PROJECT SUMMARY Hospital-acquired secondary infections are an escalating problem of global significance. Indeed, multi-drug resistant Pseudomonas aeruginosa is the leading cause of hospital-acquired infections in the USA and P. aeruginosa is now a priority pathogen on the CDC ESKAPE pathogen list. P. aeruginosa infection is a particular problem in cystic fibrosis, microbial keratitis, in third-degree burn units, and in cancer sufferers and HIV patients. P. aeruginosa virulence and biofilm development depend on the bacterial cell-to-cell communication process called quorum sensing. The known P. aeruginosa quorum-sensing circuit possesses two canonical LuxI/R type signaling pathways: LasI/R and RhlI/R, that, together, control an estimated 10% of the genes in the genome. The known circuit functions as follows: LasI produces and LasR responds to the autoinducer 3OC12-homoserine lactone. The LasR:3OC12-homoserine lactone complex activates transcription of many genes including rhlR, encoding a second quorum-sensing receptor. RhlR binds to the autoinducer C4-homoserine lactone, the product of RhlI. RhlR:C4-homoserine lactone also directs a large regulon of genes including those encoding virulence factors such as pyocyanin, elastases, and rhamnolipids. Typically, mutations in quorum-sensing luxI-type and luxR-type genes (i.e., lasI-lasR and rhlI-rhlR) confer identical phenotypes because each component of the pair needs the other to function. However, using biofilm analyses, transcriptional reporter assays, RNA-seq studies, and animal infection assays, I discovered that RhlR directs both RhlI-dependent and RhlI-independent regulons. I discovered that an alternative autoinducer, synthesized by the PqsE thioesterase enzyme, drives the RhlI- independent RhlR regulon. I demonstrated that while the canonical RhlR-RhlI system is dispensable, the RhlR- PqsE system is the crucial quorum-sensing system required for biofilm formation and for virulence in two animal models of infection. Most importantly, these studies revealed that unlike LasR that activates biofilm formation, RhlR functions as a repressor of biofilm development. Here, I propose to determine (1) how Rhl quorum sensing converges with Cbr nutrient sensing system to repress biofilm formation and (2) what factors allow biofilm formation when the sensory cues for Las, Rhl and Cbr systems are absent. The proposed research will contribute a mechanistic understanding of quorum-sensing control of biofilm formation and uncover how quorum sensing intersects with other sensory signaling systems, which is crucial for understanding basic P. aeruginosa biology and for successful development of anti-quorum-sensing strategies.