International Journal of Food Microbiology 191 (2014) 45–52

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Genetic diversity of FLO1 and FLO5 genes in wine flocculent Saccharomyces cerevisiae strains Rosanna Tofalo, Giorgia Perpetuini, Paola Di Gianvito, Maria Schirone, Aldo Corsetti, Giovanna Suzzi ⁎ Faculty of BioScience and Technology for Food, Agriculture and Environment, University of Teramo, Via C.R. Lerici 1, 64023 Mosciano S. Angelo, Italy

a r t i c l e

i n f o

Article history: Received 28 July 2014 Accepted 23 August 2014 Available online 28 August 2014 Keywords: Flocculation S. cerevisiae Wine FLO genes FLO5 Cell–cell adhesion

a b s t r a c t Twenty-eight flocculent wine strains were tested for adhesion and flocculation phenotypic variability. Moreover, the expression patterns of the main genes involved in flocculation (FLO1, FLO5 and FLO8) were studied both in synthetic medium and in presence of ethanol stress. Molecular identification and typing were achieved by PCR-RFLP of the 5.8S ITS rRNA region and microsatellite PCR fingerprinting, respectively. All isolates belong to Saccharomyces cerevisiae species. The analysis of microsatellites highlighted the intraspecific genetic diversity of flocculent wine S. cerevisiae strains allowing obtaining strain-specific profiles. Moreover, strains were characterized on the basis of adhesive properties. A wide biodiversity was observed even if none of the tested strains were able to form biofilms (or ‘mats’), or to adhere to polystyrene. Moreover, genetic diversity of FLO1 and FLO5 flocculating genes was determined by PCR. Genetic diversity was detected for both genes, but a relationship with the flocculation degree was not found. So, the expression patterns of FLO1, FLO5 and FLO8 genes was investigated in a synthetic medium and a relationship between the expression of FLO5 gene and the flocculation capacity was established. To study the expression of FLO1, FLO5 and FLO8 genes in floc formation and ethanol stress resistance qRT-PCR was carried out and also in this case strains with flocculent capacity showed higher levels of FLO5 gene expression. This study confirmed the diversity of flocculation phenotype and genotype in wine yeasts. Moreover, the importance of FLO5 gene in development of high flocculent characteristic of wine yeasts was highlighted. The obtained collection of S. cerevisiae flocculent wine strains could be useful to study the relationship between the genetic variation and flocculation phenotype in wine yeasts. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Yeast flocculation can be defined as a nonsexual, reversible (flocs can be reversible dispersed by the action of EDTA or specific sugars, like mannose) and multivalent process of aggregation of yeast cells into multicellular masses (composed by thousands or even millions of cells), called flocs, with the subsequent rapid sedimentation from the medium in which they are suspended (for a review see Soares (2011)). For this reason flocculation is an attractive property for yeast since it provides an effective, environment-friendly, simple and costfree way to separate yeast cells from the culture broth at the end of fermentation (Li et al., 2013a). The use of flocculent cells is desirable for the elaboration of sparkling wines since they allow a rapid clarification reducing the production costs (less energy consumed, becoming a ‘greener’ process) (Pretorius and Bauer, 2002). Moreover, yeast flocculation seems to be associated with the enhancement of ester production (Soares, 2011).

⁎ Corresponding author. Tel.: +39 0861266938; fax: +39 0861266940. E-mail address: [email protected] (G. Suzzi).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.08.028 0168-1605/© 2014 Elsevier B.V. All rights reserved.

Moreover, flocculation is also a cooperative protection mechanism that shields cells from stressful environments (Smukalla et al., 2008). In Saccharomyces cerevisiae specific cell surface proteins, referred as flocculins, Flo proteins or yeast adhesins are involved in flocculation. Adhesin-mediated phenotypes other than flocculation (Bester et al., 2012) include agar adhesion and/or invasion (Guo et al., 2000; Verstrepen and Klis, 2006), the formation of pseudohyphae (Lambrechts et al., 1996; Lo and Dranginis, 1998) or biofilms (Purevdorj-Gage et al., 2007), the adherence to plastic surfaces (Mortensen et al., 2007), colony morphology (Kuthan et al., 2003), as well as “flor”/“velum” formation that occurs during the aging of sherry and Vernaccia (Budroni et al., 2000; Fidalgo et al., 2006). Flocculins are encoded by unlinked dominant FLO genes, FLO1, FLO5, FLO8, FLO9, FLO10, FLO11, FLONS, FLONL and Lg-FLO1 (Govender et al., 2008; Liu et al., 2007; Verstrepen et al., 2004). FLO1, FLO5, FLO9 and FLO10 are subtelomeric genes while FLO11 (also known as MUC1) gene is the only non subtelomeric FLO family member. FLO11 is required for the formation of pseudohyphae (Lambrechts et al., 1996; Lo and Dranginis, 1998), “flor” formation (Budroni et al., 2000), “mat” formation (also referred as “biofilm formation” or “yeast sliding motility”) (Reynolds and Fink, 2001), as

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well as flocculation in S. cerevisiae var. diastaticus (Bayly et al., 2005). FLO1 is the most studied gene associated to flocculation and its regulation and expression is well known (Bester et al., 2006; Liu et al., 2007). Genetic variability related to the number of tandem repeats in this gene is responsible of the flocculation degree of yeast strains: longer repeats are associated to stronger flocculation ability (Liu et al., 2007; Sato et al., 2001). Verstrepen et al. (2005) demonstrated that size variation of FLO1 gene induced phenotypic alterations of adhesion, flocculation and biofilm formation. Recently, some authors suggested that flocculation protects the FLO1 expressing cells from environmental stresses (Verstrepen and Klis, 2006). Proteins encoded by these FLO genes share a common modular organization that consists of three domains as reported by Li et al. (2013a): an amino-terminal lectin domain responsible for the binding to carbohydrate, a central domain, and a carboxyl-terminal domain containing a glycosyl phosphatidylinositol anchoring sequence (Bauer et al., 2010). The central domain contains many tandem repeat regions of DNA sequence that can drive slippage and recombination reactions within and between FLO genes, with the generation of novel FLO alleles, conferring to yeast cells diversity and variety in flocculation ability (Verstrepen et al., 2004, 2005). FLO gene regulation and the contributing factors are far from being completely understood by winemakers and brewers limiting the control of flocculation during fermentations. Flocculent wine yeasts are rare and it was found that the frequency of strains displaying flocculation among wild Saccharomyces yeasts isolated from grape musts was low (Suzzi et al., 1984). Investigations have been made in the construction and improvement of flocculating enological strains (Govender et al., 2010; Romano et al., 1985). Those studies revealed the importance of selecting and constructing useful flocculent yeasts to facilitate yeast separation at the end of fermentation by settling, centrifugation or filtration. Coloretti et al. (2006) obtained inter-specific hybrids by crossing flocculent S. cerevisiae strains with non-flocculent strains of Saccharomyces bayanus, and the new strains produced sparkling wines with even better aromatic characteristics. Efficient wine yeast flocculation after primary alcoholic fermentation leads to the formation of compacted sediments or ‘caked’ lees, thereby reducing the handling of wines and minimizing problems associated with wine clarification (Pretorius and Bauer, 2002). Moreover flocculation is a very important process in the production of sparkling wines that undergo fermentation in bottles by the traditional method. Many studies on yeast cell flocculation have been carried out on laboratory strains such as S288c and on brewer's yeasts, but our current knowledge on the natural diversity of FLO genes in particular in wine yeasts is still very limited. In the present study we investigated 28 S. cerevisiae flocculent strains isolated about 30 years ago from different vitivinicultural areas of Northern Italy in order to select strains to be used for sparkling wine production. In particular, we focused on the adhesion and flocculation phenotypic variability and genetic variability of some FLO genes. Moreover, in some strains, selected on the basis of their flocculation degree, the expression patterns of FLO1, FLO5 and FLO8 were investigated in a synthetic medium and in presence of ethanol stress. 2. Materials and methods 2.1. Strain origin Twenty-eight S. cerevisiae strains belonging to the Department of Scienze e Tecnologie Agro-Alimentari of Bologna University were used (Table 1). They were isolated from musts and wines of North Italy as reported in a previous study (Suzzi and Romano, 1991) and they were identified in this study as described below. All isolates were routinely grown in YPD medium (1% w/v yeast extract, 2% w/v peptone, and 2% w/v glucose) for 48 h under aerobic conditions. All S. cerevisiae isolates were stored at −80 °C in YPD broth supplemented with glycerol (20%

Table 1 Set up of flocculation and adhesive properties of 28 S. cerevisiae wine strains. Strains

Flocculation degree 1991 2013

Type of flocculent yeast

Adherence Mat Invasive to formation growth polystyrene

–a Nb Fc N F F – F F N – – N – F F N N N N F F F – N – – F

− + − + + + − + + − − + + + + + + − + + − + + + − + + −

2 d 15 d 22 d FL243 FL408 F612 F1047 F6000 F6030 F6341 F6789 F6971 F7030 F7039 F7101 F7309 F7635 F10008 F10238 F10641 F10471 F10477 F10517 F10599 F10717 F10721 F10790 F10820 F10826 F10846 F10876

3 2 3 4 3 3 2 4 3 2 3 3 4 3 4 3 4 4 4 2 3 3 3 2 3 4 3 3

0 0 3 4 1 4 0 5 3 0 0 0 0 0 4 2 0 0 0 0 2 3 2 0 0 0 0 3

0 0 3 4 1 4 0 5 3 0 0 0 0 0 5 2 0 0 4 1 2 3 2 0 0 0 0 3

0 1 3 5 1 4 0 5 3 1 0 0 1 0 5 3 2 4 4 2 5 3 3 0 1 0 0 3

− − − − + − + − + + − − − − + + − − − − − − − − − − − +

+ − − + + − + + + + + − + − + + − − + + + − − − − − + +

Legend: all strains were negative for pseudohyphal and biofilm formation. a Non flocculent yeast. b NewFlo-type. c Flo type.

v/v final concentration) (Sigma-Aldrich, Milan, Italy) or on YPD agar at 4 °C for short-term storage.

2.2. Yeast identification Yeast cells were grown overnight at 28 °C aerobically in 5 mL YPD medium and DNA was subsequently extracted according to Querol et al. (1992). The 5.8S internal transcribed spacer (ITS) rRNA region was amplified in a Bio-Rad thermocycler (MyCycler, Bio-Rad Laboratories, Milan, Italy) using primers ITS1 and ITS4 and restriction enzymes CfoI, HaeIII and HinfI as described previously (Esteve-Zarzoso et al., 1999; Tofalo et al., 2009).

2.3. Microsatellite PCR fingerprinting Microsatellite PCR fingerprinting was carried out according to Vaudano and Garcia-Moruno (2008). A multiplex amplification of three microsatellite loci (SC8132X, YOR267C and SCPTSY7) was performed in 25 μL PCR reaction containing 1 × PCR buffer, 3.1 mM MgCl2, 2 U Taq polymerase (Invitrogen, Milan, Italy), 10 pmol of each primer for locus YOR267C, 15 pmol of primers for locus SC8132X and 40 pmol of primers for locus SCPTSY7. Amplification was performed on a Perkin-Elmer GeneAmpPCR System 2400 with an initial denaturation at 94 °C for 4 min followed by 28 cycles of 30 s at 94 °C, 45 s at 56 °C, 30 s at 72 °C and, finally, 10 min at 72 °C. The products were run on a 2.5% (w/v) agarose gel 1 × TAE buffer at 100 V for 80 min. Gels were stained with ethidium bromide. Two lines of 1-kb plus DNA ladder (Invitrogen) were used as molecular weight and normalization gel standards for microsatellite profiles.

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2.4. Cell–cell adhesion 2.4.1. Flocculation assay Flocculation assay was carried out as previously described by Suzzi and Romano (1991) with minor modifications. Cells were grown in 5 mL of yeast nitrogen base (YNB, Difco Laboratories, Detroit, Mich., USA) at 28 °C. Flocculation ability, scored by eye and compared with appropriate controls, was graded on a scale from 0 (non flocculent) to 5 (very flocculent), after 2, 15 and 20 days of incubation. 2.4.2. Air–liquid interfacial biofilm formation Formation of an air–liquid interfacial biofilm was performed according to Zara et al. (2005). Briefly, strains were grown in YPD broth overnight at 28 °C. Cells were washed and 10 μl were suspended in 5 ml YNB (Difco) supplemented with ethanol 4% v/v. Strains were incubated at 28 °C for 8 days under static conditions. Biofilm formation was evaluated daily. All analyses were performed in duplicate. 2.4.3. Invasive growth assay Invasive growth assay was performed as previously described (Roberts and Fink, 1994; Zara et al., 2005) with some modifications. Yeast strains were grown in YPD plates for 24 h at 28 °C. Yeast cells were isolated on YPD plates using a toothpick and incubated for 3 days at 30 °C and for 2 days at room temperature. Plates were then washed with sterile distilled water to remove cells from the agar surface, leaving subsurface cells that had effectively invaded the agar. Plates were photographed. All analyses were performed in duplicate.

47

Italy) for sequencing. The obtained sequences were compared with those available at the National Center for Biotechnology Information (NCBI) using BLASTN (Altschul et al., 1990) available at http://www. ncbi.nlm.nih.gov/BLAST/. 2.6. FLO1, FLO5, FLO8 genes expression in synthetic medium In order to evaluate FLO1, FLO5 and FLO8 genes expression F1047, F10471, F6030, F6789 and F10008 strains, having different degrees of flocculation, two strains which had lost the flocculation ability (F7039, F10826) and a non-flocculent strain (RT73) were grown in YNB at 28 °C for 5 days. Cells were harvested in the stationary phase and washed with 250 mM EDTA in order to ensure deflocculation as described by Suzzi et al. (1984) and gene expression was determined as reported below. 2.7. Stress treatments The same strains were developed in YNB for 5 days and then subjected to ethanol stress to evaluate FLO1, FLO5 and FLO8 genes expression. After deflocculation, strains were exposed to ethanol 20% (v/v) for 30 min to 1 h. Before and after the stress treatments appropriate dilutions of cells were plated on YPD, in triplicate. In all cases a control was carried out with cells unaffected by stress. Plates were incubated at 30 °C until the appearance of colonies (1 to 3 days). 2.8. qRT-PCR

2.4.4. Yeast adherence to polystyrene and MAT formation Yeast adherence to polystyrene and MAT formation was evaluated as described by Reynolds and Fink (2001). In order to assess the ability of yeast strains to adhere to polystyrene cells were grown overnight at 30 °C in YPD and harvested at an optical density (OD600nm) of 0.5–1.5. Cells were washed once in sterile water and resuspended to 1.0 OD600nm in YPD. For each strain, 100 μl of the suspension which was inoculated into individual wells of polystyrene 96-well plates YPD broth containing no inoculum was used as a negative control. The plates were incubated at 30 °C for 0–1–3–6 h. Cells adhering to polystirene were visualized by staining with crystal violet (1% w/v). Wells were rinsed three times with sterile distilled water and photographed. To evaluate MAT formation strains were inoculated onto YPD soft agar plates (0.3% agar) with a toothpick and incubated at 25 °C for 15 days. The plates were photographed after 7 and 15 days. Plates containing 2% (w/v) agar were used as negative controls. All analyses were performed in duplicate. 2.4.5. Pseudohyphal filament formation Strains tested for pseudohyphal filament formation were streaked on YNB without amino acids and ammonium sulfate, supplemented with 2% glucose and appropriate auxotrophic requirements (Lo et al., 1997). After 1 week of incubation at 30 °C plates were photographed. Analyses were performed in duplicate.

Total RNA from 10 ml of culture was extracted using the Tri reagent method (Sigma) according to the manufacturer's instructions. For reverse transcription-PCR (RT-qPCR) the absence of contaminating DNA was checked by PCR directly on the RNAs. When necessary, the DNase treatment was repeated. DNase-treated RNA samples were stored at − 80 °C until use. RNA concentration was determined spectrophotometrically (Gene Quant pro RNA/DNA Calculator, Amersham Pharmacia Biotech, Piscataway, N.J.). For each sample, 1 μg of total RNA was retrotranscribed into cDNAs using the iScript™ cDNA Synthesis Kit (Bio-Rad, Milan Italy), according to the manufacturer's instructions. Amplification, detection, and real-time analysis were performed using an iCycler IQ real-time PCR Detection System (Bio-Rad). Each reaction mixture (25 μl) contained 12.5 μl 2XIQ SYBR Green PCR SupermixTM (Bio-Rad), 0.2 μM of each primer (Invitrogen) and ultrapure water. 5 μl of cDNA (100 ng/μl) was added to each reaction mixture. FLO1, FLO8 genes and FLO5 gene expression were detected using the primers and conditions described by Van Mulders et al. (2009, 2010), respectively. The calculation of relative transcript levels (RTLs) was carried out as previously described (Govender et al., 2008). ACT1 and FBAI were used as reference genes for all qRT-PCR analyses (Cankorur-Cetinkaya et al., 2012; Smukalla et al., 2008). 3. Results and discussion

2.5. Genetic diversity of FLO1 and FLO5 genes

3.1. Strains identification and typing

Intragenic repetitive domain polymorphism in FLO1 (FLO1-reps-F 5′-CTAAGTCAATCTAACTGTACTGTCCCTGA-3′ FLO1-reps-R 5′-GATAGA GCTGGTGATTTGTCCTGAA-3′) and FLO5 (FLO5-reps-F 5′-AAGGGTACGT TTACTCTTTTGACGATGACC-3′ FLO5-reps-R 5′-ACTGAAGAAGAAATTACT GAGGAGGAAATC-3′) genes of S. cerevisiae was evaluated according to Verstrepen et al. (2005). PCR products were visualized on a 2% agarose gel and acquired using the Gel Doc 2000 (Bio-Rad, Milan, Italy). The bands were excised and put in distilled water and purified using GFX ™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences AB, Uppsala, Sweden), according to the manufacturer′s instructions. The samples were delivered to BMR Genomics (Padua University, Padua,

The majority of the studies dealing with S. cerevisiae flocculation and adhesive properties have been carried out on laboratory strains, such as S288c, or brewery yeasts (Govender et al., 2008). In this study, 28 diploid flocculent S. cerevisiae strains isolated from musts and wines of North Italy (Suzzi and Romano, 1991; Suzzi et al., 1984) about 30 years ago were subjected to molecular characterization using PCRRFLP and microsatellite analysis. PCR-RFLP analysis confirmed that all 28 isolates belong to S. cerevisiae species (data not shown). All the isolates were differentiated at strain level by microsatellite assay. In the majority of strains three bands were present; however, a number of fragments, varying from one to two for each locus, generated from

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R. Tofalo et al. / International Journal of Food Microbiology 191 (2014) 45–52

three to five bands. Relatedness between yeast strains was correlated with the similarity of the resulting electrophoretic patterns as shown in dendrogram (Fig. 1). The repeatability of the assay was 95% as measured by the correlation index between replicates of the same strain. It was possible to identify 2 main clusters using an arbitrary similarity level of 30% and the first one was made of the majority of strains. Considering a higher similarity level of 90% some other clusters, generally consisting of a single strain, were distinguishable. In particular, some strains (e.g. F10471, F10517 and F7309, F10721) showed a very similar profile even if they were isolated from fermented musts obtained with grapes from vineyards located in different North Italy areas. Strains of wine S. cerevisiae with high similarity level are quite common and have been reported by several authors (Schuller et al., 2005; for a review see Tofalo et al., 2013). 3.2. Cell–cell adhesion types It is well known that flocculation is a variable phenotype and during successive generations, flocculation tends to decrease, while other properties remain generally unchanged (Sato et al., 2001). Table 1 shows a long-term study of flocculation of the 28 wine S. cerevisiae. Only 8 strains completely lost flocculation ability, whereas in other strains the capacity of flocculate remained unchanged, decreased or increased, confirming the great variability and instability of this phenotype. This variability has been partially related to unstable tandem repeat sequences in the FLO1

100

80

60

40

Similarity (%)

F10471 F10517 F10641 F612 F10477 FL243 F10846 F1047 F7309 F10721 F10820 F10008 F6789 F7101 F10876 F10717 F10790 F10826 FL408 F10599 F10238 F6030 F6341 F6971 F7039 F7030 F6000 F7635 Fig. 1. Dendrogram showing the similarity relationship among microsatellite profiles of yeast isolates. Similarities were calculated using Unweighted Pair-Group Method with Average algorithm.

gene (Verstrepen and Klis, 2006). Expansion or contraction of the number of repeated DNA units results in stronger or weaker flocculation ability, respectively (Verstrepen et al., 2005). Generally, mutation rates of repeated DNA units are at least 100-fold greater than the average (point) mutation rates (Smukalla et al., 2008). In S. cerevisiae, lectin-like adhesion is divided into two sub-categories: Flo1 phenotype strains specifically inhibited by mannose derivatives and NewFlo phenotype strains inhibited by mannose, maltose, glucose and sucrose (Masy et al., 1992; Stratford and Assinder, 1991). To determine the carbohydrate recognition of the various adhesins, carbohydrates, at different concentrations, were added to the flocculation cultures and the effect of these sugars on the disruption of flocs through competitive inhibition of the Flo proteins was investigated. The wine yeasts showed both phenotypes (Flo1 and NewFlo) regardless of the degree of flocculation (Table 1). Flo1 phenotype strains were constitutively flocculent, whereas NewFlo phenotype strains grew and fermented as single cells showing a flocculent behavior only after 15 or 22 days. Similar results regarding the NewFlo phenotype were also reported by Sampermans et al. (2005) and Stratford and Carter (1993). A standard plate-washing assay showed particular differences between the adherences on agar of the different strains (data not shown). Several strains adhered more strongly to the agar and neither rinsing nor rubbing were able to remove the cells from the agar surface. Upon close inspection, it became clear that these cells had invaded the agar surface. S. cerevisiae can also adhere to surfaces, a process in which FLO11 was shown to play a major role (Reynolds and Fink, 2001). In our study, the tendency to form biofilms (or ‘mats’), or adhere to polystyrene was generally absent. However, some strains formed different MAT independently of flocculation degree (Fig. 2). Adhesion-encoding genes cannot be constitutively expressed and are activated by diverse environmental triggers like carbon and/or nitrogen starvation or ethanol level, conditions which are common in sparkling wine production (Sampermans et al., 2005; Verstrepen et al., 2003).

3.3. Genetic diversity of FLO1 and FLO5 genes Except for the FLO genes in the model yeast strains such as S288c, our current knowledge on the natural diversity of FLO genes is still very limited. So far little research has been dedicated to the genetic diversity of the flocculating genes from different yeast strains and in particular in wine yeasts. As reported in Fig. 3A FLO1 gene showed polymorphism with four variants. All the strains had a band at 220 bp whereas one strain showed an additional band at 500 bp (F10717), 2 strains at 650 bp (F6341, F10461), 3 strains at 750 bp (F7309, F7030, F7039) and 4 strains at 850 bp (FL243, F10471, F10517, FL408). However similar banding patterns did not correspond to the same degree and phenotype of flocculation. In fact, strains F7030, F10820 and FL408 that expressed the same degree of flocculation and phenotype showed three different patterns. In the same way strains F10846 and F10876 which had only a band at 220 bp possessed different phenotypes and degree of flocculation. In laboratory and brewery strains flocculation degree is positively related to the number of repeat sequences of FLO1 gene, which is unstable in genetics and evolves in nature rapidly (Verstrepen et al., 2005). Contrary to all expectations the wine yeast strains studied in this paper possessing the same repeat sequence profiles had a different flocculation degree or were among the strains that lost the capacity of flocculation, such as strain F7039. These data are firstly reported in different flocculent wine yeasts and are in agreement with those reported by Li et al. (2013a) who found that FLO1 allele in a flocculent strain had fewer tandem repeats of an intragenic domain than those in the nonflocculent strains. FLO genes were amplified also in strains without the flocculation phenotype (Zhao et al., 2012) and FLO1 did not necessarily confer flocculation to its carriers (Li et al., 2013b). Different profiles and biodiversity were detected also for wine yeast FLO5 gene (Fig. 3B). The

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Fig. 2. Mat formed on soft agar by F6971, F6000, F10238, F10876, and F7030 S. cerevisiae wine strains after incubation at 25 °C for 15 days.

M F6000 F7635 F10238 F6341 F10721 F7101 F10461 F10790 F6789 F612 F6030 F10599 F10846 M

A

other authors (Van Mulders et al., 2010; Zhao et al., 2012). However, template quality, PCR conditions, and primer sequences can also affect the PCR amplification results. After that, the PCR products of FLO1 and FLO5 were sequenced in order to confirm their specificity and the obtained results are reported in Tables 2A and B. The 220 bp band of FLO1 gene had a high degree of homology (100%) with S. cerevisiae strain KV361 FLO1/YAR062W fusion protein gene. This result is in agreement with Verstrepen et al. (2005). In fact these authors found 2 strains containing a FLO1 allele formed by the fusion of

M FL243 F10471 F10517 F10008 F10876 F1047 F7309 FL408 F10717 F6971 F7030 F10826 F10820 F7039 F10477 M

obtained results suggested that also in wine strains there are molecular mechanisms more complex that those involved in FLO1 gene regulation. The wine flocculent strains analyzed in this study showed smaller repeated region amplicons than brewery strains (Verstrepen et al., 2005; Zhao et al., 2012). Similar results were obtained also by Govender et al. (2010) for two commercial wine strains (VIN13 and EC1118). PCR products were not obtained for all samples. The failure to amplify the FLO genes in some strains may be due to the presence of a high number of repeated sequences in the FLO genes and to the differences in sequences at the primer binding sites as proposed also by

2000 bp 1650 bp 1000 bp 850 bp 500 bp

M F6000 F10820 F10461 F10517 FL408 F7309 F10599 F10846 F7101 F6971 M

B

M F10876 F612 F6030 F10826 F10471 F10790 F10721 F10238 F6789 F10717 F7635 F10008 F1047 F7030 F6341 F10477 Fl243 F7039 M

200 bp

2000 bp 1650 bp 1000 bp 850 bp 500 bp 200 bp

Fig. 3. FLO1 (A) and FLO5 (B) repeated sequences of the 28 wine S. cerevisiae flocculent strains. M: molecular marker.

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Table 2 Sequence analysis of bands obtained for FLO1 (A) and FLO5 (B) genes. A Strains

Band size (bp)

Taxon

Identity (%)

Accession number

F7101, FL243, F10471, F10517, F7309, FL408, F7030, F7039, F6341 F7101, F7039, F10517, F10471, F1047, F6030, F6789, F10461, F10599, F10008

500-850

220

S. cerevisiae YK gene FLO1 S. pastorianus FY-2 Lg-FLO1 S. cerevisiae S288c Flo1p S. cerevisiae S288c Flo9p S. cerevisiae strain KV361 FLO1/YAR062W fusion protein gene

96 96 90 90 100

JF909348 AB288349 NM_001178230 NM_001178205 DQ029325

Strains

Band size (bp)

Taxon

Identity (%)

Accession number

F7101

650 bp

F10471 F7039

1000 bp 850 bp

S. cerevisiae S288c Flo5p S. cerevisiae strain YK flocculin protein FLO1S gene S. cerevisiae S288c Flo5p S. pastorianus FY-2 Lg-FLO1

95 91 93 91

NM_001179342 JF909349 NM_001179342 AB288349

B

the first repeat unit of FLO1 with a repeat unit of FLO1 pseudogene YAR062W. The bands with a higher molecular weight corresponded to the FLO1 gene of S. cerevisiae and Saccharomyces pastorianus with a homology of 96%, but partially aligned also with the FLO9 gene (90%) which shared a similarity level of 94% with FLO1. Regarding FLO5, the primers used also amplified fragments of FLO1 gene. It is probably due to the fact that FLO1 gene shows a similarity of 96% and 94% with FLO5 and FLO9 gene sequences, respectively. Probably these primers developed to easily highlight the tandem repeats containing more than 40 nucleotides in brewers strains, are not specific for wine yeasts. 3.4. FLO1, FLO5 and FLO8 genes expression in synthetic medium In this part of the study the expression of FLO1, FLO5 and FLO8 genes of 5 strains with different levels of flocculation (F1047, F10471, F6030, F6789, F10008), two strains which had lost this phenotype (F10826, F7039) and a control non flocculent strain (RT73) was investigated in a synthetic medium (Fig. 4). FLO8 gene encodes a transcriptional activator of FLO1 and FLO11 (Kobayashi et al., 1999; Pan and Heitman, 1999; Rupp et al., 1999). Moreover, Kobayashi et al. (1999) suggested that 8 FLO8 7

Fold change

6

FLO1 FLO5

3.5. Stress resistance of wine flocculent S. cerevisiae strains

5 4 3 2 1 0

this gene could play a role in the invasiveness of yeast controlling the expression of some other genes that in turn alter the budding pattern, cell shape, cell polarization or other cell-surface or extracellular proteins. A part the control strain RT73, all the tested strains expressed FLO genes. FLO8 gene expression showed no difference in the tested strains. Differences were observed for FLO1 gene expression between flocculent strains and those that lost flocculation capacity. The lowest levels of expression were detected for F10471 and F6789 which showed a strong flocculation ability (scored as 4), suggesting that in wine yeasts a direct relationship between flocculation ability and FLO1 gene expression doesn't exist. Li et al. (2013b) reported that expressed FLO1 gene does not necessarily confer clumping phenotype to its carriers and demonstrated that FLO1 expression may be not the biomarker for flocculent phenotype. Other genes such as AMN1 (governing the behavior of daughter cells) and RAG1 (governing the cellular development) were found to be a more genetic determinant than FLO1 in controlling expression of flocculation (Li et al., 2013b). FLO5 gene showed a higher expression level than FLO1 in all wine strains and a strong correlation between the expression of this gene and the flocculation capacity was established; in fact the non flocculent strains had the lowest values of FLO5 expression. Govender et al. (2010) highlighted that irrespective of the promoter involved and contrarily to observations in the laboratory strain, FLO5-based constructs were observed to induce flocculation more efficiently than FLO1-based constructs in the wine yeast strains.

RT73

F10826

F7039

F10008

F6789

F10471

F6030

F1047

Fig. 4. Relative qRT-PCR expression of FLO1, FLO5, and FLO8 transcripts in YNB. The relative expression value for each sample was defined as 2−Ct(target) where Ct(target) represents the cycle number at which a sample reaches a predetermined threshold signal value for the specific target gene. Relative expression data were normalized to the relative expression value of the reference genes ACT1 and FBA1 in each respective sample. Values represent the means of experiments performed in triplicate, and error bars represent standard deviations.

A selection strategy is necessary in order to obtain a wide range of suitable strains for winemaking, capable of withstanding the adverse conditions that may occur during fermentation. In particular, during traditional sparkling wine production that involves flocculent strains, the secondary fermentation of base wine produces high alcohol content (about 10% v/v) and low fermentation temperatures (12–18 °C) and under these stress conditions, only few S. cerevisiae can grow. Flocculation has been reported as a social trait that can confer both benefits (i.e., protection from stress) and costs (i.e., slower growth due to the burden of FLO1 expression) (Smukalla et al., 2008). To investigate the role of FLO1, FLO5 and FLO8 genes in floc formation and ethanol stress resistance qRT-PCR was carried out. S. cerevisiae wine yeast strains showing a different level of flocculation and a control non flocculent strain were exposed to ethanol 20% (v/v) for 30 min to 1 h. The obtained results are shown in Fig. 5. In the presence of ethanol the expression patterns of FLO1 and FLO8 genes didn't reveal significant

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This research has received funding from Cassa di Risparmio di Teramo (Oenological microbiota: selection to identify the wine character and to improve the competitiveness of Montepulciano d'Abruzzo wineries). We thank Dr. Irene Aguzzi for technical support.

FLO5

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Fig. 5. Relative qRT-PCR expression of FLO1, FLO5 and FLO8 transcripts and survival rates of wine S. cerevisae strains in presence of ethanol stress. The relative expression value for each sample was defined as 2−Ct(target) where Ct(target) represents the cycle number at which a sample reaches a predetermined threshold signal value for the specific target gene. Relative expression data were normalized to the relative expression value of the reference genes ACT1 and FBA1 in each respective sample. Values represent the means of experiments performed in triplicate, and error bars represent standard deviations.

differences. The expression of FLO5 gene was higher than that of FLO1 gene. Generally, strains with flocculent capacity showed higher levels of FLO5 gene expression than the strains with low flocculation capacity or those that lost this ability. These results are confirmed by viable cell counts as reported in Fig. 5. RT73 strain showed the lowest values in both conditions tested, showing that flocculation confers a greater resistance to ethanol stress. Similar results were obtained by Smukalla et al. (2008) who proposed that ethanol could act as a quorum sensing molecule in S. cerevisiae probably in combination with other molecules such as tryptophol. The results obtained in this study show that the natural flocculent wine yeast population is characterized by the presence of FLO genes independent of the degree of flocculation. In particular, FLO genes are present, even if low expressed, also in strains that lost the capacity of flocculate. In this study we highlighted the role of FLO5 gene in development of high flocculent characteristic useful to select yeasts for sparkling wine production. A plethora of studies described the presence of mutated sequences present in FLO genes (Ogata et al., 2008), leading to the constant generation of novel FLO alleles and pseudogenes (Verstrepen et al., 2004, 2005). This study confirmed the high biodiversity of flocculation phenotype and genotype in wine yeasts. The knowledge regarding the molecular nature of flocculation process in S. cerevisiae wine yeasts suggests that their natural reservoir of flocculent genes represents an important source of FLO gene useful to improve sparkling wine technology but also to understand the complexity and strain-specific nature of FLO gene expression. The benefit of this attractive property is that it allows simpler and faster recovery of wines and it also promotes a greater volume recovery of fermented wine product. This improvement has significant financial cost-saving implications and can be directly attributed to the superior flocculent ability. This research provides S. cerevisiae strains to study the relationship between the genetic variation and flocculation phenotype.

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