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）：面对营养限制时，枯草芽孢杆菌采用了几种自适应策略。这些自适应机制的许多表达受到革兰氏阳性细菌中高度保守的蛋白质Cody的调节。 Cody通过与其效应子GTP和分支链氨基酸（BCAA）相互作用来感知养分的可用性。在效应子结合后，Cody能够结合并抑制其DNA靶标。尚不确定Cody中哪种氨基酸对于与这些效应子的相互作用是必需的，并且尚不清楚效应子结合如何改变Cody与DNA相互作用。该项目的目的是解决这两个基本问题。由于已经显示Cody调节了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有吸引力的靶标。但是，这种治疗方案的发展将需要检查有关Cody活动的这些基本问题。该应用的第一个目的将鉴定出对效应子结合必不可少的氨基酸。同源性搜索和X射线晶体学研究产生了一些线索，即哪些Cody残基对GTP和BCAA结合可能很重要。这些残基将被诱变，并确定它们对效应子结合的影响。该应用的第二个目的将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，在与BCAA结合时，Cody发生了构象变化，但是在Cody与GTP结合时未观察到变化。将使用近紫外圆形二角运动，光谱荧光测定法和傅立叶变换红外光谱法来确定GTP是否引起了Cody的第三级结构的细微改变。第三个目标中的实验将评估Cody效应子结合对基因表达的丧失的生理影响。这些Cody突变将引入枯草芽孢杆菌染色体，并将通过记者融合实验和微阵列分析来测定它们的作用。这项工作的结果将使我们能够理解细菌如何响应营养饥饿的基因表达。这种调节的破坏可能会导致一种新的细菌感染治疗方法。