• Journal of Microbiology and Biotechnology
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• 제25권4호
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• pp.511-520
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• 2015

Triclosan, the widely used biocide, specifically targets enoyl-acyl carrier protein reductase (ENR) in the bacterial fatty acid synthesis system. Although the fish pathogen Aeromonas salmonicida subsp. salmonicida exhibits triclosan resistance, the nature of this resistance has not been elucidated. Here, we aimed to characterize the triclosan resistance of A. salmonicida subsp. salmonicida causing furunculosis. The fosmid library of triclosan-resistant A. salmonicida subsp. salmonicida was constructed to select a fosmid clone showing triclosan resistance. With the fosmid clone showing triclosan resistance, a subsequent secondary library search resulted in the selection of subclone pTSR-1. DNA sequence analysis of pTSR-1 revealed the presence of a chromosomal-borne fabV-encoding ENR homolog. The ENR of A. salmonicida (FabVas) exhibited significant homology with previously known FabV, including the catalytic domain YX₍₈₎K. fabVas introduction into E. coli dramatically increased its resistance to triclosan. Heterologous expression of FabVas might functionally replace the triclosan-sensitive FabI in vivo to confer E. coli with triclosan resistance. A genome-wide search for fabVas homologs revealed the presence of an additional fabV gene (fabVas2) paralog in A. salmonicida strains and the fabVas orthologs from other gram-negative fish pathogens. Both of the potential FabV ENRs expressed similarly with or without triclosan supplement. This is the first report about the presence of two potential FabV ENRs in a single pathogenic bacterium. Our result suggests that triclosan-resistant ENRs are widely distributed in various bacteria in nature, and the wide use of this biocide can spread these triclosan-tolerant ENRs among fish pathogens and other pathogenic bacteria.

• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.131-137
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• 2001

Previous work has identified that only the catabolite responsive element A (creA; previously called cre-2) out of two potential cre sequences (cre-1: nucleotide +160 to +173 and cre-2: +173 to +186), recognized within the coding region of the xylanaseA gene (xynA) of Bacillus stearothermophilus No.236, was actually, was actually involved in the carbon catabolite repression(CCR) of xynA expression in B. subtilis. However, the level of CCR of xynA expression in the original B.stearothermophilus No.236 strain (70-fold repression). Therefore, to search for an additional cre element in the promoter region, the upstream region of the xynA gene was subcloned by chromosome walking, and as a result, another potential cre element (nucleotide -124∼-137; designated creB) was recognized in this region. The cre-like sequence revealed a high homology to the cre consensus sequence. The xylanase activity of B. subtilis MW15 bearing pWPBR14 (containing creA and creB) cultured in a medium containing xylose as the sole carbon source was about 7.7 times higher than that observed for the same culture containing glucose. B. subtilis MW15 bearing pWPBR23 (containing only creA) produced an activity about 2.4 times higher. This pattern of CCR was confirmed using derivatives of xynA::aprA fusion plasmids. Furthermore, a measurement of the amounts of the xynA transcript showed a similar pattern as that for the production of xylanase. In addition, the synthesis of xylanase in B. subtilis QB7115 [a catabolite control protein A (ccpA) mutant strain] carrying pWPBR14 was almost completely relieved from glucose repression. Together, these results lead to a conclusion that the CCR of the expression of the xynA gene is mediated by CcpA binding at creA and creB sites in B. subtilis.

• Journal of Ginseng Research
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• 제34권4호
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• pp.296-303
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• 2010

A peroxiredoxin cDNA (PgPrx) was isolated and characterized from the leaves of Panax ginseng. The cDNA is 716 nucleotides long and has an open reading frame of 489 base pairs with a deduced amino acid sequence of 162 residues. The calculated molecular mass of the mature protein is approximately 17.4 kDa with a predicted isoelectric point of 5.37. A GenBank BlastX search revealed that the deduced amino acid sequence of PgPrx shares a high degree homology with type II peroxiredoxin (Prx) proteins in other plants. The PgPrx gene was highly expressed in leaves, and expressed at a low level in the stem. To analyze the gene expression of PgPrx in response to various abiotic stresses, we utilized real-time quantitative RT-PCR. Our results reveal that PgPrx expression is induced by ultraviolet irradiation, low temperature, and salt. The induction of PgPrx in response to abiotic stimuli suggests that ginseng Prx may function to protect the host against environmental stresses.

• 한국패류학회지
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• 제29권3호
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• pp.181-187
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• 2013

In order to investigate environmental stress inducible genes in abalone, we analyzed differentially expressed transcripts from a Pacific abalone, Haliotis discus hannai, after exposure to heat-, cold- or hyposalinity-shock by suppression subtractive hybridization (SSH) method. 1,074 unique sequences from SSH libraries were composed to 115 clusters and 986 singletons, the overall redundancy of the library was 16.3%. From the BLAST search, of the 1,316 ESTs, 998 ESTs (75.8%) were identified as known genes, but 318 clones (24.2%) did not match to any previously described genes. From the comparison results of ESTs pattern of three SSH cDNA libraries, the most abundant EST was different in each SSH library: small heat shock protein p26 (sHSP26) in heat-shock, trypsinogen 2 in cold-shock, and actin in hyposalinity SSH cDNA library. Based on sequence similarities, several response-to-stress genes such as heat shock proteins (HSPs) were identified commonly from the abalone SSH libraries. HSP70 gene was induced by environmental stress regardless of temperature-shock or salinity-stress, while the increase of sHSP26 mRNA expression was not detected in cold-shock but in heat-shock condition. These results suggest that the suppression subtractive hybridization method is an efficient way to isolate differentially expressed gene from the invertebrate environmental stress-response transcriptome.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2000년도 국제심포지움 및 추계학술대회
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• pp.31-41
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• 2000

The major issue in the post genome sequencing era is determination of gene expression patterns in variety of biological systems. A microarray system is a powerful technology for analyzing the expression profile of thousands of genes at one experiment. In this study, we constructed cDNA microarray which carries 2,304 cDNAS derived from oligo-capped mouse cDNA library. Using this hand-made microarray we determined gene expression in various biological systems. To determine tissue specific genes, we compared Nine genes were highly-expressed in adult mouse brain compared to kidney, liver, and skeletal muscle. Tissue distribution analysis using DNA microarray extracted 9 genes that were predominantly expressed in the brain. A database search showed that five of the 9 genes, MBP, SC1, HiAT3, S100 protein-beta, and SNAP25, were previously known to be expressed at high level in the brain and in the nervous system. One gene was highly sequence similar to rat S-Rex-s/human NSP-C, suggesting that the gene is a mouse homologue. The remaining three genes did not match to known genes in the GenBank/EMBL database, indicating that these are novel genes highly-expressed in the brain. Our DNA microarray was also used to detect differentiation specific genes, hormone dependent genes, and transcription-factor-induced genes. We conclude that DNA microarray is an excellent tool for identifying differentially expressed genes.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2003년도 춘계 학술발표대회
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• pp.125-125
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• 2003

To assist genetic study of the root development in Panax ginseng, which is one of the most important medicinal plant, expressed sequence tags (EST) analysis was carried out. We constructed a cDNA library using the 14-year ginseng root. Partial sequences were obtained from 2,975 clone. The ESTs could be clustered into 1,991 (70.2%) non-redundant groups. Similarity search of the non-redundant ESTs against public non-redundant databases of both protein and DNA indicated that 1,553 groups show similarity to genes of blown function. These ESTs clones were divided into sixteen categories depending upon gene function. The most abundant transcripts were ribonuclease 1 (67) and ribonuclease 2 (65). Our extensive EST analysis of genes expressed in 14-year ginseng root not only contributes to the understanding of the dynamics of genome expression patterns in root organ but also adds data to the reperoire of all genomic genes.

• 미생물학회지
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• 제51권3호
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• pp.187-193
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• 2015

Candida (Pseudozyma로도 알려짐) antarctica lipase B(CAL-B)는 학문적으로 그리고 산업적으로 많이 활용되고 있다. CAL-B 자체에 대한 연구는 많이 진행되어온 반면, CAL-B 상동체에 관한 연구는 그리 알려진 바가 없다. 본 연구에서는 단백질 유사성 검색을 통해서 CAL-B의 상동체 탐색을 수행하였고, 6종의 단백질 서열을 찾았다. 해당하는 유전자들을 대장균에 대한 코돈 최적화를 수행하였고, 이를 바탕으로 유전자 합성을 진행하였다. 이들 유전자를 대장균 발현용 벡터에 클로닝한 후, 대장균 내에서 단백질 발현을 시도하여 이들 중 4종의 단백질이 성공적으로 발현되었다. 이들 단백질들이 가수분해 효소로서의 활성이 있는지 확인하기 위해서, 4-nitrophenyl acetate와 4-nitrophenyl butyrate를 반응기질로 하여 가수분해 반응성을 확인하였다. 이들 단백질들의 비활성(specific activity)값은 $(1.3-30){\times}10^{-2}{\mu}mol/min/mg$로 측정되었고, 이는 CAL-B의 비활성 수치보다는 다소 낮은 값에 해당하였다. (${\pm}$)-1-phenylethyl acetate의 가수분해 반응에 대한 입체선택성은 이들 상동체 효소들 중에서 Pseudozyma hubeiensis SY62에서 유래된 효소만이 CAL-B의 입체선택성과 유사함이 확인되었다.

• Journal of Microbiology and Biotechnology
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• 제30권3호
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• pp.391-397
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• 2020

In this study, we used a novel α-L-arabinopyranosidase (AbpBs) obtained from ginsenoside-converting Blastococcus saxobsidens that was cloned and expressed in Escherichia coli BL21 (DE3), and then applied it in the biotransformation of ginsenoside Rb₂ into Rd. The gene, termed AbpBs, consisting of 2,406 nucleotides (801 amino acid residues), and with a predicted translated protein molecular mass of 86.4 kDa, was cloned into a pGEX4T-1 vector. A BLAST search using the AbpBs amino acid sequence revealed significant homology with a family 2 glycoside hydrolase (GH2). The over-expressed recombinant AbpBs in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinopyranose moiety attached to the C-20 position of ginsenoside Rb₂ under optimal conditions (pH 7.0 and 40℃). Kinetic parameters for α-L-arabinopyranosidase showed apparent K_m and V_max values of 0.078 ± 0.0002 μM and 1.4 ± 0.1 μmol/min/mg of protein against p-nitrophenyl-α-L-arabinopyranoside. Using a purified AbpBs (1 ㎍/ml), 0.1% of ginsenoside Rb₂ was completely converted to ginsenoside Rd within 1 h. The recombinant AbpBs could be useful for high-yield, rapid, and low-cost preparation of ginsenoside Rd from Rb₂.

• Asian-Australasian Journal of Animal Sciences
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• 제17권3호
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• pp.309-312
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• 2004

We performed exon trapping in order to locate functional genes on chicken chromosomes (GGA) and to identify functional gene sequences from chicken cosmids. Sequence analysis of 100 clones revealed 17 putative exons, five of which were identified with known sequences in a gene database search: thymopoietin beta (TMPO), U5 snRNP-specific 40 kDa protein (HPRP8BP), dihydropyridine receptor alpha 1 subunit (CACNL1A3), cystein string protein (CPS) and C15orf4. We attempted to map the genes to chicken chromosomes by using FISH and linkage analysis. The chromosomal localizations were GGA1 (TMPO), GGA10 (C15orf4), GGA23 (HPRP8BP) and GGA28 (CPS) by FISH and linkage analysis, while that of CACNL1A3 was predicted to be on a microchromosome by FISH but not by linkage analysis. Comparative mapping analyses between chickens and humans for the genes revealed both known and new synteny. The syntenic conservation between GGA1 and human chromosome (HSA) 12q23 (TMPO) and between GGA10 and HSA15q25 (C15orf4), were consistent with a recent publication, while two new syntenies were observed between GGA28 and HSA20q13.3 in CPS and between GGA23 and HSA1p34-35 in HPRP8BP. The information of presently mapped genes can contribute as anchor markers based on functional genes and the construction of a comparative map.

• Journal of Microbiology
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• 제45권5호
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• pp.402-408
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• 2007

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around $50^{\circ}C$. Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of $Ca^{2+},\;Zn^{2+},\;Mg^{2+}$, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.