1 almeriensis sp. nov., a moderately halophilic, 2 exopolysaccharide-producing bacterium from Cabo de Gata (Almería, 3 south-east Spain). 4 5 Fernando Martínez-Checa, Victoria Béjar, M. José Martínez-Cánovas, 6 Inmaculada Llamas and Emilia Quesada. 7 8 Microbial Exopolysaccharide Research Group, Department of Microbiology, 9 Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja 10 s/n, 18071 Granada, Spain. 11 12 Running title: Halomonas almeriensis sp. nov. 13 14 Keywords: Halomonas; exopolysaccharides; halophilic ; hypersaline 15 habitats. 16 17 Subject category: taxonomic note; new taxa; γ- 18 19 Author for correspondence: 20 E. Quesada: 21 Tel: +34 958 243871 22 Fax: +34 958 246235 23 E-mail: [email protected] 24 25 26 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene 27 sequence of strain M8T is AY858696. 28 29 30 31 32 33 34 1 Summary 2 3 Halomonas almeriensis sp. nov. is a Gram-negative non-motile rod isolated 4 from a saltern in the Cabo de Gata-Níjar wild-life reserve in Almería, south-east 5 Spain. It is moderately halophilic, capable of growing at concentrations of 5% to 6 25% w/v of sea-salt mixture, the optimum being 7.5% w/v. It is chemo- 7 organotrophic and strictly aerobic, produces catalase but not oxidase, does not 8 produce acid from any sugar and does not synthesize hydrolytic enzymes. The 9 most notable difference between this microorganism and other Halomonas 10 is that it is very fastidious in its use of carbon source. It forms mucoid 11 colonies due to the production of an exopolysaccharide (EPS). Its G+C content 12 is 63.5 mol%. A comparison of 16S rRNA gene sequences confirms its 13 relationship to Halomonas species. The most closely related species is 14 Halomonas halmophila with 95.8% similarity value between their 16S rRNA 15 sequences. DNA-DNA hybridization with Halomonas halmophila is 10.1%. Its 16 major fatty acids are: 18:1 ω7c; 16:0, 16:1 ω7c/15:0 ISO 2OH; 12:0 30H, 12:0, 17 11 methyl 18:1 ω7c and 10:0. The proposed name for strain M8T is Halomonas 18 almeriensis (= CECT 7050T = LMG 22904T). 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 1 The genus Halomonas, belonging to the family within the γ- 2 Proteobacteria, contains to date 32 species of moderately halophilic bacteria, 3 most of which have been isolated from hypersaline habitats (Dobson & 4 Franzmann, 1996; Mata et al., 2002; Ventosa et al., 1998; Vreeland et al., 5 1980). Taxonomically Halomonas is a heterogeneous bacterial group. On the 6 basis of 16S and 23S rRNA gene sequences Arahal et al. (2002) have 7 established three clearly distinguishable phylogenetic groups, in addition to 8 which another three groups can also be identified by phenotypic studies, 9 according to their capacity to produce acids from glucose and their use of a 10 variety of compounds as sole source of carbon and energy (Mata et al., 2002). 11 Some of the Halomonas species, including H. eurihalina, H. maura, H. ventosae 12 and H. anticariensis, which have been isolated and characterised by our 13 research group (Quesada et al., 1990; Bouchotroch et al., 2001; Martínez- 14 Cánovas et al., 2004a; Martínez-Cánovas et al., 2004b), produce extracellular 15 polysaccharides (EPS’s) with potential biotechnological applications (Calvo et 16 al., 2002; Béjar et al., 1998; Martínez-Checa et al., 2002; Arias et al., 2003; 17 Quesada et al., 2004). 18 19 We describe here a new exopolysaccharide-producing species belonging to the 20 genus Halomonas, with the proposed name of Halomonas almeriensis. 21 22 Strain M8T was isolated from a water sample taken from a saltern in the Cabo 23 de Gata-Níjar wild-life reserve in the province of Almería in south-east Spain 24 during a wide range of samplings made by our research group in 18 hypersaline 25 habitats in Spain and Morocco (Martínez-Cánovas et al., 2004c). It was 26 routinely kept and grown at 32ºC in MY medium (Moraine & Rogovin, 1966) 27 with 7.5% w/v marine salts (Rodríguez-Valera et al., 1981). 28 29 Phenotypic characterisation, on the basis of 112 tests, was done as described 30 by Mata et al. (2002). We compared the new strain with Halomonas species 31 using the software TAXAN (Information Resources Group, Maryland 32 Biotechnology Institute, University of Maryland, College Park, USA) based on 33 numerical analysis. The dendrogram obtained by the simple-matching

34 coefficient (SSM) (Sokal & Michener, 1958) and UPGMA method (Sneath & 1 Sokal, 1973) (Supplementary Fig. A, in IJSEM Online) shows that strain M8T 2 was related to the non-acid-producing group of Halomonas species (Mata et al., 3 2002), although it shares less than 63% similarity with them. This low similarity 4 can be put down the fact that strain M8T is extremely fastidious nutritionally. The 5 main phenotypic differences between Halomonas almeriensis (M8T) and its 6 nearest philogenetically related strains of the genus Halomonas are shown in 7 Table 1. 8 9 The G+C content of the DNA of strain M8 was estimated from the midpoint

10 value (Tm) of the DNA thermal denaturation profile, as described by Marmur & 11 Doty (1962) and Ferragut & Leclerc (1976). The guanine-plus-cytosine content 12 of the DNA of the novel strain was 63.5 mol%, within the range proposed for 13 Halomonas species of 52-68 mol% (Franzmann et al., 1988). 14 15 A partial fragment of the 16S rRNA gene was amplified by PCR using the 16 protocol of Saiki et al. (1988). The forward primer, 16F27 (5´- 17 AGAGTTTGATCATGGCTCAG-3´), annealed at positions 8-27 and the reverse 18 primer, 16R1488 (5´-CGGTTACCTTGTTAGGACTTCACC-3´) (both from 19 Pharmacia), annealed at the complement of positions 1511-1488 (E. coli 20 numbering according to Brosius et al., 1978). To complete the sequence we 21 designed an internal primer, 5´-GAGGATGATCAGCCACACTG-3´, which 22 annealed at position 401-421. The PCR product was purified using the GFXTM 23 PCR DNA and Gel Band Purification Kit (Amersham Biosciences). Direct 24 sequence determinations of PCR-amplified DNAs were made with an ABI 25 PRISM dye-terminator, cycle-sequencing, ready-reaction kit (Perking-Elmer) 26 and an ABI PRISM 377 sequencer (Perking-Elmer) according to the 27 manufacturer’s instructions. The sequence obtained (1459 bp) was compared to 28 the 16S rRNA reference gene sequences found in the GenBank and EMBL 29 databases by BLAST search. Phylogenetic and molecular evolutionary analyses 30 were conducted using MEGA version 3.0 (Kumar et al., 2004) after multiple 31 alignment of the data by CLUSTALX (Thompson et al., 1997). Distances and 32 clustering were determined using the neighbour-joining and maximum- 33 parsimony algorithms, and a bootstrap analysis (1,000 replications) was made 34 to determine the stability of the clusters. The neighbour-joining tree is available 1 as supplementary material in IJSEM Online (Fig. B). A similar result (not shown) 2 was obtained using the maximum-parsimony algorithm. The taxa included in the 3 tree in Figure 1 represent only the nearest neighbours. Our analyses confirmed 4 that the new strain belongs to the genus Halomonas, is located within Group 1 5 of Halomonas species described by Arahal et al. (2002) and shares 95.8% 16S 6 rRNA sequence similarity with Halomonas halmophila (Dobson et al., 1990). 7 The 16S fragment analysed contains the 15 signature nucleotides defined for 8 Halomonadaceae family (Dobson & Franzmann, 1996). 9 10 DNA-DNA hybridization was carried out according to the method of Lind and 11 Ursing (1986) with the modifications introduced by Ziemke et al. (1998) and 12 Bouchotroch et al. (2001). The result shows the low hybridization (10.1%) with 13 Halomonas halmophila, which was chosen on the basis of our phylogenetic 14 study as being the most closely related Halomonas species. 15 16 The fatty acids were analysed at DSMZ (Deutsche Sammlung von 17 Mikroorganismen und Zellkulturen GmbH) by high-resolution GLC using a moist 18 pellet of the cells obtained from a culture in MY medium supplemented with 19 7.5% w/v sea-salt mixture. Strain M8T shows a combination of fatty acids found 20 in other species of Halomonas (Dobson & Franzmann, 1996) (see species 21 description), although it also contains a relatively high proportion of C10 22 (2.11%), 12:0 (1.22%) and 11methyl 18:1 ω7c (2.75%). 23 24 Figure C (supplementary material, in IJSEM Online) is a transmission-electron 25 micrograph showing the morphology and cell size of strain M8T and the 26 presence of an extracellular polymer that is released into the external medium. 27 The TEM method used is fully described by Bouchotroch et al. (2001). 28 29 On the basis of phylogeny, DNA-DNA hybridization, fatty-acid composition and 30 phenotypic differences between the novel and previously described species 31 within the genus Halomonas, we consider that strain M8T represents a novel 32 species, for which we propose the name Halomonas almeriensis. 33 34 1 Description of Halomonas almeriensis sp. nov. 2 3 Halomonas almeriensis (al meri en´ sis, N.L. adj. masc. = denizen of the 4 province of Almería, in south-east Spain, where the strain was isolated). 5 6 The cells are Gram-negative, non-motile rods, 2-2.5 x 0.75 µm, appearing singly 7 or in pairs. They accumulate poly-β-hydroxyalkanoates (PHB) and produce 8 exopolysaccharide. Colonies are round, convex, creamy-white and mucoid. 9 Their growth pattern is uniform in a liquid medium. They are moderately 10 halophilic, capable of growing in salt concentrations (mixture of sea salts) of 5% 11 to 25% w/v. They grow within 15ºC to 37ºC and pH values of between 6 and 10. 12 They are chemo-organotrophic. Their metabolism is respiratory with oxygen as 13 terminal electron acceptor. The cells do not grow anaerobically in the presence 14 of nitrate, nitrite or fumarate. Catalase is produced but not oxidase. They do not 15 produce acids from sugars. Indol, methyl-red and Voges-Proskauer are 16 negative. They do not hydrolyse starch, aesculin, gelatin, casein, Tween 20, 17 Tween 80, DNA or tyrosine. They produce phosphatase and grow on 18 MacConkey agar, but do not produce phenylalanine deaminase, urease, ONPG 19 or lecithinase. Gluconate is oxidised. They do not produce pigment from

20 tyrosine, H2S from L-cysteine, grow on cetrimide agar or lyse blood. D- 21 gluconate is acceptable as sole carbon energy source, whereas aesculin, L- 22 arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, 23 D-mannose, D-melezitose, salicin, starch, D-trehalose, D-xylose, acetate, 24 citrate, formate, fumarate, malonate, propionate, succinate, adonitol, ethanol, 25 glycerol, myo-inositol, D-mannitol and sorbitol are not. L-alanine and L-serine 26 are used as sole sources of carbon, nitrogen and energy, whereas L-histidine, 27 DL-isoleucine, L-lysine, L-methionine and L-valine are not. They are susceptible 28 to amoxicillin (25 µg), ampicillin (10 µg), carbenicillin (100 µg), cefotaxime (30 29 µg), cefoxitin (30 µg), chloramphenicol (30 µg), erythromycin (15 µg), 30 kanamycin (30 µg), nitrofurantoin (300 µg), rifampycin (30 µg), streptomycin (10 31 µg), tobramycin (10 µg) and trimetroprim-sulphametoxazol (1.25 µg-23.75 µg). 32 They are resistant to nalidixic acid (30 µg), polymyxin B (300 UI) and 33 sulphamide (250 µg). 1 Principal fatty acids (more than 1%) are: 18:1 ω7c (50.66%); 11 methyl 18:1 2 ω7c (2.75%); 16:0 (21.08%); 16:1 ω7c/15:0 ISO 2OH (14.16%); 12:0 30H 3 (5.64%); 12:0 (1.22%) and 10:0 (2.11%). 4

5 DNA G+C content is 63.5 mol% (Tm method). 6 7 The type strain, M8T (= CECT 7050T = LMG 22904T), was isolated from a 8 hypersaline water sample taken from a saltern at Cabo de Gata (Almería, S.E. 9 Spain). 10 11 12 Acknowledgements 13 14 This research was supported by grants from the Dirección General de 15 Investigación Científica y Técnica (BOS2003-00498) and from the Plan Andaluz 16 de Investigación, Spain. Thanks go to our colleague Dr. J. Trout for revising our 17 English text. 18 19 20 References 21 22 Arahal, D. R., Ludwig, W., Schleifer, K. H. & Ventosa, A. (2002). Phylogeny 23 of the family Halomonadaceae based on 23S and 16S rDNA sequence 24 analyses. Int J Syst Evol Microbiol 52, 241-249. 25 Arias, S., del Moral, A., Ferrer, M. R., Quesada, E. & Béjar, V. (2003). 26 Mauran, an exopolysaccharide produced by the halophilic bacterium 27 Halomonas maura, with a novel composition and interesting properties for 28 biotechnology. Extremophiles 7, 319-326. 29 Béjar, V., Llamas, I., Calvo, C., & Quesada, E. (1998). Characterization of 30 exopolysaccharides produced by 19 halophilic strains of the species Halomonas 31 eurihalina. J Biotechnol 61, 135-141. 32 Berendes, F., Gottschalk, G., Heine-Dobbernack, E., Moore, E. R. B. & 33 Tindall, B. J. (1996). Halomonas desiderata sp. nov., a new alkaliphilic, 1 halotolerant and denitrifying bacterium isolated from a municipal sewage works. 2 System Appl Microbiol 19, 158-167. 3 Bouchotroch, S., Quesada, E., del Moral, A., Llamas, I. & Béjar, V. (2001). 4 Halomonas maura sp. nov., a novel moderately halophilic, exopolysaccharide- 5 producing bacterium. Int J Syst Evol Microbiol 51, 1625-1632. 6 Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1978). Complete 7 nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc 8 Natl Acad Sci USA. 75, 4801-4805. 9 Calvo, C., Martínez-Checa, F., Toledo, F. L., Porcel, J. & Quesada, E. 10 (2002). Characteristics of bioemulsifiers synthesized in crude oil media by 11 Halomonas eurihalina and their effectiveness in the isolation of bacteria able to 12 grow in the presence of hydrocarbons. Appl Microbiol Biotechnol 60, 347-351. 13 Dobson, S.J. & Franzmann, P.D. (1996). Unification of the genera Deleya 14 (Bauman et al., 1993), Halomonas (Vreeland et al., 1980), and 15 (Fendrich, 1988) and the species Paracoccus halodenitrificans (Robinson and 16 Gibbons, 1952) into a single genus, Halomonas, and placement of the genus 17 in the family Halomonadaceae. Int J Syst Bacteriol 46, 550-558. 18 Dobson, S. J., James, S. R., Franzmann, P. D. & McMeekin, T. A. (1990). 19 Emended description of Halomonas halmophila (NCMB 1971T). Int J Syst 20 Bacteriol 40, 462-463. 21 Ferragut, C. & Leclerc, H. (1976). Étude comparative des méthodes de

22 détermination du Tm de l`ADN bacterien. Ann Microbiol 127, 223-235. 23 Franzmann, P. D., Wehmeyer, U. & Stackebrandt, E. (1988). 24 Halomonadaceae fam. nov., a new family of the class Proteobacteria to 25 accommodate the genera Halomonas and Deleya. Syst Appl Microbiol 11, 19- 26 19. 27 Kumar, S., Tamura, K. & Nei, M. (2004) MEGA3: Integrated software for 28 Molecular Evolutionary Genetics Analysis and sequence alignment. Brief 29 Bioinform 5, 150-163. 30 Lind, E. & Ursing, J. (1986). Clinical strains of Enterobacter agglomerans 31 (Synonyms, Erwinia herbicola, Erwinia milletiae) identified by DNA-DNA 32 hybridization. Acta Pathol. Microbiol. Immunol. Scand. Sect. B. 94: 205-213. 1 Marmur, J. & Doty, P. (1962). Determination of the base composition of 2 deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 3 109-118. 4 Martínez-Cánovas, M. J., Béjar, V., Martínez-Checa, F. & Quesada, E. 5 (2004a). sp. nov., from Fuente de Piedra, a saline- 6 wetland, wild-fowl reserve in Málaga (S. Spain). Int J Syst Evol Microbiol 54, 7 1329-1332 8 Martínez-Cánovas, M. J., Quesada, E., Llamas, I. & Béjar, V. (2004b). 9 sp. nov., a moderately halophilic, denitrifying, 10 exopolysaccharide-producing bacterium. Int J Syst Evol Microbiol 54, 733-737 11 Martínez-Cánovas, M. J., Quesada, E., Martínez-Checa, F. & Béjar, V. (2004c). 12 A taxonomic study to establish the relationship between exopolysaccharide- 13 producing bacterial strains living in diverse hypersaline habitats. Curr Microbiol 14 48, 348-353 15 Martínez-Checa, F., Toledo, F. L., Vilchez, R., Quesada, E, & Calvo, C. 16 (2002). Yield production, chemical composition and functional properties of 17 emulsifier H28 synthesized by Halomonas eurihalina strain H-28 in media 18 containing various hydrocarbons. Appl Microbiol Biotechnol 58, 358-363. 19 Mata, J. A., Martínez-Cánovas, M. J., Quesada, E. & Béjar, V. (2002). A 20 detailed phenotypic characterisation of the type strains of Halomonas species. 21 Syst Appl Microbiol 25, 360-375. 22 Moraine, R. A. & Rogovin, P. (1966). Kinetics of polysaccharide B-1459 23 fermentation. Biotechnol Bioeng 8, 511-524. 24 Quesada, E., Béjar, V., Ferrer, M. R., Calvo, C., Llamas, I., Martínez-Checa, 25 F., Arias, S., Ruiz-García, C., Páez, R., Martínez-Cánovas J. & Del Moral, A. 26 (2004). Moderately halophilic, exopolysaccharide-producing bacteria. In 27 Halophilic microorganisms pp. 297-314. ed. Ventosa, A. Heilderberg: Springer- 28 Verlag. 29 Quesada, E., Valderrama, M. J., Béjar, V., Ventosa, A., Gutierrez, M. C., 30 Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1990). Volcaniella eurihalina 31 gen. nov., a moderately halophilic non-motile gram-negative rod. Int J Syst 32 Bacteriol 40, 261-267. 1 Rodríguez-Valera, F., Rúiz-Berraquero, F. & Ramos-Cormenzana, A. 2 (1981). Characteristics of the heterotrophic bacterial populations in hypersaline 3 environments of different salt concentrations. Microb Ecol 7, 235-243. 4 Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. 5 T., Mullis, K. B. & Erlich, H. A. (1988). Primer-directed enzymatic amplification 6 of DNA with thermostable DNA polymerase. Science. 239, 487-491. 7 Sneath, P. H. A. & Sokal, R. R. (1973). Numerical . The Principles and 8 Practice of Numerical Classification. San Francisco: Freeman, Williams & 9 Wilkins Co. 10 Sokal, R. R. & Michener, C. D. (1958). A statistical method for evaluating 11 systematic relationships. Univ Kansas Sci Bull 38, 1409-1438. 12 Thompson, J. D., Gibson, T. J., Plewniak, K., Jeanmougin, F. & Higgins, D. 13 G. (1997). The ClustalX window interface: flexible strategies for multiple 14 sequence alignments aided by quality analysis tools. Nucleic Acids Res 24: 15 4876-4882. 16 Ventosa, A., Nieto, J. J. & Oren, A. (1988). The biology of aerobic moderately 17 halophilic bacteria. Microbiol Mol Biol Rev 62, 504-544. 18 Vreeland, R. H., Litchfield, C. D., Martin, E. L. & Elliot, E. (1980). Halomonas 19 elongata, a new genus and species of extremely salt-tolerant bacteria. Int J Syst 20 Bacteriol 30, 485-495. 21 Ziemke, F., Höfle, M. G., Lalucat, J. & Rosselló-Mora, R. (1998). 22 Reclassification of Shewanella putrefaciens Owen’s group II as Shewanella 23 baltica sp. nov. Int J Syst Bacteriol 48, 179-186. 24 25 26 27 28 29 30 31 32 33 34 1 Fig. A. Dendrogram based on the simple-matching (SSM) coefficient and 2 unweighted-pair-group clustering method (UPGMA). 3 4 Fig. B. Phylogenetic relationships between Halomonas almeriensis and other 5 Halomonas species plus other taxa of Gram-negative halophilic bacteria. The 6 tree was constructed using the neighbour-joining algorithm. Only bootstrap 7 values above 50% are shown (1000 replications). Bar, 2% estimated sequence 8 divergence. 9 10 Fig. C. Transmission electron micrograph of strain M8T stained with ruthenium 11 red (bar 1 µm). 12 13 Fig. 1. Phylogenetic relationships between Halomonas almeriensis and other 14 related Halomonas species. The tree was constructed using the neighbour- 15 joining algorithm. Only bootstrap values above 50% are shown (1000 16 replications). Bar, 1% estimated sequence divergence. 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 1 Table 1. Characteristics distinguishing between Halomonas almeriensis and other 2 phylogenetically related type strains of the genus Halomonas. 3 4 Data from this study and from Dobson et al. (1990); Mata et al. (2002); Vreeland et al. (1980). 5 ……………………………………………...... ………………………………………...... 6 1, Halomonas almeriensis CECT 7050T; 2, Halomonas halmophila ATCC 19717T; 3, Halomonas 7 elongata CECT 4279T 4, Halomonas eurihalina ATCC 49336T. Characteristic 1 2 3 4 Morphology Short Rod Rod Rod Short Rod Exopolysaccharide + - - + Motility - + + - Oxidase - + - - Sea-salt range (%, w/v) 5-25 3-25 0-20 0.5-25 Sea-salt optimum (%, w/v) 7.5 7.5 3-8 7.5 Temperature range (ºC) 15-37 15-45 4-45 4-45 Acid from adonitol - + - - L-arabinose - + + - D-fructose - + - - D-galactose - + - - D-glucose - + + - myo-inositol - + - - lactose - + + - maltose - + + - D-mannitol - + + - D-mannose - + - - D-melezitose - + - - D-rhamnose - + - - sucrose - + + - D-salicin - + + - D-sorbitol - - + - sorbose - + - - D-trehalose - + + - Hydrolysis of: aesculin - - - + gelatin - - - + Tween 20 - + - + Tween 80 - - - + tyrosine - - - + ONPG - - + + urea - - + + Pigment from tyrosine - - - +

H2S production - + + + Nitrate reduction - - + + Respiration on nitrate - - + - Gluconate oxidation + - + + 8 9 10 1 Table 1. Continued. 2 Characteristic 1 2 3 4 Growth on: agar cetrimide - - + + aesculin - + - - L-arabinose - - + + D-cellobiose - + + + D-fructose - + + + D-galactose - + + + D-glucose - + + + lactose - + + + maltose - + - + D-mannose - + + + D-melezitose - + + + D-salicin - - - + D- trehalose - + + + acetate - + + + citrate - + + + formate - + - - fumarate - - + + gluconate + - + + malonate - - + + propionate - - + + succinate - - + + adonitol - - + + ethanol - - + - glycerol - + - - myo-inositol - - + + D-mannitol - - + + sorbitol - + + + L-histidine - + + + DL-isoleucine - - + + L-lysine - - + + L-valine - + + + Susceptibility to: amoxycillin (25 µg) + + - - ampicillin (10 µg) + + - - carbenicillin (100 µg) + + - - erythromycin (15 µg) + - + + nalidixic acid (30 µg) - + + + polymyxin B (300 UI) - + - + streptomycin (10 µg) + + + - sulphamide (250 µg) - + + + G+C content (mol%) 63.5 63.0 60.5 59.1-65.7 3 4 5 6 7 1 2 3 4 5 % Similarity

55 6065 70 75 80 85 90 95 100

H. aquamarina CECT 5000T H. meridiana DSM 5425T H. cupida CECT 5001T H. pantelleriensis DSM 9661T H. halmophila ATCC 19717T H. halophila CCM 3662T H. marisflavi JCM 10873T H. elongata CECT 4279T H. eurihalina ATCC 49336T H. variabilis DSM 3051T H. anticariensis CECT 5854 T H. magadiensis NCBM 13595T H. salina ATCC 49509T H. halodenitrificans CECT 5012T H. maura CECT 5398T H. ventosae DSM 15911T H. campisalis ATCC 7000597T H. desiderata DSM 9502T H. pacifica CECT 574T H. subglaciescola DSM 4683T H. venusta ATCC 27125T H. halodurans LMG 10144T M8T 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1 2 3 4

98 H. elongata CECT 4279T (X67023) 99 H. eurihalina ATCC 49336T (X87218) H. halmophila ATCC 19717T (AJ306889) T 100 H. almeriensischaridemi M8M8 (AY858696) 85 H. maura CECT 5298T (AJ271864) H. organivorans CECT 5995T (AJ616910) 61 H. halophila CCM 3662T (M93353) 77 100 H. salina ATCC 49509T (AJ243447) 67 H. pacifica CECT 574T (L42616) 89 H. alimentaria JCM 10888T (AF211860) H. halodenitrificans CECT 5012T (L04942) 50 H. ventosae CECT 5797T (A4268080) H. cupida CECT 5001T (AY035894) 82 H. desiderata DSM 9502T (X92417) H. campisalis ATCC 700597T (AF054286) H. anticariensis LMG 22089T (AY489405) LMG 20969T (AJ320530) 62 H. muralis 70 H. pantelleriensis DSM 9661T (X93493) H. halocynthiae DSM 14573T (AJ417388) 100 H. halodurans LMG 10144T (L42619) H. subglaciescola DSM 4683T (AJ306892) 65 86 H. neptunia ATCC BAA-805T (AF212202) 98 H. boliviensis DSM 15516T (AY245449) 99 92 H. variabilis DSM 3051T (AJ306893) 56 H. sulfidaeris ATCC BAA-803T (AF212204) 100 H. venusta ATCC 27125T (AJ306894) 99 H. hydrothermalis ATCC BAA-800T (AF212218) NCMB 13595T (X92150) 79 H. magadiensis H. aquamarina CECT 5000T (AJ306888) 99 88 H. meridiana DSM 5425T (AJ306891) 100 55 H. axialensis ATCC BAA-802T (AF212206) 99 C. canadensis ATCC 43984T (AF211861) T 100 C. marismortui ATCC 17056 (X87219) T 99 C. israelensis CECT 5287 (AF211862) 100 C. salexigens DSM 3043T (AJ295146) H. marina ATCC 25374T (AJ306890) H. marisflavi JCM 10873T (AF251143) Alcanivorax borkumensis ATCC 700651T (Y12579) M. hydrocarbonoclasticus CECT 5005T (AB021372) 50 P. halophila DSM 3050T (ABO21383)

0.02 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 1 2 3 4 5 6

95 H. elongata CECT 4279T (X67023) 100 H. eurihalina ATCC 49336T (X87218) H. halmophila ATCC 19717T (AJ306889) 100 H. charidemialmeriensis M8M8T (AY858696) 55 H. salina ATCC 49509T (AJ243447) T 92 100 H. halophila CCM 3662 (M93353) H. maura CECT 5298T (AJ271864) 52 H. organivorans CECT 5995T (AJ616910) H. pacifica CECT 574T (L42616) 99 H. alimentaria JCM 10888T (AF211860) H. halodenitrificans CECT 5012T (L04942) H. ventosae CECT 5797T (A4268080) H. cupida CECT 5001T (AY035894) H. campisalis ATCC 700597T (AF054286) H. desiderata DSM 9502T (X92417) 97 H. anticariensis LMG 22089T (AY489405) 60 T 55 H. muralis LMG 20969 (AJ320530) 77 H. pantelleriensis DSM 9661T (X93493)

0.01 7 8