Identification and Characterization of the Zinc Uptake Regulator (Zur) of Corynebacterium diphtheriae

Abstract

Regulation of metal ion homeostasis is essential to bacterial cell survival and in most species 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 in response to available zinc. Although no growth phenotype was observed in differential metal conditions, a C. diphtheriae zur mutant was more sensitive to peroxide stress, suggesting that Zur has a role in protecting the bacterium from oxidative damage. By measuring promoter activities and mRNA levels, we demonstrated that Zur represses transcription of three genes, zrg, cmrA, and troA, in zinc replete conditions. Zrg and TroA have similarity to other proteins involved in zinc uptake such as Bacillus subtilis YciC and Treponema pallidium TroA, respectively. CmrA has the highest similarity to C. diphtheriae TroA. Transcription of zrg and cmrA was also regulated in response to iron and manganese, respectively, by mechanisms that are independent of Zur. In addition to these three genes, 74 genes were shown to be affected by the absence of Zur and zinc via microarray analysis of transcript levels. The microarray results were confirmed via qRT-PCR for six of these genes, including one that is seemingly activated by Zur. An adjacent gene to zur, 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. In contrast, Zur represses the activity of the zur promoter under low zinc conditions. The observed unique regulation of zur transcription has not been described for any other Zur ortholog. In addition, Zur bound the upstream region of multiple genes with varying affinity and this binding was metal dependent. A Zur binding site was determined on the upstream region of zur, cmrA, and sidA using a Dnase I protection assay. 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.