Membrane Fluidity Adjustments in Ethanol-Stressed Oenococcus oeni Cells

The effect of ethanol on the cytoplasmic membrane of Oenococcusoeni cells and the role of membrane changes in the acquiredtolerance to ethanol were investigated. Membrane tolerance toethanol was defined as the resistance to ethanol-induced leakageof preloaded carboxyfluorescein (cF) from cells. To probe thefluidity of the cytoplasmic membrane, intact cells were labeledwith doxyl-stearic acids and analyzed by electron spin resonancespectroscopy. Although the effect of ethanol was noticeableacross the width of the membrane, we focused on fluidity changesat the lipid-water interface. Fluidity increased with increasingconcentrations of ethanol. Cells responded to growth in thepresence of 8% (vol/vol) ethanol by decreasing fluidity. Uponexposure to a range of ethanol concentrations, these adaptedcells had reduced fluidity and cF leakage compared with cellsgrown in the absence of ethanol. Analysis of the membrane compositionrevealed an increase in the degree of fatty acid unsaturationand a decrease in the total amount of lipids in the cells grownin the presence of 8% (vol/vol) ethanol. Preexposure for 2 hto 12% (vol/vol) ethanol also reduced membrane fluidity andcF leakage. This short-term adaptation was not prevented inthe presence of chloramphenicol, suggesting that de novo proteinsynthesis was not involved. We found a strong correlation betweenfluidity and cF leakage for all treatments and alcohol concentrationstested. We propose that the protective effect of growth in thepresence of ethanol is, to a large extent, based on modificationof the physicochemical state of the membrane, i.e., cells adjusttheir membrane permeability by decreasing fluidity at the lipid-waterinterface.

INTRODUCTION

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Introduction

Malolactic bacteria are lactic acid bacteria that are able tocarry out malolactic fermentation (MLF). The control of theiractivity is an important aspect of the technology of commercialwine production. MLF consists of the decarboxylation of L-malicacid to L-lactic acid, which decreases total acidity and improvesthe stability and quality of wine. Oenococcus oeni is recognizedas the principal microorganism responsible for MLF under stressconditions, such as those prevailing in wine. However, inoculationof O. oeni starter cultures directly into wine leads to significantcell mortality and, consequently, failure of MLF. The reactivationand adaptation of starter cultures to wine conditions priorto inoculation into wine considerably enhance survival of thebacteria (for a review of MLF see reference 28). Ethanol toleranceappears to be a crucial parameter for the activity of O. oenicells in wine.

The role of ethanol as an agent affecting the physicochemicalstate and biological functions of various cell membranes hasbeen extensively studied. Ethanol toxicity is now generallyattributed to the interaction of ethanol with membranes at theaqueous interface, resulting in a perturbed membrane structureand function (47). In an extensive review of the biologicaleffects of ethanol, Jones (24) argues that membrane-locatedeffects of ethanol are the result of the dielectric disruptionof the aqueous phase, of competition with water for membranepolar sites, and of selective location within the polar regionof membrane surfaces or proteins. However, the exact range ofbiochemical and physiological processes affected by ethanolis, for the most part, undefined.

The importance of the membrane lipid composition with respectto ethanol tolerance has been extensively studied with Saccharomycescerevisiae (43; for a review see reference 34) and bacteria(12, 21). Ethanol tolerance has been strongly correlated withadaptive changes in plasma membrane composition, with many studiesof yeast suggesting a role for acyl-chain unsaturation (31,42, 43).

Ethanol tolerance has been associated with high plasma membranefluidity in both yeast (1, 38) and bacteria (3, 10). The fluidizationresponse can be interpreted on the basis of the hypothesis of"homeoviscous adaptation" (39) as a counteraction to the physicochemicaleffect of ethanol on membranes (21). This model, while beingwidely reported, is apparently not universally applicable toall organisms. Exceptions have been reported for Bacillus subtilis(35) and Escherichia coli (12) cells, in which plasma membranesisolated from cells grown in the presence of ethanol were morerigid than those from the control cells.

While membrane lipid composition has been considered importantfor cellular stress tolerance, other factors have also receivedextensive consideration. One widely studied aspect of the ethanol-stressresponse is the induction of heat shock proteins (hsp's) (22,29). However, the relative contribution of hsp's to ethanoltolerance has been questioned (11, 40). Although hsp's and membranecomposition are both likely to be of importance in ethanol tolerance,the relative contribution of each and the mechanisms of actionremain unresolved.

The degree to which the effects of ethanol on membrane compositionand fluidity share common features has not previously been exploredwith O. oeni. In addition, no direct measurements of the effectof growth in the presence of ethanol on the mobility of membranecomponents have been established. In this report we investigatedthe contribution of membrane fluidity changes to ethanol-stresstolerance and the relationship of those changes to the changesin fatty acid (FA) composition of O. oeni membranes. Ethanol-stresstolerance was examined by monitoring the leakage of preloadedcarboxyfluorescein (cF) from the cells. The effect of ethanolchallenge on the organization and dynamics of the plasma membranein intact cells of O. oeni was assessed by the in vivo spinlabel technique. Three nitroxide spin labels were used to obtainmotional anisotropies of the nitroxide moiety at different depthsof the plasma membrane. The molecular-order parameter S derivedfrom electron spin resonance (ESR) spectra provided a measureof membrane structural order. This parameter was studied inrelation to the concentration of ethanol in nonadapted cellsand in ethanol-adapted cells grown in the presence of 8% ethanolor after a short exposure to 12% ethanol. This study was undertakento test the hypothesis that ethanol may be toxic to O. oenibecause of its effects on the plasma membrane and that adaptationcan partially or completely reverse these membrane effects viachanges in membrane composition and/or organization. Adaptationwas assessed as the reduction in ethanol-induced cF leakage.The relationship between membrane order and acyl-chain compositionin the tolerance of O. oeni cells is discussed.

MATERIALS AND METHODS

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Materials and Methods

Strain, medium, and growth conditions.
O. oeni GM (Microlife Technics, Sarasota, Fla.) was culturedat 30°C in FT80 medium (8) at pH 4.5 without the additionof Tween 80 but containing 10 g of DL-malic acid per liter.Glucose and fructose were autoclaved separately and added tothe medium just before inoculation at final concentrations of2 and 8 g/liter, respectively. Stock cultures (kept frozen at-80°C) were grown until early stationary phase (48 h), diluted100-fold in fresh medium, incubated for 24 h, and then usedto obtain 1% inoculated cultures.

Cell adaptation.
For adaptation to ethanol during growth, O. oeni cells werecultured at 30°C in 500 ml of FT80 medium, pH 4.5, with8% (vol/vol) ethanol and recovered after 48 h (late exponentialphase). For short-term adaptation, O. oeni was cultured in 500ml of the same medium at 30°C. Exponential-phase cells (24h) were harvested by centrifugation, suspended in the same mediumcontaining 12% (vol/vol) ethanol, and incubated for 2 h at 30°C.The same procedure was repeated in the presence of chloramphenicol(CAP; 80 µg/ml) in order to inhibit de novo protein synthesisas observed by Jobin et al. for O. oeni (23).

ESR spectroscopy.
The membrane fluidity of intact O. oeni cells was studied bythe ESR spin probe technique. Spin-labeled stearic acids wereused to probe membrane fluidity. In this molecule the nitroxidedoxyl group (stable radical) is attached in a rigid, stereospecificmanner to stearic acid so that the motion of the nitroxide groupdirectly reflects the motion of the labeled part of stearicacid. The ESR spectral shape of spin-labeled stearic acids dependson the motion and angular orientation of the nitroxide groupwith respect to the membrane lipid-water interface (30). Dependingon the position of the doxyl group along the carbon chain (atthe 5th, 12th, or 16th C atom), it is possible to probe themotional freedom in membranes at the lipid-water interface,in the middle of the monolayer, and in the core of the bilayer,respectively.

5-Doxyl-stearic acid (5-DS) is commonly used to probe the membranelipid-water interface. A typical ESR spectrum of 5-DS-labeledO. oeni cells is presented in Fig. 1. The anisotropic characterof the spectral shape results from the restricted angular freedomof the radical group of 5-DS in the plasma membrane. The spectralparameters A_|| and A indicate the outer and inner hyperfinesplittings in experimental spectra (as shown in Fig. 1). Themembrane-order parameter S relates to membrane fluidity andcan be calculated as the ratio between the observed hyperfineanisotropy (A_|| - A) to the maximum theoretically obtainablevalue of 25 G, which corresponds to the completely rigid orientationof 5-DS (27). Thus, the order parameter can be calculated asfollows: S = (A_|| - A)/25.

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FIG. 1. EPR spectra of 5-DS in O. oeni cells in the absence or presence of 20% (vol/vol) ethanol. A_|| and A represent the outer and inner hyperfine splittings, respectively. The order parameter S is calculated as indicated in the figure.

From this formula it is clear that S = 1 for completely rigidorder and S = 0 for completely isotropic motion. This meansthat in the case of completely rigid orientation of 5-DS A_||is maximal and A is minimal, and in the case of completely isotropicmotion the outer and inner splittings are equal. With membranefluidization A_|| decreases and A increases, so that the orderparameter decreases.

ESR spectra were recorded at room temperature with an X-bandESR spectrometer (Bruker, Rheinstetten, Germany; model 300E).Microwave power was 5 mW, the modulation amplitude was 3 G,and the scan range was 100 G. All spin probes were from Sigma,St. Louis, Mo., and were stored as a 0.1 M stock solution inethanol at -20°C.

Cell labeling.
For labeling, 1 mM spin probe solution was freshly preparedfrom the stock solution by dilution in water. Cells (250 ml)were recovered and washed three times with 5 mM EDTA plus 0.15M KCl in P2 buffer (30 mM KP_i, 0.34 M CH₃COONH₄; pH 7.0) tochelate manganese ions. The cells were resuspended in the samebuffer, and 25 µl of the cell suspension was introducedin a 2-mm (inner-diameter) capillary tube, centrifuged, resuspendedin 20 µl of 1 mM spin probe solution, and incubated for2 min. The cells were subsequently centrifuged, and the supernatantwas removed completely. To avoid reduction of the nitroxidespin probe, the cells were washed with ferricyanide (120 mM),and after centrifugation and removal of the supernatant, thepellet was ready for ESR spectrum recording.

Stress conditions.
After the ESR measurements of the control cells (nonstressed),the pellet in the capillary was resuspended into 20 µlof 100 mM ferricyanide. From this, a 25-µl aliquot wasmixed with 25 µl of a solution with twice the desiredconcentration of ethanol in 100 mM ferricyanide. This cell suspensionwas then divided into two capillaries: one was centrifuged immediately,and the other was centrifuged after 15 min. ESR spectra wererecorded in both pellets. ESR measurements were repeated inthe same cells after the ethanol was washed out by resuspendingthe pellets in 20 µl of 100 mM ferricyanide for 10 min.

Loading cells with cF.
Cells were harvested at the end of the exponential growth phase(optical density at 600 nm [OD₆₀₀], approximately 0.4) by centrifugation,washed twice with 50 mM potassium phosphate buffer (pH 7.0),and concentrated in the same buffer to an OD₆₀₀ of 20. Cellswere deenergized with 2-deoxyglucose (at a final 2 mM concentration)by incubation at room temperature for 30 min to avoid cF extrusionfrom the cells by energy-dependent pumps (5, 6) and consequentlyto ensure that cF leakage was a consequence of ethanol-inducedmembrane damage. The cells were washed and resuspended in 50mM KP_i buffer (pH 7.0) to an OD₆₀₀ of 20. A stock solution of2.3 mg of 5(6)-carboxyfluorescein diacetate (cFDA) (MolecularProbes, Eugene, Oreg.) per ml was prepared in acetone and storedat -20°C in the dark. cFDA was added to the cell suspensionto a final 50 µM concentration and kept at 30°C for15 or 60 min in the case of cells pregrown in the presence of8% (vol/vol) ethanol. Immediately after labeling, the cellswere spun down, washed once, and resuspended in 50 mM KP_i (pH7.0) to an OD₆₀₀ of 2.0 for fluorimetric analyses. Inside cellsthe uncharged, esterified, prefluorochrome cFDA is convertedby cytoplasmic esterases into fluorescent cF that is negativelycharged at physiological pH and, consequently, will accumulateinside cells with an intact cytoplasmic membrane (R. P. Haugland,Handbook of Fluorescent Probes and Research Chemicals, 1996,Molecular Probes).

Measurement of cF efflux.
cF-loaded cells were washed twice and resuspended in 50 mM KP_ibuffer (pH 7.0) to a final OD₆₀₀ of 2.0. At time zero cell suspensionswere placed in a water bath at 30°C and incubated withoutand with ethanol (8, 12, and 16% [vol/vol]). Samples (200 µl)were withdrawn at intervals and immediately centrifuged to removethe cells. To measure the cF labeling capacity, labeled cellswere lysed by incubation at 70°C for 15 min and the debriswas removed by centrifugation. The fluorescence of the supernatantwas measured with a Perkin-Elmer LS 50B luminescence spectrometer(excitation wavelength at 490 ± 5 nm and emission wavelengthat 515 ± 5 nm). From the fluorescence of the supernatantsand the total labeling capacity, the intracellular concentrationsof cF at the sampling time points were calculated.

FA analysis.
Total lipids were extracted with chloroform-methanol-water from30 to 40 mg (dry weight) of cells according to the method ofBligh and Dyer (4) and methyl esterified by a 15-min incubationat 95°C in boron trifluoride-methanol (32). The FA methylesters were extracted with hexane, separated on a CP-Sil-88fused silica capillary column (Chrompack; 50 m by 0.25 mm by0.20 µm [film thickness]), and analyzed by gas chromatography-massspectrometry (GC-MS) (Hewlett-Packard 5970B-series gas chromatograph-massspectrometer). Electron impact spectra were obtained at 70 eVof electron energy. The following operating conditions wereused: injection temperature of 250°C and oven temperatureof 50°C initially, rising to 275°C at 6°C/min, withmaintenance at this temperature for 10 min. The FAs were identifiedwith the aid of FA methyl ester standards (Sigma), and the identitywas confirmed using the NIST Mass Spectral Library. Replicatedeterminations indicated that the relative error (standard deviationof the mean x 100%) of the values was less than 8%. The averageresults of three independent experiments are presented. In semiquantitativeanalysis, the percentage of each FA was calculated by the ratiopeak area/sum of total identified peak areas x 100. In quantitativeanalysis, peak areas were related to that of the internal standard(C₂₂) and then converted to micrograms by using the area ofthe nearest standard peak for the calculation.

RESULTS

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Results

Ethanol-induced cF leakage.
The ethanol-induced leakage of the fluorescent probe cF fromO. oeni cells into the supernatant was studied to determinethe cellular resistance to the disruptive effect of ethanol.The increased cF leakage rate values at high ethanol concentrationsindicated that cell membranes were sensitive to ethanol in allthe conditions shown (Fig. 2). Exposure of nonadapted controlcells to 16% ethanol resulted in a rapid loss of cF, suggestingan immediate disorganization of the plasma membrane. However,cells grown in the presence of 8% (vol/vol) ethanol were ableto retain cF more efficiently, for all the concentrations ofethanol tested, and even in the absence of ethanol. Leakagerates in these adapted cells were less than 50% of those innonadapted control cells. O. oeni cells preincubated with 12%(vol/vol) ethanol for 2 h were less leaky for cF during thechallenge with increasing ethanol concentrations than were thenonadapted cells. The presence of CAP during this 2-h incubationin 12% (vol/vol) ethanol did not significantly change the cF-effluxkinetics.

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FIG. 2. Effect of ethanol on the rate of cF efflux from deenergized O. oeni. The cells were loaded with cF by incubation at 30°C in 50 µM cFDA. The efflux of cF was measured by spectrofluorimetry at 30°C in 50 mM potassium buffer (pH 7.0) in cells grown without ethanol ( ${blacklozenge}$ ), preexposed to 12% (vol/vol) ethanol for 2 h in the absence ( ${circ}$ ) and in the presence (•) of CAP, and grown in the presence of 8% (vol/vol) ethanol ( ${blacktriangleup}$ ).

Ethanol stress and adaptation at the membrane lipid-water interface.
Because the interaction of ethanol with the membrane is thoughtto occur at the membrane lipid-water interface (2, 14, 20),we studied the effects of ethanol by using the lipid-water interfacemembrane probe 5-DS. Figure 3a shows the effect of additionof ethanol to intact O. oeni cells on the order parameter S,as calculated from ESR spectra of 5-DS (Fig. 1). The S valuein nonadapted cells was 0.71 and decreased to 0.58 directlyupon the addition of 20% (vol/vol) ethanol. Such behavior isindicative of significant, instantaneous disordering of themembrane lipid-water interface. The data for membrane orderclosely parallel those assessing ethanol effects on cF leakage.Interestingly, the value of 12% (vol/vol) ethanol appears tobe the point for the onset of drastic increases in both membranefluidity and permeability. The shape of the spectra did notchange when spectra were rerecorded within 15 min of the initialrecording (data not shown), indicating that ethanol causes animmediate disordering effect. Spectra of 5-DS were recordedalso from cells grown in the presence of 8% (vol/vol) ethanol.The value of S obtained from these adapted cells was approximately0.03 U higher than that from nonadapted cells, indicating ahigher order at the membrane lipid-water interface (Fig. 3a).Stressing these ethanol-adapted cells with increasing concentrationsof ethanol showed that the S values were always significantlyhigher than those in the nonadapted cells. The difference inS was particularly evident at the highest concentration of ethanoltested (20%). These results indicate that cells grown in thepresence of ethanol are more resistant to ethanol-induced disordering.

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FIG. 3. Effect of ethanol on the molecular-order parameter S calculated from ESR spectra of 5-DS-labeled intact O. oeni cells. (a) Nonadapted cells ( ${triangleup}$ ) or cells grown in the presence of 8% (vol/vol) ethanol ( ${blacktriangleup}$ ). (b) Cells preexposed to 12% (vol/vol) ethanol for 2 h in the presence ( ${circ}$ ) or in the absence (•) of CAP.

In O. oeni cells, incubation with 12% (vol/vol) ethanol inducesenhancement of the synthesis of a specific stress protein, Lo18,and a significant amount of this protein has been found to beassociated with the membrane (22). Based on this, it seemedreasonable to assume that membrane resistance to ethanol disorderingis associated with de novo protein synthesis, a possibilitythat we tested by a short adaptation of cells (without growth)to ethanol in both the presence and the absence of CAP. Valuesof S calculated from 5-DS spectra in cells preexposed to 12%(vol/vol) ethanol for 2 h were higher than those in controlcells (Fig. 3b), i.e., the lipid-water interface region of themembranes in cells preexposed to ethanol was more rigid thanthat in the nonadapted cells. The ethanol concentration-dependentdecrease of S in these ethanol-exposed cells followed the sametrend as that in the control cells, although the spin labelwas always less mobile. When cells were preexposed to 12% (vol/vol)ethanol in the presence of the protein synthesis inhibitor CAP,the results obtained were similar to those in the absence ofCAP at all concentrations of ethanol tested. These results suggestthat de novo protein synthesis does not play a significant rolein membrane adaptation to ethanol-induced disorder under nongrowingconditions.

After stressing control and ethanol-adapted cells with increasingconcentrations of ethanol, cells were resuspended in a solutionof ferricyanide in order to wash out the ethanol. The orderparameter was only partially recovered (Fig. 4), with the extentof recovery being more evident for the higher concentrationstested. In cells grown in the presence of ethanol, the extentof recovery was less pronounced than in nonadapted cells forall the ethanol concentrations tested, with no recovery forcells stressed with 20% (vol/vol) ethanol (Fig. 4b). A possibleexplanation for these observations is that preexposure to ethanoldecreases the partitioning of ethanol into O. oeni membranesas was observed for a variety of other biological membranes(26, 36). Moreover, the observation that the partition coefficientof ethanol correlates inversely with the lipid order (33) isin line with the proposed explanation.

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FIG. 4. Effect of ethanol on the molecular-order parameter S calculated from ESR spectra of 5-DS-labeled intact O. oeni cells. The measurements were made with nonadapted cells (a) or cells grown in the presence of 8% (vol/vol) ethanol (b) and with washed or nonwashed (control) cells.

Ethanol stress at the membrane interior.
To study the influence of ethanol deeper in the bilayer, welabeled O. oeni cells with 12-DS and 16-DS. These compoundsprobe membrane mobility at the level of the 12th and 16th carbonatoms of the acyl chains, respectively. Figure 5 shows a 12-DSspectrum (A, top) and a 16-DS spectrum (B, top) from cells inthe absence of ethanol. The shapes of these spectra were differentfrom that of 5-DS (Fig. 1) and are indicative of an increasingmotional freedom towards the lipid hydrocarbon core (25). Thepresence of 20% ethanol causes narrowing of the lines of boththe 12-DS and the 16-DS spectra (bottom spectra in Fig. 5A andB, respectively), which can be interpreted to mean that membranefluidity has increased (18). Thus, ethanol has a fluidizingeffect on O. oeni membranes not only at the membrane lipid-waterinterface but also deeper in the bilayer. Adaptation to ethanoldoes not cause notable changes in the spectral shape of 12-DSand 16-DS (spectra not shown), thus suggesting that the effectof adaptation mainly relates to the membrane lipid-water interface.

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FIG. 5. EPR spectra of doxyl-stearate spin probes 12-DS (A) and 16-DS (B) in O. oeni cells in the absence or presence of 20% ethanol.

Modification of fatty acyl composition of the phospholipid fraction by different treatments.
Membrane lipid FA composition has been claimed to play a majorrole in determining membrane fluidity, especially in bacteriathat typically lack sterols in their membranes (37). We determinedthe fatty acyl composition of the phospholipid fraction extractedfrom O. oeni cells grown under normal conditions (control),preexposed to 12% ethanol for 2 h in the presence or absenceof CAP, or grown in the presence of 8% (vol/vol) ethanol. Palmiticacid (C_16:0) was the major component of the FA profile followedby myristic acid (C_14:0) (data not shown). The FA profile foundwas essentially similar to the results for O. oeni reportedelsewhere (13, 16, 41), except that we could not detect lactobacillicacid. We found two minor peaks that could not be identified,which might represent lactobacillic acid; however, FA methylester standards (Sigma) and GC-MS analysis were not conclusivein this respect. Addition of 8% (vol/vol) ethanol to the growthmedium increased the degree of FA unsaturation in these ethanol-adaptedcells, while the total lipid content markedly decreased (Fig.6). This was mainly due to a strong decrease in C_16:0 and anincrease in the level of C_16:1 (data not shown). Moreover, itwas observed that two additional unsaturated short-chain FAswere present in cells grown in the presence of ethanol (datanot shown). The degree of unsaturation and the total amountof lipids were identical in control cells and in cells preincubatedin 12% (vol/vol) ethanol for 2 h, irrespective of the presenceof CAP (Fig. 6). Notably, these cells retained cF more efficiently(Fig. 2) and were more resistant to ethanol-induced membranedisordering (Fig. 3b).

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FIG. 6. Membrane lipid composition of O. oeni cells. The total lipid content (bars) and the unsaturation/saturation ratio ( ${circ}$ ) were measured in control cells (A), cells exposed to 12% ethanol for 2 h in the presence (B) or the absence (C) of CAP, and cells grown in the presence of 8% ethanol (D).

DISCUSSION

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Discussion

There is virtually universal agreement in the literature thatbiological membranes are the primary target of ethanol injury,and it is widely assumed that membrane physical properties andlipid composition are the main factors involved in ethanol tolerance(for a review see reference 24). An important question arisesas to the extent to which these aspects of membrane toleranceshare common features. In the present paper, ethanol effectson the physical and chemical properties of the cytoplasmic membraneof O. oeni cells are described, also in relation to acquiredtolerance to ethanol. We found similar trends in the ethanoldependencies of membrane permeability and fluidity. This mightbe taken as preliminary evidence that these two membrane propertiesare ruled by a common mechanism: i.e., ethanol-induced leakinessis the consequence of membrane physical disordering, and resistanceto the permeabilizing effect of ethanol would result from anadaptive increase in membrane order. However, there was no clearcorrelation between adjustment of membrane physical propertiesand changes in the unsaturated/saturated ratio of membrane FAs.

From the ESR spectra of 5-DS it is clear that the fluidity ofthe cytoplasmic membrane in O. oeni cells instantaneously increaseson addition of ethanol, in a concentration-dependent manner.5-DS allows the order to be examined at the upper methylenesegment of the lipid hydrocarbon chains, i.e., close to thelipid-water interface. From the disturbance of this relativelyimmobile membrane segment, it follows that ethanol partitionsat least into the lipid-water interface. Ethanol also increasedthe freedom of motion of spin probes that were labeled alongthe FA near the hydrophobic core of the membrane, but only forhigh concentrations of ethanol (20% [vol/vol]). These resultsare in agreement with other studies showing that ethanol moleculesreside mainly at the lipid-water interface near the lipid glycerolbackbone of the hydrocarbon chains (14, 20).

The capacity for survival under what would normally be consideredextremely adverse conditions, such as those prevailing in wine,requires specific cellular strategies that are of fundamentalimportance for microbial life in such extreme environments.For optimal biological performance, membranes should be maintainedin a fluid, liquid-crystalline state (21). We found that ethanol-adaptedO. oeni cells were able to respond to the fluidizing effectof ethanol by increasing the order at the membrane lipid-waterinterface and decreasing permeability. These results are consistentwith the theory that bacterial cells possess adaptation mechanismsto compensate for the accumulation of toxic amphiphilic compoundsin their membranes (47). Interestingly, the readdition of 8%(vol/vol) ethanol to cells grown in the presence of 8% (vol/vol)ethanol resulted in a membrane fluidity (at the position ofthe nitroxide label of 5-DS) that was similar to that in nonadaptedcells in the absence of ethanol. This result implies that ethanol-inducedadaptation in membrane fluidity is not only qualitatively butalso quantitatively consistent with the homeoviscous theoryvalidated for bacteria by Sinensky (38).

Besides long-term ethanol adaptation, achieved by cells growingin the presence of ethanol, cells need a means for rapid adjustmentof ethanol-induced membrane disorder. Cells preexposed to 12%(vol/vol) ethanol (2 h) acquired membrane ethanol tolerance,although the plasma membranes from these cells were more disturbedby ethanol than were those from cells that were grown in thepresence of 8% (vol/vol) ethanol. The tolerance included adaptivechanges in both order and permeability to negate the effectof ethanol. It has been suggested that ethanol-induced synthesisof stress proteins such as small hsp's is associated with theenhanced ethanol tolerance in bacteria (22, 29). It was recentlyshown that small hsp's interact with phospholipid bilayers andstabilize them (45). While such a mechanism may have providedtolerance to O. oeni cells that were grown in the presence of8% (vol/vol) ethanol, it is unlikely that a similar mechanismoperated during the 2-h preexposure to 12% (vol/vol) ethanol,since the adaptation was not prevented in the presence of theprotein synthesis inhibitor CAP. This leaves the possibilitythat proteins or other compounds already existing in the cytoplasmare called upon to stabilize the cytoplasmic membrane, e.g.,ethanol induces an increase in the affinity of cytoplasmic proteinsfor membranes by increasing their hydrophobicity. These resultsappear to imply that a mechanism(s) independent of de novo proteinsynthesis may be involved in the adaptive response of O. oenicells to ethanol.

Figure 7 shows the correlation between S values, calculatedfrom 5-DS spectra, and cF leakage rates in ethanol-treated cellsgrown in the presence or absence of 8% ethanol or preexposedto 12% (vol/vol) ethanol for 2 h. Although no causal relationshipbetween permeability and lipid order was established, Fig. 7shows a strong negative correlation (r = -0.93) between theseparameters, which suggests that cF leakage rates are determinedby the fluidity at the lipid-water interface. There are argumentsthat support a possible causal relationship. First, from membranedynamics simulation it appeared that the order at the membraneregion where the nitroxide moiety of 5-DS resides determinesthe ability of water and ions to diffuse across a membrane (44).Second, ethanol appears to reside at the lipid-water interface,which, together with the aforementioned permeability controlat the upper methylene segment of the acyl chains, renders adirect link between ethanol-induced disorder and leakage plausible.

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FIG. 7. Correlation between S values calculated from 5-DS spectra shown in Fig. 3 and cF leakage rate values from Fig. 2. The data relate to O. oeni cells grown without (control) or with 8% ethanol and cells preexposed for 2 h to 12% ethanol in the presence or absence of CAP. The cells were exposed to 0, 8, 12, and 16% ethanol during ESR spectrum recording and cF efflux measurements.

Adaptation of cells to ethanol would be most effective at themembrane lipid-water interface, where stress proteins are supposedto interact. Adaptive changes in the acyl-chain compositionmay adjust membrane fluidity in the core region, which mightbe less effective in the case of ethanol. This is probably thereason for the less consistent picture that emerges from theliterature concerning ethanol-induced changes in acyl-chaincomposition (for a review, see reference 24). In the presentwork we found a significant increase in the degree of unsaturationof the FAs in cells grown in the presence of 8% (vol/vol) ethanol,which is supposed to lead to a decrease in the gel-to-liquid-crystallinetransition temperature and to increased fluidity. However, asdetermined by ESR, the cytoplasmic membranes in O. oeni cellsgrown in the presence of ethanol were much more rigid than werethose in control cells. The same phenomena were observed inE. coli cells, and liposomes made from the phospholipids ofthese cells displayed, as expected, increased fluidity (12).This observation could point to an important role for the proteincontent of membranes in regulating fluidity. Actually, we foundthat O. oeni cells grown in the presence of ethanol (8%) hada reduced lipid content, i.e., half of that of the control cells.The decreased mobility of the membranes observed in these ethanol-growncells corroborates the idea that a selective decrease in thelipid content can increase the proportion of motionally hinderedlipid molecules (25).

The membrane composition of cells preexposed to 12% (vol/vol)ethanol for 2 h was identical to that of control cells. However,these preexposed cells showed decreased membrane permeabilityand disordering under ethanol-stress conditions. Thus, theseresults suggest that ethanol adaptation at the membrane leveldoes not hold for changing membrane lipid composition. Moreover,the increased level of unsaturation observed in cells grownin the presence of ethanol, more than a direct effect of ethanoladaptation, as has been suggested previously (15), is just aconsequence of ethanol-induced inhibition of saturated FA synthesis(19, 21).

In this paper we conclude that O. oeni cells adjust their membranepermeability during ethanol adaptation by decreasing fluidityat the lipid-water interface. Thus, we hypothesize that thephysical state of the membrane, rather than merely the membranecomposition, may preclude an important role during ethanol adaptationby controlling other biological process, e.g., ATPase activity(9, 17) and transport systems (7, 46).

ACKNOWLEDGMENTS

This study has been carried out with the financial support ofthe Portuguese Foundation for Science and Technology, underthe program PRAXIS XXI.

We thank Hans Dassen for assistance with GC-MS analyses.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Food Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen University and Research Center, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. Phone: 31317484981. Fax: 31317484978. E-mail: Tjakko.Abee{at}wur.nl.

REFERENCES

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