Journal of Microbiology

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Effects of Crude Oil on Marine Microbial Communities in Short Term Outdoor Microcosms: Seung Won Jung , Joon Sang Park , Oh Youn Kown , Jung-Hoon Kang , Won Joon Shim , Young-Ok Kim; J. Microbiol. 2010;48(5):594-600. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0199-2

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To assess the effects of crude oil spills on marine microbial communities, 10 L outdoor microcosms were manipulated over an exposure period of 8 days. The responses of microbial organisms exposed to five crude oil concentrations in 10 to 10,000 ppm (v/v) were monitored in the microcosms. The abundance of microalgae and copepods decreased rapidly upon the addition of crude oil at concentrations over 1,000 ppm, whereas the total density of heterotrophic bacteria increased dramatically at the higher concentrations. Bacterial diversity, determined by denaturing gradient gel electrophoresis, was increased at higher concentrations. In particular, the intensity of the bands representing Jannaschia sp. and Sulfitobacter brevis increased with the addition of oil. These results indicate that crude oil spills with concentrations over 1,000 ppm seriously affected the structure of the microbial communities.

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Green Fluorescent Protein as a Marker for Monitoring a Pentachlorophenol Degrader Sphingomonas chlorophenolica ATCC39723: Eun-Taex Oh , Jae-Seong So , Byung-Hyuk Kim , Jong-Sul Kim , Sung-Cheol Koh; J. Microbiol. 2004;42(3):243-247.; DOI: https://doi.org/2081 [pii]

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Abstract PDF: Sphingomonas chlorophenolica ATCC39723 was successfully labeled with the gfp (green fluorescent protein) gene inserted into the pcpB gene by homologous recombination. As the gfp recombinant was easily distinguished from other indigenous organisms, the population of gfp recombinant was monitored after being released into the soil microcosms. Their population density dropped from 108 to 106 (cfu/ml) in the non-sterilized soil microcosms during the first 6 days. Moreover, the gfp recombinant was not detected even at lower dilution rates after a certain time period. The recombinant, however, survived for at least 28 days in the sterilized soil microcosms. Although the gfp recombinant did not degrade pentachlorophenol (PCP), this experiment showed the possibility of using gfp as a monitoring reporter system for S. chlorophenolica ATCC39723 and potentially other species of Sphingomonas.

Structural and functional stability of the genetic recombinant plasmid pCU103 in different water environments: Kim, Chi Kyung , Kwak, Myoung Ja , Lee, Sung Gie; J. Microbiol. 1996;34(3):241-247.

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Abstract PDF: The stbility of the genetically engineered microorganisms and their recombinant plasmids released in natural environments has been regarded as one of the molecular ecological topics. In this study, the recombinant plasmids pCU103 in which the pcbCD genes involved in biodegradation of biphenyl and 4-chlorobiphenyl were cloned in pBluescript SK(+) vector, were examined for their structural and functional stability in different waters at 15℃ by the methods of electrophoresis, Southern hybridization, quantification with fluorescent dye, and transformation. The recombinant plamids maintained their stabilities for about 30 days in sterilized distilled water (SDW), 15 days in autoclaved creek water (AW), 25 days in filtered and autoclaved non-sterible creek water (FAW), 4 days in Luria-Bertani (LB) broth, and less than one day in filtered non-sterile creek water (FW). The covalently closed circular (CCC) form of the plasmid was decreased and open circular (OC) form was increased as a function of incubation time, and then linear (L) form was produced to be ultimately degraded out. The degradation rates of the plasmid were proportionally correlated to trophic level of the water, and the biological factor such as DNases was found to be one of the most critical factors affecting structural and functional stability of the plasmid in non-sterile natural water.

Construction of genetically engineered miroorganisms for overexpression of xylE gene encoding catechol 2, 3-dioxygenase and the functional stability of the recombinant plasmid pSW3a containing xylE in aquatic environment: Han, Hyo Yung , Kim, Chi Kyung , Park, yong Keun , Ka, Jong Ok , Lee, Byeong Jae , Min, Kyung Hee; J. Microbiol. 1996;34(4):341-348.

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Abstract PDF: The regulation of xylE gene expression was examined by using vector promoter and construction of genetically engineered microorganisms (GEMs) for application in microcosm. When the xylE gene wsa subcloned into pBluscript SK(+) under the control of lac promoter (pTY1) in E. coli, and the expression was induced by IPTG, the enzyme activity of catechol 2, 3-dioxygenase was increased 4.7 times more than that of the crude extracts from transformants harboring pTY1. We suggest that the xylE gene has its own promoter at the upstream portion, because it was able to be expressed even in the absence of IPTG. A recombinant plasmid, pSW3a harboring the xylE gene under the T7 promotor, showed the activity of 14.5 units/mg protein, higher than that of parental strain, E. coli PYT1. The xylE gene in recombinant plasmid pSW3a was used as reporter gene for the application in microcosm ecosystem, since it was used for detection of xylE-positive clones by catechol spray on the agar plates. The pSW3a in E. coli was introduced into Pseudomonas putida to construct GEM strain, and examined for the expression and functional stability in microcosms.

Assessing Survival and Detection of Catechol-bidegrading Strains in Waste Water microcosms: Soo-Jin Choi , Min-Sup Song , Soo-youn Lee , Seong-Karp Hong , Kyung-Hee Min , Jong-Ok Ka , Chi-Kyung Kim , Young-Keun Park; J. Microbiol. 1998;36(4):239-248.

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Abstract PDF: Catechol-degrading bacteria, Escherichia coli JM101 carrying pIB1343 (strain pIB1343) and Flavimonas orizihabitans KHi (strain KH1), were chosen as model bacteria to estimate the sur vival and catechol-degradability in waste water microcosms. Before their application to ecosystem, survival and catechol degradation of these bacteria were investigated in waste water microcosms. It was found that strain pIB1343 adapted much faster to waste water environments and degraded catechol in the case of Namdong samples, whereas the strain KH1 degraded catechol much faster in Sihwa samples. When catechol was added to the microcosms, indigenous microorganisms in Sihwa samples used catechol as a carbon and energy source much better than those in Namdong samples. A modified filter extraction technique was used to obtain the high-yield purified DNA from 50 ml of waste water samples and the extracted DNA (polymorphic DNA [20~23 kb]) was of sufficient quantity and quality for the amplification. PCR was performed with catA-specicic promers, C120U and C120L, which specifically detected the catA gene encoding catechol 1,2-dioxygenase in waste water microcosms, and then the 320 bp PCR products were amplified. PCR products were quantified by densitomenter. Using the standard curve of detection limit by catA-specific PCR products, the number of catA genes for their corresponding intensities of PCR products was obtained. the number of total catA genes PCR in waste water microcosms was correalated with catechol degradation.

Analysis of the Changes in Metabolic Diversity of Microbial Community in pH-gradient Microcosm: Ahn, YoungBeom , Cho, Hong Bum , Choi, Yong Keel; J. Microbiol. 1999;37(1):1-9.

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Abstract PDF: The Biolog redox technology was carried out for evaluation of acidification effect on microbial communities at each stage of pH gradient microcosm. While the number of heterotrophic bacterial population and activities of extracellular enzyme decreased as the pH decreased, the number of total bacteria in the microcosm was not affected. The average color development of sample at each pH-gradient showed a sigmoidal curve, and at higher pH, more overall color development appeared in Biolog plates. Average color development value in Biolog plates was stabilized at 50 hours as an optimum incubation time. The color production in the Biolog plates was caused by cell density at above pH 5.0, but by cell activity below pH 4.0. Principal component analysis of color responses revealed distinctive patterns among the pH-gradient microcosm samples.

Monitoring 4-Chlorobiphenyl-Degrading Bacteria in Soil Microcosms by Competitive Quantitative PCR: Soo Youn Lee , Min Sup Song , Kyung Man You , Bae Hoon Kim , Seong Ho Bang , In Soo Lee , Chi Kyung Kim , Yong Keun Park; J. Microbiol. 2002;40(4):274-281.

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Abstract PDF: The competitive quantitative PCR method targeting pcbC gene was developed for monitoring 4-chlorobiphenyl(4CB)-degrading bacteria, Pseudomonas sp. strain DJ-12, in soil microcosms. The method involves extraction of DNA from soil contaminated with 4CB, PCR amplification of a pcbC gene fragment from the introduced strain with a set of strain-specific primers, and quantification of the electrophoresed PCR product by densitometry. To test the adequacy of the method, Pseudomonas sp. Strain DJ-12 was introduced into both contaminated and non-contaminated soil microcosms amended with 4CB. Pseudomonas sp. strain DJ-12 was monitored and quantified by a competitive quantitative PCR in comparison with 4CB degradation and the result was compared to those obtained by using the conventional cultivation method. We successfully detected and monitored 4CB-degrading bacteria in each microcosm and found a significant linear relationship between the number of 4CB-degrading bacteria and the capacity for 4CB biodegradation. The results of DNA spiking and cell-spreading experiments suggest that this competitive quantitative PCR method targeting the pcbC gene for monitoring 4Cbdegrading bacteria appears to be rapid, sensitive and more suitable than the microbiological approach in estimating the capacity of 4CB biodegradation in environmental samples.

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