Regulation of Zinc Homeostasis in Bacillus subtilis

枯草芽孢杆菌Zur蛋白是一种锌活化抑制因子，它与涉及锌摄取的操纵子的启动子区域结合。该项目的目标是表征Zur蛋白及其与操作符DNA和金属离子的相互作用。Zur抑制两个进口锌的运输系统。一个是低亲和系统的YciC，另一个是ABC转运体。本研究将确定锌调控的启动子区域及其与Zur的相互作用。Zur与DNA的相互作用将通过电泳迁移位移测定和DNA足迹来研究。Zur可以以活性形式和非活性载脂蛋白形式纯化，这将允许确定金属离子对结合亲和力和协同性的影响。对金属感应重要的氨基酸将通过位点定向诱变和功能研究来鉴定。最后，转录谱分析，基因组搜索其他候选Zur调节基因，以及选择锌稳态改变的突变体，将允许评估锌和Zur对细胞生理的整体影响。在所有情况下，获得的结果将与其他金属调节因子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，其吸收和排出都受到严格调节。金属离子稳态的调节通常是由金属感应调节蛋白介导的，这些蛋白激活或抑制运输途径。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，土壤是一个具有高度可变金属离子可用性的环境。枯草芽孢杆菌编码四种不同的金属调节蛋白，它们感知金属离子状态并相应地调节基因表达。其中三种蛋白是大肠杆菌铁摄取抑制蛋白（Fur）的同源物。其中一种蛋白质，Zur，是一种特殊的锌传感器，可以调节锌的运输。本研究探讨了Zur与锌结合的分子基础及其转录调控。

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.