Regulation of Zinc Homeostasis in Bacillus subtilis

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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.