1 Palleronia marisminoris gen. nov., sp. nov., a moderately halophilic exopolysaccharide- 2 producing bacterium belonging to the α-, isolated from a saline soil. 3 4 Fernando Martínez-Checa, Emilia Quesada, M. José Martínez-Cánovas, Inmaculada Llamas and 5 Victoria Béjar. 6 7 Microbial Exopolysaccharide Research Group, Department of Microbiology, Faculty of 8 Pharmacy, Campus Universitario de Cartuja, University of Granada, 18071 Granada, Spain. 9 10 Running title: Palleronia marisminoris gen. nov., sp. nov. 11 12 Keywords: halophilic ; exopolysaccharides; Palleronia; Palleronia marisminoris; α- 13 Proteobacteria 14 15 Subject category: new taxa, proteobacteria 16 17 Author for correspondence: Dr. V. Béjar. 18 Tel: +34 958 243871 19 Fax: +34 958 246235. 20 e-mail: [email protected] 21 22 The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of strain B33T is 23 AY926462.

1 1 Summary 2 3 Palleronia marisminoris gen. nov., sp. nov. is a moderately halophilic, exopolysaccharide- 4 producing, Gram-negative, non-motile rod isolated from a hypersaline soil bordering a saline 5 saltern on the Mediterranean seaboard in Murcia (Spain). The bacterium is chemoheterotrophic 6 and strictly aerobic. It contains a pink pigment but does not synthesise bacteriochlorophyll a. It 7 requires 0.66 M of Na+, 0.1 M of Mg++ and 0.1 M of K+ for optimum growth. It does not 8 produce acid from carbohydrates. It cannot grow with carbohydrates, organic acids, sugars 9 alcohols or amino acids as sole sources of carbon and energy. Its major fatty-acids are 18:1 ω7c 10 (68.9%) and 19:0 cyclo ω8c (12.8%). The sole respiratory lipoquinone found in strain B33T is 11 ubiquinone-10. The G+C content is 64.2 mol%. 16S rRNA gene sequence comparisons show 12 that the isolate is a member of the Roseobacter clade within the class of α-Proteobacteria. The 13 similarity values with Roseivivax halodurans and Roseivivax halotolerans are 88.2% and 88.0% 14 respectively and 92.2% with Salipiger mucosus. DNA-DNA hybridization values with these 15 are < 30%. In the light of the polyphasic evidence gathered in this study it is proposed 16 that the isolate be classified as a new and species with the name Palleronia marisminoris 17 gen. nov., sp. nov. The proposed type strain is strain B33T (=CECT 7066T = LMG 22959T). 18 19

2 1 Moderately halophilic bacteria require from 3% to 15% w/v NaCl for satisfactory growth 2 (Kushner & Kamekura, 1988). They are widely distributed among many hypersaline habitats. 3 Taxonomically the majority of Gram-negative halophilic bacteria belong to the γ- 4 Proteobacteria class but they can also be found in other bacterial phyla (Ventosa et al.,, 1998). 5 Some halophilic microorganisms, such as those which produce exopolysaccharides, have 6 interesting industrial applications (Quesada et al., 2004). Microbial exopolysaccharides have a 7 potentially wide range of applications in such fields as pharmacy, foodstuffs, cosmetics, and the 8 petroleum industry, where emulsifying, viscosifying, suspending, and chelating agents are 9 required (Sutherland, 1990). During an extensive search of 18 hypersaline habitats in Spain and 10 Morocco, designed to obtain new exopolysaccharides, we discovered that the commonest 11 halophilic exopolysaccharide producers were various species of the genus Halomonas, most 12 importantly Halomonas maura and Halomonas eurihalina (Quesada et al., 1990; Bouchotroch 13 et al., 2001; Quesada et al., 2004; Martínez-Cánovas et al., 2004d). As a result of these searches 14 we have described the first moderately halophilic exopolysaccharide-producing microorganism 15 belonging to the α-Proteobacteria, Salipiger mucosus (Martínez-Cánovas et al.,, 2004e), three 16 new Halomonas species, H. ventosae (Martínez-Cánovas et al.,, 2004c), H. anticariensis 17 (Martínez-Cánovas et al.,, 2004a) and H. almeriensis (Martínez-Checa et al., 2005b), two new 18 Idiomarina species I. fontislapidosi and I. ramblicola (Martínez-Cánovas et al., 2004b) and a 19 new species, A. hispanica (Martínez-Checa et al., 2005a) all of which produce 20 exopolysaccharides (EPS’s). We describe and classify here an unassigned halophilic EPS- 21 producing strain also isolated in these studies and propose it as a novel genus and species 22 belonging to the α-Proteobacteria class with the name Palleronia marisminoris. 23 24 The strain in question, B33T, was isolated from a saline soil bordering a saltern on the 25 Mediterranean seaboard at Marchamalo (Murcia, SE. Spain) (Martínez-Cánovas et al., 2004d). 26 It was routinely grown at 32ºC in MY medium (Moraine and Rogovin, 1966) supplemented with a 27 5% w/v sea-salt solution (Rodríguez-Valera et al., 1981). Its phenotype was studied with 135 tests 28 by Martínez-Cánovas et al., (2004d) and it was included in phenon E along with other 29 unidentified strains. The procedures we followed for its phenotypic characterization have been 30 described by Ventosa et al., (1982), Quesada et al., (1983) and Mata et al., (2002). Salt 31 requirements and optimum salt concentration were determined in MY medium (Moraine and 32 Rogovin, 1966). The salt concentrations assayed ranged from 0.5% to 30% w/v and were 33 prepared from a mixture of sea salts according to Rodríguez-Valera et al., (1981). We also 34 tested to see whether strain B33T could survive with NaCl alone or whether it required other sea 35 salts. To determine its nutritional requirements we also assayed its growth in Koser medium 36 supplemented with yeast extract (0.1 to 3% w/v), malt extract (1 to 3% w/v) or proteose peptone 37 (1 to 5% w/v). The presence of bacteriochlorophyll a was analysed spectrophotometrically

3 1 using Cohen-Bazire and colleagues’ procedure (1957), following the recommendations of 2 Allgaier et al., (2003). DNA was purified using the technique of Marmur (1961).The guanine-

3 plus-cytosine (G+C) content of the DNA was estimated from the midpoint value (Tm) of the

4 thermal denaturation profile (Marmur and Doty, 1962). Tm was determined by the graphic method 5 described by Ferragut and Leclerc (1976) and the G+C content was calculated from this

6 temperature using Owen and Hill’s equation (1979). The Tm value of reference DNA from 7 Escherichia coli NCTC 9001 was taken to be 74.6ºC in 0.1x SSC (Owen and Pitcher, 1985). 8 9 The phenotypic characteristics and G+C content are given in the species description. 10 Phenotypic features distinguishing between strain B33T, Salipiger mucosus and the two species 11 of Roseivivax are available in Table 1, where it can be seen that strain B33T is phenotypically 12 most closely related to S. mucosus. Both species are Gram-negative, non-motile, moderately 13 halophilic rods. They are chemoheterotrophic, strictly aerobic and produce exopolysaccharides. 14 They do not produce bacteriochlorophyll α. They do not produce acids from sugars and have 15 low nutritional versatility as they cannot grow with any of the carbohydrates, alcohols, organic 16 acids or amino acids tested as sole sources of carbon and energy. For optimum growth it 17 requires yeast extract (0.3% w/v), malt extract (0.3% w/v) and proteose peptone (0.5% w/v) 18 together with Na+ (0.66 M), Mg++ (0.1M) and K+ (0.01M), and thus it flourishes in an MY 19 complex medium (Moraine and Rogovin, 1966) supplemented with 5% w/v sea salts. The most 20 important phenotypic tests distinguishing between Palleronia marisminoris and Salipiger 21 mucosus are pigment production, a negative reaction for oxidase, urease and gluconate 22 oxidation, positive reaction for ONPG, and an inability to grow with NaCl alone. The G+C 23 (mol%) content of strain B-33T (64.2) is very similar to those of S. mucosus and R. halodurans 24 (64.5 and 64.4 mol% respectively). 25 26 Fatty acids and quinones were identified by high-resolution GLC and HPLC respectively at 27 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (cf. Table 1). Strain B33T 28 contains a large quantity of cis-11 octadecenoic acid (18:1 ω7c) (68.9 %) together with 19:0 29 cyclo ω8c, 3-hydroxy 10:0, 16:0 and 18:0 (12.8%, 5%, 4.2% and 3.4% respectively). It also has 30 2.3% of an unknown component at a retention time of 4.870 min. The presence of 18:1 ω7c as 31 predominant fatty acid is a feature characteristic of taxa within the α-Proteobacteria. 32 Nevertheless, the cyclo-substituted fatty acid (19:0 cyclo ω8c) is not widely present in the 33 family; though it has been described in lesser quantities (2.2 %) in Salipiger 34 mucosus. The only respiratory lipoquinone detected was ubiquinone-10. The presence of 35 ubiquinone 10 as the dominant respiratory lipoquinone is characteristic of members of the α- 36 class of the Proteobacteria. 37

4 1 The transmission electron micrograph (Fig. 1), made using the methods described by 2 Bouchotroch et al., (2001), shows the cell morphology of strain B33T. Thin sections reveal a 3 typical Gram-negative cell-envelope profile; the cell contains poly-β-hydroxyalkanoate (PHA) 4 granules. EPS appears in the external medium. 5 6 We determined the almost complete 16S rDNA sequence of strain B33T (1351 bp) 7 corresponding to positions 46 to 1445 of the Escherichia coli 16S rRNA gene using the standard 8 protocols (Saiki et al., 1988). The forward primer was 16F27 (5´- 9 AGAGTTTGATCMTGGCTCAG-3´), annealing at positions 8-27, and the reverse primer was 10 16R1488 (5´-CGGTTACCTTGTTAGGACTTCACC-3´), annealing at the complement of 11 positions 1511-1488 (E. coli numbering according to Brosius et al., 1978.). The PCR products 12 were purified using the Qiaquick spin-gel extraction kit (Qiagen). Direct sequence 13 determinations of PCR-amplified DNA’s were carried out with the ABI PRISM dye-terminator, 14 cycle-sequencing, ready-reaction kit (Perking-Elmer) and an ABI PRISM 377 sequencer 15 (Perking-Elmer) according to the manufacturer’s instructions. The sequences obtained were 16 compared to reference 16S rRNA gene sequences available in the GenBank, EMBL and DDBJ 17 databases obtained from the National Center of Biotechnology Information database using the 18 BLAST search. Phylogenetic analysis was made using the software MEGA (Molecular 19 Evolutionary Genetics Analysis) version 3.0 (Kumar et al., 2004) after multiple alignments of 20 data by CLUSTALX (Thompson et al,, 1997). Distances and clustering were determined with 21 the neighbour-joining and maximum-parsimony methods. The stability of the clusters was 22 ascertained by performing a bootstrap analysis (1000 replications). Our phylogenetic analysis 23 with the neighbour-joining method included, along with the sequence of B33T, some 24 representatives of the Rhodobacteraceae family (Figs 1 and 2, suppplementary data). The 25 maximum-parsimony algorithm gave a similar result (data not shown). Strain B33T showed 26 92.2% similarity with Salipiger mucosus. Other phylogenetically close species were Roseivivax 27 halodurans and Roseivivax halotolerans, with which they showed 88.2% and 88.0% sequence 28 similarity respectively. Strain B33T is in the same clade as Roseivivax and Salipiger, belonging 29 to the α-3 group of the α-class of the Proteobacteria within the “Rhodobacteraceae” family 30 (Garrity, 2002). Roseivivax is taxonomically related to the Roseobacter clade, a group of genera 31 of the “Rhodobacteraceae” family (Allgaier et al., 2003), which makes up the most abundant 32 population in marine habitats (González & Moran, 1997). 33 34 DNA-DNA hybridization was done according to Lind & Ursing’s method (1986) with the 35 modifications of Ziemke et al., (1998) and Bouchotroch et al., (2001). DNA-DNA hybridization 36 values of B33T with the most phylogenetically related species, Roseivivax halodurans, 37 Roseivivax halotolerans and Salipiger mucosus, are 22, 27.7 and 29.7 respectively.

5 1 Thus, on the basis of phylogenetic evidence, DNA-DNA hybridization values, fatty-acid 2 profiles, quinones, differences in phenotypic characteristics and its inability to synthesise 3 bacteriochlorophyll a, we are of the opinion that strain B33T should be recognised as the 4 representative species of a novel genus, for which we propose the names Palleronia and 5 Palleronia marisminoris. 6 7 Description of Palleronia gen. nov. 8 9 Palleronia (Pall. er. o’nia, N.L. sb. f., in honour of Professor Norberto Palleroni, a pioneer in 10 the use of molecular identification techniques in prokaryote ). 11 12 Gram-negative, short non-motile rods, 2-2.5 µm long by 0.75-1 µm wide. Neither flagella nor 13 endospores are present. Bacterioclorophyll a is absent. Metabolism is chemoheterotrophic and 14 aerobic, the cells being unable to grow under anaerobic conditions either by fermentation, 15 nitrate or fumarate reduction or photoheterotrophy. PHA and catalase are present. Oxidase is 16 negative. Colonies contain pink pigment. The bacterium cannot produce acids from sugars and 17 has low nutritional and biochemical versatility. It is strictly halophilic, requiring Na+, Mg++ and 18 K+ for growth. The principal cellular fatty acids are 18:1 ω7c and 19:0 cyclo ω8c. It has 19 ubiquinone with ten isoprene units. The type species is Palleronia marisminoris. 20 21 Description of Palleronia marisminoris sp. nov. 22 23 Palleronia marisminoris (ma’ris, L. sb. n. gen., “of the sea”; minoris, L. adj. n. gen., “smaller”; 24 marisminoris of the smaller sea, i.e. from el Mar Menor, a shallow area of sea highly sheltered 25 from the Mediterranean sea on the S. E. coast of Spain, from whence the type strain was 26 isolated). 27 28 In addition to the traits reported for the genus, the species grows on MY solid medium in the 29 form of circular, convex, pink, mucoid colonies. In liquid medium its growth pattern is uniform. 30 The cells are encapsulated. It is moderately halophilic, capable of growing in salt concentrations 31 (mixture of sea-salts) from 0.5% to 15% w/v. Optimum growth occurs at 5% w/v sea-salt. It 32 cannot grow with NaCl as sole salt. Minimum salt requirements are 0.66 M Na+, 0.1 M Mg++ 33 and 0.01 M K+. It grows within the temperature range of 20º to 37ºC and at pH values of

34 between 5 and 10. It produces H2S from L-cysteine. Selenite reduction and phosphatase are 35 positive. Tween 20 is hydrolysed. It does not produce acids from the following sugars: adonitol, 36 D-cellobiose, D-fructose, D-galactose, D-glucose, myo-inositol, lactose, maltose, D-mannitol, 37 D-mannose, D-melezitose, L-rhamnose, sucrose, D-salicin, D-sorbitol, sorbose or D-trehalose.

6 1 ONPG is positive. O/F, indol, methyl-red, Voges-Proskauer and gluconate oxidation are 2 negative. Phenylalanine deaminase is not produced. Urea, tyrosine, Tween 80, starch, aesculin, 3 gelatine, DNA, lecithin and casein are not hydrolysed. Growth on either MacConkey or 4 cetrimide agar is inviable. Blood is not lysed. Neither nitrate nor nitrite is reduced. The 5 following compounds are not acceptable as sole carbon and energy sources: L-arabinose, D- 6 cellobiose, aesculin, D-fructose, glucose, galactose, lactose, maltose, D-mannose, D-salicin, 7 trehalose, acetate, citrate, formate, fumarate, gluconate, lactate, malonate, propionate, succinate, 8 adonitol, ethanol, glycerol, inositol, mannitol and sorbose. The following compounds are not 9 used as sole carbon, nitrogen and energy sources: L-alanine, L-cysteine, L-histidine, iso-leucine, 10 L-lysine, L-methionine, L-serine, tryptophan and L-valine. It is susceptible to (µg) amoxicillin 11 (25), ampicillin (10), carbenicillin (100), cefotaxime (30), cefoxitin (30), chloramphenicol (30), 12 erythromycin (15), kanamycin (30), streptomycin (10), nitrofurantoin (300), rifampycin (30), 13 tobramycin (10) and is resistant to nalidixic acid (30), polymixim B (300) sulfonamide (250) 14 and trimetoprim-sulphametoxazol (1.25-23.7). The major fatty acids are (%): 18:1 ω7c (68.9), 15 19:O cyclo ω8c (12.8), 3-hydroxy 10:0 (5), 16:O (4.2), 18:O (3.4). Its DNA G+C content is 64.2

16 mol % (Tm method). 17 18 The type strain is strain B33T (=CECT 7066T = LMG 22959T), isolated from a hypersaline soil 19 bordering a solar saltern in Marchamalo (Murcia, S.E. Spain). 20 21 ACKNOWLEDGEMENTS 22 23 This research was supported by grants from the Dirección General de Investigación Científica y 24 Técnica (BOS2003-00498) and from the Plan Andaluz de Investigación, Spain. The authors are 25 grateful to their colleague Dr. J. Trout for revising the English text and for his help with the 26 nomenclature and to Concepción Fernández and David Porcel for their expertise in the electron- 27 microscope studies. 28 29 REFERENCES 30 31 Allgaier, M., Uphoff, H., Feelske, A. & Wagner-Döbler, I. (2003). Aerobic anoxygenic 32 photosynthesis in Roseobacter clade bacteria from diverse marine habitats. Appl Environ 33 Microbiol 69, 5051-5059. 34 Bouchotroch, S., Quesada, E, del Moral, A., Llamas, I. & Béjar, V. (2001). Halomonas 35 maura sp. nov., a novel moderately halophilic, exopolysaccharide-producing bacterium. Int J 36 Syst Evol Microbiol 51, 1625-1632.

7 1 Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1978). Complete nucleotide 2 sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci 75, 4801- 3 4805. 4 Cohen-Bazire, G., Sistrom, W. R. & Stanier, R. Y. (1957). Kinetic studies of pigment 5 synthesis by non-sulfur purple bacteria. J Cell Comp Physiol 49, 25-68. 6 Ferragut, C. & Leclerc, H. (1976). Etude comparative des methodes de determination du Tm 7 de l’ADN bacterien. Ann Microbiol 127, 223-235. 8 Garrity, G. M. (2002). Bergey’s Manual of Systematic Bacteriology, vol. 1, 2nd Ed. New-York: 9 Springer-Verlag. 10 González, J. M. & Moran, M. A. (1997). Numerical dominance of a group of marine bacteria in 11 the α-subclass of the class Proteobacteria in coastal seawater. Appl Environ Microbiol 63, 4237- 12 4242. 13 Kumar, S., Tamura, K. & Nei, M. (2004) MEGA3: Integrated software for Molecular 14 Evolutionary Genetics Analysis and sequence alignment. Brief in Bioinformatics and sequence 15 alignments 5, 150-163. 16 Kushner, D.J. & Kamekura, M. (1988). Physiology of halophilic bacteria. In Halophilic 17 bacteria pp.109-138. Edited by F. Rodríguez-Valera. Boca Raton: CRC Press. 18 Lind, E. & Ursing, J. (1986). Clinical strains of Enterobacter agglomerans (Synonyms, Erwinia 19 herbicola, Erwinia milletiae) identified by DNA-DNA hybridization. Acta Pathol Microbiol 20 Immunol Scand Sect B 94: 205-213. 21 Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from 22 microorganisms. J Mol Biol 3, 208-212. 23 Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic 24 acid from its thermal denaturation temperature. J Mol Biol 5, 109-118. 25 Martínez-Cánovas, M. J., Béjar, V., Martínez-Checa, F. & Quesada, E. (2004a). Halomonas 26 anticariensis sp. nov., from Fuente de Piedra, a saline-wetland, wild-fowl reserve in Málaga, 27 southern Spain. Int J Syst Evol Microbiol 54, 1329-1332. 28 Martínez-Cánovas, M. J., Béjar, V., Martínez-Checa, F., Páez, R. & Quesada, E. (2004b). 29 Idiomarina fontislapidosi sp. nov. and Idiomarina ramblicola sp. nov. isolated from inland 30 hypersaline habitats in Spain. Int J Syst Evol Microbiol 54,1793-1797. 31 Martínez-Cánovas, M. J., Quesada, E., Llamas, I. & Béjar, V. (2004c). Halomonas ventosae sp. 32 nov., a moderately halophilic, denitrifying, exopolysaccharide-producing bacterium. Int J Syst Evol 33 Microbiol 54, 733-737. 34 Martínez-Cánovas, M. J., Quesada, E., Martínez-Checa, F. & Béjar, V. (2004d). A taxonomic 35 study to establish the relationship between exopolysaccharide-producing bacterial strains living in 36 diverse hypersaline habitats. Curr Microbiol 48, 348-353.

8 1 Martínez-Cánovas, M. J., Quesada, E., Martínez-Checa, F., Del Moral, A. & Béjar, V. 2 (2004e). Salipiger mucosus gen. nov. sp. nov., a moderately halophilic, exopolysacharide- 3 producing bacterium member of α-Proteobacteria isolated from hypersaline habitats. Int J Syst 4 Evol Microbiol 54, 1735-1740. 5 Martínez-Checa, F., Béjar, V., Llamas, I., del Moral, A. & Quesada. E (2005a) 6 Alteromonas hispanica sp. nov., a polyunsaturated-fatty-acid-producing, halophilic 7 bacterium isolated from Fuente de Piedra (S.E. Spain) Int J Syst Evol Microbiol Papers in 8 Press, doi:10.1099/ijs.0.63809-0 9 Martínez-Checa, F., Béjar, V., Martínez-Cánovas, M.J., Llamas, I. & Quesada, E. 10 (2005b). Halomonas almeriensis sp. nov., a moderately halophilic, exopolysaccharide-producing 11 bacterium from Cabo de Gata (Almería, south-east Spain). Int J Syst Evol Microbiol Papers in 12 Press, doi:10.1099/ijs.0.63676-0 13 Mata, J.A., Martínez-Cánovas, M. J., Quesada, E. & Béjar, V. (2000). A detailed 14 phenotypic characterisation of the type strains of Halomonas species. Syst Appl Microbiol 25, 15 360-375 16 Moraine, R. A. & Rogovin, P. (1966) Kinetics of polysaccharide B-1459 fermentation. 17 Biotechnol Bioeng 8, 511-524. 18 Nishimura, Y., Muroga, Y., Saito, S., Shiba, T., Takamiya, K. & Shioi, Y. (1994). DNA 19 relatedness and chemotaxonomic feature of aerobic bacteriochlorophyll-containing bacteria 20 isolated from coasts of Australia. J Gen Appl Microbiol 40, 287-296. 21 Owen, R. J. & Hill, L. R. (1979). The estimation of base compositions, base pairing and 22 genome size of bacterial deoxyribonucleic acids. In Identification Methods for Microbiologists, 23 pp. 277-296, 2nd ed., Edited by F.A. Skinner & D.W. Lovelock. London: Academic Press. 24 Owen R. J. & Pitcher, D. (1985). Current methods for estimating DNA composition and levels 25 of DNA-DNA hybridization. In Chemical Methods In Bacterial Systematics, pp.67-93, Edited 26 by M. Goodfellow & D.E. Minnikin. London: Academic Press. 27 Quesada, E., Valderrama, M. J., Béjar, V., Ventosa, A., Gutierrez, M. C., Ruiz- 28 Berraquero, F. & Ramos-Cormenzana, A. (1990). Volcaniella eurihalina gen. nov., a 29 moderately halophilic non-motile gram-negative rod. Int J Syst Bacteriol 40, 261-267. 30 Quesada, E., Ventosa, A., Rodríguez-Valera, F., Megías, L. & Ramos-Cormenzana, A. (1983). 31 Numerical taxonomy of moderately halophilic Gram-negative bacteria from hypersaline soils. J 32 Gen Microbiol 129, 2649-2657. 33 Quesada, E., Béjar, V., Ferrer, M.R., Calvo, C., Llamas, I., Martínez-Checa, F., Arias, S., 34 Ruiz-García, C., Páez, R., Martínez-Cánovas J. & Del Moral, A. (2004). Moderately 35 halophilic, exopolysaccharide-producing bacteria. In Halophilic Microorganisms pp. 297-314, 36 Edited by A. Ventosa. Heilderberg: Springer-Verlag.

9 1 Rodríguez-Valera, F., Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1981). Characteristics 2 of the heterotrophic bacterial populations in hypersaline environments of different salt 3 concentrations. Microbiol Ecol 7, 235-243. 4 Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. 5 & Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with thermostable 6 DNA polymerase. Science 239, 487-491. 7 Sutherland, I. W. (1990). Biotechnology of microbial exopolysaccharides. Cambridge: 8 Cambridge University Press. 9 Suzuki, T., Muroga, Y., Takahama, M. & Nishimura, Y. (1999). Roseivivax halodurans gen. 10 nov., sp. nov., and Roseivivax halotolerans sp. nov. aerobic bacteriochlorophyll-containing 11 bacteria isolated from a saline lake. Int J Syst Bacteriol 49, 629-634. 12 Thompson, J. D., Gibson, T. J., Plewniak, K., Jeanmougin, F. & Higgins D. G. (1997). The 13 ClustalX windows interface: flexible strategies for multiple sequence alignments aided by quality 14 analysis tools. Nucleic Acids Res 24, 4876-4882. 15 Ventosa, A., Quesada, E., Rodríguez-Valera, F., Ruiz-Berraquero, F. & Ramos- 16 Cormenzana, A. (1982). Numerical taxonomy of moderately halophilic gram-negative rods. J 17 Gen Microbiol 128, 1959-1968. 18 Ventosa, A., Nieto, J.J. & Oren, A. (1998). Biology of moderately halophilic aerobic bacteria. 19 Microbiol Mol Biol Rev 62, 504-544. 20 Ziemke, F., Manfred, G.H., Lalucat, J. & Roselló-Mora, R. (1998). Reclassification of 21 Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst 22 Bacteriol 48, 179-186. 23

10 1 Table 1. Characteristics that distinguish Palleronia marisminoris from related members of the 2 family “Rhodobacteraceae”. 3 4 1, Salipiger mucosus CECT 5855T (Martínez-Cánovas et al., 2004e); 2 and 3, Roseivivax 5 halodurans JCM 10272T and Roseivivax halotolerans JCM 10271T (Suzuki et al., 1999; 6 Nishimura et al., 1994).

Characteristic P. 1 2 3 marisminoris

Source of isolation Hypersaline soil Hypersaline soil Charophyte sp. Cyanobacterial in a saline lake mat in a saline lake Pigment Pink - Pink Pink Flagella* - - S, SP S, SP PHA + + ND ND Oxidase - + + + EPS production + + - - Bacteriochlorophyll a - - + + Na+ requirement + + - + Salt growth range (% 0.5-15 0.5-20 0-20 0.5-20 w/v) Optimum salt 5 3-6 ND ND concentration (%,w/v) - - NO3 to NO2 - - + - Acid from glucose - - + + Growth on glucose† - - + + Indol - - + + ONPG + - + + Urease - + - + Phosphatase + + + - Gluconate oxidation - + ND ND Major fatty acids (%)‡ 18:1ω7c (68.9) 18:1ω7c (78.0) 18:1 18:1 16:0 (4.3) 16:0 (12.4) (not quantified) (not quantified) 18:0 (3.4) 18:0 (2.0) 16:1ω7c (1.3) 3-OH fatty acids 10:0 (5.0) 12:1 (2.3) Methyl fatty acids 11-met 18:1ω7c (1.9) Cyclo-substituted fatty 19:0 cyclo ω8c 19:0 cyclo ω8c acids (12.8) (2.3) Unidentified fatty 4.870 RT (2.3) acids G+C content (mol %) 64.2 64.5 64.4 59.7 7 *S, single; SP, subpolar; †Growth on minimum medium; ‡Only percentages higher than 1% are 8 shown. 9 +, positive; -, negative; ND, no data available; RT, retention time.

11 1 Fig. 1. Transmission electron micrograph of Palleronia marisminoris strain B33T cells stained 2 with ruthenium red. Bar, 1 µm. 3 4 Fig. 2. Phylogenetic relationships between Palleronia marisminoris strain B33T and other 5 genera of the “Rhodobacteraceace” family. The tree was constructed using the neighbour- 6 joining algorithm. Only bootstrap values above 50% are shown (1000 replications). Bar, 1% 7 estimated sequence divergence. 8 9 Fig. S1. Phylogenetic relationships between Palleronia marisminoris strain B33T and other 10 genera of the “Rhodobacteraceace” family plus other taxa of Gram-negative halophilic bacteria. 11 The tree was constructed using the neighbour-joining algorithm. Only bootstrap values above 12 50% are shown (1000 replications). Bar, 2% estimated sequence divergence. 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

12 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

13 1 2

T 86 Roseisalinus antarcticus DSM 11466 (AJ605747) 79 Oceanicola granulosus HTCC 2516T (AY424896)

Ketogulonicigenium vulgare DSM 4025T (AF136849)

Lok tanella salsilacus LMG 22000T (AJ582228)

Octadecabacter arcticus CIP 106732T (U73725)

Jannaschia helgolandensis DSM 14858T (AJ438157)

64 Thalassobacter stenotrophicus CECT 5294T (AJ631302 )

T 86 Antarctobacter heliothermus DSM 11445 (Y11552) Sagittula stellata ATCC 700073T (U58356)

Sulfitobacter pontiacus DSM 10014T (Y13155)

T 85 Roseobacter litoralis ATCC 49566 (X78312) 57 Staleya guttiformis DSM 11458T (Y16427)

Leisingera methylohalidivorans DSM 14336T (AY005463) 70 Silicibacter lacuscaerulensis DSM 11314T (U77644)

99 Ruegeria atlantica DSM 5823T (D88526)

59 Roseovarius tolerans DSM 11457T (Y11551)

54 Salipiger mucosus CECT 5855T (AY527274 ) B3B333T (AY926462 ) 78 Roseivivax halodurans JCM 10272T (D85829)

85 Roseivivax halotolerans JCM 10271T (D85831)

Roseinatronobacter thiooxidans DSM 13087T (AF249749) 73 Methylarcula marina VKM B-2159 (AF030436)

Paracoccus denitrificans LMG 4218T (X69159)

Rubrimonas cliftonensis OCh 317T (D85834)

Rhodovulum sulfidophilum DSM 1374T (D16423)

Rhodobacter capsulatus ATCC 11166T (D16427)

Amaricoccus kaplicensis ACM 5099T (U88041)

Roseibium denhamense OCh 254T (D85832)

Maricaulis maris ATCC 15268T (AJ227802)

99 Hirschia baltica IFAM 1418 (X52909) 83 Hyphomonas polymorpha DSM 2665T (AJ227813)

0.01 3 4 5 6 7 8 9

14 1 2

100 Ketogulonicigenium robustum NRRL B-21627 (AF136850) 69 Ketogulonicigenium vulgare DSM 4025T (AF136849) Roseisalinus antarcticus DSM 11466T (AJ605747) T 71 Oceanicola granulosus HTCC 2516 (AY424896) Marinosulfonomonas methylotropha PSCH4T (U62894) Loktanella vestfoldensis LMG 22003T (AJ582226) Jannaschia helgolandensis DSM 14858T (AJ438157) LMG 22007T (AJ582225) 77 Loktanella fryxellensis T 100 Loktanella salsilacus LMG 22000 (AJ582228) 77 Thalassobacter stenotrophicus CECT 5294T (AJ631302 ) Ruegeria gelatinovorans ATCC 25655T (D88523) Octadecabacter arcticus CIP 106732T (U73725) T 100 Octadecabacter antarcticus CIP 106731 (U14583) 97 Roseobacter denitrificans ATCC 33942T (M96746) Roseobacter litoralis ATCC 49566T (X78312) 91 53 Sulfitobacter mediterraneus DSM 12244T (Y17387) Staleya guttiformis DSM 11458T (Y16427) T 59 Sulfitobacter pontiacus DSM 10014 (Y13155) 73 Sulfitobacter brevis DSM 11443T (Y16425) 77 Roseovarius nubinhibins DSM 15170T (AF098495) Roseovarius tolerans DSM 11457T (Y11551) Silicibacter pomeroyi DSM 15171T (AF098491) DSM 11314T (U77644) 90 Silicibacter lacuscaerulensis T 94 Ruegeria atlantica DSM 5823 (D88526) 60 52 Ruegeria algicola ATCC 51440T (X78315) 67 Leisingera methylohalidivorans DSM 14336T (AY005463) 94 Roseobacter gallaeciensis ATCC 700781T (Y13244) Antarctobacter heliothermus DSM 11445T (Y11552) T 92 Sagittula stellata ATCC 700073 (U58356) Loktanella hongkongensis JCM 12479T (AY600300) 59 Salipiger mmuuccoessucenss CECT 5855T (AY527274 ) B3B333 T (AY926462 ) T 77 Roseivivax halodurans JCM 10272 (D85829) T 80 Roseivivax halotolerans JCM 10271 (D85831) Roseinatronobacter thiooxidans DSM 13087T (AF249749) Methylarcula marina VKM B-2159 (AF030436) LMG 4218T (X69159) 70 Paracoccus denitrificans 62 T 64 Paracoccus kocurii JCM 7684 (D32241) Rhodobacter capsulatus ATCC 11166T (D16427) T 79 Rhodobacter sphaeroides ATCC 17023 (D16424) 84 Rhodovulum sulfidophilum DSM 1374T (D16423) Rubrimonas cliftonensis OCh 317T (D85834) 67 Amaricoccus kaplicensis ACM 5099T (U88041) Stappia aggregata IAM 12614 (D88520) T 100 Roseibium denhamense OCh 254 (D85832) Maricaulis maris ATCC 15268T (AJ227802) IFAM 1418 (X52909) 72 Hirschia baltica T 99 Hyphomonas jannaschiana DSM 5153 (AF082789) Idiomarina baltica DSM 15154T (AJ440214) Cobetia marina ATCC 25374T (AJ306890) 100 Halomonas elongata DSM 3043T (AJ295147) 100 T 98 Chromohalobacter marismortui ATCC 17056 (X87219)

0.02 3 4 5 6 7

15