Promoter specificity in Pseudomonas aeruginosa quorum sensing revealed by DNA binding of purified LasR

Abstract

Along with their cognate acyl-homoserine lactone signals, the quorum sensing regulators LasR and RhlR control the expression of hundreds of genes in the opportunistic human pathogen Pseudomonas aeruginosa. This extensive, overlapping regulatory network affords the opportunity to systematically investigate the sequence requirements and specificity determinants of large families of target promoters. Many of the P. aeruginosa quorum-controlled genes possess conserved palindromic promoter elements predicted to be binding sites for either one or both transcriptional regulators, but biochemical proof has not been reported. We have purified native LasR and characterized binding to various quorumcontrolled promoters in vitro. Purified LasR was a dimer in solution that irreversibly bound two molecules of 3-oxo-C12-homoserine lactone. LasR bound several las-responsive promoters specifically and with high affinity, interacting cooperatively with some promoters and noncooperatively with others. LasR recognized some, but not all, of the predicted binding sites, and also bound to several unexpected sites. In contrast to predictions from genetic data, we found that the recognition sequences of las-specific promoters showed little overall sequence conservation and did not require dyad symmetry. We found distinct differences in sequence composition between las-specific noncooperative, las-specific cooperative, and rhl-responsive promoters. These results provide the basis for defining promoter specificity elements in P. aeruginosa quorum sensing. Insights into the molecular mechanism of LasR function have implications for the development of quorum-sensing targeted antivirulence compounds.bacterial signaling ͉ cell communication ͉ homoserine lactone ͉ gene regulation

Mutation analysis of the Pseudomonas aeruginosa mvfR and pqsABCDE gene promoters demonstrates complex quorum-sensing circuitry

“…These results further elucidate the complex regulatory links between LasR and mvfR, and define the critical sequence elements of the prototypical las-specific promoter. Indeed, while the 1-C nucleotide was previously thought inconsequential for LasR recognition (Schuster et al, 2004), Fig. 1(C) shows that pGX3, in which this C is replaced by T, has greatly reduced activity, indicating the 1-C nucleotide is important for LasR activation.…”

“…1B). The las/ rhl-box sequence has nearly all the conserved sequence elements of the prototypical las-specific promoter (Schuster et al, 2004). To determine if this putative element functions in mvfR regulation, mvfR9-lacZ transcriptional fusions were generated.…”

Anaerobiosis-Induced Loss of Cytotoxicity Is Due to Inactivation of Quorum Sensing in Pseudomonas aeruginosa

“…Similar to the case of lasB, transcript levels of the mvfR, rsaL, vqsR, and rhlR genes, which encode major regulators in the P. aeruginosa QS system, were ϳ10-fold lower in bacteria grown by anaerobic respiration compared to their aerobically grown counterparts. Transcript levels of rhlA, which encodes rhamnosyltransferase (34), and PA3904, a hypothetical gene with unknown function (45), were ϳ20% and ϳ50% of the transcript levels observed under aerobic growth, respectively. In contrast, mRNA levels of PA4677 (45) and xcpP (5) were similar in cells grown under either condition.…”

“…Transcript levels of rhlA, which encodes rhamnosyltransferase (34), and PA3904, a hypothetical gene with unknown function (45), were ϳ20% and ϳ50% of the transcript levels observed under aerobic growth, respectively. In contrast, mRNA levels of PA4677 (45) and xcpP (5) were similar in cells grown under either condition. These results suggest that anaerobiosis downregulated the expression of several, but not all, LasR-regulated genes.…”

Transcriptional regulation of Pseudomonas aeruginosa rhlR: role of the CRP orthologue Vfr (virulence factor regulator) and quorum-sensing regulators LasR and RhlR

“…Thus, using this construct we can measure the binding of a protein to the DNA sequence present between the RNA polymerase 210 and 235 binding sequences, by the reduction of lacZ expression. In this experiment (Table 3) we used as positive control the DNA sequence of the lasI las box that is specifically bound by LasR/3O-C12-HSL (Schuster et al, 2004), and as negative control for LasR/3-O-C12-HSL binding and control of binding specificity we used the las box of rhlA that is only bound by RhlR in both the presence and absence of C4-HSL (in the first case it activates transcription, while in the second one it acts as a repressor: Medina et al, 2003b). It has been recently reported that some structural characteristics of LasR that affect its capacity to reversibly bind 3-O-C12-HSL, and ultimately to bind las boxes, are not maintained in the purified protein (Sappington et al, 2011).…”