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1 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
2 Université P and M Curie (Paris 6), Observatoire Océanologique de Banyuls, CNRS URA 2071, F-66650 Banyuls sur Mer, France
Correspondence
Sisinthy Shivaji
shivas{at}ccmb.res.in
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7c and C18 : 17c. The DNA G+C content of strain CK13T was 41·2 mol%. Phosphatidylethanolamine and phosphatidylglycerol were identified as the predominant phospholipids. All the above characteristics support the affiliation of strain CK13T to the genus Marinomonas. Phylogenetic analysis and phenotypic and genotypic distinctiveness confirmed that strains CK13T and CK16 are members of a novel species of the genus Marinomonas, for which the name Marinomonas polaris sp. nov. is proposed. The type strain is CK13T (=MTCC 6645T=DSM 16579T=JCM 12522T).
A supplementary table detailing the cellular fatty acid content of strain CK13T and related Marinomonas species is available in IJSEM Online.
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), as these species formed a separate and distinct rRNA branch when compared with the other true alteromonads (Van Landschoot & De Ley, 1983). Species assigned to the genus Marinomonas are rod-shaped, motile, lack amylase, gelatinase and lipase activities and have the ability to utilize glycerol, lactate and m-hydroxybenzoate, but not butyrate or valerate as carbon sources. To date, seven species of Marinomonas have been described, Marinomonas communis, M. vaga, M. mediterranea, M. primoryensis, M. ushuaiensis, M. pontica and M. aquimarina (Baumann et al., 1972; Solano & Sanchez-Amat, 1999; Romanenko et al., 2003; Ivanova et al., 2005; Macian et al., 2005; Prabagaran et al., 2005). In the present study, a bacterial strain CK13T, isolated from sea water off the subantarctic Kerguelen island, has been identified as representing a novel species of the genus Marinomonas.
Strains CK13T and CK16 were isolated from sea water collected at a site located 110 km south-west of the subantarctic Kerguelen islands (50° 40' S 68° 25' E). The strains were isolated along with 45 other pure strains by plating 200 µl sea water on marine agar 2216 (Difco) and incubating the plates at 12 °C for 10 days. Marine agar was used for growth and maintenance of the strains and for the determination of the phenotypic and chemotaxonomic characteristics as listed in Table 1 and Supplementary Table S1 (see IJSEM Online). Luria–Bertani medium [1·0 % (w/v) tryptone, 0·5 % (w/v) yeast extract and 1·0 %(w/v) NaCl] was used to test the salt tolerance of the organism. The shape, size and motility of the cells was ascertained using a Leitz Diaplan phase-contrast microscope with an oil-immersion objective (x100). The sensitivity of the cultures to antibiotics was checked by using antibiotic discs (Himedia). For biochemical tests, cultures were grown at 22 °C on marine agar 2216 and tests were performed as described by Baumann et al. (1984) and Smibert & Krieg (1994). Utilization of various carbon compounds as sole carbon sources was tested in liquid minimal medium containing (l–1) 1 g ammonium chloride, 0·075 g dipotassium hydrogen phosphate, 1·45 g calcium chloride, 30·0 g sodium chloride, 6·15 g magnesium chloride, 0·75 g potassium chloride and 0·028 g ferrous sulphate, supplemented with 0·2 % of the filter-sterilized carbon source (Romanenko et al., 2003). Fatty acid methyl esters were prepared from cells grown at 22 °C for 48 h according to the method of Sato & Murata (1988) and analysed as described by Kiran et al. (2004). The modified method of Bligh & Dyer (1959) was employed to extract polar lipids and molybdenum blue reagent was used to detect lipids containing phosphate esters. DNA was isolated and the DNA G+C content was determined as described by Gupta et al. (2004). DNA–DNA hybridization was performed by the membrane filter method of Tourova & Antonov (1987) as described by Shivaji et al. (1992). M. pontica LMG 22531T and M. primoryensis IAM 15010T were used as controls in biochemical tests, in the identification of fatty acids and polar lipids and in DNA–DNA hybridization experiments.
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. To determine the phylogenetic affiliation of the novel strains, the almost-complete 16S rRNA gene sequence (1475 bp) of strain CK13T was aligned with related species of the genus Marinomonas using CLUSTAL W (Thompson et al., 1994). Pairwise evolutionary distances were computed using the DNADIST program with the Kimura two-parameter model as developed by Kimura (1980). Phylogenetic trees were constructed using the neighbour-joining and UPGMA tree-making algorithms from the PHYLIP package (Felsenstein, 1993). The stability among clades of the phylogenetic tree was assessed by performing 1000 replicates and analysing the dataset using the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE from the PHYLIP package.
Marine isolates CK13T and CK16 are aerobic, Gram-negative, halotolerant, rod-shaped and motile bacteria which are able to grow between 4 and 37 °C. Colony appearance on marine agar is smooth, non-pigmented and convex. Morphological features and some biochemical properties characteristic of members of the genus Marinomonas, such as lack of amylase, lipase and gelatinase activities, the presence of catalase activity, but weak oxidase activity and the utilization of glycerol, but not butyrate, are also exhibited by both novel strains (Table 1
). The fatty acid content, with iso- and branched-chain fatty acids C16 : 0 (6·2 %), iso C16 : 0 (18·5 %), C16 : 17c (26·5 %) and C18 : 17c (40·0 %) as the major fatty acids, and the presence of phosphatidylethanolamine and phosphatidylglycerol as the major phospholipid constituents also indicate that CK13T and CK16 are similar to the previously described Marinomonas species (Romanenko et al., 2003; Ivanova et al., 2000, 2005; Prabagaran et al., 2005) (see Supplementary Table S1 in IJSEM Online). Other morphological and biochemical characteristics that differentiate CK13T from recognized Marinomonas species are given in Table 1 and in the description of the novel species. Fatty acid content was similar in CK13T, M. pontica LMG 22531T and M. primoryensis IAM 15010T (see Supplementary Table S1 in IJSEM Online).
Phylogenetic analysis of the almost-complete 16S rRNA gene sequence (1475 nucleotides) of strain CK13T using the neighbour-joining algorithm confirmed the affiliation of this marine strain to the genus Marinomonas. The phylogenetic tree based on the above analysis showed that strain CK13T is within the species cluster comprising the genus Marinomonas and forms a clade with M. primoryensis IAM 15010T and M. pontica LMG 22531T, with a bootstrap value of >90 % (Fig. 1). The close relationship between CK13T, M. primoryensis and M. pontica was further evident from a BLAST analysis of the 16S rRNA gene sequence of CK13T, which exhibits 97 and 96 % similarity with M. primoryensis and M. pontica, respectively. Despite the high similarity at the 16S rRNA gene sequence level, in terms of DNA–DNA hybridization strain CK13T shares only 58 and 40 % relatedness with M. pontica LMG 22531T and M. primoryensis IAM 15010T, respectively. Further, CK13T differs from these closely related species in regard to a number of phenotypic characteristics (Table 1). According to the criteria recommended for discriminating species (Stackebrandt & Goebel, 1994), strain CK13T, which exhibits >3 % difference at the 16S rRNA gene sequence level with all previously described Marinomonas species, <70 % relatedness in DNA–DNA hybridization with the closely related species M. pontica and M. primoryensis and which also differs in phenotypic traits from these two Marinomonas species, should be classified as a member of the genus Marinomonas. It is concluded that strain CK13T represents a novel species, for which the name Marinomonas polaris sp. nov. is proposed.
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Cells are Gram-negative, motile, rod-shaped and are 0·6–0·8 µm wide and 2–3·2 µm in length. On marine agar, colonies are non-pigmented, cream, circular, raised, smooth and 2–3 mm in diameter. Grows between 4 and 37 °C and with 0–11 % salt in the medium. The optimum pH and salt concentration for growth are pH 7·0 and 5 % salt, respectively. CK13T tests positive for phosphatase, catalase, 
-galactosidase, aesculinase and arginine dihydrolase activities and for citrate utilization, but negative for gelatin hydrolysis, indole production and lipase and lysine decarboxylase activities. Negative reaction in the Voges–Proskauer test, does not reduce nitrate to nitrite and no denitrification. Produces acid from sucrose, D-maltose, D-mannose, D-xylose, D-ribose and D-fructose, but not from glucose, melibiose or rhamnose. Utilizes D-glucose, D-fructose, D-xylose, D-galactose, D-ribose, D-mannose, sucrose, lactose, D-maltose, D-sorbitol, inositol, erythritol, adonitol, glycerol, D-cellobiose, dextrin, acetate, D-glucuronic acid, sodium gluconate, sodium malonate, L-glycine, L-histidine, L-threonine, L-arginine, L-asparagine and glutamine, but not L-sorbose, L-rhamnose, L-raffinose, D-arabinose, xylitol, glycogen, citrate, lactate, malate, succinate, butyrate, sodium formate, benzoate, m-hydroxybenzoate, 2-oxoglutarate, propionate, L-lysine, L-alanine, L-serine, cysteine, L-aspartic acid or L-glutamic acid as sole carbon sources. The type strain is sensitive to amikacin (30 µg), ciprofloxacin (30 µg), kanamycin (30 µg), chloramphenicol (30 µg), tobramycin (10 µg), nitrofurantoin (300 µg), vancomycin (30 µg), ampicillin (10 µg), tetracycline (30 µg), streptomycin (30 µg), erythromycin (30 µg), lincomycin (30 µg), penicillin (30 µg), nalidixic acid (30 µg), nitrofurantoin (300 µg), chloramphenicol (30 µg), gentamicin (30 µg), cephatoxime (30 µg), cefoperazone (75 µg), cefazolin (30 µg), oleandomycin (15 µg), polymyxin B (50 U), spectinomycin (100 µg), tobramycin (30 µg) and rifampicin (30 µg), but resistant to bacitracin (10 U). The predominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. Major cellular fatty acids are C14 : 0 (1·2 %), anteiso C15 : 0 (1·2 %), iso C16 : 0 (18·5 %), C16 : 0 (6·2 %), C16 : 17c (26·5 %), anteiso C17 : 0 (2·5 %), C18 : 0 (2·4 %) and C18 : 17c (40·0 %). The DNA G+C content is 41·2 mol%.
The type strain, CK13T (=MTCC 6645T=DSM 16579T=JCM 12522T), was isolated from coastal sea water collected from the subantarctic Kerguelen island.
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