Pseudomonas putida DLL-E4对硝基苯酚降解中的温敏与转录调控机制

Pseudomonas putida DLL is a high effective p-nitrophenol(PNP)degradaer. A gene cluster pnp encoding PNP catabolic pathway was cloned from DLL-E4. There existed a LysR-type transcriptional regulator PnpR in this cluster, which regulated the expression of the lower pathway of PNP degradation. Gene organization of pnpR in pnp cluster differed from other lysR-type regulator genes involved in aromatic compounds degradation. It suggests that PnpR regulates the expression of pnp cluster with a mechanism different from other LysR-type aromatic degradation regulator. We will investigate the regulation mechanism of PnpR by transcriptional start sites determination, promoter sequence determing, DNA bing tests, and DnaseI footprinting to determine the promoter sites and DNA binding site of PnpR on its own and pnpC1 regulational region. We also try to test the global regulation effects of PnpR by transcritomics and proteomics analysis. Mutant MT54 is a temeperature-sensitive mutant of DLL-E4. MT54 lost the ablity to degrade PNP at 37 ℃, but still was able to degrade PNP at 30℃ with nearly the same efficiency. MT54 is a miniTn5 insertion mutant. We will try to clone the temeperature-sensitive related gene by SEFA-PCR from both end of the Tn insertion sequence or construct the genomic DNA library of MT54 and DLL-E4 by selection of Km resitance or complement tests. The function of temeperature-sensitive related gene on the expression and stablity of PnpA will investigated. Nitroaromatic compounds have been studied as the a model for research of the evolution of degradation pathway, elucidation of the regulation mechanism of pnp cluster will supply important data for the research of catabolic pathway evolution.

恶臭假单胞菌DLL-E4是一高效对硝基苯酚（PNP）降解菌，从该菌中克隆到了编码PNP降解的pnp基因簇。pnp基因簇中存在一LysR家族调控蛋白PnpR，其基因组织方式与传统参与芳族化合物降解调控的lysR型调控基因组织方式不同，表明PnpR可能以一种新的机制调控pnp基因簇。本研究拟通过启动子分析、凝胶阻滞实验、DNA碱基保护分析、pnpR及其调控目标基因的表达分析以及转录组学和蛋白质组学等手段研究pnpR及其控制的目标基因的调控机制。DLL-E4的突变株MT54表现出30度降解而37度不降解PNP的温敏调控方式，温度影响PNP脱硝基酶PnpA，本项目拟克隆与PNP温敏调控有关的基因，研究其表达产物特性、与PnpA互作、对pnpA表达的影响，从而阐明PNP降解的温敏调控机制。硝基取代芳香族化合物已成为研究降解代谢途径进化的模式化合物，pnp调控机制的阐明将为代谢途径进化分析提供重要数据

Ps. putida DLL-E4是一株高效对硝基苯酚（PNP）降解菌，其在降解PNP过程中存在特殊的调控现象，包括不同于经典模式的LysR调控蛋白的组织结构及温敏调控现象等。基于此，本项目主要研究内容包括：（1）PNP降解途径的转录调控机制；（2）LysR调控蛋白的功能与作用机制；（3）PNP降解中的温敏调控机制；（4）PNP降解与核心碳代谢的网络关系。.项目组从以上四方面开展研究，并取得相关研究成果：（1）利用基因组测序和反转录PCR确定了两套PNP降解基因簇的组织形式：基因簇pnp由pnpA、pnpB、pnpR和pnpC1C2DECX1X2组成，基因簇pnp1由pnpAb、pnpR1和pnpC1bC2bDbEbCbX1bX2b组成；其中pnpA、pnpB，pnpR、pnpC1C2DECX1X2和pnpR1是PNP降解的关键基因，基因簇pnp1不具备降解PNP和HQ的能力；（2）结合qRT-PCR、基因敲除、诱导降解和转录组分析等技术确定了PNP降解基因簇的转录调控机制：pnpA的表达受PnpR和PnpR1的正调控，PnpR1起主要调控作用，其效应物为PNP；PnpR正调控pnpC1C2DECX1X2，操纵元pnpC1C2DECX1X2和pnpC1bC2bDbEbCbX1bX2b的效应物为HQ。（3）通过基因敲除、诱导降解和转录组分析研究了PnpR的调控功能：pnpR不参与菌株生长的调控，但对PNP/HQ的降解、碳源利用等细胞生命活动具有重要调控作用；体外结合实验和生物信息学分析表明PnpR通过TTCGGTTACCCGA序列与pnpC1的启动子区结合来调控基因的转录。（4）利用基因敲除回补和蛋白互作确定了PNP降解温敏调控的关键基因pnpB，PnpB通过解除产物抑制提高PnpA催化活性，从而提高菌株DLL-E4在37℃的降解效率；利用蛋白质结晶解析了低温酶PpnpA的三维结构，定点突变确定R179和P304为影响酶活的关键氨基酸残基；（5）采用qPCR和比较转录组学等技术阐明了PNP降解与核心碳代谢的网络关系：PNP降解通过TCA循环与核心碳代谢建立联系；PNP为全局性效应物，可诱导核心碳代谢和PNP降解相关基因的转录水平发生显著变化。.本项目的研究成果为理解微生物遗传调控机理，代谢途径进化分析提供重要理论基础，并为降解微生物的理性设计用于环境修复提供技术指导。