Archives of Microbiology (2017) 199, 627-633

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Karen S. González-Ponce, Luz E. Casados-Vázquez, Rubén Salcedo-Hernández, Dennis K. Bideshi, María C. del Rincón-Castro and José E. Barboza-Corona (2017)
Recombinant Bacillus thuringiensis subsp. kurstaki HD73 strain that synthesizes Cry1Ac and chimeric ChiA74Δsp chitinase inclusions
Archives of Microbiology 199 (4), 627-633
Abstract: In this study, the endochitinase chiA74 gene lacking its secretion signal peptide sequence (chiA74Δsp) was fused in frame with the sequence coding for the C-terminal crystallization domain and transcription terminator of cry1Ac. The chimeric gene was expressed under the strong pcytA-p/STAB-SD promoter system in an acrystalliferous Cry-B strain of Bacillus thuringiensis and B. thuringiensis subsp. kurstaki HD73. We showed that the chimeric ChiA74Δsp produced amorphous inclusions in both Cry-B and HD73. In addition to the amorphous inclusions putatively composed of the chimera, bipyramidal Cry1Ac crystals, smaller than the wild-type crystal, were observed in recombinant HD73, and chitinase activity was remarkably higher (75-fold) in this strain when compared with parental HD73. Moreover, we observed that lyophilized samples of a mixture containing Cry1Ac, amorphous inclusions, and spores maintained chitinase activity. Amorphous inclusions could not be separated from Cry1Ac crystals by sucrose gradient centrifugation. Interestingly, the chitinase activity of purified Cry1Ac/amorphous inclusions was 51-fold higher compared to purified Cry1Ac inclusions of parental HD73, indicating that the increased enzymatic activity was due primarily to the presence of the atypical amorphous component. The possibility that the chimera is occluded with the Cry1Ac crystal, thereby contributing to the increased endochitinolytic activity, cannot be excluded. Finally, bioassays against larvae of Spodoptera frugiperda with spore/crystals of HD73 or spore-crystal ChiA74Δsp chimeric inclusions of recombinant HD73 strain showed LC50s of 396.86 and 290.25 ng/cm2, respectively. Our study suggests a possible practical application of the chimera in formulations of B. thuringiensis-based lepidopteran larvicides.
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Database assignments for author(s): J. Eleazar Barboza-Corona

Research topic(s) for pests/diseases/weeds:
biocontrol - natural enemies
Research topic(s) for beneficials or antagonists:
genetical engin./transgenic plants


Pest and/or beneficial records:

Beneficial Pest/Disease/Weed Crop/Product Country Quarant.
Bacillus thuringiensis var. kurstaki (entomopathogen)
Bacillus thuringiensis Cry1A-toxin (entomopathogen)