• Journal of Microbiology and Biotechnology
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• 제27권9호
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• pp.1711-1715
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• 2017

In this study, we characterized the $bla_{NDM-5}$-bearing plasmid in a Klebsiella pneumoniae isolate that had lost the plasmid during serial passage. We determined the complete sequences of the plasmid pCC1410-2, which was extracted from a K. pneumoniae ST709 isolate collected at a Korean hospital from which two NDM-5-producing K. pneumoniae isolates were subsequently isolated. As a result, the pCC1410-2 plasmid had a backbone structure that was similar to those of two plasmids previously reported from the same hospital, but lacked some antibiotic resistance genes ($bla_{TEM-1}$, rmtB, mphR(A), mrx(A), and mph(A)). A 9-bp repeating unit encoding three amino acids (Gln-Gln-Pro) was inserted in TraD in pCC1410-2. Thus, the pCC1410-2 plasmid might be transferred from the previously identified carbapenem-resistant K. pneumoniae, but some delections and inversions might have occurred during the process. We compared the transfer frequency and stability of the plasmids. The relative frequency of conjugative transfer and stability in the host were significantly lower in pCC1410-2 than in previously reported $bla_{NDM-5}$-bearing plasmids in Korea. A low transfer frequency and instability in the host may cause underestimation of carbapenemase-producing Enterobacteriaceae in the clinical setting and in surveillance studies.

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.31-38
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• 2002

Bacteria have evolved various types of resistance mechanism to toxic heavy metals, such as arsenic and antimony. An arsenical and antimonial resistant bacterium was isolated from a shallow creek draining a coal-mining area near Taebaek City, in Kangwon-Do, Korea. The isolated bacterium was identified and named as Pseudomonas sp. KM20 after biochemical and physiological studies were conducted. A plasmid was identified and its function was studied. Original cells harboring the plasmid were able to grow in the presence of 15 mM sodium arsenite, while the plasmid-cured (plasmidless) strain was sensitive to as little as 0.5 mM sodium arsenate. These results indicated that the plasmid of Pseudomonas sp. KM20 does indeed encode the arsenic resistance determinant. In growth experiments, prior exposure to 0.1 mM arsenate allowed immediate growth when they were challenged with 5 mM arsenate, 5 mM arsenite, or 0.1 mM antimonite. These results suggested that the arsenate, arsenite, and antimonite resistance determinants of Pseudomonas sp. KM20 plasmid were indeed inducible. When induced, plasmid-bearing resistance cells showed a decreased accumulation $of\;73^As$ and showed an enhanced efflux $of\;^73As$. These results suggested that plasmid encoded a transport system that extruded the toxic metalloids, resulting in the lowering of the intracellular concentration of toxic oxyanion. In a Southern blot study, hybridization with an E. coli R773 arsA-specific probe strongly suggested the absence of an arsA cistron in the plasmid-associated arsenical and antimonial resistance determinant of Pseudomonas sp. KM20.

• 대한수의학회지
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• 제28권1호
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• pp.59-65
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• 1988

This paper deals with the 0 groups of citrate utilizing variants of Escherichia coli ($Cit^+$ E. coli) isolated from cattle, the production of colicin, hemolysin, K99 antigen, heat stable enterotoxin, and the isolation of plasmid DNA. Among 42 $Cit^+$ E. coli, 12 strains were 020, 9 strains 08, 5 strains 045, 3 strains 0115, 1 strain 064, 1 strain 0139 and remaining strains(11) were untypable. Thirty-nine(81.3%) out of 48 $Cit^+$ E. coli were produced colicin and 13(27.0%) were produced hemolysin. Of 12 $Cit^+$ E. coli bearing K99 antigen, 6(50.0%) were produced heat stable enterotoxin. In gel electrophoresis for the isolation of plasmid DNA, the number of plasmids varied from 1 to 7 in 10 $Cit^+$ E. coli. It's molecular weight ranged from 2 to 50 Mdalton, and 50 Mdalton plasmid was commonly existed in all strains.

• 한국미생물·생명공학회지
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• 제10권4호
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• pp.289-295
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• 1982

Streptomyces bobili (YS-40) 의 Hg, Ag, penicillin-G, ampicillin, chloramphenicol, oxytetracycline, streptomycin, kanamycin에 대한 생육최소저해농도는 각각 15, 10, >3,000, >100, >1,000. >100. <5, <5 $\mu\textrm{g}$/$m\ell$였으며 본 균주의 R-plasmid를 제거시키기 위하여 ethidium bromide, acriflavine, sodium dodecyl sulfate 등의 curing agent를 처리시킨 결과를 요약하면 다음과 같다. Ethidium bromide를 10$\mu\textrm{g}$/$m\ell$의 농도로 처리했을 때 98.0% 정도의 R-plasmid를 제거시킬 수 있었다. pH 7.0에서 curing시킴으로서 R-plasmid가 가장 잘 제거되었으며 분균을 24시간동안 배양해서 curing시킴으로서 R-plasmid의 제거율이 가장 높게 나타났다. Ethidium bromide에 의해서 R-plasmid가 제거된 균과 원균을 여러가지 색소생성배지에서 배양시켜 배양상의 특성을 조사해 본 결과 peptone-beef extract agar 배지에서 aerial mass color가 greyish pink에서 grey로 변했으며 tryptone-yeast extract broth에서 배지에서 soluble pigment가 pale brown에서 무색으로 변했다. 이 결과로는 aerial mycelium, melanin 생성과 R-plasmid 는 무관하다고 추측된다.

• 대한수의학회지
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• 제32권2호
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• pp.181-194
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• 1992

Two hundred and eighty nine strains of Yersinia enterocolitica isolated from healthy pigs were tested for the presence of 40~50 Megadalton virulence-associated plasmids and plasmidmediated in vitro virulence-associated properties, i.e., congo red uptake, calcium dependency, autoagglutination, CRMOX reaction, crystal violet binding and pyrazinamidase reaction. The correlationships between in vitro virulence-associated properties and the presence of 220 Kdalton outer membrane protein(OMP) were examined in strains with or without virulence-associated plasmids. The correlationships between the presence of plasmids on the production of the OMP and the expression of in vitro virulence-associated properties were studied with $CRMOX^+$ strains and acridine orangecured $CRMOX^-$ mutants. The results were as follows : 1. Of the in vitro virulence-associated tests with 289 strains of Y enterocolitica, 275 strains (95.2%) were positive for pyrazinamidase test, and followed by in order of crystal violet binding test, 226 (79.2% ) ; CRMOX test, 190 (65.7%) ; autoagglutination test, 1.85(64.0%) : calcium dependency test, 86 (29.8%) and congo red uptake test, 47(16.3%). 2. The correlationship between autoagglutination and CRMOX test(r=0.90) was highly significant (p<0.01). 3. In 190 strains(65.7%) bearing the virulence-associated plasmids(MW 40~50 Mdalton), the correlation between the presence of plasmids and their in vitro virulence-associated properties were highest with CRMOX test(r=0.93) and followed by in orders of AAG test(0.81), CV test(0.46), PYZ test(0.37) and CD test(0.18), but no correlationship between the presence of plasmids and CR test(-0.11). 4. The $CRMOX^+$ strains produced the 220 Kdalton OMP when they were cultured at $37^{\circ}C$, but not at $26^{\circ}C$. The presence of 220 Kdalton OMP was correlated significantly with in vitro virulence properties and the presence of virulence-associated plasmid, respectively. 5. In the isogenic $CRMOX^-$ mutant strains, of which plasmid were cured by treatment with acridine orange not only in vitro virulence-associated properties(CR 100%, CD 100%, AAG 82.6%, CV 58.3%) disappeared but also 220 Kdalton OMP(100%) was not produced. These results indicate that the positive CRMOX reaction is plasmid-mediated and the CRMOX test is potential as an in vitro virulence tests with Y enterocolitica.

• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1408-1414
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• 2009

The obtention of high yields of purified plasmid DNA is viewed as an essential issue to be considered towards efficient production of DNA vaccines and therapeutic plasmids. In this work, Escherichia coli $DH5\alpha$. bearing the pVAXI-LacZ plasmid was grown in a developed semi-defined medium at different temperatures and tryptone concentrations. Analysis of pDNA yields and E. coli morphology revealed that at higher temperatures (37 and $40^{\circ}C$), higher specific yields and E. coli filamentation were obtained. However, the best results were achieved when a lower tryptone concentration was used. This approach was shown to be a powerful tool to promote plasmid amplification, keeping the desirable plasmid structure, and favoring the attainment of quality. Our results suggest that by using tryptone alone as an amino acid source, pDNA amplification was improved and a specific yield of 20.43 mg pDNA/g dcw was achieved, proving that this strategy can improve pDNA yield even at a small scale.

• Journal of Microbiology
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• 제40권3호
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• pp.205-210
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• 2002

To study interactions between $C_{4}$-dicarboxylic acid transport protein D and E$\sigma$$^{54}$ in the dctA promoter regulatory region, we used the challenge phage system. An ant＇-｀lac fusion was recombined onto the challenge phage, and this ant＇-｀lac fusion along with Pant and the R. meliloti dctA promoter regulatory region were cloned onto a plasmid. The plasmid bearing the ant＇-｀lac fusion was used as a reporter plasmid in a coupled transcription-translation system. Addition of purified $\sigma$$^{54}$ to the coupled system specifically repressed transcription of the plasmid-borne ant＇-｀lac fusion. When DCTD was added along with $\sigma$$^{54}$ to the coupled system, transcription of the ant＇-｀lac fusion was even further repressed, suggesting that DCTD may stabilize closed complexes between E$\sigma$$^{54}$ and the dctA promoter.

• Animal cells and systems
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• 제2권2호
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• pp.269-275
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• 1998

The reporter plasmid pMT-lacZ containing the metallothionein (MT) promoter region (-320∼+58 with respect to the transcription initiation site) fused to the lacZ gene in a P-element vector was constructed. Transgenic Drosophila bearing the MT-lacZ fusion gene were established by P-element mediated transformation. Expression of the MT-lacZ fusion gene in transformants was examined during development. By treatment with low concentration of cadmium (>1O uM) or paraquat (>50 uM), increased expression of B-galactosidase was shown in fat body, brain lobe, and ganglion transgenic larval tissues. The results show that transformants bearing the MT-lacZ fusion gene are useful for further studies on the mechanism of regulation of MT gene expression and for monitoring toxic metals.

• 미생물학회지
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• 제21권2호
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• pp.95-102
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• 1983

The penicillin G acylase(pga) gene was cloned in the vector plasmid pKM $300(Ar^r,\;Tc^r,\;6.33kb)$ for the study of the structure and expression of the pga gene. This recombinant plasmid pPAKS-1 DNA(24.5 Kb) was cleaved into 2 fragments by restriction enzyme Eco R1.1fragment by BamH1, 4fragments by Hind III, and 2 fragments by Pst I. The pga gene was located on the Eco R1.Hind III-C fragement of pPAKS-1. The recombinant plasmids pPAKS-1 and pPAKS-2, in which the Hind III-B and Hind III-D fragments pPAKS-1 are deleted, are characterized. The results are summarized as follows : 1. Doubling times of bacterial strain bearing pPAKS-1 and pPAKS-2 are 90 and 60 minutes, respectively. 2. pPAKS-1 and pPAKS-2 are present at about 16-32 and 70 copies per cell, respectively, are 0.66 and 5.5 units, respectively, which represent 2-fold and 20-fold higher enzyme 4. pPAKS-1 is very unstable, but pPAKS-2 is stable.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.515.2-515
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• 1986

B. sphaericus 1593 K-5 synthesize a potent entomicidal toxin against mosquito larvae. B. sphaericus EcoRl DNA fragment carrying the biocidal activity was clonied and expression in E. coli JM83. For the construction of a recombinant plasmid bearing the toxin activity the DNA of B. sphaericus was partially digested by the EcoRl. The EcoRl DNA fragments were ligated to plasmid pUC 8-EcoR1 site. The transformants were selected on LB plates containing X-gall and ampicilline. The transformants were bioassayed against mosquito larvae of which two clones showed biocidal activity. The two clones were redigested with Eco R1 and analyzed by 0.7% agarose gel electrophorsis.