Introduction

The genus Roseovarius, a member of the Alphaproteobacteria, was established by Labrenz et al. (1999) with the description of Roseovarius tolerans, isolated from water samples from Ekho Lake, Antarctica, as the sole recognized species. Subsequently, nine further Roseovarius species with validly published names, Roseovarius nubinhibens (González et al. 2003), Roseovarius crassostreae (Boettcher et al. 2005), Roseovarius mucosus (Biebl et al. 2005), Roseovarius aestuarii (Yoon et al. 2008), Roseovarius pacificus (Wang et al. 2009, 2010), Roseovarius halotolerans (Oh et al. 2009), Roseovarius nanhaiticus (Wang et al. 2010), Roseovarius marinus (Jung et al. 2011) and Roseovarius indicus (Lai et al. 2011), have been described from a hypersaline lake, seawater, a dinoflagellate culture, oysters and marine sediments (Labrenz et al. 1999; González et al. 2003; Biebl et al. 2005; Boettcher et al. 2005; Yoon et al. 2008; Wang et al. 2009, 2010). In this study, we describe a bacterial strain, designated GSW-M15T, which was isolated from seawater on the southern coast of South Korea and found to be phylogenetically affiliated to the genera Roseovarius and Donghicola on the basis of comparative 16S rRNA gene sequence analysis. This study describes a polyphasic characterization, which included determination of the chemotaxonomic and other phenotypic properties, a detailed phylogenetic investigation based on 16S rRNA gene sequences and genetic analysis, to determine the exact taxonomic position of strain GSW-M15T.

Materials and methods

Bacterial strains and culture conditions

Seawater was collected from the coast of Geojedo, South Korea, and used as the source for the isolation of bacterial strains. Strain GSW-M15T was isolated by means of the standard dilution plating technique at 25 °C on marine agar 2216 (MA; Becton–Dickinson). Strain GSW-M15T was maintained on MA at 4 °C for short-term preservation and as a glycerol suspension (20 %, w/v in distilled water) at −80 °C for long-term preservation. Strain GSW-M15T has been deposited in the Korean Collection for Type Cultures (KCTC; South Korea) and the Culture Collection, University of Göteborg (CCUG; Sweden) as KCTC 23897T and CCUG 62218T, respectively. R. halotolerans KCTC 22224T and R. pacificus LMG 24575T, which were used as reference strains for fatty acid analysis, phenotypic characterization and DNA–DNA hybridization, were obtained from the Korean Collection for Type Cultures (KCTC), Daejeon, South Korea, and Laboratorium voor Microbiologie Universiteit Gent (LMG), Gent, Belgium, respectively.

Cell biomass of strain GSW-M15T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 3 days in marine broth 2216 (MB; Becton–Dickinson) at 30 °C. For fatty acid methyl ester analysis, cell mass of strain GSW-M15T, R. halotolerans KCTC 22224T and R. pacificus LMG 24575T was harvested from MA plates after incubation for 3 days at 30 °C.

Morphological, physiological and biochemical characterization

The morphological, physiological and biochemical characteristics of strain GSW-M15T were investigated using routine cultivation on MA at 30 °C. The cell morphology was examined by light microscopy (Olympus BX51) and transmission electron microscopy (Philips CM-20). The latter technique was also used to assess the presence of flagella on cells from exponentially growing cultures. For this purpose, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer’s instructions. Strain GSW-M15T was cultured at various temperatures (4, 10, 20, 25, 28, 30, 35, 37, 40 and 45 °C) to measure optimal temperature and temperature range for growth. The pH range for growth was determined in MB adjusted to pH 4.5–9.5 (using increments of 0.5 pH unit) by using sodium acetate/acetic acid and Na2CO3 buffers. The pH values were verified after autoclaving. Growth in the presence of 0–20.0 % (w/v) NaCl was investigated by using marine broth prepared according to the formula of the Becton–Dickinson medium except that NaCl was excluded. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber (1029; Forma, N2:CO2:H2, 86:7:7 %) on MA and on MA supplemented with potassium nitrate (0.1 %, w/v), both of which had been prepared anaerobically under nitrogen atmosphere. Catalase and oxidase activities were investigated as described by Cowan and Steel (1965). Hydrolysis of casein, starch, hypoxanthine, l-tyrosine and xanthine was tested on MA by using the substrate concentrations described by Cowan and Steel (1965). Hydrolysis of aesculin and Tweens 20, 40, 60 and 80 and nitrate reduction were investigated as described previously (Lányí 1987) with the modification that artificial seawater was used for preparation of media. Hydrolysis of gelatin and urea was investigated by using Nutrient gelatin and Urea agar base media (BD), respectively, with the modification that artificial seawater was used for the preparation of media. The artificial seawater contained (l−1 distilled water): 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2·6H2O, 5.94 g MgSO4·7H2O and 1.3 g CaCl2·2H2O (Bruns et al. 2001). For analysis of pigments, the absorption spectrum was determined for strain GSW-M15T that was cultivated aerobically in the dark at 30 °C in MB. The culture was centrifuged, washed twice using a MOPS buffer (MOPS/NaOH 0.01 M; KCl 0.1 M; MgCl2 0.001 M; pH 7.5) and disrupted by sonication with a Branson Sonifier 450. After removal of cell debris by centrifugation, the absorption spectrum of the supernatant was examined in 200–950 nm on a Beckman Coulter DU800 spectrophotometer. Utilization of substrates as sole carbon and energy sources was tested as described by Baumann and Baumann (1981) using supplementation with 2 % (v/v) Hutner’s mineral base (Cohen-Bazire et al. 1957) and 1 % (v/v) vitamin solution (Staley 1968). Susceptibility to antibiotics was tested on MA plates using antibiotic discs (Advantec) containing the following (μg per disc unless otherwise stated): ampicillin (10), carbenicillin (100), cephalothin (30), chloramphenicol (100), gentamicin (30), kanamycin (30), lincomycin (15), neomycin (30), novobiocin (5), oleandomycin (15), penicillin G (20 U), polymyxin B (100 U), streptomycin (50) and tetracycline (30). Enzyme activities were determined, after incubation for 8 h at 30 °C, by using the API ZYM system (bioMérieux).

16S rRNA gene sequencing and phylogenetic analysis

Chromosomal DNA was extracted and purified according to the method described previously (Yoon et al. 1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene amplification was performed using two universal primers (5′-GAGTTTGATCCTGGCTCAG-3′ and 5′-ACGGTTACCTTGTTACGACTT-3′) as described previously (Yoon et al. 1998) and the PCR products were purified by using a QIAquick PCR purification kit (Qiagen). Sequencing of the amplified 16S rRNA gene was performed as described by Yoon et al. (2003). Alignment of sequences was carried out with CLUSTAL W software (Thompson et al. 1994). Gaps at the 5′ and 3′ ends of the alignment were omitted from further analysis. Phylogenetic trees were inferred by using three tree-making algorithms, the neighbour-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Kluge and Farris 1969) methods implemented within the PHYLIP package (Felsenstein 1993). Evolutionary distance matrices for the neighbour-joining method were calculated by the algorithm of Jukes and Cantor (1969) using the program DNADIST. The stability of relationships was assessed by a bootstrap analysis based on 1,000 resampling of the neighbour-joining dataset by using the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE of the PHYLIP package.

DNA–DNA hybridization

DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values are quoted as DNA–DNA relatedness values. The DNAs of strain GSW-M15T, R. halotolerans KCTC 22224T and R. pacificus LMG 24575T were used individually as labelled DNA probes for reciprocal hybridization.

Chemotaxonomic characterization

Isoprenoid quinones were extracted according to the method of Komagata and Suzuki (1987) and analyzed using reversed-phase HPLC and a YMC ODS-A (250 × 4.6-mm) column. The isoprenoid quinones were eluted by a mixture of methanol/isopropanol (2:1, v/v), using a flow rate of 1 ml min−1 at room temperature and detected by UV absorbance at 275 nm. Fatty acids were saponified, methylated and extracted using the standard protocol of the MIDI (Sherlock Microbial Identification System, version 4.0). The fatty acids were analysed by GC (Hewlett Packard 6890) and identified by using the TSBA40 database of the Microbial Identification System (Sasser 1990). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and separated by two-dimensional TLC using chloroform/methanol/water (65:25:3.8, v/v) for the first dimension and chloroform/methanol/acetic acid/water (40:7.5:6:1.8, v/v) for the second dimension as described by Minnikin et al. (1977). Individual polar lipids were identified by spraying with the ethanolic molybdophosphoric acid, molybdenum blue, ninhydrin and α-naphthol reagents (Minnikin et al. 1984; Komagata and Suzuki 1987) and the Dragendorff’s reagent (Sigma). The DNA G+C content was determined by the method of Tamaoka and Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC equipped with a YMC ODS-A (250 × 4.6-mm) column. The nucleotides were eluted by a mixture of 0.55 M NH4H2PO4 (pH 4.0) and acetonitrile (40:1, v/v), using flow rate of 1 ml min−1 at room temperature and detected by UV absorbance at 270 nm.

Results and discussion

Morphological, cultural, physiological and biochemical characteristics

Strain GSW-M15T was found to be aerobic, gram-negative, non-spore-forming and ovoid- to rod-shaped. Strain GSW-M15T was non-motile, whereas its two closest phylogenetic neighbours, R. halotolerans KCTC 22224T and R. pacificus LMG 24575T, are non-motile and motile, respectively (Oh et al., 2009; Wang et al., 2009). Strain GSW-M15T grew optimally at 30 °C and pH 7.0–7.5. Strain GSW-M15T is a moderate halophile as it grew optimally in the presence of 2 % (w/v) NaCl; it grew in the presence of 0.5–15 % NaCl. Strain GSW-M15T showed catalase and oxidase activities but no urease activity. Strain GSW-M15T did not produce bacteriochlorophyll a and reduced nitrate to nitrite. Morphological, cultural, physiological and biochemical characteristics of strain GSW-M15T are given in the species description (see below) or in Table 1.

Table 1 Phenotypic characteristics of Roseovarius litoreus GSW-M15T and the type strains of Roseovarius halotolerans and Roseovarius pacificus
Full size table

Phylogenetic analysis

The almost complete 16S rRNA gene sequence of strain GSW-M15T determined in this study comprised 1,389 nucleotides (approximately 96 % of the Escherichia coli 16S rRNA sequence). In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain GSW-M15T clustered with the type strains of R. halotolerans and R. pacificus (Fig. 1). The relationships among strain GSW-M15T, R. halotolerans KCTC 22224T and R. pacificus LMG 24575T were also maintained in the trees constructed using the maximum-likelihood and maximum-parsimony algorithms (Supplementary Figs. 1, 2). The maximum-likelihood and maximum-parsimony phylogenetic trees revealed that strain GSW-M15T fell also within the clade comprising Roseovarius species (Supplementary Figs. 1, 2). Strain GSW-M15T exhibited highest 16S rRNA gene sequence similarity values of 98.3 and 97.5 % to the type strains of R. halotolerans and R. pacificus, respectively. Strain GSW-M15T exhibited slightly higher 16S rRNA gene sequence similarity values (96.6 and 96.2 %) to the type strains of Donghicola xiamenensis and Donghicola eburneus than to the type strains of the other Roseovarius species, with which it exhibited 92.8–96.2 % sequence similarity. In view of this and the close relationship of strain GSW-M15T to the type strains of R. halotolerans and R. pacificus (Fig. 1; Supplementary Figs. 1, 2), these three species may represent a novel genus but further studies with additional strains would be needed to confirm this.

Fig. 1
figure 1

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Roseovarius litoreus GSW-M15T, other Roseovarius species and representatives of some other related taxa. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branching points. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and maximum-parsimony algorithms. Altermonas marina SW-47T (GenBank Accession No: AF529060) was used as an outgroup (not shown). Scale bar 0.01 substitutions per nucleotide position

Full size image

DNA–DNA relatedness

Strain GSW-M15T exhibited mean DNA–DNA relatedness values of 33 ± 7 and 18 ± 5 % to R. halotolerans KCTC 22224T and R. pacificus LMG 24575T, respectively.

Chemotaxonomic characteristics

The predominant isoprenoid quinone detected in strain GSW-M15T was ubiquinone-10 (Q-10) which is compatible with other Roseovarius species (Labrenz et al. 1999; Yoon et al. 2008, Jung et al. 2011). The cellular fatty acid profiles of strain GSW-M15T, R. halotolerans KCTC 22224T and R. pacificus LMG 24575T, which were analyzed in this study, are compared in Table 2. The major fatty acids (>10 % of the total fatty acids) found in strain GSW-M15T were C18:1 ω7c and 11-methyl-C18:1 ω7c (Table 2). The fatty acid profile of strain GSW-M15T was similar with those of the two reference strains and of the type strain of R. tolerans (type species of the genus Roseovarius) analyzed previously under the same conditions (Yoon et al. 2008), even though there were differences in the proportions of some fatty acids. The major polar lipids detected in strain GSW-M15T were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and two unidentified lipids (Supplementary Fig. 3). This profile was similar with that of the type strain of R. tolerans shown by Kim et al. (2011) in that phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and two unidentified lipids exist as major polar lipids, although diphosphatidylglycerol, which is major polar lipid in the type strain of R. tolerans, was only detected as a minor component in strain GSW-M15T. The DNA G+C content of strain GSW-M15T was 62.9 mol%, a value in the range reported for Roseovarius species (Wang et al. 2009, 2010; Jung et al. 2011; Lai et al. 2011).

Table 2 Cellular fatty acid compositions (%) of Roseovarius litoreus GSW-M15T and the type strains of Roseovarius halotolerans and Roseovarius pacificus
Full size table

Conclusion

It is appropriate to classify strain GSW-M15T as a member of the genus Roseovarius as shown by the phylogenetic inference and the absence of differentiating chemotaxonomic properties from Roseovarius species, including R. halotolerans and R. pacificus (Labrenz et al. 1999; González et al. 2003; Wang et al. 2009, 2010; Jung et al. 2011; Lai et al. 2011). Phenotypic characteristics of strain GSW-M15T were compared with R. halotolerans KCTC 22224T and R. pacificus LMG 24575T i.e. related species that showed 16S rRNA gene sequence similarity of >97 % (Table 2). Strain GSW-M15T was distinguishable from R. halotolerans KCTC 22224T and R. pacificus LMG 24575T by differences in several phenotypic characteristics, including motility, nitrate reduction, utilization of some substrates, susceptibility to some antibiotics and activity of some enzymes, most of which were determined under the same conditions and methods (Table 1; Kim et al. 2011). The phylogenetic and genetic distinctiveness of strain GSW-M15T, together with the differential phenotypic properties, is sufficient to show that this strain is separate from other Roseovarius species (Wayne et al. 1987; Stackebrandt and Goebel 1994). Therefore, on the basis of the phenotypic, chemotaxonomic, phylogenetic and genetic data, strain GSW-M15T is considered to represent a novel species of the genus Roseovarius, for which the name Roseovarius litoreus sp. nov. is proposed.

Description of Roseovarius litoreus sp. nov

Roseovarius litoreus (li.to.re’us. L. masc. adj. litoreus of or belonging to the seashore).

Cells are gram-negative, non-spore-forming, non-flagellated and ovoid- to rod-shaped, 0.4–0.9 μm in diameter and 0.7–7.0 μm in length. Colonies on MA are circular, flat to raised, smooth, glistening, grayish yellow in colour and 0.5–1.0 mm in diameter after incubation for 3 days at 30 °C. Optimal growth occurs at 30 °C; growth occurs at 10 and 40 °C, but not at 4 and 45 °C. Optimal pH for growth is between 7.0 and 7.5; growth occurs at pH 5.5, but not at pH 5.0. Optimal growth occurs in the presence of 2 % (w/v) NaCl; growth occurs at 0.5–15.0 % (w/v) NaCl. Mg2+ ions are required for growth. Growth does not occur under anaerobic conditions on MA and on MA supplemented with nitrate. Bacteriochlorophyll a is not produced. Catalase- and oxidase-positive. Nitrate is reduced to nitrite. Tweens 20, 40 and 60 are hydrolysed, but aesculin, casein, gelatin, hypoxanthine, xanthine, starch, Tween 80 and l-tyrosine are not. Acetate is utilized but l-arabinose, d-cellobiose, d-fructose, d-galactose, d-glucose, maltose, d-mannose, sucrose, d-trehalose, d-xylose, citrate, benzoate, formate, l-malate, pyruvate, salicin, succinate and l-glutamate are not. Susceptible to ampicillin, carbenicillin, cephalothin, chloramphenicol, gentamicin, kanamycin, neomycin, novobiocin, penicillin G and streptomycin, but not to lincomycin, polymyxin B and tetracycline. In assays with the API ZYM system, activity of alkaline phosphatase (weak), esterase (C 4), esterase lipase (C 8), lipase (C 14) (weak) and leucine arylamidase are present, but activity of valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase are absent. The predominant ubiquinone is Q-10. The major fatty acids (>10 % of the total fatty acids) are C18:1 ω7c and 11-methyl-C18:1 ω7c. The major polar lipids are phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and two unidentified lipids. The DNA G+C content is 62.9 mol% (determined by HPLC).

The type strain, GSW-M15T (=KCTC 23897T = CCUG 62218T), was isolated from seawater of Geojedo on the South Sea, South Korea.