Complete genome sequence of Psychrobacter sp. GTC18467 isolated from snake in Japan

TL;DR

This paper presents the full genome sequence of a new Psychrobacter species found on a snake in Japan.

Contribution

The study provides the complete genome sequence of a newly identified Psychrobacter species.

Findings

The genome consists of a circular chromosome of 2,642,444 bp.

It includes three circular plasmids.

Abstract

The genus Psychrobacter belongs to the family Moraxellaceae within the class Gammaproteobacteria. Here, we report the complete genome sequence of a new Psychrobacter species obtained from snake skin in Japan. The genome comprised a circular chromosome with a length of 2,642,444 bp and three circular plasmids.

Tables

TABLE 1. Information regarding the complete genome sequence of a Psychrobacter sp. strain isolated from a snake in Japan

	Strain name
Parameter	Psychrobacter sp. GTC18467
DNBSEQ sequencinga
No. of reads	4,772,296
Size (kb)	715,844
Avg coverage (x)	271
DRA accession no.	DRR623152
ONT sequencinga
No. of reads	136,869
Size (kb)	1,494,621
Avg read length (bp)	10,920
Avg coverage (x)	566
N50	14,570
DRA accession no.	DRR623151
Assembly
Assembly N50 (bp)c	2,598,574
Estimated genome completeness (%)c	100.0
Estimated genome contamination (%)c	0.0
Genome structure	One chromosome and three plasmids
DDBJ/GenBank accession no.	AP038920 (GTC18467)AP038921 (pGTC18467-1)AP038922 (pGTC18467-2)AP038923 (pGTC18467-3)
Genome size (bp)	2,598,574 (GTC18467)26,064 (pGTC18467-1)12,994 (pGTC18467-2)4,812 (pGTC18467-3)
GC content (%)
(Chromosome/plasmid name)	45.34 (GTC18467)41.29 (pGTC18467-1)39.85 (pGTC18467-2)42.21 (pGTC18467-3)
Average read depth
	259 (GTC18467)1,037 (pGTC18467-1)778 (pGTC18467-2)1,528 (pGTC18467-3)
No. of coding sequencesb	2,275
Number of rRNAsb	12
Number of tRNAsb	48
Number of CRISPRsb	9

Full text

ANNOUNCEMENT

Identifying pet animal pathogens through genomic analysis is important for understanding pathogenic mechanisms and zoonotic infection risks (1, 2). We analyzed strain GTC18467, isolated from Python regius skin in Seki City, Gifu, Japan, in 2024. The strain was cultured on tryptic soy agar with sheep blood at 37°C for 48 h under aerobic conditions and was submitted to our laboratory as a presumptive Psychrobacter strain for further identification. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker, Bremen, Germany; 9607 MSPs) was used for identification, confirming its genus as Psychrobacter. The strain was stored at −80°C after four passages during identification.

The strain was cultured on tryptic soy agar under the same conditions. Colonies were scraped, suspended in TE buffer, and pelleted by centrifugation. Genomic DNA was extracted using NucleoBond HMW DNA (MACHEREY-NAGEL, Japan).

For identification, the 16s rRNA gene was sequenced after PCR amplification using primers 16S-8F (AGAGTTTGATCMTGGCTCAG) and 16S-1492R (GGYTACCTTGTTACGACTT). BLASTN analysis (3) identified the strain as Psychrobacter sp., most closely related to Psychrobacter ciconiae 176/10 (type strain Acc. no. NR_134796), with 97.4% similarity.

The genome was sequenced using PromethION 2i (Oxford Nanopore Technologies [ONT], Oxford, UK) for long-read sequencing and DNBSEQ (MGI Tech Co., Ltd., Shenzhen, China) for short-read sequencing (4–6). The same DNA extract was used for both sequencing processes. A library was constructed with a Native Barcode Sequencing Kit (SQK-NBD114-24; ONT) without shearing for long-read sequencing. Small DNA fragments were removed using Short Read Eliminator XS (Pacific Biosciences of California Inc., USA). Sequencing was performed on a PromethION 2i (ONT) with a FLO-PRO114M flow cell. Base calling was done with Dorado v.7.2.14 (ultra-high accuracy mode), and raw reads were trimmed and quality-filtered using NanoFilt v.2.7.1 (7) with parameters “-l 1000-q 10--headcrop 50.” For short reads, library construction was done with the MGIEasy FS DNA Library Prep Set (MGI Tech), and 2 × 150 bp paired-end sequencing was performed on the DNBSEQ-G400 platform (MGI Tech). Reads were processed with fastp v.0.20.1 (8) using “-q 30 -n 20 -t 1 -T 1.” Short-read quality was assessed using fastp (8), and long-read quality was scored using NanoPlot 1.32.1 (8). Short reads (with over 95.3% of bases >Q30) and long reads (mean read quality 15.8) were assembled using Unicycler v.0.4.8 (9) with default settings. Assembly circularization and rotation were performed automatically in Unicycler, and the assembly was rotated to start with the dnaA gene on the forward strand. The contig graph was visualized with Bandage v.0.8.1 (10), and the taxonomic integrity was confirmed using blobtools v.1.0 with short reads (11).

Table 1 summarizes the genomic information. Unicycler and Bandage indicated a complete circular genome. Quality and genome statistics were computed using CheckM (v1.2.2) (12) and seqkit (v2.9.0) (13). CheckM confirmed the genome was 100% complete with 0% contamination. The DDBJ Fast Annotation and Submission Tool (14) predicted 2,275 coding sequences, 12 rRNAs, 48 tRNAs, and 9 CRISPR sequences. Species identification using an average nucleotide identity value of the genome sequence with GTDB-tK (v.2.3.2) was unsuccessful (15).