• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.624-630
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• 2008

Terminal restriction fragment length polymorphism (T-RFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct T-RFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.

• Journal of Ecology and Environment
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• v.38 no.4
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• pp.461-476
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• 2015

Global warming has accelerated glacial retreat in the high Arctic. The exposed glacier foreland is an ideal place to study chronosequential changes in ecosystems. Although vegetation succession in the glacier forelands has been studied intensively, little is known about the microbial community structure in these environments. Therefore, this study focused on how glacial retreat influences the bacterial community structure and its relationship with soil properties. This study was conducted in the foreland of the Midtre Lovénbreen glacier in Svalbard (78.9°N). Seven soil samples of different ages were collected and analyzed for moisture content, pH, soil organic carbon and total nitrogen contents, and soil organic matter fractionation. In addition, the structure of the bacterial community was determined via pyrosequencing analysis of 16S rRNA genes. The physical and chemical properties of soil varied significantly along the distance from the glacier; with increasing distance, more amounts of clay and soil organic carbon contents were observed. In addition, Cyanobacteria, Firmicutes, and Actinobacteria were dominant in soil samples taken close to the glacier, whereas Acidobacteria were abundant further away from the glacier. Diversity indices indicated that the bacterial community changed from homogeneous to heterogeneous structure along the glacier chronosequence/distance from the glacier. Although the bacterial community structure differed on basis of the presence or absence of plants, the soil properties varied depending on soil age. These findings suggest that bacterial succession occurs over time in glacier forelands but on a timescale that is different from that of soil development.

• Korean Journal of Environmental Agriculture
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• v.34 no.2
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• pp.139-148
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• 2015

BACKGROUND: The aim of this study was to isolate and identify algicidal bacterium that tends to kill the toxic dinoflagellate Alexandrium catenella, and to determine the algicidal activity and algicidal range of algicide. METHODS AND RESULTS: Among of algicidal bacteria isolated in this study, NH-3 isolate was the strongest algicidal activity against A. catenella. NH-3 isolate was identified on the basis of biochemical characteristics and analysis of 16S rRNA gene sequences. The NH-3 isolate showed over 99% homology with Arthrobacter oxydans, and was designated as Arthrobacter sp. NH-3. The optimal culture conditions were $25^{\circ}C$, initial pH 7.0, and 2.0% (w/v) NaCl concentration. The algicidal activity of Arthrobacter sp. NH-3 was significantly increased to maximum value in the late of logarithmic phase. Arthrobacter sp. NH-3 showed algicidal activity through indirect attack, which excreted active substance into the culture filtrate. When 10% culture filtrate of NH-3 was applied to A. catenella, 100% of algal cells were destroyed within 30 h. In addition, the algicidal activities were increased in dose and time dependent manners. The pure algicide was isolated from the ethyl acetate extract of the culture filtrate of NH-3 by using silica gel column chromatography and high performance liquid chromatography (HPLC). We investigated the algicidal activity of this algicide on the growth of harmful algal bloom (HAB) species, including A. catenella. As a result, it showed algicidal activity against several HAB species at a concentration of $100{\mu}g/mL$ and had a relatively wide host range. CONCLUSION: Taken together, our results suggest that Arthrobacter sp. NH-3 and its algicide could be a candidate for controlling of toxic and harmful algal blooms.

• Korean Journal of Environmental Biology
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• v.29 no.3
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• pp.195-201
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• 2011

Today, the weather is changing continually, due to the progress of global warming. As the weather changes, the habitats of different organisms will change as well. It cannot be predicted whether or not the weather will change with each passing day. In particular, the biological distribution of the areas climate change affects constitutes a major factor in determining the natural state of indigenous plants; additionally, plants are constantly exposed to rhizobacteria, which are bound to be sensitive to these changes. Interest has grown in the relationship between plants and rhizopheric microorganisms. As a result of this interest we elected to research and experiment further. We researched the dominant changes that occur between plants and rhizospheric organisms due to global warming. First, we used temperature as a variable. We employed four different temperatures and four different sites: room temperature ($27^{\circ}C$), $+2^{\circ}C$, $+4^{\circ}C$, and $+6^{\circ}C$. The four different sites we used were populated by the following strains: Pinus densiflora, Pinus koraiensis, Quercus acutissima. We counted colonies of these plants and divided them. Then, using 16S rRNA analysis we identified the microorganisms. In conclusion, we identified the following genera, which were as follows: 24 strains of Bacillus, 6 Paenibacillus strains, 1 Pseudomonas strains. Among these genera, the dominant strains in Pinus densiflora was discovered in the same genus. Additionally, those of Pinus koraiensis and Quercus acutissima changed in both genus and strains which changed into the Bacillus genus from the Paenibacillus genus at $33^{\circ}C$.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.19
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• pp.8295-8299
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• 2014

Background: H pylori is the main causative agent of Gastric cancer and chronic gastritis. Genetic diversity of H. pylori has major contribution in its pathogenesis. We investigated the prevalence of oipA and iceA1/iceA2 positive strains of H. pylori among patients with gastric cancer and gastritis. Materials and Methods: Sampling performed by means of endoscopy from 86 patients. DNA was extracted from tissue samples using DNA extraction kit. PCR assay was performed and products were monitored by Agarose Gel Electrophoresis. Results: Urease Test and 16S rRNA PCR did not show significant differences in detection of H. pylori. The frequency of iceA1 allele in patients with gastric cancer was significantly higher than those with gastritis (p<0.05). However, there was no significant difference in prevalence of oipA and iceA2 genes among the two groups of patients (p>0.05). Conclusions: The iceA1 gene, but the oipA and iceA2 genes, is associated with H. pylori-induced gastric cancer. However, confirmatory studies must be performed in future.

• The Plant Pathology Journal
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• v.34 no.1
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• pp.23-34
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• 2018

The Ralstonia solanacearum species complex (RSSC) can be divided into four phylotypes, and includes phenotypically diverse bacterial strains that cause bacterial wilt on various host plants. This study used 93 RSSC isolates responsible for potato bacterial wilt in Korea, and investigated their phylogenetic relatedness based on the analysis of phylotype, biovar, and host range. Of the 93 isolates, twenty-two were identified as biovar 2, eight as biovar 3, and sixty-three as biovar 4. Applied to the phylotype scheme, biovar 3 and 4 isolates belonged to phylotype I, and biovar 2 isolates belonged to phylotype IV. This classification was consistent with phylogenetic trees based on 16S rRNA and egl gene sequences, in which biovar 3 and 4 isolates clustered to phylotype I, and biovar 2 isolates clustered to phylotype IV. Korean biovar 2 isolates were distinct from biovar 3 and 4 isolates pathologically as well as genetically - all biovar 2 isolates were nonpathogenic to peppers. Additionally, in host-determining assays, we found uncommon strains among biovar 2 of phylotype IV, which were the tomato-nonpathogenic strains. Since tomatoes are known to be highly susceptible to RSSC, to the best of our knowledge this is the first report of tomato-nonpathogenic potato strains. These results imply the potential prevalence of greater RSSC diversity in terms of host range than would be predicted based on phylogenetic analysis.

• Journal of Microbiology and Biotechnology
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• v.27 no.12
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• pp.2089-2093
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• 2017

The past decades have been a golden era during which great tasks were accomplished in the field of microbiology, including food microbiology. In the past, culture-dependent methods have been the primary choice to investigate bacterial diversity. However, using culturein-dependent high-throughput sequencing of 16S rRNA genes has greatly facilitated studies exploring the microbial compositions and dynamics associated with health and diseases. These culture-independent DNA-based studies generate large-scale data sets that describe the microbial composition of a certain niche. Consequently, understanding microbial diversity becomes of greater importance when investigating the composition, function, and dynamics of the microbiota associated with health and diseases. Even though there is no general agreement on which diversity index is the best to use, diversity indices have been used to compare the diversity among samples and between treatments with controls. Tools such as the Shannon-Weaver index and Simpson index can be used to describe population diversity in samples. The purpose of this review is to explain the principles of diversity indices, such as Shannon-Weaver and Simpson, to aid general microbiologists in better understanding bacterial communities. In this review, important questions concerning microbial diversity are addressed. Information from this review should facilitate evidence-based strategies to explore microbial communities.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.9
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• pp.1332-1339
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• 2017

Objective: This study investigated the effects of Bacillus subtilis CSL2 (B. subtilis CSL2) administration before Salmonella challenge on the fecal microbiota and microbial functionality of Hy-line Brown (HLB) laying hens. Methods: Fecal samples were collected from control (CON), Salmonella-infected (SAL) and Salmonella-infected, probiotic-treated (PRO) groups before and after Salmonella challenge for microbiome analysis using 16S rRNA gene pyrosequencing. Results: Infection with Salmonella led to decreased microbial diversity in hen feces; diversity was recovered with Bacillus administration. In addition, Salmonella infection triggered significant alterations in the composition of the fecal microbiota. The abundance of the phylum Firmicutes decreased while that of Proteobacteria, which includes a wide variety of pathogens, increased significantly. Bacillus administration resulted in normal levels of abundance of Firmicutes and Proteobacteria. Analysis of bacterial genera showed that Salmonella challenge decreased the population of Lactobacillus, the most abundant genus, and increased populations of Pseudomonas and Flavobacterium genera by a factor of 3 to 5. On the other hand, Bacillus administration caused the abundance of the Lactobacillus genus to recover to control levels and decreased the population of Pseudomonas significantly. Further analysis of operational taxonomic units revealed a high abundance of genes associated with two-component systems and secretion systems in the SAL group, whereas the PRO group had more genes associated with ribosomes. Conclusion: The results of this study indicate that B. subtilis CSL2 administration can modulate the microbiota in HLB laying hens, potentially acting as a probiotic to protect against Salmonella Gallinarum infection.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.918-922
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• 2001

In a previous study, we have isolated a number of lactobacilli from Korean women, and one of them (KLB46) was identified as Lactobacillus crispatus by 16S rRNA gene sequencing. For the ecological treatment of bacterial vaginosis (BV) cell suspension of L. crispatus KLB46 was instillated into BV patients. L. crispatus KLB46 was found to persist for several days in cell suspension with no nutrients. In this study, in order to assess the influence of starvation on physiological activity, we compared the viability and culturability of KLB46 following suspension in various buffer solutions. A pair of in situ fluorescent dye was used to assess viability (i.e. membrane integrity) and the culturability was examined by plate count assay. A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit $(BacLight^{TM})$ was applied to estimate both viable and total counts of bacteria in cell suspension. $BacLight^{TM}$ is composed of two nucleic acid-binding stains ($SYTO\;9^{TM}$ and propidium iodide). $SYTO\;9^{TM}$ penetrates all bacterial membranes and stains the cells green while propidium iodide only penetrates cells with damaged membranes, therefore the combination of the two stains produces red fluorescing cells. Optimal staining conditions for $BacLight^{TM}$ were found to be with 0.0835M $SYTO\;9^{TM}$ and 0.05M propidium iodide for 15 min incubation at room temperature in dark. When cells were microscopically examined during 140 hours of starvation, the culturability decreased markedly while the viability remained relatively constant, which suggests that large fraction of KLB46 cells became viable but non-culturable (VBNC) upon starvation.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.31-32
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• 2005

Neoagaro-oligosaccharides are produced only by enzymatic degradation of agarose by ${\beta}-agarase.^{1)}$ Neoagaro-oligosaccharides inhibit the growth of bacteria, slow the rate of degradation of starch, are used as low-calorie additives to improve food quality, and have macrophage-stimulating activity. Furthermore, neoagarobiose is a rare reagent that has both moisturizing effect on skin and whitening effect on melanoma $cells.^{2)}$ An agar-degrading marine bacterium was isolated from the sea water at the northeast coast in Cheju island, Korea. The strain was gram negative, aerobic, and motile rod. The 16S rRNA of the strain had the closest match of 98% homology, with that from Agarivorans albus. On the basis of several phenotypic characters and a phylogenetic analysis, this strain was designated Agarivorans sp. JA-1. In solid agar plate, Agarivorans sp. JA-1 produced a diffusible agarase that caused agar softening around the colonies. Agarivorans sp. JA-1 was cultured for 36 hr in marine broth 2216 (Difco, USA) and the supernatant that containing an extracellular ${\beta}-agarase$ was prepared by centrifugation of culture media. The enzyme exhibited relatively strong activity at $40^{\circ}C$ and was stable up to $60^{\circ}C$. Using PCR primers derived from the ${\beta}-agarase$ gene of Vibrio sp., the gene encoding ${\beta}-agarase$ from Agarivorans sp. JA-1 was cloned and sequenced. The structural gene consists of 2931 bp encoding 976 amino acids with a predicted molecular weight of 107,360 Da. The deduced amino acid sequence showed 99% and 34% homology to $agaA^{2)}$ and $agaB^{2)}$ genes for ${\beta}-agarase$ from Vibrio sp., respectively. The expression plasmid for ${\beta}-agarase$ gene of Agarivorans sp. JA-1 is being constructed and the recombinant enzyme will be biochemically characterized.