• Korean Journal of Microbiology
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• v.24 no.4
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• pp.341-351
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• 1986

R-plasmid(pSBK203, 2.5Mdal) conferring chloramphenicol resistance was isolated from mutiple antibiotic resistant Staphylococcus aureus D-H-1. Bacillus subtilis BD170 was transformed by this plasmid and restriction enzyme clevage sites of this plasmid were mapped for the cloning of chloramphenicol resistance gene. Taq I partial digested fragment of pSBK203(1.3kb) inserted into Cla I site of pBD9 appears to have both regulatory region for induction and structural gene for chloramphenicol resistance whereas Rsa I fragment (1.3kb, both ends are staggered away 0.1Kb from those of Taq I fragment) inserted into Sca I site of pBR322 showed constitutive expression in E. coli. Hinf I, Taq I, and Bgl II restriction enzyme recognition sites are found in both Rsa I fragment and Taq I fragment. Among these, Bgl II recognition site was associated with chloramphenicol resistance.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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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.

• Korean Journal of Microbiology
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• v.22 no.4
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• pp.265-269
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• 1984

For the utilization as donor cells of conjugation, DAP-Auxotrophs were isolated from C. coli cells, carrying plasmid $p^{RD1}$ with(a) drug resistance makers from Pseudononas $(Km^r, \;Carb^r, \;Tc^r)$ and (b) the nif-gene group from Klebsiela. E. coli $p^{RD1}$ cells were treated with nitrosoguanidine for the mutagenesis and cephalexin for the isolation of DAP-Auxotrophs. The nature of auxotrophs was verified by suitable biochemical test and checking with 6-cyanopurine as a color indicator for the presence of nif-gene.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1123-1126
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• 2011

The IncP plasmid R995 has been a useful self-transmissible, broad-host-range vector for a number of applications including the recombinase/conjugation-based cloning of large genomic DNA segments. However, R995 derivatives (or related plasmids) expressing a wide range of different resistance markers and Flp recombinase target sites do not exist in the literature. In addition, documented strategies for applying such plasmids in cloning applications that take advantage of conjugation for the convenient isolation and recovery of constructs are extremely limited. Here, we report a new series of R995 plasmids with alternative markers to increase options for applications in backgrounds already expressing resistance to a particular antibiotic(s). These R995 plasmids have been engineered to contain FRT sites that can be used for recombinase-based cloning. We demonstrate the utility of this approach by cloning 20 kb regions from the Salmonella Typhimurium and Escherichia coli genomes and by cloning DNA from an exogenous plasmid source. To our knowledge, this represents the first systematic engineering of an intact, self-transmissible IncP plasmid with a series of alternative antibiotic markers and FRT sites.

• Korean Journal of Veterinary Research
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• v.50 no.3
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• pp.239-246
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• 2010

Among 203 avian pathogenic Escherichia coli (APEC) isolated from poultry with colibacillosis in korea, 14 isolates were selected from total 68 isolates transferred R plasmid and classified into 5 groups on the basis of antimicrobial minimal inhibitory concentration (MIC) pattern, farm source and O serotype. An association between clonal origin and R plasmid of them was investigated by R plasmid profile, restriction endonuclease analysis and pulsed-field gel electrophoresis (PFGE). The strains that showed the same or very similar antimicrobial MIC pattern, but different farm source and O serotype, revealed different PFGE pattern, which seemed to be different clonal origin. And the strains that showed the same MIC pattern and O serotype, revealed different PFGE pattern, seemed to be originated from different clone. Also the strains showing the same MIC pattern and farm source, but different O serotype, revealed to be different clonal origin. The strains that showed the same or similar MIC pattern, farm source, and O serotype, revealed identical or similar PFGE pattern, which seemed to belong to be one clone. Meanwhile, horizontal transfer of R plasmid seems to be common in APEC with regardless of O serotype and clone of the strains. These results indicate that rapid and accurate epidemiological survey of APEC can be possible by the combination of O serotyping, plasmid profiling and PFGE analysis following the classification of them into groups of antimicrobial drug resistance pattern.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.376-383
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• 2005

Alcaligenes sp. JMP228 carrying 2,4­dichlorophenoxyacetic acid (2,4-D) degradative plasmid pJP4 was inoculated into natural soil, and transfer of the plasmid pJP4 to indigenous soil bacteria was investigated with and without 2,4-D amendment. Plasmid pJP4 transfer was enhanced in the soils treated with 2,4-D, compared to the soils not amended with 2,4-D. Several different transconjugants were isolated from the soils treated with 2,4-D, while no indigenous transconjugants were obtained from the unamended soils. Inoculation of the soils with both the donor Alcaligenes sp. JMP228/pJP4 and a recipient Burkholderia cepacia DBO 1 produced less diverse transconjugants than the soils inoculated with the donor alone. Repetitive extragenic palindromic-polymerase chain reaction (REP-PCR) analysis of the transconjugants exhibited seven distinct genomic DNA fingerprints. Analysis of 16S rDNA sequences indicated that the transconjugants were related to members of the genera Burkholderia and Pandoraea. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes revealed that inoculation of the donor caused clear changes in the bacterial community structure of the 2,4-D­amended soils. The new 16S rRNA gene bands in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D­degrading transconjugants isolated from the soil. The results indicate that introduction of the 2,4-D degradative plasmid as Alcaligenes sp. JMP228/pJP4 has a substantial impact on the bacterial community structure in the 2,4-D-amended soil.

• Korean Journal of Microbiology
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• v.27 no.3
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• pp.194-200
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• 1989

The nucleotide sequence of inducible chloramphenicol acetyl-transferase(CAT) gene isolated from a small plasmid pSBK203 of Staphylococcus aureus was determined. The base sequence shows that structural gene of pSBK203-CAT encodes a protein of 213 amino acids and has a leader region which encodes a short polypeptide of 9 amino-acids in its upstream. vertical bar /sup 35/S vertical bar-Methionine labelled CAT gene product in minicell showed almost same mobility with pC194-CAT of which molecular weight is 24Kdal on polyacrylamide gel electrophoresis. Predicted amino acid sequence of pSBK203-CAT has revealed a high degree of homology with the CATs of pC194 and pC221 than those of cat-86, Tn9 and proteus mirabilis PM13.

• Korean Journal of Veterinary Research
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• v.26 no.2
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• pp.229-235
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• 1986

1984년 5월부터 1985년 5월까지 대구(大邱), 경북(慶北), 경남(慶南) 및 충남지역(忠南地域) 7개(個) 양돈장(養豚場)의 자돈(仔豚) 및 성돈(成豚)의 분변(糞便) 및 양돈장(養豚場)의 흙, 하수(下水), 사료(飼料), 추비(推肥), 쥐 등 7,440예(例)와 대구시(大邱市) 도축장(屠畜場)의 도축돈(屠畜豚) 장간막임파절(腸間膜淋巴節) 및 직장내용물(直腸內容物) 555예(例)로부터 분리(分離)한 319주(株)의 Salmonelia속균(屬菌)을 대상으로 항균제(抗菌劑)에 대한 내성(耐性) 및 전달성(傳達性) R plasmid의 분포상황(分布狀況)을 조사(詞査)하였던 바 그 결과(結果)는 다음과 같다. 1. 공시균(供試菌) 319주(株) 중(中) 250주(株)(78.4%)가 ampicillin(An), chloramphenicol(Cm), gentamicin(Gm), kanamycin(Km), nalidixic acid(Na), rifampicin(Rf), streptomycin(Sm), sulfadimethoxine(Su), 또는 tetracycline(Tc)에 내성(耐性)을 나타내었으며, 약제별(藥劑別)로는 Su(74.9%), Sm(53.0%) 및 Tc(28.5%)에 높은 내성(耐性)이 인정(認定)되었다. 2. 내성균(耐性菌) 250주(株)의 전달성(傳達性) R plasmid 보유율(保有率)은 51.2%(128주(株))였으며, 약제별(藥劑別)로는 Am경우 100%, Tc 92. 3% 및 Cm 75.0% 순으로 보유율(保有率)이 높았다. 3. 내성균(耐性菌) 250주(株)의 내성양상(耐性樣相)은 SmSu(91주(株)), Su(59주(株)) 및 TcSmSu(50주(株))내성형(耐性型)이 대부분이었고 R plasmid 전달후(傳達後)의 내성양상(耐性樣相)은 TcSmSu(40주(株)) 및 TcSu(28주(株)) 내성형(耐性型)이 많았다. 4. 양돈장별(養豚場別) 내성균(耐性菌) 출현빈도(出現頻度)는 48.0~93.6%로 다양(多樣)하였고, 전달성(傳達性) R plasmid 보유율(保有率)은 0~77.8%로 내성균(耐性菌) 출현빈도(出現頻度)와 일치(一致)되지 않았다. 5. 공시균(供試菌) 319주(株) 중(中) 각각(各各) 2주(株)는 Rf 및 Gm에 대해 내성(耐性)을 나타내었고, 내성균(耐性菌) 250주(株) 중(中) 73.2%(183주(株))가 다제내성(多劑耐性)이었으므로 Salmonella의 다제내성화(多劑耐性化) 경향(傾向)이 있었다. 6. 내성균(耐性菌) 250주(株) 중(中) R plasmid 전달후(傳達後) 5주(株)는 TcAmCmSmSu내성형(耐性型), 1주(株)는 TcAmKmSmSu내성형(耐性型)임이 확인되었다.

• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.176-182
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• 1994

The light-organ symbiont of pine cone fish, Vibrio fischeri, senses its presence in the host and responds to environmental changes by differentially expressing its symbiosis-related luminescence genes. The V. fischeri luminescence genes are activated by LuxR protein in the presence of an autoinducer. In an effort to elucidate the mechanism of regulation of luxR, a plasmid containing luxR was mutagenized in vitro with hydroxylamine and a luxR mutant plasmid was isolated by its ability to activate luminescence genes cloned in E. coli in the absence of the autoinducer. The specific base change identified by DNA sequencing was only single base transition at ＋78 from the transcriptional start of luxR. Based on a Western immunoblot analysis, the nucleotide change directed the synthesis of much higher level of LuxR protein without any amino acid substitutions. The results suggest that the region including the ＋78th base is presumably internal operator required for autorepression of luxR, and the increased cellular level of LuxR results in activation of luminescence genes by autoinducer independent fashion.

• Microbiology and Biotechnology Letters
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• v.36 no.2
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• pp.128-134
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• 2008

In order to examine efficient L-threo-2,3-Dihydroxyphenylserine (L-threo-DOPS) synthesis process using whole cell biocatalyst, a thermostable L-threonine aldolase (L-TA), which cloned from Streptomyces coelicolor A3(2) and improved for stability, was expressed in a Corynebacterium glutamicum R strain. The constructed Corynebacterium expression vector, pCG-H44(1) successfully expressed L-TA in C. glutamicum R strain, but showed very low expression level. In order to improve the expression level, the expression vector named pCG-H44(2) was reconstructed by eliminating 1 nucleotide between SD sequence and start codon of L-TA. The pCG-H44(2) vector plasmid was able to overexpress L-TA approximately 3.2 times higher than pCG-H44(1) in C. glutamicum R strain (CGH-2). When the whole cell of CGH-2 was examined in a repeated batch system, L-threo-DOPS was successfully synthesized with a yield of 4.0 mg/ml and maintain synthesis rate constantly after 30 repeated batch reactions for 130 h.