| The high detection rate of harmful pollutants aromatic hydrocarbons in the environment.Further research on the transcriptional regulation mechanism of multiple degradation pathways of aromatic hydrocarbons will help to understand the complex regulation mechanism of aromatic hydrocarbon degradation strains and provide a theoretical basis for the construction of high-efficiency naphthalene degradation strains.There have been some studies on the cooperative regulation mechanism of regulatory proteins,but there are some differences between different strains,which requires a more extensive and deeper study.Pseudomonas putida ND6 is an aromatic hydrocarbon-degrading bacteria isolated by the research group to efficiently degrade naphthalene and benzoic acid.What is unique is the existence of some seemingly redundant repeat units such as the regulatory genes nahRp and nahRc,but these seemingly redundant repeat units actually play an important role.The NahRp,a transcripional activator of nah operon upstream of naphthalene degradation,is a LysR transcripional activator with NahRc,and highly similar protein 3D structure to the active binding site,hence possible functional overlap.In order to deeply explore the regulatory mechanism of these two transcripional activators,the regulatory protein mutant strains(ND6-ΔnahRp and ND6-ΔnahRc)and their respeonse mutant strains(ΔnahRp-c and ΔnahRc-c)were constructed.Growth curves of wild-type,mutant and remutant strains were determined under 2 g/L and 4 g/L naphthalene culture conditions,and those of ND6-ΔnahRp strains under naphthalene medium containing different concentrations cis,cismucofuroic acid.The results showed that the growth of the ND6-ΔnahRp strain was delayed by about 20 h compared to the wild-type ND6 strain,but theΔnahRp strain gradually recovered with increasing cis,cis-mucofufuric acid concentration.Therefore,nahRp is not the only transcripional activator that initiates nah operon in the cells of ND6 strain.With its high homologous nahRc in transcripional inducer cis,cis-mucous bran acid is activated transcripion and replaces the regulatory function of nahRp on the upstream naphthalene degradation pathway;Moreover,compared with ΔnahRp,nahRc had little effect on strain growth;However,under 4 g/L naphthalene culture,all strains excepΔnahRp strains showed different degrees of catephenol accumulation,andΔnahRc accumulation was lower than wild-type ND6,indicating that nahRp was more activation of naphthalene degradation upstream than nahRc,but lower than that of the two regulatory proteins.Secondly,qRT-PCR analyzed the transcripion levels of about 20 key degradation genes during naphthalene degradation in wild-type ND6 and two regulatory protein mutant strains,and showed that key genes in the naphthalene degradation pathway were rapidly transcribed,the highest in the logarithmic phase,then remained low.Meanwhile,nahRc is co-transcribed with its upstream catRⅡ gene,and nahRp and nahRc are regulating nah operon transcrip expression,but nahRp is stronger than nahRc.Finally,both NahRp and NahRc could bind to the Pnah promoter of nah operon and the binding inducer was salicylic acid.In vivo promoter reporter gene expression validated that NahRp initiated more activity against the Pnah promoter than NahRc.Established a model of cooperative regulation of NahRp and NahRc,In order to lay the theoretical foundation for enhancing the control of microbial repair process,solve the bottleneck problem of microbial repair,help to build more efficient and environmentally adaped genetically engineered bacteria and construct genetically engineered strains with high yield of intermediate metabolites. |
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