BIOSYNTHESIS OF BACTERIAL CELL WALL COMPONENTS

细菌细胞壁成分的生物合成

• 批准号: 2190282
• 负责人: FRANCIS C NEUHAUS
• 金额: $ 21.72万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190282
• 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

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-丙氨酰脂磷壁酸的生物合成和功能 酸（LTA），一种在生长中起重要作用的大两亲物， 细菌的发展。 化学性质和细胞 这种聚合物的位置表明它在组装中起作用， 细胞壁。 LTA的亲水性部分被 D-丙氨酸酯提供了一种控制阴离子电荷的机制。 我们的假设是LTA的D-丙氨酸酯在以下方面发挥作用： 通过调节膜外的细胞来决定细胞的形状和分离 细胞功能。 三个研究领域将包括： D-丙氨酸操纵子遗传结构的建立 酯形成;（B）D-丙氨酸的机制的描述 （c）建立明确的遗传系统， 可用于影响D-丙氨酸酯含量的变化。 (a)所需的遗传成分的详细表征， 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：（一）界定监管要素 和操纵子的限制;（ii）鉴定操纵子的基因;（iii） 定位D-丙氨酸载体蛋白（Dcp）的基因。 (b)D-丙氨酸-Dcp连接酶催化D-丙氨酸的活化， 连接至Dcp。 我们的目标是了解酶的机制， 的4 '-磷酸泛酰巯基乙胺辅基的硫酯化， 含D-丙氨酸的DCP。 我们的目标也是找出 接受来自D-丙氨酰的活化D-丙氨酸的蛋白质和载体脂质， Dcp. 由于载体蛋白在细胞中的核心重要性， 结合途径，其结构和功能的研究是 必要的识别特异性决定因素，调节其 作为活化D-丙氨酸的受体和供体的作用。 (c)根据（a）和（B）部分的观察，我们的目标是 设计同基因突变体，这将有助于确定基因的突变。 D-丙氨酰-LTA的体内功能。 这些结构将提供菌株 其中LTA缺乏或升高D-丙氨酸酯 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其中的作用 细胞形状的确定和分离。 本提案中所述的每一个目标都旨在提供一个 对D-丙氨酰的生物化学、遗传学和结构的详细了解， LTA生物合成。 实现这些目标将使我们深入了解 这些大分子两亲物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