In Pseudomonas aeruginosa, the production of many virulence factors and secondary metabolites is regulated in concert with cell density through quorum sensing. Two quorum-sensing regulons have been identified in which the LuxR homologues LasR and RhlR are activated by N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lactone (BHL) respectively. The lasR and rhlR genes are linked to the luxl homologues lasl and rhll, which are responsible for synthesis of OdDHL and BHL, respectively. As lasRl and rhlRl are both involved in regulating synthesis of exoenzymes such as elastase, we sought to determine the nature of their interrelationship. By using lacZ transcriptional fusions in both homologous (P. aeruginosa) and heterologous (Escherichia coli) genetic backgrounds we provide evidence that (i) lasR is expressed constitutively throughout the growth cycle, (ii) rhlR expression is regulated by LasR/OdDHL, and (iii) that RhlR/BHL regulates rhll. We also show that expression of the stationary-phase sigma factor gene rpoS is abolished in a P. aeruginosa lasR mutant and in the pleiotropic BHL-negative mutant PANO67. Furthermore, our data reveal that kin E. coli, an rpoS-lacZ fusion is regulated directly by RhlR/BHL. Taken together, these results indicate that P. aeruginosa employs a multilayered hierarchical quorum-sensing cascade involving RhlR/BHL and LasR/OdDHL, interlinked via RpoS, to integrate the regulation of virulence determinants and secondary metabolites with adaptation and survival in the stationary phase.

The opportunistic pathogen Pseudomonas aeruginosa is responsible for a wide range of acute and chronic infections. The transition to chronic infections is accompanied by physiological changes in the bacteria favoring formation of biofilm communities. Here we report the identification of LadS, a hybrid sensor kinase that controls the reciprocal expression of genes for type III secretion and biofilm-promoting polysaccharides. Domain organization of LadS and the range of LadS-controlled genes suggest that it counteracts the activities of another sensor kinase, RetS. These two pathways converge by controlling the transcription of a small regulatory RNA, RsmZ. This work identifies a previously undescribed signal transduction network in which the activities of signal-receiving sensor kinases LadS, RetS, and GacS regulate expression of virulence genes associated with acute or chronic infection by transcriptional and posttranscriptional mechanisms.biofilm ͉ pel genes ͉ small RNA ͉ two-component system ͉ type III secretion

In Pseudomonas aeruginosa PAO1, expression of elastase is dependent upon an interaction between the positive transcriptional activator LasR and the auto-inducer molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), the synthesis of which is directed by LasI. Previously we have shown that in PAN067, an elastase-negative mutant of PAO1, elastase production can be restored to some extent by addition of exogenous N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Here we report that PAN067 is also defective in the production of alkaline protease, haemolysin, cyanide, pyocyanin and autoinducer(s). As neither addition of exogenous OdDHL nor introduction of lasR restored PAN067 to the parental phenotype, we sought to complement PAN067 with PAO1 DNA. From a cosmid library, a 2 kb DNA fragment was identified which re-established production of autoinducer(s) and exoproducts in PAN067. From the nucleotide sequence of this fragment, two genes termed rhIR and rhII were identified. RhII is responsible for autoinducer synthesis and shares 31% homology with LasI; RhIR has been previously identified in P. aeruginosa strain DSM2659 as a regulator of rhamnolipid biosynthesis and shares 28% identity with LasR. These data provide clear evidence that multiple families of quorum-sensing modulons interactively regulate gene expression in P. aeruginosa.

SummaryThe global activator GacA, a highly conserved response regulator in Gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. The gacA gene of Pseudomonas aeruginosa PAO1 was isolated and its role in cell-density-dependent gene expression was characterized. Mutational inactivation of gacA resulted in delayed and reduced formation of the cell-density signal N-butyryl-L-homoserine lactone (BHL), of the cognate transcriptional activator RhlR (VsmR), and of the transcriptional activator LasR, which is known to positively regulate RhlR expression. Amplification of gacA on a multicopy plasmid caused precocious and enhanced production of BHL, RhlR and LasR. In parallel, the gacA gene dosage markedly influenced the BHL/RhlR-dependent formation of the cytotoxic compounds pyocyanin and cyanide and the exoenzyme lipase. However, the concentrations of another known cell-density signal of P. aeruginosa, N-oxododecanoyl-L-homoserine lactone, did not always match BHL concentrations. A model accounting for these observations places GacA function upstream of LasR and RhlR in the complex, cell-density-dependent signal-transduction pathway regulating several exoproducts and virulence factors of P. aeruginosa via BHL.

SummaryBiofilm formation by the opportunistic pathogen Pseudomonas aeruginosa requires the expression of a number of surface adhesive components. The expression of surface organelles facilitating biofilm formation is controlled by environmental signals acting through transcriptional regulatory networks. We analysed the expression of a family of P. aeruginosa adhesins encoded by three distinct fimbrial gene clusters ( cupA, cupB and cupC ). Using transposon mutagenesis, we have identified several regulatory loci that upregulated cupB and cupC transcription. One such locus contains three components, RocS1, RocR and RocA1, which represent a variant of a classical two-component signal transduction pathway. RocS1 is a sensor kinase, RocA1 is a DNA binding response regulator that activates cup genes, and RocR is an antagonist of RocA1 activity. Using a twohybrid assay, we have shown that RocS1 interacts with receiver domains of both RocA1 and RocR. Expression of the Cup system in response to environmental stimuli is accomplished by a novel mechanism in which the sensor kinase activates its cognate response regulator through a phosphorelay pathway, while an additional repressor protein modulates this interaction.