• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2003년도 제46회 춘계 학술연구 발표회
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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)

• Journal of Microbiology and Biotechnology
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• 제19권8호
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• pp.749-759
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• 2009

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDS-PAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.

• 한국미생물·생명공학회지
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• 제11권3호
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• pp.223-231
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• 1983

Bacillus thuringiensis 16개 균주에서 Beta-exotoxin 생산을 연구하였다. 균주는 Conner-Han-sen Mineral Salts 배지에 시험균을 접종하여 28$^{\circ}C$에서 진탕배양을 한 후 Micrococcus flava을 이용하여 감수성과 생산능을 조사했고, Spectrophotometer를 이용하여 260nM에서 생산량량과 생산속도 등을 조사했다. 1. Bacillus thuringiensis의 돌연변이 균주BTK2-T1, BTK2-T13, BTK2-T17, BTK2-T33과 BTK2-T40은 Conner-Hansen 배지에서 배양 6시간부터 Stationary Phase에 접어들어 가서 48시간 배양과 별 차이를 나타내지 않았으나, Bata-exotoxin의 생산은 6시간 배양에서는 O.D. 260nm에서 약 1.0미만(약 40$\mu\textrm{g}$/$m\ell$)이였고, 12시간 배양에서는 O.D.260nm에서 약 1.7미만(약 70$\mu\textrm{g}$/$m\ell$) 이었으며, 24시간배양부터 48시간 배양에 서는 거의 증가를 보이지 않고 O.D. 260nm에서 2.0내지 2.3(약 85$\mu\textrm{g}$/$m\ell$)을 계속 유지했다. 48시간 배양균주들은 BTK2는 배지 $m\ell$당 80$\mu\textrm{g}$(5.5$\times$$10^{8}$ Cells/$m\ell$), BTK2-T13은 84$\mu\textrm{g}$(4.3$\times$$10^{8}$ Cells/$m\ell$), BTK2-T17은 87$\mu\textrm{g}$(1.4$\times$$10^{8}$ Cells/$m\ell$), 그리고 BTK2-T33은 84$\mu\textrm{g}$ (4.9$\times$$10^{8}$ Cells/$m\ell$)을 분비했다. 2. 다른 혈청형 균주들도 모두 Beta-exotoxin을 생산했는데, 48시간 배양 배지 $m\ell$당 70$\mu\textrm{g}$을 분비한 균주는 BTK-1이고, BTK-37 균주는 $m\ell$당 88$\mu\textrm{g}$(6.1$\times$$10^{8}$ Cells/$m\ell$) BTK-35 균주는 $m\ell$당 81$\mu\textrm{g}$(5.2$\times$$10^{8}$ Cells/$m\ell$)을 생산했고. 그외는 모두 70$\mu\textrm{g}$미만이었다. 3. Beta-exotoxin과 B. thuringiensis 균체을 동시에 per os, interaperitoneal injection, subcuntaneous injection, nasal cavity inoculation, intracerebral injection을 120시간 처리했어도 치사효과를 나타내지 않았다.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.15-20
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• 2007

To clone novel cry1-type genes from the Bacillus thuringiensis K1 isolate, about 2.4-kb-long PCR fragments were amplified with two primer sets of ATG1-F/N400-R and 1BeATG1-F/N400-R. Using PCR-RFLP, three novel cry1-type genes, cry1-1, cry1-7, and cry1-44, were obtained from B. thuringiensis K1 and the complete coding sequences of these novel genes were analyzed. The Cry1-1, Cry1-7, and Cry1-44 proteins showed maximum similarities of about 78.0%, 99.7%, and 91.0% with the Cry1Ha1, Cry1Be1, and Cry1Ac2 proteins, respectively. These novel cry1-type genes were expressed using a baculovirus expression vector system and their insecticidal activities were investigated. Whereas all three novel genes were toxic to Plutella xylostella larvae, only Cry1-1 showed insecticidal activity against Spodoptera exigua larvae.

• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2005년도 제48회 춘계 학술연구 발표회
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• pp.49-49
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• 2005

• 한국응용곤충학회:학술대회논문집
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• 한국응용곤충학회 2005년도 춘계 학술발표회
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• pp.141-141
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• 2005

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.451-455
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• 1996

Physiological studies on the expression of Bacillus thuringiensis subsp. tenebrionis (Btt) gene coding for insecticidal protein in recombinant Escherichia coli 537 were carried out to identify optimal culture condition. It was necessary to shift culture temperature from 30 to $42^{\circ}C$ to express the gene. Expression of the Btt toxin gene by recombinant E. coli 537 began within one hour after induction. Complex nitrogen sources increased production of the insecticidal protein. The total insecticidal protein was 0.5 g/I when using yeast extract as a complex nitrogen source. Soybean hydrolysate showed apparently the highest induction efficiency. After induction, the cellular content of the insecticidal protein was 5.4 times higher than it had been before induction. The optimal cultivation strategy was found to grow cells for 7hours at $30^{\circ}C$ and then 5-8 hours at $42^{\circ}C$. The optimal cultivation pH for the production of insecticidal protein was 6.5. The Btt toxin produced by the recombinant E. coli 537 was found to have the same level of potency against Colorado potato beetle as the original toxin.

• BMB Reports
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• 제34권2호
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• pp.150-155
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• 2001

The mosquito-larvicidal Cry4B protein from Bacillus thuringiensis subsp. israelensds was expressed in Escherichia coli. Upon activation by trypsin, the 130-kDa protoxin was processed into the 65-kDa active toxin containing two polypeptide fragments of ca. 47 and ca. 20 kDa. These two polypeptides are products of internal cleavages on the exposed loop connecting helices 5 and 6 in the seven-helical bundle domain. PCR-based mutagenesis was employed to introduce an additional cleavage site into the loop connecting helices 3 and 4. A series of amino acid changes were introduced into the targeted loop, resulting in seven mutant protoxins. Upon digestion with trypsin, a group of mutants with arginine introduced into the loop (EPRNQ, EPNRNQ, EPRNP, ESRNP and SSRNP) produced polypeptide products similar to those of the wild type (EPNNQ). When the loop, SSRNP, was expanded by an insertion of either asparagine (NSSRNP) or valine (VSSRNP), an additional cleavage was detected with proteolytic products of 47,12 and 6 kDa. This cleavage was confirmed to be at the introduced arginine residue by N-terminal sequencing. The mosquito larvicidal assay against Aedes aegypti demonstrated a relatively unchanged toxicity for the mutants without cleavage and reduced toxicity for those with an additional cleavage.

• Journal of Microbiology and Biotechnology
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• 제22권1호
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• pp.133-140
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• 2012

A novel cry2Ab gene was cloned and sequenced from the indigenous isolate of Bacillus thuringiensis subsp. kurstaki. This gene was designated as cry2Ab25 and its sequence revealed an open reading frame of 1,902 bp encoding a 633 aa protein with calculated molecular mass of 70 kDa and pI value of 8.98. The amino acid sequence of the Cry2Ab25 protein was compared with previously known Cry2Ab toxins, and the phylogenetic relationships among them were determined. The deduced amino acid sequence of the Cry2Ab25 protein showed 99% homology to the known Cry2Ab proteins, except for Cry2Ab10 and Cry2Ab12 with 97% homology, and a variation in one amino acid residue in comparison with all known Cry2Ab proteins. The cry2Ab25 gene was expressed in Escherichia coli BL21(DE3) cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Cry2Ab25 protein is about 70 kDa. The toxin expressed in BL21(DE3) exhibited high toxicity against Malacosoma neustria and Rhagoletis cerasi with 73% and 75% mortality after 5 days of treatment, respectively.

• BMB Reports
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• 제43권6호
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• pp.427-431
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• 2010

Cyt2Aa2 is a mosquito-larvicidal protein produced as a 29 kDa crystalline protoxin from Bacillus thuringiensis subsp. darmstadiensis. To become an active toxin, proteolytic processing is required to remove amino acids from its N- and C-termini. This study aims to investigate the functional role of amino acid residues on the N-terminal ${\beta}1$ and C-terminal ${\alpha}F$ of Cyt2Aa2 protoxin. Mutant protoxins were constructed, characterized and compared to the wild type Cyt2Aa2. Protein expression data and SDS-PAGE analysis revealed that substitution at leucine-33 (L33) of ${\beta}1$ has a critical effect on dimer formation and structural stability against proteases. In addition, amino acids N230 and I233-F237 around the C-terminus ${\alpha}F$ demonstrated a crucial role in protecting the protoxin from proteolytic digestion. These results suggested that ${\beta}1$ and ${\alpha}F$ on the Nand C-terminal ends of Cyt2Aa2 protoxin play an important role in the molecular interaction and in maintaining the structural stability of the protoxin.