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Applied and Environmental Microbiology, December 1998, p. 4782-4788, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A Holistic Approach for Determining the Entomopathogenic Potential of Bacillus thuringiensis Strains

Luke Masson,1,* Martin Erlandson,2 Marianne Puzstai-Carey,3 Roland Brousseau,1 Victor Juárez-Pérez,4 and Roger Frutos4

Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec H4P 2R2,1 and Agriculture Canada Research Station, Agriculture Canada, Saskatoon, Saskatchewan S7N 0X2,2 Canada; Case Western Reserve University, Cleveland, Ohio 441063; and BIOTROP-IGEPAM, CIRAD, 34032 Montpellier Cedex 1, France4

Received 17 June 1998/Accepted 29 September 1998

The cry gene content of Bacillus thuringiensis subsp. aizawai HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR. A total of six cry genes were detected in genomic DNA purified from HD-133, four from the cry1 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the cry2B and cry1I genes, but not the cry1Aa gene, were transcribed. Cloning and sequencing of the cry1Aa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of B. thuringiensis isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total cry gene content of B. thuringiensis strains may be higher than previously thought.

* Corresponding author. Mailing address: Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montréal, Québec H4P 2R2, Canada. Phone: (514) 496-6150. Fax: (514) 496-6213. E-mail: Luke.Masson{at}NRC.Ca.

Applied and Environmental Microbiology, December 1998, p. 4782-4788, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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