title: Recombinant Bacillus thuringiensis subsp. kurstaki HD73 strain that synthesizes Cry1Ac and chimeric ChiA74∆sp chitinase inclusions.
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.
authors.affiliation: Graduate Program in Biosciences, Life Science Division, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico.
authors.forename: Karen S
authors.initials: KS
authors.lastname: González-Ponce
authors.affiliation: Graduate Program in Biosciences, Life Science Division, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico.|Food Department, Life Science División, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico.
authors.forename: Luz E
authors.initials: LE
authors.lastname: Casados-Vázquez
authors.affiliation: Graduate Program in Biosciences, Life Science Division, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico.|Food Department, Life Science División, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico.
authors.forename: Rubén
authors.initials: R
authors.lastname: Salcedo-Hernández
authors.affiliation: Department of Natural and Mathematical Sciences, California Baptist University, 8432 Magnolia Avenue, Riverside, CA, 92504, USA.|Department of Entomology, University of California, Riverside, CA, 92521, USA.
authors.forename: Dennis K
authors.initials: DK
authors.lastname: Bideshi
authors.affiliation: Graduate Program in Biosciences, Life Science Division, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico.|Food Department, Life Science División, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico.
authors.forename: María C
authors.initials: MC
authors.lastname: Del Rincón-Castro
authors.affiliation: Graduate Program in Biosciences, Life Science Division, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico. josebar@ugto.mx.|Food Department, Life Science División, University of Guanajuato Campus Irapuato-Salamanca, 36500, Irapuato, Guanajuato, Mexico. josebar@ugto.mx.
authors.forename: José E
authors.identifier: http://orcid.org/0000-0002-0704-3076
authors.initials: JE
authors.lastname: Barboza-Corona
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics* / Q000648:ultrastructure; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D061046:Biological Control Agents; D002688:Chitinases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004731:Endotoxins / Q000096:biosynthesis / Q000235:genetics*; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000235:genetics*; D002479:Inclusion Bodies / Q000737:chemistry* / Q000648:ultrastructure; D007814:Larva; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D017384:Sequence Deletion; D018411:Spodoptera / Q000254:growth & development
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D061046:Biological Control Agents; D004731:Endotoxins; D006460:Hemolysin Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D002688:Chitinases
keywords: Bacillus thuringiensis; Chimeric crystals; Cry1Ac; Endochitinase ChiA74; Spodoptera frugiperda