Utilização de substratos simples e substratos mistos por Debaryomyces hansenii: uma abordagem fisiológica e bioquímica

Poster e resumo apresentado no X Congresso Nacional de Biotecnologia, BIOTEC 2003, Lisboa, 200

Yeasts killer/sensitivity phenotypes and halotolerance

Yeast killer phenotype is not uniformly distributed within certain genera or even within some species. Salt has been described as enhancing killer action, especially in strains that have been isolated from salt environments. The aim of this work was to evaluate the extent of the correlation, if any, between killer/sensitivity and salt-stress tolerance phenotypes. A set of 58 different yeast strains (46 species) was studied. As it has been previously described, tolerance to salt stress can be classified in four major classes of tolerance: 1, 2, 3 and 4 M NaCl. Investigation of killer and sensitivity phenotypes was carried out in the absence and in the presence of NaCl from 0 to 3.5 M, in 0.5 M increments. On the basis of this study, two different groups of yeasts were established. One group was mainly composed of the more halotolerant killer yeasts, which also displayed an increase of killer spectrum in the presence of salt in the assay. The other group included the less halotolerant strains, whose killer spectrum was less significant and either did not vary consistently with salt stress in the assay or decreased in its presence. Although killer activity was found in yeasts belonging to the various classes of salt-stress tolerance, the percentage of strains showing this capacity increased significantly for the classes of higher halotolerance, while the percentage fraction of sensitive strains remained approximately constant. This suggests a phenotypic relationship between high halotolerance and killer capacity manifestation in the presence of high salt concentrations, which is not a consequence of an increase in sensitivity to salt stress of the target strains.Fenotip ubilačkog kvasca nije jednoliko rasprostranjen unutar određenih vrsta ili čak unutar nekoga soja. Poznato je da sol pojačava ubilačko djelovanje, osobito onih sojeva izoliranih iz slanog okoliša. U ovom se radu nastojao procijeniti stupanj korelacije (ako postoji) između osjetljivosti ubilačkih svojstava te otpornosti prema soli pojednih fenotipova. Kao što je prije opisano, proučena je osjetljivost prema soli 58 različitih sojeva kvasca (46 vrsta). Kvasci su prema osjetljivosti razvrstani u četiri glavne skupine, a rast je utvrđen pri 1, 2, 3 i 4 M NaCl. Ispitivanje ubilačkih svojstava i osjetljivosti fenotipova provedeno je u odsutnosti i prisutnosti NaCl, u rasponu od 0 do 3,5 M, s porastom od 0,5 M. Utvrđene su dvije različite skupine kvasaca. Jedna je uglavnom bila sastavljena od ubilačkih kvasaca jače otpornih na sol, koji su pokazivali povećane ubilačke sposobnosti u prisutnosti soli. Druga je pak obuhvaćala sojeve manje otporne na sol, čija su ubilačka svojstva bila manje izražena, a nisu se bitno mijenjala u prisutnosti soli ili su se čak smanjivala. Određujući otpornost na sol uočeno je ubilačko svojstvo kvasaca koji pripadaju raznim razredima. Postotak sojeva koji su imali to svojstvo bitno je porastao kod onih s većom otpornosti prema soli, dok je postotak osjetljivih sojeva ostao približno konstantan. To upućuje na to da postoji fenotipski odnos između velike otpornosti prema soli i pojave ubilačkih svojstava uz veliki udjel soli, što nije posljedica povećane osjetljivosti sojeva prema stresu u prisutnosti soli

Expressão dos genes GUP1 e GUP2 de Saccharomyces cerevisiae: influência da fonte de carbono e do stresse salino

Comunicação apresentada nas "9as Jornadas de Biologia de Leveduras Professor Nicolau van Uden", realizadas em Alcochete, Portugal, em 2001.Dois genes com elevado grau de homologia relacionados com um fenótipo de resistência a stress salino foram identificados em Saccharomyces cerevisiae1. A análise da actividade transportadora com glicerol radioactivo, permitiu estabelecer a função de transporte transmembranar de glicerol para estes dois genes: GUP1 e GUP2 (glycerol uptake). Em células cultivadas em etanol, a actividade de simporte com protões foi atribuida a Gup1p, enquanto que em células cultivadas em glucose nenhuma estirpe exibiu transporte de glicerol. O mutante duplo gpd1gpd2, assim como outros mutantes afectados em pelo menos um gene GPD, uma vez cultivados em glucose na presença de NaCl, exibiu uma actividade transportadora inesperadamente elevada2. Uma mutação adicional nestas estirpes no gene GUP1 reduziu esta actividade para cerca de 50%. A actividade restante foi atribuida a Gup2p. A expressão destes dois genes foi estudada por RT-PCR na estirpe selvagem e na estirpe gpd1gpd2. Os resultados revelaram níveis significativos de mRNA para GUP1 e GUP2 em células desreprimidas e em células cultivadas em glucose. Os níveis de mRNA de GUP1 aumentam em maior grau em estirpes afectadas nos genes GPD, particularmente em células cultivadas em glucose com NaCl (1M) e glicerol (15mM), enquanto que os maiores níveis de mRNA de GUP2 foram observados em células cultivadas em sal sem glicerol. Estes resultados sugerem mecanismos de regulação diferentes para a transcrição de GUP1 e GUP2. Por outro lado, os resultados também indicam a existência de mecanismos póstranscrição de regulação da actividade transportadora de glicerol. 1 - HØlst, B. et al. (2000) Mol. Microbiol, 37:98-107 2 – Lages, F. and Lucas, C. (1997). Bioch. Biophys Acta, 1322: 8-1

Saccharomyces cerevisiae genomic library screening in search for the gene responsible for inductive active glycerol uptake

Comunicação apresentada no "18th International Conference on Yeast Genetics and Molecular Biology", realizado em Stellenbosch, África do Sul em 1997.In Saccharomyces cerevisiae, physiological response to osmotic stress is done, mainly, by increased synthesis and intracellular accumulation of glycerol as compatible solute. Previous studies revealed the existence of a glycerol/H+ symport, inducible by growth under gluconeogenic conditions (Lages and Lucas) and independent of the Fps1 channel for glycerol (Sutherland et al.). In order to isolate the gene encoding for glycerol specific carrier, an isogenic strain to W303- 1A, strain YSH642, carrying gpd1 gpd2 mutations unable to synthesize glycerol, was studied for further screening of a S. cerevisiae genomic library. Physiological assays consisting on detection of extracellular alcalinization of cell suspensions upon addition of glycerol and determination of intracellular accumulation of [14C]glycerol, were performed on glucose-grown cells (repressed cells) and on ethanol-grown cells (derepressed cells). No significant differences were found between the results obtained with either YSH642 and W303-1A strains, from which we concluded that disruptions of GPD1 and GPD2 genes do not interfere with regulation of active glycerol uptake. To choose selection conditions, we assumed that the derepressed activity of the glycerol symporter will contribute to increased halotolerance in gpd1 gpd2 genetic background, provided the presence of extracellular glycerol. Thus, selective medium was designed according with previous phenotypic characterization of salt stress tolerance. Screening of a genomic library of S. cerevisiae in the multicopy plasmid YEp13 with inserts of 8-10Kb at BamHI restriction site, is underway by electroporation of strain YSH642. A field strength of 1500V and resistance of 200_ is being employed giving 0.073% viability and an efficiency of 1.1x104 trf/μgDNA, using, as selective medium, mineral medium supplemented with convenient auxotrophic requirements for both yeast strain and plasmid YEp13, glucose 2% (w/v), NaCl 1.4M and glycerol 50mM. Clones able to grow on this medium are being further characterized for osmotic tolerance and, for glycerol transport activity under conditions of repression.European Community Project - BIOTECH PL 95016

Antagonistic potential of Wickerhamomyces anomalus against phytopathogens causing Olive Anthracnose and Chestnut Ink Disease

Olive Anthracnose (OA) and Chestnut Ink Disease (CID), caused by eukaryotic organisms such as fungi and oomycetes, pose significant challenges to agricultural production, causing severe yield losses and reduced quality of harvested products. Currently, chemical pesticides are widely used to control these pathogens. However, this approach can be expensive and ineffective, and may have detrimental environmental and health effects. Thus, the development of new, greener and more effective strategies is vital for maintaining agricultural productivity and mitigating the impacts of plant diseases and plagues on food supply and economy on Portugal and other producing countries. The use of yeasts as biological control agents is an effective and environmentally friendly alternative approach for controlling plant pathogens. In this work, we explore the potential of Wickerhamomyces anomalus as a living biocontrol agent for the sustainable pre-harvest control of OA and CID, and to unveil its mode of action. We conducted antagonism assays in liquid media, co-culturing the yeast W. anomalus with three strains of Colletotrichum, causing OA, and two strains of Phytophthora, causing CID. The mode of action of W. anomalus was assessed in several aspects. (i) The viability of the mycelia resulting from the co-cultures was evaluated through re-culturing the mycelia and by microscopic inspection of Methylene Blue-staining mycelia. (ii) Additionally, two common biocontrol strategies were tested - the secretion of cell-wall degrading enzymes and siderophores. (iii) Finally, the interactions between yeast and phytopathogens were further investigated through SEM microscopy. Results confirmed the strong ability of W. anomalus to inhibit the growth of both Colletotrichum and Phytophthora strains, although in different manners. We found that (i) the fungal cells were stained with Methylene Blue, indicating cell death, although, at the same time they were all able to grow back in the presence of a reduced number of yeasts, suggesting a dose-dependent effect; and that (ii) enzymatic and siderophore activity was low/absent, indicating little influence in the mode of action. Importantly, we observed the existence of several antagonism-related features, such as coiled and emptied hyphae and yeasts cells inside the hyphae of C. godetiae and P. cinnnamomi. Results suggest that W. anomalus acts as a predatious yeast, using specific strategies according to its target, corroborating this yeast’s potential for field application in phytosanitary control.This work was supported by the “Contrato-Programa” UIDB/04050/2020 funded by national funds through the FCT I.P. M.A.-R. was the recipient of the FCT, I.P. doctoral grant SFRH/BD/145354/201

Expression studies of GUP1 and GUP2, genes involved in glycerol active transport in Saccharomyces cerevisiae, using semi-quantitative RT-PCR

Glycerol active uptake in Saccharomyces cerevisiae, characterised physiologically as a H+/symport, was previously described as repressed by glucose, induced by growth on non-fermentable carbon sources and unresponsive to growth under salt stress. GUP1 and GUP2 were identified and characterised as genes involved in glycerol active uptake. Using SQ-RT-PCR, GUP1 and GUP2 transcription was measured. Unlike active transport activity determined previously, this was shown to be constitutive and not affected by either glucose repression or growth under salt stress. Furthermore, transcription of GUP1 and GUP2 was still not affected in the gpd1gpd2 mutant strain grown under salt stress in the presence of small amounts of glycerol, in which case a very high Vmax of glycerol uptake has been reported. Intracellular compounds were determined. Glycerol, acetate and trehalose were found to be the major compounds accumulated. Surprisingly, gpd1gpd2 mutant was shown to produce significant amounts of glycerol. Yet, results do not evidence a correlation between the amount of each compound and glycerol transport activity in any of the strains

Expression of GUP1 and GUP2, Saccharomyces cerevisiae glycerol active transport genes

Poster apresentado no "2nd International Meeting on Cellular Responses to Oxidative and Osmotic Stress. Sensing, Signalling and Gene Expression", realizado na Póvoa de Varzim, Portugal de 7 a 10 de Novembro de 2001

The yeast Saccharomyces cerevisiae is sensitive to colorectal cancer routine treatment EGFR antibody Cetuximab

Cetuximab/Erbitux® (Merk Sereno), a drug used in routine treatment of colorectal cancer and other malignant pathologies, is a monoclonal antibody against the Epidermal Growth Factor Receptor (EGFR). A frequent problem affecting the clinical use of Cetuximab is the lack of effectiveness deriving from frequent mutations in K-ras (1-3). A set of mutations in K-ras gene, KRAS c.35G>A (G12D), KRAS c.38G>A (G13D) in exon 2, and KRAS c.183A>T (Q61H) in exon 3, all implicated in the development of colorectal cancer, have been recognized as impeding Cetuximab’s EGFR inhibitory action in human (1). Ras human genes have recognized counterparts in yeast, RAS1 and RAS2. The corresponding proteins belong to the PKA/cAMP MAPK pathway are involved in cell proliferation, in differentiation into hyphae and spores, in response to nitrogen starvation, and in carbon source regulation (4, 5). In opposition to Ras, yeasts do not have a recognized ortholog of EGFR. Nevertheless, yeast is sensitive to Imatinib, another drug that targets specifically EGFR in human cells (6). We generated recombinant yeast strains expressing human wild-type (wt) and mutated open reading frames (ORFs) of K-ras to use in the optimization of phenotypic tests appropriate for the assessment of cell sensitivity to Cetuximab. We observed that Saccharomyces cerevisiae, is sensitive to the treatment with this drug at identical concentration as human cell cultures. Moreover, the complementation of yeast deletions in RAS1 and/or RAS2 with wt or the above mentioned mutated forms of human K-RAS did not alter the response of the cells to the treatment. This suggests that the sensitivity of S. cerevisiae to Cetuximab is independent of the Ras/cAMP pathway. These results further indicate the existence of a paralog of EGFR protein in yeast cell surface. In view of these results, research focused on identifying the EGFR yeast counterpart, downstream effectors and target genes, and determining the correspondent Cetuximab/Erbitux® mode of action.Este trabalho é financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE e por Fundos Nacionais através da FCT – Fundação para a Ciência e a Tecnologia no âmbito do projecto PEst-C/BIA/UI4050/201

Metabolic flux response to salt induced stress in the halotolerant yeast Debaryomyces hansenii

The toxic effect of NaCl and KCI on growth of the marine yeast Debaryomyces hansenii on glucose or glycerol was studied. Above a threshold value, both salts reduced the specific growth rate, specific glucose and glycerol respiration rates and specific glucose fermentation rate, as well as biomass yields. The exponential inhibition constant, k, and minimum toxic concentration, cmin, were similar for all physiological parameters assayed. The effect of either salt on the specific activity of several glycolytic enzymes showed a similar inhibition pattern, although at much lower salt concentrations compared with the physiological parameters. In agreement with published results on glycerol phosphate dehydrogenase stimulation by salt, we present evidence that a general glycolytic flux deviation could occur naturally during salt stress, due to the intrinsic sensitivity of the glycolytic enzymes to intracellular ion concentrations.C917-B3FD-1A62 | Maria Luísa Vieira das Nevesinfo:eu-repo/semantics/publishedVersio

Programmed cell death processes in Saccharomyces cerevisiae are altered by GUP1 deletion

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. 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 life span, comparing to Wt and it is also highly sensitive to acetic acid treatment. Although both chronological aging and acetic acid lead to identical effects, the differences between the strains in the levels/types of apoptotic markers are notorious. In addition, ROS levels of gup1∆ mutant strain were extremely high. According to our results, cells lacking GUP1 seem to be incapable of undergoing apoptosis. Instead this mutant appears to be experiencing a necrotic cell death process. Gup1p has been described to have an important function on lipid rafts assembly/integrity as well as on cell lipid profile. On the other hand, in the literature, rafts have been increasingly implicated on apoptotic signaling. The present results reinforce such idea.Este trabalho é financiado por Fundos FEDER através do Programa Operacional Factores de Competitividade – COMPETE e por Fundos Nacionais através da FCT – Fundação para a Ciência e a Tecnologia no âmbito do projecto PEst-C/BIA/UI4050/201