BIOSYNTHESIS OF BACTERIAL CELL WALL COMPONENTS

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)，一种大的两亲性物质，在生物的生长和生长中起着至关重要的作用 细菌的发育。化学性质与细胞 这种聚合物的位置表明它在组装过程中起作用 细胞壁。取代LTA的亲水基团 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 通过调节膜外膜决定细胞形态和分离 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-丙氨酸的形成机理 纳入LTA；(C)构建明确的遗传系统， 可用于改变D-丙氨酸酯的含量。 (A)所需遗传成分的详细特征 D-丙氨酸酯的形成是理解LTA的先决条件 功能。因此，我们的目标是：(1)界定管制要素 和操纵子的界限；(Ii)识别操纵子的基因；(Iii) 定位D-丙氨酸载体蛋白(DCP)基因。 (B)D-丙氨酸-DCP连接酶催化D-丙氨酸及其 结扎到DCP。我们的目标是了解酶的作用机制 4‘-磷酸丙氨酸假体基团的硫代酯化反应 含D-丙氨酸的DCP。我们的目标也是确定膜 接受来自D-丙氨基的活化D-丙氨酸的蛋白质和载体脂- DCP。因为载体蛋白在人体内的核心作用 参入途径、结构和功能的研究进展 确定调节其功能的特异性决定因素所必需的 作为活性D-丙氨酸的受体和供体。 (三)根据第(一)和第(二)部分的观察，我们的目标是 设计等基因突变体，有助于In的测定 D-丙氨酰-LTA的体内作用。这些构造将提供菌株 其中LTA在D-丙氨酸酯中缺乏或升高 内容。利用基因来调节酯含量的能力 已定义的突变体将阐明这些酯在 细胞形态和分离的测定。 本提案中描述的每个目标都旨在提供 对D-丙氨基的详细生化、遗传和结构了解- LTA生物合成。实现这些目标将产生对 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。

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.