• 한국축산시설환경학회지
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• 제21권3호
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• pp.93-98
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• 2015

The objective of this study was to review application of next-generation sequencing (NGS) to investigate microbiome in the livestock sector. Since the 16S rRNA gene is used as a phylogenetic marker, unculturable members of microbiome in nature or managed environments have been investigated using the NGS technique based on 16S rRNA genes. However, few NGS studies have been conducted to investigate microbiome in the livestock sector. The 16S rRNA gene sequences obtained from NGS are classified to microbial taxa against the 16S rRNA gene reference database such as RDP, Greengenes and Silva databases. The sequences also are clustered into species-level OTUs at 97% sequence similarity. Microbiome similarity among treatment groups is visualized using principal coordinates analysis, while microbiome shared among treatment groups is visualized using a venn diagram. The use of the NGS technique will contribute to elucidating roles of microbiome in the livestock sector.

• Animal Bioscience
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• 제36권2_spc호
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• pp.364-373
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• 2023

The gastrointestinal (GI) tract of ruminants contains diverse microbes that ferment various feeds ingested by animals to produce various fermentation products, such as volatile fatty acids. Fermentation products can affect animal performance, health, and well-being. Within the GI microbes, the ruminal microbes are highly diverse, greatly contribute to fermentation, and are the most important in ruminant nutrition. Although traditional cultivation methods provided knowledge of the metabolism of GI microbes, most of the GI microbes could not be cultured on standard culture media. By contrast, amplicon sequencing of 16S rRNA genes can be used to detect unculturable microbes. Using this approach, ruminant nutritionists and microbiologists have conducted a plethora of nutritional studies, many including dietary interventions, to improve fermentation efficiency and nutrient utilization, which has greatly expanded knowledge of the GI microbiota. This review addresses the GI content sampling method, 16S rRNA gene amplicon sequencing, and bioinformatics analysis and then discusses recent studies on the various factors, such as diet, breed, gender, animal performance, and heat stress, that influence the GI microbiota and thereby ruminant nutrition.

• The Plant Pathology Journal
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• 제17권4호
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• pp.189-193
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• 2001

The relationships between the phytoplasmas infecting Zizyphus jujuba and Paulownia coreana were investigated by PCR-RELP. The 16S rRNA genes of the phytoplasmas were analyzed and compared with each other after PCR amplification. The amplified bands 1.4 kb in size were analyzed by both restriction digestion and sequencing after cloning into a plasmid vector. In some cases, two different kinds of inserts were observed in the isolates that originated from a single plant. However, many of them appeared to be the amplification products of chloroplastic 16S rRNA gene of host plants. The phytoplasma gene could be differentiated from the chloroplastic gene by restriction digestion of the plasmids carrying the amplification products. Only the recombinant plasmids carrying phytoplasma 16S rRNA gene produced a 1.4 kb band when digested with the enzyme BanII. Of the 52 recombinant plasmids analyzed, 42 appeared to contain inserts that originated from the chloroplastic 16S rRNA gene of the host plants. No variation was detected among 16S rRNA gene of nine phytoplasma isolates infecting Z. jujuba. However, the phytoplasmas infecting Z. jujuba were different from that infecting P. coreana.

• 환경생물
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• 제41권3호
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• pp.308-324
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• 2023

This study investigated unrecorded freshwater bacterial species in Korea. Water and sediment samples were collected from the Nakdong River basin from 2020-2022. Bacterial isolates obtained through the conventional culture method with commercial media were subjected to 16S rRNA gene sequencing to identify unrecorded bacterial species. Results of 16S rRNA gene sequencing of the bacterial isolates revealed that a total of 44 bacterial isolates shared 16S rRNA gene sequence similarities of more than 98.65%, with validly published bacterial species not reported in Korea yet. These isolates were phylogenetically assigned to 4 phyla, 7 classes, 21 orders, 33 families, and 42 genera. A total of 2, 6, 12, and 24 species belonged to phyla Bacillota, Bacteroidota, Actinomycetota, and Pseudomonadota, respectively. Here, we provide details of these 44 unrecorded bacterial species, including Gram staining, colony and cellular morphologies, biochemical properties, and phylogenetic position.

• 한국동물위생학회지
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• 제37권3호
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• pp.157-164
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• 2014

Clostridium chauvoei is the etiologic agent of blackleg, a high mortality rated disease infection mainly cattle. In the present study, the partial sequences of 16S rRNA and flagellin gene of C. chauvoei isolated in Jeonbuk, Korea were determined and compared with those of reference strain. Oligonucleotide primers were designed to amplify a 811 bp fragment of 16S rRNA gene and 1229 bp fragment of flagellin gene. Sequencing analysis of 16S rRNA gene showed high homology to the reference strains ranging 82.3% to 100%, while flagellin gene were different from published foreign clostridia, showing 98.7% to 72.0% nucleotide sequence homology. Phylogenetic analysis based on 16S rRNA gene revealed the close phylogenetic relationship of C. chauvoei and C. septicum in cluster I, which includes C. carnis, C. tertium, C. quinii, C. celatum, C. perfringens, C. absonum, C. botulinum B. Phylogentic analysis also revealed that flagellin gene formed a single cluster with C. chauvoei, C. septicum, C. novyi A, C. novyi B, C. tyrobutylicum, C. acetobutylicum. The genetic informations obtained from this study could be useful for the molecular study of C. chauvoei.

• Journal of Microbiology and Biotechnology
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• 제28권7호
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• pp.1112-1121
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• 2018

Jeotgal is a Korean traditional fermented seafood with a high concentration of salt. In this study, we isolated lactic acid bacteria (LAB) from galchi (Trichiurus lepturus, hairtail) and myeolchi (Engraulis japonicas, anchovy) jeotgal on MRS agar and MRS agar containing 5% NaCl (MRS agar+5% NaCl), and identified them by using 16S rRNA gene sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as culture-dependent methods. We also performed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) as a culture-independent method to identify bacterial communities. Five samples of galchi-jeotgal and seven samples of myeolchi-jeotgal were collected from different regions in Korea. A total of 327 and 395 colonies were isolated from the galchi- and myeolchi-jeotgal samples, respectively. 16S rRNA gene sequencing and MALDI-TOF MS revealed that the genus Pediococcus was predominant on MRS agar, and Tetragenococcus halophilus on MRS agar+5% NaCl. PCR-DGGE revealed that T. halophilus, Tetragenococcus muriaticus, and Lactobacillus sakei were predominant in both types of jeotgal. T. halophilus was detected in all samples. Even though the same species were identified by both culture-dependent and -independent methods, many species identified by the culture-dependent methods were not in the bacterial list identified by the culture-independent methods. The distribution of bacteria in galchi-jeotgal was more diverse than in myeolchi-jeotgal. The diverse LAB in galchi- and myeolchi-jeotgals can be further studied as candidates for starter cultures to produce fermented foods.

• Environmental Engineering Research
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• 제14권1호
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• pp.3-9
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• 2009

Microorganisms play an important role in the geochemical cycles, industry, environmental cleanup, and biotechnology among other fields. Given the high microbial diversity, identification of the microorganism is essential in understanding and managing the processes. One of the most popular and powerful method for microbial identification is comparative 16S rRNA gene analysis. Due to the highly conserved nature of this essential gene, sequencing and later comparison of it against known rRNA databases can provide assignment of the bacteria into the taxonomy, and the identity of its closest relatives. Isolation and sequencing of 16S rRNA genes directly from natural environments (either from DNA or RNA) can also be used to study the structure of the whole microbial community. Nowadays, novel sequencing technologies with massive outputs are giving researchers worldwide the chance to study the microbial world with a depth that was previously too expensive to achieve. In this article we describe commonly used research approaches for the study of individual microorganisms and microbial communities using the tools provided by Ribosomal Database Project website.

• 한국미생물·생명공학회지
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• 제39권2호
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• pp.97-103
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• 2011

GC-1과 GC-4로 명명된 두 종의 그람 양성 박테리아가 제주도 연안해수로부터 동정되었다. 이 두 균주는 16S rRNA 유전자 염기서열 분석과 생리적 특성 분석결과를 토대로 Bacillus 속의 박테리아로 규명되었다. 균주 GC-1의 16S rRNA 유전자 염기서열은 B. tequiliensis와 B. subtilis subsp. inaquosorum의 16S rRNA 유전자 염기서열과 99.91%의 상동성을 보였고, 균주 GC-4의 16S rRNA 유전자 염기서열은 B. altitudinis, B. stratosphericus 및 B. aerophilus의 16S rRNA 유전자 염기서열과 100%의 상동성을 보였다. 그러나 두 균주의 생리학적-유전학적 특성 분석 결과, 이들이 계통적 유연관계를 갖는 다른 Bacillus 속의 균주들과 상당한 차이가 있었고, 따라서 조사된 Bacillus 속과는 다른 속에 속할 가능성이 높았다. 이러한 결과는 Bacillus 속이 진화과정 중에 다양한 변종으로 진화되었음을 암시한다.

• 한국식품과학회지
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• 제53권3호
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• pp.334-343
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• 2021

본 연구에서는 16S rRNA 염기서열 분석을 통하여 시설재배 오이 내 상재균총 군집 특성을 분석하였으며, 수확 시기 및 지역에 따른 상재균총에 대한 정보를 제공하였다. 상재균총 다양성 분석(α-diversity)의 경우 5월 시료에서 더 높은 수치의 Observed OTUs와 Chao1 index가 나타났다. PCoA (β-diversity)분석을 통해서 수확 시기에 따른 상재균총의 차이가 존재함을 확인하였다. Phylum 수준에서는 Proteobacteria, Firmicutes, Actinobacteria가 우점하였고, class 수준에서는 Gammaproteobacteria, Bacilli, Alphaproteobacteria, Actinobacteria가 주로 존재하였다. Genus 수준에서는 시기적인 요인이 주로 상재균총에 영향을 끼치는 것을 확인할 수 있었으며, 일부 지역적 요인의 영향도 관찰 되었다. 5월 시료에서는 Aureimonas, Escherichia, Microbacterium이 11월 시료에서는 Enterococcus, Pseudomonas, Rhizobium이 더 높은 비율을 차지하였다. 이외에도, Acinetobacter, Aerococcus, Aureimonas, Enterobacter, Enterococcus, Escherichia, Pantoea, Pseudomonas, Staphylococcus와 같이 잠재적인 위험성을 가지는 genus가 존재함을 확인하였다.

• 한국동물위생학회지
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• 제39권1호
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• pp.1-6
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• 2016

In this study, Bacillus licheniformis which has been used as probiotics was isolated from Korean traditional fermented soybean. A total of 69 strains were presumptively identified as B. licheniformis by phenotypic methods. Based on PCR amplification and 16S rRNA gene sequencing, the multilocus sequence typing of gyrA and rpoB, followed by phylogenetic analysis was performed. The isolates were distinctly differentiated and found to be closely related to B. amyloliquefaciens, B. subtilis, and B. aerius. The partial 16S rRNA gene sequences of those strains matched those of B. sonorensis (97%) and B. aerius (98%) in the phylogenetic tree. In contrast, multilocus phylogenetic analysis (MLPA) showed that only 61 (86.9%) out of 69 strains were B. licheniformis. The rest of those strains were found to be B. subtilis (5.8%), B. amyloliquefaciens (2.9%), and B. sonorensis (2.9%), respectively. Therefore, our results suggested that since the 16S rRNA gene sequencing alone was not sufficient to compare and discriminate closely related lineages of Bacillus spp., it was required to analyze the MLPA simultaneously to avoid any misleading phenotype-based grouping of these closely related species.