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

A flor strain of S. cerevisiae, 3238-32 MATaleu2-Δ1 lys2-801 ura3-52 (12), was grown for 24 h in YEPD (2% Bacto peptone, 1% yeast extract, 2% glucose) at 30°C and 200 rpm. Cells were harvested and washed twice in sterile distilled water by centrifugation, resuspended in sterile distilled water, and diluted 500-fold into sterile-filtered YNB (Bacto yeast nitrogen base without amino acids) plus 30 μg/ml Leu, 30 μg/ml Lys, and 10 μg/ml Ura and supplemented with 4% (vol/vol) ethanol (YNB-EtOH), 3% (vol/vol) glycerol (YNB-Glyc), 1% (wt/vol) potassium acetate (YNB-Acet), 2% (wt/vol) dl-lactic acid (YNB-Lact), 2% (wt/vol) succinic acid (YNB-Succ), or 2% (vol/vol) ethyl acetate (YNB-EtAc). All YNB-based media were adjusted to pH 5.4. Four replicate 1-ml/well cultures were grown in a 24-well polystyrene microtiter plate (Becton Dickinson Labware, NJ). Biofilms were harvested by aspiration after 5 days of static incubation at 30°C by collecting the 1-ml cultures and an additional 1 ml of sterile distilled water used to rinse each well. The 2-ml samples were transferred to a cuvette and read at A₆₀₀. Biofilm biomass (dry weight) was determined from a standard curve that related A₆₀₀ values to dry weights of biofilm formed by 3238-32 grown in an independent 200-ml culture of YNB-EtOH from which the biofilm had been collected, washed with sterile distilled water, and dried under vacuum. The standard curve was generated by suspending about 60 mg of dried biofilm in 10 ml of distilled water from which a number of dilutions were prepared and read at A₆₀₀. The mean slope of two such curves (coefficient of variation, <20%) was used as the conversion factor.

Figure 1A is a representative photograph of air-liquid interfacial biofilms formed in a 24-well microtiter plate containing 2 or 3 ml of medium per well. Biofilms consistently formed in YNB-EtOH, YNB-Glyc, and YNB-EtAc but were not always observed in YNB-Succ, YNB-Lact, or YNB-Acet, in spite of subsurface growth in the latter three media. The greatest amount of biofilm formed in YNB-Glyc, followed by YNB-EtAc and then by YNB-EtOH (Table 1). Per mg of available carbon, the amounts of biofilm formed in YNB-Glyc and in YNB-EtAc were about 3.8 and 2.7 times greater, respectively, than in YNB-EtOH. Per ml of medium, the amounts formed in YNB-Glyc and in YNB-EtAc were about 3.4 and 1.8 times greater, respectively, than in YNB-EtOH. Consistent with catabolism of glycerol and ethyl acetate by flor yeasts, a decrease in both compounds during biological aging of sherry wine has been reported (2). While biofilm formation in the other media was inconsistent, subsurface growth was always observed. Cell yields in YNB-Succ, YNB-Lact, and YNB-Acet were mostly from bulk growth (Table 1). One interpretation of these data is that biofilm formation requires an energy input not needed for growth in bulk culture and growth on the more reduced substrates—glycerol, ethyl acetate, and ethanol—provided this additional energy.

To determine if biofilm-specific changes in gene expression accompanied the shift from growth in bulk culture in YNB plus 2% glucose to growth as a biofilm in YNB-EtOH, a microarray analysis was undertaken. We exploited a previous observation made when growing 3238-32. Namely, when a large inoculum (∼10%) of glucose-grown cells was shifted to YNB-EtOH, cells initially settled to the bottom of the vessel to form a visible layer that remained qualitatively unchanged in thickness over the course of 2 to 3 days while a biofilm became evident at the air-liquid interface after about 1 to 2 days. We reasoned that at a first approximation, the biofilm and nonbiofilm populations should have been genetically identical and that environmental factors were likely to explain the differential growth response. Briefly, cells grown 24 h at 30°C in YEPD to a density of 5 × 10⁸ cells/ml were pelleted, washed twice in distilled water, diluted 10-fold into 100 ml of YNB-EtOH in triplicate 250-ml beakers, and grown statically at 27°C. After 48 h, the visible biofilm covering the entire liquid surface was collected by aspiration. Once removed, cells at the bottom were collected similarly. Cells from both populations were washed once in sterile distilled water prior to RNA isolation and microarray analysis (GEO series accession number GSE19156 [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19156 ]).

Initially, a gene ontology (GO) process analysis was performed on genes upregulated in both biofilm and nonbiofilm cells (data not shown). Categories for which gene frequencies differed ≥2-fold between the two populations are listed in Table 2. Based on the centrality of these processes to cell growth (e.g., ribosome biogenesis and assembly, translation, cytokinesis), these data suggest that cells in the biofilm were growing faster than the nonbiofilm cells or that the latter had stopped growing. In support of the no-growth possibility, half of the 653 genes found to be upregulated in a stationary-phase yeast culture harvested after 5 days in YEPD (7) were also upregulated ≥2-fold in the nonbiofilm cells. Three genes whose overexpression was associated with the “quiescent” fraction of stationary-phase cells (1) were found to be highly overexpressed in the nonbiofilm cells: SIP18, 37.7-fold; GRE1, 10.7-fold; BCY1, 8.1-fold. A significant increase in expression of known biofilm formation-associated genes (FLO8, MUC1, NRG1, NRG2, SNF1, HSP12) was not observed. Expression of agglutinin-encoding genes (AGA1, AGA2, FIG2, FLO1, FLO5, FLO10, SAG1) in the biofilm cells was either the same or >2-fold greater than in the nonbiofilm cells. While expression of two genes involved in protein mannosylation was >2-fold greater than in the biofilm cells (MNN11 and MNN9), expression of others was either the same or >2-fold greater than in nonbiofilm cells (CSG2, PMI40, DPM1, VAN1, OCH1, ALG3). It is possible that because RNA was harvested well after the biofilm began to form, the primary biofilm-specific transcriptional response was missed. Alternatively, the shift to biofilm growth may not be accompanied by major shifts in transcription of known biofilm formation-related genes, or the key genes in the process may be unrecognized.

In summary, formation of an air-liquid interfacial biofilm by S. cerevisiae is not limited to aerobic growth on ethanol but occurs on other reduced nonfermentable carbon sources, glycerol and ethyl acetate. Its variable formation on less reduced substrates (succinic, acetic, and lactic acids) is consistent with an additional energy input that is not needed for growth in bulk culture.

We thank Anne-Marie Girard for technical support for the microarray analysis and Lynn G. Ketchum for photographic assistance (Fig. 1A).