Characterization of Salinivibrio socompensis sp. nov., A New Halophilic Bacterium Isolated from the High-Altitude Hypersaline Lake Socompa, Argentina

The genus Salinivibrio belongs to the family Vibrionaceae and includes Gram-stain-negative, motile by a polar flagellum, and facultatively anaerobic curved rods. They are halophilic bacteria commonly found in hypersaline aquatic habitats and salted foods. This genus includes five species and two subspecies. A presumed novel species, strain S35T, was previously isolated from the high-altitude volcanic, alkaline, and saline lake Socompa (Argentinean Andes). In this study we carried out a complete taxonomic characterization of strain S35T, including the 16S rRNA gene sequence and core-genome analysis, the average nucleotide identity (ANIb, ANIm, and orthoANI), and in silico DNA–DNA hybridization (GGDC), as well as the phenotypic and chemotaxonomic characterization. It grew at 3%–20% (w/v) NaCl, pH 6–10, and 10–42 °C, with optimum growth at 7.0%–7.5% (w/v) NaCl, pH 8.0, and 37 °C, respectively. Strain S35T was oxidase- and catalase-positive, able to produce acid from D-glucose and other carbohydrates. Hydrolysis of DNA, methyl red test, and nitrate and nitrite reduction were positive. Its main fatty acids were C16:0, C16:1 ω7c and C16:1 ω6c, and C18:1 ω7c and/or C18:1 ω6c. ANI, GGDC, and core-genome analysis determined that strain S35T constitutes a novel species of the genus Salinivibrio, for which the name Salinivibrio socompensis sp. nov. is proposed. The type strain is S35T (= CECT 9634T = BNM 0535T).


Introduction
The two main groups that inhabit hypersaline environments are the extremely halophilic archaea (also called haloarchaea) and the moderately halophilic bacteria [1,2]. The haloarchaea are classified within the class Halobacteria with three orders: Haloferacales, Halobacteriales, and Natrialbales [3]. Regarding moderately halophilic bacteria, they are very heterogeneous and are represented by a large number of species belonging to at least eight phyla of the domain Bacteria: Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, Bacteroidetes, Thermotoga, Cyanobacteria, and Tenericutes [2].
Halophilic microorganisms have an important biotechnological potential due to their exceptional physiological and biochemical characteristics. Among their most interesting applications are found the production of a novel restriction enzyme [4], agarases, with industrial and medical applications [5], or β-galactosidase, to produce lactose-free dairy products [6]. Other applications include the production of ectoines, polysaccharides, or extracellular enzymes [1].

Phylogenetic Analysis Based on 16S rRNA Gene Sequence Comparison
The DNA of strain S35 T was obtained using the commercial kit G-spinTM Total DNA Extraction Kit (INtRON Biotechnology), following the instructions of the manufacturer. The 16S rRNA gene was amplified by PCR using the primers 16F27 (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 16R1488 (5 -CGGTTACCTTGTTAGGACTTCACC-3 ) [30]. The PCR product was purified using the commercial kit MEGAquick-spinTM Plus (INtRON Biotechnology), and sequenced using the Sanger method with oligonucleotides 16F27, 16R1488, 16R343 (5 -ACTGCTGCCTCCCGTA-3 ), and 16F530 (5 -GTGCCAGCAGCCGCGG-3 ) by StabVida (Oeiras, Portugal). The gene sequences were assembled and edited by ChromasPro software Version 1.5 (Technelysium Pty) and used for initial BLAST, searched against "nt" database in GenBank. To determine the percentages of similarity between strain S35 T and the most closely related taxa we used the EzBioCloud.net server [31]. The 16S rRNA gene analysis and phylogenetic trees construction were performed with the ARB software package [32]. Phylogenetic trees were constructed using three different methods: Neighbor-joining [33], maximum parsimony [34], and maximum likelihood [35] algorithms integrated in the ARB software for phylogenetic inference. A bootstrap analysis (1000 replications) was performed to evaluate the robustness of the phylogenetic trees [36]. The 16S rRNA gene sequences of the reference type strains used for the phylogenetic comparison were obtained from GenBank database and their accession numbers are shown in Figure 1.

Phylogenetic Analysis Based on 16S rRNA Gene Sequence Comparison
The DNA of strain S35 T was obtained using the commercial kit G-spinTM Total DNA Extraction Kit (INtRON Biotechnology), following the instructions of the manufacturer. The 16S rRNA gene was amplified by PCR using the primers 16F27 (5′-AGAGTTTGATCMTGGCTCAG-3′) and 16R1488 (5′-CGGTTACCTTGTTAGGACTTCACC-3′) [30]. The PCR product was purified using the commercial kit MEGAquick-spinTM Plus (INtRON Biotechnology), and sequenced using the Sanger method with oligonucleotides 16F27, 16R1488, 16R343 (5′-ACTGCTGCCTCCCGTA-3′), and 16F530 (5′-GTGCCAGCAGCCGCGG-3′) by StabVida (Oeiras, Portugal). The gene sequences were assembled and edited by ChromasPro software Version 1.5 (Technelysium Pty) and used for initial BLAST, searched against "nt" database in GenBank. To determine the percentages of similarity between strain S35 T and the most closely related taxa we used the EzBioCloud.net server [31]. The 16S rRNA gene analysis and phylogenetic trees construction were performed with the ARB software package [32]. Phylogenetic trees were constructed using three different methods: Neighbor-joining [33], maximum parsimony [34], and maximum likelihood [35] algorithms integrated in the ARB software for phylogenetic inference. A bootstrap analysis (1000 replications) was performed to evaluate the robustness of the phylogenetic trees [36]. The 16S rRNA gene sequences of the reference type strains used for the phylogenetic comparison were obtained from GenBank database and their accession numbers are shown in Figure 1.

Phylogenomic Comparative Analysis
For the phylogenomic comparative analysis we used the genomes available from GenBank database that are shown in Table S1. These genomes correspond to the following strains used in this comparative study: Salinivibrio sp. strain 35 T , S. costicola subsp. costicola LMG 11651 T , S. costicola subsp. alcaliphilus DSM 16359 T , S. proteolyticus DSM 19052 T , S. siamensis JCM 14472 T , S. sharmensis DSM 18182 T , and S. kushneri AL184 T . The quality of these genome sequences is in accordance with the minimal standards for the use of genome data for the taxonomy of prokaryotes [37].

Phylogenomic Comparative Analysis
For the phylogenomic comparative analysis we used the genomes available from GenBank database that are shown in Table S1. These genomes correspond to the following strains used in this comparative study: Salinivibrio sp. strain 35 T , S. costicola subsp. costicola LMG 11651 T , S. costicola subsp. alcaliphilus DSM 16359 T , S. proteolyticus DSM 19052 T , S. siamensis JCM 14472 T , S. sharmensis DSM 18182 T , and S. kushneri AL184 T . The quality of these genome sequences is in accordance with the minimal standards for the use of genome data for the taxonomy of prokaryotes [37].
To determine the core genome, the Enveomics [38] tool was used to perform an all-versus-all BLAST search based on nucleotide gene sequences of strain S35 T and type strains of the genus Salinivibrio to identify clusters of orthologous genes (OGs). Those OGs shared among all taxa and present in single copy per genome were selected. They were aligned with Muscle v. 3.8.31 [39] and subsequently concatenated. An approximately maximum-likelihood tree was constructed using FastTree v. 2.1.9 [40] with the JTT replacement matrix [41] under the CAT approximation (single rate for each site) with 20 rate categories. Local support values were estimated with the Shimodaira-Hasegawa test [42]. The average nucleotide identity (ANI) among Salinivibrio sp. strain 35 T and S. costicola subsp. costicola LMG 11651 T , S. costicola subsp. alcaliphilus DSM 16359 T , S. proteolyticus DSM 19052 T , S. siamensis JCM 14472 T , S. sharmensis DSM 18182 T , and S. kushneri AL184 T was calculated using three different methods: The percentages of ANIb based on BLAST+ and ANIm based on MUMmer were performed with JSpeciesWS [43], and the orthoANI was calculated with ChunLab's Orthologous Average Nucleotide Identity Tool (OAT) [44], available on the EzBioCloud server. In-silico DDH was calculated by the bioinformatic tool Genome-to-Genome Distance Calculator (GGDC version 2.1) available from the Leibniz Institute DSMZ [45].

Morphology and Motility
Morphology and pigmentation of colonies were observed on SW7.5 solid medium at pH 7.5 after 24 h at 37 • C. Cell morphology and motility were examined by phase-contrast microscopy (Olympus CX41 with DP70 digital camera).

Physiological Characteristics
The range and optimal conditions of salinity for growth were determined by using SW liquid medium at pH 7.5 supplemented with 3%, 6%, 7%, 7.5%, 8%, 9%, 10%, 15%, and 20% (w/v) total salts respectively. In order to determine the optimal (and range) growth at different pH values of strain S35 T , the isolate was cultured under the optimal salt concentration conditions, adjusting the medium to pH 5, 6, 7, 7.5, 8, 9, and 10, respectively, with appropriate buffers. The optimal and range of temperature were determined by incubating strain S35 T under the optimal salt concentration and pH conditions, at temperatures of 4, 10, 15, 28, 37, 40, 42, 45, and 48 • C, respectively. Growth rates were determined by monitoring the increase in the optical density (O.D.) at 600 nm (ThermoSpectronics Spectronic 20D+).

Chemotaxonomic Characterization
The strain S35 T was grown on medium 1 at 37 • C for 48 h and the fatty acids composition was determined following the protocol recommended by MIDI Microbial Identification System [49]. The fatty acids were determined by gas chromatography at the Spanish Type Culture Collection (CECT), Valencia, Spain.

Phylogenetic Analysis Based on 16S rRNA Gene Sequence Comparison
The 16S rRNA gene sequence comparative analysis of strain S35 T with respect to the type strains of S. costicola subsp. costicola CECT 4059 T , S. costicola subsp. alcaliphilus DSM 16359 T , S. proteolyticus DSM 19052 T , S. siamensis JCM 14472 T , S. sharmensis DSM 18182 T , and S. kushneri AL184 T showed percentages of similarity of 99.2%, 99.4%, 97.7%, 97.7%, 97.9%, and 98.9%, respectively. These high percentages indicate that strain S35 T is a member of the genus Salinivibrio, but they are not conclusive to determinate if strain S35 T may constitute a novel species. The phylogenetic tree (Figure 1) based on the 16S rRNA gene sequences, constructed by the neighbor-joining algorithm, showed that strain S35 T clustered with the other species of the genus Salinivibrio. Topologies of phylogenetic trees inferred using the maximum likelihood and maximum parsimony were very similar to those of this tree.

Phylogenomic Comparative Analysis
Since the comparison of the 16S rRNA gene sequence does not allow us to determine in depth the phylogenetic relationships within the genus Salinivibrio [22], and in order to increase the resolution, we carried out a phylogenomic analysis based on the gene sequences obtained from the available genomes, whose characteristics are shown in Table S1, of strain S35 T , Salinivibrio costicola subsp. costicola LMG 11651 T , Salinivibrio costicola subsp. alcaliphilus DSM 16359 T , Salinivibrio proteolyticus DSM 19052 T , Salinivibrio siamensis JCM 14472 T , Salinivibrio sharmensis DSM 18182 T , and Salinivibrio kushneri AL184 T .
The core genome tree was based on 1265 common genes of the seven studied Salinivibrio strains. Figure 2 shows that strain S35 T constitutes a phylogroup different enough from the other type strains of Salinivibrio as to be considered as a new species. As shown in Figure 2, strain S35 T did not cluster together with any of the species and subspecies of Salinivibrio, being separated from all of them. Thus, we concluded that strain S35 T constitutes a new species of this genus.  Table 1 shows the ANIb, ANIm, and orthoANI percentages of strain S35 T with respect to the other species and subspecies of Salinivibrio. The current threshold value for delineating to bacterial species using the aforementioned indexes is 95%, meaning that if a result is above or equal to this value then the strains belong to the same species, but they constitute different species when this value is below 95% [44]. All ANI values obtained using the three different methods were below 95% between strain S35 T and the type strains of the species and subspecies of the genus Salinivibrio. Regarding GGCD, strain S35 T is beneath 70% threshold with respect to the type strains of the species of the genus Salinivibrio (Table 1). According to Kim et al. [50], GGDC percentages above or equal to 70% indicate that the strains can be assigned to the same species, and values under 70% indicate that the strains belong to different species.

Phenotypic Characterization
Colonies of strain S35 T showed cream-pink pigmentation and spherical shape, with a diameter lower than 3 mm. Cells were motile, Gram-stain-negative, curved rods, similar to those reported for other Salinivibrio species [14][15][16][17][18][19][20][21]. Strain S35 T was able to grow in a range of 3%-20% (w/v) NaCl, with an optimum at 7.0%-7.5% (w/v) NaCl ( Figure S1). Strain S35 T can be considered as a moderately halophilic bacterium, as are the other species of Salinivibrio ( Table 2). The pH allowing growth ranged from 6 to 10, with an optimum at pH 8.0 ( Figure S2). These values are similar to those described for other Salinivibrio strains, except for S. costicola subsp. alcaliphilus and S. sharmensis, that had optimum pH at 9.0. The temperature range for growth was 10-42 °C with an optimal growth at 37 °C. Biochemical and nutritional characteristics of strain S35 T with respect to those of the species and subspecies of the genus Salinivibrio are given in Table 2. It is noticeable that strain S35 T is not able to hydrolyze any of the substrates tested, with the exception of DNA and gelatin. On the other hand, in contrast with the other species of Salinivibrio, strain S35 T is the only one that showed a positive result  Table 1 shows the ANIb, ANIm, and orthoANI percentages of strain S35 T with respect to the other species and subspecies of Salinivibrio. The current threshold value for delineating to bacterial species using the aforementioned indexes is 95%, meaning that if a result is above or equal to this value then the strains belong to the same species, but they constitute different species when this value is below 95% [44]. All ANI values obtained using the three different methods were below 95% between strain S35 T and the type strains of the species and subspecies of the genus Salinivibrio. Regarding GGCD, strain S35 T is beneath 70% threshold with respect to the type strains of the species of the genus Salinivibrio (Table 1). According to Kim et al. [50], GGDC percentages above or equal to 70% indicate that the strains can be assigned to the same species, and values under 70% indicate that the strains belong to different species.

Phenotypic Characterization
Colonies of strain S35 T showed cream-pink pigmentation and spherical shape, with a diameter lower than 3 mm. Cells were motile, Gram-stain-negative, curved rods, similar to those reported for other Salinivibrio species [14][15][16][17][18][19][20][21]. Strain S35 T was able to grow in a range of 3%-20% (w/v) NaCl, with an optimum at 7.0%-7.5% (w/v) NaCl ( Figure S1). Strain S35 T can be considered as a moderately halophilic bacterium, as are the other species of Salinivibrio ( Table 2). The pH allowing growth ranged from 6 to 10, with an optimum at pH 8.0 ( Figure S2). These values are similar to those described for other Salinivibrio strains, except for S. costicola subsp. alcaliphilus and S. sharmensis, that had optimum pH at 9.0. The temperature range for growth was 10-42 • C with an optimal growth at 37 • C. Biochemical and nutritional characteristics of strain S35 T with respect to those of the species and subspecies of the genus Salinivibrio are given in Table 2. It is noticeable that strain S35 T is not able to hydrolyze any of the substrates tested, with the exception of DNA and gelatin. On the other hand, in contrast with the other species of Salinivibrio, strain S35 T is the only one that showed a positive result for the methyl red test. Finally, strain S35 T has predominance for the utilization of sugars instead of alcohols or organic acids as sole source of carbon and energy (Table 2). Table 2. Differential characteristics between strain S35 T and the type strains of the closely related species and subspecies of the genus Salinivibrio.
Acid production from carbohydrates: D-fructose Utilization as sole carbon and energy source of the: Amygdalin Utilization as sole carbon, nitrogen and energy source of: Alanine S. kushneri AL184 T . All strains were positive for catalase, oxidase, and hydrolysis of DNA and gelatine, and negative for Voges-Proskauer, Simmon's citrate, H 2 S production, ornithine decarboxylase, lysine decarboxylase, urease, and phenylalanine deaminase. Acid was produced from glycerol, D-glucose, and D-trehalose but not from D-arabinose. All strains were positive for raffinose, dulcitol, and propionate as sole carbon and energy source, and negative for D-galactose, lactose, melezitose, and acetate, along with lysine and methionine as sole carbon, nitrogen, and energy source. a Mellado et al. [14]. b Romano et al. [16]. c Amoozegar et al. [17]. d Chamroensaksri et al. [18]. e Romano et al. [19]. f López-Hermoso et al. [20].

Conclusions
On the basis of the results of the polyphasic taxonomic analysis, it is concluded that strain S35 T should be considered as a novel species of the genus Salinivibrio, for which the name Salinivibrio socompensis sp. nov. is proposed. We enclose the taxonomic description of this new species.
The type strain is S35 T (= CECT 9634 T = BNM 0535 T ), isolated from the volcanic saline lake Socompa, Argentina.
The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequence and complete genome sequence of the type strain S35 T are HF953987 and AQOD00000000, respectively.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/7/8/241/s1. Table S1. Genomes of the type strains of species of the genus Salinivibrio available in GenBank database used in this study, including their basic statistical information. Table S2. Cellular fatty acids composition (%) of strain S35T and the type strain of species and subspecies of the genus Salinivibrio. Figure S1. Growth curve for strain S35T at different salt concentrations. Culture media had the same composition than the medium used for routinely growth at 3, 6, 7, 7.5, 8, 9, 10, 15 and 20% (w/v) total salt, respectively. Figure S2. Growth curve for strain S35T at different pH values. Culture media had the same composition than the medium used for routinely growth and the pH was adjusted to 5, 6, 7, 7.5, 8, 9 and 10, respectively.