Abstract 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). flor yeast, flor biofilm, Ccw14p, Ygp1p, cell adherence, cell surface hydrophobicity INTRODUCTION Flor yeasts are Saccharomyces cerevisiae strains known in the enological field due to their ability to form a type of biofilm in the wine–air interface, known as ‘flor’ or ‘velum’, which confers sensorial properties during the elaboration of Sherry-like wines (López-Alejandre 2005; Peinado and Mauricio 2009; Legras et al.2016). During the wine elaboration process, the lack of sugars that have been consumed during the previous fermentation triggers flor yeast to develop the flor in the wine–air interface that allows them to reach an oxygen-rich zone where it is possible to oxidatively catabolize the remaining carbon sources from the fermentation, ethanol or glycerol (Zara et al.2010, 2011; Alexandre 2013). This feature is significantly attributed to a high cell surface hydrophobicity (Martínez, Perez-Rodriguez and Benitez 1997), increased cell buoyancy, yeast lipid content/composition (Zara et al.2009) and the presence of certain proteins such as Flocculin-11 (Flo11p), Batten disease protein 2 (Btn2p), 12 kDa heat shock protein (Hsp12p) and Transcriptional regulator NRG1 (Nrg1p) (Zara et al.2002; Ishigami et al.2004; Espinazo-Romeu et al.2008). Among them, Flo11p plays an essential role. This is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that contributes to the cell rising and hydrophobicity (Reynolds and Fink 2001; Ishigami et al.2004; Zara et al.2005; Fidalgo et al.2006; Purevdorj-Gage et al.2007). Although several traits of the flor formation have been revealed, other aspects like the composition of an extracellular matrix reported by Zara et al. (2009) or the existence of other parietal proteins besides Flo11p involved in the flor formation persist unknown. Moreno-García et al. (2016) investigated the proteome of a flor yeast, and they identified two extensively glycosylated cell surface proteins, Ccw14p (Covalently linked Cell Wall protein) and Ygp1p (Yeast GlycoProtein), that may have a crucial function in the flor formation process. In this work, we evaluated the contribution of proteins Ccw14 and Ygp1 to the biofilm ability of a flor strain using a haploid strain in which the relative ORFs have been deleted, and phenotypes related to the flor biofilm formation were analyzed. MATERIAL AND METHODS Saccharomyces cerevisiae P3-D5 MAT α strain CLIB 1772 (Jura, France), a haploid flor yeast strain was used for the genotypic/phenotypic studies (Coi et al.2016, 2017). Ccw14p and Ygp1p were selected for their ORFs deletion, according to the following election criteria: high concentration under a flor biofilm formation condition (Moreno-García et al.2016) and similarity with Flo11p on the base of hydrophobic character and cell surface location (Fig. 1). Protein hydrophobicity was determined in silico by using the Saccharomyces Genome Database (SGD) and Uniprot databases taking into account the protein hydropathicity index (positively rated proteins being more hydrophobic) and the number of glycosylation sites in the amino acid sequence. FIGURE 1. View largeDownload slide Criteria for the selection of the proteins. FIGURE 1. View largeDownload slide Criteria for the selection of the proteins. Phleomycin markers flanked by sequences homologous to the genes CCW14 and YGP1 were amplified by PCR using oligos shown in Table 1 to construct the deletion cassettes called Ble. The PCR reaction mix (50 μl) contained: 5 μl 10X PCR Buffer-MgCl2 Invitrogen (Carlsbad, California), 2 mM dNTP Mix, 50 mM MgCl2, 10 mM of each construction primer (Table 1), 25 ng pUG66 template DNA, 2.5 U Taq DNA Polymerase Invitrogen. PCR conditions were 94°C for 3 min, 94°C for 45 sec, 55°C for 30 sec, 72°C for 1 min 30 sec (30 cycles), 72°C for 10 min. The PCR product was purified (Qiagen, Inc., Hilden, Germany) and used to transform the strains P3-D5 MAT α using the protocol described by Güldener et al. (1996). Selection of transformants was performed on YPD (1% yeast extract, 2% peptone, 2% glucose and 2% agar) plates containing 150 μg/mL phleomycin. Confirmation of allele deletion was made by PCR using primers indicated in the Table 1 and consisting in a 12.5 μL PCR reaction mix: 1.25 μl 10X PCR Buffer-MgCl2 Invitrogen, 2 mM dNTP Mix, 50 mM MgCl2, 10 mM of each verification primer and 10 μM for Ble1 and Ble 2 (Table 1) (VerFw-VerRv for the first PCR verification, VerFw-Ble1 for the second and VerRv-Ble2 for the third), 15 μg template DNA, 0.625 U Taq DNA Polymerase Invitrogen. PCR conditions were the same as in the cassette construction. The proper insertion of deletion cassettes in place of CCW14 or YGP1 was verified also by Sanger sequencing (GATC-Biotech, Konstanz, Germany sequencing service) using their respective verification primers, after PCR amplification and purification (Qiagen PCR purification kit) of the corresponding regions. DNA homology searches were performed using the Basic Local Alignment Search Tool (BLAST) algorithm through the National Center for Biotechnology Information (NCBI) and SGD. TABLE 1. DNA primers. Primer Sequence Purpose CCW14PhleFw 5΄ CAGCACTACTAGACTCGTTCAACACTCGTTATATA TTATCGTACGCTGCAGGTCGACAAC 3΄ Construction of the CCW14::PHLEO cassette from pUG66 CCW14PhleRv 5΄ GATAGATACCTTAACCCATTAGAAATAAAGTGATAGATAAACTATAGGGAGACCGGCAGA 3΄ Construction of the CCW14::PHLEO cassette from pUG66 CCW14VerFw 5΄ CCAGAATACGACGAGGACGG 3΄ Verification of insertion of CCW14::PHLEO cassette CCW14VerRv 5΄ CCCAGATATGTACCGCCACC 3΄ Verification of insertion of CCW14::PHLEO cassette YGP1PhleFw 5΄ TCTACTGGATTAATCGTCAGTTAAGTAATACAGTAAT AGAAAGTACGCTGCAGGTCGACAAC 3΄ Construction of the YGP1::PHLEO cassette from pUG66 YGP1PhleRv 5΄ AAAGAATCTCTATGCTTCGCTAGATTTAATATCTATCAGTACTATAGGGAGACCGGCAGA 3΄ Construction of the YGP1::PHLEO cassette from pUG66 YGP1VerFw 5΄ CGGCTTCTCGATGCTACAGT 3΄ Verification of insertion of YGP1::PHLEO cassette YGP1VerRv 5΄ AGAAGGGGGTGAGATCCCTT 3΄ Verification of insertion of YGP1::PHLEO cassette Ble1 5΄ GTGGGCGAAGAACTCCAG 3΄ Verification of insertion of PHLEO cassette Ble2 5΄ GTTCTACCGGCAGTGCAAAT 3΄ Verification of insertion of PHLEO cassette Primer Sequence Purpose CCW14PhleFw 5΄ CAGCACTACTAGACTCGTTCAACACTCGTTATATA TTATCGTACGCTGCAGGTCGACAAC 3΄ Construction of the CCW14::PHLEO cassette from pUG66 CCW14PhleRv 5΄ GATAGATACCTTAACCCATTAGAAATAAAGTGATAGATAAACTATAGGGAGACCGGCAGA 3΄ Construction of the CCW14::PHLEO cassette from pUG66 CCW14VerFw 5΄ CCAGAATACGACGAGGACGG 3΄ Verification of insertion of CCW14::PHLEO cassette CCW14VerRv 5΄ CCCAGATATGTACCGCCACC 3΄ Verification of insertion of CCW14::PHLEO cassette YGP1PhleFw 5΄ TCTACTGGATTAATCGTCAGTTAAGTAATACAGTAAT AGAAAGTACGCTGCAGGTCGACAAC 3΄ Construction of the YGP1::PHLEO cassette from pUG66 YGP1PhleRv 5΄ AAAGAATCTCTATGCTTCGCTAGATTTAATATCTATCAGTACTATAGGGAGACCGGCAGA 3΄ Construction of the YGP1::PHLEO cassette from pUG66 YGP1VerFw 5΄ CGGCTTCTCGATGCTACAGT 3΄ Verification of insertion of YGP1::PHLEO cassette YGP1VerRv 5΄ AGAAGGGGGTGAGATCCCTT 3΄ Verification of insertion of YGP1::PHLEO cassette Ble1 5΄ GTGGGCGAAGAACTCCAG 3΄ Verification of insertion of PHLEO cassette Ble2 5΄ GTTCTACCGGCAGTGCAAAT 3΄ Verification of insertion of PHLEO cassette View Large Flo11p-related phenotypes were analyzed in different media. Biofilm formation was observed when yeasts (107 cells/mL) were grown after 5 days under static conditions at 30°C in 24-wells polystyrene microplates with 2 mL 0.67% (w/v) YNB without amino acids and 4% (v/v) ethanol (called YNB 4% ETOH medium); a diluted standard red wine with 4% (v/v) ethanol (red wine 4% ETOH) and a synthetic flor medium (Coi et al.2016) with 4% (v/v) ethanol (SFM 4% ETOH). Flor biofilms were photographed at day 5. For the flor dry weight measurement, 50 mL of medium containing 0.67% (w/v) YNB without amino acids and 3% (v/v) ETOH adjusted to pH 3.5 (YNB 3% ETOH pH 3.5) was inoculated with 6 × 107 cells/mL (Ishigami et al.2004)—to attain a high biomass—and cultivated under static conditions for 7 days at 21°C. Flor biofilm was collected by aspiration and remaining yeasts (non-biofilm forming yeasts) were collected by centrifugation and dry weight was measured. Lastly, for the adherence test, yeasts were grown overnight in SC (0.67% (w/v) YNB without amino acids) with 2% (w/v) glucose, and then in SC + 0.1% glucose and aliquoted in 96-wells microplates during 3 hr under static conditions at 25°C. Cells that adhered to plastic were colored with 100 μL crystal violet (1% w/v). After 30 min wells were washed with sterile water and 100 μL 10% SDS was added to each well. After 30 min, 100 μL of sterile water was added, and plates were photographed and adherence was quantified by measuring absorbance at 570 nm. RESULTS Our analyses show that both P3-D5 Δccw14 and P3-D5 Δygp1 flor biofilms were less visible than that of the wild type P3-D5 when cultured in YNB 4% ETOH (Fig. 2). In the other two studied conditions, differences among mutants and wild-type strain were less pronounced. It should be noted that these two conditions were less similar to the flor velum formation condition used for the proteome analyses (0.67% (w/v) YNB without amino acids, 1% w/v glycerol, 10 mM of glutamic acid and 10% (v/v) ETOH) by Moreno-García et al. (2016). FIGURE 2. View largeDownload slide Test for flor velum formation. Cells were resuspended in 2 mL of medium. The wells were photographed after 3 days of static incubation at 30°C. FIGURE 2. View largeDownload slide Test for flor velum formation. Cells were resuspended in 2 mL of medium. The wells were photographed after 3 days of static incubation at 30°C. Biofilm weight and percentage of total biomass (flor forming/flor forming + non-flor forming yeast weight) measurements indicates significant differences depending on the strain as well (F-test P-value < 0.05) (Fig. 3a and c). The two mutants formed less flor than the wild-type strain. Interestingly, the weight of yeast cells that were not forming flor (Fig. 3b) as well as the total weight (flor + non-flor cells) was higher in the case of the mutants. These results are compatible with the hypothesis that the deletion of the CCW14 and YGP1 did not affect yeast growth while determining a significant decrease of flor formation. FIGURE 3. View largeDownload slide Weight of flor biofilm (a) non-flor forming yeast or free yeast (b) and flor weight/free yeast percentage (c) and yeast adherence measured for yeast strains P3-D5 wild type, P3-D5 Δccw14 and P3-D5 Δygp1. Adherence was quantified by absorbance at 570 nm obtained from the adherence to polystyrene tests. Different letters indicate different homogeneous groups among the three strains with significant differences at 0.05 level according to the F-test. The alphabetical order indicates an increasing value. FIGURE 3. View largeDownload slide Weight of flor biofilm (a) non-flor forming yeast or free yeast (b) and flor weight/free yeast percentage (c) and yeast adherence measured for yeast strains P3-D5 wild type, P3-D5 Δccw14 and P3-D5 Δygp1. Adherence was quantified by absorbance at 570 nm obtained from the adherence to polystyrene tests. Different letters indicate different homogeneous groups among the three strains with significant differences at 0.05 level according to the F-test. The alphabetical order indicates an increasing value. The distinct appearance of flor biofilms and the weight values indicate that both, Ccw14p and Ygp1p, have a role in the process of forming flor. Fig. 3b shows the adherence to plastic of the mutant Δccw14 was significantly lower than in the parental strain (F-test P-value < 0.05). Therefore, Ccw14p is needed to achieve a strong adherence to surfaces. DISCUSSION Cell adhesion is defined as the attachment of a cell, either to another cell or to an underlying substrate such as the extracellular matrix, via cell adhesion molecules. In flor yeasts, adhesion to other cells and other inert surfaces makes the biofilm stable. Flo11p, Btn2p and Hsp12p were also reported to have a role in cell adhesion, more specifically in cell to cell adhesion (Zara et al.2002; Espinazo-Romeu et al.2008; Váchová et al.2011). On the contrary, CCW14 has never been annotated with the cell adhesion phenotype in S. cerevisiae. Covalently linked cell wall protein or Ccw14p shares several traits with the biofilm formation-related protein Flo11p: GPI-anchoring, extensively O-glycosylated mannoprotein and cell surface location (Moukadiri et al.1997; Mrsa et al.1999; Yin et al.2005; Yofe et al.2016). The deletion of CCW14 gene resulted in the decrease of the weight of biofilm formed as well as the cell adherence to polystyrene (Fig. 3). The glycosylated nature of Ccw14p, which contributes to the cell surface hydrophobicity (SGD 2017), together with adherence properties, suggest that this protein could regulate flor biofilm formation on flor yeast in a Flo11p similar fashion. Furthermore, as this protein has been reported to have a role in the cell wall organization (Moukadiri et al.1997), it can be speculated as well that Ccw14p promotes flor formation through the arrangement of constituent parts, assembly or disassembly of the cell wall. Ygp1p, on the other hand, is a cell wall-related secretory extensively N-glycosylated glycoprotein that deletion of its codifying gene revealed a significant decrease in the flor dry weight. However, the presence of this protein did not show influence on the adherence of the cells. As Ygp1p has only been localized in the extracellular region (Curwin, Fairn and McMaster 2009), we hypothesize that this protein may form a portion of the composition of an extracellular matrix not described although its presence has been revealed in flor yeasts (Zara et al.2009) and flocculent yeasts (Beauvais et al.2009). Ygp1p protein is hydrophobic not only because of the presence of attached carbohydrate groups, but also because of the hydrophobicity of the polypeptide chain itself (Uniprot 2017). If compared with Flo11p, the hydropathicity index shows that Ygp1p amino acid sequence is hydrophobic (0.12), while Flo11p is hydrophilic (−0.37). Moreover, in terms of the attached carbohydrates, Ygp1p has 14 glycosylation sites in their amino acid sequence compared to 2 in Flo11p. CONCLUSION Ccw14p and Ygp1p are two proteins that affect flor formation in a medium that simulated Sherry wine elaboration conditions, and they can be regarded as potential targets to promote the flor formation during the Sherry wine elaboration process. Moreover, these two proteins share homology with other proteins synthesized by other yeasts (with annotated genomes available in databases) such as the biofilm forming-pathogen yeast Candida glabrata (Ccw14p shares 72% and Ygp1p 51%); thus, they can also be considered potential targets to prevent biofilm formation in C. glabrata and other pathogenic biofilm yeasts. FUNDING This work was supported by the Spain's Ministry of Economy and Competitiveness and the European Community (FEDER), Grant RTA2011-00020-C02-02, MINECO-INIA-CCAA; ‘XXI Programa Propio de Fomento de la Investigación 2017’ from University of Cordoba; funding from the University of Córdoba: the scholarship ‘Ayuda para la realización de estancias para la obtención de la mención internacional en el título de doctor’ [Jaime Moreno-García]. Conflict of interest. None declared. REFERENCES Alexandre H. Flor yeasts of Saccharomyces cerevisiae—their ecology, genetics and metabolism. Int J Food Microbiol 2013; 167: 269– 75. Google Scholar CrossRef Search ADS PubMed Beauvais A, Loussert C, Prevost MC et al. Characterization of a biofilm-like extracellular matrix in FLO1-expressing Saccharomyces cerevisiae cells. FEMS Yeast Res 2009; 9: 411– 9. Google Scholar CrossRef Search ADS PubMed Coi AL, Bigey F, Mallet S et al. 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Published: Mar 1, 2018
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