International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1961–1967
DOI : 10.1099/ijs.0.02173-0
Carboxydocella thermautotrophica gen. nov.,
sp. nov., a novel anaerobic, CO-utilizing
thermophile from a Kamchatkan hot spring
Institute of Microbiology,
Russian Academy of
Sciences, Prospect 60 Let
Oktyabrya 7/2, 117811
Moscow, Russia
T. G. Sokolova, N. A. Kostrikina, N. A. Chernyh, T. P. Tourova,
T. V. Kolganova and E. A. Bonch-Osmolovskaya
Author for correspondence : Tatyana Sokolova. Tel : j7 095 135 4458. Fax : j7 095 135 6530.
e-mail : sokolova!inmi.host.ru
A novel anaerobic, thermophilic, CO-utilizing bacterium, strain 41T, was
isolated from a terrestrial hot vent on the Kamchatka Peninsula. Strain 41T was
found to be a Gram-positive bacterium, its cells being short, straight, motile
rods. Chains of three to five cells were often observed. The isolate grew only
chemolithoautotrophically on CO, producing equimolar quantities of H2 and
CO2 (according to the equation COMH2O CO2MH2). Growth was observed in the
temperature range 40–68 SC, with an optimum at 58 SC, and in the pH range
65–76, with an optimum at pH 70. The generation time under optimal
conditions for chemolithotrophic growth was 11 h. The DNA GMC content was
46O1 mol %. Growth was completely inhibited by penicillin, ampicillin,
streptomycin, kanamycin and neomycin. On the basis of the phenotypic and
phylogenetic features, it is proposed that this isolate represents a new genus
and species, Carboxydocella thermautotrophica gen. nov., sp. nov. (type strain
41T l DSM 12356T l VKM B-2282T).
Keywords : terrestrial hot vents, carbon monoxide utilization, thermophilic anaerobe,
Carboxydocella thermautotrophica
INTRODUCTION
Anaerobic oxidation of CO coupled to equimolar
hydrogen and CO formation was first found in the
purple non-sulfur, #mesophilic, phototrophic bacteria
Rubrivivax gelatinosus (Willems et al., 1991) (previously Rhodocyclus gelatinosus ; Uffen, 1976, 1983)
and Rhodospirillum rubrum (Bonam et al., 1989). Later,
it was shown that the reaction COjH O CO jH
#
#
(∆G! lk20 kJ) might be performed by# several phylogenetically diverse thermophilic bacteria. Among
them, Carboxydothermus hydrogenoformans, isolated
from terrestrial hot springs of the Kuril Islands, was
the first taxonomically described representative
(Svetlichny et al., 1991). Except for CO, the only
substrate utilized by this organism was pyruvate.
Another organism, ‘ Carboxydothermus restrictus ’,
was isolated from a terrestrial hot vent on Raoul
Island (Kermadek Archipelago, New Zealand)
.................................................................................................................................................
The GenBank accession number for the 16S rDNA sequence of strain 41T is
AY061974.
(Svetlichny et al., 1994). Carboxydobrachium pacificum
was the first isolate found to originate from the deepsea hot vents of the Okinawa Trough (Sokolova et al.,
2001). In addition to being capable of anaerobic CO
oxidation\H formation, it could also ferment diverse
#
organic substrates.
All of these organisms are obligate
or facultative lithotrophs, but have a requirement for
yeast extract for growth. Here, we report the isolation
of a novel CO-utilizing, H -producing, thermophilic
#
bacterium (from a thermal spring
of the Geyzer Valley,
Kamchatka Peninsula) that is an obligate lithoautotroph.
METHODS
Sampling. Samples of hot water, mud and cyanobacterial
mat from a hot spring (60 mC, pH 8n6) near the Giant Geyser
(Geyser Valley, Kamchatka Peninsula) were taken
anaerobically in tightly stoppered bottles and transported
(at ambient temperature) to the laboratory.
Enrichment and isolation. For enrichment and isolation of
anaerobic carboxydotrophic bacteria, the following medium
(medium 1) was used (g l−") : NH Cl (1) ; MgCl ; 6H O
%
#
#
02173 # 2002 IUMS Printed in Great Britain
1961
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T. G. Sokolova and others
(0n33), CaCl ; 6H O (0n1), KCl (0n33), KH PO (0n5),
#
#
# %
resazurin (0n001), 1 ml trace-mineral solution (Kevbrin &
Zavarzin, 1992) and 1 ml vitamin solution (Wolin et al.,
1963). After being boiled, the medium was flushed with N
#
and cooled. NaHCO (0n5 g l−") and Na S ; 9H O (1n0 g l−")
$
#
#
were then added and the pH was adjusted to 6n8 with 0n5 M
HCl. Aliquots (10 ml each) of the medium were placed into
50 ml bottles and the headspaces filled with 100 % CO at
atmospheric pressure. The incubation temperatures were 55
and 70 mC. Growth was determined using light microscopy
and GLC detection of CO utilization and the formation of
gaseous growth products. Positive enrichments were used
for further purification. After a number of serial dilution
transfers, colonies were obtained in roll-tubes prepared in
15 ml Hungate tubes on the same medium solidified with
5 % agar, with CO in the gas phase. Well-separated colonies
were transferred into the same liquid medium as that used
for enrichments.
Light and electron microscopy. Light microscopy was carried
out using a phase-contrast microscope with an oil-immersion
objective (90\1n25). For electron microscopy, negative staining of whole cells was done with 2 % phosphotungstic acid.
For the preparation of thin sections, cells were fixed with
5 % glutaraldehyde for 2 h and 1 % OsO for 4 h at 4 mC,
%
embedded in Epon-812 resin, thin-sectioned and stained
with uranyl acetate and lead citrate. Electron micrographs
were taken with a JEM-100C electron microscope.
Physiology studies. Growth of the novel isolate was tested on
different substrates in the same liquid mineral medium, with
N in the gas phase. Possible substrates were added in#
dividually at a concentration of 2 g l−" (final concentration).
Possible electron acceptors were added at 2 g l−", and
elemental sulfur at 10 g l−".
CO, CO and H were analysed using a Chrom-5 (CSFR) gas
#
#
chromatograph equipped with a thermal conductivity detector and a 1n2 m glass column filled with activated carbon
AG-3. The carrier gas was argon and the temperature in the
incubator was 25 mC. Volatile fatty acids were determined on
a Chrom-5 gas chromatograph with a flame-ionization
detector : the column was filled with Chromosorb 10 (Sigma),
the temperature was 170 mC and the carrier gas was argon at
a flow rate of 40 ml min−".
The effect of temperature on growth was studied in medium
1 under a CO atmosphere. The effect of pH on growth was
studied under a CO atmosphere in the same medium, except
that the phosphate component of the medium was added in
the form of 0n01 M phosphate buffer with the required pH.
Temperature and pH optima were determined from the
growth rates. The cell density was measured by direct cell
counting.
Sensitivity of the novel isolate to penicillin (100 µg ml−"),
ampicillin (100 µg ml−"), streptomycin (100 µg ml−"), kanamycin (50 µg ml−") and neomycin (50 µg ml−") was tested in
the same medium under a CO atmosphere.
DNA isolation and base composition. DNA was prepared as
described by Marmur (1961). The GjC content of the
DNA was determined by melting-point analysis (Marmur &
Doty, 1962) using Escherichia coli K-12 DNA as a reference.
16S rDNA sequence determination and analysis. 16S rDNA
was selectively amplified from genomic DNA by a PCR
using 5h-AGAGTTTGATCCTGGCTCAG-3h as the for-
1962
ward primer and 5h-TACGGTTACCTTGTTACGACTT3h as the reverse primer (Lane, 1991). The PCR was carried
out in 100 µl reaction mixture containing 1 µg DNA template, 200 µM (each) primers, 200 µM (each) dNTPs and 3 U
Tet-z polymerase (BioMaster) in reaction buffer (100 mM
Tris\HCl pH 8p3, 500 mM KCl, 20 mM MgCl ). The PCR
was performed using the following programme# : 30 amplification cycles of denaturation at 94 mC for 1 min, annealing
at 42 mC for 1 min and extension at 72 mC for 1 min. The final
extension was carried out at 72 mC for 6 min. The PCR
products were purified using a PCR-Prep kit (Promega) as
recommended by the manufacturer. The 16S rDNA was
sequenced in both directions by using a set of forward and
reverse universal primers (Lane, 1991). DNA sequencing
was performed by using Sequenase version 2 of the VSB kit
(USB).
The 16S rDNA sequence was aligned with a representative
set of 16S rRNA sequences obtained from the Ribosomal
Database Project or from recent GenBank releases by using
. Positions that had not been sequenced in one or
more reference organisms were omitted from the analysis.
Pairwise evolutionary distances were computed by using the
correction of Jukes & Cantor (1969). Phylogenetic trees were
constructed by the neighbour-joining method using the
programs of the  package (Van de Peer & De
Wachter, 1994), by the maximum-likelihood method using
the  program (Strimmer & von Haeseler, 1996) and
by the maximum-parsimony method using program
 of the  package (Felsenstein, 1989) with
bootstrap analysis of 100 trees.
RESULTS AND DISCUSSION
Enrichment and isolation
For the enrichment of anaerobic, carboxydotrophic,
thermophilic bacteria, bottles with liquid anaerobic
medium and a CO gas phase were inoculated with
about 1 g sample. After 2 days incubation at 55 mC,
two of the bottles showed an increase in pressure from
140 to 160–170 kPa. Growth of small rod-shaped
bacteria was observed. The CO content in the gas
phase had decreased to 40 %, and about 30 % H and
# the
30 % CO had appeared. After several transfers in
#
same medium, the enrichments were serially diluted
10-fold and transferred to solidified medium in rolltubes. After 4 days incubation at 60 mC, round, white
colonies, about 0n5 mm in diameter, were observed.
Single colonies were isolated into liquid medium and
two pure cultures were obtained. Strain 41T was
selected for further characterization.
Morphology
Cells of isolate 41T were straight rods of various
lengths from 1 to 3 µm and about 0n4–0n5 µm in width,
arranged singly or in short chains of three to five cells.
Cells were motile due to lateral flagella (Fig. 1a).
Electron microscopy of ultrathin sections revealed that
the cell envelope was of the Gram-positive type, being
composed of a cytoplasmic membrane and a doublelayer cell wall comprising an inner electron-dense layer
and outer lighter layer. Cell membrane invaginations
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Carboxydocella thermautotrophica gen. nov., sp. nov.
(a)
(b)
.................................................................................................................................................
Fig. 2. Growth of strain 41T at 60 mC in mineral medium under a
CO atmosphere. Cell density ($), CO consumption (
), H2
production (>) and CO2 production (=) were monitored.
the equation COjH O CO jH (Fig. 2). Neither
# other#metabolic
#
methane, acetate nor any
product was
detected. The generation time of strain 41T under
optimal conditions was 1n1 h.
(c)
.................................................................................................................................................
Fig. 1. Electron micrographs of cells of strain 41T. (a) Negative
staining. (b, c) Thin sections. Bars, 0n5 µm.
were often observed (Fig. 1b, c). The cells divided by
binary transverse fission (Fig. 1b).
Strain 41T did not grow on peptone, yeast extract,
starch, cellobiose, sucrose, lactose, glucose, maltose,
galactose, arabinose, fructose, acetate, formate,
pyruvate, ethanol or methanol. A H \CO gas mixture
(4 : 1, v\v) did not support growth.# No #growth was
observed on acetate, ethanol or lactate in the presence
or absence of elemental sulfur or sulfate. Neither sulfur
nor sulfate was reduced during growth with CO. No
growth or H S production occurred in the medium
# sulfur or sulfate and H in the gas
with elemental
#
phase.
Penicillin, ampicillin, streptomycin, kanamycin and
neomycin completely inhibited CO utilization and the
growth of strain 41T.
DNA base composition
Growth characteristics
Growth of strain 41T occurred within the temperature
range 40–68 mC, with an optimum at 58 mC. No growth
was observed at 37 or 70 mC. Strain 41T grew over a pH
range of 6n5–7n6, with an optimum at 7n0, but no
growth was detected at pH 6n2 or 7n8.
Strain 41T grew only under strictly anaerobic conditions. It did not grow in medium without reducing
agents, nor under a CO\air mixture (4 : 1, v\v). It grew
in an atmosphere of 100 % CO (0n23 mmol CO in the
gas phase ml−" medium) on the mineral medium
without yeast extract or other organic compounds
except vitamins. CO oxidation was coupled to H and
# to
CO formation in equimolar quantities according
#
The GjC content of the DNA of strain 41T was
46p1 mol %.
Phylogenetic analyses
We determined a partial 16S rDNA sequence (1456 nt)
for strain 41T, corresponding to positions 37–1467 of
the E. coli numbering. Preliminary comparisons (using
) with 16S rDNA sequences available in
GenBank revealed that the novel isolate 41T was a
member of the Bacillus\Clostridium subphylum of the
Gram-positive bacteria. The highest score was found
with 16S rDNA sequences of the Thermoanaerobacter–Syntrophomonas group, but it was no
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T. G. Sokolova and others
more than 89 %, so there were no sequences that were
closely related to that of strain 41T.
Several phylogenetic trees were constructed by changing the composition of reference organisms belonging
to the Clostridium group. Regions of alignment uncertainty resulting from the presence of long inserts in
16S rDNA sequences of some members of this group
(Rainey et al., 1993 ; Slobodkin et al., 1999) were
omitted from the sequence analysis. A final comparison was carried out of 1322 nt of 16S rDNA
sequences of strain 41T and 57 of the closest reference
strains of the Thermoanaerobacter–Syntrophomonas
group, combined members of clusters V–VIII of the
Clostridium spectrum (Collins et al., 1994). This group,
including species of the genera Desulfotomaculum,
Thermoanaerobacter,
Thermoanaerobacterium,
Moorella, Anaerobranca and some others, was used for
the reconstruction of a phylogenetic tree and the
calculation of sequence similarity.
The level of sequence similarity of strain 41T was
relatively low and almost equal for all reference strains
(85n3–90n7 %). In the phylogenetic tree constructed by
using the neighbour-joining algorithm (Fig. 3), strain
41T was not clustered exactly with any genus or species
of the Clostridium group. Trees constructed using
other treeing algorithms had the same topology (data
not shown).
A variety of diverse bacteria possess CO-oxidizing
enzymes, termed CO dehydrogenases, which allow
them to assimilate CO as a sole source of carbon and
energy. Aerobic carboxydotrophic bacteria grow
heterotrophically on various organic compounds or
chemolithoautotrophically on CO ; they are taxonomically diverse, consisting of more than 15 described
species from eight genera (Zavarzin & Nozhevnikova,
1977 ; Meyer et al., 1986, 1993).
In anaerobes, CO dehydrogenases are key enzymes of
the acetyl-CoA pathway found in the following phylogenetically distant micro-organisms : acetogenic bacteria (Wood & Ljungdahl, 1991 ; Ljungdahl, 1994 ;
Drake, 1994), sulfate-reducing bacteria (Schauder et
al., 1987 ; Janssen & Schink, 1995 ; Oude Elferink et al.,
1999 ; Fukui et al., 1999), photosynthetic bacteria
(Uffen, 1983 ; Ensign, 1995), methanogenic archaea
(Deppenmeier et al., 1996 ; Ferry, 1999) and nonmethanogenic archaea (Mo$ ller-Zinkhan & Thauer,
1990 ; Vorholt et al., 1995, 1997). Some of them can
grow on CO as the only energy source. During growth
on CO, homoacetogenic and methanogenic bacteria
respectively produce acetate and methane. CO is a
common component of volcanic gases in terrestrial
and submarine hot springs. Thus, the ability to utilize
CO might be assumed to be a widespread capacity of
thermophilic micro-organisms. However, only a limited number of thermophilic species are capable of
anaerobic CO utilization. Among the thermophilic
prokaryotes, CO-utilizing methanogens are represented only by Methanothermobacter thermautotrophicus (Zeikus & Wolfe 1972 ; Wasserfallen et al.,
1964
.................................................................................................................................................
Fig. 3. Phylogenetic tree showing the position of strain 41T. A
consensus 16S rDNA sequence of the actinomycete lineage was
chosen as the outgroup. The scale bar represents the expected
number of changes per sequence position. Bootstrap values
(expressed as percentages of 100 replications) are shown at
branch points ; values greater than 80 % were considered
significant. Triangles include the following sequences : Thermoanaerobacter thermohydrosulfuricus DSM 567T (L09161) ;
Thermoanaerobacter sulfurophilus L-64T (Y16940) ; Thermoanaerobacter wiegelii Rt8.B1T (X92513) ; Thermoanaerobacter
acetoethylicus ATCC 33265T (L09163) ; Thermoanaerobacter kivui
DSM 2030T (L09160) ; Thermoanaerobacter brockii subsp.
brockii DSM 1457T (L09165) ; Thermoanaerobacter mathranii A3T
(Y11279) ; Thermoanaerobacter thermocopriae JT3-3T (L09167) ;
Thermoanaerobacter ethanolicus JW 200T (L09162) ; Thermoanaerobacter brockii subsp. lactiethylicus SEBR 5268T (U14330) ;
Thermoanaerobacter siderophilus SR4T (AF120479) ; Caldicellulosiruptor owensensis OLT (U80596) ; Caldicellulosiruptor kristjanssonii DSM 12137T (AJ004811) ; Caldicellulosiruptor lactoaceticus
DSM 9545T (X82842) ; Caldicellulosiruptor saccharolyticus DSM
8903T (AF130258) ; Thermoanaerobacterium thermohydrosulfuricus E100-69T (L09161) ; Thermoanaerobacterium aotearoense
JW/SL-NZ613T (X93359) ; Thermoanaerobacterium saccharolyticum DSM 7060T (L09169) ; Thermoanaerobacterium xylanolyticum DSM 7097T (L09172) ; Thermoanaerobacterium thermosaccharolyticum ATCC 7956T (M59119) ; Moorella thermoautotrophica JW 701/5 (X58354) ; Moorella glycerini DSM 11254T
(U82327) ; Moorella thermoacetica DSM 521T (X58352) ; Syntrophomonas sapovorans DSM 3441T (AF022249) ; Syntrophomonas
wolfei LYB (AF022248) ; Desulfotomaculum halophilum SEBR
3139T (U88891) ; Desulfotomaculum nigrificans NCIMB 8395
(X62176) ; Desulfotomaculum ruminis DSM 2154T (Y11572) ;
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Carboxydocella thermautotrophica gen. nov., sp. nov.
Table 1. Characteristics of anaerobic, thermophilic, carboxydotrophic bacteria
.................................................................................................................................................................................................................................................................................................................
Data for reference species were taken from Svetlichny et al. (1991) (Carboxydothermus hydrogenoformans 2901T), Svetlichny et al.
(1994) (‘ Carboxydothermus restrictus ’ R1) and Sokolova et al. (2001) (Carboxydobrachium pacificum JMT).
Characteristic
Terrestrial or marine
Morphology
Cell-wall structure
Substrates
Temperature for growth (mC) :
Range
Optimum
pH for growth :
Range
Optimum
Minimum doubling time (h)
GjC content of DNA (mol %)
Carboxydothermus
hydrogenoformans 2901T
‘ Carboxydothermus restrictus ’ R1
Carboxydobrachium pacificum JMT
Strain 41T
Terrestrial
Short, slightly curved rods with
lateral flagella
Gram-positive, globular S-layer
CO, pyruvate
Terrestrial
Short, slightly curved rods with
peritrichous flagella
Gram-positive, globular S-layer
CO
Marine
Non-motile, long, thin rods, sometimes
branching
Gram-positive with S-layer
CO, pyruvate, peptone, yeast extract, starch,
cellobiose, glucose, galactose, fructose
Terrestrial
Straight, short rods
with lateral flagella
Gram-positive
CO
40–78
70–72
45–80
70
50–80
70
40–68
58
6n6–8n0
7n0
2n0
39–41
6n6–8n0
7n0
8n3
39
5n8–7n6
6n9
10n2
33
6n5–7n6
7n0
1n1
46
2000). Thermophilic acetogens include Moorella
thermoacetica (Fontaine et al., 1942 ; Collins et al.,
1994) and Moorella thermoautotrophica (Wiegel et al.,
1981 ; Collins et al., 1994). Anaerobic CO oxidation
coupled to molecular hydrogen formation has been
shown for four thermophilic isolates. Like strain 41T,
none of these organisms grows on a mixture of H and
# in
CO . Their comparative characteristics are given
#
Table 1. All of them are anaerobic thermophilic
bacteria with Gram-positive cell-wall structures.
Phylogenetically, they all belong to the Gram-positive
bacteria with low GjC content, but they do not form
a separate cluster. The novel isolate differs from them
in growth-temperature characteristics and in the range
of energy substrates. According to 16S rDNA analyses,
strain 41T forms a new, separate line of descent within
the low-GjC-content Gram-positive subdivision of
the Bacteria. On the basis of the 16S rRNA sequence
data, it can not be assigned to any validly described
genus.
On the basis of the physiological and phylogenetic
features of strain 41T, we propose the description of a
new genus and species, Carboxydocella thermautotrophica gen. nov., sp. nov.
Desulfotomaculum aeronauticum DSM 10349T (X98407) ;
‘ Desulfotomaculum reducens ’ MI-1 (U95951) ; Desulfotomaculum putei SMCC W459T (AF053929) ; Desulfotomaculum
acetoxidans DSM 771T (Y11566) ; Desulfotomaculum kuznetsovii
DSM 6115T (AF009646) ; Desulfotomaculum luciae SLTT
(AF069293) ; Desulfotomaculum australicum AB33T (M96665) ;
Desulfotomaculum thermocisternum ST90T (U33455) ; Desulfotomaculum thermobenzoicum DSM 6193T (L15628) ; Desulfotomaculum thermoacetoxidans DSM 5813T (Y11573) ; Desulfotomaculum geothermicum DSM 3669T (Y11567) ; Desulfotomaculum thermosapovorans DSM 6562T (Y11575) ; Anaerobranca
horikoshii JW/YL-138T (U21809) ; Anaerobranca gottschalkii DSM
13577T (AF203703) ; Desulfosporosinus orientis DSM 765T
(Y11570) ; [Desulfotomaculum] auripigmentum ORex (U85624) ;
Desulfitobacterium dehalogenans JW/IU-DC1T (U40078) ; Desulfitobacterium frappieri PCP-1T (U40078).
Description of Carboxydocella gen. nov.
Carboxydocella (Car.bo.xy.do.celhla. N.L. neut. n.
carboxydum carbon monoxide ; L. fem. n. cella cell ;
N.L. fem. n. Carboxydocella carbon monoxideutilizing bacterium).
Cells are short, straight rods. Motile. Cell wall of
Gram-positive type. Cells divide by binary transverse
fission. Bacterium. Obligate anaerobe. Thermophile.
Neutrophile. Grows chemolithoautotrophically on
CO. Utilizes CO as the sole energy source, with
equimolar formation of H and CO according to the
# GjC content
equation COjH O CO# jH . The
#
#
#
of the DNA of the type strain of the type species is
46p1 mol %. The type species is Carboxydocella
thermautotrophica.
Description of Carboxydocella thermautotrophica sp.
nov.
Carboxydocella thermautotrophica (therm.au.to.troh
phi.ca. Gr. adj. thermos hot ; Gr. adv. auto self ;
Gr. adj. trophikos one who feeds ; N.L. fem. adj.
thermautotrophica thermophilic and autotrophic,
indicating that the organism grows at elevated
temperatures and uses CO as the sole source of carbon
and energy).
The following characteristics are observed in addition
to those given in the genus description. Cells are short,
straight rods, about 0n4–0n5 µm in width and varying in
length from 1 to 3 µm, arranged singly or in short
chains of three to five cells. Motile due to lateral
flagella. On solid medium, produces round, white,
translucent colonies. Grows within the temperature
range 40–68 mC, with an optimum at 58 mC. The pH for
growth ranges from 6n5 to 7n6, with an optimum at
pH 7n0. Growth and CO consumption are inhibited by
penicillin, ampicillin, streptomycin, kanamycin and
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neomycin. Does not grow on peptone, yeast extract,
starch, cellobiose, sucrose, lactose, glucose, maltose,
galactose, arabinose, fructose, acetate, formate,
pyruvate, ethanol, methanol or a H \CO gas mixture
#
# ethanol or
(4 : 1, v\v). Does not grow on H , acetate,
#
lactate in the presence or absence of elemental sulfur or
sulfate. Does not reduce elemental sulfur or sulfate
during growth with CO. The GjC content of the
DNA of the type strain is 46p1 mol %. The type strain
is strain 41T (l DSM 12356T l VKM B-2282T), isolated from a terrestrial hot vent of the Geyzer Valley,
Kamchatka Peninsula.
ACKNOWLEDGEMENTS
The authors are grateful to G. A. Karpov, Institute of
Volcanology, Far-East Scientific Centre, for help with the
organization of the expedition and sampling. This work was
supported by the ‘ Biodiversity ’ programme of the Russian
Ministry of Industry, Science and Technology and NATO
grant LST.CLG.978269.
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