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Cysteine Scanning Mutagenesis of 4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

Groupe d'Étude des Protéines Membranaires, Université de Montréal,¹ Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada,² Peking University, Beijing, People's Republic of China,³ Horticultural Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, Quebec, Canada⁴

Received 11 January 2008/ Accepted 21 February 2008

Helix 4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the 4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.

Published ahead of print on 7 March 2008.