Complete Genome Sequence of Comamonas Sp. NLF-7-7 Isolated from Biofilter of Wastewater Treatment Plant

Complete genome sequence of Comamonas sp. NLF-7-7 isolated from biofilter of wastewater treatment plant

1 2 1 1 1 1 2 Dong-Hyun Kim , Kook-Il Han , Hae Jun Kwon , Mi Gyeong Kim , Young Guk Kim , Doo Ho Choi , Keun Chul Lee , 2 2 2,3 1 Min Kuk Suh , Han Sol Kim , Jung-Sook Lee * , and Jong-Guk Kim * 1 School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu 41566, Republic of Korea 2 Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea 3 University of Science and Technology (UST), Daejeon 34113, Republic of Korea

폐수처리장의 바이오 필터로부터 분리된 Comamonas sp. NLF-7-7 균주의 유전체 염기서열 해독

김동현1 ･ 한국일2 ･ 권해준1 ･ 김미경1 ･ 김영국1 ･ 최두호1 ･ 이근철2 ･ 서민국2 ･ 김한솔2 ･ 이정숙2,3* ･ 김종국1* 1 2 3 경북대학교, 한국생명공학연구원 생물자원센터, 과학기술연학대학원대학교

(Received August 27, 2019; Revised September 17, 2019; Accepted September 17, 2019)

Comamonas sp. NLF-7-7 was isolated from biofilter of waste- wastewater need to pass through wastewater treatment plants water treatment plant. The whole-genome sequence of Comamonas and purified. But the water purification process causes a huge sp. NLF-7-7 was analyzed using the PacBio RS II and Illumina amount of odor pollutants. Biofilters are responsible for removing HiSeqXten platform. The genome comprises a 3,333,437 bp these odor pollutants by specific microorganisms which can chromosome with a G + C content of 68.04%, 3,197 total genes, degrades pollutants (Wani et al., 1997). A sulfur oxidizing 9 rRNA genes, and 49 tRNA genes. This genome contained SoxB pollutants degradation and floc forming genes such as sulfur bacteria investigation performed that presence of a gene oxidization pathway (SoxY, SoxZ, SoxA, and SoxB) and floc indicate presence of the Sox pathway and this means presence forming pathway (EpsG, EpsE, EpsF, EpsG, EpsL, and gly- of sulfur oxidization ability in organism (Petri et al., 2001; cosyltransferase), respectively. The Comamonas sp. NLF-7-7 Anandham et al., 2008). During the study of analyzing kinds of can be used to the purification of wastewater. cultivable bacteria in Biofilter of the wastewater treatment Keywords: Comamonas sp. NLF-7-7, Illumina HiSeqXten, plants, a novel bacterial strain NLF-7-7 was isolated. Based on PacBio RS II, wastewater phylogenetic, phenotypic and chemotaxonomic data, strains NLF-7-7 (= KCTC 62943) was identified as a novel species as a member of the genus Comamonas within the family Large scale of farm cause large scale of wastewater. This Comamonadaceae of Betaproteobacteria. The genus Comamonas, proposed by De Vos et al. (1985), is Gram-negative, aerobic, *For correspondence. (J.G. Kim) E-mail: [email protected]; motile, rod shaped bacteria. And the Comamonas sp. NLF-7-7 Tel.: +82-53-950-5379; Fax: +82-53-950-5379 / is Gram-negative, aerobic, non-motile, rod shaped, and floc (J.S. Lee) E-mail: [email protected]; Tel.: +82-63-570-5618; Fax: +82-63-570-5609 forming bacteria. Here we describe the complete genome 310 ∙ Kim et al.

sequence and annotation of Comamonas sp. NLF-7-7 isolated Table 1. General features of Comamonas sp. NLF-7-7 from biofilter of wastewater treatment plant in Nonsan, Republic Property Value of Korea. Genome assembly The Comamonas sp. NLF-7-7 was grown in Trypticase Assemble method RS HGAP Assembly version 3.0 soy agar (TSA; Difco) for 3~4 days at 30°C under aerobic Genome coverage 364X condition. After lysozyme pretreatment, the genomic DNA Genome features ® was automatically extracted and purified by using Maxwell Genome size (bp) 3,333,437 G+C content (%) 68.04 16 Cell DNA Purification Kit. Nucleotides were incorporated No. of contigs 1 into a sequence by the DNA polymerase while reading a Total genes 3,197 template, like circular Single-Molecule Real-Time (SMRT) Protein-coding genes 3,079 bell template. Polymerase reads were trimmed to pick up only Pseudo genes 57 high quality region. Each polymerase read was divided to form rRNA genes (5S, 16S, 23S) 9 (3, 3, 3) one or more subreads. The DNA library was prepared using tRNA genes 49 PacBio DNA Template Prep Kit 1.0. Sequencing library was CDS assigned by COG 3,012 prepared by random fragmentation of the DNA sample and this GenBank Accession No. CP042344 library. Genome sequencing was performed using PacBio RS II and Illumina HiSeqXten platform. The sequencing data was P2 protein dmpM, Phenol hydroxylase P5 protein dmpP and converted into raw data for the analysis. tmoA. In the description, Stingele et al. (1999) reported that Eps De novo assembly was performed by using RS HGAP Assembly proteins and glycosyltransferase had a function of flocculation version 3.0 (Chin et al., 2013). Illumina raw data were filtered (exopolysaccharide biosynthesis) of bacteria. The genome by quality for error correction. The assembly was corrected sequence of this strain contained Eps protein and various using high quality HiSeqXten reads by Pilon v1.21 (Walker et glycosyltransferase gene like EpsD, EpsE, EpsF, EpsG, and al., 2014). The genome was annotated using Prokka v1.13 EpsL, and Glycosyltransferase Gtf1, Ubiquinone biosynthesis (Seemann, 2014) based on NCBI Prokaryotic Genome Annotation O-methyltransferase ubiG4, UDP-N-acetyl-D-mannosaminuronic Pipeline. For annotation, predicted protein sets were prepared acid transferase wecG, D-inositol-3-phosphate glycosyltransferase to perform InterProScan v5.30-69.0 (Jones et al., 2014) and mshA1, and O-antigen biosynthesis glycosyltransferase WbnK, psiblast v2.4.0 (Camacho et al., 2009) with EggNOG database respectively. The complete genome information of Comamonas v4.5 (Huerta-Cepas et al., 2016). Circular maps displaying sp. NLF-7-7 will contribute to understanding of the biological each contigs were generated using Circos v0.69.3. (Krzywinski functions of Comamonas sp. NLF-7-7 in the wastewater. et al., 2009). Based on the 16S rRNA gene sequence similarity and average The genome statistics are showed in Table 1. Total number of nucleotide identity, the most closely related strain with strain raw reads of strain NLF-7-7 was 1,856,190. Complete genome T NLF-7-7 is Comamonas badia KCTC 12244 with the values of Comamonas sp. NLF-7-7 was composed of a 3,333,437 bp of 95.81% and 81.91%, respectively. chromosome, genome coverage is 362X and G + C content is 68.04%. The genome contains 3,079 CDSs, 49 tRNAs and 9 Nucleotide sequence accession number rRNAs (5S, 16S, 23S) were annotated (Fig. 1). A total of 3,012 Comamonas genes were matched to EggNOG database. We found a cluster sp. NLF-7-7 has been deposited in the Korean of genes that involved in some pollutants degradation and floc Collection for Type Cultures under accession number KCTC forming pathway. The genome showed presence of SoxY, SoxZ, 62943. The GenBank/EMBL/DDBJ accession number for the Comamonas SoxA, and SoxB, which have role in sulfur oxidization pathway. genome sequence of sp. NLF-7-7 is CP042344. And the genome showed presence of phenol hydroxylation protein like Phenol hydroxylase P1 protein mphL, Phenol hydroxylase

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Fig. 1. Chromosome map of Comamonas sp. NLF-7-7. Marked characteristics are shown from outside to the center; coding sequences on forward strand, coding sequences on reverse strand, Transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), GC content, and GC skew.

적 요 Acknowledgements

본 연구에서는 폐수처리장의 바이오필터로부터 Comamonas This research was supported by project for Cooperative R&D sp. NLF-7-7 균주를 분리하고 유전체서열을 PacBio RS II와 between Industry, Academy, and Research Institute funded Illumina HiSeqXten 플랫폼을 사용하여 분석하였다. 염색체 Korea Ministry of SMEs and Startups in 2018 Grants No. 의 크기는 3,333,437 bp로 G + C 구성 비율은 68.04%, 총 유전 S2597396, and also from the Korea Research Institute of 자수는 3,197개, rRNA는 9개 및 tRNA는 49개로 구성되었다. Bioscience & Biotechnology (KRIBB) Research Initiative 본 유전체는 오염물질분해와 플록형성에 관여하는 황산화 경 Program. 로 유전자(SoxY, SoxZ, SoxA 및 SoxB)와 플록형성 경로 유전자 (EpsG, EpsE, EpsF, EpsG, EpsL 및 glycosyltransferase)를 포 함하고 있다. 이러한 Comamonas sp. NLF-7-7 균주는 폐수를 References 정화하는데 활용될 수 있다. Anandham R, Indiragandhi P, Madhaiyan M, Ryu KY, Jee HJ, and Sa TM. 2008. Chemolithoautotrophic oxidation of thiosulfate and

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phylogenetic distribution of sulfur oxidation gene (soxB) in genome-scale protein function classification. Bioinformatics 30, rhizobacteria isolated from crop plants. Res. Microbiol. 159, 1236–1240. 579–589. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer Jones SJ, and Marra MA. 2009. Circos: An information aesthetic K, and Madden TL. 2009. BLAST+: architecture and applications. for comparative genomics. Genome Res. 19, 1639–1645. BMC Bioinformatics 10, 421. Petri R, Podgorsek L, and Imhoff JF. 2001. Phylogeny and distribution Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, of the soxB gene among thiosulfate-oxidizing bacteria. FEMS Clum A, Copeland A, Huddleston J, Eichler EE, et al. 2013. Microbiol. Lett. 197, 171–178. Nonhybrid, finished microbial genome assemblies from long-read Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. SMRT sequencing data. Nat. Methods 10, 563–569. Bioinformatics 30, 2068–2069. De Vos P, Kersters K, Falsen E, Pot B, Gillis M, Segers P, and De Ley Stingele F, Newell JW, and Neeser JR. 1999. Unraveling the function J. 1985. Comamonas Davis and Park 1962, gen. nov., nom. rev. of glycosyltransferases in Streptococcus thermophilus Sfi6. J. emend., and Comamonas terrigena Hugh 1962, sp. nov., nom. Bacteriol. 181, 6354–6360. rev. Int. J. Syst. Bacteriol. 35, 443–453. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Huerta-Cepas J, Szklarczyk D, Forslund K, Cook H, Heller D, Walter Cuomo CA, Zeng Q, Wortman J, Young SK, et al. 2014. Pilon: MC, Rattei T, Mende DR, Sunagawa S, Kuhn M, et al. 2016. an integrated tool for comprehensive microbial variant detection eggNOG 4.5: a hierarchical orthology framework with improved and genome assembly improvement. PLoS One 9, e112963. functional annotations for eukaryotic, prokaryotic and viral Wani A, Branion R, and Lau AK. 1997. Biofiltration: A promising and sequences. Nucleic Acids Res. 44, D286–293. cost-effective control technology for Odors, VOCs and air Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, McWilliam toxics. J. Ferment. Technol. 64, 161–167. H, Maslen J, Mitchell A, Nuka G, et al. 2014. InterProScan 5:

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