OVERPRODUCTION OF ENCAPSULATED INSECTICIDAL CRYSTAL PROTEINS IN A BACILLUS-THURINGIENSIS SPO0A MUTANT

The spoOA gene of Bacillus subtilis encodes the key factor involved in the initiation of sporulation. It was previously shown that the B. th uringiensis (Bt) cryIIIA gene, encoding a toxin active against coleopt eran larvae, is overexpressed in an spoOA mutant of B. subtilis. In th is paper we describe the construction of a Bt spoOA mutant strain and its use to produce insecticidal crystal proteins. The spoOA gene of Bt was cloned and identified by its ability to transform a B. subtilis s poOA mutant to prototrophy. Its nucleotide sequence is homologous to t he B. subtilis gene. The spoOA gene was replaced in the Bt genome with a disrupted copy to give an Spo(-) strain unable to initiate sporulat ion. When the cryIIIA gene was cloned in the Bt spoOA mutant, large am ounts of toxins were produced and accumulated to form a large crystal inclusion which remained encapsulated within the ghost cell. These enc apsulated toxins were highly active against coleopteran larvae. We ant icipate that the cryIIIA expression system and the Bt spoOA mutant wil l provide a convenient process to generate novel formulations of stabi lized and environmentally safe Bt-based biopesticides.