Expression of ChiA74Δsp and its truncated versions in Bacillus thuringiensis HD1 using a vegetative promoter maintains the integrity and toxicity of native Cry1A toxins.
Our objective was to determine whether a recombinant chitinase ChiA74Δsp of Bacillus thuringiensis and its truncated versions (ChiA74Δsp-60, ChiA74Δsp-50) could be produced in B. thuringiensis HD1 with no detrimental effect on the size and insecticidal activity of the native bipyramidal Cry crystal. chiA-p, the promoter used to drive chitinase gene expression, was active during vegetative growth of Cry-B. HD1 recombinants showed increases from ∼7- to 12-fold in chitinase activity when compared with parental HD1 and negligible or no effect on the volume of bipyramidal crystals was observed. HD1/ChiA74Δsp-60 showed increases from 20% to 40% in the yield of Cry1A per unit of culture medium when compared with parental HD1 and HD1/ChiA74Δsp-50, HD1/ChiA74Δsp. Inclusion bodies presumably composed of the enzyme attached to native Cry1A crystals of recombinant strains were observed; these inclusions were likely responsible for the enhancements in chitinase activity. Western blot analysis using polyclonal anti-ChiA74Δsp showed a weak signal with proteins of ∼50 kDa in sporulated and lysed cells of recombinant strains. Bioassays against Spodoptera frugiperda using sporulated/lysed samples of the recombinant strains did not show statistically significant differences in LC50s when compared with HD1.