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Recombinant Bacillus thuringiensis subsp. kurstaki HD73 strain that synthesizes Cry1Ac and chimeric ChiA74∆sp chitinase inclusions

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Abstract

In this study, the endochitinase chiA74 gene lacking its secretion signal peptide sequence (chiA74∆sp) 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 ChiA74∆sp 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 ChiA74∆sp 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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Acknowledgements

This work was supported by grants 004/2014 and 015/2015 (Academic Excellence) from the University of Guanajuato and project 258220 from CONACyT, México. Karen S. González-Ponce is a student in the Bioscience graduate program supported by CONACyT. Luz E. Casados-Vázquez is a Young Associate Researcher supported by Grant 2069, “Cátedra CONACyT”. We appreciate the help of Dr. Ricardo Navarro from the University of Guanajuato-CONACYT National Laboratory for SEM-EDX microphotographs and of MSc Jonathan C. Rangel-Núñez of University of Guanajuato by his help to carry out the bioassays.

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Authors and Affiliations

  1. Graduate Program in Biosciences, Life Science Division, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico

    Karen S. González-Ponce, Luz E. Casados-Vázquez, Rubén Salcedo-Hernández, María C. del Rincón-Castro & José E. Barboza-Corona

  2. Food Department, Life Science División, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico

    Luz E. Casados-Vázquez, Rubén Salcedo-Hernández, María C. del Rincón-Castro & José E. Barboza-Corona

  3. Department of Natural and Mathematical Sciences, California Baptist University, 8432 Magnolia Avenue, Riverside, CA, 92504, USA

    Dennis K. Bideshi

  4. Department of Entomology, University of California, Riverside, CA, 92521, USA

    Dennis K. Bideshi

Authors
  1. Karen S. González-Ponce
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  2. Luz E. Casados-Vázquez
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  3. Rubén Salcedo-Hernández
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  4. Dennis K. Bideshi
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  5. María C. del Rincón-Castro
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  6. José E. Barboza-Corona
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Corresponding author

Correspondence to José E. Barboza-Corona.

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Communicated by Erko Stackebrandt.

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González-Ponce, K.S., Casados-Vázquez, L.E., Salcedo-Hernández, R. et al. Recombinant Bacillus thuringiensis subsp. kurstaki HD73 strain that synthesizes Cry1Ac and chimeric ChiA74∆sp chitinase inclusions. Arch Microbiol 199, 627–633 (2017). https://doi.org/10.1007/s00203-017-1339-4

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  • DOI: https://doi.org/10.1007/s00203-017-1339-4

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