• Journal of Microbiology and Biotechnology
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• v.23 no.1
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• pp.88-91
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• 2013

The Cyt1Aa protein of Bacillus thuringiensis subsp. israelensis is known to synergize mosquitocidal proteins of B. thuringiensis and Bacillus sphaericus strains. Cyt1Aa is highly lipophilic, and after binding in vivo to the midgut microvillar membrane serves as a "receptor" for mosquitocidal Cry proteins, which subsequently form cation channels that kill mosquito larvae. Here we report that Cyt1Aa can serve a similar function for lepidopteran-specific Cry proteins of B. thuringiensis in certain mosquito larvae. Engineering Cyt1Aa into the HD-1 isolate of B. thuringiensis subsp. kurstaki enhanced toxicity against $4^{th}$ instars of Aedes aegypti, but not against $4^{th}$ instars of Culex quinquefasciatus.

• The Korean Journal of Pesticide Science
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• v.14 no.4
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• pp.446-455
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• 2010

The characteristics of parasporal inclusion body from Bacillus thuringiensis subsp. kurstaki KB099 isolate which is high bioactive to the tobacco cutworm, Spodoptera litura, were examined. Parasporal inclusion of B. thuringiensis subsp. kurstaki KB099 isolate showed only 1 band at 130 kDa compared with B. thuringiensis subsp. kurstaki HD-l isolate producing 2 protein bands at 130 kDa and 60 kDa from by SDS-PAGE analysis without any enzyme treatment. Also, we confirmed that gut extract of sensitive S. litura KB099 isolate had digested only 60 kDa ${\delta}$-endotoxin protein. When the digestive enzyme of sensitive insect responsible for parasporal inclusion from KB099 and HD-l isolate was treated to each of them, protein band 60 KDa of KB099 was maintained up to 12 hours but all bands of HD-l were disappeared within 6 hours. In KB099 isolate, 6 genes (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1D and Cry1I) were identified by PCR analysis. Also, $Cry^-$ mutant of KB099 isolate was investigated by phase- contrast microscope, SDS-PAGE and PCR.

• Korean journal of applied entomology
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• v.28 no.2
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• pp.69-75
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• 1989

The extracellular amylase production by Bacillus thuringiensis subsp. kurstaki HD-l in amylase production media and its characteristics were investigated. The amylase production was highest in the medium composed of 0.2% soluble starch, 1.0% Bacto-peptone, 0.3% beef extract, 0.3% yeast extract, 0.5% NaCl, 0.3% $K_2HPO_4$, 0.1% $KH_2PO_4$, 0.012% $CaCl_2$.$2H_2O$, 0.005% $MnSO_4$.$H_2O$, and 0.03% $MgSO_4$.$7H_2O$. The amylase activity was inhibited by 50mM EDT A. The enzyme was optimally active from pH 6.5 to 7.0 at $55^{\circ}C$, The specific activity of the enzyme in the ethanol precipitate was 2.01 units/mg, and the Km value was approxi-mately 0.8 mg/ml.

• Journal of Life Science
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• v.12 no.3
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• pp.369-373
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• 2002

The waste-water from the industry for production of a soybean curd (the soybean curd waste-water) was investigated to use for the substrate to produce the endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 used as one of well known microbial pesticides. The pH of the soybean curd waste-water was 9.8 and its chemical oxygen demand (COD), total nitrogen (TN) and phosphate (TP) were 276.0, 71.1 and 5.5mg/$\ell$, respectively. The higher was the concentration of the soybean curd waste-water in the medium, the more endotoxin was produced. Maximal sporulation occurred at which concentration of $K_2$HPO$_4$in the medium supplied with the soybean curd waste-water was 1% (w/v). Production of the endotoxin with the optimized medium supplied with the soybean curd waste-water was 1.5 times higher than that without the soybean curd waste-water. The soybean curd waste-water was found to be suitable substrate for production of the endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1.

• Journal of Sericultural and Entomological Science
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• v.35 no.1
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• pp.36-42
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• 1993

Expression of insecticidal protein by fusion product of truncated HD-1[CryIA(a)] N-terminal and HD-73[CryIA(c)] C-Terminal fragment of Bacillus thruingiensis subsp. kurstaki was investigate. Immunological analysis of transformants by using polyclonal antisera raised against the whole-crystal protein of HD-1 revealed that SK4 and SK5 were observed cross-reaction with polypeptides of 77-kDa and 105-kDa, respectively. Bioassay of the transformant pSK5 to Plutella maculipennis and Heliothis assulta were 96% and 97%, respectively.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.647-652
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• 1990

The production of delta-endotoxin crystal and the cloning of endotoxin protein gene in Bscillus thuringiensis subsp. kurstaki HD1 strain were studied. The strain produced bipyramidal crystals ($2.9\times 1.0 \mu m$) in their cells during sporulation. The B. thuringiensis contained about 10 plasmid DNA elements ranging from 2.1 to 80 kilobases. The 73 kb plasmid DNA, the 29 kb BamHI fragment and the 7.9 kb Pstl DNA fragment hybridized to the pHL probe. The 7.9 kb fragment was eluted and cloned in the PstI site of pBR322 vector and transformed into E. coli HB101, which produced insecticidal proteins killing Bornbyx mori larvae.

• Journal of Microbiology and Biotechnology
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• v.5 no.3
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• pp.138-142
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• 1995

Bacillus thuringiensis strain BT-209 was isolated from a soybean grain dust sample in Korea. The strain BT-209 produced two different sizes of cuboidal crystals and one spore in the cell. In the biochemical characterization, the strain BT-209 showed negative reactions on the production of urease, and the utilization of citrate and sucrose. Examination of its antibiotic resistance revealed that while the strain BT-209 showed higher sensitivity than B. thuringiensis subsp. kurstaki HD-1 to ampicillin, bacitracin, chlortetracycline, gentamycin, neomycin, penicillin G, tetracycline and tobramycin, it was more resistant to methicillin than B. thuringiensis subsp. kurstaki HD-1. The $\delta$-endotoxin crystal of strain BT-209 consisted of three proteins with apparent molecular weights of appoximately 148, 135 and 62 kDa on a 10% SDS-PAGE. The strain BT-209 had at least eight different plasmids with sizes of 4.1, 5.2, 6.3, 8.6, 14.6, 24.5, 67.6 and 77.6 Kb. The strain BT-209 showed strong lethalities of 70% and 87% against Bombyx mori and Hyphantria cunea larvae. at 72 h, respectively.

• Korean journal of applied entomology
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• v.32 no.3
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• pp.300-306
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• 1993

The CryIIA gene encoding the insecticidal crystal protein of Bacillus thuringiens!s subsp. kurstalri HD-l has been cloned in Escherichia col!, and its nucleotide sequences were determined completely. 5kb Hindlli fragment harboring CryIIA gene was screened in the large ca. 225kb plasmid DNA by southern blot. HindlIT digested 5kb fragment was ligated into pUC19 and transformed in E. coli. The 4kb BamHI-HindlIT fragment containing the CryIIA gene was subcloned and named pSKIIA. DNA sequence analysis demonstrates that pSKIIA is the gene of an operon which is comprised of Lhree open reading frames (designated orn, orf2 and or£3). The CrylIA gene is composed of 3,952bp-long BamHI-Hindill DNA restriction fragment. The orf3 code for a polypeptide of 633 amino acid residues. The protoxin protein has a predicted molecular weight of 70,780. The E. coli derived protoxin gene product is biologICally active against three species of Lepidopteran (Plu.lelia maculipennis, He/iolhis assulta, Spodoptera litura) and a species of Dip Leran( Culex pipines) larvae in bioassay.

• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.37-44
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• 1991

A shuttle promoter-probe vector, pEB203, was derived from pBR322, pPL703 and pUB110. Using the vector, a useful DNA fragment, 319 bp EcoRI fragment, having strong promoter activity has been cloned from Bacillus subtills chromosomal DNA. Selection was based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The nucleotide sequence of the 319 bp fragment has been determined and the putative -35 and -10 region, ribosome binding site, and ATG initiation codon were observed. This promoter was named EB promoter and the resultant plasmid which can be used as an expression vector was named pEBP313. The crystal protein gene from B. thuringiensis subsp. kurstaki HD-73 was cloned downstream from the EB promoter without its own promoter. When the resultant plasmid, pBT313, was introduced into Escherichia coli and B. subtilis, efficient synthesis of crystal protein was observed in both cells, and the cp gene expression in B. subtilis begins early in the vegetative phase. The cell extracts from both clones were toxic to Hyphantria cunea larvae.

• Journal of Microbiology
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• v.45 no.6
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• pp.553-557
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• 2007

In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we isolated a B. thuringiensis strain (Bn1) from Balaninus nucum (Coleoptera: Curculionidae), the most damaging hazelnut pest. Bn1 was characterized via morphological, biochemical, and molecular techniques. The isolate was serotyped, and the results showed that Bn1 was the B. thuringiensis serovar, kurstaki (H3abc). The scanning electron microscopy indicated that Bn1 has crystals with cubic and bipyramidal shapes. The Polymerase Chain Reactions (PCRs) revealed the presence of the cry1 and cry2 genes. The presence of Cry1 and Cry2 proteins in the Bn1 isolate was confirmed via SDS-PAGE, at approximately 130 kDa and 65 kDa, respectively. The bioassays conducted to determine the insecticidal activity of the Bn1 isolate were conducted with four distinct insects, using spore-crystal mixtures. We noted that Bn1 has higher toxicity as compared with the standard B. thuringiensis subsp. kurstaki (HD-1). The highest observed mortality was 90% against Malacosoma neustria and Lymantria dispar larvae. Our results show that the B. thuringiensis isolate (Bn1) may prove valuable as a significant microbial control agent against lepidopteran pests.