Subtercola vilae sp. nov., a novel actinobacterium from an extremely high-altitude cold volcano lake in Chile

A novel actinobacterium, strain DB165T, was isolated from cold waters of Llullaillaco Volcano Lake (6170 m asl) in Chile. Phylogenetic analysis based on 16S rRNA gene sequences identified strain DB165T as belonging to the genus Subtercola in the family Microbacteriaceae, sharing 97.4% of sequence similarity with Subtercola frigoramans DSM 13057T, 96.7% with Subtercola lobariae DSM 103962T, and 96.1% with Subtercola boreus DSM 13056T. The cells were observed to be Gram-positive, form rods with irregular morphology, and to grow best at 10–15 °C, pH 7 and in the absence of NaCl. The cross-linkage between the amino acids in its peptidoglycan is type B2γ; 2,4-diaminobutyric acid is the diagnostic diamino acid; the major respiratory quinones are MK-9 and MK-10; and the polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol, 5 glycolipids, 2 phospholipids and 5 additional polar lipids. The fatty acid profile of DB165T (5% >) contains iso-C14:0, iso-C16:0, anteiso-C15:0, anteiso-C17:0, and the dimethylacetal iso-C16:0 DMA. The genomic DNA G+C content of strain DB165T was determined to be 65 mol%. Based on the phylogenetic, phenotypic, and chemotaxonomic analyses presented in this study, strain DB165T (= DSM 105013T = JCM 32044T) represents a new species in the genus Subtercola, for which the name Subtercola vilae sp. nov. is proposed.


Introduction
Members of the family Microbacteriaceae are widely distributed in terrestrial and aquatic environments or associated with macroorganisms (Evtushenko 2012). Some representatives, including species of the genus Subtercola, have been found in cold environments such as glacial ice (Christner et al. 2007), boreal groundwater (Männistö et al. 2000), and Antarctic sediments (Li et al. 2010). At present the genus Subtercola contains three validly named species, Subtercola boreus, Subtercola frigoramans and Subtercola lobariae; the first two were isolated from Finnish groundwater (Männistö et al. 2000) and the third from the lichen Lobaria retigera (Si et al. 2017). Based on the high similarity of 16S rRNA gene sequences ([ 96%), other isolates from cold habitats, such as Antarctic and Artic waters as well as glaciers were found to be affiliated to Subtercola (Singh et al. 2014;Zhang et al. 2013;Peeters et al. 2011).
In this study, we characterise strain DB165 T , isolated from a water sample of Llullaillaco Volcano Lake (6170 m) in Chile, one of the highest-elevation lakes on Earth. According to its distinct properties, strain DB165 T is proposed as the type strain of the new species Subtercola vilae.

Materials and methods
Isolation and cell morphology Strain DB165 T was obtained from a water sample collected at the Llullaillaco volcano lake (S24°42.878 0 , W68°33.310 0 ) on 18 January 2013, using R2A medium (DIFCO) supplemented with 18 g agar l -1 . Pure cultures were obtained after three successive transfers of single colonies to R2A medium plates. Stock cultures were maintained in SGG medium containing 10 g starch, 10 g glucose, 10 ml glycerol (99.7% v/v), 5 g soy peptone, 2.5 g corn steep solids, 2 g yeast extract, 3 g CaCO 3 , 1 g NaCl, and 18 g agar in 1 l deionised water (Goodfellow and Fiedler 2010). DB165 T was cryopreserved using CRYOBANK (Mast Diagnostica GmbH, Germany) for long term storage at -80°C.
Gram-staining was prepared using the Color Gram 2 kit (BioMérieux, France), following the manufacturer's protocol. Endospore staining was performed using the green malaquite method and light microscopy (Schaeffer and Fulton 1933). Cell morphology, shape and size were determined using scanning electron microscopy (SEM) according to Gärtner et al. (2008), after cultivation of trypticase soy medium (Trypticase Soy Broth (Becton, Dickinson and company, France) supplemented with 18 g agar l -1 .
The reference strains S. boreus DSM 13057 T and S. frigoramans DSM 13057 T were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) and cultured under the same conditions as strain DB165 T for comparative purposes.

Chemotaxonomic analyses
Polar lipids were extracted according to a modified protocol of Bligh and Dyer (1959), and the total lipid material was detected using molybdatophosphoric acid and specific functional groups were detected using spray reagents specific for defined functional groups (Tindall et al. 2007). The lipoquinones were extracted and identified using the two-stage method described by Tindall (1990a, b). After cultivation at 25°C, fatty acid methyl esters were obtained by saponification, methylation and extraction using minor modifications of the method of Miller (1982) and Kuykendall et al. (1988) The fatty acid methyl esters mixtures were separated and identified using the Sherlock Microbial Identification System (MIS) (MIDI, Microbial ID, Newark, DE 19711, USA).
The peptidoglycan was obtained from 4 g wet weight cell pellet according to the method of Schleifer (1985). The peptidoglycan analyses were performed according to Schumann (2011).
Analyses of polar lipids, respiratory quinones, whole-cell fatty acids and peptidoglycan analyses were carried out by the Identification Service of the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany).

DNA base composition
DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN). The G?C content was calculated from the genome sequence, which was determined with Nextseq 500 (Illumina). The quality of the sequences was checked and filtrated using Trimmomatic (adapters, [ Q30, [ 1000 bp) (Bolger et al. 2014). The genome was assembled using SPAdes (Kmer = 121) (Bankevich et al. 2012).

Phylogenetic analyses
DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN) with modifications. The 16S rRNA gene sequence was amplified by PCR using PureTaq Ready-To-Go PCR beads (GE Healthcare) and sequencing according to Gärtner et al. (2008).
The 16S rRNA gene sequence of strain DB165 T was aligned with sequences of 22 selected type strains of the family Microbacteriaceae, including members of the genera Subtercola, Frondihabitans and Agreia, and in addition Cellulomonas carbonis KCTC 19824 T as outgroup using SINA (Pruesse et al. 2012). Phylogenetic trees were constructed using the neighbour-joining (Saitou and Nei 1987) and maximumlikelihood algorithms using MEGA version 6.0 (Tamura et al. 2013). The tree topologies were evaluated with bootstrap analyses based on 1000 replicates.

Morphological and physiological characteristics
Colonies of strain DB165 T were observed to be sticky, golden yellow after growth at the optimal growth temperature of 10-15°C for 6-7 days (also after 7-10 days at 28°C), but are pale yellow after growth at 5°C for 2-3 weeks. Optimum growth is observed at 10-15°C (range from 5 to 28°C). No growth occurs at 30°C. Strain DB165 T tolerates only low concentrations (up to 0.9%) of NaCl and grows best in the absence of NaCl. The pH range for growth is from 5 to 8, with an optimum at pH 7. Cells show no motility and form no spores, they are irregular short rods of 0.5 lm width and 1.0-1.2 lm length, Gram-positive and have an irregular shape as seen under SEM. Some of the cells are thicker at the ends. Occasionally, coccoid cells were observed ( Fig. 1). Variable cell shapes have also been reported for S. boreus and S. frigoramans (Männistö et al. 2000).
The metabolic properties of strain DB165 T , in comparison with the type strains of S. boreus and S. frigoramans are shown in Table 1

Chemotaxonomic characteristics
The polar lipids of the strain DB165 T were found to consist of phosphatidylglycerol, diphosphatidylglycerol, 5 unidentified glycolipids, 2 unidentified phospholipids and unidentified 5 lipids. The diamino acid in the peptidoglycan was identified as 2,4diaminobutyric acid (DAB). The molar ratio of alanine:glycine:glutamic acid:DAB was 1.1:1.0:0.04:1.7. Instead of glutamic acid, high amounts of 3-hydroxyl-glutamic acid were found. Assuming that much of the glutamic acid is replaced by 3-hydroxyl-glutamic acid, and despite this Cysteine arylamidase (?) -?

16S rRNA gene sequence analyses
The 16S rRNA gene sequence of strain DB165 T (1432 bp, deposited under Genbank accession number MF276890) matches to different Subtercola species in a range of 96.1-97.4% of similarity, i.e., S. frigoramans DSM 13057 T (97.4% similarity), S. lobariae DSM 103962 T (96.7% similarity) and S. boreus DSM 13056 T (96.1% similarity). However, it also showed high similarity to Frondihabitans species, including Frondihabitans peucedani DSM 22180 T (96.8% similarity) and Frondihabitans australicus DSM 17894 T (96.6% similarity). The phylogenetic analysis (Fig. 2) based on the consensus 16S rRNA gene sequences (1416 bp) showed that strain DB165 T forms a cluster with Subtercola species and Agreia with a strong bootstrap support, while Frondihabitans species cluster together, but the separation with the Subtercola/ Agreia cluster is not well supported by bootstrap analysis. In the Subtercola clade, strain DB165 T is found in a distinct cluster with S. frigoramans DSM 13057 T with strong bootstrap support.

Genome characteristics
The genome of strain DB165 T has a size of approx. 4 Mb and give some hints on the strains' potential to adapt to the harsh conditions that are found at Llullaillaco volcano lake. These include mechanisms concerning membrane fluidity, biosynthesis of cryoprotectants, and ice-interacting proteins (data not shown).

Discussion
Subtercola species are characterised by a peptidoglycan type B2c with DAB as diamino acid, MK-9 and MK-10 as major respiratory quinones, and a similar polar lipids profile (Table 3).
Strain DB165 T has a peptidoglycan type B2c in which the glutamic acid is almost completely replaced by 3-hydroxyl-glutamic acid, as is found in other  Si et al. (2017) described Subtercola species (Männistö et al. 2000;Si et al. 2017). The metabolic characteristics that differentiate strain DB165 T from S. boreus and S. frigoramans are utilisation of methyl-b-D-xylopyranoside, arbutin salicin, D-melezitose and gentobiose as carbon sources and the enzymatic activities of trypsin, agalactosidase, tryptophanase deaminase and tryptophanase, as well as the production of acetoin. In contrast to strain DB165 T , S. frigoramans exhibited as unique features arginine dihydrolase and n-acetyl-bglucosaminidase. S. frigoramans and S. boreus can use citrate as unique carbon source and showed the enzymatic activities of alkaline phosphatase and amannosidase. S. boreus showed b-glucoronidase enzymatic activity and can use D-arabinose, D-ribose, and D-lactose, carbon sources that strain DB165 T and S. frigoramans cannot use. The fatty acid profile of strain DB165 T is similar to those of the type strains of other Subtercola species. Major differences were observed in the content of iso-C16:0, with 17% in strain DB165 T compared to 4.2-10.2% in the other Subtercola species and a low content of 3% anteiso-C15:0 DMA, compared to 9.7-11.0% in the other Subtercola species (Table 2). The phylogenetic analysis of the 16S rDNA gene sequence clearly shows the close relationship of strain DB165 T to Subtercola species rather than to Agreia and Frondihabitans species. The genus Agreia forms a sub-cluster within S. boreus (this study and Si et al. 2017) and different chemotaxonomic traits have been proposed to distinguish the two genera (Schumann et al. 2003). Though both genera have a cross-linkage replications, asterisks indicate branches of the tree that were also recovered using neighbour joining algorithm. Only bootstrap values C 50% are indicated. Genbank accession numbers are given in parentheses. Bar indicates 0.01 substitutions per site between the amino acids in the peptidoglycan of type B2c, in the case of Subtercola species, the crosslinkages have DAB, while Agreia species have L-DAB connected to D-Orn. Frondihabitans species can be clearly distinguished by their peptidoglycan, which is of the B2b type (Zhang et al. 2007). Fatty acids play an important role in the differentiation of the genera. The presence of 1,2 dimethyl acetals (iso-C16:0 DMA and anteiso-C17:0 DMA) is observed in all Subtercola species, while Agreia only contains iso-C15:0 DMA in low proportions (B 4%) (Schumann et al. 2003;Behrendt et al. 2002). Frondihabitans species have a fatty acid profile very distinct from those of Subtercola and Agreia species, having C18:1 and C14:0 2-OH as major fatty acids but lacking 1,2 dimethyl acetals. The major menaquinones of Subtercola species, including the strain DB165 T , comprise MK-9 and MK-10, while in Agreia bicolorata DSM 14575 T MK-10 and in Agreia pratensis DSM 4246 T MK-10 and MK-11 are dominant, and in Frondihabitans spp. MK-8 and MK-7. The presence of MK-9 as a major component can be used as a marker to differentiate these three genera (Table 2). It should be mentioned that A. pratensis, which was originally classified as Subtercola pratensis, contains MK-10 (51%) and MK-11 (21%) as major menaquinones, but in addition 13% of MK-9 (Behrendt et al. 2002, Evtushenko et al. 2001. Irrespective of the problematic taxonomic position of Agreia species and the similarity of 16S rRNA gene sequences with Frondihabitans, the phylogenetic relationships (Fig. 2) and chemotaxonomic criteria clearly support the classification of strain DB165 T as a member of the genus Subtercola (Table 3). Based on the phenotypic and genetic analyses presented in this work, strain DB165 T is considered to represent a new species of the genus Subtercola, for which the name Subtercola vilae sp. nov. is proposed. The Digital Protologue database (Rosselló-Móra et al. 2017) TaxoNumber for strain DB165 T is TA00217.

Description of Subtercola vilae sp. nov.
Subtercola vilae (vi'lae, of Vila, named in honour of Irma Vila, a Chilean limnologist with outstanding contributions to the microbiology and ecology of lakes in the Chilean Altiplano and Atacama Desert).