Pseudomonas aeruginosa contains two transcription regulators (LasR and RhlR) that, when complexed with their specific autoinducers (3-oxo-dodecanoyl-homoserine lactone and butanoyl-homoserine lactone, respectively) activate transcription of different virulence-associated traits. We studied the RhlR-dependent transcriptional regulation of the rhlAB operon encoding rhamnosyltransferase 1, an enzyme involved in the synthesis of the surfactant monorhamnolipid, and showed that RhlR binds to a specific sequence in the rhlAB regulatory region, both in the presence and in the absence of its autoinducer. Our data suggest that in the former case it activates transcription, whereas in the latter it acts as a transcriptional repressor of this promoter. RhlR seems to repress the transcription of other quorum-sensing-regulated genes; thus, RhlR repressor activity might be of importance in the finely regulated expression of P. aeruginosa virulence-associated traits.

The Pseudomonas aeruginosa rhlR gene encodes the transcriptional regulator RhlR which has a central role in the quorum-sensing response. Different gene products involved in bacterial pathogenesis are regulated at the transcriptional level by two quorum-sensing response systems, Las and Rhl. The expression of rhlR has been reported to be under the control of the Las system, but its transcriptional regulation has not been studied in detail. Here, the rhlR promoter region has been characterized and shown to present four different transcription start sites, two of which are included in the upstream gene (rhlB) coding region. It was found that rhlR expression is not only dependent on LasR but also on different regulatory proteins such as Vfr and RhlR itself, and also on the alternative sigma factor σ 54. It is reported that rhlR expression is partially LasR-independent under certain culture conditions and is strongly influenced by environmental factors.

The Pseudomonas aeruginosa rhlAB operon encodes the enzyme rhamnosyltransferase 1, which produces the biosurfactant mono-rhamnolipid; rhlAB induction is dependent on the quorum-sensing transcription activator RhlR complexed with the autoinducer N-butyryl-homoserine lactone (C 4 -HSL). In this work we studied rhlAB induction in a P. aeruginosa and Escherichia coli background. We found that, in both bacteria, its expression is not induced during the logarithmic phase of growth even in the presence of RhlR and C 4 -HSL. Additionally, we found that rhlAB expression is partially s dependent.Pseudomonas aeruginosa is an opportunistic pathogen causing serious nosocomial infections (5). The production of several of the virulence-associated traits created by this bacterium is regulated at the level of transcription by the so-called quorum-sensing response (8,30).Rhamnolipids are biosurfactants produced by P. aeruginosa (13) that are regulated by the quorum-sensing response dependent on the transcription activator RhlR (16) and the autoinducer (AI) N-butyryl-homoserine lactone (C 4 -HSL) synthesized by RhlI (17). The rhlAB operon encodes rhamnosyltransferase 1, which is responsible for monorhamnolipid production from TDP-L-rhamnose and ␤-hydroxy-fatty acids (15), and rhlC encodes rhamnosyltransferase 2, which produces dirhamnolipid by using monorhamnolipid and TDP-L-rhamnose as substrates (22). The model for the transcription activation of rhlAB and rhlC is that with increasing bacterial cell density the concentration of C 4 -HSL reaches a threshold level and then attaches to the transcription activator RhlR (16). The RhlR-C 4 -HSL complex activates the transcription of the rhlAB operon (18, 20) and rhlC (22).P. aeruginosa contains a second quorum-sensing regulon, consisting of the transcription regulator LasR, which is activated by the AI N-3-oxododecanoyl homoserine lactone (3-o-C 12 -HSL). The transcription of several genes encoding virulence-associated traits is activated by the Las system (8, 31), and it has a central role in the transcription of rhlR (12,20).The aim of this work was to determine whether the expression of the rhlAB operon is dependent only on the presence of RhlR and C 4 -HSL or whether other regulatory elements participate. To do this, we studied rhlAB expression along the growth curve of P. aeruginosa PAO1/pECP61.5 (rhlA::lacZ ptac-rhlR) (Table 1) adding C 4 -HSL and 0.1 mM isopropyl-␤-

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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