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

SummaryBacterial quorum sensing using acyl-homoserine lactones (acyl-HSLs) as cell-density dependent signalling molecules is important for the transcriptional regulation of many genes essential in the establishment and the maintenance of bacteria-host associations. Vibrio fischeri , the symbiotic partner of the Hawaiian bobtail squid Euprymna scolopes , possesses two distinct acyl-HSL synthase proteins, LuxI and AinS. Whereas the cell density-dependent regulation of luminescence by the LuxI-produced signal is a well-described phenomenon, and its role in light organ symbiosis has been defined, little is known about the ain system. We have investigated the impact of the V. fischeri acyl-HSL synthase AinS on both luminescence and symbiotic colonization. Through phenotypic studies of V. fischeri mutants we have found that the AinS-signal is the predominant inducer of luminescence expression in culture, whereas the impact of the LuxI-signal is apparent only at the high cell densities occurring in symbiosis. Furthermore, our studies revealed that ainS regulates activities essential for successful colonization of E. scolopes , i.e. the V. fischeri ainS mutant failed to persist in the squid light organ. Mutational inactivation of the transcriptional regulator protein LuxO in the ainS mutant partially or completely reversed all the observed phenotypes, demonstrating that the AinS-signal regulates expression of downstream genes through the inactivation of LuxO. Taken together, our results suggest that the two quorum-sensing systems in V. fischeri, ain and lux , sequentially induce the expression of luminescence genes and possibly other colonization factors.

The bacterium Proteus mirabilis is capable of movement on solid surfaces by a type of motility called swarming. Boundaries form between swarming colonies of different P. mirabilis strains but not between colonies of a single strain. A fundamental requirement for boundary formation is the ability to discriminate between self and non-self. We have isolated mutants that form boundaries with their parent. The mutations map within a six-gene locus that we term ids for identification of self. Five of the genes in the ids locus are required for recognition of the parent strain as self. Three of the ids genes are interchangeable between strains and two encode specific molecular identifiers.

The Escherichia coli gcvB gene encodes a small RNA transcript that is not translated in vivo. Transcription from the gcvB promoter is activated by the GcvA protein and repressed by the GcvR protein, the transcriptional regulators of the gcvTHP operon encoding the enzymes of the glycine cleavage system. A strain carrying a chromosomal deletion of gcvB exhibits normal regulation of gcvTHP expression and glycine cleavage enzyme activity. However, this mutant has high constitutive synthesis of OppA and DppA, the periplasmic‐binding protein components of the two major peptide transport systems normally repressed in cells growing in rich medium. The altered regulation of oppA and dppA was also demonstrated using oppA–phoA and dppA–lacZ gene fusions. Although the mechanism(s) involving gcvB in the repression of these two genes is not known, oppA regulation appears to be at the translational level, whereas dppA regulation occurs at the mRNA level.