Quorum sensing enables bacteria to coordinate gene expression in response to population density through the detection of small-molecule signals. In gram-negative bacteria, LuxR-type transcription factors bind acyl-homoserine lactones to regulate collective behaviors, yet how ligand sensitivity is tuned to shape transcriptional outcomes remains poorly defined. In Pseudomonas aeruginosa, the quorum-sensing receptor RhlR responds to N-butyryl-L-homoserine lactone (C4HSL) and controls late-stage quorum-sensing behaviors, including phenazine biosynthesis. Here, we use a chemical-genetic and structure-guided mutational approach to define how RhlR ligand sensitivity regulates promoter-specific transcription. We identify substitutions within the RhlR ligand-binding pocket that enhance sensitivity to C4HSL without altering ligand specificity, generating hypersensitive receptor variants. Increased ligand sensitivity selectively represses phenazine biosynthetic gene expression, reduces pyocyanin production, and alters phenazine output, while leaving other RhlR-dependent quorum-sensing traits unaffected. Transcriptomic and chromatin immunoprecipitation analyses reveal that these effects arise from reduced expression of the RhlR co-regulator PqsE, leading to decreased RhlR occupancy at phenazine gene promoters. These findings support a coincidence-detection mechanism in which ligand sensing and co-regulator availability jointly determine transcriptional output. Together, our results demonstrate that ligand sensitivity is a critical regulatory determinant that tunes quorum-sensing gene expression. This work reveals how changes in signal detection can reshape transcriptional hierarchies and metabolic outputs, providing insight into the fine control of bacterial collective behaviors and virulence-associated metabolism.IMPORTANCEQuorum sensing is often described as an on/off regulatory switch, yet many bacterial behaviors require more nuanced control. This study shows that the sensitivity of a quorum-sensing receptor to its signal molecule is a key regulatory parameter that shapes downstream gene expression. By tuning the ligand sensitivity of the Pseudomonas aeruginosa quorum-sensing regulator RhlR, we demonstrate that changes in signal detection selectively reprogram quorum-sensing transcriptional outputs, particularly for phenazine biosynthesis, without broadly disrupting quorum-sensing functions. Our findings reveal how ligand sensing is integrated with accessory regulatory factors to control promoter selection and metabolic outcomes. This work highlights signal sensitivity as an important mechanism by which bacteria fine-tune collective behaviors and virulence-associated metabolism.

Quorum sensing (QS) enables Pseudomonas aeruginosa to coordinate virulence and biofilm formation through cell density-dependent signaling. In clinical isolates from patients with cystic fibrosis (pwCF), mutations in canonical QS systems such as lasR and rhlI often lead to altered signaling hierarchies that complicate our understanding of QS regulation during chronic infection. Here, we dissect the relative contributions of the autoinducer N-butyryl-L-homoserine lactone (C4HSL) and the protein binding partner PqsE to RhlR-dependent transcription in CF clinical isolates. Using site-directed mutagenesis to generate RhlR and PqsE variants incapable of responding to C4HSL (RhlR A44M) or dimerizing to interact with RhlR (PqsENI), we show that both inputs are essential for the full expression of QS-regulated virulence factors, including pyocyanin and rhamnolipids. Transcriptomic analyses revealed that C4HSL and PqsE co-regulate a conserved set of 28 RhlR-dependent genes, encompassing canonical virulence loci as well as uncharacterized genes that are likely important for adaptation to the CF airway environment. These findings establish that clinical isolates maintain functional QS circuitry reliant on dual activation of RhlR by both C4HSL and PqsE, revealing a conserved regulatory module that underpins pathogenic behavior across genetically diverse isolates.

In Vibrio species, quorum sensing signaling culminates in the production of the master transcription factor SmcR that regulates group behavior genes in a density-dependent manner. Previously, we identified a small-molecule thiophenesulfonamide inhibitor called PTSP [3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole] that targets the SmcR family of proteins in multiple Vibrio species and blocks activity in vivo. Here, we used structure-function analyses to identify eight PTSP-interacting residues in the ligand-binding pocket that are required for PTSP inhibition of Vibrio vulnificus SmcR. Binding of PTSP to SmcR drives allosteric unfolding of the N-terminal DNA-binding domain, and, in this state, SmcR is specifically degraded by the ClpAP protease. This mechanism of PTSP inhibition was observed for all thiophenesulfonamide compounds tested against V. vulnificus as well as Vibrio parahaemolyticus and Vibrio campbellii. We show that Vibrio cells expressing degradation-resistant smcR alleles are impervious to changes in cell density state. These studies implicate ligand binding as a mediator of SmcR protein stability and function, which dictates the timing of quorum-sensing gene expression in three Vibrio pathogens.IMPORTANCESmcR family proteins were discovered in the 1990s as central regulators of quorum-sensing gene expression and later discovered to be conserved in all studied Vibrio species. SmcR homologs regulate a wide range of genes involved in pathogenesis, including but not limited to genes involved in biofilm production and toxin secretion. As archetypal members of the broad class of TetR-type transcription factors, each SmcR-type protein has a predicted ligand-binding pocket. However, no native ligand has been identified for these proteins that control their function as regulators. Here, we used SmcR-specific chemical inhibitors to determine that ligand binding drives proteolytic degradation in vivo, providing the first demonstration of SmcR function connected to ligand binding for this historical protein family.

Quorum sensing (QS) enables bacteria to coordinate gene expression in response to population density, with LuxR-type transcription factors playing a central role in this process for many Gram-negative species. Traditionally understood as ligand-activated transcriptional regulators, LuxR-type proteins are increasingly recognized as targets of diverse protein-protein interactions (PPIs) that modulate their activity, stability, and specificity. This review synthesizes emerging insights into the regulatory landscape of LuxR-type receptors, focusing on direct PPIs that expand the functional repertoire of LuxR-type receptors. We classify LuxR-interacting partners into negative regulators, dual or context-dependent regulators, and global regulators, highlighting the well-characterized system of PqsE-RhlR in Pseudomonas aeruginosa as well as emerging candidates. We discuss how these interactions influence LuxR-type receptor outputs beyond canonical ligand-mediated gene regulation, through stabilization of active conformations, inhibition of dimerization, proteolytic regulation, and potential recruitment of transcriptional machinery. By examining functional parallels, structural determinants, and environmental integration, we highlight a framework in which LuxR-type receptors are subject to diverse regulatory PPI that allow for the fine-tuning of QS output. Understanding these regulatory mechanisms offers promising alternatives to conventional QS disruption strategies, for targeted interference with bacterial virulence and communication.

Abstract Pseudomonas aeruginosa is an opportunistic human pathogen that is hospital-endemic, forming biofilms on medical equipment and causing thousands of hospital-acquired infections each year. The success of P. aeruginosa as an opportunistic pathogen is linked to its phenotypic and genotypic adaptability. Relatedly, P. aeruginosa forms a small colony subpopulation in response to stresses like oxygen limitation and antibiotic exposure. Additionally, P. aeruginosa coordinates population-level decisions using a mechanism of cell-cell communication called quorum sensing. P. aeruginosa uses these signaling pathways to control virulence factor production and biofilm formation in the host. We show that certain quorum-sensing mutations promote phenotypic variation; specifically, deletion of lasR and autoinducer modulating mutations in rhlI enhanced small colony formation in a time course-dependent manner. Using transcriptome analyses of isogenic small and large colony variants, we show that small colony formation is driven in part by the PhoPQ two-component signal transduction system in quorum-sensing mutant backgrounds. Specifically, our data show that unphosphorylated PhoP represses rhlR gene expression, and that subsequent de-repression of quorum sensing contributes to the production of virulence factors and the small colony phenotype. In total, these findings provide insight on how mutations evolved by clinical strains might serve as a bet-hedging strategy to promote the formation of a small colony phenotype and alter quorum-sensing signaling within a subpopulation of a community.

Bacteria control individualistic and group behaviors using a form of cell-cell communication called quorum sensing. Quorum sensing relies on the production of chemical signals called autoinducers and the subsequent detection of those signals by a cognate receptor. Many Gram-negative bacteria use the LuxR-type family of transcription factor receptors that bind to acyl-homoserine lactone autoinducer signals to regulate their function as DNA-binding proteins. A subclass of this family of transcription factor receptors requires their cognate autoinducer to fold and dimerize to bind DNA to regulate gene expression and, thus, traits associated with quorum sensing, such as biofilm formation and virulence factor production. Here, we use a chemical-genetic approach to determine the structural basis for ligand selection by the quorum-sensing receptor RhlR from Pseudomonas aeruginosa . The native ligand for RhlR is N -butyryl-L-homoserine lactone, and this protein-ligand interaction is important for initiating gene expression in P. aeruginosa . We determine key residues that drive ligand specificity and selectivity of RhlR to define the role of ligand-driven RhlR-dependent gene regulation of quorum-sensing traits, namely the differential expression of the phenazine genes, which encode the enzymes responsible for the synthesis of the redox-sensitive virulence factor pyocyanin, among other phenazines. Furthermore, we provide a chemical-genetic framework for future studies aimed at disrupting the RhlR-ligand interaction to suppress virulence in P. aeruginosa , an important nosocomial pathogen with widespread antimicrobial resistance.

In Vibrio species, quorum sensing signaling culminates in the production of the master transcription factor SmcR that regulates group behavior genes in a density-dependent manner. Previously, we identified a small molecule thiophenesulfonamide inhibitor called PTSP that targets the SmcR family of proteins and blocks activity in vivo . Here, we used structure-function analyses to identify eight PTSP-interacting residues in the ligand binding pocket that are required for PTSP inhibition of Vibrio vulnificus SmcR. Binding of PTSP to SmcR drives allosteric unfolding of the N-terminal DNA-binding domain and, in this state, SmcR is degraded by the ClpAP protease. SmcR degradation controls the timing of the phenotypic switch between high and low cell density, and strains expressing degradation-resistant smcR alleles are impervious to changes in cell density state. These studies implicate ligand binding as a mediator of SmcR protein stability and function, which dictates the timing of quorum sensing gene expression in three Vibrio pathogens. Significance Statement:SmcR family proteins were discovered in the 1990s as central regulators of quorum sensing gene expression and later discovered to be conserved in all studied Vibrio species. SmcR homologs regulate a wide range of genes involved in pathogenesis, including but not limited to genes involved in biofilm production and toxin secretion. As archetypal members of the broad class of TetR-type transcription factors, each SmcR type protein has a predicted ligand binding pocket. However, no ligand has been identified for these proteins that control their function as regulators. Here, we used SmcR-specific chemical inhibitors to determine that ligand binding drives proteolytic degradation in vivo , the first demonstration of SmcR function connected to ligand binding for this historical protein family.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that poses a significant public health threat, particularly in healthcare settings. A key determinant of P. aeruginosa virulence is the regulated synthesis and release of extracellular products, which is controlled by a cell density-dependent signaling system known as quorum sensing (QS). P. aeruginosa uses a complex QS network, including two systems that rely on diffusible N-acylhomoserine lactone (AHL) signal molecules. The LuxR-type receptor RhlR is unique in that it requires not only its cognate AHL but also the accessory protein PqsE to maximally bind to promoter DNA and initiate transcription. Our group previously demonstrated that PqsE physically interacts with RhlR, enhancing its affinity for target promoters across the P. aeruginosa genome. Although LuxR-type receptors are widespread in Gram-negative bacteria and important for pathogenesis, PqsE orthologs are restricted to Pseudomonas and Burkholderia species. This study explored the conservation of PqsE and examined PqsE ortholog structure-function across different species. Our results show that PqsE in Pseudomonas retains their functional interactions with RhlR homologs, unlike PqsE orthologs in Burkholderia spp., which do not interact with their respective LuxR-type receptors. Additionally, we assessed the AHL preferences of different receptors and hypothesized that the PqsE-RhlR interaction evolved to stabilize the inherently unstable RhlR, preventing its degradation. Indeed, we observe higher levels of RhlR protein turnover in a strain lacking pqsE compared to a wild-type strain of PA14, which can be partially rescued in a strain of P. aeruginosa lacking the Lon protease. IMPORTANCE:Pseudomonas aeruginosa, a major pathogen for patients with cystic fibrosis and a primary constituent of healthcare-associated infections, relies on a complex quorum-sensing (QS) network to coordinate virulence factor production. Central to this system is the interaction between two proteins, PqsE and RhlR, which drive gene expression essential for pathogenesis. Our study investigates the conservation of the PqsE-RhlR interaction across related bacterial species, revealing that PqsE in Pseudomonas can enhance RhlR activity, while orthologs in Burkholderia lack this capacity. These findings offer new insights into the specificity and evolution of QS mechanisms, highlighting the PqsE-RhlR interaction as a potentially selective target for treating P. aeruginosa infections.

ABSTRACT The bacterium Pseudomonas aeruginosa is an opportunistic pathogen that can cause lung, skin, wound, joint, urinary tract, and eye infections. While P. aeruginosa is known to exhibit a robust competitive response toward other bacterial species, this bacterium is frequently identified in polymicrobial infections where multiple species survive. For example, in prosthetic joint infections, P. aeruginosa can be identified along with other pathogenic bacteria including Staphylococcus aureus , Enterococcus faecalis , and Corynebacterium striatum . Here, we have explored the survival and behavior of such microbes and find that E. faecalis readily survives culturing with P. aeruginosa while other tested species do not. In each of the tested conditions, E. faecalis growth remained unchanged by the presence of P. aeruginosa , indicating a unique mutualistic interaction between the two species. We find that E. faecalis proximity leads P. aeruginosa to attenuate competitive behaviors as exemplified by reduced production of Pseudomonas quinolone signal and pyocyanin. Reduced alkyl quinolones are important to E. faecalis as these will grow in supernatant from a quinolone mutant but not P. aeruginosa wild-type in planktonic culture. The reduced pyocyanin production of P. aeruginosa is attributable to production of ornithine by E. faecalis , which we recapitulate by adding exogenous ornithine to P. aeruginosa monocultures. Similarly, co-culture with an ornithine-deficient strain of E. faecalis leads P. aeruginosa to yield near monoculture amounts of pyocyanin. Here, we directly demonstrate how notorious pathogens such as P. aeruginosa might persist in polymicrobial infections under the influence of metabolites produced by other bacterial species. IMPORTANCE While we now appreciate that many infections are polymicrobial, we understand little of the specific actions between a given set of microbes to enable combinatorial survival and pathogenesis. The bacteria Pseudomonas aeruginosa and Enterococcus faecalis are both prevalent pathogens in wound, urinary tract, and bacteremic infections. While P. aeruginosa often kills other species in standard laboratory culture conditions, we present here that E. faecalis can be reliably co-cultured with P. aeruginosa . We specifically detail that ornithine produced by E. faecalis reduces the Pseudomonas quinolone signal response of P. aeruginosa . This reduction of the Pseudomonas quinolone signal response aids E. faecalis growth.

Quorum sensing is a mechanism of bacterial cell-cell communication that relies on the production and detection of small molecule autoinducers, which facilitate the synchronous expression of genes involved in group behaviors, such as virulence factor production and biofilm formation. The Pseudomonas aeruginosa quorum sensing network consists of multiple interconnected transcriptional regulators, with the transcription factor, RhlR, acting as one of the main drivers of quorum sensing behaviors. RhlR is a LuxR-type transcription factor that regulates its target genes when bound to its cognate autoinducer, C4-homoserine lactone, which is synthesized by RhlI. RhlR function is also regulated by the metallo-β-hydrolase enzyme, PqsE. We recently showed that PqsE binds RhlR to alter its affinity for promoter DNA, a new mechanism of quorum-sensing receptor activation. Here, we perform ChIP-seq analyses of RhlR to map the binding of RhlR across the P. aeruginosa genome, and to determine the impact of C4-homoserine lactone and PqsE on RhlR binding to different sites across the P. aeruginosa genome. We identify 40 RhlR binding sites, all but three of which are associated with genes known to be regulated by RhlR. C4-homoserine lactone is required for maximal binding of RhlR to many of its DNA sites. Moreover, C4-homoserine lactone is required for maximal RhlR-dependent transcription activation from all sites, regardless of whether it impacts RhlR binding to DNA. PqsE is required for maximal binding of RhlR to many DNA sites, with similar effects on RhlR-dependent transcription activation from those sites. However, the effects of PqsE on RhlR specificity are distinct from those of C4-homoserine lactone, and PqsE is sufficient for RhlR binding to some DNA sites in the absence of C4-homoserine lactone. Together, C4-homoserine lactone and PqsE are required for RhlR binding at the large majority of its DNA sites. Thus, our work reveals three distinct modes of activation by RhlR: i) when RhlR is unbound by autoinducer but bound by PqsE, ii) when RhlR is bound by autoinducer but not bound by PqsE, and iii) when RhlR is bound by both autoinducer and PqsE, establishing a stepwise mechanism for the progression of the RhlR-RhlI-PqsE quorum sensing pathway in P. aeruginosa.

Quorum sensing is a mechanism of bacterial communication that controls virulence gene expression. Pseudomonas aeruginosa regulates virulence via two synthase/transcription factor receptor pairs: LasI/R and RhlI/R. LasR is considered the master transcriptional regulator of quorum sensing, as it upregulates rhlI/R. However, clinical isolates often have inactivating mutations in lasR, while maintaining Rhl-dependent signaling. We sought to understand how quorum sensing progresses in isolates with lasR mutations, specifically via activation of RhlR. We find that clinical isolates with lasR inactivating mutations often harbor concurrent mutations in rhlI. Using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry, we discover that strains lacking lasR overproduce the RhlI-synthesized autoinducer and that RhlI variants re-calibrate autoinducer concentrations to wild-type levels, restoring virulent phenotypes. These findings provide a mechanism for the plasticity of quorum sensing progression in an acute infection niche.

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).

Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of extracellular signal molecules called autoinducers (AI). Quorum sensing is required for virulence and biofilm formation in the human pathogen Pseudomonas aeruginosa. In P. aeruginosa, LasR and RhlR are homologous LuxR-type soluble transcription factor receptors that bind their cognate AIs and activate the expression of genes encoding functions required for virulence and biofilm formation. While some bacterial signal transduction pathways follow a linear circuit, as phosphoryl groups are passed from one carrier protein to another ultimately resulting in up- or down-regulation of target genes, the QS system in P. aeruginosa is a dense network of receptors and regulators with interconnecting regulatory systems and outputs. Once activated, it is not understood how LasR and RhlR establish their signaling hierarchy, nor is it clear how these pathway connections are regulated, resulting in chronic infection. Here, we reviewed the mechanisms of QS progression as it relates to bacterial pathogenesis and antimicrobial resistance and tolerance.

Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for thousands of deaths every year in the United States. P. aeruginosa virulence factor production is mediated by quorum sensing, a mech-anism of bacterial cell-cell communication that relies on the production and detection of signal molecules called autoinducers. In P. aeruginosa, the transcription factor receptor RhlR is activated by a RhlI-synthe-sized autoinducer. We recently showed that RhlR-dependent transcription is enhanced by a physical inter-action with the enzyme PqsE via increased affinity of RhlR for promoter DNA. However, the molecular basis for complex formation and how complex formation enhanced RhlR transcriptional activity remained unclear. Here, we report the structure of ligand-bound RhlR in complex with PqsE. Additionally, we determined the structure of the complex bound with DNA, revealing the mechanism by which RhlR-mediated transcription is enhanced by PqsE, thereby establishing the molecular basis for RhlR-dependent virulence factor produc-tion in P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for thousands of deaths every year in the United States. P. aeruginosa virulence factor production is mediated by quorum sensing, a mechanism of bacterial cell-cell communication that relies on the production and detection of signal molecules called autoinducers. In P. aeruginosa, the transcription factor receptor RhlR is activated by a RhlI-synthesized autoinducer. We recently showed that RhlR-dependent transcription is enhanced by a physical interaction with the thioesterase enzyme PqsE via increased affinity of RhlR for promoter DNA. However, the molecular basis for complex formation and how complex formation enhanced RhlR transcriptional activity remained unclear. Here, we report the structure of ligand bound RhlR in complex with PqsE. Additionally, we determined the structure of the complex bound with DNA, revealing the mechanism by which RhlR mediated transcription is enhanced by PqsE, thereby establishing the molecular basis for RhlR-dependent virulence factor production in P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes fatal infections. There exists an urgent need for new antimicrobial agents to combat P. aeruginosa. We conducted a screen for molecules that bind the virulence-controlling protein PqsE and characterized hit compounds for inhibition of PqsE enzymatic activity. The binding conformations of two inhibitory molecules, BB391 and BB393, were identified by crystallography, and inhibitor binding was mimicked by the substitution of PqsE residues E182 and S285 with tryptophan. Comparison of the inhibitor-mimetic mutations to the catalytically inactive PqsE D73A protein demonstrated that catalysis is not responsible for the role PqsE plays in driving virulence factor production. Rather, the PqsE E182W protein fails to interact with the quorum-sensing receptor, RhlR, and our results suggest that it is this interaction that is responsible for promoting virulence factor production in P. aeruginosa. These findings provide a new route for drug discovery efforts targeting PqsE.

Quorum sensing is a bacterial communication process whereby bacteria produce, release, and detect extracellular signaling molecules called autoinducers to coordinate collective behaviors. In the pathogen Vibrio cholerae, the quorum-sensing autoinducer 3,5-dimethyl-pyrazin-2-ol (DPO) binds the receptor and transcription factor VqmA. The DPO-VqmA complex activates transcription of vqmR, encoding the VqmR small RNA, which represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA is soluble and properly folded and activates basal-level transcription of its target vqmR in the absence of DPO. VqmA transcriptional activity is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae quorum-sensing transition at high cell densities. We solved the DPO-VqmA crystal structure to 2.0 Å resolution and compared it with existing structures to understand the conformational changes VqmA undergoes upon DNA binding. Analysis of DPO analogs showed that a hydroxyl or carbonyl group at the 2'-position is critical for binding to VqmA. The proposed DPO precursor, a linear molecule, N-alanyl-aminoacetone (Ala-AA), also bound and activated VqmA. Results from site-directed mutagenesis and competitive ligand-binding analyses revealed that DPO and Ala-AA occupy the same binding site. In summary, our structure-function analysis identifies key features required for VqmA activation and DNA binding and establishes that, whereas VqmA binds two different ligands, VqmA does not require a bound ligand for folding or basal transcriptional activity. However, bound ligand is required for maximal activity.

Bacteria use a cell cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150 degrees. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and A1a127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors.

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 (QS) is a cell-cell communication process that bacteria use to orchestrate group behaviors. QS is mediated by signal molecules called autoinducers. The major human pathogen Vibrio cholerae possesses multiple, converging quorum-sensing pathways. At high cell density, when autoinducer concentrations are high, biofilm formation and virulence are repressed. V. cholerae uses two transmembrane histidine sensor kinase quorum-sensing receptors called CqsS and LuxPQ, which respond to the autoinducers called CAI-1 and AI-2, respectively. A new third QS pathway functions in parallel with these two systems. This new QS circuit is composed of a transcription factor receptor, VqmA, which activates the expression of a gene encoding a small regulatory RNA called VqmR. VqmR represses biofilm and pathogenesis genes. The autoinducer that drives VqmA activity is 3,5-dimethylpyrazin-2-ol (DPO), which is synthesized from threonine and alanine. The requirement for threonine is particularly interesting because mucin, found in the gut of the human host, is especially rich in threonine, and DPO can be made from mucin, suggesting a link between the eukaryotic niche of pathogenic V. cholerae and activation of VqmA. Unlike other cytoplasmic quorum-sensing receptors, VqmA can function without its autoinducer, albeit with reduced efficacy. To understand the structural basis underpinning DPO-enhancement of VqmA activity, we determined the crystal structure of full-length VqmA bound to DPO. The VqmA N-terminal ligand-binding region contains a PAS domain and represents the dimerization interface. The VqmA DNA-binding domain is a canonical helix-turn-helix fold. Mutational and biochemical analyses of VqmA reveal that large scale macromolecular changes occur upon ligand binding. We suggest that basal, DPO-independent VqmA activity “primes” this pathway to respond to an influx of threonine in the gut when biofilm formation needs to be repressed to enable V. cholerae to disperse from the host.