Open AccessCysteine Scanning Mutagenesis of α4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa
Author(s) -
F. Girard,
Vincent Vachon,
Gabrielle Préfontaine,
Lucie Marceau,
Yanhui Su,
Geneviève Larouche,
C. Vincent,
Jean-Louis Schwartz,
Luke Masson,
Raynald Laprade
Publication year - 2008
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.00094-08
Subject(s) - manduca sexta , midgut , bacillus thuringiensis , brush border , mutant , cysteine , biology , biochemistry , toxin , mutagenesis , bombyx mori , amino acid , wild type , vesicle , microbiology and biotechnology , biophysics , insect , bacteria , larva , enzyme , membrane , genetics , gene , botany
Helix alpha4 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 alpha4 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.