engleski

Characterization of CCW7 cell wall protein of film-forming Saccharomyces cerevisiae wine yeast strains at gene and protein level

Aging of Hungarian botrytized wine Tokaji szamorodni results aromatic compounds produced as by-products of the oxidative metabolism of yeasts taht are forming a biofilm at the surface of wines. Film-forming strains show a planktonic growth during the main fermentation phase but the cells rise to the surface afetrward and form a multylayerd biofilm on it. The cells metabolize mainly ethanol and glycerol during maturation and produce caracteristic aromatic compounds like acetaldehyde. However, the mechanism of attachment and the role of cell surface moleculs in film formationare not clarified at all. Our aim was to determine the possible role of certain cell wall proteins in film formation, therefore we looked for differences in the cell wall proteins of sedimenting and film-forming Saccharomyces cerevisiae strains. We found different protein patterns among alkali extractable cell wall proteins of laboratory and film-forming Saccharomyces cerevisiae strins. In the case of film-forming strains a smaller ccw7 protein was observed and isolated. For further investigation we designed a specific primer pair for the amplification of the Ccw7p encoding gene HSP150. PCR amplification of the gene confirmed the size difference between the ccw7 proteins of the laboreatory and film-forming strains. Almost all the film-forming isolates generated a single PCR product of the same size but two of them contained additional amplicons, a shorter or a longer one. Sequence analysis of the amplified common PCR products of film-forming yeasts revealed two deletions in the internal repetitive region of the gene. In the case of TDO4 strain that has an additional longer PCR amplicon we induced meiotic segregation aiming to check whether there is any correlation between the film forming ability and the CCW7 alleles. The alleles segregated during meiosis, however, both types of the segregants maintained the film forming ability. CCW7 amplicons of different lenghts. By the use of RFLP analysis of the CCW7 amplicons we found that majority of film forming yeasts formed a separate cluster of 100% similarity, while sedimenting wine yeast strains showed an extended length polymorphism.

yeast; cell wall; cell wall proteins; CCW7

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