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1 Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Prospekt 100 let Vladivostoku 159, 690022, Vladivostok, Russia
2 Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan
3 BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Faculteit Wetenschappen, Ghent University, B-9000 Ghent, Belgium
4 Institute of Microbiology of the Russian Academy of Sciences, Prospekt 60 let October 7/2, Moscow, 117811, Russia
Correspondence
Olga I. Nedashkovskaya
olganedashkovska{at}yahoo.com
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Published online ahead of print on 5 March 2004 as DOI 10.1099/ijs.0.63091-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Zobellia amurskyensis KMM 3526T, Zobellia laminariae KMM 3676T and Zobellia russellii KMM 3677T are AB121974, AB121975 and AB121976, respectively.
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and contains Gram-negative, aerobic, gliding, agarolytic bacteria that produce flexirubin-type pigments. To date, two species of the genus Zobellia have validly published names, Zobellia galactanivorans and Zobellia uliginosa, and both originate from marine environments. The latter species was isolated from beach sand in California and was originally classified as Flavobacterium uliginosum (ZoBell & Upham, 1944). Later, it was transferred to the genus Cytophaga as Cytophaga uliginosa (Reichenbach, 1989), reclassified in the genus Cellulophaga as Cellulophaga uliginosa (Bowman, 2000) and finally assigned to the new genus Zobellia on the basis of a higher DNA G+C content (mol%), its maximum growth temperature, the presence of flexirubin pigments and its unique phylogenetic position (Barbeyron et al., 2001). Z. galactanivorans, a marine species isolated from a red alga, was chosen as the type species. In the course of a study on 450 strains isolated from sea water and bottom sediment samples, the sea urchin Strongulocentrothus intermedius and red (Polysiphonia japonica), green (Acrosiphonia sonderi and Ulva fenestrata) and brown (Chorda filum and Laminaria japonica) algae, the taxonomic position of four isolates was unclear and was further investigated in the present study. We report the results of phenotypic, physiological and genomic analyses on the latter strains, which led to the description of three novel species of the genus Zobellia, for which the names Zobellia amurskyensis sp. nov., Zobellia laminariae sp. nov. and Zobellia russellii sp. nov. are proposed.
The isolates investigated in this study were obtained during sampling in the Gulf of Peter the Great, Sea of Japan, Pacific Ocean, in June 2000. Strain KMM 3526T was isolated from a sea-water sample collected in Amursky Bay. Strains KMM 3676T, KMM 3926 and KMM 3677T were recovered from the brown alga L. japonica (KMM 3676T, KMM 3926) and the green alga A. sonderi (KMM 3677T), collected in Troitsa Bay. For the isolation, 0·1 ml sea water or algal tissue homogenates was transferred to marine agar 2216 (Difco). After primary isolation and purification, strains were cultivated at 28 °C on the same medium and stored at –80 °C in marine broth (Difco) supplemented with 20 % (v/v) glycerol.
The almost complete 16S rRNA gene sequences of isolates KMM 3526T, KMM 3676T and KMM 3677T were determined by PCR amplification and direct sequencing (Hiraishi, 1992). The conditions and reagents used for PCR amplification and sequencing of 16S rRNA gene were as described previously (Suzuki et al., 2001). The sequences were aligned on the secondary-structure model, maintained by the SSU rRNA database (Van de Peer et al., 2000), using the profile-alignment program of the CLUSTAL W software (Thompson et al., 1994). Evolutionary distances were then computed with the DNADIST program in the PHYLIP 3.572 package (Felsenstein, 1995) with the two-parameter model (Kimura, 1980); a phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987). To evaluate the phylogenetic trees, a bootstrap analysis with 1000 sample replications was performed with the SEQBOOT and CONSENSE programs in the PHYLIP 3.572 package.
The 16S rRNA gene-based analysis revealed that KMM 3526T, KMM 3676T and KMM 3677T formed a coherent cluster within the genus Zobellia of the family Flavobacteriaceae (Fig. 1). The level of 16S rRNA gene sequence similarity between the KMM strains and Z. galactanivorans and Z. uliginosa ranged from 97·4 to 98·3 %. The 16S rRNA gene sequence similarities between strains KMM 3526T, KMM 3676T and KMM 3677T ranged from 98·2 to 99·3 %.
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, as modified by Leisner et al. (2002). DNA–DNA hybridizations were performed using the microplate method as described by Ezaki et al. (1989). Hybridizations were performed at 37 °C in a hybridization mixture containing 50 % formamide (2x SSC, 5x Denhardt's solution, 2·5 % dextran sulphate, 50 % formamide, 100 µg denaturated salmon sperm DNA ml–1 and 1250 ng biotinylated probe DNA ml–1). The DNA G+C content was determined using two methods: (i) the HPLC method of Mesbah et al. (1989) on DNA extracted as indicated above and (ii) the thermal denaturation method of Marmur & Doty (1962) on DNA extracted according to the protocol of Marmur (1961). The G+C contents of the DNA of strains KMM 3526T, KMM 3676T and KMM 3677T were 37·1, 36·1 and 38·6 mol%, respectively, when determined by the thermal denaturation method. Slightly higher values were observed when determined by HPLC: 37·7 mol% for KMM 3526T, 36·3 mol% for KMM 3676T and 38·8 mol% for KMM 3677T. DNA–DNA relatedness between strains KMM 3526T, KMM 3676T and KMM 3677T and the type strains Z. galactanivorans DsijT and Z. uliginosa CIP 104808T ranged from 8 to 29 %. The DNA–DNA binding value for strains KMM 3676T and KMM 3926 was 93 %.
To obtain whole-cell fatty acid profiles, the strains studied were grown at 28 °C for 24 h on marine agar 2216 (Difco). Analysis of fatty acid methyl esters was carried out according to the standard protocol of the Microbial Identification System (Microbial ID).
The dominant cellular fatty acids of the novel isolates and the type strains of Z. galactanivorans and Z. uliginosa were the straight-chain and branched-chain saturated and unsaturated fatty acids 15 : 0, iso-15 : 0, iso-15 : 0 3-OH, iso-15 : 1 and iso-17 : 0 3-OH (Table 1). No significant differences were found between the species.
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. The major lipoquinone of the novel isolates, Z. galactanivorans DsijT and Z. uliginosa CIP 104808T was MK-6.
Gram-staining, hydrolysis of agar, starch, casein, elastin, gelatin, cellulose [filter paper and carboxymethylcellulose (CM-cellulose)], chitin, DNA, urea and alginic acids, flexirubin production, growth at different pH values, production of acid from carbohydrates, hydrolysis of Tweens 20, 40 and 80, nitrate reduction, production of hydrogen sulphide and indole and 
-galactosidase, oxidase, catalase and alkaline phosphatase activities were tested according to Gerhardt et al. (1994). Oxidative versus fermentative utilization of glucose was determined using Hugh & Leifson medium modified for marine bacteria (Lemos et al., 1985). Susceptibility to antibiotics was tested as described earlier (Nedashkovskaya et al., 2003). To examine carbon-source utilization, a medium containing 0·2 g NaNO3, 0·2 g NH4Cl, 0·05 g yeast extract (Difco) and 0·4 % (w/v) carbon source in 1000 ml artificial sea water was used. The carbon sources tested were L-arabinose, D-glucose, D-lactose, D-mannose, D-sucrose, inositol, sorbitol, mannitol, fumarate, citrate and malonate. Spreading growth was observed with cultivation on medium B containing (l–1) 1 g Bactopeptone (Difco), 1 g yeast extract (Difco), 15 g agar and half-strength natural sea water under high moisture conditions. Gliding motility was determined as described by Bowman (2000).
The physiological, morphological and biochemical characteristics of the strains studied are listed in the species descriptions and in Table 2. The presence of oxidase, catalase, -galactosidase, agarase and alkaline phosphatase activities, the absence of urease activity, flexirubin-type pigment production, the reduction of nitrate to nitrite and the oxidation of carbohydrates, the absence of crystalline and amorphous cellulose hydrolysis and the respiratory quinone and fatty acid compositions of strains KMM 3526T, KMM 3676T and KMM 3677T are consistent with the characteristics of Z. galactanivorans DsijT and Z. uliginosa CIP 104808T (Table 1
). However, the isolates differed clearly from Z. galactanivorans and Z. uliginosa by their inability to grow at 42 °C and to hydrolyse casein, their ability to oxidize L-rhamnose and the lower G+C content of their DNA (Table 2). Strain KMM 3526T is distinguished from strain KMM 3677T by the inability to grow with 8 % NaCl or at 37 °C or to hydrolyse Tween 40, by the absence of acid production from L-arabinose, D-cellobiose, DL-xylose and mannitol, by susceptibility to streptomycin and by resistance to tetracycline (Table 2). It is possible to differentiate strains KMM 3526T and KMM 3676T by the hydrolysis of starch, alginate, DNA, Tweens 20, 40 and 80, by the oxidation of L-arabinose, D-cellobiose, D-raffinose and mannitol, and by susceptibility to streptomycin. KMM 3677T is distinguished from KMM 3676T by the ability to grow with 10 % NaCl and at 37 °C, the ability to oxidize DL-xylose and the ability to hydrolyse starch, alginate, DNA, Tween 20 and Tween 80.
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Description of Zobellia amurskyensis sp. nov.
Zobellia amurskyensis (a.mur.sky.en'sis. N.L. fem. adj. amurskyensis of Amursky Bay, in which the type strain was isolated).
Cells range from 0·4 to 0·5 µm in width and from 1·2 to 1·4 µm in length. On marine agar, colonies are 2–4 mm in diameter, circular, shiny with entire edges, pigmented dark orange and sunken in the agar. Growth occurs at 4–32 °C, with the optimum at 23–25 °C, and at 1–6 % NaCl, with the optimum at 2 % NaCl. Decomposes agar, gelatin, starch, alginate, DNA, Tween 20 and Tween 80. Does not hydrolyse casein, cellulose (CM-cellulose and filter paper), chitin or Tween 40. Forms acid from D-glucose, L-fucose, D-maltose, L-rhamnose and D-sucrose, but not from L-arabinose, D-cellobiose, D-galactose, D-lactose, D-melibiose, L-sorbose, L-raffinose, DL-xylose, N-acetylglucosamine, citrate, adonitol, dulcitol, glycerol, inositol or mannitol. Utilizes L-arabinose, D-lactose, D-mannose and mannitol, but not inositol, sorbitol, malonate or citrate. Nitrate is reduced. H2S, indole and acetoin (Voges–Proskauer reaction) are not produced. Susceptible to carbenicillin, lincomycin, oleandomycin and streptomycin, but resistant to ampicillin, benzylpenicillin, gentamicin, kanamycin, neomycin, polymyxin B and tetracycline. The predominant fatty acids are 15 : 0 (14·4 %), i15 : 0 (22·5 %), i15 : 0 3-OH (4·6 %), i15 : 1 (10·4 %) and i17 : 0 3-OH (15·1 %). The major lipoquinone is MK-6. The G+C content of the DNA is 37·1 mol%.
The type strain is KMM 3526T (=LMG 22069T=CCUG 47080T). Isolated from sea water.
Description of Zobellia laminariae sp. nov.
Zobellia laminariae (la.mi.na'ri.ae. N.L. gen. n. laminariae of Laminaria, the generic name of the brown alga Laminaria japonica, from which the bacteria were isolated).
Cells range from 0·4 to 0·5 µm in width and from 1·2 to 1·4 µm in length. On marine agar, colonies are 2–4 mm in diameter, circular, shiny with entire edges, pigmented dark red and sunken in the agar. Growth occurs at 4–30 °C, with the optimum at 21–23 °C, and at salt concentrations from 1·5 to 6 % NaCl, with an optimum at 2 %. Decomposes agar, gelatin and Tween 40. Does not hydrolyse casein, starch, alginate, DNA, Tween 20, Tween 80, cellulose (CM-cellulose and filter paper) or chitin. Forms acid from L-arabinose, D-cellobiose, D-glucose, L-fucose, D-maltose, D-raffinose, L-rhamnose, D-sucrose and mannitol, but not from D-galactose, D-lactose, D-melibiose, L-sorbose, DL-xylose, N-acetylglucosamine, citrate, adonitol, dulcitol, glycerol or inositol. Utilizes D-lactose and D-mannose, but not inositol, sorbitol, malonate or citrate. Nitrate is reduced. H2S, indole and acetoin (Voges–Proskauer reaction) are not produced. Susceptible to carbenicillin, lincomycin and oleandomycin, but resistant to ampicillin, benzylpenicillin, gentamicin, kanamycin, neomycin, polymyxin B, streptomycin and tetracycline. The predominant fatty acids are 15 : 0 (12·5 %), i15 : 0 (16·8 %), i15 : 0 3-OH (6·1 %), i15 : 1 (12·3 %) and i17 : 0 3-OH (22·4 %). The major lipoquinone is MK-6. The G+C content of the DNA is 36–37 mol%.
The type strain is KMM 3676T (=LMG 22070T=CCUG 47083T). Isolated from the brown alga Laminaria japonica.
Description of Zobellia russellii sp. nov.
Zobellia russellii (rus'sel.li.i. N.L. gen. n. russellii of H. L. Russell, the American scientist, for his contribution to the development of marine microbiology).
Cells range from 0·4–0·5 µm in width and from 1·2 to 1·4 µm in length. On marine agar, colonies are 2–4 mm in diameter, circular, shiny with entire edges, pigmented dark orange and sunken in the agar. Growth occurs at 4–38 °C, with the optimum at 25–28 °C, and at salt concentrations between 1 and 10 % NaCl, with the optimum at 2–3 %. Decomposes agar, gelatin, starch, alginate, DNA, Tween 20, Tween 40 and Tween 80. Does not hydrolyse casein, cellulose (CM-cellulose and filter paper) or chitin. Forms acid from L-arabinose, D-cellobiose, D-glucose, L-fucose, D-maltose, L-rhamnose, D-sucrose, DL-xylose and mannitol, but not from D-galactose, D-lactose, D-melibiose, L-sorbose, D-raffinose, N-acetylglucosamine, citrate, adonitol, dulcitol, glycerol or inositol. Utilizes D-lactose and D-mannose, but not inositol, sorbitol, malonate or citrate. Nitrate is reduced. H2S, indole and acetoin (Voges–Proskauer reaction) are not produced. Susceptible to carbenicillin, lincomycin, oleandomycin and tetracycline, but resistant to ampicillin, benzylpenicillin, gentamicin, kanamycin, neomycin, polymyxin B and streptomycin. The predominant fatty acids are 15 : 0 (11·0 %), i15 : 0 (20·1 %), i15 : 0 3-OH (5·9 %), i15 : 1 (14·9 %) and i17 : 0 3-OH (19·7 %). The major lipoquinone is MK-6. The G+C content of the DNA is 38·6 mol%.
The type strain is KMM 3677T (=LMG 22071T=CCUG 47084T). Isolated from the green alga Acrosiphonia sonderi.
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ACKNOWLEDGEMENTS |
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