Table Of ContentSPRINGER
LAB MANUALS
Springer-Verlag Berlin Heidelberg GmbH
NATALIE EYNARD JUSTIN TEISSIE (EDS.)
Electrotransformation
of Bacteria
With 14 Figures
Springer
NATALIE EYNARD, PHD
JUSTIN TEISSIE, DR., DIRECTEUR DE RECHERCES AU CNRS
Institut de Pharmacologie et Biologie Structurale
CNRS UPR 9062
118 route de Narbonne
31062 Toulouse cedex
France
Library of Congress Cataloging-in-Publication Data
Electrotransformation of bacteria I Natalie Eynard, Justin Teissie (eds.)
p. cm. - (Springer Iab manuals)
Includes bibliographical references and index.
ISBN 978-3-642-08593-2 ISBN 978-3-662-04305-9 (eBook)
DOI 10.1007/978-3-662-04305-9
1. Bacterial Iransformation - Labaratory manuals.
2. Electric fields. I. Eynard, Natalie, 1964 - II. Teissie Justin, 1947- III. Series.
QH448.4.E43 2000
571.9'648-dc21 99-086708
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© Springer-Verlag Berlin Heidelberg 2000
Originally published by Springer-Verlag Berlin Heidelberg New York in 2000
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Preface
Electrotransformation of intact Bacteria was described for the
first time only 12 years ago. Since then it has been proved to
be a highly efficient technique and easy to use. Gram-positive
as well as Gram-negative bacteria can be transformed. Several
manufacturers provide reliable electropulsers andin some cases
also protocols. But as the molecular mechanisms supporting the
introduction of macromolecules into the bacterial cell remain
unknown, empirism and adaptation of methods are still the
most effective tools to obtain transformed species. Parameters
are clearly dependent on the species, which are used. An im
pressive number of protocols on various species has by now
been published and can be used as a data base of protocols
for various research fields.
The idea of this lab manual is to provide scientists with pro
tocols for a wide spectrum of species - protocols which had been
carefully checked and proved to be effective. They can be used as
they are or might be slightly modified to improve the yield.
About 40 different species are considered in this manual. Chap
ters are selected and ordered byfields of applications. The choice
can sometimes be rather arbitray since some species could be
treated under different application fields. A major part of
each chapter is devoted to the specific problems of the species
in use and, of course, suggestions of how to find solutions.
Only procedures where intact bacteria are used are included in
the manual. Protocols in which protoplasts were first prepared,
mixed with plasmids and then electropulsed, are not described.
They were observed to be less effective because the survival rate
was always very low. This appears tobe due to the lethal effect of
the field when the plasma membrane is not protected by a wall.
Furthermore, these procedures aretime consuming. Neverthe
less, such an approach may be considered with reluctant species
\Tl Preface
where the integrity of the cell envelope obviously prevents an
effective transport.
In the first part of the book, general and technical aspects are
treated. Taking into account that the molecular aspects respon
sible for the induction of electrocompetence of pulsed intact bac
teria remain unresolved, we have not tried to describe the
different hypothesis currently discussed. One of their limits is
that they are often based on the simple assumption that a cell
is a sphere built from a lipid bilayer. \Tery few descriptions
take the presence of proteins into account, the ellipsoidal shape
and the presence of a wall. How the bacterial envelope is affected
remained unknown. In the first chapter we therefore describe
our present experimental knowledge on the processes driving
plasmids into the cytoplasm when bacteria are pulsed. The
second chapter gives a description of the physical, mainly elec
trical processes, which take place when a field pulse is applied.
This should allow the user to estimate the physicallimits of an
experiment and to avoid the occurence of arcing.
The first protocols are on the laboratory workhorse, E. coli.
The, with respect to the importance of bacterial transformation
for the biotechnological industry, techniques using strains rele
vant to Biotechnology and Food Technology are described,
followed by protocols for species involved in Medical and Ve ter
inary Applications. The next section deals with the electrotrans
formation of strains with relevance for the agroindustry. Here,
protocols dealing with the modification of Bacteria interacting
with plants are given. In the final chapters, procedures on the
genetic modifications of bacteria present in the environment,
which are now of growing importance, are described in detail.
In Appendix A some practical notes on the preparation of plas
mids are made, followed by Appendix B, a list of suppliers of the
equipment.
We would like to thank all contributors for their works and
express our gratitude to many scientists in the CNRS community
in Toulouse for their helpful comments and friendly discussions
during the last 15 years. This local network was a strong support
for the development of our contribution to bacterial electro
transformation.
Toulouse, April 2000 NATALIE EYNARD
AND JUSTIN TEISSIE
Contents
Part I lntroduction
Chapter 1
General Principles of Bacteria Electrotransformation:
Key Steps
NATALIE EYNARD and JUSTIN TEISSIE . . . . . . . . . . . . . . 1
Chapter 2
A Critical Introduction to the Technology
of Bacterial Electrotransformation
JUSTIN TEISSIE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Chapter 3
Electrotransformation of Escherichia coli
NATALIE EYNARD and JUSTIN TEISSIE . . . . . . . . . . . . . . 35
Chapter 4
Transformation of Bacillus subtilis PB 1424
by Electroporation
PATRIZIA BRIGIDI, MADDALENA ROSSI
and DIEGO MATTEUZZI . . . . . . . . . . . . . . . . . . . . . . . . . 42
Part II Biotechnology and Food Technology
Chapter 5
Clostridium in Biotechnology and Food Technology
HANS P. BLASCHEK, JOSEPH FORMANEK and C.K. CHEN 47
Subprotocol 1: Electroporation-Induced Transformation
of Clostridium perfringens . . . . . . . . . . . 50
Subprotocol 2: Electroporation Induced Transformation
of Clostridium beijerinckii . . . . . . . . . . . 53
VIII Contents
Chapter 6
Electrotransformation of Lactococcus lactis
PASCAL LE BOURGEOIS, PHILIPPE LANGELLA
and PAUL RITZENTHALER . . . . . . . . . . . . . . . . . . . . . . . . 56
Chapter 7
Electrotransformation of Salmonella typhimurium
NATALIE EYNARD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Chapter 8
Electroporation of bifidobacteria
MADDALENA ROSSI, PATRIZIA BRIGIDI and DIEGO
MATTEUZZI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Chapter 9
Electrotransformation of Listeria species
}ANET E. ALEXANDER . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Subprotocol 1: Preparation of Competent Cells for the
Electroporation of Listeria Species . . . . 80
Subprotocol 2: Electroporation of Listeria Species
with Vectors Greater than 10 kb . . . . . . 85
Chapter 10
Transformation of Methylobacterium extorquens
with a Broad-Host-Range Plasmid by Electroporation
SHUNSAKU UEDA and TSUNEO YA MANE . . . . . . . . . . . . 88
Chapter 11
Electrotransformation of Acidophilic, Heterotrophie,
Gram-negative Bacteria
THOMAS E. wARD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Chapter 12
Acetobacter xylinum - Biotechnology
and Food Technology
ROBERT E. CANNON . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Chapter 13
Electrotransformation of Sphingomonas pancimobilis
ISABEL SA-CORREIA and ARSENIG M. FIALHO ........ 108
Contents IX
Chapter 14
Bacillus arnyloliquefaciens - Production Host
for Industrial Enzymes
JARl VEHMAANPERÄ ............................ 119
Part 111 Medical and Veterinary Applications
Chapter 15
Electrotransformation of Yersinia ruckeri
JUAN M. CUTRIN, ALICIA E. TORANZO and JUAN L. BARJA 125
Chapter 16
Electrotransformation of Enterococci
ALBRECHT MUSCHOLL-SILBERHORN
and REINHARD WIR TH . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Chapter 17
Prevotella bryantii, P. rurninicola and Bacteroides Strains
HARRY J. FLINT, JENNIFER C. MARTIN
and ANDREW M. THOMSON . . . . . . . . . . . . . . . . . . . . . . 140
Chapter 18
Electrotransformation of Bordetella
GA VIN R. ZEALEY and REZA K. YA COOB . . . . . . . . . . . . 150
Chapter 19
Transformation of Carnpylobacter jejuni
BEN N. FRY, MARC M.S.M. WÖSTEN,
TRUDY M. WASSENAAR and
BERNARD A.M. V AN DER ZEIJST . . . . . . . . . . . . . . . . . . 157
Chapter 20
Slow-Growing Mycobacteria
BARRY J. W ARDS and DESMOND M. COLLINS . . . . . . . . 168
Chapter 21
Electrotransformation of Photobacteriurn darnselae
subsp. piscicida
JUAN M. CUTRIN, JUAN L. BARJA and ALICIA E. TORANZO 175
