The Gram negative, opportunistically pathogenic Gammaproteobacterium Pseudomonas aeruginosa has a propensity to form biofilms. Biofilms are surface associated or aggregated bacterial communities, embedded in a self-derived, extracellular matrix (EPS). One major component of the P. aeruginosa biofilm EPS is the polysaccharide Psl. Psl plays a structural and protective role in P. aeruginosa biofilms by mediating attachment to abiotic surfaces and mammalian cells, conferring protection from components of the host immune response and a variety of antimicrobials, as well as promoting cell-cell adhesion. More recently, Psl has also been shown to function as a cell-cell signaling molecule that activates two diguanylate cyclases through an unknown mechanism, which results in an increase in intracellular levels of c-di-GMP. The secondary messenger c-di-GMP is central to the P. aeruginosa virulence regulatory network, and promotes biofilm formation, in part, by inducing the production of EPS components, including Psl. Thus, presence of environmental Psl appears to act as a feed-forward signaling molecule that enhances biofilm formation. In order to further understand this function, this investigation will employ mini-Tn5 mutagenesis to define the signal transduction pathway(s) responsible for activating the diguanylate cyclases through a novel feed-forward mechanism of Psl-dependent signaling in P. aeruginosa.