Introduction

The number of genera within the family Rhodobacteraceae [12] has increased considerably in recent years. At the time of writing, more than 80 genera have been identified. Over three quarters of the species in the family originate from marine environments, such as seawater, sediment, marine algae, invertebrates, vertebrates, hypersaline microbial mats, and coastal biofilm [3]. Many novel genera of this clade have been described recently, for example Agaricicola [4], Celeribacter [17], Gaetbulicola [35], Hwanghaeicola [19], and Pontibaca [20].

Materials and Methods

Bacterial Strains Isolation and Cultivation

During a survey of the biodiversity of bacteria off the coast of Guishan island, with a hydrothermal vent nearby, Taiwan, a novel strain, designated JLT2003T, was isolated from a surface seawater sample collected during the cruise in September 2009 after using a standard dilution-to-extinction culturing method. After incubation on RO plates [36] at 30°C for 2 weeks, strain JLT2003T was purified as single colonies. The culture was maintained routinely on RO plates and was preserved as glycerol suspensions (15%, v/v) at −80°C.

Phenotypic and Chemical Characterization

Morphological, physiological, and biochemical characteristics of strain JLT2003T were investigated using routine cultivation aerobically on RO medium or MA at 30°C for 3 days. Cell morphology and motility were examined by high-resolution confocal microscopy (BX61; OLYMPUS) with cells grown for 3 days. Cell motility was determined by transmission electron microscopy (JEM-1230; JEOL USA) after negatively stained with 1% (w/v) phosphotungstic acid. The Gram staining was determined as described by Gerhardt et al. [13].

Oxidase activity was determined using Bactident Oxidase strips (MERCK) according to Smibert and Krieg [31] and catalase activity was tested using 3% H2O2 [8]. Nitrite and nitrate reduction were tested in RO medium by growing the strain in the presence of NO2 − and NO3 −, respectively [8]. The presence of poly-β-hydroxybutyrate (PHB) granules were determined by Nile blue A staining [27]. Hydrolysis of casein, starch, gelatin, Tween 80 and cellulose, and urease activity were tested as described by Cowan and Steel [7]. H2S production was tested on RO medium supplemented with 0.05% l-cysteine, with a strip of 5–10% lead acetate impregnated paper as indicator placed in the neck of the tube [5, 26]. Methyl red and Voges–Proskauer reaction tests were performed by using methyl red and Barritt’s reagent [2, 26]. The presence of bacteriochlorophyll a was tested according to Pukall [28]. Glucose fermentation was tested using the fermentation medium reported by Leifson [24]. The results are summarized in Table 1. Growth on sole carbon sources and nitrogen sources was examined by using Microlog GN2 plates (Biolog) according to the procedures of Garland [11]. Other physiological and biochemical tests were performed with the API ZYM (bioMérieux) systems according to the manufacturer’s instructions.

Table 1 Differential characteristics of strain JLT2003T and other related taxa within the family Rhodobacteraceae
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Growth at various NaCl concentrations was investigated on MA medium with final NaCl concentrations of 0, 0.5, and 1–12%, at intervals of 1% (at 30°C and pH 7.8). Growth at various temperatures (4, 16, 20, 25, 30, 35, and 40°C, at pH 7.8, 2% NaCl) was measured on MA medium. Growth at different pH values was determined by adjusting the final pH of MA medium to 4, 5, 6, 7, 8, 9, and 10 with HCl or NaOH (2% NaCl, 30°C). Tests of antimicrobial susceptibility were performed by using the disk-diffusion plate (Kirby–Bauer) method according to Fraser and Jorgensen [10] and Andrews [1].

Cellular Fatty Acid and Polar Lipids Analysis

Cellular fatty acid analysis was carried out as described by Komagata and Suzuki [21]. The fatty acid composition on the growth phase of strain JTL2003T was tested (see Table 3) according to Kim [20]. Polar lipids were texted by two-dimensional thin-layer chromatography according to Collins et al. [6], using Merck silica gel 60F254 plates (10 by 20 cm) and chloroform–methanol–water (65:25:4, vol/vol) in the first dimension and chloroform–methanol–acetic acid–water (80:12:15:4, vol/vol) in the second dimension. Lipids were revealed by spraying with 10% molybdophosphoric acid in ethanol, followed by heating at 150°C for 3–5 min.

G+C Content and Isoprenoid Quinones Analysis

The genomic DNA G+C content of strain JLT2003T was estimated from the midpoint value (T m) of the thermal denaturation profile, as described by Mandel et al. [25]. Isoprenoid quinones were analyzed according to Hiraishi et al. [16] by using a UPLC–Q-TOF–MS spectrometer and electrospraying ionization [30].

16S rRNA Gene Analysis and PCR Amplification

Genomic DNA was extracted in accordance with the method of Rainey et al. [29] from cells grown on RO medium for 2 days at 30°C and subsequently washed and resuspended in TE buffer [37]. Purity was assessed by A 280/A 260 and A 230/A 260 ratios [18], and phylogenetic analysis based on 16S rRNA gene sequences was performed as described by Ying et al. [32]. The 16S rRNA gene was amplified with universal bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) [9]. The 16S rRNA gene sequence of strain JLT2003T was compared with those available from the GenBank database by using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi; NCBI) to determine approximate phylogenetic affiliation. Phylogenetic analysis was performed using BioEdit [14] and phylogenetic trees were constructed by using the neighbor-joining and maximum-parsimony methods within the MEGA software [22].

Results and Discussion

Phenotypic Properties

Strain JLT2003T was Gram negative, ovoid or coccoid, non-motile, 0.9- to 1.2-μm long and 0.8- to 1.0-μm wide (Fig. 1). Colonies were pink, 0.5–1.5 mm in diameter, uniformly circular, convex, and opaque on RO or MA medium. Cells grew at 16–40°C, optimally at 20–30°C. Growth occurs at pH 4–10 (optimum 6–9) and at 0.5–12% NaCl (optimum 4–5%).Catalase and oxidase positive. Strain JLT2003T was susceptible to kanamycin, gentamicin, tetracycline, medemycin, polymyxin B, vancomycin, cephalothin, and azteonam. The DNA G+C content of strain JLT2003T was 62.3 mol%.

Fig. 1
figure 1

Micrographs of negatively stained cells of strain JLT2003T observed using transmission electron microscopy

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The dominant fatty acids are C18:1ω7c, cyclo C19:0, and C16:0. During the cultivation the percentage of cyclo19:0ω8c increased until the seventh day but then decreased slightly, while the predominant fatty acid (C18:1ω7c) decreased with length of cultivation. The fatty acid 15:0 3-OH and 11-methyl 18:1ω7c was only detected at early exponential growth (day 2) (Tables 2, 3). The polar lipid profile consisted of diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG) (Fig. 2). The major respiratory ubiquinone was Q-10.

Table 2 Cellular fatty acids of strains JLT2003T and the type strains of related species
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Table 3 Cellular fatty acids of strain JLT2003T grown on tryptic soy agar harvested after different lengths of time
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Fig. 2
figure 2

Two-dimensional TLC of polar lipids of strain O. guishaninsula JLT2003T (a) and P. methylaminivorans GRP21T (b) detection with 10% molybdophosphoric acid in ethanol, followed by heating at 150°C for 3 min. PC Phosphatidylcholine, PE phosphatidylethanolamine, PG phosphatidylglycerol, AL unknown aminolipid, PL unknown phospholipid, L1–3 unknown polar lipids that were not stainable with any of the specific spray reagents applied to detect a phosphate group, an amino group or a sugar moiety

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Additional phenotypic properties are given in Table 1 and in the genus and species descriptions below.

Phylogenetic Analysis

Phylogenetic analysis of the 16S rRNA gene sequences showed that strain JLT2003T was affiliated with the “Rhodobacteraceae” (Fig. 3), most closely related to species of various genera: Pontibaca methylaminivorans GRP21T (94.8% similarity), Maribius salinus CL-SP27T (94.5%), Tranquillimonas alkanivorans A34T (94.4%), Pseudoruegeria aquimaris SW-255T (94.1%), Maritimibacter alkaliphilus HTCC2654T (94.0%), and Donghicola eburneus SW-277T (94.0%).

Fig. 3
figure 3

Neighbor-joining phylogenetic tree, based on 16S rRNA gene sequences showing the relationship between strain JLT2003T and representatives of the order Rhodobacterales. Bootstrap percentages from neighbor-joining (above nodes) and maximum-parsimony (below nodes) analyses based on 1,000 replications are shown at nodes; only values >50% are shown. Rubribacterium polymorphum LMG 25349T was used to root the tree. Filled circles indicate nodes recovered reproducibly by using the two treeing methods. Bar 0.01 substitutions per nucleotide position

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In conclusion, on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic characteristics, strain JLT2003T is considered to represent a novel species of a new genus and species, for which the name Oceaniovalibus guishaninsula gen. nov., sp. nov. is proposed.

Description of Oceaniovalibus gen. nov.

Oceaniovalibus (Oceaniovalibus Arbitrary Name from oceani n. ocean; ovalibus adj. oval)

Cells are Gram negative, non-motile, ovoid or coccoid. PHB granules are not produced, and bacteriochlorophyll a is absent. Colonies are circular, convex, opaque, and pink formed on RO or MA medium. Catalase and oxidase positive. Nitrate is reduced but nitrite is not. The dominant fatty acids are C18:1ω7c, cyclo C19:0, and C16:0. The predominant respiratory ubiquinone is Q-10.

The type species is Oceaniovalibus guishaninsula .

Description of Oceaniovalibus guishaninsula sp. nov.

Oceaniovalibus guishaninsula (n. guishaninsula guishan Island)

The type strain exhibits the following properties in addition to those given in the genus description. Cells are 0.8–1.0 × 0.9–1.2 μm. Colonies (about 0.8–1.0 mm in diameter) are circular, smooth, convex, opaque, and pink. Growth occurs at 16–40°C (optimum 20–30°C), at pH 4–10 (optimum 6–9) and at 0.5–12% NaCl (optimum 4–5%). Positive for hydrolysis of starch and nitrate reduction. Negative for aesculin, casein, and gelatin hydrolysis. The Voges–Proskauer test is negative, the methyl red test is positive. Indole and H2S are not produced. In tests with Biolog GN2 microplates, the following carbon substrates are utilized: l-arabinose, d-cellobiose, d-glucose, d-fructose, sucrose, d-raffinose, d-mannitol, d-gluconic, mono-methyl-succinate, d, l-lactic acid, γ-amino butyric acid, 2-aminoethanol, glycerol, glucose-6-phosphate, m-inositol, starch, l-alanine, l-proline, maltose, l-rhamnose, and d-galactose are weakly utilized. The following carbon substrates are not utilized: α-cyclodextrin, dextrin, Tween 40 and 80, d-arabitol, l-fucose, α-d-lactose, d-mannose, d-sorbitol, d-trehalose, xylitol, acetic acid, cis-aconitic acid, citric acid, formic acid, d-glucosaminic acid, α-keto-glutaric acid, malonic acid, succinic acid, succinamic acid, l-asparagine, l-aspartic acid, l-glutamic acid, l-histidine, l-leucine, l-ornithine, l-serine, l-threonine, inosine, and uridine. Acid is produced from sucrose, d-fructose, d-raffinose, d-cellobiose, l-arabinose, m-inositol, maltose, d-galactose, l-rhamnose, but not from d-glucose, l-threonine, l-alanine, and sodium pyruvate. Sensitive to kanamycin, gentamicin, tetracycline, medemycin, polymyxin B, vancomycin, cephalothin, azteonam, but was resistant to penicillin. According to API ZYM tests, α-glucosidase, β-glucosidase, alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), ONPG, naphthol-AS-BI-phosphohydrolase are positive. The predominant fatty acid is C18:1ω7c, other significant fatty acids are cyclo C19:0, C16:0, C18:0, C12:0 3-OH, C10:0 3-OH, C12:0, 11-methyl C18:1 ω7c, and C17:0. The DNA G+C content of the type strain is 62.3 mol%. The type strain, JLT2003T (=JCM 17765T = CGMCC 1.10827T), was isolated from surface seawater off the coast of Guishan island, Taiwan.