B-182. Identification and Characterization of the Zinc Uptake Regulator (Zur) in Corynebacterium diphtheriae

K. F. Smith1, F. M. Jobling2, R. K. Holmes2, D. M. Oram1;
1Univ. of Maryland, Baltimore, MD, 2Univ. of Colorado Sch. of Med., Aurora, CO.

The Ferric Uptake Regulator (Fur) and the Diphtheria Toxin Repressor (DtxR) are two families of metal-dependent transcriptional regulators that directly bind metal and repress transcription from promoters containing specific recognition sequences (operators). The genome of Corynebacterium diphtheriae, the causative agent of diphtheria, includes genes that encode two DtxR-like proteins and one Fur-like protein. The Fur-like protein has not been characterized. In this work, we show that the Fur-like protein is a metalloregulatory protein that represses transcription of genes in response to zinc (Zn) and is therefore designated a Zinc Uptake Regulator (Zur). Zn is essential for structural stability of many proteins and as an enzyme cofactor. However, Zn can also block important thiols or compete with other metals for the active site of enzymes at high concentrations. Therefore, strict regulation on the uptake of Zn is essential. We hypothesize that Zur is a metal-dependent transcriptional regulator that represses a variety of zinc uptake and mobilization genes in the presence of zinc. We utilized bioinformatics, ß-galactosidase reporter assays, and qRT-PCR (quantitative Real Time Polymerase Chain Reaction) to identify several genes regulated by the Fur-like protein. Approximately 30 additional genes were identified as Zur regulated using a microarray. Growth curves with a zur deletion mutant and its wild-type parent strain showed that the absence of Zur has no significant effect on growth. Interestingly, we identified two promoters for zur. One promoter is located between zur and the gene located directly upstream, predicted to encode an ArsR-like regulator. The second promoter is required for a transcript that includes both zur and the arsR-like gene. The co-transcript of zur and arsR-like gene is repressed by Zur as shown by RT-PCR. To demonstrate in vitro DNA binding activity of Zur, the recombinant Zur protein has been purified and DNA binding assays are being performed. In summary, we have characterized the first Zn specific transcriptional regulator (Zur) in C. diphtheriae.