Molecular Mechanisms Of Zinc Homeostasis In Bacillus Subtilis Mediated By Zur, A Fur Family Member.

Zinc is an essential nutrient due to its role as a structural co-factor for protein folding and as a catalytic co-factor for many enzymes. However, if this nutrient accumulates over a given threshold, it can become toxic to the cell. For these reasons, it is absolutely critical for cell survival that zinc homeostasis be tightly controlled. In Bacillus subtilis, a model Gram positive organism, the response to zinc limitation is mediated by Zur which acts as a classical repressor when Zinc is present. The Zur regulon has been characterized and currently contains ten genes all suspected or known to contribute to the zinc starvation response. Classically this response was thought of in terms of obtaining external zinc by high affinity pumps. In part, the work presented within will show that our understanding bacterial metal ion homeostasis is expanding. We now understand that in addition to uptake as a metal limitation response, cells also create duplicates of zinc requiring proteins which have altered metal co-factor specificity, a mechanism we classify as substitution. Finally, bacteria use mobilization as a response mechanism by creating conditions in which zinc containing proteins are replaced and the zinc which they contained is used for continued growth in zinc limiting conditions. The work presented here shows a cross-section of the molecular mechanisms Zur employs to achieve zinc homeostasis in Bacillus subtilis. Starting at the protein biochemistry level, I will present studies aimed at addressing how Zur senses zinc. This work uses a site directed mutagenesis approach to identify amino acid residues which make up the three conserved zinc binding pockets within the Zur protein and how each contributes to Zur's function. Second, after understanding how the protein senses zinc, I sought to understand how Zur regulated one of the uncharacterized members of its regulon, the yciC gene. In addition to elucidating the regulation of the unique promoter structure of yciC, I also show that Zur binds a consensus 9-1-9 inverted repeat. Finally at a physiological level, I studied the three ribosomal genes under the control of Zur to understand their contribution towards the zinc starvation response