Mycobacteria Protocols

METHODS IN MOLECULAR BIOLOGY

105 . Phospholipid Signaling Protocols, edited by Ian M. Bird,

1998

104 . Mycoplasma Protocols, edited by Roger J. Miles and Robin

A. J. Nicholas, 1998

103 . Pichia Protocols, edited by David R. Higgins and James M.

Cregg, 1998

102 . Bioluminescence Methods and Protocols, edited by Robert

A . LaRossa, 1998

101 . Mycobacteria Protocols, edited by Tanya Parish and Neil G .

Stoker, 1998

100 . Nitric Oxide Protocols, edited by Michael A . Titheradge,1998

99 . Human Cytokines and Cytokine Receptors, edited by Reno

Debets and Huub Savelkoul, 1999

98 . Forensic DNA Profiling Protocols, edited by Patrick J. Lin-

coln and James M. Thomson, 1998

97 . Molecular Embryology : Methods and Protocols, edited byPaul T. Sharpe and Ivor Mason, 1998

96. Adhesion Proteins Protocols, edited by Elisabetta Dejana,

1998

95 . DNA Topoisomerases Protocols : II. Enzymology and Drugs,

edited by Mary-Ann Bjornsti and Neil 0sheroff, 1998

94 . DNA Topoisomerases Protocols : I. DNA Topology and En-

zymes, edited by Mary-Ann Bjornsti and Neil Osheroff, 1998

93. Protein Phosphatase Protocols, edited by John W. Ludlow, 199892 . PCR in Bioanalysis, edited by Stephen J. Meltzer, 1998

91 . Flow Cytometry Protocols, edited by Mark J. Jaroszeski, Ri-chard Heller, and Richard Gilbert, 1998

90 . Drug-DNA Interaction Protocols, edited by Keith R. Fox,

1998

89. Retinoid Protocols, edited by Christopher Redfern, 1998

88 . Protein Targeting Protocols, edited by Roger A . Clegg, 1998

87 . Combinatorial Peptide Library Protocols, edited by Shmuel

Cabilly, 1998

86. RNA Isolation and Characterization Protocols, edited byRalph Rapley and David L. Manning, 1998

85 . Differential Display Methods and Protocols, edited by Peng

Liang and Arthur B. Pardee, 1997

84. Transmembrane Signaling Protocols, edited by Dafna Bar-

Sagi, 1998

83 . Receptor Signal Transduction Protocols, edited by R . A. John

Challiss, 1997

82 . Arabidopsis Protocols, edited by Jose M Martinez-Zapater

and Julio Salinas, 1998

81 . Plant Virology Protocols : From Virus Isolation to Transgenic

Resistance, edited by Gary D. Foster and Sally Taylor, 1998

80.1mmunochemical Protocols (2nd . ed .), edited by

John Pound, 1998

79 . Polyamine Protocols, edited by David M. L. Morgan, 1998

78 . Antibacterial Peptide Protocols, edited by William M . Shafer,

1997

77. Protein Synthesis : Methods and Protocols, edited by Robin

Martin, 1998

76 .Glycoanalysis Protocols (2nd . ed.), edited by Elizabeth F.

Hounsell, 1998

75 . Basic Cell Culture Protocols (2nd. ed.), edited by Jeffrey W.

Pollard and John M. Walker, 1997

John M. Walker, SERIES EDITOR

74 . Ribozyme Protocols, edited by Philip C. Turner, 1997

73 . Neuropeptide Protocols, edited by G . Brent Irvine and Carvell

H. Williams, 1997

72 . Neurotransmitter Methods, edited by Richard C. Rayne, 1997

71 . PRINS and In Situ PCR Protocols, edited by John R. Golden,

1996

70. Sequence Data Analysis Guidebook, edited by Simon R.

Swindell, 1997

69. cDNA Library Protocols, edited by Ian G . Cowell

and Caroline A. Austin, 1997

68. Gene Isolation and Mapping Protocols, edited by Jacqueline

Boultwood, 1997

67 . PCR Cloning Protocols : From Molecular Cloning to Genetic

Engineering, edited by Bruce A . White, 1997

66 . Epitope Mapping Protocols, edited by Glenn E. Morris,

1996

65 . PCR Sequencing Protocols, edited by Ralph Rapler, 1996

64 . Protein Sequencing Protocols, edited by Bryan J. Smith, 1997

63 . Recombinant Protein Protocols : Detection and Isolation, ed-

ited by Rocky S . Tuan, 1997

62 . Recombinant Gene Expression Protocols, edited by Rocky

S. Tuan, 1997

61 . Protein and Peptide Analysis by Mass Spectrometry, ed-

ited by John R . Chapman, 1996

60 . Protein NMR Techniques, edited by David G . Reid, 1997

59 . Protein Purification Protocols, edited by Shawn Doonan,

1996

58 . Basic DNA and RNA Protocols, edited byAdrianJ. Harwood,

1996

57. I n Vitro Mutagenesis Protocols, edited by Michael K. Trower, 1996

56 . Crystallographic Methods and Protocols, edited by Chris-

topher Jones, Barbara Malloy, and Mark R. Sanderson, 1996

55 . Plant Cell Electroporation and Electrofusion Protocols, ed-ited by Jac A . Nickoloff 1995

54 . YAC Protocols, edited by David Markie, 1996

53 . Yeast Protocols : Methods in Cell and Molecular Biology,

edited by Ivor H. Evans, 1996

52 . Capillary Electrophoresis Guidebook : Principles, Opera-

tion, and Applications, edited by Kevin D. Altria, 1996

51 . Antibody Engineering Protocols, edited by Sudhir Paul, 1995

50 . Species Diagnostics Protocols : PCR and Other Nucleic Acid

Methods, edited by Justin P . Clapp, 1996

49 . Plant Gene Transfer and Expression Protocols, edited by

Heddwyn Jones, 1995

48 . Animal Cell Electroporation and Electrofusion Protocols,

edited by Jac A . Nickoloff, 1995

47 . Electroporation Protocols for Microorganisms, edited byJac A . Nickoloff, 1995

46 . Diagnostic Bacteriology Protocols, edited by Jenny Howardand David M. Whitcombe, 1995

45 . Monoclonal Antibody Protocols, edited by William C. Davis,

1995

44 . Agrobacterium Protocols, edited by Kevan M. A. Gartland

and Michael R . Davey, 1995

METHODS IN MOLECULAR BIOLOGY TM

MycobacteriaProtocols

Edited by

Tanya Parishand

Neil G . StokerLondon School of Hygiene and Tropical Medicine, London, UK

Humana Press Totowa, New Jersey

© 1998 Humana Press Inc .999 Riverview Drive, Suite 208Totowa, New Jersey 07512

All rights reserved . No part of this book may be reproduced, stored in a retrieval system, or transmitted inany form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwisewithout written permission from the Publisher . Methods in Molecular Biology"' is a trademark of TheHumana Press Inc .

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This publication is printed on acid-free paper.ANSI Z39 .48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials .

Cover illustration : Fig . 3E from Chapter 20, "Green Fluorescent Protein : A Tool for Gene Expression andCell Biology in Mycobacteria, "by Laura E . Via, Subramanian Dhandayuthapani, Dusanka Deretic, and V .Deretic.

Cover design by Patricia F . Cleary .

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Main entry under title : Methods in molecular biologyTM .

Mycobacteria protocols / edited by Tanya Parish and N . G . Stoker .p. cm.-(Methods in molecular biology ; vol . 101)

Includes index .ISBN 0-89603-471-2 (alk. paper)1 . Mycobacteria-Laboratory manuals . 2 . Bacterial genetics-Laboratory manuals . I. Parish, Tanya. 11 .

Stoker, N . G. (Neil G .) III . Series : Methods in molecular biology (Totowa, N.J.) ; v . 101 .[DNLM : 1 . Mycobacterium-genetics . 2. Mycobacterium-growth & development . 3. DNA-

physiology . 4 . Molecular Biology-methods. QW I25 .5 .M9 M9952 1998]QR82.2.M8M93 1998579.3'74--dc2l 98-22177

CIP

Preface

Mycobacteria include species of great medical importance, being re-sponsible for both human and animal disease, including tuberculosis and lep-rosy. The use of molecular techniques to explore the biology of these organismshas exploded in the past few years . Mycobacteria pose particular problems inthe laboratory, most notably their slow growth rate, their tendency to clump,and their resistance to standard methods for lysis ; thus special adaptations ofcommonly used methods are required . Mycobacteria Protocols provides de-scriptions of both basic and recently developed techniques for studying themolecular biology of mycobacteria . Although many genetic tools are still re-quired, there is much that can be accomplished using those available, and thisbook provides a starting point for anyone interested in working on these in-triguing, but difficult bacteria .

The first eight chapters describe the basic techniques required for anystudy of mycobacteria, including culture and safety aspects, and preparationof nucleic acids, proteins, and cell-wall fractions . The introduction of DNAinto mycobacteria was a major advance in enabling the development of ge-netic systems . Two chapters deal with introduction of DNA by electroporationand conjugation. Phages have also been used as the basis for genetic systems,and a comprehensive list of protocols for their use is presented .

It is becoming difficult to handle the rapidly increasing body of knowl-edge in the mycobacterial field. The MycDB database is one approach; thisuses the powerful AceDB software, originally developed for the Caenorhabditiselegans genome project, which include sequences, immunological reagents,and research publications, to integrate these data . One chapter introduces theuse of this software on the World Wide Web .

The creation of mycobacterial mutants is an essential genetic tool,and various methods for achieving this are presented, including chemicalmutagenesis and transposon mutagenesis . Several chapters deal with obtain-ing defined gene knockouts by homologous recombination in fast and slowgrowing species. Although technically difficult in slow growers, this can nowbe achieved and will become increasingly important as more genome dataemerge. The study of gene regulation and expression is dealt with in chapterscovering the use of expression vectors to produce both mycobacterial and

V

vi Preface

mammalian proteins, the use of reporter genes, and the study of gene regula-tion at the mRNA level using RT-PCR and RAP-PCR .

Finally, the application of molecular biology to diagnostic techniquesis growing and methods for speciation using both biochemical methods andrDNA sequencing, detection of Mycobacterium tuberculosis complex by PCR,spoligotyping, and analysis of RFLP patterns, the detection of drug resistanceusing PCR-SSCP are all covered .

Although Mycobacteria Protocols does not cover all techniques thathave been used with success in mycobacteria, it should provide a basic ground-ing in the application of most techniques today . We hope that it will providean easy introduction to those wishing to carry out genetic studies of mycobac-teria, whether in basic or applied research . Since mycobacteria are sometimesdifficult to work with, the special value of our book is that it will allow readersto benefit from the advice and experience of researchers already in this field.

Our thanks should go to all the contributors for their good work and toJohn Walker and Tom Lanigan for their support of this project .

Tanya Parish

Neil G. Stoker

Contents

Preface v

Contributors xi

1 Mycobacteria : Bugs and BugbearsTanya Parish and Neil G. Stoker 1

2 Mycobacteria : General Culture Methodology and SafetyConsiderations

Bryan W. Allen 15

3 Isolation of Genomic DNA from MycobacteriaJohn T. Belisle and Michael G. Sonnenberg31

4 Nucleic-Acid Extraction : Plasmid/Cosmid DNABonnie B. Plikaytis and C. Harold King 45

5 Pulsed Field Gel Electrophoresis for MycobacteriaWolfgang J. Philipp,Stephen Gordon, Amalio Telenti, and

Stewart T. Cole 51

6 Extraction of RNA from MycobacteriaEshwar Mahenthiralingam 65

7 Preparation of Cell-Free Extracts from Mycobacteria

Tanya Parish and Paul R. Wheeler 77

8 Preparation of Cell-Wall Fractions from MycobacteriaGurdyal S. Besra 91

9 Using MycDB on the World-Wide WebStaffan Bergh and Stewart T. Cole 109

10 Conjugating DNA into MycobacteriaJulie-Ann Gavigan and Carlos Martin 119

11 Electroporation of MycobacteriaTanya Parish and Neil G. Stoker 129

12 Mycobacteriophages

Gary J. Sarkis and Graham F. Hatfull 145

vii

viii Contents

13 Chemical Mutagenesis of MycobacteriaLucy A . Brooks 175

14 Tn611 Transposon Mutagenesis in Mycobacterium smegmatisUsing a Temperature-Sensitive Delivery System

Esther Perez, Julie-Ann Gavigan, Isabel Otal,Christophe Guilhot, Vladimir Pelicic, Brigitte Giguel,and Carlos Martin 187

15 Homologous Recombination in Mycobacterium smegmatis :Screening Methods for Detection of Gene Replacement

Robert N. Husson 199

16 Gene Replacement in Mycobacterium smegmatis Using aDominant Negative Selectable Marker

Peter Sander and Erik C. Bottger 207

17 Gene Replacement in Mycobacterium intracellulareBritt-lnger Marklund and Richard W. Stokes217

18 Gene Replacement in Mycobacterium bovis BCGElizabeth Norman 225

19 The Application of Bacterial Luciferase as a Reporter Genein Mycobacteria

Stephen Gordon, Gavin Chung, and Peter Andrew235

20 Green Fluorescent Protein : A Tool for Gene Expression andCell Biology in Mycobacteria

Laura E. Via, Subramanian Dhandayuthapani,Dusanka Deretic, and V. Deretic 245

21 Expression of Genes in MycobacteriaPeadar O Gaora 261

22 Secretion of Mammalian Proteins from MycobacteriaPeter J. Murray and Richard A. Young 275

23 Intracellular Gene Expression : Analysis of RNA from Mycobacteriain Macrophages Using RT-PCR

Philip D. Butcher, Joseph A . Mangan, and Irene M. Monahan . .. . 285

24 Analysis of Mycobacterial Differential Gene Expressionby RAP-PCR

Joseph A. Mangan and Philip D. Butcher307

25 Mycobacterial SpeciationFrancis A. Drobniewski and Anne H . C. Uttley323

26 Species Identification of Mycobacteria Using rDNA SequencingPhilip Kirschner and Erik C. Bottger 349

Contents ix

27 Detection of Mycobacterium tuberculosis by a ColorimetricPolymerase Chain Reaction

Stuart M. Wilson 363

28 Rapid Detection and Simultaneous Strain Differentiation ofMycobacterium tuberculosis Complex Bacteria by Spoligotyping

Henri 0. F. Molhuizen, Annelies E. Bunschoten, Leo M. Schouls,and Jan D. A. van Embden 381

29 Computer Analysis of IS6110 RFLP Patterns of Mycobacteriumtuberculosis

Herre F. Heersma, Kristin Kremer, and Jan D. A. van Embden . .. 395

30 Detection of Mutations in Mycobacteria by PCR-SSCP(Single-Strand Conformation Polymorphism)

Amalio Telenti, Nadine Honore, and Stewart T. Cole423

31 The Use of Luciferase-Reporter Phage for Antibody-SusceptibilityTesting of Mycobacteria

Paul F. Riska and William R. Jacobs, Jr 431

Index 457

Contributors

BRYAN W . ALLEN • Department of Medical Microbiology, St. George'sHospital Medical School, London

PETER ANDREW -Department of Microbiology and Immunology, Universityof Leicester, Leicester, UK

SYLVIE G. BERNIER • Department of Pharmacology, Faculty of Medicine,University of Sherbrooke, Quebec, Canada

JOHN T. BELISLE • Department of Microbiology and Immunology, Universityof Leicester, Leicester, UK

STAFFAN BERGH • Department of Microbiology, Colorado State University,Fort Collins, CO

GURDYAL S . BESRA • Department of Microbiology, Colorado StateUniversity, Fort Collins, CO

ERIK C . BOTTGER • Institut fur Medizinische Mikrobiologie, MedizinischeHochschule Hannover, Hannover, Germany

Lucy A . BROOKS • Division of Infectious and Immunological Diseases,Department of Pediatrics, University of British Columbia, Vancouver,BC, Canada

ANNELIES E. BUNSCHOTEN • National Institute of Public Health andEnvironment, Research Laboratory for Infectious Diseases, Bilthoven,The Netherlands

PHIILIP D . BUTCHER • Division of Molecular Microbiology, Department ofCellular and Molecular Sciences, St. George's Hospital Medical School,London, UK

GAVIN CHUNG • Chemotherapy Department, Glaxo-Wellcome MedicalResearch Centre, Stevenage, UK

STEWART T. COLE • Unite de Genetique Mycobacterienne, Departement deBacteriologie et Mycologie, Institut Pasteur, Paris, France

DUSANKA DERETIC • Department ofAnatomy and Cell Biology, University ofMichigan Medical School, Ann Arbor, MI

VoJo DERETIC • Department of Microbiology and Immunology, University ofMichigan Medical School, Ann Arbor, MI

xi

xii Contributors

SUBRAMANIAN DHANDAYUTHAPANI • Department of Microbiology, Universityof Texas Health Science Center at San Antonio, San Antonio, TX

FRANCIS A. DROBNIEwsKI • PHLS Mycobacterium Reference Unit, DulwichHospital, London, UK

JAN D . A. VAN EMBDEN • National Institute for Public Health and the Environ-ment, Research Laboratory for Infectious Diseases, Bilthoven, The Netherlands

JULIE-ANN GAVIGAN -Department of Microbiology, University College,Cork, Ireland

BRIGITTE GICQUEL • Unite de Genetique Mycobacteriennee, Departement deBacteriologie et Mycologie, Institut Pasteur, Paris, France

STEPHEN GORDON • Unite de Genetique Moleculaire Bacterienne, InstitutPasteur, Paris, France

CHRISTOPHE GUILHOT • Unite de Genetique Mycobacterienne, Department deBacteriologie et Mycologie, Institut Pasteur, Paris, France

GRAHAM F. HATFULL • Department of Biological Sciences, University ofPittsburgh, Pittsburgh, PA

HERRE F. HEERSMA • National Institute for Public Health and the Environ-ment, Division ofPublic Health Research, Bilthoven, The Netherlands

NADINE HONORE • Unite de Genetique Mycobacterienne, Departement deBacterilogie et Mycologie, Institut Pasteur, Paris, France

ROBERT N. HUSSON • Children's Hospital, Harvard Medical School, Boston, MAWILLIAM R. JACOBS, JR . • Howard Hughes Medical Institute, Albert Einstein

College ofMedicine, Bronx, NYC . HAROLD KING • Division ofAIDS, STD, and TB Laboratory Research,

Centers for Disease Control and Prevention, Atlanta, GAPHILLIP KIRSCHNER • Institut fur Medizinische Mikrobiologie, Medizinische

Hochschule Hannover, Hannover, GermanyKRISTIN KREMER • National Institute for Public Health and the Environment,

Division ofPublic Health Research, Bilthoven, The NetherlandsESHWAR MAHENTHIRALINGAM • Division ofInfectious and Immunological

Diseases, Department ofPediatrics, University ofBritish Columbia,Vancouver, British Columbia, Canada

JOSEPH A. MANGAN • Division ofMolecular Microbiology, Department of Cellularand Molecular Sciences, St. George's Hospital Medical School, London, UK

BRITT-INGER MARKLUND • Department ofBacteriology, Swedish Institute forInfectious Disease Control, Stockholm, Sweden

CARLOS MARTIN • Departmento de Microbiologia, Facultad de Medicina,Universidad de Zaragoza, Zaragoza, Spain

HENRI 0. F. MOLHUIZEN • Research Laboratory for Infectious Diseases, NationalInstitute for Public Health and Environment, Bilthoven, The Netherlands

Contributors xiii

IRENE M. MONAHAN • Division ofMolecular Microbiology, Department of Cellularand Molecular Sciences, St. George's Hospital Medical School, London, UK

PETER J. MURRAY • Whitehead Institute for Biomedical Research, Cambridge, MAELIZABETH NORMAN • Molecular Microbiology Group, School ofBiological

Sciences, University of Surrey, Guilford, Surrey, UKPEADAR O GAORA • Department of Medical Microbiology, Imperial College

School ofMedicine at St . Mary's, London, UKISABEL OTAL • Departmento de Microbiologia, Medicina, Preventiva y Salud

Publica, Facultad de Medicina, Universidad de Zaragoza, Zaragoza, SpainTANYA PARISH • Department ofInfectious and Tropical Diseases, London

School ofHygiene & Tropical Medicine, London, UKVLADIMIR E. PELICIC • Unite de Genetique Mycobacterienne, Departement de

Bacteriologic et Mycologie, Institut Pasteur, Paris, FranceESTHER PEREZ • Departamento de Microbiologia, Medicina, Preventiva y Salud

Publica, Facultad de Medicina, Universidad de Zaragoza, Zaragoza, SpainWOLFGANG J. PHILIPP • Institute ofMedical Microbiology, University of

Berne, Berne, SwitzerlandBONNIE B. PLIKAYTIS • Centers for Disease Control and Prevention, Atlanta, GAPAUL F. RISKA • Division ofInfectious Diseases, Albert Einstein College of

Medicine, Bronx, NYPETER SANDER • Institut fur Medizinische Mikrobiologie, Medizinische

Hochschule Hannover, Hannover, GermanyGARY J . SARKIS • Department of Biological Sciences, University ofPitts-

burgh, Pittsburgh, PALEO M . SCHOULS • Research Laboratory for Infectious Diseases, National

Institute ofPublic Health and Environment, Bilthoven, The NetherlandsMICHAEL G. SONNENBERG • Department ofMicrobiology, Colorado State

University, Fort Collins, CONEIL G. STOKER • Department ofInfectious and Tropical Diseases, London

School ofHygiene and Tropical Medicine, London, UKRICHARD W. STOKES • Division ofInfectious and Immunological Diseases,

British Columbia's Children's Hospital and Department ofPaediatrics,University of British Columbia and Canadian Bacterial Diseases Net-work, BC Research Institute for Child and Family Health, Vancouver,British Columbia, Canada

AMALio TELENTI • Institute ofMedical Microbiology, University of Berne,Berne, Switzerland

ANNE C. UTTLEY • Dulwich Public Health Laboratory, Dulwich Hospital,London, UK

xiv Contributors

LAURA E. VIA • Department ofMicrobiology and Immunology, University ofMichigan Medical School, Ann Arbor, MI

PAUL R. WHEELER • Department ofInfectious and Tropical Diseases, LondonSchool of Hygiene and Tropical Medicine, London, UK

STUART M. WILSON • PHLS Mycobacterium Reference Unit and RegionalCentre for Mycobacteriology, Dulwich Public Health Laboratory andDepartment of Microbiology, London, UK

RICHARD YOUNG • Whitehead Institute for Biomedical Research, Cambridge,MA and Department ofBiology, Massachusetts Institute of Technology,Cambridge, MA