RESEARCH ARTICLE
A proteomic view of the facultatively
chemolithoautotrophic lifestyle of Ralstonia
eutropha H16
Edward Schwartz1, Birgit Voigt2, Daniela Z .uhlke2, Anne Pohlmann1, Oliver Lenz1,Dirk Albrecht2, Alexander Schwarze1, Yvonne Kohlmann1, Cornelia Krause1,Michael Hecker2 and B .arbel Friedrich1
1 Institut f .ur Biologie, Mikrobiologie, Humboldt-Universit .at zu Berlin, Berlin, Germany2 Institut f .ur Mikrobiologie, Ernst-Moritz-Arndt-Universit .at Greifswald, Greifswald, Germany
Received: May 20, 2009
Revised: August 21, 2009
Accepted: August 26, 2009
Ralstonia eutropha H16 is an H2-oxidizing, facultative chemolithoautotroph. Using 2-DE in
conjunction with peptide mass spectrometry we have cataloged the soluble proteins of this
bacterium during growth on different substrates: (i) H2 and CO2, (ii) succinate and (iii) glycerol.
The first and second conditions represent purely lithoautotrophic and purely organoheterotrophic
nutrition, respectively. The third growth regime permits formation of the H2-oxidizing and CO2-
fixing systems concomitant to utilization of an organic substrate, thus enabling mixotrophic
growth. The latter type of nutrition is probably the relevant one with respect to the situation faced
by the organism in its natural habitats, i.e. soil and mud. Aside from the hydrogenase and Calvin-
cycle enzymes, the protein inventories of the H2-CO2- and succinate-grown cells did not reveal
major qualitative differences. The protein complement of the glycerol-grown cells resembled that
of the lithoautotrophic cells. Phosphoenolpyruvate (PEP) carboxykinase was present under all
three growth conditions, whereas PEP carboxylase was not detectable, supporting earlier findings
that PEP carboxykinase is alone responsible for the anaplerotic production of oxaloacetate from
PEP. The elevated levels of oxidative stress proteins in the glycerol-grown cells point to a
significant challenge by ROS under these conditions. The results reported here are in agreement
with earlier physiological and enzymological studies indicating that R. eutropha H16 has a
heterotrophic core metabolism onto which the functions of lithoautotrophy have been grafted.
Keywords:
Autotrophy / Hydrogenase / Lithotrophy / Microbiology / Oxidative stress /
Phosphoenolpyruvate-pyruvate-oxaloacetate node
1 Introduction
Ralstonia eutropha H16 is a strictly respiratory, facultatively
chemolithoautotrophic representative of the b-proteo-
bacteria. The soil- and mud-dwelling organism grows well
on simple organic acids and a few sugars. In the absence of
organic substrates, it thrives on H2 as sole energy source,
fixing CO2 via the Calvin–Benson–Bassham cycle. With
regard to these alternative growth modes, R. eutropha H16 is
a typical representative of the aerobic H2 oxidizers [1, 2].
Recently, the deciphering of the entire 7 416 678-bp genome
of R. eutropha H16 was completed [3, 4]. The genome
consists of three circular replicons: chromosome 1
(4.05 Mbp), chromosome 2 (2.91 Mbp) and the megaplasmid
pHG1 (0.45 Mbp). A total of 6626 coding sequences were
predicted. A disproportionately large fraction of the total
coding capacity is devoted to transport systems – another
indication that the organism in its natural habitat is
confronted with and can grow on a wide variety of
substrates.
Abbreviations: FBA, fructose-1,6-bisphosphate aldolase; FBP,
fructose-1,6-bisphosphatase; GAPDH, glyceraldehyde 3-phos-
phate dehydrogenase; GN, glycerol minimal medium; MBH,
membrane-bound hydrogenase; PEP, phosphoenolpyruvate;
PGK, phosphoglycerate kinase; PK, pyruvate kinase; RH, regula-
tory hydrogenase; SH, soluble hydrogenase; SN, succinate
minimal medium; TCA, tricarboxylic acid
Correspondence: Dr. Edward Schwartz, Institut f .ur Biologie,
Mikrobiologie, Humboldt-Universit .at zu Berlin, Chausseestr.
117, 10115 Berlin, Germany
E-mail: [email protected]
Fax: 149-3020938102
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
5132 Proteomics 2009, 9, 5132–5142DOI 10.1002/pmic.200900333
Oxidation of H2 in R. eutropha H16 is catalyzed by two
NiFe hydrogenases. One of them, a dimeric membrane-
bound hydrogenase (MBH), is the first component of an
H2-dependent electron transport chain which conserves
energy during lithotrophic growth. The MBH is coupled –
both physically and electronically – to the respiratory
chain by a specialized b-type cytochrome. The hydrogenase
dimer is located on the periplasmic side of the cytoplasmic
membrane. The second energy-conserving hydrogenase is a
hexameric protein located in the cytoplasm. This
soluble hydrogenase (SH) couples H2 oxidation to the
reduction of NAD1. It consists of a dimeric hydrogenase
module, a dimeric diaphorase module and a homodimer of
a 19-kD protein responsible for the NADPH-dependent
reductive activation of the enzyme [5]. The SH supplies
the organism with reductant for, e.g. fixation of CO2, giving
R. eutropha a great advantage over lithotrophs that synthe-
size NADH via reverse electron flow. A remarkable aspect of
the hydrogenases is the complex pathway of post-transla-
tional maturation steps required for the assembly and
insertion of the bimetallic center at the active site of each
enzyme.
The MBH and SH are encoded along with accessory
proteins in two large operons on the 450-kb megaplasmid
pHG1. The expression of the hydrogenase genes is
co-ordinately controlled by an H2-dependent signal trans-
duction chain. The H2-sensing component is itself an
hydrogenase-like dimer called a regulatory hydrogenase
(RH). When H2 becomes available, the RH transmits a
signal, the nature of which is presently not well understood,
to a histidine kinase, which in turn interacts with an NtrC-
like response regulator, causing the latter to activate tran-
scription from the hydrogenase promoters [6–8].
Early enzymological studies revealed that the production
of hydrogenases in R. eutropha H16 is not strictly H2-
dependent [9]. Hydrogenase expression is tightly repressed
on succinate (SN) and pyruvate. However, during growth on
other carbon/energy sources hydrogenase expression is
derepressed to varying degrees. The degree of derepression
mediated by the different substrates is more or less corre-
lated with the growth rates they support. Thus, hydrogenase
expression is susceptible to a form of catabolite repression.
During lithoautotrophic growth, CO2 is fixed by ribulose-
1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The
RuBisCO is encoded along with phosphoribulokinase and
the other Calvin-cycle enzymes in a 12-gene operon present
in two copies, one located on the megaplasmid pHG1, the
other on chromosome 2 [10, 11].
The expression patterns of the key components of litho-
trophic metabolism in R. eutropha H16, the hydrogenases
and the enzymes of the Calvin–Benson–Bassham cycle,
have been the subject of detailed investigations in the past
years, which have provided us with a basic understanding of
the underlying regulatory mechanisms [6, 9, 11–13]. Hence,
this is not the main interest of this study. Rather, our aim
was to obtain information about the global metabolic char-
acteristics correlated with the typical growth modes of
R. eutropha H16. Specifically, we cultivated R. eutropha H16
under three different regimes: lithoautotrophically on H2
and CO2, heterotrophically on glycerol (a substrate that
supports only slow growth and leads to high-level expression
of hydrogenase and Calvin-cycle enzymes) and SN (tight
repression of these enzymes). Using 2-D gels we cataloged
the proteins present in cells grown under these three
conditions.
This study was also conceived with more general ques-
tions regarding the bases of lithoautotrophic metabolism
and the underlying features of facultative and obligate
lithoautotrophy in mind. During the 1960s and 1970s, these
questions prompted numerous studies and were the subject
of vigorous debate among microbiologists [14, 15]. With the
advent of molecular genetic techniques, attention shifted to
the characterization of the specialized enzymes and proteins
involved in the various forms of lithoautotrophy. Although
great progress has been made in this area, the adaptations of
core metabolism to lithoautotrophic lifestyles remain to be
characterized [16]. The holistic approaches of the genomic
era provide tools for such studies that were unimaginable 20
years ago. With the availability of genomic sequences of
both facultative and obligate lithoautotrophs, interest in
their general metabolism is reawakening [17, 18]. This study
is a step toward understanding one such organism in the
hope that it will contribute to our knowledge of lithoauto-
trophy in general.
2 Materials and methods
2.1 Strain and culture conditions
R. eutropha H16 (DSM428, ATCC 17699) was cultivated at
301C in mineral salts medium (100 mL of culture in 500-mL
baffled flasks with shaking at 120 rpm) [19]. Lithoautotrophic
cultures were grown under an atmosphere of hydrogen,
oxygen and carbon dioxide at a ratio of 8:1:1 v/v/v. For
heterotrophic experiments the medium was supplemented
with either 0.4% w/v succinate (succinate minimal medium
(SN)) or 0.4% glycerol (glycerol minimal medium (GN)) as
carbon source. Growth was monitored by measuring
turbidity at 436 nm (Supporting Information Fig. S1).
2.2 Preparation of soluble proteins
Cells were harvested during the exponential growth phase at
OD436 5 1 by centrifugation (6000� g, 41C, 15 min), washed
twice with TEP buffer (10 mM Tris, 1 mM EDTA, pH 7.5,
0.3 mg/mL PMSF), resuspended in 0.5 mL TEP buffer, and
disrupted by four passages through a French pressure cell
(SLM, Rochester, NY, USA) at 900 psi. Crude protein
extracts were separated from cellular debris by ultra-
centrifugation (90 000� g, 41C, 30 min). The resulting
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supernatant consisting of the soluble protein fraction was
stored at �801C until use.
2.3 2-DE, imaging and protein identification
The protein content of the soluble fraction was determined
using the RotiNanoquant Kit (Roth). In total, 500mg protein
extract were separated by 2-DE as described previosly [20]. The
IPG reswelling solution for R. eutropha H16 protein extracts
contained 2% w/v CHAPS, 8 M urea, 0,5% v/v Pharmalyte
3–10 and 13 mM DTT. Isoelectric focusing was performed
using non-linear IPGs in the pH range 3–10 (Amersham
Biosciences). 2-D gels were fixed and stained with colloidal
Coomassie Brilliant Blue G-250 (Amersham Biosciences). For
the processing of gel images and gel-based relative quantita-
tion, Decodon Delta 2D software was used. Protein identifi-
cation was performed as described by Voigt et al. [21].
Quantitative protein data presented here are the arithmetic
means of three sets of data obtained from two biological
replicates. For the quantitative comparison of expression
patterns for the three growth conditions investigated, ratios of
mean normalized spot volumes were calculated.
2.4 RT-PCR
Total RNA from lithoautotrophically and heterotrophically
grown cells was isolated with the ribopure bacteria
system (Applied Biosystems, Darmstadt) after quenching
the cells with 50% �801C methanol and stabilizing the
RNA with RNAprotect (Qiagen, Hilden). The integrity
of the RNA was checked using RNA 6000 Nano assay
chips on a Bioanalyzer 2100 (Agilent). In total, 2mg of total
RNA were reverse transcribed with the high-capacity RNA-
to-cDNA kit (Applied Biosystems). Diluted cDNA samples
were used as templates in Real-time qPCR analysis
using specific primer pairs and SYBR Green fluorescent
dye. Real-time PCR was performed using PowerSYBR
Green PCR Mastermix on a 7500 Fast PCR Cycler (Applied
Biosystems). Uniformity of the product was checked
for every PCR by the determination of a dissociation
curve. Pairs of primers with lengths of 19–21 nucleotides
were optimized for use at an annealing temperature of
58–601C. Each primer pair amplified a fragment of
150–200 bp. All primer pairs showed the same efficiency
(100710%) in a control qPCR experiment with serially
diluted cDNA templates. Relative expression ratios were
determined by the ddCt method using gyrB as a constitutive
control. Primers used for qPCR analysis were ppc 301
(50-TGTTCAACCGCATCAGCAA-30), ppc 302 (50-CAGCAA
CTCAACCTGCAAGTG-30), pepck 311 (50-CAACGCCATTC
CAGTTCAAGT-30), pepck 312 (50-CCGAGGATTTACTGCG
TCAAC-30), ppsA 321 (50-GGTGGACGCCGATGTTGT-30),
ppsA 322 (50-GGAAACCGAAGTGACCGAAGT-30), pyk1
331 (50-CAGCTCGACACCGATTTCCT-30), pyk1 332 (50-GG
TGACGGCTCATCCACAAT-30), gyrB 151 (50-GCCTGCAC-
CACCTTGTCTTC-30) and gyrB 152 (50-TGTGGAGGGAC
CTGACT-30).
2.5 Generation of an unmarked knockout mutation
A mutant of R. eutropha H16 with an in-frame lesion in the
prkA gene was obtained using a gene replacement protocol as
described previously [22]. Briefly, a segment of genomic DNA
encompassing prkA was amplified by polymerase chain
reaction using the primer pair 50-CGGACAGTACCGGTCG-
CCTGTTCATTTTCTCG-30 and 50-ACCGCGCGCTTGGCCA-
GGGCGGTGGCGGGACGACG-30. The purified amplicon
was treated with AgeI and inserted into vector plasmid
Litmus28 (NEB). The resulting plasmid was cut with BglII to
excise a 930-bp internal fragment. The prkA deletion allele
was transferred to suicide vector pLO2 [22]. The resulting
mobilizable, deletogenic plasmid was introduced into
R. eutropha H16 by conjugative transfer from the donor strain
Escherichia coli S17-1. Sucrose-resistant transconjugants
were screened by PCR for the desired deletions. This yielded
the R. eutropha derivative HFJ74 ( 5 H16 prkAD).
3 Results and discussion
3.1 General features and statistics
A total of 292 proteins were identified during 2-D gel-based
proteome analysis of three different growth modes
(Supporting Information Tables S1–S3). The protein profiles
of the three growth modes are summarized in a multi-color
protein map (Fig. 1). The false-color map differentiates
between (i) proteins up-regulated in cells growing on one of
the substrates, (ii) proteins identified in cells growing on two
substrates and (iii) constitutively expressed proteins present
under all three growth conditions. Surprisingly, 81% of all
proteins identified in the present study are encoded on
chromosome 1 versus 13% for chromosome 2. This is
remarkable since the latter represents nearly 40% of the total
coding capacity (Table 1). The underrepresentation of
proteins derived from chromosome 2 is, however, in accor-
dance with the finding, that chromosome 1 encodes key
functions of general metabolism involved in e.g. DNA repli-
cation, transcription and translation. Chromosome 2, on the
other hand, harbors genes for alternative metabolic pathways
including various pathways for the decomposition of
aromatic compounds and the utilization of alternative nitro-
gen sources. The majority of these genes are probably not
expressed under the growth conditions tested in our study.
The proteins identified in our 2-D gels were classified
according to function. The major functional groups represent
amino acid biosynthesis (19% of all identified proteins),
carbohydrate metabolism (12%) and energy metabolism
(11%) (Supporting Information Fig. S2).
5134 E. Schwartz et al. Proteomics 2009, 9, 5132–5142
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3.2 Key enzymes of lithoautotrophic growth
Proteins belonging to both of the energy-conserving hydro-
genases were identified in 2-D gels of lithoautotrophically
(H2-CO2-) grown cells of R. eutropha H16 (Table 2). All five
subunits of the SH were found in the protein complement
of the H2-CO2-grown cells. The subunits HoxF, HoxU,
HoxY and HoxH were up-regulated by the factors of 9.4, 2.4,
2.6 and 11.3, respectively, compared with SN-grown cells.
The previous physiological studies on R. eutropha H16
grown in SN and H2-CO2 cultures showed an approx.
200-fold induction of both MBH and SH activities under the
latter conditions [9]. These values reflect similarly strong
induction of promoter activity of the PMBH and PSH
promoters and corresponding increases in the abundance of
the MBH and SH transcripts [12].
The fifth subunit of the SH, the HoxI protein, was
identified early on as a dominant species in SDS-PAGE gels
of lithoautotrophically grown cells of R. eutropha H16.
Under these conditions, HoxI (formerly designated
‘‘B-protein’’) represents 4% of the total protein content [23].
In this study we identified HoxI as a dominant protein
species in 2-D gels of H2-CO2-grown cells and found it to be
91-fold up-regulated with respect to SN-grown cells. The
physiological role of the highly abundant HoxI protein was
for a long-time enigmatic. Recently, it was shown that HoxI
is a subunit of the SH (two HoxI monomers/SH holo-
enyzme) and is responsible for the reductive activation of
the enzyme by NADPH [5].
Proteins of the MBH are not necessarily to be expected
in the soluble fraction. Nevertheless, one of the two
subunits of the MBH, the [Ni-Fe] center-containing HoxG,
H2-CO2 H2-CO2 + H2-CO2 + constitutive(H CO +
Chr1Glycerol
Glycerol
Succinate
Glycerol Succinate
Glycerol + Succinate
(H2-CO2 +Glycerol + Succinate)
Chr2pHG1
H2-CO2 Succinate
Figure 1. False-color map of the
proteins detected in soluble
extracts of R. eutropha H16
cells grown under three differ-
ent conditions. In the first
dimension proteins were sepa-
rated in a pH gradient in the
range 3–10. The color coding
used for the protein spots is
defined in the lower part of the
figure. Selected proteins are
labelled. The color of the label
indicates the location of the
corresponding gene in the
genome.
Table 1. Statistics for the R. eutropha H16 genome
Feature Chromosome 1 Chromosome 2 Megaplasmid pHG1 Total
Size (bp) 4 052 032 2 912 490 452 156 7 416 678Coding capacity coding sequence 3651 (55%) 2555 (39%) 420 (6%) 6626 (100%)Proteins identified 236 (81%) 37 (13%) 19 (6%) 292 (100%)
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was present and sixfold up-regulated compared with SN-
grown cells. The small subunit (HoxK), which is anchored
in the membrane via an hydrophobic C-terminal segment,
was not detected.
In addition to the subunits of the energy-conserving
hydrogenases, the products of three other hydrogenase-
related genes were also identified in H2-CO2-grown cells.
These were the large subunit (HoxC) of the RH, the
hydrogenase accessory protein HypB2 and a maturation
protease (HoxW). The formation of active hydrogenases
requires a complex maturation apparatus composed of
multiple proteins. Since the latter proteins catalyze the
formation of active-site metallocomplexes in the maturing
hydrogenases, they are probably much less abundant than
their substrates, the hydrogenases.
Not surprisingly, hydrogenase proteins were also identi-
fied in 2-D gels of the glycerol-grown cells of R. eutrophaH16. This is in accord with the previous findings demon-
strating a 4100-fold induction of hydrogenases in glycerol
cultures [9]. The SH subunits HoxF, HoxU, HoxY, HoxH
and HoxI and the MBH protein HoxG were up-regulated by
the factors of 65.1, 6.2, 12.5, 22.3, 288.5 and 13.5, respec-
tively, relative to SN cultures.
In the absence of organic growth substrates, R. eutrophaH16 can fix CO2 by means of a RuBisCO enzyme and the
other enzymes of the Calvin–Benson–Bassham cycle [10].
Both the chromosomal and megaplasmid cbb operons
(designated cbbc and cbbp, respectively) are actively tran-
scribed during growth on H2 [11]. Like the hydrogen-
oxidizing system, the Calvin-cycle enzymes are also expres-
sed under conditions of energy limitation, e.g. during
growth on glycerol as carbon and energy source. We iden-
tified and quantified 11 Cbb proteins in our 2-D gels: CbbEp,
CbbEc, CbbFp, CbbFc, CbbPp, CbbGp, CbbGc, CbbKp,
CbbKc, CbbAp and CbbAc (Table 3 and Supporting Infor-
mation Table S1). The dual R. eutropha H16 cbb operons are
coordinately controlled by CbbR, a LysR-type activator
protein. The absence of CbbR from our 2-D gels is not
surprising, since the previous studies using reporter gene
fusions to monitor promoter activity showed that tran-
scription from the chromosomal cbbR promoter is weak and
constitutive, pointing to low levels of CbbR in the cell [11].
For all but two proteins, the induction ratio H2-CO2 versusSN is higher than glycerol versus SN. We note that this is the
opposite of the trend observed for the hydrogenase-related
proteins.
Table 2. Hydrogenase-related proteins identified in 2-D gels
Gene Locus tag Product Ratio H2-CO2/SNa) Ratio GNb)/SN
hoxG PHG002 Membrane-bound [NiFe] hydrogenase, large subunit 6.0 13.5hoxC PHG021 Regulatory [NiFe] hydrogenase, large subunit 4.0 51.0hoxF PHG088 NAD-reducing hydrogenase diaphorase moiety, large subunit 9.4 65.1hoxU PHG089 NAD-reducing hydrogenase diaphorase moiety, small subunit 2.4 6.2hoxY PHG090 NAD-reducing hydrogenase, H2ase moiety, small subunit 2.6 12.5hoxH PHG091 NAD-reducing hydrogenase, H2ase moiety, large subunit 11.3 22.3hoxW PHG092 [NiFe] hydrogenase C-terminal protease (HoxH-specific) 6.2 15.8hoxI PHG093 NAD-reducing hydrogenase subunit 91.2 288.5hypB2 PHG095 [NiFe] hydrogenase maturation protein 2.6 5.5
a) Minimal medium containing succinate.b) Minimal medium containing glycerol.
Table 3. Proteins of the Calvin cycle identified in 2-D gels
Gene Locus tag Product Ratio H2-CO2/SNa) Ratio GNb)/SN
cbbEp PHG423 Ribulose-phosphate 3-epimerase 0.7 1.4cbbEc H16_B1391 Ribulose-phosphate 3-epimerase 1.8 1.1cbbFp PHG422 FBP 1.0 1.1cbbFc H16_B1390 FBP 4.3 0.5cbbPp PHG421 Phosphoribulokinase 26.7 16.0cbbGp PHG418 GAPDH 44.1 9.5cbbGc H16_B1386 GAPDH 145.9 8.5cbbKp PHG417 PGK 13.9 3.2cbbKc H16_B1385 PGK 7.7 2.9cbbAp PHG416 FBA 4.4 0.8cbbAc H16_B1384 FBA 17.3 1.6
a) Minimal medium containing succinate.b) Minimal medium containing glycerol.
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3.3 Central metabolic pathways
The comparison of protein complements of cells grown
under lithoautotrophic and organoheterotrophic conditions
showed surprisingly little difference in central pathways.
Among the enzymes of these pathways present in our 2-D
gels, we found phosphoenolpyruvate (PEP) carboxykinase
(H16_A3711), PEP synthetase (H16_A2038) and pyruvate
kinase (PK) (H16_A0567) under all three growth conditions.
These proteins belong to a network of enzymes inter-
connecting the metabolites PEP, oxaloacetate, pyruvate and
malate, which is known as the ‘‘PEP-pyruvate-oxaloacetate
node’’ and is a major switchboard controlling the distribu-
tion of carbon among different metabolic pathways (Fig. 2)
[24]. Two other enzymes of the network responsible for
interconverting C3 and C4 intermediates, PEP carboxylase
and pyruvate carboxylase, were not detected in our 2-D gels,
although corresponding genes are present in the R. eutrophaH16 genome (H16_A2921 and H16_A1251). In many
bacteria PEP carboxylase and pyruvate carboxylase catalyze
the carboxylation of PEP and pyruvate, respectively, to
oxaloacetate and, thus, contribute to the anaplerotic regen-
eration of the key C4 intermediate. The results of our 2-D
gels are interesting in light of an earlier study by Schobert
F6P E4P
S7PS1,7BP GAP
GAP Xu5P
CbbF /CbbF /Fbp CbbA /CbbA
CbbF /CbbF
G6PPgi1/Pgi2
CbbT /CbbT /TktAA
GAPTpiAGlpK
glycerol glycerol -3-P
GlpD
DHAP
Ru1,5BP
Ru5P
DHAP
Xu5P R5P
p
F1,6BP
CbbP /CbbPCbbS L /
CbbS L
CbbE /CbbE /Rpe
CbbT /CbbT /TktA
CbbG /CbbG /GapA
CbbK /CbbK /Pgk
CbbA /CbbA
CbbA /CbbA /FbaA/FbaB
RpiA
3PGA
CbbE /CbbE /Rpe1,3PGA
pyruvate
acetyl CoA
PEP
Pgm1/Pgm2/A1100
Eno
oxalo-acetate
Pck Ppc
Pyk1/Pyk2/Pyk3
PdhA1BL
CO
CbbS L
PpsA
2PGA
Pyc
B
acetyl-CoA
citrateCisY/A1229/B0357/B0414/B2211
AcnA/AcnB
FumA/FumC
fumarate
Icd2/icd1/icd3
Mdh1/Mdh2
isocitrate
malate
oxalo-acetate
MaeA/MaeB
y
α– keto-glutarateOdhABL
SucCD
SdhABCD
succinate
succinyl-CoA
Figure 2. Central metabolic pathways of R. eutropha H16. (A) Schematic map representing the segments of metabolism relevant for this
study. Arrows indicate the entry points of the carbon sources used in our cultures. Enzyme designations are based on the gene anno-
tations of the R. eutropha H16 genome project. Proteins identified in our 2-D gels are underlined. Metabolites are given in italics. DHAP,
dihydroxyaceton phosphate; E4P, erythrose-4-phosphate; F1,6BP, fructose-1,6-bisphosphate; F6P, fructose-6-phosphate; GAP, glycer-
aldehyde-3-phosphate; G6P, glucose-6-phosphate; 1,3PGA, 1,3-diphosphoglyceric acid; 2PGA, 2-phosphoglyceric acid; 3PGA, 3-phos-
phoglyceric acid; R5P, ribose-5-phosphate; Ru1,5BP, ribulose-1,5-bisphosphate; Ru5P, ribulose-5-phosphate; S1,7BP, sedoheptulose-1,7-
bisphosphate; S7P, sedoheptulose-7-phosphate and Xu5P, xylulose-5-phosphate. (B) The PEP-pyruvate-oxaloacetate node of R. eutropha
H16. Enzymatic reactions discussed in the text are represented by solid black arrows. Other relevant reactions are indicated by grey
arrows. The values for relative transcript abundance (fold difference H2-CO2 versus SN) as determined by RT-PCR are given for four
enzymes: PEP carboxykinase (PEPCK), PEP carboxylase (PPC), PEP synthetase (PPS) and PK. The thickness of the respective arrows is
proportional to these values. The values in parentheses give the fold difference (H2-CO2 versus SN) of the corresponding proteins in 2-D
gels. PC, pyruvate carboxylase; ME, malic enzyme; MDH, malate dehydrogenase and OADC, oxaloacetate decarboxylase.
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and Bowien [25], who measured enzyme activities of the
PEP-pyruvate-oxaloacetate node in cells of R. eutropha H16
grown under various conditions. Neither PEP carboxylase
activity nor pyruvate carboxylase activity was detectable
under any of the conditions tested, including lithoauto-
trophic growth on H2 and CO2 and organoheterotrophic
growth on SN. Schobert and Bowien concluded that in
R. eutropha H16 PEP carboxykinase is, in addition to its
activity as an oxaloacetate decarboxylase, the sole enzyme
capable of carboxylating C3 intermediates to replenish the
oxaloacetate pool.
The levels of the three enzymes of the PEP-pyruvate-
oxaloacetate node were slightly higher (�twofold) in 2-D
gels of lithoautotrophically grown cells compared with
SN-grown cells. In order to obtain more reliable information
on the expression of these important enzymes, we decided
to monitor the relative abundance of the respective tran-
scripts. Using RT-PCR we measured relative transcript
levels for four enzymes in H2-CO2- and SN-grown cells: PEP
carboxykinase, PEP carboxylase, PEP synthetase and PK.
The results of these experiments are shown in Fig. 2B. PEP
carboxykinase expression was tenfold higher in the
lithoautotrophically grown cells versus SN cultures. PK and
PEP synthetase transcripts were 4.5- and 7.6-fold more
abundant, respectively, in the H2-CO2-grown cells. PEP
carboxylase transcripts were present in cells under both
growth conditions but the levels were low. Taken together,
the above results indicate that expression of PEP carbox-
ykinase, PEP synthetase and PK is in fact elevated in cells
growing on H2 and CO2. Furthermore, they suggest
that the gene for PEP carboxylase (ppc) is transcribed,
but that an active enzyme is not formed. The reasons for
higher expression in the lithoautotrophically growing cells
are not clear.
We also identified proteins corresponding to a nearly
complete set of enzymes of gluconeogenesis (fructose-1,6-
bisphosphatase (FBP), fructose-1,6-bisphosphate aldolase
(FBA), triosephosphate isomerase, glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH), phosphoglycerate kinase
(PGK), phosphoglycerate mutase, enolase and PK) in 2-D
gels of lithoautotrophically grown cells. The enzymes FBP,
FBA, GAPDH and PGK are present in multiple isoenzymes
encoded by the duplicate genes of the two cbb operons and
one or two additional chromosomal alleles not associated
with the cbb operons. Clearly, the organism must synthesize
hexoses from C3 building blocks during both lithoauto-
trophic growth and growth on glycerol or SN, requiring the
activities of some or all of the above enzymes. During
growth on SN the cbb genes are tightly repressed. With the
exception of the fbaA gene for FBA, the non-cbb alleles were
expressed at roughly the same levels in H2-CO2-grown cells
as in SN-grown cells, and thus appear to be constitutive.
Expression of the non-cbb gene for FBA is sevenfold up-
regulated. The reason for this is not apparent. During
lithoautotrophic growth, the activities of FBP, FBA, triose-
phosphate isomerase, GAPDH and PGK are required for the
regeneration of the carbon acceptor ribulose-1,5-bispho-
sphate. Under these conditions, the dual cbb operons are
fully induced, providing additional enzymatic capacity to
meet the demands of CO2 fixation. In other words, for
each of the above-named enzymes with the exception of
triosephosphate isomerase, at least three isoenzymes are
formed. In each case, at least two of them were detected in
2-D gels from lithoautotrophic cultures. Proteins corre-
sponding to the specialized enzymes of glycolysis, which in
R. eutropha H16 proceeds via the Entner–Doudoroff Path-
way, were absent from 2-D gels for our three growth
conditions. This is not surprising, since none of the
substrates used in these studies is catabolized by Entner–
Doudoroff enzymes.
Most of the tricarboxylic acid (TCA)-cycle enzymes were
resolved in the 2-D gels of lithoautotrophically grown cells.
Aconitate hydratase, isocitrate dehydrogenase, a-ketogluta-
rate dehydrogenase (subunits E1 and E3), succinyl-CoA
synthetase, succinate dehydrogenase, fumarase, malate
dehydrogenase and citrate synthase were present, although
not all of them could be quantified. This finding is in line
with the results of earlier enzymological and isotopic label-
ling studies [26–30] which demonstrated activities corre-
sponding to a complete set of TCA cycle enzymes in
lithoautotrophically grown cells of R. eutropha H16. The
comparison of H2-CO2- and SN-grown cells revealed that
two enzymes were up-regulated under lithoautotrophic
conditions: aconitate hydratase (sevenfold) and isocitrate
dehydrogenase 1 (3.5-fold).
3.4 Growth on glycerol
In general, the expression of proteins representing the
central metabolic pathways was not markedly different in
lithotrophically grown and glycerol-grown cells. Interest-
ingly, several proteins involved in the biosynthesis of
macromolecules were significantly down-regulated in the
glycerol-grown cells relative to the SN cultures (Supporting
Information Table S3). These include enzymes of pathways
for the biosynthesis of amino acids (IlvC, MetE and CysD),
ribosomal proteins (RplJ, RplI and RpsA) and a component
of the transcriptional apparatus (RpoA). This pattern of
down-regulation is symptomatic for stress responses
observed in E. coli and other bacteria [31]. The curtailment of
expression of this set of genes is triggered by various stress
conditions including nutrient shift-down and exposure to
oxygen radicals. The glycerol-grown cells of R. eutropha H16
revealed the overproduction of four proteins that are
involved in the detoxification of ROS. Catalase encoded by
the gene katG (H16_A2777) was 13.6-fold up-regulated in
glycerol-grown cells. The organic hydroperoxide resistance
protein Ohr (H16_B0157) [32], was up-regulated ninefold
relative to SN-grown cells. Two other proteins, a peroxi-
redoxin (H16_A0306) and a glutathione peroxidase
(H16_A3102), were only detected in cells growing on
5138 E. Schwartz et al. Proteomics 2009, 9, 5132–5142
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
glycerol. A peroxiredoxin (H16_A1460) and a superoxide
dismutase, the product of gene sodA (H16_A0610), were
present, but not significantly up-regulated. The function of
the above-named proteins is to protect the cell against ROS.
ROS arise in organisms during normal aerobic metabolism
e.g. via the reaction of O2 with flavoproteins [33]. Certain
conditions lead to increased production of superoxide and
thus increase the incidence of damage to DNA and other cell
components. This state of oxidative stress provokes the
induction of protective proteins, which in E. coli constitute
the soxRS and oxyR regulons [34]. However, some of these
proteins are also produced under other stress conditions
such as nutrient limitation. Furthermore, the response to
oxidative stress is not identical in all bacteria [35, 36]. Thus,
the diagnosis of oxidative stress based on the proteomic data
is not always clear-cut. Two lines of evidence support the
assumption that R. eutropha H16 is subject to exacerbated
oxidative stress during growth on glycerol. First, the catalase
that is up-regulated in the glycerol-grown cells of R. eutrophaH16 is a heme-containing, bifunctional catalase/peroxidase
similar to the hydroperoxidase I of E. coli. In contrast to the
hydroperoxidase II, the hydroperoxidase I is not usually
present in aerobically growing cells, but is induced when
cells are challenged with H2O2 [37]. Another hallmark of the
response to oxidative stress is a shift in the relative levels of
aconitase isoenzymes [38, 39]. Like other bacteria, R. eutro-pha H16 encodes two distinct aconitases: an oxidation-
resistant type (AcnA; H16_A2638) and an oxidation-labile
form (AcnB; H16_B0568). Although the former is present in
the same amounts in glycerol- and SN-grown cells of
R. eutropha H16, the expression of the oxidation-labile form
is curtailed tenfold during growth on glycerol.
What is the cause of elevated levels of ROS in the
glycerol-grown cells? The hydrogenases are formed during
growth on glycerol as well as during growth on H2.
However, the levels of SH under the former conditions are
significantly higher (ca. 4% of total cell protein; [23]) than
during lithoautotrophic growth. Furthermore, the glycerol
cultures are exposed to twice as much O2 as the lithoauto-
trophic cultures. It has been known for some time that
the SH produces superoxide in vitro in the presence
of O2 and electron donors, leading to its own destruction [40,
41]. The copious amounts of SH in the glycerol-grown cells
may produce enough ROS to elicit an oxidative stress
response.
3.5 Regulatory proteins
In total, 12 proteins with functions in signal transduction
and regulation were identified in the proteome of R. eutro-pha cells (Table 4). Three of these proteins, Rho, NusA and
NusG, are transcription termination factors that mediate
termination at both constitutive and variable termination
sites. TypA belongs to the family of TypA/BipA proteins that
are ribosome-binding GTPases controlling both general
housekeeping processes, such as cellular response to stress
[42], as well as strain-specific behavior, such as virulence in
Salmonella typhimurium [43] and symbiotic interaction with
plants in Sinorhizobium meliloti [44]. The proteins GreA1
and DksA1 act in concert with ppGpp and NTPs to shift the
equilibrium between free RNA polymerase and closed RNA
polymerase–promoter complex, thereby regulating the
activity of rRNA promoters, many tRNA promoters and
some mRNA promoters [45]. TypA and GreA1 are down-
regulated tenfold during growth on glycerol. One of the two
isoforms of the nitrogen regulatory protein PII encoded in
the genome was identified in the 2-D gels. This protein was
up-regulated in both the H2-CO2- and the glycerol-grown
cells.
Two response regulators and a serine/threonine protein
kinase-designated PrkA (H16_B0700) were identified in the
protein profiles (Table 4). PrkA is up-regulated twofold and
threefold in H2-CO2-grown cells and glycerol-grown cells,
respectively (ratio H2-CO2/SN: 2.0; ratio glycerol/SN: 3.0).
PrkA belongs to the family of regulators that control cellular
Table 4. Proteins with functions in transcription and regulation identified in 2-D gels
Gene Locus tag Product Ratio H2-CO2/SNa) Ratio GNb)/SN
nusA H16_A2307 Transcription pausing factor L 1.2 0.2H16_A1372 H16_A1372 Response regulator, NarL-family 4.5 3.8rho H16_A2395 Transcription termination factor Rho 1.8 0.1prkA H16_B0700 Serine protein kinase 2.0 3.0H16_A0750 H16_A0750 Nitrogen regulatory protein PII 9.1 3.1hoxC PHG021 Regulatory [NiFe] hydrogenase, large subunit 4.0 51.0H16_A1463 H16_A1463 Response regulator, OmpR-family 7.1 10.6nusG H16_A3502 Transcription antitermination protein 1.1 0.3rpoA H16_A3458 DNA-directed RNA polymerase, a-subunit 0.3 0.3typA H16_A2294 GTP-binding elongation factor family protein 0.3 0.1greA1 H16_A2451 Transcription elongation factor GreA 0.0 0.1dksA1 H16_A0194 DnaK suppressor protein 1.3 0.9
a) Minimal medium containing succinate.b) Minimal medium containing glycerol.
Proteomics 2009, 9, 5132–5142 5139
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
processes by phosphorylating a target protein or proteins at
serine, threonine or tyrosine residues (serine/threonine
protein kinases). Originally discovered and characterized in
eukaryotes, recent years have seen numerous reports of
these proteins in bacteria [46–53].
With the aim of gaining new insights into regulatory
processes involved in reprogramming the metabolism of
R. eutropha H16 for the transition between lithoautotrophic
and organoheterotrophic growth, we generated a knockout
mutant for prkA. The resulting strain, designated HFJ74,
carried an unmarked in-frame deletion in the prkA gene.
The mutant was not significantly affected in lithoauto-
trophic growth on H2-CO2 or organoheterotrophic growth
on SN (data not shown). The role of the prkA gene product
remains unclear.
4 Concluding remarks
The protein inventories obtained in this study provide a
wide-angle albeit static picture of the metabolism of
R. eutropha H16 for three different growth modes. These
results offer a framework for the reassessment of the data of
earlier enzymological studies in light of the recently
published genomic sequences [3, 4]. One interesting finding
is the observation that lithoautotrophically grown cells
contain PEP carboxykinase but no pyruvate carboxylase.
This feature is not correlated with facultative lithoauto-
trophy, since in the facultative lithotroph Paracoccus versutusA2 the anaplerotic formation of oxaloacetate is catalyzed by
pyruvate carboxylase [54].
The previously published enzymological studies revealed
that lithoautotrophically growing cells of R. eutropha H16
contain the enzymatic activities corresponding to a complete
TCA cycle [29, 30]. In accordance with these findings, this
study showed that most of the proteins of TCA cycle
enzymes are detectable in the lithoautotrophically grown
cells. A hallmark of obligate lithoautotrophs is the incom-
plete or interrupted TCA cycle, which becomes a bifurcated
pathway with a biosynthetic but not a bioenergetic role.
These organisms usually lack a functional a-ketoglutarate
dehydrogenase [14, 15, 55]. This type of adaptation has been
documented for Thiobacillus denitrificans, Acidithiobacillusferrooxidans, Hydrogenobacter thermophilus TK-6, and Nitro-somonas europaea [56–59]. Facultative lithoautotrophs, on the
other hand, are able to stop the TCA cycle during the phases
of lithoautotrophic growth. This is achieved by repressing
the expression of a-ketoglutarate dehydrogenase. A repre-
sentative of this group of organisms is P. versutus A2 [54, 56].
Unlike P. versutus A2, R. eutropha H16 maintains a func-
tional TCA cycle during lithoautotrophic growth. Thus,
inactivation of the TCA cycle is not a prerequisite for
lithoautotrophic growth.
The finding that R. eutropha H16 retains a functional
TCA cycle during lithoautotrophic growth is of major
importance in the context of the mixotrophic capabilities of
this organism. Mixotrophy, i.e. the capacity to utilize inor-
ganic and organic substrates concomitantly, has been
reported for R. eutropha H16 and for some other aerobic H2
oxidizers including Aquaspirillum autotrophicum [60], for the
carboxidotroph Hydrogenophaga pseudoflava [61] and for
the facultative sulfur oxidizer P. versutus A2 [54, 62]. For the
latter three organisms there is convincing evidence that
mixotrophic growth does not involve CO2 fixation. In a
series of elegant experiments based on mutants of
R. eutropha H16 and carbon-limited chemostat cultures,
K.arst and Friedrich showed that, unlike the mixotrophic
bacteria named above, R. eutropha H16 does in fact rely on
CO2 fixation for mixotrophic growth [63]. For a chemostat
culture of R. eutropha H16 growing in the presence of H2
and limited for succinate as the sole carbon source, the
authors observed an increase in yield of 135%. Since there
was no exogenous CO2 available to the culture, but the yield
increase was largely dependent on the activity of the Calvin-
cycle enzymes, the authors concluded that the carbon
substrate was respired and the resulting CO2 then fixed and
assimilated to form cell material. A small but significant
increase in growth yield (14%) was observed when a mutant
defective for CO2 fixation was cultivated on succinate and
H2, indicating that, to a limited extent, both substrates were
used concomitantly as energy sources.
Taken together, the different lines of evidence show that
R. eutropha H16 maintains a basically heterotrophic core
metabolism during lithoautotrophic growth on H2 and CO2.
This is compatible with the notion that R. eutropha H16 is a
heterotroph, which has relatively recently acquired the
megaplasmid-encoded capacity for lithoautotrophic growth.
The authors thank Susanne Paprotny und Enrico Klotz forexpert technical assistance and Decodon GmbH (Greifswald,Germany) for providing the Delta2D software. E. S. is grateful toBotho Bowien for enlightening discussions. This work wassupported by grants from the Bundesministerium f .ur Bildungund Forschung in the framework of the program ‘‘GenomicResearch on Bacteria (GenoMik)’’ to B. F and M. H. and fromthe Fonds der chemischen Industrie to B. F.
The authors have declared no conflict of interest.
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