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. Saccharomyces cerevisiae killer strains produce and secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. These toxins have been grouped into three types, K1, K2, or K28, based on their killing profiles and lack of cross-immunity. Members of each group can kill nonkiller yeasts as well as killer yeasts belonging to the other types. They are immune, however, to their own toxin or to toxins produced by strains of the same killer type (for reviews, see references 21, 32, 33, and 47).K1, K2, and K28 killer toxins are genetically encoded by medium-size double-stranded RNA (dsRNA) viruses grouped into three types, M1, M2, and M28, of 1.6, 1.5, and 1.8 kb, respectively. Only one strand (the positive strand) has coding capacity. In each case, the 5Ј-end region contains an open reading frame (ORF) that codes for the toxin precursor, or preprotoxin (pptox), which also provides immunity. The three toxin-coding M dsRNAs show no sequence homology to each other (35). M viruses depend on a second large (4.6-kb) dsRNA helper virus, L-A, for maintenance and replication. L-A provides the capsids in which both L-A and M dsRNAs are separately encapsidated (reviewed by Schmitt and Breinig [33]). L-BC virus is an L-A-related virus, with a similar 4.6-kb genome size, which coexists with L-A in most killer and nonkiller S. cerevisiae strains (1, 37). L-BC shows no sequence homology with L-A, and it has no known helper activity. L-A and L-BC, however, share the same genomic organization. They code for two proteins, the major coat protein Gag and a minor Gag-Pol fusion protein translated by a Ϫ1 ribosomal frameshifting mechanism (7,10,17,26). These viruses, called Saccharomyces cerevisiae viruses (ScVs), belong to the Totiviridae family and are cytoplasmically inherited, spreading horizontally by cell-cell mating or by heterokaryon formation (47). In addition to the M dsRNA-encoded killer toxins, other S. cerevisiae killer toxins, named KHR and KHS, showing weak killer activity, are encoded on chromosomal DNA (13,14).The positive strands of both L-A and M viruses contain cis signals in their 3Ј-terminal regions essential for packaging and replication (46). The signal for transcription initiation has been proposed to be present in the first 25 nucleotides (nt) of L-A, probably in the 5Ј-terminal sequ...

Wine Torulaspora delbrueckii strains producing a new killer toxin (Kbarr-1) were isolated and selected for wine making. They killed all the previously known Saccharomyces cerevisiae killer strains, in addition to other non-Saccharomyces yeasts. The Kbarr-1 phenotype is encoded by a medium-size 1.7 kb dsRNA, TdV-Mbarr-1, which seems to depend on a large-size 4.6 kb dsRNA virus (TdV-LAbarr) for stable maintenance and replication. The TdV-Mbarr-1 dsRNA was sequenced by new generation sequencing techniques. Its genome structure is similar to those of S. cerevisiae killer M dsRNAs, with a 5′-end coding region followed by an internal A-rich sequence and a 3′-end non-coding region. Mbarr-1 RNA positive strand carries cis acting signals at its 5′ and 3′ termini for transcription and replication respectively, similar to those RNAs of yeast killer viruses. The ORF at the 5′ region codes for a putative preprotoxin with an N-terminal secretion signal, potential Kex2p/Kexlp processing sites, and N-glycosylation sites. No relevant sequence identity was found either between the full sequence of Mbarr-1 dsRNA and other yeast M dsRNAs, or between their respective toxin-encoded proteins. However, a relevant identity of TdV-Mbarr-1 RNA regions to the putative replication and packaging signals of most of the M-virus RNAs suggests that they are all evolutionarily related.

Wine killer yeasts such as killer strains of Torulaspora delbrueckii and Saccharomyces cerevisiae contain helper large-size (4.6 kb) dsRNA viruses (V-LA) required for the stable maintenance and replication of killer medium-size dsRNA viruses (V-M) which bear the genes that encode for the killer toxin. The genome of the new V-LA dsRNA from the T. delbrueckii Kbarr1 killer yeast (TdV-LAbarr1) was characterized by high-throughput sequencing (HTS). The canonical genome of TdV-LAbarr1 shares a high sequence identity and similar genome organization with its Saccharomyces counterparts. It contains all the known conserved motifs predicted to be necessary for virus translation, packaging, and replication. Similarly, the Gag-Pol amino-acid sequence of this virus contains all the features required for cap-snatching and RNA polymerase activity, as well as the expected regional variables previously found in other LA viruses. Sequence comparison showed that two main clusters (99.2–100% and 96.3–98.8% identity) include most LA viruses from Saccharomyces, with TdV-LAbarr1 being the most distant from all these viruses (61.5–62.5% identity). Viral co-evolution and cross transmission between different yeast species are discussed based on this sequence comparison. Additional 5′ and 3′ sequences were found in the TdV-LAbarr1 genome as well as in some newly sequenced V-LA genomes from S. cerevisiae. A stretch involving the 5′ extra sequence of TdV-LAbarr1 is identical to a homologous stretch close to the 5′ end of the canonical sequence of the same virus (self-identity). Our modeling suggests that these stretches can form single-strand stem loops, whose unpaired nucleotides could anneal to create an intramolecular kissing complex. Similar stem loops are also found in the 3′ extra sequence of the same virus as well as in the extra sequences of some LA viruses from S. cerevisiae. A possible origin of these extra sequences as well as their function in obviating ssRNA degradation and allowing RNA transcription and replication are discussed.

We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae. Spontaneous cyh2 R /cyh2 R mutants resistant to high levels of cycloheximide can be directly isolated from cyh2 S /cyh2 S wine yeasts. Heterozygous cyh2 R /cyh2 S 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 cyh2 R /cyh2 R 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.

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