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


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Article Info

DATE

2017-02-09

AUTHORS

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

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. More... »

PAGES

627-633

References to SciGraph publications

  • 2009-04-01. Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of Bacillus thuringiensis in ANTONIE VAN LEEUWENHOEK
  • 2006-03. Carboxy-terminal extension effects on crystal formation and insecticidal properties of Colorado potato beetle-active Bacillus thuringiensis δ-endotoxins in MOLECULAR BIOTECHNOLOGY
  • 2000-10. Domain I plays an important role in the crystallization of Cry3A in Bacillus thuringiensis in MOLECULAR BIOTECHNOLOGY
  • 2008-02-08. Improving the Insecticidal Activity by Expression of a Recombinant cry1Ac Gene with Chitinase-Encoding Gene in Acrystalliferous Bacillus thuringiensis in CURRENT MICROBIOLOGY
  • 2015-09-24. Enhanced nematicidal potential of the chitinase pachi from Pseudomonas aeruginosa in association with Cry21Aa in SCIENTIFIC REPORTS
  • 2010-07-13. Integration of a Recombinant Chitinase into Bacillus thuringiensis Parasporal Insecticidal Crystal in CURRENT MICROBIOLOGY
  • 2014-01-24. Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use in MICROBIAL CELL FACTORIES
  • 2009-03-10. Efficient constitutive expression of chitinase in the mother cell of Bacillus thuringiensis and its potential to enhance the toxicity of Cry1Ac protoxin in APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
  • 1998-07. Cloning and expression of the cry1Ea4 gene of Bacillus thuringiensis and the comparative toxicity of its gene product in WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
  • Identifiers

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    http://scigraph.springernature.com/pub.10.1007/s00203-017-1339-4

    DOI

    http://dx.doi.org/10.1007/s00203-017-1339-4

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/28184966


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    30 schema:description 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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