• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.308-312
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• 1991

The delta-endotoxin from B. thuringiensis subsp. damstadienszs 73E10-2 was resistant to high concentration of salt (4M NaBr), organic solvents (50% acetone), denaturants (4 M urea), and neutral detergents (10% triton X-100). In contrast, the toxin was inactivated by treating with charged detergents as well as guanidine hydrochloride or carbon tetra-chloride. The delta-endotoxin is not a sulfhydryl activated toxin, but modification of the lysine side chains eliminated toxicity against mosquito larvae.

• International Journal of Industrial Entomology and Biomaterials
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• v.8 no.1
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• pp.89-93
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• 2004

Expression of a fusion protein between B. thuringiensis crystal protein, Cry1Ac1 and a scorpion insect toxin (AaIT, Androctonus australis Hector insect toxin) in acrystalliferous B. thuringiensis strain (Cry-B strain) was examined. The cry 1Ac1 gene was cloned in B. thuringiensis-E coli shuttle vector, pHT3101, under the control of the native cry 1Ac1 gene promoter (pProAc) and a gene encoding AaIT was inserted in XhoI site in the middle of the cry 1Ac1 gene (pProAc-ScoR). B. thuringiensis Cry-B strain carrying pProAc-ScoR (PyoAc-ScoR/CB) produced an inclusion body of irregular shape and the expressed fusion protein is approximately 65 kDa in size. Sporulated cells and spore-crystal mixtures of ProAc-ScoR/CB had insecticidal activity against Plutella xylostella larvae, showing $LT_50$ of ProAc-ScoR/CB (22.59 hrs) lower than that of ProAc/CB (30.06 hrs) at $1{\times}{10^7} {CEU/cm^2}$. These results suggest that the fusion protein including a B. thuringiensis crystal protein and an AaIT may be functionally expressed in B. thupingiensis. Moreover, we verified the additive toxicity of AaIT, which is a new feasible candidate for insect control.

• Korean journal of applied entomology
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• v.33 no.3
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• pp.178-183
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• 1994

The prolein components of each crystal toxin of Bacillus thuringiensis kurstaki and alzawai were separated by SDS-PAGE. The major clqistal proteins from both shins were composed of paiypeptides having molecular weights of 130 kd and 64 kd. Digestive mixture of both. toxins with t~ypsin and gut juices shared 62 kd polypeptide which may be major actwe toxh. However, aizawal produced much less amount of 62 kd polypeptide than kurstaki did. On ingestion with Bt &-endotoxin, larvae of Hyphantria cunea developed 45 kd stress protein in the several tissues including fat body, which was induced by heat and cold shock.

• BMB Reports
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• v.47 no.10
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• pp.546-551
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• 2014

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae.

• Animal cells and systems
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• v.1 no.3
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• pp.507-513
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• 1997

Sequential observations of binding patterns and structural effects of Bacillus thuringiensis var. kurstaki were made on fat body tissue of the fall webworm, Hyphantria cunea Drury. Fat body was cultured in vitro in the presence of purified 62 kDa endotoxin and then examined for protein synthesis and the localization of membrane-bound toxin detected by an antibody against the 62 kDa endotoxin. Protein synthesis was mostly inhibited at concentrations of 15 ${\mu}$g/ml and higher. Immunocytochemical observations suggest that the toxin binds to all exposed basal lamina surrounding the fat body without apparent specificity. The cytopathic effect delectable by scanning electron microscope is disintegration rather than cell swelling. The basal lamina bound toxin was eventually detached from the fat body and followed by an extrusion of cell contents like lipid granules.

• BMB Reports
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• v.36 no.5
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• pp.508-513
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• 2003

The bacterium Bacillus thuringiensis produces toxin inclusions that are deleterious to target insect larvae. These toxins are believed to interact with a specific receptor protein(s) that is present on the gut epithelial cells of the larvae. In various insect species (in particular those belonging to the lepidopteran class), aminopeptidase N (APN) is one of the two receptor proteins that are considered to be involved in toxin-receptor interactions. However, in mosquitoes, the nature and identity of the receptor protein is unknown. Here, using RT-PCR, we identified two isoforms of the APN transcripts in the Aedes aegypti mosquito larval midgut. These results are congruent with a previous report of multiple isoforms of the APN gene expression in lepidopteran larvae. Which of the two isoforms (or other yet unidentified receptor proteins) is involved in the killing of mosquito larvae remains to be elucidated.

• Journal of Sericultural and Entomological Science
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• v.17 no.1
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• pp.55-62
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• 1975

In the studies of characterization of crystal prototoxin isolated from Bacillus thuringinensis var. alesti and its bioassay to the silkworm larvae, Bombyx mori L., the following results were obtained. 1. The complete lysis of bacteria grown on the nutrient agar at 30$^{\circ}C$ in an incubator took 30 days, but the period could be reduced by a half in a devised broth media in a fermentation jar. 2. The protein toxin extracted directly from a mixture of crystals and spores without separation of crystals and spores was pure as same as the protein toxin extracted from only crysals separated. 3. By SLS polyacrylamide gel electrophoresis of prototoxin released from crystals in alkali (pH 9.5) three different proteins in molecular weights of 120,000, 87,000, 74,000 were separated. 4. In the bioassay of the toxin to the silkworm larvae, LD$\sub$50/ per gram of the larvae in the 4th instar was 0.080 $\mu\textrm{g}$.

• BMB Reports
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• v.46 no.3
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• pp.175-180
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• 2013

Cyt2Aa2 is a mosquito larvicidal and cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. The toxin becomes inactive when isoleucine at position 150 was replaced by alanine. To investigate the functional role of this position, Ile150 was substituted with Leu, Phe, Glu and Lys. All mutant proteins were produced at high level, solubilized in carbonate buffer and yielded protease activated product similar to those of the wild type. Intrinsic fluorescence spectra analysis suggested that these mutants retain similar folding to the wild type. However, mosquito larvicidal and hemolytic activities dramatically decreased for the I150K and were completely abolished for I150A and I150F mutants. Membrane binding and oligomerization assays demonstrated that only I150E and I150L could bind and form oligomers on lipid membrane similar to that of the wild type. Our results suggest that amino acid at position 150 plays an important role during membrane binding and oligomerization of Cyt2Aa2 toxin.

• Journal of Sericultural and Entomological Science
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• v.36 no.2
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• pp.162-167
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• 1994

Surface changes of tissues caused by B. thuringiensis var. kurstaki-$\delta$-endotoxin intoxication of Helicoverpa assulta were observed by scanning electron microscopy. Bt-endotoxin crystals induced the erosion and disruption on the surface of all tissues tested. The results revealed that the toxin binds to all exposed plasma membranes without apparent specificity for particular membrane domains.

• Microbiology and Biotechnology Letters
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• v.13 no.1
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• pp.51-57
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• 1985

Delta-endotoxin in Bacillus thuringiensis var. finitimus, HD-1, HD-9 and HD-73 strains were isolated by NaBr, CsCl and Renografin density gradients. The purity of the toxin was about 98%. The purified o-endotoxin was analyzed by SDS-PAGE, electron microscopic observation and bioassay. According to SDS-PAGE, the molecular weight of subunits of the o-endotoxin were about 66,000 and 130,000 daltons. The shapes of the crystal toxin observed by TEM except finitimus strain were bipyramidal. When the purified endotoxin was bioassayed against tobacco horn worm, the entomocidal activities ($1{\mu}g/ml$) of HD-1 and HD-73 strains were, respectively, 60% and 100% at nine days after treated. The molecular weights of the plasmids isolated from B. thuringiensis were various from 0.5 to 120 Kb. The numbers of plasmids in HD-1, HD-9 and HD-73 strains were 12, 3 and 11, respectively, but B. thuringiensis var. finitimus strain had no plasmid.