Complete genome sequence of Kosakonia pseudosacchari RX.G5M8, a putative methylotroph isolated from Hong Kong soil

ABSTRACT

Kosakonia pseudosacchari RX.G5M8, a putative methylotroph, was isolated from garden soil in Hong Kong. Its complete genome, a single chromosome of 4,953,935 bp (GC content 53.91%), was established through hybrid assembly.

ANNOUNCEMENT

Genus Kosakonia, designated in 2013 after reassignment of four species of Enterobacter (1), currently comprises at least nine species (2). These include K. sacchari, first isolated from Saccharum officinarum (3, 4); and, K. pseudosacchari, found as a Zea mays endophyte (5). While Kosakonia spp. are rarely human pathogens, K. quasisacchari and K. cowanii have been isolated from sites of infection (6, 7). Nevertheless, the significant antimicrobial resistance and virulence genes of K. cowanii strain 888-76T (8) seem uncommon in the other species. Instead, these are more often recognized as plant growth-promoting rhizobacteria with plant-beneficial genes enabling phosphate solubilization, siderophore production, nitrogen fixation, as well as indole-3-acetic acid and ethylene synthesis (9–13). Here, RX.G5M8 was isolated from soil screened for methylotrophy, a plant-associated trait previously observed in K. arachidis (14).

A 0.1 g sample of garden soil collected in Hong Kong (22.3038099 N, 114.1616503 E; 7 November 2021) was vortexed with 900 µL 0.9% (wt/vol) saline and a 10-µL aliquot used to inoculate 1 mL of a minimal salts medium (MSM) (15) containing 5% (vol/vol) methanol as the only carbon source. Following incubation at 27°C for 7 days with shaking, the mixture was streaked onto Luria agar (16), giving cream-colored colonies after overnight growth. Selected colonies were passaged to purity on Luria agar and reassessed for growth in MSM containing 1% (vol/vol) methanol by plate counts of aliquots withdrawn daily over 5 days. For DNA extraction (Qiagen DNeasy PowerSoil Pro Kit), a single colony of RX.G5M8, which showed moderate growth in 1% methanol, was spread on Luria agar and incubated for 24 h before harvesting.

A paired-end short-read sequencing library (NEB Next Ultra DNA Library Prep Kit) was sequenced via the NovaSeq 6000 platform using an SP PE250 flowcell and v1.5 Reagent Kit. Reads were quality filtered and trimmed using TrimGalore! v0.6.7 (https://github.com/FelixKrueger/TrimGalore) (stringency: 3; -e: 0.2), giving 2,137,624 read pairs (mean length, 250  bp) totaling ~534 Mbp. Long-read libraries, prepared from the same extracted DNA using the Rapid Barcoding Kit SQK-RBK004 (without size selection), were sequenced via Oxford Nanopore’s Spot-ON Flow Cell (vR9.4.1) and MinION sequencer, with MinKNOW v20.06.2 software and basecalling by Guppy v6.1.5 high-accuracy mode. The final long-read data set, trimmed by Filtlong v0.2.1 (https://github.com/rrwick/Filtlong) (min_length: 2,000; target_bases: 500 Mbp; length_weight: 10), totaled 63,710 reads (984 Mbp) with mean length 15,448 bp (N50 19,020). Default parameters were used for all software unless otherwise specified.

Unicycler v0.4.3 (17) combined the Illumina and MinION data sets to yield a circular chromosome of 4,953,935 bp (53.91% G + C, mean coverage 199×), which was submitted to NCBI PGAP v5.0 (18) for annotation. JSpeciesWS (19) found RX.G5M8 closest to Kosakonia pseudosacchari strain BDA62-3 (CP063425), with an average nucleotide identity of 98.67%.

RX.G5M8 possesses a glutathione-dependent formaldehyde detoxification pathway for methanol metabolism (20). The nitrogen-fixing Nif-regulon is at locus PF050_12345 to PF050_12440, and the recently identified non-mobile colistin resistance enzyme (NMCR-2) (21) is encoded at locus PF050_10580.

DATA AVAILABILITY

The complete genome sequence and raw sequence data for Kosakonia pseudosacchari RX.G5M8 are available through NCBI under BioProject PRJNA922486, with GenBank and SRA accession numbershttps://www.ncbi.nlm.nih.gov/nuccore/CP082785.1CP115712, SRR24734531 (MinION), and SRR24734532 (NovaSeq), respectively.