• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
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• pp.143-147
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• 2005

The non-pathogenic, non-glycopeptide-producing actinomycete Streptomyces coelicolor carries a cluster of seven genes (vanSRJKHAX) that confers inducible, high-level resistance to vancomycin. The van genes are organised into four transcription units, vanRS, vanJ, vanK and vanHAX, and these transcripts are induced by vancomycin in a vanR-dependent manner. vanHAX are orthologuous to genes found in vancomycin resistant enterococci that encode enzymes predicted to reprogramme peptidoglycan biosynthesis such that cell wall precursors terminate in D-Ala-D-Lac, rather than D-Ala-D-Ala. vanR and vanS encode a two-component signal transduction system that mediates transcriptional induction of the seven van genes. vanJ and vanK are novel genes that have no counterpart in previously characterised vancomycin-resistance clusters from pathogens. VanK is essential for vancomycin resistance in S. coelicolor and it is required for adding Gly branch to stem peptides terminating D-Ala-D-Lac. Because VanK can recognise D-Lac-containing precursors but the constitutively expressed femX enzyme, encoded elsewhere on the chromosome, cannot recognize D-Lac-containing precursors as a substrate, vancomycin-induced expression of VanHAX in a vanK mutant is lethal. Further, femX null mutants are viable in the presence of glycopeptide antibiotics but die in their absence. Bioassay using vanJp-neo fusion reporter system also showed that all identified inducers for van genes expression were glycopeptide antibiotics, but teicoplanin, a membrane-anchored glycopeptide, failed to act as an inducer.

• 대한의생명과학회지
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• 제10권1호
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• pp.35-42
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• 2004

Liver and serum $\beta$-D-mannosidase activities were determined in ethanol intoxicated rats with extrahepatic cholestasis induced by common bile duct ligation (CBD) to manifest the biochemical background of alcohol drinking hazard under the hepatobiliary disease. Liver $\beta$-D-mannosidase activity and its Vmax value in CBD ligated rats with chronic ethanol intoxication were found to be significantly decreased than that in CBD ligation alone. However, the difference of Km value on above hepatic enzyme was not found between the experimental groups. On the other hand, serum $\beta$-D-mannosidase activity in CBD ligated rats with chronic ethanol intoxication was increased more than that in CBD ligation alone. These results indicate that the biosynthesis of the hepatic $\beta$-D-mannosidase decreases and the serum $\beta$-D-mannosidase activity increases in cholestasis combined with chronic ehtanol intoxication, reflecting damage of aggravated hapatocytic membrane. Accordingly, the resulting data supported the fact that alcoholic drinks were enzymologically harmful to the hepatobiliary disease.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.512.1-512
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• 1986

The cdd gene of Bacillus subtilis, encoding the deoxycytidinecytidine deaminase of pyrimidine nucleotide biosynthesis has been cloned into the EcoRl site of pBR322. The recombinant plasmid, pSol, promoted the synthesis of 100-140 fold elevated levels of the enzyme. A comparison of the polypeptides encoded by cdd complementing and non-complementing plasmids in the mini cell showed the gene product to have a molecular mass of approximately 14 kDa. The nucleotide sequence of the gene and 460 base pairs upstream from the coding region was determined. An open-reading frame, encoding a protein with a calculated molecular mass of 14337 Da, was deduced to be the coding region for cdd. However, the enzyme has an apparent molecular mass of 54 kDa as determined by gel filteration, whereas sucrose density gradient centrifugation shows 58 kDa. It means that the enzyme could be forming a tetramer in a physiological state. About 28 amino acids of the N-tetramer in a physiological state. About 28 amino acids of the N-terminal presumably form a signal for membrane translocation and six cystein residues are contained in the structure. S1 nuclease mapping indicated that transcription of cdd is initiated 17 base pairs upstream from the translational start. The structural characterization of the odd gene was performed.

• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1125-1131
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• 2006

A kan'::luxCDABE fusion strain that was both highly bioluminescent and responsive to benzoic acid was constructed by transforming E. coli strain W3110 with the plasmid pUCDK, which was constructed by digesting and removing the 7-kb KpnI fragment from the promoterless luxCDABE plasmid pUCD615. Experiments using buffered media showed that this induction was dependent on the pH of the media, which influences the degree of benzoic acid protonation, and the expression levels seen are likely due to acidification of the cytoplasm by uncoupling of benzoic acid. Consequently, the sensitivity of this strain for benzoic acid was increased by nearly 20-fold when the pH was shifted from 8.0 to 6.5. Benzoic acid derivatives and several phenolics also resulted in significantly increased bioluminescent signals. Although these compounds are known to damage membranes and induce the heat-shock response within E. coli, bacterial strains harboring mutations in the fadR and rpoH genes, which are responsible for fatty acid biosynthesis during membrane stress and induction of the heat-shock response, respectively, showed that these mutations had no effect on the responses observed.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.2028-2036
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• 2017

The Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for scientific research and biotechnological applications. However, omics analyses have revealed that E. coli K-12 and B exhibit notably different genotypic and phenotypic attributes, even though they were derived from the same ancestor. In a previous study, we identified a limited number of proteins from the two strains using two-dimensional gel electrophoresis and tandem mass spectrometry (MS/MS). In this study, an in-depth analysis of the physiological behavior of the E. coli K-12 and B strains at the proteomic level was performed using six-plex isobaric tandem mass tag-based quantitative MS. Additionally, the best lysis buffer for increasing the efficiency of protein extraction was selected from three tested buffers prior to the quantitative proteomic analysis. This study identifies the largest number of proteins in the two E. coli strains reported to date and is the first to show the dynamics of these proteins. Notable differences in proteins associated with key cellular properties, including some metabolic pathways, the biosynthesis and degradation of amino acids, membrane integrity, cellular tolerance, and motility, were found between the two representative strains. Compared with previous studies, these proteomic results provide a more holistic view of the overall state of E. coli cells based on a single proteomic study and reveal significant insights into why the two strains show distinct phenotypes. Additionally, the resulting data provide in-depth information that will help fine-tune processes in the future.

• 생명과학회지
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• 제11권5호
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• pp.415-422
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• 2001

Many secreted proteins have disulfide bonds that are important for their structure and function. Protein disulfide isomerase (PDI, EC 5.3.1.4.), an enzyme that catalyzes the formation and rearrangement of thiol/disulfide exchange reactions, is a resident of the endoplasmic reticulum (ER). The subcellular localization and its function as catalyst of disulfide bond formation in the biosynthesis of secretory and cell membrane proteins suggest that PDI plays a key role in the secretory pathway. We have isolated a cDNA encoding protein disulfide isomerase from Bombyx mori(bPDI). It has been characterized under ER stress conditions (dominantly induced by calcium ionophore A23187, tunicamycin and DTT), which is known to cause an accumulation of unfolded proteins in the ER. Furthermore, It has also been examined for tissue distribution(pronounced at the fat body), hormonal regulation (juvenile hormone, insulin and juvenile +transferrin; however, it is not effected by transferrin alone), and the effect of exogenous bacteria (peak at 16 h after infection) on the bPDI mRNA expression. The results suggest that bPDI is a member of the ER stress protein group, and it may play an important role in exogenous bacterial infection in fat body, and that homones regulate its expression.

• Plant Biotechnology Reports
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• 제3권1호
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• pp.95-101
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• 2009

Tea leaves are major source of catechins—antioxidant flavonoids. Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is one of the important enzymes that catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key ''late'' step in the biosynthesis of catechins. This manuscript reports characterization of DFR from tea (CsDFR) that comprised 1,413 bp full-length cDNA with ORF of 1,044 bp (115-1,158) and encoding a protein of 347 amino acids. Sequence comparison of CsDFR with earlier reported DFR sequences in a database indicated conservation of 69-87% among amino acid residues. In silico analysis revealed CsDFR to be a membrane-localized protein with a domain (between 16 and 218 amino acids) resembling the NAD-dependent epimerase/dehydratase family. The theoretical molecular weight and isoelectric point of the deduced amino sequence of CsDFR were 38.67 kDa and 6.22, respectively. Upon expression of CsDFR in E. coli, recombinant protein was found to be functional and showed specific activity of 42.85 nmol $min^{-1}$ mg $protein^{-1}$. Expression of CsDFR was maximum in younger rather than older leaves. Expression was down-regulated in response to drought stress and abscisic acid, unaffected by gibberellic acid treatment, but up-regulated in response to wounding, with concomitant modulation of catechins content. This is the first report of functionality of recombinant CsDFR and its expression in tea.

• 한국응용곤충학회지
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• 제49권3호
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• pp.241-249
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• 2010

Xenorhabdus 및 Photorhabdus 두 곤충병원세균은 곤충의 아이코사노이드 생합성을 억제하여 면역작용을 저하시키는 인자로 알려지고 있다. 본 연구는 이들 세균의 배양액을 이용하여 배추좀나방(Plutella xylostella)을 방제하는 새로운 생화학농약을 개발하려 추진되었다. 두 세균 배양액의 단독 처리는 배추좀나방의 생존력에 뚜렷한 영향을 주지 않았다. 그러나 비티(Bacillus thuringiensis) 생물농약과 혼합하여 처리할 경우 비티 단독 처리에 비해 배추좀나방 4령충에 대해서 현격하게 높은 병원성 증가효과를 주었다. 배양액의 세균 활성을 조사하기 위해 이 세균 배양액을 고온 멸균 및 $0.2\;{\mu}m$ 여과 멸균 처리하였다. 이렇게 처리된 멸균 배양액은 세균이 생존했던 배양액과 차이 없이 비티 생물농약의 병원력을 상승시켰다. X. nematophila 배양액에서 유래된 세 가지 대사물질도 세균배양액과 유사한 비티 병원력 상승효과를 보였다. 야외 배추좀나방 방제 시험에서도 세균배양액은 비티 생물농약의 방제가를 상승시키는 효과를 나타냈다.

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.789-797
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• 2010

The limited information on differential gene expression in the different serotypes of Actinobacillus pleuropneumoniae has significantly hampered the research on the pathogenic mechanisms of this organism and the development of multivalent vaccines against A. pleuropneumoniae infection. To compare the gene expressions in the A. pleuropneumoniae strains CVCC259 (serotype 1) and CVCC261 (serotype 3), we screened the differentially expressed genes in the two strains by performing representational difference analysis (RDA). Northern blot analyses were used to confirm the results of RDA. We identified 22 differentially expressed genes in the CVCC259 strain and 20 differentially expressed genes in the CVCC261 strain, and these genes were classified into 11 groups: (1) genes encoding APX toxins; (2) genes encoding transferrin-binding protein; (3) genes involved in lipopolysaccharide (LPS) biosynthesis; (4) genes encoding autotransporter adhesin; (5) genes involved in metabolism; (6) genes involved in the ATP-binding cassette (ABC) transporter system; (7) genes encoding molecular chaperones; (8) genes involved in bacterial transcription and nucleic acid metabolism; (9) a gene encoding protease; (10) genes encoding lipoprotein/membrane protein; and (11) genes encoding various hypothetical proteins. This is the first report on the systematic application of RDA for the analysis of differential gene expression in A. pleuropneumoniae serotypes 1 and 3. The determination of these differentially expressed genes will serve as an indicator for future research on the pathogenic mechanisms of A. pleuropneumoniae and the development of a multivalent vaccine against A. pleuropneumoniae infection.

• BMB Reports
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• 제39권5호
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• pp.626-635
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• 2006

Lysophosphatidic acid acyltransferase (LPAAT) is an intrinsic membrane protein that catalyzes the synthesis of phosphatidic acid (PA) from lysophosphatidic acid (LPA). It is well known that LPAAT is involved in lipid biosynthesis, while its role in tumour progression has been of emerging interest in the last few years. To date, seven members of the LPAAT gene family have been found in human. Here we report a novel LPAAT member, designated as LPAAT-theta, which was 2728 base pairs in length and contained an open reading frame (ORF) encoding 434 amino acids. The LPAAT-theta gene consisted of 12 exons and 11 introns, and mapped to chromosome 4q21.23. LPAAT-theta was ubiquitously expressed in 18 human tissues by RT-PCR analysis. Subcellular localization of LPAAT-theta-EGFP fusion protein revealed that LPAAT-theta was distributed primarily in the endoplasmic reticulum (ER) of COS-7 cells. Furthermore, we found that the overexpression of LPAAT-theta can induce mTOR-dependent p70S6K phosphorylation on Thr389 and 4EBP1 phosphorylation on Ser65 in HEK293T cells.