Regulation of glutamate synthesis in Bacillus subtilis

描述（由申请人提供）：谷氨酸的生物合成位于碳和氮代谢的交叉点，通过谷氨酰胺合成酶将克雷布斯柠檬酸循环与氮同化联系起来。在枯草芽孢杆菌中，谷氨酸合成基因和谷氨酸前体通路受到一系列蛋白质的严格调控，这些蛋白质对各种代谢信号作出反应。该项目的长期目标是揭示和理解基因、酶和调节蛋白的网络，这些网络允许细胞保持对谷氨酸积累的严格控制。基于从先前的工作中获得的知识，本提案旨在关注其中两个调节蛋白CcpC和GItC的作用。CcpC功能的两个方面将被研究：与诱导剂柠檬酸盐的相互作用，以及多聚在抑制中的作用。对于GItC，将确定调节其活性的代谢物或蛋白质，并探索GltC在基因调控中的广泛作用及其与其他调节蛋白的功能相互作用。其中一种克雷布斯循环酶，乌头酸酶，可能具有第二种非酶活性，可能作为RNA结合蛋白。假定的乌头酶的二级活性将通过寻找这种功能的目标和通过创造保留酶活性但失去非酶活性的突变体来测试。这第二种活性对枯草芽孢杆菌产孢的影响将受到特别的关注。由于枯草芽孢杆菌是细菌界革兰氏阳性分支的模式生物，因此在这里获得的知识将应用于一个相关的致病性物种，单核细胞增生李斯特菌。因此，本研究旨在利用枯草芽孢杆菌和单核增生乳杆菌之间的调控蛋白、基因组织和调控位点的明显保守性，从而在一种重要病原体的生命中尚未探索的方面取得快速进展。

DESCRIPTION (provided by applicant): The biosynthesis of glutamate lies at the intersection of carbon and nitrogen metabolism, linking the Krebs citric acid cycle to nitrogen assimilation through glutamine synthetase. In Bacillus subtilis, the genes for glutamate synthesis and for the pathways leading to the precursors of glutamate are tightly regulated by a host of proteins that respond to a variety of metabolic signals. The long-term goal of this project is to unravel and understand the network of genes, enzymes, and regulatory proteins that allow the cell to maintain tight control over glutamate accumulation. Building on knowledge gained from previous work, this proposal aims to focus on the roles of two of these regulatory proteins, CcpC and GItC. Two aspects of CcpC function will be investigated: interaction with the inducer, citrate, and the role of multimerization in repression. For GItC, the metabolite or protein that regulates its activity will be identified, in addition, the broad role of GltC in gene regulation and its functional interaction with other regulatory proteins will be explored. One of the Krebs cycle enzymes, aconitase, may have a second, non-enzymatic activity, perhaps as an RNA binding protein. The putative secondary activity of aconitase will be tested by seeking targets of such a function and by creating mutants that retain enzymatic activity but have lost the non-enzymatic activity. The implications of this second activity for sporulation in B. subtilis will receive particular attention. Since B. subtilis is a model organism for the gram-positive branch of the bacterial world, the knowledge gained here will be applied to a related, pathogenic species, Listeria monocytogenes. Thus, this proposal seeks to take advantage of the apparent conservation of regulatory proteins, gene organization and regulatory sites between B. subtilis and L. monocytogenes and thereby make rapid progress in an unexplored aspect of the life of an important pathogen.