The dissolution and degradation of 8-endotoxin (crystal) of Bacillus thuringiensis subsp. kurstaki strain HD-1 were investigated. Crystals were dissolved in 0.1 M phosphate-carbonate-NaOH buffer at pH > 12. Swelling of crystals occurred in the buffer between pH 10 and 11, and crystals dissolved in the same buffer supplemented with gut juice protease of the silkworm Bombyx mori. The proteolytic dissolution of crystals occurred after a time lag of several minutes in 0.1 M carbonate-NaOH buffer, pH 10.2. The time lag was not observed when crystals were suspended in the buffer for 30 min before the addition of protease. After the dissolution of the crystals and further degradation of the solubilized protein, the appearance of a toxic protein with a molecular weight of 59,000, designated P-59, was observed. Lower-molecular-weight peptides (less than 40,000) showed no toxicity to the silkworm larvae on feeding. Digestion of the 120,000-dalton subunit of the crystal by gut juice protease also produced P-59. These observations suggest the occurrence of a similar process in vivo, i.e., the swelling of crystals due to the alkalinity of gut juice and the production of P-59, dependent on the hydrolysis of swollen crystals by gut juice protease.

The structure of the subunit protein of the bipyramidal

Three proteases designated P-I, P-II, and P-III were separated from larval gut juice of the silkworm, Bombyxmori, and the effects of these proteases on the two morphologically different inclusions produced by Bacillus thuringiensis kurstaki HD-1 strain were investigated. Bipyramidal inclusions were solubilized by each protease at pH10.2, and proteins with a molecular weight of about 60,000 were produced, while cuboidal inclusions were resistant to the proteases. Protease P-III appeared to be most important in the dissolution of the bipyramidal inclusions. Based on the affinity to various synthesized substrates, P-III was considered to be a chymotrypsin-like protease. After dissolution of inclusions by P-III, dissolved and undissolved fractions were separated, and the insecticidal activity of each fraction was tested. Results indicate that bipyramidal inclusions are preferentially toxic to the silkworm and the cuboidal ones carry the mosquitocidal activity.

The mode of action of the toxic fragment (P-59) derived from bipyramidal-shaped 5-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 on the silkworm Bombyx mori was investigated. An enzyme-linked immunosorbent assay showed that there was no translocation of P-59 from the gut lumen to the hemocoel. When membrane vesicles prepared from silkworm midgut were incubated with P-59, normally smooth surface of vesicles became rough, and patch formation was observed on the surface. Vesicles treated with P-59 tended to agglutinate. The vesicle-denaturing activity of a 130,000-dalton subunit protein of bipyramidal toxin was enhanced by treatment with a gut juice protease of the silkworm. P-59 did not cause any uncoupling effect on mitochondria of the silkworm midgut. These results suggest that the attacking site of this toxin is not the mitochondrion but the cell membrane of the susceptible cell.

Bacillus thuringiensis kurstaki strain HD-1 produces two morphologically different parasporal inclusions of (5-endotoxin, bipyramidal and cuboidal inclusions. We found that the bipyramidal inclusion was composedof a 130,000-dalton subunit protein and was toxic to the silkworm, Bombyxmori (a lepidopteran insect), but not to the mosquito, Aedes aegypti (a dipteran insect) and that the cuboidal inclusion was composedof a 65,000-dalton subunit protein and was associated with the mosquitocidal activity. The distribution of the two proteins, the toxic fragment with a molecular weight of about 60,000 derived from the bipyramidal inclusions and the cuboidal toxin, was studied in various strains of B. thuringiensis by an enzyme-linked immunosorbentassay.