Heterologous N- and C-terminal domains of 3D-cry proteins form a functional operon enabling natural crystallization and nematicidal activity.

Bacillus thuringiensis (Bt) is an insect pathogen that primarily relies on pore-forming toxins known as Cry proteins to kill its insect larval hosts. The effectiveness of Cry proteins has driven a worldwide search for Bt strains to identify and characterize novel insecticidal proteins with different specificities. In this study, Bt genome analysis revealed two consecutive open reading frames that are highly similar to the N-terminal of Cry14Aa1 and the C-terminal of Cry21Ca2, both of which target nematodes. The two genes can be cotranscribed as a functional operon; however, when expressed individually, neither gene forms crystalline inclusions. In contrast, operon-based co-expression restores crystal formation and confers nematicidal activity comparable to that of the full-length Cry14Aa protein. These findings demonstrate that the Bt genome encodes fragmented yet structurally complementary toxin genes that functionally reconstitute via operon-mediated co-expression. This work provides important insights into the functional recombination of Bt toxin genes and offers a novel model for the engineering of biopesticides.