# In vivo 23Na nuclear magnetic resonance study of maintenance of a sodium gradient in the ruminal bacterium Fibrobacter succinogenes S85

V. Schwaab, C. Matheron, A.M. Delort, Gérard Gaudet, Evelyne Forano

► To cite this version:

V. Schwaab, C. Matheron, A.M. Delort, Gérard Gaudet, Evelyne Forano. In vivo 23Na nuclear magnetic resonance study of maintenance of a sodium gradient in the ruminal bacterium Fibrobacter succinogenes S85. Applied and Environmental Microbiology, 2001, 67 (9), pp.4390-4392.
10.1128/AEM.67.9.4390-4392.2001. hal-02672169

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# In Vivo $^{23}$Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium

## *Fibrobacter succinogenes* S85

VÉRONIQUE SCHWAAB,¹,² CHRISTELLE MATHERON,¹ ANNE-MARIE DELORT,¹*

GENEVIEVE GAUDET,² AND EVELYNE FORANO²

Laboratoire de Synthèse, Electrosynthèse et Etude de Systèmes à Intérêt Biologique, UMR 6504, Université Blaise Pascal—CNRS, 63177 Aubière cedex,¹ and Unité de Microbiologie, INRA, Centre de Recherches de Clermont-Ferrand- Theix, 63122 Saint-Genès-Champanelle,² France

Received 26 January 2001/Accepted 7 June 2001

Sodium gradients ($\Delta p$Na) were measured in resting cells of *Fibrobacter succinogenes* by in vivo $^{23}$Na nuclear magnetic resonance using $T_m(DOTP)^{5-}$ [thulium(III) 1,4,7,10-tetraazacyclododecane-*N*′, *N*′′, *N*′′′-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a $\Delta p$Na of -55 to -40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of $Na^+$ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).

In the ruminal bacterium *Fibrobacter succinogenes* S85, glucose and cellobiose uptake was shown to be driven by an artificial electrical gradient ($\Delta\Psi$) or sodium gradient ($\Delta p$Na) 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 $^{23}$Na 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 (Dy$^{3+}$, $Tm^{3+}$, and Tb$^{3+}$) 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 $T_m(DOTP)^{5-}$ 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 · liter$^{-1}$. The cells were harvested and suspended under anaerobic con-

ditions (8, 11) in variable sodium buffers at a concentration of 10 mg of protein · ml$^{-1}$. $T_m(DOTP)^{5-}$ (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 $[Na_{10}^+Dy^{3+}(PPPi_2)^{7-}]$, used for intensity calibration. In vivo $^{23}$Na NMR experiments were carried out anaerobically at 39°C with a Bruker MSL 300 spectrometer operating at 79.39 MHz. $^{23}$Na 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 $^{23}$Na NMR spectra of *F. succinogenes* resting cells, prepared in 75 mM $Na^+$-containing buffer and incubated in the presence of $[Na_5^+Tm(DOTP)^{5-}]$ with increasing concentrations of sodium (50, 75, 100, and 150 mM). Intracellular $Na^+$ concentrations were measured from $^{23}$Na 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:

$$C_{\text{in}} = (I_{\text{in}} \times I'_{\text{ex}} \times 2.2)/(I_{\text{cap}} \times I'_{\text{in}} \times 884) \quad (1)$$

where $C_{\text{in}}$ is the intracellular $Na^+$ concentration; $I_{\text{cap}}$ is the integral of the $^{23}$Na NMR signal of the reference capillary, 2.2 is the calibration factor of the capillary (millimolar), 884 µl is the extracellular volume of the sample (in microliters), $I_{\text{in}}$ is the integral of intracellular $^{23}$Na NMR signals in the absence of ionophores (Fig. 1A), and $I'_{\text{ex}}$ and $I'_{\text{in}}$ are the integrals of extracellular and intracellular $^{23}$Na NMR signals, respectively, in the presence of ionophores (Fig. 1B).

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

<footnote>* Corresponding author. Mailing address: Laboratoire de Synthèse, Electrosynthèse et Etude de Systèmes à Intérêt Biologique, UMR 6504, Université Blaise Pascal-CNRS, 63177 Aubière cedex, France. Phone: 33 04 73 40 77 14. Fax: 33 04 73 40 77 17. E-mail: amdelort@chimtp.univ-bpclermont.fr.</footnote>
<figure><img src="image_2.png" /></figure>

FIG. 1. In vivo <sup>23</sup>Na NMR experiments. *F. succinogenes* S85 resting cells (10 mg of protein · ml<sup>-1</sup>) washed in 75 mM Na<sup>+</sup> solutions were incubated in buffers containing 50 mM Na<sup>+</sup> and 5 mM Tm(DOTP)<sup>5-</sup> (a), 75 mM Na<sup>+</sup> and 6 mM Tm(DOTP)<sup>5-</sup> (b), 100 mM Na<sup>+</sup> and 6 mM Tm(DOTP)<sup>5-</sup> (c), and 150 mM Na<sup>+</sup> and 6 mM Tm(DOTP)<sup>5-</sup> (d). (A) In vivo <sup>23</sup>Na 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).

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<sub>in</sub>, calculated by using equation 1 under these
differing conditions, are reported in Fig. 2. Intracellular Na<sup>+</sup>

concentrations of *F. succinogenes* resting cells slightly in-
creased, from about 4 to 12 mM, when extracellular sodium
was increased from 30 to 100 mM. The resulting sodium gra-
dient (ΔpNa) was calculated as ΔpNa = 2.3 × RT/ZF × log
C<sub>in</sub>/C<sub>ex</sub>, where R is 8.32J · K<sup>-1</sup> · mol<sup>-1</sup>, T is 312 K, F is 96,500,
Z is 1 (cation charge), C<sub>in</sub> is the intracellular sodium concen-

<figure><img src="image_3.png" /></figure>

FIG. 2. Intracellular sodium concentrations (♦) measured by in vivo <sup>23</sup>Na NMR and calculated corresponding ΔpNa (○). *F. succinogenes* S85 resting cells (10 mg of protein · ml<sup>-1</sup>) washed in solutions containing 25 mM Na<sup>+</sup>, 75 mM Na<sup>+</sup>, and 100 mM, Na<sup>+</sup>, were incubated in buffers containing 30 to 200 mM Na<sup>+</sup> in the presence of 1, 5, or 6 mM Tm(DOTP)<sup>5-</sup>. Intracellular sodium concentrations were calculated from integrals measured in <sup>23</sup>Na NMR spectra (see Fig. 1).
<figure><img src="image_4.png" /><figcaption>FIG. 3. In vivo ¹³C NMR experiments. Time-dependent changes of ¹³C NMR signal integrals of β and α [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 · ml⁻¹). 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⁺.</figcaption></figure>

tration (millimolar), and C<sub>ex</sub> is the extracellular sodium con- centration (millimolar).

The calculated sodium gradient (ΔpNa) was rather constant at about −55 mV for external sodium concentrations less than or equal to 100 mM. For higher extracellular [Na⁺], the intra- cellular sodium concentration increased and ΔpNa 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]glu- cose utilization was monitored by in vivo ¹³C NMR as previ- ously described (11). The rate of glucose metabolism was de- creased 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).

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⁺]; ΔpNa was around −55 mV; it decreased slightly when the extracellular sodium con- centration 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 mod- erately halophilic *Vibrio costicola* (9), were similar to those measured in *F. succinogenes* for the same extracellular [Na⁺]. As a result, the calculated ΔpNa values were comparable for

these bacterial systems, although the ΔpNa 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 ΔpNa in the range of physiological extracellular sodium concentrations (4). The ΔpNa 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⁺ con- centration 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, ΔpNa could be essential when ΔΨ 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 require- ment for growth of *F. succinogenes*, as shown in some marine bacteria (16).

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