Abstract
The insecticidal crystal protein (Cry) genes of Bacillus thuringiensis are a key gene resource for generating transgenic crops with pest resistance. However, many cry genes cannot be expressed or form crystals in mother cells. Here, we report a novel Cry protein gene, cry65Aa1, which exists in an operon that contains a downstream gene encoding a hypothetical protein ORF2. We demonstrated that ORF2 is required for Cry65Aa1 expression and crystallization by function as a C-terminal crystallization domain. The orf2 sequence is also required for Cry65Aa expression, because orf2 transcripts have a stabilizing effect on cry65Aa1 transcripts. Furthermore, we found that the crystallization of Cry65Aa1 required the Cry65Aa1 C-terminus in addition to ORF2 or a typical Cry protein C-terminal region. Finally, we showed that Cry65Aa1 has a selective cytotoxic effect on MDA-MB231 cancer cells. This report is the first description of a 130-kDa mass range Cry protein requiring two C-termini for crystallization. Our findings reveal a novel evolutionary strategy of Cry proteins and provide an explanation for the existence of Cry protein genes that cannot form crystals in B. thuringiensis. This study also provides a potential framework for isolating novel cry genes from “no crystal” B. thuringiensis strains.
Highlights
The expression and crystallization of Cry65Aa require two C-termini, revealing a novel evolutionary strategy of Bacillus thuringiensis crystal protein (Cry) proteins
This study provides a potential framework for isolating novel cry genes from ‘‘no crystal’’ B. thuringiensis strains
The SDS-PAGE profiles of sporulated cultures showed that all analyzed recombinant strains lacked distinct bands corresponding to Cry65Aa1 (Fig. 6C, lanes 1–3). These results indicated that Cry65A-C, ORF2, or a typical C-terminal domain does not contribute to the expression or crystallization of Cry65Aa1 when fused in-frame, such as those in 130-kDa crystal proteins
Summary
The expression and crystallization of Cry65Aa require two C-termini, revealing a novel evolutionary strategy of Bacillus thuringiensis Cry proteins. The insecticidal crystal protein (Cry) genes of Bacillus thuringiensis are a key gene resource for generating transgenic crops with pest resistance. Our findings reveal a novel evolutionary strategy of Cry proteins and provide an explanation for the existence of Cry protein genes that cannot form crystals in B. thuringiensis. Bacillus thuringiensis is a Gram-positive, spore-forming soil bacterium that can produce parasporal crystals during the sporulation phase. These parasporal crystals consist of proteins (Cry) that exhibit specific toxicity against a variety of insects, such as Lepidoptera, Coleoptera, and Diptera, and against some nematodes, mites, and protozoa[1,2]. The cry genes of B. thuringiensis are considered to be a key gene resource for generating transgenic crops with pest resistance[4]
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