The long-term goals of this research are to gain a more complete understanding of the function of the outer membrane (OM) of gram-negative bacteria and the mechanisms involved in its biogenesis. As an approach to these goals, we have investigated the biochemistry and genetics of enterobacterial common antigen (ECA) synthesis and assembly in Escherichia coli. In addition, we have investigated the function of ECA in E. coli, Shigellaflexnerii, and Salmonella enterica serovar typhimurium. ECA is an OM glycolipid that is unique to the Enterobacteriaceae, and it is present in all members of this family. In spite of the ubiquitous occurrence of ECA in gram-negative enteric bacteria, the function of ECA has remained unknown. Our previous endeavors to study ECA synthesis and and assembly have been facilitated by the isolation and characterization of mutants defective in these processes. The characterization of these mutants has resulted in the identification of biosynthetic intermediates involved in ECA synthesis that have, in turn, led to the development of in vitro and in vivo experimental systems to demonstrate specific enzymatic steps in ECA assembly. Nevertheless, several important steps in the assembly of ECA remain to be established. We propose to continue this combined biochemical and genetic approach to complete the characterization of the assembly process. In addition, we have obtained exciting new data that strongly supports the conclusion that ECA plays an important role in the mechanism responsible for the resistance of gram-negative enteric bacteria to bile salts. Thus, the specific aims for the requested period of support are: (i) identification of the genetic determinant of ECA polysaccharide chain elongation in E. coil K-12, and biochemical characterization of the polymerization mechanism, (ii) determination of the role of o416 of the E. coli wec gene cluster in ECA assembly, (iii) isolation of E. coil mutants defective in the synthesis of the ECApG polysaccharide-aglycone linkage and biochemical characterization of the mechanism involved in linkage formation, and (iv) determination of the role of ECA in the resistance of gram-negative enteric bacteria to bile salts. It is anticipated that these studies will provide valuable insights into the assembly of other important membrane glycolipids and polysaccharides. Such information will also provide a rationale for the development of new antimicrobial agents.