Introduction

During a screening of novel bacteria from marine environments around Geoje island, the second largest island in the South Korea, many novel bacterial taxa have been isolated and characterized taxonomically (Park et al. 2013b, c; Yoon et al. 2013a, b). One of these bacterial strains, designated GJSW-22T, which was isolated from seawater around Geoje island, is described in this study. Comparative 16S rRNA gene sequence analysis indicated that the novel strain is phylogenetically most closely associated with the members of the genera Thalassobius, Shimia and Epibacterium of the class Alphaproteobacteria. The genera Thalassobius, Shimia and Epibacterium were proposed by Arahal et al. (2005), Choi and Cho (2006) and Penesyan et al. (2013), respectively. Currently, the genera Thalassobius, Shimia and Epibacterium comprise four, four, and one species with validly published names, respectively (Euzéby 1997). Members of the three genera have been isolated from a variety of marine environments (Rüger and Höfle 1992; Choi and Cho 2006; Yi and Chun 2006; Chen et al. 2011; Hyun et al. 2013; Penesyan et al. 2013). The aim of the present work was to determine the exact taxonomic position of strain GJSW-22T by using a polyphasic characterization that included chemotaxonomic and other phenotypic properties, detailed phylogenetic investigations based on 16S rRNA gene sequences and DNA–DNA hybridization.

Materials and methods

Bacterial strains and culture conditions

Seawater was collected from coast at Geoje island on the South Sea, South Korea, and used as a source for the isolation of bacterial strains. Strain GJSW-22T was isolated by the standard dilution plating technique on marine agar 2,216 (MA; Becton, Dickinson and Company) at 25 °C and cultivated routinely on MA at 30 °C. Strain GJSW-22T 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 GJSW-22T has been deposited in the Korean Collection for Type Cultures (KCTC; South Korea) and the NITE Biological Resource Centre (NBRC; Japan) under the accession numbers KCTC 42115T and NBRC 110378T, respectively.

The type strains of four Thalassobius species were used for reference strains for fatty acid analysis and DNA–DNA hybridization. Thalassobius aestuarii KCTC 12049T and Thalassobius gelatinovorus KCTC 22092T were obtained from the Korean Collection for Type Cultures (KCTC), Daejeon, South Korea. Thalassobius mediterraneus CCUG 49438T was obtained from the Culture Collection, University of Göteborg (CCUG), Göteborg, Sweden. Thalassobius maritimus GSW-M6T was obtained from our previous study (Park et al. 2012).

Cell biomass of strain GJSW-22T for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultures grown for 3 days in marine broth 2,216 (MB; Becton, Dickinson and Company) at 30 °C. For cellular fatty acid analysis, cell mass of strain GJSW-22T, T. aestuarii KCTC 12049T, T. gelatinovorus KCTC 22092T, T. mediterraneus CCUG 49438T and T. maritimus GSW-M6T was harvested from MA plates after cultivation for 5 days at 30 °C. The physiological age of the cell masses was standardized by observing the growth development of colonies on the agar plates followed by harvesting them from the same quadrant on the agar plates according to the standard MIDI protocol (Sherlock Microbial Identification System, version 6.1).

Morphological, physiological and biochemical characterization

The cell morphology, Gram reaction, pH range for growth, anaerobic growth, activity of catalase and oxidase, nitrate reduction, H2S production, hydrolysis of several substrates and utilization of various substrates were determined as described by Park et al. (2013a). Growth at 4, 10, 15, 20, 25, 30 and 37 °C was measured on MA to measure the optimal temperature and temperature range for growth. Growth at various concentrations of NaCl (0, 0.5 and 1.0–9.0 %, at increments of 1.0 %) was investigated by supplementing with appropriate concentrations of NaCl in MB prepared according to the formula of the Becton, Dickinson and Company medium except that NaCl was excluded. Requirement of Mg2+ ions was investigated by using MB, prepared according to the formula of the Becton, Dickinson and Company medium, that comprised all of the constituents except MgCl2 and MgSO4. 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); the strip was inoculated with cells suspended in artificial seawater (Bruns et al. 2001) from which CaCl2 was excluded.

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 (5′-GAGTTTGATCCTGGCTCAG-3′ and 5′-ACGGTTACCTTGTTACGACTT-3′). 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 and the remaining part was verified and corrected manually by using BioEdit software (Hall 1999). Phylogenetic analysis was performed as described by Yoon et al. (2012).

DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes in microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded and the means of the remaining three values are quoted as DNA–DNA relatedness values.

Chemotaxonomic characterization

Isoprenoid quinones were extracted and analysed as described by Komagata and Suzuki (1987), using reversed-phase HPLC equipped with 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 270 nm. Fatty acids were saponified, methylated and extracted using the standard protocol of the MIDI (Sherlock Microbial Identification System, version 6.1). The fatty acids were analysed by GC (Hewlett Packard 6890) and identified by using the TSBA6 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, by vol.) for the first dimension and chloroform/methanol/acetic acid/water (40:7.5:6:1.8, by vol.) for the second dimension as described by Minnikin et al. (1977). Individual polar lipids were identified by spraying with 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 as described elsewhere (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 as a standard.

Results and discussion

Morphological, cultural, physiological and biochemical characteristics

Strain GJSW-22T was found to be aerobic, Gram-negative, non-flagellated, non-spore-forming and rod-shaped or ovoid. Strain GJSW-22T was observed to grow optimally at 30 °C and at pH 7.0–8.0. It was found to grow in the presence of 0.5–6.0 % (w/v) NaCl with an optimum of approximately 2.0 % (w/v). While strain GJSW-22T was found to be able to hydrolyse aesculin, Tween 60 and xanthine, the type strains of Thalassobius species were unable to hydrolyse these three substrates (Table 1). Strain GJSW-22T was found to be able to utilize D-galactose and unable to utilize succinate, whereas the type strains of Thalassobius species showed inverse results (Table 1). Strain GJSW-22T was found to be resistant to gentamicin and kanamycin, whereas the type strains of Thalassobius species were susceptible to gentamicin and kanamycin (Table 1). Strain GJSW-22T was also found to be susceptible to ampicillin, carbenicillin, cephalotin, chloramphenicol, neomycin, novobiocin, oleandomycin, penicillin G, streptomycin and tetracycline, but resistant to lincomycin and polymyxin B.

Table 1 Differential characteristics of strain GJSW-22T and the type strains of Thalassobius species
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Morphological, physiological and biochemical characteristics of strain GJSW-22T are given in the species description (see below) and in Table 1 or Supplementary Fig. 1.

Phylogenetic analysis and DNA–DNA relatedness

The almost-complete 16S rRNA gene sequence (1,386 nucleotides; GenBank/EMBL/DDBJ accession number KJ729030) of strain GJSW-22T, approximately 95 % of the Escherichia coli 16S rRNA sequence, was determined in this study. Strain GJSW-22T exhibited the highest 16S rRNA gene sequence similarity values to the type strains of Thalassobius aestuarii (98.0 %) and Shimia isoporae (97.2 %). In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain GJSW-22T clustered with the type strain of T. aestuarii by a bootstrap resampling value of 83.2 %, and this cluster joined the cluster comprising the type strains of three other Thalassobius species (Fig. 1). The relationship between strain GJSW-22T and the type strain of T. aestuarii was found in the tree constructed using the maximum-parsimony algorithm (Fig. 1). Strain GJSW-22T exhibited 16S rRNA gene sequence similarity values of 95.6–96.1 % to the type strains of the other Thalassobius species, of 95.9–96.3 % to the type strains of the other Shimia species, of 96.7 % to the type strain of Epibacterium ulvae, and of <96.2 % to the type strains of the other recognized species.

Fig. 1
figure 1

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Thalassobius aquaeponti GJSW-22T, the type strains of Thalossobius 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, while open circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm. Stappia stellulata IAM 12621T (GenBank accession number, D88525) was used as an outgroup. Scale bar, 0.01 substitutions per nucleotide position

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Mean DNA–DNA relatedness value between strain GJSW-22T and T. aestuarii KCTC 12049T was 23 ± 5.7 %.

Chemotaxonomic characteristics

The predominant isoprenoid quinone detected in strain GJSW-22T was ubiquinone-10 (Q-10), which is typical of the vast majority of the class Alphaproteobacteria as well as the genus Thalassobius (Uchino et al. 1998; Yi and Chun 2006; Park et al. 2012). In Table 2, the fatty acid profile of strain GJSW-22T is compared with those of the type strains of T. aestuarii, T. gelatinovorus, T. mediterraneus and T. maritimus, grown and analysed under identical conditions in this study. The major fatty acids (>10 % of the total fatty acids) found in strain GJSW-22T were C18:1 ω7c (61.4 %) and 11-methyl C18:1 ω7c (16.3 %). The fatty acid profile of strain GJSW-22T was similar with those of the four Thalassobius species, although their profiles were distinguishable by the differences in the proportions of some fatty acids (Table 2). The major polar lipids detected in strain GJSW-22T were found to consist of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid; minor amounts of diphosphatidylglycerol, two additional unidentified lipids, one unidentified phospholipid and one unidentified aminophospholipid were also present (Supplementary Fig. 2). The polar lipid profile of strain GJSW-22T is similar to those of the type strains of T. aestuarii, T. gelatinovorus, T. mediterraneus and T. maritimus in that phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and one unidentified lipid are major polar lipids (Supplementary Fig. 2; Park et al. 2012). The G + C content of strain GJSW-22T is 60.3 mol %, a value in the range reported for members of the genus Thalassobius (Table 1).

Table 2 Cellular fatty acid compositions (%) of strain GJSW-22T and the type strains of Thalassobius species
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Conclusion

In the phylogenetic analyses based on 16S rRNA gene sequences, strain GJSW-22T is phylogenetically closely related to the genera Thalassobius and Shimia. The type strains of Shimia species were found to form a coherent cluster in the neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees (Fig. 1). Moreover, the type strains of Shimia species were described as not having phosphatidylethanolamine (Hameed et al. 2013; Hyun et al. 2013), which is found as a major component in the type strains of Thalassobius species as well as strain GJSW-22T.

The cluster comprising strain GJSW-22T and the type strain of T. aestuarii may not join the cluster comprising the type strains of the other Thalassobius species or forms an independent phylogenetic lineage, depending on which reference strains are included or excluded and depending on treeing methods used. The phylogenetic position of the type strain of T. maritimus has been also found to be unstable or undefined (Penesyan et al. 2013; Park and Yoon 2014). The taxonomic statuses of strain GJSW-22T and Thalassobius species may have to be reevaluated after including additional species belonging to the genus Thalassobius. However, it is reasonable to classify strain GJSW-22T as a member of the genus Thalassobius at this time, since there are no distinct phenotypic, particularly chemotaxonomic, properties between strain GJSW-22T and T. aestuarii and the other Thalassobius species. Strain GJSW-22T was distinguished from the four Thalassobius species by differences in phenotypic characteristics, including motility, nitrate reduction, hydrolysis of and utilization of some substrates and susceptibility to antibiotics (Table 1). These differences, in combination with the phylogenetic and genetic distinctiveness between strain GJSW-22T and other Thalassobius species, are sufficient to show that the novel strain is separated from all other recognized Thalassobius species (Wayne et al. 1987; Stackebrandt and Goebel 1994). Therefore, on the basis of the data presented, strain GJSW-22T is considered to represent a novel species of the genus Thalassobius, for which the name Thalassobius aquaeponti sp. nov. is proposed.

Description of Thalassobius aquaeponti sp. nov

Thalassobius aquaeponti (a.quae.pon’ti. L. n. aqua water; L. gen. n. ponti of the sea; N.L. gen. n. aquaeponti of the water of the sea, from where the type strain was isolated).

Cells are Gram-stain-negative, non-spore-forming, non-flagellated and rod-shaped or ovoid, approximately 0.2–0.5 μm in diameter and 0.7– >10.0 μm in length; a few cells greater than 10 μm in length are also observed. Colonies on MA agar are circular, slightly convex, smooth, glistening, greyish-yellow in colour and 0.7–1.0 mm after incubation for 5 days at 30 °C. Optimal growth temperature is 30 °C; growth occurs at 10 and 35 °C, but not at 4 and 37 °C. Optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 6.0, but not at pH 5.5. 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. Anaerobic growth does not occur on MA and on MA supplemented with nitrate. Catalase- and oxidase-positive. Nitrate is reduced to nitrite. H2S is not produced. Aesculin, hypoxanthine, xanthine, l-tyrosine and Tweens 20, 40 and 60 are hydrolysed, but casein, gelatin, starch, Tween 80 and urea are not. D-Cellobiose, D-galactose, d-glucose, D-xylose, acetate, citrate, L-malate and pyruvate are utilized as carbon and energy sources, but L-arabinose, d-fructose, maltose, d-mannose, sucrose, D-trehalose, benzoate, formate, succinate, L-glutamate and salicin are not. In the API ZYM system, alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and acid phosphatase activities are present, but lipase (C 14), valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, 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, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The DNA G + C content of the type strain is 60.3 mol %.

The type strain, GJSW-22T (=KCTC 42115T = NBRC 110378T), was isolated from a seawater collected at Geoje island in the South Sea, South Korea. The GenBank/EMBL/DDBJ accession number of the 16S rRNA gene sequence of strain GJSW-22T is KJ729030.