International Journal of Systematic and Evolutionary Microbiology (2003), 53, 847–851
DOI 10.1099/ijs.0.02503-0
Thermococcus gammatolerans sp. nov., a
hyperthermophilic archaeon from a deep-sea
hydrothermal vent that resists ionizing radiation
Edmond Jolivet,13 Stéphane L’Haridon,1 Erwan Corre,2 Patrick Forterre3
and Daniel Prieur1
Correspondence
Edmond Jolivet
ejolivet@lsu.edu
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
Enrichments for anaerobic organotrophic hyperthermophiles were performed with hydrothermal
chimney samples collected at the Guaymas Basin (27˚ 019 N, 111˚ 249 W). Positive enrichments
were submitted to c-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 l21. 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).
All members of the archaeal order Thermococcales are
strictly anaerobic hyperthermophiles. The Thermococcaceae,
the single family of this order, is composed of three genera,
Thermococcus (Zillig et al., 1983), 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 uC for
Thermococcus and Palaeococcus species and between 96
and 100 uC for Pyrococcus members) and the clustering of
their 16S rRNA sequences as separate clades within the
Published online ahead of print on 29 November 2002 as DOI
10.1099/ijs.0.02503-0.
3Present address: Dept of Biological Sciences, Louisiana State
University and A & M College, Baton Rouge, LA 70803, USA.
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.
02503 G 2003 IUMS
Printed in Great Britain
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
847
E. Jolivet and others
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 c-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
(27u 019 N, 111u 249 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 uC 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 uC. 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 %.
The positive enrichments obtained after 2 days incubation
consisted of irregular motile and nonmotile coccoid cells.
Aliquots of these cultures were irradiated at 30 kGy on ice
with a c-ray source (137Cs) at a rate of 60 Gy min21 (Institut
Curie, Orsay, France). After irradiation, cultures were
transferred in YPS medium and incubated at 85 uC for
3 days. Creamy colonies were obtained on YPS medium
solidified with 1 % (w/v) gelrite and incubated in an
anaerobic jar at 80 uC (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 c-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
Table 1. Characteristics that distinguish strain EJ3T from its closest phylogenetic relatives
+, Positive; 2, negative;
Property
ND,
not determined;
NR,
not reported; R, required; S, stimulatory.
Thermococcus
celer*
Thermococcus
profundusD
Thermococcus
hydrothermalisd
Thermococcus
guaymasensis§
Thermococcus
gorgonarius||
Strain EJ3T
+
+
+
2
+
+
+
2
+
+
+
R
+
ND
2
2
2
Weak
R
ND
2
2
2
2
2
R
2
Mobility
Energy substrate
Casein
Amino acids
Starch
Maltose
Pyruvate
Sulfur requirement
Rifampicin resistance
Growth temp. (uC)
Range
Optimum
NaCl concn (g l21)
Range
Optimum
pH
Range
Optimum
G+C content (mol%)
+
+
ND
ND
ND
+
S
+
+
+
2
R
2
+
+
2
2
+
S
+
¡93
88
50–90
80
55–100
85
56–90
88
68–95
80–88
55–95
88
ND
10–60
20
20–80
30–40
ND
18
10–50
20–35
10–40
20
4?4–8?5
7?5
52?2
3?5–9?5
6?0
58
5?6–8?1
7?2
46
5?8–8?5
6?5–7?2
50?6
4–8?5
6?0
51?3
ND
40
NR
5?8
57
*Zillig et al. (1983).
DKobayashi et al. (1994).
dGodfroy et al. (1997).
§Canganella et al. (1998).
||Miroshnichenko et al. (1998).
848
International Journal of Systematic and Evolutionary Microbiology 53
Thermococcus gammatolerans sp. nov.
The diameter of the cells ranged from 0?6 to 1?4 mm and
remained relatively constant around 1 mm under optimal
growth conditions. Cells appeared to divide by constriction.
Fig. 1. Gamma-radiation survival curves. The new isolate, EJ3T
(%), 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’ (m)
and Deinococcus radiodurans (X) were taken from Kopylov
et al. (1993), Battista (1997) and Gérard et al. (2001).
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).
Cells of strain EJ3T formed regular cocci occurring singly or
in pairs. They were motile by means of polar flagella (Fig. 2).
Fig. 2. Electron micrograph of a negatively stained cell of
strain EJ3T prepared as previously described (L’Haridon et al.,
1998). Bar, 1 mm.
http://ijs.sgmjournals.org
Unless otherwise stated, YPS medium was used for growth
experiments. The optimal pH for growth was determined
at 85 uC as described by Marteinsson et al. (1999). 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 uC and the optimum temperature for growth was
88 uC. No growth was detected at 50 and 96 uC. 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 l21. No growth was detected at NaCl concentrations of 0 and 40 g l21. 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 l21) (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,
Su 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 mg ml21, 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 uC (Huber et al., 1986).
849
E. Jolivet and others
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: 59-TCC GGT TGA TCC TGC CGG-39) and one
universal (1492R primer: 59-GGT TAC CTT GTT ACG ACT
T-39). PCR reactions were typically carried out in a volume
of 50 ml containing 50–100 ng template, 100 ng of each of
the two specific primers, 250 mM dNTP, 1±5 mM MgCl2,
16 buffer (Promega) and 2?5 U polymerase. The different
steps of PCR were as follows: 5 min at 95 uC; then 25 cycles
of 1?5 min at 95 uC, 1?5 min at 53 uC and 2?5 min at 72 uC;
then finally a polymerization step of 8 min at 72 uC. 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 %.
Considering the high levels of similarity (more than 98 %)
existing between strain EJ3T and Thermococcus gorgonarius,
Thermococcus celer, Thermococcus guaymasensis, Thermococcusprofundus and Thermococcus hydrothermalis, quantitative DNA–DNA hybridizations between the isolate and its
closest relatives were performed as described by Jeanthon
et al. (1998). 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 c-irradiation.
Description of Thermococcus gammatolerans
sp. nov.
Thermococcus gammatolerans (ga.mma.to9le.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 c-rays).
Cells are cocci (diameter 0?6–1?4 mm) that are motile by the
presence of polar flagella. Cell division occurs by constriction. Obligately anaerobic. Growth occurs at 55–95 uC, and
the optimum temperature is 88 uC. Grows optimally in the
presence of 20 g NaCl l21 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,
Fig. 3. Phylogenetic position of strain EJ3T
(in boldface) amongst some representatives
of the family Thermococcaceae; 1320 nucleotides were used in the phylogenetic analysis.
Numbers after the strain names are
GenBank accession numbers of 16S rDNA
sequences. The topology shown is the tree
obtained using the neighbour-joining method
(Jukes and Cantor distance correction).
Numbers at the nodes refer to the bootstrap
values (100 replicates) in distance, maximumlikelihood and maximum-parsimony analyses,
respectively. Bootstrap values below 50 %
were not represented or represented by
dashes. The scale bar represents the expected
number of changes per sequence position.
850
International Journal of Systematic and Evolutionary Microbiology 53
Thermococcus gammatolerans sp. nov.
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 mg ml21, but sensitive to 150 mg rifampicin ml21.
The results of 16S rDNA sequence comparisons place
Thermococcus gammatolerans in the Thermococcales.
T
T
T
The type strain, EJ3 (=DSM 15229 =JCM 11827 ), was
isolated from an active chimney recovered from a hydrothermal site in Guaymas Basin (27u 019 N and 111u 249 W)
at a depth of 2616 m.
Acknowledgements
We thank Dr Vincent Favaudon for the use of the 137Cs c-ray source
(Institut Curie, Orsay, France). We also thank Dr Christian Jeanthon
for critical reading of the manuscript and for useful discussions.
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