Mosquitoes are medically important insect pests that act as vectors of various diseases when they feed on humans. Insect growth regulators (IGRs) could become an effective alternative to control mosquitoes because of their high specificity and relatively low toxicity to environment. Recently, we have developed high-throughput juvenile hormone antagonist (JHAN) screening system based on yeast-two hybrid assay. Actinomycetes have been reported to produce various bioactive compounds including insect growth regulators (IGRs). In this study, culture filtrates and mycelia extracts of 2,875 actinomycetes isolates screened for their IGR activities and mosquitocidal activities. Among 75 culture filtrates and 17 mycelia extracts with JHAN activities, 19 culture filtrate and 3 mycelia extracts showed high level of mosquitocidal activities against 3rd instar larvae of Aedes albopictus. These results suggested that secondary metabolites of actinomycetes could be used as environmentally benign mosquitocidal agents.

Entomopathogenic fungi have been widely studied for their potential as the effective biological control agents. Theyproduce variety of secondary metabolites with insecticidal activities, and it is reasonable to assume that entomopathogenicfungi might produce secondary metabolites modulating juvenile hormone for their survival against defense mechanismsof host insect. In this study, Acetone extracts of 189 entomopathogenic fungi cultured on unpolished rice medium werescreened for their juvenile hormone antagonist (JHAN) activities using the yeast-two hybrid system. Among them, 14extracts showed high level of JHAN activities and their insecticidal activities against Aedes albopictus were investigated.

The novel serogroup of Bacillus thuringiensis serovar mogi (H3a3b3d) was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. Plasmids from B. thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases in mammals and insects. In this study, the genome sequence of the strain was determined. The 6.0-Mb genome of B. thuringiensis mogi contains three replicons: a circular chromosome (5.40-Mb) encoding 5,652 predicted open reading frames (ORFs), and two megaplasmids, pMOGI364 (364 564 bp) and pMOGI222 (222 348 bp). The G+C contents of these replicons ranged from 31.3% to 34.2% for pMOGI364 and pMOGI222, respectively. There are six putative cry genes, cry19Bb1, cry73Aa, cry20Bb1, cry27Ab1, cry4Aa and cry56Ba1, distributed on these two megaplasmids. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative PCR (qPCR) from the wild type strain. Also, these cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K under the control of its own promoter and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization.

Plasmids are crucial for determining the pathogenicity and host range of organisms of the Bacillus thuringiensis strains. In this research, a novel serogroup of B. thuringiensis serovar mogi (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained two megaplasmids (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that there are 7 putative cry genes, cry19Bb1, cry73Aa, cry40orf2, cry20Bb1, cry27Ab1, cry56Ba1 and cry39orf2, distributed on the two different megaplasmids, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K under the control of its own promoter and p1KSD, which is a recombinant expression vector containing cyt1Aa promoter combined with the STAB-SD sequence, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative PCR (qPCR) from the wild type strain. These results clearly indicate that the cry39orf2 was the dominant ingredient in the crystal. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.

Bacillus thuringiensis serovar mogi of a novel serogroup (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained only megaplasmid (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that the megaplasmid harbored at least seven putative cry genes, showing about 84%, 75%, 73%, 58%, 84%, 39% and 75% similarities in amino acid sequences with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative real-time PCR (qrtPCR) from the wild type strain as well as transformant strains. The results clearly indicate that the cry39orf2 was the dominant ingredient in the crystal. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases in mammals and insects. B. thuringiensis serovar mogi of a novel serogroup (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained only megaplasmid (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that the megaplasmid harbored at least seven putative cry genes, showing about 84%, 75%, 73%, 58%, 84%, 39% and 75% similarities in amino acid sequences with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that in mammals and insects. A novel serogroup (H3a3b3d), B. thuringiensis strain K4 which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the strain K4 (designated as serovar mogi) had only one large plasmid (>200kb) on which the toxin genes were occasionally located. A 454 pyrosequencing was used for the complete sequencing of the large plasmid. The sequence analysis showed that k4 plasmid had at least seven putative cry genes, ending up to showing 84%, 75%, 73%, 58%, 84%, 39% and 75% homology with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2 toxins in amino acids, respectively. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, can be used as a good resource for studying unknown mosquitocidal cry genes. The E. coli-B. thuringiensis shuttle vector, pHT1K was used to clone these cry genes for characterization. In each clone, the level of transcription and production of crystal proteins will be investigated in near the future.

Bacillus thuringiensis (Bt) strain K4 was isolated from fallen leaves which had been collected at a forest stand in Mungyeong city, Republic of Korea. The flagellated vegetative cells of Bt K4 were agglutinated with the H3 reference antiserum among 55 reference H-antisera. In a further test to identify subfactors, 3b and 3d monospecific antisera were reactive to the cells, followed up with introducing a novel serogroup of 3a3b3d, designated as serovar mogi. The strain K4 had mosquitocidal activity against Dipteran larvae, Anopheles sinensis and Culex pipiens pallens, with no Lepidopteran toxicity observed. The SDS-PAGE profile of K4 crystal protein, ovoidal-shaped, included several bands ranging from 30-75 kDa. Four putative peptides, Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected from the bands by a nano-LC-ESI-IT MS analysis. Through a thermal asymmetric interlaced PCR, cry19Ba, cry40ORF2 and cry27Aa genes were partially cloned from K4 strain. Three cry genes were further found in the strain by a 454 pyrosequencing, ending up to showing 58%, 39% and 84% homology in amino acids with Cry56Aa, Cry8Ba and Cry39ORF2 toxins, respectively. This novel 3a3b3d type strain, B. thuringiensis subsp. mogi, can be used as a good resource for studying unknown mosquitocidal cry genes.

The Bacillus thuringiensis strain K4 was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. The flagellated vegetative cells of B. thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific antisera but non-reactive for 3c and 3e factor sera. These results create a new serogroup with flagellar antigenic structure of 3a3b3d, designated serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced a single ovoidal-shaped parasporal crystal whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected. In contrast to the plasmid profile of B. thuringiensis H3 serotype strains, the strain K4 contained only a large plasmid (~100 kb) and we cloned partial cry27Aa, cry19Ba and cry40ORF2 genes from it by thermal asymmetric interlaced PCR. Sequencing analysis showed 87%, 88% and 88% homologous with known cry27Aa, cry19Ba and cry40ORF2 genes, respectively. The new type strain, B. thuringiensis subsp. mogi (H3a3b3d) will be a good resource for new mosquitocidal cry genes.

국내에서 분리된 Bacillus thuringiensis NT0423은 나비목과 파리목 곤충에 독성을 보이는 130 kDa의 전형적인 다이아몬드형 내독소 단백질을 생성한다. 이 균주의 파리목에 대한 독성을 강화하기 위하여, B. thuringeinsis NT0423에 모기 유충에 강한 독성을 보이는 B. thuringiensis subsp. morrisoni PG-14의 cryVID유전자를 가지고 있는 pCG10 플라스미드를 electroporation 방법을 이용하여 형질전환하였다. 형질전환체인 B. thuringiensis PT1227내에서 cryIVD와 숙주가 생성하는 원래의 다이아몬드형 130kDa 내독소 단백질 유전자는 그 자신의 형태로 잘 발현되었다. 형질전환체의 모기 유충에 대한 독성은 원래 숙주의 내 독소 단백질과ㅏ 도입된 CryIVD의 상승효과에 의해 현저히 증가하였다.

Bacillus thuringiensis subsp. morrisoni PG-14의 cryIVD 유전자를 포함하고 있는 발현벡터 pCYASK5-1을 제작하여 Synechocystis PCCC6803에 형질전환시킨 후, 학질모기(Anopheles sinensis)유충에 대한 독성을 검정하였다. Kanamycin이 포함된 BG-11배지에서 선발된 형질전환체의 cryIVD 유전자 발현은 SDS-PAGE와 West-ern blot분석으로 확인하였다. 형질전환체는 A. sinensis 유충에 대해 높은 독성을 나타내었으며, 성장은 야생주인 Synechocystis Pccc6803과 유사하였다. 또 수심에 따른 형질전환체의 분포도 조사에서 전체적으로 살충농도의 세포수로 분포함을 확인하였다. 이러한 결과들은 B. thuringiensis subsp. morrisoni PG-14의 cryIVD 유전자로 형질전환된 Synechocystis PCCC6803이 모기유충 방제에 효율적으로 이용될 수 있음을 나타내었다.