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Significant increase in toxicity of the Cry1Ac1 protein of Bacillus thuringiensis by the exchange of two amino acids in its domain III

Reinoso-Pozo, Yaritza, Del Rincón-Castro, Ma. Cristina, Ibarra, Jorge E.
Biological control 2018 v.125 pp. 1-6
Bacillus thuringiensis, Manduca sexta, Trichoplusia ni, amino acid substitution, amino acids, biological control, crystal proteins, crystals, genetic similarity, insecticidal proteins, larvae, mutants, site-directed mutagenesis, soil, toxicity, toxins
The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed high biochemical and genetic similarities with the HD-73 standard strain, besides expressing only a Cry1Ac protein: Cry1Ac39. However, LBIT-1200 was 6.4 and 9.5 times more toxic against Manduca sexta and Trichoplusia ni larvae, respectively, than HD-73, which expresses the Cry1Ac1 protein. Sequencing of Cry1Ac1 and Cry1Ac39 proteins showed only four amino acid differences, including two amino acid replacements in domain III, as compared to the rest of Cry1Ac proteins reported to date: N547Y and R602G. These two amino acid substitutions in domain III were introduced in the Cry1Ac1 protein from HD-73 through site directed mutagenesis. Three mutants were obtained: two single and one double mutant, which were expressed in an acrystaliferous B. thuringiensis strain, as well as the native Cry1Ac1 and Cry1Ac39 toxins. Bipyramidal crystals produced by the single mutant strains were larger than the original Cry1Ac1 protein; however, the high toxicity levels previously observed in the strain LBIT-1200 were restored in the three mutants, when tested against M. sexta and T. ni. The substitution N547Y seems to have a higher contribution to LBIT-1200 toxicity.