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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,
1
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