Introduction

Bacteria belonging to the Roseobacter clade (order Rhodobacterales, class Alphaproteobacteria) [8] are likely one of the most abundant groups of microbial communities associated with marine and saline environments, being isolated from seawater, sediments, polar sea ice, microbial mats, seaweeds, and animals [3]. In recent years, the Roseobacter clade has been considerably expanded with an inclusion of the genera Thalassobius [1], Thalassobacter [16, 22], Shimia [4], Phaeobacter and Marinovum [18], Donghicola [33], Pseudoruegeria [12, 34], and Marivita [10].

During the investigation of microorganisms inhabiting the shallow sediments of the Sea of Japan Alphaproteobacteria-like bacteria were found abundantly in many samples. We have recently proposed two novel genera within class Alphaproteobacteria to accommodate some of them [25, 26]. Here we describe the phenotypic and phylogenetic characterization of three strains, designated KMM 9008, KMM 9017, and KMM 9024T, which were isolated from the shallow sediments of the Sea of Japan. On the basis of the phenotypic and molecular data obtained, a novel genus and a novel species, V. arenosi gen. nov., sp. nov., is described.

Materials and Methods

Strains, Isolation, Cultivation, and Physiological Tests

Strains KMM 9008, KMM 9017, and KMM 9024T were isolated from a sandy sediment sample, collected from the Sea of Japan seashore, Russia, as described previously [23, 24]. Bacteria were grown aerobically on marine 2216 agar (MA) or marine broth (MB), and stored at −80°C in the MB supplemented with 30% (v/v) glycerol. Motility was determined by the hanging drop method as described [9]. The Gram staining, oxidase and catalase, and hydrolysis of gelatin, casein, chitin, DNA, Tween 80, 40, 20, and H2S production from thiosulfate were tested according to the standard methods [29]. Acid production from carbohydrates was examined using the oxidation/fermentation medium of Leifson [15]. Requirement for and tolerance of sodium chloride was tested on the artificial sea water (ASW)-based medium using various concentrations of NaCl ranging 0–20%, supplemented with 10.0 g l−1 Bacto peptone, 2.0 g l−1 yeast extract, 0.028 g l−1 FeSO4, and 15.0 g l−1 agar. ASW was prepared according to the composition as described [2]. In addition, bacterial growth was tested on above medium, containing NaCl alone without any of the other sea salts components, MgCl2, Mg2SO4, KCl, or CaCl2. The ability of the strains to grow in the presence of organic substrates as a sole carbon and energy source was tested for 3 weeks on the ASW-based medium supplemented with 1 g NH4Cl l−1, 0.5 g yeast extract l−1, and 0.4% carbon source. Growth was considered as negative if it was equal, or lesser, compared to that of that of the source-free control.

Growth at different temperatures (4–40°C) and pH values (4.0–12.0), and antibiotic resistance were studied as described previously [23, 24]. In addition, biochemical tests were carried out using API ZYM, API 32GN, and API 20NE test kits (bioMérieux) according to the manufacturer’s instructions, except that the cultures were suspended in ASW.

Lipid Analyses

For the analyses of polar lipids, cellular fatty acids and respiratory lipoquinone, the strains were grown on MA and MB at 28°C for 3 days. Lipids were extracted as described [7]. Two-dimensional thin layer chromatography of polar lipids was carried out on Silica gel 60 F254 (10 × 10 cm, Merck, Germany) using chloroform–methanol–water (65:25:4, v/v) for the first direction, and chloroform–methanol–acetic acid–water (80:12:15:4, v/v) for the second one [5].

Fatty acid methyl esters (FAMEs) were prepared according to a procedure of the Microbial Identification System (MIDI) [27]. The analysis of FAMEs was performed using the GC-17A chromatograph (Shimadzu, Kyoto, Japan) equipped with a capillary column (30 m × 0.25 mm I.D.) coated with Supecowax-10 and SPB-5 phases (Supelco, USA). Identification of FAMEs was accomplished by equivalent chain length values and comparing the retention times of the samples to those of standards. In addition, FAMEs were analyzed using a GLC-MS Shimadzu GC-MS model QP5050 (column MDM-5S, the temperature program from 140 to 250°C, at a rate of 2°C/min). Isoprenoid quinones were extracted and analyzed by HPLC as described [5, 19].

Production of bacteriochlorophyll α (Bchl α) was spectrophotometrically tested in methanolic extracts of cells grown on MA and MB in the dark as described [14].

Isolation of DNA and DNA–DNA Hybridization

The DNA base composition was determined as described [17, 20]. DNA–DNA hybridization between strains KMM 9008, KMM 9017, and KMM 9024T was carried out by the photobiotin-labelled DNA probe microplate method [6].

DNA Sequence Analysis

The 16S rRNA gene sequences of 1440, 1445, and 1439 nucleotides were determined for strains KMM 9008, KMM 9017, and KMM 9024T, respectively, as described [28]. The sequences obtained were compared with 16S rRNA gene sequences retrieved from the DDBJ/GenBank/EMBL databases by using the FASTA program [21].

Phylogenetic analysis of 16S rRNA gene sequences was performed using the software package MEGA 4 [30] after multiple alignments of data by CLUSTALX (version 1.83) [31]. Phylogenetic trees were constructed by the neighbor-joining and maximum-parsimony methods and the distances were calculated according to the Kimura two-parameter model. The robustness of phylogenetic trees was estimated by the bootstrap analysis of 1,000 replicates.

Results and Discussion

Comparative 16S rRNA gene sequence analysis showed that the novel isolates were closely related to each other (99.6–100% sequence similarity) and formed a separate branch within the class Alphaproteobacteria (Fig. 1). In the neighbor-joining and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences strains KMM 9008, KMM 9017, and KMM 9024T were positioned as a distinct phylogenetic line adjacent to Celeribacter neptunius. The three strains shared highest sequence similarities to the members of genera, Celeribacter (96.2–95.9%), Pseudoruegeria (95.6–94.3%), and Thalassobacter (95.2–93.1%). The low sequence similarities found relatively to validly described species of the Roseobacter clade demonstrate that novel strains can be considered to represent a novel genus. Strains KMM 9008, KMM 9017, and KMM 9024T showed a high level of DNA relatedness (88–96%), confirming their affiliation to the same species in accordance with the discriminative value of 70% recognized for the delineation of bacterial species [32].

Fig. 1
figure 1

Neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences available from the GenBank/EMBL/DDBJ databases (accession numbers are given in parentheses) showing relationship of strains KMM 9008, KMM 9017, and KMM 9024T and related genera of the class Alphaproteobacteria. Bootstrap values based on 1,000 replications are given as percentages at the branching points. Numbers indicate percentages greater than 70%. Filled circles indicate that the corresponding nodes were recovered in the tree generated with maximum-parsimony algorithm. Bar, 0.01 substitutions per nucleotide position

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Cultural, physiological, and metabolic properties of strains KMM 9008, KMM 9017, and KMM 9024T are listed in Table 1 and in the genus and species descriptions. Novel strains were similar in their phenotypic characteristics, except for differences in carbohydrate utilization patterns and sensitivity to antibiotics (Table 1), and in their chemotaxonomic traits (Table 2). They gave negative results for the assimilation of carbon sources in API 20NE tests, but could utilize a number of substrates, preferring carbohydrates and organic acids, but not amino acids, when they were cultivated on ASW-based media containing substrates as a sole carbon and energy source.

Table 1 Differential characteristics of V. arenosi gen. nov., sp. nov. and related members of the Alphaproteobacteria
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Table 2 Fatty acid composition (%) and polar lipids of V. arenosi gen nov., sp., nov. and related members of the Alphaproteobacteria
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The analysis of respiratory lipoquinone revealed ubiquinone Q-10, and polar lipids included PC, PG, PA, an unknown aminolipid and an unknown lipid as the major components and PE in a minor amount in all strains (Supplementary material). Chemotaxonomic properties obtained for the novel isolates (ubiquinone Q-10, the predominance of C18:1ω7c, 11-Methyl C18:1ω7c , 3-OH C10:0, and the presence of PC, PG, and PE) are in line with characteristics reported for the members of the Roseobacter clade. Together with the common characteristics, novel strains revealed some differences in their polar lipid and fatty acid profiles as compared with those of phylogenetically related bacteria. As seen from Table 2, unlike Thalassobacter stenotrophicus [16], Thalassobacter arenae [13], Pseudoruegeria aquimaris [34], and Pseudoruegeria lutimaris [12], the novel strains did not contain DPG, nor did they contain PL and GL which were found in P. aquimaris and P. lutimaris. Novel strains differed from T. stenotrophicus and P. aquimaris in the absence of C19:1 or/and cyclo C19:0; from P. lutimaris in the presence of 3-OH C10:0 and in the absence of 3-OH C12:0; and from T. arenae in the absence of C20:1 and C18:1. In contrast to their closest relative, C. neptunius [11], the novel strains contained 11-Methyl C18:1ω7c , a high amount of 3-OH C10:0, C12:1, a small amount of C20:1, PA and L, but did not contain LPE (Table 2). The DNA G+C contents of 56.7, 57, and 60 mol% in strains KMM 9008, KMM 9017, and KMM 9024T, respectively, were close to the G+C values reported for C. neptunius [11], T. stenotrophicus [16], and T. arenae [13], but clearly distinguished from those of P. aquimaris [34] and P. lutimaris [12] (Table 1).

The phylogenetic distinctiveness found for strains KMM 9008, KMM 9017, and KMM 9024T was supported by a combination of phenotypic characteristics differentiating the novel strains from related Alphaproteobacteria (Table 1). Main phenotypic differential characteristics between novel strains and their closest relative C. neptunius [11] were the absence of flagella and non-motility, growth at 4 and 37°C, the presence of oxidase and β-galactosidase, positive (or weakly positive) reaction for hydrolysis of DNA, and negative reactions for hydrolysis of Tween-80 and urea, and utilization of sucrose and d-xylose (Table 1). Based on distinctive phenotypic and chemotaxonomic characteristics and phylogenetic evidence, strains KMM 9008, KMM 9017, and KMM 9024T are considered to represent a novel genus and species, for which the name V. arenosi gen. nov., sp. nov. is proposed.

Description of Vadicella gen. nov

Vadicella (Va.di. cel’la. L. n. vadum, a shallow place, a shallow; L. fem. n. cella, a chamber, a cell; N. L. fem. n., Vadicella, a cell from a shallow place)

Gram-negative, strictly aerobic, oxidase- and catalase-positive, rod-shaped bacteria. Chemoorganoheterotrophic. Sodium ions are essential for growth. The predominant isoprenoid quinone is Q-10. Bchl α is not produced. Polar lipids include phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, an unknown aminolipid, and an unknown lipid as major and phosphatidylethanolamine as a minor component. The major fatty acids were C18:1ω7c , followed by 11-Methyl C18:1ω7c , C12:1, and C10:0 3-OH. Isolated from the marine environments. On basis of the 16S rRNA gene sequence analysis the genus represents a separate branch within the Alphaproteobacteria, closely related to the genera Celeribacter, Pseudoruegeria, and Thalassobacter. The type species of the genus is V. arenosi.

Description of V. arenosi gen. nov., sp. nov.

V. arenosi (a.re.no’si. L. gen. n. arenosi, of a sandy place, dwelling in marine sand)

In addition to properties given in the genus description the species is characterized as follows: cells are rods 0.6–0.8 μm in diameter and 2.5–4.5 μm in length. Non-motile. Strains formed non-pigmented, translucent, smooth and shiny colonies with the regular edges of 2–3 mm in diameter on MA. Required NaCl for growth; growth occurred at concentrations of 1–7% NaCl (w/v) and is optimal in 3–4%, weak growth in 0.5–1% NaCl, and no growth in 8% NaCl. Grow in/on basal media, containing NaCl alone without any of sea salts components, MgCl2, KCl, CaCl2, MgSO4 addition. The temperature range for growth was 4–37°C with optimum growth at 25–30°C; growth at 4 and 37°C is weak and strain-dependent, and no growth occurred above 37°C. The pH range is 5.5–9.5 with an optimum of 7.0–8.0. Positive or weakly positive for nitrate reduction. Negative for hydrolysis of casein, starch, gelatin, chitin, l-tyrosine, Tweens 20, 40, 80, and for H2S production. On the l-tyrosine containing medium, strains did not produce any pigments or/and clearance zones. Hydrolysis of DNA is positive for the type strain and weakly positive for the rest strains. No acid production observed from d-glucose, d-maltose, d-galactose, sucrose, d-lactose, d-mannose, d-cellobiose, d-xylose, l-arabinose, l-rhamnose, d-melibiose, d-ribose, fructose, l-sorbose, raffinose, N-acetylglucosamine, glycerol, d-myo-inositol, and d-mannitol. According to the API 20NE, positive for PNPG test, hydrolysis of aesculin and nitrate reduction, and negative for indole production, d-glucose acidification under anaerobic conditions, arginine dihydrolase, urease, gelatin hydrolysis, and assimilation of d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetylglucosamine, maltose, d-gluconate, caprate, adipate, l-malate, citrate, and phenylacetate. According to the API 32 GN, assimilation of 3-hydroxybutyric acid and lactic acid is strain-dependent (the reaction of the type strain is positive for 3-hydroxybutyric acid and negative for assimilation of lactic acid); negative for assimilation of d-ribose, d-sucrose, d-maltose, itaconic acid, suberic acid, sodium malonate, sodium acetate, inositol, d-melibiose, l-proline, l-rhamnose, N-acetylglucosamine, l-alanine, potassium 5-ketogluconate, glycogen, 3-hydroxybenzoic acid, l-serine, d-mannitol, d-glucose, salicin, l-fucose, d-sorbitol, l-arabinose, propionic acid, capric acid, valeric acid, trisodium citrate, l-histidine, potassium 2-ketogluconate, and 4-hydroxybenzoic acid. According to the API ZYM tests, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, β-galactosidase, α-glucosidase, and β-glucosidase, and negative for lipase (C14), valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. Production of acid phosphatase and naphthol-AS-BI-phosphohydrolase is strain-dependent (the reaction of the type strain for acid phosphatase is positive, and negative for naphthol-AS-BI-phosphohydrolase).

Utilized d-glucose, maltose, d-cellobiose, citrate, acetate, fumarate, and malate as a carbon and energy source; some strains could weakly utilized glycerol, fructose, d-galactose; did not utilize l-rhamnose, l-arabinose, d-mannose, d-xylose, sucrose, raffinose, d-mannitol, inositol, aminoacetic acid, l-tyrosine, ornithine, dl-leucine, l-α-alanine, dl-β-phenylalanine, dl-lysine, l-arginine, l-asparagine, and l-methionine. A detailed fatty acid composition and polar lipids are displayed in Table 2 and Supplementary Fig. 1. All strains were susceptible to antibiotics (content per disk): ampicillin (10 μg), benzylpenicillin (10 U), carbenicillin (100 μg), gentamicin (10 μg), rifampicin (5 μg), streptomycin (30 μg), vancomycin (30 μg), oxacillin (10 μg), neomycin (30 μg), kanamycin (30 μg), oleandomycin (15 μg), erythromycin (15 μg), cephazolin (30 μg), cephalexin (30 μg), tetracycline (30 μg), and chloramphenicol (30 μg). Sensitivity to nalidixic acid (30 μg), lincomycin (15 μg), ofloxacin (5 μg), and polymyxin (300 U) is strain-dependent; the type strain is susceptible to lincomycin (15 μg), ofloxacin (5 μg), and polymyxin (300 U) and resistant to nalidixic acid (30 μg). The DNA G+C content was 56.7–60 mol% (T m). Isolated from a sandy sediment sample collected from the Sea of Japan seashore, Russia. The type strain of the species is strain KMM 9024T (=NRIC 0787T = JCM 17190T).