Download New insight into the photoheterotrophic growth of the

Microbiology (2015), 161, 1061–1072
DOI 10.1099/mic.0.000067
New insight into the photoheterotrophic growth of
the isocytrate lyase-lacking purple bacterium
Rhodospirillum rubrum on acetate
B. Leroy,1 Q. De Meur,1 C. Moulin,1 G. Wegria2 and R. Wattiez1
Correspondence
1
B. Leroy
[email protected]
2
Received 24 September 2014
Accepted 27 February 2015
Laboratory of Proteomics and Microbiology, Research Institute for Biosciences, University of Mons,
Place du Parc 20, 7000 Mons, Belgium
Biotech Materia Nova, Parc Initialis, Avenue Copernic 1, 7000 Mons, Belgium
Purple non-sulfur bacteria are well known for their metabolic versatility. One of these bacteria,
Rhodospirillum rubrum S1H, has been selected by the European Space Agency to ensure the
photoheterotrophic assimilation of volatile fatty acids in its regenerative life support system,
MELiSSA. Here, we combined proteomic analysis with bacterial growth analysis and enzymatic
activity assays in order to better understand acetate photoassimilation. In this isocitrate lyaselacking organism, the assimilation of two-carbon compounds cannot occur through the glyoxylate
shunt, and the citramalate cycle has been proposed to fill this role, while, in Rhodobacter
sphaeroides, the ethylmalonyl-CoA pathway is used for acetate assimilation. Using proteomic
analysis, we were able to identify and quantify more than 1700 unique proteins, representing
almost one-half of the theoretical proteome of the strain. Our data reveal that a
pyruvate : ferredoxin oxidoreductase (NifJ) could be used for the direct assimilation of acetyl-CoA
through pyruvate, potentially representing a new redox-balancing reaction. We additionally
propose that the ethylmalonyl-CoA pathway could also be involved in acetate assimilation by the
examined strain, since specific enzymes of this pathway were all upregulated and activity of
crotonyl-CoA reductase/carboxylase was increased in acetate conditions. Surprisingly, we also
observed marked upregulation of glutaryl-CoA dehydrogenase, which could be a component of a
new pathway for acetate photoassimilation. Finally, our data suggest that citramalate could be an
intermediate of the branched-chain amino acid biosynthesis pathway, which is activated during
acetate assimilation, rather than a metabolite of the so-called citramalate cycle.
INTRODUCTION
Rhodospirillum rubrum belongs to the purple non-sulfur
bacteria, a highly metabolically versatile group of bacteria.
The S1H strain of this bacterium has been selected by
the European Space Agency as a major component of its
regenerative life support system MELiSSA (Micro-Ecological
Life Support System Alternative) (Hendrickx et al., 2006). In
this artificial ecosystem, bioreactors inhabited by different
organisms will recycle crew-produced waste to generate
oxygen, water and food. The first compartment, which treats
crude waste through thermophilic fermentation, will produce large amounts of volatile fatty acids, the most abundant
Abbreviations: EMC, ethylmalonyl-CoA; Icl2, isocytrate lyase-lacking;
ILV, isoleucine, leucine and valine; IlvC, ketol-acid reductoisomerase;
IlvD, dihydroxy-acid dehydratase; MELiSSA, Micro-Ecological Life
Support System Alternative; NifJ, pyruvate : ferredoxin oxidoreductase;
PHB, poly(3-hydroxybutyrate); XIC, extracted ion chromatogram.
Two supplementary tables are available with the online Supplementary
Material.
000067 G 2015 The Authors
of which will be acetate. Rs. rubrum, which is included in the
second compartment of the MELiSSA loop, will be used to
photoassimilate these volatile fatty acids.
The assimilation of carbon sources that enter central carbon
metabolism through acetyl-CoA requires an alternative cycle
to replenish the TCA cycle intermediates used for the
synthesis of all cell components (Dunn et al., 2009). During
photoheterotrophic metabolism, the TCA cycle is primarily
employed for intermediate metabolite production, rather
than acetyl-CoA oxidation and energy production. The first
identified shunt allowing two-carbon compound assimilation was the glyoxylate pathway (Kornberg & Krebs, 1957),
through which two acetyl-CoA molecules are converted into
malate to replenish the intermediates consumed. The key
enzyme involved in the glyoxylate shunt is isocitrate lyase,
which converts isocitrate into succinate and glyoxylate.
Glyoxylate is then combined with acetyl-CoA by malate
synthase to produce malate. Through the consumption of
one citrate molecule and one acetyl-CoA molecule, this cycle
results in the net production of two molecules of malate,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
Printed in Great Britain
1061
B. Leroy and others
which will be converted into oxaloacetate and 2-oxoglutarate,
which are at the root of several biosynthetic pathways.
A number of bacteria, including Rs. rubrum, lack a functional isocitrate lyase and are thus designated Icl2 (Kornberg
& Lascelles, 1960). In these organisms, an alternative to the
glyoxylate shunt is required to produce oxaloacetate and 2oxoglutarate to feed the reactions of cell component biosynthesis (Anthony, 2011). The methylaspartate cycle was
recently demonstrated to occur in haloarchaea (Khomyakova
et al., 2011). Moreover, a second pathway allowing acetylCoA assimilation, the ethylmalonyl-CoA (EMC) pathway,
has been identified in Rhodobacter sphaeroides by Alber and
colleagues (Erb et al., 2009a). All the enzymes belonging to
this pathway in Rb. sphaeroides have been identified, purified
and extensively characterized (Alber et al., 2006; Erb et al.,
2007, 2008, 2009b; Meister et al., 2005). Schneider et al.
(2012) recently showed that Methylobacterium extorquens
also employs the EMC pathway. On the other hand, Rs.
rubrum has been proposed to assimilate acetate through the
citramalate cycle (Ivanovsky et al., 1997), based on the early
finding that acetate assimilation is accompanied by the
production of a compound identified as citramalate (Hoare,
1963) and on the presence of enzymatic activity allowing
the production of glyoxylate and propionyl-CoA from C5
compounds (Osumi & Katsuki, 1977). Berg and Ivanovsky’s
group also reported the detection of enzymatic activity related
to this pathway in Rs. rubrum cell extracts (Berg & Ivanovskiı̆,
2009; Berg et al., 2002; Filatova et al., 2005), but the enzyme
responsible for citramalate synthesis or conversion to
mesaconate has not been identified/purified to date, nor has
a putative gene encoding this enzyme been detected in the
genome. Recently, the same group observed the expression of
crotonyl-CoA carboxylase/reductase, the key enzyme of the
EMC pathway, in Rs. rubrum grown photoheterotrophically
on acetate, but the authors concluded that EMC involvement
was not significant in this strain (Berg & Ivanovskiı̆, 2009). In
both the citramalate and EMC pathways, glyoxylate and
propionyl-CoA are produced from mesaconyl-CoA, with 3methylmalyl-CoA generated as an intermediate, but the two
pathways differ in the first step, leading to production of
mesaconyl-CoA as well as propionyl-CoA assimilation.
Here, we used growth analysis, proteomics and enzymatic
activity tests to analyse acetate photoassimilation in Rs.
rubrum strain S1H. More than 1700 proteins, covering a
significant part of the expressed proteome, were identified
and quantified. Except for those shared with poly(3-hydroxybutyrate) (PHB) formation, all the proteins belonging to the
EMC pathway exhibited a higher abundance under the
conditions tested, suggesting that the EMC pathway could
effectively be operational in our strain. In addition, activity of
crotonyl-CoA carboxylase/reductase was also higher under
acetate conditions. An enzyme that could be responsible for
detected citramalate synthesis has also been observed in
higher abundance in acetate-growing culture. Citramalate
synthesis is here proposed to be a consequence of operation of
part of the branched-chain amino acid biosynthetic pathway.
In addition, several other proteins showed higher abundances
1062
and could be linked to the cellular redox balance of the cells
and also to a potential assimilation pathway through glutarylCoA dehydrogenase that has not so far been identified.
METHODS
Bacterial strain, culture medium and growth conditions. Rs.
rubrum strain S1H (ATCC 25903), hereafter named S1H, was used to
study acetate assimilation under anaerobic and phototrophic conditions, in accordance with the requirements of the MELiSSA project. The
S1H strain differs from the S1 type strain only in its lower sensitivity to
growth inhibition by L-threonine. The bacterium was cultivated on the
basal salt medium of Segers & Verstraete (1983) as described by Suhaimi
et al. (1987), with acetate (62 mM) or succinate (124 mM) and
ammonium chloride (35 mM) as the sources of C and N, respectively,
and biotin as the only vitamin provided. Carbonate was included in the
medium at a concentration of 3 mM under both conditions. When
acetate and propionate were used as a mixed carbon source, both
components were supplied at an equivalent net carbon concentration
(acetate 31 mM, propionate 20.6 mM). The cultures were grown in
50 ml serum bottles containing 40 ml medium. Pure nitrogen was used
to remove oxygen from the upper gas phase and the flasks were then
hermetically sealed. Unless otherwise stated, an illumination intensity of
50 mmol m22 s21, as determined with an underwater quantum meter
(LI-193; LI-COR), was supplied with a halogen light (10 W, 100 lm;
2650 K; Sencys). The cultures were incubated at 30 uC with rotary
shaking at 150 r.p.m. Growth was monitored by measuring OD680.
Proteomic analysis. Bacteria were acclimated and subcultivated in
acetate or succinate starting at OD680 0.4–0.5. On reaching OD680 0.9–
1.2, bacteria were harvested via centrifugation at 16 000 g at 4 uC, and
proteins were extracted using guanidinium chloride (6 M) and
ultrasonication (3610 s, amplitude 40 %, IKA U50 sonicator). The
protein concentration was determined using the Bradford method
(Bradford, 1976), with bovine gamma-globulin as a standard. Aliquots
of protein (50 mg) were reduced, alkylated and precipitated with
acetone. The protein pellets obtained were solubilized using 50 mM
ammonium bicarbonate containing 1 mg trypsin, and incubated
overnight at 37 uC. Digestion was stopped with 0.1 % formic acid (v/
v, final concentration).
Protein identification and quantification were performed following a
label-free strategy on a UHPLC-HRMS platform (Eksigent 2D UltraAB
SCIEX Triple TOF 5600). Peptides (2 mg) were separated in a 25 cm C18
column (Acclaim PepMap100, 3 mm; Dionex) using a linear acetonitrile
(ACN) gradient [5–35 % (v/v), in 120 min] in water containing 0.1 % (v/
v) formic acid, at a flow rate of 300 nl min21. To achieve the greatest
possible retention time stability, which is required for label-free
quantification, the column was equilibrated with 10 vols 5 % ACN
prior to each injection. Mass spectra were acquired over the range 400–
1500m/z in high-resolution mode (resolution .35 000), with a 500 ms
accumulation time. The instrument was operated in data-dependent
acquisition mode, and MS/MS spectra were acquired over the range
100–1800m/z. The precursor selection parameters were as follows:
intensity threshold, 200 c.p.s.; maximum precursors per cycle, 50;
accumulation time, 50 ms; exclusion time after one spectrum, 15 s.
These parameters lead to a duty cycle of 3 s per cycle to ensure that highquality extracted ion chromatograms (XICs) are obtained for peptide
quantification.
ProteinPilot software (v4.1) was used to perform database searches
against the UniProt database, restricted to Rhodospirillum entries
(ATCC 11170 plus F11 strains). The search parameters included
differential amino acid mass shifts for carbamidomethyl cysteine,
oxidized methionine, all biological modifications, amino acid
substitutions and missed trypsin cleavage sites.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
Microbiology 161
Acetate photoassimilation in Rhodospirillum rubrum
For peptide quantification, the ‘quant’ application of PeakView was
employed to calculate XICs for the five top peptides from all proteins
identified at a false discovery rate below 1 %. Only unmodified and
unshared peptides were subjected to quantification. Peptides were
also excluded if the confidence in their identification was below 0.99,
as determined in ProteinPilot. A retention time window of 2 min and
a mass tolerance of 0.015m/z were applied. The calculated XICs were
exported into MarkerView, which was used to normalize the
chromatograms based on the summed area of the entire run. Only
proteins exhibiting a fold change greater/less than 1.5/0.66 with a Pvalue lower than 0.05 across the three biological replicates and
quantified with at least two peptides were employed for metabolic
characterization.
with 1 % (w/v) OsO4/cacodylate buffer at room temperature, and
washed again. They were subsequently dehydrated in a graded ethanol
series up to 100 %, and finally embedded in Spurr resin (TAAB
Laboratories Equipment). Ultrathin slices (70–90 mm) obtained with
a Leica Ultracut UCT ultramicrotome were stained with uranyl
acetate and lead citrate. Prepared samples were observed with a Zeiss
LEO 906E transmission electron microscope operated at 60 kV, and
transmission electron microscopy images were acquired with analySIS
software (Soft Imaging System).
Monitoring of volatile fatty acid consumption by HPLC
analysis. Culture supernatants were obtained from culture samples
after centrifugation at 16 000 g for 10 min at 4 uC. Aliquots (50 ml) of
Rs. rubrum growth with acetate as unique carbon
source – light stress sensitivity
culture supernatants were analysed in isocratic mode using a Shodex
Sugar SH1011 column (30068 mm) with aqueous H2SO4 (5 mM) as
the mobile phase. Acetate and propionate were assayed by integrating
their specific peaks (retention times 11 and 13 min, respectively) and
comparison with a reference curve constructed for acetate and
propionate standards. Detection was performed using a refractometer.
Enzymatic activity tests. Cells were harvested by centrifugation at
16 000 g for 10 min at 4 uC in the middle of the exponential growth
phase (OD680 ~1). Cells were mechanically lysed in Tris/HCl buffer
(50 mM pH 7.9) using bead-beating [bead size ,106 mm (Sigma
Aldrich); 1 : 3 beads : sample ratio; 3000 r.p.m., 5 min at 4 uC). Cellfree extract was obtained by centrifugation at 16 000 g for 10 min at
4 uC. The protein concentration was determined using the Bradford
method (Bradford, 1976), with bovine gamma-globulin as standard.
Acetolactate synthase activity was tested as described previously
(Muhitch, 1988), using leucine and valine (10 mM each) as inhibitors
of acetolactate synthase to obtain specific activity as recommended.
The specific acetolactate synthase activity is defined as the part of the
measured activity that can be inhibited by the presence of leucine and
valine. The final activity observed in the presence of leucine and valine
(the non-specific activity is attributed to pyruvate decarboxylase) was
subtracted from the activity measured in their absence. Acetolactate
produced after 1 h was assayed at a single end point by conversion to
acetoin, which was detected by the reaction described by Westerfield
(1945). The amount of acetoin produced was determined through the
use of a standard curve. Values were normalized using the amount of
protein determined by Bradford assay. Crotonyl-CoA carboxylase/
reductase activity was measured as described previously (Erb et al.,
2007) by following crotonyl-CoA-dependent oxidation of NADPH
spectrophotometrically at 380 nm (e51210 l mol21 cm21) for 3 min.
The test started when protein extract (20–40 mg) was added to the
reaction mixture. Glutaryl-CoA dehydrogenase activity was measured
as described previously (Blázquez et al., 2008) by following glutarylCoA-dependent reduction of ferricenium hexafluorophosphate spectrophotometrically at 280 nm (e54300 l mol21 cm21) for 50 min,
the test starting when protein extract (20–40 mg) was added to the
reaction mixture. Values obtained for each enzymatic activity were
normalized using the amount of protein determined by Bradford
assay. All tests were also carried out in the absence of protein extract
to ensure the protein dependence of the test (data not shown).
Transmission electron microscopy. Cells were harvested by
centrifugation at 16 000 g for 10 min at 4 uC in the middle of the
exponential growth phase (OD680 ~1). Cells were washed with a
cacodylate buffer at pH 7.8 [0.1 M cacodylate, 8 mM NaCl, 0.05 %
(w/v) ruthenium red] and immediately fixed for 2 h at 4 uC with 3 %
(v/v) glutaraldehyde in cacodylate buffer. After two additional 10 min
washing steps with cacodylate buffer, samples were post-fixed for 2 h
http://mic.sgmjournals.org
RESULTS AND DISCUSSION
To better characterize the growth behaviour of our strain
when using acetate as the carbon source and to define
culture conditions allowing almost parallel growth on
acetate and succinate, which is a requirement for unbiased
proteomic analysis, we first established growth curves for
the acetate and succinate cultures under various conditions. In contrast with succinate, we observed a particularly
long lag phase (more than 150 h) for the acetate cultures
under conditions involving a small amount of succinateobtained inoculum (starting OD680 ~0.1) and high light
intensity (150 mmol m22 s21) (Fig. 1a). Because this difference would bias the proteomic analysis, we decided to
optimize culture conditions to decrease the lag phase for
acetate photoassimilation. Even though such a long lag
phase is unlikely to be attributable to metabolic adaptation,
we tested the effect of using acetate-grown inocula without
any decrease in the lag phase (data not shown). No
improvement was observed when the carbonate concentration was doubled (data not shown), suggesting that the
long lag phase observed did not result from the already
described dependence of acetate assimilation on the presence of CO2 (Hoare, 1962; Porter & Merrett, 1972). In
contrast, we were able to significantly decrease the length of
the lag phase by decreasing the light intensity from 150 to
50 mmol m22 s21 (Fig. 1b). A second major improvement
was achieved by increasing the size of the inoculum so that
the culture started with OD680 0.5–0.6 (Fig. 1c), in place of
0.2 initially. Based on these results, it appears likely that the
long lag phase could be due to a higher sensitivity to light
stress when S1H is cultivated with acetate as unique carbon
source. Indeed, dilution of the culture at the time of inoculation significantly increases the light intensity experienced
at the cellular level. In contrast, increasing the OD at
inoculation and lowering the light intensity reduce the level
of ‘inoculation stress’. To confirm the higher sensitivity to light stress of S1H growing on acetate vs succinate
as the carbon source, cultures reaching OD680 1.1 (the
middle of the exponential growth phase) were subjected to
a twofold increase in the light intensity (from 50 to
100 mmol m22 s21). As shown in Fig. 2, the increase in the
light intensity had almost no effect on the growth rate of
the bacteria on succinate but completely abolished growth
on acetate. This experiment demonstrates that Rs. rubrum
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
1063
B. Leroy and others
(a)
2.5
1.5
1.0
Time (h)
2.0
Log OD680
0
200
–0.5
300
400
600
OD680
0.5
1.5
Succinate
1.0
Acetate
–1.0
0.5
–1.5
Succinate
–2.0
Acetate
–50
–2.5
0
50
Time, t (h)
100
150
–3.0
(b) 1.0
Time (h)
0.5
Log OD680
0
50
–0.5
100
150
250
200
–1.0
–1.5
succinate
–2.0
acetate
–2.5
–3.0
–3.5
(c) 1.0
0.8
Log OD680
0.6
–0.4
Time (h)
0.2
0
–0.2
20
40
60
80
100
120
140
–0.4
succinate
–0.6
acetate
–0.8
explain the difference in sensitivity observed between these
two culture conditions. A rapid increase in the light intensity
would trigger an increase in the photosynthetically produced
proton gradient, which would, in turn, increase the rate of
NAD+ photoreduction to NADH through reverse NADH
dehydrogenase activity (Golomysova et al., 2010; Herter et al.,
1998; McEwan, 1994). Acetate is a more reduced carbon
source than succinate (McKinlay & Harwood, 2010), and the
NAD+/NADH ratio could be more difficult to maintain if
the intensity of the light received is suddenly increased. In
support of this hypothesis, the elemental composition of
acetate-grown Rs. rubrum exposed to different lighting
regimes clearly indicates that a more reduced biomass is
produced when the bacterium is cultivated at a biomass
concentration of 1 g l21 (high light) than at 4 g l21 (low light)
(Favier-Teodorescu et al., 2003).
Proteomic analysis of acetate photoassimilation
by Rs. rubrum S1H
Fig. 1. Optimization of culture conditions for Rs. rubrum when
using acetate as the sole carbon source. Growth was followed by
measuring OD680 (expressed as log OD680) for cultures submitted
to high light intensity (150 mmol m”2 s” 1) and inoculated at OD680
0.1 (a), submitted to low light intensity (50 mmol m”2 s”1) and
inoculated at OD680 0.1 (b), and submitted to low light intensity
(50 mmol m”2 s”1) and inoculated at OD680 0.5–0.6 (c). All inocula
were taken from precultures already acclimated to the corresponding carbon source. Means±SD; n53 for all conditions.
S1H shows greater sensitivity to light stress when growing
on acetate as the sole carbon source. We assume that a
redox imbalance is responsible for this light stress and can
1064
Fig. 2. Comparison of the effect of light stress on Rs. rubrum S1H
grown on acetate or succinate as the sole carbon source. Acetate
and succinate cultures submitted to low light intensity (50 mmol
m”2 s”1) and inoculated at OD680 0.5–0.6 were subjected when
they reached the middle of the exponential growth phase (OD680
~1.1) to a twofold increase in light intensity (t50). Growth was
followed by measuring OD680. All inocula were taken from
precultures already acclimated to the corresponding carbon
source. Means±SD; n53 for all conditions.
For proteomic analysis, cultures were grown under a low
light intensity (50 mmol m22 s21), starting at OD680
between 0.4 and 0.5, to reduce the delay in the growth of
acetate cultures as much as possible. To avoid bias due to
the different light intensities effectively perceived by the
cells, biomass samples were collected at a constant cell
density (OD680 1.1–1.4) from the cultures grown on acetate
and succinate.
Three biological replicates, originating from different clonal
populations, were analysed under acetate and succinate
conditions. Altogether, more than 1700 unique proteins,
representing almost 50 % of the theoretical proteome (3461
and 3878 entries for ATCC 11170 and strain F11, respectively,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
Microbiology 161
Acetate photoassimilation in Rhodospirillum rubrum
in UniProt), were identified and quantified. The complete
dataset can be found in Table S1, available in the online
Supplementary Material. Among the proteins for which a
significant (P,0.05) fold change (.1.5 or ,0.66) was
detected between the acetate and succinate conditions, 352
and 225 proteins exhibited lower and higher abundances,
respectively, under acetate conditions. For further interpretation, only proteins quantified with at least two peptides
were taken into account.
Ethylmalonyl-CoA pathway involvement in
acetate assimilation by Rs. rubrum S1H
Proteomic data revealed that all the enzymes proposed to
belong to the EMC pathway (Table 1) were effectively
upregulated (Fig. 3b), suggesting that this pathway could
be involved in acetate assimilation in our strain. These
enzymes were often badly annotated in the database but
comparison to sequences of EMC enzymes studied in Rb.
sphaeroides (Alber, 2011) allowed us to correct their
annotation and clearly link them to this pathway. (Table
S2 presents the best homologue candidates of all the
enzymes of the EMC pathway of Rb. sphaeroides in the
genome of Rs. rubrum.)
The data obtained through differential proteomic analyses
are therefore largely in favour of the significant use of the
EMC pathway for the assimilation of acetate in our strain.
As the upregulation of an enzyme does not evidence its
higher activity, crotonyl-CoA carboxylase/reductase (Ccr)
activity has been measured in cell-free extracts of Rs.
rubrum S1H grown with acetate or succinate as unique
carbon source. As shown in Fig. 4b, Ccr activity was
significantly higher (8.8 times higher; P,0.01) in acetate
than in succinate conditions. This result is in contradiction
with that obtained previously by Berg & Ivanovskiı̆ (2009),
who observed no increase in Ccr activity in acetate conditions compared with malate conditions. This difference
between the two studies could arise from the different
experimental set-up (such as light intensity, which was not
sufficiently detailed to be compared with our study) and
also in Ccr activity test procedures. Notably, Ivanovsky’s
group used incorporation of radiolabelled bicarbonate
while we used cofactor oxidation, and their extracts were
produced using a French press while we used bead-beating.
Also, the exact control used in their study is unclear.
Citramalate involvement in acetate assimilation
by Rs. rubrum S1H
In the Icl2 purple bacterium Rs. rubrum, Ivanovsky and coworkers proposed that the citramalate cycle takes place, in
which acetate is assimilated via the condensation of acetylCoA with pyruvate through the action of citramalate synthase
(Ivanovsky et al., 1997). This pathway was proposed based on
the observation that citramalate is the earliest molecule to be
labelled when 13C-acetate is photoassimilated (Porter &
Merrett, 1972). Here, we observed slight upregulation of a
http://mic.sgmjournals.org
protein that is currently annotated as 2-isopropylmalate
synthase (LeuA) (Rru_A0695; fold change, 1.6; P-value, 0.01;
identified peptides, 12), but for which the PRIAM method
[for automated enzyme detection (Claudel-Renard et al.,
2003)] predicts citramalate synthase activity with a higher
probability (E-values, 6e254 for citramalate synthase versus
1e241 for isopropylmalate synthase). Both 2-isopropylmalate synthase and citramalate synthase are members of the
LeuA dimer superfamily (Frantom, 2012). The best-characterized citramalate synthase among proteobacteria is that
of Geobacter sulfurreducens (Risso et al., 2008a), and this
protein shares 51 % identity with Rru_A0695. These data
favour the annotation of this protein as a true citramalate
synthase. Nevertheless, upregulation of another group of
enzymes leads us to propose a new hypothesis regarding
involvement of citramalate in acetate assimilation by Rs.
rubrum S1H. Indeed, among the identified upregulated
proteins, we detected a cluster of three genes encoding ketolacid reductoisomerase (IlvC) (Rru_A0469; fold change, 4.1;
P-value, 0.0005; identified peptides, 54) and the acetolactate
synthase small (Rru_A0468; fold change, 3.1; P-value, 0.003;
identified peptides, 22) and large subunits (Rru_A0467; fold
change, 3.1; P-value, 0.003; identified peptides, 33). These
proteins are involved in the first step of branched-chain
amino acid (isoleucine, leucine and valine, ILV) synthesis
(Fig. 3a). Dihydroxy-acid dehydratase (IlvD), which is also
involved in this pathway, was also more abundant under
acetate conditions (Rru_A1786; fold change, 1.6; P-value,
0.04; identified peptides, 32). Citramalate is an intermediate
in the atypical isoleucine synthesis pathway, which is
independent of threonine supply (Wu et al., 2010). In this
alternative pathway, citramalate is converted to 2-oxobutanoate by the action of 3-isopropylmalate/(R)-2-methylmalate dehydratase large and small subunits (LeuC and LeuD,
Rru_A1189 and Rru_A1190) and 3-isopropylmalate dehydrogenase (LeuB, Rru_A1191). These three proteins were
identified in this study but were not significantly differentially
regulated under acetate condition (Fig. 3a). Acetolactate
synthase can equivalently transfer an acetaldehyde from
pyruvate to 2-oxobutanoate, forming 2-ethyl-2-hydroxy-3oxobutanoate, a reaction involved in the ILV biosynthesis
pathway. As the upregulation of an enzyme does not evidence
its higher activity, we measured the acetolactate synthase
activity in cell-free extracts of Rs. rubrum. As shown in Fig. 4a,
acetolactate synthase activity was slightly but significantly
higher in acetate conditions than in the succinate control
(P,0.05), suggesting this enzyme could be involved in
acetate photoassimilation.
However, we observed a decrease in abundance of some
branched-chain amino acid transaminases (Rru_A2223; fold
change 0.4, P-value 0.01, identified peptides 16; Rru_A1040,
fold change 0.03, P-value 0.001, identified peptides 16),
suggesting that the outcome of acetate assimilation through
reactions belonging to the ILV pathway is not the production
of branched-chain amino acids. 3-Methyl-2-oxopentanoate
and/or 2-oxoisovalerate could, for example, be directed to
another metabolic pathway, such as the valine degradation
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
1065
B. Leroy and others
Table 1. Differentially expressed proteins
UniProt accession
number
Locus tag
P-value
Fold change
(acetate/succinate)*
No. identified
peptidesD
Description
tr|Q2RYD8
tr|Q2RYD7
tr|Q2RXX3
tr|Q2RXR7
tr|Q2RXR6
tr|Q2RXR4
tr|Q2RV44
tr|Q2RV43
tr|Q2RU23
tr|Q2RT18
tr|Q2RRG6
tr|Q2RQI1
tr|Q2RQ36
tr|Q2RPT8
tr|Q2RPT7
tr|Q2RPT6
tr|Q2RWQ6
sp|P80448
tr|Q2RX73
tr|Q2RX72
tr|G2T8X1
tr|Q2RWJ6
tr|Q2RVK4
tr|G2TF50
tr|G2TF51
tr|G2TFQ4
tr|Q2RTR3
tr|G2T9T2
tr|Q2RT67
tr|Q2RT66
tr|Q2RT64
tr|Q2RS72
sp|Q53046
tr|G2T7S3
tr|G2T7S5
tr|Q2RU35
tr|G2T7T0
tr|Q2RU29
tr|G2TF86
tr|Q2RRP3
sp|Q2RRP2
tr|Q2RRP1
tr|Q2RRP0
tr|Q2RW98
tr|Q2RW96
tr|Q6US84
tr|Q6US85
tr|Q6US86
tr|Q6US87
tr|Q2RSU0
tr|Q2RST9
Rru_A0052
Rru_A0053
Rru_A0217
Rru_A0273
Rru_A0274
Rru_A0276
Rru_A1200
Rru_A1201
Rru_A1572
rru_A1927
Rru_A2479
Rru_A2817
Rru_A2964
Rru_A3062
Rru_A3063
Rru_A3064
Rru_A0635
Rru_A0077
Rru_A0467
Rru_A0468
Rru_A0469
Rru_A0695
Rru_A1040
Rru_A1189
Rru_A1190
Rru_A1191
Rru_A1682
Rru_A1786
Rru_A1878
Rru_A1879
Rru_A1881
Rru_A2223
Rru_A2398
Rru_A1556
Rru_A1558
Rru_A1560
Rru_A1563
Rru_A1566
Rru_A2400
Rru_A2402
Rru_A2403
Rru_A2404
Rru_A2405
Rru_A0793
Rru_A0795
Rru_A2264
Rru_A2265
Rru_A2266
Rru_A2267
Rru_A2005
Rru_A2006
0.000003
0.000045
0.002630
0.070980
0.001060
0.022770
0.001320
0.000340
0.008110
0.004190
0.005550
0.000870
0.489180
0.000002
0.002520
0.006950
0.000630
0.002830
0.003230
0.003480
0.000500
0.012260
0.001140
0.080750
0.341380
0.017120
0.02998
0.041030
0.000130
0.000059
0.000310
0.012260
0.010130
0.000280
0.033260
0.032290
0.014250
0.000009
0.037140
0.017310
0.001050
0.002590
0.001440
0.011820
0.001720
0.000320
0.002080
0.000720
0.000720
0.000007
0.000900
4.75
5.14
2.08
0.86
0.53
1.60
2.31
4.46
1.58
1.83
1.98
0.66
0.81
8.80
3.20
2.79
0.21
0.26
3.12
3.07
4.07
1.59
0.03
1.38
1.15
1.36
1.70
1.62
0.16
0.21
0.40
0.44
1.83
2.43
1.54
1.82
1.91
80.97
0.52
2.12
4.82
2.05
2.63
0.06
0.02
6.23
4.15
5.35
6.14
176.27
8.05
54
40
39
51
119
10
25
20
9
34
28
10
15
7
27
35
5
8
33
22
54
12
16
22
19
31
27
32
13
20
20
16
80
12
8
42
5
3
54
8
22
24
39
3
12
6
28
25
32
26
26
Biotin carboxylase
Carboxyltransferase
L-Malyl-CoA/b-methylmalyl-CoA lyase
3-Oxoacyl-(acyl-carrier-protein) reductase
Acetyl-CoA acetyltransferase
Polyhydroxyalkanoate synthesis repressor
Malyl-CoA thioesterase
Mesaconyl-CoA hydratase
Ethylmalonyl-CoA/methylmalonyl-CoA epimerase
Acetyl-CoA hydrolase
Methylmalonyl-CoA mutase
Phasin
MaoC-like dehydratase
Ethylmalonyl-CoA mutased
Crotonyl-CoA carboxylase/reductase
Methylsuccinyl-CoA dehydrogenase
Na+/solute symporter
Ferredoxin-2
Acetolactate synthase, large subunit
Acetolactate synthase, small subunit
Ketol-acid reductoisomerase
2-Isopropylmalate synthase
Leucine dehydrogenase
3-Isopropylmalate dehydratase, large subunit
3-Isopropylmalate dehydratase,small subunit
3-Isopropylmalate dehydrogenase
Branched-chain amino acid aminotransferase
Dihydroxy-acid dehydratase
Dihydrolipoamide dehydrogenase
Dihydrolipoamide acetyltransferase
Pyruvate dehydrogenase (lipoamide)
Branched-chain amino acid aminotransferase
Pyruvate : ferredoxin oxidoreductase
NADH-quinone oxidoreductase subunit B
NADH dehydrogenase subunit D
NADH-quinone oxidoreductase, subunit F
NADH-quinone oxidoreductase subunit I
NADH-plastoquinone oxidoreductase, chain 5
Ribulose bisphosphate carboxylase
Ribulose-phosphate 3-epimerase
Fructose-1,6-bisphosphatase class 1
Phosphoribulokinase
Transketolase
Nitrogenase
Nitrogenase iron protein 1
FixX§
FixC§
FixB§
FixA§
Glutaryl-CoA dehydrogenase
L-Carnitine dehydratase/bile acid-inducible protein F
The complete dataset can be found in the supplementary data.
*The fold change is the ratio of the abundance of a protein under acetate conditions to the abundance under succinate conditions.
D‘No. identified peptides’ represents the number of identified peptides with a confidence higher than 95 %.
dDue to interference in MS signal, fold change for this protein was manually corrected and results from quantification of four peptides rather than five.
§Fix, Electron transfer flavoprotein.
1066
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
Microbiology 161
Acetate photoassimilation in Rhodospirillum rubrum
(b)
O
OH
O HO CH3
D-Citramalate
Rru_A1190
#19; x1.15
#22; x1.4
CH3
D-Erythro-3-
methylmalate
Rru_A1191
S CoA
O
H3C
CoA
S
Crotonyl-CoA
Rru_A2398
O
H 3C
H3C
2× Acetyl-CoA
#15; x1
CO2
NADH + H+
CO2
#31; x1.4
#34; x1.8
O
Rru_A2964
S CoA
Acetyl-CoA
NAD+
Rru_A1927
CoA
S
OH
Acetate
(R)-3-Hydroxybutyryl-CoA
O
H3C
O
H3C
#34; x1.8
#12; x1.6
O
OH
Rru_A1927
OH
NADP+
#51; x0.85
Acetate
O
NADPH + H+
Rru_A0273
OH
H3C
Rru_A0695
OH
O
H3C
CoA
S
Acetoacetyl-CoA
O
Rru_A1189
HO
O
H3C
#22; x0.8
O
HO
Rru_A1380
(a)
CO2
NADPH + H+
NADP+
Rru_A3063
#80; x1.8
#27; x3.2
Rru_A2005
CO2
#26; x176
OH
O
O
2-Oxobutanoate
OH
H3C
O
CO2
O
O
OH
H3C
(2S)-Ethylmalonyl-CoA
#33; x3/#22; x3.1
#9; x1.6
CO2
O
OH
O
HO
O
OH
OH
CH3
NADPH + H+
FAD
Rru_A3062
Acetolactate
FADH2
#7; x8.8
O
HO
NADPH + H+
Rru_A0469
#54; x4
NADP+
(2R)-Ethylmalonyl-CoA
OH
HO CH3
(S)-2-Aceto-2hydroxybutanoate
CoA
S
O
H3C
CH3
CoA
S
Glutaryl-CoA
Rru_A1572
Rru_A0467/Rru_A0468
H 3C
O
HO
CH3
Pyruvate
O
CoA
S
O
Pyruvate
O
O
HO
S
CoA
CH3
O
NADP+
Methylsuccinyl-CoA
HO CH3 O
HO CH3 O
H 3C
Rru_A3064
#35; x2.8
OH
H3C
OH
O
OH
OH
(R)-2,3-Dihydroxy-3methylpentanoate
HO
(R)-2,3-Dihydroxy-3methylbutanoate
CoA
Mesaconyl-CoA
Rru_A1786
H
Rru_A1201
#32; x1.6
#20; x4.4
CH3 O
OH
CH3 O
H3C
S
CH3
O
O
CH3
β-Methylmalyl-CoA
2-Oxoisovalerate
(S)-3-Methyl-2oxopentanoate
S CoA
OH
O
H
O
HO
H3C
OH
O
O
Rru_A0217
O
#39; x2.1
Rru_A1040
Rru_A1040
#16; x0.03
#16; x0.03
CH3 O
H3C
O
OH
Isoleucine
CH3
HO
CH3
NH2 CH3
NH2
Valine
Glyoxylate
H3C
O
CH3
HO
NH2
OH
O
O
Leucine
S CoA
Propionyl-CoA
HCO3–
ATP
ADP + Pi
Rru_A0052/0053
#54; x4.7/#40; x5
O
Rru_A0217
#39; x2.1
OH
O
HO
S CoA
HO
CH3
(R)-Methylmalonyl-CoA
Rru_A1572
O
S CoA
O
Malyl-CoA
Rru_A1200
#25; x2.3
#9; x1.6
O
Fold change
<0.2
<0.5 <0.66 <1.5
<2
<5
>5
OH
O
S CoA
HO
O
HO
CH3
(S)-Methylmalonyl-CoA
OH
O
Malate
Rru_A2479
#28; x2
O
HO
S CoA
O
Succinyl-CoA
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
1067
B. Leroy and others
Fig. 3. Schematic representation of the metabolic pathways highlighted through differential proteomic analysis. (a) Metabolic
reactions linked to branched-chain amino acid (ILV) biosynthesis. (b) Ethylmalonyl-CoA pathway. Water molecules were
omitted to preserve clarity of the schematic representation. x: fold change observed in differential proteomic analysis; #, number
of peptides detected for the protein. Colour indicates the fold change: red represents downregulation under the acetate
conditions, green represents upregulation under the acetate conditions, and light grey represents not significantly affected by
carbon source. Arrows in blue represent enzymatic reactions that have been monitored by an enzymatic activity test.
pathway (Sykes et al., 1987), which is conducive to propionylCoA production. However, despite intensive researches for
an enzyme similar to the already described keto-acid
dehydrogenase in the genome of Rs. rubrum, we could not
find any candidate fulfilling this function. Further investigation, notably including metabolite analyses, will thus be
needed in order to clarify the involvement of reactions
belonging to the ILV synthesis pathway in acetate photoassimilation in Rs. rubrum.
(a)
35
Specific activity
(nmol min–1 mg–1)
30
25
20
15
10
5
(b)
60
Specific activity
(nmol min–1 mg–1)
0
50
Acetate
Succinate
Acetate
Succinate
Acetate
Succinate
40
30
20
10
(c)
25
Specific activity
(nmol min–1 mg–1)
0
20
15
10
5
0
–5
Fig. 4. Enzymatic activity test. Cell-free extracts of Rs. rubrum
S1H grown with acetate or succinate as unique carbon source
were tested for acetolactate synthase activity (n56) (a), crotonylCoA reductase/carboxylase activity (n55) (b), and glutaryl-CoA
dehydrogenase activity (n54) (c). Means±SE.
1068
Acetolactate synthase combines two molecules of pyruvate
to produce one molecule of acetolactate and releases one
molecule of CO2. Substantial activation of this pathway
would result in enhanced consumption of pyruvate, which
would need to be replenished continuously. We detected
the presence of a pyruvate : ferredoxin oxidoreductase, also
known as NifJ (Rru_A2398; fold change, 1.8; P-value, 0.01;
identified peptides, 80), which is commonly reported to
catalyse oxidative decarboxylation of pyruvate to generate
acetyl-CoA. It has been shown that this enzyme is able
to catalyse the reverse reaction as well, thus producing
pyruvate via the reductive carboxylation of acetyl-CoA
(Furdui & Ragsdale, 2000; St Maurice et al., 2007). At
physiological concentrations, Furdui & Ragsdale (2000)
measured a significant carboxylating activity and showed
that the rate of the reaction depends only on the concentration of acetyl-CoA and pyruvate. As the intracellular
concentration of acetyl-CoA is likely to be high in cells
grown on acetate and as pyruvate level should be kept low
through consumption by acetolactate synthase, the net flux
through this enzyme is expected to favour pyruvate synthesis
when the bacteria are grown on acetate. Noticeably, the high
level of acetyl-CoA can be maintained in the cell through the
upregulation of acetyl-CoA transferase (Rru_A1927; fold
change, 1.8; P-value, 0.004; identified peptides, 34), which
could be responsible for acetate activation, as mentioned by
Alber et al. (2006). The preference for reductive carboxylation
activity is further supported by the observation of major
downregulation of all subunits of the pyruvate dehydrogenase
(lipoamide) complex (Rru_A1878, Rru_A1879, Rru_A1881).
Indeed, pyruvate dehydrogenase (lipoamide), dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase were all clearly downregulated under acetate conditions,
suggesting that oxidative decarboxylation of pyruvate activity
is lowered in cells grown on acetate. Importantly, reductive
carboxylation activity relies on the presence of an electron
supplier, which has been proposed to be ferredoxin (Furdui &
Ragsdale, 2000). In our dataset, we identified 4Fe–4S ferredoxin as differentially regulated under acetate conditions, but
its abundance was greatly decreased (Rru_A0077; fold change,
0.25; P-value, 0.003; identified peptides, 8). Nevertheless,
a second ferredoxin encoded in the fix gene cluster was
clearly upregulated under acetate conditions (Rru_A2264;
fold change, 6.2; P-value, 0.0003; identified peptides, 6) and
could therefore serve as the electron supplier for reductive
carboxylation of acetate. Altogether, these data are in favour
of a major involvement of carboxylation of acetyl-CoA to
pyruvate, which in turn feeds the branched-chain amino acid
synthesis pathway. This interpretation of our result is also in
agreement with the data of Porter & Merrett (1972), who
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
Microbiology 161
Acetate photoassimilation in Rhodospirillum rubrum
Differential proteomic analysis also revealed the intense
upregulation of the clustered glutaryl-CoA dehydrogenase
(Rru_A2005; fold change, 176; P-value, 76106; identified
peptides, 26) and carnitine hydratase (best annotated as a
CoA transferase) (Rru_A2006; fold change, 8; P-value,
96104; identified peptides, 26). Glutaryl-CoA dehydrogenase is generally observed to be involved in the anaerobic
degradation of benzoate in bacteria (Härtel et al., 1993;
Wischgoll et al., 2009), in which it catalyses the decarboxylation of glutaryl-CoA to produce crotonyl-CoA. Under our
acetate conditions, this compound could be easily assimilated
through the EMC pathway (Fig. 3b), but the origin of
glutaryl-CoA and the role of the linked CoA transferase
remain enigmatic. Glutaryl-CoA dehydrogenase has been
shown to use electron transfer flavoproteins as electron
acceptors (Ramsay et al., 1987). Under acetate conditions, we
observed a large increase of the abundance of the four proteins encoded by the fix gene cluster (FixABCX; Rru_A2267,
Rru_A2266, Rru_A2265 and Rru_A2264, respectively). FixA
and FixB are electron transfer flavoproteins that are usually
responsible for electron transfer to nitrogenase in diazotrophically growing Rs. rubrum (Edgren & Nordlund, 2004).
However, nitrogenase was clearly downregulated in response
to the use of acetate as the carbon source for growth
(Rru_A0793, fold change 0.06, P-value 0.01, identified
peptides 3; and nitrogenase iron protein Rru_A0795, fold
change 0.02, P-value 0.0017, identified peptides 12). We thus
analysed the glutaryl-CoA dehydrogenase activity in cell-free
extracts of Rs. rubrum S1H grown in acetate and succinate
conditions. As shown in Fig. 4(c), glutaryl-CoA dehydrogenase activity was not detected in the succinate conditions
but was readily detected in the acetate conditions [17 nmol
min21 (mg protein)21, P,0.01]. The detection of glutarylCoA dehydrogenase activity only in acetate-grown cells is in
total agreement with the very high fold change observed in
proteomic data which reveal absence/presence of the enzyme.
We also tested (data not shown) glutaryl-CoA dehydrogenase
activity in cultures assimilating butyrate and propionate as
sole carbon source in the same conditions but neither showed
glutaryl-CoA dehydrogenase activity. Non-decarboxylating
glutaryl-CoA dehydrogenase exists, which releases the
http://mic.sgmjournals.org
Acetate transport in Rs. rubrum S1H
Among the observed downregulated proteins, we identified
a Na+/solute symporter (Rru_A0635; fold change, 0.2; Pvalue, 0.0006; identified peptides, 5) that is highly similar
(70 % ID) to the acetate permease (ActP) of Escherichia
coli. Downregulation of the acetate transporter in response
to acetate in the medium could appear to be counterproductive, but Gimenez et al. (2003) clearly demonstrated
that, in E. coli, ActP was only needed for growth at low
acetate concentration. In addition, Risso et al. (2008b) have
shown that acetate transporter genes in Geobacter species
were only expressed at low concentration of acetate.
Moreover, we observed that propionate, which has been
shown to inhibit acetate transport through ActP in E. coli
(Gimenez et al., 2003), had no effect on acetate assimilation
in Rs. rubrum when the two carbon sources were present
simultaneously and at the same concentration in the
medium (Fig. 5). Altogether, these observations lead us to
35
2.5
30
2.0
25
20
1.5
15
1.0
10
0.5
5
OD680
Glutaryl-CoA dehydrogenase involvement in
acetate assimilation by Rs. rubrum S1H
intermediate glutaconyl-CoA in place of crotonyl-CoA.
While the released compound was not identified in our
enzymatic test, we consider Rru_A2005 most probably
belongs to the decarboxylating group of glutaryl-CoA
dehydrogenases, based on conservation of key amino acids
highlighted by the Boll group (Schaarschmidt et al., 2011;
Wischgoll et al., 2009), namely E91, S99 and Y373. Our data
thus suggest that glutaryl-CoA dehydrogenase could play a
previously unsuspected role in acetate photoassimilation. It is
clear that only a detailed flux analysis could definitely identify
the relative importance of the multiple pathways proposed
here. Nevertheless, comparison of specific activities obtained
for crotonyl-CoA carboxylase/reductase [47 nmol min21
(mg protein)21] and glutaryl-CoA dehydrogenase [17 nmol
min21 (mg protein)21] suggests that the latter potentially
contributes significantly to acetate assimilation.
VF A in medium (mM)
reported that citramalate was one of the earliest and most
abundant radiolabelled compounds in short-term exposure
experiments with NaH14CO3 and acetate-assimilating Rs.
rubrum. Altogether, these data lead us to propose that the
extensively reported contribution of citramalate synthase in
acetate assimilation by Rs. rubrum is linked to the use of part
of the ILV biosynthesis pathway (Fig. 3a) rather than to the
so-called citramalate cycle, and that the ILV biosynthesis
pathway plays a significant but not yet fully understood role
in acetate assimilation. Further experiments such as flux
analyses will be needed to definitely demonstrate and
quantify the involvement of this proposed pathway in acetate
photoassimilation in Rs. rubrum.
Acetate
Propionate
OD680
0
0
0
20
40
Time (h)
60
80
Fig. 5. Photoassimilation of acetate and propionate when supplied
together at an equivalent net carbon concentration. Rs. rubrum
S1H was cultivated with a 1 : 1 mixture of acetate and propionate
as carbon source. Abundances of acetate and propionate in the
culture supernatant were monitored by HPLC during growth.
Means±SD; n53. VFA, volatile fatty acid.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
1069
B. Leroy and others
(a)
(b)
2 mm
5 mm
2 mm
5 mm
Fig. 6. PHB production in Rs. rubrum S1H cultivated in acetate or succinate conditions. Transmission electron microscopy
photomicrographs were obtained from Rs. rubrum S1H grown in the presence of succinate (a) or acetate (b). Arrows indicate
PHB granules.
suggest that ActP is not involved in acetate transport in our
strain under our conditions.
Respiratory chain modification during acetate
assimilation by Rs. rubrum S1H
Five of the ten members of the NADH-quinone oxidoreductase complex identified in the present study were
detected at higher abundances under acetate conditions.
The activation of respiratory chain electron transport
makes sense only if a final diffusible/excretable electron
acceptor is present, such as O2 or nitrate. In this study, cells
were grown under anaerobic conditions with ammonium
as the nitrogen source, and neither O2 nor nitrate was present
as a final electron acceptor. The absence of O2 was confirmed
based on pigment expression, which is immediately repressed
at a very low O2 partial pressure (Sistrom, 1965). Upregulation
of members of NADH-quinone oxidoreductase in acetate
conditions will need further investigation.
Redox balance during acetate assimilation by Rs.
rubrum S1H
Photoheterotrophic growth of purple bacteria has been
extensively reported to depend on the Calvin–Benson–
Bassham (CBB) cycle playing the role of an ‘electron sink’
(Falcone & Tabita, 1991; McKinlay & Harwood, 2010; Wang
et al., 2010). In our dataset, we also observed a decreased
abundance of RuBisCO when acetate is used as sole carbon
source (Rru_A2400; fold change, 0.52; P-value, 0.04;
identified peptides, 54) as compared with succinate-containing cultures. This finding seems to be in agreement with the
conclusion of Laguna et al. (2011) that, in Rb. sphaeroides,
CO2 fixation through the EMC pathway is sufficient to
equilibrate redox balance even in the absence of a functional
CBB cycle. Nevertheless, we observed that other redoxbalancing mechanisms could be playing a role during acetate
assimilation. Notably, we observed that, as already mentioned by Kim et al. (2012) for Rb. sphaeroides, our strain
produced larger amounts of PHB during acetate assimilation
(Fig. 6). Hauf et al. (2013) have shown that the reduction state
1070
of the cofactor pool can influence PHB production. PHB
production observed here could be the result of an increased
level of NAD(P)H and indirectly be a way to deal with NAD+
photoreduction (Herter et al., 1998), notably occurring at the
beginning of the growth curve. Further experiments are
needed to confirm this hypothesis, notably by following PHB
accumulation along the complete growth curve in acetate
conditions. NifJ catalyses the reductive carboxylation of
acetyl-CoA, which could also play the role of redox-balancing
reaction. Interestingly, in a kinetic experiment in which we
compared the proteome of a culture in lag phase with the
proteome of the same culture just after the initiation of
growth on acetate as a carbon source (data not shown), NifJ
was the most highly overexpressed protein when growth was
initiated (fold change, 11.2; P-value, 0.003; identified
peptides, 86). This result suggests that NifJ plays a major
role at the beginning of the growth curve, corresponding to
the period during which light stress due to culture dilution
likely triggered the previously mentioned redox imbalance
(Favier-Teodorescu et al., 2003). CO2 fixation through
acetyl-CoA carboxylation could therefore represent a new
redox-balancing reaction developed by bacteria to combine
acetate assimilation and maintenance of the redox balance.
CONCLUSIONS
In conclusion, our proteomic data suggest that Rs. rubrum
S1H assimilates acetate through multiple pathways.
Combination of proteomic analysis and enzymatic activity
testing reveal that the EMC pathway could be operational
in Rs. rubrum when growing photoheterotrophically on
acetate. Indeed, all the enzymes specific to the EMC
pathway were detected in higher abundances and crotonylCoA carboxylase/reductase activity was increased under
acetate culture conditions. Our data also lead us to propose
a new outcome for citramalate. Indeed, citramalate could be
an intermediate of an alternate route of acetate assimilation
through pyruvate and part of the ILV biosynthesis pathway.
NifJ, which has been shown to produce pyruvate through
acetyl-CoA reductive carboxylation, was upregulated in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
Microbiology 161
Acetate photoassimilation in Rhodospirillum rubrum
acetate conditions. This could represent a new redoxbalancing reaction, and the pyruvate generated could be
directed to acetolactate synthase and the IlvC and IlvD
enzymes, which were also detected in higher abundances in
acetate conditions. The outcome of the activation of part of
the ILV biosynthesis pathway during acetate assimilation is
still not understood and requires further investigation.
Finally, glutaryl-CoA dehydrogenase could play a previously
unsuspected but important role in photoheterotroph acetate
assimilation since this protein was the mostly upregulated in
this study.
Erb, T. J., Fuchs, G. & Alber, B. E. (2009a). (2S)-Methylsuccinyl-CoA
dehydrogenase closes the ethylmalonyl-CoA pathway for acetyl-CoA
assimilation. Mol Microbiol 73, 992–1008.
Erb, T. J., Brecht, V., Fuchs, G., Müller, M. & Alber, B. E. (2009b).
Carboxylation mechanism and stereochemistry of crotonyl-CoA
carboxylase/reductase, a carboxylating enoyl-thioester reductase.
Proc Natl Acad Sci U S A 106, 8871–8876.
Falcone, D. L. & Tabita, F. R. (1991). Expression of endogenous and
foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO)
genes in a RubisCO deletion mutant of Rhodobacter sphaeroides.
J Bacteriol 173, 2099–2108.
Favier-Teodorescu, L., Cornet, J. F. & Dussap, C. G. (2003).
Modelling continuous culture of Rhodospirillum rubrum in photobioreactor under light limited conditions. Biotechnol Lett 25, 359–364.
ACKNOWLEDGEMENTS
This research was supported by the European Space Agency and
BELSPO (GSTP ‘Melgen-3’). This work was also partially financed by
the FNRS under grant ‘Grand Equipement’ no. 2877824.
Filatova, L. V., Berg, I. A., Krasil’nikova, E. N., Tsygankov, A. A.,
Laurinavichene, T. V. & Ivanovskiı̆, R. N. (2005). [The mechanism of
acetate assimilation in purple nonsulfur bacteria lacking the
glyoxylate pathway: acetate assimilation in Rhodobacter sphaeroides
cells]. Mikrobiologiia 74, 313–318 (in Russian).
REFERENCES
Frantom, P. A. (2012). Structural and functional characterization of
a-isopropylmalate synthase and citramalate synthase, members of the
Alber, B. E. (2011). Biotechnological potential of the ethylmalonyl-
Furdui, C. & Ragsdale, S. W. (2000). The role of pyruvate ferredoxin
CoA pathway. Appl Microbiol Biotechnol 89, 17–25.
oxidoreductase in pyruvate synthesis during autotrophic growth by
the Wood-Ljungdahl pathway. J Biol Chem 275, 28494–28499.
LeuA dimer superfamily. Arch Biochem Biophys 519, 202–209.
Alber, B. E., Spanheimer, R., Ebenau-Jehle, C. & Fuchs, G. (2006).
Study of an alternate glyoxylate cycle for acetate assimilation by
Rhodobacter sphaeroides. Mol Microbiol 61, 297–309.
Anthony, C. (2011). How half a century of research was required to
understand bacterial growth on C1 and C2 compounds; the story of the
serine cycle and the ethylmalonyl-CoA pathway. Sci Prog 94, 109–137.
Berg, I. A. & Ivanovskiı̆, R. N. (2009). [Enzymes of the citramalate cycle in
Rhodospirillum rubrum]. Mikrobiologiia 78, 22–31 (in Russian).
Berg, I. A., Filatova, L. V. & Ivanovsky, R. N. (2002). Inhibition
of acetate and propionate assimilation by itaconate via propionylCoA carboxylase in isocitrate lyase-negative purple bacterium
Rhodospirillum rubrum. FEMS Microbiol Lett 216, 49–54.
Blázquez, B., Carmona, M., Garcı́a, J. L. & Dı́az, E. (2008).
Gimenez, R., Nuñez, M. F., Badia, J., Aguilar, J. & Baldoma, L. (2003).
The gene yjcG, cotranscribed with the gene acs, encodes an acetate
permease in Escherichia coli. J Bacteriol 185, 6448–6455.
Golomysova, A., Gomelsky, M. & Ivanov, P. S. (2010). Flux balance
analysis of photoheterotrophic growth of purple nonsulfur bacteria
relevant to biohydrogen production. Int J Hydrogen Energy 35, 12751–
12760.
Härtel, U., Eckel, E., Koch, J., Fuchs, G., Linder, D. & Buckel, W.
(1993). Purification of glutaryl-CoA dehydrogenase from Pseudo-
monas sp., an enzyme involved in the anaerobic degradation of
benzoate. Arch Microbiol 159, 174–181.
Hauf, W., Schlebusch, M., Hüge, J., Kopka, J., Hagemann, M. &
Forchhammer, K. (2013). Metabolic changes in Synechocystis
Identification and analysis of a glutaryl-CoA dehydrogenase-encoding
gene and its cognate transcriptional regulator from Azoarcus sp. CIB.
Environ Microbiol 10, 474–482.
PCC6803 upon nitrogen-starvation: excess NADPH sustains polyhydroxybutyrate accumulation. Metabolites 3, 101–118.
Bradford, M. M. (1976). A rapid and sensitive method for the
Hendrickx, L., De Wever, H., Hermans, V., Mastroleo, F., Morin, N.,
Wilmotte, A., Janssen, P. & Mergeay, M. (2006). Microbial ecology of
quantitation of microgram quantities of protein utilizing the principle
of protein–dye binding. Anal Biochem 72, 248–254.
Claudel-Renard, C., Chevalet, C., Faraut, T. & Kahn, D. (2003).
Enzyme-specific profiles for genome annotation: PRIAM. Nucleic
Acids Res 31, 6633–6639.
Dunn, M. F., Ramı́rez-Trujillo, J. A. & Hernández-Lucas, I. (2009).
Major roles of isocitrate lyase and malate synthase in bacterial and
fungal pathogenesis. Microbiology 155, 3166–3175.
Edgren, T. & Nordlund, S. (2004). The fixABCX genes in
Rhodospirillum rubrum encode a putative membrane complex
participating in electron transfer to nitrogenase. J Bacteriol 186,
2052–2060.
Erb, T. J., Berg, I. A., Brecht, V., Müller, M., Fuchs, G. & Alber, B. E.
(2007). Synthesis of C5-dicarboxylic acids from C2-units involving
the closed artificial ecosystem MELiSSA (Micro-Ecological Life
Support System Alternative): reinventing and compartmentalizing
the Earth’s food and oxygen regeneration system for long-haul space
exploration missions. Res Microbiol 157, 77–86.
Herter, S. M., Kortlüke, C. M. & Drews, G. (1998). Complex I of
Rhodobacter capsulatus and its role in reverted electron transport.
Arch Microbiol 169, 98–105.
Hoare, D. S. (1962). The photometabolism of [1-14C]acetate and
[2-14C]acetate by washed-cell suspensions of Rhodospirillum rubrum.
Biochim Biophys Acta 59, 723–725.
Hoare, D. S. (1963). The photo-assimilation of acetate by Rhodo-
spirillum rubrum. Biochem J 87, 284–301.
Ivanovsky, R. N., Krasilnikova, E. N. & Berg, I. A. (1997). A proposed
crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway.
Proc Natl Acad Sci U S A 104, 10631–10636.
citramalate cycle for acetate assimilation in the purple non-sulfur
bacterium Rhodospirillum rubrum. FEMS Microbiol Lett 153, 399–404.
Erb, T. J., Rétey, J., Fuchs, G. & Alber, B. E. (2008). Ethylmalonyl-
Khomyakova, M., Bükmez, O., Thomas, L. K., Erb, T. J. & Berg, I. A.
(2011). A methylaspartate cycle in haloarchaea. Science 331, 334–337.
CoA mutase from Rhodobacter sphaeroides defines a new subclade of
coenzyme B12-dependent acyl-CoA mutases. J Biol Chem 283, 32283–
32293.
http://mic.sgmjournals.org
Kim, M. S., Kim, D. H., Cha, J. & Lee, J. K. (2012). Effect of carbon and
nitrogen sources on photo-fermentative H2 production associated with
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
1071
B. Leroy and others
nitrogenase, uptake hydrogenase activity, and PHB accumulation in
Rhodobacter sphaeroides KD131. Bioresour Technol 116, 179–183.
A dehydrogenase by site-directed mutagenesis. FEBS Lett 585, 1317–
1321.
Kornberg, H. L. & Krebs, H. A. (1957). Synthesis of cell constituents from
Schneider, K., Peyraud, R., Kiefer, P., Christen, P., Delmotte, N.,
Massou, S., Portais, J. C. & Vorholt, J. A. (2012). The ethylmalonyl-
C2-units by a modified tricarboxylic acid cycle. Nature 179, 988–991.
Kornberg, H. L. & Lascelles, J. (1960). The formation of isocitratase
by the Athiorhodaceae. J Gen Microbiol 23, 511–517.
Laguna, R., Tabita, F. R. & Alber, B. E. (2011). Acetate-dependent
CoA pathway is used in place of the glyoxylate cycle by Methylobacterium extorquens AM1 during growth on acetate. J Biol Chem 287,
757–766.
photoheterotrophic growth and the differential requirement for the
Calvin–Benson–Bassham reductive pentose phosphate cycle in
Rhodobacter sphaeroides and Rhodopseudomonas palustris. Arch
Microbiol 193, 151–154.
Segers, L. & Verstraete, W. (1983). Conversion of organic acids to H2
McEwan, A. G. (1994). Photosynthetic electron transport and
of bacteriochlorophyll in Rhodospirillum molischianum. J Bacteriol 89,
403–408.
anaerobic metabolism in purple non-sulfur phototrophic bacteria.
Antonie van Leeuwenhoek 66, 151–164.
McKinlay, J. B. & Harwood, C. S. (2010). Carbon dioxide fixation as a
central redox cofactor recycling mechanism in bacteria. Proc Natl
Acad Sci U S A 107, 11669–11675.
Meister, M., Saum, S., Alber, B. E. & Fuchs, G. (2005). L-Malylcoenzyme A/b-methylmalyl-coenzyme A lyase is involved in acetate
assimilation of the isocitrate lyase-negative bacterium Rhodobacter
capsulatus. J Bacteriol 187, 1415–1425.
by Rhodospirillaceae grown with glutamate or dinitrogen as nitrogen
source. Biotechnol Bioeng 25, 2843–2853.
Sistrom, W. R. (1965). Effect of oxygen on growth and the synthesis
St Maurice, M., Cremades, N., Croxen, M. A., Sisson, G., Sancho, J. &
Hoffman, P. S. (2007). Flavodoxin : quinone reductase (FqrB): a
redox partner of pyruvate : ferredoxin oxidoreductase that reversibly
couples pyruvate oxidation to NADPH production in Helicobacter
pylori and Campylobacter jejuni. J Bacteriol 189, 4764–4773.
Suhaimi, M., Liessens, J. & Verstraete, W. (1987). NH+
4 -N
assimilation by Rhodobacter capsulatus ATCC 23782 grown axenically
and non-axenically in N and C rich media. J Appl Bacteriol 62, 53–64.
Muhitch, M. J. (1988). Acetolactate synthase activity in developing
Sykes, P. J., Burns, G., Menard, J., Hatter, K. & Sokatch, J. R. (1987).
maize (Zea mays L.) kernels. Plant Physiol 86, 23–27.
Osumi, T. & Katsuki, H. (1977). A novel pathway for L-citramalate
Molecular cloning of genes encoding branched-chain keto acid
dehydrogenase of Pseudomonas putida. J Bacteriol 169, 1619–1625.
synthesis in Rhodospirillum rubrum. J Biochem 81, 771–778.
Wang, D., Zhang, Y., Welch, E., Li, J. & Roberts, G. P. (2010).
Porter, J. & Merrett, M. J. (1972). Influence of light intensity on
reductive pentose phosphate cycle activity during photoheterotrophic
growth of Rhodospirillum rubrum. Plant Physiol 50, 252–255.
Elimination of Rubisco alters the regulation of nitrogenase activity
and increases hydrogen production in Rhodospirillum rubrum. Int J
Hydrogen Energy 35, 7377–7385.
Ramsay, R. R., Steenkamp, D. J. & Husain, M. (1987). Reactions of
Westerfield, W. W. (1945). A colorimetric determination of blood
electron-transfer flavoprotein and electron-transfer flavoprotein :
ubiquinone oxidoreductase. Biochem J 241, 883–892.
Risso, C., Van Dien, S. J., Orloff, A., Lovley, D. R. & Coppi, M. V.
(2008a). Elucidation of an alternate isoleucine biosynthesis pathway
acetoin. J Biol Chem 161, 495–502.
Wischgoll, S., Taubert, M., Peters, F., Jehmlich, N., von Bergen, M. &
Boll, M. (2009). Decarboxylating and nondecarboxylating glutaryl-
in Geobacter sulfurreducens. J Bacteriol 190, 2266–2274.
coenzyme A dehydrogenases in the aromatic metabolism of obligately
anaerobic bacteria. J Bacteriol 191, 4401–4409.
Risso, C., Methé, B. A., Elifantz, H., Holmes, D. E. & Lovley, D. R.
(2008b). Highly conserved genes in Geobacter species with expression
Wu, B., Zhang, B., Feng, X., Rubens, J. R., Huang, R., Hicks, L. M.,
Pakrasi, H. B. & Tang, Y. J. (2010). Alternative isoleucine synthesis
patterns indicative of acetate limitation. Microbiology 154, 2589–2599.
pathway in cyanobacterial species. Microbiology 156, 596–602.
Schaarschmidt, J., Wischgoll, S., Hofmann, H. J. & Boll, M. (2011). Con-
version of a decarboxylating to a non-decarboxylating glutaryl-coenzyme
1072
Edited by: P. O’Toole
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 15:02:18
Microbiology 161