Molecular biological study on the mechanism of a novel Fe^<2+>-dependent mercury reducing system of a mercury resistant iron-oxidizing bacterium

汞对几乎所有生物体都有剧毒，因此，从汞污染的土壤和废水中去除汞是一个世界性的问题。本研究项目的最终目标是使用高抗汞铁氧化细菌A.ferrooxidans菌株MON-1从汞污染土壤和废水中挥发和回收有毒汞化合物。嗜酸氧化亚铁硫杆菌MON-1菌株是一株高抗汞的菌株，能在含20 μM氯化汞的铁培养基（pH 2.5）中生长。作者报道了MON-1菌株在细胞中具有一个新的依赖Fe^<2+>的汞挥发酶系统和一个依赖NADPH的汞还原酶。在第一部分中，作者首次分析了以TMPD为电子给体的Fe^<2+>依赖的汞挥发体系。结果表明，在厌氧条件下，菌株MON-1细胞能以TMPD为电子供体挥发Hg^0，且该活性可被1 mM KCN完全抑制，表明细胞色素c氧化酶参与了Hg^0的挥发。作者从MON-1细胞中分离出细胞色素c氧化酶。以TMPD为电子供体时，高纯度的MON-1细胞色素c氧化酶比以Fe^<2+>为电子供体时能更快地挥发出Hg^0。目前还没有氧化亚铁硫杆菌对有机汞化合物产生抗性的报道，可能是因为还没有关于该菌中有机汞裂解酶基因（merB）的报道。在第二部分中，作者首次发现在11株氧化亚铁不动杆菌中对有机汞化合物抗性最强的菌株MON-1的静息细胞和胞浆具有类有机汞裂解酶活性，在NADPH、β-巯基乙醇和内源汞还原酶存在下，该酶能催化Hg^0从反应混合物中挥发。

Mercury is highly toxic for almost all organisms and thus, the removal of mercury from mercury polluted soil and wastewater is a worldwide problem. The final goal of this research project is volatilization and recovery of toxic mercury compounds from mercury polluted soil and wastewater using a highly mercury-resistant iron-oxidizing bacterium A.ferrooxidans strain MON-1. Acidithiobacillus ferrooxidans strain MON-1 is a highly mercury resistant strain that can grow in an iron medium (pH 2.5) with 20 μM mercuric chloride. Author has reported that strain MON-1 has a novel Fe^<2+>-dependent mercury volatilization enzyme system as well as a NADPH-dependent mercury reductase in the cells. In section 1, author analyzed for the first time the Fe^<2+>-dependent mercury volatilization system with TMPD as an electron donor for mercury volatilization reaction. It was found that strain MON-1 cells can volatilize Hg^0 with TMPD as an electron donor under anaerobic conditions and the activity was completely inhibited by 1 mM KCN, indicating that cytochrome c oxidase is involved in Hg^0 volatilization. Author isolated cytochrome c oxidase from MON-1 cells. A highly purified MON-1 cytochrome c oxidase could volatilize Hg^0 more rapidly when TMPD was used as electron donor compared with Fe^<2+>.There have been no reports on A.ferrooxidans strains resistant to organomercurial compounds probably because there has been no report on the organomercurial lyase gene (merB) in the bacterium. In section 2, author shows for the first time that the resting cells and the cytosol from strain MON-1, the most resistant to the organomercurial compounds among the 11 strains of A.ferrooxidans tested, had organomercurial lyase-like activity which catalyzed the volatilization of Hg^0 from the reaction mixture in the presence of NADPH, β-mercaptoethanol and the intrinsic mercuric reductase.