Regulation of glutamate synthesis in Bacillus subtilis

描述(由申请人提供)：关键代谢交叉点的管理是细菌经济中的一个关键问题。其中一个交叉点，a-酮戊二酸和谷氨酸的相互转化，特别重要和有趣，因为它代表了中枢碳代谢和中枢氮代谢之间的耦合点。Krebs循环中产生α-酮戊二酸的三羧酸酶(柠檬酸合成酶、乌头酸酶和异柠檬酸脱氢酶)和氮同化酶(谷氨酰胺合成酶、谷氨酸合成酶和谷氨酸脱氢酶)的合成是由受多种形式调控的基因编码的。本项目旨在详细了解枯草芽孢杆菌和单核细胞增生性李斯特菌中调控这些基因的分子机制。枯草杆菌是一种研究得很好的革兰氏阳性模式细菌，而李斯特菌是一种代谢调控相对未知的细胞内模式病原菌。该提案侧重于具有转录后调控功能的三种转录调控因子(CCPC、GLTC和GlnR)和两种酶(谷氨酸脱氢酶和乌头酸酶)。这项建议的目的是：(A)确定控制三羧酸支链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；以及(C)确定乌头酸酶作为枯草杆菌产孢量的转录后调节因子以及铁代谢和单核细胞增生性乳杆菌毒力基因的作用。这些问题将使用遗传和生化技术的组合来解决。将确定在每个调控因子中存在缺陷的突变体的表型，并将通过体外转录以及DNA和RNA结合实验来纯化和检测蛋白质。由于缺乏Krebs循环基因两个抑制子的单核细胞增多性乳杆菌突变株在小鼠模型中的毒力存在显著缺陷，因此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、存活和致病的关键因素。这项提议旨在揭示两个相关细菌物种-枯草芽孢杆菌(非病原体)和单核细胞增生性李斯特菌(李斯特菌病的病原体)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对致病机制的更深层次的理解，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.