ABSTRACT
The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism. Therefore, the site-specific location of proteins involved in ethanol adaptation, including cytoplasmic, membrane-associated, and integral membrane proteins, was investigated. Ethanol triggers alterations in protein patterns of O. oeni cells stressed with 12% ethanol for 1 h and those of cells grown in the presence of 8% ethanol. Levels of inosine-5'-monophosphate dehydrogenase and phosphogluconate dehydrogenase, which generate reduced nicotinamide nucleotides, were decreased during growth in the presence of ethanol, while glutathione reductase, which consumes NADPH, was induced, suggesting that maintenance of the redox balance plays an important role in ethanol adaptation. Phosphoenolpyruvate:mannose phosphotransferase system (PTS) components of mannose PTS, including the phosphocarrier protein HPr and EII(Man), were lacking in ethanol-adapted cells, providing strong evidence that mannose PTS is absent in ethanol-adapted cells, and this represents a metabolic advantage to O. oeni cells during malolactic fermentation. In cells grown in the presence of ethanol, a large increase in the number of membrane-associated proteins was observed. Interestingly, two of these proteins, dTDT-glucose-4,6-dehydratase and D-alanine:D-alanine ligase, are known to be involved in cell wall biosynthesis. Using a proteomic approach, we provide evidence for an active ethanol adaptation response of O. oeni at the cytoplasmic and membrane protein levels.
Subject(s)
Adaptation, Physiological , Bacterial Proteins/metabolism , Cytoplasm/metabolism , Ethanol/metabolism , Gram-Positive Cocci/growth & development , Membrane Proteins/metabolism , Proteome , Amino Acid Sequence , Bacterial Proteins/chemistry , Culture Media , Electrophoresis, Gel, Two-Dimensional , Gene Expression Regulation, Bacterial , Gram-Positive Cocci/physiology , Heat-Shock Response , Leuconostoc/growth & development , Leuconostoc/physiology , Mass Spectrometry , Membrane Proteins/chemistry , Molecular Sequence DataABSTRACT
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.
