A typical conception of bacteria represents them as individual and autonomous cells, yet microbes often team up to enact collective behaviors. One such behavior is biofilm formation, in which a community of cells lives within and is protected by a self-produced extracellular matrix. Bacterial biofilms can be a nuisance to industry when they form in pipes or in liquid-handling apparatuses, but they are an even greater hazard in human infections. Because the biofilm matrix shields cells and because cells within biofilms display altered biological functions, infectious biofilms are highly resistant to host immune responses and to antibiotic therapies. The opportunistic human pathogen Pseudomonas aeruginosa is notorious for its ability to form treatment-resistant biofilms in burn wounds, diabetic ulcers, and in the lungs of cystic fibrosis patients. Because such biofilm infections are so treatment-resistant, effective methods to prevent biofilm infections are urgently needed. Such treatment modalities will be made possible by the identification and characterization of new control points within the signaling network that initiates biofilm formation; such control points might then be exploited to shut off biofilm formation. A sensitive visual screening method developed by the PI recently identified two proteins with strong but previously unappreciated roles in P. aeruginosa biofilm signaling. Deletions of these two proteins have opposite effects, stimulating or suppressing biofilm formation. As these proteins potentially represent new control points, this proposal aims to characterize two of the proteins, precisely defining their functions and fitting them in with to known biofilm signaling pathways. What other proteins or molecules do they interact with to stimulate or suppress the generation of a biofilm? These aims will be achieved by combining transcriptomics, visual genetic screening, protein interaction screening, and other genetic and biochemical approaches. This work will define the roles of newly discovered biofilm regulators and is also likely to generate new leads with respect to the pathways that connect different nodes of the biofilm signaling network. The network control points revealed by these data will in turn act as a foundation for devising new strategies to effectively combat biofilm infections.