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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 Tokyo Research Laboratories, Kyowa Hakko Kogyo Company Ltd, 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan
3 Institute of Microbiology of the Russian Academy of Sciences, Pr. 60 let October 7/2, Moscow, 117811, Russia
4 BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ledeganckstraat 35, B 9000 Ghent, Belgium
5 Institute of Marine Biology of the Far-Eastern Branch of the Russian Academy of Sciences, Pal'chevskogo St 17, 690032, Vladivostok, Russia
Correspondence
Olga I. Nedashkovskaya
olganedashkovska{at}piboc.dvo.ru
or
olganedashkovska{at}yahoo.com
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Published online ahead of print on 31 October 2003 as DOI 10.1099/ijs.0.02737-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of Cellulophaga pacifica KMM 3664T, KMM 3669 and KMM 3915 are AB100840, AB100841 and AB100842, respectively.
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TOPABSTRACTMAIN TEXTREFERENCES |
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as a separate branch in the family Flavobacteriaceae, to comprise three species: Cellulophaga baltica and Cellulophaga fucicola, two agarolytic, yellow–orange-pigmented bacteria associated with the brown alga Fucus serratus, and Cellulophaga lytica, a common representative of coastal microbial communities that was formerly misclassified as [Cytophaga] lytica (Lewin, 1969; Reichenbach, 1989). The latter species was assigned as the type species of the genus. A fourth member of the genus, Cellulophaga algicola, which originated from the surface of algal species from Antarctic marine coasts and sea ice, was described relatively recently (Bowman, 2000). This author also reclassified [Cytophaga] uliginosa (Reichenbach, 1989), formerly Flavobacterium uliginosum (ZoBell & Upham, 1944), in the genus Cellulophaga as Cellulophaga uliginosa. Recently, C. uliginosa has been transferred to a novel genus, Zobellia, based on DNA G+C content, maximum growth temperature, presence of flexirubin and phylogenetic position (Barbeyron et al., 2001). Three gliding, agarolytic, strictly aerobic, Gram-negative and yellow-pigmented bacterial strains were isolated from a sea water sample that was collected in the Sea of Japan, Pacific Ocean. Phenotypic, chemotaxonomic and genotypic characteristics assigned these bacteria to the family Flavobacteriaceae. Phylogenetic and phenotypic data indicated that the unknown organisms comprised a distinct species within the genus Cellulophaga. The name Cellulophaga pacifica sp. nov. is proposed for these sea water isolates; the type strain is KMM 3664T (=JCM 11735T=LMG 21938T).
Strains KMM 3664T, KMM 3669 and KMM 3915 were isolated from a sea water sample that was collected during June 2000 in Amursky Bay, Gulf of Peter the Great, Sea of Japan, Pacific Ocean, from a depth of 5 m (salinity, 33 
; temperature, 15 °C). After primary isolation and purification, strains were cultivated at 28 °C on marine agar 2216 (Difco) and stored at -80 °C in marine broth (Difco) supplemented with 20 % (v/v) glycerol. The bacteria isolated in this study and reference strains are shown in Table 1.
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), using conditions and reagents that were described previously (Suzuki et al., 2001). The determined sequences were aligned to an alignment based on a secondary structure model, maintained by the SSU rRNA database (Van de Peer et al., 2000), by 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 Kimura two-parameter correction (Kimura, 1980); a phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987). To evaluate the phylogenetic tree, bootstrap analysis with 1000 sample replications was performed with the SEQBOOT and CONSENSE programs in the PHYLIP 3.572 package.
Comparative analysis of the sequences obtained in this study with published sequences of representatives of the phylum Cytophaga–Flavobacterium–Bacteroides that have been described to date revealed that the sea water isolates were members of the family Flavobacteriaceae and formed a distinct lineage within the genus Cellulophaga (Fig. 1). 16S rDNA sequence similarity values of strains KMM 3664T, KMM 3669 and KMM 3915 to the type strains of C. lytica and C. fucicola were too low (91·9–92·1 and 93·0–93·1 %, respectively) to consider the new isolates as members of these species. However, the new isolates shared a level of 16S rDNA sequence similarity that may indicate possible species-level relatedness to the type strains of C. algicola and C. baltica (97·1 and 97·5 %, respectively).
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for strains KMM 3664T, KMM 3669 and KMM 3915, using the DNA extraction protocol of Marmur (1961); and (ii) the HPLC method of Mesbah et al. (1989) for C. baltica NN015840T, KMM 3664T and KMM 3669, following the DNA extraction protocol of Pitcher et al. (1989) as modified by Leisner et al. (2002) and, for C. algicola ACAM 630T, by using the DNA extraction protocol of Wilson (1987) as modified by Cleenwerck et al. (2002). These high-molecular-mass DNA extracts were used to perform DNA–DNA hybridizations, using the microplate method and fluorescence measurements for calculation of binding values as described by Ezaki et al. (1989). Hybridizations were performed at 35 °C in hybridization mixture (2x SSC, 5x Denhardt's solution, 2·5 % dextran sulphate, 50 % formamide, 100 µg denaturated salmon sperm DNA ml-1, 1250 ng biotinylated probe DNA ml-1).
DNA–DNA hybridization experiments indicated that strains KMM 3664T, KMM 3669 and KMM 3915 shared only 11–12 % DNA–DNA homology with C. baltica NN015840T and C. algicola ACAM 630T. Furthermore, the three new isolates represented one genomic group, with DNA–DNA binding values of 72–90 % between them. DNA–DNA relatedness between C. algicola and C. baltica was 32 %; this confirms the results of Bowman (2000). DNA G+C contents were 33·2, 33·0 and 34·3 mol% for strains KMM 3664T, KMM 3669 and KMM 3915, respectively, when determined by the thermal denaturation method. Slightly lower values of 31·6, 32·0, 33·4 and 34·6 mol% for KMM 3664T, KMM 3669, C. algicola ACAM 630T and C. baltica NN015840T, respectively, were observed when determined by HPLC.
For determination of whole-cell fatty acid composition, strains were cultivated at 21 °C for 48 h on marine agar 2216 (Difco). Analysis of fatty acid methyl esters was performed by GLC [30 mx0·25 mm Supelcowax 10 column, 205 °C] as described by Svetashev et al. (1995).
The predominant cellular fatty acids of strains KMM 3664T, KMM 3669 and KMM 3915 were branched-chain saturated and unsaturated fatty acids and straight-chain saturated and monounsaturated fatty acids, namely i-C15 : 0 (9·1, 6·1 and 9·6 %, respectively), i-C15 : 1 (20·9, 13·1 and 19·6 %, respectively), C15 : 0 (16·6, 12·0 and 16·9 %, respectively), C16 : 1
7 (13·3, 9·1 and 17·4 %, respectively) and i-C17 : 0 3-OH (3·7, 7·8 and 2·8 %, respectively). Isoprenoid quinones were extracted and analysed by the method of Nakagawa & Yamasato (1993). The major lipoquinone was MK-6. The results of chemotaxonomic analyses support the affiliation of the sea water isolates to the family Flavobacteriaceae, all members of which are characterized by the presence of menaquinone 6 as the only or major respiratory quinone and the predominance of fatty acids C15 : 0, i-C15 : 0 and i-C17 : 0 3-OH (Bernardet et al., 1996, 2002).
Gram-staining reaction, oxidase, catalase and alkaline phosphatase activities, degradation of agar, starch, casein, gelatin, cellulose (filter paper and CM-cellulose), chitin, DNA, Tweens 20, 40 and 80, urea and alginic acids, flexirubin production, growth at different temperatures, NaCl concentrations and pH, production of acid from carbohydrates, nitrate reduction and production of H2S, indole and acetoin (Voges–Proskauer reaction) were tested according to the methods of Gerhardt et al. (1994). Susceptibility to antibiotics was determined as described previously (Nedashkovskaya et al., 2003). Gliding motility and spreading growth were determined by cultivation of strains on a medium that contained (l-1): 1 g Bactopeptone (Difco), 1 g yeast extract (Difco), 15 g agar and half-strength artificial sea water. The results are summarized in Table 2 and in the species description (see below).
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Based on the results of the polyphasic taxonomic analysis that is presented in this work, the new environmental isolates clearly represent a novel species in the genus Cellulophaga. We propose that strains KMM 3664T, KMM 3669 and KMM 3915 should be placed in this genus as members of a novel species, Cellulophaga pacifica sp. nov.
Description of Cellulophaga pacifica sp. nov.
Cellulophaga pacifica (pa.ci'fi.ca. N.L. adj. pacifica still; referring to the Pacific Ocean, from which the organism was isolated).
Cells are Gram-negative, strictly aerobic, chemo-organotrophic, motile by gliding, asporogenic rods that are 0·5–0·7 µm wide and 2·7–5·3 µm long. Oxidase-, catalase- and alkaline phosphatase-positive. Colonies are circular, low convex, shiny with entire edges, weakly sunken into agar and 1–3 mm in diameter on marine agar 2216. Yellow, non-diffusible pigments are produced. No growth is observed without Na+. Growth occurs at 1–8 % NaCl. pH range for growth is 5·5–10·0, with optimum growth at pH 7·5–8·5. Flexirubin pigments are absent. Growth is detected at 4 and 34 °C. Agar, gelatin, starch and Tweens 20, 40 and 80 are hydrolysed, but casein, cellulose (CM-cellulose and filter paper), alginic acids, chitin and DNA are not. Nitrate reduction is positive. Indole, acetoin (Voges–Proskauer reaction) and H2S are not produced. Acid is formed from arabinose, cellobiose, galactose, glucose, lactose, maltose, raffinose, sucrose and xylose, but not from melibiose, rhamnose, sorbose, N-acetylglucosamine, adonitol, dulcitol, glycerol, inositol, sorbitol or mannitol. Strains are susceptible to carbenicillin, oleandomycin and lincomycin and resistant to kanamycin, benzylpenicillin, neomycin, tetracycline, gentamicin and polymyxin B. Predominant cellular fatty acids are i-C15 : 0, i-C15 : 1, C15 : 0, C16 : 17 and i-C17 : 0 3-OH. Major lipoquinone is MK-6. DNA G+C content is 32–34 mol% (Tm and HPLC).
The type strain is KMM 3664T (=JCM 11735T=LMG 21938T). Isolated from sea water.
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ACKNOWLEDGEMENTS |
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