Transcription And Regulationof Cellobiose Transport Pathway In Bacillus Thuringiensis

Bacillus thuringensis（Bt）as a microbial insecticide,the active ingredient for the insecticidal crystal protein,because of its friendly to environmental and the human body become the world’s most widely used microbial pesticides.In the previous study,the deletion of Sigma54 factor was found in the HD73 strain to reduce the insecticidal crystal protein.Further analysis of cry1 Ac gene transcription was not controlled by Sigma54.Sigma 54 factor controls a variety of metabolic pathways,and Sigma54-controlled gene transcription also need enhancer binding protein（EBP）.In HD73 strain foundeight EBPs that which may be involved in the different metabolic pathway,so we try to know the mechanism of these metabolic pathway and analyze themechanism of insecticidal crystal proteins formation,which is benefit to optimize the fermentation conditions,carbon source and nitrogen source,in Bt production,thence effectively improving the production of insecticidal crystal proteins.In this study,the transcriptional regulation mechanism of cel gene cluster in Bt HD73 strain was studied.The transcriptional unit of cel gene cluster is cel A-cel B-cel C-cel D-cel E by RT-PCR in HD73.According to the annotations of the cel gene cluster found which encode of PTS system（phosphotransferase system）components,such as cel A（HD73₅614）encode the cellobiose-specific EIIB component of PTS,cel B（HD73₅613）encodes cellobiose-specific EIIC component of PTS,cel C encodes the cellobiose-specific EIIA component of PTS,cel D（HD73₅612）encodes β-6-P-glucosidase,cel E encode lactose/cellobiose-specific EIIA subunit of PTS and cel R（HD73₅607）encodes the transcriptional regulator of CelR（Sigma54 factor interaction domain-containing protein）which is EBPs.Conserved domain analysis showed that the CelR contained the HTH domain for DNA binding,a domain homologous to the central AAA+ domain of ATPase associated for the interaction with the RNA polymerase associated with Sigma54,a domain corresponding to Enzyme IIAman of the PTS,and two PTS regulation domains（PRD）.All these features suggested that the CelR protein is Sigma54-dependent transcription activator factor.The promoter sequences of the cel gene cluster analysis showed that it contains Sigma54-binding site,the CelR-binding site and a Ccp A-binding siteon the DBTBs database（http://dbtbs.hgc.jp/）.The 5’-RACE results showed the transcriptional start sites（TSS）of the cel gene cluster is located 55 bp upstream from the beginning of the cel A coding sequence,and it has 13 bp between the TSS and the-12 of the Sigma54-binding site in the Pcel A.The β-galactosidase assay showed the transcription of cel gene cluster is induced by cellobiose and repressed by glucose.The induced transcriptional activity of cel gene cluster is controlled by Sigma54 factor and activated through the Sigma54-dependent transcriptional regulator CelR.However,the transcription of cel operon is repressed by glucose,and Ccp A mediated this repression.The Δcel A,Δcel B,Δcel C,Δcel D and Δcel E mutants were obtained through the method of homologous recombination technology.According to PTS activity and utilization of cellobiose showed that cellobiose only be used by HD73 strain rather than the other cel gene cluster mutants,Δsig L and Δcel R,which illustrates the enzymes encoded by the cel gene cluster participate in the transport and metabolism of cellobiose,and this process is controlled by Sigma54 and positive regulatedby CelR.Cellobiose is potential carbon source as the component of cellulose widely existing in the nature.So confirming the mechanism of cellobiose utilization and the function of genes involved in the celoperon is contribute to the carbon source effectively utilizing in the Bt industry fermentation and production.We also make a base for improving the yield of insecticidal crystal proteins in Bt strain from the view of metabolism regulation.