Comparative Genomic Analysis of Lactobacillus rhamnosus BFE5264, a Probiotic Strain Isolated from Traditional Maasai Fermented Milk

정해영,최상행,강지희,지요셉,박소영,Wilhelm Heinrich Holzapfel,Julius Maina Mathara,박건석 한국미생물·생명공학회 2019 한국미생물·생명공학회지 Vol.47 No.1

Lactobacillus rhamnosus BFE5264, isolated from a Maasai fermented milk product (“kule naoto”), was previously shown to exhibit bile acid resistance, cholesterol assimilation, and adhesion to HT29-MTX cells in vitro. In this study, we re-annotated and analyzed the previously reported complete genome sequence of strain BFE5264. The genome consists of a circular chromosome of 3,086,152 bp and a putative plasmid, which is the largest one identified among L. rhamnosus strains. Among the 2,883 predicted protein-coding genes, those with carbohydrate-related functions were the most abundant. Genome analysis of strain BFE5264 revealed two consecutive CRISPR regions and no known virulence factors or antimicrobial resistance genes. In addition, previously known highly variable regions in the genomes of L. rhamnosus strains were also evident in strain BFE5264. Pairwise comparison with the most studied probiotic strain L. rhamnosus GG revealed strain BFE5264-specific deletions, probably due to insertion sequence-mediated recombination. The latter was associated with loss of the spaCBA pilin gene cluster and exopolysaccharide biosynthetic genes. Comparative genomic analysis of the sequences from all available L. rhamnosus strains revealed that they were clustered into two groups, being within the same species boundary based on the average nucleotide identities. Strain BFE5264 had a sister group relationship with the group that contained strain GG, but neither ANI-based hierarchical clustering nor core-gene-based phylogenetic tree construction showed a clear distinctive pattern associated with the isolation source, implying that the genotype alone cannot account for their ecological niches. These results provide insights into the probiotic mechanisms of strain BFE5264 at the genomic level.

UbC gene allele frequency in Korean population and novel UbC mosaic repeat unit formation

김룡남,강아람,최상행,김대수,김동욱,남성혁,김애리,박건향,윤병하,이강선,박홍석 한국유전학회 2012 Genes & Genomics Vol.34 No.4

The genomic structural organization of human UbC CDS repeat units could be representative of concerted evolution. The structure of the UbC gene and its repeat unit number frequency at scales of different human ethnic populations remain to be sufficiently determined. In this study, we performed comparative analysis of UbC CDS regions in genomes from 140Korean individuals. We found that the UbC gene allele types 9, 8 and 7 are present in the Korean population in proportions of 97.1%, 0.4% and 2.5%, respectively. Interestingly, we discovered that the allele types 7 and 8 harbor the novel UbC gene mosaic repeat units 3^5 (combined between sequence parts derived from standard repeat units 3 and 5) and 8^9(combined between sequence parts derived from standard repeat units 8 and 9) within their sequence structures,respectively. Our analysis showed that the novel mosaic repeat unit 3^5 lacks the highly human-specific amino acid S38, implying a functional consequence. These results suggest that the genomic organization of UbC repeat units is still undergoing dynamic structural changes due to concerted evolution through unequal crossing-over. Our results could represent valuable data for future investigations related to treating genetic diseases caused by UbC gene mutations and variations.

Bioinformatic analysis and annotation of expressed sequence tags (ESTs) generated from Anopheles sinensis mosquitoes challenged with apoptosis-inducing chemical, actinomycin-D

조용훈,이용석,강세원,고원규,박홍석,최상행,김유정,홍영석,노미영,오승한,김익수,한연수 한국곤충학회 2011 Entomological Research Vol.41 No.2

In 1993, after 10 years of absence, malaria re-emerged in the Republic of Korea in Gyeonggi and Gangwon Provinces near the Demilitarized Zone (DMZ). Anopheline mosquitoes are known to play a critical role in malaria transmission. Several studies have used gene expression data to examine the transcription responses of Anopheles gambiae after microbial and Plasmodium infections; however, as of now, there have been no reports of studies concerning the innate immune response and apoptosis in Anopheles sinensis. We have constructed two independent cDNA libraries from An. sinensis challenged with actinomycin-D (T, treated with apoptosis-inducing chemical) and without actinomycin-D (C, control). Analysis of the 5704 (C, 2848; T, 2856) expression sequence tags (ESTs) generated from these libraries identified 884 contigs (C, 421; T, 463) and 2180 singletons (C, 1038; T, 1142) with average lengths of 831 and 817 bp, respectively. Furthermore, comparison of our EST sequences against the eukaryotic orthologous group database showed that 4324 (75.82%) of 5704 sequences could be grouped into three major functional categories. These EST data provide the basis for further studies characterizing the molecular mechanisms of midgut apoptosis.

연체동물 전용 서열 블라스트 서버구축

이용석,박홍석,연제오,정계헌,김대원,최상행,조용훈,김대수,김민영,변인선,강보라 한국패류학회 2004 The Korean Journal of Malacology Vol.20 No.2

The BLAST server for the mollusk was constructed on the basis of the Intel Server Platform SC-5250 dual Xeon 2.8 GHz cpu and Linux operating system. After establishing the operating system, we installed NCBI (National Center for Biotechnology Information) WebBLAST package after web server configuration for cgi (common gate interface) (http://chimp.kribb. re.kr/mollusks). To build up the stand alone blast, we conducted as follows: First, we downloaded the genome information (mitochondria genome information), DNA sequences, amino acid sequences related with mollusk available at NCBI. Second, it was translated into the multifasta format that was stored as database by using the formatdb program provided by NCBI. Finally, the cgi was used for the Stand Alone Blast server. In addition, we have added the vector, Escherichia coli, and repeat sequences into the server to confirm a potential contamination. Finally, primer3 program is also installed for the users to design the primer. The stand alone BLAST gave us several advantages: (1) we can get only the data that agree with the nucleotide sequence directly related with the mollusks when we are searching BLAST; (2) it will be very convenient to confirm contamination when we made the cDNA or genomic library from mollusks; (3) Compared to the current NCBI, we can quickly get the BLAST results on the mollusks sequence information. 본 연구를 통해서 http://chimp.kribb.re.kr/mollusks 에 연체동물 전용 서열 BLAST 데이터베이스가 구축되었다. 예비 실험을 통해 본 결과와 마찬가지로 연체동물을 대상으로 한 유전자 정보만을 매우 빠른 속도로 얻을 수 있었다. 본 시스템을 사용하여 앞으로 많은 연구가 진행되어질 연체동물 유전자 연구 및 EST 연구에 많은 도움이 되리라고 사료된다.

왼돌이물달팽이(Physa acuta)의 Metallothionein 유전자 클로닝 및 발현양상

조용훈,백문기,강세원,이재봉,변인선,최상행,채성화,강정하,한연수,박홍석,이용석 한국패류학회 2009 The Korean Journal of Malacology Vol.25 No.3

Metallothioneins (MTs) play a key role in metallic homeostasis and detoxification in most living organisms. In an attempt to study the biological functions and significance of MT in a snail, we cloned and partially characterized the MT gene from the left-handed snail, Physa acutawhich has been regarded as a potential biomonitering species for fresh water. The complete cDNA sequence of PaMT cDNA was identified from the expressed sequence tag (EST) sequencing project of Physa acuta. The coding region of 180 bp gives 60 amino acid residues including the initiation methionine and termination codon. Clustering and phylogenic analysis of PaMT with other MT amino acid sequences show that it has some identities to Helix pomatia (60%), Arianta arbustorum (58%), Perna viridis (49%), Mytilus edulis (49%), Bathymodiolus azoricus (49%), Bathymodiolus azoricus (48%) and Bathymodiolus sp. FD-2002 (48%). Time dependent induction for PaMT from P. acuta exposed with cadmium (50 ppb) indicated that PaMT was induced at 4-8 hr after exposure. It remains to further develop PaMTas a potential biomarker for water contamination in fresh water.

EST Knowledge Integrated Systems (EKIS): An Integrated Database of EST Information for Research Application

김대원,정태성,최영상,권혁렬,김동욱,최한석,최상행,박홍석 한국유전체학회 2009 Genomics & informatics Vol.7 No.1

The EST Knowledge Integrated System, EKIS (http://ekis. kribb.re.kr), was established as a part of Korea's Ministry of Education, Science and Technology initiative for genome sequencing and application research of the biological model organisms (GEAR) project. The goals of the EKIS are to collect EST information from GEAR projects and make an integrated database to provide transcriptomic and metabolomic information for biological scientists. The EKIS constitutes five independent categories and several retrieval systems in each category for incorporating massive EST data from high-throughput sequencing of 65 different species. Through the EKIS database, scientists can freely access information including BLAST functional annotation as well as Genechip and pathway information for KEGG. By integrating complex data into a framework of existing EST knowledge information, the EKIS provides new insights into specialized metabolic pathway information for an applied industrial material.

바지락 (Ruditapes philippinarum) 의 중금속 축적에 관한 연구

이용석,조용훈,변인선,강세원,조은미,한연수,최상행,박홍석,고원규,안인영,정계헌 한국패류학회 2006 The Korean Journal of Malacology Vol.22 No.2

The present study was conducted to confirm that a bivalve Ruditapes philippinarum can be used as a biomarker for the monitoring of the heavy metal pollution in the silt of the marine environment. The clams were collected from the silt of Cheonsu-bay, Buheung-ri, and Tan-island of the West Sea, Korea. To observe the normal structures of the target organs (hepatopancreas and gill), they were dissected out for the immunohistochemical study and the electron microscopy with TEM, SEM, and SEM-EDS. The immunohistochemical study showed that the interdiverticular connective tissues of the hepatopancreas, and the outer epithelium of the gill lamellae was strongly reacted to anti-metallothionein (MT), indicating the presence of MT, a metal-binding protein, involved in metal detoxifying process. According to the examinations under the TEM, the epithelial cells of the hepatopancreas of the clams collected from polluted area (Tan-island) showed certain changes such as swollen rER, swollen nuclear envelope and inclusion bodies in the nulcei. In the SEM-EDS analysis, tissue of the hepatopancreas showed relatively higher concentration of S, Zn, and Cd. These elements are supposed to be concerning with the MT-reaction in the hepatopancreas. Considering that the coastal bivalve R. philippinarum showed immediate subcellular responses to heavy metal pollution in the overall experiments conducted, this species might act as one of efficient biomarkers for the heavy metal contamination in the marine environment.

Profiling of the Bacteria Responsible for Pyogenic Liver Abscess by 16S rRNA Gene Pyrosequencing

송윤규,심상군,김광민,이동해,김대수,최상행,송재영,강형련,백승철,이우곤,조명제,이광호 한국미생물학회 2014 The journal of microbiology Vol.52 No.6

Pyogenic liver abscess (PLA) is a severe disease with considerablemortality and is often polymicrobial. Understandingthe pathogens that cause PLA is the basis for PLA treatment. Here, we profiled the bacterial composition in PLA fluid bypyrosequencing the 16S ribosomal RNA (rRNA) gene basedon next-generation sequencing (NGS) technology to identifyetiological agents of PLA and to provide information oftheir 16S rRNA sequences for application to DNA-basedtechniques in the hospital. Twenty patients with PLA whounderwent percutaneous catheter drainage, abscess culture,and blood culture for isolates were included. Genomic DNAsfrom abscess fluids were subjected to polymerase chain reactionand pyrosequencing of the 16S rRNA gene with a454 GS Junior System. The abscess and blood cultures werepositive in nine (45%) and four (20%) patients, respectively. Pyrosequencing of 16S rRNA gene showed that 90% of thePLA fluid samples contained single or multiple genera ofknown bacteria such as Klebsiella, Fusobacterium, Streptococcus,Bacteroides, Prevotella, Peptostreptococcus, unassignedEnterobacteriaceae, and Dialister. Klebsiella was predominantlyfound in the PLA fluid samples. All samples thatcarried unassigned bacteria had 26.8% reads on average. We demonstrated that the occurrence of PLA was associatedwith eight known bacterial genera as well as unassignedbacteria and that 16S rRNA gene sequencing was more usefulthan conventional culture methods for accurate identificationof bacterial pathogens from PLA.

한국 고유종 Pisidium (Neopisidium)coreanum (산골조개)의 metallothionein 유전자를 기초로 한 분자계통 분류학적 연구

백문기,이준서,강세원,이재봉,강현정,조용훈,노미영,한연수,최상행,채성화,박홍석,이준상,이용석 한국패류학회 2009 The Korean Journal of Malacology Vol.25 No.2

Pisidium (Neopisidium) coreanum is a freshwater snail and lives in spring water near mountain areas. Interestingly, this snail has traditionally regarded as medicinal food, and thus has been used as folk remedies for healing broken bones. Recently, alpha classification on Pisidium (Neopisidium) coreanum through re-description has been conducted. However, not much attention has so far been made in beta classification. In this study, we performed the beta classification based on metallothionein (MT) genes found from various organisms. To this end, the complete cDNA sequences were obtained from the Expressed Sequence Tag (EST) sequencing project of Pisidium (Neopisidium) coreanum. The coding region (315 bp) gives an amino acid sequence of 105 residues. The combined results from BLAST analyses, multiple sequence alignment and molecular phylogenetic study of Pc-MT gene indicate that Pisidium (Neopisidium) coreanum has similarity to freshwater bivalve such as Dreissena polymorpha (zebra mussel), Unio tumidus (swollen river mussel) and Crassostrea ariakensis (suminoe oyster). Pisidium (Neopisidium) coreanum is a freshwater snail and lives in spring water near mountain areas. Interestingly, this snail has traditionally regarded as medicinal food, and thus has been used as folk remedies for healing broken bones. Recently, alpha classification on Pisidium (Neopisidium) coreanum through re-description has been conducted. However, not much attention has so far been made in beta classification. In this study, we performed the beta classification based on metallothionein (MT) genes found from various organisms. To this end, the complete cDNA sequences were obtained from the Expressed Sequence Tag (EST) sequencing project of Pisidium (Neopisidium) coreanum. The coding region (315 bp) gives an amino acid sequence of 105 residues. The combined results from BLAST analyses, multiple sequence alignment and molecular phylogenetic study of Pc-MT gene indicate that Pisidium (Neopisidium) coreanum has similarity to freshwater bivalve such as Dreissena polymorpha (zebra mussel), Unio tumidus (swollen river mussel) and Crassostrea ariakensis (suminoe oyster).

하동 천년차나무의 엽록체 유전체 분석

심두보,황정규,강은주,조경환,손용휘,송성화,김지민,김윤미,최상행,김종철 한국차학회 2019 한국차학회지 Vol.25 No.2

In this study, we present the results of performing the chloroplast genome analysis of the Hadong Cheonnyeon cha (HCNC), Camellia sinensis var. sinensis (CSS), and we achieved this by employing illumina sequencing technology. The total nucleotide sequence of 156 Gbp was sequenced and the size of the genome was measured by k-mer analysis, and was found to be about 3,109 Mbp. The sequence corresponding to the chloroplast was obtained, and the analysis was performed again to finally complete the chloroplast of 157,025 bp. The chloroplast had 126 protein coding genes with an average length of 2.4 kb and 40 non-coding RNA. It was confirmed that there were almost no structural differences from the 11 NCBI registries and there was only a difference in the nucleotide sequence. As a result, HCNC was clearly distinguished from assamica (GenBank JQ975030.1, MH394407.1 ∼ MH394410.1), and HCNC was most similar to KF562708.1. The GenBank registered KF562708.1 is one of the Chinese representative varieties knows as Longjing 43. Although the Chinese tea cultivars were registered in GenBank and they were compared with the HCNC, the Japanese cultivars were not registered in there. Further analysis of chloroplast genomes such as Yabukita, Hushun and Samidori were conducted to further investigate the relationship between the HCNC and Japanese tea cultivars. 차세대염기서열(NGS) 분석과 illumina sequencing 기술을 활용하여 하동 천년차나무의 엽록체 유전체 서열을 조사하고 NCBI에 등재된 차나무 11종의 염기서열을 비교하였다. 생산된 총 염기서열은 156 Gbp, GC 비율은 38.3%로 식물에서 전형적으로 나타나는 분포를 보였다. 등록된 11종의 서열에 mapping하여 확보한 최종 엽록체 서열은 157,025 bp를 확인하였다. 이 서열에 대한 유전자 예측결과 총 166개의 유전자를 확인하였고 이중 단백질 암호화 유전자의 평균 길이는 2.4 kb, 비암호화 유전자는 40개 정도였다. Circle map 구조는 다른 11개 서열과 거의 동일하였으며, 염기서열의 크기에서 근소한 차이를 보였다. Phylogenetic tree로 근연관계를 확인해본 결과 대엽종(assamica)인 JQ975030.1, MH394407.1 ∼ MH394410.1과 뚜렷한 차이를 보였으며, Longjing 43 (GenBank : KF562708.1)과 유사하였다. 중국 차나무 품종은 GenBank에 등록되어 있어 비교가 가능하였으나 일본 품종 중에 등록된 것은 없어 Yabukita, Hushun, Samidori 등의 엽록체 유전체 분석을 추가로 실시하여 하동천년차나무와 일본 차나무 품종의 근연관계를 추가로 연구하고자 한다.