Draft Genome Sequences of 16 Halophilic Prokaryotes Isolated from Diverse Environments

ANNOUNCEMENT

Halophiles have the ability to survive and thrive in high salinity. Halophile-specific enzymes can be harnessed for use in a variety of commercial and industrial applications, making them a valuable target for study (1, 2). As part of a two-quarter course series, undergraduate students isolated and cultured microbes obtained from several high-salinity substrates (Table 1). All samples from Puerto Rico were from salt flats in Cabo Rojo. Halobacterium medium 372 was used as the culturing medium (https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium372.pdf). To isolate a wider variety of strains, samples were plated in medium 372 at multiple salinity levels, either 100, 150, 200, or 250 g NaCl per 1,000 ml of medium. To grow a diversity of strains, we used both direct-plated samples and samples plated from liquid enrichment cultures. For solid samples, direct plating required hydrating the sample with H₂O at a density of 200 g of sample per liter. To create enrichment cultures, we combined 100 μl of sample with 3 ml of liquid medium and incubated it at 37°C until turbid in ≤7 days. Colonies were grown by spread plating of the prepared samples. Pure liquid cultures were created from isolated plate colonies and grown at 37°C until turbid in ≤7 days. Purity was assessed using microscopy and visual analysis of streak-plated pure cultures.

Genomic DNA was isolated from the liquid cultures using a Wizard genomic DNA purification kit (Promega, Madison, WI, USA). MicrobesNG (Birmingham, UK) performed library preparation, sequencing, adapter trimming, and assembly (full protocol available at https://microbesng.com/documents/5/MicrobesNG_Methods_Document_-_PDF.pdf). DNA was quantified using the Quant-iT double-stranded DNA (dsDNA) high-sensitivity (HS) assay (Thermo Fisher Scientific, Waltham, MA, USA). Libraries were prepared using a Nextera XT library prep kit (Illumina, San Diego, CA, USA) with the following two protocol modifications: using 2 ng of DNA instead of 1 ng and adjusting the PCR elongation time to 1 minute. Libraries were sequenced using an Illumina HiSeq instrument with a 250-bp paired-end protocol. A mean of 1,122,706 reads per strain were generated by the sequencing. Reads were trimmed using Trimmomatic 0.30 with a sliding window quality cutoff of Q15 (3). SPAdes 3.7 (4) was used for de novo assembly with recommended settings for 250-bp paired-end reads. The mean coverage ranged from 35 to 289×. The mean N₅₀ value was 442,887 bp with a mean L₅₀ of 14.1.

The taxonomy was estimated with BLAST (5) using the 16S rRNA gene sequences of each strain. We identified 14 bacteria (genera Halobacillus, Pontibacillus, Virgibacillus, Bacillus, and Halomonas) and 2 archaea (genus Halorubrum). The top query results all had above a 97% identity (99.4% mean identity).