Gramella echinicola gen. nov., sp. nov., a novel halophilic bacterium of the family Flavobacteriaceae isolated from the sea urchin Strongylocentrotus intermedius

A novel marine bacterium, strain KMM 6050^T, was isolated from the sea urchin Strongylocentrotus intermedius, which inhabits the Sea of Japan. The strain studied was strictly aerobic, heterotrophic, yelloworange-pigmented, motile by gliding, Gram-negative and oxidase-, catalase-, β-galactosidase- and alkaline phosphatase-positive. The results of 16S rRNA gene sequence analysis showed that strain KMM 6050^T occupies a distinct lineage within the family Flavobacteriaceae and is most closely related to the species Mesonia algae and Salegentibacter salegens (sequence similarity of 92·592·6 %). The DNA G+C content of KMM 6050^T was 39·6 mol%. The major respiratory quinone was MK-6. The predominant fatty acids were i15 : 0, a15 : 0, 15 : 0, i16 : 1, i16 : 0, i16 : 0 3-OH and i17 : 0 3-OH. On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacterium has been assigned to the genus Gramella gen. nov., as Gramella echinicola sp. nov. The type strain is KMM 6050^T (=KCTC 12278^T=NBRC 100593^T=LMG 22585^T).

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Published online ahead of print on 10 September 2004 as DOI 10.1099/ijs.0.63314-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Gramella echinicola strain KMM 6050^T is AY608409.

Flavobacteria are often found in different marine habitats, including sea water, sediments or algae (Hanzawa et al., 1998; Glöckner et al., 1999; Patel et al., 2003). Also, the representatives of this group are considered to be an important part of marine-animal microbial populations, as indicated by data from culture-independent molecular analyses (Cooney et al., 2002; López-García et al., 2002; Webster et al., 2004). For example, Alain et al. (2002) examined the microbial assemblage associated with the hydrothermal vent polychaete Paralvinella palmiformis of the north-eastern Pacific by using 16S rRNA gene sequence-based phylogenetic analysis; they reported that members of the CytophagaFlavobacterium group were identified frequently in 16S rRNA gene clone libraries.

In the course of a study on the microbial population of the sea urchin Strongylocentrotus intermedius (a common member of the fauna of the Sea of Japan), a novel heterotrophic, aerobic, yelloworange-pigmented, gliding, Gram-negative bacterium was isolated. 16S rRNA gene sequence-based phylogenetic analysis of strain KMM 6050^T revealed it to be a potentially distinct and novel member of the family Flavobacteriaceae. The nearest neighbours of the strain studied were Mesonia algae and Salegentibacter salegens (92·6 and 92·5 % sequence similarity, respectively). On the basis of the results of 16S rRNA gene sequence phylogenetic analysis and the chemotaxonomic and phenotypic features of strain KMM 6050^T, obtained and presented in this work, we propose the description of a new genus of the family Flavobacteriaceae, Gramella gen. nov., and novel species, Gramella echinicola sp. nov.

Strain KMM 6050^T was isolated from the sea urchin Strongylocentrotus intermedius, which was collected at a depth of 3 m in Troitsa Bay, Gulf of Peter the Great, Sea of Japan, Pacific Ocean, in September 2002. For strain isolation, 0·1 ml homogenates of sea-urchin tissues were transferred onto plates of 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.

DNA extraction, PCRs and sequencing of the 16S rRNA gene followed procedures described previously (Kim et al., 1998). The sequences obtained 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 from the PHYLIP package (Felsenstein, 1993). Phylogenetic distances were calculated from the models of Kimura (1980) and the trees were constructed on the basis of the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1993) algorithms. A bootstrap analysis was performed with 1000 resampled datasets by using the SEQBOOT and CONSENSE programs of the PHYLIP package.

Phylogenetic 16S rRNA gene sequence analysis indicated that strain KMM 6050^T is a member of the family Flavobacteriaceae and forms a cluster with species of the genera Psychroflexus, Salegentibacter and Mesonia (Fig. 1). The closest relatives of this strain are M. algae KMM 3909^T and Salegentibacter salegens DSM 5424^T, there being 16S rRNA gene sequence similarities of 92·6 and 92·5 %, respectively, without significant bootstrap support.

(15K): Fig. 1. Phylogenetic tree based on the 16S rRNA gene sequences of strain KMM 6050T and related members of the family Flavobacteriaceae. The asterisks indicate branches that were also recovered with the maximum-likelihood algorithm, while the numbers at the nodes are the levels of bootstrap support (%) from the 1000 resamplings in the dataset. Bar, 0·01 substitution per nucleotide position.

DNA was isolated according to the method of Marmur (1961) and its G+C content was determined by the thermal denaturation method (Marmur & Doty, 1962). The DNA G+C content of strain KMM 6050^T was 39·6 mol%.

Analysis of fatty acid methyl esters was carried out according to the standard protocol of the Microbial Identification System (Microbial ID). The main cellular fatty acids of KMM 6050^T were i15 : 0, a15 : 0, 15 : 0, i16 : 1, i16 : 0, i16 : 0 3-OH, i17 : 0 3-OH and summed feature 3, comprising 16 : 1ω7 or i15 : 0 2-OH fatty acids (Table 1). Isoprenoid quinones were extracted from lyophilized cells and analysed as described previously (Nedashkovskaya et al., 2003a). The major respiratory quinone was MK-6.

Table 1. Cellular fatty acid compositions (percentage content) of Gramella echinicola KMM 6050T and its closest relatives Species: 1, G. echinicola KMM 6050T; 2, M. algae KMM 3909T; 3, Salegentibacter salegens DSM 5424T. Data are from Nedashkovskaya et al. (2003a) and this study.

A phenotypic analysis was performed by using previously described methods (Nedashkovskaya et al., 2003a, b). Gliding motility was determined as described by Bowman (2000).

The physiological, biochemical and morphological characteristics of strain KMM 6050^T are listed in the species description and are shown in Table 2. The results of the phenotypic examination demonstrated that the strain studied and M. algae had many traits in common. However, strain KMM 6050^T differs sufficiently from its closest relative by having the ability to move by gliding, by producing acid from carbohydrates, by growing at 37 °C, by hydrolysing starch and DNA, by producing hydrogen sulfide and by having a higher G+C content (Table 2). The sea-urchin isolate can be clearly differentiated from the other nearest neighbour, Salegentibacter salegens, by the presence of gliding motility and by the ability to degrade casein (Table 2). Significant differences in the cellular fatty acid composition of the strain studied and type strains of the genera Mesonia and Salegentibacter support the creation of a new genus for strain KMM 6050^T (Table 1). The low sequence similarities between the strain tested and other flavobacteria described to date (89·391·3 %) demonstrate clearly that the bacterium isolated in this study represents a new genus.

Table 2. Differential characteristics of Gramella gen. nov. and related genera of the family Flavobacteriaceae Genera: 1, Gramella; 2, Mesonia; 3, Salegentibacter; 4, Psychroflexus. Data are from Dobson et al. (1993), McCammon & Bowman (2000), Bowman et al. (1998), Nedashkovskaya et al. (2003a, 2004) and this study. , Negative; +, positive; V, variable.

It is, therefore, evident from the phylogenetic distinctness in combination with significant differences in the phenotypic findings and cellular fatty acid composition that the strain studied should be differentiated from the nearest neighbours M. algae and Salegentibacter salegens. Thus, the results of the polyphasic analysis presented in this work clearly demonstrate that the strain studied cannot be assigned to any of the currently described taxa of the family Flavobacteriaceae, and support the placement of strain KMM 6050^T in a new genus, Gramella gen. nov., as Gramella echinicola sp. nov.

Description of Gramella gen. nov.
Gramella [Gra.mel'la. L. dim. ending -ella; N.L. fem. n. Gramella of Gram. Named in honour of the famous Danish pharmacologist and pathologist Hans Christian Gram (18531938), who proposed the differentiating staining of bacteria by gentian violet].

Rod-shaped cells, motile by gliding. Gram-negative. Do not form endospores. Strictly aerobic. Produce non-diffusible carotenoid pigments. Chemo-organotrophic. Cytochrome oxidase-, catalase- and alkaline phosphatase-positive. The major respiratory quinone is MK-6. The main cellular fatty acids are straight-chain unsaturated and branched-chain unsaturated fatty acids i15 : 0, a15 : 0, 15 : 0, i16 : 1, i16 : 0, i16 : 0 3-OH and i17 : 0 3-OH and summed feature 3, comprising fatty acids 15 : 0 iso 2-OH and/or 16 : 1ω7 or both. As determined by 16S rRNA gene sequence analysis, the genus Gramella is a member of the family Flavobacteriaceae.

The type species is Gramella echinicola.

Description of Gramella echinicola sp. nov.
Gramella echinicola (e.chi.ni.co'la. L. n. echinus a hedgehog, a sea urchin; L. suffix -cola dweller; N.L. n. echinicola a sea-urchin-dweller).

The main characteristics are the same as those given for the genus. In addition, cells are 0·50·7 µm in width and 2·12·7 µm in length. On marine agar, colonies are 24 mm in diameter, circular, shiny with entire edges and yelloworange in colour. Growth is observed at 437 °C. Optimal temperature for growth is 2325 °C. Growth occurs in 115 % (w/v) NaCl. β-Galactosidase-positive. Casein, gelatin, starch, DNA, Tween 40 and Tween 80 are decomposed. Agar, urea, Tween 20, cellulose (CM-cellulose and filter paper) and chitin are not degraded. Acid is formed from D-galactose, D-glucose, D-maltose, D-sucrose and L-raffinose, but not from L-arabinose, D-cellobiose, L-fucose, D-lactose, D-melibiose, L-rhamnose, DL-xylose, citrate, adonitol, dulcitol, glycerol, inositol or mannitol. L-Arabinose, D-glucose and D-sucrose are utilized, but D-lactose, D-mannose, mannitol, inositol, sorbitol, malonate and citrate are not utilized. Nitrate is not reduced. H₂S, indole and acetoin (VogesProskauer reaction) are not produced. The G+C content of the DNA is 39·6 mol%.

The type strain is KMM 6050^T (=KCTC 12278^T=NBRC 100593^T=LMG 22585^T). Isolated from the sea urchin Strongylocentrotus intermedius, collected in Troitsa Bay, Gulf of Peter the Great, Sea of Japan.