Effect of adaptation to ethanol on cytoplasmic and membrane protein profiles of Oenococcus oeni

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.

FIG. 1.

2D-E of total protein extracts of O. oeni cells grown in normal conditions (a), stressed with 12% ethanol for 1 h (b), and grown in the presence of 8% ethanol (c). Molecular masses (in kilodaltons) of marker bands (left side) and pI ranges (bottom) are indicated. Selected proteins are boxed and numbered (see also Tables 1 and 2).

FIG. 2.

2D separation of hydrophobic membrane proteins by 16-BAC-SDS-PAGE from O. oeni cells grown under normal conditions (a), stressed with 12% ethanol for 1 h (b), and grown in the presence of 8% ethanol (c). Molecular masses (in kilodaltons) of marker bands (left side) are indicated. Analyzed proteins are boxed and numbered (see also Table 3).

FIG. 3.

2D-E of membrane-associated proteins of O. oeni cells grown in the absence (a) and in the presence (b) of 8% ethanol. Proteins were extracted from the same amount of cells. The same samples were used to extract integral membrane proteins (see above and Materials and Methods). Molecular masses (in kilodaltons) of marker bands (left side) and pI ranges (bottom) are indicated. Selected proteins are boxed and numbered (see also Table 4).