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Applied and Environmental Microbiology, May 1999, p. 1941-1948, Vol. 65, No. 5
Laboratoire de Synthèse,
Received 20 October 1998/Accepted 18 February 1999
The effect of the presence of ammonia on
[1-13C]glucose metabolism in the rumen fibrolytic
bacterium Fibrobacter succinogenes S85 was studied by
13C and 1H nuclear magnetic resonance (NMR).
Ammonia halved the level of glycogen storage and increased the rate of
glucose conversion into acetate and succinate 2.2-fold and 1.4-fold,
respectively, reducing the succinate-to-acetate ratio. The
13C enrichment of succinate and acetate was precisely
quantified by 13C-filtered spin-echo difference
1H-NMR spectroscopy. The presence of ammonia did not modify
the 13C enrichment of succinate C-2 (without ammonia,
20.8%, and with ammonia, 21.6%), indicating that the isotopic
dilution of metabolites due to utilization of endogenous glycogen was
not affected. In contrast, the presence of ammonia markedly decreased
the 13C enrichment of acetate C-2 (from 40 to 31%),
reflecting enhanced reversal of the succinate synthesis pathway. The
reversal of glycolysis was unaffected by the presence of ammonia as
shown by 13C-NMR analysis. Study of cell extracts showed
that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine
dehydrogenase. Glutamine synthetase activity was not detected.
Glutamate dehydrogenase was active with both NAD and NADP as cofactors
and was not repressed under ammonia limitation in the culture.
Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities
were evidenced by spectrophotometry and 1H NMR. When cells
were incubated in vivo with [1-13C]glucose, only
13C-labeled aspartate, glutamate, alanine, and valine were
detected. Their labelings were consistent with the proposed amino acid
synthesis pathway and with the reversal of the succinate synthesis pathway.
Fibrobacter succinogenes
is a major rumen fibrolytic bacterium. It uses cellulose, glucose, and
cellobiose as carbon and energy sources and ammonia as its sole
nitrogen source (21). Ammonia is the prime source for
protein synthesis in the rumen, accounting for the synthesis of 50 to
70% of bacterial nitrogen (13). Most of the studies of
ammonia assimilation in the rumen have dealt with total ruminal
content, and only a few papers have given detailed information on
individual species (18, 23). In particular, the pathways of
ammonia assimilation and amino acid synthesis in F. succinogenes are still unknown, although its carbon metabolism has
been extensively studied (14, 15, 17). Glucose is
metabolized through glycolysis into succinate, acetate, and a little
formate. A part of the carbohydrates metabolized is stored as glycogen. Analysis by 13C and 1H nuclear magnetic
resonance (NMR) was previously used to monitor in vivo the storage and
degradation of glycogen in resting cells of different strains of
F. succinogenes (8, 16), and a futile cycling of
glycogen was shown: glycogen was simultaneously stored and degraded
when bacteria were supplied with an exogenous carbon source.
Furthermore, F. succinogenes was shown to accumulate
glycogen throughout the exponential growth phase even when ammonia was not limiting (8, 16), whereas bacteria usually accumulate glycogen when ammonia (or another factor) limits growth
(19). In addition to the futile glycogen cycle, a reversal
of glycolysis and of the succinate synthesis pathway was found in
F. succinogenes (16). These unusual features
prompted us to investigate the relationship between ammonia
assimilation and glycogen storage and also the effect of the presence
of ammonia on the futile cycles. The enzymes responsible for ammonia
assimilation and amino acid synthesis from intermediates of glucose
metabolism were also sought.
The main pathways of ammonia assimilation were glutamate dehydrogenase
(GLDH) and alanine dehydrogenase (ADH). The results obtained by
13C-NMR and 13C-filtered spin-echo difference
(13C-FSED) 1H-NMR spectroscopy suggest a
modulation of carbon metabolism by ammonia in F. succinogenes S85.
Culture conditions.
F. succinogenes S85 (ATCC 19169)
was grown for 15 h on a chemically defined medium (8)
with 3 g of cellobiose per liter.
Preparation and incubation of cells.
For in vivo NMR
experiments, cells were prepared as described by Matheron et al.
(16). The cells harvested in the late log phase were spun
(6,000 × g, 10 min, 4°C) and resuspended in a reduced 50 mM potassium phosphate-0.4%
Na2CO3-0.05% cysteine buffer (pH 7.1). The
cells at a final concentration of 5 mg of protein · ml Preparation of cell extracts.
For 13C-FSED
1H-NMR and 13C-NMR experiments, samples taken
at the end of the incubation were frozen in liquid nitrogen and thawed three times. After being spun (15,000 × g, 10 min,
4°C) to remove the cell debris, the supernatants were analyzed by NMR.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Interactions between Carbon and Nitrogen Metabolism
in Fibrobacter succinogenes S85: a 1H and
13C Nuclear Magnetic Resonance and Enzymatic
Study
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 were incubated with 32 mM
[1-13C]glucose, with or without 13 mM
(NH4)2SO4, depending on the
experiment. The cells were incubated at 37°C either in the
spectrometer (in vivo NMR) or in a water bath and then sampled.
NMR spectroscopy. NMR spectra were recorded on a Bruker MSL 300 spectrometer operating at 300.13 MHz for 1H NMR and 75.4 MHz for 13C NMR. 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, with a 10-mm-diameter probe (14). In vitro 13C-NMR experiments were performed with a 5-mm-diameter probe (60° pulse, 2-s relaxation delay, 8 kilobytes of data, 1,000 scans). The low-power Waltz-16 proton-decoupling program was used to avoid sample heating. A pure benzene capillary, centered in the NMR tube, was used as an external reference resonating at 128.5 ppm from tetramethylsilane and to normalize the integrals of 13C-NMR spectra. 1H-NMR spectra, unless otherwise stated, were acquired with a 13C-FSED pulse sequence and a 5-mm-diameter inverse probe (1H-13C-15N) (16) by the following formula: preparation
(90°)H 
/2 (180°)H/(
)X /2 (90°)X FID(1H), where the subscripts
denote the nucleus experiencing the pulse (H, proton;
X, 13C), is the evolution interval, is
pulse angle, and FID is free induction decay. The last carbon pulse is
a purging pulse. Spectra with an of 0 or 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) and 180° pulse imperfections (EXORCYCLE). In a
preparation period the solvent resonance was saturated by the DANTE
pulse train [(
/2
)B1 = 5,500 Hz; pulse duration, 15 µs; pulse repetition, 250 µs] for a period of 2 s. was
adjusted according to the one-bond C,H constant for succinate and
acetate [ = 1/1J(C,H) = 7.2 ms]. After 8 dummy scans, 256 scans were accumulated in 8 kilobytes 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.
The determination of percentages of 13C enrichment of
succinate and acetate is fully described in the work of Matheron et al. (16).
Enzyme activities.
The experimental conditions for the
enzymatic assays are reported in Table 1.
The assays for GLDH (reaction I), glutamine synthetase (GS) (reaction
II), glutamate-pyruvate transaminase (reaction V), and
glutamate-oxaloacetate (OAA) transaminase (reaction VI) activities were
adapted from the work of Erfle et al. (7), except that the
products of reactions II, V, and VI were detected by 1H
NMR. Activity of ADH was assayed in a system similar to that in
reaction I except that pyruvate was used in place of -ketoglutarate.
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Metabolite assays. Protein concentration was determined by the Bradford method (3), with bovine serum albumin as the standard.
Succinate, acetate, and glucose were assayed with a Boehringer kit. Ammonia was assayed by the phenol-hypochlorite reaction described by Weatherburn (24). Glycogen was quantified by a glucose assay after hydrolysis with amyloglucosidase as previously described (16).Chemicals. [1-13C]glucose (99% labeled) was purchased from Eurisotop (Saint Aubin, France). All enzymes and chemicals were purchased from Sigma or Boehringer.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of ammonia on glucose metabolism in F. succinogenes S85 cells.
Resting cells of F. succinogenes S85 (5 mg of protein · ml1) were
incubated with 32 mM [1-13C]glucose at 37°C in the
absence of ammonia or in the presence of 26 mM ammonia. Kinetics of
[1-13C]glucose utilization were monitored in vivo by
13C-NMR spectroscopy. Figure
1A presents a spectrum recorded after 27 min of incubation in the presence of ammonia. As in the absence of
ammonia, disappearance of the two glucose anomer signals,
-[1-13C]glucose (96.4 ppm) and
-[1-13C]glucose (92.6 ppm), was associated with
detection of signals of [2-13C]succinate (34.5 ppm),
[2-13C]acetate (23.7 ppm), [6-13C]glycogen
(61.0 ppm), and [1-13C]glycogen (100.1 ppm); the labeling
at the C-6 position of glycogen results from glycolysis reversal after
isomerization at the triose-phosphate level (8, 16). The
ratio of the integrals of [1-13C] and
[6-13C]glycogen (about 3) was not significantly
affected by the presence of ammonia, indicating that ammonia did not
modulate this reversal.
|
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) or in the absence
(
) of 26 mM ammonia, measured from in vivo 13C-NMR
spectra.
1 · min1) was reduced by 33%
(0.20 µmol · mg of protein1 · min1) in the presence of 26 mM ammonia. The ammonia
consumption was 60 nmol · mg of protein1 · min1. The presence of ammonia had no effect on the
kinetics of succinate synthesis, while the synthesis of acetate was
increased 1.5-fold. The accumulation of glycogen was decreased twofold
during the first 10 min and threefold by the end of the incubation.
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= 181.8 ppm) was increased in the presence
of ammonia. This result is consistent with the increase in the reversal
of the succinate pathway. We previously explained that because of
scrambling at the fumarate level, the reversal gives rise to both
[1-13C]acetate and [2-13C]acetate
(16).
Ammonia assimilation and amino acid synthesis. (i) Enzyme
activities.
Enzymes involved in the synthesis of amino acids from
intermediates of carbon metabolism (-ketoglutarate, pyruvate, OAA, and fumarate), either by direct amination from ammonia or by
transamination, were sought in F. succinogenes sonicated
extracts. Activities of dehydrogenases or transaminases were monitored
by either spectrophotometry or 1H NMR (Table
4).
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-ketoglutarate by GLDH (reaction I) and the synthesis
of glutamine from glutamate by GS (reaction II). The ammonia
concentration in the culture medium may affect the respective contributions of the two enzymes to ammonia assimilation (18, 23). We therefore assayed the activities of the two enzymes in
F. succinogenes cells cultured with two different ammonia
concentrations: 0.5 and 13 mM. The specific activities of NAD-GLDH were
80 nmol · mg of protein1 · min1 in extracts of cells grown with 13 mM ammonia and
120 nmol · mg of protein1 · min1 in extracts of cells grown with 0.5 mM ammonia
(Table 4). GLDH activities were equivalent with NADH or NADPH as the
cofactor in extracts of cells grown with 13 mM ammonia. Under this
condition, the reverse reaction was 10 times less efficient and
occurred only with NADP as the cofactor (Table 4). Our results extend the previous conclusions of Joyner and Baldwin (12), who
found only an NADP-GLDH activity in extracts of F. succinogenes S85.
The GS (reaction II) activities in extracts incubated with ammonia and
ATP or GTP were investigated by 1H NMR. No resonance
corresponding to the signals of glutamine was detected on the spectrum
(not shown).
Activity of ADH (reaction III) was detected in the presence of NADPH
(15 nmol · mg of protein1 · min1) (Table 4). The reverse reaction was not found.
Aspartase (reaction IV) activity in extracts incubated with fumarate
and ammonia was investigated by 1H NMR. Under these
conditions, only malate, resulting from the activity of fumarase, was
detected on the spectrum (not shown).
Activities of the reverse reactions of glutamate-pyruvate transaminase
(reaction V) and glutamate-OAA transaminase (reaction VI), measured by
spectrophotometry, were 17.3 and 36 nmol · mg of
protein1 · min1, respectively (Table
4). Both direct and reverse reactions were shown by 1H NMR
(Table 4).
The spectra of incubations performed to show transaminations V and VI
in the forward direction are presented in Fig.
4. On the spectrum in Fig. 4A, obtained
at the end of an incubation with pyruvate and glutamate (reaction V), a
singlet at 2.37 ppm, a multiplet at 2.09 ppm, and a triplet at 2.34 ppm
were assigned to the protons bound to pyruvate C-3 and glutamate C-3
and C-4. Resonances centered at 1.46 ppm (doublet), 2.44 ppm (triplet), and at 3.01 ppm (triplet) were assigned, respectively, to protons borne
by alanine C-3 and -ketoglutarate C-3 and C-4, the transamination products. The doublet observed at 1.46 ppm is not symmetric, because it
overlaps the singlet signal of a proton borne by C-3 of hydrated pyruvate at 1.48 ppm. The resonances of protons borne by the glutamate C-2 overlapped those of sugars (not shown).
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-ketoglutarate and also by
aspartate C-3 (doublets centered at 2.79 and 2.68 ppm, corresponding to
the AB part of the ABX system). The resonances corresponding to OAA
(initial substrate) were not observed on the spectrum in Fig. 4B owing
to the very fast exchange between a proton and deuterium from
D2O (enol form of OAA). An unexpected doublet centered at
1.46 ppm was assigned to the protons bound to alanine C-3. This signal
was observed in a control experiment in which extracts were incubated
only with aspartate (not shown). Alanine may result from
decarboxylation of aspartate by an aspartate decarboxylase. Finally,
the signal at 2.37 ppm, assigned to pyruvate, can be explained by
transamination (reaction V) of the alanine coming from decarboxylation
of aspartate and reacting with the -ketoglutarate present in the
incubation mixture.
(ii) In vivo observations. In vivo 13C-NMR spectra of cells incubated with [1-13C]glucose in the presence of ammonia (Fig. 1A) showed signals that were identified as [3-13C]aspartate (37.0 ppm), [2-13C]aspartate (52.8 ppm), [3-13C]alanine (17.0 ppm), and [4-13C]valine (17.2 and 18.5 ppm). These resonances increased for the first 10 min and then remained at a constant level. No other amino acids were detected on the in vivo 13C-NMR spectra, suggesting that their intracellular concentrations were very low owing to their rapid incorporation into proteins. Additional signals were detected on spectra of extracts of the cells at the end of the incubation, recorded under conditions allowing higher sensitivity (Fig. 1B). They were assigned to [2-13C] and [3-13C]glutamate (55.08 and 27.35 ppm, respectively) and to [2-13C]alanine (51.4 ppm).
Modifications of the ammonia concentration in the incubation mixture and addition of essential volatile fatty acids (isobutyrate and valerate [5]) did not promote amino acid accumulation in the cells. Preincubation of cells for 20 min in the presence of tetracycline or chloramphenicol (1 mg/ml) did not change the observed amino acid pattern. No modification was induced even when cells were incubated in culture medium instead of buffer. The labeling of C-3 of alanine and aspartate and of C-2 of glutamate was as expected from the metabolism of [1-13C]glucose via reactions III and V, reaction VI, and reaction I, respectively (Fig. 5). The labeling of C-4 of valine is consistent with the classical pathway of valine synthesis from two [3-13C]pyruvates as building blocks (9) (Fig. 5). Labeling of C-4 of glutamate was expected, resulting from the incorporation of [2-13C]acetyl coenzyme A and leading to the formation of-ketoglutarate. The signal of the
[4-13C]glutamate probably overlapped the signal of
[2-13C]succinate.
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,
13C enrichment obtained via forward pathway; 
,
13C enrichment obtained after reversion of the succinate
synthesis pathway.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Our results show the activity of a reversible GLDH in F. succinogenes S85. In the reaction leading to glutamate synthesis, GLDH activity was measured with NADH or NADPH as the cofactor in sonicated extracts of cells grown in a medium containing 0.5 or 13 mM ammonia.
We found no GS activity in F. succinogenes S85. The enzyme
may be present in the cells, but its activity may be very low and therefore undetected by 1H NMR. It is also possible that
the synthesis of glutamine in F. succinogenes occurs via
asparagine synthetase, replacing GS in a coupled system similar to that
of GS-glutamine:-oxoglutarate aminotransferase (GOGAT), as shown
previously for other rumen bacteria (7).
We showed the synthesis of alanine from pyruvate and ammonia (ADH activity [reaction III]), with NADPH as the cofactor. The reverse reaction was not detected.
GLDH and ADH thus seem to be the main pathways of direct assimilation of ammonia in F. succinogenes. These systems are generally considered low-affinity systems for the integration of ammonia, while the GS-GOGAT couple is the highest-affinity enzyme system (23). In the rumen, GS is thought to play a major role when the ammonia concentration is low while GLDH is thought to be predominant when the rumen ammonia concentration is high (18, 23). The rumen ammonia concentration varies with diet, from 1 mM with a low-protein diet to 40 mM, transiently, with a nitrogen-rich diet (23). However, enzyme activities measured in individual rumen bacterial species do not always fit this general assumption and suggest that GLDH may be the main pathway of ammonia assimilation. For example, in Selenomonas ruminantium cultured with low concentrations of ammonia, GS activity was predominant but GLDH was still active and never repressed and GOGAT was not overexpressed (20). In Ruminobacter amylophilus, although ammonia limitation resulted in repression of GLDH and stimulation of GS, GOGAT was not detected (11). In Ruminococcus flavefaciens, GLDH was induced in ammonia-limited cultures (6), and in Streptococcus bovis, GLDH activity was much higher than GS activity (10).
In the nonruminal species Bacteroides fragilis, NAD- and NADP-dependent GLDH activities with high affinities for ammonia (Km = 0.8 mM) and a negligible GS activity were found. GLDH activity was not repressed under ammonia limitation (25). These results recall those obtained with F. succinogenes.
The role of ADH in ammonia assimilation in the rumen is not yet fully understood, although enriched alanine was the main labeled amino acid when rumen microbial cells were incubated with 15NH4+ (2). However, results obtained with pure cultures may differ significantly from results obtained with rumen content. Assimilation of 15NH4+ by pure culture of the predominant noncellulolytic bacteria Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1 was shown to be affected by the addition of amino acids or peptides to the culture medium, but glutamate and aspartate were always formed de novo to a greater extent than the other amino acids, including alanine (1).
The two transaminases most commonly found in rumen microorganisms (23), i.e., glutamate-pyruvate and glutamate-OAA transaminases, were found in F. succinogenes by both enzymatic assays and 1H NMR. This last approach is very easy and straightforward, allowing unambiguous identification of the reaction products.
In in vivo incubations in the presence of ammonia and
[1-13C]glucose, only a few amino acids were detected by
13C NMR, suggesting a very efficient incorporation of amino
acids into proteins in F. succinogenes cells. The detected
amino acids were aspartate, glutamate, alanine, and valine. Aspartate
results from the activity of glutamate-OAA transaminase (reaction VI), glutamate results from GLDH activity (reaction I) or from
transaminations (reactions V and VI), alanine can be synthesized from
ADH (reaction III) or glutamate-pyruvate transaminase (reaction V)
activity or from decarboxylation of aspartate, and valine can be
synthesized, as in many bacteria, from the condensation of two
pyruvates (9) via -acetolactate. This intermediate was
previously identified in F. succinogenes extracts
(17).
The most important result in this work concerns the modification of fluxes of carbon metabolism induced by the presence of ammonia. When ammonia was added to resting cells metabolizing glucose, the flux to glycogen synthesis was decreased and those to acetate and succinate syntheses were increased. We showed that, under these conditions, NH4+ was consumed and that amino acids and macromolecules were synthesized, probably in place of glycogen. The presence of ammonia also modified the ratio of succinate to acetate in favor of acetate. The increase in the rate of conversion of glucose into acetate may be explained by the need of ATP for the synthesis of amino acids, ATP being directly provided by the acetate synthesis pathway. This increase in acetate production at the expense of succinate may result from the reversal of the succinate synthesis pathway (see below).
Although glycogen synthesis was decreased by 50%, the reversal of glycolysis from triose-phosphate (16) was not modified by the presence of ammonia, as the ratio of the integrals of [1-13C] and [6-13C]glycogen (about 3) was not affected by the presence of ammonia. The storage of [1-13C]glycogen results from incorporation of [1-13C]glucose-6-phosphate (G 6P) synthesized either directly from [1-13C] glucose or after reversal of glycolysis, whereas the storage of [6-13C]glycogen results only from reversal of glycolysis. The constancy of the ratio of [1-13C]glycogen to [6-13C]glycogen indicates that the flux of G 6P entering the pathway of glycogen synthesis with or without reversal of glycolysis is not modulated by ammonia, suggesting a mechanism of regulation of this flux.
The analysis of the percentage of 13C enrichment of succinate C-2, measured by 13C-FSED 1H NMR, indicated that the contribution of glycogen to the synthesis of the metabolites was not modified by the presence of ammonia and therefore that futile glycogen cycling was still effective under this condition. With or without ammonia, about 16% of the glucose molecules entering glycolysis come from prestored glycogen. However, the percentage of 13C enrichment of C-2 of acetate was decreased from 40 to 31%. This result was explained by an increase (sevenfold) in the flux through the reverse succinate pathway. This reversal was confirmed (i) by the labeling of the carboxylate of acetate and (ii) by the labeling of the amino acids. The labeling of aspartate C-2 reflects the labeling of OAA C-2 and thus proves the reversal from fumarate to OAA (Fig. 5). The labeling of acetate C-1, previously observed in the absence of ammonia (16), reflects the labeling of pyruvate C-2 (Fig. 5). Pyruvate can be synthesized from OAA via two routes: either directly through the activity of OAA-decarboxylase or indirectly through the combined activities of phosphoenolpyruvate-carboxykinase and pyruvate kinase. OAA-decarboxylase and phosphoenolpyruvate-carboxykinase activities could not be measured in F. succinogenes extracts owing to the very high activity of malate dehydrogenase (unpublished results). Consequently, we do not yet know which of these two enzymes is involved in this step.
Such a reversal from OAA to pyruvate was also observed in rabbit kidney tubule (4). It was due to OAA-decarboxylase activity and was greatly stimulated by the addition of ammonia to the cells. In these cells, the addition of NH4Cl also increased glucose conversion into metabolites (4). These results recall those obtained with F. succinogenes cells and reflect the regulation of carbon metabolism by ammonia.
Until now, no information concerning nitrogen metabolism in F. succinogenes has been available. Our work shows some of the enzymes involved in ammonia assimilation and amino acid synthesis in this bacterium. Furthermore, the use of 13C and 1H NMR enabled us to quantify the modulation of carbon fluxes by ammonia. Also, we showed that the futile cycles previously found in F. succinogenes, i.e., reversal of glycolysis, simultaneous synthesis and degradation of glycogen, and reversal from fumarate to pyruvate, were still effective in the presence of ammonia, under conditions closer to physiological conditions. It would now be interesting to determine whether these futile cycles are present under conditions where succinate does not accumulate, such as in the rumen. For this purpose, we could apply a similar approach to coincubations of cells of F. succinogenes and of a succinate-utilizing strain such as Selenomonas ruminantium (22).
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Microbiologie, INRA, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France. Phone: 33 (0)4 73 62 42 75. Fax: 33 (0)4 73 62 45 81. E-mail: gaudet{at}clermont.inra.fr.
Present address: Slovak Technical University, Central Laboratories,
81237 Bratislava, Slovakia.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, May 1999, p. 1941-1948, Vol. 65, No. 5
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