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Applied and Environmental Microbiology, January 2002, p. 194-200, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.194-200.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mutagenic Analysis of a Conserved Region of Domain III in the Cry1Ac Toxin of Bacillus thuringiensis

Luke Masson,^1* Bruce E. Tabashnik,² Alberto Mazza,¹ Gabrielle Préfontaine,¹ Léna Potvin,¹ Roland Brousseau,¹ and Jean-Louis Schwartz^1,3

Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2,¹ Department of Entomology, University of Arizona, Tucson, Arizona 85721,² Groupe de recherche en transport membranaire, Université de Montréal, Montreal, Quebec H3C 3J7, Canada³

Received 10 April 2001/ Accepted 30 October 2001

We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4- to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with Plutella xylostella larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.

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

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Applied and Environmental Microbiology, January 2002, p. 194-200, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.194-200.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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