• The Plant Pathology Journal
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• v.34 no.1
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• pp.11-22
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• 2018

Type VI secretion system (T6SS) has been discovered in a variety of gram-negative bacteria as a versatile weapon to stimulate the killing of eukaryotic cells or prokaryotic competitors. Type VI secretion effectors (T6SEs) are well known as key virulence factors for important pathogenic bacteria. In many Burkholderia species, T6SS has evolved as the most complicated secretion pathway with distinguished types to translocate diverse T6SEs, suggesting their essential roles in this genus. Here we attempted to detect and characterize T6SSs and potential T6SEs in target genomes of plant-associated and environmental Burkholderia species based on computational analyses. In total, 66 potential functional T6SS clusters were found in 30 target Burkholderia bacterial genomes, of which 33% possess three or four clusters. The core proteins in each cluster were specified and phylogenetic trees of three components (i.e., TssC, TssD, TssL) were constructed to elucidate the relationship among the identified T6SS clusters. Next, we identified 322 potential T6SEs in the target genomes based on homology searches and explored the important domains conserved in effector candidates. In addition, using the screening approach based on the profile hidden Markov model (pHMM) of T6SEs that possess markers for type VI effectors (MIX motif) (MIX T6SEs), 57 revealed proteins that were not included in training datasets were recognized as novel MIX T6SE candidates from the Burkholderia species. This approach could be useful to identify potential T6SEs from other bacterial genomes.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.69.1-69
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• 2003

RSH (relA/spoT homolog) has been known to determine the level of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effector nucleotide of the prokaryotic stringent response and also play a role in antibiotic production and differentiation in Streptomyces species but not a little in eukaryotic organism, especially in plant. Salicylic acid (SA), a critical signal molecule of establishing systemic acquired resistance (SAR), could induce SAR in Pepper (Capcicum annuum) against Phytophthora capsici. And the extent of SAR induction was in proportion to the dosage of SA (or BTH). Suppression subtractive hybridization (SSH), a PCR-based method for cDNA subtraction, was carried out between SA-treated and non-SA-treated pepper leaves to isolate genes which may be responsible for defense signaling against pathogens. Early upregulated gene was selected from reverse northern and kinetics of SSH-genes transcripts in SA-treated pepper leaves upon SA treatment. Full-length cDNA of the gene (PepRSH； Pepper RelA / SpoT homolog) had an open reading frame (ORF) of 2166 bp encoding a protein of 722 amino acids and a significant homology with (p)ppGpp phosphohydrolase or synthetase. Genomic DNA gel blot analysis showed that pepper genome has at least single copy of PepRSH. PepRSH transcripts was very low in untreated pepper leaves but strongly induced by SA and methyljasmonic acid (MeJA), indicating that PepRSH may share common SA and MeJA-mediated signal transduction pathway Functional analysis in E. coli showed PepRSH confers phenotypes associated with (p)ppGpp synthesis through a complementation using active site mutagenesis.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.65-72
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• 2004

Instability of some eukaryotic sequence propagated in prokaryotic hosts is a frequently observed phenomenon. It is well documented that long inverted repeats, AT-rich sequences with structures like Z-DNA are extremely unstable in E. coli. These sequences may either be under-represented or even lost when cloned in E. coli. When we analyzed the polymorphic pattern for several tandom repeat (TR) in human SCKI gene, we found some TR regions were frequently deleted from plasmids and had difficult problem for their sequencing. These regions may result in non-clonability of the DNA sequence. Here we have cloned two difficult TR regions under low temperature and made two library for DNA sequencing using a nebulizer or sonicator. This study will help to determine the unstable genomic elements in complex mammalian genome.

• Journal of Life Science
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• v.25 no.9
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• pp.1007-1013
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• 2015

A COG (Cluster of Orthologous Groups of proteins) algorithm was applied to detect conserved genes in 711 prokaryotes. Only COG0080 (ribosomal protein L11) was common among all the 711 prokaryotes analyzed and 58 COGs were common in more than 700 prokaryotes. Nine COGs among 58, including COG0197 (endonuclease III) and COG0088 (ribosomal protein L4), were conserved in a form of one gene per one organism. COG0008 represented 1356 genes in 709 of the prokaryotes and this was the highest number of genes among 58 COGs. Twenty-two COGs were conserved in more than 708 prokaryotes. Of these, two were transcription related, four were tRNA synthetases, eight were large ribosomal subunits, seven were small ribosomal subunits, and one was translation elongation factor. Among 58 conserved COGs in more than 700 prokaryotes, 50 (86.2%) were translation related, and four (6.9%) were transcription related, pointing to the importance of protein-synthesis in prokaryotes. Among these 58 COGs, the most conserved COG was COG0060 (isoleucyl tRNA synthetase), and the least conserved was COG0143 (methionyl tRNA synthetase). Archaea and eubacteria were discriminated in the genomic analysis by the average distance and variation in distance of common COGs. The identification of these conserved genes could be useful in basic and applied research, such as antibiotic development and cancer therapeutics.

• Korean Journal of Plant Tissue Culture
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• v.25 no.6
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• pp.477-485
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• 1998

To underatand in vitro regulation of differentiation, the effects of growth regulators and nitrogen source on metabolism of cell wall polysaccharides in suspension culture of kidney bean (Phaseolus vulgaris L.) were investigated. The suspension cells (cell clusters) were directly induced from the epicotyl segments of the seedlings, which were cultivated in MS medium supplemented with 1.0mg/L of 2,4-D and 0.5 mg/L of kinetin. When compared with cell wall sugar contents of the epicotyl segments, the cellulose content of the suspension-cultured cells decreased; while the pectin and hemicellulose content increased; suggesting increases of rhamnogalacturonan I and arabinogalactan IIduring the dedifferentiation, respectively, The effects of growth regulators(2,4-D, 1.0mg/L and kinetin, 0.5mg/L) and nitrogen source (potasium nitrate, 19.0mg/L and ammonium nitrate, 16.5 g/L) in the medium on the proliferation and the turnover of the cell wall polysaccharides were investigated for 30 days. In the medium with growth regulators and without nitrogen source, the proliferation rate was extremely high (16 folds). Growth regulators and nitrogen source increased the pectin content. Analysis of neutral sugar composition of pectin fraction showed that nitrogen source enhanced rhamnose level remarkably, suggesting that rhamnogalacturonan I was the one most likely synthesized. In hemicellulose fraction, growth regulators reduced arabinose level, suggesting that arabinogalactan II was degraded. And nitrogen source reduced galactose level, suggesting that xyloglucan was also degraded.

• Korean Journal of Microbiology
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• v.39 no.4
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• pp.207-215
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• 2003

Gene structure prediction, which is to predict protein coding regions in a given nucleotide sequence, is the most important process in annotating genes and greatly affects gene analysis and genome annotation. As eukaryotic genes have more complicated stuructures in DNA sequences than those of prokaryotic genes, analysis programs for eukaryotic gene structure prediction have more diverse and more complicated computational models. We have developed EGSP, a eukaryotic gene structure program, using duration hidden markov model. The program consists of two major processes, one of which is a training process to produce parameter values from training data sets and the other of which is to predict protein coding regions based on the parameter values. The program predicts multiple genes rather than a single gene from a DNA sequence. A few computational models were implemented to detect signal pattern and their scanning efficiency was tested. Prediction performance was calculated and was compared with those of a few commonly used programs, GenScan, GeneID and Morgan based on a few criteria. The results show that the program can be practically used as a stand-alone program and a module in a system. For gene prediction of eukaryotic microbial genomes, training and prediction analysis was done with Saccharomyces chromosomes and the result shows the program is currently practically applicable to real eukaryotic microbial genomes.

• Bulletin of the Korean Chemical Society
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• v.34 no.8
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• pp.2419-2424
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• 2013

Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In this study, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGpp-binding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensional gel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). The binding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA with ppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as $5.2{\pm}2.0{\mu}M$, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression of yjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgA deletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the over-expression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell division under stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, that are expected to be expressed but there is no experimental report on their functions. The approach utilized in this study may serve as an effective mean to probe the functions of hypothetical proteins.

• Journal of Life Science
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• v.32 no.3
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• pp.249-255
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• 2022

Metabolism is essential for survival and reproduction, and there is a metabolic pathways entry in the clusters of orthologous groups of proteins (COGs) database, updated in 2020. In this study, the metabolic pathways of 1309 prokaryotes were analyzed using COGs. There were 822 COGs associated with 63 metabolic pathways, and the mean for each taxon was between 200.50 (mollicutes) and 527.07 (cyanobacteria) COGs. The metabolic pathway composition ratio (MPCR) was defined as the number of COGs present in one genome in relation to the total number of COGs constituting each metabolic pathway, and the number of pathways with 100% MPCR ranged from 0 to 26 in each prokaryote. Among 1309 species, the 100% MPCR pathways included murein biosynthesis associated with cell wall synthesis (922 species); glycine cleavage (918); and ribosomal 30S subunit synthesis (903). The metabolic pathways with 0% MPCR were those involving photosystem I (1263 species); archaea/vacuolar-type ATP synthase (1028); and Na⁺-translocation NADH dehydrogenase (976). Depending on the prokaryote, three to 49 metabolic pathways could not be performed at all. The sequence of most highly conserved metabolic pathways was ribosome 30S subunit synthesis (96.1% of 1309 species); murein biosynthesis (86.8%); arginine biosynthesis (80.4%); serine biosynthesis (80.3%); and aminoacyl-tRNA synthesis (82.2%). Protein and cell wall synthesis have been shown to be important metabolic pathways in prokaryotes, and the results of this study of COGs related to such pathways can be utilized in, for example, the development of antibiotics and artificial cells.

• Journal of Life Science
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• v.32 no.12
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• pp.956-961
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• 2022

Plant Chloroplast have several advantages as an expression platform of biopharmaceuticals over conventional expression platforms such as mammalian cells, yeast and bacteria. First, plants do not serve as a host for mammalian infectious virus and have endotoxin like bacteria which can cause anaphylactic shock. In addition, high copy number of chloroplast genome allows for chloroplast transformants to reach the high level of expression of heterologous genes. Moreover, the integration of transgenes into specific region of chloroplast genomes makes chloroplast transformants unaffected by positional effect which can be frequently observed from nuclear transformants, resulting in loss of transgene expressions. Antimicrobial peptides (AMPs) are a kind of innate immunity which is found from bacteria to humans. Unlike conventional antibiotics, very less dosage of AMPs can have catastrophic effect on bacterial survival. Further, the repeated use of AMPs does not trigger the development of bacterial resistance. Moricin, one of the AMPs, was isolated from Bombyx mori, a silkworm moth. The C-terminal of moricin consists largely of basic amino acids, and the N-terminal has an α-helix structure. Moricin was chosen and expressed in a SUMO/SUMOase without leaving any unwanted amino acids which could potentially affect the anti-bacterial activity of the moricin. The transformation vector used in this study has already been created in this lab for the expression in both prokaryotic systems such as E. coli and chloroplast. The expressed moricin was purified using Ni columns and SUMOase, and the antibacterial activity of the purified moricin was confirmed using an agar diffusion assay.