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. ...

In Saccharomyces cerevisiae, FLO11 encodes an adhesin that is associated with different phenotypes, such as adherence to solid surfaces, hydrophobicity, mat and air-liquid biofilm formation. In the present study, we analysed FLO11 allelic polymorphisms and FLO11-associated phenotypes of 20 flor strains. We identified 13 alleles of different lengths, varying from 3.0 to 6.1 kb, thus demonstrating that FLO11 is highly polymorphic. Two alleles of 3.1 and 5.0 kb were cloned into strain BY4742 to compare the FLO11-associated phenotypes in the same genetic background. We show that there is a significant correlation between biofilm-forming ability and FLO11 length both in different and in the same genetic backgrounds. Moreover, we propose a multiple regression model that allows prediction of air-liquid biofilm-forming ability on the basis of transcription levels and lengths of FLO11 alleles in a population of S. cerevisiae flor strains. Considering that transcriptional differences are only partially explained by the differences in the promoter sequences, our results are consistent with the hypothesis that FLO11 transcription levels are strongly influenced by genetic background and affect biofilm-forming ability.

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The most important dogma in white-wine production is the preservation of the wine aroma and the limitation of the oxidative action of oxygen. In contrast, the aging of Sherry and Sherry-like wines is an aerobic process that depends on the oxidative activity of flor strains of Saccharomyces cerevisiae. Under depletion of nitrogen and fermentable carbon sources, these yeast produce aggregates of floating cells and form an air–liquid biofilm on the wine surface, which is also known as velum or flor. This behavior is due to genetic and metabolic peculiarities that differentiate flor yeast from other wine yeast. This review will focus first on the most updated data obtained through the analysis of flor yeast with -omic tools. Comparative genomics, proteomics, and metabolomics of flor and wine yeast strains are shedding new light on several features of these special yeast, and in particular, they have revealed the extent of proteome remodeling imposed by the biofilm life-style. Finally, new insights in terms of promotion and inhibition of biofilm formation through small molecules, amino acids, and di/tri-peptides, and novel possibilities for the exploitation of biofilm immobilization within a fungal hyphae framework, will be discussed.

A biofilm-forming strain of Pichia fermentans proved to be most effective in controlling brown rot on apple fruit when coinoculated into artificial wounds with a phytopathogenic isolate of Monilinia fructicola. Culture filtrates and autoclaved cells had no significant influence on the disease. When sprayed onto the apple fruit surface, this yeast formed a thin biofilm but failed to colonize the underlying tissues. When inoculated into wounds artificially inflicted to peach fruit or when sprayed onto the surface of peach fruit, the same strain showed an unexpected pathogenic behaviour, causing rapid decay of fruit tissues even in the absence of M. fructicola. Both optical and scanning electron microscopy were used to evaluate the pattern of fruit tissue colonization by P. fermentans. While on apple surface and within the apple wound the antagonist retained its yeast-like shape, colonization of peach fruit tissue was always characterized by a transition from budding growth to pseudohyphal growth. These results suggest that pseudohyphal growth plays a major role in governing the potential pathogenicity of P. fermentans, further emphasizing the importance of a thorough risk assessment for the safe use of any novel biocontrol agent.