• Korean Journal of Veterinary Research
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• v.41 no.2
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• pp.157-165
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• 2001

Amplified fragment length polymorphism(AFLP) technique is based on the polymorphism detection through selective PCR amplification of restriction fragments from digested genomic DNA and thus includes the procedures of the total DNA digestion by endonucleases, ligation of adapters to the ends of the fragments, and following the selective amplification of the restricted DNA fragments. This study were aimed to : (1) determine the genetic variability of S aureus strains, (2) estimate genetic diversity within and among these strains, (3) compare phylogenetic relationships among these strains as genetic markers using AFLP techniques. Genomic DNA was digested with a particular combination of three restriction enzymes with specific recognition sites and the DNA fragments were ligated to restriction specific adapters and amplified using the selective primer combinations. In the S aureus strain, the number of scorable AFLP bands detected per each primer combination varied from 29 to 102, with an average of 61.59 using 27 primer combinations. A total of 1,663 markers were generated, 904 bands of which were polymorphic, showing a 33.48% level of polymorphism with these primer combinations. Among the primer combinations, E02/T02, E02/T03, E04/H02, E02/T01 and E04/H03 primer combinations showed a high level of polymorphism with 0.78, 0.76, 0.74, 0.71 and 0.70, respectively. But T03/H01, E01/T02 and E01/T03 primer combinations showed a low level of polymorphism with 0.38, 0.37 and 0.15, respectively, Therefore, the former primer combinations will be the most effective for AFLP analysis of S aureus. In SA1 sub-types the level of polymorphism of S aureus KCTC 1927 was similar to that of S aureus CU 01(0.825) and higher than those of other strains such as S aureus CU 02 (0.715), S aureus KCTC 2199(0.625), S aureus KCTC 1916(0.607) and S aureus KCTC 1621 (0.553). In SA2 sub-types the level of polymorphism of S aureus CU 07 was similar to that of S aureus CU 08(0.935) and higher than those of both S aureus CU 04(0.883) and S aureus CU 05(0.883) and lower than those of S aureus CU 03(0.583). In SA3 subtypes the level of polymorphism of S aureus CU 11 was similar to that of S aureus CU 12(0.913) and lower than that of S aureus CU 15(0.623). The results proved that AFLP marker analysis of S aureus strain could be used to study the epidemiology of mastitis and in addition, common genotype in geographic region could be useful for the development of an effective vaccine or DNA marker for easy diagnosis of mastitis caused by S aureus infection.

• Journal of Life Science
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• v.24 no.9
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• pp.995-1000
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• 2014

The studies of marine viruses in terms of viral isolation and detection have been limited due to the high mutation rate and genetic diversity of marine viruses. Of the modern methods currently used to detect marine viruses, serological methods based on enzyme-linked immunosorbent assay (ELISA) are the most common. They depend largely on the quality of the antibodies and on highly purified suitable antigens. Recently, a new experimental system for using viral capsid protein as an antigen has been developed using the yeast surface display (YSD) technique. In the present study, the capsid protein gene of the red-spotted grouper nervous necrosis virus (RGNNV) was expressed and purified via YSD and HA-tagging systems, respectively. Two regions of the RGNNV capsid protein gene, RGNNV1 and RGNNV2, were individually synthesized and subcloned into a yeast expression vector, pCTCON. The expressions of each RGNNV capsid protein in the Saccharomyces cerevisiae strain EBY100 were indirectly detected by flow cytometry with fluorescently labeled antibodies, while recognizing the C-terminal c-myc tags encoded by the display vector. The expressed RGNNV capsid proteins were isolated from the yeast surface through the cleavage of the disulfide bond between the Aga1 and Aga2 proteins after ${\beta}$-mercaptoethanol treatment, and they were directly detected by Western blot using anti-HA antibody. These results indicated that YSD and HA-tagging systems could be applicable to the expressions and purification of recombinant RGNNV capsid proteins.

• Journal of Life Science
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• v.28 no.12
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• pp.1477-1488
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• 2018

This study investigated the muscles, intestines, and gonads of Sulculus diversicolor supertexta to examine the diversity of microbial communities within examples collected from the Jeju Coast. Using different media, initial pure isolation in MA, 1% BHIA, and 1% TSA indicated that the muscles, intestines, and gonads supported more communities, respectively. In analysis of relative similarity with 16s rRNA sequencing, 190 pure colonies were isolated, and further analysis with NBLAST identified 71 species, 39 genera, 25 families, and five phyla. Homogeny with the reference strain was 91-100%. Microbial communities in S. supertexta consisted of gamma and alpha Proteobacteria (48%), Actinobacteria (32.5%), Firmicutes (16.9%), Deinococcus-Thermus (1.3%), and Bacteroides (1.3%). In all tissue, Psychrobacter cibarius in Moraxellaceae was dominant. Alteromonadaceae, Enterobacteriaceae, Pasturellaceae, Moraxellaceae, Rhodobacteraceae, Geminicoccaceae, Dietziaceae, Intrasporangiaceae, Microbacteriaceae, Micrococcaceae, Micromonosporaceae, Streptomycetaceae, Aerococcaceae, Bacillaceae, Paenibacillaceae, Planococcaceae, and Staphylcoccaceae were commonly isolated across all tissues, and Flavobacteriaceae, Corynebacteriaceae, Yesiniaceae, Vibrionaceae, Hahellaceae, Pseudomonadaceae were also identified from the intestines. In microbial monitoring of four harmful bacteria, Streptomyces albus (96%) showed antibacterial activity against all four strains, and Agrococcus baldri (99%) and Psychrobacter nivimaris (99%) presented against E. Coli and E. aerogens. In addition, some strains with low homogeny were isolated and further experiments are therefore required, for example to refine the antimicrobial substances including new strain investigations. These additional experiments would aim to establish generic resources for the microbial communities in S. Supertexta and provide basic data for applied microbiological research.

• The Korean Journal of Malacology
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• v.30 no.4
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• pp.383-390
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• 2014

The multiplex PCR system including six microsatellites from Haliotis discus hannai, consisting of dinucleotide and trinucleotide repeat units, is developed. The six loci were coamplified in a single reaction employing dye-labeled primers. Alleles from these loci were sized using an internal standard by automated sample processing in an ABI3100 Genetic Analyser. Amplified alleles in profiles containing selected microsatellites were typed clearly, providing easily interpretable results. In this results suggest that the presented multiplex PCR system may be a useful tool in a selective breeding program of H. discus hannai in which genetic identification will allow different genotypes to be reared together from fertilization. This should have a great impact as it will make selective breeding more efficient. Moreover, it will be useful in a variety of applications, including strain and hybrid identification, parentage assignment, pedigree reconstruction, estimating genetic diversity and/or inbreeding.

• Journal of Species Research
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• v.5 no.1
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• pp.1-13
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• 2016

To discover and characterize indigenous species in Korea, a total of 31 bacterial strains that belong to the phylum Actinobacteria were isolated from various niches in Korea. Each strain showed the high sequence similarity (>99.1%) with the closest bacterial species, forming a robust phylogenetic clade. These strains have not been previously recorded in Korea. According to the recently updated taxonomy of the phylum Actinobacteria based upon 16S rRNA trees, we report 25 genera of 13 families within 5 orders of the class Actinobacteria as actinobacterial species found in Korea. Cellular morphology, Gram staining, basic biochemical characteristics are described in the species description.

• ALGAE
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• v.30 no.3
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• pp.183-195
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• 2015

The dinoflagellate genus Pelagodinium is genetically classified in distinct sub-clades and subgroups. However, it is difficult to determine whether this genetic diversity represents intra- or interspecific divergence within the genus since only the morphology of the type strain of the genus Pelagodinium, Pelagodinium bei, is available. An isolate associated with the genus Pelagodinium from Shiwha Bay, Korea, was recently cultured. This isolate was clustered with 3 to 4 strains from the Atlantic Ocean, Mediterranean Sea, and Indian Ocean. This cluster was distinct from the subgroup more closely associated with P. bei. The morphology of the isolate was analyzed using optical and scanning electron microscopy and was almost identical to that of P. bei except that this isolate had two series of amphiesmal vesicles (AVs) in the cingulum, unlike P. bei that has one series. When the pigment compositions of the isolate and P. bei were analyzed using high-performance liquid chromatography, these two strains had peridinin as a major accessory pigment and their pigment compositions were almost identical. In addition, the swimming behaviors of these two strains were very similar. The reexamination of the type culture of P. bei revealed two series in the cingulum as for the isolate. The new findings on the number of series of AVs in the cingulum, the pigment composition, and the swimming behaviors suggest that P. bei and the isolate are conspecific despite their genetic divergence. This study provides a basis to further understand the molecular classification within Pelagodinium combining genetic, morphological, pigment, and behavioral data.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.68-76
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• 2006

Despites many attempts to explore the microbial diversity in kimchi fermentation, the predominant flora remains controversial to date. In the present study, major lactic acid bacteria (LAB) were investigated in Chinese cabbage kimchi in the early phase of fermention. For the samples over pH 4.0, viable cell counts of Leuconostoc and Pediococcus were $10^6\;cfu/ml$ and below $10^2\;cfu/ml$, respectively, and 20 isolates out of 172 were subjected to a biochemical identification (API 50 CH kit) as well as molecular-typing methods including ITSPCR with a RsaI digestion and 16s rRNA gene sequence analysis for species confirmation. Seven isolates were nicely assigned to Lb. brevis, 6 to Leuconostoc spp. (2 mesenteroides, 2 citreum, I carnosum, I gasicomitatum), 4 to Weissella (3 kimchii/cibaria, 1 hanii) and 2 to other Lactobacillus spp. (1 farciminis, 1 plantarum). On the other hand, the biochemical identification data revealed 9 strains of Lb. brevis, 6 strains of Leuconostocs,2 strains of Lb. plantarum and 1 strain each of Lb. coprophilus and Lactococcus lactis. However, a single isolates, YSM 16, was not matched to the ITS-PCR database constructed in the present study. Two Lb. brevis strains by API 50 CH kit were reassigned to W kimchii/cibaria, Lb. coprophilus or W hanii, respectively, judging from the results by the above molecular typing approaches. As a whole, the identification data obtained by the biochemical test were different from those of ITS-PCR molecular method by about $63\%$ at genus-level and $42\%$ at species-level. The data by the ITS-PCR method conclusively suggest that predominant LAB species is probably heterolactic Lb. brevis, followed by W kimchii/cibaria, Leuc. mesenteroides, and Leuc. citreum, in contrast to the previous reports [3] that Leuc. mesenteroides is the only a predominant species in the early phase kimchi fermentation.

• Journal of Microbiology and Biotechnology
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• v.26 no.5
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• pp.891-899
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• 2016

The recently isolated aniline-degrading bacterium Pseudomonas migulae AN-1 was tagged with green fluorescent protein (GFP) to investigate its bioaugmentation potential against aniline-contaminated groundwater through microcosm experiments. The survival and cellular response of GFP-tagged AN-1 introduced in a lab-scale aquifer corresponded directly with aniline consumption. During the process, the GFP-tagged AN-1 biomass increased from 7.52 × 10⁵ cells/ml to 128 × 10⁵ cells/ml and the degradation rate of aniline was 6.04 mg/l/h. GFP-tagged AN-1 was moderately hydrophobic (41.74%-47.69%) when treated with 20-100 mg/l aniline and exhibited relatively strong hydrophobicity (55.25%-65.78%) when the concentration of aniline was ≥100 mg/l. The membrane permeability of AN-1 increased followed by a rise in aniline below 100 mg/l and was invariable with aniline above 100 mg/l. Pyrosequencing analysis showed that the relative abundance of Proteobacteria (accounted for 99.22% in the non-bioaugmentation samples) changed to 89.23% after bioaugmentation with GFP-tagged AN-1. Actinobacteria increased from 0.29% to 2.01%, whereas the abundance of Firmicutes barely changed. These combined findings demonstrate the feasibility of removing aniline in aquifers by introducing the strain AN-1 and provide valuable information on the changes in the diversity of dominant populations during bioaugmentation.

• Journal of Microbiology and Biotechnology
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• v.19 no.8
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• pp.749-759
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• 2009

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDS-PAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.

• Journal of Microbiology and Biotechnology
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• v.20 no.8
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• pp.1210-1214
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• 2010

Many bacteria are involved in the fermentation of doenjang, and Bacillus species are known to perform significant roles. Although SDS-PAGE has been frequently used to classify and identify bacteria in various samples, the microbial diversity in doenjang has not yet been investigated. This study aims to determine the identity and distribution of dominant Bacillus species in doenjang using SDS-PAGE profiles of whole-cell proteins and 16S rRNA gene sequencing. Reference Bacillus strains yielded differential SDS-PAGE banding patterns that could be considered to be highly specific fingerprints. Grouping of bacterial strains isolated from doenjang samples by whole-cell protein patterns was confirmed by analysis of their 16S rRNA gene sequences. B. subtilis was found to be the most dominant strain in most of the samples, whereas B. licheniformis and B. amyloliquefaciens were less frequently found but were also detected in several samples. The results obtained in this study show that a combined identification method using SDS-PAGE profiles of whole-cell proteins and subsequent 16S rRNA gene sequence analysis could successfully identify Bacillus species isolated from doenjang.