Effect Of The Cysteines Located In C-terminal Half Of Bacillus Thuringiensis Crystal Protein Cry7Ba1 On Its Crystal Solubility And Insecticidal Activity

The formation of a large crystalline inclusion containing insecticidal toxins in the mother cell of Bacillus thuringiensis during sporulation is an important aspect of this bacterium’s pathogenicity. The crystal can dissolve in the alkaline insect gut, allowing the soluble toxins to exert their pathogenic effect. In previous study, cry7Bal was isolated from Bacillus thuringiensis subsp. huazhonggiensis YBT-978, that encoded protein Cry7Bal cannot be dissolved and have no insecticidal activity at low pH (9.5), while can be dissolved and exhibit high toxicity against to Plutella xylostella larvae at higher pH (12.5), After the C-terminal of Cry7Bal were replaced by that of Cry1Ac and Cry1C, the recombinant crystal proteins could be dissolved at pH 9.5, and exhibited toxicity to P. xylostella larvae.Sequence analysis showed there are 7 cysteines (cys) locate in Cry7Bal C-terminal, which is more than that of conventional 130-to 140-kDa crystal protein. The abundance of cys may play a critical role for Cry7Bal solubility. Further,6 single cysteine mutations (C834S, C854S, C697S, C1059S, C1101S, and C1154S) were successful constructed by substituting with serine. The expression and crystal formation results showed no differences among these six mutants and Cry7Ba1. Solubility results found that three mutations (C834S, C854S and C697S) can be dissolved at lower pH (9.5-10.5). Bioassay results showed that mutation C834S recovers the toxicity against P. xylostella (LC50=3.07μg/mL). Our discovery suggest a single cys changes in C-terminal of Cry protein has a major practical significance with respect to recover the solubility and toxicity of Cry proteins which like Cry7Ba1, it also has important implications related to the application of such Cry proteins.