Regulation and activity of a zinc uptake regulator, Zur, in Corynebacterium diphtheriae

Abstract

Regulation of metal ion homeostasis is essential to bacterial cell survival, and in most species it is controlled by metal-dependent transcriptional regulators. In this study, we describe a Corynebacterium diphtheriae ferric uptake regulator-family protein, Zur, that controls expression of genes involved in zinc uptake. By measuring promoter activities and mRNA levels, we demonstrate that Zur represses transcription of three genes (zrg, cmrA, and troA) in zinc-replete conditions. All three of these genes have similarity to genes involved in zinc uptake. Transcription of zrg and cmrA was also shown to be regulated in response to iron and manganese, respectively, by mechanisms that are independent of Zur. We demonstrate that the activity of the zur promoter is slightly decreased under low zinc conditions in a process that is dependent on Zur itself. This regulation of zur transcription is distinctive and has not yet been described for any other zur. An adjacent gene, predicted to encode a metal-dependent transcriptional regulator in the ArsR/SmtB family, is transcribed from a separate promoter whose activity is unaffected by Zur. A C. diphtheriae zur mutant was more sensitive to peroxide stress, which suggests that zur has a role in protecting the bacterium from oxidative damage. Our studies provide the first evidence of a zinc specific transcriptional regulator in C. diphtheriae and give new insights into the intricate regulatory network responsible for regulating metal ion concentrations in this toxigenic human pathogen.

FIG. 1.

Genetic arrangement of the C. diphtheriae and M. tuberculosis zur loci. (A) The dark gray arrow represents glyS, a glycyl-tRNA synthetase beta subunit. The gray striped arrow represents an arsR-like gene, a putative metal-dependent transcriptional regulator. The black arrow represents zur, a zinc-dependent transcriptional regulator. dip1711 and Rv2360c are genes with unknown function. The insertion site of the IS element in C. diphtheriae C7(β) is shown as a triangle, and the region deleted in the Δzur strain is indicated as a dashed line. The percentages of amino acid sequence similarities between genes are indicated between the loci. (B) The sequences of the 28-bp inverted repeats at the ends of the IS element are shown. Bases that are interruptions in the inverted repeat are shown in lowercase, and the rest of the IS element is indicated as a dashed line.

FIG. 2.

arsR and zur promoter activity. NCTC13129 (indicated as wild type [WT]) and NCTC13129Δzur (indicated as Δzur) strains with either vector control, pKPIM, or complementing wild-type zur, pKPIMzur (shown as +zur), were inoculated into zinc-replete (+Zn) or -depleted (-Zn) conditions and tested for arsR and zur promoter activity. β-Galactosidase activity is expressed in Miller units. The cmrA promoter is a positive control for zinc-depleted conditions. The asterisks denote statistical significance (<0.05) between zinc-replete and -depleted conditions, as determined by a one-way analysis of variance, followed by the Holm-Sidak method.

FIG. 3.

Detection of arsR and zur transcripts. RNA isolated from NCTC13129 (WT) and NCTC13129Δzur (Δzur) in zinc-replete and -depleted conditions was analyzed for relative transcript levels (normalized to gyrB mRNA levels) of arsR (A) and zur (B). (C) Fold change of these genes from NCTC13129 in zinc-replete conditions.

FIG. 4.

Activity of Zur-regulated promoters. NCTC13129 (indicated as WT) and NCTC13129Δzur (indicated as Δzur) strains with either vector control, pKPIM, or complementing wild-type zur, pKPIMzur (shown as +zur), inoculated into zinc-replete (+Zn) or -depleted (-Zn) conditions were used to test the activity of promoters (zrg, cmrA, and troA) in the reporter plasmid pSPZ. (A) The β-galactosidase activity was determined under differential conditions. The activity of the zrg promoter under iron and zinc stress conditions (B) and activity of the cmrA promoter under manganese and zinc stress conditions (C) are also presented. The promoters of tox, mntA, and cmrA genes (cloned into the reporter vector) were used as positive controls for iron-, manganese-, and zinc-depleted conditions, respectively.

FIG. 5.

RNA levels of Zur-regulated genes. RNA isolated from NCTC13129 and NCTC13129Δzur under zinc-replete and -depleted conditions was analyzed by using qRT-PCR. (A) Relative level of transcripts (normalized to gyrB mRNA levels) of cmrA, zrg, and troA. (B) Fold change of these genes from NCTC13129 under zinc-replete conditions.

FIG. 6.

Regulator binding sites in the upstream regions of zrg, cmrA, and troA. The locations of the putative binding sites for Zur (diamonds), DtxR (triangles), and MntR (black and white circles) are indicated for the zrg, cmrA, and troA promoter regions. The black circles indicate the inverted repeat, and the white circles indicate the direct repeat in the MntR binding site The lengths of the DNA fragment used in the promoter assays (Fig. 5) are shown in boldface below each region, and the distance between adjacent genes is shown in normal typeface above each region. The diagram is not drawn to scale.