• Food Science and Biotechnology
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• v.18 no.2
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• pp.343-349
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• 2009

Inhibitory effect of Escherichia coli O157 adhered to Caco-2 cells by the cells of Lactobacillus plantarum K11 and the cell-free culture supernatant (CFCS) and bacteriocin prepared from this strain was investigated. As the cell counts of viable L. plantarum K11 previously adhered to Caco-2 were increased, the rate of adhesion and adherent cell counts of E. coli O157 was lower. However, because the heated L. plantarum K11 rarely have the adhesion ability to Caco-2, the adhesion rate and adherent cell counts of E. coli O157 were high. In addition, the inhibitory effects of E. coli O157 adhesion by the CFCS and bacteriocin of L. plantarum K11 were dose-dependent manner. Therefore, the inhibition of adhesion of E. coli O157 to Caco-2 may result from the antimicrobial substances such as lactic acid and bacteriocin. Moreover the inhibitory activity of adhesion by the heated bacteriocin for 30 min at 100oC was similar to activity of non-treated bacteriocin, but the activity was disappeared by treatment with protease.

• Food Science and Biotechnology
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• v.17 no.1
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• pp.191-194
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• 2008

The antagonistic activity of probiotic strains (Bifidobacterium animalis BB-12, Bifidobacterium bifidum A, Bifidobacterium longum B6, Lactobacillus acidophilus ADH, Lactobacillus paracasei ATCC 25598, and Lactobacillus rhamnosus GG) against nalidixic acid resistant ($NA^R$) Escherichia coli O157:H7 MF1847, E. coli O157:H7 H2439, E. coli O157:H7 ATCC 43894, and E. coli O157:H7 C7927 was investigated using the agar-overlay, well diffusion, and broth culture tests. L. paracasei ATCC 25598 was the most effective probiotic strain in terms of in vitro antagonistic activity against $NA^R$ E. coli O157:H7, followed by L. rhamnosus GG, B. longum B6, and L. acidophilus ADH. The use of selected probiotic strains could be an effective pre-harvest intervention strategy to reduce the risk of $NA^R$ E. coli O157:H7 by maintaining a balanced microflora in animals and might provide many potential benefits in lieu of using antimicrobials.

• Environmental Engineering Research
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• v.14 no.1
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• pp.63-67
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• 2009

Escherichia coli K-12 (E. coli K-12) is a representative indicator globally used for distinguishing and monitoring dynamic fates of pathogenic microorganisms in the environment. This study investigated how to most critically quantify lacZ ($\beta$-galactosidase) gene in E. coli K-12 by two different real-time polymerase chain reaction (real-time PCR) in association with three different DNA extraction practices. Three DNA extractions, i.e., sodium dodecyl sulfate (SDS)/proteinase K, magnetic beads and guanidium thiocyanate (GTC)/silica matrix were each compared for extracting total genomic DNA from E. coli K-12. Among them, GTC/silica matrix and magnetic beads beating similarly worked out to have the highest (22-23 ng/${\mu}L$) concentration of DNA extracted, but employing SDS/proteinase K had the lowest (10 ng/${\mu}L$) concentration of DNA retrieved. There were no significant differences in the quantification of the copy numbers of lacZ gene between SYBR Green I qPCR and QProbe-qPCR. However, SYBR Green I qPCR obtained somewhat higher copy number as $1{\times}10^8$ copies. It was decided that GTC/silica matrix extraction or magnetic beads beating in combination with SYBR Green I qPCR can be preferably applied for more effectively quantifying specific gene from a pure culture of microorganism.

• Proceedings of the Microbiological Society of Korea Conference
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• 2008.05a
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• pp.135-135
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• 2008

Various biosensors were evaluated for identifying and detecting foodborne pathogens in a rapid and effective manner. First, five strains of Escherichia coli and six strains of Salmonella were identified using Fourier transform infrared spectroscopy and a statistical program. For doing this, lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) were extracted from a cell wall of each bacterial strain. As a result, each strain was identifed at the level of 97% for E. coli and 100% for Salmonella. Second, E. coli O157:H7, S. Enteritidis, and Listeria monocytogenes were identified by multiplex PCR products from four specific genes of each bacteria using a capillary electrophoresis (CE). Also, ground beef for E. coli O157:H7, lettuce for S. Enteritidis, and hot dog for L. monocytogenes were used to determine the possibility of detecting pathogens in foods. Foods inoculated with respective pathogen were cultivated for six hours and multiplex PCR products were obtained and assessed. The minimum detection levels of tested bacteria were <10 cells/g, <10 cells/g, and $10^4$ cells/g for E. coli O157:H7, S. Enteritidis, and L. monocytogenes, respectively. Third, it was possible to detect S. Typhimurium in a pure culture and lettuce by a bioluminescence-based detection assay using both recombinant bacteriophage P22::luxI and a bioluminescent bioreporter. In addition, bacteriophage T4 was quantitatively monitored using E. coli including luxCDABE genes.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.226.2-226.2
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• 2003

The FimH subunit of type 1-fimbriated Escherichia coli has been determined as a major cause of urinary tract infection. To produce a possible vaccine antigen against urinary tract infection, the fimH gene was genetically linked to the Itxa2b gene, which was then cloned into the pMAL -p2E expression vector. The chimaeric construction of pMALfimH/Itxa2b was transformed into Escherichia coli TB1 and its N-terminal amino acid sequence was analyzed. (omitted)

• Microbiology and Biotechnology Letters
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• v.27 no.3
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• pp.260-265
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• 1999

The structural $\beta$-galactosidase gene (lacZ) from Lactococcus lactis ssp. lactis 7962 was cloned into plamid vector pKF18, which was designated as pKF-gal. Expression of the lacZ from L. lactis 7962 was found to be higher when cells were grown at 3$0^{\circ}C$ than 37$^{\circ}C$. Maximum $\beta$-galactosidase activity was obtained when E. coli/pKF-gal was cultivated for 6hr at 3$0^{\circ}C$ and for 3hr at 37$^{\circ}C$, and L. lactis 7962 was grown for 8hr at 3$0^{\circ}C$. Enzyme induction was achieved by the addition of lactose, galactose, or lactose+IPTG to growing culture. The addition of glucose had no effect on enzyme induction.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.93-97
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• 2008

RraA is a recently discovered protein inhibitor that regulates the enzymatic activity of RNase E, which plays a major role in the decay and processing of RNAs in Escherichia coli. It has also been shown to regulate the activity of RNase ES, a functional Streptomyces coelicolor ortholog of RNase E, which has 36% identity to the amino-terminal region of RNase E. There are two open reading frames in S. coelicolor genome that can potentially encode proteins having more than 35.4% similarity to the amino acid sequence of RraA. DNA fragment encoding one of these RraA orthologs, designated as RraAS2 here, was amplified and cloned in to E. coli vector to test whether it has ability to regulate RNase E activity in E. coli cells. Co-expression of RraAS2 partially rescued E. coli cells over-producing RNase E from growth arrest, although not as efficiently as RraA, induced by the increased ribonucleolytic activity in the cells. The copy number of ColEl-type plasmid in these cells was also decreased by 14% compared to that in cells over-producing RNase E only, indicating the ability of RraAS2 to inhibit RNase E action on RNA I. We observed that the expression level of RraAS2 was lower than that of RraA by 4.2 folds under the same culture condition, suggesting that because of inefficient expression of RraAS2 in E. coli cells, co-expression of RraAS2 was not efficiently able to inhibit RNase E activity to the level for proper processing and decay of all RNA species that is required to restore normal cellular growth to the cells over-producing RNase E.

• Journal of Korean Society of Water and Wastewater
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• v.23 no.2
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• pp.199-205
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• 2009

It is very important to differentiate of DNA derived from live or dead bacteria within mixed microbial communities in aquatic environments. Ethidium monoazide (EMA) is a DNA intercalating agent and the treatment of EMA with strong visible light cleaves the genomic DNA of bacteria. In dead bacterial cells, EMA intercalates into the genomic DNA, induces the cleavage of DNA, and inhibits the PCR amplification. In this study, we developed the EMA-PCR and EMA real-time PCR to detect the DNA derived from viable Escherichia coli (E.coli) in mixed cultures of live and dead E.coli. The treatment of EMA, $50{\mu}g/mL$, and 650 W visible halogen light exposure for 2 minutes cleaved the genomic DNA derived from heat killed E.coli but did not those of live E.coli. EMA-PCR could detect the DNA from live E.coli in mixed culture samples of live and dead E.coli at various ratio and there was no DNA amplification in only dead E.coli cultures. Similar results were observed in EMA real-time PCR. Further studies are needed to develop various EMA-PCR methods to detect viable waterborne pathogens such as Helicobacter pylori, Giardia lamblia, and so on.

• KSBB Journal
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• v.31 no.1
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• pp.27-32
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• 2016

Several CoA transferases from Clostridium beijerinckii, C. perfringens and Klebsiella pneumoniae were examined for biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) in recombinant Escherichia coli XL1-Blue strain. The CB3819 gene and the CB4543 gene from C. beijerinckii, the pct gene from C. perfringens and the pct gene from K. pneumoniae, which encodes putative CoA transferase gene, respectively, was co-expressed with the Pseudomonas sp. MBEL 6-19 phaC1437 gene encoding engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) to examine its activity for the construction of key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli XL1-Blue expressing the phaC1437 gene and CB3819 gene synthesized poly(3-hydroxybutyrate) [P(3HB)] homopolymer to the P(3HB) content of 60.5 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 3-hydroxybutyrate. Expression of the phaC1437 gene and CB4543 gene in recombinant E. coli XL1-Blue also produced P(3HB) homopolymer to the P(3HB) content of 51.2 wt% in the same culture condition. Expression of the phaC1437 gene and the K. pneumoniae pct gene in recombinant E. coli XL1-Blue could not result in the production of PHAs in the same culture condition. However, the recombinant E. coli XL1-Blue expressing the phaC1437 gene and the C. perfringens gene could produce poly(3-hydroxybutyrate-co-lactate [P(86.4mol%3HB-co-13.7 mol%LA) up to the PHA content of 10.6 wt% in the same culture condition. Newly examined CoA transfereases in this study may be useful for the construction of engineered E. coli strains to produce PHA containing novel monomer such lactate.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.10
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• pp.1550-1554
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• 2003

One hundred of lactic cultures were evaluated for their ability to inhibit hazardous microbes, such as Salmonella enteritidis, Salmonella typhimurium, Escherichia coli, Listeria monocytogenes, and Bacillus cereus by agar well diffusion method. None of them showed inhibitory halo against S. enteritidis, while 27 strains showed inhibitory activity against S. typhimurium, 6 against E. coli, 9 against ampicillin resistant E. coli, 31 against L. monocytogens, 10 against B. cereus. pH of the culture does not explain for the inhibitory activity except against B. cereus. A neutralized culture from corn silage showed highest inhibitory activity against S. typhimurium, and the size of inhibitory halo was same as 10 ug/mL of ampicillin. The culture was identified to be Lactobacillus buchneri on the basis of biochemical characteristics and utilization of substrates. Using the culture as probiotics could be expected to reduce antibiotics for animal feeding.