Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa

Abstract

A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhiR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.

Pseudomonas aeruginosa las and rhl quorum-sensing systems are important for infection and inflammation in a rat prostatitis model

“…In the las system, LasI synthesizes the AHL signal, N-3-oxo-dodecanoylhomoserine lactone, and LasR acts as the transcriptional regulator (Gambello & Iglewski, 1991;Passador et al, 1993;Pearson et al, 1994). For the rhl system, RhlI and RhlR are the synthase and transcriptional regulator, respectively, for the AHL signal, N-butyrylhomoserine lactone (Ochsner et al, 1994;Ochsner & Reiser, 1995;Pearson et al, 1995). Binding of the transcriptional regulators in both the las and rhl quorum-sensing systems induces or represses the expression of many genes, including virulence factors.…”

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

“…In P. aeruginosa, QS has been proposed as a global mechanism for controlling virulence factor expression and the development of biofilms (Davies et al, 1998). Several genes are regulated by QS, including toxA (exotoxin A; Gambello et al, 1993), lasB (elastase; Brint & Ohman, 1995;Latifi et al, 1995;Passador et al, 1993;Pearson et al, 1997) and lasA (LasA protease; Toder et al, 1991), aprA (alkaline metalloproteinase; Gambello et al, 1993;Latifi et al, 1995), rhlAB, encoding proteins involved in the synthesis of rhamnolipid (Ochsner et al, 1994;Pearson et al, 1997), katA and sodA (catalase and superoxide dismutase; Hassett et al, 1999), lecA (lectin; Winzer et al, 2000) and genes involved in pyocyanin formation (Brint & Ohman, 1995;Latifi et al, 1995) and twitching motility (Glessner et al, 1999). However, studies of QS-regulated gene expression, utilizing las and/or rhl mutants, may be inaccurate due to the accumulation of secondary mutations in global regulators such as vfr and algR that are directly responsible for some of these phenotypes (e.g.…”

Control of cell division in Escherichia coli: regulation of transcription of ftsQA involves both rpoS and SdiA-mediated autoinduction.

“…The autoinduction mechanism in V. fischeri is mediated through a transcriptional activator protein, LuxR, and LuxI, which produces autoinducer from cytoplasmic components (6). There are at least eight other proteins that show extensive similarity to LuxR: SdiA from Escherichia coli (12,13), LasR and RhlR from Pseudomonas aeruginosa (1,14), the nopaline-and octopine-type TraR proteins from Agrobacterium tumefaciens (4,15), RhiR from Rhizobium leguminosarum (16), ExpR from Erwinia carotovora (5), and PhzR from Pseudomonas aureofaciens (3). The structures of seven autoinducers have been determined: V. fischeri autoinducer, N-(3-oxohexanoyl)-Lhomoserine lactone (9); V. harveyi autoinducer, N-(3-hydroxybutanoyl)-L-homoserine lactone (17); A. tumefaciens autoinducer, N-(3-oxooctanoyl)-L-homoserine lactone (18); P. aeruginosa autoinducer, N-(3-oxododecanoyl)-L-homoserine lactone (19); P. aeruginosa factor 2, N-(3-butyryl)-L-homoserine lactone (20); and two other autoinducers produced by V. fischeri-the luxI-independent compound N-octanoyl-Lhomoserine lactone (AI-2), and N-hexanoyl-L-homoserine lactone (AI-3) which is dependent on luxI for its synthesis (21,22).…”