• YAKHAK HOEJI
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• v.39 no.6
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• pp.676-680
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• 1995

The nucleotide sequence of Xbal-Mbol fragment of pKH7, a chloramphenicol-resistant($Cm^{r}$) plasmid isolated from multidrug-resistant S. aureus SA2, has been determined. Xbal-Mbol fragment of pKH7 was found to contain two ORFs. One ORF encoded Rap and the other encoded CAT protein. The deduced amino acid sequences of Rep and CAT of pKH7 were compared to those of pUB112 and pC221. Comparisons revealed that there was one amino acid difference in CAT between pKH7 and pUB112. CAT of pKH7 exhibited 98.6% amino acid identity to that of pC221. In case of Rep proteins, a slightly lower homology of 96.4% and 86.7% in amino acid sequences was observed between pKH7 and pUB112 and between pKH7 and pC221, respectively.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.31-36
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• 1991

A 7.5 kilobases hybrid plasmid, designated as pCE1301, was constructed by combining Eschurichia cwli plasmid pBELl which carries the kanamycin resistance gene of Tn5 with a cryptic plasmid, pSRl of Corynebacterium glutamicum. pCE1301 was transformed C. glutaicum by PEG-mediated protoplast method and its transformation efficiency was about $3.0\times 10^3$ transformants per $\mu g$ of the hybrid plasmid DNA. The physical map reveals that pCE1301 has single restriction sites for SalI and EcoRl, respectively. 'The kanamycin resistance of pCE1301 was stably maintained in C. glutamicum up to 25 generations and any segregation was not detected. pCI31301 was also introduced into Brevibacterium flavum and E coil, and replicated in those strains. pCE1301 was proved to be useiul in cloning the homoscrine dehydrogenase gene from C. glutamicum.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.516.2-516
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• 1986

DNA subfragments, sopA, sopB, and sopC supporting stable maintenance of an oriC plasmid, were derived from mini-F plasmid DNA (EcoRI restriction fragment, f5) after digestion with restriction endonucleases, and cloned in vector plasmid pBR322. The recombinant plasmid obtained were introduced into E. coli KY7231 and E. coli CSR603, and proteins specified by the mini-F fragments were analysed by SDS-polyacrylamide gel electrophoresis. Two proteins encoded by the F fragments were detected, having molecular weights of 41,000 and 37.000. The sopA protein (41K) encoded by a plasmid pXX288 was observed in the cytoplasm, whereas the sopB protein (37K) encoded by a plasmid pXX157 was in the membrane fraction. There was no novel protein band detected in the cell with a plasmid pXX300, which contained sopC fragment. Gene products of a plasmid pXX167, which is comprised of sopA, sopB, and sopC, were not detectable. Fluorography after one and two dimensional gel electrophoresis of the lysates showed that these two proteins were overproduced in the cells which were allowed to incorporate radioactive amino acid after plasmid amplification by chloramphenicol treatment. The isoelectric points of the sopA and sepB proteins were 6.6 and 7.0, respectively.

• Korean Journal of Microbiology
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• v.30 no.2
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• pp.88-95
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• 1992

The genetically engineered microorganisms (GEMS) could be released accidentally or ii)rexperimental purposes, as the genetic engineering, technique ha:. become very popular inany laboratories of biological sciences. But there have been littlt: informations on transkrbehavior of the genetically ~nodified genes in the natural en\ironmentx. In this stutly.antibiotic resistant bacteria were isolated from nat.ural waters. and then GEM strains wereconstructed i'rom the natural isolate (NI) by ~noclification oi' the Km' plasmitl. Thetransferability of the plasmids in the GEM and NI strains were examinetl by con-jugationin Luria-Bertani broth :it 30$^{\circ}$C. Also the cff'ccts 01' mating strain and pH on their transferfrequency and rearrangement of the plasmids in tl-~ec o~ijugantsM ere comp:irati\ely stuclictl.I'hc transkr frequency of Km' plasmid in donor of GEM and N1 strains wah similar a.;about 10 ' when co~ljugation was conducted wit11 M'I'I strain is recipient at pH 7. butthat of 1)KCOOI was lowered to 1.2X 10 '. And when the lab. stlain was uhccl as recipient.the transfer tendency of the plasmid was about same in both (;EM and NI strains usedas donor. All thc tionor 5trains. except for I)KC601. showecl the Ilighcs~ frequency of about10 ' at pH 7 and the frequcncics were lowered at both pH 5 and 9. Hut the mocliliedKm' plasmid in the cloned strain of DKC601 was transferred hy very low frequency of10 "at pH 5 ant1 7 comparing to other GEM strains. especiall! any co~~.jugantws ere notobtained at pH 4 and 9 even after conjugation for 6 hours. Rearrangement of the plasmidstranskrred into the lab. strain was not found in the conjugants. I\ulcornerut a lot of rearrangclncntwas ohservecl nlhen they were transferred into the NI strain. Such a rearrangement wasmore severe when donor was GEM strain rather then NI strain Hut such ;r phenomenonwas less affected by p!-l values.r phenomenon was less affected by p!-l values.

• Korean Journal of Microbiology
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• v.30 no.3
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• pp.218-224
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• 1992

Recombinant plasmid shuttle vectors were constructed for genetic studies on the oxidation of carbon monoxide by carboxydobacteria. Two vectors. pYK322 (7.2 kb, Ap'. Tc') and pYK 324 (7.2 kb, Ap', Tc'), were constructed using pBR322 and pYK100. a small plasmid in Pseudomonas carbo,xydovorans. Four plasmids. pYK2IO (5.2 kb. Cm'), pYK220 (5.2 kb, Cmr), pYK230 (5.2 kb, Cm'), and pYK232 (5.2 kb. Cm'), were constructed using pACYC184 and pYK100. Transformation of several carboxydobacteria with pYK322 and pYK220 was round to be efficient when the cells were transformed by the methoti of Bagdasarian and Timmis (Curr. Top. Microbiol. Immunol. 96:47-67. 1982) with several modifications; cells growing on 0.2% succinate were harvested at the mid-exponential phase. 10 mM RbCl in transformation solution was substituted with 100 mM KCI. cclls in transformation solution were incubated for 12 h at 4'C before addition of DNA and heat shock was carried out for 3 min at 45$^{\circ}$C. Plasmid vectors used for transformation, however. were not detected from antibiotics-resistant transformants, suggesting that the vectors may be integrated into the chromosomal DNA.

• Journal of Life Science
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• v.7 no.3
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• pp.212-218
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• 1997

The studies were conducted to explore the dffects of growth or survival against various factors and plasmid profiles of 49 Y. enterocolitica isolated from springs water. In the presence of calcium hypochlorite, y. enterocolitica was entirely extinguished by exposure for 33 hours at 0.8 ppm concentration, and was grown up to 7% NaCl, but not at 95 NaCI. Y. enterocolitica was presented optimal growth at pH 7.0 anad 9.0, and not allowth the growth at pH3.0, 5.0 and 11.0. The optimal temperature for growth of Y. enterocolitica was 25$\circ$C and 35$\circ$C, and allowed the growth at refrigerant temperature, 5$\circ$C. Y. enterocolitica was remarkably decreased by exposure for 30 seconds under UV light, and entirely extinguished by exposure for 90 seconds. Therefore, UV light was effective for sterilization of Y. enterocolitica. Fourty-nine strains of Y. enterocolitica were harbor plasmid DNA of approximately 46 Kb molecular weight.

• Journal of Microbiology
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• v.44 no.5
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• pp.473-479
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• 2006

A well characterized naphthalene-degrading strain, Pseudomonas putida PpG7 was observed to utilize limonin, a highly-oxygenated triterpenoid compound as a sole source of carbon and energy. Limonin concentrations evidenced a 64% reduction over 48 h of growth in batch cultures. Attempts were made to acquire a plasmid-less derivative via various methods (viz. Ethidium Bromide, SDS, elevated temperature & mitomycin C), among which the method involving mitomycin C ($20{\mu}g/ml$) proved successful. Concomitant with the loss of plasmid in P. putida PpG7 strain, the cured derivative was identified as a $lim^-$ phenotype. The $lim^+$ phenotype could be conjugally transferred to the cured derivative. Based on the results of curing with mitomycin C, conjugation studies and presence of ndo gene encoding naphthalene 1,2 dioxygenase, it was demonstrated that genes for the limonin utilization were encoded on an 83 kb indigenous transmissible Inc. P9 NAH plasmid in Pseudomonas putida PpG7 strain.

• Microbiology and Biotechnology Letters
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• v.11 no.3
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• pp.163-168
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• 1983

The penicillin resistant plasmid DNA was prepared from Streptomyces bobili YS-40, producing penicillinase, by the phenol extraction method and introduced into Bocillus subtilis IAM 12118 by the transformation procedure of Mahler method. The optimal pH and temperature on the transformation was 7.0, 3$0^{\circ}C$ respectively. Above 20 minutes contact of plasmid DNA and recipient cell was shown the high transformation frequency. The transformant of penicillin resistance was proportionally increased as increase of the DNA concentration. The addition of lysine in transformation system increased the transformation frequency about 6-fold and the addition of the chloramphenicol did not affect the transformation frequency.

• Microbiology and Biotechnology Letters
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• v.14 no.3
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• pp.213-218
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• 1986

Hybrid plasmid pEA24, shuttle vector YRp7 carrying amylase gene of Bacillus amyloliquefaciens, was transformed to yeast Saccharomyces cerevisiae, and the expression of B. amyloliquefaciens amylase gene in yeast was investigated. The frequency of transformation to S. cerevisiae DBY747 with YRp7 was increased by treatment of 40% polyethylene glycol (MW 4, 000), PH 7.0, at 3$0^{\circ}C$, and by regeneration used 2% top agar. The amount of cellular amylase activity produced by S. cerevisiae containing pEA24 was 2% of that secreted from B. amyloliquefaciens, but in case of S. cerevisiae transformant, the amylase secreted was not detected. A comparison of genetic stability of pEA24 and YRp7 plasmids in yeast was carried out by cultivation of transformants in tryptophan-supplement-medium. The pEA24 plasmid was more unstable than YRp7 in S. cerevisiae. The size of pEA24 extracted from S. cerevisiae transformants was found to be identical with that from E. coli transformants by agarose gel electrophoresis.

• Korean Journal of Microbiology
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• v.31 no.1
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• pp.44-47
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• 1993

A replication initiation gene was identified and its nucleotide sequence has been determined from a 3.8 kb, chloramphenicol acethyltransferase conferring R-plasmid pSBK203 of Staphylococcus aures. Location of the replication related region of pSBK 203 was determined by interuption with pUC 119 at XBaI and MspI sites which resulted in inactivation of replication in Bacilius subtilis. Base sequence of this region revealed on open reading frame of 942 base pairs, which encoded a 314 amino acid protein. Base sequence homology with other rep of pT181 family plasmids such as pT181, pC221, pC223, pS194, pU112, and pCW7 was ranged from 78% to 97% and the predicted amino acid sequence homology was from 72% to 95%.