Bacillus thuringiensis subsp kurstaki HD1 as a factory to synthesize alkali-labile ChiA74Δsp chitinase inclusions, Cry crystals and spores for applied use

Background: The endochitinase ChiA74 is a soluble secreted enzyme produced by Bacillus thuringiensis that synergizes the entomotoxigenecity of Cry proteins that accumulate as intracellular crystalline inclusion during sporulation. The purpose of this study was to produce alkaline-soluble ChiA74 Delta sp inclusions in B. thuringiensis, and to determine its effect on Cry crystal production, sporulation and toxicity to an important agronomical insect, Manduca sexta. To this end we deleted the secretion signal peptide-coding sequence of chiA74 (i.e. chiA74 Delta sp) and expressed it under its native promoter (pEHchiA74 Delta sp) or strong chimeric sporulation-dependent cytA-p/STAB-SD promoter (pEBchiA74 Delta sp) in Escherichia coli, acrystalliferous B. thuringiensis (4Q7) and B. thuringiensis HD1. Results: Based on mRNA analyses, up to similar to 9-fold increase in expression of chiA74 Delta sp was observed using the cytA-p/STAB-SD promoter. ChiA74 Delta sp (similar to 70 kDa) formed intracellular inclusions that frequently accumulated at the poles of cells. ChiA74 Delta sp inclusions were dissolved in alkali and reducing conditions, similar to Cry crystals, and retained its activity in a wide range of pH (5 to 9), but showed a drastic reduction (similar to 70%) at pH 10. Chitinase activity of E. coli-pEHchiA74 Delta sp was similar to 150 mU/mL, and in E. coli-pEBchiA74 Delta sp, 250 mU/mL. 4Q7-pEBchiA74 Delta sp and 4Q7-pEHchiA74 Delta sp had activities of similar to 127 mU/mL and similar to 41 mU/mL, respectively. The endochitinase activity in HD1-pEBchiA74 Delta sp increased 42x when compared to parental HD1 strain. HD1-pEBchiA74 Delta sp and HD1 harbored typical bipyramidal Cry inclusions, but crystals in the recombinant were similar to 30% smaller. Additionally, a 3x increase in the number of viable spores was observed in cultures of the recombinant strain when compared to HD1. Bioassays against first instar larvae of M. sexta with spore-crystals of HD1 or spore-crystal-ChiA74 Delta sp inclusions of HD1-pEB-chiA74 Delta sp showed LC(50)s of 67.30 ng/cm(2) and 41.45 ng/cm(2), respectively. Conclusions: Alkali-labile ChiA74 Delta sp inclusion bodies can be synthesized in E. coli and B. thuringiensis strains. We demonstrated for the first time the applied utility of synthesis of ChiA74 Delta sp inclusions, Cry crystals and spores in the same sporangium of HD1, a strain used successfully worldwide to control economically significant lepidopteran pests of agriculture. Our findings will allow to us develop strategies to modify expression of ChiA74 Delta sp while maximizing Cry crystal synthesis in commercial strains of B. thuringiensis.