| Bacillus thuringiensis(Bt)is a gram-positive bacterium belonging to the B.cereus group.The most significant feature of Bt is that it produces one or more parasporal crystals in the same mother cell during sporulation.Bt preparations have a broad insecticidal spectrum,are harmless to humans and animals,and do not pollute the environment,which are the most widely used microbial insecticides in the world.However,compared with traditional chemical pesticides,Bt preparations have poor field stability,short duration,and relatively low insecticidal efficiency,which have become a bottleneck restricting their further promotion.Ultraviolet(UV)radiation in sunlight is considered to be the critical influencing factor affecting the effectiveness of Bt preparations.Studies have shown that the encapsulation of crystals in mother cells can significantly reduce the sensitivity to UV and significantly increase the insecticidal activity.The development of engineered bacteria that the mother cells without lysis is a good strategy to improve the persistence of Bt preparations.However,the mechanism of mother cell lysis in Bacillus is only the discovery and research of hydrolase,it is still unknown whether other proteins are involved.This study screened and identified a new mutant HD(ΔmclX),the deletion of mclX gene completely blocked the lysis of mother cells.Therefore,this study started with mclX as the target gene,and explored the role of mclX gene in the process of mother cell lysis.The mclX gene encodes an unknown functional protein with a size of 32 k Da,and Mcl X has no known domains and no transmembrane structure.By measuringβ-galactosidase activity,we found that mclX is controlled byσ~K and negatively regulated by Ger E.Through optical microscope observation,it was found that the deletion of the mclX gene strain still not lysed on the third and fifth day,and the genetic complement strain could recover the phenotype.The deletion of mclX gene did not affect the sporulation,crystal protein production and insecticidal activity of the Bt HD73 strain.We expressed the Mcl X-His protein with histidine tag in Escherichia coli,and tested the cell wall hydrolysis activity with the purified Mcl X-His protein,we found that Mcl X-His could not hydrolyze the Bt HD73 cell wall in vitro.Western blot results showed that CwlC,the key hydrolase for mother cell lysis,was not detected in the HD(ΔmclX)cell lysis.Subsequently,the fusion expression vector of cwlC promoter and lac Z was transferred into HD(ΔmclX)mutant,β-galactosidase assay revealed that the transcription activity of cwlC promoter in mclX mutant was significantly lower than that of the wild type.Using the xylose-inducible promoter Pxyl to express cwlC in HD(ΔmclX),we found that cells can be lysed in HD(ΔmclX)strain,indicating that Mcl X affected the expression of cwlC gene at the transcriptional level.By measuring the promoter activity of sig K and ger E in HD(ΔmclX),we demonstrated that Mcl X did not affect the transcription activity of sig K and ger E.The bxp B gene was controlled byσ~K and positively regulated by Ger E.Its transcriptional activity in HD(ΔmclX)is not affected.Therefore,Mcl X did not affect the protein functions of Sig K and Ger E.This study identified the biological function of the mclX gene involved in the lysis of mother cells,and explored its relationship with the key hydrolase gene cwlC for the mother cell lysis,which laying a foundation for in-depth understanding of the mother cell lysis mechanism and obtain high-efficiency insecticidal strains. |
