Regulation of Nitrogen Metabolism in Bacillus subtilis

描述(申请人提供)：细胞生理学中的一个基本问题是细胞如何识别和响应环境的变化。申请者的总体目标是了解控制模式低G+C革兰氏阳性细菌枯草芽孢杆菌中氮化合物利用的调控系统网络。这些研究将为如何控制农业和商业上重要的芽孢杆菌的氮代谢提供洞察力。以及重要的低G+C革兰氏阳性病原菌，如葡萄球菌、链球菌和肠球菌。 两个非常相似的全局转录因子GInR和TnrA负责在枯草杆菌氮限制生长过程中激活基因表达。TnrA的活性受一种新的信号转导系统的调节，其中反馈抑制形式的GS与TnrA结合，阻断其DNA结合活性。这个项目的目标之一是了解TnrA和GS之间蛋白质-蛋白质相互作用的分子机制。将进行广义和定点突变，以确定GS反馈抑制以及TnrA和GS之间相互作用所需的氨基酸残基。将测定GS与TnrA相互作用的化学计量比和平衡结合常数。TnrA结合位点将由蛋白质足迹来定义。所有现有的遗传证据表明，反馈抑制形式的GS也调节GInR的活性。将确定GS是否直接与GInR结合，激活其DNA结合活性。反馈性抑制GS可能通过伙伴转换机制间接调节GlnR。将使用生化和遗传方法来寻找谷氨酰胺合成酶调节GInR活性所需的因素。

DESCRIPTION (provided by applicant): A fundamental question in cellular physiology is how cells recognize and respond to changes in their environment. The applicant's overall goal is to understand the network of regulatory systems controlling the utilization of nitrogen compounds in the model low G+C Gram-positive bacterium, Bacillus subtilis. These studies will provide insight into how nitrogen metabolism is controlled in the agriculturally and commercially important Bacillus spp. as well as in important low G+C Gram-positive pathogens such as Staphylococcus, Streptococcus and Enterococcus. Two very similar global transcription factors, GInR and TnrA, are responsible for the activation of gene expression during nitrogen-limited growth in B. subtilis. The activity of TnrA is regulated by a novel signal transduction system where the feedback-inhibited form of GS binds TnrA, blocking its DNA binding activity. One goal of this project is to understand the molecular mechanism responsible for the protein-protein interaction between TnrA and GS. Generalized and site-directed mutagenesis will be performed to identify amino acid residues required for feedback inhibition of GS and for the interaction between TnrA and GS. The stoichiometry and equilibrium binding constant of the interaction between GS and TnrA will be determined. The TnrA binding site will be defined by protein footprinting. All the available genetic evidence indicates that the feedback-inhibited form of GS also regulates the activity of GInR. It will be determined whether GS directly binds to GInR, activating its DNA binding activity. Alternatively feedback inhibited GS may indirectly regulate GlnR by a partner switching mechanism. Biochemical and genetic approaches will be used to search for factors required for the regulation of GInR activity by glutamine synthetase.