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

   McCready, A. R.; Paczkowski, J. E.; Cong, J-P.; Bassler, B. L. (July 2019, PLOS pathogens)

   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. 

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2. The PqsE-RhlR Interaction Regulates RhlR DNA Binding to Control Virulence Factor Production in Pseudomonas aeruginosa

   https://doi.org/10.1128/spectrum.02108-21  

   Simanek, Kayla A.; Taylor, Isabelle R.; Richael, Erica K.; Lasek-Nesselquist, Erica; Bassler, Bonnie L.; Paczkowski, Jon E. (February 2022, Microbiology Spectrum)

   LaRock, Christopher N. (Ed.)

   ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes disease in immunocompromised individuals and individuals with underlying pulmonary disorders. P. aeruginosa virulence is controlled by quorum sensing (QS), a bacterial cell-cell communication mechanism that underpins transitions between individual and group behaviors. In P. aeruginosa , the PqsE enzyme and the QS receptor RhlR directly interact to control the expression of genes involved in virulence. Here, we show that three surface-exposed arginine residues on PqsE comprise the site required for interaction with RhlR. We show that a noninteracting PqsE variant [PqsE(NI)] possesses catalytic activity, but is incapable of promoting virulence phenotypes, indicating that interaction with RhlR, and not catalysis, drives these PqsE-dependent behaviors. Biochemical characterization of the PqsE-RhlR interaction coupled with RNA-seq analyses demonstrates that the PqsE-RhlR complex increases the affinity of RhlR for DNA, enabling enhanced expression of genes encoding key virulence factors. These findings provide the mechanism for PqsE-dependent regulation of RhlR and identify a unique regulatory feature of P. aeruginosa QS and its connection to virulence. IMPORTANCE Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI). QS is required for virulence in the human pathogen Pseudomonas aeruginosa , which can cause fatal infections in patients with underlying pulmonary disorders. In this study, we determine the molecular basis for the physical interaction between two virulence-driving QS components, PqsE and RhlR. We find that the ability of PqsE to bind RhlR correlates with virulence factor production. Since current antimicrobial therapies exacerbate the growing antibiotic resistance problem because they target bacterial growth, we suggest that the PqsE-RhlR interface discovered here represents a new candidate for targeting with small molecule inhibition. Therapeutics that disrupt the PqsE-RhlR interaction should suppress virulence. Targeting bacterial behaviors such as QS, rather than bacterial growth, represents an attractive alternative for exploration because such therapies could potentially minimize the development of resistance. 

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3. Structure of the RhlR-PqsE complex from Pseudomonas aeruginosa reveals mechanistic insights into quorum-sensing gene regulation

   https://doi.org/10.1016/j.str.2022.10.008  

   Feathers, J. Ryan; Richael, Erica K.; Simanek, Kayla A.; Fromme, J. Christopher; Paczkowski, Jon E. (December 2022, Structure)
4. The PqsE and RhlR proteins are an autoinducer synthase–receptor pair that control virulence and biofilm development in Pseudomonas aeruginosa

   https://doi.org/10.1073/pnas.1814023115  

   Mukherjee, Sampriti; Moustafa, Dina A.; Stergioula, Vasiliki; Smith, Chari D.; Goldberg, Joanna B.; Bassler, Bonnie L. (September 2018, Proceedings of the National Academy of Sciences)
5. N -Acyl l -Homocysteine Thiolactones Are Potent and Stable Synthetic Modulators of the RhlR Quorum Sensing Receptor in Pseudomonas aeruginosa

   https://doi.org/10.1021/acschembio.8b01079  

   Boursier, Michelle E.; Combs, Joshua B.; Blackwell, Helen E. (January 2019, ACS Chemical Biology)