INTRODUCTION

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Propionic acid bacteria, especially Propionibacterium freudenreichii subsp. shermanii, is the main ripening flora of Swiss-type cheeses. Two interesting features of propionibacterial metabolism are the central carbon metabolic pathway of the Wood-Werkman cycle, part of the central carbon metabolic pathway, and the presence of a multimeric transcarboxylase (EC 2.1.3.1). This transcarboxylase catalyzes the reversible transfer of a carboxyl group from methylmalonyl coenzyme A (CoA) to pyruvate to form propionyl-CoA and oxaloacetate. The carboxyl group transferred is never released nor exchanged with the CO₂ dissolved in the medium (20).

During the warm-room period (the last stage of the cheese-making process), the growth of propionibacteria occurred, the concentration of lactate changed from 150 to 50 mmol/kg of cheese, and that of pyruvate increased from about 1 to 10 mmol/kg of cheese (6, 16).

The fermentation of lactate to propionate, acetate, and CO₂ is usually represented as follows: 3 lactate  2 propionate + 1 acetate + 1 CO₂ + 1 H₂O. In the fermentation of pyruvate, the acids produced are qualitatively the same but quantitatively the reverse (11). Moreover, production of significant amounts of other products, like succinate, has also been reported (15). The propionate-to-acetate ratios are then often different from the theoretical values of 2 (lactate fermentation) and 0.5 (pyruvate fermentation). The reason for such discrepancies from the theoretical values is still unknown.

Since propionibacteria are involved in lactate breakdown in cheese and are known to be able, in complex media or in resting cells, to ferment pyruvate (1, 9, 10, 11, 15) and to excrete pyruvate (4, 5, 19) during lactate fermentation, it would be interesting to know how propionibacteria manage the fermentation of both substrates when they are available together.

In a previous work, it was shown that aspartate and alanine are intermediates or products of pyruvate metabolism in Propionibacterium spp. (9). We conducted this study with a larger initial concentration of pyruvate in order to determine if pyruvate is the key to the control of central carbon metabolism in P. shermanii and how the cells regulate the influxes and outfluxes at this node. Cometabolism experiments were conducted in order to observe how the cells regulate the flux of pyruvate in the presence of the most-used and preferred substrate in Swiss-type cheese, namely, lactate.

	MATERIALS AND METHODS

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Culture conditions. P. freudenreichii subsp. shermanii (CIP 103027) was grown on a modified yeast extract-lactate medium (9).

Cell suspension preparation. For in vivo nuclear magnetic resonance (NMR) experiments, cells were prepared as described by Deborde et al. (8). One gram (wet weight) of cell pellet was resuspended in 2 ml of sterile water (9 g · liter¹) and immediately transferred to an NMR tube. Argon was bubbled through the suspension to maintain anaerobiosis.

NMR experiments. All NMR experiments were performed by using an Avance DMX500 spectrometer system (Bruker, Wissembourg, France) operating at 11.75 T (125.7 MHz for ¹³C and 500.1 MHz for ¹H).

(i) In vivo natural-abundance ¹³C NMR spectra. Natural-abundance ¹³C NMR spectra were determined at 297 K on an NMR spectrometer with a 10-mm probe. A cell suspension (3 ml) was transferred to an NMR tube containing a capillary full of HMPA (hexamethylphosphoramide; Sigma) in H₂O. Chemical shifts were measured relative to the HMPA capillary centered in the NMR tube as an external reference resonating at 36.8 ppm from tetramethylsilane (Spectrométrie Spin et Techniques). The capillary was present throughout the recording of spectra. ¹³C natural-abundance spectra were recorded as previously described (8). For experiments with [2-¹³C(99%)]pyruvic acid sodium salt, FID (free induction decay) was acquired with the same acquisition parameters but with 64 or 512 scans. Peak areas were determined by using the interactive integration package of XWINNMR spectrometer software (Bruker).

(ii) Quantification of end products by ¹H and ¹³C NMR analyses of supernatants. Following the NMR experiments with labeled-substrate addition, the cell suspension was diluted with 3 ml of sterile water and immediately centrifuged (9,000 × g, 10 min, ambient temperature, Heraeus Biofuge 15). The supernatant was collected, and the cell pellet was resuspended with 6 ml of sterile water and centrifuged again. The two supernatants were pooled and then filtered through a sterile disposable 0.45-µm syringe filter and kept like the cell pellet at 20°C until analyzed. Analysis of supernatants was performed at 298 K on an NMR spectrometer with a 5-mm probe. The sample supernatant (400 µl) was supplemented with 50 µl of tetradeuterio-2,2,3,3-(trimethylsilyl)-3-propionic acid sodium salt (TSP; Spectrométrie Spin et Techniques) in D₂O and 50 µl of glycine (internal standard; 20 mM final concentration). ¹H spectra were recorded with the following parameters: 9-s repetition time, 6-s relaxation delay, 90° pulse angle, 7-kHz spectral width, 2-s presaturation time of the water signal, and 512 scans. For each sample, two ¹H NMR spectra were run, one without (¹H NMR) and a second with broadband ¹³C decoupling using the GARP (18a) composite pulse decoupling scheme (¹H{¹³C} NMR). A 0.3-Hz broadening factor was applied to the FID signal before Fourier transformation. Peak areas were determined by interactive integration by using the spectrometer software.

Miscellaneous. Total cell mass was determined by A₆₅₀ measurements (Beckman DU 7400) correlated with the wet-cell concentration. One A₆₅₀ unit was equivalent to 0.28 (dry weight) or 2.5 (wet weight) mg (r² = 0.99).

Chemicals. [2-¹³C]sodium pyruvate (99% ¹³C) was purchased from Isotec, and sodium L-[1-¹³C]lactate (99% ¹³C) was purchased from Cambridge Isotope Laboratories, A.R.C. All of the other chemicals used were of reagent grade.

	RESULTS

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Metabolic pathway of [2-¹³C]pyruvate by P. freudenreichii subsp. shermanii. A series of ¹³C NMR spectra obtained after feeding of Na [2-¹³C]pyruvate (360 µmol, corresponding to a final concentration of 124 mM in the NMR tube) to a suspension of P. shermanii at 24°C are shown in Fig. 1. Pyruvate (resonance at 206 ppm) was consumed at an apparent initial rate of 148 µmol · min¹ · g¹ (cell dry weight). In the first spectrum after addition, resonances due to pyruvate and its hydrate (95 ppm; not shown), acetate, propionate, and succinate are already clearly observed. The intensities of resonances due to carbons 2 and 3 of the alanine molecule (C-2 and C-3 in the following text), malate (C-2, 71.2 ppm [not shown]; C3, 43.3 ppm), and pyruvate (C-3) and its dimer increased with time, reached a maximum, and decreased at the onset of exhaustion of the added pyruvate, while the intensities of resonances due to propionate, acetate, succinate, glutamate, and glutamine continued to increase (Fig. 1 and 2a, b, and c).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Spectra of in situ kinetics of [2-13C]pyruvate metabolism by resting cells of P. freudenreichii subsp. shermanii CIP 103027. [2-13C]pyruvate (360 µmol, corresponding to a final concentration of 124 mM in the NMR tube) was added at time zero to a cell suspension (1 g [wet weight] in 2 ml of sterile saline water) at 24°C (297 K), and proton-decoupled 13C NMR spectra were collected every 104 s (64 scans). Chemical shifts were referred to an HMPA capillary. Only five spectra, selected from 33 experiments, are presented. The intensities of the stackplot on the right were multiplied by 0.5.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Time courses of the various metabolites observed by in vivo 13C NMR spectroscopy during [2-13C]pyruvate metabolism by P. freudenreichii subsp. shermanii cells. (a) Symbols: , pyruvate C-2; , acetate C-1; , acetate C-2; , propionate C-3; , propionate C-2; , succinate C-2; , pyruvate C-3. (b) Symbols: , pyruvate C-2; , pyruvate C-3; , glutamate C-3; , glutamate C-2. (c) Symbols: , pyruvate C-2; , pyruvate C-3; , alanine C-2; , malate C-3. au, arbitrary units.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Expanded spectra of the 95- to 10-ppm region of the series of spectra shown in Fig. 1 but before and 86 min after addition of [2-13C]pyruvate to resting cells of P. shermanii. Each spectrum was collected with 512 scans.

Pyruvate-lactate cometabolism in P. shermanii. The metabolism of Na [2-¹³C]pyruvate (194 µmol, corresponding to a final concentration of 67 mM in the NMR tube) and Na L-[1-¹³C]lactate (270 µmol, corresponding to a final concentration of 95 mM in the NMR tube) by a suspension of P. shermanii was investigated by in vivo ¹³C NMR at 24°C (Fig. 4). Five minutes after addition, four major resonances were observed: the strong one at 183 ppm was due to [1-¹³C]lactate, the second at 206 ppm was due to [2-¹³C]pyruvate, the third at 95 ppm was due to its hydrate, and finally and more interestingly, the fourth at 69.5 ppm was due to [2-¹³C]lactate. The latter signal was composed of a singlet (the biggest peak in this pattern) and a doublet around this singlet. Since the isotopic purity of [1-¹³C]lactate was checked by NMR, the doublet was due to naturally abundant L-[1,2-¹³C (99%, 1.1%)]lactate; no singlet was detected (not shown). From this, the singlet due to [2-¹³C]lactate might be biosynthesized during the first 5 min after the addition. This hypothesis was confirmed by the fact that only the singlet of the lactate C-2 resonance increased for the next 10 min while the doublet disappeared.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Spectra of in-situ kinetics of [2-13C]pyruvate and [1-13C]lactate cometabolism by resting cells of P. freudenreichii subsp. shermanii CIP 103027. Na [2-13C]pyruvate (194 µmol, corresponding to a final concentration of 67 mM in the NMR tube) and Na L-[1-13C]lactate (270 µmol, corresponding to a final concentration of 95 mM in the NMR tube) were added at time zero to a cell suspension (1 g [wet weight] in 2 ml of sterile saline water) at 24°C (297 K), and proton-decoupled 13C NMR spectra were collected every 104 s (64 scans). Chemical shifts were referred to an HMPA capillary. Only eight spectra, selected from 40 experiments, are presented. Abbreviations: Pyr C1, pyruvate C-1; Pyr C2 imp, C-2 of pyruvate hydrate; lact C2; lactate C-2. HMPA, hexamethylphosphoramide.

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Expanded spectra of the 95.5- to 93.5-, 74.3- to 69.7-, 70- to 68.5-, and 63- to 60-ppm regions of the series of spectra shown in Fig. 4. Each spectrum resulted from FID addition and thus 256 scans.

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View larger version (35K): [in this window] [in a new window]   FIG. 6.   Time courses of the various metabolites observed by in vivo 13C NMR spectroscopy during [2-13C]pyruvate and [1-13C]lactate cometabolism by P. shermanii cells. (a) Symbols: , lactate C-1 (broken line) and pyruvate C-2 (solid line); , lactate C-2; , pyruvate C-1; , pyruvate C-3; , alanine C-2. (b) Symbols: , lactate C-1 (broken line) and pyruvate C-2 (solid line); , propionate C-2; , propionate C-3; , propionate C-1 (broken line). (c) Symbols:  lactate C-1 (broken line) and pyruvate C-2 (solid line); , pyruvate C-3; , acetate C-1; , acetate C-2. (d) Symbols: , lactate C-1 (broken line) and pyruvate C-2 (solid line); , pyruvate C-1; , CO2; , HCO3; , succinate C-2. au, arbitrary units.

End products of metabolism as analyzed by ¹H NMR and ¹³C NMR. The end products of metabolism were measured in the supernatant by NMR as described in Materials and Methods. A comparison of the spectra analyzed by ¹H NMR and ¹H{¹³C} NMR was used to identify the satellites due to ¹³C-¹H coupling. Quantification of the end products was performed by measuring the ¹H{¹³C} NMR spectrum with glycine as the internal standard.

View larger version (16K): [in this window] [in a new window]   FIG. 7.   Expanded spectra of the 1.22- to 1.14-, 1.1- to 1.02-, and 0.97- to 0.89-ppm regions of 1H NMR spectra of a supernatant cometabolism experiment with P. shermanii after exhaustion of [2-13C]pyruvate and [1-13C]lactate showing the pattern of the methyl group of propionate. The intensities of the spectra on the right and left were multiplied by 4. The coupling constants involved in the splitting are 3J H3-H2 = 8 Hz, 1J 13C3-H2 = 127 Hz, 2J 13C2-H3 = 5 Hz, and 3J 13C1-H3 = 5 Hz.

	DISCUSSION

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The most important result of this work about pyruvate-lactate cometabolism of P. shermanii, studied by in vivo ¹³C NMR, concerns the modification of fluxes of pyruvate metabolism induced by the presence of lactate. Pyruvate was temporarily converted to lactate and alanine (Fig. 4 to 6); the flux to acetate synthesis was maintained, but the flux to propionate synthesis was increased; and the reverse flux of the first part of the Wood-Werkman cycle, up to acetate synthesis, was decreased.

Metabolic pathway of [2-¹³C]pyruvate by P. freudenreichii subsp. shermanii. According to currently known P. shermanii metabolic pathways, several intermediates, like [3-¹³C]pyruvate and [2-¹³C]- and [3-¹³C]malate, and final products, like [2-¹³C]- and [3-¹³C]propionate, [2-¹³C]- and [1-¹³C]acetate, [2-¹³C]succinate, and [2-¹³C]- and [3-¹³C]glutamate, are expected to be produced from [2-¹³C]pyruvate (9, 15, 20) and were effectively observed. [3-¹³C]pyruvate, [3-¹³C]alanine, and [3-¹³C]malate evidenced the active reversibility or bidirectional reactions of the Wood-Werkman cycle up to pyruvate (9).

Pyruvate-lactate cometabolism experiments. The pyruvate pool must be regulated in order to be under the toxic concentration, as observed by Hugenholtz (12) for lactic acid bacteria. In this study, we found that P. shermanii is able to manage the carbon fluxes at the pyruvate node in several ways (Fig. 8).

View larger version (18K): [in this window] [in a new window]   FIG. 8.   Schematic diagram of the proposed regulation of central carbon metabolism in P. freudenreichii subsp. shermanii. OAA, oxaloacetate; PEP, phosphoenolpyruvate.

(i) Transport and excretion of pyruvate. To control the size of the pyruvate pool, the cell reduced the apparent consumption rate of [2-¹³C]pyruvate from 148 to 90 µmol · min¹ · g¹ in cometabolism. Another way to decrease the size of the pyruvate pool is excretion, but that was not observed in our experiments. When Crow (5) measured intracellular pyruvate concentrations of up to 137 mM during the initial stages of lactate fermentation by P. shermanii, an extracellular pyruvate concentration of up to 3.7 mM was observed during the last stages of lactate fermentation, i.e., lactate exhaustion.

(ii) Transport and synthesis of lactate. It is noteworthy that [1-¹³C]lactate was also metabolized to [1-¹³C]pyruvate at the same time as [2-¹³C]pyruvate. In P. shermanii, Schilliger-Devriend (18) demonstrated the existence of two lactate transport systems, one which had affinity for the anion and another which transported the unionized form of lactate. The transport system was not further characterized in terms of pyruvate competition. Nevertheless, if [1-¹³C]lactate was rapidly transported into the cells and further metabolized to [1-¹³C]pyruvate, this increased the size of the pyruvate pool already largely bred by [2-¹³C]pyruvate.

(iii) Propionate, acetate, and succinate syntheses. In monoaddition experiments, the reverse flow of the first part of the Wood-Werkman cycle, which is visualized by the formation of [3-¹³C]malate and [3-¹³C]pyruvate and finally by the formation of [2-¹³C]acetate, took place immediately. In cometabolism experiments, this reverse flow was delayed and really took place when lactate had almost disappeared and when [2-¹³C]pyruvate was in the second step of its evolution (12 min after addition; Fig. 6a and c.). In cometabolism experiments, the flow of [2-¹³C]pyruvate through the acetate pathway was the same as in monoaddition experiments, which led us to think that the conversion rate could not be greater. Moreover, [2-¹³C]- and [3-¹³C]propionate were observed and appeared at the very beginning of the cometabolism experiment, underlining the fact that the direct flow of the first part of the Wood-Werkman cycle was largely predominant. In such a situation, it could be postulated that the labeled-malate pool was small. In fact, it was under our limit of detection.

(iv) Alanine synthesis. Alanine was biosynthesized for either anabolic processes or toxic reasons or both. Pyruvate was temporarily converted to alanine to constitute an intracellular pool which lowered the intracellular concentration of pyruvate and did not interfere with cell metabolism. In experimental cheeses, the alanine concentration increased up to 10 to 14 mmol/kg of cheese during ripening but the role of propionibacteria was not clear (6).

(v) Trehalose synthesis. Another way to reduce the pyruvate pool is gluconeogenesis, and both [2-¹³C]pyruvate and [1-¹³C]pyruvate are precursors of trehalose.

(vi) Glutamate and glutamine syntheses. In cometabolism experiments, the first oxaloacetate to be produced would be enriched at position 1, which has no impact on the carbon skeleton of glutamate. When [2,4-¹³C]oxaloacetate was produced, then [1,3-¹³C]glutamate would be synthesized. Since the amount of glutamate produced was low and the coupling constant J₁₃ is small, then it could not be detected. Moreover, the initial amount of intracellular glutamate is known to be high (17) and will participate in the dilution of the biosynthesized glutamate. Furthermore, in P. shermanii, the TCA cycle is probably dedicated to anabolism and is not used for energetic purposes (3).