Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis

Abstract

Zinc is an essential nutrient for all cells, but remarkably little is known regarding bacterial zinc transport and its regulation. We have identified three of the key components acting to maintain zinc homeostasis in Bacillus subtilis. Zur is a metalloregulatory protein related to the ferric uptake repressor (Fur) family of regulators and is required for the zinc-specific repression of two operons implicated in zinc uptake, yciC and ycdHIyceA. A zur mutant overexpresses the 45-kDa YciC membrane protein, and purified Zur binds specifically, and in a zinc-responsive manner, to an operator site overlapping the yciC control region. A similar operator precedes the ycdH-containing operon, which encodes an ABC transporter. Two lines of evidence suggest that the ycdH operon encodes a high-affinity zinc transporter whereas YciC may function as part of a lower-affinity pathway. First, a ycdH mutant is impaired in growth in low-zinc medium, and this growth defect is exacerbated by the additional presence of a yciC mutation. Second, mutation of ycdH, but not yciC, alters the regulation of both the yciC and ycdH operons such that much higher levels of exogenous zinc are required for repression. We conclude that Zur is a Fur-like repressor that controls the expression of two zinc homeostasis operons in response to zinc. Thus, Fur-like regulators control zinc homeostasis in addition to their previously characterized roles in regulating iron homeostasis, acid tolerance responses, and oxidative stress functions.

FIG. 1

Multiple sequence alignment of Fur-like regulatory proteins. B. subtilis encodes three Fur-like regulatory proteins: Fur (BsuFur), PerR (BsuPerR), and Zur (BsuZur) (5, 23). These proteins are aligned against the E. coli Fur (EcoFur) (32) and Zur (EcoZur) (33) proteins and a Fur-like regulatory protein from S. epidermidis (SepFur) (16) that is closely related to B. subtilis Zur. The amino-terminal domain contains a proposed helix-turn-helix motif with a conserved recognition helix. The carboxyl-terminal metal-binding domain contains a cluster of conserved His and Cys residues.

FIG. 2

Regulation of YciC by Zur. Membrane protein fractions from wild-type (WT) and zur mutant strains grown in minimal medium were fractionated by SDS-PAGE (12% gel) and stained with Coomassie blue. The abundant 45-kDa protein present in the zur mutant strain is indicated. Apart from YciC, no other changes in protein expression were visible on this gel.

FIG. 3

Regulation of yciC as determined by using a yciC-cat-lacZ transcriptional fusion. Cells were grown overnight in MM containing either no additional metal ions or 5 μM indicated divalent cation, and β-galactosidase (Beta-gal) activity was determined. Expression of the yciC-cat-lacZ fusion is constitutive in the zur mutant strain HB8011 (right).

FIG. 4

Purification and DNA-binding selectivity of Zur. (A) SDS-PAGE analysis of Zur apoprotein purified from E. coli. Lanes: M, molecular weight markers (the last five bands are 66, 45, 31, 21, and 14 kDa; Zur runs just below the 21-kDa marker); 1, uninduced cell extract; 2, induced cell extract; 3, pooled fractions from QAE-Sepharose; 4, fraction from Superdex-75. (B) Electrophoretic mobility shift analysis of Zur apoprotein in the presence of yciC promoter fragments (top band) and an unrelated DNA fragment (from the B. subtilis yknW gene). Lane 1, DNA alone. Lanes 2 to 10 contain DNA plus 200 ng of Zur with 100 μM metal ions as follows: lane 2, none; lane 3, Zn²⁺; lane 4, Zn²⁺ plus 25 mM EDTA; lane 5, Cd²⁺; lane 6, Cu²⁺; lane 7, Mn²⁺; lane 8, Ni²⁺; lane 9, Co²⁺; lane 10, Fe³⁺. The formation of a protein-DNA complex is evident from slower migration of the yciC promoter fragment in lanes amended with Zn or Mn.

FIG. 5

(A) The yciC promoter region contains a Fur box-like sequence overlapping a DraI site (TTTAAA; site underlined) that is protected against digestion in presence of Zur. A similar operator-like sequence is found in the promoter region of the ycdH operon. (B) The genomic context of the yciC and ycdH ycdI yceA transcription units is illustrated. Each line represents a 5-kbp segment of the B. subtilis genome with boundaries indicated in kilobase pairs, based on the complete genome sequence (20). The DNA sequences preceding the yciC and ycdH genes are shown to illustrate putative ς^A-like promoter elements (in bold) and the relative positions of the operator-like sequences (underlined). Proposed transcription terminator sites (T) and a possible termination site within the ycdH-containing operon (t) are indicated.

FIG. 6

Effects of Zn and EDTA on growth of wild-type (WT) and yciC, ycdH, and yciC ycdH mutant strains. (A) Cell optical density (OD) after overnight growth in LZMM (light bars) or LZMM supplemented with 10 μM Zn²⁺ (dark bars) is indicated for each strain. The results are representative of experiments performed three or more times. (B) Growth inhibition by EDTA. For each strain, the diameter of the zone of growth inhibition was determined on MM plates surrounding a 0.6-cm-diameter filter paper disk containing 10 μl of EDTA at either 10, 100, or 500 mM. Strains are wild type (○), yciC (•), ycdH (□), and yciC ycdH (▴). Values represent the overall diameter of inhibition minus the diameter of the filter and are reproducible to within 0.2 cm.

FIG. 7

Effects of yciC and ycdH mutations on regulation. β-Galactosidase (β-gal) assays were performed for strains containing the yciC-cat-lacZ (A) or ycdH-cat-lacZ (B) transcriptional fusion after overnight growth in LZMM supplemented with Zn at the indicated concentration. The fusions were assayed in the wild-type (○) and the ycdH (□) and yciC ycdH (▴) mutant backgrounds. In the yciC mutant background, the results were indistinguishable from those for the wild type (not shown). The curves shown are best fits to the experimental data as described in Materials and Methods.