Analysis Of Strong Promoters In SpoIIID Mutant Of Bacillus Thuringiensis

Bacillus thuringiensis?Bt?belongs to Bacillus cereus group?Bc?,a Gram-positive bacterium that can produce spores and parasporal crystals.Bt is widely used as a microbial insecticide while some problems still need to be sloved during application.The released Cry proteins are rapidly inactivated by exposure to sunlight when Bt is applied in the field,the large number of the released spores in the environment may cause ecological risks.Previous studies have proved that the deletion of the spoIIID gene results in no spore formation.However the effect of the spoIIID gene on the mother cell lysis and the crystal yield havenot been reported.This study carried out the following research work on the spoIIID mutant:1.We demonstrated that the sigK gene was positively regulated by SpoIIID and?^K negatively regulated the expression of sigE by using of?-galactosidase assay,which is similar to the regulation network in Bacillus subtilis.HD??spoIIID?had exhibited no mother cell lysis in sporulation medium Schaeffer's sporulation medium?SSM?and Luria-Bertani?LB?medium,but not in 1/2 LB medium.?-galactosidase assay revealed that the transcriptional activity of the cwlC gene in spoIIID mutant significantly decrease,compared to that in wild strain HD73 in SSM and 1/2 LB mediums.The transcriptional activity of cwlC promoter in 1/2 LB was higher than that in SSM medium.HD??spoIIID???cwlC?mutant had been obtained by knocking out the cwlC gene in HD??spoIIID?and displayed no mother cell lysis in both the SSM and 1/2 LB mediums.2.The deletion of spoIIID gene decreased the crystal protein production in HD73 which promoter rely on both and?^E?^K.P_orf1cry8E and P₅₀₁₄ directed by?^E were introduced into spoIIID mutant.The transcriptional activity of the two promoters in the spoIIID mutant was similar to that in the HD73wild-type strain.SDS-PAGE and bioassay results showed that the orf1cry8E and 5014 promoters in HD^-??spoIIID?can direct cry1Ac to produce the similar amount of crystal protein to the HD73wild-type strain,as well as with a highly insecticidal activity.3.Highly expression genes in HD??spoIIID?were screened by gene chip data analysis and their promoters was inserted into the linearized pHT304-18Z plasmid which harbors a promoterless lacZ gene.?-galactosidase assay showed that no promoter was found with stronger activity than orf1cry8E and 5014 promoters under the genetically background of spoIIID deletion.Then some genes regulated by SpoIIID in Bt were obtained through bioinformatics.SpoIIID protein have been expressed and purified for further determining SpoIIID regulon.In this study,we analyzed the promoters'activity and the mother cell lysis under the genetically background of spoIIID deletion.Two strong promoters were screened for the expression elements in this genetic background.The double mutant of both spoIIID and cwlC with no spore and mother cell lysis has been obtained,which provides a new strategy for the improvement of Bt engineering strains.