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Draft genome sequence of Mesosutterella multiformis KGMB 02707 isolated from a healthy Korean feces
Korean J. Microbiol. 2019;55(4):415-418
Published online December 31, 2019
© 2019 The Microbiological Society of Korea.

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

1Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea
2Seoul National University Bundang Hospital, Gyeonggi-do 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: juhuck@kribb.re.kr;
Tel.: +82-63-570-5634
Received July 23, 2019; Revised September 30, 2019; Accepted October 17, 2019.
Abstract

The genus of Mesosutterella has been isolated from human feces. Mesosutterella multiformis KGMB 02707 was isolated from a healthy Korean feces. The whole-genome sequence of Mesosutterella multiformis KGMB 02707 was analyzed using the PacBio Sequel platform. The genome comprises a 2,702,150 bp chromosome with a G + C content of 56.85%, 2,285 open reading frames, 29 rRNA genes, and 55 tRNA genes. Interestingly, unlike its similar genus Sutterella we found that strain KGMB 02707 had all nine genes involved in the biosynthesis of Kdo2-Lipid A, which is part of lipopolysaccharide (LPS) structure, in its genome based on the result of genome analysis.

Keywords : Mesosutterella multiformis KGMB 02707, human feces, LPS structure
Body

The gut microbiota play critical roles in maintaining the gut homeostasis, including enhancing resistance to infection, stimulating immunological development. Additionally, they metabolize nutrients and vitamins, and function as structural gut barrier in host. Therefore, balanced gut microbiota is important for health and gut homeostasis (Macpherson and Harris, 2004; O'Hara and Shanahan, 2006). The acquisition of gut microbiota including their genetic and metabolic profiles is becoming gradually important for therapeutic approaches to a wide range of diseases (Bolan et al., 2016). Recently, we isolated one bacterial strain designated KGMB 02707 from a healthy Korean feces who had not taken any medicine and had normal BMI (body mass index). Based on 16S rRNA sequences strain KGMB 02707 (= KCTC 15688 = DSM 107829) is most closely related to Mesosutterella multiformis 4NBBH2T (99% 16S rRNA gene sequence similarity), which was recently proposed as novel strain, and a member of the genus Mesosutterella within the family Sutterellaceae which belongs to the order Burkholderiales in the class Betaproteobacteria (Sakamoto et al., 2018).

The genus Mesosutterella of family Sutterellaceae was firstly proposed by Sakamoto et al. (2018). Based on the phylogenetic location, the genus Mesosutterella is similar to the genus Sutterella. In addition, the genus Mesosutterella is obligately anaerobic, Gram-stain-negative, non-motile and non-spore forming bacteria (Sakamoto et al., 2018). While the role of Mesosutterella sp. as part of the human gut microbiota has been unreported, the Sutterella sp. have been suspected to play a role in the pathogenesis of inflammatory bowel disease (IBD) and metabolic syndrome (Mangin et al., 2004; Williams et al., 2012). Another study, however, showed no significant difference in the microbial composition including the abundance of Sutterella sp. in metabolic syndrome (Lim et al., 2017). Here, we describe the draft genome sequence and annotation of Mesosutterella multiformis KGMB 02707 isolated from a healthy Korean feces.

To isolate strain KGMB 02707, the fecal sample was collected from Seoul National University Bundang Hospital. The isolation and culture of bacteria from the fecal sample were performed in the anaerobic chamber (Coy Laboratory Products) filled with 86% N2, 7% CO2, and 7% H2. One gram of the fecal sample was suspended in sterilized phosphate-buffered saline (PBS), then the fecal suspension was serially diluted in PBS, followed by cultivation onto tryptic soy agar plate supplemented with 5% sheep blood (TSAB).

To prepare the genomic DNA of KGMB 02707, the isolate was cultured on TSAB agar plate at 37°C for 3 days in anaerobic condition. The genomic DNA was extracted by using a Wizard genomic DNA purification kit (Promega) following the manufacturer’s instructions. The purified genomic DNA was fragmented by using a g-TUBETM device according to the manufacturer’s instructions (Covaris). The fragmented DNA quantity and the size were analyzed by a Qubit 2.0 fluorometer with a Qubit dsDNA HS Assay Kit (Invitrogen) and the Agilent 2100 Bioanalyzer with the DNA 12000 assay (Agilent), respectively. Single-Molecule Real-Time (SMRT) bell library was prepared according to the manufacturer’s instructions (Pacific Biosciences) without a non-size selection. Whole genome sequencing was executed by a Pacific Biosciences Sequel (Pacific Biosciences) with 2.0 sequencing chemistry and 600 min movies.

Hierarchical Genome Assembly Process (HGAP4) pipeline in the SMRT Analysis version 4.0 with default parameters was employed for de novo genome assembly, generating 13 contigs. Potential contamination in genome assembles were inspected by the Contamination Estimator by 16S (ContEst16S) and CheckM tools. The coding DNA sequences (CDSs) and tRNA were predicted using prodigal and tRNAscan-SE, respectively.

The clustered regularly interspaced short palindromic repeats (CRISPRs) were found using PILER-CR and CRISPR Recognition Tool (CRT). The rRNAs and other non-coding RNAs were searched by covariance model search with inference of Rfam 12.0. The annotation of each CDS was performed by homology search against Swiss-prot, EggNOG 4.5, SEED and KEGG databases.

The genome statistics are summarized in Table 1. The draft genome of strain KGMB 02707 consists of a 2,702,150 bp chromosome with a G + C content of 56.85%. In addition, N50 and N90 size are 1,455,315 bp and 270,518 bp, respectively. The genome is showed to contains 2,285 CDSs, 29 rRNAs (5S, 16S, 23S), and 55 tRNAs by using CLgenomics (Fig. 1). Total of 2,052 genes were functionally assigned to categories based on clusters of orthologous group (COG) assignments.

General features of Mesosutterella multiformis KGMB 02707

PropertyValue
Genome assembly
Assemble methodSMRT Analysis version 4.0
Genome coverage250X
Genome features
Genome size (bp)2,702,150
G + C content (%)56.85
No. of contigs13
N50 and N90 (bp)1,455,315 and 270,518
rRNA genes (5S, 16S, 23S)29 (8, 10, 11)
tRNA genes55
Open reading frame2,285
CDS assigned by COG2,052
CRISPR arrays6
GenBank Accession No.BHWA01000000

Fig. 1.

Graphical circular map of Mesosutterella multiformis KGMB 02707. Marked characteristics are shown from the center to the outside: GC skew (red and green), G + C content (yellow and blue), CDSs on the reverse strand (colored by COG categories), CDS on the forward strand (colored by COG categories), and RNA genes (rRNAs-red and tRNAs-blue).


The previous report showed that Sutterella sp. moderately stimulated IL-8 release and did not impact on the epithelial barrier function. Furthermore, it suggested that the mild pro- inflammatory capacity may assist in keeping the host’s immune system at an appropriate level, and the capacity may be due to their the particular structure of Kdo2-Lipid A (Hiippala et al., 2016). LPS on the surface of Gram-negative bacteria is composed of Kdo2-Lipid A, which is the active component of the LPS to stimulates potent host immune responses (Wang et al., 2015), core-oligosaccharide and O-antigen repeats. Unlike E. coli having nine genes for complete Kdo2-lipid A biosynthesis, which were LpxA (UDP-N-acetylglucosamine O-acetyltransferase), LpxC (UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase), LpxD (UDP-3-O-[3-hydroxymyristoyl]glucosamine N-acyltransferase), LpxH (UDP-2,3-diacylglucosamine pyrophosphohydrolase), LpxB (lipid-A-disaccharide synthase), LpxK (lipid A 40-kinase), WaaA (3-deoxy-D-manno-octulosonate lipid A transferase), LpxL (Kdo2-lipid IV(A) lauroyl-ACP acyltransferase) and LpxM (Kdo2-lauroyl-lipid IV(A)-myristoyl-ACP acyltransferase), the Sutterella sp. posseses only seven genes except for LpxL and LpxM (Hiippala et al., 2016). Interestingly, contrary to other Sutterella sp., strain KGMB 02707 has all nine genes as E. coli, suggesting this may be the distinct feature of Mesosutterella sp. compared to Sutterella sp.. Thus, we assume that strain KGMB 02707 may cause a strong IL-8 response, because the strain has all genes involved in Kdo2- lipid A biosynthesis as E. coli, which is a strong inducer of cytokine reponse (Suzuki et al., 2000). Furthermore, this finding suggests that the difference of LPS structure between Sutterella sp. and Mesosutterella sp. probably determines their pathogenesis. However, it is necessary to determine whether the strain KGMB 02707 casuses strong induction of IL-8 in enterocyte. The draft genome sequence of Mesosutterella multiformis KGMB 02707 will contribute to understanding the physiological functions of strain KGMB 02707 in the gut.

Nucleotide sequence accession number

Mesosutterella multiformis KGMB 02707 has been deposited in the Korean Collection for Type Cultures under accession number KCTC 15688. The GenBank/EMBL/DDBJ accession number for the genome sequence of Mesosutterellamultiformis KGMB 02707 is BHWA01000000.

적 요

Mesosutterella 속 균주는 사람의 분변에서 분리되었다. 본 연구에서는 건강한 한국인 분변으로부터 Mesosutterella multiformis KGMB 02707 균주를 분리하였으며 PacBio Sequel 플랫폼을 이용하여 KGMB 02707 균주의 유전체염기서열을 분석하였다. 유전체는 G + C 구성 비율이 56.85%이고, 2,285개의 유전자와 rRNA 29개 tRNA 55개로 구성되었으며, 염색체의 크기는 2,702,150 bp였다. 또한, Mesosutterella 속과 유사한 Sutterella 속과는 다르게 KGMB 02707 균주는 유전체 분석 결과를 통해 LPS 구조의 부분인 Kdo2-Lipid A의 생합성과 관련된 아홉 가지 모든 유전자를 가지고 있음을 발견하였다.

Acknowledgements

This work was supported by the Bio & Medical Technology Development program (Project No. NRF-2016M3A9F3947 962) 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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