Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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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.
Mol Microbiol 2004 Aug
PMID:Structure of the Pseudomonas aeruginosa acyl-homoserinelactone synthase LasI. 1530 17

Agrobacterium tumefaciens C58 communicates using N-acyl-homoserine lactones (acyl-HSL) and contains two lactonase-encoding genes, attM and aiiB, the products of which are capable of inactivating the acyl-HSL signal. In A. tumefaciens A6, the expression of the attKLM operon is controlled by the transcriptional repressor encoded by an adjacent gene, attJ. An attJ::Tn5 mutant does not accumulate acyl-HSL because of the constitutive expression of the lactonase AttM, the activity of which inactivates acyl-HSL. In this work, the attKLM operon of A. tumefaciens C58 was shown to be involved in an assimilative pathway of gamma-butyrolactone (GBL), gamma-hydroxybutyrate (GHB), and succinate semialdehyde (SSA), in which AttM and AttL are key enzymes for GBL and GHB assimilation. The expression of the attKLM promoter was activated in the presence of GBL, GHB, and SSA. Under these conditions, A. tumefaciens C58 did not accumulate the acyl-HSL that it naturally synthesizes, and also became able to inactivate exogenous acyl-HSL signals. Therefore, in A. tumefaciens C58, the assimilative pathway of gamma-butyrolactone interferes with the acyl-HSL signaling.
Mol Plant Microbe Interact 2004 Sep
PMID:The assimilation of gamma-butyrolactone in Agrobacterium tumefaciens C58 interferes with the accumulation of the N-acyl-homoserine lactone signal. 1538 85

TraM, an 11.2 kDa antiactivator, modulates the acyl-homoserine lactone-mediated autoinduction of Ti plasmid conjugative transfer by interacting directly with TraR, the quorum-sensing transcriptional activator. Most antiactivators and antisigma factors examined to date act in dimer form. However, whether, and if so, how TraM dimerizes is unknown. Analyses based on a genetic assay using fusions of TraM to the lambda cI DNA binding domain, and biochemical assays using chemical crosslinking and gel filtration chromatography showed that TraM forms homodimers. Although SDS-PAGE studies suggested that the lone cysteine residue at position 71 was involved in interprotomer disulfide-bridging in TraM, altering Cys-71 to a serine did not significantly affect dimerization or the antiactivator activity of this mutant protein when expressed at wild-type levels in vivo. Analysis of N-terminal, C-terminal, and internal deletion mutants of TraM identified two regions of the protein involved in dimerization; one located within a segment between residues 20 and 50, and the other located to a segment between residues 67 and 96. Both regions are required for formation of fully stable dimers. Analysis of the activity of these deletion mutants in vivo, and their ability to bind TraR and to disrupt TraR-DNA complexes in vitro, suggests that while the internal segment of the protein is required for dimerization, determinants located at the far C-terminus and beginning at between residues 10 and 20 at the N-terminus play a role in TraR binding and antiactivator function. When co-expressed with lambda cI'::TraR fusions, wild-type TraM mediated quormone-independent dimerization of the transcriptional activator, suggesting that dimers of TraM can multimerize TraR.
Mol Microbiol 2004 Sep
PMID:Dimerization properties of TraM, the antiactivator that modulates TraR-mediated quorum-dependent expression of the Ti plasmid tra genes. 1538 23

Burkholderia glumae BGR1 produces a broad-host range phytotoxin, called toxoflavin, which is a key pathogenicity factor in rice grain rot and wilt in many field crops. Our molecular and genetic analyses of toxoflavin-deficient mutants demonstrated that gene clusters for toxoflavin production consist of four transcriptional units. The toxoflavin biosynthesis genes were composed of five genes, toxA to toxE, as Suzuki et al. (2004) reported previously. Genes toxF to toxI, which are responsible for toxoflavin transport, were polycistronic and similar to the genes for resistance-nodulation-division (RND) efflux systems. Using Tn3-gusA reporter fusions, we found that ToxR, a LysR-type regulator, regulates both the toxABCDE and toxFGHI operons in the presence of toxoflavin as a coinducer. In addition, the expression of both operons required a transcriptional activator, ToxJ, whose expression is regulated by quorum sensing. TofI, a LuxI homologue, was responsible for the biosynthesis of both N-hexanoyl homoserine lactone and N-octanoyl homoserine lactone (C8-HSL). C8-HSL and its cognate receptor TofR, a LuxR homologue, activated toxJ expression. This is the first report that quorum sensing is involved in pathogenicity by the regulation of phytotoxin biosynthesis and its transport in plant pathogenic bacteria.
Mol Microbiol 2004 Nov
PMID:Quorum sensing and the LysR-type transcriptional activator ToxR regulate toxoflavin biosynthesis and transport in Burkholderia glumae. 1552 77

Candida albicans is an opportunistic pathogen that is commonly found as a member of the human microflora. The ability of C. albicans to alter its cellular morphology has been associated with its virulence; yeast cells are more prevalent in commensal interactions whereas filamentous cells appear important in opportunistic infections. C. albicans encounters a multitude of other microbial species in the host environment and it is likely that they impact the C. albicans transition between virulent and non-virulent states. Here, we report that C. albicans morphology is significantly affected by the presence of Pseudomonas aeruginosa, another opportunistic pathogen. In a screen using a C. albicans HWP1-lacZ strain to indicate regions of filamentous growth, we identified P. aeruginosa mutants incapable of inhibiting C. albicans filamentation. Through these studies, we found that 3-oxo-C12 homoserine lactone, a cell-cell signalling molecule produced by P. aeruginosa, was sufficient to inhibit C. albicans filamentation without affecting fungal growth rates. Both microscopic analysis and real-time reverse transcription polymerase chain reaction analysis of morphology-specific markers confirmed that filamentation was suppressed by 200 microM 3-oxo-C12 homoserine lactone. Structurally related compounds with a 12-carbon chain length, e.g. C12-acyl homoserine lactone and dodecanol also affected C. albicans filamentation at similar concentrations. In contrast, other acylated homoserine lactones of different chain lengths did not affect fungal morphology. The activity of 3OC12HSL is compared with that of farnesol, a C. albicans-produced molecule also with a C12-backbone. The effects that bacteria have on the morphology of C. albicans represents one of the ways by which bacteria can influence the behaviour of fungal cells.
Mol Microbiol 2004 Dec
PMID:A Pseudomonas aeruginosa quorum-sensing molecule influences Candida albicans morphology. 1555 63

Erwinia carotovora produces the beta-lactam antibiotic, carbapenem, in response to a quorum sensing signalling molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). We have mapped the OHHL-dependent promoter upstream of the first of the biosynthetic genes, carA. We have also analysed the effect on this promoter of the known genetic regulators of carbapenem expression, carR, carI (encoding homologues of LuxR and LuxI respectively) and hor (encoding a SlyA/MarR-like transcriptional regulator). We describe a previously unknown promoter located within the carA-H operon. This promoter does not respond to CarR and is required for quorum sensing-independent expression of the carbapenem resistance determinants encoded by the carFG genes. We have mapped the carR, carI and hor transcription start points, shown that CarR is positively autoregulated in the presence of OHHL, and have demonstrated negative feedback affecting transcription of carI. In addition, various environmental and physiological factors were shown to impinge on the transcription of the car biosynthetic genes. The nature of the carbon source and the temperature of growth influence carbapenem production by modulating the level of the OHHL signalling molecule, and thereby physiologically fine-tune the quorum sensing regulatory system.
Mol Microbiol 2005 Jan
PMID:Carbapenem antibiotic biosynthesis in Erwinia carotovora is regulated by physiological and genetic factors modulating the quorum sensing-dependent control pathway. 1565 68

LuxR-type transcriptional regulators play key roles in quorum-sensing systems that employ acyl-homoserine lactones (acyl-HSLs) as signal molecules. These proteins mediate quorum control by changing their interactions with RNA polymerase and DNA in response to binding their cognate acyl-HSL. The evolutionarily related LuxR-type proteins exhibit considerable diversity in primary sequence and in their response to acyl-HSLs having acyl groups of differing length and composition. Little is known about which residues determine acyl-HSL specificity, and less about the evolutionary time scales required to forge new ones. To begin to examine such issues, we have focused on the LuxR protein from Vibrio fischeri, which activates gene transcription in response to binding its cognate quorum signal, 3-oxohexanoyl-homoserine lactone (3OC6HSL). Libraries of luxR mutants were screened for variants exhibiting increased gene activation in response to octanoyl-HSL (C8HSL), with which wild-type LuxR interacts only weakly. Eight LuxR variants were identified that showed a 100-fold increase in sensitivity to C8HSL; these variants also displayed increased sensitivities to pentanoyl-HSL and tetradecanoyl-HSL, while maintaining a wild-type or greater response to 3OC6HSL. The most sensitive variants activated gene transcription as strongly with C8HSL as the wild type did with 3OC6HSL. With one exception, the amino acid residues involved were restricted to the N-terminal, 'signal-binding' domain of LuxR. These residue positions differed from critical positions previously identified via 'loss-of-function' mutagenesis. We have demonstrated that acyl-HSL-dependent quorum-sensing systems can evolve rapidly to respond to new acyl-HSLs, suggesting that there may be an evolutionary advantage to maintaining such plasticity.
Mol Microbiol 2005 Feb
PMID:Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. 1566 Sep 98

The transcriptional regulator MvfR is required for full Pseudomonas aeruginosa virulence, the function of multiple quorum sensing (QS)-regulated virulence factors and the synthesis of 4-hydroxy-2-alkylquinolines (HAQs), including the Pseudomonas quinolone signal (PQS). Here we investigate the role of MvfR in the QS circuitry and P. aeruginosa pathogenesis. We demonstrate using a combination of biochemical and molecular approaches, including transcription profiling, that MvfR is involved in the regulation of multiple P. aeruginosa QS-controlled genes without altering the expression of lasRI/rhlRI or the production of N-acyl-L-homoserine lactone (AHL) signals. Dissection of how mvfR is interwoven into the P. aeruginosa QS circuitry reveals that the MvfR system, through the essential contribution of PqsE, positively regulates a subset of genes dependant on both LasR and RhlR. Animal studies show that MvfR contributes to P. aeruginosa virulence by controlling the transcription of genes not under RhlR regulation, and that reduced virulence of a mvfR mutant is caused by the loss of pqsE expression and not only a deficiency in HAQs/PQS production. This study provides novel insights into the unique role of the MvfR system in AHL-mediated QS and further supports its importance in P. aeruginosa pathogenesis.
Mol Microbiol 2005 Feb
PMID:The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple quorum sensing-regulated genes are modulated without affecting lasRI, rhlRI or the production of N-acyl-L-homoserine lactones. 1568 49

The LuxR-type quorum-sensing transcription factor TraR regulates replication and conjugal transfer of the tumour-inducing (Ti) plasmid in the plant pathogen Agrobacterium tumefaciens. TraR is a two-domain protein with an N-terminal domain that binds to the quorum-sensing signal N-3-oxooctanoyl- l-homoserine lactone (OOHL) and a C-terminal domain that binds to specific DNA sequences called tra boxes. TraR-OOHL complexes form homodimers that activate transcription of at least seven promoters on the Ti plasmid. At five promoters, a tra box overlaps the binding site of core RNA polymerase (class II promoters), while in the other two promoters, this site is located farther upstream (class I promoters). In this study, we performed saturating point mutagenesis of the surface residues of the TraR C-terminal domain. Each mutant was tested for proteolytic stability and transcription activity in vivo, and for DNA binding activity in vitro. Mutants of TraR with single substitutions at positions W184, V187, K189, E193Q, V197 and D217 have wild-type levels of accumulation and DNA binding, but are defective in transcription of both types of promoters. These residues constitute a patch on the surface of the DNA-binding domain. We propose that this patch is an activating region that recruits RNA polymerase to TraR-dependent promoters through direct contact. As residues of this patch are critical for activation at both a class I and a class II promoter, we predict that these residues may contact the C-terminal domain of the RNA polymerase alpha-subunit.
Mol Microbiol 2005 Mar
PMID:Identification of amino acid residues of the Agrobacterium tumefaciens quorum-sensing regulator TraR that are critical for positive control of transcription. 1572 May 54

The LuxR-type quorum-sensing transcription factor EsaR functions as a repressor of exopolysaccharide (EPS) synthesis in the phytopathogenic bacterium Pantoea stewartii ssp. stewartii. The cell density-dependent expression of EPS is critical for Stewart's wilt disease development. Strains deficient in the synthesis of a diffusible acyl-homoserine lactone inducer remain repressed for EPS synthesis and are consequently avirulent. In contrast, disruption of the esaR gene leads to hypermucoidy and attenuated disease development. Ligand-free EsaR functions as a negative autoregulator of the esaR gene and responds to exogenous acyl-homoserine lactone for derepression. The focus of this study was to define the mechanism by which EsaR governs the expression of the cps locus, which encodes functions required for stewartan EPS synthesis and membrane translocation. Genetic and biochemical studies show that EsaR directly represses the transcription of the rcsA gene. RcsA encodes an essential coactivator for RcsA/RcsB-mediated transcriptional activation of cps genes. In vitro assays identify an EsaR DNA binding site within the rcsA promoter that is reasonably well conserved with the previously described esaR box. We also describe that RcsA positively controls its own expression. Interestingly, promoter proximal genes within the cps cluster are significantly more acyl-homoserine lactone responsive than genes located towards the middle or 3' end of the gene cluster. We will discuss a possible role of EsaR-mediated quorum sensing in the differential expression of the cps operon.
Mol Microbiol 2005 Apr
PMID:The cell density-dependent expression of stewartan exopolysaccharide in Pantoea stewartii ssp. stewartii is a function of EsaR-mediated repression of the rcsA gene. 1577 89


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