Flocculation onset in Saccharomyces cerevisiae: effect of ethanol, heat and osmotic stress

AIMS: To examine the effect of different stress conditions on the onset of flocculation in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]; plasma membrane integrity was accessed using propidium iodide and the staining of the yeast cell wall was performed using calcofluor white M2R. Cells in exponential phase of growth were subjected to different stress conditions. The addition of 1%, 3% and 5% (v/v) ethanol, 1% and 3% (v/v) isopropanol or a brief heat shock (52 degrees C, 5 min), did not induce an early flocculation phenotype when compared with control cells. The addition of 10% (v/v) ethanol, a continuous mild heat-stress (37 degrees C) or an osmotic stress (0.5 or 1 mol l(-1) of NaCl) did not induce a flocculent phenotype. CONCLUSIONS: Flocculation seems not to be induced as a response to different chemical (ethanol and isopropanol) and physical (heat and osmotic) stress conditions. Conversely, osmotic and ethanol [10% (v/v)] stress, as well as a continuous mild heat shock (37 degrees C), have a negative impact on the phenotype expression of flocculation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to the elucidation of the control of yeast flocculation. This information might be useful to the brewing industry, as the time when the onset of flocculation occurs can determine the fermentation performance and the beer quality, as well as in other biotechnological industries where flocculation can be used as a cell separation process.     

The phosphatase Ptc6 is involved in virulence and MAPK signalling in Fusarium oxysporum

Mitogen-activated kinase (MAPK) signalling pathways are involved in several important processes related to the development and virulence of Fusarium oxysporum. Reversible phosphorylation of the protein members of these pathways is a major regulator of essential biological processes. Among the phosphatases involved in dephosphorylation of MAPKs, type 2C protein phosphatases (PP2Cs) play important roles regulating many developmental strategies and stress responses in yeasts. Nevertheless, the PP2C family is poorly known in filamentous fungi. The F. oxysporum PP2C family includes seven proteins, but only Ptc1 has been studied so far. Here we show the involvement of Ptc6 in the stress response and virulence of F. oxysporum. Expression analysis revealed increased expression of ptc6 in response to cell wall and oxidative stresses. Additionally, targeted inactivation of ptc6 entailed enhanced susceptibility to cell wall stresses caused by Calcofluor White (CFW). We also demonstrate that the lack of Ptc6 deregulates both the Mpk1 phosphorylation induced by CFW and, more importantly, the Fmk1 dephosphorylation induced by pH acidification of the extracellular medium, indicating that Ptc6 is involved in the regulation of these MAPKs. Finally, we showed, for the first time, the involvement of a phosphatase in the invasive growth and virulence of F. oxysporum.     

Novel insights into the osmotic stress response of yeast

Response to hyperosmolarity in the baker's yeast Saccharomyces cerevisiae has attracted a great deal of attention of molecular and cellular biologists in recent years, from both the fundamental scientific and applied viewpoint. Indeed the underlying molecular mechanisms form a clear demonstration of the intricate interplay of (environmental) signalling events, regulation of gene expression and control of metabolism that is pivotal to any living cell. In this article we briefly review the cellular response to conditions of hyperosmolarity, with focus on the high-osmolarity glycerol mitogen-activated protein kinase pathway as the major signalling route governing cellular adaptations. Special attention will be paid to the recent finding that in the yeast cell also major structural changes occur in order to ensure maintenance of cell integrity. The intriguing role of glycerol in growth of yeast under (osmotic) stress conditions is highlighted.     

Differential response to UV stress and DNA damage during the yeast replicative life span

The yeast Saccharomyces cerevisiae is mortal. Before they die, individual yeasts bud repeatedly producing a finite number of progeny, which have the capacity for a full life span. A feature of aging in many species is the waning of resistance to stress. To determine whether this is the case in yeast, we have examined the survival (viability) of age-synchronized populations of yeasts of various ages, spanning youth, midlife, and old age, after irradiation with ultraviolet light (UV). Resistance to UV was biphasic. There was an increase through midlife, followed by a precipitous decline. For comparison, another mutagenic agent, ethyl methanesulfonate (EMS), was tested in the same way. The response was very different. A uniphase decrease in resistance to this DNA-alkylating agent was found with a plateau later in life. The results argue that the increase in resistance to UV with age is an active process and not simply a monotonic age change. RAS2 is among the genes that determine yeast longevity. This gene is preferentially expressed in young cells and has a life span-extending effect on yeasts. One known function of RAS2 is to mount a protective response to irradiation by UV, which occurs independently of DNA damage. The distinction between UV and EMS found here is consistent with the notion that resistance to UV plays a role in yeast longevity in a manner not related to DNA damage. Furthermore, it suggests that RAS2 may participate in this response. We have found that RAS2 expression and UV resistance coincide in middle-aged yeasts bolstering this possibility. These data and the eclipse in activity of several longevity determining genes at midlife in yeasts also raise the possibility that active life maintenance processes function through this period, after which the organism operates on any remaining reserves until death. © 1996 Wiley-Liss, Inc.     

Biotechnological impact of stress response on wine yeast

Wine yeast deals with many stress conditions during its biotechnological use. Biomass production and its dehydration produce major oxidative stress, while hyperosmotic shock, ethanol toxicity and starvation are relevant during grape juice fermentation. Most stress response mechanisms described in laboratory strains of Saccharomyces cerevisiae are useful for understanding the molecular machinery devoted to deal with harsh conditions during industrial wine yeast uses. However, the particularities of these strains themselves, and the media and conditions employed, need to be specifically looked at when studying protection mechanisms.     

Limited stress response in Streptococcus pneumoniae.

In Streptococcus pneumoniae, heat shock induces the synthesis of 65-, 73-, and 84-kDa proteins, and ethanol shock induces a 104-kDa protein. In this study, the 65-, 84-, and 104-kDa proteins were identified as members of the GroEL, ClpL and alcohol dehydrogenase families, respectively, and the general properties of the stress response of S. pneumoniae to several other stresses were characterized. However, several stresses which are known to induce stress responses in Escherichia coli and Bacillus subtilis failed to induce any high molecular weight heat-shock proteins (HSPs) such as GroEL and DnaK homologues. A minor temperature shift from 30 to 37 C triggered induction of the homologues of DnaK and GroEL of E. coli. These features may provide a foundation for evaluating the role of heat-shock proteins relative to the physiology and pathogenesis of pneumococcus.     

Cellular stress response and pulmonary inflammation

Innate immunity as the first line of the immune system, provides initial protection against various pathogens and infections. Recent studies suggest a link between cell stress response and immune response upon exogenous insults in the lung. The key proteins in cellular stress responses were demonstrated to be involved in the activation and regulation of the immune signaling pathways. Further research on the function of these stress proteins in innate immunity defenses, particularly in pulmonary diseases and inflammation may help to clarify the disease pathogenesis and provide potential therapeutic treatments for various infectious and inflammatory lung diseases.     

Phenotypic heterogeneity in the bacterial oxidative stress response is driven by cell-cell interactions

1. Download : Download high-res image (242KB)
2. Download : Download full-size image

     

Cell wall integrity maintenance in plants: Lessons to be learned from yeast?

The plant cell wall is involved in different biological processes like cell morphogenesis and response to biotic/abiotic stress. Functional integrity of the wall is apparently being maintained during these processes by changing structure/composition and coordinating cell wall with cellular metabolism. In S.cerevisiae a well-characterized mechanism exists that is maintaining functional integrity of yeast the cell wall during similar processes. During the last years it has become obvious that plants have evolved a mechanism to monitor and maintain functional integrity of their cell walls. However, our understanding of the mechanism is rather limited. The available evidence suggests that similar signaling cascades may be involved and particular protein activities may be conserved between plants and yeast. Here we review the available evidence briefly and highlight similarities between yeast and plants that could help us to understand the mode of action of the signaling cascades maintaining plant cell wall integrity.     

Critical role of RPI1 in the stress tolerance of yeast during ethanolic fermentation

Stress tolerance of yeast Saccharomyces cerevisiae during ethanolic fermentation is poorly understood due to the lack of genetic screens and conventional plate assays for studying this phenotype. We screened a genomic expression library of yeast to identify gene(s) that, upon overexpression, would prolong the survival of yeast cells during fermentation, with the view to understand the stress response better and to use the identified gene(s) in strain improvement. The yeast RPI1 (Ras-cAMP pathway inhibitor 1) gene was identified in such a screen performed at 38 °C; introducing an additional copy of RPI1 with its native promoter helped the cells to retain their viability by over 50-fold better than the wild type (WT) parent strain, after 36 h of fermentation at 38 °C. Disruption of RPI1 resulted in a drastic reduction in viability during fermentation, but not during normal growth, further confirming the role of this gene in fermentation stress tolerance. This gene seems to improve viability by fortifying the yeast cell wall, because RPI1 overexpression strain is highly resistant to cell lytic enzyme zymolyase, compared with the WT strain. As the RPI1 overexpression strain substantially retains cell viability at the end of fermentation, the cells can be reused in the subsequent round of fermentation, which is likely to facilitate economical production of ethanol.     

MAPK转导通路在白念珠菌菌体抗氧化应激中的作用

目的探讨MAPK通路在念珠菌抗氧化应激中的作用。方法采用不同浓度过氧化氢刺激白念珠菌,通过流式细胞仪检测念珠菌的凋亡率,并计算其增殖指数;通过实时荧光定量PCR检测MAPK通路中8种基因的表达水平。结果随着过氧化氢的刺激浓度增高,白念珠菌的凋亡率逐渐升高,而其增殖指数下降。在不同的过氧化氢浓度刺激下,MAPK通路中各基因表达水平基本一致,即在较低的过氧化氢浓度刺激下,各基因表达水平均有一定的上升,而随着浓度增高,在高浓度的过氧化氢刺激下,各基因表达水平趋于稳定。结论在低浓度的过氧化氢刺激下,白念珠菌的凋亡率虽有所上升,但其相应的增殖指数也有所上升,即生长加快。这可能与其MAPK通路中各基因表达增强有一定的关系。     

Cell integrity signaling activation in response to hyperosmotic shock in yeast

Current progress highlights the role of the yeast cell wall as a highly dynamic structure that responds to many environmental stresses. Here, we show that hyperosmotic shock transiently activates the PKC signaling pathway, a response that requires previous activation of the HOG pathway. Phosphorylation of Slt2p under such conditions is related to changes in the glycerol turnover and is mostly Mid2p dependent, suggesting that changes in cell turgor, mediated by intracellular accumulation of glycerol, are sensed by PKC sensors to promote the cell integrity response. These observations, together with previous results, suggest that yeast cells respond to changes in cellular turgor by remodeling their cell walls.     

Leaf morphology and water status changes in Triticum durum under water stress

Water relations, leaf morphology and the chemical composition of cell walls in irrigated and unirrigated plants of three durum wheat eultivars were measured at two growth stages (booting and flowering). Plant response to water stress differed at the two stages: cell wall elasticity increased at booting and osmotic potential values decreased at flowering; this may be due to the changes in stress history, leaf development and plant growth stage between the two harvests. Leaf tissue characteristics were modified by water stress only at flowering: accumulation of fibrous constituents and hemicellulose in the cell walls, reduction of acid detergent fiber (ADF) per unit of leaf area, increase in specific leaf weight (SLW), decrease in turgid weight/dry weight ratio (TW/DW) and alteration in mesophyll cell morphology (cell area / ceil perimeter ratio) were observed.
Generally, cv. Valforte (the less drought-resistant cultivar) had the greatest mesophyll cell area and perimeter and it had greater values of neutral detergent fiber (NDF) at the booting stage than cv. Appulo. Reactivity to water stress differed in the eultivars: Valforte showed the greatest increase in hemicellulose content and decrease in cell dimensions under drought at flowering.
No significant relationships between osmotic potential and mesophyll cell characters were observed; there were no correlations among cell wall elasticity, cell morphology and the chemical components of leaf tissue. The total fiber content and the hemicellulose per unit of leaf area were correlated with the TW/DW ratio at flowering. This parameter decreased more in plants subjected to water stress owing to accumulation of hemicellulose. Correlations between leaf structural constituents and $$ suggest that the absorptive capacity of the cell wall may significally affect the osmotic volume of the cell.     

The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae

Saccharomyces cerevisiae is traditionally used for alcoholic beverage and bioethanol production; however, its performance during fermentation is compromised by the impact of ethanol accumulation on cell vitality. This article reviews studies into the molecular basis of the ethanol stress response and ethanol tolerance of S. cerevisiae; such knowledge can facilitate the development of genetic engineering strategies for improving cell performance during ethanol stress. Previous studies have used a variety of strains and conditions, which is problematic, because the impact of ethanol stress on gene expression is influenced by the environment. There is however some commonality in Gene Ontology categories affected by ethanol assault that suggests that the ethanol stress response of S. cerevisiae is compromised by constraints on energy production, leading to increased expression of genes associated with glycolysis and mitochondrial function, and decreased gene expression in energy‐demanding growth‐related processes. Studies using genome‐wide screens suggest that the maintenance of vacuole function is important for ethanol tolerance, possibly because of the roles of this organelle in protein turnover and maintaining ion homoeostasis. Accumulation of Asr1 and Rat8 in the nucleus specifically during ethanol stress suggests S. cerevisiae has a specific response to ethanol stress although this supposition remains controversial.     

Stress-induced growth rate reduction restricts metabolic resource utilization to modulate osmo-adaptation time

1. Download : Download high-res image (132KB)
2. Download : Download full-size image