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Microbial biofilms are undesired in food manufacturing, drinking water distribution systems, and clinical realms. Yeast biofilms are particularly problematic because of the strong capacity of yeast cells to adhere to abiotic surfaces, cells, and tissues. Novel approaches have been developed over recent years to prevent the establishment of microbial biofilms, such as through the use of small molecules with inhibiting and dispersing properties. Here, we studied the inhibitory activity of 11 different amino acids on the biofilm formation ability of three wild-type Saccharomyces cerevisiae strains and the reference strain ∑1278b. Subsequent evaluation of different concentrations of the two most effective amino acids, namely, arginine and cysteine, revealed that they acted in different ways. Arginine prevented biofilm formation by reducing FLO11 gene expression; its addition did not affect cell viability and was even found to enhance cell metabolism (vitality marker) as determined by phenotype microarray (PM) analysis. On the contrary, the addition of cysteine reduced both cell viability and vitality as well as FLO11 expression. Thus, the use of cysteine and arginine as agents against biofilm formation can be diversified depending on the most desired action towards yeast growth.

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., (© 2017 John Wiley & Sons Ltd.)

Flor yeasts of Saccharomyces cerevisiae have been extensively studied for biofilm formation, however the lack of specific haploid model strains has limited the application of genetic approaches such as gene knockout, allelic replacement and Quantitative Trait Locus mapping for the deciphering of the molecular basis of velum formation under biological ageing. The aim of this work was to construct a set of flor isogenic haploid strains easy to manipulate genetically. The analysis of the allelic variations at 12 minisatellite loci of 174 Saccharomyces cerevisiae strains allowed identifying three flor parental strains with different phylogenic positions. These strains were characterized for sporulation efficiency, growth on galactose, adherence to polystyrene, agar invasion, growth on wine and ability to develop a biofilm. Interestingly, the inability to grow on galactose was found associated with a frameshift in GAL4 gene that seems peculiar of flor strains. From these wild flor strains, isogenic haploid strains were constructed by deleting HO gene with a loxP-KanMX-loxP cassette followed by the removal of the kanamycin cassette. Haploid strains obtained were characterized for their phenotypic and genetic properties and compared with the parental strains. Preliminary results showed that the haploid strains represent new tools for genetic studies and breeding programs on biofilm formation., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The deliberate inoculation of yeast strains isolated from food matrices such as wine or bread, could allow the transfer of novel properties to beer. In this work, the feasibility of the use of baker's yeast strains as starters for craft beer production has been evaluated at laboratory and brewery scale. Nine out of 12 Saccharomyces cerevisiae strains isolated from artisanal sourdoughs metabolized 2 % maltose, glucose and trehalose and showed growth rates and cell populations higher than those of the brewer's strain Safbrew-S33. Analysis of allelic variation at 12 microsatellite loci clustered seven baker's strains and Safbrew-S33 in the main group of bread isolates. Chemical analyses of beers produced at a brewery scale showed significant differences among the beers produced with the baker's strain S38 or Safbrew-S33, while no significant differences were observed when S38 or the brewer's strain Safbrew-F2 was used for re-fermentation. The sensory profile of beers obtained with S38 or the brewer's yeasts did not show significant differences, thus suggesting that baker's strains of S. cerevisiae could represent a reservoir of biodiversity for the selection of starter strains for craft beer production.

Flor yeasts of Saccharomyces cerevisiae have an innate diversity of Flo11p which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling Flo11p alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce Flo11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to Flo11p expression. Accordingly, L-histidine did not affect the viability of the Δflo11 and S288c strains. Also, L-histidine dramatically decreased air-liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the Flo11p gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts [corrected].

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., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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., (Copyright © 2011 John Wiley & Sons, Ltd.)

During fermentation of high-sugar-containing medium lacking lipid nutrients, wine yeasts undergo oxidative stress and oxidative damage to cell membranes and proteins. Considering that cell membranes are important stress sensors, and that under hypoxic conditions wine yeasts modulate cell membranes composition by incorporating lipids available in the growth medium, in the present work, the effects of lipid nutrition on wine yeast oxidative stress response were evaluated on two strains of Saccharomyces cerevisiae. Biomarkers of oxidative stress, oxidative damage and antioxidant response were evaluated together with viability and acetic acid production during fermentation of a synthetic must lacking lipid nutrients as compared to added oleic acid and ergosterol. The results show that the availability of lipid nutrients causes a significant reduction in the intracellular content of reactive oxygen species and in the oxidative damage to membranes and proteins, as indicated by flow cytometry of cells stained with dihydroethidum (DHE) and propidium iodide (PI) and by Western blot of protein carbonyls. Accordingly, lipid nutrients feeding results in the increase in cell viability and superoxide activity, and the reduction in trehalose accumulation, proteinase A activity and production of acetic acid. In summary, these results are compatible with the hypothesis that the supplementation of lipid nutrients mitigates oxidative stress and oxidative damage in wine strains of S. cerevisiae during growth under unfavourable conditions., (2010 Elsevier B.V. All rights reserved.)

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.

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.

To further elucidate the biosynthesis of lipids in flor strains under fermentative conditions, the transcription levels of the lipid biosynthetic genes ACS1, ACS2, ACC1, OLE1, ERG1, ERG11, ARE1 and ARE2, as well as the lipid composition and cell viability of a flor strain were compared with that of a non-flor strain during hypoxic and aerobic fermentations in the absence of lipid nutrients. While no significant differences in transcription levels or lipid compositions were observed between the two strains when oxygen was not limiting, significant differences were seen during hypoxic fermentation. In this last condition, the flor strain, in spite of higher levels of transcription of hypoxic genes, lost the abilities to desaturate fatty acids and complete ergosterol biosynthesis, and showed a dramatic loss of viability. In contrast, the non-flor strain, which showed lower transcription levels, was able to reach a balanced lipid composition and maintained a higher cell viability. One possible explanation is that the flor strain requires a higher amount of oxygen than the non-flor strain in order to carry out the oxygen-dependent steps of lipid biosynthesis under fermentative conditions.

A strain of Saccharomyces cerevisiae having desirable winemaking properties and high spore viability was bred from a semi-homothallic parent strain with similar winemaking properties but that produced sixfold fewer viable spores. Because the parent was homozygous for HO and for the MATa allele at both silent HMR and HML loci, it produced two MATa and two nonmating progeny per ascus. To obtain a segregant able to mate with the stable MATa progeny, a strain of the nonmating progeny, previously subjected to HO distruption with a KanMX4 cassette, was used. The resultant MATalphaho::KanMX4 transformant was mated to a MATa HO segregant and the diploid produced was sporulated to allow the isolation of a semi-homothallic diploid segregant designated 2D that lacked the KanMX4-disrupted HO allele as confirmed by sequence analysis. Genetic analysis indicated greater homozygosity in 2D than in the parent as assessed by PCR at five loci. The sugar consumption profiles of both 2D and the parent in grape juice fermentations were the same. Acetaldehyde levels and postfermentation biofilm formation were higher in 2D than in the parent. Because 2D has acceptable winemaking characteristics but produces significantly more viable spores than the parent strain, it will be useful in future breeding efforts.

Aim: To verify a possible correlation between cell lipid composition, expression of key genes in lipid metabolism and fermentative behaviour of Saccharomyces cerevisiae wine strains., Methods and Results: The fermentative abilities of two commercial wine strains of S. cerevisiae were tested under stressful conditions. Cell number, glucose and fructose concentrations, expression of ACS1, ACS2, ACC1, OLE1, ERG9, ERG10, ARE1 and ARE2 and lipid content were evaluated. The strain that failed to complete the fermentation had lower amounts of C16:1 and C16:0 fatty acids at the beginning of fermentation (0 h) and late logarithmic phase (72 h). While the amount of C18:1 in this strain was lower than that in the strain that completed the fermentation at 0 h, same levels were observed for both strains at 72 h. The sterol levels were generally higher in the strain that failed to complete the fermentation. Gene expression generally increased from the beginning of the fermentation to the late logarithmic phase in both strains., Conclusion: A positive correlation between good fermentative ability, elevated fatty acid content and ACC1 gene expression has been identified., Significance and Impact of the Study: The cell lipid content at the time of inoculum and expression of ACC1 gene of starter strains should be carefully considered in order to identify the possible stuck/sluggish fermentations.

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.

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 yeasts are Saccharomyces cerevisiae strains noted by their ability to create a type of biofilm in the air-liquid interface of some wines, known as 'flor' or 'velum', for which certain proteins play an essential role. Following a proteomic study of a flor yeast strain, we deleted the CCW14 (covalently linked cell wall protein) and YGP1 (yeast glycoprotein) genes--codifying for two cell surface glycoproteins--in a haploid flor yeast strain and we reported that both influence the weight of the biofilm as well as cell adherence (CCW14). [ABSTRACT FROM AUTHOR]

Background and Aims Interactions between different yeast species used as starters may lead to inconsistent results in mixed fermentations. The aim of this study was to assess the influence of different nutrients on the association between a wine strain of C andida zemplinina ( CDZ1) and a commercial wine strain of S accharomyces cerevisiae ( EC1118) in mixed culture fermentations. Methods and Results Laboratory-scale fermentations were carried out by inoculating CDZ1 and, after 3 days, EC1118 with the simultaneous addition of diammonium phosphate, yeast hulls, ergosterol and oleic acid, each provided separately. The addition of yeast hulls resulted in a higher cell population of CDZ1 and in a higher glycerol concentration of wine as compared with that of the other fermentations. Pilot-scale fermentations, carried out in a commercial winery, confirmed that the winemaking protocol based on the use of the mixed starters CDZ1/ EC1118 with added yeast hulls results in wine with a higher glycerol content, more structured and persistent. Conclusions Addition of yeast hulls improved the fermentative performance of the mixed starters CDZ1/ EC1118. Significance of the Study The high glycerol content of wines made with the combined use of yeast hulls and CDZ1/ EC1118 mixed starters increases wine softness and body. [ABSTRACT FROM AUTHOR]