Characterization of a Bacillus thuringiensis delta-endotoxin which is toxic to insects in three orders

We report here the first Bacillus thuringiensis (Bt) toxin which is toxic to insects from three insect orders (Diptera, Coleoptera, and Lepidoptera). An oligonucleotide probe based on the delta-endotoxin N-terminal sequence was used to detect the gene. A 23-kb BamHI fragment containing the intact gene was identified and cloned from Bt strain YBT-226 plasmid DNA into the vector pBluescript II. Through a series of DNA manipulations the size of this fragment was reduced and the gene sequenced. The deduced amino acid sequence gave a predicted molecular mass of 137 kDa and was identical to a cry1Ba protein from Bt subsp. thuringiensis HD-2, which is now designated as Cry1Ba1 under a new classification scheme. This protein also showed 81.6% similarity with the Cry1B protein (Cry1Bb1) from Bt strain EG 5847. When the YBT-226 cry1Ba1 gene was expressed in an acrystalliferous Bt subsp. israelensis strain it produced irregular bipyramidal crystals during sporulation, which reacted specifically with anti-Cry1Ba antiserum. Bioassays using these crystals after purification resulted in significant mortality at low to moderate concentrations to larvae of the house fly (Musca domestica, Diptera), cottonwood leaf beetle (Chrysomela scripta, Coleoptera), and tobacco hornworm (Manduca sexta, Lepidoptera). This broad-spectrum toxicity was not dependent on presolubilization. In assays with insect cell lines not derived from midgut cells, the soluble toxin killed CH1t (Manduca sexta cells) but was inactive against CF1 (Choristoneura fumiferana cells), Aa(s) (Aedes aegypti), and C2 (Culex quinquefasciatus) mosquito cells.