Handbook of MolecularMicrobial Ecology IMetagenomics and ComplementaryApproaches

Edited by

Frans J. de Bruijn

A John Wiley & Sons, Inc., Publication

Handbook of MolecularMicrobial Ecology I

Handbook of MolecularMicrobial Ecology IMetagenomics and ComplementaryApproaches

Edited by

Frans J. de Bruijn

A John Wiley & Sons, Inc., Publication

Copyright 2011 by Wiley-Blackwell. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data:

Bruijn, F. J. de (Frans J. de)Handbook of molecular microbial ecology I : metagenomics and complementary approaches / Frans J. de Bruijn.

p. cm.Includes index.ISBN 978-0-470-64479-9 (hardback)

1. Molecular microbiology. 2. Microbial ecology. I. Title.QR74.B78 2011576–dc22

2010042169

Printed in Singapore

Set ISBN: 978-0-470-92418-1oBook ISBN: 978-1-118-01051-8ePDF ISBN: 978-1-118-01044-0ePub ISBN: 978-1-118-01049-5

10 9 8 7 6 5 4 3 2 1

To my two daughters, Waverly and Vanessa de Bruijn, for their support even from a distance

Contents

Preface xv

Contributors xvii

1. Introduction 1

Frans J. de Bruijn

Part 1 Background Chapters

2. DNA Reassociation Yields Broad-Scale Information on Metagenome Complexityand Microbial Diversity 5

Vigdis L. Torsvik and Lise Øvreas

3. Diversity of 23S rRNA Genes Within Individual Prokaryotic Genomes 17

Anna Pei, William E. Oberdorf, Carlos W. Nossa, Pooja Chokshi, Martin J. Blaser,Liying Yang, David M. Rosmarin, and Zhiheng Pei

4. Use of the rRNA Operon and Genomic Repetitive Sequences for the Identificationof Bacteria 29

Andrea Maria Amaral Nascimento

5. Use of Different PCR Primer-Based Strategies for Characterization of NaturalMicrobial Communities 41

James I. Prosser, Shahid Mahmood, and Thomas E. Freitag

6. Horizontal Gene Transfer and Recombination Shape Mesorhizobial Populationsin the Gene Center of the Host Plants Astragalus Luteolus and Astragalus Ernestiiin Sichuan, China 49

Qiongfang Li, Xiaoping Zhang, Ling Zou, Qiang Chen, David P. Fewer,and Kristina Lindstrom

7. Amplified rDNA Restriction Analysis (ARDRA) for Identification and PhylogeneticPlacement of 16S-rDNA Clones 59

Menachem Y. Sklarz, Roey Angel, Osnat Gillor, and Ines M. Soares

vii

viii Contents

8. Clustering-Based Peak Alignment Algorithm for Objective and Quantitative Analysisof DNA Fingerprinting Data 67

Satoshi Ishii, Koji Kadota, and Keishi Senoo

Part 2 The Species Concept

9. Population Genomics Informs Our Understanding of the Bacterial Species Concept 77

Margaret A. Riley

10. The Microbial Pangenome: Implications for Vaccine Development 83

Annalisa Nuccitelli, Claudio Donati, Michele A. Barocchi, and Rino Rappuoli

11. Metagenomic Insights into Bacterial Species 89

Konstantinos T. Konstantinidis

12. Reports of Ad Hoc Committees for the Reevaluation of the Species Definitionin Bacteriology 99

Erko Stackebrandt

13. Metagenomic Approaches for the Identification of Microbial Species 105

David M. Ward, Melanie C. Melendrez, Eric D. Becraft, Christian G. Klatt, Jason M. Wood,and Frederick M. Cohan

Part 3 Metagenomics

14. Microbial Ecology in the Age of Metagenomics 113

Jianping Xu

15. The Enduring Legacy of Small Subunit rRNA in Microbiology 123

Susannah G. Tringe and Philip Hugenholtz

16. Pitfalls of PCR-Based rRNA Gene Sequence Analysis: An Update on Some Parameters 129

Erko Stackebrandt

17. Empirical Testing of 16S PCR Primer Pairs Reveals Variance in Target Specificityand Efficacy not Suggested by In Silico Analysis 135

Sergio E. Morales and William E. Holben

18. The Impact of Next-Generation Sequencing Technologies on Metagenomics 143

George M. Weinstock

19. Accuracy and Quality of Massively Parallel DNA Pyrosequencing 149

Susan M. Huse and David B. Mark Welch

Contents ix

20. Environmental Shotgun Sequencing: Its Potential and Challenges for Studyingthe Hidden World of Microbes 157

Jonathan A. Eisen

21. A Comparison of Random Sequence Reads Versus 16S rDNA Sequencesfor Estimating the Biodiversity of a Metagenomic Sample 163

Chaysavanh Manichanh, Charles E. Chapple, Lionel Frangeul, Karine Gloux,Roderic Guigo, and Joel Dore

22. Metagenomic Libraries for Functional Screening 171

Trine Aakvik, Rahmi Lale, Mark Liles, and Svein Valla

23. GC Fractionation Allows Comparative Total Microbial Community Analysis, EnhancesDiversity Assessment, and Facilitates Detection of Minority Populations of Bacteria 183

William E. Holben

24. Enriching Plant Microbiota for a Metagenomic Library Construction 197

Ying Zeng, Hao-Xin Wang, Zhao-Liang Geng, and Yue-Mao Shen

25. Towards Automated Phylogenomic Inference 205

Martin Wu and Jonathan A. Eisen

26. Integron First Gene Cassettes: A Target to Find Adaptive Genes in Metagenomes 217

Lionel Huang and Christine Cagnon

27. High-Resolution Metagenomics: Assessing Specific Functional Types in ComplexMicrobial Communities 225

Ludmila Chistoserdova

28. Gene-Targeted Metagenomics (GT Metagenomics) to Explore the Extensive Diversityof Genes of Interest in Microbial Communities 235

Shoko Iwai, Benli Chai, Ederson da C. Jesus, C. Ryan Penton, Tae Kwon Lee,James R. Cole, and James M. Tiedje

29. Phylogenetic Screening of Metagenomic Libraries Using Homing EndonucleaseRestriction and Marker Insertion 245

Torsten Thomas, Staffan Kjelleberg, and Pui Yi Yung

30. ArrayOme- and tRNAcc-Facilitated Mobilome Discovery: ComparativeGenomics Approaches for Identifying Rich Veins of Bacterial Novel DNA Sequences 251

Hong-Yu Ou and Kumar Rajakumar

31. Sequence-Based Characterization of Microbiomes by Serial Analysisof Ribosomal Sequence Tags (SARST) 265

Zhongtang Yu and Mark Morrison

x Contents

Part 4 Consortia and Databases

32. The Metagenomics of Plant Pathogen-Suppressive Soils 277

Jan Dirk van Elsas, Anna Maria Kielak, and Mariana Silvia Cretoiu

33. Soil Metagenomic Exploration of the Rare Biosphere 287

Tom O. Delmont, Laure Franqueville, Samuel Jacquiod, Pascal Simonet,and Timothy M. Vogel

34. The BIOSPAS Consortium: Soil Biology and Agricultural Production 299

Luis Gabriel Wall

35. The Human Microbiome Project 307

George M. Weinstock

36. The Ribosomal Database Project: Sequences and Softwarefor High-Throughput rRNA Analysis 313

James R. Cole, Qiong Wang, Benli Chai, and James M. Tiedje

37. The Metagenomics RAST Server: A Public Resource for the AutomaticPhylogenetic and Functional Analysis of Metagenomes 325

Elizabeth M. Glass and Folker Meyer

38. The EBI Metagenomics Archive, Integration and Analysis Resource 333

C. Hunter, G. Cochrane, R. Apweiler, S. Hunter

Part 5 Computer-Assisted Analysis

39. Comparative Metagenome Analysis Using MEGAN 343

Daniel H. Huson and Suparna Mitra

40. Phylogenetic Binning of Metagenome Sequence Samples 353

Alice Carolyn McHardy and Kaustubh Patil

41. Gene Prediction in Metagenomic Fragments with Orphelia: A Large-ScaleMachine Learning Approach 359

Katharina H. Hoff, Maike Tech, Thomas Lingner, Rolf Daniel,Burkhard Morgenstern, and Peter Meinicke

42. Binning Metagenomic Sequences Using Seeded GSOM 369

Ching-Hung Tseng, Chon-Kit Kenneth Chan, Arthur L. Hsu, Saman K. Halgamuge,and Sen-Lin Tang

Contents xi

43. Iterative Read Mapping and Assembly Allows the Use of a More DistantReference in Metagenome Assembly 379

Bas E. Dutilh, Martijn A. Huynen, Jolein Gloerich, and Marc Strous

44. Ribosomal RNA Identification in Metagenomic and Metatranscriptomic Datasets 387

Ying Huang, Weizhong Li, Patricia W. Finn, and David L. Perkins

45. SILVA: Comprehensive Databases for Quality Checked and Aligned RibosomalRNA Sequence Data Compatible with ARB 393

Elmar Prusse, Christian Quast, Pelin Yilmaz, Wolfgang Ludwig, Jorg Peplies,and Frank Oliver Glockner

46. ARB: A Software Environment for Sequence Data 399

Ralf Westram, Kai Bader, Elmar Prusse, Yadhu Kumar, Harald Meier,Frank Oliver Glockner, and Wolfgang Ludwig

47. The Phyloware Project: A Software Framework for Phylogenomic Virtue 407

Daniel N. Frank and Charles E. Robertson

48. MetaSim: A Sequencing Simulator for Genomics and Metagenomics 417

Daniel C. Richter, Felix Ott, Alexander F. Auch, Ramona Schmid, and Daniel H. Huson

49. ClustScan: An Integrated Program Package for the Detection andSemiautomatic Annotation of Secondary Metabolite Clusters in Genomicand Metagenomic DNA Datasets 423

John Cullum, Antonio Starcevic, Janko Diminic, Jurica Zucko, Paul F. Long,and Daslav Hranueli

50. MetaGene: Prediction of Prokaryotic and Phage Genesin Metagenomic Sequences 433

Hideki Noguchi

51. Primers4clades: A Web Server to Design Lineage-Specific PCR Primersfor Gene-Targeted Metagenomics 441

Bernardo Sachman-Ruiz, Bruno Contreras-Moreira, Enrique Zozaya,Cristina Martınez-Garza, and Pablo Vinuesa

52. A Parsimony Approach to Biological Pathway Reconstruction/Inferencefor Metagenomes 453

Yuzhen Ye and Thomas G. Doak

53. ESPRIT: Estimating Species Richness Using Large Collectionsof 16S rRNA Data 461

Yijun Sun, Yunpeng Cai, Li Liu, Fahong Yu, and William Farmerie

xii Contents

Part 6 Complementary Approaches

54. Metagenomic Approaches in Systems Biology 475

Marıa-Eugenia Guazzaroni and Manuel Ferrer

55. Towards “Focused” Metagenomics: A Case Study Combining DNA Stable-IsotopeProbing, Multiple Displacement Amplification, and Metagenomics 491

Yin Chen, Marc G. Dumont, Joshua D. Neufeld, and J. Colin Murrell

56. Suppressive Subtractive Hybridization Reveals Extensive Horizontal Transferin the Rumen Metagenome 497

Elizabeth A. Galbraith, Dionysios A. Antonopoulos, Karen E. Nelson, and Bryan A. White

Part 6A Microarrays

57. GeoChip: A High-Throughput Metagenomics Technology for DissectingMicrobial Community Functional Structure 509

Joy D. van Nostrand, Zhili He, and Jizhong Zhou

58. Phylogenetic Microarrays (PhyloChips) For Analysis of ComplexMicrobial Communities 521

Eoin L. Brodie

59. Phenomics and Phenotype Microarrays: ApplicationsComplementing Metagenomics 533

Barry R. Bochner

60. Microbial Persistence in Low-Biomass, Extreme Environments: The Great Unknown 541

Parag Vaishampayan, James N. Benardini, Myron T. La Duc, and Kasthuri Venkateswaran

61. Application of Phylogenetic Oligonucleotide Microarrays in Microbial Analysis 551

Pankaj Trivedi and Nian Wang

Part 6B Metatranscriptomics

62. Isolation of mRNA From Environmental Microbial Communitiesfor Metatranscriptomic Analyses 569

Peer M. Schenk

63. Comparative Day/Night Metatranscriptomic Analysis of Microbial Communitiesin the North Pacific Subtropical Gyre 575

Rachel S. Poretsky and Mary Ann Moran

Contents xiii

64. The “Double-RNA” Approach to Simultaneously Assess the Structure and Functionof a Soil Microbial Community 587

Tim Urich and Christa Schleper

65. Soil Eukaryotic Diversity: A Metatranscriptomic Approach 597

Roland Marmeisse, Julie Bailly, Coralie Damon, Frederic Lehembre, Marc Lemaire,Micheline Wesolowski-Louvel, and Laurence Fraissinet-Tachet

Part 6C Metaproteomics

66. Proteomics for the Analysis of Environmental Stress Responses in Prokaryotes 605

Ksenia J. Groh, Victor J. Nesatyy, and Marc J.-F. Suter

67. Microbial Community Proteomics 627

Paul Wilmes

68. Synchronicity between Population Structure and Proteome Profiles:A Metaproteomic Analysis of Chesapeake Bay Bacterial Communities 637

Jinjun Kan, Thomas E. Hanson, and Feng Chen

69. High-Throughput Cyanobacterial Proteomics: Systems-Level ProteomeIdentification and Quantitation 645

Saw Yen Ow and Phillip C. Wright

70. Protein Expression Profile of an Environmentally Important Bacterial Strain:The Chromate Response of Arthrobacter Species Strain FB24 663

Kristene L. Henne, Joshua E. Turse, Cindy H. Nakatsu, and Allan E. Konopka

Part 6D Metabolomics

71. The Small-Molecule Dimension: Mass-spectrometry-based Metabolomics,Enzyme Assays, and Imaging 677

Trent R. Northen

72. Metabolomics: High-Resolution Tools Offer to Follow Bacterial Growthon a Molecular Level 683

Lucio Marianna, Agnes Fekete, Moritz Frommberger, and Philippe Schmitt-Kopplin

73. Metabolic Profiling of Plant Tissues by Electrospray Mass Spectrometry 697

Heather Walker

74. Metabolite Identification, Pathways, and Omic Integration Using OnlineDatabases and Tools 709

Matthew P. Davey

xiv Contents

Part 6E Single-Cell Analysis

75. Application of Cytomics to Separate Natural Microbial Communitiesby their Physiological Properties 727

Susann Muller and David R. Johnson

76. Capturing Microbial Populations for Environmental Genomics 735

Martha Schattenhofer and Annelie Wendeberg

77. Microscopic Single-Cell Isolation and Multiple DisplacementAmplification of Genomes from Uncultured Prokaryotes 741

Peter Westermann and Thomas Kvist

Index 747

Preface

In the last 25 years, microbiology and molecular micro-bial ecology have undergone drastic transformationsthat changed the microbiologist’s view of how to studymicroorganisms. Previously, the main problem was theassumption that microorganisms needed to be culturable,in order to classify them and study their metabolic andorganismal diversity. The heart of this transformation wasthe convincing demonstration that the yet-unculturableworld was far greater than the culturable one. In fact, thenumber of microbial genomes has been estimated from2000 to 18,000 genomes per gram of soil. In 1985, anexperimental advance radically changed our perception ofthe microbial world. After Carl Woese showed that rRNAgenes could be used to derive evolutionary relationships,phylogenetic “trees” and evolutionary chronometers,Norman Pace and colleagues created a new chapter inmolecular microbial ecology, using the direct analysisof rRNA sequences in the environment to describe thediversity of microorganisms without culturing (Han-delsman, 2004). The next major step forward was thedevelopment of the PCR reaction, to amplify rRNA genesfor subsequent sequence analysis and classification. Thesubsequent major advance was the notion that one couldextract total DNA or RNA from environmental samples,including culturable and yet unculturable organisms, andclone it into a suitable vector for introduction into aculturable organism, followed by analysis by using highthroughput shotgun DNA sequencing of cloned DNA, orby direct sequencing The idea of cloning DNA directlyfrom environmental samples was first proposed by Page;this method was coined “metagenomics” by Handelsmanet al. in 1994, and is now used in many laboratoriesworldwide to study diversity and for the isolation ofnovel medical and industrial compounds.

These recent studies are reviewed in this book andthe companion book, Handbook of Molecular MicrobialEcology II: Metagenomics in Different Habitats . Insteadof relying only on a limited number of (long) reviewarticles on selected topics, this book provides reviews

as well as a large number of case studies, mostly basedon original publications and written by expert “at-the-bench” scientists from more than 20 different countries.Both books highlight the databases and computer pro-grams used in each study, by listing them at the end ofthe chapter, together with their sites. This special featureof both books, facilitates the computer-assisted analysis ofthe vast amount of data generated by metagenomic studies.In addition, metagenomic studies in a variety of habitatsare described, primarily in Volume II, which present alarge number of system dependent different approaches ingreatly differing habitats. The latter also results in the pre-sentation of multiple biological systems which are inter-esting to microbial ecologists and microbiologists in theirown right. Both books should be of interest to scien-tists in the fields of soil, water, medicine and industrywho are or are contemplating using metagenomics andcomplementary approaches to address academic, medical,or industrial questions about bacterial communities fromvaried habitats, but also to those interested in particularbiological systems in general.

ACKNOWLEDGMENTS

For their support of this project, I gratefully acknowledge:

The Laboratory for Plant Microbe Interactions (LIPM),

the Institut National de Recherche de Agri-culture (INRA), and

the Centre National de Recherche Scien-tifique (CNRS).

I would like to thank Claude Bruand for his help withthe computer work.

Frans J. de BruijnCastanet, Tolosan, FranceMarch 2011

xv

Contributors

Editor

Frans J. de Bruijn , Laboratory of Plant Micro-organism Interaction, CNRS-INRA, Castanet Tolosan,France

Authors

Trine Aakvik , Norwegian University of Science and Technology, Trondheim, Norway

Roey Angel , Ben-Gurion University of the Negev, Israel

Dionysios A. Antonopoulos , Argonne National Laboratory, Argonne, Illinois

R. Apweiler , The Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom

Alexander F. Auch , University of Tubingen, Tubingen, Germany

Kai Bader , Technical University of Munchen, Freising, Germany

Julie Bailly , University of Lyon, Villeurbanne Cedex, France

Michele A. Barocchi , Novartis Vaccines and Diagnostics, Siena, Italy

Eric D. Becraft , Montana State University, Bozeman, Montana

James N. Benardini , California Institute of Technology, Pasadena, California

Martin J. Blaser , New York University School of Medicine, New York, New York

Barry R. Bochner , Biolog, Inc., Hayward, California

Eoin L. Brodie, Lawrence Berkeley National Laboratory, Berkeley, California

Christine Cagnon , Universite de Pau et des Pays de l’Adour, Pau, France

Yunpeng Cai , University of Florida, Gainesville, Florida

Benli Chai , Michigan State University, East Lansing, Michigan

Chon-Kit Kenneth Chan , University of Melbourne, Melbourne, Victoria, Australia

Charles E Chapple, Center for Genomic Regulation, Barcelona, Spain

Feng Chen , Biotechnology Institute, University of Maryland, Baltimore, Maryland

Qiang Chen , Sichuan Agricultural University, Ya’an Sichuan, China

Yin Chen , University of Warwick, Coventry, United Kingdom

Ludmila Chistoserdova , University of Washington, Seattle, Washington

Pooja Chokshi , College of Arts and Sciences, Tufts University, Medford, Massachusetts

G. Cochrane, The Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom

Frederick M. Cohan , Wesleyan University, Middletown, Connecticut

xvii

xviii Contributors

James R. Cole, Michigan State University, East Lansing, Michigan

Bruno Contreras-Moreira, Upper Counsel of Scientific Investigations, Zaragoza, Spain

Mariana Silvia Cretoiu , University of Groningen, Haren, The Netherlands

John Cullum, University of Kaiserslautern, Kaiserslautern, Germany

Coralie Damon , University of Lyon, Villeurbanne, Lyon, France

Rolf Daniel , Georg August University at Gottingen, Gottingen, Germany

Matthew P. Davey , University of Cambridge, Cambridge, United Kingdom

Tom O. Delmont , Environmental Microbial Genomics Group, Ecully, France.

Janko Diminic, University of Zagreb, Zagreb, Croatia

Thomas G. Doak , Indiana University, Bloomington, Indiana

Claudio Donati , Novartis Vaccines and Diagnostics, Siena, Italy

Joel Dore, INRA/CNRS, Jouy-en-Josas, France

Marc G. Dumont , University of Warwick, Coventry, United Kingdom

Bas E. Dutilh , Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands

Jonathan A. Eisen , University of California—Davis, Davis, California

Jan Dirk van Elsas , University of Groningen, Haren, The Netherlands

William Farmerie, University of Florida, Gainesville, Florida

Agnes Fekete, Institute of Ecological Chemistry, Neuherberg, Germany

Manuel Ferrer , Institute of Catalysis, Madrid, Spain

David P. Fewer , University of Helsinki, Helsinki, Finland

Patricia W. Finn , University of California—San Diego, La Jolla, California

Laurence Fraissinet-Tachet , University of Lyon, Villeurbanne, Lyon, France

Daniel N. Frank , University of Colorado, Boulder, Colorado

Lionel Frangeul , Genopole, Pasteur Institute, Paris, France

Laure Franqueville, Environmental Microbial Genomics Group, Ecully, France

Thomas E. Freitag , Uppsala BioCenter, Uppsala, Sweden

Moritz Frommberger , Institute of Biological Chemistry, Neuherberg, Germany

Elizabeth A. Galbraith , Agtech Products, USA Inc., Waukesha, Wisconsin

Zhao-Liang Geng , Kunming Institute of Botany, the Chinese Academy of Sciences, Yunnan, China

Osnat Gillor , Ben-Gurion University of the Negev, Beersheba, Israel

Elizabeth M. Glass , The University of Chicago, Chicago, Illinois

Frank Oliver Glockner , Max Planck Institute for Marine Microbiology, Bremen, Germany; JacobsUniversity, Bremen, Germany

Jolein Gloerich , Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands

Karine Gloux , INRA-CNRS, Jouy-en-Josas, France

Ksenia J. Groh , Swiss Federal Institute of Science and Technology, Duebendorf, Switzerland

Marıa-Eugenia Guazzaroni , Institute of Catalysis, Madrid, Spain

Roderic Guigo, Center for Genomic Regulation, Barcelona, Spain

Saman K. Halgamuge, The University of Melbourne, Melbourne, Victoria, Australia

Thomas E. Hanson , University of Delaware, Newark, Delaware

Kristene L. Henne, Purdue University, West Lafayette, Indiana

Zhili He, University of Oklahoma, Norman, Oklahoma

Katharina H. Hoff , Medical Center Gottingen, Gottingen, Germany

Contributors xix

William E. Holben , The University of Montana, Missoula, Montana

Daslav Hranueli , University of Zagreb, Zagreb, Croatia

Arthur L. Hsu , The University of Melbourne, Melbourne, Victoria, Australia

Lionel Huang , Universite de Pau et des Pays de l’Adour, Pau, France

Ying Huang , University of California—San Diego, La Jolla, California

Philip Hugenholtz , Department of Energy Joint Genome Institute, Walnut Creek, California

Daniel H. Huson , University of Tubingen, Tubingen, Germany

C. Hunter , The Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom

S. Hunter , The Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom

Susan M. Huse, Marine Biological Laboratory at Woods Hole, Woods Hole, Massachusetts

Martijn A. Huynen , Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands

Satoshi Ishii , The University of Tokyo, Tokyo, Japan

Shoko Iwai , Michigan State University, East Lansing, Michigan

Samuel Jacquiod , Environmental Microbial Genomics Group, Ecully, France

Ederson da C. Jesus , Michigan State University, East Lansing, Michigan; University of Para, Belem,Brazil

David R. Johnson , Swiss Federal Institute of Technology Zurich (ETHZ), Zurich, Switzerland

Koji Kadota , The University of Tokyo, Tokyo, Japan

Jinjun Kan , University of Southern California, Los Angeles, California

Anna Maria Kielak , University of Groningen, Haren, The Netherlands

Staffan Kjelleberg , The University of New South Wales, Sydney, Australia

Christian G. Klatt , Montana State University, Bozeman, Montana

Konstantinos T. Konstantinidis , Georgia Institute of Technology, Atlanta, Georgia

Allan E. Konopka , Pacific Northwest National Laboratory, Richland, Washington

Yadhu Kumar , Technical University of Munchen, Freising, Germany

Thomas Kvist , BioGasol ApS, Ballerup, Denmark

Myron T. La Duc, California Institute of Technology, Pasadena, California

Rahmi Lale, Norwegian University of Science and Technology, Trondheim, Norway

Tae Kwon Lee, Yonsei University, Seoul, Republic of Korea

Frederic Lehembre, University of Lyon, Villeurbanne, Lyon, France

Marc Lemaire, University of Lyon, Villeurbanne, Lyon, France

Qiongfang Li , Sichuan Agricultural University, Ya’an Sichuan, China

Weizhong Li , University of California—San Diego, La Jolla California

Li Liu , University of Florida, Gainesville, Florida

Mark Liles , Auburn University, Auburn, Alabama

Kristina Lindstrom, University of Helsinki, Helsinki, Finland

Thomas Lingner , Georg August University of Gottingen, Gottingen, Germany

Paul F. Long , University of London, London, United Kingdom

Wolfgang Ludwig , Technical University Munich, Freising, Germany

Shahid Mahmood , Uppsala BioCenter, Uppsala, Sweden

Chaysavanh Manichanh , University Hospital Vall d’Hebron, Barcelona, Spain

Lucio Marianna, Institute of Ecological Chemistry, Neuherberg, Germany

Roland Marmeisse, University of Lyon, Villeurbanne, Lyon, France

xx Contributors

Cristina Martınez-Garza , Autonomous University of the State of Morelos, Morelos, Mexico

Alice Carolyn McHardy , Max-Planck Institut for Information, Saarbrucken, Germany

Harald Meier , Technical University of Munchen, Freising, Germany

Peter Meinicke, Georg August University of Gottingen, Gottingen, Germany

Melanie C. Melendrez , Montana State University, Bozeman, Montana

Folker Meyer , The University of Chicago, Chicago, Illinois

Suparna Mitra, Tubingen University, Tubingen, Germany

Mary Ann Moran , University of Georgia, Athens, Georgia

Sergio E. Morales , The University of Montana, Missoula, Montana

Burkhard Morgenstern , Georg August University of Gottingen, Gottingen, Germany

Mark Morrison , The Ohio State University, Columbus, Ohio

Susann Muller , Helmholtz Center for Environmental Research, UFZ, Leipzig, Germany

J. Colin Murrell , University of Warwick, Coventry, United Kingdom

Cindy H. Nakatsu , Purdue University, West Lafayette, Indiana

Andrea Maria Amaral Nascimento, Federal University of General Mines, Minas Gerasis, Brazil

Karen E. Nelson , J. Craig Venter Institute, Rockville, Maryland

Victor J. Nesatyy , EPFL, Lausanne, Switzerland

Joshua D. Neufeld , University of Warwick, Coventry, United Kingdom; University of Waterloo,Ontario, Canada

Hideki Noguchi , Tokyo Institute of Technology, Yokohama, Japan

Trent R. Northen , Lawrence Berkeley National Laboratory, Berkeley, California

Carlos W. Nossa , New York University School of Medicine, New York, New York

Joy D. van Nostrand , University of Oklahoma, Norman, Oklahoma

Annalisa Nuccitelli , Novartis Vaccines and Diagnostics, Siena, Italy

William E. Oberdorf , New York University School of Medicine, New York, New York

Felix Ott , Max-Planck Institute for Developmental Biology, Tubingen, Germany

Hong-Yu Ou , Shanghai Jiaotong University, Shanghai, China

Lise Øvreas , University of Bergen, Bergen, Norway

Saw Yen Ow , The University of Sheffield, Sheffield, United Kingdom

Kaustubh Patil , Max-Planck Institut fur Informatik, Saarbrucken, Germany

Anna Pei , Washington University College of Arts and Sciences, St. Louis, Missouri

Zhiheng Pei , New York University School of Medicine, New York, New York

C. Ryan Penton , Michigan State University, East Lansing, Michigan

Jorg Peplies , Ribocon GmbH, Bremen, Germany

David L. Perkins , University of California–San Diego, La Jolla, California

Rachel S. Poretsky , University of Georgia, Athens, Georgia

James I. Prosser , University of Aberdeen, Aberdeen, Scotland

Elmar Prusse, Max Planck Institute for Marine Microbiology, Bremen, Germany

Christian Quast , Max Planck Institute for Marine Microbiology, Bremen, Germany

Kumar Rajakumar , University of Leicester, Leicester, United Kingdom

Rino Rappuoli , Novartis Vaccines and Diagnostics, Siena, Italy

Daniel C. Richter , University of Tubingen, Tubingen, Germany

Margaret A. Riley , University of Massachusetts, Amherst, Massachusetts

Contributors xxi

Charles E. Robertson , University of Colorado, Boulder, Colorado

David M. Rosmarin , New York University School of Medicine, New York, New York

Bernardo Sachman-Ruiz , Autonomous National Vniversity of Mexico, Cuernavaca, Morelos, Mexico

Martha Schattenhofer , Helmholtz Centre for Environmental Research, Leipzig, Germany

Peer M. Schenk , The University of Queensland, St. Lucia, Queensland, Australia

Christa Schleper , University of Vienna,Vienna, Austria; University of Bergen, Bergen, Norway

Ramona Schmid , Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany

Philippe Schmitt-Kopplin , Institute of Ecological Chemistry, Neuherberg, Germany

Keishi Senoo, The University of Tokyo, Tokyo, Japan

Yue-Mao Shen , Kunming Institute of Botany, the Chinese Academy of Sciences, Yunnan, China

Pascal Simonet , Environmental Microbial Genomics Group, Ecully, France

Menachem Y. Sklarz , Ben-Gurion University of the Negev, Beersheba, Israel

Ines M. Soares , Ben-Gurion University of the Negev, Beersheba, Israel

Erko Stackebrandt , German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig,Germany

Antonio Starcevic, University of Zagreb, Zagreb, Croatia

Marc Strous , University of Bielefeld, Bielefeld, Germany

Yijun Sun , University of Florida, Gainesville, Florida

Marc J.-F. Suter , Swiss Federal Institute of Science and Technology, Duebendorf, Switzerland

Sen-Lin Tang , Academia Sinica, Taiwan

Maike Tech , Georg August University of Gottingen, Gottingen, Germany

Torsten Thomas , The University of New South Wales, Sydney, Australia

James M. Tiedje, Michigan State University, East Lansing, Michigan

Vigdis L. Torsvik , University of Bergen, Bergen, Norway

Susannah G. Tringe, U. S. Departement of Energy Joint Genome Institute, Walnut Creek, California

Pankaj Trivedi , University of Florida, Lake Alfred, Florida

Ching-Hung Tseng , Academia Sinica, Taiwan

Joshua E. Turse, Pacific Northwest National Laboratory, Richland, Washington

Tim Urich , University of Vienna,Vienna, Austria; University of Bergen, Bergen, Norway

Parag Vaishampayan , California Institute of Technology, Pasadena, California

Svein Valla , Norwegian University of Science and Technology, Trondheim, Norway

Kasthuri Venkateswaran , California Institute of Technology, Pasadena, California

Pablo Vinuesa , Autonomous National University of Mexico, Cuernavaca, Morelos, Mexico

Timothy M. Vogel , Environmental Microbial Genomics Group, Ecully, France

Heather Walker , University of Sheffield, Western Bank, Sheffield, United Kingdom

Luis Gabriel Wall , National University of Quilmes, Bernal, Buenos Aires, Argentina

Hao-Xin Wang , Kunming Institute of Botany, the Chinese Academy of Sciences, Yunnan, China

Nian Wang , University of Florida, Lake Alfred, Florida

Qiong Wang , Center for Microbial Ecology, Michigan State University, Michigan

David M. Ward , Montana State University, Bozeman, Montana

George M. Weinstock , Washington University School of Medicine, St. Louis, Missouri

David B. Mark Welch, Marine Biological Laboratory at Woods Hole, Woods Hole, Massachusetts

Annelie Wendeberg , Helmholtz Center for Environmental Research, Leipzig, Germany

xxii Contributors

Micheline Wesolowski-Louvel , University of Lyon, Villeurbanne, Lyon, France

Peter Westermann , Aalborg University, Ballerup, Denmark

Ralf Westram , Technical University of Munchen, Freising, Germany

Bryan A. White, University of Illinois at Urbana-Champaign, Urbana, Illinois

Paul Wilmes , Department of Environment and Agro-Biotechnologies; Gabriel Lippmann PublicResearch Center, Luxembourg, Belgium

Jason M. Wood , Montana State University, Bozeman, Montana

Phillip C. Wright , The University of Sheffield, Sheffield, United Kingdom

Martin Wu , University of Virginia, Charlottesville, Virginia

Jianping Xu, McMaster University, Ontario, Hamilton, Canada

Liying Yang , New York University School of Medicine, New York, New York

Yuzhen Ye, Indiana University, Bloomington, Indiana

Pelin Yilmaz , Max Planck Institute for Marine Microbiology, Bremen, Germany; Jacobs University,Bremen, Germany

Fahong Yu , University of Florida, Gainesville, Florida

Pui Yi Yung , The University of New South Wales, Sydney, Australia

Zhongtang Yu , Ohio State University, Columbus, Ohio

Ying Zeng , Kunming Institute of Botany, the Chinese Academy of Sciences, Yunnan, China

Xiaoping Zhang , Sichuan Agricultural University, Ya’an Sichuan, China

Ling Zou , Sichuan Agricultural University, Ya’an Sichuan, China

Jizhong Zhou , University of Oklahoma, Norman, Oklahoma

Enrique Zozaya , Autonomous National University of Mexico, Cuernavaca, Morelos, Mexico

Jurica Zucko, University of Kaiserslautern, Kaiserslautern, Germany; University of Zagreb, Zagreb,Croatia

Chapter 1

Introduction

Frans J. de Bruijn

In this first volume of the Handbook, metagenomics isintroduced, together with computer-assisted analysis,information on consortia and databases, and as a numberof complementary methods, such as microarrays,metatranscriptomics, metaproteomics, metabolomics,phenomics (the “omics”), and single-cell analysis.

Part 1, “Background Chapters,” contains a number ofchapters on nonmetagenomic methods, such as differentgenomic fingerprinting techniques and their analysis andlevel of resolution, as well as the first approach to metage-nomics (Chapter 2). All these methods are still used today.

In Part 2, “The Species Concept,” several expertsexamine the parameters to call something a new speciesand provide suggestions to authors when it is proper tocall a novel isolate [operating taxonomic unit (OTU)] anew species. The recommendations of two expert meet-ings on the topic are summarized in another chapter in thispart describing the 70% DNA–DNA hybridization levelas essential in the species concept. This discussion is veryrelevant to all phylogenetic studies in both volumes of theHandbook.

In Part 3, metagenomics is introduced and a numberof practical parameters of this technique are outlined. Anintroduction to metagenomics and the other “omics” ispresented in Chapter 14. Three subsequent chapters dealwith the 16S rRNA gene as phylogenetic marker and alsoexamine the pitfalls of its use. Three chapters describe theimpact of next-generation sequencing on metagenomics,examine its accuracy and quality of reads, and reviewthe potential and challenges of environmental shotgunsequences for studying the hidden world of microbes.Metagenomics can involve (a) the generation and analy-sis of clone libraries which can be screened for particularproperties and (b) random sequencing of metagenomic

Handbook of Molecular Microbial Ecology, Volume I: Metagenomics and Complementary Approaches, First Edition. Edited by Frans J. de Bruijn. 2011 Wiley-Blackwell. Published 2011 by John Wiley & Sons, Inc.

DNA. The former is discussed in an article on vectortools and functional screening of metagenomic libraries(see also Parts 6 and 7, Vol. II). The latter is used inmany other articles in the Handbook. The remaining arti-cles in this section introduce various technical aspectsof metagenomics, as well as novel approaches such asgene-targeted metagenomics, using homing endonucleaserestriction and marker insertion for phylogenetic stud-ies, finding integrons, arrayOme- and tRNAcc-facilitatedmobilome discovery, and improved serial analysis of V1ribosomal sequence tags (SARST-V1) to study bacterialdiversity. A plethora of other studies in various habitatsare presented in Volume II of this Handbook.

In Part 4, some consortia and databases are discussed,including the Metacontrol consortium focusing on themetagenomics of suppressive soils, the Terragenomeconsortium to provide a metagenomic shotgun andphosmid sequencing analysis of a “reference” soil, andthe Argentinian BIOSPAS consortium aimed at bringingtogether a group of scientists employing metagenomic andassociated approaches. This is followed by a descriptionof the Human Gut Microbiome Initiative (HGMI) and therelated Human Microbiome Project (HMP). Chapter 36in this part describes the Ribosomal Database Project, anirreplaceable source for phylogenetic studies, using therRNA genes as target (see Chapter 15, Vol. I). The finalchapter in this part describes the Metagenomics RASTserver a a public resource for automated phylogeneticand functional analysis of Metagenomes.

In Part 5, a smorgasbord of computer programs ispresented essential for the analysis of (meta)genomicdata. Clearly, computer-assisted analysis is a crucialcomponent of every metagenomic project, and progressin the field is dependent on creating programs anddatabases for ever-growing datasets and can be thelimiting factor for large metagenomic, transcriptomic,

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2 Chapter 1 Introduction

proteomic, and metabolomic projects. It equals in impor-tance to the development of higher throughput novelsequencing methods (see Chapter 18, Vol. I). The authorsin Part 5, as well as all other authors, have been askedto highlight the programs and web sites used in theirchapters; therefore in addition to the limited programshighlighted in Part 5, a wealth of further information andother programs can be found in the chapters in VolumesI and II.

In Part 6 a number of complementary approches tometagenomics are presented, including metagenomicsapproaches in systems biology, the use of stable isotopeprobing, and subtractive hybridization.

In Part 6A the use of microarrays, includingphylochips and geochips and metagenomic arrays, isdiscussed and examples in different habitats, such asNASA rocket cleanrooms, are given. This part alsocontains a chapter on phenotypic arrays or “phenomics,”another “omic” technique, which can reveal the metaboliccapacity of microbes in microplates.

In Part 6B, some examples of metatranscriptomicanalysis are presented, which permit a glimpse into themetagene expression profile in various environments,such as the symbiotic protist community in Reticulitermesand comparative day and night metatranscriptomics ofmicrobial communities in the North Pacific. In additiona “double RNA” approach is presented to simultane-ously assess the structure and function of microbial

communities, and one chapter on the metatranscriptomicsof eukaryotes is included.

In Part 6C, metaproteomics approaches are high-lighted, and examples are presented on the proteomics ofmicrobial stress responses, the metaproteomic analysis ofChesapeake Bay microbial communities, high-throughputproteomics in cyanobacteria, and global proteomicanalysis of the chromate response in Arthrobacter .

In Part 6D, metabolomics is highlighted, whichrequires more sophisticated tools such as mass spec-trometry. Examples include (a) two chapters that reviewthe small molecule dimension and high-resolution toolsto monitor bacterial growth on a molecular level, (b)one chapter on metabolomics in plants, where themetabolomics techniques are well established, and (c)a chapter on metabolite identification, pathways and“omic” integration using databases and other tools.

In Part 6E a highly specialized complementaryapproach is described, namely the isolation and use ofsingle cells for metagenomic and other analysis.

None of the parts described above are comprehensive.They mainly give a short insight about what one can doin addition to metagenomics to extract more functionaldata from the system under study to answer the followingquestions: “Who is there?” and “What are they doing?”An attempt was made to select studies in very differenthabitats, and a variety of approaches are highlighted. Thisis continued and expanded upon in Volume II.

Part 1

Background Chapters

Chapter 2

DNA Reassociation Yields Broad-ScaleInformation on MetagenomeComplexity and Microbial Diversity

Vigdis L. Torsvik and Lise Øvreas

2.1 INTRODUCTION

2.1.1 Evolution and Developmentof DiversityThere are close relationships between microbial evo-lution, diversity, and ecology. Prokaryotic organismshave evolved through 3.8 billion years [Rosing, 1999]in response to varying geological, geochemical, andclimatic conditions. For approximately half of theirlife’s history, they resided alone on Earth. Due totheir great metabolic flexibility, short generation time,and ability to exchange genes over deep phylogeneticbarriers, their ability to adapt and evolve are superior.This means that virtually every (micro) environmenton Earth with physical–chemical conditions that cansustain life is occupied by prokaryotic organisms [seeVol. II]. It is therefore not surprising that the biodiver-sity on Earth is dominated by these organisms, whichconstitute two of the three primary domains of life, theArchaea and Bacteria [Woese, 1987; Woese and Fox,1977]. Their ecological consequences are huge, becauseecosystem processes to a large extent are regulated bymicrobial communities. Important for understandingcomplex ecosystem functioning is to identify the primarydrivers of microbial diversity and community structure.According to ecological theories, relationships betweenecosystem functioning and diversity can partly beexplained by the resource heterogeneity hypothesis andthe “insurance hypothesis” [Yachi and Loreau, 1999]. The

Handbook of Molecular Microbial Ecology, Volume I: Metagenomics and Complementary Approaches, First Edition. Edited by Frans J. de Bruijn. 2011 Wiley-Blackwell. Published 2011 by John Wiley & Sons, Inc.

insurance hypothesis suggests that high diversity protectscommunities from unstable environmental conditionsbecause the presence of diverse subpopulations notonly increases the range of conditions in which thecommunity as a whole can succeed, but also ensureslong-term attainment of the community [Boles et al.,2004].

2.1.2 Methodological Advances,Discoveries, and Issues thatPromoted Exploring theEnvironmental Community DNABefore the introduction of molecular methods in microbialecology, it was only possible to study the compositionand diversity of microbial communities by investigatingcultivated isolates. This traditional reductionist approachhas limited our understanding of microbial ecology. Ina holistic approach, the microorganisms in a communityhave been treated as one “black box.” The aims were to(a) measure collective variables like biomass, populationsizes, process rates, and diversity of cultured microor-ganisms and (b) integrate these to better understandmicrobial ecosystems. This approach was hampered bythe lack of conceptual models linking biomasses, rateof functions, and diversity to the underlying controllingfactors. During the 1970s, methods for direct countsof microorganisms using fluorescence microscopy weredeveloped [Hobbie et al., 1977]. It was then realized

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6 Chapter 2 DNA Reassociation Yields Broad-Scale Information on Metagenome Complexity

that the microbial biomass in natural environments wasorders of magnitude higher than previously anticipated,one gram of soil and sediment could harbor morethan 1010 cells. It was demonstrated that there was afactor of 2–3 orders of magnitude between the numbersof microorganisms estimated by direct counts and bycolony-forming units (cfu) [Fægri et al., 1977]. A mainquestion was why there was such a discrepancy. Oneassumption was that the majority of the microorganismsobserved in natural environments like soils and sedimentswere inactive and that those growing in the laboratoryrepresented the active populations. To investigate this,a fractionated centrifugation method for separating thebacteria from soil was developed. By microscopic countsit was estimated that the bacterial fractions contained50–80% of the bacteria present in the soil samples andthat no eukaryotic cells were present. Respiration wasused to measure the activity in the bacterial fraction,and the specific oxygen uptake rates (qO2) calculatedon the basis of microscopic counts ranged from 3 to300 µl O2 mg−1 dry weight h−1, indicating that mostof the microbial cells observed in the microscope weremetabolically active [Fægri et al., 1977]. Furthermore, theamount of DNA in the bacterial fractions (washed withsodium pyrophosphate to remove extracellular DNA)corresponded to an average DNA content per microscopiccounted cell of 8.4 fg (10−15 g). This is approximatelythe same as in Escherichia coli cells in stationary growthphase [Ritz et al., 1997; Torsvik and Goksoyr, 1978].It was therefore concluded that virtually all the cellsobserved in the microscope were viable and belongedto the metabolically active microbial community. Amain issue was then whether the cultured bacterialisolates were representative for the total environmentalcommunity or whether they constituted a small, exoticsubpopulation of microorganisms that could easily be“domesticated” and grown in the laboratory.

Early in the 1980s, ideas emerged that led to arevolution and paradigm shift in microbial ecology. Thebasic idea was that if it was possible to retrieve DNAfrom the entire microbial community, this DNA wouldin principle contain genetic information about nearly allthe organisms in the community, including both culturedand uncultured microorganisms. Major problems were(a) the lack of methods for extracting ultrapure DNAfrom “dirty” samples like soil and sediments and (b)finding tools to analyze and interpret the informationharbored in such community metagenomes. During the1980s, developments of techniques for nucleic acidanalyses advanced rapidly. The possibility to studymicrobial communities at a genomic level led to newavenues of research strategies and made it possibleto attach problems that were previously regarded asunsolvable. An advantage of analyzing nucleic acids from

microorganisms was that it was a growth-independentapproach and that the information could be used toinvestigate and compare microorganisms at differentbiological organization levels, from infraspecies andtaxon to community level.

2.1.3 Microbial Biodiversityand Metagenome DiversityDiversity can be defined at different level of biologicalorganization ranging from genomic diversity within anorganism, species diversity, and variability within andbetween species population, to community diversity[Bull, 1992; Harper and Hawksworth, 1994]. Ecologicaldiversity includes community parameters like variabilityin community structure, the number of guilds (functionaldiversity), the number of trophic levels, and complexityof interactions. Traditionally, microbial biodiversity hasbeen used to describe the variability among the organismsin an assemblage or a community. Phenotypic diversity isrelated to the variation in microbial traits, which reflectsthe expression of genes under a given set of conditions.Genetic diversity measures the total genetic potentialin the assemblage or community independent of theenvironmental conditions.

Commonly, the diversity concept based on taxaincludes both the richness (e.g., number of species) andthe evenness—that is, how evenly the individuals aredistributed among the taxa. The diversity can also beregarded as an expression of the amount of information ina biological assemblage or community [Atlas, 1984]. Thisdefinition is adopted from information technology andtakes into account both the amount of information andhow the information is distributed among the individualsin a community. It can be applied directly to geneticdiversity.

Metagenome has been defined as the collectionof genomes from the total number of microorganismsin an environmental assemblage or in a whole naturalcommunity [Handelsman et al., 1998]. Metagenomicsrefers to extraction of DNA from natural environmentalsamples and analyses of this DNA in order to gaininformation about the organisms the DNA originatedfrom. Our rationale for exploring DNA retrieved frommicrobial communities in natural environments wasthat this metagenome, being a mixture of genomesfrom an unknown number of different microorganismsin amounts corresponding to their relative abundance,ought to provide information about the microbialdiversity at the community level. DNA reassociationkinetics was expected to provide such informationbecause it could be used to assess total DNA com-plexity, and it might therefore be used as a measure