The Extracellular Matrix of Yeasts: A Key Player in the Microbial Biology Change of Paradigm.

Microbes are traditionally regarded as planktonic organisms, individual cells that live independently from each other. Although this is true, microbes in nature mostly live within large multi-species communities forming complex ecosystems. In these communities, microbial cells are held together and organised spatially by an extracellular matrix (ECM). Unlike the ECM from the tissues of higher eukaryotes, microbial ECM, mostly that of yeasts, is still poorly studied. However, microbial biofilms are a serious cause for concern, for being responsible for the development of nosocomial infections by pharmacological drugs-resistant strains of pathogens, or for critically threatening plant health and food security under climate change. Understanding the organization and behaviour of cells in biofilms or other communities is therefore of extreme importance. Within colonies or biofilms, extremely large numbers of individual microbial cells adhere to inert surfaces or living tissues, differentiate, die or multiply and invade adjacent space, often following a 3D architectural programme genetically determined. For all this, cells depend on the production and secretion of ECM, which might, as in higher eukaryotes, actively participate in the regulation of the group behaviour. This work presents an overview of the state-of-the-art on the composition and structure of the ECM produced by yeasts, and the inherent physicochemical properties so often undermined, as well as the available information on its production and delivery pathways.

Moniliophthora perniciosa, the Causal Agent of Cacao Witches' Broom Disease Is Killed in vitro by Saccharomyces cerevisiae and Wickerhamomyces anomalus Yeasts.

Cacao plantations from South America have been afflicted with the severe fungal disease known as Witches' Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça, and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa, originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts' antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecting yeast to hypha. This evidence suggests W. anomalus cells form a network of yeast cells connecting with each other and with hyphae, supporting a possible cooperative collective killing and feeding strategy. The present results provide an initial step toward the formulation of a new eco-friendly and effective alternative for controlling cacao WBD using live yeast biocides.

Physical, genetic and functional interactions between the eisosome protein Pil1 and the MBOAT O-acyltransferase Gup1.

The Saccharomyces cerevisiae MBOAT O-acyltransferase Gup1 is involved in many processes, including cell wall and membrane composition and integrity, and acetic acid-induced cell death. Gup1 was previously shown to interact physically with the mitochondrial membrane VDAC (Voltage-Dependent Anion Channel) protein Por1 and the ammonium transceptor Mep2. By co-immunoprecipitation, the eisosome core component Pil1 was identified as a novel physical interaction partner of Gup1. The expression of PIL1 and Pil1 protein levels were found to be unaffected by GUP1 deletion. In ∆gup1 cells, Pil1 was distributed in dots (likely representing eisosomes) in the membrane, identically to wt cells. However, ∆gup1 cells presented 50% less Pil1-GFP dots/eisosomes, suggesting that Gup1 is important for eisosome formation. The two proteins also interact genetically in the maintenance of cell wall integrity, and during arsenite and acetic acid exposure. We show that Δgup1 Δpil1 cells take up more arsenite than wt and are extremely sensitive to arsenite and to acetic acid treatments. The latter causes a severe apoptotic wt-like cell death phenotype, epistatically reverting the ∆gup1 necrotic type of death. Gup1 and Pil1 are thus physically, genetically and functionally connected.

Draft genome sequence of Wickerhamomyces anomalus LBCM1105, isolated from cachaça fermentation.

Wickerhamomyces anomalus LBCM1105 is a yeast isolated from cachaça distillery fermentation vats, notable for exceptional glycerol consumption ability. We report its draft genome with 20.5x in-depth coverage and around 90% extension and completeness. It harbors the sequences of proteins involved in glycerol transport and metabolism.

Potential of Yeasts as Biocontrol Agents of the Phytopathogen Causing Cacao Witches' Broom Disease: Is Microbial Warfare a Solution?

Plant diseases caused by fungal pathogens are responsible for major crop losses worldwide, with a significant socio-economic impact on the life of millions of people who depend on agriculture-exclusive economy. This is the case of the Witches' Broom Disease (WBD) affecting cacao plant and fruit in South and Central America. The severity and extent of this disease is prospected to impact the growing global chocolate market in a few decades. WBD is caused by the basidiomycete fungus Moniliophthora perniciosa. The methods used to contain the fungus mainly rely on chemical fungicides, such as copper-based compounds or azoles. Not only are these highly ineffective, but also their utilization is increasingly restricted by the cacao industry, in part because it promotes fungal resistance, in part related to consumers' health concerns and environmental awareness. Therefore, the disease is being currently tentatively controlled through phytosanitary pruning, although the full removal of infected plant material is impossible and the fungus maintains persistent inoculum in the soil, or using an endophytic fungal parasite of Moniliophthora perniciosa which production is not sustainable. The growth of Moniliophthora perniciosa was reported as being antagonized in vitro by some yeasts, which suggests that they could be used as biological control agents, suppressing the fungus multiplication and containing its spread. Concurrently, some yeast-based products are used in the protection of fruits from postharvest fungal spoilage, and the extension of diverse food products shelf-life. These successful applications suggest that yeasts can be regarded a serious alternative also in the pre-harvest management of WBD and other fungal plant diseases. Yeasts' GRAS (Generally Recognized as Safe) nature adds to their appropriateness for field application, not raising major ecological concerns as do the present more aggressive approaches. Importantly, mitigating WBD, in a sustainable manner, would predictably have a high socioeconomic impact, contributing to diminish poverty in the cacao-producing rural communities severely affected by the disease. This review discusses the importance/advantages and the challenges that such a strategy would have for WBD containment, and presents the available information on the molecular and cellular mechanisms underlying fungi antagonism by yeasts.

High-affinity transport, cyanide-resistant respiration, and ethanol production under aerobiosis underlying efficient high glycerol consumption by Wickerhamomyces anomalus.

Wickerhamomyces anomalus strain LBCM1105 was originally isolated from the wort of cachaça (the Brazilian fermented sugarcane juice-derived Brazilian spirit) and has been shown to grow exceptionally well at high amounts of glycerol. This paramount residue from the biodiesel industry is a promising cheap carbon source for yeast biotechnology. The assessment of the physiological traits underlying the W. anomalus glycerol consumption ability in opposition to Saccharomyces cerevisiae is presented. A new WaStl1 concentrative glycerol-H+ symporter with twice the affinity of S. cerevisiae was identified. As in this yeast, WaSTL1 is repressed by glucose and derepressed/induced by glycerol but much more highly expressed. Moreover, LBCM1105 aerobically growing on glycerol was found to produce ethanol, providing a redox escape to compensate the redox imbalance at the level of cyanide-resistant respiration (CRR) and glycerol 3P shuttle. This work is critical for understanding the utilization of glycerol by non-Saccharomyces yeasts being indispensable to consider their industrial application feeding on biodiesel residue.

Conditions promoting effective very high gravity sugarcane juice fermentation.

BACKGROUND: Applying very high gravity (VHG) fermentation conditions to the sugarcane juice (SCJ) bioethanol industry would improve its environmental and economic sustainability without the need for major infrastructure changes or investments. It could enable a decrease in the consumption of biological and natural resources (cane/land, water and energy) while maintaining acceptable production parameters. The present study attempts to demonstrate and characterise an effective industrially relevant SCJ-VHG fermentation process. RESULTS: An industry-like SCJ-VHG bioethanol production process with 30 and 35 °Bx broth was employed to investigate the effects of both the yeast strain used and nitrogen source supplementation on process yield, process productivity, biomass viability, glycerol concentration and retention-associated gene expression. Process performance was shown to be variably affected by the different process conditions investigated. Highest process efficiency, with a 17% (w/v) ethanol yield and only 0.2% (w/v) sugar remaining unfermented, was observed with the Saccharomyces cerevisiae industrial strain CAT-1 in 30 °Bx broth with urea supplementation. In addition, efficient retention of glycerol by the yeast strain was identified as a requisite for better fermentation and was consistent with a higher expression of glycerol permease STL1 and channel FPS1. Urea was shown to promote the deregulation of STL1 expression, overcoming glucose repression. The consistency between Fps1-mediated ethanol secretion and ethanol in the extracellular media reinforces previous suggestions that ethanol might exit the cell through the Fps1 channel. CONCLUSIONS: This work brings solid evidence in favour of the utilisation of VHG conditions in SCJ fermentations, bringing it a step closer to industrial application. SCJ concentrated up to 30 °Bx maintains industrially relevant ethanol production yield and productivity, provided the broth is supplemented with a suitable nitrogen source and an appropriate industrial bioethanol-producing yeast strain is used. In addition, the work contributes to a better understanding of the VHG-SCJ process and the variable effects of process parameters on process efficiency and yeast strain response.

Saccharomyces cerevisiae mitochondrial Por1/yVDAC1 (voltage-dependent anion channel 1) interacts physically with the MBOAT O-acyltransferase Gup1/HHATL in the control of cell wall integrity and programmed cell death.

Gup1 is the yeast counterpart of the high eukaryotes HHATL. This and the close homologue Gup2/HHAT regulate the Hedgehog morphogenic, developmental pathway. In yeasts, a similar paracrine pathway is not known though the Δgup1 mutant is associated with morphology and proliferation/death processes. As a first step toward identifying the actual molecular/enzymatic function of Gup1, this work identified by co-immunoprecipitation the yeast mitochondria membrane VDAC1/Por1 as a physical partner of Gup1. Gup1 locates in the ER and the plasma membrane. It was now confirmed to further locate, as Por1, in the mitochondrial sub-cellular fraction. The yeast Por1-Gup1 association was found important for (i) the sensitivity to cell wall perturbing agents and high temperature, (ii) the differentiation into structured colonies, (iii) the size achieved by multicellular aggregates/mats and (iv) acetic-acid-induced Programmed Cell Death. Moreover, the absence of Gup1 increased the levels of POR1 mRNA, while decreasing the amounts of intracellular Por1, which was concomitantly previously known to be secreted by the mutant but not by wt. Additionally, Por1 patchy distribution in the mitochondrial membrane was evened. Results suggest that Por1 and Gup1 collaborate in the control of colony morphology and mat development, but more importantly of cellular integrity and death.

The Yeast Saccharomyces cerevisiae as a Model for Understanding RAS Proteins and their Role in Human Tumorigenesis.

The exploitation of the yeast Saccharomyces cerevisiae as a biological model for the investigation of complex molecular processes conserved in multicellular organisms, such as humans, has allowed fundamental biological discoveries. When comparing yeast and human proteins, it is clear that both amino acid sequences and protein functions are often very well conserved. One example of the high degree of conservation between human and yeast proteins is highlighted by the members of the RAS family. Indeed, the study of the signaling pathways regulated by RAS in yeast cells led to the discovery of properties that were often found interchangeable with RAS proto-oncogenes in human pathways, and vice versa. In this work, we performed an updated critical literature review on human and yeast RAS pathways, specifically highlighting the similarities and differences between them. Moreover, we emphasized the contribution of studying yeast RAS pathways for the understanding of human RAS and how this model organism can contribute to unveil the roles of RAS oncoproteins in the regulation of mechanisms important in the tumorigenic process, like autophagy.

Yeast Gup1(2) Proteins Are Homologues of the Hedgehog Morphogens Acyltransferases HHAT(L): Facts and Implications.

In multiple tissues, the Hedgehog secreted morphogen activates in the receiving cells a pathway involved in cell fate, proliferation and differentiation in the receiving cells. This pathway is particularly important during embryogenesis. The protein HHAT (Hedgehog O-acyltransferase) modifies Hh morphogens prior to their secretion, while HHATL (Hh O-acyltransferase-like) negatively regulates the pathway. HHAT and HHATL are homologous to Saccharomyces cerevisiae Gup2 and Gup1, respectively. In yeast, Gup1 is associated with a high number and diversity of biological functions, namely polarity establishment, secretory/endocytic pathway functionality, vacuole morphology and wall and membrane composition, structure and maintenance. Phenotypes underlying death, morphogenesis and differentiation are also included. Paracrine signalling, like the one promoted by the Hh pathway, has not been shown to occur in microbial communities, despite the fact that large aggregates of cells like biofilms or colonies behave as proto-tissues. Instead, these have been suggested to sense the population density through the secretion of quorum-sensing chemicals. This review focuses on Gup1/HHATL and Gup2/HHAT proteins. We review the functions and physiology associated with these proteins in yeasts and higher eukaryotes. We suggest standardisation of the presently chaotic Gup-related nomenclature, which includes KIAA117, c3orf3, RASP, Skinny, Sightless and Central Missing, in order to avoid the disclosure of otherwise unnoticed information.

Quantitative differential proteomics of yeast extracellular matrix: there is more to it than meets the eye.

BACKGROUND: Saccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation. METHODS: S. cerevisiae W303-1A wt strain and gup1∆ mutant were used as previously described to generate biofilm-like mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis. RESULTS: The proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133. CONCLUSIONS: yECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1∆ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.

Two glycerol uptake systems contribute to the high osmotolerance of Zygosaccharomyces rouxii.

The accumulation of glycerol is essential for yeast viability upon hyperosmotic stress. Here we show that the osmotolerant yeast Zygosaccharomyces rouxii has two genes, ZrSTL1 and ZrSTL2, encoding transporters mediating the active uptake of glycerol in symport with protons, contributing to cell osmotolerance and intracellular pH homeostasis. The growth of mutants lacking one or both transporters is affected depending on the growth medium, carbon source, strain auxotrophies, osmotic conditions and the presence of external glycerol. These transporters are localised in the plasma membrane, they transport glycerol with similar kinetic parameters and besides their expected involvement in the cell survival of hyperosmotic stress, they surprisingly both contribute to an efficient survival of hypoosmotic shock and to the maintenance of intracellular pH homeostasis under non-stressed conditions. Unlike STL1 in Sa. cerevisiae, the two Z. rouxii STL genes are not repressed by glucose, but their expression and activity are downregulated by fructose and upregulated by non-fermentable carbon sources, with ZrSTL1 being more influenced than ZrSTL2. In summary, both transporters are highly important, though Z. rouxii CBS 732(T) cells do not use external glycerol as a source of carbon.

Elemental biochemical analysis of the polysaccharides in the extracellular matrix of the yeast Saccharomyces cerevisiae.

In yeast multicellular aggregates, such as biofilms and colonies, cells are supported by a yeast extracellular matrix (yECM) of glycosidic nature, the composition of which is mostly unknown. Saccharomyces cerevisiae ECM was produced, extracted and partitioned. An analytical-grade pure glycoside fraction was obtained, fractionated by anionic exchange liquid chromatography and analyzed by gas chromatography-mass spectrometry and polyacrylamide gel electrophoresis. Two different molecular weight polysaccharides were found, composed of glucose, mannose and small relative amounts of galactose. One of the polysaccharides had a low molecular weight, compatible with the association with glycoproteins abundantly occurring in yECM. In addition, these polysaccharide species were separated by diaminopropane agarose gel electrophoresis and induced metachromatic shift, suggesting chemical substitution, which was corroborated by anticoagulation activity. This was shown to be associated with the double deletion of the yeast homologues of the mammalian Hedgehog modulators Hhatl and Hhat, respectively yeast Gup1 and Gup2. These results pioneer the study of the molecular biology of the ECM supporting S. cerevisiae multicellular aggregates such as biofilms.

Biotechnological potential of yeast isolates from cachaça: the Brazilian spirit.

This study identified phenotypic traits appropriate for biotechnological applications of 118 yeasts isolated from cachaça distilleries. Different properties were verified: capacity to use alternative carbon sources; ability to tolerate high concentrations of sucrose, ethanol, methanol, aluminum and zinc as well as different pH values and foam production. Pichia guilliermondii and Pichia anomala strains were identified as the most promising ones for application in the second-generation biofuel industry, showing ability to grow on high glycerol concentrations. Other isolates, identified as Saccharomyces cerevisiae, produced bioethanol comparable to the industrial strains, and were therefore ideal for use in the first-generation ethanol industry. Some of these strains also showed high resistance to aluminum, as observed in sugarcane juice, and to inter-cycle washings with diluted sulphuric acid, as performed in the industrial bioethanol production process. In summary, yeast isolates from cachaça distilleries displayed robustness and phenotypic plasticity, which makes them interesting for biotechnological applications.

Methodologies to generate, extract, purify and fractionate yeast ECM for analytical use in proteomics and glycomics.

BACKGROUND: In a multicellular organism, the extracellular matrix (ECM) provides a cell-supporting scaffold and helps maintaining the biophysical integrity of tissues and organs. At the same time it plays crucial roles in cellular communication and signalling, with implications in spatial organisation, motility and differentiation. Similarly, the presence of an ECM-like extracellular polymeric substance is known to support and protect bacterial and fungal multicellular aggregates, such as biofilms or colonies. However, the roles and composition of this microbial ECM are still poorly understood. RESULTS: This work presents a protocol to produce S. cerevisiae and C. albicans ECM in an equally highly reproducible manner. Additionally, methodologies for the extraction and fractionation into protein and glycosidic analytical pure fractions were improved. These were subjected to analytical procedures, respectively SDS-PAGE, 2-DE, MALDI-TOF-MS and LC-MS/MS, and DAE and FPLC. Additional chemical methods were also used to test for uronic acids and sulphation. CONCLUSIONS: The methodologies hereby presented were equally efficiently applied to extract high amounts of ECM material from S. cerevisiae and C. albicans mats, therefore showing their robustness and reproducibility for yECM molecular and structural characterization. yECM from S. cerevisiae and C. albicans displayed a different proteome and glycoside fractions. S. cerevisiae yECM presented two well-defined polysaccharides with different mass/charge, and C. albicans ECM presented a single different one. The chemical methods further suggested the presence of uronic acids, and chemical modification, possibly through sulphate substitution. All taken, the procedures herein described present the first sensible and concise approach to the molecular and chemical characterisation of the yeast ECM, opening the way to the in-depth study of the microbe multicellular aggregates structure and life-style.

XYLH encodes a xylose/H+ symporter from the highly related yeast species Debaryomyces fabryi and Debaryomyces hansenii.

The closely related yeasts Debaryomyces fabryi and Debaryomyces hansenii are excellent xylose consumers. We previously described the activity of a high-affinity xylose/H(+) symport from an industrial strain of D. hansenii subsequently reclassified as D. fabryi. We now report the identification of the gene encoding this permease, AY347871.2. This was retrieved from D. fabryi gDNA using a degenerate primer PCR strategy, based on conserved regions from the amino acid sequences of three well-characterized bacterial xylose/H(+) symporters. This sequence is 86% identical to another, DEHA2C11374p from D. hansenii type strain. DEHA2C11374p was conceptually ascribed to the major facilitator superfamily. The putative amino acid sequence of AY347871.2 and DEHA2C11374p presented a hydrophobicity pattern compatible with plasma membrane proteins. The last was functionally expressed in Saccharomyces cerevisiae. The sensitivity of transport activity to a protonophore confirmed its dependence on proton motive force, as expected from a symporter. We named D. fabryi AY347871.2 and D. hansenii DEHA2C11374p as XYLH from Xylose/H(+) symport. Based on the very high similarity, we suggested that Scheffersomyces stipitis Xut3 and Aspergillus nidulans AN8400.2 may also encode xylose high-affinity permeases.

Programmed cell death in Saccharomyces cerevisiae is hampered by the deletion of GUP1 gene.

BACKGROUND: During the past years, yeast has been successfully established as a model to study mechanisms of programmed cell death regulation. Saccharomyces cerevisiae commits to cell death showing typical hallmarks of metazoan apoptosis, in response to different stimuli. Gup1p, an O-acyltransferase, is required for several cellular processes that are related to apoptosis development, such as rafts integrity and stability, lipid metabolism including GPI anchor correct remodeling, proper mitochondrial and vacuole function, bud site selection and actin dynamics. Therefore, we hypothesize that apoptotic process would be affected by GUP1 deletion. RESULTS: In the present work we used two known apoptosis inducing conditions, chronological aging and acetic acid, to assess several apoptotic markers in gup1∆ mutant strain. We found that this mutant presents a significantly reduced chronological lifespan as compared to Wt and it is also highly sensitive to acetic acid treatment. In addition, it presents extremely high levels of ROS. There were notorious differences on apoptotic markers between Wt and gup1∆ mutant strains, namely on the maintenance of plasma membrane integrity, on the phosphatidylserine externalization, on the depolarization of mitochondrial membrane and on the chromatin condensation. Those suggested that the mutant, under either condition, probably dies of necrosis and not from apoptosis. CONCLUSIONS: To Gup1p has been assigned an important function on lipid rafts assembly/integrity, lipid metabolism and GPI anchor remodeling. Our results provide, for the first time, the connection of the integrity of yeast lipid rafts and apoptosis induction and/or signaling, giving new insights into the molecular mechanisms underlying this process in yeast.

Saccharomyces cerevisiae glycerol/H+ symporter Stl1p is essential for cold/near-freeze and freeze stress adaptation. A simple recipe with high biotechnological potential is given.

BACKGROUND: Freezing is an increasingly important means of preservation and storage of microbial strains used for many types of industrial applications including food processing. However, the yeast mechanisms of tolerance and sensitivity to freeze or near-freeze stress are still poorly understood. More knowledge on this regard would improve their biotechnological potential. Glycerol, in particular intracellular glycerol, has been assigned as a cryoprotectant, also important for cold/near-freeze stress adaptation. The S. cerevisiae glycerol active transporter Stl1p plays an important role on the fast accumulation of glycerol. This gene is expressed under gluconeogenic conditions, under osmotic shock and stress, as well as under high temperatures. RESULTS: We found that cells grown on STL1 induction medium (YPGE) and subjected to cold/near-freeze stress, displayed an extremely high expression of this gene, also visible at glycerol/H+ symporter activity level. Under the same conditions, the strains harbouring this transporter accumulated more than 400 mM glycerol, whereas the glycerol/H+ symporter mutant presented less than 1 mM. Consistently, the strains able to accumulate glycerol survive 25-50% more than the stl1Δ mutant. CONCLUSIONS: In this work, we report the contribution of the glycerol/H+ symporter Stl1p for the accumulation and maintenance of glycerol intracellular levels, and consequently cell survival at cold/near-freeze and freeze temperatures. These findings have a high biotechnological impact, as they show that any S. cerevisiae strain already in use can become more resistant to cold/freeze-thaw stress just by simply adding glycerol to the broth. The combination of low temperatures with extracellular glycerol will induce the transporter Stl1p. This solution avoids the use of transgenic strains, in particular in food industry.

Candida albicans virulence and drug-resistance requires the O-acyltransferase Gup1p.

BACKGROUND: GUP1 gene was primarily identified in Saccharomyces cerevisiae being connected with glycerol uptake defects in association with osmotic stress response. Soon after, Gup1p was implicated in a complex and extensive series of phenotypes involving major cellular processes. These include membrane and wall maintenance, lipid composition, bud-site selection, cytoskeleton orientation, vacuole morphology, secretory/endocytic pathway, GPI anchors remodelling, and lipid-ordered domains assembly, which is compatible with their inclusion in the Membrane Bound O-acyl transferases (MBOAT) family. In mammals, it has been described as a negative regulator of the Sonic hedgehog pathway involved in morphogenesis, differentiation, proliferation, among other processes. RESULTS: We show that Candida albicans Gup1p strongly interferes with the capacity of cells to develop hyphae, to adhere, to invade, and to form a biofilm, all of which are significant virulence factors. Furthermore, the mutant colonies exhibited an aberrant morphology/differentiation pattern. Identically to S. cerevisiae, Cagup1Δ null mutant was more resistant to antifungals like fluconazole, ketoconazole, and clotrimazole, and displayed an abnormal even sterol distribution at the plasma membrane. CONCLUSIONS: This work is the first study in the opportunistic yeast Candida albicans, showing a role for the GUP1 gene in virulence as well as in the mechanisms underlying antifungal resistance. Moreover, its impact is even more significant since these results, taken together with all the knowledge about GUP1 gene (from S. cerevisiae and mammals) give consistence to the possibility that Gup1p may be part of a yeast morphogenic pathway parallel to the mammalian Hedgehog.

Protective effect of ions against cell death induced by acid stress in Saccharomyces.

Saccharomyces boulardii is a probiotic used to prevent or treat antibiotic-induced gastrointestinal disorders and acute enteritis. For probiotics to be effective they must first be able to survive the harsh gastrointestinal environment. In this work, we show that S. boulardii displayed the greatest tolerance to simulated gastric environments compared with several Saccharomyces cerevisiae strains tested. Under these conditions, a pH 2.0 was the main factor responsible for decreased cell viability. Importantly, the addition of low concentrations of sodium chloride (NaCl) protected cells in acidic conditions more effectively than other salts. In the absence of S. boulardii mutants, the protective effects of Na(+) in yeast viability in acidic conditions was tested using S. cerevisiae Na(+)-ATPases (ena1-4), Na(+)/H(+) antiporter (nha1Delta) and Na(+)/H(+) antiporter prevacuolar (nhx1Delta) null mutants, respectively. Moreover, we provide evidence suggesting that this protection is determined by the plasma membrane potential, once altered by low pH and low NaCl concentrations. Additionally, the absence or low expression/activity of Ena proteins seems to be closely related to the basal membrane potential of the cells.