BIOSYNTHESIS OF ENTEROBACTERIAL COMMON ANTIGEN

本研究的长期目标是阐明 参与外膜的生物发生和组装的机制， 革兰氏阴性菌。 据估计，这些知识 原核生物中的过程将提供对一般 真核细胞膜生物发生和组装的机制 和细胞器。 这里提出的具体研究涉及确定 肠杆菌共同抗原（ECA）的生物合成机制。 ECA 是存在于所有细胞外膜的细胞表面抗原， 属于肠杆菌科的革兰氏阴性细菌。 的 ECA的血清学特异性通过线性 由N-乙酰基-D-葡糖胺（G1 cNAc）组成杂多糖， N-乙酰基-D-甘露糖胺糖醛酸（ManNAcUA）和4-乙酰氨基-4，6- 双脱氧-D-半乳糖（Fuc 4 NAc）。 这些氨基糖形成了 大于4）-β-D-ManpNAc UA-（1大于 4）-α-D-Glcp-NAc-（1大于3）-D-Fucp 4 NAc-（1大于. 虽然这种分子的狭隘分类学分布表明， 功能与革兰氏阴性肠道细菌独特相关， 已知的是关于生物合成的功能或机制， 他们的分子。 我们在这里提出的实验设计，以确定在初始步骤， ECA生物合成途径。 这些实验将进行初步的 这些观察结果表明，G1 cNAc-焦磷酸十一肾上腺素 （GlcNAc-PP-lipid）是ECA合成的早期中间体。 因此，委员会认为， 我们将研究最初的三糖重复序列 通过将ManNAcUA和Fuc 4 NAc依次转移到 来自核苷酸糖供体UDP-ManNAcUA的GlcNAc-PP-脂质和 TDP-Fuc4NAc。 后续实验将针对 证明了脂质连接的杂多糖的体外合成 链含有多个重复单位，我们也将确定 链伸长的机制。 将关注更多实验 通过鉴定rfe突变体中ECA合成被阻断的步骤 色葡萄鼠伤寒。 拟议的研究将利用分子生物学技术， 生物化学，如体内脉冲标记，体外多糖 的合成、分离、分馏和结构表征 生物合成中间体

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.