An autoinducer-independent RhlR quorum-sensing receptor enables analysis of RhlR regulation

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. Pseudomonas aeruginosa, an opportunistic pathogen, employs multiple quorum-sensing systems to control behaviors including virulence factor production and biofilm formation. One P. aeruginosa quorum-sensing receptor, called RhlR, binds the cognate autoinducer N-butryl-homoserine lactone (C4HSL), and the RhlR:C4HSL complex activates transcription of target quorum-sensing genes. Here, we use a genetic screen to identify RhlR mutants that function independently of the autoinducer. The RhlR Y64F W68F V133F triple mutant, which we call RhlR*, exhibits ligand-independent activity in vitro and in vivo. RhlR* can drive wildtype biofilm formation and infection in a nematode animal model. The ability of RhlR* to properly regulate quorum-sensing-controlled genes in vivo depends on the quorum-sensing regulator RsaL keeping RhlR* activity in check. RhlR is known to function together with PqsE to control production of the virulence factor called pyocyanin. Likewise, RhlR* requires PqsE for pyocyanin production in planktonic cultures, however, PqsE is dispensable for RhlR*-driven pyocyanin production on surfaces. Finally, wildtype RhlR protein is not sufficiently stabilized by C4HSL to allow purification. However, wildtype RhlR can be stabilized by the synthetic ligand mBTL (meta-bromo-thiolactone) and RhlR* is stable without a ligand. These features enabled purification of the RhlR:mBTL complex and of RhlR* for in vitro examination of their biochemical activities. To our knowledge, this work reports the first RhlR protein purification. Author summary The human pathogen Pseudomonas aeruginosa uses a chemical communication process called quorum sensing to orchestrate group behaviors including virulence factor production and biofilm formation. Thus, quorum sensing is essential for P. aeruginosa to be a pathogen. Quorum sensing relies on the production, release, and detection of extracellular signal molecules called autoinducers. Autoinducers are bound by partner receptor proteins, and together, autoinducer-receptor complexes control gene expression. Here, we identify, purify, and characterize a mutant version of the P. aeruginosa RhlR quorum-sensing receptor that we call RhlR*, which, remarkably, does not require its partner autoinducer to function. We show that P. aeruginosa carrying RhlR* can properly form biofilms, produce virulence factors, and infect a nematode animal used as a model for pathogenesis. Because RhlR* does not rely on an autoinducer to function, biochemical and genetic analyses that were previously not possible with RhlR could be performed with RhlR*. Indeed, our studies of RhlR* provide new insight into the workings of other P. aeruginosa quorum-sensing components, most notably, the PqsE enzyme that functions together with RhlR to control virulence factor production. We propose that RhlR* is an especially valuable tool for learning about cell-cell communication and virulence in P. aeruginosa, a pathogen of high clinical relevance.