Yeast programed cell death and aging

[Excerpt] Similarly to metazoans, yeast cells can exhibit several characteristics of apoptosis, including chromatin condensation, DNA breakage, flipping of phosphatidylserine to the outer leaflet of the plasma membrane, accumulation of reactive oxygen species (ROS), and release of pro-death factors such as cytochrome c or Endonuclease G from mitochondria. Yeast programed cell death has been shown to occur in response to a variety of stimuli, such as oxidative stress, exposure to acetic acid, and expression of mammalian pro-apoptotic proteins. This program is also inherent to the yeast life cycle, as aged mother cells and cells exposed to pheromone also display an apoptotic and necrotic phenotype. Yeast therefore comprises a conserved core programed cell death process that shares several regulators with mammalian cells, which play major roles in the pathogenesis of human diseases. At the same time, it lacks many of the cell death regulators that have evolved in higher eukaryotes, probably due to the invention of multicellularity. The simplicity of the yeast model allows elucidating the basic molecular pathways of programed cell death without interference from multifaceted regulation, due to various protein isoforms or cellular specificity often observed in studies using mammalian systems. In addition, yeast heterologous expression systems offer the opportunity to exploit the individual functional and mechanistic properties of mammalian apoptotic regulators. [...

New observations on the integrity, structure and physiology of flesh cells from fully ripened grape berry

The physiological/structural status of the soft ripened berry is still a matter of debate. In this paper isolated mesocarp cells from ripened berries of both wine and table varieties were studied by bright-field, fluorescence and confocal microscopy and flow cytometry to highlight the organization of berry flesh cell, function and viability. Flow cytometry analysis confirmed that protoplasting from grape berry mesocarp tissue yields a single heterogenous population of intact and viable cells. Also, the integrity of the plasma membrane and the architecture and complexity of the intracellular membranous system were shown by FM1-43 staining coupled to confocal microscopy imaging. The observed incorporation of the fluorescent glucose analogue 2-NBDG suggests that endocytosis is involved in the transport and intracellular compartmentation of apoplastic sugars. Neutral Red staining confirmed the intricate organization, size, diversity and integrity of the vacuolar apparatus that is probably related to the multifaceted roles of the vacuoles in the developing fruit.This work was supported in part by the Fundação para a Ciência e a Tecnologia (research projects PTDC/AGR-ALI/100636/2008 and PTDC/BIA-BCM/69448/2006 and grant no. SFRH/BD/23169/2005 to N.F)

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Production of renewable liquid biofuels that can substitute fossil fuel, has emerged as a major challenge for applied biology. Biodiesel, in the form of fatty acid esters, produced by oleaginous microorganisms could be an attractive alternative, since the utilization of diesel fuel is more efficient than for example ethanol. Oleaginous algae and yeasts may accumulate very high (60%) levels of intracellular lipids but two drawbacks are the relatively complicated extraction process and the subsequent transesterification with the accompanying glycerol by-product formation. The objective of this work is to apply metabolic engineering of fatty acid synthesis and secretion in the model yeast S. cerevisiae in order to create a microorganism able to produce and secrete free fatty acids or fatty acid esters. S. cerevisiae is a proper model, since lipid metabolism has been studied extensively and all genes encoding enzymes directly involved in lipid synthesis are known. This organism has also been reported to acquire oleaginous properties by no more than three genetic modifications (1). In the yeast S. cerevisiae, activation of exogenous long-chain fatty acids to coenzyme A derivatives, prior to metabolic utilization, proceeds through the fatty acyl-CoA synthetases Faa1p and Faa4p. It has been shown that free fatty acids are secreted from a FAA1,4 double mutant (2). This modification will be combined with modifications of core fatty acid elongation, such as overexpression of acetyl-CoA synthetase Acs1p in an attempt to improve fatty acid production rate. Essential for this work is to gain a deeper understanding of the dynamics of fatty acid synthesis and export, and hopefully facilitate a biological platform for efficient fatty acid or lipid production. In this work, the “delitto perfetto” method (3) is applied to delete these two fatty acyl-CoA synthetases generating genetically clean strains without markers or bacterial DNA. Results show that this technique can be used to generate multiple knockouts by recycling the marker gene. 1. Kamisaka Y et al. Biochem J. 2007 Nov 15;408(Pt 1):61–68. 2. Michinaka Y et al. Journal of Bioscience and Bioengineering. 2003 ;95(5):435-440. 3. Storici F, Resnick MA. Methods Enzymol. 2006 ;409329-45

Selective media as a rapid method for the differentiation of the spoilage yeast Zygosaccharomyces bailii from food and beverages

Resumo da comunicação apresentadal no "X Congresso Nacional de Bioquímica", 31.10. - 02.11.1996, Universidade do Minho, Braga, Portugal.A collection of spoilage yeasts, isolated mostly from wines, was used in order to develop selective media as a rapid method for the differentiation of such yeasts from food and beverages. The strains tested belonged to species of Pichia membranaefaciens, Pichia anomala, Torulaspora delbrueckii, Dekkera anomala, Dekkera bruxellensis, Debaryomyces hansenii, Rhodotorula mucilaginosa, Zygosaccharomyces rouxii, Zygosaccharomyces florentinus and Zygosaccharomyces bailii, the yeast Saccharomyces cerevisiae being used as reference. The culture media tested included a weak carboxylic acid or a mixture of a sugar plus a weak carboxylic acid as the only carbon and energy sources. Depending on the pH, the acid concentration and the proportion of the concentration of the two substrates, the species displayed different ability to transport and metabolize the substrate(s), being possible to differentiate distinct groups of yeasts. The incorporation of an acid-base indicator in the media allowed to visualize those distinct behavior patterns and hence to develop a rapid colorimetric method for the differentiation of Z. bailii from food and beverages, with potential industrial application

A differential medium for the enumeration of the spollage yeast zygosaccharomyces bailii in wine

A collection of yeasts, isolated mostly from spoiled wines, was used in order to develop a differential medium for Zygosaccharomyces bailii. The 118 selected strains of 21 species differed in their origin and resistance to preservatives and belonged to the genera Pichia, Torulaspora, Dekkera, Debaryomyces, Saccharomycodes, Issatchenkia, Kluyveromyces, Kloeckera, Lodderomyces, Schizosaccharomyces, Rhodotorula, Saccharomyces and Zygosaccharomyces. The design of the culture medium was based on the different ability of the various yeast species to grow in a mineral medium with glucose and formic acid (mixed-substrate medium) as the only carbon and energy sources and supplemented with an acid-base indicator. By manipulating the concentration of the acid and the sugar it was possible to select conditions where only Z. bailii strains gave rise to alkalization, associated with a color change of the medium (positive response). The final composition of the mixed medium was adjusted as a compromise between the percentage of recovery and selectivity for Z. bailii. This was accomplished by the use of pure or mixed cultures of the yeast strains and applying the membrane filtration methodology. The microbiological analysis of two samples of contaminated "Vinho Verde" showed that the new medium can be considered as a differential medium to distinguish Z. bailii from other contaminating yeasts, having potential application in the microbiological control of wines and probably other beverages and foods.EU project AIR-2-CT93-830

Desenho de um meio diferencial para detecção da levedura de contaminação alimentar Zygosaccharomyces bailii

Resumo da comunicação oral apresentada no encontro científico "5as Jornadas de Biologia de Leveduras Professor Nicolau van Uden", em 1997, Viseu, Portugal.Zygosaccharomyces bailii é uma levedura frequentemente associada a problemas de contaminação alimentar dada a sua capacidade de sobreviver a ambientes ácidos na presença de ácidos orgânicos fracos normalmente utilizados como preservativos químicos. Com vista a desenvolver um meio diferencial para Z. bailii recorreu-se a uma colecção de leveduras isoladas preferencialmente de vinhos contaminados. Assim as estirpes seleccionadas para este estudo diferem na sua origem e resistência a preservativos ácidos e pertencem espécies de Pichia membranaefaciens, Pichia anomala, Torulaspora delbrueckii, Dekkera anomala, Dekkera bruxellensis, Debaryomyces hansenii, Saccharomycodes ludwigii, Issatchenkia orientalis, Kluyveromyces marxianus, Kloeckera apiculata, Lodderomyces elongisporus, Schizosaccharomyces pombe, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Zygosaccharomyces rouxii, Zygosaccharomyces florentinus and Z. bailii. O desenho do meio de cultura baseou-se na diferente capacidade de crescimento, que as diferentes espécies de levedura apresentam em meio mineral simples (ácido carboxílico fraco) ou misto (açúcar e ácido carboxílico fraco) como única fonte de carbono e energia. Quando os ensaios foram conduzidos num meio líquido simples a maior parte das estirpes apresentou capacidade de utilizar pelo menos um um dos vários ácidos carboxílicos testados. A natureza do ácido e a sua concentração bem como a manipulação do pH do meio, associada à incorporação de um indicador ácido-base, permitiu seleccionar condições em que somente as estrpes de Z. bailii originavam variação de cor do meio (resposta positiva). Contudo, esta resposta só foi observada após 115 a 168 h. Quando se utilizou o meio misto, em todas as estirpes de Z. bailii strains observou-se resposta positiva após um periódo de tempo consideravelmente mais reduzido (cerca de 48 h) quando comparado com o meio simples para a mesma densidade de inóculo e para as mesmas condições de incubação. Utilizando quer microplacas quer meio sólido procedeu-se, de seguida, ao teste deste meio misto com as outras espécies da colecção. Em ambos os casos somente as espécies de Z. bailii strains apresentaram resposta positiva durante as primeiras 48 h de incubação. Concluímos que este meio misto apresenta-se como um meio diferencial, para distinguir Z. bailii de outras leveduras de contaminação, com potencial aplicação prática no controlo microbiológico de alimentos e bebidas.União Europeia (UE) - project AIR-2-CT93-830

Differential medium for the detection of the food and beverage spoilage yeast Zygosaccharomyces bailii

Resumo da comunicação apresentada sob forma de painel, "18th International Specialized Symposium on Yeasts : Yeast Nutrition and Natural Habitats", 1997, Bled, Eslovénia.Zygosaccharomyces bailii is a frequent food and beverage spoilage yeast, which is able to survive at low pH in the presence of weak organic acid preservatives. A collection of yeasts, isolated mostly from spoiled wines, was used in order to develop a differential medium for Z. bailii.. The strains selected differed in their origin and resistance to preservatives, belonging to the species Pichia membranaefaciens, Pichia anomala, Torulaspora delbrueckii, Dekkera anomala, Dekkera bruxellensis, Debaryomyces hansenii, Saccharomycodes ludwigii, Issatchenkia orientalis, Kluyveromyces marxianus, Kloeckera apiculata, Lodderomyces elongisporus, Schizosaccharomyces pombe, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Zygosaccharomyces rouxii, Zygosaccharomyces florentinus and Z. bailii. The design of the culture media was based on the different ability of the various yeast species to grow in a mineral medium containing a single substrate (weak carboxylic acid) or mixed substrate (a sugar plus a weak carboxylic acid) as the only carbon and energy sources. When the assays were carried out in liquid medium with single substrate most of the strains displayed ability to utilize at least one of the several weak carboxylic acids tested. The nature of the acid and its concentration as well as the manipulation of the pH of the medium, associated to the incorporation of an acid-base indicator, allowed to select conditions where only Z. bailii strains gave rise to change in colour of the medium (positive response). However, these positive responses were only obtained after about 115 to 168 h. When the mixed substrate medium (weak acid plus sugar) was used, all the Z. bailii strains gave positive response after a considerably lower time (about 48 h) when compared with the single substrate medium for the same inoculum density and incubation conditions. Following these results the mixed substrate medium was tested for the other species using either microplate wells or solid medium. In both cases, only the Z. bailii strains gave positive response during the first 48 h of incubation. We concluded that such mixed substrate medium could be considered as a differential medium to distinguish Z. bailii from other dangerous yeast in food and beverages, with potential application.União Europeia (UE) - project AIR-2-CT93-830

Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii

Here we show that 320-800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XX