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. thuringiensis (Bt) cryIIIA gene, encoding a toxin active against coleopteran larvae, is overexpressed in an spoOA mutant of B. subtilis. In this 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 spoOA mutant to prototrophy. Its nucleotide sequence is homologous to the B. subtilis gene. The spoOA gene was replaced in the Bt genome with a disrupted copy to give an Spo(-) strain unable to initiate sporulation. When the cryIIIA gene was cloned in the Bt spoOA mutant, large amounts of toxins were produced and accumulated to form a large crystal inclusion which remained encapsulated within the ghost cell. These encapsulated toxins were highly active against coleopteran larvae. We anticipate that the cryIIIA expression system and the Bt spoOA mutant will provide a convenient process to generate novel formulations of stabilized and environmentally safe Bt-based biopesticides.