Abstract: Cry4Aa produced by Bacillus thuringiensis subsp. israelensis (Bti) exhibits a specific toxicity to Anopheles, Aedes, and Culex larvae, which are vectors of serious diseases, and formulations of Bti are used worldwide for mosquito control. In general, domain II of the Cry toxin is believed to be important for target specificity, and three loops (loops 1, 2, and 3) in domain II have been studied extensively. In this report, to analyze the biological functions of loops 1, 2, and 3 of Cry4Aa, mutants were constructed in which one of the loops was replaced with either of the other two loops. A bioassay using Culex pipiens larvae revealed that the mosquitocidal activity was virtually lost upon replacement of loop2. The mutants in which loops 1 and/or 3 were replaced also showed decreased activity, but they still maintained some activities. This suggested that loop2, but not loops 1 and 3, was essential for the mosquitocidal activity of Cry4Aa. Proteolytic digestion revealed the involvement of loops in the stability of the Cry4Aa structure. No significant differences were observed in the amount of wild-type and mutant Cry4Aa bound to the BBMVs prepared from the C. pipiens larvae.

Abstract: Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is, therefore, of great interest for developing a bioinsecticide to control mosquitoes. However, the expression of Cry4Aa in Escherichia coli is relatively low, which is a major disadvantage in its development as a bioinsecticide. In this study, to establish an effective production system, a 1,914-bp modified gene (cry4Aa-S1) encoding Cry4Aa was designed and synthesized in accordance with the G + C content and codon preference of E. coli genes without altering the encoded amino acid sequence. The cry4Aa-S1 gene allowed a significant improvement in expression level, over five-fold, compared to that of the original cry4Aa gene. The product of the cry4Aa-S1 gene showed the same level of insecticidal activity against Culex pipiens larvae as that from cry4Aa. This suggested that unfavorable codon usage was one of the reasons for poor expression of cry4Aa in E. coli, and, therefore, changing the cry4Aa codons to accord with the codon usage in E. coli led to efficient production of Cry4Aa. Efficient production of Cry4Aa in E. coli can be a powerful measure to prepare a sufficient amount of Cry4Aa protein for both basic analytical and applied researches.

Abstract: Cry1C, one of the lepidopteran-specific insecticidal proteins from Bacillus thuringiensis, exhibits potent cytotoxicity against Sf9, an insect cell line. Cry1Aa and Cry4A, which are lepidopteran- and dipteran-specific insecticidal proteins, respectively, show no cytotoxicity against Sf9. When domain III of Cry1C was replaced with that of Cry1Aa or Cry4A, the hybrid Cry1C protein retained the cytotoxicity. These results suggest that domain III of Cry1C is not crucial in determining the cytocidal specificity of Cry1C against Sf9.