Proteins in gram-negative bacteria destined for the extracellular environment are met with the additional challenge of crossing two membranes in order to reach the exterior of the cell. Unique among the several pathways evolved for this purpose is the type II secretion (T2S) pathway, which has the ability to transport folded proteins through the outer membrane. As many of the substrates secreted via the T2S system constitute toxins and degradative enzymes, this secretion system is considered a major virulence determinant. An exciting role for the involvement of the T2S system in biofilm formation is also emerging. T2S substrates are produced with N-terminal signal peptides, which target them to the Sec pathway for inner membrane translocation. Following removal of the signal peptides and folding in the periplasm the proteins engage with the T2S system, a multiprotein complex that spans the entire cell envelope. While many T2S substrates are released to the extracellular space once transported through the outer membrane, a subset of proteins remain surface associated or may reattach to the bacterial cell surface following extracellular release. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, the main virulence factor cholera toxin is an example of a completely secreted substrate, while the trypsin-like protease VesB is primarily localized to the cell surface. VesB is produced with a conserved C-terminal extension that contains two prominent glycines and a hydrophobic helix followed by positively charged residues (GlyGly-CTERM domain), a tripartite pattern that is reminiscent of the sortase targeting motif that is required for surface anchoring of proteins in Gram positive bacteria. As VesB transits the cell envelope, the GlyGly-CTERM domain is cleaved off by the rhomboid-like intramembrane protease, rhombosortase. When the rhombosortase gene is inactivated or when VesB is expressed without its GlyGly-CTERM domain, surface associated VesB activity is no longer detected. In addition to the defect in VesB cleavage, biofilm formation is negatively affected in the rhombosortase mutant. Using two model organisms, V. cholerae and the nosocomial pathogen Acinetobacter baumannii, the experiments described in this proposal are designed to test the hypothesis that the T2S system, in collaboration with rhombosortase, promotes the maturation and surface localization of GlyGly-CTERM proteins that support biofilm development and maintenance. Specifically, this proposal will determine the mechanism of maturation and surface localization of proteins with GlyGly-CTERM extensions, resolve the relationship between rhombosortase and the T2S apparatus, and identify the role(s) of GlyGly-CTERM proteins in biofilm formation, growth and maintenance. The findings will facilitate understanding of the function and specificity of rhombosortase as well as the broader class of medically relevant rhomboid proteases and may identify ways to manipulate the T2S/rhombosortase system for preventative and therapeutic use.