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 is the primary factor in flor formation caused by cell surface hydrophobicity in wild-type flor yeast.

Some strains of Saccharomyces cerevisiae form a biofilm called a "flor" on the surface of wine after ethanolic fermentation, but the molecular mechanism of flor formation by the wild-type flor strain involved in wine making is not clear. Previously, we found that expression of the C-terminally truncated form of NRG1 (NRG1(1-470)) on a multicopy plasmid increases the hydrophobicity of the cell surface, conferring flor formation on the non-flor laboratory strain. Here we show that in Ar5-H12, a wild-type flor haploid strain, flor formation is regulated by NRG1(1-470). Moreover, the disruptant of the wild-type flor diploid strain (Deltaflo11/Deltaflo11) show a weak ability to form the flor. The expression of FLO11 is always high in the wild-type flor strain, regardless of carbon source. Thus FLO11 is primary factor for wild-type flor strains. Furthermore, the disruptant (Deltaflo11) shows lower hydrophobicity of cell surface than the wild type. However, the hydrophobicity of the wild-type flor strains grown in ethanol medium was much higher than those grown in glucose medium. These results indicate that cell surface hydrophobicity is closely related to flor formation in wild-type flor yeasts.

Ishigami M ，Nakagawa Y ，Hayakawa M ，Iimura Y ... -  《BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY》

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》

The role of FLO11 in Saccharomyces cerevisiae biofilm development in a laboratory based flow-cell system.

A role of the FLO11 in Saccharomyces cerevisiae biofilm development in a flow cell system was examined. We carried out an ectopic FLO11 expression in the wild type (wt) BY4741 strain that has low levels of endogenous FLO11 transcript. In contrast to the nonadhesive wt, the FLO11 overexpression strain (BY4741 FLO11(+)) readily adhered to both liquid-hydrophobic and liquid-hydrophilic solid interfaces and was able to grow as a biofilm monolayer in a flow system. Cellular features associated with FLO11 were examined and found to be consistent with the previous studies conducted in different strains of S. cerevisiae. When grown in suspended liquid culture, BY4741 FLO11(+) formed larger cellular aggregates (clumps), consisting of from five to 60 cells, and displayed an increased cell surface hydrophobicity, without changes in the cell size or growth rate, compared to wt. However, the invasive growth associated with FLO11 expression was not observed in BY4741 FLO11(+). The significance of these findings is discussed in the context of clinically and industrially relevant biofilms.

Purevdorj-Gage B ，Orr ME ，Stoodley P ，Sheehan KB ，Hyman LE ... -  《FEMS YEAST RESEARCH》

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 ... -  《-》