Microbiology (2013), 159, 1760–1769
DOI 10.1099/mic.0.067868-0
Function of formate dehydrogenases in
Desulfovibrio vulgaris Hildenborough energy
metabolism
Sofia M. da Silva,1 Johanna Voordouw,2 Cristina Leitão,1 Mónica Martins,1
Gerrit Voordouw2 and Inês A. C. Pereira1
Correspondence
Inês A. C. Pereira
1
Instituto de Tecnologia Quimica e Biologica, António Xavier, Universidade Nova de Lisboa, Oeiras,
Portugal
[email protected]
2
Received 19 March 2013
Accepted 28 May 2013
The genome of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough encodes
three formate dehydrogenases (FDHs), two of which are soluble periplasmic enzymes (FdhAB
and FdhABC3) and one that is periplasmic but membrane-associated (FdhM). FdhAB and
FdhABC3 were recently shown to be the main enzymes present during growth with lactate,
formate or hydrogen. To address the role of these two enzymes, DfdhAB and DfdhABC3, mutants
were generated and studied. Different phenotypes were observed in the presence of either
molybdenum or tungsten, since both enzymes were important for growth on formate in the
presence of Mo, whereas in the presence of W only FdhAB played a role. Both DfdhAB and
DfdhABC3 mutants displayed defects in growth with lactate and sulfate providing the first direct
evidence for the involvement of formate cycling under these conditions. In support of this
mechanism, incubation of concentrated cell suspensions of the mutant strains with lactate and
limiting sulfate also gave elevated formate concentrations, as compared to the wild-type strain. In
contrast, both mutants grew similarly to the wild-type with H2 and sulfate. In the absence of
sulfate, the wild-type D. vulgaris cells produced formate when supplied with H2 and CO2, which
resulted from CO2 reduction by the periplasmic FDHs. The conversion of H2 and CO2 to formate
allows the reversible storage of reducing power in a much more soluble molecule. Furthermore,
we propose this may be an expression of the ability of some sulfate-reducing bacteria to grow by
hydrogen oxidation, in syntrophy with organisms that consume formate, but are less efficient in H2
utilization.
Department of Biological Sciences, University of Calgary, Calgary, AB, Canada
INTRODUCTION
Formate is an important metabolite in anoxic ecosystems. It
is formed by fermentative organisms in the degradation of
complex organic matter, and it is a common growth
substrate for many bacteria and archaea. It is also a cellular
intermediate in processes like acetogenesis or methylotrophy
and a possible substrate for methanogenesis (Chistoserdova
et al., 2007; Müller, 2003; Stams & Plugge, 2009; Thauer &
Shima, 2008). Together with hydrogen, formate is also
involved in interspecies electron transfer by syntrophic
communities of bacteria and archaea in methanogenic or
sulfate-reducing environments (McInerney et al., 2009;
Müller et al., 2010; Schink & Stams, 2006; Stams & Plugge,
2009). The ability to use formate depends on formate
dehydrogenases (FDHs), the enzymes that catalyse the
reversible conversion of formate to CO2. FDHs display a
Abbreviations: FDH, formate dehydrogenase; PFL, pyruvate-formate
lyase; SRB, sulfate-reducing bacteria.
1760
large diversity in quaternary structure, cofactor composition,
cellular localization and electron donors and acceptors,
reflecting the different physiological roles of formate (Ferry,
1990; Vorholt & Thauer, 2002). Most FDHs contain a
molybdenum or tungsten pterin cofactor, but some aerobic
bacteria possess nicotinamide adenine dinucleotide
(NAD+)-dependent FDHs that lack a prosthetic group
(Gonzalez et al., 2013; Popov & Lamzin, 1994; Vorholt &
Thauer, 2002).
The cellular location of FDHs is linked to their function. The
FDHs that function mainly as CO2-reductases are cytoplasmic enzymes found in acetogens, in symbionts and other
fermentative organisms, where they produce formate in the
first step of the Wood–Ljungdahl pathway for the biosynthesis of C1 compounds (Ragsdale, 2008). In methanogens,
cytoplasmic FDHs oxidize formate as an energy substrate,
which is converted to CO2 to be used in the methanogenic
pathway (Jones & Stadtman, 1981; Schauer & Ferry, 1982;
Wood et al., 2003). In Escherichia coli a cytoplasmic FDH
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Formate dehydrogenases in sulfate-reducing bacteria
(FDH-H) associates with a membrane-bound hydrogenase
forming a formate hydrogen-lyase complex (FHL) (Sawers,
2005). FDH-H is expressed during fermentative growth of E.
coli. When the formate concentration builds up it oxidizes
formate to CO2, coupled to reduction of protons to H2 by
the hydrogenase partner. Periplasmic FDHs generally act to
oxidize formate and are usually membrane-bound through
an integral membrane subunit that transfers electrons to the
quinone pool (Jormakka et al., 2002; Kröger et al., 1979). In
most micro-organisms this subunit is a b-type cytochrome
that may be involved in a redox loop mechanism with
another membrane-bound enzyme, resulting in electron
transfer to the cytoplasm and proton release in the
periplasm, thus contributing to the proton motive force
and, consequently, energy conservation (Simon et al., 2008).
However, in the deltaproteobacterial sulfate reducers most
periplasmic FDHs (and hydrogenases) are soluble, and
transfer electrons to cytochrome c3 (Matias et al., 2005;
Pereira et al., 2011). These FDHs either include a dedicated
cytochrome c3 subunit (as in FdhABC3) (Costa et al., 1997;
Sebban et al., 1995), or have only two subunits (FdhAB)
(Almendra et al., 1999; Raaijmakers et al., 2002), and
transfer electrons to the periplasmic cytochrome c network
and from this to membrane-bound complexes such as Qrc,
which reduce the menaquinone pool (Venceslau et al.,
2010). Sulfate-reducing bacteria (SRB) are metabolically
versatile and in the absence of sulfate can also grow
syntrophically with other organisms, which consume their
fermentation products: H2, CO2, acetate or formate (Bryant
et al., 1977; Muyzer & Stams, 2008; Plugge et al., 2011;
Schink & Stams, 2006; Stams & Plugge, 2009; Stolyar et al.,
2007). A high number of FDH genes is usually present in
SRB genomes (Pereira et al., 2011), which agrees with
formate playing an important role as a syntrophic
intermediate. In addition, intracellular cycling of formate
has also been suggested to contribute to energy conservation
in SRB (Pereira et al., 2008; Voordouw, 2002). Here, formate
formed intracellularly by the pyruvate-formate lyase (PFL)
would be transported to the periplasm where it would be
oxidized by FDHs, and electrons and channelled back to the
cytoplasm for sulfate reduction leading to a proton motive
force across the membrane. However, this proposal has not
yet been confirmed.
To further study the function of FDHs and the role of
formate in SRB metabolism we chose the model organism
Desulfovibrio vulgaris Hildenborough. This bacterium has
three periplasmic FDHs, two of which are soluble
(FdhABC3 and FdhAB), and one that is associated with
the membrane (da Silva et al., 2011; Sebban et al., 1995).
No cytoplasmic FDHs are encoded in the genome. The
FdhABC3 and FdhAB are the two main FDHs detected in
D. vulgaris, and they are differentially regulated by the
metals Mo and W (da Silva et al., 2011). Increased
expression of both FDHs is observed during growth with
formate/sulfate (relative to lactate/sulfate) (da Silva et al.,
2011; Zhang et al., 2006). In addition, increased transcription of all FDHs was observed during growth with H2/CO2/
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sulfate (Pereira et al., 2008), and in particular for the WFdhAB (da Silva et al., 2011). The function of FDHs during
growth of D. vulgaris with H2/CO2/sulfate is not immediately obvious. During this type of growth CO2 and acetate
are also supplied as carbon sources and one or more of
the FDHs may be acting to reduce CO2 to formate. The
formate may then act as a reservoir of stored hydrogen,
which can be released by reversible conversion of formate
to H2 and CO2. Alternatively, the formate is transported
into the cell and converted to pyruvate by the PFL, acting
in reverse. In order to get further insight on the role of
formate in SRB metabolism, we constructed deletion
mutants for the two main enzymes detected in D. vulgaris,
FdhAB (DfdhAB) and the FdhABC3 (DfdhABC3), and
studied their physiology under different growth conditions.
METHODS
Construction of mutant strains. The 500 bp regions located within
and downstream of the gene encoding the catalytic subunit of
FdhABC3 (fndG3; DVU2812) were PCR-amplified using primer pairs
p290f/p291r and p292f/p293r, respectively (Table 1). The amplified
fragments were cloned sequentially into pNOT19 (Schweizer, 1992)
by digestion with SacI and BamHI and PstI and BamHI and ligation
to obtain pNOTDfndG3. The latter was cleaved with BamHI and
ligated to the cat gene-containing 1.4 kb BamHI fragment from
pUC19Cm to obtain pNOTDfndG3Cm. NotI digestion of the latter
and insertion of the 4.2 kb NotI fragment from pMOB2 (Schweizer,
1992) gave pNOTDfndG3CmMob, which was transformed into E. coli
S17-1. Following conjugation of E. coli S17-1 (pNOTDfndG3CmMob)
and D. vulgaris, single crossover integrants were obtained on medium E
plates containing chloramphenicol (Cm), as described elsewhere (Fu &
Voordouw, 1997). Growth of the integrant in defined medium
containing Cm and 5 % (w/v) sucrose produced the double crossover
mutant D. vulgaris DfdhABC3 in which 1029 bp of the 39 end of the
3039 bp fndG3 coding region were replaced with the cat gene.
Likewise, the 500 bp regions located up- and downstream of the
fdhAB genes (DVU0587 and DVU0588) were PCR-amplified using
primer pairs p411f/p412r and p413f/p414r, respectively (Table 1) and
cloned following digestion with HindIII and BamHI and KpnI and
BamHI, respectively. The procedures used were similar to those for
construction of the DfdhABC3 mutant, except that the nptii gene,
encoding kanamycin (Km) and G418 resistance, was used as
the selectable marker (Bender et al., 2007; Caffrey et al., 2007).
This resulted in construction of the suicide plasmid
pNOTDFdhABKmMob and the double crossover mutant D. vulgaris
DfdhAB, which had the fdhAB genes replaced with the nptii gene.
Table 1. PCR primers used for mutant construction
Primer
p290f
p291r
p292f
p293r
p411f
p412r
p413f
p414r
Sequence
tcgagagctcTCAGCAGGCTGGCGACGTACT
tcgaggatccAGTTCGAGAAGGGACCCGACG
tcgaggattcGCGTCGAAGGTCGCCTTTCAG
tcgactgcagCTGTCAGGTCTGTCGCCGATG
tcgaaagcttAGCGCCTGACGCACCCTGTGA
tcgaggatccCGTGCCTCCTCTGGGGTTCAG
tcgaggatccGGTGGAGGGACCGGTAACGGT
tcgaggtaccAGTGCCGGACATCCTCCGGTC
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S. M. da Silva and others
Culture media and growth conditions. D. vulgaris wild-type (wt)
and mutant strains were grown in modified Postgate medium C
containing 0.2 g yeast extract l21, 25 mM iron, 1 mM nickel, 1mM
selenium and 38 mM sulfate. Either molybdenum or tungsten were
added at a final concentration of 0.1 mM. Electron donors used were
lactate, formate or hydrogen. For organic acids, a final concentration
of 40 mM was used. When hydrogen was the electron donor a
mixture of 80 % hydrogen/20 % CO2 was used as the gas phase, to a
final overpressure of 1 bar (105 Pa). Acetate (10 mM) was also present
in formate- or hydrogen-containing media. Growth was performed
under anaerobic conditions in 500 ml flasks half filled with culture
medium. The cells were grown at 37 uC and optical density was
measured at 600 nm with a Shimadzu spectrophotometer. All optical
density measurements are the mean of two biologically independent
experiments.
Analytical procedures. For metabolite analysis 1 ml samples of
culture were taken at regular intervals, immediately centrifuged for 10
min at 10000 g and the supernatants frozen at 220 uC. Lactate,
formate and acetate concentrations were determined by high pressure
liquid chromatography (HPLC), with a Waters chromatograph and
an LKB 2142 Differential Refractometer (LKB Bromma) detector. An
Aminex HPX-87H column (Bio-Rad) was used at 60 uC and data
were collected with the Millenium 32 software, version 3.05.01
(Waters). Samples (20 ml) were eluted isocratically at a flow rate of
0.5 ml min21, with 2.5 mM H2SO4. Sulfate concentrations were
determined with a PRP-x100 column (Hamilton) on a Waters
Acquity Ultra-Performance Liquid Chromatograph with indirect UV
detection at 310 nm, and data were collected and processed by
Empower 2 software (Waters). Samples (10 ml) were injected at 25 uC
and eluted isocratically at a flow rate of 2 ml min21, with a mobile
phase of 3 % (v/v) methanol and 97 % (v/v) of 6 mM hydroxybenzoic
acid (pH 10).
Formate quantification in cell suspensions. Cells grown with
either lactate or H2 were collected at mid-exponential phase,
centrifuged for 15 min at 6 000 g and suspended in a 10-fold smaller
volume of fresh culture medium containing a limiting concentration
of sulfate (10 mM). Lactate (40 mM) or H2 [1 bar (105 Pa)
overpressure; 80 % H2, 20 % CO2] were provided as electron donors.
Cells were incubated at 37 uC and samples taken at several intervals
and treated as described above. For formate quantification, 20 ml of
supernatant from each sample was dispensed per well in a 96-well
plate (Greiner). The reaction was started by adding a solution
containing 1 mM NAD+, 40 mM Tris/HCl buffer pH 8 (at 25 uC)
and 0.5 U of Candida boidinii formate dehydrogenase (Sigma) to a
final volume of 200 ml per well. Absorbance was read at 340 nm at the
start and after 1 h of incubation at 37 uC, in a 96-well plate reader
(BioTek). When necessary the samples were appropriately diluted.
RESULTS
In a previous study, we showed that FdhABC3 is the main
FDH present when Mo is available, and is particularly
upregulated during growth with formate and sulfate (da
Silva et al., 2011). On the other hand, FdhAB is the main
FDH in the presence of W, and fdhAB is particularly
upregulated during growth with H2 and sulfate, while W
causes repression of the fdhABC3 genes. The metals have no
effect on growth rate during growth with H2 and sulfate,
whereas a small increase in growth rate was observed in
formate/sulfate and lactate/sulfate in the presence of W
relative to Mo. These results, obtained with wild-type (wt)
cells, were again confirmed in the present work.
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Growth with formate/sulfate
When formate (40 mM) was used as electron donor with
excess sulfate (38 mM) significant differences were
observed between wt and mutant strains, and strikingly,
different phenotypes were revealed in the presence of Mo
or W (Fig. 1). In the presence of Mo (Fig. 1a, b) the wt and
DfdhABC3 strains had similar initial growth rate, but this
slowed down over time for the mutant, and its final cell
density was considerably lower than for wt. The DfdhAB
strain had an even lower growth rate but the final cell
density was not much different from that of the DfdhABC3
mutant. In the presence of W (Fig. 1c, d), the DfdhABC3
strain had only a slightly lower growth rate than the wt,
and the final cell density was similar for both, while growth
of the DfdhAB strain was severely impaired. Metabolite
analysis showed that four formate molecules were consumed per sulfate, as expected. Formate consumption (Fig.
1b, d) and sulfate reduction (data not shown) followed the
growth pattern in all experiments including for the DfdhAB
mutant in the presence of W, where practically no growth
occured.
D. vulgaris does not possess all the enzymes necessary for
the complete Wood–Ljungdahl pathway, namely a formyltetrahydrofolate synthase, which would allow the use
of CO2 or formate as carbon sources for C1 metabolism
(Maden, 2000). For this reason, when growing with
formate or H2 as electron donors, acetate or acetate and
CO2 have to be added as carbon sources. Acetate is
activated to acetyl-CoA, which can be reductively carboxylated to pyruvate (Tang et al., 2007), and is only used for
anabolic reactions, with 70 % of cell carbon being derived
from acetate and 30 % from CO2 (Badziong et al., 1979).
Acetate was not detectably metabolized and remained
constant (data not shown), due to the low biomass
produced.
Growth with lactate/sulfate
When lactate (40 mM) was used as an electron donor with
excess sulfate (38 mM) both mutant strains, DfdhAB and
DfdhABC3, grew at a lower rate than the wt and reached a
lower final cell density. Both effects were more pronounced
for the DfdhABC3 strain, and this behaviour was identical
irrespective of whether Mo or W were present in the
medium (Fig. 2a, c). These results indicate that the FDHs
play a role in energy metabolism during growth on lactate/
sulfate. Metabolite analysis showed that, in cultures of the
wt, 40 mM lactate and 20 mM sulfate were consumed, in
accordance with the expected stoichiometry. In contrast,
the mutants were not able to metabolize all lactate present
in the medium (Fig. 2b, d). Both mutants consumed only
30 mM lactate and reduced 15 mM of sulfate, in the
presence of Mo or W. Most of the lactate used was oxidized
to acetate and CO2 for energy generation, and only a small
part was incorporated into biomass (Stolyar et al., 2007;
Tang et al., 2007). No extracellular formate was detected
during growth.
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Microbiology 159
Formate dehydrogenases in sulfate-reducing bacteria
0.4
(a)
DvH wt
0.4
OD600nm
0.3
OD600nm
Mo
ΔfdhAB
ΔfdhABC3
0.2
0.1
W
(d)
W
0.3
0.2
0.1
0.0
0.0
50
(b)
Mo
50
40
Formate (mM)
Formate (mM)
(c)
30
20
10
40
30
20
10
0
0
0
10
20
30
40
50
60
0
10
Time (h)
20
30
40
50
60
Time (h)
Fig. 1. Growth curves (a, c) and formate consumption (b, d) of the wt, DfdhAB and DfdhABC3 strains in formate/acetate
(40 mM/10 mM) plus sulfate (38 mM) medium, in the presence of either Mo (0.1 mM; a and b) or W (0.1 mM; c and d). Data are
means from duplicate experiments; error bars indicate SE.
an additional carbon source. No significant differences in
growth rate or final cell densities were observed between wt
and mutant strains, except for a small delay to start growth,
and a slightly reduced cell density in the DfdhABC3 mutant
Growth with H2/sulfate
When hydrogen was used as electron donor, a headspace
overpressure of 1 bar (105 Pa) of H2/CO2 was maintained
during growth and acetate was again present to be used as
(a)
OD600nm
0.6
(c)
W
(d)
W
0.8
0.4
0.6
0.4
0.2
0.2
0.0
0.0
60
50
50
50
50
40
40
40
40
30
30
30
30
20
20
20
20
10
10
10
10
0
0
0
(b)
0
Mo
10
20
30
40
Time (h)
50
60
Lactate (mM)
60
60
Acetate (mM)
OD600nm
1.0
DvH wt
ΔfdhAB
ΔfdhABC3
0.8
Lactate (mM)
Mo
60
Acetate (mM)
1.0
0
0
10
20
30
40
Time (h)
50
60
Fig. 2. Growth curves (a, c) and metabolite production/consumption (b, d) of the wt, DfdhAB and DfdhABC3 strains in lactate
(40 mM) plus sulfate (38 mM) medium, in the presence of either Mo (0.1 mM; a and b) or W (0.1 mM; c and d). (b, d) Full
symbols indicate lactate and open symbols indicate acetate. Data are means from duplicate experiments; error bars indicate SE.
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S. M. da Silva and others
(Fig. 3a, c). This suggests that the FDHs are not essential
for growth in H2/CO2, despite the increase in their
expression levels under these conditions (da Silva et al.,
2011; Pereira et al., 2008). Approximately 10 mM sulfate
was consumed by all strains before growth stopped, which
was possibly due to inhibition of hydrogenases caused by
the accumulation of sulfide (Fig. 3b, d). As with growth in
lactate/sulfate medium, no extracellular formate was
detected during growth on H2/sulfate medium.
reduction of 10 mM sulfate, about 20 mM of lactate was
consumed and 20 mM of acetate was produced (data not
shown), according to the expected stoichiometry. After
sulfate is depleted, lactate can be fermented, to acetate, CO2
and H2 (Pankhania et al., 1988; Voordouw, 2002), but
formate can also be produced by PFL. In fact, very low
levels of formate were detected transiently after sulfate was
consumed. These levels were higher in the mutants than in
the wt, which agrees with a role of the FDHs in oxidizing
formate under these conditions, pointing to a cycling
mechanism during growth with lactate and sulfate.
Formate quantification in cell suspensions
With H2 as the electron donor, both wt and mutants took
longer to consume sulfate than with lactate as the electron
donor, but it was eventually depleted after 40 h for wt in
medium with Mo, and was reduced slightly faster in
medium with W (Fig. 5b, d). In medium with Mo, a low
level of formate started to be detected at 20 h incubation,
both in wt and in DfdhAB cells, and at 40 h in DfdhABC3
cells (Fig. 5a). After sulfate was depleted, formate started to
accumulate, and at the end of the incubation period the
formate concentration was higher for the wt (10 mM) than
for both mutants, indicating that FdhAB and FdhABC3
were functioning as CO2-reductases under these conditions. The rate of CO2 reduction was similar for wt and
DfdhABC3, but was considerably slower for the DfdhAB
strain. DfdhABC3 cells accumulated more formate than
DfdhAB cells (8 and 5 mM, respectively). In medium with
W, formate started to accumulate at 20 h incubation for
both wt and DfdhABC3, and at a similar rate in both (Fig.
5c). At the end of the incubation there was 11 mM formate
The growth phenotype of the FDH mutants in lactate/
sulfate medium suggests that intracellular formate cycling
is occurring under these conditions. In contrast, no
phenotype was observed for growth with H2/CO2, which
upregulates FdhAB expression. The fact that extracellular
formate is not being detected in either growth condition
does not mean that it is not being formed and consumed
by the cells at significant rates. The excreted concentration
could be so low that it is below the HPLC detection limit.
In order to try to detect low levels of formate, we carried
out experiments with concentrated cell suspensions, and
measured formate enzymically, which is a more sensitive
method. In these experiments sulfate was present in
limiting amounts (10 mM) relative to the substrate.
With lactate as electron donor (40 mM) sulfate was
consumed in the first three hours of incubation by the
wt and DfdhABC3 mutant, whereas it took longer for the
DfdhAB mutant to consume all the sulfate (Fig. 4). For
0.4
Mo
(a)
0.4
(c)
W
(d)
W
0.2
DvH wt
0.1
ΔfdhAB
ΔfdhABC3
0.0
0.2
0.1
Mo
(b)
45
Sulfate (mM)
Sulfate (mM)
0.3
0.0
45
40
35
40
35
30
30
25
OD600nm
OD600nm
0.3
0
10
20
30
Time (h)
40
50
60
25
0
10
20
30
40
50
60
Time (h)
Fig. 3. Growth curves (a, c) and sulfate consumption (b, c) of the wt, DfdhAB and DfdhABC3 strains in 1 bar (105 Pa)
overpressure of H2/CO2 (80 %/20 %) and sulfate (38 mM), in the presence of either Mo (0.1 mM; a and b) or W (0.1 mM; c and
d). Data are means from duplicate experiments; error bars indicate SE.
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Microbiology 159
Formate dehydrogenases in sulfate-reducing bacteria
2.0
(a)
Mo
DvH wt
ΔfdhAB
ΔfdhABC3
1.5
1.0
0.5
W
(d)
W
1.0
0.5
0.0
0.0
Mo
(b)
15
Sulfate (mM)
15
Sulfate (mM)
(c)
1.5
Formate (mM)
Formate (mM)
2.0
10
5
10
5
0
0
0
20
40
60
Time (h)
80
0
100
20
40
60
Time (h)
80
100
Fig. 4. Formate production (a, c) and sulfate consumption (b, d) in wt, DfdhAB and DfdhABC3 cell suspensions incubated with
lactate (40 mM) and limiting sulfate (10 mM), in the presence of Mo (a, b) or W (c, d). Results are means from duplicate
experiments; error bars indicate SE.
for the wt and 10 mM for the DfdhABC3 mutant. In
contrast, only 0.4 mM formate accumulated in the
incubation with DfdhAB cells.
14
(a)
Formate (mM)
Formate (mM)
14
8
6
4
W
(d)
W
10
8
6
4
2
2
0
0
Mo
(b)
15
Sulfate (mM)
15
Sulfate (mM)
(c)
12
ΔfdhAB
ΔfdhABC3
10
Many bacterial and archaeal genomes code for multiple
FDHs. Isoenzymes with different functions, expressed
Mo
DvH wt
12
DISCUSSION
10
5
10
5
0
0
0
20
40
60
Time (h)
80
100
0
20
40
60
Time (h)
80
100
Fig. 5. Formate production (a, c) and sulfate consumption (b, d) in wt, DfdhAB and DfdhABC3 cell suspensions incubated with
a headspace of hydrogen/CO2 (2 bar (2¾105 Pa)) and limiting sulfate (10 mM), in the presence of Mo (a, b) or W (c, d). Results
are means from duplicate experiments; error bars indicate SE.
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S. M. da Silva and others
under different conditions, with different cell locations, or
incorporating different metals in the active site often
coexist in the same organism (Chistoserdova et al., 2007;
Matson et al., 2010; Pierce et al., 2008; Sawers, 2005; Wood
et al., 2003). This allows the use of different pathways
for energy conservation and adaptation to environmental
constrains, such as substrate or metal availability. The D.
vulgaris genome codes for three periplasmic FDHs,
suggesting a function mainly in formate oxidation. The
two main enzymes detected (FdhABC3 and FdhAB) (da
Silva et al., 2011) are soluble and interact with the
cytochrome c pool. In the present work we constructed
deletion mutants of each of these enzymes to gain insight
into their role in energy metabolism during growth with
formate, but also with lactate or hydrogen, two of the main
energy substrates for SRB. In interpreting the results it is
important to keep in mind that these substrates, as well as
the metals Mo and W, are involved in regulating FDH
expression (da Silva et al., 2011). In the presence of Mo, the
main FDH expressed is FdhABC3, and this is significantly
upregulated by formate. In the presence of W the FdhABC3
is downregulated and FdhAB is the main enzyme, and
this is upregulated with both formate and H2. Similar
observations have been made for FDHs from Desulfovibrio
alaskensis (Mota et al., 2011).
With formate as energy source, the role of FDHs is rather
straightforward. They oxidize formate to CO2, releasing
protons in the periplasm and electrons that are transferred
through membrane-bound carriers to the cytoplasm to
reduce sulfate, thus generating a proton motive force.
Upon growth with formate the two mutants revealed
different phenotypes in medium with Mo or W. DfdhAB
was most impaired, showing a lower growth rate than wt
and the DfdhABC3 mutant in medium with Mo, whereas in
the presence of W this mutant was unable to grow with
formate. This is due to the downregulation of FdhABC3 by
W and its inability to incorporate this metal. Thus, FdhAB
is essential for growth in the presence of W. However, it
also plays a role in Mo conditions, since the DfdhAB
mutant did not grow identically to the wt in the presence
of Mo.
The DfdhABC3 mutant grew slower than the wt in Mo
conditions, and the final biomass yield was considerably
lower, which agrees with the fact that FdhABC3 is the main
FDH present in formate/Mo conditions (da Silva et al.,
2011). In the presence of W, the DfdhABC3 mutant grew at
the same rate as and with similar cell yield to the wt,
indicating that FdhABC3 does not play a role in these
conditions, where FdhAB is the main FDH present. Thus,
when Mo is available both soluble FDHs play a role during
growth with formate, whereas only FdhAB is involved in
the presence of W. These results highlight the importance
of considering the effect of trace elements in the
phenotypic analysis of mutants. This effect is often
neglected, but as shown here can be very significant if
these elements are involved in regulating the expression of
the deleted genes.
1766
When lactate is used as an electron donor for sulfate
reduction, substrate level phosphorylation alone is not
sufficient to generate energy, since the same number of
ATP molecules is produced through this mechanism as is
consumed for sulfate activation. Alternative mechanisms
involving the cycling of intermediates, such as hydrogen
(Odom & Peck, 1981), CO (Voordouw, 2002) or formate
(Heidelberg et al., 2004; Pereira et al., 2008; Voordouw,
2002), were proposed. Lactate is first oxidized to pyruvate,
whose metabolism can proceed via pyruvate : ferredoxin
oxidoreductase (PFOR) or via the PFL. PFL produces
formate that can be transported across the membrane and
oxidized to CO2 by the periplasmic FDHs. Both mutants
showed impaired growth in lactate and sulfate in the
presence of either Mo or W, in agreement with the less
pronounced regulatory effect of these metals upon lactate
growth (da Silva et al., 2011). This result shows for the first
time that both FdhAB and FdhABC3 play a role during
growth on this substrate, which provides strong evidence
for the formate cycling hypothesis during growth with
lactate and sulfate. This hypothesis is further supported
by the observation that in D. alaskensis G20 (formerly
Desulfovibrio desulfuricans G20) a mutant containing a
deletion of the gene coding for cytochrome c3, an electron
acceptor for the periplasmic FDHs, accumulates formate
during growth with lactate and sulfate (Li et al., 2009).
When lactate is present in excess, as in the experiments
with concentrated cell suspensions, cells turn to a
fermentative metabolism after sulfate is consumed. Here,
we could barely detect accumulation of formate, which
agrees with previous reports indicating that H2 rather than
formate is produced in lactate fermentation, and is the
main metabolite responsible for interspecies electron
transfer in syntrophic growth (Pankhania et al., 1988;
Stolyar et al., 2007). However, formate also plays a role,
albeit less important, as indicated by reduced growth of a
syntrophic association between D. vulgaris and a mutant of
Methanococcus maripaludis containing a deletion of two
FDHs (Stolyar et al., 2007), or by the reduced ability for
syntrophic growth on lactate of a PFL deletion mutant of
D. alaskensis G20 (Li et al., 2011).
Regarding growth in H2/CO2, it was previously shown that
expression of FDHs and PFL activating enzyme is higher in
D. vulgaris when cells are grown with H2/CO2 relative
to lactate (Pereira et al., 2008), suggesting that in these
conditions FDHs could also be involved in energy
conservation. However, the role played by FDHs during
growth with H2/CO2 is not clear. No significant differences
in growth were observed between the mutants and the
wild-type during growth in H2/sulfate, indicating that the
FDHs are not essential. If FDHs are functioning as CO2reductases, then less formate would be expected to
accumulate in the mutants versus the wt. During growth
with H2/CO2/sulfate, no formate production was detected,
but with concentrated cell suspensions placed under H2/
CO2, formate accumulation was observed as sulfate became
depleted, and this accumulation was faster and reached
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Microbiology 159
Formate dehydrogenases in sulfate-reducing bacteria
higher concentrations in wt cells, indicating that formate is
indeed being produced by the FDHs from reduction of
CO2. When sulfate becomes depleted, an excess of
reductive power accumulates. In this situation, the FDHs
may function in reverse to reduce CO2, which replaces
sulfate as electron acceptor, providing an electron and
proton sink for H2 oxidation. The results indicate that the
main FDH acting as CO2-reductase in these conditions is
FdhAB, although in Mo the FdhABC3 also plays a role.
Previously, we tested both purified enzymes for CO2
reduction activity and could not detect activity for FdhAB,
while a low activity was detected for FdhABC3 (da Silva
et al., 2011). However, the present results indicate that in
vivo, FdhAB does indeed function as CO2-reductase. Wcontaining FDHs are proposed to function mainly as CO2reductases because of the lower redox potential of W(IV)/
W(VI) compared to Mo(IV)/Mo(VI), and many of them
are extremely sensitive to oxygen, although Desulfovibrio
gigas FdhAB appears to be an exception (Almendra et al.,
1999; Hagen et al., 2009). Most likely the CO2-reductase
activity of isolated D. vulgaris FdhAB was lost upon
purification.
The observed production of formate from hydrogen and
CO2 may have biological and environmental significance.
When growing syntrophically, SRB ferment organic acids
and alcohols, as long as the end products (acetate, CO2, H2
and possibly formate) are consumed by other organisms,
keeping their concentration low (Bryant et al., 1977;
Muyzer & Stams, 2008). Although SRB are generally
thought to compete with methanogens for common
substrates, they do in fact coexist in habitats with low
sulfate concentrations, where fermentative growth of the
sulfate reducers is sustained by the acetate-, H2- and CO2consuming methanogens (Leloup et al., 2009; Plugge et al.,
2011). In syntrophic associations, both H2 and formate are
thought to play a role in interspecies electron transfer
(Schink & Stams, 2006; Stams & Plugge, 2009). They have
similar midpoint redox potentials (E52414 mV and
E52432 mV, respectively), allowing interconversion
between the two, and H2 production from formate has
been reported in methanogens (Lupa et al., 2008), and also
SRB (Dolfing et al., 2008). The conversion of formate and
H+ to H2 and CO2 (DG523 kJ mol21) was not
considered to provide enough energy for microbial growth.
However, growth on formate was shown to be possible
by syntrophic associations of both an acetogen and a
methanogen, and of a sulfate reducer and a methanogen
(Dolfing et al., 2008). More recently, growth of a single
organism on formate by production of H2 was demonstrated with the hyperthermophilic archaeon Thermococcus
onnurineus NA1, by using a high temperature and high
formate concentration that resulted in DG values in the
order of 28 to 220 kJ mol21 (Kim et al., 2010). Clearly,
the interconversion between formate and H2 can sustain
microbial life, depending on the relative concentrations of
the two. Given the high affinity of SRB for hydrogen we
propose that in certain habitats with high H2 partial
http://mic.sgmjournals.org
pressures and no available sulfate, these organisms may be
able to grow by converting H2 to formate in syntrophic
association with methanogens or other organisms that
consume formate, but are not as efficient as the SRB in the
use of H2. In typical sediment conditions where the H2
partial pressure ranges from 1 to 10 Pa, the CO2 partial
pressures are of the order of 30 000 Pa, and formate
concentrations are in the order of 1 to 10 mM (Schink &
Stams, 2006), the conversion of H2+CO2 to formate+H+
has a DG at 25 uC between 233.5 kJ mol21 (at 1 Pa H2 and
10 mM formate) and 244.9 kJ mol21 (at 10 Pa H2 and
1 mM formate). These values are well below the minimum
free energy required for microbial growth of 220 kJ mol21
from culture studies (Schink, 1997), or the 219 kJ mol21
determined for SRB in anoxic marine sediments (Hoehler
et al., 2001), suggesting that growth of SRB from
conversion of H2 and CO2 to formate is possible. This
would be another expression of the increasingly recognized
metabolic flexibility displayed by anaerobic micro-organisms, which is particularly apparent in the case of the
deltaproteobacterial SRB (Pereira et al., 2011). In addition,
given the extremely low solubility of H2 in water (0.8 mM
for 1 atm (101.3 kPa) of 100 % H2 at 25 uC), converting it
to formate may allow the storage of reducing power in a
reversible process that can rapidly regenerate H2, if
necessary. Our experiments, with cell suspensions, 2 bar
(26105 Pa) of H2/CO2, corresponding to 1.6 bar (1.66105
Pa) of H2 or 1.2 mM of dissolved H2, resulted in
production of 10 mM formate, increasing the availability
of aqueous H2 by ninefold. In practice, formate can act as a
soluble carrier of H2, as it is already considered in chemical
terms (Boddien et al., 2011; Hull et al., 2012).
In conclusion, this work provides evidence for the
occurrence of intracellular formate cycling during growth
of D. vulgaris Hildenborough with lactate and sulfate, as one
of several metabolic pathways the organism uses for energy
conservation. Both soluble FDHs were found to play a role
in this process. During growth with H2 and sulfate (in the
presence of CO2 and acetate), the FDHs are apparently nonessential, but when sulfate is exhausted they reduce CO2 to
formate, in what may be a newly recognized pathway to
allow syntrophic growth of SRB on hydrogen. Further work
will be required to test this hypothesis.
ACKNOWLEDGEMENTS
This work was supported by research grants PTDC/QUI/68486/2006
and PTDC/QUI-BIQ/100591/2008 to I. A. C. P. and Pest-OE/EQB/
LA0004/2011 to Instituto de Tecnologia Quimica e Biologica funded by
Fundação para a Ciência e Tecnologia (FCT, Portugal) and the FEDER
program, as well as by an NSERC Discovery grant to G. V. S. M. da S. was
supported by an FCT PhD fellowship SFRH/BD/24312/2005.
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