Publications

• Functional sites and evolutionary connections of acylhomoserine lactone synthases.

  Chakrabarti S., Sowdhamini R.

  Acylhomoserine lactone (AHL) synthases act as chemical communication signals or pheromones in Gram-negative bacteria and regulate diverse physiological events in a cell density-dependent manner. The recent crystal structure determination of EsaI, a key enzyme in this pathway, shows that the AHL sy ... >> More

  Acylhomoserine lactone (AHL) synthases act as chemical communication signals or pheromones in Gram-negative bacteria and regulate diverse physiological events in a cell density-dependent manner. The recent crystal structure determination of EsaI, a key enzyme in this pathway, shows that the AHL synthase superfamily members adopt the fold of the N-acetyltransferase superfamily. We suggest, by the identification of intermediate sequences, that the two superfamilies are evolutionarily related. Evolutionary trace analyses of aligned sequences and docking studies have been used to discuss functionally important residues of EsaI homologues. << Less

  Protein Eng 16:271-278(2003) [PubMed] [EuropePMC]

• Acyl homoserine-lactone quorum-sensing signal generation.

  Parsek M.R., Val D.L., Hanzelka B.L., Cronan J.E. Jr., Greenberg E.P.

  Acyl homoserine lactones (acyl-HSLs) are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression. These signals are synthesized by members of the LuxI family of proteins. To understand the mechanism of acyl- ... >> More

  Acyl homoserine lactones (acyl-HSLs) are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression. These signals are synthesized by members of the LuxI family of proteins. To understand the mechanism of acyl-HSL synthesis we have purified the Pseudomonas aeruginosa RhlI protein and analyzed the kinetics of acyl-HSL synthesis by this enzyme. Purified RhlI catalyzes the synthesis of acyl-HSLs from acyl-acyl carrier proteins and S-adenosylmethionine. An analysis of the patterns of product inhibition indicated that RhlI catalyzes signal synthesis by a sequential, ordered reaction mechanism in which S-adenosylmethionine binds to RhlI as the initial step in the enzymatic mechanism. Because pathogenic bacteria such as P. aeruginosa use acyl-HSL signals to regulate virulence genes, an understanding of the mechanism of signal synthesis and identification of inhibitors of signal synthesis has implications for development of quorum sensing-targeted antivirulence molecules. << Less

  Proc Natl Acad Sci U S A 96:4360-4365(1999) [PubMed] [EuropePMC]

• Quorum quenching: enzymatic disruption of N-acylhomoserine lactone-mediated bacterial communication in Burkholderia thailandensis.

  Ulrich R.L.

  Many species of gram-negative bacteria communicate by synthesizing, secreting, and responding to N-acylhomoserine lactones (AHLs), a mechanism termed quorum sensing. Several investigations have characterized numerous AHL-degrading enzymes (AiiA lactonases) encoded by environmental isolates of Baci ... >> More

  Many species of gram-negative bacteria communicate by synthesizing, secreting, and responding to N-acylhomoserine lactones (AHLs), a mechanism termed quorum sensing. Several investigations have characterized numerous AHL-degrading enzymes (AiiA lactonases) encoded by environmental isolates of Bacillus spp. The Burkholderia thailandensis quorum system is comprised of at least three AHL synthases (AHSs) and five transcriptional regulators belonging to the LuxIR class of proteins. Expression of the Bacillus anthracis (Ames strain) AiiA lactonase in B. thailandensis completely abolished the accumulation of N-decanoylhomoserine lactone (C(10)-HSL) and N-octanoylhomoserine lactone (C(8)-HSL), reduced N-hexanoylhomoserine lactone (C(6)-HSL) levels, altered both swarming and twitching motility, caused a significant increase in generation time, and affected carbon metabolism. In contrast, heterologous expression of the Bacillus cereus strain A24 AiiA lactonase in B. thailandensis reduced the concentrations of C(6)-HSL, C(8)-HSL, and C(10)-HSL to nondetectable levels; altered both swarming and twitching motility; and caused fluctuations in carbon utilization. Individual disruption of the B. thailandensis AHSs, specifically disruption of the btaI1 and btaI3 genes, which encode the proteins that direct the synthesis of C(8)-HSL and C(6)-HSL, respectively, caused the hyper-beta-hemolysis of sheep erythrocytes on blood agar plates. In contrast, AHL cleavage in B. thailandensis by the Bacillus AiiA lactonases failed to enhance beta-hemolytic activity. The results of this study demonstrate that heterologous expression of Bacillus sp. AiiA lactonases in B. thailandensis reduced AHL accumulation, affected both swarming and twitching motility, increased generation time, altered substrate utilization, and prevented the beta-hemolysis of sheep erythrocytes. << Less

  Appl Environ Microbiol 70:6173-6180(2004) [PubMed] [EuropePMC]

• Acylhomoserine lactone synthase activity of the Vibrio fischeri AinS protein.

  Hanzelka B.L., Parsek M.R., Val D.L., Dunlap P.V., Cronan J.E. Jr., Greenberg E.P.

  Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone from an acyl-acyl carrier protein and S-adenosy ... >> More

  Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone from an acyl-acyl carrier protein and S-adenosylmethionine. Another V. fischeri gene, ainS, directs the synthesis of N-octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the Vibrio harveyi LuxM protein. The luxM gene is required for the synthesis of N-(3-hydroxybutyryl)-L-homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of N-octanoylhomoserine lactone from S-adenosylmethionine and either octanoyl-acyl carrier protein or, to a lesser extent, octanoyl coenzyme A. With the exception that octanoyl coenzyme A served as an acyl substrate for the MBP-AinS fusion protein, the substrates for and reaction kinetics of the MBP-AinS fusion protein were similar to those of the several LuxI family members previously studied. We conclude that AinS is an acylhomoserine lactone synthase and that it represents a second family of such enzymes. << Less

  J Bacteriol 181:5766-5770(1999) [PubMed] [EuropePMC]

• Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

  Schaefer A.L., Val D.L., Hanzelka B.L., Cronan J.E. Jr., Greenberg E.P.

  Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesi ... >> More

  Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate. << Less

  Proc Natl Acad Sci U S A 93:9505-9509(1996) [PubMed] [EuropePMC]

• Structure of the Pseudomonas aeruginosa acyl-homoserinelactone synthase LasI.

  Gould T.A., Schweizer H.P., Churchill M.E.

  The LasI/LasR quorum-sensing system plays a pivotal role in virulence gene regulation of the opportunistic human pathogen, Pseudomonas aeruginosa. Here we report the crystal structure of the acyl-homoserine lactone (AHL) synthase LasI that produces 3-oxo-C12-AHL from the substrates 3-oxo-C12-acyl- ... >> More

  The LasI/LasR quorum-sensing system plays a pivotal role in virulence gene regulation of the opportunistic human pathogen, Pseudomonas aeruginosa. Here we report the crystal structure of the acyl-homoserine lactone (AHL) synthase LasI that produces 3-oxo-C12-AHL from the substrates 3-oxo-C12-acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine. The LasI six-stranded beta sheet platform, buttressed by three alpha helices, forms a V-shaped substrate-binding cleft that leads to a tunnel passing through the enzyme that can accommodate the acyl-chain of acyl-ACP. This tunnel places no apparent restriction on acyl-chain length, in contrast to a restrictive hydrophobic pocket seen in the AHL-synthase EsaI. Interactions of essential conserved N-terminal residues, Arg23, Phe27 and Trp33, suggest that the N-terminus forms an enclosed substrate-binding pocket for S-adenosyl-L-methionine. Analysis of AHL-synthase surface residues identified a binding site for acyl-ACP, a role that was supported by in vivo reporter assay analysis of the mutated residues, including Arg154 and Lys150. This structure and the novel explanation of AHL-synthase acyl-chain-length selectivity promise to guide the design of Pseudomonas aeruginosa-specific quorum-sensing inhibitors as antibacterial agents. << Less

  Mol Microbiol 53:1135-1146(2004) [PubMed] [EuropePMC]

• Crystallization and rhenium MAD phasing of the acyl-homoserinelactone synthase EsaI.

  Watson W.T., Murphy F.V., Gould T.A., Jambeck P., Val D.L., Cronan J.E. Jr., Beck von Bodman S., Churchill M.E.

  Acyl-homoserine-L-lactones (AHLs) are diffusible chemical signals that are required for virulence of many Gram-negative bacteria. AHLs are produced by AHL synthases from two substrates, S-adenosyl-L-methionine and acyl-acyl carrier protein. The AHL synthase EsaI, which is homologous to the AHL syn ... >> More

  Acyl-homoserine-L-lactones (AHLs) are diffusible chemical signals that are required for virulence of many Gram-negative bacteria. AHLs are produced by AHL synthases from two substrates, S-adenosyl-L-methionine and acyl-acyl carrier protein. The AHL synthase EsaI, which is homologous to the AHL synthases from other pathogenic bacterial species, has been crystallized in the primitive tetragonal space group P4(3), with unit-cell parameters a = b = 66.40, c = 47.33 A. The structure was solved by multiple-wavelength anomalous diffraction with a novel use of the rhenium anomalous signal. The rhenium-containing structure has been refined to a resolution of 2.5 A and the perrhenate ion binding sites and liganding residues have been identified. << Less

  Acta Crystallogr D Biol Crystallogr 57:1945-1949(2001) [PubMed] [EuropePMC]

• Specificity of acyl-homoserine lactone synthases examined by mass spectrometry.

  Gould T.A., Herman J., Krank J., Murphy R.C., Churchill M.E.

  Many gram-negative bacteria produce a specific set of N-acyl-L-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-A ... >> More

  Many gram-negative bacteria produce a specific set of N-acyl-L-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine by the AHL synthase enzyme. The appearance of specific AHLs is due in large part to the intrinsic specificity of the enzyme for subsets of acyl-ACP substrates. Structural studies of the Pantoea stewartii enzyme EsaI and AHL-sensitive bioassays revealed that threonine 140 in the acyl chain binding pocket directs the enzyme toward production of 3-oxo-homoserine lactones. Mass spectrometry was used to examine the range of AHL molecular species produced by AHL synthases under a variety of conditions. An AHL selective normal-phase chromatographic purification with addition of a deuterated AHL internal standard was followed by reverse-phase liquid chromatography-tandem mass spectrometry in order to obtain estimates of the relative amounts of different AHLs from biological samples. The AHLs produced by wild-type and engineered EsaI and LasI AHL synthases show that intrinsic specificity and different cellular conditions influence the production of AHLs. The threonine at position 140 in EsaI is important for the preference for 3-oxo-acyl-ACPs, but the role of the equivalent threonine in LasI is less clear. In addition, LasI expressed in Escherichia coli produces a high proportion of unusual AHLs with acyl chains consisting of an odd number of carbons. Furthermore, these studies offer additional methods that will be useful for surveying and quantitating AHLs from different sources. << Less

  J Bacteriol 188:773-783(2006) [PubMed] [EuropePMC]

• Chemical mechanism and substrate specificity of RhlI, an acylhomoserine lactone synthase from Pseudomonas aeruginosa.

  Raychaudhuri A., Jerga A., Tipton P.A.

  The enzyme RhlI catalyzes the formation of N-butyrylhomoserine lactone from S-adenosylmethionine and N-butyrylacyl carrier protein. N-Butyrylhomoserine lactone serves as a quorum-sensing signal molecule in Pseudomonas aeruginosa, and is implicated in the regulation of many processes involved in ba ... >> More

  The enzyme RhlI catalyzes the formation of N-butyrylhomoserine lactone from S-adenosylmethionine and N-butyrylacyl carrier protein. N-Butyrylhomoserine lactone serves as a quorum-sensing signal molecule in Pseudomonas aeruginosa, and is implicated in the regulation of many processes involved in bacterial virulence and infectivity. The P. aeruginosa genome contains three genes encoding acyl carrier proteins. We have cloned all three genes, expressed the acyl carrier proteins, and characterized each as a substrate for RhlI. A continuous, spectrophotometric assay was developed to facilitate kinetic and mechanistic studies of RhlI. Acp1, which has not been characterized previously, was a good substrate for RhlI, with a K(m) of 7 microM; the reaction proceeded with a k(cat) value of 0.35 s(-1). AcpP, which supports fatty acid biosynthesis, was also a good substrate in the RhlI reaction, where k(cat) was 0.46 s(-1), and the K(m) for AcpP was 6 microM. The third acyl carrier protein, Acp3, was a poor substrate for RhlI, with a K(m) of 280 microM; k(cat) was 0.03 s(-1). Taken together with microarray data from the literature which show that expression of the gene encoding Acp1 is under the control of the quorum-sensing system, our data suggest that Acp1 is likely to be the substrate for RhlI in vivo. Isotope labeling studies were conducted to investigate the chemical mechanism of the RhlI-catalyzed lactonization reaction. Solvent deuterons were not incorporated into product, which implicates a direct attack mechanism in which the carboxylate oxygen of the presumptive N-butyryl-SAM intermediate attacks the methylene carbon adjacent to the sulfonium ion. Alternative mechanisms, in which N-butyrylvinylglycine is formed via elimination of methylthioadenosine, were ruled out on the basis of the observation that RhlI failed to convert authentic N-butyrylvinylglycine to N-butyryl-L-homoserine lactone. << Less

  Biochemistry 44:2974-2981(2005) [PubMed] [EuropePMC]

• Isolation and characterization of an autoinducer synthase from Acinetobacter baumannii.

  Niu C., Clemmer K.M., Bonomo R.A., Rather P.N.

  The opportunistic human pathogen Acinetobacter baumannii strain M2 was found to produce distinct acyl-homoserine lactone (AHL) signals based on the use of an Agrobacterium tumefaciens traG-lacZ biosensor. An A. baumannii gene, designated abaI, was cloned and directed AHL production in recombinant ... >> More

  The opportunistic human pathogen Acinetobacter baumannii strain M2 was found to produce distinct acyl-homoserine lactone (AHL) signals based on the use of an Agrobacterium tumefaciens traG-lacZ biosensor. An A. baumannii gene, designated abaI, was cloned and directed AHL production in recombinant Escherichia coli. The AbaI protein was similar to members of the LuxI family of autoinducer synthases and was predicted to be the only autoinducer synthase encoded by A. baumannii. The primary AHL signal directed by AbaI was identified by mass spectrometry as being N-(3-hydroxydodecanoyl)-L-HSL (3-hydroxy-C(12)-HSL). Minor amounts of at least five additional AHLs were also identified. The expression of abaI at the transcriptional level was activated by ethyl acetate extracts of culture supernatants or by synthetic 3-hydroxy-C(12)-HSL. An abaI::Km mutant failed to produce any detectable AHL signals and was impaired in biofilm development. << Less

  J. Bacteriol. 190:3386-3392(2008) [PubMed] [EuropePMC]