• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.15-21
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• 2002

Terminal-restriction fragment length polymorphism (T-RFLP) analysis has been optimized by using in vitro model community composed of genomic DNAs of known bacterial strains and has been applied to assess the bacterial community structure in marine sediments. The specific fluorescence-labeled terminal restriction fragments (T-RFs) between 39 and 839 base long specifying each strain were precisely measured for known bacterial strains. The addition of a co-solvent (dimethylsulfoxide or glycerol) into PCR reactions has reduced differential PCR amplification. Comparative bacterial community structure was investigated for pristine and polluted sediments. A complex T-RFLP pattern showing complex bacterial community structure was obtained in the pristine sediment, whereas simple T-RFLP pattern (low bacterial diversity) was shown in polluted sediments where caged aquaculture has been conducted for several years. The results of T-RFLP analysis were compared with that of cloning and sequencing 16S rDNA clones from the same sediments. Sequence analysis of 16S rDNA clones (72) of the pristine sediment revealed a diverse collection of lineages, largely of the class Proteobacteria ($6\%$ alpha subdivision, $46\%$ gamma subdivision, $13\%$ delta subdivision, and $3\%$ epsilon subdivision), Nitrospina $(8\%)$, high G+C gram positive $(8\%)$, Verrucomicrobia $(7\%)$, and Planctomycetes $(6\%)$. In the contaminated sediments, 17 $(59\%)$ of the 16S rDNA clones (29) were related to Campylobacter and symbiont of Rimicaris exoculata belonging to epsilon subdivision of Proteobacteria. The results obtained indicated that T-RFLP analysis is a rapid and precise technique for comparative bacterial community analysis.

• Korean Journal of Microbiology
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• v.45 no.2
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• pp.170-176
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• 2009

Culture-independent 16S rDNA-DGGE profiling and phylogenetic analysis were used to examine the predominant bacterial communities associated with the two sponges, Dictyonella sp. and Spirastrella abata from Jeju island. The culture-independent approach involved extraction of total bacterial DNA, PCR amplification of the 16S ribosomal DNA using primer pair 341f-GC and 518r, and separation of the amplicons on a denaturing gradient gel. Denaturing gradient gel electrophoresis banding patterns indicated 8 and 7 bands from the two sponge species, Dictyonella sp. and Spirastrella abata, respectively. There were not common major bands in two different sponges. Comparative sequence analysis of variable DGGE bands revealed from 93% to 98% similarity to the known published sequences. The dominant bacterial group of Dictyonella sp. belonged to uncultured Gammaproteobacteria, while, that of Spirastrella abata belonged to uncultured Alphaproeobacteria and Firmicutes. DGGE analysis indicated predominant communities of the sponge-associated bacteria differ in the two sponges from the same geographical location. This result revealed that bacterial community profiles of the sponges were host species-specific.

• Fisheries and Aquatic Sciences
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• v.5 no.4
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• pp.258-262
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• 2002

Effects of Escherichia coli genomic DNA on the production of phagocyte activating supernatants by the head kidney leucocytes isolated from olive flounder (Paralichthys olivaceus) were investigated. Phagocyte activating activity of the supernatants was estimated by. measuring reactive oxygen species (ROS) production in target head kidney phagocytes. All supernatants from olive flounder head kidney leucocytes-stimulated with E. coli DNA induced significantly (P<0.01) higher ROS production from target pagocytes than the unstimulated control supernatant. Maximum enhancement of chemiluminescent response was observed $5.0-10.0{\mu}g\;mL^{-1}$ of bacterial DNA while the increment ability was decreased significantly (P<0.01) at the concentration of $20.0{\mu}mL^{-1}$. The results demonstrate that olive flounder head-kidney leucocytes stimulated with bacterial DNA release a soluble phagocyte activating cytokines capable of enhancing the respiratory burst activity from target phagocytes.

• Korean Journal of Microbiology
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• v.48 no.4
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• pp.275-283
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• 2012

Culture-dependent RFLP and culture-independent DGGE were employed to investigate the bacterial community associated with the marine sponge Asteropus simplex collected from Jeju Island. A total of 120 bacterial strains associated with the sponge were cultivated using modified Zobell and MA media. PCR amplicons of the 16S rDNA from the bacterial strains were digested with the restriction enzymes HaeIII and MspI, and then assigned into different groups according to their restriction patterns. The 16S rDNA sequences derived from RFLP patterns showed more than 94% similarities compared with known bacterial species, and the isolates belonged to five phyla, Alphaproteobacteria, Gammaproteobacteria Actinobacteria, Bacteroidetes, and Firmicutes, of which Gammaproteobacteria was dominant. DGGE fingerprinting of 16S rDNAs amplified from the sponge-derived total gDNA showed 12 DGGE bands, and their sequences showed more than 90% similarities compared with available sequences. The sequences derived from DGGE bands revealed high similarity with the uncultured bacterial clones. DGGE revealed that bacterial community consisted of seven phyla, including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Actinobacteira, Chloroflexi, and Nitrospira. Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were commonly found in bacteria associated with A. simplex by both RFLP and DGGE methods, however, overall bacterial community in the sponge differed depending on the analysis methods. Sponge showed more various bacterial community structures in culture-independent method than in culture-dependent method.

• Korean Journal of Environmental Biology
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• v.18 no.1
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• pp.53-61
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• 2000

In order to characterize the genetic diversity of bacterial community in groundwater, samples were collected from used for drinking water and polluted with heavy metal wastewater in Seoul city and natural cave of Kangwondo. The DNA was amplified with 165 rDNA-based primers by use of the PCR, and then analysed ARDRA (amplified ribosomal DNA restriction analysis). Restriction endonuclease analysis patterns of amplified 165 rDNA in drinking water and wastewater relatively showed high genetic diversity in situ and drinking groundwater. The number of DNA fragments varied with in situ and drinking water. This method of ARDRA of bacterial communities in groundwater could be used for a quick assessment of genotypic changes between different locations reflecting different environmental conditions and the diversity reflected pollution of groundwater (natural cave water>drinking water>waste water, as in order of grade). [Genetic diversity, Groundwater, 165 rDNA, PCR, ARDRA].

• Korean Journal Plant Pathology
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• v.14 no.2
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• pp.177-183
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• 1998

A DNA fragment which is involved in tolassin production was cloned to obtain a molecular marker of Pseudomonas tolaasii, a casual agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). Tolaasin is a lipodepsipeptide toxin and known as a primary disease determinant of the P. tolaasii. It is responsible for formation of white line in agar when P. tolaasii were cultured against white line reacting organisms (WLROs). White line negative mutants (WL-) were generated by conjugation between rifampicin resistant strain of P. tolaasii and E. coli carrying suicidal plasmid pSUP2021 : : Tn5. The ability of tolaasin production of the WL- mutants was examined by hemolysis test, pathogenicity test, and high pressure liquid chromatography (HPLC) analysis of culture filtrate. All of the WL- mutants were lost the ability of tolaasin production (Tol-). Genomic library of the Tol- mutant was constructed in pLAFR3 and the cosmid clone containing Tn5 was selected. DNA fragment fro franking region of Tn5 was cloned from the plasmid and used as a probe in Southern blot. DNA-DNA hybridization with the probe to total DNA from group of bacteria ecologically similar to P. tolaasii including WLORs, fluorescent Pseudomonads isolated from oyster mushroom, P. agarici, P. gingeri, and some of other species of Psedomonas showed that some of the tested bacteria do not have any hybridized band and others have bands sowing RFLP. The cloned DNA fragment or its nucleotide sequence will be useful in detection and identification of the P. tolaasii.

• Korean Journal of Microbiology
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• v.46 no.3
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• pp.255-261
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• 2010

The bacterial community structure associated with two marine sponges, Hyrtios sp. 604 and Callyspongia sp. 612 collected from the South Pacific Ocean were analyzed by 16S rDNA-denaturing gradient gel electrophoresis (DGGE). The phylogenetic analysis showed that the bacterial community associated with Hyrtios sp. 604 contained diverse bacterial groups such as Chloroflexi, Firmicutes, Cyanobacteria, Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, and Acidobacteria. Callyspongia sp. 612 harbored Chloroflexi, Cyanobacteria, Alphaproteobacteria, and Gammaproteobacteria. Hyrtios sp. 604 belonging to genus Hyrtios known to produce natural products showed greater bacterial diversity than Callyspongia sp. 612. Phylum Actinobacteria was shown to be one of dominant bacterial groups in Hyrtios sp. 604. Although the same phyla of bacteria were found in both sponge species, the spongeassociated predominant bacterial groups differed between the two sponges with different chemical characteristics from the same geographical location. Uncultured bacteria represented over 90% of the bacteria diversity present in all bacterial communities of the sponges.

• Korean Journal of Microbiology
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• v.46 no.4
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• pp.366-374
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• 2010

Culture-dependent RFLP and culture-independent DGGE were employed to investigate the bacterial community associated with the marine sponge Spirastrella abata. A total of 164 bacterial strains associated with the sponge were cultivated using Zobell and Natural sea salt media. PCR amplicons of the 16S rDNA from the bacterial strains were digested with the restriction enzymes HaeIII and MspI, and then assigned into different groups according to their restriction patterns. The 16S rDNA sequences derived from RFLP patterns showed more than 95% similarities compared with known bacterial species, and the isolates belonged to four phyla, Proteobacteria (Alphaproteobacteria, Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteriodetes, of which Alphaproteobacteria was dominant. DGGE fingerprinting of 16S rDNAs amplified from the sponge- derived total gDNA showed five major DGGE bands, and their sequences showed more than 96% similarities compared with available sequences. The sequences derived from DGGE bands revealed high similarity with the uncultured bacterial clones. DGGE revealed that bacterial community consisted of four phyla, including Proteobacteria (Alphaproteobacteria, Gammaproteobacteria), Actinobacteria, Spirochetes, and Chloroflexi. Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were commonly found in bacteria associated with S. abata by both RFLP and DGGE methods; however, overall bacterial community in the sponge differed depending on the analysis methods.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.22-26
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• 2002

There are a number of ways in which environmental microbiology and microbial ecology will benefit from DNA micro array technology. These include community genome arrays, SSU rDNA arrays, environmental functional gene arrays, population biology arrays, and there are clearly more different applications of microarray technology that can be applied to relevant problems in environmental microbiology. Two types of the applications, bacterial identification chip and functional gene detection chip, will be presented. For the bacterial identification chip, a new approach employing random genome fragments that eliminates the disadvantages of traditional DNA-DNA hybridization is proposed to identify and type bacteria based on genomic DNA-DNA similarity. Bacterial genomes are fragmented randomly, and representative fragments are spotted on a glass slide and then hybridized to test genomes. Resulting hybridization profiles are used in statistical procedures to identify test strains. Second, the direct binding version of microarray with a different array design and hybridization scheme is proposed to quantify target genes in environmental samples. Reference DNA was employed to normalize variations in spot size and hybridization. The approach for designing quantitative microarrays and the inferred equation from this study provide a simple and convenient way to estimate the target gene concentration from the hybridization signal ratio.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.945-951
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• 2007

DNA-DNA hybridization has been established as an important technology in bacterial species taxonomy and phylogenetic analysis. In this study, we analyzed how the efficiency with which the genomic DNA from one species hybridizes to the genomic DNA of another species (DNA-DNA hybridization) in microarray analysis relates to the similarity between two genomes. We found that the predicted DNA-DNA hybridization based on genome sequence similarity correlated well with the experimentally determined microarray hybridization. Between closely related strains, significant numbers of highly divergent genes (>55% identity) and/or the accumulation of mismatches between conserved genes lowered the DNA-DNA hybridization signal, and this reduced the hybridization signals to below 70% for even bacterial strains with over 97% 16S rRNA gene identity. In addition, our results also suggest that a DNA-DNA hybridization signal intensity of over 40% indicates that two genomes at least shared 30% conserved genes (>60% gene identity). This study may expand our knowledge of DNA-DNA hybridization based on genomic sequence similarity comparison and further provide insights for bacterial phylogeny analyses.