Air-liquid biofilm formation is dependent on ammonium depletion in a Saccharomyces cerevisiae flor strain.

Air-liquid biofilm formation appears to be an adaptive mechanism that promotes foraging of Saccharomyces cerevisiae flor strains in response to nutrient starvation. The FLO11 gene plays a central role in this phenotype as its expression allows yeast cells to rise to the liquid surface. Here, we investigated the role of ammonium depletion in air-liquid biofilm formation and FLO11 expression in a S. cerevisiae flor strain. The data obtained show that increasing ammonium concentrations from 0 to 450 m m reduce air-liquid biofilm in terms of biomass and velum formation and correlate with a reduction of FLO11 expression. Rapamycin inhibition of the TOR pathway and deletion of RAS2 gene significantly reduced biofilm formation and FLO11 expression. Taken together, these data suggest that ammonium depletion is a key factor in the induction of air-liquid biofilm formation and FLO11 expression in S. cerevisiae flor strains.

Zara G ，Budroni M ，Mannazzu I ，Zara S ... -  《-》

FLO11 expression and lipid biosynthesis are required for air-liquid biofilm formation in a Saccharomyces cerevisiae flor strain.

Air-liquid biofilm formation is largely dependent on Flo11p and seems related to cell lipid content and composition. Here, it is shown that in the presence of cerulenin, a known inhibitor of the fatty acid synthase complex, biofilm formation is inhibited together with FLO11 transcription in a flor strain of Saccharomyces cerevisiae, while the administration of saturated fatty acids to cerulenin-containing medium restores biofilm formation and FLO11 transcription. It is also shown that, in biofilm cells, the FLO11 transcription is accompanied by the transcription of ACC1, ACS1 and INO1 key genes in lipid biosynthesis and that biofilm formation is affected by the lack of inositol in flor medium. These results are compatible with the hypothesis that the air-liquid biofilm formation depends on FLO11 transcription levels as well as on fatty acids biosynthesis.

Zara G ，Goffrini P ，Lodi T ，Zara S ，Mannazzu I ，Budroni M ... -  《-》

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

The flor strains of Saccharomyces cerevisiae form a flor on the surface of wine after alcoholic fermentation. High hydrophobicity of the cell surface is suggested to be important for flor formation by the flor wine yeasts. However, the molecular mechanism of flor formation is not clear. We found that expression of C-terminal deleted NRG1 lacking its two C2H2 zinc finger motifs (NRG1(1-470)) on the multicopy plasmid conferred the ability to form a flor to a non-flor laboratory strain. The cell surface hydrophobicity of NRG1(1-470) was higher than of the non-flor strain. Disruption of the Nrg1p-repressed gene FLO11, which encodes a cell surface glycoprotein that functions as a flocculin or an adhesin, abolished flor formation. Moreover, expression of FLO11 on a multicopy plasmid could also cause flor formation. These results indicate that FLO11 is essential for flor formation by NRG1(1-470). In addition, the results suggest that the C-terminal truncated form of Nrg1p exerts a dominant negative effect on FLO11 repression, resulting in FLO11 expression and, thus, flor formation.

Ishigami M ，Nakagawa Y ，Hayakawa M ，Iimura Y ... -  《FEMS MICROBIOLOGY LETTERS》

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

Saccharomyces 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 tryptophan-rich 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-to-cell 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.

Bou Zeidan M ，Carmona L ，Zara S ，Marcos JF ... -  《-》

Btn2p is involved in ethanol tolerance and biofilm formation in flor yeast.

Flor yeasts are a particular kind of Saccharomyces cerevisiae strains involved in Sherry wine biological ageing. During this process, yeasts form a film on the wine surface and use ethanol as a carbon source, producing acetaldehyde as a by-product. Acetaldehyde induces BTN2 transcription in laboratory strains. Btn2p is involved in the control of the subcellular localization of different proteins. The BTN2 gene shows a complex expression pattern in wine yeast, increasing its expression by acetaldehyde, but repressing it by ethanol. A flor yeast strain transcribes more BTN2 than a first fermentation yeast during growth, but less under different stress conditions. BTN2 deletion decreases flor yeast resistance to high ethanol concentrations. Surprisingly, this effect is suppressed by the addition of high amounts of amino acids to the growth medium, indicating that the role of Btn2p protein in amino acid transport is important for ethanol resistance. Btn2p deletion increases the fermentative capacity of flor yeast and its overexpression prevents its growth on nonfermentable carbon sources. BTN2 deletion also affects the biofilm formation ability of flor yeast, and it increases its sliding motility, resulting in increased mat formation. This correlates with an increased transcription of the FLO11 gene, a gene essential for biofilm formation.

Espinazo-Romeu M ，Cantoral JM ，Matallana E ，Aranda A ... -  《FEMS YEAST RESEARCH》