Applied and Environmental Microbiology, June 1999, p. 2503-2507, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
Received 25 November 1998/Accepted 15 March 1999
During sporulation, Bacillus thuringiensis produces
crystalline inclusions comprised of a mixture of
-endotoxins.
Following ingestion by insect larvae, these inclusion proteins are
solubilized, and the protoxins are converted to toxins. These bind
specifically to receptors on the surfaces of midgut apical cells and
are then incorporated into the membrane to form ion channels. The steps required for toxin insertion into the membrane and possible
oligomerization to form a channel have been examined. When bound to
vesicles from the midguts of Manduca sexta larvae, the
Cry1Ac toxin was largely resistant to digestion with protease K. Only
about 60 amino acids were removed from the Cry1Ac amino terminus, which
included primarily helix
1. Following incubation of the Cry1Ab or
Cry1Ac toxins with vesicles, the preparations were solubilized by
relatively mild conditions, and the toxin antigens were analyzed by
immunoblotting. In both cases, most of the toxin formed a large,
antigenic aggregate of ca. 200 kDa. These toxin aggregates did not
include the toxin receptor aminopeptidase N, but interactions with
other vesicle components were not excluded. No oligomerization occurred
when inactive toxins with mutations in amphipathic helices (
5) and known to insert into the membrane were tested. Active toxins with other
mutations in this helix did form oligomers. There was one exception; a
very active helix
5 mutant toxin bound very well to membranes, but
no oligomers were detected. Toxins with mutations in the loop
connecting helices
2 and
3, which affected the irreversible binding to vesicles, also did not oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the
Heliothis virescens midgut than with those from the
M. sexta midgut, which correlated with observed differences
in toxicity. Tight binding of virtually the entire toxin molecule to
the membrane and the subsequent oligomerization are both important
steps in toxicity.
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