Biofilms are surface-attached microbial communities found in clinical, industrial and natural environments. Numerous studies suggest that biofilm bacteria can be distinguished from their planktonic counterparts in terms of morphology and physiology, and also differ in gene and protein expression profiles. We have proposed that the formation of biofilms is a well-regulated developmental system. If biofilm formation is analogous to other developmental systems we hypothesize that there should be distinct regulatory pathways that control the transition between planktonic and biofilm growth, and this regulatory cascade should be controlled in response to environmental signals. Using Pseudomonas aeruginosa as a model organism, we have identified a 3-component regulatory system (designated SadARS) comprised of a sensor histidine kinase and two associated response regulators required for biofilm development. The SadARS system is similar to the Bvg virulence gene regulatory locus of Bordetella pertussis both in terms of DNA sequence and genomic organization. Preliminary studies have demonstrated that the SadARS locus is required for maturation of biofilms on abiotic surfaces. Strains lacking a functional SadARS locus also show altered colonization of airway-derived epithelial cells. The Specific Aims of this proposal are to: Specific Aim 1. Identify & functionally characterize the targets of the SadARS 3-component regulatory system. Specific Aim 2. Investigate the role of electron transport system (ETS) signaling via the SadARS system in biofilm development. Specific Aim 3. Determine the small molecule(s) and receptor determinant(s) required to signal via the periplasmic domain of the SadS sensor histidine kinase. The experiments proposed here are part of a multi-faceted Lung Biology Program at Dartmouth. The specific contribution of my laboratory to this program is to study the virulence mechanisms of the respiratory pathogen P. aeruginosa. Specifically, these experiments will provide insight into a regulatory cascade required for biofilm development, especially as it pertains to colonization of airway-derived epithelial cells from both CF and non-CF individuals. Information from these studies may lead to identification of new targets for anti-biofilm therapies to treat these respiratory infections.