ln (

34

)

(21.6)

a AB .

Similarly, Miller (2002) recommends e xpressing observ ed mass-dependent fractionation in logarithmic form and def ines the factor

( l= ln ⎡⎣(1 + d

) (1 + d 1000 ) (1 + d

ln ⎡⎣ 1 + d 33 SA 1000 34

SA

) )

SB 1000 ⎤⎦ . 34 SB 1000 ⎤⎦ 33

(21.7)

In many cases λ = θ, but because measured v alues of λ represent “net” quantities that can include the fractionation ef fects of different processes with different fractionation factors (e.g. biological networks), and material transfer, λ ≠ θin all cases. Values for 33–34q in mass-dependent fractionations range from 0.500 to 0.516 for

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different types of sulfur isotope fractionation processes (e.g., equilibrium, kinetic, gravitational). Values of 33–34θ produced by equilibrium isotope exchange of sulfur between sulfur species ha ve been theoretically constrained to be near 0.515 for 33– 34 θ, while kinetic processes are more v ariable and f all between 0.500 and 0.515 depending on the nature of the fractionation process producing isotopic fractionations (Farquhar et al. 2003; Johnston et al. 2005a). Because l values depend on the structure of sequential chemical reactions in a reaction netw ork and on material transfer, they can be more variable than values of q for the different steps in a given network. It is the small dif ferences between q and l that pro vide the theoretical framework for modeling mass-dependent fractionations of multiple sulfur isotopes in biological systems. In a recent study , Farquhar et al. (2003) e xplored the isotope fractionations of the four sulfur isotopes during dissimilatory sulf ate reduction. The y developed a theoretical framework that describes the mass flow of sulfur through the metabolic reaction network (see Eq. 21.4) from Rees (1973), using a set of flux terms, f3 and f5. f3 = j3/(j3 + j2) and f5 = j5/(j5 + j3), where j is the mass flow of material along a particular path of the network. f3 indicates the amount of material leaving the cell as hydrogen sulf ide (pathway 3 in Eq. 21.4) and f5 represents the internal backflo w of sulfur (pathw ays 5a and 5b in Eq. 21.4). These flux terms are similar to those used by Canfield et al. (2006) discussed in Sect. 21.3; however, Canfield et al. (2006) def ined f5 as the fraction of sulfur that is further reduced to sulf ide. Therefore, when the f5 def ined by Canf ield et al. (2006) is 1, the f5 from Farquhar is 0. As discussed in Sect. 21.3, the v alues of f3 and f5 depend on dif ferent en vironmental f actors, such as temperature and electron donor and electron acceptor concentrations, as well as on inherent differences between or ganisms, such as the fractionation f actors and acti vity of the different enzymatic reduction steps. Because these factors affect the magnitude and direction of material flo w through the netw ork (quantified by f3 and f5), they directly influence values of l. Farquhar et al. (2003) introduced a flo w net that is contoured by f3 and f5 (Fig. 21.2) to illustrate the dependence of l on the intracellular c ycling of sulfur (f5) and the exchange of sulfate across the cell membrane (f3) during dissimilatory sulfate reduction. The contour at f5 = 0 reflects the case where transport of sulfate into the cell and acti vation of sulf ate to APS is limiting and the contour at f3 = 0 reflects the case where the enzymatic reduction of sulf ite to hydrogen sulf ide is limiting. Fractionation of 33S and 34S by Archaeoglobus fulgidus produced a range of 33–34l a veraging around 0.5117. These v alues are distinct from 33–34l v alues calculated for equilibrium e xchange and v alues observ ed in nature, b ut can be accounted for by the reaction netw ork summarized in Sect. 21.3 (F arquhar et al. 2003). Interestingly, the isotope results for A. fulgidus plot along the contour of f3=0.4 and v ariable f5 for e xperiments which were run with dif ferent sulf ate concentrations, suggesting that f5 responds to changing concentrations of internal sulfate. Furthermore, f5 approached 1 as sulf ate concentrations increased, and f5 decreased as sulf ate concentrations decreased. The implications of these results are that internal enzymatic reaction rates rather than sulf ate exchange across the

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Fig. 21.2 The effect of the dissimilatory sulf ate reduction network (Rees 1973) on multiple isotope fractionations between hydrogen sulfide and sulfate. Fractionation data from several different sulfate reduction e xperiments are plotted along the contours for dif ferent v alues of f3 and f5. (Modified from Johnston et al. 2005a)

cell membrane control the net fractionation. Similar results were obtained by Johnston et al. (2005a) with Desulfovibrio jorgensii grown in batch culture, while the isotopic relationships produced by batch culturexperiments e withDesulfovibrio autotrophicum e volved in a dif ferent w ay. Fractionation patterns from D. autotrophicum follo w the contour of f5 = 0.6 and variable f3 (Fig. 21.2), which suggests that transport of sulfate across the cell membrane varied more than was observed with A. fulgidus and D. jorgensii. Johnston et al. (2005a) suggest that this may result from constantly changing sulf ate concentrations as the gro wth in the batch cultures progressed. Interestingly , one of the data points for D. jorgensenii f alls outside the predicted flo w net de veloped by F arquhar et al. (2003), which suggests that at least one of the fractionation f actors used in the reaction network (Eq. 21.4) is inaccurate. An important consequence from this analysis is that l will be less than q for all values of f other than 0 and 1 if the fractionation f actors are less than 1.00. Conversely, when the fractionation f actors are greater than 1, l will be greater than θ for all v alues of f other than 0 and 1. In more comple x networks where some fractionation factors are greater than 1 and others are less than 1, such as in sulfur-compound disproportionation, l can be greater than or less than q depending on the values of f and the relative magnitude of the different fractionation factors. One of the implications of this treatment is that dif ferent sulfur metabolisms, such as dissimilatory sulf ate reduction and sulfur -compound disproportionation,

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produce resolvable and dif ferent 33S/32S fractionations for similar magnitudes of 34 32 S/ S fractionations (Johnston et al. 2005b). This provides an important tool for distinguishing the relati ve contrib ution to the sulfur c ycle of biological sulf ate reduction and sulfur-compound disproportionation. This application has subsequently been used by Johnston et al. (2005b) to sho w that sulfur -compound disproportionation was an active part of the sulfur cycle by 1,300 million years ago, predating earlier estimates by several hundred million years (Canfield and Teske 1996).

21.5

Conclusions and Future Research

Although the classic literature from the 1950s and 1960s laid the foundation for our understanding of sulfur isotope fractionation during dissimilatory sulf ate reduction, recent e xperimental and modeling w ork has pro vided signif icant advances in our understanding of the controls on fractionation during sulf ate reduction. While early work focused almost exclusively on pure-culture experiments with a fe w Desulfovibrio species, recent w ork has pro vided a broader survey of fractionations imposed during sulfate reduction by pure cultures under a wider range of gro wth conditions. Furthermore, much w ork has also focused on measuring fractionation by natural populations of sulf ate reducers. These experiments ha ve impro ved our understanding of the en vironmental f actors controlling fractionation as well as placing a possible limit on the e xtent of fractionation during sulf ate reduction in nature. Additional critical adv ances in our understanding of isotope fractionation during sulf ate reduction ha ve come from incorporating multiple isotope analyses in fractionation e xperiments. Although technically challenging, results from these studies ha ve pro vided a unique perspecti ve on the f actors controlling fractionation. This w ork has stimulated serious efforts to develop predictive models for the e xtent of isotope fractionation during sulf ate reduction as well as for sulfur -compound disproportionation. These models have already been used to decipher the relative contributions of dissimilatory sulf ate reduction and sulfur -compound disproportionation to the c ycling of sulfur preserved in the geologic record. Despite the critical adv ances made in recent years, the ne w models that ha ve been proposed highlight se veral uncertainties that still remain unanswered. F or example, fractionation f actors for indi vidual enzymatic reduction steps during sulfate reduction ha ve ne ver been precisely measured despite the critical reliance of an y quantitati ve fractionation model on accurate fractionation values. Furthermore, the extent to which sulf ate transport into the cell is re versible and whether or not the dissimilatory sulfite reductase operates in reverse have never been tested. All these factors play a critical role in controlling the e xtent of isotope fractionation during sulf ate reduction and seriously af fect the predicti ve success of any isotope fractionation model.

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References Bigeleisen J, Mayer MG (1947) Calculation of equilibrium constants for isotopic e xchange reactions. J Chem Phys 15:261–267 Bigeleisen J, W olfsberg M (1958) Theoretical and e xperimental aspects of isotope ef fects in chemical kinetics. Adv Chem Phys 1:15–76 Bolliger C, Schroth MH, Bernasconi SM, Kleik emper J, Zeyer J (2001) Sulfur isotope fractionation during microbial sulf ate reduction by toluene-de grading bacteria. Geochim Cosmochim Acta 65:3289–3298 Böttcher ME, Sie vert S, Küv er J (1999) Fractionation of sulfur isotopes during dissimilatory reduction of sulfate at 60°C. Arch Microbiol 172:125–128 Brüchert V (2004) Ph ysiological and ecological aspects of sulfur isotope fractionation during bacterial sulfate reduction. In: Amend JP, Edwards KJ, Lyons TW (eds) Sulfurbiogeochemistry. Special paper 379. Geological Society of America, Boulder , p 205 Brüchert V, Knoblauch C, Jor gensen BB (2001) Controls on stable sulfur isotope fractionation during bacterial sulf ate reduction in Arctic sediments. Geochim Cosmochim Acta 65:763–776 Brunner B, Bernasconi SM (2005) A revised isotope fractionation model for dissimilatory sulfate reduction in sulfate reducing bacteria. Geochim Cosmochim Acta 69:4759–4771 Canfield DE (2001a) Biogeochemistry of sulfur isotopes. In: V alley JW, Cole DR (eds) Stable isotope geochemistry, vol 43. Mineralogical Society of America, Washington, pp 607–636 Canfield DE (2001b) Isotope fractionation by natural populations of sulf ate-reducing bacteria. Geochim Cosmochim Acta 65:1117–1124 Canfield DE, Teske A (1996) Late Proterozoic rise in atmospheric oxygen concentration inferred from phylogenetic and sulphur-isotope studies. Nature 382:127–132 Canfield DE, Habicht KS, Thamdrup B (2000) The Archean sulfur c ycle and the early history of atmospheric oxygen. Science 288:658–661 Canfield DE, Olesen CA, Cox RP (2006) T emperature and its control of isotope fractionation by a sulfate-reducing bacterium. Geochim Cosmochim Acta 70:548–561 Chambers LA, T rudinger PA, Smith JW , Burns MS (1975) Fractionation of sulfur isotopes by continuous cultures of Desulfovibrio desulfuricans. Can J Microbiol 21:1602–1607 Cypionka H (1995) Solute transport and cell ener getics. In: Barton LL (ed) Sulf ate-reducing bacteria. Plenum, New York, pp 151–184 Dahl C, T rüper HG (1994) Enzymes of dissimilatory sulf ide oxidation in phototrophic sulfur bacteria. Methods Enzymol 243:400–421 Detmers J, Bruchert V, Habicht KS, Kuever J (2001) Diversity of sulfur isotope fractionations by sulfate-reducing prokaryotes. Appl Environ Microbiol 67:888–894 Farquhar J, Johnston DT, Wing BW, Habicht KS, Canfield DE, Airieau S, Thiemens MH (2003) Multiple sulphur isotopic interpretations of biosynthetic pathways: implications for biological signatures in the sulphur isotope record. Geobiology 1:27–36 Habicht KS, Canfield DE (1996) Sulphur isotope fractionation in modern microbial mats and the evolution of the sulphur cycle. Nature 382:342–343 Habicht KS, Canfield DE (1997) Sulfur isotope fractionation during bacterial sulfate reduction in organic-rich sediments. Geochim Cosmochim Acta 61:5351–5361 Habicht KS, Canfield DE (2001) Isotope fractionation by sulfate-reducing natural populations and the isotopic composition of sulfide in marine sediments. Geology 29:555–558 Habicht KS, Gade M, Thamdrup B, Berg P, Canfield DE (2002) Calibration of sulfate levels in the Archean ocean. Science 298:2372–2374 Habicht KS, Salling LL, Thamdrup B, Canfield DE (2005) Effect of low sulfate concentrations on lactate oxidation and isotope fractionation during sulfate reduction by Archaeoglobus fulgidus strain Z. Appl Environ Microbiol 71:3770–3777 Harrison AG, Thode HG (1957) The kinetic isotope ef fect in the chemical reduction of sulphate. Trans Faraday Soc 53:1648–1651

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Harrison AG, Thode HG (1958) Mechanism of the bacterial reduction of sulphate from isotope fractionation studies. Trans Faraday Soc 54:84–92 Hoek J, Re ysenbach AL, Habicht KS, Canf ield DE (2006) Ef fect of h ydrogen limitation and temperature on the fractionation of sulfur isotopes by a deep-sea h ydrothermal vent sulfatereducing bacterium. Geochim Cosmochim Acta 70:5831–5841 Johnston DT, F arquhar J, W ing B A, Kaufman A, Canf ield DE, Habicht KS (2005a) Multiple sulfur isotope fractionations in biological systems: a case study with sulf ate reducers and sulfur disproportionators. Am J Sci 305:645–660 Johnston DT, Wing BA, Farquhar J, Kaufman AJ, Strauss H, L yons TW, Kah LC, Canf ield DE (2005b) Acti ve microbial sulfur disproportionation in the Mesoproterozoic. Science 310:1477–1479 Kaplan IR, Rittenber g SC (1964) Microbiological fractionation of sulphur isotopes. J Gen Microbiol 34:195–212 Kemp ALW, Thode HG (1968) Mechanism of bacterial reduction of sulphate and of sulphite from isotope fractionation studies. Geochim Cosmochim Acta 32:71–91 Kleikemper J, Schroth MH, Bernasconi SM, Brunner B, Ze yer J (2004) Sulfur isotope fractionation during gro wth of sulf ate-reducing bacteria on v arious carbon sources. Geochim Cosmochim Acta 68:4891–4904 McCready RGL (1975) Sulfur isotope fractionation by Desulfovibrio and Desulfotomaculum species. Geochim Cosmochim Acta 39:1395–1401 Miller MF (2002) Isotopic fractionation and the quantif ication of O-17 anomalies in the oxygen three-isotope system: an appraisal and geochemical signif icance. Geochim Cosmochim Acta 66:1881–1889 Mook WG (2000) Environmental isotopes in the h ydrological cycle, vol I. Introduction – theory, methods, review. UNESCO/IAEA, Geneva, p 280 Peck HD (1961) Evidence for re versibility of reaction catalyzed by adenosine 5 ′-phosphosulfate reductase. Biochim Biophys Acta 49:621–624 Rees CE (1973) A steady state model for sulphur isotope fractionation in bacterial reduction processes. Geochim Cosmochim Acta 37:1141–1162 Smejkal V, Cook FD, Krouse HR (1971) Studies of sulfur and carbon isotope fractionation with microorganisms isolated from springs of western Canada. Geochim Cosmochim Acta 35:787–800 Thode HG, Kleerekoper H, McElcheran DE (1951) Sulphur isotope fractionation in the bacterial reduction of sulphate. Res Lond 4:581–582 Urey HC (1947) The thermodynamic properties of isotopic substances. J Chem Soc May 562–581

Chapter 22

Bioprocess Engineering of Sulfate Reduction for Environmental Technology Piet N.L. Lens, Roel J.W. Meulepas, Ricardo Sampaio, Marcus Vallero, Giovanni Esposito

Abstract Sulfate reduction can be used in a lar ge number of en vironmental technologies. Methanogenic bioreactors treating or ganic w astewater containing sulfate can be negatively affected by the sulfide produced; however, it is possible to combine methanogenesis and sulf ate reduction when adequate measures are applied. F or the treatment of inor ganic w astewaters containing sulf ate, or ganic substrates or H 2/CO2 are added as electron donors. Alternati vely synthesis gas or methane can be used; ho wever, the sulf ate reduction rates with methane are still extremely low. Heavy metals such as Cu, Zn, Cd, Pb, Ni and Fe can be remo ved from waste streams by precipitation with biogenic sulf ide. Because of dif ferences in solubility products the metals can be selectively precipitated. The insoluble metal sulfides formed can be recovered in order to be reused.

22.1 Intr oduction Several of the microbial conversions of the sulfur cycle can be implemented for pollution control (Table 22.1). This chapter overviews environmental technology applications that utilize the metabolism of sulfate-reducing bacteria (SRB) as the key process. Technological utilization of SRB sounds at f irst somewhat controversial, as sulfate reduction has for many years been considered unwanted, since the production of H 2S causes a multitude of problems, such as toxicity , corrosion, odour, increase of the liquid ef fluent chemical oxygen demand (COD), as well as reduced quality and amount of biogas (Lens et al. 1998a). The emphasis of the research in the 1970–1980s w as therefore mainly on the pre vention or minimization of sulf ate reduction during methanogenic w astewater treatment (Colleran et al. 1995). From the 1990s, interest has gro wn in applying sulf ate reduction for the treatment of specif ic waste streams, e.g. inorganic sulfate rich wastewaters such as acid mine drainage, metal-polluted groundw ater and flue gas scrubbing waters.

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Table 22.1 Overview of applications in environmental biotechnology that mainly utilize conversions from the microbial sulfur cycle Application

Sulfur conversion utilized

Typical waste stream

Wastewater treatment Removal of oxidized sulfurous compounds (sulfate, sulfite and thiosulfate) Sulfide removal Heavy metal removal

S-oxyanion reduction to S2−, followed by sulfideremoval step Partial S2− oxidation to S o SO42− reduction

Industrial wastewaters, acid mine drainage and spent sulfuric acid Industrial wastewaters Extensive treatment in wetlands or anaerobic ponds High-rate reactors for process water, acid mine drainage and ground water Domestic wastewater Textile wastewaters Domestic sewage

Nitrogen removal Removal of xenobiotics Microaerobic treatment Off-gas treatment Biofiltration of gases Treatment of scrubbing waters

Solid-waste treatment Reduction of waste sludge production Desulfurization of resources Bioleaching of metals Gypsum processing Treatment of soils and sediments Bioleaching of metals Phytoextraction Degradation of xenobiotics

S2−, S0 and S 2O32− oxidation SO42− reduction Internal sulfur cycle in a biofim Oxidation of S 2− and organosulfur compounds SO42− and/or SO 32−reduction, plus partial S 2− oxidation to S0 Internal sulfur cycle in a biofim Organo-sulfur oxidation S2− oxidation SO42− reduction S2− oxidation SO42− uptake by plants SO42−reduction

Biogas, malodorous gases from composting and farming Scrubbing waters of SO 2-rich gasses

Sulfur cycle in biofilms Waste rubber, coal, oil, LPG, spent caustic acid Sewage sludge, compost Waste gypsum depots

Dredged sediments and spoils Dredged sediments and spoils PCB-contaminated soil slurries

22.2 Sulfate Reduction in Methanogenic Wastewater Treatment Despite the problems associated with sulf ate reduction in anaerobic w astewater treatment, methanogenic treatment of sulfate-rich wastewater is possible if adequate measures that allo w the inte gration of sulf ate reduction with methanogenesis are applied (Fig. 22.1, Table 22.2). Trends in industries to close w ater cycles lead to the accumulation of salts (including sulfates) and heat in the wastewaters, and thus

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Fig. 22.1 Process configurations integrating methanogenesis with sulf ate reduction and sulf ide removal

Table 22.2 Process technological measures to reduce the reactor sulf ide concentration, thus allowing the integration of methanogenesis and sulfate reduction in anaerobic bioreactors Dilution of the influent Non-sulfate-containing process water Recycle of effluent after a sulfide removal step by sulfide stripping, sulfide precipitation, biological sulfide oxidation to elemental sulfur ( Thiobacillus sp., oxygen; Thiobacillus denitrificans, nitrate; Chlorobium limicola, sunlight), chemical oxidation to elemental sulfur (ferric sulfate/silicone supported reactor) Decrease of the unionized sufide concentration Elevation of the reactor pH Elevation of the reactor temperature Precipitation of sulfide, e.g. with iron salts Stripping of the reactor liquid using high de gree of mixing inside the reactor, recirculation of biogas after scrubbing, other stripping g as (e.g. N 2) Separation of sufide production and methanogenesis Two-stage anaerobic digestion with sulfate-reducing bacteria in the acidifying stage Upflow staged sludge bed with methanogenic bacteria in the bottom and sulf ate-reducing bacteria in the top compartment Selective inhibition of sulfate-reducing bacteria Sulfate analogues (e.g. mobybdate) Transition elements (e.g. copper addition) Antibiotics

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impose the need for the methanogenic treatment of hot and saline w astewaters that also contain moderate (1–3 g l −1) sulfate levels. Several studies ha ve reported the feasibility of thermophilic treatment of sulf ate-rich w astewaters containing low-energy substrates, e.g. a 1:1:1 mixture of acetate–propionate–butyrate at 55°C, acetate at 70°C, methanol at 65 and 70°C, and formate at 75°C (Vallero et al. 2007). For w astewaters rich in unacidif ied or ganic matter , additional precautions are required to cope with the potential reactor acidif ication or deterioration of the granular sludge quality due to the e xcessive growth of acidif iers (Verstraete et al. 1996). One w ay to o vercome these problems is to separate the acidifying and methanogenic activities in phased or staged reactor designs. If sulf ate is present in the w astewater, sulf ate reduction will occur together with acidif ication in the acidification phase (Reis et al. 1995) or in the f irst stages of upflo w staged sludge bed reactors (Lens et al. 1998b). A complete sulf ate reduction in the f irst stage or phase together with high gas (CO2) production rates during acidification may result in high H 2S-stripping efficiencies, and thus in high sulfur -removal efficiencies in the acidifying reactor or compartment. Studies on thermophilic (55°C) granular sludge reactors operated under acidifying (pH 6) conditions sho wed that SRB can coexist with acidif iers during the treatment of a sucrose–propionate–b utyrate mixture (ratio 2:1:1 on a COD basis) with a COD-to-sulf ate ratio of 6.7 (Sipma et al. 2000) or in synthetic cardboard production wastewater with a COD-to-sulfate ratio of 10 (Lens et al. 2001, 2002) at or ganic loading rates up to, respecti vely, 46 and 35 g COD l −1 reactor day −1.

22.3 Sulfate-Reducing 22.3.1 High-Rate

Bioreactors

Sulfate-Reducing Bioreactors

22.3.1.1 Inocula Initially, the e xperience that sulf ate reduction de velops spontaneously during anaerobic w astewater treatment supported the adoption of bioreactor conf igurations commonly used in methanogenic wastewater treatment, i.e. upflow anaerobic sludge bed (UASB) reactors, for high-rate sulf ate reduction bioreactors. In U ASB reactors, sulfidogenic granules can be obtained by feeding methanogenic granular sludge with a sulfate-rich wastewater; however, it can take a very long time before the sulf ate reducers outcompete the methanogens. Using a mathematical model, Omil et al. (1998) sho wed that the competition between acetate-utilizing SRB and methanogenic bacteria (MB) is very time consuming. For a granular sludge with an inoculum size of 10 3 and 10 9 cells of, respecti vely, acetotrophic SRB and MB, it was calculated that it will take over 1,000 days before the sizes of both populations are equal. This time period can be shortened by manipulating the population size of SRB and MB in the inoculum sludge, i.e. deacti vating methanogens or by bioaugmentation with pure cultures of SRB.

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22.3.1.2 Electr on Donor For the treatment of inor ganic wastewaters, the choice of the electron donor is an important design parameter . One can, for e xample, supply an or ganic substrate (e.g. molasses) as the electron donor , although this increases the risk of residual pollutants. F or high-rate sulf ate reduction bioreactors supplied with a H 2/CO2 mixture, high conversion rates can be obtained in mesophilic (30°C; v an Houten et al. 1994) or thermophilic (55°C; v an Houten et al. 1997) gas-lift reactors in a short (10-day) start-up period. In H2/CO2-fed reactor systems, a consortium of SRB (Desulfovibrio sp.) and homoacetogens (Acetobacterium sp.) develops (van Houten et al. 1995). In cases where pure hydrogen gas is not a vailable, one can use synthesis g as (a mixture of H2, CO2 and CO), either directly (van Houten et al. 1997) or after enriching its H 2 content by means of a w ater-gas-shift reaction, either chemically or biologically with anaerobic granular sludge (Sipma et al. 2004). P arshina et al. (2005) isolated Desulfotomaculum carboxydivor ans, the f irst sulf ate reducer capable of hydrogenogenic growth on CO. In the presence of sulf ate, the hydrogen formed is used for sulfate reduction. This organism grows rapidly at 200 kPa CO, pH 7.0 and 55°C, with a generation time of 100 min, producing nearly equimolar amounts of H2 and CO 2 from CO and H 2O. High specific CO conversion rates, e xceeding 0.8 mol CO (g protein) −1 h−1, make it an interesting candidate for a biological alternati ve to the currently employed chemical catalytic water-gas-shift reaction to purify synthesis gas (contains mainly H 2, CO and CO 2). Furthermore, as D. carboxydivorans is capable of hydrogenotrophic sulf ate reduction at partial CO pressures e xceeding 100 kPa, it is also a good candidate for biodesulfurization processes at elevated temperatures, e.g. in biological flue gas desulfurization (Sipma et al. 2006). H2 is currently produced by reforming methane supplied by natural gas or biogas (Fig. 22.2). However, the emission of the greenhouse gas CO 2 and the costs of the wastewater treatment w ould be greatly reduced if methane could be used directly as an electron donor for biological sulf ate reduction (T able 22.3). The f irst clear evidence for anaerobic oxidation of methane (AOM) came from in situ geochemical studies of marine sediments (Krüger 2005). These studies re vealed that methane diffusing upwards from deep sites of sediments often disappears long before an y contact with oxygen is possible. In such anoxic zones of sediments, sulf ate is the only electron acceptor that can account for methane oxidation. A OM was demonstrated by the formation of radiolabelled CO 2 upon injection of [ 14C]methane into anoxic marine sediments. A OM has been detected at temperatures from 4 to o ver 30°C and at dif ferent locations lik e lak es and seashores. Stable isotope analysis showed that archaea and sulfate reducers were both involved in AOM. The main drawback of gas-lift bioreactors is the high pressure drop of the w ater column that needs to be o vercome when supplying the gaseous substrate (H 2). Cell-suspension bioreactors (Lens et al. 2003) or bubbleless H2 supply by hydrophobic membranes (Fedoro vich et al. 2000) might be ele gant alternative reactor designs. Cell-suspension bioreactors also allo w, via the dilution rate, control of the competition between SRB and MB on the basis of their gro wth kinetics (P aulo et al. 2005).

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Fig. 22.2 Wastewater (consisting mainly of ZnSO 4) treatment process at Zinife x (Budel, The Netherlands). Full-scale plant ( a) and flo w sheet of the plant in its present situation and when methane is used as the electron donor ( b)

Alternatively, under thermophilic (55–65°C) conditions, methanol can be supplied as this substrate is con verted to H 2/CO2 at these high temperatures (Vallero et al. 2003). In cases where soluble substrates (such as methanol) are supp lied, no H 2S stripping occurs and therefore H2S removal needs to be adopted to prevent its accumulation in toxic concentrations. This can be done by stripping using an e xternal gas stream (e.g. N 2) or via extractive H2S membranes (De Smul and Verstraete 1999).

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Table 22.3 Comparison of h ydrogen-utilizing sulf ate-reducing process with the process with direct methane utilization Sulfate reduction with H 2 Sulfate reduction with CH 4 via CH4 directly Temperature required

900°C

Pressure required 16 (bar) CH4 required per 1.88 mole of SO 42− (mol) CO2 emission per ton 0.9 of SO42− (t)

Wastewater temperature 1 1 0.45

Table 22.4 Solubility products of metal sulfides and hydroxides logKsp (metal hydroxide) Metal ion logKsp (metal sulfide) Hg(II) −52.4 −52.4 Ag(II) −49.7 −7.71 Cu(I) −48.0, −48.5a – Cu(II) −35.1, −36.2b −20.4, −19.7b a Cd(II) −27.7, −25.8 −14.4 Pb(II) −27.0, −27.5a −15.3 Zn(II) −23.8, −24.7b −16.7, −16.9b Co(II) −21.3 −14.8 Ni(II) −20.7, −19.5b −17.2, −13.8b Fe(II) −17.3 −15.2 Data from Peters et al. (1984) e xcept where indicated. a Data from Smith and Martell (1976). b Data from Brown et al. (1997).

22.3.2

Passive Sulfate-Reducing Systems

Microbial sulf ate reduction is also re garded as an ef fective basic mechanism for treating acid or neutral w aters contaminated with hea vy metals and sulf ate, which might simultaneously remove acidity and metals owing to, respectively, the alkalinity produced during sulf ate reduction and the v ery lo w solubility of metal sulf ides (Table 22.4). Application of high-rate sulfate reduction systems for the treatment of mine waters is hampered by too high in vestment and operating costs. Attempts to overcome these problems have essentially focused on two strategies. Firstly, established industrial technologies can be adapted for the purpose of mine drainage treatment, e.g. by choosing particular lo w-cost substrates such as whe y, methanol or even wastewaters (Rose et al. 1998). The second approach uses SRB in passi ve processes, e.g. constructed wetlands (Gibert et al. 2004; Mark ewitz et al. 2004) or reactive walls (Waybrant et al. 1998; Benner et al. 2002). P assive processes ha ve been developed on the basis of naturelik e habitats such as marshes and wetlands and use both chemical and biological processes, thereby reducing the need for sophisticated process technology (Barton and Karathanasis 1999).

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Sulfate Reduction for Metal Recovery/Reuse

22.4.1 Metal

Sulfide Precipitation

Heavy metals such as Cu, Zn, Cd, Pb, Ni and Fe precipitate with biogenic sulf ide to form insoluble metal sulf ides (Table 22.4), thereby concentrating the metals into an easy separable and sometimes v aluable form. In engineered systems, metal sulf ide precipitation can be optimized with respect to the rate of biogenic sulf ide production, metal precipitate product quality and selective precipitation of metal sulfides. Treatment processes should focus on recovery of the metals also, as metal resources are depleting. Reuse of metals can only become economically and technically feasible when metals are removed selectively and relatively pure metal sludges are produced. In industry, hydroxide precipitation w as by f ar the most widely used method in the past for wastewater treatment. However, it is also well known that technologies based on metal precipitation with sulf ide ha ve some fundamental adv antages over hydroxide precipitation (Kim and Amodeu 1983; Peters et al. 1984; Veeken et al. 2003): 1. Effluent concentrations are orders of magnitude lo wer: micrograms per litre vs. milligrams per litre. 2. The interference of chelating agents in the w astewater is less problematic. 3. Selective metal removal gives better opportunities for metal reuse. 4. Metal sulfide sludges have better settling, thickening and dewatering characteristics than hydroxide sludges. 5. Existing smelters can process sulf ide precipitates, thus enabling metal reco very and eliminating the need for sludge disposal.

22.4.2

Biogenic Sulfide for Metal Sulfide Precipitation

Earlier objections against the use of sulf ide, i.e. that it is toxic, malodorous and corrosive, can today be overcome by adequate safety measures and the use of modern corrosion-resistant construction materials. Chemical forms of sulfide such as Na2S, NaHS, CaS and H 2S can be used, but these need to be transported to the treatment site. In general, these sulf ide sources are more e xpensive than lime or limestone. Moreover, the hazards that accompan y transport, handling and storage of the chemical sulfides lead to additional costs for safety measures. These drawbacks can be o vercome by the on-site production of biogenic sulf ide in bioreactors as described in Sect. 22.3.1. Several studies have focused on the use of SRB for precipitating metal sulf ides in the same reactor systems where the sulfate reduction activity occurs; however, a problem associated with this is the metal toxicity to SRB (Chen et al. 2000). Another problem associated with the use of SRB biomass in the metal-precipitation

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reactor is that the precipitated metals are located on the biomass together with the microbial population, thus increasing the v olume of metal-contaminated sludge. A tw o-stage process in which the metal-precipitation step is separated from the SRB bioreactor system is a good alternati ve that uncouples the process conditions of the bioreactor and the precipitator (Esposito et al. 2006).

22.4.3

Selective Metal Precipitation

The metal sulf ides formed are highly insoluble at neutral pH, while some metal sulfides (e.g. CuS) are highly insoluble at pH values as low as 2. The great advantage of sulf ide precipitation is the possibility of selecti ve precipitation. T abak et al. (2003) have shown the possibility of selecti ve precipitation in acid mine drainage only by changing pH and temperature. Veeken et al. (2003) showed that stoichiometric addition of sulfide to a hea vy metal (Cu, Zn, Cd and Ni) solution can be achie ved by controlling the sulfide concentration in the precipitator by means of combining a pH and a sulfide ion selective electrode. This results in very low effluent concentrations of both metals and sulf ide. Ev ery precipitating metal precipitates at a unique S2− concentration (pS), which is directly related to the solubility product of the metal sulf ide. The uniqueness of the pS le vel for each metal w as successfully applied as a control parameter to precipitate metals selecti vely and to obtain pure metal sulfide, which have better chances for reuse (König et al. 2006). The success of the precipitation process not only depends on the removal of metal ions from the soluble phase, but also on the separation of the solid phase (metalsulfide precipitate) from the liquid phase. Therefore, solid–liquid separation processes such as sedimentation or filtration are of key importance in efficient metal-removal processes (Esposito et al. 2006).

References Barton CD, Karathanasis AD (1999) Renovation of a failed constructed wetland treating acid mine drainage. Environ Geol 39:39–50 Benner SG, Blo wes DW, Ptacek CJ, Mayer KU (2002) Rates of sulphate reduction and metal sulfide precipitation in a permeable reactive barrier. Appl Geochem 17:301–320 Brown TL, Lemay HE, Bursten BE (1997) Chemistry: the central science, 7th edn. Prentice Hall, Upper Saddle River Chen BY, Utgikar VP, Harmon SM, Tabak HH, Bishop DF, Govind R (2000) Studies on biosorption of zinc(II) and copper (II) on Desulfovibrio desulfuricans . Int Biodeterior Biode grad 46:11–18 Colleran E, Finnegan S, Lens P (1995) Anaerobic treatment of sulphate-containing waste streams. Antonie Van Leeuwenhoek 67:29–46 De Smul A, V erstraete W (1999) The phenomenology and the mathematical modelling of the silicone-supported chemical oxidation of aqueous sulfide to elemental sulfur with ferric sulfate. J Chem Technol Biotechnol 74:456–466

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Esposito G, Veeken A, Weijma J, Lens PNL (2006) Effect of the use of biogenic sulphide on ZnS precipitation under different process conditions. Sep Purif Technol 51:31–39 Fedorovich V, Greben M, Kalyuzhn yi S, Lens P , Hulshof f Pol L, Letting a G (2000) Use of membranes for hydrogen supply in a sulfate reducing reactor. Biodegradation 11:295–303. Gibert O, de P ablo J, Cortina JL, A yora C (2004) Chemical characterisation of natural or ganic substrates for biological mitigation of acid mine drainage. Water Res 38:4186–4196 König J, K eesman KJ, Veeken A, Lens PNL (2006) Dynamic modelling and process control of ZnS precipitation. Sep Sci Technol 41:1025–1042 Krüger M, T reude T, Wolters H, Nauhaus K, Boetius A (2005) Microbial methane turno ver in different marine habitats. Palaeogeogr Palaeoclimatol Palaeoecol 227:6–17 Kim BM, Amodeo PA (1983) Calcium sulf ide process for treatment of metal-containing w astes. Environ Prog 2:175–180 Lens P, Visser A, Janssen A, Hulshof f Pol L, Letting a G (1998a) Biotechnological treatment of sulfate rich wastewaters. Crit Rev Environ Sci Technol 28:41–88 Lens P, van den Bosch M, Hulshof f Pol L, Letting a G (1998b) Ef fect of staging on v olatile fatty acid degradation in a sulfidogenic granular sludge reactor. Water Res 32:1178–1192 Lens P, Gastesi R, Hulshof f Pol L, Letting a G (2003) Use of sulphate reducing cell suspension bioreactors for the treatment of SO 2 rich flue gases. Biodegradation 14:229–240 Lens PNL, Korthout D, van Lier JB, Hulshoff Pol LW, Lettinga G (2001) Effect of upflow velocity on thermofilic sulfate reduction under acidifying conditions. Environ Technol 22:183–193 Lens PNL, Klijn R, van Lier JB, Hulshoff Pol LW, Lettinga G (2002) Effect of specific gas loading rate on thermofilic sulfate reduction under acidifying conditions. Water Res 37:1033–1047 Markewitz K, Cabral AR, P anarotto CT , Lefeb vre G (2004) Anaerobic biode gradation of an organic by-products leachate by interaction with dif ferent mine tailings. J Hazard Mater 110:93–104 Omil F, Lens P, Visser A, Hulshof f Pol LW, Lettinga G (1998) Long term competition between sulfate reducing and methanogenic bacteria in U ASB reactors treating v olatile f atty acids. Biotechnol Bioeng 57:676–685 Parshina SN, Sipma J, Nakashimada Y, Henstra HM, Smidt H, L ysenko AM, Lens PNL, Letting a G, Stams AJM (2005) Desulfotomaculum carboxydivorans sp. nov., a novel sulfate reducing bacterium capable of growth at 100% CO. Int J Syst Ev ol Microbiol 55:2159–2165 Paulo P, Kleerebezem R, Lettinga G, Lens PNL (2005) Cultivation of high-rate sulphate reducing sludge by pH-based electron donor dosage. J Biotechnol 118:107–116 Peters RW, Ku Y, Battacharyya D (1984) Ev aluation of recent treatment techniques for remo val of heavy metals from industrial wastewaters. Paper presented at AIChE meeting, Philadelphia, pp 19–22 Reis MAM, Lemos PC, Carrondo MJT (1995) Biological sulf ate removal of industrial ef fluents using the anaerobic digestion. Med F ac Landbouwwet Univ Gent 60:2701–2707 Rose PD, Boshof f GA, v an Hille RP, Wallace LC, Dunn KM, Duncan JR (1998) An inte grated algal sulphate reducing high rate ponding process for the treatment of acid mine drainage wastewaters. Biodegradation 9:247–257 Sipma J, Lens PNL, V ieira A, Miron Y , v an Lier JB, Hulshof f Pol L W, Lettinga G (2000) Thermofilic sulfate reduction in UASB reactors under acidifying conditions. Process Biochem 35:509–522 Sipma J, Meulepas RJW, Parshina SN, Stams AJM, Lettinga G, Lens PNL (2004) Effect of carbon monoxide, h ydrogen and sulf ate on thermophilic (55°C) h ydrogenogenic carbon monoxide conversion in two anaerobic bioreactor sludges. Appl Microbiol Biotechnol 64:421–428 Sipma J, Lettinga G, Stams AJM, Lens PNL (2006)Hydrogenogenic CO conversion in a moderately thermophilic (55°C) sulf ate-fed g as lift reactor: competition for CO-deri ved H 2. Biotechnol Progr 22:1327–1334 Smith RM, Martell AE (1976) Critical stability constants, v ol. 4. Inor ganic lig ands. Plenum, New York

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Tabak HH, Scharp R, Burckle J, Kawahara FK, Govind R (2003) Advances in biotreatment of acid mine drainage and bioreco very of metals: 1. Metal precipitation for reco very and rec ycle. Biodegradation 14:423–436 Vallero MVG, Lens PNL, Hulshof f Pol L W, Lettinga G (2003) Ef fect of NaCl on thermophilic (55°C) methanol degradation in sulfate reducing reactors. Water Res 37:2269–2280 Vallero MVG, Camarero E, Lettinga G, Lens PNL (2007) Hyperthermophilic sulfate reduction in methanol and formate fed UASB reactors. Appl Environ Microbiol (in press) van Houten RT, Hulshoff Pol LW, Lettinga G (1994) Biological sulphate reduction using g as-lift reactors fed with h ydrogen and carbon dioxide as ener gy and carbon source. Biotechnol Bioeng 44:586–594 van Houten R T, Oude Elferink SJWH, v an Hamel SE, Hulshof f Pol L W, Lettinga G (1995) Sulphate reduction by aggregates of sulphate-reducing bacteria and homo-acetogenic bacteria in a lab-scale gas-lift reactor. Bioresour Technol 54:73–79 van Houten RT, Yun SY, Lettinga G (1997) Thermophilic sulphate and sulfite reduction in lab-scale gas-lift reactors using H 2 and CO 2 as ener gy and carbon source. Biotechnol Bioeng 55:807–814 Veeken AHM, de Vries S, v an der Mark A, Rulk ens WH (2003) Selective precipitation of heavy metals as controlled by a sulf ide-selective electrode. Sep Sci Technol 38:1–19 Verstraete W, de Beer D, Pena M, Letting a G, Lens P (1996) Anaerobic bioprocessing of w aste. World J Microbiol Biotechnol 12:221–238 Waybrant KR, Blowes DW, Ptacek CJ (1998) Selection of reactive mixtures for use in permeable reactive walls for treatment of acid mine drainage. En viron Sci Technol 32:1972–1979

Chapter 23

Impact of Nitrate on the Sulfur Cycle in Oil Fields Gerrit Voordouw

Abstract Production of oil from subsurf ace reservoirs requires injection of w ater or gas to maintain reserv oir pressure. Sea water is usually injected on of fshore platforms (as in the North Sea). The combination of ab undant electron donors (selected oil components) and electron acceptors (30 mM sulfate in sea w ater) can lead to significant production of sulfide in the subsurface through action of resident or injected sulf ate-reducing bacteria (SRB). Lo wering sulfide concentrations in the produced oil–water mixture is desirable to reduce corrosion risk. Injection of nitrate has recently emer ged as a ne w technology that can reduce sulf ide le vels reliably. Adding low concentrations (50–100 ppm) of nitrate continuously to all injected water can eliminate sulf ide from produced w ater and oil. The mechanism underlying this technology appears to be lar gely microbial. Nitrate-reducing, sulf ide-oxidizing bacteria remove sulfide with production of nitrite and other reacti ve nitrogen species. Nitrite is a po werful SRB inhibitor that specif ically af fects dissimilatory sulf ite reductase, the enzyme that produces the sulf ide. Heterotrophic nitrate-reducing bacteria can directly oxidize oil components with the injected nitrate, outcompeting SRB. This results in a desirable subsurf ace microbial community change that pre vents the formation of sulfide, improving oil quality. Nitrate injection is one of the first reliable, microbe-based processes that is becoming widely used in oil production to control the oil field sulfur cycle, making microbiologists partners in discovering how we can continue to produce the world’s most significant energy resource.

23.1 Intr oduction Our society depends heavily on the use of fossil fuels for its ener gy supply, with oil, gas and coal contrib uting 40, 24 and 22% of the w orld’s gro wing ener gy needs, respectively. Nuclear energy and renewable forms of energy (wind, hydropower, biomass) contrib ute 6 and 8% w orldwide. The ne gative en vironmental impact of this hea vy reliance on fossil fuels is becoming increasingly apparent and includes significant increases in the CO 2 concentration of the Earth’s atmosphere, with associated global w arming. In seeking solutions for these problems we should aim to reduce per capita ener gy consumption, to increase the contribution 296 C. Dahl and C.G. Friedrich (eds.), Microbial Sulfur Metabolism. © Springer 2008

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of renewables to our energy supply and to use and extract fossil fuels as efficiently as possible. Microbiology is one of the disciplines that can help impro ve the efficiency of fossil-fuel e xtraction. Although its potential in this re gard has been recognized for over 50 years, microbial processes in fossil-fuel extraction or upgrading are not yet common. Potential or proven processes include the microbial desulfurization of coal and oil, tar geting p yrite and or ganic sulfur compounds such as dibenzothiophene, respecti vely (Monticello and Finnerty 1985). A more recent development has been the use of nitrate to manage the sulfur c ycle in oil and gas fields. This application is now being used field-wide in several oil fields, especially in the North Sea to remo ve sulfide from oil and associated produced w ater. The microbial basis of this process and its application will be re viewed in this chapter.

23.2

The Oil Field Sulfur Cycle

Much of the w orld’s oil is produced by w ater injection to maintain reserv oir pressure. As a result an oil–w ater mixture is produced which is separated into produced water and produced oil. Depending on w ater availability, the produced water is reinjected (produced w ater reinjection, PWRI) or dischar ged. PWRI is common in landlocked reservoirs, but rare in of fshore situations where sea water is plentiful. Oil production by w ater injection often results in increased sulf ide levels (souring), because sulfate-reducing bacteria (SRB) couple the oxidation of degradable oil organics present in the w ater in the reserv oir (“formation water”) to the reduction of sulf ate to sulf ide (Fig. 23.1). The problem can be especially severe when sea water, which has a high sulf ate concentration of 30 mM, is injected. An example is provided by seawater flooding of the Skjold f ield in the Danish sector of the North Sea. T otal daily production of sulf ide increased from 100 kg day−1 initially to up to 1,100 kg day −1 after 5 years of sea water injection (Larsen 2002). The sulf ide was produced by SRB, presumably in the zone where sulfate-containing, injected seawater mixes with oil organics-containing formation water. High concentrations of sulfide are unwanted because of the toxicity, and the associated risk of corrosion of pipes and aboveground equipment processing the oil, as well as the potential for reserv oir plugging by precipitated sulfides. The reserv oir SRB are either indigenous (Magot 2005) or introduced with the injection water. They are mesophilic or thermophilic, depending on reserv oir depth. Although some are incomplete oxidizers, con verting oil organics to CO 2 and acetate (Fig. 23.1), complete oxidizers, producing CO 2, only are also common. The incomplete oxidizers include the mesophilic Desulfovibrio spp., which are well known and easy to isolate, but may represent a minor fraction of the SRB found in mesophilic oil field en vironments (Rab us et al. 1996). Thermophilic oil f ield sulf ate-reducing prokaryotes (SRP) include the completely oxidizing Thermodesulforhabdus and Archaeoglobus spp. (Beeder et al. 1994, 1995). Souring can be pre vented or re versed by nitrate, which can be added to the injection w ater in the appropriate concentration. Nitrate injection stimulates

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(A)

lactate SRB

acetate+CO2 lactate (B)

2

SO4 HS-

S0

N2 NO2NR-SOB

NH3

NO3-

NO3-

hNRB

acetate+CO2

NO2-

NH3 N2

Fig. 23.1 Survey of microbial groups impacting the sulfur c ycle in oil f ields. a Sulfate-reducing bacteria ( SRB) couple incomplete oxidation of oil or ganics (to acetate and CO 2), or complete oxidation of oil organics to CO2 (not shown) to the reduction of sulfate to sulfide. Nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) oxidize sulfide to sulfur or sulfate, with nitrate being reduced to nitrite and then to either nitrogen (with NO and N 2O as intermediates) or to ammonia (without intermediates). b Heterotrophic nitrate-reducing bacteria ( hNRB) couple incomplete (as sho wn) or complete oxidation of oil or ganics to reduction of nitrate to nitrite and then to either nitrogen or ammonia. Note that some NR-SOB/hNRB do not reduce nitrate be yond nitrite. Also nitrite is a powerful SRB inhibitor, as explained in the text

nitrate-reducing, sulf ide-oxidizing bacteria (NR-SOB) and heterotrophic n itratereducing bacteria (hNRB). The activities of these two groups are also outlined in Fig. 23.1. The hNRB oxidize degradable oil organics, which would otherwise be used by SRB. Such “competitive exclusion” was initially postulated as the main mechanism through which souring w as prevented (Hitzman and Dennis 1997). Ho wever, early studies on nitrate injection in Cole ville, a medium-temperature f ield in western Canada, indicated that Thiomicrospira sp. strain CV O became a major community component, both in injector and in producing wells, when nitrate was injected (Telang et al. 1997). Strain CVO is an autotroph, deriving energy for growth from the oxidation of sulfide to sulfate with sulfur as an intermediate, while reducing nitrate to nitrogen with nitrite, nitric oxide and nitrous oxide as intermediates. Sulf ide concentrations decreased on average by 70% and this w as credited primarily to strain CV O, i.e., the souring control mechanism at Coleville was as indicated in Fig. 23.1a. A more e xtensive survey of the presence of SRB, hNRB and NR-SOB in western Canadian oil ields f has indicated the presence of all three microbial groups in many oilfields (Eckford and Fedorak 2002). The hNRB often appeared to outnumber the NR-SOB, i.e., the microbial ecology found at Cole ville appeared to be the e xception rather than the rule and souring control by competitive exclusion is also a likely mechanism.

23.3

Effect of Nitrate Injection on SRB Ph ysiology

By def inition, SRB reduce sulf ate, although there are some strains that also reduce nitrate, e.g., Desulfovibrio desulfuricans strain ATCC 27774 (Gonzalez et al. 2006). Peculiarly, such strains may not downregulate genes for enzymes involved

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in sulfate reduction when the y are reducing nitrate; hence the use of alternati ve electron acceptors by SRB serv es a v ery different purpose than, for instance, in Escherichia coli, where distinct gene-expression patterns are established for cells grown with oxygen, nitrate or fumarate as the electron acceptor. In these different gene-expressing states genes encoding oxidoreductases for the electron acceptor present in the medium are “on,” whereas genes for electron acceptors not present in the medium are “of f”. In contrast, in Desulfovibrio spp. genes for sulf ate reduction are on all the time, indicating this to be the primary lifestyle of the organism. Hence, the function of alternate electron acceptor (nitrate, oxygen) reduction in Desulfovibrio spp. appears primarily to pre vent inhibition of sulfate reduction. Although the physiology and gene-e xpression pattern of the majority of SRB, that do not reduce nitrate, are not af fected by the addition of millimolar concentrations of nitrate, these are strongly af fected by nitrite, which is a strong SRB inhibitor (Haveman et al. 2004; He et al. 2006). Nitrite is bound tightly by dissimilatory sulf ite reductase (DsrAB), the terminal reductase of SRB (W olfe et al. 1994), which slo wly reduces nitrite to ammonia. These properties mak e nitrite a strong competiti ve inhibitor, preventing reduction of sulf ite to sulf ide, the normal physiological function of DsrAB. Addition of millimolar concentrations of nitrite to mid-log-phase cultures ofD. vulgaris halts sulfate reduction and associated growth and do wnregulates expression of genes for enzymes in volved in sulf ate reduction (sulf ate aden ylyltransferase, p yrophophatase, adenosine 5′-phosphosulfate reductase) with the exception of DsrAB (Haveman et al. 2004; He et al. 2006). Genes for ATP synthase, as well as genes for two membrane-bound redox protein comple xes, QmoABC and DsrMKJOP , are also do wnregulated. This indicates that a proton-moti ve force allowing phosphorylation of ADP to ATP is lacking under conditions of nitrite inhibition. It also indicates in volvement of QmoABC and DsrMJK OP in sulfate respiration, i.e., electrons for the APS reductase and DsrAB catalyzed reactions are lik ely provided by QmoABC and DsrMJKOP, respectively. Although the detailed bioenergetic mechanism through which D. vulgaris and other SRB deri ve energy for growth from sulfate respiration is by no means solved, these studies provided strong evidence for involvement of membrane-bound comple xes in sulf ate respiration. Hence, when D. vulgaris derives energy for growth from coupling the oxidation of lactate to the reduction of sulf ate, reducing equi valents (H +, e−) c ycle from the c ytoplasm to the periplasm to return to the cytoplasm through QmoABC and DsrMJKOP (Haveman et al. 2004; Mussmann et al. 2005; He et al. 2006). As a consequence, these comp lexes appear strongly conserv ed in all SRP , including in the thermophilic archaeon Archaeoglobus fulgidus . Hence inhibition of sulf ate reduction by nitrite has, in addition to being of practical signif icance, given us insight into the mechanism of sulfate reduction by SRP . In order to pre vent inhibition of DsrAB by nitrite, SRB can ha ve a periplasmic nitrite reductase (NrfHA), which reduces nitrite to ammonia. The nrfHA genes of D. vulgaris Hildenborough are upre gulated upon addition of nitrite; hence, when nitrite is added, reducing equi valents deri ved from lactate oxidation are temporary di verted from sulf ate reduction to nitrite reduction by periplasmic NrfHA, as well as at a slo wer rate by c ytoplasmic

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DsrAB. The lethality of nitrite depends on the time required for an SRB population to reduce all nitrite to ammonia. This depends on the biomass concentration and the presence or absence of NrfHA. Mid-log-phase cultures of D. vulgaris Hildenborough can survive addition of 5–10 mM nitrite, but an nrfHA mutant can survive addition of only 0.5 mM nitrite. F or single cells on plates, the lo west possible biomass concentration, the inhibitory nitrite concentration, is only 0.04 mM (Haveman et al. 2004). Interestingly , this lo w inhibitory concentration is the same for wild-type and nrfHA-mutant cells, because the binding af finity (Km) of NrfHA for nitrite is quite high (millimolar); hence, NrfHA does not contribute to nitrite detoxif ication at 0.04 mM, which is instead reduced by the target DsrAB under these conditions. NrfHA thus allows dense SRB populations to survive millimolar concentrations of nitrite by its rapid reduction to ammonia. Thermophilic SRP (tSRP; including members of Archaeoglobus) appear to lack nitrite reductase and are, as a result, much more sensiti ve to inhibition by nitrite than are mesophilic SRB.

23.4

Mechanism of Souring Control

Having established that SRP are strongly inhibited by nitrite, the question arises to what extent this inhibition contributes to souring control. hNRB and NR-SOB, collectively referred to as NRB, reduce nitrate to nitrite, which is then further reduced to either nitrogen or ammonia (Fig. 23.1). Many NRB excrete nitrite and it is not uncommon to f ind nitrite in produced w aters of oil f ields subjected to nitrate injection or in the ef fluent of upflo w bioreactors that aim to model such fields (Reinsel et al. 1996; Myhr et al. 2002). Studies in which the NR-SOB Thiomicrospira sp. strain CVO was added to growing cultures of mesophilic SRB in the presence of nitrate indicated rapid formation of millimolar concentrations of nitrite under these conditions (Greene et al. 2003). This led to either permanent or transient inhibition of SRB acti vity, depending on the presence of nitrite reductase in the SRB strain. However, because in mesophilic oil field populations some of the SRB present are lik ely to ha ve nitrite reductase, it is unlik ely that such a population could ever become permanently inhibited by nitrite. The nitrate dose required to eliminate sulfide appears dictated by the concentration of degradable oil organics in such systems, as w as demonstrated in bioreactor studies by Hubert et al. (2003). The situation may be dif ferent in thermophilic oil f ield communities. Nitrite reductase has so f ar not been demonstrated in tSRP . As a result tSRP-containing enrichments from Ek ofisk, a North Sea oil f ield with an in situ temperature of 80–90°C, were inhibited by v ery lo w concentrations of nitrite (0.25–0.5 mM). Nitrate does not af fect sulf ate reduction rates at Ek ofisk, because thermophilic NRB also appear to be absent; hence, nitrate injection may not w ork, but injection of nitrite could be effective in this field (Kaster et al. 2007).

23 Impact of Nitrate on the Sulfur Cycle in Oil Fields

23.5

301

Prospects for Nitrate Injection

Nitrate injection is the f irst reliable, microbe-based process that is being applied continuously and f ield-wide to impro ve the production of oil (Thorstenson et al. 2002; Larsen et al. 2004). The nitrate dose required to pre vent souring needs to be determined by trial and error . In mesophilic systems the dose is dictated by the concentration of oxidizable electron donors (sulf ide, sulfur and de gradable oil organics), whereas in thermophilic systems inhibition of thermophilic SRB by nitrite may also contrib ute, lowering the ef fective dose required. Adoption of this successful technology is currently being considered in many fields, including those subjected to PWRI. Successful adoption of the technology in PWRI situations still requires considerably more research. Also the ef fects of continuous, long-term, field-wide nitrate injection need to be considered. So far the experiences with up to 6 years of continuous injection have been positive and preliminary reports, indicating that this practice leads to production of additional oil through microbially enhanced oil recovery, are further f anning interest in this technology . Nitrate injections are here to stay and may well prove the ideal stepping stone to further expand petroleum microbiology as a contributing science towards improving the production efficiency of oil, the most important ener gy supply in the world today. Acknowledgements. Research in the author’s laboratory has been supported through Strategic Grants of the Natural Science and Engineering Research Council of Canada (NSERC) with ConocoPhillips, Bak er Petrolite and the Computer Modelling Group as industrial partners. The research contrib utions of graduate students Casey Hubert and Krista Kaster , as well as of postdoctoral fello ws Anne Greene, Alexander Grigoriyan and Mehdi Nemati are gratefully ackno wledged.

References Beeder J, Nilsen RK, Rosnes JT , Torsvik T, Lien T (1994) Archaeoglobus fulgidus isolated from hot North Sea oil field waters. Appl Environ Microbiol 60:1227–1231 Beeder J, Torsvik T, Lien T (1995) Thermodesulforhabdus norvegicus gen. nov., sp. nov., a novel thermophilic sulfate reducing bacterium from oil f ield water. Arch Microbiol 164:331–336 Eckford RE, Fedorak PM (2002) Planktonic nitrate-reducing bacteria and sulf ate-reducing bacteria in some western Canadian oil field waters. J Ind Microbiol Biotechnol 29:83–92 Greene EA, Hubert C, Nemati M, Jenneman G, V oordouw G (2003) Nitrite reductase acti vity of sulfate-reducing bacteria pre vents their inhibition by nitrate-reducing, sulf ide-oxidizing bacteria. Environ Microbiol 5:607–617 Gonzalez PJ, Rivas MG, Brondino CD, Bursak ov SA, Moura I, Moura JJ (2006) EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774. J Biol Inorg Chem. 11:609–616 Haveman SA, Greene EA, Stil well CP, Voordouw JK, V oordouw G (2004) Ph ysiological and gene expression analysis of inhibition of Desulfovibrio vulgaris Hildenborough by nitrite. J Bacteriol 186:7944–7950 He Q, Huang KH, He Z, Alm EJ, Fields MW , Hazen TC, Arkin AP , Wall JD, Zhou J (2006) Energetic consequences of nitrite stress in Desulfovibrio vulgaris Hildenborough, inferred from global transcriptional analysis. Appl Environ Microbiol 72:4370–4381

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Hitzman DO, Dennis DM (1997) Ne w technology for pre vention of sour oil and g as. In: Proceedings SPE/DOE e xploration and production en vironmental conference, Dallas, pp 406–411 Hubert C, Nemati M, Jenneman GE, V oordouw G (2003) Containment of biogenic sulf ide production in continuous up-flow, packed-bed bioreactors with nitrate or nitrite. Biotechnol Prog 19:338–345 Kaster KM, Grigoryan A, Jenneman G, Voordouw G (2007) Effect of nitrate and nitrite on sulfide production by tw o thermophilic, sulf ate-reducing enrichments from an oil f ield in the North Sea. Appl Microbiol Biotechnol 75:195–203 Larsen J (2002) Do wnhole nitrate applications to control sulf ate reducing bacteria acti vity and reservoir souring. Corrosion 2002. Paper 02025. NACE International, Houston Larsen J, Rod MH, Zwolle S (2004) Prevention of reservoir souring in the Halfdan field by nitrate injection. Corrosion 2004. Paper 04761. NACE International, Houston Magot M (2005) Indigenous microbial communities in oil f ields. In: Olli vier B, Magot M (eds) Petroleum microbiology. ASM, Washington, pp 21–33 Monticello DJ, Finnerty WR. 1985. Microbial desulfurization of fossil fuels. Annu Rev Microbiol 39:371–389 Mussmann M, Richter M, Lombardot T, Meyerdierks A, Kuever J, Kube M, Glockner FO, Amann R (2005) Clustered genes related to sulf ate respiration in uncultured prokaryotes support the theory of their concomitant horizontal transfer. J Bacteriol 187:7126–7137 Myhr S, Lillebo BLP, Sunde E, Beeder J, Torsvik T (2002) Inhibition of microbial H2S production in an oil reservoir model column by nitrate injection. Appl Microbiol Biotechnol 58:400–408 Rabus R, Fukui M, W ilkes H, W iddel F (1996) De gradative capacities and 16S rRN A-targeted whole-cell h ybridization of sulf ate-reducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil. Appl Environ Microbiol 62:3605–3613 Reinsel MA, Sears JT, Steward PS, McInerney MJ (1996) Control of microbial souring by nitrate, nitrite or glutaraldehyde injection in a sandstone column. J Industr Microbiol 17:128–136 Telang AJ, Ebert S, Foght JM, Westlake DWS, Jenneman GE, Gevertz D, Voordouw G (1997) The effect of nitrate injection on the microbial community in an oil f ield as monitored by re verse sample genome probing. Appl Environ Microbiol 63:1785–1793 Thorstenson T, Bødtker G, Sunde E, Beeder J (2002) Biocide replacement by nitrate in sea w ater injection in sea w ater injection systems. Corrosion 2002. P aper 02033. N ACE International, Houston Wolfe B, Lui SM, Co wan J (1994) Desulfo viridin, a multimeric-dissimilatory sulf ite reductase from Desulfovibrio vulgaris (Hildenborough). Purification, characterization, kinetics and EPR studies. Eur J Biochem 233:79–89

Index

A Acetate kinase, 177 Acid mine drainage, 184 Acidianus A. ambivalens, 185, 187, 190, 217–220, 222, 223 A. brierleyi, 185, 217–220 A. tengchongensis, 188–191, 217–220, 222, 223 Acidithiobacillus, 165, 218–219, 221, 222 A. ferrooxidans, 77–85, 198 A. thiooxidans, 187 Adenosine-5’-phosphosulfate (adenylylsulfate, APS), 14, 18, 19, 245, 247 Adenosine-5’-phosphosulfate (adenylylsulfate, APS) reductase, 14, 18, 24, 26, 28, 32, 66, 69, 94, 112, 152, 196, 197, 248 Adenylate kinase, 187, 197 Adenylylsulfate:phosphate adenylyltransferase (adenosine-5’-phosphosulfate: phosphateadenylyltransferase APAT), 187, 197 ADP sulfurylase, 95 Alkalilimnicola ehrlichei,71, 109 Allochromatium vinosum, 66, 69, 71, 88, 89, 92, 94–96, 106, 109, 259, 263, 268 Allochromatium, 247, 248 Amoebobacter, 90, 91 Anaerobic, 117, 119, 122, 286–289 Anaerobic oxidation of methane (AOM), 203, 206, 212, 289 aprAB, 69, 73 APS reductase, 24, 26, 28, 32, 94, 112, 152, 248 Aquifex aeolicus, 189, 190, 218 Archaea, 184, 186, 188, 198, 204, 208, 212, 213 Archaeoglobus fulgidus, 25, 26, 29, 30, 93, 110, 203, 206, 209, 210, 211, 280 Arcobacter, 244, 245, 251–253

Arenicola marina, 37–39 Assimilatory sulfate reduction (Asr), 69, 71–73, 204, 206, 209 Assimilatory sulfur metabolism, 71 ATP sulfurylase, 14, 60, 95, 112, 113, 197, 245, 248 ATP, 2 7 ATP synthesis, 38, 39, 42 Axial volcano, 252, 253 B Bacteriochlorophyll a, 121 Bacteriochlorophyll c, 120 Beggiatoa, 88–91, 102, 103, 241–243, 248, 249 Bioleaching, 184, 186, 187, 189 Biomining, 78 Bioprocess engineering, 285, 287, 289, 291, 293, 295 Bioreactor, 287–289, 292, 293 C Caldariella quinone, 187, 197 Calvin-Benson-Bassham cycle, 245 Canfield oceans, 41, 42 Capnine, 173 Chemolithoautotrophy, 238, 241, 246–248, 254 Chlorobaculum parvum, 103 Chlorobaculum tepidum, 62, 64, 67, 109, 117–124, 248, 249 Chlorobaculum thiosulfatiphilum, 128, 129, 134 Chlorobii, 61, 73 Chlorobium chlorochromatii, 26 Chlorobium clathratiforme, 109 Chlorobium ferrooxidans, 64–67, 69, 72, 73 Chlorobium limicola f. thiosulfatophilum, 128

303

304 Chlorobium limicola, 94, 109 Chlorobium phaeobacteroides, 109 Chlorobium tepidum (syn. Chlorobaculum tepidum), 26, 29, 62, 64, 67, 117–124 Chloroherpeton thalassium, 64, 66, 67, 70, 72, 73 Chlorosomes, 62 Chromatiaceae, 95, 96, 102, 103 Chromatium, 88, 90–94 Clostridium thermocellum, 72 Coenzyme F420 reducing hydrogenase, 208, 212 Coenzyme F420, 8-hydroxy-5 deazariboflavin derivative, 208 Coenzyme F420-dependent sulfite reductase (Fsr), 204, 208–213 Coenzyme M, 173, 174, 213 Complementation, 122 Crystallographic analysis, 192 CysH, 69, 72 Cysteine persulfide, 195 Cysteine sulfinate, 173 Cysteine, 189, 190, 194, 195, 198 Cysteine-aminotransferase, 39 Cyteine-disulfide transporter SoxV, 141 Cytochrome c oxidase, 38 Cytochrome c3, 14, 30, 31 Cytochrome, 25, 26, 29–33 Cytoplasm, 24, 25, 27–32 D Deep-sea hydrothermal vents, 238, 239, 241, 246, 247, 249, 251, 253, 254 Desulfotalea psychrophila, 25, 26, 30, 110 Desulfitobacterium hafniense, 29, 72, 110 Desulfotomaculum reducens, 26, 27 Desulfovibrio D. autotrophicum, 281 D. desulfuricans, 25, 26, 29, 31, 32, 275 D. jorgensii, 281 D. vulgaris, 2–11, 25, 26, 31, 33, 109 Desulfovibrio spp, 70 Dimethylsulfoxide reductase, 158 Disproportionation, 187–189, 196, 198, 199 Dissimilatory sulfate reduction, 202, 204, 206 factors controlling isotope fractionation, 273, 275, 277, 279–281 isotope fractionation by natural populations, 276 isotope fractionation models, 277–279

Index minor sulfur isotopes, 274, 279 sulfur isotope fractionation during, 273, 275, 282 Dissimilatory sulfite reductase (Dsr), 28–33, 47, 52, 66, 72, 144, 177, 204, 206, 209–211, 213, 247, 248 DNA macroarray, 80, 81, 83 dsr gene cluster, 66 dsr genes, 95 dsr operon, 107, 111 Dsr proteins phylogeny, 108 DsrA, A subunit of Dsr, 67, 68, 73, 204, 209 DsrAB, 47, 49–56 DsrB, B subunit of Dsr, 204, 209 DsrMKJOP, 67 E East Pacific Rise (EPR), 242, 244, 245, 252, 253 Ecology, 53, 56 Ectothiorhodospira, 90, 93 Electron acceptor, 24, 26, 30–32 Electron donor, 27, 28, 32 Electron paramagnetic resonance, 14 Elemental sulfur, 13, 14, 88, 89, 95, 118, 119, 122, 123, 259, 265, 268–270 Endosymbiotic theory, 40 Energy conservation, 25, 26 Environmental technology, 285 Epsilon proteobacteria, 239, 241, 245, 246, 249, 251, 253, 254 EPR spectroscopy, 189 9°N EPR, 244, 245, 252 Eukaryotes, 36–40, 42 Evolution, 40, 41, 47, 49, 52, 53, 56 Extremophile, 235 Extremophilic, 202 F F1 ATPase, 123 FAD, 14, 204 FCSD, 141, 144 Ferredoxin, 204, 209 Ferroplasma acidarmanus, 189, 190 Filamentous sulfur, 251–253 Flavocytochrome c, 66, 69, 71, 73, 95, 106, 111 Flavocytochrome c-sulfide dehydrogenase (FCSD), 141, 144 Flavoprotein component of Sir (SirFP), 204 Flavoprotein SoxF, 140, 141, 144

Index Fluorescence, 121 FMN, 204 FmoA Protein, 123 Formate dehydrogenase, 25, 30, 33 Formate, 25, 30, 33 FqoF, H2F420 dehydrogenase subunit of H2F420: quinone oxidoreductase complex, 204, 209 Fsr, 204, 208–213 Fumarate reductase, 17, 42 G Gammaproteobacteria, 231–235, 239, 241, 245–248, 253, 254 Gene expression, 1 Geukensia demissa, 37, 38 Giant tubeworm. See Riftia pachyptila Glutathioneamide, 111 Green sulfur bacteria, 61–64, 70–73, 91, 92 H H2F420 dehydrogenase, 204, 209, 210 H2F420, reduced F420, 208 H2F420:quinone oxidoreductase, 204, 209 Halophilic, 225, 226, 228–236 Halorhodospira halophila, 62, 109 Halothiobacillus, 225, 228–232, 236 9Hc, 31 Hdr, 26 Heavy metal, 286, 291–293 Heme enzyme SoxXA, 140 Heme-protein component of Sir (SirHP), 204 Hemoglobins, sulfide-transporting, 37 Hemoproteins, 124 Heterodisulfide reductase, 26, 66, 70, 73, 108 Hexahistidine tag, 122 History of sulfur metabolism, 87, 93 2-His 1-Carboxylate facial triad, 194 Hmc, 31–33 Hme Hydrogen cycling, 25 Hydrogen oxidation, 66 Hydrogen sulfide H2S, 188, 196, 198, 199 Hydrogen, 25, 30–32 Hydrogenase, 14, 25, 26, 29, 30, 32, 248, 250 Hydrogenosomes, 41, 42 Hydrothermal environment, 184 Hydrothermal vent, 37, 206, 207 Hypersaline, 225–231, 234–236 Hyperthermophile, 185, 188, 189 Hyperthermophilic, 207

305 I Inorganic wastewaters, 289 Intermediate oxidation state, 41 Interprotein disulfide, 140, 145, 146 Iron oxidation, 63 Iron-sulfur cluster, 15, 16, 20, 21 Isotope fractionation equilibrium, 274, 280 kinetic, 274, 280 J Juan de Fuca Ridge, 252, 253 K Kulunda Steppe, 226, 227 L Lactate oxidation, 6, 7 Lamprocystis, 90, 91 Lateral gene transfer, 67, 72 L-cysteate sulfo-lyase, 175, 176 L-cysteate, 173–176, 180 Linear alkylbenzenesulfonate, 173, 175 Lugworm. See Arenicola marina M Magnetococcus sp., 109 Magnetospirillum magnetotacticum, 109 Magnetotactic bacteria, 102 Marine sediments, 37, 38 Membrane complexes, 25, 30, 33 Menaquinol, 27, 28, 32 Menaquinone, 25, 27–33 3-mercaptopyruvate-sulfurtransferase, 39 Metal sulfide, 291–293 Metallosphaera, 184 Methanesulfonate, 171, 176 Methanocaldococcus igneus, 207 Methanocaldococcus jannaschii, 202, 204, 207–211 Methanococcoides burtonii, 212 Methanococcus maripaludis, 207 Methanogen, 206, 207, 211–213 Methanogenesis, 26, 202, 203, 206, 207, 209, 210, 212, 213 Methanogenic archaea, 204, 208, 212, 213 Methanogenic, 285–288 Methanoplanus limicola, 207 Methanopyrus kandleri, 207, 212 Methanosarcina acetivorans, 207 Methanosarcina barkeri, 212 Methanosarcina, 26, 207, 210, 212 Methanothermobacter marburgensis, 26, 207

306 Methanothermobacter thermautotrophicus, 207, 210, 212 Methanothermococcus thermolithotrophicus, 207 Methylcoenzyme M reductase, 203, 206, 211 Methylcoenzyme M, 203, 206, 211 Methylsulfonate monooxygenase, 175 Mine(s), 185 Mitochondria, 38–40, 42 Mitochondria, anaerobic, 36 Mitochondria, denitrifying, 42 Mitosomes, 41, 42 MmpL proteins, 162 Molybdenum cofactor, 153 Moorella thermoacetica, 29, 110 Mutagenesis, 120 N N-acetyltaurine, 182 NADH, 25 NADH:quinone oxidoreductase, 198 Natronorubrum sp. HG 1, 197 Nitrate reductase, 246 Nitrate, 297–301 Nitrate-reducing bacteria, 296, 298 Nitrite, 298–301 Nitrogenase, 118 Nonaheme c, 14 Non-heme iron, 189, 194 O Oil field, 297, 298, 300 Ore, 185, 186 Oscillatoria limnetica, 67 OsmC-like protein, 165 Outer membrane, 119, 123, 124 Oxidation, 61 Oxidative phosphorylation, 24 Oxidoreductase SAOR, 187, 196, 197 Oxygen, 37, 38, 40–42 Oxygenase, 184, 186–189, 194, 196 P Paracoccus pantotrophus, 70, 73, 96, 102–105, 119, 139–142, 144–147 Paracoccus versutus, 103 Paracoccus, 247, 250 Periplasm, 25, 28, 29, 30, 33 Periplasmic proteins, 83, 84 Persephonella, 245, 246 Phage green sulfur bacteria, 72, 73 Phospho-adenylylphosphosulfate, 151 Phosphosulfolactate synthase, 173

Index Photosynthesis, 89, 91, 92, 94 Photosynthetic electron transport, 118 Phototrophic sulfur bacteria, 92–94 Phototrophic sulfur oxidation, 127, 128 Picrophilus torridus, 189, 190 Plant nitrate reductases, 161 Polysulfide reductase, 66, 70 Polysulfide-reductase-like complex, 3, 70 Polysulfides, 111, 186, 194, 196 Polythionates, 184, 186, 198 Precipitation, 287, 292, 293 Prostecochloris aestuarii, 109 Prostecochloris vibrioformis, 109 γ-proteobacteria, 231–235, 239, 241, 245–248, 253, 254 ε-proteobacteria, 239, 241, 245, 246, 249, 251, 253, 254 Proteomics, 78, 81–83, 85 Proton motive force, 24, 26 PscD, 123 Psychrophilic, 212 Purple non-sulfur bacteria, 91, 92 Purple sulfur bacteria, 61, 88–92, 102, 104, 112 Pyrite, 78, 79, 81, 83, 85 Pyrococcus furiosus, 186 Q Qmo complex, 66, 70 Qmo, 26, 28, 32 qmoABC, 70, 73 R Redox loop, 25, 28 Redox titration, 189 Reduction potential, 189, 194 Reductive tricarboxylic acid (TCA) cycle, 118, 245 Respiratory chain, 37, 38, 42 Respiratory NADH dehydrogenase complex I, 210 Reverse methanogenesis pathway, 203, 209, 210 Rhodanese, 70, 79–81, 104, 105 Rhodobacter capsulatus, 67, 92 Rhodobacter sphaeroides, 92 Rhodoquinone, 42 Rhodospirillum rubrum, 90–92 Rhodovulum sulfidophilum, 102, 105 Ribbed mussel. See Geukensia demissa Riftia pachyptila, 37 RNA virus, 72 RNA-directed DNA polymerase, 72 RuBisCO-like protein, 67

Index S Salmonella enterica, 206 Saltern, 226–228, 230, 232, 233, 236 Schizosaccharomyces pombe, 38, 39 Sir, Asr of Escherichia coli, 204 Siroamide, 107, 112, 204 Siroheme, 20, 21, 95, 107, 204, 206, 210, 212 Small sulfite reductase, 202, 212, 213 Snowblower vents, 251, 252 Solfatara, 184, 185 SorB cytochrome c, 163 Souring, 297, 298, 300, 301 Sox complex, 87, 103, 105, 186, 196, 197, 248 Sox enzyme system, 139–141, 144–146 sox gene cluster, 70, 73 Sox genes abundance, 142–144 complementation, 106 inactivation, 106 phototrophic bacteria green sulfur bacteria, 70, 71, 142 purple sulfur bacteria, 144 SoxAX, 30 soxCD soxJ, 70 soxK, 70 SoxY component of complex SoxYZ, 127 conformation, 131, 132 crystal structure, 128 intersubunit disulfide, 128, 132, 134–136 tetramerization, 129, 134 thiosulfate binding, 127 homodimer, 145, 146 SoyYZ, 71, 73 SQR. See Sulfide:quinone oxidoreductase Starkeya novella, 96, 105 Stygiolobusi, 184, 185 Subseafloor biosphere, 251, 254 Succinate, 42 Sulfate reducing bacteria, 285, 287, 296–298 Sulfate reduction, 1, 2, 5, 6, 8, 285–289, 291, 292 Sulfate respiration, 14 Sulfate thioesterase SoxB, 139–141 Sulfate, 13–16, 20, 24–26, 28–33, 186, 198, 202–204, 206, 208, 211–213, 285–289, 291, 292 Sulfate-reducing microorganisms, 47, 48, 50

307 Sulfide:quinone oxidoreductase, 66–68, 87, 96, 106, 111, 187, 188, 198, 242, 245 Sulfide, 14, 20, 61–69, 71–73, 118, 119, 123, 203, 204, 207, 208, 210, 211, 213, 286, 287, 291–293, 297–300 oxidation, 92, 94–96, 102–104, 106, 107, 111 Sulfite, 13–17, 19, 20, 24, 26, 29, 30, 32, 33, 186–188, 191, 196–199, 202–204, 206–213 redox potential, 152 Sulfite:acceptor oxidoreductase (SOR), 112, 113, 187, 196, 245, 247 Sulfite assimilation, 202 Sulfite dehydrogenase, 96, 102, 178–181 Sulfite detoxification, 207, 211 Sulfite oxidase family Archaea, 152, 155, 164, 165 bacterial enzymes, 153, 155, 164, 166 electron acceptors, 152, 153 electron transfer, 154, 160 phylogenetic analysis, 155, 165 phylogenetic groups, 155, 163 plant SO, 152, 154, 164 properties, 154 protein structure, 157–159, 162 Roseobacter lineage, 157, 159 SorAB, 154, 155, 158–161, 163 SoxCD, 155, 160–164 sulfite oxidases, 161 Thiobacillus denitrificans, 157, 159 YedY-like enzymes, 157 Sulfite reductase, 14, 24, 26, 29, 30, 32, 33, 95, 102, 107, 108, 112, 202–204, 206–213 Sulfiting agents, 151 Sulfoacetaldehyde acetyltransferase, 175 Sulfolactate sulfo-lyase, 176 Sulfolactate, 173–176 Sulfolobales, 184, 185, 198 Sulfolobus S. acidocaldarius, 185, 190, 198 S. metallicus, 219, 221, 223 S. solfataricus, 185, 190, 198 S. tokodaii, 185, 189, 190, 198, 217–221 Sulfopyruvate, 173 Sulfoquinovose, 173 Sulfur, 60–67, 69, 71–73 sulfur disproportionation, 187–189 Sulfur bacteria, 259, 266, 267 Sulfur cycle, 296–298

308 Sulfur dehydrogenase, 104, 107 SoxCD, 139–140, 144, 155, 160–163 SoxD, 162, 163 absence of, Sulfur globules, 63, 66, 67 envelope, 95, 102, 106 intracellular, 102, 106 oxidation, 101, 104, 107 Sulfur isotopes, 273–277, 279, 280 Sulfur metabolism, 78, 80, 82–85 Sulfur oxidation chemotrophic, 141, 142 phototrophic, 141, 142 Sulfur oxygenase reductase (SOR), 184, 186, 187, 191 diversity, 218, 219 physiological function, 219, 223 Sulfur oxygenase, 187–189, 191 Sulfur speciation, 260, 261, 266–268 Sulfur-binding protein SoxYZ, 141 Sulfur-binding protein, 127, 128 Sulfuric acid, 184, 186 Sulfurimonas, 241, 244–246, 249, 250, 253 Sulfurisphaera, 184 Sulfur-oxidizing bacteria (SOB) free-living, 241–249 SOB, 225, 226, 228–236 symbiotic, 239, 240 Sulfur-oxidizing microorganisms, 51 Symbionts endosymbionts, 239, 241, 248, 254 epibionts, 239, 241 Synchrotron radiation, 260, 261 Syntrophobacter fumaroxidans, 110 T Tapes philippinarum, 39 Taurine dehydrogenase, 177, 178 Taurine, 171, 173–180 Taurine:pyruvate aminotransferase, 177 Tetrathionate 63, 64, 94 formation, 102 hydrolase, 184, 187, 188, 197, 198 Thermophiles, 207 Thermothrix, 103 Thiobacillus denitrificans, 26, 29, 93, 95, 102, 109, 196 Thiocapsa roseopersicina, 94 Thiocapsa, 90, 91, 94, 96 Thiocyanate, 227–229, 234–236

Index Thiocystis, 90, 91 Thiodenitrifyers, 232, 233, 235 Thiohalomonas thiohalophilus, 228–230, 234, 235 Thiohalorhabdus, 228–230, 234, 235 Thiohalospira, 228–230, 232–235 Thiomargarita, 90 Thiomicrospira denitrificans, 143, 144 Thiomicrospira, 228–231, 234, 241, 242, 249, 250, 253 Thioploca, 90 Thioredoxin SoxS, 139, 141, 144, 146, 147 SoxW, 141, 144 Thiospirillum, 90, 91 Thiosulfate, 30, 38, 39, 61–66, 69–73, 78–85, 118, 119, 121, 177, 184, 186–188, 197–199, 226–229, 232–234, 236, 245, 248, 249 oxidation, 94, 101–104, 106, 107, 111, 113 dehydrogenase, 102 oxidizing multienzyme complex (TOMES), 159, 162–164 reductase, 104 sulfur transferase, 79, 81 Thiosulfate:quinone oxidoreductase, TQO, 82, 187, 197 Thiothrix, 90, 91, 102, 103 Tmc, 31–33 4-toluenesulfonate, 175 TpIc3, 30–32 Transcript quantity, 1 Transport systems, 82 U Ubiquinone, 38, 39 V Volcanic eruptions, 251 W Wastewater, 285, 286, 288–292 X XANES spectroscopy, 87, 96, 262–264, 266, 267, 269, 270 X-ray crystallography, 191, 192