Maryline Foglino

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.

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.

Interactions of the quorum sensing regulator QscR: interaction with itself and the other regulators of Pseudomonas aeruginosa LasR and RhlR.

Pseudomonas aeruginosa controls the production of many exoproteins and secondary metabolites via a hierarchical quorum sensing (QS) regulatory cascade involving the LuxR‐like proteins LasR, RhlR and their cognate signal molecules N‐(3‐oxododecanoyl)‐l‐homoserine lactone (3O‐C12‐HSL) and N‐(butanoyl)‐l‐homoserine lactone (C4‐HSL). The finding of a third LuxR‐type protein in P. aeruginosa, QscR, adds further complexity to this regulatory network. It has been shown previously that QscR represses transcription of three QS‐controlled gene clusters, phz (phenazine), hcn (hydrogen cyanide) and qsc105 (Chugani, Whiteley, Lee, D’Argenio, Manoil, and Greenberg, 2001, Proc Natl Acad Sci USA 98: 2752–2757). In this study, we identify two novel QscR targets these are lasB, encoding the extracellular elastase, and the second phenazine gene cluster, both of which are downregulated by QscR. In addition, we show that QscR synthesis is regulated by the two‐component response regulator GacA. Taking advantage of the in vivo fluorescence anisotropy technology that we have developed, we show that QscR can be found in several different types of association. Indeed, we identify QscR multimers in the absence of any acyl‐HSL, lower order QscR oligomers associated either with C4‐HSL or 3O‐C12‐HSL and QscR‐containing heterodimers with LasR or RhlR. The formation of heterodimers between QscR and LasR or RhlR, in the absence of acyl‐HSLs, is a very exciting, new result that should improve our understanding of the QscR network and its relationship to the production of P. aeruginosa virulence factors.

A direct sulfhydrylation pathway is used for methionine biosynthesis in Pseudomonas aeruginosa.

The relationship between genes and enzymes in the methionine biosynthetic pathway has been studied in Pseudomonas aeruginosa. The first step is catalysed by an O-succinylhomoserine synthase, the product of the metA gene mapped at 20 min on the chromosome. The second step is achieved by direct sulfhydrylation, involving the enzyme encoded by a metZ gene that we have identified and sequenced, located at 40 min. Thus Pseudomonas appears to be the only organism so far described that uses O-succinylhomoserine as substrate for a direct sulfhydrylation. As in yeast, the two transsulfuration pathways between cysteine and homocysteine, with cystathionine as an intermediate, probably exist in parallel in this organism.

Dimerization of the quorum sensing regulator RhlR: development of a method using EGFP fluorescence anisotropy

Of considerable interest in the biology of pathogenic bacteria are the mechanisms of intercellular signalling that can lead to the formation of persistent infections. In this article, we have examined the intracellular behaviour of a Pseudomonas aeruginosa quorum sensing regulator RhlR believed to be important in this process. We have further examined the modulation of this behaviour in response to various auto‐inducers. For these measurements, we have developed an assay based on the fluorescence anisotropy of EGFP fusion proteins that we use to measure protein–protein interactions in vivo. We show that the transcriptional regulator, RhlR, expressed as an EGFP fusion protein in Escherichia coli, forms a homodimer. This homodimer can be dissociated into monomers by the auto‐inducer N‐(3‐oxododecanoyl)‐l‐homoserine lactone (3O‐C12‐HSL) whereas N‐(butanoyl)‐l‐homoserine lactone (C4‐HSL) has little effect. These observations are of particular interest as RhlR modulation of gene expression depends on the presence of C4‐HSL, whereas 3O‐C12‐HSL modulates the expression of genes regulated by LasR. These observations thus provide a framework for understanding the regulatory network that links the various different QS regulators in P. aeruginosa. Furthermore, the technique we have developed should permit the study of numerous protein/protein or protein/nucleic acid interactions in vivo and so shed light on natural protein function.

Resistance to Bacitracin in Bacillus subtilis: Unexpected Requirement of the BceAB ABC Transporter in the Control of Expression of Its Own Structural Genes

The Bacillus subtilis BceAB ABC transporter involved in a defense mechanism against bacitracin is composed of a membrane-spanning domain and a nucleotide-binding domain. Induction of the structural bceAB genes requires the BceR response regulator and the BceS histidine kinase of a signal transduction system. However, despite the presence of such a transduction system and of bacitracin, no transcription from an unaltered bceA promoter is observed in cells lacking the BceAB transporter. Expression in trans of the BceAB transporter in these bceAB cells restores the transcription from the bceA promoter. Cells possessing a mutated nucleotide-binding domain of the transporter are also no longer able to trigger transcription from the bceA promoter in the presence of bacitracin, although the mutated ABC transporter is still bound to the membrane. In these cells, expression of the bceA promoter can no longer be detected, indicating that the ABC transporter not only must be present in the cell membrane, but also must be expressed in a native form for the induction of the bceAB genes. Several hypotheses are discussed to explain the simultaneous need for bacitracin, a native signal transduction system, and an active BceAB ABC transporter to trigger transcription from the bceA promoter.

Nucleotide sequence of the pepN gene encoding aminopeptidase N of Escherichia coli

We have sequenced a 3.3-kb fragment of the Escherichia coli chromosome that contains pepN gene encoding aminopeptidase N. This gene codes for a protein of 870 amino acid residues. From the size of the pepN transcript and the presence of inverted repeats in the nucleotide (nt) sequence, a putative transcription terminator has been identified. The N-terminal amino acid sequence deduced from the pepN nt sequence corresponds to the N-terminal sequence of the purified protein; the amino acid composition of the protein is also in good agreement with that deduced from the gene sequence. No obvious homology with previously sequenced peptidases has been detected.

Global regulation in Pseudomonas aeruginosa: the regulatory protein AlgR2 (AlgQ) acts as a modulator of quorum sensing

The Pseudomonas aeruginosa protein AlgR2 (AlgQ) was originally identified as a regulatory protein implicated in alginate production. It also regulates the synthesis of polyphosphate as well as of a variety of secretable virulence factors, upregulating neuraminidase and siderophore synthesis and downregulating rhamnolipid biosurfactant and extracellular protease synthesis. In this study, we show that the regulatory effect of AlgR2 on elastase protease synthesis is exerted at transcriptional level on the lasB gene. We also demonstrate that AlgR2 negatively modulates the expression of quorum sensing regulatory genes lasR and rhlR. Finally, results obtained from DNA retardation assays provide evidence that AlgR2 can bind specifically to the lasR and rhlR promoters. Altogether, these data provide strong support for the hypothesis that AlgR2 is a global transcriptional regulator in P. aeruginosa.