Title | An autoinducer-independent RhlR quorum-sensing receptor enables analysis of RhlR regulation. |
Publication Type | Journal Article |
Year of Publication | 2019 |
Authors | McCready, AR, Paczkowski, JE, Cong, J-P, Bassler, BL |
Journal | PLoS Pathog |
Volume | 15 |
Issue | 6 |
Pagination | e1007820 |
Date Published | 2019 06 |
ISSN | 1553-7374 |
Keywords | Amino Acid Substitution, Animals, Bacterial Proteins, Caenorhabditis elegans, Mutation, Missense, Pseudomonas aeruginosa, Pyocyanine, Quorum Sensing, Receptors, Cell Surface |
Abstract | <p>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.</p> |
DOI | 10.1371/journal.ppat.1007820 |
Alternate Journal | PLoS Pathog |
PubMed ID | 31194839 |
PubMed Central ID | PMC6564026 |
Grant List | R01 GM065859 / GM / NIGMS NIH HHS / United States R37 GM065859 / GM / NIGMS NIH HHS / United States T32 GM007388 / GM / NIGMS NIH HHS / United States / HHMI / Howard Hughes Medical Institute / United States |