Bioassay of Solubilized \u3ci\u3eBacillus thuringiensis\u3c/i\u3e var. \u3ci\u3eisraelensis\u3c/i\u3e Crystals by Attachment to Latex Beads

Solubilized crystals of Bacillus thuringiensis var. israelensis were 7,000 times less toxic to Aedes aegypti larvae than intact crystals, presumably because mosquito larvae are filter feeders and selectively concentrate particles while excluding water and soluble molecules. A procedure is described whereby soluble toxins are adsorbed to 0.8- micrometer latex beads, with retention of toxicity. The latex bead assay should make it possible to analyze the structure and mode of action of the mosquito toxin

Resistance of Agrotricums to wheat streak mosaic

Digitized by Kansas Correctional Industrie

Stability of the Larvicidal Activity of \u3ci\u3eBacillus thuringiensis\u3c/i\u3e subsp. \u3ci\u3eisraelensis\u3c/i\u3e: Amino Acid Modification and Denaturants

The Bacillus thuringiensis subsp. israelensis mosquito larvicidal toxin is not a sulfhydryl-activated toxin. The protein disulfide bonds were cleaved and blocked without loss of toxicity. In contrast, modification of the lysine side chains eliminated toxicity. Additionally, the toxin was resistant to high concentrations of salt (8 M NaBr), organic solvents (40% methanol), denaturants (4 M urea), and neutral detergents (10% Triton X-100). However, it was inactivated by both positively and negatively charged detergents and by guanidine hydrochloride

Amino Sugars in the Glycoprotein Toxin from \u3ci\u3eBacillus thuringiensis\u3c/i\u3e subsp. \u3ci\u3eisraelensis\u3c/i\u3e

The carbohydrate content of purified Bacillus thuringiensis subsp. israelensis crystal toxin was determined by six biochemical tests, column chromatography on an amino acid analyzer, and the binding of 11 fluorescent lectins. The crystals contained approximately 1.0% neutral sugars and 1.7% amino sugars. The anmino sugars consisted of 70% glucosamine and 30% galactosamine. No N-acetylneuraminic acid (sialic acid) was detected. The presence of amino sugars was confirmed by the strong binding of fluorescent wheat germ agglutinin and the weak binding of fluorescent soybean agglutinin. These lectins recognize N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, respectively. The lectin-binding sites appeared evenly distributed among the protein subunits of the crystal. The sugars were covalently attached to the crystal toxin because wheat germ agglutinin still bound alkali-solubilized toxin which had been boiled in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. This study demonstrates the covalent attachment of amino sugars and indicates that the B. thuringiensis subsp. israelensis protein toxins should be viewed as glycoprotein toxins. The crystals used in the present study were purified on sodium bromide density gradients. Studies employing crystals purified on Renografin density gradients can give artificially high values for the anthrone test for neutral sugars

Immunological Relationships among Proteins Making Up the \u3ci\u3eBacillus thuringiensis \u3c/i\u3esubsp. \u3ci\u3eisraelensis\u3c/i\u3e Crystalline Toxin

The immunological relationships among the proteins of the mosquito larvicidal toxin produced by Bacillus thuringiensis subsp. israelensis have been investigated by using polyclonal antisera specific for the 28-, 70-, and 135-kilodalton proteins. Each of these proteins was immunologically distinct. There was no cross-reaction among the three proteins and the two non-homologous antisera. Treatment of toxin proteins with larval gut enzymes for 20 h identified protease-resistant domains at approximately 65, 38, and 22 kilodaltons. Similar domains were generated by treatment with trypsin and chymotrypsin. Our immunological and kinetic data indicate that the 28-kilodalton protein is degraded successively to protein bands at 26, 25, 23, and 22 kilodaltons, the 70-kilodalton protein is degraded to a protein at 38 kilodaltons, and the 135-kilodalton protein is degraded successively to protein bands at 94, 72, and, probably, 65 kilodaltons. Solubilized toxin possesses two biological activities, larvicidal and general cytolytic (hemolytic). We used nondenaturing gel electrophoresis to show that the hemolytic activity resides in the 28-kilodalton protein. However, higher-molecular-weight proteins are required to achieve the level of toxicity observed in intact toxin

Toxicity of Protease-Resistant Domains from the Delta-Endotoxin of \u3ci\u3eBacillus thuringiensis\u3c/i\u3e subsp. \u3ci\u3eisraelensis\u3c/i\u3e in \u3ci\u3eCulex quinquefasciatus\u3c/i\u3e and \u3ci\u3eAedes aegypti\u3c/i\u3e Bioassays

The mosquitocidal glycoprotein endotoxin of Bacillus thuringiensis subsp. israelensis was digested with chymotrypsin to yield protease-resistant domains which were then separated from smaller protease digestion products by high-performance liquid chromatography. Once purified, the domains no longer bound wheat germ agglutinin, a lectin which binds N-acetylglucosamine (GlcNAc) and GlcNAc oligomers. Purified protease-resistant domains were as toxic for Culex quinquefasciatus larvae as intact solubilized toxin. In separate experiments, the toxicity of chymotrypsin-digested endotoxin for Aedes aegypti larvae was reduced fivefold or more. A model is presented in which GlcNAc-containing oligosaccharides are required for toxicity for A. aegypti larvae but not C. quinquefasciatus larvae

Analysis of Mosquito Larvicidal Potential Exhibited by Vegetative Cells of \u3ci\u3eBacillus thuringiensis\u3c/i\u3e subsp. \u3ci\u3eisraelensis\u3c/i\u3e

Vegetative Bacillus thuringiensis subsp. israelensis cells (6 x 105/ml) achieved 100% mortality of Aedes aegypti larvae within 24 h. This larvicidal potential was localized within the cells; the cell-free supernatants did not kill mosquito larvae. However, they did contain a heat-labile hemolysin which was immunologically distinct from the general cytolytic (hemolytic) factor released during solubilization of B. thuringiensis subsp. israelensis crystals. The larvicidal potential of the vegetative cells was not due to poly-3-hydroxybutyrate. Instead, it correlated with the ability of vegetative cells to sporulate during the bioassays. No toxicity was observed when bioassays were conducted in the presence of chloramphenicol or streptomycin. It is unlikely that the vegetative cells sporulate in the alkaline (pH 9.5 to 10.5) larval guts after ingestion. B. thuringiensis subsp. israelensis is not an alkalophile; we have been unable to grow it in culture at pH values of .9.5. Moreover, we have been unable to demonstrate formation of a protective capsule. However, bacteria may replicate in the gut fluids of dead or dying mosquito larvae because their alkaline gut pH values drop markedly after exposure to the B. thuringiensis subsp. israelensis crystal toxins