Isolation and Characterization of Extremely Thermophilic

Archaebacteria Related to the Genus Thermococcus from

Deep-Sea Hydrothermal Guaymas Basin
Abstract. During the "Guaynaut" oceanographic cruise performed by IFREMER in
November 1991, sediment cores were collected from high-temperature and
petroleum-rich deposits in an active hydrothermal zone, at the Guaymas basin
(Central gulf of California). Those samples were collected by the French deep-sea
manned submersible "Nautile" at a depth of 2000 meters. Four sediment cores of
20-40-cm length were drilled at the bottom of a block assemblage of active
smokers inside sediments whose temperatures were 3.5C on the top to 105C at
20 cm depth. They were subsampled in 22 slices of 5-cm thickness and used for
isolation of heterotrophic hyperthermophilic microorganisms, after inoculation in
sulfur-free SME liquid medium. From those enrichments 18 isolates were
obtained, 2 growing at 95C and 16 at 80C and their taxonomic characterization
was undertaken. Lipid analysis indicated the presence of diethers and tetraethers
in the cell walls and membranes, characteristic of Archaebacteria. Examinations
by scanning electron microscopy showed that isolates were cocci of
heterogeneous sizes (diameter from 0.1 to 0.5 micrometers) or thick, piled-up
discs 0.5 m thick and 1 m in diameter. Both forms were embedded in a dense
fiber network. Physiologically they were found to be anaerobic, heterotrophic, and
hyperthermophilic (80C 95C). Determination of the DNA base composition
resulted in G + C mol % values ranging from 36 to 57. Qualitative hybridizations
of the 18 isolate DNAs with hyperthermophilic Archaebacteria reference strain
DNAs showed that hybridizations occurred neither with the two species of
Pyrococcus nor with the two species of Desulfurococcus, nor with
Staphylothermus marinus. On the other hand, all isolates hybridized with at least
one of the three species of Thermococcus tested (T. celer, T. stetteri, T. litoralis).
Restriction polymorphism on a PCR-amplified fragment of the rrn operon showed
that 12 isolates had the same profile as T. celer and T. stetteri, 4 isolates had the
same profile as T. litoralis, and 2 had new profiles, suggestive that they are new
species.
Deep-sea hydrothermal vents at sea floor spreading centers display high
pressures, high temperatura gradients, and high concentrations of unusual
elements (heavy metals, sulfides). The vents are colonized by an exuberant
fauna of invertebrates and by heterotrophic microorganisms, supported by
autotrophic microorganisms whose chemosynthesis is the bottom of the food
web. The Guaymas basin vent site (Central gulf of California) differs from other
hydrothermal systems by the presence of a 300- to 400-m-thick sediment layer.
This rich organic matter layer is formed by a substantial sedimentation of
terrigenous and pelagic detritus. Chemical composition of the hydrothermal fluid
is affected by its passage through this sediment layer before it reaches the sea
floor. It differs from the East Pacific Rise (21C) hydrothermal fluid by a higher pH
(5.9), a strong alkalinity (3 = 11 mEq/L), a significant presence of ammonium (10
= 16 mM), a weaker concentration of reduced elements, and by the formation of
hydrocarbons. This creates a unique deep-sea hydrothermal environment by the
presence of organic-rich and highly reducing muds with temperatures ranging
from 3C up to 100C [19, 27].

During the "Guaynaut" oceanographic cruise, sediment cores were collected from
these hightemperature and petroleum-rich hydrothermal deposits by means of
tubular core samplers operated by the French submersible "Nautile". They were
used for the isolation of heterotrophic, hyperthermophilic, anaerobic
Archaebacteria. The aim of this work was to compare the Archaebacteria
obtained from those samples with the previously described deep-sea
hydrothermal environment Archaebacteria, and more precisely with
Archaebacteria described from the Guaymas basin. The discovery of new species
could be anticipated in order to extend the knowledge of that ecosystem
biodiversity.

In this paper we report the taxonomic characterization of the 18 isolates obtained

from hot sediments of the hydrothermal Guaymas basin, from morphological
observations, lipid analysis, and biomolecular properties.

Materials and Methods

Collection of samples. Four sediment cores, 20 = 40 cm length, of hot

sediments were sampled at the bottom of a block assemblage of active
smokers at a depth of 2000 m in the Guaymas basin. Temperatures inside
the sediment were in the range of 3.5C on the top to 105C at a 20-cm
depth as measured by a temperatura probe. The sediment cores were
subsampled in 22 5-cm-thick slices, immediately upon retrieval. They were
then transferred to sterile glass bottles, filled with sterile sea water
containing resazurine, sealed, reduced until colorless with Na2S 2.5%
(wt/vol), and finally stored at ambient temperature.

Enrichment and cultivation medium. Strict anaerobic procedures were

followed according to Balch and Wolfe [1]. Enrichment cultures and
cultivations were carried out in 20 ml penicillin-like sealed bottles containing
10 ml of slightly modified sulfur-free SME medium [2, 28]. The pH was
adjusted to 6 and buffered with Tris-HCl (2.5 M). The medium was
autoclaved and introduced, after cooling, into an anaerobic chamber where
oxygen was reduced by the addition of 20 ml (per liter) of a 2.5% (wt/vol)
solution of Na2S in distilled water. Final gas atmosphere was N2-H2-CO2
(90%:5%:5%). Incubation of the enrichment cultures was made both at 80C
and 95C and further pure isolate cultures were made at 80C or 95C
according to their isolation temperature.

Isolation. Enrichments were checked by microscopic observations of

filtered acridine orange-stained preparations. Positive enrichments were
then submitted to purification. Pure cultures of the isolates were obtained by
serial dilutions of the first enrichment cultures in SME medium; the cultures
were finally plated onto Gelrite solid medium plates. After incubation of the
plates, each isolated colony was individually transferred and grown in liquid
SME medium, then plated again. This purification step was repeated two or
three times. Purity of the isolates was checked on the basis of microscopic
examination and the presence of a single colony type after repeated
transfers in liquid and onto solid medium. The pure isolate was transferred
and stored in liquid medium at room temperature.

Microscopy. Cells from pure cultures were observed with an BH2-Olympus

microscope equipped with a vertical illuminator after acridine orange
staining [11]. Scanning electron microscopy was also performed on a
Cambridge S 100 electron microscope (Cambridge, UK).
DNA extraction and base composition. DNA of harvested cells was
extracted according to the procedure of Marmur [20]. G + C mol % was
determined by thermo-denaturation according to Marmur and Doty [18, 21].

Lipid analysis. Lipids from lyophitized cells (100 rag) were extracted by a
modified Bligh-Dyer method [3, 30]. The lipidextracted residue was
recovered from the aqueous phase. The lipids were fractionated into neutral
lipids, glycolipids, and polar lipids by silicic acid column chromatography
with appropriate volumes of chloroform, acetone, and methanol respectively.
The fractions were dried under vacuum and stored under N2 until further
analysis. The polar lipids and residual fractions were treated with a strong
acid methanolysis to cleave the ether lipids from phosphate or saccharide
head groups or from the cell walls.
Alkyl iodides were prepared according to the procedures detaiied elsewhere
[I5, I7]. Authentic samples of C20 diether and C40 tetraether were prepared
from Sulfolobus acidophilus, and 1,2 di-O-hexadecylglycerol (Sigma) was
used as internal standard.

Comparison with reference strains. Archaebacteria reference strains

had been obtained from the German collection of microorganisms and ceils
cultures (DSM). They were chosen according to their taxonomic proximity
with our isolates and their ability to grow under the same conditions. They
were Pyrococcus furiosus, DSM 3638 [9]; Pyrococcus woesei, DSM 3773
[32]; Staphylothe~rnus magnus, DSM 3639 and DSM 3666 [10];
Thermococcus celer, DSM 2476 [31]; Thermococcus litoralis DSM 5473 [24];
and Thetznococcus stetteri, DSM 5262 [23]. To identify possible relatives, we
probed DNA of the Guaymas isolates against DNA of those known marine
hyperthermophiles by dot-blot hybridizations [12, 26]. Afterwards, restriction
polymorphism on a PCR-amplified fragment of the rrn operon [22] was
performed, a different profile corresponding to different species of
Thermococcales, as confirmed by the 16S rRNA sequences and quantitative
DNA hybridizations [22].

Results
Enrichment and isolation. Incubation of the 22 sample enrichments were
made in SME liquid medium, as defined previously. Temperatures of
incubation were 80C and 95C Eight samples gave positive enrichment at
80C, and 2 at 95C. From those positive enrichments, 16 isolates were
obtained at 80C (GY 857, GY 858, GY 859, GY 860, GY 861, GY 862, GY 863,
GY 864, GY 865, GY 866, GY 867, GY 868, GY 869, GY 870, GY 872, GY 873)
and 2 at 95C (GY 871, GY 874). No correlation was observed between the
presence of isolates and the depth in the sediment (i.e., the temperature in
the sediment).
 Fig. 1. Scanning electron
photomicrographs of the
Guaymas Basin isolates. (a)
Cells of GY 859 strung in a
tubular sheath. Bar
represents 5 m. (b) Cells of
GY 863 showing size
heterogeneity and the fiber
network. Bar represents
2 m.

Morphology. Examinations of the acridine orangestained cells showed that

they were irregular cocci arranged in necklaces, the general effect looking
like a mosaic on the filters. It seemed that the cocci were strung in a tubular
sheath. Scanning electron microscopy confirmed this examination. All the
isolates appeared embedded in a dense fiber network, this network being
tubular sheaths including the cocci, as seen on Fig. 1. This sheath could be
retracted owing to the mode of desiccation of the sample and looked like a
string holding the cocci. Two ranges of sizes have been observed. The
majority of the isolates had a homogeneous size of 1 m in diameter. The
other cocci had very heterogeneous sizes from 0.1 to 0.5 m in diameter.

DNA base composition. Determination ot the DNA base composition of

hyperthermophilic isolates resulted in G + C mol% values ranging from 36 to
57 (Fig. 2). Three main groups could be identified: a group of 12 isolates
with a G + C mol% ranging from 55 to 57 (857, 858, 859, 860, 861, 862,
864, 865, 866, 867, 870, 873), a group of 4 isolates with a G + C mol% of 42
= 43 (863, 871, 872, 874), and 2 isolates with a low G + C tool% (36 and
38) (868, 869).

Lipid analysis. The lipid composition of nine isolates was studied: six from
the high G + C mol% group (859, 861,862, 864, 867, 870), one from the
mdium group (863), and the two isolates from the lowest group (868 and
869). The presence of ether lipids indicated that those isolates belong to the
Archaebacteria. The ether Iipids found in the different isolates were the
glycerol diether (DE), glycerol tetraether (TE), and tetraethers modified by
formation of pentacyclic moieties. For all isolates analyzed in this study,
more lipids were found to be present in the residual phase than in the polar
phase. Very small amounts of ether lipids were determined in the glycolipid
fraction.
The six isolates of the high G + C mol% group were characterized by large
amounts (up to 80% of the total ether lipids) of 2,3 di-O-phytanyl glicerol
(C20) in the dichloromethane-soluble phase. Other isolates (863, 868, 869)
showed the same gtyceroI ether as the predominant lipid, but significant
amounts of C40 di-biphytanyl ethers were also analyzed. Conversely, the
residual phase of all isolates demonstrated large amounts of tetraethers
including tetraethers modified with pentacyclic moities along with unknown
ether lipids. All isolates exhibited in the residual phase the same unknown
component, tentatively identified as a C30 ether.

Dot-blot hybridizations. Dot-blot hybridizations of the 18 isolate DNAs

with hyperthermophilic Archaebacteria reference strain DNAs were
performed. Hybridizations occurred neither with the two species of
Pyrococcus nor with the two species of Desulfurococcus, nor with
Staphylothermus marinus. On the other hand, all the isolates hybridized
with at least one of the three species of Thermococcus, and consequently it
may be assumed that they belong to this genus [12].
The intensity of the blots was assessed from 0 for no hybridization (blank on
the paper) to 4 for strong hybridization (high black blot of the probe versus
itself giving the 100% hybridization). A nonparametric scale was created: 0
for no hybridization, I for very low hybridization, 2 for low hybridization, 3 for
medium hybridization, and 4 for strong hybridization. A three-dimensional
representation of those estimated values in an axe system corresponding to
the three reference strains of the genus Thermococcus is represented in Fig.
3. Isolates clustered in three groups similar to the G + C mol% defined
groups:

The high G + C mol% group (55 - 57) is very close to T. celer and
T. stetteri, whose G + C mol% are respectively 56.6 and 50.2.
The intermediary G + C mol% group (42 = 43) is closest to T.
litoralis, whose G + C mol% is 38.
Two isolates hybridize only and very weakly with T. litoralis. Their
G + C mol% are 36 and 38.
RFLP/PCR 16S:23S. Restriction polymorphism analysis on a PCR-amplified
fragment of the rrn operon was performed. Figure 4 reports profiles obtained
for the Guaymas isolates and for reference strains of Thermococcales (P.
woesei, Pyrococcus abyssi, T. stetteri, T. litoralis, T. celer).
Profiles obtained for the Guaymas hyperthermophilic isolates showed that
the same group of 12 isolates that clustered close to T. celer and T. stetteri
by dot-blot hybridizations and by their G + C mol% base composition
effectively had the same profile as T. celer and T. stetteri. The 4 isolates
closest to T. litoralis effectively had the same profile that T. litoralis. The last
two isolates showing a low G + C mol% and a different response to the
hybridization with the Thermococcus genus effectively had a new profile. It
can be hypothesized that they are a new species, probably of the
Thermococcus genus.

Fig. 2. DNA base composition of the hyperthermophilic isolates

Fig. 3. Semi-quantitative dot-blot hybridizations with reference
strains. 0, no hybridization; 1, very low hybridization; 2, low
hybridization; 3, mdium hybridization; 4, strong hybridization.
Discussion
Microbial diversity of deep-sea hydrotherrnal vents has been a topic of
interest since the discovery of those ecosystems in 1977 [6]. Research
expeditions during the last 15 years have provided the description of many
new species of hyperthermophilic Archaebacteria coming from these
environments. They belong to a previously known genus primarily
discovered in terrestrial aquatic sources such as hot springs or shallow
marine hydrothermal environments, like the chemolithotrophic methanogen
Methanococcus jannaschii [14], the sulfate reducer Archeoglobus profundus
[4], or heterotrophic sulfur-dependent Archaebacteria like Desulfurococcus
[13], Pyrodictium abyssi [25], and Pyrococcus abyssi [8]. A new genus of the
hyperthermophilic methanogen, Methanopyrus kandleri [16], and a new
genus of sulfur-dependent Archaebacteria, Staphylothermus marinus [10],
have also been described.
 Fig. 4. RFLP/PCR
16S:23S analyzed
by agarose (4%) gel
electrophoresis.

In this paper,
characterization
of 18
hyperthermophilic heterotroph Archaebacteria isolated from the Guaymas
Basin hydrothermal vent sediments were presented. Those 18 isolates
appeared to be closely related to the Thermococcus genus, as indicated by
cell morphology, metabolism, DNA base compositions, lipid compositions,
qualitative hybridizations, and RFLP/PCR 16S:23S.
On the basis of qualitative dot-blot hybridizations and RFLP/PCR 16S:23S,
three groups were distinguished: a group of 12 isolates similar to T. celer-T.
stetteri species, a group of 4 isolates similar to T. litoralis species, and a new
group of 2 isolates.

The lipid profiles of all isolates were characterized by the presence of ether
lipids with the diphytanyl glycerol ether (C20) predominating in the polar
phase and tetraethers in the residual phase. Regarding the
dichloromethane-soluble phase, two main lipid composition groups were
distinguished: the first one corresponding to the 12 isolates similar to T.
celer-T, stetteri, characterized by large amounts of C20, and the second one,
regrouping to the two other groups, with higher amounts of tetraethers. In
addition, the residual phase of all isolates contained large amounts of
tetraethers with significant amounts of pentacyclic modified tetraethers in
the second lipid composition group.
For the 12 isolates related to T. celer-T, stetteri, their G + C mol% (55 = 57)
permitted relating them more precisely to T. celer, whose G + C mol% is
56.6, instead of T. stetteri, whose G + C mol% is 50.2. The fact that they
could grow without elemental sulfur as T. celer, when T. stetteri was unable
to grow without this element, confirmed this hypothesis. Furthermore, the
lipid profiles of this group were in agreement with that of T. celer [7]. In fact,
their lipid profiles, like those of T. celer, showed the 2,3-di-Ophytanyl- sn-
glycerol ester of myo-inositol phosphate, the major ether lipid of that sulfur-
dependent Archaebacteria. However, the physiological properties of this
group were slightly different from those of T. celer, (T opt. and pH opt. being
respectively 80C 85C and 6.5-7.5 versus 88C and 5.8 for T. celer; data
not shown), suggesting they might be different species.
For the four isolates related to T. litoralis by qualitative dot-blot
hybridizations and RFLP/PCR 16S:23S, the G + C mol% (42 - 43) were not in
agreement with the G + C mol% of T. litoralis (38), and unfortunately, to our
best knowledge, no data are available on the lipids of this species. This
group also might be different species.
The latter two isolates were very different on the basis of RFLP profiles and
the fact that they hybridize only and very weakly with T. litoralis. Although
they had a G + C mol% of 36-38, close to the G + C mol% of T. litoralis, the
probability of this group representing a new species of Thermococcales was
very high.
In conclusion, the taxonomic characterization of the 18 isolates from
Guaymas Basin hot sediments indicated clearly that they belong to the
Thermococcus genus, with a strong homology to T. celer or T. litoralis for 16
isolates and strong arguments for a new species of Thermococcales for the
quite different later group of two isolates (also probably close to the
Thermococcus genus). The exact phylogenetic position of all isolates awaits
16S rRNA sequence analysis and quantitative DNA/DNA hybridizations.
Up to now the hyperthermophilic Archaebacteria described for the Guaymas
Basin include the methanogen M. kandleri, the sulfate reducerA.profundus,
and the heterotrophic sulfur-dependent Pyrodictium abyssii. Recently a
thermophilic methanogen similar to Methanococcus and two thermophilic
heterotrophs related to Desulfurococcus have also been reported from this
site [5]. This study showed that there is now evidence for the presence of
relatives of the Thermococcus genus in this site. No species description is
planned until further experiments based on quantitative DNA/DNA
hybridizations and 16S rRNA sequence analysis are completed to give the
exact phylogenetic position of the Guaymas new Thermococcus.

The novelty of those results is that at this time the genus Thermococcus is
very poorly mentioned in the hydrothermal deep-sea environment. The only
observation of deep-sea hydrothermal Thermococcus related up to now is T.
profundus [29]. The knowledge about the biodiversity and the ecology of
the hydrothermal sites is still incomplete, and the discovery and
characterization of novel microorganisms from such environments provides
information regarding the ubiquity and distribution of such organisms in
deepsea hydrothermal vent systems.
ACKNOWLEDGMENTS
The authors thank the chief scientist A.M. Alayse and the crews of the N.O.
NADIR and of the submersible NAUTILE for organization of the cruise and
collection of samples. We thank also IFREMER and REGION BRETAGNE who
supported this work.