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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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The Korean Journal of Microbiology
Journal Basic Information
Journal DOI :
The Microbiological Society of Korea
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Volume & Issues
Volume 31, Issue 4 - Aug 1993
Volume 31, Issue 3 - Jun 1993
Volume 31, Issue 2 - Apr 1993
Volume 31, Issue 1 - Feb 1993
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Characteristics of a New Obligate Methanol-Oxidizing Bacterium
Kim, Si-Wook ; Park, Yong-Ha ;
The Korean Journal of Microbiology, volume 31, issue 4, 1993, Pages 261~266
A new methyltrophic bacterium which utilizes methanol as a sole source of carbon and energy was isolated from soil. It was Gram-negative, nonmotile, nonspore-forming rod, and strictly aerobic bacterium. Catalase and oxidase activities were present. Nitrate was reduced to nitrite. Vitamins and other growth factors were not required. Generation time was 1.6 hr under the optimal condition. The isolate assimilated methanol via the ribulose mono-phosphate pathway (Enter-Doudoroff varient) and did not have .alpha.-ketoglutarate dehydrogenase. It assimilated ammonia through glutamate dehydrogenase. The guanine plus cytosine content of the DNA was 61.0 mol%. The celular fatty acid composition was primarily straight-chain saturated
acids (palmitic acids) and unsaturated
acid (palmitoleic acids), and the isolate also contained two unidentified
branched fatty acids. The major ubiquinone was Q-8, and Q-6 and Q-7 were present as minor components. Phosphatidylethanolamine and phosphatidylglycerol were predominantly present, and diphosphatidyglycerol was also detected. Based on the physiological and biochemical properties, the isolate was assigned to a novel species of the genus Methylobacillus, Methylobacillus methanolovorus sp. nov.
The Screening and Characterization of Promoters Inducible by Superoxide Radical in Escherichia coli
The Korean Journal of Microbiology, volume 31, issue 4, 1993, Pages 267~273
We screened promoters inducible by superoxide radical from Escherichia coli. For this. we constructed random promoter library from E. coli MG 1655 using a promoter-probing plasmid. pJAC4. Six hundred and sixty clones in this library were classified based on their promoter strength by ampicillin gradient plate assay. Three hundred and eighty three clones with relatively weak to medium promoter strength were selected and then screened for their inducibility by superoxide radical on ampicillin gradient plate containing paraquat. Three clones (clones 5. 15 and 34) were detected to be induced by paraquat treatment and the level of induction were between 1.4 and 4 folds. Comparison of nucleotide sequences of the cloned promoter fragment with registered sequences in GENBANK and EMBL databases suggests that the cloned DNA fragments have not been yet characterized in E. coli. Transcription start sites in these clones were determined by rrimer extension and S I nuclease protection analysis. S 1 analysis of clones 5 and IS indicated that the mRNA levels were increased by paraquat treatment. Especially. clone 5 \vas found to have two transcription start sites. the upstream start site of which was selectively used by paraquat treatment. Searching for promoter clements. we found that only the downstream promoter of clone 5 has -10 and - 35 promoter elements recognized by RNA polymerase (
) and the others have no conserved promoter elements. This suggests that these superoxideinducible promoters may require transcription initiation protein(s) other than
Solubilities and Activities of Chloramphenicol Acetyltransferase and
-Lactamase Overproduced by the T7 Expression System in Escherichia coli
Kim, Han-Bok ;
The Korean Journal of Microbiology, volume 31, issue 4, 1993, Pages 274~278
Overproduced proteins in many cases result in forming insoluble inclusion bodies, and their formation might be due to high concentration of protein. To investigate how proteins become insoluble, chloramphenicol acetyltransferase (CAT) and .betha.-lactamase were overproduced, and their solubilities and activities were determined. CAT was accumulated from 9 to 45% of total cellular protein in a fully soluble form without inclusion body formation. CAT specific activity was shown to be proportional to the amount of the protein produced. Moderately produced .betha.-lactamase by the phase T7 expression system at 30.deg.C comprised only mature forms in a soluble form. However, overproduced .betha.-lactamase at 37.deg.C became insoluble. Most precursor forms of .betha.-lactamase in the cytoplasm were insoluble, whereas majority of the mature forms in the periplasm space were soluble. Also, chaperone GroE proteins which assist proper protein folding and translocation did not increase .betha.-lactamase solubility significantly under the experimental condition. It seems that the formation of inclusion bodies in the cell is related to the nature of protein itself rather than just to high concentration of protein.
Expression of Human p53 Gene as Glutathione S-transferase Fusion Proteins in Escherichia coli
The Korean Journal of Microbiology, volume 31, issue 4, 1993, Pages 279~285
Alterations of the p53 gene arc among the most frequent genetic changes in human cancer and often result in increased levels of p53 protein within the malignant cells. Detection of accumulated p53 protein can be a useful prognostic tool in human cancer. In order to make polyclonal antibodies for immunohistochemical screening. human p53 gene was expressed in E. coli in the form of GST (glutathione S-transfi.:rase) fusion proteins. Two p53 gene fragments. which were N('()I small fragment encoding amino acid residues of 1-151-: and Ncol large fragment of 159-393. were subeloned into the unique BamHI site present within the pGEX-2T vector using BamHI linker and recombinant plasmids pGTNS and pGTNL were constructed. respectively. The p53 cDNA fragment (from pC53-
,) encoding amino acid 38-145 (proline at residue 72) was amplified by polymerase chain reaction(PCR). The amplified DNA was digested with BamHI and Prull and inserted into the BamHI-Smal sites of pG EX-2T and recombinant plasmid pGTBP was constructed. After IPTG induction of these plasmids for 4 hours. fusion proteins were purified from E. coli extracts with glutathione Sepharose beads. The bound proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and the molecular weights were 54 kDa. 53 kDa and 40 kDa. respectively. Approximately one milligram of fusion proteins were purified from 1 -liter cultures.
Gene and Plasmid by Conjugal Transfer in aquatic Environments
The Korean Journal of Microbiology, volume 31, issue 4, 1993, Pages 286~291
gene and plasmid of natural isolate and genetically modified microorganisms (GMM) rearranged by conjugation in water environments were comparatively analyzed by agarose gel electrophoresis and Southern analysis. The transfer rates of the
gene from GMM strains were generally 100 times higher than thosc of natural iso]ate(DKI) under laboratory cnvironments, but their transfer rate was not much different in Moosimcheon River water. The conjugants obtained in LB(Luria-Bertani broth) and FW(filtered river water) water under laboratory conditions showed same number of the plasmids. but the sizes of the plasmids were changed. The
gene in the conjugants was found in the same position as the pDKJO]
plasmid. In case of the GMM strains as donor. the large plasmids of 180 kb appeared in conjugants obtained in LB and FW water. Especially, the
gene in the donor of DKC600 was found to be inserted into chromosome of the conjugant obtained in FW water. However. in the conjugants obtained from DKl and DKB 701 in Moosimcheon River water, the plasmids were rearranged by 4 and 8. respectively, and all of them showed hybridization by the
probe. But the small plasmids of the recipient disappeared in the conjugant from DKC600 as donor, and the rearranged plasm ids and chromosome in the conjugants were observed to be hybridized with the
probe. Therefore, rearrangement of
gene and plasmids by conjugation was found to be afTected diversely by cellular characteristics as well as by environmental factors.
Identification of Octopine Type Ti Plasmid in Agrobacterium tumefaciens KU12
The Korean Journal of Microbiology, volume 31, issue 4, 1993, Pages 292~299
Agrobacterium tumefaciens KU12 isolated from Korea is able to induce tumors on various plants and catabolize octopine as a sole carbon and nitrogen source. A, tumefaciens KU12 contains three plasmids. Their sizes are 45.5 kb. 240 kb. and > 240 kb. respectively. For the purpose of identification of octopine type Ti plasmid, avirulent A, tumefacients A136 is transformed with plasmids isolated from KU12 by direct transformation. Transformants containing Ti plasmid were grown on AB medium containing octopine as a sole nitrogen source. The isolated strain, named KU911, contains only 240 kb plasmid. As a result of induction of crown gall and Southern hybridization with other octopine Ti plasmid pTiAch5, 240 kb plasmid named pTiKU12 was Ti plasmid.
Heavy Metal Accumulation in Neurospora crassa
The Korean Journal of Microbiology, volume 31, issue 4, 1993, Pages 300~305
Neurospora crassa accumulated cadmium. iron, manganese and lead in the mycelium. The growth of N. crassa was inhibited in the presence of cadmium but not in the presence of iron or manganese. In the presence of lead. the growth of N. crassa was accelerated. In the presence of cadmium. mycelium became thick and a conidiospore grew into a mycelial ball instead of normal threadlike gro~1h. Each metal wa'i accumulated in different subcellular organelles bound to protein and induced different proteins.
Induction of Methanol Tolerance in Rhizopus nigricans Ehrenberg
The Korean Journal of Microbiology, volume 31, issue 4, 1993, Pages 306~311
The effects of methanol. used as a solvent for the hydrophobic substrate progesterone. on the morphology of Rhizopus nigricans and 11
-hydroxylation of progesterone was investigated. The methanol tolerance of the 11
-hydroxylase system in polyacrylamide immobilized R. nigricans mycelia as well as in free mycelia has been induced by adding various unsaturated fatty acids. biotin and ions into the cultivation medium. Immobilization of the cell seemed to protect the cells from denaturation by methanol. It gave higher reaction rate of progesterone than the free mycelia in the presence of methanol.500
g/l of biotin was found to be the most effective induction agent for the methanol tolerance among tested chemicals. R. nixricans cells sustained its enzymatic activity at higher methanol concentrations as a result of accumulation of unsaturated fatty acids. especially oleic acid. in the membrane phospholipid.
Variation in Trichothecene and Zearalenone Production by Fusarium graminearum Isolates form Corn and Barley in Korea
Kim, Jin-Cheol ; Park, Ae-Ran ; Lee, Yin-Won ; Youn, Hee-Ju ; Cha, Seung-Hee ;
The Korean Journal of Microbiology, volume 31, issue 4, 1993, Pages 312~317
A total of 110 Fusarium graminearum isolates were obtained from corn and barley samples which were collected from Kangwon province and the southern part of Korea, respectively. The isolates were tested for trichothecene and zearalenone (ZEA) production in rice culture. The incidences of trichothecene production by 51 isolates of F. graminearum from corn were 64.7% for deoxynivalenol (DON), 7.8% for 3-acetyldeoxynivalenol (3-ADON),33.3% for 15-acetylde-oxynivalenol (15-ADON), 21.6% for invalenol (NIV), and 13.7% for 4-acetylnivalenol (4-ANIV). DON producers frequently co-produced 15-ADON rather than 3-ADON. On the other hand, the incidences of trichothecene production by 59 isolates of F. graminearum from barley were 71.2% for NIV, 61.0% for 4-ANIV, and only one isolate produced DON and 3-ADON. The incidences and mean levels of ZEA producers were 32.0% and 71.
g/g for the isolates from corn, and 29.0% and 74 .
g/g for the isolates from barley. There was a great regional difference in trichothecene production of F. graminearum isolates between Kangwon province and the southern part of Korea.
Spheroplast Formation, Regeneration and Fusion of Flavimonas oryzihabitans KU21
The Korean Journal of Microbiology, volume 31, issue 4, 1993, Pages 318~325
The optima] conditions for the formation, the regeneration. and the spheroplast fusion of Flavimonas aryz/habitans spheroplasts were investigated. Cells were transformed to spherop]asts effectively by treatment of 0.5% volume (v/v) of 0.] M EDTA and ]00 flg/ml lysozyme at
for 30 min without shaking. Magnesium chloride and calcium chloride were effective on the stabilization of spheroplasts. and 20 mM calcium chloride in the rich regeneration medium improve the yield of regenerants as much as 3.5-fo]d. Addition of 0.8% bovine serium albumine (BSA) in dilution buffer for spheroplast formation improved the stabilization of spheroplasts over extended periods (4-6 hr) at room temperature. and thus increased the yield of recombinants to 4.5-fold. The spheroplast formation frequency and regeneration frequency of F aryzihabitans strain was 90.10% and 3.800/." respectively. The first regenerated cell of F. aryzihabitans spheroplasts were appeared 6 hours after plating. By I I hours after plating, 80% of spheroplasts were regenerated on thc rich regeneration medium containing 0.5 M sucrose. The intraspeci11c spheroplast fusion of F urvz/habitans was carried out and the properties of obtained fusants were investigated. Formation of fusion products was effective when the Flav/munas spheroplast mixture was treated with 40%(w/v) PEG6000 and 20 mM CaCl, for 10 min at room temperature. and thc formation of frequency of recombinants were
. All tested recombinant clones were very stable on further propagation.