• Applied Biological Chemistry
• /
• v.19 no.4
• /
• pp.189-201
• /
• 1976

In an effort to develop a microbial in secticide, B. thdringiensis var. thuringiensis was cultured in the medium composed of cocoon-cooked water from a filature. The results obtained are summarized as followss : (1) Bacillus thuringiensis is a bacterium producing a ${\delta}-endotoxin$ especially toxic to lepidopterous insects and a thermostable exotoxin harmful to dipterous insects. (2) With a view to utilizing the cocoon-cooked water discarded from the filature, as a nutrient source in the B. thuingiensis culture, it was analyzed to contain large amounts of various minerals and protein (7.5 mg/ml) believed to be extracted from the pupae. (3) A large amount of the ${\delta}-endotoxin$ can be obtained most cheeply by using cocoon-cooked water instead of distilled water in preparing GYS and citrate salts media. (4) The largest amount of a mixture of the vegetative cells, spores, and crystals was obtained by addition of 8 gr/l of glucose to the GYS medium. (5) The growth of the bacterium was far better, when leucine, isoleucine, and valine were added all together to the citrate salts medium to the concentration of $1.25{\times}10^{-3}M$. (6) The best growth was observed by addition of Na-glutamate to the citrate salts medium to the concentration of $2.5{\times}10^{-3}M$. (7) The optimal culture time ranged from 9 to 15 days. (8) The highest mortality was shown in Pieris rapae Linne with a pH of the total body extract of 8.4, whereas Dendrolimus spectabilis Butler and Bombyx mori Linne with lower pH's were less susceptible to the ${\delta}-endotoxin$. (9) The presence of the thermo stable exotoxin was confirmed by the fact that the supernatant of the culture was very toxic to the Drosophila melanogaster tested.

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

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

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

• Journal of environmental and Sanitary engineering
• /
• v.8 no.1
• /
• pp.129-138
• /
• 1993

The marked spread of the cockraches of recent years has raised a great social problem in urban areas. The cockroach have to remove1 because transmit a disease to human as pest insect, but particulars are not yet reported on biological control agent for the cockroach removal. This study was tried for the first time on biological control for the cockroach removal. The obtained results were as follows : 1. The isolated were spore-forming bacillus 1098 strain in soil. The No. 109(TH 109) strain of the among spore-forming bacillus was showed the poisonous against Cockroach. 2. The biological characteristics and flagella antigenicity of the strain is similar to Bacillus thur-ingiensis subsp. indiana. 3. TH 109 strain have the delta-endotoxin of cuboid shap. 4. This delta-endotoxin of product by TH 109 strain was toxic to the cockroach(Blattella gemzanica. L).

• Microbiology and Biotechnology Letters
• /
• v.16 no.2
• /
• pp.168-173
• /
• 1988

Immunological analysis between endotoxin proteins produced by Bacillus thuringiensis serovar. kurstaki HD1 and HD73 have been investigated by using polyclonal antibodies. The antisera against the endotoxin proteins were prepared from rabbits injected with the endotoxin protein antigens. When about 2mg/$m\ell$ of the antigens were injected for 7 times, the titers were highest. The stability of the antigens was reduced to about 50% after 9 days incubation at 4$^{\circ}C$. The sensitibity of endotoxin protein from B. thuringiensis HD1 and HD73 by indirect ELISA was 50ng/$m\ell$ and 400ng/$m\ell$, respectively. The cross reaction of antiserum appeared that anti-HD1 partialy reacted with crystal protein but anti-HD73 reacted with HD1 endotoxin about 100%.

• Animal cells and systems
• /
• v.1 no.3
• /
• pp.507-513
• /
• 1997

Sequential observations of binding patterns and structural effects of Bacillus thuringiensis var. kurstaki were made on fat body tissue of the fall webworm, Hyphantria cunea Drury. Fat body was cultured in vitro in the presence of purified 62 kDa endotoxin and then examined for protein synthesis and the localization of membrane-bound toxin detected by an antibody against the 62 kDa endotoxin. Protein synthesis was mostly inhibited at concentrations of 15 ${\mu}$g/ml and higher. Immunocytochemical observations suggest that the toxin binds to all exposed basal lamina surrounding the fat body without apparent specificity. The cytopathic effect delectable by scanning electron microscope is disintegration rather than cell swelling. The basal lamina bound toxin was eventually detached from the fat body and followed by an extrusion of cell contents like lipid granules.

• Microbiology and Biotechnology Letters
• /
• v.23 no.6
• /
• pp.730-736
• /
• 1995

The solubilization and proteolytic process of $\delta $-endotoxin was analvsed to compare the biochemical property of the toxin isolated from B. thuringiensis subsp. sotto. The purified crystals were dissolved in 50 mM carbonate buffer containing 10 mM dithiothreitol at pH 10 for various times. The electrophoretic pattern showed that a rapid disappearance of 138 kDa protein band. This disappearance of protein with high molecular weight was accompanied by the appearance of new protein fragment with 104 kDa, 60 kDa, and 25 kDa. For proteolvtic processing, the soluble crystals were digested with trypsin for various times. The soluble crystal protein of 104 kDa was completely disappeared. However, the protein fragment of 60 kDa and 25 kDa still remained after complete proteolysis. The comparative immunoblot analysis showed that the antiserum against intact crystals showed strong immunoreactivity to the homologous inclusion protein of 138 kDa, 104 kDa, and 25 kDa, and to the intact spores of 221 kDa and 138 kDa, but not to the vegetative cell homogenate. The sera against crystals and spores had no immunoreactivity to the vegetative cell homogenate.

• Journal of Microbiology and Biotechnology
• /
• v.4 no.4
• /
• pp.322-326
• /
• 1994

Bacillus thuringiensis strain BT-14 was isolated from alfalfa dust in Korea. The strain BT-14 produced one bipyramidal crystal and one spore in the cell. The biochemical characteristics of the strain BT-14 were similar to those of Bacillus thuringiensis subsp. kurstaki HD-l. Examination of its antibiotic resistance revealed that while the strain BT-14 was less resistant than BTK HD-l to ampicillin, gentamycin, neomycin and tobramycin, it was more resistant to amikacin than BTK HD-l. The $\delta$-endotoxin crystal of strain BT-14 consisted of a single protein with a high molecular weight of ca 135 KD on a 10% SDS-PACE. The strain BT-14 contained at least nine different plasmids with sizes of 2.9, 5.3, 5.8, 6.2, 9.4, 15.1, 18.1, 23.1 and 79 Kb. In insect bioassay, the isolated strain BT-14 showed lethality of 67% against Plutella xylostella larvae at dilution of 5$\times$$l0^{-4}$ (5$\times$l0 to 3$\times$$l0^2$ spores/ml), which is, almost equivalent to that of BTK HD-l.

• The Journal of Natural Sciences
• /
• v.6 no.1
• /
• pp.41-48
• /
• 1993

A cloned delta-endotoxin gene from Bacillus thuringiensis var. kurstaki NRD 6 was mutated at N-terminal toxic portion of the protoxin by site-directed mutagenesis. A Stu 1 restriction site was created in protoxin region of the mutant B.t.kurstaki NRD 6-Stu 1 which changed A to C in 177 region of the protoxin(silent mutation). Antigenic property of crystalline inclusion (delta-endotoxin) of the mutant was no difference to intact strain by the western blot analysis and its toxidity threshold value was also showed 0.015~0.030ng against Choristoneura femiferana-1 insect, similar with 0.01~0.024ng of parent strain, but stronger than that of B.t.kurstaki NRD 5 (500~1000ng) and B.t.kurstaki NRD 4(none toxic).