The genomic sequence of Exiguobacterium chiriqhucha str. N139 reveals a species that thrives in cold waters and extreme environmental conditions

Introduction

The high altitude Andean Lakes (HAALs) from Puna, Argentina, are a group of lakes located at 3,000–6,000 m above sea level which are characterized by high ultraviolet (UV) radiation and salinity, broad temperature variations, low nutrient concentrations and high contents of metals and metalloids, mainly arsenic (Fernández-Zenoff et al., 2006; Fernández-Zenoff, Siñeriz & Farías, 2006; Dib et al., 2008; Flores et al., 2009; Ordoñez et al., 2009; Albarracín et al., 2011; Belfiore, Ordoñez & Farías, 2013). These environmental conditions are considered to be extreme and might resemble those of the Earth’s early atmosphere, as has been stated by NASA (Cabrol et al., 2007; Farías et al., 2009). Hence, these geographical areas have been proposed for studies on astrobiology (Farías et al., 2009). Despite being oligotrophic and hostile, a great microbial diversity has been found in the HAALs, where bacteria from the genus Exiguobacterium are one of the dominant taxa (Ordoñez et al., 2009; Ordoñez et al., 2013; Sacheti et al., 2013).

The Exiguobacterium genus, a sister clade to the Bacillus genus, is currently underexplored, and molecular studies of this genus from different sources are limited (Vishnivetskaya, Kathariou & Tiedje, 2009). Exploring Exiguobacterium strains is of great significance because understanding their strategies to adapt to diverse and extreme environmental conditions will likely place them as model organisms involved in the remediation of organic and inorganic pollutants. In particular, Exiguobacterium strains isolated from the HAALs have the potential of becoming an attractive model system to study environmental stress responses, as these microorganisms are able to grow efficiently in the laboratory (Ordoñez et al., 2009; Belfiore, Ordoñez & Farías, 2013). Moreover, Dib et al. (2008) suggested that these microorganisms could harbor various stress defense associated systems.

The Exiguobacterium chiriqhucha str. N139 was selected for genome sequencing due to its stress defense mechanisms such as its tolerance to high UV-B radiation, salinity and metalloids, particularly arsenic. This strain was isolated from the water column of Laguna Negra, which belongs to the ‘Salar de la Laguna Verde’, a system of five shallow oligotrophic lakes originated in the Tertiary (65 million to 1.8 million years ago) (Ericksen & Salas, 1987).

In the present study we characterized the genome of E. chiriqhucha str. N139, in order to identify the strategies that this organism employs to cope with the extreme environmental factors present in the aforementioned lake, mainly those related to metal and UV-B resistance. We also performed comparative genomics focusing on the two strains that would comprise the same species as Exiguobacterium chirqhucha str. N139; E. pavilionensis str. RW-2, isolated from the permanently cold Pavilion Lake in Canada (White III, Grassa & Suttle, 2013) and Exiguobacterium str. GIC31 isolated from a glacier in Greenland (Vishnivetskaya et al., 2014). Since the three strains were isolated from cold lakes, we propose the name ‘chiri qhucha’, which means ‘cold lake’ in Quechua, the Andean prehispanic language.

Classification and features

Members of the Exiguobacterium genus are Firmicutes, Gram-positive, facultative anaerobes with a low G + C content (Vishnivetskaya, Kathariou & Tiedje, 2009). Exiguobacterium is widely distributed all over the world (Karami et al., 2011) and has been isolated and typified from a wide variety of environments including hot springs (Vishnivetskaya, Kathariou & Tiedje, 2009; Vishnivetskaya et al., 2011), hydrothermal vents (Crapart et al., 2007), permafrost (Vishnivetskaya & Kathariou, 2005; Vishnivetskaya et al., 2006; Rodrigues et al., 2008), marine sediment (Kim et al., 2005), oligotrophic environments (Rebollar et al., 2012), biofilms (Carneiro et al., 2012), alkaline methanogenic microcosms (Rout, Rai & Humphreys, 2015) and more recently in water and microbial mats from high-altitude desert wetlands (Ordoñez et al., 2013). The Exiguobacterium genus is divided in two main phylogenetic clades (Vishnivetskaya, Kathariou & Tiedje, 2009); clade I is composed of temperate and cold-adapted strains, whereas clade II includes alkaliphilic species, with a marine origin and/or from high-temperature habitats (Fig. 1A).

E. chiriqhucha str. N139, which belongs to clade II, was isolated from the water column of Laguna Negra, in the HAALs (GPS: 27°38′49″S, 68° 32′43″W) and in laboratory conditions can uptake a wide variety of carbon sources (Table S1). Its cells are short rods and do not sporulate (Fig. 2, Table 1).

Table 1:

Classification and general features of Exiguobacterium chiriqhucha str. N139.

Property	Term	Evidence codea
Classification	Domain Bacteria	TAS (Woese, Kandler & Wheelis, 1990)
	Phylum Firmicutes	TAS (Gibbons & Murray, 1978)
	Class Bacilli	TAS (De Vos et al., 2009)
	Order Bacillales	TAS (De Vos et al., 2009)
	Family Bacillales Family XII. Incertae Sedis	TAS (De Vos et al., 2009)
	Genus Exiguobacterium	TAS (De Vos et al., 2009; Vishnivetskaya, Kathariou & Tiedje, 2009)
	Species Exiguobacteriumchiriqhucha	TAS (White III, Grassa & Suttle, 2013)
	Strain: N139 (Accession: JMEH00000000)
Gram stain	Positive	IDA
Cell shape	Short rods	IDA
Motility	Motile	IDA
Sporulation	Non-sporulating	EXP
Temperature range	Mesophilic (30–37 °C)	IDA
Optimum temperature	30 °C	IDA
pH range; Optimum	7–9	IDA
Carbon source	β—Methylglucoside, Galacturonic acid, L-asparagine, Tween 40, L-Serine, N-acetyl-glucosamine, Hydroxybutyric acid, Itaconic acid, Ketobutyric acid, Putrescine (See Table S1)	EXP
Habitat	Aquatic	TAS (Flores et al., 2009)
Salinity	0.11%–10% NaCl (w/v)	IDA
Oxygen requirement	Facultatively anaerobic	TAS (De Vos et al., 2009)
Biotic relationship	free-living	IDA
Pathogenicity	non-pathogen	NAS
Geographic location	Laguna Negra, Catamarca, Argentina	IDA
Sample collection	2006	IDA
Latitude	27°39′20.17′′S	IDA
Longitude	68°33′46.18′′W	IDA
Altitude	4100 masl	IDA

DOI: 10.7717/peerj.3162/table-1

Notes:

aEvidence codes—IDA, Inferred from Direct Assay; TAS, Traceable Author Statement (i.e., a direct report exists in the literature); NAS, Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence); EXP, Inferred from Experiment. These evidence codes are from the Gene Ontology project (Gene Ontology Evidence Codes).

Materials and Methods

Clustering of Strains into a Single Species

Phylogenetic analyses, ANI and AAI calculations, synteny analyses, as well as pangenome reconstruction were performed in order to a better understanding of the taxonomic position of the strain N139 isolated from Laguna Negra, relative to all the Exiguobacterium genomes available at the time of analysis.

ANI and AAI calculations (Goris et al., 2007) were done with default parameters for N139 versus all other complete genomic sequences of Exiguobacterium as well as pairwise comparisons for the three closest strains using the web server from Kostas lab http://enve-omics.ce.gatech.edu/ with default parameters. Comparisons to Exiguobacterium str. N139 can be seen in Table S2.

To explore the genomic rearrangements present on E. chiriqhucha str. N139 in comparison to other Exiguobacterium species, nucleotide syntenic blocks were obtained with Mauve version 2.3.1 (Darling et al., 2004). Syntenic block permutations were exported and used as input for MGR (Bourque & Pevzner, 2002). MGR was used to calculate the minimum number of rearrangements between the species analyzed, and to recover the rearrangement dendrogram. genoPlotR (Guy, Kultima & Andersson, 2010) was used to plot the syntenic blocks (Fig. 1B).

The pangenome of the nine Exiguobacterium genomes from clade II available at the time was reconstructed to aid in the taxonomic positioning of the strain N139. Orthologs were first calculated following the OrthoMCL pipeline (Li, Jr & Roos, 2003; Fischer et al., 2011), and the pangenome and the core genome were elucidated using ad hoc perl scripts. The selected strains were E. str. AT1b isolated from a hot spring in Yellowstone, USA (Vishnivetskaya et al., 2011); E. marinum, isolated from the Yellow Sea, South Korea (Vishnivetskaya et al., 2014); E. aurantiacum, from a potato processing plant, in the UK (Vishnivetskaya et al., 2014); E. str. 8-11-1 isolated from a salt lake in Inner Mongolia, China (Jiang et al., 2013); E. sp. S17 from the Laguna Socompa, another HAAL, Argentina (Ordoñez et al., 2013); E. mexicanum isolated from a brine shrimp Artemia franciscana (López-Cortés et al., 2006); E. pavilionensis (now chiriqhucha) str. RW-2 isolated from Pavilion Lake, Canada (White III, Grassa & Suttle, 2013) and E. (now chiriqhucha) str. GIC31, isolated from glacier ice in Greenland (Vishnivetskaya et al., 2014).

UV Resistance Assays: Determination of Survival Rate

The strains Exiguobacterium sp. S17, isolated from Laguna Socompa (HAAL), and Exiguobacterium aurantiacum str. DSM 6208, from the German Collection of Microorganisms and Cell Cultures (DSM), were used in these assays for comparison as external controls. These strains and E. chiriqhucha str. N139 were grown in 40 mL of LB medium under shaking (150 rpm) at 30 °C and cells were harvested by mid log phase (OD_600 nm 0.5) by centrifugation at 8,000 rpm for 10 min at 4 °C. The pellets were washed twice with 30 mL of 0.9% NaCl, and resuspended in the same volume of 40 mL. 20 mL of cell suspensions were transferred into sterile quartz tubes (16 cm long and 1.8 cm diameter) and placed horizontally to ensure maximal exposure and incubated at 15 °C under gentle shaking (150 rpm).

Tubes were irradiated from a distance of 30 cm with UV-B doses between 2,0 - 3,0 W/m2 during 240 min (09815-06 lamps, Cole Parmer Instrument Company; major emission line at 312 nm). Tubes were covered with an acetate sheet to block out UV-C. Irradiance was quantified with a radiometer (09811-56, Cole Parmer Instrument Company) at 312 nm with half bandwidth of 300 to 325 nm. Aliquots of 0.1 mL were taken at different exposure times (0, 60, 120, 180 and 240 min). Samples were then serially diluted in LB broth and spread in duplicate on Petri dishes with the same medium to determine the number of colony forming units (CFU). Controls of unexposed samples were run simultaneously in darkness and the percentage of cell survival after each treatment was calculated relative to these controls.

Results and Discussion

Genome rearrangements

Genome rearrangements within clades I and II are scarce, showing high conservation of the genomic structure within clades. However, several genomic rearrangements occurred as both clades diverged.

In order to determine which contigs of E. chiriqhucha str. N139 belong to plasmids, the plasmid sequences of pEspA and pEspB from E. arabatum RFL1109 (Jakubauskas et al., 2009) were retrieved from NCBI. This strain was selected for comparison because their plasmids have been widely studied (Jakubauskas et al., 2009) and because it is phylogenetically close to E. chiriqhucha str. N139. Jakubauskas and colleagues identified the regions hr-A1, hr-AB and hr-A2 in plasmid pEspA as capable of replicating the plasmid in Bacillus. For plasmid pEspB, they hypothesized that the regions hr-B1, hr-AB and hr-B2 are involved in a theta replication mechanism (Jakubauskas et al., 2009). BLAST searches of these regions were performed against all Exiguobacterium genome sequences available to date. For the strains E. MH3, E. antarcticum and E. sp. AT1b, which are described as genomes without plasmids, no significant hits were found. Conversely, hits to the E. arabatum sequences hr-B1, hr-AB and hr-B2 (a fragment of 39 kb) were found in the genomes of E. chiriqhucha str. GIC31 (56 kb) and E. chiriqhucha str. N139 (contig000014 of size 25 kb). It was concluded that the sequences present in the plasmids are shared within different Exiguobacterium strains, displaying a highly dynamic behavior. Therefore it was not possible to determine which of our contigs correspond to the three megaplasmids observed in the PFGE experiments (see Supplemental Information 1). Furthermore, contig 14 in our assembly corresponds to the smallest contig of E. chiriqhucha str. GIC31, so it could be a plasmid in both Exiguobacterium strains. Genes belonging to contig 14 are mostly hypothetical proteins, only 11 genes could be annotated. Of these, 9 correspond to genes involved in mobile elements (antirestriction proteins, integrases and transposases), conjugal transfer proteins and competence factors; one antibiotic resistance gene and a RNaseH. However, contig 14 lies adjacent to contig 13, both accounting for a total size of 100 kb when synteny was evaluated against E. chiriqhucha (pavillionensis) str. RW-2. It is worth mentioning that contig 13 possesses most of the genes responsible for metals resistance, but this region appears to be integrated in the chromosome of E. chiriqhucha str. GIC31. This highly dynamic behavior across strains, along with the presence of several genes involved in mobility, suggests that, if both contigs belong to a plasmid, it might be an integrative one. A MAUVE analysis performed between the three E. chiriqhucha strains, N139, GIC31 and RW-2, shows high synteny across their chromosomes. This idea that contigs 12, 13 and 14 might belong to the plasmids is supported by their shifts in GC skew (Fig. 3). To all appearances, the chromosomes within each of the two main clades of the Exiguobacterium species are very similar, but quite distinct when compared between these clades (Fig. 1).

Resistance to metals and metalloids

In Laguna Negra, ubiquitous Arsenic enters the E. chiriqhucha str. N139 cells through existing transporters due to its high structural similarity with other molecules (Rosen, 1999) and induces oxidative stress responses (Oremland & Stolz , 2003). Furthermore, arsenite (AsO₂H) and arsenate (AsO₄³⁻), are both toxic molecules. Arsenite binds to reduced cysteines in proteins inactivating them, and arsenate is a molecular analog of phosphate and therefore inhibits oxidative phosphorylation (Oremland & Stolz , 2003). Arsenate is far less toxic than arsenite, hence the oxidation of arsenite is considered a detoxification process. However, the oxidation of arsenite to arsenate, when coupled to the reduction of oxygen to water, is an exergonic process, and it has been suggested that at least some bacteria may derive energy out of this process (vanden Hoven & Santini, 2004). E. chiriqhucha str. N139 has an arsenite oxidase, AioB, enabling it to oxidize arsenite. This is an important metabolic capability, because it uses arsenite as an electron donor. Moreover, from a bioremediation point of view, this former metabolic feature is important since arsenite is more soluble than arsenate, so it can facilitate the removal of As in solution. E. chiriqhucha str. N139 also has an arsenite efflux pump, ArsB, as well as an ATPase that provides energy to ArsB for extrusion of arsenite and antimonite, ArsA, co-transcribed with ArsD, an arsenic chaperone for the ArsAB pump (Páez-Espino et al., 2009). Hence, this bacterium can probably detoxify and extrude As, as well as oxidize arsenite acquiring energy from this process. These ArsAB and ArsD proteins are also present in Salinivibrio strains isolated from the Laguna Socompa. However, these Salinivibrio strains also have ArsC, a cytoplasmic oxidoreductase that reduces arsenate to arsenite in a ATP-glutathione-glutaredoxin dependent way (Gorriti et al., 2014). E. chiriqhucha str. N139 lacks significant homologs to this gene as well as significant homologs to B. subtilis’ arsC gene.

E. chiriqhucha str. N139 also possesses redundancy for mercury detoxification, harboring four paralogous copies of merA. Briefly, MerA is the key detoxification enzyme of the mercury resistance system, reducing Hg²⁺ to Hg⁰ (Silver & Phung, 2005). Hg is toxic due to its high affinity to sulfur (Nies, 2003) and usually, mer resistance genes are co-transcribed in an operon whose dissemination is common by horizontal gene transfer (HGT) (Barkay, Miller & Summers, 2003). In this organism, two copies of merA are present in a monocystronic fashion; a third one is transcribed with a hypothetical protein. A fourth copy is co-transcribed with merR, the regulatory protein of the system. Two mer transporters which uptake Hg and merP, a transporter with a Sec-type signal, which could import Hg as a neutral chloride or hydroxide and deliver it to the other Mer transporters, which will finally transfer it to MerA.

The most common mechanism of resistance to metals consists of efflux pumps for inorganic ions. However, As and Hg resistance mechanisms are unique in the sense that these elements are reduced to lower their toxicity (Silver & Phung, 2005), instead of being exported. E. chiriqhucha str. N139 is resistant to cadmium, zinc, cobalt, and copper by pumping it out from the cell. It has two membrane embedded Cd²⁺ efflux pumps, one of which can also extrude zinc and cobalt; two paralogous copies of copA and copB, two P-type ATPase systems for exporting copper, and cueR, a sensing cytoplasmic Cu that protects periplasmic proteins from copper-induced toxicity (Orell et al., 2010). copB is transcribed monocystronically, and each of the copA genes form an operon co-transcribed with a copy of copZ, a copper chaperone, but one is co-transcribed with a glutaredoxin, whilst the other is co-transcribed with csoR, a copper-sensitive operon repressor.

Additionally, this microorganism lives in a low phosphorous environment, and relies on strategies for phosphorous uptake, like the presence of high-affinity Pi transporters and its regulation (pstS, pstCAB, phoB, phoR, dedA and ptrA) and genes for polyphosphate storage and breakdown (ppk and ppx). Organisms that scavenge phosphate can sometimes uptake the structurally similar arsenate ion, and hence also depend on arsenate detoxification mechanisms. It is also able to thrive in the alkaline environment of Laguna Negra since its genome code for all the typical antiporters present in alkaliphilic bacilli (nhaC, nhaP, the mpr operon, yhaU, norM and mleN). These antiporters present also contribute to a moderate salinity resistance this could also be related with the maintenance of metal resistance strategies in its genome, since it has been shown that lowering the salinity can lead to enhanced sensitivity to cadmium, cobalt and copper.

Resistance to UV radiation

Another extreme environmental condition in Laguna Negra is high ultraviolet radiation, particularly UV-B (Flores et al., 2009; Ordoñez et al., 2009). In order to determine if E. chiriqhucha str. N139 can cope with this constant stress, we measure the effect of colony survival of different Exiguobacterium strains exposed to UV-B radiation (Fig. 4). More than 25% of the colonies survive after 3 hrs of constant UV-B radiation. This contrasts with the other Exiguobacterium strains which rapidly start to decay, even though one of them, str. S17 was isolated from a neighbor lake, Laguna Socompa, in the HAALs (Ordoñez et al., 2013).

Bacteria have different UV damage repair pathways, including photoenzymatic repair (PER), nucleotide excision repair (NER) also called dark repair, and recombinational repair (PRR) or post-replication repair (Goosen & Moolenaar, 2008). E. chiriqhucha str. N139 has three genes (exiN139_00335 (phrB), exiN139_01768 and exiN139_00235) related to photolyases, which are involved in PER. They use UV as energy source (using FADH and transferring electrons) and catalyze the monomerization of cyclobutyl pyrimidine dimers. The gene exiN139_00335 only has homologues in Firmicutes including other known Exiguobacterium, and exiN139_ 01768 has homologues in Firmicutes, Cyanobacteria, α- and γ- Proteobacteria, and Euryarchaeotes. Additionally, exiN139_ 00235 is a cryptochrome, which are flavoproteins related to photolyases. Cryptochromes do not repair DNA and are presumed to act in other (unknown) processes, such as entraining circadian rhythms (Yuan et al., 2012). It is worth to remark that from these proteins only exiN139_01768, annotated as Deoxyribodipyrimidine photo-lyase-related protein, contains significant homologs in the genomes of the two Exiguobacterium compared in the UV-B radiation resistant assay (Fig. 4).

E. chiriqhucha str. N139 has also genes for NER. Its genome encodes the UvrABC endonuclease, a complex that recognizes DNA damage, binds to the damaged segment and cleaves it. Additionally, it codes for PcrA (also known as UvrD), a helicase in charge of removing the excised segment recognized and cleaved by UvrABC. These genes are regulated by the SOS response, which uses LexA as a repressor inactivated by RecA (Minko et al., 2001). E. aurantiacum and E. str. S17, contain homologs for the uvrB/uvrC gene, and the pcrA and recA regulatory genes.

Regarding the PRR, E. chiriqhucha str. N139 encodes for RecA, which recognizes SSB and cleaves UmuD, which becomes UmuD’ and binds UmuC to generate polymerase V, which in turn repairs damages, sometimes causing mutations. The gene exiN139_03003 may produce polymerase IV that is also involved in DNA damage repair (Sommer et al., 1998). UmuC is found in the genome, however UmuD is missing. It is possible that a protein highly similar to an existing copy of LexA may be taking its role, given that both can be cleaved by RecA and are present in Exiguobacterium. UmuD and UmuC are not present in the genomic sequences of E. aurantiacum and E. str. S17.

E. chiriqhucha str. N139 appears robust towards UV-B radiation, possessing several mechanism to cope with this constant stress. Moreover, the two strains proposed to be part of the same chiriqhucha species, E. pavilionensis str. RW-2 and E. str. GIC31, contain the same set of genes described above to cope with UV-B radiation, another unifying property of these strains. The strains RW-2 and N139 contain two homologs for Deoxyribodipyrimidine photo-lyases, while GIC31 contains three.

Living in syntrophy?

Finally, it is likely that E. chiriqhucha str. N139 participates in biofilm formation in Laguna Negra along with other bacteria (unpublished results). Analyses of other Exiguobacterium have shown that they participate in marine biofilms interacting with other Firmicutes and Proteobacteria (López et al., 2006; Carneiro et al., 2012). Evidence of possible biofilms associated genes originates from two loci present in the genome of E. chiriqhucha str. N139. The first locus encoding a protein capable of producing alginate, a linear co-polymer of two uronic acids that is produced in its acetylated form by some bacteria for adherence of these bacteria to target cell walls by the creation of a biofilm (Ramphall & Pier, 1985). The second locus codes for the arginine deiminase system, which can function at very low pH and is thought to be a critical factor in oral biofilm pH homeostasis (Burne & Marquis, 2000).

Conclusions

E. chiriqhucha str. N139 lives in a high-altitude, salted lake exposed to intense UV radiation, about 300 km away from the nearest ocean, the Pacific. Many factors in E. chiriqhucha str. N139 metabolism, such as the its needs to uptake certain intermediates like phenylalanine and BCAAs, and the possible excretion of the overproduced anthranilate, suggest that it is a key player in the amino acid metabolism of a microbial consortium that inhabit Laguna Negra. Moreover, the excess of anthranilate that it may produce could be directed to some novel pathway that remains to be uncovered, such as a new antibiotic, a new pigment or a new quorum sensing molecule.

The genome of E. chiriqhucha str. N139 contains all the necessary strategies to cope with all the environmental stresses that simultaneously co-occur in Laguna Negra. This Exiguobacterium is able to detoxify metals like arsenic, mercury, cadmium, zinc, cobalt, and copper; it has a complete defense system against UV damage; and it is also able to thrive in the alkaline environment of Laguna Negra. (Ventosa, Nieto & Oren, 1998). With all these characteristics, E. chiriqhucha str. N139 is an excellent candidate for future biotechnological research.

Although our study generates more questions than the ones it could solve, by sequencing its genome we have gained insights on the strategies the strain N139 employs for thriving in its habitat. From its Strain Specific set of genes, only 23 out of 59 could be annotated and classified to a COG, and still, most of the COG-classified genes belong to the poorly characterized category. This set of genes of unknown function require further experimental work to completely unveil how the strain N139 is adapting to the extreme environment of Laguna Negra.

Description of Exiguobacterium chiriqhucha sp. nov.

Exiguobacterium chiriqhucha (chi.ri.qhu.cha. (/ʃi ri ku tʃa/)) Quechua. Adj. chiri: cold, freezing; Quechua. Noun. qhucha: lake, pond. chiriqhucha of or belonging to a cold lake, referring to the common habitat of these three species). Members of the species Exiguobacterium chiriqhucha; inhabitants of freshwater ponds, saline ponds; distinguishable by their 16S rRNA sequences; accession numbers are: JMEH00000000 for the str. N139 genome, ATCL00000000 for the RW-2 genome (White III, Grassa & Suttle, 2013) and JNIP00000000 for the GIC31 genome (Vishnivetskaya et al., 2014). The three strains that so far comprise this species form orange shiny colonies and are Gram-positive, rod-shaped, facultative anaerobes and motile via peritrichous flagella (Miteva, Sheridan & Brenchley, 2004; Vishnivetskaya, Kathariou & Tiedje, 2009; White III, Grassa & Suttle, 2013). Two of them, str. RW-2 and str. GIC31 were isolated from permanently cold environments (Pavilion Lake and Glacier Ice, Greenland; (White III, Grassa & Suttle, 2013; Vishnivetskaya et al., 2014)) whilst the str. N139 was isolated from Laguna Negra, a HAAL which temperature can drop to −30 °C. Their temperature range of growth is from a minimum (str. GIC31) of 2 °C to a maximum (str. RW-2) of 50 °C; the pH range for growth is from 5 to 11 in str. RW-2 and from 7 to 9 in str. N139 (Miteva, Sheridan & Brenchley, 2004; White III, Grassa & Suttle, 2013). The three strains possess cold-shock proteins and have a G + C content of 52% (White III, Grassa & Suttle, 2013; Vishnivetskaya et al., 2014). The type strain is RW-2.

Supplemental Information