Study On Molecular Mechanism Of Insecticidal Crystal Formation In Bacillus Thuringiensis

Bacillus thuringiensis(Bt)has become the most widely used microbial insecticide worldwide,its biological activities are mainly attributed to the insecticidal crystal proteins.It is important to study the molecular mechanism of insecticidal crystal formation in Bacillus thuringiensis for improving the yield of insecticidal crystal protein,enhancing the virulence of Bt and constructing recombinant Bt strain with high efficiency and broad spectrum.As previous reports indicated that the cannibalism behavior in Bacillus subtilis could affect the spore fornation period,we carried out the research about whether there existed such similar behavior in Bt.According to genome sequencing data of our screened Bt4.0718 and bioinformatics comparison analysis,we found that this strain contains homologous sequence to the gene modules involved in cannibalism behavior of Bs.It was revealed that sdpRI-sdpABC module is present on the plasmid and sdpRⅡ module is present on the chromosome in Bt.0718.Meanwhile,we also found that the acrystalliferous strain XBU001 of Bt contains only sdpRⅡ module on its chromosome.Fluorescent staining and laser confocal microscope revealed that in logarithmic growth phase,Bt4.0718 populations were all viable cells,while in early and middle stationary growth phase,viable and dead cells existed in Bt4.0718 populations simultaneously.These results suggested that there existed cannibalism phenomenon during the culture of Bt4.0718.Furthermore,we constructed the sdpRⅡ module deletion mutant of XBU001.The sdpRⅠ-sdpABC module was amplified from Bt4.0718 and cloned into pHT315 vector.The recombinant plasmid was transformed into XBU001 to analysis the preliminary function of its gene modules involved in cannibalism behavior.The 130～140 kDa Cry1 protoxins contained multiple cysteine(Cys)residues in the non-toxic C-terminal half.It is generally assumed that these Cys residues contributed to crystal assembly through forming disulfide bonds.In addition,the N-terminal non-toxic portion cleaved by proteolytic in the activation process in insect midgut also contains Cys residues.In order to further study the function of non-toxic area Cys residues in crystal formation,we conducted serine(Ser)scanning mutagenesis on Cry1Ac protoxin of Bt subsp.kurstaki HD73.The production and assembly of Cry1Ac was analyzed by SDS-PAGE and phase-contrast microscope.The 16 cysteine(Cys)gene in cry1Ac non-toxin C-terminal half and N-terminal portion were replaced by serine(Ser),cloned into pHT315 vector,then expressed in XBU001.All 15 single and 15 accumulated mutant recombinants could still form typical diamond-shaped crystals through phase-contrast microscope observation.SDS-PAGE analysis of Cry1Ac showed that all the single and accumulated mutant recombinants strains could aslo able to express the expected size protein of 130 kDa.These results indicated that individually and simultaneously replacement of cysteine residues in cry1Ac gene did not affect the crystal formation and expression.Furthermore,Bioassay results showed that the wild-type and accumulated Cys residues mutantion in non-toxin region of Cry1Ac recombinant strains had moderate toxicity against Helicoverpa armigera.We also evaluated the function of ORF2 accessory protein from Bt subsp.jegathesanon the expression of Cry11A.By using genetic engineering techniques,the cry11A individually expression vector or p20,orf2-30Ca with cry11A co-expression vector under the control of cytlAP/STAB sequence were constructed.Polyacrylamide gel block coupled to mass spectrometry showed that the accessory proteins P20,ORF2-30Ca were co-expressed with Cry11A.All three difference recombinant strains could produce visible inclusions through phase-contrast microscope observation.When ORF2-30Ca and Cry11A were co-expressed,SDS-PAGE analysis showed that the expression level of Cry11A was only one second compared to that expressed Cry11A alone or P20 co-expressed with Cry 11 A.The virulent against fourth instar Culex quinquefasciatushas also reduced.These results illustrated that the presence of ORF2-30Ca affected the correct folding or stability of Cry11A,resulting in the degradation of inclusions in or after the strain lysis.In summary,this paper found that the cannibalism behavior existed in Bt4.0718 strain.The preliminary function analysis of its gene module of the behavior laid the foundation of further study on insecticidal crystal formation and provides a basis for the study of Bt fermentation period.Meanwhile,site-directed mutagenesis studies of non-toxic region of cysteine of Cry toxin and accessory proteins P20,ORF2-30C on the Cry11A expression in the study enrich people’s understanding on insecticidal crystal formation and regulation mechanismof Bt,it provides a guideline for the future to enhance the expression level of crystal protein and build efficient engineering Bt strains.