• Journal of Microbiology and Biotechnology
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• 제30권3호
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• pp.404-416
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• 2020

Bacteria that are resistant to high temperatures and alkaline environments are essential for the biological repair of damaged concrete. Alkaliphilic and halotolerant Bacillus sp. AK13 was isolated from the rhizosphere of Miscanthus sacchariflorus. Unlike other tested Bacillus species, the AK13 strain grows at pH 13 and withstands 11% (w/v) NaCl. Growth of the AK13 strain at elevated pH without urea promoted calcium carbonate (CaCO₃) formation. Irregular vaterite-like CaCO₃ minerals that were tightly attached to cells were observed using field-emission scanning electron microscopy. Energy-dispersive X-ray spectrometry, confocal laser scanning microscopy, and X-ray diffraction analyses confirmed the presence of CaCO₃ around the cell. Isotope ration mass spectrometry analysis confirmed that the majority of CO₃^2- ions in the CaCO₃ were produced by cellular respiration rather than being derived from atmospheric carbon dioxide. The minerals produced from calcium acetate-added growth medium formed smaller crystals than those formed in calcium lactate-added medium. Strain AK13 appears to heal cracks on mortar specimens when applied as a pelletized spore powder. Alkaliphilic Bacillus sp. AK13 is a promising candidate for self-healing agents in concrete.

• Journal of Microbiology and Biotechnology
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• 제23권8호
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• pp.1060-1069
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• 2013

Genome organization near cyclomaltodextrinases (CDases) was analyzed and compared for four different hyperthermophilic archaea: Thermococcus, Pyrococcus, Staphylothermus, and Thermofilum. A gene (CL1_0884) encoding a putative CDase from Thermococcus sp. CL1 (tccd) was cloned and expressed in Escherichia coli. TcCD was confirmed to be highly thermostable, with optimal activity at $85^{\circ}C$. The melting temperature of TcCD was determined to be $93^{\circ}C$ by both differential scanning calorimetry and differential scanning fluorimetry. A size-exclusion chromatography experiment showed that TcCD exists as a monomer. TcCD preferentially hydrolyzed ${\alpha}$-cyclodextrin (${\alpha}$-CD), and at the initial stage catalyzed a ring-opening reaction by cleaving one ${\alpha}$-1,4-glycosidic linkage of the CD ring to produce the corresponding single maltooligosaccharide. Furthermore, TcCD could hydrolyze branched CDs (G1-${\alpha}$-CD, G1-${\beta}$-CD, and G2-${\beta}$-CD) to yield significant amounts (45%, 40%, and 46%) of isomaltooligosaccharides (panose and $6^2$-${\alpha}$-maltosylmaltose) in addition to glucose and maltose. This enzyme is one of the most thermostable maltogenic amylases reported, and might be of potential value in the production of isomaltooligosaccharides in the food industry.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.217-228
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• 2009

Mobile genetic segments, or transposons, are also referred to as jumping genes as they can shift from one position in the genome to another, thus inducing a chromosomal mutation. According to the target site-specificity of the transposon during a transposition event, the result is either the insertion of a gene of interest at a specific chromosomal site, or the creation of knockout mutants. The former situation includes the integration of conjugative transposons via site-specific recombination, several transposons preferring a target site of a conserved AT-rich sequence, and Tn7 being site-specifically inserted at attTn7, the downstream of the essential glmS gene. The latter situation is exploited for random mutagenesis in many prokaryotes, including IS (insertion sequence) elements, mariner, Mu, Tn3 derivatives (Tn4430 and Tn917), Tn5, modified Tn7, Tn10, Tn552, and Ty1, enabling a variety of genetic manipulations. Randomly inserted transposons have been previously employed for a variety of applications such as genetic footprinting, gene transcriptional and translational fusion, signature-tagged mutagenesis (STM), DNA or cDNA sequencing, transposon site hybridization (TraSH), and scanning linker mutagenesis (SLM). Therefore, transposon-mediated genetic engineering is a valuable discipline for the study of bacterial physiology and pathogenesis in living hosts.

• 약학회지
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• 제50권2호
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• pp.93-98
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• 2006

Human foamy virus (HFV) integrase mediates integration of viral c-DNA into cellular DNA. In this process, HFV prointegration complex (PIC) in which integrase is a key component moves to nuclei of the infected cells and leads to integration of viral DNA to the cellular genome, which is essential in viral life cycle. In general nuclear localization signals (NLS) have been suggested to be involved in localizing retroviral PIC to nuclei, but the mechanisms for nuclear localization of the HFV PIC remains unclear. To functionally identify the NLS of HFV integrase, various subdomains of the protein were expressed as GFP fusions and their subcellular locations were analyzed with confocal laser scanning microscopy. Wild type HFV integrase was karyophilic by targeting the fusion protein to nuclei of the COS-1 and 293T cells. Our results showed that strong NLS of HFV integrase was mapped to the C-terminal regions. In addition the karyophilic properties of N-terminal and central regions are not individually strong enough to direct localization of the fusion proteins to nuclei, but their cooperative activity for nuclear import was confirmed.

• ALGAE
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• 제33권3호
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• pp.269-278
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• 2018

Ocean warming can have significant negative impacts on population genetic diversity, local endemism and geographical distribution of a wide range of marine organisms. Thus, the identification of conservation units with high risk of extinction becomes an imperative task to assess, monitor, and manage marine biodiversity for policy-makers. Here, we surveyed population structure and genetic variation of the red seaweed Gracilaria vermiculophylla along the coast of China using genome-based amplified fragment length polymorphism (AFLP) scanning. Regardless of analysis methods used, AFLP consistently revealed a south to north genetic isolation. Populations at the southern coast of China showed unique genetic variation and much greater allelic richness, heterozygosity, and average genetic diversity than the northern. In particular, we identified a geographical barrier that may hinder genetic exchange between the two lineages. Consequently, the characterized genetic lineage at the southern coast of China likely resulted from the interplay of post-glacial persistence of ancestral diversity, geographical isolation and local adaptation. In particular, the southern populations are indispensable components to explore evolutionary genetics and historical biogeography of G. vermiculophylla in the northwestern Pacific, and the unique diversity also has important conservation value in terms of projected climate warming.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.16-24
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• 2021

Hepatitis B virus (HBV) genome P-encoded protein HBV DNA polymerase (Pol) has long been known as a reverse transcriptase during HBV replication. In this study, we investigated the impact of HBV Pol on host cellular processes, mainly apoptosis, and the underlying mechanisms. We showed a marked reduction in apoptotic rates in the HBV Pol-expressed HepG2 cells compared to controls. Moreover, a series of assays, i.e., yeast two-hybrid, GST pull-down, co-immunoprecipitation, and confocal laser scanning microscopy, identified the host factor eEF1A2 to be associated with HBV Pol. Furthermore, knockdown of eEF1A2 gene by siRNA abrogated the HBV Pol-mediated anti-apoptotic effect with apoptosis induced by endoplasmatic reticulum (ER) stress-inducer thapsigargin (TG), thus suggesting that the host factor eEF1A2 is essential for HBV Pol's anti-apoptosis properties. Our findings have revealed a novel role for HBV Pol in its modulation of apoptosis through integrating with eEF1A2.

• Journal of Plant Biotechnology
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• 제37권1호
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• pp.77-83
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• 2010

국립원예특작과학원에서 분양받은 토마토 18계통을 공시하여 재분화가 잘되는 적정 품종을 탐색한 결과, 계통번호 2001-58에서의 재분화율이 93%로 양호하였다. 또한 식물체로의 재분화 과정에서 보여 지는 갈변현상과 phenolic compound에 의한 식물조직의 괴사현상을 막기 위하여 항산화제인 ascorbic acid와 cystein을 단용 또는 혼용으로 첨가한 후 토마토 재분화에 미치는 영향을 살펴 본 결과, ascorbic acid $200{\sim}300\;{\mu}M/L$ 처리구에서 줄기형성율 및 생체중이 증가되는 현상을 관찰할 수 있었다. 토마토 엽록체 형질전환체 선발을 위해 spectinomycin의 적정 농도를 살펴본 결과, 재분화배지에 spectinomycin 20~25 mg/L 농도가 첨가되어진 처리구에서 재분화가 거의 이루어지지 않았다. 토마토 엽록체 형질전환을 위해 토마토 엽록체 게놈 일부를 분리하여 염기서열을 분석하여 담배와 비교 분석한 결과, homology가 매우 높음을 알 수 있었다. Homologous recombination에 의한 엽록체 형질전환이 되기 위해서 분리한 토마토 엽록체 게놈 일부를 border sequence로 이용하였고, transient assay를 위해 GFP 유전자가 포함된 토마토 엽록체 형질전환용 운반체 pKRT22-AG를 제작하였다. Bombardment을 한 후 원형질체를 나출하여 공초점 현미경하에서 관찰한 결과 엽록체 내에서만 GFP가 발현됨을 알 수 있었으며, DNA 농도 $1\;{\mu}g$, $0.6\;{\mu}m$ gold particle 1 mg, target distance 9 cm 조건이 가장 좋았다.

• 미생물학회지
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• 제39권4호
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• pp.207-215
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• 2003

주어진 염기서열에서 단백질로 코딩되는 영역을 예측하는 유전자 구조 예측은 유전자 annotation의 가장 핵심적인 부분으로 유전자 분석 및 유전체 프로젝트 전체에 큰 영향을 준다. 진핵생물의 유전자가 원핵생물의 유전자에 비해 더 복잡한 구조를 가지기 때문에 진핵생물의 유전자 구조 예측 모델 역시 원핵생물에 비해 다양하고 복잡한 모델로 구성되어 있다. 본 연구팀은 duration hidden markov model을 기본형태로 하여 진핵생물의 유전자 구조 예측 프로그램인 EGSP를 개발하였다. 이 프로그램은 각 생명체의 유전자 구조 예측에 필요한 파라메터를 생성하는 학습기능과, 이를 기반으로 핵산 서열을 입력으로 해서 단백질을 코딩하는 부위를 예측하여 출력하는 기능으로 구성되며, 최근의 프로그램들의 추세대로 복수 개 유전자 예측의 기능을 갖추고 있다. EGSP의 학습과 예측에 사용되는 각 파라메터의 전체 성능에 대한 효과 분석 등을 위해 여러 개 signal에 대한 개별 모델이 주는 효과 등을 분석하였다. 진핵생물의 유전자 구조 예측에 가장 많이 연구되는 human dataset을 이용하여 현재 개발된 유전자 구조 예측 프로그램인 GenScan과 GeneID, Morgan 등 보편적으로 사용되는 프로그램들과의 성능을 여러 가지 기준에서 비교한 결과, 본 프로그램이 실용성 있는 수준을 보여주는 것을 확인하였다. 그리고 진핵 미생물인 Saccharomyces cerevisiae로 성능을 테스트한 결과 만족할 만한 수준의 성능을 나타내는 것을 알 수 있었다.

• Molecules and Cells
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• 제23권3호
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• pp.323-330
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• 2007

The mature wheat embryo is arguably one of the best explants for genetic transformation because of its unlimited availability and lack of growth season restriction. However, an efficient regeneration system using mature wheat embryos (Triticum aestivum L.) is still not available. To identify genes related to the tissue culture response (TCR) of wheat, QTLs for callus induction from mature embryos and callus regeneration were mapped using an RIL population derived from the cross of 'Wangshuibai' with 'Nanda2419', which has a good TCR. By whole genome scanning we identified five, four and four chromosome regions conditioning, respectively, percent embryos forming a callus (PEFC), percent calli regenerating plantlets (PCRP), and number of plantlets per regenerating callus (NPRC). The major QTLs QPefc.nau-2A and QPcrp.nau-2A were mapped to the long arm of chromosome 2A, explaining up to 22.8% and 17.6% of the respective phenotypic variance. Moreover, two major QTLs for NPRC were detected on chromosomes 2D and 5D; these together explained 51.6% of the phenotypic variance. We found that chromosomes 2A, 2D, 5A, 5B and 5D were associated via different intervals with at least two of the three TCR indexes used. Based on this study and other reports, the TCRs of different explant types of wheat may be under the control of shared or tightly linked genes, while different genes or gene combinations may govern the stages from callus induction to plantlet regeneration. The importance of group 2 and 5 chromosomes in controlling the TCRs of Triticeae crops and the likely conservation of the corresponding genes in cereals are discussed.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2005년도 2005 Annual Meeting & International Symposium
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• pp.154-164
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• 2005

Saccharomyces cerevisiae KNU5377 is a thermotolerant strain, which can ferment ethanol from wasted papers and starch at 40$^{\circ}C$ with the almost same rate as at 30$^{\circ}C$. This strain showed alcohol fermentation ability to convert wasted papers 200 g (w/v) to ethanol 8.4% (v/v) at 40$^{\circ}C$, meaning that 8.4% ethanol is acceptable enough to ferment in the industrial economy. As well, all kinds of starch that are using in the industry were converted into ethanol at 40$^{\circ}C$ with the almost same rate as at 30$^{\circ}C$. Hyperthermic cell killing kinetics and differential scanning calorimetry (DSC) revealed that exponentially growing cells of this yeast strain KNU5377 were more thermotolerant than those of S. cerevisiae ATCC24858 used as a control. This intrinsic thermotolernace did not result from the stability of entire cellular components but possibly from that of a particular target. Heat shock induced similar results in whole cell DSC profiles of both strains and the accumulation of trehalose in the cells of both strains, but the trehalose contents in the strain KNU5377 were 2.6 fold higher than that in the control strain. On the contrary to the trehalose level, the neutral trehalase activity in the KNU5377 cells was not changed after the heat shock. This result made a conclusion that though the trehalose may stabilize cellular components, the surplus of trehalose in KNU5377 strain was not essential for stabilization of whole cellular components. A constitutively thermotolerant yeast, S. cerevisiae KNU5377, was compared with a relatively thermosensitive control, S. cerevisiae ATCC24858, by assaying the fluidity and proton ATPase on the plasma membrane. Anisotropic values (r) of both strains were slightly increased by elevating the incubation temperatures from 25$^{\circ}C$ to 37$^{\circ}C$ when they were aerobically cultured for 12 hours in the YPD media, implying the membrane fluidity was decreased. While the temperature was elevated up to 40$^{\circ}C$, the fluidity was not changed in the KNU5377 cell, but rather increased in the control. This result implies that the plasma membrane of the KNU5377 cell can be characterized into the more stabilized state than control. Besides, heat shock decreased the fluidity in the control strain, but not in the KNU5377 strain. This means also there's a stabilization of the plasma membrane in the KNU5377 cell. Furthermore, the proton ATPase assay indicated the KNU5377 cell kept a relatively more stabilized glucose metabolism at high temperature than the control cell. Therefore, the results were concluded that the stabilization of plasma membrane and growth at high temperature for the KNU5377 cell. Genome wide transcription analysis showed that the heat shock responses were very complex and combinatory in the KNU5377 cell. Induced by the heat shock, a number of genes were related with the ubiquitin mediated proteolysis, metallothionein (prevent ROS production from copper), hsp27 (88-fold induced remarkably, preventing the protein aggregation and denaturation), oxidative stress response (to remove the hydrogen peroxide), and etc.