Cloning, Expression and Characterization of β-agarase Gene from a Marine Bacterium, Pseudoalteromonas sp. JT-6

Xueying Tao(도설영),Wu Jing(오청),Kyoung-Sook Kim(김경숙),Ziniu Yu(유자우),Young-Choon Lee(이영춘) 한국생명과학회 2008 생명과학회지 Vol.18 No.5

해양으로부터 분리한 한천분해효소를 생산하는 미생물을 분리하여 동정한 결과 이 세균은 Pseudoalteromonas sp.로 판명되어 Pseudoalteromonas sp. JT-6로 명명하였다. 또한 이 균주로부터 한천분해효소 유전자를 클로닝하여 염기서열을 결정하였던 바, 이 유전자는 445개의 아미노산을 코드하는 1338 bp의 염기서열로 이루어져 있었다. 이 유전자는 Janthinobacterium sp. SY12와 99%의 높은 아미노산 서열 상동성을 나타내었고 N-말단으로부터 신호서열, 당분해효소 family 16에 속하는 β-agarase 촉매영역 및 탄수화물 결합영역으로 이루어진 도메인 구조를 나타내었다. 대장균에서 생산되어 정제된 재조합 한천분해효소는 40℃와 pH 9.0에서 최대 활성을 나타내었으며, 기질인 한천분해 형태로 볼 때 본 효소는 endo-type의 β-agarase임이 증명되었다. A gene (agaA6) encoding an extracellular agarase from a marine bacterium, Pseudoalteromonas sp. JT-6, was cloned, sequenced and expressed in Escherichia coli. It comprised an open reading frame of 1338 base pairs and encodes a protein of 445 amino acids with a predicted molecular weight of 50,150 daltons. The entire amino acid sequence of this agarase gene showed 99% identity with the agaA gene from Janthinobacterium sp. SY12. It consists of a signal peptide, a glycoside hydrolase family 16 β-agarase domain and a carbohydrate-binding domain. The recombinant agarase was overexpressed and purified to homogeneity. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant enzyme were around 40℃ and 9.0. The enzyme was an endo-type β-agarase and hydrolyzed agarose to several oligosaccharides.

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.

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.

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.

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.

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.

Cloning and Molecular Characterization of a Novel Rolling-Circle Replicating Plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1

Ming Shun Li,Jong Yul Roh,Xueying Tao,Zi Niu Yu,Zi Duo Liu,Qin Liu,Hong Guang Xu,Hee Jin Shim,Yang-Su Kim,Yong Wang,Jae Young Choi,Yeon Ho Je 한국응용곤충학회 2009 한국응용곤충학회 학술대회논문집 Vol.2009 No.05

Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki K1 using PCS which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pK1S-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pK1S-1, ORF2 (MobK1) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8-25.2%) with the Rep protein coded by RCR plasmids, however. The putative double-strand origin of replication (dso) and single-strand origin of replication (sso) of pK1S-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pK1S-1, seven subclones were contructed in the B. t huringiensis ori-negative pHT1K vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (Rep1K), dso and ORF4, exhibited replication ability. These findings identified pK1S-1 as a new RCR group VII plasmid, and determined its replication region.

Development of an Specific Antibodies for the detection of Rice black-streaked dwarf virus using Baculovirus Expression Vectors

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

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus within the family Reoviridae, is the causative agent of maize rough dwarf and rice black-streaked dwarf diseases, both of which can lead to severe yield losses in east Asia. Although molecular approaches such as RT-PCR have potential for detection and diagnosis of this virus infections, their impact on high throughput certification is still limited. Therefore, the development of an antibody-based assay for rapid and effective diagnosis of RBSDV is preferable. In this study, we collected RBSDV from rice with rough dwarf disease and its complete nucleotide sequences of 10 genomic segments encoding 12 non-overlapping ORFs were determined. Among 12 ORFs, ORF1, 2 and 12 showed high level of similarities with the RdRp, major core protein and major outer shell protein, respectively. These ORFs were expressed as polyhedrin fusion protein or full-length soluble protein using baculovirus expression system for the preparation of specific antibody against RBSDV, which could be useful for the detection and diagnosis of this virus.

Synergistic Effect of Beauveria bassiana SFB-205 Culture Broth on the Insecticidal Activity against Lepidopteran Pests

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

Entomopathogenic fungi are widely available as biological control agents for controlling insect pests in agriculture and forestry. The fungal culture broth contains various pathogenesis-related components such as blastospores, mycelium and insecticidal enzymes such as chitinase, Pr1- and Pr2-proteases, which have been reported to play an important role in penetrating insect cuticles. In this study, we tried to evaluate the utility of culture broth from Beauveria bassiana SFB-205 to control lepidopteran pests. High level of insecticidal activity correspond to over 90% of mortality were observed when the culture broth of B. bassiana SFB-205 was inoculated to the Spodoptera litura larvae together with the B. thuringiensis K1. The freeze-dried culture broth showed synergistic effects in insecticidal activity against larvae of S. exigua and S. litura when treated with corresponding baculoviruses, SeNPV and SlNPV. Active ingredient of the B. bassiana SFB-205 culture broth was identified to chitinase, which have truncated form by insertional mutation compared to previously reported chitinases.

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.