search for




 

Draft genome sequence of Ruminococcus sp. KGMB03662 isolated from healthy Korean human feces
Korean J. Microbiol 2019;55(3):274-277
Published online September 30, 2019
© 2019 The Microbiological Society of Korea.

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

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: jslee@kribb.re.kr; Tel.: +82-63-570-5618; Fax: +82-63-570-5609
Received July 3, 2019; Revised July 16, 2019; Accepted July 16, 2019.
Abstract
Ruminococcus sp. KGMB03662 was isolated from fecal samples obtained from a healthy Korean. The whole-genome sequence of Ruminococcus sp. KGMB03662 was analyzed using the PacBio Sequel platform. The genome comprises a 2,707,502 bp chromosome with a G + C content of 43.09%, 2,484 total genes, 2,367 protein-coding gene, 14 rRNA genes, and 53 tRNA genes. In the draft genome, genes involved in the hydrolysis enzyme, fatty acid biosynthesis, fatty acid metabolite, antibiotic biosynthesis, and antibiotic resistance have been identified. Those genes of KGMB03662 may be related to the regulation of human health and disease.
Keywords : Ruminococcus sp. KGMB03662, antibiotic biosynthesis, fatty acid biosynthesis, hydrolytic enzyme
Body

The gut microbiome is associated closely with health and disease (Sekirov et al., 2010). The short chain fatty acids are the primary end-products of fermentation of non-digestible carbohydrates that become available to the gut microbiome (Morrison and Preston, 2016). Therefore, many novel species remain to be identified and characterized from the human gut (Lau et al., 2016). Recently, strain KGMB03662 was isolated during the investigation of the bacterial diversity of Korean gut microbiome. On the basis of the phylogenetic, phenotypic and chemotaxonomic characteristics, strains KGMB03662T (= KCTC 15720T = CCUG 726234T) was found to belong to a novel species as a member of the genus Ruminococcus within the family Ruminococcaceae of Clostridia.

Members of the genus Ruminococcus are strictly anaerobic, Gram-positive, non-motile cocci bacteria (Rainey, 2009). These species belong to the phyla Firmicutes which are the predominant bacterial groups in the human gut microbiota. The Ruminococcus species have been isolated from human gut (Chassard et al., 2012), feces (Simmering et al., 2002; Domingo et al., 2008; Kim et al., 2011) and cattle rumen (Sijpesteijn, 1949). The Ruminococcus is less abundant in human with inflammatory bowel disease (Nagao-Kitamoto and Kamada, 2017). Also, genus of Ruminococcus is less abundant in Parkinson’s disease (Hill-Burns et al., 2017). Here, we describe the draft genome sequence and annotation of Ruminococcus sp. KGMB03662 isolated from healthy Korean feces.

The study was approved by the institutional review board (IRB) of Korea Research Institute of Bioscience and Biotechnology (KRIBB, Approval number: P01-201702-31-007). The fecal sample was collected from Seoul National University Bundang Hospital, Republic of Korea. The Ruminococcus sp. KGMB03662 was grown in Ruminococcus albus broth for 5 days at 37°C under a N2/H2/CO2 (8.6:0.7:0.7, by volume) gas mixture. The composition of Ruminococcus albus medium was as follows: 5.0 g Tryptone, 2.0 g Yeast extract, 3.0 g Glucose, 2.0 g Cellobiose, 1.0 mg Resazurin, 920.0 g Distilled water, 40.0 ml Mineral solution 1 (per L: 0.06 g K2HPO4) and 40.0 ml Mineral solution 2 [per L: 0.06 g KH2PO4, 0.2 g (NH4)2SO4, 0.12 g NaCl, 0.025 g MgSO4·7H2O and 0.016 g CaCl2·2H2O] at pH 7.0. After, media was autoclave-sterilize. Additionally, 4.0 g Na2CO3, 1.0 ml fatty acid mixture (10 ml Isobutyric acid, 10 ml Isovaleric acid, 10.0 ml 2-Methylbutyric acid, and 70.0 ml DW) and 500 mg L-cysteine·HCl·H2O were added.

The genomic DNA was obtained from the cultivated cells on Ruminococcus albus agar during 3 days using the Wizard genomic DNA purification kit (Promega). The purified genomic DNA sheared to a size of 10 kb using a g-TUBE™ device according to the manufacturer’s instructions (Covaris). The fragmented DNA quantity was analyzed by a Qubit 2.0 fluorometer with a Qubit dsDNA high sensitivity assay kit (Invitrogen). The DNA size was measured by the Agilent 2100 Bioanalyzer with the DNA 12000 assay kit (Agilent). The Single-Molecule Real-Time (SMRT) bell library was prepared according to the manufacturer’s instructions (Pacific Biosciences) without a non-size selection. The genome sequencing was performed using a Pacific Biosciences Sequel (Pacific Biosciences) with 2.0 sequencing chemistry and 600-min movies.

De nove assembly of the sequencing reads was performed through the Hierarchical Genome Assembly Process (HGAP4, version 4.0, Pacific Biosciences). Pipeline in the SMRT Analysis (version 4.0, Graphical User Interface) used default parameters. Potential contamination in genome assemblies was checked by the ContEst16S. The tRNAs were searched by using tRNAscan-SE. The CRISPRs were predicted using PILER-CR and CRISPR Recognition Tool (CRT). The rRNAs and other non-coding RNAs were predicted by covariance model search with the inference of Rfam 12.0 (Nawroki and Eddy, 2013). The annotation of each CDS was performed by homology search against Swiss-prot (UniProt, 2015), EggNOG 4.5 (Powell et al., 2014), SEED (Overbeek et al., 2005), and KEGG (Kanehisa et al., 2014) databases. The functional assignment of genes was performed by searching translated coding DNA sequences (CDSs) against sequences in the clusters of orthologous group (COG) databases (Tatusov et al., 2000).

The genome statistics are shown in Table 1. The draft genome of Ruminococcus sp. KGMB03662 was composed of a 2,707,502 bp chromosome with a G + C content of 43.09%. The genome features of Ruminococcus sp. KGMB03662 are summarized in Fig. 1. The genome is showed to contain 2,367 Protein-coding genes, 14 rRNAs (5S, 16S, 23S), and 53 tRNAs were annotated. A total of 2,023 genes were functionally assigned to categories based on COG assignments.

General features of Ruminococcus sp. KGMB03662

Property Value
Genome assembly
 Assemble method SMRT Analysis version 4.0
 Genome coverage 376X
Genome features
 Genome size (bp) 2,707,502
 G+C content (%) 43.09
 No. of contigs 7
 Total genes 2,484
 Protein-coding genes 2,367
 Pseudo genes 45
 rRNA genes (5S, 16S, 23S) 14 (2, 6, 6)
 tRNA genes 53
 CDS assigned by COG 2,023
 GenBank Accession No. VCGV00000000

Fig. 1.

Graphical circular map of Ruminococcus sp. KGMB03662. Marked characteristics are shown from outside to the center; coding DNA sequences (CDS) on forward strand, CDS on reverse strand, tRNA, rRNA, GC content, and GC skew.


The majority of the genes are related to replication, recombination and repair [163 genes (8.1%)], amino acid transport and metabolism [157 genes (7.8%)] and translation, ribosomal structure and biogenesis [143 genes (7.1%)].

We found that effective genes involved in hydrolysis enzymes, fatty acid biosynthesis, fatty acid metabolism, antibiotic biosynthesis, and antibiotic resistance were identified in the genome. The genome sequence contained genes for hydrolysis enzymes such as cellulose, chitinase, β-glucosidase bglX, chitooligosaccharide deacetylase pdaA, fructan β-fructosidase fruA, β-fructofuranosidase INV|sacA, mannan endo-1,4-β-mannosidase gmuG, α-galactosidase galA|rafA, and β-galactosidase lacZ. The genome contained the fatty acid biosynthesis and metabolism genes such as 3-oxoacyl-[acyl-carrier-protein] reductase fabG, Holo-[acyl-carrier-protein] synthase acpS, Lysophospholipase pldB, Acyl carrier protein, Acetyl-CoA carboxylase accA, Acetyl-CoA carboxylase accD, Biotin carboxyl carrier protein of acetyl-CoA carboxylase, 3-oxoacyl-(acyl-carrier-protein) synthase nodE, Beta-ketoacyl-[acyl-carrier-protein] synthase III fabH, [Acyl-carrier-protein] S-malonyltransferase fabD. The genome contained the antibiotic biosynthesis genes such as GTP diphosphokinase relA. Also, the genome has several antibiotic resistance factor genes, such as Zinc D-Ala-D-Ala carboxypeptidase vanY, Macrolide export ATP-binding/permease protein MacB macB, Undecaprenyl-diphosphate phosphatase bacA, Phosphinothricin acetyltransferase pat, Multidrug export protein MepA, Bacitracin transport ATP-binding protein BcrA. The draft genome sequence of Ruminococcus sp. KGMB03662 will contribute to understanding the physiological functions of Ruminococcus sp. KGMB03662 in the gut.

Based on the 16S rRNA gene sequence similarity and average nucleotide identity (ANI), the strain KGMB03662 is most closely related to Ruminococcus albus 7T with the values of 94.05% and 71.35%, respectively.

Nucleotide sequence accession number

Ruminococcus sp. KGMB03662 has been deposited in the Korean Collection for Type Cultures under accession number KCTC 15720. The GenBank/EMBL/DDBJ accession number for the genome sequence of Ruminococcus sp. KGMB03662 is VCGV00000000.

적 요

본 연구에서는 건강한 한국인 분변으로부터 Ruminococcus sp. KGMB03662 균주를 분리하고 유전체서열을 PacBio Sequel 플랫폼을 사용하여 분석하였다. 염색체의 크기는 2,707,502 bp 로 G + C 구성 비율은 43.09%, 총 유전자수는 2,484개, 단백질 코딩 유전자는 2,367개, rRNA는 14개 및 tRNA는 53개로 구성되었다. 본 유전체로부터 가수분해효소, 지방산생합성 및 대사와 항생제생합성 및 내성 관련 유전자를 확인하였다. 이러한 유전체의 분석은 KGMB03662 균주가 사람의 건강 및 질병에 관여할 것으로 여겨진다.

Acknowledgements

This work was supported by the Bio & Medical Technology Development program (Project No. NRF-2016M3A9F3947962) 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.

References
  1. Chassard C, Delmas E, Robert C, Lawson PA, and Bernalier-Donadille A. 2012. Ruminococcus champanellensis sp. nov., a cellulose-degrading bacterium from human gut microbiota. Int. J. Syst. Evol. Microbiol 62, 138-143.
    Pubmed CrossRef
  2. Domingo MC, Huletsky A, Boissinot M, Bernard KA, Picard FJ, and Bergeron MG. 2008. Ruminococcus gauvreauii sp. nov., a glycopeptide-resistant species isolated from a human faecal specimen. Int. J. Syst. Evol. Microbiol 58, 1393-1397.
    Pubmed CrossRef
  3. Hill-Burns EM, Debelius JW, Morton JT, Wissemann WT, Lewis MR, Wallen ZD, Peddada SD, Factor SA, Molho E, and Zabetian CP, et al. 2017. Parkinson's disease and Parkinson's disease medications have distinct signatures of the gut microbiome. Mov. Disord 32, 739-749.
    Pubmed CrossRef
  4. Kanehisa M, Goto S, Sato Y, Kawashima M, Furumichi M, and Tanabe M. 2014. Data, information, knowledge and principle:back to metabolism in KEGG. Nucleic Acids Res 42, D199-205.
    Pubmed CrossRef
  5. Kim MS, Roh SW, and Bae JW. 2011. Ruminococcus faecis sp. nov., isolated from human faeces. J. Microbiol 49, 487-491.
    Pubmed CrossRef
  6. Lau JT, Whelan FJ, Herath I, Lee CH, Collins SM, Bercik P, and Surette MG. 2016. Capturing the diversity of the human gut microbiota through culture-enriched molecular profiling. Genome Med 8, 72.
    Pubmed CrossRef
  7. Morrison DJ, and Preston T. 2016. Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes 7, 189-200.
    Pubmed CrossRef
  8. Nagao-Kitamoto H, and Kamada N. 2017. Host-microbial cross-talk in inflammatory bowel disease. Immune Netw 17, 11-12.
    Pubmed CrossRef
  9. Nawrocki EP, and Eddy SR. 2013. Infernal 1.1:100-fold faster RNA homology searches. Bioinformatics 29, 2933-2935.
    Pubmed CrossRef
  10. Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY, Cohoon M, de Crécy-Lagard V, Diaz N, Disz T, and Edwards R, et al. 2005. The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res 33, 5691-5702.
    Pubmed CrossRef
  11. Powell S, Forslund K, Szklarczyk D, Trachana K, Roth A, Huerta-Cepas J, Gabaldon T, Rattei T, Creevey C, and Michael K, et al. 2014. eggNOG v4.0:nested orthology inference across 3686 organisms. Nucleic Acids Res 42, D231-239.
    Pubmed CrossRef
  12. Rainey F. 2009. Springer. Family VIII. Ruminococcaceae fam. nov, pp. 1016-1043. Bergey's Manual of Systematic Bacteriology, 3, 2nd edn, Vol. 3. Dordrecht, Heidelberg, London and New York, USA.
  13. Sekirov I, Russell SL, Antunes LC, and Finlay BB. 2010. Gut microbiota in health and disease. Physiol. Rev 90, 859-904.
    Pubmed CrossRef
  14. Sijpesteijn A. 1949. Cellulose-decomposing bacteria from the rumen of cattle. Thesis. Leiden University. With a summary in Antonie van Leeuwenhoek. J. Microbiol. Serol 15, 49.
    CrossRef
  15. Simmering R, Taras D, Schwiertz A, Le Blay G, Gruhl B, Lawson PA, Collins MD, and Blaut M. 2002. Ruminococcus luti sp. nov., isolated from a human faecal sample. Syst. Appl. Microbiol 25, 189-193.
    Pubmed CrossRef
  16. Tatusov RL, Galperin MY, Natale DA, and Koonin EV. 2000. The COG database:a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res 28, 33-36.
    Pubmed CrossRef
  17. UniProt Consortium. 2015. UniProt:a hub for protein information. Nucleic Acids Res 43, D204-212.
    Pubmed CrossRef


September 2019, 55 (3)
Full Text(PDF) Free

Social Network Service
Services

Author ORCID Information