Date Published: June 13, 2019
Publisher: Public Library of Science
Author(s): Amelia R. McCready, Jon E. Paczkowski, Jian-Ping Cong, Bonnie L. Bassler, Vincent T. Lee.
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.
Quorum sensing is a process of intercellular communication that bacteria use to coordinate group behaviors [1–4]. Quorum sensing relies on the production, release, and group-wide detection of signaling molecules called autoinducers [5–7]. At low concentrations of autoinducer, bacteria act as individuals. At high concentrations of autoinducer, bacteria act as collectives, initiating behaviors that are beneficial when undertaken in unison by the group. Many species of Gram-negative bacteria use LuxR-type quorum-sensing receptors to orchestrate group behaviors [8–10]. LuxR-type receptors are transcription factors that, as they fold, typically bind to and are stabilized and activated by cognate homoserine lactone (HSL) autoinducers [6, 11]. The opportunistic pathogen Pseudomonas aeruginosa employs two LuxR-type quorum-sensing receptors, LasR and RhlR, that interact with the cognate autoinducers N-3-oxo-dodecanoyl-L-homoserine lactone (3OC12HSL) and N-butyryl-L-homoserine lactone (C4HSL), respectively [8, 10]. 3OC12HSL and C4HSL are produced by the LasI and RhlI synthases, respectively. LasR activates the genes encoding RhlR and RhlI, in addition to its own regulon, so the two quorum-sensing systems function in tandem [12, 13]. RhlR also responds to a second autoinducer, designated the “alternative autoinducer”, whose identity remains unknown . The alternative autoinducer is produced by PqsE , a thioesterase involved in alkylquinolone synthesis . When bound to either C4HSL or the alternative autoinducer, RhlR activates transcription of many genes, including those required for virulence factor production and biofilm formation [17, 18].
RhlR, a central component of the P. aeruginosa quorum-sensing system, controls many genes, including those required for biofilm formation and virulence factor production. Here, we report RhlR*, a constitutive RhlR allele that is stably produced and that functions without an agonist bound. There are dozens of studied LuxR-type receptors, of which RhlR is one. Almost all are unstable and inactive absent a ligand. We note that EsaR and a few other LuxR-type receptors are exceptions in that they operate by a mechanism distinct from the vast majority of LuxR-type receptors, RhlR included. Specifically, EsaR binds DNA and activates transcription when no ligand is bound and EsaR is inactive when bound to an autoinducer [43, 44]. Amongst the ligand-dependent LuxR-receptors, RhlR* represents a new type of mutant and its lack of ligand-dependence enables previously inaccessible possibilities for its study.