PROJECT SUMMARY/ABSTRACT Within host environments, ethanolamine (EA) can serve as a key source of carbon, nitrogen and/or energy for those bacteria that encode the EA-utilizing (eut) genes necessary for its catabolism. In many of the pathogens in which it has been studied, EA utilization positively contributes to survival and pathogenicity. Many EA- utilizing bacteria also encode for the formation of an organelle-like, protein-bound structure called a bacterial microcompartment (BMC) in which EA catabolism takes place. Currently, there is a critical lack of knowledge regarding how EA is derived and when and where EA catabolism is utilized in host cells. Our long-term goal is to understand EA utilization in bacteria. The objective of this application is to elucidate key gaps in Eut BMC dynamics and EA catabolism with a focus on the intracellular environment. The central hypothesis is that BMCs assemble within the host cell phagosome and derive EA by catabolism of PEA. Specific Aim #1 will identify when and where BMCs are formed within host cells. L. monocytogenes is a natural intracellular pathogen that escapes the phagocytic phagosome to replicate in the host cell cytoplasm. Based on strong preliminary data, it is postulated that the Eut BMCs form in the phagosome. Specific Aim #2 will elucidate how EA is derived from phospholipid sources. The hypothesis is that bacteria that utilize EA obtain it by breakdown of PEA and therefore encode the enzymes necessary for PEA catabolism. Candidate genes were identified and preliminary studies of one demonstrated its role in EA utilization. Overall, this study will significantly impact the fields of BMC biology, EA utilization and host-pathogen interactions. Additionally, because it will identify the bacterial enzymes necessary to generate EA, potential targets for antimicrobial development will be pinpointed. The proposed research is conceptually innovative in that it seeks to decipher BMC dynamics in host cells. Moreover, it will examine how EA becomes available in host cells, an unasked question that has largely been swept under the rug by the field.