BBA - Proteins and Proteomics 1865 (2017) 1455–1469

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BBA - Proteins and Proteomics

journal homepage: www.elsevier.com/locate/bbapap

Understanding the response of Desulfovibrio desulfuricans ATCC 27774 to the MARK

electron acceptors nitrate and sulfate - biosynthetic costs modulate substrate
selection
Joana R. Sousaa, Célia M. Silveiraa,b, Pedro Fontesa, Catarina Roma-Rodriguesc,
Alexandra R. Fernandesc, Gonzalez Van Driesschec, Bart Devreesed, Isabel Mouraa,
José J.G. Mouraa, M. Gabriela Almeidaa,e,⁎
a
UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Monte de Caparica, Portugal
b
Instituto de Tecnologia Química e Biológica António Xavier, Universidade NOVA de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
c
UCIBIO, Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Monte de Caparica, Portugal
d
Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Gent University, 9000 Gent, Belgium
e
Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Instituto Superior de Ciências da Saúde Egas Moniz, Campus Universitário, Quinta da Granja, 2829-511
Caparica, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that obtain their energy from
Desulfovibrio desulfuricans ATCC 27774 dissimilatory sulfate reduction. Some SRB species have high respiratory versatility due to the possible use of
Proteomics alternative electron acceptors. A good example is Desulfovibrio desulfuricans ATCC 27774, which grows in the
Respiratory flexibility presence of nitrate (end product: ammonium) with higher rates and yields to those observed in sulfate containing
Nitrate reduction
medium (end product: sulfide). In this work, the mechanisms supporting the respiratory versatility of D. de-
Ammonium assimilation
Energy metabolism
sulfuricans were unraveled through the analysis of the proteome of the bacterium under different experimental
conditions. The most remarkable difference in the two-dimensional gel electrophoresis maps is the high number
of spots exclusively represented in the nitrate medium. Most of the proteins with increase abundance are in-
volved in the energy metabolism and the biosynthesis of amino acids (or proteins), especially those participating
in ammonium assimilation processes. qPCR analysis performed during different stages of the bacterium's growth
showed that the genes involved in nitrate and nitrite reduction (napA and nrfA, respectively) have different
expressions profiles: while napA did not vary significantly, nrfA was highly expressed at a 6 h time point. Nitrite
levels measured along the growth curve revealed a peak at 3 h. Thus, the initial consumption of nitrate and
concomitant production of nitrite must induce nrfA expression. The activation of alternative mechanisms for
energy production, aside several N-assimilation metabolisms and detoxification processes, solves potential
survival problems in adapting to different environments and contributes to higher bacterial growth rates.

1. Introduction
Sulfate reducing bacteria (SRB) are a large and diverse group of
anaerobic microorganisms that obtain their energy through dissim-
ilatory sulfate reduction (DSR), a pathway of the sulfur cycle where
sulfate is reduced to sulfide as terminal electron acceptor [1–3]. How-
ever, the successive discovery of many other inorganic compounds
(sulfite, thiosulfate, elemental sulfur and even nitrate and nitrite) that
can also serve as final electron acceptors attested the high flexibility of
the energy metabolism of these organisms [1,2,4,5]. This enables them
to easily adapt to changes in the environment and inhabit a multitude
of both natural and artificial locations, such as marine sediments, hy-
drothermal vents, oil fields, freshwater sediments and anoxic waste-
water treatment plants [1]. There are several examples in the literature
of SRB species that reduce nitrate as an alternative electron acceptor,
including Desulfobulbus propionicus, Desulforhopalus singaporensis, De-
sulfobacterium catecholicum, Desulfotomaculum thermobenzoicum, De-
sulfovibrio oxamicus, Desulfovibrio termitidis, Desulfovibrio furfuralis, De-
sulfovibrio profundus, Desulfovibrio simplex and Desulfovibrio
desulfuricans, thereby involving these bacteria in the biological nitrogen
cycle as well [6]. Nitrogen is an essential component in living organ-
isms and can be found in several oxidation states [7]. The nitrogen

⁎
Corresponding author at: UCIBIO, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Monte de Caparica, Portugal.
E-mail address: mg.almeida@fct.unl.pt (M.G. Almeida).

http://dx.doi.org/10.1016/j.bbapap.2017.07.021
Received 4 April 2017; Received in revised form 12 July 2017; Accepted 21 July 2017
Available online 25 August 2017
1570-9639/ © 2017 Elsevier B.V. All rights reserved.
J.R. Sousa et al.
cycle includes six oxidation/reduction reactions wherein the nitrogen
atom passes from the fully oxidized form (nitrate, NO3−, + 5) to its
completely fully reduced form (ammonium, NH4+, −3) [7].
The interest on SRB is extended over different research areas. These
microorganisms have been linked to health issues since they can have a
role in inflammatory bowel diseases [8]. They are involved in corrosion
of metal equipment and souring of petroleum reservoirs, which can
cause serious economic problems in the oil industry [9]. Furthermore,
SRB's ability to reduce toxic heavy metals might be valuable for bior-
emediation [10]. The implications in such different areas are surely a
consequence of the adaptability of the bacteria to different substrates
and strongly compel studies regarding the understanding of its versatile
metabolism.
In this paper, we will focus on the bacterium Desulfovibrio de-
sulfuricans ATCC 27774 (D. desulfuricans), which grows in medium
containing nitrate with rates and yields ca. 2 times superior to those
observed in a sulfate rich environment [11]. The changes in cellular
metabolism that enable the switch in oxidizing substrates are not
completely understood. However, it is well known that the nitrate
(NapA) and nitrite reductases (NrfA), the two enzymes involved in the
dissimilatory reduction of nitrate to ammonia (DNRA), are over-
expressed when nitrate is the sole electron acceptor. In this case, DNRA
is considered a two-step respiratory process where nitrate is the term-
inal electron acceptor, initially converted into nitrite, and then reduced
to ammonia. Interestingly, in the presence of nitrate and sulfate, the
thermodynamically less-favorable sulfate is preferred [11–13]. Nitrate
reduction seems to occur only in the absence of sulfate and sulfide (the
product of sulfate reduction) [11,14,15]. In fact, opposing to the sulfate
reduction pathway, which is constitutively expressed when the cells
grow in nitrate, Marietou et al. have shown that nitrate respiration is
regulated at the transcriptional level with the nitrate reductase operon
nap being induced by nitrate but repressed by sulfate [11]. Although
the nitrate reduction pathway is not completely expressed while sulfate
is available, the nrf genes coding for NrfA seem to be expressed con-
stitutively in SRB and are present in sulfate grown bacteria [11,15,16].
In addition to the role in nitrate respiration, NrfA is thought to be in-
volved in nitrite and nitric oxide detoxification processes. The latter has
been reported in enteric bacteria that use NrfA to reduce NO [17,18].
Nitrite has been shown to inhibit SRB growth and therefore the pre-
sence of NrfA can sustain the cells in environments with millimolar
concentrations of nitrite (e.g. cohabitated by nitrate-reducing and sul-
fide-oxidizing bacteria) [19,20]. This detoxification effect has been
shown, for example, in Desulfovibrio vulgaris Hildenborough, which is
unable to use nitrate as respiratory substrate; however, it can cope with
nitrite through the expression of nrf genes [21,22]. Besides catalyzing
the six-electron reduction of nitrite to ammonia, some NrfAs were also
shown to reduce sulfite to sulfide, thus forming the only known link
between the nitrogen and sulfur biogeochemical cycles. The sulfite re-
ductase activity in these enzymes is considerably lower than their ni-
trite reductase activity; though, it is sometimes higher than that of
sulfite reductase enzymes themselves [23,24]. Regarding the peri-
plasmic nitrate reductase (NapA), which is involved in the reduction of
nitrate to nitrite, this enzyme participates in several processes, such as
redox balancing, nitrate scavenging, if in residual amounts, and aerobic
or anaerobic denitrification [6,25].
To identify the molecular tools involved in the respiration flexibility
of D. desulfuricans cells induced by different electron acceptors (nitrate
or sulfate), we performed a differential proteomic analysis based in
two-dimensional gel electrophoresis (2DE) combined with mass spec-
trometry (MS) based protein identification. Moreover, we have ana-
lyzed the expression of key genes involved in the respiratory chain.
Additionally, we monitored the levels of nitrite present in the extra-
cellular medium and the NrfA and NapA enzyme activities throughout
the bacterial growth, until reaching the stationary phase.
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BBA - Proteins and Proteomics 1865 (2017) 1455–1469
2. Materials and methods
2.1. Cell growth
D. desulfuricans cells were grown under anoxic conditions at 37 °C in
a lactate containing medium supplemented with either nitrate or sulfate
as electron acceptor substrates. The medium was composed of (in g L− 1
distilled water): KH2PO4, 0.5; NH4Cl, 1.0; CaCl2·2H2O, 0.04 (Merck);
sodium lactate, 6.0 (Sigma); sodium citrate, 0.3 (Ridel-de-Haen); ca-
saminoacids, 2.0 (Dfico); tryptone, 2.0 (Scharlau); Wolfe's Elixir
(1 mL L− 1) and vitamins solution (2 mL L− 1). Please note that tryptone
and casaminoacids are digestion products of casein, resulting in a
mixture of peptides and amino acids (in the case of casaminoacids),
which makes their exact composition unknown (semi-defined). The
culture medium was supplemented with Na2SO4, 4.5; FeSO4·7H2O,
0.004 (Merck) and MgSO4·7H2O, 0.060 (Panreac) for sulfate and with
NaNO3, 2.4; FeCl2·4H2O, 0.003 (Merck) and MgCl2·6H2O, 0.050
(Panreac) for nitrate and prepared at pH 7.5. The vitamins solution
contained (in mg L- 1 distilled water): vit. B2, 0.02; vit. B3, 0.05; vit. B1,
0.06; vit. B5, 0,06; vit. B6, 0.06; vit. C, 0.2 (Merck); vit. B12, 0.05
(Roche) and vit. H, 0.01 (Fluka). The solution was sterilized with
0.2 μm filters. The Wolfes's Elixir contained (in g L− 1 distilled water):
nitriloacetic acid, 1.5 (Fluka); MgSO4·7H2O, 3.0 (Panreac); MnSO4·H2O,
0.5; FeSO4·7H2O, 0.1; CoSO4·7H2O, 0.1; NiCl2·6H2O, 0.1; CuCl2·2H2O,
0.1; ZnSO4·7H2O, 0.1; CuSO4·5H2O, 0.01; AlK(SO4)2·12H2O, 0.01;
H3BO3,·0.01; Na2MoO4·2H2O, 0.01; Na2MoO4·2H2O, 0.01,
Na2SeO3·5H2O, 0.01 (Merck) and NaCl, 1.0 (Ridel-de Haen). The
medium was purged with argon for 30 min, to remove dissolved oxygen
and autoclaved at 121 °C for 20 min. The cells were grown in 1 or 2 L
flasks, specially adapted for anoxic cultures. The nitrate and sulfate cell
cultures were performed in triplicate. The inoculums (cells grown up to
the stationary phase) were 10% of the total volume. The growth rate
was traced by measuring the optical density (OD) at 600 nm using a
Shimadzu UV160A spectrophotometer. The specific growth rates (μ)
and culture doubling times (Td) were determined using the slopes of the
growth curves (semi logarithmic plots) in the exponential phase. Cells
were harvested after 16 h of growth by centrifugation (Beckman Avanti
J-25) of the culture media at 10000 × g (15 min, 10 °C). The pellet was
then washed in 10 mM phosphate buffer, pH 7.6. Finally, the cells were
suspended in the same buffer (1 g pellet per 3 mL buffer) and stored at
− 80 °C.
2.2. Sample preparation
The D. desulfuricans cells were homogenized in a Potter-Elvejhem
homogenizer (Multifix) in the presence of a protease inhibitor cocktail
(Complete EDTA - free, Roche) and a mix of endonucleases (DNase I,
Roche and RNase A, Sigma; 5 mg/L each). The cell suspensions were
disrupted in a French-Press (Thermo-FA-080A) at ca. 16000 psi and
centrifuged at 8000 × g for 15 min at 4 °C to remove non-lysed cells and
debris in a Sigma 3K30 centrifuge. The obtained protein samples were
stored at − 80 °C.
2.3. Protein quantification
Protein quantification was performed with the BioRad Protein Assay
kit and the 2D Quant kit (GE Healthcare). Bovine serum albumin
(Sigma) was used as protein standard solution.
2.4. Two-dimensional gel electrophoresis
Immobiline Drystrip gel (IPG) strips, 18 cm, pH gradient 4–7 (GE
Healthcare) were rehydrated overnight with 340 μL of a 2D rehydration
solution containing 7 M urea (Merck), 2 M thiourea (Merck), 2% (m/v)
CHAPS (Calbiochem), 0.5% (v/v) IPG buffer (GE Healthcare), 0.002%
(m/v) bromophenol blue (Merck), 0.28% (m/v) DTT (GE Healthcare)
J.R. Sousa et al.
and 350 μg of the protein samples.
The first dimension separation (isoelectric focusing) was performed
in an Ettan IPGphor 3 system (GE Healthcare) at 20 °C with a four step
program: step 1–500 V fixed voltage for 4 h, step 2 – voltage gradient
up to 1000 V in 3 h, step 3 – voltage gradient up to 10,000 V in 3 h and
step 4–10,000 V fixed voltage for 3 h, adding up to a total of 61.5 kV
ran in 15 h.
After isoelectric focusing the IPG strips were saturated with the
buffer system required for the second dimension separation in two
consecutive equilibration steps performed at room temperature. The
IPG strips were soaked and rocked for 15 min in SDS equilibration
buffer [6 M urea, 50 mM Tris-HCl pH 8.8, 30% (v/v) glycerol (Panreac),
2% (m/v) SDS (Sigma) and 0.002% of bromophenol blue] containing i)
10 mg/mL of DTT and ii) 25 mg/mL of iodoacetamide (Merck). For the
second dimension, the proteins were separated by SDS-PAGE in 12.5%
acrylamide gels on a vertical unit (EttanDalt Six, GE Healthcare). The
IPG strips were applied on top of the gel and the system was sealed with
1% agarose (GE Healthcare) solution prepared in electrophoresis buffer.
The SDS-PAGE was carried out at 350 V for ca. 3:30 h after an initial
period of 30 min at 80 V to allow the sample to migrate from the IPG
strip to the second dimension gel. The separated proteins were visua-
lized by the colloidal Coomassie Blue G-250 (Merck) staining method
[26]. The stained gels were scanned with the ImageScanner II (GE
Healthcare).
Gel image analysis and statistical quantification of relative protein
abundances was performed with Melanie 7.0 software (GeneBio). Three
replicate gels of three independent growths (biological replicates) from
each condition (nitrate and sulfate) were used in the analysis and
evaluated as a single set of 9 gels. The relative abundance of each
protein spot was compared between the nitrate and sulfate growth
conditions: the spots with a relative volume ratio higher than 2 and
ANOVA p-value bellow than 0.05 were considered differentially ex-
pressed. These spots and those observed only in one experimental
condition were selected for protein identification by Matrix-Assisted
Laser Desorption/Ionization - Time-of-Flight (MALDI-TOF) mass spec-
trometry.
2.5. Protein identification
Coomassie-stained spots were destained and washed twice with
200 mM ammonium bicarbonate in 50% acetonitrile, and subsequently
dried in a Speedvac Concentrator. The gel was incubated in 50 μL of a
0.003 μg/μL modified trypsin solution (Promega) in 50 mM ammonium
bicarbonate, pH 8.6, and incubated at 37 °C overnight. The supernatant
was transferred to another tube. Peptides that were retained in the gel
spot were extracted using 80% acetonitrile (ACN) containing 0.5%
trifluoroacetic acid. The pooled tryptic peptide extracts were lyophi-
lized and resuspended in 15 μL of 0.1% formic acid for mass spectro-
metric analysis.
Tryptic digests from each spot of interest were analyzed using
MALDI-TOF MS. Therefore, 1 μL of the peptide mixture was mixed with
an equal volume of matrix solution, i.e. 50 mM α-cyano hydro-
xycinnamic acid (Sigma) in 50% acetonitrile containing 0.1% formic
acid and deposited onto the MALDI target plate. MALDI mass spectro-
metric analyses were performed on a 4800 Plus MALDI TOF/TOF
Analyzer (Absciex) in MS and MS/MS mode (both in reflectron mode),
both calibrated with a standard calibration mixture provided by the
manufacturer. The MALDI data were processed with MASCOT software
searching against the non-redundant NCBI protein database
(20150726) selecting for ‘bacteria’, using the public Matrix Science
server (www.matrixscience.com). Variable modifications (oxidation
(M) and deamidation (NQ)) were selected. Other parameters were: 1
missed cleavage, peptide tolerance 0.35 Da and MS/MS tolerance
0.25 Da.
Proteins that could not be identified using MALDI-TOF MS were
further analyzed using LC-MS. Therefore, peptide mixtures were
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BBA - Proteins and Proteomics 1865 (2017) 1455–1469
separated using a 2-dimensional NanoAcquity UPLC® system (Waters
Corporation) in pseudo 1D mode. For the first dimension (high pH),
solvent A1 and B1 were composed of 20 mM ammonium formate in
water and ACN, respectively. For the second dimension (low pH), sol-
vent A2 and B2 were composed of 0.1% formic acid in water and 0.1%
formic acid in ACN, respectively. The sample (5 μL) was loaded onto an
XbridgeTM BEH130 C18 column (300 μm × 50 mm, 5 μm; Waters) at
3% solvent B1 at 2 μL/min. Peptides were eluted from the first di-
mension column in 50% of solvent B, and trapped on a Symmetry® C18
trapping column (180 μm × 20 mm, 5 μm; Waters). The trapped pep-
tides were separated on a HSS T3 C18 analytical column
(75 μm × 250 mm, 1.8 μm; Waters) at 40 °C at 250 nL/min by in-
creasing the acetonitrile concentration from 5 to 50% B2 over 60 min.
The eluting peptides were directly ionized using nano-electropray and
analyzed with a SYNAPT G1 HDMS (Waters) using a PicoTip Emitter
from New Objective (uncoated SilicaTipTM 10+/− 1 μm, Woburn).
The TOF analyzer was externally calibrated with MS/MS fragments of
human [glu1]-fibrinopeptide B (Glu-fib) from m/z 72 to 1285, and the
data was corrected post-acquisition using the monoisotopic mass of the
doubly charged precursor of Glu-fib (m/z 785.8426) (lock mass cor-
rection). Accurate mass data were collected in a data independent po-
sitive mode of acquisition (MSE) by alternating between low (5 V) and
high (ramping from 15 to 35 V) energy scan functions. The selected m/z
range was 125 to 2000 Da. The capillary voltage was set to 3.0 kV, the
sampling cone voltage was 26 V and the extraction cone voltage on
2.65 V. The source temperature was set on 65 °C.
The resulting LCMSE data were processed using ProteinLynx Global
SERVER v3.0 (PLGS, Waters Corporation). In brief, lock mass-corrected
spectra (0.250 Da window allowed) were automatically centroided,
deisotoped and charge-state reduced to produce a single monoisotopic
peak for each peptide and associated fragment ion. The correlation of a
precursor and a potential fragment ion was achieved by means of time
alignment, in the first instance. The following parameters were used for
the data processing in PLGS: the chromatographic peak width, the TOF
resolution and retention time window were determined automatically
by the software, and the low energy, high energy, and intensity
thresholds, which were set to 250, 100 and 1500 counts respectively. A
database containing Desulfovibrio protein sequences, downloaded from
Uniprot in April 2015, was used. The precursor and fragment ion tol-
erance were determined automatically. The default protein identifica-
tion criteria used included a maximal protein mass of 250,000 Da, a
detection of minimal 3 fragment ions per peptide, minimal 7 fragment
ions per protein and minimal 1 peptide per protein. Variable mod-
ifications (oxidation (M) and deamidation (NQ)) were selected.
Maximally one missed cleavages and a false positive rate of 4% was
allowed.
2.6. Nitrite quantification
The quantification of nitrite in the growth media of D. desulfuricans
cells (nitrate or sulfate based) was performed by the Griess colorimetric
assay [27]. The sample (50 μL) was mixed with 0.45 mL of 0.2 M
phosphate buffer, pH 7.6, followed by sequential addition of 0.25 mL of
sulphanilamide (Merck) [1% (m/v)] and 0.25 mL of n-(1-naphtil)
ethylenediamine (NEDA, Merck) [0.02% (m/v)]. This solution was
mixed in a vortex and incubated at room temperature for 10 min, after
which the absorbance at 540 nm was recorded using a Shimadzu
UV1800 spectrophotometer.
2.7. Nitrite and nitrate reductases activities
The nitrite and nitrate reductase activities of the D. desulfuricans
protein samples were tested in native polyacrylamide gels. Samples
were incubated in sample buffer [0.0625 M tris-HCl, pH 6.8, 10% (v/v)
glycerol and 0.01% bromophenol blue] and loaded on native 7.5%
polyacrylamide gels. Electrophoresis was carried out at 100 V for 1 h
J.R. Sousa et al.
after an initial period of 15 min at 80 V. Gels were incubated for 10 min
in three different solutions: 1) control − 100 mM phosphate buffer,
pH 7.6, 0.6 mM methyl viologen (Aldrich), 6 mM sodium dithionite
(Merck); 2) nitrate activity - same solution as control with 20 mM so-
dium nitrate (Merck) as substrate 3) nitrite activity - same solution as
control with 20 mM sodium nitrite (Merck) as substrate. The nitrite (or
nitrate) reductase activity was identified by the appearance of a col-
orless blot in the gels, which indicates that the immobilized enzyme
consumes the reduced methyl viologen (blue colored), the co-substrate
of the catalytic reaction with nitrite (or nitrate). No activity could be
detected when either the purified NrfA or the protein extracts were
incubated in the absence of nitrite or nitrate (i.e. with the control so-
lution).
2.8. mRNA Expression levels
Cell pellets were resuspended in NZYol (NZYtech) in a proportion of
100 μg pellet/500 μL NZYol. After incubation at room temperature for
15 min, the RNA was extracted according to the manufacturer in-
structions. To ensure total DNA removal, RNA samples were treated for
20 min with DNase I (Fermentas, ThermoFisher Scientific) using the
procedure recommended by the manufacturer. The reaction was
stopped by adding 2 volumes ethanol 99% (AGA), followed by an
overnight incubation and a centrifugation of 15,000 × g, 20 min at 4 °C.
After removal of the supernatant, the pellet was washed with 1 volume
ethanol 75% (v/v), centrifuged at 15000 × g, 15 min at 4 °C and the air-
dried pellet was resuspended in water treated with diethyl pyr-
ocarbonate 0.1 % (v/v) (DEPC, Sigma). cDNA synthesis was performed
with 500 μg RNA using the NZY M-MuLV First-Strand cDNA synthesis
kit (NZyTech) according to the manufacturer instructions. The expres-
sion of genes involved in nitrate metabolism, cytochrome c nitrite re-
ductase catalytic subunit NrfA (nrfA) and the periplasmic nitrate re-
ductase (napA), sulfate metabolism, dissimilatory sulfite reductase
alpha subunit (Ddes_0921) and adenylyl-sulfate reductase (apsA), and as
an internal control, DNA polymerase I (polA) (primer sequences in
Table S1) were evaluated by quantitative real time PCR (qPCR) using
the Hot FirePol Evagreen qPCR Mix Plus (ROX) (Solis BioDyne) per-
formed in a Corbett Research Rotor-Gene RG3000 (Qiagen), using the
program described in Table S2. The analysis of the relative gene ex-
pression variation in each sample was performed using the 2− ΔΔCt
method [28]. Significant differential expression was considered for
samples with 2− ΔΔCt values higher than 1.5 (2− ΔΔCt > 1.5) or lower
than 0.7 (2− ΔΔCt < 0.7).
3. Results and discussion
3.1. Cell growth
To evaluate the effects of the oxidizing substrate on cell growth, D.
desulfuricans was cultured in a lactate containing medium supple-
mented with either nitrate or sulfate salts. As expected, the cell growth
in nitrate medium is faster than in sulfate medium (Fig. 1) [11]. Ac-
cordingly, the specific growth rates (μ) and culture doubling times (Td)
are ca. 1.5 times lower in the former case (Fig. 1, inset).
The maximum OD in the presence of sulfate, observed in the sta-
tionary phase, is about 50% of the one obtained in the presence of ni-
trate. Moreover, the biomass harvested after 16 h of inoculation is al-
most doubled in the nitrate containing media. These results have been
attributed to the fact that nitrate reduction is thermodynamically more
favorable than sulfate reduction and so the cells grow faster when using
this electron acceptor. Besides, the final products of sulfate reduction
(sulfide) might be toxic to the cells and therefore, could inhibit their
growth [11,16].
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BBA - Proteins and Proteomics 1865 (2017) 1455–1469
Fig. 1. Growth curves of D. desulfuricans cells in (◆) nitrate and (■) sulfate based media.
Inset: specific growth rates (μ) and doubling times (Td). Error bars represent the standard
deviations from the mean values for three independent cultures.
3.2. Differential protein expression
To identify the changes induced by the different respiratory sub-
strates at the molecular level, 2DE proteomic studies were performed
on total protein extracts of D. desulfuricans cells grown in sulfate or
nitrate containing media harvested at 16 h (stationary phase). Due the
complexity of proteome maps and the high number of proteins visua-
lized in the pH range from 3 to 10 (not shown), we chose to analyze the
samples in a narrower interval (4–7) where most proteins are found; at
the same time, gel resolution is increased.
The gels obtained from protein extracts of sulfate and nitrate grown
cells were compared in terms of the number of spots and their relative
intensities (Fig. 2). To obtain reliable results, three gels (i.e. technical
replicates) were analyzed from three different growths cultures of each
medium (i.e. biological replicates), summing up to a total of nine gels
per condition. Overall, most protein spots are observed across the range
of pH 5 to 7, with relative molecular masses between 40 and 100 kDa.
Image analysis detects 604 spots in nitrate gels and 519 spots in sulfate
gels (average values), whereas the overlap between the spots of gels
from the same condition is over 98%. Because the total protein extracts
were analyzed, which should encompass all expressed proteins (soluble
and membrane) in the cell, we expected a higher number of protein
spots in the gels. Several reasons may contribute for this. First, it must
be considered that the analysis presented here refers only to the pH
range of 4–7. Second, sampling was restricted to the stationary phase,
which means that proteins expressed in other time windows are not
represented in the proteomes. Third, the inherent limitations of 2DE
could also explain the relatively small number of spots detected. For
instance, incomplete protein solubilization or cell lysis may well lead to
the loss of some proteins, namely membrane proteins [29]. Proteins
with a low copy number in the cell will also be underrepresented in the
gel maps, both due to the difficulties in extraction/solubilization pro-
cess and staining detection limits. The staining method used (CCB) is
adequate for the quantification of spot intensities and their relationship
with protein concentration, but is less sensitive than, for example,
fluorescent staining, which provides lower detection limits [30].
Next, a comparative analysis on the 2DE gels was conducted to
identify the changes in the protein profiles of D. desulfuricans cells
grown with either sulfate or nitrate. A high number of protein spots
could not be matched between the gels of the two conditions. These
spots, which we designate “exclusive”, are detected mainly in the gels
of nitrate grown cells (81 vs 7 found solely in the gels from sulfate
obtained cells). Note that, some of these spots may in fact have a very
low abundance in the gel in which they were not detected, thus being
below the detection limits of the staining method and image analysis
software. By using a 2-fold spot volume intensity ratio and p-value
(ANOVA) lower than 0.05 as criteria, we found that 40 additional
J.R. Sousa et al. BBA - Proteins and Proteomics 1865 (2017) 1455–1469

Fig. 2. 2D gel maps of total protein fractions of D. de-

sulfuricans grown for 16 h in (A) nitrate and (B) sulfate
containing media. Circles indicate protein spots with in-
creased intensity in each condition. The spot numbers
marked with * represent proteins that are exclusively de-
tected in one growth condition. Proteins (350 μg) were se-
parated by 2D gel electrophoresis (12.5% acrylamide) in a
pH gradient of 4–7. Gels were stained with colloidal
Coomassie blue. Gel images were acquired from Melanie 7.0
software.

1459
 Table 1
Proteins with changed abundance in the total proteome extracts of D. desulfuricans grown in nitrate or sulfate containing media. *Theoretical values.

Spot nr Gene identifier Protein name and response in nitrate medium Functional categories Molecular mass pI* Fold change
J.R. Sousa et al.

(Da)*

Down-expressed
504 Ddes_0477 Dihydrodipicolinate reductase Amino acid biosynthesis: Aspartate family 38,638 5.68 − 3.68
164 Ddes_2166 Thiazole synthase Biosynthesis of cofactors, prosthetic groups, and carriers: Thiamine 27,537 5.98 − 4.43
10 Ddes_0134 Methyl accepting chemotactic protein Cell motility, Signal transduction mechanisms 87,676 5.21 − 4.50
528 Ddes_1494 Catalase Cellular processes: Detoxification 55,226 6.17 Exclusive
129 Ddes_0602 Peroxiredoxin Cellular processes: Detoxification 20,896 5.33 − 4.08
194 Ddes_0048 Glutaminase Energy metabolism: Amino acids and amines 32,762 5.07 − 2.59
307 Ddes_0173 NAD(P)(+) transhydrogenase (AB-specific) Energy metabolism: Electron transport 41,358 5.95 − 2.08
320 Ddes_0173 NAD(P)(+) transhydrogenase (AB-specific) Energy metabolism: Electron transport 41,332 5.95 − 2.20
340 Ddes_0174 NAD(P) transhydrogenase subunit alpha Energy metabolism: Electron transport 41,331 5.91 − 3.83
523 Ddes_1038 [NiFe] hydrogenase large subunit (Fragment) Energy metabolism: Electron transport 60,026 6.74 Exclusive
529 Ddes_1531 Succinate dehydrogenase or fumarate reductase, flavoprotein subunit Energy metabolism: TCA cycle 67,486 6.07 Exclusive
527 Ddes_1616 3-Oxoacyl-[acyl-carrier-protein] synthase 2 Fatty acid and phospholipid metabolism: Biosynthesis 43,771 5.44 Exclusive
524 Ddes_1751 Hypothetical protein Hypothetical proteins 8825 6.23 Exclusive
120 Ddes_1402 Intracellular protease, PfpI family Protein fate: Degradation of proteins, peptides, and glycopeptides 21,056 5.93 − 2.40
460 Ddes_1785 Elongation factor G Protein synthesis: Translation factors 76,332 5.29 − 2.73
411 Ddes_1338 AMP dependent synthetase and ligase Secondary metabolites biosynthesis, transport and catabolism 61,339 5.06 − 2.05
179 Ddes_1328 Methyltransferase type 12 Secondary metabolites biosynthesis, transport and catabolism 27,581 4.98 − 3.04
68 Ddes_0648 Nickel transporter Transport and binding proteins 30,429 6.54 − 3.14
448 Ddes_1343 TonB-dependent receptor Transport and binding proteins 83,615 4.78 − 2.45

Overexpressed
522 Ddes_1152 Glutamate dehydrogenase Amino acid biosynthesis: Glutamate family 48,913 5.97 Exclusive
275 Ddes_1676 Ornithine carbamoyl transferase Amino acid biosynthesis: Glutamate family 38,392 5.07 2.65
285 Ddes_1676 Ornithine carbamoyltransferase Amino acid biosynthesis: Glutamate family 38,405 5.00 2.48

1460
568 Ddes_1811 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide Amino acid biosynthesis: Histidine family 26,667 4.91 Exclusive
isomerase
564 Ddes_1017 Phosphoserine phosphatase Amino acid biosynthesis: Serine family 22,638 5.23 Exclusive
233 Ddes_1898 Pyridoxal biosynthesis protein; Pyridoxal 5′-phosphate synthase subunit PdxS Biosynthesis of cofactors, prosthetic groups, and carriers: 31,489 5.85 2.81
Pyridoxine
544 Ddes_0164 Cell shape determining protein, MreB/Mrl family Cell envelope: Biosynthesis and degradation of murein sacculus 37,306 5.43 Exclusive
and peptidoglycan
374 Ddes_1108 UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase Cell envelope: Biosynthesis and degradation of murein sacculus 52,646 5.49 2.99
and peptidoglycan
534 Ddes_2048 NAD-dependent epimerase/dehydratase Cell wall/membrane/envelope biogenesis 33,603 5.15 Exclusive
575 Ddes_2281 Flagellin Cellular processes: Chemotaxis and motility 41,718 4.84 Exclusive
587 Ddes_2281 Flagellin Cellular processes: Chemotaxis and motility 31,603 5.21 Exclusive
589 Ddes_1028 Flagellin Cellular processes: Chemotaxis and motility 32,110 5.23 Exclusive
396 Ddes_1829 Hydroxylamine reductase Cellular processes: Detoxification 58,499 6.04 2.69
601 Ddes_1829 Hydroxylamine reductase Cellular processes: Detoxification 58,622 5.98 Exclusive
135 Ddes_0538 Superoxide dismutase Cellular processes: Detoxification 22,526 5.43 2.57
494 Ddes_0298 Pyruvate synthase Energy metabolism: Electron transport 128,159 5.32 2.11
267 Ddes_0132 Glyceraldehyde 3-phosphate dehydrogenase Energy metabolism: glycolysis, gluconeogenesis 36,242 5.89 2.67
536 Ddes_0480 Transketolase Energy metabolism: Pentose phosphate pathway 71,527 5.57 Exclusive
314 Ddes_0101 Isocitrate dehydrogenase Energy metabolism: TCA cycle 46,840 5.26 4.28
333 Ddes_0101 Isocitrate dehydrogenase Energy metabolism: TCA cycle 46,840 5.26 3.01
586 Ddes_2012 Lactamase Energy production and conversion 45,380 5.98 Exclusive
554 Ddes_1704 Hypothetical protein Hypothetical proteins 13,641 5.14 Exclusive
420 Ddes_1548 GroEL chaperone Protein fate: Protein folding and stabilization 56,997 4.82 3.00
546 Ddes_2248 GTPase Protein synthesis (ribossome biogenesis) 40,419 4.91 Exclusive
499 Ddes_1785 Translation elongation factor G Protein synthesis: Translation factors 76,437 5.04 2.45
359 Ddes_1628 Elongation factor Tu Protein synthesis: Translation factors 43,293 4.87 3.39
608 Ddes_0010 Aspartyl-tRNA synthetase Protein synthesis: tRNA aminoacylation 70,434 5.60 Exclusive
588 Ddes_2264 Phosphoribosylaminoimidazolecarboxamide formyltransferase Purines, pyrimidines, nucleosides, and nucleotides: Purine 45,565 4.99 Exclusive
BBA - Proteins and Proteomics 1865 (2017) 1455–1469

(continued on next page)

J.R. Sousa et al. BBA - Proteins and Proteomics 1865 (2017) 1455–1469

Fold change protein spots display a different relative intensity in the gels from the

Exclusive
Exclusive

Exclusive
two conditions. Among these, 17 have lower abundance and 23 have

2.28

2.05
2.19
higher abundance in the cells grown in nitrate medium. Furthermore, a
careful analysis of the fold change values (Tables 1 and S3) shows that,

5.10
5.89
6.86

5.23
4.89
5.28
in general, they are higher for the proteins increased in sulfate grown
pI*

cells, which is a logical result since the total amount of protein loaded
in the gels, from both samples, sulfate vs nitrate, is the same, but the
number of proteins observed is much lower in the former case. Overall,
Molecular mass

the 128 differentially expressed proteins represent about 25% of the

total spots identified in 2D gels (4–7 pH range), which is a clear in-
41,851
41,054
47,667

38,859

12,404

dication that the respiratory substrates have important consequences in

(Da)*

8793

the cellular composition of D. desulfuricans, considerably affecting its

protein profile. These striking differences and the high number of
protein spots that are present almost exclusively in nitrate respiring
Purines, pyrimidines, nucleosides, and nucleotides: Pyrimidine

Secondary metabolites biosynthesis, transport and catabolism

Secondary metabolites biosynthesis, transport and catabolism

cells reveals that the bacterium requires an additional metabolic effort

to produce the proteins responsible for its adaptation to a nitrate rich
environment. We propose that while growing in sulfate containing
medium, the cells only need to produce the proteins necessary for their
base metabolism. This provides a rationale for the preference that D.
desulfuricans has demonstrated for sulfate reduction in the presence of
both substrates (nitrate and sulfate) [11–13]. To understand the pro-
cesses involved in the adaptation of D. desulfuricans to a nitrate based
culture medium the identity and function of the changed proteins was
ribonucleotide biosynthesis

further assessed in the next section. One should mention that our pre-
liminary work showed that samples collected at 8 and 16 h exhibited
Functional categories

similar proteomic profiles. That is why we opted to analyze cells har-

Unknown function

vested at 16 h (stationary phase), when higher cell yields (particularly

Transcription
Transcription

for sulfate grown cells) could be obtained [31].

metabolism

3.3. Identification and functional categorization of differential proteins

The protein spots showing different intensities (increasing or de-

creasing) and those exclusive found in a single growing condition (ni-
trate vs sulfate) were excised from the 2DE gels for analysis by mass
spectrometry (Tables S3 and S4). The identified proteins were listed
and grouped into functional role categories according to the annotation
provided by TIGRFAMs (Table 1) [32–34].
In general, there is a good agreement between theoretical molecular
mass and pI values and the actual electrophoretic mobility of the pro-
teins in the 2D gels. Discrepancies can be attributed to spots re-
presenting degradation products of larger protein species, protein ag-
gregates that do not disintegrate during electrophoresis and proteins
with extensive post-translational modifications [35,36]. Dissimilar 2D
spots representing the same protein or protein complex are observed for
isocitrate dehydrogenase, ornithine carbamoyl transferase, flagellin,
DNA-directed RNA polymerase subunit omega
Protein name and response in nitrate medium

DNA-directed RNA polymerase subunit alpha

hydroxylamine reductase, NAD(P) transhydrogenase, elongation factor

Pyridine nucleotide-disulfide oxidoreductase

DegT/DnrJ/EryC1/StrS aminotransferase

G and DNA-directed RNA polymerase. The identification of different

forms of a protein species with different expression levels may com-
2-Hydroxyglutaryl-CoA dehydratase

plicate interpretation of proteomic data. This is not the case in our work
since for most spots identified as the same protein the relative abun-
dance values follow the same trend, i.e., they are increased in the same
cell extract. Furthermore, they are usually found in closely related
positions in the 2D gels (same molecular mass and different pIs). This
suggests the spots represent isoforms of the same protein species or the
proteins have undergone post-translational modifications.
The proteins that show significant expression changes belong to
cupin

diverse functional categories, major groups being related with amino

acid or protein biosynthesis and energy metabolism. This is consistent
Gene identifier

with significant metabolism adaptations. No reliable identifications

were obtained for half of all tested spots (Table S3), which can be at-
Ddes_1416

Ddes_0545
Ddes_2174
Ddes_0686
Ddes_2161
Ddes_0206
Table 1 (continued)

tributed to insufficient quality of the MS spectra and/or a low abun-

dance of peptides. In fact, most of the unidentified proteins represent
faint spots, in particular the ones that were detected exclusively in the
Spot nr

gels of nitrate grown cells. In the next sections, we discuss the proteins
582

301
590
302
518
556

and cellular processes affected in D. desulfuricans by the use of different

electron acceptors.

1461
J.R. Sousa et al.
3.3.1. Energy and carbon metabolism
Several proteins involved in central carbon metabolic pathways
display relative abundance differences. For instance, glyceraldehyde-3-
phosphate dehydrogenase and transketolase are overexpressed during
growth of D. desulfuricans on nitrate medium. These are key enzymes in
glycolysis and the pentose phosphate pathway, respectively, and pro-
vide links between the two metabolic pathways. Glyceraldehyde-3-
phosphate dehydrogenase catalyzes the interconversion of 1,3-dipho-
sphoglycerate and glyceraldehyde-3-phosphate (in glycolysis and glu-
coneogenesis), whereas transketolase is responsible for two separate
reactions, in the non-oxidative branch of the pentose phosphate
pathway, both yielding glyceraldehyde-3-phosphate [37,38]. The
higher expression of these proteins suggests increased flow through
these metabolic pathways. The up-regulation of pyridoxal 5′-phosphate
synthase (cf. Table 1 and Section 3.3.2) indicates that glyceraldehyde-3-
phosphate may be directed to the production of vitamin B6, an im-
portant cofactor in many enzyme reactions [39,40].
Another protein with increased expression in nitrate grown cells and
involved in carbon metabolism is pyruvate synthase, which catalyzes
the decarboxylation of pyruvate to acetyl-CoA. It has been proposed
that this enzyme is very important in the energy metabolism in SRB,
since it is involved in energy conservation through substrate level
phosphorylation [41,42]. The enzyme is central to the lactate oxidation
pathway as pyruvate is a key intermediate of the oxidation of organic
substrates (Fig. 3) [43]. Therefore, we can associate the higher abun-
dance of pyruvate synthase with the faster cell division rates and con-
sequent higher energy demand of the cells grown in nitrate medium.
SRB species usually oxidize their organic substrates incompletely to
acetate to generate energy (NAD(P)H and ATP production) [44,45].
Numerous species, including D. vulgaris, display incomplete pentose
phosphate pathway and TCA cycles, which are thought to be used
mainly for the biosynthesis of amino acids and nucleic acids [34,45].
Nevertheless, complete oxidation of acetate to carbon dioxide has been
observed in some SRB [45]. Certain Desulfobacter species have full TCA
cycles (slightly different from aerobic bacteria cycles), whereas species
that do not possess complete TCA cycles can use the carbon monoxide
dehydrogenase pathway to oxidize acetate [44–46]. Higher production
of carbon monoxide dehydrogenase was previously observed in D. de-
sulfuricans growing with lactate and sulfate and under molybdate stress
1462
BBA - Proteins and Proteomics 1865 (2017) 1455–1469
Fig. 3. Proposed lactate oxidation/energy transduction
pathway in D. desulfuricans cells grown in nitrate or sulfate
containing media. Orange and dark green boxes indicate
proteins with higher or lower abundance in nitrate grown
cells, respectively. Beige and light green boxes indicate the
proteins involved in nitrate or sulfate reduction, respec-
tively. Gray boxes indicate proteins not identified in this
study. PFOR: pyruvate synthase; Hases: hydrogenases;
APSR: adenylyl-sulfate reductase; SR: sulphite reductase;
[NiFe]Hase:[NiFe] hydrogenase; NrfA: cytochrome c nitrite
reductase, catalytic subunit; NrfH: nitrite reductase, small
subunit; NapA: periplasmic nitrate reductase. (For inter-
pretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
[46]. In addition, we did not identify genes of some TCA enzymes in the
genome sequence of D. desulfuricans (e.g. citrate synthase and succinyl-
coA synthetase). These evidences indicate that the bacterium does not
express a full TCA cycle. Hence, the higher abundance of the TCA en-
zyme isocitrate dehydrogenase, is not directly correlated with an in-
creased energy production via TCA cycle. Instead, the up-regulation of a
partial TCA cycle (possibly citrate to 2-ketoglutarate) is related with
biosynthetic pathways. Isocitrate dehydrogenase catalyzes the re-
versible oxidative decarboxylation of isocitrate to 2-ketoglutarate,
coupled to the reduction of NAD(P)+ to NAD(P)H; but the reaction is
also an important branch-point for amino acid biosynthesis, since 2-
ketoglutarate is used as a carbon skeleton for the production of amino
acids from the glutamate family [47], pathways that are also up-regu-
lated in the cells obtained from nitrate culture medium (cf. Section
3.3.2). In contrast with isocitrate dehydrogenase, succinate dehy-
drogenase/fumarate reductase, which also takes part in the TCA cycle
(succinate/fumarate reversible conversion), has lower expression in the
cells grown in nitrate. The enzyme was previously shown to function as
fumarate reductase in D. desulfuricans (strain Essex) enabling fumarate
respiration or disproportionation [48,49]. The decrease of fumarate
reductase expression suggests the reductive branch of the TCA cycle is
more important for the energy metabolism of D. desulfuricans while
reducing sulfate.
Other proteins involved in energy metabolism displaying abundance
changes, also with lower expression in nitrate grown cells, include
[NiFe] hydrogenase, NAD(P)(+) transhydrogenase and glutaminase. In
fact, high levels of periplasmic [NiFe] hydrogenases were previously
observed in D. desulfuricans strains growing on sulfate and lactate. This
supports a hydrogen cycling model of energy conservation, according to
which cytoplasmatic hydrogenases convert the protons and electrons
derived from lactate oxidation into hydrogen. This molecule then dif-
fuses across the membrane and is reoxidized by periplasmic hydro-
genases, generating a proton gradient that can be used for ATP synth-
esis [34,50,51]. Therefore, [NiFe] hydrogenases are considered key
players for energy production in SRB growing on sulfate and lactate
(Fig. 3). Our proteomic data show that in the absence of sulfate the
protein is not detected, suggesting that the bacterium favors other
mechanisms for energy production. The increased flow through the
pyruvate synthase reaction, for example, points towards ATP synthesis
J.R. Sousa et al.
via substrate level phosphorylation.
NAD(P) transhydrogenases regulate the NADPH/NADP+ ratio in-
side cells. Under normal conditions, the balance is kept high since
NADPH is required for the biosynthesis of cell components [52]. The
sequence of D. desulfuricans reveals 6 genes for NAD(P) transhy-
drogenase and we have identified three protein spots of similar mole-
cular mass and pI, with lower abundance in nitrate grown cells, which
represent 2 protein species (Ddes_174 and 2 isoforms of Ddes_173). We
associate the decrease of NAD(P) transhydrogenase with the higher
flow through NADPH generating reactions, such as the ones catalyzed
by glyceraldehyde-3-phosphate and isocitrate dehydrogenases [52,53].
In contrast, the reduced flow through energy generating pathways in
sulfate respiring cells may be compensated by a higher abundance of
NAD(P) transhydrogenases, that increase the levels of NADPH, and thus
maintain the cellular redox equilibrium.
Glutaminase, catalyzes the deamination of glutamine to glutamate
and ammonium. It has been proposed that the enzyme controls the
cellular levels of glutamine, which is a central nitrogen metabolite.
Glutamine is essential to numerous cellular processes, such as the
synthesis of both purine and pyrimidine nucleotides [54–56]. We as-
sociate the decreased abundance of glutaminase, in cells obtained from
nitrate medium, with the need to maintain adequate levels of in-
tracellular glutamine to be used in biosynthetic processes. Additionally,
because the enzyme reaction releases ammonium, its down-expression
may prevent further accumulation of this product, already being gen-
erated from the respiration of nitrate (Fig. 4).
3.3.2. Biosynthesis of amino acids, nucleotides and cofactors
A majority of the identified protein spots belonging to functional
categories related to biosynthesis of amino acids, nucleotides and sec-
ondary metabolites, display higher relative abundances in D. desulfur-
icans cells grown with nitrate as electron acceptor. The result is in line
with the higher cell yields and overall faster growth observed in nitrate
medium.
As shown in Table 1, at least 4 proteins belonging to the biosyn-
thetic pathways of different amino acids have increased expression
when nitrate is the respiratory substrate of D. desulfuricans. The in-
creased levels of glutamate dehydrogenase and ornithine carbamoyl
transferase are particularly interesting. The first enzyme plays a role in
glutamate synthesis converting 2-ketoglutarate and ammonium into
glutamate; thus, this reaction is well suited for assimilating the
1463
BBA - Proteins and Proteomics 1865 (2017) 1455–1469
expectedly high concentration of ammonium generated from nitrate
respiration (Fig. 4). By taking up a TCA intermediary, glutamate de-
hydrogenase is also an important link between the carbon and nitrogen
metabolisms. The earlier mentioned up-regulation of isocitrate dehy-
drogenase can therefore be associated with 2-ketoglutarate derived
amino acids biosynthesis. Similarly, higher arginine production may be
inferred from augmented ornithine carbamoyl transferase levels. This
protein is involved in an intermediary step in the arginine biosynthetic
pathway, where it catalyzes the reversible conversion of carbamoyl
phosphate and ornithine (the product of glutamate degradation) into
citrulline [57]. Our proteomic data point to the assimilation of am-
monium derived from nitrate reduction as a driver of amino acid bio-
synthesis, thereby contributing to the higher bacterial growth rates and,
at the same time, dealing with potentially toxic elevated levels of am-
monium inside the cell (Fig. 4). In addition to the amino acids from the
glutamate family, enhanced production of serine and histidine is in-
ferred from the higher abundance of phosphoserine phosphatase and 1-
(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino]
imidazole-4-carboxamide isomerase, respectively (cf. Table 1) [58,59].
In contrast, dihydrodipicolinate reductase, a component of lysine bio-
synthetic pathway from aspartate, which is also ammonium assim-
ilating, has lower levels in the cells obtained from nitrate media. Thus
far, we could not find an explanation for this apparently contradictory
result.
Increased biosynthesis of nucleotides in nitrate grown cells is sug-
gested by the up-regulation of phosphoribosylaminoimidazolecarbox-
amide formyltransferase. The protein catalyzes the two final reactions
of the purine ribonucleotide biosynthetic pathway yielding inosine
monophosphate (IMP), a central intermediate in purine metabolism
[60]. The enzyme substrate, 5-aminoimidazole-4-carboxamide ribonu-
cleotide (AICAR), is a byproduct of histidine biosynthesis also shown to
be augmented [61]. Furthermore, 5-phosphoribosyl diphosphate
(PRPP), the precursor of purine, pyrimidine and histidine metabolisms
is derived from ribose-5-phosphate, one of the products of the reaction
of the pentose phosphate pathway enzyme transketolase [38,59], which
is up-regulated under the same growing conditions (cf. Table 1 and
Section 3.3.1).
Aside from amino acids and nucleotides, our study shows different
expression levels of proteins involved in cofactor biosynthesis, namely
pyridoxal 5′-phosphate synthase (subunit PdxS), augmented in nitrate
grown cells. The protein is involved in vitamin B6 metabolism; this
Fig. 4. Proposed ammonia assimilation pathways in D. de-
sulfuricans cells grown in nitrate or sulfate containing
media. Orange and dark green boxes indicate proteins with
higher or lower abundance in nitrate grown cells, respec-
tively. Beige boxes indicate the proteins involved in nitrate
reduction. Gray boxes indicate proteins not identified in
this study. IDH: isocitrate dehydrogenase; GDH: glutamate
dehydrogenase. (For interpretation of the references to
color in this figure legend, the reader is referred to the web
version of this article.)
J.R. Sousa et al.
cofactor has an important role in several enzyme processes, including
amino acid and fatty acid metabolism [62,63]. Pyridoxal 5′-phosphate
synthase contains a second subunit with glutaminase activity, which
yields ammonia from glutamine hydrolysis that is incorporated into the
pyridine ring of pyridoxal 5′-phosphate [64]. Consequently, higher
abundance of pyridoxal 5′-phosphate synthase is consistent with in-
creased activities of enzymes involved in the biosynthesis of the glu-
tamate family of amino acids and with glyceraldehyde-3-phosphate
dehydrogenase and transketolase from carbon metabolic pathways.
3.3.3. Transcription and protein synthesis
Higher levels of DNA-directed RNA polymerase, which takes part in
the gene transcription process, are observed in the cells obtained from
nitrate medium. This agrees with the overall up-regulation of protein
biosynthesis observed in that condition. Additionally, several proteins
belonging to the functional category of protein synthesis have increased
cellular levels. Obg GTPase and the elongation factors G and Tu are
translational GTPases involved in protein biosynthesis at the ribosome
[65–67]. The latter two have a concerted action during the elongation
phase of protein synthesis. The elongation factor Tu's main functions
are to bind GTP and aminoacyl-tRNA and deliver it to the ribosome.
Upon matching to the mRNA, and GTP hydrolysis, the peptide bond is
formed and elongation factor G translocates the mRNA one codon to
allow for the arrival of a new aminoacyl-tRNA [66,67]. D. desulfuricans
genome has multiple genes for elongation factors Tu and G. This could
explain why our data also shows one down-expressed elongation factor
G protein and suggests different patterns of gene regulation in response
to the available electron acceptor.
3.3.4. Stress response and detoxification
The stress response proteins peroxiredoxin and catalase are more
abundant in cells grown in sulfate medium. These proteins are im-
portant for the protection and survival of cells as they play an important
role in antioxidative defense mechanisms from damage induced by
reactive oxygen species (ROS). Catalase catalyzes the reduction of hy-
drogen peroxide to water and oxygen, while peroxiredoxins are able to
eliminate peroxynitrite, organic hydroperoxides as well as hydrogen
peroxide [68–70]. Some Desulfovibrio species, including desulfuricans
strains, can utilize oxygen as respiratory substrate. The SRB oxygen
reducing systems are mainly thought to be involved in protective
functions and should enable the bacteria to survive in the oxic en-
vironments of by lowering the local oxygen concentrations [2,71,72].
Aside from oxygen reduction, these systems can also generate ROS.
Within SRB, D. desulfuricans has a particularly high tolerance to oxygen,
being able to grow under nearly atmospheric oxygen levels. Accord-
ingly, the bacterium expresses not only oxygen reductases but also
proteins that allow the efficient removal of ROS [12]. Under the con-
ditions employed in this work (anoxic batch cultures), oxygen con-
centrations should be very low. Still, the residual levels may induce the
auto-oxidation of some reduced proteins, such as cytochromes and
flavoproteins, which can generate ROS [2,73]. More importantly, the
auto-oxidation of sulfide, the end-product of sulfate reduction, should
significantly increase the oxidative stress in the cells in comparison
with the ones growing in nitrate containing medium [73,74]. There-
fore, the increased level of antioxidant proteins is consistent with the
end of the exponential phase and the accumulation of stress products,
especially sulfide [71,75]. In contrast with peroxiredoxin and catalase,
superoxide dismutase is down-expressed in the samples from sulfate
cultures. This protein protects cells from free superoxide radicals by
converting them to hydrogen peroxide and oxygen. Previous studies
suggested that superoxide dismutases are constitutively expressed in
some Desulfovibrio species and that activities do not increase sig-
nificantly with exposure to oxygen [12,76,77]. The presence of me-
chanisms that defend the cells against oxidative stress, even under
anoxic conditions, certainly confers a major adaptive advantage to SRB,
facilitating a rapid adjustment to different environmental conditions.
1464
BBA - Proteins and Proteomics 1865 (2017) 1455–1469
Herein we propose that decreased superoxide dismutase levels may be
compensated by the other active oxidative stress defenses. Conversely,
the protein could play a more significant role in the scavenging of
oxygen radicals during growth of D. desulfuricans in the presence of
nitrate. This activity seems particularly relevant in these conditions in
order to prevent the formation of the highly reactive peroxynitrite ion.
Our study reveals two protein spots with increased intensity in ni-
trate grown cells, which were both identified as hydroxylamine re-
ductase. The protein reduces the highly toxic and mutagenic hydro-
xylamine to ammonium. The reaction product can subsequently be
assimilated through amino acid biosynthesis, as discussed in earlier
sections. Hence, hydroxylamine reductase is likely involved in ni-
trosative stress response [78]. An increased production of nitrogen re-
active species is expected during nitrate respiration. The activation of
hydroxylamine reductase may indicate an incomplete reduction of ni-
trite to hydroxylamine by NrfA, the enzyme responsible for the final
step of the dissimilatory nitrate reduction to ammonium; however,
previous work has shown that NrfA enzymes do not release detectable
intermediaries in the course of their catalytic cycle [79]. Still, NrfA is
capable of reducing nitric oxide and hydroxylamine, which also sup-
ports its active role in the protection against nitrogenous stressors [80].
In addition to the detoxification of hydroxylamine, hydroxylamine re-
ductase may be involved in hydrogen peroxide stress defense. Previous
studies, with the purified enzyme from D. desulfuricans demonstrated
that its KM values for hydrogen peroxide are within the range of other
peroxidases [81].
3.3.5. Other cellular processes
Cells grown in nitrate containing medium show higher abundance
of flagellin, a main component of the bacterial flagellum. Improved
motility allows the cell to reach available nutrients easily, which sup-
ports the faster growth observed in nitrate respiring cells. Similarly,
increased levels of a cell shape determining protein from MreB/Mrl
family, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopime-
late ligase and NAD-dependent epimerase/dehydratase, all involved in
the formation of the cell wall, is in line with the rapid cell division rates.
Moreover, a GroEL chaperone is overexpressed in nitrate cultured
cells. The protein is a component of the GroEL/GroES system, which is
essential for protein folding. Increased abundance of GroEL chaperone
assures the maintenance of optimal conditions for the formation of the
quaternary structure of proteins and the assembly of oligomeric com-
plexes [82]. This is consistent with the overall high protein expression
in nitrate grown cells. In addition, these molecular chaperones are
important to prevent nonspecific association of polypeptides under
stress conditions (e.g. end of the exponential growth phase).
Interestingly, two transporter proteins are down-expressed in the
samples obtained from nitrate medium, a TonB-dependent receptor and
a nickel transporter. TonB-dependent receptors are outer membrane
transporters that couple proton gradient with the uptake of iron che-
lates, vitamin B12, carbon substrates and nickel complexes into the
periplasmic space [83,84]. As previously reported, the presence of
nickel transport systems is mostly related with the occurrence of nickel-
dependent metalloenzymes [85]. In this case, decreased abundance of
the TonB-dependent receptor and nickel transporter proteins is in
agreement with the earlier discussed lower expression of [NiFe] hy-
drogenase, which is one of the major nickel-binding enzymes in bac-
terial cells [85,86].
3.4. The terminal enzymes of nitrate reduction
The enzymes involved in the nitrate reduction pathway in D. de-
sulfuricans, namely the periplasmic NapA and the membrane-bound
NrfA, could not be identified in the proteomic analysis. This is not
surprising, considering the intrinsic limitations of 2D electrophoresis to
analyze hydrophobic proteins, such as NrfA. In fact, our previous
findings suggested that NrfA cannot be loaded and/or separated in the
J.R. Sousa et al.
Fig. 5. Relative expression of genes involved in nitrate metabolism (periplasmic nitrate
reductase (napA) and cytochrome c nitrite reductase, catalytic subunit (nrfA)) and sulfate
metabolism (adenylyl-sulfate reductase (apsA) and dissimilatory sulfite reductase alpha
subunit (dsrA)), when D. desulfuricans is grown for 16 h in nitrate vs sulfate medium. Data
was normalized to the expression of polymerase A (polA) of cells growing in the same
condition. Error bars represent standard error of at least three independent experiments.
The dotted lines represent the cut-off where overexpression (2− ΔΔCt > 1.5) or down-
expression (2− ΔΔCt < 0.7) was considered.
2D gels [31]. In the case of NapA, it is possibly excluded from the
analysis because its pI (theoretical value 7.97) is outside the range
studied in this work (4–7). Therefore, we have tested NapA and NrfA
catalytic activities as well as their gene expression by mRNA quantifi-
cation.
3.4.1. Gene expression
To compare the transcript levels between the nitrate and sulfate
cultures, we used a normalized quantity (compared to sulfate) that is
directly proportional to transcript abundance (Fig. 5).
The analysis revealed that, at the stationary phase, napA is up-
regulated in the nitrate grown cells, which agrees with the activation of
the nitrate respiration pathway in the presence of that electron ac-
ceptor, as reported for several SRB species [44]. Surprisingly, the
transcript levels of nrfA, responsible for the second step in bacterial
nitrate reduction, were decreased. In previous works, it was shown that
while napA genes were strongly induced in the presence of nitrate, as
also observed in the present study, nrfA may be constitutively expressed
in D. desulfuricans (nitrite or nitrate only slightly increased its transcript
levels) [16,87]. The constitutive expression of nrf genes guarantees an
immediate response upon cell exposure to nitrite. Still, this does not
explain why nrfA gene was repressed when the cells are grown with
nitrate as electron acceptor in comparison with the cells respiring sul-
fate. Different authors have discussed the proteome/transcriptome re-
lations in cells. It has been established that although some genes have
similar transcript and protein trends, these parameters are not ne-
cessarily correlated [88–91]. To get a better understanding of the reg-
ulation of the nitrate reduction pathway in D. desulfuricans, we eval-
uated the gene expression levels during different stages of the
bacterium's growth in the nitrate containing medium. Particularly, the
napA and nrfA levels were reported at 4 different time points (3, 6, 12
and 16 h of growth) as a relative ratio compared to the 3 h of growth
(Fig. 6).
The napA gene abundance did not vary significantly, as the tran-
script level ratios were maintained between 1.5 and 0.7 throughout the
growth curve (Fig. 6). These results suggest napA transcript levels were
always kept high inside the cells because the gene is induced by the
elevated nitrate concentrations in the medium [11,87]. Furthermore,
NapA has a considerably lower turnover when compared to NrfA [4], so
the cells may need to produce the protein continuously to preserve a
high nitrate reduction rate. On the other hand, the data showed that
nrfA was highly expressed around 6 h, during the exponential growth
phase, and significantly down-regulated at later stages of cell devel-
opment (Fig. 6). nrfA appears to be induced later, possibly after initial
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BBA - Proteins and Proteomics 1865 (2017) 1455–1469
Fig. 6. Relative expression of genes involved in nitrate metabolism (periplasmic nitrate
reductase (napA) and cytochrome c nitrite reductase, catalytic subunit (nrfA)) at different
stages of D. desulfuricans growth curve. Data was normalized to the expression of poly-
merase A (polA) of cells collected after 3 h growth. Error bars represent standard error of
at least three independent experiments. The dotted lines represent the cut-off where
overexpression (2− ΔΔCt > 1.5) or down-expression (2− ΔΔCt < 0.7) was considered.
nitrate reduction to nitrite by NapA. The gene was expressed in the
early exponential phase, which is consistent with increased energy
demands in the cells to support the accelerated growth rates measured.
Upon entering the stationary phase, cell division is significantly lower
and the NrfA accumulated earlier was enough to process nitrite and
maintain an adequate flow through energy production pathways.
3.4.2. In-gel activity
In the next step, the nitrate and nitrite reductases activities of the
protein extracts of D. desulfuricans cells were evaluated in native
polyacrylamide gels. The colorless activity blot observed in the gel
containing the nitrate grown cells was considerably larger than the one
present in the gel of sulfate cell extracts, both obtained after 16 h of
growth (Fig. 7A). This indicated a much higher nitrite reducing activity
in the former cell samples, which apparently contradicts the nrfA
transcript levels measured in that experimental condition, but supports
our proposal that after being highly expressed and accumulated during
the exponential phase, NrfA remains active inside the nitrate grown
cells. Therefore, after being up-regulated, nrfA expression decreases
explaining why the abundance of the gene transcript is no longer ele-
vated after 16 h. The results obtained with cells harvested from nitrate
medium after 3, 6 and 12 h of culture also back this conclusion; they
reveal increased enzymatic activity, hence higher NrfA concentration in
the cells, at 6 h and 12 h (Fig. 7B).
The residual nitrite reducing activity detected in sulfate respiring
cells is in line with previous studies where NrfA was shown to be
constitutively produced in D. desulfuricans [16]. Unfortunately, the ni-
trate reducing activity in the cell extracts was below the detection limit
and therefore could not be monitored in this assay. As above men-
tioned, this is probably due to the low turnover of NapA in comparison
with NrfA [4].
3.4.3. Nitrite levels
As a final step to understand the expression of the enzymes re-
sponsible for the dissimilatory nitrate reduction in D. desulfuricans, we
measured the nitrite levels in the nitrate and sulfate cultures over a 24 h
period, accompanying the complete cell growth curves (Fig. 8). These
values should enable us to assess the activity of both NrfA and NapA. In
sulfate containing medium, the values of nitrite concentration are in
general very low. A small increase is observed between 10 and 15 h of
cell growth for which, thus far, we have not found an explanation.
Thereafter, during the stationary phase of cell growth, nitrite con-
centration stabilizes at a residual value of ca. 35 μM. According to these
results, the comparatively higher transcript levels of nrfA (at 16 h) in
the cells from sulfate medium can be explained by the accumulation of
J.R. Sousa et al.
Fig. 7. In gel nitrite reductase activities of protein extracts from D. desulfuricans. A) Gels
loaded cells grown for 16 h in medium containing A1) nitrate, A2) sulfate and A3) pur-
ified NrfA. B) Gels loaded with total protein extracts of cells grown in medium containing
nitrate harvested at different time points: B1) 3 h, B2) 6 h; B3) 12 h and B4) purified NrfA.
Native polyacrylamide gels (7.5%) were loaded with 16.8 μg of each protein extract and
10 μg of NrfA. Gels were incubated for 15 min in staining solution containing 0.6 mM MV,
6 mM sodium dithionite and 20 mM sodium nitrite in 100 mM phosphate buffer pH 7.6.
nitrite during later stages of cell growth. In addition, the produced NrfA
may contribute to increased sulfite reduction rates [23,24]. Interest-
ingly, the variation of nitrite concentration during D. desulfuricans
growth in nitrate medium is non-progressive, i.e., there is an initial
peak of nitrite concentration around 3 h (during the lag phase) and
after that nitrite levels decrease to residual values (25 μM) until 10 h of
growth, when a new progressive increase is detected, similar to the one
observed in the sulfate containing medium, that stabilizes around
250 μM, after 16 h. These results suggest that in the early stages of
cellular growth D. desulfuricans must sequentially produce high levels of
NapA to metabolize the excess of nitrate the cells must deal with upon
inoculation of the nitrate containing medium. Subsequently, the gen-
erated nitrite induces nrfA expression, which leads to a fast decrease in
nitrite concentration. Nitrite quantification results are also in agree-
ment with in-gel activity assays, which show a lower abundance of NrfA
in the cells harvested at 3 h of growth, when the outburst of nitrite
concentration is observed. Then, the nrfA gene is up-regulated so that
nitrite may be reduced. This is consistent with the cell requirements at
the exponential phase, when energy demands are higher due to the
rapid cell division rate.
3.5. The terminal enzymes of sulfate reduction
Unlike the enzymes involved in nitrate reduction, the transcript
levels of APS reductase and sulfite reductase genes do not change sig-
nificantly between the cell extracts obtained from nitrate and sulfate
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Fig. 8. Variation of nitrite concentrations in the culture media during D. desulfuricans
cellular growth. Nitrate (A) and sulfate (B) based media. Error bars represent the standard
deviation of mean values of at least three independent measurements.
based medium (Fig. 5). This data is in agreement with a previous work
also demonstrating that genes involved in the sulfate reduction
pathway are constitutively expressed when the cells grow in the pre-
sence of nitrate [11].
4. Conclusions
As published earlier by others [11], our work clearly shows that D.
desulfuricans is able to grow in nitrate medium with growth rates and
yields higher than those obtained in the presence of its natural electron
acceptor, sulfate. To activate the alternative dissimilatory nitrate re-
duction pathway, the genes encoding the terminal reductases NapA and
NrfA are up-regulated at key points of the cell growth. According to
comparative measurements of the transcripts levels, the nitrate re-
ductase gene napA, is overexpressed in a nitrate rich environment since
the lag phase, with no significant variations over the growth curve. In
what concerns the nitrite reductase NrfA, its gene expression is induced
when considerable amounts of nitrite are already produced (3 h). Ap-
parently, this enzyme is sufficiently stable and catalytically active over
the remaining growth stages, so its expression is silenced thereafter.
The differentiated set of reactions underlying nitrate reduction (ni-
trate → nitrite → ammonium) is thermodynamically more favorable
[11], allowing D. desulfuricans to grow faster. However, the differential
analysis (nitrate vs sulfate) of the total proteomes in the pI range 4–7,
demonstrates that the bacterium's adaptation to the alternative electron
acceptor (nitrate) implies the increased production of a large number
(ca. 80) of proteins that are exclusively detected in this condition. It is
possible that these proteins are below the detection limits of the ex-
perimental technique for the samples obtained from sulfate medium,
but they are clearly less significant for D. desulfuricans growth with
J.R. Sousa et al.
sulfate as terminal electron acceptor. Overall, these increased proteins
are involved in non-energetic metabolisms, with a special prominence
of those related with ammonium assimilation, such as amino acid
biosynthesis. Besides contributing to the higher bacterial growth rates,
this helps to detoxify the elevated levels of ammonium derived from
nitrate reduction.
Therefore, we propose that in order to adapt to the nitrate medium,
D. desulfuricans activates additional pathways involving amino acid
and/or protein biosynthesis, whereas in the presence of sulfate the
bacterium expresses predominantly the proteins required for its con-
stitutive energy metabolism. Such drastic changes in the cellular com-
position indicate that the metabolism shifting from sulfate to nitrate
respiration has high biosynthetic costs that may well justify the pre-
ference of this SRB for sulfate, when in the presence of both electron
acceptor substrates [11].
Transparency document
The http://dx.doi.org/10.1016/j.bbapap.2017.07.021 associated
with this article can be found, in online version.
Acknowledgements
The authors acknowledge funding from UCIBIO@REQUIMTE Pest-
C/EQB/LA0006/2013. The authors thank Ana Teresa Lopes for assis-
tance with cell cultures. CMS thanks the financial support from
Fundação para a Ciência e Tecnologia (Fellowship SFRH/BPD/79566/
2011).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbapap.2017.07.021.
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