Institut für Genomforschung und Systembiologie, Centrum für Biotechnologie, Universität Bielefeld, D-33615 Bielefeld, Germany.
BMC Genomics. 2010 Jan 7;11:12. doi: 10.1186/1471-2164-11-12.
Zinc 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.
The 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.
Whole-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.
锌被认为是所有生物的必需元素，但在高浓度下也可能有毒。因此，细菌严格控制锌的代谢。谷氨酸棒杆菌的 Cg2502 蛋白是控制该物种锌代谢的候选蛋白，因为它被归类为铁摄取调节剂（Fur）家族 DNA 结合转录调节剂中锌摄取调节剂（Zur）亚组的金属调节剂。
通过等位基因交换程序，在谷氨酸棒杆菌 ATCC 13032 的染色体中删除了 cg2502（zur）基因，生成了 zur 缺陷突变体谷氨酸棒杆菌 JS2502。比较谷氨酸棒杆菌 JS2502 与野生型菌株的全基因组 DNA 微阵列杂交和实时 RT-PCR 检测到 18 个基因在 zur 突变体中表达增强。表达数据与共享调控位点的跨基因组比较结果相结合，揭示了映射的五个潜在锌 ABC 型转运体（cg0041-cg0042/cg0043；cg2911-cg2912-cg2913）、一种假定分泌蛋白（cg0040）、一种假定氧化还原酶（cg0795）和 COG0523 蛋白家族的假定 P 环 GTPase（cg0794）的转录单元编码组件的候选 Zur 结合位点的存在。通过实时 RT-PCR 验证了相应基因在谷氨酸棒杆菌 JS2502 中的转录水平增强，并用野生型 zur 基因互补突变体逆转了差异基因表达的影响。用 GFP 报告系统在体内检测到假定的 cg0042 和 cg2911 操纵子的锌依赖性表达。此外，通过 DNA 带迁移分析体外证明了纯化的 Zur 蛋白与包含候选 Zur 结合位点的双链 40 -mer 寡核苷酸的锌依赖性结合。
全基因组表达谱分析和 DNA 带迁移分析表明，Zur 以锌依赖性方式直接抑制五个转录单元中九个基因的表达。因此，Zur（Cg2502）蛋白是谷氨酸棒杆菌中参与锌稳态的基因的关键转录调节剂。
