Enhanced toxicity of Bacillus thuringiensis Cry3A delta-endotoxin in coleopterans by mutagenesis in a receptor binding loop.
We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 350-354. Two mutant toxins, A1 (R(345)A,Y(350)F,Y(351)F) and A2 (R(345)A,DeltaY(350), DeltaY(351)), showed significantly improved toxicity against Tenebrio molitor (yellow mealworm). The mutant toxin A1 was also more potent against both Leptinotarsa decemlineata (Colorado potato beetle) and Chrysomela scripta (cottonwood leaf beetle), while A2 displayed enhanced toxicity only in L. decemlineata. Competitive binding assays of L. decemlineata brush border membrane vesicles (BBMV) revealed that binding affinities for the A1 and A2 mutant toxins were ca. 2.5-fold higher than for the wild-type Cry3 toxin. Similar binding assays with C. scripta BBMV revealed a ca. 5-fold lower dissociation rate for the A1 mutant as compared to that of Cry3A.
Wu
S J
SJ
Department of Biochemistry, The Ohio State University, 484 W. 12th Ave., Columbus, OH, USA.
Koller
C N
CN
Miller
D L
DL
Bauer
L S
LS
Dean
D H
DH
D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D001517:Coleoptera / Q000187:drug effects* / Q000254:growth & development; D004274:DNA, Recombinant / Q000235:genetics; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D015870:Gene Expression; D006460:Hemolysin Proteins; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013712:Tenebrio / Q000187:drug effects / Q000254:growth & development; D018675:Toxicity Tests
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004274:DNA, Recombinant; D004731:Endotoxins; D006460:Hemolysin Proteins; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis