We report the complete 8.94-Mb genome sequence of the type strain of Cupriavidus basilensis (DSM 11853 = CCUG 49340 = RK1), formed by two chromosomes and six putative plasmids, which offers insights into its chloroaromatic-biodegrading capabilities.
ABSTRACT
We report the complete 8.94-Mb genome sequence of the type strain of Cupriavidus basilensis (DSM 11853 = CCUG 49340 = RK1), formed by two chromosomes and six putative plasmids, which offers insights into its chloroaromatic-biodegrading capabilities.
ANNOUNCEMENT
The complete genome sequence of the type strain of Cupriavidus basilensis (← Wautersia basilensis ← Ralstonia basilensis ← Ralstonia sp.) (1–4) has been determined. Strain RK1T (= DSM 11853T = CCUG 49340T) was isolated from sediment from a freshwater pond in Amponville, France, with 2,6-dichlorophenol as the sole carbon and energy source (1).
Strain DSM 11853T was cultivated on Reasoner’s 2A (R2A) broth, at 30°C, for 48 h. Genomic DNA was isolated, using a GenElute bacterial genomic DNA kit (Sigma-Aldrich) and a Wizard genomic DNA purification kit (Promega) for Illumina sequencing and a previously described protocol (5) for Oxford Nanopore sequencing. A DNA library was prepared, using a Nextera XT kit (Illumina) and sequenced on an Illumina HiSeq platform at MicrobesNG (Birmingham, UK), generating 3,305,358 paired-end reads of 251 bp. Another library was prepared, using a TruSeq Nano DNA sample preparation kit (Illumina), and sequenced on an Illumina MiSeq platform at ChunLab, Inc. (Seoul, South Korea), resulting in 4,445,298 paired-end reads of an average length of 292 bp. The reads were trimmed using Sickle v1.33 (Phred quality cutoff, Q30) (6) and assessed using CLC Genomics Workbench v12.0.3 (Qiagen).
Two Oxford Nanopore libraries were prepared, using a rapid barcoding sequencing kit (SQK-RBK004), and sequenced on a MinION device (Oxford Nanopore). The Nanopore reads were base called, using Guppy v2.3.7 and v3.1.5 (Oxford Nanopore) and evaluated, using NanoPlot v1.26.3 (7). The sequencing runs yielded 1.82 and 1.72 Gb, distributed in 291,236 and 243,691 reads, with N50 values of 11,574 and 12,956 bp, respectively.
The Illumina and Nanopore reads were assembled de novo using Unicycler v0.4.7 (8), resulting in complete circular sequences for all replicons except for chromosome 1, which was completed by assembling all Nanopore reads de novo, using Canu v1.5 (9). Subsequently, the sequence was polished with Illumina reads, using the tool Polish with Reads in CLC Genomics Workbench v20 (one round) and Pilon v1.20 (10) (two rounds). For Pilon, the reads were mapped using BWA v0.7.17 (11). The assembly statistics were obtained, using QUAST v5.0.2 (12). The complete genome sequence is composed of eight circular replicons, two chromosomes, and six putative plasmids, totaling 8,942,610 bp (Table 1). The sequence was annotated, using PGAP v4.13 (13) and BlastKOALA v2.2 (14), revealing 8,060 coding sequences (including 1,082 hypothetical proteins), 7 ribosomal operons, 67 tRNAs, and 236 pseudogenes, with a G+C content of 65.0 mol%.
TABLE 1.
General features of the eight replicons of the complete genome sequence of C. basilensis DSM 11853T (= CCUG 49340T = RK1T)
| Replicon | GenBank accession no. | Length (bp) | G+C content (mol%) | No. of CDSa | No. of ribosomal RNAs | No. of operons | No. of tRNAs | No. of hypothetical proteins (percentage of CDS)a |
|---|---|---|---|---|---|---|---|---|
| Chromosome 1 | CP062803 | 4,566,734 | 65.3 | 4,123 | 12 | 4 | 54 | 464 (11) |
| Chromosome 2 | CP062804 | 3,303,026 | 65.8 | 2,908 | 9 | 3 | 12 | 357 (12) |
| Plasmid pRK1-1 | CP062805 | 425,364 | 61.0 | 387 | 0 | 0 | 1 | 90 (23) |
| Plasmid pRK1-2 | CP062806 | 355,033 | 62.0 | 322 | 0 | 0 | 0 | 62 (19) |
| Plasmid pRK1-3 | CP062807 | 125,309 | 60.2 | 132 | 0 | 0 | 0 | 54 (41) |
| Plasmid pRK1-4 | CP062808 | 82,842 | 62.6 | 104 | 0 | 0 | 0 | 40 (38) |
| Plasmid pRK1-5 | CP062809 | 81,787 | 62.3 | 81 | 0 | 0 | 0 | 14 (17) |
| Plasmid pRK1-6 | CP062810 | 2,515 | 59.2 | 3 | 0 | 0 | 0 | 1 (33) |
| Total | NAb | 8,942,610 | 65.0 | 8,060 | 21 | 7 | 67 | 1,082 (13) |
CDS, coding DNA sequences.
NA, not applicable.
The key genes involved in chloroaromatic degradation, encoding chlorophenol monooxygenases (GenBank accession number QOT82435 and QOT82420), chlorohydroquinone 1,2-dioxygenase (QOT82419), and chlorocatechol 1,2-dioxygenases (QOT82433 and QOT82442), are located on plasmid pRK1-5 (CP062809). Additionally, C. basilensis DSM 11853T has extensive catabolic potential, harboring nearly all major central pathways for aromatic compounds (15), including catechol 1,2-dioxygenase (QOT79538), catechol 2,3-dioxygenase (QOT80779), protocatechuate 3,4-dioxygenase (QOT80900 and QOT81306), homoprotocatechuate 2,3-dioxygenase (QOT78968), gentisate 1,2-dioxygenase (QOT81130), and homogentisate 1,2-dioxygenase (QOT81322), all of them located on chromosome 2 (CP062804), among other ring-cleavage enzymes.
This complete genome sequence represents a valuable taxonomic reference within the genus Cupriavidus and the family Burkholderiaceae and offers a genetic basis for elucidating the catabolic pathways for chloroaromatic compounds in this specialized bacterium.
Data availability.
This complete genome sequence has been deposited in DDBJ/ENA/GenBank under the accession numbers CP062803, CP062804, CP062805, CP062806, CP062807, CP062808, CP062809, and CP062810. The versions described in this paper are the first versions. The Illumina and Oxford Nanopore raw sequence reads are available in the Sequence Read Archive under the accession numbers SRR12739612, SRR12739613, SRR12739614, and SRR12739615.
ACKNOWLEDGMENTS
This work was funded by FONDECYT 1201741, ANID PIA/Anillo ACT172128, and ANID PIA/BASAL FB0002 from the Chilean Government (D.P-P.). F.S.-S. was supported by the Genomics and Proteomics Research on Bacterial Diversity Programme of the Culture Collection University of Gothenburg (CCUG), through the Department of Clinical Microbiology, Sahlgrenska University Hospital, and the Department of Infectious Disease, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg. Illumina HiSeq genome sequencing was provided by MicrobesNG, which is supported by the BBSRC (grant number BB/L024209/1). The computations were partially performed on resources provided by the Swedish National Infrastructure for Computing (SNIC) through the Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) under project SNIC 2019/8-176.
We thank Christel Unosson for providing assistance with DNA extraction and the staff at the CCUG for providing technical assistance with strain cultivation and maintenance.
REFERENCES
- 1.Steinle P, Stucki G, Stettler R, Hanselmann KW. 1998. Aerobic mineralization of 2,6-dichlorophenol by Ralstonia sp. strain RK1. Appl Environ Microbiol 64:2566–2571. doi: 10.1128/AEM.64.7.2566-2571.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Goris J, De Vos P, Coenye T, Hoste B, Janssens D, Brim H, Diels L, Mergeay M, Kersters K, Vandamme P. 2001. Classification of metal-resistant bacteria from industrial biotopes as Ralstonia campinensis sp. nov., Ralstonia metallidurans sp. nov. and Ralstonia basilensis Steinle et al. 1998 emend. Int J Syst Evol Microbiol 51:1773–1782. doi: 10.1099/00207713-51-5-1773. [DOI] [PubMed] [Google Scholar]
- 3.Vaneechoutte M, Kämpfer P, De Baere T, Falsen E, Verschraegen G. 2004. Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov. Int J Syst Evol Microbiol 54:317–327. doi: 10.1099/ijs.0.02754-0. [DOI] [PubMed] [Google Scholar]
- 4.Vandamme P, Coenye T. 2004. Taxonomy of the genus Cupriavidus: a tale of lost and found. Int J Syst Evol Microbiol 54:2285–2289. doi: 10.1099/ijs.0.63247-0. [DOI] [PubMed] [Google Scholar]
- 5.Salvà-Serra F, Gomila M, Svensson-Stadler L, Busquets A, Jaén-Luchoro D, Karlsson R, Moore ERB. 2018. A protocol for extraction and purification of high-quality and quantity bacterial DNA applicable for genome sequencing: a modified version of the Marmur procedure. Protoc Exch doi: 10.1038/protex.2018.084. [DOI] [Google Scholar]
- 6.Joshi NA, Fass JN. 2011. Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (version 1.33). https://github.com/najoshi/sickle.
- 7.De Coster W, D'Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34:2666–2669. doi: 10.1093/bioinformatics/bty149. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595. doi: 10.1371/journal.pcbi.1005595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722–736. doi: 10.1101/gr.215087.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. doi: 10.1371/journal.pone.0112963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760. doi: 10.1093/bioinformatics/btp324. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. doi: 10.1093/bioinformatics/btt086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI Prokaryotic Genome Annotation Pipeline. Nucleic Acids Res 44:6614–6624. doi: 10.1093/nar/gkw569. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Kanehisa M, Sato Y, Morishima K. 2016. BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol 428:726–731. doi: 10.1016/j.jmb.2015.11.006. [DOI] [PubMed] [Google Scholar]
- 15.Pérez-Pantoja D, Donoso R, Agulló L, Córdova M, Seeger M, Pieper DH, González B. 2012. Genomic analysis of the potential for aromatic compounds biodegradation in Burkholderiales. Environ Microbiol 14:1091–1117. doi: 10.1111/j.1462-2920.2011.02613.x. [DOI] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
This complete genome sequence has been deposited in DDBJ/ENA/GenBank under the accession numbers CP062803, CP062804, CP062805, CP062806, CP062807, CP062808, CP062809, and CP062810. The versions described in this paper are the first versions. The Illumina and Oxford Nanopore raw sequence reads are available in the Sequence Read Archive under the accession numbers SRR12739612, SRR12739613, SRR12739614, and SRR12739615.