ROLES OF PSEUDOMONAS-AERUGINOSA LAS AND RHL QUORUM-SENSING SYSTEMS INCONTROL OF ELASTASE AND RHAMNOLIPID BIOSYNTHESIS GENES

Two quorum-sensing systems (las and rhl) regulate virulence gene expre ssion in Psendomonas aeraginosa. The las system consists of a transcri ptional activator, LasR and LasI, which directs the synthesis of the a utoinducer N-(3-oxododecanoyl) homoserine lactone (PAI-1). Induction o f lasB (encoding elastase) and other virulence genes requires LasR and PAI-1. The rhl system consists of a putative transcriptional activato r, RhlR, and RhlI, which directs the synthesis of N-butyryl homoserine lactone (PAI-2). Rhamnolipid production in P. aeruginosa has been rep orted to require both the rhl system and rhlAB (encoding a rhamnosyltr ansferase), Here we report the generation of a Delta lasI mutant and b oth Delta lasI Delta rhlI and Delta lasR rhlR::Tn501 double mutants of strain PAO1. Rhamnolipid production and elastolysis were reduced in t he Delta lasI single mutant and abolished in the double-mutant strains . rhlAB mRNA,vas not detected in these strains at mid-logarithmic phas e but was abundant in the parental strain. Further RNA analysis of the wild-type strain revealed that rhlAB is organized as an operon. The r hlAB transcriptional start was mapped, and putative sigma(54) and sigm a(70) promoters were identified upstream, To define components require d for rhlAB expression, we developed a bioassay in Escherichia coli an d demonstrated that PAI-2 and RhlR are required and sufficient for exp ression of rhlA. To characterize the putative interaction between PAI- 2 and RhlR, we demonstrated that [H-3]PAI-2 binds to E. coil cells exp ressing RhlR and not to those expressing LasR, Finally, the specificit y of the las and rhl systems was examined in E. coil bioassays. The In s system was capable of mildly activating rhlA, and similarly, the rhl system partly activated lasB. However, these effects were much less t han the activation of rhlA by the rhl system and lasB by the [as syste m, The results presented here further characterize the roles of the rh l and las quorum-sensing systems in virulence gene expression.