Killer systems in Saccharomyces cerevisiae: three distinct modes of exclusion of M2 double-stranded RNA by three species of double-stranded RNA, M1, L-A-E, and L-A-HN.

M1 and M2 double-stranded RNAs (dsRNAs) code for the K1R1 and K2R2 killer toxin and resistance functions, respectively. Natural variants of a larger dsRNA (L-A) carry various combinations of the [EXL], [HOK], and [NEX] genes, which affect the K1 and K2 killer systems. Other dsRNAs, the same size as L-A, called L-B and L-C, are often present with L-A. We show that K1 killer strains have [HOK] and [NEX] but not [EXL] on their L-A (in disagreement with Field et al., Cell 31:193-200, 1982). These strains also carry other L-size molecules detectable after heat-curing has eliminated L-A. The exclusion of M2 dsRNA observed on mating K2 strains with K1 strains is due to the M1 dsRNA (not the L-A dsRNA as claimed by Field et al.) in the K1 strains. Four independent mutants of a [KIL-k2] [NEX-o] [HOK-o] strain were selected for resistance to [EXL] exclusion of M2 ([EXLR] phenotype). The [EXLR] phenotype showed non-Mendelian inheritance in each case, and these mutants had simultaneously each acquired [HOK]. The mutations were located on L-A and not on M2, and did not confer resistance to M1 exclusion of M2.     

酿酒酵母两种不同嗜杀菌的比较研究

以酿酒酵母两种不同类型的嗜杀菌株ＳＫ４（Ｋ１型）和ＥＲＲ１（Ｋ２型）为材料,分析了不同嗜杀酵母的嗜杀特性,两株嗜杀酵母具有相互杀死作用,其嗜杀活性与菌体生长有关。ＳＫ４和ＥＲＲ１的嗜杀质粒的比较表明：Ｍ１－ｄｓＲＮＡ质粒和Ｍ２－ｄｓＲＮＡ质粒分子量分别为１．７ｋｂ和１．５ｋｂ,两株菌的Ｌ－ｄｓＲＮＡ质粒均为４．０ｋｂ。用高温和紫外线处理嗜杀酵母,嗜杀活性随之消失,消除菌中的Ｍ－ｄｓＲＮＡ质粒也相应     

Virus-like particle capsid proteins encoded by different L double-stranded RNAs of Saccharomyces cerevisiae: their roles in maintenance of M double-stranded killer plasmids.

The plasmid determinants of killer phenotypes in type K1 and K2 killer yeast cells are the 1.9-kilobase (kb) M1 and 1.7-kb M2 double-stranded RNAs (dsRNAs), respectively. These are dependent for their maintenance and encapsidation, in Saccharomyces cerevisiae virus ScV-M1 or ScV-M2 virus-like particles, on the capsid provided by one of a group of moderately related 4.7-kb dsRNAs called LA. The L1A and L2A dsRNAs found in naturally isolated K1 and K2 killers encode 88-kilodalton VL1A-P1 and 86-kilodalton VL2A-P1 capsids, respectively. These are competent for encapsidating homologous LA dsRNAs as well as M dsRNAs. Most strains of S. cerevisiae, including killers, contain one of a second group of closely related 4.7-kb dsRNAs called LBC. These encode their own 82-kilodalton capsid protein, VLBC-P1, which, at least in strains containing only LBC, encapsidates homologous dsRNA in ScV-LBC virus-like particles. In a K1 killer strain containing both L1A and LBC, ScV-M1 particles contain only VL1A-P1. In such strains it is probable that each virus-like particle contains a single capsid type and that each L dsRNA is encapsidated by a homologous capsid.     

Mutual antagonism among killer yeasts: competition between K1 and K2 killers and a novel cDNA-based K1-K2 killer strain of Saccharomyces cerevisiae

Mutually antagonistic K1 and K2 killer strains compete when mixed and serially subcultured. At pH 4.6, where the K1 killer toxin is more stable in vitro, the K1 strain outcompeted the K2 strains at both 18 and 30 degrees C. At pH 4.0, closer to the in vitro pH optimum of the K2 killer toxin, the K1 strain again predominated at 18 degrees C, but at 30 degrees C the K2 strains became the sole cell type on subculture. To show more clearly that these results were dependent upon the respective killer toxins, control experiments were conducted with isogenic, nonkiller strains cured of the dsRNA-based killer virions. Such nonkiller strains were unable to compete with antagonistic killers under conditions where their isogenic killer parents could, strongly suggesting that the killer phenotype was important in these competitions. Double K1-K2 killer strains cannot stably exist, as their dsRNA genomes compete at a replicative level. Using recombinant DNA methodology, a stable K1-K2 killer strain was constructed. This strain outcompeted both K1 and K2 killers when serially subcultured under conditions where either the K1 or the K2 strains would normally predominate in mixed cultures. Such a double killer may be useful in commercial fermentations, where there is a risk of contamination by killer yeasts.     

Killer toxin producing strains of the yeasts Hanseniaspora uvarum and Pichia kluyveri

By heat treatment killer strains of the type K1 of Saccharomyces cerevisiae that are known to harbour dsRNA plasmids were completely cured, whereas only a small fraction of the clones of the killer type K2 had lost the dsRNA dependent killer character. The K2 killers but not the strains of killer type K1 were easily cured by cycloheximide. Killer strains of Hanseniaspora uvarum were not curable by heat treatment. Curing was successfull with cycloheximide or 5-fluorouracil. Two double-stranded RNA plasmids were detected in the killer strains of H. uvarum. The smaller dsRNA plasmid was absent in the strains that were cured of their killer character by 5-fluorouracil. The killer character of H. uvarum was transferred to S. cerevisiae by spheroplast fusion. The fusion products showing the killer character contained both dsRNA plasmids, obviously the smaller plasmid (M-dsRNA) carries the genes for killer toxin formation. Killer strains of Pichia kluyveri were not curable of their killer character, in these strains no dsRNA plasmids were detected.This paper was kindly supported by a grant from the Deutsche Forschungsgemeinschaft     

Presence of non-suppressive, M2-related dsRNAs molecules in Saccharomyces cerevisiae strains isolated from spontaneous fermentations

Total dsRNA extractions in five killer K2 strains of Saccharomyces cerevisiae isolated from spontaneous fermentations revealed the presence of a novel dsRNA fragment (which we named NS dsRNA) of approximately 1.30 kb, together with L and M2 dsRNAs. NS dsRNA appeared to be encapsidated in the same kind of viral particles as L and M2 dsRNA. Northern blot hybridization experiments indicated that NS dsRNA was derived from M2 dsRNA, likely by deletion of the internal A+U-rich region. However, unlike S dsRNAs (suppressive forms derived from M1 dsRNA in K1 killers), NS dsRNA did not induce exclusion of the parental M2 dsRNA when the host strain was maintained for up to 180 generations of growth.     

Construction and properties of K1 type killer wine yeasts

Summary With the use of a protoplast fusion technique the killer character of K1 type was transferred into four industrial Saccharomyces wine yeasts. The prototrophic yeast strains active against standard sensitive and K2 killer Saccharomyces strains, resistant to K1 killer toxin were constructed with no changes in technological properties.     

Different action of killer toxins K1 and K2 on the plasma membrane and the cell wall of Saccharomyces cerevisiae

Study of Saccharomyces cerevisiae killer toxin-sensitive strains with the deltakre2 phenotype (resistant to toxin K1, sensitive to toxin K2) showed that the phenotype is complemented by the KRE2 gene not only in intact cells but also in spheroplasts, and resistance to K1 thus resides very probably in the plasma membrane. deltakre1 deletant displays a faulty interaction with both K1 and K2 toxin. Hence, Kre1p probably serves as plasma membrane receptor for both toxins. Deletants in seven other genes (GDA1, SAC1, LUV1, KRE23, SAC2, KRE21, ERG4) exhibit different degrees of the deltakre2-like resistance pattern, but the phenotype in deltagda1 and deltasac1 is not connected with a defect in K1 toxin interaction with the plasma membrane, similarly as in deltakre6 and deltakre11 strains with a higher resistance to K2 toxin. Differences between the K1 and K2 killer toxin thus occur on the level of both the plasma membrane and the cell wall.     

Kinetic study and mathematical modelling of killer and sensitive S. Cerevisiae strains growing in mixed culture

This paper presents a kinetic study of two yeasts growing in pure and mixed batch cultures. Two winemaking strains were used: S. cerevisiae K1 possessing the K2 killer character and S. cerevisiae 522D sensitive to the K2 killer toxin. Initially the kinetics of growth of the two strains were analysed in pure culture. In this case, the kinetic profiles of biomass production have shown that the growth rate of the K1 strain is slightly superior to the 522D strain. During the fermentation, the viability for both populations was higher than 90%. Fermentations in mixed culture with an initial percentage in killer strain of 5 and 10% with respect to the total population were carried out. The results showed a more important decrease in the percentage of total viable yeasts when the initial concentration of killer yeast increased. However, the kinetic profiles of total biomass (killer plus sensitive yeasts) were very similar for both fermentations. A mathematical model was proposed to simulate the microbial growth of the killer and sensitive strain developing in pure and mixed cultures. This mathematical model consists in three main reactions: the evolution of the killer toxin in the culture medium, the duplication and the mortality rates for each microbial population. The results of the simulation appeared in agreement with the experimental data.     

Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain

Killer yeasts secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. Among the four types of Saccharomyces killer yeasts already described (K1, K2, K28, and Klus), we found K2 and Klus killer yeasts in spontaneous wine fermentations from southwestern Spain. Both phenotypes were encoded by medium-size double-stranded RNA (dsRNA) viruses, Saccharomyces cerevisiae virus (ScV)-M2 and ScV-Mlus, whose genome sizes ranged from 1.3 to 1.75 kb and from 2.1 to 2.3 kb, respectively. The K2 yeasts were found in all the wine-producing subareas for all the vintages analyzed, while the Klus yeasts were found in the warmer subareas and mostly in the warmer ripening/harvest seasons. The middle-size isotypes of the M2 dsRNA were the most frequent among K2 yeasts, probably because they encoded the most intense K2 killer phenotype. However, the smallest isotype of the Mlus dsRNA was the most frequent for Klus yeasts, although it encoded the least intense Klus killer phenotype. The killer yeasts were present in most (59.5%) spontaneous fermentations. Most were K2, with Klus being the minority. The proportion of killer yeasts increased during fermentation, while the proportion of sensitive yeasts decreased. The fermentation speed, malic acid, and wine organoleptic quality decreased in those fermentations where the killer yeasts replaced at least 15% of a dominant population of sensitive yeasts, while volatile acidity and lactic acid increased, and the amount of bacteria in the tumultuous and the end fermentation stages also increased in an unusual way.     

Structure and expression of the M2 genomic segment of a type 2 killer virus of yeast.

The M2 double-stranded (ds) RNA species encodes toxin and resistance functions in Saccharomyces cerevisiae strains with the K2 killer specificity. RNA sequence analysis reveals the presence of a large open reading frame on the larger heat-cleavage product of M2 dsRNA, which is translated in vitro to yield a 28 kd polypeptide as a major product. The postulated translation initiator AUG triplet is located within a stem and loop structure near the 5' terminus of the positive strand, which also contains plausible 18S and 5.8S ribosomal RNA binding sites. These features may serve to regulate the translation of the K2 toxin precursor. The M1 (from type 1 yeast killers) and M2 dsRNA species lack extensive sequence homology, although specific features are shared, which may represent structural elements required for gene expression and replication.     

Yeast killer plasmid mutations affecting toxin secretion and activity and toxin immunity function.

M double-stranded RNA (MdsRNA) plasmid mutants were obtained by mutagenesis and screening of a diploid killer culture partially heat cured of the plasmid, so that a high proportion of the cells could be expected to have only on M plasmid. Mutants with neutral (nonkiller [K-], immune [R+]) or suicide (killer [K+], sensitive [R-] phenotypes were examined. All mutants became K- R- sensitives on heat curing of the MdsRNA plasmid, and showed cytoplasmic inheritance by random spore analysis. In some cases, M plasmid mutations were indicated by altered mobility of the MdsRNA by agarose gel electrophoresis or by altered size of in vitro translation products from denatured dsRNA. Neutral mutants were of two types: nonsecretors of the toxin protein or secretors of an inactive toxin. Of three neutral nonsecretors examined, one (NLP-1), probably a nonsense mutation, made a smaller protoxin precursor in vitro and in vivo, and two made full-size protoxin molecules. The in vivo protoxin of 43,000 molecular weight was unstable in the wild type and kinetically showed a precursor-product relationship to the processed, secreted 11,000-molecular-weight toxin. In one nonsecretor (N1), the protoxin appeared more stable in a pulse-chase experiment, and could be altered in a recognition site required for protein processing.     

Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains.

The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations. The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity. A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation. The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions. Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100). A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown. An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations. In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis. The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed.     

Characterization of killer-resistant strains of Saccharomyces cerevisiae isolated from spontaneous fermentations

A study of 26 killer-resistant wine strains of Saccharomyces cerevisiae, isolated during spontaneous fermentations in three vineyards in NW Spain, was carried out employing several methods that included a spheroplast-killing assay and analysis of chromosomal DNA patterns by pulse-field agarose electrophoresis. The results showed that 92% of the strains were derivatives of K2 killer toxin producing wine strains isolated from the same fermentations, and that they could be grouped into four different karyotypes. The remaining strains were killer-resistant at cell-wall level and were not related to the others, as was demonstrated by the absence of L and M ds-RNAs and by their different karyotypes.     

Nuclear mutants of Saccharomyces cerevisiae K2 yeasts with decreased killer activity

Recessive mutations in two chromosomal unlinked genes kir1 and kir2 of Saccharomyces cerevisiae K2 result in weak killer activity or in complete loss of killer capacity. Kir1 is located on chromosome 7 and is linked to ade7 and ski6. The kir1 and kir2 mutants reveal no alteration of cell membrane. They normally excrete acid phosphatase and have a normal level of mating and sporulation. The analysis of the plasmid nucleic acid in two strains containing the mutant alleles kir1-12 and kir2-23 shows the increased content of L double-stranded DNA, the content of M double-stranded RNA being increased.     

Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character.

Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267. pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively. Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI. pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromyces thermotolerans, and K. vanudenii. All K. lactis strains lacking the pGK1 plasmids were nonkillers. A hybrid was constructed between K. lactis IFO 1267 and a nonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation. The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids. The double-stranded ribonucleic acid killer plasmid could not be detected in any K. lactis killer strains. It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids. A single chromosomal gene was found which was responsible for the resistance to the K. lactis killer.     

Killer activity of Saccharomyces cerevisiae strains: partial characterization and strategies to improve the biocontrol efficacy in winemaking

Killer yeasts are considered potential biocontrol agents to avoid or reduce wine spoilage by undesirable species. In this study two Saccharomyces cerevisiae strains (Cf8 and M12) producing killer toxin were partially characterized and new strategies to improve their activity in winemaking were evaluated. Killer toxins were characterized by biochemical tests and growth inhibition of sensitive yeasts. Also genes encoding killer toxin were detected in the chromosomes of both strains by PCR. Both toxins showed optimal activity and production at conditions used during the wine-making process (pH 3.5 and temperatures of 15–25 °C). In addition, production of both toxins was higher when a nitrogen source was added. To improve killer activity different strategies of inoculation were studied, with the sequential inoculation of killer strains the best combination to control the growth of undesired yeasts. Sequential inoculation of Cf8–M12 showed a 45 % increase of killer activity on sensitive S. cerevisiae and spoilage yeasts. In the presence of ethanol (5–12 %) and SO₂ (50 mg/L) the killer activity of both toxins was increased, especially for toxin Cf8. Characteristics of both killer strains support their future application as starter cultures and biocontrol agents to produce wines of controlled quality.