Pseudomonas fluorescens菌株LZ-4同时降解萘与还原六价铬的机理研究

Microorganisms that can degrade polycyclic aromatic hydrocarbons （PAHs） and reduce heavy metals are often utilized in microbial remediation. However, microbes that can aerobically degrade PAHs and reduce heavy metals simultaneously have not been discovered yet.A Pseudomonas fluorescens strain LZ-4 was previously isolated from contaminated soil samples of Lanzhou Xigu district. This strain can grow using naphthalene as sole carbon source, while reducing hexavalent chromium (Cr(VI)) to trivalent chromium (Cr(III)). Strain LZ-4 bears a native plasmid pHWL10 with a size of 8.1Kb encoding three genes. Plasmid curing and complementation experiments indicated that pHWL10 is involved in naphthalene degradation pathway. In this project, we will perform whole genome sequencing on strain LZ-4 to predict the genes involved in naphthalene degradation and Cr(VI) reduction. Then we will use quantitative PCR, gene knockout and complementation and physiological and biochemical techniques to further confirm the functions of the genes and elucidate the complete pathways. Next, by transcriptome sequencing, we will identify the genes that are involved in both naphthalene degradation and Cr(VI) reduction. Thereby, we will determine whether the naphthalene degradation and Cr(VI) reduction process are associated to each other. Meanwhile, we will identify the gene(s) in pHWL10 that is related to naphthalene degradation and further clarify the roles of pHWL10 in naphthalene degradation pathway in strain LZ-4.

微生物能降解多环芳烃，也能还原重金属，这是微生物治理环境污染的重要依据。在好氧条件下，尚未发现有细菌可以同时还原重金属并降解多环芳烃。本实验室在兰州西固污染地区土壤中分离出一株Pseudomonas fluorescens菌株，命名为LZ-4，该菌株能以萘为唯一碳源生长并同时将六价铬还原成三价铬。该菌株还拥有一个天然质粒pHWL10，该质粒全长8.1Kb,携带三个基因，质粒去除与回补实验表明该质粒与萘降解途径相关。本项目希望通过全基因组测序与生物信息学预测菌株LZ-4 中与萘降解、六价铬还原相关的基因。并通过荧光定量PCR、基因敲除回补及生理生化方法进一步鉴定这些基因的功能，绘制完整的萘降解、六价铬通路图。然后通过转录组测序的方法鉴定与萘降解及六价铬还原两个过程均相关的基因，分析萘降解与铬还原两个过程是否相互关联。同时研究质粒pHWL10在菌株LZ-4 萘降解途径中的具体作用。

复合污染尤其是多环芳烃和重金属的污染已经成为急需解决的问题。本项目从兰州一处污水排放口筛选到一株假单胞杆菌 Pseudomonas fluorescens LZ-4可以同时降解萘和还原六价铬。菌株在不同碳源下还原六价铬的能力的检测结果表明当加入六价铬进入培养基后，以萘为唯一碳源的LZ-4号菌株能显著的促进六价铬还原为三价铬。全基因组测序分析表明LZ-4 菌株通过萘降解途径基因降解萘，包含两个操纵子，分别能够将萘降解到水杨酸然后继续降解至丙酮酸至进入柠檬酸循环。通过化学反应实验，基因敲除，RNA干扰以及纯化结晶及酶活实验等手段证明部分参与降解萘的酶如NahG，NahE，NahA具有还原金属铬的能力，表明萘促进铬的还原主要是由于在萘降解过程中产生的中间产物邻苯二酚和邻苯二甲酸能够提高还原六价铬的能力。本项目中筛选到的菌株LZ-4将在复合污染治理领域具有很大的应用前景。通过对菌株降解萘耦合铬还原机理的探究将为未来筛选改造工程菌株提供指导，同时也能为未来复合污染提供新见解。