Complete genome sequences of two Pseudoalteromonas undina strains isolated from a marine nematode (Oncholaimidae) collected at Tybee Island

TL;DR

This paper presents the full genome sequences of two bacteria found in a marine nematode, helping to study their interactions.

Contribution

The novelty lies in reporting genome sequences of Pseudoalteromonas strains from a marine nematode, a less-studied symbiotic relationship.

Findings

Two Pseudoalteromonas undina strains were isolated from a marine nematode.

The complete genome sequences of these strains are reported for future interaction studies.

Abstract

Pseudoalteromonas is known to form symbiotic relationships with various marine invertebrates, but association with nematodes has not been well-explored. Here, we report the genome sequences of two Pseudoalteromonas strains isolated from a predatory marine nematode (Oncholaimidae) collected from Tybee Island, GA, that will facilitate the study of nematode-bacterial interactions.

Tables

TABLE 1. Sampling location and genome statistics for strains US3C1013 and US3C1004

Parameter	US3C1013	US3C1004
Sample location
Host	Nematode (Oncholaimidae)
Location	Tybee Island, GA
Latitude	32.0157
Longitude	−80.8911
Depth	0.5 m
Sample collection	December 2020
Isolation experiment	March 2021
Genome summary
Taxonomy (GTDB-tk)	Pseudoalteromonas undina	Pseudoalteromonas undina
Reference (GTDB-tk)	GCF_000238275.3	GCF_000238275.3
Genome size (bps)	4,118,019(3,320,003 + 788,016)	4,107,746(3,319,730 + 788,016)
Number of replicons	2	2
Circularized?	Yes	Yes
GC content	40%	40%
Number coding sequences	3,774	3,773
Completeness (CheckM)	100%	100%
Contamination (CheckM)	0.71%	0.71%
Coding density (CheckM)	0.883	0.884
Coverage (Flye)	100×	83×
Num read pairs Illumina	1,773,627	2,582,410
Num reads Nanopore/N50	226,322/35,802	262,460/40,362
Assembly accession	GCA_048401115.1	GCA_048401065.1
SRA accession Illumina	SRX27591692	SRX27591694
SRA accession Nanopore	SRX27591693	SRX27591695

Full text

ANNOUNCEMENT

Pseudoalteromonas bacteria are ubiquitous in marine ecosystems and have been found in both free-living and host-associated contexts, including microbiomes of several marine invertebrate phyla (1–4). Recently, there has been evidence that Pseudoalteromonas may occur in the core microbiome of marine nematodes in the family Oncholaimidae (5); however, nematode-associated Pseudoalteromonas have eluded whole-genome sequencing.

We isolated two Pseudoalteromonas strains from an oncholaimid nematode collected from muddy sediments at Tybee Island, GA, USA (Table 1). Nematodes were isolated from the sediment using a decantation-flotation method (6) and decanted over a 45 µm sieve using sterile artificial seawater (Instant Ocean, Spectrum Brands, Blacksburg, VA). Nematodes were picked under a dissecting microscope (Olympus SZX16, Olympus Corporation, Tokyo, Japan) and rinsed in sterile molecular-grade water, as described (7). We mounted the worms on temporary slides and identified them to the morphospecies level using the appropriate taxonomic keys (8) under a compound microscope (Olympus BX63, Olympus Corporation, Tokyo, Japan). Several nematodes from the same morphospecies (Family: Oncholaimidae) were subsequently transferred to a polymerase chain reaction (PCR) tube with 1 mL SJB1 sterile artificial seawater medium (9) and ground to a slurry using a sterile pipette tip in a biosafety cabinet. The slurry was treated with 0.008% Tween-20, vortexed for 5 min, and centrifuged at 500× g for 55 min at room temperature (RT) to dissociate microbial cells from the nematode biomass. We quantified the supernatant cell concentration using an Accuri C6 Plus flow cytometer (BD Biosciences) and inoculated SJB1 medium for high-throughput dilution culturing and identification as described (10). We grew strains US3C1013 and US3C1004 for sequencing in SJB1 at RT and extracted DNA using our phenol-chloroform method (11).

DNA from the same extractions was prepared for hybrid Oxford Nanopore and Illumina sequencing with an Oxford Nanopore Technologies native barcoding kit (#SQK-NBD114) and an Illumina DNA Prep tagmentation kit (#20060059) at SeqCoast Genomics. The Long Fragment Buffer was used to promote longer read lengths. No physical size selection was done. Sequencing was performed on an Illumina NextSeq2000 using a 300-cycle flow cell kit, producing 2 × 150 bp paired-end reads. A 1%–2% PhiX control was spiked into the run to support optimal base calling. Nanopore sequencing was completed using a FLO-PRO114M version R10.4.1 flow cell (translocation speed 400 bps) on a PromethION 2 Solo sequencer. Base-calling was performed using the super-accurate model with barcode trimming enabled with MinKNOW v24.02.16, Dorado v7.3.11 (Oxford Nanopore). Reads were demultiplexed and trimmed using DRAGEN v3.10.2. We assembled contigs using Flye v2.9.1-b1780 (12) with four iterations and polished with short reads using Polypolish v0.5.0 (13). This generated circularized genomes, including one primary chromosome and one extrachromosomal element in each. No separate rotations of the genomes were done. Percent completeness and contamination were assessed using CheckM2 v1.0.0 predict (14). Genomes were classified as Pseudoalteromonas undina using GTDB-tk v2.1.1 classify_wf (15) and annotated via the NCBI Prokaryotic Genome Annotation Pipeline (16). Default settings were used for all software unless otherwise stated. Genome statistics are summarized in Table 1.