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Interdelta sequence typing was used to investigate the genetic diversity of 54 Chinese indigenous wine strains of Saccharomyces cerevisiae selected on the basis of preliminary sequence analysis from 349 strains that were isolated previously from 15 spontaneous fermentations in Shanshan, Xinjiang, and Qing Tongxia, Ningxia, China. Of the 54 strains tested, 78% (42/54) were confirmed as genetically distinct. Dendrograms based on strain similarity revealed differences in the genetic relationships of Xinjiang yeast populations between table and wine grape varieties, in addition to differences between red and white grape varieties in Ningxia (Dice coefficients of 0.448 and 0.674, respectively). When data from Saccharomyces strains collected from California, France, Italy, northern Europe, and Spain were included in the analysis, the dendrogram revealed five groups containing 51, 4, 48, 3, and 1 strain, respectively. Ningxia and Xinjiang provinces displayed local specific S. cerevisiae biotas that show a clear separation from other strains. Cluster XJ19 isolated from Xinjiang displayed a high level of similar­ity with UCD587, UCD2515, and UCD2516 from California. Clusters XJ2, XJ7, XJ20, and XJ3, also isolated from Xinjiang, had a lower degree of similarity with other Chinese indigenous genotypes and strains from other regions. This study compares, for the first time, the genetic diversity and relationships between indigenous S. cerevisiae wine strains collected from Xinjiang and Ningxia provinces in China with wine strains from different geographic regions.

The yeast Saccharomyces cerevisiae has evolved to dominate grape juice fermentation. A suite of cellular properties, rapid nutrient depletion, production of inhibitory compounds and the metabolic narrowing of the niche, all enable a minor resident of the initial population to dramatically increase its relative biomass in the ecosystem. This dominance of the grape juice environment is fueled by a rapid launch of glycolysis and energy generation mediated by transport of hexoses and an efficient coupling of transport and catabolism. Fermentation occurs in the presence of molecular oxygen as the choice between respiratory or fermentative growth is regulated by the availability of sugar a phenomenon known as glucose or catabolite repression. Induction of the [GAR + ] prion alters the expression of the major hexose transporter active under these conditions, Hxt3, reducing glycolytic capacity. Bacteria present in the grape juice ecosystem were able to induce the [GAR + ] prion in wine strains of S. cerevisiae. This induction reduced fermentation capacity but did not block it entirely. However, dominance factors such as the rapid depletion of amino acids and other nitrogen sources from the environment were impeded enabling greater access to these substrates for the bacteria. Bacteria associated with arrested commercial wine fermentations were able to induce the prion state, and yeast cells isolated from arrested commercial fermentations were found to be [GAR + ] thus confirming the ecological relevance of prion induction. Subsequent analyses demonstrated that the presence of environmental acetic acid could lead to [GAR + ] induction in yeast strains under certain conditions. The induction of the prion enabled yeast growth on non-preferred substrates, oxidation and reduction products of glucose and fructose, present as a consequence of bacterial energy production. In native ecosystems prion induction never exceeded roughly 50-60% of the population of yeast cells suggesting that the population retains the capacity for maximal fermentation. Thus, the bacterial induction of the [GAR + ] prion represents a novel environmental response: the query of the environment for the presence of competing organisms and the biological decision to temper glucose repression and dominance and enter a metabolic state enabling coexistence.

The yeast Saccharomyces cerevisiae has evolved to dominate grape juice fermentation. A suite of cellular properties, rapid nutrient depletion, production of inhibitory compounds and the metabolic narrowing of the niche, all enable a minor resident of the initial population to dramatically increase its relative biomass in the ecosystem. This dominance of the grape juice environment is fueled by a rapid launch of glycolysis and energy generation mediated by transport of hexoses and an efficient coupling of transport and catabolism. Fermentation occurs in the presence of molecular oxygen as the choice between respiratory or fermentative growth is regulated by the availability of sugar a phenomenon known as glucose or catabolite repression. Induction of the [GAR + ] prion alters the expression of the major hexose transporter active under these conditions, Hxt3, reducing glycolytic capacity. Bacteria present in the grape juice ecosystem were able to induce the [GAR + ] prion in wine strains of S. cerevisiae. This induction reduced fermentation capacity but did not block it entirely. However, dominance factors such as the rapid depletion of amino acids and other nitrogen sources from the environment were impeded enabling greater access to these substrates for the bacteria. Bacteria associated with arrested commercial wine fermentations were able to induce the prion state, and yeast cells isolated from arrested commercial fermentations were found to be [GAR + ] thus confirming the ecological relevance of prion induction. Subsequent analyses demonstrated that the presence of environmental acetic acid could lead to [GAR + ] induction in yeast strains under certain conditions. The induction of the prion enabled yeast growth on non-preferred substrates, oxidation and reduction products of glucose and fructose, present as a consequence of bacterial energy production. In native ecosystems prion induction never exceeded roughly 50-60% of the population of yeast cells suggesting that the population retains the capacity for maximal fermentation. Thus, the bacterial induction of the [GAR + ] prion represents a novel environmental response: the query of the environment for the presence of competing organisms and the biological decision to temper glucose repression and dominance and enter a metabolic state enabling coexistence.

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.