Regulation of the Bacillus subtilis yciC Gene and Insights into the DNA-Binding Specificity of the Zinc-Sensing Metalloregulator Zur

Zinc is an essential nutrient used both as a structural cofactor for protein folding and as a catalytic cofactor for many enzymes. However, at high levels, zinc can be toxic (3). It is therefore crucial that cells tightly regulate zinc levels within the cell. This involves, in part, the regulated expression of uptake and efflux proteins. It has been noted that the equilibrium levels of free zinc, as sensed by the well-characterized Escherichia coli zinc metalloregulators Zur and ZntR, are in the femtomolar range (25). In Bacillus subtilis, we have observed a similar high affinity for the homologous Zur protein (unpublished results). Since this corresponds to essentially no free zinc in the cell, zinc is presumably chaperoned within the cell by one or more metallochaperones analogous to those that direct intracellular copper trafficking (9). To date, however, no zinc metallochaperone has been described in detail. One candidate for such a function is B. subtilis YciC, an abundant cytosolic protein regulated by Zur. The B. subtilis Zur protein represses expression of at least 10 genes in response to zinc sufficiency (14, 26). Orthologs of Zur have been found in a wide range of species, including E. coli, Mycobacterium tuberculosis, Salmonella enterica, and Staphylococcus aureus (4, 20, 21, 27). In B. subtilis, the genetic response to zinc starvation includes, as expected, the derepression of a high-affinity zinc uptake system, an ABC transporter encoded by the ycdHI-yceA operon (13). Zur also represses three genes (ytiA, rpmGC, and yhzA) encoding paralogs of ribosomal proteins (26). The ytiA gene encodes an alternative form of L31 lacking zinc. L31 (encoded by rpmE) is a small, zinc-containing protein that associates with the large ribosomal subunit (23). When zinc is limiting in the cell, YtiA is expressed and displaces L31 (RpmE) from the ribosome, which is then postulated to liberate zinc for essential cellular functions (1). The RpmGC protein, encoding a zinc-free L33 paralog, is postulated to play a similar role by displacement of one or both of the other two zinc-containing L33 proteins (encoded by rpmGA and rpmGB). Finally, the Zur-regulated YhzA protein can functionally replace S14, a zinc metalloprotein required for ribosomal assembly. Expression of YhzA provides a “fail-safe” mechanism to allow continued ribosome assembly even under severe zinc limitation (24). Recently, insights have begun to emerge into the functions of the remaining Zur-regulated genes. ZinT is postulated to function in zinc trafficking, but few details are understood. YciA represents a novel class of GTP cyclohydrolases, enzymes typically requiring a zinc metal cofactor (11). When there is insufficient zinc to support the catalytic activity of MtrA, a zinc-containing GTP cyclohydrolase essential for folate biosynthesis, derepression of YciA allows continued growth (unpublished results). YciC was originally identified as an abundant, membrane-associated protein in extracts of zur mutant cells. Early studies suggested that a yciC mutation further exacerbated the growth defect of a transporter-deficient strain under zinc limitation (13). This observation led to the speculation that this protein might play a secondary role in zinc uptake. However, protein sequence comparisons indicated that YciC has similarities with factors implicated in protein metallation reactions, suggesting that YciC may instead be involved as a metallochaperone (19). It is not yet known whether YciC functions as a zinc metallochaperone or perhaps as a chaperone for one or more other metal ions. Here, we report a series of studies performed to define the genetic requirements for the zinc-dependent repression of yciC transcription. Although also expressed as part of the Zur-regulated yciABC operon, the bulk of yciC transcripts initiate from within the yciB-yciC intergenic region. We demonstrate that this region contains a σA-dependent promoter and two Zur boxes separated by nearly 200 bp. While either Zur box can mediate zinc-responsive repression of yciC, complete repression requires the promoter-proximal C2 box. DNA-binding studies demonstrated that Zur recognizes a minimal operator site (a 9-1-9 inverted sequence motif) that is somewhat larger than that reported for other characterized Fur family members.