1 Alteromonas hispanica sp. nov., a polyunsaturated-fatty-acid-producing,
2 halophilic bacterium isolated from Fuente de Piedra (S.E., Spain)
3
4 Fernando Martínez-Checa, Victoria Béjar, Inmaculada Llamas, Ana del Moral
5 and Emilia Quesada.
6
7 Microbial Exopolysaccharide Research Group, Department of Microbiology,
8 Faculty of Pharmacy, Cartuja Campus, University of Granada, 18071 Granada,
9 Spain.
10
11 Key words: Alteromonas, Alteromonas hispanica, halophiles,
12 exopolysaccharides, polyunsaturated fatty acids.
13
14 Subject category: taxonomic note, new taxa, γ-Proteobacteria.
15
16 Author for correspondence: E. Quesada, Department of Microbiology, Faculty of
17 Pharmacy, Cartuja Campus, University of Granada, 18071 Granada, Spain.
18 Tel: +34 958 243871
19 Fax: +34 958 246235
20 e-mail: [email protected]
21
22 The GenBank/EMB/DDBJ accession number for the 16S rRNA gene sequence
23 of strain Alteromonas hispanica F-32T is AY926460.
1 1 Abstract
2
3 Strain F-32T, which produces exopolysaccharides and contains polyunsaturated
4 fatty acids, was isolated from a hypersaline water sample collected from Fuente
5 de Piedra (S.E. Spain). Phylogenetic analyses indicated conclusively that the
6 strain in question belonged to the genus Alteromonas. Phenotypic tests showed
7 that it could be assigned to the genus Alteromonas although it had a number of
8 distinctive characteristics; it is moderately halophilic, growing best with 7.5 -10%
o 9 w/v NaCl; it grows at 4 C and produces H2S; it does not grow with D-cellobiose,
10 D-fructose, D-galactose, D-glucose or lactose as sole sources of carbon and
11 energy; its fatty-acid profile is typical of Alteromonas but it also contains a large
12 amount of an unusual acid with three double bonds (18:3 ω6c [6, 9, 12]; 5.01%,
13 w/v). The major isoprenoid quinone is Q8. The DNA G+C composition is 46.3
14 mol%. The phylogenetic, phenotypic and genetic properties of strain F32T place
15 it within a novel species, for which we propose the name Alteromonas hispanica
16 sp. nov. The type strain is F-32T (= CECT 7067T = LMG 22958T).
17
18
2 1 The genus Alteromonas was isolated and named by Baumann et al. (1972)
2 (emended description by Gauthier et al., 1995; and later Van Trappen et al.,
3 2004) and originally contained a phylogenetically and phenotypically
4 heterogeneous group of Gram-negative, heterotrophic, marine bacteria, motile
5 by a single polar flagellum. Many of its species, however, have gradually been
6 reclassified into other genera such as Marinomonas, Pseudoalteromonas, and
7 Shewanella (Van Landschoot & de Ley, 1983; MacDonnell & Colwell, 1985;
8 Coyne et al., 1989; Gauthier et al., 1995; Sawabe et al., 2000; Ivanova et al.,
9 2000; 2001). Nowadays Alteromonas comprises only four valid species: A.
10 macleodii (Baumann et al., 1972 and 1984; Gauthier et al., 1995; Yi et al.,
11 2004), A. marina (Yoon et al., 2003), A. stellipolaris (Van Trappen et al., 2004)
12 and A. litorea (Yoon et al., 2004). Together with the genus Glaciecola (Bowman
13 et al., 1998) and Aestuariibacter (Yi et al., 2004), it is included within the family
14 Alteromonadaceae (Ivanova et al., 2004).
15
16 Alteromonadaceae are Gram-negative, rod-shaped, motile bacteria that
17 do not form endospores or microcysts. They are chemo-organotrophs, have a
18 respiratory metabolism and use oxygen as electron acceptor. They do not
19 denitrify or have dihydrolase activity. All the species require Na+ for growth and
20 in most of them the major isoprenoid quinone is Q8. The major fatty acids are
21 16:0, 16:1 ω7c, and 18:1 ω7c. All the species have been isolated from marine
22 habitats (coastal sea waters and marine invertebrates). The family is a member
23 of the γ-proteobacteria with the following nucleotide sequence characteristics:
24 304 (A), 734 (A), 736 (T), 770 (T), 809 (A). The type genus is Alteromonas
25 (Ivanova et al., 2004).
3 1 Van Trappen et al. (2004) made the last emended description of
2 Alteromonas, which was based on Gauthier et al (1995), when they discovered
3 that members of the genus were prosthecate, budding bacteria. In addition to
4 the traits reported for the family, the genus also includes bacteria which are
5 catalase and oxidase positive, unpigmented and not luminescent. Species of
6 the genus do not usually grow at 4oC, do not accumulate poly-β-
7 hydroxybutyrate and require a sea-water base for growth but not organic growth
8 factors. A. macleodii, A. marina and A. stellipolaris produce buds and prostheca
9 when they grow at low temperatures (12º - 20oC) for 3 or more days in complex
10 media with added sea salts. The G+C content of the DNA is 44 to 47 mol%. The
11 type species is A. macleodii.
12
13 In this study we describe strain F32T of Alteromonas, for which we
14 propose the name Alteromonas hispanica. This strain is the only representative
15 of the genus Alteromonas identified so far that has been isolated from an inland
16 hypersaline habitat and produces polyunsaturated fatty acids (PUFAs) at a
17 relatively high incubation temperature (32oC), which contradicts the notion that
18 only barophilic and psychrophilic marine species are able to produce significant
19 levels of PUFAs (Nogi et al., 1998; Russell and Nichols, 1999).
20
21 The strain studied here was isolated in 1998 from a hypersaline water sample
22 taken from Fuente de Piedra (Málaga, S. Spain), an inland, hypersaline
23 wetland, during a wide research program aimed at discovering new halophilic
24 bacteria for biotechnological purposes (Martínez-Cánovas et al., 2004;
25 Quesada et al., 2004). Strain F-32T was isolated using MY medium (Moraine &
4 1 Rogovin 1966), supplemented with 10% w/v marine salts (Rodriguez-Valera et
2 al., 1981). The strain was kept and routinely grown in MY medium, with the
3 addition of 7.5% w/v marine salts for optimum growth. Strain F32T was originally
4 characterized phenotypically by Martínez-Cánovas et al. (2004) by means of
5 135 tests. Its flagellation pattern was determined in this work by transmission
6 electron microscopy of negatively stained cells. The phenotypic data are given
7 in the species description. Table 1 shows the main phenotypic differences
8 between strain F-32T and the other four species of the genus Alteromonas. The
9 same table contains the G+C content of strain F32T estimated from the midpoint
10 value (Tm) of the DNA thermal denaturation profile, as described in Martínez-
11 Cánovas et al. (2004).
12
13 Colonies used for the analysis of prostheca and buds were grown on MY (7.5%
14 w/v) for 7 days at 12oC, as recommended by Van Trappen et al. (2004). EPS
15 and poly-β-hydroxybutyrate granules were observed in cells grown on the same
16 medium after 1 day’s incubation at 32oC. Transmission electron micrographs
17 were taken using the methods described by Bouchotroch et al. (2001).
18
19 Phylogenetic analyses were made according to Bouchotroch et al. (2001). We
20 determined the near complete 16S rRNA gene sequence of strain F-32T (1479
21 bp). The sequence obtained was compared to reference 16S rRNA gene
22 sequences available in the GenBank, EMBL and DDBJ databases obtained
23 from the National Centre of Biotechnology Information database using the
24 BLAST search. Phylogenetic analysis was made using the software MEGA
25 version 3.0 (Kumar et al., 2004) after multiple data alignments by CLUSTALX
5 1 (Thompson et al., 1997). Distances and clustering were determined using the
2 neighbour-joining and maximum-parsimony methods. The stability of clusters
3 was ascertained by performing a bootstrap analysis (1000 replications). The
4 phylogenetic tree obtained by neighbour joining is shown in Figure 1. The
5 maximum-parsimony algorithm gave a similar result (data not shown). Our
6 results indicate conclusively that strain F32T belongs to the genus Alteromonas,
7 showing from 95.3% to 97.4% similarity with the other four species within the
8 genus. As expected, the next closest neighbours were the other members of the
9 Alteromonadaceae family, Glaciecola (Ivanova et al., 2004) and Aestuariibacter,
10 (Yi et al., 2004). The nucleotide-sequence characteristics of the family
11 Alteromonadaceae, 304 (A), 734 (A), 736 (T), 770 (T), 809 (A) (Ivanova et al.,
12 2004) were present in strain F32T.
13
14 The fatty acids and quinones were analysed at the DSMZ (Deutsche Sammlung
15 von Mikroorganismen und Zellkulturen GmbH) in a culture of strain F-32T made
16 at 32oC in MY 7.5% w/v (see Table 2). The fatty-acid profile of strain F32T was
17 typical of Alteromonas species, with a predominance of 16:0, 16:1 ω7c, 18:1
18 ω7c, but it also contains large amounts of 16:0 N alcohol, 17:0 10 methyl, 18:0,
19 and an unusual unsaturated fatty acid (18:3 ω6c [6, 9, 12]). Unsaturated fatty
20 acids (PUFAs) are rare in mesophilic bacteria and have not been found so far in
21 any Alteromonas species. The predominant respiratory quinone was Q8
22 (ubiquinone 8, 96.5%; ubiquinone 7, 3.5%).
23 In conclusion, polyphasic analyses demonstrate that the new isolate
24 belongs to a consistent taxon and represents a novel species within the genus
25 Alteromonas, for which we propose the name Alteromonas hispanica.
6 1 Description of Alteromonas hispanica sp. nov.
2
3 Alteromonas hispanica (his pa’ ni ca: L. fem. adj. = Spanish)
4
5 The cells are straight rods 1-2 µm long and 0.75 µm wide, appearing either
6 singly or in pairs. They stain Gram negative and are motile by one polar
7 flagellum. They produce buds, prostheca and PHB. No spores are observed
8 under any conditions. Colonies are cream coloured, round, convex and mucoid.
9 Exopolysaccharide is produced. Growth pattern is uniform in a liquid medium.
10 The bacterium is chemo-organotrophic and strictly aerobic, i.e. anaerobic
11 respiration with nitrate, nitrite or fumarate is negative. Catalase and oxidase are
12 positive. It produces acids from maltose but not from any of the following
13 sugars: adonitol, L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose,
14 myo-inositol, lactose, D-mannitol, mannose, D-melezitose, L-rhamnose, D-
15 salicin, D-sorbitol, sorbose, sucrose or trehalose. It is moderately halophilic,
16 capable of growing in NaCl concentrations of 7.5% to 15% w/v (optimum 7.5% -
17 10%). It does not require additional magnesium or potassium salts. It grows
18 within the temperature range of 4ºC to 40ºC (optimum 32ºC) and at pH values
19 of between 5 and 10 (optimum 7 - 8). It shows positive activity for ONPG,
20 phosphatase, selenite reduction, H2S production from cysteine, and hydrolysis
21 of aesculin, casein, gelatine, Tween 20, Tween 80, starch and DNA. It shows
22 negative activity for nitrate and nitrite reduction, urease, lecithinase
23 phenylalanine deaminase, gluconate oxidation, growth on cetrimide agar,
24 growth on MacConkey agar, indol, methyl red, Voges Proskauer and
25 haemolysis. It grows in synthetic media supplemented with maltose and
7 1 mannitol as sole source of carbon and energy. It does not grow in synthetic
2 media supplemented with the following sole sources of carbon and energy, or
3 carbon, nitrogen and energy: aesculin, L-arabinose, D-cellobiose, D-fructose, D-
4 galactose, D-glucose, lactose, D-mannose, D-melezitose, L-rhamnose, D-
5 salicin, starch, D-trehalose, citrate, formate, fumarate, gluconate, lactate,
6 malonate, propionate, succinate, adonitol, ethanol, myo-inositol, sorbitol, L-
7 alanine, L-cysteine, L-histidine, DL-isoleucine, L-lysine, L-methionine, L-serine
8 and L-valine. It is susceptible to amoxicillin (25 µg), ampicillin (10 µg),
9 carbenicillin (100 µg), cefotaxime (30 µg), chloramphenicol (30 µg),
10 erythromycin (15 µg), kanamycin (30 µg), nalidixic acid (30 µg), nitrofurantoin
11 (300 µg), polymyxin B (300 UI), rifampycin (30 µg), streptomycin (10 µg),
12 sulphamide (250 µg), tobramycin (10 µg), and trimetroprim-sulphametoxazol
13 (1.25 µg-23.75 µg). It is resistant to cefoxitin (30 µg). The principal fatty acids
14 are (%): 16:0 N alcohol (7.35); 15:0 ISO 2OH/16: 1ω7c (22.10); 16:0 (13.77);
15 17:0 10 methyl (15.63); 18:0 (5.89); 18:3 ω6c (6, 9, 12) (5.01); and 18:1 ω7c
16 (14.30).
17
18 DNA G+C content is 46.3 mol% (Tm method).
19
20 The type strain, F-32T (= CECT 7067T = LMG 22958T), was isolated from a
21 hypersaline water sample taken at Fuente de Piedra (Málaga, S. Spain).
22
23
24
25
8 1 ACKNOWLEDGEMENTS
2
3 This research was supported by grants from the Dirección General de
4 Investigación Científica y Técnica (BOS2003-00498) and from the Consejería
5 de Innovación, Ciencia y Empresa de la Junta de Andalucía, Spain. The
6 authors are very grateful to Concepción Fernández and David Porcel for their
7 expertise in the electron-microscope studies and to their colleague Dr. J. Trout
8 for revising the English text.
9
10 REFERENCES
11
12 Baumann, L., Baumann, P., Mandel, M. & Allen, R. D. (1972). Taxonomy of
13 aerobic marine eubacteria. J Bacteriol 110, 402-429.
14 Baumann, P., Baumann, L., Bowditch, R. D. & Beaman, B. (1984).
15 Taxonomy of Alteromonas: A. nigrifaciens sp. nov., nom. rev.; A. macleodii; and
16 A. haloplanktis. Int J Syst Bacteriol 34, 145-149.
17 Bouchotroch, S., Quesada, E., del Moral, A., Llamas, I. & Béjar, V. (2001).
18 Halomonas maura sp. nov., a novel moderately halophilic, exopolysaccharide-
19 producing bacterium. Int J Syst Evol Microbiol 51, 1625-1632.
20 Bowman, J. P., McCammon, S. A., Brown, J. L. & McMeekin, T. A. (1998).
21 Glaciecola punicea gen. nov., sp. nov. and Glaciecola pallidula gen. nov., sp.
22 nov.: psychrophilic bacteria from Antarctic sea-ice habitats. Int J Syst Bacteriol
23 48, 1213-1222.
24 Coyne, V. E., Pillidge, C. J., Sledjeski, D. D., Hori, H., Ortiz-Conde, B. A.,
25 Muir, D. G., Weiner, R. M. & Colwell, R. R. (1989). Reclassification of
9 1 Alteromonas colwelliana to the genus Shewanella by DNA-DNA hybridization,
2 serology and 5S ribosomal RNA sequence data. Syst Appl Microbiol 12, 275-
3 279.
4 Gauthier, G., Gauthier, M. & Christen, R. (1995). Phylogenetic analysis of the
5 genera Alteromonas, Shewanella, and Moritella using genes coding for small-
6 subunit rRNA sequences and division of the genus Alteromonas into two
7 genera, Alteromonas (emended) and Pseudoalteromonas gen. nov., and
8 proposal of twelve new species combinations. Int J Syst Bacteriol 45, 755-761.
9 Ivanova, E. P., Chun, J., Romanenko, L. A., Matte, M. E., Mikhailov, V. V.,
10 Frolova, G. M., Huq, A. & Colwell, R. R. (2000). Reclassification of
11 Alteromonas distincta Romanenko et al. 1995 as Pseudoalteromonas distincta
12 comb. nov. Int J Syst Evol Microbiol 50, 141-144.
13 Ivanova, E. P., Flavier, S. & Christen, R. (2004). Phylogenetic relationships
14 among marine Alteromonas-like proteobacteria: emended description of the
15 family Alteromonadaceae and proposal of Pseudoalteromonadaceae fam. nov.,
16 Colwelliaceae fam. nov., Shewanellaceae fam. nov., Moritellaceae fam. nov.,
17 Ferrimonadaceae fam. nov., Idiomarinaceae fam. nov. and
18 Psychromonadaceae fam. nov. Int J Syst Evol Microbiol 54, 1773-1788.
19 Ivanova, E. P., Romanenko, L. A., Matté, M. H & 10 other authors (2001).
20 Retrieval of the species Alteromonas tetraodonis Simidu et al. 1990 as
21 Pseudoalteromonas tetraodonis comb. nov. and emendation of description. Int J
22 Syst Evol Microbiol 51, 1071-1078.
23 Kumar, S., Tamura, K. & Nei, M. (2004) MEGA3: Integrated software for
24 Molecular Evolutionary Genetics Analysis and sequence alignment. Brief
25 Bioinform 5, 150-163.
10 1 MacDonell, M. T. & Colwell, R. R. (1985). Phylogeny of the Vibrionaceae, and
2 recommendation for two new genera, Listonella and Shewanella. Syst Appl
3 Microbiol 6, 171-182.
4 Martínez-Cánovas, M. J., Quesada, E., Martínez-Checa, F. & Béjar, V. 2004). A
5 taxonomic study to establish the relationship between exopolysaccharide-
6 producing bacterial strains living in diverse hypersaline habitats. Curr Microbiol
7 48, 348-353
8 Moraine, R. A. & Rogovin, P. (1966). Kinetics of polysaccharide B-1459
9 fermentation. Biotechnol Bioeng 8, 511-524.
10 Nogi, Y., Kato, C. & Horikoshi, K. (1998). Taxonomic studies of deep-sea
11 barophilic Shewanella strains and description of Shewanella violacea sp. nov.
12 Arch Microbiol 170, 331-338.
13 Quesada, E., Béjar, V., Ferrer, M. R., Calvo, C., Llamas, I., Martínez-Checa,
14 F., Arias, S., Ruiz-García, C., Páez, R., Martínez-Cánovas J. & Del Moral, A.
15 (2004). Moderately halophilic, exopolysaccharide-producing bacteria. In
16 Halophilic microorganisms pp. 297-314. ed. Ventosa, A. Heilderberg: Springer-
17 Verlag.
18 Rhodes, M. E. (1958). The cytology of Pseudomonas spp. as revealed by a
19 silver-plating staining method. J Gen Microbiol 18, 639-648.
20 Rodríguez-Valera, F., Rúiz-Berraquero, F. & Ramos-Cormenzana, A.
21 (1981). Characteristics of the heterotrophic bacterial populations in hypersaline
22 environments of different salt concentrations. Microb Ecol 7, 235-243.
23 Russell, N. J. & Nichols, D. S. (1999). Polyunsaturated fatty acids in marine
24 bacteria – a dogma rewritten. Microbiology 145, 767-779.
11 1 Sawabe, T., Tanaka, R., Iqbal, M. M., Tajima, K., Ezura, Y., Ivanova, E. P. &
2 Christen, R. (2000). Assignment of Alteromonas elyakovii KMM 162T and five
3 strains isolated from spot-wounded fronds of Laminaria japonica to
4 Pseudoalteromonas elyakovii comb. nov. and the extended description of the
5 species. Int J Syst Evol Microbiol 50, 265-271.
6 Thompson, J. D., Gibson, T. J., Plewniak, K., Jeanmougin, F. & Higgins, D.
7 G. (1997). The ClustalX window interface: flexible strategies for multiple
8 sequence alignments aided by quality analysis tools. Nucleic Acids Res 24:
9 4876-4882.
10 Van Landschoot, A. & de Ley, J. (1983). Intra- and intergeneric similarities of
11 the rRNA cistrons of Alteromonas, Marinomonas (gen. nov.) and some other
12 Gram-negative bacteria. J Gen Microbiol 129, 3057-3074.
13 Van Trappen, S., Tan, T-L., Yang, J., Mergaert, J., & Swings, J. (2004).
14 Alteromonas stellipolaris sp. nov., a novel, budding, prosthecate bacterium from
15 Antarctic seas, and emended description of the genus Alteromonas. Int J Syst
16 Evol Microbiol 54, 1157-1163.
17 Wilson, P. W. & Knight, S. C. (1952) Experiments in bacterial physiology.
18 Burguess, Minneapolis.
19 Yi, H., Bae, K. S. & Chun, J. (2004). Aestuariibacter salexigens gen. nov., sp.
20 nov. and Aestuariibacter halophilus sp. nov., isolated from tidal flat sediment,
21 and amended description of Alteromonas macleodii. Int J Syst Evol Microbiol
22 54, 571-576.
23 Yoon, J-H., Kim, I. G., Kang, K. H., Oh, T-K. & Park, Y-H. (2003).
24 Alteromonas marina sp. nov., isolated from sea water of the East Sea in Korea.
25 Int J Syst Evol Microbiol 53, 1625-1630.
12 1 Yoon, J-H., Yeo, S-H., Oh, T-K. & Park, Y-H. (2004). Alteromonas litorea sp.
2 nov., a slightly halophilic bacterium isolated from an intertidal sediment of the
3 Yellow Sea in Korea. Int J Syst Evol Microbiol 54, 1197-1201.
4
13 1 Table 1. Phenotypic characteristics and DNA G+C content distinguishing Alteromonas hispanica from the other species of Alteromonas. 2 Data from Baumann et al., 1972; 1984; Gauthier et al., 1995; Ivanova et al., 2004; Van Trappen et al., 2004; Yi et al., 2004; Yoon et al., 2003; 2004 ; 3 and from this study. 4 Characteristic F-32T A. macleodii A. marina A. stellipolaris A. litorea Origin of the strain Hypersaline water Sea water Sea water Antarctic sea Intertidal sediment Fuente de Piedra East Sea in water Yellow Sea of Korea (Málaga, S. Spain) Korea Cellular size (µm) 1-2 x 0.75 1.8-2.5 x 0.9-1.3 2.5-4 x 1-1.2 2-7 x 0.4 2-4 x 0.9-1.2 Bud/prosthecum + + + + ND Pigmentation Cream Cream Cream Brown diffuse Cream PHB + - ND - ND EPS + ND - ND ND NaCl range (% w/v) 7.5-15 ND 2-15 1-10 2-14 NaCl optimum (% w/v) 7.5-10 ND 2-5 ND 2-5 Sea-salt range (% w/v) 3-20 ND ND ND ND Sea-salt optimum (% w/v) 5-15 ND ND ND ND Growth at 4 oC + - + + - Acid from: Glucose - + - - - Maltose + + - ND + Sucrose - + + +* - D-trehalose - + + + + H2S from cysteine + ND - - - Growth on D-cellobiose - + ND (+) ND D-fructose - + + + + D-galactose - + + + + D-glucose - + + + + Lactose - + + (+) + D-mannitol + + - + - L-serine - - ND +* ND G+C (mol%) 46.3 44.9-46.4 44-45 43-45 46 5 *, weak reaction; ND, not determined; (+) the majority of the strains are positive.
14 1 Table 2. Major fatty acids in the Alteromonas species.
2 Data from this work and from Van Trappen et al., 2004; Yoon et al., 2004
Fatty acid F-32T A. macleodii A. marina A. stellipolaris A. litorea
10:0 3OH 1.20 1.5 1.3 - 1.4 11:0 3OH - - 1.0 - 1.2 12:0 1.77 2.5 2.9 - 2.7 12 :0 3OH 1.06 1.2 1.1 - 1.1 14:0 2.07 2.5 2.6 - 3.6 15:0 - 2.5 2.8 - 2.1 15:1 ω8c - 1.7 1.1 - 1.0 16:0 13.77 23.8 21.2 12.6 20.0 16:0 iso - 1.1. - - - 16:0 N alcohol 7.35 6.6 3.2 - 5.6 16:1 ω7c alcohol 1.167 4.3 1.9 - 5.8 17:0 - 2.6 3.2 - 2.9 17:0 10 methyl 15.63 2.9 1.5 - 4.5 17:1 ω8c - 4.3 5.6 9.4 3.6 18:1 ω7c 14.30 9.9 11.8 18.0 12.5 18:0 5.89 - - - - 18:3 ω6c (6, 9, 12) 5.01 - - - - 15.0 iso 2OH/16:1ω7c 22.10 24.6 28.5 27.3 20.0 16:1 iso I/14:0 3OH 2.57 3.3 3.6 - 3.0 3 -, negative or less than 1%;
15 1 Fig.1. Phylogenetic relationship between strain F-32T of Alteromonas hispanica
2 and the other species of the genus Alteromonas, together with members of the
3 Alteromonadaceae family and other related Gammaproteobacteria. The tree
4 was constructed using the neighbour-joining algorithm. Only bootstrap values
5 above 50% are shown (1000 replications). Bar, 2% estimated sequence
6 divergence.
7
8 Fig. 2. Electron micrographs of negatively stained preparations of cells of strain
9 F-32T: a) showing a polar flagellum (F), b) prostheca (P) and buds (B). Cells
10 were stained with 1% uranyl acetate in 0.4% sucrose. Bars, 0.3 µm.
11
12 Fig. 3. Electron micrographs of thin-section preparations of cells of strain F-32T:
13 a) showing EPS and PHB granule, b) bud formations (B) and c) prostheca (P).
14 Thin-section preparations were stained with lead citrate and 1% uranyl acetate.
15 Bars, 0.3 µm.
16
17
18
19
20
21
22
23
24
25
16 1
2
3
4 KCCM 41638T (AF529060) 85 Alteromonas marina
T 5 58 Alteromonas macleodii DSM 6062 (Y18228) 72 Alteromonas litorea KCCM 41775T (AY428573) 100 6 Alteromonas stellipolaris LMG 21861T (AJ295715)
61 F32 (AY926460 )
7 Aestuariibacter salexigens DSM 15300T (AY207502) 100 72 Aestuariibacter halophilus DSM 15266T (AY207503) 8 Glaciecola punicea ACAM 611T (U85853)
73 9 Glaciecola pallidula ACAM 615T (U85854) Colwellia rossensis ACAM 608T (U14581) 10 76 Colwellia maris JCM 10085T (AB002630)
74 Colwellia psychrotropica ACAM 179T (U85846)
11 Colwellia psychrerythraea ATCC 27364T (AF001375) 100 57 Colwellia demingiae ACAM 459T (U85845) 12 Colwellia hornerae ACAM 607T (U85847) Pseudoalteromonas haloplanktis ATCC 14393T (X67024) 13 82 Pseudoalteromonas prydzensis ACAM 620T (U85855) 14 Pseudoalteromonas citrea DSM 8771T (X82137) 100 Pseudoalteromonas denitrificans ATCC 43337T (X82138)
15 Pseudoalteromonas rubra ATCC 29570T (X82147)
T 57 Psychromonas antarctica DSM 10704 (Y14697) 16 Moritella marina NCIMB 1144T (X82142)
56 T 17 Shewanella putrefaciens LMG 26268 (X81623) Vibrio cholerae CECT 514T (X76337) 18 0.02 19
20
21
22
23
24
25
17 1
2 B
3
4 B
P P 5
6
7 F a) b) 8
9
10
11
12 B
B P 13 P
14 B
15
16
17 a) b) c)
18
19
20
21
22
18