Salt stress response of the extremely halotolerant yeast candida halophila (syn versatilis) CBS 4019 : biochemical and physiological studies

摘要

Halotolerant yeasts have been described as being able to grow in the presence of salt concentrations as high as 4 M NaCl, but yeasts can survive environments with higher salt concentrations like in salines. On the other hand, physiological mechanisms underlying extreme salttolerance in yeasts are still poorly understood. For this reason we selected Candida halophila from a salt stress resistance survey performed on 42 different yeast species, as a potentially good model to unveil some of the physiological mechanisms underlying long-term extreme tolerance to salt. The physiological studies here presented were performed in yeast cells surviving under heavy stress, i.e., growing on synthetic medium in the presence of increasing salt concentrations up to close to the limits of salt solubility (4 to 5 M). These studies included (1) the determination of kinetic growth parameters, (2) the measurement of intracellular accumulation of osmolyte and other compounds, (3) glucose, glycerol and mannitol plasma membrane transport characterization, (4) the determination of intracellular volume and pH, (5) the study of glycerol and mannitol pathways, (6) the measurement of fermentative and respiratory fluxes, (7) in vitro assays of several enzymes from different central metabolic pathways like glycolysis or Krebs cycle between others, (8) the measurement of intracellular bulk redox equivalents and (9) the determination of sodium and potassium intracellular concentrations.C. halophila grows better in the presence of a certain amount of salt than in its absence, although, unlike halophilic bacteria, it does not depend on the presence of salt to survive. It is a slow growing yeast, not severely affected by salt, but rather by external pH under stress. When growing on glucose as carbon source, mannitol and glycerol were produced. Glycerol is the osmolyte, since its accumulation increased exponentially with increasing concentrations of sodium chloride in the media. Mannitol is produced in cells using either carbon source tested, yet, its physiological role is still unknown. Data suggest that both glycerol and mannitol are actively transported. On the other hand, glucose is transported by facilitated diffusion. While both active transport Vmax were not affected by growth under stress, glucose facilitated diffusion Vmax was. Internal pH was constant during growth at all initial pH/salt combinations. H+-ATPase activity was not affected by salt. Furthermore, we have shown that C. halophila maintains and even increases slightly intracellular volume when cultivated under stress, though it decreases significantly upon the same degrees of stress shock.Glycerol and mannitol pathways were not under glucose repression but still metabolically regulated. In particular, glycerol kinase and mitochondrial glycerol 3-P dehydrogenase activities were not detected, suggesting glycerol dissimilation operates through dihydroxyacetone. Glycerol-3Pdehydrogenase (Gpdp) is the key enzyme in glycerol production. Unlike Gpd1p and Gpd2p from S.cerevisiae, in C. halophila it proved to be able to use either co-factor, being NADPH preferred during growth under salt. This co-factor interchange ability, an increased fermentation rate and mannitol pathway activity, are suggested to contribute to redox stability as found.Our results show a generalised increase in enzyme expression under stress-growth conditions, though enzymes per se were not salt-tolerant in vitro. This increase had a correspondence in respiratory and fermentative fluxes increment. Consistently, intracellular sodium was low and potassium high. Potassium concentrations closely correlate with growth rates in the presence of salt and thus can be considered as a requirement for growth. In view of the results from this work, the concept of halophily and its applicability to yeasts is discussed.