Studies on the Mode of Action of Reutericyclin

The mode of action of reutericyclin was determined with fluorescentdyes that probed the permeability of the cytoplasmic membraneby large molecules, protons, and potassium. A comparison ofreutericyclin activity with those of nisin, nigericin, and valinomycindemonstrated that reutericyclin does not form pores but selectivelydissipates the transmembrane proton potential.

INTRODUCTION

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Introduction

Reutericyclin is a tetramic acid derivative produced by sourdoughisolates of Lactobacillus reuteri. It is inhibitory for a broadrange of gram-positive bacteria at concentrations ranging from0.1 to 1 mg liter^-1 (2, 4). Yeasts, fungi, and gram-negativebacteria are resistant to reutericyclin, and the permeabilitybarrier of gram-negative bacteria mediates resistance to reutericyclin(2). Reutericyclin is produced at active concentrations duringsourdough fermentation and has been suggested to contributeto the stable persistence of L. reuteri in industrial sourdoughfermentation (3). It was the aim of this study to elucidatethe mode of action of reutericyclin. Because reutericyclin isan amphiphilic molecule consisting of a hydrophilic, negativelycharged head group with two hydrophobic side chains, the experimentswere based on the hypothesis that the membrane is the cellulartarget.

Bacterial strains and growth conditions.

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Bacterial strains and growth...

Lactobacillus plantarum TMW 1.460 and Lactobacillus sanfranciscensisLTH2581 were grown at 30°C in MRS medium (mMRS) modifiedaccording to Stolz et al. (8). The cultures were subculturedtwice with a 1% inoculum for 24 and 16 h prior to each experiment.

Chemicals.

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Chemicals.

Reutericyclin was purified from cultures of L. reuteri LTH2584as previously described (3). The concentration of the stocksolution in propanol-water (4:1, vol/vol) was determined byhigh-performance liquid chromatography (3) with synthetic reutericyclin(EMC, Tübingen, Germany) used as an external standard.Stock solutions of nisin, valinomycin, and nigericin (Sigma,Deisenhofen, Germany) were prepared by dissolving the compoundsto concentrations of 0.5 mg ml^-1, 100 µM, and 100 µM,respectively, in 50% ethanol. Propidium iodide (PI), 5 (and6-)-carboxyfluorescein diacetate succinimidyl ester (cFDASE),and 3,3-dipropylthiacarbocyanine [DiSC₃(5)] were purchased fromMolecular Probes (Eugene, Oreg.).

Determination of the MICs.

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Determination of the mics.

The MICs of reutericyclin and nisin for L. sanfranciscensisand L. plantarum were determined by critical-dilution assays(2) with mMRS with the pH values adjusted to 6.2 and 5.0. TheMIC of reutericyclin or nisin in mMRS at pH 6.2 and 5.0 foreither strain ranged from 0.1 to 1 mg liter^-1.

Evaluation of pore-forming activity of reutericyclin.

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Evaluation of pore-forming...

The pore-forming activity of reutericyclin was assayed by determinationof the permeability of the cells to PI. Nisin served as thecontrol. The cells were washed twice with 50 mM sodium phosphatebuffer (pH 6.5) and resuspended in the same buffer containing13 µM PI. Reutericyclin (final concentrations of 8, 17,and 33 mg liter^-1) and nisin (final concentrations of 2.5, 5,and 10 mg liter^-1) were added to aliquots of this suspension,and the PI fluorescence was determined at intervals over a periodof 3 h at 30°C. During incubation of L. plantarum with nisinand PI, a dose-dependent increase in PI fluorescence was observed(Fig. 1), in accordance with the pore-forming activity of thisantibiotic (5).The cells were not found to be permeable to PIwhen reutericyclin was added at levels by far in excess of theMIC. Corresponding results were obtained with L. sanfranciscensis(data not shown).

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FIG. 1. PI fluorescence of L. plantarum during incubation with nisin and reutericyclin. Control lines represent the fluorescence of viable and heat-killed (80°C, 10 min) cells. Inhibitors were added to obtain the following concentrations: 10 ( ${circ}$ ), 5 ( ${square}$ ), and 2.5 ( ${triangleup}$ ) mg of nisin liter^-1 and 33 mg of reutericyclin liter^-1 (•). The results are representative for two independent experiments. Corresponding results were obtained with L. sanfranciscensis (data not shown). RFU, relative fluorescence units.

Dissipation of transmembrane proton potential ( ${Delta}$ pH).

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Dissipation of transmembrane...

The effects of reutericyclin and nisin on the transmembraneproton potential (measured as the transmembrane pH difference[ ${Delta}$ pH]) of L. plantarum and L. sanfranciscensis were determinedby labeling the cells with cFDASE and calibrating each batchof labeled cells as previously described (1). For experimentswith L. sanfranciscensis, maltose and fructose were used inplace of glucose as energy sources. cFDASE-labeled cells wereresuspended in 50 mM citrate buffer (pH 5.0) containing 10 mMglucose or maltose and fructose and 0.4 mM MgSO₄ and 0.3 mMMnSO₄. Nisin and reutericyclin were added at concentrationsranging from 0 to 2 mg liter^-1. The internal pH was determinedafter 15 min of incubation at 30°C. If they were added atthe MIC, nisin and reutericyclin dissipated the ${Delta}$ pH in cellsof L. plantarum (Fig. 2) and L. sanfranciscensis (data not shown).Comparable results were obtained if the experiment was carriedout at a buffer pH of 6.5 (data not shown).

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FIG. 2. Decrease of transmembrane ${Delta}$ pH of L. plantarum upon incubation with reutericyclin ( ${circ}$ ) or nisin (•). The buffer pH was 5.0, and the internal pH of L. plantarum in the absence of inhibitors was 6.6 ± 0.3. The results indicate the means ± standard deviations of three independent experiments. Corresponding results were obtained with L. sanfranciscensis (data not shown).

Dissipation of transmembrane potassium potential ( ${Delta}$ ${psi}$ ).

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The transmembrane potassium potential was determined with thefluorescent probe DiSC₃(5). Fluorescence quenching of DiSC₃(5)is observed if the dye becomes concentrated in the cells asa response to a ${Delta}$ ${psi}$ (9). Cells were washed twice with potassiumphosphate buffer (50 mM, pH 6.5) containing 10 mM glucose asthe energy source and then were resuspended to an optical density(at 578 nm) of 0.5. DiSC₃(5) was added to a final dye concentrationof 5 µM, and the cells were incubated for 10 min to equilibratethe internal and external dye concentrations. Fluorescent traceswere measured in a fluorescence spectrophotometer (model LB50,Perkin-Elmer, Überlingen, Germany) at the excitation andemission wavelengths of 643 and 666 nm, respectively. The temperaturewas maintained at 30°C during measurements. Reutericyclinwas added during the measurements, and the proton ionophorenigericin and the potassium ionophore valinomycin served asthe controls. The addition of nigericin to cells of L. plantarumresulted in decreased DiSC₃(5) fluorescence, corresponding toan increased ${Delta}$ ${psi}$ as a response to the nigericin-mediated dissipationof the transmembrane ${Delta}$ pH (Fig. 3A). The subsequent addition ofvalinomycin dissipated the transmembrane ${Delta}$ ${psi}$ , resulting in therelease of the dye from the cells and increased fluorescence.The addition of reutericyclin at levels of 2 and 8 mg liter^-1increased the ${Delta}$ ${psi}$ to a level comparable to that observed afterthe addition of nigericin (Fig. 3B). The addition of nigericinafter the addition of 8 mg of reutericyclin liter^-1 inducedno further changes in the fluorescence intensity, and the additionof valinomycin after the addition of reutericyclin increasedthe fluorescence intensity to those levels observed in the presenceof nigericin and valinomycin (data not shown).

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FIG. 3. Fluorescent traces of DiSC₃(5) in stained cells of L. plantarum (at excitation and emission wavelengths of 643 and 666 nm, respectively). (A) In a control experiment, valinomycin and nigericin were added to a final concentration of 1 µM each at time points indicated by the arrows. (B) Reutericyclin was added to a final concentration of 2 (upper trace) and 8 (lower trace) mg liter^-1 at the time points indicated by the arrows. The results are representative for four independent experiments. RFU, relative fluorescence units.

The determination of reutericyclin's effects on the permeabilityof the cytoplasmic membrane to large molecules, protons, andpotassium has shown that reutericyclin at its MIC selectivelydissipates the transmembrane ${Delta}$ pH. Thus, the mode of action ofreutericyclin is essentially comparable to that of other weakorganic acids that are widely used in food preservation, suchas acetic acid and sorbic acid. The much lower MIC of reutericyclinis explained by increased partitioning into the cytoplasmicmembrane due to its high hydrophobicity. Comparable to reutericyclin,the hop bitter acids isohumulone, humulone, and humulinic acidact as proton ionophores and at micromolar concentrations inhibitthe growth of lactic acid bacteria in but are not inhibitoryto yeasts (7). Isohumulone has a pK_a of 3.1, and the undissociatedacid is 100-fold more active than the corresponding salt (6).Reutericyclin is a weak acid with a pK_a between 2 and 3 as determinedby isoelectric focusing (M. G. Gänzle and W. P. Hammes,unpublished data). Its activity is increased at low pH (2; presentstudy), suggesting an effect of environmental conditions onits activity comparable to those known for hop bitter acids.

In conclusion, reutericyclin acts as a proton ionophore, selectivelydissipating the transmembrane ${Delta}$ pH in sensitive cells. This informationabout the mode of action of reutericyclin will allow the assessmentof mechanisms of resistance towards this compound and will facilitatethe development of food or drug applications.

ACKNOWLEDGMENTS

We acknowledge our students Micha Hoffmann, David Walerius,and Clarissa Schwab, who contributed to the work during theirlaboratory courses.

FOOTNOTES

* Corresponding author. Mailing address: TU München, Lehrstuhl für Technische Mikrobiologie, Weihenstephaner Steig 16, 85350 Freising, Germany. Phone: 49 8161 71 3959. Fax: 49 8161 71 3327. E-mail: michael.gaenzle{at}wzw.tum.de.

REFERENCES

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