INFLUENCE OF (+)-CATECHIN ON BIOFILM GROWTH OF OENOLOGICAL SACCHAROMYCES
CEREVISIAE STRAINS
Yeast ‘mats’ are defined as complex multicellular structures composed of yeast-form cells (1). This morphological event is, together with others, closely related to the genes encoding for cell wall glycoproteins. In Saccharomyces cerevisiae, proteins encoded by FLO genes, such as FLO1, 5, 9, 10 and 11, confer adherence to agar, solid surfaces and other yeast cells (2,1); so, the expression of each member of FLO family produces distinct cell-surface properties (3). A comparable bacterial sliding motility derived from the friction between cells and substrate was found in S. cerevisiae grown on low agar concentration substrate as consequence of FLO11 expression; this confers hydrophobic characteristic responsible for cell movement on soft agar surface and growth resulting in a confluent ‘mat’ (1). Pseudohyphal growth was related to nitrogen starvation, carbon availability and other stress (4, 5) even if factors such as medium viscosity, Flo11p proteins on the yeast cell surfaces, and nutrients in the medium contribute to the development of unusual structures (1). Epigenetic regulation of FLO genes family was demonstrated to determine the transition to filamentous development and to explain both the variation in filamentation within a genetically homogeneous colony of yeast and the presence of overlapping phenotypes (3).
Recently, it was reported that FLO11 expression increased the cell surface hydrophobicity in S. cerevisiae sherry strains (6); it seems that the Candida albicans mannan components of the cell wall glycoproteins are somehow related to the cell hydrophobicity (7) and it has been suggested that a reduction in the amount of phosphodiester-linked -1,2-oligomannosyl branches of cell surface mannoproteins lead to an increase of the C. albicans cell hydrophobicity (8). Moreover, differences in mannosylphosphorilation degree of yeast cell wall glycoproteins were associated to the different wine yeast ability to adsorb wine phenolic compounds (9), and this character was demonstrated to be inheritable (10). Catechin exhibits antioxidant activity (11) and it plays a role as protectant against osmotic and thermal stress in wine yeast resulting in improved fermentation performance (12,13).
AIM OF THE WORK
This work aims to explore the oenological S. cerevisiae phenotypic feature ‘mat’ formation and to study how it is affected by the addition of (+)-catechin.
INTRODUCTION
REFERENCES
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4. Gimeno C.J., Ljungdahl P.O., Styles C.A., Fink G.R. (1992) Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS2. Cell 68: 1077–1090. 5. Gagiano M., Bauer F.F., Pretorius I.S. (2002) The sensing of nutritional status and relationship to filamentous growth in Saccharomyces cerevisiae. FEMS Yeast Res 2: 433–470.
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8. Masuoka J., Hazen K.C. (1999) Differences in the acid-labile component of Candida albicans mannan from hydrophobic and hydrophilic cells. Glycobiology 9: 1281–1286. 9. Rizzo M., Ventrice D., Varone M.A., Sidari R., Caridi A. (2006) HPLC determination of phenolics adsorbed on yeasts. J Pharm Biomed Anal 42: 46–55.
10. Caridi A., Sidari R., Solieri L., Cufari A., Giudici P. (2007) Wine colour adsorption phenotype: an inheritable quantitative trait loci of yeasts. J Appl Microbiol 103: 735–742.
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13. Caridi A. (2003) Effect of protectants on the fermentation performance of wine yeasts subjected to osmotic stress. Food Technol Biotechnol. 41: 145–148.
MATERIALS AND METHODS
The study was carried out using 46 oenological S. cerevisiae strains - isolated from Calabrian and Sicilian grapes and musts, respectively 37 and 9 strains - belonged to the microorganism collection of the Unit of Microbiology, Department of Scienze e Tecnologie Agro-Forestali e Ambientali, Faculty of Agricultural Sciences of the Mediterranea University of Reggio Calabria, Italy. These strains were tested in the framework of PRIN 2007 for their ability to adsorb wine phenolic compounds based on cell wall glycoprotein phosphorilation. S. cerevisiae Σ1278b, haploid and MATa, which has filamentous phenotypes and S. cerevisiae BY4742, haploid, MATα and Δflo8, which has not an adhesive phenotype (EUROSCARF), were used as control strains. These strains belonged to the Department of Agriculture and Food Systems, Melbourne School of Land and Environment, University of Melbourne, Victoria, Australia.
Soft YPD agar plates (1% yeast extract, 2% peptone, 2% glucose, 0.3% agar) with or without 100 mg/l of (+)-catechin were prepared. After 1 day, overnight yeast cultures (0.1 μl) grown in YPD broth (1% yeast extract, 2% peptone, 2% glucose) at 28 °C were inoculated in the centre of both soft YPD agar and soft YPD agar supplemented with (+)-catechin. The plates were wrapped with Parafilm and incubated at 25 °C for 13 days. To monitor the development of each yeast strain biomass throughout the 13 days, the plates were photographed after 3, 5, 7, 10, and 13 days by Canon EOS 350D digital camera. Photographs were elaborated using the computer program ArchiCAD 8.1 to determine the biomass area - expressed as cm2
-of yeast biomass grown both on s-oft YPD agar and s-oft YPD agar supplemented with (+)-catechin.
RESULTS
Figure 1 shows the 46 yeast strains and the negative control strain Sc BY4742 growth, expressed as cm2 of biomass area, on soft agar throughout 13 days of
incubation at 25 °C; after this time, the 41.30% of strains produced ‘mat’ structures while the 58.69% did not.
Differences in ‘mat’ structures was observed among the strains; in particular, some of them showed rough texture with central hub and radial spokes while others were either less rough or smooth in texture. This could be due to the possible different yeast strains mating type and MAT ploidy.
The medium supplementation with (+)-catechin determined a changing in the yeast behaviour towards ‘mat’ formation (Figure 2). In fact, strains forming ‘mat’ when grown on soft agar - Sc 329, Sc 456, Sc 1128, Sc 1240, Sc 1321, Sc 1340, Sc 1349, Sc 1871, and Sc 2319 - did not exhibit the same aptitude when grown on soft agar supplemented with (+)-catechin; analogously, strains not forming ‘mat’ when grown on soft agar - Sc 76, Sc 157, Sc 396, Sc632, Sc 1326, Sc 1526, Sc 1721, Sc 1741, Sc 1798, Sc 1803, Sc1864, and Sc 2485 - exhibited this aptitude when grown on soft agar supplemented with (+)-catechin. Three strains forming ‘mat’ on soft agar - Sc 1541, Sc 1591, and Sc 1687 - exhibited an increase in ‘mat’ diameter on medium supplemented with (+)-catechin.
Moreover, the presence of (+)-catechin induced the transition to pseudohyphal growth for strains Σ1278b, Sc 251, Sc 384, Sc 1240, Sc 1798, Sc 1871, Sc 2306, Sc 2319, Sc 2363, and Sc 2360. It is interesting to note that pseudohyphal formation was heterogeneous; within a genetically homogeneous strain colony some cells initiated the filamentation program whereas other adjacent cells did not (Figure 3). (+)-Catechin could be considered as an environmental signal that regulates epigenetically the expression of the cell surface proteins of the FLO family by the access to the silent FLO genes, whose mechanisms need to be explored.
The possible correlation between the abilities to form ‘mat’ and to adsorb coloured phenolic compounds was explored hypothesizing that they may be considered two not fully correlated yeast phenotypes, both depending on cell wall glycoproteins. As result, different yeast feature combinations were observed which could be explained considering that on yeast cell walls exist different mannoproteins, among which Flo11p, with various phosphorilation degree, that confers ability both/either to adsorb phenolics and/or form ‘mat’ structures. Therefore, phosphorilated mannoproteins, together with proteins of the FLO family or only one of these class of proteins alternatively, may be expressed on yeast cell surface.
PERSPECTIVES
This work has highlighted an interesting variability among the oenological yeast strains tested, that needs to be genetically explored and elucidated. A knowledge that go into the epigenetic phenomena supposed to regulate the FLO gene family members seems to be important to understand the environmental role in generate yeast strain diversity.
Moreover, this work has led to hypothesize that the simultaneous or not expression of various classes of glycoproteins with different degree of phosphorilation determines combination of the two phenotypes: ability to form ‘mat’ and ability to adsorb coloured phenolic compounds.
Future work will be carried out to characterize yeast strains both for mating type and MAT ploidy, and to understand the mechanisms regulating, in presence of (+)-catechin, the yeast behaviour towards ‘mat’ and pseudohyphal formation.
R. SIDARI
1
, K. HOWELL
2
, A. CARIDI
1
1
Department of Scienze e Tecnologie Agro-Forestali e Ambientali (DISTAFA), "Mediterranea" University of Reggio
Calabria, Via Feo di Vito, I-89122 Reggio Calabria, Italy
2
Department of Agriculture and Food Systems, Melbourne School of Land and Environment, University of Melbourne,
Parkville 3010, Melbourne, VIC, Australia
Figure 2 – Comparison between the ‘mat’ formation assay of S. cerevisiae strains and strain Sc BY4742 grown both on soft agar and on soft agar supplemented with (+)-catechin after 13 days of incubation at 25 °C. 0 5 10 15 20 25 30 35 S c B Y 4 7 4 2 S c 7 6 S c 1 5 7 S c 2 5 1 S c 3 2 9 S c 3 8 4 S c 3 9 6 S c 4 2 8 S c 4 5 6 S c 4 6 8 S c 6 3 2 S c 7 8 6 S c 1 0 5 5 S c 1 1 2 8 S c 1 2 4 0 S c 1 3 2 1 S c 1 3 2 6 S c 1 3 4 0 S c 1 3 4 9 S c 1 5 2 6 S c 1 5 4 1 S c 1 5 7 2 S c 1 5 9 1 S c 1 6 7 4 S c 1 6 8 7 S c 1 7 2 1 S c 1 7 4 1 S c 1 7 5 1 S c 1 7 5 8 S c 1 7 7 1 S c 1 7 9 8 S c 1 8 0 3 S c 1 8 6 4 S c 1 8 7 1 S c 1 9 0 5 S c 1 9 3 6 S c 2 3 0 6 S c 2 3 1 9 S c 2 3 6 1 S c 2 3 6 3 S c 2 3 6 6 S c 2 4 8 5 S c 2 6 1 6 S c 2 6 2 1 S c 2 6 3 0 Yeast strains A re a ( c m 2 )
soft agar soft agar + C
Figure 1 – ‘Mat’ formation assay of oenological S. cerevisiae strains and strain Sc BY4742 grown on soft agar throughout 13 days of incubation at 25 °C.
0 5 10 15 20 25 S c B Y 4 7 4 2 S c 7 6 S c 1 5 7 S c 2 5 1 S c 3 2 1 S c 3 2 9 S c 3 8 4 S c 3 9 6 S c 4 2 8 S c 4 5 6 S c 4 6 8 S c 6 3 2 S c 7 8 6 S c 1 0 5 5 S c 1 1 2 8 S c 1 2 4 0 S c 1 3 2 1 S c 1 3 2 6 S c 1 3 4 0 S c 1 3 4 9 S c 1 5 2 6 S c 1 5 4 1 S c 1 5 7 2 Yeast strains A re a ( c m 2 ) 3d 5d 7d 10d 13d 0 5 10 15 20 25 S c B Y 4 7 4 2 S c 1 5 9 1 S c 1 6 7 4 S c 1 6 8 7 S c 1 7 2 1 S c 1 7 4 1 S c 1 7 5 1 S c 1 7 5 8 S c 1 7 7 1 S c 1 7 9 8 S c 1 8 0 3 S c 1 8 6 4 S c 1 8 7 1 S c 1 8 9 7 S c 1 9 0 5 S c 1 9 3 6 S c 2 3 0 6 S c 2 3 1 9 S c 2 3 6 1 S c 2 3 6 3 S c 2 3 6 6 S c 2 4 8 5 S c 2 6 1 6 S c 2 6 2 1 S c 2 6 3 0 Yeast strains A re a ( c m 2 ) 3d 5d 7d 10d 13d Figure 3 – Heterogeneous initiation of filamentation at the colony periphery in strains Σ1278b (a) and Sc 1240 (b) grown on soft agar supplemented with (+)-catechin.
