<i>Tetrapisispora phaffii</i> killer toxin is a highly specific β-glucanase that disrupts the integrity of the yeast cell wall

Background. Killer yeasts have been used to combat contaminating wild yeasts in food, to control pathogenic fungi in plants, and in the medical field, to develop novel antimycotics for the treatment of human and animal fungal infections. Among these killer yeasts, Tetrapisispora phaffii (formerly known as Kluyveromyces phaffii) secretes a glycoprotein known as Kpkt that is lethal to spoilage yeasts under winemaking conditions. In the present study, the mode of action of Kpkt, and the specific damage produced by this toxin on sensitive yeasts is investigated. Results. The use of castanospermine, a β-glucanase inhibitor, demonstrated that β-glucanase activity is essential for the Kpkt killer activity in vivo. Accordingly, Kpkt has no killer activity on either sensitive yeast spheroplasts or whole sensitive cells in the presence of isosmothic medium (0.8 molar sorbitol). Kpkt induces ultrastructural modifications in the cell wall of sensitive strains, as shown by confocal microscopy, laser-scanning electron microscopy, and atomic force microscopy. The Kpkt killer action is mediated by the glucidic portion of the toxin. This, in turn, appears to be involved both in the stronger cytocidal activity and in the selectivity for the sensitive strain shown by Kpkt compared to laminarinase. Conclusion. Collectively, these data indicate that the mode of action of Kpkt is directed towards the disruption of cell-wall integrity, and that this is mediated by a highly specific β-glucanase activity. In this, Kpkt differs from other microbial β-glucanases that do not show killer activities

Exploitation of the semi-homothallic life cycle of Saccharomyces cerevisiae for the development of breeding strategies.

A strain of Saccharomyces cerevisiae having desirable winemaking properties and high spore viability was bred from a semi-homothallic parent strain with similar winemaking properties but that produced sixfold fewer viable spores. Because the parent was homozygous for HO and for the MATa allele at both silent HMR and HML loci, it produced two MATa and two nonmating progeny per ascus. To obtain a segregant able to mate with the stable MATa progeny, a strain of the nonmating progeny, previously subjected to HO distruption with a KanMX4 cassette, was used. The resultant MATalphaho::KanMX4 transformant was mated to a MATa HO segregant and the diploid produced was sporulated to allow the isolation of a semi-homothallic diploid segregant designated 2D that lacked the KanMX4-disrupted HO allele as confirmed by sequence analysis. Genetic analysis indicated greater homozygosity in 2D than in the parent as assessed by PCR at five loci. The sugar consumption profiles of both 2D and the parent in grape juice fermentations were the same. Acetaldehyde levels and postfermentation biofilm formation were higher in 2D than in the parent. Because 2D has acceptable winemaking characteristics but produces significantly more viable spores than the parent strain, it will be useful in future breeding efforts

Occurrence of wine yeasts on grapes subjected to different pesticide treatments

As a contribution to the study and preservation of indigenous wine yeast populations, we initiated a programme of isolation and characterization of yeast strains from grapes of the Italian region of Marche. During the 1996 vintage, grape samples were collected from three groups of vineyards differing in the pesticide treatments given. Of the 279 yeasts isolated, thirty-nine were assigned to the species Saccharomyces cerevisiae. The majority of the isolates and all thirty-nine S. cerevisiae came from vineyards which had not been treated with systemic pesticides, indicating that these pesticides may exert a negative effect on the biodiversity of the grape microflora, and in particular on the occurrence of S. cerevisiae on grapes. The thirty-nine S. cerevisiae isolated were subjected to microfermentation mals, in which ten of them showed promising fermentative capabilities

Lactic acid bacteria and yeasts from wheat sourdoughs of the Marche region

The need for a greater diversification of baked products has given rise to the on-going search for yeast and lactic acid bacteria (LAB) strains with optimal baking potential. Thirty-six yeasts and 118 LAB, isolated from nine type I sourdoughs that were sampled in bakeries located in the Marche region (central Italy), were molecularly and phenotypically characterized. The polyphasic approach used revealed the biodiversity of the microbial communities investigated and two yeasts and ten LAB cultures with the potential to be used in sourdough bread-making processes were identified

Vanadium detoxification and resistance in yeast: a minireview

The study of the effects exerted by vanadium on living systems has recently became an object of growing interest. In fact, the possibility to develop vanadium as a new drug in the treatment of diabetes and cancer calls for the individuation of the cellular vanadate- sensitive targets. Moreover, the fact that vanadium can be an environmental pollutant in urban and industrial areas, as a consequence of the combustion of petroleum, coal, and heavy oils, requires the elucidation of the mechanisms by which living organisms protect themselves from the toxic effects of this metal ion. Studies on the cellular response to vanadate in yeast have shown that the activation of different concerted mechanisms is involved in the detoxification of vanadium. Moreover, the isolation and characterisation of genes involved in vanadate-sensitivity and resistance have shed light on some of the numerous cellular vanadate sensitive targets

Copper sensitive mutants of <i>Hansenula polymorpha</i>

Copper is an essential trace element as it is an important cofactor in electron transport and for many redox-actjve metalloenzymes. However, due to its redox activity, copper can be highly toxic when present in excess intracellularly, because of its ability to generate reactive oxygen species and damage proteins, membranes and nucleic acids. The thermotolerant yeast Hansenula polymorpha represents an interesting model for investigating the mechanisms evolved by cells in response to potentially cytotoxic metal ions. H. polymorpha can easily adapt to high copper concentrations by repeated culturing of wild type cells in the presence of successively increasing concentrations of copper sulphate. Moreover, this yeast is able to grow on vanadate concentrations in the millimolar range, while most cells are inhibited by micromolar concentrations of this metal ion. As part of an investigation programme concerning the understanding of the mechanisms responsible for heavy metal resistance in the yeast H. polymorpha, we have isolated 75 cos (copper sensitive) recessive mutants, 29 of which harbour pleiotropic mutations. Twenty-six of these that present vas (vanadate sensitive) cos or vas cos vpa (vacuolar proteolitic activity defective) phenotypes are part of the vas collection of mutants described in the accompanying abstract (see Tagliapietra Orciani et al.). The remaining 3 mutants present a cos vpa phenotype. All these mutants are currently being subjected to complementation analysis. Moreover, in two of them, one cos and one cos vpa, the wild type phenotype has been restored by means of transformation with a H. polymorpha genomic library. The complementing plasmids are under study

Impact of mother sediment on yeast growth, biodiversity, and ethanol production during fermentation of Vinsanto wine

The aim of this study was to determine the impact of Vinsanto mother sediment both on the growth and biodiversity of the yeast microflora and on the production of ethanol under natural and inoculated fermentation of Vinsanto wines. To achieve this ten fermentation trials were carried out in 50-L barrels, five without added mother sediment and five with. Moreover, eight of the ten barrels were inoculated with four Saccharomyces cerevisiae wine strains, while the remaining two barrels were not inoculated and were used as controls to study the behaviour of the natural yeast microflora in the absence and presence of mother sediment. The counts of viable yeasts at three different sampling times indicated that the mother sediment had a positive influence on yeast growth and persistence during fermentation. Molecular characterization of the Saccharomyces type colonies isolated after three months of fermentation showed that the addition of mother sediment had no effects on the dominance of the wine starters. In contrast, the mother sediment had a positive influence on the biodiversity of the spontaneous S. cerevisiae yeasts. Moreover, possibly due to its content of fatty acids and sterols and other nutrients, the addition of mother sediment also showed a positive effect on the fermentative activities of wine yeasts as measured by their ethanol production

Identification of killer factor in the yeast genus <i>Metschnikowia</i>

Out of 260 Metschnikowia pulcherrima strains isolated from Sardinian grapes and musts, 6 proved to be killer yeasts. Maximal killing effect occurred between 3.6 and 5.2 pH, and with 48h or 72h cultures