Marilena Budroni

FLO11-Based Model for Air-Liquid Interfacial Biofilm Formation by Saccharomyces cerevisiae

ABSTRACT 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.

FLO11 gene length and transcriptional level affect biofilm-forming ability of wild flor strains of Saccharomyces cerevisiae.

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.

HSP12 is essential for biofilm formation by a Sardinian wine strain of S. cerevisiae

Sardinian sherry strains of S. cerevisiae form a biofilm on the surface of wine at the end of the ethanolic fermentation, when grape sugar is depleted and when further growth becomes dependent on access to oxygen. A point mutation in HSP12 or deletion of the entire gene results in inability to form this film. HSP12 encodes a heat‐shock protein previously foundby others to be active during stationary phase, in cells depleted for glucose, and in cells metabolizing ethanol and fatty acids, all conditions associated with sherry biofilms. The DNA sequence of HSP12 allele of strain Ar5‐H12 has GenBank Accession No. AY046957. Copyright

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

ABSTRACT 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.

SED1 gene length and sequence polymorphisms in feral strains of Saccharomyces cerevisiae.

ABSTRACT The SED1 gene (YDR077W), coding for the major cell wall glycoprotein of Saccharomyces cerevisiae stationary-phase cells, contains two blocks of tandem repeat units located within two distinct regions of the nucleotide sequence. A PCR survey of the SED1 open reading frames (ORFs) of 186 previously uncharacterized grape must isolates of S. cerevisiae yielded 13 PCR profiles arising from different combinations of seven SED1 length variants in individuals homozygous or heterozygous for the gene. Comparison of the nucleotide sequences of a group of representatives of each of the seven length variants with those of S288C and the type strain, CBS1171, unequivocally identified them as SED1 alleles and provided evidence for the presence of two minisatellite-like sequences, variable in length, within the ORF of an S. cerevisiae gene. The segregation analyses of the SED1 length variants and other genetic markers in 13 isolates representative of each PCR profile suggested that molecular mechanisms involved in minisatellite expansion and contraction may be responsible for SED1 heterozygosities within a population of homothallic must isolates of S. cerevisiae.

A genetic study of natural flor strains of Saccharomyces cerevisiae isolated during biological ageing from Sardinian wines.

In this study, three flor strains of Saccharomyces cerevisiae were genetically characterized. They were isolated from biofilms on Sardinian sherry‐like wines produced at family‐run wineries where pure cultures of yeasts were not used. The study aimed to investigate the life cycle of these naturally‐occurring flor strains, using a genetic procedure supplemented by analysis of subsequent meiotic generations. A semi‐homothallic life cycle was found in three strains that could be helpful in a genetic improvement programme.

Cellular fatty acid composition in film‐forming strains of two physiological races of Saccharomyces cerevisiae

Eleven strains belonging to two physiological races of Saccharomyces cerevisiae endowed with different abilities of forming films at air‐liquid interfaces were analysed in relation to cell fatty acid composition and cell hydrophobicity. Extensive individual differences in fatty acid profiles were observed both in the film and in the non‐film phase. The ability of the cells to form a floating film seems to be an implicit strain character associated with an elevated unsaturation level and a mean chain length of fatty acid residues, as well as cellular hydrophobicities higher than those shown by non‐film‐forming strains belonging to the same species.

Identification of killer factor in the yeast genus Metschnikowia

SummaryOut of 260Metschnikowia pulcherrima strains isolated from Sardinian grapes and musts, 6 proved to be killer yeasts. Maximal killing effect occurred between 3.6 and 5.2 pH, and with 48h or 72h cultures.

Genomic signatures of adaptation to wine biological ageing conditions in biofilm‐forming flor yeasts

The molecular and evolutionary processes underlying fungal domestication remain largely unknown despite the importance of fungi to bioindustry and for comparative adaptation genomics in eukaryotes. Wine fermentation and biological ageing are performed by strains of S. cerevisiae with, respectively, pelagic fermentative growth on glucose and biofilm aerobic growth utilizing ethanol. Here, we use environmental samples of wine and flor yeasts to investigate the genomic basis of yeast adaptation to contrasted anthropogenic environments. Phylogenetic inference and population structure analysis based on single nucleotide polymorphisms revealed a group of flor yeasts separated from wine yeasts. A combination of methods revealed several highly differentiated regions between wine and flor yeasts, and analyses using codon‐substitution models for detecting molecular adaptation identified sites under positive selection in the high‐affinity transporter gene ZRT1. The cross‐population composite likelihood ratio revealed selective sweeps at three regions, including in the hexose transporter gene HXT7, the yapsin gene YPS6 and the membrane protein coding gene MTS27. Our analyses also revealed that the biological ageing environment has led to the accumulation of numerous mutations in proteins from several networks, including Flo11 regulation and divalent metal transport. Together, our findings suggest that the tuning of FLO11 expression and zinc transport networks are a distinctive feature of the genetic changes underlying the domestication of flor yeasts. Our study highlights the multiplicity of genomic changes underlying yeast adaptation to man‐made habitats and reveals that flor/wine yeast lineage can serve as a useful model for studying the genomics of adaptive divergence.

Flor Yeast: New Perspectives Beyond Wine Aging

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.