Kim E J, Chung H J, Suh B, Hah Y C, Roe J H
Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University, Korea.
J Bacteriol. 1998 Apr;180(8):2014-20. doi: 10.1128/JB.180.8.2014-2020.1998.
Streptomyces coelicolor Müller contains two superoxide dismutases (SODs), nickel-containing (NiSOD) and iron- and zinc-containing SOD (FeZnSOD). The sodF gene encoding FeZnSOD was isolated by using PCR primers corresponding to the N-terminal peptide sequence of the purified FeZnSOD and a C-terminal region conserved among known FeSODs and MnSODs. The deduced amino acid sequence exhibited highest similarity to Mn- and FeSODs from Propionibacterium shermanii and Mycobacterium spp. The transcription start site of the sodF gene was determined by primer extension. When the sodF gene was cloned in pIJ702 and introduced into Streptomyces lividans TK24, it produced at least 30 times more FeZnSOD than the control cells. We disrupted the sodF gene in S. lividans TK24 and found that the disruptant did not produce any FeZnSOD enzyme activity but produced more NiSOD. The expression of the cloned sodF gene in TK24 cells was repressed significantly by Ni, consistent with the regulation pattern in nonoverproducing cells. This finding suggests that the cloned sodF gene contains the cis-acting elements necessary for Ni regulation. When the sodF mRNA in S. coelicolor Muller cells was analyzed by S1 mapping of both 5' and 3' ends, we found that Ni caused a reduction in the level of monocistronic sodF transcripts. Ni did not affect the stability of sodF mRNA, indicating that it regulates transcription. S. lividans TK24 cells overproducing FeZnSOD became more resistant to oxidants such as menadione and lawsone than the control cells, suggesting the protective role of FeZnSOD. However, the sodF disruptant survived as well as the wild-type strain in the presence of these oxidants, suggesting the complementing role of NiSOD increased in the disruptant.
天蓝色链霉菌 Müller 含有两种超氧化物歧化酶（SOD），即含镍超氧化物歧化酶（NiSOD）和含铁锌超氧化物歧化酶（FeZnSOD）。通过使用与纯化的 FeZnSOD 的 N 端肽序列相对应的 PCR 引物以及已知 FeSOD 和 MnSOD 中保守的 C 端区域，分离出了编码 FeZnSOD 的 sodF 基因。推导的氨基酸序列与谢氏丙酸杆菌和分枝杆菌属的 MnSOD 和 FeSOD 具有最高的相似性。通过引物延伸确定了 sodF 基因的转录起始位点。当 sodF 基因克隆到 pIJ702 中并导入变铅青链霉菌 TK24 时，它产生的 FeZnSOD 比对照细胞至少多 30 倍。我们破坏了变铅青链霉菌 TK24 中的 sodF 基因，发现破坏株不产生任何 FeZnSOD 酶活性，但产生了更多的 NiSOD。克隆的 sodF 基因在 TK24 细胞中的表达受到镍的显著抑制，这与非过量生产细胞中的调控模式一致。这一发现表明，克隆的 sodF 基因包含镍调控所需的顺式作用元件。当通过对 5' 和 3' 末端进行 S1 作图分析天蓝色链霉菌 Müller 细胞中的 sodF mRNA 时，我们发现镍导致单顺反子 sodF 转录本水平降低。镍不影响 sodF mRNA 的稳定性，表明它调节转录。与对照细胞相比，过量生产 FeZnSOD 的变铅青链霉菌 TK24 细胞对诸如甲萘醌和胡桃醌等氧化剂的抗性更强，这表明 FeZnSOD 具有保护作用。然而，在这些氧化剂存在的情况下，sodF 破坏株与野生型菌株一样存活，这表明破坏株中增加的 NiSOD 具有互补作用。
