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1 Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch, Russian Academy of Sciences, 690022 Vladivostok, Prospekt 100 Let Vladivostoku, 159, Russia
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
3 Institute of Marine Biology, Far-Eastern Branch, Russian Academy of Sciences, 690041 Vladivostok, Russia
4 GBF – Gesellschaft für Biotechnologische Forschung GmbH, D-38124 Braunschweig, Germany
Correspondence
E. Stackebrandt
Erko{at}dsmz.de
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ABSTRACT |
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MAIN TEXT |
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Cell morphology was examined by transmission and scanning electron microscopy of cells grown in MB after 30 h incubation. Cells were fixed with 1 % (v/v) glutaraldehyde and negatively stained with 4 % (w/v) aqueous uranyl acetate and carbon film. Samples were examined by using a Zeiss transmission electron microscope (model TEM910) at an acceleration voltage of 80 kV at calibrated magnifications. Gram-reaction, oxidase, catalase and hydrolysis of starch, casein, DNA, chitin and gelatin were tested as described by Baumann et al. (1972)
and Smibert & Krieg (1994). Growth at 4–45 °C was determined on MA and TSA. Tolerance of NaCl was tested by using glucose–peptone nutrient medium, prepared with artificial sea water and supplemented with NaCl concentrations of 0, 0·5, 1, 3, 5, 8, 10, 12 and 15 % (w/v). Ability to use phenol as the sole carbon source was determined on minimal media that contained 2 or 5 % NaCl (w/v) and 4 mM phenol for up to 7 days incubation. Leifson's oxidation–fermentation medium for marine bacteria (Leifson, 1963) was used to test acid production from carbohydrates with 1 % (w/v) of each compound. Other biochemical tests were carried out by using API 20NE test kits (bioMérieux) according to the manufacturer's instructions, except that the culture was suspended in 2 % (w/v) NaCl solution, and by the Biolog GN MicroPlate panel. For the latter tests, strain KMM 3654T was grown on MA at 28 °C for 24 h and Biolog microtitre plates were inoculated with cells suspended in 2 % (w/v) NaCl solution. Results were read automatically with a spectrophotometer after 24 and 48 h incubation at 28 °C. For lipid analysis, the strain was cultivated on MA at 28 °C for 2 days. Fatty acid methyl esters were prepared from cells by acid-catalysed transmethylation and analysed by GLC. Whole-cell fatty acids and phospholipids were examined according to procedures described previously (Svetashev et al., 1995; Ivanova et al., 2000). DNA G+C content was determined by HPLC according to the method of Mesbah et al. (1989).
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and sequencing of PCR products were carried out as described by Rainey et al. (1996). Purified PCR products were sequenced directly by using a Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) according to the manufacturer's instructions. An Applied Biosystems 310 Genetic Analyzer was used for electrophoresis of sequence reaction products. The 16S rDNA sequence of strain KMM 3654T was aligned manually with nucleotide sequences obtained from GenBank/EMBL. The method of Jukes & Cantor (1969) was used to calculate evolutionary distances. Accession numbers of reference strains used in the phylogenetic analysis are shown in Fig. 1.
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) were the closest phylogenetic neighbours, respectively displaying 96·7 and 95·6 % sequence similarity to strain KMM 3654T. Ferrimonas balearica DSM 9799T (Rosselló-Mora et al., 1995), Tolumonas auensis DSM 9187T (Fischer-Romero et al., 1996) and members of the families Vibrionaceae, Enterobacteriaceae and Aeromonadaceae were more distantly related (<92 % sequence similarity). This relationship is displayed in the 16S rRNA gene sequence dendrogram based on the additive treeing algorithm of DeSoete (1983) (Fig. 1
). Based on moderate sequence similarity of <97 %, the novel isolate can be considered to be a separate taxonomic entity from the two described species of the genus Oceanimonas (Stackebrandt & Goebel, 1994), which share only 38·6 % DNA similarity (Brown et al., 2001).
Chemotaxonomically, isolate KMM 3654T shared properties that were also reported for the two species of the genus Oceanimonas (Brown et al., 2001), i.e. whole-cell fatty acids, phospholipids and DNA G+C content of 56·4 mol%, which is similar to those reported for O. doudoroffii DSM 7028T and O. baumannii ATCC 700832T (each 54 mol%). High amounts (up to 90 % of the total) of fatty acids C16 : 0, C16 : 1cis and C18 : 1cis found in strain KMM 3654T were also reported for Oceanimonas species (Brown et al., 2001). Quantitative analysis is given in the species description. KMM 3654T contained phosphatidylethanolamine and phosphatidylglycerol (43·7 and 46·9 %, respectively) as the main phospholipids. These compounds were also found in the two Oceanimonas species; KMM 3654T differed from these species in the lack of fatty acid C12 : 0 and in a higher proportion (9·4 %) of diphosphatidylglycerol.
Strain KMM 3654T was aerobic, Gram-negative, moderately halophilic and oxidase- and catalase-positive. Cells were coccoid, 1·0–1·2 µm in diameter, encapsulated and motile by a single polar flagellum (Fig. 2a). Cells grown on hard media occurred as single motile or non-motile cells, due to their ability to lose flagella easily, and formed aggregates and some fibrillar structures when cultivated in liquid media (Fig. 2b). The isolate required sodium ions for growth and grew in 0·5–10 % NaCl at 4–42 °C, with optima between 1 and 3 % NaCl and 28 and 35 °C, respectively. After 24 h on MA, strain KMM 3654T produced smooth, shining, slightly yellow colonies with regular edges that were 3–5 mm in diameter. Diffusion of yellowish pigment into the medium was observed. Growth was weak or delayed for 5–7 days when cultivated in minimal salts medium that contained 2 % NaCl and phenol as the sole carbon and energy source, which was used to cultivate strains of Oceanimonas (Brown et al., 2001), but growth did not occur in this medium when the salinity was raised to 5 %. The main phenotypic characteristics of strain KMM 3654T and related Oceanimonas species are shown in Table 1.
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observed production of exopolymer and, in addition, fibrillar material that connected marine bacterial cells. The capsular envelope has been hypothesized to play a basic role in bacterial attachment to surfaces in marine environments. Taking into consideration the fact that the habitat of KMM 3654T is marine sand sediment, the formation of cell aggregates and capsular and fibrillar-like materials may be a useful cellular adaptation for attachment to surfaces. Strain KMM 3654T lacked the ability to degrade phenol, which is positive for O. doudoroffii and O. baumannii (Brown et al., 2001). The new isolate exhibited weak reactions for utilization of Tween 40, Tween 80, 
-ketobutyric acid, -ketovaleric acid and L-glutamic acid and did not utilize most of the carbohydrates, organic acids or amino acids provided by the API 20NE and Biolog substrate panels (Table 1).
Strain KMM 3654T can be distinguished from members of the genus Oceanimonas by using a combination of biochemical characteristics (Table 1) and phylogenetic distance (Fig. 1). Based on these results, we propose to classify isolate KMM 3654T in a novel genus and species, Oceanisphaera litoralis gen. nov., sp. nov., with the type strain KMM 3654T (=DSM 15406T).
Description of Oceanisphaera gen. nov.
Oceanisphaera (O.ce.a.ni.sphae'ra. L. masc. n. oceanus ocean; L. fem. n. sphaera ball, globe, sphere; N.L. fem. n. Oceanisphaera oceanic sphere).
Spherical cells, 1·0–1·2 µm in diameter and motile by means of flagella. Gram-negative chemoorganotroph with absolute requirement for sodium ions. Aerobic. Moderately halophilic and oxidase- and catalase-positive. Major phospholipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Major fatty acids are C16 : 0, C16 : 1
7c and C18 : 17c. DNA G+C content is 56·4 mol% (HPLC). Isolated from a marine environment. Phylogenetically related to the genus Oceanimonas in the 
-Proteobacteria. The type species is Oceanisphaera litoralis.
Description of Oceanisphaera litoralis sp. nov.
Oceanisphaera litoralis (li.to.ra'lis. L. fem. adj. litoralis of or belonging to the seashore).
Gram-negative, aerobic, oxidase- and catalase-positive, motile by a single polar flagellum, spherical, encapsulated single cells that are 1·0–1·2 µm in diameter. May be non-motile. Able to form aggregates and fibrillar structures when cultivated in liquid media. Moderately halophilic, does not grow without sodium ions; grows in 0·5–10 % NaCl at 4–42 °C, but not at 44 °C. On MA, strain KMM 3654T produces smooth, shining, yellowish colonies with regular edges that are 3–5 mm in diameter after 24 h incubation at 28 °C. Gelatin, casein, aesculin, starch, DNA and chitin are not hydrolysed. No acid is produced from glucose, sucrose, maltose, lactose, N-acetylglucosamine, arabinose, rhamnose, galactose, glycerol or mannitol. Positive for nitrate reduction, urease activity and utilization of malate, citrate and phenylacetate. Metabolic properties are indicated in Table 1. According to Biolog system identification tests, KMM 3654T weakly utilizes Tween 40, Tween 80, -ketobutyric acid, -ketovaleric acid and L-glutamic acid. The rest of the organic substrates included in the Biolog panel are not utilized. Main fatty acids are C16 : 0 (21·6 %), C16 : 17c (41·0 %) and C18 : 17c (27·5 %); minor amounts of C15 : 0 (1·8 %), iso-C16 : 0 (1·6 %), C17 : 0 (1·9 %) and C17 : 18c (1·9 %) are present. Phospholipids consist of phosphatidylethanolamine (43·7 %), phosphatidylglycerol (46·9 %) and diphosphatidylglycerol (9·4 %). DNA G+C content is 56·4 mol% (HPLC).
The type strain is KMM 3654T (=DSM 15406T). Isolated from a bottom sand sample, Peter the Great Bay, Sea of Japan, Pacific Ocean, Russia.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brown, G. R., Sutcliffe, I. C. & Cummings, S. P. (2001). Reclassification of [Pseudomonas] doudoroffii (Baumann et al. 1983) into the genus Oceanomonas gen. nov. as Oceanomonas doudoroffii comb. nov., and description of a phenol-degrading bacterium from estuarine water as Oceanomonas baumannii sp. nov. Int J Syst Evol Microbiol 51, 67–72.[Abstract]
DeSoete, G. (1983). A least squares algorithm for fitting additive trees to proximity data. Psychometrika 48, 621–626.[CrossRef]
Fischer-Romero, C., Tindall, B. J. & Jüttner, F. (1996). Tolumonas auensis gen. nov., sp. nov., a toluene-producing bacterium from anoxic sediments of a freshwater lake. Int J Syst Bacteriol 46, 183–188.
Heissenberger, A., Leppard, G. G. & Herndl, G. J. (1996). Relationship between the intracellular integrity and the morphology of the capsular envelope in attached and free-living marine bacteria. Appl Environ Microbiol 62, 4521–4528.[Abstract]
Ivanova, E. P., Zhukova, N. V., Svetashev, V. I., Gorshkova, N. M., Kurilenko, V. V., Frolova, G. M. & Mikhailov, V. V. (2000). Evaluation of phospholipid and fatty acid compositions as chemotaxonomic markers of Alteromonas-like Proteobacteria. Curr Microbiol 41, 341–345.[CrossRef][Medline]
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Leifson, E. (1963). Determination of carbohydrate metabolism of marine bacteria. J Bacteriol 85, 1183–1184.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt, E. (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46, 1088–1092.
Rosselló-Mora, R., Ludwig, W., Kämpfer, P., Amann, R. & Schleifer, K. H. (1995). Ferrimonas balearica gen. nov., sp. nov, a new marine facultative Fe(III)-reducing bacterium. Syst Appl Microbiol 18, 196–202.
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–655. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Svetashev, V. I., Vysotskii, M. V., Ivanova, E. P. & Mikhailov, V. V. (1995). Cellular fatty acids of Alteromonas species. Syst Appl Microbiol 18, 37–43.
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