3. Results and discussion
3.1 Characteristics of the SRP core genome and
pan-genome
The pan-genomes of three SRP genera are assembled to highlight the
genetic and metabolic diversity of SRPs. Based on the sequence
similarity analysis, a phylogenetic tree created for all 24 SRPs
together shows a clear separation of Archaeoglobus ,Desulfovibrio , and Desulfotomaculum (Figure S1),
demonstrating that they belong justifiably to three different genera.
Pan-genome of every genus can be subdivided into three subsets: (i) the
core genome (genes shared among all species and mostly play a
housekeeping role), (ii) the accessory genome (genes present in some but
not all species and contribute to species adaptation to different
conditions), and (iii) the unique genome (species-specific genes).
Pan-genome subset distribution and function profile ofArchaeoglobus , Desulfovibrio , and Desulfotomaculumare shown in Figure 2, and pan-genome and core genome development plot
projections are provided in supplementary Figure S2. The core genomes of
all three genera display a similar evolution, which rapidly stagnates at
close to 800 genes. The proportion of core genome in the pan-genome
varies significantly (from 3.7% to 25.8%) due to the differences in
pangenome size, but remains relatively low, revealing a rather high
inter-species diversity of each genus.
In terms of functional annotation
according to COGs, almost half of the genes from the core genomes of the
three genera are devoted to translation, energy metabolism, and the
metabolism of building blocks (carbohydrates, amino acids, nucleotides,
coenzymes, and lipids) (Figure 2), which supports Ouzounis and Kyrpides
(1996)’s hypothesis that metabolism and translation are conserved and
close to the last universal ancestor. Besides, a significant fraction
(more than 20%) of core genes of Archaeoglobus andDesulfotomaculum encodes poorly characterized proteins
(categories R and S) whose function is hypothetical, while that fraction
of Desulfovibrio is only 8.2% because Desulfovibriostrains have been intensively studied (Barton & Fauque, 2009).
Key genes and associated functional proteins involved in central carbon
and energy metabolism are provided in the ortholog table generated from
the pan-genome analysis (Table S1). Overall, all SRP species contain
complete gene sets for glycolysis, while adopting an incomplete pentose
phosphate pathway and an incomplete TCA cycle. A complete set of genes
for the Wood-Ljungdahl pathway (Schauder et al., 1986; Spormann &
Thauer, 1988) was identified in Archaeoglobus . The essential
enzymes for DSR are conserved in all studied SRPs, including sulfate
transporters, ATP sulfurylase (Sat), APS reductase (AprAB), and
dissimilatory sulfite reductase
(DsrAB). Two electron-transporting enzyme complexes, dissimilatory
sulfite reductase (DsrMKJOP) and quinone-interacting membrane-bound
oxidoreductase (QmoABC), are conserved across SRPs, which are assumed to
be involved in electron transfer from electron donors to the terminal
reductases AprAB and DsrAB respectively. In addition,Desulfovibrio contains several other electron transfer complexes
such as Hmc (high-molecular-weight complex), Tmc (transmembrane
complex), Rnf (ferredoxin: NAD
reductase), Nuo (NADH: quinone oxidoreductase complex), and
energy-conserving hydrogenases (Ech or Coo), but few of them are found
in Archaeoglobus or Desulfotomaculum , suggesting that
electron transfer for sulfate reduction is substantially different among
SRPs.
3.2 Model construction and
validation
Based on the orthologous relationships resulted from pan-genome
analysis, core metabolic models of the 24 SRPs are constructed and used
to simulate their metabolic capabilities and growth characteristics
under different growth conditions. The resulting core metabolic models
consist of 91-110 reactions (Figure 3). They share most of the reactions
related to central carbon metabolism but vary significantly in electron
transfer pathways. Clustering of the models shows that the
phylogenetically closely related SRP species have similar metabolic
capabilities. Detailed information of all the reactions and the GPR
associations, as well as the model files of 24 SRPs can be found on
Github (https://github.com/TANG-Wentao/SRP_MetabolicModel).
To demonstrate the predictive accuracy of the core metabolic model,
steady-state growth simulation was performed on the metabolic model
under two different growth conditions: lactate-limited and
hydrogen-limited sulfate respiration (LS and HS, respectively). The
predicted growth yields (YX/S) and specific growth rates
(μ) are compared to the experimentally determined values. Multiple
literature datasets describing growth yields of DvH are available. The
growth yields are dependent on the specific growth rate (μ) and the
maintenance coefficient (m) (Russell & Cook, 1995); thus, growth rates
and growth yields from both experimental results and model predictions
are compiled according to Eq. 1 (Pirt, 1965). As shown in Figure 4,
under LS and HS conditions, the predicted growth yields and specific
growth rates of DvH are in close agreement with the experimental data.
\begin{equation}
\frac{1}{Y}\ =\ \ \frac{m}{\mu}\ +\ \ \frac{1}{Y_{\max}}\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ Eq.1\nonumber \\
\end{equation}
For HS growth, a net synthesis of 1.21 ATP molecule per sulfate reduced
is predicted from metabolic modeling, which is close to the theoretical
value proposed by a previous study (Badziong & Thauer, 1978). By
comparing the growth yield data and the growth energy requirement ofD. vulgaris under HS growth, a net yield of 1 ATP molecule per
sulfate reduced was proposed by Badziong and Thauer (1978). The
stoichiometry of cell synthesis and energy reactions for growth ofDesulfovibrio under LS growth is provided in Table 2. More than
90% of electron donor is used for energy generation, and the rest is
used for cell synthesis, which is in agreement with previous studies
(Noguera et al., 1998; Traore et al., 1981). Our results demonstrate
that the high-quality core metabolic model equipped with improved
annotations in ETC pathways can accurately predict the growth
characteristics and energy yield of SRPs, providing a basis for further
quantitative analysis of energy metabolism.
3.3 Lactate-sulfate (LS)
respiration
Lactate is the most widely used
growth substrate by known SRPs and often applied for cultivation
purposes. The central carbon metabolism and the predicted metabolic flux
of essential reactions associated with energy generation under LS growth
are shown in Figure 5 and Figure 6a. Lactate oxidation to acetate is
mediated by lactate dehydrogenase (Ldh), and the produced pyruvate from
lactate oxidation is then converted to acetyl-CoA via pyruvate: formate
lyase (Pfl) and/or pyruvate: ferredoxin oxidoreductase (Por). The
predicted pathway of lactate oxidation agrees well with previous studies
(Keller & Wall, 2011; P. M. Pereira et al., 2008). Acetyl-CoA is mainly
used to produce ATP through substrate-level phosphorylation by
Acetyl-CoA synthetase (ADP-forming, Acs), or phosphotransacetylase
(Pta)/acetate kinase (Ack) couple. Consistent with previous genome
analysis, this pathway is highly conserved in SRPs (Vita et al., 2015).
For SRPs without Acs or Pta, such as Desulfovibrio magneticus andDesulfovibrio gigas , acetyl-CoA is predicted to be converted to
acetaldehyde by aldehyde:ferredoxin oxidoreductase (Aor) or to citrate
by citrate synthase (CIT), which are in turn converted to ethanol and
succinate respectively.
For Desulfovibrio and Desulfotomaculum species except forD.magneticus and D. gigas , a theoretical ratio of acetate
excretion to lactate utilization of 1:1 is expected in energy metabolism
based on the stoichiometry of this pathway, and an average
acetate-to-lactate ratio of 0.907:1 is predicted (Figure 6b & Table 2).
A difference exists between the theoretical ratio and the predicted
value because a small amount of acetyl-CoA is used for the synthesis of
the building blocks, and this part of acetyl-CoA plays a non-energetic,
assimilatory role in SRPs. For the complete oxidizers, e.g.Archaeoglobus species, acetyl-CoA is predicted to be further
utilized by the Wood–Ljungdahl pathway
(Schauder et al., 1986; Spormann
& Thauer, 1988) for energy generation by coupling the endergonic
oxidation of acetate to CO2, thus no acetate is excreted
and the ratio of CO2 to lactate is triple that of
complete oxidizers (Figure 6b). The ATP yield (ATP/sulfate ratio) varies
significantly across different SRPs, with a predicted average of 2.35.
Electrons generated through lactate oxidation are assumed to be used
indirectly for sulfate reduction by the hydrogen (H2)
cycling pathway (Odom & Jr, 1981). Another study (Lupton et al., 1984)
argued that H2 is not an obligatory intermediate, but is
produced only as a mechanism to control the redox state of internal
electron carriers, and the electrons transport directly through
membrane-bound electron carriers to sulfate. Experimental evidence
partially supports each hypothesis (Keller & Wall, 2011; Noguera et
al., 1998). Based on our modeling work (Figure 6a and Figure 7), these
two electron transport mechanisms can complement—rather than compete
with—each other for Desulfovibrio . As shown in Figure 7, for
the direct pathway, electrons flow from lactate to the membrane-bound
electron carrier
menaquinone
(MQ) and then transfer to sulfate reduction (via Qmo and DsrMK) without
H2 involvement. In general, the consumption and
evolution of H2 are mediated by hydrogenases (Hase),
which are redox metalloenzymes that catalyze the reversible oxidation of
H2 (Vignais & Colbeau, 2004). For the
H2 cycling pathway, electron equivalents
(Fdred and MQH2) and protons generated
from Ldh will react with membrane-bound Hase Ech and/or Coo to form
H2, which will diffuse to the periplasm and be oxidized
back to protons and electrons by periplasmic Hase (Hase_p). The
electrons from H2 oxidation are transported to the
electron equivalent Type I cytochrome c3(TpIc3), and then transferred back to the cytoplasm via
Hmc and Qrc for sulfate reduction, creating a transmembrane proton
gradient for ATP synthesis. This predicted phenomenon is supported by a
previous study of Desulfovibrio gigas , where only one periplasmic
and one cytoplasmic Hase are present. Single deletion of mutants for
each of these proteins showed slightly lower growth rates with
lactate-sulfate or pyruvate-sulfate than the wild-type strain,
suggesting that H2 cycling can be compensated by the
direct MQ mediated electron transport (Morais-Silva et al., 2013). Based
on our simulation, the so-called H2-cycling mechanism is
responsible for around half of the electron transfer inDesulfovibrio under LS growth, except Desulfovibrio
alaskensis G20 who contains no Ech or Coo.
Genera Archaeoglobus and Desulfotomaculum do not use the
H2 cycling pathway for energy conservation under LS
growth due to the lack of a periplasmic space for H2oxidation and the absence of transmembrane electron transfer proteins
such as Qrc and Hmc. For Desulfotomaculum species,
H2 generated from Ldh is predicted to involve in the
flavin-based electron bifurcation mechanism (see section 3.5 for
details) for the generation of reduced ferredoxin
(Fdred) and NADH via cytoplasmic Hase (Hase_c), thus
serving as a redox balancing strategy as suggested by Lupton et al.
(1984). NADH and Fdred further drive sulfite reduction
via heterodisulfide reductase (Hdr) and/or dissimilatory sulfite
reductase (DsrC). For Archaeoglobus species, H2generation is not predicted during LS growth, and a new kind of electron
equivalent, reduced coenzyme F420(F420H2), is introduced.
F420H2 is generated as a result of
complete oxidation of lactate into CO2 via the
Wood-Ljungdahl pathway, and is further converted to MQH2by the membrane-bound electron carriers Fqo
(F420H2: quinone oxidoreductase). NADH
is somewhat a substitute for H2 in Archaeoglobusand Desulfotomaculum . It not only works as the redox currency,
but also directly drives energy conservation by Nuo (NADH: quinone
oxidoreductase).
3.4 Hydrogen-sulfate (HS)
respiration
Molecular hydrogen (H2), an electron sink product of
anaerobic fermentation, can be utilized via DSR, methanogenesis, and
acetogenesis to enable more efficient energy recovery from organic
substrates (Rowland et al., 2018). When H2 is the only
available electron donor, most SRPs will grow heterotrophically on
acetate, and carbon dioxide can serve as a supplementary carbon source.
The average carbon dioxide-to-acetate ratio of SRPs is predicted to be
0.96, which means 67.6% of the carbon is derived from acetate and the
other 32.4% comes from carbon dioxide (Figure 8 &Table 2). This result
agrees well with experimental findings from previous work (Badziong &
Thauer, 1978), which reported that 70% of the carbon is derived from
acetate, and the remaining 30% comes from carbon dioxide.Archaeoglobus sulfaticallidus are capable of coupling
chemolithoautotrophic growth on H2/CO2to sulfate reduction in addition to heterotrophic growth. ForArchaeoglobus other than A. sulfaticallidus , although the
machinery for CO2 fixation via the Wood–Ljungdahl
pathway exists, autotrophic growth with
H2/CO2 is not feasible. This is because
Fdred generated from Hase is completely used up for APS
reduction (Qmo), CO2 fixation through the
Wood–Ljungdahl pathway cannot be promoted without
Fdred.
Metabolic fluxes predicted by FBA for essential reactions related to
central carbon metabolism and energy generation are shown in Figure 8a.
As expected, a strikingly different flux distribution is observed under
HS growth condition as compared to LS growth condition. With
H2 serving as the electron donor for growth,Desulfovibrio is predicted to establish a pmf directly by
periplasmic H2 oxidation (Hase_p). Besides, the
membrane-bound Hase Ech can function in reverse to oxidize
H2 and generate Fdred for carbon
fixation during growth with H2 for severalDesulfovibrio species. Archaeoglobus andDesulfotomaculum species are predicted to use soluble cytoplasmic
Hases (MvH and Hase_c) that are not present in Desulfovibrio ,
and these Hases are involved in electron bifurcation mechanisms to
produce Fdred and balance the redox state of internal
electron carriers (see 3.5 for details). Por (pyruvate: ferredoxin
oxidoreductase) involves in energy metabolism of all examined SRPs under
both LS and HS growth. It can work bidirectionally to oxidize pyruvate
into acetyl-CoA and CO2 accompanied with
Fdred generation under LS growth, or to synthesize
pyruvate via CO2 fixation fueled by
Fdred under HS growth. For several SRPs (Figure 8a),
formate is predicted to be generated from pyruvate via Pfl and is then
oxidized to protons and electrons by periplasmic formate dehydrogenase
(Fdh). This formate cycling pathway performs a similar function to the
H2 cycling pathway, which is supported by the
transcriptome study of P. M. Pereira et al. (2008).
3.5 Energy conservation
mechanisms
In sulfate respiration, energy is mainly generated through the
conversion of the free energy of a redox reaction into a pmf, which
drives ATP production by ATP synthase (ATPase). Despite intensive study,
the mechanism of energy conservation during sulfate respiration is still
incompletely understood. By simulating the steady-state growth of 24
SRPs under different growth conditions, three mechanisms are proposed
for energy conservation via oxidative phosphorylation in this study, as
shown in Figure 9.
The MQ-based redox loop is the universal machine used for energy
conservation in SRPs. In this scenario, proton translocation is achieved
via charge separation performed by coupling MQ reduction with
MQH2 oxidation (Simon et al., 2008). A MQ reductase such
as Qrc, Hmc, Nuo, and Fqo, together with a MQH2 oxidase
such as Qmo, Coo, and DsrMK, constitute the two arms of an
energy-conserving redox loop (Figure 9a). The charge separation is
established by electron extraction from the positive (P) side and proton
uptake on the negative (N) side of the membrane by MQ reductases, paired
with electron transfer to the N side and proton release to the P side by
MQH2 oxidases (Figure 9a). The second energy
conservation mechanism is to build up a pmf via direct proton pumping
(Figure 9b). Several electron transfer complexes capable of proton
pumping are found in SRPs, including Nuo, Fqo, Rnf, Ech, Coo, and PPi
(inorganic pyrophosphate), which provides SRPs with great flexibility in
terms of energy metabolism. The Ech and Coo Hases are involved in both
direct proton pumping and indirect H2 cycling (Figure
9c). Ech and Coo can convert protons and electron equivalents
(Fdred and MQH2) that are generated by
the oxidation of organic compounds (e.g., lactate) to
H2, simultaneously translocate protons. The
H2 is then reoxidized to protons and electrons
(TpIc3 pool) by periplasmic Hase
(Hase_p). The H2 cycling has to be operated at very low
H2partial pressure to allow H2 generation through Ech and
Coo thermodynamically feasible. This is because the
H-/H2 couple has lower redox potential
(E°’= -414 mV) than MQ/MQH2 (E°’= -74 mV) and
Fdred/Fdox (E°’= -398 mV) at standard
conditions (Table 3), and the decrease of H2 partial
pressure to 1 Pa will increase the lower redox potential of
H-/H2 couple to -270 mV. Formate
cycling across the membrane works as an alternative energetic pathway of
H2 cycling, and carbon monoxide (CO) cycling is also
proposed during fermentative growth (Voordouw, 2002).
Flavin-based electron bifurcation (FBEB) is a novel mechanism by which a
hydride electron pair from H2, NAD(P)H,
F420H2, or formate is split by
flavoproteins into one-electron with lower redox potential and one with
a higher redox potential than that of the electron pair (Buckel &
Thauer, 2018). FBEB is not directly involved in energy conservation;
instead, it supports energy conservation through Qmo, Rnf, Coo, and Ech
by generating Fdred from relatively high-potential
electron donors such as NADH (Figure 9d). The discovery of electron
bifurcation also helps to resolve the physiological role of MQ in DSR.
MQ pool is widely accepted as a critical mediator in the electron
transfer and energy generation in SRPs. However, the redox potential of
MQ/MQH2 (E°’= -74 mV) is higher than most of the redox
couples found in SRPs (Table 3) and is very similar to
APS/HSO3- (E°’= -65 mV). Therefore, MQ
can readily receive electrons from other electron donors, whereas
donating electrons from MQH2 to other electron acceptors
seems thermodynamically unfavorable. Via electron bifurcation,
MQH2 oxidation can be achieved by introducing a second
electron donor (e.g., Fdred) with lower redox potential,
so that electron transfer via Qmo and Coo can proceed (Figure 9 a and
c).
Overall, MQ-based redox loop and FBEB are widely used to conserve energy
in SRPs. H2 cycling is restricted toDesulfovibrio , and Nuo induced proton pumping plays a crucial
role in Archaeoglobus and Desulfotomaculum . A wide variety
of proteins are involved in energy conservation of SRPs, and our
simulations show that alternative energy-conserving proteins and
different energy-conserving mechanisms can be used in response to
shifting nutrient availability, which enables SRPs to thrive in a range
of different environmental conditions.