Regulation of glutamate synthesis in Bacillus subtilis

枯草芽孢杆菌谷氨酸合成的调控

• 批准号: 7526791
• 负责人: BORIS R BELITSKY
• 金额: $ 28.79万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7526791
• 关键词: Aconitate Hydratase; Address; Affinity; Assimilations; Bacillus subtilis; Bacteria; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biological Assay; Carbon; Cells; Citrate; Citrate (si)-Synthase; Citrates; Citric Acid Cycle; Complex; Coupling; DNA; Defect; Depth; Enzymes; Equilibrium; Event; Feedback; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Glutamate Dehydrogenase; Glutamate Synthase; Glutamate-Ammonia Ligase; Glutamates; Glutamine; Goals; Gram-Positive Bacteria; Growth; In Vitro; Iron; Isocitrate Dehydrogenase; Knowledge; Life; Life Style; Listeria; Listeria monocytogenes; Listeriosis; Measures; Messenger RNA; Metabolic; Metabolism; Modeling; Molecular; Molecular Conformation; Molecular Models; Mutate; Nitrogen; Nitrogen Fixation Genes; Nutritional; Operon; Pathogenesis; Pathway interactions; Personal Satisfaction; Phenotype; Physiological; Physiology; Protein Overexpression; Proteins; Proteolysis; Public Health; RNA Binding; Regulation; Resistance; Role; Series; Source; Staging; Stress; Structure; Study models; Techniques; Testing; Transcript; Tricarboxylic Acids; Virulence; Virulence Factors; Work; base; cell type; design; enzyme activity; in vivo; inhibitor/antagonist; interest; iron metabolism; mouse model; mutant; nitrogen metabolism; novel; pathogen; research study; response

DESCRIPTION (provided by applicant): Management of key metabolic intersections is a critical issue in bacterial economy. One such intersection, the interconversion of a-ketoglutarate and glutamate, is particularly important and interesting because it represents the point of coupling between central carbon metabolism and central nitrogen metabolism. Synthesis of the enzymes of the tricarboxylic acid branch of the Krebs cycle (citrate synthase, aconitase and isocitrate dehydrogenase) that produce a-ketoglutarate and the enzymes of nitrogen assimilation (glutamine synthetase, glutamate synthase and glutamate dehydrogenase) are encoded by genes that are subject to multiple forms of regulation. This project seeks to understand in detail the molecular mechanisms that regulate these genes in Bacillus subtilis, a well studied model Gram-positive bacterium, and Listeria monocytogenes, a model intracellular pathogen whose metabolic regulation is relatively unexplored. The proposal focuses on three transcriptional regulators (CcpC, GltC and GlnR) and two enzymes (glutamate dehydrogenase and aconitase) that have post-transcriptional regulatory functions. The aims of this proposal are: (a) to determine the molecular mechanisms that control the synthesis and activity of the tricarboxylic acid branch enzymes; (b) to reveal the molecular mechanisms that control the synthesis of glutamate and glutamine; and, (c) to determine the role of aconitase as a post-transcriptional regulator of sporulation in B. subtilis and of iron metabolism and virulence genes in L. monocytogenes. These issues will be addressed using a combination of genetic and biochemical techniques. The phenotypes of mutants defective in each of the regulatory factors will be determined and the proteins will be purified and tested using in vitro transcription and DNA and RNA binding experiments. Since L. monocytogenes mutants that lack two repressors of the Krebs cycle genes have a significant defect in virulence in a mouse model, the molecular basis for this defect will be explored. PUBLIC HEALTH RELEVANCE: Maintaining the proper balance in intracellular metabolism is a critical factor in bacterial growth, survival and pathogenesis. This proposal aims to unravel the intricate mechanisms by which two related bacterial species, Bacillus subtilis (a non-pathogen) and Listeria monocytogenes (the causative agent of listeriosis), control the flow of metabolites between central carbon and central nitrogen pathways. The knowledge gained will provide a deeper understanding of pathogenic mechanisms and may aid in designing novel inhibitors of bacterial growth and pathogenesis.

描述（由申请人提供）：关键代谢交叉点的管理是细菌经济中的关键问题。这样的交叉点之一，即酮戊二酸和谷氨酸的相互转换，特别重要且有趣，因为它代表了中央碳代谢和中央氮代谢之间的耦合点。克雷布斯循环的三羧酸分支的酶的合成（柠檬酸酸合成酶，刺刺酶和异位酸脱氢酶）产生a-酮戊二酸酯，氮的酶，以及氮的同在（通过谷氨酸合酶，谷氨酸酶合成酶）的多种形式，该酶的多种形式是谷氨酸合成酶，该酶是该酶的多种形式，该酶是谷氨酸合成酶的， 规定。该项目试图详细了解调节枯草芽孢杆菌中这些基因的分子机制，一种研究了经过良好研究的革兰氏阳性细菌和单核细胞增生李斯特菌，这是一种模型的细胞内病原体，其代谢调节相对尚未探索。该提案侧重于具有转录后调节功能的三个转录调节剂（CCPC，GLTC和GLNR）和两种酶（谷氨酸脱氢酶和粘结酶）。该提案的目的是：（a）确定控制三羧酸分支酶的合成和活性的分子机制； （b）揭示控制谷氨酸和谷氨酰胺合成的分子机制； （c）确定痤疮酶在枯草芽孢杆菌中的孢子体和铁代谢和毒力基因中的孢子孢子的转录后调节剂的作用。这些问题将通过遗传和生化技术的结合来解决。将确定每个调节因素中突变体有缺陷的表型，并使用体外转录以及DNA和RNA结合实验对蛋白质进行纯化和测试。由于缺乏两个克雷布斯循环基因的两个阻遏物的单核细胞增生李斯特菌突变体在小鼠模型中具有显着的毒力缺陷，因此将探索该缺陷的分子基础。公共卫生相关性：保持细胞内代谢的适当平衡是细菌生长，生存和发病机理的关键因素。该提案旨在揭示两种相关的细菌物种，即枯草芽孢杆菌（非病原体）和单核细胞增生李斯特菌（李斯特氏病的病因），控制中央碳和中央氮途径之间的代谢流动。获得的知识将提供对致病机制的更深入的了解，并可能有助于设计新型细菌生长和发病机理的抑制剂。