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1 Institute of Microbiology, Russian Academy of Sciences, Prospekt 60-letiya Oktyabrya 7/2, Moscow 117811, Russia
2 UMR 6539, Centre National de la Recherche Scientifique and Université de Bretagne Occidentale, Institut Universitaire Européen de la Mer, 29280 Plouzané, France
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, 38124 Braunschweig, Germany
Correspondence
M. L. Miroshnichenko
alfamirr{at}mail.ru
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ABSTRACT |
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). The family Deferribacteraceae (Huber & Stetter, 2002) is composed of three genera, Deferribacter, Flexistipes and Geovibrio, which form one separate lineage on the 16S-rRNA-based phylogenetic tree with sequence similarity values of around 89 %. Currently, the only two species within the genus Deferribacter are Deferribacter thermophilus (Greene et al., 1997) and Deferribacter desulfuricans (Takai et al., 2003). The type species of the genus, D. thermophilus, was isolated from the production water of Beatrice oil field, a high-temperature, sea-water-flooded oil reservoir located in the North Sea, UK. The recently described second species of the genus, D. desulfuricans, was obtained from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Izu-Bonin Arc, Japan. Both described species of the genus Deferribacter are strictly anaerobic, thermophilic organisms capable of the oxidation of a variety of complex organic compounds and organic acids in the presence of diverse electron acceptors. In this report, we describe a novel species of this genus, two representatives of which were obtained from two different deep-sea hydrothermal vent fields of the Mid-Atlantic Ridge.
Strains JRT and DR were isolated from hydrothermal samples collected in May 2001 at the Rainbow (36° 16' N, 33° 54' W) and Menez Gwen (37° 50' N, 31° 50' W) vent fields of the Mid-Atlantic Ridge. At Rainbow (at a depth of 2400 m), an in situ growth chamber or vent cap (Reysenbach et al., 2000) designed to concentrate the micro-organisms discharged by hydrothermal emissions was deployed using the hydraulic arm of the remotely operated vehicle (ROV) Victor. After in situ incubation for 2 days, the vent cap was closed by the hydraulic arm of the ROV, before transportation to the surface. The fluid from the cap was used in further work. At Menez Gwen (at a depth of 850 m), a chimney fragment was collected and placed in an insulated container for the trip to the surface. No temperature measurements were done at either site. Once on board the ship, samples were immediately transferred into 50 ml glass vials and flooded with a sterile solution of 3 % (w/v) Sea Salts (Sigma). The vials were then closed tightly with butyl-rubber stoppers (Bellco), pressurized with N2 (100 kPa), reduced with sodium sulfide and stored at 4 °C until further processing at the laboratory.
For the enrichment of organisms, the following basal medium was used (g l-1, unless indicated otherwise): NH4Cl, 0·33; KCl, 0·33; KH2PO4, 0·33; CaCl2.2H2O, 0·33; MgCl2.6H2O, 0·33; NaCl, 25·0; yeast extract, 0·1; NaHCO3, 0·3; sodium acetate, 2; trace elements (Balch et al., 1979), 10 ml l-1; vitamins (Wolin et al., 1963), 10 ml l-1. Elemental sulfur (10 g l-1) or nitrate (2 g l-1) was added as the electron acceptor. The media were prepared anaerobically and dispensed into 17 ml Hungate tubes; the headspace (7 ml) was filled with N2. The pH of the media before sterilization was 6·5.
After 3–5 days incubation at 60 °C, abundant growth of motile, straight- to vibrio-shaped organisms was detected in both types of enrichment medium. From the enrichment on sulfur-containing medium, strain JRT was obtained by the agar-shake dilution technique in the same basal medium supplemented with polysulfides (Widdel & Pfennig, 1992) and solidified with 1·5 % agar (Difco). Strain DR was obtained by the same technique, but on the solid medium supplemented with sodium nitrate (1 g l-1) from the corresponding enrichment.
Strain JRT was studied in detail. Cells of this strain were small rods of about 1·5–2 µm in length and 0·4–0·5 µm in width, which occurred singly, in pairs or in small chains. They were motile by means of one polar flagellum (Fig. 1a). In the stationary phase of growth, some rods took a spherical shape. Spores were never observed. Thin sections (Bonch-Osmolovskaya et al., 1990) revealed that the cell wall of strain JRT had a typical Gram-negative structure (Fig. 1b). Strain JRT grew over a temperature range of 45–65 °C, with optimal growth observed at 60 °C. At 60 °C, the strain grew between pH 6·0 and 7·2, with an optimum around pH 6·5. Strain JRT required NaCl for growth and grew at NaCl concentrations ranging from 10 to 50 g l-1, with an optimum at 30 g NaCl l-1. When air was added to the headspace of the tubes filled with basal medium (5 ml), supplemented with 2 g yeast extract l-1 and elemental sulfur, up to final oxygen concentrations of 0·1–1 %, no growth of strain JRT was observed.
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) in the course of sulfur reduction. Nitrate was reduced to nitrite; NO, N2O or ammonium were not detected by methods described elsewhere (Miroshnichenko et al., 2003c). The doubling time under the optimal growth conditions, on the medium with yeast extract and elemental sulfur, was around 55 min, with the final cell concentration exceeding 108 cells ml-1. No growth on fermentable substrates occurred in the absence of electron acceptors. Strain JRT could also grow chemolithoautotrophically on the basal medium in the absence of yeast extract, using molecular hydrogen as the electron donor, sulfur or nitrate as electron acceptors and CO2 as the carbon source. The strain was also shown to be able to grow with amorphous Fe(III) oxide (90 mM) (Slobodkin et al., 1999) or Fe(III) citrate (20 mM) as electron acceptors and acetate, succinate or molecular hydrogen as electron donors with approximately the same doubling time and cell yield as with other electron acceptors. Growth of strain JRT with molecular hydrogen and amorphous Fe(III) oxide was, however, possible only in the presence of yeast extract (50 mg l-1 minimal concentration). Amorphous Fe(III) oxide was reduced to a black magnetic precipitate with a high Fe(II) content. Sulfate, thiosulfate, nitrite, malate (sodium salts, 1 g l-1) and Mn(IV) supplied as 20 mM of MnO2 were not used as electron acceptors and did not support the growth on any of the substrates. Growth of strain JRT was inhibited by chloramphenicol, penicillin, rifampicin, streptomycin but not by tetracycline (all tested at a concentration of 100 µg ml-1 at 60 °C). Because of its unstable growth, strain DR was characterized only tentatively. Nevertheless, it was found to have the same morphology, optima and ranges of pH, temperature and salinity and used the same electron acceptors as strain JRT.
The G+C content of strain JRT DNA, determined according to Miroshnichenko et al. (1994), was 30·8 mol%. Analysis of the almost-complete 16S rRNA gene sequences of strains JRT and DR (1517 nucleotides), done as described previously (Rainey et al., 1995; Maidak et al., 2001; Jukes & Cantor 1969), indicated that their 16S sequences were identical and 95·3 and 95·2 % similar to those of D. thermophilus BMAT and D. desulfuricans DSM 14783T, respectively. The binary similarity value for the latter two strains was 97·9 %. A phylogenetic dendrogram (Fig. 2), displaying the position of strain JRT in relation to the two described species of the genus Deferribacter, was reconstructed from a distance matrix using the treeing algorithm of De Soete (1983). Most branching points are supported by high bootstrap values. The three Deferribacter species were distantly related to Flexistipes sinusarabici DSM 4947T (87·1–87·6 % sequence similarity), the two species of Geovibrio (84·6–85·9 % sequence similarity) and Denitrovibrio acetiphilus N2460T (83·3–84·4 % sequence similarity).
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, b), are able to reduce nitrate to nitrite, members of the genus Persephonella (Götz et al., 2002) are able to produce nitrogen in the course of nitrate reduction, whereas Caldithrix abyssi (Miroshnichenko et al., 2003c), Caminibacter hydrogeniphilus (Alain et al., 2002) and Thermovibrio ruber (Huber et al., 2002) are able to reduce nitrate to ammonium as the end product of denitrification. The presence in deep-sea hydrothermal vents of thermophilic dissimilatory Fe(III)-reducing micro-organisms, including representatives of the genus Deferribacter, was first demonstrated by denaturing-gradient gel electrophoresis (DGGE) experiments performed on enrichment cultures (Slobodkin et al., 2001). However, to date, the archaeon Geoglobus ahangari (Kashefi et al., 2002) is the only example of a deep-sea hydrothermal vent micro-organism that is capable of dissimilatory Fe(III) reduction in growing cultures. Strain JRT, one of the new isolates described in this report, represents another group of micro-organisms inhabiting deep-sea hydrothermal vents – moderately thermophilic bacteria able to reduce Fe(III) compounds as well as nitrate and sulfur. Strain JRT shares many common phenotypic features with the type species of the genus Deferribacter, D. thermophilus (Table 1). However, strain JRT differs from D. thermophilus and D. desulfuricans by its ability to grow lithoautotrophically, utilizing hydrogen as an electron donor, CO2 as a carbon source and elemental sulfur or nitrate as electron acceptors. Other significant characteristics that differentiate strain JRT from D. thermophilus are its ability to reduce sulfur and inability to use Mn(IV) as an electron acceptor. In contrast to D. desulfuricans, strain JRT is able to reduce Fe(III). On the basis of the results of phylogenetic, genosystematic and physiological studies that clearly differentiate strain JRT from known species of the genus Deferribacter, we propose to describe it as the type strain of a new species of the genus, namely, Deferribacter abyssi. Strain DR (=DSM 14927) represents the second strain of the same species.
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Small rods that are straight- to vibrio-shaped. Cells are 1·5–2·0 µm long and 0·4–0·5 µm wide, with the Gram-negative type of cell wall. Motile by means of one polar flagellum. Anaerobic. Moderately thermophilic, growing between 45 and 65 °C, with optimum growth at 60 °C. Neutrophilic, growing between pH 6·0 and 7·2, with optimum growth at pH 6·5–6·7. Grows at NaCl concentrations ranging from 10 to 50 g l-1, with optimum growth at 30 g NaCl l-1. Capable of chemolithoautotrophic growth with hydrogen as an electron donor, elemental sulfur or nitrate as electron acceptors and CO2 as a carbon source. Anaerobic oxidation of acetate, pyruvate, succinate and proteinaceous substrates by using sulfur, nitrate or Fe(III) as electron acceptors. Not capable of fermentation. Sensitive to rifampicin, chloramphenicol, vancomycin, penicillin and streptomycin but resistant to tetracycline. Isolated from hydrothermal vent fields of the Mid-Atlantic Ridge.
The type strain is JRT (=DSM 14873T=JCM 11955T). The G+C content of its DNA is 30·8 mol%. Isolated from the Rainbow (36° 16' N, 33° 54' W) hydrothermal vent field of the Mid-Atlantic Ridge.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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