Download Autotrophic CO2 fixation via the reductive tricarboxylic acid cycle in

Environmental Microbiology (2007) 9(1), 81–92
doi:10.1111/j.1462-2920.2006.01118.x
Autotrophic CO2 fixation via the reductive tricarboxylic
acid cycle in different lineages within the phylum
Aquificae: evidence for two ways of citrate cleavage
Michael Hügler,1 Harald Huber,2 Stephen J.
Molyneaux,1 Costantino Vetriani3 and
Stefan M. Sievert1,4*
1
Biology Department, Woods Hole Oceanographic
Institution, Woods Hole, MA 02543, USA.
2
Lehrstuhl für Mikrobiologie, Universität Regensburg,
Universitätsstrasse 31, 93053 Regensburg, Germany.
3
Department of Biochemistry and Microbiology and
Institute of Marine and Coastal Sciences, Rutgers
University, New Brunswick, NJ 08901, USA.
4
NASA Astrobiology Institute, Marine Biological
Laboratory, Woods Hole, MA 02543, USA.
Summary
Autotrophic carbon fixation was characterized in
representative members of the three lineages of the
bacterial phylum Aquificae. Enzyme activity measurements and the detection of key genes demonstrated
that Aquificae use the reductive tricarboxylic acid
(TCA) cycle for autotrophic CO2 fixation. This is the
first time that strains of the Hydrogenothermaceae
and ‘Desulfurobacteriaceae’ have been investigated
for enzymes of autotrophic carbon fixation. Unexpectedly, two different mechanisms of citrate cleavage
could be identified within the Aquificae. Aquificaceae
use citryl-CoA synthetase and citryl-CoA lyase,
whereas Hydrogenothermaceae and ‘Desulfurobacteriaceae’ use ATP citrate lyase. The first mechanism is
likely to represent the ancestral version of the reductive TCA cycle. Sequence analyses further suggest
that ATP citrate lyase formed by a gene fusion of
citryl-CoA synthetase and citryl-CoA lyase and subsequently became involved in a modified version of
this pathway. However, rather than having evolved
within the Aquificae, our phylogenetic analyses indicate that Aquificae obtained their ATP citrate lyase
through lateral gene transfer. Aquificae play an important role in biogeochemical processes in a variety of
high-temperature habitats. Thus, these findings sub-
Received 25 January, 2006; accepted 27 June, 2006. *For
correspondence. E-mail [email protected]; Tel. (+1) 508 289 2305;
Fax (+1) 508 457 2076.
stantiate the hypothesis that autotrophic carbon fixation through the reductive TCA cycle is widespread
and contributes significantly to biomass production
particularly in hydrothermal habitats.
Introduction
Autotrophy is a requirement for self-sustaining life and
chemolithoautotrophs – i.e. organisms that acquire energy
from the oxidation of inorganic compounds and that use
inorganic carbon as the source for cell carbon – are likely
to have been among the first types of organisms on Earth
(e.g. Huber and Wächtershäuser, 1997; Russell and Hall,
1997). Phylogenetic analyses based on 16S rRNA and
whole genomes place autotrophic hyperthermophiles at
the base of the evolutionary tree of life, lending evidence to
the hypothesis that in addition to being autotrophs, the first
life forms might have been hyperthermophilic (Pace, 1991;
House and Fitz-Gibbon, 2002). Microorganisms carrying
out autotrophic carbon fixation at high temperatures, i.e.
above 70°C, utilize CO2 fixation pathways other than the
Calvin-Benson-Bassham (Calvin) cycle, which is well
known from cyanobacteria, plants and a variety of chemolithoautotrophic Proteobacteria (Fuchs, 1989; Madigan
et al., 2003). Presently, three of these so-called alternative
pathways are known: the 3-hydroxypropionate cycle, the
reductive acetyl-CoA pathway and the reductive tricarboxylic acid (TCA) cycle (Madigan et al., 2003). The
reductive TCA cycle, which has been proposed as a likely
candidate for the earliest autotrophic pathway that evolved
on Earth (Wächtershäuser, 1990; Cody et al., 2001; Smith
and Morowitz, 2004), was first shown to function in the
green sulfur bacterium Chlorobium limicola (Evans et al.,
1966; Fuchs et al., 1980; Ivanovski et al., 1980). Subsequently, the operation of the reductive TCA cycle was also
confirmed in a variety of anaerobic and microaerobic bacteria, including the sulfate-reducing bacterium Desulfobacter hydrogenophilus (Schauder et al., 1987), and most
recently various e-Proteobacteria (Hügler et al., 2005;
Takai et al., 2005). Interestingly, two species of the
Aquificaceae, Aquifex pyrophilus (Beh et al., 1993) and
Hydrogenobacter thermophilus (Shiba et al., 1985)
have also been shown to use the reductive TCA cycle
for autotrophic carbon fixation.
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd
82 M. Hügler et al.
Fig. 1. 16S rRNA based tree showing the
phylogenetic position of Aquificaceae,
Hydrogenothermaceae and
‘Desulfurobacteriaceae’. The tree was
constructed by using the phylogenetic
software ARB (Ludwig et al., 2004), using
Archaea as an outgroup. Strains used in this
study are indicated in bold.
Escherichia coli
Candidatus Arcobacter sulfidicus
Thiomicrospira denitrificans
Desulfobacter hydrogenophilus
Chlorobium limicola
Chlorobium tepidum
Chloroflexus aurantiacus
Bacillus subtilis
Streptomyces griseus
Thermus aquaticus
Thermoanaerobacter brockii
Fervidobacterium islandicum
Thermosipho africanus
Thermotoga maritima
Aquifex aeolicus
Aquifex pyrophilus
Hydrogenivirga caldilitoris
Thermocrinis albus
Hydrogenobacter subterraneus
Hydrogenobacter hydrogenophilus
Hydrogenobacter thermophilus
Thermocrinis ruber
Hydrogenobaculum acidophilum
Aquificaceae
Sulfurihydrogenibium subterraneum
Sulfurihydrogenibium azorense
Hydrogenothermus marinus
Hydrogenothermaceae
Persephonella hydrogeniphila
Persephonella marina
Persephonella guaymasensis
Desulfurobacterium thermolithotrophum
Desulfurobacterium crinifex
'Desulfurobacteriaceae'
Thermovibrio ammonificans
Balnearium lithotrophicum
Thermovibrio ruber
Archaeoglobus fulgidus
Methanocaldococcus jannaschii
Sulfolobus solfataricus
0.1 nucleotide substitutions / site
Within the bacteria, the phylum Aquificae is the only
group that contains hyperthermophilic autotrophs. Presently it is divided into three major lineages (Eder and
Huber, 2002; Takai et al., 2003a), (i) the Aquificaceae
comprising the genera Aquifex, Hydrogenobacter, Hydrogenobaculum, Hydrogenivirga and Thermocrinis; (ii) the
Hydrogenothermaceae comprising the genera Hydrogenothermus, Persephonella and Sulfurihydrogenibium;
and (iii) several genera incertae sedis (‘Desulfurobacteriaceae’) including the genera Balnearium, Desulfurobacterium and Thermovibrio (Fig. 1). Whereas the
Aquificaceae and Hydrogenothermaceae contain predominantly microaerophilic chemolithoautotrophs that
obtain energy by oxidizing molecular hydrogen or reduced
sulfur compounds, all members of the ‘Desulfurobacteriaceae’ are strict anaerobes, obtaining energy by coupling
the oxidation of H2 either to the reduction of elemental
sulfur or nitrate.
In recent years, several studies have shown
that members of the Aquificae are likely to play an
important role as primary producers in various hightemperature environments (e.g. Reysenbach et al.,
1994; 2000a,b; Harmsen et al., 1997; Yamamoto et al.,
1998; Eder and Huber, 2002; Blank et al., 2002; Inagaki
et al., 2003; Spear et al., 2005), suggesting that carbon
fixation through the reductive TCA cycle might be sig-
nificant in these habitats. However, at present no information exists on what carbon fixation pathway members
of the ‘Desulfurobacteriaceae’ might use and only
limited information, in form of carbon isotopic analyses,
exists for the Hydrogenothermaceae (Zhang et al.,
2002).
The reductive TCA cycle is essentially a reversal of the
well-known oxidative TCA or Krebs cycle. Three molecules of CO2 are fixed, forming acetyl-CoA and subsequently pyruvate, the precursor of all other central
metabolites. While most of the enzymes are shared
between the reductive and oxidative TCA cycle, three
enzymes are essential to run this cycle in reverse:
2-oxoglutarate : ferredoxin oxidoreductase, fumarate
reductase and ATP citrate lyase. The latter enzyme
catalyses the ATP- and the CoA-dependent cleavage of
citrate into acetyl-CoA and oxaloacetate. ATP citrate lyase
is encoded by two separated genes aclA and aclB,
arranged in the same way in all organisms studied to date
(Fig. 2A). ATP citrate lyase genes have so far only been
found in prokaryotes using the reductive TCA cycle for
autotrophic carbon fixation, such as the green sulfur bacteria (Antranikian et al., 1982; Wahlund and Tabita, 1997;
Kanao et al., 2001), and e-Proteobacteria (Campbell
et al., 2003; Hügler et al., 2005; Takai et al., 2005), and
thus can at present be considered as indicator genes
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92
Autotrophic CO2 fixation in Aquificae
83
Fig. 2. Reaction schemes of the citrate
cleavage reactions within the reductive TCA
cycle.
A. Reaction catalysed by ATP citrate lyase
(ACL) and gene organization of the genes
coding for ACL (aclB and aclA).
B. Reaction catalysed through the combined
action of citryl-CoA synthetase (CCS) and
citryl-CoA lyase (CCL) and gene organization
of the genes coding for CCS (ccsA and ccsB)
and CCL (ccl).
for this pathway. In contrast to Chlorobiaceae and
e-Proteobacteria, the ATP-dependent citrate cleavage in
H. thermophilus is catalysed by two enzymes, citryl-CoA
synthetase and citryl-CoA lyase, which catalyse the ATPdependent formation of citryl-CoA from citrate and CoA
and the subsequent cleavage of citryl-CoA into acetylCoA and oxaloacetate respectively (Aoshima et al.,
2004a,b) (Fig. 2B). The sequence similarity between
citryl-CoA synthetase, succinyl-CoA synthetase and ATP
citrate lyase on one hand and between citryl-CoA lyase,
citrate synthase and ATP citrate lyase on the other hand
suggests that these enzymes share an evolutionary
history (Aoshima et al., 2004b). Furthermore, it has been
argued that the reductive TCA cycle as identified in
H. thermophilus constitutes the ancient form, whereas the
reductive TCA cycle in Chlorobium involving ATP citrate
lyase represents a secondary adaptation that evolved
from the oxidative TCA cycle (Aoshima et al., 2004b). In
this context, we were interested in investigating which
citrate cleavage mechanism might operate in the two
other lineages of the Aquificae.
In the present study we have investigated the carbon
fixation pathways in representative organisms of all major
lineages within the phylum Aquificae. We could measure
the activities of the enzymes of the reductive TCA cycle
and amplify the genes coding for the respective citrate
cleaving enzymes, suggesting that all investigated
members of the Aquificae utilize the reductive TCA cycle
for autotrophic carbon fixation. Unexpectedly, we found
that there exist two ways of citrate cleavage within the
Aquificae. The evolutionary and ecological implications of
these findings are discussed.
Results and discussion
Activities of enzymes of the reductive TCA cycle in
Aquificae
We studied carbon fixation in representatives of the three
different lineages of the phylum Aquificae. Table 1 provides an overview of the strains that were used in this
investigation, including their isolation site and growth
requirements. Cell extracts of Aquifex aeolicus, Thermocrinis ruber (both Aquificaceae), Thermovibrio ammonificans, Thermovibrio ruber (both ‘Desulfurobacteriaceae’)
and Sulfurihydrogenibium subterraneum (Hydrogenothermaceae) were tested for the activity of enzymes of the
reductive and oxidative TCA cycle. In addition, cell extract
of Desulfurobacterium thermolithotrophum (‘Desulfurobacteriaceae’) was tested for citrate cleavage,
2-oxoglutarate and pyruvate : benzyl viologen (BV) oxidoreductase activities. The results of these enzyme measurements are presented in Table 2. In general, the
activities of all enzymes of a reductive TCA cycle could be
detected in all tested organisms, including the key enzymatic activities of ATP-dependent citrate cleavage, fuma-
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92
84 M. Hügler et al.
Table 1. Strains used in this study and some characteristics.
Strain
Family
Isolation site
Aquifex aeolicus
Aquificaceae
Balnearium
lithotrophicum
Desulfurobacterium
crinifex
Desulfurobacterium
thermolithotrophum
Hydrogenobacter
hydrogenophilus
Hydrogenobacter
thermophilus
Persephonella marina
‘Desulfurobacteriaceae’
Hydrothermal system
(Vulcano, Italy)
Deep-sea hydrothermal vent
chimney (Suiyo Seamount, Japan)
Deep-sea hydrothermal vent
chimney (Juan de Fuca Ridge)
Deep-sea hydrothermal vent
chimney (Mid-Atlantic Ridge)
Hot spring (Geyser Valley,
Kamchatka, Russia)
Hot spring
(Mine, Izu, Japan)
Deep-sea hydrothermal
vent chimney (East Pacific Rise)
Octopus Spring
(Yellowstone National Park)
Deep-sea hydrothermal vent
chimney (East Pacific Rise)
Submarine hydrothermal vent system
(Lihir Island, Papua New Guinea)
Hot spring
(Furnas, Azores, Portugal)
Terrestrial, subsurface hot aquifer water
(Hishikari gold mine, Japan)
‘Desulfurobacteriaceae’
‘Desulfurobacteriaceae’
Aquificaceae
Aquificaceae
Hydrogenothermaceae
Thermocrinis ruber
Aquificaceae
Thermovibrio
ammonificans
Thermovibrio ruber
‘Desulfurobacteriaceae’
Sulfurihydrogenibium
azorense
Sulfurihydrogenibium
subterraneum
Hydrogenothermaceae
‘Desulfurobacteriaceae’
Hydrogenothermaceae
Growth
temperature
(°C) (optimum)
Electron
donor/
carbon source
Electron
acceptor
85
H2/CO2
O2
H2/CO2
S
H2/CO2
S
H2/CO2
O2
H2/CO2
O2
Formate
O2
H2/CO2
NO3–
H2/CO2
NO3–
H2/CO2
NO3–
45–80
(70–75)
50–70
(60–65)
40–75
(70)
50–82
(74–76)
50–79
(70)
55–80
(73)
44–89
(80)
60–80
(75)
50–80
(75)
50–73
(68)
40–70
(65)
only very low rates. These enzymes are 2-oxoglutarate,
succinate and pyruvate dehydrogenase. In the following
sections, the results for the three families are described
and discussed in more detail.
rate reductase, 2-oxoglutarate : BV oxidoreductase, and
pyruvate : BV oxidoreductase. Enzymes of the oxidative
TCA cycle, which are not shared with the reductive
version of the pathway, could not be detected or exhibited
Table 2. Specific activities [nmol min-1 (mg cell protein)-1] of enzymes of the reductive and oxidative TCA cycle.a
Enzyme activity tested
Assay temperature (°C)
Citrate cleavage
2-Oxoglutarate : BV oxidoreductase
Pyruvate : BV oxidoreductase
Fumarate reductase
Isocitrate dehydrogenase
NAD+
NADP+
Malate dehydrogenase
NADH
NADPH
Fumarate hydratase
Fumarate
Malate
Succinyl-CoA synthase
2-Oxoglutarate dehydrogenase
NAD+
NADP+
Pyruvate dehydrogenase
NAD+
NADP+
Succinate dehydrogenase
Formate dehydrogenase
Aquifex
aeolicus
Thermocrinis
ruber
Sulfurihydrogenibium
subterraneum
85
71
420
160
1300
80
82
345
140
1010
65
535
1600
160
2690
385
11
145
8
400
52 100
9500
1310
3350
1710
n.d.
100
600
Desulfurobacterium
thermolithotrophum
75
485
330
360
–
Thermovibrio
ammonificans
Thermovibrio
ruber
75
275
565
175
635
75
340
400
160
1200
–
–
115
11 750
185
45 500
6040
2920
–
–
9120
4500
13 780
8560
n.d.
81
975
455
235
380
–
–
–
190
77
580
4500
350
1460
10
11
11
7
n.d.
n.d.
–
–
n.d.
n.d.
67
52
n.d.
7
n.d.
n.d.
1
2
n.d.
140
n.d.
n.d.
n.d.
n.d.
–
–
–
–
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
a. Mean values were obtained from at least five measurements. Standard errors were less than ⫾ 20%. n.d., no activity detected, detection limit
< 1 nmol min-1 (mg cell protein)-1; –, not determined.
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92
Autotrophic CO2 fixation in Aquificae
Aquificaceae. The operation of the reductive TCA cycle
for carbon fixation had previously been demonstrated in
A. pyrophilus and H. thermophilus, two members of the
Aquificaceae (Shiba et al., 1985; Beh et al., 1993). We
could measure all the enzymes of the reductive TCA cycle
in A. aeolicus and Tc. ruber. Based either on the whole
genome sequence (A. aeolicus) or carbon isotopic measurements (Tc. ruber) both organisms were previously
suspected to use this pathway, but direct evidence had
been lacking (Deckert et al., 1998; Jahnke et al., 2001).
Isocitrate dehydrogenase, as well as malate dehydrogenase, was found to be NAD(H)-dependent. ATPdependent citrate lyase activity in cell extracts was found
to be optimal around pH 8.3 (data not shown) exhibiting
values of 70 nmol min-1 (mg protein-1) for A. aeolicus and
80 nmol min-1 (mg protein-1) for Tc. ruber. These activities
are low compared with the values obtained for
S. subterraneum, D. thermolithotrophum or the Thermovibrio species. This might reflect difficulties in measuring
the coupled reactions of three different enzymes in vitro,
namely citryl-CoA synthetase, citryl-CoA lyase and malate
dehydrogenase rather than two as in the case of ATP
citrate lyase and malate dehydrogenase. Although
Tc. ruber was grown with formate as carbon source, the
organism still uses the enzymes of the reductive TCA
cycle to fix the CO2 derived from formate. In addition,
formate dehydrogenase activity could be measured, probably catalysing the formation of CO2 from formate.
Hydrogenothermaceae. Sulfurihydrogenibium subterraneum, a representative member of the Hydrogenothermaceae, showed activities of all enzymes of the reductive
TCA cycle, including high activities of ATP-dependent
citrate lyase and 2-oxoglutarate : BV oxidoreductase. The
pH optimum of ATP citrate lyase was determined to be
pH 8.3 (data not shown). In contrast to members of the
Aquificaceae, isocitrate dehydrogenase was NADPdependent. Activities of the enzymes of the oxidative TCA
cycle were not detected. The demonstration of the reductive TCA cycle in a member of the Hydrogenothermaceae
is in agreement with stable carbon isotopic analyses and
the amplification of a small fragment of aclB from Persephonella marina (Zhang et al., 2002; Campbell et al.,
2003).
‘Desulfurobacteriaceae’. All tested members of this family
showed high activities of ATP-dependent citrate lyase and
2-oxoglutarate/pyruvate : BV oxidoreductase. The pH
optimum of ATP citrate lyase was determined to be
pH 8.3 in both Tv. ammonificans and Tv. ruber (data not
shown). In both strains isocitrate dehydrogenase was
NADP-dependent. This is the first report describing the
autotrophic carbon fixation mode in this lineage, which has
been proposed to represent an additional order within the
85
phylum Aquificae (L’Haridon et al., 1998; Huber et al.,
2002). Based on our data, the use of the reductive TCA
cycle for autotrophic carbon fixation emerges as a common
feature among the different lineages within the Aquificae.
Amplification of citryl-CoA lyase and ATP citrate lyase
genes
Citrate cleavage is a key reaction of the reductive TCA
cycle. It can be catalysed either through ATP citrate lyase
(e.g. Chlorobium, Thiomicrospira denitrificans) (Kanao
et al., 2001; Hügler et al., 2005), or by the combined
action of citryl-CoA synthetase and citryl-CoA lyase
(H. thermophilus) (Aoshima et al., 2004a,b) (Fig. 2). To
determine which mechanism is present in the different
lineages of the Aquificae, we isolated DNA and amplified,
cloned and sequenced both subunits of ATP citrate lyase
as well as large parts of citryl-CoA lyase using previously
published and newly designed primer pairs (see Experimental procedures). Genomic DNA of A. aeolicus, Hydrogenobacter hydrogenophilus, H. thermophilus, Tc. ruber
(all Aquificaceae); S. subterraneum (Hydrogenothermaceae); Balnearium lithotrophicum, Desulfurobacterium
crinifex, D. thermolithotrophum, Tv. ammonificans and
Tv. ruber (all ‘Desulfurobacteriaceae’) was used. In addition, the draft genomes of P. marina and Sulfurihydrogenibium azorense were searched for ATP citrate lyase,
citryl-CoA lyase and citryl-CoA synthetase genes (preliminary sequence data were obtained from The Institute for
Genomic Research through the website at http://www.tigr.
org). Surprisingly, both mechanisms for citrate cleavage
seem to be present within the Aquificae.
Aquificaceae. As expected, we were not able to amplify
fragments of the ATP citrate lyase genes (acl) from
A. aeolicus, H. hydrogenophilus, H. thermophilus and
Tc. ruber. Instead, we could amplify a fragment of the
gene coding for citryl-CoA lyase (ccl) from all four strains
using the newly designed primer pairs cclf1/cclr1 or cclf2/
cclr2. This resulted in ~620 bp of sequence for ccl (representing ~80% of the expected ~780 bp). A phylogenetic
tree based on the alignment of the protein sequence of
citryl-CoA lyase is presented in Fig. 3. The ccl gene has
previously been sequenced from H. thermophilus
(Aoshima et al., 2004b). Based on this sequence, gene
aq_150 (AAC06486) from A. aeolicus (annotated as
coding for citrate synthase) could be identified as a ccl
gene (Deckert et al., 1998; Aoshima et al., 2004b). The
sequenced gene fragments from H. hydrogenophilus and
Tc. ruber are closely related to these genes. As no acl
gene could be amplified from H. hydrogenophilus and
Tc. ruber and no acl genes are found in the genome of
A. aeolicus, we assume that these three organisms use
the combined action of citryl-CoA synthetase and citryl-
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92
86 M. Hügler et al.
Fig. 3. Phylogenetic tree based on ~205
amino acids of the citryl-CoA lyase protein.
The tree depicts the phylogenetic
relationships between the newly sequenced
and translated ccl fragments and sequences
obtained from the data base representing
citryl-CoA lyase, ATP citrate lyase and citrate
synthase sequences.
ATP citrate lyase (aclA)
P. marina
S. azorense
T. denitrificans
C. limicola
S. azorense
S. subterraneum
A.aeolicus
C. tepidum
citryl-CoA lyase
H. hydrogenophilus
H. thermophilus
Tc. ruber
T. denitrificans
P. marina
E. coli
P. marina
C. tepidum
S. azorense
citrate synthase
0.1 amino acid substitutions / site
CoA lyase for the cleavage of citrate into oxaloacetate and
acetyl-CoA. Genes coding for citryl-CoA synthetase can
be found within the genome sequence of A. aeolicus.
They have been assigned to encode succinyl-CoA synthetase [sucC1 (AAC07285) and sucD1 (AAC07686)].
Consequently, A. aeolicus, H. hydrogenophilus and
Tc. ruber seem to fix CO2 via the same set of enzymes as
H. thermophilus. These include citryl-CoA synthetase,
citryl-CoA lyase as well as a NAD-dependent isocitrate
dehydrogenase (Aoshima et al., 2004a,b,c).
Hydrogenothermaceae. Like in Aquificaceae, we could
successfully amplify and sequence a gene fragment of
citryl-CoA lyase (~620 bp, representing ~80% of the
expected ~780 bp). In addition, we also amplified, cloned
and sequenced the genes coding for the a- and b-subunit
of ATP citrate lyase (aclA and aclB) from S. subterraneum
using the primer pairs F2/R5, 892F/R5 and F1/R1. This
way we obtained the complete gene sequence for aclB
and a ~1.600 bp fragment of aclA (~90% of the expected
~1.800 bp). The gene arrangement is identical to all
known ATP citrate lyase genes (aclBA). BLAST searches of
the draft genomes of P. marina and S. azorense revealed
that both organisms also possess genes coding for both
ATP citrate lyase subunits, strongly suggesting that they
also use the reductive TCA cycle for carbon fixation. The
ATP citrate lyase genes of all three investigated members
of Hydrogenothermaceae show high sequence identities,
clustering together in the phylogenetic tree. Interestingly,
BLAST searches also revealed a putative gene in the draft
genomes of P. marina and S. azorense that could code for
citryl-CoA lyase. However, a homologue of the citryl-CoA
synthetase could not be identified, indicating that these
organisms use ATP citrate lyase to catalyse citrate
cleavage. The physiological role of citryl-CoA lyase is
presently unclear and warrants further investigations.
‘Desulfurobacteriaceae’. We could amplify the ATP
citrate lyase genes from all isolated strains of the
‘Desulfurobacteriaceae’. The entire sequence of aclB and
~90% of aclA was obtained from B. lithotrophicum,
D. crinifex and D. thermolithotrophum. In addition, we
amplified and sequenced a ~1000 bp fragment of aclA
(primer pair F2/R5) from Tv. ammonificans, as well as
~450 bp of the 5′ end of aclB and ~90% of aclA from
Tv. ruber (primer pair 892F/R5). The translated
sequences obtained from these strains show high identities between each other, as well as high identities and
similarities to the sequences from Persephonella and
Sulfurihydrogenibium. Using our newly designed primers
we failed to amplify a citryl-CoA lyase gene from members
of the ‘Desulfurobacteriaceae’. However, at this point we
cannot exclude the presence of a citryl-CoA gene within
this lineage, as the primers were designed only on a
limited number of ccl sequences and thus might not be
appropriate for this group. Regardless, the presence of
aclBA and the high citrate cleavage activities strongly
suggest that ‘Desulfurobacteriaceae’ use ATP citrate
lyase to accomplish citrate cleavage.
ATP citrate lyase (aclBA) and citryl-CoA lyase (ccl) as
phylogenetic and functional gene markers
Phylogenetic analyses of available aclA and aclB
sequences reveal that the sequences obtained from
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92
Autotrophic CO2 fixation in Aquificae
B. lithotrophicum
(86/100)
Tv. ruber
(100/100)
D. crinifex
(100/100)
(100/100)
D. thermolithotrophum
S. subterraneum
(100/100)
(100/100)
'Desulfurobacteriaceae'
S. azorense
(63/-)
Hydrogenothermaceae
P. marina
T. denitrificans
(100/100)
(100/100)
(92/83)
6C6
ε-Proteobacteria
7G3
Neurospora
(100/100)
87
Fig. 4. Phylogenetic tree based on ~970
amino acids of both subunits of ATP citrate
lyase. The tree depicts the phylogenetic
relationships of the newly obtained ATP citrate
lyase sequences from members of the
Aquificae with sequences from
e-Proteobacteria, Chlorobiaceae and
eukaryotes. A hypothetical ancestral ATP
citrate lyase, constructed by concatenating
citryl-CoA synthetase and citryl-CoA lyase
from H. thermophilus and A. aeolicus,
respectively, to one protein, was used as
outgroup. Bootstrap values from both distance
analysis (first number) and parsimony
analysis (second number) are shown as
percentages of 1000 bootstrap replications.
Schizosaccharomyces
(100/100)
(100/100)
(100/100)
Rattus
Eukaryotes
Danio
(100/100)
Arabidopsis
Oryza
(100/100)
C. limicola
Chlorobiaceae
C. tepidum
(100/100)
H. thermophilus
A. aeolicus
0.05 amino acid substitutions / site
members of the phylum Aquificae form a monophyletic
group
clearly
separated
from
sequences
of
e-Proteobacteria, Chlorobiaceae and eukaryotes (fungi,
plants, animals) (Fig. 4). The ATP citrate lyase sequences
from Aquificae are most closely related to sequences from
e-Proteobacteria, with which they form a clade of their
own. Phylogenetic trees calculated for the individual subunits of ATP citrate lyase show the same overall pattern
(data not shown). The ATP citrate lyase based tree is
largely in congruence with 16S rRNA phylogeny, with
e-Proteobacteria and Aquificae clearly separated from
each other, and the sequences from ‘Desulfurobacteriaceae’ (Balnearium, Desulfurobacterium, Thermovibrio)
and Hydrogenothermaceae (Persephonella, Sulfurihydrogenibium) forming two branches within the Aquificae.
Our expanded data set of ATP citrate lyase genes from
well-characterized strains of the Aquificae builds a framework that allows us to assign acl sequences obtained from
the environment to particular lineages. Along these lines,
we compared the acl sequences obtained in this study with
environmental sequences obtained from different hydrothermal vent sites (Campbell and Cary, 2004) (Fig. 5). This
way we can clearly classify some sequences as
Persephonella-like, while Sulfurihydrogenibium species
apparently seem to be absent. This is not too surprising,
considering that all Sulfurihydrogenibium species to date
have been identified in terrestrial environments. However,
no sequences were found that could be assigned to the
‘Desulfurobacteriaceae’, although these organisms have
been isolated from hydrothermal vents, and were found to
account for 40% of the microorganisms present in
chimney walls (e.g. Harmsen et al., 1997). An explanation
for this unexpected finding could be the specificity of the
primers used by Campbell and Cary (2004), which in our
hands failed to amplify aclB from ‘Desulfurobacteriaceae’.
In addition, our sequence analyses revealed a cluster of
acl sequences that seem to be affiliated with Hydrogenothermaceae, but cannot be directly linked to sequences
obtained from either Persephonella or Sulfurihydrogenibium. This strongly suggests that these sequences
represent a new group of so far uncultivated Aquificae.
Thus, our data provide further evidence for the usefulness
of the ATP citrate lyase genes as both phylogenetic and
functional gene markers. Based on our limited number of
sequences, it appears that citryl-CoA lyase may have the
potential to be used in the same way (Fig. 3).
Evolutionary implications
Surprisingly, two different pathways of citrate cleavage
exist within the Aquificae. Members of the genera Aquifex,
Hydrogenobacter and Thermocrinis use two enzymes,
namely citryl-CoA synthetase and citryl-CoA lyase to
accomplish the ATP-dependent cleavage of citrate into
acetyl-CoA and oxaloacetate. This has been proposed to
represent the ancestral version of the reductive TCA cycle
(Aoshima et al., 2004a,b). Furthermore, succinyl-CoA
synthetase and citryl-CoA synthetase are believed to
have evolved via gene duplication and diversification of
an enzyme that possibly catalysed both reactions
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92
88 M. Hügler et al.
Tv. ruber
B. lithotrophicum
'Desulfurobacteriaceae'
D. crinifex
D. thermolithotrophum
2E08 Guay
694-12
Fig. 5. Phylogenetic tree based on a ~330 bp
fragment of the gene coding for the small
subunit of ATP citrate lyase (aclB). The tree
depicts the phylogenetic relationships of the
newly obtained sequences from Aquificae with
other bacterial sequences derived either from
the environment (Campbell and Cary, 2004)
or from other cultures.
694-4
690-42
Persephonella
729-4 A1
P. marina
729-4 C2
729-4 A5
S. azorense
S. subterraneum
Sulfurihydrogenibium
820-A8
729-4 C1
820-C7
820-D8
uncultured Aquificae ?
820-C1
820-A12
729-4 H5
820-D5
T. denitrificans
Candidatus A. sulfidicus
Nautilia sp. Am-H
899-1
ε-Proteobacteria
899-3
6C6
7G3
C. tepidum
C. limicola
0.05 nucleotide substitutions / site
(Aoshima et al., 2004a). On the other hand, members of
the genera Persephonella, Sulfurihydrogenibium (both
Hydrogenothermaceae), Balnearium, Desulfurobacterium
and Thermovibrio (all ‘Desulfurobacteriaceae’) are using
ATP citrate lyase for citrate cleavage. Based on sequence
similarity, the origin of the ATP citrate lyase might be the
result of a gene fusion of citryl-CoA synthetase and citrylCoA lyase, which themselves are homologues of succinylCoA synthetase and citrate synthase (Fatland et al., 2002;
Aoshima et al., 2004a,b). The small subunit of ATP citrate
lyase is a homologue of the large subunit of citryl-CoA
synthetase, whereas the large subunit of ATP citrate
lyase seems to have formed by a fusion of the small
subunit of citryl-CoA synthetase and citryl-CoA lyase
(Fig. 2). These findings raise the question on the origin
of ATP citrate lyase in Hydrogenothermaceae and
‘Desulfurobacteriaceae’. In one possible scenario, ATP
citrate lyase might have evolved within the Aquificae
from an ancient form of the reductive TCA cycle
involving a two-step citrate cleavage, as exemplified
presently by H. thermophilus, to a reductive TCA cycle
utilizing only one enzyme, like it is operating in the
‘Desulfurobacteriaceae’. In another scenario, ATP citrate
lyase might have been transferred into Hydrogenothermaceae and ‘Desulfurobacteriaceae’ via lateral gene
transfer. Based on our data, the second scenario seems
more likely, because ATP citrate lyase from Hydrogenothermaceae and ‘Desulfurobacteriaceae’ appears to be a
derived form of the ATP citrate lyase since it is more
similar to the one from e-Proteobacteria than to the evolutionary ancestral enzyme (Figs 3 and 4). Our phylogenetic analyses confirm earlier results of Fatland and
colleagues (2002), who concluded that the sequence from
C. limicola appears to be closest to the evolutionary
ancestral form. Thus, ATP citrate lyase appears to have
been acquired by Aquificae via horizontal gene transfer,
most likely from e-Proteobacteria. Along these lines it is
interesting to note that both groups occur in similar environments, providing an ecological basis for such an event.
Ecological implications
The results obtained in this study lend further support to
the hypothesis that autotrophic carbon fixation through
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92
Autotrophic CO2 fixation in Aquificae
the reductive TCA cycle is widespread and contributes
significantly to biomass production in certain environments, particularly in hydrothermal habitats. Members of
the Aquificae are important in many hot environments,
e.g. shallow and deep-sea marine vents, terrestrial hot
springs, as well as the terrestrial subsurface (e.g.
Harmsen et al., 1997; Reysenbach et al., 2000a,b; Blank
et al., 2002; Takai et al., 2002; Huber et al., 2003;
Inagaki et al., 2003), and have been isolated from all
around the world (Table 1). Extensive research has been
particularly carried out at Yellowstone National Park,
USA, where Tc. ruber has been identified as an ubiquitous member of microbial communities in silicadepositing hot springs (Blank et al., 2002) and Aquificae
in general may dominate the microbial communities at
temperatures higher than 70°C (Spear et al., 2005). Our
finding that Tc. ruber and other Aquificae do fix CO2 via
the reductive TCA cycle suggests that primary production
at temperatures higher than 70°C in hot springs in Yellowstone and probably elsewhere occurs mainly via the
reductive TCA cycle.
Members of the Aquificae have also been shown to
constitute an important component of the microbial communities at deep-sea hydrothermal vents, where these
organisms seem to be mainly associated with sulfide
structures and most likely also colonize the subsurface
portion of vents (Harmsen et al., 1997; L’Haridon et al.,
1998; Reysenbach et al., 2000a; Götz et al., 2002; Alain
et al., 2003; Huber et al., 2003; Takai et al., 2003a; Vetriani et al., 2004). Besides members of the Aquificae,
e-Proteobacteria have been identified as a dominant
bacterial group at deep-sea vents (e.g. Reysenbach
et al., 2000b; Huber et al., 2003; Nakagawa et al.,
2005). Interestingly, autotrophic members of this group
also use the reductive TCA cycle for carbon fixation
(Campbell et al., 2003; Hügler et al., 2005; Takai et al.,
2005). As a whole, both groups exhibit similar metabolisms – i.e. the oxidation of reduced sulfur compounds
and hydrogen with both oxygen and nitrate or the oxidation of hydrogen with elemental sulfur coupled to the
fixation of inorganic carbon – and thus occupy a similar
ecological niche. However, they seem to be partitioned
by their temperature preference, with e-Proteobacteria
dominating the microbial communities at temperatures
from 20°C to 60°C, whereas Aquificae seem to be the
predominant autotrophs at temperatures higher than
60°C. Taken together, this suggests that a significant
fraction of the biomass at deep-sea hydrothermal vents
might be produced via carbon fixation through the reductive TCA cycle. It remains to be determined whether this
production rivals the fixation occurring through the
Calvin cycle, which in the current paradigm forms the
base of deep-sea hydrothermal ecosystems. Ongoing
studies in our laboratory that aim to couple the identity
89
of the microorganisms carrying out CO2 fixation in situ
with concurrent rate measurements are addressing this
question.
Experimental procedures
Bacteria, growth conditions and preparation of
cell extracts
Table 1 gives an overview about the strains used in this
investigation, including their isolation site and growth
requirements. Aquifex aeolicus VF5 (Eder and Huber, 2002),
B. lithotrophicum 17S (DSMZ 16304) (Takai et al., 2003a),
D. crinifex NE1206 (DSMZ 15218) (Alain et al., 2003),
D. thermolithotrophum BSA (DSMZ 11699) (L’Haridon et al.,
1998), H. hydrogenophilus Z-829 (DSMZ 2913) (Kryukov
et al., 1983), H. thermophilus TK-6 (DSMZ 6543) (Kawasumi
et al., 1984), Tv. ammonificans HB-1 (DSMZ 15698) (Vetriani
et al., 2004), Tv. ruber ED11/3LLK (DSMZ 14644) (Huber
et al., 2002) and S. subterraneum HGMK1 (DSMZ 15120)
(Takai et al., 2003b) were grown autotrophically as described
in references. Thermocrinis ruber OC1/4 (DSMZ 12173)
(Huber et al., 1998) was grown with formate as sole carbon
source. Biomass was harvested under the exclusion of
oxygen. Cell extracts were prepared using a mixer mill (type
MM2; Retsch, Haare, Germany) according to Hügler and
colleagues (2005). Protein concentration in cell extracts was
determined by the method of Bradford (1976) using bovine
serum albumin as standard.
Enzyme assays
Enzyme assays (0.5 ml assay mixture) were performed in
stoppered 0.5 ml glass cuvettes. The assay temperature
differed according to the organisms and is given in Table 2.
Reactions involving pyridine nucleotides were followed spectrophotometrically at 365 nm [e365 nm NAD(P)H = 3.4 ¥ 103 M-1
cm-1]. Reactions involving BV were followed spectrophotometrically at 578 nm (e578 nm BV = 8.6 ¥ 103 M-1 cm-1).
The citrate cleavage activity of ATP citrate lyase or the
combined activities of citryl-CoA synthetase and citryl-CoA
lyase were determined by coupling the reactions to malate
dehydrogenase, which reduces the produced oxaloacetate
with NADH. The citrate-, CoA- and MgATP-dependent
oxidation of NADH was monitored. The assay mixture contained 100 mM HEPES (N-2-Hydroxyethylpiperazine-N⬘-2ethanesulfonic acid)/NaOH, pH 8.3, 5 mM DTE, 5 mM MgCl2,
3 mM ATP, 0.5 mM CoA, 0.4 mM NADH, and 3 mM
D-citrate. The reaction was started by the addition of citrate.
Buffers used to determine the pH optimum were HEPES/
NaOH (pH 6.5–8.5), and MOPS [3-(N-Morpholino)-2hydroxypropanesulfonic acid]/NaOH (pH 6.5–8.5). 2Oxoglutarate : BV oxidoreductase, pyruvate : BV oxidoreductase, fumarate reductase, malate dehydrogenase, isocitrate
dehydrogenase, 2-oxoglutarate dehydrogenase, and pyruvate dehydrogenase were measured according to references
(Hügler et al., 2003; 2005). Formate dehydrogenase and
succinyl-CoA synthetase were measured as described
in Beh and colleagues (1993). Succinate dehydrogenase
was determined spectrophotometrically as the reduction of
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92
90 M. Hügler et al.
2,6-dichloroindophenol with succinate at 578 nm according to
Schauder and colleagues (1987). Fumarate hydratase was
measured in a direct spectrophotometrical assay by following
the consumption or the production of fumarate at 240 nm
using 0.5 mM fumarate or 5 mM malate as substrate
(Schauder et al., 1987; Hügler et al., 2003).
DNA extraction, PCR amplification, cloning and
sequencing of aclBA and ccl and phylogenetic analyses
DNA was extracted with the UltraClean microbial DNA Isolation kit (Mo Bio Laboratories, Solana Beach, CA) according to
the manufacturer’s protocol. For amplification of the ATP
citrate lyase genes, different primer sets were used in a
35-cycle PCR at an annealing temperature of 42–50°C. The
used primers were F2/R5 (annealing at 42°C) (Hügler et al.,
2005), 892f/R5 (annealing at 50°C) (Campbell et al., 2003;
Hügler et al., 2005) and F1 (5′-ATG GCT CAA AAG GCA ATT
AGA GAA T-3′)/R1 (5′-GCA CCA GCA TGA CCA AAC TGA
-3′) (annealing at 46°C). For the amplification of the citryl-CoA
lyase gene we used newly designed primers cclf1 (5′-CGG
CTT CTT GAT CTT GTA GG-3′)/cclr1(5′-TTC TCT GTG GTT
AGT TCT TC-3′) or cclf2(5′-CCT TTA ACC CAG ATG ATA
GG-3′)/cclr2(5′-GGT TCT TGA GTT AGT TCT TC-3′) in a
30-cycle PCR at an annealing temperature of 50°C. PCR
products were purified from agarose gels using a QIAquick
gel extraction kit (QIAGEN, Chatswood, CA) and cloned
using the TOPO TA cloning kit for sequencing (Invitrogen,
Carlsberg, CA) as described by the manufacturer. Colonies
were picked, and the plasmid DNA was purified with the
QIAprep spin miniprep kit (QIAGEN) as described by the user
manual. Plasmids were sent to the BioResource Center at
Cornell University (Ithaca, NY) for sequencing. DNA
sequencing was performed using the Applied Biosystems
Automated 3730 DNA analyser. The big dye terminator chemistry and AmliTaq-FS DNA polymerase were used. Preliminary sequence data of P. marina and S. azorense were
obtained from The Institute for Genomic Research through
the website at http://www.tigr.org. The sequences were analysed using MacVector (Accelrys, San Diego, CA) and PAUP,
version 4.0b10, as described previously (Hügler et al., 2005).
Nucleotide sequence accession numbers
The nucleotide sequences have been deposited in GenBank.
The accession numbers for the ATP citrate lyase genes
are: DQ853417 (B. lithotrophicum), DQ853418 (D. crinifex),
DQ853419
(D. thermolithotrophum),
DQ853420
(Tv.
ammonificans), DQ853421 (Tv. ruber), and DQ853422
(S. subterraneum). The accession numbers for the citryl-CoA
lyase gene fragments are: DQ853423 (H. hydrogenophilus),
DQ853424 (S. subterraneum) and DQ853425 (Tc. ruber).
Acknowledgements
This study was supported by the NASA Astrobiology Institute
(‘From Early Biospheric Metabolism to the Evolution of
Complex Systems’; Grant NNA04CC04A), as well as NSF
Grants MCB 04-56676 (C.V.), MCB 04-56689 (S.M.S.) and
OCE 04-52333 (S.M.S.). M.H. was supported through a post-
doctoral scholarship from the Woods Hole Oceanographic
Institution. Preliminary sequence data were obtained from
The Institute for Genomic Research through the website at
http://www.tigr.org. Sequencing of P. marina and S. azorense
was accomplished with support from the National Science
Foundation. We thank Virginia Edgcomb (WHOI) for help with
DNA extractions, James Voordeckers (Rutgers University) for
growing Tv. ammonificans, and Ken Takai (Japan Agency for
Marine-Earth Science and Technology) for kindly making
DNA of B. lithotrophicum available to us.
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© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92