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 σS

Abstract

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

The effect of specificrhlA-las-box mutations on DNA binding and gene activation byPseudomonas aeruginosaquorum-sensing transcriptional regulators RhlR and LasR

“…In addition, we found that the rhlA‐ las‐box mutant C12T is no longer bound by RhlR in the absence of C4‐HSL. This is similar to the way that RhlR interacts with the lecA‐ las‐box (Table ), but differs with the reported ability of this protein to interact with the rhlA‐ and rmlB‐ las‐boxes in the absence of this autoinducer (Medina et al ., ; Aguirre‐Ramírez et al ., ). As both the rhlA‐ and the rmlB‐ las‐boxes contain a C in position 12, it is possible that RhlR without autoinducer only binds to las‐boxes that contain a C in position 12, and not to those containing a T in this position, while RhlR/C4‐HSL can interact with las‐boxes containing either of these nucleotides in position 12.…”

“…The 5′ untranslated region (5′‐UTR) of the rmlBDAC operon that encodes the enzymes that convert glucose‐1‐phosphate to TDP‐L‐rhamnose (the substrate for the synthesis of rhamnolipids) contains a sequence where RhlR binds specifically (rhl‐box), both in the presence and in the absence of C4‐HSL; this rhl‐box has the sequence: AC CT ACCA G ATCTGGGGTTG (Aguirre‐Ramírez et al ., ). Thus, the rmlB‐ rhl‐box lacks the A at position 17 and the G at position 18 that was proposed to be invariable for las‐boxes (Whiteley & Greenberg, ).…”

The OpdQ porin of Pseudomonas aeruginosa is regulated by environmental signals associated with cystic fibrosis including nitrate‐induced regulation involving the NarXL two‐component system

“…; Aguirre‐Ramirez et al. ). These sites could serve as recognition sequences for effectors that downregulate opdQ transcription during hypoxic growth.…”

Semi‐rational evolution of the 3‐(3‐hydroxyalkanoyloxy)alkanoate (HAA) synthase RhlA to improve rhamnolipid production in Pseudomonas aeruginosa and Burkholderia glumae

“…This is attributed to the complexity of the genetically regulated pathways controlling in vivo RL production and/or the inability of efficiently controlling heterologous gene expression leading to metabolic perturbations. In fact, expression of rmlBDAC , rhlAB and rhlC in P. aeruginosa is mainly regulated by the rhl quorum sensing system , which in turn is part of a complex genetic regulation cascade . According to a previous report , RhlA is overexpressed in cultures optimized for the overproduction of RLs, suggesting that RhlA acts as a key enzyme providing the lipidic flux toward the RL biosynthesis pathway.…”