ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of HertfordshireHatfield, Hertfordshire, UK

For further volumes:http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols andmethodologies in the critically acclaimedMethods in Molecular Biology series. The series wasthe first to introduce the step-by-step protocols approach that has become the standard in allbiomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagentsneeded to complete the experiment, and followed by a detailed procedure that is supportedwith a helpful notes section offering tips and tricks of the trade as well as troubleshootingadvice. These hallmark features were introduced by series editor Dr. John Walker andconstitute the key ingredient in each and every volume of the Methods in Molecular Biologyseries. Tested and trusted, comprehensive and reliable, all protocols from the series areindexed in PubMed.

Horizontal Gene Transfer

Methods and Protocols

Edited by

Fernando de la Cruz

Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria-CSIC, Santander,Cantabria, Spain

EditorFernando de la CruzInstituto de Biomedicina y Biotecnologıa de CantabriaUniversidad de Cantabria-CSICSantander, Cantabria, Spain

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-4939-9876-0 ISBN 978-1-4939-9877-7 (eBook)https://doi.org/10.1007/978-1-4939-9877-7

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Preface

There is a fundamental difference between prokaryotes and eukaryotes in their evolutionmechanisms and, consequently, in their genetic organization. Eukaryotes evolve withinspecies, limited by sexual reproduction, while prokaryotes largely evolve by horizontalgene transfer (HGT), limited by the boundaries of gene exchange between species. Inprokaryotes, HGT is pervasive, occurs contemporarily, and is an all-important mechanismof genetic originality. It can even be stated that bacterial genome structure and evolutioncannot be understood unless we contemplate the direct effects of HGT. Conversely, HGT ineukaryotes is an evolutionary mechanism that operates at a different timescale. Mosteukaryotic species are confined within the limits of their own species by sexual reproduction.Consequently, and with due respect to affected actors, active interspecific HGT is presentlyresidual in eukaryotic organisms (not its cumulative effects, which are of extraordinaryinterest).

Not surprisingly, therefore, the technologies used to study HGT in prokaryotes andeukaryotes are vastly different and their audiences quite separate. This book concentrates ontechnologies used to study HGT in prokaryotes. The elements involved in prokaryotic HGTare called mobile genetic elements (MGEs). There is a need for individualizing and func-tionally describing active MGEs if we attempt to understand their structure, epidemiology,and evolution. In order to achieve it, updated methods for their detection and isolation areshown in Part I of this book. The main molecular processes involved in HGT are conjuga-tion, transformation, and transduction. Part II deals with methods involved in the analysis ofthese molecular processes. Again, quantitative description of these processes is required toattempt a comparative, functional description of the different processes. There is a myriad ofMGEs, and each one must have differential specifications that justify their existence in ever-evolving microbial ecosystems. Because of HGT, and the ups and downs of particularMGEs, bacterial populations adapt and evolve. Part III puts together a series of methodsto analyze the adaptation and evolution of MGEs, with special attention to bioinformaticstools, as well as methods derived from the activity of MGEs that found utilities elsewhere.Methods for the metagenomic analysis of MGEs will hopefully be the subject of a later book.

Santander, Spain Fernando de la Cruz

v

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I METHODS FOR THE DETECTION AND ISOLATION

OF MOBILE GENETIC ELEMENTS

1 Plasmid DNA Isolation and Visualization: Isolation and Characterizationof Plasmids from Clinical Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Kaitlin A. Tagg, Carola Venturini, Muhammad Kamruzzaman,Andrew N. Ginn, and Sally R. Partridge

2 Isolation and Visualization of Plasmids from Gram-PositiveBacteria of Interest in Public Health. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Ana R. Freitas, Carla Novais, Luısa Peixe, and Teresa M. Coque

3 Detection, Isolation, and Characterization of Plasmids in the Environment . . . . 39Khald Blau, Sven Jechalke, and Kornelia Smalla

4 Bacteriophage Isolation and Characterization: Phagesof Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Juan Jofre and Maite Muniesa

5 Detection and Characterization of Transposons in Bacteria. . . . . . . . . . . . . . . . . . . 81Catherine Guynet, Phan Thai Nguyen Le, Michael Chandler,and Bao Ton-Hoang

PART II METHODS TO QUANTIFY AND CHARACTERIZE HORIZONTAL

GENE TRANSFER

6 Measuring Plasmid Conjugation Using Antibiotic Selection . . . . . . . . . . . . . . . . . . 93Jorge Rodriguez-Grande and Raul Fernandez-Lopez

7 Measuring Plasmid Conjugation Using Fluorescent Reporters. . . . . . . . . . . . . . . . 99Carolina Palencia-Gandara, Raul Ruiz, and Raul Fernandez-Lopez

8 Methods to Quantify DNA Transfer in Enterococcus . . . . . . . . . . . . . . . . . . . . . . . . . 111Carla Novais, Ana R. Freitas, Ricardo Le�on-Sampedro, Luısa Peixe,and Teresa M. Coque

9 Quantifying and Characterizing Distributive Conjugal Transferin Mycobacterium smegmatis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123Ryan R. Clark, Todd A. Gray, and Keith M. Derbyshire

10 Spectrophotometric Assays to Quantify the Activity of T4SS ATPases . . . . . . . . . 135Elena Cabez�on and Ignacio Arechaga

vii

11 Identification of Relaxase-DNA Covalent Complexes and DNA StrandTransfer Reaction Products by Polyacrylamide Gel Electrophoresis . . . . . . . . . . . 145Marıa Lucas and Gabriel Moncalian

12 First Biochemical Steps on Bacterial Transposition Pathways . . . . . . . . . . . . . . . . . 157Catherine Guynet, Emilien Nicolas, Bao Ton-Hoang, Jean-Yves Bouet,and Bernard Hallet

13 Natural Transformation in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179Yoko Komiyama and Sumio Maeda

14 Integron Identification in Bacterial Genomes and CassetteRecombination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189Claire Vit, Celine Loot, Jose Antonio Escudero, Aleksandra Nivina,and Didier Mazel

15 Methods to Identify and Analyze Vesicle-Protected DNA Transfer . . . . . . . . . . . . 209Alba Blesa and Jose Berenguer

16 Measuring Plasmid Stability in Gram-Negative Bacteria. . . . . . . . . . . . . . . . . . . . . . 223Damian Lobato-Marquez

17 Methods for the Analysis and Characterization of DefenseMechanisms Against Horizontal Gene Transfer: CRISPR Systems . . . . . . . . . . . . 235Alicia Calvo-Villamanan, Aude Bernheim, and David Bikard

PART III OMICS, BIOINFORMATICS, MATHEMATICAL MODELING,AND TECHNOLOGICAL APPLICATIONS OF HGT

18 The Mobilome: Metagenomic Analysis of Circular Plasmids,Viruses, and Other Extrachromosomal Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . 253Patrick Denis Browne, Witold Kot, Tue Sparholt Jørgensen,and Lars Hestbjerg Hansen

19 Identifying Conjugative Plasmids and Integrative ConjugativeElements with CONJscan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265Jean Cury, Sophie S. Abby, Olivia Doppelt-Azeroual, Bertrand Neron,and Eduardo P. C. Rocha

20 PlasmidFinder and In Silico pMLST: Identification and Typingof Plasmid Replicons in Whole-Genome Sequencing (WGS) . . . . . . . . . . . . . . . . . 285Alessandra Carattoli and Henrik Hasman

21 MOBscan: Automated Annotation of MOB Relaxases . . . . . . . . . . . . . . . . . . . . . . . 295M. Pilar Garcillan-Barcia, Santiago Redondo-Salvo, Luis Vielva,and Fernando de la Cruz

22 Plasmid Typing and Classification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309Laura Villa and Alessandra Carattoli

23 Plasmid Reconstruction from Next-Gen Data: A Detailed Protocolfor the Use of PLACNETw for the Reconstruction of Plasmidsfrom WGS Datasets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323Marıa de Toro, Val F. Lanza, Luis Vielva, Santiago Redondo-Salvo,and Fernando de la Cruz

viii Contents

24 Statistical Analysis of Accessory Genome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341Miguel D. Fernandez-de-Bobadilla, Teresa M. Coque,and Val F. Lanza

25 Inferring Horizontal Gene Transfer with DarkHorse, Phylomizer,and ETE Toolkits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355Luis Delaye, Carlos Vargas, Amparo Latorre, and Andres Moya

26 Methods to Study Fitness and Compensatory Adaptationin Plasmid-Carrying Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371Javier DelaFuente, Jeronimo Rodriguez-Beltran, and Alvaro San Millan

27 A Broad Host Range Plasmid-Based Roadmap for ssDNA-BasedRecombineering in Gram-Negative Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383Tomas Aparicio, Vıctor de Lorenzo, and Esteban Martınez-Garcıa

28 Conjugative Assembly Genome Engineering (CAGE) . . . . . . . . . . . . . . . . . . . . . . . 399Marc Guell

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411

Contents ix

Contributors

SOPHIE S. ABBY � Universite Grenoble Alpes, CNRS, Grenoble INP, TIMC-IMAG, Grenoble,France

TOMAS APARICIO � Systems and Synthetic Biology Program, Centro Nacional de Biotecnologıa(CNB-CSIC), Madrid, Spain

IGNACIO ARECHAGA � Departamento de Biologıa Molecular, Instituto de Biomedicina yBiotecnologıa de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander,Spain

JOSE BERENGUER � Centro de Biologıa Molecular Severo Ochoa, Universidad Aut�onoma deMadrid-Consejo Superior de Investigaciones Cientıficas, Madrid, Spain

AUDE BERNHEIM � Synthetic Biology Group, Microbiology Department, Institut Pasteur,Paris, France; Ecole Doctorale FIRE—Programme Bettencourt, Centre de RecherchesInterdisciplinaires, Paris, France; Microbial Evolutionary Genomics, Department ofGenomes and Genetics, Institut Pasteur, Paris, France; AgroParisTech, Paris, France

DAVID BIKARD � Synthetic Biology Group, Microbiology Department, Institut Pasteur, Paris,France

KHALD BLAU � Institute for Epidemiology and Pathogen Diagnostics, Julius Kuhn-Institut(JKI), Federal Research Centre for Cultivated Plants, Braunschweig, Germany

ALBA BLESA � Centro de Biologıa Molecular Severo Ochoa, Universidad Aut�onoma deMadrid-Consejo Superior de Investigaciones Cientıficas, Madrid, Spain; Department ofBiotechnology, Faculty of Experimental Sciences, Universidad Francisco de Vitoria,Madrid, Spain

JEAN-YVES BOUET � Laboratoire de Microbiologie et Genetique Moleculaires, Centre deBiologie Integrative (CBI), Centre National de la Recherche Scientifique (CNRS),Universite de Toulouse, UPS, Toulouse, France

PATRICK DENIS BROWNE � Department of Environmental Science, Aarhus University,Roskilde, Denmark; Department of Plant and Environmental Sciences,University of Copenhagen, Frederiksberg, Denmark

ELENA CABEZON � Departamento de Biologıa Molecular, Instituto de Biomedicina yBiotecnologıa de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander,Spain

ALICIA CALVO-VILLAMANAN � Synthetic Biology Group, Microbiology Department, InstitutPasteur, Paris, France; Ecole Doctorale FIRE—Programme Bettencourt, Centre deRecherches Interdisciplinaires, Paris, France

ALESSANDRA CARATTOLI � Department of Molecular Medicine, Sapienza University of Rome,Rome, Italy

MICHAEL CHANDLER � Laboratoire de Microbiologie et Genetique Moleculaires, Centre deBiologie Integrative (CBI), Centre National de la Recherche Scientifique (CNRS),Universite de Toulouse, UPS, Toulouse, France; Department of Biochemistry, Molecular andCellular Biology, Georgetown University Medical Center, Washington, DC, USA

RYAN R. CLARK � New York State Department of Health, Wadsworth Center, Albany, NY,USA

xi

TERESA M. COQUE � Department of Microbiology, Ram�on y Cajal Research Institute(IRYCIS), Madrid, Spain; CIBER in Epidemiology and Public Health, (CIBERESP),Madrid, Spain

JEAN CURY � Microbial Evolutionary Genomics, Institut Pasteur, Paris, France; CNRS,UMR3525, Paris, France

FERNANDO DE LA CRUZ � Instituto de Biomedicina y Biotecnologıa de Cantabria,Universidad de Cantabria CSIC, Santander, Cantabria, Spain

VICTOR DE LORENZO � Systems and Synthetic Biology Program, Centro Nacional deBiotecnologıa (CNB-CSIC), Madrid, Spain

MARIA DE TORO � Plataforma de Gen�omica y Bioinformatica, Centro de Investigaci�onBiomedica de La Rioja (CIBIR)—Fundaci�on Rioja Salud (FRS), Logrono, Spain

JAVIER DELAFUENTE � Department of Microbiology, Hospital Universitario Ramon y Cajal(IRYCIS), Madrid, Spain; Network Research Centre for Epidemiology and Public Health(CIBERESP), Madrid, Spain

LUIS DELAYE � Departamento de Ingenierıa Genetica, CINVESTAV Irapuato, Irapuato,Guanajuato, Mexico

KEITH M. DERBYSHIRE � New York State Department of Health, Wadsworth Center, Albany,NY, USA; Department of Biomedical Sciences, University at Albany, Albany, NY, USA

OLIVIA DOPPELT-AZEROUAL � Bioinformatics and Biostatistics Hub—C3BI, Institut Pasteur,Paris, France; CNRS, USR 3756, Paris, France

JOSE ANTONIO ESCUDERO � Unite Plasticite du Genome Bacterien, Departement Genomes etGenetique, Institut Pasteur, Paris, France; CNRS, UMR3525, Paris, France

MIGUEL D. FERNANDEZ-DE-BOBADILLA � Department of Microbiology, Ram�on y CajalUniversitary Hospital (IRYCIS), Madrid, Spain

RAUL FERNANDEZ-LOPEZ � Instituto de Biomedicina y Biotecnologıa de Cantabria,Universidad de Cantabria-Consejo Superior de Investigaciones Cientıficas, Santander,Cantabria, Spain

ANA R. FREITAS � Laboratory of Microbiology, Biological Sciences Department, Faculty ofPharmacy, UCIBIO/REQUIMTE, University of Porto, Porto, Portugal

M. PILAR GARCILLAN-BARCIA � Instituto de Biomedicina y Biotecnologıa de Cantabria,Universidad de Cantabria-Consejo Superior de Investigaciones Cientıficas, Santander,Cantabria, Spain

ANDREW N. GINN � Centre for Infectious Diseases and Microbiology, The Westmead Institutefor Medical Research, The University of Sydney, Westmead, NSW, Australia; Centre forInfectious Diseases and Microbiology Laboratory Services, NSW Health Pathology,Westmead Hospital, Westmead, NSW, Australia

TODD A. GRAY � New York State Department of Health, Wadsworth Center, Albany, NY,USA; Department of Biomedical Sciences, University at Albany, Albany, NY, USA

MARC GUELL � Departament de Ciencies Experimentals i de la Salut, Universidad PompeuFabra, Barcelona, Spain

CATHERINE GUYNET � Laboratoire de Microbiologie et Genetique Moleculaires, Centre deBiologie Integrative (CBI), Centre National de la Recherche Scientifique (CNRS),Universite de Toulouse, UPS, Toulouse, France

BERNARD HALLET � Louvain Institute of Biomolecular Science and Technology (LIBST),Universite Catholique de Louvain (UCLouvain), Louvain-la-Neuve, Belgium

LARS HESTBJERG HANSEN � Department of Environmental Science, Aarhus University,Roskilde, Denmark; Department of Plant and Environmental Sciences, University ofCopenhagen, Frederiksberg, Denmark

xii Contributors

HENRIK HASMAN � Department of Microbiology and Infection Control, Statens SerumInstitut, Copenhagen, Denmark

SVEN JECHALKE � Institute for Epidemiology and Pathogen Diagnostics, Julius Kuhn-Institut(JKI), Federal Research Centre for Cultivated Plants, Braunschweig, Germany

JUAN JOFRE � Department of Microbiology, University of Barcelona, Barcelona, SpainTUE SPARHOLT JØRGENSEN � Department of Environmental Science, Aarhus University,

Roskilde, Denmark; Department of Science and Environment, Roskilde University,Roskilde, Denmark; DTU BIOSUSTAIN, Novo Nordisk Foundation Center forBiosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark

MUHAMMAD KAMRUZZAMAN � Centre for Infectious Diseases and Microbiology, The WestmeadInstitute for Medical Research, The University of Sydney, Westmead, NSW, Australia

YOKO KOMIYAMA � Graduate School of Humanities and Sciences, Nara Women’s University,Nara, Japan

WITOLD KOT � Department of Environmental Science, Aarhus University, Roskilde,Denmark; Department of Plant and Environmental Sciences, University of Copenhagen,Frederiksberg, Denmark

VAL F. LANZA � Department of Microbiology, Ram�on y Cajal Universitary Hospital(IRYCIS), Madrid, Spain; CIBER in Epidemiology and Public Health, (CIBERESP),Madrid, Spain; Bioinformatics Unit, Ram�on y Cajal Universitary Hospital (IRYCIS),Madrid, Spain

AMPARO LATORRE � Institute of Integrative Systems Biology, University of Valencia and CSIC,Valencia, Spain; Joint Unit in Genomics and Health, FISABIO-Public Health—I2Sysbio,University of Valencia and CSIC, Valencia, Spain; CIBER in Epidemiology and PublicHealth, Madrid, Spain

PHAN THAI NGUYEN LE � Laboratoire de Microbiologie et Genetique Moleculaires, Centre deBiologie Integrative (CBI), Centre National de la Recherche Scientifique (CNRS),Universite de Toulouse, UPS, Toulouse, France

RICARDO LEON-SAMPEDRO � Department of Microbiology, Ram�on y Cajal Health ResearchInstitute (IRYCIS), Unidad de Resistencia a Antibi�oticos y Virulencia Bacteriana (RYC-CSIC), CIBER en Epidemiologıa y Salud Publica (CIBER-ESP), Ram�on y CajalUniversity Hospital, Madrid, Spain

DAMIAN LOBATO-MARQUEZ � Department of Infection Biology, Faculty of Infectious andTropical Diseases, London School of Hygiene and Tropical Medicine, Keppel, London, UK

CELINE LOOT � Unite Plasticite du Genome Bacterien, Departement Genomes et Genetique,Institut Pasteur, Paris, France; CNRS, UMR3525, Paris, France

MARIA LUCAS � Departamento de Biologıa Molecular, Instituto de Biomedicina yBiotecnologıa de Cantabria (IBBTEC), Universidad de Cantabria-Consejo Superior deInvestigaciones Cientıficas, Santander, Spain

SUMIO MAEDA � Graduate School of Humanities and Sciences, Nara Women’s University,Nara, Japan

ESTEBAN MARTINEZ-GARCIA � Systems and Synthetic Biology Program, Centro Nacional deBiotecnologıa (CNB-CSIC), Madrid, Spain

DIDIER MAZEL � Unite Plasticite du Genome Bacterien, Departement Genomes et Genetique,Institut Pasteur, Paris, France; CNRS, UMR3525, Paris, France

ALVARO SAN MILLAN � Department of Microbiology, Hospital Universitario Ramon y Cajal(IRYCIS), Madrid, Spain; Network Research Centre for Epidemiology and Public Health(CIBERESP), Madrid, Spain

Contributors xiii

GABRIEL MONCALIAN � Departamento de Biologıa Molecular, Instituto de Biomedicina yBiotecnologıa de Cantabria (IBBTEC), Universidad de Cantabria-Consejo Superior deInvestigaciones Cientıficas, Santander, Spain

ANDRES MOYA � Institute of Integrative Systems Biology, University of Valencia and CSIC,Valencia, Spain; Joint Unit in Genomics and Health, FISABIO-Public Health—I2Sysbio,University of Valencia and CSIC, Valencia, Spain; CIBER in Epidemiology and PublicHealth, Madrid, Spain

MAITE MUNIESA � Department of Microbiology, University of Barcelona, Barcelona, SpainBERTRAND NERON � Bioinformatics and Biostatistics Hub—C3BI, Institut Pasteur, Paris,

France; CNRS, USR 3756, Paris, FranceEMILIEN NICOLAS � Laboratory of Evolutionary Genetics and Ecology (LEGE), Department of

Biology—URBE, University of Namur (UNamur), Namur, BelgiumALEKSANDRA NIVINA � Unite Plasticite du Genome Bacterien, Departement Genomes

et Genetique, Institut Pasteur, Paris, France; CNRS, UMR3525, Paris, France;Paris Descartes, Sorbonne Paris Cite, Paris, France

CARLA NOVAIS � Laboratory of Microbiology, Biological Sciences Department, Faculty ofPharmacy, UCIBIO/REQUIMTE, University of Porto, Porto, Portugal

CAROLINA PALENCIA-GANDARA � Instituto de Biomedicina y Biotecnologıa de Cantabria,Universidad de Cantabria-Consejo Superior de Investigaciones Cientıficas, Santander,Cantabria, Spain

SALLY R. PARTRIDGE � Centre for Infectious Diseases and Microbiology, The WestmeadInstitute for Medical Research, The University of Sydney, Westmead, NSW, Australia

LUISA PEIXE � Laboratory of Microbiology, Biological Sciences Department, Faculty ofPharmacy, UCIBIO/REQUIMTE, University of Porto, Porto, Portugal

SANTIAGO REDONDO-SALVO � Instituto de Biomedicina y Biotecnologıa de Cantabria,Universidad de Cantabria CSIC, Santander, Cantabria, Spain

EDUARDO P. C. ROCHA � Microbial Evolutionary Genomics, Institut Pasteur, Paris, France;CNRS, UMR3525, Paris, France

JERONIMO RODRIGUEZ-BELTRAN � Department of Microbiology, Hospital UniversitarioRamon y Cajal (IRYCIS), Madrid, Spain; Network Research Centre for Epidemiology andPublic Health (CIBERESP), Madrid, Spain

JORGE RODRIGUEZ-GRANDE � Instituto de Biomedicina y Biotecnologıa de Cantabria,Universidad de Cantabria-Consejo Superior de Investigaciones Cientıficas, Santander,Cantabria, Spain

RAUL RUIZ � Instituto de Biomedicina y Biotecnologıa de Cantabria, Universidad deCantabria-Consejo Superior de Investigaciones Cientıficas, Santander, Cantabria, Spain

KORNELIA SMALLA � Institute for Epidemiology and Pathogen Diagnostics, Julius Kuhn-Institut (JKI), Federal Research Centre for Cultivated Plants, Braunschweig, Germany

KAITLIN A. TAGG � Centre for Infectious Diseases and Microbiology, The Westmead Institutefor Medical Research, The University of Sydney, Westmead, NSW, Australia

BAO TON-HOANG � Laboratoire de Microbiologie et Genetique Moleculaires, Centre deBiologie Integrative (CBI), Centre National de la Recherche Scientifique (CNRS),Universite de Toulouse, UPS, Toulouse, France

CARLOS VARGAS � Institute of Integrative Systems Biology, University of Valencia and CSIC,Valencia, Spain

CAROLAVENTURINI � Centre for Infectious Diseases andMicrobiology, The Westmead Institutefor Medical Research, The University of Sydney, Westmead, NSW, Australia

xiv Contributors

LUIS VIELVA � Departamento de Ingenierıa de Comunicaciones, Universidad de Cantabria,Santander, Cantabria, Spain

LAURA VILLA � Department of Infectious Diseases, Istituto Superiore di Sanita, Rome, ItalyCLAIRE VIT � Unite Plasticite du Genome Bacterien, Departement Genomes et Genetique,

Institut Pasteur, Paris, France; CNRS, UMR3525, Paris, France; Sorbonne Universite,College doctoral, Paris, France

Contributors xv