Korean Journal of Microbiology (2019) Vol. 55, No. 4, pp. 411-414 pISSN 0440-2413
DOI https://doi.org/10.7845/kjm.2019.9112 eISSN 2383-9902
Copyright ⓒ 2019, The Microbiological Society of Korea
Draft genome sequence of Anaerotignum faecicola KGMB03357 isolated from human faeces
Seung-Hyeon Choi
1, Ji-Sun Kim
1, Jam-Eon Park
1, Byeong Seob Oh
1, Seoung Woo Ryu
1, Seung Yeob Yu
1, Se Won Kang
1, Kook-Il Han
1, Min Kuk Suh
1, Han Sol Kim
1, Keun Chul Lee
1, Mi Kyung Eom
1, Dong Ho Lee
2, Hyuk Yoon
2, Byung-Yong Kim
3, Je Hee Lee
3, Ju Huck Lee
1, Jung-Sook Lee
1,4, and Seung-Hwan Park
1*
1
Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea
2
Seoul National University Bundang Hospital, Seongnam 13620, Republic of Korea
3
ChunLab, Inc., Seoul 06725, Republic of Korea
4
University of Science and Technology (UST), Daejeon 34113, Republic of Korea
인간 분변으로부터 분리된 Anaerotignum faecicola KGMB03357 균주의 유전체 염기서열 초안
최승현
1･ 김지선
1･ 박잠언
1･ 오병섭
1･ 유승우
1･ 유승엽
1･ 강세원
1･ 한국일
1･ 서민국
1･ 김한솔
1･ 이근철
1･ 엄미경
1･ 이동호
2･ 윤혁
2･ 김병용
3･ 이제희
3･ 이주혁
1･ 이정숙
1,4･ 박승환
1*
1
한국생명공학연구원 생물자원센터,
2분당서울대학교병원,
3천랩,
4과학기술연합대학원대학교
(Received September 24, 2019; Revised October 11, 2019; Accepted October 11, 2019)
*For correspondence. E-mail: [email protected];
Tel.: +82-63-570-5636; Fax: +82-63-570-5609
The genus of Anaerotignum was isolated from a methanogenic reactor, black mud, anoxic digestor sludge, chicken caeca, and human faeces. Anaerotignum faecicola KGMB03357 was isolated from faeces of a healthy Korean. The whole-genome sequence of Anaerotignum faecicola KGMB03357 was analyzed using the PacBio Sequel platform. The genome consists of a 2,520,658 bp chromosome with a DNA G + C content of 47.3 mol%, 2,357 total genes, 28 rRNA genes, and 74 tRNA genes. Also, we found that strain KGMB03357 had various genes for biosynthesis of amino acid and antibiotic resistance in its genome based on the result of genome analysis.
Keywords: Anaerotinum faecicola, draft genome sequence, human faeces, PacBio Sequel
The human gut microbiome plays an important role in
nutrient metabolism and the immune system (Kau et al., 2011).
Imbalance of gut microbial system is referred to as “dysbiosis”,
and may cause various diseases such as diabetes, colon cancer,
liver disease, inflammatory bowel diseases, irritable bowel
syndrome, metabolic syndromes, and obesity (Bolan et al.,
2016). Along with the genetic and metabolic profiles of intestinal
microorganisms, the collection and storage, called the biobanking
of gut microbes, are becoming increasingly important for
therapeutic approaches to a wide range of diseases (Bolan et al.,
2016). Therefore, we launched the Korean gut microbiome
bank (KGMB) at Korea Research Institute of Bioscience and
Biotechnology in 2016. A novel bacterial strain designated
KGMB03357 was isolated from faeces of a healthy Korean
selected by Bundang Seoul National University based on health
status (antibiotic use, blood test, body mass index, smoking,
alcohol use, drug use, and Bristol stool chart). Based on the
phenotypic, phylogenetic and chemotaxonomic characteristics,
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Table 1. General features of Anaerotignum faecicola KGMB03357
Property Value
Genome assembly
Assemble method SMRT Analysis version 4.0
Genome coverage 176.7X
Genome features
Genome size (bp) 2,520,658
DNA G + C content (%) 47.3
No. of contigs 14
rRNA genes (5S, 16S, 23S) 28 (9, 9, 10)
tRNA genes 74
Open reading frame 2,357
CDS assigned by COG 2,118
GenBank Accession No. BHVZ00000000
strain KGMB03357 (= KCTC 15736 = DSM 107953) represent to a novel species of the genus Anaerotignum belonging to the family Lachnospiraceae in the class Clostridia.
The genus Anaerotignum was firstly proposed by Ueki et al. (2017) with validly published names (www.bacterio.net/
anaerotignum.html), which proposed that Clostridium propionicum, Clostridium neopropionicum, and Clostridium lactatifermentans be reclassified within the new genus Anaerotignum and assigned to Clostridium cluster XIVb belonging to the family Lachnospiraceae.
Members of the genus Anaerotignum are strictly anaerobic rods, Gram-positive-type cell walls, chemoorganotrophs (van der Wielen et al., 2002; Ueki et al., 2017). Here, we describe the draft genome sequence and annotation of Anaerotignum faecicola KGMB03357.
The Anaerotignum faecicola KGMB03357 was grown on EG medium (2.4 g meat extract, 10.0 g proteose peptone No. 3, 5.0 g yeast extract, 4.0 g Na
2HPO
4, 1.5 g glucose, 0.5 g soluble starch, 0.2 g
L-cystine, 0.5 g
L-cysteine hydrochloride mono- hydrate, and 15.0 g agar per 1 L with 5% [v/v] sheep blood at pH 7.6–7.8) in anaerobic chamber (Coy Laboratory Products) containing 90% N
2, 5% H
2, and 5% CO
2.
The genomic DNA was extracted using a Wizard genomic DNA purification kit (Promega). Purified genomic DNA sheared to a size of 10 kb using a g-TUBE
TMdevice according to the manufacturer’s instructions (Covaris). Fragmented DNA quantity was analyzed by a Qubit 2.0 fluorometer with a Qubit dsDNA HS Assay Kit (Invitrogen) and size was measured by the Agilent 2100 Bioanalyzer with the DNA 12000 assay (Agilent). Single- Molecule Real-Time (SMRT) bell library was prepared according to the manufacturer’s instructions (Pacific Biosciences) without a non-size selection. Genome sequencing was performed using a Pacific Biosciences Sequel (Pacific Biosciences) with 2.0 sequencing chemistry and 600 min movies.
De novo genome assembly was performed with the Hier- archical Genome Assembly Process (HGAP4) pipeline in the SMRT Analysis version 4.0 using default parameters. Potential contamination in genome assembles were checked by the Contamination Estimator by 16S (ContEst16S) and CheckM tools (Parks et al., 2015; Lee et al., 2017). The coding DNA sequences (CDSs) and tRNAs were predicted using prodigal and tRNAscan-SE, respectively. The functional annotation of each CDS analyzed by homology search against UniProt
(UniProt, 2015), KEGG (Kanehisa et al., 2014) databases. To calculate the average nucleotide identity (ANI) values, a ChunLab’s online ANI calculator using the OrthoANI algorithm was used (Lee et al., 2016).
The genome statistics are showed in Table 1. The draft genome of Anaerotignum faecicola KGMB03357 was consist of a 2,520,658 bp chromosome with a DNA G + C content of 47.3 mol%. The genome is showed to contain 2,357 CDSs, 28 rRNAs (5S, 16S, 23S), and 74 tRNAs were annotated (Fig. 1).
A total of 2,118 genes were functionally assigned to categories based on clusters of orthologous group (COG) assignments.
We found that various genes involved in amino-acid biosynthesis were identified in the genome. Aspartokinase, aspartate-semialdehyde dehydrogenase (asd), homoserine de- hydrogenase and homoserine kinase (thrB) genes involved in
L
-threonine biosynthesis from
L-aspartate. Acetolactate synthase (ilvB), Acetolactate synthase (ilvB-2), ketol-acid reductoisomerase (ilvC), dihydroxy-acid dehydratase (ilvD) and branched-chain amino acid aminotransferase (ilvE) genes involved in
L-valine,
L
-isoleucine biosynthesis from pyruvate. 2-isopropylmalate
synthase (leuA), 2-isopropylmalate dehydratase small subunit
(leuD), 2-isopropylmalate dehydratase large subunit (leuC),
3-isopropylmalate dehydrogenase (leuB), and branched-chain
amino acid aminotransferase (ilvE) involved in
L-leucine from
2-oxoisovalerate. Such amino acids are essential amino acids
for human. Additionally, we found that the genome has several
beta-lactam resistance genes, such as bla regulator protein
(blaR1), BlaI family transcriptional regulator and penicillinase
Draft genome sequence of A. faecicola KGMB03357 ∙
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Korean Journal of Microbiology, Vol. 55, No. 4
Fig. 1. Graphical circular map of Anaerotignum faecicola KGMB03357. From outside to the center; ring 1 shows tRNA and rRNA, as well as coding DNA sequences (CDS) on the forward strand and reverse strand from the outside; ring 2 shows G + C% content and ring 3 shows GC skew.repressor (blaI), penicillin-binding protein 1A (mrcA), penicillin- binding protein 2 (mrdA).
The strain KGMB03357 is most closely related to Anaerotignum lactatifermentans G17
Tbased on the 16S rRNA gene sequence similarity (96.1%) and average nucleotide identity (ANI, 73.3%).
Comparison with the A. lactatifermentans G17
Trevealed that meso-DAP d-dehydrogenase (ddh) involved in
L-lysine bio- synthesis, hydroxymethylbilane synthase (hemC) and precorrin-2 dehydrogenase (sirC) involved in porphyrin-containing com- pound metabolism, and shikimate dehydrogenase (aroE) involved in chorismate biosynthesis are only present in A. faecicola KGMB03357.
The draft genome sequence of Anaerotignum faecicola KGMB03357 will contribute to understanding the physiological functions of Anaerotignum faecicola KGMB03357.
Nucleotide sequence accession number
Anaerotignum faecicola KGMB03357 has been deposited in the Korean Collection for Type Cultures under accession number KCTC 15736. The GenBank/EMBL/DDBJ accession number for the genome sequence of Anaerotignum faecicola KGMB03357 is BHVZ00000000.
적 요
Anaerotigum 속 균주들은 메탄 생성 반응기, 검은 진흙, 무 산소 소화조 슬러지, 닭의 맹장, 인간 분변 등에서 분리된 것으 로 알려져 있다. 본 연구에서는 건강한 한국인 분변으로부터 Anaerotigum faecicola KGMB03357 균주를 분리하였으며 PacBio Sequel 플랫폼을 이용하여 Anaerotigum faecicola
KGMB03357 균주의 유전체서열을 분석하였다. 유전체는
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DNA G + C 구성 비율이 47.3 mol%이고, 2,357개의 유전자와 rRNA 28개, tRNA 74개로 구성되었으며, 염색체의 크기는 2,520,658 bp였다. 또한, 유전체 분석 결과를 통해 아미노산 생합성 그리고 항생제 내성과 관련된 다양한 유전자를 발견 하였다.
Acknowledgements
This work was supported by the Bio & Medical Technology Development program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT) of the Republic of Korea and a grant from the Korea Research Institute of Bioscience & Biotechnology (KRIBB) Research initiative program.
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