Effector binding by Bacillus subtilit CodY

枯草芽孢杆菌 CodY 的效应子结合

• 批准号: 7484169
• 负责人: LUKE D HANDKE
• 金额: $ 1.47万
• 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 2008-11-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7484169
• 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; 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; Site-Directed Mutagenesis; Spectroscopy, Fourier Transform Infrared; Starvation; Structure; Virulence Factors; Work; antimicrobial; base; detection of nutrient; 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)：枯草芽孢杆菌在面对营养限制时采用了几种适应性策略。其中许多适应机制的表达受到Cody的调控，Cody是革兰氏阳性细菌中的一种高度保守的蛋白质。Cody通过与其效应器、GTP和支链氨基酸(BCAAs)相互作用来感知营养的有效性。在效应器结合后，Cody能够结合并抑制其DNA靶标。目前尚不确定Cody中哪些氨基酸是与这些效应器相互作用所必需的，也不清楚效应器结合如何改变Cody，使其与DNA相互作用。该项目的目标是解决这两个基本问题。由于Cody已被证明可以调节几种细菌病原体的毒力因子的表达，因此它是新的抗菌治疗的一个有吸引力的靶点。然而，这条治疗路线的发展将需要检查这些与Cody活动有关的基本问题。这项应用的第一个目标是识别效应器结合所必需的氨基酸。同源性搜索和X射线结晶学研究已经得出了一些线索，即哪些Cody残基可能分别对GTP和BCAA结合重要。这些残基将被诱变，它们对效应器结合的影响将被确定。这项应用的第二个目的将研究效应器结合如何增加Cody对DNA的亲和力。部分蛋白质降解表明Cody与支链氨基酸结合时发生构象变化，但Cody与GTP结合时没有观察到变化。近紫外圆二色谱、荧光分光光度和傅里叶变换红外光谱将用于鉴定GTP是否会导致Cody的三级结构发生细微的变化。第三个目标的实验将评估Cody效应器结合丢失对基因表达的生理影响。这些Cody突变将被引入枯草杆菌染色体，并将通过报告融合实验和微阵列分析来检测它们的效果。这项工作的结果将使我们能够理解细菌如何调节基因表达来应对营养饥饿。打破这一规定可能会导致一种治疗某些细菌感染的新方法。