The extent to which ATP demand controls the glycolytic flux depends strongly
on the organism and conditions for growth.
Brian J. Koebmann1, Hans V. Westerhoff2, Jacky L. Snoep2,3, Christian Solem1, Martin B. Pedersen4
Dan Nilsson5, Ole Michelsen1, and Peter R. Jensen1*
1 Section of Molecular Microbiology, BioCentrum-DTU, Technical University of Denmark,
Building 301, DK-2800 Kgs. Lyngby, Denmark.
2Department of Molecular Cell Physiology, Vrije Universiteit, De Boelelaan 1087, NL-1081 HV
Amsterdam, The Netherlands
3Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland 7602, South
Africa
4 Department of Genomics and Strain Development, Chr. Hansen A/S, Bøge Allé 10-12, DK-2970
Hørsholm, Denmark.
5 CH Bio Ingredients, Chr. Hansen A/S, Bøge Allé 10-12, DK-2970 Hørsholm, Denmark
Running title: Control of glycolysis in Escherichia coli and Lactococcus lactis
Key words: ATPase, coli, glycolysis, lactococcus, MCA, metabolic control analysis
*Correspondent footnote. Mailing address: Section of Molecular Microbiology, BioCentrumDTU, Technical University of Denmark, Building 301, DK-2800 Kgs. Lyngby. Tel +45 45252510.
Fax. +45 45932809. E-mail: [email protected]
Abstract
Using molecular genetics we have introduced uncoupled ATPase activity in two different
bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the
growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth
in batch cultures of E. coli the ATP demand was found to have almost full control on the glycolytic
flux (FCC=0.96) and the flux could be stimulated by 70%. In contrast to this, in L. lactis the control
by ATP demand on the glycolytic flux was close to zero. However, when we used non-growing cells
of L. lactis (which have a low glycolytic flux) the ATP demand had a high flux control and the flux
could be stimulated more than two fold. We suggest that the extent to which ATP demand controls
the glycolytic flux depends on how much excess capacity of glycolysis is present in the cells.
Introduction
The control of the glycolytic flux in living cells has been investigated for several decades.
Most of the glycolytic enzymes have been overexpressed individually or in combinations of several
enzymes together with virtually no effect on the glycolytic flux (Schaaff et al., 1989; Snoep et al.,
1996; Müller et al., 1997; Hauf et al., 2000). This could be due to distribution of the flux control
over many enzymes in the pathway, which would then require the simultaneous overexpression of
many enzymes in order to achieve a higher glycolytic flux. Alternatively, the flux control could also
reside outside the glycolytic reactions, for instance in the transport of sugar into the cell or in the
reactions that consume the products of glycolysis. Indeed, early computer simulations indicated that
the ATP consuming reactions could play an important role in controlling the glycolytic flux in
erythrocytes (Rapoport et al., 1976; Heinrich and Schuster, 1996). Such a distribution where most
flux control lies outside the pathway has also been favoured from a functional point of view
(Hofmeyr and Cornish-Bowden, 2000).
In this paper we discuss experimental data on the importance of the ATP consuming
reactions for control of glycolysis in two microbial systems. We show that the ATP consumption
does indeed have almost full control over the glycolytic flux in aerobic E. coli while in anaerobic L.
lactis it has virtually no control at all.
Materials and Methods
The materials and methods used throughout the current paper are described in appendix I
Results
Introduction of uncoupled ATPase activity in bacteria.
The cytoplasmic F1-domain of the F1F0-H+ -ATPase contains the catalytic site for
synthesis/hydrolysis of ATP. We previously described the cloning of atpAGD genes encoding the
a-, g-, and b-subunits of the F1-part, respectively downstream synthetic constitutive promoters,
which expressed the genes to different extents (Fig. I; Koebmann et al., 2002a, 2002b). The E. coli
and L. lactis atp genes were cloned in transcriptional fusions with lacLM coding for b-galactosidase,
which allowed for an indirect measurement of transcription of the ATPase genes in both organisms
E. coli BOE270
L. lactis MG1363
1
0,8
0,6
0,4
0,2
0
-0,2
-0,4
-0,6
-0,8
-1
Elasticity of glyc. flux towards [ATP]/[ADP]
1
Elasticity of glyc. flux towards [ATP]/[ADP]
Elasticity of growth towards [ATP]/[ADP]
Elasticity of growth towards [ATP]/[ADP]
0,8
0,6
Elasticities
Elasticities
(A)
0,4
0,2
0
-0,2
-0,4
-0,6
-0,8
-1
0
2
4
6
8
10
5
6
7
8
9
[ATP]/[ADP] ratio
[ATP]/[ADP] ratio
(B)
1
Control by ATP demand on
glycolytic flux
Control by ATP demand on
glycolytic flux
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0
2
4
6
8
[ATP]/[ADP] ratio
10
0
-1
5
6
7
8
9
[ATP]/[ADP] ratio
Figure 1: Elasticities and flux control by ATP demand on the glycolytic fluxes in E. coli and L.
lactis. (A) Elasticities of the glycolytic flux and the growth rate towards the intracellular
[ATP]/[ADP] ratio. The elasticities are the slopes of the scaled fluxes towards the [ATP]/[ADP]
ratio in (Fig. IIIB (Appendix III)), calculated from the fitted equations. (B) Flux control by the
demand for ATP on the glycolytic flux as a function of the [ATP]/[ADP] ratio, calculated from
eq. 1. (From Koebmann et al., 2002a, 2002b)
(Koebmann et al., 2002a). The gradual increase in ATPase activity resulted in a concomitant gradual
decrease in biomass yield. In combination with direct in vitro measurements of the ATPase activity
in E. coli (Koebmann et al., 2002a) this showed that ATP was indeed being hydrolyzed in the
bacterial cells.
Impact of increased ATP demand on the energy state.
We then studied how the extra ATP consumption affected the energy state of the cells. In E.
coli the concentration of ATP decreased slightly, with increased ATPase activity to 25% lower
concentration at the highest ATPase activity, while the ADP concentration increased by more than
65% (Fig. II (Appendix III); Koebmann et al., 2002a). This was associated with an approximately
18% decrease in total ATP+ADP concentration and a drop in [ATP]/[ADP] ratio from 11 to 5 (Fig.
II). The intracellular energy level was also affected in L. lactis, where the [ATP]/[ADP] ratio
dropped from 9 to 5 (Fig. II (Appendix III); Koebmann et al., 2002b). But in contrast to E. coli,
there was no significant change in the total ATP+ADP concentration in L. lactis.
Impact of uncoupled ATPase activity on anabolic and catabolic fluxes.
The increased ATP demand had different impacts on the glycolytic fluxes in E. coli and L.
lactis. In both organisms we observed a decrease in growth rate to 76% (E. coli) and 69% (L. lactis)
of wild-type level (Table I (Appendix II)). The uncoupled ATPase activity resulted in a significant
decrease in biomass yield in E. coli to 45% of the wild-type yield (Koebmann et al., 2002a) and in L.
lactis the yield decreased to 69% of the wild-type yield (Koebmann et al., 2002b). The glycolytic
fluxes were measured as the steady state consumption rate of glucose during exponential growth.
Interestingly, the glycolytic flux in E. coli increased gradually to 170% of wild-type flux (Koebmann
et al., 2002a), whereas in L. lactis no change in the glycolytic flux was observed (Koebmann et al.,
2002b).
A possible explanation for the lack of stimulation of the glycolytic flux in L. lactis could be
that glycolysis in growing L. lactis is already running close to its maximal capacity. We therefore
also measured the effect of ATPase activity in non-growing cells of L. lactis. L. lactis cells
containing different F1-ATPase activities were resuspended in SA medium without vitamins and
amino acids, which lead to a decrease in the ATP demanding anabolic reactions as a result of the lack
of essential nutrients. Thus, in non-growing wild-type cells the glycolytic flux is reduced to 37% of
steadily growing cells (Koebmann et al., 2002b). The introduction of F1-ATPase activity stimulated
the glycolytic flux until the flux in growing cells was approached, but not above this flux (data not
shown).
Flux control of the ATP demanding processes.
In order to quantity the extent of which ATP demand controls glycolysis we simplified the
cellular free-energy metabolism into a supply module and a demand module for ATP, by assuming
that the fluxes are only linked via ATP:
e1
Substrate
e2
DGp
Growth
When Metabolic Control Analysis is applied the flux control coefficients can be expressed in
terms of the block elasticities as:
CeJ2 =
- e ep1
e ep2 - e ep1
(eq. 1)
The elasticities of the blocks can be obtained from the slopes of the fluxes as functions of the
energy level (Fig. IIIA (Appendix III)). After the data points have been fitted to algebraic equations
in a log-log plot (Fig. IIIB (Appendix III)), the elasticities can be calculated (Fig. 1A). For E. coli the
elasticity of the catabolic block was quite high, -0.89, in the absence of ATPase and dropped to
–0.42 at the highest ATPase activity. In contrast, the elasticity of the anabolic block was rather low,
0.04, in the absence of ATPase and increased gradually to 0.65 at the highest ATPase activity
(Koebmann et a., 2002a). For L. lactis different curves (logarithmic, linear, exponential, power) were
fitted to the data points with small changes in the [ATP]/[ADP] ratio as compared to the wild type.
The elasticity of the catabolic block was estimated to be close to zero, -0.02, which indicates that
glycolysis is very insensitive towards changes in the energy level. The elasticity of the anabolic
block (growth rate) ranged from 0.22 to 0.26 in the absence of ATPase, depending on the applied
curve fit, and increased slightly with higher ATPase expression.
The control by the ATP demanding processes on the glycolytic flux was then calculated
from eq. 1 (Fig. 1B). For E. coli essentially all control resided in the ATP demanding processes
( CeJ21 =0.96). Even if calculation of the anabolic flux is based on a linear fit (which leads to
overestimation of the anabolic elasticity in the wild-type cell) flux control by ATP demand is still
CeJ21 =0.75. In L. lactis the ATP consuming reactions were found to have less than 10% of flux
control on glycolysis ( CeJ21 =0.1).
Discussion
We have measured to what extent ATP demand contributes to the control of glycolytic flux in
bacteria. By expressing three subunits, a, b and g, of the catalytic part (F1) of the H+ -ATPase from
a series of promoters with increasing strength we varied the ATP hydrolysis reaction. The
introduction of uncoupled ATPase activity in the cytoplasm resulted in a gradual decrease in the
intracellular [ATP]/[ADP] ratio and in a decrease of the biomass yield.
In aerobic E. coli cells growing in minimal medium with glucose as the sole carbon- energy
source, the ATP demand turned out to have almost full control over the glycolytic flux with a flux
control coefficient of 0.96. The high flux control was the result of a high (negative) elasticity of
glycolysis and a low elasticity of the anabolic reactions towards the phosphorylation potential. The
results also demonstrate that the growth rate of E. coli is controlled mainly by anabolic reactions and
not by ATP production. Experiments have been performed earlier with E. coli cells in which the
activity of the coupled H+ -ATPase (ATP synthase) was being modulated (genetically), which
amounts to modulating the ATP supply (Jensen et al., 1993b). In those experiments it was found
that the ATP synthase had zero control on the growth rate, which is therefore in good agreement
with the results reported in the current paper.
An interesting observation was that the flux control by ATP demand remained relatively
high as the activity of F1-ATPase increased and an overall increase in the glycolytic flux by 70%
could be achieved. This result shows that there is a relatively large excess of glycolytic capacity,
which can be mobilized upon demand.
In anaerobic Lactococcus lactis cells growing in defined medium supplemented with glucose,
the control by ATP demand was found to be very close to zero. The anabolic reactions in L. lactis
had a low elasticity towards the phosphorylation potential similarly to what was observed for E.
coli but in the case of L. lactis the elasticity of glycolysis was virtually zero. These data suggest that
the rate of glycolysis in growing L. lactis is already close to its maximal capacity. Indeed, when the
experiment was repeated with non-growing L. lactis cells (cells resuspended in buffer which have 3
fold lower glycolytic flux compared to growing cells) the uncoupled ATPase resulted in more than
two fold increase in the glycolytic flux. Thus, in non-growing cells the ATP demand does appear to
have a high control on the glycolytic flux, also in L. lactis. Apparently, the extent to which the ATP
demand controls glycolysis depends on how much excess capacity of glycolysis is present in the
cells.
Acknowledgement: This work was supported by The Danish Academy of Technical Sciences
(ATV), The Danish Research Agency, and Chr. Hansen A/S. We thank Regina Schürmann for expert
technical assistance, Jannie Hofmeyr, David Fell, and Reinhardt Heinrich for discussions.
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