Draft Genome of a Heavy-Metal-Resistant Bacterium, Cupriavidus Sp

Draft genome of a heavy-metal-resistant bacterium, Cupriavidus sp. strain SW-Y-13, isolated from river water in Korea

Kiwoon Baek , Young Ho Nam , Eu Jin Chung , and Ahyoung Choi* Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Republic of Korea

강물에서 분리한 중금속 내성 세균 Cupriavidus sp. SW-Y-13 균주의 유전체 해독

백기운 ･ 남영호 ･ 정유진 ･ 최아영* 국립낙동강생물자원관 담수생물연구본부

(Received July 6, 2020; Revised September 18, 2020; Accepted September 18, 2020)

Cupriavidus sp. strain SW-Y-13 is an aerobic, Gram-negative, found to survive in close association with pollution-causing rod-shaped bacterium isolated from river water in South Korea, heavy metals, for example, Cupriavidus metallidurans, which in 2019. Its draft genome was produced using the PacBio RS II successfully grows in the presence of Cu, Hg, Ni, Ag, Cd, Co, platform and is thought to consist of five circular chromosomes Zn, and As (Goris et al., 2001; Vandamme and Coenye, 2004; with a total of 7,307,793 bp. The genome has a G + C content Janssen et al., 2010). Several bacteria found in polluted of 63.1%. Based on 16S rRNA sequence similarity, strain SW-Y-13 is most closely related to Cupriavidus metallidurans environments have been shown to adapt to the presence of toxic (98.4%). Genome annotation revealed that the genome is heavy metals. Identification of novel bacterial mechanisms comprised of 6,613 genes, 6,536 CDSs, 12 rRNAs, 61 tRNAs, facilitating growth in heavy-metal-polluted environments and 4 ncRNAs. Resistance to Co2+ is primarily mediated by the provides useful information for the development of novel efflux system encoded by the SW-Y-13 genome, which includes technologies (Ayangbenro and Babalola, 2017). In the present the czcCBA operons, czcD genes, and czcN genes, among others. study, we describe the draft genome sequence of a heavy- This study may provide useful information on the heavy-metal metal-resistant bacterium, Cupriavidus sp. strain SW-Y-13. resistance mechanisms of strain SW-Y-13. Cupriavidus sp. strain SW-Y-13 is a novel organism and was Keywords: Cupriavidus sp., draft genome, heavy-metal-resistant isolated from the surface of the Nakdong River in South Korea bacterium, PacBio RS II (36.432178 N, 128.250770 E) using a standard dilution plating method on YPD agar (BD Difco). This collection was screened 2+ 2+ Cupriavidus belongs to the family Burkholderiaceae, from for heavy metal (Co and Cu ) resistance. Genomic DNA was which the type species Cupriavidus necator was isolated from extracted using the DNeasy Blood and Tissue kit (Qiagen) soil (Makkar and Casida, 1987). Currently, the genus comprises according to the manufacturer’s instructions. Phylogenetic 16 species that inhabit diverse environments (http://www. analysis based on 16S rRNA gene sequences revealed that bacterio.net/cupriavidus). Some Cupriavidus species have been strain SW-Y-13 is closely related to Cupriavidus metallidurans strain CH34T with 98.4% sequence similarity (Fig. 1). *For correspondence. E-mail: [email protected]; To produce sequencing data for the generation of a high- Tel.: +82-54-530-0712; Fax: +82-54-530-0719 344 ∙ Baek et al.

Fig. 1. Neighbor-joining phylogenetic tree based on 16S rRNA sequences showing the relationship among isolates belonging to the Cupriavidus sp. SW-Y-13 and related taxa. Numbers at each node indicate bootstrap values (above 50%) based on 1,000 resampled data sets. Scale Bar represents 0.005 substitutions per nucleotide position. quality genome, the genomic DNA from strain SW-Y-13 was (PGAP v4.8) (Tatusova et al., 2016). Potential coding sequences extracted and further purified from a pure culture isolate grown were evaluated using the Basic Local Alignment Search Tool on YPD agar at 25°C, using the DNeasy Blood and Tissue kit (BLAST) and the UniProt (Wu et al., 2006), Pfam (Punta et al., and Wizard Genomic DNA Purification Kit (Promega), 2012), and COG (Tatusov et al., 2003) databases. rRNA and respectively. Whole-genome sequencing was performed on the tRNA genes as well as other miscellaneous features, were PacBio RS II platform (Menlo Park). Single-molecule real-time predicted using the RNAmmer 1.2 (Lagesen et al., 2007) and sequencing (SMRT) yielded a total of 136,681 subreads (1,4 tRNAscan-SE 1.21 (Lowe and Eddy, 1997) servers and the Gb, mean subread length: 10,333 bp, N50 value: 14,631) and Rfam v. 12.0 database (Nawrocki et al., 2015). de novo assembly of the SW-Y-13 genome was completed The basic genome statistics are provided in Table 1. The using the Hierarchical Genome Assembly Process 3 (HGAP3) draft genome of strain SW-Y-13 comprised 5 contigs with a G within the PacBio SMRT analysis 2.3.0 software (Chin et al., + C content of 63.1% and a genome size of 7,307,793 bp (N50, 2013). Contigs were annotated via Rapid Annotation using the 2,677,351 bp; sequencing depth of coverage, 68.7×). The Subsystem Technology (RAST v2.0) server (Aziz et al., 2008) genome contained 6,536 CDSs, 12 rRNAs (5S, 16S, 23S), 61 and the NCBI Prokaryotic Genome Annotation Pipeline tRNAs, and 4 ncRNAs genes (Fig. 2). A total of 4,088 genes

미생물학회지 제56권 제3호 Draft genome sequence of Cupriavidus sp. SW-Y-13 ∙ 345

Table 1. General genomic features of Cupriavidus sp. SW-Y-13 06057), cobalt efflux system protein (czcA; CUSW19178_

Features Value 04268, czcD; CUSW19178_03032, and CUSW19178_06063), Genome size (bp) 7,307,793 copper resistance protein (copD; CUSW19178_06088), nickel G + C content (mol%) 63.1 –cobalt–cadmium resistance protein (nccB; CUSW19178_ No. of contigs 5 04822), nickel and cobalt resistance protein (cnrR; CUSW Genes (total) 6,613 19178_04825 and cnrY; CUSW19178_04826), and arsenic CDSs (total) 6,536 resistance protein (arsB; CUSW19178_04162). The draft Genes (coding) 6,409 genome sequence of SW-Y-13 will contribute to our under- CDSs (with protein) 6,409 standing of the underlying mechanism of heavy metal resistance No. of rRNA genes (5S, 16S, 23S) 12 (4, 4, 4) in Cupriavidus species. No. of tRNA genes 61 were functionally assigned to COG categories. Nucleotide sequence accession number The genome annotation analysis revealed several genes The whole genome sequence of Cupriavidus sp. strain known to be involved with metal resistance, including cobalt– SW-Y-13 was deposited in DDBJ/ENA/GenBank under the zinc–cadmium resistance protein (czcB; CUSW19178_03307, accession number WHLX00000000. The strain was deposited czcC; CUSW19178_03308, CUSW19178_03498, CUSW19 at the Korean Culture Center for Microorganisms under the 178_04266, CUSW19178_06060, and czcN; CUSW19178_ accession number KCCM 43367.

Fig. 2. Circular map of the Cupriavidus sp. strain SW-Y-13 genome. From outside to the center; the colored bands in ring 1 represent contigs; ring 2 represents the annotated genes on the forward strand (color determined by COG category); ring 3 shows the annotated genes on the reverse strand (color determined by COG category); ring 4 displays the RNA genes (rRNAs are displayed in red and tRNAs are displayed in purple); ring 5 shows the GC skew (higher-than-average values are displayed in green, while lower-than-average values are displayed in red) and ring 6 shows the GC ratio (higher-than-average values in blue and lower-than-average values in yellow).

Korean Journal of Microbiology, Vol. 56, No. 3 346 ∙ Baek et al.

적 요 Clum A, Copeland A, Huddleston J, Eichler EE, et al. 2013. Nonhybrid, finished microbial genome assemblies from long- Cupriavidus sp. SW-Y-13 균주는 2019년 한국의 강물에서 read SMRT sequencing data. Nat. Methods 10, 563–569. Goris J, De Vos P, Coenye T, Hoste B, Janssens D, Brim H, Diels L, 분리된 호기성 그람 음성 막대 모양의 세균이다. PacBio RS II 플 Mergeay M, Kersters K, and Vandamme P. 2001. Classification 랫폼을 사용하여 초안 유전체를 얻었으며, 5개의 원형 염색체로 of metal-resistant bacteria from industrial biotopes as Ralstonia 구성되어있다. SW-Y-13 균주의 유전체 크기는 총 7,307,793 bp, campinensis sp. nov., Ralstonia metallidurans sp. nov. and G + C 함량은 63.1%이다. 16S rRNA 유사도에 따르면 SW-Y- Ralstonia basilensis Steinle et al. 1998 emend. Int. J. Syst. Evol. 13 균주는 Cupriavidus metallidurans (98.4%)와 가장 밀접한 Microbiol. 51, 1773–1782. Janssen PJ, Van Houdt R, Moors H, Monsieurs P, Morin N, Michaux 관련이 있다. 유전체 분석을 통해 총 6,613개의 유전자와 6,539 A, Benotmane MA, Leys N, Vallaeys T, Lapidus A, et al. 2010. 개의 CDS, 12개 rRNA, 61개 tRNA, 4개 ncRNA를 포함하였 The complete genome sequence of Cupriavidus metallidurans 다. CO2+에 대한 내성은 주로 czcCBA 오페론, czcD 유전자 및 strain CH34, a master survivalist in harsh and anthropogenic czcN 유전자를 포함하는 SW-Y-13 게놈에 의해 인코딩된 유 environments. PLoS ONE 5, e10433. Lagesen K, Hallin P, Rødland EA, Staerfeldt HH, Rognes T, and 출 시스템에 의해 매개된다. 이 연구는 SW-Y-13 균주의 중금 Ussery DW. 2007. RNAmmer: consistent and rapid annotation 속 저항 메커니즘에 대한 유용한 정보를 제공할 수 있다. of ribosomal RNA genes. Nucleic Acids Res. 35, 3100–3108. Lowe TM and Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964. Acknowledgments Makkar NS and Casida LE, Jr. 1987. Cupriavidus necator gen. nov., sp. nov.: a nonobligate bacterial predator of bacteria in soil. Int. This work was supported by a grant from the Nakdonggang J. Syst. Bacteriol. 37, 323–326. National Institute of Biological Resources (NNIBR), funded Nawrocki EP, Burge SW, Bateman A, Daub J, Eberhardt RY, Eddy by the Ministry of Environment (MOE) of the Republic of SR, Floden EW, Gardner PP, Jones TA, Tate J, et al. 2015. Rfam Korea (grant NNIBR202002103). 12.0: updates to the RNA families database. Nucleic Acids Res. 43, D130–D137. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, et al. 2012. The Pfam Conflict of Interest protein families database. Nucleic Acids Res. 40, D290–D301. Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, There are no conflicts of interest to declare. Koonin EV, Krylov DM, Mazumdr R, Mekhedov SL, Nikolskaya AN, et al. 2003. The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4, 41. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, References Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, and Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Ayangbenro AS and Babalola OO. 2017. A new strategy for heavy Acids Res. 44, 6614–6624. metal polluted environments: A review of microbial biosorbents. Vandamme P and Coenye T. 2004. Taxonomy of the genus Cupriavidus: Int. J. Environ. Res. Public Health 14, 94. a tale of lost and found. Int. J. Syst. Evol. Microbiol. 54, 2285– Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, 2289. Formsma K, Gerdes S, Glass EM, Kubal M, et al. 2008. The Wu CH, Apweiler R, Bairoch A, Natale DA, Barker WC, Boeckmann RAST server: rapid annotations using subsystems technology. B, Ferro S, Gasteiger E, Huang H, Lopez R, et al. 2006. The BMC Genomics 9, 75. universal protein resource (UniProt): an expanding universe of Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, protein information. Nucleic Acids Res. 34, D187–D191.

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