A Novel Serogroup of Bacillus thuringiensis serovar mogi (flagellar serotype 3a3b3d) with Mosquitocidal Activity

Qin Liu,Yong Wang,Jae Young Choi,Xueying Tao,Jong Bin Park,Jae Su Kim,Yeon Ho Je 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.05

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.

Characterization and Cloning of cry Genes from a Novel Serogroup Mosquitocidal Strain, Bacillus thuringiensis Serovar mogi

Qin Liu,Jae Young Choi,Jae Su Kim,Xueying Tao,Jong Bin Park,Joo Hyun Lee,Yeon Ho Je 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.10

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.

Molecular Characterization of A Genomic DNA of Bacillus thuringiensis Bacteriophage and Its Distribution in Bacillus thuringiensis Type Strains

Jong Yul Roh,Qin Liu,Yong Wang,Xueying Tao,Jae Young Choi,Jong Bin Park,Hee Jin Shim,Soo Dong Woo,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.05

Through an application of plasmid capture system (PCS) to Bacillus thuringiensis plasmid DNAs, we acquired 21 polymorphic clones of putative genomic DNA of bacteriophage. The genome size of phage 1-3 (PhBT1-3) was determined to be 46,517 base pairs (bp) with 35.43% G + C content and 83% coding region. Sixty-five putative open reading frames (ORFs) with more than 50 codons were found in the new phage genome. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Morphological characterization and infectivity assay demonstrated that PhBT1-3 belongs to the family Siphoviridae and it showed infectivity to three B. thuringiensis type strains, galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phages in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and the similarity of those putative amino acids was more than 70%. Furthermore, we verified the existence of various shaped phages from the supernatants of 10 B. thuringiensis type cultures. In conclusion, we characterized a putative genome of phage, PhBT1-3 from B. thuringiensis 1-3, and confirmed the distribution of phages in the group of 67 B. thuringiensis type strains.

Plasmid Capture System and Its Applications

Jong Yul Roh,Yong Wang,Qin Liu,Xueying Tao,Jae Young Choi,Hee Jin Shim,Hong Guang Xu,Soo Dong Woo,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2009 한국응용곤충학회 학술대회논문집 Vol.2009 No.05

Plasmid capture system (PCS) was developed for easy cloning and manipulation of circular double-stranded DNA from various sources. Recently, we improved PCS system (named PCS-LZ) to clone relatively large-sized DNA molecules (30-150 kb). PCS-LZ donor consists of a Mini-F replicon and a kanamycin resistance marker between Tn7L and Tn7R regions. Both replicon and marker gene of PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition and the transposed DNAs can replicate in E. coli cells by transformation. White/blue screening by LacZ expression is also available to avoid backgrounds. Up to now, we acquired various circular DNA clones from four sources such as plasmids of B. thuringiensis, bacteriophage genome isolated from B. thuringiensis, genome segments of Cotesia glomerata bracovirus, and polymorphic genomes of Autographa californica nucleopolyhedrovirus. Among them, interestingly, the genome clones of bacteriphage (Ph1-3) were screened from the PCS transposition with plasmids of B. thuringiensis 1-3 strain. The genome of Ph1-3 was fully sequenced (46517 bp) and open reading frames were analyzed. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Ph1-3 showed infectivity to B. thuringiensis type strains such as subsp. galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phage in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and their sequence similarity was more than 70%. Conclusively, we expect this PCS-LZ system would be a powerful tool for genomic analysis and mutagenesis study at the area of invertebrate pathology and further its application will be enlarged to the vertebrate pathology area.

Cloning of circular DNAs from microorganisms associated with insects using a novel plasmid capture system

Jong Yul Roh,Yong Wang,Qin Liu,Xueying Tao,Jae Young Choi,Hee Jin Shim,Hong Guang Xu,Seungdon Lee,Soo Dong Woo,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2009 한국응용곤충학회 학술대회논문집 Vol.2009 No.10

Plasmid capture systems (PCS) facilitate cloning and manipulation of circular double-stranded DNA. We recently developed an improved PCS (PCS-LZ) to clone relatively large DNA molecules of 30-150 kb. The PCS-LZ donor consists of a mini-F replicon and a kanamycin resistance marker between Tn7 left and Tn7 right ends. Both the replicon and marker gene of the PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition, followed by replication in E. coli. Colonies are tested for lacZ expression by blue/white screening. Circular DNAs were obtained from plasmids of Bacillus thuringiensis, genome segments of Cotesia glomerata bracovirus and polymorphic genomes of Autographa californica nucleopolyhedrovirus. PCS-LZ is a powerful tool for use in genomic analysis and mutagenesis in invertebrate pathology, and we are extending its application to include vertebrate research.

Characterization of a strain of Bacillus thuringiensis serovar aizawai which harbors a Rolling-Circle Replicating Plasmid, pBt1-3

Qin Liu,Jong Yul Roh,Yong Wang,Jae Young Choi,Xueying Tao,Jong Bin Park,Hee Jin Shim,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.05

Bacillus thuringiensis 1-3 (Bt 1-3) which was isolated from a Korean soil sample showed high insecticidal activity against Aedes aegypti as well as Plutella xylostella. The isolate was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins with a molecular weight of 130 kDa. PCR analysis with cry gene specific primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A gene, differing from spp. aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D. We modified the plasmid capture system (PCS) to clone plasmid from Bt 1-3 through in vitro transposition. Fifty-three clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified according to similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its 20 putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family.

Characterization of new B. thuringiensis plasmids and construction of E.coli-Bt shutter vectors using a modified plasmid capture system

Qin Liu,Jong Yul Roh,Yong Wang,Jae Young Choi,Xueying Tao,Hee Jin Shim,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2009 한국응용곤충학회 학술대회논문집 Vol.2009 No.10

Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles from type strain and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. To clone its plasmids and construct E.coli-Bt shuttle vector, we constructed the plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy). Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified in four groups showing similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily clone Bt plasmids and construct novel shuttle vectors.

Generation of Autographa californica multicapsid nucleopolyhedrovirus gene knock-out mutants using plasmid capture system

Xueying Tao,Jong Yul Roh,Jae Young Choi,Yong Wang,Qin Liu,Hee Jin Shim,Yeon Ho Je 한국응용곤충학회 2009 한국응용곤충학회 학술대회논문집 Vol.2009 No.10

The baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), a large circular double-stranded DNA virus whose genome encodes at least 155 open reading frames (ORFs), is highly pathogenic to a number of lepidopteran insects and widely used to transduce various cells for exogenous gene expression. Although many genes of AcMNPV have been identified, the genome-wide study related to viral replication has not been well announced. In this study, to elucidate DNA replication cascade of AcMNPV, we firstly developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, ORF knock-out mutants were generated by random insertion into bAc-MK genome. These mutants will be suffered DNA microarray to elucidate AcMNPV replication cascade.

Cloning of B. thuringiensis Plasmids using a Modified Plasmid Capture System

Qin Liu,Jong Yul Roh,Yong Wang,Jae Young Choi,Hee Jin Shim,Hong Guang Xu,Xueying Tao,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2009 한국응용곤충학회 학술대회논문집 Vol.2009 No.05

Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. This strain exhibited dual insecticidal activity against Aedes aegypti as well as Plutella xylostella. Recently, we improved the donor-s of plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy), to construct E.coli-Bt shuttle vector. Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their range of sizes were approximately 10 kb. Based on the sequence analysis, they were classified in 4 groups showing similarity with 4 known plasmids, pGI1, pGI2, pGI3 and pBMB175, respectively. One of pGI3-like clones was fully sequenced and its open reading frames were analyzed. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily construct novel shuttle vectors with any Bt plasmids and this combined procedure can introduce foreign gengs into various circular DNA molecular.

Molecular characterization of Bacillus thuringiensis serovar mogi (flagellar serotype 3a3b3d), a novel serogroup with mosquitocidal activity

Qin Liu,Jong Yul Roh,Yong Wang,Jae Young Choi,Xueying Tao,Jong Bin Park,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.10

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.