Autoinducer-Independent Control of Quorum Sensing by RhlR in Pseudomonas aeruginosa Biofilms

Abstract

Pseudomonas aeruginosa is a potent human pathogen that infects host tissues by forming multicellular communities called biofilms. Quorum sensing (QS) is a mechanism of bacterial cell-cell communication that relies on the production, release, and group-wide detection of extracellular signaling molecules called autoinducers (AIs). The P. aeruginosa QS circuit consists of two AI synthase/receptor pairs, LasI/R and RhlI/R, which produce and detect the homoserine lactones (HSLs), 3OC12-HSL and C4-HSL, respectively. At high cell density, LasR and RhlR bind their cognate AIs, dimerize, bind DNA, and activate expression of genes required for virulence and biofilm formation. While wild-type P. aeruginosa forms rugose-center, smooth-periphery colony biofilms on Congo red semi-solid agar, here, it is shown that ΔrhlI and ΔrhlR mutants form smooth and hyper-rugose colony biofilms, respectively. One would expect ΔrhlI and ΔrhlR mutants to have identical biofilm phenotypes, since there is an obligate relationship between QS AIs and receptors. The hypothesis examined in this thesis is that RhlR binds to an alternate ligand in the absence of its cognate AI C4-HSL to direct a RhlI-independent regulon. Tn5 transposon mutagenesis in the ΔrhlI mutant was used to identify gene(s) for an alternate ligand synthase. Biofilm analyses were used in a genetic screen for mutants that form hyper-rugose colony biofilms in a ΔrhlI mutant background. Reporter assays were employed in a screen in the ΔrhlI strain to identify mutants with altered expression of rhlA, a RhlR-dependent QS gene. In parallel, a short flexible region in RhlR was probed with epitope tags and single alanine point mutations to garner evidence for an alternate ligand binding pocket. Together, these findings provided insight into how RhlR controls P. aeruginosa biofilm formation, most notably, in the absence of the RhlI ligand. The work also identified potential targets for novel therapeutic compounds that inhibit biofilm formation.