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Journal of Bacteriology, October 1999, p. 6103-6107, Vol. 181, No. 19
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Analysis of Mutations in the Pore-Forming Region Essential for Insecticidal Activity of a Bacillus thuringiensis delta -Endotoxin

A. S. Manoj Kumar and A. I. Aronson*

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Received 8 March 1999/Accepted 29 July 1999

The Bacillus thuringiensis insecticidal delta -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 alpha 4 and the loop connecting helices alpha 4 and alpha 5, 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 alpha 3 in order to increase its hydrophobicity. Among 12 random mutations in helix alpha 4, 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 alpha 4 and alpha 5 did not affect toxicity, nor did mutations in alpha 3, which should have enhanced the hydrophobic properties of this helix. In contrast to mutations in helix alpha 5, those in helix alpha 4 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 alpha 5 is important for oligomerization and perhaps has other functions, whereas helix alpha 4 must have a more direct role in establishing the properties of the channel.

* Corresponding author. Mailing address: Department of Biological Sciences, Purdue University, W. Lafayette, IN 47906. Phone: (765) 494 4992. Fax: (765) 494 0876. E-mail: aaronson{at}bilbo.bio.purdue.edu.

Journal of Bacteriology, October 1999, p. 6103-6107, Vol. 181, No. 19
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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