HSP12 is essential for biofilm formation by a Sardinian wine strain of S. cerevisiae

We used transcriptional profiling to investigate the response of the fungal pathogen Candida albicans to temperature and osmotic and oxidative stresses under conditions that permitted Ͼ60% survival of the challenged cells. Each stress generated the transient induction of a specific set of genes including classic markers observed in the stress responses of other organisms. We noted that the classical hallmarks of the general stress response observed in Saccharomyces cerevisiae are absent from C. albicans; no C. albicans genes were significantly induced in a common response to the three stresses. This observation is supported by our inability to detect stress cross-protection in C. albicans. Similarly, in C. albicans there is essentially no induction of carbohydrate reserves like glycogen and trehalose in response to a mild stress, unlike the situation in S. cerevisiae. Thus C. albicans lacks the strong general stress response exhibited by S. cerevisiae. INTRODUCTIONUnicellular organisms have developed different strategies, termed the adaptive response (Ruis and Schuller, 1995), to respond appropriately to environmental changes. Sudden and dramatic changes are defined as stresses and can directly affect the survival of the cells. In general, cells respond to stresses with transient changes in the expression of genes encoding products that serve to protect the cell against the encountered stress (Estruch, 2000).The model unicellular eukaryotic yeast Saccharomyces cerevisiae has been extensively studied to determine the molecular basis for its responses to different stresses. Following a heat shock, yeast cells rapidly produce a large set of chaperones, the heat shock proteins (HSPs), which can help stabilize cellular proteins and block their thermal denaturation (Boy-Marcotte et al., 1999). When challenged with a hyperosmotic stress, S. cerevisiae cells rapidly accumulate osmoprotective molecules like glycerol and produce more salt transporters to adapt their internal ionic strength to the new environment (Boy-Marcotte et al., 1999;Proft and Serrano, 1999;Hohmann, 2002). Another stress response involves the production of detoxification enzymes when the cells are faced with an increase in reactive oxygen species (Lee et al., 1999). S. cerevisiae is also able to induce a general stress response; in addition to the pattern of genes induced specifically by the stress, a general set of genes is induced that is common to all the stresses (Ruis and Schuller, 1995). This ability is linked to the function of the general stress response transcription factors Msn2p and Msn4p that recognize the stress responsive element (STRE; consensus "CCCCT") in the promoters of the stress response genes (Marchler et al., 1993;Martinez-Pastor et al., 1996;Schmitt and McEntee, 1996). This regulation is of great importance in the cross-protection to stress. It allows cells that have been challenged with a mild stress to acquire resistance to a stronger stress, even if the second stress is different from the first moderate one (Lewis et al., 1...

Section: Discussionmentioning

confidence: 96%

Stress-induced Gene Expression inCandida albicans: Absence of a General Stress Response

Enjalbert

Nantel

Whiteway

2003

MBoC

248

257

“…Biofilm cells have been found to have an elevated and/or altered lipid content and increased surface hydrophobicity (3,5,8,9,11). While both Hsp12, a small heat shock protein (13), and Muc1 (also known as Flo11), a hydrophobic cell wall mannoprotein (4,6), have been shown to be required for the flor biofilm (10,12,14), other genetic or environmental requirements, other than an absence of glucose and the presence of ethanol and oxygen, have not been demonstrated. Here, we asked whether flor formation could be induced during growth on nonfermentable substrates other than ethanol.…”

mentioning

confidence: 99%

Ethanol-Independent Biofilm Formation by a Flor Wine Yeast Strain ofSaccharomyces cerevisiae

Groß

et al. 2010

Appl Environ Microbiol

Self Cite

Flor strains of Saccharomyces cerevisiae form a biofilm on the surface of wine at the end of fermentation, when sugar is depleted and growth on ethanol becomes dependent on oxygen. Here, we report greater biofilm formation on glycerol and ethyl acetate and inconsistent formation on succinic, lactic, and acetic acids.Flor or velum formation by certain wine strains of Saccharomyces cerevisiae (flor strains) is a form of cellular aggregation observed as an air-liquid interfacial biofilm at the end of the alcoholic fermentation. Formation of the biofilm appears to be an adaptive mechanism because it ensures access to oxygen and therefore permits continued growth on nonfermentable ethanol. In general, nonbuoyant cells cease growth at the end of completed wine fermentations not for lack of carbon but for lack of oxygen. Biofilm cells have been found to have an elevated and/or altered lipid content and increased surface hydrophobicity (3,5,8,9,11). While both Hsp12, a small heat shock protein (13), and Muc1 (also known as Flo11), a hydrophobic cell wall mannoprotein (4, 6), have been shown to be required for the flor biofilm (10,12,14), other genetic or environmental requirements, other than an absence of glucose and the presence of ethanol and oxygen, have not been demonstrated. Here, we asked whether flor formation could be induced during growth on nonfermentable substrates other than ethanol. On the basis of dry weight of biofilm formed per mg of available carbon, the best carbon sources were found to be glycerol, ethyl acetate, and ethanol, in descending order. While subsurface growth occurred on acetic, DL-lactic, and succinic acids, an air-liquid interfacial biofilm did not always form. Microarray analysis of cells shifted from growth on glucose to growth on ethanol did not detect significant changes in expression of known biofilm formation-associated genes.Yeast strain, growth conditions, and quantitation of biofilm. A flor strain of S. cerevisiae, 3238-32 MATa leu2-⌬1 lys2-801 ura3-52 (12), was grown for 24 h in YEPD (2% Bacto peptone, 1% yeast extract, 2% glucose) at 30°C and 200 rpm. Cells were harvested and washed twice in sterile distilled water by centrifugation, resuspended in sterile distilled water, and diluted 500-fold into sterile-filtered YNB (Bacto yeast nitrogen base without amino acids) plus 30 g/ml Leu, 30 g/ml Lys, and 10 g/ml Ura and supplemented with 4% (vol/vol) ethanol (YNBEtOH), 3% (vol/vol) glycerol (YNB-Glyc), 1% (wt/vol) potassium acetate (YNB-Acet), 2% (wt/vol) DL-lactic acid (YNBLact), 2% (wt/vol) succinic acid (YNB-Succ), or 2% (vol/vol) ethyl acetate (YNB-EtAc). All YNB-based media were adjusted to pH 5.4. Four replicate 1-ml/well cultures were grown in a 24-well polystyrene microtiter plate (Becton Dickinson Labware, NJ). Biofilms were harvested by aspiration after 5 days of static incubation at 30°C by collecting the 1-ml cultures and an additional 1 ml of sterile distilled water used to rinse each well. The 2-ml samples were transferred to a cuvette and read at A 600 . Biofilm bio...

“…Biofilm cells have been found to have an elevated and/or altered lipid content and an increased surface hydrophobicity (7,9,15,16,24). Recently, Zara et al (35) found that the small heat shock protein Hsp12 is required for biofilm formation in a Sardinian flor strain. Reynolds and Fink (28) reported that a laboratory strain of S. cerevisiae could be induced to form a biofilm at a liquid-hydrophobic solid interface and that such formation was dependent on FLO11.…”

mentioning

confidence: 99%

FLO11-Based Model for Air-Liquid Interfacial Biofilm Formation bySaccharomyces cerevisiae

Bakalinsky

et al. 2005

Appl Environ Microbiol

Self Cite

106

109

Sardinian wine strains of Saccharomyces cerevisiae used to make sherry-like wines form a biofilm at the air-liquid interface at the end of ethanolic fermentation, when grape sugar is depleted and further growth becomes dependent on access to oxygen. Here, we show that FLO11, which encodes a hydrophobic cell wall glycoprotein, is required for the air-liquid interfacial biofilm and that biofilm cells have a buoyant density greater than the suspending medium. We propose a model for biofilm formation based on an increase in cell surface hydrophobicity occurring at the diauxic shift. This increase leads to formation of multicellular aggregates that effectively entrap carbon dioxide, providing buoyancy. A visible biofilm appears when a sufficient number of hydrophobic cell aggregates are carried to and grow on the liquid surface.Flor or velum formation by certain wine strains of Saccharomyces cerevisiae (flor strains) is a form of cellular aggregation that manifests as an air-liquid interfacial biofilm at the end of alcoholic fermentation. Increased cell buoyancy and the resultant biofilm that forms on the wine surface appear to be an adaptive mechanism because the biofilm assures access to oxygen and therefore permits continued growth on nonfermentable ethanol. In general, nonbuoyant cells cease growth at the end of completed wine fermentations not for lack of carbon, but for lack of oxygen. In contrast to other microbial biofilms, those formed by flor strains appear to consist of a layer of buoyant cells without a suspending extracellular polysaccharide or protein matrix, as no evidence for such extracellular material has been reported. Biofilm cells have been found to have an elevated and/or altered lipid content and an increased surface hydrophobicity (7,9,15,16,24). Recently, Zara et al. (35) found that the small heat shock protein Hsp12 is required for biofilm formation in a Sardinian flor strain. Reynolds and Fink (28) reported that a laboratory strain of S. cerevisiae could be induced to form a biofilm at a liquid-hydrophobic solid interface and that such formation was dependent on FLO11. In addition, flo11⌬ mutants were reported to be less hydrophobic than the wild type.FLO11 has an open reading frame (ORF) of 4,104 bp, which encodes a hydrolase belonging to the glycosylphosphatidylinositol-anchored class of cell wall proteins rich in serine and threonine. The central domain of Flo11 is similar to that of the flocculins Flo1, Flo5, and Flo10 (33). The FLO11 promoter is at least 2,800 bp (22) and is complex, consisting of four upstream activating sequences and at least nine upstream repressing sequences, the activities of which depend upon growth stage and nutritional conditions (30). In the present study, we demonstrate that FLO11 is required for yeast biofilm formation at an air-liquid interface and that the biofilm cells are not less dense than the suspending medium, and we propose a model to explain the role of FLO11 in biofilm formation. MATERIALS AND METHODSYeast strains, media, and genetic methods. ...