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.     

(1-->6)-Beta-D-glucan as the cell wall binding site for Debaryomyces hansenii killer toxin

AIMS: The aims of this study were to characterize the cell wall binding site of Debaryomyces hansenii killer toxin to provide a simple purification method and to determine some characteristics of this toxin. METHODS AND RESULTS: Various linear (1-->6)-beta-D-glucans of different origins were effective competitive inhibitors of the toxin action. Periodate oxidation and 1H-NMR was used to determine the receptor nature. Affinity chromatography on pustulan-Sepharose column was used to purify D. hansenii killer toxin, probably a 23-kDa protein. The killer toxin character was cureless. CONCLUSIONS: The investigation revealed that the killer toxin was mainly adsorbed by (1-->6)-beta-D-glucans. This is a low molecular weight protein, probably encoded by chromosomal genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The specificity of the killer toxin for its receptor provides an effective means to purify the killer toxin. This study is the first to identify the cell wall binding site of this killer toxin, a toxin with properties of industrial relevance.     

Killer toxin of Kluyveromyces phaffii DBVPG 6076 as a biopreservative agent to control apiculate wine Yeasts.

The use of Kluyveromyces phaffii DBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin of K. phaffii DBVPG 6076 showed extensive anti-Hanseniaspora activity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40 degrees C. These biochemical properties would allow the use of K. phaffii killer toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain of Hanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin to H. uvarum cells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that the K. phaffii DBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.     

Mannoprotein of the yeast cell wall as primary receptor for the killer toxin of Saccharomyces cerevisiae strain 28

The killer toxin KT 28 of Saccharomyces cerevisiae strain 28 is primarily bound to the mannoprotein of the cell wall of sensitive yeasts. The mannoprotein of S. cerevisiae X 2180 was purified; gel filtration and SDS-PAGE indicated an estimated Mr of 185,000. The ability to bind killer toxin KT 28 increased during purification of the mannoprotein. Removing the protein part of the mannoprotein by enzymic digestion or removing the alkali-labile oligosaccharide chains by beta-elimination did not destroy the ability to bind killer toxin KT 28. However, binding activity was lost when the 1,6-alpha-linkages of the outer carbohydrate backbone were hydrolysed by acetolysis. The separated oligomannosides of the side chains also failed to bind toxin, indicating that the main mannoside chains were essential for the receptor activity. The reversible adsorption of killer toxin to mannoprotein was demonstrated by linking it covalently to Sepharose and using this material for affinity chromatography. A 90-fold increase in the specific activity of a preparation of killer toxin KT 28 was achieved in this way.     

Killer toxin of Hanseniaspora uvarum

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000.Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by -1, 6-d-glucans.     

Killer Toxin of Kluyveromyces phaffii DBVPG 6076 as a Biopreservative Agent To Control Apiculate Wine Yeasts

The use of Kluyveromyces phaffii DBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin of K. phaffii DBVPG 6076 showed extensive anti-Hanseniaspora activity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40°C. These biochemical properties would allow the use of K. phaffii killer toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain of Hanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin to H. uvarum cells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that the K. phaffii DBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.     

Volatile and Nonvolatile Maillard Reaction Products between l-Lysine and d-Glucose

The killer toxin produced by the Pichia farinosa KK1 strain was purified by ammonium sulfate precipitation, gel filtration, ion-exchange chromatography and reverse-phase HPLC. The molecular weight of the killer toxin was about 25 kd and its isoelectric point was 6.4. A significant amount of carbohydrate was not detected in the purified killer toxin, suggesting that the toxin is not glycosylated. Its N-terminal amino acid sequence showed no homology with other proteins. The stability and efficacy of the toxin’s killer activity was examined. The toxin completely retained activity at pH 2.5 ~ 4.0 and 5°C, but lost activity at higher temperatures. Killer activity increased with increasing NaCl or KC1 concentration, although NaCl was more effective than KCl.     

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.     

Yeast killer toxin: purification and characterisation of the protein toxin from Saccharomyces cerevisiae.

Killer toxin from killer strains of Saccharomyces cerevisiae was isolated from concentrates of extracellular medium by precipitation in poly(ethylene glycol) and chromatography through glyceryl-controlled-pore glass. The toxin migrated as a single protein band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A molecular weight of 11470 was determined for the toxin protein from its electrophoretic mobility and amino acid composition. Gel filtration of the active toxin indicated that the 11,470-Mr monomer was the active unit. Electrophoretic comparison of extracellular concentrates from a killer strain and an isogenic non-killer showed the presence of the toxin protein only in the killer-derived material. The activity of the toxin was most stable between pH 4.2 and 4.6. At 30 degrees C toxin from a superkiller strain was more stable than that from a normal killer.     

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.     

Production,purification, and characterization of a novel killer toxin from <Emphasis Type="BoldItalic">Kluyveromyces siamensis</Emphasis> 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.     

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.     

(1-->6)-beta-D-glucan as cell wall receptor for Pichia membranifaciens killer toxin

The killer toxin from Pichia membranifaciens CYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1-->6)-beta-D-glucan of the cell wall of a sensitive yeast (Candida boidinii IGC 3430). The (1-->6)-beta-D-glucan was purified from cell walls of C. boidinii by alkali and hot-acetic acid extraction, a procedure which solubilizes glucans. The major fraction of receptor activity remained with the alkali-insoluble (1-->6)-beta- and (1-->3)-beta-D-glucans. The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and (1)H nuclear magnetic resonance) analyses of the fractions obtained showed that (1-->6)-beta-D-glucan was a receptor. Adsorption of most of the killer toxin to the (1-->6)-beta-D-glucan was complete within 2 min. Killer toxin adsorption to the linear (1-->6)-beta-D-glucan, pustulan, and a glucan from Penicillium allahabadense was observed. Other polysaccharides with different linkages failed to bind the killer toxin. The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines. This toxin shows its maximum killer activity in the presence of NaCl. This report is the first to identify the (1-->6)-beta-D-glucan as a receptor for this novel toxin.     

Viral induced yeast apoptosis

In an analogous system to mammals, induction of an apoptotic cell death programme (PCD) in yeast is not only restricted to various exogenous factors and stimuli, but can also be triggered by viral killer toxins and viral pathogens. In yeast, toxin secreting killer strains are frequently infected with double-stranded (ds)RNA viruses that are responsible for killer phenotype expression and toxin secretion in the infected host. In most cases, the viral toxins are either pore-forming proteins (such as K1, K2, and zygocin) that kill non-infected and sensitive yeast cells by disrupting cytoplasmic membrane function, or protein toxins (such as K28) that act in the nucleus by blocking DNA synthesis and subsequently causing a G1/S cell cycle arrest. Interestingly, while all these virus toxins cause necrotic cell death at high concentration, they trigger caspase- and ROS-mediated apoptosis at low-to-moderate concentration, indicating that even low toxin doses are deadly by triggering PCD in enemy cells. Remarkably, viral toxins are not solely responsible for cell death induction in vivo, as killer viruses themselves were shown to trigger apoptosis in non-infected yeast. Thus, as killer virus-infected and toxin secreting yeasts are effectively protected and immune to their own toxin, killer yeasts bear the intrinsic potential to dominate over time in their natural habitat.     

The costs and benefits of killer toxin production by the yeast Pichia kluyveri

Numerous yeast species in many genera are able to produce and excrete extracellular toxic proteins (mycocins) that can kill other specific sensitive yeasts. Natural distributions of killer yeasts suggest that they may be important in maintaining community composition and provide a benefit to the toxin producing cells. The fact that not all yeasts are killers and that polymorphisms exist within some killer species suggests there may be a cost associated with killer toxin production. This study focuses on the costs and benefits associated with toxin production by the yeast Pichia kluyveri. Strains differing in their ability to kill were obtained by tetrad dissection. One parent strain produced spores that exhibited a trade-off between killing ability and intrinsic growth rate. A killer clone from this strain was able to maintain a higher proportion of cells than a non-killer when grown with the same sensitive yeast under laboratory-simulated natural conditions. On the other hand, when grown with a yeast not sensitive to Pichia kluyveri toxin, the non-killer maintained a higher proportion of the total community than did the killer clone. The data support the hypothesis that there are both costs and benefits to producing killer toxin, and based on this, selection may favor different phenotypes in different conditions.     

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 degrees C. The killer protein was chromosomally encoded. Mannan, but not beta-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.     

Pichia acaciae Killer System: Genetic Analysis of Toxin Immunity

The gene responsible for self-protection in the Pichia acaciae killer plasmid system was identified by heterologous expression in Saccharomyces cerevisiae. Resistance profiling and conditional toxin/immunity coexpression analysis revealed dose-independent protection by pPac1-2 ORF4 and intracellular interference with toxin function, suggesting toxin reinternalization in immune killer cells.     

Transfer of DNA killer plasmids from Kluyveromyces lactis to Kluyveromyces fragilis and Candida pseudotropicalis.

Killer plasmids pGKL1 and pGKL2 of double-stranded linear DNAs were transferred from Kluyveromyces lactis to strains of Kluyveromyces fragilis and Candida pseudotropicalis. The resultant killer strains produced 17-fold and 6-fold larger amounts of killer toxin than K. lactis did, respectively. The killer toxin produced by each species appeared to be a glycoprotein.     

Production and characteristics of Debaryomyces hansenii killer toxin

The optimal conditions for the production of the killer toxin of Debaryomyces hansenii CYC 1021 have been studied. The lethal activity of the killer toxin increased with the presence of NaCl in the medium used for testing the killing action. Production of the killer toxin was stimulated in the presence of proteins of complex culture media. Addition of nonionic detergents and other additives, such as dimethylsulfoxide enhanced killer toxin production significantly. Killer toxin secretion pattern followed the growth curve and reached its maximum activity at the early stationary phase. Optimal stability was observed at pH 4.5 and temperatures up to 20 °C. Above pH 4.5 a steep decrease of the stability was noted. The activity was hardly detectable at pH 5.1.