Matthias Sipiczki

Where does fission yeast sit on the tree of life

The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are as different from each other as either is from animals: their ancestors separated about 420 to 330 million years ago. Now that S. pombe is poised to join the post-genome era, its evolutionary position should become much clearer.

The endo-β-1,3-glucanase eng1p is required for dissolution of the primary septum during cell separation in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission throughout contraction of an actomyosin ring and deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomoysin ring and septum assembly, little information is available concerning the mechanism of cell separation. Here we describe the characterization of eng1+, a new gene that encodes a protein with detectable endo-β-1,3-glucanase activity and whose deletion is not lethal to the cells but does interfere in their separation. Electron microscopic observation of mutant cells indicated that this defect is mainly due to the failure of the cells to degrade the primary septum, a structure rich in β-1,3-glucans, that separates the two sisters cells. Expression of eng1+ varies during the cell cycle, maximum expression being observed before septation, and the protein localizes to a ring-like structure that surrounds the septum region during cell separation. This suggests that it could also be involved in the cleavage of the cylinder of the cell wall that covers the division septum. The expression of eng1+ during vegetative growth is regulated by a C2H2 zinc-finger protein (encoded by the SPAC6G10.12c ORF), which shows significant sequence similarity to the Saccharomyces cerevisiae ScAce2p, especially in the zinc-finger region. Mutants lacking this transcriptional regulator (which we have named ace2+) show a severe cell separation defect, hyphal growth being observed. Thus, ace2p may regulate the expression of the eng1+ gene together with that of other genes whose products are also involved in cell separation.

Metschnikowia Strains Isolated from Botrytized Grapes Antagonize Fungal and Bacterial Growth by Iron Depletion

ABSTRACT Noble-rotted grapes are colonized by complex microbial populations. I isolated pigment-producing Metschnikowia strains from noble-rotted grapes that had antagonistic activity against filamentous fungi, yeasts, and bacteria. A red-maroon pigment was formed from a diffusible colorless precursor released by the cells into the medium. The conversion of the precursor required iron and could occur both in the cells (red colonies) and in the medium (red halos around colonies). The intensity of pigmentation was correlated with the intensity of the antimicrobial activity. Mutants that did not form pigment also lacked antifungal activity. Within the pigmented halos, conidia of the sensitive fungi did not germinate, and their hyphae did not grow and frequently lysed at the tips. Supplementation of the medium with iron reduced the size of the halos and the inhibition zones, while it increased the pigment accumulation by the colonies. The iron-binding agent tropolone had a similar effect, so I hypothesize that pigmented Metschnikowia isolates inhibit the growth of the sensitive microorganisms by pigment formation, which depletes the free iron in the medium. As the pigment is a large nondiffusible complex produced in the presence of both low and high concentrations of ferric ions, the proposed mechanism is different from the mechanisms operating in microbes that release siderophores into the environment for iron acquisition.

Interspecies hybridization and recombination in Saccharomyces wine yeasts

The ascomycetous yeasts traditionally referred to as the Saccharomyces sensu stricto complex are a group of closely related species that are isolated by a postzygotic barrier. They can easily hybridize; and their allodiploid hybrids propagate by mitotic divisions as efficiently as the parental strains, but can barely divide by meiosis, and thus rarely produce viable spores (sterile interspecies hybrids). The postzygotic isolation is not effective in allotetraploids that are able to carry out meiosis and produce viable spores (fertile interspecies hybrids). By application of molecular identification methods, double (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces kudriavzevii) and triple (S. cerevisiae x S. uvarum x S. kudriavzevii) hybrids were recently identified in yeast populations of fermenting grape must and cider in geographically distinct regions. The genetic analysis of these isolates and laboratory-bred hybrids revealed great variability of hybrid genome structures and demonstrated that the alloploid genome of the zygote can undergo drastic changes during mitotic and meiotic divisions of the hybrid cells. This genome-stabilization process involves loss of chromosomes and genes and recombination between the partner genomes. This article briefly reviews the results of the analysis of interspecies hybrids, proposes a model for the mechanism of genome stabilization and highlights the potential of interspecies hybridization in winemaking.

Protein O-mannosylation is crucial for cell wall integrity, septation and viability in fission yeast.

Protein O‐mannosyltransferases (PMTs) initiate the assembly of O‐mannosyl glycans, which are of fundamental importance in eukaryotes. The PMT family, which is classified into PMT1, PMT2 and PMT4 subfamilies, is evolutionarily conserved. Despite the fact that PMTs are crucial for viability of bakers yeast as well as of mouse, recent studies suggested that there are significant differences in the organization and properties of the O‐mannosylation machinery between yeasts and mammals. In this study we identified and characterized the PMT family of the archaeascomycete Schizosaccharomyces pombe. Unlike Saccharomyces cerevisiae where the PMT family is highly redundant, in S. pombe only one member of each PMT subfamily is present, namely, oma1+ (protein O‐mannosyltransferase), oma2+ and oma4+. They all act as protein O‐mannosyltransferases in vivo. oma1+ and oma2+ form heteromeric protein complexes and recognize different protein substrates compared to oma4+, suggesting that similar principles underlie mannosyltransfer reaction in S. pombe and budding yeast. Deletion of oma2+, as well as simultaneous deletion of oma1+ and oma4+ is lethal. Characterization of the viable S. pombe oma1Δ and oma4Δ single mutants showed that a lack of O‐mannosylation results in abnormal cell wall and septum formation, thereby severely affecting cell morphology and cell–cell separation.

Saccharomyces bayanus var. uvarum in Tokaj wine-making of Slovakia and Hungary

Abstract. Using genetic hybridisation analysis and molecular karyotyping we revealed an association of Saccharomyces bayanus var. uvarum species with Tokaj wine-making. Along with identification of Saccharomyces strains isolated by E. Minárik in Slovakia, the composition of Tokaj populations in Hungary was studied. Twenty-eight Hungarian Saccharomyces strains were analysed in terms of karyotype. The majority of strains belong to S. bayanus var. uvarum. Two non-identified Saccharomyces strains were found to be polyploid according to their complex karyotype patterns.

Analysis of yeasts derived from natural fermentation in a Tokaj winery

The diversity of yeast flora was investigated in a spontaneously fermenting sweet white wine in a Tokaj winery. The non-Saccharomyces yeasts dominating the first phase of fermentation were soon replaced by a heterogeneous Saccharomycespopulation, which then became dominated by Saccharomyces bayanus. Three Saccharomyces sensu stricto strains isolated from various phases of fermentation were tested for genetic stability, optimum growth temperature, tolerance to sulphur dioxide, copper and ethanol as well as for the ability to produce hydrogen sulphide and various secondary metabolites known to affect the organoleptic properties of wines. The analysis of the single-spore cultures derived from spores of dissected asci revealed high stability of electrophoretic karyotypes and various degrees of heterozygosity for mating-types, the fermentation of galactose and the production of metabolic by-products. The production levels of the by-products did not segregate in a 2:2 fashion, suggesting that the synthesis of these compounds is under polygenic control.

Taxonomic reclassification of Candida stellata strains reveals frequent occurrence of Candida zemplinina in wine fermentation

Yeasts identified as Candida stellata are frequently associated with overripe and botrytized grapes and can survive in the fermenting must until the completion of vinification. The molecular taxonomic examination of 41 strains deposited in six culture collections or described in the literature as C. stellata revealed that most of those isolated from grapes or wines belonged to Candida zemplinina and related species. This confusion around the taxonomic position of the strains may account for the rather controversial descriptions of the oenological properties of C. stellata in the literature. Because the authors did not find it among strains newly isolated from botrytized grapes and wines, it was proposed that it is usually C. zemplinina rather than C. stellata that occurs on grapes and in wine fermentation.

The yeast Starmerella bacillaris (synonym Candida zemplinina) shows high genetic diversity in winemaking environments

The yeast Candida zemplinina (Starmerella bacillaris) is frequently isolated from grape and wine environments. Its enological use in mixed fermentation with Saccharomyces cerevisiae has been extensively investigated these last few years, and several interesting features including low ethanol production, fructophily, glycerol and other metabolites production, have been described. In addition, molecular tools allowing the characterization of yeast populations have been developed, both at the inter- and intraspecific levels. However, most of these fingerprinting methods are not compatible with population genetics or ecological studies. In this work, we developed 10 microsatellite markers for the C. zemplinina species that were used for the genotyping of 163 strains from nature or various enological regions (28 vineyards/wineries from seven countries). We show that the genetic diversity of C. zemplinina is shaped by geographical localization. Populations isolated from winemaking environments are quite diverse at the genetic level: neither clonal-like behaviour nor specific genetic signature were associated with the different vineyards/wineries. Altogether, these results suggest that C. zemplinina is not under selective pressure in winemaking environments.

sep1+ Encodes a transcription-factor homologue of the HNF-3/forkhead DNA-binding-domain family in Schizosaccharomyces pombe

We report on the cloning of sep1+, a gene whose mutation causes filamentous growth in Schizosaccharomyces pombe. Since cell growth and propagation are not affected by the mutation, it could not be cloned using selective conditions for the identification of the positive transformants. Instead, we cloned it from a cosmid of a contig (Hoheisel et al., Cell 73, 109-1120, 1993) supposed to cover the chromosomal region where the sep1-1 mutation mapped. The 1761 bp long ORF codes for a protein containing a sequence similar to the DNA-binding domains of the HNF-3/forkhead family of transcription factors.