Killer Yeasts Research Articles

Evidence for DNA Cleavage Caused Directly by a transfer RNA-Targeting Toxin

The killer yeast species Pichia acaciae produces a heteromeric killer protein, PaT, that causes DNA damage and arrests the cell cycle of sensitive Saccharomyces cerevisiae in the S phase. However, the mechanism by which DNA damage occurs remains elusive. A previous study has indicated that Orf2p, a subunit of PaT, specifically cleaves an anticodon loop of an S. cerevisiae transfer RNA (tRNAGln mcm5s2UUG). This finding raised a question about whether the DNA damage is a result of the tRNA cleavage or whether Orf2p directly associates with and cleaves the genomic DNA of sensitive yeast cells. We showed that Orf2p cleaves genomic DNA in addition to cleaving tRNA in vitro. This DNA cleavage requires the same Orf2p residue as that needed for tRNA cleavage, His299. The expression of Orf2p, in which His299 was substituted to alanine, abolished the cell cycle arrest of the host cell. Moreover, the translation impairment induced by tRNA cleavage enabled Orf2p to enter the nucleus, thereby inducing histone phosphorylation.

Analysis of cancer cell death in hepatoma cell line after the treatment of lethal culture extract from Taiwan Barnettozyma spp.

Cancer is the leading cause of death in the world, and therefore, identifying advanced anticancer drugs is the most important objective of cancer research. However, at present, most synthetic anticancer drugs developed cause serious side effects thereby reducing their therapeutic efficiency. In a previous study, we have reported that culture filtrates from Barnettozyma spp. can not only maintain their lethal effect under physical environment, but can also exert stronger cytotoxicity to the hepatoma cell line HepG2 than to the normal Chang's liver cell. Herein, we further classify the types of liver cancer cell death, and identify those proteins responsible for the cell death by matrix-assisted laser desorption/ionization–time-of-flight spectra to study the possible mechanism of lethal culture filtrate from a special Taiwan killer yeast Barnettozyma spp. (EN14S14). Results of our preliminary study indicated that autophagic induction is most likely the major form of HepG2 cell death, which is induced by the “killing” culture filtrate. Proteins differentially expressed after treatment fell into three major categories, namely, metabolism, cytoskeleton, and signal transduction.

Role of killer factors in the inhibitory activity of bio-control yeasts against Penicillium expansum and Aspergillus ochraceus

This work evaluated the antagonism of killer positive yeast strains (isolated from 11 samples of different frozen fruit pulps) against the strains of Penicillium expansum and Aspergillus ochraceus. Of the total 41 killer yeasts tested in YM agar, 19 showed antibiosis against P. expansum and A. ochraceus, with inhibition zone ranging from 10 to 18 mm and 10 to 19 mm, respectively. In the following step, the extracellular activity of Kluyveromyces sp. FP4(13) was tested performing the assay in YM broth. The antifungal activity of Kluyveromyces sp. FP4(13) cell-free culture supernatant (25ºC/96 h) was more effective against the conidia germination, showing inhibition rates of 93.33 and 86.44% for P. expansum and A. ochraceus, respectively. The micelial growth inhibition was 28.45 and 21.0%, respectively. The antagonism showed by the selected yeasts could be used as a promising alternative tool to reduce and control the postharvest fungal spoilage of the fruits. However, further studies should be carried out in order to better elucidate the role of innocuous characters in antagonistic microorganisms, as well as the purification and characterization of new killer toxins.

Susceptibility of species within the Sporothrix schenckii complex to a panel of killer yeasts

The Sporothrix schenckii complex is the etiologic agent of sporotrichosis, a subacute or chronic mycosis which can affect humans and animals. Killer yeasts have been used in the medical field for development of novel antimycotics and biotyping of pathogenic fungi. The action of 18 killer yeasts on the growth of 88 characterized S. schenckii, Sporothrix globosa, Sporothrix brasiliensis, and Sporothrix mexicana clinical and environmental isolates was evaluated. Killer studies were performed on Petri dishes containing cheese black starch agar. The yeasts Candida catenulata (QU26, QU31, QU127, LV102); Trichosporon faecale (QU100); Trichosporon japonicum (QU139); Kluyveromyces lactis (QU30, QU99, QU73); Kazachstania unispora (QU49), Trichosporon insectorum (QU89), and Kluyveromyces marxianus (QU103) showed activity against all strains of the S. schenckii complex tested. Observation by optical microscopy of S. brasiliensis 61 within the inhibition haloes around the colonies of the killer yeasts QU100, QU139, and LV102 showed that there was no conidiation, but there was hyphal proliferation. The toxins were fungistatic against S. brasiliensis 61. There was no difference in susceptibility to the toxins among the S. schenckii species complex. Further investigations are necessary to clearly establish the mechanism of action of the toxins.

Use of killer yeast in the management of postharvest papaya anthracnose

The efficiency of two killer yeast strains, Wickerhamomyces anomalus (strain 422) and Meyerozyma guilliermondii (strain 443), as biocontrol agents against Colletotrichum gloeosporioides, a postharvest anthracnose agent of papaya and other tropical fruit, was assessed. These strains were previously selected through in vitro assays, but in the present study, their in vivo action was assessed. In addition, the influence of phytopathogen inoculation time on the fruit in combination with the use of the biocontrol agent was also assessed. We assessed mycoparasitism as an antagonistic mechanism of action by scanning electron microscopy (SEM). In addition, two hydrolytic enzymes, chitinase and β-1,3 glucanase, were assayed. Our results indicated that W. anomalus (strain 422) and M. guilliermondii (strain 443) reduced disease incidence by 24.62% and 20.68%, respectively, for up to 6d after inoculation, when applied 3h before the phytopathogen and incubated in a wet chamber (95% RH) at 28°C. The time of yeast inoculation had a significant effect on its antagonistic action. Application of the yeasts 12 or 24h before the phytopathogen inoculation resulted in 13.75% and 30% of disease reductions for W. anomalus (strain 422) and 31.35% and 41.17% reductions for M. guilliermondii (strain 443), respectively. Electron micrographs confirmed mycoparasitism by showing the interaction of the yeasts with C. gloeosporioides hyphae, causing in some cases, a loss of turgor and yeast penetration of walls with marked concavity formation on hypha cell walls.

Killer toxin from a novel killer yeastPichia kudriavzeviiRY55 with idiosyncratic antibacterial activity

The killer phenomenon of yeast may have technological implications in many areas like beverage fermentation, food technology, biological control in agriculture, and in medicine. In the present study the killer phenomenon in Pichia kudriavzevii (P. kudriavzevii RY55) is being reported for the first time. The P. kudriavzevii RY55 toxin exhibited excellent antibacterial activity against several pathogens of human health significance such as Escherichia coli, Enterococcus faecalis, Klebsiella sp., Staphylococcus aureus, Pseudomonas aeruginosa and Pseudomonas alcaligenes. Killer toxin was purified to homogeneity by using ammonium sulphate precipitation and ion exchange chromatography and characterized for few properties. P. kudriavzevii RY55 killer toxin may be of vast significance in the development of novel antimicrobial chemotherapeutic agents, new bio-based safer candidates for food preservation and biocontrol, and starter cultures for fermentation industries.

Monitoring of killer yeast populations in mixed cultures: influence of incubation temperature of microvinifications samples

Killer yeasts are frequently used to combat and prevent contamination by wild-type yeasts during wine production and they can even dominate the wine fermentation. Stuck and sluggish fermentations can be caused by an unbalanced ratio of killer to sensitive yeasts in the bioreactor, and therefore it is important to determine the proportion of both populations. The aim of this study was to provide a simple tool to monitor killer yeast populations during controlled mixed microvinifications of killer and sensitive Saccharomyces cerevisiae. Samples were periodically extracted during vinification, seeded on Petri dishes and incubated at 25 and 37°C; the latter temperature was assayed for possible inactivation of killer toxin production. Colonies developed under the described conditions were randomly transferred to killer phenotype detection medium. Significant differences in the killer/sensitive ratio were observed between both incubation temperatures in all microvinifications. These results suggest that 37°C seems a better option to determine the biomass of sensitive yeasts, in order to avoid underestimation of sensitive cells in the presence of killer yeasts during fermentations. Incubation at a toxin-inhibiting temperature clearly showed the real ratio of killer to sensitive cells in fermentation systems.

Pseudozyma spp. and Barnettozyma spp. effectively kill cancer cells in vitro

Cancer is the overall leading cause of death in developed countries and also worldwide, and being able to exploit an effective anticancer drug is the aim of all cancer scientists. However, many of the synthetic drugs produced so far usually cause serious side effects, which reduces their therapeutic efficacy. Discovering new drugs or auxiliary therapies derived from natural products might thus provide a novel opportunity for cancer therapy. A recent study reported that some lethal toxins can maintain their activity after being injected into mice. We therefore used two Pseudozyma spp. and three Barnettozyma spp. to examine whether these killer yeasts can preserve their lethal effect on cancer cells under the physical environment (optimum pH, temperature and osmolality, supporting a living cell accomplishes to proliferate, metabolize, differentiate and survive). Our preliminary results showed that both Barnettozyma spp. and Pseudozyma spp. have stronger cytotoxicity against HepG2 than Chang’s liver cells. According to the results of two-dimensional difference gel electrophoresis (2D-DIGE), a total of 115 and 27 proteins differentially expressed by 1.5-fold or more were observed for HepG2 and Chang’s liver cells, respectively. Furthermore, we explored the mechanism involved in the effect of the lethal yeast filtrates on liver cancer cells using 2D-DIGE and mass spectrometry.

Purification, characterization and gene cloning of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2

As the killer toxin produced by Williopsis saturnus WC91-2 could kill many sensitive yeast strains, including the pathogenic ones, the extracellular killer toxin in the supernatant of cell culture of the marine yeast strain was purified and characterized. The molecular mass of the purified killer toxin was estimated to be 11.0kDa according to the data from SDS-PAGE. The purified killer toxin had killing activity, but could not hydrolyze laminarin. The optimal conditions for action of the purified killer toxin against the pathogenic yeast Metschnikowia bicuspidate WCY were the assay medium with 10% NaCl, pH 3–3.5 and temperature 16°C. The gene encoding the killer toxin from the marine killer yeast WC91-2 was cloned and the ORF of the gene was 378bp. The deduced protein from the cloned gene encoding the killer toxin had 125 amino acids with calculated molecular weight of 11.6kDa. It was also found that the N-terminal amino acid sequence of the purified killer toxin had the same corresponding sequence deduced from the cloned killer toxin gene in this marine yeast, confirming that the purified killer toxin was indeed encoded by the cloned gene.

Efficacy of killer yeasts in the biological control of Penicillium digitatum on Tarocco orange fruits (Citrus sinensis)

Killer Saccharomyces cerevisiae and Wickerhamomyces anomalus yeast strains were tested as biocontrol agents against Penicillium digitatum, one the most important causes of postharvest decay in orange fruits.W. anomalus, grown on acidified medium, demonstrated micocinogenic activity against P. digitatum, as indicated by large inhibition halos and hyphal damage resulting from β-glucanase activity.Oranges that had been deliberately inoculated with pathogens were protected from decay by W. anomalus. Inoculation of oranges with W. anomalus strains BS 91 and BS 92 reduced disease severity to 1 and 4%, respectively, for up to 10 days in storage.

Yeasts Producing Killer Toxins: An Overview

The production of exotoxins with antimicrobial activity on susceptible microorganisms by yeasts is a relatively common phenomenon. Exotoxins (generally proteins or glycoproteins) that are able to kill susceptible cells belonging to the same or congeneric species have been defined as killer toxins. Since first discovered in Saccharomyces cerevisiae , killer strains have been isolated from several yeast genera, including Candida, Cryptococcus, Hanseniaspora , Kluyveromyces, Pichia , Torulopsis , Ustilago , Williopsis and Zygosaccharomyces. Many types of killer toxins have been reported and their genomes were mapped on double-stranded RNA (S. cerevisiae K1, K2, K28,Ustilage maydis and Hanseniaspora uvarum) , a linear double-stranded DNA plasmid (Kluyveromyces lactis, Pichia acaciae and Pichia inositovora) or carried on a chromosome (S. cerevisiae KHS, KHR and Williopsis mrakii). During the last two decades, secreted killer toxins and toxin-producing killer yeasts have found several applications. For instance in the food and fermentation industries,killer yeasts have been used to combat contaminating wild-type yeasts which can occur during the production of wine, beer and bread . Killer yeasts have also been used as bio-control agents in the preservation of foods , in the bio-typing of medically important pathogenic yeasts and yeast-like fungi , in the development of novel antimycotics for the treatment of human and plant fungal infections, and finally in the field of recombinant DNA technology.

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.

Simultaneous production of single cell protein and killer toxin by Wickerhamomyces anomalus HN1-2 isolated from mangrove ecosystem

The yeast Wickerhamomyces anomalus (the previous name was Pichia anomala) HN1-2 isolated from the mangrove ecosystem was found to be able to produce high level of both killer toxin and single cell protein. When the killer yeast cells were grown by batch cultivation in 5-l fermentor, crude protein in the cells, cell mass, reducing sugar, and diameter of the inhibition zone reached 56.0 g per 100 g of cell dry weight, 7.3 g per liter, 9.5 g per liter, and 19.0 mm, respectively within 12 h and this yeast synthesized a large amount of the essential amino acids, such as lysine (7.8%), methionine (1.8%), and leucine (9.0%). The crude killer toxin produced by the killer yeast isolate HN1-2 could kill the cells of Lodderomyces elongisporus, Candida albicans, Metschnikowia bicuspidata, Pichia guilliermondii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Kluyveromyces aestuarii, which were widely distributed in natural marine environments. The results also showed that the undesirable yeast could be avoided during cell growth of the killer yeast.

Compatibility with Killer Explains the Rise of RNAi-Deficient Fungi

The RNA interference (RNAi) pathway is found in most eukaryotic lineages but curiously is absent in others, including that of Saccharomyces cerevisiae. We show that reconstituting RNAi in S. cerevisiae causes loss of a beneficial double-stranded RNA virus known as killer virus. Incompatibility between RNAi and killer viruses extends to other fungal species in that RNAi is absent in all species known to possess double-stranded RNA killer viruses, whereas killer viruses are absent in closely related species that retained RNAi. Thus, the advantage imparted by acquiring and retaining killer viruses explains the persistence of RNAi-deficient species during fungal evolution.

Killer phenotype of indigenous yeasts isolated from Argentinian wine cellars and their potential starter cultures for winemaking

Of 31 yeasts, from different surfaces of two cellars from the northwest region of Argentina, 11 expressed killer activity against the sensitive strain Saccharomyces cerevisiae P351. Five of these killer yeasts were identified as S. cerevisiae by phenotypic tests and PCR-RFLP analysis. Two S. cerevisiae killer strains, Cf5 and Cf8, were selected based on their excellent kinetic and enological properties as potential autochthonous mixed starter cultures to be used during wine fermentation. They could dominate the natural microbiota in fermentation vats and keep the typical sensorial characteristics of the wine of this region.

Production, purification, and characterization of a novel killer toxin from Kluyveromyces siamensis against a pathogenic yeast in crab

The yeast Kluyveromyces siamensis HN12-1 isolated from mangrove ecosystem was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the killer yeast was grown in the medium with pH 4.0 and 0.5% NaCl and at 25 °C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The killing activity of the purified killer toxin against the pathogenic yeast M. bicuspidata WCY was the highest when it was incubated at 25 °C in the assay medium without added NaCl and pH 4.0. The molecular weight of the purified killer toxin was 66.4 kDa. The killer toxin produced by the yeast strain HN12-1 could kill only the whole cells of M. bicuspidata WCY among all the yeast species tested in this study. This is the first time to report that the killer toxin produced by the yeast K. siamensis HN12-1 isolated from the mangrove ecosystem only killed pathogenic yeast M. bicuspidata WCY.

Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis

Brettanomyces bruxellensis is one of the most damaging species for wine quality, and tools for controlling its growth are limited. In this study, thirty-nine strains belonging to Saccharomyces cerevisiae and B. bruxellensis have been isolated from wineries, identified and then tested against a panel of thirty-nine killer yeasts. Here, for the first time, the killer activity of Ustilago maydis is proven to be effective against B. bruxellensis. Mixed cultures in winemaking conditions show that U. maydis CYC 1410 has the ability to inhibit B. bruxellensis, while S. cerevisiae is fully resistant to its killer activity, indicating that it could be used in wine fermentation to avoid the development of B. bruxellensis without undesirable effects on the fermentative yeast. The characterization of the dsRNAs isolated and purified from U. maydis CYC 1410 indicated that this strain produces a KP6-related toxin. Killer toxin extracts were active against B. bruxellensis at pH values between 3.0 and 4.5 and temperatures comprised between 15 °C and 25 °C, confirming their biocontrol activity in winemaking and wine aging conditions. Furthermore, small amounts (100 AU/ml) of killer toxin extracts from U. maydis significantly reduced the amount of 4-ethylphenol produced by B. bruxellensis, indicating that in addition to the growth inhibition observed for high killer toxin concentrations (ranging from 400 to 2000 AU/ml), small amounts of the toxin are able to reduce the production of volatile phenols responsible for the aroma defects in wines caused by B. bruxellensis.

Inhibition of bacteria contaminating alcoholic fermentations by killer yeasts

The aim of this work was to study the in vitro antibacterial activity possessed by killer yeast strains against bacteria contaminating alcoholic fermentation (Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides), in cell X cell and cell X crude toxin preparations. The bacteria were not inhibited by any S. cerevisiae killer strains (5 out of 11). The inhibition caused by two crude toxin preparations (Trichosporon figueirae and Candida sp) against L. plantarum was surprisingly high but not in the same extent for B. subtilis, especially with three killer strains (Candida glabrata, Pichia anomala and Candida sp). L. mesenteroides and L. fermentum strains were neither inhibited in cell X cell nor crude toxin X cell tests. The results suggested that killer activity of yeasts might operate over bacteria and it could be used for the biocontrol of contaminating bacteria from alcoholic fermentation if additional tests on toxin application in fermentation shown to be successful. A wider panel of S. cerevisiae killer strains should be used to confirm that they were really unable to control the growth of these Gram-positive bacteria.

Optimization of killer assays for yeast selection protocols

A new optimized semiquantitative yeast killer assay is reported for the first time. The killer activity of 36 yeast isolates belonging to three species, namely, Metschnikowia pulcherrima, Wickerhamomyces anomala and Torulaspora delbrueckii, was tested with a view to potentially using these yeasts as biocontrol agents against the wine spoilage species Pichia guilliermondii and Pichia membranifaciens. The effectiveness of the classical streak-based (qualitative method) and the new semiquantitative techniques was compared. The percentage of yeasts showing killer activity was found to be higher by the semiquantitative technique (60%) than by the qualitative method (45%). In all cases, the addition of 1% NaCl into the medium allowed a better observation of the killer phenomenon. Important differences were observed in the killer capacity of different isolates belonging to a same killer species. The broadest spectrum of action was detected in isolates of W. anomala NPCC 1023 and 1025, and M. pulcherrima NPCC 1009 and 1013. We also brought experimental evidence supporting the importance of the adequate selection of the sensitive isolate to be used in killer evaluation. The new semiquantitative method proposed in this work enables to visualize the relationship between the number of yeasts tested and the growth of the inhibition halo (specific productivity). Hence, this experimental approach could become an interesting tool to be taken into account for killer yeast selection protocols.

Non-Saccharomyces wine yeasts have a promising role in biotechnological approaches to winemaking

Biotechnology as applied to winemaking includes several aspects of the fermentation industry, such as monitoring of microbial populations, use of selected starter cultures, and control of undesired yeasts. Over the last few decades, the control of microorganisms using biotechnological approaches has become of increasing importance in the winemaking field. The profusion of selected starter strains has allowed more extensive use of inoculated fermentations, with a consequent improvement in the control of fermentation combined with the use of new biotechnological processes in winemaking. As a consequence of this re-evaluation of the role of non-Saccharomyces yeasts in winemaking over the last few years, several studies have evaluated the use of controlled mixed fermentations using Saccharomyces and different, non-Saccharomyces, yeast species that are a part of the winemaking environment. In this context, mixed fermentations using controlled inoculations of Saccharomyces cerevisiae starter cultures and non-Saccharomyces yeasts represent a practical way towards improving wine complexity and enhancing specific characteristics of a wine. Another trait in the use of non-Saccharomyces yeasts in winemaking relates to the control of spoilage microorganisms. Indeed, more strict control of undesirable yeasts is required during the various phases of wine fermentation. Moreover, there is now increasing interest in the use of natural antimicrobial agents in foods and, in this context, killer yeasts might have important roles in the control of spontaneous and/or spoilage microflora. Thus, killer toxins appear to represent an attractive solution for use as antimicrobial agents, to partially, or even completely, substitution for chemical agent use even if application costs could limit their use in winemaking.