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Applied and Environmental Microbiology, July 2009, p. 4661-4667, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.00409-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa Synergism{triangledown} ,{dagger}

Alejandro Hernández-Soto,1 M. Cristina Del Rincón-Castro,2,3 Ana M. Espinoza,1 and Jorge E. Ibarra2*

Centro de Investigación en Biología Celular y Molecular, Ciudad de la Investigación, Universidad de Costa Rica, San José, Costa Rica,1 Departamento de Biotecnología y Bioquímica, CINVESTAV, Irapuato, Guanajuato, México,2 División de Ciencias de la Vida, Universidad de Guanajuato, Irapuato, Guanajuato, México3

Received 18 February 2009/ Accepted 14 May 2009

Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-µm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

* Corresponding author. Mailing address: CINVESTAV-IPN, Apartado Postal 629, 36500 Irapuato, Gto., Mexico. Phone: 52-462-623-9643. Fax: 52-462-624-5996. E-mail: jibarra{at}ira.cinvestav.mx

{triangledown} Published ahead of print on 22 May 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, July 2009, p. 4661-4667, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.00409-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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