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Regulation of Nitrogen Metabolism in Bacillus subtilis

枯草芽孢杆菌氮代谢的调节

基本信息

批准号：
8294687
负责人：
SUSAN H. FISHER
金额：
$ 38.54万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-09-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8294687
关键词：
Active Sites Alanine Amino Acids Bacillus subtilis Bacteria Binding Sites Biochemical C-terminal Cells Collaborations Complex Cysteine DNA DNA Binding Deoxyribonucleases Development Dimerization Enterococcus Enzymes Feedback Fluorescence Resonance Energy Transfer Gene Expression Genetic Genetic Screening Genus staphylococcus Glutamate-Ammonia Ligase Glutamine Goals Gram-Positive Bacteria Grant Growth Health Human In Vitro Laboratories LacZ Genes Listeria Maps Mediating Metabolism Microbial Biofilms Modeling Modification Molecular Molecular Analysis Molecular Chaperones Molecular Conformation Mutagenesis Mutation N-terminal NMR Spectroscopy Nitrogen Orthologous Gene Peptide Hydrolases Phosphorylation Phosphotransferases Physiology Process Production Protein Conformation Protein Footprinting Proteins Proteobacteria Regulation Research Role Signal Transduction Site-Directed Mutagenesis Source Streptococcus Structure Surface System Testing Transcriptional Regulation X-Ray Crystallography in vivo insight mutant nitrogen metabolism novel pathogen pathogenic bacteria prevent protein protein interaction research study response transcription factor

项目摘要

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.

描述（由申请人提供）：低 G+C 革兰氏阳性细菌枯草芽孢杆菌中的氮代谢受新型调节系统控制，其中谷氨酰胺合成酶是谷氨酰胺合成和直接控制两种转录因子 GlnR 和 TnrA 活性所必需的。谷氨酰胺合成酶的反馈抑制形式通过形成稳定的 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短暂结合而激活的，从而稳定 GlnR-DNA 复合物。有趣的是，这种相同的 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型谷氨酰胺合成酶调节 GlnR 和 TnrA 活性的分子机制。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 端区域自动抑制 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质-蛋白质界面将使用突变、生化和结构方法进行表征。定点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该过程受到细胞氮状态的影响。通过确定生物膜基质蛋白 TasA 的氮调节和 sinR 突变体中谷氨酰胺合成酶表达减少的机制，将探索氮代谢和生物膜形成之间的相互关系。遗传实验表明，谷氨酰胺合成酶突变体中观察到的组成型生物膜形成是由于 Spo0A 磷酸化水平增加所致。这将通过检查野生型和突变细胞中 Spo0A~P 依赖性 lacZ 融合体的表达来证实。公共卫生相关性：生理学的一个基本问题是细菌如何适应不同氮源的生长。拟议的研究将研究低 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，因此这些研究将深入了解这些细菌中氮代谢的调节方式。