Table Of ContentBacteriocins, Microcins and Lantibiotics
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Series H: Cell Biology, Vol. 65
Bacteriocins, Microcins
and Lantibiotics
Edited by
Richard James
School of Biological Sciences
University of East Anglia
Norwich NR4 7T J
Norfolk, United Kingdom
Claude Lazdunski
Centre National de la Recherche Scientifique
Centre de Biochimie et de Biologie Moleculaire
31, chemin Joseph Aiguier, B. P. 71
13402 Marseille Cedex 9, France
Franc Pattus
European Molecular Biology Laboratory
Meyerhofstrasse 1, Postfach 10 22 09
6900 Heidelberg, Germany
Springer-Verlag
Berlin Heidelberg New York London Paris Tokyo
Hong Kong Barcelona Budapest
Published in cooperation with NATO Scientific Affairs Division
Proceedings of the NATO Advanced Research Workshop on Bacterial
Plasmid-Coded Toxins: Bacteriocins, Microcins and Lantibiotics, held at lie de
Bendor, France, September 22-26, 1991
ISBN-13: 978-3-642-76976-4 e-ISBN-13: 978-3-642-76974-0
001: 10.1007/978-3-642-76974-0
Library of Congress Cataloging-in-Publication Data
Bacteriocins. microcins, and lantibiotics / edited by Richard James, Claude Lazdunski, and Franc Pattus.
(NATO ASI series. Series H, Cell biology; vol. 65)
"Proceedings of the NATO Advanced Research Workshop on Bacterial Plasmid-Coded Toxins:
Bacteriocins, microcins, and lantibiotics, held at ile de Bendor, France, September 22-26, 1991" --Copr.
Includes bibliographical references and index.
1. Bacteriocins--Congresses.1. James, Richard. II. Lazdunski, Claude. III. Pattus, Franc. IV. NATO Advanced
Research Workshop on lantibiotics (1991 : ile de Bendor, France) V. Series.
QR92.B3B33 1992 589.9'019246--dc20
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Preface
The EMBO-FEMS-NATO Advanced Research Workshop on Bacteriocins, Microcins and
Lantibiotics was held on the Isle de Bendor, France from September 22nd-26th, 1991.
Bacteriocins are protein antibiotics produced by bacteria. They differ from traditional antibiotics
because they only kill bacteria which are closely related to the producing strain. The producing strain
usually shows immunity towards the bacteriocin which it produces. Bacteriocins are of high molecular
weight and are typically plasmid-encoded proteins. The most intensively studied group ofbacteriocins,
the colicins, are antibiotic proteins produced by some strains of Escherichia coli and closely related
enterobacteria such as Shigella sonnei. Colicins are subdivided into groups based largely upon the
receptors to which they bind on the surface of sensitive E.coli cells; ie. the E colicins bind to the product
of the btuB gene of E.coli.
B acteriocins have been subjected to intensive study in the last ten years and it has now become clear
that they have a contribution to make in addressing some fundamental problems in modem biology.
1. Being large proteins, how are bacteriocins secreted across both the cytoplasmic and outer membrane
of a Gram-vehost cell, and how do they get into and then kill sensitive cells? A considerable amount
is known about the uptake mechanism into sensitive cells but there are considerable unresolved
questions about how colicins are secreted from the producing cell.
2. The mechanism of insertion of the pore-forming colicins E1 and A into E.coli membranes has
generated considerable interest amongst membrane physiologists as well as those who are more
interested in these molecules as colicins.
3. What is the mechanism of immunity to a bacteriocin? The specific nature of the interaction between
a colicin and its cognate immunity protein provide a model system for studying protein-protein
interactions.
4. Families ofE colicins which show close homology have been identified. How have these proteins,
and their cognate immunity proteins, evolved?
These are some of the interesting questions addressed by the later sessions of the workshop in Bendor.
I have worked with colicins for 12 years since being introduced to the subject by Pearl Cooper here
in Norwich. In that time I have not been to a conference at which more than a handful of other bacteriocin
workers had been present, usually as a peripheral part of the main theme of the conference. I thought for
some time as to why there had been so few bacteriocin meetings and concluded that, although there were
a large number of interesting problems being studied which involved bacteriocins, no single problem had
the critical mass of scientists required to organise and attract funding for a meeting. A related problem to
this was that researchers working with colicins did not have much contact with those working with the
VI
bacteriocins produced by Gram +ve bacteria. I therefore decided to try and organize a workshop with the
aim to encompass the breadth of research problems being addressed using bacteriocins. I wrote to as many
people as I knew worlting in the field and was gratified to receive an enthusiastic response to the idea of
a workshop. I was lucky that Claude Lazdunski and Franc Pattus agreed to be co-organisers. They had
considerable input into the structure of the workshop programme, the selection of the invited speakers and
the general planning of the workshop. Without them the workshop would have been impossible to
organise. Not least in importance was their inspired choice of the Isle de Bendor as the workshop location.
Everyone who attended the workshop will I am sure have happy memories of Bendor and Provence. Like
me, I am sure that many will hope to return.
In order to broaden the scope of the workshop, we also included sessions on microcins and
lantibiotics. Microcins are a family of low molecular weight antibiotics « 10,000 daltons) produced by
diverse strains of Enterobacteriaceae. Microcins, like other traditional antibiotics, are produced as the
cultures enter the stationary phase of growth. They can therefore serve as excellent model systems in which
to study stationary phase phenomena. As a bonus, microcins are secreted into the medium by dedicated
export mechanisms and are therefore model systems for studying signal-sequence independent protein
secretion.
Lantibiotics are distinguished from other bacteriocins of Gram-positive bacteria because of their
unique structural features. The term lantibiotic is an abbreviation for lanthionine-containing peptide
antibiotic. Besides lanthionine and its analogue 3-methyllanthionine, all lantibiotics contain
didehydroalanine and/or didehydrobutyrine. There is considerable interest in determining the solution
structures of these unusual molecules and the mechanisms by which the unusual amino-acid residues are
introduced into lantibiotics. It is of particular interest that one member of the lantibiotics, nisin, is now
approved for use as a food preservative. This is perhaps the first example of an important commercial use
for a member of the bacteriocins, microcins and lantibiotics. Epidermin, another lantibiotic, has potential
as a highly specific therapeutic drug against acne. If we consider that the primary role of bacteriocins in
nature is as "agents of bacteriological warfare" against other bacteria, then it is possible to envisage
bacteriocins, other than nisin, being used as biological control agents. In the event this would dramatically
alter the funding prosepects for work with these molecules, and would also ensure that there are many more
workshops like Bendor in the future.
The workshop co-organisers invited some 40 participants and then selected 36 others from those
who applied in response to advertizements. The participants came from a total of 17 countries, with most
of the major research groups in the world being represented. A list of participants is included at the back
of this book. I am sure that all participants will agree with me that the quality of the invited lectures was
very high, with considerable discussion following each one. The workshop timetable did not try to pack
VII
in too many lectures. I believe that this proved to be a suitable fonnat for the exchange of views, techniques
and data betwen the participants. The workshop was also enhanced by the large number ofv ery high quality
posters on display. It is unfortunate that the rival attractions of the French cuisine, especially at dinner,
restricted the time for participants to view the posters. I am therefore glad that some of the poster
presentations have been written up as manuscripts and are included in the second half of this book.
The reader will hopefully notice that the contributions to this book have been type-set. I personally
do not like to see the array of typefaces and fonnats which appear in the typical Proceedings of a workshop.
I therefore decided to use an Apple Mac desk top publishing system, available in Norwich, to impose a
common fonnat and typeface on the contributions. With the benefit of hindsight, considering the amount
of work involved, I would probably not do this again unless the authors provided their contributions in a
suitable electronic fomat for direct input into the desk top publishing system. However, I hope that both
contributors and readers appreciate the efort involved and agree that it improves the appearance and
readibility of the book. I hope that the publication of this volume will remind those who were there what
a marvellous workshop it was, and will perhaps persuade someone that it would be useful to organize a
follow up meeting in two years time. In addition I hope that it will attract new workers to this wonderful
research field.
Lastly I should like on behalf of my co-organisers to thank Deborah Oemitshaw the AR W secretary.
Without her considerable organisational and secretarial skills, both in Norwich and in Bendor, the planning
and smooth running of this Workshop, as well as the production of this book, would have been made
impossible.
Richard James
Norwich
Acknowledgements
In addition to the major financial support from EMB 0, FEMS and NATO for this AR W, we are also
grateful to the CNRS, France for providing financial support for the travel expenses of French-based
speakers.
Contents
Introduction to the Microcin Session
Roberto Kolter and Felipe Moreno .......................................................................................................... 1
Escherichia coli genes regulating the production ofmicrocins MCCBl7 and MCCC7
F. Moreno, J. L. San Millan, I. del Castillo, J. M. G6mez, M. C. Rodriguez-Sainz, J. E. Gonzalez-
Pastor and L. Diaz-Guerra ........................................................................................................................ 3
Uptake and mode of action of the peptide antibiotic microcin Bl7
C. Hernandez-Chico, O. Mayo, J. L. Vizan, M. Lavinal, and F. Moreno ............................................. 15
The structure and maturation pathway ofmicrocin Bl7
Peter Yorgey, Jonathan Lee and Roberto Kolter ................................................................................... 19
Bacteriocins of Gram-positive bacteria: an opinion regarding their nature, nomenclature and
numbers
J. R. Tagg ............................................................................................................................................... 33
Molecular properties of Lactobacillus bacteriocins
T.R. Klaenhammer, C. Ahn, C. Fremaux and K. Milton ....................................................................... 37
Lactococcal bacteriocins: genetics and mode of action
MJ. van Belkum, B.J. Hayema, J. Kok, G. Venema, H. Ho10, I.F. Nes, W.N. Konings
and T. Abee ............................................................................................................................................ 59
Introduction to the Lantibiotics session
H.G. Sahl ................................................................................................................................................ 71
Lantibiotics : An overview and conformational studies on Gallidermin and PepS
Stefan Freund and GUnther Jung ............................................................................................................ 75
Biosynthesis of the lantibiotic PepS and mode of action of type A lantibiotics
H.-G. Sahl ............................................................................................................................................... 93
Identification of genes involved in lantibiotic synthesis
K.-D. Entian, C. Klein and C. Kaletta ................................................................................................. 107
Pore forming bacteriocins
D. Baty, F. Pattus and A. Finkelstein ................................................................................................... 117
In vivo properties of colicin A: channel activity and translocation
L. Letellier, C. Lazdunski, H. Benedetti, J.P. Bourdineaud, P. Boulanger .......................................... 119
Site-directed fluorescence spectroscopy as a tool to study the membrane insertion of colicin A
J.H. Lakey, D. Baty, JM Gonzalez-Manas, D. Duche and F. Pattus .................................................... 127
Structure-function of the colicin El ion channel: voltage-driven translocation and gating of a
tetra-(or hexa-) helix channel
W. A. Cramer, F. S. Cohen, C. V. Stauffacher, Y.-L. Zhang, A. R. Merrill, H. Y. Song
and P. Elkins ......................................................................................................................................... 139
Voltage-dependent gating of colicin El channels in planar bilayers
S.L. Slatin, K.S. Jakes, c.K. Abrams & A. Finkelstein ....................................................................... 151
Immunity to colicins
Karen S. Jakes and Claude Lazdunski ................................................................................................. 163
x
Immunity protein to pore forming colicins
Vincent Geli & Claude Lazdunski ....................................................................................................... 171
Specificity determinants for the interaction of colicin E9 with its immunity protein
R. James, M.D. Curtis, R. Wallis, M. Osborne, C. Kleanthous and G.R. Moore ............................... 181
Structural studies on colicin E3 and its immunity protein
M. Shoham and A. Djebli .................................................................................................................... 203
Study of the import mechanisms of colicins through protein engineering and K+ effiux kinetics
Helene Benedetti, Lucienne Letellier, Roland Lloures, Vincent Geli, Daniel Baty
and Claude Lazdunski .......................................................................................................................... 215
Import and export of colicin M
V. Braun, S. Gaisser, C. Glaser, R. Harkness, T. Olschliger and 1. Mende ......................................... 225
ToIA: structure, location and role in the uptake of colicins
Robert E. Webster and Sharyn K. Levengood ..................................................................................... 243
Domains of the Escherichia coli BtuB protein involved in outer membrane association and
interaction with colicin translocation components
Robert J. Kadner, Bei-Yang Wei and Wolfgang Koster ...................................................................... 255
A structure-function analysis of BtuB, the E.coli vitamin B12 outer membrane
transport protein
RJ.Ward, S.E.Hufton, N.A.C.Bunce, A.J.P.Fletcher and R.E.Glass .................................................. 271
General introduction to the secretion of bacteriocins
D. Cavard and B. Oudega .................................................................................................................... 297
Functioning of the pCloDF13 encoded BRP
J. Luirink, O. Mol and B. Oudega ....................................................................................................... 307
Structure/function relationships in the signal sequence of the colicin A lysis protein
S. P. Howard and L. Lindsay ............................................................................................................... 317
The secretion of colicin V
Michael J. Fath, Rachel Skvirsky, Lynne Gilson, Hare Krishna Mahanty and Roberto Kolter .......... 331
Introduction to the session on the evolution of bacteriocins
Richard James ...................................................................................................................................... 349
Molecular evolution of E colicin plasmids with emphasis on the endonuclease types
Peter C.K. Lau, Michael Parsons and Tai Uchimura ........................................................................... 353
Immunity specificity and evolution of the nuclease-type E colicins
H. Masaki, S. Yajima, A. Akutsu-Koide, T. Ohta and T. Uozumi ...................................................... 379
Replicon evolution of ColE2-related plasmids
T. Itoh and S. Hiraga ............................................................................................................................ 397
Manuscripts of poster presentations
Genetic determinants for microcin H47, an Escherichia coli chromosome-encoded antibiotic
M. Lavifia and C. Gaggero ................................................................................................................... 413
BLIS production in the genus Streptococcus
J.R. Tagg .............................................................................................................................................. 417
XI
A new Leuconostoc bacteriocin, Mesentericin YIOS, bactericidal to Listeria monocytogenes
Y. Hechard, C. Jayat, F. Letellier, M.H. Ratinaud, R. Julien and Y. Cenatiempo .............................. .421
Cloning and characterisation of a lysin gene from a Listeria bacteriophage
John Payne and Michael Gasson .......................................................................................................... 427
Transformation of Enterococcus jaecalis OGIX with the plasmid pMB2 encoding for the peptide
antibiotic AS-48, by protoplast fusion and regeneration on calcium alginate
M. Martinez-Bueno, I. Guerra, M.Maqueda, A.GaIvez and E.Valdivia ............................................. .433
NMR studies of lantibiotics: the three-dimensional structure of nisin in aqueous solution
Frank J.M. van de Yen, Henno W. van den Hooven, Cornelis W. Hilbers
and Ruud N.H. Konings ....................................................................................................................... 435
Localization and phenotypic expression of genes involved in the biosynthesis of the Lactococcus
lactis subsp. lactis lantibiotic nisin
Luc De Vuyst and Erick J. Vandamme ............................................................................................... .449
Expression of nisin in Bacillus subtllis
Helena Rintala, Lars Paulin, Tytti Graeffe, Tiina Immonen and Per E.J. Saris .................................. .463
Tn5301, a Lactococcal transposon encoding genes for nisin biosynthesis
H.M. Dodd, N. Horn. S. Swindell and M.J. Gasson ........................................................................... .473
Development of yeast inhibitory compounds for incorporation into silage inoculants
S.A.Goodman, C.Orr, P.J.Warner ........................................................................................................ 479
The excC and excD genes of Escherichia coli K-12 encode the peptidoglycan-associated
lipoprotein (PAL) and the TolQ protein, respectively
J.C. Lazzaroni, A. Vianney, C. Amouroux and R. Portalier ............................................................... .487
Resistance and tolerance of bacteria to E colicins
J. Smarda .............................................................................................................................................. 493
Construction and characterization of chimeric proteins between pyocins and colicin E3
M. Kageyama , M. Kobayashi, Y. Sano , T. Uozumi and H. Masaki ................................................. 503
Colicins as anti-tumour drugs
J. Smarda .............................................................................................................................................. 505
List of Participants ............................................................................................................................. 511
Subject Index ...................................................................................................................................... 513
