The Function Of A Hydrolase,CwlC,Essential For Mother Cell Lysis In Bacillus Thuringiensis

Bacillus thuringiensis is used worldwide as an effective pesticide that can produce toxic proteins known as crystal proteins during sporulation.However,crystal proteins released from mother cells can be inactivated in the field when exposed to environmental stresses,such as UV-light.Studies have shown that crystal encapsulation in the mother cell can effectively degrade the sensitivity to UV.Currently,little is known about how cell wall hydrolases function in B.thuringiensis.In this study,we will study the essential enzymes involved in mother cell lysis and explore the new genetic improvement strategy for B.thuringiensis strain.Previously,it was demonstrated that deletion of the cwlB gene can delay,but not block mother cell lysis in B.thuringiensis.In this study,a novel gene(tentatively named cwlC)involved in mother cell lysis of B.thuringiensis was identified and characterized.The CwlC protein consists of an N-terminal N-acetylmuramoyl-L-alanine amidase(MurNAc-LAA)domain and an amidase02-C domain,which was deduced a cell wall binding domain.Furthermore,NCBI blastP in the protein nr database showed that many orthologs of the CwlC protein were found in the B.cereus group,with the amino acid sequence identities more than 90%.Reverse transcription-PCR and 5?-rapid amplification of cDNA ends-PCR analysis indicated that cwl C is transcribed as a monocistronic operon from a“G,”located 21 nucleotides upstream of its translational start codon,by a?~K-dependent RNA polymerase.Deletion of cwlC completely blocked mother cell lysis in the late sporulation stage without affecting the sporulation frequency or Cry1Ac protein production.The purified histidine-tagged CwlC protein produced in Escherichia coli cells was 27.9 k Da in size and bound to the B.thuringiensis cell wall.Furthermore,CwlC hydrolyzed living B.thuringiensis and B.cereus cells at exponential growth phase.Laser scanning confocal microscopy(CLSM)was used to observe the lysis of mutant HD(?cwlC)and wild-type strain HD73 cell membrane.The result showed that Bt cell membrane first ruptured and then cell wall collapsed.CLSM observation of GFP-CwlC fusion proteins revealed that CwlC protein was expressed in the cytoplasm of late sporulation cells,not expressed in the spores,and the presence of CwlC protein was found at the site of cell membrane rupture.It is more likely that after the rupture of cell membrane,CwlC proteins enter into cell envelope to lyse cell wall.Our data provide evidence that CwlC is not only critical for B.thuringiensis mother cell lysis during sporulation,which suggests the potential for agricultural applications with new effective engineered B.thuringiensis strains,but that CwlC also shows potential for applications in biomedicine and food industry.