Complete Genome Sequence of Sutterella Sp. KGMB03119 Isolated from Healthy Korean Feces

Complete genome sequence of Sutterella sp. KGMB03119 isolated from healthy Korean feces

1 1 1 1 1 1 1 Seoung Woo Ryu , Byeong Seob Oh , Seung Yeob Yu , Seung-Hwan Park , Se Won Kang , Ji-Sun Kim , Kook-Il Han , 1 1 1 1 1 1 2 Keun Chul Lee , Mi Kyung Eom , Jam-Eon Park , Seung-Hyeon Choi , Min Kuk Suh , Han Sol Kim , Dong Ho Lee , 2 3 3 1,4 1 Hyuk Yoon , Byung-Yong Kim , Je Hee Lee , Jung-Sook Lee , and Ju Huck Lee * 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

건강한 한국인 분변으로부터 분리된 Sutterella sp. KGMB03119 균주의 유전체 염기서열

유승우1 ･ 오병섭1 ･ 유승엽1 ･ 박승환1 ･ 강세원1 ･ 김지선1 ･ 한국일1 ･ 이근철1 ･ 엄미경1 ･ 박잠언1 ･ 최승현1 ･ 서민국1 ･ 김한솔1 ･ 이동호2 ･ 윤혁2 ･ 김병용3 ･ 이제희3 ･ 이정숙1,4 ･ 이주혁1* 1한국생명공학연구원 생물자원센터, 2분당서울대학교병원, 3천랩, 4과학기술연합대학원대학교

(Received November 19, 2019; Revised December 16, 2019; Accepted December 17, 2019)

The genus of Sutterella was isolated from human and dog feces, Gut microbiota affect human fitness in a variety of ways; one except Sutterella wadsworthensis was isolated from human way is that the production of metabolites by microbes contri- appendiceal and peritoneal fluid. Sutterella sp. KGMB03119 butes to the health of the host (Nicholson et al., 2012). The was isolated from healthy human feces. The whole-genome imbalance of the gut microbiota called dysbiosis may be sequence of the isolate was analyzed by PacBio Sequel associated with diverse diseases, not only metabolic diseases platform. The genome consists a 2,987,884 bp chromosome with a G + C content of 58.30%, 2,436 total genes, 18 rRNA but also mental disorders like autism (Backhed et al., 2012). genes and 63 tRNA genes. In addition, we found that strain Despite the increasing knowledge about gut microbiome due to KGMB03119 had several genes for antibiotic resistance and the development of metagenome analysis technology, it is still bile acid resistance in its genome based on the result of genome difficult to study gut microbiota because of the lack of gut analysis. microbe resources. As the need for collection and storage of gut Keywords: Sutterella sp. KGMB03119, antibiotic resistance, bile microbiota for therapeutic applications increases (Bolan et al., acid resistance, human feces 2016), Korean gut microbiome bank launched in 2016. Recently, a novel bacterial strain designated KGMB03119 was isolated from a Korean fecal sample. Based on phylogenic, phenotypic and chemotaxonomic characteristics, strain KGMB03119 (= KCTC 15823T) was identified to a novel species as a member of *For correspondence. E-mail: [email protected]; Tel.: +82-63-570-5634; Fax: +82-63-570-5609 the genus Sutterella within the family Sutterellaceae which 66 ∙ Ryu et al.

belongs to the order Burkholderiales in the class Betaproteo- Table 1. General features of Sutterella sp. KGMB03119 bacteria. Based on the 16S rRNA gene sequence similarity and Property Value average nucleotide identity, strain KGMB03119 is most Genome assembly closely related to Sutterella wadsworthensis KCTC 15691T Assemble method SMRT Analysis version 4.0 with the values of 96.9%. Genome coverage 133X The genus Sutterella was first isolated and proposed by Genome features Wexler et al. (1996). Members of the genus Sutterella are Gram- Genome size (bp) 2,987,884 G+C content (%) 58.3 negative, non-spore forming, anaerobic or microaerophilic No. of contigs 1 bacteria (Wexler et al., 1996; Greetham et al., 2004; Sakon et rRNA genes (5S, 16S, 23S) 18 (6, 6, 6) al., 2008). Sutterella genus was isolated from human and dog tRNA genes 63 feces while S. wadsworthensis was isolated from patients with Open reading frame 2,436 a variety of gastrointestinal infections (Wexler et al., 1996). CDS assigned by COG 2,160 Here, we described the complete genome sequence and anno- GenBank Accession No. CP040882 tation of Sutterella sp. KGMB03119 isolated from healthy Korean feces. searched by covariance model search with inference of Rfam We collected the fecal samples from the people who had not 12.0. Each CDS was annotated through a homology search taken any medicine and had normal BMI (Body Mass Index) against Swiss-prot, EggNOG 4.5, SEED, and KEGG databases. from Seoul National University Bundang Hospital for Korean The genome statistics are described in Table 1. The complete gut microbiome bank, and strain KGMB30119 was isolated genome of Sutterella sp. KGMB03119 consists of a single from one of the collected fecal samples. One gram of fecal circular 2,987,884 bp chromosome with a G + C content of sample suspended in oxygen free sterilized phosphate-buffered 58.30%. Furthermore, the genome contains 2,436 CDSs, 18 saline (PBS) was diluted using the PBS by decuple dilution up rRNAs (5S, 16S, 23S), and 63 tRNAs based on CLgenomics to 10-6. The diluted fecal samples were spread and cultured on (Fig. 1). Total 2,160 genes were functionally allocated to cate- tryptic soy agar supplemented with 5% sheep blood (TSAB). gories based on clusters of orthologous group (COG) assignments. All procedures for the isolation of stain KGMB03119 were Previously it was reported that S. wadsworthensis was performed in anaerobic chamber (Coy Laboratory Products) intermediately susceptible or resistant to some antibiotic agents. containing 90% N2, 5% H2, and 5% CO2. For example, S. wadsworthensis was resistant to piperacillin, For the whole genome sequencing of strain KGMB03119, which is β-lactam antibiotic of the ureidopenicillin class the genomic DNA was extracted using a Wizard genomic DNA (Molitoris et al., 1997). Interestingly, the genome sequence of purification kit (Promega), a 10 kb Single-Molecule Real-Time Sutterella sp. KGMB03119 contains gene for β-lactamase. (SMRT) bell library was prepared according to the manu- Moreover, we found the genes for multidrug efflux transporter facturer’s instructions (Pacific Biosciences) without a non-size proteins such as multidrug resistance protein MdtA, MdtB, selection, and Pacific Biosciences Sequel was used with 2.0 MdtC, MdtK, and probable multidrug resistance protein NorM sequencing chemistry and 600-minute movies. De novo genome which play a predominant role in the multidrug resistance of of strain KGMB03119 was assembled by the Hierarchical gram-negative bacteria (Nikaido, 2009); all these suggest that Genome Assembly Process (HGAP4) pipeline in the SMRT strain KGMB03119 possibly has the ability of the antibiotic Analysis version 4.0 using default parameters. Probable con- resistance. In addition, all of three species in Sutterella genus tamination in genome assembles was checked by the Contami- possess 20% bile acid resistance activity (Wexler et al., 1996; nation Estimator by 16S (ContEst16S) and CheckM tools (Parks Greetham et al., 2004; Sakon et al., 2008). Same as the other et al., 2015; Lee et al., 2017). The coding DNA sequences species, the genome of KGMB03119 revealed the presence of (CDSs) and tRNAs were predicted using prodigal and tRNAscan- ileal sodium/bile acid cotransporter, indicating that Sutterella SE, respectively. The rRNAs and other non-coding RNAs were sp. KGMB03119 may also have the resistance to bile acid. All

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Fig. 1. Graphical circular map of Sutterella sp. KGMB03119. 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 and forward strand (colored by COG categories) and RNA genes (rRNAs-red and tRNAs-blue). these predicted functions, however, need to be experimentally 분석결과를 토대로 다양한 항생제 내성 유전자와 담즙산 내성 verified. Nevertheless, the complete genome sequence generated 관련 유전자를 가지고 있는 것을 발견하였다. in this study will contribute to understanding the physiological functions of Sutterella sp. KGMB03119 in the gut. Acknowledgments Nucleotide sequence accession number This work was supported by the Bio & Medical Technology Sutterella sp. KGMB03119 has been deposited in the Development program (Project No. NRF-2016M3A9F3947 Korean Collection for Type Cultures under accession number 962) of the National Research Foundation of Korea (NRF) KCTC 15823. The GenBank/EMBL/DDBJ accession number funded by the Ministry of Science and ICT (MSIT) of the for the genome sequence of Sutterella sp. KGMB03119 is Republic of Korea and a grant from the Korea Research CP040882. Institute of Bioscience & Biotechnology (KRIBB) Research initiative program.

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Sutterella 속 균주들은 사람과 개의 분변에서 분리된 것으 References 로 알려져 있다. 본 연구에서는 건강한 한국인 분변으로부터 Backhed F, Fraser CM, Ringel Y, Sanders ME, Sartor RB, Sherman Sutterella sp. KGMB03119 균주를 분리하였으며 PacBio Se- PM, Versalovic J, Young V, and Finlay BB. 2012. Defining a quel 플랫폼을 이용하여 Sutterella sp. KGMB03119 균주의 유 healthy human gut microbiome: Current concepts, future direc- 전체서열을 분석하였다. 유전체는 G + C 구성 비율이 58.3%이 tions, and clinical applications. Cell Host Microbe 12, 611–622. Bolan S, Seshadri B, Talley NJ, and Naidu R. 2016. Bio-banking gut 고, 염색체의 크기는 2,987,884 bp이며, 2,436개의 유전자와 microbiome samples. EMBO Rep. 17, 929–930. 18개의 rRNA, 63개의 tRNA로 구성되어 있다. 또한, 유전체 Greetham HL, Collins MD, Gibson GR, Giffard C, Falsen E, and

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Lawson PA. 2004. Sutterella stercoricanis sp. Nov., isolated 78, 119–146. from canine faeces. Int. J. Syst. Evol. Microbiol. 54, 1581–1584. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, and Tyson GW. Lee I, Chalita M, Ha SM, Na SI, Yoon SH, and Chun J. 2017. 2015. CheckM: Assessing the quality of microbial genomes Contest16s: An algorithm that identifies contaminated proka- recovered from isolates, single cells, and metagenomes. Genome ryotic genomes using 16S RNA gene sequences. Int. J. Syst. Res. 25, 1043–1055. Evol. Microbiol. 67, 2053–2057. Sakon H, Nagai F, Morotomi M, and Tanaka R. 2008. Sutterella Molitoris E, Wexler HM, and Finegold SM. 1997. Sources and parvirubra sp. Nov. and Megamonas funiformis sp. Nov., isolated antimicrobial susceptibilities of Campylobacter gracilis and from human faeces. Int. J. Syst. Evol. Microbiol. 58, 970–975. Sutterella wadsworthensis. Clin. Infect. Dis. 25 Suppl 2, S264– Wexler HM, Reeves D, Summanen PH, Molitoris E, McTeague M, 265. Duncan J, Wilson KH, and Finegold SM. 1996. Sutterella Nicholson JK, Holmes E, Kinross J, Burcelin R, Gibson G, Jia W, and wadsworthensis gen. Nov., sp. Nov., bile-resistant microaero- Pettersson S. 2012. Host-gut microbiota metabolic interactions. philic Campylobacter gracilis-like clinical isolates. Int. J. Syst. Science 336, 1262–1267. Bacteriol. 46, 252–258. Nikaido H. 2009. Multidrug resistance in bacteria. Annu. Rev. Biochem.

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