多元调控因子在4-硝基酚分解代谢中的作用

The ever-increasing pollutants have caused devastating damage to the natural environment and mankind. It is of significance to study the microbial degradation of environmental pollutants as well as the transcriptional regulation for such process, as it will certainly help us understand better how microbes adapt to the polluted-environment and also improve their bioremediation capacity. Pseudomonas strain WBC-3 metabolizes the environmental priority pollutant para-nitrophenol (PNP) via hydroquinone and the enzymes involved in the PNP catabolism are located on three different operons. Our previous study revealed that, in addition to the LysR-type PnpR, an extra transcriptional regulator was probably also involved in the PNP catabolism in this strain. Now we have demonstrated that, like PnpR, a pnpM-encoded LysR-type regulator was also necessary for the growth of this strain. By using approaches including EMSA, DNaseI foot printing and ChIP, this proposed project is to conduct a comprehensive and systematic research on the regulation of PNP degradation at biochemical and physiological levels, particularly aiming to identify the transcriptional regulators in the catabolism of PNP and the catabolic intermediate hydroquinone respectively, as well as their regulatory mechanisms and possible interaction between regulators PnpR and PnpM in vitro and in vivo. This will illustrate an example of the involvement of multiple regulators in controlling the same catabolic operon, and will also help reveal the molecular mechanisms underlying microbial adaptive evolution in the PNP catabolism.

日益增长的污染物给环境和人类带了严重的破坏，研究微生物对其降解及调控，对认识微生物如何适应污染物以及提高其生物修复的能力具有重要意义。假单胞菌WBC-3经对苯二酚代谢环境优先污染物4-硝基酚，其编码代谢的基因簇位于3个不同的操纵子上。本课题组前期研究表明除了LysR家族的PnpR外，该菌株中另一个LysR家族蛋白PnpM也可能参与该代谢途径的调控。因此本项目拟通过包括EMSA、DNaseI 足迹、甲醛交联及染色质免疫沉淀等研究手段，在体内和体外研究PnpM参与调控4-硝基酚的代谢机理，以及它与PnpR之间可能的相互作用，从而在分子生物学、生物化学和生理代谢等水平上完整地阐述WBC-3菌株中4-硝基酚经由对苯二酚代谢途径的转录调控机制，并揭示多元调控因子协同调控同一代谢操纵子的机理。这对于进一步阐明微生物分解代谢硝基酚污染物的调控机理及其适应性进化机制具有重要意义。

假单胞菌WBC-3利用4-硝基酚（PNP）生长，且通过对苯二酚（HQ）降解PNP。LysR家族调控蛋白PnpR参与调控三个操纵子pnpA、pnpB和pnpCDEFG。但当启动子突变之后，发现负责PNP下游代谢的pnpCDEFG的启动子活力与上游代谢的pnpA、pnpB表现明显不同。在2-氯-4-硝基酚（2C4NP）诱导下，PNP代谢调控蛋白PnpR不能激活基因pnpA和pnpB转录，继而限制菌株WBC-3利用2C4NP生长。本研究通过基因敲除，实时定量PCR，凝胶阻滞实验，DNase I足迹分析等一系列体内和体外实验揭示了调控蛋白PnpM参与调控PNP的下游代谢机理，以及它与PnpR之间可能的相互作用。从而证明了PnpM参与操纵子pnpCDEFG的调控。又通过定向进化手段获得了可以利用2C4NP生长的变异菌株假单胞菌WBC-3M，发现基因pnpR的第6位密码子发生错义突变继而导致半胱氨酸取代丝氨酸，生成突变体PnpRS6C；其以接近组成型形式激活基因pnpA和pnpB 的启动子进行转录且PNP或2C4NP能提高其诱导基因转录的能力1-2倍。PnpRS6C和PnpR都只能在PNP诱导下激活对苯二酚降解途径编码基因pnpCDEFG转录；调控蛋白PnpM能够同时响应PNP或者2C4NP诱导去激活基因pnpC（代表操纵子pnpCDEFG）转录。.同时还开展了4-硝基酚类似物2-溴-4-硝基酚(2B4NP)和4-氯-2-硝基酚(4C2NP)的微生物代谢研究。2B4NP被用于合成杀虫剂和细胞毒素抑制剂，其对水生生物有毒害。贪铜菌NyZ375能以2B4NP作为唯一碳源、氮源和能源生长。2B4NP单加氧酶BnpAB催化2B4NP生成 2-溴-对苯二酚（BHQ，63%）和1, 2, 4-苯三酚（BT，37%）。1, 2, 4-苯三酚1,2-双加氧酶BnpC催化1,2,4-苯三酚氧化开环形成马来酰乙酸，最终进入三羧酸循环。2B4NP是一种毒性较强的异生物质，是多种农药及医药的合成前体物质。在其他碳源存在的情形下，贪铜菌NyZ417能够利用4C2NP作为唯一的氮源进行生长。单加氧酶OnpANyZ417催化4C2NP经历单加氧反应生成4-氯邻苯二酚（4CC）。上述研究结果完善了微生物代谢PNP调控机的制理解并阐明了微生物对PNP类似物2B4NP和4C2NP的代谢机理。

内容获取失败，请点击重试