• Journal of Microbiology and Biotechnology
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• v.23 no.2
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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 Biosystems Engineering
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• v.43 no.4
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• pp.394-400
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• 2018

Purpose: This research evaluated five types of nanoparticles to develop a surface-enhanced Raman spectroscopy (SERS) method for the rapid detection of two Bacillus species (Bacillus cereus and Bacillus thuringiensis) that are commonly found on fresh produce, which can cause food poisoning. Methods: Bacterial concentrations were adjusted to a constant turbidity, and a total of $30{\mu}L$ of each Bacillus cell suspension was prepared for each nanoparticle. A point-scan Raman system with laser light source of wavelength 785 nm was used to obtain SERS data. Results: There was no qualitative difference in the SERS data of B. cereus and B. thuringiensis for any of the five nanoparticles. Three gold nanoparticles, stabilized in either citrate buffer or ethanol, showed subtle differences in Raman intensities of two Bacillus species at $877.7cm^{-1}$. Conclusions: Among the three types of nanoparticles, the gold nanoparticles stabilized in citrate buffer showed the lowest standard deviation, followed by gold nanoparticles stabilized in ethanol. This result supports the potential application of gold nanoparticles for SERS-based detection of B. cereus and B. thuringiensis.

• Microbiology and Biotechnology Letters
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• v.21 no.3
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• pp.193-199
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• 1993

Effects of the five fermenation media on the growth and the production of endotoxin of twenty-five Bacillus thuringiensis isolates were investigated. Among the five different media, the growth and the production of endotoxin of B. thuringiensis were highest in the M4 and M5 media containing high amounts of nitrogen and carbohydrate. But the production of endotoxin was highest in the M1 and M3 media containing high amounts of nitrogen sources limiting carbohydrate. The average number of endotoxin and growth(O.D.) in the M1 and M3 medial were 4.4*107/ml and 2.42 in M1 medium and 1.3*107/ml and 1.46, respectively.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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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.

• The Korean Journal of Pesticide Science
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• v.13 no.4
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• pp.315-324
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• 2009

For the environmental friendly management of diamondback moth, Plutella xylostella (L.), Bacillus thuringiensis subsp. kurstaki ($30{\times}10^8\;cfu/mg$) and neem oil (0.5% azadirachtin) were used as green control agencies with mixed and alternative treatments on the chinese cabbage. When Bacillus thuringiensis subsp. kurstaki was applied to 1st and 2nd larva of P. xylostella with recommended concentration, their mortalities were reached to 100% by 2 days after treatment. In case of azadirachtin, its effect of mortality was continued for 7 days, and reached to 100% mortality. $LC_{50}$ values of Bacillus thuringiensis subsp. kurstaki and azadirachtin against 1st, 2nd, 3rd and 4th larva of P. xylostella were $2.8{\times}10^4$, $3.1{\times}10^4$, $3.4{\times}10^4$ and $1.5{\times}10^5\;cfu/ml$, and 2.7, 3.9, 4.7 and 7.1 ppm, respectively. The number of laid eggs of P. xylostella was reduced to 57.5 at 25 ppm of azadirachtin compared with control treatment. The hatch ratio was not significantly different with Bacillus thuringiensis subsp. kurstaki treatment in comparison with control treatment. However, when azadirachtin was applied, their hatch ratio were reduced to 25.8 and 45.4% at 25 and 50 ppm, respectively. On the other hand, emergence rate of eggs was not different with Bacillus thuringiensis subsp. kurstaki treatment, but 45.4% was shown in azadirachtin treatment with 50 ppm in comparison with control. When the mixture with Bacillus thuringiensis subsp. kurstaki and azadirachtin was applied to adults of P. xylostella, their mortality was higher than Bacillus thuringiensis subsp. kurstaki treatment only. These results are supposed that the mixture of Bacillus thuringiensis subsp. kurstaki and azadirachtin might be used as green control agents for reducing the demage of diamondback moth in the Chinse cabbage.

• 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.

• Korean journal of applied entomology
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• v.49 no.2
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• pp.151-158
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• 2010

Among 18 Bacillus thuringiensis isolates with spherical parasporal inclusion from soils, B. thuringiensis subsp. israelensis CAB199 was selected. It was showing over 90% mortality against Aedes aegypti and Culex pipiens moletus. It was confirmed that this B. thuringiensis subsp. israelensis CAB199 isolate also had a insecticidal activity against Culex inatomii that was occurred in the marsh. Because most of mosquito larva were primarily situated or shifted from under- to surface water, we need to select long floating formulations on surface water for controlling mosquito larva. It was tested the pesticidal and control effects in the laboratory and wetland with two formulation types of B. thuringiensis subsp. israelensis, for example, wettable power (WP) and suspension concentrate type (SC). Laboratory test showed that SC formulation type was relatively faster and more effective against 3 tested mosquito species, C. pipiens, Aedes aegypti, and C. inatomii. Otherwise, the control efficacy of SC formulation type was more rapidly appeared against C. inatomii in the wetland.

• International Journal of Industrial Entomology and Biomaterials
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• v.6 no.1
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• pp.81-85
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• 2003

To verify importance of the intervening sequence between the ribosome binding site (RBS) and the initiation codon for expression of Bacillus thuringiensis $\delta$-endotoxin, the pProMu, containing SphI and NcoIsites between RBS and the initiation codon of the cry1Ac gene, and the deletion derivatives of pProMu were constructed and transformed into the B. thuringiensis subsp. kurstaki $Cry^{-B}$ strain. The pProMu-ΔSphIhad identical six bases of intervening sequence to pProAc though the arrangement of sequence was different. Other mutants containing pProMu had 1 or 10 or 14 bases between RBS and the initiation codon. Among deletion mutants, only ProMu-ΔSphI/CB only produced 130 kDa typical bipyramidal crystals like those seen for ProAc/CB. However, ProMu/CB, $ProMu-{\Delta}NcoI$, and ProMu-ΔSphI＋NcoIdid not produce Cry1Ac crystals. In conclusion, the results suggest that 6-base intervening sequence was important for expression of cry1-type class gene. Furthermore, spacing effect of the intervening sequences may play an important role in expression of individual crystal proteins in B. thuringiensis without doubt.

• Microbiology and Biotechnology Letters
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• v.13 no.1
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• pp.51-57
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• 1985

Delta-endotoxin in Bacillus thuringiensis var. finitimus, HD-1, HD-9 and HD-73 strains were isolated by NaBr, CsCl and Renografin density gradients. The purity of the toxin was about 98%. The purified o-endotoxin was analyzed by SDS-PAGE, electron microscopic observation and bioassay. According to SDS-PAGE, the molecular weight of subunits of the o-endotoxin were about 66,000 and 130,000 daltons. The shapes of the crystal toxin observed by TEM except finitimus strain were bipyramidal. When the purified endotoxin was bioassayed against tobacco horn worm, the entomocidal activities ($1{\mu}g/ml$) of HD-1 and HD-73 strains were, respectively, 60% and 100% at nine days after treated. The molecular weights of the plasmids isolated from B. thuringiensis were various from 0.5 to 120 Kb. The numbers of plasmids in HD-1, HD-9 and HD-73 strains were 12, 3 and 11, respectively, but B. thuringiensis var. finitimus strain had no plasmid.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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• pp.788-792
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• 2012

We report the computational structural simulation of the Cry1Ab19 toxin molecule from B. thuringiensis BtX-2 based on the structure of Cry1Aa1 deduced by x-ray diffraction. Validation results showed that 93.5% of modeled residues are folded in a favorable orientation with a total energy Z-score of -8.32, and the constructed model has an RMSD of only $1.13{\AA}$. The major differences in the presented model are longer loop lengths and shortened sheet components. The overall result supports the hierarchical three-domain structural hypothesis of Cry toxins and will help in better understanding the structural variation within the Cry toxin family along with facilitating the design of domain-swapping experiments aimed at improving the toxicity of native toxins.