• Journal of Microbiology and Biotechnology
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• v.20 no.9
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• pp.1307-1313
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• 2010

An extracellular ${\alpha}$-amylase from Lactococcus lactis IBB500 was purified and characterized. The optimum conditions for the enzyme activity were a pH of 4.5, temperature of $35^{\circ}C$, and enzyme molecular mass of 121 kDa. The genome analysis and a plasmid curing experiment indicated that $amy^+$ genes were located in a plasmid of 30 kb. An analysis of the phylogenetic relationships strongly supported a hypothesis of horizontal gene transfer. A strong homology was found for the peptides with the sequence of ${\alpha}$-amylases from Ralstonia pikettii and Ralstonia solanacearum. The protein with ${\alpha}$-amylase activity purified in this study is the first one described for the Lactococcus lactis species, and this paper is the first report on a Lactococcus lactis strain belonging to the amylolytic lactic acid bacteria (ALAB).

• 한국가시화정보학회:학술대회논문집
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• 2003.11a
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• pp.81-82
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• 2003

We have developed the method for the conjugation of biotinylated DNA to streptavidin-coated QDs. QD-DNA conjugates and a high-sensitive fluorescence imaging technique are adopted to visualize gene transport across the membrane of the live cell in real time. Endocytotic cellular uptake of oligonucleotide and electrically-mediated plasmid DNA transfer into the live cell are monitored by a quantitative microscopic imaging system. Long-term kinetic study enables us to reveal the unknown mechanisms and rate-limiting steps of extracellular and intracellular transport of biomolecules. We designed experimental protocols to conjugate the oligonucleotide or the plasmid DNA to commercially available streptavidin-coated QDs. Gel electrophoresis is used to verify the effect of incubation time and the molar ratio of QDs and DNA on the conjugation efficiency. It is possible to fractionate the QD-DNA conjugates according to the DNA concentration and obtain the purified conjugates by a gel extraction technique.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.211-220
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• 1986

Nitrogen fixation (nif) genes of Rhizobium leguminosarum were transferred to nif Klebsiella pneumoniae and E. coli by conjugation after partial heat induction of $RP_4$ :: Mu cts in Rhizobium $R^+$ transconjugant, and the hybrid plasmids in the transconjugant strains were isolated and characterized. In order to transfer the nif genes from Rhizobium, the hybrid plasmid $RP_4$ :: Mu cts was transferred by conjugation from E. coil to the symbiotic nitrogen fixer, R. leguminosarum. After stabillity test, the $RP_4$ :: Mu cts in Rhixobium $R^+$ transconjugant was subjected to partial heat induction by culturing it statically at $38^{\circ}C$ for 16 hours, and then conjugated with the nif defective mutant strains of K. pneumoniae or nif mutant strains of E. coli having whole nif gene plasmid. Recombinant strains of K. pneumoniae, which could grow in a N-free medium and exhibit the nitrogenase activity were selected. However, in the case of E. coli, they could grow well in a NA medium containing antibiotices, but hardly frow in a N-free medium. The hybrid plasmids in these transconjugal strains were isolated by gel electrophoresis and compared their molecular sizes.

• Journal of Microbiology
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• v.42 no.2
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• pp.87-93
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• 2004

Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide meco-prop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)-and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MPll, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired dif-ferent mecoprop-degradative plasmids in different soils through natural gene transfer.

• The Journal of the Korean Society for Microbiology
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• v.17 no.1
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• pp.21-34
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• 1982

One hundred and twenty-one strains each of Escherichia coli isolated from stools of 60 patients who received various antimicrobial drugs in hospital for more than one week and apparently healthy 60 students who have no history of taking antimicrobial drugs during recent one month, were tested for their resistance to 13 antimicrobial drugs. The frequency of resistance strains was highest to tetracycline with 69.2%, and followed by streptomycin(Sm), sulfisomidine(Su), chloramphenicol(Cm), ampicillin(Ap), and carbenicillin(Cb) in the decreasing order, ranging from 61.2% to 39.3%. Strains resistant to kanamycin(Km), cephaloridine(Cr), and trimethoprim(Tp) occupied about one-fourth of strains, and only four strains were resistant either one or more of nalidixic acid, gentamicin and amikacin, and no strain was resistant to rifampicin. The frequency of resistant strains to Cm, Ap, Km, Cr, and Cb was much higher among patient isolates than student strains, but strains resistant to the other drugs showed almost the same frequencies between patient and student isolates. There was a marked difference in average minimum inhibitory concentrations of between resistant and susceptible strains, suggesting that the resistance to drugs is the plasmid origin. Seventy-six percent of strains were resistant to one to 10 drugs tested, and no much difference was observed between strains from patients and students. However, strains resistant to four or more drugs were much more frequently found among patient isolates than student strains, with the increasing tendency of multiply resistant strains among patient isolates following the increase in the number of resistant drugs. The transfer of drug resistance by conjugation was tested and 98 strains(67.5%) among 145 which were resistant to two or more drugs were found to transfer their drug resistance to E. coli. Among 74 strains resistant to 7 or more drugs, all except one transferred the resistance, and the number of strains with transferable resistance decreased, as the number of resistant drugs decrease. A R plasmid from randomly selected p13 strain was tested for the incompatibility group, and the plasmid was classified into Inc F II. R plasmM DNA bands were identified by polyacrylamide gel electrophoresis.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1279-1287
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• 2012

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, $10^{-5}-10^{-3}\;recipient^{-1}$). pSTE33T showed lower efficiency ($10^{-7}-10^{-6}\;recipient^{-1}$) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.154-159
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• 1989

Chromosomal DNA fragments of Bacillus sp. YA-14 isolated from soil as a potent xylan hydrolyzing bacterium, were ligated to a vector plasmid, pBR322, and used to transfer Escherichia coli HB101 cells. The recombinant plasmid pYDC21 was found to enable the transformants to produce xylanase. pYDC21 was found to contain the 3 kb HindIII fragment originated from the Bacillus sp. YA-14 chromosomal DNA by southern hybridization. The optimum temperature and pH for the reaction of xylanse produced by E. coli (pYDC21) were appeared at 50$_7$C and pH 7.0, respectiveiy. the xylanase enzyme was stable between pH 5.0 and 7.0 and maintained stably up to 4$0^{\circ}C$.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.427.2-428
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• 2002

Previously we formulated new cationic liposomes, DDC, composed of DOTAP. DOPE, and cholesterol (Chol) in 1 : 0.7 : 0.3 molar ratios, and showed that DDC efficiently deliver the plasmid DNA into ovarian cancer cell lines. Here, wild type p53 DNA was transfected into ovarian cancer cells, using the DOC as a nonviral vector and the expression and activity of p53 gene were evaluated in vitro and in vivo. The complexes of plasmid DNA (pp53-EGFP) and DDC were transfected into OVCAR-3 cells. The gene expression was determined by RT -PCR and western blot analysis. (omitted)

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.454-460
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• 1989

Antibiotics resistance genes both in natural bacterial isolates and the genetically cloned bacteria were comparatively studied for their transfer frequencies by the method of conjugation in several different water environments. The Kmr genes in both kinds of bacteria were transferred more frequently in autoclaved wastewater of laboratory environment than in natural river water, but in Luria Bertani (LB) broth medium under the laboratory conditions the transfer frequences of the genes were much higher than in the autoclaved wastewater. The transfer frequencies at 2$0^{\circ}C$ and 3$0^{\circ}C$ were not much different in any water environments. The Km$^{${\gamma}$}$ genes of the genetically cloned bacteria and the natural isolates were transferred at the same frequency both in natural river water and in the autoclaved wastewater of laboratory environment, but in LB broth under laboratory conditions the transfer frequencies were lowered by 10$^{-3}$ to 10$^{-4}$ in the genetically cloned cells than the natural isolates. When donors of different cloned cells were conjugated with recipient of a natural isolates, the Km$^{${\gamma}$}$ genes of different donor cells were transferred at the about same frequency, but the same donor of the cloned cell were conjugated with recipients of different natural isolates, the transfer of Km$^{${\gamma}$}$ gene of the cloned cell showed some differences of 101 to 102 in frequency.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.189-194
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• 2006

Sperm-mediated gene transfer(SMGT) can be used to transfer exogenous DNA into the oocyte at fertilization. The main objective of this study was to assess efficiency of transferring mitochondrial DNA(mtDNA) fragment into boar spermatozoa in either presence or absence of liposome and quality of transfected spermatozoa. The mtDNA of chicken liver was isolated and purified by phenol and alkaline lysis extraction, and it was inserted to plasmid. The genome of transfected spermatozoa treated with DNase I was purified by alkaline lysis, and then amplified by the PCR analysis. After electrophoresis, DNA quantitation of each well was calculated by comparison of the band intensity with standard. As a result, exogenous DNA was composed of mtDNA fragment(1.2 kb) and plasmid(2.7 kb). On the other hand, efficiency of transfection by liposome($9.0{\pm}0.34ng/{\mu}l$) in SMGT was higher than that by DNA solution($6.9{\pm}0.53ng/{\mu}l$). However, there was no significant difference. Transfering exogenous DNA into spermatozoa was completed within 90 min of incubation. In another experiment, there were significant (p<0.05) differences between transfected spermatozoa using both DNA solution and DNA/liposome completes with unheated spermatozoa for viability ($70.8{\pm}1.80$ and $68.0{\pm}2.16%$ vs. $83.3{\pm}1.69%$, respectively) and motility($78.7{\pm}1.59$ and $79.3{\pm}2.14%$ vs. $86.7{\pm}1.59%$, respectively). This study indicates that exogenous mtDNA can be efficiently transferred into boar spermatozoa regardless of the presence of liposome, and transfected spermatozoa can also use insemination and in vitro fertilization to generate transgenic pig.