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1 UMR 6539, Centre National de la Recherche Scientifique et Université de Bretagne Occidentale, Technopôle Brest-Iroise, Place Nicolas Copernic, 29280 Plouzané, France
2 UMR 7127, Station Biologique, CNRS et Université Pierre et Marie Curie, Place Georges Teissier, 29682 Roscoff Cedex, France
3 UMR 8621, Institut de Génétique et Microbiologie, CNRS et Université Paris-Sud, Bât 409, 91405 Orsay Cedex, France
Correspondence
Edmond Jolivet
ejolivet{at}lsu.edu
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-irradiation at a dose of 30 kGy. One of the resistant strains, designated strain EJ3T, formed regular motile cocci. The new strain grew between 55 and 95 °C, with an optimum growth temperature of 88 °C. The optimal pH for growth was 6·0, and the optimum NaCl concentration for growth was around 20 g l-1. Strain EJ3T was an obligately anaerobic heterotroph that utilized yeast extract, tryptone and peptone. Elemental sulfur or cystine was required for growth and reduced to hydrogen sulfide. The G+C content of the genomic DNA was 51·3 mol%. As determined by 16S rRNA gene sequence analysis, the organism was most closely related to Thermococcus celer, Thermococcus guaymasensis, Thermococcus hydrothermalis, Thermococcus profundus and Thermococcus gorgonarius. However, no significant homology was observed between them by DNA–DNA hybridization. The novel organism also possessed phenotypic traits that differ from those of its closest phylogenetic relatives. Therefore, it is proposed that this isolate, which constitutes the most radioresistant hyperthermophilic archaeon known to date, should be described as the type strain of a novel species, Thermococcus gammatolerans sp. nov. The type strain is EJ3T (=DSM 15229T=JCM 11827T).
The GenBank accession number for the 16S rRNA sequence of Thermococcus gammatolerans strain EJ3T is AF479014.
Data on the effect of temperature, pH and NaCl concentration on EJ3T are available as supplementary material in IJSEM Online.
Present address: Dept of Biological Sciences, Louisiana State University and A & M College, Baton Rouge, LA 70803, USA. 
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), Pyrococcus (Fiala & Stetter, 1986) and Palaeococcus (Takai et al., 2000). The main characteristics that distinguish these genera are their optimal growth temperature (between 75 and 88 °C for Thermococcus and Palaeococcus species and between 96 and 100 °C for Pyrococcus members) and the clustering of their 16S rRNA sequences as separate clades within the Thermococcales (Zillig & Reysenbach, 2001). The genus Thermococcus includes at present 20 species that share similar physiological characteristics and can be divided into two groups on the basis of their G+C content. Members of this genus grow heterotrophically by fermentation or sulfur respiration on a variety of organic compounds such as peptone, yeast extract, meat extract, casein, peptides, Casamino acids and starch. Some of them are able to grow in the absence of elemental sulfur, but this compound significantly stimulates their growth. Representative species of Thermococcus are widely distributed at deep-sea and shallow marine hydrothermal vents and have also been isolated from terrestrial thermal springs in New Zealand and deep oil reservoirs (Miroshnichenko et al., 2001; Zillig & Reysenbach, 2001).
In the deep-sea hydrothermal environments of the East Pacific Rise, the polychaete Alvinella colonizes the walls of active chimneys and is exposed to natural radioactivity levels (210Pb, 210Po, 222Rn) a hundred times higher than received on the surface of the Earth (Cherry et al., 1992). Deep-sea hydrothermal vents could therefore represent an attractive milieu for studying the effects of ionizing radiation on thermophilic micro-organisms.
In this paper, a deep-sea hydrothermal vent chimney collected at the Guaymas Basin was used to isolate and characterize a novel Thermococcus species that resists high levels (30 kGy) of -irradiation.
The new organism was isolated from chimney samples collected by the submersible Nautile during the cruise ‘Guaynaut’ in 1991 in the Guaymas Basin [Gulf of California (27° 01' N, 111° 24' W)] at a depth of 2616 m. Samples were immediately transferred into flasks filled with sterile reduced artificial seawater. The vials were then closed tightly with butyl rubber stoppers and stored at 4 °C until used for further experiments.
Anaerobic procedures were performed as described by Balch & Wolfe (1976). Enrichment cultures were performed anaerobically in Hungate tubes containing 10 ml YPS medium and incubated at 85 °C. The same conditions were used to cultivate routinely the reference strains and the new isolate (Table 1). The YPS medium contained per litre of distilled water: 35 g Sea Salts (Sigma), 3·46 g PIPES, 1 g yeast extract, 4 g peptone, 5 g elemental sulfur, 0·5 g NH4Cl, 0·35 g KH2PO4, 0·2 g CaCl2, 6·7 mg FeCl3, 2·9 mg Na2WO4 and 0·1 mg resazurin. The pH was adjusted to 6·8 before autoclaving. Final anaerobiosis was achieved by adding sterile 5 % (w/v) Na2S.9H2O to a final concentration of 0·025 %.
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-ray source (137Cs) at a rate of 60 Gy min-1 (Institut Curie, Orsay, France). After irradiation, cultures were transferred in YPS medium and incubated at 85 °C for 3 days. Creamy colonies were obtained on YPS medium solidified with 1 % (w/v) gelrite and incubated in an anaerobic jar at 80 °C (gas phase N2/CO2, 80 : 20, 1 bar) (Erauso et al., 1995). One colony was randomly picked and streaked on YPS-gelrite plates four times successively. The purity of the isolate (designated EJ3T) was checked microscopically by a serial dilution step.
After purification, the survival rate to -irradiation of isolate EJ3T was evaluated and compared to that of ‘Pyrococcus abyssi’ GE5T and Thermococcus stetteri DSM 5262T. After irradiation at increasing doses, the surviving fraction was enumerated by the most probable number technique. The new isolate was found to resist 3 kGy without loss of cultivatability (Fig. 1). Contrary to ‘P. abyssi’ and Thermococcus stetteri, its survival curve was close to that determined for Deinococcus radiodurans (Battista, 1997). Like D. radiodurans, a fraction of an end-exponential culture of the new isolate was able to grow after irradiation at 30 kGy. When tested for this ability, cells of ‘P. abyssi’ and Thermococcus stetteri could not be cultivated after irradiation doses exceeding 11 and 18 kGy, respectively (data not shown).
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). The diameter of the cells ranged from 0·6 to 1·4 µm and remained relatively constant around 1 µm under optimal growth conditions. Cells appeared to divide by constriction.
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. To determine the optimal NaCl concentration for growth, increasing concentrations of NaCl were added to a medium that contained per litre of distilled water: 10·77 g MgCl2.6H2O, 3·97 g Na2SO4, 0·20 g NaHCO3, 0·09 g KBr, 0·025 SrCl2.6H2O, 0·671 g KCl, 0·26 g H3BO3, 0·003 g NaF, 3·46 g PIPES, 1 g yeast extract, 4 g peptone, 5 g elemental sulfur, 0·5 g NH4Cl, 0·35 g KH2PO4, 0·2 g CaCl2, 6·7 mg FeCl3, 2·9 mg Na2WO4 and 0·1 mg resazurin. The pH was adjusted to 6·8 before autoclaving. Final anaerobiosis was achieved by adding sterile 5 % (w/v) Na2S.9H2O to a final concentration of 0·025 %. All the experiments were performed in duplicate. Under these conditions, isolate EJ3T grew between 55 and 95 °C and the optimum temperature for growth was 88 °C. No growth was detected at 50 and 96 °C. The optimum pH was between 5·5 and 6·5. No growth occurred at pH 3·0 and 8·5. The optimum NaCl concentration was 20 g l-1. No growth was detected at NaCl concentrations of 0 and 40 g l-1. Under optimal growth conditions (temperature, pH and NaCl), the doubling time of the isolate was around 95 min. See the supplementary data available in IJSEM Online at http://ijs.sgmjournals.org
The ability of the isolate to use single carbon sources for growth was tested at optimal growth temperature on YPS medium in which yeast extract and peptone were omitted. A filter-sterilized solution of vitamins (10 ml l-1) (Widdel & Bak, 1992) was added and N2 was used as headspace. Since no growth was observed in aerated conditions and in mineral medium supplemented with vitamins and a H2/CO2 (80 : 20) headspace, strain EJ3T appeared to be an obligately anaerobic organotroph. Under anaerobic conditions, S° and cystine were necessary for growth and reduced to hydrogen sulfide. No growth was detected in the presence of thiosulfate (10 mM), sulfate (20 mM) or sulfite (10 mM). Significant growth was observed on yeast extract, peptone and tryptone (all at 0·2 %, w/v). Strain EJ3T was not able to grow on a mixture of 20 amino acids. No growth was observed on Casamino acids, acetate, succinate, propionate, pyruvate (all at 0·2 %, w/v), vitamins, gelatin (0·5 %, w/v), sucrose, cellobiose, lactose, maltose, glycogen, xylose or starch (all at 0·5 %, w/v). No growth was observed in the basal medium using H2/CO2 (80 : 20; 200 kPa) as headspace.
Isolate EJ3T was resistant to chloramphenicol, ampicillin, penicillin, kanamycin, vancomycin and streptomycin at a concentration of 150 µg ml-1, but this isolate was sensitive to rifampicin at the same concentration. Thermotoga maritima, used as control, exhibited the expected pattern of antibiotic susceptibility at 80 °C (Huber et al., 1986).
The G+C content of the DNA of isolate EJ3T determined by the thermal denaturation method as described by Jeanthon et al. (1998) was 51·3 mol%.
16S rDNA was amplified by PCR with Taq polymerase (Promega), using the genomic DNA from strain EJ3T as template and two primers: one specific for archaea (4F primer: 5'-TCC GGT TGA TCC TGC CGG-3') and one universal (1492R primer: 5'-GGT TAC CTT GTT ACG ACT T-3'). PCR reactions were typically carried out in a volume of 50 µl containing 50–100 ng template, 100 ng of each of the two specific primers, 250 µM dNTP, 1±5 mM MgCl2, 1x buffer (Promega) and 2·5 U polymerase. The different steps of PCR were as follows: 5 min at 95 °C; then 25 cycles of 1·5 min at 95 °C, 1·5 min at 53 °C and 2·5 min at 72 °C; then finally a polymerization step of 8 min at 72 °C. PCR products were cloned in vector PCRII2.1 and several clones were sequenced to ensure the sequence quality, using Texas-red-labelled primers, a Thermosequenase kit (RPN 2444; Amersham) and a Vistra 725 automated sequencer. Phylogenetic analysis of the 16S rDNA gene sequence was realized as described by Corre et al. (2001). The sequence of strain EJ3T has been deposited in the GenBank database under accession number AF479014.
The 16S rDNA sequence analysis placed strain EJ3T within the genus Thermococcus (Fig. 3). The highest levels of similarity between the 16S rDNA sequence of EJ3T and those of other Thermococcus species were as follows: Thermococcus gorgonarius, 98·9 %; Thermococcus celer and Thermococcus guaymasensis, 98·4 %; Thermococcus profundus, 98·3 %; and Thermococcus hydrothermalis, 97·6 %, Pyrococcus furiosus, 96·4 %, and Palaeococcus ferrophilus, 94·0 %.
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. When strain EJ3T was used as the labelled probe, the levels of DNA reassociation were as follows: Thermococcus gorgonarius, 18·3 %; Thermococcus celer, 19·2 %; Thermococcus guaymasensis, 23·2 %; Thermococcus profundus, 19·5 %; and Thermococcus hydrothermalis, 22·1 %.
When a number of different taxonomic parameters were compared, strain EJ3T differed from its closest phylogenetic relatives (Table 1
). It differs from most of them by its temperature range for growth and its inability to grow on casein and pyruvate. Moreover, strain EJ3T differs strongly from Thermococcus celer, Thermococcus hydrothermalis and Thermococcus guaymasensis in its G+C content and its rifampicin sensitivity, and from Thermococcus gorgonarius in its salinity range and its optimum pH for growth. Finally, it can be distinguished from Thermococcus profundus by its salinity range and its inability to use starch and maltose.
On the basis of its phenotypical and genetic characteristics, strain EJ3T represents a novel species within the genus Thermococcus. We propose to name it Thermococcus gammatolerans according to its high degree of tolerance to -irradiation.
Description of Thermococcus gammatolerans sp. nov.
Thermococcus gammatolerans (ga.mma.to'le.rans. gamma referring to gamma rays used as selection pressure for isolation; L. pres. part. tolerans tolerating; N.L. adj. gammatolerans referring to its ability to tolerate high levels of -rays).
Cells are cocci (diameter 0·6–1·4 µm) that are motile by the presence of polar flagella. Cell division occurs by constriction. Obligately anaerobic. Growth occurs at 55–95 °C, and the optimum temperature is 88 °C. Grows optimally in the presence of 20 g NaCl l-1 and at pH around 6·0. Obligately organotrophic. Grows preferentially on proteolysis products such as yeast extract, tryptone and peptone. Does not grow on Casamino acids, acetate, succinate, propionate, pyruvate, gelatin, glucose, maltose or starch. Sulfur or cystine are necessary for growth and reduced to hydrogen sulfide. Thiosulfate, sulfate and sulfite are not used as electron acceptors. Resistant to chloramphenicol, ampicillin, penicillin, kanamycin, vancomycin and streptomycin at 150 µg ml-1, but sensitive to 150 µg rifampicin ml-1. The results of 16S rDNA sequence comparisons place Thermococcus gammatolerans in the Thermococcales.
The type strain, EJ3T (=DSM 15229T=JCM 11827T), was isolated from an active chimney recovered from a hydrothermal site in Guaymas Basin (27° 01' N and 111° 24' W) at a depth of 2616 m.
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ACKNOWLEDGEMENTS |
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-ray source (Institut Curie, Orsay, France). We also thank Dr Christian Jeanthon for critical reading of the manuscript and for useful discussions. |
REFERENCES |
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), was irradiated at the end of the exponential growth phase in YPS growth medium under anaerobic conditions. These values are a mean of two independent experiments. Survival curves of Thermococcus stetteri (
), ‘Pyrococcus abyssi’ (
) and Deinococcus radiodurans (
) were taken from Kopylov et al. (1993)
