Genome Sequence and Methylation Patterns of Halorubrum sp. Strain BOL3-1, the First Haloarchaeon Isolated and Cultured from Salar de Uyuni, Bolivia.

Halorubrum sp. strain BOL3-1 was isolated from Salar de Uyuni, Bolivia, and sequenced using single-molecule real-time sequencing. Its 3.7-Mbp genome was analyzed for gene content and methylation patterns and incorporated into the Haloarchaeal Genomes Database (http://halo.umbc.edu). The polyextremophilic character and high-elevation environment make the microbe of interest for astrobiology.

ANNOUNCEMENT

Halorubrum sp. strain BOL3-1 is the first haloarchaeon from Bolivia to be cultured and sequenced. It was isolated from salt samples from Salar de Uyuni, Department of Potosí, Bolivia, the largest salt flat in the world and an environment remarkable for its high elevation and high albedo and UV radiation exposure (1). The environment is unique and of significant interest to the astrobiology community due to its multiple extremes (2).

Stratified salt crust was sampled from the Salar in March 2015 at a remote site, (20°33′28.58ʺS and 67°12′29.56ʺW [–20.5579389°, –067.2082111°]), 3,647 m above sea level. Typical conditions are pH 7.3 to 7.6, ≥28% NaCl (wt/vol) concentration, and temperatures of –15 to 22°C. Salt samples were dissolved in CM+ medium (3), and growth was stimulated under illumination at 37°C with shaking at 220 rpm (Innova 4230 refrigerated incubator shaker; New Brunswick, NJ, USA). The enrichment culture was plated on CM+ agar plates and purified by 3 rounds of streaking. The isolated strain, BOL3-1, formed biofilms in liquid culture, and colonies were bright red and translucent.

Nucleic acids were extracted using standard methods (3), and sequencing was performed using the PacBio RS II platform. A SMRTbell sequencing library was prepared from 3 μg genomic DNA randomly sheared to 20 kb with a Megaruptor instrument (Diagenode, Denville, NJ). The library was sequenced using a single-molecule real-time (SMRT) cell with C4-P6 chemistry and a 360-min collection time. Sequencing reads were filtered (quality, ≥0.80; length, ≥100 bp) and assembled de novo (98,158 reads with a mean subread length of 3,908 bp) using RS_HGAP_Assembly.3 (4) in the SMRT Analysis 2.3.0 environment (minimum seed read length, 5,000 bp; minimum coverage for correction, 8×). Error correction and closure were performed using RS_BridgeMapper.1, and methylation patterns were determined using RS_Modification_and_Motif_Analysis.1 within SMRT Analysis using default settings (minimum modification quality value [QV], 30).

Genome annotation was performed in-house using EMBOSS version 6.6.0.0 (5), GeneMark.hmm version 2 (6), and tRNAscan version 1.3 (7), as well as the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) build 3190 (8). The annotated genome sequence was analyzed on the Haloarchaeal Genomes Database (HaloWeb version r1555192846) (9). The 3,668,425-bp genome is GC rich (65.9%) and consists of a large circular chromosome (66.8% GC content) and 3 plasmids, p163 (55.1% GC content), p117 (54.8% GC content), and p13 (67.6% GC content) based on computer assembly. Three complete rRNA operons (two on the chromosome and one on p117) and 57 tRNA genes are present. The closest relatives based on 16S rRNA similarity (≤97% identity) are Halorubrum ezzemoulense and Halorubrum chaoviator (10, 11).

Genome annotation predicted 3,266 encoded proteins with a calculated mean isoelectric point (pI) value of 4.58, a highly acidic proteome characteristic of haloarchaea (12). The genome contains the great majority of conserved haloarchaeal groups (HOGs), including 775 core (cHOGs) and 77 signature (ucHOGs) groups (12, 13). Expanded gene families common in haloarchaea include 9 origin recognition complex (Orc/Cdc6) proteins, 4 TATA-binding and 7 TFB proteins, and 5 photolyase/cryptochrome family proteins (14). Genes encoding retinal proteins, including bacteriorhodopsin, halorhodopsin, and sensory rhodopsin 2, were found. Bacteriorhodopsin can be observed spectroscopically and constitutes a remotely detectable biosignature (15). A catabolic gene cluster is present, with a GH-42 β-galactosidase likely responsible for o-nitrophenyl-β-d-galactopyranoside (ONPG)-hydrolytic activity (16–18).

Over 100 transposase genes are present, suggesting a large number of insertion sequences in the genome. There are 2 clustered regularly interspaced short palindromic repeat (CRISPR) arrays (a type I-B CRISPR-associated protein, Cas5, on p163 and a type I-B CRISPR-associated protein, Cas7/Csh2, on p117). The methylated DNA motifs and the methyltransferases (MTases) predicted to be responsible for some of these proteins are shown in Table 1.

TABLE 1

Methylated motifs in Halorubrum species strain BOL3-1 detectable by SMRT sequencing

Motifa	Type	% sites methylated	Putative MTase responsible
GGAG(N5)TTC	m6A	100	EKH57_07125 (M.HspBol31I)
GATC	m6A	100	EKH57_00995 (M.HspBol31II)
GTCAGT	m6A	100	Undetermined
CAGAGC	m6A	100	Undetermined
GCGGAGG	m6A	99	Undetermined
CATTC	m6A	98	Undetermined

Locations of methylated bases on either the top or bottom strands are underlined.

Data availability.

The Halorubrum sp. strain BOL3-1 genome sequence has been deposited in GenBank with the accession numbers CP034692, CP034691, CP034690, and CP034693 and is also available on HaloWeb (https://halo.umbc.edu/cgi-bin/haloweb/haloweb.pl). The raw data are available in the NCBI Sequence Read Archive with the accession number SRP175004.