Virginie Galeote

Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118

Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non-Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations.

FSY1, a horizontally transferred gene in the Saccharomyces cerevisiae EC1118 wine yeast strain, encodes a high-affinity fructose/H+ symporter

Transport of glucose and fructose in the yeast Saccharomyces cerevisiae plays a crucial role in controlling the rate of wine fermentation. In S. cerevisiae, hexoses are transported by facilitated diffusion via hexose carriers (Hxt), which prefer glucose to fructose. However, utilization of fructose by wine yeast is critically important at the end of fermentation. Here, we report the characterization of a fructose transporter recently identified by sequencing the genome of the commercial wine yeast strain EC1118 and found in many other wine yeasts. This transporter is designated Fsy1p because of its homology with the Saccharomyces pastorianus fructose/H(+) symporter Fsy1p. A strain obtained by transformation of the V5 hxt1-7Δ mutant with FSY1 grew well on fructose, but to a much lesser extent on glucose as the sole carbon source. Sugar uptake and symport experiments showed that FSY1 encodes a proton-coupled symporter with high affinity for fructose (K(m) 0.24±0.04mM). Using real-time RT-PCR, we also investigated the expression pattern of FSY1 in EC1118 growing on various carbon sources. FSY1 was repressed by high concentrations of glucose or fructose and was highly expressed on ethanol as the sole carbon source. The characteristics of this transporter indicate that its acquisition could confer a significant advantage to S. cerevisiae during the wine fermentation process. This transporter is a good example of acquisition of a new function in yeast by horizontal gene transfer.

A novel fungal family of oligopeptide transporters identified by functional metatranscriptomics of soil eukaryotes

Functional environmental genomics has the potential to identify novel biological functions that the systematic sequencing of microbial genomes or environmental DNA may fail to uncover. We targeted the functions expressed by soil eukaryotes using a metatranscriptomic approach based on the use of soil-extracted polyadenylated messenger RNA to construct environmental complementary DNA expression libraries. Functional complementation of a yeast mutant defective in di/tripeptide uptake identified a novel family of oligopeptide transporters expressed by fungi. This family has a patchy distribution in the Basidiomycota and Ascomycota and is present in the genome of a Saccharomyces cerevisiae wine strain. High throughput phenotyping of yeast mutants expressing two environmental transporters showed that they both displayed broad substrate specificity and could transport more than 60–80 dipeptides. When expressed in Xenopus oocytes one environmental transporter induced currents upon dipeptide addition, suggesting proton-coupled co-transport of dipeptides. This transporter was also able to transport specifically cysteine. Deletion of the two copies of the corresponding gene family members in the genome of the wine yeast strain severely reduced the number of dipeptides that it could assimilate. These results demonstrate that these genes are functional and can be used by fungi to efficiently scavenge the numerous, low concentration, oligopeptides continuously generated in soils by proteolysis.

Amplification of a Zygosaccharomyces bailii DNA Segment in Wine Yeast Genomes by Extrachromosomal Circular DNA Formation

We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations.

Sfl1p acts as an activator of the HSP30 gene in Saccharomyces cerevisiae

In the yeast, environmental challenges are known to induce both specific and general stress response. The HSP30 gene is strongly induced when cells are exposed to various stresses but this activation is largely independent of the major stress-related transcription factor Hsf1p and partly independent from Msn2p/Msn4p. In order to identify new potential regulators of HSP30 we isolated insertion mutants affected in HSP30 expression. We identified SFL1 gene encoding a protein previously shown to repress several genes. We show that Sfl1 is involved in the transcriptional activation of HSP30. Mutation of sfl1 reduces HSP30-lacZ expression under both basal and stress-induced conditions. We also show, using site-directed mutagenesis, that HSL motifs (Heat-Shock-Like putative DNA binding sequence) located in HSP30 promoter are required for HSP30 activation. Finally, a genome-wide analysis of the effects of SFL1 deletion on gene expression revealed that Sfl1p controls the expression of a small number of genes, with some being activated by the protein and others repressed. As a whole our data show that Sfl1p is a key component of the transcriptional control of the stress responsive gene HSP30. Moreover, we show that Sfl1, which was previously described as being a transcriptional repressor, can also act as an activator.

The Saccharomyces cerevisiae zinc factor protein Stb5p is required as a basal regulator of the pentose phosphate pathway

In Saccharomyces cerevisiae, the oxidative stress-activated zinc cluster protein Stb5p activates genes involved in NADPH production and most genes of the pentose phosphate (PP) pathway. To gain insight into the role of Stb5p, we studied the behaviour of stb5 deletion mutants during aerobic and anaerobic growth on glucose. stb5 mutants were auxotrophic for methionine and pyrimidine nucleotides. The methionine auxotrophy phenotype was air dependent, suggesting an impaired aerobic NADPH status. Consistent with this, the acetate level was reduced and the α-ketoglutarate level was increased in the stb5 mutant. stb5 cells also required pyrimidine nucleotides for aerobic and anaerobic growth, consistent with a reduction in 5-phosphoribosyl-1-pyrophosphate production caused by a reduced flux through the PP pathway. Strains overexpressing STB5 could not grow on glucose. This growth defect was restored by overproduction of an NADPH-butanediol dehydrogenase, which reoxidizes the excess NADPH in the oxidative PP pathway. These findings suggest a major role for the transcription factor Stb5p in maintaining a basal flux through the PP pathway to meet the NADPH requirements for aerobic growth, and to provide the nucleotide precursors. Our data also demonstrate the potential use of a system based on overproduction of this transcription factor to increase flux through the PP pathway.

Horizontally acquired oligopeptide transporters favour adaptation of Saccharomyces cerevisiae wine yeast to oenological environment.

In the past decade, horizontal gene transfer (HGT) has emerged as a major evolutionary process that has shaped the genome of Saccharomyces cerevisiae wine yeasts. We recently showed that a large Torulaspora microellipsoides genomic island carrying two oligopeptide transporters encoded by FOT genes increases the fitness of wine yeast during fermentation of grape must. However, the impact of these genes on the metabolic network of S. cerevisiae remained uncharacterized. Here we show that Fot-mediated peptide uptake substantially affects the glutamate node and the NADPH/NADP(+) balance, resulting in the delayed uptake of free amino acids and altered profiles of metabolites and volatile compounds. Transcriptome analysis revealed that cells using a higher amount of oligopeptides from grape must are less stressed and display substantial variation in the expression of genes in the central pathways of carbon and nitrogen metabolism, amino acid and protein biosynthesis, and the oxidative stress response. These regulations shed light on the molecular and metabolic mechanisms involved in the higher performance and fitness conferred by the HGT-acquired FOT genes, pinpointing metabolic effects that can positively affect the organoleptic balance of wines.