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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 .
The content and opinions expressed in this book are the sole work of the authors and editors, who havewarranted due diligence in the creation and issuance of their work . The publisher, editors, and authors arenot responsible for errors or omissions or for any consequences arising from the information or opinionspresented in this book and make no warranty, express or implied, with respect to its contents .
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 .
For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contactHumana at the above address or at any of the following numbers : Tel : 973-256-1699 ; Fax : 973-256-8341 ;E -mail : humana@humanapr .com , or visit our Website at www.humanapress .com
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Library of Congress Cataloging in Publication Data
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