Andrée Lazdunski

A hierarchical quorum‐sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary‐phase sigma factor RpoS

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 RhIR are activated by N‐(3‐oxododecanoyl)‐l‐homo‐serine lactone (OdDHL) and N‐butanoyl‐l‐homoserine lactone (BHL) respectively. The lasR and rhIR genes are linked to the luxl homologues last and rhll, which are responsible for synthesis of OdDHL and BHL, respectively. As lasRI and rhlRI 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) rhIR expression is regulated by LasR/OdDHL, and (iii) that RhIR/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 PAN067. Furthermore, our data reveal that in E. coli, an rpoS‐lacZ fusion is regulated directly by RhIR/BHL. Taken together, these results indicate that P. aeruginosa employs a multilayered hierarchical quorum‐sensing cascade involving RhIR/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.

Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, expression of elastase is dependent upon an interaction between the positive transcriptional activator LasR and the autoinducer 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 IasR 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.

The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N‐butyryl‐homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase

The 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.

The chaperone/usher pathways of Pseudomonas aeruginosa: Identification of fimbrial gene clusters (cup) and their involvement in biofilm formation

Pseudomonas aeruginosa, an important opportunistic human pathogen, persists in certain tissues in the form of specialized bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surfaces, resulting in an organized structure. By screening a library of Tn5 insertions in a nonpiliated P. aeruginosa strain, we identified genes involved in early stages of biofilm formation. One class of mutations identified in this study mapped in a cluster of genes specifying the components of a chaperone/usher pathway that is involved in assembly of fimbrial subunits in other microorganisms. These genes, not previously described in P. aeruginosa, were named cupA1–A5. Additional chaperone/usher systems (CupB and CupC) have been also identified in the genome of P. aeruginosa PAO1; however, they do not appear to play a role in adhesion under the conditions where the CupA system is expressed and functions in surface adherence. The identification of these putative adhesins on the cell surface of P. aeruginosa suggests that this organism possess a wide range of factors that function in biofilm formation. These structures appear to be differentially regulated and may function at distinct stages of biofilm formation, or in specific environments colonized by this organism.

Involvement of the twin-arginine translocation system in protein secretion via the type II pathway.

The general secretory pathway (GSP) is a two‐step process for the secretion of proteins by Gram‐negative bacteria. The translocation across the outer membrane is carried out by the type II system, which involves machinery called the secreton. This step is considered to be an extension of the general export pathway, i.e. the export of proteins across the inner membrane by the Sec machinery. Here, we demonstrate that two substrates for the Pseudomonas aeruginosa secreton, both phospholipases, use the twin‐arginine translocation (Tat) system, instead of the Sec system, for the first step of translocation across the inner membrane. These results challenge the previous vision of the GSP and suggest for the first time a mosaic model in which both the Sec and the Tat systems feed substrates into the secreton. Moreover, since P.aeruginosa phospholipases are secreted virulence factors, the Tat system appears to be a novel determinant of bacterial virulence.

Advancing the Quorum in Pseudomonas aeruginosa: MvaT and the Regulation of N-Acylhomoserine Lactone Production and Virulence Gene Expression

Pseudomonas aeruginosa regulates the production of many exoproteins and secondary metabolites via a hierarchical quorum-sensing cascade through LasR and RhlR and their cognate signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-(butanoyl)-L-homoserine lactone (C4-HSL). In this study, we found that transcription of the quorum sensing-regulated genes lecA (coding for PA-IL lectin), lasB (coding for elastase), and rpoS appeared to be growth phase dependent and their expression could not be advanced to the logarithmic phase in cells growing in batch culture by the addition of exogenous C4-HSL and 3O-C12-HSL. To identify novel regulators responsible for this growth phase dependency, a P. aeruginosa lecA::lux reporter strain was subjected to random transposon mutagenesis. A number of mutants affected in lecA expression were found that exhibited altered production of multiple quorum sensing-dependent phenotypes. While some mutations were mapped to new loci such as clpA and mvaT and a putative efflux system, a number of mutations were also mapped to known regulators such as lasR, rhlR, and rpoS. MvaT was identified as a novel global regulator of virulence gene expression, as a mutation in mvaT resulted in enhanced lecA expression and pyocyanin production. This mutant also showed altered swarming ability and production of the LasB and LasA proteases, 3O-C12-HSL, and C4-HSL. Furthermore, addition of exogenous 3O-C12-HSL and C4-HSL to the mvaT mutant significantly advanced lecA expression, suggesting that MvaT is involved in the growth phase-dependent regulation of the lecA gene.

A novel two‐component system controls the expression of Pseudomonas aeruginosa fimbrial cup genes

Biofilm 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 two‐hybrid 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.

Regulatory circuits and communication in Gram-negative bacteria

It is increasingly apparent that, in nature, bacteria function less as individuals and more as coherent groups that are able to inhabit multiple ecological niches. The increased awareness of the role of cell–cell communication in the ecology of Gram-negative bacteria is matched by an understanding of both the physiology and the molecular biology that underlie this process. In particular, the regulatory circuits and the structure of one of the important regulatory proteins have recently been described. Here, we review the current understanding of quorum-sensing circuits in bacteria, and the role of the regulatory LuxR-type proteins in particular.

Cell signalling by oligosaccharides. Two-component systems in Pseudomonas aeruginosa: why so many?

Screening the Pseudomonas aeruginosa genome has led to the identification of the highest number of putative genes encoding two-component regulatory systems of all bacterial genomes sequenced to date (64 and 63 encoding response regulators and histidine kinases, respectively). Sixteen atypical kinases, among them 11 devoid of an Hpt domain, and three independent Hpt modules were retrieved. These data suggest that P. aeruginosa possesses complex control strategies with which to respond to environmental challenges.

Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase

The xcp genes are required for the secretion of most extracellular proteins by Pseudomonas aeruginosa. The products of these genes are essential for the transport of exoproteins across the outer membrane after they have reached the periptasm via a signal sequence‐dependent pathway. To date, analysis of three xcp genes has suggested the conservation of this secretion pathway in many Gram‐negative bacteria. Furthermore, the xcpA gene was shown to be identical to pilD, which encodes a peptidase involved in the processing of fimbrial (pili) subunits, suggesting a connection between pili biogenesis and protein secretion. Here the nucleotide sequences of seven other xcp genes, designated xcpR to ‐X, are presented. The N termini of four of the encoded Xcp proteins display similarity to the N‐termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstrated in vivo. Furthermore, two other proteins, XcpR and XcpS, show similarity to the PilB and PilC proteins required for fimbriae assembly. Since XcpR and PilB display a canonical nucleotide‐binding site, ATP hydrolysis may provide energy for both systems.