Introduction

The global sulfur cycle is closely connected to the cycling of carbon, nitrogen, phosphorus and iron, which depends on the activities of metabolically and phylogenetically diverse microorganisms, most of which reside in the ocean (Sievert et al. 2007). One of these sulfur related metabolic groups is the sulfur-oxidizing bacteria (SOB), of which marine SOB thrive in low-oxygen environments (Marshall and Morris 2013). In an attempt to investigate SOB in the deep sea environment of the Atlantic Ocean, many bacterial strains were isolated and characterized taxonomically. This study focuses on one of those isolates, designated strain 13D2W-2T. Comparative 16S rRNA gene sequence analysis indicated that strain 13D2W-2T belongs to the genus Thioclava, which belongs to the family Rhodobacteraceae. The genus Thioclava was proposed by Sorokin and colleagues (Sorokin et al. 2005) and currently includes two species Thioclava pacifica, the type species, and Thioclava dalianensis (Zhang et al. 2013). Accordingly, the aim of the present work was to determine the exact taxonomic position of strain 13D2W-2T using polyphasic characterization.

Materials and methods

Bacterial strains, isolation, and cultivation

Sediment was sampled from the deep sea of the Atlantic Ocean in 2011 by a TV-guided grab sampler. The sampling site was DY22III-S013-TVG7 (W13.9°, S26.0°). The sediment was used for enrichment of SOB. The enrichment and isolation were conducted in DSMZ medium 1121 (MMJS medium) with modification of the NaCl concentration to be 30 g/L. The last four sterile concentrated components (Vitamin solution, NaHCO3, Na2S2O3·5H2O and sulfur) were added to the medium after the other medium components (adjusted with NaOH to be pH 6.8) were autoclaved. The microaerobic condition for sample enrichment was under a gas phase of 80 % H2/19 % CO2/1 % O2 (200 kPa) in a glass bottle tightly sealed with a butyl-rubber cap at 25 °C. After successfully enrichment, the isolation of the bacterial consortium was attempted on plates of the same medium solidified with agar (1.5 % w/v) in a closed jar (Anoxomat Mart II) using a standard microaerobic condition by filling with 6 % O2 (N2 78 %, CO2 10 % and H2 6 %) according to the manufacturer’s instructions. Then the strains were incubated on 216L agar medium (CH3COONa 1.0 g, tryptone 10.0 g, yeast extract 2.0 g, sodium citrate 0. 5 g, NH4NO3 0.2 g, 1 L sea water, 15 g agar, pH 7.5) (Lai et al. 2009) at normal atmosphere conditions for morphological and biochemical characterization.

Phenotypic characterization

Gram reaction, oxidase, catalase and lipase (Tween 80) activities, tolerance to NaCl, the optimal growth temperature and pH, hydrolysis of aesculin and starch, general cell morphology and electron microscopy were studied as previously described (Lai et al. 2009). Antibiotic susceptibility tests were performed by the disc diffusion method as described previously (Shieh et al. 2003). Other biochemical tests were carried out using API 20NE and API ZYM strips (bioMérieux) and the Biolog GN2 MicroPlate test panel according to the manufacturer’s instructions, except adjusting the NaCl concentration in all tests to 3.0 %. The oxidization of inorganic sulfur compounds (thiosulfate, sulfur and sulfide) were tested in mixotrophic growth medium supplied with acetate (2.72 g/L) and yeast extract (0.2 g/L) according to the method described previously (Sorokin et al. 2005). T. pacifica TL 2T and T. dalianensis DLFJ1-1T were obtained as described in our earlier study and were cultured under the same conditions (Zhang et al. 2013).

Determination of 16S rRNA gene sequence and phylogenetic analysis

Genomic DNA was extracted according to a previously described method (Ausubel et al. 2002). The 16S rRNA gene was amplified by PCR using primers that have been described previously (Liu and Shao 2005) and also extracted from the draft genome sequence. The 16S rRNA gene sequence similarity was determined using the EzTaxon-e server (Kim et al. 2012). Sequences of related taxa were obtained from the GenBank database. Phylogenetic analysis was performed using MEGA version 5.0 (Tamura et al. 2011) with distance options according to the Kimura two-parameter model and clustering with the neighbour-joining (Saitou and Nei 1987), maximum likelihood (Felsenstein 1981), and minimum evolution (Rzhetsky and Nei 1992) methods, and supported with bootstrap values based on 1,000 replications.

Genome sequencing, G + C content, dDDH and ANI estimation

The draft genome sequences of strain 13D2W-2T, T. dalianensis DLFJ1-1T and T. pacifica TL 2T were determined by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China), using Solexa paired-end (500 bp library) sequencing technology. A total of 500 Mbp clean data for each strain was generated to reach about 100-fold depth of coverage with an Illumina/Solexa Genome Analyzer IIx (Illumina, SanDiego, CA). The clean data was assembled by SOAPdenovo2 (Luo et al. 2012). The G+C content of the chromosomal DNA was determined by analysis of the draft genome sequences. The digital DNA:DNA hybridization (dDDH) estimate values between the three strains were analyzed using the genome-to-genome distance calculator (GGDC2.0) (Auch et al. 2010a, 2010b; Meier-Kolthoff et al. 2013). The average nucleotide identity (ANI) was calculated using the algorithm of Goris et al. (2007) using the EzGenome web service.

Determination of fatty acid, isoprenoid quinone, and polar lipid compositions

Fatty acids in whole cells grown on marine agar 216L medium at 28 °C for 48 h were saponified, methylated and extracted using the standard MIDI (Sherlock Microbial Identification System, version 6.0B) protocol. The fatty acids were analysed by gas chromatography (Agilent Technologies 6850) and identified using the TSBA6.0 database of the Microbial Identification System (Sasser 1990). Analysis of the respiratory quinone and polar lipid were carried out by the Identification Service of DSMZ and Dr. B. J. Tindall, DSMZ, Braunschweig, Germany.

Results and discussion

Phenotypic characteristics

Strain 13D2W-2T was observed to be a Gram-negative, non-pigmented, short rod-shaped bacterium that was motile by means of a flagellum (see Fig S1). Moderately halophilic, 13D2W-2T was found to be able to grow in 0.5–12 % of NaCl (optimum 3–5 %) and at 4–41 °C (optimum 28–37 °C), but not at 45 °C. The strain was found to be sensitive to ampicillin (10 μg/per disk, OXOID), carbenicillin (100), cefalexin (30), cefazolin (30), cefobid (30), cephradin (30), chloromycetin (30), ciprofloxacin (5), erythromycin (15), gentamicin (10), kanamycin (30), minomycin (30), ofloxacin (5), oxacillin (1), penicillin G (10), piperacillin (100), polymyxin B (30IU), rifampicin (5), rocephin (30), streptomycin (10), tetracycline (30), vancomycin (30), and vibramycin (30); and resistant to clindamycin (2), Co-trimoxazole (25), lincomycin (2), norfloxacin (10 μg) and metronidazole (5). Strain 13D2W-2T can oxidize reduced sulfur compounds such as thiosulfate, sulfur and sulfide as energy source. Sulfate was the most oxidised product from thiosulfate, while sulfite was the main product from sulfide and sulfur used by this strain after culture for 3 days.

16S rRNA gene sequence analysis

A nearly full-length 16S rRNA gene sequence (1433 nt, GenBank accession number KJ755834) of strain 13D2W-2T was determined. As shown in Fig. 1, phylogenetic trees based on 16S rRNA gene sequences showed that strain 13D2W-2T and the two species of the genus Thioclava formed an independent monophyletic cluster separated from other genera, with high a bootstrap (90 %) value support. Clustering results using the maximum likelihood and minimum-evolution approach were similar to that obtained using the neighbour joining method (Fig. S2 and S3). Strain 13D2W-2T shared the highest 16S rRNA gene sequence similarity of 97.8 % to T. dalianensis DLFJ1-1T, follow by T. pacifica TL 2T (97.7 %), while the similarities to other species were all below 97.0 %.

Fig. 1
figure 1

Neighbour-joining tree showing the phylogenetic positions of strain 13D2W-2T and representatives of some other related taxa based on 16S rRNA gene sequences. Filled circles indicate nodes that were also recovered in maximum-likelihood and minimum evolution trees based on the same sequences. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branch points. Bar, 0.005 nucleotide substitution rate (Knuc) units. Stappia stellulata IAM 12621T (D88525) as used as the outgroup

Full size image

Genome sequencing, DNA G+C content, dDDH and ANI estimation

A total of 500 Mbp clean sequence data for strains 13D2W-2T, T. pacifica TL 2T and T. dalianensis DLFJ1-1T were generated to reach about 100-fold depth of coverage with an Illumina/Solexa Genome Analyzer IIx. The clean data was assembled by SOAPdenovo2 (Luo et al. 2012). The draft genome accession number for strains 13D2W-2T, T. pacifica TL 2T and T. dalianensis DLFJ1-1T are AQRC00000000, AUND00000000 and JHEH00000000 respectively.

The chromosomal DNA G+C content of strain 13D2W-2T was determined to be 65.3 mol%, which is close to the values of the other two species of genus Thioclava (62.5–63.9 mol%). The estimated dDDH values between strain 13D2W-2T and, respectively, T. dalianensis DLFJ1-1T and T. pacifica TL 2T were 22.60 ± 2.36 and 25.60 ± 2.41 %, values which are below the standard criteria (70 %) for delineation of bacterial species (Wayne et al. 1987). The ANI values between strain 13D2W-2T and the related type species T. dalianensis DLFJ1-1T and T. pacifica TL 2T were 78.49 and 81.91 %, respectively, which are below standard ANI criteria for species identity (95–96 %; Richter and Rossello-Mora 2009). These data confirm that strain 13D2W-2T is a novel species of the genus Thioclava.

A complete sulfur oxidation gene cluster (soxTRSVWXYZABCD) was found to present in the genomes of strain 13D2W-2T and T. Pacifica TL 2T but not in that of T. dalianensis DLFJ1-1T. This is in accordance with their features i.e. that strain 13D2W-2T and T. pacifica TL 2T can oxidize inorganic sulfur compounds, but T. dalianensis DLFJ1-1T cannot (Zhang et al. 2013). A Form I ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (cbbL) was also found in the draft genome sequences of strain 13D2W-2T and T. pacifica TL 2T, but not in that of T. dalianensis DLFJ1-1T.

Chemotaxonomic characteristics

The fatty acid profile of 13D2W-2T was obtained at the same time as the data was obtained for the two reference species of the genus Thioclava during our earlier study of T. dalianensis (Zhang et al. 2013). The results for the three strains are shown in Table 1, which shows that the three profiles are qualitatively similar. The major fatty acids identified in strain 13D2W-2T were Summed Feature 8 (C18:1 ω7c/ω6c as defined by MIDI). The isoprenoid quinone detected in strain 13D2W-2T was Q10 (100 %), as is the case with the two species of the genus Thioclava Query.

Table 1 Cellular fatty acid content of strain 13D2W-2T and related members of the genus Thioclava
Full size table

The major polar lipids of strain 13D2W-2T were found to comprise phosphatidylethanolamine, phosphatidylglycerol, an aminophospholipid, a glycolipid and three unknown phospholipids, as shown in Fig. S4. This profile is the same as that obtained for T. dalianensis DLFJ1-1T (Zhang et al. 2013).

Taxonomic conclusion

The high 16S similarity and the results of phenotypic analysis and chemotaxonomic studies presented above support the view that strain 13D2W-2T should be assigned to the genus Thioclava. However, strain 13D2W-2T can be differentiated from the two current species of the genus Thioclava with the low values of dDDH (22.60–25.60 %) and ANI (78.49–81.91 %) and the different characteristics given in Table 2. On the basis of data described above, strain 13D2W-2T should be placed into a new species of genus Thioclava, for which a name Thioclava atlantica sp. nov. is proposed.

Table 2 Physiological characteristics of strain 13D2W-2T and the members of the genus Thioclava
Full size table

Description of Thioclava atlantica sp. nov.

Thioclava atlantica (at.lan’ti.ca. L. fem. adj. atlantica referring to the Atlantic Ocean, where the strain was isolated).

Cells are Gram-negative short rod-shaped, 0.7–0.8 μm wide and 1.2–2.0 μm long, motile by means of a flagellum. Positive for oxidase, catalase, β-glucosidase (aesculin hydrolysis), urease, beta-galactosidase, d-glucose fermentation and nitrate reduction, but negative for indole production, gelatinase and arginine dihydrolase. Can oxidize reduced sulfur compounds such as thiosulfate, sulfur and sulfide as energy source. On 216L agar plates, forms smooth grey-white colonies with regular edges and of 2–3 mm in diameter after 72 h incubation at 28 °C, non-pigmented and slightly raised in the center. Moderately halophilic, grows in 0.5–12 % of NaCl (optimum 3–5 %) and at 4–41 °C (optimum 28–37 °C), but not at 45 °C. The principal fatty acid is Summed Feature 8 (C18:1 ω7c/ω6c). The only quinone is Q10. The polar lipids are comprised of phosphatidylethanolamine, phosphatidylglycerol, an aminophospholipid, a glycolipid and three unknown phospholipids. API ZYM test strip results indicate the type strain is positive for alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), leucine aminopeptidase, valine aminopeptidase, α-glucosidase, β-glucosidase; weakly positive for cystine aminopeptidase, lipase (C14), naphtol-AS-Bl-phosphoamidase, trypsin, β-glucuronidase; and negative for N-acetyl-β-glucosaminidase, α-chymotrypsin, α-fucosidase, α-galactosidase, α-mannosidase, β-galactosidase. According to API 20NE test, can utilize d-glucose, d-maltose, d-mannitol, d-mannose, l-arabinose, malic acid, potassium gluconate and trisodium citrate, but not adipic acid, capric acid, N-acetyl-glucosamine or phenylacetic acid. Of the 95 substrates in the Biolog GN2 system, the type strain is positive for d-alanine, d-fructose, d-melibiose, glucose-1-phosphate, glycerol, Hydroxy-l-proline, l-alanine, l-asparagine, l-fucose, l-glutamic acid, l-histidine, malonic acid, sebacic acid, succinic acid, β-methyl-d-glucoside; weakly positive for acetic acid, d,l-α-glycerol phosphate, d-cellobiose, itaconic acid, l-aspartic acid, N-acetyl-d-galactosamine, p-Hydroxy phenylacetic acid and uridine. The G+C content of the DNA of the type strain is 65.3 mol%. Table 2 shows the characteristics used to distinguish the type strain from the other two members of the genus.

The type strain, 13D2W-2T ( = MCCC 1A02612T = LMG 27145T) was isolated from deep sea sediment of the Atlantic Ocean. The GenBank accession number for the 16S rRNA gene sequence of T. atlantica 13D2 W-2T is KJ755834 and that of the draft genome sequence is AQRC00000000.