FLO5 gene controls flocculation phenotype and adhesive properties in a Saccharomyces cerevisiae sparkling wine strain

Abstract

Flocculation is an important feature for yeast survival in adverse conditions. The natural diversity of flocculating genes in Saccharomyces cerevisiae can also be exploited in several biotechnological applications. Flocculation is mainly regulated by the expression of genes belonging to the FLO family. These genes have a similar function, but their specific contribution to flocculation ability is still unclear. In this study, the distribution of FLO1, FLO5 and FLO8 genes in four S. cerevisiae wine strains was investigated. Subsequently, both FLO1 and FLO5 genes were separately deleted in a flocculent S. cerevisiae wine strain. After gene disruption, flocculation ability and agar adhesion were evaluated. FLO1 and FLO5 genes inheritance was also monitored. All strains presented different lengths for FLO1 and FLO5 genes. Results confirm that in S. cerevisiae strain F6789, the FLO5 gene drives flocculation and influences adhesive properties. Flocculation ability monitoring after a cross with a non-flocculent strain revealed that FLO5 is the gene responsible for flocculation development.

Figure 1

(A) Specific PCR detection of FLO1, FLO5 and FLO8 genes in S. cerevisiae strains. (B) Intragenic repetitive domains of gene FLO5. PCR mix without DNA was used as negative control.

Figure 2

Flocculation level of parental (F6789), haploid (F6789A), transformants (F6789A-Δflo1 F6789A-Δflo5) and V5 strains. V5 is a laboratory haploid yeast derivative from a strain isolated from Champagne. This yeast was previously used as negative control for flocculation (Bidard et al.) and we are sure that it has no adhesive properties. Flocculation was determined in YNB + glucose (2% w/v) (A), in YEPD (B) and in synthetic must (C). (D) Sedimentation without CaCl₂ of non-flocculent strain V5 and F6789A-Δflo5. The data reported are mean values for triplicates. Vertical error bars represent standard deviations.

Figure 3

(A) Photographs of the parental (F6789), haploid (F6789A) and transformants F6789A-Δflo1 and F6789A-Δflo5 strains. Petri dishes were filled with equivalent volumes of YEPD medium originating from a liquid cell culture in stationary phase. (B) Agar invasion before and after the wash. Strains were spreaded onto YEPD plates and were cultivated for 3 days at 28 °C, followed by 2 days at room temperature. Plates were documented before and after non-adhesive cells were washed off the agar with a gentle stream of water. (C) Optical microscope photos (40X objective) of the parental strain (F6789), haploid strain (F6789A) and transformants.

Figure 4

(A) Primary-structure of FLO genes. (B) Structure of Flo proteins. (C) Protein alignment of FLO5 protein between S288C and F6789A strains. Alignment with SPSC02 and YJW6 FLO5 sequences was not shown because these were not available in public databases. (D) Protein alignment of FLO1 protein between F6789A, SPSC01 (Accession n. JQ629938/protein id. AFJ20718.1), S288C and YJWE6 (Accession n.  X78160/protein id. CAA55024.1) strains.

Figure 5

Phenotypic characterization of hybrids derived from the cross between 59A and F6789A strains. Column (A) sedimentation test: liquid cultures were vigorously mixed and placed in vertical position. Tubes were photographed after 30 s. Column (B) microscope observation (objective 40X).

Figure 6

FLO5 gene repeated regions among segregants.

Figure 7

Localization of cell wall flocculins by FITC-labelled specific Anti-Flop antibody of parental (F6789), haploid (F6789A), F6789A-Δflo1, F6789A-Δflo5 and V5 strains. The S. cerevisiae cells were grown in YNB+glucose. After 48 h (A_{600 nm} = 1), cells were stained with labelled lectin-specific IgGs and examined under a fluorescence microscope (100X oil objective). No fluorescence was observed in V5 strain nor in cells incubated with TBS only.