High-quality draft genome sequence of Cryobacterium sp. AP23 isolated from ancient Alaskan permafrost

ABSTRACT

The high-quality draft genome of AP23, a novel Cryobacterium species isolated from Alaskan permafrost with aromatic compound degradation genes, is presented here. Additional analyses are required to confirm its placement within the Cryobacterium genus; nevertheless, AP23 represents a useful candidate for industrially relevant low-temperature protein research.

ANNOUNCEMENT

The expanding human footprint in Arctic regions has led to waste accumulation (e.g., plastics, fuels, lubricants, paper-based products, and other organic contaminants commonly treated at wastewater treatment plants) (1–4). The low temperatures in those regions represent a significant challenge for established wastewater treatment methods (5, 6). Psychrophilic microbes such as those within the Cryobacterium genus (7) offer a potential solution given their known aromatic and petroleum-derived compound degradation capabilities (8–11).

Permafrost core samples were collected in June 2024 from the U.S. Army Cold Regions Research and Engineering Laboratory Permafrost Tunnel in Fox, AK, USA (64.9528, −147.6178) as previously described (12, 13), with the exception that a 0.5 m stainless steel barrel was used (cleaned with 70% ethanol, DNA Away, and RNase Away), rendering sub-sampling unnecessary. Single-colony samples were suspended in sterile phosphate-buffered saline, plated on LB agar, and incubated at 4°C. After the third passage, samples were preserved in glycerol and stored at −80°C. Fresh axenic culture was grown at room temperature (shaking, 200 RPM, LB broth). After 18 h, 1 mL of culture was centrifuged (13,000 × RCF, 1 min). Genomic DNA was isolated from the recovered pellet using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions (14). DNA was quantified using the Qubit dsDNA HS Quantification Assay Kit (Life Technologies, Carlsbad, CA, USA). This extracted DNA was used for both libraries. Long-read libraries were prepared using the Oxford Nanopore Rapid Barcoding Kit (Oxford Nanopore Technologies, Oxford, UK) with 200 ng of DNA as input according to the manufacturer’s instructions. DNA was not size-selected prior to enzymatic fragmentation using the kit. This library was sequenced on a Nanopore GridION Mk1 equipment and an R10.4.1 flow cell. Basecalling was done onboard the GridION (Dorado V. 7.4.13); a total of 50,798 raw reads, with an N50 of 3,354, were obtained according to NanoStat (v.1.6.0) (15). Short-read libraries were prepared using the Illumina DNA Prep kit (Illumina, California, USA) with 150 ng of DNA as input, and sequenced using a NextSeq 2000 P4 flowcell using PE150 chemistry, producing 12,386,740 reads, with a read length of 35–151 bp, as determined by FastQC (v.0.12.1) (16).

Default parameters were used for all listed software, unless otherwise specified. Raw reads were trimmed using fastp (v.0.22.0) (17). Long-reads were error corrected (--only-error-correction), then paired with the short-read data for hybrid assembly (--only-assembler –isolate -k 31,51,71,91,121) using SPAdes (v.4.2.0) (18) (Fig. 1). Post-assembly quality and marker-gene completion were evaluated using QUAST (v5.3.0) (19) and CheckM2 (v1.1.0; database Uniref100/KO) (20), respectively. Taxonomic identification via 16S rRNA gene BLAST (21) returned Cryobacterium soli GCJ02 (GCF_003611035.1) (22) as the best hit (100% query coverage; 99.80% identity). Nevertheless, average nucleotide identity analysis of AP23 via skani (v0.2.2; database r226 04/2025) (23) returned Cryobacterium sp. RYN_54 (GCA_025463825.1) (24) as best hit (88.36% identity; over GCJ02, with 86.16%), highlighting the need for further chemotaxonomic characterization.

Fig 1.

Circular representation of the Cryobacterium sp. AP23 genome. This scheme is purely illustrative; no circularization of the genome was performed. From outermost to innermost ring, the features displayed are (i) genomic position (Mb), (ii) genes encoded on the forward strand, (iii) genes encoded on the reverse strand, (iv) GC content (red = above average, green = below average), and (v) GC skew (orange/blue). This map was generated using pyCirclize (v1.9.0) (25) through Bakta (v1.11.0) (26).

Annotation through the Prokaryotic Genome Annotation Pipeline (2025-05-06.build7983, see Table 1) (27) revealed aromatic compound degradation enzymes like aromatic ring-opening dioxygenase, protocatechuate 3,4-dioxygenase, and extradiol aromatic ring-opening family dioxygenase.

TABLE 1.

General assembly and annotation overview

Features	Description
Size	4,242,373 Mb
Topology	Linear
Min. contig length	1 kbp
Contigs	1
Coverage	77.89×
N 50	4,242,373
CheckM2 completion	99.99%
CheckM2 contamination	0.98%
G + C content	68.55%
CDSs (with protein)	3,904
RNA	62
rRNA (5S, 16S, 23S)	4, 3, 3
tRNA	49
ncRNA	3

DATA AVAILABILITY

This Whole Genome Shotgun project has been deposited in GenBank under accession number JBSQBY000000000, as BioProject PRJNA1333436, and Biosample SAMN51795667. The assembly is available at GenBank through accession number GCA_054075385.1. Short- and long-raw reads are available at SRX32022346 and SRX32115231, respectively. The isolate has been deposited in the CRREL ICE COLD (Innovative, Collaborative, Exploratory Cold Regions Organism Library for Discovery in Biotechnology; https://icecold-library.github.io/).