Maria C. Loureiro-Dias

Flow Cytometric Assessment of Membrane Integrity of Ethanol-Stressed Oenococcus oeni Cells

ABSTRACT The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into the mechanisms involved in ethanol toxicity and tolerance in this organism. Exposure to ethanol resulted in an increase in the permeability of the cytoplasmic membrane, enhancing passive proton influx and concomitant loss of intracellular material (absorbing at 260 nm). Cells grown in the presence of 8% (vol/vol) ethanol revealed adaptation to ethanol stress, since these cells showed higher retention of compounds absorbing at 260 nm. Moreover, for concentrations higher than 10% (vol/vol), lower rates of passive proton influx were observed in these ethanol-adapted cells, especially at pH 3.5. The effect of ethanol on O. oeni cells was studied as the ability to efficiently retain carboxyfluorescein (cF) as an indicator of membrane integrity and enzyme activity and the uptake of propidium iodide (PI) to assess membrane damage. Flow cytometric analysis of both ethanol-adapted and nonadapted cells with a mixture of the two fluorescent dyes, cF and PI, revealed three main subpopulations of cells: cF-stained intact cells; cF- and PI-stained permeable cells, and PI-stained damaged cells. The subpopulation of O. oeni cells that maintained their membrane integrity, i.e., cells stained only with cF, was three times larger in the population grown in the presence of ethanol, reflecting the protective effect of ethanol adaptation. This information is of major importance in studies of microbial fermentations in order to assign bulk activities measured by classical methods to the very active cells that are effectively responsible for the observations.

Effects of salts on Debaryomyces hansenii and Saccharomyces cerevisiae under stress conditions.

The effect of Na+ and K+ on growth and thermal death of Debaryomyces hansenii and Saccharomyces cerevisiae were compared under stress conditions as those commonly found in food environments. At the supraoptimal temperature of 34 degrees C both cations at concentrations of 0.5 M stimulated growth of D. hansenii, while K+ had no effect and Na+ inhibited growth of S. cerevisiae. At 8 degrees C, close to the minimum temperature for growth in both species, both cations inhibited both yeasts, this effect being more pronounced with Na+ in S. cerevisiae. At extreme pH values (7.8 and 3.5) both cations at concentrations of 0.25 M stimulated D. hansenii while Na+ inhibited S. cerevisiae. K+ inhibited this yeast at pH 3.5. Thermal inactivation rates, measured at 38 degrees C in D. hansenii and at 48 degrees C in S. cerevisiae, decreased in the presence of both cations. This protective effect could be observed in a wider range of concentrations in D. hansenii. These results call the attention to the fact that not all yeasts have the same behaviour on what concerns synergy or antagonism of salt together with other stress factors and should be taken into consideration in the establishment of food preservation procedures.

Cyanide-resistant respiration, a very frequent metabolic pathway in yeasts

It has recently been shown that cyanide-resistant respiration (CRR) is very common in Crabtree-negative yeasts (incapable of aerobic fermentation) and in non-fermentative yeasts. It is conferred by a salicylhydroxamic acid-sensitive alternative oxidase that transfers electrons from ubiquinol to oxygen, bypassing the cytochrome chain. An interesting finding is that, in general, whenever CRR is present, complex I is also present. In this article we briefly review the occurrence of CRR, the biochemistry and molecular biology of the alternative oxidase, and summarise the putative functions that have been attributed to this ubiquitous metabolic pathway, whose usefulness for the yeast cells still remains obscure.

Cloning and Expression of Two Genes Coding for Sodium Pumps in the Salt-Tolerant Yeast Debaryomyces hansenii

Two genes encoding Na(+)-ATPases from Debaryomyces hansenii were cloned and sequenced. The genes, designated ENA1 from D. hansenii (DhENA1) and DhENA2, exhibited high homology with the corresponding genes from Schwanniomyces occidentalis. DhENA1 was expressed in the presence of high Na(+) concentrations, while the expression of DhENA2 also required high pH. A mutant of Saccharomyces cerevisiae lacking the Na(+) efflux systems and sensitive to Na(+), when transformed with DhENA1 or DhENA2, recovered Na(+) tolerance and also the ability to extrude Na(+).

Extrusion of benzoic acid in Saccharomyces cerevisiae by an energy-dependent mechanism

When grown in the presence of benzoic acid, Saccharomyces cerevisiae was able to extrude [(14)C]benzoic acid when a pulse a glucose was given to preloaded cells. While octanoic, sorbic, hexanoic, salicylic, butyric and propionic acids were also inducers, ethanol and acetic acid were not. The mechanism of extrusion required energy and prior growth in the presence of the inducers. Diethylstilbestrol, an inhibitor of ATPases, prevented benzoic acid extrusion. Propionic acid was not actively extruded in cells adapted to either benzoic or propionic acid, behaving as an appropriate probe to measure intracellular pH. Even though the extrusion mechanism was active, benzoic acid entered the cells by a simple diffusion mechanism.

Kinetics and regulation of fructose and glucose transport systems are responsible for fructophily in Zygosaccharomyces bailii

A strain of Zygosaccharomyces bailii was selected for studies on fructose and glucose transport to determine the basis of the fructophilic behaviour of this species. Fructose was transported by a specific low-affinity, high-capacity transport system with a K m of 65.6 mM and a V max of 6.7 mmol g-1 h-1 for cells grown on 2% (w/v) fructose, while the transport of glucose showed a K m of 7 mM and a V max of 1.7 mmol g-1 h-1 for cells grown on 2% (w/v) glucose. The transporter of glucose also fructose as a substrate. Fructose inactivated the glucose transporter; inactivation was faster at higher concentrations. Both transporters were partially inductive. Measurements of metabolic fluxes and respiration and fermentation rates supported the general features identified by transport measurements. The kinetics and regulation of transport of the two sugars confirm the fructophilic behaviour previously described by other authors.

The osmotolerant fructophilic yeast Zygosaccharomyces rouxii employs two plasma-membrane fructose uptake systems belonging to a new family of yeast sugar transporters.

Owing to its high resistance to weak-acid preservatives and extreme osmotolerance, Zygosaccharomyces rouxii is one of the main spoilage yeasts of sweet foods and beverages. In contrast with Saccharomyces cerevisiae, Z. rouxii is a fructophilic yeast; it consumes fructose faster than glucose. So far, to our knowledge, no specific Z. rouxii proteins responsible for this fructophilic behaviour have been characterized. We have identified two genes encoding putative fructose transporters in the Z. rouxii CBS 732 genome. Heterologous expression of these two Z. rouxii ORFs in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic analysis of sugar transport showed that both proteins are functionally expressed at the plasma membrane: ZrFfz1 is a high-capacity fructose-specific facilitator (K(m)∼400 mM and V(max)∼13 mmol h(-1) g(-1)) and ZrFfz2 is a facilitator transporting glucose and fructose with similar capacity and affinity (K(m)∼200 mM and V(max)∼4 mmol h(-1) g(-1)). These two proteins together with the Zygosaccharomyces bailii Ffz1 fructose-specific transporter belong to a new family of sugar transport systems mediating the uptake of hexoses via the facilitated diffusion mechanism, and are more homologous to drug/H(+) antiporters (regarding their primary protein structure) than to other yeast sugar transporters of the Sugar Porter family.

Membrane tension regulates water transport in yeast

Evidence that membrane surface tension regulates water fluxes in intact cells of a Saccharomyces cerevisiae strain overexpressing aquaporin AQY1 was obtained by assessing the osmotic water transport parameters in cells equilibrated in different osmolarities. The osmotic water permeability coefficients (P(f)) obtained for yeast cells overexpressing AQY1 incubated in low osmolarity buffers were similar to those obtained for a double mutant aqy1aqy2 and approximately three times lower (with higher activation energy, E(a)) than values obtained for cells incubated in higher osmolarities (with lower E(a)). Moreover, the initial inner volumes attained a maximum value for cells equilibrated in lower osmolarities (below 0.75 M) suggesting a pre-swollen state with the membrane under tension, independent of aquaporin expression. In this situation, the impairment of water channel activity suggested by lower P(f) and higher E(a) could probably be the first available volume regulatory tool that, in cooperation with other osmosensitive solute transporters, aims to maintain cell volume. The results presented point to the regulation of yeast water channels by membrane tension, as previously described in other cell systems.

Energy conversion coupled to cyanide-resistant respiration in the yeasts Pichia membranifaciens and Debaryomyces hansenii

Cyanide-resistant respiration (CRR) is a widespread metabolic pathway among yeasts, that involves a mitochondrial alternative oxidase sensitive to salicylhydroxamic acid (SHAM). The physiological role of this pathway has been obscure. We used the yeasts Debaryomyces hansenii and Pichia membranifaciens to elucidate the involvement of CRR in energy conversion. In both yeasts the adenosine triphosphate (ATP) content was still high in the presence of antimycin A or SHAM, but decreased to low levels when both inhibitors were present simultaneously, indicating that CRR was involved in ATP formation. Also the mitochondrial membrane potential (Delta Psi(m)), monitored by fluorescent dyes, was relatively high in the presence of antimycin A and decreased upon addition of SHAM. In both yeasts the presence of complex I was confirmed by the inhibition of oxygen consumption in isolated mitochondria by rotenone. Comparing in the literature the occurrence of CRR and of complex I among yeasts, we found that CRR and complex I were simultaneously present in 12 out of 13 yeasts, whereas in six out of eight yeasts in which CRR was absent, complex I was also absent. Since three phosphorylating sites are active in the main respiratory chain and only one in CRR, we propose a role for this pathway in the fine adjustment of energy provision to the cell.

Grapevine Aquaporins: Gating of a Tonoplast Intrinsic Protein (TIP2;1) by Cytosolic pH

Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops being considered as a fruit ligneous tree model system in which the water status appears crucial for high fruit and wine quality, controlling productivity and alcohol level. V. vinifera genome contains 28 genes coding for aquaporins, which acting in a concerted and regulated manner appear relevant for plant withstanding extremely unfavorable drought conditions essential for the quality of berries and wine. Several Vv aquaporins have been reported to be expressed in roots, shoots, berries and leaves with clear cultivar differences in their expression level, making their in vivo biochemical characterization a difficult task. In this work V. vinifera cv. Touriga nacional VvTnPIP1;1, VvTnPIP2;2 and VvTnTIP2;1 were expressed in yeast and water transport activity was characterized in intact cells of the transformants. The three aquaporins were localized in the yeast plasma membrane but only VvTnTIP2;1 expression enhanced the water permeability with a concomitant decrease of the activation energy of water transport. Acidification of yeast cytosol resulted in loss of VvTnTIP2;1 activity. Sequence analysis revealed the presence of a His131 residue, unusual in TIPs. By site directed mutagenesis, replacement of this residue by aspartic acid or alanine resulted in loss of pHin dependence while replacement by lysine resulted in total loss of activity. In addition to characterization of VvTn aquaporins, these results shed light on the gating of a specific tonoplast aquaporin by cytosolic pH.