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.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Killer toxin from Hansenula mrakii selectively inhibits cell wall synthesis in a sensitive yeast

Hansenula mrakii secretes extracellularly a killer toxin which kills sensitive Saccharomyces cerevisiae. In protoplasts of this yeast, the killer toxin selectively inhibited the synthesis of alkali-insoluble acid-insoluble polysaccharides consisting mainly of beta-glucan, but did not inhibit either the synthesis of other cell wall polysaccharides, such as mannan, chitin and alkali-insoluble acid-soluble polysaccharides, or the synthesis of protein. Consistent with these results, the toxin was inhibitory to the beta-(1,3)-glucan synthetase activity of a cell-free extract from sensitive S. cerevisiae.     

Characterization of a novel killer toxin encoded by a double-stranded linear DNA plasmid of Kluyveromyces lactis

A novel killer toxin, encoded by a double-stranded linear DNA plasmid pGK l-1 (5.4 MDa) in Kluyveromyces lactis IFO 1267 was purified 320 000-fold from the culture broth of yeast. The toxin was obtained in an electrophoretically homogeneous state with a yield of 24% by hydroxyapatite column chromatography, chromatofocusing and polyacrylamide gel electrophoresis. The purified toxin was dissociated into two subunits with molecular masses of 27 kDa and above 80 kDa, as estimated by Laemmli's sodium dodecylsulfate gel electrophoresis; the exact composition ratio of the two subunits remains unestablished. The isoelectric point was between 4.4 and 4.8. As compared with the reported narrow pH range of action and instability of k1 killer toxin encoded by a double-stranded RNA plasmid of Saccharomyces cerevisiae, the K. Lactis toxin was effective with sensitive strains of S. cerevisiae in a relatively wider pH range between 4 and 8; it was stable for several months at pH 6.0 when stored below -20 degrees C. In contrast to the simple protein nature of the k1 killer toxin with a molecular mass of 11.47 kDa, the K. lactis toxin maintained a mannoprotein nature, as it was absorbed by a ConA-Sepharose column and eluted by methyl alpha-D-mannoside. The growth inhibitory activity of K. lactis toxin was enhanced 2-35-fold by the presence of 4-60% glycerol.     

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.     

The ecological role of killer yeasts in natural communities of yeasts

The killer phenomenon of yeasts was investigated in naturally occurring yeast communities. Yeast species from communities associated with the decaying stems and fruits of cactus and the slime fluxes of trees were studied for production of killer toxins and sensitivity to killer toxins produced by other yeasts. Yeasts found in decaying fruits showed the highest incidence of killing activity (30/112), while yeasts isolated from cactus necroses and tree fluxes showed lower activity (70/699 and 11/140, respectively). Cross-reaction studies indicated that few killer-sensitive interactions occur within the same habitat at a particular time and locality, but that killer-sensitive reactions occur more frequently among yeasts from different localities and habitats. The conditions that should be optimal for killer activity were found in fruits and young rots of Opuntia cladodes where the pH is low. The fruit habitat appears to favor the establishment of killer species. Killer toxin may affect the natural distribution of the killer yeast Pichia kluyveri and the sensitive yeast Cryptococcus cereanus. Their distributions indicate that the toxin produced by P. kluyveri limits the occurrence of Cr. cereanus in fruit and Opuntia pads. In general most communities have only one killer species. Sensitive strains are more widespread than killer strains and few species appear to be immune to all toxins. Genetic study of the killer yeast P. kluyveri indicates that the mode of inheritance of killer toxin production is nuclear and not cytoplasmic as is found in Saccharomyces cerevisiae and Kluyveromyces lactis.     

A molecular target for viral killer toxin: TOK1 potassium channels.

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes.     

Occurrence of killer yeast strains in industrial and clinical yeast isolates

The secretion of proteinaceous toxins is a widespread characteristic in environmental and laboratory yeast isolates, a phenomenon called "killer system". The killer phenotype (K+) can be encoded by extrachromosomal genetic elements (EGEs) as double stranded DNA or RNA molecules (dsDNA, dsRNA) or in nuclear genes. The spectrum of action and the activity of killer toxins are influenced by temperature, salinity and pH of media. In the present work we determined the existence of K+ in a collection of S. cerevisiae and P. anomala yeasts isolated from environmental, industrial and clinical sources. The assays were performed in strains belonging to three yeast genera used as sensitive cells and under a wide range of pH and temperatures. Approximately 51 % of isolates tested showed toxicity against at least one sensitive yeast strain under the conditions tested. The K+ P. anomala isolates showed a wide spectrum of action and two of them had toxic activity against strains of the three yeast genera assayed, including C. albicans strains. In all S. cerevisiae K+ isolates an extrachromosomal dsRNA molecule (4.2 Kb) was observed, contrary to P. anomala K+ isolates, which do not possess any EGEs. The K+ phenotype is produced by an exported protein factor and the kinetics of killer activity production was similar in all isolates with high activity in the log phase of growth, decaying in the stationary phase.     

Membrane-mediated killing of Saccharomyces cerevisiae by glycoproteins from Torulopsis glabrata.

Cell-free supernatants from cultures of Torulopsis glabrata contained glycoprotein toxins that killed sensitive and killer strains of Saccharomyces cerevisiae with single-hit kinetics. Growing S. cerevisiae treated with the toxins showed a leakage of cellular potassium, partial dissipation of the adenosine triphosphate pool, and a coordinate shutdown of macromolecular synthesis. These pool efflux-stimulating toxins have been partially purified and at least three toxic glycoproteins have been separated. Pool efflux-stimulating toxin activity was stable from pH 3 through 7, though killing was maximal close to pH 4.     

Immunity to K1 killer toxin: internal TOK1 blockade

K1 killer strains of Saccharomyces cerevisiae harbor RNA viruses that mediate secretion of K1, a protein toxin that kills virus-free cells. Recently, external K1 toxin was shown to directly activate TOK1 channels in the plasma membranes of sensitive yeast cells, leading to excess potassium flux and cell death. Here, a mechanism by which killer cells resist their own toxin is shown: internal toxin inhibits TOK1 channels and suppresses activation by external toxin.     

Genetic and fermentation properties of the K2 killer yeast, Saccharomyces cerevisiae T206

Saccharomyces cerevisiae T206 K⁺R⁺, a K₂ killer yeast, was differentiated from other NCYC killer strains of S. cerevisiae on the basis of CHEF-karyotyping and mycoviral RNA separations. Genomic DNA of strain T206 was resolved into 13 chromosome bands, ranging from approximately 0.2 to 2.2 Mb. The resident virus in strain T206 yielded L and M RNA species of approximately 5.1 kb and 2.0 kb, respectively. In micro-scale vinifications, strain T206 showed a lethal effect on a K^-R^- mesophilic wine yeast. Metabolite accumulation and toxin activity were measured over a narrow pH range of 3.2 to 3.5. Contrary to known fermentation trends, the challenged fermentations were neither stuck nor protracted although over 70% of the cell population was killed. Toxin-sensitive cells showed cytosolic efflux.     

P-type ATPase spf1 mutants show a novel resistance mechanism for the killer toxin SMKT

SMKT, a killer toxin produced by the halotolerant yeast Pichia farinosa KK1, consists of alpha and beta subunits with folding remarkably similar to that of the fungal killer toxin KP4, a Ca2+ channel inhibitor. The budding yeast Saccharomyces cerevisiae is sensitive to SMKT. To understand the killing mechanism of SMKT, we isolated SMKT-resistant mutants of S. cerevisiae and characterized them. Five spf mutants (sensitivity to the P. farinosa killer toxin) fell into a single genetic complementation group, designated spf1. The SPF1 gene was cloned by complementation of the mutant phenotype. The SPF1 gene encodes a putative P-type ATPase of 1215 amino acid residues that contains 12 membrane-spanning regions. Gene disruption revealed that the SPF1 gene is not essential for viability but is required for the sensitivity to SMKT. The spf1 disruptant showed some phenotypes characteristic of glycosylation-defective mutants and secreted underglycosylated invertase. Fluorescence-activated cell-sorting analysis and indirect immunofluorescence microscopy showed that SMKT interacts with the cell surface of the resistant cells but not with that of sensitive cells, suggesting a novel resistance mechanism for this toxin. The glycosylation-defective phenotype and possible killer-resistant mechanisms are discussed in comparison with the Golgi Ca2+ pump Pmr1p.     

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.     

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.     

Mixed culture of killer and sensitive Saccharomyces cerevisiae strains in batch and continuous fermentations

This paper presents a kinetic study of the dynamics of the population of two Saccharomyces cerevisiae strains (designated K1 and 522D) in mixed culture. These two strains are commonly used in wine making. The K1 strain (killer yeast) secretes a glycoprotein (killer toxin) which causes the death of the 522D strain (sensitive yeast). Initially, the mixed cultures were realized in batch fermentations. Initial concentrations of killer yeast were 5 and 10% of the total population. The influence of the killer strain on the sensitive cultures was measured in comparison with a reference fermentation. The reference fermentation was inoculated only with the sensitive strain. Results show that an initial concentration of 10% of killer strain affects the microbial population balance and the rate of ethanol production. However the fermentation was only slightly disturbed when the proportion of killer to sensitive yeast at the beginning of mixed culture was 5%. To achieve total displacement by the killer yeast at low concentrations, the mixed cultures were carried out in a continuous system. The results obtained in continuous fermentations with the same strains have shown that a level of contamination as low as 0.8% of killer strain was sufficient to completely displace the original sensitive population after 150 h incubation.     

Mixed culture of killer and sensitive Saccharomyces cerevisiae strains in batch and continuous fermentations

This paper presents a kinetic study of the dynamics of the population of two Saccharomyces cerevisiae strains (designated K1 and 522D) in mixed culture. These two strains are commonly used in wine making. The K1 strain (killer yeast) secretes a glycoprotein (killer toxin) which causes the death of the 522D strain (sensitive yeast). Initially, the mixed cultures were realized in batch fermentations. Initial concentrations of killer yeast were 5 and 10% of the total population. The influence of the killer strain on the sensitive cultures was measured in comparison with a reference fermentation. The reference fermentation was inoculated only with the sensitive strain. Results show that an initial concentration of 10% of killer strain affects the microbial population balance and the rate of ethanol production. However the fermentation was only slightly disturbed when the proportion of killer to sensitive yeast at the beginning of mixed culture was 5%. To achieve total displacement by the killer yeast at low concentrations, the mixed cultures were carried out in a continuous system. The results obtained in continuous fermentations with the same strains have shown that a level of contamination as low as 0.8% of killer strain was sufficient to completely displace the original sensitive population after 150 h incubation.     

Involvement of [beta]-glucans in the wide-spectrum antimicrobial activity of Williopsis saturnus var. mrakii MUCL 41968 killer toxin

BACKGROUND: Williopsis saturnus var. mrakii MUCL 41968 secretes a 85-kDa glycoprotein killer toxin (WmKT) that displays a cytocidal activity against a wide range of microorganisms, making WmKT a promising candidate for the development of new antimicrobial molecules. Although the killing mechanism of WmKT is still unknown, the toxin was recently proposed to bind to the surface of sensitive microorganisms through the recognition of beta-glucans. Indeed, Saccharomyces cerevisiae strains sensitive to the toxin become resistant when mutated in their beta-glucan synthesis pathway. MATERIALS AND METHODS: To investigate the interaction of WmKT with beta-glucans, we examined in agar diffusion assays the WmKT activity in the presence of enzymes displaying beta-glucanase activity. The toxin activity was also investigated using spheroplasts derived from sensitive yeast cells. The hydrolytic activity of WmKT was studied using specific glucosidase inhibitors as well as various sugar molecules covalently linked to p-nitrophenyl as potential substrates. Finally, the ultrastructural modifications induced by WmKT activity on sensitive yeasts were assessed by scanning electron microscopy. RESULTS: The data reported here support the hypothesis that WmKT binds to sensitive cells using surface-exposed beta-glucans. Indeed beta-glucanase exerts an antagonistic effect on WmKT activity and spheroplasts derived from WmKT-sensitive yeast cells are shown to be resistant to WmKT, suggesting that cell wall beta-glucans are required for WmKT lethal effect. Because WmKT exhibits amino acid sequence similarities with proteins suspected to be glucanase, we also investigated the effect of castanospermine, a potent glucosidase inhibitor, on WmKT activity. Castanospermine completely abolished WmKT killer activity as well as its hydrolytic enzymatic activity against p-nitrophenyl beta-D-glucopyranoside. The scanning electron microscopy analysis of sensitive yeast cells treated with the toxin reveals that WmKT causes cell wall modifications similar to those observed with zymolyase. CONCLUSION: The results reported in this study show that WmKT activity requires an interaction between the mycocin and the cell wall beta-glucans. Moreover, they indicate that WmKT acts on sensitive yeast cells through a hydrolytic activity directed against cell wall beta-glucans that disrupts the yeast cell wall integrity leading to death.     

Expression of pGKL killer 28K subunit in Saccharomyces cerevisiae: identification of 28K subunit as a killer protein.

Saccharomyces cerevisiae and other yeast cells harboring the linear double stranded (ds) DNA plasmids pGKL1 and pGKL2 secrete a killer toxin consisting of 97K, 31K and 28K subunits into the culture medium (EMBO J. 5, 1995-2002 (1986), Nucleic Acids Res., 15, 1031-1046 (1987]. The 28K subunit of the killer toxin was successfully expressed in S. cerevisiae when it was cloned on a circular plasmid with its putative promoter region replaced with that of S. cerevisiae chromosomal genes. The expression of the 28K subunit of the killer toxin in killer-sensitive cells resulted in the death of the host cells. This killing activity by the 28K subunit was prevented by the expression of the killer immunity, indicating that the killing activity of the killer toxin complex was carried out by the 28K subunit. Although the 28K subunit was synthesized as a intact precursor protein with its own signal sequence, it was not secreted into the culture medium but remained in the host cells. This indicated that 28K subunit killed host cells from inside of the cells rather than from outside. We further suggested that 28K killer subunit without 97K and 31K subunits did not kill the killer-sensitive cells from outside.