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Draft genome sequence of Anaerotignum faecicola KGMB03357 isolated from human faeces
Korean J. Microbiol. 2019;55(4):411-414
Published online December 31, 2019
© 2019 The Microbiological Society of Korea.

Seung-Hyeon Choi1, Ji-Sun Kim1, Jam-Eon Park1, Byeong Seob Oh1, Seoung Woo Ryu1, Seung Yeob Yu1, Se Won Kang1, Kook-Il Han1, Min Kuk Suh1, Han Sol Kim1, Keun Chul Lee1, Mi Kyung Eom1, Dong Ho Lee2, Hyuk Yoon2, Byung-Yong Kim3, Je Hee Lee3, Ju Huck Lee1, Jung-Sook Lee1,4, and Seung-Hwan Park1*

1Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea
2Seoul National University Bundang Hospital, Seongnam 13620, Republic of Korea
3ChunLab, Inc., Seoul 06725, Republic of Korea
4University of Science and Technology (UST), Daejeon 34113, Republic of Korea
Correspondence to: *E-mail: biopark@kribb.re.kr;
Tel.: +82-63-570-5636; Fax: +82-63-570-5609
Received September 24, 2019; Revised October 11, 2019; Accepted October 11, 2019.
Abstract

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
Body

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, 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 Anaerotignumfaecicola KGMB03357.

The Anaerotignumfaecicola 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 Na2HPO4, 1.5 g glucose, 0.5 g soluble starch, 0.2 g L-cystine, 0.5 g L-cysteine hydrochloride monohydrate, 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% N2, 5% H2, and 5% CO2.

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-TUBETM device 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 Hierarchical 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 Anaerotignumfaecicola 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.

General features of Anaerotignum faecicola KGMB03357

PropertyValue
Genome assembly
Assemble methodSMRT Analysis version 4.0
Genome coverage176.7X
Genome features
Genome size (bp)2,520,658
DNA G + C content (%)47.3
No. of contigs14
rRNA genes (5S, 16S, 23S)28 (9, 9, 10)
tRNA genes74
Open reading frame2,357
CDS assigned by COG2,118
GenBank Accession No.BHVZ00000000

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.


We found that various genes involved in amino-acid biosynthesis were identified in the genome. Aspartokinase, aspartate-semialdehyde dehydrogenase (asd), homoserine dehydrogenase 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 repressor (blaI), penicillin-binding protein 1A (mrcA), penicillin- binding protein 2 (mrdA).

The strain KGMB03357 is most closely related to Anaerotignum lactatifermentans G17T based on the 16S rRNA gene sequence similarity (96.1%) and average nucleotide identity (ANI, 73.3%). Comparison with the A. lactatifermentans G17T revealed that meso-DAP d-dehydrogenase (ddh) involved in L-lysine biosynthesis, hydroxymethylbilane synthase (hemC) and precorrin-2 dehydrogenase (sirC) involved in porphyrin-containing compound metabolism, and shikimate dehydrogenase (aroE) involved in chorismate biosynthesis are only present in A. faecicola KGMB03357.

The draft genome sequence of Anaerotignumfaecicola KGMB03357 will contribute to understanding the physiological functions of Anaerotignumfaecicola 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, 무산소 소화조 슬러지, 닭의 맹장, 인간 분변 등에서 분리된 것으로 알려져 있다. 본 연구에서는 건강한 한국인 분변으로부터 Anaerotigumfaecicola KGMB03357 균주를 분리하였으며 PacBio Sequel 플랫폼을 이용하여 Anaerotigumfaecicola KGMB03357 균주의 유전체서열을 분석하였다. 유전체는 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 발견하였다.

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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