Analysis of mutations in the pore-forming region essential for insecticidal activity of a Bacillus thuringiensis δ-endotoxin

Abstract

The Bacillus thuringiensis insecticidal δ-endotoxins have a three- domain structure, with the seven amphipathic helices which comprise domain I being essential for toxicity. To better define the function of these helices in membrane insertion and toxicity, either site-directed or random mutagenesis of two regions was performed. Thirty-nucleotide segments in the B. thuringiensis cry1Ac1 gene, encoding parts of helix α4 and the loop connecting helices α4 and α5, were randomly mutagenized. This hydrophobic region of the toxin probably inserts into the membrane as a hairpin. Site- directed mutations were also created in specific surface residues of helix α3 in order to increase its hydrophobicity. Among 12 random mutations in helix α4, 5 resulted in the total loss of toxicity for Manduca sexta and Heliothis virescens, another caused a significant increase in toxicity, and one resulted in decreased toxicity. None of the nontoxic mutants was altered in toxin stability, binding of toxin to a membrane protein, or the ability of the toxin to aggregate in the membrane. Mutations in the loop connecting helices α4 and α5 did not affect toxicity, nor did mutations in α3, which should have enhanced the hydrophobic properties of this helix. In contrast to mutations in helix α5, those in helix α4 which inactivated the toxin did not affect its capacity to oligomerize in the membrane. Despite the formation of oligomers, there was no ion flow as measured by light scattering. Helix α5 is important for oligomerization and perhaps has other functions, whereas helix α4 must have a more direct role in establishing the properties of the channel.