The long term goals of this research are directed at elucidating the mechanisms involved in the biogenesis and assembly of the outer membrane in Gram-negative bacteria. It is anticipated that a knowledge of these processes in prokaryotic organisms will provide insights into general mechanisms involved in membrane biogenesis and assembly in eukaryotic cells and cell organelles. The specific research proposed here is concerned with determining the mechanism of biosynthesis of the enterobacterial common antigen (ECA). ECA is a cell surface antigen present in the outer membrane of all Gram-negative bacteria belonging to the family Enterobacteriaceae. The serological specificity of ECA is determined by a linear heteropolysaccharide comprised of N-acetyl-D-glucosamine (G1cNAc), N-acetyl-D-mannosaminuronic acid (ManNAcUA), and 4-acetamido-4,6- dideoxy-D-galactose (Fuc4NAc). These amino sugars form the repeating trisaccharide unit Greater than 4)-Beta-D-ManpNAcUA-(1 Greater than 4)-Alpha-D-Glcp-NAc-(1 Greater than 3)-D-Fucp4NAc-(1 Greater than. Although the parochial taxanomic distribution of this molecule suggests a function uniquely associated with Gram-negative enteric bacteria, nothing is known concerning either the function or mechanism of biosynthesis of theis molecule. We propose here experiments designed to define the initial steps in the pathway of ECA biosynthesis. These experiments will pursue preliminary observations which suggest that G1cNAc-pyrophosphorylundecaprenol (G1cNAc-PP-lipid) is an early intermediate in ECA synthesis. Accordingly, we will investigate the possibility that the initial trisaccharide repeat unit is synthesized by the sequential transfer of ManNAcUA and Fuc4NAc into G1cNAc-PP-lipid from the nucleotide sugars donors UDP-ManNAcUA and TDP-Fuc4NAc, respectively. Subsequent experiments will be directed at demonstrating the in vitro synthesis of lipid-linked heteropolysaccharide chains containing multiple repeat units, and we will also determine the mechanism of chain elongation. Additional experiments will be concerned with identifying the step at which ECA synthesis is blocked in rfe mutants of S. typhimurium. The proposed research will utilize techniques of molecular biology and biochemistry such as in vivo pulse-labeling, in vitro polysaccharide synthesis, and isolation, fractionation, and structural characterization of biosynthetic intermediates.
