Draft Genome Sequence of the Marine Flavobacteriaceae sp. Strain LSUCC0859

ABSTRACT

A new marine Flavobacteriaceae sp. strain, LSUCC0859, was isolated off the coast of Louisiana with artificial seawater via high-throughput dilution-to-extinction (DTE) cultivation. The 2,168,862-bp genome sequence provides opportunities to investigate the biology of a poorly understood lineage within the Bacteroidetes.

ANNOUNCEMENT

LSUCC0859 was isolated from Bay de Pomme d’Or (Louisiana, Gulf of Mexico; 29.35°N, −89.54°W) in January 2017 using dilution-to-extinction (DTE) cultivation with artificial seawater (1, 2). Source water salinity and temperature were 11.7 and 12.38°C, respectively (2). The 16S rRNA gene was previously Sanger sequenced (GenBank accession number MK603739.1) (2), and a BLASTn search of the NCBI nt database found that the closest related cultured organisms were Bizionia psychrotolerans strain sea water13 (99.17% identity; MT112327.1) and Flavobacteria bacterium Yb008 (99.17% identity; AB496663.1). However, there were no close hits to genome-sequenced organisms. We therefore sequenced the genome of LSUCC0859 because of its presence within an understudied group of Flavobacteriaceae.

In preparation for genome sequencing, one LSUCC0859 cryostock was inoculated into 50 mL MWH3 medium (2) and grown at 25°C for 4 days until the cell density reached ~10⁶ cells mL⁻¹. This seed culture was subsequently transferred into three 300-mL and two 600-mL replicates in polycarbonate flasks. After 2 days of growth, cultures reached cell densities of ~1.5 × 10⁶ cells mL⁻¹, after which they were each vacuum filtered through a 0.2-μm polyethersulfone filter (Millipore Sigma, USA). A combination of proteinase K digestion, phenol-chloroform-isoamyl alcohol extraction, and ethanol precipitation (https://dx.doi.org/10.17504/protocols.io.b5iiq4ce) was performed to obtain genomic DNA that was quantified using a Qubit fluorometer. Library preparation and sequencing were performed at the Microbial Genome Sequencing Center (https://www.migscenter.com/). Illumina sequencing libraries were constructed with an Illumina DNA prep kit and integrated DNA technologies 10-bp unique dual indexes. Libraries were sequenced on an Illumina NextSeq 2000, producing 2,879,268 2 × 151-bp read pairs, and demultiplexed, adaptor trimmed, and quality controlled with bcl2fastq (v2.20.0422) (3).

We trimmed paired-end sequence data with Trimmomatic v0.38 (4) using LEADING:20 TRAILING:20 SLIDINGWINDOW:13:20 MINLEN:40 and assembled the reads with SPAdes v3.13.0 (5). Scaffolds of <500 bp were manually removed. We mapped raw reads to the genome using the Burrows-Wheeler Aligner (BWA) v0.7.17 (r1188) (6) and samtools v18.0.4 (7) for polishing with Pilon v1.22 (8). The final assembly resulted from the Pilon output. We also used CheckM v1.1.3 (9) for additional quality control, and the genome was annotated using the Prokaryotic Genome Annotation Pipeline (PGAP) (10). Whole-genome taxonomy was performed with GTDB-tk v1.5.0 and the release 202 database using classify_wf (11). Default settings were used for all software unless otherwise noted.

The draft genome was 2,168,862 bp long in 12 scaffolds (N₅₀, 468,311 bp), with a GC content of 40.16% and mean coverage of 201×. Of the 1,919 predicted genes, 1,870 were protein coding, and 1 each of the 5S, 16S, and 23S rRNA genes. CheckM estimated that the genome coding density was 93.39%, with 96.26% completion and 0% contamination. GTDB-tk placed the LSUCC0859 genome within the Flavobacteriaceae, with the nearest neighbor being the metagenome-assembled genome (MAG) “MAG-120531 sp000173115” (average nucleotide identity, 78.71%). The organism had a mean growth rate of 2.4 doublings day⁻¹ (calculated as described in reference 12) at room temperature in MWH3 medium (Fig. 1).

FIG 1.

Growth curves (left) and calculated growth rates (right) for LSUCC0859 growing at room temperature on MWH3 medium. Output is from sparse-growth-curve (12).

Data availability.

The accession numbers PRJNA804496, JAKOGP000000000, and SRX14114391 are the NCBI BioProject, GenBank, and SRA accession numbers, respectively. The genome version described in this paper is version JAKOGP010000000. Live cultures or cryostocks of the organism will be provided upon request.