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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
Anne Postec
Anne.Postec{at}ifremer.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of AT1271T is AJ786363.
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; Lien et al., 1998; Windberger et al., 1989). The order Thermotogales includes six genera: Thermotoga, Thermosipho, Fervidobacterium, Petrotoga, Geotoga and Marinitoga (Davey et al., 1993; Huber et al., 1986, 1989; Patel et al., 1985; Wery et al., 2001a). To date, only two species belonging to the genus Marinitoga have been described, Marinitoga camini (Wery et al., 2001a) and Marinitoga piezophila (Alain et al., 2002), both originating from deep-sea hydrothermal vents. Members of the order Thermotogales are fermentative bacteria that produce hydrogen, the accumulation of which has been widely reported to inhibit their growth (Van Ooteghem et al., 2002). This inhibition could be overcome by addition of elemental sulfur or thiosulfate (Alain et al., 2002; Antoine et al., 1997; Jeanthon et al., 1995; Lien et al., 1998). Here we report the description of a novel thermophilic bacterium belonging to the genus Marinitoga that is able to tolerate 100 % hydrogen in the headspace when growing on organic substrates.
Samples were collected by the ROV Victor in 2001 during the ATOS cruise on the Mid-Atlantic Ridge. The deep-sea vent field Rainbow (36° 13' N 33° 54' W) at 2275 m depth was explored and part of an active black smoker chimney was brought to the surface in a decontaminated insulated box. On board, eight subsamples representative of the whole chimney were crushed in an anaerobic chamber and stored in sterile serum vials filled with sterile sea water containing 0·5 mg Na2S l–1. The subsamples were pooled and used as inoculum at 2 % (v/v). A continuous enrichment culture was performed on a modified SME medium (Postec et al., 2005; Sharp & Raven, 1997) in a 2 litre gas-lift bioreactor (Godfroy et al., 2000; Raven et al., 1992) at 60 °C at atmospheric pressure. A culture sample collected on day 7 permitted the isolation of several strains by three serial dilutions to extinction (Baross, 1995). Single colonies were obtained by streaking on the same medium solidified with 15 g Gelrite l–1 (Scott Laboratories). Plates were incubated for 2 days at 60 °C in anaerobic jars (Godfroy et al., 1997). Colonies were subsequently picked and streaked twice under the same conditions. One isolate was referenced as strain AT1271T and was chosen for further characterization.
Microscopic observations indicated that cells of isolate AT1271T were weakly motile rods surrounded by a ‘toga’, an outer sheath-like structure. Cells were approximately 0·6 µm wide and 1·5 µm long during the exponential phase and appeared single or in short chains within the envelope. Cells became elongated, forming long chains, during stationary phase (Fig. 1). Cells were negatively stained for examination under a transmission electron microscope (Raguénès et al., 1997): a polar flagellum and the presence of a toga were observed (Fig. 1). The Ryu KOH reaction (Powers, 1995) leading to immediate cell lysis, as confirmed by phase-contrast microscopy, was positive, indicating that cells of strain AT1271T were Gram-negative.
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. Bacterial growth was directly monitored in culture tubes by spectrometry at 600 nm (a Spectronic 401 instrument; Milton Roy). Previous correlation between the optical density at 600 nm and cell numeration was simultaneously established by direct counting of the cells using a Thoma chamber viewed with an Olympus model BH-2 microscope. Isolate AT1271T grew at 35–65 °C; the optimum temperature was 60 °C. Growth occurred at pH 4·5–8·5, the optimum being around pH 6. No growth was observed at pH 4 or pH 9. The strain grew at sea salt concentrations ranging from 10 to 65 g l–1 (corresponding to 8–50 g NaCl l–1). The optimum sea salt concentration for growth was approximately 35 g l–1 (corresponding to 27 g NaCl l–1). Under optimal conditions, the maximal cell density obtained was 6·0x108 cells ml–1 and the maximal observed growth rate was 0·37 h–1 (equivalent to a doubling time of 1·9 h).
Strain AT1271T is an obligate chemo-organotroph. No growth was detected on mineral media complemented with a H2/CO2 (80 : 20) headspace. The ability of the isolate to use various carbon sources was tested in medium containing (per litre): 30 g sea salts, 3·9 g MES buffer, 10 ml vitamin solution (Balch et al., 1979), 10 ml mineral solution (Balch et al., 1979), 0·1 g yeast extract as the nitrogen source and complex carbon sources. The following carbohydrates were tested at 0·5 % (w/v): cellobiose, glucose, maltose, fructose, galactose, ribose, starch, cellulose, glycogen, chitin, pectin and carboxymethylcellulose. Ethanol was tested at 0·5 % (v/v). The following organic acids and proteinaceous substrates were tested at 0·2 % (w/v): acetate, pyruvate, yeast extract, brain heart infusion, peptone, tryptone, casein, Casamino acids and keratin. A solution of 20 amino acids, each at 0·1 g l–1, was also tested. Positive cultures were confirmed by subcultures on the same medium. Compared with growth on the medium without added carbon source, growth was enhanced by addition of glucose, starch, glycogen, chitin, yeast extract, brain heart infusion, peptone and casein. To extend the investigation of carbohydrate consumption, cellobiose, glucose, maltose, fructose, galactose, ribose, acetate, pyruvate and rhamnose were tested at 1 g l–1 in the presence 1 g yeast extract l–1. A negative control was performed without added sugar. Growth was monitored by spectrometry and carbohydrate concentrations were estimated by HPLC after 6 and 13 h of culture. At these conditions, growth of strain AT1271T was enhanced when pyruvate or maltose was added. Nitrogen sources were tested in medium containing (per litre): 30 g sea salts, 3·9 g MES buffer, 10 ml Balch vitamins, 10 ml mineral solution and glucose (5 g l–1). Growth occurred in the presence of yeast extract (0·2 g l–1), whereas urea (0·2 g l–1) and NH4Cl (1 g l–1) alone did not support growth.
The end-products of glucose metabolism were acetate, ethanol, formate, hydrogen and CO2. In the presence of elemental sulfur, no hydrogen was detected. Growth in the presence of different electron acceptors was tested on GYPS medium. A small increase in the final cell concentration was obtained when thiosulfate (20 mM), cystine (50 mM) or sulfur (10 g l–1) was added. Addition of sulfate (20 mM), sulfite (1 mM), nitrate (20 mM) or nitrite (1 mM) did not enhance growth. Strong production of H2S was revealed by using lead acetate strips (Whatman) on the culture with cystine and sulfur, indicating the use of these electron acceptors.
Various gas phases were tested in the presence and absence of sulfur (10 g l–1) on GYPS medium: N2/H2/CO2 (90 : 5 : 5), H2/CO2 (80 : 20), N2/CO2 (80 : 20) and N2 (100 %). Cell density was above 1x108 ml–1 in the absence of sulfur, and above 4x108 ml–1 in the presence of sulfur, regardless of the gas phase.
The effect of H2 in the gas phase was tested using the following N2/H2 ratios: 100 : 0, 80 : 20, 60 : 40, 40 : 60, 20 : 80 and 0 : 100. M. camini, M. piezophila and strain AT1271T were grown for comparison in triplicate on GYPS at 50, 65 and 60 °C, respectively. The initial gas phase of the culture medium (N2/H2/CO2 90 : 5 : 5) was replaced and 10 cycles of vacuum extraction/addition of the different calibrated mixtures of N2/H2 were performed. Cell densities were estimated after 4, 8, 12 and 24 h of incubation by counting. No significant difference appeared between controls made with N2/H2/CO2 (90 : 5 : 5). Maximal cell concentrations for M. camini and M. piezophila were obtained with 0 % H2. Congruently, no growth was observed for either species with 80 % H2 (Alain et al., 2002; Wery et al., 2001a). M. camini and M. piezophila displayed a linear decrease in maximal cell concentration with H2 increase, and total inhibition of growth at 40 and 60 % H2, respectively. In contrast, strain AT1271T grew at up to 2–4x108 cells ml–1 regardless of H2 concentration. Strain AT1271T tolerates up to 2 % oxygen in the gas phase. Growth was inhibited in the presence of 4 % oxygen.
Genomic DNA was extracted as described by Wery et al. (2001a). The G+C content was determined by thermal denaturation (Marmur & Doty, 1962) with the modifications described by Raguénès et al. (1997). The G+C content of the genomic DNA of strain AT1271T was 28±1 mol%. The 16S rRNA gene was selectively amplified as described by Wery et al. (2001b), and the PCR product was sequenced with the primers described by Raguénès et al. (1996). The sequence was then compared with available sequences in the GenBank database using a BLAST search (Altschul et al., 1990). A multiple sequence file was obtained by using the Wisconsin Package version 10.3 (Accelrys Inc.). Alignments and similarity levels were obtained by the CLUSTAL W method with weighted residues (Thompson et al., 1994). Alignments were manually refined 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 (Felsenstein, 1985) were determined. Strain AT1271T was phylogenetically affiliated to the genus Marinitoga (Fig. 2), the nearest recognized relatives being M. camini and M. piezophila with respective 16S rRNA gene sequence similarities of 96 and 94 %. The positioning of strain AT1271T was supported by the results of the three algorithms used.
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). Moreover, unlike M. camini, strain AT1271T does not use sucrose, fructose, cellobiose, cellulose, carboxymethylcellulose or pectin, and it does ferment casein. It is noteworthy that strain AT1271T tolerates 100 % hydrogen in the gas phase whereas the growth of other Marinitoga species is inhibited at lower hydrogen concentrations. This tolerance to hydrogen has never been reported to date among the order Thermotogales, and could be related to the high hydrogen concentrations measured (16 mmol kg–1) in all vent fluids at the Rainbow field, the site from where strain AT1271T was recovered (Charlou et al., 2002).
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Description of Marinitoga hydrogenitolerans sp. nov.
Marinitoga hydrogenitolerans (hy.dro.ge.ni.to'le.rans. N.L. hydrogenum hydrogen; L. pres. part. tolerans tolerating; N.L. adj. hydrogenitolerans hydrogen-tolerating, referring to its ability to tolerate a high level of hydrogen in the headspace).
Rod-shaped, weakly motile, Gram-negative bacteria surrounded by a sheath-like structure. Growth occurs at 35–65 °C (optimum 60 °C), pH 4·5–8·5 (optimum pH 6·0) and 10–65 g sea salts l–1 (optimum 30–40 g l–1). The doubling time under optimal conditions is 1·9 h and the maximum cell yield is 6·0x108 cells ml–1. Anaerobic; resistant to concentrations of oxygen up to 2 % and hydrogen up to 100 %. Chemo-organotrophic; able to ferment glucose, maltose, pyruvate, starch, glycogen, chitin, yeast extract, brain heart infusion, peptone and casein. Fermentation products identified on glucose are acetate, ethanol, formate, hydrogen and CO2. Sulfur, cystine and thiosulfate stimulate growth slightly and H2S production is observed with sulfur and cystine. The G+C content of the genomic DNA of the type strain is 28 mol%. 16S rRNA gene sequence analysis indicates that the species is classified within the genus Marinitoga, order Thermotogales in the bacterial domain. 16S rRNA gene sequence similarity of the type strain to M. camini MV1075T is 96 %.
The type strain, AT1271T (=DSM 16785T=JCM 12826T), was isolated from a sample collected on the Rainbow hydrothermal site on the Mid-Atlantic Ridge (36° 13' N 33° 54' W).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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