Regulation of Nitrogen Metabolism in Bacillus subtilis

描述(申请人提供)：低G+C革兰氏阳性细菌枯草杆菌的氮代谢由一种新的调节系统控制，其中谷氨酰胺合成酶是谷氨酰胺合成所必需的，并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体来抑制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瞬时结合激活的，谷氨酰胺合成酶稳定GlnR-DNA复合体。有趣的是，这个相同的GlnR-谷氨酰胺合成酶氮调节系统存在于许多重要的低G+C革兰氏阳性病原体中。下一个项目期的主要重点将是详细分析反馈抑制的谷氨酰胺合成酶调节GlnR和TnrA活性的分子机制。通过鉴定GlnRC-末端和N-末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，研究Gln R C-末端区域自动抑制Gln R二聚的机制(S)。TnrA-谷氨酰胺合成酶和GlnR-谷氨酰胺合成酶复合体中的蛋白质-蛋白质界面将用突变、生化和结构方法进行表征。定点突变将被用来鉴定反馈抑制所需的谷氨酰胺合成酶活性部位的氨基酸残基。对枯草杆菌生物膜发育的研究表明，这一过程受细胞氮素状态的影响。通过确定生物膜基质蛋白Tasa的氮素调节机制和sinR突变体中谷氨酰胺合成酶的表达减少，将探索氮代谢和生物膜形成之间的相互关系。遗传学实验表明，谷氨酰胺合成酶突变体中结构性生物膜的形成是由于Spo0A磷酸化水平增加所致。这将通过检测Spo0A~P依赖的LacZ融合在野生型和突变细胞中的表达而得到证实。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。这项研究将探索低G+C革兰氏阳性细菌枯草杆菌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革兰氏阳性病原菌中，这些研究将为深入了解这些细菌的氮代谢是如何调节的提供依据。

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.