Tanja Hanke, Katharina Nöh, Stephan Noack, Tino Polen,
Stephanie Bringer, Hermann Sahm, Wolfgang Wiechert and
Michael Bott
Appl. Environ. Microbiol. 2013, 79(7):2336. DOI:
10.1128/AEM.03414-12.
Published Ahead of Print 1 February 2013.
Updated information and services can be found at:
http://aem.asm.org/content/79/7/2336
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Combined Fluxomics and Transcriptomics
Analysis of Glucose Catabolism via a
Partially Cyclic Pentose Phosphate
Pathway in Gluconobacter oxydans 621H
Combined Fluxomics and Transcriptomics Analysis of Glucose
Catabolism via a Partially Cyclic Pentose Phosphate Pathway in
Gluconobacter oxydans 621H
Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, Jülich, Germany
In this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes in Gluconobacter oxydans 621H with
glucose were studied by 13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays.
For 13C-MFA, cells were cultivated with specifically 13C-labeled glucose, and intracellular metabolites were analyzed for their
labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized
periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional
gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30%
was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the
periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. Since G. oxydans lacks
phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the
Entner-Doudoroff pathway (EDP). 13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in
both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II.
T
he strictly aerobic acetic acid bacterium Gluconobacter oxydans
is industrially applied for the production of vitamin C, L-sorbose, 2-ketogulonic acid, dihydroxyacetone, and 6-amino-L-sorbose. However, G. oxydans grows only to low cell densities, which
has been explained by an inefficient respiratory chain (1). Membrane-bound dehydrogenases enable G. oxydans to oxidize sugars
and sugar alcohols in two or more steps in the periplasm (1, 2)
(Fig. 1). Intermediates and products of these reactions accumulate
in the medium. In parallel, part of the substrates or its oxidation
products is taken up into the cytoplasm. In the case of glucose,
uptake occurs by a yet-unknown transport system, and the oxidation product gluconate is imported by gluconate permease
(GOX2188) (3).
As G. oxydans lacks phosphofructokinase, the Embden-Meyerhof-Parnas pathway (EMP) is interrupted. Intracellular glucose
is either oxidized to gluconate and 5-ketogluconate by NAD(P)linked dehydrogenases or glucose and gluconate are phosphorylated by glucose and gluconate kinases, respectively, and further
catabolized via the pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP) (4–6). In the absence of phosphofructokinase, the PPP is expected to operate partly cyclic, as fructose 6-phosphate formed by transaldolase or transketolase is
isomerized to glucose 6-phosphate, which enters the oxidative
PPP again (7, 8). Formally, this conversion can be described as
follows: glucose ⫹ ADP ⫹ Pi ⫹ NAD⫹ ⫹ 6 NADP⫹ ¡ ATP ⫹
NADH ⫹ 6 NADPH ⫹ 3 CO2 ⫹ pyruvate⫺ ⫹ 8 H⫹. Due to
absence of genes coding for succinate dehydrogenase, the tricarboxylic acid (TCA) cycle is also incomplete and fulfills exclusively
biosynthetic functions (3, 9, 10).
Growth of G. oxydans on glucose proceeds in two different
metabolic phases. In phase I, glucose is rapidly oxidized to gluco-
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nate by the membrane-bound glucose dehydrogenase (GdhM). In
growth phase II, gluconate is further oxidized to ketogluconates.
Depending on the pH of the culture medium, 5-ketogluconate
and 2-ketogluconate are formed in the periplasm by the membrane-bound major polyol dehydrogenase and gluconate 2-dehydrogenase, respectively (11). At pH 6, the pH value of the culture
medium applied in the present work, formation of 2-ketogluconate is predominant in the periplasm (1).
In the present study, 13C-based metabolic flux analysis (13CMFA) was applied in order to elucidate the relative contributions
of the PPP and the EDP to cytoplasmic glucose catabolism and to
test the proposed cyclic flux of carbon through the oxidative PPP.
The most common variant of 13C-MFA uses gas chromatographymass spectrometry (GC-MS) to derive labeling information available in protein-bound amino acids. In this case, the utilization of
a defined minimal medium is imperative to avoid erroneous interpretation of the mass isotopomer fractions (12). As a suitable
minimal medium is not yet available for G. oxydans and in order to
minimize influences of the medium, highly sensitive liquid chromatography-mass spectrometry (LC-MS) was applied in this
Received 6 November 2012 Accepted 14 January 2013
Published ahead of print 1 February 2013
Address correspondence to Michael Bott, [email protected], or Katharina Nöh,
[email protected].
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AEM.03414-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.03414-12
Applied and Environmental Microbiology
p. 2336 –2348
April 2013 Volume 79 Number 7
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Tanja Hanke, Katharina Nöh, Stephan Noack, Tino Polen, Stephanie Bringer, Hermann Sahm, Wolfgang Wiechert, Michael Bott
Fluxomics and Transcriptomics of G. oxydans 621H
yeast extract in a bioreactor at a constant pH of 6 and a constant dissolved
oxygen concentration of 15%. In panel A, the concentration profiles in g liter⫺1 of glucose (, GLC, black solid line, initial concentration of 80 g liter⫺1),
gluconate (}, GLCN, blue dashed line), 2-ketogluconate (Œ, 2KGA, red dotted
line), 5-ketogluconate (, 5KGA, magenta dot-dashed line), and biomass dry
weight (scaled by a factor of 10, , DW, green solid line) are shown. The two
vertical dashed lines indicate the times (I, 8.00 h; II, 14.25 h) at which samples
were taken for 13C-based metabolic flux analysis. In panel B, the oxygen consumption rates of the two 13C-labeled cultures (䊐, }) and the nonlabeled
culture () are shown. In panel C, the carbon dioxide production rates of the
13
C-labeled culture (䊐) and of the nonlabeled culture (}) are indicated.
study for the detection of intracellular isotopomer concentrations
of almost all intermediates of central metabolism down to the
nanomolar range (13). Utilization of these primary metabolites
for 13C-MFA rather than protein-bound amino acids prevents an
increased uncertainty in the metabolic network, e.g., due to incomplete metabolic reconstruction, yet unknown carbon-atom
transitions, or the need for considering putative carbon source
uptake routes.
Complementary to 13C-MFA, we used genome-wide DNA microarrays to study the changes of global gene expression between
growth phases I and II. Despite the long-time use in biotechnology, the regulation of carbon and energy metabolism in G. oxydans is largely unknown. Based on the genome sequence of strain
621H (DSM2343) (3), we recently applied DNA microarrays to
analyze the influence of pH and oxygen limitation on global gene
expression and to study mutants defective in the PPP or the EDP
(14, 15).
MATERIALS AND METHODS
Chemicals and enzymes. [1-13C]glucose and [U-13C]glucose were obtained from Deutero GmbH (Kastellaun, Germany). Other chemicals and
auxiliary enzymes (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from yeast) for enzyme activity assays were purchased from Sigma-Aldrich (Taufkirchen, Germany) and Merck (Darmstadt, Germany).
Bacterial strains, culture conditions, and bioreactor system. Gluconobacter oxydans DSM 2343 (ATCC 621H) was obtained from the Deutsche
Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). Precultures of the strain were cultivated on complex medium
containing 5 g liter⫺1 yeast extract, 2.5 g liter⫺1 MgSO4 · 7 H2O, 1 g liter⫺1
(NH4)2SO4, 1 g liter⫺1 KH2PO4, 0.5 g liter⫺1 glycerol, and 8% (wt/vol)
April 2013 Volume 79 Number 7
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FIG 1 Biphasic growth of G. oxydans with 80 g liter⫺1 glucose and 5 g liter⫺1
sorbitol. The initial pH value of the medium was 6.0. G. oxydans possesses
a natural resistance toward cefoxitin; as a precaution to prevent bacterial
contaminations, cefoxitin was added to the medium at a concentration of
50 ␮g ml⫺1. Precultures were grown in baffled shaking flasks at 30°C and
140 rpm. For DNA microarray analysis and enzyme activity measurements, cells were cultivated in 250 ml of the same medium containing 8%
glucose instead of sorbitol in a bioreactor system (DASGIP, Jülich, Germany) composed of four 400-ml vessels, each equipped with electrodes
for measuring the dissolved oxygen concentration (DO) and the pH value.
The system allows to constantly control these two parameters. The pH was
kept at pH 6.0 by automatic titration of 2 M NaOH. The oxygen availability was kept constant at 15% DO by mixing air, O2, and N2. Calibration
was performed by gassing with air (100% DO) and N2 (0% DO). The
agitation speed was kept constant at 900 rpm. The carbon dioxide concentration in the exhaust air was measured continuously by an infrared
spectrometer, and the oxygen concentration was measured with a zirconium dioxide sensor.
Control and recording of all data were carried out by the Fedbatch Pro
software (DASGIP, Jülich, Germany). For metabolic flux analysis, cells
were grown in a 200-ml volume of the same medium containing 8%
glucose of the following optimized composition: 4.0% naturally labeled
glucose, 7.7% [1-13C]glucose, and 88.3% [U-13C]glucose. A reference
culture with 100% naturally labeled glucose was used for comparison.
HPLC analysis and glucose determination. Gluconate, 5-ketogluconate (5-KGA), and 2-ketogluconate (2-KGA) were analyzed by highperformance liquid chromatography (HPLC). One milliliter of culture
was centrifuged for 5 min at 13,000 ⫻ g, and the supernatant was filtered
through a 0.2-␮m-pore-size filter (Millipore, MA) prior to HPLC analysis. The substances were separated using a Shodex DE 613 column (150
mm long, 6.0 mm internal diameter; Phenomenex, Aschaffenburg, Germany) using 2 mM HClO4 as the eluant at a flow rate of 0.5 ml min⫺1 and
detected by a UV spectrophotometer at 210 nm. Glucose coeluted with
gluconate, but at this wavelength, glucose does not absorb, and therefore
gluconate analysis was not distorted. Glucose concentrations were determined enzymatically with glucose dehydrogenase by application of a kit
(DiaSys Diagnostic Systems GmbH, Holzheim, Germany) according to
the instructions of the manufacturer.
Determination of extracellular rates. A bioreactor model of the batch
cultivation process was set up to calculate rates for growth on glucose and
gluconate (␮GLC and ␮GLCN, respectively) and consumption and formation rates of glucose, gluconate, 2- and 5-ketogluconates, as well as carbon
dioxide from the glucose enzymatic test results and HPLC data (see Fig.
S1.1, Fig. S1.2, Table S1.1, and Table S1.2 in the supplemental material).
This process model contains only two compartments, divided by an outer
membrane, i.e., the extracellular compartment [ex] and the remainder,
periplasm plus cytoplasm ([p] ⫹ [c]) (see Fig. S1.1 in the supplemental
material). O2 consumption rates and CO2 production rates of growing
cells were measured by the DASGIP bioreactor system. Because 13C-labeled carbon dioxide was not quantitatively detectable by the employed
infrared spectroscopy method, carbon dioxide production rates were obtained from the reference culture grown with naturally labeled glucose.
Rate estimations were then used to calculate specific extracellular rates,
which were, in turn, used for 13C-MFA.
Sampling and sample processing for LC-MS analysis. For LC-MS
analysis, 50 ml culture was harvested in the first growth phase (after 8 h;
sample point I) and 15 ml culture was harvested in the second growth
phase (after 14.25 h; sample point II) and mixed immediately with 150 ml
or 45 ml 60% methanol at ⫺80°C in order to stop metabolism. The mixtures were centrifuged for 5 min at 10,000 ⫻ g and ⫺20°C, and each cell
pellet was resuspended in 1 ml pure methanol (⫺70°C) and 1 ml TE buffer
(pH 7.0). After the suspension was vortexed, 2 ml chloroform (⫺20°C)
was added. The suspension was shaken at ⫺20°C for 2 h and then centrifuged for 10 min at 10,000 ⫻ g at ⫺20°C. The upper methanol phase was
filtrated through a 0.2-␮m-pore-size filter (Millipore, MA) and frozen at
⫺80°C for subsequent LC-MS analysis.
Hanke et al.
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enzyme assay according to reference 22. Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured at
30°C by a standard method (23). Again, different dilutions of cell extract
were applied. Since 6-phosphogluconate dehydrogenase is an NAD⫹-preferring enzyme in G. oxydans, 0.1 mM NAD⫹ was used instead of NADP⫹
for activity determination (24, 25). The protein concentration of the cell
extracts was determined by the method of Bradford (26) by using bovine
serum albumin as the standard.
DNA microarray analysis. RNA preparation, cDNA labeling, and
DNA microarray analysis were performed as described recently by Hanke
et al. (14). The samples for DNA microarray analysis were taken after 8 h
and 14.25 h, and three biological replicates were performed.
Microarray data accession number. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are
accessible through GEO Series accession number GSE42223.
RESULTS
Substrate conversion in the periplasm, oxygen consumption,
and carbon dioxide production during growth of G. oxydans
621H with glucose. In Fig. 1A, growth of G. oxydans in a medium
containing 80 g liter⫺1 glucose, 0.5 g liter⫺1 glycerol, and 5 g liter⫺1 yeast extract is shown. Two independent cultures containing
13
C-labeled glucose (for details see Materials and Methods)
showed growth behavior and substrate oxidation rates identical to
those of the reference culture containing naturally labeled glucose
(Fig. 1A). Samples for carbon flux analysis were taken after 8.0 h
(sample point I) and after 14.25 h (sample point II). After 8 h, the
cultures had an optical density at 600 nm (OD600) of ⬃3.6 and
used glucose as the major substrate. After 14.25 h, the cultures had
an OD600 of ⬃7.6 and used gluconate as the major substrate. With
the exception of CO2 production, which could be quantified only
for the culture containing naturally labeled glucose, triplicate data
sets were available for extracellular rates, whereas 13C labeling data
were obtained in duplicate. Because isotopic substitution will affect the distribution of vibrational and rotational energy states of a
molecule, each distinct isotopomer of CO2 has its own rotationalvibrational infrared spectrum (27). Therefore, 13C-labeled carbon
dioxide was not quantitatively detectable by the employed infrared spectroscopy method, and a reference culture with unlabeled
glucose was necessary to obtain correct carbon dioxide production rates, which could be applied for flux analysis. As shown by
the measurements’ standard deviations, parallel cultivation under
controlled conditions allowed for collection of comprehensive
and reproducible data (biological replicates) suitable for modelbased 13C-MFA. The carbon balance of the averaged cultivations
amounted to 101% (see Table S4.1 in the supplemental material).
Extracellular rates of substrate consumption and product formation were estimated consistently with a batch bioreactor process model (see Fig. S1.2A in the supplemental material). In the
first growth phase, a ␮GLC of 0.27 h⫺1 was measured until an
OD600 of 6.6 was reached after 10 h, which corresponds to 80% of
the maximal OD observed at the end of the cultivation.
At the end of growth phase I after 10 h, the extracellular concentrations of glucose, gluconate, and 2-ketogluconate were 49
mM, 264 mM, and 63 mM, respectively. 2-Ketogluconate was
formed in the periplasm by the membrane-bound gluconate 2-dehydrogenase (Gad2) (28) and accumulated in the medium. Gluconate 2-dehydrogenase has its optimum pH at 6 (28). Besides
2-ketogluconate, 5-ketogluconate was also detected (30 mM),
which presumably was formed intracellularly by gluconate 5-dehydrogenase and then exported into the medium. The alternative
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LC-MS analysis of intracellular metabolites. Cell extraction samples
were analyzed with an Agilent 1100 HPLC system (Agilent Technologies,
Waldbronn, Germany) coupled with an API4000 mass spectrometer (Applied Biosystems, Concord, Canada) equipped with a TurboIon spray
source. Detailed information on separation methods have been reported
previously (13, 17). The LC-MS data were analyzed as described previously (18). Briefly, the mass isotopomer fractions of the intermediates
1,3-bisphosphoglycerate, 2-phosphoglycerate, 2-oxoglutarate, 6-phosphogluconate, aconitate, citrate-isocitrate, dihydroxyacetone phosphate,
erythrose 4-phosphate, fructose 1,6-diphosphate, fructose 6-phosphate,
fumarate, gluconolactone, glucose 6-phosphate, glyceraldehyde 3-phosphate, malate, phosphoenolpyruvate, pyruvate, ribose 5-phosphate, ribulose 5-phosphate/xylulose 5-phosphate, sedoheptulose 7-phosphate,
succinate, and the free amino acids alanine, arginine, aspartate, glutamate,
histidine, leucine, lysine, methionine, phenylalanine, proline, tyrosine,
and valine were determined from the respective mass spectra (see Table
S2.1, Table S2.2, and Fig. S2 in the supplemental material).
13
C metabolic flux analysis. For 13C labeling experiments, cells were
cultivated with the 13C-labeled glucose mixture described above. Metabolic stationarity in the cells was approximately maintained. Samples
taken after 8 h and 14.25 h were analyzed by LC-MS to determine the mass
isotopomer patterns of the intracellular intermediates listed above. Accompanied by the estimations of consumption and formation rates, 13CMFA enabled a detailed quantification of intracellular in vivo carbon
fluxes from intracellular metabolites’ labeling patterns. 13C-MFA is a
model-based approach, and for more details the reader is referred to recent review papers (19, 20). Here, we differentiated between three compartments: the medium external to the cells (termed extracellular, [ex]),
the cytoplasm ([c]), and periplasm ([p]).
Based on the genome information for G. oxydans 621H, a metabolic
network model of central metabolism was formulated (see Fig. S3 and
Table S3.1 in the supplemental material). This model included reactions
of the EMP, the PPP, the EDP, and the TCA cycle, as well as all membranebound and cytoplasmic dehydrogenase reactions involved in glucose catabolism that are characteristic for G. oxydans (Fig. 1). All together, the
model covers 57 reactions, of which 18 are reversible. Additional uptake
reactions for naturally labeled fumarate (FUM), acetyl coenzyme A
(acetyl-CoA), and glutamate (GLUT) were added to the metabolic network, allowing the utilization of components of the yeast extract in central
metabolism. Aiming at a more detailed quantification of the metabolic
conversion rates, a focused network without the reactions of the TCA
cycle was deduced. The focused network contained 40 reactions, of which
15 are reversible (see Fig. 3 and Table S3.1 in the supplemental material).
In total, up to 34 model parameters (degrees of freedom) had to be estimated using 80 labeling measurements (mass isotopomers of intracellular
metabolites detected by LC-MS; see Fig. S2 in the supplemental material)
and five carbon exchange flux values (glucose uptake, gluconate uptake,
ketogluconate excretion, and carbon dioxide production rate) estimated
from the process model (see Table S1.1 in the supplemental material). The
metabolite pools of phosphoglycerates (1,3-bisphosphoglycerate [1,3PG],
2-phosphoglycerate [2PG], and 3-phosphoglycerate [3PG]) were not separable by LC-MS and, thus, lumped into a single phosphoglycerate pool
(PGP). The software toolbox 13CFLUX2 (http://www.13cflux.net) (21)
was used for all modeling and evaluation steps (20).
Preparation of cell extracts and enzyme assays. For in vitro determinations of enzyme activities, culture samples (50 ml) were taken after 8 h
and 14.25 h, i.e., at the same time points used to take samples for LC-MS
analysis, and centrifuged at 8,000 ⫻ g for 10 min. After being washed once
in 0.9% NaCl, cells were resuspended in 60 mM Tris-HCl, 13 mM MgCl2,
1 mM dithiothreitol (DTT), pH 7.5 (10 ml g⫺1 cell wet weight). Cells were
disrupted by sonication (3 min using cycle 0.5 and amplitude 70; UP 200s
sonifier; Hielscher, Stuttgart, Germany) in an ice bath. After centrifugation at 10,000 ⫻ g for 2 min, the supernatant was used as the cell extract.
The determination of glucose kinase and gluconate kinase activities
was performed with different dilutions of cell extract at 30°C by a coupled
Fluxomics and Transcriptomics of G. oxydans 621H
April 2013 Volume 79 Number 7
unstable and can decompose spontaneously, or by conversion of
␣-ketoglutarate to succinate semialdehyde by ␣-ketoglutarate decarboxylase (GOX0882) and subsequent oxidation of succinate
semialdehyde to succinate by succinate semialdehyde dehydrogenase (GOX1122, GOX0499). To summarize, the isotope labeling
data indicated that the TCA cycle intermediates were predominantly derived from the catabolism of amino acids in the yeast
extract. All measurable amino acids were found to be purely naturally labeled in both phases (Fig. 2), which can be expected during cultivation in a medium containing yeast extract.
Although we incorporated into our model (Fig. 2) reactions in
which TCA cycle intermediates were formed from naturally labeled yeast extract components, the low 13C labeling of citrateisocitrate, aconitate, 2-oxoglutarate, and succinate could not be
reasonably explained. This discrepancy might be accounted for,
e.g., by assuming that part of the pyruvate is converted acetate or
by assuming that labeling was not yet equilibrated in TCA intermediates at the sampling time point (8.0 h after the switch from
naturally labeled glucose to 13C-labeled glucose mixture). The latter assumption is supported by the fact that most TCA cycle intermediates showed a higher labeling enrichment after 14.25 h than
after 8 h. Consequently, the TCA cycle intermediates were excluded from metabolic flux analysis. Instead, a reduced model was
used where effluxes from PEP and pyruvate represent fluxes to
TCA, biomass, and by-products like acetate (see Figure 3). STD,
standard deviation.
Branching of intracellular carbon fluxes between nonphosphorylated and phosphorylated compounds. For sample points I
and II, intracellular fluxes from glucose to pyruvate were estimated using the extracellular flux rates determined with the bioreactor model (see Fig. S1.2 in the supplemental material) and the
13
C labeling patterns of EMP and PPP intermediates of sample
points I and II, respectively (Fig. 1A; Fig. 2). Repeated flux estimation with randomly chosen initial values showed a reproducibly
well agreement of measurements and model predictions for all
reactions of the EMP, PPP, and EDP.
The small amount of glucose taken up by the cells (9.8%) at 8.0
h (sample point I) was primarily converted to gluconate (91.3%)
by the cytoplasmic glucose dehydrogenase (GdhS) and gluconolactonase (Pgl) (Fig. 3A). Only 8.7% of the glucose taken up was
phosphorylated to glucose 6-phosphate by glucose kinase (Glk). A
total of 69.9% of the intracellular gluconate was converted to 5-ketogluconate by the NADP-dependent gluconate 5-dehydrogenase
(31) and exported out of the cell. A smaller fraction was phosphorylated by gluconokinase (GntK) to 6-phosphogluconate, an intermediate common to both the PPP and the EDP. Hence, the sum of
fluxes through Glk and GntK is the amount of carbon captured by
the cells, i.e., only 5.3% of the total glucose metabolized.
Measured in vitro enzyme activities revealed a Glk activity of 86
nmol min⫺1 mgprotein⫺1, agreeing well with the value of 60 nmol
min⫺1 mgprotein⫺1 reported by Pronk et al. (32). The same authors
reported activities of 4,000 and 150 nmol min⫺1 mgprotein⫺1 for
GdhM and NADP-dependent GdhS, respectively, indicating that
the cytoplasmic capacity for glucose dehydrogenation is only 3.8%
of the membrane-bound capacity. Thus, in vitro determinations
of enzyme activities are in agreement with our model prediction of
the in vivo situation of carbon flux at sample point I.
At sample point II, glucose uptake almost ceased due to the
depletion of glucose from the culture medium. Instead, at 14.25 h,
gluconate was consumed with 16.9% of the glucose consumption
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possibility that 5-ketogluconate was formed in the periplasm by
the membrane-bound major polyol dehydrogenase (GOX0854
and GOX0855) is unlikely, as this enzyme has its optimum activity
for this reaction at acidic pH values of 3.5 to 4.0 (11, 29, 30). The
cytoplasmic formation of 5-ketogluconate is in accordance with
the 13C-MFA-based prediction.
In the second growth phase, which was characterized by a very
low growth rate (␮GLC ⫽ 0.002 h⫺1), gluconate was oxidized to
2-ketogluconate. This reaction proceeded at a lower velocity than
the oxidation of glucose to gluconate (Fig. 1A). The specific activity of gluconate 2-dehydrogenase indeed is 70 to 80% lower than
that of the membrane-bound glucose dehydrogenase, as determined with intact cells using a Clark oxygen electrode (results not
shown). As discussed above, the oxidation to 5-ketogluconate by the
major polyol dehydrogenase was prevented by keeping the pH at 6. In
both the first and second oxidation phases, 337 mM 2-ketogluconate
was formed and 432 mM O2 was consumed. NADH generated in the
cytoplasm probably reduced the residual 95 mM O2.
Hence, the main energy supply of the cells originated from
substrate oxidation in the periplasm. Biomass production in the
second growth phase was only one-fourth (0.38 gcdw liter⫺1) of that
formed in the first growth phase (1.5 gcdw liter⫺1), although the concentration of accumulated gluconate at the beginning of the second
growth phase was more than two-thirds of the initial 80 g liter⫺1
glucose. This indicates that energy generation by gluconate oxidation
to ketogluconate is much lower than by oxidation of glucose to gluconate. Growth stopped before gluconate oxidation was completed.
Parallel to biphasic growth, the oxygen consumption rate also
showed two maxima (Fig. 1B). In growth phase I, the cells rapidly
consumed oxygen, whereas in growth phase II, the oxygen consumption rate was much slower. This correlates with the fast oxidation of glucose to gluconate and the slow oxidation of gluconate
to 2-ketogluconate (Fig. 1B). The carbon dioxide production rate
was also biphasic (Fig. 1C), but its course was contrary to that of
the oxygen consumption rate, as the rate was higher in growth
phase II than in growth phase I, for which a constant rate of 6.5
mmol carbon dioxide h⫺1 gcdw⫺1 was estimated. The total CO2
produced in phase II (213 mM) was 8-fold higher than that in
phase I (27 mM) (Fig. 1B). This increase in carbon dioxide production was an outcome of an activated, cyclic PPP as shown by
13
C-MFA described below.
13
C labeling patterns of intracellular metabolites. As described before, cells were fed with specifically labeled [13C]glucose, and mass isotopomer distributions of intracellular metabolites were quantified at the two sample points using LC-MS (cf.
Tables S2.1 and S2.2 in the supplemental material). The MS analysis showed that labeling information was predominantly distributed in intermediates of the EMP and the PPP (Fig. 2). Notably,
for these metabolites, no significant changes in 13C labeling patterns between sample points I and II were observed. In contrast to
EMP and PPP intermediates, almost no 13C enrichment from labeled glucose was measured for TCA cycle intermediates. Considerable amounts of unlabeled fumarate and malate were detected.
This indicates formation of these metabolites from amino acids
present in the yeast extract. Despite the fact that G. oxydans 621H
lacks genes for succinyl-CoA synthetase and succinate dehydrogenase, mainly unlabeled succinate was detected by the LC-MS analysis. It might be formed from glutamate or glutamine present in
the yeast extract by conversion to ␣-ketoglutarate, which then is
either oxidatively decarboxylated to succinyl-CoA, which is quite
Hanke et al.
rate at 8.0 h, whereas the glucose consumption rate dropped to
almost zero (Fig. 3B). Contrary to the situation at sample point I,
almost all intracellular gluconate was captured by phosphorylation to 6-phosphogluconate (98.8%).
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Intracellular carbon fluxes of phosphorylated compounds.
High fluxes in the oxidative part of the PPP, mediated by glucose
6-phosphate dehydrogenase (Zwf), 6-phosphogluconate dehydrogenase (Gnd), transaldolase (Tal), and transketolase (Tkt) in
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FIG 2 Mass isotopomer labeling measurements of intracellular metabolites (red bars, after 8.0 h in growth phase I; green bars, after 14.25 h in growth phase II)
arranged by pathways: fractional abundance over mass in context of the metabolic network. Error bars indicate standard deviations derived from two technical
replicates (phase I) and two independent biological replicates with two technical replicates each (phase II). For abbreviations of flux and metabolite names used
in the model, see Table S3.2 and S3.3 in the supplemental material. A switch from predominantly fully labeled mass isotopomers in the EMP and the PPP
intermediates to almost naturally labeled TCA cycle intermediates is evident. Analysis of unphosphorylated glucose in the cytoplasm was rendered unfeasible by
the large excess of extracellular glucose. Therefore, the labeling pattern of intracellular glucose is not included in the analysis. STD, standard deviation.
Downloaded from http://aem.asm.org/ on March 24, 2013 by FORSCHUNGSZENTRUM JULICH GMBH
FIG 3 In vivo flux distribution of G. oxydans during growth with glucose at 8.0 h (sample point I, panel A) and 14.25 h (sample point II, panel B) after switch of
naturally labeled to specifically 13C-labeled glucose (GLC). The network diagram shows the metabolic pathways in the periplasm and the cytoplasm that were in
the focus of the 13C-MFA study. Metabolites are represented by rectangles (white, extracellular; pink, periplasmic; orange, cytoplasmic). Flux values (yellow
hexagons) at sampling times are related to 100% glucose (sample point I, panel A) or gluconate (sample point II, panel B) uptake. The width of each flux edge
is scaled proportional to its underlying value; flux arrows are pointing in net flux direction. For abbreviations of flux and metabolite names used in the model,
see Table S3.2 and S3.3 in the supplemental material. Absolute net flux values are given in the supplemental material (see Table S3.4).
April 2013 Volume 79 Number 7
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Hanke et al.
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ratory chain showed increased mRNA levels at sample point II,
such as those for a PQQ-containing myo-inositol dehydrogenase
(GOX1857, mRNA ratio of 12.0) (34), for the membrane-bound
gluconate 2-dehydrogenase (GOX1230 and GOX1231, mRNA ratios of 2.8 and 2.3), for the type II NADH dehydrogenase
(GOX1675, mRNA ratio of 3.0), and for the membrane-bound
glycerol 3-phosphate dehydrogenase (GOX2088, mRNA ratio of
4.5). One of the two terminal oxidases of the respiratory chain of
G. oxydans, the cytochrome bd ubiquinol oxidase (GOX0278 and
GOX0279, mRNA ratios of 2.7 and 1.8), was also upregulated in
growth phase II.
The genes encoding pyridine nucleotide transhydrogenase
(pntA1A2B, GOX0310 to GOX0312, mRNA ratios of 4.4 to 6.0)
belonged to the most strongly upregulated genes at sample point
II. Transhydrogenase PntAB is located in the cytoplasmic membrane of many bacteria and couples the reduction of NADP⫹ by
NADH to the import of protons across the membrane. Transhydrogenase can operate reversibly, i.e., either consumes the electrochemical proton gradient across the cytoplasmic membrane for
NADP⫹ reduction or builds up an electrochemical proton gradient at the expense of NADPH oxidation (35). The genes downstream of pntB, GOX0313 and GOX0314, showed comparable
mRNA ratios (5.5 and 4.8) as the pntA1A2B genes, suggesting that
these five genes might form an operon. The two genes encode zinccontaining alcohol dehydrogenases, of which one (GOX0313) was
recently characterized (36).
The G. oxydans genome contains three gene clusters coding for
subunits of F1F0-ATP synthases. The clusters GOX1110 to
GOX1113 and GOX1310 to GOX1314 encode the subunits of the
F0 part and the F1 part of an ATP synthase, which is an ortholog of
the ATP synthases of Acetobacter pasteurianus IFO 3283-01, Gluconacetobacter diazotrophicus PAl 5, and other alphaproteobacteria. Both these clusters showed a decreased expression at sample
point II (mRNA ratio of 0.4 to 0.5). The genes of the third cluster,
GOX2167 to GOX 2175, might code for an Na⫹-translocating
F1F0-ATP synthase (33) and showed an increased expression at
sample point II (mRNA ratio of 1.4 to 3.3).
Genes involved in metabolism. Differentially expressed genes
encoding enzymes involved in central metabolism all showed elevated mRNA ratios during growth on gluconate in phase II: glucose 6-phosphate dehydrogenase (zwf, GOX0145, ratio of 2.8),
6-phosphogluconate dehydrogenase (gnd, GOX1705, ratio of
2.7), bifunctional transaldolase/phosphoglucose isomerase (pgi/
tal, GOX1704, ratio of 2.9), transketolase (tkt, GOX1703, ratio of
2.7), and fructose 1,6-bisphosphate aldolase (fba, GOX1540, ratio
of 3.2) (Fig. 4). Also the genes for triosephosphate isomerase (tpi)
and the four adjacent genes, encoding ribose 5-phosphate isomerase, a ribose ABC transporter, and dihydroxyacetone kinase
(GOX2217 to GOX2222, mRNA ratios of 10.8 to 1.9) showed
increased expression in growth phase II.
The genes encoding a glycerol facilitator (GOX 2089, mRNA
ratio of 3.9) and glycerol kinase (GOX3090, mRNA ratio of 4.6),
which presumably form an operon with GOX2088 (the membrane-bound glycerol 3-phosphate dehydrogenase, GOX2088,
mRNA ratio of 4.5), also showed increased mRNA levels, which
could suggest that the glycerol in the cultivation medium (0.5 g
liter⫺1) is metabolized preferably in the second growth phase. A
model variant allowing for glycerol uptake estimated a vanishing
low uptake rate of glycerol, whereas the fit was not improved (data
not shown). Therefore, this model variant was not regarded fur-
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the direction of fructose 6-phosphate formation, were indicative
of a major role of the PPP in glucose catabolism of G. oxydans.
Setting the carbon fluxes at sample point I to 100% at the level of
6-phosphogluconate, 62% of the carbon flux was directed to Gnd
and 38% was directed to 6-phosphogluconate dehydratase (Edd)
(Fig. 3A). Consequently, the metabolic activity of the EDP was
much lower than that of the PPP at sample point I. At sample
point II, the same calculation resulted in an even stronger preference for the PPP, since here 93% of the carbon flux was directed to
Gnd and only 7% to Edd (Fig. 3B).
Operation of a cyclic PPP in G. oxydans. 13C-MFA revealed a
high flux for the glucose 6-phosphate isomerase-catalyzed reaction (Pgi) in the direction from fructose 6-phosphate to glucose
6-phosphate, i.e., contrary to the direction used in glycolysis. At
sample points I and II, the net negative fluxes through Pgi were
91% and 176% of the sum of fluxes via glucose kinase and gluconate kinase. This result shows that due to the lack of phosphofructokinase, fructose 6-phosphate formed in the PPP is isomerized to
glucose 6-phosphate and enters the oxidative PPP again. Consequently, a partially cyclic flow of carbon through the PPP occurs
and 1 mol glucose 6-phosphate is converted to 1 mol pyruvate and
three mol carbon dioxide (Fig. 3A and B). Since at sample points I
and II 38% and 7% of the total 6-phosphogluconate was diverted
to the EDP, in which no carbon dioxide is produced, the actual
CO2 per glucose/gluconate yield should be lower than three. However, the measured amount of carbon dioxide was 20% higher
than the one calculated from the cytoplasmic metabolism of glucose described above (see Table S4.2 in the supplemental material). This discrepancy is most likely due to the metabolism of
components present in the yeast extract and of glycerol.
In accordance with the fact that the carbon flux downstream of
the PPP is continued at the level of glyceraldehyde 3-phosphate,
very low fluxes were observed for the reactions of fructose 1,6bisphosphatase (Fbp), fructose 1,6-bisphosphate aldolase (Fba),
and triosephosphate isomerase (Tpi) (Fig. 3A and B).
Comparison of global gene expression and selected enzyme
activities in growth phases I and II. The results described above
showed that glucose is oxidized mainly in the periplasm to gluconate (growth phase I) and subsequently to 2-ketogluconate
(growth phase II). Glucose and gluconate taken up into the cytoplasm are predominantly catabolized via a partially cyclic PPP,
notably in growth phase II. To correlate the carbon fluxes with
global gene expression and in vitro activities of selected enzymes,
cells were harvested for RNA isolation and cell extracts were prepared after 6.5 h (OD600 ⫽ 2.2, sample point I) and after 14 h
(OD600 ⫽ 6.0, sample point II), respectively (see Fig. S5 in the
supplemental material). The cultivations were performed in triplicate starting from independent precultures. RNA was prepared
from the six samples and used for comparative DNA microarray
analysis as described recently (14). In total, 454 genes showed
differential expression: 227 genes had an mRNA ratio (sample
point II to sample point I) of ⱖ2.0, and 227 genes had an mRNA
ratio of ⱕ0.5. These genes are listed in Table S6 in the supplemental material. Selected genes (including operons) are described in
the following paragraphs based on a functional categorization
(Table 1).
Genes involved in respiration and energy metabolism. Many
of the genes whose expression was influenced after the transition
to growth phase II are involved in respiration and ATP synthesis.
Several genes encoding proteins that feed electrons into the respi-
Fluxomics and Transcriptomics of G. oxydans 621H
TABLE 1 Genome-wide comparison of mRNA levels in G. oxydans during growth on glucose in sample point II versus sample point Ia
Annotation or protein
Respiration and energy metabolism
GOX1857
GOX0310
GOX0311
GOX0312
GOX0313
GOX0314
GOX2087
GOX2088
GOX2089
GOX2090
GOX2167
GOX2168
GOX2169
GOX2170
GOX2171
GOX2172
GOX2173
GOX2174
GOX2175
GOX1675
GOX1230
GOX1231
GOX1232
GOX0278
GOX0279
GOX1310
GOX1311
GOX1312
GOX1313
GOX1314
GOX1110
GOX1111
GOX1112
GOX1113
mRNA ratio of 14 genes, ⱖ2.0; 9 genes, ⱕ0.5
myo-Inositol dehydrogenase
NAD(P) transhydrogenase subunit ␣1
NAD(P) transhydrogenase subunit ␣2
NAD(P) transhydrogenase subunit ␤
Alcohol:NAD⫹ oxidoreductase
Probable alcohol:NAD(P)⫹ oxidoreductase
Glycerol-3-phosphate regulon repressor
Glycerol-3-phosphate dehydrogenase
Glycerol uptake facilitator protein
Glycerol kinase
F1F0-ATP synthase subunit ␤
F1F0-ATP synthase subunit ε
F1F0-ATP synthase subunit q
F1F0-ATP synthase subunit r
F1F0-ATP synthase subunit a
F1F0-ATP synthase subunit c
F1F0-ATP synthase subunit b
F1F0-ATP synthase subunit ␣
F1F0-ATP synthase subunit ␥
NADH dehydrogenase type II
Gluconate 2-dehydrogenase cytochrome c subunit
Gluconate 2-dehydrogenase subunit ␣
Gluconate 2-dehydrogenase subunit ␥
Cytochrome bd ubiquinol oxidase subunit I
Cytochrome bd ubiquinol oxidase subunit II
F1F0-ATP synthase subunit ␦
F1F0-ATP synthase subunit ␣
F1F0-ATP synthase subunit ␥
F1F0-ATP synthase subunit ␤
F1F0-ATP synthase subunit ε
F1F0-ATP synthase subunit b=
F1F0-ATP synthase subunit b
F1F0-ATP synthase subunit c
F1F0-ATP synthase subunit a
Metabolism
GOX2217
GOX2218
GOX2219
GOX2220
GOX2221
GOX2222
GOX1540
GOX1703
GOX1704
GOX1705
GOX0145
GOX1381
GOX1643
GOX0431
GOX1335
GOX1336
mRNA ratio of 45 genes, ⱖ2.0; 66 genes, ⱕ0.5
Triosephosphate isomerase
Ribose-5-phosphate isomerase B
Ribose ABC transporter, periplasmic binding protein
Ribose ABC transporter, ATP-binding protein
Ribose ABC transporter, permease protein
Dihydroxyacetone kinase
Fructose-1,6-bisphosphate aldolase
Transketolase
Bifunctional transaldolase/phosphoglucose isomerase
6-Phosphogluconate dehydrogenase-like protein
Glucose-6-phosphate 1-dehydrogenase
Gluconolactonase
Fumarate hydratase
Phosphogluconate dehydratase
Aconitate hydratase
Isocitrate dehydrogenase
Transport
GOX2188
GOX0812
GOX0813
GOX0814
GOX0815
GOX0816
mRNA ratio of 11 genes, ⱖ2.0; 36 genes, ⱕ0.5
Gluconate permease
Phosphoenolpyruvate-protein phosphotransferase
Phosphocarrier protein HPr
PTS system IIA component
Hypothetical protein GOX0815
HPr kinase
Gene
pntA1
pntA2
pntB
glpR
glpD
glpT
glpK
atpD
atpC
atpQb
atpRb
atpB
atpE
atpF
atpA
atpG
ndh
gndA
gndB
gndC
cydA
cydB
atpH
atpA
atpG
atpD
atpC
atpF’
atpF
atpE
atpB
tpi
rpi
fba
tkt
pgi/tal
gnd
zwf
fumC
edd
aco
icd
ptsI
ptsH
ptsIIA
ptsK
mRNA ratio of
phases II/I
P value
11.96
4.38
6.02
5.06
5.53
4.84
1.84
4.50
3.93
4.58
2.09
3.27
2.09
1.60
2.39
1.83
1.75
1.63
1.41
2.96
2.75
2.33
2.17
2.70
1.75
0.35
0.44
0.43
0.49
0.51
0.37
0.41
0.44
0.41
1.69 ⫻10⫺2
2.88 ⫻10⫺4
3.74 ⫻10⫺3
1.46 ⫻10⫺3
6.79 ⫻10⫺4
9.01 ⫻10⫺4
7.62 ⫻10⫺2
4.00 ⫻10⫺3
1.32 ⫻10⫺2
9.33 ⫻10⫺3
4.12 ⫻10⫺2
NA
1.04 ⫻10⫺2
NA
1.39 ⫻10⫺2
2.84 ⫻10⫺2
3.89 ⫻10⫺2
6.11 ⫻10⫺2
NA
2.13 ⫻10⫺3
4.98 ⫻10⫺3
1.10 ⫻10⫺2
6.59 ⫻10⫺2
1.17 ⫻10⫺3
6.67 ⫻10⫺2
2.40 ⫻10⫺3
4.94 ⫻10⫺3
1.09 ⫻10⫺2
3.67 ⫻10⫺2
1.34 ⫻10⫺2
6.03 ⫻10⫺4
3.28 ⫻10⫺3
9.45 ⫻10⫺5
1.36 ⫻10⫺3
10.78
4.96
4.08
1.95
3.24
1.85
3.18
2.71
2.85
2.72
2.75
2.56
0.50
0.44
0.38
0.29
2.33 ⫻10⫺3
1.16 ⫻10⫺3
7.58 ⫻10⫺3
6.41 ⫻10⫺6
NA
5.13 ⫻10⫺3
3.58 ⫻10⫺4
4.39 ⫻10⫺3
2.50 ⫻10⫺2
4.73 ⫻10⫺2
1.97 ⫻10⫺2
2.89 ⫻10⫺3
9.48 ⫻10⫺3
5.70 ⫻10⫺3
6.51 ⫻10⫺3
3.99 ⫻10⫺3
3.46
1.42
2.07
2.79
3.12
1.13
7.93 ⫻10⫺4
3.19 ⫻10⫺2
1.16 ⫻10⫺2
2.46 ⫻10⫺3
4.49 ⫻10⫺5
7.27 ⫻10⫺2
(Continued on following page)
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Function and locus tag
Hanke et al.
TABLE 1 (Continued)
Annotation or protein
Regulation and signal transduction
GOX0506
GOX0498
GOX2735
GOX2565
GOX2564
GOX0577
GOX0974
GOX1693
GOX0135
GOX2405
GOX1946
GOX0563
GOX2114
GOX1600
GOX0132
GOX2579
GOX0394
GOX0395
GOX0772
mRNA ratio of 14 genes, ⱖ2; 5 genes, ⱕ0.5
RNA polymerase sigma factor H (␴32)
Transcriptional regulator, HxlR family
Transcriptional regulator, PemK
Transcriptional regulator, PemI
Transcriptional regulator, toxin ChpA
Transcriptional regulator
Transcriptional regulator, Crp/Fnr family
Cell cycle transcriptional regulator CtrA
Transcriptional regulator, Ros/MucR family
Two-component response regulator, PhyR homolog
Two-component response regulator
Transcriptional regulator, TetR family
Transcriptional regulator
Two-component response regulator
Transcriptional regulator
Transcriptional regulator, TetR family
Two-component sensor histidine kinase
DNA-binding response regulator
Transcriptional regulator, Ros/MucR family
Stress
GOX2079
GOX1329
GOX2397
GOX0833
GOX1879
GOX2163
GOX1837
GOX0608
GOX0609
GOX1107
GOX0857
GOX1414
GOX0707
mRNA ratio of 13 genes, ⱖ2.0; 1 gene, ⱕ0.5
General starvation protein
Small heat shock protein
Small heat shock protein
Cold shock protein
Superoxide dismutase
Cold shock protein
Small heat shock protein HspA
ATP-dependent Clp protease adaptor protein ClpS
ATP-dependent Clp protease ATP-binding subunit ClpA
O antigen biosynthesis protein RfbC
Chaperone protein DnaK
Chaperone protein DnaJ
DNA starvation/stationary phase protection protein Dps
Motility
GOX0697
Transcriptional and translational machinery
Predicted functions
Hypothetical proteins
mRNA ratio of 1 gene, ⱖ2.0; 0 genes, ⱕ0.5
Flagellar FliL protein
mRNA ratio of 6 genes, ⱖ2.0; 66 genes, ⱕ0.5
mRNA ratio of 30 genes, ⱖ2.0; 18 genes, ⱕ0.5
mRNA ratio of 93 genes, ⱖ2.0; 26 genes, ⱕ0.5
mRNA ratio of
phases II/I
P value
4.83
4.62
4.49
4.17
3.27
3.23
2.68
2.58
2.45
2.40
2.39
2.35
2.14
2.03
0.24
0.49
0.46
0.44
0.43
6.65 ⫻10⫺3
1.67 ⫻10⫺3
1.08 ⫻10⫺3
8.51 ⫻10⫺4
9.09 ⫻10⫺4
2.03 ⫻10⫺3
3.00 ⫻10⫺3
2.00 ⫻10⫺2
3.47 ⫻10⫺2
6.46 ⫻10⫺3
3.45 ⫻10⫺4
8.20 ⫻10⫺4
4.78 ⫻10⫺3
7.60 ⫻10⫺6
1.03 ⫻10⫺4
4.38 ⫻10⫺2
4.70 ⫻10⫺4
4.53 ⫻10⫺19
3.35 ⫻10⫺5
hsd
hsd
csp
sod
csp
hspA
clpS
clpA
rfbC
dnaK
dnaJ
dps
31.69
18.31
4.84
4.74
3.58
2.97
2.82
2.37
1.75
2.33
2.31
2.26
2.03
2.71 ⫻10⫺4
1.04 ⫻10⫺3
1.40 ⫻10⫺3
1.42 ⫻10⫺4
2.19 ⫻10⫺4
4.13 ⫻10⫺3
5.99 ⫻10⫺3
8.09 ⫻10⫺3
2.79 ⫻10⫺3
6.22 ⫻10⫺3
5.98 ⫻10⫺3
2.54 ⫻10⫺2
1.32 ⫻10⫺3
fliL
2.68
4.04 ⫻10⫺3
Gene
rpoH
Cells were harvested for RNA isolation after 8 h (OD600 ⫽ 3.6, sample point I) and after 14.25 h (OD600 ⫽ 7.6, sample point II). Selected genes with an mRNA ratio of ⱖ2.0
(lower ones allowed in the case of operons) or ⱕ0.5 (higher ones allowed in the case of operons) and a P value of ⱕ0.05 are listed. The data shown represent mean values from
three biological replicates. The genes were grouped into different functional categories within which they were ordered according to their mRNA ratios except in the case of
operons, which were ordered according to their locus tag. A list of all genes with an mRNA ratio of ⱖ2.0 or ⱕ0.5 in the respective functional categories is given in Table S6 in the
supplemental material. NA, not available.
b
Nomenclature according to Dibrova et al. (33).
a
ther. Possibly, glycerol catabolism is repressed in the presence of
glucose in growth phase I, which could be due to a regulatory
function of the rudimentary PTS system (see below).
Among the genes with a lowered expression level at sample
point II was phosphogluconate dehydratase (edd, GOX0431,
mRNA ratio of 0.4), the first of the two key enzymes of the EDP.
Furthermore, three genes encoding enzymes of the TCA cycle
showed decreased mRNA ratios, aconitate hydratase (acn,
GOX1335, ratio of 0.4), isocitrate dehydrogenase (icd, GOX1336,
ratio of 0.3), and fumarate hydratase (fumC, GOX1643, ratio
of 0.5).
Genes involved in transport. In accordance with gluconate
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being the carbon source in growth phase II, the gene encoding
gluconate permease (GOX2188, mRNA ratio of 3.5) showed increased expression. Due to the lack of the EIIB and EIIC components, the PTS system in G. oxydans is considered to be inactive as
a transport system (3), and the function of the remaining components, EI, HPr, EIIA, is not yet clear. The genes encoding the latter
proteins (GOX0812 to GOX0816) had increased expression levels
(mRNA ratios of 1.4 to 2.8) in growth phase II, showing that they
are subject to transcriptional regulation.
Genes involved in regulation and signal transduction. Thirteen genes coding for transcriptional regulators had increased
mRNA levels at sample point II, 10 of them coding for one-com-
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Function and locus tag
Fluxomics and Transcriptomics of G. oxydans 621H
activity ratios, and rounded rectangles the mRNA ratios. For abbreviations of flux and metabolite names used in the model, see Table S3.2 and S3.3 in the
supplemental material.
ponent transcriptional regulators of different families and three
coding for response regulators of two-component signal transduction systems. In addition, expression of the gene coding for
sigma factor ␴32 (sigH, GOX0506, mRNA ratio of 4.8) was in-
April 2013 Volume 79 Number 7
creased in growth phase II. Five transcriptional regulator genes
showed decreased expression. Whereas the target genes of the
transcriptional regulators and the stimuli they respond to have not
been identified yet, ␴32 of other bacteria is known to be induced
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FIG 4 Multiomics comparison of ratios at sampling time point II versus sampling time point I. Arrows represent the carbon flux ratios, diamonds the enzyme
Hanke et al.
DISCUSSION
In this study, the first 13C-based metabolic flux analysis was performed for G. oxydans and combined with a comparative transcriptome analysis and enzyme activity assays. Although the genome sequence indicates that G. oxydans 621H contains all genes
required for the de novo synthesis of all nucleotides, amino acids,
phospholipids, and most vitamins (3), it apparently cannot provide sufficient quantities of certain building blocks, as there are
2346 aem.asm.org
TABLE 2 Comparison of in vitro enzyme activities of Glk (EC 2.7.1.1),
Gntk (EC 2.7.1.12), Zwf (EC 1.1.1.49), and Gnd (EC 1.1.1.44) of
glucose-grown cells at sample points I and IIa
Activity
(nmol min⫺1
mgprotein⫺1) at
sample point ⫾ SD
Enzyme
I
II
Ratio
II/I ⫾ SD
Glucose kinase (Glk)
Gluconate kinase (Gntk)
Glucose-6-phosphate dehydrogenase (Zwf)
6-Phosphogluconate dehydrogenase (Gnd)
86 ⫾ 4
34 ⫾ 3
280 ⫾ 6
180 ⫾ 2
86 ⫾ 4
68 ⫾ 1
940 ⫾ 9
420 ⫾ 8
1.0 ⫾ 0.1
2.0 ⫾ 0.2
3.4 ⫾ 0.1
2.3 ⫾ 0.1
Initial concentration of glucose, 80 g liter⫺1. Mean values and standard deviations
derived from three independent cultures are shown.
a
several reports in the literature that growth of G. oxydans on defined medium without yeast extract results in a very low biomass
formation (39–41). Thus, in the 13C-MFA, the utilization of yeast
extract components had to be taken into account, which complicates the analysis. A second challenge for 13C-MFA was the low
growth rate in growth phase II, which requires long experimentation times in order to reach an isotopically pseudo-equilibrated
state that is characteristic for a growth phase. If cellular conditions
change considerably within this period (e.g., in the case of substrate depletion), isotopic stationarity cannot be reached in less
active parts of metabolism. These problems might be solved in the
future by an isotope-based untargeted analysis, recently termed
the TARDIS (time and relative differences in systems) approach,
which provides an option to address both metabolic pathway elucidation and flux determination (42).
Despite the hurdles described above, 13C-MFA led to a number
of important results with respect to the cytoplasmic sugar catabolism of G. oxydans. Glucose taken up into the cytoplasm at sample point I was predominantly oxidized to gluconate, which then
was either further oxidized to 5-ketogluconate or phosphorylated
to 6-phosphogluconate. About 10% of the glucose was phosphorylated and oxidized to 6-phosphogluconate. The major route for
6-phosphogluconate metabolism was the PPP, 62% and 93% in
growth phases I and II, respectively. This preference of the PPP
was also observed by us in a recent analysis of ⌬gnd and ⌬edd-eda
mutants of G. oxydans during growth on mannitol (15) or on
glucose (43). The prevalence of the PPP as the major catabolic
pathway is a feature not often observed in bacteria. In a comparative 13C-MFA of glucose metabolism in seven bacterial species
(Agrobacterium tumefaciens, two pseudomonads, Sinorhizobium
meliloti, Rhodobacter sphaeroides, Zymomonas mobilis, and Paracoccus versutus), Fuhrer and Sauer (44) showed that glucose was
predominantly degraded via the EDP, and the PPP had a solely
anabolic function.
Another important result of 13C-MFA was the demonstration
of the cyclic nature of the carbon flux through the oxidative part of
the PPP, as shown by the strong net flux from fructose 6-phosphate to glucose 6-phosphate in the Pgi-catalyzed reaction. This
cyclic flux is caused by the absence of 6-phosphofructokinase.
Carbon dioxide production in growth phase II was in accordance
with the amount calculated based on the stoichiometry of a cyclized PPP from the experimentally determined carbon uptake values. A partially cyclic operation of the PPP was expected from
theoretical considerations (7) and has recently also been demon-
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under different stress conditions and to activate expression of
genes required to counteract these stresses (37). As shown below,
expression of several stress genes was induced during growth with
gluconate in phase II, which might be due to the activity of ␴32.
Genes involved in stress responses. The highest expression
change in all of this genome-wide transcriptional analysis was displayed by GOX2079 (mRNA ratio of 31.7). The gene product
possesses a GsiB domain often found in stress-induced proteins
and might be related to a general starvation protein, as found in
Bacillus species (38). Several other genes involved in different
types of stress responses, such as heat shock (GOX1329, ratio of
18.3; GOX2397, ratio of 4.8; GOX1837, ratio of 2.8), cold shock
(GOX0833, ratio of 4.7; GOX2163, ratio of 3.0), or oxidative stress
(GOX1879, ratio of 3.6), the chaperons DnaK (GOX0857, ratio of
2.3) and DnaJ (GOX1414, ratio of 2.3), as well as two genes encoding components of Clp ATPases (GOX0608, ratio of 2.4;
GOX0609, ratio of 1.8) were found to have increased mRNA levels
at sample point II, which might be related to the increased expression of the ␴32 gene. As the cells face neither heat nor cold stress
under the cultivation conditions used, the induction of these
genes might represent a kind of general stress response triggered
by the decreased growth rate with gluconate.
Genes involved in motility. Only one of the 29 genes involved
in flagellum biosynthesis and function showed an increased expression (fliL, GOX0697, mRNA ratio of 2.7), and none of the 13
chemotaxis-assigned genes present in the genome of G. oxydans
621H were differentially regulated, suggesting that G. oxydans
does not respond with increased motility when growing on gluconate.
Genes involved in the transcriptional and translational machinery. Many genes encoding proteins involved in transcription
and translation showed lower expression at sample point II (see
Table S6 in the supplemental material). This response probably
presents an adaptation to the reduced, linear growth observed
after transition of the cells from glucose to gluconate as the carbon
source.
Genes with predicted and unknown functions. Forty-eight
genes encoding proteins with predicted functions were differentially expressed, of which 30 showed an mRNA ratio of ⱖ2.0 and
18 an mRNA ratio of ⱕ0.5 (see Table S6 in the supplemental
material). A total of 119 genes with unknown functions encoding
hypothetical proteins were differentially expressed.
In vitro enzyme assays. The in vitro activities of glucose kinase
(Glk), gluconate kinase (GntK), glucose 6-phosphate dehydrogenase (Zwf), and 6-phosphogluconate dehydrogenase (Gnd) were
determined in cell extracts derived from sample points I and II
(Table 2). Whereas the activity of Glk remained constant in both
phases, the activity of the other three enzymes increased 2- to
3.4-fold in the second growth phase, which correlates with their
increased mRNA levels and the increased carbon flux through the
PPP in growth phase II (Fig. 4).
Fluxomics and Transcriptomics of G. oxydans 621H
April 2013 Volume 79 Number 7
MFA, in vitro enzyme assays, and genome-wide transcription analysis that the PPP is the predominant pathway for intracellular sugar
catabolism in G. oxydans. In addition, 13C-MFA verified the cyclic
nature of the carbon flux through the oxidative part of the PPP.
ACKNOWLEDGMENTS
This work was funded by the German Ministry of Education and Research
within the GenoMik-Plus program (BMBF 0313751H). We thank DSM
Nutritional Products, Kaiseraugst, Switzerland, for financial support.
We thank Petra Simić and Hans-Peter Hohmann (DSM Nutritional
Products) for their scientific input and their continued disposition for
discussion. We also thank Bianca Klein for expert LC-MS analysis, Jana
Tillack for providing process model simulations, and Janine Richhardt for
her support during manuscript writing.
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