Abstract
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.
Highlights
Quorum sensing is a process of intercellular communication that bacteria use to coordinate group behaviors [1,2,3,4]
The opportunistic pathogen Pseudomonas aeruginosa employs two LuxR-type quorum-sensing receptors, LasR and RhlR, that interact with the cognate autoinducers N3-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
We used our previously reported E. coli reporter assay that harbors rhlR on one plasmid, and a RhlR-activated prhlA-lux transcriptional fusion on a second plasmid [26]. rhlA encodes a rhamnolipid biosynthetic enzyme required for virulence [27, 28]
Summary
Quorum sensing is a process of intercellular communication that bacteria use to coordinate group behaviors [1,2,3,4]. 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 [8,9,10]. 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 N3-oxo-dodecanoyl-L-homoserine lactone (3OC12HSL) and N-butyryl-L-homoserine lactone (C4HSL), respectively [8, 10]. 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]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
