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1 Laboratoire de Microbiologie et de Biotechnologie des Extrêmophiles, IFREMER, Centre de Brest, BP 70, 29280 Plouzané, France
2 Laboratoire IRD de Microbiologie des Anaérobies, UR 101, Universités de Provence et de la Méditerranée, CESB-ESIL, case 925, 163 Avenue de Luminy, 13288 Marseille, France
Correspondence
Georges Barbier
Georges.Barbier{at}ifremer.fr
or
Georges.Barbier{at}univ-brest.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DV1140T is AJ577471.
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MAIN TEXT |
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), Thermosipho japonicus (Takai & Horikoshi, 2000), Thermosipho melanesiensis (Antoine et al., 1997) and Thermosipho geolei (L'Haridon et al., 2001). Thermosipho africanus Ob7T was isolated from a coastal hydrothermal spring. Thermosipho japonicus IHB1T and Thermosipho melanesiensis BI429T were isolated from deep marine hydrothermal vents. Thermosipho geolei SL31T was isolated from a deep continental oil reservoir. In this study we report the description of a novel thermophilic bacterium isolated from a deep-sea hydrothermal vent chimney, and assign this to a novel species within the genus Thermosipho. Different types of samples were collected by the human-operated submersible Nautile in 1994 during the DIVA2 cruise on deep-sea vent fields of the Mid-Atlantic Ridge: Lucky Strike (32° 16' W 37° 17' N; 1600–1700 m water depth) and Menez-Gwen (31° 31' W 37° 51' N; 800–1000 m water depth). Fragments of an active black smoker chimney were collected by the DSV Nautile at Menez-Gwen and placed in an insulated box previously filled with sterile sea water. The box was opened in an on-board laboratory under sterile conditions. Friable pieces of chimney wall were crushed in an anaerobic chamber after addition of sterile sea water. Subsamples were transferred in cryotubes with 5 % DMSO and in serum vials stored, respectively, at –70 °C and 4 °C.
For inoculations, contents from one serum vial (10 ml) and one cryotube (1·8 ml) were mixed and diluted to 60 ml with 23 g l–1 NaCl. Enrichments (1 ml inoculum in 20 ml culture medium) and isolations were performed using PEXS medium (Urios et al., 2004) at different temperatures (50, 65 and 80 °C). Positive cultures were determined by microscopic observations and then purified. One isolate obtained at 65 °C was referenced as strain DV1140T. Single colonies of this isolate were obtained by streaking on PEXS medium solidified with 15 g l–1 Gelrite (Scott Laboratories). Plates were incubated in anaerobic jars for 3 days at 65 °C. Colonies were subsequently picked and streaked twice under the same conditions.
Microscope observations indicated that cells of isolate DV1140T were non-motile straight rods surrounded by a sheath-like structure. Cells were approximately 1·8±0·8 µm long and 0·4±0·2 µm wide (mean±95 % confidence interval) and appeared singly or in short chains. Cells were negatively stained for transmission electron microscopy (Raguénès et al., 1997). No flagella were observed. The presence of a toga was observed (Fig. 1). The Ryu KOH reaction (Powers, 1995), leading to immediate cell lysis as confirmed by phase-contrast microscopy, was positive, indicating that the cells were Gram-negative.
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. Growth was observed at 45–80 °C with the optimum temperature 65 °C. The strain required marine salts for growth, and grew at sea salt concentrations of 20–60 g l–1 (corresponding to 15–46 g NaCl l–1). No significant growth was observed at concentrations of 10 or 80 g l–1. The optimum sea salt concentration was approximately 30 g l–1 (corresponding to 23 g NaCl l–1). Growth occurred at pH 5·0–9·0, and the optimum pH was 6·0. Growth rate decreased to 50 % at pH 7·0 and to 60 % at pH 8·0 in comparison with that at pH 6·0. Under optimal conditions, the maximum cell concentration obtained was 2x108 cells ml–1 and the shortest generation time observed was 72 min.
The ability to use different carbon sources was investigated by adding one of the following compounds to a final concentration of 0·5 % (w/v) instead of glucose to the GYPS medium: sucrose, cellobiose, xylose, starch, lactate, maltose, mannose, trehalose, lactose, arabinose, galactose, mannitol, peptone, Casamino acids, casein, gelatin and brain heart infusion (BHI). Weak growth was observed in the presence of yeast extract and peptone (YPS). Growth yield of strain DV1140T was enhanced either by replacing yeast extract or peptone with 2 g BHI l–1 or by the addition of gelatin, starch, galactose, arabinose, glucose, trehalose or cellobiose to YPS. Amino acids and organic acids were analysed as metabolic end products by means of HPLC as described by Wery et al. (2001b). Production of H2S from elemental sulfur was investigated using lead acetate paper and 5 mM CuSO4/50 mM HCl as indicated by Alain et al. (2002). H2 and H2S were also quantified as described by Cord-Ruwisch (1985) and Fardeau et al. (1993). H2S production was observed and compared with that for controls (sterile GYPS medium with or without elemental sulfur) in the presence of 10 g l–1 elemental sulfur in GYPS medium. During GYPS fermentation until stationary phase, 5·6 mM glucose was consumed, and 1·7 mM acetate, 0·14 mM isovalerate and 12·5 mM H2 were produced. H2S was not detected. Fermentation in the presence of 10 g elemental sulfur l–1 in GYPS led to consumption of 6·0 mM glucose and production of 1·9 mM acetate, 0·15 mM isovalerate, 7·5 mM H2 and 1·3 mM H2S. Acetate and isovalerate were the only organic acids detected in both experiments. Amino acid analysis revealed an increase of 10 mg glycine l–1, 3 mg alanine l–1 and 10 mg proline l–1 in the culture medium as compared with controls.
The requirement for an external electron acceptor was tested. Only a slight enhancement of growth yield (16 %) was observed in the presence of 50 mM cystine. No significant differences with regard to growth kinetics and maximum cell concentrations (2x108 cells ml–1) were noticed during cultures on GYPS medium with or without elemental sulfur (10 g l–1). Polysulfides (Blumentals et al., 1990), sodium thiosulfate (20 mM), sodium sulfite (20 mM), sodium sulfate (20 mM), sodium nitrite (20 mM) and sodium nitrate (20 mM) did not enhance growth. No difference in growth was noticed between GYPS medium conditioned in an anaerobic chamber with or without Na2S. In this last case, a N2 flow was applied for 10 min after vacuum extraction.
The effect of H2 concentrations in the gas phase (N2/H2 at 100 : 0, 90 : 10, 75 : 25, 50 : 50, 25 : 75 and 10 : 90) was studied for DV1140T and Thermosipho geolei SL31T when grown in GYPS and in the medium described by L'Haridon et al. (2001), respectively. For both strains, the highest mean maximal concentrations from triplicate experiments at the end of the exponential phase were observed with 0 % H2 (respectively 3·6x108 and 9·6x107 cells ml–1) and decreased linearly with an increase in H2. We estimated from linear regressions that growth was completely inhibited with 87 % H2 for Thermosipho geolei SL31T and with 29 % H2 for DV1140T. Congruently, no significant growth was observed for Thermosipho geolei SL31T with 80 % H2 (L'Haridon et al., 2001).
Susceptibility to oxygen was investigated by incubating DV1140T in GYPS medium, with or without elemental sulfur, under O2 concentrations up to 12 % of the gas phase. The initial gas phase of the culture medium (N2/H2/CO2 90 : 5 : 5) was replaced after vacuum extraction by different calibrated mixtures of N2 and N2/O2 (80 : 20). In GYPS medium, growth was noticed up to 4 % O2. A decrease of 43 % of the maximum cell concentration occurred at 2 % O2 and of 63 % at 4 % O2, in comparison with a control comprising the same medium with a gas phase containing only N2. No growth was observed for a concentration of 6 %. In GYPS medium with elemental sulfur (10 g l–1), significant growth was obtained up to 8 % O2. The maximum cell concentration decreased by 15 % at 4 % O2, 25 % at 8 % O2 and 97 % at 10 % O2 in comparison with the control comprising the same medium with a gas phase containing only N2. No significant difference appeared between controls with N2 or N2/H2/CO2 (90 : 5 : 5) gas phases. This resistance to O2 was comparable with the results obtained for Thermotoga strains (Van Ooteghem et al., 2001).
Genomic DNA was extracted as described by Wery et al. (2001a). The DNA was purified by CsCl gradient centrifugation (Wery et al., 2001b) and the G+C content was determined by thermal denaturation according to the method of Marmur & Doty (1962) under the conditions reported by Raguénès et al. (1997). The G+C content of the genomic DNA of strain DV1140T was 33 mol%. The 16S rRNA gene was selectively amplified as described by Wery et al. (2001b) and the PCR product was sequenced using the primers described by Raguénès et al. (1996). This almost complete sequence of 1511 bp was then compared with others available in GenBank using BLAST (Altschul et al., 1997). A multiple sequence file was obtained by using the MEGALIGN program of the DNASTAR package (Promega). Alignments and similarity levels were obtained by the CLUSTAL W method with weighted residues (Thompson et al., 1994). Alignments were manually corrected using the multiple sequence alignment editor SEAVIEW and the phylogenetic reconstruction was produced using PHYLO_WIN (Galtier et al., 1996) with the following algorithms: Jukes–Cantor distance matrix and successively the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony and maximum-likelihood methods (Felsenstein, 1981). Bootstrap values were determined according to Felsenstein (1985). Strain DV1140T was phylogenetically affiliated to the genus Thermosipho (Fig. 2). The nearest recognized relatives were Thermosipho africanus Ob7T (=DSM 5309T), Thermosipho melanesiensis BI429T (=DSM 12029T), Thermosipho japonicus IHB1T (=JCM 10495T) and Thermosipho geolei SL31T (=DSM 13256T), with sequence similarity values of 91, 92, 94 and 96 %, respectively. Pairwise evolutionary distances were computed by use of Kimura's two-parameter model (Kimura, 1980) and a dendrogram was constructed from these distances by use of the neighbour-joining method. The positioning of strain DV1140T was supported by the results of the three algorithms used.
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; Hofstad, 1980; Kilian, 1978; Tharagonnet et al., 1977). The test was performed at 65 °C in duplicate. Three differences were revealed between the two strains: leucine arylamidase, valine arylamidase and 
-chymotrypsin reactions were only positive for strain DV1140T.
Strain DV1140T corresponds with the major characteristics of the Thermotogales. Strain DV1140T and its nearest relative Thermosipho geolei SL31T (L'Haridon et al., 2001) present a similar cell morphology, but they differ with regard to production of H2S in the presence of elemental sulfur, growth on glucose, peptone and yeast extract, a (slight) stimulation of growth with cystine and 96 % 16S rRNA gene sequence similarity (Table 1). Their geographical origins are very dissimilar. No flagella were found with DV1140T cells, regardless of the growth phase. The pH optimum is clearly different (DV1140T has the lowest pH observed for Thermosipho species). Under usual anaerobic culture conditions, the presence of elemental sulfur had no visible effect on growth kinetics and maximum cell concentrations, but a minor effect on glucose fermentation products. Consequently, strain DV1140T is the only Thermosipho strain that remains unaffected by elemental sulfur for growth. Strain DV1140T is able to grow at O2 concentrations up to 8 % in the presence of elemental sulfur and up to 4 % O2 without elemental sulfur, whereas growth of Thermosipho geolei SL31T is inhibited at lower concentrations (0·2–1·0 % O2). Growth of DV1140T and Thermosipho geolei SL31T are inhibited with 29 and 87 % H2 in the gas phase, respectively. Strain DV1140T was able to grow on galactose, arabinose, starch and gelatin, which are not used by Thermosipho geolei SL31T. Leucine, valine arylamidase and -chymotrypsin reactions tested with the API ZYM system were only positive for strain DV1140T. The G+C content of the genomic DNA of strain DV1140T is 3 mol% higher than that of Thermosipho geolei SL31T and is the highest yet found for the genus Thermosipho (29–31·4 mol%).
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Description of Thermosipho atlanticus sp. nov.
Thermosipho atlanticus (at.lan'ti.cus. L. masc. adj. atlanticus from the Atlantic Ocean, referring to the site of isolation of the type strain).
Rod-shaped, non-motile, Gram-negative bacteria surrounded by a sheath-like structure. Growth occurs at 45–80 °C (optimum 65 °C), at pH 5·0–9·0 (optimum pH 6·0) and at sea salt concentrations of 20–60 g l–1 (optimum 30 g l–1). Anaerobic, resistant to concentrations of oxygen up to 4 %, heterotrophic, able to ferment BHI and also starch, galactose, arabinose, glucose, trehalose, cellobiose and gelatin in the presence of peptone and yeast extract. Elemental sulfur does not enhance growth. The G+C content of the genomic DNA is 33 mol%.
The type strain, DV1140T (=CIP 108053T=DSM 15807T), was isolated from a sample collected on the Menez-Gwen hydrothermal site on the Mid-Atlantic Ridge (31° 31' W 37° 51' N; 800–1000 m water depth).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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