土壤细菌生物氧化Mn(Ⅱ)的分子机制研究

项目名称： 土壤细菌生物氧化Mn(Ⅱ)的分子机制研究

项目编号： No.31270158

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 生物科学

项目作者： 李林

作者单位： 华中农业大学

项目金额： 78万元

中文摘要： 以1株高锰氧化活性土壤大肠杆菌MB266为对象，拟研究其在细胞表面氧化Mn(Ⅱ)生成锰氧化物的分子机制及基本代谢网络。首先通过构建该菌株的转座突变文库，从中筛选导致锰氧化活性减弱与丧失的负变突变子，再利用锚定PCR和原位杂交技术确定其效应分子及编码基因；然后用Real-time qPCR和蛋白质组学技术，对比分析MB266菌株和负变菌株，确定直接参与锰氧化作用与起调控作用的蛋白质（酶）系分子。再利用细胞表面展示技术，以多铜氧化酶MCO等主要锰氧化酶为目标酶，通过Pull-down技术探究与MCO在细胞壁上发生耦联的电子传递蛋白，并通过体外纯化不同的细胞外膜组分和锰氧化蛋白组成多重体系，分析确定参与Mn(Ⅱ)细胞外膜电子传递体，结合构建基因中断突变株和进行质粒表达补偿作用，测定氧化Mn(Ⅱ)时与细胞膜电子传递体耦联的电子传递流向，来解析细菌MCO氧化Mn(Ⅱ)电子传递的反应体系与分子机制。

中文关键词： 锰氧化细菌；锰氧化作用；分子机制；代谢网络；信号转导

英文摘要： Although Mn(II)-oxidizing bacteria are found to be widely distributed naturally, little information is available on their metabolisms of biochemically oxidizing Mn(II) into Mn(III)/Mn(IV) oxides. In the current proposal, a naturally occurring soil-borne Escherichia coli with high Mn-oxidizing activity, MB266, was used as the target bacterium to probe and elucidate the molecular mechanism of manganese oxidization on the cell surface of this strain as well as the preliminary metabolic network. A transposition mutagenesis system will be initially performed to create the mutation library of MB266, which allows screening the mutants that were attenuated or fully losed the Mn(II)-oxidizing activities by using routine Mn-oxidization determinative assays. Subsequently, the anchor PCR and in situ hybridization technologies will be used to analyze the related effectors and their encoding genes from the MB266 genome, and the Real-time qPCR and proteomics technologies will be further employed to probe and confirm the functioning and regulative molecules involving in Mn(II) oxidization by comparatively investigating both MB266 and those negative mutant strains. Based on these results, the specific cell-surface display technology will be projected to immobilize the main Mn(II) oxidases including the common multicopper oxidase

英文关键词： Manganese-oxidizing bacterium；Manganese oxidation；Molecular mechanism；Metabolic network；Signal transduction