Single amino acid changes in domain II of Bacillus thuringiensis CryIAb delta-endotoxin affect irreversible binding to Manduca sexta midgut membrane vesicles

Abstract

Deletion of amino acid residues 370 to 375 (D2) and single alanine substitutions between residues 371 and 375 (FNIGI) of lepidopteran-active Bacillus thuringiensis CryIAb ␦-endotoxin were constructed by site-directed mutagenesis techniques. All mutants, except that with the I-to-A change at position 373 (I373A), produced ␦-endotoxin as CryIAb and were stable upon activation either by Manduca sexta gut enzymes or by trypsin. Mutants D2, F371A, and G374A lost most of the toxicity (400 times less) for M. sexta larvae, whereas N372A and I375A were only 2 times less toxic than CryIAb. The results of homologous and heterologous competition binding assays to M. sexta midgut brush border membrane vesicles (BBMV) revealed that the binding curves for all mutant toxins were similar to those for the wild-type toxin. However, a significant difference in irreversible binding was observed between the toxic (CryIAb, N372A, and I375A) and less-toxic (D2, F371A, and G374A) proteins. Only 20 to 25% of bound, radiolabeled CryIAb, N372A, and I375A toxins was dissociated from BBMV, whereas about 50 to 55% of the less-toxic mutants, D2, F371A, and G374A, was dissociated from their binding sites by the addition of excess nonlabeled ligand. Voltage clamping experiments provided further evidence that the insecticidal property (inhibition of short-circuit current across the M. sexta midgut) was directly correlated to irreversible interaction of the toxin with the BBMV. We have also shown that CryIAb and mutant toxins recognize 210-and 120-kDa peptides in ligand blotting. Our results imply that mutations in residues 370 to 375 of domain II of CryIAb do not affect overall binding but do affect the irreversible association of the toxin to the midgut columnar epithelial cells of M. sexta.

All Domains of Cry1A Toxins Insert into Insect Brush Border Membranes

“…2 and by earlier studies (23). We are further investigating the specific role of Phe-371 in mediating membrane insertion.…”

“…Uracil-containing template for Cry1Ab was obtained as described (23). Primers for site-directed mutagenesis were obtained from either Integrated DNA Technologies, Inc., or Bioneer Inc. Site-directed mutagenesis was carried out using mutagene M13 in vitro mutagenesis kit as described in the manufacturer's manual (Bio-Rad).…”

Bioactivities of cry gene positive Bacillus thuringiensis (Berliner) (Bacillales: Bacillaceae) strains on Ephestia kuehniella Zeller, 1879 and Plodia interpunctella (Hübner, 1813) (Lepidoptera: Pyralidae)

“…In addition, plasmid patterns, protein profiles and cry2-type genes of the most toxic strains were identified. Substantial evidence indicates that B. thuringiensis from different regions produce genetically and functionally different toxins (Palma et al, 2014) and a single amino acid changes in insecticidal Cry proteins can significantly alter their toxicity (Udayasuriyan et al, 1994;Rajamohan et al, 1995). The fact that some insects develop resistance against B. thuringiensis toxins after repeated exposure (Tabashnik, 1994;Ferre et al, 1995) makes it necessary to detect and identify new B. thuringiensis strains for the management of resistant insect populations.…”

Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

“…A previous study [33], in which residues in loop 2 of Cry1Ab toxin were replaced by alanine, showed that mutant Phe 371 Ala had reduced toxicity for M. sexta larvae. Competitions experiments revealed that this mutant had similar binding parameters as Cry1Ab, but dissociation binding experiments showed that the percentage of irreversible binding (membrane insertion) was lower.…”