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Appl Environ Microbiol, January 1998, p. 74-81, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

13C and 1H Nuclear Magnetic Resonance Study of Glycogen Futile Cycling in Strains of the Genus Fibrobacter

Christelle Matheron,1 Anne-Marie Delort,1,* Geneviève Gaudet,2,3 Evelyne Forano,3,* and Tibor Liptaj1,dagger

Laboratoire de Synthèse, Electrosynthèse et Etude de Systèmes à Intérêt Biologique, UMR 6504-Centre National de la Recherche Scientifique,1 and Centre Universitaire des Sciences et Techniques,2 Université Blaise-Pascal, 63177 Abière, and Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle,3 France

Received 30 May 1997/Accepted 19 September 1997

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. 13C nuclear magnetic resonance (NMR) analysis of [1-13C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by 13C NMR by following the simultaneous metabolism of labeled [13C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH2 of succinate and the CH3 of acetate when the resting cells were metabolizing [1-13C]glucose and unlabeled glycogen were precisely quantified by using 13C-filtered spin-echo difference 1H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Fibrobacter succinogenes is a major fibrolytic bacterium found in the rumens of cattle and sheep (36). It uses cellulose, glucose, and cellobiose as carbon and energy sources and produces succinate, acetate, and a little formate. A portion of the carbohydrates metabolized may be stored as glycogen, which can account for as much as 70% of the dry mass of the bacteria (35), and the intracellular glycogen concentration has been shown to be related to cell viability (39). 13C nuclear magnetic resonance (NMR) and 1H NMR have been used to monitor in vivo the storage and degradation of glycogen in resting cells of F. succinogenes S85 (10). This study showed that simultaneous storage and degradation of glycogen occurred when bacteria were supplied with an exogenous carbon source. Also, glycogen was synthesized during all growth phases (10), even though bacteria usually accumulate glycogen during periods of slow or no growth, such as the stationary phase, in the presence of excess carbon source (28). This finding, together with the presence of futile cycling of glycogen in resting cells, suggested that deficient regulation of glycogen metabolism occurs in F. succinogenes S85.

Strain S85 was originally isolated from the bovine rumen by Bryant and Doetsch in 1954 (5) and has been maintained as a pure culture ever since. Cultivation of this bacterium in laboratory media for more than 40 years may have led to genotypic changes in the organism. There is evidence which suggests that when wild strains of F. succinogenes are grown under laboratory conditions, physiological changes occur and variants or mutants are selected (35, 38). In this study, the glycogen metabolism and general features of carbon metabolism of two other strains of F. succinogenes and one strain of Fibrobacter intestinalis were analyzed by using in vivo 13C NMR. The F. succinogenes strains were isolated from different animals (cow, sheep, and buffalo) (2, 15, 16), and one of them (strain 095) was chosen because it has been subcultured little since its isolation. The F. intestinalis strain was isolated from a rat and shows less than 8% total DNA homology with strain S85, indicating that these organisms are only distantly related (2, 22). A new method, 13C-filtered spin-echo difference 1H NMR spectroscopy, was developed to measure the 13C enrichment of the end products succinate and acetate for all of the strains. This study was undertaken (i) to find out whether futile cycling of glycogen is due to deregulation in strain S85 resulting from extended cultivation under laboratory conditions or is a property of the genus or species and (ii) to look for metabolic differences between different strains of the genus Fibrobacter. In addition, complementary experiments were carried out to check the hypothesis that there is reversal of the succinate synthesis pathway in strain S85.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacterial strains and culture conditions. The strains used were F. succinogenes S85 (= ATCC 19169), the type strain of this species, which was isolated from the bovine rumen; F. succinogenes HM2 (= ATCC 43856), which was isolated from the sheep rumen and belongs to a different group in the species (2, 15, 16); F. succinogenes 095, an unclassified strain isolated from the buffalo rumen (obtained from K.-J. Cheng, Lethbridge, Alberta, Canada); and F. intestinalis NR9 (= ATCC 43854), the type strain of the second known species of the genus Fibrobacter, which was isolated from the rat cecum (22). The bacteria were grown on a medium containing 40% rumen fluid (12) and 3 g of cellobiose per liter.

Quantitative DNA-DNA hybridization. DNAs were isolated from the strains as described previously (8).

The relative DNA concentration for each strain was first determined by hybridization at 46°C to oligonucleotide ACGGGCGGTGTGTRC, which is complementary to all genes of 16S-like rRNAs characterized so far (25). The oligonucleotide probe was synthesized by Eurogentec (Seraing, Belgium) and was labeled with [gamma -32P]ATP by using T4 polynucleotide kinase. The hybridization and washing conditions used were the conditions described previously (15).

DNA (2.1 µg) from each isolate was denatured, divided into three 0.7-µg aliquots, and spotted onto nylon membranes (Hybond N+) by using a dot blot device (Bio-Rad). The membranes were then hybridized with 32P-radiolabeled (Ready-to-Go kit; Pharmacia) total DNA from each strain and washed under conditions similar to those used previously (2). After autoradiography, the membranes were cut and counted by scintillation spectrometry with a Packard model 2000CA apparatus.

Preparation of cells for NMR spectroscopy. For in vivo experiments, cells were prepared as described by Matheron et al. (18). Cells harvested in the late log phase were centrifuged (6,000 × g, 10 min, 4°C) and resuspended in reduced 50 mM potassium phosphate-0.4% Na2CO3-0.05% cysteine buffer (pH 7.3). The cells, at a final concentration of 5 mg of protein · ml-1, were incubated with 32 mM labeled or unlabeled glucose depending on the experiment. The cells were incubated at 37°C either in a spectrometer (in vivo NMR) or in a water bath and then sampled.

Sequential incubations were performed as follows. The first incubation of cells was performed in the presence of 32 mM [1-13C]glucose or unlabeled glucose. After the resting cells were washed, a second incubation was then performed with unlabeled glucose or [1-13C]glucose (17). The kinetics were monitored by in vivo 13C NMR.

For preparation of cell extracts, samples were frozen in liquid nitrogen and thawed three times. After centrifugation (15,000 × g, 10 min, 4°C) to remove the cell debris, the supernatants were used for 1H NMR or in vitro NMR.

Each experiment was carried out at least three times by using three different cultures for each strain.

NMR spectroscopy. Unless otherwise stated, NMR spectra were recorded with a Bruker model MSL 300 spectrometer operating at 300.13 MHz for 1H and at 75.4 MHz for 13C. The 2H resonance of D2O (10%) was used to lock the field and for shimming.

In vivo 13C NMR experiments were performed at 37°C as previously described by using a 10-mm-diameter probe (17). 1H-decoupled 13C NMR spectra were registered by using a Waltz 16 program to avoid sample heating.

In vitro 13C NMR experiments were performed by using an inverse-gated sequence with a Bruker model AM400 spectrometer (100.61 MHz; 90° pulse, 5.8 µs; relaxation delay, 60 s; 32,000 data points; 1,200 scans; 5-mm-diameter probe).

1H NMR spectra were acquired with an X-filtered spin-echo difference (XFSED) pulse sequence (9) by using a 5-mm inverse probe (1H/13C/15N) and the following equation: preparation - (90°)H - tau /2 - (180°)H/(alpha )X - tau /2 - (90°)X - FID(1H), where tau  is the evolution interval and the subscripts denote the nuclei experiencing the pulse (H, proton; X, 13C). The last carbon pulse was a purging pulse. Spectra with alpha = 0° or alpha  = 180° were acquired in subsequent scans and were stored independently in two blocks of memory. Extensive phase cycling was used to compensate for quadrature detection artifacts (CYCLOPS [13]) and 180° pulse imperfections (EXORCYCLE [23]). In a preparation period the solvent resonance was saturated by the DANTE pulse train (3) (gamma /2pi B1 = 5,500 Hz; pulse duration, 15 µs; pulse repetition, 250 µs) for a period of 2 s. tau  was adjusted by using the one-bond C,H constant for succinate and acetate (tau = 1/1J(C,H) = 7.2 ms). After eight dummy scans, 256 scans were accumulated in 8K of memory. The acquisition time was 1.024 s, the spectral width was 4,000 Hz, and the scan repetition time was 3.03 s. This repetition time was not sufficient for full relaxation of all protons, so for quantitative analysis a calibration measurement was made with an additional 10-s free relaxation delay inserted before the DANTE pulse. Both time domain spectra (alpha  = 0°, alpha  = 180°) were processed under identical conditions (same window function, same phase correction). After Fourier transformation the spectra were edited. The sum of the two spectra yielded the subspectrum of protons directly bonded to 12C nuclei or another heteronucleus (O or N), and their difference gave the subspectrum of protons directly bonded to 13C nuclei. We designated the first subspectrum the 12C subspectrum and the second subspectrum the 13C subspectrum. Examples of both subspectra are shown below (see Fig. 4). Before quantitative analysis both subspectra were corrected for the presence of metabolites at the beginning of the incubation with glucose. The sample for zero incubation time also contained an internal standard [3-(trimethylsilyl)propionic-2,2,3,3 D4 acid (TSPD4)] for quantitative comparison of integral intensities. After the zero time correction, relative integral intensities were determined for all of the relevant signals in both subspectra by using a standard integration procedure. Normalized intensities (TSPD4 = 100) were then corrected for the differences in T1 relaxation times by using correction factors determined by the calibration experiment. In addition, 13C integrals were corrected for the natural abundance contribution (1.1%). Isotopic enrichment (ei) at the position of the i-th carbon was then determined from the corrected integral intensities [Ii(13C) and Ii(12C)] of relevant signals in 13C and 12C subspectra with the equation ei = Ii(13C)/[Ii(13C) + Ii(12C)].

Precision of the method. Introduction of the 13C label into succinate molecules breaks the original symmetry of the proton spin system. The highly symmetrical A4 proton spin system (-12CH2-12CH2-) is replaced by the less symmetrical AA'BB' spin system (-13CHAA'-12CHBB'-) due to the isotopic effect of the 13C nucleus. As a consequence, different spin-spin interactions affect the evolution of the two spin systems during the spin-echo period. This change of evolution was manifested as small artifact signals present in both subspectra B and C (see Fig. 4). For quantitative determination of isotopic enrichment by using spin-echo spectra, it is therefore important to evaluate this effect. We analyzed the evolution of spins during a spin-echo experiment by using product spin operator formalism (34), and we found that the systematic error of the spin-echo method for determination of the enrichment of succinate CH2 groups was less than 1% and negative; i.e., the spin-echo method slightly underestimates the enrichment of succinate. The same results were obtained experimentally by carefully comparing the results obtained from standard spectra (10) and XFSED spectra. In the case of acetate, the introduction of the 13C label does not change the symmetry of the proton spin system, and no artifacts were observed in the 12C or 13C subspectra for this molecule.

Metabolite assays. Protein concentration was determined by the Bradford method (4) by using bovine serum albumin as the standard.

Succinate, acetate, formate, and glucose were assayed by using a Boehringer kit.

For glycogen determination, cells were harvested by centrifugation (15,000 × g, 15 min, 4°C), and the pellets were suspended in 0.25% sodium dodecyl sulfate. The suspension was then diluted 1:10 in 50 mM potassium phosphate buffer (pH 4.5) and incubated with 80 µg of amyloglucosidase (1,4-alpha -D-glucan glucohydrolase; EC 3.2.1.3) from Rhizopus mold per ml for 60 min at 55°C. Samples were centrifuged (15,000 × g, 5 min), and the glucose in the supernatant was assayed.

Chemicals. [1-13C]glucose and [2-13C]glucose (99% labeled) were purchased from Eurisotop (Saint-Aubin, France). All enzymes and chemicals were purchased from Sigma or Boehringer.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Classification of strain 095. The relationships between strain 095 and the other Fibrobacter strains studied were analyzed by performing DNA-DNA hybridization by using 32P-radiolabeled total DNA from each strain as a probe. The estimated levels of genomic DNA similarity determined in our hybridization experiments for S85, HM2, and NR9 were consistent with the values obtained previously by using the same technique or by 16S rRNA similarity analysis (2). About 55% DNA similarity was obtained between strains S85 and 095, suggesting that strain 095 belongs to group 1 of the F. succinogenes subspecies (2, 16).

Metabolite production by growing cells. Fibrobacter strains produce succinate, acetate, and a little formate from carbohydrate metabolism (18, 21). The concentrations of the metabolites produced by the strains studied and the ratios of the metabolites were first compared by using cultures grown for 15 h in a rumen fluid-containing medium supplemented with cellobiose as the carbon source (Table 1). The four strains produced the same metabolites at similar concentrations, and the ratios of succinate, acetate, and formate were similar (3.7/1/0.3).

                              
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  		TABLE 1.   Concentrations of metabolites produced by Fibrobacter strains

The abilities of the different strains to store glycogen during growth were also tested. Glycogen was stored during all growth phases by the four strains cultivated with 3 g of either glucose or cellobiose per liter (data not shown). Glycogen accumulation expressed as the glycogen-to-protein mass ratio was stable during the exponential growth phase; the mean value was 0.6 for all four Fibrobacter strains.

Glucose metabolism in resting cells. Resting cells (5 mg of protein · ml-1) of Fibrobacter strains were incubated with [1-13C]glucose at 37°C, and the kinetics of glucose utilization were monitored in vivo by 13C NMR spectroscopy. Figure 1 shows 13C NMR spectra for glucose metabolism by the four strains used, obtained at the end of incubation. Disappearance of the two glucose anomers beta -[1-13C] (96.4 ppm) and alpha -[1-13C] (92.6 ppm) was associated with the production of [2-13C]succinate (34.5 ppm), [2-13C]acetate (23.7 ppm), [1-13C]glycogen (100.1 ppm), and [6-13C]glycogen (61.0 ppm). The spectra clearly showed that all of the strains produced the same metabolites from glucose catabolism and that the positions of the labeled carbons in succinate and acetate were as expected for glucose metabolized via the Embden-Meyerhof-Parnas (EMP) pathway (18, 21). Additionally, [3-13C]malate (43.03 ppm), an intermediate in the succinate synthesis pathway (18), was found in all four strains. Formate was never observed on 13C NMR spectra recorded during in vivo experiments or in cell extracts from [1-13C]glucose catabolism, and it was clearly present on 1H spectra for all of the strains (data not shown). Thus, formate is likely formed from C-3 or C-4 of glucose. Similar incubations were carried out in parallel in a water bath at 37°C, and succinate and acetate were enzymatically assayed. The succinate-to-acetate concentration ratios ranged between 3.5 and 3.7 for the four Fibrobacter strains (data not shown), and these ratios agreed with those calculated for growing cells (Table 1). The same ratios were also obtained between the areas of the succinate and acetate signals, obtained from cell extracts by 1H NMR under quantitative conditions.


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  		FIG. 1.   13C NMR spectra of in vivo [1-13C]glucose utilization by resting cells of Fibrobacter species. [1-13C]glucose (32 mM) was added to a cell suspension (5 mg of protein · ml-1) in a 10-mm tube, and proton-decoupled 13C NMR spectra were obtained every 4.5 min; the spectra shown were recorded 22.5 min after glucose was added. AC, acetate; GLC, glucose; GLY, glycogen; MAL, malate; SUC, succinate.

The ability of the strains to store glycogen was also shown by the 13C NMR spectra, in which high [1-13C]glycogen and [6-13C]glycogen peaks were observed. Labeling at position C-6 of glycogen was observed previously for strain S85 and results from a reversal of glycolysis at the triose phosphate level. This characteristic was observed here for the three other strains studied. To quantify the extent of the glycolysis reversal in resting cells, the ratios of [1-13C]glycogen to [6-13C]glycogen were calculated from the areas of the signals obtained by in vivo 13C NMR. These ratios were approximately constant throughout incubation, and their means ranged from 2.5 to 3. These values suggest that about 50% of the labeled glycogen is synthesized after reversal of the glycolytic pathway.

Glycogen futile cycling. We first determined by in vivo 13C NMR that all of the strains were able to metabolize endogenous glycogen when the exogenous carbon source was limiting or exhausted. Figure 2A shows the kinetics of [1-13C]glucose utilization by F. intestinalis NR9. During the first 25 min, [1-13C]glucose was consumed and [2-13C]succinate, [2-13C] acetate, [1-13C]glycogen, and [6-13C]glycogen were synthesized. For clarity, only the [1-13C]glucose, [1-13C]glycogen, and [2-13C]succinate signal intensities are shown in Fig. 2A. After 25 min, when approximately 6 mM glucose was left, the amounts of [1-13C]glycogen and [6-13C]glycogen started to decrease, whereas synthesis of [2-13C]succinate and [2-13C]acetate continued. These results show that in the presence of a low concentration of glucose, glycogen was metabolized. Similar results were obtained for strains S85, 095, and HM2 of F. succinogenes (data not shown).


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  		FIG. 2.   Time-dependent changes in signal integrals of labeled metabolites during [1-13C]glucose or prestored [1-13C]glycogen and [6-13C]glycogen utilization by resting cells of F. intestinalis NR9. (A) Resting cells of F. intestinalis (5 mg of protein · ml-1) were incubated with 32 mM [1-13C]glucose, and metabolite production was monitored during glucose utilization and 25 min after exogenous carbon sources were exhausted. (B) Resting cells of F. intestinalis (5 mg of protein · ml-1) were first incubated with 32 mM [1-13C]glucose for 22.5 min, washed, and incubated with 32 mM unlabeled glucose. (C) Resting cells of F. intestinalis (5 mg of protein · ml-1) were first incubated with 32 mM unlabeled glucose for 20 min, washed, and incubated with 32 mM [1-13C]glucose. Symbols: square , total [1-13C]glucose; black-triangle, total [1-13C]glucose 6-phosphate; bullet , [1-13C]glycogen; open circle , [6-13C]glycogen; black-square, total [1-13C]glycogen and [6-13C]glycogen; triangle , [2-13C]succinate.

To test for futile cycling of glycogen in the strains studied, sequential incubation experiments were carried out. Glycogen was first labeled by incubating the cells with 32 mM [1-13C]glucose at 37°C, and then the cells were washed in reduced potassium phosphate buffer containing unlabeled glucose (32 mM) at 4°C and incubated in the same buffer at 37°C. In vivo 13C NMR spectra were collected every 4.5 min for 30 min. This experiment allowed the time course of the signals of prestored [1-13C]glycogen and [6-13C]glycogen to be followed during metabolism of exogenous unlabeled glucose by the cells. Typical spectra for Fibrobacter strains S85, 095, and NR9 are shown in Fig. 3; these spectra show that [2-13C]succinate, [1-13C]glucose 6-phosphate, and [6-13C]glucose 6-phosphate were present. Accumulation of glucose 6-phosphate under similar conditions has been observed previously for strain S85 (17). Time courses of the relative integrals of [1-13C]glycogen, [6-13C]glycogen, [alpha -1-13C]glucose 6-phosphate, [beta -1-13C]glucose 6-phosphate, and [2-13C]succinate obtained for strain NR9 are presented in Fig. 2B. The [1-13C]glycogen and [6-13C]glycogen signal intensities decreased during the 30 min of incubation. At the same time, [2-13C]succinate, [1-13C]glucose 6-phosphate, and [6-13C]glucose 6-phosphate were synthesized, confirming that glycogen was consumed while bacteria were metabolizing exogenous glucose. In parallel, we checked whether the cells were still metabolizing exogenous glucose into glycogen and succinate by performing the complementary reverse experiment; the cells were first incubated with unlabeled glucose, washed, and then incubated with [1-13C]glucose (Fig. 2C).


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  		FIG. 3.   13C NMR spectra for in vivo prestored [1-13C]glycogen and [6-13C]glycogen utilization by resting cells of Fibrobacter species. Resting Fibrobacter cells (5 mg of protein · ml-1) were first incubated with 32 mM [1-13C]glucose for 22.5 min, washed, and incubated with 32 mM unlabeled glucose. The proton-decoupled 13C NMR spectra shown were recorded 27 min after unlabeled glucose was added. GLY, glycogen; G6P, glucose 6-phosphate; SUC, succinate.

The same time courses were obtained with F. succinogenes S85 and 095 (data not shown). It was not possible to perform similar experiments with HM2 as this strain did not tolerate repeated centrifugation.

The presence of futile cycling was also shown during single incubations of bacteria with [1-13C]glucose by quantifying the isotopic enrichment of succinate and acetate. Metabolism of unlabeled endogenous glycogen along with exogenous [1-13C]glucose results in isotopic dilution of the metabolites. Thus, to check for futile cycling in strain HM2 and to confirm the results obtained by sequential incubations of the other strains, percentages of labeling of acetate and succinate were determined for cell extracts from the four strains incubated with [1-13C]glucose. Percentages of labeling can be determined by standard 1H NMR (10). However, the disadvantage of standard 1H NMR is that signals from labeled and unlabeled molecules can overlap and this can seriously impair the precision of the results. As a low level of isotopic dilution is expected (10), precision is critical in drawing a conclusion. Consequently, we decided to develop a 13C-filtered spin-echo difference pulse sequence to separate 13C and 12C proton subspectra and thereby increase the precision of the quantification. An example of the spectra obtained for strain HM2 is presented in Fig. 4. Spectrum A represents a standard 1H spectrum, with the central peaks corresponding to protons bound to 12C atoms and the satellite peaks corresponding to protons bound to 13C atoms. Spectra B and C correspond to subspectra of protons bound to 12C and 13C atoms, respectively. The percentage of enrichment of the CH2 of succinate, as determined by this method, varied from 20.2 to 21% for the four strains (Table 2). The systematic error of the spin-echo method was less than 1% and negative, as estimated by using product spin operator formalism (see above) (34). The values obtained (20 to 21%) can thus be considered significantly different from the theoretical enrichment value, 25%. Taking into account the 1% underestimation, our results indicate that the lack of enrichment of CH2 (3 to 4%) reflects isotopic dilution due to futile cycling of glycogen. In the case of acetate, the theoretical enrichment value is 50%, and the values obtained were much lower (33 to 36%) (Table 2). The dilution of the acetate molecules results from two phenomena, glycogen futile cycling and another mechanism that we decided to quantify and elucidate in the case of strain S85.


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  		FIG. 4.   Separation of 12C and 13C subspectra in 1H NMR spectra from strain HM2. The spectra were acquired with an X-filtered spin-echo pulse sequence as described in Materials and Methods. Spectrum A represents the output of an experiment with no 180° (13C) pulse in the middle of the spin-echo period and is similar to a standard 1H NMR spectrum. Subspectra B and C were obtained after subtraction (subspectrum B) or addition (subspectrum C) of the two original spectra acquired with the spin-echo pulse sequence (with and without central inversion of 13C spin). Subspectrum C is a pure 13C isotopomer subspectrum of a standard 1H spectrum.

                              
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  		TABLE 2.   Percentages of 13C enrichment of the CH3 of acetate and the CH2 of succinate in the metabolites produced by Fibrobacter strains

Complementary experiments. The following different hypotheses can explain the synthesis from [1-13C]glucose of acetate not labeled on the CH3: (i) the oxidative part of the pentose phosphate pathway is connected with a phosphoketolase or with the Entner-Dodouroff pathway; (ii) there is reversion of the pathway of succinate synthesis (from fumarate to phosphoenolpyruvate [PEP] or pyruvate); or (iii) there is another metabolic pathway. In a recent paper, we showed that the first hypothesis was not supported by data; although a phosphoketolase was present, the oxidative part of the pentose phosphate pathway was not shown (18). On the contrary, we clearly found that fumarase activity was reversible even in vivo (18), and this was the first indication which supported the second hypothesis. Complementary experiments were carried out to check this hypothesis.

First, 13C NMR experiments were performed with extracts from cells incubated with [1-13C]glucose under conditions allowing detection of carboxylates (Fig. 5, spectrum A). Direct metabolism of [1-13C]glucose produces [2-13C]acetate and no [1-13C]acetate. However, if PEP or pyruvate is formed from fumarate by the reversal of this metabolic route, it can give rise to [1-13C]acetate. Indeed, because of the scrambling at the fumarate level, labeling is distributed between [2-13C]malate and [3-13C]malate (18), giving rise to [1-13C]pyruvate and [2-13C]pyruvate and finally to [1-13C]acetate and [2-13C]acetate. In Fig. 5, spectrum A, a signal resonating at 181.8 ppm was assigned to [1-13C]acetate. This signal was not present when the cells were incubated with [12C]glucose (Fig. 5, spectrum C). On the contrary, the signal at 182.7 ppm, corresponding to the carboxylate of succinate, was present in Fig. 5, spectra A and C; it was thus attributed to a natural abundance contribution.


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  		FIG. 5.   Proton-decoupled 13C NMR spectra obtained with extracts of F. succinogenes S85 incubated with [1-13C]glucose (spectrum A), [2-13C]glucose (spectrum B), or [12C]glucose (spectrum C) by using an inverse-gated sequence.

In parallel, the cells were incubated under the same experimental conditions but with [2-13C]glucose (Fig. 5, spectrum B). In this case, only [1-13C]acetate was expected from direct synthesis. Because of the scrambling phenomenon described above, reversal from fumarate should give rise to [1-13C]acetate and [2-13C]acetate. In Fig. 5, spectrum B, resonance of [2-13C]acetate (delta  = 23.6 ppm) was clearly detected, while it was absent in Fig. 5, spectrum C; thus, this resonance corresponded to significant labeling of the methyl group of acetate.

The results of these 13C NMR experiments are consistent with the formation of part of acetate via reversal from fumarate. Thus, this process could be responsible for the unexpected dilution of enrichment of acetate shown in Table 2 when the cells were incubated with [1-13C]glucose, in addition to the dilution due to futile cycling of glycogen.

To quantify the contributions of this phenomenon and of futile cycling to the final isotopic dilution of acetate, precise measurements of the labeling of acetate (CH3) and succinate (CH2) were obtained by 1H NMR by using the XFSED method with extracts from cells incubated with [2-13C]glucose. The results are reported in Table 2. The percentage of 13C enrichment of the CH3 of acetate resulting from putative reversal from fumarate was 9.8%. Consequently, when cells are incubated with [1-13C]glucose, this phenomenon gives rise to 9.8% of the acetate that is not labeled on CH3, and so the total amount of acetate coming from the degradation of [1-13C]glucose is 33.4% plus 9.8% (43.2%). The neat contribution of futile cycling to the labeling of the acetate molecule is 6.8% (about 7%).

The values obtained for 13C enrichment of the CH2 of succinate (Table 2) were similar when the cells were incubated with [1-13C]glucose (21.0%) or [2-13C]glucose (20.8%), equivalent to about 42% of the labeled succinate molecules. This confirms that in the case of succinate, isotopic dilution is only due to the futile cycling of glycogen. The futile cycle contributes to the synthesis of about 8% of the unlabeled succinate or about 7% if we take into account the possible systematic error of the spin-echo method.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this work, we investigated the carbon metabolism of three strains of F. succinogenes, type strain S85, strain 095, which we classified in the same group, and strain HM2, belonging to another group (15, 16), and of the type strain of F. intestinalis, NR9. These four strains produced the same amounts of metabolites (succinate, acetate, and formate) in the same ratios and via the same metabolic pathways. Monitoring of [1-13C]glucose utilization by in vivo 13C NMR revealed equivalent reversals of glycolysis for the four strains studied. In addition, all of the strains were able to store glycogen throughout the growth phase with the same constant glycogen/protein ratio during the exponential growth phase. Usually, storage polymers such as glycogen are synthesized in cells growing under conditions of carbon input exceeding the capacity of the central pathways, while Fibrobacter strains always store glycogen. Simultaneous storage and degradation of glycogen were previously found in resting cells of F. succinogenes S85 (10). As this feature was unusual, we wanted to determine whether it was a specific characteristic of strain S85 or a property of the genus Fibrobacter.

The analysis was based on precise determination of the isotopic dilution of the CH2 of succinate and the CH3 of acetate by using 13C heteronuclear spin-echo difference 1H NMR spectroscopy. This technique was first introduced by Freeman et al. (9), but has not been used frequently in metabolic studies. Its main applications in this field have been concerned with indirect 15N detection (20, 33, 37). In this work, we used this approach for indirect 13C detection. This method, when it was applied to the four strains metabolizing [1-13C]glucose, revealed a deficit of 3 to 4% in the enrichment of the CH2 groups of succinate molecules (i.e., a shortfall of enrichment of the succinate molecule of 6 to 8%), indicating that 12 to 16% of the succinate came from unlabeled glucose. In the case of strain S85, quantification of the percentage of enrichment of succinate with cells incubated with [2-13C]glucose confirmed this estimate; a shortfall of enrichment of the succinate molecule of at least 7% was obtained (i.e., at least 14% of the succinate came from unlabeled glucose).

For acetate, the values obtained for the four strains incubated with [1-13C]glucose were much smaller than the theoretical values (Table 2), due to an additional phenomenon that was investigated with strain S85. This mechanism was quantified, and it accounted for 9.8% of the labeling of acetate, indicating that the total level of labeling of acetate was about 43%. Consequently, the shortfall of enrichment of 7% for the acetate molecule was due to the futile cycling of glycogen, indicating that 14% of the acetate came from unlabeled glucose (Fig. 6).


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  		FIG. 6.   Expected enrichment of succinate and acetate produced from 100% [1-13C]glucose. The asterisk indicates the 13C atom.

In conclusion, converging values from two different experiments showed that 12 to 16% of the glucose entering the EMP pathway is derived from prestored glycogen (Fig. 6).

The results of an analysis of the distribution of the 13C label of acetate when S85 cells were incubated with [1-13C]glucose or [2-13C]glucose are consistent with reversal from fumarate to PEP or pyruvate. Under our experimental conditions, reversal from fumarate to malate was clearly shown previously (18). Different enzymes can catalyze the reactions from malate to PEP or pyruvate; the activity of these enzymes should be tested in F. succinogenes to confirm the occurrence of this reversal under our experimental conditions and to exclude the possibility that there is any other unidentified metabolic pathway leading to the same labels on acetate. Whatever its origin, this phenomenon was precisely quantified and allowed us to determine the significant contribution of futile cycling to carbon metabolism in resting cells.

To our knowledge, futile cycles have not yet been reported for glycogen in other microorganisms. Furthermore, in this study futile cycling was quantified in resting cells, and we do not know its contribution in growing cells. It has been suggested that futile cycles could serve to dissipate ATP under conditions of energy excess. F. succinogenes was also shown to synthesize cellodextrins (either from the activity of a cellobiose phosphorylase [40] or from the activity of a cellobiase [17]) that were released into the culture medium and could be utilized by other bacteria (40). This cellodextrin efflux represents a potential loss of carbon and energy, and its role is not known (40). Thus, it seems that F. succinogenes is able to dissipate excess energy in the form of cellodextrins or a glycogen futile cycle. As bacteria usually use futile cycles to consume unneeded ATP when their growth is limited by nutrients other than the carbon source, for example, it would be interesting to quantify glycogen futile cycling in F. succinogenes under conditions that are not limiting (e.g., in the presence of ammonia).

Futile cycles have been identified in several systems, although their role in metabolism is not fully understood (30-32); for example, futile cycling was unequivocally observed in liver parenchymal cells and kidney cortex tubules in vitro at the following three major sites: between pyruvate and PEP, between fructose 1,6-diphosphate and fructose 6-phosphate, and between glucose 6-phosphate and glucose (31). Futile cycles have also been described in Streptococcus cremoris and yeasts (7, 24), in which simultaneous activity of phosphofructokinase (PFK) and fructose 1,6-diphosphatase, resulting in dissimilation of ATP, was found. As stated above, a reversal of glycolysis was observed in F. succinogenes, implying that fructose 6-phosphate is synthesized from fructose 1,6-diphosphate. However, in F. succinogenes, as in some other bacteria, plants, and protozoa (1, 19), a PPi-dependent PFK activity was observed along with a very weak ATP-dependent PFK activity (29). In contrast to the ATP-dependent PFK, the PPi-dependent PFK is reversible (19). It is thus likely that no futile cycling occurs at this level in F. succinogenes.

Futile cycles may be difficult to detect with classical techniques. Overexpression of enzymes by using multicopy plasmids was used to induce such cycles. In Escherichia coli, a futile cycle between PEP and oxaloacetate or between PEP and pyruvate was created by overexpression of the corresponding enzymes (6, 26). The presence of these futile cycles induced an increase in the excretion of fermentation end products and a loss of energy. Another way to reveal such metabolic or enzyme futile cycles is to use 13C NMR spectroscopy. Analysis of the isotopomer composition by using in vivo 13C NMR showed that there is futile cycling in different types of cells (7, 14, 27, 31). It is likely that the increasingly common utilization of NMR spectroscopy to study carbon metabolism will reveal additional futile cycles, and screening of glycogen metabolism in microorganisms able to store it may well demonstrate that glycogen futile cycling is widespread.

DNA hybridization and 16S rRNA similarity analysis indicated that there is broad genetic diversity in the genus Fibrobacter (2). This work aimed to determine if certain specific metabolic features of F. succinogenes S85 were due to phenotypic alteration with time. Although strains S85 and 095 were not closely related to strains HM2 and NR9 and strain 095 has not been subcultured much compared with S85, the following physiological properties tested were found to be similar for all four strains: the general metabolic pathway and the more specific routes, such as futile cycling of glycogen and reversal of glycolysis. These results indicate that these phenotypic characteristics are intrinsic properties of the genus Fibrobacter. Also, other unusual features were previously found in strains representative of the two Fibrobacter species, such as the production of a GTP-dependent hexokinase found in F. succinogenes S85 and F. intestinalis C1A (11) or the activity of a pentose-phosphate phosphoketolase observed in the four strains studied here (18). Although genetically diverse, the genus Fibrobacter appears to exhibit marked homogeneity in carbon metabolism.

    ACKNOWLEDGMENTS

This work was supported by a grant to C.M. from the Centre National de la Recherche Scientifique and the Région Auvergne. T.L. was an invited professor at the Université Blaise-Pascal of Clermont-Ferrand.

We thank Y. Ribot for performing the hybridization experiments.

    FOOTNOTES

* Corresponding author. Mailing address for Anne-Marie Delort: Laboratoire de Synthèse, Electrosynthèse et Etude de Systèmes à Intérêt Biologique, UMR 6504-CNRS, Université Blaise-Pascal, 63177, Aubière, France. Phone: (33) 04 73 40 77 14. Fax: (33) 04 73 40 77 17. E-mail: amdelort{at}chimtp.univ-bpclermont.fr. Mailing address for Evelyne Forano: Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France. Phone: (33) 04 73 62 42 48. Fax: (33) 04 73 62 45 81. E-mail: forano{at}clermont.inra.fr.

dagger Present address: Central Laboratories, Slovak Technical University, 81237 Bratislava, Slovakia.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Appl Environ Microbiol, January 1998, p. 74-81, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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