PROJECT SUMMARY Nearly 3 million antibiotic resistant infections occur per year in the United States. This problem is especially acute in gram-negative bacteria, where the outer membrane (OM) which surrounds the aqueous periplasm acts as a permeability barrier capable of excluding many antibiotics. We are interested in the OM of Enterobacterales (e.g., Escherichia, Salmonella, Klebsiella), which are adapted to an enteric environment rich in toxic molecules, such as bile salts, necessitating an especially strong OM. It has become clear that the permeability of the OM can be altered by the physiological state of the cell. Specifically, stresses such as nutrient limitation can result in strengthening of the OM permeability barrier. Elucidation of the pathways responsible for this strengthening will lead to new targets for the development of small molecules that can weaken the OM permeability barrier. We have found enterobacterial common antigen (ECA), a conserved component of the Enterobacterales OM and periplasm, to be important for OM impermeability under stress. Two forms of ECA (phospholipid-linked ECA (ECAPG), and cyclic ECA (ECACYC)) have different roles related to OM permeability; however, their precise functions remain unknown, in part, because many steps in their biogenesis are poorly understood. Our long-term goal is to understand the biogenesis of ECA to facilitate functional studies and identify potential antimicrobial targets. Specifically, this project aims to elucidate, in Escherichia coli K12, the regulation of and unknown steps in biogenesis of the forms of ECA contributing to antibiotic resistance. Biochemical reactions are required for these forms of ECA to be produced and yet the genes responsible for these steps and the regulation of these steps are largely unknown. The central hypothesis is that ECAPG and ECACYC can be differentiate through their unique biosynthetic genes and regulatory roles. This hypothesis will be addressed with the following aims: identify the genes and substrate necessary for ECA to become a phospholipid head group forming ECAPG using genetic interactions with other biosynthesis pathways (Aim 1); elucidate factors and mechanisms involved in ECACYC biogenesis using an antibiotic sensitivity suppression phenotype we discovered (Aim 2); and uncover the mechanisms of the two novel pathways of ECA regulation we discovered (Aim 3). These conceptually innovative aims will be approached through a blend of high-throughput genomics, genetic screens and selections, and biochemical techniques. Completion of this project will identify genes and residues important for biogenesis of ECAPG and ECACYC, which represent targets for development of small molecules weakening the OM. In addition, this will allow genetic analyses of ECA function, providing insights into Enterobacterales biology.