Regulation of Zinc Homeostasis in Bacillus subtilis

枯草芽孢杆菌中锌稳态​​的调节

• 批准号: 9905682
• 负责人: John Helmann
• 金额: $ 27万
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9905682&HistoricalAwards=false
• 关键词: Regulation; Zinc; Homeostasis; Bacillus; subtilis

Helmann The Bacillus subtilis Zur protein is a zinc activated repressor that binds to the promoter regions of operons implicated in zinc uptake. The goal of this project is to characterize the Zur protein and its interactions with both operator DNA and metal ions. Zur represses two transport systems that import zinc. One is YciC, part of a low affinity system, while the other is an ABC transporter. This research will define the zinc-regulated promoter regions and their interactions with Zur. The interaction of Zur with DNA will be studied using electrophoretic mobility shift assays and DNaseI footprinting. Zur can be purified both in an active form and an inactive apo-protein form which will allow the effects of metal ions on binding affinity and cooperativity to be determined. Amino acids important for metal-sensing will be identified by site-directed mutagenesis followed by functional studies. Finally, transcriptional profiling, genome searches for other candidate Zur-regulated genes, and selection for mutants altered in zinc homeostasis, will allow an assessment of the global effects of zinc and Zur on cell physiology. In all cases, the results obtained will be compared with analogous studies of other metalloregulators to better understand the evolution of this family of proteins. Metal ions are essential nutrients for all organisms and both uptake and efflux are tightly regulated. The regulation of metal ion homeostasis is often mediated by metal-sensing regulatory proteins that activate or repress transport pathways. Bacillus subtilis provides an attractive model system for these studies since it is a genetically well-characterized microbe found in soil, an environment with highly variable metal ion availability. B. subtilis encodes four distinct metalloregulatory proteins that sense metal ion status and regulate gene expression accordingly. Three of these proteins are homologs of the ferric uptake repressor (Fur) protein of Escherichia coli. One of these proteins, Zur, is a specific sensor of zinc that regulates zinc transport. This research investigates the molecular basis of zinc-binding and transcriptional control by Zur.

赫尔曼 枯草芽孢杆菌Zur蛋白是锌激活的阻遏物，其结合到涉及锌摄取的操纵子的启动子区。该项目的目标是表征Zur蛋白及其与操纵基因DNA和金属离子的相互作用。Zur抑制了两个输入锌的运输系统。一个是YciC，低亲和力系统的一部分，而另一个是ABC转运蛋白。本研究将确定锌调控的启动子区域及其与Zur的相互作用。将使用电泳迁移率变动分析和DNaseI足迹法研究Zur与DNA的相互作用。Zur可以以活性形式和非活性脱辅基蛋白形式纯化，这将允许确定金属离子对结合亲和力和协同性的影响。对金属传感重要的氨基酸将通过定点诱变随后进行功能研究来鉴定。最后，转录分析，基因组搜索其他候选的锌调节基因，并选择突变体改变锌稳态，将允许锌和Zur对细胞生理学的全球影响的评估。在所有情况下，所获得的结果将与其他金属调节剂的类似研究进行比较，以更好地了解该蛋白质家族的进化。金属离子是所有生物体必需的营养物质，其吸收和流出都受到严格的调节。金属离子稳态的调节通常由激活或抑制转运途径的金属敏感调节蛋白介导。枯草芽孢杆菌为这些研究提供了一个有吸引力的模型系统，因为它是一种在土壤中发现的遗传特征良好的微生物，土壤是一种具有高度可变的金属离子可用性的环境。B。枯草芽孢杆菌编码四种不同的金属调节蛋白，其感测金属离子状态并相应地调节基因表达。这些蛋白质中的三个是大肠杆菌的铁摄取抑制蛋白（Fur）的同源物。这些蛋白质之一，Zur，是一种特定的锌传感器，调节锌的运输。本研究探讨Zur的锌结合和转录调控的分子基础。