潘多拉菌中氯苯代谢的两个基因簇的转录调控研究

Chlorobenzene is one of highly toxic and carcinogenic halogenated organic compounds. When a pure bacterial strain of Burkholderia sp. JS150 was inculcated into the bioreactor (continuous culture) fed with chlorobenzene, strain JS150 disappeared after a week and a new strain of Pandoraea sp. MCB032 appeared and persisted in the reactor. In a previous study, two gene clusters (clc and cbs) were found to be involved in the chlorobenzene metabolism in strain MCB032. In addition to the genes encoding the enzymes for the chlorobenzene degradation in these two clusters, clcR encodes a putative LysR family regulator and cbsR encodes a putative XylR family regulator. Our preliminary evidences showed that the clcR-deleted strain lost the activity of chlorocatechol-l, 2-dioxygenase encoded by clcA gene, indicating that the ClcR positively regulates chlorobenzene metabolism. In this proposed study, the interaction between regulatory proteins ClcR /CbsR and each promoter of the operons will be performed by electrophoretic mobility shift assay and DNase I footprinting. The structures of each promoter will be resolved and the regulatory mechanisms of each operon together with their auto-regulation will be revealed. This study will be the first to interpret the involvement of a LysR family regulator and a XylR family regulator in the regulation of chlorobenzene metabolism.

氯苯是高毒性和潜在致癌性的卤代有机化合物。接入菌Burkholderia sp. JS150的氯苯生物处理反应器（连续培养）中，JS150菌株消亡而潘多拉菌MCB032演替出现并持续存在。MCB032菌株的氯苯代谢途径已阐明，参与其代谢的基因位于clc和cbs两个基因簇上：除编码氯苯代谢酶的基因以外，clcR基因编码一个LysR 家族调控蛋白，cbsR编码一个XylR 家族调控蛋白；初步试验表明，clcR基因缺失后，clcA基因编码的氯邻苯二酚-1，2-双加氧酶活力丧失，这表明ClcR蛋白是该代谢途径的正调控因子。本项目将以MCB032菌株中氯苯代谢的两个基因簇为对象，研究调控蛋白ClcR 和XylR 与每个启动子的结合情况，并解析启动子的结构，揭示它们对每个操纵子的调控及自调控机制，这将首次在分子水平阐释LysR家族和XylR家族的两个调控蛋白同时参与氯苯代谢的调控机理。

氯苯是高毒性和潜在致癌性的卤代有机化合物。潘多拉菌MCB032可以以氯苯为唯一碳源和能源生长，参与其氯苯代谢的基因位于clc和cbs两个基因簇上。本项目研究了该菌株中氯苯代谢途径在转录水平上的调控。在其基因组上，参与氯苯代谢基因由cbsFAaAbAcAdB、cbsRX、orf56cbsXR、clcABCDE和clcR等五个操纵子组成，其中，cbsR编码一个XylR家族调控蛋白，clcR基因编码一个LysR家族调控蛋白。cbsR和clcR基因缺失后，clcA基因编码的氯邻苯二酚-1，2-双加氧酶活力基本丧失，这表明CbsR和ClcR蛋白是该代谢途径的正调控因子。本研究通过引物延伸解析了启动子，体外表达了CbsR和ClcR蛋白，并通过实时荧光定量分析了cbs和clc基因簇的转录情况，揭示cbsR和clcR基因对每个操纵子的调控及自调控机制，从而初步在分子水平阐释LysR家族和XylR家族的两个调控蛋白同时参与氯苯代谢的调控机理。