The production of many virulence factors by Pseudomonas aeruginosa is regulated by the quorum-sensing (QS) response. In this regulatory network LasR and RhlR, bound to their corresponding autoinducers, play a central role. The QS response has a hierarchical structure: LasR/3O-C12-HSL activates the transcription of rhlR, and RhlR/C4-HSL activates the transcription of several genes, including the rhlAB operon, which encodes the enzymes responsible for rhamnolipid synthesis. The rhlAB operon is located immediately upstream of the rhlR gene. rhlR has four transcription start sites, two of which are located in the rhlB coding region. Vfr directly activates transcription of lasR, and has been reported to be also involved in rhlR expression. The aim of this work was to characterize the details of the mechanism of rhlR transcriptional regulation. We show that Vfr directly regulates rhlR transcription through its binding to several Vfr-binding sites (VBSs) present in the rhlR promoter region, one of which has a negative effect on transcription. Two of the VBSs overlap with las boxes where LasR/3O-C12-HSL binds to activate rhlR transcription. We also show that rhlR transcription is subject to positive-feedback autoregulation through RhlR/C4-HSL activation of the rhlA promoter. This positive autoregulation plays a major role in rhlR expression.

The Pseudomonas aeruginosa rmlBDAC operon, encoding dTDP-L-rhamnose biosynthetic enzymes, is regulated by the quorum-sensing transcriptional regulator RhlR and the alternative sigma factor s Pseudomonas aeruginosa produces as biosurfactants rhamnolipids, containing one (monorhamnolipid) or two (di-rhamnolipid) L-rhamnose molecules. The rhamnosyltransferase RhlB catalyses the synthesis of mono-rhamnolipid using as precursors dTDP-L-rhamnose and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) produced by RhlA, while the rhamnosyltransferase RhlC synthesizes di-rhamnolipid using mono-rhamnolipid and dTDP-L-rhamnose as substrates. The Las and Rhl quorum-sensing systems coordinately regulate the production of these surfactants, as well as that of other exoproducts involved in bacterial virulence, at the transcriptional level in a cell density-dependent manner. In this work we study the transcriptional regulation of the rmlBDAC operon, encoding the enzymes involved in the production of dTDP-Lrhamnose, the substrate of both rhamnosyltransferases, RhlB and RhlC, and also a component of P. aeruginosa lipopolysaccharide. Here we show that the rmlBDAC operon possesses three promoters. One of these transcriptional start sites (P2) is responsible for most of its expression and is dependent on the stationary phase sigma factor s S and on RhlR/C 4 -HSL through its binding to an atypical 'las box'. INTRODUCTIONPseudomonas aeruginosa is an opportunistic human pathogen that causes serious nosocomial infections. The secretion of numerous toxic compounds and hydrolytic enzymes that are involved in its pathogenicity is coordinately regulated at the transcriptional level by the so-called quorum-sensing Besides being a rhamnolipid precursor, dTDP-L-rhamnose is a lipopolysaccharide (LPS) precursor in a variety of bacteria, including P. aeruginosa (Rahim et al., 2000). This activated sugar has been implicated in bacterial pathogenicity (Ma et al., 1997(Ma et al., , 2001Bozue et al., 2005; EngelsDeutsch et al., 2003). The dTDP-L-rhamnose biosynthetic pathway consists of the conversion of glucose 1-phosphate to dTDP-L-rhamnose via dTDP-glucose, dTDP-6-deoxy-Dxylo-4-hexulose and dTDP-6-deoxy-L-xylo-4-hexulose Abbreviations: C 4 -HSL, butanoyl-homoserine lactone; 3O-C 12 -HSL, 3-oxo-dodecanoyl homoserine lactone; RNAP, RNA polymerase; QS, quorum sensing; QSR, quorum-sensing response.A supplementary table, showing oligonucleotides used in this study, is available with the online version of this paper.Microbiology ( 2012), 158, 908-916

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