The long term goals of this research are to elucidate the mechanisms involved in the biogenesis and function of outer membrane components of gram-negative bacteria. As an approach to this goal, we have investigated the biochemistry and genetics of enterobacterial common antigen (ECA) synthesis in Escherichia coli and Salmonella typhimurium. ECA is an outer membrane glycolipid unique to the Enterobacteriaceae, and it is present in all bacteria belonging to this family. Specific emphasis will be placed on determining the mechanism of biosynthesis of ECA and on identifying and characterizing the genes involved in this process. This endeavor has been facilitated by the isolation and characterization of mutants of E. coli and S. typhimurium defective in several of the genetic determinants of ECA. The characterization of these mutants has resulted in the identification of biosynthetic intermediates involved in ECA synthesis and the development of in vitro and in vivo experimental systems that allow demonstration of specific enzymatic steps involved in ECA assembly. We propose here experiments for the continuation of our studies, and the specific aims for the requested period of support are to (i) determine the mechanism involved in the synthesis of phospholipid-linked ECA polysaccharide chains (ECA-PL), (ii) determine the mechanism involved in the assembly of ECA polysaccharide chains, (iii) determine the mechanism involved in the transfer of ECA polysaccharide chains to lipopolysaccharide acceptors to form ECA-LPs, and (iv) investigate the possible function of open reading frame o348 in the assembly of ECA molecules. The proposed research will utilize techniques of molecular biology, biochemistry, and genetics. It is anticipated that a knowledge of the mechanism of ECA assembly will provide insights into the processes of membrane biogenesis and assembly in bacteria. Such information will provide a rational basis for the development of new antimicrobial agents, and it will also provide insights into the mechanisms involved in the biogenesis and assembly of membranes in eukaryotic cells and cell organelles.
