Download From CO2 to cell: energetic expense of creating biomass using the

FEMS Microbiology Letters, 363, 2016, fnw054
doi: 10.1093/femsle/fnw054
Advance Access Publication Date: 2 March 2016
Research Letter
R E S E A R C H L E T T E R – Physiology & Biochemistry
From CO2 to cell: energetic expense of creating
biomass using the Calvin–Benson–Bassham and
reductive citric acid cycles based on genome data
Department of Integrative Biology, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620, USA
∗
Corresponding author: Department of Integrative Biology, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620, USA.
Tel: 813-974-5173; Fax: 813-974-3263; E-mail: [email protected]
One sentence summary: The first genome sequence-based estimate of the cost to build cells from CO2 using two common autotrophic pathways
predicts that the Calvin–Benson–Bassham cycle is 122% as expensive as the reductive citric acid cycle.
Editor: Rich Boden
ABSTRACT
The factors driving the dominance of the Calvin–Benson–Bassham cycle (CBB) or reductive citric acid cycle (rCAC) in
autotrophic microorganisms in different habitats are debated. Based on costs for synthesizing a few metabolic
intermediates, it has been suggested that the CBB poses a disadvantage due to higher metabolic cost. The purpose of this
study was to extend this estimate of cost from metabolite synthesis to biomass synthesis. For 12 gammaproteobacteria
(CBB) and five epsilonproteobacteria (rCAC), the amount of ATP to synthesize a gram of biomass from CO2 was calculated
from genome sequences via metabolic maps. The eleven central carbon metabolites needed to synthesize biomass were all
less expensive to synthesize via the rCAC (66%–89% of the ATP needed to synthesize them via CBB). Differences in cell
compositions did result in differing demands for metabolites among the organisms, but the differences in cost to
synthesize biomass were small among organisms that used a particular pathway (e.g. rCAC), compared to the difference
between pathways (rCAC versus CBB). The rCAC autotrophs averaged 0.195 moles ATP per g biomass, while their CBB
counterparts averaged 0.238. This is the first in silico estimate of the relative expense of both pathways to generate biomass.
Keywords: autotroph; deep-sea; hydrothermal; Calvin cycle; reductive citric acid cycle; carbon fixation
INTRODUCTION
The vast majority of biomass carbon begins its transit through
the biosphere via autotrophic carbon fixation pathways. Given
the habitat, phylogenetic and physiological diversity of autotrophic organisms, the diversity of biochemistries that can
catalyze autotrophic carbon fixation is perhaps unsurprising;
six pathways have been described (reviewed in Berg 2011).
The Calvin–Benson–Bassham cycle (CBB) and reductive citric acid cycle (rCAC) are present in several phyla of Bacteria; the hydroxypropionate bicycle is present in Chloroflexi;
the 3-hydroxypropionate/butyrate and the dicarboxylate/4-
hydroxybutarate cycles operate in Crenarchaea; and the WoodLjungdahl pathway is present in both Archaea and Bacteria (Berg
2011). Each autotrophic pathway dovetails with central carbon
metabolism to synthesize all of the metabolic intermediates
necessary for cellular biosynthesis (e.g. pyruvate, oxaloacetate,
et cetera; Fig. 1).
In autotrophs, all of the carbon atoms in these metabolic intermediates originated from CO2 and/or HCO3 − , with electrons
provided from cellular reductant pools (e.g. NADH, NADPH, reduced ferredoxin). Regardless of pathway, each metabolic intermediate carries the same number of carbon atoms and acquires the same number of electrons from cellular reductant
Received: 22 January 2016; Accepted: 29 February 2016
C FEMS 2016. All rights reserved. For permissions, please e-mail: [email protected]
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Mary Mangiapia and Kathleen Scott∗
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FEMS Microbiology Letters, 2016, Vol. 363, No. 7
pools (e.g. three carbons and 10 electrons for pyruvate). However, the enzymes responsible for the synthesis of each compound from CO2 /HCO3 − vary among the different autotrophic
pathways, and introduce differences in the energetic requirements for the synthesis of these molecules. For example,
phosphoenolpyruvate synthesis from CO2 via the CBB consumes ATP due to the activities of two kinases unique to
the CBB (phosphofructokinase and sedoheptulose-7-phosphate
kinase), while its synthesis via the rCAC requires ATP consumption via pyruvate:water dikinase or phosphoenolpyruvate
carboxykinase (Fig. 1; Tables S1 and 2, Supporting Information). Additionally, the energetic requirements for the synthesis of each compound vary based on the identity of the
cellular electron donor used by the enzymes catalyzing its synthesis; electron donors with more negative redox potentials
(e.g. ferredoxin) are more energetically expensive to reduce
(Fuchs 2011). Given these differences in energetic expense, it has
been suggested that some pathways might be more favorable
in energy-limited or energy-replete habitats (Berg 2011; Boyle
2011; Fuchs 2011; Bar-Even, Noor and Milo 2012; Konneke et al.
2014).
Deep-sea hydrothermal vents are inhabited by autotrophic
microorganisms that use a diversity of biochemistries for carbon fixation, and therefore provide a context for comparing the
potential advantages and disadvantages of these biochemistries
for their host organisms. The microbes responsible for primary
productivity at these sites rely on geothermally-generated electron donors (e.g. H2 S, H2 ) supplied by hydrothermal fluid emitted
from the crust (Sievert and Vetriani 2012). Sulfur-oxidizing autotrophic gamma- and epsilonproteobacteria are some of the
dominant microorganisms at these sites. The gammaproteobacterial sulfur oxidizers include Thiomicrospiras, which
are common isolates from many sulfide-rich habitats, including
deep-sea hydrothermal vents in the Atlantic and Pacific Oceans
(Ruby, Wirsen and Jannasch 1981; Muyzer et al. 1995; Takai et al.
2004), shallower marine sediments from temperate (Kuenen
and Veldkamp 1972; Brinkhoff et al. 1998; Brinkhoff et al. 1999c),
Arctic (Knittel et al. 2005) and Antarctic sites (Mikucki and
Priscu 2007) and hypersaline lakes and springs (Brinkhoff and
Muyzer 1997; Sorokin et al. 2006). The epsilonproteobacterial
sulfur oxidizers inhabit many of these same habitats (Campbell
et al. 2006). At hydrothermal vents, the epsilonproteobacterial
sulfur-oxidizers typically are numerically dominant (LopezGarcia et al. 2003; Campbell et al. 2013); however, sometimes
the gammaproteobacterial sulfur-oxidizers prevail (Brazelton
and Baross 2010). The factors proposed to drive the relative
dominance of these two groups of bacteria include epsilonproteobacterial sensitivity to oxygen and versatility with respect to
electron donors and acceptors (Campbell et al. 2006).
Another trait that distinguishes these two groups of bacteria
are the pathways they use for autotrophic carbon fixation. Most
of the sulfur-oxidizing vent gammaproteobacteria use the CBB,
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Figure 1. Central carbon metabolism for autotrophic microorganisms using the rCAC (left) or CBB cycle (right) for carbon fixation. Key central carbon metabolites are
in red, and for CBB autotrophs, the citric acid cycle is designated as a ‘cycle’ since many autotrophs do not operate a complete oxidative citric acid cycle.
Mangiapia and Scott
MATERIALS AND METHODS
Sulfur-oxidizing autotrophic gamma- and epsilonproteobacterial organisms were selected based on the quality of their sequence data (complete genome or high-quality draft). Repre-
senting gammaproteobacterial CBB autotrophs, Thiomicrospira
crunogena, KP2, JR2, Milos T1 and T2 and MA2-6 were isolated
from hydrothermal vents (Jannasch et al. 1985; Muyzer et al.
1995; Brinkhoff et al. 1999c), while T. pelophila, arctica, chilensis
and kuenenii originated from coastal sediments (Kuenen and
Veldkamp 1972; Brinkhoff et al. 1999a,b; Knittel et al. 2005),
T. halophila was isolated from a hypersaline lake (Sorokin et al.
2006), and Hydrogenovibrio marinus was isolated from surface
ocean water (Nishihara, Igarashi and Kodama 1991). The epsilonproteobacterial rCAC autotrophs included in this study are
hydrothermal vent isolates Sulfurimonas autotrophica, Nitratiruptor Sb155-2 and Sulfurovum NBC37-1 (Inagaki et al. 2003; Nakagawa et al. 2005), pelagic isolate S. gotlandica GD1 (Labrenz et al.
2013) and sediment isolate S. denitrificans (Timmer-Ten Hoor
1975).
Overview of calculations
Estimating the cost of synthesizing a cell from CO2, using the
CBB or rCAC, was informed by genome data via KEGG maps
(http://www.genome.jp/kegg/ and http://img.jgi.doe.gov/; Kanehisa and Goto 2000; Markowitz et al. 2010; Table S3, Supporting
Information), and supplemented with relevant literature. This
estimate began by calculating the cost (ATP, reducing equivalents, CO2 ) for synthesizing a mole of each of the key central
carbon metabolites from CO2 (Fig. 1; Tables S1–3, Supporting Information). This was followed by calculating the cost of synthesizing building blocks (e.g. amino acids, nucleotides, fatty
acids) from these metabolites, and then the cost of polymerizing
these building blocks into macromolecules (Fig. 2). To calculate
the final cost of synthesizing the cells, the costs of the macromolecules were normalized to their relative amounts in a cell.
The macromolecular composition of the cells was based on data
from E. coli (Neidhardt, Ingraham and Schaechter 1990), since
these analyses have not been conducted on the taxa included
in this study. Further, details of the calculations are provided in
Supplementary Methods.
RESULTS AND DISCUSSION
Central carbon metabolites from CO2
The amount of ATP and reducing equivalents needed to synthesize central carbon metabolites from CO2 is consistent with
those calculated previously (Tables S1 and 2, Supporting Information; (Berg et al. 2010; Boyle 2011; Bar-Even, Noor and Milo
2012; Konneke et al. 2014). For organisms using either carbon
fixation pathway, the number of electrons per mol of metabolite produced is the same, but the cellular electron donors differ
(Table 1). For example, all the electrons for pyruvate synthesis
are provided by NADH when the CBB cycle is used, while use of
the rCAC requires other electron donors (e.g. PFOR utilizes reduced ferredoxin).
Once electron donors are converted to ‘ATP equivalents’
to account for the energetic expense of reducing them (see
Supplementary Methods), it is apparent that the central carbon metabolites are more expensive to synthesize for the
gammaproteobacteria (Thiomicrospiras and H. marinus), which
use the CBB, compared to the epsilonproteobacteria, which
use the rCAC (Fig. 3). Energetic expense for central carbon
metabolites averaged ∼80% (rCAC/CBB), with largest differences (rCAC/CBB) for 2-oxoglutarate (66%) and acetyl-CoA (68%),
and smallest for fumarate (88%; Fig. 4). Driving this increased
expense are the dephosphorylations of bisphosphorylated
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while the vent epsilonproteobacteria use the rCAC (Fig. 1; Felbeck 1981; Hügler et al. 2005; Takai et al. 2005; Scott et al. 2006).
The enzymes of the CBB cycle are oxygen-tolerant, but oxygen
is a competitive substrate with CO2 for RubisCO and results in
a wasteful oxygenase reaction. Aerobic cells must minimize the
chances of the oxygenase reaction, which results in the loss of
a carbon dioxide molecule as well as the consumption of ATP
to regenerate RuBP (Tabita et al. 2008). The rCAC is even more
sensitive to oxygen than the CBB. The two oxidoreductases necessary for this pathway (pyruvate: ferredoxin oxidoreductase,
PFOR and 2-oxoglutarate: ferredoxin oxidoreductase) are typically inactivated by oxygen, which generally restricts this cycle
to anaerobes and microaerobes (Berg 2011). However, some rCAC
autotrophs (Hydrogenbacter thermophilus and Aquifex pyrophilus)
live in microaerobic environments and may have oxygen tolerant versions of these enzymes (Shiba et al. 1985; Beh et al. 1993).
Accordingly, sensitivity to oxygen has also been suggested to be
a major driver in the distribution of these two autotrophic pathways (Berg 2011).
At hydrothermal vents, multiple factors are likely to dictate the distribution of the autotrophs and their biochemistries.
Given that dissolved oxygen concentrations can vary from 0
to ∼100 μM over spatial scales of less than 1 cm and temporal scales ranging from seconds to days, oxygen sensitivity is
likely to play a role. Additionally, electron donor availabilities
are also quite variable; for example, sulfide concentrations also
range from 0 to ∼100 μM and vary over the same distances and
timescales as oxygen (Fisher et al. 1988; Johnson, Childress and
Beehler 1988; Goffredi et al. 1997). Given the variability in redox
substrate availability, energetic expense of autotrophic pathways may also factor into the success of different organisms at
the vents.
The degree to which carbon fixation pathways are more or
less expensive (in terms of ATP and cellular reductant use) has
been addressed previously, to a limited extent. Previous efforts
limited their calculations to the biosynthesis of pyruvate (Berg
et al. 2010; Fuchs 2011; Bar-Even, Noor and Milo 2012), calculated the costs of other metabolites in terms of ATP consumption without adding in the cost of the cellular electron donors
(Konneke et al. 2014), or kept the tallies of ATP and cellular electron donors separate (Boyle 2011), which complicates comparisons between different autotrophic pathways, since the different pathways utilize different cellular reductants in differing amounts, and these cellular reductants differ in the energy
needed to regenerate them (Fuchs 2011). These approaches have
been illuminating; however, the objective of autotrophy is the
synthesis of cells, not metabolic intermediates, so the application of these prior estimates to understanding physiology or
ecology is very problematic.
Happily, the recent sequencing of many autotrophic bacterial genomes makes it possible to expand this initial calculation
to include all of the metabolic intermediates, building blocks
(e.g. amino acids) and macromolecules, while accounting for
variations in pathways among organisms. The objective of this
work was to use genome data from 17 species of sulfur-oxidizing
gamma- and epsilonproteobacteria to compare the expense of
synthesizing autotrophic cells from CO2 using either the CBB or
rCAC.
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FEMS Microbiology Letters, 2016, Vol. 363, No. 7
Table 1. ATP and cellular electron donors needed to synthesize central carbon metabolites from CO2 using the CBB and rCAC cyclesa .
Metabolite
Acetyl-CoA
Pyruvate
Oxaloacetate
Fumarate
2-oxoglutarate
Phosphoenolpyruvated
3-phosphoglycerate
Erythrose-4-P
Ribose-5-P
Fructose 6-P
Sedoheptulose 7-P
ATP, CBB
ATP, rCAC
Electrons, CBB
Electrons, rCAC
7
7
8
8
15
8
8
12.33
15
18
21
2
2
3.33
3.33
4.33
4
4
6.67
8.33
10
11.67
4 NADH
5 NADH
5 NADH
5 NADH, 1 Qred b
–1 NADPH, 9 NADH
5 NADH
5 NADH
8 NADH
10 NADH
12 NADH
14 NADH
2 Fdred , 1 NADPH, 2 NADH
4 Fdred , 1 NADPH, 2 NADH
4 Fdred , 1 NADPH, 2 NADH
4 Fdred , 1 NADPH, 3 NADH
6 Fdred , 1 NADPH, 4 NADHc
4 Fdred , 1 NADPH, 2 NADH
4 Fdred , 1 NADPH, 2 NADH
5.33 Fdred , 1.33 NADPH, 4 NADH
6.67 Fdred , 1.67 NADPH, 5 NADH
8 Fdred , 2 NADPH, 6 NADH
9.33 Fdred , 2.33 NADPH, 7 NADH
a
Steps to synthesize these compounds are detailed in Tables S1 and 2 (Supporting Information). Amounts in table are the moles necessary to synthesize 1 mol of each
central carbon metabolite.
b
Qred = quinol (ubiquinol for Thiomicrospiras and H. marinus).
c
For Sulfurovum sp. NBC37-1 and S. gotlandica the electrons needed for 2-oxoglutarate synthesis are 6 Fdred , 1 NADPH, 3 NADH and 1 Qred (Table S2, Supporting Information).
d
For S. denitrificans, phosphoenolpyruvate synthesis requires 4.33 ATP, 4 Fdred , 1 NADPH and 2 NADH (Table S2, Supporting Information).
compounds necessary to drive the CBB forward (fructose
1,6-bisphosphate, 1,3-bisphosphoglycerate, sedoheptulose 1,7phosphate; File 1, Supporting Information). While dephosphorylation of two of these compounds (fructose 1,6-bisphosphate,
1,3-bisphosphoglycerate; Table S1, Supporting Information) is
also necessary for rCAC autotrophs to synthesize the six
metabolites produced via gluconeogenesis and the pentose
phosphate pathway (Fig. 1), CBB autotrophs have the added bur-
den of having to send all of the carbon they fix through multiple
dephosphorylation steps in the CBB cycle before feeding them
into central carbon metabolism to synthesize all of the 11 central carbon metabolites needed for biosynthesis.
Another added expense for CBB autotrophs arises from the
syntheses of acetyl-CoA and 2-oxoglutarate, which require oxidative decarboxylations to synthesize them from pyruvate and
isocitrate, respectively (Fig. 1). Some of the losses are recouped
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Figure 2. Methods overview. Steps used to determine the amount of ATP required to build a cell from CO2 via the rCAC or CBB from CO2 .
Mangiapia and Scott
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as NADH and NADPH via pyruvate dehydrogenase and isocitrate dehydrogenase, but these central carbon metabolites remain more expensive for CBB autotrophs. Interestingly, some of
the Thiomicrospiras have genes encoding an oxoacid oxidoreductase (T. crunogena, halophile, JR2 and MA2-6; Tcr 1709 and 1710
in T. crunogena). Cell extracts of T. crunogena reduce benzyl viologen when pyruvate is added, consistent with the presence of
PFOR (Thomas Hanson, unpublished data). Use of PFOR could
make acetyl-coA and 2-oxoglutarate less expensive, as the reduced ferredoxin is worth more ATP-equivalents (see above).
Bucking this trend, a few steps for rCAC-mediated central carbon metabolite synthesis are a bit profligate relative
to the CBB. Use of ferredoxin electrons to synthesize pyruvate and 2-oxoglutarate is expensive as described above. The
synthesis of phosphoenolpyruvate has the added expense of
using phosphoenolpyruvate carboxykinase (S. denitrificans) or
pyruvate, water dikinase (Table S2, Supporting Information),
which hydrolyzes ATP to AMP and results in a two ATP difference in expense between phosphoenolpyruvate and pyru-
vate in rCAC autotrophs. For the CBB autotrophs, the reverse
reaction is catalyzed by pyruvate kinase, with a 1 ATP difference in expense between these two compounds (Table S1, Supporting Information). Lastly, some of the rCAC autotrophs (S.
autotrophica, S. denitrificans, Nitratiruptor sp. SB155-2) have a fumarate reductase whose predicted subunit composition is consistent with using NADH instead of quinol as the reductant,
adding to the expense for synthesis of 2-oxoglutarate and other
compounds downstream of this reaction (Table S2, Supporting
Information).
Macromolecules from central carbon metabolites
As expected, protein synthesis has a high demand for pyruvate, oxaloacetate, 2-oxoglutarate, phosphoenolpyruvate and
3-phosphoglycerate for carbon skeletons for amino acids
(Table 2; Fig. S1, Supporting Information). Metabolite requirements are high relative to the other macromolecules due in part
to the relative abundance of proteins compared to other macro-
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Figure 3. Moles of ATP to synthesize each central carbon metabolite. The ATP and cellular reductant necessary for metabolite synthesis from CO2 were tallied (Table 1).
As described in Supplementary Methods, to facilitate comparison, cellular reductant (Fdred , NADPH, NADH, Qred ) was converted to equivalents of ATP by estimating
their cost to proton potential (3H+ = 1 ATP) if reduced via reverse electron transport (NADH), membrane-bound transhydrogenase (NADPH) and Rnf or NfnAB complex
(Fdred ). Values for gammaproteobacteria are depicted as blue-hued bars (Thiomicrospira spp., H. marinus), while those for epsilonproteobacteria are indicated with
red-hued bars (S. autotrophica, gotlandica and denitrificans; Nitratiruptor Sb155-2; Sulfurovum NBC37-1).
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FEMS Microbiology Letters, 2016, Vol. 363, No. 7
molecules in cells. NADPH is required for reductive amination of
alphaketoacids, while NADH is produced as a byproduct of some
amino acid synthesis pathways (e.g. those synthesized from 3phosphoglycerate: glycine, serine, cysteine).
Nucleic acid synthesis draws heavily on oxaloacetate, 3phosphogycerate and ribose 5-phosphate pools, with NADH produced from GMP and pyrimidine synthesis (Table 2; Fig. S1, Supporting Information). With respect to the thioredoxin required
for deoxynucleotide synthesis, since the electrons carried by
thioredoxin for deoxyribonucleotide biosynthesis originate from
NADPH via thioredoxin reductase (Arner and Holmgren 2000),
their cost in ATP equivalents is equivalent to that for NADPH
electrons.
The other macromolecules require heavy input of metabolites needed for their synthesis [polar lipids: acetyl-CoA, 3phosphoglycerate and cellular reductant (NADH and NADPH);
peptidoglycan: acetyl-coA, phosphoenolpyruvate and fructose
6-phosphate for the glycans, with pyruvate, oxaloacetate, 2ketoglytarate for the tetrapeptide linkers; lipooligosaccharide:
acetyl-CoA and reductant for fatty acids, phosphoenolpyruvate,
sugar phosphates for KDO2 and core oligosaccharide; Table 2;
Fig. S1, Supporting Information].
Differences among species in the moles of metabolites
needed to synthesize proteins, nucleic acids and polar lipids sufficient for 1 g of cells are most apparent for polar lipids (Table 2;
Fig. S1, Supporting Information). These differences between taxa
are, however, quite small compared to the differences apparent
for central carbon metabolite synthesis (Table 1). No difference
between species is shown for peptidoglycan or lipooligosaccharide synthesis (Table 2; Fig. S1, Supporting Information), as it
is not known how these macromolecules vary among the taxa
studied here.
Biomass from macromolecules
When the metabolites necessary for synthesizing macromolecules were tallied to estimate the amounts necessary for
synthesizing a gram of biomass, differences between taxa were
small, despite accommodating species-specific differences in
macromolecule composition whenever these data were available (Table 2; Fig. S1, Supporting Information). However, when
the moles of metabolite needed to synthesize 1 g biomass are
multiplied by the moles of ATP needed to synthesize each (Table 1; Fig. 3), differences become apparent (Fig. 4). The added
expense of central carbon metabolite synthesis from the CBB
(Table 1) is substantial enough to propagate through the differences in cell composition, resulting in calculated expense for
1 g biomass production by rCAC autotrophs that is 82% of the average calculated for CBB autotrophs (Table S4, Supporting Information). Differences among rCAC and CBB autotrophs are quite
small (0.194–0.196 moles ATP per g biomass for rCAC; 0.237–0.239
for CBB; Table S4, Supporting Information), as within these two
subclusters the differences in macromolecule composition did
not have a substantial impact on the relative amounts of central
carbon metabolites necessary to synthesize a cell (Table 2).
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Figure 4. Moles of ATP needed to synthesize sufficient metabolites for 1 g biomass (dry weight). The moles of metabolites needed to synthesize 1 g biomass (Fig.
S1, Supporting Information) were multiplied by the moles of ATP necessary to synthesize them in each organism based on carbon fixation pathway and central
carbon metabolism (Table 1). Values for gammaproteobacteria are depicted as blue-hued bars (Thiomicrospira spp., H. marinus), while those for epsilonproteobacteria
are indicated with red-hued bars (S. autotrophica, gotlandica and denitrificans; Nitratiruptor Sb155-2; Sulfurovum NBC37-1).
Mangiapia and Scott
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Table 2. Millimoles of metabolites needed to synthesize 1g biomass from CO2 using the CBB and rCAC cyclesa .
Proteins
Metabolite
Acetyl-CoA
Pyruvate
Oxaloacetate
Fumarate
2-oxoglutarate
Phosphoenolpyruvate
3-phosphoglycerate
Erythrose-4-P
Ribose-5-P
Fructose 6-P
Sedoheptulose 7-P
ATP
NADPH
NADH
Nucleic acids
Polar lipids
Peptidoglycan
LOSb
Biomass
CBB
rCAC
CBB
rCAC
CBB
rCAC
CBB and rCAC
CBB and rCAC
CBB
rCAC
0.44
2.34
1.55
− 0.29
0.85
0.71
0.58
0.36
0.10
0
0
23.40
8.60
− 1.46
0.41
2.34
1.71
− 0.24
0.71
0.81
0.55
0.41
0.09
0
0
23.25
9.01
− 1.36
0
0
0.87
− 0.56
0
0
0.38
0
0.69
0
0
8.75
1.42
7.36
0
0
0.88
− 0.57
0
0
0.38
0
0.69
0
0
8.74
1.44
7.36
1.97
0.01
0
0
0
0
0.23
0
0.03
0
0
2.34
2.03
1.63
1.91
0
0
0
0
0
0.23
0
0
0
0
2.16
1.91
1.58
0.05
0.07
0.02
0
0.02
0.02
0
0
0
0.05
0
0.44
0.19
0
0.54
0
0
0
0
0.03
0
0
0.03
0.03
0.05
0.80
0.47
0.47
3.00
2.42
2.45
−0.85
0.87
0.76
1.20
0.36
0.85
0.08
0.05
35.72
12.71
−0.25
2.91
2.42
2.62
−0.80
0.74
0.86
1.16
0.41
0.80
0.08
0.05
35.38
13.03
−0.20
a
CONCLUSIONS
This work substantially strengthens the argument for a key advantage of the rCAC, given that it is based not on a few intermediates, but the whole process of cellular biosynthesis, tailored to
each organism using genome sequence-informed predictions of
all pathways from CO2 to biomass, supplemented with all available information from the literature. Based on this in silico study,
biomass production from CO2 is less energetically expensive using the rCAC than the CBB. This difference in expense reflects
biosynthetic expense of the central carbon metabolites; surprisingly, differences in biomass composition exerted a relatively
weak effect on the final tally of ATP equivalents per g biomass.
Given a low-oxygen habitat where it can operate unhindered,
the rCAC is predicted to provide a substantial advantage over
any CBB competitors, which begs empirical verification: Given
identical growth conditions, do rCAC autotrophs have greater
growth yields than CBB autotrophs? In order to make such a
comparison, organisms would need to be selected based on similarities in their electron transport chains to keep the potential
yield per reductant constant. Further, growth conditions would
need to be tailored to avoid activating energetically expensive
auxiliary capabilities that cells would need to induce to maintain either the rCAC or CBB (e.g. oxidative damage response for
rCAC autotrophs; carbon concentrating mechanisms for CBB autotrophs). One possible comparison would be T. crunogena (CBB)
and S. autotrophica (rCAC), given the similarities in their electron transport chains: both genomes encode the Sox complex
for thiosulfate oxidation (soxABCXYZ genes: Tcr 0601, 1549, 0156,
0604, 0603, 0602; Saut 0994, 0995, 2096, 0991–3; Scott et al. 2006;
Sikorski et al. 2010), and both are capable of robust growth using oxygen as a sole terminal electron acceptor (Jannasch et
al. 1985; Inagaki et al. 2003), presumably by using cbb3 -type cytochrome c oxidase complexes (Pitcher and Watmough 2004, cbb
I – III:Tcr 1965–1963; Saut 1965, 1964, 1962). If both species were
cultivated in the presence of elevated concentrations of CO2 and
low-oxygen tensions, their growth yields could be compared to
the results from this calculation. If indeed the measured growth
yields match those predicted from these calculations, they could
explain why epsilonproteobacterial autotrophs dominate under
anoxic coditions (Campbell et al. 2006; Han and Perner 2015),
while Thiomicrospira can dominate in habitats perfused with oxygenated water, e.g. the Lost City hydrothermal field (Brazelton
and Baross 2010), at which any advantage in rCAC-mediated
growth yield is presumably negated by inactivation by oxygen.
An explanation for the dominance of CBB or rCAC in different
habitats is key to understanding primary productivity in these
habitats, and could be helpful in synthetic biology-based efforts
to engineer organisms to generate compounds of industrial interest from CO2 .
SUPPLEMENTARY DATA
Supplementary data are available at FEMSLE online.
ACKNOWLEDGEMENTS
The authors are grateful to Sydney Russell for her assistance in
assessing metabolic maps in IMG, to Thomas Hanson for use of
unpublished data and to anonymous reviewers for their helpful
suggestions.
FUNDING
Sequencing and annotating the Thiomicrospiras genomes was
performed by the United States Department of Energy Joint
Genome Institute, under contract no. DE-AC02-05CH11231. This
work was supported by the National Science Foundation [NSFMCB-0643713 and NSF-IOS-1257532 to KMS].
Conflict of interest. None declared.
REFERENCES
Arner ESJ, Holmgren A. Physiological functions of thioredoxin
and thioredoxin reductase. Eur J Biochem 2000;267:6102–9.
Bar-Even A, Noor E, Milo R. A survey of carbon fixation pathways
through a quantitative lens. J Exp Bot 2012;63:2325–42.
Beh M, Strauss G, Huber R et al. Enzymes of the reductive citricacid cycle in the autotrophic eubacterium Aquifex pyrophilus
and in the Archaebacterium Thermoproteus neutrophilus. Arch
Microbiol 1993;160:306–11.
Downloaded from http://femsle.oxfordjournals.org/ by guest on April 5, 2016
Averages from 11 species of gammaproteobacterial CBB autotrophs and five epsilonproteobacterial rCAC autotrophs are presented here. Data from individual species
are depicted in (Figs 1 and 2, Supporting Information).
b
LOS = lipooligosaccharides.
8
FEMS Microbiology Letters, 2016, Vol. 363, No. 7
Jannasch H, Wirsen C, Nelson D et al. Thiomicrospira crunogena sp.
nov., a colorless, sulfur-oxidizing bacterium from a deep-sea
hydrothermal vent. Int J Syst Bacteriol 1985;35:422–4.
Johnson KS, Childress JJ, Beehler CL. Short term temperature
variability in the Rose Garden hydrothermal vent field. DeepSea Res 1988;35:1711–22.
Kanehisa M, Goto S. KEGG: Kyoto encyclopedia of genes and
genomes. Nucleic Acids Res 2000;28:27–30.
Knittel K, Kuever J, Meyerdierks A et al. Thiomicrospira arctica sp.
nov. and Thiomicrospira psychrophila sp. nov., psychrophilic,
obligately chemolithoautotrophic, sulfur-oxidizing bacteria
isolated from marine Arctic sediments. Int J Syst Evol Micr
2005;55:781–6.
Konneke M, Schubert DM, Brown PC et al. Ammonia-oxidizing
archaea use the most energy-efficient aerobic pathway for
CO2 fixation. P Natl Acad Sci USA 2014;111:8239–44.
Kuenen JG, Veldkamp H. Thiomicrospira pelophila, gen. n., sp. n.,
a new obligately chemolithoautotrohic colorless sulfur bacterium. Anton Van Lee 1972;38:241–56.
Labrenz M, Grote J, Mammitzsch K et al. Sulfurimonas gotlandica
sp. nov., a chemoautotrophic and psychrotolerant epsilonproteobacterium isolated from a pelagic redoxcline, and an
emended description of the genus Sulfurimonas. Int J Syst Evol
Micr 2013;63:4141–8.
Lopez-Garcia P, Duperron S, Philippot P et al. Bacterial diversity
in hydrothermal sediment and epsilonproteobacterial dominance in experimental microcolonizers at the Mid-Atlantic
Ridge. Environ Microbiol 2003;5:961–76.
Markowitz VM, Chen I-MA, Palaniappan K et al. The integrated
microbial genomes system: an expanding comparative analysis resource. Nucleic Acids Res 2010;38(Database issue):D382–
90.
Mikucki JA, Priscu JC. Bacterial diversity associated with Blood
Falls, a subglacial outflow from the Taylor Glacier, Antarctica.
Appl Environ Microb 2007;73:4029–39.
Muyzer G, Teske A, Wirsen CO et al. Phylogenetic relationships of Thiomicrospira species and their identification in
deeop-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol
1995;164:165–72.
Nakagawa S, Takai K, Inagaki F et al. Distribution, phylogenetic diversity and physiological characteristics of epsilonProteobacteria in a deep-sea hydrothermal field. Environ Microbiol 2005;7:1619–32.
Neidhardt FC, Ingraham JL, Schaechter M. Composition and organization of the bacterial cell. In: Physiology of the Bacterial Cell: A Molecular Approach. Neidhardt FC, Ingraham JL,
Schaechter M (eds). Sunderland: Sinauer Associates, Inc.,
1990, 1–29.
Nishihara H, Igarashi Y, Kodama T. Hydrogenovibrio marinus
gen. nov., sp. nov., a marine obligately chemolithoautotrophic hydrogen-oxidizing bacterium. Int J Syst Bacteriol
1991;41:130–3.
Pitcher RS, Watmough NJ. The bacterial cytochrome cbb3 oxidases. Biochim Biophys Acta 2004;1655:388–99.
Ruby EG, Wirsen CO, Jannasch HW. Chemolithotrophic SulfurOxidizing Bacteria from the Galapagos Rift Hydrothermal
Vents. Appl Environ Microb 1981;42:317–24.
Scott KM, Sievert SM, Abril FN et al. The genome of deep-sea
vent chemolithoautotroph Thiomicrospira crunogena. PLoS Biol
2006;4:1–17.
Shiba H, Kawasumi T, Igarashi Y et al. CO2 assimilation
via the reductive tricarboxylic-acid cycle in an obligatley autotrophic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus. Arch Microbiol 1985;141:198–203.
Downloaded from http://femsle.oxfordjournals.org/ by guest on April 5, 2016
Berg I. Ecological aspects of the distribution of different
autotrophic CO2 fixation pathways. Appl Environ Microb
2011;77:1925–36.
Berg I, Kockelkorn D, Ramos-Vera W et al. Autotrophic carbon
fixation in archaea. Nat Rev Microbiol 2010;8:447–59.
Boyle NR. Computation of metabolic fluxes and efficiencies for
biological carbon dioxide fixation. Metab Eng 2011;11:150–8.
Brazelton WJ, Baross JA. Metagenomic comparison of two Thiomicrospira lineages inhabiting contrasting deep-sea hydrothermal environments. PLoS One 2010;5:e13530.
Brinkhoff T, Muyzer G. Increased species diversity and extended
habitat range of sulfur-oxidizing Thiomicrospira spp. Appl Environ Microb 1997;63:3789–96.
Brinkhoff T, Muyzer G, Wirsen CO et al. Thiomicrospira chilensis sp.
nov., a mesophilic obligately chemolithoautotrophic sulfuroxidizing bacterium isolated from a Thioploca mat. Int J Syst
Evol Micr 1999a;49:875–9.
Brinkhoff T, Muyzer G, Wirsesn CO et al. Thiomicrospira kuenenii
sp. nov. and Thiomicrospira frisia sp. nov., two mesophilic
obligately chemolithoautotrophic sulfur-oxidizing bacteria
isolated from an intertidal mud flat. Int J Syst Evol Micr
1999b;49:385–92.
Brinkhoff T, Santegoeds CM, Sahm K et al. A ployphasic approach to study the diversity and vertical distribution of
sulfur-oxidizing Thiomicrospira species in coastal sediments
of the german wadden sea. Appl Environ Microb 1998;64:
4650–7.
Brinkhoff T, Sievert SM, Kuever J et al. Distribution and diversity of sulfur-oxidizing Thiomicrospira spp. at a shallow-water
hydrothermal vent in the Aegean Sea (Milos, Greece). Appl
Environ Microb 1999c;65:3843–9.
Campbell BJ, Engel AS, Porter ML et al. The versatile epsilonproteobacteria: key players in sulphidic habitats. Nat Rev Microbiol 2006;4:458–68.
Campbell BJ, Polson SW, Allen LZ et al. Diffuse flow environments within basalt- and sediment-based hydrothermal
vent ecosystems harbor specialized microbial communities.
Front Microbiol 2013;4:15.
Felbeck H. Chemoautotrophic potential of the hydrothermal
vent tube worm, Riftia pachyptila Jones (Vestimentifera). Science 1981;213:336–8.
Fisher C, Childress J, Arp A et al. Microhabitat variation in the
hydrothermal vent mussel, Bathymodiolus thermophilus, at
the Rose Garden vent on the Galapagos Rift. Deep-Sea Res
1988;35:1769–91.
Fuchs G. Alternative pathways of carbon dioxide fixation: insights into the early evolution of life? Annu Rev Microbiol
2011;65:631–58.
Goffredi SK, Childress JJ, Desaulniers NT et al. Inorganic carbon acquisition by the hydrothermal vent tubeworm Riftia pachyptila depends upon high external P-CO2 and upon
proton-equivalent ion transport by the worm. J Exp Biol
1997;200:883–96.
Han Y, Perner M. The globally widespread genus Sulfurimonas:
versatile energy metabolisms and adaptations to redox
clines. Front Microbiol 2015;6:989.
Hügler M, Wirsen CO, Fuchs G et al. Evidence for autotrophic CO2
fixation via the reductive tricarboxylic acid cycle by members of the epsilon subdivision of proteobacteria. J Bacteriol
2005;187:3020–7.
Inagaki F, Takai K, Kobayashi H et al. Sulfurimonas autotrophica gen. nov., sp nov., a novel sulfur-oxidizing epsilonproteobacterium isolated from hydrothermal sediments in
the Mid-Okinawa Trough. Int J Syst Evol Micr 2003;53:
1801–5.
Mangiapia and Scott
Sievert SM, Vetriani C. Chemoautotrophy at deep-sea vents.
Oceanography 2012;25:219–33.
Sikorski J, Munk C, Lapidus A et al. Complete genome sequence
of Sulfurimonas autotrophica type strain (OK10). Stand Genomic
Sci 2010;3:194–202.
Sorokin DY, Tourova TP, Kolganova TV et al. Thiomicrospira
halophila sp. nov., a moderately halophilic, obligately
chemolithoautotrophic, sulfur-oxidizing bacterium from
hypersaline lakes. Int J Syst Evol Micr 2006;56:2375–80.
Tabita FR, Hanson TE, Satagopan S et al. Phylogenetic and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms. Philos T Roy Soc B 2008;363:2629–40.
9
Takai K, Campbell BJ, Cary SC et al. Enzymatic and genetic characterization of carbon and energy metabolisms by deep-sea
hydrothermal chemolithoautotrophic isolated of Epsilonproteobacteria. Appl Environ Microb 2005;71:7310–20.
Takai K, Hirayama H, Nakagawa T et al. Thiomicrospira thermophila sp nov., a novel microaerobic, thermotolerant,
sulfur-oxidizing chemolithomixotroph isolated from a deepsea hydrothermal fumarole in the TOTO caldera, Mariana Arc, Western Pacific. Int J Syst Evol Micr 2004;54:
2325–33.
Timmer-Ten Hoor A. A new type of thiosulphate oxidizing, nitrate reducing microorganism: Thiomicrospira denitrificans sp.
nov. Neth J Sea Res 1975;9:344–50.
Downloaded from http://femsle.oxfordjournals.org/ by guest on April 5, 2016