Elucidating the parameters of intracellular ethanolamine utilization - 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, but not all, 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 which eut genes are required and at what stage they are expressed during intracellular infection of host cells. Our long-term goal is to understand EA utilization in bacteria. The objective of this application is to elucidate key gaps in the understanding EA metabolism, with a focus on EA’s role in promoting the intracellular replication of the human pathogen, L. monocytogenes (LMO). The central hypothesis is that EA utilization occurs in the phagosome but does not absolutely require all the eut genes, specifically not all the BMC and enzyme encoding genes. Specific Aim #1 will identify the intracellular location of EA utilization by LMO. L. mono- cytogenes 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 EA utilization occurs in the phagosome. Spe- cific Aim #2 will identify the eut genes required for LMO EA utilization and intracellular replication. The L. monocytogenes eut locus contains 17 genes encoding for the full array of regulatory, enzymatic, and structural proteins involved in EA utilization. However, we postulate that intracellular EA utilization does not require all the eut genes; specifically, it may not require all the encoded Eut enzymes and Eut BMC structural proteins. Overall, this study will significantly impact the fields of EA utilization, BMC biology, and host-pathogen interactions. Ad- ditionally, because it studies a process necessary for optimal intracellular survival, potential targets for antimi- crobial development will be identified. The proposed research is conceptually innovative in that it seeks to deci- pher the dynamics of eut gene expression and BMC formation in host cells in addition to clarifying the core genetic components required for EA metabolism in the intracellular niche.