• Carbohydrate metabolism;
  • Metabolic cycle;
  • Polysaccharide;
  • 13C-Nuclear magnetic resonance
  • DQF-COSY, double-quantum-filter-correlation spectroscopy;
  • ED, Entner–Doudoroff;
  • EMP, Embden–Meyerhof–Parnas;
  • NMR, nuclear magnetic resonance;
  • PEP, phosphoenolpyruvate;
  • PHB, polyhydroxybutyrate;
  • PP, pentose-phosphate (pathway);
  • PPi, pyrophosphate;
  • TCA, tricarboxylic acid (cycle);
  • TQF-COSY, triple-quantum-filter-correlation spectroscopy


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Definitions and diversity of carbohydrate cycles
  5. 3General considerations on the isotopic investigation of carbohydrate cycles
  6. 4Reversibility
  7. 5Metabolic cycles
  8. 6Substrate or ‘futile’ cycles
  9. 7Concluding comments
  10. Acknowledgements
  11. References

The extension of 13C-nuclear magnetic resonance (NMR) techniques to study cellular metabolism over recent years has provided valuable data supporting the occurrence, diversity and extent of carbon cycling in the carbohydrate metabolism of micro-organisms. The occurrence of such cycles, resulting from the simultaneous operation of different and sometimes opposite individual steps, is inherently related to the network organisation of cellular metabolism. These cycles are tentatively classified here as ‘reversibility’, ‘metabolic’ and ‘substrate’ cycles on the basis of their balance in carbon and cofactors. Current hypotheses concerning the physiological relevance of carbohydrate cycles are discussed in light of the 13C-NMR data. They most likely represent system-level mechanisms for coherent and timely partitioning of carbon resources to fit with the various biosynthetic, energetic or redox needs of cells and/or additional strategies in the adaptive capacity of micro-organisms to face variation in environmental conditions.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Definitions and diversity of carbohydrate cycles
  5. 3General considerations on the isotopic investigation of carbohydrate cycles
  6. 4Reversibility
  7. 5Metabolic cycles
  8. 6Substrate or ‘futile’ cycles
  9. 7Concluding comments
  10. Acknowledgements
  11. References

Cyclic processes are key features in carbon metabolism. Some individual pathways with cyclic organisation (such as the tricarboxylic acid (TCA) cycle) occupy a central role in the metabolism of a wide variety of organisms. Other cycles are more intimately related to the network organisation of cellular metabolism, since they result from the simultaneous operation of different – and sometimes opposite – individual pathways. They induce unbalanced stoichiometries through metabolism within which extreme situations exist, such as substrate or ‘futile’ cycles, i.e. pure energy-dissipating processes. Such carbon cycles are known to operate in the carbohydrate metabolism of micro-organisms, plants [1] and animals [2], although they differ in both nature and significance. Carbohydrate metabolism is central to all metabolic processes and carbohydrates serve as the primary source of carbon and energy for a wide range of micro-organisms. The occurrence of carbohydrate cycling, where opposite processes operate simultaneously, in sugar-utilising organisms may significantly affect the carbon, energetic and redox status of the cells (including the loss of carbon). Such features seem at first to contrast with the tight regulation of metabolism usually assumed to provide coherent partitioning of resources over the various energetic and synthetic requirements, although some of these carbohydrate cycles may themselves play regulatory functions [3]. The knowledge of the occurrence and extent of carbohydrate cycling is therefore essential for an accurate appreciation of both organisation and function of cellular metabolism and also of the physiological significance of these processes. It is of particular significance in the field of metabolic engineering, where increased metabolic efficiencies are desired.

The occurrence of carbohydrate cycling has been proposed from both genetic and radiolabelling studies. Though these methods are still widely useful in the field [4], detailed investigations of these processes have long suffered from the lack of reliable methods to probe unambiguously their occurrence and extent. Genetic studies provide indirect and pleiotropic [5] information while radiolabelling requires cumbersome experimental procedures to access most cycles [6]. The effectiveness of 13C-nuclear magnetic resonance (NMR) techniques to trace the metabolic fate of individual carbons has provided an accurate tool for carbon cycling. NMR spectrometry is a relatively new technique to study microbial metabolism and initial studies primarily focussed on the elucidation of metabolic routes. The accuracy of the method to probe cycles was later demonstrated through extensive investigations of the TCA cycle [7–9]. In the last decade, a growing number of studies have provided evidence that carbohydrate cycling is a widespread feature in sugar-utilising micro-organisms. Most of these studies were not particularly centred on this topic but mainly on polysaccharide biosynthesis.

The aim of this review is to show how 13C-labelling studies – in conjunction with NMR – have provided new and consistent insight into the occurrence, diversity and extent of carbon cycling within carbohydrate metabolism (later referred to as ‘carbohydrate cycling’) of micro-organisms (Fig. 1). Carbohydrate metabolism includes a large number of reactions and pathways. Therefore a wide range of carbon cycling processes may occur within carbohydrate metabolism. We focus here on the main cycles associated with the central carbohydrate pathways in micro-organisms (Fig. 1). These include interconversion of (mono-)saccharides and of intermediates of carbohydrate pathways, the cycling of storage carbohydrates, and the cyclic operation of pathways (e.g. the pentose-phosphate (PP), Entner–Doudoroff (ED) and Embden–Meyerhof–Parnas (EMP) pathways). The particular case of substrate cycles will be emphasised. The mannitol [10–12], glycerol [13] and pentitol [14] cycles will not be discussed here. The organisms considered are mainly bacteria, but also include fungi.


Figure 1. Main cyclic processes within the carbohydrate metabolic pathways.

Download figure to PowerPoint

In this review, it is not intended to present a complete description of the use of NMR for metabolic studies or give a detailed account of isotopic studies of metabolism. The reader should refer to excellent reviews previously published on the topic [7,8,15–18].

2Definitions and diversity of carbohydrate cycles

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Definitions and diversity of carbohydrate cycles
  5. 3General considerations on the isotopic investigation of carbohydrate cycles
  6. 4Reversibility
  7. 5Metabolic cycles
  8. 6Substrate or ‘futile’ cycles
  9. 7Concluding comments
  10. Acknowledgements
  11. References

2.1Carbon cycling

Carbon cycling is defined as the re-formation of a metabolite using carbon atoms that were previously part of the same metabolite. Cycling is achieved by the occurrence of at least one forward and one backward step.


In this review, reversibility (Fig. 2a) refers to a carbon cycling process in which the backward step is strictly the reverse of the forward step. It is linked to the general property of (near-equilibrium) reactions to occur in both directions. Reversibility can be the property of one single reaction (catalysed by a single enzyme) or of a set of connected reversible reactions. The net balance of a reversible cycle is null. Two key examples will be further detailed in this review: (i) the aldolase/triose-phosphate isomerase cycle (see Section 4.1) and (ii) the non-oxidative branch of the PP pathway (see Section 4.2).


Figure 2. Definitions of carbohydrate cycles.

Download figure to PowerPoint

2.3Metabolic cycle

A metabolic cycle (Fig. 2b) is a carbon cycle in which one (or more) backward step(s) differ(s) from its forward counterpart (or has no forward counterpart). These metabolic cycles are made thermodynamically possible when both sets of reactions are exergonic. The forward and backward reactions differ in their cofactor requirements and are catalysed by distinct enzymes. A metabolic cycle is therefore a multi-enzymatic system and is inherent in the network organisation of cellular metabolism. A distinguishing difference from reversibility is that the net balance of a metabolic cycle is not null.

The cyclic operation of a pathway is a metabolic cycle involving the breakdown of a carbohydrate via that catabolic pathway followed by the re-formation of the carbohydrate. Each of the three main pathways of carbohydrate catabolism found in micro-organisms – the PP, ED and EMP pathways – may operate in a cyclic fashion.

2.4Substrate or ‘futile’ cycle

A substrate cycle (Fig. 2c) is a metabolic cycle for which the net balance consists solely of the dissipation of energy. In carbon cycles, the dissipation occurs mainly but not exclusively through the net hydrolysis of ATP (or any NTP). The term ‘futile’ was previously used in reference to the waste of energy, although some putative roles for these processes can now be proposed (see Section 6).


The term recycling expresses the possibility of a metabolite that has ever gone through a carbon cycle being able to enter this cycle (or another one) again (see Fig. 2d). Where the net balance of the cycle is not null, recycling is responsible for a loss of stoichiometry between the net number of metabolites used and the net number of cofactors used or formed.

3General considerations on the isotopic investigation of carbohydrate cycles

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Definitions and diversity of carbohydrate cycles
  5. 3General considerations on the isotopic investigation of carbohydrate cycles
  6. 4Reversibility
  7. 5Metabolic cycles
  8. 6Substrate or ‘futile’ cycles
  9. 7Concluding comments
  10. Acknowledgements
  11. References

Isotope studies of carbohydrate cycling rely on the same general basis as the investigation of any other metabolic processes: their occurrence causes either isotopic dilution or isotopic redistribution or both. Classically, isotope studies of metabolism include the examination of label incorporation from a labelled substrate into the end-product(s) – or intermediate(s) in some cases – of the investigated pathway(s). To establish the occurrence of a carbon cycle, it has to be shown that label goes from the substrate to the intermediate/end-product and back to the initial compound. Therefore, investigations on carbon cycling should rely principally on the collection of isotopic data from the starting, or cycled, metabolite, although ‘classical’ end-products may be helpful, provided that specific isotopic features can be seen therein.

Hexoses are the most commonly used carbohydrates in micro-organisms, in which they are efficiently converted into hexose-phosphates, mainly glucose-6-phosphate and fructose-6-phosphate. The latter compounds are further processed through the various catabolic or anabolic processes and may be considered the starting point of carbohydrate pathways, including carbohydrate cycles (‘hexose-phosphate cycling’), for carbohydrate-utilising micro-organisms. The hexose skeleton can be divided into two halves (carbons 1/2/3, the ‘top half’, and carbons 4/5/6, the ‘bottom half’) that undergo separate behaviour within a given catabolic pathway and/or between pathways (see Figs. 3 and 4). The bottom half forms glyceraldehyde-3-phosphate in all three catabolic pathways and may be additionally recycled in the PP pathway. The top half forms dihydroxyacetone-1-phosphate (EMP), pyruvate (ED), or is recycled with carbon losses (PP). Such differential behaviour provides not only the basis for discrimination of the pathways, but may also be responsible for specific isotopic redistributions within the carbohydrate cycles (Figs. 3 and 4). Care in the interpretation of isotopic redistributions has to be taken where symmetrical substrates or intermediates (such as mannitol) are concerned [19,20].


Figure 3. Isotopic transfers via the PP pathway from [1-13C]-, [2-13C]-, and [6-13C]glucose. 1: Glucose-6-phosphate dehydrogenase; 2: 6-phosphogluconolactonase; 3: 6-phosphogluconate dehydrogenase; 4: pentose-5-phosphate isomerase; 5: pentose-5-phosphate-3-epimerase; 6: transketolase; 7: transaldolase; 8: transketolase; 9: phosphoglucoisomerase; 10: triose-phosphate isomerase; 11: fructose-1,6-bisphosphate aldolase; 12: fructose-1,6-bisphosphatase.

Download figure to PowerPoint


Figure 4. Isotopic transfers via the EMP (top) and ED pathways (bottom) from [1-13C]- and [6-13C]glucose. 1: Glucose-6-phosphate dehydrogenase; 9: glucose-6-phosphate isomerase; 10: triose-phosphate isomerase; 11: fructose-1,6-bisphosphate aldolase; 12: fructose-1,6-bisphosphatase; 13: ATP-phosphofructokinase; 14: 6-phosphogluconate dehydratase; 15: 2-keto-3-deoxygluconate aldolase.

Download figure to PowerPoint

Both radioisotopes (14C, 3H, 32P) and stable isotopes (2H, 13C, 18O, etc.) are useful to probe the occurrence of cyclic processes. Radiolabelling approaches have the advantage of a high sensitivity (nM) but they present severe drawbacks (security, sample destruction, etc.) among which is the need for long and tedious operations to obtain positional information [4,6]. By contrast, the use of stable isotopes in conjunction with the non-destructive NMR techniques provides an efficient way to trace the metabolic fate of individual atoms (carbon or others) both in vitro and in vivo. Advantageously, NMR provides different parameters that enable detailed isotopic fingerprinting of molecules (see Fig. 5), giving much easier access to positional information. In addition, NMR can be applied to complex biological media, such as cellular extracts or culture media (in vitro NMR) or even whole cells (in vivo NMR). This last aspect is particularly important for detection of intracellular polymers that are not soluble and also for monitoring real-time kinetics. On the other hand, in vitro samples being rather homogeneous, direct or inverse 1H and 13C one-dimensional (1D) or more sophisticated two-dimensional (2D) sequences can give more detailed and quantitative data [8]. The main limitation of the NMR technique is its low sensitivity (in the mM range within the NMR tube), preventing the detection of low levels of accumulated compounds. The NMR detectability of a compound depends not only on its amount but also on its mobility. For some high-molecular-mass polysaccharides, which may be valuable isotopic markers for carbohydrate cycling (see below), their low mobility results in low detectability, especially in vivo. In addition to NMR, mass spectrometry is also increasingly used to perform stable isotopic investigations of cellular metabolism [21,22].


Figure 5. Isotopomer analysis by 13C- and 1H-NMR. A: 13C-NMR allows the determination of isotopic transfers as each 13C isotopomer gives rise to a specific 13C signature. A simple example is given for a molecule that contains two carbons. There are four possible isotopomers due to the combination of 13C (black circle) and 12C (unfilled circle) atoms being present in various amounts (12.5, 25, 50%) in the sample. Each carbon (C1 and C2) gives rise to a specific resonance, when two 13C enriched carbons are neighbours the signal is split into a doublet due to J13C–13C coupling, otherwise it gives a singlet. The final spectrum contains the combination of all resonances, the integrals of the signals correspond to the relative amount of each isotopomer. However, 13C-NMR presents some limitations: 12C atoms are not detected, thus isotopic dilution cannot be assessed directly. Complementary assays (enzymatic assays, high-performance liquid chromatography, etc.) must be performed to quantify the total amount of metabolites. In addition, some factors (relaxation, nuclear Overhauser effect, etc.) alter the signal intensity and must be considered to correct initial 13C areas and obtain quantitative data. B: Measurement of fractional labelling by means of 1H-NMR spectroscopy. The 1H-NMR resonance from protons directly linked to 13C atoms is split by one bond coupling constant 1J13C–1H. This gives rise to satellites that differ from signals exhibited by 12C-linked protons. This is shown in the very basic example given here, where two isotopomers are present in equal amounts. In more complex situations other types of coupling can be encountered, including J1H–1H couplings or two bonds coupling constant 2J13C–1H. Different 1D or 2D NMR pulse sequences can be used to detect and quantify these various types of signals and thus assess the isotopic dilution of metabolites but also (because of these long-range couplings) a possible isotopic distribution.

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Definitions and diversity of carbohydrate cycles
  5. 3General considerations on the isotopic investigation of carbohydrate cycles
  6. 4Reversibility
  7. 5Metabolic cycles
  8. 6Substrate or ‘futile’ cycles
  9. 7Concluding comments
  10. Acknowledgements
  11. References

Reversibility refers first to the same reaction (enzyme) functioning simultaneously in both forward and backward directions, but it can also apply to a set of reversible reactions that are interconnected. Reversible reactions generally represent neither metabolic bottlenecks nor targets for regulation. Moreover, the net balance of a reversibility cycle is null. Therefore one should question the interest in reversible processes. The first reason is that the reversibility of a reaction in vivo is often deduced from the standard free energy for the reaction measured in vitro or from the observation that a pathway operates that requires the reaction to be reversible. It may be important in some cases to verify that the reaction is actually reversible in vivo. Such confirmation can be achieved by means of labelling experiments. But the main reason for the current interest in reversible processes is the extension of the 13C-labelling techniques for the elucidation of metabolic fluxes. Combined with mass balances, these techniques provide a detailed picture of the partition of carbon resources within metabolic networks of increasing size and complexity. This knowledge is of major significance for a comprehensive understanding of metabolism at the whole-cell level and how metabolism is related to the cell physiology or function. Knowledge of metabolic fluxes is also particularly significant in the field of metabolic engineering to provide a basis for directed improvement of the performance of industrially exploited organisms. Indeed, measurement of metabolic fluxes has become a major achievement for both physiologists and bioengineers.

Metabolic fluxes can be evaluated from the 13C-labelling pattern of metabolites provided the underlying metabolic network is known and can be computed. The reader should refer to [23–27] for more details on these aspects. Complete information on the fate of single carbons throughout the network under study should be known to account accurately for the observed labelling patterns. In that respect, some reversible reactions play a key role, either because they themselves affect the labelling state of metabolites, or because they enable isotopic events generated elsewhere in the metabolic network to be detected in analysed metabolites, or both. These reactions and their effects on the labelling state of metabolites should be accounted for properly to avoid misinterpretation of labelling data and errors in the calculated metabolic fluxes.

Two key examples will be further detailed for carbohydrate metabolism. One (the aldolase/triose-phosphate isomerase triangle) is illustrative of how positional information in labelling experiments can resolve a simple reversibility cycle, whereas the other (the non-oxidative steps of the PP pathway) puts emphasis on a process that has profound isotopic effects that are still being debated.

4.1The aldolase/triose-phosphate isomerase triangle

Fructose-1,6-bisphosphate aldolase catalyses the cleavage of fructose-1,6-bisphosphate into dihydroxyacetone-phosphate and glyceraldehyde-3-phosphate, which are further isomerised by the triose-phosphate isomerase (Fig. 1). The former reaction is readily endergonic in vitro (ΔG°′=+27 kJ mol−1) but its known involvement in either gluconeogenesis or glycolysis indicates that it functions at near equilibrium in vivo. Most direct evidence for reversibility in this triangle has been obtained from the scrambling of 13C (or 14C) label between the C1 and C6 positions of the hexose skeleton. For instance [1-13C]glucose will be converted into C1-labelled dihydroxyacetone-1-phosphate and unlabelled glyceraldehyde-3-phosphate. The label can be transferred to the C3 of glyceraldehyde-3-phosphate via the triose-phosphate isomerase activity. Finally, after the reverse reaction of aldolase, label can be found on either C1 or C6 of the regenerated fructose-1,6-bisphosphate. Similarly C6-labelled glucose should result in transfer of label to the C1 position.

As previously mentioned, the purification of the compounds and their chemical or enzymatic dismutation is required to obtain positional information in radiolabelling experiments. In the case of fructose-1,6-bisphosphate, a seven-step procedure is necessary after the compound is purified to obtain such information [28]. It is demonstrative of the power of NMR techniques that the very first NMR studies of metabolism provided direct evidence for reversibility of the triangle. In their pioneering work, den Hollander et al. [29] demonstrated that the two triose-phosphates freely equilibrated in Saccharomyces cerevisiae. Later they showed that the resulting scrambling of the 13C label was more important during aerobiosis than anaerobiosis [30] as clearly shown in Fig. 6. In this experiment, S. cerevisiae cells incubated with [6-13C]glucose, [6-13C]fructose-1,6-bisphosphate and [1-13C]fructose-1,6-bisphosphate were clearly detected on 13C-NMR spectra of cell extracts but the ratio C1/C6 was lower under anaerobic conditions compared to aerobic conditions. In addition, it was observed that label scrambling was about a factor of two with [6-13C]glucose compared to [1-13C]glucose. This last point was due to the interference of the PP pathway (see Section 4.2). The scrambling of label from C1 to C6 reported at the level of fructose-1,6-bisphosphate was also observed in S. cerevisiae[31] and in other micro-organisms including Escherichia coli[32], Corynebacterium melassecola[33] and Lactococcus lactis[11,34].


Figure 6. Expanded view of the 60–70 ppm part of the 13C-NMR spectrum of extracts prepared during aerobic and anaerobic glycolysis of [6-13C]glucose by S. cerevisiae. The ratio [1-13C]fructose-1,6-bisphosphate/[6-13C]fructose-1,6-bisphosphate is higher in aerobic conditions. This spectrum was recorded by den Hollander et al. [30]. G-C6: [6-13C]glucose; G6P-C6: [6-13C]glucose-6-phosphate; GLY: glycerol; α-GP: α-glycerophosphate; FRU-P2-C1: [1-13C]fructose-1,6-bisphosphate; FRU-P2-C6: [6-13C]fructose-1,6-bisphosphate.

Download figure to PowerPoint

Unfortunately, the concentration of fructose-1,6-bisphosphate is too low in many organisms to be detected, especially in NMR experiments in vivo. Evidence for the operation of the reversible cycle was mainly obtained from the labelling patterns in upstream metabolites such as fructose-6-phosphate or glucose-6-phosphate, or in storage carbohydrates. The observation of the labelling patterns in these compounds indicates the occurrence of not only the reversible cycle but also the triose-phosphate cycle. Indeed, label scrambling due to the aldolase/triose-phosphate isomerase triangle is a key feature providing evidence of the latter cycle, as will be emphasised in Section 5.2.3.

4.2Non-oxidative branch of the PP pathway

The PP pathway is widely distributed in micro-organisms, plants and animals [35]. The pathway provides the cell with anabolic requirements such as NADPH (for most biosynthetic processes), ribose-5-phosphate (for nucleic acid synthesis), or others (amino acids, secondary metabolites, etc.). In the PP pathway (Fig. 3), the first series of non-reversible reactions – the oxidative steps – ensures oxidative decarboxylation of glucose-6-phosphate into ribulose-5-phosphate. A second series – the non-oxidative steps – allows the conversion of ribulose-5-phosphate into other sugars, including ribose-5-phosphate or glycolytic intermediates (fructose-6-phosphate and glyceraldehyde-3-phosphate).

The reactions of the non-oxidative steps of the PP pathway have profound effects on the labelling state of key intermediates, not only in the carbohydrate pathways, but also in the overall metabolic network. This is mainly because:

  • 1
    The reactions include cleavage and reassembly of carbon backbones, resulting in significant redistribution of label inside the molecules; these redistributions can be deduced from the carbon atom transitions in the reactions and concern only the upper part of the hexose molecule in the classical PP pathway (see Fig. 3).
  • 2
    All the reactions – isomerisation, epimerisation, transaldolisation and transketolisation – are assumed to be reversible, enabling the non-oxidative branch as a whole to function in both directions (from ribulose-5-phosphate to fructose-6-phosphate+glyceraldehyde-3-phosphate and from fructose-6-phosphate+glyceraldehyde-3-phosphate to ribulose-5-phosphate). This allows isotopic exchange (Fig. 3) to occur inside both pools of hexose-6-phosphates, triose-phosphates and pentose-5-phosphates [36,37]. The extent of the exchanges depends on the extent of reversibility.
  • 3
    The non-oxidative branch is the part of the PP pathway that interconnects the oxidative steps to other glycolytic pathways (ED or EMP pathways) and to numerous anabolic processes. Therefore, in feeding experiments the range of metabolites for which the labelling state is affected by the non-oxidative steps of the PP pathways goes far beyond simple PP intermediates.

For the above reasons the isotopic redistribution resulting from the non-oxidative steps should be accounted for properly to avoid misinterpretation of labelling data and/or errors in the measurement of metabolic fluxes. Indeed, a number of studies in recent years have concentrated on the in-depth analysis of isotopic effects resulting from these non-oxidative steps [36–39]. Because the nature of the isotopic redistributions can be easily determined, most of the ongoing debate has focussed on the appropriate way to account for reversibility [38–40].

To assess the reversibility within all the non-oxidative steps, one should be able to monitor separately the individual fluxes, i.e. the flux from ribulose-5-phosphate to fructose-6-phosphate+glyceraldehyde-3-phosphate and the flux from fructose-6-phosphate+glyceraldehyde-3-phosphate to ribulose-5-phosphate. For the sake of clarity, the former will be referred to as ‘forward’ flux (or direction) and the latter as ‘backward’ flux (or direction).

4.2.1Use of monolabelled hexose

The most common labelled substrate for investigating microbial metabolism, [1-13C]glucose, is not a suitable substrate to provide direct evidence for the forward flux in the non-oxidative steps because the C1 of glucose is lost in the oxidative part of the pathway, and there is no input of label at the level of ribulose-5-phosphate. However, it provides a specific isotopic signature in the opposite direction, since [1-13C]fructose-6-phosphate – generated from exogenous [1-13C]glucose – used in the backward direction results in labelling at the C1 of ribulose-5-phosphate (or ribose-5-phosphate). Unfortunately there are poor reports of such labelling patterns for ribose-5-phosphate in micro-organisms. Some direct evidence for the operation of both backward and forward directions in feeding experiments with [1-13C]glucose were nonetheless obtained for two Rhizobiaceae, Agrobacterium and Sinorhizobium meliloti, a family known to possess both PP and ED pathways [46,47]. These bacteria synthesise polysaccharides (curdlan, β-glucans) made of glucose units. In feeding experiments performed with [1-13C]glucose, an unexpected labelling was observed at the C3 position of the glycosidic units. The breakdown of the labelled glucose into pyruvate via the ED pathway and recycling of the labelled pyruvate into hexose-phosphates could explain such labelling. But in that case labelling at C4 would be observed first (see Fig. 4), which was not the case. The labelling at C3 was most probably the result of a backward flux in the non-oxidative steps, yielding [1-13C]ribulose-5-phosphate, followed by the use of the latter in the forward direction. This isotopic exchange process enables the labelling of hexose at both C1 (again) and C3 [36]. The opportunity to observe the isotopic exchange in these bacteria probably reflected the extent of the transketolase and transaldolase activities therein.

From both theoretical considerations [36,38] and experimental observations [6,37,48–50], a well-suited substrate for the assessment of the forward flux within the non-oxidative steps is [2-13C]glucose [51]. Breakdown of [2-13C]glucose via the oxidative steps of the pathway forms [1-13C]ribulose-5-phosphate, which, in turn, forms hexose-phosphates labelled at C1 and/or C3 in the forward reactions (as above). Such labelling patterns were reported for a number of polysaccharide-producing organisms utilising the PP and ED pathways [41–45,51,52], including the two Rhizobiaceae mentioned above. Carbohydrate metabolism in these species will be discussed in more detail later in this review (Section 5.2). The same strategy was used to assess the contribution of the PP pathway to glycolysis in S. cerevisiae[53].

The choice of a hexose labelled in the bottom half of the molecule does not itself provide direct evidence for reversibility within the non-oxidative steps, as the bottom half of the hexose molecule has the same behaviour within all three glycolytic pathways (yielding glyceraldehyde-3-phosphate) and is transferred intact within the non-oxidative steps of the PP pathway. However, this provides a reliable control to experiments with label in the top half, in that isotopic redistribution observed in such experiments is likely to be due to the PP pathway if the symmetric redistribution is not observed from label in the bottom half.

4.2.2Use of uniformly labelled hexose

Recent years have seen extension of the use of uniformly labelled substrates in 13C-labelling experiments. This strategy was first introduced by Gagnaire and Taravel [52] and perfected by Szyperski [8,54]. In these experiments, only a part of the substrate is uniformly labelled, whereas the remaining substrate can be totally unlabelled or made of a mixture of unlabelled and monolabelled molecules. These molecules are extensively cleaved and reassembled throughout the metabolic pathways. Because reassembly is between carbon blocks that are uniformly – or partially – labelled and unlabelled, it generates a variety of new molecules with specific labelling patterns. Each of the new molecules is called an isotopomer and there are 2n isotopomers for a metabolite with n carbons. The isotopomeric composition of a metabolite can be determined from NMR data by exploiting scalar couplings (see Fig. 5). This composition is not randomly determined but depends uniquely on the nature and extent of the processes through which the metabolite is formed. The large number of different isotopomers that can be generated provides valuable data to discriminate between biochemical pathways or reactions that cannot be assessed when monolabelled substrates are used. The isotopomer approach provides rich information on the overall metabolic network since the isotopomeric composition can be evaluated for a large number of metabolites. The interpretation of such complex data necessitates mathematical description of the underlying metabolic network and iterative computing methods to solve the equations. Initially developed for 1-13C-labelled hexose experiments [55,56], such interpretation methods were further extended to uniformly labelled substrates and successfully applied to a growing number of organisms including mainly E. coli[54,57,58], Corynebacterium glutamicum[59], Bacillus subtilis[60] or S. cerevisiae[61].

The non-oxidative steps of the PP pathway play a key role in the accuracy of isotopomer-based strategies to provide valuable information on metabolic fluxes. The set of molecular rearrangements therein generates numerous combinations of label redistribution when mixtures of uniformly labelled and unlabelled substrates are used. The resulting isotopomeric patterns provide the basis for accurate assessment of fluxes within the PP pathway. But it was recently emphasised [38,62] that the reversibility of the non-oxidative steps in the PP pathway should be kept in mind to avoid misinterpretation and errors in flux measurements. Indeed, considerable variation in the measurement of fluxes through the PP pathway could be obtained from the same set of experimental data depending on the treatment applied to account for reversibility [54,62]. Recent modelling of data from carbon-labelled studies given in the literature has shown the operation and significance of this exchange process in several cellular systems [38].

It was recently outlined that the enzymes in the non-oxidative steps can accept a broader range of substrate than is usually assumed [39]. This is particularly the case for transaldolase and transketolase, i.e. the enzymes that are responsible for cleavage and reassembly of intermediates. For example, transketolase accepts glucose as the donor of a C2 fragment and hydroxypyruvate as an acceptor, in addition to its usual substrates. Different combinations of donors and acceptors are possible. Van Winden et al. [39] have calculated that transketolase could catalyse at least six different reactions instead of the two usually considered. The observations apply to transaldolase as well, which can catalyse three different reactions. Therefore the range of possible carbon atom transition – and, thus, of label redistribution in labelling experiments – may be much wider than usually assumed.

5Metabolic cycles

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Definitions and diversity of carbohydrate cycles
  5. 3General considerations on the isotopic investigation of carbohydrate cycles
  6. 4Reversibility
  7. 5Metabolic cycles
  8. 6Substrate or ‘futile’ cycles
  9. 7Concluding comments
  10. Acknowledgements
  11. References

This section considers the cyclic operation of carbohydrate pathways, i.e. metabolic cycles where a breakdown of the hexose (catabolic part of the cycle) is followed by its re-formation (anabolic part of the cycle). Breakdown of the hexose within the carbohydrate pathways generates 5-, 4- or 3-carbon compounds, with pentose-phosphates and triose-phosphates being the key intermediates and pyruvate being the end-product. Each of these compounds can be recycled into a hexose, although by different routes. Direct evidence for cycling of pyruvate (into hexose-phosphates) is hampered by isotopic interferences from non-carbohydrate pathways (TCA cycle, anaplerotic reactions, glyoxylate pathway). Given that the processes involved in pyruvate recycling are more related to metabolism around the TCA cycle, they will not be considered in this review dealing with purely carbohydrate processes, except for some specific aspects. The reader should refer to the current literature on the subject (as an example, see [63]).

5.1Cycling of pentose-phosphates

PP cycling (Figs. 1 and 3) is obtained by the consecutive operation of the oxidative and non-oxidative steps of the PP pathway. It results in the conversion of three glucose-6-phosphate molecules into two fructose-6-phosphate molecules, one glyceraldehyde-3-phosphate molecule and three CO2 molecules. The cycle is achieved by isomerisation of fructose-6-phosphate into glucose-6-phosphate. From a quantitative point of view, cycling concerns the total number of glucose-6-phosphate molecules converted into ribose-5-phosphate minus the part of PP intermediates used for anabolic purposes. The PP cycle is well-known [6] and appears in biochemistry textbooks as a mechanism enabling efficient synthesis of NAD(P)H at the expense of carbon losses.

In organisms devoid of phosphofructokinase or an equivalent enzyme (i.e. in non-EMP-utilising species), the fructose-6-phosphate molecules cannot be converted further into triose-phosphates. They can, however, be used for other metabolic processes (anabolism) or they can be converted into glucose-6-phosphate, achieving a complete cycle. As previously mentioned, the occurrence of complete PP cycling was reported to occur in some ED-utilising, polysaccharide-producing bacteria from the observation of the isotopic transfer from [2-13C]hexose towards C1 and C3 of the glycosidic units in the polymers (see Fig. 3). More details about these organisms are given in Section 5.2.

In organisms (EMP-utilising species) where the phosphofructokinase is active, fructose-6-phosphate may be converted into triose-phosphates. This enables a greater number of different metabolic routes to occur compared to previous organisms. A complete description of these numerous metabolic routes (up to 13) was given in [64] using the ‘elementary flux modes’ description. When considering the occurrence of PP cycling in these species, one should discriminate among the molecules of fructose-6-phosphate generated by the PP pathway:

  • 1
    The molecules that are converted into triose-phosphates. In this case, there is no cycling since the PP pathway provides an alternative route for the provision of glycolytic fructose-6-phosphate (and triose-phosphates).
  • 2
    The molecules that are actually converted back into glucose-6-phosphate. This is true PP cycling.

Both processes result in the formation of NADPH but only the former enables the formation of ATP and NADH from fructose-6-phosphate.

5.1.1Contribution of the PP pathway to glycolysis

The contribution of the PP pathway to glycolysis is a widespread phenomenon in EMP-utilising organisms. A significant number of studies have been devoted to determining the partitioning of glycolytic flux through both PP and EMP pathways. The first evaluations were obtained from experiments using [1-13C]glucose, where the label is lost as CO2 via the PP pathway [65]. The unlabelled fructose-6-phosphate formed by the pathway generates an isotopic dilution, which is generally assessed at the level of glycolytic end-products or further metabolites. Various considerations have shown the limitations of this approach, including isotopic interferences with other pathways and dilution arising from unlabelled endogenous compounds. A more accurate estimate of the PP contribution was obtained from parallel experiments using C6-labelled hexose, where the label is incorporated into glycolytic end-products via both pathways. In that case, incorporation of label in the end-products represents the total glycolytic flux from the substrate. The contribution of the PP pathway can be evaluated from the excess in label compared to experiments with C1-labelled hexose. As illustrative examples, Rollin et al. [33] and Campbell-Burk et al. [66] used this strategy for studying Corynebacterium melassecola and S. cerevisiae, respectively. A further step was made by using a mixture of uniformly labelled and unlabelled hexose – i.e. an isotopomer approach – enabling detailed analysis of the formation and breakage of covalent bonds via the analysis of coupling patterns.

As previously mentioned, detailed quantitative analysis of metabolic fluxes by means of mathematical modelling of metabolism has been achieved for a few organisms (E. coli, C. glutamicum, B. subtilis, S. cerevisiae). These selected but illustrative examples emphasised the extent of the contribution of the PP pathway to glycolysis in bacteria (in the range of 20–30% of the glucose input flux in B. subtilis[60], 66–88% in C. glutamicum[56,59] and 53–78% in E. coli[62]). In contrast to bacteria, the contribution of the PP pathway is very low in S. cerevisiae (<4%) [67]. In the amino acid producer, C. glutamicum, the strategy was applied to compare various physiological situations (lysine production, glutamate production and growth) [68] indicating that the PP contribution correlated with the demands for NADPH for biosynthetic purposes. Such observations were later confirmed by experiments where a three-fold reduction in PP contribution was observed in cells where NADPH-dependent enzymes involved in amino acid synthesis were replaced by NADH-dependent enzymes [69].

All these data are consistent with the role of the PP pathway in the supply of NADPH for anabolic purposes, although other systems may be involved, depending on the sugar [70]. Because the EMP pathway provides only NADH, whereas the PP pathway provides either NADPH only (when pentose-phosphates are used for anabolism) or both NADPH and NADH (through the glycolytic PP route), the splitting of hexose-phosphates between the three processes plays a key role in controlling the flow of reductive power between the NADP and NAD redox systems. Two studies reporting on the metabolism in cells lacking phosphoglucoisomerase [53,71]– a situation where direct feeding of the EMP pathway is not allowed and where the PP pathway is the only glycolytic process – provided supportive data [53,71]. In such a situation, cells are affected for growth on glucose, and it is assumed that this is because the PP pathway flux is too low to support growth. In S. cerevisiae[53], spontaneous revertants able to grow on glucose but still lacking the enzyme were obtained. By using [2-13C]glucose as substrate, it was shown that the activity of the PP pathway was significantly increased in the revertants. This observation indicated that the defect in EMP glycolysis was compensated by increased carbon fluxes through the PP pathway. It suggests that the unique operation of the PP pathway can sustain growth on glucose provided the flow of sugar in the pathway can increase. Interestingly, the increased flux in the PP pathway in the revertants was associated with increased activities of both NADP- and NAD-dependent enzymes. Moreover, the significance of NADH formation in these cells was emphasised by a large accumulation of glycerol that provided a route for regenerating NAD+. Similarly to S. cerevisiae, E. coli cells defective in phosphoglucoisomerase have reduced growth rates [71]. Detailed analysis of metabolic fluxes in these cells indicated that glucose was primarily routed via the PP pathway, resulting in disturbed redox balances, given the process strongly favours the formation of NADPH. Interestingly, improved growth was observed in cells overexpressing a transhydrogenase enabling the transfer of electrons from NADPH to NAD+, thereby providing a sink for extra NADPH. In this case the glycolytic flow via the PP pathway was increased. Taken together, these studies outline the negative control exerted by the NADPH/NADP+ on the PP pathway flux. They emphasise also that a primary role for the EMP pathway might be the efficient control of the cellular redox balances in coordination with the PP pathway.

5.1.2True PP cycling

Note that in the two studies, the lack of phosphoglucoisomerase prevented the conversion of fructose-6-phosphate back into glucose-6-phosphate, i.e. prevented recycling of pentose-phosphates occurring. But it is not obvious to what extent this feature may contribute to the observed phenotypes. In most studies where metabolic fluxes were calculated, the sum of the glucose-6-phosphate molecules entering the two pathways determined the rate of sugar consumption. This meant that PP-derived fructose-6-phosphate fed only the bottom half of glycolysis, i.e. there was no recycling. Indeed, it is generally assumed that all fructose-6-phosphate generated through the PP pathway is used for glycolytic purposes. Interestingly, PP recycling was recently observed in C. glutamicum during co-utilisation of glucose and acetate [59]. Growth on acetate was characterised by significant gluconeogenic activities to generate hexose-phosphates that entered the PP pathway. In cells grown on glucose, glucose-6-phosphate was processed mostly (nearly 90%) via the PP pathway and the remainder via the EMP pathway. When growth was carried out on glucose plus acetate, both substrates were utilised. Acetate was the predominant substrate but there was no net gluconeogenesis. Glucose was catabolised only via the PP pathway. A part of the fructose-6-phosphate so formed was routed towards the bottom half of glycolysis – i.e. there was a net glycolytic flux in these cells – whereas the major part (more than 70%) was recycled into glucose-6-phosphate. Such a recycling situation resulted in the rate of PP activity being 1.7-fold higher than the rate of glucose consumption.

5.2Triose-phosphate cycling

Triose-phosphate cycling can be associated with any of the carbohydrate pathways. It describes situations where the triose-phosphate molecules generated through catabolism are converted back into hexose-phosphates. The anabolic part of the cycle includes the consecutive operation of the triose-phosphate isomerase, aldolase, fructose bisphosphatase and phosphoglucoisomerase (Fig. 3) and is the common to all triose-phosphate cycles. The key reaction in these steps is the non-reversible dephosphorylation of fructose-1,6-bisphosphate into fructose-6-phosphate catalysed by fructose bisphosphatase. Diversity in triose-phosphate cycling processes relies on the catabolic part of the cycle and the effects of cycling differ significantly from one carbohydrate pathway to another (see Fig. 7). They will be considered separately in the following sections.


Figure 7. Comparing the effects of triose-phosphate cycling in organisms utilising the EMP (left) or ED (right) pathways. Due to their respective pathway structures and cofactor requirements, the effects of triose-phosphate cycling are different in EMP- and ED-utilising species. Two types of effects can be distinguished: (i) effects on the total flow in the overall glycolytic process; (ii) effects on the cofactor balances. The cofactors internal to the cycle and the cofactors associated with the cycle should both be considered. The latter cofactors that those used or formed in the linear branches directly connected to the cycles. The assumptions made are:

• The system is at steady state.

• The rate of hexose uptake is 100 arbitrary units (AU).

• The hexose is phosphorylated via an ATP-dependent system.

• The glyceraldehyde-3-phosphate dehydrogenase is NAD-dependent.

• Pyruvate is the end-product of the carbohydrate pathways and the overall glycolytic flux is equivalent to the rate of pyruvate formation.

Definitions: r is the recycling flux, i.e. the number of glucose-6-phosphate molecules that are reformed from triose-phosphate per time unit. vNADPH is the rate of NADPH formation. vNADH is the rate of NADH formation. vATP is the rate of ATP formation. vNADPH is the rate of NADPH formation.

Download figure to PowerPoint

In EMP-utilising species, triose-phosphate cycling via the EMP pathway (a simplified scheme is shown on the left of Fig. 7) is a carbon-conservative process, i.e. one glucose-6-phosphate is reformed from one hexose entering the cycle. There is one linear path downstream of the cycle. The effects are (see table on the left of Fig. 7):

  • 1
    The net rate through the EMP pathway is independent of the rate of recycling (this is because there is no carbon loss along the process).
    • image
  • 2
    The balance for internal cofactors (ATP) is affected.
    • image
  • 3
    The balance for external cofactors (NADH) is not affected.
    • image

Because the only metabolite affected is ATP, this triose-phosphate cycle is a substrate cycle.

In ED-utilising species, triose-phosphate cycling via the ED pathway (a simplified scheme is shown on the right of Fig. 7) is a semi-conservative process, in which only the bottom half of the hexose molecule entering the process is recycled. Only one glucose-6-phosphate molecule can be reformed from one hexose entering the cycle. The different processes downstream of the cycle can be split into two distinct and linear branches: (i) the direct formation of pyruvate via aldolase cleavage and (ii) the conversion of triose-phosphates into pyruvate. The effects are (see table on the right of Fig. 7):

  • 1
    The net rate of pyruvate formation appears to be independent of the rate of recycling.
    • image
  • 2
    The balance for internal cofactors (NADPH) is affected.
    • image
  • 3
    The balance for external cofactors (NADH, ATP) is affected as well.
    • image
    • image

Note that the effects on NADPH and NADH are opposite and that the extent to which ATP is affected in ED species is higher than for the EMP-related substrate cycle given the same recycling rate.

5.2.1Triose-phosphate cycling in the PP pathway

Triose-phosphate cycling is a further step in the cyclic operation of the PP pathway where the glyceraldehyde-3-phosphate molecules generated in the PP cycle are converted back into glucose-6-phosphate (Fig. 1). Given that glyceraldehyde-3-phosphate is derived from the bottom half of the hexose skeleton and is further isomerised into dihydroxyacetone-1-phosphate to deliver the top half of the reformed hexose-phosphate, this results in the incorporation of label in the top half of reformed hexose-phosphate from hexose labelled in the bottom half (e.g. label at C1 from C6-labelled hexose), whereas the reciprocal redistribution (e.g. label at C6 from C1-labelled hexose) can be obtained via the PP pathway, except for ED-utilising species.

The key enzyme in the process is the gluconeogenic enzyme fructose-bisphosphatase. Therefore triose-phosphate cycling may be expected to occur in situations where gluconeogenesis predominates rather than glycolysis. Clear isotopic evidence for triose-phosphate cycling through the PP pathway under glycolytic conditions was nonetheless obtained in micro-organisms also utilising the ED pathway [41–43,52] and will be detailed in Section 5.2.2. Operation of the cycle in EMP-utilising organisms is more difficult to evaluate because triose-phosphate cycling may occur simultaneously via the EMP pathway (see Fig. 1), leading to the same redistribution of label from C6 to C1 of hexose. As a distinguishing difference, triose-phosphate cycling via the EMP pathway also results in the reciprocal redistribution from 1-labelled hexose to the C6 of hexose-phosphate (Fig. 4). Ambiguity remains in those organisms where both PP cycling and EMP triose-phosphate cycling were shown to occur, as part of the recycled triose-phosphates may have been formed through the PP pathway. This point will be further discussed in Section 5.2.3.

5.2.2The ED cycle

The ED pathway is mainly found in Gram-negative bacteria and in Archaea but is also found in some Gram-positive bacteria and fungi [71,72]. The pathway shares with the PP pathway the dehydrogenation of glucose-6-phosphate into 6-phosphogluconate, catalysed by the glucose-6-phosphate dehydrogenase (Fig. 4). The first committed step of the ED pathway is the dehydration of 6-phosphogluconate into 2-keto-3-deoxy-6P-gluconate, catalysed by a 6-phosphogluconate dehydratase. The 2-keto-3-deoxygluconate is further cleaved by a 2-keto-3-deoxygluconate aldolase into pyruvate and glyceraldehyde-3-phosphate. The ED pathway can operate in different modes [71]– i.e. phosphorylated, non-phosphorylated, etc. – including a cyclic mode (see below). All alternative ED pathways involve both dehydrogenation and aldolase cleavage. Triose-phosphate cycling in ED-utilising organisms (see Figs. 1 and 3) is achieved when the glycolytic glyceraldehyde-3-phosphate molecules are recycled into hexose-phosphates. This process – referred to as the ED cycle – is a semi-conservative cycle where only the bottom half of the hexose molecule is recycled. Recycling of pyruvate has not been shown so far in these organisms.

The occurrence of the ED cycle was initially demonstrated in Xanthomonas phaseoli[73]. It is known to operate also in other proteobacteria – including Acetobacter, Agrobacterium, Azotobacter, Pseudomonas, Rhizobium, Thiobacillus[72], and other Xanthomonas species (Letisse and Lindley, personal communication) – but it has not been reported so far in other ED-utilising species, including the archaebacteria currently under investigation [74,75], or in other bacteria and fungi. Bacteria utilising the ED cycle have common metabolic characteristics, including the preferential utilisation of organic acids over carbohydrates and the ability to synthesise exopolysaccharides. Most, but not all, of them are devoid of phosphofructokinase or similar enzymes, and therefore do not catabolise sugars via the EMP pathway. They often utilise a complete PP pathway. ED cycle in Pseudomonas

The occurrence of the ED cycle was particularly well documented in the genus Pseudomonas where the predominance of the ED pathway for sugar catabolism has been widely described [72,74–77]. In alginate-producing Pseudomonas (P. aeruginosa, P. mendocina), and in other alginate-producing bacteria such as Azotobacter vinelandii, the cyclic ED was shown to contribute to alginate biosynthesis when glucose was the carbon source [78]. Therefore the metabolic pathways involved in the conversion of glucose into alginate were studied in detail in these organisms. Alginate is made of mannuronic and l-guluronic acids, both derived from fructose-6-phosphate. In these species, the direct conversion of glucose into the glycosidic precursor can be achieved by active uptake of glucose, intracellular phosphorylation and isomerisation, [77]. But two key observations were reported to explain the contribution of the ED cycle to the conversion of glucose into the glycosidic units of alginate [79]:

  • The activity of phosphoglucoisomerase, catalysing the isomerisation of glucose-6-phosphate into fructose-6-phosphate, was reported to be low in the organisms concerned [72,76,80]. Moreover, the enzyme is subject to inhibition by 6-phosphogluconate and the rapid conversion of glucose into the latter can result in efficient inhibition of the enzyme [81], thereby limiting or preventing the isomerisation of glucose-6-phosphate into fructose-6-phosphate.
  • Most of the ED-utilising bacteria can oxidise glucose into gluconate in the periplasm. The resulting gluconate molecules can enter the cells [46] and feed the catabolic pathway at the level of 6-phosphogluconate. This pathway (the ‘gluconate by-pass’) does not allow the formation of glucose-6-phosphate because the reaction catalysed by glucose-6-phosphate dehydrogenase is not reversible. In Pseudomonas putida, glucose catabolism occurs almost totally via the gluconate by-pass [80]

According to these observations, both the formation of glucose-6-phosphate and its isomerisation into fructose-6-phosphate appear to be limited in glucose-grown cells. Therefore the direct polymerisation of introduced glucose cannot satisfy the anabolic demands for fructose-6-phosphate without the additional involvement of the ED cycle. The latter process acts as an alternative route for the conversion of glucose into fructose-6-phosphate in these bacteria (Fig. 8A).


Figure 8. Comparison of carbohydrate cycling in Pseudomonas and A. vinelandii (A) and in Rhizobiaceae (B).

Download figure to PowerPoint incomplete cycle?

One should note that the inhibition of phosphoglucoisomerase might prevent not only the conversion of glucose-6-phosphate into fructose-6-phosphate but also the opposite conversion of fructose-6-phosphate into glucose-6-phosphate. This is further supported by the fact that the inhibition of the enzyme by 6-phosphogluconate was demonstrated in vitro using fructose-6-phosphate as the substrate for the phosphoglucoisomerase assays [81]. This means that, under in vivo conditions, the fructose-6-phosphate molecules generated via the ED cycle may be not actually be converted into glucose-6-phosphate, thereby preventing recycling to occur. In other words, the cycle may be not complete (Fig. 8A).

Most of the studies reported above were performed before NMR became commonplace in metabolic studies. Although radiolabelling techniques in conjunction with genetic studies have provided unequivocal evidence for the occurrence of the ED cycle, the difficulty in obtaining positional information when using radiotracers may have precluded fine analysis of the contribution of ED cycling to the metabolism of glucose. In 1996, a very detailed NMR study of alginate synthesis in A. vinelandii[51] provided supportive evidence that all alginate units were generated via the ED cycle on glucose. By feeding the organism with variously monolabelled glucose, the authors first ruled out both the EMP and PP pathways for the catabolism of the sugar. To determine if parts of the polymer units were formed by direct incorporation of the precursor rather than via the ED cycle, they fed the bacteria with a mixture of unlabelled (87.5%) and uniformly 13C-labelled (12.5%) glucose. Direct incorporation of the exogenous substrate should result in uniformly labelled glycosidic units, whereas cleavage and reassembling of labelled and unlabelled three-carbon units within the ED cycle should lead to half-labelled glycosidic units. Since 12.5% of the precursor was labelled, the probability of uniformly labelling glycosidic units by reassembling two labelled three-carbon blocks was only 1.6%. To probe the reassembly of three-carbon blocks, Beale and Foster [51] used ‘filtering’ NMR techniques, enabling the specific detection of molecules bearing two (double-quantum-filter-correlation spectroscopy; DQF-COSY) or three (triple-quantum-filter-correlation spectroscopy; TQF-COSY) adjacent 13C nuclei. While 3/4 13C2 blocks were not detected in DQF-COSY experiments, only 1/2/3 and 4/5/6 13C3 blocks could be reported in TQF-COSY experiments. The occurrence of uniformly labelled glycosidic units would result in 3/4/5 13C3 blocks, which were not detectable, showing that almost all alginate units were processed through the cyclic ED. Furthermore, the two 1/2/3 and 4/5/6 blocks had equal 13C abundance, thereby indicating that the dihydroxyacetone-1-phosphate and glyceraldehyde-3-phosphate pools had fully equilibrated prior to conversion into alginate units.

The observations made by Beale and Foster [51] have provided definitive evidence that the isomerisation is almost completely blocked in A. vinelandii. The ‘cyclic’ ED in this organism – and, presumably, in some alginate-producing Pseudomonas– seems nothing but a long route for the conversion of glucose-6-phosphate into fructose-6-phosphate to fulfil anabolic demands. The net balance of such an alternative pathway is:

  • image

The biosynthesis of polysaccharides requires the availability of glycosidic – and, in some cases, substituent – precursors and energy in the form of ATP or other NTPs. The balance given above shows that the reformation of one fructose-6-phosphate from two molecules (a semi-conservative cycle) occurs at the expense of a phosphoester bond whereas NADPH is formed. From the energetic point of view, the ED cycle does not seem favourable because of the loss of phosphoester bonds. Moreover, the anabolic rather than catabolic use of the triose-phosphates results in smaller ATP, NADH and pyruvate yields within the carbohydrate pathways [82]. The ‘cycle’ may therefore have significant effects on the carbon, energetic and redox status in these sugar-utilising bacteria. However, polysaccharides are generally synthesised under carbon excess conditions – where the energetic demands for cell growth or, more likely, cell maintenance are fulfilled. Under such conditions the energetic expenses arising from the cycle may be well supported. The pathway appears to link catabolism (pyruvate formation) to anabolism since it makes sense with anabolic requirements for both fructose-6-phosphate and NADPH. on other sugars

The ED cycle in Pseudomonas can contribute to the metabolism of sugars other than glucose, including gluconate, mannitol, fructose and glycerol [77]. The cases of fructose and glycerol are particularly interesting because both compounds feed the carbohydrate pathways downstream of the reaction catalysed by phosphoglucoisomerase. Fructose is brought into the cells via the phosphoenolpyruvate (PEP)-dependent phosphotransferase system producing fructose-1-phosphate that is further phosphorylated into fructose-1,6-bisphosphate. Glycerol is taken up by an active system, phosphorylated and dehydrogenated, resulting in the formation of dihydroxyacetone-1-phosphate. There is clear genetic evidence for the contribution of the ED cycle to the catabolism of these two carbon sources [77]. Mutants lacking phosphoglucoisomerase or ED enzymes are affected in both growth and alginate synthesis. Mutants lacking the glyceraldehyde-3-phosphate dehydrogenase – and thus unable to catabolise triose-phosphates into pyruvate – can grow on glycerol. This is because the triose-phosphate molecules generated from glycerol can be catabolised via the ED cycle.

Though genetic studies suggest that the metabolism of fructose or glycerol may proceed via the ED cycle, they do not provide direct evidence for the actual operation of the cycle. Indeed, radiotracer studies in P. aeruginosa, P. mendocina and A. vinelandii cells grown with fructose as the carbon source have indicated that the conversion of this sugar into alginate does not involve the ED cycle [78]. In 1990, Narbad et al. [83] used in vivo and in vitro 13C-NMR to rule out definitively the involvement of triose-phosphate cycling of fructose in P. aeruginosa. When [1-13C]- or [2-13C]fructose was used as substrate, the enrichment in alginate monomers could only be detected in C1 or C2 respectively, indicating that the hexose molecule was retained intact. The analysis of the labelling pattern of catabolic end-products (lactate) showed also that a significant part (40%) of fructose was catabolised through EMP in addition to ED, while glucose is degraded uniquely via the ED pathway in these bacteria. Such differential behaviour for glucose and fructose in the same organism echoed the comment made by Conway [72] that the cyclic ED operates in micro-organisms unable to catabolise sugars via the EMP pathway. For both sugars, contribution from the PP pathway was not detected. cycling of both pentose-phosphates and triose-phosphates

A common feature of the above micro-organisms (P. aeruginosa, P. mendocina, A. vinelandii) is the lack of the oxidative PP pathway [72]. Most micro-organisms – including some Pseudomonas species – utilising the ED pathway also possess the PP pathway. As discussed above, the PP pathway enables both PP and triose-phosphate cycles to occur and the question of the simultaneous cyclic operation of both pathways should be raised. In his review on the ED pathway, Conway [72] reported that three organisms (namely, Pseudomonas cepacia, S. meliloti, and Thiobacillus ferrooxidans) for which the ED cycle was proposed were known to possess the PP pathway. The contribution of the PP pathway was mentioned only for the latter organism. By using radiorespirometric approaches to study glucose metabolism in T. ferrooxidans, Tabita and Lundgren [84] have observed an unexpected early release of label from the C6 of glucose, suggesting that glyceraldehyde-3-phosphate molecules generated through the ED pathway were converted into glucose-6-phosphate molecules labelled at C1 that further entered the PP pathway (this is recycling, indeed). The same observation was reported for another Thiobacillus species [85]. One should note that the cycling of triose-phosphates through the unique PP pathway might also generate such results. Unfortunately, the situation in Thiobacillus with respect to carbohydrate cycling was not further investigated.

In their pioneering 13C-NMR work on the biosynthesis of cellulose in Acetobacter xylinum, Gagnaire and Taravel [52] have given clear evidence for the simultaneous operation of both ED and PP cycling in this species. Cellulose is a homopolymer of glucose and its glycosidic units are metabolically derived from glucose-6-phosphate. Recycling is not expected to contribute to the formation of the glycosidic precursors because glucose-6-phosphate can be generated directly from the exogenous glucose. In their experiments, Gagnaire and Taravel [52] observed labelling at C1 of cellulose units from [6-13C]glucose while no label was found at C6 from [1-13C]glucose. These labelling patterns showed the occurrence of triose-phosphate cycling through the dehydrogenating pathways (Figs. 3 and 4) and ruled out the involvement of the EMP pathway (Fig. 4). PP cycling was also demonstrated to occur. The key feature in these experiments was the use of uniformly labelled glucose as the carbon source. By developing a statistical analysis of the coupling figures observed in the 1D NMR spectra of cellulose units and mathematical modelling, Gagnaire and Taravel [52] evaluated that 30% of the total units derived from the exogenous glucose were processed through triose-phosphate cycling, whereas 26% were processed through PP cycling. Their results were later confirmed by a monolabelling approach [41,42], where [2-13C]- and [4-13C]glucose were used in addition to [1-13C]- and [6-13C]glucose. Some quantitative differences exist between the two studies. in the Rhizobiaceae family

As previously mentioned, the cyclic ED has been proposed to occur in S. meliloti[46]. The occurrence of cycling through the PP pathway was not investigated although the bacteria possess the PP enzymes [47]. Recently, both PP and triose-phosphate cycling were ascertained by a 13C-labelling approach not only for S. meliloti but also for Agrobacterium, a member of the Rhizobiaceae family [41–43,45,49].

Similar to the studies reported above, clear evidence for the occurrence of carbohydrate cycling in Rhizobiaceae (Fig. 8) was obtained from studies of poly- or oligosaccharide biosynthesis – curdlan in Agrobacterium sp. [41,42] and β-glucans and succinoglycan in S. meliloti[43], using a monolabelling approach. These polysaccharides are made of glucose (curdlan, β-glucans) or glucose+galactose (succinoglycan) and derive metabolically from glucose-6-phosphate. In 13C-labelling experiments, the isotopic patterns in polysaccharides showed remarkable homologies for the two species. The occurrence of triose-phosphate cycling was deduced from differential labelling patterns from [1-13C]- and [6-13C]glucose. The contribution of triose-phosphate cycling was about 25%. A significant redistribution of label from exogenous [2-13C]glucose towards both the C1 and C3 of glycosidic units ascertained the occurrence of PP cycling and indicated that 40% of the labelled hexose units on this sugar had been processed through the cycle.

Although both ED and PP enzymes are found in these species [47,72], all the isotopic features reported above could be explained by the unique operation of the PP pathway (see Section 4). In S. meliloti, the isotopic analysis of polyhydroxybutyrate (PHB), another polymer accumulated simultaneously with the polysaccharides, supports the operation of the ED pathway [45]. This polymer is made of β-hydroxybutyrate units derived from acetyl-CoA, itself derived from glycolytic pyruvate. No labelling was found in PHB from [1-13C]glucose while a labelling at the positions derived from the carbonyl group of acetyl-CoA was observed from [2-13C]- or [1,2-13C2]glucose. This was indicative of glucose breakdown through the ED pathway (see Fig. 4). Therefore both PP and ED pathways provided glyceraldehyde-3-phosphate molecules that could be recycled to hexose-phosphates. The amount of label found in PHB from [2-13C]glucose was small, suggesting that the ED pathway may not be predominant for both glucose catabolism and triose-phosphate cycling. Consistently, predominance of the PP pathway could be seen from (i) the significant labelling at C1 and C3 from [2-13C]glucose and (ii) the extent of reversibility through the non-oxidative branch of the pathway, as reported in Section 4.2[43,45].

The occurrence of PP cycling in these PP-utilising species is not surprising. As previously mentioned, they usually do not utilise sugars via an EMP-like pathway, i.e. they do not convert fructose-6-phosphate into fructose-1,6-bisphosphate. The labelling data reported above are consistent with this observation. The PP-derived fructose-6-phosphate molecules can either enter specific pathways or be converted back into glucose-6-phosphate (recycling). The occurrence of PP (re)cycling seems therefore to be structurally related to the PP pathway in these organisms. But the simultaneous operation of triose-phosphate cycling – whether the triose-phosphates are formed by the ED or PP pathway – makes the situation in these organisms analogous to that described in alginate-producing species. In both situations, triose-phosphate cycling allows for the preservation of hexose-phosphates. gluconate by-pass

In alginate-producing species the conversion of glucose into fructose-6-phosphate via the ED cycle was made necessary because of the limited isomerisation of glucose-6-phosphate into fructose-6-phosphate and/or the occurrence of the gluconate by-pass. The organisms where both PP and triose-phosphate cycling was shown to occur synthesise polysaccharides (cellulose, curdlan, succinoglycan, β-glucans) that are metabolically derived from glucose-6-phosphate. The isomerisation step is not required for direct polymerisation of introduced glucose (indeed, the labelling data obtained therein are consistent with free isomerisation of hexose-phosphates). For the organisms studied so far by 13C-NMR, A. xylinum, Agrobacterium sp. and S. meliloti, the labelling data have provided evidence for the direct polymerisation of introduced glucose to occur. But these species are known to possess the gluconate by-pass as well. Given the latter pathway does not feed the glucose-6-phosphate pool, the preferential utilisation of glucose via the oxidative pathway may lead to a situation where the generation of hexose-6-phosphate is inconsistent with the anabolic demands unless cycling occurs.

The role of the glucose by-pass in carbohydrate cycling was further investigated in S. meliloti. First, in vivo NMR experiments have shown that glucose was actually oxidised into gluconate simultaneously with polymer biosynthesis [45,86]. The net consumption of gluconate was observed at the onset of glucose exhaustion [87], providing data supporting the previous hypothesis. A number of arguments suggested however that the gluconate by-pass might not be responsible for the occurrence of carbohydrate cycling in S. meliloti. (i) Labelling data were consistent with direct polymerisation of a part of the exogenous glucose. (ii) Significance of glucose uptake and phosphorylation was sustained by the opportunity to observe isotopic exchange within the non-oxidative PP steps. (iii) Similar labelling patterns in polysaccharides were obtained from non-growing cells that no longer produce gluconate [87].

More direct evidence was obtained recently [88]. Both PP and triose-phosphate cycling were shown to occur for glucose in a S. meliloti mutant lacking the ability to convert glucose into gluconate. Moreover, it was shown that both cycles occurred for fructose [88], a sugar that is brought directly into the cells by an active transport system [89]. These findings indicate that in S. meliloti, carbohydrate cycling is not due to the gluconate by-pass nor is it specific for glucose. Though carbohydrate cycling was observed for all situations, the extent of cycling was higher when glucose could be converted into gluconate, i.e. in the wild-type [88], indicating that the occurrence of the gluconate by-pass favours cycling. Interestingly, the extent of the enhancement was more pronounced for PP cycling than for triose-phosphate cycling. comments

Most of the comments so far on the occurrence of carbohydrate cycling in ED (+PP)-utilising species were more or less driven by the idea that cycling was justifiable by the significant demands for hexose-phosphates in polysaccharide biosynthesis. It is likely that the ED cycle plays a key role in the provision of glycosidic precursors in alginate-producing species grown on glucose. One of the main contributions of NMR studies to that field was to provide data indicating that cycling of both pentose-phosphates and trioses-phosphates could occur in some of these bacteria. In such situations the link between carbohydrate cycling and polysaccharide synthesis is not that clear, because there is no obvious limitation for the direct formation of glycosidic precursors. The accumulation of polysaccharides may have offered the only valuable markers in sufficient amounts to obtain evidence for the operation of the cycles. Alternative considerations may help to explain the occurrence of carbohydrate species. demands in NADPH

It is likely that in the ED+PP-utilising species PP cycling plays its well-known role in the optimisation of NADPH production for sustaining anabolic demands. Interestingly, the operation of the ED cycle may also be seen as a process enabling the optimisation of NADPH synthesis [82]. In the ‘linear’ ED pathway, one NADPH molecule is generated per molecule of glucose-6-phosphate entering the process. In the cyclic version, the yield of NADPH can increase by a factor up to 2 depending on the extent of recycling. Such a role may be particularly relevant in non-PP-utilising organisms.

One argument against the above hypothesis is that cycling was observed mainly in conditions favourable to the production of polysaccharides, i.e. in non-growth situations [79] where the NADPH demands are much lower than during growth. One exception is the case of bacteria, such as S. meliloti, that synthesise PHB during the stationary phase of growth because the biosynthesis of this polymer requires two NADPH per monomer. In S. meliloti, PHB can accumulate up to 80% of the dry cell weight [90,91], and the requirements for NADPH are elevated. The recycling of carbohydrates may be a way to sustain such high demands. But the reverse situation may be considered as well, i.e. the biosynthesis of PHB may provide a sink for the excess in NADPH resulting from cycling. Interestingly enough, both PP (NADPH optimisation) and triose-phosphate (hexose-phosphate conservation) were observed in S. meliloti, in situations of sugar excess (sustained acetyl-CoA formation). This seems to provide favourable conditions for the simultaneous and significant synthesis of both polysaccharides and PHB [90,91]. redox status

The comments above outline the effects of carbohydrate cycling on the NADPH/NADP+ ratio that plays a key role in the control of anabolic processes (the ‘anabolic redox charge’). Triose-phosphate cycling also affects the NADH/NAD+ ratio (the ‘catabolic redox charge’) but in an opposite manner. In sugar-utilising species glyceraldehyde-3-phosphate dehydrogenase in the bottom part of glycolysis plays a major role in the establishment and maintenance of the NADH/NAD+ ratio. The utilisation of triose-phosphate molecules for carbohydrate cycling decreases the flow of molecules entering the dehydrogenation process, and, thereby, the rate of NADH formation. Therefore, splitting triose-phosphates between recycling and further glycolysis may contribute to triggering the distribution of reducing power between the anabolic and catabolic redox charges.

In P. aeruginosa, there are two distinct glyceraldehyde-3-phosphate dehydrogenases [77]. One is NADP-dependent and appears to be constitutive whereas the other is NAD-dependent and is inducible. The bottom part of glycolysis does not contribute to maintaining the catabolic redox charge when only the NADPH-dependent enzyme is present. In this case, the triose-phosphates contribute to NADPH formation whether they enter glycolysis or are recycled, although with different yields. The link between splitting triose-phosphates into glycolysis and recycling and the balance between the two redox charges seems irrelevant in such a situation. But, interestingly, the gene encoding the NAD-dependent enzyme appears to be member of the same regulon as the key genes of the ED cycle [77]. Therefore, the operation of the ED cycle in this organism is intimately related to a situation where the glycolytic utilisation of triose-phosphates includes the NAD-dependent dehydrogenation.

Linton proposed a hypothesis – the lack of control of flux – from the observation of an inverse relationship between growth efficiency and the rate of exopolysaccharide production in producing bacteria [79]. The ED-utilising bacteria maintain high glycolytic fluxes when sugars are available, even under non-growing situations. This is consistent with the efficiency of the key enzyme in the pathway, namely glucose-6-phosphate dehydrogenase. The high glycolytic rates are favourable under growth conditions to sustain the elevated energy requirements, but they result in excess energy (and carbon) in other situations. In Linton's view, the biosynthesis of excreted compounds (such as polysaccharides) may provide a sink for excess carbon and energy. Both the loss of phosphoester bonds via gluconeogenic activities and the decreased rates of formation of pyruvate (from which most of the energy is derived under aerobiosis) resulting from triose-phosphate cycling are consistent with this hypothesis. Accordingly, the extent of polysaccharide synthesis is related to the activity of the fructose-bisphosphatase [79], i.e. to the recycling capability.

This hypothesis also makes sense from the redox point of view. A high flux through catabolism is likely to result in a high NADH/NAD+ ratio that exerts negative control on NAD+-consuming reactions, including glyceraldehyde-3-phosphate dehydrogenase. The result should be the accumulation of triose-phosphates that are made available for anabolic purpose. Recent observations in Xanthomonas campestris are consistent with this hypothesis (Letisse and Lindley, personal communication).

13C-labelling strategies have proved to give strong and direct evidence for the occurrence of carbohydrate cycling in ED-utilising organisms and it is likely that the number of species (at least among related proteobacteria) where such cycles are observed will be further extended in the near future. From all the considerations given above, it can be seen that the role of carbohydrate cycling in these species may go far beyond the ‘simple’ alternative pathway for provision of anabolic precursors. To gain further insights into the role and physiological relevance of these processes, more must be known about the physiological situations in which they actually operate and their extent.

5.2.3Triose-phosphate cycling in the EMP pathway

Cycling of triose-phosphates via the EMP pathway results from backward fluxes through the entire upper half of the pathway and includes the aldolase/triose-phosphate isomerase triangle, the phosphofructokinase/fructose-biphosphatase cycle – or reversibility through the pyrophosphate (PPi):fructose-6-phosphate phosphotransferase (see Section 6.1) – and, finally, reversibility of phosphoglucoisomerase. The cycle is conservative and the net balance is the hydrolysis of one ATP molecule (see Fig. 7). As mentioned above, the occurrence of triose-phosphate cycling can be seen from the observation of isotopic features due to the aldolase/triose-phosphate isomerase triangle at the level of hexose-phosphates or derived compounds. Classically, cyclic operation of the pathway is assumed to occur when a label is found at C6 (and C1) of hexose-phosphates from a hexose labelled at C1 (Fig. 4). Such labelling patterns were clearly detected in glucose-6-phosphate by in vivo 13C-NMR in S. cerevisiae[30] and in various strains of the genus Fibrobacter[92–94].

In ED-utilising species, triose-phosphate cycling was observed mainly in those producing polysaccharide. In contrast, such cycling was reported not to occur in L. lactis, a polysaccharide-producing, EMP-utilising organism [95]. However, some evidence was obtained from storage carbohydrate. For instance, detection of [1-13C]glycogen and [6-13C]glycogen signals in 13C-NMR in vivo spectra of Fibrobacter resting cells [92,93,96,97] showed that about 50% of glycogen monomers were obtained after reversion of the glycolytic pathway. Trehalose is also a good marker of triose-phosphate cycling in the EMP pathway. Various 13C precursors were used to assess 13C scrambling in this disaccharide from [1-13C]glucose in S. cerevisiae[66,98] or [2-13C]pyruvate in dairy Propionibacterium[99]; under these conditions [1-13C]- and [6-13C]trehalose or [2-13C]- and [5-13C]trehalose were detected respectively. In S. cerevisiae[66], it was found via the 13C labelling of trehalose that the flux through fructose-bisphosphatase was null under anaerobic conditions while it was approximately 20% of the phosphofructokinase flow under aerobiosis. These fluxes could be calculated by introducing corrections due to the contribution of the PP pathway. Finally, Kai et al. [44] detected C1[RIGHTWARDS ARROW]C6, C6[RIGHTWARDS ARROW]C1 or C2[RIGHTWARDS ARROW]C5 isotopic transfers in branched polysaccharides in Pestalotiopsis strains incubated with [1-13C]-, [6-13C]- or [2-13C]glucose respectively.

In the fungus Phycomyces blakesleeanus, evidence for cycling within the EMP pathway was obtained from a secondary metabolite, gallic acid [100]. The authors used 13C-NMR strategies to elucidate the biosynthetic pathway of this metabolite, for which controversy existed, and showed that gallic acid was formed via the shikimate pathway rather than from phenylalanine. Interestingly, via the shikimate pathway, a four-carbon block in gallic acid is derived without alteration from the PP intermediate erythrose-4-phosphate. This offered the opportunity to access isotopic features due to carbohydrate pathways. More specifically, the results indicated that that two-thirds of the hexose-phosphates converted into erythrose-4-phosphate had been processed through the EMP triose-phosphate cycle prior to conversion. This work emphasised the usefulness of secondary metabolites as markers of central processes, a strategy that has been further extended [15].

It is noteworthy that the opportunity to monitor cycling of triose-phosphates in the EMP pathway relies uniquely on isotopic features resulting from the aldolase/triose-phosphate isomerase triangle. PP and triose-phosphate cycling

There is so far no clear evidence for simultaneous operation of both PP and triose-phosphate cycling in EMP-utilising organisms. Furthermore, isotopic evidence for such a situation is hampered by label redistribution occurring within the non-oxidative part of the PP pathway (see Section 4.2). First, the dilution effect of PP cycling may minimise the evaluation of triose-phosphate cycling. Second, the PP pathway can be responsible for isotopic transfer from C1 to C6 in EMP species. EMP degradation of [1-13C]glucose results in C1-labelled dihydroxy-acetone-1-phosphate that is further isomerised into C3-labelled glyceraldehyde-3-phosphate. If the latter enters the non-oxidative steps of the PP pathway, C6-labelled fructose-6-phosphate is obtained. The redistribution of label from [1-13C]glucose to the C6 of hexose-phosphates is the result of glycolytic breakdown followed by isotopic exchange between the triose-phosphates and hexose-phosphate pools. It is therefore not a triose-phosphate cycle.

Some studies were performed to discriminate the backward flux in the EMP pathway from reversion of the PP pathway. The synthesis of the trehalose from l-[U-13C]aspartate was studied in Streptomyces parvulus[101]. The detection of α,α-[4,5,6]trehalose by 13C-NMR as the only isotopomer clearly showed that this disaccharide was formed by reversion of PP pathway and transaldolase activity. In this organism, [1,2,3-13C]glyceraldehyde-3-phosphate issued from l-[U-13C]aspartate was not isomerised into dihydroxyacetone-1-phosphate entering gluconeogenesis, a process that would have led to both α,α-[4,5,6]trehalose and α,α-[1,2,3]trehalose.

6Substrate or ‘futile’ cycles

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Definitions and diversity of carbohydrate cycles
  5. 3General considerations on the isotopic investigation of carbohydrate cycles
  6. 4Reversibility
  7. 5Metabolic cycles
  8. 6Substrate or ‘futile’ cycles
  9. 7Concluding comments
  10. Acknowledgements
  11. References

The carbon cycles resulting solely in the loss of energy were initially referred to as ‘futile cycles’, because they appeared to be wasteful. The more neutral term ‘substrate cycles’ was recently introduced to account for possible roles for these processes (see below). The concept of a ‘substrate cycle’ was detailed by Fell [102] who provided a definition enabling their classification. A metabolic network is assumed to contain a substrate cycle when:

  • 1
    “The flux pattern in the network cannot be fully described as the combination of the minimum number of linear paths needed to account for the mass flows connecting the inputs and outputs.”
  • 2
    “One of the additional fluxes needed to complete the description is a feasible, internal cycle.”
  • 3
    “There is one step of the cycle that can be deleted in principle and still leave a network capable of connecting the observed input fluxes to the observed output fluxes. (This is the criterion that shows that the cycle is intrinsically unnecessary […]).”

According to Fell's definition, the occurrence of substrate cycling is examined at the network level and substrate cycles are necessarily embedded into one or more linear pathways. In the present review, the definitions of the various cycles are only based on the structure and balance of the cycle itself, independently of the connected reactions. This leads to a broader acceptance for the term substrate cycle. As an illustrative example, the simultaneous synthesis and degradation of storage compounds (see Fig. 2) is an external (or dead-end) cycle and therefore is outside the scope of Fell's definition, whereas it fits with the definition given here.

The purpose of the above comments is not to provide a general definition for substrate cycle that differs from that given by Fell, but to utilise this term in the present review in a manner consistent with the level of examination of the metabolic processes therein. Indeed, the definition of substrate cycles given here is less restrictive but less integrative than Fell's.

Though the former term ‘futile’ cycle referred to the ‘waste’ of energy for the organisms, a number of roles were proposed for substrate cycles. The suggested roles for substrate cycles include mainly [102,103]:

  • 1
    Generation of heat. This function was reported concerning adipose tissue of mammals and flight muscle of bumble bees [102] but is not usually evoked in the case of micro-organisms.
  • 2
    Buffering of metabolite concentrations. The glucose/glucose-6-phosphate cycling in the liver of animals was proposed to contribute to buffering the blood glucose concentration, and this reasoning can be extended to other cycles. There are, however, arguments indicating that substrate cycling may not be an efficient buffering system [102].
  • 3
    Improved sensitivity in metabolic regulation [103]. If the rate of cycling is assumed to be much higher than the net flux through the pathway containing the cycle, then the value of the net flux can be finely tuned to a new one by small increments in one of the forward or backward fluxes of the cycle.
  • 4
    Switch mechanism. The maintained capability to perform both forward and backward reactions makes possible rapid changes in the direction of the net flux. Cycling is a mechanism enabling the control of the direction of flow in bidirectional pathways [102].
  • 5
    Safety valve. There is current regain in interest for this explanation that relies on the dissipation of energy, which is advantageous in providing an efficient mechanism in situations where a sudden elevation of the energetic charge occurs that may have negative effects.

The occurrence and physiological relevance of substrate cycles is still controversial. The main reason has long been the lack of reliable tools to provide unambiguous proof, since it is not so easy to probe such cycles. The main contribution of NMR to this field is to provide an accurate tool by the ease in obtaining positional information in carbon-labelling experiments.

6.1Phosphofructokinase/fructose-bisphosphatase cycle

The carbohydrate pathways may contain three distinct substrate cycles: (i) cycling of glucose and glucose-6-phosphate through the glucokinase/glucose-6-phosphate phosphatase cycle, (ii) cycling of fructose-6-phosphate and fructose-1,6-bisphosphate through the phosphofructokinase/fructose-bisphosphatase cycle, and (iii) cycling of PEP and pyruvate, a process that includes at least three enzymes: pyruvate kinase, pyruvate carboxylase and PEP carboxykinase. All three processes fit with Fell's definition of a substrate cycle. As previously mentioned, pyruvate cycling will not be discussed in this review. The two other cycles are similar in structure (two enzymes) and result (hydrolysis of one ATP molecule). The glucokinase/glucose-6-phosphate phosphatase was observed in higher eukarya but has not been reported so far in micro-organisms where the release of glucose is not usual, at least under laboratory conditions. Therefore only the phosphofructokinase/fructose-bisphosphatase cycle will be further discussed.

The phosphofructokinase/fructose-bisphosphatase cycle has been described in many organisms, including prokaryotes and eukaryotes [103]. It is exemplary of the difficulty in assessing the occurrence and extent of a substrate cycle. The cycle induces no net variation in the amounts of the two phosphorylated sugars and the rate of cycling is independent of the net flux through the process (whatever its direction). The cycle results in the net hydrolysis of ATP, but it is not easy to measure at the whole-cell level the effects on the ATP balance resulting specifically from the cycle. The effects of cycling on the energetic status of the cells were analysed in cells over-expressing one of the cycling enzymes. The observed effects included lower growth yields or higher respiration rates [104], suggesting that the energetic status was affected to a significant extent. However, the main problem of this approach is that genetic manipulation can widely affect the cell machinery; therefore, the observed effects might not solely be related to the occurrence of futile cycling.

Finally, isotopic investigations do not necessarily provide evidence for the cycle. The cycle in itself is not responsible for a specific isotopic signature in carbon-labelling experiments since no symmetrical intermediate is formed nor does cleavage of the carbon backbone occur. Its activity can only be revealed if isotopic features due to the aldolase/triose-phosphate isomerase triangle cycle can be observed at the level of fructose-6-phosphate – or at a higher level. Many studies were based on the use of radiolabelled compounds [105], but difficulty in obtaining accurate positional information did not allow precise evaluation of the extent of substrate cycling. More recently, Torres et al. [106] proposed a method for assessing the rate of cycling in vitro in a system where glycolysis and gluconeogenesis were reconstituted by adding the relevant enzymes. They could measure forward and backward fluxes in such a system by adding very small amounts of radiolabelled substrates to a reaction at equilibrium without changing the substrate/product ratio. The rate at which the labelled substrate appeared reflected the isotopic exchange due to the cycling. Though interesting, this method seems difficult to apply in vivo.

In such conditions, the ease in obtaining positional information makes the NMR technique particularly relevant to that field. Unfortunately, in most cases fructose-6-phosphate does not accumulate sufficiently to be detected by NMR, and valuable data on the operation of the cycle were mainly derived from the analysis of glucose-6-phosphate or storage compounds (glycogen, trehalose) as illustrated in Section 5.2.3 for Saccharomyces, Fibrobacter, Propionibacterium, Pestalotiopsis, Phycomyces and Streptomyces, showing its wide distribution. It was shown not to occur in some other situations [32].

6.1.1Alternative enzymes

The occurrence of alternative enzymes for the phosphorylation of fructose-6-phosphate may lead to misinterpretation with respect to substrate cycling [107]. A first alternative enzyme, the ADP-dependent phosphofructokinase, is known to function in some bacteria [108]. Its involvement in fructose-6-phosphate/fructose-1,6-bisphosphate cycling – which has not been reported so far – may result in the net hydrolysis of ADP and therefore can be treated in a similar way to the ATP-dependent enzyme with respect to substrate cycling. Another alternative enzyme is the PPi:fructose-6-phosphate phosphotransferase, which catalyses a reaction that does not consume ATP and is reversible. The forward and backward reactions of the PPi:fructose-6-phosphate phosphotransferase cycle therefore generate a reversibility cycle, independently of the fructose-bisphosphatase. Such a cycle was shown to operate in plants [1], in Fibrobacter succinogenes[93] and in the methylotrophic actinomycete Amycolatopsis methanolica[109].

The availability of complete genome sequences allows the possible enzymes in a growing number of species to be predicted. But such knowledge does not provide information on the enzyme(s) actually operating under particular conditions, either because genes for many alternative enzymes are present, or because they might have alternative enzymes encoded from yet unassigned open reading frames. Definitive conclusions on the operation of substrate cycling from 13C-labelling studies should be possible in light of enzymatic assays of ATP- or ADP- phosphofructokinase.

Though NMR is a valuable tool to provide evidence for the phosphofructokinase/fructose-bisphosphatase cycle, there have been very few NMR studies that have focused on the investigation of this process [31]. Therefore most of the NMR studies performed so far have contributed more to ascertaining the occurrence of the cycle rather than illuminating its physiological role. From the roles mentioned above, only the latter few – improved sensitivity in control, switch mechanism and safety valve – were considered reasons for the phosphofructokinase/fructose-bisphosphatase cycle. Some observations suggest that the gain in sensitivity may be not relevant [103]. The two remaining explanations seem to be possible from a theoretical point of view. They correspond to situations where cells have to adapt rapidly to varying conditions. There is increasing evidence for such a role for cycling of storage carbohydrates (see below), but its relevance for the phosphofructokinase/fructose-bisphosphatase cycle remains to be investigated.

6.2Cycling of storage compounds

As stated above, the simultaneous synthesis and breakdown of storage carbohydrates (see Fig. 1) constitute a substrate cycle. Storage carbohydrates studied so far by NMR include oligo- (trehalose) and polysaccharides (glycogen). It is not easy to obtain isotopic evidence for the occurrence of storage carbohydrate cycling [110]. The carbon skeleton is retained unchanged all along the process, and the cycle does not by itself generate isotopic redistribution (inside the carbon skeleton) but only by dilution. To demonstrate the occurrence of cycling, it has to be shown that a transfer of label can occur in the direction opposite to the net flux, or, if there is no net flux, that the label can be transferred in both directions at the same time. The isotopic analysis of storage carbohydrate cycling is made more complicated by the fact that the glycosidic units contained in the storage material may have been formed through processes that generate isotopic redistribution and/or dilution. Specific approaches must be undertaken to discriminate isotopic features (redistribution and dilution) generated via other processes from those (dilution only) specifically related to the cycling of storage compounds.

Two approaches have been developed to assess the occurrence of these cycles: (i) kinetic approaches by in vivo 13C-NMR and (ii) detailed analysis of dilution phenomena by means of 1H-NMR performed on extracts or supernatants. In both cases the easiest way to ‘visualise’ futile cycles is to incubate the cells in the presence of an exogenous soluble substrate (usually glucose) which is labelled differently from the storage compound. The different strategies will be illustrated in detail by the study of the futile cycling of glycogen in F. succinogenes; another example will be given for Glomus species. Although 13C-NMR studies have focused on the elucidation of the biosynthetic pathways for trehalose in various micro-organisms [19,31,66,98,101,111], trehalose cycling was studied only in Pichia pastoris and Glomus species.

6.2.1Cycling of glycogen in F. succinogenes

F. succinogenes, a strictly anaerobic bacterium of the rumen, degrades cellulose into glucose and cellobiose, which are further metabolised into succinate, acetate and some formate [92–94,96,97,112,113]. It is able to store up to 70% of its dry weight as glycogen. This storage is continuous and in all growth phases, the glycogen/protein ratio remaining constant [96]. This unusual feature suggested that storage and degradation of glycogen was not strictly regulated by limitations in nitrogen or glucose source as often stated. NMR experiments allowed the demonstration of a futile cycle for glycogen in this bacterium.

In vivo kinetics: The use of 13C-NMR in vivo is particularly suited to the study of glycogen cycling. Glycogen is not fully soluble in cellular extracts but can easily be detected in whole cells by NMR. In contrast to other high-molecular-mass polymers, the molecular mobility of glycogen is high, a feature that favours its detection by NMR [114]. Additionally, the opportunity to perform in vivo kinetic analysis of glycogen labelling enables the monitoring of its simultaneous synthesis and breakdown. Adapted experimental set-ups (‘sequential incubations’) have been developed for such purposes in F. succinogenes. During a first incubation period, resting cells were incubated with exogenous [1-13C]glucose so that intracellular glycogen became labelled on the C1 or C6 (due to triose-phosphate cycling, see Section 5) positions. Then the pre-loaded cells were washed and incubated with [2-13C]glucose. During this second incubation period, both the loss of label at C1 and C6 and incorporation of label in C2 and C5 in glycogen units were observed, showing the simultaneous breakdown (loss of loaded label) and synthesis (new label incorporation) [96]. As an alternative, unlabelled glucose could be used during the second incubation period [93,97], and in that case the degradation of pre-stored 13C-labelled glycogen can be observed. A parallel experiment, where the cells are pre-loaded with [12C]glucose and then incubated with [1-13C]glucose, must be performed to monitor the simultaneous synthesis of glycogen.

The ‘sequential incubation’ experiments have provided direct evidence for glycogen cycling in different strains of the genus Fibrobacter[93], indicating that this phenomenon was a general and common feature in this genus. However, the approach presents some drawbacks: (i) bacteria can be affected by repeated washings – a lowered metabolism was observed for some strains and one did not tolerate the treatment [93]; and (ii) only qualitative observations or rough quantitative estimations can be made.

Detailed quantitative analysis: More quantitative information was obtained from 1H-NMR analysis of glycolytic end-products. During the first incubation period in the sequential incubations, the utilisation of exogenous [1-13C]glucose resulted in the incorporation of label into hexose-phosphates, glycogen and glycolytic products (succinate and acetate). Simultaneous breakdown of prestored endogenous [12C]glycogen resulted in the isotopic dilution of the hexose-phosphate pools and, consequently, of glycolytic products. In F. succinogenes, the major product of glucose metabolism is succinate, which provided an accurate isotopic marker. Each of the two triose-phosphate molecules obtained by glycolytic cleavage of one [1-13C]glucose-6-phosphate molecule may be converted into succinate, with only one being labelled. Given the symmetry of succinate, the label is found at either C2 or C3. If the totality of succinate is derived from exogenous 100% labelled [1-13C]glucose, both positions are 25% labelled. This value is the maximal percentage of labelling that can be observed in each position in such conditions. Now, if parts of the succinate molecules result from the breakdown of unlabelled glycogen, the labelling of [2-13C]succinate should be less than 25%. Therefore, the extent of glycogen cycling can be evaluated from analysis of the dilution effects observed at the level of succinate. Note that in using this approach, there was no need for second incubation.

1H-NMR was used to obtain detailed quantification of the fractional labelling in succinate C2 (see Fig. 5B). However, the extent of the isotopic dilution was too low (a few per cent) to be accurately evaluated by basic 1D 1H-NMR because of possible overlapping of non-labelled substrates under 13C satellites. The analysis of satellite signals arising from protons bound to a 13C enabled proper assessment (see Fig. 5B). By using 13C-filtered spin echo difference sequences [93], the spectra of exclusively 12C-linked (central signal) or 13C-linked (satellite signal) protons were specifically edited. This technique allowed very precise measurements (1% error) of the 13C enrichment in succinate C2. It could be concluded from the data that 16% of hexose-phosphate units converted into succinate were derived from unlabelled glycogen. Although other metabolic fluxes such as glucose consumption, acetate or glycogen synthesis were modified in parallel, the contribution of glycogen cycling to glycolysis was shown to be rather constant under various conditions, for instance when resting cells were incubated with [1-13C]glucose in the presence of NH4+[97] or when A. xylinum cellulose was added as substrate [113].

The isotopic dilution resulting from cycling of glycogen was also assessed from the fractional labelling of other bacterial metabolites, particularly sugar derivatives such as glucose-1-phosphate, glucose-6-phosphate, cellodextrins and maltodextrins using 2D 1H-NMR experiments (DQF-COSY) [115].

6.2.2Cycling of both trehalose and glycogen in Glomus strains

It has been shown by in vivo 13C-NMR that cycling of both glycogen and trehalose could take place during arbuscular mycorrhiza symbiosis (i.e. plant roots colonised by the fungus Glomus). It was first demonstrated to occur in leek roots colonised by Glomus etunicatum[116]. Under perfusion of roots with medium containing [1-13C]glucose it was shown that pre-stored [1-13C]trehalose and [1-13C]glycogen signals decreased and then increased again with time. Another study was performed with carrot roots colonised by Glomus intraradices, using an in vitro dual mycorrhizal culture system in divided Petri plates [117,118]. Addition of 13C-labelled compound to either the root compartment or fungal compartment allowed monitoring of the uptake, metabolism and translocation of carbon by the fungal partner. It was shown that G. intraradices was able to convert hexose to trehalose and glycogen that could be rapidly synthesised and degraded in the short term.

6.2.3Trehalose cycling in P. pastoris

In the sporulating yeast P. pastoris, trehalose is a storage compound accumulated during sporulation and mobilised at the onset of germination. Shulman and collaborators [119] studied the mobilisation of endogenous trehalose in P. pastoris ascospores. By using in vivo 13C-NMR at natural abundance [119], they showed that both added glucose and internal trehalose were utilised during the induction of germination, meanwhile both glycerol and ethanol accumulated as the end-products. These observations suggested breakdown of glucose and trehalose occurred simultaneously. Consistently, the activity of trehalase, the enzyme responsible for trehalose breakdown in P. pastoris, increased significantly during the early stage of germination. In these experiments the absence of 13C labelling did not allow monitoring of the possible synthesis of trehalose from the added substrate. In a second study, P. pastoris asci were first pre-incubated with [1-13C]acetate, resulting in the loading of trehalose labelled at the C3 and/or C4 positions, as expected from the metabolic routes for conversion of acetate into hexose, involving the glyoxylate and TCA cycles [120]. A subsequent incubation was performed where the pre-loaded asci were incubated with [1-13C]glucose. The simultaneous consumption of endogenous trehalose and exogenous glucose was easily monitored in the spore during the course of germination by in vivo 13C-NMR due to the differential carbon labelling of the two carbon sources. In addition, these NMR experiments allowed the measurement of newly synthesised trehalose by integrating the [1-13C]trehalose signal resulting from direct integration of [1-13C]glucose in this molecule.

6.2.4Physiological relevance of storage carbohydrate cycling

Storage compounds, as readily metabolisable food, provide energy (and carbon) reserves for cells under a variety of environmental conditions. The formation and degradation of storage carbohydrates are controlled by complex regulatory systems [121–123] so that the reserves can properly serve the metabolic needs of the organism. Therefore, it is not expected that simultaneous synthesis and breakdown of the storage carbohydrates would be observed, especially in cells facing unfavourable conditions.

There is now a great wealth of genetic, radiotracer, stoichiometric analysis and stable isotope labelling data supporting the occurrence of glycogen cycling in a number of micro-organisms, including both yeasts (S. cerevisiae[121] and Glomus species [116–118]) and bacteria (F. succinogenes[92–94,96,97], Mycobacterium smegmatis[124] and Clostridium cellulolyticum[125,126]). The detailed NMR investigations of glycogen cycling in F. succinogenes show not only that cycling occurs independently of the strain and under various physiological situations but they also emphasise the extent of cycling that represents up to 16% of the glycolytic flow. It is likely that the screening of glycogen cycling in micro-organisms by the NMR approach will demonstrate that glycogen cycling is a widespread phenomenon. Indeed, glycogen cycling is widely distributed in nature, from bacteria to mammals – where NMR studies have emphasised its occurrence in different tissues, e.g. liver [2,105,127,128], muscle [129] and myocardium [130]. A question remains about the physiological relevance of these cycles in vivo. Rognstad [105] reports controversial considerations on this topic, stating that in whole animals, the operation of such a cycle is often exaggerated. For micro-organisms it is almost impossible to assess the occurrence of futile cycling of glycogen in their ecosystems, which are far too complex. However, the strikingly constant contribution of this cycle observed in the case of Fibrobacter, whatever the strains or the external factors studied, suggests it might be an important metabolic feature for this organism.

Most observations on glycogen cycling are consistent with a role for the cycle as a switch mechanism. Cells storing reserve compounds must be able to utilise these reserves very rapidly in adapting to sudden environmental changes. The limitation or inhibition of the ability to degrade glycogen under glycogen-accumulating conditions may prevent the efficient mobilisation of the reserve when the energy demands increase abruptly. Conversely, the limitation or inhibition of glycogen synthesis under glycogen-utilising conditions may prevent the adaptation to overflow metabolism upon restoration of favourable conditions. Therefore cycling of storage compounds may be a key element for the response to changes in the environment over very short timescales.

A recent theory considers that glycogen cycling is basically linked to the fractal structure of this polymer [131]. Due to this fractal structure, the chain ends are regularly distributed so that the attack domains, accessible to glycogen phosphorylase, are all structurally equivalent and represent about 33% of the total stored glucose, independently of the size of the molecule. As a consequence it allows a quick release of stored fuel as well as a fast recovery. However, this regular structure of glycogen must be maintained under certain constraints. In particular to avoid an over-extension of linear chains during glycogen synthesis, two regulatory mechanisms are active: (i) the concentration of branching enzyme is high compared to that of glycogen synthase, (ii) glycogen phosphorylase constantly degrades glycogen as a kind of ‘repair enzyme’. This last factor would be responsible for glycogen cycling. These types of arguments cannot apply to trehalose cycling (see below), trehalose being a simple disaccharide.

Trehalose cycling was also firmly established to occur in different micro-organisms, using either radiolabelling [132] or stable isotope labelling experiments [19,117–119]. The role of trehalose cycling in P. pastoris seems analogous to that discussed for glycogen, for both sugars act as storage compounds. But, in addition to acting as a storage compound, trehalose can also play a role as a protective agent under stress conditions such as heat shock or hyper-osmosis [120,133,134]. This is a major difference with glycogen. Indeed, in S. cerevisiae a fast turnover of trehalose was found in heat-shocked cells incubated with [1-14C]glucose, indicating that trehalose cycling took place under these stress conditions [132]. Also, Breedveld et al. [19] clearly showed an increase of trehalose content in two strains of Rhizobium under osmotic shock by using in vivo 13C-NMR experiments. The authors demonstrated that trehalose synthesis resulted from integration of [1-13C]mannitol (15–20%) and from the breakdown of endogenous [12C]glycogen and/or [12C]PHB (80–85%).

The physiological role of trehalose cycling is not fully understood, but the hypothesis of a switch enabling the rapid inversion of flow from accumulation to utilisation makes sense with the need for an immediate response to sudden – and unfavourable – changes. Blomberg [134] more recently proposed an alternative, attractive model based on the ‘turbo design’ of glycolysis described by Teusink et al. [135]. This theory considers that glycolysis is divided into two parts: in the upper part two molecules of ATP are invested while four molecules of ATP are yielded in the lower part; this situation creates a surplus production of ATP and thus an imbalance in the rates in these two parts of glycolysis. As a consequence hexose-phosphates and/or fructose-1,6-bisphosphate may accumulate to large extents, resulting in phosphate depletion and cell death. Regulation must take place to prevent accumulation. Trehalose cycling may be a response by decreasing the ATP pools. This explanation makes sense with osmotic stress conditions. The delayed growth under osmotic stress conditions decreases the ATP demands for overall anabolism whereas high glycolytic flux must be maintained to sustain the biosynthesis and accumulation of the osmoprotectant glycerol. In such a situation ATP may accumulate to very high extents, resulting in the same effects as above. Blomberg [134] proposed that ATP futile cycle via trehalose cycling would act as a “safety valve to avoid substrate-accelerated death under stress”. François and Parrou repeated the same arguments [121] to explain both trehalose and glycogen cycling in stressed yeasts.

7Concluding comments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Definitions and diversity of carbohydrate cycles
  5. 3General considerations on the isotopic investigation of carbohydrate cycles
  6. 4Reversibility
  7. 5Metabolic cycles
  8. 6Substrate or ‘futile’ cycles
  9. 7Concluding comments
  10. Acknowledgements
  11. References

Most of the 13C-NMR studies performed so far were not specifically directed towards the investigation of carbohydrate cycling. They have nonetheless provided unambiguous evidence for the occurrence of a wide range of cyclic processes within carbohydrate metabolism. Because of this diversity, it is likely that both the relevance and significance of carbohydrate cycles for cellular metabolism cannot be discussed on a general basis. There are, however, two – non-exhaustive – general comments that can be made.

  • 1
    Metabolic cycles in metabolic networks. Because metabolic cycles are inherently linked to the network organisation of cellular metabolism, their roles should be examined at the system level. In the face of the large scale of metabolic networks, metabolic cycles are quite small but they occupy a central place by interconnecting most other processes – or a large number of them. The recent introduction of graphical analysis methods in the field of metabolism [136] has enabled Gleiss et al. [137] to propose that central, small cyclic structures may provide adaptive advantages by reducing the length of detours when a connection is clipped, thereby shortening the transition times in responding to external perturbations. The physiological relevance of such observations to some metabolic cycles may have to be considered.
  • 2
    Timescales. The possible role for substrate cycles as mechanisms enabling the rapid switching of the direction of metabolic flows or as a safety valve for adapting to abrupt elevation of the energetic charge has put emphasis on the timescale for the operation of these processes. Such consideration may be extended to other cycles as well. Timescales for metabolic reactions are <1 s whereas those for 13C-labelling experiments are usually in the range of minutes to hours. Such differential timescales may lead to inaccurate time resolution for monitoring sequential operations of unidirectional processes. Interestingly enough, the paradoxical cycling of glycogen in exercising human muscle was recently explained by taking timescales into account [129]. Given the rapidity of contraction (∼10–40 ms) the energy cannot be supplied from the breakdown of exogenous glucose but from glycogenolysis whereas between contractions glycogenesis refills the pools. Although this explanation cannot apply directly to micro-organisms, it emphasises that timescales of events should be considered for accurate assessment of the physiological relevance of metabolic cycles in the latter species.

It is therefore clear that further studies should focus more specifically on the investigation of cycles per se. Two main questions should be addressed: when do these cycles occur? and to what extent? A more quantitative investigation of the extent of these cycles should provide an accurate evaluation of their effects, not only on carbohydrate metabolism, but also on the overall metabolic status of the cells (energetic status, redox potential, carbon balance, etc.). The effectiveness of 13C-labelling strategies in conjunction with mathematical models enabling metabolic fluxes to be calculated will be particularly useful. A main limitation is that, at the current state of progress, the measurement of metabolic fluxes by means of 13C-labelling strategies can only be made under steady-state conditions and, therefore, they cannot be applied as such to investigate the role of cycles in adapting to sudden changes – i.e. states of transition.

If the present review outlines the accuracy of NMR for the assessment of carbohydrate cycles, its low sensitivity may limit the investigation of cycling to situations where relevant metabolites accumulate to large extents. More specifically, direct assessment of carbohydrate cycling at the level of hexose-phosphates – or other relevant intermediates – is often prevented by insufficient amounts of the metabolites. Increased NMR sensitivity can be achieved by means of 1H-detected methods [8], use of magnetic fields of higher intensity than usually employed for metabolic studies and use of sensitivity-improving cold-metal NMR probes that have recently appeared. An important issue in the field is the recent development of the highly sensitive mass spectrometry techniques that enable both the analysis of low-concentrated metabolites and generation of (mass) isotopomer data [21,22]. These techniques will allow extension of investigations to situations that are unfavourable for NMR studies, whereas ‘in vivo’ NMR remains unique in providing in situ information on the kinetics of metabolic responses to varying environmental conditions.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Definitions and diversity of carbohydrate cycles
  5. 3General considerations on the isotopic investigation of carbohydrate cycles
  6. 4Reversibility
  7. 5Metabolic cycles
  8. 6Substrate or ‘futile’ cycles
  9. 7Concluding comments
  10. Acknowledgements
  11. References

We are grateful to Ramnheek Bhogal for careful reading of the manuscript and to Anne-Marie Raynaud for technical assistance. Part of this work was supported by Grants from ‘Alternatech’ (formerly ‘Biopôle Végétal’, Amiens, France).


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Definitions and diversity of carbohydrate cycles
  5. 3General considerations on the isotopic investigation of carbohydrate cycles
  6. 4Reversibility
  7. 5Metabolic cycles
  8. 6Substrate or ‘futile’ cycles
  9. 7Concluding comments
  10. Acknowledgements
  11. References
  • [1]
    Dieuaide-Noubhani, M, Raffard, G, Canioni, P, Pradet, P, Raymond, P (1995) Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- or 14C-labelled glucose. J. Biol. Chem. 270, 1314713159.
  • [2]
    Landau, B.R (1999) Quantifying the contribution of gluconeogenesis to glucose production in fasted human subjects using stable isotopes. Proc. Nutr. Soc. 58, 963972.
  • [3]
    Newsholme, E.A., Parry-Billings, M (1992) Some evidence for the existence of substrate cycles and their utility in vivo. Biochem. J. 285, 340341.
  • [4]
    Torres, J.C., Guixé, V, Babul, J (1997) A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli. Biochem. J. 327, 675684.
  • [5]
    Gueirreiro, N, Ksenzenko, V.N., Djordjevic, M.A., Ivashina, T.V., Rolfe, B.G (2000) Elevated levels of synthesis of over 20 proteins results after mutation of the Rhizobium leguminosarum exopolysaccharide synthesis gene pssA. J. Bacteriol. 182, 45214532.
  • [6]
    Katz, J, Rognstad, R (1967) The labelling of pentose phosphate from glucose-14C and estimation of the rates of transaldolase, transketolase, the contribution of the pentose-phosphate cycle, and ribose-phosphate synthesis. Biochemistry 6, 22272247.
  • [7]
    Sherry, A.D., Malloy, C.R (1996) Isotopic methods for probing organisation of cellular metabolism. Cell Biochem. Funct. 14, 259268.
  • [8]
    Szyperski, T (1998) 13C-NMR, MS and metabolic flux balancing in biotechnology research. Q. Rev. Biophys. 31, 41106.
  • [9]
    Klapa, M.I., Park, S.M., Sinskey, A.J., Stephanopoulos, G (1999) Metabolite and isotopomer balancing in the analysis of metabolic cycles: I theory. Biotechnol. Bioeng. 62, 375391.
  • [10]
    Rager, M.N., Binet, M.R.B., Bouvet, O.M.M (1999) 31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida. Characterisation of a new mannitol-producing metabolic pathway. Eur. J. Biochem. 263, 695701.
  • [11]
    Neves, A.R., Ramos, A, Shearman, C, Gasson, M.J., Almeida, J.S., Santos, H (2000) Metabolic characterisation of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR. Eur. J. Biochem. 267, 38593868.
  • [12]
    Christensen, B, Nielsen, J (2000) Metabolic network analysis. A powerful tool in metabolic engineering. Adv. Biochem. Eng. Biotechnol. 66, 209231.
  • [13]
    Hondman, D.H.A., Busink, R, Witteveen, C.F.B., Visser, J (1991) Glycerol catabolism in A. nidulans. J. Gen. Microbiol. 137, 629636.
  • [14]
    Witteveen, C.F.B., Busink, R, van der Vondervoort, P, Dijkema, C, Swart, K.D., Visser, J (1989) L-arabinose and D-xylose catabolism in A. niger. J. Gen. Microbiol. 135, 21632171.
  • [15]
    Bacher, A, Rieder, C, Eichinger, D, Arigoni, D, Fuchs, G, Eisenrich, W (1999) Elucidation of novel biosynthetic pathways and metabolite flux patterns by retrobiosynthetic NMR analysis. FEMS Microbiol. Rev. 22, 567598.
  • [16]
    Grivet, J.P (2001) NMR and microorganisms. Curr. Issues Mol. Biol. 3, 714.
  • [17]
    Lundberg, P, Harmsen, E, Ho, C, Vogel, H.J (1990) Nuclear magnetic resonance studies of cellular metabolism. Anal. Biochem. 19, 193222.
  • [18]
    Barbotin, J.-N. and Portais, J.-C. (Eds.) (2000) NMR in Microbiology, Theory and Applications. Horizon Scientific Press, Norfolk.
  • [19]
    Breedveld, M.W., Dijkema, C, Zevenhuizen, L.P.T.M., Zhender, A.J.B (1993) Response of intracellular carbohydrates to a NaCl shock in Rhizobium leguminosarum biovar trifolii TA-1 and Rhizobium meliloti Su47. J. Gen. Microbiol. 139, 31573163.
  • [20]
    Cherniak, R, O'Neill, E.B., Sheng, S (1998) Assimilation of xylose, mannose and mannitol for synthesis of glucoronoxylomannan of Cryptococcus neoformans determined by 13C nuclear magnetic resonance spectroscopy. Infect. Immun. 66, 29962998.
  • [21]
    Christensen, B, Nielsen, J (1999) Isotopomer analysis using GC-MS. Metab. Eng. 1, 282290.
  • [22]
    Dauner, M, Sauer, U (2000) GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnol. Prog. 16, 642649.
  • [23]
    Schmidt, K, Carlsen, M, Nielsen, J, Villadsen, J (1997) Modeling isotopomer distributions in biochemical networks using isotopomer mapping matrices. Biotechnol. Bioeng. 55, 831840.
  • [24]
    Szyperski, T, Glaser, R.W., Hochuli, M, Fiaux, J, Sauer, U, Bailey, J.E., Wuthrich, K (1999) Bioreaction network topology and metabolic flux ratio analysis by biosynthetic fractional 13C labeling and two-dimensional NMR spectroscopy. Metab. Eng. 1, 189197.
  • [25]
    Stephanopoulos, G (1999) Metabolic fluxes and metabolic engineering. Metab. Eng. 1, 111.
  • [26]
    Wiechert, W, Mollney, M, Petersen, S, DeGraaf, A.A (2001) A universal framework for 13C metabolic flux analysis. Metab. Eng. 3, 265283.
  • [27]
    Wiechert, W (2002) Modeling and simulation: tools for metabolic engineering. J. Biotechnol. 94, 3763.
  • [28]
    Babul, J, Clifton, D, Kretschmer, M, Fraenkel, G (1993) Glucose metabolism in Escherichia coli and the effect of increased amount of aldolase. Biochemistry 32, 46854692.
  • [29]
    den Hollander, J.A., Brown, T.R., Ugurbil, K, Shulman, R.G (1979) 13C nuclear magnetic resonance studies of anaerobic glycolysis in suspensions of yeast cells. Proc. Natl. Acad. Sci. USA 76, 60966100.
  • [30]
    den Hollander, J.A., Ugurbil, K, Shulman, R.G (1986) 31P and 13C studies of intermediates of aerobic and anaerobic glycolysis in Saccharomyces cerevisiae. Biochemistry 25, 212221.
  • [31]
    Navas, M.A., Cerdàn, S, Gancedo, J.M (1993) Futile cycles in Saccharomyces cerevisiae strains expressing the gluconeogenic enzymes during growth on glucose. Proc. Natl. Acad. Sci. USA 90, 12901294.
  • [32]
    Shulman, R.G., Brown, T.R., Ugurbil, K, Ogawa, S, Cohen, S.M., den Hollander, J.A (1979) Cellular applications of 31P and 13C nuclear magnetic resonance. Science 205, 160166.
  • [33]
    Rollin, C, Morgant, V, Guyonvarch, A, Guerquin-Kern, J.L (1995) 13C-NMR studies of Corynebacterium melassecola metabolic pathways. Eur. J. Biochem. 227, 488493.
  • [34]
    Neves, A.R., Ramos, A, Nunes, M, Kleerebezem, M, Hugenholtz, J, de Vos, W.M., Almeida, J.S., Santos, H (1999) In vivo nuclear magnetic resonance studies of glycolytic kinetics in Lactococcus lactis. Biotechnol. Bioeng. 64, 200212.
  • [35]
    Wood, T (1986) Distribution of the pentose-phosphate pathway in living organisms. Cell Biochem. Funct. 4, 235240.
  • [36]
    Schuster, R, Holzhütter, H, Schuster, S (1992) Simplification of complex kinetic models used for the quantitative analysis of nuclear magnetic resonance or radioactive tracer. J. Chem. Soc. Faraday Trans. 88, 28372844.
  • [37]
    Berthon, H.A., Bubb, W.A., Kuchel, P (1993) 13C nmr isotopomer and computer-simulation studies of the non-oxidative pentose phosphate pathway of human erythrocytes. Biochem. J. 296, 379387.
  • [38]
    Follstad, B.D., Stephanopoulos, G (1998) Effect of reversible reactions on isotope label redistribution. Analysis of the pentose phosphate pathway. Eur. J. Biochem. 252, 360371.
  • [39]
    Van Winden, W, Verheijen, P, Heijnen, S (2001) Possible pitfalls of flux calculations based on 13C-labelling. Metab. Eng. 3, 151162.
  • [40]
    Wiechert, W. and de Graaf, A.A. (1997) Bidirectional steps in metabolic networks: I. Modelling and simulation of carbon isotope labelling experiments. Biotechnol. Bioeng. 55, 101–117, and following papers in the series: Biotechnol. Bioeng. 55 (1997) 118–135, Biotechnol. Bioeng. 66 (1999) 69–85, Biotechnol. Bioeng. 66 (1999) 86–103.
  • [41]
    Kai, A, Ishido, T, Arashida, T, Hatanaka, K, Hatanaka, K, Akaike, T, Matsuzaki, K, Kaneko, Y, Mimura, T (1993) Biosynthesis of curdlan from culture media containing 13C-labelled glucose as the carbon source. Carbohydr. Res. 240, 153159.
  • [42]
    Kai, A, Arashida, T, Hatanaka, K, Akaike, T, Matsuzaki, K, Mimura, T, Kaneko, Y (1994) Analysis of the biosynthetic process of cellulose and curdlan using 13C-labelled glucose. Carbohydr. Polym. 23, 235239.
  • [43]
    Portais, J.C., Tavernier, P, Gosselin, I, Barbotin, J.N (1999) Cyclic organisation of the carbohydrate metabolism in Sinorhizobium meliloti. Eur. J. Biochem. 265, 473480.
  • [44]
    Kai, A, Karasawa, H, Kikawa, M, Hatanaka, K, Matsuzaki, K, Mimura, T, Kaneko, Y (1998) Biosynthesis of 13C-labelled branched polysaccharides by pestalotiopsis from 13C-labelled glucoses and the mechanism of formation. Carbohydr. Polym. 35, 271278.
  • [45]
    Gosselin, I., Barbotin, J.-N. and Portais, J.-C. (2000) in: NMR in Microbiology, Theory and Applications (Barbotin, J.-N. and Portais, J.-C., Eds.), pp. 331–348. Horizon Scientific Press, Norfolk.
  • [46]
    Stowers, M.D (1985) Carbon metabolism in Rhizobium species. Annu. Rev. Microbiol. 39, 89108.
  • [47]
    Irigoyen, J.J., Sanchez-Diaz, M, Emerich, D.W (1990) Carbon metabolism enzymes of Rhizobium meliloti cultures and bacteroids and their distribution within alfalfa nodules. Appl. Environ. Microbiol. 56, 25872589.
  • [48]
    Jones, D.N.M., Sanders, J.K.M (1989) Biosynthetic studies using 13C-COSY: the Klebsiella K3 serotype polysaccharide. J. Am. Chem. Soc. 111, 51325137.
  • [49]
    Tavernier, P, Portais, J.C., Besson, I, Courtois, J, Courtois, B, Barbotin, J.N (1998) A 13C-NMR study of exopolysaccharide synthesis in Rhizobium meliloti Su47 strain. J. Chim. Phys. 95, 256259.
  • [50]
    Rijhwani, S.K., Ho, C.H., Shanks, J.V (1999) In vivo 31P and 13C NMR measurements for evaluation of plant metabolic pathways. Metab. Eng. 1, 1225.
  • [51]
    Beale, J.M., Foster, J.L (1996) Carbohydrate fluxes into alginate biosynthesis in Azotobacter vinelandii NCIB 8789: NMR investigation of the triose pools. Biochemistry 35, 44924501.
  • [52]
    Gagnaire, D.Y., Taravel, F.R (1980) Biosynthesis of bacterial cellulose from d-glucose uniformly enriched in 13C. Eur. J. Biochem. 103, 133143.
  • [53]
    Dickinson, J.R., Sobanski, M.A., Hewlins, M.J (1995) In Saccharomyces cerevisiae deletion of phosphoglucose isomerase can be suppressed by increased activities of enzymes of the hexose monophosphate pathway. Microbiology 141, 385391.
  • [54]
    Szyperski, T (1995) Biosynthetically directed 13C-fractional labelling of proteinogenic amino acids. Eur. J. Biochem. 232, 433448.
  • [55]
    Portais, J.C., Schuster, R, Merle, M, Canioni, P (1993) Metabolic flux determination in C6 glioma cells using carbon-13 distribution upon [1-13C]glucose incubation. Eur. J. Biochem. 217, 457468.
  • [56]
    Marx, A, De Graaf, A.A., Wiechert, W, Eggeling, L, Sahm, H (1996) Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing. Biotechnol. Bioeng. 49, 111129.
  • [57]
    Frey, A.D., Fiaux, J, Szyperski, T, Wuthrich, K, Bailey, J.E., Kallio, P.T (2001) Dissection of central carbon metabolism of hemoglobin-expressing Escherichia coli by 13C nuclear magnetic resonance flux distribution analysis in microaerobic bioprocesses. Appl. Environ. Microbiol. 67, 680687.
  • [58]
    Schmidt, K, Nielsen, J, Villadsen, J (1999) Quantitative analysis of metabolic fluxes in Escherichia coli, using two-dimensional NMR spectroscopy and complete isotopomer models. J. Biotechnol. 71, 175190.
  • [59]
    Wendish, V.F., De Graaf, A.A., Sahm, H, Eikmanns, B (2000) Quantitative determination of metabolic fluxes during co-utilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose. J. Bacteriol. 182, 30883096.
  • [60]
    Dauner, M, Bailey, J.E., Sauer, U (2001) Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis. Biotechnol. Bioeng. 76, 144156.
  • [61]
    Fiaux, J, Cakar, Z.P., Bailey, J.E., Sauer, U, Szyperski, T (2001) Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids. Eur. J. Biochem. 268, 24642479.
  • [62]
    Schmidt, K, Nielsen, J, Villadsen, J (1999) Quantitative analysis of metabolic fluxes in Escherichia coli, using two-dimensional NMR spectroscopy and complete isotopomer models. J. Biotechnol. 71, 175190.
  • [63]
    Petersen, S, de Graaf, A.A., Eggeling, L, Mollney, M, Wiechert, W, Sahm, H (2000) In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum. J. Biol. Chem. 275, 3593235941.
  • [64]
    Schuster, S, Fell, D.A., Dandekar, T (2000) A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic networks. Nat. Biotechnol. 18, 326332.
  • [65]
    Walker, T.E., Han, C.H., Kollman, V.H., London, R.E., Matwiyoff, N.A (1982) 13C nuclear magnetic resonance studies of the biosynthesis by Microbacterium ammoniaphilum of l-glutamate selectively enriched with carbon-13. J. Biol. Chem. 257, 11891195.
  • [66]
    Campbell-Burk, S.L., den Hollander, J.A., Alger, J.R., Shulman, R.G (1987) 31P NMR saturation-transfer and 13C NMR kinetic studies of glycolytic regulation during anaerobic and aerobic glycolysis. Biochemistry 26, 74937500.
  • [67]
    Maaheimo, H, Fiaux, J, Çakar, Z.P., Baley, J.E., Sauer, U, Szyperski, T (2001) Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional 13C labeling of common amino acids. Eur. J. Biochem. 268, 24642479.
  • [68]
    Guillouet, S., Lessard, P.A. and Sinskey, A.J. (2000) in: NMR in Microbiology, Theory and Applications (Barbotin, J.-N. and Portais, J.-C., Eds.), pp. 259–282. Horizon Scientific Press, Norfolk.
  • [69]
    Marx, A, Eikmanns, B.J., Sahm, H, De Graaf, A.A., Eggeling, L (1998) Response of central metabolism in Corynebacterium glutamicum to the use of an NADH-dependent glutamate dehydrogenase. Metab. Eng. 1, 3548.
  • [70]
    Dominguez, H, Rollin, C, Guyonvarch, A, Guerguin-Kern, J.L., Cocaign-Bousquet, M, Lindley, N (1998) Carbon-flux distribution in the central metabolic pathways of Corynebacterium glutamicum during growth on fructose. Eur. J. Biochem. 254, 96102.
  • [71]
    Canonaco, F, Hess, T.A., Heri, S, Wang, T, Szyperski, T, Sauer, U (2001) Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA. FEMS Microbiol. Lett. 204, 247252.
  • [72]
    Conway, T (1992) The Entner-Doudoroff pathway: history, physiology and molecular biology. FEMS Microbiol. Rev. 103, 128.
  • [73]
    Hochster, R.M., Katznelson, H (1958) On the mechanism of glucose-6-phosphate oxidation in cell-free extracts of Xanthomonas phaseoli (XP8). Can. J. Biochem. Physiol. 36, 669689.
  • [74]
    Selig, M, Xavier, K.B., Santos, H, Schonheit, P (1997) Comparative analysis of Embden-Meyerhof and Entner-Doudoroff glycolytic pathways in hyperthermophilic archae and the bacterium Thermogata. Arch. Microbiol. 167, 217232.
  • [75]
    Adams, M.W (1994) Biochemical diversity among sulfur-dependent, hyperthermophilic microorganisms. FEMS Microbiol. Rev. 15, 261277.
  • [76]
    Lessie, T.G (1984) Alternate pathways of carbohydrate utilization in Pseudomonads. Annu. Rev. Microbiol. 38, 359387.
  • [77]
    Temple, L.M., Sage, A.E., Schweizer, H.P. and Phibbs, P.V. Jr. (1998) Carbohydrate metabolism in Pseudomonas aeruginosa. In Pseudomonas (Montie, T.C., Ed.), Biotechnology Handbooks 10. Plenum Press, New York.
  • [78]
    Lynn, A.R., Sokatch, J.R (1984) Incorporation of isotope from specifically labelled glucose into alginates of Pseudomonas aeruginosa and Azotobacter vinalandii. J. Bacteriol. 158, 11611162.
  • [79]
    Linton, J.D (1990) The relationship between metabolite production and the growth efficiency of the producing organism. FEMS Microbiol. Lett. 75, 118.
  • [80]
    Schleissner, C, Reglero, A, Luengo, J.M (1997) Catabolism of d-glucose by Pseudomonas putida U occurs via extracellular transformation into d-gluconic acid and induction of a specific gluconate transport system. Microbiology 143, 15971603.
  • [81]
    Anderson, A.J., Hacking, A.J., Dawes, E.A (1987) Alternate pathways for the biosynthesis of alginate from fructose and glucose in Pseudomonas mendocina and Azotobacter vinelaandii. J. Gen. Microbiol. 133, 10451052.
  • [82]
    Portais, J.C., Tavernier, P, Gosselin, I, Barbotin, J.-N (2000) Relevance and isotopic assessment of hexose-6-phosphate recycling in microorganisms. J. Biotechnol. 77, 4964.
  • [83]
    Narbad, A, Hewlins, M.J.E., Gacesa, P, Russel, N.J (1990) The use of 13C-n.m.r. spectroscopy to monitor alginate biosynthesis in mucoid Pseudomonas aeruginosa. Biochem J. 267, 579584.
  • [84]
    Tabita, R, Lundgren, D.G (1971) Heterotrophic metabolism of the chemolithotroph Thiobacillus ferrooxidans. J. Bacteriol. 108, 334342.
  • [85]
    Wood, A.P., Kelly, D.P., Thurston, C.F (1977) Simultaneous operation of three catabolic pathways in the metabolism of glucose by Thiobacillus A2. Arch. Microbiol. 113, 265274.
  • [86]
    Tavernier, P, Besson, I, Portais, J.C., Courtois, J, Courtois, B, Barbotin, J.N (1998) In vivo 3C-NMR studies of polymer synthesis in Rhizobium meliloti M5N1 strain. Biotechnol. Bioeng. 58, 250253.
  • [87]
    Portais, J.C., Tavernier, P, Besson, I, Courtois, J, Courtois, B, Barbotin, J.N (1997) Mechanism of gluconate synthesis in Rhizobium meliloti by using in vivo NMR. FEBS Lett. 412, 485489.
  • [88]
    Gosselin, I, Wattraint, O, Riboul, D, Barbotin, J.N., Portais, J.C (2001) A deeper investigation of hexose-6-phosphate recycling in Sinorhizobium meliloti. FEBS Lett. 499, 4549.
  • [89]
    Lambert, A, Osteras, M, Mandon, K, Poggi, M.-C, Le Rudulier, D (2001) Fructose uptake in Sinorhizobium meliloti is mediated by a high-affinity ATP-binding cassette transport system. J. Bacteriol. 183, 47094717.
  • [90]
    Zevenhuizen, L.P.T.M (1981) Cellular glycogen, β-(1,2)-glucan, poly-β-hydroxybutyric acid and extracellular polysaccharides in fast growing species of Rhizobium. Antonie van Leeuwenhoek 47, 481497.
  • [91]
    Tavernier, P, Portais, J.-C, Nava Saucedo, J.E., Courtois, J, Courtois, B, Barbotin, J.-N (1997) Exopolysaccharide and poly-β-hydroxybutyrate coproduction in two Rhizobium meliloti strains. Appl. Environ. Microbiol. 63, 2126.
  • [92]
    Matheron, C, Delort, A.-M, Gaudet, G, Forano, E (1996) Simultaneous but differential metabolism of glucose and cellobiose in cells, studied by in vivo 13C-NMR. Evidence of glucose 6-phosphate accumulation. Can. J. Microbiol. 42, 10911099.
  • [93]
    Matheron, C, Delort, A.-M, Gaudet, G, Forano, E, Liptaj, T (1998) 13C- and 1H-NMR study of glycogen futile cycling in strains of the genus Fibrobacter. Appl. Environ. Microbiol. 64, 7481.
  • [94]
    Matheron, C, Delort, A.-M, Gaudet, G, Forano, E (1998) In vivo 13C NMR study of glucose and cellobiose metabolism by four cellulolytic strains of the genus Fibrobacter. Biodegradation 9, 451461.
  • [95]
    Ramos, A, Boels, IC, de Vos, WM, Santos, H (2001) Relationship between glycolysis and exopolysaccharide biosynthesis in Lactococcus lactis. Appl. Environ. Microbiol. 67, 3341.
  • [96]
    Gaudet, G, Forano, E, Dauphin, G, Delort, A.-M (1992) Futile cycling of glycogen in Fibrobacter succinogenes as shown by in situ 13C- and 1H-NMR investigation. Eur. J. Biochem. 207, 155162.
  • [97]
    Matheron, C, Delort, A.-M, Gaudet, G, Liptaj, T, Forano, E (1999) Interaction between carbon and nitrogen metabolism in Fibrobacter succinogenes S85: a 13C- and 1H nuclear magnetic resonance and enzymatic study. Appl. Environ. Microbiol. 65, 19411948.
  • [98]
    Tran-Dinh, S, Hervé, M, Wietzerbin, J (1991) Determination of flux through different metabolite pathways in Saccharomyces cervisiae by 1H-NMR and 13C-NMR spectroscopy. Eur. J. Biochem. 201, 715721.
  • [99]
    Deborde, C, Corre, C, Rolin, D.B., Nadal, L, de Certaines, J.D., Boyaval, P (1996) Trehalose biosynthesis in dairy Propionibacterium. J. Magn. Reson. Anal. 2, 297304.
  • [100]
    Werner, I, Bacher, A, Eisenreich, W (1997) Retrobiosynthetic NMR studies with 13C-labelled glucose. Formation of gallic acid in plants and fungi. J. Biol. Chem. 272, 2547425482.
  • [101]
    Inbar, L, Lapidot, A (1991) 13C nuclear magnetic resonance and gas chromatography-mass spectrometry study of carbon metabolism in the actinoycin D producer Streptomyces parvulus by use of 13C-labelled precursors. J. Bacteriol. 173, 77907801.
  • [102]
    Fell, D. (1997) Understanding the Control of Metabolism, pp. 213–225. Portland Press, London.
  • [103]
    Newsholme, E.A., Challis, R.A.J., Crabtree, B (1984) Substrate cycles: their role in improving sensitivity in metabolic control. Trends Biochem. Sci. 9, 277280.
  • [104]
    Steinbuchel, A (1986) Expression of the Escherichia coli pfkA gene in Alcaligenes eutrophus and in other Gram-negative bacteria. J. Bacteriol. 107, 570573.
  • [105]
    Rognstad, R (1996) Futile cycling in carbohydrate metabolism I. Background and current controversies on pyruvate cycling. Biochem. Arch. 12, 7183.
  • [106]
    Torres, J.C., Guixé, V, Babul, J (1995) A new method for assessing rates of the futile cycle during glycolytic and gluconeogenic metabolism. Arch. Biochem. Biophys. 321, 517525.
  • [107]
    Ronimus, R.S., Morgan, H.W (2001) The biochemical properties and phylogenies of phosphofructokinases from extremophiles. Extremophiles 5, 357373.
  • [108]
    Kengen, S, de Bok, F.A.M., van Loo, N.D., Dijkema, C, Stams, A.J.M., de Vos, W (1994) Evidence for the operation of a novel Embden-Meyerhof pathway that involves ADP-dependant kinases during sugar fermentation by Pyrococcus furiosus. J. Biol. Chem. 269, 1753717541.
  • [109]
    Alves, A.M., Euverink, G.J., Santos, H, Dijkhuizen, L (2001) Different physiological roles of ATP- and PP(i)-dependent phosphofructokinase isoenzymes in the methylotrophic actinomycete Amycolatopsis methanolica. J. Bacteriol. 183, 72317240.
  • [110]
    Landau, B.R (2001) Methods for measuring glycogen cycling. Am. J. Physiol. Endocrinol. Metab. 281, E413E419.
  • [111]
    Inbar, L, Kahana, Z.E., Lapidot, A (1985) Natural-abundance 13C Nuclear magnetic resonance studies of regulation and overproduction of l-lysine by Brevibacterium flavum. Eur. J. Biochem. 162, 621633.
  • [112]
    Matheron, C, Delort, A.-M, Gaudet, G, Forano, E (1997) Re-investigation of glucose metabolism in Fibrobacter succinogenes using NMR and enzymatic assays. Evidence of pentose phosphate phosphoketolase and pyruvate formate lyase activity. Biochim. Biophys. Acta 1335, 5060.
  • [113]
    Bibollet, X, Bosc, N, Matulova, M, Delort, A.-M, Gaudet, G, Forano, E (2000) 13C- and 1H-NMR study of cellulose metabolism by Fibrobacter succinogenes S85. J. Biotechnol. 77, 3747.
  • [114]
    Sillerud, L.O., Shulman, R.G (1983) Structure and metabolism of mammalian liver glycogen monitored by carbon-13 nuclear magnetic resonance. Biochemistry 22, 10871094.
  • [115]
    Matulova, M, Delort, A.M., Nouaille, R, Gaudet, G, Forano, E (2001) Concurrent maltodextrin and cellodextrin synthesis by Fibrobacter succinogenes S85 as identified by 2D NMR spectroscopy. Eur. J. Biochem. 268, 39073915.
  • [116]
    Schachar-Hill, Y, Pfeffer, P.E D.D. Doubs Jr., Osman, S.F., Doner, L.W., Ratcliffe, R.G (1995) Partitioning of intermediate carbon metabolism in vesicular-arbuscular mycorrhizal colonized leek. Plant Physiol. 108, 715.
  • [117]
    Bago, B, Pfeffer, P.E D.D. Doubs Jr., Brouillette, J, Bécard, G, Schachar-Hill, Y (1999) Carbon metabolism in spores of the Arbuscar Mycorrhizal fungus Glomus intraradices as revealed by nuclear magnetic resonance spectroscopy. Plant Physiol. 121, 263271.
  • [118]
    Pfeffer, P.E D.D. Doubs Jr., Bécard, G, Schachar-Hill, Y (1999) Carbon uptake and the metabolism and transport of lipids in an arbuscar mycorrhiza. Plant Physiol. 120, 587598.
  • [119]
    Thevelein, J.M., den Hollander, J.A., Shulman, R.G (1982) Changes in the activity and properties of trehalase during early germination of yeast ascospores: correlation with trehalose breakdown as studied by in vivo 13C NMR. Proc. Natl. Acad. Sci. USA 79, 35033507.
  • [120]
    Barton, J.K., den Hollander, J.A., Hopfield, J.J., Shulman, R.G (1982) 13C Nuclear magnetic resonance study of trehalose mobilisation in yeast spores. J. Bacteriol. 151, 177185.
  • [121]
    François, J, Parrou, J (2000) Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol. Rev. 25, 125145.
  • [122]
    Romeo, T, Black, J, Preiss, J (1990) Genetic regulation of glycogen biosynthesis in Escherichia coli: in vivo effects of the catabolic repression and stringent response systems in glg gene expression. Curr. Microbiol. 264, 39303934.
  • [123]
    Roach, P.J., Cheng, C, Huang, D, Lin, A, Mu, J, Skurat, A.V., Wilson, W, Zhai, L (1998) Novel aspects of the regulation of glycogen storage. J. Basic Clin. Physiol. Pharmacol. 9, 139151.
  • [124]
    Belanger, A.E., Hatfull, G.F (1999) Exponential-phase glycogen recycling is essential for growth of Mycobacterium smegmatis. J. Bacteriol. 181, 66706678.
  • [125]
    Guedon, E, Desvaux, M, Petitdemange, H (2000) Kinetic analysis of Clostridium cellulolyticum carbohydrate metabolism: importance of glucose-1-phosphate and glucose-6-phosphate branch points for distribution of carbon fluxes inside and outside cells as revealed by steady-state continuous culture. J. Bacteriol. 182, 20102017.
  • [126]
    Desvaux, M, Guedon, E, Petitdemange, H (2001) Carbon flux distribution and kinetics of cellulose fermentation in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium. J. Bacteriol. 183, 119130.
  • [127]
    Shulman, G.I., Rothman, D.L., Chung, Y, Rosseti, L W.A. Petit Jr., Barett, E.J., Shulman, R.G (1988) 13C NMR studies of glycogen turnover in the perfused rat liver. J. Biol. Chem. 263, 50275029.
  • [128]
    Massillon, D, Bollen, M, De Wulf, H, Overloop, K, Vanstapel, F, Van Hecke, P, Stalmans, W (1995) Demonstration of a glycogen/glucose-1-phosphate cycle in hepatocytes from fasted rats. Selective inactivation of phosphorylase by 2-deoxy-2-fluoro-α-d-glucopyranosyl fluoride. J. Biol. Chem. 270, 1935119356.
  • [129]
    Shulman, R.G., Rothman, D.L (2001) The ‘glycogen shunt’ in exercising muscle: A role for glycogen in muscle energetics and fatigue. Proc. Natl. Acad. Sci. USA 98, 457461.
  • [130]
    Laughlin, M.R., Petit, W.A., Dizon, J.M., Shulman, R.G., Barrett, E.J (1988) NMR measurement of in vivo myocardial glycogen metabolism. J. Biol. Chem. 263, 22852291.
  • [131]
    Melendez, R, Melendez-Hevia, E, Canela, E.I (1999) The fractal structure of glycogen: a clever solution to optimize cell metabolism. Biophys. J. 77, 13271332.
  • [132]
    Hottiger, T, Schmutz, P, Wiemken, A (1987) Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae. J. Bacteriol. 169, 55185522.
  • [133]
    Arguelles, J.C (2000) Physiological roles of trehalose in bacteria and yeasts: a comparative study. Arch. Microbiol. 174, 217224.
  • [134]
    Blomberg, A (2000) Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions: questions, some answers and a model. FEMS Microbiol. Lett. 182, 18.
  • [135]
    Teusink, B, Walsh, M.C., Van Dam, K, Westerhoff, V (1998) The danger of metabolic pathways with turbo design. Trends Biochem. Sci. 23, 162169.
  • [136]
    Jeong, R, Tombor, B, Albert, R, Oltvai, Z.N., Barabasi, A.-L (2000) The large-scale organization of metabolic networks. Nature 407, 651654.
  • [137]
    Gleiss, P.M., Stadler, P.F., Wagner, A. and Fell, D.A. (2000) Small cycles in small worlds. The Santa Fe Institute, electronic publications at: