Roseivirga echinicomitans sp. nov., a novel marine bacterium isolated from the sea urchin Strongylocentrotus intermedius, and emended description of the genus Roseivirga

A novel strictly aerobic, heterotrophic, pink-pigmented, non-motile, Gram-negative, oxidase-, catalase-, β-galactosidase- and alkaline phosphatase-positive marine bacterium, designated strain KMM 6058^T, was isolated from the sea urchin Strongylocentrotus intermedius and studied using a polyphasic taxonomic approach. The G+C content of the DNA of the isolate was 41·3 mol%. The predominant fatty acids were i15 : 1, i15 : 0, a15 : 0 and i17 : 0 3-OH. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KMM 6058^T formed a monophyletic clade with Roseivirga ehrenbergii, with 99 % similarity. On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacterium should be assigned to the genus Roseivirga as Roseivirga echinicomitans sp. nov. The type strain is KMM 6058^T (=KCTC 12370^T=LMG 22587^T).

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Published online ahead of print on 15 April 2005 as DOI 10.1099/ijs.0.63621-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Roseivirga echinicomitans KMM 6058^T is AY753206.

The genus Roseivirga accommodates Gram-negative, strictly aerobic, heterotrophic, pink-pigmented, non-motile marine bacteria that belong to the phylum Bacteroidetes (Nedashkovskaya et al., 2005). The single strain of this genus was isolated from the green alga Ulva fenestrata and described as Roseivirga ehrenbergii. The genus Roseivirga forms a phylogenetic cluster with the species Reichenbachia agariperforans and the misclassified strains [Flexibacter] aggregans IFO 15974 and [Flexibacter] tractuosus IFO 16035. The heterogeneity of the genera joining the flexibacteria has been discussed previously (Bernardet et al., 1996; Nakagawa et al., 1997; Sly et al., 1998). The phylogenetic position of 40 strains belonging to the genera Flexibacter, Microscilla and Flexithrix has been analysed in detail by Nakagawa et al. (2002). On the basis of fresh 16S rRNA gene sequencing, the strains studied were divided into 24 groups. These authors suggested that the genera Flexibacter and Microscilla, currently represented by phylogenetically distant strains, should be restricted to the type species Flexibacter flexilis and Microscilla marina, respectively. Also, strains [Flexibacter] aggregans IFO 15974 and [Flexibacter] tractuosus IFO 16035, the closest relatives of the genus Roseivirga, may be classified as two species of a novel genus, because 16S rRNA gene sequence similarity between the strains is 94·5 %. The type strains of the heterogenic species [Flexibacter] tractuosus and [Flexibacter] aggregans, IFO 15989^T (=ATCC 23168^T) and IFO 15976^T (=ATCC 23162^T), are the nearest neighbours of Microscilla sericea IFO 15983 and Flexithrix dorotheae IFO 15987^T, respectively, with respective similarities of 99·3 and 100 %. It is expected that the classification of the above-mentioned strains will be improved by further study of their genomic and phenotypic characteristics.

During September 2002, we isolated an unknown bacterium from the sea urchin Strongylocentrotus intermedius collected in Troitsa Bay, Gulf of Peter the Great, East Sea (also known as the Sea of Japan). Polyphasic taxonomic study of the phenotypic, chemotaxonomic and genotypic characteristics and the phylogenetic position of strain KMM 6058^T cultured on marine agar 2216 (Difco) at 25 °C for 48 h, presented here, indicates that the isolate represents a novel species of the genus Roseivirga.

Genomic DNA extraction, a PCR and sequencing of the 16S rRNA gene were performed according to procedures described previously (Kim et al., 1998). The sequence data obtained were aligned with those of representative members of selected genera that belong to the phylum Bacteroidetes, using PHYDIT version 3.2 (). Phylogenetic trees were inferred using suitable programs of 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. Bootstrap analysis was performed with 1000 resampled datasets by using the SEQBOOT and CONSENSE programs of the PHYLIP package.

Phylogenetic analysis of the almost-complete 16S rRNA gene sequence (1415 nt) revealed that strain KMM 6058^T forms a distinct lineage within the genus Roseivirga (Fig. 1). The 16S rRNA gene sequence similarity between the strain studied and Roseivirga ehrenbergii KMM 6017^T was 99 %.

(50K): Fig. 1. Phylogenetic tree based on the 16S rRNA gene sequences of KMM 6058T and other representative taxa belonging to the phylum Bacteroidetes. Numbers at nodes indicate levels of bootstrap support (%) obtained using 1000 resampled datasets. Asterisks indicate branches that were also recovered with the maximum-likelihood algorithm. Bar, 0·1 substitutions per nucleotide position.

DNA was isolated according to the method of Marmur (1961) and its DNA G+C content was determined by using the thermal denaturation method (Marmur & Doty, 1962). The G+C content of the DNA of strain KMM 6058^T was 41·3 mol%. DNADNA hybridization experiments were performed using the method of De Ley et al. (1970). The DNADNA relatedness level between strain KMM 6058^T and Roseivirga ehrenbergii KMM 6017^T was 35 %. The DNA hybridization results clearly showed that the strain under study belongs to a novel species of the genus Roseivirga (Wayne et al., 1987).

Analysis of fatty acid methyl esters was carried out according to the standard protocol of the Sherlock Microbial Identification System (Microbial ID). The following cellular fatty acids accounted for more than 1·0 % of the total: i13 : 0 (2·9 %), i14 : 0 (1·9 %), i15 : 1 (20·2 %), a15 : 1 (2·4 %), i15 : 0 (20·2 %), a15 : 0 (13·1 %), i16 : 1 (2 %), i16 : 0 (1·8 %), i15 : 0 3-OH (4·1 %), i17 : 0 (1 %), i17 : 1ω9c (1·1 %), i16 : 0 3-OH (4·2 %), 16 : 0 3-OH (1·4 %), i17 : 0 3-OH (12·1 %), 17 : 0 2-OH (2 %), summed feature 3 (1 %) (comprising 16 : 1ω7 and/or i15 : 0 2-OH) and unidentified fatty acids (5·3 %).

Phenotypic analysis was performed using methods described previously (Nedashkovskaya et al., 2003a, b). API 20E, API 20NE, API ZYM (bioMérieux) and Microlog GN2 plates (Biolog) were also used for physiological and biochemical testing, performed according to the manufacturers' instructions, except that a solution for bacterial suspension consisting of 1·5 % NaCl was used. Gliding motility was determined as described by Bowman (2000).

The physiological, biochemical and morphological characteristics of strain KMM 6058^T are given in the species description and in Table 1. The results of phenotypic examination demonstrated many common traits between the strain studied and its closest relative, Roseivirga ehrenbergii. However, strain KMM 6058^T clearly differs from the above-mentioned species by the inability to grow at 37 °C and to hydrolyse DNA and Tween 20 (Table 1). Strain KMM 6058^T can also be readily differentiated from Roseivirga ehrenbergii by its ability to decompose Tween 40, to utilize D-galacturonic acid, glycerol, α-DL-glycerol phosphate, inosine, thymidine and citrate, to produce α- and β-galactosidases, β-glucosidase and N-acetyl-β-glucosaminidase, by its higher DNA G+C content and by its susceptibility to benzylpenicillin, streptomycin and tetracycline.

Table 1. Phenotypic characteristics of Roseivirga species Both strains gave positive results for the following: respiratory metabolism; oxidase, catalase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, α-glucosidase, alkaline phosphatase and acid phosphatase activities; Na+ requirement for growth; growth at 48 % NaCl and at 31 °C; hydrolysis of gelatin; and susceptibility to ampicillin, carbenicillin, lincomycin and oleandomycin. Both strains were negative for the following: gliding motility; flexirubin production; H2S, indole and acetoin production; hydrolysis of agar, casein, starch, Tween 80, cellulose (CM-cellulose and filter paper) and chitin; acid production from L-arabinose, L-fucose, D-galactose, D-glucose, D-lactose, D-maltose, D-melibiose, L-rhamnose, L-sorbose, D-sucrose, D-cellobiose, L-raffinose, DL-xylose, N-acetylglucosamine, glycerol, adonitol, inositol, mannitol, sorbitol and citrate; utilization of D-lactose, D-mannose, inositol, sorbitol and mannitol; α-glucuronidase and α-fucosidase activities; and susceptibility to gentamicin, kanamycin, neomycin and polymixin B.

Consequently, on the basis of the combination of significant molecular, genotypic and phenotypic differences between the strain studied and Roseivirga ehrenbergii presented here, we propose that strain KMM 6058^T should be placed in the genus Roseivirga, as Roseivirga echinicomitans sp. nov.

Since this study was completed, Yoon et al. (2005) have described a novel genus, Marinicola. We have found that the level of 16S rRNA gene sequence similarity between Marinicola seohaensis SW-152^T and Roseivirga ehrenbergii KMM 6017^T is 99·8 %. Consequently, on the basis of the phylogenetic relationship, the genus Marinicola should be placed in the genus Roseivirga. This conclusion is supported by the results of genomic, chemotaxonomic and phenotypic analyses, which revealed many common features between members of the genera Marinicola and Roseivirga. These data have been incorporated in the emended description of the genus Roseivirga.

Description of Roseivirga echinicomitans sp. nov.
Roseivirga echinicomitans [e.chi.ni.co'mi.tans. L. n. echinus -i sea urchin; L. pres. part. comitans (from L. v. comito) accompanying; N.L. part. adj. echinicomitans accompanying a sea urchin].

The main characteristics are the same as those given for the genus. In addition, cells range from 0·3 to 0·5 µm in width and 2·1 to 3·2 µm in length. Gliding motility not observed. On marine agar, colonies are 23 mm in diameter, circular, shiny with entire edges and pink-pigmented. Growth is observed at 431 °C. Optimal temperature for growth is 2123 °C. Growth occurs at 18 % NaCl. β-Galactosidase-positive. Decomposes aesculin, gelatin and Tween 40. Does not degrade agar, casein, starch, DNA, Tweens 20 or 80, cellulose (CM-cellulose and filter paper) or chitin. Does not form acid from L-arabinose, D-cellobiose, L-fucose, D-galactose, D-glucose, D-lactose, D-maltose, D-melibiose, L-raffinose, L-rhamnose, L-sorbose, D-sucrose, DL-xylose, N-acetylglucosamine, citrate, adonitol, dulcitol, glycerol, inositol or mannitol. Does not utilize L-arabinose, D-lactose, D-mannose, mannitol, inositol, sorbitol, malonate or citrate. Positive results for citrate utilization, acid formation from amygdalin, assimilation of glucose, N-acetylglucosamine, maltose and adipate and activity of esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, α-glucosidase and alkaline and acid phosphatases are obtained with API 20E, API 20NE and API ZYM kits. According to Microlog GN2 (Biolog), utilizes D-galacturonic acid, glycerol, α-DL-glycerol phosphate, inosine, thymidine, i-erythritol, D-galactose, D-sorbitol, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid and 2,3-butanediol and does not utilize α-cyclodextrin, dextrin, glycogen, Tweens 40 and 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, D-arabitol, cellobiose, D-fructose, L-fucose, gentiobiose, α-D-glucose, myo-inositol, α-lactose, α-D-lactose lactulose, maltose, D-mannitol, D-mannose, D-melibiose, methyl β-D-glucoside, psicose, D-raffinose, L-rhamnose, sucrose, D-trehalose, turanose, xylitol, methylpyruvate, monomethyl succinate, acetic acid, cis-aconitic acid, citric acid, formic acid, D-galactonic acid, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, α-ketobutyric acid, α-ketoglutaric acid, α-ketovaleric acid, DL-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, D-serine, L-serine, L-threonine, DL-carnitine, γ-aminobutyric acid, urocanic acid, uridine, phenylethylamine, putrescine, 2-aminoethanol, glucose 1-phosphate and glucose 6-phosphate. Nitrate is reduced. Results are negative for H₂S, indole and acetoin (VogesProskauer reaction) production. Fatty acids accounting for more than 1·0 % are i13 : 0 (2·9 %), i14 : 0 (1·9 %), i15 : 1 (20·2 %), a15 : 1 (2·4 %), i15 : 0 (20·2 %), a15 : 0 (13·1 %), i16 : 1 (2 %), i16 : 0 (1·8 %), i15 : 0 3-OH (4·1 %), i17 : 0 (1 %), 17 : 1ω9c (1·1 %), i16 : 0 3-OH (4·2 %), i17 : 0 3-OH (12·1 %), 17 : 0 2-OH (2 %) and summed feature 3 (1 %), comprising 16 : 1ω7 and/or i15 : 0 2-OH. The G+C content of the DNA of the type strain is 41·3 mol%.

The type strain, KMM 6058^T (=KCTC 12370^T=LMG 22587^T), was isolated from the sea urchin Strongylocentrotus intermedius collected in Troitsa Bay, Gulf of Peter the Great, East Sea.

Emended description of the genus Roseivirga Nedashkovskaya et al. 2005
Roseivirga (Ro.se.i.vir'ga. L. adj. roseus -a -um pink-coloured; L. fem. n. virga rod; N.L. fem. n. Roseivirga a pink-coloured and rod-shaped marine bacterium).

Rod-shaped cells that can move by gliding. Gram-negative. Do not form endospores. Strictly aerobic. Produce non-diffusible carotenoid pigments and can produce flexirubin pigments. Chemo-organotrophic. Cytochrome oxidase-, catalase- and alkaline phosphatase-positive. Can hydrolyse DNA and Tweens 20 and 40. The major respiratory quinone is MK-7. The main cellular fatty acids are straight-chain unsaturated and branched-chain unsaturated fatty acids i15 : 1, i15 : 0, a15 : 0 and i17 : 0 3-OH. As determined by 16S rRNA gene sequence analysis, the genus Roseivirga is a member of the phylum Bacteroidetes. The type species is Roseivirga ehrenbergii.