Biochemical Engineering Division, GBF National Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany
Correspondence
Ursula Rinas
URI{at}gbf.de
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ABSTRACT |
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Present address: Aventis Pharma Deutschland GmbH (ein Unternehmen der sanofi-aventis Gruppe), 65926 Frankfurt, Germany.
Present address: DSM Biologics, 6000 av. Royalmount, Montréal, Quebec, Canada H4P 2T1.
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INTRODUCTION |
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One important group of regulatory compounds involved in the coupling of catabolic and anabolic reactions are the adenosine nucleotides AMP, ADP and ATP. The level of these nucleotides reflects the energetic status of the cell. High ATP levels in conjunction with low AMP levels will inhibit enzymes of the ATP-replenishing pathways and vice versa (Atkinson, 1968). cAMP, in conjunction with the cAMP receptor protein, is another adenosine-nucleotide-derived molecule that plays a key regulatory role. Synthesis of cAMP is catalysed by adenylate cyclase and, after the first discovery of cAMP in Escherichia coli (Makman & Sutherland, 1965
), numerous studies have been carried out to unravel its not yet fully understood function in the regulation of gene expression and anabolic and catabolic pathway utilization [for a review, see Saier et al. (1996)
and references cited therein, for example]. Most notably, cAMP formed after depletion of a preferred carbon substrate such as glucose plays a key role in restructuring carbon catabolism to allow the utilization of less-preferred carbon substrates. Under conditions of glucose excess, adenylate cyclase activity is repressed but expression of the gene encoding adenylate cyclase, cya, is maximal, thereby increasing the potential for cAMP formation once glucose is depleted.
One pathway that is often neglected in the evaluation of unbalanced conditions is the methylglyoxal (MG) pathway, which exists in many organisms including E. coli (Tempest & Neijssel, 1992; Inoue & Kimura, 1995
; Ferguson et al., 1998
; Kalapos, 1999
). In Klebsiella aerogenes, it was shown that MG formation is activated when a slow-growing culture is pulsed with glucose (Teixeira de Mattos et al., 1984
). In E. coli, activation of the MG pathway has been confirmed when the intracellular uptake of glucose 6-phosphate or other carbon substrates such as xylose, lactose, arabinose, glycerol or gluconate are deregulated by mutation and/or by addition of cAMP (Ackerman et al., 1974
; Puskas et al., 1983
; Kadner et al., 1992
). MG is produced from dihydroxyacetone phosphate (DHAP) and the MG pathway represents an energetically unfavourable bypass to the glycolytic reactions of the lower EmbdenMeyerhofParnas (EMP) pathway (Cooper & Anderson, 1970
). Synthesis of MG is induced by high concentrations of DHAP and inhibited by high phosphate concentrations (Hopper & Cooper, 1971
). It has been suggested that utilization of the MG pathway relieves the cells from stress caused by elevated levels of sugar phosphates (Cooper & Anderson, 1970
; Kadner et al., 1992
; Ferguson et al., 1998
; Tötemeyer et al., 1998
). However, MG is a very toxic compound that arrests growth of E. coli at millimolar concentrations (Együd & Szent-Györgyi, 1966
). It can react with the nucleophilic centres of macromolecules such as DNA, RNA and proteins (Lo et al., 1994
, Papoulis et al., 1995
) and it has been proposed that MG inhibits growth by interfering with protein synthesis and consequently preventing initiation of DNA replication (Fraval & McBrien, 1980
). Due to the cytotoxicity of MG, its production is tightly controlled. Even a 900-fold overexpression of MG synthase in E. coli caused the accumulation of only very low levels of MG (Tötemeyer et al., 1998
).
In this study, the metabolic dynamics of E. coli in response to feast and famine were studied in glucose-limited steady-state cultures by up- or downshifts of the dilution rates, respectively. In particular, transients in the extracellular rates of oxygen uptake and formation of carbon dioxide, MG, D- and L-lactate, pyruvate and acetate were followed. Moreover, several intracellular metabolites including the adenosine nucleotides and the key glycolytic intermediates at the branch-point to the MG pathway [fructose 1,6-diphosphate (FDP), DHAP and glyceraldehyde 3-phosphate (GAP)] were measured to provide information about regulation of the cellular network during the adaptation to sudden changes of nutrient supply. In addition, the uncoupling of anabolism and catabolism and the activation of the MG pathway were simulated using a stoichiometry-based metabolic model.
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METHODS |
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Analytical techniques.
For analysis of MG, 910 ml culture liquid was sprayed into vacuum-sealed precooled test tubes using a rapid sampling technique as described in the accompanying article (Kayser et al., 2005). The test tubes were filled prior to sampling with 1 ml of a precooled solution of 5 mol perchloric acid l1 (0 °C). Conversion of MG to 2-methylquinoxaline with o-phenylenediamine was performed according to Chaplen et al. (1996a
, b)
. 2-Methylquinoxaline was analysed by HPLC using a C-18 column (Alltech) with 5-methylquinoxaline as internal standard. The column was eluted at 20 °C with 68 % v/v 10 mmol KH2PO4 l1 and 32 % v/v acetonitrile at a flow rate of 1 ml min1.
For analysis of DHAP, GAP and FDP, 34 ml culture liquid was sprayed into precooled test tubes filled with 1 ml of 5 mol perchloric acid l1 (25 °C). After one freezethaw cycle, the extracts were neutralized on ice with a solution containing 2 mol KOH and 0·4 mol imidazole l1 (pH 7·2±0·1). The KClO4 precipitate was removed by centrifugation (14 000 r.p.m., 30 s) and filtration (pore size 0·45 µm). Quantification of DHAP, GAP and FDP was carried out according to Michal (1984). For conversion to intracellular concentrations, an intracellular volume of 2·15 ml corresponding to 1 g cell dry mass was assumed (Pramanik & Keasling, 1997
).
Analytical techniques for the determination of glucose, cell dry mass, organic acids, adenosine nucleotides (AMP, ATP, AMP and cAMP) and outlet-gas composition are described in the accompanying article (Kayser et al., 2005). For analysis of pyruvic acid, test kits from Roche Diagnostics were employed. D- and L-lactic acid were also analysed separately by enzymic test kits from Roche Diagnostics.
Calculation of rates.
The glucose uptake and biomass formation rates were calculated from dynamic mass balances. For the j-th compound, it can be written as:
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Flux analysis.
Metabolic flux analysis applying a linear programming technique was used to predict by-product formation during carbon-overflow conditions and to simulate the activation of the MG pathway. The metabolic model and the methodology are described in the accompanying article (Kayser et al., 2005). To simulate pathway reorganization in response to carbon-overflow conditions, an upper limit for the biomass formation rate was given and, with known glucose and oxygen uptake and carbon dioxide formation rates, the metabolic model determined the excreted by-products as a function of the discrepancy between the catabolic and anabolic reactions.
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RESULTS AND DISCUSSION |
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Changes in the specific concentrations of the adenosine nucleotides (AMP, ADP, ATP and cAMP) were also followed during the adaptation period after the up- and downshift of the dilution rate (Fig. 2). After the upshift, the ATP level increased instantaneously, reaching a peak within 2 min and subsequently decreased during the next hour until a new steady-state level was reached (Fig. 2a
). The adenylate energy charge (AEC) closely followed the time profile of the ATP concentration, also peaking 2 min after the dilution rate upshift and then decreasing within the next hour until the new steady-state value of 77 % was reached (Fig. 2c
; see Kayser et al., 2005
). The rapidly increasing AEC within the first 2 min after the dilution rate upshift coincided with the immediate increase in the glucose uptake rate, which was not accompanied by a corresponding increase in the biomass formation rate (compare Fig. 1c
). Thus, the production of ATP in the catabolic pathway exceeded the consumption of ATP for anabolic purposes. This is also confirmed by the immediate increase in the respiratory activity (Fig. 1a
), which reflects the ATP formation rate, since most of the ATP is generated through oxidative phosphorylation in the respiratory chain. Subsequently, pathways generating less energy were activated and the ATP level and, correspondingly, the AEC decreased again.
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The cAMP level rose quickly after the dilution rate downshift (Fig. 2d), but also increased after the dilution rate upshift within the first 2 min from zero to more than 1 µmol (g cell dry mass)1, a third of the level that was reached during the downshift experiment (Fig. 2c
). In both experimental set-ups, the cAMP level declined again after the change in the dilution rate and a steady-state level of zero was reached in all experiments. Thus, the immediate response of the cells to a sudden carbon-overflow situation resembles the typical response that cells exhibit when they need to reorganize metabolism while adapting to the utilization of a less-preferred carbon substrate. Apparently, cAMP is involved in restructuring carbon catabolism not only after nutrient downshift but also after nutrient upshift.
Accumulation of extra- and intracellular metabolites in response to feast conditions
During the dilution rate downshift experiment, extracellular by-product formation was not detectable, in agreement with the finding that there was no uncoupling of anabolic and catabolic reactions during the adaptation period to reduced carbon supply. During the upshift experiment, however, D- and L-lactate, acetate and, in small quantities, pyruvate and formate were excreted into the culture fluid (Fig. 3). Acetate accumulated to significantly higher concentrations than the other by-products; a maximum of 450 mg acetate l1 was reached during the transient in comparison to about 100 mg lactate l1 (sum of D- and L-lactate). However, the excretion of acetate started considerably later than the excretion of the other organic acids. The most rapid by-product formation was observed for lactate and MG; just 14 min after the dilution rate upshift, more than 0·6 mg MG l1 was detected in the culture fluid (Fig. 3
). The maximum concentration of MG was 0·9 mg l1; this is well below the threshold value of 20 mg l1 that is reported to cause growth inhibition (Ferguson et al., 1996
). The accumulation of MG indicates the activation of the MG pathway, which bypasses the EMP pathway at the level of DHAP (Fig. 4
). DHAP is converted to pyruvate via MG and D- and L-lactate, suggesting that excretion of D- and L-lactate may originate from degradation of MG.
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A decrease in GAP with a concomitant increase in DHAP can be explained by a strong increase in the flux of the upper part of the EMP pathway up to the triose level, where equal amounts of GAP and DHAP are produced from FDP by aldolase, concomitant with a reduced drain in the lower part of the EMP pathway (from GAP to PEP) and the induction of the MG pathway. The short-term increase in the AEC and elevated levels of FDP contribute to the activation of MG synthase and inhibition of GAP dehydrogenase by decreasing the level of available free phosphate. The triosephosphate-isomerase-catalysed conversion of GAP to DHAP and vice versa favours the formation of DHAP (95 % DHAP at equilibrium; Veech et al., 1969; Richard, 1993
), thereby contributing to an increased DHAP level and induction of the MG pathway at the expense of the lower part of the EMP pathway. Prior to the dilution rate upshift, intracellular GAP and DHAP concentrations did not differ very much (2·1±0·1 mmol GAP l1; 1·67±0·04 mmol DHAP l1); during the transient, however, intracellular GAP levels decreased to 1·2 mmol l1 whereas DHAP levels reached 3 mmol l1, thus approaching more closely, with 70 % DHAP, the favourable formation of DHAP (Fig. 5
).
Simulation of MG pathway activation and excretion of by-products
The key event for the activation of the MG pathway is the excessive uptake of glucose, which leads to an imbalance of catabolic and anabolic reactions. This imbalance is manifest as increasing respiration and glucose uptake rates, while the biomass formation rate did not increase accordingly (Fig. 1). To simulate the activation of the MG pathway and the excretion of by-products during carbon-overflow conditions, by-product excretion rates normalized with respect to the glucose uptake rate were calculated at various fixed ratios of biomass formation to glucose uptake rates (Fig. 6
). The ratio variation was used to approach the dynamic response that cells exhibit when going through a carbon-overflow transient, characterized by more rapid adaptation of catabolic reactions compared with anabolic reactions. All excreted by-products that were determined experimentally were also predicted to accumulate by the metabolic model. The model predicted that, at high ratios of biomass formation to glucose uptake rate, rx rs1, formate would be formed and, at lower values of rx rs1, pyruvate, acetate, MG and finally D- and L-lactate (in equal amounts) would be excreted. To see how the entire network responds to the restricted anabolic rate, a flux distribution map for rx rs1=30 % mol mol1 is shown as an example (Fig. 7
). The calculation revealed that triosephosphate isomerase (r6) favours the formation of DHAP, which is subsequently converted to MG (r42). Half of the MG is degraded through the MG reductase and aldehyde dehydrogenase system (r43 and r49) and the other half through the glyoxylase I/II and glyoxylase III pathways (r44/r45 and r46). The fluxes through the glyoxylase I/II or glyoxylase III pathways cannot be discriminated by the model. The resulting D- and L-lactate is the major source for pyruvate in addition to the phosphotransferase system (PTS; r1); the excess of pyruvate is excreted. These results also indicate that the carbon flow from pyruvate to DL-lactate (r47 and r50) is smaller than the flow from DL-lactate to pyruvate (r48 and r51), suggesting that the observed excretion of DL-lactate is connected to the activation of the MG pathway (r43, r44 and r46). The pyruvate kinase (r9) of the EMP pathway carries no flux. In the PTS system, glucose enters the cell with concomitant consumption of PEP to form glucose 6-phosphate and pyruvate (r1). The high PEP consumption due to the high glucose uptake rate is balanced by PEP synthase (r10), which converts pyruvate to PEP at the cost of 1 mol ATP. In general, it is found that pathways are utilized that yield less ATP: (i) the MG bypass is used, which circumvents substrate-level phosphorylation; (ii) the glyoxylate bypass (r26r29) is active instead of the entire TCA cycle; (iii) the less-energy-yielding enzymes of the respiratory chain, i.e. NADH dehydrogenase without proton-translocating capability (r32), FADH reductase (r34) and formate dehydrogenase (r35), exhibit elevated activities; and (iv) ATP-dissipating futile cycles are observed, e.g. between PEP carboxylase (r11) and ATP-consuming PEP carboxykinase (r12). Recently, the existence of the PEP carboxylase and PEP carboxykinase futile cycle has been established experimentally (Sauer et al., 1999
). The preferential formation of MG and lactate instead of acetate predicted by the model at low rx rs1 is in line with the measured late formation of acetate during the transient, indicating that low-energy-yielding reactions are utilized preferentially in the very first part of the adaptation period, since acetate formation (r15) is the only by-product formation reaction that results in ATP generation. However, these calculations represent a rough estimate, since the pseudo-steady-state approximation for intracellular metabolites is not warranted under these conditions.
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The maximum MG concentration excreted into the medium upon the upshift was dependent on the extent of the dilution rate change (Fig. 9). At dilution rate shifts where no oscillations were observed (from D=0·066 h1 to D<0·28 h1), the maximum concentration of MG which was reached during the adaptation period increased nearly linearly with the dilution rate change. At dilution rate shifts beyond 0·28 h1, where oscillations of respiratory activity occurred, the maximum MG concentration did not surpass 1·5 mg l1. This seems to be the threshold value that affects the cellular vitality of E. coli at a given biomass concentration, resulting in an unstable oscillatory behaviour. Oscillations in cell density in connection with MG formation have been reported so far only for the ruminal bacterium Prevotella ruminicola grown in nitrogen-limited continuous culture with an excess of glucose (Russell, 1993
). In this case, the cell density changed between 0·5 and 2·5 OD units with an oscillatory cycle of approximately 3 days.
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The metabolic reorganization during the transient also became apparent through the time profiles of the intracellular concentrations of key glycolytic pathway intermediates (FDP, GAP, DHAP) and co-metabolites (adenosine nucleotides). The rapidly increasing concentration of FDP reflects the bottleneck in the upper part of the EMP pathway, while the simultaneously increasing level of DHAP is considered to play a key role in the activation of the MG pathway. Activation of the MG pathway may also be stimulated by the transient increase in the cAMP concentration and by the increase in the AEC, resulting from elevated ATP concentrations, which are most likely accompanied by a transiently reduced level of intracellular available inorganic phosphate. The depletion of the intracellular inorganic phosphate pool is probably further amplified by the elevated level of sugar phosphates.
With a more substantial increment in the dilution rate upshift, the culture exhibited oscillatory behaviour, most likely caused by critical levels of MG in the culture broth. In this context, activation of the MG pathway must be regarded as a high-risk strategy, since formation and consumption of MG must be balanced very precisely. However, it has been shown for many substrates that their uptake capabilities are not fully exploited in low-nutrient environments (Teixeira de Mattos & Neijssel, 1997); for example, E. coli and K. aerogenes can rapidly increase the glucose transport capacity when grown in a glucose-limited chemostat culture even when growing close to the maximum growth rate (Neijssel et al., 1977
), indicating that cells always maintain a reserve in capacity to pick up a substrate that will then become unavailable to a potential competitor. It is thus tempting to speculate that cells are predisposed to harm themselves rather than to share a feast with a rival organism.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 July 2004;
revised 3 December 2004;
accepted 6 December 2004.
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