Cloning and Nucleotide Sequence of the KHS Killer Gene of Saccharomyces cerevisiae.

Goto, Kuniyasu; Fukuda, Hisashi; Kichise, Kouji; Kitano, Kazuyoshi; Hara, Shodo

doi:10.1271/bbb1961.55.1953

Cited by 27 publications

(27 citation statements)

References 2 publications

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“…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 sequence itself (5Ј-GAAAAA).…”

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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

Rodríguez-Cousiño

Maqueda

Ambrona

et al. 2011

Appl Environ Microbiol

113

149

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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...

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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

Rodríguez-Cousiño

Maqueda

Ambrona

et al. 2011

Appl Environ Microbiol

113

149

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“…Most killer systems involve either cytoplasmic dsRNA ("killer viruses," such as the M1 virus of S. cerevisiae) or cytoplasmic dsDNA ("killer plasmids," such as the pGKL1 plasmid of Kluyveromyces lactis). More rarely, polymorphic nuclear genes have been implicated in killer phenotypes ("chromosomal killers"); the best known of these are the KHS1 and KHR1 genes of S. cerevisiae (17,18) and the SMK1 gene of Pichia farinosa (56). Killer yeasts produce and secrete extracellular toxin proteins to which they are immune or resistant but which can kill sensitive strains of the same or different species.…”

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“…CELL same family of plasmids is also genuine; otherwise, it would be an extraordinary coincidence. The 25 locus is a relatively long (974 bp) but degraded pseudogene with similarity to a family of chromosomal genes that includes the S. cerevisiae chromosomal killer gene KHS1 (17). The KHS (killer, heat-sensitive) phenotype involves a chromosomally encoded thermolabile toxin that kills sensitive strains.…”

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“…We believe that there are errors in the original report of the sequence of KHS1, from S. cerevisiae strain no. 115 (17), because it differs from all other sequences by many frameshifts and by a 1.4-kb inversion that ends at a restriction site used in cloning. More interestingly, the coding region of KHS1 from S. cerevisiae strains YJM789 and M22 is much more similar to its ortholog in Saccharomyces paradoxus (99% DNA sequence identity) than to the null alleles in other strains of S. cerevisiae (89% identity).…”

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Evolutionary Capture of Viral and Plasmid DNA by Yeast Nuclear Chromosomes

Frank

Wolfe

2009

Eukaryot Cell

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A 10-kb region of the nuclear genome of the yeast Vanderwaltozyma polyspora contains an unusual cluster of five pseudogenes homologous to five different genes from yeast killer viruses, killer plasmids, the 2m plasmid, and a Penicillium virus. By further database searches, we show that this phenomenon is not unique to V. polyspora but that about 40% of the sequenced genomes of Saccharomycotina species contain integrated copies of genes from DNA plasmids or RNA viruses. We propose the name NUPAVs (nuclear sequences of plasmid and viral origin) for these objects, by analogy to NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) of organellar origin. Although most of the NUPAVs are pseudogenes, one intact and active gene that was formed in this way is the KHS1 chromosomal killer locus of Saccharomyces cerevisiae. We show that KHS1 is a NUPAV related to M2 killer virus double-stranded RNA. Many NUPAVs are located beside tRNA genes, and some contain sequences from a mixture of different extrachromosomal sources. We propose that NUPAVs are sequences that were captured by the nuclear genome during the repair of double-strand breaks that occurred during evolution and that some of their properties may be explained by repeated breakage at fragile chromosomal sites.It is well known that the nuclear genomes of most eukaryotes contain integrated fragments of organellar DNA called NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) (26,29,44,45,57). These fragments are usually pseudogenes, although some NUMTs and NUPTs have become incorporated into functional nuclear genes (38). The NUMTs present in the nuclear genomes of Saccharomycotina yeast species were recently analyzed by Sacerdot et al. (48).In addition to their mitochondrial genomes, yeast species contain a variety of other extranuclear DNA and RNA elements, including viruses and plasmids. These extrachromosomal elements are usually considered to be autonomous entities that do not interact with nuclear DNA. When our laboratory sequenced the genome of the yeast Vanderwaltozyma polyspora (synonym: Kluyveromyces polysporus) (49), we were therefore surprised to find the genomic region we describe here, which contains integrated fragments of several plasmid-and virus-like sequences. We propose that this region was formed by the capture of plasmid and viral sequences by the same mechanism that captures mitochondrial DNA to form NUMTs (43, 65). In a literature search, we could find only one previous report of a similar finding: Utatsu et al. (59) reported the sequences of two regions of nuclear DNA from Zygosaccharomyces rouxii that were highly similar to parts of the 2m-like plasmid pSR1 from that species, but rearranged.Before describing the V. polyspora region, and similar regions found in other species, we will first briefly introduce the extrachromosomal RNA and DNA entities that are known to exist in yeasts. Extrachromosomal nucleic acids are relatively uncommon in yeasts: a broad...

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“…Since first discovered in Saccharomyces cerevisiae (2), killer strains have been isolated from several yeast genera, including Candida (46), Cryptococcus (10), Hanseniaspora (33), Kluyveromyces (14), Pichia (27), Torulopsis (7), Ustilago (30), Williopsis (45), and Zygosaccharomyces (32). Based on killing and immunity interactions among killer yeasts, killer phenotypes are classified into at least 10 groups (48) and the responsible genes may be carried on a chromosome (S. cerevisiae KHS, KHR, Williopsis mrakii) (11,12,21), on a double-stranded RNA (dsRNA) (S. cerevisiae K1, K2, K28, Ustilage maydis, Hanseniaspora uvarum) (5,15,22,35,49), or on a linear double-stranded DNA (dsDNA) (Kluyveromyces lactis, Pichia inositovora, Pichia acaciae) (14,16,44).Schwanniomyces occidentalis produces amylolytic enzymes, including ␣-amylase and glucoamylase (8). It is one of the few yeasts capable of completely hydrolyzing soluble starch.…”

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Isolation, Purification, and Characterization of a Killer Protein fromSchwanniomyces occidentalis

Chen¹,

Han²,

Jong

et al. 2000

Appl Environ Microbiol

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The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. Killer yeasts secrete toxins lethal to sensitive yeasts but are immune to their own toxins. Since first discovered in Saccharomyces cerevisiae (2), killer strains have been isolated from several yeast genera, including Candida (46), Cryptococcus (10), Hanseniaspora (33), Kluyveromyces (14), Pichia (27), Torulopsis (7), Ustilago (30), Williopsis (45), and Zygosaccharomyces (32). Based on killing and immunity interactions among killer yeasts, killer phenotypes are classified into at least 10 groups (48) and the responsible genes may be carried on a chromosome (S. cerevisiae KHS, KHR, Williopsis mrakii) (11,12,21), on a double-stranded RNA (dsRNA) (S. cerevisiae K1, K2, K28, Ustilage maydis, Hanseniaspora uvarum) (5,15,22,35,49), or on a linear double-stranded DNA (dsDNA) (Kluyveromyces lactis, Pichia inositovora, Pichia acaciae) (14,16,44).Schwanniomyces occidentalis produces amylolytic enzymes, including ␣-amylase and glucoamylase (8). It is one of the few yeasts capable of completely hydrolyzing soluble starch. Moreover, it can grow to high cell mass by utilizing cheap starch from plants such as cassava, corn, potato, sorghum, and wheat as the carbon source (40). S. occidentalis has been used to produce ethanol and single cell protein from starch fermentation (19,42). S. occidentalis has no detectable extracellular proteases and can secrete large proteins (40). It also has an established transformation system and available inducible promoters, which could make it a commercially important system for the production of heterologous proteins (40). For example, endoglucanase D recently has been successfully expressed and secreted in this system (31).Wild killer yeasts sometimes contaminate cultures of industrial yeasts, resulting in lagging or stopped fermentation and poor product quality (39). To avoid such complications, the use of industrial killer strains as starters has been suggested (17). Commercially interesting killer strains for sake brewing, wine making, alcohol fermentation, and lager, beer, and ale production have been constructed (29,36,39,47). Furthermore, the W. mrakii mycocin expressed by Aspergillus niger can reduce silage and yogurt spoilage caused by yeasts (25).The killer phenotype of S. occidentalis has not been described previously. Thus, our objectives in this study were as follows: (i) to screen killer strains from S. occidentalis for a killer phenotype; (ii...

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Cloning and Nucleotide Sequence of the KHS Killer Gene of Saccharomyces cerevisiae.

Cited by 27 publications

References 2 publications

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

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

Evolutionary Capture of Viral and Plasmid DNA by Yeast Nuclear Chromosomes

Isolation, Purification, and Characterization of a Killer Protein fromSchwanniomyces occidentalis

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