Journal of Microbiology

Volume 37(2); June 1999

Isolation of an Autonomously Replicating DNA Sequence from Aspergillus nidulans: Jang, Seung Hwan , Jahng, Kwang Yeop; J. Microbiol. 1999;37(2):51-58.

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Abstract: Using yeast, Saccharomyces cerevisiae, and the integrate vector system, we have isolated and characterized an autonomously replicating sequence (ARS) from Aspergillus nidulans. The DNA fragment, designated ANR1, is 5.0 kb in size and maintained free from the chromosome in S. cerevisiae. The YIplac211-ANR1 recombinant plasmid, which consists of sequences derived from the yeast integrative vector YIplac211 and 5.0 kb ANR1 fragment, showed a 10⁴-fold enhancement in transformation efficiency over that found for YIplac211, and was easily recovered from the transformed yeast. Genetic analysis of transformants showed that YIplac21-ANR1 could be over 96% cured when cultured over 20 generations in complete medium and thus suggests that this sequence is mitotically unstable. In A. nidulans, recombinant plasmid PILJ16-4.5 which carries the 4.5 kb EcoRI fragment of ANR1 showed a 170-fold enhancement in transformation efficiency compared to that of the integrative vector PILJ16.

Physiological Relevance of Salt Environment for in vitro recA System: Kim , Jong Il; J. Microbiol. 1999;37(2):59-65.

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Abstract: RecA protein can promote strand assimilation, homologous pairing, and strand exchange. All these reactions require DNA-dependent ATP hydrolysis by recA protein, and the activities of recA protein are affected by the ionic environment. In this experiment, DNA-dependent ATPase activity showed different sensitivity to anionic species. ATP hydrolysis and strand exchange were relatively sensitive to salt in the reactions with NaCl, strongly inhibited at 100 mM NaCl. However, the inhibition by sodium acetate or sodium glutamate was not observed at 50∼100 mM concentration. Addition of sodium glutamate to the standard reaction condition increased the apparent efficiency of ATP hydrolysis during strand exchange. The condition including 50∼100 mM sodium-glutamate might be similar to the physiological condition.

Mycolic Acid-Containing Actinomycetes Associated with Activated Sludge Foam: Seong, Chi Nam , Kim, Young Sook , Baik, Keun Shik , Lee, Soon Dong , Hah, Yung Chil , Kim, Seung Bum , Goodfellow, Michael; J. Microbiol. 1999;37(2):66-72.

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Abstract: Mycolic acid-containing actinomycetes associated with extensive foaming in the aeration basin of the activated sludge process were isolated and analyzed by phenotypical, chemotaxonomical and phylogenetic methods. Whole cell sugar patterns of two isolates were pattern A. The nearly complete sequences of the 16S rRNA genes (rDNAs) of the isolates were determined and compared by using several tree-making algorithms. With polyphasic methods, strain SCNU1 was identified as Gordona sputi, and strain SCNU5 assigned to the genus Tsukamurella. The presence of opportunistic pathogens of chronic lung infections within foams can cause public health problems and render waste-treatment processes inefficient.

Degradation of Anthracene by a Pseudomonas strain, NGK1: Manohar, Shinde , Kim, Chi Kyung , Karegoudar, Timmanagouda B.; J. Microbiol. 1999;37(2):73-79.

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Abstract: Pseudomonas sp. NGK1, isolated by naphthalene enrichment culture technique, is capable of degrading anthracene as a sole source of carbon and energy. The organism degraded anthracene through the intermediate formation of 1,2-dihydroxyanthracene, 2-hydroxy-3-naphthoic acid, salicylate, and catechol. The intermediates were isolated and characterized by TLC, spectrophotometry, and HPLC analysis. The cell free extract of anthracene-grown cells showed activities of anthracene dioxygenase, 2-hydroxy-3-naphthylaldehyde dehydrogenae, 2-hydroxy-3-naphthoate hydroxylase, salicylate hydroxylase and catechol 2,3-dioxygenase. The formed catechol as a metabolite is degraded through meta-cleavage with the formation of α-hydroxymuconic semi-aldehyde.

Purification and Characteristics of Glucoamylase in Aspergillus oryzae NR 3-6 Isolated from Traditional Korean Nuruk: Yu, Tae Shick , Kim, Tae Hyoung , Joo, Chong Yoon; J. Microbiol. 1999;37(2):80-85.

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Abstract: The purification system of glucoamylase (glucan 1,4-α-glucosidase, EC 3. 2. 1. 3), some characteristics of the purified enzyme and hydrolysis rate of various raw starch were investigated through several experiments. The enzyme was produced on a solid, uncooked wheat bran medium of Aspergillus oryzae NR 3-6 isolated from traditional Korean Nuruk. The enzyme was homogeneously purified 6.8-fold with an overall yield of 28.3% by the criteria of disc- and SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated to be 48 kDa by SDS-PAGE. The optimum temperature and pH were 55℃ and 4.0, respectively. The enzyme was stable at a pH range of 3.0∼10.0 and below 45℃. Enzyme activity was inhibited about 27% by 1mM Hg^2+. The hydrolysis rate of raw wheat starch was shown to be 17.5-fold faster than the hydrolysis rate of soluble starch. The purified enzyme was identified as glucoamylase because the product of soluble starch by the purified enzyme was mainly glucose by thin layer chromatography.

An OTHBVS Cell Line Expresses the Human HBV Middle S Protein: Park, Sung Gyoo , Jung, Gu Hung; J. Microbiol. 1999;37(2):86-89.

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Abstract: An OTHBVS cell line from HepG2 was established. This cell line stably expresses the human hepatitis B virus (HBV) middle S protein that includes the preS2 region which is important for HBV particle entry into the hepatocyte. To establish this cell line, the middle S open reading frame (ORF), with a promoter located in the 5' region and enhancer located in the 3' region, was cloned downstream from the metallothionine (MT) promoter of the OT1529 vector. In this vector, expression of the middle S protein was constructed to be regulated by its own promoter and enhancer. Expression of the large S protein which contains the preS1 region in addition to the middle S protein was designed to be regulated by the MT promoter. When extracts of OTHBVS cells were examined with an S protein detection kit (RPHA, Korea Green Cross Co.), an S protein was detected. Total mRNA of OTHBVS cell examined by northern blot analysis with an S ORF probe revealed small/middle S transcripts (2.1 kb). When the MT promoter was induced by Zn, large S transcripts (2.4 kb) were detected. The GP36 and GP33 middle S proteins were presumably detected, but large S proteins were not detected by immunostain analysis using anti-preS2 antibody.

Hepatitis C Virus Core Protein Sensitizes Cells to Apoptosis Induced by Anti-Cancer Drug: Kang, Mun Il , Cho, Mong , Kim, Sun Hee , Kang, Chi Dug , Kim, Dong Wan; J. Microbiol. 1999;37(2):90-96.

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Abstract: The core protein of the hepatitis C virus (HCV) is a multifunctional protein. The HCV core protein was reported to regulate cellular gene expression and transform primary rat embryo fibroblast cells. However, the role of the core protein in the pathogenesis of HCV-associated liver diseases is not well understood. To investigate the functional role of the core protein in cytophathogenicity, we have constructed stable expression systems of full length or truncated HCV core protein lacking the C-terminal hyderophobic domains and established HepG2 cell clones constitutively expressing the core protein. The full length core protein was localized in the cytoplasm and the C-terminal truncated core protein was localized in the nucleus. HepG2 cells expressing nuclear, truncated core protein showed elevated cell death during cultivation compared to untransfected cells and full length core-expressing cells. In the treatment with bleomycin, both cell clones expressing full length or truncated core protein appeared to be more sensitive to bleomycin than the parental HepG2 cells. These results suggest that the core protein may play a role in HCV pathogenesis promoting apoptotic cell death of infected cells.

Construction of a Hexapeptide Library using Phage Display for Bio-panning: Cho, Won Hee , Yoo, Seung Ku; J. Microbiol. 1999;37(2):97-101.

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Abstract: Random hexapeptide library on the surface of filamentous bacteriophage was constructed using the SurfZAP vector. The size of the library was approximately 105. The peptide insert was flanked by two cysteines to constrain the peptide structure with a disulfide bond. This library was screened for the topoisomerase II binding peptide. Dramatic enrichment of the fusion phage over the VCS M13 helper phage was demonstrated by bio-panning affinity selection.

Cloning and Characterization of a Heterologous Gene Stimulating Antibiotic Production in Streptomyces lividans TK-24: Kwon, Hyung Jin , Lee, Seung Soo , Hong, Soon Kwang , Park, Uhn Mee , Suh, Joo Won; J. Microbiol. 1999;37(2):102-110.

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Abstract: Genetic determinant for the secondary metabolism was studied in heterologous expression in Streptomyces lividans TK-24 using Streptomyces griseus ATCC 10137 as a donor strain. Chromosomal DNA of S. griseus was ligated into the high-copy number Streptomyces shuttle plasmid, pWHM3, and introduced into S. lividans TK-24. A plasmid clone with 4.3-kb BamHI DNA of S. griseus (pMJJ201) was isolated by detecting for stimulatory effect on actinorhodin production by visual inspection. The 4.3-kb BamHI DNA was cloned into pWHM3 under the control of the strong constitutive ermEp promoter in both directions (pMJJ202); ermEp promoter-mediated transcription for coding sequence reading right to left: pMJJ203; ermEp promoter-mediated transcription for coding sequence reading left to right) and reintroduced into S. lividans TK-24. The production of actinorhodin was markedly stimulated due to introduction of pMJJ202 on regeneration agar. The introduction of pMJJ202 also stimulated production of actinorhodin and undecylproidigiosin in submerged culture employing the actinorhodin production medium. Introduction of pMJJ203 resulted in a marked decrease of production of the two pigments. Nucleotide sequence analysis of the 4.3-kb region revealed three coding sequences: two coding sequences reading left to right, ORF1 and ORF2, one coding sequence reading right to left, ORF3. Therefore, it was suggested that the ORF3 product was responsible for the stimulation of antibiotic production. The C-terminal region of ORF3 product showed a local alignment with Myb-related transcriptional factors, which implicated that the ORF3 product might be a novel DNA-binding protein related to the regulation of secondary metabolism in Streptomyces.

Tumor Necrosis Factor Receptor (TNFR)-associated factor 2 (TRAF2) is not Involved in GM-CSF mRNA Induction and TNF-Mediated Cytotoxicity: Kim, Jung Hyun , Cha, Myung Hoon , Lee, Tae Kon , Seung, Hyo Jun , Park, Choon Sik , Chung, Il Yup; J. Microbiol. 1999;37(2):111-116.

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Abstract: Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is known to act as a signal transducer that connects TNFR2 to its downstream effector functions such as proliferation of thymocytes, regulation of gene expression, and cell death. TRAF2 consists of largely two domains, the N-terminal half that contains a signal-emanating region and the C-terminal half that is responsible for binding to the intracellular region of TNFR2. In this study, we examined the possible roles of TRAF2 in granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression and cell death. A truncated mutant of TRAF2 (Δ2-263) that contains only a C-terminal half was generated, and transiently transfected to the A549 cell, a human lung cancer cell line, and L929 cell, a murine TNF-sensitive cell line. GM-CSF mRNA was induced in untransfected A540 cells both in dose- and time-dependent manner upon the exposure of TNF. However, neither the full length TRAF2 nor the mutant altered GM-CSF mRNA production regardless of the presence or absence of TNF. Furthermore, neither TRAF2 versions significantly changed the cytotoxic effect of TNF on L929 cells. These data suggest that TRAF2 may not be involved in the signal transduction pathway for GM-CSF gene induction and cell death mediated by TNF.

Protective Effects of Antoxidant Enzymes of Candida albicans against Oxidative Killing by Macrophages: Kim, Hye Jin , Na, Byoung Kuk , Kim, Moon Bo , Choi, Duk Young , Song, Chul Yong; J. Microbiol. 1999;37(2):117-122.

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Abstract: Protective roles of antioxidant enzymes, copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and catalase of Candida albicans against exogenous reactive oxygens and oxidative killing by macrophages were investigated. The initial growth of C. albicans was inhibited by reactive, oxygen-producing chemicals such as hydrogen peroxide, pyrogallol, and paraquat, but it was restored as the production of antioxidant enzymes were increased. The growth inhibition of C. albicans by reactive, oxygen-producing chemicals was reduced by treating the purified candidal SOD and catalase. Also, in the presence of SOD and catalase, the oxidative killing of C. albicans by macrophages was significantly inhibited. These results suggest that antioxidant enzymes, CuZnSOD, MnSOD, and catalase of C. albicans may play important roles in the protection of C. albicans not only from exogenous oxidative stress but also from oxidative killing by macrophages.

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