Catarina Prista

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.

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(+).

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.

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.

Effect of ethanol on fluxes of water and protons across the plasma membrane of Saccharomyces cerevisiae.

Plasma membrane integrity, ability to transport substrates and maintenance of homeostasis represent obligatory requirements for efficient ethanol production by Saccharomyces cerevisiae. The effect of ethanol on water diffusion through the bilayer and on mediated water movements was evaluated by stopped flow spectroscopy. Ethanol stimulated water diffusion and inhibited mediated water transport. In a strain overexpressing AQY1, the activation energy for water transport increased progressively (from 5.9 to 12.7 kcal mol(-1)) for increasing ethanol concentrations (up to 12% v/v), indicating that mediated water transport lost importance as compared with water diffusion through the bilayer. The effect of ethanol on proton movements (inward by passive diffusion and outward through the PMA1 H(+)-ATPase) was evaluated by measuring the rate of extracellular alcalinization and acidification of unbuffered cell suspensions at different temperatures. Above 10% ethanol, H(+) diffusion was strongly increased at 30 degrees C, but no effect was observed at 20 degrees C up to 12%, indicating the existence of a threshold above which ethanol has a marked effect. On H(+) extrusion, ethanol had no effect at 20 degrees C, but induced a monotonous decrease at higher temperatures. Our results support the view that above a threshold of ethanol concentration, the membrane structure is disrupted, becoming very leaky to H(+).

Yeast water channels: an overview of orthodox aquaporins

In yeast, the presence of orthodox aquaporins has been first recognized in Saccharomyces cerevisiae, in which two genes (AQY1 and AQY2) were shown to be related to mammal and plant water channels. The present review summarizes the putative orthodox aquaporin protein sequences found in available genomes of yeast and filamentous fungi. Among the 28 yeast genomes sequenced, most species present only one orthodox aquaporin, and no aquaporins were found in eight yeast species. Alignment of amino acid sequences reveals a very diverse group. Similarity values vary from 99% among species within the Saccharomyces genus to 34% between ScAqy1 and the aquaporin from Debaryomyces hansenii. All of the fungal aquaporins possess the known characteristic sequences, and residues involved in the water channel pore are highly conserved. Advances in the establishment of the structure are reviewed in relation to the mechanisms of selectivity, conductance and gating. In particular, the involvement of the protein cytosolic N‐terminus as a channel blocker preventing water flow is addressed. Methodologies used in the evaluation of aquaporin activity frequently involve the measurement of fast volume changes. Particular attention is paid to data analysis to obtain accurate membrane water permeability parameters. Although the presence of aquaporins clearly enhances membrane water permeability, the relevance of these ubiquitous water channels in yeast performance remains obscure.

The grapevine tonoplast aquaporin TIP2;1 is a pressure gated water channel

In plants, the vacuole is a multifunctional organelle with an important role in the maintenance of the intracellular space. Tonoplast membranes are highly permeable to water due to their content in aquaporins TIPs (Tonoplast Intrinsic Proteins) that allow the rapid water influx creating an internal turgor pressure responsible for cell expansion, elongation and shape. The aim of the present study was to evaluate if the grapevine Vitis vinifera TIP2;1 would operate as a possible volume regulator gated by membrane surface tension. For that, the wild type VvTIP2;1 and a non-functional mutated form were heterologous expressed in yeast. Using an experimental strategy in which cells are incubated in external media that induce an increase in internal hydrostatic pressure and consequently membrane surface tension, we were able to compare the osmotic permeability (Pf) and the activation energy for water transport (Ea) of yeast strains expressing the functional and a non-functional TIP2;1. We found Pf and Ea dependence on internal turgor pressure only for the strain harboring the functional aquaporin indicating that TIP2;1 activity is regulated by membrane tension changing from an open to a closed state in an internal pressure dependent manner. This turgor dependent gating of TIP2;1 might be a mechanism to regulate vacuolar size and shape in plants withstanding hostile drought conditions such as grapevine.

ZrFsy1, a High-Affinity Fructose/H+ Symporter from Fructophilic Yeast Zygosaccharomyces rouxii

Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H+ symporter, with Km 0.45±0.07 mM and Vmax 0.57±0.02 mmol h−1 (gdw) −1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.

Heterologous Expression Implicates a GATA Factor in Regulation of Nitrogen Metabolic Genes and Ion Homeostasis in the Halotolerant Yeast Debaryomyces hansenii

ABSTRACT The yeast Debaryomyces hansenii has a remarkable capacity to proliferate in salty and alkaline environments such as seawater. A screen for D. hansenii genes able to confer increased tolerance to high pH when overexpressed in Saccharomyces cerevisiae yielded a single gene, named here DhGZF3, encoding a putative negative GATA transcription factor related to S. cerevisiae Dal80 and Gzf3. Overexpression of this gene in wild-type S. cerevisiae increased caffeine and rapamycin tolerance, blocked growth in low glucose concentrations and nonfermentable carbon sources, and resulted in lithium- and sodium-sensitive cells. Sensitivity to salt could be attributed to a reduced cation efflux, most likely because of a decrease in expression of the ENA1 Na+-ATPase gene. Overexpression of DhGZF3 did not affect cell growth in a gat1 mutant but was lethal in the absence of Gln3. These are positive factors that oppose both Gzf3 and Dal80. Genome-wide transcriptional profiling of wild-type cells overexpressing DhGZF3 shows decreased expression of a number of genes that are usually induced in poor nitrogen sources. In addition, the entire pathway leading to Lys biosynthesis was repressed, probably as a result of a decrease in the expression of the specific Lys14 transcription factor. In conclusion, our results demonstrate that DhGzf3 can play a role as a negative GATA transcription factor when expressed in S. cerevisiae and that it most probably represents the only member of this family in D. hansenii. These findings also point to the GATA transcription factors as relevant elements for alkaline-pH tolerance.

Characterization of new polyol/H+ symporters in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast that produces and assimilates a wide variety of polyols. In this work we evaluate polyol transport in D. hansenii CBS 767, detecting the occurrence of polyol/H+ (and sugar/H+) symporter activity, through the transient extracellular alkalinization of unbuffered starved cell suspensions. From the D. hansenii genome database, we selected nine ORFs encoding putative transporter proteins to clone in a centromeric plasmid with C-terminal GFP tagging and screened for polyol/H+ symporters by heterologous expression in Saccharomyces cerevisiae. Five distinct D. hansenii polyol/H+ symporters were identified and characterized, with different specificities and affinities for polyols, namely one glycerol-specific (DhStl1), one D-galactitol-specific (DhSgl1, Symporter galactitol/H+ 1), one D-(+)-chiro-inositol-specific (DhSyi1, Symporter D-(+)-chiro-inositol/H+ 1), one for D-sorbitol/D-mannitol/ribitol/D-arabitol/D-galactitol (DhSyl1, Symporter Polyols 1) and another for D-sorbitol/D-mannitol/ribitol/D-arabitol (DhSyl2, Symporter Polyols 2). This work contributed to the annotation of new yeast polyol transporters, including two specific for uncommon substrates as galactitol and D-(+)-chiro-inositol.