• Title/Summary/Keyword: Strain Amplification

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Isolation and Characterization of 2-Methyl-4-Chlorophenoxyacetic Acid-Degrading Bacteria from Agricultural Soils

  • Cho, Seung-Hee;Ka, Jong-Ok
    • Journal of Applied Biological Chemistry
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    • v.42 no.2
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    • pp.57-61
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    • 1999
  • Seven numerically dominant 2-methyl-4-chlorophenoxyacetic acid (MCPA)-degrading bacteria were isolated from agricultural soils. The isolates utilized the herbicide MCPA as a sole carbon source, producing significant biomass in MCPA mineral medium. They exhibited diverse herbicide degradation capabilities, but most of them grew very slowly in mineral medium containing herbicide. The chromosomal DNA patterns of the isolates obtained by polymerase chain reaction amplification of repetitive extragenic palindromic sequences were distinct from each other. One isolate, SH3, which was identified as Sphingomonas species by fatty acid methyl ester analysis, was able to degrade 5 different phenoxyacetic acid herbicides within 4 days. This strain contains two plasmids, and the smaller one has a crucial role in herbicide degradation. MCPA treated into agricultural soils without indigenous MCPA-degraders persisted for a long time, but the application of the isolate SH3 resulted in rapid decline of MCPA concentration in the soil.

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Genetic and Phenotypic Diversity of Dichlorprop-Degrading Bacteria Isolated from Soils

  • Park, Hae-Dong;Ka, Jong-Ok
    • Journal of Microbiology
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    • v.41 no.1
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    • pp.7-15
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    • 2003
  • Nine dichlorprop-degrading bacteria and three pairs of bacteria showing syntrophic metabolism of the herbicide were isolated from soils, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genera, Sphingomonas, Herbaspirillum, and Bradyrhizobium. Twelve different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 15 isolates. The isolates were able to utilize the herbicide dichlorprop as a sole source of carbon and energy and their dichlorprop derogative pathways were induced by the presence of dichlorprop. Most of the isolates and syntrophic pairs were able to degrade both (R)- and (S)-dichlorprop, but strain DP522 exhibited enantioselective degradation of (S)-dichlorprop. The isolates degraded 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid , and mecoprop, in addition to dichlorprop. Oxygen uptake experiments indicated that most of the isolates degraded dichlorprop through 2,4-dichlorophenol.

Development of L-Threonine Producing Recombinant Escherichia coli using Metabolic Control Analysis (대사 조절 분석 기법을 이용한 L-Threonine 생산 재조합 대장균 개발)

  • Choi, Jong-Il;Park, Young-Hoon;Yang, Young-Lyeol
    • KSBB Journal
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    • v.22 no.1
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    • pp.62-65
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    • 2007
  • New strain development strategy using kinetic models and metabolic control analysis was investigated. In this study, previously reported mathematical models describing the enzyme kinetics of intracellular threonine synthesis were modified for mutant threonine producer Escherichia coli TF5015. Using the modified models, metabolic control analysis was carried out to identify the rate limiting step by evaluating the flux control coefficient on the overall threonine synthesis flux exerted by individual enzymatic reactions. The result suggested the production of threonine could be enhanced most efficiently by increasing aspartate semialdehyde dehydrogenase (asd) activity of this strain. Amplification of asd gene in recombinant strain TF5015 (pCL-$P_{aroF}$-asd) increased the threonine production up to 23%, which is much higher than 14% obtained by amplifying aspartate kinse (thrA), other gene in threonine biosynthesis pathway.

The Virulence Factors of Vibrio spp. (비브리오의 병원성 인자에 관한 연구)

  • Oh, Yang-Hyo;Kim, Yung-Bu;Park, Young-Min;Kim, Min-Jung;Cha, Mi-Sun;Kim, Young-Hee;Lim, Eun-Gyoung
    • The Journal of the Korean Society for Microbiology
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    • v.34 no.6
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    • pp.513-518
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    • 1999
  • A total of 113 Vibrio sp. strains were examined for plasmid content which were subjected to digestion with restriction enzymes. About the 55% Vibrio spp. have the plasmid more than one. Most of these plasmid various derivatives ranged from $2.4\;kb{\sim}23\;kb$, especially two strains of V. mimicus and one strain of V. furnissii carried one high-molecular weight plasmid (molecular weight ranging between $70\;kb{\sim}100\;kb$). Results of restriction analysis for plasmid of this three strains were by no means the rule. For detection of tdh and ctx gene, the virulence factor involved in the pathogenesis, we carried out the TDH and CT assay, PCR amplification, and hybridization. A total 11 strains were produced TDH, involved in 9 strains of V. parahaemolyticus and 1 strain of V. alginolyticus from clinical isolates and 1 strains of V. mimicus from environmental isolates. In the experiments of tdh gene detection, in all, 3 strains of V. parahaemolyticus from clinical isolates and 2 strains from environmental isolates could be successfully amplified in 400 bp by PCR. The PCR results were consistent with DNA hybridization tests. In the experiments of CT assay, in all, 3 strains of V. cholerae from clinical isolate and 1 strain of V. cholerae from environmental isolates were observed CT-producing. These CT-producing strains amplified in 302 bp by PCR for the detection of ctx gene. All CT-producing strains hybridized with digoxigenin-labeled DNA probe, while CT-negative strains did not hybridize. Also hybridization tests results for detection of ctx gene consistent with PCR.

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Screening and Characterization of Probiotic Strains for Prevention of Bacterial Fish Diseases (어류의 세균성 질병 예방을 위한 Probiotic균주의 선발 및 특성)

  • 허문수;양병규;전유진
    • Microbiology and Biotechnology Letters
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    • v.31 no.2
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    • pp.129-134
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    • 2003
  • The purpose of the present study was to screen the effective of lactic acid bacteria (LAB) as probiotics, which are able to protect aquacultural fish pathogenic bacteria, and investigate their characterization. Twenty strains of lactic acid bacteria were isolated from fish intestine, fermented fish foods and kimchis. These bacteria were screened for antagonistic activity against fish pathogenic bacteria. Seven tested LAB strains were able to inhibit the fish pathogenic bacteria, including Vibrio anguillarum, Edwardsiella tarda, and Streptococcus sp.. Of the probiotic candidates, BK19 strain isolated from fermented pollack viscera indicated the largest inhibition activity. Moreover, this strain showed a resistance over low pH and antibiotic agents. Therefore this probiotic candidate BK19 was finally selected and identified as a probiotic strain. This particular probiotic bacteria was identified as Lactobacillus sakei BK19 by biochemical characteristics and 165 rRNA PCR amplification.

Characterization of a Ligninase Producing Strain, Serratia marcescens HY-5 isolated from Sympetrum dopressiusculum (고추좀잠자리 (Sympetrum depressiusculum)로부터 분리한 리그닌 분해균주, Serratia marcescens HY-5의 특성)

  • Kim, Ki-Duck;Park, Doo-Sang;Shin, Dong-Ha;Han, Bo-Na;Oh, Hyun-Woo;Youn, Young-Nam;Park, Ho-Yong
    • Korean journal of applied entomology
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    • v.45 no.3 s.144
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    • pp.301-307
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    • 2006
  • A ligniolytic bacterial strain was isolated from the digestive tract of a red dragonfly, Sympetrum dopressiusculum. It was identified as a Serratia marcescens HY-5 by 16S rDNA sequence analysis and physiological and biochemical analysis. The isolated strain showed proportional increase of ligninolytic activity to the cell growth in the culture media which include lignin compounds. It showed about 25-45% decomposition of lignin compound by 48 hr incubation especially, showed effective decomposition of monomer lignin compounds, vanillin and guaiacol, and a dimer, dealkaline lignin. PCR amplification of 16S rDNA followed by denaturing gradient gel electrophoresis analysis showed high density of S. marcescens HY-5 in the gut of the S. depressiusculum at both gut samples which collected at different site.

Induction by Carvone of the Polychlorinated Biphenyl (PCB)-Degradative Pathway in Alcaligenes eutrophus H850 and Its Molecular Monitoring

  • Park, Young-In;So, Jae-Seong;Koh, Sung-Cheol
    • Journal of Microbiology and Biotechnology
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    • v.9 no.6
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    • pp.804-810
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    • 1999
  • There is a possibility that carvone, a monoterpene from spearmint (Mentha spicata), could induce the bph degradative pathway and genes in Alcaligenes eutrophus H850, which is a known Gram-negative PCB degrader with a broad substrate specificity that was thoroughly investigated with Arthrobacter sp. BIB, a Gram-positive PCB degrader. The strains BIB and H850 were unable to utilize and grow on the plant terpene [(R)-(-)-carvone] (50ppm) to be recognized as a sole carbon source. Nevertheless, the carvone did induce 2,3-dihydroxybiphenyl 1,2-dioxygenase (encoded by bphC) in the strain B lB, as observed by a resting cell assay that monitors accumulation of a yellow meta ring fission product from 4,4'-dichlorobiphenyl (DCBp). The monoterpene, however, did not appear to induce the meta cleavage pathway in the strain H850. Instead, an assumption was made that the strain might be using an alternative pathway, probably the ortho-cleavage pathway. A reverse transcription (RT)-PCR system, utilizing primers designed from a conserved region of the bphC gene of Arthrobacter sp. M5, was employed to verify the occurrence of the alternative pathway. A successful amplification (182bp) of mRNA transcribed from the N-terminal region of the bphC gene was accomplished in H850 cells induced by carvone (50ppm) as well as in biphenyl-growth cells. It is, therefore, likely that H850 possesses a specific PCB degradation pathway and hence a different substrate specificity compared with B1B. This study will contribute to an elucidation of the dynamic aspects of PCB bioremediation in terms of roles played by PCB degraders and plant terpenes as natural inducer substrates that are ubiquitous and environmentally compatible.

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Potential Evaluation and Health Fostering Intrinsic Traits of Novel Probiotic Strain Enterococcus durans F3 Isolated from the Gut of Fresh Water Fish Catla catla

  • Alshammari, Eyad;Patel, Mitesh;Sachidanandan, Manojkumar;Kumar, Prashant;Adnan, Mohd
    • Food Science of Animal Resources
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    • v.39 no.5
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    • pp.844-861
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    • 2019
  • Over the last few years, marine environment was found to be a source of surplus natural products and microorganisms with new bioactive secondary metabolites of interest which can divulge nutritional and biological impact on the host. This study aims to assess the possible, inherent and functional probiotic properties of a novel probiotic strain Enterococcus durans F3 (E. durans F3) isolated from the gut of fresh water fish Catla catla. Parameters for evaluating and describing the probiotics described in FAD/WHO guidelines were followed. E. durans F3 demonstrated affirmative results including simulated bile, acid and gastric juice tolerance with exhibited significant bactericidal effect against pathogens Staphylococcus aureus, Salmonella Typhi, Escherichia coli and Pseudomonas aeruginosa. This can be due to the enterocin produced by E. durans F3 strain, which was resolute by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel with amplification of the anticipated fragment of a structural gene; enterocin A, followed by antibiotic susceptibility assessment. Effective antioxidant potentiality against ${\alpha}$-diphenyl-${\alpha}$-picrylhydrazyl free radicals including lipase, bile salt hydrolase activity with auto-aggregation and cell surface hydrophobicity was similarly observed. Results are proving the potentiality of E. durans F3, which can also be used as probiotic starter culture in dairy industries for manufacturing new products that imparts health benefits to the host. Finding the potent and novel probiotic strains will also satisfy the current developing market demand for probiotics.

Genetic and Phenotypic Diversity of Fenitrothion-Degrading Bacteria Isolated from Soils

  • Kim, Kyung-Duk;Ahn, Jae-Hyung;Kim, Tae-Sung;Park, Seong-Chan;Seong, Chi-Nam;Song, Hong-Gyu;Ka, Jong-Ok
    • Journal of Microbiology and Biotechnology
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    • v.19 no.2
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    • pp.113-120
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    • 2009
  • Twenty-seven fenitrothion-degrading bacteria were isolated from different soils, and their genetic and phenotypic characteristics were investigated. Analysis of the 16S rDNA sequence showed that the isolates were related to members of the genera Burkholderia, Pseudomonas, Sphingomonas, Cupriavidus, Corynebacterium, and Arthrobacter. Among the 27 isolates, 12 different chromosomal DNA fingerprinting patterns were obtained by polymerase chain reaction(PCR) amplification of repetitive extra genic palindromic(REP) sequences. The isolates were able to utilize fenitrothion as a sole source of carbon and energy, producing 3-methyl-4-nitrophenol as the intermediate metabolite during the complete degradation of fenitrothion. Twenty-two of 27 isolates were able to degrade parathion, methyl-parathion, and p-nitrophenol but only strain BS2 could degrade EPN(O-ethyl-O-p-nitrophenyl phenylphosphorothioate) as a sole source of carbon and energy for growth. Eighteen of the 27 isolates had plasmids. When analyzed with PCR amplification and dot-blotting hybridization using various specific primers targeted to the organophosphorus pesticide hydrolase genes of the previously reported isolates, none of the isolates showed positive signals, suggesting that the corresponding genes of our isolates had no significant sequence homology with those of the previously isolated organophosphate pesticide-degrading bacteria.

PCR technique for detection of toxigenic Pasteurella multocida in mixed bacterial cultures from pigs (Polymerase chain reaction을 이용한 독소생산성 Pasteurella multocida의 검출)

  • Chi, Yongzhe;Lee, Dong-seok;Han, Jeong-hee;Han, Kyung-soo;Hahn, Tae-wook
    • Korean Journal of Veterinary Research
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    • v.40 no.1
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    • pp.56-62
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    • 2000
  • Pasteurella multocida is kind of commensal bacteria in the upper respiratory tract of pigs. It is classified toxigenic and nontoxigenic strains based on the production of dermonecrotic toxin. Toxigenic strain is most associated with atrophic rhinitis which brings great economical loss in swine industry. However, toxigenic and nontoxigenic strains do not differ by diagnostic biochemical reaction or morphology. One of recently developed techniques, PCR detects the toxigenic P multocida. Amplification of an 846-nucleotide fragment of toxA gene was developed. The fragment amplified by PCR was detected in P multocida type D not type A. The PCR amplification was as sensitive as it could detect 1 pg of P multocida DNA. We compared the result of the PCR with the enzyme linked immunosorbent assay (ELISA) in a test for 40 swine nasal swabs. All of these isolates were toxin negative based on the ELISA while 2 isolates were detected in the PCR technique. in addition to accuracy, as required for rapid detection from contaminated nasal swabs, toxigenic P multocida was recovered efficiently from contaminated culture without inhibition of the PCR. The results show that the PCR detection of toxigenic P multocida directly form nasal swabs are feasible.

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