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J. Bacteriol. doi:10.1128/JB.01978-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Regulation of the Bacillus subtilis yciC gene and insights into the DNA-binding specificity of the zinc-sensing metalloregulator Zur

Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA

^* To whom correspondence should be addressed. Email: dh9{at}cornell.edu.

	Abstract

The Bacillus subtilis Zur protein regulates zinc homeostasis by repressing at least ten genes in response to zinc sufficiency. One of these genes, yciC, encodes an abundant protein postulated to function as a metallochaperone. Here, we used a genetic approach to identify the cis-acting elements and trans-acting factors contributing to the tight repression of yciC. Initial studies led to the identification of only trans-acting mutations and, when the selection was repeated using a transposon library, all recovered mutants contained insertionally inactivated zur. Using a zur merodiploid strain, we obtained two cis-acting mutations that contained large deletions in the yciC regulatory region. We demonstrate that the yciC regulatory region contains two functional Zur boxes: a primary site (C2) overlapping a ^A promoter 200 bp upstream of yciC, and a second site near the translational start point (C1). Zur binds to both of these sites to mediate strong, zinc-dependent repression of yciC. Deletion studies indicate that either Zur box is sufficient for repression, although repression by Zur bound to C2 is more efficient. Binding studies demonstrate that both sites bind Zur with high affinity. Sequence alignment of these and previously described Zur boxes suggest that Zur recognizes a more extended operator than other Fur family members. We have used synthetic oligonucleotides to identify bases critical for DNA-binding by Zur. Unlike Fur and PerR, which bind efficiently to sequences containing a core 7-1-7 repeat element, Zur requires a 9-1-9 inverted repeat for high affinity binding.