• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.167-176
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• 2013

A novel bioactive molecule produced by Bacillus thuringiensis subsp. kurstaki Bn1 (Bt-Bn1), isolated from a common pest of hazelnut, Balaninus nucum L. (Coleoptera: Curculionidae), was determined, purified, and characterized in this study. The Bt-Bn1 strain was investigated for antibacterial activity with an agar spot assay and well diffusion assay against B. cereus, B. weinhenstephenensis, L. monocytogenes, P. savastanoi, P. syringae, P. lemoignei, and many other B. thuringiensis strains. The production of bioactive molecule was determined at the early logarithmic phase in the growth cycle of strain Bt-Bn1 and its production continued until the beginning of the stationary phase. The mode of action of this molecule displayed bacteriocidal or bacteriolytic effect depending on the concentration. The bioactive molecule was purified 78-fold from the bacteria supernatant with ammonium sulfate precipitation, dialysis, ultrafiltration, gel filtration chromatography, and HPLC, respectively. The molecular mass of this molecule was estimated via SDS-PAGE and confirmed by the ESI-TOFMS as 3,139 Da. The bioactive molecule was also determined to be a heat-stable, pH-stable (range 6-8), and proteinase K sensitive antibacterial peptide, similar to bacteriocins. Based on all characteristics determined in this study, the purified bacteriocin was named as thuricin Bn1 because of the similarities to the previously identified thuricin-like bacteriocin produced by the various B. thuringiensis strains. Plasmid elution studies showed that gene responsible for the production of thuricin Bn1 is located on the chromosome of Bt-Bn1. Therefore, it is a novel bacteriocin and the first recorded one produced by an insect originated bacterium. It has potential usage for the control of many different pathogenic and spoilage bacteria in the food industry, agriculture, and various other areas.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1498-1503
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• 2007

To identify novel crystal proteins, Bacillus thuringiensis 2385-1 was isolated from Korean soil samples and characterized. The H-serotype of 2385-1 was identical to that of subsp. kenyae (H4a4c), and its crystal toxin was bipyramidal-shaped. However, 2385-1 showed a much higher toxicity towards Plutella xylostella and Spodoptera exigua larvae than subsp. kenyae. In addition, the crystal protein profile and plasmid DNA pattern of 2385-1 differed from those of subsp. kenyae. To verify the crystal protein gene types of 2385-1, a PCR-RFLP analysis was performed, and the results revealed that 2385-1 contained two novel cry1-type crystal protein genes, cryl-5 and cry1-12, in addition to the crylJal gene. The deduced amino acid sequences of cryl-5 and cry1-12 showed a 97.9% and 75.7% sequence similarity with the CrylAb and CrylJa crystal proteins, respectively. Among the novel crystal proteins, Cry1-5 showed a high toxicity towards P. xylostella and S. exigua larvae. In conclusion, B. thuringiensis 2385-1 is a new isolate in terms of its gene types, and should be a promising source for an insecticide to control lepidopteran larvae.

• Journal of Microbiology
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• 제42권4호
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• pp.340-345
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• 2004

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis $Cry^-B$ strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the crylAc gene (pProAc-GFP). The B. thuringiensis $Cry^-B$ strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immu­noblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single spe­cies, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis $Cry^-B$ strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuring­iensis.

• The Plant Pathology Journal
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• 제35권5호
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• pp.486-497
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• 2019

Citrus canker is a devastating disease of citrus caused by Xanthomonas citri subsp. citri (Xcc). A total of 134 endophytic bacteria were isolated from various gymnospermic and angiospermic plants. They were screened for their antagonistic activities against three wild-type and six streptomycin-resistant Xcc strains. TbL-22 and TbL-26, both later identified as Bacillus thuringiensis, inhibited all the wild and resistant Xcc strains. TbL-22 exerted the highest antagonistic activity against XccW3 and XccM6 with inhibition zones of $20.64{\pm}0.69$ and $19.91{\pm}0.87mm$, respectively. Similarly ethyl acetate extract of TbL-22 showed highest inhibition zones $15.31{\pm}2.08$ and $19.37{\pm}3.17mm$ against XccW3 and XccM6, respectively. TbL-22 reduced canker incidence on infected leaves by 64.05% relative to positive controls. Scanning electron microscopy revealed that the cell membranes of Xcc treated with ethyl acetate extract of TbL-22 were ruptured, lysed, and swollen. B. thuringiensis TbL-22 can effectively and sustainably controls streptomycin-resistant citrus canker.

• 한국미생물·생명공학회지
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• 제33권2호
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• pp.154-158
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• 2005

Twenty-three Bacillus thuringiensis strains isolated from Korea were screened to detect the cry-type genes using PCR with 21 specific oligonucleotide primers. Eight strains contained distinct multiple crystal genes; cry1Aa2, cry1Ab1, cry1Ac1 and cry2Aa1. These results indicate that the strains coincided with the B. thuringiensis subsp. kurstaki strain. The other 15 strains were not recognised to the 21 specific primers.

• 한국미생물·생명공학회지
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• 제22권3호
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• pp.227-232
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• 1994

In order to microbially control root-knot nematode(Meloidogyne incognita) in tomato, a strain BtTH109 of Bacillus thuringiensis producing root-knot nematocidal toxin was isolated. The strain BtTH109 was identified B. thuringiensis subsp. indiana(serotype 16) based on flagella antigenicity, biochemical properties, and morphological charcateristics. The strain BtTH109 have extracellularly produced a root-knot nematocidal toxin, which was very toxic against not only egghatch but also the 2nd-nematode larva of root-knot nematode in vitro.

• Journal of Microbiology and Biotechnology
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• 제5권6호
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• pp.359-363
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• 1995

The antigenic variation of B. thuringiensis subsp. satto have been investigated for 120 hours during sporulation and crystallization by using SDS-PAGE and Western blot. Most antigens of a vegetative cell were found to disappear as it was in sporulation and crystallization, but protein antigens of 46, 29, 27, and 21 kDa continued to be expressed. The new protein bands of 293, 138, 119, 75, and 68 kDa appeared on days 2 through 5 in modified GYS medium. They were thought to be involved in sporulation and crystallization. The protein of 138 kDa was found to be a major protein of both crystal and spore. The expression patterns were immunologically analyzed by Western blot. The polyclonal antisera against the intact crystal showed strong immunoreactivity to proteins with molecular masses of 293, 138, 68, and 46 kDa. The polyclonal antisera against the spore recognized proteins of 293, 138, 68, and 46 kDa. Both crystals and spores appeared to express the common protein antigens.

• 한국미생물·생명공학회지
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• 제35권3호
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• pp.250-254
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• 2007

식물 뿌리혹 선충인 Meloidogyne incognita에 살충성이 있는 Bacillus thuringiensis 108 균주를 분리 선별하였다. 이 균주는 Gram양성이며, 포자를 형성하고, 운동성이 활발하며, lecithinase 및 catalase활성을 나타내었다. 이 균주가 생산하는 결정형 내독소(d-endotoxin)는 bipyramid형이었고, 편모항원성에 따른 균주의 동정 결과 serotype 3으로 표준 균주인 B. thuringiensis subsp. kurstaki의 편모항원성과 일치하였다. B. thuringiensis 108균주의 항선충성 물질은 Sephadex G-25 gel filtration, 활성탄 흡착 chromatography, Silica gel 흡착 chromatography 및 Sephadex G-10 gel filtration을 이용하여 정제를 하였다. 최종 정제단계인 Sephadex G-10 gel filtration 후에 항선충 물질의 $LC_{90}$는 $1.2\;{\mu}g/ml$이었다. B. thuringiensis 108 균주가 생산하는 항선충성 물질은 $100^{\circ}C$ 열처리에서 안정하였으나 autoclave($110^{\circ}C$ 30 min)를 했을 때는 그 독성이 완전히 소실되었다.

• 한국잠사곤충학회지
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• 제35권1호
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• pp.36-42
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• 1993

Bacillus thruingiensis subsp. kurstaki HD-1의 내독소단백질 유전자인 CryIA(a)의 N-말단 부분과 B.tk HD-73의 내독소단백질 유전자인 CryIA(c)의 C-말단 부분의 융합에 따른 독성발현 여부에 관하여 조사하였다. 융합생산물인 pSK3, pSK4 및 pSK5의 플라스미드는 각각 4.5kb, 4.8kb 그리고 5.5kb로 구성되어 있다. 형질전환체의 내독소단백질에 대한 Western blotting은 SK4 및 SK5가 77-kDa 그리고 105-kDa에서 B.t k HD-1 항체에 대하여 반응을 나타내었다. 독성검정의 공시충인 배추좀나방 및 담배나방을 사용한 결과 pSK5를 포함하는 형질전환체만이 각각 96% 및 97%의 높은 치사율을 나타냈다.

• 한국잠사곤충학회지
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• 제40권2호
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• pp.126-130
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• 1998

A noninsecticidal strain, Bacillus thuringiensis NTB-88, isolated from Korean soil, had a typical bipyramidal parasporal inclusion and its serotype is identical to B. thuringiensis subspmorrisoni (H8a8b). To elucidate differences between insecticidal and noninsecticidal strains, we compared strain NTB-88 to other toxic B. thuringiensis subsp. morrisoni strains (HD-12 and PG-14). Restriction endonucleases digested plasmid DNA patterns showed that strain NTB-88 was different from lepidopteran-toxic strain, HD-12, but it was similar to dipteran-toxic strain, PG-14. The gene type of strain NTB-88 was different from those of other insecticidal strains, Furthermore, the NH2-terminal amino acid sequence of crystal protein of strain NTB-88 had no relation to those of the previously known $\delta$-endotoxins in other toxic strains as well as HD-12 and PG-14 strains. Therefore, the noninsecticidal crystal protein in strain NTB-88 is novel and its property is different from insecticidal ones.