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Paul Williams - One of the best experts on this subject based on the ideXlab platform.
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simultaneous quantitative profiling of n acyl l Homoserine Lactone and 2 alkyl 4 1h quinolone families of quorum sensing signaling molecules using lc ms ms
Analytical and Bioanalytical Chemistry, 2011Co-Authors: Catharine A Ortori, Miguel Cámara, Siri Ram Chhabra, Kim R. Hardie, Paul Williams, Jeanfrederic Dubern, David A BarrettAbstract:An LC-MS/MS method, using positive mode electrospray ionization, for the simultaneous, quantitative and targeted profiling of the N-acyl-l-Homoserine Lactone (AHL) and 2-alkyl 4-(1H)-quinolone (AQ) families of bacterial quorum-sensing signaling molecules (QSSMs) is presented. This LC-MS/MS technique was applied to determine the relative molar ratios of AHLs and AQs produced by Pseudomonas aeruginosa and the consequences of mutating individual or multiple QSSM synthase genes (lasI, rhlI, pqsA) on AHL and AQ profiles and concentrations. The AHL profile of P. aeruginosa was dominated by N-butanoyl-l-Homoserine Lactone (C4-HSL) with lesser concentrations of N-hexanoyl-l-Homoserine Lactone (C6-HSL) and 3-oxo-substituted longer chain AHLs including N-(3-oxodecanoyl)-l-Homoserine Lactone (3-oxo-C10-HSL) and N-(3-oxododecanoyl)-l-Homoserine Lactone (3-oxo-C12-HSL). The AQ profile of P. aeruginosa comprised the C7 and C9 long alkyl chain AQs including 2-heptyl-4-hydroxyquinoline (HHQ), 2-nonyl-4-hydroxyquinoline, the “pseudomonas quinolone signal” (2-heptyl-3-hydroxy-4-quinolone) and the N-oxides, 2-heptyl-4-hydroxyquinoline N-oxide and 2-nonyl-4-hydroxyquinoline N-oxide. Application of the method showed significant effects of growth medium type on the ratio and the nature of the QSSMs synthesized and the dramatic effect of single, double and triple mutations in the P. aeruginosa QS synthase genes. The LC-MS/MS methodology is applicable in organisms where either or both AHL and AQ QSSMs are produced and can provide comprehensive profiles and concentrations from a single sample.
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Quorum Quenching by an N-Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1
Infection and immunity, 2006Co-Authors: Charles Frederik Sio, Miguel Cámara, Paul Williams, Linda G. Otten, Robbert H. Cool, Stephen P. Diggle, Peter Braun, Rein Bos, Mavis Daykin, Wim J. QuaxAbstract:The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-Homoserine Lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the Homoserine Lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3′ position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-Homoserine Lactone (C4-HSL) and N-(3-oxododecanoyl)-l-Homoserine Lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.
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In vitro biosynthesis of the Pseudomonas aeruginosa quorum‐sensing signal molecule N‐butanoyl‐L‐Homoserine Lactone
Molecular microbiology, 2002Co-Authors: Yan Jiang, Miguel Cámara, Siri Ram Chhabra, Kim R. Hardie, Barrie W. Bycroft, Andrée Lazdunski, George P. C. Salmond, Gordon S. A. B. Stewart, Paul WilliamsAbstract:In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-Homoserine Lactone (BHL) and N-hexanoyl-L-Homoserine Lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or Homoserine Lactone (HSL) but not Homoserine as the source of the Homoserine Lactone moiety. As we were unable to detect Homoserine Lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylHomoserine Lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.
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the pseudomonas aeruginosa quorum sensing signal molecule n 3 oxododecanoyl l Homoserine Lactone has immunomodulatory activity
Infection and Immunity, 1998Co-Authors: Gary Telford, Barrie W. Bycroft, Gordon S. A. B. Stewart, Paul Williams, David A Wheeler, P T Tomkins, P Appleby, Herbert F Sewell, D I PritchardAbstract:Diverse gram-negative bacterial cells communicate with each other by using diffusible N-acyl Homoserine Lactone (AHL) signal molecules to coordinate gene expression with cell population density. Accumulation of AHLs above a threshold concentration renders the population "quorate," and the appropriate target gene is activated. In pathogenic bacteria, such as Pseudomonas aeruginosa, AHL-mediated quorum sensing is involved in the regulation of multiple virulence determinants. We therefore sought to determine whether the immune system is capable of responding to these bacterial signal molecules. Consequently the immunomodulatory properties of the AHLs N-(3-oxododecanoyl)-L-Homoserine Lactone (OdDHL) and N-(3-oxohexanoyl)-L-Homoserine Lactone (OHHL) were evaluated in murine and human leukocyte immunoassays in vitro. OdDHL, but not OHHL, inhibited lymphocyte proliferation and tumor necrosis factor alpha production by lipopolysaccharide-stimulated macrophages. Furthermore, OdDHL simultaneously and potently down-regulated the production of IL-12, a Th-1-supportive cytokine. At high concentrations (>7 x 10(-5) M) OdDHL inhibited antibody production by keyhole limpet hemocyanin-stimulated spleen cells, but at lower concentrations (<7 x 10(-5) M), antibody production was stimulated, apparently by increasing the proportion of the immunoglobulin G1 (IgG1) isotype. OdDHL also promoted IgE production by interleukin-4-stimulated human peripheral blood mononuclear cells. These data indicate that OdDHL may influence the Th-1-Th-2 balance in the infected host and suggest that, in addition to regulating the expression of virulence determinants, OdDHL may contribute to the pathogenesis of P. aeruginosa infections by functioning as a virulence determinant per se.
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n acyl Homoserine Lactone mediated regulation of phenazine gene expression by pseudomonas aureofaciens 30 84 in the wheat rhizosphere
Journal of Bacteriology, 1997Co-Authors: Derek W Wood, Paul Williams, Mavis Daykin, Fangcheng Gong, Leland S PiersonAbstract:Pseudomonas aureofaciens 30-84 is a soilborne bacterium that colonizes the wheat rhizosphere. This strain produces three phenazine antibiotics which suppress take-all disease of wheat by inhibition of the causative agent Gaeumannomyces graminis var. tritici. Phenazines also enhance survival of 30-84 within the wheat rhizosphere in competition with other organisms. Expression of the phenazine biosynthetic operon is controlled by the phzR/phzI N-acyl-Homoserine Lactone (AHL) response system (L. S. Pierson III et al., J. Bacterial 176:3966-3974, 1994; D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). By using high-pressure liquid chromatography coupled with high-resolution mass spectrometry, the AHL produced by PhzI has now been identified as N-hexanoyl-Homoserine Lactone (HHL). In addition, the ability of HHL to serve as an interpopulation signal molecule in the wheat rhizosphere has been examined by using isogenic reporter strains. Disruption of phzI reduced expression of the phenazine biosynthetic operon 1,000-fold in the wheat rhizosphere. Coinoculation of an isogenic strain which produced the endogenous HHL signal restored phenazine gene expression in the phzI mutant to wild-type levels in situ. These results demonstrate that HHL is required for phenazine expression in situ and is an effective interpopulation signal molecule in the wheat rhizosphere.
Barrie W. Bycroft - One of the best experts on this subject based on the ideXlab platform.
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In vitro biosynthesis of the Pseudomonas aeruginosa quorum‐sensing signal molecule N‐butanoyl‐L‐Homoserine Lactone
Molecular microbiology, 2002Co-Authors: Yan Jiang, Miguel Cámara, Siri Ram Chhabra, Kim R. Hardie, Barrie W. Bycroft, Andrée Lazdunski, George P. C. Salmond, Gordon S. A. B. Stewart, Paul WilliamsAbstract:In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-Homoserine Lactone (BHL) and N-hexanoyl-L-Homoserine Lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or Homoserine Lactone (HSL) but not Homoserine as the source of the Homoserine Lactone moiety. As we were unable to detect Homoserine Lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylHomoserine Lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.
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n acyl Homoserine Lactone binding to the carr receptor determines quorum sensing specificity in erwinia
The EMBO Journal, 2000Co-Authors: Martin Welch, Barrie W. Bycroft, Daniel E Todd, Neil A Whitehead, Simon J Mcgowan, George P. C. SalmondAbstract:Quorum sensing via an N-acyl Homoserine Lactone (HSL) pheromone controls the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR-type activator protein, CarR. Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays. CarR bound the most physiologically relevant ligand, N-(3-oxohexanoyl)-L-Homoserine Lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 microM, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the ligand causes the protein to multimerize. Chemical cross-linking analysis supported this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein.
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the pseudomonas aeruginosa quorum sensing signal molecule n 3 oxododecanoyl l Homoserine Lactone has immunomodulatory activity
Infection and Immunity, 1998Co-Authors: Gary Telford, Barrie W. Bycroft, Gordon S. A. B. Stewart, Paul Williams, David A Wheeler, P T Tomkins, P Appleby, Herbert F Sewell, D I PritchardAbstract:Diverse gram-negative bacterial cells communicate with each other by using diffusible N-acyl Homoserine Lactone (AHL) signal molecules to coordinate gene expression with cell population density. Accumulation of AHLs above a threshold concentration renders the population "quorate," and the appropriate target gene is activated. In pathogenic bacteria, such as Pseudomonas aeruginosa, AHL-mediated quorum sensing is involved in the regulation of multiple virulence determinants. We therefore sought to determine whether the immune system is capable of responding to these bacterial signal molecules. Consequently the immunomodulatory properties of the AHLs N-(3-oxododecanoyl)-L-Homoserine Lactone (OdDHL) and N-(3-oxohexanoyl)-L-Homoserine Lactone (OHHL) were evaluated in murine and human leukocyte immunoassays in vitro. OdDHL, but not OHHL, inhibited lymphocyte proliferation and tumor necrosis factor alpha production by lipopolysaccharide-stimulated macrophages. Furthermore, OdDHL simultaneously and potently down-regulated the production of IL-12, a Th-1-supportive cytokine. At high concentrations (>7 x 10(-5) M) OdDHL inhibited antibody production by keyhole limpet hemocyanin-stimulated spleen cells, but at lower concentrations (<7 x 10(-5) M), antibody production was stimulated, apparently by increasing the proportion of the immunoglobulin G1 (IgG1) isotype. OdDHL also promoted IgE production by interleukin-4-stimulated human peripheral blood mononuclear cells. These data indicate that OdDHL may influence the Th-1-Th-2 balance in the infected host and suggest that, in addition to regulating the expression of virulence determinants, OdDHL may contribute to the pathogenesis of P. aeruginosa infections by functioning as a virulence determinant per se.
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Bacterial N-acyl-Homoserine-Lactone-dependent signalling and its potential biotechnological applications
Trends in biotechnology, 1997Co-Authors: Neil D. Robson, Barrie W. Bycroft, Simon J Mcgowan, Anthony Cox, George P. C. SalmondAbstract:N-acyl Homoserine Lactones are bacterial signalling molecules involved in regulating diverse metabolic functions, particularly those relating to virulence, in concert with cell density. Each aspect of the signalling pathway, from production and recognition of the signal to expression of the target genes, offers a potential opportunity for exploitation. Attention is now focusing on the development of novel methods for bacterial enumeration, modulation of bacterial virulence and flexible, coordinated expression of heterologous genes through the use of N-acyl-Homoserine-Lactone-based systems.
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involvement of n acyl l Homoserine Lactone autoinducers in controlling the multicellular behaviour of serratia liquefaciens
Molecular Microbiology, 1996Co-Authors: Leo Eberl, Siri Ram Chhabra, Barrie W. Bycroft, Gordon S. A. B. Stewart, Paul Williams, Michael K. Winson, Claus Sternberg, Gunna Christiansen, Søren Molin, Michael GivskovAbstract:Summary Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer-cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high-performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N-butanoyl-l-Homoserine Lactone (BHL) and N-hexanoyl-l-Homoserine Lactone (HHL) in cell-free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrlswarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the Luxl family of putative Nacyl Homoserine Lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.
Mair E. A. Churchill - One of the best experts on this subject based on the ideXlab platform.
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Structural basis of acyl-Homoserine Lactone-dependent signaling.
Chemical reviews, 2010Co-Authors: Mair E. A. Churchill, Lingling ChenAbstract:Acyl-Homoserine Lactones are small neutral lipid molecules that bacteria in the Proteobacteria group use to sense and signal their cell density. This signaling process, known as quorum sensing, activates differentiation to diverse community oriented lifestyles within the bacterial population. Since the first description of autoinduction 40 years ago 1 and quorum sensing systems in the light producing gram-negative bacterium Vibrio fischeri 2,3, which is a symbiont of the Hawaiian bobtail squid Euprymna scolopes, much has been learned about the molecular mechanism of signaling in these and many related bacteria (reviewed in 4 and Chapters 5-25 in 5). The primary signals used by gram negative bacteria to sense cell density are acyl-Homoserine Lactone (AHL) molecules, which are composed of a Homoserine Lactone ring (HSL) with an acyl chain (Figure 1). The acyl-chain length generally varies from C4 to C18 6,7 and this can be modified by a 3-oxo substituent, or in some cases, a 3-hydroxy substituent, a terminal methyl branch, or varied degrees of unsaturation 8. AHLs are synthesized by enzymes known as the AHL synthases 7,9. Once produced, the AHLs diffuse in and out of the cell by passive 10 as well as active transport mechanisms 11. The concentration of AHL eventually reaches a sufficiently high concentration at a given threshold cell number or bacterial “quorum”, and it is then recognized by a receptor protein, which is the second component of the system. The AHLresponsive receptors include a wide variety of transcriptional regulators called “R proteins”, such as LuxR or LasR, which are DNA-binding transcription factors 4,12 and a small family of sensor kinases related to LuxN 13. The binding of the AHL by most of the characterized R-proteins initiates the activation and repression of target genes, and in some cases AHL binding leads to target gene derepression 14-18.
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Defining the structure and function of acyl-Homoserine Lactone autoinducers.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Mair E. A. Churchill, Hiruy M. Sibhatu, Charis L. UhlsonAbstract:Quorum sensing plays a central role in regulating many community-derived symbiotic and pathogenic relationships of bacteria, and as such has attracted much attention in recent years. Acyl-Homoserine Lactones (AHLs) are important signaling molecules in the quorum sensing gene-regulatory processes found in numerous gram-negative species of bacteria that interact with eukaryotic organisms. AHLs are produced by acyl-Homoserine Lactone synthases. Bacteria can have multiple genes for AHL synthase enzymes, and such species are likely to produce several different types of AHLs. Determination of the types and the relative amounts of AHLs produced by AHL synthases in bacteria under varied conditions provides important insights into the mechanism of AHL synthase function and the regulation of transcriptional cascades initiated by quorum sensing signaling. This chapter describes a mass spectrometry method for determining the types and relative amounts of AHLs present in a sample.
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Quorum-Sensing Signal Synthesis by the Yersinia pestis Acyl-Homoserine Lactone Synthase YspI
Journal of bacteriology, 2006Co-Authors: J. Paul Kirwan, Ty A. Gould, Herbert P. Schweizer, Scott W. Bearden, Robert C. Murphy, Mair E. A. ChurchillAbstract:The acyl-Homoserine Lactone molecular species (AHLs) produced by the Yersinia pestis AHL synthase YspI were identified by biochemical and physical/chemical techniques. Bioassays of extracts from culture supernatants of the recombinant YspI and wild-type Yersinia pestis showed similar profiles of AHLs. Analysis by liquid chromatography-mass spectrometry revealed that the predominant AHLs were N-3-oxooctanoyl-l-Homoserine Lactone and N-3-oxo-hexanoyl-l-Homoserine Lactone.
Siri Ram Chhabra - One of the best experts on this subject based on the ideXlab platform.
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simultaneous quantitative profiling of n acyl l Homoserine Lactone and 2 alkyl 4 1h quinolone families of quorum sensing signaling molecules using lc ms ms
Analytical and Bioanalytical Chemistry, 2011Co-Authors: Catharine A Ortori, Miguel Cámara, Siri Ram Chhabra, Kim R. Hardie, Paul Williams, Jeanfrederic Dubern, David A BarrettAbstract:An LC-MS/MS method, using positive mode electrospray ionization, for the simultaneous, quantitative and targeted profiling of the N-acyl-l-Homoserine Lactone (AHL) and 2-alkyl 4-(1H)-quinolone (AQ) families of bacterial quorum-sensing signaling molecules (QSSMs) is presented. This LC-MS/MS technique was applied to determine the relative molar ratios of AHLs and AQs produced by Pseudomonas aeruginosa and the consequences of mutating individual or multiple QSSM synthase genes (lasI, rhlI, pqsA) on AHL and AQ profiles and concentrations. The AHL profile of P. aeruginosa was dominated by N-butanoyl-l-Homoserine Lactone (C4-HSL) with lesser concentrations of N-hexanoyl-l-Homoserine Lactone (C6-HSL) and 3-oxo-substituted longer chain AHLs including N-(3-oxodecanoyl)-l-Homoserine Lactone (3-oxo-C10-HSL) and N-(3-oxododecanoyl)-l-Homoserine Lactone (3-oxo-C12-HSL). The AQ profile of P. aeruginosa comprised the C7 and C9 long alkyl chain AQs including 2-heptyl-4-hydroxyquinoline (HHQ), 2-nonyl-4-hydroxyquinoline, the “pseudomonas quinolone signal” (2-heptyl-3-hydroxy-4-quinolone) and the N-oxides, 2-heptyl-4-hydroxyquinoline N-oxide and 2-nonyl-4-hydroxyquinoline N-oxide. Application of the method showed significant effects of growth medium type on the ratio and the nature of the QSSMs synthesized and the dramatic effect of single, double and triple mutations in the P. aeruginosa QS synthase genes. The LC-MS/MS methodology is applicable in organisms where either or both AHL and AQ QSSMs are produced and can provide comprehensive profiles and concentrations from a single sample.
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In vitro biosynthesis of the Pseudomonas aeruginosa quorum‐sensing signal molecule N‐butanoyl‐L‐Homoserine Lactone
Molecular microbiology, 2002Co-Authors: Yan Jiang, Miguel Cámara, Siri Ram Chhabra, Kim R. Hardie, Barrie W. Bycroft, Andrée Lazdunski, George P. C. Salmond, Gordon S. A. B. Stewart, Paul WilliamsAbstract:In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-Homoserine Lactone (BHL) and N-hexanoyl-L-Homoserine Lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or Homoserine Lactone (HSL) but not Homoserine as the source of the Homoserine Lactone moiety. As we were unable to detect Homoserine Lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylHomoserine Lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.
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involvement of n acyl l Homoserine Lactone autoinducers in controlling the multicellular behaviour of serratia liquefaciens
Molecular Microbiology, 1996Co-Authors: Leo Eberl, Siri Ram Chhabra, Barrie W. Bycroft, Gordon S. A. B. Stewart, Paul Williams, Michael K. Winson, Claus Sternberg, Gunna Christiansen, Søren Molin, Michael GivskovAbstract:Summary Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer-cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high-performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N-butanoyl-l-Homoserine Lactone (BHL) and N-hexanoyl-l-Homoserine Lactone (HHL) in cell-free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrlswarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the Luxl family of putative Nacyl Homoserine Lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.
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Involvement of N‐acyl‐l‐Homoserine Lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens
Molecular microbiology, 1996Co-Authors: Leo Eberl, Siri Ram Chhabra, Barrie W. Bycroft, Gordon S. A. B. Stewart, Paul Williams, Michael K. Winson, Claus Sternberg, Gunna Christiansen, Søren Molin, Michael GivskovAbstract:Summary Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer-cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high-performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N-butanoyl-l-Homoserine Lactone (BHL) and N-hexanoyl-l-Homoserine Lactone (HHL) in cell-free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrlswarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the Luxl family of putative Nacyl Homoserine Lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.
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multiple n acyl l Homoserine Lactone signal molecules regulate production of virulence determinants and secondary metabolites in pseudomonas aeruginosa
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Michael K. Winson, Miguel Cámara, Siri Ram Chhabra, George P. C. Salmond, Mavis Daykin, Amel Latifi, Maryline Foglino, Marc Bally, Virginie Chapon, Barrie W. BycroftAbstract:Pseudomonas aeruginosa produces a spectrum of exoproducts many of which have been implicated in the pathogenesis of human infection. Expression of some of these factors requires cell-cell communication involving the interaction of a small diffusible molecule, an "autoinducer," with a positive transcriptional activator. In P. aeruginosa PAO1, LasI directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-Homoserine Lactone (OdDHL), which activates the positive transcriptional activator, LasR. Recently, we have discovered a second signaling molecule-based modulon in PAO1, termed vsm, which contains the genes vsmR and vsmI. Using HPLC, mass spectrometry, and NMR spectroscopy we now establish that in Escherichia coli, VsmI directs the synthesis of N-butanoyl-L-Homoserine Lactone (BHL) and N-hexanoyl-L-Homoserine Lactone (HHL). These compounds are present in the spent culture supernatants of P. aeruginosa in a molar ratio of approximately 15:1 and their structures were unequivocally confirmed by chemical synthesis. Addition of either BHL or HHL to PAN067, a pleiotropic P. aeruginosa mutant unable to synthesize either of these autoinducers, restored elastase, chitinase, and cyanide production. In E. coli carrying a vsmR/vsmI'::lux transcriptional fusion, BHL and HHL activated VsmR to a similar extent. Analogues of these N-acyl-L-Homoserine Lactones in which the N-acyl side chain has been extended and/or oxidized at the C-3 position exhibit substantially lower activity (e.g., OdDHL) or no activity (e.g., dDHL) in this lux reporter assay. These data indicate that multiple families of quorum sensing modulons interactively regulate gene expression in P. aeruginosa.
Miguel Cámara - One of the best experts on this subject based on the ideXlab platform.
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simultaneous quantitative profiling of n acyl l Homoserine Lactone and 2 alkyl 4 1h quinolone families of quorum sensing signaling molecules using lc ms ms
Analytical and Bioanalytical Chemistry, 2011Co-Authors: Catharine A Ortori, Miguel Cámara, Siri Ram Chhabra, Kim R. Hardie, Paul Williams, Jeanfrederic Dubern, David A BarrettAbstract:An LC-MS/MS method, using positive mode electrospray ionization, for the simultaneous, quantitative and targeted profiling of the N-acyl-l-Homoserine Lactone (AHL) and 2-alkyl 4-(1H)-quinolone (AQ) families of bacterial quorum-sensing signaling molecules (QSSMs) is presented. This LC-MS/MS technique was applied to determine the relative molar ratios of AHLs and AQs produced by Pseudomonas aeruginosa and the consequences of mutating individual or multiple QSSM synthase genes (lasI, rhlI, pqsA) on AHL and AQ profiles and concentrations. The AHL profile of P. aeruginosa was dominated by N-butanoyl-l-Homoserine Lactone (C4-HSL) with lesser concentrations of N-hexanoyl-l-Homoserine Lactone (C6-HSL) and 3-oxo-substituted longer chain AHLs including N-(3-oxodecanoyl)-l-Homoserine Lactone (3-oxo-C10-HSL) and N-(3-oxododecanoyl)-l-Homoserine Lactone (3-oxo-C12-HSL). The AQ profile of P. aeruginosa comprised the C7 and C9 long alkyl chain AQs including 2-heptyl-4-hydroxyquinoline (HHQ), 2-nonyl-4-hydroxyquinoline, the “pseudomonas quinolone signal” (2-heptyl-3-hydroxy-4-quinolone) and the N-oxides, 2-heptyl-4-hydroxyquinoline N-oxide and 2-nonyl-4-hydroxyquinoline N-oxide. Application of the method showed significant effects of growth medium type on the ratio and the nature of the QSSMs synthesized and the dramatic effect of single, double and triple mutations in the P. aeruginosa QS synthase genes. The LC-MS/MS methodology is applicable in organisms where either or both AHL and AQ QSSMs are produced and can provide comprehensive profiles and concentrations from a single sample.
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Quorum Quenching by an N-Acyl-Homoserine Lactone Acylase from Pseudomonas aeruginosa PAO1
Infection and immunity, 2006Co-Authors: Charles Frederik Sio, Miguel Cámara, Paul Williams, Linda G. Otten, Robbert H. Cool, Stephen P. Diggle, Peter Braun, Rein Bos, Mavis Daykin, Wim J. QuaxAbstract:The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-Homoserine Lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the Homoserine Lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3′ position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-Homoserine Lactone (C4-HSL) and N-(3-oxododecanoyl)-l-Homoserine Lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.
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In vitro biosynthesis of the Pseudomonas aeruginosa quorum‐sensing signal molecule N‐butanoyl‐L‐Homoserine Lactone
Molecular microbiology, 2002Co-Authors: Yan Jiang, Miguel Cámara, Siri Ram Chhabra, Kim R. Hardie, Barrie W. Bycroft, Andrée Lazdunski, George P. C. Salmond, Gordon S. A. B. Stewart, Paul WilliamsAbstract:In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-Homoserine Lactone (BHL) and N-hexanoyl-L-Homoserine Lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or Homoserine Lactone (HSL) but not Homoserine as the source of the Homoserine Lactone moiety. As we were unable to detect Homoserine Lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylHomoserine Lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.
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multiple n acyl l Homoserine Lactone signal molecules regulate production of virulence determinants and secondary metabolites in pseudomonas aeruginosa
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Michael K. Winson, Miguel Cámara, Siri Ram Chhabra, George P. C. Salmond, Mavis Daykin, Amel Latifi, Maryline Foglino, Marc Bally, Virginie Chapon, Barrie W. BycroftAbstract:Pseudomonas aeruginosa produces a spectrum of exoproducts many of which have been implicated in the pathogenesis of human infection. Expression of some of these factors requires cell-cell communication involving the interaction of a small diffusible molecule, an "autoinducer," with a positive transcriptional activator. In P. aeruginosa PAO1, LasI directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-Homoserine Lactone (OdDHL), which activates the positive transcriptional activator, LasR. Recently, we have discovered a second signaling molecule-based modulon in PAO1, termed vsm, which contains the genes vsmR and vsmI. Using HPLC, mass spectrometry, and NMR spectroscopy we now establish that in Escherichia coli, VsmI directs the synthesis of N-butanoyl-L-Homoserine Lactone (BHL) and N-hexanoyl-L-Homoserine Lactone (HHL). These compounds are present in the spent culture supernatants of P. aeruginosa in a molar ratio of approximately 15:1 and their structures were unequivocally confirmed by chemical synthesis. Addition of either BHL or HHL to PAN067, a pleiotropic P. aeruginosa mutant unable to synthesize either of these autoinducers, restored elastase, chitinase, and cyanide production. In E. coli carrying a vsmR/vsmI'::lux transcriptional fusion, BHL and HHL activated VsmR to a similar extent. Analogues of these N-acyl-L-Homoserine Lactones in which the N-acyl side chain has been extended and/or oxidized at the C-3 position exhibit substantially lower activity (e.g., OdDHL) or no activity (e.g., dDHL) in this lux reporter assay. These data indicate that multiple families of quorum sensing modulons interactively regulate gene expression in P. aeruginosa.
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Multiple N-acyl-L-Homoserine Lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa
Microbiology, 1995Co-Authors: Michael Winson, Miguel Cámara, Mavis Daykin, Amel Latifi, Maryline Foglino, Marc Bally, Virginie Chapon, Ram Siri Chhabrat, George Salmond, Barrie BycroftAbstract:Pseudomonas aeruginosa produces a spectrum of exoproducts many of which have been implicated in the pathogenesis of human infection. Expression of some of these factors requires cell-cell communication involving the interaction of a small diffusible molecule, an "autoinducer," with a positive transcriptional activator. In P. aeruginosa PAO1, LasI directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-Homoserine Lactone (OdDHL), which activates the positive transcriptional activator, LasR. Recently, we have discovered a second signaling molecule-based modulon in PAO1, termed vsm, which contains the genes vsmR and vsmI. Using HPLC, mass spectrometry, and NMR spectroscopy we now establish that in Escherichia coli, VsmI directs the synthesis of N-butanoyl-L-Homoserine Lactone (BHL) and N-hexanoyl-L-Homoserine Lactone (HHlL). These compounds are present in the spent culture supernatants of P. aeruginosa in a molar ratio of-15:1 and their structures were unequivocally confirmed by chemical synthesis. Addition of either BHL or HHL to PAN067, a pleiotropic P. aeruginosa mutant unable to synthesize either of these autoinducers, restored elastase, chitinase, and cyanide production. In E. coli carrying a vsmR/vsmI'::lux transcriptional fusion, BHL and HHL activated VsmR to a similar extent. Analogues of these N-acyl-L-Homoserine Lactones in which the N-acyl side chain has been extended and/or oxidized at the C-3 position exhibit substantially lower activity (e.g., OdDHL) or no activity (e.g., dDHL) in this lux reporter assay. These data indicate that multiple families of quorum sensing modulons interactively regulate gene expression in P. aeruginosa. The recognition that N-(3-oxohexanoyl)-L-Homoserine Lactone (OHHL; Fig. 1), previously termed the lux autoinducer (AI) (1), regulates both secondary metabolism (2, 3) and virulence in the plant pathogen Erwinia carotovora (4, 5) has been followed by a surge of interest in the role of N-acyl-L-Homoserine Lactones (AHLs) in bacterial gene expression (6-8). Diverse Gram-negative bacteria are now known to synthesize AHLs which form part of a cell-cell communication system that facilitates the induction of genetic regulons only when a significant cell population density has been attained (2, 6-8). This cell density dependency reflects the accumulation of the signal molecule to a critical threshold concentration (1, 6-8) and is termed quorum sensing (6). Quorum sensing systems depend upon the interaction of an Al, the synthesis of which is directed by a LuxI homologue, with a positive N HH