The RhlR quorum-sensing receptor controls <i>Pseudomonas aeruginosa</i> pathogenesis and biofilm development independently of its canonical homoserine lactone autoinducer

<div><p>Quorum sensing (QS) is a bacterial cell-to-cell communication process that relies on the production, release, and response to extracellular signaling molecules called autoinducers. QS controls virulence and biofilm formation in the human pathogen <i>Pseudomonas aeruginosa</i>. <i>P</i>. <i>aeruginosa</i> possesses two canonical LuxI/R-type QS systems, LasI/R and RhlI/R, which produce and detect 3OC12-homoserine lactone and C4-homoserine lactone, respectively. Here, we use biofilm analyses, reporter assays, RNA-seq studies, and animal infection assays to show that RhlR directs both RhlI-dependent and RhlI-independent regulons. In the absence of RhlI, RhlR controls the expression of genes required for biofilm formation as well as genes encoding virulence factors. Consistent with these findings, Δ<i>rhlR</i> and Δ<i>rhlI</i> mutants have radically different biofilm phenotypes and the Δ<i>rhlI</i> mutant displays full virulence in animals whereas the Δ<i>rhlR</i> mutant is attenuated. The Δ<i>rhlI</i> mutant cell-free culture fluids contain an activity that stimulates RhlR-dependent gene expression. We propose a model in which RhlR responds to an alternative ligand, in addition to its canonical C4-homoserine lactone autoinducer. This alternate ligand promotes a RhlR-dependent transcriptional program in the absence of RhlI.</p>