PROJECT SUMMARY Escherichia coli and Proteus mirabilis are important urinary tract pathogens and the most common causes of catheter-associated urinary tract infections (CAUTIs). CAUTIs are one of the most common health care- associated infections and contribute to nearly double the mortality rate of catheterized compared to non- catheterized patients. CAUTIs are frequently polymicrobial and inter-species interactions contribute to the development of catheter biofilms and increase the risk of sepsis. Polymicrobial environments are shaped by both cooperative and competitive interactions between species. However, specific interactions between different urinary tract colonizers currently remain poorly defined. Characterizations of competitive interactions is additionally hampered by the fact that a large number of competition systems remain undiscovered. Our overall goal is to study the role of competition systems for successful colonization of Enterobacteriaceae in the urinary tract. This proposal specifically aims to identify and further characterize a novel inter-species competition system employed by Proteus mirabilis to kill competitor species. We showed that clinical and commensal P. mirabilis strains quickly and drastically (2-million-fold) reduced viability of E. coli and other Enterobacteriaceae during co-culture. Cell-free P. mirabilis culture supernatant alone did not reduce viability of target cells. Killing instead required direct contact between cells. However, a heat-labile component in stationary phase P. mirabilis supernatant was sufficient to induce killing, suggesting that the system is likely regulated via quorum sensing. Killing also occurred on solid surfaces, where P. mirabilis was able to penetrate and kill all cells in established microcolonies of E. coli. This highlights a potential role of the killing system in mixed biofilms, which is an important component of urinary catheter colonization and persistence. The only known contact-dependent competition system in P. mirabilis is the Type 6 Secretion System (T6SS), but a mutant deficient in this system still killed E. coli. We thus hypothesize that P. mirabilis employs a novel contact-dependent system to kill E. coli cells. The objective of this study is to identify the killing system and its regulation, define the mechanism of action, and elucidate the role of the killing system for competition of P. mirabilis with E. coli during polymicrobial infections in vivo. In Aim 1, we will use a transposon library of P. mirabilis and a bioluminescence-based assay to screen for P. mirabilis mutants unable to kill E. coli and to identify genes coding for effector molecules, delivery system and regulatory molecules of the killing system. We will also test the hypothesis that quorum sensing molecules regulate the activity of the system and investigate the mechanism of how E. coli cells die after contact with P. mirabilis. In Aim 2 we will assess the role of the P. mirabilis killing system...