Complete Genome Sequence of Rhodococcus qingshengii N9T-4

ABSTRACT

Rhodococcus qingshengii N9T-4 can grow on media without added carbon sources. Here, we report the complete nucleotide sequence of the N9T-4 genome, consisting of a chromosome (6,439,972 bp), a linear plasmid (pN9T4-1 [565,206 bp]), and two circular plasmids (pN9T-4-2 [99,662 bp] and pN9T-4-3 [30,419 bp]).

ANNOUNCEMENT

Rhodococcus strains can be isolated from various environments (1). Previously, bacterial strains that can grow on media without added carbon sources, belonging to the genera Rhodococcus and Streptomyces, were isolated (2). Rhodococcus qingshengii (formerly Rhodococcus erythropolis) N9T-4, which was isolated from the stocked crude oil in an oil stockpile in Japan, showed the best growth among our isolates, and CO₂ is required for its growth (3). Although several genes that were upregulated under conditions without added carbon sources were identified (4, 5), complete genome sequencing is necessary to understand the carbon metabolism in N9T-4.

Total DNA of N9T-4 was extracted from 1 mL of overnight culture in Luria broth (6) using the NucleoBond high-molecular-weight (HMW) kit (Takara Bio, Shiga, Japan). The complete genome sequence of N9T-4 was determined using a combination of Pacific Biosciences (PacBio) Sequel and Illumina MiSeq sequencers. A PacBio 20-kb sequencing library was prepared using the SMRTbell template preparation kit v. 1.0 and sequenced with the Sequel platform (Macrogen, Inc., Seoul, South Korea). An Illumina sequencing library was constructed using the TruSeq Nano DNA library preparation kit and sequenced with the MiSeq platform. Sequel subreads were filtered (read lengths, ≥6,000 bp) using SeqKit v. 0.8.0 (7), and the resulting subreads were used for de novo assembly with Flye v. 2.8.3 (8) with the –plasmids option. Three circular contigs and one linear contig were obtained. Circularized contigs were rotated to start with the gene encoding the replication protein using Circlator v. 1.1.1 (9). Illumina paired-end (2 × 250-bp) reads were cleaned (read lengths, ≥150 bp; quality scores, ≥15) using Trimmomatic v. 0.39 (10), and the resulting high-quality short reads were used to correct Sequel errors using BWA-MEM v. 0.7.15 (11) and Pilon v. 1.23 (12). Mapping of short reads to all contigs indicated that there were no reads that mapped in pairs with the other contigs at either end of the linear contig, suggesting that the contig is a linear plasmid. Gene prediction and annotation were performed using DFAST v. 1.2.15 (13) by running GeneMarkS2 v. 1.14_1.25 (14), RNAmmer v. 1.2 (15), and tRNAscan-SE v. 2.0.5 (16). Default parameters were used for all software unless otherwise specified. Information on the PacBio and Illumina reads is summarized in Table 1.

TABLE 1.

Summary of reads generated by Illumina and PacBio sequencing

Parameter	Data for sequencing with:
	Illumina MiSeq	PacBio Sequel
Raw reads
No. of reads	1,558,816	73,378
Total size (bp)	390,155,166	779,091,421
N50 (bp)		13,738
Filtered reads
No. of reads	1,385,380	54,401
Total size (bp)	339,591,845	723,937,732
N50 (bp)		14,230
Mean coverage (×)a	48	101
Accession no.	DRR402011	DRR402012

^aTotal size of filtered reads (bp)/genome size of N9T-4 (bp).

The genome of N9T-4 consists of a circular chromosome (6,439,972 bp, with a G+C content of 62%), a linear plasmid (pN9T4-1 [565,206 bp], with a G+C content of 62%), and two circular plasmids (pN9T4-2 [99,662 bp] and pN9T4-3 [30,419 bp], with G+C contents of 62% and 63%, respectively). The genome has 6,738 coding sequences, 5 sets of rRNA genes, and 58 tRNA genes. Average nucleotide identity (ANI) analysis was performed with calcANI.pl (https://github.com/Computational-conSequences/SequenceTools) with the ANIb method using 586 genome sequences of Rhodococcus strains in the NCBI RefSeq database (as of 30 March 2022). The ANI analysis of N9T-4 showed high ANI values (>95%) with respect to the type strains R. qingshengii djl-6 (98.49%) (accession number CP096563) (17) and R. erythropolis JCM 3201 (accession number GCA_003990875) (18) (95.27%), suggesting that N9T-4 could be recognized as R. qingshengii.

Data availability.

The nucleotide sequences of the N9T-4 chromosome and plasmids have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers AP026691 (chromosome) and AP026692, AP026693, and AP026694 (plasmids pN9T4-1, pN9T4-2, and pN9T4-3, respectively). The raw data are available under DRA accession number DRA014768 for Illumina (accession number DRR402011) and PacBio (accession number DRR402012) sequencing. All project data are available under BioProject accession number PRJDB13588.