• Microbiology and Biotechnology Letters
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• v.35 no.3
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• pp.250-254
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• 2007

Bacillus thringiensis strain 108 was isolated from soil and had nematicidal activity against second stage juvenile of plant root-knot nematode, Meloidogyne incognita. The strain 108, a rod shape, spore forming and Gram positive bacterium, produced lecithinase, catalase, and ${\delta}$-endotoxin. The strain 108 belongs to H serotype 3, Bacillus thuringiensis var. kurstaki. A nematicidal substance of the strain 108 was partially purified on Sephadex G-25 gel filtration, activated carbon adsorption, silica gel adsorption, and Sephadex G-10 gel filtration. $LC_{90}$ of the partially purified substance against M incognita was $1.2\;{\mu}g/ml$. The nematicidal substance was stable by heat treatment at $100^{\circ}C$ for 1hr, but was perfectly lost nematicidal activity after autoclave ($110^{\circ}C$, 30 min).

• The Korean Journal of Pesticide Science
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• v.14 no.4
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• pp.446-455
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• 2010

The characteristics of parasporal inclusion body from Bacillus thuringiensis subsp. kurstaki KB099 isolate which is high bioactive to the tobacco cutworm, Spodoptera litura, were examined. Parasporal inclusion of B. thuringiensis subsp. kurstaki KB099 isolate showed only 1 band at 130 kDa compared with B. thuringiensis subsp. kurstaki HD-l isolate producing 2 protein bands at 130 kDa and 60 kDa from by SDS-PAGE analysis without any enzyme treatment. Also, we confirmed that gut extract of sensitive S. litura KB099 isolate had digested only 60 kDa ${\delta}$-endotoxin protein. When the digestive enzyme of sensitive insect responsible for parasporal inclusion from KB099 and HD-l isolate was treated to each of them, protein band 60 KDa of KB099 was maintained up to 12 hours but all bands of HD-l were disappeared within 6 hours. In KB099 isolate, 6 genes (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1D and Cry1I) were identified by PCR analysis. Also, $Cry^-$ mutant of KB099 isolate was investigated by phase- contrast microscope, SDS-PAGE and PCR.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.142-147
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• 1989

Six plasmids of B. thuringiensis serovar. kurstaki HD73 were detected, with approximate sizes of 7.4, 7.8, 8.1, 11.3, and 75 Kb, as well as a low copied plasmid of similar length to 75 Kb. Partially cured mutants from B. thuringiensis HD73 were obtained either by the treatment of the curing agent, ethidium bromide(0.02 $\mu\textrm{g}$/$m\ell$) or by spontaneous curing, Acrystalliferous mutants(Cry$^-$) were identified by microscopic observation and immunoblotting with polyclonal antibody against 133 KD deltaendotoxin of HD73. Ten Cry$^-$ mutants were found to be lack of 75 Kb plasmid. These results implicated that this plasmid was associated with delta-endotoxin production, After isolating the mutants, we streaked them on potato dextrose agar, spizizen casamino acid glucose, starch agar, and nutrient agar. Only on starch agar medium did morphologies of Cry$^-$ appear translucent and light greyish. On the other hand, the mutants of B. thuringiensis isolated from Korean soil, designated KBS722, were obtained by the treatment of novobiocin (3 $\mu\textrm{g}$/$m\ell$). Acrystalliferous mutants of KBS722 were less translucent than HD73 mutants' only on nutrient agar medium. Compared the plasmid profile of the mutants with delta-endotoxin production, the results seemed to indicate that the insecticidal protein gene of B. thuringiensis isolates KBS722 located on about 225 Kb plasmid DNA.

• BMB Reports
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• v.40 no.5
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• pp.783-790
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• 2007

Receptor binding plays an important role in determining host specificity of the Bacillus thuringiensis Cry $\delta$-endotoxins. Mutations in domains II and III have suggested the participation of certain residues in receptor recognition and insect specificity. In the present study, we expressed the cloned domain II-III fragment of Cry4Ba and examined its binding characteristics to mosquito-larval midgut proteins. The 43-kDa Cry4Ba-domain II-III protein over-expressed in Escherichia coli as inclusion bodies was only soluble when carbonate buffer, pH 10.0 was supplemented with 4M urea. After renaturation via stepwise dialysis and subsequent purification, the refolded domain II-III protein, which specifically reacts with anti Cry4Ba-domain III monoclonal antibody, predominantly exists as a $\beta$-sheet structure determined by circular dichroism spectroscopy. In vitro binding analysis to both histological midgut tissue sections and brush border membrane proteins prepared from susceptible Aedes aegypti mosquito-larvae revealed that the isolated Cry4Ba-domain II-III protein showed binding functionality comparable to the 65-kDa full-length active toxin. Altogether, the data present the 43-kDa Cry4Ba fragment comprising domains II and III that was produced in isolation was able to retain its receptor-binding characteristics to the target larval midgut proteins.

• BMB Reports
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• v.40 no.2
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• pp.163-171
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• 2007

Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry $\delta$- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, $Trp^{243}$, $Phe^{246}$, $Tyr^{249}$ and $Phe^{264}$, in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at $Tyr^{249}$ and $Phe^{264}$ still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of $Tyr^{249}$ and $Phe^{264}$ within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.

• BMB Reports
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• v.37 no.3
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• pp.292-297
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• 2004

The current model for the mechanism of action of the Bacillus thuringiensis Cry $\delta$-endotoxins involves the penetration of the ${\alpha}4-{\alpha}5$ hairpin into the target midgut epithelial cell membranes, followed by pore formation. In this study, PCR-based mutagenesis was employed to identify a critical residue within the ${\alpha}4-{\alpha}5$ loop of the 130-kDa Cry4A mosquito-larvicidal protein. Alanine-substitutions of two charged (Asp-198 and Asp-200) and four polar (Asn-190, Asn-195, Tyr-201 and Tyr-202) residues in the ${\alpha}4-{\alpha}5$ loop were performed. Like the wild-type, all of the mutant toxins were over-expressed as inclusion bodies in Escherichia coli. When E. coli cells expressing each mutant toxin were bioassayed against Aedes aegypti larvae, larvicidal activity was completely abolished for the substitution of only Tyr-202, while replacements at the other positions still retained a high level of toxicity. Further replacement of Tyr-202 with an aromatic side chain, phenylalanine, did not affect the toxicity. These results revealed a crucial role in toxin activity for the conserved aromatic residue at the 202 position within the ${\alpha}4-{\alpha}5$ loop of the Cry4A toxin.

• BMB Reports
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• v.34 no.4
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• pp.293-298
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• 2001

Tryptic activation of the 130-kDa Bacillus thuringiensis Cry4B $\delta$-endotoxin produced protease-resistant products of ca. 47 kDa and ca. 21 kDa. The 21-kDa fragment was identified as the N-terminal five-helix bundle (${\alpha}1-{\alpha}5$,) which is a potential candidate for membrane insertion and pore formation. In this study, we constructed the recombinant clone over-expressing this putative pore-forming (PPF) fragment as inclusion bodies in Escherichia coli. The partially purified inclusions were composed of a 23-kDa protein, which cross-reacted with Cry4B antibodies, and whose N-terminus was identical to that of the 130-kDa protein. Dissimilar to protoxin inclusions, the PPF inclusions were only soluble when the carbonate buffer, pH 9.0, was supplemented with 6 M urea. After renaturation via a stepwise dialysis, the refolded PPF protein appeared to exist as an oligomer and was structurally stable upon trypsin treatment. Unlike the 130kDa protoxin, the refolded protein was able to release entrapped glucose from liposomes, and showed comparable activity to the full-length activated toxin, although it lacks larvicidal activity These results, therefore, support the notion that the PPF fragment that consists of ${\alpha}1-{\alpha}5$ of the activated Cry4B toxin is involved in membrane pore-formation.

• BMB Reports
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• v.34 no.5
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• pp.402-407
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• 2001

Based on the currently proposed toxicity model for the different Bacillus thuringiensis Cry $\delta$-endotoxins, their pore-forming activity involves the insertion of the ${\alpha}4-{\alpha}5$ helical hairpin into the membrane of the target midgut epithelial cell. In this study, a number of polar or charged residues in helix 4 within domain I of the 65-kDa dipteranactive Cry11A toxin, Lys-123, Tyr-125, Asn-128, Ser-130, Gln-135, Arg-136, Gln-139 and Glu-141, were initially substituted with alanine by using PCR-based directed mutagenesis. All mutant toxins were expressed as cytoplasmic inclusions in Escherichia coli upon induction with IPTG. Similar to the wild-type protoxin inclusion, the solubility of each mutant inclusion in the carbonate buffer, pH 9.0, was relatively low When E. coli cells, expressing each of the mutant proteins, were tested for toxicity against Aedes aegypti mosquito-larvae, toxicity was completely abolished for the alanine substitution of arginine at position 136. However, mutations at the other positions still retained a high level of larvicidal activity Interestingly, further analysis of this critical arginine residue by specific mutagenesis showed that conversions of arginine-136 to aspartate, glutamine, or even to the most conserved residue lysine, also abolished the wild-type activity The results of this study revealed an important determinant in toxin function for the positively charged side chain of arginine-136 in helix 4 of the Cry11A toxin.

• BMB Reports
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• v.40 no.1
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• pp.58-64
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• 2007

Similar to the other known structures of Bacillus thuringiensis Cry $\delta$-endotoxins, the crystal structure of the 65-kDa activated Cry4Ba toxin comprises three domains which are, from the N- to C-terminus, a bundle of $\alpha$-helices, a three-$\beta$-sheet domain, and a $\beta$-sandwich. To investigate the properties of the C-terminal domain III in isolation from the rest of the toxin, the cloned Cry4Ba-domain III was over-expressed as a 21-kDa soluble protein in Escherichia coli, which cross-reacted with anti-Cry4Ba domain III monoclonal antibody. A highly-purified domain III was obtained in a monomeric form by ion-exchange and size-exclusion FPLC. Circular dichroism spectroscopy indicated that the isolated domain III fragment distinctly exists as a $\beta$-sheet structure, corresponding to the domain III structure embodied in the Cry4Ba crystal structure. In vitro binding analysis via immuno-histochemical assay revealed that the Cry4Ba-domain III protein was able to bind to the apical microvilli of the susceptible Stegomyia aegypti larval midguts, albeit at lower-binding activity when compared with the full-length active toxin. These results demonstrate for the first time that the C-terminal domain III of the Cry4Ba mosquito-larvicidal protein, which can be isolated as a native folded monomer, conceivably participates in toxin-receptor recognition.

• Journal of Sericultural and Entomological Science
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• v.37 no.2
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• pp.176-180
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• 1995

To characterize expression and formation of three type crystal proteins in transformant, Bacillus thruingiensis PT0529 was analysed by transmission electron microscope and SDS-PAGE according to growth. The results showed that the introduced crystal protein genes, rcyIVD and cytA, were well expressed at earlier stage than resident crystal proteins were also expressed with their own morphology. However, resident crystal protein of B. thuringiensis PT0529 was smaller than that of wild type B. thuringiensis NT0423, suggesting that resident crystal protein production was interfered with introduced two type crystal protein genes.