The occurrence of killer character in yeasts of various genera.

Species of 7 of the 28 yeast genera in the National Collection of Yeast Cultures exhibited killing activity against Saccharomyces cerevisiae. The highest incidence of killer yeasts was found in the genus Hansenula (12 of the 29 strains examined). Saccharomyces, the best represented genus in the Collection, showed a low incidence of killer activity and many of the killer strains are hybrids with a common S. cerevisiae parent. The activities of culture filtrates of the 59 killer yeast isolated responded differently to pH and four types of response were recognised.     

Isolation, Purification, and Characterization of a Killer Protein from Schwanniomyces occidentalis

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. The protein was stable between pH 2.0 and 5.0 and inactivated at temperatures above 40°C. The killer protein was chromosomally encoded. Mannan, but not β-glucan or laminarin, prevented sensitive yeast cells from being killed by the killer protein, suggesting that mannan may bind to the killer protein. Identification and characterization of a killer strain of S. occidentalis may help reduce the risk of contamination by undesirable yeast strains during commercial fermentations.     

Construction of Killer Wine Yeast Strain

A double-stranded RNA plasmid which confers the superkiller phenotype was transferred into a wine yeast (Montrachet strain 522) and its leucine-requiring derivative (strain 694) by cytoduction, using the protoplast fusion technique. The killer wine yeast constructed completely suppressed the growth of killer-sensitive strains of Saccharomyces cerevisiae in yeast extract-peptone-glucose medium at pH 4.5, whereas the killer effect was somewhat decreased at pH 3.5. The wine yeast harboring the killer factor also inhibited the growth of killer-sensitive cells satisfactorily when it was grown in grape juice.     

A novel killer toxin produced by the marine-derived yeast Wickerhamomyces anomalus YF07b

In our previous study, it was found that the killer toxin produced by the marine-derived yeast Wickerhamomyces anomalus YF07b has both killing activity and β-1,3-glucanase activity and the molecular mass of it is 47.0 kDa. In this study, the same yeast strain was found to produce another killer toxin which only had killing activity against some yeast strains, but had no β-1,3-glucanase activity and the molecular mass of the purified killer toxin was 67.0 kDa. The optimal pH, temperature and NaCl concentration for action of the purified killer toxin were 3.5, 16 °C and 4.0 % (w/v), respectively. The purified killer toxin could be bound by the whole sensitive yeast cells, but was not bound by manann, chitin and β-1,3-glucan. The purified killer toxin had killing activity against Yarrowia lipolytica, Saccharomyces cerevisiae, Metschnikowia bicuspidata WCY, Candida tropicalis, Candida albicans and Kluyveromyces aestuartii. Lethality of the sensitive cells treated by the newly purified killer toxin from W. anomalus YF07b involved disruption of cellular integrity by permeabilizing cytoplasmic membrane function.     

Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts

An excellent wine yeast, Saccharomyces cerevisiae W3, which had KHR killer, was added as a starter yeast into grape must and behavior of the starter strain and wild yeasts was investigated during fermentation by using KHR killer as a genetic marker. The KHR killer was detected only in the strain W3 and not in other wine and wild yeast strains. Accordingly, the frequency of starter yeast W3 was monitored throughout the fermentation of grape musts by using KHR killer, W3 was discriminated efficiently from wild yeasts during fermentation by KHR killer activity and proved to lead the fermentation as a dominant yeast until their termination.     

Occurrence of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> killer yeasts in wine-producing areas of Greece

The occurrence of the killer character was studied in 840 Saccharomyces cerevisiae isolates at the end of spontaneous fermentations in four wine-producing regions of Greece, Mantinia and Nemea in Peloponnese and the islands Limnos and Santorini. The incidence of killer strains varied from one region to another. Sensitive and neutral strains were also found among the S. cerevisiae strains. Using the plate bioassay at pH 4.5 two different killer phenotypes were detected among the killer isolates which differed in their degree of killer activity. They were designated as SK (strong killer) and WK (weak killer). The proportion of SK to WK phenotypes differed from one area to another. All killer isolates were assayed for expression of killer activity also at pH 3.5. The lower pH decreased the killer activity of all isolates and changed the proportion of SK to WK phenotypes. The percentage of WK phenotypes increased in all cases and some killer isolates lost their killer activity completely, but generally the killer activity remained significant, especially in the area of Mantinia where the SK phenotype remained dominant at the low pH found in musts and wine.     

Influence of killer strains of Saccharomyces cerevisiae on wine fermentation

The effect of killer strains of Saccharomyces cerevisiae on the growth of sensitive strains during must fermentation was studied by using a new method to monitor yeast populations. The capability of killer yeast strains to eliminate sensitive strains depends on the initial proportion of killer yeasts, the susceptibility of sensitive strains, and the treatment of the must. In sterile filtered must, an initial proportion of 2-6% of killer yeasts was responsible for protracted fermentation and suppression of isogenic sensitive strains. A more variable initial proportion was needed to get the same effect with non-isogenic strains. The suspended solids that remain in the must after cold-settling decreased killer toxin effect. The addition of bentonite to the must avoided protracted fermentation and the suppression of sensitive strains; however, the addition of yeast dietary nutrients with yeast cell walls did not, although it decreased fermentation lag.     

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.     

Scent of a killer: How could killer yeast boost its dispersal?

Vector‐borne parasites often manipulate hosts to attract uninfected vectors. For example, parasites causing malaria alter host odor to attract mosquitoes. Here, we discuss the ecology and evolution of fruit‐colonizing yeast in a tripartite symbiosis—the so‐called “killer yeast” system. “Killer yeast” consists of Saccharomyces cerevisiae yeast hosting two double‐stranded RNA viruses (M satellite dsRNAs, L‐A dsRNA helper virus). When both dsRNA viruses occur in a yeast cell, the yeast converts to lethal toxin‑producing “killer yeast” phenotype that kills uninfected yeasts. Yeasts on ephemeral fruits attract insect vectors to colonize new habitats. As the viruses have no extracellular stage, they depend on the same insect vectors as yeast for their dispersal. Viruses also benefit from yeast dispersal as this promotes yeast to reproduce sexually, which is how viruses can transmit to uninfected yeast strains. We tested whether insect vectors are more attracted to killer yeasts than to non‑killer yeasts. In our field experiment, we found that killer yeasts were more attractive to Drosophila than non‐killer yeasts. This suggests that vectors foraging on yeast are more likely to transmit yeast with a killer phenotype, allowing the viruses to colonize those uninfected yeast strains that engage in sexual reproduction with the killer yeast. Beyond insights into the basic ecology of the killer yeast system, our results suggest that viruses could increase transmission success by manipulating the insect vectors of their host.     

A novel yeast gene, RHK1, is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits β-1,3-glucan synthesis

The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall β-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast. A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin. These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups. A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated. DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids. Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S. cerevisiae cells with complete killer resistance. A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of β-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin.     

The Species-Specific Acquisition and Diversification of a K1-like Family of Killer Toxins in Budding Yeasts of the Saccharomycotina

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.     

Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of M_r=16,000. The amino acid composition of the toxin type K₂ of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.     

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.     

Isolation and identification of killer yeasts from Agave sap (aguamiel) and pulque

Wild killer yeasts have been identified as inhibitory to strains used as starters in the production of alcoholic beverages such as beer and wine; therefore, killer or killer-resistant strains have been sought for use in alcoholic fermentations. In the current paper a total of 16 strains belonging to six species were isolated. From two samples of Agave sap (aguamiel) the following yeast strains were isolated: Candida lusitaneae (1), Kluyveromyces marxianus var. bulgaricus (2), and Saccharomyces cerevisiae (capensis) (1). Additionally, in seven samples of pulque (the fermented product), the species C. valida (six strains), S. cerevisiae (chevalieri) (4), S. cerevisiae (capensis) (1), and K. marxianus var. lactis (1) were found. The killer strains were C. valida and K. marxianus var. lactis from pulque and K. marxianus var. bulgaricus from aguamiel. One strain of S. cerevisiae (chevalieri) isolated from pulque which did not show killer activity was, on the other hand, resistant to other killer strains and it had a remarkable ethanol tolerance, suggesting that this strain could be used for alcohol production.     

Killer yeasts of Kluyveromyces and Hansenula genera with potential application in fermentation and therapy

The killing/immunity interactions among killer strains of the genera Kluyveromyces, Hansenula and Saccharomyces from the Czechoslovak Collection of Yeasts were studied with the aim to find the strains with broad specificity and killer activity targeted against a range of undesirable wild yeasts causing stuck fermentations. Among 49 tested Kluyveromyces strains, five strains were found, and among 55 Hansenula strains, ten yeast strains were found with activity against a sensitive strain of Saccharomyces. Hansenula mrakii CCY 38-7-1 and Hansenula saturnus var. subsufficiens CCY 38-4-2 showed exceptional activity against the wine contaminants, Zygosaccharomyces bailii, as well as against pathogenic Candida species within a broad range of pH 2.9–5.1. Their potential biotechnological application is discussed.     

Characterization of yeast starter cultures used in household alcoholic beverage preparation by a few ethnic communities of Northeast India

In this study, we report the characterization of traditional household starter cultures from a few ethnic groups of northeast India. Pure cultures obtained from the study have been deposited in the Microbial Type Culture Collection (MTCC), India. These isolates have been analyzed for their growth characteristics, sensitivity to temperature and pH, alcohol tolerance, alcohol production, and alcohol dehydrogenase (ADH) content. The pure cultures obtained from different starter cultures revealed the presence of Debaryomyces, Wickerhamomyces, and Candida along with the fermenting yeast Saccharomyces. The growth behavior at different temperatures, pH and alcohol tolerance revealed numerous facts and behavior of the yeast strains associated with traditional alcoholic fermentation. All the isolates were found to be thermotolerant up to 37 °C, fairly pH-resistant, good in ADH secretion, and with appreciable alcohol production. For all the strains studied, the Saccharomyces cerevisiae MTCC 3976 strain from the Tea Tribes of Assam and the Wickerhamomyces anomalus MTCC 3979 from the Apatani Tribe of Arunachal Pradesh were found to be exceptional in terms of thermotolerance, alcohol tolerance, alcohol production, and ADH activity, and hence may be identified as potential strains for industrial fermentation.     

Occurrence and Growth of Killer Yeasts during Wine Fermentation

Sixteen wine fermentations were examined for the presence of killer yeasts. Killer property and sensitivity to killer action were found in isolates of Saccharomyces cerevisiae but not in isolates of Kloeckera, Candida, Hansenula, and Torulaspora spp. Several killer and killer-sensitive strains of S. cerevisiae were differentiated by colony morphology, and this property was used to monitor their growth kinetics in mixed cultures in grape juice. Killer-sensitive strains died off within 24 to 48 h during mixed-strain fermentation. Killer action was demonstrated at pH 3.0 and pH 3.5 and over the range of 15 to 25°C but depended on the proportion of killer to killer-sensitive cells at the commencement of fermentation. The dominance of killer strains in mixed-strain fermentations was reflected in the production of ethanol, acetic acid, and glycerol.     

Protective and reactivating effect of the protein exometabolite on yeast cells inactivated by the ultraviolet irradiation

It has been shown that Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida utilis strains produce the protein exometabolites, which has a protective and reactivating effect on the ultraviolet irradiated yeast cells. The protective effect of the preliminary ultraviolet irradiated (activated) protein exometabolite of all strains increased 2–3 times, though its reactivating activity did not change. Yarrowia lipolytica yeast cells, isolated from the areas with the high daily irradiation, and Endomyces magnusii, the obligate fungi parasites, were characterized by the highest ultraviolet tolerance in comparison with the other strains. However, they did not produce the exometabolites with the antistress effect. Luteococcus casei reactivating factor demonstrated protective and reactivating cross-action in relation to the ultraviolet irradiated S. cerevisiae, K. lactis, and C. utilis cells and were inactive in relation to Y. lipolytica and E. magnusii. Using killer and nonkiller S. cerevisiae strain, it has been shown that the peptide exometabolite accumulation was not associated with toxin production.     

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background

Virus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/β heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants.

Results

In this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the β-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding.

Conclusion

Successful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.

     

A population study of killer viruses reveals different evolutionary histories of two closely related Saccharomyces sensu stricto yeasts

Microbes have evolved ways of interference competition to gain advantage over their ecological competitors. The use of secreted killer toxins by yeast cells through acquiring double‐stranded RNA viruses is one such prominent example. Although the killer behaviour has been well studied in laboratory yeast strains, our knowledge regarding how killer viruses are spread and maintained in nature and how yeast cells co‐evolve with viruses remains limited. We investigated these issues using a panel of 81 yeast populations belonging to three Saccharomyces sensu stricto species isolated from diverse ecological niches and geographic locations. We found that killer strains are rare among all three species. In contrast, killer toxin resistance is widespread in Saccharomyces paradoxus populations, but not in Saccharomyces cerevisiae or Saccharomyces eubayanus populations. Genetic analyses revealed that toxin resistance in S. paradoxus is often caused by dominant alleles that have independently evolved in different populations. Molecular typing identified one M28 and two types of M1 killer viruses in those killer strains. We further showed that killer viruses of the same type could lead to distinct killer phenotypes under different host backgrounds, suggesting co‐evolution between the viruses and hosts in different populations. Taken together, our data suggest that killer viruses vary in their evolutionary histories even within closely related yeast species.