Beijerinck Centennial
.
Microbial Physiology and Gene Regulation:
Emerging Principles and Applications
10, 14 December 1995
The Hague, The N etherlands
Microbial Physiology and Gene Regulation I
Beijerinck Centennial
Microbial Physiology and Gene Regulation:
Emerging Principles and Applications
libliotheek TU Delft
1111111111111111111111111111111111 C Oi03!1391 i2414
534
1
Microbial Physiology and Gene Regulation 111
Beijerinck Centennial
Microbial Physiology and Gene Regulation:
Emerging Principles and Applications
The Hague, The Netherlands, 10 - 14 December 1995
Editors: W.A. Scheffers and J.P. van Dijken
The Beijerinck Centennial has been organized jointly by: The American Society for Microbiology The Netherlands Society for Microbiology
The Netherlands Biotechnological Society The Delft University of Technology
IV Beijerinck Centennial
Published and distributed by: Delft University Press Stevinweg 1
2628 CN Delft The Netherlands
Telephone +31 152783254 Telefax + 31 15 2781661
Graphic image of Beijerinck: J.P. de Ruiter
CIP-DATA Koninklijke Bibliotheek, Den Haag Beijerinck
Beijerinck Centennial ; Microbial Physiology and Gene Regulation: Emerging Principles and Applications. Book of Abstracts / ed. by W.A. Scheffers and J.P. van Dijken - Delft: University Press. -III. -With index
ISBN 90-407-1213-1 NUGI 841
Subject heading: Microbial Physiology / Gene Regulation / Biodiversity & Evolution Copyright c 1995 by Delft University Press
All rights reserved.
No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, eletronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without permission from the publisher: Delft University Press, Stevinweg 1,2628 CN Delft, The Netherlands.
Microbial Physiology and Gene Regulation V
Preface
The Beijerinck Centennial is bringing together over 300 scientists from all over the world, contributing more than 250 lectures and posters. With the founders of the Delft School of Microbiology they share their fascination with microbiology in all its facets: from diversity to physiology, from interaction to application. Like Beijerinck, Kluyver and Van Niel, we all want to know why and how.
We are ready to use all of the modern techniques available to hand, and do not care whether it is called molecular biology, NMR, biochemistry, ecology or technology.
Today, much of our fundamental understanding of how the intact organism functions can be obtained by studying genes and the way they are expressed and regulated in a complex, yet concerted way. One of the main challenges of modern microbial physiology is thus to integrate knowledge gained from molecular biology and biochemistry with information gained from more classical lines of research. Only the combination will allow us to develop a full picture of how organisrns survive in their naturalor artificial environments, how they play a role in rnaintaining the balanees on this earth, and how they can be controlled for beneficial applications. The founders of the Delft School of Microbiology would have been fascinated by the present possibilities. No doubt they would agree with Beijerinck's statement: "Gelukkig ZIJ die NU beginnen, - Happy THOSE who are starting NOW".
J.G. Kuenen
Professor of Microbiology Delft University of Technology
Microbial Physiology and Gene Regulation 1
Contents
Page Organizing Committee 3 Scientific Committee 3 Sponsors 3Program & Lectures 5
Poster Contributions 173
Classification Poster Contributions 175
Index Poster Contributions 511
Organizing Committee
J.G. Kuenen (chairman) R.H. Baltz J . W. Bennett P. Bos A.M. Breure S. Kaplan S.A. Lerner L.A. RobertsonW.A. Scheffers (secretary) C.P. van der Beek J.P. van Dijken
Sponsors
The following sponsors supported the symposium:
Abbott Laboratories
AKZO Nobel Pharma/Diosynth American Society for Microbiology Applikon B.V.
Avebe B.V.
Delft Faculty of Chemical' Engineering and Materials Science
Delft University of Technology DSM Research
Eli Lilly and Company European Commission Witb special thanks to:
Microbial Physiology and Gene Regulation 3
Scientific Committee
J.G. Kuenen (chairrnan) R.H. Baltz J . W. Bennett P. Bos A.M. Breure J.A.M. de Bont E.J.J. Lugtenberg W. A. Scheffers A.H. Stouthamer J.P. van Dijken J. Verhoef G.D. VogelsFoundation Antonie van Leeuwenhoek Glucona B.V.
Heineken Nederland N. V. Koninklijke Gist-brocades N.V. Lepetit Research Centre Lilly Research Laboratory Nestlé S.A.
Netherlands Society for Microbiology TNO Biotechnology
Unilever Research Laboratory
Microbial Physiology and Gene Regulation 5
Program
Sunday 10 December 1995 17.00 - 20.00 hrs. 18.00 - 19.15 hrs. 19.15 - 20.30 hrs. RegistrationChair: J.G. Kuenen, Delft, The Netherlands
Opening of the Beijerinck Centennia! by the Rector Magnificus of Delft University of Technology, K.F. Wakker
J.W.M. la Rivière, Delft, The Netherlands
The Delft School of Microbiology in historica! perspective
Get-together party
Offered by the Delft University of Technology Monday 11 December 1995
8.00 - 9.00 hrs.
9.00 - 9.20 hrs.
Registration
Opening by the Chairman of the Symposium, J.G. Kuenen, Delft, The Netherlands
Al Molecular Physiology of Microbial Interactions
(Plenary session) 9.20 - 10.00 hrs. 10.00 - 10.40 hrs. 10.40 - 11.10 hrs. 11.10 - 11.50 hrs. 11.50 - 12.30 hrs. 12.30 - 14.00 hrs.
Chair: J.W. Bennett, New Orleans, USA Co-chair: P. Bos, Delft, The Netherlands S. Normark, Stockholm, Sweden
Bacteria! adhesins and their role in infectious disease H.P. Spaink, B.J.J. Lugtenberg, Leiden, The Netherlands Molecular basis of nodulation of leguminous plants by rhizobia
Coffee break/ Posters
E.P. Greenberg, Iowa City, USA Quorum sensing in bacteria J. W. Costerton, Bozeman, USA Microbia! interactions in biofilrns
Poster exhibition and lunch
14 15 16 17 19 20
6 Beijerinck Centennial
Monday 11 December 1995
A2 Quorum Sensing in Micro-Organisms (parallel session) 14.00 - 14.10 hrs. 14.10 -14.40 hrs. 14.40 - 15.10 hrs. 15.10 - 15.40 hrs. 15.40 - 16.00 hrs. 16.00 - 16.20 hrs. 16.20 - 16.40 hrs. 16.40 -17.00 hrs.
Chair: E.P. Greenberg, Iowa City, USA
Co-chair: K.J. Hellingwerf, Amsterdam, The Netherlands Chairman' s introduction
H.H. Iglewski, L. Passador, J. Pearson, E. Pesci, P. Seed, Rochester, USA
Quorum sensing and regulation of virulence genes in Pseudomonas
aeruginosa
B.L. Bassier, Princeton, USA
Intercellular signalling in Vibrio harveyi: Regulation of the expression of bioluminescence
Tea break/ Posters
K.L. Visick, K.J. Boetteher, E.G. Ruby, Los Angeles, USA The role of symbiotic signaling during colonization of the squid light organ by Vibrio fischeri
B.A.B. Leonard, A. ColweIl, A. Podbielski, G.M. Dunny, Minneapolis, USA Regulation of endogenous pheromone to prevent self-induction of conjugation and its possible role in plasmid replication in Enterococcus faecalis
M.B. Brurberg, D.B. Diep, V. Eijsink, L.S. Hävarstein, I.F. Nes, Às, Norway
Quorum sensing and the regulation of bacteriocin production in Lactobacillus A.A.N. van Brussel, Leiden, The Netherlands
Identification of acylated homoserine lactone molecules in Rhizobium
leguminosarum; possible functions.
Cl 8acterial Metabolism of Inorganic Compounds (Parallel session)
Chair: E.R. Leadbetter, Storrs, USA
Co-chair: A.M. Breure, Bilthoven, The Netherlands 14.00 -14.10 hrs. Chairman's introduction
14.10 - 14.40 hrs. A.B. Hooper, St. Paul, USA
Enzymology of the oxidation of ammonia to nitrite by bacteria 14.40 - 15.10 hrs. D.P. Kelly, Coventry, Warwick, United Kingdom
Oxidative metabolism of inorganic sulfur compounds by bacteria
15.10 - 15.40 hrs. Tea break/ Posters
23 24 25 27 28 30 32 3S 36 38
Microbial Physiology and Gene Regulation 7
Monday 11 December 1995
Cl Bacterial Metabolism of Inorganic Compounds
(Parallel session, continued)
15.40 - 16.10 hrs.
16.10 - 16.30 hrs.
16.30 - 16.50 hrs.
16.50 - 17.10 hrs.
M.C.M. van Loosdrecht, G.J. Smolders, T. Kuba, J.J. Heijnen, Delft, The Netherlands
Ecophysiology of polyphosphate accumulating bacteria: Need for pure cultures?
J.B. UtAker, L. Bakken, Q.Q. Jiang, I.F. Nes, Às, Norway Phylogeny of the cJosely related ammonia-oxidizing bacteria
L. Crossman, J. Moir, J.-M. Wehrfritz, A. Keech, A. Thomson, S. Spiro, D. Richardson, Norwich, United Kingdom
Heterotrophic nitrification in Paraeoeeus denitrifieans
R.P. Gunsalus, R. Cavicchioli, R. Chiang, I. Shroder, Los AngeJes, USA MolecuJar biology of nitrate sensing and regulation of the anaerobic electron transport related genes in Eseheriehia eoU
Dl Metabolic Regulation in Industrial Yeasts
(Parallel session) 14.00 - 14.10 hrs. 14.10 - 14.40 hrs. 14.40 - 15.10 hrs. 15.10 - 15.40 hrs. 15.40 - 16.10 hrs. 16.10 - 16.40 hrs. 16.40 - 17.00 hrs. 17.00 - 17.20 hrs.
Chair: W. Harder, Delft, The Netherlands
Chairman's introduction
J.T. Pronk, LP. van Dijken, H.Y. Steensma, Delft, The Netherlands Modification of metabolic fluxes at the pyruvate node in Saeeharomyees eerevisiae
C.T. Verrips, J. Chapman, Vlaardingen, The Netherlands Present and future use of yeast in industry
Tea break/ Posters
1.1. van der Klei, M. Veenhuis, Haren, The Netherlands
Peroxisome biogenesis and degradation in the methylotrophic yeast
Hansenula polymorpha
S.G. Oliver, Manchester, United Kingdom Future steps in the yeast genome project
M. Elskens, C. Jaspers, K. Medhi, M. Penninckx, Brussels, Belgium Glutathione, a major non-protein thiol in rnicro-organisms: roles in the cellular maintenance of yeast Saeeharomyees eerevisiae and drug bioreduction M.S. van Dyk, I.P.B. Rensburg, V.N. Thanh, Bloemfontein, South Africa The reduction of monoterpenoids by yeasts
Plenary Evening Session - Kluyver Memorial Lecture
17.40 - 18.40 hrs. Chair: J.G. Kuenen, Delft, The Netherlands
R. Thauer, Marburg, Germany
Biodiversity and unity in rnicrobial biochernistry
18.40 - 19.40 hrs. Reeeption offered by the Netherlands Society for Mierobiology
40 42 44 46 49 52 54 57 59 60 62 65 66
8 Beijerinck Centennial
Tuesday 12 December 1995 BI Biodiversity and Evolution
(Plenary session) 9.00 - 9.40 hrs. 9.40 - 10.20 hrs. 10.20 - 11.00 hrs. 11.00 - 11.40 hrs. 11.40 - 12.20 hrs. 12.20 - 14.00 hrs.
Chair: R.K. Thauer, Marburg, Gerrnany
Co-chair: G.O. Vogels, Nijmegen, The Netherlands
F. Aeckersberg, P. Rueter, R. Rabus, F. Widdel, Bremen, Gerrnany Hydrocarbon degradation by anaerobic bacteria
J.R. van der Meer, J.H.I. Leveau, R. Ravatn, R. Tchelet, C. Werlen,
A.J.B. Zehnder, Duebendorf, Switzerland
Evolution of novel metabolic pathways for the degradation of chloroaromatic compounds
Coffee break/ Posters
L. Margulis, Amherst, USA
Symbiogenesis: Evolution of cell organelles A. Tomasz, New York, USA
Assembly of new genotypes in multidrug-resistant clones of staphylococci and pneumococci
Poster exhibition and lunchbuffet
A3 Microbial Interactions in tbe Hmnan and Animal Host
(Parallel session) 14.00 - 14.10 hrs. 14.10 - 14.40 hrs. 14.40 - 15.10 hrs. 15.10 - 15.40 hrs. 15.40 - 16.10 hrs. 16.10 - 16.30 hrs. 16.30 - 16.50 hrs. 16.50 - 17.10 hrs.
Chair: 1. Verhoef, Utrecht, The Netherlands Chairrnan' s introduction
A. Camilli, 1.1. Mekalanos, Boston, USA
In vivo expression technology: Probing the pathogen-host interface D.B. Young, London, United Kingdom
New developments in tuberculosis
Tea break/ Posters
A. Pettersson, 1.T. Poolman, P. van der Ley, 1. Tommassen, Utrecht, The Netherlands
Response of Neisseria meningitidis to iron limitation in the host R. Wirth, A. Muscholl, G. Wanner, Regensburg, Gerrnany Interactions of Enterococcus faecalis with its environment 1.R. Leadbetter, 1.A. Breznak, East Lansing, USA
Isolation, characterization, and in situ localization of termite gut methanogens
P.S. Langendiik, C.W.A. van den Kieboom, l.S. van der Hoeven,
Nijmegen, The Netherlands
Sulfate-reducing bacteria in the periodontal pocket
69 70 72 74 76 79 80 82 83 85 86 88
Microbial Physiology and Gene Regulation 9
Tuesday 12 December 1995
B2 Biodiversity in Complex Microbial Systems
(Parallel session) Chair: H.J. Laanbroek, Nieuwersluis, The Netherlands 14.00 - 14.10 hrs. 14.10 - 14.40 hrs. 14.40 - 15.10 hrs. 15.10 -15.40 hrs. 15.40 - 16.10 hrs. 16.10 - 16.30 hrs. 16.30 - 16.50 hrs. 16.50 -17.10 hrs. Chairman' s introduction R.1. Amann, München, Germany
In situ hybridization with rRNA-targeted probes as a tooi to study the composition and dynamics of microbial communities
O.M. Ward, S.C. Nold, C.M. Santegoeds, Bozeman, USA
Biodiversity within a hot spring microbial mat community: molecular monitoring of enrichment cultures
Tea break/ Posters
N.R. Pace, S.M. Barns, A.-L. Reysenbach, G.S. Wickharn, M. Ehringer, Bloomington, USA
Analysis of YeUowstone hot spring communities by 16S rRNA sequences without cultivation
P. Westbroek, Leiden, The Netherlands Calcifying phytoplankton and world climate G.S. Roberts, G. Lewis, Auckland, New Zealand
A novel method for the detection of bacterial ceUs in cryosectioned biofilms by in situ hybridisation and PCR produets
J.M. Wood, G. Avgustin, K.P. Scott, H.l. Flint, Aberdeen, United Kingdom
Assessment of the relative abundance of Prevotella ruminicola strains in the rumen ecosystem by restriction enzyme profiling of PCR produets D2 lndustrial Production of Antibiotics and Proteins
(Parallel session) 14.00 - 14.10 hrs. 14.10 - 14.40 hrs. 14.40 - 15.10 hrs. 15.10 -15.40 hrs. 15.40 - 16.10 hrs. 16.10 - 16.30 hrs.
Chair: R.H. Baltz, Indianapolis, USA
Co-chair: C.P. van der Beek, Delft, The Netherlands Chairman' s introduction
R.H. Baltz, M.A. McHenney, C.A. CantweU, E.T. Seno, S.W. Queener, P.J. Solenberg, Indianapolis, USA
Applications of transposition mutagenesis in antibiotic producing streptomycetes O.A. Hopwood, Norwich, United Kingdom
Genetic manipulation of polyketide biosynthesis in Streptomyces for aromatic antibiotic production
Tea break/ Posters
W.J. Quax, R.WJ. Hommes, Delft, The Netherlands New concepts in industrial enzyme production
F. Marinelli, L. Gastaldo, C. Quarta, E. Selva, Varese, Italy
Antibiotic GE37468A: A new inhibitor of bacterial protein synthesis. III. Strain and fermentation studies
91 92 93 94 96 98 100 103 104 105 106 108
10 Beijerinck Centennial
Tuesday 12 December 1995
D2 Industrial Production of Antibiotics and Proteins (Parallel session, continued)
16.30 - 16.50 hrs. K. Ylihonko, J. Tuikkanen, S. Jussila, J. Hakala, P. Mäntsälä, Turku, Finland
Characterization of a Streptomyces nogalater gene cluster producing a biosynthetic intermediate of anthracyclines
110
16.50 -17.10 hrs. K. Mitsushima, A. Takimoto, S. Yagi, Osaka, Japan 112 Gene cloning and expression of a cephalosporin-C deacetylase from Bacillus subtiUs
Plenary Evening Session - Historical Lecture
17.30 - 18.15 hrs.
18.30 - 20.00 hrs.
Chair: J. Verhoef, Utrecht, The Netherlands M.C. Horzinek, Utrecht, Tbe Netherlands Tbe birth of virology
Reception offered by Heineken Breweries, at the Municipal Museum of The Hague
Wednesday 13 December 1995
C2 Metabolism of Inorganic Nitrogen Compounds: Emerging Principles and Applications (Plenary session) 9.00 - 9.20 hrs. 9.20 - 10.00 hrs. 10.00 - 10.40 hrs. 10.40 - 11.20 hrs. 11.20 -12.00 hrs. 12.00 - 12.40 hrs. 12.40 - 14.00 hrs. 14.00 -23.00 hrs.
Chair: A.H. Stouthamer, Amsterdam, The Netherlands A.H. Stouthamer, Amsterdam, The Netherlands
Introduction to the metabolism of inorganic nitrogen compounds W.G. Zumft, H. Körner, Karlsruhe, Germany
Diversity of gene organization and enzymes of denitrification H. Hennecke,
o.
Preisig, R. Zufferey, L. Tböny-Meyer, Zürich, SwitzerlandTbe role of respiration in symbiotic nitrogen fixation
Coffee break/ Posters
M. Jetten, S. Logemann, G. Muyzer, M. van Loosdrecht, S. de Vries, L. Robertson, J.G. Kuenen, Delft, The Netherlands
Novel principles and processes in the removal of nitrogen from waste water N.P. Revsbech, Aarhus, Denmark
Ecology of nitrification and denitrification
Poster exhibition and buffetlunch Excursions to Delft (optional)
114 116 119 120 122 124 126 128
Microbial Physiology and Gene Regulation II
Thursday 14 December 1995
D3 Industrial Use of Micro-Organisms
9.00 - 9.40 hrs. 9.40 - 10.20 hrs. JO.20 - 11.00 hrs. 11.00 - 11.40 hrs. 11.40 -12.20 hrs. 12.20 - 14.00 hrs.
Chair: J.A.M. de Bont, Wageningen, The Netherlands
H. Sahm, Jülich, Germany
Metabolic aspects of L-lysine and L-isoleucine production with Corynebacterium glutamicum
J.F. Martin, León, Spain
Classical and modem aspects of penicillin production and its regulation
Coffee break/ Posters
W.N. Konings, B. Poolman, J. Lolkema, A.J.M. Driessen, Haren, The Netherlands
Transport of low and high molecular weight compounds across the cytoplasmic membranes of bacteria and archaea
T. Stachelhaus, A. Schneider, M.A. Marahiel, Marburg, Germany Molecular structure of peptide synthetases involved in antibiotic production
Poster exhibition and buffetlunch
A4 Interactions of Microbes with Plants
(Parallel session) 14.00 - 14.10 hrs. 14.10 - 14.40 hrs. 14.40 - 15.10 hrs. 15.JO - 15.40 hrs. 15.40 - 16.10 hrs. 16.10 - 16.30 hrs. 16.30 - 16.50 hrs.
Chair: EJ.J. Lugtenberg, Leiden, The Netherlands
Chairman's introduction
P.J.G.M. de Wit, R. Laugé, G. Honée, M.H.AJ. Joosten, P. Vossen, M. Kooman-Gersmann, R. Vogelsang, J. Vervoort, Wageningen, The Netherlands
Molecular and biochemical basis of communication between tomato and its fungal pathogen Cladosporium fulvum
F. Q'Gara, Cork, Ireland
Plant growth-promoting rhizobacteria: Progress and potential
Tea break/ Posters
F.J. de Bruijn, A. Milcamps, D. Ragatz, East Lansing, USA Environmental control of gene expression in Rhizobium meliloti
C.C. Tebbe, F. Schwieger, B. Willke, M. Keiler, A. Piihler, J.C. Munch, Braunschweig, Germany
Field release of genetica1ly engineered Rhizobium meliloti strains to study the survival, spread and ecological interactions of an introduced soil
bacterium
G.Muii.oz, E. Agosin, M. Penttila, Santiago, Chile
lsolation of hydrophobin-like spore proteins involved in the biocontrol competence of Trichoderma ha17.ianum
131 132 134 136 138 141 142 144 146 148 150
12 Beijerinck Centennia1
Thursday 14 december 1995
B3 Biodiversity and Endosymbiotic Interactions (Parallel session) 14.00 - 14.10 hrs. 14.10 - 14.40 hrs. 14.40 - 15.10 hrs. 15.10 - 15.40 hrs. 15.40 - 16.10 hrs. 16.10 - 16.30 hrs. 16.30 - 16.50 hrs.
Chair: J.C. Gottschal, Haren, The Netherlands Chairman' s introduction
G.D. Vogels, J.H.P. Hackstein, Nijmegen, The Netherlands Endosymbiotic interactions in anaerobic protozoa
C.M. Cavanaugh, Cambridge, USA
Chemosynthetic bacteriaJmarine invertebrate symbioses Tea break/ Posters
T. Fenchel, HelsingliJr, Denmark
Ecophysiology of protozoa with endo- and ectosymbionts
M. van der Giezen, J.C. Gottschal, R.A. Prins, Haren, The Netherlands The evolutionary origin of hydrogenosomes in anaerobic fungi
D.M. Krueger, C.M. Cavanaugh, Cambridge, USA
Diversity of bacterial symbionts from bivalve hosts: Phylogenetic evidence for multiple origins of the Solemya symbiosis
D4 Industrial Application of Metabolic Engineering (Parallel session)
14.00 - 14.10 hrs. 14.10 -14.40 hrs.
14.40 - 15.10 hrs.
15.10 - 15.40 hrs.
Chair: S. Kaplan, Houston, USA
Co-chair: J.A. den Hollander, Delft, The Netherlands Chairman' s introduction
J .E. Bailey, Zürich, Switzerland Engineering of metabolic networks L. Katz, Abbott Park, USA
Genetic engineering of macrolide antibiotic production Tea break/ Posters
153 154 156 158 159 160 163 164 165
15.40 -16.10 hrs. A.A. de Graaf, A. Marx, W. Wiechert, L. Eggeling, H. Sahm, Jülich, 167
16.10 -16.30 hrs.
16.30 - 16.50 hrs.
Plenary Closing session 17.00 - 17.30 hrs.
Germany
Determination of fluxes in the central metabolism of microorganisms by
NMR spectroscopy combined with metabolite balancing: principles and application to L-lysine overproduction with Corynebaeterium glutamieum
M. Zinn, R. Dumer, T. Egli, B. Witholt, Zürich, Switzerland Double-nutrient-limited growth of Pseudomonas oleovorans: Application of a transient feed technique in continuous culture
M.R. de Graef, J.L. Snoep, M.J. Teixeira de Mattos, H.V. Westerhoff, O.M. Neijssel, Amsterdam, The Netherlands
Anaerobic/aerobic transitions in Eseheriehia eoU: Effects on catabolism, NADH/NAD ratio, ATP/ADP ratio and DNA supercoiling
Closing session
169
Microbial Physiology and Gene Regulation 13
14 Beijerinck Centennial
The Delft School of Microbiology in historical perspective.
J.W.M. Ia Rivière, International Institute for Infrastructural, Hydraulic and Environmental Engineering, P.O. Box 3015, 2601 DA Delft, The Netherlands, fax: +31-15-2122921.
Microbiology has been practised in Delft for a long time. This paper examines how out of this work the Delft School of Microbiology developed itself. This is done by discussing the relevant contributions of its founding fathers Beijerinck, Kluyver and van Niel and an attempt is made to define contents and principles of the Delft School as it is understood today.
The paper further analyses wh at significance can be ascribed to the Delft School in the development of molecular biology, biotechnology, microbial ecology, microbial taxonomv and the study of biodiversity.
It also tries to explain the considerable resonance the Delft School enjoys in the microbiological scientific community and the interest it receives from historians of science. The author finally ventures to propose th at the principles of the Delft School have not only provided guidance in the past but also constitute a message th at is relevant and valuable for present and future microbiologists.
Microbial Physiology and Gene Regulation 15
16 Beijerinck Centennial
Bacterial adhesins and their role in infectious disease
s.
NormarkMicrobiology and Tumorbiology Center, Karolinska Institute, S-105 21 Stockholm, Sweden; Telefax: +468735.10.40
Abstract
The ability of a bacterial pathogen to colonise and cause infectious disease depends upon a number of interaction events between the pathogen and host cells. The microbe responds to signals evoked by the host environment as weIl as by other microbes and is also able to trigger various responses in the host that may result in cell death, bacteriaI entry into cells or an effect on unspecific and specific immune responses. Interaction events are described in this talk that are aimed to increase our molecular understanding of infectious diseases such as urinary tract infection, uIcer disease and salmonellosis. For Escherichia coti associated with urinary tract infections the receptor-binding Gala(I-4)Gal specific adhein is a protein located at the tip of so-called P-funbriae. Isogenic mutants show that the tip adhesin on E. coti P-fimbriae is essential for the microbe to generate kidney but not bladder infection in primates. Data also show that bacteriaI interaction to the Gai-Gai receptor evokes a signal in the bacterium upregulating a sensory-regulatory protein. In the gastric pathogen Heticobacter pylori interaction with the gastric mucosa is specified by the expression of a stationary phase induced adhesin mediating binding to gastric surface mucous cells. The receptor on these gastric cells is believed to be the fucosylated Leb antigen that is present on one dominating protein species the nature of which will be discussed. BacteriaI adhesins may not only play a role in the interaction with surface epithelium. The ability to express stationary phase induced curli organelles in Salmonella are required for the bacteria to cause salmonellosis in a murine model both via the oral route and intraperitoneally. The latter, suggests that this type of fiber may play a role in bacterial interaction with macrophages. These curli organelles are assembied via a novel pathway that involves the extracellular nucleation of secreted subunits.
Microbial Physiology and Gene Regulation 17
MOLECULAR BASIS OF NODULATION OF LEGUMINOUS
PLANTS BY RHIZOBIA
Herman P. Spaink and Ben
J.J.
Lugtenberg
Leiden
University,
Institute
of Molecular
Plant
Sciences,
Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.
FAX:3171-275088.
After the discovery by HeIlriegel and Wilfarth (1888), that root
nodules of leguminous plants are able to fix atmospheric nitrogen, it
soon became c1ear by the work of Beijerinck (1888) that bacteria
inside the nodules we re the actual nitrogen fixers. These bacteria are
presently being c1assified as be10nging to the genera
Rhizobium,
Bradyrhizobium
and
Azorhizobium,
collectively called rhizobia. The
interaction between rhizobia in the soil and the roots of leguminous
plants results in the induction of the root nodule. After invading
these root nodules
via
infect ion threads the bacterium differentiates
into a new form, the bacteroid, which is able to fix nitrogen under the
special physiological conditions inside cells of the root nodule. This
symbiotic interaction is highly host-specific in that each rhizobial
strain is able to associate with only a limited number of host plant
species.
The subject of this presentation is the molecular mechanism by
which the bacterium can induce the formation of root nodules as weIl
as the host-specific characteristics of this process. This mechanism
appears to be based on at least two stages of molecular signaling
between the bacterium and the plant host. In the first stage,
fIavonoids secreted by the plant root induce, often in a host specific
way, the transcription of bacterial genes which are involved in
nodulation, the so-called
nod
genes. This leads to the second step of
the signaling system: the production and secretion of Nod factors by
the
Rhizobium
bacteria. The latter signal molecules, which are
acylated forms of small fragments of chitin (called lipo-chitin
oligosaccharides), have many biological effe cts on the roots of the
host plants which are also observed in the interaction of the
bacterium with the root. One of these effects is the dedifferentiation
of groups of cells located in the cortex, which leads to the formation
of nodule meristems. With their mitogenic activity the bacterial Nod
factors resembie several well-known plant hormones like auxins and
cytokinins. However, there are two major differences: (i) the bacterial
signals lead to the induction of a specific organ and
(ii)
they are
host-specific in that only the signals produced by compatible bacteria are
able to induce meristems. The
nod
genes determine this stage of host
18 Beijerinck Centennial
specificity by their role
in
the biosynthesis or modification of the
signal molecules. They appear to encode enzymes which are involved
in the processes of fatty acid biosynthesis, fatty acid transfer, chitin
synthesis and chitin modification (e.g. acetyl-, fucosyl-, sulpho-,
carbamyl-, or methyltransferases). It will be illustrated that the
nod
gene products are ideal model enzymes for the study of these
important processes because they are not needed in the free-living
state of the bacteria.
It
has been suggested that lipo-oligosaccharide signals are also
produced by higher plants and animals and therefore their
biosynthesis might be a process which is common to many organisms
(Spaink and Lugtenberg, 1994; Semino and Robbins, 1995). Our
knowledge of the rhizobial system which is described in tbis
presentation will be an excellent basis to undertake the difficult task
of studying analogous signal molecules in higher organisms.
References
Beyerinck MW: Die bacterien der Papilionacaeeen-knollchen. Bot.
Zeitung 46-50: 725-804 (1888).
Hellriegel H, Wilfarth H: Untersuchungen über die stickstoffnahrung
der gramineen und leguminosen. Beilageheft Z. Ver.
Rübenzucker-Ind. D. Reichs (1988).
Semino
CE,
Robbins PW:
Synthesis of "Nod"-like
chitin
oligosaccharides by the
Xenopus
developmental protein DG42. Proc.
Natl. Acad. Sci. USA 92: 3498-3501 (1995).
Spaink HP, Lugtenberg BJJ: Role of rhizobial lipo-chitin
oligosaccharide signal molecules in root nodule organogenesis. Plant
Mol. Biol. 26: 1413-1422 (1994)
Microbial Physiology and Gene Regulation 19
Quorum sensing in bacteria E. Peter Greenberg
Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA (Fax: 00 1 319 335 7949).
A phenomenon termed quorum sensing and response is common to a number of gram-negative bacterial species including a variety of animal and plant pathogens. The model quorum sensing system is the cell density dependent activation of the Vibrio fiseheri luminescence genes. There are two
quorum sensing genes dedicated to regulation of V. fiseheri luminescence, luxl, which codes for production of an intercellular signal, the autoinducer,
and luxR. which codes for an autoinducer-dependent activator of the luminescence genes. Homologous regulatory genes exist in other bacteria and control such functions as extracellular virulence factors, antibiotic synthesis, and conjugation. The V. fiseheri autoinducer is N-(3-oxohexanoyl)
homoserine lactone. The autoinducers from different bacterial species can differ in acyl side chain length and in substitutions in the side chain. Some bacteria produce autoinducers with a hydroxyl in position 3, some have rio substitution in position 3, and at least one bacterium, Rhizobium leguminosarum produces an autoinducer with a double bond in the acyl side
chain. Some bacteria such as Pseudomonas aeruginosa have multiple
quorum sensing modulons.
The LuxR polypeptide is a two-domain transcriptional activator. Binding of the autoinducer to the N-terminal domain is required for an interaction of the C-terminal domain with lux regulatory DNA. The C-terminal domain binds
to this DNA in synergy with RNA polymerase. A variety of autoinducer analogs can bind to LuxR. Some of these analogs can stimulate luminescence gene expression. Others interfere with the ability of the autoinducer to stimulate luminescence gene expression.
Little is known about how the luxl product catalyzes synthesis of autoinducer.
Studies with whole cells of recombinant E. eoti indicate that s-adenosyl
methionine and not homoserine or homoserine lactone is a substrate for autoinducer synthesis. There is some evidence to suggest that the fatty acyl moiety is derived from the fatty acid biosynthetic rather than the degradative pathway.
20 Beijerinck Centennial
MICROBIAL INTERACTIONS IN BIOFILMS
J. William Costerton Director
Center for Biofilm Engineering Montana State University Bozeman MT USA 59717
Fax: I 406 994 6098
It is obvious from direct microscopic studies of living microbial biofilms that these sessile communities achieve a structural complexity and a sophistication of intercellular relationships that have never been seen in the hundreds of years during which Microbiologists have examined planktonic bacteria in pure cultures. Direct examinations of living biofilms show unequivocally that these attached populations consist of cells in slime-enclosed microcolonies separated by open water channels. The microcolonies that contain virtually all ofthe biofilm cells are often seen to assume complex shapes that clearly indicate that some measure of cell-cell communication is involved in their formation. The water channels have been shown to convey liquids by convective flow and we presurne that this anastomosing system of channels constitutes a primitive circulatory system that delivers nutrients and removes waste products. Thus, even in a simple monospecies biofilms, each cell occupies a special niche in which it is protected and in which it may develop optimal conditions in a "customized microniche". As bacterial cells adhere to a surf ace to initiate the process of biofilm formation they undergo a very profound phenotypic change that is directed by a special sigma factor and involves a large number of genes in the polysaccharide synthesis and cell wall synthesis loci. For these reasons bacterial cells within biofilms are substantially structurally different from planktonic cells ofthe same species. The fact that bacterial cells within biofilms extrude protons into the surrounding matrices, and not into the bulk fluid, mayalso profoundly alter the energy metabolism of sessile cells as contrasted with that of planktonic cells.
While the monospecies biofilm may achieve quite remarkable levels of complexity and sophistication these levels are far exceeded by the complexity ofthe multi species biofilms that dominate virtually all natural aquatic ecosystems. In these multi species biofilms cells of different species often cross-feed each other by the exchange of organic nutrients, or by direct hydrogen transfer, and cells that find themselves in optimal nutrient circumstances obviously divide to produce mixed microcolonies whose cooperative metabolic capabilities far exceed those of either biofilm or planktonic cells of single species. The biofilm communities that mediate the decomposition of complex organic matter in leaf litter or in the romen clearly achieve their impressive efficiency by the cooperative interrelations of their component species that are held in stabie juxtaposition by their growth within these highly structured biofilms. In fact it is difficuit to imagine how bacterial cells could cooperate metabolically, in the same way, ifthese organisms were all independently mobile in the bulk tluid phase. Because ofthe structural and metabolic heterogeneity and complexity of microbial biofilms it is not surprising that these sessile
Microbial Physiology and Gene Regulation 21
electrochemical heterogeneity, especially when they grow on nonconductive surfaces. If these electrochemical heterogeneities develop in biofilms on conductive surf aces these potential differences may generate a corrosion current and mediate microbially influenced corrosion.
Recent studies of microbial biofilms have revealed a level of complexity in structure and in cell-cell interrelationships that is impossible in planktonic communities in which all members live in the bulk fluid. We expect that further direct observations of living biofilms by modern methods of microscopy will reveal even more complex structures and interrelationships.
Microbial Physiology and Gene Regulation 23
24 Beijerinck Centennia!
Quorum Sensing and Regulation of Virulence Genes in Pseudomonas aeruginosa. B.H. 19lewski, L. Passador,
J.
Pearson, E. Pesci and P. SeedDepartment of Microbiology and Immunology, University of Rochester, Rochester, New York 14642
Pseudomonas aeruginosa is an important opportunistic pathogen of humans
. Virulence of this organism is multifactorialland involves both c;ell-associated and extracellular products. The extracellular virulence factors include a large number of toxins and enzymes such as toxin A, exoenzyme 5, hemolysins and at least three proteases; elastase, LasA elastase and alkaline protease. We and others have shown that the genes encoding many of these virulence factors are regulated by a process termed autoinduction or quorum-sensing. In P.aeruginosa two pairs of quorum-sensing components exist; the LuxR-like proteins
LasR and RhlR and the two autoinducers PAl and Factor 2. The twoautoinducers are produced by the autoinducer synthases LasI and RhlI respectively. LasRjPAI and RhlRjFactor 2 form non-interchangeable pairs which stimulate the expression of various virulence factor genes. Mutations in either the lasR, las!, rhlR or
rhl! genes in P.
aerugillosa result in pleiotropie
mutants. For example, a lasR null mutant is defieient or decreased in production of PAl, elastase, LasA elastase, alkaline protease and toxin A. Similarly, a rhlR null mutant is defieient in the production of rhamnolipid, elastase and alkaline protease. These observations suggest that these two autoinduction systems are somehow interrelated. Using transcriptional reporter fusions of various
virulence factor structural genes we have shown that there is a hierarchy of gene recognition by both of these quorum sensing pairs. RhlRjFactor2 preferentially activate expression of rhlA over [asB while expression of las! is not stimulated by this pair. LasRjPAI however, preferentially activates las! over InsB with even less significant effects on aprA and toxA. Autoinduction hierarchy is likely dictated by the recognition element for each cognate pair found in close proximity to the promoter elements of each responsive gene. Activation of lasB by LasRjPAI requires an intact lux-like box within the operator of lasB.
However, a different sequence is recognized in the las! operator suggesting that both the specificity and sensitivity of P. aerugillosa genes to activation by LasRjPAI is sequence specific.
Microbial Physiology and Gene Regulation 25
Intercellular Signalling in
Vibrio harveyi:Regulation of the
Expression of Bioluminescence. Bonnie L. Bassier
Department of
Molecular Biology.
Princeton University.
Princeton, New Jersey,
08544-1014 USA. Fax
609-:L58-6175The mechanisms that marine bacteria use to perceive their
environment and adapt to life in the ocean are being explored. One of
the objectives of these studies is to genetically and biochemically
characterize the regulatory components that control gene expression in
response to environmental and intercellular signais.
A model
luminous bacterium,
Vibrio harveyi , is under investigation.
V
.
harveyi is found free-living in the ocean where it exists in the water
column, in the sediments, and on the surfaces of various marine
animals.
Itis not understood what specific benefit V
.
harveyi derives
from producing light.
We do know, however, that luminescence
expression in V.
harveyi is strongly regulated by cues from its
environment. These inputs include ceB density, and the availability of
iron, oxygen, and glucose
.
Presumably, bioluminescence is important
for survival of V.
harveyi and the cues controlling its expression are
indicative of environmental circumstances.
V.
harveyi senses and
relays this information to effect an appropriate change in luminescence
(lux) phenotype in order to adapt and survive in a complex and
changing habitat.
The
luxCDABEGH operon of V
.
harveyi encodes the enzymatic
functions required for light production. The
luxA and luxB genes
encode the subunits of the luciferase enzyme and
luxC, luxD, and luxE
encode the fatty acid reductase complex which is involved in substrate
recycling. The
luxG and luxH genes are hypothesized to be involved
in flavin biosynthesis
.
However, defects in
lu xC and luxH do not
result in a reduction in light production As mentioned
,
one of the
inputs regulating light production by V
.
11l1rveyi is the
density of the
cell culture
.
Light emission per cell can be as much as one thousand
fold higher in dense cultures than in dilute cultures.
V.
harveyi
synthesizes two extracellular signal molecules, called autoinducers,
which accumulate in the growth medium and induce the expression of
luminescence
.
Itis the concentration of autoinducers and not ceB
density
per se that directly affects expression of luminescence
.
Sensing
and responding to cell density
via autoinducers is called Quorum
Sens ing
.
In the V.
harveyi system, one of the autoinducers (AI-I), is a
product of the functions encoded by the
luxL and luxM genes and is a
homoserine lnctone (N-(3-hydroxybutanoyl)-L-homoserine lactone ).
The gene for the second autoinducer (AI-2) has not been identified and
the structure of this compound is unknown.
The apparatus for
detection of and response to the two autoinducers in V
.
harveyi is
complex and appears to consist of two interconn
c
cted pnthways of
signal transduction (System 1 and System 2) which modulate the
26 Beijerinck Centennia!
transcription of the
luxCDABEGHoperon.
Density-dependent
regulation of luminescence in V. harveyi begins with binding of the
signalling system 1 and 2 autoinducers by their cognate sensors LuxN
and LuxPQ respectively. The LuxP sensor protein is similar to the
periplasmic ribose binding protein of
E.
coli
and it acts in conjunction
with the LuxQ sensor protein.
The LuxN and LuxQ sensors are
members of the bacterial "two-component" family of adaptive response
regulators and each contains a histidine-protein kinase sensor domain
and a response regulator domain. Signa I relay is proposed to occur bya
series of phospho-relay reactions. Coupling and integration of the
System 1 and System 2 signals is accomplished by LuxO, a negative
regulator of luminescence. LuxO is similar to the response regulator
domain of two-component regulatory proteins and it contains a DNA
binding domain, so it could directly interact with
luxDNA. A positive
transcription factor called LuxR is also required for the expression of
the
luxCDABEGHoperon.
A model for density dependent
luminescence expression in
V.harveyi is shown.
v.
harveyi
Signalling System 1 Signalling System 2
AI-l
0
AI-2
0
t
t
t
t
Signal 1
1
Sensor 11
luminescence Signal21
Sensor21
Microbial Physiology and Gene Regulation 27
The role of symbiotic signaling during colonization
of the squid light organ by
Vibrio fischeri
Karen
L.
Visick, Katherine J. Boettchert, and Edward G. Ruby Department of Biological Sciences, University of Southem CaliforniaLos Angeles, California, USA 90089-0371 FAX (213)-740-9123
Symbiotic interactions between bacteria and their animal hosts are believed to involve a complex exchange of signaIs. This communication results in a reciprocal modulation of gene expression that expresses itself as a morphological and physiological differentiation by both organisms. Such communication can occur not only between the bacterium and host, but also among the bacteria themselves. For instance, a farnily ofbacterial
homoserine lactones (or "autoinducers") has recently been implicated in several monospecific bacterial infections as molecules that signa! a process termed "quorum sensing". Unfortunately, the role of such signaIs is generally very difticult to exarnine experimentally in either pathogenic or cooperative animal-bacterial associations due to either the inability to culture and genetically manipulate the bacterium, the difficulty or inappropriateness of the animal model, or the confounding effects of host tissue damage.
Symbiotic colonization of the light -emitting organ of the sepiolid squid Euprymna scolopes by the luminous bacterium Vibrio fischeri can be examined without these problems. The process is easily studied by exposing newly hatched, uninfected, squids to specific strains of V.
fischeri, and monitoring the initiation and subsequent persistence of the
symbiotic infection simply by observing the development of animal bioluminescence. This system affords many advantages for examining symbiotic signaling because: (i) the association exhibits a high degree of species-specificity; (ii) molecular genetic techniques can be applied to V.
fischeri, and; (iii) the bacterial colonization triggers a developmental
program of host differentiation that unfolds in a program of distinct and easily assayed stages.
We have investigated the role of signaling by V. fische ri cells in this symbiosis using three approaches. The first is designed to specifically exarnine the role of V. fischeri autoinducer
CV
AI-I) in the establishment of the symbiotic colonization using mutants defective in VAl -1 production. The second approach involves a broad search for mutations in colonization-induced bacterial genes to study their effects on symbiotic competency. Finally, we have used co-inoculation studies, performed with different combinations of these mutant and wildtype strains, to revea1 the presence of either competitive dominance or functiona! complementation. These studies have allowed us to begin to describe the genetic and physiologicalcomponents underlying a stabie bacterial colonization of host tissue. t Present address: Department of Microbiology, University of Maine, Orono, Maine
28 Beijerinek Centennial
REGULATION OF ENDOGENOUS PHEROMONE TO PREVENT SELF-INDUCTION OF CONJUGATION AND lTS POSSIBLE ROLE IN PLASMID REPLICATION IN ENTEROCOCCUS FAECALIS.
B.A.H. Leonard1, A. ColweUl, A. Podbielski,2 and G.M. Dunny1.
Dept. of Microbiology, UMHC Box 196, Univ. ofMN, Minneapolis, MN, 55455, USA, Fax: 1-612-626-06231 and Institut f. Med. Mikrobiologie, Klinikum RWTH, Pauwelstr. 78, D-52057 Aaehen, Germany, Fax: 49-241-8888-483 2. lotroductioo
Conjugative transfer ofthe plasmid, pCFlO, by Enterococcusfaecalis oeeurs in response to a specific peptide pheromone, eCFlO, seereted by recipient eells. When deteeted by donor eells, eCFlO initiates a cascade of events leading to plasmid transfer including the induction of a eell surface adhesin, Aggregation Substanee. The expression of aggregation substanee is under both positive and negative control (for review see 2). Sinee both donor and recipient eells ean produce eCFlO, donor eells must beeome insensitive to endogenous pheromone and then transmit a signal upon eneountering reeipient-seereted eCFlO. Recent studies on signalling in response to exogenous eCFl 0 suggest: 1) that processing of the pheromone signal involves recruitment of a ehromosomal Opp system by the eCFlO binding protein, PrgZ, and 2) that signalling oeeurs by direct
interaction of internalized pheromone with intracellular effectors (Leonard et al., submitted for publieation).
Previous studies indieated the role of a hydrophobie peptide, iCFlO, in inhibition of donor-seereted eCFlO (4). Present studies suggest that eCFlO is also present in E. faecalis membranes and inhibition of eell-assoeiated eCFlO aetivity requires both iCFlO and a putative transmembrane protein, PrgY. During the studies on eCFlO signalling, it was found that Opp- strains eould not maintain pCFlO. In addition, a putative replication protein, PrgW, was isolated by eCFlO affinityehromatography. Taken together with the eontinued presenee of eCFlO in eells eontaining pCFlO, this suggested a earefully regulated bi-funetional role of eCFlO in both plasmid replication and eonjugative transfer.
Experimeotal Procedures
Construction of Strains. The construction of most strains utilized in these studies have been previously reported (1, Hedberg et al., submitted). The Opp-strain was eonstrueted by integration of pG+HOST5 into the oppD gene of E. faecalis whieh was identified by degenerative PCR. eCFlO-producing Lactococcus lactis were ereated by introduction of a eCFlO produeing plasmid (5) into L. lactis by eleetroporation. pCFlO was introdueed into L. lactis by either conjugation or eleetroporation (5)
Detection of eell associated cCFIO activity. Membranes from log-phase E.
faecalis were prepared by lysis and ultraeentrigation. The presenee of aetive eell assoeiated eCFlO was deteeted by the induetion of OG 1 RF(pCF 10) clumping in a microtiter assay (3).
cCF 10 affinity chromatagraphy. Fully aetive modified cCF1 0 (L VTL VFL Y) was tethered to Sephadex beads by the earboxy end. Proteins interacting with the free arnino end were isolated by affinity chromatography of whole cell extracts and analyzed by SDS PAGE.
Results aod Discussioo
Inhibition of cell-associated cCF 10 requires bath Prg Y and iCF 10.
Donor eells eontaining pCFlO still seerete active eCFlO (4) yet these eells do not self-induee. Previous studies have demonstrated the role of inhibitor molecule (iCFlO), eneoded by prgQ, to bloek secreted pheromone activity (4)
To determine whether the production of iCFlO by the eell was sufficient to inhibit all cell-associated cCFlO aetivity, the eells themselves were assayed for
Microbial Physiology and Gene Regulation 29
the presence of active pheromone. OG lRF showed significant concentrations of active cell-associated cCFlO, while OGIRF containing pCFlO showed no detectable cCFlO activity. Interestingly, the OGIRF strain containing only cloned prgQ (iCFI0) had an intermediate level of cCFlO activity (8-fold less than OG lRF), indicating an additional gene may be necessary to inhibit all cell-associated cCFI0 activity. Analysis of various subclones and insertional mutants indicated a role of prgY in endogenous cCFlO inhibition. Whenever PrgY was missing or disrupted an intermediate level of cCFlO activity was detected. Taken together these data indicate that complete inhibition of cell associated cCFlO activity requires the products of at least prgQ and prg Y. Attempts are currently being made to HPLC quantitate cell-associated cCFlO.
To begin to deterrnine whether iCFlO and PrgY inhibit cell associated cCFlO activity by distinct mechanims, additional iCFlO was introduced both
exogenously (addition of 1 ng/rnl synthetic iCFlO) and endogenously (cloned copy of prgQ) to one of the prg Y mutants (pCF389). Addition of the endogenous or exogenous iCFlO did not affect the constitutive clumpy phenotype of pCF389, indicating aggregation substance was still induced, and iCFlO could not
compensate for a mutation in the predicted membrane spanning inhibitor molecule, prgY.
Opp function is required for maintenance of pCF 10. Numerous attempts to create an Opp- mutation in pCFlO containing strains were unsuccessful. pCFlO is very stabie in E. faecalis and is maintained at greater than 95% even af ter 100 generations without antibiotic selection. However, if Opp- integrates were selected solely on erythromycin (pG+HOST5 selection), the integrates obtained were cured for pCFlO. If tetracycline selection for pCFlO was used in addition to erm, no integrates were obtained in 4 independent experiments. In addition, conjugation experiments carried out on the Opp- derivative failed to establish pCFlO while the Opp+ signalling strain easily obtained pCFI0. These data suggested that Opp was essential for pCFI0 maintenance.
Since cCFlO production continues after plasmid acquistion, and the host range of pCFlO appears to be limited to cCFlO-producing enterococci, it is suggested that cCFl 0 signalling through Opp may be required for plasmid maintenance. cCFlO was cloned into Lactococcus lactis and production of pheromone was confirmed. pCFlO could be introduced by conjugation into the cCFlO-producing L. lactis strain. In addition, cCFlO affinity chromatography experiments detect the binding of cCFlO to a putative pCFI0 replication protein, Prg W. These observations are being confirmed with a miniplasmid containing just the pCFlO replicon and the cCFlO gene.
Conc1usion
Signalling of exogenous cCFI0 leading to conjugative transfer of the plasmid has been shown to be dependent on the intemalization of cCFl 0 through the chromosomal Opp. The inhibition of all cell associated cCFlO activity in pCFlO eontaining cells relies on the independent aetion of two plasmid eneoded proteins, iCFlO and PrgY. The eontinued produetion of eCFlO by the eells af ter obtaining pCFlO may be physiologieally relevant sinee it appears that replieation of the plasmid depends on the produetion of eCFlO and the presenee of a funetion Opp. This indicates a tightly regulated dual role of cCFlO signalling through Opp in both conjugative transfer and replieation of pCF 10.
References
1. Christie, P.J., and G.M. Dunny. 1986. Plasmid 15:230-241.
2. Dunny, G.M., B.A.B. Leonard, and P.l Hedberg. 1995. 1 Bact. 177:871-876 3. Dunny, G.M., et al. 1979. Plasmid 2:454-465.
4. Nakayama, J., et al. 1994. J. Baet. 176: 7405-7408.
5. Ruhfel, R., et al. In Bacteria as Multieellular Organisms, J. Sharpiro and M. Dworkin, eds. Oxford University Press. In press.
30 Beijerinck Centennial
Quorum sensing and the regulation of bacteriocin
production in
LactobaciUus
MA Y BENTE BRURBERG, DZUNG BAO DIEP, VINCENT EIJSINK,
LEIV SIGVE HÁVARSTEIN, AND INGOLF F. NES
Laboratory for Microbial Gene Technology, Department of Biotechno-logical Sciences, Agricultural University of Norway, P.O. Box
5051, N-1432 As, Norway; fax: 47-64941465.
Lactic acid bacteria produce antimicrobial peptides called bacteriocins. These ribosomally sythesized peptides usually have a basic character, and pans of their sequence show amphiphilicity when projected onto a helical wheel. Bacteriocins are produced as prepeptides, of ten with a characteristic so-called 'double-glycine' type leader peptide (8). Little is known about the regulation of
bacteriocin production, but it is c1ear that regulatory mechanisms exist:
bacteriocin production is growth-phase dependent and genes encoding the histidine kinase and response regulator of two-component regulatory systems were shown to be essential for bacteriocin production in all cases tested (e.g. 1). We describe a mechanism for the regulation of bacteriocin production in Lactobacillus employing secreted peptides for intercellular signalling.
MATERlALS AND METHODS
The bacteriocin producing strains were Lb. plantarum CII (3) and Lb. sake L TH673 (9). Peptides were purified from culture supernatants by ammonium sulfate precipitation followed by various chromatographic steps
(usually: cation exchange -> hydrophobic interaction -> revyrsed phase).
Genetic studies were conducted using well-established techniques. All methods will be or have been described elsewhere (2, 4-7, 9).
RESULTS AND DISCUSSION
Bacteriocin producing cultures of Lb. plantarum C11 or Lb. sake L TH673 loose the Bac+ phenotype upon plating or upon extreme dilution. This indicates that the supernatants of Bac+ cultures contain factors that are indispensible for bacteriocin production. These factors were purified and found
to be non-modified, amphiphatic peptides (-20 residues). Addition of these
peptides (purified or synthetic) to non-producing cultures resulted in the
induction of bacteriocin production. Likewise, the peptides induced their own
production and they restored immunity (both these phenotypes had been lost
concomitantly with the loss of bacteriocin production). The inducing peptides
did not exhibit significant bacteriocin activity.
Analysis of the genetic determinants of bacteriocin production revealed that the gene encoding the inducing peptides is co-transcribed with genes encoding a two-component regulatory system (2, 4). The inducing peptides are initially produced with a typical double-glycine leader peptide, meaning that
Microbial Physiology and Gene Regulation 31
exports the bacteriocin (8). Northem analysis showed that the transcription of the genes involved in baeteriocin production is switched on by adding the appropriate inducing peptide to non-producing cultures of Lb. plantarum CII (5, 6) and Lb. sake LTH673 (2, 7). Non-producing cells remained sensitive for induction till they reached the late-Iogarithmic phase.
Bacteriocins inhibit the growth of bacterial species closely related to the producing organism and thus provide the latter with a selective advantage over its natural competitors. The sensing of its own growth, which is likely to be eomparable to that of eompeting species in the same medium, would enable the producing organism to switch-on the production of anti-microbial activity at higher eell densities, when the competition for nutrients becomes more severe. Such quorum sensing could be based on a slowaccumulation of the inducing peptide in the early stages of growth as a result of low eonstitutive production. At a certain point the aceumulated peptide then triggers the autoinduction pathway resulting in increased expres sion of genes involved in the production of itself and the cognate bacteriocin.
Fully functional autoinduction pathways that initiate bacteriocin production at an early stage during growth may easily be overlooked under laboratory conditions. Autoinduction in Lb. sake LTH673 and Lb. plantarum
CH could easily be discovered, since extemally added inducing peptide was needed for the onset of bacteriocin production in non-producing cells. Possibly, constitutive expres sion of the IF is too low, either as a consequence of a mutation or as a consequence of the growth conditions used. Genes encoding regulatory systems similar to the one described here were shown to be essential for bacteriocin produetion in what seem to be constitutive producers. We are currently studying the regulation of bacteriocin production in these apparently constitutive producers in more detail.
REFERENCES
1. AxeJsson, L., and A. Holck. 1995. The genes involved in prodcution of and immunity to sakacin A, a bacteriocin from Lactobacillus sake Lb706. J. Bacteriol. 177:2125-2137.
2. Brurberg, M. B., I. F. Nes, and V. G. H. Eijsink. Manuscript in preparation.
3. Daeschel, M. A., M. C. McKenney, and L. C. McDonald. 1990. Bacteriocidal activity of Lactobacillus plantarum Cll. Food Microbiol. 7:91-99.
4. Diep, D. B., L. S. Havarstein, J. Nissen-Meyer, and I. F. Nes. 1994. The gene encoding plantaricin A, a bacteriocin Crom Lac/obacillus plantarum Cll, is located on the same transcriptional unit as an agr-like regulatory system. Appl. Environ. Microbiol. 60:160-166.
5. Diep, D. B., L. S. Hävarstein, and I. F. Nes. 1995. A bacteriocin-like peptide induces
bacteriocin synthesis in Lactobacillus plantarum Cll. Mol. Microbiol., in press. 6. Diep, D. B., L. S. Hävarstein, M. B. Brurberg, and I. F. Nes. Manuscript in
preparation.
7. Eijsink, V. G. H., M. B. Brurberg, P. J. Middelhoven, D. B. Diep, and I. F. Nes. 1995. Induction of bacteriocin production in Lactobacillus sake by a secreted peptide.
J. Bacteriol., submitted for publication.
8. Hävarstein, L. S., D. B. Diep, and I. F. Nes. 1995. A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export. Mol. Microbiol. 16:229-240.
9. Tichaczek, P. S., J. Nissen-Meyer, I. F. Nes, R. F. Vogel, and W. P. Hammes. 1992. Characterization of the bacteriocins curvacin A from Lactobacillus curvatus LTH1l74 and sakacin P from L. sake LTH673. System. Appl. Microbiol. 15:460-468.
32 Beijerinck Centennia}
Identification of acylated homoserine lactone molecules
in Rhizobium leguminosarum; possible functions.
A.A.N. van Brussel, Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands, Telefax: 31715275088
Rhizobium leguminosarum
is a bacterium weIl known for its ability to induce formation of symbiotic nitrogen-flXÏng root nodules on certain legumi-nous plants. The species is subdivided, according to the subset of leguminous plants it can nodulate, in three biovars, viciae, trifolii, and phaseoli.In two recent papers dealing with
R. leguminosarum
(5,7), the identification of the autoinducer (RLAI) of therhi
genes (5) and a bacteriocin called Small bacteriocin(Small)
(7) has been described. Both molecules appear to be the same N-acylated homoserine lactone molecule (aHSL), N-(3R-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone. Several aHSL molecules have already been described as quorum sensing signal molecules (4), which function as co-transcription factors of genes encoding proteins required at higher cell densities and which are often involved in interactions between bacteria and higher organisms. Since the regulatory RhiR protein has homology with LuxR fromVibrio fischeri
(1), which requires an aHSL as a co-transcription factor, the identification of RLAI as an aHSL molecule did not come as an surprise. However, (7) was the fust report of an aHSL active as abacteriocin.Small
producing strains have been found between a number of strains of each biovar ofR. leguminosarum
but not in other species (6,9). In some strains ofR. leguminosarum, Small
is not produced, although the presence of a gene forSmall
production could be demonstrated (9). Strains investigated for latter feature all contained a self-transmissable plasmid, and curing of strains for this plasmid resulted in cells producingSmalI.
This indicated that a locus repressing the I2roduction ofS.mall (rps
locus) is present on these plasmids (9). As theSmall
production gene is present in allR
.
leguminosarum
strains, it most probable has a chromosomal location and it represents aspecific trait of this species. InSmall
sensitive strains,Small
could interfere with stress- or stationary phase-signalling thereby inhibiting growth.Production of Rhi proteins is an almost restricted to
R. leguminosarum
bv. viciae strains (2,3). RhiA is a cytoplasmic protein encoded by the
rhiA
gene, which, in strain 248, is located on Sym plasmid pRLlJI in the
rhiABC
operon and is regulated by the
