• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1614-1620
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• 2015

Plasmid-cured derivative strains of Bacillus anthracis are frequently used in laboratory studies. Plasmid incompatibility, which does not increase the risk of chromosomal mutation, is a useful method for plasmid curing. However, in bacteria containing multiple plasmids, it often requires the sequential introduction of multiple, specific incompatibility plasmids. This lengthy process renders the traditional plasmid incompatibility method inefficient and mutation-prone. In this study, we successfully cured plasmids pXO1 and pXO2 from B. anthracis A16 simultaneously using only one recombinant incompatible plasmid, pKORT, to obtain a plasmid-free strain, designated A16DD. This method may also be useful for the simultaneous, one-step curing of multiple plasmids from other bacteria, including Bacillus thuringiensis and Yersinia pestis.

• Microbiology and Biotechnology Letters
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• v.10 no.4
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• pp.289-295
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• 1982

Streptomyces bobili (YS-40) isolated from soil was tested that it had drug resistance against penicillin, cephalosporin series antibiotics and other antibiotics in the previous paper. The treatment of Streptomyces bobili, (YS-40) with ethidium bromide (EtBr), acriflavine and sodium dodecyl sulfate. (SDS) resulted in the elimination of R-plasmid from the host strain. Minimum growth inhibitory concentrations (MIC) of Hg, Ag, penicillin-G, ampicillin, chloramphenicol, oxytetracycline, streptomycin and kanamycin were found to be 15, 10, > 3, 000, > 100, > 1, 000, > 100, < 5 and < 5$\mu\textrm{g}$/$m\ell$ respectively. Among the curing agents, EtBr was proved to be the most powerful compound for the elimination of R-plasmid in the strain and the elimination rate with EtBr(10$\mu\textrm{g}$/$m\ell$) was about 98%. Optimal pH to. the elimination of R-plasmid was pH 7.0 and the R-plasmid in the cells incubated for 24 hrs was proved to be eliminated most effectively. Aerial mass color, soluble pigment formation and reverse side color were reported to be often the plasmid associated characteristics of the R-plasmid bearing bacteria. But these characteristics of the uncured and cured Streptomyces bobili, (YS-40) showed no changes in the most of the pigment formation media tested in this work.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.142-147
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• 1989

Six plasmids of B. thuringiensis serovar. kurstaki HD73 were detected, with approximate sizes of 7.4, 7.8, 8.1, 11.3, and 75 Kb, as well as a low copied plasmid of similar length to 75 Kb. Partially cured mutants from B. thuringiensis HD73 were obtained either by the treatment of the curing agent, ethidium bromide(0.02 $\mu\textrm{g}$/$m\ell$) or by spontaneous curing, Acrystalliferous mutants(Cry$^-$) were identified by microscopic observation and immunoblotting with polyclonal antibody against 133 KD deltaendotoxin of HD73. Ten Cry$^-$ mutants were found to be lack of 75 Kb plasmid. These results implicated that this plasmid was associated with delta-endotoxin production, After isolating the mutants, we streaked them on potato dextrose agar, spizizen casamino acid glucose, starch agar, and nutrient agar. Only on starch agar medium did morphologies of Cry$^-$ appear translucent and light greyish. On the other hand, the mutants of B. thuringiensis isolated from Korean soil, designated KBS722, were obtained by the treatment of novobiocin (3 $\mu\textrm{g}$/$m\ell$). Acrystalliferous mutants of KBS722 were less translucent than HD73 mutants' only on nutrient agar medium. Compared the plasmid profile of the mutants with delta-endotoxin production, the results seemed to indicate that the insecticidal protein gene of B. thuringiensis isolates KBS722 located on about 225 Kb plasmid DNA.

• Journal of Microbiology
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• v.43 no.3
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• pp.251-256
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• 2005

In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, $17\%,\;58\%,\;and\;25\%$ were found to exhibit a high degree of resistance to $200\;{\mu}g/ml$ of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least $50\;{\mu}g/ml$ of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least $50\;{\mu}g/ml$ of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.

• Korean Journal of Microbiology
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• v.27 no.3
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• pp.181-187
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• 1989

Plasmid DNA was isolated from Lactobacillus casei SW-M1($Lac^{+}$strain). The curing frequencies of pPLac plasmid from L. casei SW-M1 showed 43% for acriflavin treatment and 53% for ethidium bromide treatment after 3 times transfer. On the charaterization of pPLac plasmid, it was found that the plasmid contained gene encoding phospho-$\beta$-galactosidase for lactose utilization. Lactose-PTS(phosphotransferase system)was involved in membrane transport system in $Lac^{+}$ strain. Induction of phospho-$\beta$-galactosidase was specially effective by galactose, lower effect with lactose and glucose but not by IPTG(isopropyl-$\beta$-D-thiogalactoside). This result showed that induction of phospho-$\beta$-galactosidase by IPTG did not appeared. The catabolite repression of phospho-$\beta$-galactosidase synthesis by glucose was not found in L. casei.

• Korean Journal of Microbiology
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• v.37 no.2
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• pp.120-123
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• 2001

Sphingomonas chungbukensis DJ77 is able to use phenanthrene and biphenyl as the sole carbon and energy source. Mitomycin C curing experiment suggested that polyaromatic hydrocarbon (PAH) utilization in strain DJ77 was plasmid-encoded. The plasmid cured strains were failed to grow on the minimal medium sprayed with biphenyl or phenanthrene. This was evident from southern hybridizations using a previously cloned DNA segment as a probe. There were positive signals in the palsmid DNA of the wild-type strain DJ77 and the absence of hybridizations with chromosomal DNA from the plasmid DNA of the wild-type strain DJ77 and the absence of hybridizations with chromosomal DNA from the palsmid-cured mutant strains.

• Biomolecules & Therapeutics
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• v.3 no.2
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• pp.154-158
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• 1995

We isolated Streptomyces spry. from Korean soil, which showed high cytotoxicity against tumor cell lines, L1210 and P388Dl. Among 30 strains, three strains (DKM 104, DKM 128, DKM 409) were appeared to possess plasmid. Strain DKM 104 and DKM 128 had two CCC(Covalently Closed Circular) form plasmid, about 20 Kb in size and about 1 Kb compared with λ Hind III DNA size marker. And strain DKM 409 had three plasmids, among which two plasmic were CCC form about 20 Kb and about 20 Kb compared with same size marker. To find out whether plasmid involved in production of antitumor agent or not, we performed to curing experiment. Comparing cytotoxicity between culture filtrate of plasmid-containing strains and cured strains, we knew that only the cytotoxic activity of the strain DKM 128 was involved in plasmid.

• Korean Journal of Microbiology
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• v.20 no.1
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• pp.11-20
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• 1982

A study was undertaken to examine the effect of curing agents on the stability, curing and segregation of R plasmid pSL100. And also the stability, transfer frequency, and recombination of its segregants obtained from curing agent treatment were studied. Ethidium bromide, acridine orange, and mitomycin-C were used as curing agent. The results obtained were as follows ; 1. The curing agent ethidium bromide, acridine orange, and mitomycin-C were not effective for curing the multiple antibiotic resistant determinant of pSL100 in Salmonella typhimurium and Escherichia coli. However, they induced plasmid segregation with high frequency in S.typhimuruim LT-2strains. TcApSmCm, TcSmCmKm, TcApCm, TcAp, TcKm, Tc segregants were obtained. 2. The resistant markers of the segregents were transferred to S.typhimurium LT-2 strains with high frequencies whereas they were transferred to E.coli K-12 only with low frequencies. 3. The transconjugants obtained from conjugation between two different S.typhimurium segregants were similar to the phenotype of the original R factor pSL100 and the resistant markers were transferred to the S.typhimurium LT-2 or E.coli strain with equal frequencies, indicating that they are recombinants. 4. The transconjugants obtained from conjugation between pSL100 segrgants and pKM101, or pBR322 possessed the resistant markers of the two parental plasmids and they were transferred to both S.typhimurium and E.coli K-12 strains with the same frequencies and maintained stably, suggesting that they are also recombinants. 5. The recombinant pSL100 could be also obtained in rec A-strains of E.coli, suggesting that the gene function of rec A is required for the recombination of pSL100 segregants in E.coli.

• YAKHAK HOEJI
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• v.36 no.3
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• pp.255-258
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• 1992

The clinical isolate Staphylococcus aureus SA8 was resistant to tetracycline(Tc) and harboured a plasmid pKH1(24.82 kb). pKH1 was shown by curing and by transformation to specify resistance to Tc. The cleavage map of a pKH1 was determined by restricction enzyme mapping techniques. Cleavage map is given for BglII, EcoRI, HpaII, PvuII and SalI. Restriction endonuclease BamHI, BglI, BstEII, HpaI, PstI, and XhoI have no sites on this plasmid. HaeIII, XbaI, and HindIII have 5, 6, 14 sites, respectively.

• Korean Journal of Microbiology
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• v.17 no.1
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• pp.25-41
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• 1979

A strain able to grow up m-toluate minimal medium has been isolated after selective enrichment and given the name T81X, which was later identified as pseudomonase putida according to its morphological and biochemical characteristics. After treatment with plasmied specific curing agent, mitomycin C, followed by replica plating on m-toluate and xylene minimal agar plate, T81Xstrain has been shown to harbour a curable plasmid relating to the m-toluate and xylene metabolism. Spontaneous curing frquency of this plasmid was also greatly enhanced by growing on benzoate minimal medium. After then, it was also xylene metabogrowing on benzoate minimal medium. After then, it was found to be conjugally nontransmissible. From the comparative investigation of catechol 1,2-oxygenase and catechol 2,3-oxygenase activities in wild type and cured strain on various growth substrate, it appeared that T81X strain has both of these two enzymes while cured strain has catechol 1,2-oxygenase only. Growing on m-toluate minimal medium T81X strain should carry the genetic information necessary for coding the catechol 1,2-oxygense induced by m-toluate or benzoate, on that curable plasmid.