Regulation of Zinc Homeostasis in Bacillus subtilis

赫尔曼，枯草杆菌Zur蛋白，是一种锌激活的阻遏蛋白，与锌摄取相关操纵子的启动子区域结合。该项目的目标是表征Zur蛋白及其与操纵者DNA和金属离子的相互作用。Zur压制了两个进口锌的运输系统。一个是YciC，低亲和力系统的一部分，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。Zur与DNA的相互作用将用电泳迁移率改变分析和DNaseI足迹法来研究。Zur既可以以活性形式纯化，也可以以非活性脱辅蛋白形式纯化，这将允许确定金属离子对结合亲和力和协作性的影响。对金属感应重要的氨基酸将通过定点突变和功能研究来鉴定。最后，转录图谱、其他候选Zur调控基因的基因组搜索以及锌稳态改变突变的选择，将使我们能够评估锌和Zur对细胞生理学的整体影响。在所有情况下，获得的结果都将与其他金属调节剂的类似研究进行比较，以更好地了解这一蛋白质家族的进化。金属离子是所有生物体必需的营养物质，吸收和排出都受到严格的调控。金属离子动态平衡的调节通常是由激活或抑制转运通路的金属感应调节蛋白介导的。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具有良好遗传学特征的微生物，土壤是一个高度可变的金属离子可获得性环境。枯草杆菌编码四种不同的金属调节蛋白，它们可以感知金属离子状态，并相应地调节基因表达。这些蛋白中有三个是大肠杆菌铁吸收抑制蛋白(Fur)的同源物。其中一种蛋白质Zur是锌的特殊感受器，调节锌的运输。本研究探讨了Zur对锌结合和转录调控的分子基础。

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.