Regulation of Nitrogen Metabolism in Bacillus subtilis

描述（由申请人提供）：低G+C革兰氏阳性细菌枯草芽孢杆菌的氮代谢由一个新的调节系统控制，其中谷氨酰胺合成酶既需要谷氨酰胺合成，也需要直接控制两个转录因子GlnR和TnrA的活性。反馈抑制形式的谷氨酰胺合成酶通过形成稳定的TnrA-谷氨酰胺合成酶复合物来抑制TnrA的活性。相反，GlnR DNA结合是通过与反馈抑制的谷氨酰胺合成酶的短暂结合而激活的，从而稳定了GlnR-DNA复合物。有趣的是，同样的glnr -谷氨酰胺合成酶氮调控系统存在于许多重要的低G+C革兰氏阳性病原体中。下一个项目阶段的主要重点将是对反馈抑制谷氨酰胺合成酶调节GlnR和TnrA活性的分子机制进行详细分析。通过鉴定GlnR c端和n端结构域之间发生的分子内相互作用以及这些相互作用所需的氨基酸残基，将研究GlnR c端区域自身抑制GlnR二聚化的机制。tnra -谷氨酰胺合成酶和glnr -谷氨酰胺合成酶复合物中存在的蛋白-蛋白界面将通过突变，生化和结构方法进行表征。位点定向诱变将用于鉴定反馈抑制所需的谷氨酰胺合成酶活性位点的氨基酸残基。枯草芽孢杆菌生物膜发育的特征表明，这一过程受细胞氮状态的影响。通过确定sinR突变体中氮调控生物膜基质蛋白TasA和谷氨酰胺合成酶表达减少的机制，探索氮代谢与生物膜形成之间的相互关系。遗传实验表明，在谷氨酰胺合成酶突变体中看到的组成型生物膜的形成是由于Spo0A磷酸化水平的增加。这将通过检测Spo0A~ p依赖性lacZ融合在野生型和突变型细胞中的表达来证实。公共卫生相关性：生理学中的一个基本问题是细菌如何适应不同氮源的生长。本研究将探索低G+C革兰氏阳性细菌枯草芽孢杆菌中氮信号转导的新机制，其中谷氨酰胺合成酶直接控制转录因子TnrA和GlnR的活性。由于glnr -谷氨酰胺合成酶调节系统存在于许多重要的低G+C革兰氏阳性病原体中，这些研究将为了解这些细菌中氮代谢是如何调节的提供见解。

DESCRIPTION (provided by applicant): Nitrogen metabolism in the low G+C Gram-positive bacterium Bacillus subtilis is controlled by a novel regulatory system where the enzyme glutamine synthetase is required for both glutamine synthesis and the direct control of the activity of two transcription factors GlnR and TnrA. The feedback-inhibited form of glutamine synthetase inhibits the activity of TnrA by forming a stable TnrA-glutamine synthetase complex. In contrast, GlnR DNA binding is activated by a transient association with feedback-inhibited glutamine synthetase which stabilizes the GlnR-DNA complexes. Interestingly, this same GlnR-glutamine synthetase nitrogen regulatory system is present in a number of important low G+C Gram-positive pathogens. The major focus of the next project period will be directed toward a detailed analysis of the molecular mechanisms by which feedback-inhibited glutamine synthetase regulates the activity of GlnR and TnrA. The mechanism by which the C-terminal region of GlnR autoinhibits GlnR dimerization will be investigated by identifying the intramolecular interactions that occur between the GlnR C-terminal and N-terminal domains and the amino acid residues required for these interaction(s). The protein-protein interfaces present in the complexes of both TnrA-glutamine synthetase and GlnR-glutamine synthetase will be characterized using mutational, biochemical and structural approaches. Site-directed mutagenesis will be used to identify amino acid residues in the active site of glutamine synthetase required for feedback inhibition. Characterization of biofilm development in B. subtilis indicates that this process is influenced by the nitrogen status of the cell. The interrelationship between nitrogen metabolism and biofilm formation will be explored by identifying the mechanisms responsible for nitrogen regulation of the biofilm matrix protein TasA and the reduced expression of glutamine synthetase in sinR mutants. Genetic experiments suggest that constitutive biofilm formation seen in glutamine synthetase mutants results from increased levels of Spo0A phosphorylation. This will be confirmed by examining expression of Spo0A~P-dependent lacZ fusions in wild-type and mutant cells. PUBLIC HEALTH RELEVANCE: A fundamental question in physiology is how bacteria adapt to growth on different sources of nitrogen. The proposed research will investigate a novel mechanism of nitrogen signal transduction in the low G+C Gram-positive bacterium Bacillus subtilis where the enzyme glutamine synthetase directly controls the activity of the transcription factors TnrA and GlnR. Since the GlnR-glutamine synthetase regulatory system is present in a number of important low G+C Gram-positive pathogens, these studies will provide insight into how nitrogen metabolism is regulated in these bacteria.