Jesús Ambrona

A New Wine Saccharomyces cerevisiae Killer Toxin (Klus), Encoded by a Double-Stranded RNA Virus, with Broad Antifungal Activity Is Evolutionarily Related to a Chromosomal Host Gene

ABSTRACT Wine Saccharomyces cerevisiae strains producing a new killer toxin (Klus) were isolated. They killed all the previously known S. cerevisiae killer strains, in addition to other yeast species, including Kluyveromyces lactis and Candida albicans. The Klus phenotype is conferred by a medium-size double-stranded RNA (dsRNA) virus, Saccharomyces cerevisiae virus Mlus (ScV-Mlus), whose genome size ranged from 2.1 to 2.3 kb. ScV-Mlus depends on ScV-L-A for stable maintenance and replication. We cloned and sequenced Mlus. Its genome structure is similar to that of M1, M2, or M28 dsRNA, with a 5′-terminal coding region followed by two internal A-rich sequences and a 3′-terminal region without coding capacity. Mlus positive strands carry cis-acting signals at their 5′ and 3′ termini for transcription and replication similar to those of killer viruses. The open reading frame (ORF) at the 5′ portion codes for a putative preprotoxin with an N-terminal secretion signal, potential Kex2p/Kexlp processing sites, and N-glycosylation sites. No sequence homology was found either between the Mlus dsRNA and M1, M2, or M28 dsRNA or between Klus and the K1, K2, or K28 toxin. The Klus amino acid sequence, however, showed a significant degree of conservation with that of the product of the host chromosomally encoded ORF YFR020W of unknown function, thus suggesting an evolutionary relationship.

Genetic Instability of Heterozygous, Hybrid, Natural Wine Yeasts

ABSTRACT We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae. Spontaneous cyh2R/cyh2R mutants resistant to high levels of cycloheximide can be directly isolated from cyh2S/cyh2S wine yeasts. Heterozygous cyh2R/cyh2S hybrid clones vary in genetic instability as measured by loss of heterozygosity at cyh2. There were two main classes of hybrids. The lawn hybrids have high genetic instability and generally become cyh2R/cyh2R homozygotes and lose the killer phenotype under nonselective conditions. The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype. The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported. Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity. This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations.

Rapid asymmetrical evolution of Saccharomyces cerevisiae wine yeasts

Genetic instability causes very rapid asymmetrical loss of heterozygosity (LOH) at the cyh2 locus and loss of killer K2 phenotype in some wine yeasts under the usual laboratory propagation conditions or after long freeze‐storage. The direction of this asymmetrical evolution in heterozygous cyh2R/CYH2S hybrids is determined by the mechanism of asymmetrical LOH. However, the speed of the process is affected by the differences in cell viability between the new homozygous yeasts and the original heterozygous hybrid cells. The concomitant loss of ScV‐M2 virus in the LOH process may increase cell viability of cyh2R/cyh2R yeasts and so favour asymmetrical evolution. The presence of active killer K2 toxin, however, abolishes the asymmetrical evolution of the hybrid populations. This phenomenon may cause important sudden phenotype changes in industrial and pathogenic yeasts. Copyright

Construction of Sterile ime1Δ-Transgenic Saccharomyces cerevisiae Wine Yeasts Unable To Disseminate in Nature

ABSTRACT The use of new transgenic yeasts in industry carries a potential environmental risk because their dispersal, introducing new artificial genetic combinations into nature, could have unpredictable consequences. This risk could be avoided by using sterile transgenic yeasts that are unable to sporulate and mate with wild yeasts. These sterile yeasts would not survive the annual cyclic harvesting periods, being condemned to disappear in the wineries and vineyards in less than a year. We have constructed new ime1Δ wine yeasts that are unable to sporulate and mate, bear easy-to-detect genetic markers, and quickly disappear in grape must fermentation immediately after sporulation of the yeast population. These sterile yeasts maintained the same biotechnological properties as their parent yeasts without any detectable deleterious effect of the ime1Δ mutation. These yeasts are therefore interesting biotechnologically for food industry applications and for genetically modified microorganism environmental monitoring studies.

Analysis of homothallic Saccharomyces cerevisiae strain mating during must fermentation.

ABSTRACT Genetic instability and genome renewal may cause loss of heterozygosity (LOH) in homothallic wine yeasts (Saccharomyces cerevisiae), leading to the elimination of the recessive lethal or deleterious alleles that decrease yeast fitness. LOH was not detected in genetically stable wine yeasts during must fermentation. However, after sporulation, the heterozygosity of the new yeast population decreased during must fermentation. The frequency of mating between just-germinated haploid cells from different tetrads was very low, and the mating of haploid cells from the same ascus was favored because of the physical proximity. Also, mating restriction between haploid cells from the same ascus was found, leading to a very low frequency of self spore clone mating. This mating restriction slowed down the LOH process of the yeast population, maintaining the heterozygote frequency higher than would be expected assuming a fully random mating of the haploid yeasts or according to the Mortimer genome renewal proposal. The observed LOH occurs because of the linkage of the locus MAT to the chromosome III centromere, without the necessity for self spore clone mating or the high frequency of gene conversion and rapid asymmetric LOH observed in genetically unstable yeasts. This phenomenon is enough in itself to explain the high level of homozygosis found in natural populations of wine yeasts. The LOH process for centromere-linked markers would be slower than that for the nonlinked markers, because the linkage decreases the frequency of newly originated heterozygous yeasts after each round of sporulation and mating. This phenomenon is interesting in yeast evolution and may cause important sudden phenotype changes in genetically stable wine yeasts.