International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1331–1339
DOI : 10.1099/ijs.0.02068-0
Marinitoga piezophila sp. nov., a rod-shaped,
thermo-piezophilic bacterium isolated under
high hydrostatic pressure from a deep-sea
hydrothermal vent
1
2
3
UMR 6539, Centre National
de la Recherche
Scientifique et Universite!
de Bretagne Occidentale,
Institut Universitaire
Europe! en de la Mer, Place
Nicolas Copernic, 29280
Plouzane! , France
Prokaria, Gylfaflot 5, IS-112
Reykjavik, Iceland
Institute of Microbiology,
Russian Academy of
Sciences, Prospect 60-letiya
Oktyabrya 7/2, 117811
Moscow, Russia
Karine Alain,1† Viggo! Tho! r Marteinsson,2 Margarita L. Miroshnichenko,3
Elisaveta A. Bonch-Osmolovskaya,3 Daniel Prieur1 and Jean-Louis Birrien1
Author for correspondence : Jean-Louis Birrien. Tel : j33 298 498 751. Fax : j33 298 498 705.
e-mail : birrien!univ-brest.fr
A thermophilic, anaerobic, piezophilic, chemo-organotrophic sulfur-reducing
bacterium, designated as KA3T, was isolated from a deep-sea hydrothermal
chimney sample collected at a depth of 2630 m on the East-Pacific Rise (13S N).
When grown under elevated hydrostatic pressure, the cells are rod-shaped
with a sheath-like outer structure, motile, have a mean length of 1–15 µm and
stain Gram-negative. They appear singly or in short chains. When grown at
lower, or atmospheric, pressures, the cells elongate and become twisted.
Growth is enhanced by hydrostatic pressure ; the optimal pressure for growth
is 40 MPa (26 MPa pressure at sampling site). The temperature range for
growth is 45–70 SC, the optimum being around 65 SC (doubling time is
approximately 20 min at 40 MPa). Growth is observed from pH 5 to pH 8, the
optimum being at pH 6. The salinity range for growth is 10–50 g NaCl lN1, the
optimum being at 30 g lN1. The isolate is able to grow on a broad spectrum of
carbohydrates or complex proteinaceous substrates, and growth is stimulated
by L-cystine and elemental sulfur. The GMC content of the genomic DNA is
29O1 mol %. According to phylogenetic analysis of the 16S rDNA gene, the
strain is placed within the order Thermotogales, in the bacterial domain. On
the basis of 16S rDNA sequence comparisons and morphological, physiological
and genotypic characteristics, it is proposed that the isolate be described as a
novel species of the genus Marinitoga, with Marinitoga piezophila sp. nov. as
the type species. The type strain is KA3T (l DSM 14283T l JCM 11233T).
Keywords : deep-sea hydrothermal vent, thermophile, Thermotogales, piezophile,
Marinitoga piezophila
INTRODUCTION
Members of the order Thermotogales are rod-shaped
bacteria characterized by a sheath-like outer structure
called the ‘ toga ’. This order comprises six genera :
Thermotoga (eight species) (Huber et al., 1986 ;
Jannasch et al., 1988 ; Jeanthon et al., 1995 ; Ravot et
al., 1995a ; Takahata et al., 2001 ; Windberger et al.,
.................................................................................................................................................
† Present address : Laboratoire de Microbiologie et de Biotechnologie des
Extre# mophiles, De! partement de Valorisation des Produits, Centre IFREMER
de Brest, BP 70, 29280 Plouzane! , France.
The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of
Marinitoga piezophila strain KA3T (l DSM 14283T l JCM 11233T) is
AF326121.
1989) ; Thermosipho (four species) (Antoine et al.,
1997 ; Huber et al., 1989 ; L ’Haridon et al., 2001 ; Takai
& Horikoshi, 2000) ; Petrotoga (two species) (Davey et
al., 1993) ; Geotoga (two species) (Davey et al., 1993) ;
Fervidobacterium (four species) (Andrews & Patel,
1996 ; Bertoldo et al., 1999 ; Huber et al., 1990 ; Patel et
al., 1985) ; and Marinitoga (one species) (Wery et al.,
2001). All these genera have been isolated from
extreme environments such as brines from oilfields or
oil reservoirs, and from continental or submarine
volcanic areas. All of them contain moderate thermophiles, except the genus Thermotoga, which contains
hyperthermophiles. The members of Geotoga and
Petrotoga are also characterized by tolerance to high
salt concentrations. Some strains belonging to the
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K. Alain and others
Thermotogales have been isolated from high-pressure
environments (L ’Haridon et al., 1995 ; Marteinsson et
al., 1997 ; Takai & Horikoshi, 2000 ; Wery, 2000), but
none was obtained by cultivation under in situ pressure
conditions.
To date, only the piezophilic and hyperthermophilic
vent archaeon Thermococcus barophilus (Marteinsson
et al., 1999) has been obtained by enrichment and
isolation under in situ hydrostatic pressure. However,
several studies of high pressure have been performed
on thermophilic micro-organisms previously isolated
under atmospheric pressure (Erauso et al., 1995 ;
Canganella et al., 1997 ; Holden & Baross, 1995 ;
Jannasch et al., 1992 ; Marteinsson et al., 1997, 1999 ;
Miller et al., 1988 ; Nelson et al., 1991, 1992 ; Pledger et
al., 1994 ; Reysenbach & Deming, 1991 ; Takai et al.,
2000).
Various microbial growth responses to in situ pressure
conditions have been observed in thermophilic strains
that were also piezotolerant (Reysenbach & Deming,
1991), piezosensitive (Jannasch et al., 1992) and
piezophilic (Canganella et al., 1997 ; Marteinsson et
al., 1997 ; Takai & Horikoshi, 2000 ; Takai et al., 2000),
but no obligate piezophilic thermophile has been
isolated so far.
During the French ‘ AMISTAD ’ cruise in June 1999,
samples were collected from deep-sea-vent fields
(13m N) at the East-Pacific Rise. In this paper, we
report the first isolation of a thermophilic and piezophilic bacterium under in situ hydrostatic pressure.
METHODS
Collection of samples. Samples were collected by the manned
submersible DSV Nautile during the French oceanographic
‘ AMISTAD ’ cruise. A deep-sea-vent field located at the
East-Pacific Rise at a depth of 2630 m was explored. Active
chimney rocks and fluids were collected. A bacterial
tray, made of iron mesh (Marteinsson et al., 1997) was deployed near an active chimney for 74 h at a site named
‘ Grandbonum ’ (PP52 ; 12m 48721h N, 103m 56351h W).
Aboard the ship NO LhAtalante, solid samples from the trap
and small chimney pieces were immediately transferred into
an anaerobic chamber and then inoculated either into
10–30 ml sterile glass syringes (Ultrafit ; Heinke-Sass-Wolf,
as described by Marteinsson et al. (1999), or into serum vials
filled with sterile sea water (under anaerobic conditions : N
headspace gas and 0n5 g sodium sulfide l−"). Syringes were#
transferred into high-pressure vessels aboard the ship and
pressurized to a hydrostatic pressure of up to 30 MPa.
Samples were kept under pressure and at 4 mC until enrichment in the laboratory.
Enrichment and isolation under in situ hydrostatic pressure.
All manipulations preceding pressure enrichment and isolation experiments were performed in an anaerobic chamber.
Samples, stored at 4 mC under a hydrostatic pressure of
30 MPa, were depressurized at room temperature, and 0n5 ml
rock suspensions were transferred into 10 ml syringes. Each
syringe contained RS medium, which had the following
composition (l−" distilled water) : 1n0 g NH Cl, 0n2 g
MgCl .6H O, 0n1 g CaCl .2H O, 0n1 g KCl, 20% g NaCl,
# COO.3H O,#3n45# g PIPES buffer (Sigma ; pH
0n83 g#NaCH
$
# (Difco), 5 g Bio-trypcase (Difco),
6n5–7n0), 5 g yeast
extract
1332
0n3 g K HPO , 0n3 g KH PO and 1 mg resazurin (Sigma).
# adjusted
%
The pH# of the% medium was
to 7n0 with 5 M NaOH
at room temperature. About 0n1 g elemental sulfur was
added to the syringes, which had been sealed as described by
Marteinsson et al. (1997). The syringes were then transferred
into the high-pressure and high-temperature incubation
system, custom-built by Top Industrie (Industrial zone ‘ Le
Plateau de Bie' re ’, Dammarie-les-Lys, France), pressurized
to 30 MPa and heated to 65 mC until the culture became
turbid. To obtain a pure culture, the dilution-to-extinction
technique was employed (Baross, 1995). The isolate was
purified by using six serial dilutions to extinction performed
under 30 MPa at 65 mC. The purity of the isolate was
confirmed by microscopic observations and by cloning and
sequencing of 10 16S rDNA-clone genes.
Culture conditions. The new isolate was routinely cultivated
under 40 MPa in modified RS medium, designated RCj,
which had the same composition as RS medium except that
-cystine (12 g l−") had been added instead of sulfur, the pH
had been adjusted to 6n0 with 10 mM MES buffer (Sigma)
instead of PIPES buffer, and it contained 30 g NaCl l−" and
20 mM maltose (Sigma).
The medium was autoclaved for 20 min, reduced with 0n5 g
sodium sulfide (Na S.9H O) l−" and transferred into an
# \CO , 90 : 5 : 5) before distrianaerobic chamber # (N \H
#
# containing 12 g sterile
bution of the medium into #syringes
-cystine l−".
Experiments with high pressures were performed in 10 ml
syringes loaded anaerobically with 10 ml reduced RCj
medium and inoculated with 0n2 ml culture in late exponential phase grown under 40 MPa pressure at 65 mC. All
samples were made in duplicate and cells were fixed with
0n25 % (v\v) glutaraldehyde to be counted using a flow
cytometer.
Determination of cell numbers. Growth was measured by
either flow cytometry or microscopy. For flow cytometry,
samples were fixed with 0n25 % (v\v) glutaraldehyde for
20 min at ambient temperature before storage at k80 mC.
Cell DNA was stained with SYBR Green (Molecular
Probes) at ambient temperature in Sea Salts buffer (30 g l−" ;
Sigma) at a final concentration of 1 : 10 000 of the commercial
solution. Cells were analysed with 488 nm excitation and
enumerated as described by Marteinsson et al. (1999) (for
the protocol, see Marie et al., 2000). For microscopy, cells
were counted in a Thoma chamber (depth 0n02 mm) using a
light microscope (model CX 40 ; Olympus) equipped with a
phase-contrast oil-immersion objective. A good correlation
was found between microscopy and flow-cytometry counts.
Growth rates were calculated using linear regression analysis
from three to five points along the logarithmic portions of
the resulting growth curves, and confidence intervals were
calculated as described by Barbier et al. (1999).
Microscopic observations. Bacto 3-step and Gram stain SetS (Difco) were used for Gram staining. SpotTest Flagella
stain (Difco) was used for flagella detection. The presence of
spores was investigated by phase-contrast microscopy.
Cells cultivated at atmospheric pressure and under high
hydrostatic pressure were observed by light microscopy and
scanning electron microscopy. The samples for scanning
electron microscopy were prepared as follows : 2 ml culture
was filtered on polycarbonate filters (0n2 µm ; Nucleopore)
and immediately enclosed in 3i3 cm squares of clean
aluminium foil (this step was completed in 30 s to avoid airdrying of the filters), then the foil wrappers containing the
filters were immersed in a 2 % (v\v) glutaraldehyde solution
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Marinitoga piezophila sp. nov.
buffered with filtered sea water for 1 h at 4 mC. Samples were
then passed through decreasing concentrations of filtered
sea water. After 1 h fixation, the samples were dehydrated by
transfer through a series of vials of increasing concentrations
of ethyl alcohol (50, 70, 75 and 100 % for 20 min each) and
transferred to amyl acetate for 2–3 h. Amyl acetate was then
replaced with carbon dioxide, using a pressure-bomb apparatus (Top Industrie). Finally, dried filters were mounted
on scanning electron microscopy sample stubs and coated in
a vacuum with two layers of gold, each at a thickness of
100 AH . Samples were examined with a scanning electron
microscope (KL-30 LaB6 ; Philips).
Determination of growth parameters. To determine the
optimum temperature at atmospheric pressure, cells were
grown in serum bottles containing 50 ml RCj medium in a
temperature-controlled oven. Bottles were inoculated with
cultures adapted to atmospheric pressure (nine subcultures
at atmospheric pressure after enrichment and isolation
under 30 MPa hydrostatic pressure) with gas (gas phase
N \H \CO , 90 : 5 : 5) as the headspace. As cells grew better
# # high# hydrostatic pressure, optimum pH and salt
under
concentrations were determined under a hydrostatic pressure of 40 MPa as described below. To determine the effect of
pH on the growth, RCj medium was modified by using the
following buffers (Sigma), each at a concentration of
10 mM : for pH 2, 3 and 4, no buffer ; for pH 5, 5n5 and 6,
MES buffer ; for pH 6n5 and 7, PIPES buffer ; for pH 7n5,
HEPES buffer ; for pH 8 and 8n5, Tris buffer ; and for pH 9,
no buffer. Sodium sulfide was added and the pH was
controlled at room temperature and adjusted, if necessary,
with 0n1 M HCl and 0n1 M NaOH. To determine the salt
requirement, RCj medium was prepared with different
concentrations of NaCl. All assays (temperature, pH and
salinity) were carried out in duplicate and repeated twice.
Determination of growth rates under hydrostatic pressures.
To determine growth rates at different temperatures under
high and low pressures, cells were grown in 10 ml syringes
containing 10 ml RCj medium. Cells grown at high
pressure were used as the inoculum in all experiments.
Heating started at 22 mC, and it took approximately 15 min
to obtain stable test temperatures (40, 45, 50, 55, 60, 65, 70,
75 and 80 mC) and stable test pressures (0n3, 10, 20, 30, 40, 50
and 60 MPa) in the high-pressure and high-temperature
incubation system. To determine the optimum temperature
for growth under pressure, each temperature was tested at
high (40 MPa) and low (0n3 MPa) pressure in parallel
experiments. To collect samples, the incubation system was
depressurized gently through a valve, whereas the temperature remained more or less stable. Samples (approx.
0n4 ml) were immediately collected anaerobically and
injected into Venoject blood-collecting tubes (Terumo)
before the syringes were again pressurized to the test pressure
(approx. 7–8 min). Each sample was processed in duplicate
and was fixed immediately in 0n25 % (v\v) glutaraldehyde.
At each time-point, analyses were carried out in duplicate,
and the experiment was repeated four times. The optimum
pressure for growth at 65 mC (the optimum growth temperature) was determined from different experiments performed at 0n3, 10, 20, 30, 40, 50 and 60 MPa.
Determination of growth requirements and substrate
utilization. Growth requirements and substrate utilization
were tested in serum vials incubated at atmospheric pressure
under the anaerobic gas mixture at 65 mC. Various carbon
sources were added to RCj basal medium (pH 6n0) prepared
without carbon sources. The following carbon sources were
tested : maltose, starch, glycogen, (j)-cellobiose, (k)-
ribose, (j)-glucose, (k)-fructose and (j)-galactose
(each at a final concentration of 0n5 %, w\v), yeast extract,
peptone, tryptone, pyruvate, casein, brain–heart infusion,
Casamino acids, succinate, propionate and acetate (each at
a final concentration of 0n2 %, w\v). This experiment was
performed, on the one hand, with these substrates tested as
sole carbon sources in the RCj basal medium, and on the
other hand, in the presence of a small amount of yeast
extract (0n02 %, w\v) used for culture induction. Nitrogen
sources were tested on RCj medium prepared without
NH Cl, Bio-trypcase and -cystine but with 40 mM acetate
% carbon and energy source and 12 g sulfur l−" as the
as the
electron acceptor. Urea, glutamate and gelatin were all
tested at 0n2 % (w\v), while NH Cl, NaNO and NaNO
were tested at 20 mM. Tests were%performed #in serum vials$
with H \CO (80 : 20) as the headspace. To avoid growth on
#
# brought with the inoculum, positive cultures
the substrates
were transferred once (10 % inoculum) into the test media
for confirmation of growth. The final concentration of the
cells was determined, by direct counting, and compared with
the concentration in the control without the added carbon or
nitrogen source.
Growth in the presence of different electron acceptors was
tested. Elemental sulfur and -cystine were tested at 12 g l−",
thiosulfate at 20 mM and NaNO and NaNO at 20 mM.
Growth in the absence of -cystine# was tested by$ cultivating
the cells in RCj medium from which sulfur compounds had
been omitted. In these experiments, N was used as the
#
headspace and titanium(III) citrate (1 mM
final concentration) was used as reducing agent, instead of Na S.9H O.
#
#
Tests were performed in serum vials ; positive cultures
were
transferred once for confirmation of growth. Growth was
determined by direct cell counting in a Thoma chamber
(depth 0n02 mm) with a phase-contrast microscope.
The influence of hydrogen on growth was investigated by
using Rj medium with and without elemental sulfur (l
RSj medium) and N \CO (80 : 20, v\v ; 200 kPa) or
# kPa)
# as the headspace gas. The
H \CO (80 : 20, v\v ; 200
#
#
H S formation was detected by the addition of 500 µl 5 mM
#
CuSO
and 50 mM HCl to 0n2 ml of the culture. A brown
%
precipitate
demonstrated the presence of H S. Autotrophic
growth was tested in RCj mineral basal# medium with
H \CO (80 : 20, v\v ; 200 kPa) as the gas phase.
#
#
Susceptibility to antibiotics. The sensitivity to antibiotics was
tested at atmospheric pressure. It was estimated by using 10,
25, 50, 75, 100, 150 and 200 µg ml−" solutions of the following
antibiotics : ampicillin, chloramphenicol, fusidic acid, kanamycin, nalidixic acid, penicillin G, rifampicin, streptomycin,
spectinomycin, tetracycline, vancomycin and gentamicin.
Antibiotic solutions were added to RCj medium, just
before inoculation. When the antibiotic was diluted in
ethanol (chloramphenicol) or dimethylsulfoxide (rifampicin), the same volume of solvent was added to the control
cultures.
Controls were performed with an antibiotic-sensitive bacterium, Thermus thermophilus HB8T (l DSM 579T),
obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) and
cultivated under the same conditions to establish the
efficiency of the antibiotics at 65 mC.
DNA extraction. Genomic DNA of strain KA3T was
extracted using a modification of the procedure described by
Charbonnier & Forterre (1994). The DNA was purified by
caesium chloride gradient centrifugation (Sambrook et al.,
1989), and purity was checked spectrophotometrically.
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K. Alain and others
(a)
(b)
(c)
(d)
.....................................................................................................
Fig. 1. (a) Scanning electron micrograph of
strain KA3T in the mid-exponential phase
of growth, showing a polar flagellum and
division by constriction (cells were cultivated
at a hydrostatic pressure of 40 MPa). The
sheath-like outer structure is arrowed ; bar,
1 µm. (b–d) Phase-contrast micrographs of
strain KA3T. (b) Rod-shaped cells, in the midexponential phase of growth, cultivated at
40 MPa ; bar, 10 µm. (c) Elongated cells,
cultivated at 10 MPa ; bar, 1 µm. (d) Twisted
cells, cultivated at atmospheric pressure
(after culture at high hydrostatic pressure) ;
bar, 1 µm.
DNA base composition. The GjC content (mol %) of the
genomic DNA was determined from the melting point,
according to Marmur & Doty (1962) with the modifications
described by Rague! ne' s et al. (1996). Ultrapure DNAs from
Escherichia coli strain B (50 mol % GjC), Clostridium
perfringens (26n5 mol % GjC) and Micrococcus luteus
(72 mol % GjC) were used as standards (Sigma).
Amplification and 16S rRNA gene sequence analysis. The
PCR amplifications were as described by Wery et al. (2001).
The 16S rDNA was selectively amplified from purified
genomic DNA by using a PCR with oligonucleotide primers
designed to anneal to conserved positions in the 3h and 5h
regions of the 16S rRNA genes. The bacterial forward
primer, SAdir (5h-AGAGTTTGATCATGGCTCAGA-3h),
corresponded to positions 8–28 of E. coli 16S rRNA, and the
bacterial reverse primer, S17rev (5h-GTTACCTTGTTACGACTT-3h), corresponded to the complement of positions
1493–1509 of E. coli 16S rRNA.
The 16S rRNA gene was double-strand sequenced. The PCR
products were sequenced, on the one hand by utilizing
primers F9, F515, R357, R805, R1195, R1544 and with an
ABI 377 DNA sequencer by using the Rhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Applied
Biosystems), and on the other hand by Genome Express
(Meylan, France) with the primers described by Rague! ne' s et
al. (1996).
Sequences were manually aligned with closely related
sequences obtained from the Ribosomal Database Project
after  searches (Altschul et al., 1990). Sequence
alignments and phylogenetic analysis were performed
with the  program (http :\\www.mikro.biologie.tumuenchen.de), omitting regions of sequence ambiguity.
Phylogenetic trees based on three algorithms [neighbour
1334
joining (Saitou & Nei, 1987), maximum parsimony (Lake,
1987) and maximum likelihood (Felsenstein, 1981)] were
constructed. A distance tree was constructed by using
neighbour-joining algorithms with the Jukes–Cantor corrections, and a maximum-likelihood tree was constructed by
the fastDNAml software included in the  package.
Homologous nucleotide positions, based on the filter of the
 database, were included in the alignment and used for
the comparison analysis. Bootstrap analysis (Felsenstein,
1985) included in the  package was used to provide
confidence estimates for phylogenetic tree topologies. The
16S rDNA sequences used for phylogenetic analysis are
given in Fig. 3.
RESULTS
Enrichment and isolation
Enrichment cultures from various samples were grown
on RS medium. Growth was observed at 65 mC under
a hydrostatic pressure of 30 MPa. The positive enrichment consisted of dense populations of short, rodshaped cells that were motile and single or in chains
and was successfully subcultured. One isolate was
purified with six serial dilutions to extinction and
designated as strain KA3T (l DSM 14283T l JCM
11233T).
Morphology
Microscopic observations indicated that cells of isolate
KA3T were motile, Gram-negative rods with polar
flagella (visible by scanning electron microscopy ; Fig.
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Marinitoga piezophila sp. nov.
Specific growth rate (h–1)
2
pressure became very twisted, deformed and elongated
(Fig. 1d). However, after nine transfers at atmospheric
pressure (after numerous generations), the cells did not
again show morphological deformations. No spore
formation by physiological induction was observed in
any phase of growth under any growth conditions
tested.
(a)
1·5
1
Determination of growth parameters
The isolate grew under anaerobic conditions over a
temperature range of about 45–70 mC, optimum
growth occurring at about 65 mC. The generation time
at this temperature was around 21 min at pH 6n0 and a
hydrostatic pressure of 40 MPa (Fig. 2a). No growth
was observed at 75 mC. Growth was observed at salt
concentrations ranging from 10 to 50 g l−", the optimum salinity being around 30 g l−" ; no growth was
observed below 10 g l−" or above 50 g l−". Growth was
observed from pH 5 to pH 8, the optimum being
around pH 6. No growth was detected below pH 5 or
above pH 8. The morphology of the cells changed
when grown at pH values or salt concentrations close
to the minimum or maximum values allowing growth.
0·5
0
40
50
60
70
80
Temperature (ºC)
Specific growth rate (h–1)
2
(b)
1·5
Determination of pressure effects on growth
1
0·5
0
0
20
40
60
80
Hydrostatic pressure (MPa)
.................................................................................................................................................
Fig. 2. (a) Effect of temperature and hydrostatic pressure on
the specific growth rate of strain KA3T. The cells were grown in
RCj medium (pH 6n0, 30 g NaCl l−1) at 40 MPa (triangles) and at
a hydrostatic pressure of 0n3 MPa, either after culture at high
hydrostatic pressure (circles) or after nine subcultures at
atmospheric pressure (squares). Bars indicate confidence
intervals. (b) Effect of hydrostatic pressure on growth of strain
KA3T cultivated on RCj medium (65 mC, pH 6n0, 30 g NaCl l−1).
Bars indicate confidence intervals.
1a). The cells were surrounded by a ‘ toga ’, an outer
sheath-like structure specific to members of the order
Thermotogales (Fig. 1a). This envelope was visible by
phase-contrast microscopy in all phases of growth.
The cells appeared singly or in chains within the
sheath. The smaller single rods seemed to move more
rapidly than the longer cells. When the isolate was
grown under optimal growth conditions, the cells were
short rods 1–1n5 µm long and 0n5 µm wide (Fig. 1b).
The morphology changed when the isolate was grown
under unfavourable growth conditions (Fig. 1c). The
cells appeared elongated, and filaments with chains
of up to 10 cells were occasionally observed. Cells
cultivated at 65 mC under a hydrostatic pressure of
40 MPa then subcultured at 65 mC at atmospheric
The growth rate of the isolate increased at all
temperatures tested when the cells were grown under
high-pressure conditions. The optimum growth temperature was around 65 mC under conditions of both
high- and low-hydrostatic pressure (Fig. 2a). Growth
was observed between 0n3 and 50 MPa at 65 mC, but no
growth was observed at 60 MPa (Fig. 2b). The growth
rate of the isolate was enhanced by increasing the
hydrostatic pressure at 65 mC, and the optimum pressure for growth was 40 MPa.
Determination of growth requirements and substrate
utilization
Strain KA3T grew very well in the RCj medium
containing 0n5 % (w\v) Bio-trypcase and 0n5 % (w\v)
yeast extract, with a generation time of about 21 min
and a cell density of 5n6i10) cells ml−" at 65 mC and
40 MPa. Only brain–heart infusion and yeast extract
were able to support good growth when provided
alone in the basal medium, while acetate used as the
sole carbon source supported only poor growth.
Utilization of other substrates required the addition of
0n02 % (w\v) yeast extract. Growth was observed on
both proteinaceous substrates and carbohydrates
when yeast extract was added. Under these conditions,
several complex substrates such as casein, Casamino
acids, peptone and tryptone strongly improved growth
( 5i10) cells ml−"), while lower maximum cell
densities were obtained with a variety of sugars and
organic acids when combined with 0n02 % (w\v)
yeast extract. Growth of strain KA3T was weakly
improved by starch, (k)-fructose, (j)-glucose,
(j)-galactose,
maltose,
(j)-cellobiose
( 5i10( cells ml−" 10)) and very weakly improved
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K. Alain and others
.....................................................................................................
KA3T (AF326121: DSM 14283T)
Marinitoga camini (AJ250439: DSM 13578T)
100
Geotoga subterranea (L10659: ATCC 51225T)
100
Geotoga petraea (L10658: ATCC 51226T)
Petrotoga miotherma (L10657: ATCC 51224T)
100
Fervidobacterium islandicum (M59176: DSM 5733T)
Fervidobacterium nodosum (M59177: DSM 35602T)
100 Thermosipho japonicus (AB024932: JCM 10495T)
Thermosipho africanus (M83140: DSM 5309T)
Thermosipho melanesiensis (Z70248: CIP 104789T)
Thermotoga hypogea (U89768: DSM 11164T)
50
100 Thermotoga subterranea (U22664: DSM 9912T)
90
Thermotoga elfii (X80790: DSM 9442T)
Thermotoga maritima (Z11839: DSM 3109T)
100
100
100
94
0·067
by (k)-ribose and acetate ( 5i10( cells ml−"). The
other carbon sources tested did not support growth,
even when combined with yeast extract.
Growth occurred with NH Cl and urea as a nitrogen
%
source, but no growth was supported
by glutamate or
gelatin. The isolate does not grow under autotrophic
culture conditions and is therefore heterotrophic. In
the presence of any added electron acceptor, cultivation under an H gas phase resulted in complete
# whereas H inhibition was
inhibition of growth,
#
overcome in the presence of elemental
sulfur or cystine.
Very weak growth occurred in the RCj medium
prepared without sulfur compounds and reduced by
titanium(III) citrate under a N \CO (80 : 20) gas
#
#
headspace. The strain can therefore
grow
only by
fermentation of organic substrates, and sulfur compounds, as electron acceptors, are not absolutely
required. Addition of NaNO or NaNO to the culture
$
# increase in
medium did not stimulate growth.
A small
the final cell concentration was obtained with thiosulfate (Na S O ). Optimal growth yields were
# $presence of elemental sulfur or obtained in #the
cystine acting as electron acceptors. In all cases, when
elemental sulfur or -cystine was present in the culture
medium, the growth of strain KA3T was accompanied
by the production of H S, which was not detected in
# incubated under the same
the uninoculated control
conditions.
Susceptibility to antibiotics
Growth of isolate KA3T was inhibited by the addition
of vancomycin, fusidic acid and chloramphenicol at
10 µg ml−", by penicillin G, rifampicin and streptomycin at 25 µg ml−", by nalidixic acid at 100 µg ml−",
and by spectinomycin and ampicillin at 150 µg ml−".
The new isolate was insensitive to kanamycin and
gentamicin at a concentration of 200 µg ml−".
DNA base composition
The DNA GjC content of the genomic of isolate
KA3T was 29p1 mol %.
1336
Fig. 3. Phylogenetic position of strain KA3T
within the order Thermotogales. Alignment
was performed with 13 species representatives of the Fervidobacterium, Thermotoga,
Thermosipho, Geotoga, Petrotoga and
Marinitoga genera. Thermus thermophilus
ATCC 27634T (TTHB27) was chosen as the outgroup. The topology shown is an unrooted
tree obtained by means of a neighbourjoining algorithm (Jukes–Cantor correction)
established
using
the
ARB
package. This topology was confirmed by
maximum-parsimony and maximum-likelihood methods. Bootstrap values are
shown on the branches. Scale bar, 6n7 nt
substitutions per 100 nt.
16S rDNA sequence analysis
A phylogenetic analysis revealed that the new isolate
was a member of the order Thermotogales. The 16S
rRNA gene sequence was almost completely sequenced
and consisted of 1443 bp. The sequences were aligned
(positions 8–1449 ; E. coli numbering in the 
program) with the sequences of representatives of the
order Thermotogales (Fig. 3). The closest relative of
strain KA3T was Marinitoga camini (having a 16S
rDNA sequence similarity of 94 %) and members of
the genera Petrotoga (mean 81 % similarity), Geotoga
(mean 82 % similarity), Thermotoga (mean 81 % similarity), Thermosipho (mean 81 % similarity) and
Fervidobacterium (mean 80 % similarity).
DISCUSSION
The novel marine strain KA3T, isolated from a deepsea hydrothermal vent at the East-Pacific Rise at a
depth of 2630 m, is a Gram-negative, obligately
anaerobic and heterotrophic, thermo-piezophilic bacterium capable of reducing elemental sulfur to hydrogen sulfide. Physiological features and morphological characteristics, such as the sheath-like outer
structure, suggest that the isolate belonged to the order
Thermotogales. The strain is able to ferment complex
proteinaceous substrates such as yeast extract, casein,
peptone, tryptone and brain–heart infusion, and
growth is stimulated by -cystine and elemental sulfur.
However, thiosulfate does not have a stimulatory
effect on growth rates and therefore differs from certain
species belonging to the Thermotogales (Ravot et al.,
1995b, 1996).
Affiliation with the order Thermotogales was confirmed by 16S rRNA analysis. On the basis of the
results of 16S rDNA sequencing, the new isolate,
KA3T, is most closely related to the newly described
genus Marinitoga. This new genus originated from a
hydrothermal vent situated on the Mid-Atlantic Ridge,
and contains only one species, Marinitoga camini, type
strain MV1075T (Wery et al., 2001). The new isolate
can be distinguished from Marinitoga camini by many
important phenotypic and genetic criteria : (1) the two
stains share only 94 % 16S rRNA gene sequence
International Journal of Systematic and Evolutionary Microbiology 52
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Marinitoga piezophila sp. nov.
similarity and less than 83 % with respect to other
genera in the order Thermotogales ; (2) the new isolate
is a piezophile, whereas Marinitoga camini is unable to
grow under hydrostatic pressure (Wery, 2000) (unsuccessful attempts were made in our laboratory to
cultivate Marinitoga camini at 30 and 40 MPa) ; (3) the
optimal growth temperature for strain KA3T is 65 mC,
whereas that for Marinitoga camini is at 55 mC ; (4) the
generation time of the new isolate is also approximately five times faster [21 min for KA3T (at hydrostatic pressure) and 1n7 h for Marinitoga camini (at
atmospheric pressure)] ; (5) unlike Marinitoga camini,
strain KA3T is able to utilize (k)-ribose and (j)galactose in the presence of yeast extract and is also
able to grow on acetate, Casamino acids and casein.
Moreover, the new isolate shows unusual morphological deformations when grown under conditions of
stress. When the cells are stressed by culture conditions
such as growth at atmospheric pressure or under nonoptimal culture conditions, they become very elongated and twisted. Similar changes have been observed
when the psychrophilic, obligately barophilic bacterium MT-41, isolated from the Mariana Trench
(10 476 m depth), was isothermally decompressed
(Chastain & Yayanos, 1991 ; Yayanos et al., 1981), but
this is the first time that such morphological changes
have been observed as a result of pressure on a
thermophilic bacterium. However, it seems that the
isolate can adapt to unfavourable growth conditions
after several subcultures. After nine subcultures at
atmospheric pressure, the cells do not show any
morphological deformations and are ‘ adapted ’ to
atmospheric pressure. All thermophilic members of
the Archaea that have been studied under elevated
hydrostatic pressures show increases in their minimal,
optimal and\or maximal growth temperatures when
cultivated under elevated pressures (Canganella et al.,
1997 ; Jannasch et al., 1992 ; Marteinsson et al., 1997,
1999 ; Pledger et al., 1994), except in the case of the
baro-thermophilic archaeon Thermococcus barophilus
– for which the optimum temperature was not shifted
(Marteinsson et al., 1999) (for a review, see Prieur &
Marteinsson, 1998). When isolate KA3T is cultivated
at 40 MPa, the minimal temperature for growth
decreases by 5 mC while the optimal and maximal
temperatures for growth remain unchanged. This is
the first report of a thermo-piezophilic bacterium, with
clear piezophilic behaviour, obtained after enrichment
and isolation at high temperature and under elevated
hydrostatic pressure.
KA3T strain was sampled at a site where the pressure
is around 26 MPa, but its optimal pressure for growth
is 40 MPa and the cells show deformations when
cultivated at low pressures. As has already been
reported for several baro-thermophilic members of the
Archaea, this suggests that strain KA3T originates
from a deeper place than its sampling site, or from the
deep subsurface beneath the sea floor (Gold, 1992 ;
Deming & Baross, 1993 ; Summit & Baross, 1998).
On the basis of its phenotypic and genetic charac-
teristics, we propose that KA3T be assigned to a new
species of the genus Marinitoga. Because of its piezophilic behaviour, we give it the species name piezophila.
Description of Marinitoga piezophila sp. nov. Alain
et al.
Marinitoga piezophila (pie.zohphi.la. Gr. v. piezo to
press ; Gr. adj. philos loving ; N.L. fem. adj. piezophila
referring to its best growth under pressure).
The cells are rod-shaped with a sheath-like outer
structure, motile with polar flagella, and stain Gramnegative. Cell division occurs by constriction. Under
optimal conditions, cells appear as short rods (1–
1n5 µm longi0n5 µm wide), singly or in short chains of
fewer than five cells. On the other hand, they show
cellular deformations, becoming twisted and elongated
when they are cultivated at decreased pressures.
Obligate anaerobe. Grows optimally at 3 % (w\v)
NaCl and pH 6n0. Growth occurs at 40–75 mC, the
optimum growth temperature being 65 mC. Piezophile
at its growth temperature range, optimum growth
occurring at 40 MPa. Optimal doubling time (at 65 mC
and 40 MPa) is 21 min ; maximum cell yield in vials
is 5n6i10) cells ml−". Obligate chemoorganoheterotroph. Grows on complex organic compounds
and several carbohydrates in the presence of yeast
extract. -Cystine and elemental sulfur greatly enhance
growth but are not absolutely required. The GjC
content of the genomic DNA is 29p1 mol %. The
16S rDNA gene sequence similarity to Marinitoga
camini is 94 %. Isolated from a hydrothermal vent
sample, under 30 MPa hydrostatic pressure at 65 mC,
from the East-Pacific Ridge, 13m N, at a depth of
2630 m. The type strain is strain KA3T (l DSM
14283T l JCM 11233T). The GenBank accession number for the 16S rDNA sequence of the type strain is
AF326121.
ACKNOWLEDGEMENTS
We thank the chief scientist of the French oceanographical
cruise ‘ AMISTAD ’ (1999), Christian Jeanthon, the captain
and crew of the NO LhAtalante and the DSV Nautile pilots
and support crew. We gratefully acknowledge Dominique
Marie (CNRS, Station Biologique, Roscoff, France) for
flow-cytometry assistance, Ge! rard Rague! ne' s for measurement of the GjC content of genomic DNA and Philippe
Crassous (IFREMER, Centre de Brest, France) for assistance with the scanning electron microscopy. The work
of M. M. and E. B.-O. was supported by RFBR grant
no. 00-04-48924.
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