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 mammalian oocytes, meiosis arrests at prophase I. Meiotic resumption requires activation of Maturation-Promoting Factor (MPF), comprised of a catalytic Cyclin-dependent kinase-1 (Cdk1) and a regulatory subunit cyclin B and results in germinal vesicle breakdown (GVBD). Cyclic AMP (cAMP)-mediated Protein Kinase A (PKA) activity sustains prophase arrest by inhibiting Cdk1. However, the link between PKA activity and MPF inhibition remains unclear. Cdc25 phosphatases can activate Cdks by removing inhibitory phosphates from Cdks. Thus one method for sustaining prophase arrest could be inhibition of the activity of the Cdc25 protein required for MPF activation. Indeed, studies in Xenopus identify Cdc25C as a target of PKA activity in meiosis. However, in mice, studies suggest that Cdc25B is the phosphatase essential for GVBD and, therefore, the likely target of PKA activity. To assess these questions, we targeted a potential PKA substrate, a highly conserved serine 321 residue of Cdc25B and evaluated the effect on oocyte maturation. A Cdc25B-Ser321Ala point mutant mRNA induces GVBD when injected into prophase-arrested oocytes more rapidly than wild type mRNA. Using fluorescently-tagged proteins we also determined that the mutant protein enters the nucleus more rapidly than its wildtype counterpart. These data suggest that phosphorylation of the Ser321 residue plays a key role in the negative regulation and localization of Cdc25B during prophase arrest. PKA also phosphorylates a wildtype Cdc25B protein but not a Ser321Ala mutant protein in vitro. Mutation of Ser321 in Cdc25B also affects its association with a sequestering protein, 14-3-3. Our studies suggest that Cdc25B is a direct target of PKA in prophase-arrested oocytes and that Cdc25B phosphorylation results in its inhibition and sequestration by the 14-3-3 protein.

AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals. AMP is believed to control the activity of AMPK by binding to the gamma subunit of this heterotrimeric enzyme. This subunit contains two Bateman domains, each of which is composed of a tandem pair of cystathionine beta-synthase (CBS) motifs. No structural information is currently available on this subunit, and the molecular basis for its interactions with AMP is not well understood. We report here the crystal structure at 1.9 Angstrom resolution of the Bateman2 domain of Snf4, the gamma subunit of the yeast ortholog of AMPK. The structure revealed a dimer of the Bateman2 domain, and this dimerization is supported by our light-scattering, mutagenesis, and biochemical studies. There is a prominent pocket at the center of this dimer, and most of the disease-causing mutations are located in or near this pocket.

The impact of a prerequisite course was examined using both quantitative (exam data analyzed with a “familiarity” scale) and qualitative (surveys and semistructured interviews) data. These data affirmed a recent program change and highlighted the importance of collecting and analyzing student feedback in development of curricula.

Saccharomyces cerevisiae flor yeasts, which are subjected to stressful conditions during wine ageing, exhibit a number of characteristics which distinguish them from non-flor S. cerevisiae wine strains. In the present work, 22 flor and 14 non-flor S. cerevisiae wine strains are compared, in order to elucidate other possible peculiarities of these yeasts. The results obtained demonstrate that in contrast to the homothallic nature of the non-flor strains, 77% of the flor strains exhibit two variants of a semi-homothallic life cycle. Moreover, the flor-forming ability is shown to be inversely correlated to spore viability and the utilisation of maltose and galactose.

Several mutations in genes involved in Saccharomyces mating type switching may affect the homothallic behaviour in wine yeasts. In this study the semi-homothallic (Hq) segregation of a flor wine yeast strain was analysed. We aimed to understand the molecular basis of this behaviour in a flor autochthonous strain, verifying the MAT locus status by a PCR-based HO gene disruption and sequencing of the Y region of the HML, HMR and MAT loci, after nested PCR. Presence of ORFs a1 and a2 in the Y region of the HML locus was found. At the ORF a2 at HML locus, a mutation in the stop codon was found, so the a2 ORF contains 33 more bases.

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