Effector binding by Bacillus subtilit CodY

枯草芽孢杆菌 CodY 的效应子结合

• 批准号: 7331685
• 负责人: LUKE D HANDKE
• 金额: $ 4.68万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7331685
• 关键词: Address; Affinity; Amino Acids; Bacillus (bacterium); Bacillus subtilis; Bacteria; Bacterial Infections; Binding; Biochemical; Biological Assay; Branched-Chain Amino Acids; Cells; Chromosomes; Circular Dichroism; Crystallography; DNA; DNA Binding; DNA Microarray Chip; DNA Microarray format; DNase-I Footprinting; Development; Disruption; Essential Amino Acids; Fluorescence Spectrometry; GTP Binding; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Gram-Positive Bacteria; Guanosine Triphosphate; In Vitro; Isoleucine; Microarray Analysis; Molecular; Mutation; Nutrient; Physiological; Proteins; Proteolysis; Regulation; Reporter; Resolution; Site-Directed Mutagenesis; Spectroscopy, Fourier Transform Infrared; Starvation; Structure; Virulence Factors; Work; antimicrobial; base; detection of nutrient; mutant; novel; pathogen; research study; response

DESCRIPTION (provided by applicant): Bacillus subtilis employs several adaptive strategies when faced with nutrient limitation. Expression of many of these adaptive mechanisms is under regulation by CodY, a highly conserved protein in Gram-positive bacteria. CodY senses nutrient availability by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon effector binding, CodY is able to bind to and repress its DNA targets. It is uncertain which amino acids in CodY are necessary for interaction with these effectors, and it is unclear how effector binding alters CodY such that it interacts with DNA. The goal of this project is to address both of these underlying questions. As CodY has been shown to regulate expression of virulence factors in several bacterial pathogens, it is an attractive target for novel antimicrobial therapy. However, development of this line of therapy will require examination of these basic issues concerning CodY activity. The first aim of this application will identify amino acids essential for effector binding. Homology searches and x-ray crystallography studies have yielded some clues about which CodY residues may be important for GTP and BCAA binding, respectively. These residues will be mutagenized, and their impact on effector binding will be determined. The second aim of this application will investigate how effector binding increases CodY's affinity for DNA. Partial proteolysis has indicated that a conformational change occurs in CodY upon binding to BCAAs, but no change was observed upon CodY binding to GTP. Near-UV circular dichroism, spectrofluorometry, and Fourier transform infrared spectroscopy will be used to identify whether GTP induces a subtle alteration in the tertiary structure of CodY. Experiments in the third aim will assess the physiological impact of the loss of CodY effector binding on gene expression. These codY mutations will be introduced into the B. subtilis chromosome, and their effects will be assayed by reporter fusion experiments and microarray analysis. The results of this work will allow us to understand how bacteria regulate gene expression in response to nutrient starvation. Disruption of this regulation may result in a novel means of treatment of certain bacterial infections.

描述（由申请人提供）：枯草芽孢杆菌在面临营养限制时采用多种适应性策略。许多这些适应性机制的表达都受到 CodY 的调节，CodY 是革兰氏阳性细菌中高度保守的蛋白质。 CodY 通过与其效应器、GTP 和支链氨基酸 (BCAA) 相互作用来感知营养物质的可用性。效应子结合后，CodY 能够结合并抑制其 DNA 靶标。目前尚不清楚 CodY 中的哪些氨基酸是与这些效应子相互作用所必需的，也不清楚效应子结合如何改变 CodY 使其与 DNA 相互作用。该项目的目标是解决这两个根本问题。由于 CodY 已被证明可以调节几种细菌病原体中毒力因子的表达，因此它是新型抗菌治疗的一个有吸引力的靶点。然而，该疗法的开发需要检查有关 CodY 活性的这些基本问题。该应用的首要目标是鉴定效应子结合所必需的氨基酸。同源性搜索和 X 射线晶体学研究已经提供了一些线索，说明哪些 CodY 残基可能分别对 GTP 和 BCAA 结合很重要。这些残基将被诱变，并确定它们对效应子结合的影响。该应用的第二个目标是研究效应子结合如何增加 CodY 对 DNA 的亲和力。部分蛋白水解表明 CodY 在与 BCAA 结合后发生构象变化，但在 CodY 与 GTP 结合后未观察到任何变化。近紫外圆二色性、荧光分光光度法和傅里叶变换红外光谱将用于鉴定 GTP 是否会引起 CodY 三级结构的细微改变。第三个目标的实验将评估 CodY 效应子结合丧失对基因表达的生理影响。这些 codY 突变将被引入枯草芽孢杆菌染色体，并通过报告基因融合实验和微阵列分析来测定其影响。这项工作的结果将使我们能够了解细菌如何调节基因表达以应对营养匮乏。破坏这种调节可能会产生治疗某些细菌感染的新方法。