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Applied and Environmental Microbiology, October 2003, p. 5826-5832, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.5826-5832.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Membrane Fluidity Adjustments in Ethanol-Stressed Oenococcus oeni Cells

M. Graça Da Silveira,1 Elena A. Golovina,2 Folkert A. Hoekstra,2 Frank M. Rombouts,1 and Tjakko Abee1*

Wageningen University and Research Center, 6700 EV Wageningen,1 Laboratory of Plant Physiology, The Graduate School of Experimental Plant Sciences, Wageningen University, 6703 BD Wageningen, The Netherlands2

Received 7 May 2003/ Accepted 28 July 2003

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.

* 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.

Applied and Environmental Microbiology, October 2003, p. 5826-5832, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.5826-5832.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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