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BIOSYNTHESIS OF BACTERIAL CELL WALL COMPONENTS

细菌细胞壁成分的生物合成

基本信息

批准号：
2444853
负责人：
FRANCIS C NEUHAUS
金额：
$ 22.51万
依托单位：
NORTHWESTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-07-01 至 2000-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2444853
关键词：
Lactobacillus casei Streptococcus alanine bacterial genetics carbohydrate biosynthesis cell wall enzyme mechanism esters genetic regulatory element membrane proteins membrane reconstitution /synthesis microorganism metabolism operon protein purification site directed mutagenesis teichoate thiols

项目摘要

The primary goal of our research program is directed towards an understanding of the biosynthesis and function of D-alanyl-lipoteichoic acid (LTA), a macroamphiphile which plays a vital role in the growth and development of the bacterium. The chemical properties and cellular location of this polymer indicate that it functions in the assembly of the cell wall. The substitution of the hydrophilic moiety of LTA with D-alanine ester provided a mechanism for controlling the anionic charge. Our hypothesis is that the D-alanine esters of LTA play a role in determining cell shape and separation by regulating extramembranal cellular functions. Three areas of research will include: (a) establishment of the genetic organization of the operon for D-alanine ester formation; (b) delineation of the mechanism of D-alanine incorporation into LTA; (c) construction of defined genetic systems which can be used to effect changes in the D-alanine ester content. (a) The detailed characterization of the genetic components required for D-alanyl ester formation is a prerequisite for understanding LTA function. Thus, it is our aim to: (i) define the regulatory elements and limits of the operon; (ii) identify the genes of the operon; (iii) locate the gene for the D-alanine carrier protein (Dcp). (b) D-Alanine-Dcp ligase catalyzes the activation of D-alanine and its ligation to Dcp. Our goal is to understand the enzymic mechanism for the thiolesterification of the 4'-phosphopantetheine prosthetic group of Dcp with D-alanine. It is also our goal to identify the membrane proteins and carrier lipid that accept activated D-alanine from D-alanyl- Dcp. Because of the central importance of the carrier protein in the incorporation pathway, studies of its structure and function are necessary for identifying the specificity determinants that regulate its role as an acceptor and as a donor of activated D-alanine. (c) Based on the observations form part (a) and (b), our goal is to design isogenic mutants which will aid in the determination of the in vivo function of D-alanyl-LTA. These constructions will provide strains in which the LTA is either deficient or elevated in D-alanine ester content. The ability to modulate the ester content using genetically defined mutants will clarify the role of these esters in the determination of cell shape and separation. Each of the aims described in this proposal is designed to provide a detailed biochemical, genetic and structural understanding of D-alanyl- LTA biosynthesis. Achieving these goals will yield insights into the role of these macroamphiphiles in the physiology of the gram-positive bacterium. The elucidation of the D-alanine incorporation system will identity new targets which may be used for the rational design of antibacterial agents.

我们研究计划的主要目标是了解 D-丙氨酰脂磷壁的生物合成和功能酸（LTA），一种大分子两亲物，在生长和生长过程中起着至关重要的作用细菌的发育。化学性质和细胞该聚合物的位置表明它在组装中发挥作用细胞壁。 LTA的亲水部分被取代 D-丙氨酸酯提供了控制阴离子电荷的机制。我们的假设是 LTA 的 D-丙氨酸酯在通过调节膜外确定细胞形状和分离细胞功能。三个研究领域将包括：(a) D-丙氨酸操纵子遗传组织的建立酯的形成； (b) D-丙氨酸作用机制的描述纳入 LTA； (c) 构建明确的遗传系统可用于影响 D-丙氨酸酯含量的变化。 (a) 所需遗传成分的详细特征 D-丙氨酰酯的形成是理解 LTA 的先决条件功能。因此，我们的目标是： (i) 定义监管要素和操纵子的限制； (ii) 鉴定操纵子的基因； (三) 找到 D-丙氨酸载体蛋白 (Dcp) 的基因。 (b) D-丙氨酸-Dcp连接酶催化D-丙氨酸及其衍生物的活化连接至 Dcp。我们的目标是了解酶的机制 4'-磷酸泛硫氨酸辅基的硫酯化 DCP 与 D-丙氨酸。鉴定膜也是我们的目标接受来自 D-丙氨酰-的活化 D-丙氨酸的蛋白质和载体脂质直流电。由于载体蛋白在掺入途径，对其结构和功能的研究确定调节其特异性的决定因素是必要的作为活化 D-丙氨酸的受体和供体。 (c) 根据 (a) 和 (b) 部分的观察结果，我们的目标是设计同基因突变体，这将有助于确定 D-丙氨酰-LTA 的体内功能。这些结构将提供应变其中 LTA 中 D-丙氨酸酯缺乏或升高内容。利用基因调节酯含量的能力定义的突变体将阐明这些酯在细胞形状和分离的测定。本提案中描述的每个目标旨在提供 D-丙氨酰-的详细生化、遗传和结构理解 LTA生物合成。实现这些目标将深入了解这些大两亲分子在革兰氏阳性菌生理学中的作用细菌。 D-丙氨酸掺入系统的阐明将确定可用于合理设计的新目标抗菌剂。