Abstract
BackgroundZinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators.ResultsThe cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays.ConclusionWhole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum.
Highlights
Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations
Annotation of the corynebacterial zinc uptake regulator Zur The Cg2502 (Zur) protein of C. glutamicum ATCC 13032 has a predicted size of 144 amino acids, a theoretical molecular mass of 15.7 kDa and belongs to the small core set of 24 transcription regulators that were detected in all hitherto sequenced corynebacterial genomes [5,19]
Protein domain predictions performed with the SUPERFAMILY [20] and the Conserved Domain Database tools [21] showed that the Zur protein contains an amino-terminal helix-turn-helix motif of the winged-helix type and is a member of the Zur subgroup of the ferric uptake regulator (Fur) family of metalloregulatory proteins [14]
Summary
Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators. Transcription regulators represent key components in the control of bacterial gene expression and permit the cell to sense and respond to environmental changes [6]. Metal ion homeostasis in bacterial cells is tightly regulated by specific metal-sensing transcription regulators. These metalloregulatory proteins, in principle, sense the intracellular levels of specific metal ions by binding them to a metal binding site, which leads to conformational changes affecting the regulator’s ability to bind operator sites in regulatory DNA regions [7]. Numerous studies indicated a tremendous diversity in metal selectivity and biological function within the Fur protein family that can be divided into sensors of iron (Fur), manganese (Mur), nickel (Nur), and zinc (Zur) [14]
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