Cellulophaga pacifica sp. nov.

Three marine, heterotrophic, aerobic, agarolytic, pigmented and gliding bacteria were isolated in June 2000 from a sea water sample that was collected in the Gulf of Peter the Great, Sea of Japan, and analysed in a polyphasic taxonomic study. 16S rDNA sequence analysis indicated that strains KMM 3664^T, KMM 3669 and KMM 3915 were members of the family Flavobacteriaceae. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, the isolates were classified in the genus Cellulophaga as members of a novel species, Cellulophaga pacifica sp. nov. The type strain is KMM 3664^T (=JCM 11735^T=LMG 21938^T).

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Abbreviations: ACAM, Australian Collection of Antarctic Microorganisms, University of Tasmania, Hobart, Tasmania, Australia; ATCC, American Type Culture Collection, Manassas, VA, USA; JCM, Japan Collection of Microorganisms, Institute of Physical and Chemical Research (RIKEN), Wako, Japan; KMM, Collection of Marine Microorganisms of the Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia; LMG, BCCM/LMG Bacteria Collection, Laboratorium Microbiologie, Universiteit Gent, Belgium; NN, Enzyme Research, Novo Nordisk A/S, Bagsvaerd, Denmark

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 3664^T, KMM 3669 and KMM 3915 are AB100840, AB100841 and AB100842, respectively.

The genus Cellulophaga was proposed by Johansen et al. (1999) as a separate branch in the family Flavobacteriaceae, to comprise three species: Cellulophaga baltica and Cellulophaga fucicola, two agarolytic, yelloworange-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 3664^T (=JCM 11735^T=LMG 21938^T).

Strains KMM 3664^T, 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.

Table 1. Bacterial strains used in this study

To detect the precise taxonomic position of the strains studied, the almost-complete 16S rDNA sequences of strains KMM 3664^T, KMM 3669 and KMM 3915 were determined by PCR amplification and direct sequencing (Hiraishi, 1992), 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 CytophagaFlavobacteriumBacteroides 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 3664^T, KMM 3669 and KMM 3915 to the type strains of C. lytica and C. fucicola were too low (91·992·1 and 93·093·1 %, 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).

(49K): Fig. 1. Phylogenetic position of strains KMM 3664T, KMM 3669 and KMM 3915 among species of the family Flavobacteriaceae on the basis of 16S rDNA sequence comparison. The phylogenetic tree was generated by the neighbour-joining method (Saitou & Nei, 1987). The 16S rDNA sequence of Bacteroides fragilis (X83935) was used as the outgroup. Numbers next to nodes indicate percentage bootstrap values from 1000 replicates (only values of 70 % or higher are cited). Bar, 0·02 genetic distance (Knuc).

For determination of DNA G+C content and DNADNA binding values, DNA was prepared from cells that had been cultivated on marine agar (Difco) for 2448 h at 25 °C. The DNA G+C content was determined by using two approaches: (i) the thermal denaturation method of Marmur & Doty (1962) for strains KMM 3664^T, 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 NN015840^T, KMM 3664^T 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 630^T, 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 DNADNA 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).

DNADNA hybridization experiments indicated that strains KMM 3664^T, KMM 3669 and KMM 3915 shared only 1112 % DNADNA homology with C. baltica NN015840^T and C. algicola ACAM 630^T. Furthermore, the three new isolates represented one genomic group, with DNADNA binding values of 7290 % between them. DNADNA 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 3664^T, 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 3664^T, KMM 3669, C. algicola ACAM 630^T and C. baltica NN015840^T, 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 3664^T, KMM 3669 and KMM 3915 were branched-chain saturated and unsaturated fatty acids and straight-chain saturated and monounsaturated fatty acids, namely i-C_{15 : 0} (9·1, 6·1 and 9·6 %, respectively), i-C_{15 : 1} (20·9, 13·1 and 19·6 %, respectively), C_{15 : 0} (16·6, 12·0 and 16·9 %, respectively), C_{16 : 1}ω7 (13·3, 9·1 and 17·4 %, respectively) and i-C_{17 : 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 C_{15 : 0}, i-C_{15 : 0} and i-C_{17 : 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 (VogesProskauer 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).

Table 2. Phenotypic properties of Cellulophaga pacifica and other Cellulophaga species Strains: 1, C. pacifica KMM 3664T; 2, C. pacifica KMM 3669; 3, C. pacifica KMM 3915; 4, C. lytica ATCC 23178T; 5, C. algicola ACAM 630T; 6, C. baltica NN015840T; 7, C. fucicola NN015860T. Data are from Johansen et al. (1999), Bowman (2000) and this study. All strains tested were positive for the following characteristics: motion by gliding, respiratory metabolism, oxidase, catalase and alkaline phosphatase production, Na+ requirement for growth, growth at 4 °C, growth at 6 % NaCl, hydrolysis of agar, gelatin and starch and susceptibility to lincomycin and oleandomycin. All strains were negative for the following characteristics: flexirubin pigments, growth at 10 % NaCl, hydrolysis of cellulose (CM-cellulose and filter paper) and chitin, production of H2S, indole and acetoin (VogesProskauer reaction), acid from rhamnose, sorbose, N-acetylglucosamine, adonitol, dulcitol, glycerol, inositol and sorbitol and susceptibility to benzylpenicillin, gentamicin, kanamycin and neomycin.

Strains KMM 3664^T, KMM 3669 and KMM 3915 have some traits in common with currently described Cellulophaga species, but can be differentiated from all of them by arabinose and raffinose oxidation and the absence of DNase production, alginate hydrolysis and susceptibility to carbenicillin. The strains do not grow at 37 °C, in contrast to C. lytica strains. Reduction of nitrates to nitrites is noted for the strains studied, but not for strains of C. lytica or C. fucicola. Strains KMM 3664^T, KMM 3669 and KMM 3915 differ from the type strains of C. baltica (NN015840^T) and C. fucicola (NN015860^T) by the absence of casein hydrolysis.

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 3664^T, 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 0·50·7 µm wide and 2·75·3 µm long. Oxidase-, catalase- and alkaline phosphatase-positive. Colonies are circular, low convex, shiny with entire edges, weakly sunken into agar and 13 mm in diameter on marine agar 2216. Yellow, non-diffusible pigments are produced. No growth is observed without Na⁺. Growth occurs at 18 % NaCl. pH range for growth is 5·510·0, with optimum growth at pH 7·58·5. 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 (VogesProskauer 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-C_{15 : 0}, i-C_{15 : 1}, C_{15 : 0}, C_{16 : 1}ω7 and i-C_{17 : 0} 3-OH. Major lipoquinone is MK-6. DNA G+C content is 3234 mol% (T_m and HPLC).

The type strain is KMM 3664^T (=JCM 11735^T=LMG 21938^T). Isolated from sea water.