Halovenus salina sp nov., an extremely halophilic archaeon isolated from a saltern

1 Halovenus salina sp. nov., an extremely halophilic archaeon isolated from a saltern 1 2 Carmen Infante-Domínguez, Paulina Corral, Cristina Sánchez-Porro and Antonio 3 Ventosa 4 5 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of 6 Sevilla, 41012 Sevilla, Spain. 7 8 9 Running title: Halovenus salina sp. nov. 10 Subject category: New taxa, Archaea 11 12 Author for correspondence: Antonio Ventosa, Department of Microbiology and 13 Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain. E-mail: 14 [email protected]s 15 16 The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, atpB, ef-2, glnA and 17 rpoB gene sequences of strain ASP54T are KJ661744, LN851866, LN851867, 18 LN851868 and LN851869, and for the atpB, ef-2 and glnA gene sequences of 19 Halovenus aranensis IBRC-M 10015T are LN851870, LN851871, and LN851872, 20 respectively. 21 22 Two supplementary figures are available with the online Supplementary Material. 23 24 Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Halovenus salina sp. nov. rev.doc 2 An extremely halophilic archaeon was isolated from a sample of Isla Bacuta 25 saltern in Huelva, Spain. Strain ASP54T is a novel red-pigmented, motile, rod 26 shaped, Gram-stain-negative and strictly aerobic haloarchaeon. ASP54T grew in 27 media containing 15-30 % (w/v) salts and optimally at 25 % (w/v) salts. It grew 28 between pH 5.0 and 9.0 (optimally at pH 7.5) and at 20-40 ºC (optimally at 37 ºC). 29 Phylogenetic analysis based on multilocus sequence analysis (MLSA) and the 30 comparison of 16S rRNA gene sequences revealed that strain ASP54T is most 31 closely related to the genus Halovenus. The closest relatives to this strain were 32 Halovenus aranensis IBRC-M 10015T (92.1 % 16S rRNA gene sequence similarity), 33 Halorientalis regularis JCM 16425T (92.1 %), and Halorientalis persicus IBRC-M 34 10043T (92.0 %). The polar lipid pattern of strain ASP54T consisted of 35 biphosphatidylglycerol, phosphatidylglycerol, phosphatidylglycerol phosphate 36 methyl ester, phosphatidylglycerol sulfate, sulfated mannosyl glucosyl diether and 37 a minor-phospholipid. The predominant respiratory quinone was menaquinone-8 38 (MK-8) (83 %), and a minor amount of MK-8(VIII-H2) (17 %) was also detected. 39 The G+C content of the genomic DNA of this strain was 63.1 mol%. Based on the 40 phenotypic, chemotaxonomic and phylogenetic data presented in this study, the 41 strain ASP54T is proposed as a novel species of the genus Halovenus, for which the 42 name Halovenus salina sp. nov. is suggested. The type strain is ASP54T (= CECT 43 8749T = IBRC-M 10946T = JCM 30072T). 44 45 3 The genus Halovenus was proposed by Makhdoumi-Kakhki et al. (2012) and currently, 46 it includes only one species: Halovenus aranensis, isolated from Aran-Bigdol 47 hypersaline lake in Iran. The only species of Halovenus includes Gram-stain-negative, 48 strictly aerobic, non-motile and pleomorphic (rods to triangles, squares or disc-shaped) 49 cells. Catalase and oxidase are positive. Extremely halophilic with growth occurring in 50 media containing 15-30 % (w/v) NaCl and optimal growth at 25 % (w/v) NaCl. The 51 polar lipids pattern consists on phosphatidylglycerol, phosphatidylglycerol phosphate 52 methyl ester and two minor phospholipids. MK-8(II-H2) is the only lipoquinone present. 53 The DNA G+C content is 61 mol% (Makhdoumi-Kakhki et al., 2012). 54 Currently the phylogenetic analyses in prokaryotes are mainly based on the comparison 55 of the 16S rRNA gene sequences. However, haloarchaea are known for their frequent 56 recombination across great genetic distances (Williams et al., 2012; Nelson-Sathi et al., 57 2012) and in many cases their rRNA operons undergo intragenic recombination 58 (Boucher et al., 2004). For these reasons, a multilocus sequence analysis (MLSA) 59 approach is also being used. Recent MLSA studies recommended the use of the atpB, 60 ef-2, glnaA and rpoB’ housekeeping genes. These genes provide a lower frequency of 61 recombination, are single copy in haloarchaeal genomes, are more highly conserved 62 than 16S rRNA genes, and are more suitable for distinguishing among the species 63 (Papke et al., 2011; Oren & Ventosa, 2013; Fullmer et al., 2014; Ram Mohan et al., 64 2014). 65 Recently, we have studied the microbial diversity of the marine saltern ponds in Santa 66 Pola, Alicante, and Isla Cristina, Huelva, Spain, by metagenomic analyses. These 67 studies determined that haloarchaea are dominant in intermediate to salt-saturation 68 ponds of these salterns and new groups not yet characterized were reported (Ghai et al., 69 2011; Fernandez et al., 2014a; 2014b). We have developed strategies to isolate and 70 4 characterize these previously undescribed haloarchaeal groups. In this study, a novel, 71 red-pigmented, Gram-stain-negative extremely halophilic archaeon, strain ASP54T, was 72 isolated from a water sample obtained from a saltern of Isla Bacuta, Huelva, Spain. At 73 the time of sampling, the salinity of the water was 22 % (w/v) total salts, the pH was 7.4 74 and the water temperature was 29 ºC. Preliminary data indicated that strain ASP54T was 75 phylogenetically related to the genus Halovenus and to other halophilic members of the 76 family Halobacteriaceae. In this paper we describe the taxonomic features of this novel 77 archaeon and propose it as a new species of the genus Halovenus, as Halovenus salina 78 sp. nov. For this purpose, we have followed the minimal standards recommended by 79 Oren et al. (1997) for the description of new taxa of the order Halobacteriales. In 80 addition, MLSA was used to further resolve the phylogenetic position of the new 81 isolate. 82 The isolation of strain ASP54T was performed by plating aliquots of the water sample 83 previously diluted with sterile salt solution; 100 µl of the sample were inoculated after 84 serial dilutions up to 10-4 from the water sample, on saline media and incubated 85 aerobically at 37 ºC for one month. The isolation medium used was prepared with a 25 86 % (g l-1) salts mixture: NaCl, 195; MgCl2.6H2O, 32.5; MgSO4.7H2O, 50.8; CaCl2.2H2O, 87 0.5; KCl, 5; NaHCO3, 0.1; NaBr, 0.38 (Subow, 1931), supplemented with 0.1% (w/v) 88 yeast extract (Difco), 0.1 % (w/v) sodium pyruvate (Aldrich) and 1 % (w/v) agarose 89 (Lonza). The pH of the medium was adjusted to 7.2-7.4 with 1 M KOH. For routine 90 growth, the strain was cultivated in the same medium prepared with 25 % (w/v) total 91 salts and 0.1 % (w/v) yeast extract, but without sodium pyruvate and solidified with 1.8 92 % (w/v) agar (Difco) when necessary. The strain was maintained in the routine medium 93 in slant tubes, and for long term preservation in cryotubes prepared for freezing at -80 94 ºC as follows: 750 µl of the fresh culture (OD600 0.8-1.0) and 250 µl of glycerol/25 % 95 5 salt mixture (80:20, v/v) (Dyall-Smith, 2009). Halovenus aranensis IBRC-M 10015T 96 and Halorientalis persicus IBRC-M 10043T, obtained from IBRC (Microorganisms 97 Bank, Iranian Biological Resource Centre) and Halorientalis regularis JCM 16425T, 98 obtained from JCM (Japan Collection of Microorganisms) were used as reference 99 strains for comparison with our strain. They were grown following the 100 recommendations of the culture collections. 101 Cell morphology and motility were examined using an Olympus CX41 microscope 102 equipped with phase-contrast optics. Cells were motile rods with a size of 0.5-1 x 1.0- 103 3.0 µm and occurred as single cells, without any regular arrangement (Supplementary 104 Fig. S1, available at IJSEM online). Colony morphology was observed on routine 105 medium under optimal growth conditions after incubation at 37 ºC for 3 weeks. 106 Colonies of strain ASP54T on agar plates were circular, smooth, entire, opaque, red- 107 pigmented, with a size of 1 mm of diameter. 108 Optimal conditions for growth and range were determined by growing the strain in the 109 routine medium prepared at the following salinity values: 0.5, 1, 3, 5, 7, 7.5, 8, 10, 15, 110 20, 25 and 30 % (w/v) total salts and at temperatures from 5 to 45 ºC (in increments of 5 111 ºC) and from 35 to 40 ºC (in increments of one unit). The pH range was determined in 112 the routine medium at optimal salinity adjusted to pH values ranging from 4.5 to 9.5 (in 113 increments of one pH unit), by the addition of MES (pH 4.5–6.0), PIPES (pH 6.5–7.0), 114 Tricine (pH 7.5–8.5) or CHES (pH 9.0–9.5) to a final concentration of 50 mM to the 115 isolation medium (Scorpio, 2000). 116 Strain ASP54T grows in media containing 15-30 % (w/v) salts and optimally in media 117 containing 25 % (w/v) salts. No growth was observed in the absence of NaCl. It grew at 118 temperatures in the range 20-40 ºC (optimally at 37 ºC) and at a pH range between 5.0- 119 6 9.0 (optimally at pH 7.5). The strain was not able to grow anaerobically, determined 120 after incubation in an anaerobic chamber (GasPak Anaerobic system, BBL). 121 The genomic DNA of strain ASP54T was obtained using the method described by 122 Marmur (1961). The 16S rRNA gene was amplified by PCR (Sambrook & Russell, 123 2001) using universal primers 21F (5´-TTCCGGTTGATCCTGCCGGA-3´) (DeLong, 124 1992) and 1492R (5´-GGTTACCTTGTTACGACTT-3´) (DeLong, 1992). The PCR 125 product was sequenced using the Sanger method. The nucleotide sequence of 16S rRNA 126 gene of strain ASP54T (1402 bp) was aligned with ChromasPro software version 1.7.6 127 and ARB 5.5 software package (Ludwig et al., 2004). The identification of the closest 128 phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence 129 similarity were achieved using the EzTaxon-e tool (Kim et al., 2012). The 16S rRNA 130 gene sequence was checked against both primary and secondary structures of the 16S 131 rRNA molecule using the alignment tool of the ARB software package (Ludwig et al., 132 2004). Phylogenetic trees were constructed using three different methods, namely the 133 maximum-likelihood (Felsenstein, 1985), maximum-parsimony (Fitch, 1971) and 134 neighbour-joining (Saitou & Nei, 1987) algorithms integrated in the ARB software. The 135 analysis of the 16S rRNA gene sequences studied revealed that the most closely related 136 species of strain ASP54T were Halovenus aranensis EB27T (92.1 % similarity), 137 Halorientalis regularis TNN28T (92.1 %), Halorientalis persicus IBRC-M 10043T (92.0 138 %). Lower similarities were obtained with respect to Halomicrobium zhoui TBN51T 139 (91.9 %), Halorhabdus utahensis DSM 12940T (91.8 %), Halorhabdus tiamatea SARL 140 48T (91.8 %), Halomicrobium mukohataei DSM 12286T (91.8 %), and Halosimplex 141 carlsbadense 2-9-1T (91.5 %). The phylogenetic tree (Fig. 1) based on the 16S rRNA 142 gene sequence comparison and constructed by maximum-likelihood showed that strain 143 ASP54T constituted a single branch, which clustered with the species Halovenus 144 7 aranensis EB27T; then, it was related to other species belonging to the family 145 Halobacteriaceae. Similar tree topologies were obtained using neighbour-joining and 146 maximum-parsimony treeing methods (Fig. 1). 147 In order to complete the phylogenetic study we carried out a MLSA approach, based on 148 four housekeeping genes: atpB, ef-2, glnA and rpoB’, that were amplified by PCR, using 149 primers designed for each locus. For each primer, the sequencing primer M13 (18 bp) 150 was added to the 5´ end of the primer (Fullmer et al., 2014; Ram Mohan et al., 2014). 151 Each PCR reaction was 20 microliters in volume. The PCR reaction was run on a 152 Mastercycler Ep Thermocycler (Eppendorf) using the following PCR cycle protocol: 30 153 s initial denaturation at 98 ºC, followed by 40 cycle of 30 s at 98 ºC, 5 s at the annealing 154 temperature for each set of primers and 15 s at 72 ºC. Final elongation occurred at 72 ºC 155 for 1 min. The PCR products were separated by gel electrophoresis with agarose (1.5 156 %). Gels were stained with ethidium bromide. A 1 kb Blue DNA marker (Intron) was 157 used to estimate the size of the amplicons. The PCR product was sequenced using the 158 Sanger method. The concatenation of the four loci (atpB, ef-2, glnA and rpoB’) 159 produced an alignment of 2128 bp. 160 The comparison of the sequences of the genes atpB, ef-2, glnA and rpoB’ of strain 161 ASP54T with those of Halovenus aranensis IBRC-M 10015T were 92.0 %, 92.0 %, 84.0 162 % and 89.0 %, respectively. Lower similarity values were obtained with respect to other 163 members of the Halobacteriaceae. These similarity values are much lower than the cut- 164 off percentages that are currently considered for the delineation of species. The 165 phylogenetic tree (Fig. 2) constructed by neighbour-joining based on the four 166 concatenated housekeeping genes was carried out on amplified MLSA target gene 167 sequences (atpB, ef-2, glnA and rpoB’) of the new isolate and Halovenus aranensis 168 IBRC-M 10015T, Halorientalis regularis JCM 16425T, and Halorientalis persicus 169 8 IBRC-M 10043T. Other related sequences were obtained from GenBank database. 170 Multiple-sequence alignments (MSAs) were created taking into account the 171 corresponding amino acid alignments for protein-coding genes generated by translating 172 the genes to protein sequences, in addition the alignments were edited using BioEdit 173 (Hall, 1999). The genes were concatenated in the order atpB, ef-2, glnA and rpoB’. 174 Using MEGA5 (Tamura et al., 2011), the MLSA concatenated phylogenetic tree of the 175 four loci (atpB, ef-2, glnA and rpoB’) showed that strain ASP54T clustered with the 176 species Halovenus aranensis but forming an independent branch, clearly separated from 177 this species (Fig. 2). These data are in agreement with those obtained from the 16S 178 rRNA gene sequence analysis and clearly support the placement of strain ASP54T as a 179 separate species of the genus Halovenus. 180 For the phenotypic characterization all features were determined growing the strains in 181 the medium prepared at 25 % (w/v) total salts, pH 7.5 and at an incubation temperature 182 of 37 ºC. The catalase activity was determined by adding a 1 % (w/v) H2O2 solution to 183 colonies on solid medium. The oxidase test was performed using the Dry Slide assay 184 (Difco). The tests for the hydrolysis of starch, gelatin, casein, Tween 80, nitrate and 185 nitrite reduction were carried out as described by Barrow & Feltham (2003). The methyl 186 red and Voges-Proskauer tests, Simmons’s citrate and anaerobic growth in the presence 187 of nitrate, arginine or DMSO were performed as described by Oren et al. (1997). Tests 188 for indole production from tryptophan and urease were performed as described by 189 Gerhardt et al. (1994). The production of H2S was determined by a strip of lead acetate 190 paper held in the top of the inoculated tube prepared on the liquid medium (Clarke, 191 1953). The production of acid from different carbohydrates was tested in a medium with 192 0.5 % (w/v) yeast extract and supplemented with 1 % (w/v) of the carbohydrate assayed 193 and sterilized separately as described by Ventosa et al. (1982). To determine the 194 9 utilization of different organic substrates as carbon and energy sources, a medium 195 containing 0.05 % (w/v) yeast extract and supplemented with 1 % (w/v) of the tested 196 substrate was used (Ventosa et al., 1982). 197 Strain ASP54T was catalase and oxidase positive. Methyl red was positive. Voges- 198 Proskauer, indole production from tryptone, Simmons’s citrate, and urease were 199 negative; nitrate was reduced but nitrite was not reduced. Gelatin was hydrolyzed; 200 Tween 80, starch, casein and aesculin were not hydrolyzed. H2S was produced. Other 201 phenotypic characteristics of strain ASP54T are included in Table 1 and in the species 202 description. 203 Antimicrobial compounds sensitivity tests were performed by spreading the culture 204 suspension on solid medium plates and applying discs impregnated with the 205 antimicrobial compounds according to the conventional Bauer-Kirby method (Bauer et 206 al., 1966) and incubated at 37 ºC for 3 weeks and inhibition zone was interpreted. The 207 following antimicrobial compounds were tested: ampicillin (10 µg), neomycin (10 µg), 208 novobiocin (30 µg), rifampicin (30 µg), gentamycin (10 µg), 209 trimethoprim+sulfametoxazol (23.75 ng/1.25 ng), erythromycin (15 µg), 210 chloramphenicol (30 µg), nalidixic acid (30 µg) kanamycin (30 µg) and penicillin G (10 211 UI). Strain ASP54T was sensitive to novobiocin and trimethoprim+sulfametoxazol, but 212 resistant to ampicillin, neomycin, gentamycin, erythromycin, chloramphenicol, nalidixic 213 acid, kanamycin, rifampicin and penicillin G. In contrast to strain ASP54T, Halovenus 214 aranensis IBRC-M 10015T was sensitive to rifampicin. 215 The genomic DNA G+C content was determined from the mid-point (Tm) of the 216 thermal desnaturation profile (Marmur & Doty, 1962), using the equation of Owen & 217 Hill (1979). The DNA G+C content of strain ASP54T was 63.1 mol%. This value is 218 16 Makhdoumi-Kakhki, A., Amoozegar, M. A. & Ventosa A. (2012). Halovenus 361 aranensis gen. nov., sp. nov., an extremely halophilic archaeon from Aran-Bidgol salt 362 lake. Int J Syst Evol Microbiol 62, 1331-1336. 363 Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from 364 microorganisms. J Mol Biol 3, 208–218. 365 Marmur, J. & Doty, P. (1962). Determination of the base composition of 366 deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109-118. 367 Nelson-Sathi, S., Dagan, T., Landan, G., Janssen, A., Steel, M., McInerney, J. O., 368 Deppenmeir, U. & Martin, W. (2012). Acquisition of 1,000 eubacterial genes 369 physiologically transformed a methanogen at the origin of haloarchaea. Proc Natl Acad 370 Sci USA 50, 20537-20542. 371 Oren, A. & Ventosa, A. (2013). Minutes of the joint open meeting of the 372 Subcommittee on the taxonomy of Halobacteriaceae and Subcommittee on the 373 taxonomy of Halomonadaceae. Int J Syst Evol Microbiol 63, 3540-3544. 374 Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposed minimal standards for 375 description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47, 233-238. 376 Owen, R. J. & Hill, L. R. (1979). The estimation of base compositions, base pairing 377 and genome sizes of bacterial deoxyribonucleic acids. In: F. A. Skinner and D. W. 378 Lovelock (eds.). Identification Methods for Microbiologists, 2nd edition. Academic 379 Press, London, pp. 277-296. 380 Papke, R.T., White, E., Reddy, P., Weigel, G., Kamekura, M., Minegishi, H., 381 Usami, R. & Ventosa, A. (2013). A multilocus sequence approach to the phylogeny 382 and taxonomy of the Halobacteriales. Int J Syst Evol Microbiol 61, 2984-2995. 383 17 Ram Mohan, N., Fullmer, M. S., Makkay, A. M., Wheeler, R., Ventosa A., Naor, 384 A., Gogarten, J. P. and Papke, R. T. (2014). Evidence from phylogenetic and genome 385 fingerprinting analyses suggests rapidly changing variation in Halorubrum and 386 Haloarcula populations. Front Microbiol 5, 143. 387 Saitou, N. & Nei, M. (1987). The neighbor joining method: a new method for 388 reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425. 389 Sambrook, J. & Russell, D. W. (eds.) (2001). Molecular Cloning: a Laboratory 390 Manual. Cold Spring Harbor, NY: Laboratory Press. 391 Scorpio, R. (2000). Fundamentals of Acids, Bases, Buffers and Their Application to 392 Biochemical Systems. Kendall/Hunt Publishing Company. 393 Subov, N. N. (1931). Oceanographical tables. Comissariat of Agriculture of URSS, 394 Hydro-Meteorological Commitee of URSS, Oceanographical Institute of URSS, 395 Moscow. 396 Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). 397 MEGA5: molecular evolutionary genetics analysis using maximum likelihood, 398 evolutionary distance and maximum parsimony methods. Mol Biol Evo 28, 2731–2739. 399 Ventosa, A., Quesada, E., Rodríguez-Valera, F., Ruiz-Berraquero, F. & Ramos- 400 Cormenzana, A. (1982). Numerical taxonomy of moderately halophilic Gram-negative 401 rods. J Gen Microbiol 128, 1959–1968. 402 Williams, D., Gogarten, J. P. & Papke, R. T. (2012). Quantifying homologous 403 replacement of loci between haloarchaeal species. Genome Biol Evol 4, 1223-1244. 404 405 18 Table 1. Characteristics that differentiate strain ASP54T from the closely related species 406 of the genera Halovenus and Halorientalis. 407 Strains: 1, Halovenus salina sp. nov. ASP54T; 2, Halovenus aranensis IBRC-M 408 10015T; 3, Halorientalis regularis JCM 16425T; 4, Halorientalis persicus IBRC-M 409 10043T. All data are from this study except those marked with a, obtained from 410 Makhdoumi-Kakhki et al. (2012); b, obtained from Cui et al. (2011) and c, obtained 411 from Amoozegar et al. (2014). +, Positive reaction; -, negative reaction. 412 413 Characteristic 1 2 3 4 Cell size (width x length; µm) 0.5-1.0 x 1.0-3.0 0.6-1.3 x 0.7-4.9 a 0.5-1.0 x 1.0-6.0 b 0.7-1.0 x 1.0-2.0 c Temperature range (ºC) 20-45 25-50 a 20-50 b 30-50 c NaCl optimal (%, w/v) 25 25 a 18-20 b 25 c pH range 5.0-9.0 6.0-8.0 a 6.0-9.0 b 6.5-9.0 c H2S production + - + - Hydrolysis of gelatin + - - - Acid production from: D-Arabinose D-Glucose + + - + + + + - Utilization of compounds as carbon and energy sources: D-Galactose D-Glucose Lactose D-Manose D-Raffinose D-Xylose D-Melezitose L-Valine L-Threonine Glycerol Myo-inositol D-Mannitol D-Sorbitol - + + + + + + + + + + + + -(+ a) -(+ a) - - - - - - - - - - - -(+ b) + +(- b) -(+ b) + +- - + + + - + + - + + + + + + - + +(- c) - + + Polar lipids* BPG, PG, PGP-Me, PGS, S-DGD, PL BPG, PG, PGP-Me, S-DGD BPG, PG, PGP-Me BPG, PG, PGP-Me 19 Source Saltern Saline lakea Marine solar salternB Saline lakeC DNA G+C content (mol%) 63.1 60.0 (61.0)a 62.1 (61.9)b 63.5 (62.8) c 414 *BPG, biphosphatidylglycerol; PG, phosphatidylglycerol; PGP-Me, phosphatidylglycerol phosphate methyl ester; 415 PGS, phosphatidylglycerol sulfate; S-DGD, sulfated mannosyl glucosyl diether; PL, minor phospholipid. 416 417 20 Legend to figures 418 419 Fig. 1. Maximum-likehood phylogenetic tree based on 16S rRNA gene sequences, 420 showing the relationships between Halovenus salina ASP54T, related species of the 421 genus Halovenus and other representatives of the class Halobacteria. Bootstrap 422 percentages (based on 1000 replications) ≥ 70 % are shown at branch points. Filled 423 circles indicate that the corresponding nodes were also obtained in trees generated with 424 the maximum-parsimony and neighbour-joining algorithms. Reference sequences were 425 retrieved from GenBank under the accession numbers indicated. Methanospirillum 426 hungatei JF-1T was used as outgroup. Bar represents 0.02 substitutions per nucleotide 427 position. 428 429 430 Fig. 2. Neighbour-joining phylogenetic tree based on the four-gene (atpB, ef-2, glnA 431 and rpoB’) concatenated sequence showing the phylogenetic relationship between 432 Halovenus salina ASP54T, related species of the genus Halovenus and other 433 representatives of the class Halobacteria. Bootstrap percentages (based on 1000 434 replications) ≥ 70 % are shown at branch points. Haloquadratum walsbyi was used as 435 outgroup. Bar represents 0.01 substitutions per nucleotide position. 436 Natronorubrum bangense A33T(Y14028) Natronorubrum tibetense GA33T(AB005656) Natrialba chahannaoensis C112T(AJ004806) Natrialba hulunbeirensis X21T(AJ003193) Haloterrigena saccharevitans AB14T(AY820137) Haloterrigena turkmenica VKM B-1734T (AB004878) Natronobacterium gregoryi NCIMB 2189T(D87970) Natronococcus amylolyticus Ah-36T(D43628) Halalkalicoccus jeotgali B3T(EF077632) Halalkalicoccus tibetensis DS12T(AF435112) Haladaptatus litoreus RO1-28T(EU887285) Halorientalis persicus D108T(KF313134) Halorientalis regularis TNN28T(GQ282621) Halorhabdus tiamatea SARL4BT(EF127229) Halorhabdus utahensis AX-2T(AF071880) Halovenus salina ASP54T(KJ661744) Halovenus aranensis EB27T(HQ197980) Halomicrobium mukohataei JCM 9738T (EF645690) Halobacterium salinarum DSM 3754T(AJ496185) Haloarcula marismortui ATCC 43049T(AY596297) Haloarcula vallismortis JCM 8877T(EF645688) Haloarcula japonica JCM 7785T(EF645685) Haloarcula amylolytica BD-3T (DQ826512) Halosimplex carlsbadense 2-9-1T(AJ586107) Halogranum amylolyticum TNN58T (GQ282623) Halogranum rubrum RO2-11 T (EU887283) Halopelagius inordinatus RO5-2T (EU887284) Haloquadratum walsbyi C23T(AY676200) Haloferax larsenii ZJ206T(AY838278) Haloferax lucentense JCM 9276T(AB081732) Methanospirillum hungatei JF-1T(AY196683) 0.02 94 100 99 82 100 96 100 82 98 100 100 87 85 99 85 92 85 96 100 100 100 100 100 73 Figure 1 Click here to download Figure: Figure 1 revised.pptx Natrialba chahannaoensis Natrialba hulunbeirensis Natronobacterium gregoryi Natronolimnobius baerhuensis Natronorubrum tibetense Natronococcus amylolyticus Natronococcus occultus Natronolimnobius innermongolicus Halostagnicola larsenii Natronorubrum bangense Halobiforme haloterrestris Halobiforme lacisalsi Halobiforme nitratireducens Natrinema ejinorense Haloterrigena turkmenica Haloterrigena saccharevitans Natrinema pellirrubrum Natrinema versiforme Halobaculum gomorrense Natronococcus jeotgali Haloarcula marismortui Haloarcula vallismortis Halomicrobium mukohataei Halosimplex carlsbadense Halovenus salina ASP54T Halovenus aranensis Halobacterium salinarum Halalkalicoccus jeotgali Halalkalicoccus tibetensis Halorubrum saccharovorum Halorientalis regularis Halorientalis persicus Haloferax larsenii Haloferax lucentense Halogeometricum borinquense Halosarcina pallida Haloquadratum walsbyi 100 100 100 100 100 100 96 85 93 93 100 87 98 73 78 79 80 100 79 70 74 0.01 Figure 2 Click here to download Figure: Figure 2 rev.ppt Supplementary Material File Click here to download Supplementary Material Files: Halovenus salina supplementary material rev.pdf Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY on 2015, available at: https://doi.org/10.1099/ijs.0.000370”