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

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.