Hwanghaeicola aestuarii gen. nov., sp. nov., a moderately halophilic bacterium isolated from a tidal flat of the Yellow Sea

Since a Gram-negative bacterium belonging to the Roseobacter clade within the class Alphaproteobacteria was first reported by Shiba (1991), many new genera of the Roseobacter clade have been isolated from various marine environments such as seawater, tidal flats, marine algae, and hypersaline microbial mats (Choi et al., 2007; Lee et al., 2007; Y.-G. Kim et al., 2008; Vandecandelaere et al., 2009; Wang et al., 2009; Yoon et al., 2009; Zheng et al., 2010). The Roseobacter clade is known as one of the most abundant groups in marine environments and its members show diverse physiological and morphological features. The Yellow Sea coasts of the Korean peninsula consist of vast tidal flats known as getbol, which are marine habitats having valuable biological resources such as micro-organisms and marine animals. Therefore, bacteria were isolated from these tidal flats and characterized in our laboratory (Kim et al., 2010). Here, we describe the taxonomic characterization of a novel genus isolated from a flat of the Yellow Sea belonging to the Roseobacter clade, for which the name Hwanghaeicola aestuarii gen. nov., sp. nov. is proposed.

Strain Y26^T was isolated from a tidal flat of the Taean coast of the Yellow Sea, South Korea, using a previously described procedure with some modifications (J. M. Kim et al., 2008). Briefly, a sediment sample was serially diluted with marine broth (MB; Difco), spread on marine agar 2216 (MA; Difco) plates and incubated at 25 °C for 5 days. Colonies were randomly selected and crude lysates containing genomic DNA from respective colonies were prepared by boiling a small amount of cell material in 100 μl 5 % Chelex 100 solution (Bio-Rad) for 10 min followed by centrifugation at 15 000 g for 10 min. PCR amplification of 16S rRNA genes from the crude lysates was performed using the universal primers F1 (5′-AGAGTTTGATCMTGGCTCAG-3′) and R13 (5′-TACGGYTACCTTGTTACGACTT-3′), as described previously (Lu et al., 2006). The amplicons were double-digested with HaeIII and HhaI. Restriction fragment length polymorphism (RFLP) patterns were analysed on 2.5 % MetaPhore agarose (BioWhittaker) gels and representative PCR products containing distinct RFLP patterns were sequenced. The resulting 16S rRNA gene sequences were analysed using the blast program () in GenBank. From the analysis, a novel strain belonging to the Roseobacter clade, designated strain Y26^T, was selected for further phenotypic and phylogenetic analysis. The strain was routinely grown aerobically on MA at 30 °C for 3 days, except where indicated otherwise. The strain was stored at −80 °C in MB supplemented with 10 % (v/v) glycerol for preservation. The type strains of related taxa, including Maribius pelagius B5-6^T, Maribius salinus CL-SP27^T, Palleronia marisminoris LMG 22959^T and Maritimibacter alkaliphilus HTCC2654^T, were used as reference strains for the biochemical tests or fatty acid analysis.

The 16S rRNA gene sequence (1350 nt) of strain Y26^T was compared with available 16S rRNA gene sequences from GenBank using the blast program to determine an approximate phylogenetic affiliation. Sequence similarity values between the novel isolate and related taxa were evaluated using the Nucleotide Similarity Search program (:8080/; Chun et al., 2007) and aligned by using the clustal w software program (Thompson et al., 1994). Phylogenetic trees were reconstructed by using the neighbour-joining, maximum-likelihood and maximum-parsimony algorithms available in the phylip software package, version 3.6 (Felsenstein, 2002). The resulting tree topology reconstructed by using the neighbour-joining method was evaluated using a bootstrap analysis based on 1000 resampled datasets with the phylip package. Comparative analysis of the 16S rRNA gene sequences showed that strain Y26^T was most closely related to Maribius pelagius B5-6^T, Maritimibacter alkaliphilus HTCC2654^T and Maribius salinus CL-SP27^T with similarities of 94.6, 94.4 and 94.3 %, respectively. These levels of 16S rRNA gene sequence similarity are sufficient to potentially define a new genus (Rosselló-Mora & Amann, 2001). Phylogenetic analysis based on 16S rRNA gene sequences also indicated that strain Y26^T formed a distinct phyletic lineage from other related taxa (Fig. 1⇓). The topologies of phylogenetic trees built using the maximum-likelihood and maximum-parsimony algorithms supported the notion that there was no genus group showing a clear phylogenetic relationship with strain Y26^T within the Roseobacter clade (Supplementary Fig. S1, available in IJSEM Online).

Temperature and pH for growth of strain Y26^T were examined by growing the isolate at different temperatures (5–45 °C at 5 °C intervals) on MA and at different pH (5.0–10.5 at 0.5 pH unit intervals) in MB. The pH was adjusted prior to sterilization by the addition of HCl or NaOH and measured again after sterilization. Gram staining was performed using the bioMérieux Gram Stain kit according to the instructions of the manufacturer. Oxidase activity was tested by oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck), and catalase activity was evaluated by the production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution (Smibert & Krieg, 1994). Cell morphology, motility and the presence of flagella were studied using phase-contrast microscopy and transmission electron microscopy (JEM-1010, JEOL) with 2-day-old cells grown on MA as described previously (Jeon et al., 2004). Requirement and tolerance of NaCl were determined using synthetic ZoBell broth (0.5 % Bacto peptone, 0.1 % yeast extract and 0.01 % ferric citrate) with modified artificial seawater [ASW; 0–15 % (w/v) NaCl at 0.5 % intervals, 5.94 g MgSO₄ . 7H₂O, 4.53 g MgCl₂ . 6H₂O, 0.64 g KCl and 1.3 g CaCl₂ per litre] (Kahng et al., 2009). Ionic requirements were determined using synthetic ZoBell broth with the following combinations of salts (all w/v): (i) 3.0 % NaCl; (ii) 3.0 % NaCl, 0.6 % MgCl₂ . 6H₂O and 0.3 % MgSO₄ . 7H₂O; (iii) 3.0 % NaCl, 0.6 % MgCl₂ . 6H₂O, 0.3 % MgSO₄ . 7H₂O and 0.06 % KCl; and (iv) 3.0 % NaCl, 0.6 % MgCl₂ . 6H₂O, 0.3 % MgSO₄ . 7H₂O, 0.06 % KCl and 0.2 % CaCl₂ . 2H₂O (Choi et al., 2007). Nitrate reduction was assessed according to the method of Lányí (1987). Biochemical features were characterized by using the API 20E and API 20NE kits as recommended by the manufacturer (bioMérieux) except that the kits were incubated for 3 days at 30 °C; all suspension media were supplemented with ASW containing 3.0 % (w/v) NaCl. Additional enzyme activities were determined by the API ZYM system (bioMérieux) at 30 °C based on the manufacturer's instructions. Antibiotic susceptibility tests were performed on MA in duplicate using filter-paper discs (8 mm diameter) containing the following antibiotics: ampicillin (10 μg), polymyxin B (100 U), streptomycin (50 μg), penicillin G (10 IU), gentamicin (30 μg), chloramphenicol (100 μg), tetracycline (30 μg), kanamycin (30 μg), lincomycin (15 μg), oleandomycin (15 μg), carbenicillin (100 μg) and novobiocin (50 μg). Anaerobic growth was assessed on MA under anaerobic (with 4–10 % CO₂) conditions using the GasPak Plus system (BBL) at 30 °C for 15 days.

Strain Y26^T grew at temperatures between 15 and 35 °C (optimum 25–30 °C), at pH 6.0–8.0 (optimum 6.5–7.5) and in the presence of 1.5–6.0 % (w/v) NaCl (optimum 2.0–3.0 %). Bacterial cells were Gram-stain negative, strictly aerobic, motile cocci (0.8–1.2 μm in diameter) with a single flagellum at 30 °C on MA (Fig. 2⇓). The strain was not able to grow in media with NaCl as a sole salt, but was able to grow in media containing Na⁺ and Mg⁺ ions. Detailed phenotypic features of strain Y26^T are presented in the description and compared with those of closely related taxa in Table 1⇓.

Isoprenoid quinones were analysed using an HPLC (model LC-20A, Shimadzu) equipped with a diode array detector (SPD-M20A, Shimadzu) and a reversed-phase column (250×4.6 mm, Kromasil, Akzo Nobel) as described by Komagata & Suzuki (1987). For analysis of fatty acid methyl esters, cells of strain Y26^T were harvested after incubation at 30 °C for 3 days on MA. Analysis of fatty acid methyl esters was performed according to the instructions of the Microbial Identification System (MIDI; Microbial ID). The DNA G+C content of strain Y26^T was determined by using an HPLC fitted with a reversed-phase column (250×4.6 mm, GROM-SIL 100 ODS-2FE, GROM) according to the method of Tamaoka & Komagata (1984). The major respiratory lipoquinone of strain Y26^T was ubiquinone Q-10 (Q-10). The major cellular fatty acids (>2 % of the total fatty acids) were C_18 : 1ω7c (66.2 %), C_16 : 0 (12.4 %), C_10 : 0 3-OH (5.0 %), C_19 : 0 cyclo ω8c (3.7 %), unknown 11.799 (3.4 %), 11-methyl C_18 : 1ω7c (2.6 %) and C_18 : 0 (2.7 %). Although the overall fatty acid profile of strain Y26^T was similar to those of phylogenetically related taxa (Table 2⇓), significant differences in the respective proportions of several components clearly distinguished the novel strain from related taxa (Table 2⇓). The DNA G+C content of strain Y26^T was 61.0 mol%. Therefore, the physiological, biochemical and phylogenetic properties of strain Y26^T support its description as a novel genus within the family Rhodobacteraceae of the class Alphaproteobacteria, for which the name Hwanghaeicola aestuarii gen. nov., sp. nov. is proposed.