• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1099-1106
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• 2013

Tuta absoluta (Povolny, 1994) is a devastating moth to the Solanaceae plants. It is a challenging pest to control, especially on tomatoes. In this work, we studied the entomopathogenic activity of the Cry-forming ${\delta}$-endotoxins produced by Bacillus thuringiensis strain KS and B. thuringiensis kurstaki reference strain HD1 against T. absoluta. These strains carried the cry2, cry1Ab, cry1Aa/cry1Ac, and cry1I genes, and KS also carried a cry1C gene. The ${\delta}$-endotoxins of KS were approximately twofold more toxic against the third instar larvae than those of HD1, as they showed lower 50% and 90% lethal concentrations (0.80 and 2.70 ${\mu}g/cm^2$ (${\delta}$-endotoxins/tomato leaf)) compared with those of HD1 (1.70 and 4.50 ${\mu}g/cm^2$) (p < 0.05). Additionally, the larvae protease extract showed at least six caseinolytic activities, which activated the KS and HD1 ${\delta}$-endotoxins, yielding the active toxins of about 65 kDa and the protease-resistant core of about 58 kDa. Moreover, the histopathological effects of KS and HD1 ${\delta}$-endotoxins on the larvae midgut consisted of an apical columnar cell vacuolization, microvillus damage, and epithelial cell disruption. These results showed that the KS strain could be a candidate for T. absoluta control.

• Microbiology and Biotechnology Letters
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• v.18 no.3
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• pp.301-304
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• 1990

In the mosquitocidal delta-endotoxins from Bacillus thuringiensis subsp, isruelensis and B. thuringiensis subsp. darmstudiensis 73E10-2, were contained an immunologically homologous protein. The homologous protein was confirmed from Ouchterlony test, irnmuno-electrophoresis, and enzyme linked immunoassay by polyclonal antibodies against the delta-endotoxins of both strains.

• Microbiology and Biotechnology Letters
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• v.27 no.5
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• pp.359-363
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• 1999

To prevent appearance of resistant mosquitoes against $\delta$-endotoxin of bacillus thuringiensis subsp. israelensis (Bti) in field, a mosquitocidal Bacillus thuringiensis strain 21-2(Bt21-2) producing a new type of $\delta$-endotoxin was isolated. The strain Bt 21-2 belongs to H serotype 43, B. thuringiensis subsp. guiyangiensis (Btg). The $\delta$-endotoxins from the strain Bt 21-2 and the strain Bti were a cuboid shape morphologically, but the $\delta$-endotoxin of the strain Bt 21-2 was composed of 150, 90 and 70kDa proteins on SDS-PAGE, and the antigenicity of $\delta$-endotoxin of the strain Bt 21-2 was different from that of the strain Bti on immunoblot. The $\delta$-endotoxin gene of the strain Bt 21-2 was not amplified with specific primers of $\delta$-endotoxin gene (cry4A and cry4B) of the strain Bti on PCR.

• Journal of Microbiology and Biotechnology
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• v.6 no.5
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• pp.326-330
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• 1996

To illustrate whether a hemolysin in $\delta$-endotoxins of Bacillus thuringiensis strain 73E10-2 and subsp. israelensis had immunological identity, a cyt gene of the strain 73E10-2 which encodes a hemolysin was cloned to B. subtilis (transformant 2753). The transformant 2753 containing cyt gene produced the hemolysin which lysed sheep erythrocytes after treatment of proteinase K. The hemolysin was proved also to be toxic against mosquito larvae (Aedes aegypti). The molecular weight of the hemolysin produced from the transformant 2753 was determined to be about 25 kDa by SDS-PAGE and immunoblot. The hemolysin in $\delta$-endotoxin of subsp. israelensis and subsp. kyushensis did not react on immunoblot using polyclonal anti-$\delta$-endotoxin of the strain 73E10-2, but 70-140 kDa mosquitocidal toxins in $\delta$-endotoxin of subsp. kyushuensis reacted.

• 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.36 no.3
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• pp.294-298
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• 2003

The pathological effect of the Bacillus thuringiensis Cry $\delta$-endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.

• 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.