Regulation of glutamate synthesis in Bacillus subtilis

描述(申请人提供)：谷氨酸的生物合成位于碳代谢和氮代谢的交汇点，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，谷氨酸合成和谷氨酸前体途径的基因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做出反应。这个项目的长期目标是解开和了解基因、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。基于从以前的工作中获得的知识，这项提案旨在关注其中两种调节蛋白CCPC和GItC的作用。CCPC功能的两个方面将被研究：与诱导剂柠檬酸的相互作用，以及多聚化在抑制中的作用。对于GItC，将确定调节其活性的代谢物或蛋白质，此外，还将探索GLTC在基因调节中的广泛作用及其与其他调节蛋白的功能相互作用。克雷布斯循环酶之一的乌头酸酶可能具有第二种非酶活性，可能是作为一种RNA结合蛋白。乌头酸酶假定的二级活性将通过寻找这一功能的靶标和创造保持酶活性但失去非酶活性的突变体来测试。这第二个活动对枯草杆菌产孢量的影响将受到特别关注。由于枯草杆菌是细菌界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物种--单核细胞增生性李斯特菌。因此，这一建议试图利用枯草杆菌和单核细胞增多性乳杆菌之间调节蛋白、基因组织和调节位点的明显保守性，从而在一个重要病原体生命的未知方面取得快速进展。

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.