Combinatorial control of biofilm development by quorum-sensing and nutrient-sensing regulators.

The human pathogen Pseudomonas aeruginosa, a leading cause of hospital-acquired infections, inhabits and forms sessile antibiotic-resistant communities called biofilms in a wide range of biotic and abiotic environments. In this study, we examined how two global sensory signaling pathways - the RhlR quorum-sensing system and the CbrA/CbrB nutritional adaptation system - intersect to control biofilm development. Previous work has shown that individually these two systems repress biofilm formation. Here, we used biofilm analyses, RNA-seq, and reporter assays to explore the combined effect of information flow through RhlR and CbrA on biofilm development. We find that the {Delta}rhlR{Delta}cbrA double mutant exhibits a biofilm morphology and an associated transcriptional response distinct from wildtype and the parent {Delta}rhlR and {Delta}cbrA mutants indicating codominance of each signaling pathway. The {Delta}rhlR{Delta}cbrA mutant rapidly gains suppressor mutations that map to the carbon catabolite repression protein Crc. The combined absence of RhlR and CbrA leads to drastic reduction in the abundance of the Crc antagonist small RNA CrcZ. Thus, CrcZ acts as the molecular convergence point for quorum- and nutrient-sensing cues. Furthermore, in the absence of antagonism by CrcZ, Crc promotes the expression of biofilm matrix components - Pel exopolysaccharide, and CupB and CupC fimbriae. Therefore, this study uncovers a regulatory link between nutritional adaption and quorum sensing with potential implications for anti-biofilm targeting strategies. AUTHOR SUMMARYBacterial pathogens often form multicellular communities encased in an extra cytoplasmic matrix called biofilms as a virulence strategy. Biofilm development is controlled by various environmental stimuli that are decoded and converted into appropriate cellular responses. How information from two or more stimuli is integrated is poorly understood. Using Pseudomonas aeruginosa biofilm formation as a model, we studied the intersection of two global sensory signaling pathways - quorum sensing and nutritional adaptation. We find parallel regulation by each pathway that converges on the abundance of a small RNA. Thus, we describe a regulatory link between P. aeruginosa quorum-sensing and nutritional adaptation pathways that allows integration of information from each system into the control of biofilm development. These results expand our understanding of the genetic regulatory strategies that allow P. aeruginosa to successfully colonize host during chronic infections.