Functional analysis of the Bacillus subtilis Zur regulon

Abstract

The Bacillus subtilis zinc uptake repressor (Zur) regulates genes involved in zinc uptake. We have used DNA microarrays to identify genes that are derepressed in a zur mutant. In addition to members of the two previously identified Zur-regulated operons (yciC and ycdHI-yceA), we identified two other genes, yciA and yciB, as targets of Zur regulation. Electrophoretic mobility shift experiments demonstrated that all three operons are direct targets of Zur regulation. Zur binds to an approximately 28-bp operator upstream of the yciA gene, as judged by DNase I footprinting, and similar operator sites are found preceding each of the previously described target operons, yciC and ycdHI-yceA. Analysis of a yciA-lacZ fusion indicates that this operon is induced under zinc starvation conditions and derepressed in the zur mutant. Phenotypic analyses suggest that the YciA, YciB, and YciC proteins may function as part of the same Zn(II) transport pathway. Mutation of yciA or yciC, singly or in combination, had little effect on growth of the wild-type strain but significantly impaired the growth of the ycdH mutant under conditions of zinc limitation. Since the YciA, YciB, and YciC proteins are not obviously related to any known transporter family, they may define a new class of metal ion uptake system. Mutant strains lacking all three identified zinc uptake systems (yciABC, ycdHI-yceA, and zosA) are dependent on micromolar levels of added zinc for optimal growth.

FIG. 1.

Identification of the Zur regulon by microarray analysis. (A) zur/WT expression ratios of B. subtilis genes are compared for two independent measurements made with RNAs from separate cultures. As an arbitrary cutoff value, genes showing expression ratios below threefold were considered unchanged (gray diamonds). Genes that constitute the Zur regulon are represented by closed squares, genes in the sbo operon are represented by closed circles, and genes with lowered expression in the zur mutant are represented by open squares. The apparent up-regulation of yckA (closed triangle) is likely an artifact due to convergent transcription from the strongly expressed yciC gene. (B) The zur/WT expression ratios of selected genes are illustrated. The results shown are averages of two measurements, and the error bars represent the ranges.

FIG. 2.

Effects of zinc ions on the expression of sbo and yciA were determined with cat-lacZ transcriptional fusions. Wild type (closed squares) or zur mutant (closed circles) cells containing an sbo′-cat-lacZ (A) or a yciA′-cat-lacZ (B) transcriptional fusion were grown to mid-logarithmic phase in LZMM with different concentrations of zinc, and β-galactosidase activity was determined.

FIG. 3.

Primer extension mapping of the transcription start sites of yciA (A) and ycdH (B). The primer extension product was generated with RNA isolated from either wild type (WT) or zur mutant cells grown in LB medium. The sequence ladders were generated by PCR cycle sequencing with the same primer as in the primer extension reaction.

FIG. 4.

The yciABC and ycdHI-yceA regions of B. subtilis. Open reading frames are indicated by open arrows, promoter sites are indicated by bent arrows, and Zur boxes are indicated by closed circles. Sequences of the three Zur-controlled promoters are shown with σ^A-dependent recognition sites (bold), and the residue corresponding to the predominant transcription start site is indicated by +1. Note that the yciC transcript initiates more than 200 bases upstream of the yciC start codon. Regions of Zur protection in the operators of yciA, yciC, and ycdH are underlined (see Fig. 5; data not shown).

FIG. 5.

Interaction of Zur with its target operons. (A) EMSA of Zur binding to the promoter regions of yciC, yciA, and ycdH. Zur protein (concentrations indicated in nanomolar monomer) was incubated with ³²P-end-labeled fragments and analyzed by 6% polyacrylamide gel electrophoresis. (B) DNase I footprinting analysis of Zur binding to the yciA promoter region. Zur protein was incubated with a ³²P-end-labeled DNA fragment, and a G+A chemical cleavage ladder was included for localization of the binding sites. The concentration of Zur (monomer) in each reaction was (from left to right) 0, 20, 40, or 100 nM. (C) Alignment of the Zur-protected DNA regions in the promoters of yciA, yciC, and ycdH. The bottom part of panel C compares the core region protected by Zur (a 7-1-7 inverted repeat motif designated a Zur box) with similar operator sites recognized by the Fur homolog PerR and by Fur (1, 17).

FIG. 6.

Effects of EDTA on the growth of the wild type and zinc uptake mutants. Inhibition of the growth of the wild type (♦) and the ycdH (▵), ycdH yciA (○), ycdH yciC (•), and yciA ycdH zosA (⋄) mutants by EDTA is shown. The zosA, yciA, and yciC single mutants and the yciA-yciC double mutant were indistinguishable from the wild type. For each strain, the diameter of the zone of growth inhibition was determined on MM plates overlaid with a 0.6-cm-diameter filter paper disk containing 10 μl of EDTA at either 10, 100, or 500 mM. Values represent the average of three independent measurements with an average error of ± 0.1 cm.

FIG. 7.

Effect of zinc limitation on the growth of the wild type and zinc uptake mutants. (A) Growth curves of the wild type (♦) and the yciA (▪), ycdH (▴), ycdH yciA (○), yciA zosA (□), ycdH zosA (▵), and yciA ycdH zosA (⋄) mutants in LZMM with 500 nM Zn(II). (B) Effect of zinc concentration on the growth yield (10 h after 1:100 dilution into LZMM supplemented as indicated) of the wild type (♦) and the yciA ycdH zosA mutant (⋄). OD₆₀₀, optical density at 600 nm.