Complete genome sequence of Cupriavidus sp. Agwp_2, isolated from soil in an iron mining area

TL;DR

This paper presents the full genome sequence of a new Cupriavidus bacterium found in soil from an iron mining area.

Contribution

The complete genome sequence of Cupriavidus sp. Agwp_2 is newly reported, including its two chromosomes and a plasmid.

Findings

The genome consists of two chromosomes totaling 6,608,576 bp and a plasmid of 847,469 bp.

The average GC content of the genome is 66.17%.

Abstract

Cupriavidus spp. is a Gram-negative bacterium belonging to the Burkholderia family. Here, we report the complete genome sequence of Cupriavidus sp. Agwp_2, whose genome consists of two chromosomes of 3,823,900 bp and 2,784,676 bp, respectively, and one plasmid of 847,469 bp with an average GC content of 66.17%.

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ANNOUNCEMENT

The genus Cupriavidus includes Gram-negative bacteria belonging to the Burkholderia family within the β-proteobacteria class. Strain Agwp_2 was isolated from a soil sample that was gathered from a deserted iron mining site (26.46 N 106.38 E) situated in Doupeng Mountain, Guiyang, China in July 2021. The soil sample was processed into a suspension and vigorously agitated for 2 hours. Then, 200 µL of the supernatant was plated onto a selective Luria-Bertani (LB, excluding NaCl) agar plate supplemented with 50 µmol/L AgNO3. The inoculated plate was inverted and incubated at 28°C for 48 hours. Ultimately, we obtained a colony named Agwp_2 that exhibited resistance to silver nitrate, as confirmed by its successful growth in NaCl-free LB liquid medium supplemented with 50 µmol/L AgNO_3_. The 16S rRNA gene sequence was amplified using the colony PCR method with the primer pair 27F and 1492R. A BLAST search for the 16S rRNA gene of the Agwp_2 strain showed the highest sequence identity of 99.15% to that of Cupriavidus necator strain N-1 (NR_102851.1); thus, it was designated as Cupriavidus sp. Agwp_2. This genome sequence is a valuable resource for defining the relatedness among species within the genus Cupriavidus.

Strain Agwp_2 was cultured in 100 mL LB medium at 28°C for 24 hours. Genomic DNA was extracted using the SDS method (1), and its quality and quantity were assessed by agarose gel electrophoresis and Qubit 2.0 Fluorometer (Thermo Fisher Scientific), respectively. The library with an insert size of ≥10 kb was prepared with the SQK-LSK109 rapid barcoding kit (Oxford Nanopore, Oxford, UK) with the native barcoding expansion kit (EXP-NBD104) following the manufacturer’s protocol. The constructed library was loaded onto a PromethION Flow Cell FLO-PRO002 (Oxford Nanopore Technologies) and sequenced on a PromethION Beta sequencer. Base calling was performed using Guppy v6.2.7 (2) with the parameter “-c dna_r9.4.1_450bps_hac_prom.cfg.” The data underwent quality control using NanoPlot (v1.38.0) (3) with the minimum Q score cutoff of 7. A total of 262,814 filtered reads (2,928,675,317 bp) were obtained, with a mean length of 11,143.50 bp and an N50 value of 12,155 bp, providing an average coverage of 392.79×. Sequence assembly was performed using Unicycler (Version 0.4.7) (4) with default parameters and polished using Medaka (v1.8.0) with basecall model “r941_prom_hac_g507.” The genome sequences were circularized using Unicycler that automatically identified and trimmed the overlapping ends, and the genome was not rotated. Assembly quality was assessed using both checkM v5.0.2 (5) and Quast (6) with default parameters. Taxonomic classification by average nucleotide identity analysis, using a local version of PGAP (version 2024-04-27.build7426) (7), showed a score below the cutoff value (>95%) with the closest relative (92.917%), C. oxalaticus NBRC 13593^T^ (GCA_001592245.1).

Two chromosomes of 3,823,900 bp and 2,784,676 bp and the plasmid of 847,469 bp are all circular, with GC content of 66.44%, 67.15%, and 61.73%, respectively. We annotated Agwp_2’s genome using the PGAP v5.3 (7). A total of 6,832 genes were identified across all three replicons, including 6,537 coding sequences, 93 RNA genes, and 202 pseudogenes.