Draft Genome of Spiribacter salinus M19-40, an Abundant Gammaproteobacterium in Aquatic Hypersaline Environments.

We have previously used a de novo metagenomic assembly approach to describe the presence of an abundant gammaproteobacterium comprising nearly 15% of the microbial community in an intermediate salinity solar saltern pond. We have obtained this microbe in pure culture and describe the genome sequencing of the halophilic photoheterotrophic microbe, Spiribacter salinus M19-40.

GENOME ANNOUNCEMENT

Hypersaline systems, e.g., solar salterns, are extreme environments of salinity higher than that of seawater. Microbes adapt to high salinities using two strategies. In the salt-out strategy, cells accumulate compatible solutes (e.g., ectoine and betaine) in the cytoplasm to exclude inorganic salts (mostly Na+). However, in the salt-in strategy, they actively maintain a high internal ionic concentration of potassium ions and have a significantly higher proportion of positively charged amino acids (arginine and lysine) in their proteins to maintain solubility. A range of metagenomic data sets from different salinities has been analyzed previously, and novel microbes were identified using a de novo metagenomic assembly approach (1). In that previous work, in a metagenomic data set from a solar saltern pond of 19% salinity, metagenomic assembly yielded 15 contigs (a total of 94 kb; G+C content, 63% to 67%) that could clearly be attributed to a gammaproteobacterium related to the genera Alkalilimnicola and Nitrococcus. However, due to the lack of a complete genome, not many observations could be made, except that the isoelectric points of the predicted proteins suggested that the microbe uses a salt-out strategy.

In an effort to culture these novel microbes, we undertook further sampling and culturing from the same saltern (Santa Pola, Spain) and from another with similar salinity (Isla Cristina, Spain), using 16S rRNA PCR for colony identification. A slow growing microbe, with a 16S rRNA sequence related to Alkalilimnicola, was obtained from the Isla Cristina saltern. In liquid culture, the cells appeared as spiral rods and clustered into aggregates, likely due to polysaccharide production. On solid medium, the cells appeared as slender small rods. We have named this microbe Spiribacter salinus M19-40, and a formal taxonomic description is in progress.

Whole-genome shotgun (WGS) sequencing of S. salinus M19-40 was performed using Illumina HiSeq 2× 100-bp paired-end (PE) reads and Pacific Biosciences 3- to 5-kb reads. Illumina reads at 100× coverage and long PacBio error-corrected reads (2) at a coverage of ~50× were assembled using MIRA (3) into 6 contigs (N50, 1,369,469). The total genome size was 1.74 Mb (1,742,247 bp), with a G+C content of 62.65%. We identified 1,706 protein-coding genes (4) and a single rRNA operon (5). Forty-five tRNAs for all canonical 20 amino acids were identified (6). Protein-coding genes were compared to NCBI nonredundant (NR) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases for functional annotation.

Analysis of the global predicted proteome pI, and the presence of ectoine biosynthesis genes, confirm that S. salinus appears to have a salt-out strategy. No flagellar genes could be found, indicating the absence of swimming motility. The presence of a rhodopsin gene indicated that S. salinus is probably a photoheterotroph. The most similar 16S rRNA gene sequences in the Ribosomal Database Project (7) appeared to be uncultured Ectothiorhodospiraceae bacteria and Alkalilimnicola species. The identity similarity of the single 16S rRNA gene to that of the Alkalilimnicola 16S rRNA gene was 95%. However, the average nucleotide identity between the genomes was only 67.92%, which is indicative of the microbe belonging to a new genus.

Nucleotide sequence accession numbers.

This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. ANKZ00000000. The version described in this article is the first version, ANKZ01000000.