Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells

Abstract

The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.

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.

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 (⧫), preexposed to 12% (vol/vol) ethanol for 2 h in the absence (○) and in the presence (•) of CAP, and grown in the presence of 8% (vol/vol) ethanol (▴).

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 (▵) or cells grown in the presence of 8% (vol/vol) ethanol (▴). (b) Cells preexposed to 12% (vol/vol) ethanol for 2 h in the presence (○) or in the absence (•) of CAP.

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.

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.

FIG. 6.

Membrane lipid composition of O. oeni cells. The total lipid content (bars) and the unsaturation/saturation ratio (○) 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).

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.