The expression and crystallization of Cry65Aa require two C-termini, revealing a novel evolutionary strategy of Bacillus thuringiensis Cry proteins

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.

In Vivo Crystallization of Three-Domain Cry Toxins

“…Another interesting example for this type of operon organization is the 118 kDa Cry65Aa1 toxin which reportedly requires two C-termini for crystallization [30]. The N-terminus of Cry65Aa1 resembles a typical three-domain Cry protein.…”

“…This downstream ORF was shown to be required for the efficient expression and crystallization of the Cry65Aa toxin. Peng et al [30] claimed that the unusual C-terminus of Cry65Aa is also required for crystallization, although this was not clearly demonstrated. Interestingly this report described how a stem-loop structure within the orf2 gene sequence can aid expression by stabilizing the cry65Aa mRNA.…”

Dissimilar Crystal Proteins Cry5Ca1 and Cry5Da1 Synergistically Act against Meloidogyne incognita and Delay Cry5Ba-Based Nematode Resistance

“…Although its genome harbors four potential nematicidal cry genes, only two of the four have the ability to target nematodes, with the exception of the cancer cell-killing parasporin Cry65Aa1 (39). Based on an analysis of other strains, such as B. thuringiensis L366 with naturally truncated Cry5Ad1 (40); B. thuringiensis DB27 products Cry21Fa1, Cry21Ha1, and naturally interceptive Cry21Ga1 (6); and YBT1518 products Cry6Aa2, Cry55Aa1, and silent Cry5Ba2 (26), we believe that the different characteristic crystal proteins that exist in one Bt may be the result of coevolution between the host and pathogen (41) and that the outcome arises from the natural selection pressure of antagonist nematodes and Bt itself.…”

Toxin stability improvement and toxicity increase against dipteran and lepidopteran larvae ofBacillus thuringiensiscrystal protein Cry2Aa

“…They demonstrated that the Cry2Aa production level was enhanced, but the increment did not exceed 15%. The variability of Cry2Aa production enhancement levels could mainly be due to differences in gene combination strategies …”