Regulation of Nitrogen Metabolism in Bacillus subtilis

描述（由申请人提供）：细胞生理学中的一个基本问题是细胞如何识别和响应其环境的变化。 申请人的总体目标是了解控制模型低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。 这些研究将为了解农业和商业上重要的芽孢杆菌属物种中氮代谢是如何控制的提供见解。以及重要的低G+C革兰氏阳性病原体如葡萄球菌、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B中氮限制生长期间基因表达的激活。枯草杆菌。 TnrA的活性受一种新的信号转导系统调节，其中GS的反馈抑制形式结合TnrA，阻断其DNA结合活性。 该项目的一个目标是了解负责TnrA和GS之间的蛋白质-蛋白质相互作用的分子机制。 将进行广义和定点诱变，以鉴定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和TnrA之间相互作用的化学计量和平衡结合常数。 TnrA结合位点将通过蛋白质足迹法确定。 所有可用的遗传证据表明，GS的反馈抑制形式也调节GInR的活性。 将确定GS是否直接结合GInR，从而激活其DNA结合活性。 另外，反馈抑制型GS可能通过伴侣转换机制间接调节GlnR。 生物化学和遗传学的方法将被用来寻找所需的调节因子的GInR活性的谷氨酰胺合成酶。

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.