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Query: UNIPROT:P06889 (Mol)
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The opportunistic pathogen Pseudomonas aeruginosa undergoes genetic change during chronic airway infection of cystic fibrosis (CF) patients. One common change is a mutation inactivating lasR, which encodes a transcriptional regulator that responds to a homoserine lactone signal to activate expression of acute virulence factors. Colonies of lasR mutants visibly accumulated the iridescent intercellular signal 4-hydroxy-2-heptylquinoline. Using this colony phenotype, we identified P. aeruginosa lasR mutants that emerged in the airway of a CF patient early during chronic infection, and during growth in the laboratory on a rich medium. The lasR loss-of-function mutations in these strains conferred a growth advantage with particular carbon and nitrogen sources, including amino acids, in part due to increased expression of the catabolic pathway regulator CbrB. This growth phenotype could contribute to selection of lasR mutants both on rich medium and within the CF airway, supporting a key role for bacterial metabolic adaptation during chronic infection. Inactivation of lasR also resulted in increased beta-lactamase activity that increased tolerance to ceftazidime, a widely used beta-lactam antibiotic. Loss of LasR function may represent a marker of an early stage in chronic infection of the CF airway with clinical implications for antibiotic resistance and disease progression.
Mol Microbiol 2007 Apr
PMID:Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients. 1749 32

A comparative molecular modelling study of acyl homoserine lactones-dependent transcriptional regulators (TraR, SdiA, LuxR and LasR) involved in bacterial quorum sensing (QS) revealed a high structural homology of their active site. Docking studies within the active site of TraR of fixed conformations obtained using molecular mechanics calculations showed that TraR, for which the crystalline structure is known, is a relevant model for the study of other protein-ligand interactions in the same protein family. Structure-activity relationships of AHLs derived QS modulators including carboxamides, sulfonamides and ureas were thus investigated. The results show that Tyr61, a residue conserved in the LuxR-proteins family, is involved in attractive interactions with aromatic carboxamide antagonists. Tyr53, Tyr61 and Asp70, conserved residues, are implicated in both the development of additional hydrogen bonds and attractive interactions with the N-sulfonyl homoserine lactones and AHLs derived ureas antagonists.
J Mol Graph Model 2007 Sep
PMID:Exploring the active site of acyl homoserine lactones-dependent transcriptional regulators with bacterial quorum sensing modulators using molecular mechanics and docking studies. 1752 90

The quorum sensing (QS) signalling system of Pseudomonas aeruginosa controls many important functions, including virulence. Although the production of the QS signal molecule N-3-oxo-dodecanoyl-homoserine lactone (3OC(12)-HSL) is positively autoregulated, its concentration reaches a steady level long before stationary phase. The RsaL protein represses transcription of the lasI signal synthase gene, and thus reduces QS signal production. We show that RsaL binds simultaneously with LasR to the rsaL-lasI bidirectional promoter thereby preventing the LasR-dependent activation of both genes. In an rsaL mutant, 3OC(12)-HSL production continues to increase throughout growth. Thus RsaL provides homeostasis by functioning in opposition to LasR and limiting 3OC(12)-HSL production to a physiological concentration. Furthermore, transcription profiling revealed that RsaL regulates 130 genes independent of its effect on QS signal molecule production, including genes involved in virulence. We show that RsaL can repress pyocyanin and hydrogen cyanide virulence genes in two ways: directly, by binding to their promoters, and indirectly, by decreasing levels of the signals for their QS signal-dependent transcription. These investigations highlight the importance of RsaL as a global regulator of P. aeruginosa physiology that provides a counterbalance to 3OC(12)-HSL-dependent gene activation via multiple mechanisms.
Mol Microbiol 2007 Dec
PMID:RsaL provides quorum sensing homeostasis and functions as a global regulator of gene expression in Pseudomonas aeruginosa. 1804 85

Despite significant advances in the development of sensitive tools for studying genetics and signal exchange in legume-rhizobium symbioses, many uncertainties remain about the in vivo role of bacterial and plant signals in symbiotic gene regulation. In this study, we adapted TnpR recombinase-based in vivo expression technology (RIVET) to document gene regulation in Sinorhizobium meliloti. The substrate for TnpR, the res1-tet-res1 cassette, is stably inherited when cloned into a neutral site of the S. meliloti genome. Bicistronic promoterless tnpR-beta-glucuronidase (GUS) reporters were constructed to track expression ("resolution") of symbiotically relevant S. meliloti genes during different stages of the interaction. In proof of principle experiments, the resolution of the nodC::tnpR reporter was detected within 4 h of exposure to micromolar levels of the nod operon inducer luteolin and after overnight incubation in the rhizosphere. RIVET demonstrated that cell division gene ftsZ2 was not strongly expressed in the rhizosphere but was activated inside the nodules and on agar surfaces. Rhizosphere expression of the N-acyl homoserine lactone (AHL) synthase sinI::tnpR-GUS reporter was modest in prequorate microcolonies, and then increased with time. AHL synthase sinI and an AHL-regulated gene, expG, were activated inside the nodules.
Mol Plant Microbe Interact 2008 Feb
PMID:RIVET-a tool for in vivo analysis of symbiotically relevant gene expression in Sinorhizobium meliloti. 1818 60

Many bacteria can sense their population density. This has been termed "quorum sensing." The bacteria use this information to coordinate their behavior, essentially behaving as multicellular organisms. The paradigm of Gram-negative quorum sensing is the LuxL/LuxR-type system employed by Vibriofischeri to regulate luminescence. The LuxR transcription factor detects the presence of N-acylhomoserine lactones (AHLs) produced by LuxI. The AHL diffuses freely across the cell wall, and its accumulation signals a high population density within a confined space. Upon binding AHL, the LuxR transcription factor activates the luminescence genes. Homologous systems are used by numerous Gram-negative pathogens to regulate host interaction genes. The AHLs produced by different LuxI homologs can vary in the length and modification of their acyl side chain. In the first section of this chapter, we describe the use of bacterial biosensors to determine whether a particular bacterial species synthesizes AHLs. The second section describes how to identify AHL-responsive genes in Salmonella typhimurium, an organism that detects but does not synthesize AHLs. The approach described can be modified for use with any organism that responds to AHLs but does not synthesize them. The third section describes the use of recombination-based in vivo expression technology (RIVET) to study AHL detection in vitro and in vivo, in this case the mouse gut.
Methods Mol Biol 2007
PMID:Methods in cell-to-cell signaling in Salmonella. 1836 42

Many bacteria use quorum sensing (QS) as an intercellular signaling mechanism to regulate gene expression in local populations. Plant and algal hosts, in turn, secrete compounds that mimic bacterial QS signals, allowing these hosts to manipulate QS-regulated gene expression in bacteria. Lumichrome, a derivative of the vitamin riboflavin, was purified and chemically identified from culture filtrates of the alga Chlamydomonas as a QS signal-mimic compound capable of stimulating the Pseudomonas aeruginosa LasR QS receptor. LasR normally recognizes the N-acyl homoserine lactone (AHL) signal, N-3-oxo-dodecanoyl homoserine lactone. Authentic lumichrome and riboflavin stimulated the LasR receptor in bioassays and lumichrome activated LasR in gel shift experiments. Amino acid substitutions in LasR residues required for AHL binding altered responses to both AHLs and lumichrome or riboflavin. These results and docking studies indicate that the AHL binding pocket of LasR recognizes both AHLs and the structurally dissimilar lumichrome or riboflavin. Bacteria, plants, and algae commonly secrete riboflavin or lumichrome, raising the possibility that these compounds could serve as either QS signals or as interkingdom signal mimics capable of manipulating QS in bacteria with a LasR-like receptor.
Mol Plant Microbe Interact 2008 Sep
PMID:The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum-sensing receptor. 1870 Aug 23

In Pseudomonas aeruginosa quorum sensing (QS), the transcriptional regulator LasR controls the expression of more than 300 genes. Several of these genes are activated indirectly via a second, subordinate QS regulator, RhlR. Conserved sequence elements upstream of individual other genes have been shown to bind LasR in vitro. To comprehensively identify all regions that are bound by LasR in vivo, we employed chromatin immunoprecipitation in conjunction with microarray analysis. We identified 35 putative promoter regions that direct the expression of up to 74 genes. In vitro DNA binding studies allowed us to distinguish between cooperative and non-cooperative LasR binding sites, and allowed us to build consensus sequences according to the mode of binding. Five promoter regions were not previously recognized as QS-controlled. Two of the associated transcript units encode proteins involved in the cold-shock response and in Psl exopolysaccharide synthesis respectively. The LasR regulon includes seven genes encoding transcriptional regulators, while secreted factors and secretion machinery are the most over-represented functional categories overall. This supports the notion that the core function of LasR is to co-ordinate the production of extracellular factors, although many of its effects on global gene expression are likely mediated indirectly by regulatory genes under its control.
Mol Microbiol 2009 Sep
PMID:Global position analysis of the Pseudomonas aeruginosa quorum-sensing transcription factor LasR. 1968 64

The Sin quorum sensing system of Sinorhizobium meliloti depends upon at least three genes, sinR, sinI and expR, and N-acyl homoserine lactones (AHLs) as signals to regulate multiple processes in its free-living state in the rhizosphere and in the development towards symbiosis with its plant host. In this study, we have characterized novel mechanisms of transcription control through which the system regulates itself. At low AHL levels a positive feedback loop activates expression of sinI (AHL synthase), resulting in amplification of AHL levels. At high AHL levels, expression of sinI is reduced by a negative feedback loop. These feedback mechanisms are mediated by the LuxR-type regulators ExpR and SinR. Expression of sinR and expR is regulated by ExpR in the presence of AHLs. A novel ExpR binding site in the promoter of sinR is responsible for the reduction of expression of this gene. In addition, expression of sinR, upon which sinI expression is dependent, is induced by phoB during growth under phosphate-limiting conditions. This indicates that this response ensures quorum sensing in phosphate-restricted growth.
Mol Microbiol 2009 Dec
PMID:Novel Sinorhizobium meliloti quorum sensing positive and negative regulatory feedback mechanisms respond to phosphate availability. 1988 97

The ability of LuxR-type proteins to regulate transcription is controlled by bacterial pheromones, N-acylhomoserine lactones (AHLs). Most LuxR-family proteins require their cognate AHLs for activity, and at least some of them require AHLs for folding and protease resistance. However, a few members of this family are able to fold, dimerize, bind DNA, and regulate transcription in the absence of AHLs; moreover, these proteins are antagonized by their cognate AHLs. Complexes between some of these proteins and their DNA binding sites are disrupted by AHLs in vitro. All such proteins are fairly closely related within the larger LuxR family, indicating that they share a relatively recent common ancestor. The 3' ends of the genes encoding these receptors invariably overlap with the 3' ends of the cognate AHL synthase genes, suggesting additional antagonism at the level of mRNA synthesis, stability or translation.
Mol Microbiol 2010 Sep
PMID:LuxR-type quorum-sensing regulators that are detached from common scents. 2062 21

In many pathogenic bacteria, quorum sensing (QS) controls expression of genes that are involved in virulence, production and resistance to antibiotics, formation and maintenance of microbial multicellular consortia on biotic and abiotic surfaces of medical and industrial importance. N-acyl homoserine lactones (AHL) are the best characterized quorum sensing signals in Gram-negative bacteria. Interference with AHL-mediated QS, therefore, is considered an attractive strategy for controlling virulence in pathogens. The search for AHL signals and their mimics has been facilitated by the development of sensitive bioassays, in which QS reporters luminesce in response to AHL signals. These bioassays have already led to the identification of dozens of compounds with QS modulating activities. The characterization of the mode of action of QS signals and their mimics requires follow-up biochemical studies. Here, we describe a set of luminescent reporters, which could be used in high, medium or low throughput format, for the discovery and validation of agonists or antagonists of the Las QS system of Pseudomonas aeruginosa. These nearly isogenic reporters contain truncations or point mutations in the AHL binding domain of the AHL receptor LasR, as well as mutations in the promoter for the gene encoding LasI AHL synthase. We also developed reporters for documenting the regulation of lasI and lasR promoters. The use of these reporters significantly streamlines identification and characterization of the Las QS signal agonists and antagonists prior to biochemical experiments. To test the usefulness of these reporters, we carried out bioassays with patulin, a known inhibitor of Las QS.
Methods Mol Biol 2011
PMID:Luminescent reporters and their applications for the characterization of signals and signal-mimics that alter LasR-mediated quorum sensing. 2103 8


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