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In the ruminal bacterium Fibrobacter succinogenes S85, glucose and cellobiose uptake was shown to be driven by an artificial electrical gradient () or sodium gradient (pNa) in de-energized cells (5), suggesting a Na⁺ cotransport of these sugars that could explain the requirement of sodium for growth of the bacterium (2, 7, 10). However, the presence of a sodium transmembrane gradient has never been demonstrated in F. succinogenes as well as it has been demonstrated in other rumen bacteria. The objective of this work was to demonstrate and measure such a sodium gradient in F. succinogenes and to monitor its variation according to that of the extracellular sodium concentration.

For that purpose, an in vivo ²³Na nuclear magnetic resonance (NMR) methodology that distinguishes the resonances of intracellular and extracellular sodium has been used to measure sodium gradients directly on living cells. The technique consists of using shift reagents. The reagents are anionic complexes of lanthanides (Dy³⁺, Tm³⁺, and Tb³⁺) whose paramagnetic properties, when the reagents are exchanged with external sodium, induce a chemical shift of external Na⁺ resonance. As these reagents cannot cross the cytoplasmic membrane, the intracellular sodium is not shifted. These reagents have been widely used on various biological systems, but few studies have been performed using them with bacteria. In this work we present the application of the Tm(DOTP)⁵ complex thulium(III) 1,4,7,10-tetraazacyclododecane-N',N'',N'''-tetramethylenephosphonate (1) to the study of sodium gradients in a bacterium.

F. succinogenes S85 (ATCC 19169) was grown for 15 h on a chemically defined medium (8) with 3 g of cellobiose · liter¹. The cells were harvested and suspended under anaerobic conditions (8, 11) in variable sodium buffers at a concentration of 10 mg of protein · ml¹. Tm(DOTP)⁵ (Macrocyclics, Richardson, Tex.) was freshly added at a final concentration of 1, 5, or 6 mM just before the experiments were performed. The bacterial suspension was transferred to 10-mm-diameter tubes in each of which was centered a capillary containing [Na₁₀⁺Dy³⁺(PPPi)₂⁷], used for intensity calibration. In vivo ²³Na NMR experiments were carried out anaerobically at 39°C with a Bruker MSL 300 spectrometer operating at 79.39 MHz. ²³Na NMR spectra (60°pulse: 16.5 µs; repetition time, 350 ms; 900 scans, 1K) were collected every 5 min for 15 min, and then ionophores (monensin at 90 µM and valinomycin and [carbonyl cyanide m-chlorophenylhydrazone] CCCP at 30 µM each) were added and three extra spectra were collected. In Fig. 1A are presented the in vivo ²³Na NMR spectra of F. succinogenes resting cells, prepared in 75 mM Na⁺-containing buffer and incubated in the presence of [Na₅⁺Tm(DOTP)⁵] with increasing concentrations of sodium (50, 75, 100, and 150 mM). Intracellular Na⁺ concentrations were measured from ²³Na NMR spectra registered on a single bacterial sample before (Fig. 1A) and after (Fig. 1B) addition of ionophores, and they were calculated from the following equation:

(1)

where C_in is the intracellular Na⁺ concentration; I_cap is the integral of the ²³Na NMR signal of the reference capillary, 2.2 is the calibration factor of the capillary (millimolar), 884µl is the extracellular volume of the sample (in microliters), I_in is the integral of intracellular ²³Na NMR signals in the absence of ionophores (Fig. 1A), and I'_ex and I'_in are the integrals of extracellular and intracellular ²³Na NMR signals, respectively, in the presence of ionophores (Fig. 1B).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   In vivo 23Na NMR experiments. F. succinogenes S85 resting cells (10 mg of protein · ml1) washed in 75 mM Na+ solutions were incubated in buffers containing 50 mM Na+ and 5 mM Tm(DOTP)5 (a), 75 mM Na+ and 6 mM Tm(DOTP)5 (b), 100 mM Na+ and 6 mM Tm(DOTP)5 (c), and 150 mM Na+ and 6 mM Tm(DOTP)5 (d). (A) In vivo 23Na NMR spectra collected after 15 min in the absence of ionophore. (B) Spectra collected after a further 15 min of incubation of the same samples in the presence of monensin (90 µM), valinomycin (30 µM), and FCCP (30 µM).

The addition of ionophores allowed us to equilibrate intracellular and extracellular Na⁺ concentrations and thus to avoid measurement of the intracellular volume, which is often difficult to ascertain (a detailed calculation using this equation will be published elsewhere). Experiments (not shown) were also performed with cells prepared in various buffers (25, 50, and 100 mM) and resuspended to reach the final external sodium concentrations of interest: 30, 50, 75, 100, 150, and 200 mM.

The values of C_in, calculated by using equation 1 under these differing conditions, are reported in Fig. 2. Intracellular Na⁺ concentrations of F. succinogenes resting cells slightly increased, from about 4 to 12 mM, when extracellular sodium was increased from 30 to 100 mM. The resulting sodium gradient (pNa) was calculated as pNa = 2.3 × RT/ZF × log C_in/C_ex, where R is 8.32J · K¹ · mol¹, T is 312 K, F is 96,500, Z is 1 (cation charge), C_in is the intracellular sodium concentration (millimolar), and C_ex is the extracellular sodium concentration (millimolar).

View larger version (11K): [in this window] [in a new window]   FIG. 2.   Intracellular sodium concentrations () measured by in vivo 23Na NMR and calculated corresponding pNa (). F. succinogenes S85 resting cells (10 mg of protein · ml1) washed in solutions containing 25 mM Na+, 75 mM Na+, and 100 mM, Na+, were incubated in buffers containing 30 to 200 mM Na+ in the presence of 1, 5, or 6 mM Tm(DOTP)5. Intracellular sodium concentrations were calculated from integrals measured in 23Na NMR spectra (see Fig. 1).

The calculated sodium gradient (pNa) was rather constant at about 55 mV for external sodium concentrations less than or equal to 100 mM. For higher extracellular [Na⁺], the intracellular sodium concentration increased and pNa decreased to 44 and 38 mV for 150 and 200 mM extracellular [Na⁺], respectively.

The tight regulation of sodium gradients by F. succinogenes in the range of 30 to 100 mM Na⁺ suggests that sodium ions play a very important role in its metabolism. In addition, we show that depletion of Na⁺ ions during the washing steps led to irreversible damage to the cells that cannot be repaired by further addition of Na⁺ (75 mM). The kinetics of [1-¹³C]glucose utilization was monitored by in vivo ¹³C NMR as previously described (11). The rate of glucose metabolism was decreased by a factor of 2 (Fig. 3) and the amount of glucose was finally degraded by a factor of 3 when the cells were prepared in the absence of sodium. Moreover, under these conditions the intracellular sodium concentration increased from 18 mM at 5 min to 54 mM at 30 min after the addition of sodium (75 mM), as shown by in vivo ²³Na NMR experiments, indicating that the cells were unable to maintain a stable Na⁺ gradient (data not shown).

View larger version (14K): [in this window] [in a new window]   FIG. 3.   In vivo 13C NMR experiments. Time-dependent changes of 13C NMR signal integrals of  and  [1-13C]glucose (circles), [2-13C]succinate (squares), and [1-13C]glycogen (triangles) during 64 mM [1-13C]glucose utilization by F. succinogenes S85 resting cells (10 mg of protein · ml1). Cells were washed in a buffer containing 75 mM Na+ solution (solid symbols) or a buffer lacking sodium (open symbols) and then incubated in a buffer containing 75 mM Na+.

In this work we have shown that F. succinogenes is able to maintain a high transmembrane sodium gradient. In the range of 30 to 100-mM extracellular [Na⁺]; pNa was around 55 mV; it decreased slightly when the extracellular sodium concentration was increased. The intracellular free (visible) Na⁺ concentrations, measured by ²³Na NMR in Escherichia coli (3, 12), in the halotolerant Brevibacterium sp. (12), or in the moderately halophilic Vibrio costicola (9), were similar to those measured in F. succinogenes for the same extracellular [Na⁺]. As a result, the calculated pNa values were comparable for these bacterial systems, although the pNa value was slightly higher in the case of E. coli (76 mV for 75 mM extracellular [Na⁺]) (3). E. coli was shown to maintain a constant pNa in the range of physiological extracellular sodium concentrations (4). The pNa measured in F. succinogenes also appeared constant for concentrations of 30 to 100 mM extracellular Na⁺, with the Na⁺ concentration in the rumen ranging from 75 to 100 mM. Regulation of the intracellular sodium concentration is necessary, particularly in bacteria growing in a sodium-rich environment, in order to maintain an intracellular Na⁺ concentration low enough not to interfere with enzyme activities, Na⁺ often being inhibitory to these activities (15), and to utilize efficiently the ion gradient across the membrane for Na⁺-driven systems (6). In particular, pNa could be essential when is low, i.e., at more acidic pHs (13).

In this work we also showed that lack of sodium during cell-washing steps might lead to damage to cell membranes that cannot be reversed. A nonspecific effect on F. succinogenes cells of Na⁺ as previously found on marine bacteria, such as osmotic effect or lysis prevention caused by interactions of the cations with the outer membrane (14, 15), might explain these results. This effect might also contribute to the sodium requirement for growth of F. succinogenes, as shown in some marine bacteria (16).