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，谷氨酸合成基因和导致谷氨酸前体的途径的基因受到响应于各种代谢信号的大量蛋白质的严格调控。该项目的长期目标是解开和理解基因、酶和调节蛋白的网络，这些网络使细胞能够保持对谷氨酸积累的严格控制。从以前的工作中获得的知识的基础上，本建议的目的是集中在这些调节蛋白，CcpC和GItC的作用。CcpC功能的两个方面将进行研究：与诱导剂，柠檬酸盐的相互作用，以及多聚化在阻遏中的作用。对于GItC，将鉴定调节其活性的代谢产物或蛋白质，此外，将探索GltC在基因调控中的广泛作用及其与其他调控蛋白的功能相互作用。三羧酸循环酶之一乌头酸酶可能具有第二种非酶活性，可能作为RNA结合蛋白。将通过寻找这种功能的靶标和通过产生保留酶活性但丧失非酶活性的突变体来测试乌头酸酶的推定次级活性。这第二种活性对B中孢子形成的影响。枯草芽孢杆菌将受到特别关注。从B开始。枯草杆菌是细菌世界的革兰氏阳性分支的模式生物，这里获得的知识将应用于相关的致病性物种单核细胞增生李斯特菌。因此，该提议试图利用B之间的调节蛋白、基因组织和调节位点的明显保守性。subtilis和L.单核细胞增多症，从而在一个重要病原体生命的未探索方面取得快速进展。

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.