FLO11 is essential for flor formation caused by the C-terminal deletion of NRG1 in Saccharomyces cerevisiae

et al. 2013

Appl Environ Microbiol

bSaccharomyces cerevisiae "flor" yeasts have the ability to form a buoyant biofilm at the air-liquid interface of wine. The formation of biofilm, also called velum, depends on FLO11 gene length and expression. FLO11 encodes a cell wall mucin-like glycoprotein with a highly O-glycosylated central domain and an N-terminal domain that mediates homotypic adhesion between cells. In the present study, we tested previously known antimicrobial peptides with different mechanisms of antimicrobial action for their effect on the viability and ability to form biofilm of S. cerevisiae flor strains. We found that PAF26, a synthetic tryptophanrich cationic hexapeptide that belongs to the class of antimicrobial peptides with cell-penetrating properties, but not other antimicrobial peptides, enhanced biofilm formation without affecting cell viability in ethanol-rich medium. The PAF26 biofilm enhancement required a functional FLO11 but was not accompanied by increased FLO11 expression. Moreover, fluorescence microscopy and flow cytometry analyses showed that the PAF26 peptide binds flor yeast cells and that a flo11 gene knockout mutant lost the ability to bind PAF26 but not P113, a different cell-penetrating antifungal peptide, demonstrating that the FLO11 gene is selectively involved in the interaction of PAF26 with cells. Taken together, our data suggest that the cationic and hydrophobic PAF26 hexapeptide interacts with the hydrophobic and negatively charged cell wall, favoring Flo11p-mediated cell-tocell adhesion and thus increasing biofilm biomass formation. The results are consistent with previous data that point to glycosylated mucin-like proteins at the fungal cell wall as potential interacting partners for antifungal peptides.

mentioning

confidence: 99%

mentioning

confidence: 99%

FLO11 Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide

Zeidan

Carmona

et al. 2013

Appl Environ Microbiol

“…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.…”

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confidence: 99%

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

Groß

et al. 2010

Appl Environ Microbiol

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...

“…The common laboratory strain background S288C does not express FLO11 due to a nonsense mutation in the transcriptional activator FLO8 (28). In some industrial strains, FLO11 mediates formation of the specialized biofilms called flors that are necessary for the production of sherry wine (19,48). The common feature of all these phenotypes is adhesion.…”

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confidence: 99%

Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

Douglas

Yang

et al. 2007

Eukaryot Cell

The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background ⌺1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to ⌺1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.