Desulfurobacterium atlanticum sp. nov.,
Desulfurobacterium pacificum sp. nov. and
Thermovibrio guaymasensis sp. nov., three
thermophilic members of the
Desulfurobacteriaceae fam. nov., a deep branching
lineage within the Bacteria
S. L’Haridon,1A.-L. Reysenbach,2B. J. Tindall,3P. Scho ¨nheit,4A. Banta,2
U. Johnsen,4P. Schumann,3A. Gambacorta,5E. Stackebrandt3
and C. Jeanthon13
1UMR 6197, Centre National de la Recherche Scientifique, IFREMER and Universite ´ de
Bretagne Occidentale, IFREMER Centre de Brest, BP 70, 29280 Plouzane ´, France
2Portland State University, Department of Biology, Portland, OR 97201, USA
3German Collection of Microorganisms and Cell Cultures DSMZ, Inhoffenstrasse 7b, D–38124
4Institut fu ¨r Allgemeine Mikrobiologie, Christian-Albrechts-Universita ¨t Kiel, Am Botanischen
Garten 1-9, 24118 Kiel, Germany
5Istituto di Chimica Biomolecolare, Via Campi Flegrei 34, 80078 Puzzuoli, Napoli, Italy
Three thermophilic, anaerobic, strictly chemolithoautotrophic, sulphur- and/or thiosulphate-
reducing bacteria, designated SL17T, SL19Tand SL22T, were isolated from deep-sea
hydrothermal samples collected at 13 6N (East Pacific Rise), Guaymas Basin (Gulf of California)
and 23 6N (Mid-Atlantic Ridge), respectively. These strains differed in their morphology,
G+C content of the genomic DNA was 41 mol% (SL22T), 42 mol% (SL17T) and 46 mol%
(SL19T). Comparative analysis of phenotypic and phylogenetic traits indicated that strains SL17T
and SL22Trepresented two novel species of the genus Desulfurobacterium and that strain SL19T
should be considered as a novel species of the genus Thermovibrio. The names
Desulfurobacterium pacificum sp. nov. (type strain SL17T=DSM 15522T=JCM 12127T),
Desulfurobacterium atlanticum sp. nov. (type strain SL22T=DSM 15668T=JCM 12129T) and
Thermovibrio guaymasensis sp. nov. (type strain SL19T=DSM 15521T=JCM 12128T) are
analyses correlated with the significant phenotypic differences between members of the lineage
encompassing the genera Desulfurobacterium, Thermovibrio and Balnearium and that of the
families Aquificaceae and Hydrogenothermaceae. It is therefore proposed that this lineage
represents a new family, Desulfurobacteriaceae fam. nov., within the order Aquificales.
3Present address: UMR 7144, Equipe Prokaryotes Photosynthe ´tiques Marins, Station Biologique, Place Georges Teissier, 29680 Roscoff, France.
The GenBank/EMBL/DDBJ accession numbers for the almost complete 16S rRNA gene sequences of strains SL17T, SL19Tand SL22Tare
AY268936, AY268937 and AY268939, respectively.
Supplementary tables detailing the respiratory lipoquinone composition and cellular fatty acid content of the three novel strains and the reductive citric
acid cycle enzymes of Desulfurobacterium thermolithotrophum are available in IJSEM Online. Electron micrographs of cells of the three novel strains,
TLCs of polar lipids and a figure depicting the structure of the aminophospholipid of D. thermolithotrophum are also available as supplementary figures.
Abbreviations: bm, broad multiplet; b or bs, broad signal; BV, benzyl viologen; cm, complex multiplet; d, doublet; dd, double doublet; ddm, double doublet
multiplet; dt, double triplet; m, multiplet; TEA, triethanolamine.
63994 G 2006 IUMSPrinted in Great Britain2843
International Journal of Systematic and Evolutionary Microbiology (2006), 56, 2843–2852
the phylum Aquificae (Reysenbach, 2001a) is generally
considered to be one of the deepest and earliest branching
groups with the Bacteria. It encompasses two families within
2001b). The family Aquificaceae is composed of five genera,
Hydrogenobacter, Aquifex, Thermocrinis, Hydrogenobaculum
1998; Sto ¨hr et al., 2001; Nakagawa et al., 2004), while the
family Hydrogenothermaceae (Eder & Huber, 2002) is
formed by the genera Hydrogenothermus, Persephonella and
Sulfurihydrogenibium (Sto ¨hr et al., 2001; Go ¨tz et al., 2002;
Takai et al., 2003a).
Members of the order Aquificales are Gram-negative
thermophilic rods capable of chemolithotrophic microaer-
ophilic growth using H2, O2 and CO2. Cultivated
representatives of the Aquificales have been isolated from
terrestrial hydrothermal systems, deep gold mines and
shallow and deep-sea hydrothermal vents.
In contrast, strains of the genera Desulfurobacterium,
Thermovibrio and Balnearium are strictly anaerobic chemo-
lithoautotrophs using hydrogen exclusively as the electron
donor and sulphur or nitrate as the main electron acceptors
(L’Haridon et al., 1998; Huber et al., 2002; Alain et al., 2003;
Takai et al., 2003b; Vetriani et al., 2004). These deeply
branching thermophilic bacteria have been isolated exclu-
sively from marine hydrothermal systems and form a
monophyletic cluster on the basis of their 16S rRNA gene
a single species, Desulfurobacterium thermolithotrophum, and
wasplaced withinthephylumAquificae asgenusincertae sedis
(L’Haridon & Jeanthon, 2001). 16S rRNA gene sequences
related to those of this organism were detected in environ-
mental samples obtained from deep-sea hydrothermal vents
on the Juan de Fuca Ridge and the Mid-Atlantic Ridge, the
et al., 2003). In situ hybridization experiments demonstrated
that D. thermolithotrophum and phylogenetically closely
related species could represent up to 40% of the bacterial
(Harmsen et al., 1997).
In this study, we report the isolation and characterization of
novel, extremely thermophilic, strictly anaerobic chemo-
lithoautotrophic strains obtained from geographically
distant deep-sea hydrothermal vents. Based on 16S rRNA
gene sequence analyses, the novel strains clustered within
the lineage encompassing the genera Desulfurobacterium,
Thermovibrio and Balnearium.
Reference strains. D. thermolithotrophum DSM 11699Twas iso-
lated in our laboratory (L’Haridon et al., 1998) and we recently
updated the GenBank 16S rRNA gene sequence (GenBank accession
no. AJ001049) of the type strain. Thermovibrio ruber DSM 14644T
was obtained from the Deutsche Sammlung von Mikroorganismen
und Zellkulturen (DSMZ, Brauschweig, Germany).
Collection of hydrothermal samples, enrichment cultures
and purification. Chimney structures and/or sediment cores were
collected in the Guaymas Basin (27u 019 N 111u 249 W) at a depth
of 2000 m, on the East Pacific Rise (EPR; 12u 499 N 103u 569 W) at
a depth of 2600 m and on the Mid-Atlantic Ridge (Snake Pit; 23u
229 N 44u 579 W) vent fields at a depth of 3500 m. Using the port
manipulator of the submersible Nautile, these samples were placed
in a submersible insulated basket for the trip to the surface. On
board, subsamples were transferred to 50 ml glass vials and flooded
with a sterile solution of 3% (w/v) sea salts (Sigma). The vials were
then closed tightly with butyl rubber stoppers (Bellco), pressurized
with N2(100 kPa), reduced with sodium sulphide and stored at 4uC
until processed further.
Enrichment, isolation and cultivation of thermophilic chemolitho-
trophic bacteria were performed in a basal medium containing
(distilled water l21): 20 g NaCl; 1 g NH4Cl; 0?35 g KH2PO4, 1?95 g
MES, 1 g NaHCO3, 1 ml trace element mixture (Widdel & Bak, 1992),
1 ml selenite-tungstate solution (Widdel & Bak, 1992), 1 ml vitamin
mixture (Widdel & Bak, 1992), 1 ml thiamine solution (Widdel & Bak,
1992), 1 ml vitamin B12solution (Widdel & Bak, 1992), 1 ml growth-
stimulating factors (distilled water 100 ml21: 0?5 g isobutyric acid,
0?5 g valeric acid, 0?5 g 2-methylbutyric acid, 0?5 g 3-methylbutyric
acid, 0?2 g caproic acid and 0?6 g of succinic acid; Pfennig et al., 1981)
and 1 mg resazurin. The enrichment medium was supplemented with
10 g sulphur or 20 mM thiosulphate. The pH of the medium was
adjusted to 6 using 1 M NaOH before autoclaving. H2/CO2(80:20;
200 kPa) was used as the gas phase. Unless otherwise indicated,
cultures were incubated at 65uC and the pH of the medium was
readjusted after 1 h incubation. Enrichments were performed
anaerobically in 50 ml vials according to Miller & Wolin (1974) and
and purified by streaking onto the basal medium supplemented with
thiosulphate (20 mM) and polysulphides and solidified with 0?7%
(w/v) Phytagel (a gellan gum from Sigma). Plates were incubated in
anaerobic jars at 65uC for 3 days under a H2/CO2atmosphere (80:20;
200 kPa). Stock cultures of the isolates were stored in culture medium
at 4uC. For long term storage, pure cultures were stored at 280uC in
the same medium containing 10% (w/v) DMSO.
Determination of growth parameters and requirements. The
influence of pH on growth, growth requirements and antibiotic sus-
ceptibility were determined as described previously (L’Haridon et al.,
1998). In order to determine the salt requirement, medium was
prepared with increasing amounts of NaCl and incubated at the
optimal temperature and pH for growth. Growth was determined by
measuring changes in turbidity at 600 nm in a spectrophotometer
(Spectronic 401; Bioblock). All growth experiments were performed
Light and electron microscopy experiments were performed as
described previously (L’Haridon et al., 1998).
H2S production was evaluated by adding 500 ml CuSO4 solution
(5 mM) and HCl (50 mM) to 250 ml culture grown at 65uC. A dark
brown precipitate demonstrated the presence of sulphide and was
compared with the uninoculated medium incubated under the same
conditions. The production of ammonium was evaluated by adding
0?1 ml of a freshly prepared mixture of 0?5 ml NaOH (27% w/v) and
0?5 ml potassium tetraiodomercurate (II) solution (Nessler’s reagent)
to 0?5 ml culture medium. An orange precipitate indicated the
presence of ammonium.
2844 International Journal of Systematic and Evolutionary Microbiology 56
S. L’Haridon and others
Extraction and analysis of respiratory lipoquinones, polar
lipids and fatty acids. Respiratory lipoquinones and polar lipids
were extracted from 100 mg freeze-dried cell material using the two
stage method described by Tindall (1990a, b).
Respiratory lipoquinones were separated into their different classes
(menaquinones and ubiquinones) by TLC on silica gel, eluted and
further analysed by HPLC.
Polar lipids were separated by two dimensional silica gel TLC as
described by Tindall (1990a, b). Total lipid material and specific
(total lipids), Zinzadze reagent (phosphate), ninhydrin (free amino
groups), periodate-Schiff (a-glycols), Dragendorff (quaternary nitro-
gen) and anisaldehyde-sulphuric acid (glycolipids).
Fatty acids were analysed as the methyl ester derivatives prepared from
10 mg dry cell material using methods described by Labrenz et al.
Structure analysis of an aminophospholipid of D. thermo-
lithotrophum strain BSAT. Wet cells (15 g) were extracted and
analysed by TLC as described by De Rosa & Gambacorta (1994).
The total lipid extract was firstly purified by flash chromatography
on silica gel and eluted with chloroform/methanol/water (65:25:4,
by vol.). The final purification was achieved by TLC developed with
chloroform/methanol/water (65:25:4, by vol.). The spots, visualized
by iodine vapour, were scraped from the plates and eluted from the
silica gel by chloroform/methanol (1:1). The compounds were
L’Haridon et al. (1998). The hydrolysed compounds were purified
and analysed by
et al. (1998). The NMR spectra were recorded on a Bruker AMX
500 (500?13 MHz for
Chemical shifts are given in p.p.m. (d) the chloroform signal was
used as an internal standard (d 7?601H; d 77?013C). The spectra
were performed in deuterated chloroform (CDCl3)–methanol (1:1)
for polar lipids and in CDCl3for fatty acid methyl esters (FAME).
Distortionless enhancement by polarization transfer (DEPT) experi-
ments were performed according to the methods of Doddrell et al.
(1982). NMR experiments included1H-1H COSY (correlation spec-
troscopy) and HMQC (heteronuclear multiple quantum coherence).
13C-NMR and hydrolysed as reported by
1H-NMR and GC-MS as described by L’Haridon
1H and 125?75 MHz for
Isolation of DNA, RFLP analysis, sequencing and phyloge-
netic analysis of the 16S rRNA genes. Genomic DNA was iso-
lated after disruption of cells using a French pressure cell (Thermo
Spectronic)and purified by
(Cashion et al., 1977). The DNA was hydrolysed with P1 nuclease
and the nucleotides dephosphorylated with bovine alkaline phospha-
tase (Mesbah et al., 1989). The G+C content of the DNA was deter-
mined by the HPLC method described by Tamaoka & Komagata
A total of about 1500 nucleotides were sequenced using a previously
described suite of primers (Go ¨tz et al., 2002). Sequence alignment
and phylogenetic analyses were done using 1399 homologous
nucleotides as described previously (Jeanthon et al., 2002). Using
only Desulfurobacteriaceae sequences included in the analysis, all
nucleotides (and gaps) were used to construct distance matrices by
pairwise analysis with the Jukes and Cantor correction (Jukes &
Cantor, 1969). Comparisons using a more conserved subset of
nucleotides (only 1242 positions) did not change the distances
significantly. Maximum-likelihood, maximum-parsimony and neigh-
bour-joining analyses were performed as described previously (Go ¨tz
et al., 2002).
Reductive citric acid cycle for autotrophic CO2fixation in D.
thermolithotrophum. D. thermolithotrophum BSATwas grown
anaerobically at 65uC at pH 6?5 in a 100 l Biostat fermenter in
the medium described by L’Haridon et al. (1998). Cells were
harvested in the late exponential growth phase at cell densities of
Frozen cells were suspended in 0?1 M Tris/HCl pH 7 containing
10 mM MgCl2. The suspension was passed through a French pressure
cell at 137 MPa. Cell debris and unbroken cells were removed by
centrifugation (20 min at 10000 g). The supernatant (cell-free extract)
contained about 8 mg protein ml21. Protein was determined by the
method of Bradford (1976) using bovine serum albumin as standard.
All enzyme assays were performed in cuvettes containing 1 ml assay
mixtures. Reactions involving NADH or NAD were followed spectro-
photometrically at 365 nm (e=3?4 cm21mM21). One unit (U) of
enzyme activity is defined as 1 mmol substrate consumed or product
formed per minute. Enzyme assays following benzyl viologen (BV)
reduction were carried out under anoxic conditions in stoppered glass
cuvettes with N2. Assay mixtures were slightly reduced by the
addition of a sodium dithionite solution prior to the reaction
start. BV reduction was followed spectrophotometrically at 578 nm
(e=17?2 cm21mM21).Oneunitofenzymeactivityisequalto2 mmol
BV reduced per minute.
ATP citrate lyase was determined according to Beh et al. (1993) at
50uC. The assay mixture contained 100 mM triethanolamine (TEA)
pH 7, 5 mM MgCl2, 5 mM citrate, 0?3 mM NADH, 2 mM ATP,
0?5 mM CoA, 10 mM dithioerythritol (DTE) and 2 U malate
dehydrogenase. Malate dehydrogenase was determined at 50uC in
contained 100 mM TEA pH 7, 5 mM MgCl2, 3 mM oxaloacetate and
0?3 mM NADH. Fumarase was measured at 65uC by following the
formation of fumarate at 250 nm (e=1?44 cm21mM21). The assay
mixture contained 100 mM TEA pH 7 and 10 mM malate. Fumarate
reductase was determined at 65uC by measuring fumarate-dependent
oxidation ofreduced BVat 578 nm,according to Behet al.(1993). The
assay mixture contained 100 mM TEA pH 7, 2 mM BV, 1 mM
fumarate and 5 mM DTE. Succinyl-CoA synthetase was determined at
55uC according to Selig & Scho ¨nheit (1994). The assay mixture
contained 100 mM TEA pH 7, 0?3 mM NADH, 5 mM MgCl2, 2 U
pyruvate kinase, 1 U lactate dehydrogenase, 2 mM phosphoenol
pyruvate, 0?5 mM CoA, 5 mM succinate and 2 mM ATP. 2-
the CoA dependent reduction of BV with 2-oxoglutarate at 578 nm.
The assay mixture contained 100 mM TEA pH 7, 10 mM DTE,
0?5 mM CoA, 5 mM 2-oxoglutarate and 5 mM BV. 2-Oxoglutarate
dehydrogenase was determined at 65uC by using the 2-oxoglutarate:
BV oxidoreductase assay, containing 1 mM NAD+instead of BV.
Isocitrate dehydrogenase was assayed at 70uC. The assay mixture
contained 100 mM TEA pH 6?6, 5 mM MgCl2, 1 mM NAD+and
1 mM isocitrate. Aconitase was determined at 50uC by using the ATP
citrate lyase assay except that the mixture contained 5 mM isocitrate
instead of citrate.
RESULTS AND DISCUSSION
Enrichment and isolation of strains
In order to enrich for chemolithoautotrophic, sulphur- and
thiosulphate-reducing thermophiles, 10 ml enrichment
medium was inoculated with approximately 1 ml of
chimney or sediment suspensions. The enrichments were
performed in 50 ml vials with H2/CO2as the gas phase
(80:20; 200 kPa) without shaking at 65uC. Within
2–3 days, turbidity caused by cell growth was observed
Desulfurobacteriaceae fam. nov., with three novel species
and H2S was produced with the reduction of sulphur or
thiosulphate. Subcultures were streaked onto solidified
phase at 65uC. On thiosulphate-polysulphide medium,
round yellow to orange colonies (1 mm in diameter) were
visible after incubation for up to 3 days. Three strains,
designated SL17T, SL19Tand SL22T, were obtained in pure
cultures after three successive streakings on plates and were
selected for further detailed characterization.
Morphology, physiology and growth
requirements of strains SL17T, SL19Tand SL22T
Cells of strains SL17T, SL19Tand SL22Tstained Gram-
negative and occurred singly or in pairs. Chains of 5–6 cells
were formed by cells of strains SL19Tand SL22T. Cells of
strains SL17T(straight rods about 1–2 mm long and
0?4–0?5 mm wide) and SL19T(coccoid to lemon-shaped
rods about 1–2 mm long and 1–2 mm wide), appeared to be
highly motile and up to 3 and 4 monopolar flagella,
respectively, could be observed by negative staining (see
Supplementary Fig. S1 in IJSEM Online). Cells of strain
SL22Tformed straight to curved motile rods, about
2?5–3?5 mm long and 0?4–0?5 mm wide. Up to three
monopolar flagella could be observed by negative staining
of cells. Some cells of the three strains became spherical in
the stationary growth phase.
The three strains showed differences in their temperature
optima and ranges for growth. Strain SL17Tgrew between
55 and 85uC with an optimum around 75uC, but no growth
was detected at 50uC or 88uC after 48 h incubation. Strain
SL19Tgrew between 50 and 88uC with an optimum around
75–80uC, but no growth was detected at 45uC or 90uC after
48 h incubation. Strain SL22Tgrew between 50 and 80uC
with an optimum around 70–75uC, but no growth was
detected at 45uC or 85uC after 48 h incubation. Growth was
pH 6-6?2, for strains SL17Tand SL19T. No growth was
detected for either strains at pH 5?4 or 8 after 48 h
incubation at 75uC. Strain SL22Tgrew between pH 5?5
and 7 with an optimum around 5?8–6, but no growth was
detected at pH 5?4 or 7?5 after 48 h incubation at 75uC.
Growth of the three novel strains could be observed at NaCl
concentrations ranging from 15 to 50 g l21, with an
optimum of approximately 30 g l21. The novel strains did
not grow at 10 or 60 g NaCl l21after 48 h incubation at
The three novel strains were unable to grow in the culture
medium with sulphur or thiosulphate in the presence of
oxygen, even at low concentrations (0?2–1%). These strict
anaerobes were autotrophic organisms that utilized sulphur
and nitrate (SL17Tand SL19T) or thiosulphate (SL17Tand
SL22T) as the electron acceptor in the presence of H2for
growth. They did not utilize sulphite, cystine, sulphate or
nitrite. Growth on sulphur compounds and nitrate was
accompanied by exponential H2S and ammonium produc-
tion, respectively, which paralleled growth. No growth was
observed on acetate, formate, methanol, monomethylamine
or yeast extract with a N2/CO2or H2headspace, with or
without sulphur, thiosulphate or nitrate. Nitrate, tryptone,
glutamate and yeast extract were used as nitrogen sources.
The three novel strains were inhibited by chloramphenicol,
penicillin G and rifampicin (all at 10 mg l21), but not by
streptomycin (200 mg l21) at 70uC.
DNA G+C content and 16S rRNA gene sequence
The G+C contents of the DNA of strains SL17T, SL19Tand
SL22Tas determined by liquid chromatography were 42, 46
and 41 mol%, respectively. Using this method, the DNA
G+C content of D. thermolithotrophum BSATwas deter-
mined to be 36 mol% (35 mol% by the Tm method;
L’Haridon et al., 1998).
On the basis of the 16S rRNA gene sequence analysis, the
three strains belonged to a robust phylogenetic cluster that
consisted of species of the genera Desulfurobacterium,
Thermovibrio and Balnearium. Strain SL22Twas most
closely related to strain SL17Tand Desulfurobacterium
species (95?5–95?9% similarity). Species of the genus
Desulfurobacterium were also the closest relatives of strain
SL17T(~ 95?0% similarity). Strain SL19Tgrouped with
Thermovibrio ammonificans (96?5% similarity), however
the bootstrap support for this was low (42%) (Fig. 1). A
comprehensive resolution of the group may evolve from
alternative gene and genome phylogenies. Nevertheless, the
distance matrices and the physiological differences concur.
Furthermore, there are certain regions within the 16S rRNA
gene that appear to be useful diagnostic markers. For
example the 992–1031 region (Escherichia coli numbering)
has diagnostic sequences for the strain SL19T–Thermovibrio
lineage andthe443–487region(E.colinumbering)may bea
good target for developing probes that target the different
groups. In this latter case, strain SL19Tcan be distinguished
from the genus Thermovibrio.
Examination of the respiratory lipoquinone composition of
D. thermolithotrophum BSAT, T. ruber ED11/3LLKTand
strains SL17T, SL19Tand SL22Trevealed that naphthoqui-
none-like components were the sole respiratory quinones
of the compounds eluted from the TLC plates co-chromato-
graphed within known menaquinone standards. Mass
were present in D. thermolithotrophum BSATand in strains
SL17Tand SL19T(typical fragmentation of the menaquinone
ring nucleus at m/z 187 and 225). The mole peak gave a
value of m/z 654, which is 6 mass units higher than authentic
MK-7 (mole peak m/z at 648). Given the presence of the
menaquinone ring nucleus, this would indicate that a hexa-
hydrogenated derivative of MK-7 was present (i.e. MK-7H6).
Thetypicalhomologousfragmentationseries found in MK-7
2846International Journal of Systematic and Evolutionary Microbiology 56
S. L’Haridon and others
was not observed in the high mass region of the novel
compounds, suggesting that unsaturation occurred at the
end of the isoprenoid chain. The typical fragments at m/z
187 and 225 were not observed in strain SL22T. Although
fragments at higher mass were observed in this strain, it was
not possible to assign any of them unambiguously to a
known structure (i.e. monomethyl- or dimethyl-menaqui-
nones). The mole peak was at m/z 638, indicating that the
major peak had a mass 10 units less than authentic MK-7.
The retention time of the major compound on reverse-
phase HPLC also suggested that the isoprenoid chain length
is shorter than seven isoprene units. T. ruber ED11/3LLKT
was unique in that it appeared to contain a mixture of the
novel MK-7 (MK-7H6) derivative found in D. thermolitho-
trophum BSAT, SL17Tand SL19Tas well as a menathioqui-
none derivative. The presence of a fragment at m/z 257
confirmed the presence of the latter compound and its mole
peak at m/z 686 was two mass units higher than that of
authentic MTK-7H4, indicating that the compound was
probably MTK-7H6. This was also consistent with the
retention time of this compound, which was longer than
that of authentic MTK-7H4.
The cellular fatty acids comprised both saturated and
unsaturated straight chains, as well as hydroxylated fatty
acids (see Supplementary Table S2). The presence of
hydroxylated fatty acids is indicative of the presence of
lipopolysaccharides. For all strains, the major straight chain
fatty acids present were 18:0 and 18:1v7c, but differences
were observed between the strains. Strains SL17Tand SL22T
could be distinguished by the presence of high amounts of
16:0. Significant amounts of 19:1 were also a differentiat-
ing characteristic of strain SL17T. Along with D. thermo-
lithotrophum and T. ruber, the three novel strains contained
no, or small, amounts of 20:1. Among the novel strains,
only strain SL19Tcontained 20:0.
The polar lipids of the strains were predominantly phos-
pholipids. The two major lipids were identified on the basis
of their RFvalues and staining behaviour as phosphatidyli-
nositol and phosphatidylaminopentatetrol (see Supple-
mentary Fig. S2 in IJSEM Online). Additional phospholipids
(typically phosphatidylglycerol) were present in some, but
not all strains, examined. Other phospholipids, present in
small amounts could not be unambiguously identified.
Comparison of the novel strains with related
species and justification for the creation of a
As the 16S rRNA gene sequence divergence of the three
isolated strains from their closest phylogenetically related
species was > 3%, this supports the proposal that they may
represent novel species (Wayne et al., 1987; Stackebrandt &
Goebel, 1994). Table 1 shows the differentiating character-
genera Desulfurobacterium, Thermovibrio and Balnearium.
Strain SL22Twas most closely related to strain SL17Tand
known Desulfurobacterium species (95?5–95?9% 16S rRNA
gene sequence similarity). Strain SL17Tdiffered from strain
SL22Tby its cell shape and size, temperature range for
growth, the electron acceptors used for energy production
and quinonecomposition (Supplementary Table S1). These
phenotypic features and the G+C content of their genomic
DNA also distinguish both strains from D. thermolitho-
trophum and ‘D. crinifex’. Based on phylogenetic considera-
tions, strain SL19Tis most closely related to T. ruber. Strain
SL19Tcould be differentiated from T. ruber by its
morphology, temperature, pH and NaCl ranges for
growth and quinone and fatty acid composition (see
Supplementary Tables S1 and S2). Most of these traits
and the G+C content distinguished strain SL19Tfrom T.
ammonificans. On the basis of the combination of their
maximum-likelihood analysis showing the posi-
tion of the novel strains SL17T, SL19Tand
SL22T. Bootstrap values > 70% obtained for a
Additional sequences used to generate the tree
were Thermotoga maritima MSB8T(GenBank
accession no. M21774), Thermosipho melane-
siensis BI429T(Z70248), Thermus thermo-
philus HB8T(X07998), Deinococcus radio-
durans UWO298 (M21413), Bacillus subtilis
(J01695) and Flexibacter flexilis DSM 6793T
JAL-1T(M59126) was used as the outgroup
(not shown). Bar, 0?10 fixed mutations per
Phylogenetic tree generated using
Desulfurobacteriaceae fam. nov., with three novel species
distinct morphological and physiological characters and
their distant phylogenetic positions relative to previously
described organisms, we propose that strains SL17T, SL19T
and SL22Trepresent novel bacterial species. We propose to
name them Desulfurobacterium pacificum (strain SL17T),
Thermovibrio guaymasensis (strain SL19T).
As supported by 16S rRNA gene phylogenetic analyses,
species of the genera Desulfurobacterium, Thermovibrio and
Balnearium form a strongly supported cluster with inter
strain gene sequence similarity ranging from 93 to 96?6%.
Within the order Aquificales, this lineage is a separate clade
from the genera Hydrogenobacter, Aquifex, Thermocrinis,
Hydrogenobaculum and Hydrogenivirga, forming the family
maceae. Phylogenetic distances between these organisms and
those belonging to the families Aquificaceae and the
Hydrogenothermaceae are > 20%. Based on the distinct
propose to group species of the genera Desulfurobacterium,
Thermovibrio and Balnearium into a new family. The
phylogenetic distinctiveness of the new family from the
families Aquificaceae and Hydrogenothermaceae is also sup-
ported by physiological and chemotaxonomic data. In
contrast to members of these two families, species of the
genera Desulfurobacterium, Thermovibrio and Balnearium are
strict anaerobes unable to grow under microaerophilic
conditions and contain no or low levels of fatty acid 20:1
(Jahnke et al., 2001; Sto ¨hr et al., 2001; Eder & Huber, 2002).
Desulfurobacterium, Thermovibrio and Balnearium form a
separate branch within the Aquificales and are well defined
phenotypically, we propose to create the family Desulfu-
robacterium as the type genus.
intoaccount that speciesof the genera
In order to gain more insight into the biochemical
characteristics of D. thermolithotrophum, the type species
of this genus, we analysed the structure of an aminopho-
spholipid previously identified in the type strain (L’Haridon
et al., 1998) and investigated the possible presence of the
reductive citric acid cycle for autotrophic CO2fixation in
Table 1. Comparison of properties of the novel strains and related species
Strains: 1, strain SL17T; 2, strain SL22T; 3, Desulfurobacterium thermolithotrophum; 4, ‘Desulfurobacterium crinifex’; 5, strain SL19T; 6,
Thermovibrio ruber; 7, Thermovibrio ammonificans; 8, Balnearium lithotrophicum. Data are taken from L’Haridon et al. (1998), Huber et al.
(2002), Alain et al. (2003), Takai et al. (2003b) and Vetriani et al. (2004).
Cell shape Straight
to curved rods
in the middle
Cell size (mm)
Flagellation Monopolar Monopolar
Up to 6
Number of flagella
Temperature range (uC)
NaCl range (%)
Optimum NaCl (%)
DNA G+C content
Up to 3Up to 3 Up to 32 Up to 4Up to 2Several
*As measured by the HPLC method.
DAs measured by the thermal denaturation method.
2848International Journal of Systematic and Evolutionary Microbiology 56
S. L’Haridon and others
Chemical structure of an aminophospholipid of
D. thermolithotrophum strain BSAT
The lipids of D. thermolithotrophum strain BSATwere
previously found to be characterized by the presence of an
aminophospholipid and a phospholipid, in a relative ratio of
2?5:2?2 (L’Haridon et al., 1998). In this previous study, the
structure of the phospholipid was fully defined. Here, we
0?7). For structural definition, the heteronuclear correlation
with two dimensional proton–proton correlation was
diagnostic. The1H-NMR showed signals at d 5?25 (1H, m),
d 4?46 (1H, dd, J=3?1 and 12?0 Hz) and d 4?20 (1H, dd,
J=6?6 and 12?0 Hz) due to the ABX system of a diacylated
glycerol, (2, 1,respectively; see Supplementary Fig. S3),while
the other glycerol methylene linked to the phosphate group
resonated asa multiplet at d 4?05 (3; Supplementary Fig. S3).
The signals of the aminopentanetetrol were found at d 3?37
(CH2, dd, 19), d 4?6 (CH, bm, 29), d 3?99 (CHOH, dd, 39), d
3?73 (CHOH, ddm, 49), d 3?7 (CH2OH, dd, 59). The
remaining signals are due to the acyl chains, d 0?89 overlapp-
ing triplets (terminal CH3, K; Supplementary Fig. S3), d 1?3
(bs, terminal methylenes, J), d 1?55 (bs, methylenes b to the
ester carbonyl group, D), d 2?30 (methylenes a to the above-
bonds, dt, F), d 5?34 (CH of the double bonds, triplet, H and
G), d 5?4 and d 5?6 (CH in trans positions, cm, I, L). In the
13C-NMR, the signals due to the acyl chains were observable
B; Supplementary Fig. S3). At d 130, are resonances of CH in
cis double bonds (H and G), d 132 and d 134, CH in trans
double bonds (I, L). At d 14?0, terminal methyls, d 30?1
methylenes in chains, d 37?2 methylenes a to the double
bond, d 24?0 CH2b to carbonyl group and d 37?5, CH2a to
the carbonyl group (K, J, F, D, C; Supplementary Fig. S3). At
d 41?1 CH2-NH2methylene on amino group. In the region
d 62?6–71?0, seven signals are present that were methine
and methylene carbons from the DEPT experiment. At d
62?5 CH2 of glycerol linked to the acyl chain (1;
Supplementary Fig. S3), at d 64?0 of the terminal CH2OH
in the pentanetetrol, d 65?0, glycerol methylene coupled with
phosphorus (59 and 3). The methine carbons resonated at d
a methylene linked to a phosphate group and at d 74?0 with a
coupling constant of 3?5 Hz (2 and 39; Supplementary
Fig. S3). The other two methines were at d 74?0 and d 72?5,
was the last CHOH of the pentanetetrol (29 and 49;
Supplementary Fig. S3). NMR experiments of1H-1H and
1H-13C correlation fully confirmed the assignments reported
above. Although the stereochemistry of the glycerol of the
novel aminolipid is not known, the compound can be
defined as 1,2-diacyl-3-O(phospho-29-O(19-amino)-29-39-
49-59-pentanetetrol-sn-glycerol), with acyl chains that also
have monounsaturation with different stereochemistry and
positions on the chains.
This compound was first identified in H. thermophilus
strain TK-6 (Yoshino et al., 2001). A similar structure was
characterized in Methanothrix concillii strain GP6 by
Ferrante et al. (1987) and has been found in members of
Methanomicrobiaceae (Koga et al., 1993).
Enyzme activities of the reductive citric acid
cycle in D. thermolithotrophum strain BSAT
Cell extracts of D. thermolithotrophum BSATcontained all
the enzymes of the reductive citric cycle, including the key
enzyme of the pathway, ATP citrate lyase (citrate + ATP +
CoA R acetyl-CoA + oxaloacetate + ADP + P). The data
indicate that acetyl-CoA synthesis from two CO2in this
organism proceeds via the reductive citric acid cycle (see
Supplementary Table S3).
The reductive citric acid cycle for autotrophic CO2fixation
has been reported for members of both the domains of
Bacteria and Archaea (Beh et al., 1993, Scho ¨nheit & Scha ¨fer,
1995). The pathwayhas beendescribedfor thephototrophic
green bacterium Chlorobium limicola and a few sulphate-
reducing bacteria, which belong to the genus Desulfobacter.
The pathway is also present in the genera Hydrogenobacter
and Aquifex. Thus, the presence of the reductive citric acid
cycle in both the genera Desulfurobacterium and Aquificales
to the lithotrophic microaerophilic genera Aquifex and
Hydrogenobacter, Desulfurobacterium is an anaerobic sul-
phur-reducing lithoautotroph. This CO2fixation pathway
has also been reported in sulphur-dependent lithoauto-
Scha ¨fer et al., 1986). This is the first report of the operation
of the reductive citric acid cycle in a sulphur-dependent
lithoautotroph of the bacterial domain.
Emended description of the order Aquificales
et al. (1992). The order was described by Reysenbach
(2001b) and the name was validly published by Reysenbach
(2002). With the new results obtained in this study, we
propose the following emended description. Thermophilic
motile and non-motile rods that vary from 0?2 to 6 mm in
length. Gram-negative. Spores not formed. Long filamen-
tous forms may develop under some growth conditions. All
members are capable of chemolithotrophic growth under
microaerophilic or strict anaerobic conditions. All isolates
grow best at 70uC or above and are found in terrestrial,
shallow and deep-sea marine thermal springs. The type
genus is Aquifex.
Emended description of the genus
The genus Desulfurobacterium was described by L’Haridon
et al. (1998) and an emended description has since been
proposed (Alain et al., 2003). With the new results obtained
in this study, we propose the following emended descrip-
tion. Cells are Gram-negative rods. Spores are not
produced. Anaerobic and thermophilic. Strictly chemo-
lithotrophic. Sulphur-reducing and/or sulphite-reducing
Desulfurobacteriaceae fam. nov., with three novel species
and/or thiosulphate-reducing and/or nitrate-reducing. May
form macroscopic coloured cell masses encased in a
polymeric matrix. CO2 is fixed via the reductive citric
acid cycle. Main cellular fatty acids are 18:0 and 18:1v7c.
In most species, the major quinone is MK-7H6. The G+C
content of the DNA ranges from 36 to 42 mol% (HPLC
method). The type species is Desulfurobacterium thermo-
Description of Desulfurobacterium pacificum
Desulfurobacterium pacificum (pa.ci9fi.cum. L. neut. adj.
pacificum peaceful; pertaining to the Pacific Ocean).
Straight rods of about 1–2 mm length and 0?4–0?5 mm in
width. Highly motile by means of up to three monopolar
flagella. Occur singly or in pairs. Some cells become
spherical in the stationary growth phase. Gram-negative.
Yellow to orange colonies about 1 mm in diameter formed
on Phytagel plates containing thiosulphate and polysul-
phides. Growth occurs between 55 and 85uC, with an
optimum at approximately 75uC. Growth occurs between
pH 5?5 and 7?5 with an optimum of pH between 6 and 6?2
and at NaCl concentrations ranging between 15 and
70 g l21with an optimum of 30 g l21. Strictly anaerobic.
Obligately chemolithoautotrophic. Sulphur, thiosulphate
and nitrate serve as electron acceptors in the presence of H2
with the formation of H2S and ammonium, respectively.
electron acceptors. Growth is inhibited by chloramphenicol,
penicillin G and rifampicin at 10 mg ml21, but not by
streptomycin at 200 mg ml21. The major cellular fatty acids
are 18:0, 18:1v7c 16:0 and 3-OH 14:0 (ester linked) (see
also supplementary Table S2). The DNA G+C content of
the type strain is 42 mol% (as determined by HPLC).
The type strain, Desulfurobacterium pacificum SL17T
(=DSM 15522T=JCM 12127T), was obtained from a
deep-sea hydrothermal vent chimney at the East Pacific Rise
Description of Desulfurobacterium atlanticum
Desulfurobacterium atlanticum (at.lan9ti.cum. L. neut. adj.
atlanticum of or pertaining to the Atlantic Ocean).
Straight to curved rods of about 2?5–3?5 mm long and 0?4–
0?5 mm wide. Motile by means of up to three monopolar
flagella. Occur singly, in pairs and in chains of 5–6 cells.
Some cells become spherical in the stationary growth phase.
Gram-negative. Yellow to orange colonies about 1 mm in
diameter formed on Phytagel plates containing thiosulphate
and polysulphides. Growth occurs between 50 and 80uC,
with an optimum between 70 and 75uC. Growth occurs
between pH 5 and 7?5, with an optimum of pH between 6
and 6?2, and at NaCl concentrations ranging between 15
and 70 g l21, with an optimum of 30 g l21. Strictly
anaerobic. Obligately chemolithoautotrophic. Thiosulphate
and nitrate serve as electron acceptors in the presence of H2
with the formation of H2S and ammonium, respectively.
Sulphur, sulphate, sulphite, cystine, nitrite and oxygen are
not used as electron acceptors. Growth is inhibited by chlo-
ramphenicol,penicillinGandrifampicinat10 mg ml21,but
notbystreptomycinat200 mg ml21.Themajorcellularfatty
(see also supplementary Table S2). The respiratory lipo-
quinone composition is atypical. DNA G+C content of the
type strain is 41 mol% (as determined by HPLC).
The type strain, Desulfurobacterium atlanticum SL22T
(=DSM 15668T=JCM 12129T), was obtained from a
deep-sea hydrothermal vent chimney at the Mid-Atlantic
Description of Thermovibrio guaymasensis
Thermovibrio guaymasensis (gua.y.mas9en.sis. N.L. masc.
adj. guaymasensis pertaining to Guaymas Basin).
Coccoid to lemon-shaped rods of about 1–2 mm in length
and 1–2 mm in width. Highly motile by means of up to four
monopolar flagella. Occurs singly, in pairs and in chains of
5–6 cells. Some cells become spherical in the stationary
growth phase. Gram-negative. Yellow to orange colonies
about 1 mm in diameter formed on Phytagel plates
containing thiosulphate and polysulphides. Growth occurs
between 50 and 88uC, with an optimum between 75 and
80uC. Growth occurs between pH 5?5 and 7?5, with an
optimum pH between 6 and 6?2 and at NaCl concentrations
ranging between 15 and 70 g l21, with an optimum of
30 g l21. Strictly anaerobic. Obligately chemolithoauto-
trophic. Sulphur and nitrate serve as electron acceptors in
the presence of H2 with the formation of H2S and
ammonium, respectively. Sulphate, thiosulphate, sulphite,
cystine, nitrite and oxygen are not used as electron
acceptors. The major cellular fatty acids are 18:0,
18:1v7c, 16:0 and 3-OH 14:0 (ester linked) (see also
supplementary Table S2). Growth is inhibited by chlor-
amphenicol, penicillin G and rifampicin at 10 mg ml21, but
not by streptomycin at 200 mg ml21. DNA G+C content of
the type strain is 46 mol% (as determined by HPLC).
The type strain, Thermovibrio guaymasensis SL19T(=DSM
15521T=JCM 12128T) was obtained from a deep-sea
hydrothermal vent chimney at Guaymas Basin.
Description of Desulfurobacteriaceae fam. nov.
neut. n. Desulfurobacterium type genus of the family; suff.
-aceae ending to denote a family; N.L. fem. pl. n.
Desulfurobacteriaceae the Desulfurobacterium family).
Rods that vary from 1 to 3?5 mm in length. Gram-negative.
Spores not produced. Cell masses of isolates have an intense
red colouration. Long filaments may develop under some
growthconditions. Strictlyanaerobic. Thermophilicwith an
2850International Journal of Systematic and Evolutionary Microbiology 56
S. L’Haridon and others
optimum of 60–80uC. Chemolithoautotrophic growth in
the presence of hydrogen and carbon dioxide with sulphur,
thiosulphate, sulphite or nitrate as electron acceptors.
Isolated from deep-sea hydrothermal vents. The major
phospholipids are phosphatidylinositol and phosphatidyl-
aminopentatetrol. Polar lipid side chains are typically of the
of C18 chain lengths. Unsaturated C18:1 fatty acids are
present. The major respiratory quinones are naphthoqui-
none derivatives, typically with relatively short, partially
saturated isoprenoid side chains (e. g. MK-7H6). Sulphur
The G+C content of the DNA is 36–55 mol%. The 16S
rRNA gene sequences differ by> 20%betweenmembers of
this family and members of the families Aquificaceae and
Hydrogenothermaceae. Members of this family have been
isolated from deep-sea hydrothermal vents. The type genus
The authors also thank Eduardo Pagnotta and Raffaele Turco for lipid
analysis. We thank the captains and the support crews of N. O. Le
Nadir, L’Atalante and the D. S. V. Nautile pilots for skilful operations
during the cruises Guaynaut (1992), Hero (1993) and Microsmoke
(1995). This work, performed at Plouzane ´, was supported by grants
from CNRS-Rho ˆne-Poulenc and the Conseil Re ´gional de Bretagne
(PRIR) and INTAS grant 99-1250.
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