• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.152-161
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• 2015

CodY is a highly conserved protein in low G+C gram-positive bacteria that regulates genes involved in sporulation and stationary-phase adaptation. Bacillus thuringiensis is a grampositive bacterium that forms spores and parasporal crystals during the stationary phase. To our knowledge, the regulatory mechanism of CodY in B. thuringiensis is unknown. To study the function of CodY protein in B. thuringiensis, BMB171codY^- was constructed in a BMB171 strain. A shuttle vector containing the ORF of cry1Ac10 was transformed into BMB171 and BMB171codY^-, named BMB171cry1Ac and BMB171codY^-cry1Ac, respectively. Some morphological and physiological changes of codY mutant BMB171codY^-cry1Ac were observed. A comparative proteomic analysis was conducted for both BMB171codY^-cry1Ac and BMB171cry1Ac through two-dimensional gel electrophoresis and MALDI-TOF-MS/MS analysis. The results showed that the proteins regulated by CodY are involved in microbial metabolism, including branched-chain amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, and energy metabolism. Furthermore, we found CodY to be involved in sporulation, biosynthesis of poly-β-hydroxybutyrate, growth, genetic competence, and translation. According to the analysis of differentially expressed proteins, and physiological characterization of the codY mutant, we performed bacterial one-hybrid and electrophoretic mobility shift assay experiments and confirmed the direct regulation of genes by CodY, specifically those involved in metabolism of branched-chain amino acids, ribosomal recycling factor FRR, and the late competence protein ComER. Our data establish the foundation for in-depth study of the regulation of CodY in B. thuringiensis, and also offer a potential biocatalyst for functions of CodY in other bacteria.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.04a
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• pp.73-73
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• 2003

Bacillus thuringiensis 2385-1, which showed toxicity to lepidopteran but not to dipteran was isolated from Korean soil sample and characterized. The H-serotype of 2385-1 was identical to that of serovar kenyae (H4a4c), and its crystal toxin was bipyramidal-shaped with a molecular weight of 130 kDa. However, the plasmid profile of 2385-1 was different from that of serovar kenyae. (omitted)

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.10a
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• pp.114-115
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• 2003

A Bacillus thuringiensis isolate, Bt 2385-1, which showed toxicity to lepidopteran, was isolated from Korean soil sample and characterized. PCR-RFLP showed that this isolate contains two novel cryl-type crystal protein genes. In this study, we designed cryl-type specific primer set (ATG1-F and N400-R) to clone the toxic domain of the all cryl-type genes. The two novel rlyl-type toxin genes in addition to crylJal gene were cloned and sequenced. (omitted)

• Journal of Microbiology and Biotechnology
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• v.21 no.9
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• pp.937-946
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• 2011

Several isolates of Bacillus thuringiensis (Bt) were screened for the vegetative insecticidal protein (Vip) effective against sap-sucking insect pests. Screening results were based on $LC_{50}$ values against cotton aphid (Aphis gossypii), one of the dangerous pests of various crop plants including cotton. Among the isolates, the Bt#BREF24 showed promising results, and upon purification the aphidicidal protein was recognized as a binary toxin. One of the components of this binary toxin was identified by peptide sequencing to be a homolog of Vip2A that has been reported previously in other Bacillus spp. Vip2 belongs to the binary toxin group Vip1-Vip2, and is responsible for the enzymatic activity; and Vip1 is the translocation and receptor binding protein. The two genes encoding the corresponding proteins of the binary toxin, designated as vip2Ae and vip1Ae, were cloned from the Bt#BREF24, sequenced, and heterologously expressed in Escherichia coli. Aphid feeding assay with the recombinant proteins confirmed that these proteins are indeed the two components of the binary toxins, and the presence of both partners is essential for the activity. Aphid specificity of the binary toxin was further verified by ligand blotting experiment, which identified an ~50 kDa receptor in the brush border membrane vesicles of the cotton aphids only, but not in the lepidopteran insects. Our finding holds a promise of its use in future as a candidate gene for developing transgenic crop plants tolerant against sap-sucking insect pests.

• International Journal of Industrial Entomology and Biomaterials
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• v.11 no.2
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• pp.125-127
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• 2005

In order to improve the transformation efficiency of the Bacillus thuringiensis (Bt)-Escherichia coli (E. coli) shuttle vector, pHT3101, we intended to minimize replication origin of Bt in pHT3101. For this, two modified shuttle vectors, pHT1K and pHT261, in which 2.9 kb of replication origin of Bt were shortened to 1 kb and 261 bp, respectively as previously reported. Whereas the pHT1K could efficiently transform Bt into the antibiotic resistant, no transformants were obtained with pHT261. Furthermore, pHT1K showed higher transformation efficiency compared to that of parent vector, pHT3101. Therefore, pHT1K might be a very useful Bt-E. coli shuttle vector carrying minimal replication origin of Bt.

• Microbiology and Biotechnology Letters
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• v.46 no.1
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• pp.45-50
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• 2018

The chitosanase gene (btbchito) of Bacillus thuringiensis BMB171 was cloned and heterologously expressed in the yeast Pichia pastoris. After purification, about 300 mg of recombinant chitosanase was obtained from the 1-1 culture medium with a specific activity of 240 units/mg. Results determined by the combined use of thin layer chromatography (TLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) showed that the chitooligosaccharides (COSs) obtained by chitosan (N-deacetylated by 70%, 80%, and 90%) hydrolysis by rBTBCHITO were comprised of oligomers, with degrees of polymerization (DP) mainly ranging from trimers to heptamers; high molecular weight chitopentaose, chitohexaose, and chitoheptaose were also produced. Hydrolysis products was also deduced using MS since the COSs (n) are complex oligosaccharides with various acetyl groups from one to two, so the non-acetyl COSs (GlcN)n and COSs with more acetyls (> 2) were not detected. The employment of this method in the production of high molecular weight COSs may be useful for various industrial and biological applications, and the activity of chitosanase has great significance in research and other applications.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.358-364
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• 1998

New microbial-control agents were prepared with B. thuringiensis strain NT0423 having unique properties which are different with other B. thuringiensis strains belonging to serotype 7[Kor. J. Appl. Entomol. 32: 426-432.]. Three B. thuringiensis formulations designated as BioBact 10%, 20% and 40%, were made with various combinations of adjuvants. These formulations showed good physical properties in wettability, suspensibility, particle size and adherence. In addition the result of SDS-PAGE analysis indicated that $\delta$-endotoxins remain stably in all formulations. Among the tested formulations, two wettable powder formulations, BioBact 20% and 40%, comprising 20% and 40% of B. thuringiensis technical powder showed the effective control against diamondback moth larvae (Plutella xylostella) in laboratory and field tests. Especially, when compared with commercial B. thuringiensis formulations (A and B commercial formulations) in field evaluation, BioBact 20% and 40% formulations showed equal activity up to 80% lethality and a good persistence effect which remain on leaves at least 7 days.

• Journal of Sericultural and Entomological Science
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• v.28 no.1
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• pp.43-53
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• 1986

Among many microbial pesticides, Bacillus thuringiensis is one of the most hopeful pesticide and some commercial products have been appearing on the market. Because these commercial products contain living spores and toxins of the organism, there is a danger that living spores of B. thuringiensis may be scattered by wind and cause a great damage in the sericulture areas. In order to avoide these risks it is desirable to select the strain which has low pathogenicity to the silkworm, and at the sometime being highly pathogenic to the pest insects. Thus this study has been carried out to acquire some basic informations about the procedure of desicable strain selection. Three strains of B. thuringiensis var. kurstaki, var. dendrolimus and var. aizawai were used for the pathogenicity test on the silkworm, Bombux mori and the fall webwarm, Hyphantria cunea. Those strains were investigated by the agarose gel electrophoresis patterns of plasmid DNA determine whether mutation had occured. Pathogenicity tests were carried out of using isolated crystal proteins and spore-crystal protein to mixtures of each strain, seperatively. In case of using spore-crystal protein mixture, the order of pathogenicity in varities of B. thuringiensis against B.mopri and H.cunea were kurstaki, aizawai, dendrolimus and kurstaki, dendrolimus, aizawai, respectively. But using isolated crystal proteins, dendrolimus had the highest toxicity to H. cunea and the lowest toxicity to B. mori among tested three strains. From the above results, dendrolimus was presumed the most desirable straing for using microbial pesticide.

• 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.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.843-849
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• 2013

A dye-decolorizing bacterium was isolated from a soil sample and identified as Bacillus thuringiensis using 16S rRNA sequencing. The bacterium was able to decolorize three different textile dyes, namely, Reactive blue 13, Reactive red 58, and Reactive yellow 42, and a real dyehouse effluent up to 80-95% within 6 h. Some non-textile industrially important dyes were also decolorized to different extents. Fourier transform infrared spectroscopy and gas chromatography-mass spectrometer analysis of the ethyl acetate extract of Congo red dye and its metabolites showed that the bacterium could degrade it by the asymmetric cleavage of the azo bonds to yield sodium (4-amino-3-diazenylnaphthalene-1-sulfonate) and phenylbenzene. Sodium (4-amino-3-diazenylnaphthalene-1-sulfonate) was further oxidized by the ortho-cleavage pathway to yield 2-(1-amino-2-diazenyl-2-formylvinyl) benzoic acid. There was induction of the activities of laccase and azoreductase during the decolorization of Congo red, which suggests their probable role in the biodegradation. B. thuringiensis was found to be versatile and could be used for industrial effluent biodegradation.