Study On The Regulation Of Chlorobenzene Metabolism In Pandoraeapnomenusa MCB032

Chlorobenzene is widely used as solvent,degreasing agent,flavoring agent and intermediate in the synthesis of various pesticides and dyes.The widespread use of chlorobenzene has resulted in their release into the environment in large quantities.Chlorobenzene has attracted much attention because of its persistent toxicity and accumulation in the food chain.So far,the research on the microbial metabolism of chlorobenzene has focused on its catabolic genes and enzymology.However,there are few reports on how chlorobenzene responds to the environment and its metabolic regulation mechanism.Pandoraeapnomenusa MCB032 is a new dominant strain in chlorobenzene bioreactor.It completely degrades chlorobenzene,and its metabolic pathway is encoded by two gene clusters,cbs and clc.These two gene clusters may contain multiple operons,and there are two possible regulatory proteins Cbs R and Clc R.Whether different induction conditions exist in their expression process,and whether these two potential regulatory proteins play a role in the succession and emergence of strain MCB032 are worthy of further study.This paper mainly studies the mechanism of the possible regulatory proteins Cbs R and Clc R in MCB032 strain on the metabolism of chlorobenzene,attempts to explain the mechanism of the emergence of the strain succession,and lays a foundation for further elucidation of the biological phenomenon of microbial tolerance to chlorobenzene and its metabolism.In this paper,the ability of strain MCB032 to degrade chlorobenzene was verified,and the growth curve and degradation curve of strain MCB032 were drawn.The results showed that strain MCB032 could completely degrade 1 m M of chlorobenzene within 24 hours,and the OD₆₀₀ of bacterial solution increased from 0to 0.4.Strain MCB032 was resistant to kanamycin,gentamicin and ambenomycin,but not to tetracycline and chloramphenicol.Whole genome sequencing confirmed the existence of cbs gene cluster in plasmid seq0002 and clc gene cluster in plasmid seq0003.The genome sequence is helpful to further study the mechanism of chlorobenzene degradation.RT-PCR showed that 6 genes of cbs gene clustercbs F,cbs Aa,cbs Ab,cbs Ac,cbs Adand cbs Bwere transcribed successively and located on the same operon cbs FAa Ab Ac Ad B.Five genes ofclc gene cluster,clc A,clc B,clc C,clc D and clc E,were sequentially transcribed and localized to the same operon,clc ABCDE.The clc R gene constituted an operon and formed an independent transcription unit.The transcriptional levels of cbs R and cbs FAa Ab Ac Ad B operon and clc R and clc ABCDE operon were also increased by RT-q PCR.The results indicated that the expression of genes involved in chlorobenzene degradation in strain MCB032 was induced by chlorobenzene.Through gene knockout and callback experiments,it was found that the regulatory protein Cbs R positively regulates the transcription of the operoncbs FAa Ab Ac Ad B,which encodes enzymes involved in the metabolic pathway from chlorobenzene to 3-chloro-catecholol.At the same time,Clc R positively regulates transcription of clc ABCDE operons,which encode enzymes involved in the3-chloro-catechol metabolic pathway.The transcription start sites of cbs FAa Ab Ac Ad B and cbs R operon were determined by primer extension analysis,and the transcription start site of cbs FAa Ab Ac Ad B operon was determined to be located on guanine（G）residues 590bp upstream of the start codon（ATG）of cbs F gene.The TSS of the cbs R operon is localized to guanine residues（G）66bp upstream of the start codon（ATG）of the cbsgene.Bioinformatics analyzed the possible binding sites of regulatory proteins,designed two DNA probes,and purified Cbs R protein for electrophoretic mobility shift assay（EMSA）,the results showed that Cbs R protein can bind to two DNA probes.In conclusion,this study verified the ability of MCB032 strain to degrade chlorobenzene.The composition of chlorobenzene metabolizing gene cluster was analyzed by whole genome sequencing and RT-PCR.The expression of genes involved in chlorobenzene degradation in strain MCB032 was induced by chlorobenzene by RT-q PCR.Through gene knockout and callback experiments and electrophoretic mobility shift assay,it was proved that the regulatory protein Cbs R positively regulates the transcription of the cbs FAa Ab Ac Ad B operon,and Clc R positively regulates the transcription of the clc ABCDE operon.Through these experimental studies,this paper lays a foundation for the in-depth elaboration of the biological phenomenon of microbial tolerance to chlorobenzene and its metabolism.