Background: Bacillus thuringiensis
Cry toxins bind with different insect midgut proteins leading to toxin
oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to
readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction.
The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against
different Lepidoptera, and no-cross resistance with Cry1A.
Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to
SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if
Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal
end that is processed during activation participate in this oligomerization. To determine the role of these
residues on oligomerization and in toxicity single and double alanine substitution were constructed. In
contrast to single C14A and C16A mutants, the double C14A–C16A mutant did not form oligomers in solution.
Toxicity assays against
Plutella xylostella
showed that the C14A–C16A mutant had a similar insecticidal activity
as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor
binding are not related with toxicity.
Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and
C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action
of Cry9Aa leading to toxicity.
