重金属膜輸送遺伝子とキナーゼ遺伝子の共クローニングによる重金属とリン除去の研究

该研究项目的目的是开发一种生物技术同时去除磷和重金属的技术。研究人员重点研究了细菌在细胞内聚积多磷酸盐时，会聚积Mg^<2+>和Ca^<2+>等二价阳离子的现象。因此，研究了细菌对Hg^<2+>和Cd^<2+>等二价重金属阳离子的潜在生物富集能力。本课题在第一阶段对细菌质粒Hg <2+>和Cd^<2+>抗性进行了表征。在该项目的第二阶段，在大肠杆菌菌株最稳定的细胞中克隆了磷酸转运基因，一种后操纵子。随后，利用假单胞菌的抗汞操纵子与多磷酸激酶基因在同一株大肠杆菌中共克隆汞内流转运基因。本课题的研究成果如下：(1) cadC基因产物的转录调控蛋白被鉴定为Cd抗性的抑制蛋白。(2)其中一株大肠杆菌能稳定地携带后操纵子和多磷酸激酶基因，并能在稀释后的LB肉汤中活跃生长。该菌株从培养基中吸收磷酸盐。(3)通过定位缺失汞还原基因（merA），构建了对氯化汞和甲基汞敏感的大肠杆菌菌株。(4)在共克隆的大肠杆菌细胞中发现了多磷酸盐和汞同时积累的可能性。

The purpose of the research project was to develop a technology for simultaneous removal of phosphorus and heavy metals with biotechnological methods. The researcher focused on the phenomenon that bacteria accumulate divalent cations such as Mg^<2+> and Ca^<2+> when they are accumulate polyphosphate in the cells. Therefore, bacterial potential capability of bio-accumulation of divalent heavy metal cations such as Hg^<2+> and Cd^<2+> was investigated.Hg^<2+> and Cd^<2+> resistance of bacterial plasmids were characterized at the first stage of this research project. Phosphate transport genes, a pst operon, were cloned in the most stable cells of an E.coli strain, in the second stage of this project. Thereafter, a mercury resistance operon of Pseudomonas strain were challenged to co-clone the mercury influx transport genes with a polyphosphate kinase gene in a same E.coli strain. The research results obtained from this project were as follows.(1) Transcriptional regulatory protein of the product of cadC gene was characterized as a repressor protein for Cd resistance.(2) One of E.coli strain can possess pst operon and polyphosphate kinase gene stably and can grow actively in diluted LB broth. This bacterial strain uptook phosphate from the medium constitutively.(3) Hyper-sensitive E.coli strain for mercury chloride and methyl mercury was constructed by sitederected deletion of mercury reducing gene (merA).(4) The possibility of simultaneous accumulation of polyphosphate and mercury in the co-cloned E.coli cells was recognized.