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1 Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Pr. 100 Let Vladivostoku 159, 690022 Vladivostok, Russia
2 BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
3 Department of Microbiology, Chungnam National University, 220 Gung-dong, Yusong, Daejon 305-764, Republic of Korea
4 Institute of Microbiology of the Russian Academy of Sciences, Pr. 60 Let October 7/2, 117811 Moscow, Russia
5 Department of Applied Microbiology, College of Agriculture and Life Sciences, Chungnam National University, 220 Gung-dong, Yuseong, Daejeon 305-764, Republic of Korea
6 Korea Institute of Bioscience and Biotechnology, 52 Oun-dong, Yusong, Daejon 305-333, Republic of Korea
Correspondence
Olga I. Nedashkovskaya
olganedashkovska{at}yahoo.com
or
olganedashkovska{at}piboc.dvo.ru
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) and currently comprises three species: Arenibacter latericius, Arenibacter troitsensis and Arenibacter certesii (Ivanova et al., 2001; Nedashkovskaya et al., 2003a, 2004). Members of the genus are Gram-negative, aerobic, heterotrophic and dark-orange-pigmented marine bacteria. These organisms were isolated from various marine environments, including bottom-sediment samples, the brown alga Chorda filum, the green alga Ulva fenestrata and the edible holothurian Apostichopus japonicus. The genera Muricauda (Bruns et al., 2001) and Zobellia (Barbeyron et al., 2001) are the closest phylogenetic relatives of the Arenibacter species. Novel phenotypic findings and improved determination of some previously described phenotypic properties justify emended descriptions of the genus Arenibacter and A. latericius. During June 2000 we isolated three novel strains, KMM 3961T, KMM 3979 and KMM 3980, from the green alga Ulva fenestrata, which was collected in Pallada Bay, Gulf of Peter the Great, Sea of Japan. A polyphasic taxonomic study of the algal isolates, cultured on marine agar 2216 (Difco), indicated that they represent a novel species of the genus Arenibacter.
The phylogenetic position of strain KMM 3961T was determined by analysis of the complete 16S rRNA gene sequence. Genomic DNA was prepared according to the protocol of Niemann et al. (1997). 16S rRNA gene amplification, purification and sequencing were performed as described by Vancanneyt et al. (2004) but with the following modifications: PCR-amplified 16S rRNA genes were purified by using a NucleoFast 96 PCR Clean-up kit (Macherey-Nagel). Sequencing reactions were performed by using a BigDye terminator cycle sequencing kit (Applied Biosystems) and purified using a Montage SEQ96 Sequencing Reaction Clean-up kit (Millipore). Electrophoresis of sequence reaction products was performed by using an ABI Prism 3100 genetic analyser (Applied Biosystems). Sequence assembly was performed using the program AutoAssembler (Applied Biosystems). The 16S rRNA gene sequence (continuous stretch of 1476 bp) and sequences of strains retrieved from EMBL were aligned and a phylogenetic tree was constructed by the neighbour-joining method, using the BIONUMERICS software package, version 3.50 (Applied Maths). Unknown bases were excluded from the analyses. Bootstrapping analysis was undertaken to test the statistical reliability of the topology of the neighbour-joining tree: 500 bootstrap resamplings of the data were performed (Fig. 1). A comparative analysis revealed that strain KMM 3961T is affiliated to the genus Arenibacter, a member of the family Flavobacteriaceae (Fig. 1). The levels of 16S rRNA gene sequence similarity between strains KMM 3961T and A. certesii KMM 3941T, A. latericius KMM 426T and A. troitsensis KMM 3674T were 94·8, 95·1 and 99·7 %, respectively.
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, as modified by Cleenwerck et al. (2002). Cells were lysed in a Tris/EDTA buffer (10 mM Tris/HCl with up to 200 mM EDTA, pH 8·0) containing RNase A (Sigma), SDS (Serva) and proteinase K (Merck) to final concentrations of 400 µg ml–1, 2 % (w/v) and 200 µg ml–1, respectively. NaCl (5 M stock solution) and CTAB/NaCl solution (10 % w/v CTAB in 0·7 M NaCl) were added to final concentrations of 1 M and 13·3 %, v/v, respectively. For determination of the DNA G+C content, DNA was enzymically degraded into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture obtained was then separated by HPLC using a Waters Symmetry Shield C8 column maintained at a temperature of 37 °C. The solvent was 0·02 M NH4H2PO4 (pH 4·0) with 1·5 % acetonitrile. Non-methylated phage 
DNA (Sigma) was used as the calibration reference.
The DNA G+C contents for all strains tested were in the range 37–39 mol%. Slightly higher values (39–40 mol%) were obtained when the DNAs of strains KMM 3961T, KMM 3979 and KMM 3980 were isolated using the method of Marmur (1961) and when the DNA G+C contents were determined by the thermal denaturation method (Marmur & Doty, 1962).
DNA–DNA hybridizations between strains KMM 3961T, A. certesii KMM 3941T, A. latericius KMM 426T and A. troitsensis KMM 3674T were performed; the DNA was prepared as described above. The microplate method was used as described by Ezaki et al. (1989) and Goris et al. (1998), using an HTS7000 BioAssay Reader (Perkin Elmer) for the fluorescence measurements. Biotinylated DNA was hybridized with single-stranded unlabelled DNA non-covalently bound to microplate wells. Hybridizations were performed at 35 °C in a hybridization mixture [2x SSC, 5x Denhardt's solution, 2·5 % dextran sulfate, 50 % formamide, denatured low-molecular-mass salmon sperm DNA (100 µg ml–1) and biotinylated probe DNA (1250 ng ml–1)]. Each hybridization experiment was performed in triplicate. A binding level of 62 % was found between strains KMM 3961T and A. troitsensis KMM 3674T, indicating that KMM 3961T represents a separate species. The latter two strains had low values for binding (6–20 %) with A. certesii KMM 3941T and A. latericius KMM 426T. To determine the levels of DNA relatedness between strains KMM 3961T, KMM 3979 and KMM 3980, DNA was isolated by the method of Marmur (1961) and DNA–DNA hybridizations were performed spectrophotometrically using the initial renaturation rate method described by De Ley et al. (1970). The levels of DNA–DNA binding between strains KMM 3961T, KMM 3979 and KMM 3980 were found to be 96–99 %. The results of the DNA–DNA hybridization experiments indicate that the strains under study represent a separate and novel Arenibacter species (Wayne et al., 1987).
To determine their whole-cell fatty acid profiles, strain KMM 3961T, A. certesii strain KMM 3941T, A. latericius strains KMM 426T, KMM 3522, KMM 3523, KMM 3528 and KMM 3557 and A. troitsensis strain KMM 3674T were grown at 25 °C for 48 h on marine agar. Analysis of the fatty acid methyl esters was carried out according to the standard protocol of the Microbial Identification System (Microbial ID). The predominant cellular fatty acids of KMM 3961T were of the straight-chain unsaturated, branched-chain unsaturated and saturated types: iso-C15 : 0 (8·7 %), iso-C15 : 1 (12·7 %), C15 : 0 (15·0 %), iso-C17 : 0 3-OH (17·4 %) and summed feature 3 (11·1 %; comprising iso-C15 : 0 2-OH and/or C16 : 1
7) (Table 1). The presence of a significant amount of iso-C17 : 0 3-OH (6·9–21·9 %) in all strains tested should be emphasized as it is one of the characteristic fatty acids of members of the family Flavobacteriaceae. Previously, Ivanova et al. (2001) had reported the absence of hydroxy fatty acids in Arenibacter strains.
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. Menaquinones were detected by using monitoring at 270 nm and were identified by comparison with known quinones from the reference strain Salegentibacter salegens DSM 5424T. The main isoprenoid quinone was MK-6.
The physiological and biochemical properties of strains KMM 3961T, KMM 3979 and KMM 3980 were examined as described by Nedashkovskaya et al. (2003b, 2004). Physiological and biochemical properties of strain KMM 3961T were also determined using API 20E, API 20NE and API ZYM galleries (bioMérieux) and the Biolog GN2 Microplate system according to the manufacturers' instructions.
The physiological, morphological and biochemical characteristics of the strains studied are listed in the species description and in Table 2. Similarities in the phenotypic characteristics support the inclusion of strains KMM 3961T, KMM 3979 and KMM 3980 in the genus Arenibacter. However, the three strains differ clearly from currently described Arenibacter species by their ability to move on substrate surfaces by means of gliding and to grow in media containing no sea water or Na+ ions. Also, the novel isolates cannot reduce nitrates to nitrites or form acid from D-lactose or L-raffinose, in contrast to the other Arenibacter strains tested in this study. Moreover, only strains KMM 3961T, KMM 3979 and KMM 3980 produced acid from DL-xylose and were resistant to oleandomycin. The novel bacteria can be further differentiated from A. latericius and A. certesii by the absence of urea hydrolysis, by the lack of susceptibility to ampicillin and by the higher G+C content of the DNA (Table 2). Other phenotypic characteristics, such as growth at 8 % NaCl, the maximum growth temperature (38 °C), oxidation of D-galactose, D-glucose, D-melibiose and N-acetylglucosamine, resistance to tetracycline and the absence of Tween 40 hydrolysis and H2S production also distinguish the novel isolates from the closest relative, A. troitsensis (Table 2).
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), in association with molecular divergence, support strongly the differentiation of the strains studied from the Arenibacter species with validly published names.
Originally, the genus Arenibacter was described as consisting of non-gliding bacteria requiring Na+ ions for growth and unable to decompose gelatin (Ivanova et al., 2001). In the course of our study of these novel algal isolates and A. troitsensis KMM 3674T, several phenotypic traits were found to be helpful for the differentiation of Arenibacter species.
Thus, we propose the placement of strains KMM 3961T, KMM 3979 and KMM 3980 in the genus Arenibacter, as Arenibacter palladensis sp. nov., and the emendation of the description of the genus Arenibacter.
Emended description of the genus Arenibacter Ivanova et al. 2001
This description is based on that of Ivanova et al. (2001). Some species may display gliding motility and grow without sea water or Na+ ions. Aerobic. Produce non-diffusible carotenoid pigments. Cytochrome oxidase-, catalase- and alkaline phosphatase-positive. The major respiratory quinone is MK-6. The predominant cellular fatty acids are straight-chain saturated and unsaturated and branched-chain unsaturated fatty acids C15 : 0, iso-C15 : 0, iso-C15 : 1, iso-C17 3-OH and summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 17). The main polar lipid is phosphatidylethanolamine. The type species is Arenibacter latericius.
Description of Arenibacter palladensis sp. nov.
Arenibacter palladensis (pal.la.den'sis. N.L. masc. adj. palladensis pertaining to Pallada Bay, where the first strains were isolated).
The main characteristics are the same as those given for the genus. In addition, cells range from 0·4 to 0·5 µm in width and from 1·6 to 2·3 µm in length, and move by means of gliding. On marine agar, colonies are 2–4 mm in diameter, circular with entire edges and dark orange in colour. Growth is observed at 4–38 °C. The optimal temperature for growth is 23–25 °C. Growth occurs at 0–10 % NaCl. Does not hydrolyse agar, casein, gelatin, alginate, starch, Tween 20, DNA, urea, cellulose (CM-cellulose and filter paper) or chitin. Forms acid from D-glucose, D-lactose, D-maltose and D-sucrose, but not from L-arabinose, D-galactose, L-sorbose, N-acetylglucosamine, citrate, adonitol, dulcitol, glycerol, inositol or mannitol. Utilizes L-arabinose and D-mannose, but not mannitol, inositol, sorbitol, malonate or citrate. Nitrate is not reduced. Indole, H2S and acetoin (Voges–Proskauer reaction), arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are not produced. According to the results of testing with the Biolog GN2 Microplate system, the type strain utilizes dextrin, cellobiose, D-fructose, L-fucose, D-galactose, gentiobiose, 
-D-glucose, -lactose, -D-lactose, lactulose, maltose, D-mannose, D-melibiose, methyl 
-D-glucoside, D-raffinose, sucrose, D-trehalose, turanose, methylpyruvate, -ketobutyric acid, DL-lactic acid, N-acetyl-D-glucosamine, L-glutamic acid, L-threonine and glucose 1-phosphate. Does not utilize -cyclodextrin, Tween 40 or 80, adonitol, L-arabinose, D-arabitol, i-erythritol, myo-inositol, D-mannitol, psicose, L-rhamnose, xylitol, monomethyl succinate, acetic acid, citric acid, formic acid, D-galactonic acid lactone, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, - and -hydroxybutyric acids, p-hydroxyphenylacetic acid, itaconic acid, -ketoglutaric acid, -ketovaleric acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, alaninamide, D-alanine, L-alanyl glycine, L-asparagine, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, L-pyroglutamic acid, D- and L-serine, DL-carnitine, 
-aminobutyric acid, uronic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol or glucose 6-phosphate. The predominant fatty acids in the type strain are straight-chain saturated and unsaturated and branched-chain unsaturated fatty acids C15 : 0 (15·0 %), iso-C15 : 0 (8·7 %), iso-C15 : 1 (12·7 %), iso-C17 3-OH (17·4 %) and summed feature 3 (11·1 %; comprising iso-C15 : 0 2-OH and/or C16 : 17). The G+C content of the DNA is 39–40 mol%.
The type strain is KMM 3961T (=LMG 21972T=CIP 108849T), isolated from the green alga U. fenestrata, collected in Pallada Bay, Sea of Japan.
Emended description of Arenibacter latericius Ivanova et al. 2001
The description is as for the genus and is based on that of Ivanova et al. (2001). In addition, cells range from 0·4 to 0·6 µm in width and from 2·1 to 5·0 µm in length. Gliding motility not observed. Growth is detected at 10–42 °C and in 1–8 % NaCl. Decomposes urea. Some strains may hydrolyse DNA and Tweens 20 and 40. Does not hydrolyse agar, casein, gelatin, starch, alginic acids, Tween 80, cellulose (CM-cellulose and filter paper) or chitin. Forms acid from D-cellobiose, D-galactose, D-glucose, D-lactose, D-maltose, L-raffinose, D-sucrose and glycerol. Can oxidize D-melibiose, L-fucose, L-rhamnose and N-acetylglucosamine. Does not produce acid from L-arabinose, L-sorbose, DL-xylose, adonitol, inositol, dulcitol, mannitol, malate, fumarate or citrate. According to the Biolog system, -D-glucose, L-glutamic acid, L-ornithine, uridine, glycerol, DL--glycerol phosphate, glucose 1-phosphate, glucose 6-phosphate are utilized. Dextrin, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, cellobiose, D-fructose, -D-lactose, -D-lactose lactulose, glycogen, maltose, D-mannitol, D-mannose, D-melibiose, methyl -D-glucoside, D-raffinose, sucrose, D-trehalose, DL-lactic acid, turanose, succinic acid, glucuronamide, alaninamide, L-alanine, L-alanyl-glycine, L-asparagine, L-aspartic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-proline, D- and L-serine and L-threonine are weakly utilized. Susceptible to erythromycin, ampicillin, carbenicillin, oleandomycin, cephaloridin and lyncomycin. Not susceptible to kanamycin, benzylpenicillin, oxacillin, neomycin, streptomycin, gentamicin, polymyxin B or tetracycline. Hydrogen sulfide, indole and acetoin are not produced. Nitrates are reduced to nitrites. The cellular fatty acids are predominantly odd-numbered and iso-branched (about 70 %): C15 : 0 (4·2–16·0 %), iso-C15 : 0 (6·9–15·8 %), anteiso-C15 : 0 (4·8–13·5 %), iso-C15 : 1 (4·9–14·0 %), iso-C15 : 0 3-OH (4·6–5·7 %), iso-C17 : 0 3-OH (6·9–14·4 %) and summed feature 3 (9·8–11·9 %; comprising C16 : 17 and/or iso-C15 : 0 2-OH).
The type strain is KMM 426T (=VKM B-2137DT=LMG 19693T=CIP 106861T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Barbeyron, T., L'Haridon, S., Corre, E., Kloareg, B. & Potin, P. (2001). Zobellia galactanovorans gen. nov., sp. nov., a marine species of Flavobacteriaceae isolated from red alga, and classification of [Cytophaga] uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Zobellia uliginosa gen. nov., comb. nov. Int J Syst Evol Microbiol 51, 985–997.[Abstract]
Bernardet, J.-F., Nakagawa, Y. & Holmes, B. (2002). Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, 1049–1070.[Abstract]
Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006.[Abstract]
Cleenwerck, I., Vandemeulebroecke, K., Janssens, D. & Swings, J. (2002). Re-examination of the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int J Syst Evol Microbiol 52, 1551–1558.[Abstract]
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a microplate DNA-DNA hybridization method compared with the initial renaturation method. Can J Microbiol 44, 1148–1153.[CrossRef]
Ivanova, E. P., Nedashkovskaya, O. I., Chun, J. & 7 other authors (2001). Arenibacter gen. nov., new genus of the family Flavobacteriaceae and description of a new species, Arenibacter latericius sp. nov. Int J Syst Evol Microbiol 51, 1987–1995.[Abstract]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
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.
Nedashkovskaya, O. I., Suzuki, M., Vysotskii, M. V. & Mikhailov, V. V. (2003a). Arenibacter troitsensis sp. nov., isolated from marine bottom sediment. Int J Syst Evol Microbiol 53, 1287–1290.
Nedashkovskaya, O. I., Suzuki, M., Vysotskii, M. V. & Mikhailov, V. V. (2003b). Reichenbachia agariperforans gen. nov., sp. nov., a novel marine bacterium in the phylum Cytophaga–Flavobacterium–Bacteroides. Int J Syst Evol Microbiol 53, 81–85.
Nedashkovskaya, O. I., Kim, S. B., Han, S. K., Lysenko, A. M., Mikhailov, V. V. & Bae, K. S. (2004). Arenibacter certesii sp. nov., a novel marine bacterium isolated from the green alga Ulva fenestrata. Int J Syst Evol Microbiol 54, 1173–1176.
Niemann, S., Puehler, A., Tichy, H.-V., Simon, R. & Selbitschka, W. (1997). Evaluation of the resolving power of three different DNA fingerprinting methods to discriminate among isolates of a natural Rhizobium meliloti population. J Appl Microbiol 82, 477–484.[CrossRef][Medline]
Vancanneyt, M., Mengaud, J., Cleenwerck, I. & 7 other authors (2004). Reclassification of Lactobacillus kefirgranum Takizawa et al. 1994 as Lactobacillus kefiranofaciens subsp. kefirgranum subsp. nov. and emended description of L. kefiranofaciens Fujisawa et al. 1988. Int J Syst Evol Microbiol 54, 551–556.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Bacterial Systematics. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Wilson, K. (1987). Preparation of genomic DNA from bacteria. In Current Protocols in Molecular Biology, pp. 2.4.1–2.4.5. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Green Publishing and Wiley-Interscience.
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