Abstract
Published online ahead of print on 19 July 2004 as DOI 10.1099/ijs.0.63307-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Winogradskyella thalassocola KMM 3907T, Winogradskyella epiphytica KMM 3906T and Winogradskyella eximia KMM 3944T are AY521223, AY521224 and AY521225, respectively.
Footnotes
†Present address: Department of Microbiology, School of Bioscience and Biotechnology, Chungnam National University, Yusong, Daejon 305-764, Republic of Korea.Bacteria of the family Flavobacteriaceae are often found attached to the surfaces of a diverse range of marine algae (Chan & McManus, 1969; Bolinches et al., 1988; Hanzawa et al., 1998). The novel marine bacteria Arenibacter latericius, Cellulophaga fucicola, Cellulophaga baltica, Cellulophaga algicola, Formosa algae, Mesonia algae, Maribacter ulvicola, Tenacibaculum amylolyticum, Ulvibacter litoralis and Zobellia galactanivorans, associated with different algae, have been isolated and described (Johansen et al., 1999; Bowman, 2000; Barbeyron et al., 2001; Suzuki et al., 2001; Ivanova et al., 2001, 2004; Nedashkovskaya et al., 2003b, 2004a, b). The above-mentioned flavobacteria are commonly characterized by rod-shaped cells. During studies on microbial communities of algae inhabiting the Sea of Japan, we recovered three novel isolates belonging to the family Flavobacteriaceae from the algae frond surfaces; these isolates were able to form cellular network-like structures or aggregates that can be considered to serve for an attachment adaptation.
Based on a polyphasic study of the algal isolates, including phylogenetic, genotypic, chemotaxonomic and phenotypic data, we propose a new genus, Winogradskyella gen. nov., containing three novel species.
Strains KMM 3906T, KMM 3907T and KMM 3944T were isolated from the green alga Acrosiphonia sonderi, and the brown algae Chorda filum and Laminaria japonica, respectively, collected in the Gulf of Peter the Great of the Sea of Japan during June 2000. Strains were cultivated at 28 °C on marine agar 2216 (MA; Difco) and stored at 80 °C in marine broth 2216 (MB; Difco) supplemented with 20 % (v/v) glycerol. On MA, colonies of strains studied were round, 24 mm in diameter, yellow-pigmented, shiny, viscous and with entire edges.
The phylogenetic position of the three isolates was determined using previously described procedures for DNA extraction, PCR and 16S rRNA gene sequence analysis (Kim et al., 1998). The sequence data were aligned with those of representative members of selected genera of the family Flavobacteriaceae by using PHYDIT version 3.2 (). Phylogenetic trees were inferred by using suitable programs of the PHYLIP package (Felsenstein, 1993). Phylogenetic distances were calculated from the two-parameter model of Kimura (1980), and trees were constructed on the basis of the neighbour-joining (Saitou & Nei, 1987), least-squares (Fitch & Margoliash, 1967) and maximum-likelihood (Felsenstein, 1993) algorithms. Bootstrap analysis was performed with 1000 resampled datasets using the SEQBOOT and CONSENSE programs of the PHYLIP package.
Phylogenetic analysis of almost-complete 16S rRNA gene sequences of strains KMM 3906T, KMM 3907T and KMM 3944T revealed that they form a distinct lineage within the family Flavobacteriaceae (Fig. 1). Psychroserpens burtonensis was found to be the nearest neighbour; this relationship was supported by a high bootstrap value and also by the different tree-making algorithms used. However, 16S rRNA gene sequence similarity between the three strains and P. burtonensis was only 93·593·8 %. 16S rRNA gene sequence similarity values of the three strains to other close relatives, Gelidibacter algens and Formosa algae, were 90·891·3 and 92·792·9 %, respectively. The low sequence similarity values of KMM 3906T, KMM 3907T and KMM 3944T to other Cytophaga-like bacteria described to date (85·692·1 %) demonstrate that the bacteria isolated in this study represent a new genus.
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The three strains had 16S rRNA gene sequence similarities in the range 96·397·1 %.
For DNADNA hybridizations and determination of the G+C content, DNA was isolated following the method of Marmur (1961). The G+C content was determined by the thermal denaturation method of Marmur & Doty (1962). DNADNA hybridization was performed spectrophotometrically and initial renaturation rates were recorded as described by De Ley et al. (1970).
The DNA G+C contents of strains KMM 3906T, KMM 3907T and KMM 3944T were 35·2, 34·6 and 36·1 mol%, respectively. DNADNA relatedness between the strains was 3445 %. These values indicated that the strains represent three separate species. Phenotypic data distinguishing the strains are given in Table 1.
Table 1. Phenotypic properties of the Winogradskyella gen. nov. species Taxa: 1, W. thalassocola KMM 3907T; 2, W. epiphytica KMM 3906T; 3, W. eximia KMM 3944T. All were positive for: respiratory metabolism; gliding motility; oxidase, catalase and alkaline phosphatase activities; requirement for Na+ ions for growth; growth at 33 °C and in 15 % NaCl; hydrolysis of agar, gelatin and Tween 40; susceptibility to carbenicillin and lincomycin. All were negative for: flexirubin pigments; nitrate reduction; urease and β-galactosidase activities; hydrolysis of cellulose (carboxymethylcellulose, filter paper) and chitin; acid formation from L-arabinose, D-galactose, D-lactose, D-melibiose, L-rhamnose, DL-xylose, adonitol, dulcitol, inositol, sorbitol and citrate; utilization of L-arabinose, D-lactose, D-sucrose, inositol, mannitol, sorbitol, malonate and citrate; indole, acetoin and H2S production; susceptibility to benzylpenicillin, gentamicin, kanamycin, neomycin, polymyxin B and streptomycin.
Analysis of fatty acid methyl esters was carried out according to the standard protocol of the Microbial Identification System (Microbial ID). Isoprenoid quinones were extracted from lyophilized cells and analysed as described by Akagawa-Matsushita et al. (1992). Isoprenoid quinone composition was characterized by HPLC (Shimadzu Instruments) using a reversed-phase type Zorbax ODS column (250x4·6 mm) and acetonitrile/2-propanol (65 : 35, v/v) as a mobile phase at a flow rate of 0·5 ml min1. The column was kept at 40 °C. Menaquinones were detected by monitoring at 270 nm and were identified by comparison with known quinones from reference strain Salegentibacter salegens DSM 5424T.
Predominant cellular fatty acids of the strains studied were branched-chain unsaturated and straight-chain saturated, namely iso-C15 : 0, anteiso-C15 : 0, iso-C15 : 1, iso-C16 : 0-3OH and iso-C17 : 0-3OH (Table 2). The major isoprenoid quinone was MK-6.
Table 2. Whole-cell fatty acid profiles (percentage composition) of the Winogradskyella species Taxa: 1, W. thalassocola KMM 3907T; 2, W. epiphytica KMM 3906T; 3, W. eximia KMM 3944T.
Phenotypic characterization was performed using the tests described previously (Nedashkovskaya et al., 2003a, b). Gliding motility was determined as described by Bowman (2000). Scanning electron microscopy was used to examine the bacteria, which were fixed with a solution containing 2 % glutaraldehyde and 3 % formaldehyde in cacodylate buffer (0·1 M cacodylate, 0·09 M sucrose, 0·01 M CaCl2, 0·01 M MgCl2, pH 6·9) for 1 h on ice and washed with cacodylate buffer. After washing several times in TE buffer (20 mM Tris, 1 mM EDTA, pH 7·0), samples were dehydrated through a graded series of acetone (10, 30, 50, 70, 90, 100 %) on ice, each step for 15 min, followed by critical-point drying with liquid CO2. Samples were sputter-coated with an approximately 10 nm thick gold film before examination in a Zeiss field-emission scanning electron microscope (DSM982 Gemini) at an acceleration voltage of 5 kV using the Everhart Thornley secondary electron (SE) detector and the Inlens-SE detector at a 50 : 50 ratio.
The three bacteria described in this study were Gram-negative, chemo-organotrophic with respiratory-type metabolism, non-motile single flexible rods, 0·40·6 µm in diameter and 1·01·3 µm in length. All three strains formed unique network-like structures or aggregates (Fig. 2AC). Growth of strain KMM 3944T was observed at 15 % NaCl; strains KMM 3906T and KMM 3907T grew in media containing 18 % NaCl. Optimal growth was observed at 1·52 % NaCl. The maximum growth temperature for strain KMM 3906T was 37 °C, and that for strains KMM 3907T and KMM 3944T was 33 °C. Strain KMM 3906T was able to oxidize carbohydrates, but the other two strains were not. Other physiological characteristics of the three strains are given in the species description and in Table 1.
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The three isolates occupy a distinct phylogenetic branch and share many common phenotypic traits with other members of the family Flavobacteriaceae. Differential features of strains studied and their close relatives are given in Table 3. The algal isolates can be distinguished from their closest relative P. burtonensis by the presence of gliding motility, oxidase activity and hydrolysis of agar and casein. The ability to produce oxidase and agarase distinguishes the strains studied and members of the genus Gelidibacter. The requirement of Na+ ions for growth and casein hydrolysis separate the strains studied from their close neighbour Formosa algae.
Table 3. Differential characteristics of the genus Winogradskyella and allied genera of the family Flavobacteriaceae Genera: 1, Winogradskyella; 2, Psychroserpens; 3, Gelidibacter; 4, Formosa. Data from Bowman et al. (1997), Macián et al. (2002), Ivanova et al. (2004) and this study. , Negative; +, positive; V, variable; ND, not determined.
Polyphasic data on the strains studied, including their separate phylogenetic branching, morphological characteristics, fatty acid composition, phenotypic features and low levels of DNADNA relatedness, indicate that they can not be assigned to any of the taxa currently included in the family Flavobacteriaceae. Consequently, we propose that strains KMM 3907T, KMM 3906T and KMM 3944T be placed in a new genus, Winogradskyella gen. nov., as Winogradskyella thalassocola sp. nov., Winogradskyella epiphytica sp. nov. and Winogradskyella eximia sp. nov., respectively.
Description of Winogradskyella gen. nov.
Winogradskyella [Wi.no.grad'sky.el.la. N.L. fem. n. Winogradskyella named after Sergey Winogradsky (18561953), a Russian microbiologist who made a considerable contribution to the taxonomy of bacteria of the phylum CytophagaFlavobacteriumBacteroides].
Rod-shaped cells, motile by gliding. The cells can form network-like structures. Gram-negative. Do not form endospores. Strictly aerobic. Produce non-diffusible yellow pigments. No flexirubins are formed. Chemo-organotrophic. Cytochrome oxidase-, catalase- and alkaline phosphatase-positive. Can hydrolyse gelatin, starch and DNA. The main cellular fatty acids are straight-chain saturated, branched-chain saturated and unsaturated fatty acids iso-C15 : 0, anteiso-C15 : 0, iso-C15 : 1, iso-C16 : 0-3OH and iso-C17 : 0-3OH. On the basis of 16S rRNA gene sequence analysis, the genus Winogradskyella is a member of the family Flavobacteriaceae, phylum Bacteroidetes.
The type species is Winogradskyella thalassocola.
Description of Winogradskyella thalassocola sp. nov.
Winogradskyella thalassocola (tha.las.so.co'la. Gr. n. thalassa the sea; L. suffix -cola dweller; N.L. n. thalassocola a sea-dweller).
Main characteristics are as given for the genus. In addition, cells are 0·50·7 µm in width and 47·3 µm in length. On MA, colonies are 24 mm in diameter, circular, shiny with entire edges, yellow-pigmented and viscous. Growth occurs at 433 °C. Optimal temperature for growth is 2123 °C. Growth occurs in 18 % NaCl. Decomposes gelatin and Tween 40. Does not hydrolyse starch, DNA, Tween 20, Tween 80, urea, cellulose (carboxymethylcellulose and filter paper) or chitin. Forms acid from D-glucose, D-maltose and D-cellobiose, but not from L-arabinose, D-galactose, D-lactose, D-melibiose, L-rhamnose, D-sucrose, DL-xylose, citrate, adonitol, dulcitol, inositol or mannitol. Utilizes D-glucose and D-mannose, but not L-arabinose, D-lactose, D-sucrose, mannitol, inositol, sorbitol, malonate or citrate. β-Galactosidase activity is negative. Nitrate is not reduced. H2S, indole and acetoin (VogesProskauer reaction) production are negative. Susceptible to carbenicillin, lincomycin and oleandomycin, but resistant to ampicillin, benzylpenicillin, gentamicin, kanamycin, neomycin, polymyxin B, streptomycin and tetracycline. The DNA G+C content is 34·6 mol%.
The type strain, KMM 3907T (=KCTC 12221T=LMG 22492T=DSM 15363T), was isolated from the brown alga Chorda filum, collected in Troitsa Bay, Gulf of Peter the Great, Sea of Japan.
Description of Winogradskyella epiphytica sp. nov.
Winogradskyella epiphytica (e.pi.phy'ti.ca. epiphiticus -a -um adj. derived from Gr. epi on and phyt- relating to plants; N.L. epiphytica onto plant, pertaining to the original isolation from the surface of the algal fronds).
Main characteristics are as given for the genus. In addition, cells are 0·50·7 µm in width and 47·3 in length. On MA, colonies are 24 mm in diameter, circular, shiny with entire edges, yellow-pigmented and viscous. Growth occurs at 437 °C. Optimal temperature for growth is 2325 °C. Growth occurs in 18 % NaCl. Decomposes agar, gelatin, DNA, and Tween 20, Tween 40 and Tween 80. Does not hydrolyse starch, urea, cellulose (carboxymethylcellulose and filter paper) or chitin. Does not form acid from L-arabinose, D-cellobiose, D-galactose, D-glucose, D-lactose, D-maltose, D-melibiose, L-rhamnose, D-sucrose, DL-xylose, citrate, adonitol, dulcitol, inositol or mannitol. Does not utilize L-arabinose, D-glucose, D-lactose, D-mannose, D-sucrose, mannitol, inositol, sorbitol, malonate or citrate. β-Galactosidase activity is negative. Nitrate is not reduced. H2S, indole and acetoin (VogesProskauer reaction) production are negative. Susceptible to ampicillin, carbenicillin, lincomycin, oleandomycin and tetracycline, but resistant to benzylpenicillin, gentamicin, kanamycin, neomycin, polymyxin B and streptomycin. The DNA G+C content is 35·2 mol%.
The type strain, KMM 3906T (=KCTC 12220T=LMG 22491T=CCUG 47091T), was isolated from the green alga Acrosiphonia sonderi, collected in Troitsa Bay, Gulf of Peter the Great, Sea of Japan.
Description of Winogradskyella eximia sp. nov.
Winogradskyella eximia (e.xi'mi.a. L. fem. adj. eximia excellent).
Main characteristics are as given for the genus. In addition, cells are 0·50·7 µm in width and 47·3 in length. On MA, colonies are 24 mm in diameter, circular, shiny with entire edges, yellow-pigmented and viscous. Growth occurs at 433 °C. Optimal temperature for growth is 2123 °C. Growth occurs in 15 % NaCl. Decomposes casein, gelatin, starch, Tween 20 and Tween 40. Does not hydrolyse DNA, urea, Tween 80, cellulose (carboxymethylcellulose and filter paper) or chitin. Forms acid from D-glucose, D-maltose, D-sucrose and mannitol, but not from L-arabinose, D-cellobiose, D-galactose, D-lactose, D-melibiose, L-rhamnose, DL-xylose, citrate, adonitol, dulcitol or inositol. Utilizes D-glucose and D-mannose, but not L-arabinose, D-lactose, D-sucrose, mannitol, inositol, sorbitol, malonate or citrate. β-Galactosidase activity is negative. Nitrate is not reduced. H2S is produced but indole and acetoin (VogesProskauer reaction) are not. Susceptible to lincomycin, but resistant to ampicillin, benzylpenicillin, carbenicillin, gentamicin, kanamycin, oleandomycin, neomycin, polymyxin B, streptomycin and tetracycline. The DNA G+C content is 36·1 mol%.
The type strain, KMM 3944T (=KCTC 12219T=LMG 22474T), was isolated from the brown alga Laminaria japonica, collected in the Gulf of Peter the Great, Sea of Japan.
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