Cloning Of Chloramphenicol Downstream Catabolism Genes In Strain Nocardioides Sp. LMS-CY And The Study Of Enzymatic Properties And Transcriptional Regulation

Chloramphenicol(CAP),a broad-spectrum antibiotic,was isolated from Streptomyces venezuelae.CAP is used widely in medicine,aquaculture and other industries in the wide-world.Due to the large amount and stable nature of CAP,it has caused the severe residual of CAP in the environment.High concentrations of CAP have been detected in water bodies in many areas of China in recent years,which is directly endangers human health.Besides,the accumulation of CAP in the environment leads to the generation of antibiotic-resistant bacteria and the transfer of resistance genes,which indirectly causes hidden dangers to human health and safety.Catabolism of CAP by microorganism is an efficient and economical way,to date,there is a lack of reports on microbial degradation of CAP and related metabolic pathways,and metabolic genes are uncertain.In this study,a lab-stocked CAP-degrading strain Nocardioides sp.LMS-CY was used as the research material.The metabolic pathway of CAP was partially identified,and the genes of nitroreductase and hydroxylamine lyase were cloned,and the related transcriptional regulation mechanism was studied.The main results of this study are as follows:1.Identification of the metabolic pathway of CAPThe samples in the degradation process were detected by LC-MS/MS,and it was found that the intermediate product 4-nitrobenzoate(4NBA)accumulates first and then degrades completely,which indicated that CAP was metabolized to 4NBA through one or more reactions and 4NBA was further wholly degraded.The glucose(G),CAP,and 4NBA were used to induce the strain LMS-CY,and the induced strain was used to degrade 4NBA.The results showed that the degradation rate of the strain induced by CAP and 4NBA were much faster than that of the strain-induced by G.We speculated that the degradation of genes of 4NBA might be regulated by transcription through this phenomenon.2.Cloning of 4NBA degrading genes and characteristics of enzymesThe genome completion map of strain LMS-CY was determined.Afterward,the transcriptomes of the strains induced by CAP and G were determined,and two genes,nfnA and nfnB,which may be involved in 4NBA catabolism,were found through comparative transcriptomics.The genes nfnA and nfnB were cloned into the broad-host-range cloning vector pBBR1MCS-2,and the recombinant plasmid was electrotransformed into the engineering strain Pseudomonas putida KT2440.The degradation results showed that the recombinant strain could degrade 4NBA.According to the control of standard protocatechuic acid(PCA),the function of genes nfnA and nfnB was speculated to convert 4NBA to PCA.The genes nfnA and nfnB were heterologously expressed in Escherichia coli BL21(DE3),and NfnA and NfnB were purified.The enzymatic reaction results showed that NfnB could reduce 4NBA to 4-hydroxyaminobenzoate(4HABA),and NfnA could further convert 4HABA to PCA.Studies on enzyme characteristics show that the optimal reaction temperature of NfnB is 35℃ and the optimal pH is 7.5.The addition of metal ions does not increase enzyme activity.While Ag2+,Fe2+,Fe 3+5 Co2+,and Mn2+have moderate inhibition of the enzyme activity.And Zn2+ and Cu2+ have strong inhibition on enzyme activity.The optimal reaction temperature of NfnA is 30℃,and the optimal pH is 7.0.The addition of metal ions will strongly inhibit the enzyme activity.The Substrate spectrum research shows that NfnB could not only catalyze the reduction of 4NBA,but also catalyze some aromatic nitro compounds,such as the isomer 3-nitrobenzoate(3NBA).NfnA could only specifically catalyze the conversion of 4HABA to PCA.3.Transcriptional regulator of 4NBA degrading genesA transcription regulator gene nfnR was found upstream of nfnA and nfnB.NfnR was proposed as the TetR family transcriptional regulator through amino acid sequence alignment.Co-transcription analysis showed that nfnA and nfnB are in one transcriptional unit.Vector pSRGFP-18 was used to verify the repression regulation of nfnA and nfnB by NfnR,which could respond to effector 4NBA.The relative expression of nfnA and nfnB was increased 60-70 times in the 4NBA-induced strains than G-induced.NfnR was heterologously expressed in Escherichia coli BL21(DE3),and purified protein could bind to the promoter DNA of nfnA and nfnB.NfnR loses the ability to bind to promoter DNA when the 4NBA exists.The DNaseI footprinting results identified a 28-bp binding protected area,which almost completely covered the predicted nfnA and nfnB promoter position.After deleting this protected area,NfnR could not bind to the mutant promoter DNA.After analyzing the protected area sequence,it was found that there is a palindrome-like sequence "TACAANNNN NNNNNNNNATGTT".After deleting this 10-bp sequence,NfnR was also basically lost the ability to bind to promoter DNA,which indicated that this palindrome-like sequence might play a key role in regulating protein recognition or binding to DNA.