Regulation of glutamate synthesis in Bacillus subtilis

描述（由申请方提供）：关键代谢交叉点的管理是细菌经济学中的关键问题。一个这样的交叉点，α-酮戊二酸和谷氨酸的相互转化，是特别重要和有趣的，因为它代表了中心碳代谢和中心氮代谢之间的耦合点。产生α-酮戊二酸的克雷布斯循环的三羧酸分支的酶（柠檬酸合酶、顺乌头酸酶和异柠檬酸脱氢酶）的合成和氮同化的酶（谷氨酰胺合成酶、谷氨酸合酶和谷氨酸脱氢酶）由受到多种形式调节的基因编码。该项目旨在详细了解枯草芽孢杆菌（一种经过充分研究的革兰氏阳性菌模型）和单核细胞增生李斯特菌（一种代谢调节相对未探索的细胞内病原体模型）中调节这些基因的分子机制。该提案的重点是三个转录调控因子（CcpC，GltC和GlnR）和两个酶（谷氨酸脱氢酶和顺乌头酸酶），具有转录后调节功能。本提案的目的是：（a）确定控制三羧酸分支酶合成和活性的分子机制;（B）揭示控制谷氨酸和谷氨酰胺合成的分子机制;（c）确定乌头酸酶作为B中孢子形成的转录后调节因子的作用。枯草芽孢杆菌的铁代谢基因和毒力基因。单核细胞增多症这些问题将使用遗传和生物化学技术相结合来解决。将确定每种调节因子缺陷突变体的表型，并纯化蛋白质，并使用体外转录和DNA及RNA结合实验进行检测。自L.缺乏克雷布斯循环基因两个抑制子的单核细胞增生突变体在小鼠模型中的毒力存在显着缺陷，将探索这种缺陷的分子基础。公共卫生相关性：维持细胞内代谢的适当平衡是细菌生长、存活和发病的关键因素。该提案旨在揭示两种相关细菌物种，枯草芽孢杆菌（非病原体）和单核细胞增生李斯特菌（弧菌病的病原体）控制中心碳和中心氮途径之间代谢物流动的复杂机制。所获得的知识将提供更深入的了解致病机制，并可能有助于设计新的细菌生长和发病机制的抑制剂。

DESCRIPTION (provided by applicant): Management of key metabolic intersections is a critical issue in bacterial economy. One such intersection, the interconversion of a-ketoglutarate and glutamate, is particularly important and interesting because it represents the point of coupling between central carbon metabolism and central nitrogen metabolism. Synthesis of the enzymes of the tricarboxylic acid branch of the Krebs cycle (citrate synthase, aconitase and isocitrate dehydrogenase) that produce a-ketoglutarate and the enzymes of nitrogen assimilation (glutamine synthetase, glutamate synthase and glutamate dehydrogenase) are encoded by genes that are subject to multiple forms of regulation. This project seeks to understand in detail the molecular mechanisms that regulate these genes in Bacillus subtilis, a well studied model Gram-positive bacterium, and Listeria monocytogenes, a model intracellular pathogen whose metabolic regulation is relatively unexplored. The proposal focuses on three transcriptional regulators (CcpC, GltC and GlnR) and two enzymes (glutamate dehydrogenase and aconitase) that have post-transcriptional regulatory functions. The aims of this proposal are: (a) to determine the molecular mechanisms that control the synthesis and activity of the tricarboxylic acid branch enzymes; (b) to reveal the molecular mechanisms that control the synthesis of glutamate and glutamine; and, (c) to determine the role of aconitase as a post-transcriptional regulator of sporulation in B. subtilis and of iron metabolism and virulence genes in L. monocytogenes. These issues will be addressed using a combination of genetic and biochemical techniques. The phenotypes of mutants defective in each of the regulatory factors will be determined and the proteins will be purified and tested using in vitro transcription and DNA and RNA binding experiments. Since L. monocytogenes mutants that lack two repressors of the Krebs cycle genes have a significant defect in virulence in a mouse model, the molecular basis for this defect will be explored. PUBLIC HEALTH RELEVANCE: Maintaining the proper balance in intracellular metabolism is a critical factor in bacterial growth, survival and pathogenesis. This proposal aims to unravel the intricate mechanisms by which two related bacterial species, Bacillus subtilis (a non-pathogen) and Listeria monocytogenes (the causative agent of listeriosis), control the flow of metabolites between central carbon and central nitrogen pathways. The knowledge gained will provide a deeper understanding of pathogenic mechanisms and may aid in designing novel inhibitors of bacterial growth and pathogenesis.