Acetaldehyde and ethanol are responsible for mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) in flor yeasts.

Flor yeasts grow and survive in fino sherry wine where the frequency of respiratory-deficient (petite) mutants is very low. Mitochondria from flor yeasts are highly acetaldehyde- and ethanol-tolerant, and resistant to oxidative stress. However, restriction fragment length polymorphism (RFLP) of mtDNA from flor yeast populations is very high and reflects variability induced by the high concentrations of acetaldehyde and ethanol of sherry wine on mtDNA. mtDNA RFLP increases as the concentration of these compounds also increases, but is followed by a total loss of mtDNA in petite cells. Yeasts with functional mitochondria (grande) are target of continuous variability, so that flor yeast mtDNA can evolve extremely rapidly and may serve as a reservoir of genetic diversity, whereas petite mutants are eventually eliminated because metabolism in sherry wine is oxidative.

Castrejón F ，Codón AC ，Cubero B ，Benítez T ... -  《SYSTEMATIC AND APPLIED MICROBIOLOGY》

Physiological and molecular characterization of flor yeasts: polymorphism of flor yeast populations.

Yeast strains which form velum on the surface of Sherry wine during the aging process have been isolated and characterized. According to their metabolic and molecular features most of the yeasts that were isolated belong to different races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis and rouxii). Due to the conditions under which these yeasts were isolated, all strains have in common the capacity to develop a film as an adaptive mechanism which allows them to grow and survive in 15.5% vol. ethanol. All strains were prototrophs for amino acids and most vitamins but they gave different responses to the killer factor. However, whereas their physiological features were similar, they showed a great heterogeneity with regards to the nuclear and mitochondrial genome (mtDNA): DNA content per cell was quite variable (1.3 to 2n), electrophoretic karyotypes of nuclear genomes indicated a main pattern with some variations, and polymorphism shown by the mtDNA was very high. Under extreme conditions such as Sherry wine with 15.5% vol. ethanol, no fermentable sugar and an exclusively oxidative metabolism, cells hardly grow and the maintenance of a live population depends on survival and respiration, which in turn depend on the mtDNA. At the same time these environmental conditions are mutagenic for the mtDNA, causing an increase in variation. Thus, the polymorphism observed might reflect the enormous variability induced by the ethanol followed by the selection of those mtDNA sequences which make the mitochondria metabolically active under these conditions.

Martínez P ，Codón AC ，Pérez L ，Benítez T ... -  《YEAST》

Indigenous yeast population from Georgian aged wines produced by traditional "Kakhetian" method.

The yeast microbiota present in wines produced by the ancient "Kakhetian" method in Georgia (EU) was studied. This technique involves the use of terracotta vessels (amphoras), during spontaneous fermentation, maceration phase and wine ageing. The analysed yeasts were collected from wines after maturation for one year in ten amphoras from a Georgian winery. The 260 isolates were all identified as Saccharomyces cerevisiae, and the majority were classified as flor yeasts by restriction analysis of ITS region. A first technological and molecular screening was used to select 70 strains for further characterization. Both genetic and metabolic characterization discriminated flor from non-flor strains. The combined results obtained by analysis of interdelta region and mtDNA-RFLP yielded 23 different biotypes; no biotype was common to flor and non-flor strains. The wines produced by flor yeasts showed a high content in acetaldehyde, acetic acid, acetoin, whereas the level of other compounds was similar to wines obtained by non-flor strains. This study represents the first report on the composition of yeast microbiota involved in the maturation of this traditional wine. These flor strains represent an interesting yeast population, in possession of peculiar characteristics allowing them to survive during wine ageing, becoming the dominant flora in the final wine.

Capece A ，Siesto G ，Poeta C ，Pietrafesa R ，Romano P ... -  《-》

Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts.

Saccharomyces flor yeasts proliferate at the surface of sherry wine, which contains over 15% (vol) ethanol. Since ethanol is a powerful inducer of respiration-deficient mutants, this alcohol has been proposed to be the source of the high diversity found in the mitochondrial genomes of flor yeasts and other wine yeasts. Southern blot analysis suggests that mitochondrial DNA (mtDNA) polymorphic changes are due to minor lesions in the mitochondrial genome. As determined in this work by pulsed-field gel electrophoresis, restriction analysis, and Southern blot analysis, ethanol-induced petite mutants completely lack mtDNA (rho zero). Ethanol-induced changes in the mitochondrial genome that could explain the observed mtDNA polymorphism in flor yeasts were not found. The transfer of two different mtDNA variants from flor yeasts to a laboratory strain conferred in both cases an increase in ethanol tolerance in the recipient strain, suggesting that mtDNAs are probably subjected to positive selection pressure concerning their ability to confer ethanol tolerance.

Ibeas JI ，Jimenez J 《-》

Correlation between acetaldehyde and ethanol resistance and expression of HSP genes in yeast strains isolated during the biological aging of sherry wines.

In the production of sherry wines, the process of biological aging is essential for the development of their organoleptic properties. This process involves velum formation by "flor" yeasts. Several of these yeast strains have been isolated and characterized with regard to their genetic, physiological and metabolic properties. In this work, we studied their resistance to cold-, osmotic-, oxidative-, ethanol- and acetaldehyde-stress, and found, in most cases, a correlation between resistance to acetaldehyde stress and ethanol stress and isolation from "soleras." Moreover, gene expression analysis revealed induction of the heat shock protein (HSP) genes HSP12, HSP82, and especially HSP26 and HSP104, under acetaldehyde stress in most of the strains. In strain C, there was a clear correlation between resistance to ethanol and acetaldehyde, the high induction of HSP genes by these compounds and its presence as the predominant strain in most levels of several soleras.

Aranda A ，Querol A ，del Olmo Ml 《ARCHIVES OF MICROBIOLOGY》