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1 UMR 6539, Centre National de la Recherche Scientifique, and Université de Bretagne Occidentale, Institut Universitaire Européen de la Mer, F-29280 Plouzané, France
2 Portland State University, Department of Biology, Portland, OR 97201, USA
3 Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur, A-1180 Wien, Austria
4 DSMZ – German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
C. Jeanthon
jeanthon{at}univ-brest.fr
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ABSTRACT |
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The GenBank accession number for the 16S rDNA sequence of Methanocaldococcus indicus SL43T is AF547621.
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MAIN TEXT |
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70 °C) and the hyperthermophilic members of the Methanocaldococcaceae (Whitman et al., 2001
). The family Methanocaldococcaceae includes two new genera, Methanocaldococcus and Methanotorris (Whitman & Jeanthon, 2002). Members of these genera possess less than 93 % 16S rRNA gene sequence similarity and have been exclusively isolated from deep-sea hydrothermal vents. Within the genus Methanocaldococcus, the 16S rRNA sequence similarity between species is greater than 95 %.
To date, Methanocaldococcus species have been isolated from actively venting sulfide deposits at deep-sea hydrothermal vents at the East Pacific Rise (13°N and 21°N) (Jones et al., 1983; Jeanthon et al., 1999a) and the Mid-Atlantic Ridge (14°45'N and 23°N) (Jeanthon et al., 1998, 1999b) and from hydrothermally heated sediments from Guaymas Basin in the Gulf of California (Zhao et al., 1988; Jones et al., 1989; Jeanthon et al., 1999b).
Because of the paucity of readily identifiable phenotypic characteristics, taxonomic distinction between Methanocaldococcus species on this basis is difficult. Reliable comparison of strains can be made by RFLP analysis of the 16S rDNA (Jeanthon et al., 1999b). This method was used to reveal a novel species isolated from sulfide deposits of a deep-sea hydrothermal vent on the Central Indian Ridge. In this paper, we describe the isolation and characterization of this novel organism.
The novel strain was isolated from chimney samples collected in April 2001 at the Kairei vent field on the Central Indian Ridge (25°19'S, 70°02'E) at a depth of 2420 m (Van Dover et al., 2001). Using the hydraulic arm of the remotely operated vehicle Jason, the chimney samples were placed in insulated containers for the trip to the surface. Once on board, samples were ground anaerobically and stored in serum vials at 4 °C with N2 in the headspace.
Initial enrichments were done on-board ship in MSH medium (Boone et al., 1989; http://methanogens.pdx.edu/OCM_media.html). Seven chimney samples were ground under a stream of N2. Chimney samples were hard and contained pyrite, pyrrhotite, some chalcopyrite, anhydrite and iron oxides. Each sulfide slurry (0·5 ml) was inoculated into 5 ml MSH medium and the serum tubes were incubated at 80 °C for 2–3 days or until turbidity was noted. When growth occurred, it consisted of motile coccoid cells that fluoresced intense green at 420 nm. Positive enrichments were transferred in a culture medium that contained the following (l-1 distilled water): 30 g NaCl, 3 g MgCl2.2H2O, 4 g Na2SO4, 0·5 g KCl, 0·25 g NH4Cl, 0·2 g KH2PO4, 3·46 g PIPES, 0·15 g CaCl2.2H2O, 0·5 g Difco yeast extract, 1 ml trace-element mixture (Widdel & Bak, 1992), 0·2 mg sodium tungstate, 50 µg sodium selenate, 1 ml vitamin mixture (Widdel & Bak, 1992), 1 ml thiamin solution (Widdel & Bak, 1992), 0·05 mg vitamin B12, 1 ml growth-stimulating factors (Pfennig et al., 1981) and 1 mg resazurin. Prior to autoclaving, the pH of the medium was adjusted to 6·5 using 5 M HCl. After autoclaving, the pH of the medium was readjusted to 6·5 at room temperature. H2/CO2 (80 : 20; 200 kPa) was used as the gas phase. Pure cultures were obtained and purified on the same medium solidified with 0·7 % (w/v) Phytagel (Sigma) and reduced with a titanium(III) citrate solution (Zehnder & Wuhrmann, 1976).
Pure cultures were obtained by streaking subcultures onto solidified culture medium. Pale-yellow, round colonies (1 mm in diameter) were visible on plates after 3 days at 80 °C in anaerobic jars under a H2/CO2 atmosphere (80 : 20; 300 kPa). Seven colonies were randomly picked and subcultured. Analysis of the RFLP profiles of the 16S rRNA genes showed that all the patterns were identical and were distinct from those of the described species of the genera Methanothermococcus, Methanocaldococcus and Methanotorris (Jeanthon et al., 1999b). One of the pure cultures, designated isolate SL43T, was selected for further characterization. Stock cultures of isolate SL43T were stored in culture medium at 4 °C. For long-term storage, pure cultures were stored at -80 °C in the same medium containing 10 % (w/v) DMSO.
Cells of strain SL43T were irregular cocci, about 1–3 µm in diameter depending on the growth phase (Fig. 1). The cells stained Gram-negative (Murray et al., 1994), occurred singly or in pairs and exhibited tumbling motility. As reported for Methanococcales (Whitman & Jeanthon, 2002), cells from the mid-exponential to late-exponential growth phase lysed rapidly in SDS (0·01 %) and in distilled water (Boone & Whitman, 1988). Ultrathin sections of cells, prepared as described previously (Sleytr et al., 1988), revealed a delicate, hexagonal S-layer with a lattice constant of approximately 13·1 nm. The S-layer could not be recognized clearly all over the cell surface, in particular, in areas of heavy flagellation. Negative staining revealed that flagellation was mainly restricted to one specific region of the cell body, where numerous flagella originated (Fig. 1).
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6·5) and 20 mM MES (for pH 6). Since they may vary with incubation temperature, the pH values of these media were checked at room temperature after overnight incubation of uninoculated tubes under H2/CO2 at 85 °C. Final OD600 values of cultures after 6–8 h incubation were used to determine the growth ranges and optima for temperature, pH and NaCl concentration for strain SL43T. The effects of pH and NaCl concentration were determined at the optimal temperature for growth. Under these conditions, strain SL43T grew between 50 and 86 °C, with optimum growth at around 85 °C; no growth was detected at 45 or 88 °C. Growth was observed between pH 5·5 and 6·7, with optimum growth at around pH 6·5. No growth was observed at pH 5·2 or 7. Growth occurred in NaCl concentrations ranging from 15 to 50 g l-1, with optimum growth at 30 g l-1. No growth was observed at 10 or 60 g NaCl l-1. Under the optimal conditions for growth, the doubling time of the novel organism was 25–30 min.
Strain SL43T is a strictly anaerobic, autotrophic organism. Growth was prevented in the presence of low levels of oxygen, and H2 and CO2 served as the only substrates for growth. No growth was observed on acetate (2 g l-1), formate (5 g l-1), methanol (0·5 %, v/v), monomethylamine (2 g l-1) or yeast extract (2 g l-1) with a N2/CO2 (80 : 20; 200 kPa) or H2 (100 %; 200 kPa) headspace. In the presence of H2 and CO2, methane production paralleled growth (Jeanthon et al., 1998). Ammonium (10 mM) was the preferred nitrogen source, but significant growth also occurred in the presence of yeast extract, tryptone, urea, glutamate (all at 2 g l-1) and nitrate (10 mM). When supplemented individually, selenate, tungstate and yeast extract stimulated the growth rate. When sulfur (5 g l-1) was added to the sulfate-free medium in the presence of CO2 and H2, growth occurred and H2S was produced. No dissimilatory reduction of cystine (5 g l-1), sulfate or thiosulfate (at 20 mM) was observed.
Sensitivity to antibiotics supplemented at 25, 50, 75, 100 and 200 µg ml-1 was tested in the culture medium at 80 °C. Strain SL43T was resistant to ampicillin, penicillin, streptomycin and kanamycin (200 µg ml-1) and was sensitive to chloramphenicol (75 µg ml-1) and rifampicin (25 µg ml-1).
DNA was isolated after disruption of cells using a French pressure cell (Thermo Spectronic) and purified by hydroxyapatite chromatography (Cashion et al., 1977). The DNA was hydrolysed with P1 nuclease and the nucleotides were dephosphorylated with bovine alkaline phosphatase (Mesbah et al., 1989). The G+C content of the DNA of strain SL43T was 30·7 mol%, as determined by the HPLC method (Tamaoka & Komagata, 1994), and is in the range reported for other members of the genus Methanocaldococcus (Whitman et al., 2001).
A total of 1409 nucleotides of the 16S rRNA gene were sequenced as described previously (Götz et al., 2002). Distance and maximum-likelihood trees (De Soete, 1983; Olsen et al., 1994) (1354 nucleotides were used) placed strain SL43T as a relative of Methanocaldococcus infernus MET (3·2 % distance), Methanocaldococcus jannaschii JAL-1T (4·1 % distance), Methanocaldococcus vulcanius M7T and Methanocaldococcus fervens AG86T (5·3 % distance) (Fig. 2). However, the level of 16S rDNA sequence similarity between strain SL43T and these organisms is lower than the limit (97 %) used to define distinct species at the DNA level without the requirement for DNA–DNA reassociation tests (Stackebrandt & Goebel, 1994). This significant phylogenetic distinctiveness was confirmed by RFLP analysis of the 16S rDNA (Jeanthon et al., 1999b), a reliable and useful method for distinguishing species of Methanocaldococcus from each other (Whitman et al., 2001).
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). Strain SL43T is distinct from other Methanocaldococcus species, and particularly from Methanocaldococcus infernus, its closest phylogenetic relative, in its maximum pH and temperature for growth and in the numbers of regions of insertion of flagella into the cell body. This distinctiveness was confirmed by comparing whole-cell protein patterns of strain SL43T with those of type strains of described Methanocaldococcus and Methanotorris species (Fig. 3). We propose that strain SL43T represents a novel Methanocaldococcus species, named Methanocaldococcus indicus sp. nov.
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Cells exhibit a tumbling motility by means of numerous flagella, predominantly inserted at one specific region of the cell body. They are cocci (1–3 µm in diameter), covered with a hexagonal S-layer lattice, and occur singly and in pairs. Cells lyse rapidly in SDS (0·01 %) and in distilled water. Pale-yellow, round colonies about 1 mm in diameter form on Phytagel plates. Growth occurs at between 50 and 86 °C, with an optimum at 85 °C, between pH 5·5 and 6·7, with the optimum at around pH 6·5, and between 15 and 50 g NaCl l-1, with an optimum of 30 g l-1. Obligately anaerobic. Chemolithotrophic. Uses H2 and CO2 as energy and carbon sources to produce methane. Growth is stimulated by selenate, tungstate and yeast extract. Sulfur is reduced to H2S in the presence of CO2 and H2. Growth is inhibited by chloramphenicol (75 µg ml-1) and rifampicin (25 µg ml-1) but not by streptomycin, penicillin G, kanamycin or ampicillin (200 µg ml-1). The DNA G+C content of the type strain is 30·7 mol% (as determined by HPLC). Distinguished from other Methanocaldococcus strains by comparison of whole-cell protein patterns.
The type strain, strain SL43T (=DSM 15027T=JCM 11886T), was obtained from a deep-sea hydrothermal vent chimney in the Kairei vent field on the Central Indian Ridge.
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ACKNOWLEDGEMENTS |
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