Quorum sensing signal interference within and across the kingdoms

QUORUM SENSING SIGNAL INTERFERENCE WITHIN AND ACROSS THE KINGDOMS YANG FAN (B.Sci., ZhongShan University) A THESIS SUBMITED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CIVIL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS I would like to express my greatest appreciation and admiration to my supervisors, Associate Professor Hu Jiangyong and Associate Professor Zhang Lianhui, for their invaluable guidance, advice and encouragement throughout my study. They have shown me the true meaning of research and science, which influence me deeply. I am very grateful to Assistant Professor Wang Lianhui and Assistant Professor Dong Yihu for their invaluable advice and suggestions, and sharing with me their excellent experiences in both biochemistry and molecular microbiology. My great appreciation is also given to all the members in the Laboratory of Microbial Quorum Sensing, including Dr. Zhang Haibao, Dr. Jiang Zide, Dr. Liu Ziduo, Dr. Weng Lixing, Dr. Wang Jing, Dr. Wu Ji’en, Dr. Boon Calvin, Dr. He Yawen, Dr. Wang Chao, Ms. Xu Jinling, Ms. Zhang Xifen, Ms. Zhou Lian, Ms. Hussain Mumtaz, Ms. Tan Aitee, Mr. Teng Raymond, Ms. An Shuwen, Mr. Tao Fei, Mr. Deng Yinyue, Mr. Lim Likai, Ms. Seet Qihui, and Ms. Lee Jasmine, for their practical discussions and unreserved help . Finally, I would like to thank my parents and my sister, who never failed to stand by me and gave utmost support and love thorough all my life. Special thanks to my wife for her faithful understanding and endless love. I TABLE OF CONTENTS ACKNOWLEDGEMENTS . I  TABLE OF CONTENTS II  SUMMARY . V  ABBREVIATIONS .VIII  LIST OF TABLES . XII  LIST OF FIGURES XIII  CHAPTER . 1  INTRODUCTION AND LITERATURE REVIEW 1  1.1  QUORUM SENSING IN BACTERIA 1  1.1.1  Discovery of bacterial quorum sensing 2  1.1.2  Quorum sensing is conserved in diverse bacterial species 3  1.1.3  Classification of quorum-sensing signals and biological functions 5  1.2  QUORUM SENSING MECHANISM 8  1.2.1  Generation of AHLs . 8  1.2.2  Accumulation of AHLs 12  1.2.3  Transcriptional regulation of target genes 13  1.2.4  QS signal turnover 16  1.3  QUORUM SENSING AND SIGNAL INTERFERENCE 17  1.3.1  AHL signal degradation . 18  1.3.1.1  AHL-Lactonase 18  1.3.1.2  AHL-Acylase . 20  1.3.2  Interruption and suppression of AHL biosynthesis . 22  1.3.3  Interference with the bacterial membrane efflux pump (AHL transportation) . 23  1.3.4  Small molecules interfering with AHL signal receptor 24  1.3.4.1  Natural QSIs . 24  1.3.4.2  Synthetic QSIs 26  1.4  RESEARCH STATEMENT . 30  1.5  AIM AND SCOPE OF THIS STUDY 32  CHAPTER . 34  QUORUM SENSING AND SIGNAL INTERFERENCE IN MULTI-SPECIES BIOFILMS 34  2.1  INTRODUCTION 34  2.2  MATERIALS AND METHODS 36  2.2.1  Zeolite biofilter system and operation conditions . 36  2.2.2  Sample collection and SEM observation 36  2.2.3  Bacterial isolation and identification 37  2.2.4  AHL bioassay . 38  II 2.3  2.4  2.2.5  Bioassay of AHL degradation 39  2.2.6  Thin-layer Chromatography (TLC) bioassay of AHL signals 39  2.2.7  Assays for pyocyanin production, swarming motility, and biofilm formation . 40  2.2.8  Nematode killing assay . 41  RESULTS 42  2.3.1  The biofilm from a water reclamation system comprises multi bacterial species . 42  2.3.2  AHL signal production and AHL-degrading activities among bacterial isolates 44  2.3.3  Characterization of P. aeruginosa HL43 44  DISCUSSION . 51  CHAPTER . 54  PUTATIVE SIGNAL INTERFERENCE MOLECULE PRODUCED BY PSEUDOMONAS AERUGINOSA 54  3.1  INTRODUCTION 54  3.2  MATERIALS AND METHODS 56  3.3  3.4  3.2.1  Chemicals . 56  3.2.2  AHL activity bioassay 56  3.2.3  QSI activity bioassay 56  3.2.4  Extraction and purification of the putative QSI 56  3.2.5  TLC-overlay bioassay of the putative QSI . 57  3.2.6  HPLC analysis 57  3.2.7  NMR analysis . 57  3.2.8  Mass spectrometry (MS) analysis . 58  3.2.9  Ninhydrin test . 58  3.2.10  Quantitative β-galactosidase assay . 59  RESULTS 60  3.3.1  P. aeruginosa produced a QS inhibitory compound . 60  3.3.2  Characterization and purification of the putative quorum sensing inhibitor (QSI) . 62  3.3.3  Structural elucidation of PAi 66  3.3.4  The dosage effect of PAi on expression of the QS-dependent gene 73  DISCUSSION . 77  CHAPTER . 81  MOLECULAR MECHANISMS OF PAI PRODUCTION 81  4.1  INTRODUCTION 81  4.2  MATERIALS AND METHODS 82  4.3  4.2.1  Chemicals, media and bacterial strains . 82  4.2.2  AHL and QSI bioassay . 83  4.2.3  TLC and overlay QSI bioassay . 83  4.2.4  Tn5 transposon mutagenesis . 83  4.2.5  Gene deletion and complementation 84  RESULTS 86  4.3.1  Effect of medium composition on PAi production in PAO1 86  III 4.4  4.3.2  PA2305 is essential for PAi generation 89  4.3.3  PAi production was impaired in QS dual mutants 93  DISCUSSION . 95  CHAPTER . 99  SIGNAL INTERFERENCE MECHANISMS IN EUKARYOTES . 99  5.1  INTRODUCTION 99  5.2  MATERIALS AND METHODS 101  5.3  5.4  5.2.1  Chemicals . 101  5.2.2  Bacterial strains and media . 101  5.2.3  Animal sera and purified AHL-lactonase . 101  5.2.4  Bioassay of AHL inactivation activity . 102  5.2.5  AHL activity recovering by acidification . 102  5.2.6  HPLC and electrospray ionization (ESI)-MS analysis 102  5.2.7  Expression of mouse PON genes in Chinese hamster ovary (CHO) cell line . 103  RESULTS 104  5.3.1  Rabbit serum degrades AHL signals 104  5.3.2  Rabbit serum lactonase activity 107  5.3.3  Substrate specificity of serum lactonase . 109  5.3.4  AHL degradation activities varied among animal sera . 109  5.3.5  Animal cell line CHO expressing PONs enzymes showed strong AHL degradation activity 111  DISCUSSION . 113  CHAPTER . 116  GENERAL CONCLUSIONS AND RECOMMENDATIONS . 116  6.1  MAIN CONCLUSIONS 116  6.2  RECOMMENDATIONS FOR FUTURE STUDY 118  REFERENCE . 120  PUBLICATIONS . 150  IV SUMMARY Bacteria were historically considered as individuals they proliferate independently and are unable to interact with each other or collectively respond to environmental stimuli, as typically for multi-cellular organisms. Over the past two decades, however, our understanding of bacteria has dramatically changed. People now realize that many bacterial cells are in fact, highly communicative via a dedicated cell-cell communication system. This type of cell-cell communication is also known as “quorum sensing” (QS), which emphasizes the fact that a sufficient number of bacteria, the bacterial “quorum”, is needed to switch on or off the expression of target genes and to coordinate different biological functions. Given the fact that QS is now recognized as playing a major role in the virulence of many pathogenic bacteria, antiQS approaches, also known as “quorum quenching” (QQ) or “signal interference” (SI), have recently been proposed as a promising strategy for preventing and controlling bacterial diseases. Therefore, better understanding of QS signal interference might provide new insights on how to uncouple bacterial QS and significantly facilitate the development of novel antimicrobial agents. In this study, I explored the widespread existence of QS signal interference within and across the kingdoms and identified several QQ factors from bacteria and mammals. Microbial diversity has been investigated in multi-species biofilms from a water reclamation system. At least 11 bacterial species were revealed by 16S ribosomal RNA gene sequencing analysis, including the frequently encountered bacterial pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae, and several rare pathogens. Among them, Pseudomonas isolate HL43 has been further characterized. It was shown virulent against animal model Caenorhabditis elegans V and generated 2-6 folds more pyocyanin cytotoxin than Pseudomonas strains PA01 and PA14, the two commonly used laboratory strains. We also found that bacterial isolates Agrobacterium tumefaciens XJ01, Bacillus cereus XJ08 and Ralstonia sp. XJ12 could produce N-acyl homoserine lactone (AHL) degradation enzymes. The fact that AHL-producing and AHL-degradating bacterial species coexisted in biofilms may indicate the sophisticated dynamics of QS signaling and signal interference in the determination of microbial composition in multi-species biofilms. A putative quorum sensing inhibitor (QSI), tentatively named as “PAi”, has been isolated and purified from P. aeruginosa PAO1. This QSI compound showed strong inhibitory activity against the QS-dependent lacZ reporter gene expression. The inhibitory activity could be partially overcome by supplementation of AHL, suggesting that this QSI compound may interfere with QS regulation in a dosagedependent competitive manner. Unlike AHL-type signals, the PAi compound was highly polar and cannot be dissolved or extracted by organic solvents such as ethyl acetate, chloroform and hexane. The PAi was fairly stable, resistant to high temperature and acid- or alkaline-treatment. Nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis indicated that PAi is very likely to be 2-amino-4methoxy-but-3-enoic acid, an amino acid containing an enol ether group. The molecular mechanisms of PAi production have also been investigated. King’s A medium which favors pyocyanin production resulted in the best PAi production among the tested media. The gene PA2305, which encodes a putative nonribosomal peptide synthetase (NRPS), was identified essential for the production of PAi. According to microarray analysis, the transcription of PA2305 is likely under the regulation of QS. Consistently, the QS dual mutants MW1 (lasI::tetA, rhlI::Tn501), and DMR (∆lasR::Tcr, ∆rhlR::Gmr) , which defected in the AHLs synthesis or the VI corresponding reception, respectively, were defective in PAi production. These results pointed to the involvement of QS system in the regulation of PAi production. AHL enzymatic inactivation activity, albeit with variable efficiencies, has been found conserved in a range of mammalian serum samples, including human, rabbit, mouse, horse, goat, and bovine, but not in chicken and fish. High-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESIMS) analyses showed that these mammalian sera hydrolyzed the lactone ring of AHLs to produce acyl homoserines, through the action of enzymes reminiscent of paraoxonases (PONs). Animal cell lines expressing mouse PON genes displayed strong AHL degradation activities. Further analysis revealed that mammalian sera PONs possess a catalytic mechanism different from bacterial AHL-lactonase, although they share a same function in degrading AHL signals. The QQ occurrence among eukaryotes may represent an innate defense mechanism of host organisms. VII ABBREVIATIONS :: fusion or insertion µg microgram µl microlitre µM micromolar 5-MTA 5-methyathioadenosine ACP acyl carrier protein AHL N-acyl homoserine lactone AI autoinducer ATP adenosine triphosphate BLAST Basic Local Alignment and Search Tool bp base pair Cb carbenicillin CHO Chinese hamster ovary cm centimeter CoA coenzyme A COSY correlation spectroscopy DEPT Distortionless Enhancement by Polarization Transfer DMEM Dulbecco's Modified Eagle's Medium DNA deoxyribonucleic acid DTT dithiothreitol EDTA ethylenediamine tetraacetic acid ELISA enzyme-linked immunosorbent assay EPS exopolysaccharide VIII ESI-MS Electrospray Ionisation Mass Spectrometry FabI enyol-ACP reductase FBS Fetal Bovine Serum fmol femtomolar FRT Flp recombinase target g gram GFP green fluorescence protein Gm gentamycin h hour HMBC Heteronuclear Multiple Bond Correlation HMQC heteronuclear multiple quantum coherence HPLC high performance liquid chromatograghy HS homoserine HSL homoserine lactone Kb kilobase Kda kiloDalton Kn kanamycin L liter lacZ a repoter gene that codes for β-galactosidase LB Luria-Bertani (medium) Met methionine mg milligram minute ml milliliter mm millimeter IX McClean, K.H., Winson, M.K., Fish, L., Taylor, A., Chhabra, S.R., Camara, M., Daykin, M., Lamb, J.H., Swift, S., Bycroft, B.W., Stewant G.S. and Williams, P. (1997a) Quorum-sensing and Chromobacterium violaceum: Exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143, 3703-3711. McClean, R.J.C., Whiteley, M., Stickler, D.J. and Fuqua, W.C. (1997b) Evidence of autoinducer activity in naturally occurring biofilms. FEMS Microbiol. Lett. 154, 259263. McGowan, S., Sebaihia, M., Jones, S., Yu, S., Bainton, N., Chan, P. F., Bycroft, B.W., Stewart, G.S.A.B., Salmond, G.P.C. and Williams, P. (1995) Carbapenem antibiotic production in Erwinia carotovora is regulated by CarR, a homologue of the LuxR transcriptional activator. Microbiology 141,541-550. McKnight, S.L., Iglewski, B.H. and Pesci, E.C. (2000) The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 182, 2702–2708. McMurry, L., Petrucci, R.E. Jr., and Levy, S.B. (1980) Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. Proc. Natl. Acad. Sci. USA 77, 3974-3977. Micek, S.T., Lloyd, A.E., Richie, D.J., Reichley, R.M., Fraser, V.J. and Kollef, M.H. (2005) Pseudomonas aeruginosa bloodstream infection: importance of appropriate initial antimicrobial treatment. Antimicrob. Agents Chemother. 49, 1306-1311. Miles, EW. (1978). In Enzyme-Activated Irreversible Inhibitors: proceedings of the International Symposium on Substrate-Induced Irreversible Inhibition of Enzymes, held in Strasbourg, France, July 24 and 25, 1978 (Seiler N, Jung MJ and Koch-Weser J. eds.), pp. 73-85, Elsevier/North-Holland, Amsterdam. Miller, M.B. and Bassler, B.L. (2001) Quorum sensing in bacteria. Annu. Rev. Microbiol. 55, 165-199. 135 Milton, D.L., Hardman, A., Camara, M., Chhabra, S.R., Bycroft, B.W., Stewart, G.S. and Williams, P. (1997) Quorum sensing in Vibrio anguillarum: characterization of the vanI/vanR locus and identification of the autoinducer N-(3-oxodecanoyl)-Lhomoserine lactone. J. Bacteriol. 179, 3004-3012. Minogue, T.D., Trebra, M.W., Bernhard, F. and Beck von Bodman, S. (2002) The autoregulatory role of EsaR, a quorum-sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function. Mol. Microbiol. 44, 1625-1635. Molina, L., Constantinescu, F., Michel, L., Reimmann, C., Duffy, B. and Défago, G. (2003) Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism. FEMS Microbiol. Ecol. 45, 7181. Moré, M.I., Finger, D., Stryker, J.L., Fuqua, C., Eberhard, A. and Winans, S.C. (1996) Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates. Science 272, 1655–1658. Nasser, W., Bouillant, M.L., Salmond, G. and Reverchon, S. (1998). Characterization of the Erwinia chrysanthemi expI-expR locus directing the synthesis of two N-acylhomoserine lactone signal molecules. Mol. Microbiol. 29, 1391-1405. Nealson, K.H., Platt, T. and Hastings, J.W. (1970) Cellular control of the synthesis and activity of the bacterial luminescent system. J. Bacteriol. 104, 313–322. Olsen, J. A., Severinsen, R., Rasmussen, T.B., Hentzer, M., Givskov, M. and Nielsen, J. (2002) Synthesis of new 3- and 4-substituted analogues of acyl homoserine lactone quorum sensing autoinducers. Bioorg. Med. Chem. Lett. 12, 325–328. O’Toole, G.A. and Kolter, R. (1998) Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol. Microbiol. 28, 449-461. 136 Park, S.Y., Lee, S.J., Oh, T.K., Oh, J.W., Koo, B.T., Yum, D.Y. and Lee, J.K. (2003) AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology 149, 1541–1550. Parsek, M.R., Schaefer, A.L. and Greenberg, E.P. (1997) Analysis of random and sitedirected mutations in rhlI, a Pseudomonas aeruginosa gene encoding an acylhomoserine lactone synthase. Mol. Microbiol. 26, 301-310. Parsek, M.R., Val, D.L., Hanzelka, B.L., Cronan, J.E., Jr. and Greenberg, E.P. (1999) Acyl homoserine-lactone quorum-sensing signal generation. Proc. Natl. Acad. Sci. USA 96, 4360-4365. Parsek, M.R. and Greenberg, E.P. (2000) Acyl-homoserine lactone quorum sensing in Gram-negative bacteria: A signaling mechanism involved in associations with higher organisms. Proc. Natl. Acad. Sci. USA 97, 8789–8793. Passador, L., Cook, J.M., Gambello, M.J., Rust, L. and Iglewski, B.H. (1993) Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260, 1127-1130. Passador, L., Tucker, K.D., Guertin, K.R., Journet, M.P., Kende, A.S. and Iglewski, B.H. (1996) Functional analysis of the Pseudomonas aeruginosa autoinducer PAI. J. Bacteriol. 178, 5995–6000. Pearson, J.P., Gray, K.M., Passador, L., Tucker, K.D., Eberhard, A., Iglewski, B.H. and Greenberg, E.P. (1994) Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc. Natl. Acad. Sci. USA, 91, 197-201. Pearson, J.P., Passador, L., Iglewski, B.H. and Greenberg, E.P. (1995) A Second NAcylhomoserine Lactone Signal Produced by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA, 92, 1490-1494. 137 Pearson, J.P., Pesci, E.C. and Iglewski, B.H. (1997) Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J. Bacteriol. 179, 5756-5767. Pearson, J.P., Delden, C.V. and Iglewski, B.H. (1999) Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J. Bacteriol. 181, 1203-1210. Persson, T., Hansen, T.H., Rasmussen, T.B., Skinderso, M.E., Givskov, M. and Nielsen, J. (2005) Rational design and synthesis of new quorum-sensing inhibitors derived from acylated homoserine lactones and natural products from garlic. Org. Biomol. Chem. 3, 253–262. Pesci, E.C. and Iglewski, B.H. (1997) The chain of command in Pseudomonas quorum sensing. Trends Microbiol. 5, 132–134. Pesci, E.C., Pearson, J.P., Seed, P.C. and Iglewski, B.H. (1997) Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179, 3127-3132. Pesci, E.C., Milbank, J.B., Pearson, J.P., McKnight, S., Kende, A.S., Greenberg, E.P. and Iglewski, B.H. (1999) Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 96, 11229-11234. Pierson, L.S.III, Keppenne, V.D. and Wood, D.W. (1994) Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density. J. Bacteriol. 176, 3966-3974. Piper, K.R., Beck von Bodman, S. and Farrand, S.K. (1993) Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature 362, 448–450. 138 Piper, K.R., Beck Von Bodman, S., Hwang, I. and Farrand, S.K. (1999) Hierarchical gene regulatory systems arising from fortuitous gene associations: controlling quorum sensing by the opine regulon in Agrobacterium. Mol. Microbiol. 32, 1077-1089. Piper, K.R. and Farrand, S.K. (2000) Quorum sensing but not autoinduction of Ti plasmid conjugal transfer requires control by the opine regulon and the antiactivator TraM. J. Bacteriol. 182, 1080-1088. Pirhonen, M., Flego, D., Heikinheimo, R. and Palva, E.T. (1993) A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora. EMBO J. 12, 2467-2476. Plotnikova, J.M., Rahme, L.G. and Ausubel, F.M. (2000) Pathogenesis of the human opportunistic pathogen Pseudomonas aeruginosa PA14 in Arabidopsis. Plant Physiol. 124, 1766-1774. Pratt, L.A. and Kolter, R. (1998) Genetic analysis of Escherichia coli biofilm formation: defining the roles of flagella, motility, chemotaxis and type I pili. Mol. Microbiol. 30, 285-294. Pratt, L.A. and Kolter, R. (1999) Genetic analyses of bacterial biofilm formation. Curr. Opin. Microbiol. 2, 598–603. Puskas, A., Greenberg, E.P., Kaplan, S. and Schaefe,r A.L. (1997) A quorum-sensing system in the free-living photosynthetic bacterium Rhodobacter sphaeroides. J. Bacteriol. 179, 7530-7537. Qin, Y., Luo, Z.Q., Smyth, A.J., Gao, P., Beck von Bodman, S. and Farrand, S.K. (2000) Quorum-sensing signal binding results in dimerization of TraR and its release from membranes into the cytoplasm. EMBO J. 19, 5212-5221. Rahme, L.G., Stevens, E.J., Wolfort, S.F., Shao, J., Tompkins, R.G. and Ausubel, F.M. (1995) Common virulence factors for bacterial pathogenicity in plants and animals. Science 268, 1899-1902. 139 Rando, R.R. (1974) β, γ Unsaturated amino acids as irreversible enzyme inhibitors. Nature 250, 586–587. Rando, R.R., Relyea, N. and Cheng, L. (1976) Mechanism of the irreversible inhibition of aspartate aminotransferase by the bacterial toxin L-2-amino-4-methoxytrans-3-butenoic acid. J. Bio . Chem. 251, 3306–3312. Rashid, M.H. and Kornberg, A. (2000) Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 97, 4885–4890. Rasmussen, T.B., Manefield, M., Andersen, J.B., Eberl, L., Anthoni, U., Christophersen, C., Steinberg, P., Kjelleberg, S. and Givskov, M. (2000) How Delisea pulchra furanones affect quorum sensing and swarming motility in Serratia liquefaciens MG1. Microbiology 146, 3237-3244. Rasmussen, T.B., Bjarnsholt, T., Skindersoe, M.E., Hentzer, M., Kristoffersen, P., Kote, M., Nielsen, J., Eberl, L. and Givskov, M. (2005a) Screening for quorumsensing inhibitors (QSI) by use of a novel genetic system, the QSI selector. J. Bacteriol. 187, 1799–1814. Rasmussen, T.B., Skindersoe, M.E., Bjarnsholt, T. and 10 other authors. (2005b) Identity and effects of quorum-sensing inhibitors produced by Penicillium species. Microbiology 151, 1325–1340. Rasmussen, T.B., and Givskov M. (2006) Quorum sensing inhibitors: a bargain of effects. Microbiology 152, 895-904. Reimmann, C., Ginet, N., Michel, L., Keel, C., Michaux, P., Krishnapillai, V., Zala, M., Heurlier, K., Triandafillu, K., Harms, H., Défago, G. and Haas, D. (2002) Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1. Microbiology 148, 923932. 140 Renders, N., Verbrugh, H. and Belkum, A.V. (2001) Dynamics of bacterial colonisation in the respiratory tract of patients with cystic fibrosis. Infect. Genet. Evol. 1, 29–39. Reverchon, S., Bouillant, M.L., Salmond, G. and Nasser, W. (1998) Integration of the quorum-sensing system in the regulatory networks controlling virulence factor synthesis in Erwinia chrysanthemi. Mol. Microbiol. 29, 1407-1418. Reverchon, S., Chantegrel, B., Deshayes, C., Doutheau, A. and Cotte-Pattat, N. (2002) New synthetic analogues of N-acyl homoserine lactones as agonists or antagonists of transcriptional regulators involved in bacterial quorum sensing. Bioorg. Med. Chem. Lett. 12, 1153–1157. Rhodius, V.A. and Busby, S.J.W. (1998) Positive activation of gene expression. Curr. Opin. Microbiol. 1, 152–159. Riedel, K., Ohnesorg, T., Krogfelt, K.A., Hansen, T.S., Omori, K., Givskov, M. and Eberl, L. (2001) N-Acyl-L-homoserine lactone-mediated regulation of the lip secretion system in Serratia liquefaciens MG1. J. Bacteriol. 183, 1805-1809. Roche, D.M., Byers, J.T., Smith, D.S., Glansdorp, F.G., Spring, D.R. and Welch, M. (2004) Communications blackout? Do N-acylhomoserine-lactone-degrading enzymes have any role in quorum sensing?. Microbiology 150, 2023-2028. Rosemeyer, V., Michiels, J., Verreth, C. and Vanderleyden, J. (1998) luxI- and luxRhomologous genes of Rhizobium etli CNPAF512 contribute to synthesis of autoinducer molecules and nodulation of Phaseolus vulgaris. J. Bacteriol. 180, 815821. Ruimy, R., Genauzeau, E., Barnabe, C., Beaulieu, A., Tibayenc, M. and Andremont, A. (2001) Genetic diversity of Pseudomonas aeruginosa strains isolated from ventilated patients with nosocomial pneumonia, cancer patients with bacteremia, and environmental water. Infect. Immunol. 69, 584–588. 141 Sahm, U., Knobloch, G. and Wagner, F. (1973). Isolation and characterization of the methionine antagonist L-2-amino-4-methoxy-trans-3-butenoic acid from Pseudomonas aeruginosa grown on n-paraffin. J. Antibiotics 26, 389-390. Saleh, A., Figarella, C., Kammouni, W., Marchand-Pinatel, S., Lazdunski, A., Tubul, A., Brun, P. and Merten, M.D. (1999) Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone inhibits expression of P2Y receptors in cystic fibrosis tracheal gland cells. Infect. Immun. 67, 5076-5082. Scannell, J.P., Pruess, D.L., Demny, T.C., Sello, L.H., Williams, T. and Stempel, A. (1972) Antimetabolites produced by microorganisms. V. L-2-Amino-4-methoxytrans-3-butenoic acid. J. Antibiotics 25, 122-127. Schaefer, A.L., Hanzelka, B.L., Eberhard, A. and Greenberg, E.P. (1996) Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactions with autoinducer analogs. J. Bacteriol. 178, 2897–2901. Schuster, M., Lostroh, C.P., Ogi, T. and Greenberg, E.P. (2003) Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J. Bacteriol. 185, 2066–2079. Schwarzer, D., Finking, R. and Marahiel, M.A. (2003) Nonribosomal peptides: from genes to products. Nat. Prod. Rep. 20, 275-287. Schweizer, H.P. (1991) Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene 103, 109-112. Schweizer, H.P. (2001) Triclosan: a widely used biocide and its link to antibiotics. FEMS Microbiol. Lett. 202, 1–7. Seed, P.C., Passador, L. and Iglewski, B.H. (1995) Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy. J. Bacteriol. 177, 654–659. 142 Shadel, G.S., Devine, J.H. and Baldwin, T.O. (1990) Control of the lux regulon of Vibrio fischeri. J. Biolumin. Chemilumin. 5, 99-106. Shadel, G. S. and Baldwin, T.O. (1991) The Vibrio fischeri LuxR protein is capable of bidirectional stimulation of transcription and both positive and negative regulation of the luxR gene. J. Bacteriol. 173, 568-574. Shapiro, J.A. (1998) Thinking about bacterial populations as multicellular organisms. Annu. Rev. Microbiol. 52, 81-104. Shaw, P.D., Ping, G., Daly, S.L., Cha, C., Cronan, Jr. J.E., Rinehart, K.L. and Farrand, S.K. (1997) Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. USA 94, 6036-6041. Slack, M.P.E. and Nichols, W.W. (1981) The penetration of antibiotics through sodium alginate and through the exopolysaccharide of a mucoid strain of Pseudonmonas aeruginosa. Lancet 11, 502-503. Slock, J., van Riet, D., Kolibachuk, D. and Greenberg, E.P. (1990) Critical regions of the Vibrio fischeri LuxR protein defined by mutational analysis. J. Bacteriol. 172, 3974–3979. Smith, R.S. and Iglewski, B.H. (2003) Pseudomonas aeruginosa quorum sensing as a potential antimicrobial target. J. Clin. Invest. 112, 1460-1465. Smith, K.M., Bu, Y. and Suga, H. (2003a). Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs. Chem. Biol. 10, 81–89. Smith, K.M., Bu, Y. and Suga, H. (2003b). Library screening for synthetic agonists and antagonists of a Pseudomonas aeruginosa autoinducer. Chem. Biol. 10, 563–571. Spangerberg, C., Heuer, T., Bürger, C. and Tümmler, B. (1996) Genetic diversity of flagellins of Pseudomonas aeruginosa. FEBS Lett. 396, 213–217. 143 Steidle, A., Allesen-Holm, M., Riedel, K., Berg, G., Givskov, M., Molin, S. and Eberl, L. (2002) Identification and Characterization of an N-Acylhomoserine LactoneDependent Quorum-Sensing System in Pseudomonas putida Strain IsoF. Appl. Environ. Microbiol. 68, 6371-6382. Stevens, A.M., and Greenberg, E.P. (1997) Quorum sensing in Vibrio fischeri: essential elements for activation of the luminescence genes. J. Bacteriol. 179, 557562. Stevens, A.M., Dolan, K.M. and Greenberg, E.P. (1994) Synergistic binding of the Vibrio fischeri LuxR transcriptional activator domain and RNA polymerase to the lux promoter region. Proc. Natl. Acad. Sci. USA 91, 12619–12623. Stickler, D.J., Morris, N.S., McLean, R.J.C. and Fuqua, C. (1998) Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro. Appl. Environ. Microbiol. 64, 3486–3490. Stover, C.K., Pham, X.Q., Erwin, A.L., Mizoguchi, S.D., Warrener, P., Hickey, M.J. and 20 other authors. (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959-964. Sufrin, J.R., Lombardini, J.B. and Keith, D.D. (1982) L-2-Amino-4-methoxy-cis-but3-enoic acid, a potent inhibitor of the enzymatic synthesis of S-adenosylmethionine. Biochem. Biophys. Res. Commun. 106, 251–255. Swift, S., Karlyshev, A.V., Fish, L., Durant, E.L., Winson, M.K., Chhabra, S.R., Williams, P., Macintyre, S. and Stewart, G.S.A.B. (1997) Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J. Bacteriol. 179, 5271-5281. 144 Swift, S., Lynch, M.J., Fish, L., Kirke, D.F., Tomas, J.M., Stewart, G.S.A.B. and Williams, P. (1999) Quorum sensing-dependent regulation and blockade of exoprotease production in Aeromonas hydrophila. Infect. Immun. 67, 5192-5199. Tan, M.W., Mahajan-Miklos, S. and Ausubel, F.M. (1999) Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc. Natl. Acad. Sci. USA 96, 715–720. Teplitski, M., Robinson, J.B. and Bauer, W.D. (2000) Plants secrete substances that mimic bacterial N-acyl homoserine lactone signal activities and affect population density-dependent behaviors in associated bacteria. Mol. Plant Microbe Interact. 13, 637–648. Telford, G., Wheeler, D., Williams, P., Tomkins, P.T., Appleby, P., Sewell, H., Stewart, G.S., Bycroft, B.W. and Pritchard, D.I. (1998) The Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone has immunomodulatory activity. Infect. Immun. 66, 36-42. Throup, J., Camara, M., Briggs, G., Winson, M.K., Chhabra, S.R., Bycroft, B.W., Williams, P. and Stewart, G.S.A.B. (1995) Characterization of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N-acylhomoserine lactone signal molecules. Mol. Microbiol. 17, 345–356. Tisdale, M.J. (1980) The effect of the methionine antagonist L-2-amino-4-methoxytrans-3-butenoic acid on the growth and metabolism of Walker carcinosarcoma in vitro. Biochem. Pharmacol. 29, 501–508. Uroz, S., D'Angelo-Picard, C., Carlier, A., Elasri, M., Sicot, C., Petit, A., Oger, P., Faure, D. and Dessaux, Y. (2003) Novel bacteria degrading N-acylhomoserine lactones and their use as quenchers of quorum-sensing-regulated functions of plantpathogenic bacteria. Microbiology 149, 1981–1989. Van Bambeke, F., Glupczynski, Y., Plésiat, P., Pechère,J.C. and Tulkens, P.M. (2003) Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and 145 strategies for the future of antimicrobial therapy. J. Antimicrob. Chemother. 51, 1055– 1065. Vidal, O., Longin, R., Prigent-Combaret, C., Dorel, C., Hooreman, M. and Lejeune, P. (1998) Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: Involvement of a new ompR allele that increases curli expression. J. Bacteriol. 180, 2442–2449. von Bodman, S.B., Majerczak, D.R., Coplin, D.L. (1998) A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii. Proc. Natl. Acad. Sci. USA 95, 7687– 92. Wagner, V.E., Bushnell, D., Passador, L., Brooks, A.I. and Iglewski, B.H. (2003) Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J. Bacteriol. 185, 2080–2095. Wang, L.H., Weng, L.X., Dong, Y.H. and Zhang, L.H. (2004) Specificity and enzyme kinetics of the quorum-quenching N-acyl homoserine lactone lactonase (AHLlactonase). J. Biol. Chem. 279, 13645–13651. Watnick, P.I., Fullner, K.J. and Kolter, R. (1999) A role for the mannose-sensitive hemagglutinin in biofilm formation by Vibrio cholerae El Tor. J. Bacteriol. 181, 3606-3609. Watnick, P.I. and Kolter, R. (1999) Steps in the development of a Vibrio cholerae El Tor biofilm. Mol. Microbiol. 34, 586-95. Watson, W.T., Minogue, T.D., Val, D.L., Beck von Bodman, S. and Churchill, M.E.A. (2002) Structural basis and specificity of acyl-homoserine lactone signal production in bacterial quorum sensing. Mol. Cell 9, 685– 694. Wei, J.R., Tsai, Y.H., Horng, Y.T., Soo, P.C., Hsieh, S.C., Hsueh, P.R., Horng, J.T., Williams, P. and Lai, H.C. (2006) A mobile quorum-sensing system in Serratia marcescens. J. Bacteriol. 188, 1518–1525. 146 Welch, M., Todd, D.E., Whitehead, N.A., McGowan, S.J., Bycroft, B.W. and Salmond, G.P.C. (2000) N-Acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia. EMBO J. 19, 631–641. Whiteley, M., Lee, K.M. and Greenberg, E.P. (1999) Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 96, 13904-13909. Whiteley, M., Parsek, M.R. and Greenberg, E.P. (2000) Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa. J. Bacteriol. 182, 4356–4360. Whiteley, M., Bangera, M.G., Bumgarner, R.E., Parsek, M.R. and Teitzel, G.M. (2001) Gene expression in Pseudomonas aeruginosa biofilms. Nature 413, 860-64. Williams, P. (2002) Quorum sensing: an emerging target for antibacterial chemotherapy?. Expert. Opin. Ther. Targets. 6, 257–274. Willams, P., Bainton, N.J., Swift, S., Chhabra, S.R., Winson, M.K., Stewart, G.S.A.B., Salmond, G.P.C. and Bycroft B.W. (1992) Small molecule-mediated, densitydependent control of gene expression in prokaryotes: bioluminescence and biosynthesis of carbapenem antibiotics. FEMS Microbiol. Lett. 100, 161-168. Williams, P., Winzer, K., Chan, W. and Camara, M. (2007) Look who's talking: communication and quorum sensing in the bacterial world. Philos. Trans. R Soc. Lond. B Biol. Sci., 362, 1119–1134. Winson, M.K., Camara, M., Latifi, A., Foglino, M., Chhabra, S.R., Daykin, M., Bally, M., Chapon, V., Salmond, G.P., Bycroft, B.W., Lazdunski, A., Stewart, G.S.A.B. and Willams, P. (1995) Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 92, 9427-9431. 147 Winstanley, C., Coulson, M.A., Wepner, B., Morgan, J.A.W. and Hart, C.A. (1996) Flagellin gene and protein variation amongst clinical isolates of Pseudomonas aeruginosa. Microbiology 142, 2145–2151. Winzer, K., Falconer, C., Garber, N.C., Diggle, S.P., Camara, M. and Williams, P. (2000) The Pseudomonas aeruginosa lectins PA-IL and PA-IIL are controlled by quorum sensing and by RpoS. J. Bacteriol. 182, 6401–6411. Wisplinghoff, H., Seifert, H., Tallent, S.M., Bischoff, T., Wenzel, R.P. and Edmond, M.B. (2003) Nosocomial bloodstream infections in pediatric patients in United States Hospitals: epidemiology, clinical features and susceptibilities. Pediatr. Infect. Dis. J. 22, 686-691. Wisniewski-Dye, F. and Downie, J.A. (2002) Quorum-sensing in Rhizobium. Antonie van Leeuwenhoek 81, 397-407. Wolff, J.A., MacGregor, C.H., Eisenberg, R.C. and Phibbs, P.V. (1991) Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO. J. Bacteriol. 173, 4700-4706 Yates, E.A., Philipp, B., Buckley, C., Atkinson, S., Chhabra, S.R., Sockett, R.E., Goldner, M., Dessaux, Y., Camara, M., Smith, H., Williams, P. (2002) NAcylhomoserine Lactones Undergo Lactonolysis in a pH-, Temperature-, and Acyl Chain Length-Dependent Manner during Growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa. Infect. Immun. 70, 5635-5646. Zhang, H.B., Wang, L.H. and Zhang, L.H. (2002.) Genetic control of quorumsensing signal turnover in Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA 99, 9427–9431. Zhang, L.H. and Kerr, A. (1991) A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens. J. Bacteriol. 173, 1867-1872. 148 Zhang, L.H., Murphy, P.J., Kerr, A. and Tate, M.E. (1993) Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature 362, 446447. Zhang, L.H. (2003). Quorum quenching and proactive host defense. Trends Plant Sci. 8, 238-244. Zhang, L.H. and Dong, Y.H. (2004) Quorum sensing and signal interference: diverse implications. Mol. Microbiol. 53, 1563-1571. Zhang, R., Pappas, T., Brace, J.L., Miller, P.C., Oulmassov, T., Molyneaux, J.M., Anderson, J.C., Bashkin, J.K., Winans, S.C., and Joachimiak, A. (2002) Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA. Nature 417, 971-974. Zhang, Z. and Pierson L.S.III. (2001). A second quorum-sensing system regulates cell surface properties but not phenazine antibiotic production in Pseudomonas aureofaciens. Appl. Environ. Microbiol. 67, 4305-4315. Zhu, J. and Winans, S.C. (1998) Activity of the quorum-sensing regulator TraR of Agrobacterium tumefaciens is inhibited by a truncated, dominant defective TraR-like protein. Mol. Microbiol. 27, 289-297. Zhu, J. and Winans, S.C. (1999) Autoinducer binding by the quorum-sensing regulator TraR increases affinity for target promoters in vitro and decreases TraR turnover rates in whole cells. Proc. Natl. Acad. Sci. USA 96, 4832-4837. Zhu, J., and Winans, S.C. (2001) The quorum-sensing transcriptional regulator TraR requires its cognate signaling ligand for protein folding, protease resistance, and dimerization. Proc. Natl. Acad. Sci. USA 98, 1507–1512. 149 PUBLICATIONS Hu J.Y., Yang F., Lin Y.H., Zhang H.B., Ong S.L., Dong N., Xu J.L., Ng W.J. and Zhang L.H. (2003) Microbial diversity and prevalence of virulent pathogens in biofilms developed in a water reclamation system. Research in Microbiology, 154 (9), 623-629. Hu J.Y., Yang F., Lin Y.H., Ong S.L., Dong N., and Zhang L.H. (2004) Detection and inactivation of quorum sensing signal molecules associated with biofilm developed in a water reclamation system. International Symposium on biotechnology for environmental pollution control, Aug. 14-15, 2004, Beijing, China. Yang F., Wang L.H., Wang J., Dong Y.H., Hu J.Y. and Zhang L.H. (2005) Quorum quenching enzyme activity is widely conserved in the sera of mammalian species. FEBS Letters, 579 (17), 3713- 3717. Yang F., Dong Y.H., Wang L.H., Wu J.E., Lee J., Hu J.Y. and Zhang L.H. (in preparation) A putative quorum sening inhibitor (QSI) produced by Pseudomonas aeruginosa. 150 [...]... phenomenon to him during a family gathering at Christmas (Greenberg, 1996) The quorum sensing bacteria produce, detect and respond to small signal molecules The most characterized quorum- sensing signals in Gram-negative bacteria are the N-acyl homoserine lactones (AHLs) (Fuqua et al., 2001), while Gram-positive bacteria produce small peptides as quorum- sensing signals (Dunny and Leonard, 1997) Up to now,... AHL-mediated quorum sensing systems 1 1.1.1 Discovery of bacterial quorum sensing The phenomenon of quorum sensing, originally termed autoinduction, was initially discovered in the luminescent marine bacteria Vibrio fischeri and Vibrio harveyi in the early 1970s (Nealson et al., 1970; Eberhard, 1972) It was noted that the bioluminescence of these bacteria exhibits a lag to their growth When these bacteria... as AinS and LuxM proteins, were also found to synthesize AHLs (Gilson et al., 1995; Bassler et al., 1993) The acyl chain is synthesized via the common fatty acid biosynthesis pathway and the homoserine lactone is derived from SAM (Fig.1.2) Several proteins and enzymes have been identified in the biosynthesis of AHLs, including the acyl carrier proteins (ACPs), the enoyl-ACP reductase FabI and AHL synthase... the luminescence autoinduction via luxR and luxI At low cell densities, luxI is transcribed at a basal level and VAI 2 accumulates slowly in the bacterial culture As bacterial cells grow, the bacterial population density increases, and so do the VAI signal molecules When VAI accumulates to a sufficiently high concentration, the signals interact with LuxR, which then activates the transcription of the. .. known as quorum sensing , which emphasizes the fact that a sufficient number of bacteria, the bacterial quorum , is needed to switch on or off the expression of target genes The term quorum sensing first appeared in a minireview written by Clay Fuqua et al (1994) It originated with a lawyer who was trying to understand what they were studying as Steve Winan (the 2nd author) explained the phenomenon... physical and chemical factors can also influence the dynamics of AHL accumulation in a bacterial community One of the important factors is the diffusion ability of AHLs Generally, it is assumed that AHL signals can passively diffuse across bacterial membranes based on the study that V fischeri quorum- sensing signal, 3OC6HSL, freely diffuses in and out of V fischeri and E coli cells (Kaplan and Greenberg,... cells to sense a change in growth and adjust their cellular activities accordingly Byers et al (2002) provide another view in quorum sensing signal turnover They showed that the concentration of 3OC6HSL in E carotovora spp carotovora culture supernatants rapidly decreases in the stationary phase and the decrease is due to non-enzymatic turnover of the signal The non-enzymatic degradation of 3OC6HSL is... suggesting that the regulation mechanism of EsaR is different from other known LuxR-type transcriptional activators 1.2.4 QS signal turnover A signal turnover system is an essential component of many genetic regulatory mechanisms Given the role played by the quorum sensing signal in the bacterial gene expression regulatory system, we may expect that its concentration is tightly regulated and a signal turnover... However, there is also evidence that 3OC12HSL, one of the two P areuginosa quorum- sensing signals, is not freely diffusible (Pearson et al., 1999) The mexA-mexB-oprM-encoded efflux pump is involved in active transport of 3OC12HSL out of P aeruginosa cells (Evans et al., 1998; Pearson et al., 1999) It has been reported that the length of the acyl chain is the major specificity determinant in the quorum- sensing. .. the –35 sequence, then recruits RNAP and initiates the transcription of target genes (Egland and Greenberg, 1999) In addition, different LuxR-type proteins probably have different mechanisms of transcription activation Site-directed mutagenesis of LuxR indicated that residues within the carboxy-terminal DNA-binding domain are required for transcription activation (Egland and Greenberg, 2001), but the . QUORUM SENSING SIGNAL INTERFERENCE WITHIN AND ACROSS THE KINGDOMS YANG FAN (B.Sci., ZhongShan University) A THESIS SUBMITED FOR THE DEGREE OF DOCTOR. agents. In this study, I explored the widespread existence of QS signal interference within and across the kingdoms and identified several QQ factors from bacteria and mammals. Microbial diversity. 13 1.2.4 QS signal turnover 16 1.3 QUORUM SENSING AND SIGNAL INTERFERENCE 17 1.3.1 AHL signal degradation 18 1.3.1.1 AHL-Lactonase 18 1.3.1.2 AHL-Acylase 20 1.3.2 Interruption and suppression