INVOLVEMENT OF 2 AMINO-ACID-RESIDUES IN THE LOOP REGION OF BACILLUS-THURINGIENSIS CRY1AB TOXIN IN TOXICITY AND BINDING TO LYMANTRIA-DISPAR

Two amino acids, Gly and Ser, at positions 282 and 283 in the loop reg ion of domain II of Cry1Ab2 toxin are substituted with AIa and Leu in the Cry1Ab9-033 toxin. Cry1Ab2 exhibited about a 10-fold increase in t oxicity and a 9-fold increase in binding affinity to Lymantria dispar compared to Cry1Ab9-033. However, these toxins showed similar toxicity and binding affinity to Manduca sexta and Spodoptera exigua. Heterolo gous competition assays and brush border membrane vesicle (BBMV) Ligan d blotting experiments demonstrated that Cry1Ab2 and Cry1Ab9-033 toxin s recognized the same 210-kDa L. dispar BBMV protein. No measurable di fferences in dissociation binding assays were observed between these t wo toxins. Digestion of these toxins with L. dispar gut enzymes and BB MV proteases indicated no differences in stability. Ala and Leu residu es in Cry1Ab9-033 were substituted with Gly and Ser by site-directed m utagenesis to produce mutant Cry1Ab alpha 8. This toxin exhibited full recovery of toxicity and binding affinity for L. dispar. These data s uggested that the residues Gly and Ser in the loop region might be dir ectly involved in receptor binding and toxicity in L. dispar. (C) 1996 Academic Press, Inc.