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.
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).
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 |
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.
ACKNOWLEDGMENTS
We also acknowledge the Center for Advanced Research Computing (CARC) at the University of Southern California (https://carc.usc.edu) and the Georgia Advanced Computing Resource Center (GACRC) at UGA (https://gacrc.uga.edu/) for computational resources that have contributed to the results in this publication.
Funding for this study was provided by the Gordon and Betty Moore Foundation (Symbiosis in Aquatic Systems Initiative, grant #9326 to H.M.B. and J.C.T.). Research support for A.D.S. was provided by the University of Georgia Research Foundation and the National Institute of General Medical Sciences of the National Institute of Health under award number 1T32GM142623.
Contributor Information
Holly M. Bik, Email: hbik@uga.edu.
J. Cameron Thrash, Email: thrash@usc.edu.
Julie C. Dunning Hotopp, University of Maryland School of Medicine, Baltimore, Maryland, USA
DATA AVAILABILITY
Sequencing reads and genomes (accession numbers in Table 1) are available at NCBI under BioProject number PRJNA1219708.
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Associated Data
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Data Availability Statement
Sequencing reads and genomes (accession numbers in Table 1) are available at NCBI under BioProject number PRJNA1219708.