RhlR quorum-sensing receptor ligand sensitivity regulates the differential expression of phenazine genes in Pseudomonas aeruginosa

ABSTRACT 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.