Download Acidilobus aceticus gen. nov., sp. nov., a novel anaerobic

International Journal of Systematic and Evolutionary Microbiology (2000), 50, 2001–2008
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Acidilobus aceticus gen. nov., sp. nov., a novel
anaerobic thermoacidophilic archaeon from
continental hot vents in Kamchatka
M. I. Prokofeva,1 M. L. Miroshnichenko,1 N. A. Kostrikina,1
N. A. Chernyh,1 B. B. Kuznetsov,2 T. P. Tourova1
and E. A. Bonch-Osmolovskaya1
Author for correspondence : E. A. Bonch-Osmolovskaya. Tel : j7 95 135 44 58. Fax : j7 95 135 65 30.
e-mail : lbo!inmi.host.ru
1
Institute of Microbiology,
Russian Academy of
Sciences, Prospect 60 Let
Oktyabrya 7/2, 117811,
Moscow, Russia
2
Bioengineering Centre,
Russian Academy of
Sciences, Prospect 60 Let
Oktyabrya 7/1, 117811,
Moscow, Russia
New thermoacidophilic organisms that were able to grow anaerobically on
starch were isolated from the acidic hot springs of Kamchatka. Strain 1904T,
isolated from a hot spring of the Moutnovski volcano, was characterized in
detail. Its cells were regular or irregular cocci that were 1–2 µm in diameter,
non-motile, and had a cell envelope consisting of one layer of subunits. The
new organism was a hyperthermophile, growing in the temperature range
60–92 SC (with an optimum at 85 SC), an acidophile, having the pH range for
growth of 20–60 (with an optimum at 38), and an obligate anaerobe. It
fermented starch, forming acetate as the main growth product. Other growth
substrates were yeast extract, beef extract and soya extract. Growth on yeast
extract, beef extract and soya extract was stimulated by elemental sulfur,
which was reduced to H2S. Acetate, arabinose, cellulose, formate, fructose,
galactose, glucose, glycine, guar gum, lichenan, malate, maltose, methanol,
pectin, pyruvate, propionate, xylan, xylose or a mixture of amino acids failed
to support growth both in the presence and the absence of sulfur. When starch
was used as the growth substrate, yeast extract (100 mg lV1) was required as a
growth factor. The GMC content of the DNA was found to be 538 mol %.
Comparison of the complete 16S rDNA sequence with databases revealed that
the new isolate belonged to the kingdom Crenarchaeota. It was not closely
related to any described genera (showing sequence similarity below 908 %)
and formed a separate branch of the Crenarchaeota. On the basis of
physiological differences and rRNA sequence data, a new
genus – Acidilobus – is proposed, the type species being Acidilobus aceticus
strain 1904T (l DSM 11585T).
Keywords : Acidilobus aceticus, Archaea, hyperthermophiles, anaerobes, acidophiles
INTRODUCTION
Hyperthermophilic prokaryotes have a temperature
optimum for growth of greater than 80 mC and are
represented by both the Bacteria and the Archaea
(Stetter, 1996). Although only neutrophilic species are
known among hyperthermophilic bacteria, hyperthermophilic archaea include neutrophiles, alkaliphiles
.................................................................................................................................................
The GenBank accession number for the 16S rDNA sequence of strain 1904T
is AF191225.
and acidophiles. Alkaliphilic hyperthermophilic
archaea are currently represented by a few species of
the genus Thermococcus (Keller et al., 1995 ; Dirmeier
et al., 1998), which have a pH optimum for growth of
9n0. Thermoacidophilic isolates are much more numerous and comprise mostly organisms with respiratory metabolism, i.e. obligate or facultative aerobes
(Scho$ nheit & Scha$ fer, 1995), and just one obligate
anaerobe, Stigiolobus azoricus (Segerer et al., 1991),
which can grow at pH 1n0–5n5 by means of lithotrophic
sulfur respiration by utilizing molecular hydrogen as
the sole growth substrate. Among the acidophiles,
01268 # 2000 IUMS
2001
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M. I. Prokofeva and others
fermentative metabolism has been detected only in
Thermoplasma acidophilum (Segerer et al., 1988), a
facultative aerobe and moderate thermophile. Here we
report the isolation of a new obligately anaerobic
organism capable of fermentative growth at 85 mC and
pH 3n8.
METHODS
Sampling and enrichment. Samples of water, mud and soil
were taken from solfataric fields (in Kamchatka) and were
shown to have pH values of 4n0 or less. The samples were put
in 100 ml flasks with screw caps, closed hermetically,
transported to the laboratory and used without delay for
inoculation of the basal medium, which contained the
following (mg lV") : KCl, 330 ; NH Cl, 330 ; KH PO , 330 ;
# O,% 500 ;
MgCl .6H O, 330 ; CaCl .2H O, %330 ; Na S.9H
#
#
#
#
#
starch, 5000 ; yeast extract (Difco), 100 ; resazurin, #1. Traceelement (Kevbrin & Zavarzin, 1992) and vitamin (Wolin
et al., 1963) stock solutions were added at 1 ml lV". The
medium was prepared anaerobically under a flow of oxygenfree CO . After the reduction of the medium by boiling and
#
consequent
addition of Na S.9H O (700 mg lV") and de#
#
colourization of resazurin, the pH was adjusted to 3.0 by the
addition of 5 M H SO . Portions of the medium (20 ml) were
# bottles
%
dispensed into 50 ml
with screw caps, filled with CO
and sterilized at 110 mC. Each bottle was inoculated with 1 g#
of sample and incubated at 80 mC. Microbial growth was
monitored using light microscopy.
Isolation of pure cultures. For isolation of colonies, flat
100 ml bottles (Bellco) containing 3 ml of the same medium
solidified with 0n8 % (w\v) Gelrite and 0n05 % (w\v)
MgSO .7H O were used. Individual colonies were picked up
under a% flow# of oxygen-free CO and transferred to the liquid
# was tested by using light
medium. The purity of isolates
microscopy of cultures in different growth conditions.
Morphology and ultrastructure studies. The morphology of
new isolates was examined using phase-contrast light microscopy. The ultrastructure was studied in a JEM-100
electron microscope, with samples prepared as described
elsewhere (Bonch-Osmolovskaya et al., 1990).
Metabolic studies. Potential growth substrates (5000 mg lV")
were added to the basal medium prepared without starch.
Organic acids were added as their sodium salts, and a
mixture of 20 amino acids was used (SAG-II). The headspace
was filled with 100 % CO ; when molecular hydrogen was
# it was used in a mixture with
tested as a growth substrate,
CO (8 : 2, v\v). Growth was measured by direct cell counting
# the light microscope. Growth products were detected
under
using methods described earlier (Miroshnichenko et al.,
1994). The temperature range and optimum for growth were
determined on the medium with yeast extract and elemental
sulfur at pH 3n8. The pH range and optimum for growth
were determined at 80 mC. Possible electron acceptors added
to the basal medium included elemental sulfur as sulfur
flower powder (10 000 mg lV"), nitrate as the sodium salt,
(500, 1000 and 2500 mg lV") and Fe(III) as amorphous ferric
oxide [90 mM Fe(III) ; Slobodkin et al., 1999] or Fe(III)
citrate (20 mM).
Sensitivity to antibiotics. The influence of penicillin, strep-
tomycin (both at 500 mg lV") and chloramphenicol
(100 mg lV") on the growth of the new isolate was tested. The
stability of the antibiotics at high temperature and low pH
was checked by observing their effects on the growth of
2002
Thiobacillus ferrooxidans VKM B-458, after preliminary
incubation for 3 d at 80 mC and pH 2n5.
Determination of DNA GjC content. The GjC content of
the genomic DNA was determined as reported earlier
(Miroshnichenko et al., 1994).
Sequencing and analysis of 16S rDNA. The 16S rRNA gene
was selectively amplified from the genomic DNA by a PCR
using 5h-AGAGTTTGATCCTGGCTCAG-3h as the forward primer and 5h-TACGGTTACCTTGTTACGACTT-3h
as the reverse primer (Lane, 1991). The PCR reaction was
carried out in 100 µl reaction mixture containing 1 µg DNA
template, 200 µM (each) primer, 200 µM (each) dNTP and 3
U Tet-z polymerase (BioMaster) in reaction buffer (100 mM
Tris\HCl pH 8n3, 500 mM KCl, 20 mM MgCl ). The temperature regime involved 30 amplification cycles# of 1 min at
94 mC, 1 min at 42 mC and 1 min at 72 mC. The final extension
was carried out at 72 mC for 6 min. The PCR products were
purified using the PCR-prep kit (Promega) as recommended
by the manufacturer. The 16S rRNA gene was sequenced in
both directions using the forward and reverse primers. The
DNA sequencing was performed by using USB Sequenase
version 2 kit.
The 16S rDNA sequence was aligned with a representative
set of analogous sequences obtained from the latest versions
of the Ribosomal Database Project (RDP) or from GenBank
by using the  software (Corpet, 1988). Positions
that had not been sequenced in one or more reference
organisms were omitted and a total of 1247 nucleotides were
used in the analysis. Pairwise evolutionary distances were
computed by using the correction of Jukes & Cantor (1969).
The phylogenetic tree was constructed by the neighbourjoining method (Saitou & Nei, 1987) with bootstrap analysis
of 100 trees using the programs of the  package (Van
de Peer & De Wachter, 1994), with Methanococcus vannielii
as the outgroup.
Nucleotide sequence accession numbers. The accession
numbers of the sequences used as references are as follows :
Sulfurisphaera ohwakuensis TA1T, D85507 ; Sulfolobus acidocaldarius DSM 639T, D14053 ; Thermosphaera aggregans
M11TLT, X99556 ; Staphylothermus marinus F1 DSM 3639T,
X99560 ; Hyperthermus butylicus DSM 5456T, X99553 ;
Sulfophobococcus zilligii K1T, X98064 ; Stygiolobus azoricus
DSM 6296T, D85520 ; Acidianus infernus DSM 3191T,
D85505 ; Aeropyrum pernix K1T, D83259 ; Metallosphaera
sedula DSM 5348T, X90481 ; Stetteria hydrogenophila
4ABCT, Y07963 ; Pyrodictium occultum PL19, M21087 ;
Desulfurococcus mobilis, M36474 ; Thermofilum pendens
Hvv3T, X14835 ; Thermoproteus tenax, M35966 ; Pyrobaculum islandicum geo3T, L07511 ; Caldivirga maquilingensis IC-167T, AB013926 ; Thermocladium modestius IC-125T,
AB005296 ; and ‘ Caldococcus noboribetus ’ NC12, D85038.
RESULTS
Enrichment and isolation
Samples obtained from acidic hot springs from different areas in Kamchatka were used to inoculate
anaerobic starch medium at pH 3n0. After 2–5 d
incubation at 80 mC, eight samples showed microbial
growth (Table 1). Cells of irregular coccoid shape were
observed in all cultures. When transferred to Gelritesolidified medium, smooth white colonies approximately 1 mm in diameter appeared after 7 d incubation. By isolating single colonies, strains 1904T, 1919
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Acidilobus aceticus gen. nov., sp. nov.
Table 1. Characteristics of the Kamchatka sampling sites used for enrichment of anaerobic organotrophic
thermoacidophiles
Sample no.
1904T
1919
1920
301
310
321
322
345
Location
Description
Temp. (mC)
pH
Mutnovski volcano
Mutnovski volcano
Mutnovski volcano
Geyser valley
Geyser valley
Uzon caldera, Orange thermal field
Uzon caldera, Orange thermal field
Moutnovski volcano
Hot spring with ferric iron deposits
Hot well surrounded with grass
Hot spring with ferric iron deposits
Black mud near the Giant geyser
Hot well near the Giant geyser
Mud hole
Mud hole
Mud hole
87
90
90
91
80
70
87
82
4n0
3n0
3n0
4n5
4n5
4n0
4n5
4n0
(a)
(b)
S
ol
(c)
cm
.................................................................................................................................................
Fig. 2. Electron micrographs of ultrathin sections of isolate
1904T cells in the late-exponential phase of growth.
Abbreviations : s, subunit ; cm, cytoplasmic membrane ; ol,
osmiophilic layer. Bars, 0n5 µm.
Morphology and ultrastructure
.................................................................................................................................................
Fig. 1. Electron micrographs of isolate 1904T whole cells in the
late-exponential phase of growth. Bars, 0n5 µm.
and 1920 were obtained. Isolate 1904T, obtained from
the Moutnovski volcano sample, showed the best
growth and was selected for further experiments.
Cells of isolate 1904T were regular to irregular cocci
approximately 1–2 µm in diameter (Fig. 1, top). Active
motility was never observed in light microscope, and
no flagella were ever seen on the electron micrographs
of the whole cells. Double or triple cells were frequently
observed in young cultures (Fig. 1, middle and bottom). Electron microscopy of thin sections revealed
that the cell envelope comprised an S-layer attached to
the cytoplasmic membrane (Fig. 2, top). The S-layer
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2003
7
Cells (i10 –6 ml –1)
Doubling time (h)
6
5
4
3
2
70
75
80
85
90
Temperature (°C)
95
T
Fig. 3. Influence of temperature on the growth of isolate 1904
on the medium with yeast extract and elemental sulfur at pH
3n8.
25
2·5
20
2·0
15
1·5
10
1·0
5
0·5
2
3
4
5
Time (d)
6
7
8
0
.................................................................................................................................................
Fig. 5. Production of H2S (
) and acetate (>), and growth ($),
of isolate 1904T on the medium with yeast extract and S0.
was 2n5 h. The maximum cell concentration achieved
was 2–2n5i10( cells mlV". Isolate 1904T was found to
be a strict anaerobe unable to grow without prereduction of the medium.
13
Isolate 1904T was found to grow on starch, yeast
extract, beef extract and soya extract. None of the
following supported growth : acetate, arabinose, cellulose, formate, fructose, galactose, glucose, glycine,
guar gum, lichenan, malate, maltose, methanol, pectin,
pyruvate, propionate, xylan, xylose or a mixture of 20
amino acids. Yeast extract (100 mg lV") was required
as a growth factor when starch was used as the growth
substrate.
11
Doubling time (h)
3
1
100
.................................................................................................................................................
9
7
5
3
1
1
30
Acetate, H2S (µmol ml –1)
M. I. Prokofeva and others
2
3
4
5
6
pH
.................................................................................................................................................
Fig. 4. Influence of pH on the growth of isolate 1904T on the
medium with yeast extract and elemental sulfur (incubation
temperature, 80 mC).
Growth of isolate 1904T on a medium with yeast
extract, beef extract and soya extract as the growth
substrates was stimulated by elemental sulfur, which
was reduced to hydrogen sulfide. On a medium with
starch, the stimulatory effect of sulfur was less prominent. Nitrate, at all concentrations tested, did not
influence growth on either medium. Fe(III), added as
ferric oxide or ferric citrate, did inhibit growth.
was composed of a single layer of subunits covered
with a thin osmophilic layer that was probably
proteinaceous in nature (Fig. 2, bottom).
The main metabolic product of isolate 1904T, both on
yeast extract (Fig. 5) and on starch, was acetate.
Neither H nor ethanol was formed in the course of
growth on# any of the substrates used. Isolate 1904T
was shown to be resistant to penicillin, streptomycin
and chloramphenicol.
Physiology
The GjC content of the DNA
Isolate 1904T grew at temperatures between 60 and
92 mC, with an optimum at 85 mC (Fig. 3), and within a
pH range of 2n0–6n0, with an optimum at 3n8 (Fig. 4).
The minimum doubling time under optimal conditions
The GjC content of the total DNA of isolate 1904T
was 53n8 mol %. Similar values were obtained for other
isolates : 54n5 mol % (isolate 1919), 55n1 mol % (isolate
1920).
2004
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Acidilobus aceticus gen. nov., sp. nov.
.................................................................................................................................................
Fig. 6. Phylogenetic tree showing the relationship between
novel strain 1904T and other archaea, based on a comparison of
16S rDNA sequences.
compared with the corresponding sequence data from
the RDP. This analysis revealed that new isolate 1904T
was a member of the kingdom Crenarchaeota of the
domain Archaea. Additional sequence alignments and
phylogenetic analyses performed with type species of
the validly published genera of this kingdom showed
that strain 1904T was not closely related to any
reference organism (82n5–90n8 % sequence similarity).
In the phylogenetic tree (Fig. 6), strain 1904T formed a
single cluster with Aeropyrum pernix (90n8 % sequence
similarity), but the bootstrap probability of this
branching point was not high (80 %). More detailed
analysis of the 16S rRNA gene of strain 1904T revealed
unique deviation from the known crenarchaeotal
sequences at positions where all other Crenarchaeota
exhibited conserved bases (Table 2). Of these changes,
the deviations at positions 592 : 647, 1308 : 1329 and
1310 : 1327 occurred (using the inferred secondary
structure) in helixes and involved paired bases (compensatory changes). When the 16S rRNA sequence of
isolate 1904T was compared with all available sequences, it exhibited a high degree of 16S rRNA
sequence similarity (98n3 %) to crenarchaeotal strain
NC12 (‘ Caldococcus noboribetus ’) and had the same
signature nucleotides.
Table 2. Sequence signatures of the 16S rRNA from
isolate 1904T
DISCUSSION
Sequence
position*
321 : 332
592 : 647
678
913
1302
1308 : 1329
1310 : 1327
1335
1393
1414
Corresponding bases†
Isolate 1904T
Common to
crenarchaeotal species‡
C:G
C:G
C
U
C
U:A
G:C
C
U
U
A:G
G (u,a) : C (a,u)
U
A
U (a)
C:G
A:U
G
C
C
* Numbering according to Escherichia coli nomenclature.
† Base pairing was deduced from secondary-structure assignment.
‡ All of the crenarchaeotal 16S rRNA sequences were from the
Ribosomal Database Project ; lower-case letters indicate bases
found in less than 15 % of assayable cases.
Analysis of 16S rDNA
The almost complete (1411-nucleotide) sequence of the
16S rRNA gene (corresponding to positions 27–1479
using Escherichia coli numbering) of isolate 1904T was
determined. It was found to have a high GjC content
(67n3 mol %), like the 16S rRNA genes of other
thermophilic prokaryotic organisms. In an initial
analysis, the 16S rDNA sequence of strain 1904T was
Hot springs with low-pH water are quite common in
different volcanic areas of the world (Brock, 1978 ;
Stetter et al., 1990). The first representative of extremely thermophilic archaea, Sulfolobus solfataricus,
was isolated in 1972 and was a thermoacidophile
growing optimally at 70–75 mC and pH 2n0–3n0 (Brock
et al., 1972). More recently, significant progress has
been made in investigations of both the phenotypic
and the phylogenetic diversity of hyperthermophiles
(Blo$ chl et al., 1995 ; Stetter, 1996) ; 10 genera of
thermoacidophilic archaea have been described (Table
3). Most of the genera belong to the Crenarchaeota,
with the exception of Thermoplasma (Segerer et al.,
1988) and Picrophilus (Schleper et al., 1995), which
belong to the order Thermoplasmales of the kingdom
Euryarchaeota. The majority of the thermoacidophilic
archaea are aerobes capable of lithotrophic growth
with elemental sulfur, sulfides or molecular hydrogen
as electron donors (Stetter, 1996 ; Scho$ nheit & Scha$ fer,
1995). Some of them, like Acidianus infernus (Segerer
et al., 1986), are facultative anaerobes and are capable
of anaerobic growth with molecular hydrogen by using
elemental sulfur as an alternative electron acceptor.
The metabolism of Thermoproteus tenax (Zillig et al.,
1981) and Stigiolobus azoricus (Segerer et al., 1991),
which are obligate anaerobes, is also based on this
reaction. Whilst most of the organisms mentioned
above have coccoid or flat irregular cells, representatives of the genus Thermoproteus have rod-shaped
cells (Zillig et al., 1981). In this genus, it is only the type
species, Thermoproteus tenax, that is moderately acidophilic, growing at pH values in the range 2n5–6n0 (with
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M. I. Prokofeva and others
Table 3. Characteristics of thermoacidophilic archaea
.................................................................................................................................................................................................................................................................................................................
Abbreviations : R, respiration ; F, fermentation ; Org, organic substrates.
Genus/type species
Topt. (mC) pHopt.
Type of
metabolism
Sulfolobus\S. acidocaldarius
70–75
2–3
R
Thermoproteus\T. tenax
Sulfurococcus\S. mirabilis
Acidianus\A. infernus
Thermoplasma\T. acidophilum
Metallosphaera\M. sedula
Stigiolobus\S. azoricus
Picrophilus\P. oshimae
Sulfurisphaera\S. ohwakuensis
Thermocladium\T. modestius
90
70–75
90
59
75
80
60
84
75
5
2–3
2
1–2
1–4n5
2n5–3
0n7
2n0
4n0
R
R
R
F\R
R
R
R
R
R
85
3n8
F(R?)
Isolate 1904T
an optimum at pH 5n0). Other species, as well as
representatives of the genus Pyrobaculum (Huber et
al., 1987), grow optimally at pH 6n0–7n0. Members of
these two genera also have a respiratory metabolism
and are capable of lithotrophic or organotrophic
growth via sulfur respiration (Scho$ nheit & Scha$ fer,
1995) or, in the case of Pyrobaculum aerophilum (Vo$ lkl
et al., 1993), via aerobic respiration or nitrate reduction. The fermentative growth capacity was
observed only with Thermoproteus uzoniensis (BonchOsmolovskaya et al., 1991), which is an obligate
anaerobe and neutrophile. This type of metabolism is
quite common among representatives of the neutrophilic, hyperthermophilic archaea (Scho$ nheit &
Scha$ fer, 1995). In the Euryarchaeota kingdom, they
are represented by the genera Thermococcus (Zillig
et al., 1983) and Pyrococcus (Fiala & Stetter, 1986). In
the Crenarchaeota, obligately anaerobic fermentative
micro-organisms belong to the genera Desulfurococcus
(Zillig et al., 1982), Staphylothermus (Fiala et al.,
1986), Hyperthermus (Zillig et al., 1990), Thermosphaera (Huber et al., 1998), Sulphophobococcus
(Hensel et al., 1997) and Pyrodictium (Pley et al., 1991).
All these organisms are capable of fermenting peptides
and\or polysaccharides and produce volatile fatty
acids, hydrogen and CO . Stetteria hydrogenophila
#
(Jochimsen et al., 1997) requires
molecular hydrogen
and elemental sulfur for its organotrophic growth and
thus was considered to possess a respiratory type of
metabolism. All these organisms are extreme thermophiles or hyperthermophiles, and neutrophiles.
Our findings show that obligately anaerobic organisms
with fermentative metabolism are widespread in
Kamchatka hot springs with low-pH water. Organic
substrates for their growth might be synthesized by
lithotrophic components of the same microbial community (Bonch-Osmolovskaya et al., 1999) or might
2006
Electron
donors
Electron
acceptors
H , org, S!,
O
#
#
Fe#V, S O#V
% '
S!
H ,org
#
Org, S!, Fe#V, S O#V
O
% '
#
S!, Fe#V, H
S!, O
#
#
Org
O
#
V
H , org, S!, S O#
O
#
% '
#
H
S!
#
Org
O
#
H , org, S!
S!, O
#
V #
Org
S!, SO# , S O#V
%
# $
-cystine
Org
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Reference
Brock et al. (1972)
Zillig et al. (1981)
Golovacheva et al. (1985)
Segerer et al. (1986)
Segerer et al. (1988)
Huber et al. (1989)
Segerer et al. (1991)
Schleper et al. (1995)
Kurosawa et al. (1998)
Itoh et al. (1998)
This work
come from surrounding areas with abundant vegetation. Isolate 1904T, described in this work, is a true
hyperthermophile, having a temperature optimum for
growth of 85 mC, and is a true acidophile, having a pH
optimum for growth of 3n8. Isolate 1904T produced
significant growth on organic substrates in the absence
of elemental sulfur ; acetate was the main metabolic
product. The metabolism of this isolate, therefore,
could be characterized as fermentative. The growthstimulating action of sulfur could be explained by the
need for an additional electron sink for the fermentation of some of the yeast-extract components. This
phenomenon was shown for several organotrophic,
hyperthermophilic archaea (Fiala & Stetter, 1986 ;
Bonch-Osmolovskaya & Miroshnichenko, 1994).
However, the possibility of respiratory metabolism in
this organism cannot be excluded until additional
experiments have been performed. Three other strains,
isolated from different terrestrial thermal habitats of
low pH, phenotypically resemble isolate 1904T and
have similar GjC DNA contents. All of the isolates
probably represent the same species.
Comparison of an almost complete 16S rDNA sequence from isolate 1904T with nucleotide sequences
of reference micro-organisms showed that it does not
belong to any validly described Crenarchaeota genera.
Isolate 1904T exhibited a high degree of 16S rDNA
sequence similarity (98n3 %) and shared signature
nucleotides only with crenarchaeotal strain NC12 (‘ C.
noboribetus ’ ; Aoshima et al., 1996). The description of
this organism, however, has never been published and
strain NC12 was not available for the comparison. Of
the representatives of the validated Crenarchaeota
taxa, the closest to our isolate was A. pernix (90n8 %
similarity). This organism is a neutrophile and an
aerobe (Sako et al., 1996). On the basis of the
differentiating phenotypic and genomic features of
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Acidilobus aceticus gen. nov., sp. nov.
isolate 1904T, we propose that this isolate should be
assigned to a new genus – Acidilobus – the type species
being Acidilobus aceticus.
Description of Acidilobus gen. nov.
Acidilobus (A.ci.di.lohbus. L. masc. adj. acidus acid ;
Gr. masc. n. lobos lobe ; M.L. masc. n. Acidilobus acid
lobe).
Cells are regular to irregular cocci. The cell envelope
consists of an S-layer attached to the cytoplasmic
membrane. Archaeon. Hyperthermophile. Acidophile.
Obligate anaerobe. Organotroph. Peptides and polysaccharides serve as energy and carbon sources.
Acetate is the major growth product. Elemental sulfur
stimulates growth and is reduced to hydrogen sulfide.
Resistant to antibiotics. The type species is Acidilobus
aceticus. The habitat is terrestrial acidic hot springs.
nov. – a new thermophilic sulfur-reducing eubacterium. Arch
Microbiol 153, 151–155.
Bonch-Osmolovskaya, E. A., Miroshnichenko, M. L., Kostrikina,
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influence of molecular hydrogen and elemental sulfur on the
metabolism of extremely thermophilic archaea of genus Thermococcus. Microbiology (English translation of Mikrobiologiya)
63, 777–782.
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A. I. & 7 other authors (1999). Biodiversity of anaerobic
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High Temperatures. New York : Springer.
Brock, T. D., Brock, K. M., Belly, R. T. & Wiess, R. L. (1972).
Description of Acidilobus aceticus sp. nov.
Acidilobus aceticus (a.cehti.cus. L. masc. adj. aceticus
producing acetate).
Cells are non-motile, regular to irregular cocci approximately 1–2 µm in diameter. The cell envelope consists
of an S-layer attached to the cytoplasmic membrane.
Growth occurs in the temperature range 60–92 mC
(optimum at 85 mC) and in the pH range 2n0–6n0
(optimum at 3n8). Strictly anaerobic. Heterotrophic.
Yeast extract, beef extract, soya extract and starch
may serve as growth substrates. Elemental sulfur
stimulates growth on yeast extract but is not obligately
required. Growth products are acetate and, in the
presence of S!, H S. Resistant to penicillin, strep#
tomycin and chloramphenicol.
The source of isolation
was a solfataric field near the Moutnovski volcano,
Kamchatka. The GjC content of the DNA is
53n8 mol %. The type strain is Acidilobus aceticus 1904T
(l DSM 11585T).
ACKNOWLEDGEMENTS
This work was supported by the Russian Foundation for
Basic Research, grants no. 96-04-49463 and 99-04-48360
and by the ‘ Biodiversity Programme ’ of the Russian
Ministry of Science and Technology. We also thank T. A.
Pivovarova (Institute of Microbiology, Russian Academy of
Sciences) for the strain of Thiobacillus ferrooxidans used in
this work.
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