Role of the NiFe Hydrogenase Hya in Oxidative Stress Defense in
Pier-Luc Tremblay and Derek R. Lovley
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, USA
Geobacter sulfurreducens, an Fe(III)-reducing deltaproteobacterium found in anoxic subsurface environments, contains 4 NiFe
binant strain better fitted for exposure to oxidative stress than wild-type G. sulfurreducens. These results demonstrate that one
of the physiological roles of the O2-resistant Hya is to participate in the oxidative stress defense of G. sulfurreducens.
position of their active site: NiFe hydrogenases, FeFe hydroge-
nases, and Fe hydrogenases (40, 42). NiFe and FeFe hydrogenases
are involved in hydrogen respiration, hydrogen production dur-
ing fermentation, methanogenesis, and the recovery of H2gener-
ated during nitrogen fixation (42). Fe hydrogenases are found in
certain methanogenic archaea and catalyze a step in the conver-
sion of carbon dioxide to methane (44).
Geobacteraceae, a family in the Deltaproteobacteria, are mostly
found in anoxic subsurface environments in which insoluble
Fe(III) is the main electron acceptor (3, 4, 11, 19, 35–39). Many
Geobacteraceae can respire hydrogen, a source of electrons made
available in the subsurface by fermentative species (27, 30). There
are 4 NiFe hydrogenases encoded in the genome of Geobacter sul-
other periplasmic hydrogenase, Hya, is not essential for this pro-
cess (10). No function was attributed to the two cytoplasmic hy-
drogenases, Hox and Mvh (8).
Throughout evolution, anaerobic bacteria have developed de-
togenic bacteria isolated from termite guts, hydrogen oxidation
role in a protective mechanism against oxidative stress was pro-
posed for a periplasmic FeFe hydrogenase of the deltaproteobac-
terium Desulfovibrio vulgaris (5, 16).
Like Desulfovibrio species (1, 12, 13, 15), G. sulfurreducens is
able to survive exposure to O2and even to respire it at low con-
centrations (23). In this study, we employed a set of mutants to
evaluate whether the periplasmic hydrogenases of G. sulfurredu-
cens participate in the defense against oxidative stress. We dem-
onstrate that the NiFe hydrogenase Hya is more resistant to oxi-
dative stress than Hyb. We show that Hya has a function in the
overall protection against reactive oxygen species (ROS) and,
interconversion of H2with 2H?and 2e?(40, 42). Hydroge-
more specifically, in preventing deactivation of the hydrogenase
the electron donor.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The bacterial strains and plas-
routinely maintained anaerobically (N2-CO2, 80:20) at 30°C in NBF me-
dium with acetate (10 mM) as the electron donor and fumarate (40 mM)
dent growth, 10 ml of hydrogen gas was injected into the headspace of a
27-ml pressure tube containing 10 ml of donor-free NBF medium, and 1
mM acetate was added as a carbon source (10). Cultures grown with a
limiting concentration of acetate as the electron donor contained 1 mM
oxidative stress, only 0.4 ?M cysteine was added. For cultures exposed to
containing 10 ml of medium prior to inoculation. Escherichia coli was
cultivated in Luria-Bertani medium. Appropriate antibiotics were added
construct a ?hyaSLB::Kmr?hybL::Cmrdouble mutant, the ?hybL::Cmr
mutant allele was introduced into DL7. hybL::Cmrwas amplified by PCR
with primers MChyd21for (5=-GCACGAACTGTCCGGCATCG-3=) and
genomic DNA as the template. Electroporation of the 2-kb PCR product
and double mutant isolation were performed as previously described (9,
26). The double mutant was verified by PCR and Sanger sequencing.
Received 10 January 2012 Accepted 15 February 2012
Published ahead of print 24 February 2012
Address correspondence to Pier-Luc Tremblay, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jb.asm.org0021-9193/12/$12.00 Journal of Bacteriologyp. 2248–2253
Replacement of native hya promoter by Phyb. A 0.1-kb chromo-
somal region containing the native promoter of the hya operon was re-
placed by the 0.6-kb hyb promoter region. The Phyb promoter was fused
to the 5= part of the hyaS gene by recombinant PCR with the primers
GC-3=), recphybhyadn2g (5=-TTGCCATCGTCGTTACCTGCCATAAA
TCCCCCTGTCGGTATGAT), recphybhyaup (5=-ATCATACCGACAG
GGGGATTTATGGCAGGTAACGACGATGGC), and hya500dn (5=-GA
product was cloned into pCR2.1-TOPO with a TOPO TA cloning kit
(Invitrogen, Carlsbad, CA), resulting in pPLT158. The 3= part of Gsul
0124 (0.5 kb) was amplified by PCR with primers hyaup500BHI (5=-CTC
GGATCCAGTACGGCCACAAGGCTCACCTG-3=) and hyaupAvrII 5=-
CTGCCTAGGCTTTCCGCGATGGTCCTGGTGTTG-3=), digested with
BamHI-AvrII (NEB, Beverly, MA) and ligated with the T4 DNA ligase
(NEB) upstream of Phyb into pPLT158, resulting in pPLT159. pUC19-
was cloned into the AvrII restriction site located between the 3= part of
Gsul 0124 and Phyb of pPLT159, resulting in pPLT160. pPLT160 was
ducens and the DL8 strain as described previously (9, 26). Recombinant
strains were verified by PCR and Sanger sequencing.
extracted with an RNeasy Minikit (Qiagen, Valencia, CA) from mid-log-
phase cultures grown with hydrogen, 10 or 1 mM acetate as an electron
source, 20 or 0.4 ?M cysteine, no O2, or 0.15 ml of O2in the headspace.
The Enhanced Avian First-Strand Synthesis kit (Sigma-Aldrich, St-Louis,
and hybS (GSU0782) transcripts were amplified and quantified with the
SYBR green PCR master mix (Applied Biosystems, Foster City, CA) and
hyaS825r (5=-GCCCATTTTGTAGAGGCAGT-3=). Primers used for the
amplification of hybS were hybS229f (5=-CTTGACATGATTTCGCTGG
of these genes was normalized to expression of proC, a constitutively ex-
pressed gene in G. sulfurreducens (18). proC was amplified with primers
proc1f (5=-ATGCTGAAGGGAAGCACTCT-3=) and proc75r (5=-GGCC
AGCAGCCCTTTGAT-3=). Relative levels of expression of the studied
genes were calculated by the 2???CTmethod (25).
Benzyl viologen assay. G. sulfurreducens hydrogen-fumarate cultures
The resulting1.1-kb PCR
nm of 0.1 (mid-log phase). Cells were collected by centrifugation and
washed twice with anaerobic buffer A (50 mM Tris-Cl [pH 8.0], 150
?g/ml chloramphenicol). Cells were resuspended in buffer A to concen-
trate them 7.5-fold. O2-exposed cell suspensions were maintained for 5
min in the presence of atmospheric oxygen just before starting the assay.
Reduction of benzyl viologen was assayed under anaerobic conditions as
previously described (21). At time zero, 500 ?l of cell suspension was
added to 9.5 ml of H2-saturated buffer A containing 2 mM benzyl violo-
gen. Reduction of benzyl viologen was followed at room temperature by
OD at 550 nm. At this wavelength, the extinction coefficient of reduced
benzyl viologen is 9 mM?1cm?1(21). Protein concentrations were de-
termined with the bicinchoninic acid assay (Sigma-Aldrich). Hydrogen
oxidation activity was expressed as nmol of reduced benzyl viologen per
mg of proteins.
by the xanthine oxidase reaction and H2O2. Mid-log-phase G. sulfurre-
ducens hydrogen-fumarate cultures reduced with 20 ?M cysteine were
collected by centrifugation, washed twice, and resuspended in 4 ml of
anaerobic phosphate-buffered saline (PBS). The cell suspension was sep-
arated in two sealed pressure tubes filled with H2. In order to evaluate the
impact of hydrogen peroxide exposure, 1 mM was added to one of cell
suspensions, which were then incubated for 2 h at 30°C. The cell suspen-
sions were then serially diluted in PBS and plated on acetate (10 mM)-
fumarate (40 mM) medium. To calculate the survival rate, number of
CFU obtained from the cell suspension exposed to H2O2was divided by
the number of CFU obtained from the cell suspension not exposed to
The same experimental design was adopted to establish the ability of
G. sulfurreducens strains to survive superoxide exposure. O2
O2(10 ml) and 1 mM xanthine were added along with 1000 U/ml of
bovine liver catalase to eliminate H2O2. Xanthine oxidase was added to
initiate superoxide production.
RESULTS AND DISCUSSION
The NiFe hydrogenase Hya protects G. sulfurreducens against
was deleted grew poorly in medium in which hydrogen was pro-
vided as the primary electron donor, whereas a strain in which
only hya was deleted grew well (Fig. 1A). Slow growth of the hyb-
deficient strains was observed over time (Fig. 1A), but this was
TABLE 1 Bacterial strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s)Source or reference
E. coli Top10recA1 endA1 gyrA96 thi-1 hsdR17 (rK
?) supE44 relA1 ?lacU169
Invitrogen, Carlsbad, CA
PCA (ATCC 51573)
hyb Phyb mutant
Maddalena V. Coppi
PCR cloning vector; AprKmr
pUC19 carrying GmrloxP; AprGmr
pCR2.1 carrying Phyb upstream of the 5= part of hyaS; AprKmr
pPLT158 carrying the 3= part of Gsul 0124; AprKmr
pPLT159 carrying a gentamicin resistance cassette between the 3=
part of Gsul 0124 and Phyb; AprKmrGmr
Invitrogen, Carlsbad, CA
Oxidative Stress and G. sulfurreducens Hya Hydrogenase
May 2012 Volume 194 Number 9jb.asm.org 2249
attributed to growth with the low concentrations of acetate (1
mM) that were included in the medium as a carbon source, be-
with acetate alone (Fig. 1B). When the cysteine concentration in
the medium was decreased 50-fold, growth at these low acetate
concentrations was substantially diminished (Fig. 1D). When hy-
was comparable to growth in the presence of hydrogen with the
higher concentration of reductant (Fig. 1C). However, strains in
medium than in the medium with the higher concentration of
cysteine. In contrast, if the cultures were grown with a higher
concentration of acetate (10 mM) that supported rapid growth,
deleting hya had no impact on growth in low-cysteine medium
(Fig. 1D, inset).
These results demonstrated that when sufficient electron do-
nor was present, in the form of either hydrogen or high concen-
trations of acetate, wild-type cells did not require the higher con-
centrations of medium reductant. Increased electron donor
availability might provide more electrons to combat oxidative
stress. The surprising finding was the relative importance of hya
and hyb in overcoming oxidative stress with hydrogen. Whereas
hya, which is not required for growth on hydrogen and has been
reported to encode almost no hydrogenase uptake capacity (10),
was required in order for the cells to overcome the stress, deleting
hyb, which is required for growth on hydrogen and encodes sub-
stantial hydrogen uptake capacity, had no impact. In contrast to
the substantial impact of deleting hya on growth in low-cysteine
medium in the presence of hydrogen, the hya deletion had had
only a slight negative impact, if any, during growth with low ace-
tate and had no impact in high-acetate medium. These results
suggested that hya might be specifically responsible for providing
is available as an electron donor.
In order to further evaluate the potential role of Hya in oxida-
tive stress defense, G. sulfurreducens growth with hydrogen in the
presence of oxygen in the headspace was evaluated. Oxygen had a
slight effect on the growth of wild-type cells (Fig. 2A), increasing
more substantial impact on growth of the hya mutant (Fig. 2A),
increasing the doubling time 2.3-fold (Fig. 2B).
Sensitivity of G. sulfurreducens hya to exogenous O2
H2O2. Enzymes expected to contribute to the defense against ox-
idative stress encoded in the G. sulfurreducens genome (33) in-
clude those that degrade superoxide (superoxide dismutase
[GSU1158] and superoxide reductase [GSU0720]), as well as
cytochrome c peroxidase (GSU2813), catalase (GSU2100), vari-
ous peroxiredoxins (GSU0066, GSU0352, GSU0893, GSU3246,
and GSU3447), and rubrerythrins (GSU2612 and GSU2814) (33,
34). In order to determine if Hya might be directly or indirectly
involved in the promotion of these activities, the impact of delet-
ing hya on the response to superoxide or hydrogen peroxide ex-
FIG1 Growth of G. sulfurreducens hydrogenase mutants exposed to oxidative stress. The wild-type strain and hya, hyb, and ?hyaSLB::Kmr?hybL::Cmr(hyab)
Bars designate one standard deviation of the mean.
Tremblay and Lovley
jb.asm.org Journal of Bacteriology
of Hya (Fig. 3).
the gene for the small subunit of Hyb, hybS, were 71- to 143-fold
more abundant than transcripts for hyaS under all of the tested
conditions. The difference in the expression of hyb and hya might
explain the difference between the hydrogen oxidation activities
of the mutants observed previously (10). There was no significant
variation in hyaS or hybS transcript levels when cells were grown
with hydrogen or acetate as the electron donor, at a high concen-
tration versus a low concentration of cysteine, or in the presence
or absence of O2.
Replacement of the native hya promoter by the stronger hyb
promoter increased the transcript level of hyaS by two orders of
magnitude to a level comparable to that for hybS (Fig. 4A). Over-
expression of hya permitted a hyb-deficient mutant to grow with
hydrogen as the sole electron donor at low as well as high cysteine
concentrations (Fig. 4B). These results demonstrate that Hya can
support hydrogen-dependent growth when expressed at higher
levels. The facts that cells relying on Hya for hydrogen uptake can
grow at low cysteine concentrations but that cells that only have
Hyb cannot suggest that Hya is more resistant to oxidative stress
the hyb-deficient mutant overexpressing hya was 4 times slower
with high cysteine concentrations and 2 times slower with low
cysteine concentrations (Fig. 4C). These results indicate that even
when expression levels are comparable, Hyb is more competent
comparable to that of the wild type with high cysteine concentra-
with low cysteine concentrations. These results demonstrate that
genetically modifying G. sulfurreducens to overexpress Hya while
conserving the Hyb hydrogen oxidation capacity created a strain
better suited to grow under oxidative stress when hydrogen is the
Hydrogen uptake attributable to Hya. A role of Hya in con-
which hydrogen-dependent reduction of anthraquinone-2,6-dis-
ulfonate (AQDS), Fe(III)-nitrilotriacetic acid [Fe(III)-NTA], or
fumarate was investigated, because the hya-deficient mutant re-
duced AQDS and Fe(III) as well as the wild type with only a mar-
ginal diminution of the fumarate reduction rate (10). In order to
investigate hydrogenase activity in a more sensitive manner, ben-
zyl viologen reduction assays in the presence of chloramphenicol
to block de novo protein synthesis (24) were conducted with con-
centrated cell suspensions.
The bulk of the hydrogen oxidation activity in the wild type
could be attributed to Hyb (Table 2), but there was detectable
hydrogenase activity in the hyb-deficient mutant that was not
found in the hya hyb double mutant, suggesting that Hya could
account for low rates of hydrogen uptake. Since participation of
it is possible that this hydrogen oxidation by Hya is central to its
newly discovered function.
Hya prevents inactivation of Hyb caused by O2. Exposure of
cells to atmospheric O2for 5 min partially reduced the hydroge-
nase activity in the wild type, which can be attributed to Hyb
(Table 2). The marginal hydrogenase activity detected in the hyb
hydrogenase activity attributable to Hyb was completely inhib-
ited, illustrating the protective effect of Hya on Hyb activity. The
strain which lacked hyb and in which hya was overexpressed had
comparable hydrogenase activity before and after oxygen expo-
sure, further indicating that Hya is more resistant to oxidative
stress than Hyb.
The hydrogenase activity of the hyb-deficient mutant overex-
pressing hya, although substantial, is lower than that of the wild
H2but can do so under oxidative stress. Hydrogen oxidation ac-
FIG 3 Survival of wild-type and Hya-deficient strains of G. sulfurreducens
following superoxide and hydrogen peroxide exposure for 2 h. Survival rates
are the means from at least three independent experiments.
FIG 2 Growth of the G. sulfurreducens hya mutant exposed to O2. The wild
fumarate as the electron acceptor. Where indicated, 0.15 ml of O2was intro-
duced into the headspace prior to inoculation. Growth curves (A) and dou-
bling times (B) are the means from at least three independent replicate cul-
Oxidative Stress and G. sulfurreducens Hya Hydrogenase
May 2012 Volume 194 Number 9jb.asm.org 2251
tivity of a strain overexpressing Hya with a functional Hyb was
the wild type. Therefore, the observed improvement in the dou-
bling time of this recombinant strain under oxidative stress com-
hydrogen oxidation capacity.
Implications. The O2tolerance of the NiFe hydrogenases
is active at higher redox potential (32), and Hyd-2 is more sensi-
difference in the oxygen sensitivities of hydrogenases in G. sul-
furreducens. Two of the three Hya subunits have high homology
(HyaL, 49%; HyaB, 34%) to the respective E. coli Hyd-1 subunits,
the accessory protein of E. coli. The Hya small subunit, HyaS, has
the Hyb subunit and accessory proteins have high homology (be-
results suggest that Hya is less oxygen sensitive than Hyb and that
even though Hya is expressed at relatively low levels, its activity
helps protect Hyb from oxygen inactivation.
Fe(III) is generally most abundant near the oxic-anoxic inter-
face, where physical perturbations are likely to result in oxygen
incursions into the anoxic habitat of Geobacteraceae (41, 43), and
in most of these environments metabolism is likely to be limited
by electron donor availability (14, 28, 29, 31). Hya might aid in
survival under such conditions. Homologs of Hya are found in
multiple anaerobic bacteria (8, 42) and might have a similar pro-
enhance hydrogen-dependent growth under oxygen stress by
overexpressing Hya suggests a strategy for genetic engineering
Geobacter species for bioremediation or other applications in
which oxidative stress may be a concern.
We thank Maddalena V. Coppi for the construction of strain DLMC10.
This research was supported by the Office of Science (BER), U.S. De-
partment of Energy, Cooperative Agreement no. DE-FC02-02ER63446.
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FIG 4 Overexpression of the hydrogenase Hya from the hyb promoter. (A) Quantitative RT-PCR assay of hyaS and hybS under the control of their respective
native promoters and of hyaS under the control of Phyb. (B) Growth on hydrogen of the G. sulfurreducens hyb mutant with hya under the control of its native
promoter or Phyb with a high or low cysteine concentration. (C) Doubling times on hydrogen of the wild type, a recombinant strain with hya under the control
of Phyb, and the hyb mutant with hya under the control of Phyb (C). Each value or curve is the mean from at least three replicates.
TABLE 2 Hydrogen oxidation activity
Reduced benzyl viologen (nmol/min/mg
protein) in cellsa:
Not exposed to O2
7.2 ? 0.3
0.1 ? 0.0
8.4 ? 0.7
7.7 ? 2.3
2.5 ? 0.4
Exposed to O2
4.7 ? 0.5
0.1 ? 0.0
7.5 ? 0.4
2.7 ? 0.1
hya hyb mutant
hyb Phyb mutant
aEach value is the mean and standard deviation of at least three replicates.
Chloramphenicol (150 ?g/ml) was added to block protein synthesis.
bCells were exposed to atmospheric oxygen for 5 min.
cND, not detected.
dThe native hya promoter was replaced by the hyb promoter (Phyb).
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