Halorubrum halodurans sp. nov., an extremely halophilic archaeon isolated from a hypersaline lake

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in International Journal of Systematic and Evolutionary Microbiology on 01 January 2016, available at: https://doi.org/10.1099/ijsem.0.000738 .” 1 Halorubrum halodurans sp. nov., an extremely halophilic archaeon 1 isolated from a hypersaline lake 2 3 Paulina Corral1, Rafael R. de la Haba1, Cristina Sánchez-Porro1, Mohammad Ali 4 Amoozegar2, R. Thane Papke3 and Antonio Ventosa1 5 6 1Department of Microbiology and Parasitology, Faculty of Pharmacy, University 7 of Sevilla, 41012 Sevilla, Spain. 8 2Department of Microbiology, Faculty of Biology and Center of Excellence in 9 Phylogeny of Living Organisms, College of Science, University of Tehran, 10 Tehran, Iran. 11 3Department of Molecular and Cell Biology, University of Connecticut, 06269 12 Storrs, CT, USA. 13 Running title: Halorubrum halodurans sp. nov. 14 Category: New Taxa, Archaea. 15 16 Corresponding author: Antonio Ventosa, Department of Microbiology and 17 Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain. 18 Tel. +34 95 455 67 68; Fax +34 95 462 81 62. E-mail: [email protected] 19 20 The GenBank/EMBL/DDBJ accession number for the 16S rRNA and rpoB’ gene 21 sequences of strain Cb34T are HG421007 and KJ152360, respectively. 22 Manuscript Including References (Word document)Click here to download Manuscript Including References (Word document) Hrr halodurans revised.doc 2 Two extremely halophilic archaea, strains Cb34T and C170 belonging to 23 the genus Halorubrum were isolated from the brine of the hypersaline lake 24 Aran-Bidgol in Iran. Cells of the two strains were motile pleomorphic rods, 25 stained Gram-variable and produced red-pigmented colonies. Strains 26 Cb34T and C170 required for optimal growth 25 % (w/v) salts, pH 7.0 and 27 37 ºC in aerobic conditions, 0.3 M Mg2+ is required. The cells of both 28 isolates were lysed in distilled water and the hypotonic treatment with < 29 10 % NaCl provoked cell lysis. Phylogenetic analysis based on 16S rRNA 30 gene sequence similarities showed that these two strains were closely 31 related to Halorubrum cibi B31T (98.8 %) and other Halorubrum species. 32 Besides, studies based on the rpoB′ gene revealed that strains Cb34T and 33 C170 are placed among the species of Halorubrum and are closely related 34 to Halorubrum cibi B31T with values lower or equal to 95.7 % gene 35 sequence similarity. The polar lipids pattern of both strains consisted of 36 phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester 37 (PGP-Me), phosphatidylglycerol sulfate (PGS) and sulfated mannosyl 38 glucosyl diether (S-DGD). The DNA G+C content was 62.1–62.4 mol%. 39 DNA-DNA hybridization studies confirmed that strains Cb34T and C170 40 constitute a distinct species. Data obtained in this study show that both 41 strains represent a novel species, for which the name Halorubrum 42 halodurans sp. nov. is proposed. The type strain is Cb34T (= CECT 8745T = 43 IBRC-M 10233T). 44 45 46 3 Haloarchaea is a group of extremely halophilic aerobic archaea included on the 47 class Halobacteria, that comprises 40 genera including the genus Halorubrum 48 established by McGenity & Grant in 1995. Currently it is the largest 49 haloarchaeal genus with 30 validly described species names (Parte, 2015). 50 Species of the genus Halorubrum are isolated from diverse natural and artificial 51 Hhypersaline environments such as salterns, salt lakes, coastal sabkhas, soda 52 lakes, saline soils or salt-fermented and salt-preserved food. It is often reported 53 as dominant genus present in many hypersaline environments, their ubiquity 54 and adaptability to nearly all-possible hypersaline conditions has been 55 demonstrated by cultivation and culture-independent methodologies (Ghai et 56 al., 2011; Makhdoumi-Kakhki et al., 2012a; Fernández et al., 2014a; 2014b). 57 Species of this genus are aerobic, chemoorganotrophic and require for growth 58 high concentrations of NaCl in media (e.g., 1.0-5.2 M), the genus is 59 phenotypically highly variable: they use many carbon substrates including a 60 wide range of sugars as sources of energy. However, some species use only 61 single carbon sources for growth. They produce bacterioruberin carotenoid 62 pigments which colors colonies pink to red. Most of the described species are 63 neutrophilic but some are alkaliphilic and grow optimally at pH 9.5. The major 64 polar lipids are C20C20 and sometimes C20C25 glycerol diether derivatives of 65 phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP66 Me) derived from both C20C20 and C20C25 archaeol, phosphatidylglycerol sulfate 67 (PGS) and a sulfated mannosyl glucosyl diether (S-DGD), but alkaliphilic 68 species lack PGS and glycolipids (McGenity & Grant 1995). The DNA G+C 69 content range is between 60.2 and 71.2 mol%. The type species is Halorubrum 70 saccharovorum (Oren et al., 2009; Roh & Bae, 2009). 71 4 Modern classification of the haloarchaea requires using phenotypic 72 characterization, a chemotaxonomic approach based on the polar lipid 73 composition as well as the comparison of the 16S rRNA gene, which is 74 considered a universal phylogenetic and taxonomic molecular marker (Oren, 75 2012). The usefulness of this molecular marker however, is problematic for 76 haloarchaeal taxonomic studies, as over the years studies have demonstrated it 77 has many qualities that make it difficult to distinguish species like recombination 78 between genera (Boucher et al., 2004; Papke et al., 2015) and closely related 79 species (Papke et al., 2004; 2007); a high sequence conservation that does not 80 discriminate among closely related species (Pesenti et al., 2008; Mancinelli et 81 al., 2009); their rRNA operons undergo intragenic recombination (Boucher et 82 al., 2004); and many genera (e.g., Haloarcula, Halosimplex, Halomicrobium) 83 contain multiple gene copies with greater than 5% sequence divergence 84 (Boucher et al., 2004). The high number of strains frequently isolated as 85 members of Halorubrum and the issues concerning establishing a phylogeny 86 and therefore taxonomy makes the 16S rRNA gene unsuitable for these 87 purposes (Ram-Mohan et al., 2014). To overcome some of these limitations, the 88 analysis of several housekeeping genes using a multilocus sequencing analysis 89 (MLSA) approach is being used to construct phylogenies within the family 90 Halobacteriaceae, since it resolves and defines with sensitivity the phylogeny of 91 newly discovered strains of haloarchaea. The MLSA approach is typically a 92 reliable method that differentiates individual strains, groups strains into species, 93 and species into genera, as well as family-like relationships within the 94 Halobacteriales (Papke et al., 2011). Among the single copy protein-encoding 95 genes previously investigated for evolutionary and taxonomic studies in 96 5 haloarchaea, the rpoB’ has been reported to be very successful in overcoming 97 the above 16S rRNA gene marker disadvantages (Walsh et al., 2004; Enache 98 et al., 2007; Minegishi et al., 2010; Papke et al., 2011; Fullmer et al., 2014; 99 Ram-Mohan et al., 2014). 100 Aran-Bidgol hypersaline lake is a natural ecosystem located in the central 101 desert area of Iran, formed in the Pliocene period. The pH of the lake brine is 102 neutral, the predominant salts are NaCl, Na2SO4, MgCl2 and MgSO4 with trace 103 amounts of carbonates, and it is considered a thalassohaline lake (Makhdoumi104 Kakhki et al., 2012a). In previous studies of the haloarchaeal population in this 105 lake, several new genera and species of this group of microorganisms were 106 isolated and characterized (Amoozegar et al., 2012, 2013, 2014a, 2014b, 107 2014c; Makhdoumi-Kakhki et al., 2012b, 2012c). 108 In the present study is described the properties of two extremely halophilic 109 archaea, strains Cb34T and C170 isolated from Aran-Bidgol hypersaline lake, on 110 the basis of standard taxonomic methods following the proposed minimal 111 standards recommended by Oren et al. (1997) for describing novel taxa of the 112 order Halobacteriales. Additionally, we have included a rpoB` gene phylogeny 113 to complement and reinforce our results. 114 Strains Cb34T and C170 were isolated from the brine of the hypersaline lake 115 Aran-Bidgol (34°18'–34°45' N, 51°33' 52°10' E) in Iran. The isolation of 116 haloarchaea was performed by plating directly 100 µl of brine sample, as well 117 as using serial dilutions up to 10−6 from sediment sample on Hv-YPC solid 118 medium (Allers et al., 2004). The plates were incubated aerobically at 37 °C in 119 sealed plastic bags for up to 4 weeks. Representative colonies were evidenced 120 and transferred to the same medium, after successive streaking were obtained 121 6 two pure cultures designed as strains Cb34T and C170. They produced convex, 122 smooth, round and red-pigmented colonies (0.5-1.0 mm diameter) with an entire 123 edge. Routinely the strains were grown in M1 20 % medium (Rodriguez-Valera 124 et al., 1980), prepared using a salt mixture designated as SW 30 % (w/v) stock 125 solution (Subow, 1931) which consists of (per litre): 234 g NaCl, 39 g 126 MgCl2 . 6H2O, 61 g MgSO4 .7H2O, 1 g CaCl2, 6 g KCl, 0.2 g NaHCO3 and 0.7 g 127 NaBr. This solution was supplemented with 0.2 % (w/v) yeast extract, 0.1 % 128 (w/v) casaminoacids and 0.005 % (w/v) pyruvic acid sodium salt. The pH of 129 media was adjusted to 7.0–7.2. For solid media 2.0 % (w/v) agar was used 130 when necessary. The maintenance of strains were on the same medium in slant 131 tubes, for long term preservation were prepared cryotubes for freezing at -80 ºC 132 in suspensions as follows: to 750 µl of fresh culture (OD600 0.8-1.0) 250 µl of 133 glycerol/SW 30 (80:20, v/v) was added (Dyall-Smith, 2009). 134 Cell morphology and motility was examined in liquid medium after seven days 135 of growth by optical and phase-contrast microscopy (BX41; Olympus). Gram 136 staining was performed using acetic acid-fixed samples, as described by 137 Dussault (1955). Cells of strains Cb34T and C170 were observed as rod-shaped 138 and flattened pleomorphic cells (Supplementary Fig. S1), motile and stained 139 Gram-variable. 140 The growth and optimum requirements for NaCl, Mg2+, pH and temperature 141 were determined in the routine medium M1 changing the recipe for testing 142 growth at different concentrations. The range of NaCl (5–30 %, w/v) was tested 143 at intervals of 5 units. Strains Cb34T and C170 grow between 20-30 % (w/v) of 144 total salts (optimum 25 %, w/v). The cells of both isolates are lysed in distilled 145 water as well as in hypotonic treatment with less than 10 % (w/v) NaCl. 146 7 Magnesium range was tested using MgCl2 (0-10 %, w/v) at intervals of 1 % 147 (w/v). Routine cultivation was performed at 37 °C and pH 7.2. Strains Cb34T 148 and C170 have not a minimal magnesium ion requirement, but exhibited a poor 149 growth in the absence of Mg2+. Over 0.3 M MgCl2 occurs a low or no growing. 150 The pH range of 5.5–10.0 was assayed at intervals of 0.5 units in liquid medium 151 M1 with various pH buffers: MES (pH 5.5-6.0), PIPES (pH 6.5–7.0), Tricine (pH 152 7.5–8.5), CHES (pH 9.0–9.5) or CAPS (pH 10.0) at a concentration of 50 mM to 153 the isolation medium. Strains Cb34T and C170 grow between pH 6.0-8.0 and 154 the optimum is 7.0. The optimum temperatures were determined incubating at 4 155 ºC, 10 ºC, 20 ºC, 30 ºC, 37 ºC and 45 ºC in M1 medium with optimal NaCl and 156 Mg2+ concentrations and pH. The growth was evidenced from 20 ºC to 45 ºC 157 and the optimum is 37 ºC in aerobic conditions. 158 All phenotypic tests were carried out using the routine medium M1 prepared at 159 20 % (w/v) total salts, pH 7.0 and at 37 ºC. The following type strains of 160 Halorubrum were used as reference for comparative purposes for subsequent 161 testing: Halorubrum cibi JCM 15757T, Halorubrum aquaticum EN-2T, 162 Halorubrum kocurii CECT 7322T, Halorubrum alkaliphilum JCM 121358T, 163 Halorubrum tibetense JCM 11889T, Halorubrum lipolyticum JCM 13559T and 164 Halorubrum saccharovorum JCM 8865T. 165 Anaerobic growth was tested in the presence of nitrate and L-arginine by adding 166 to the medium 3 % KNO3 or 4% L-arginine, respectively in filled stoppered 167 tubes, as well the plates of cultures incubated for 10 days at 37 ºC in an 168 anaerobic jar (Oren et al., 1997). Strains Cb34T and C170 were unable to use 169 nitrate (with or without L-arginine) or DMSO as alternative electron acceptors 170 under anaerobic conditions. Catalase activity was determined by adding a 1 % 171 8 (w/v) H2O2 solution to colonies on solid medium. The oxidase test was 172 performed using a DrySlide assay (Difco). Both strains were oxidase and 173 catalase negative. The hydrolysis of starch, gelatin, esculin, casein and Tween 174 80 were carried out as described by Barrow & Feltham (2003). Test for indole 175 production from tryptophan and urea, were performed as described by Gerhardt 176 (1994). The methyl red, Voges-Proskauer test and Simmons citrate were 177 performed as described by Oren et al. (1997). H2S formation was determined by 178 monitoring the production of a black sulfide precipitate in solid M1 medium 179 containing 0.5 % (w/v) sodium thiosulfate. The reduction of nitrate was detected 180 by using sulfanilic acid and α-naphthylamine reagents (Smibert & Krieg, 1981). 181 To determine the utilization of different organic substrates such as 182 carbohydrates, alcohols, amino acids and organic acids as the only source of 183 carbon, nitrogen and energy, a medium containing 0.05 % (w/v) yeast extract 184 and supplemented with 1 % (w/v) of the tested substrate (sterilized separately) 185 was assessed as described by Ventosa et al. (1982). The production of acid 186 from different carbohydrates was tested in a medium with 0.5 % (w/v) yeast 187 extract and supplemented with 1 % (w/v) of the carbohydrate tested (Oren et 188 al., 1997). Hydrolysis of different compounds and utilization of several 189 substrates is shown in the species description. Strains Cb34T and C170 share 190 similar growth conditions and metabolic requirements; there are not significant 191 phenotypic differences between them. Additionally, there were no discrepancies 192 in phenotypic tests determined in our laboratory and those reported for the 193 closely related species. The differential features of both strains Cb34T and C170 194 from other closely related species are shown in Table 1. 195 15 species is in agreement with the genotypic data and provides further evidence 344 indicating that these strains may represent a different species. 345 The polyphasic approach including phylogenetic analyses using 16S rRNA and 346 rpoB’ gene sequence comparisons, polar lipid profiles, DDH and detailed 347 phenotypic characterization confirm that both strains represent a novel species 348 of the genus Halorubrum, for which the name Halorubrum halodurans sp. nov. 349 is proposed. 350 351 Description of Halorubrum halodurans sp. nov. 352 Halorubrum halodurans (ha. lo. du’rans. Gr. n. hals halos, salt; L. part. adj. 353 durans, enduring; N.L. part. adj. halodurans, salt-enduring, resisting). 354 Cells are motile, pleomorphic rods, with a size of 5.0 -10 x 1.0-4.0 µm and 355 display separately without grouping. Gram-stain-variable, in young cultures 356 most cells are Gram-stain-negative while few cells are observed as Gram-stain357 positive. Gas vesicles inside the cells are not observed. Colonies on solid 358 medium after incubation at 37 ºC for 10 days are circular, with regular edges, 359 smooth, convex and 0.5–1 mm in diameter. During the first five days, colonies 360 appear pink and gradually the color intensifies to red upon incubation for next 361 days. The cells are lysed in distilled water; hypotonic treatment with less than 362 10 % (w/v) NaCl induces cell lysis. The strains had no minimal magnesium ion 363 requirement, but poor growth was observed in the absence of Mg2+ ions, and 364 low to no growth above 0.3 M MgCl2 was observed. 0.3 M Mg2+ is required for 365 growth. The species is neutrophilic, growing at pH 6.0–8.0 with an optimum pH 366 between 7.0–7.2. Requirements for growth on NaCl are 20–30 % (w/v) with an 367 optimum of 25 %, (w/v), and for temperature it needs 20–45 ºC (optimum, 37 368 16 ºC). Chemoorganotrophic and strictly aerobic. Oxidase and catalase activities 369 are positive. Anaerobic growth does not occur with nitrate or L-arginine. 370 Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase are not 371 produced. H2S is produced from sodium thiosulfate or cysteine; indole is not 372 produced from tryptophan. Nitrate is reduced to nitrite, but nitrite is not reduced 373 further and no gas is formed. Methyl red and Voges-Proskauer tests are 374 negative, and citrate is not utilized. Extracellular hydrolysis of Tween 80, starch, 375 DNA, gelatin and casein was not observed. Tests for urease and phosphatase 376 activities are negative. The following substrates are utilized for growth as sole 377 source of carbon and energy: D-glucose, maltose, sucrose, D-mannose, 378 trehalose, glycerol, D-mannitol, acetate, glutamate, lactate, malate, pyruvate, 379 succinate and propionate. D-galactose, D-fructose, Dribose, Dxylose, 380 lactose, sorbose, sorbitol, raffinose, L-arabinose, rhamnose, fumarate and 381 citrate are not used as sole source of carbon and energy. The following amino 382 acids are used as sole source of carbon, nitrogen and energy: L-serine, 383 threonine, glycine, asparagine and L-lysine. Isoleucine, alanine, arginine and 384 ornithine are not used as sole source of carbon, nitrogen and energy. 385 The polar lipid pattern consists of phosphatidylglycerol (PG), 386 phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol 387 sulfate (PGS) and sulfated mannosyl glucosyl diether (S-DGD) as major lipids. 388 Bi-phosphatidyl glycerol (BPG) and minor phospholipidic components are 389 weakly present. The DNA G+C content ranges from 62.1 to 62.4 mol% (Tm). 390 The type strain is Cb34T (= CECT 8745T = IBRC-M 10233T), isolated from 391 sediment of the hypersaline lake Aran-Bidgol in Iran. The DNA G+C content of 392 the type strain is 62.1 mol% (Tm). 393 17 394 Acknowledgements 395 This work was supported by grants from Spanish Ministry of Economy and 396 Competitiveness (CGL2013-46941-P) and from the Andalusian Council (P10397 CVI-6226), both with European Funds (FEDER) (to A. Ventosa), the National 398 Science Foundation (award numbers DEB0919290 and DEB0830024 ) and 399 NASA Astrobiology: Exobiology and Evolutionary Biology Program (Grant 400 Number NNX12AD70G) (to R. T. Papke) and the Iranian Biological Resource 401 Centre (IBRC) (MI-1388-04) (to M. A. Amoozegar). 402 403 404 18 References 405 Akaike, H. (1974). A new look at the statistical model identification. IEEE T 406 Automat Contr 19, 716-723. 407 Allers, T., Ngo, H. P., Mevarech, M. & Lloyd, R. G. (2004). Development of 408 additional selectable markers for the halophilic archaeon Haloferax volcanii 409 based on the leuB and trpA genes. Appl Environ Microbiol 70, 943–953. 410 Amoozegar, M. A., Makhdoumi-Kakhki, A., Mehrshad, M., Rasooli, M., 411 Shahzadeh Fazeli, S. A., Spröer, C. & Ventosa, A. (2014a). Halovivax cerinus 412 sp. nov., an extremely halophilic archaeon from a hypersaline lake. Int J Syst 413 Evol Microbiol (in press). 414 Amoozegar, M. 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Prokaryotic Diversity in Aran-Bidgol Salt Lake, the largest 544 hypersaline playa in Iran. Microbes Environ 27, 87-93. 545 Makhdoumi-Kakhki, A., Amoozegar, M. A., Bagheri, M., Ramezani, M. & 546 Ventosa, A. (2012b). Haloarchaeobius iranensis gen. nov., sp. nov., an 547 extremely halophilic archaeon isolated from the saline lake Aran-Bidgol, Iran. Int 548 J Syst Evol Microbiol 62, 1021-1026. 549 Makhdoumi-Kakhki, A., Amoozegar, M. A. & Ventosa, A. (2012c). Halovenus 550 aranensis gen. nov., sp. nov., a novel extremely halophilic archaeon from Aran551 Bidgol salt lake, Iran. Int J Syst Evol Microbiol 62, 1331-1336. 552 Mancinelli, R. L., Landheim, R., Sánchez-Porro, C., Dornmayr553 Pfaffenhuemer, M., Gruber, C., Legat, A., Ventosa, A., Radax, C., Ihara, K. 554 31 Figure legends 656 657 Fig. 1. Neighbour-joining (NJ) phylogenetic tree based on the 16S rRNA gene 658 sequence comparison, showing the relationships between Halorubrum 659 halodurans sp. nov. (strains Cb34T and C170) and members of the genus 660 Halorubrum and other related haloarchaea. The sequence data used were 661 obtained from GenBank database (accession numbers are given in 662 parentheses). Bootstrap values (%) are based on 1000 replicates and are 663 shown for branches with more than 70 % bootstrap support. Filled circles 664 indicate that the corresponding nodes were also obtained in the trees generated 665 with the maximum-parsimony (MP) and maximum-likelihood (ML) algorithms. 666 Bar, 2 % substitution per nucleotide position. 667 668 Fig. 2. Maximum-likelihood tree based on rpoB’ gene sequence showing the 669 phylogenetic relationship between members of the genus Halorubrum and 670 Halorubrum halodurans sp. nov. (strains Cb34T and C170) isolated from the 671 lake Aran-Bidgol. Bootstrap values >70 % are indicated. The species 672 Halogeometricum borinquense DSM 11551T and Haloferax volvanii NCIMB 673 2287T were used as outgroup. The scale bar represents 5 % substitutions per 674 nucleotide position. 675 94 99 100 96 72 100 72 96 86 Halorubrum trapanicum JCM 10477T (AB663424) Strain C170 (KT825566) Halorubrum californiense SF3-213T (EF139654) Halorubrum ezzemoulense 5.1T (DQ118426) Halorubrum chaoviator HALO-G*T(AM048786) Halorubrum distributum JCM 9100T (D63572) Halorubrum litoreum Fa-1T (EF028067) Halorubrum coriense Ch2T(L00922) Halorubrum xinjiangense BD-1T (AY510707) Halorubrum sodomense ATCC 33755T(X82169) Halorubrum tebenquichense CECT 5317T(AJ276887) Halorubrum ejinorense EJ-32T (AM491839) Halorubrum terrestre VKM-B 1739T (AB090169) Halorubrum arcis AJ201T(DQ355793) Halorubrum salinum GX71T(HM063951) Halorubrum saccharovorum NCIMB 208T(X82167) Halorubrum halophilum B8T (HM063951) Halorubrum aidingense 31-hongT(DQ355813) Halorubrum kocurii BG-1T (AM900832) Halorubrum lacusprofundi ACAM 34T (X82170) Halorubrum lipolyticum 9-3T (DQ35581) Halorubrum orientale EJ -52T(AM235786) Halorubrum alkaliphilum DZ1T (AY510708) Halorubrum tibetense 8W8T (AY149598) Halorubrum aquaticum EN2T (AM268115) Halorubrum rubrum YC87T (JQ237124) Halorubrum cibi B31T (EF077639) Halorubrum vacuolatum JCM 9060T(D87972) Halobacterium salinarum DSM 3754T (AJ496185) Strain Cb34T(HG421007) Halorubrum persicum C49T(HG421000) 0.02 Figure 1 Click here to download Figure Figure 1 tree (16S rRNA) revised.pptx Halorubrum chaoviator DSM 19316T(AB820293) Halorubrum ezzemoulense CECT 7099T(AB820292) Halorubrum coriense JCM 9275T (AB477179) Halorubrum distributum JCM 9100T (AB477180) Halorubrum terrestre JCM 10247T (AB477187) Halorubrum litoreum JCM 13561T (AB477183) Halorubrum arcis JCM 13916T (AB477178) Halorubrum californiense JCM 14715T (AB820294) Halorubrum xinjiangense JCM 12388T (AB477191) Halorubrum ejinorense JCM 14265T (AB820295) Halorubrum sodomense JCM 8880T (AB477185) Halorubrum trapanicum JCM 10477T (AB477189) Halorubrum tebenquichense JCM 12290T (AB477186) Halorubrum luteum CECT 7303T(AB820300) Halorubrum vacuolatum JCM 9060T (AB477190) Halorubrum alkaliphilum JCM 12358T (AB477177) Halorubrum tibetense JCM 11889T (AB477188) Strain Cb34T(KT935295) Strain C170 (KJ152359) Halorubrum cibi JCM 15757T (AB820297) Halorubrum aquaticum JCM 14031T (AB820296) Halorubrum lipolyticum JCM 13559T (AB477182) Halorubrum lacusprofundi JCM 8891T (AB477181) Halorubrum persicum C49T (KJ152361) Halorubrum halophilum B8T (KF700332) Halorubrum saccharovorum JCM 8865T (AB477184) Halorubrum kocurii JCM 14978T (AB820299) Halorubrum yunnanense Q85T(KR813313) Halorubrum aidingense JCM 13560T (AB477176) Halorubrum salinum GX71T(KF680550) Halorubrum gandharaense MK13-1T(AB820320) Haloferax volcanii JCM 8879T(AB477170) Halogeometricum borinquense JCM 10706T(AB477171) 100 100 93 100 93 73 95 100 84 85 72 92 0.1 100 100 Halorubrum orientale CECT 7145T(AB820298) rpoB’ 1830 bp Figure 2 Click here to download Figure Figure 2 (rpoB) revised.ppt Supplementary Material International Journal of Systematic and Evolutionary Microbiology Halorubrum halodurans sp. nov., an extremely halophilic archaeon isolated from sediment of a hypersaline lake Paulina Corral1, Rafael R. de la Haba1, Cristina Sánchez-Porro1, Mohammad Ali Amoozegar2, Thane R. Papke3 and Antonio Ventosa1 1Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain. 2Department of Microbiology, Faculty of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran, Iran. 3Department of Molecular and Cell Biology, University of Connecticut, 06269 Storrs, CT, USA. Supplementary Material FilesClick here to download Supplementary Material Files Supplementary Material Hrr. halodurans Cb34.pdf Supplementary Fig. S1. Phase-contrast photomicrograph of cells of strain Cb34T cultured in liquid medium under optimal conditions. Scale bar, 10 μm. Supplementary Fig. S2. High performance thin layer chromatography (HPTLC) of polar lipids extracted from Halorubrum halodurans sp. nov. (strains Cb34T and C170 in green box) and some other haloarchaeal species. The plate was developed with sulfuric acid 5 % in water, and charred by heating at 160 ºC. Lanes: 1, Halobacterium salinarum DSM 3754T; 2, Halorubrum cibi JCM 15757T; 3, Halorubrum aquatiucm EN2T; 4, strain Cb34T; 5, strain C170; 6, Halorubrum tibetense JCM 11889T. Abbreviations: BPG, biphosphatidylglycerol; PG, phosphatidylglycerol; PGPMe, phosphatidylglycerol phosphate methyl ester; PGS, phosphatidylglycerol sulfate; S-DGD, sulfated mannosyl, glucosyl diether; S-TGD-1-PA, glycocardiolipin (sulfated triglycosyl diphytanyl archaeol ester linked to phosphatidic acid); S-TGD-1, sulfated triglycosyl diphytanyl archaeol.