• 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.

• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1107-1115
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• 2013

The Cyt1Aa protein of Bacillus thuringiensis susbp. israelensis elaborates demonstrable toxicity to mosquito larvae, but more importantly, it enhances the larvicidal activity of this species Cry proteins (Cry11Aa, Cry4Aa, and Cry4Ba) and delays the phenotypic expression of resistance to these that has evolved in Culex quinquefasciatus. It is also known that Cyt1Aa, which is highly lipophilic, synergizes Cry11Aa by functioning as a surrogate membrane-bound receptor for the latter protein. Little is known, however, about whether Cyt1Aa can interact similarly with other Cry proteins not primarily mosquitocidal; for example, Cry2Aa, which is active against lepidopteran larvae, but essentially inactive or has very low toxicity to mosquito larvae. Here we demonstrate by ligand binding and enzyme-linked immunosorbent assays that Cyt1Aa and Cry2Aa form intermolecular complexes in vitro, and in addition show that Cyt1Aa facilitates binding of Cry2Aa throughout the midgut of C. quinquefasciatus larvae. As Cry2Aa and Cry11Aa share structural similarity in domain II, the interaction between Cyt1Aa and Cry2Aa could be a result of a similar mechanism previously proposed for Cry11Aa and Cyt1Aa. Finally, despite the observed interaction between Cry2Aa and Cyt1Aa, only a 2-fold enhancement in toxicity resulted against C. quinquefasciatus. Regardless, our results suggest that Cry2Aa could be a useful component of mosquitocidal endotoxin complements being developed for recombinant strains of B. thuringiensis subsp. israelensis and B. sphaericus aimed at improving the efficacy of commercial products and avoiding resistance.

• 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.

• BMB Reports
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• v.34 no.2
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• pp.150-155
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• 2001

The mosquito-larvicidal Cry4B protein from Bacillus thuringiensis subsp. israelensds was expressed in Escherichia coli. Upon activation by trypsin, the 130-kDa protoxin was processed into the 65-kDa active toxin containing two polypeptide fragments of ca. 47 and ca. 20 kDa. These two polypeptides are products of internal cleavages on the exposed loop connecting helices 5 and 6 in the seven-helical bundle domain. PCR-based mutagenesis was employed to introduce an additional cleavage site into the loop connecting helices 3 and 4. A series of amino acid changes were introduced into the targeted loop, resulting in seven mutant protoxins. Upon digestion with trypsin, a group of mutants with arginine introduced into the loop (EPRNQ, EPNRNQ, EPRNP, ESRNP and SSRNP) produced polypeptide products similar to those of the wild type (EPNNQ). When the loop, SSRNP, was expanded by an insertion of either asparagine (NSSRNP) or valine (VSSRNP), an additional cleavage was detected with proteolytic products of 47,12 and 6 kDa. This cleavage was confirmed to be at the introduced arginine residue by N-terminal sequencing. The mosquito larvicidal assay against Aedes aegypti demonstrated a relatively unchanged toxicity for the mutants without cleavage and reduced toxicity for those with an additional cleavage.

• BMB Reports
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• v.39 no.3
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• pp.270-277
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• 2006

Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba $\delta$-endotoxin have been shown to be important determinants for mosquito-larvicidal activity, likely being involved in membrane-pore formation. In this study, the Cry4Ba mutant protein containing an additional engineered tryptic cleavage site was used to produce the $\alpha4$-$\alpha5$ hairpin peptide by an efficient alternative strategy. Upon solubilization of toxin inclusions expressed in Escherichia coli and subsequent digestion with trypsin, the 130-kDa mutant protoxin was processed to protease-resistant fragments of ca. 47, 10 and 7 kDa. The 7-kDa fragment was identified as the $\alpha4$-loop-$\alpha5$ hairpin via N-terminal sequencing and mass spectrometry, and was successfully purified by size-exclusion FPLC and reversed-phase HPLC. Using circular dichroism spectroscopy, the 7-kDa peptide was found to exist predominantly as an $\alpha$-helical structure. Membrane perturbation studies by using fluorimetric calcein-release assays revealed that the 7-kDa helical hairpin is highly active against unilamellar liposomes compared with the 65-kDa activated full-length toxin. These results directly support the role of the $\alpha4$-loop-$\alpha5$ hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.

• BMB Reports
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• v.41 no.11
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• pp.820-825
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• 2008

Bacillus thuringiensis Cyt2Aa toxin is a mosquito-larvicidal and cytolytic $\delta$-endotoxin, which is synthesized as a protoxin and forms crystalline inclusions within the cell. These inclusions are solubilized under alkaline conditions and are activated by proteases within the larval gut. In order to assess the functions of the N-and C-terminal regions of the protoxin, several N- and C-terminal truncated forms of Cyt2Aa were constructed. It was determined that amino acid removal at the N-terminal, which disrupts the $\beta$1 structure, might critically influence toxin production and inclusion formation. The deletion of 22 amino acids from the C-terminus reduced the production and solubility of the toxin. However, the removal of more than 22 amino acids from the C-terminus or the addition of a bulky group to this region could result in the inability of the protein to adopt the proper folding. These findings directly demonstrated the critical roles of N- and C-terminal amino acids on the production and folding of the B. thuringiensis cytolytic $\delta$-endotoxin.

• Korean journal of applied entomology
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• v.43 no.1
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• pp.35-41
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• 2004

Bacillus thuringiensis produces crystal proteins toxic to medically and agriculturally important pests during sporulation. To improve the activity of insecticidal crystal protein in applying to mosquito larval control, an expression vector, pSyn4D harboring the mosquitocidal cry11Aa gene under control of psbA promoter of Amaranthus hybridus was constructed. This expression vector was transformed into Synechocystis PCC6803 and a transformant, Tr2C was selected with kanamycin. The mosquitocidal cry11Aa gene was stably integrated Into genomic DNA of Tr2C in PCR detection using cry11Aa-specific primers. The transformant expressed 72-kDa Cry11Aa protein and median lethal time (LT$\sub$50/) was approximately 2.1 days for Culex tritaeniorhynchus larvae and 0.7 day for Anopheles sinensis larvae, respectively. These results suggest this transformant can be used for mosquito larval control as a biological control agent.

• 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.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.156-163
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• 1995

Bacillus sphaericus 1593 is pathogenic to the larvae of a number of mosquito species that are known as important vectors for the transmission of certain human and animal diseases. As a preliminary experiment for developing a multfunctional B. sphaericus 1593 as a potent antagonist, we investigated the conditions for the protoplast transformation system of B. sphaericus 1593 using the plasmid pGB215-110$\Delta$B. The protoplast of B. sphaericus 1593 were obtained most efficiency by treating the cells with 500 $\mu$g/ml of lysozyme in the SMM buffer containing 0.5 M sucrose at pH 8.0 and 40$\circ$C for 60 minutes. The cell wall was regenerated on the plate containing 1.2% agar and 0.8 M mannitol. Under the best condition for protoplast formation and regeneration established in the work the highest frequency of transformation was achieved with the 40% PEG (M.W 4,000) treatment for 15 minutes of incubation at 4$\circ$C, and subsequently for 120 minutes incubation at 30$\circ$C for phenotypic expression. The highest transformation efficiency were observed at 1.0 $\mu$g/ml of the final concentration of the plasmid DNA and the plasmids were found to be fairly stable since about 70% of the plasmids were maintained after 8 successive daily transfers onto the fresh medium.

• BMB Reports
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• v.43 no.1
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• pp.23-28
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• 2010

Binary toxin consisting of BinA and BinB from Bacillus sphaericus is toxic to mosquito larvae. BinB is responsible for specific binding to the larval gut cell membrane while BinA is crucial for toxicity. To investigate functional role of cysteine in BinB, three cysteine residues at positions 67, 161, and 241 were replaced by alanine or serine. Mutations at these positions did not affect protein production and overall structure of BinB. These cysteine residues are not involved in disulfide bond formation between BinB molecules. Mosquito-larvicidal assays revealed that C67 and C161 are essential for toxicity, whereas C241 is not. Mutations at C67 and C161 resulted in weaker BinA-BinB interaction. The loss of toxicity may be due to the reduction of interactions between BinA and BinB or BinB and its receptor. C67 and C161 could also play a part during conformational changes or internalization of the binary toxin into the target cell.