Soortia roseihalophila gen. nov., sp. nov., a new taxon in the order Balneolales isolated from a travertine spring, and description of Soortiaceae fam. nov.

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 2017, available at: https://doi.org/10.1099/ijsem.0.001583 .” 1 Soortia roseihalophila gen. nov., sp. nov., a new taxon isolated from a travertine spring in 1 the order Balneolales, and description of Soortiaceae fam. nov. 2 3 Mohammad Ali Amoozegar,1 Javad Khansha,2 Maliheh Mehrshad,1,2 Seyed Abolhassan 4 Shahzadeh Fazeli,2,3 Mohaddaseh Ramezani,2 Rafael R. de la Haba,4 Cristina Sánchez-Porro,4 5 and Antonio Ventosa 4 6 7 1Extremophiles Laboratory, Department of Microbiology, Faculty of Biology and Center of 8 Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, 9 Tehran, Iran 10 2Microorganisms Bank, Iranian Biological Resource Centre (IBRC), ACECR Tehran, Iran 11 3Department of Molecular and Cellular Biology, Faculty of Basic Sciences and Advanced 12 Technologies in biology, University of Science and Culture, Tehran, Iran 13 4Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 14 41012 Sevilla, Spain 15 16 Running title: Soortia roseihalophila gen. nov., sp. nov. 17 Subject category: New taxa, Bacteroidetes 18 Author for correspondence: M. A. Amoozegar, Extremophiles Laboratory, Dept. of 19 Microbiology, Faculty of Biology, College of Science, University of Tehran, Tehran, Iran; P. 20 O. Box 14155-6455; Phone: +98-21-61113557; Fax: +98-21-66492992; E. mail: 21 [email protected].ir 22 23 Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document) Sortia roseahalophila article-revision all final 2 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Bsw24 2bT is KU695464. 25 26 A novel Gram-stain-negative, slightly halophilic motile curved rod with horseshoe shape, 27 designated strain Bsw-2bT, was isolated from Badab-Soort travertine spring in Iran. 28 Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain Bsw-2bT 29 belongs to the order Balneolales, showing 84.6% sequence similarity to Gracilimonas 30 tropica DSM 19535T and 84.4 % and 83.9 % sequence similarity to Gracilimonas rosea 31 CL-KR2T and Balneola vulgaris DSM 17893T, respectively. Besides, phenotypic and 32 physiological features could clearly differentiate strain Bsw-2bT from species of the most 33 closely related genera Gracilimonas, Balneola, Aliifodinibius and Fodinibius. The strain 34 was able to grow at 1–3 % (w/v) (optimum at 2 %) NaCl, at temperatures of 28–34 ℃ 35 (optimum at 30 ℃) and between pH 6.0 and 8.0 (optimum at pH 7.0). The major cellular 36 fatty acids of strain Bsw-2bT were iso-C15:0, iso-C13:0 and iso-C14:0. The polar lipid profile 37 of strain Bsw-2bT was composed predominantly of phosphatidylethanolamine, 38 diphosphatidylglycerol, phosphatidylglycerol, an unknown glycolipid and four unknown 39 phospholipids. The DNA G+C content was 40.5 mol %. Based on the polyphasic evidence, 40 strain Bsw-2bT represents a novel species in a novel genus within a new family, for which 41 the name Soortia roseihalophila gen. nov., sp. nov. is proposed, within the new family, 42 Soortiaceae fam. nov. The type strain is strain Bsw-2bT (= IBRC-M 10915T = LMG 43 28547T). 44 45 46 47 3 The order Balneolales belongs to the class Balneolia, phylum Rhodothermaeota and, at the 48 time of writing the order includes four genera: Aliifodinibius, Balneola, Fodinibius, and 49 Gracilimonas which all belong to the family Balneolaceae (Munoz et al., 2016). So far all 50 representatives of the genera Aliifodinibius and Fodinibius have been isolated from hypersaline 51 environments and are considered to be moderately halophilic bacteria (Wang et al., 2012; 2013; 52 Xia et al., 2016); however, members of genera Balneola and Gracilimonas mostly belong to 53 the marine environments (Urios et al., 2006; Urios et al., 2008; 2009; Cho et al., 2013). The 54 order Balneolales accommodates Gram-staining-negative non-endospore-forming rod or 55 curved rod shaped bacteria, pigmented, chemoheterotrophic, catalase positive, aerobic, non56 motile or motile by flagella, which have DNA G+C content between 39 and 49 mol % (Munoz 57 et al., 2016). 58 Badab-Soort spring is one of the most beautiful travertine springs in the world and is located 59 on N 36° 21' 30.5" E 53° 51' 38.0" in North of Iran and at an altitude of 1840 m. This spring 60 has iron minerals, which causes red, pink, white and gray colors to spring's structures that could 61 be radial or laminated (Supplementary figure S1). This spring has some springheads with 62 different physical and chemical features that are very close together, springheads based on 63 water source, type of origin's underground layer and upper layers have unique features. 64 Geological maps of this area report some faults like other travertines (Pentecost, 2005) which 65 allow discharge of the water through these faults. Precipitated carbonate deposits are observed. 66 During a study of the diversity of prokaryotic microorganisms in Badab-Soort spring, a 67 collection of bacterial strains was isolated. One of them was a slightly halophilic bacterium 68 which was believed to represent a novel bacterial taxon. The aim of the present work was to 69 determine the exact taxonomic position of this strain by using a polyphasic taxonomic 70 characterization that combines phenotypic, including chemotaxonomic, phylogenetic and 71 genotypic analyses. 72 4 The strain Bsw-2bT was isolated by plating the water samples taken from Badab-Soort spring 73 on modified nutrient agar (MNA) medium which contains 2.5 % (w/v) total salt (g l-1): NaCl, 74 20.25; peptone, 5.0; yeast extract, 2.0; meat extract, 1.0; KCl, 0.5; MgSO4.7H2O, 2.4; 75 CaCl2.2H2O, 0.007; MgCl2.6H2O, 1.6; NaHCO3, 0.0045 and agar, 15.0; distilled water, 1000 76 ml. The pH was adjusted to 7.3 with NaOH 1 N and the inoculated plates were incubated at 30 77 °C for two weeks. The strain was subsequently purified three times by plating on the same 78 medium. The strain was maintained on MNA medium and also at -80 °C in MNA medium 79 without agar and supplemented with 30 % (v/v) glycerol. 80 In order to phenotypically characterize strain Bsw-2bT, standard phenotypic tests were selected 81 according to the recommendations of the notes on the characterization of prokaryote strains for 82 taxonomic purposes (Tindall et al., 2010). For comparison in our study Gracilimonas tropica 83 DSM 19535T and Balneola vulgaris DSM 17893T were used as reference strains. They were 84 cultured following the recommendations of the culture collection. 85 Cell morphology was examined with an Olympus BX51 microscope equipped with phase86 contrast optics and a KYKY model 3200 scanning electron microscope using cells from 87 exponentially growing cultures. Gram staining was performed by the Burke method (Murray 88 et al., 1994). The presence of endospores was investigated by using the Schaeffer-Fulton 89 staining method (Murray et al., 1994). Production of flexirubin pigments was investigated by 90 using bathochromatic shift test with KOH 20 % (Bernardet et al., 2005). Motility was analyzed 91 by the wet-mount method (Murray et al., 1994). Catalase and oxidase activities, nitrate 92 reduction, hydrolysis of aesculin, casein, gelatin, starch, production of indole and Voges– 93 Proskauer tests were carried out as recommended by Smibert & Krieg (1994), using media with 94 2 % NaCl. Hydrolysis of Tween 20, 40 or 80 was examined as described by Harrigan & 95 McCance (1976) on media with 2 % NaCl. The ability of strain Bsw-2bT to grow anaerobically 96 was tested in MNA medium with 2 % NaCl in a GasPack anaerobic container system. 97 5 The miniaturized identification systems API ZYM and API 20E kits (bioMerieux) were used 98 for examination of enzymatic activities and acid production from carbohydrates, respectively. 99 Utilization of carbon sources was performed as recommended by Ventosa et al. (1982). 100 Antimicrobial susceptibility tests were performed on Mueller–Hinton agar plus 2 % (w/v) sea 101 salts (Ventosa et al., 1982) seeded with a bacterial suspension containing 1.5×106 c.f.u. ml-1 102 using discs (HiMedia) impregnated with various antimicrobial compounds. The plates were 103 incubated at 30 °C for 48 h and the inhibition zone was interpreted according to the 104 manufacturer’s manual. 105 To determine the range and optimal temperature for growth of the strain, modified nutrient 106 broth with 2 % NaCl was incubated at 4, 10, 15, 20, 28, 30, 34 and 40 ℃ for 2 weeks. For 107 growth experiments at different pH values a range of 5.0–9.0 at intervals of 0.5 pH units was 108 tested; the buffers MES (pH 5-6.5), HEPES (pH 7-8) and CHES (pH 8.5-9) were added at a 109 concentration of 50 mM. Growth at different NaCl concentrations (0, 1, 2, 3, 5, 7 and 10 %, 110 w/v) was tested on modified nutrient broth at pH 7.0. Growth was monitored by turbidity at 111 OD600 using a spectroscopic method (model UV-160 A; Shimadzu). Other physiological and 112 biochemical tests were performed as described previously (Mata et al., 2002; Quesada et al., 113 1984; Ventosa et al., 1982). 114 Strain Bsw-2bT was Gram-stain-negative, strictly aerobic, curved and motile rod; horseshoe 115 shape, spirillum, coccoid and crescentus forms were also observed (Fig. 1). Colonies formed 116 on agar plates were small (about 0.5 mm diameter), smooth, translucent, circular, convex, 117 round, with an entire edge and pale red-pigmented. The organism produced flexirubin. The 118 strain was a slightly halophilic bacterium, growing in media containing 1.0-3.0 % (w/v) NaCl 119 and optimally in media containing 2.0 % (w/v) NaCl. No growth was observed in the absence 120 of NaCl. Strain Bsw-2bT grew at a temperature range of 28-34 °C (optimum temperature at 30 121 °C), pH 6-8 (optimum pH at 7.0). This isolate was sensitive to chloramphenicol (30 µg), 122 6 tetracycline (30 µg), amikacin (30 µg), rifampicin (5 µg), cephoxitin (30 μg), polymixin B (300 123 U) erythromycin (5 µg), gentamycin (10 µg), and bacitracin (10 µg), but resistant to penicillin 124 G (10 U), ampicillin (10 µg), nalidixic acid (30 µg), tobramycine (10 µg) and kanamycin (5 125 µg). The detailed physiological and biochemical characteristics of strain Bsw-2bT are listed in 126 Table 1 and in the species description. 127 Genomic DNA of strain Bsw-2bT was extracted using the method described by Marmur (1961). 128 The 16S rRNA gene was amplified using the bacterial universal primers 27F and 1492R 129 (Mehrshad et al., 2013). PCR products were purified with the DNA purification kit (Roche, 130 Germany), according to the manufacturer’s protocol. The purified PCR products were then 131 electrophoresed on a 1 % agarose gel to check their quality. Ligation of the PCR products with 132 the pGEM-T vector, transformation of Escherichia coli DH5α, and selection of the 133 transformants were carried out with pGEM-T TA cloning kit (Promega, USA), used according 134 to the manufacturer’s protocol. Clones were randomly picked and then sequenced by the 135 service of Macrogen Company, South Korea. 136 The 16S rRNA gene sequence was aligned with the published sequences of the type species of 137 the phyla Bacteroidetes and Rhodothermaeota, and the alignment was confirmed and checked 138 against both primary and secondary structures of the 16S rRNA molecule using the alignment 139 tool of the ARB software package (Ludwig et al., 2004). A specific ARB database 140 for Bacteroidetes and Rhodothermaeota (including 1155 species) was used (Munoz et al., 141 2016). Phylogenetic trees were constructed using neighbour-joining (Saitou & Nei, 1987) and 142 maximum-likelihood (Felsenstein, 1981) algorithms integrated in the ARB software using a 40 143 % maximum frequency conservational filter. The stability of the topology of the neighbour144 joining phylogenetic tree was determined using the bootstrap method with 1000 repetitions 145 (Felsenstein, 1985). Due to the computational cost the bootstrap analysis for the maximum146 likelihood tree could not be calculated. 147 7 An almost-complete 16S rRNA gene sequence (1516 bp) of strain Bsw-2bT was obtained and 148 used for BLAST searches in GenBank and phylogenetic analysis. The identification of 149 phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarity were 150 achieved using the EzTaxon-e server (Kim et al., 2012). The 16S rRNA gene sequence analysis 151 showed that strain Bsw-2bT is a member of the order Balneolales but there were very low 152 similarities to type strains of other members of this order. The closest relative of strain Bsw153 2bT was Gracilimonas tropica DSM 19535T, with a sequence similarity of 84.6 %. Other strains 154 most closely related to strain Bsw-2bT were Gracilimonas rosea CL-KR2T (84.4 %), Balneola 155 vulgaris DSM 17893T (83.9 %), Balneola alkaliphila CM41-14bT (83.8 %), Gracilimonas 156 mengyeensis YIM J14T (83.5 %), Aliifodinibius roseus YIM D15T (83.2 %), Fodinibius salinus 157 YIM D17T (83 %), Aliifodinibius sediminis YIM J21T (82.9 %), and Rubricoccus marinus SG158 29T (80.8 %). The level of 16S rRNA gene sequence similarity between strain Bsw-2bT and 159 other taxa was lower than 80.0 %. 160 Phylogenetic analysis using the neighbour-joining algorithm revealed that strain Bsw-2bT 161 represents a separate lineage (Fig. 2). The phylogenetic position was also confirmed in the tree 162 obtained using the maximum-likelihood algorithm (Fig. 2). Based on the sequence divergence, 163 it was evident that strain Bsw-2bT clustered as a separate clade within the order Balneolales, 164 showing very low 16S rRNA gene sequence similarity with the taxa of this order previously 165 described. 166 Cell biomass for fatty acid analysis was obtained by cultivation on MNA medium with 2 % 167 NaCl at pH 7.0 and 30 °C. Cells were harvested in the mid-exponential growth phase. The 168 whole-cell fatty acid composition of strain Bsw-2bT was determined according to the standard 169 protocol of the Microbial Identification System (MIDI, Version 6.1; Identification Library 170 TSBA40 4.1; Microbial ID). Extracts were analyzed using a Hewlett Packard model HP6890A 171 gas chromatograph equipped with a flame-ionization detector as described by Kämpfer and 172 8 Kroppenstedt (1996). Fatty acid peaks were identified using the TSBA5.0 database. The 173 cellular fatty acid profile of strain Bsw-2bT was characterized by the fatty acids iso-C15:0 (28.2 174 %), iso-C13:0 (17.4 %), anteiso-C17:0 (16.1 %), iso-C14:0 (13.5 %), summed feature 3 (comprising 175 iso-C15: 2OH and/or C16:1 ω7c) (11.4 %), as the major fatty acids. The fatty acids profile was 176 different than those of the related type species of the genera Gracilimonas and Balneola. The 177 new isolate could be distinguished by the absence of C17:1 ω8с, C15:1 ω6с, C15:0, iso-C17:0 and 178 C16:1 ω5с in contrast to species of the genera Gracilimonas and Balneola. Besides, the 179 percentages of several other fatty acids were different (Table 2). 180 Cell biomass for isoprenoid quinone and polar lipid analyses was obtained by cultivation on 181 MNA medium with 2 % NaCl at pH 7.0 and 30 °C. For polar lipid analysis strain Bsw-2bT and 182 Gracilimonas tropica DSM 19535T were cultivated under identical conditions. Polar lipids 183 were analyzed as described by Groth et al. (1996). Isoprenoid quinone analysis was carried out 184 as described by Monciardini et al. (2003). Strain Bsw-2bT contained MK-7 as the only 185 isoprenoid quinone which was similar to what reported for the related genera: Gracilimonas, 186 Aliifodinibius and Fodinibius, which contain MK-7 as the only or major isoprenoid quinone 187 (Cho et al., 2013; Wang et al., 2012, 2013). The polar lipids of strain Bsw-2bT were 188 phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), an 189 unknown glycolipid and four unknown lipids (Fig. 3). The polar lipid profile is similar to that 190 obtained for Gracilimonas tropica DSM 19535T but there were differences on the number of 191 unknown lipids and the absence of phospholipid in strain Bsw-2bT. 192 For determination of DNA base composition, cells were disrupted by using a Constant Systems 193 TS 0.75 KW (IUL Instruments, Germany) and the DNA in the crude lysate was purified by 194 chromatography on hydroxyapatite as described by Cashion et al. (1977). The DNA G+C 195 content was determined by reversed-phase HPLC of nucleosides according to the protocol of 196 Mesbah et al. (1989). The G+C content of the DNA of strain Bsw-2bT was 40.5 mol %. This 197 15 Tindal, B.J., Rosselló-Móra, R., Busse, H.J., Ludwig, W. & Kämpfer, P. (2010). Notes on 339 the characterization of prokaryote strains for taxonomic purposes. Int. J. Syst. Evol. 340 Microbiol. 60: 249–266. 341 Urios, L., Agogué, H., Lesongeur, F., Stackebrandt, E., Lebaron, P. (2006). Balneola 342 vulgaris gen. nov., sp. nov., a member of the phylum Bacteroidetes from the north-western 343 Mediterranean Sea. Int. J. Syst. Evol. Microbiol. 56, 1883-7. 344 Urios, L., Intertaglia, L., Lesongeur, F. & Lebaron, P. (2008). Balneola alkaliphila sp. 345 nov., a marine bacterium isolated from the Mediterranean Sea. Int. J. Syst. Evol. Microbiol. 346 58, 1288-1291. 347 Ventosa, A., Quesada, E., Rodriguez-Valera, F., Ruiz-Berraquero, F. & Ramos348 Cormenzana, A. (1982). Numerical taxonomy of moderately halophilic Gram-negative rods. 349 J Gen Microbiol 128, 1959–1968. 350 Wang, Y.-X., Liu, J.-H., Xiao, W., Ma,X.-L., Lai, Y.-H., Li, Z.-Y., Ji, K.-Y., Wen, M.-L. 351 & Cui, X.-L. (2013). Aliifodinibius roseus gen. nov., sp. nov., and Aliifodinibius sediminis 352 sp. nov., two moderately halophilic bacteria isolated from salt mine samples. Int J Syst Evol 353 Microbiol 63, 2907-2913. 354 Wang, YX., Liu, JH., Xiao, W., Ma, XL., Lai, YH., Li, ZY., Ji, KY., Wen, ML., Cui, XL. 355 (2013). Aliifodinibius roseus gen. nov., sp. nov., and Aliifodinibius sediminis sp. nov., two 356 moderately halophilic bacteria isolated from salt mine samples. Int. J. Syst. Evol. Microbiol. 357 63, 2907-13. 358 Wang, Y.-X., Liu, J.-H., Xiao, W., Zhang, X.-X., Li, Y.-Q., Lai, Y.-H., Ji, K.-Y., Wen, 359 M.-L. & Cui, X.-L. (2012). Fodinibius salinus gen. nov., sp. nov., a moderately halophilic 360 bacterium isolated from a salt mine. Int. J. Syst. Evol. Microbiol. 62, 390-396. 361 16 Xia, J., Ling, SK., Wang, XQ., Chen, GJ., Du, ZJ. (2016). Aliifodinibius halophilus sp. nov., 362 a moderately halophilic member of the genus Aliifodinibius, and proposal of Balneolaceae fam. 363 nov. Int. J. Syst. Evol. Microbiol. 66, 2225-33. 364 365 17 Table 1. Differential characteristics between strain Bsw-2bT and closely related species within 366 the order Balneolales. Strains: 1, Bsw-2bT (this study); 2, Gracilimonas tropica DSM 19535T 367 (this study); 3, Balneola vulgaris DSM 17893T (this study); 4, Aliifodinibius roseus YIM D15T 368 (Wang et al., 2013); 5, Fodinibius salinus YIM D17T (Wang et al., 2012). Symbols: +, positive; 369 -, negative. 370 Characteristic 1 2 3 4 5 Cell size (µm) 0.4×1.5-2.5 0.2-0.4×4.0-11.5 0.3×2.03.5 0.3×0.5-0.6 0.3×1.0-3.0 Cell shape Curved rods Long rods Rods Rods Curved rods Colony colour Orange Orange Orange Pinkred Pink Endospore like formation - + - - - Motility + - + - - Anaerobic growth - + - + - NaCl concentration for growth (%, w/v): Range 1-3 1.0-20 0-5.0 4.0-20 4.0-23 Optimum 2 3-5 2 6-10 10-15 Temperature for growth (°C): Range 28-34 20-40 10-40 20-42 25-45 Optimum 30 30 35 28 37 pH for growth: Range 6-8 6-10 5-10 6.5-8 6-9 Optimum 7 7-8 8 7 7.5-8 Oxidase - + - + + Nitrate reduction - - - - + Hydrolysis of: Aesculin + + - + + Tween 40 + + - - + Tween 80 + + - + + Gelatin + + - + + Starch + + - - - Utilization of: Acetate - - + + - D-Mannitol - - + - - D-Galactose + - - + - Enzyme activity: Esterase (C4) - + - + - Lipase (C14) - + - + + β-Glucosidase + + - + - β-Galactosidase + - + + - 18 a Data from Choi et al. (2009), b Data from Urios et al. (2006). 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 Polar lipids PE, PC, PG, DPG, PLs, PE, PG, DPG, PLs, GL PE, PG, DPG, GL, L PE, PC, DPG, GL, L, PL PE, PC, PG, DPG, GL, AL, PL, L Major cellular fatty acid iso-C15:0, iso-C13:0, anteiso-C17:0 and iso-C14:0 iso C15:0, C16:1 ω7с/ iso-C15:0 2OH, isoC17:1 ω9с iso-C15:0, iso-C15:0 2-OH, C17:1 ω8с anteisoC15:0, isoC15:0, isoC17:1 ω9с iso-C17:1 ω 9c/10-methyl-C16 : 0, iso-C15:0 , C16:1 ω 7c/C16:1 ω 6c DNA G+C content (mol%) 40.5 42.7a 41.8b 49.0 43.0 Isolation source Spring Seawater Seawater Salt mine Salt mine 19 Table 2. Cellular fatty acid compositions of strain Bsw-2bT, Gracilimonas tropica DSM 388 19535T, Balneola vulgaris DSM 17893T and Chitinophaga pinensis ACM 2034T. 389 Taxa : strains: 1, strain Bsw-2bT 2 ; Gracilimonas tropica DSM 19535T; 3, Balneola vulgaris 390 DSM17893T. 391 Cells of all strains were harvested after cultivation at 30 ºC on MNA medium with 2 % NaCl. 392 Values are percentages of total fatty acids; fatty acids accounting less than 1.0 % of the total 393 content in all strains are omitted. 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 Fatty acids 1 2 3 iso-C13:0 17.4 3.2 5.0 iso-C14:0 13.5 2.9 4.1 iso-C15:1 3.9 3.1 - iso-C15:0 28.2 40.5 25.1 anteiso-C15:0 1.2 6.2 3.2 C15:1 ω6с - 4.5 8.5 C15:0 - 3.6 8.2 iso-C16:0 2.2 1.0 2.3 C16:0 1.1 - 1.0 C16:1 ω5с - 1.1 1.0 C16:1 ω9с 1.1 - - iso-C17:0 - 2.0 1.2 anteiso-C17:0 16.1 1.0 1.4 C16:1 ω7с/ iso-C15:0 2OH 11.4 11.8 10.2 isoC17:1 ω9с 1.8 11.1 7.0 C17:1 ω8с - 4.2 13.2 20 Legends to figures 410 411 Fig. 1. Phase contrast microscopy image (a) and scanning electron micrograph of strain Bsw412 2bT grown on modified nutrient agar for 2 days at 30 ºC, (bar, 2 µm). 413 414 Fig. 2. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the 415 position of Soortia roseihalophila sp. nov. with respect to the existing species of the phyla 416 Bacteroidetes and Rhodothermaeota. Filled circles indicate branches found in phylogenetic 417 tree generated with the maximum-likelihood algorithm. The sequences of species of the 418 phylum Chlorobi were used as outgroup. Numbers at node are bootstrap values (%) based on 419 1000 replicates. Numbers within the collapsed clades correspond to the number of species 420 included in the respective clade. Bar, 0.02 substitutions per nucleotide position. 421 Fig. 3. Polar lipid profile of strain Bsw-2bT and Gracilomonas tropica DSM 19535T. The first 422 direction was developed in chloroform: methanol: water (65:25:4, v/v/v), and the second in 423 chloroform: methanol: acetic acid: water (80:12:15:4, v/v/v/v). Total lipid material and specific 424 functional groups were detected using molybdenum–blue reagent (phospholipids), 425 molybdophosphoric acid (total lipids), ninhydrin (free amino groups), Dragendorff (quaternary 426 nitrogen), and a-naphthol-sulphuric acid (glycolipids). DPG, diphosphatidulglycerol; PG, 427 phosphatidylglycerol; PE, phosphatidylethanolamine; GL, glycolipid; PL’ phospholipid; L1428 L4, lipids 429 430 431 432 433 21 Fig. 1 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 22 Fig. 2 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 23 Fig. 3. 475 476 477 478 479 Figure 1a Click here to download Figure Fig.1 a.jpg