Regulation of glutamate synthesis in Bacillus subtilis

描述（申请人提供）：关键代谢交叉点的管理是细菌经济学的关键问题。其中一个交集，a-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。克雷布斯循环中产生a-酮戊二酸的三羧酸分支酶（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。该项目旨在详细了解枯草芽孢杆菌和单核增生李斯特菌中这些基因的调控分子机制，前者是一种研究得很好的革兰氏阳性细菌模型，后者是一种代谢调控相对未被探索的细胞内病原体模型。该提案侧重于三种转录调节因子（CcpC， GltC和GlnR）和两种具有转录后调节功能的酶（谷氨酸脱氢酶和乌头碱酶）。本提案的目的是：(a)确定控制三羧酸分支酶的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)确定乌头酶作为枯草芽孢杆菌产孢和单核增生乳杆菌铁代谢和毒力基因转录后调节剂的作用。这些问题将使用遗传和生化技术的组合来解决。将确定每个调节因子中有缺陷的突变体的表型，并使用体外转录和DNA和RNA结合实验纯化和测试蛋白质。由于缺乏克雷布斯循环基因两种抑制因子的单核增生乳杆菌突变体在小鼠模型中具有明显的毒力缺陷，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、生存和发病的关键因素。本研究旨在揭示两种相关细菌——枯草芽孢杆菌（Bacillus subtilis，非病原体）和单核增生李斯特菌（Listeria monocytogenes，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.