Introduction

The genus Ruegeria, a member of the class Alphaproteobacteria, was established by Uchino et al. (1998) with the reclassification of Agrobacterium atlanticum, Agrobacterium gelatinovorum and Roseobacter algicola as Ruegeria atlantica, Ruegeria gelatinovora and Ruegeria algicola, respectively. However, Ruegeria gelatinovorans corrig and Ruegeria algicola were subsequently reclassified as members of two other genera (Arahal et al. 2005; Martens et al. 2006), whilst Silicibacter lacuscaerulensis (Petursdottir and Kristjansson 1997) and Silicibacter pomeroyi (González et al. 2003) were transferred into the genus Ruegeria as two different species (Yi et al. 2007). It was recently shown that Ruegeria pelagia (Lee et al. 2007) is a later synonym of Ruegeria mobilis (Lai et al. 2010). The genus Ruegeria currently comprises seven species with validly published names: R. atlantica (Uchino et al. 1998), Ruegeria lacuscaerulensis and Ruegeria pomeroyi (Yi et al. 2007), R. mobilis (Muramatsu et al. 2007), Ruegaria scottomollicae (Vandecandelaere et al. 2008), Ruegeria marina (Huo et al. 2011) and Ruegeria faecimaris (Oh et al. 2011). In this study, we describe a Ruegeria-like bacterial strain, designated G-M8T, which was isolated from seashore sand around a seaweed farm at Geoje island, South Korea. The aim of the present work was to determine the exact taxonomic position of strain G-M8T by using a polyphasic approach.

Materials and methods

Bacterial strains and culture conditions

Marine sand was collected from the surface of the seashore around a seaweed farm at Geoje island in the South Sea, South Korea, and used as a source for the isolation of bacterial strains. Strain G-M8T was isolated by the standard dilution plating technique on marine agar 2216 (MA; Becton–Dickinson) at 25 °C and cultivated routinely on MA at 30 °C. Strain G-M8T 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 G-M8T has been deposited in the Korean Collection for Type Cultures (KCTC; South Korea) and the Culture Collection, University of Göteborg (CCUG; Sweden) as KCTC 23960T and CCUG 62412T, respectively. R. atlantica (the type species of the genus Ruegeria) KCTC 12424T and R. lacuscaerulensis KCTC 2953T, which were used as reference strains for DNA–DNA hybridization, were obtained from the Korean Collection for Type Cultures (KCTC), Daejeon, South Korea.

Cell biomass of strain G-M8T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids, and cell biomass of R. atlantica KCTC 12424T and R. lacuscaerulensis KCTC 2953T for DNA extraction, were obtained from cultures grown for 3 days in marine broth 2216 (MB; Becton–Dickinson) at 30 °C. For cellular fatty acids analysis, cell mass of strain G-M8T was harvested from MA plates after incubation for 3 days at 30 °C.

Morphological, physiological and biochemical characterization

The cell morphology and flagellation were examined by using light microscopy (BX51; Olympus) and transmission electron microscopy (CM-20; Philips), respectively. To assess the flagellation on cells from an exponentially growing MA culture, 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. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber (1029; Forma, N2:CO2:H2, 86:7:7 %) on MA which had been prepared anaerobically under nitrogen atmosphere. Strain G-M8T was cultured at various temperatures (4, 10, 20, 25, 28, 30, 35, 37, 40, 45 and 50 °C) to measure optimal temperature and temperature range for growth. Growth in the absence of NaCl and in the presence of 0.5, 1.0, 2.0 and 3.0 % (w/v) NaCl was investigated by using trypticase soy broth prepared according to the formula of the Becton–Dickinson medium except that NaCl was excluded and that 0.45 % (w/v) MgCl2·6H2O or 0.06 % (w/v) KCl was added. Growth at various NaCl concentrations (2.0–10.0 %, w/v, at increments of 1.0 %) was investigated in MB. The pH range for growth was determined in MB adjusted to pH 4.5–9.5 (using increments of 0.5 pH units) by using sodium acetate/acetic acid and Na2CO3 buffers. The pH values were verified after autoclaving. Catalase and oxidase activities were determined as described by Cowan and Steel (1965). Hydrolysis of casein, hypoxanthine, l-tyrosine and xanthine was tested on MA, using the substrate concentrations described by Cowan and Steel (1965). Nitrate reduction and hydrolysis of aesculin and Tweens 20, 40, 60 and 80 were investigated as described previously (Lányí 1987) with the modification that artificial seawater was used for the preparation of media. Hydrolysis of gelatin and urea were investigated by using Nutrient gelatin and Urea agar base media (Becton–Dickinson), 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). Acid production from carbohydrates was tested as described by Leifson (1963). Utilization of various substrates for growth was tested according to Baumann and Baumann (1981), supplemented with 1 % (v/v) vitamin solution (Staley 1968) and 2 % (v/v) Hutner’s mineral salts (Cohen-Bazire et al. 1957). Susceptibility to antibiotics was investigated 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).

Molecular studies

Chromosomal DNA was extracted and purified according to the method described by Yoon et al. (1996), with the modification that RNase T1 was used in combination with RNase A to minimize contamination of RNA. The 16S rRNA gene was amplified by PCR as described previously (Yoon et al. 1998) using two universal primers, 9F (5′-GAGTTTGATCCTGGCTCAG-3′) and 1512R (5′-ACGGTTACCTTGTTACGACTT-3′), 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 analysis was performed as described by Yoon et al. (2012). The amplification of the DNA gyrase B subunit gene (gyrB) was performed by using two primers, UP-1 and UP-2r, according to the method described previously (Yamamoto and Harayama 1995) and the PCR products were purified with the QIAquick PCR purification kit (Qiagen). The amplified gyrB was cloned into pGEM T-easy vector (Promega) according to the manufacturer’s instructions. Sequence of the gyrB was determined for both strands by extension from vector-specific priming sites (T7 and SP-6 primers from pGEM T-easy vector). Phylogenetic analysis of the gyrB sequence was performed as described by Yoon et al. (2007). 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 G-M8T, R. atlantica KCTC 12424T and R. lacuscaerulensis KCTC 2953T 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/v) for the first dimension and chloroform/methanol/acetic acid/water (40:7.5:6:1.8, v/v/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 with 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. Escherichia coli DNA was used a standard.

Results and discussion

Morphological, cultural, physiological, and biochemical characteristics

Strain G-M8T was found to be a facultatively aerobic, Gram-negative, non-spore-forming rod shaped bacterium. Strain G-M8T was indicated to be motile by means of peritrichous flagella (Supplementary Fig. 1), whereas the type strains of R. atlantica and R. lacuscaerulensis are non-motile as determined by the absence of flagella (Uchino et al. 1998; Petursdottir and Kristjansson 1997). Strain G-M8T grew optimally at 30–37 °C, pH 7.0–7.5 and was found to be a moderate halophile as it grew optimally in the presence of 2 % (w/v) NaCl; the strain grew in the presence of 0.5–6.0 % NaCl. Mg2+ ions were required for growth, but K+ ions were not required for growth. Strain G-M8T showed catalase and oxidase activities but no urease activity. Strain G-M8T reduced nitrate to nitrite. Strain G-M8T was susceptible to ampicillin, carbenicillin, cephalothin, chloramphenicol, neomycin (weak), oleandomycin, penicillin G, polymyxin B and streptomycin but not to gentamicin, kanamycin, lincomycin, novobiocin and tetracycline. Morphological, cultural, physiological and biochemical characteristics of strain G-M8T are given in the species description (see below) and in Table 1.

Table 1 Differential phenotypic characteristics of Ruegeria arenilitoris G-M8T and the type strains of Ruegeria atlantica and Ruegeria lacuscaerulensis
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Phylogenetic analysis

The almost-complete 16S rRNA gene sequence of strain G-M8T comprised 1384 nucleotides. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain G-M8T fell within the clade comprising Ruegeria species, joining the cluster comprising the type strains of R. atlantica and R. lacuscaerulensis by a bootstrap resampling value of 85.8 % (Fig. 1). Strain G-M8T exhibited 16S rRNA gene sequence similarity values of 97.5 and 97.5 % to R. atlantica IAM 14463T and R. lacuscaerulensis ITI-1157T, respectively and 95.4–96.7 % to the type strains of the other validly named Ruegeria species. The gyrB sequence of strain G-M8T determined in this study comprised 1,180 nucleotides. In the neighbour-joining phylogenetic tree based on gyrB sequences, strain G-M8T clustered with the type strain of R. lacuscaerulensis, supported by a bootstrap resampling value of 99.7 %, with which it exhibited the highest sequence similarity (88.5 %) (Fig. 2). Strain G-M8T exhibited 78.8–83.8 % gyrB sequence similarity values to the type strains of the other validly named Ruegeria species.

Fig. 1
figure 1

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Ruegeria arenilitoris G-M8T, Ruegeria species and representative of some other related taxa. Only bootstrap values (expressed as percentages of 1,000 replications) of >50 % 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. Stappia stellulata IAM 12621T (GenBank accession no. D88525) was used as an outgroup. Bar 0.01 substitutions per nucleotide position

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Fig. 2
figure 2

Neighbour-joining phylogenetic tree based on gyrB sequences showing the positions of Ruegeria arenilitoris G-M8T, some Ruegeria species and representative of some other related taxa. Only bootstrap values (expressed as percentages of 1,000 replications) of >50 % 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. Oceanospirillum linum ATCC 11336T (GenBank accession number, AB014935) was used as an outgroup. Scale bar 0.02 substitutions per nucleotide position

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DNA–DNA relatedness

Strain G-M8T exhibited mean DNA–DNA relatedness values of 18 ± 5.3 and 10 ± 3.6 % to R. atlantica KCTC 12424T and R. lacuscaerulensis KCTC 2953T, respectively.

Chemotaxonomic characteristics

The predominant isoprenoid quinone detected in strain G-M8T was ubiquinone-10 (Q-10), which is compatible with those of members of the genus Ruegeria (Yi et al. 2007; Muramatsu et al. 2007; Oh et al. 2011). In Table 2, the fatty acid profile of strain G-M8T is compared with those of the type strains of two phylogenetically related Ruegeria species, R. atlantica and R. lacuscaerulensis, which were analysed under identical conditions and methods. The predominant fatty acid found in strain G-M8T was C18:1 ω7c in line with Ruegeria species and the vast majority members of the Alphaproteobacteria (Yi et al. 2007; Muramatsu et al. 2007; Oh et al. 2011). The fatty acid profile of strain G-M8T was similar to those of R. atlantica KCTC 12424T and R. lacuscaerulensis KCTC 2953T, although there were differences in the proportions of some fatty acids (Table 2). In particular, strain G-M8T was distinguished from R. lacuscaerulensis KCTC 2953T in that some fatty acids which are present as significant amounts in strain G-M8T are minor components or not detected in R. lacuscaerulensis KCTC 2953T. The major polar lipids detected in strain G-M8T were phosphatidylcholine, phosphatidylglycerol, one unidentified aminolipid and two unidentified lipids (Supplementary Fig. 2). The polar lipid profile of strain G-M8T was compared with that of R. atlantica KCTC 12424T grown and analysed under identical conditions by Kim et al. (2012). The profile of strain G-M8T was similar with that of R. atlantica KCTC 12424T in that phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and an unidentified lipid are major polar lipids, but was distinguishable from that of R. atlantica KCTC 12424T in that a minor amount of diphosphatidylglycerol and one additional unidentified lipid are absent. The G+C content of strain G-M8T was 64.6 mol%, a value in the range reported for Ruegeria species (Huo et al. 2011; Oh et al. 2011).

Table 2 Cellular fatty acid compositions (%) of Ruegeria arenilitoris G-M8T and the type strains of Ruegeria atlantica and Ruegeria lacuscaerulensis
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Conclusion

The results obtained from the chemotaxonomic analyses are sufficient to support the result of the phylogenetic analysis based on 16S rRNA gene sequences, suggesting that strain G-M8T is a member of the genus Ruegeria. Phenotypic characteristics of strain G-M8T were compared with those of R. atlantica KCTC 12424T and R. lacuscaerulensis KCTC 2953T, i.e. related species that showed 16S rRNA gene sequence similarity of >97 % (Table 1). Strain G-M8T was clearly distinguishable from R. atlantica KCTC 12424T and R. lacuscaerulensis KCTC 2953T by differences in several phenotypic characteristics, including motility, anaerobic growth, growth at 4 °C, xanthine hydrolysis and acid production from some substrates, most of which were determined under the same conditions and methods (Table 1). These differences, together with the phylogenetic distinctiveness and genetic distinctiveness of strain G-M8T, are sufficient to show that this strain is separate from recognized species of the genus Ruegeria (Wayne et al. 1987; Stackebrandt and Goebel 1994). Therefore, on the basis of the phenotypic, chemotaxonomic, phylogenetic and genetic data, strain G-M8T is considered to represent a novel species of the genus Ruegeria, for which the name Ruegeria arenilitoris sp. nov. is proposed.

Description of Ruegeria arenilitoris sp. nov

Ruegeria arenilitoris (a.re.ni.li.to’ris. L. n. arena sand; L. n. litus -oris the seashore, coast; N.L. gen. n. arenilitoris of sand of seashore, from which the type strain was isolated).

Cells are Gram-negative, non-spore-forming and rods-shaped, approximately 0.2–0.6 μm in diameter 0.8–4.0 μm in length. Motile by means of peritrichous flagella. Colonies on marine agar are circular to slightly irregular, flat to raised, glistening, smooth, grayish yellow in colour and 2.0–3.0 mm after incubation for 3 days at 30 °C. Anaerobic growth occurs on marine agar. Optimal growth occurs at 30–37 °C; growth occurs at 4 and 45 °C, but not at 50 °C. Optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 5.5, but not at pH 5.0. Optimal growth occurs in the presence of 2.0 % (w/v) NaCl; growth occurs in the presence of 0.5–6.0 % (w/v) NaCl. Mg2+ ions are required for growth but K+ ions are not. Catalase- and oxidase-positive. Nitrate reduction is positive. Hypoxanthine and l-tyrosine are hydrolysed but aesculin, casein, gelatin, starch, Tweens 20, 40, 60 and 80, urea and xanthine are not. d-Cellobiose, d-fructose, d-galactose, d-glucose, d-mannose, d-xylose, acetate, citrate, l-malate, pyruvate and succinate are utilized as carbon and energy sources but l-arabinose, maltose, sucrose, d-trehalose, benzoate, formate, salicin and l-glutamate are not. Acid is produced from l-arabinose, d-cellobiose (weak) and d-galactose but not from d-fructose, d-glucose, myo-inositol, lactose, maltose, d-mannitol, d-mannose, d-melezitose, melibiose, d-raffinose, l-rhamnose, d-ribose, d-sorbitol, sucrose, d-trehalose and d-xylose. In assays with the API ZYM system, alkaline phosphatase activity is present and esterase (C 4) and leucine arylamidase activities are weakly present, but esterase lipase (C 8), lipase (C 14), 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 activities 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, one unidentified aminolipid and two unidentified lipids. The DNA G+C content of the type strain is 64.6 mol% (determined by HPLC).

The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequence and gyrB sequence of strain G-M8T are JQ807219 and JQ807221, respectively. The type strain, G-M8T (=KCTC 23960T = CCUG 62412T), was isolated from seashore sand around a seaweed farm at Geoje island, South Korea.