The bacterial and yeast microbiota in livestock forages in Hungary.

BACKGROUND: Along bacteria, yeasts are common in forages and forage fermentations as spoilage microbes or as additives, yet few studies exist with species-level data on these fungi's occurrence in feedstuff. Active dry yeast and other yeast-based products are also common feed additives in animal husbandry. Here, we aimed to characterize both fermented and non-fermented milking cow feedstuff samples from Hungary to assess their microbial diversity in the first such study from Central Europe. RESULTS: We applied long-read bacterial metabarcoding to 10 fermented and 25 non-fermented types of samples to assess bacterial communities and their characteristics, surveyed culturable mold and yeast abundance, and identified culturable yeast species. Fermented forages showed the abundance of Aerococcaceae, Bacillaceae, Brucellaceae, Lactobacillaceae, Staphylococcaceae, and Thermoactinomycetaceae, non-fermented ones had Cyanothecaceae, Enterobacteriaceae, Erwiniaceae, Gomontiellaceae, Oxalobacteraceae, Rhodobiaceae, Rickettsiaceae, and Staphylococcaceae. Abundances of bacterial families showed mostly weak correlation with yeast CFU numbers, only Microcoleaceae (positive) and Enterococcaceae and Alcaligenaceae (negative correlation) showed moderate correlation. We identified 14 yeast species, most commonly Diutina rugosa, Pichia fermentans, P. kudriavzevii, and Wickerhahomyces anomalus. We recorded S. cerevisiae isolates only from animal feed mixes with added active dry yeast, while the species was completely absent from fermented forages. The S. cerevisiae isolates showed high genetic uniformity. CONCLUSION: Our results show that both fermented and non-fermented forages harbor diverse bacterial microbiota, with higher alpha diversity in the latter. The bacterial microbiome had an overall weak correlation with yeast abundance, but yeasts were present in the majority of the samples, including four new records for forages as a habitat for yeasts. Yeasts in forages mostly represented common species including opportunistic pathogens, along with a single strain of Saccharomyces used as a feed mix additive.

Novel Saccharomyces uvarum x Saccharomyces kudriavzevii synthetic hybrid with enhanced 2-phenylethanol production.

BACKGROUND: Over the last two decades, hybridization has been a powerful tool used to construct superior yeast for brewing and winemaking. Novel hybrids were primarily constructed using at least one Saccharomyces cerevisiae parent. However, little is known about hybrids used for other purposes, such as targeted flavor production, for example, 2-phenylethanol (2-PE). 2-PE, an aromatic compound widely utilised in the food, cosmetic, and pharmaceutical industries, presents challenges in biotechnological production due to its toxic nature. Consequently, to enhance productivity and tolerance to 2-PE, various strategies such as mutagenesis and genetic engineering are extensively explored to improved yeast strains. While biotechnological efforts have predominantly focused on S. cerevisiae for 2-PE production, other Saccharomyces species and their hybrids remain insufficiently described. RESULTS: To address this gap, in this study, we analysed a new interspecies yeast hybrid, II/6, derived from S. uvarum and S. kudriavzevii parents, in terms of 2-PE bioconversion and resistance to its high concentration, comparing it with the parental strains. Two known media for 2-PE biotransformation and three different temperatures were used during this study to determine optimal conditions. In 72 h batch cultures, the II/6 hybrid achieved a maximum of 2.36 ± 0.03 g/L 2-PE, which was 2-20 times higher than the productivity of the parental strains. Our interest lay not only in determining whether the hybrid improved in productivity but also in assessing whether its susceptibility to high 2-PE titers was also mitigated. The results showed that the hybrid exhibited significantly greater resistance to the toxic product than the original strains. CONCLUSIONS: The conducted experiments have confirmed that hybridization is a promising method for modifying yeast strains. As a result, both 2-PE production yield and tolerance to its inhibitory effects can be increased. Furthermore, this strategy allows for the acquisition of non-GMO strains, alleviating concerns related to additional legislative requirements or consumer acceptance issues for producers. The findings obtained have the potential to contribute to the development of practical solutions in the future.

MAT heterozygosity and the second sterility barrier in the reproductive isolation of Saccharomyces species.

The genetic analysis of large numbers of Saccharomyces cerevisiae × S. uvarum ("cevarum") and S. kudriavzevii × S. uvarum ("kudvarum") hybrids in our previous studies revealed that these species are isolated by a postzygotic double-sterility barrier. We proposed a model in which the first barrier is due to the abruption of the meiotic process by the failure of the chromosomes of the subgenomes to pair (and recombine) in meiosis and the second barrier is assumed to be the result of the suppression of mating by allospecific MAT heterozygosity. While the former is analogous to the major mechanism of postzygotic reproductive isolation in plants and animals, the latter seems to be Saccharomyces specific. To bolster the assumed involvement of MAT in the second sterility barrier, we produced synthetic alloploid two-species cevarum and kudvarum hybrids with homo- and heterothallic backgrounds as well as three-species S. cerevisiae × S. kudvarum × S. uvarum ("cekudvarum") hybrids by mass-mating and examined their MAT loci using species- and cassette-specific primer pairs. We found that the allospecific MAT heterozygosity repressed MAT switching and mating in the hybrids and in the viable but sterile spores produced by the cevarum hybrids that had increased (allotetraploid) genomes. The loss of heterozygosity by meiotic malsegregation of MAT-carrying chromosomes in the latter hybrids broke down the sterility barrier. The resulting spores nullisomic for the S. uvarum chromosome produced vegetative cells capable of MAT switching and conjugation, opening the way for GARMe (Genome Autoreduction in Meiosis), the process that leads to chimeric genomes.

Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae.

Molecular combing and gel electrophoretic studies revealed endogenous nicks with free 3'OH ends at ∼100 kb intervals in the genomic DNA (gDNA) of unperturbed and G1-synchronized Saccharomyces cerevisiae cells. Analysis of the distribution of endogenous nicks by Nick ChIP-chip indicated that these breaks accumulated at active RNA polymerase II (RNAP II) promoters, reminiscent of the promoter-proximal transient DNA breaks of higher eukaryotes. Similar periodicity of endogenous nicks was found within the ribosomal rDNA cluster, involving every ∼10th of the tandemly repeated 9.1 kb units of identical sequence. Nicks were mapped by Southern blotting to a few narrow regions within the affected units. Three of them were overlapping the RNAP II promoters, while the ARS-containing IGS2 region was spared of nicks. By using a highly sensitive reverse-Southwestern blot method to map free DNA ends with 3'OH, nicks were shown to be distinct from other known rDNA breaks and linked to the regulation of rDNA silencing. Nicks in rDNA and the rest of the genome were typically found at the ends of combed DNA molecules, occasionally together with R-loops, comprising a major pool of vulnerable sites that are connected with transcriptional regulation.

Non-introgressive genome chimerisation by malsegregation in autodiploidised allotetraploids during meiosis of Saccharomyces kudriavzevii x Saccharomyces uvarum hybrids.

Saccharomyces strains with chimerical genomes consisting of mosaics of the genomes of different species ("natural hybrids") occur quite frequently among industrial and wine strains. The most widely endorsed hypothesis is that the mosaics are introgressions acquired via hybridisation and repeated backcrosses of the hybrids with one of the parental species. However, the interspecies hybrids are sterile, unable to mate with their parents. Here, we show by analysing synthetic Saccharomyces kudriavzevii x Saccharomyces uvarum hybrids that mosaic (chimeric) genomes can arise without introgressive backcrosses. These species are biologically separated by a double sterility barrier (sterility of allodiploids and F1 sterility of allotetraploids). F1 sterility is due to the diploidisation of the tetraploid meiosis resulting in MAT a /MAT α heterozygosity which suppresses mating in the spores. This barrier can occasionally be broken down by malsegregation of autosyndetically paired chromosomes carrying the MAT loci (loss of MAT heterozygosity). Subsequent malsegregation of additional autosyndetically paired chromosomes and occasional allosyndetic interactions chimerise the hybrid genome. Chromosomes are preferentially lost from the S. kudriavzevii subgenome. The uniparental transmission of the mitochondrial DNA to the hybrids indicates that nucleo-mitochondrial interactions might affect the direction of the genomic changes. We propose the name GARMe (Genome AutoReduction in Meiosis) for this process of genome reduction and chimerisation which involves no introgressive backcrossings. It opens a way to transfer genetic information between species and thus to get one step ahead after hybridisation in the production of yeast strains with beneficial combinations of properties of different species.

Synthetic two-species allodiploid and three-species allotetraploid Saccharomyces hybrids with euploid (complete) parental subgenomes.

Combination of the genomes of Saccharomyces species has great potential for the construction of new industrial strains as well as for the study of the process of speciation. However, these species are reproductively isolated by a double sterility barrier. The first barrier is mainly due to the failure of the chromosomes to pair in allodiploid meiosis. The second barrier ensures that the hybrid remains sterile even after genome duplication, an event that can restore fertility in plant interspecies hybrids. The latter is attributable to the autodiploidisation of the allotetraploid meiosis that results in sterile allodiploid spores (return to the first barrier). Occasionally, mating-competent alloaneuploid spores arise by malsegregation of MAT-carrying chromosomes. These can mate with cells of a third species resulting in aneuploid zygotes having at least one incomplete subgenome. Here we report on the construction of euploid three-species hybrids by making use of "rare mating" between a sterile S. kudriavzevii x S. uvarum allodiploid hybrid and a diploid S. cerevisiae strain. The hybrids have allotetraploid 2nScnSk nSu genomes consisting of complete sets of parental chromosomes. This is the first report on the production of euploid three-species Saccharomyces hybrids by natural mating, without genetic manipulation. The hybrids provide possibilities for studying the interactions of three allospecific genomes and their orthologous genes present in the same cell.

Double sterility barrier between Saccharomyces species and its breakdown in allopolyploid hybrids by chromosome loss.

The analysis of 57 synthetic interspecies hybrids revealed that Saccharomyces cerevisiae and Saccharomyces uvarum ( Saccharomyces bayanus var. uvarum) are isolated by a double sterility barrier: by hybrid sterility (hybrid cells cannot produce viable spores) operating in allodiploids and by F1 sterility (F1 cells cannot produce viable spores) operating in allopolyploids. F1-sterility is caused by mating-type heterozygosity. It can be overcome by eliminating chromosome 2 of the S. uvarum subgenome that carries a MAT locus. The loss of this MAT gene abolishes the repression of mating activity. In cultures of the resulting fertile alloaneuploid F1 segregants, the cells can conjugate with each other like haploids and form zygotes capable of performing meiotic divisions producing viable and fertile F2 spores. To the best of our knowledge, this is the first report on breaking down interspecies hybrid sterility by chromosome loss in eukaryotic organisms. The filial generations are genetically unstable and can undergo additional changes mainly in the S. uvarum subgenome (directional changes). It is proposed that regaining fertility and subsequent preferential reduction in one of the subgenomes may account for the formation of chimerical ('natural hybrid') genomes found among wine and brewery strains and may also play roles in speciation of hybrid taxa in the Saccharomyces genus.

Diversity and Postzygotic Evolution of the Mitochondrial Genome in Hybrids of Saccharomyces Species Isolated by Double Sterility Barrier.

Eukaryotic species are reproductively isolated by sterility barriers that prevent interspecies fertilization (prezygotic sterility barrier) or the fertilization results in infertile offspring (postzygotic sterility barrier). The Saccharomyces species are isolated by postzygotic sterility barriers. Their allodiploid hybrids form no viable gametes (ascospores) and the viable ascospores of the allotetraploids cannot fertilize (conjugate). Our previous work revealed that this mechanism of reproductive isolation differs from those operating in plants and animals and we designated it double sterility barrier (the failure of homeologous chromosomes to pair and the repression of mating by mating-type heterozygosity). Other studies implicated nucleo-mitochondrial incompatibilities in the sterility of the Saccharomyces hybrids, a mechanism assumed to play a central role in the reproductive isolation of animal species. In this project the mitochondrial genomes of 50 cevarum (S. cerevisiae × S. uvarum) hybrids were analyzed. 62% had S. cerevisiae mitotypes, 4% had S. uvarum mitotypes, and 34% had recombinant mitotypes. All but one hybrid formed viable spores indicating that they had genomes larger than allodiploid. Most of these spores were sterile (no sporulation in the clone of vegetative descendants; a feature characteristic of allodiploids). But regardless of their mitotypes, most hybrids could also form fertile alloaneuploid spore clones at low frequencies upon the loss of the MAT-carrying chromosome of the S. uvarum subgenome during meiosis. Hence, the cevarum alloploid nuclear genome is compatible with both parental mitochondrial genomes as well as with their recombinants, and the sterility of the hybrids is maintained by the double sterility barrier (determined in the nuclear genome) rather than by nucleo-mitochondrial incompatibilities. During allotetraploid sporulation both the nuclear and the mitochondrial genomes of the hybrids could segregate but no correlation was observed between the sterility or the fertility of the spore clones and their mitotypes. Nucleo-mitochondrial incompatibility was manifested as respiration deficiency in certain meiotic segregants. As respiration is required for meiosis-sporulation but not for fertilization (conjugation), these segregants were deficient only in sporulation. Thus, the nucleo-mitochondrial incompatibility affects the sexual processes only indirectly through the inactivation of respiration and causes only partial sterility in certain segregant spore clones.

Vinification without Saccharomyces: Interacting Osmotolerant and "Spoilage" Yeast Communities in Fermenting and Ageing Botrytised High-Sugar Wines (Tokaj Essence).

The conversion of grape juice to wine starts with complex yeast communities consisting of strains that have colonised the harvested grape and/or reside in the winery environment. As the conditions in the fermenting juice gradually become inhibitory for most species, they are rapidly overgrown by the more adaptable Saccharomyces strains, which then complete the fermentation. However, there are environmental factors that even Saccharomyces cannot cope with. We show that when the sugar content is extremely high, osmotolerant yeasts, usually considered as "spoilage yeasts", ferment the must. The examination of the yeast biota of 22 botrytised Tokaj Essence wines of sugar concentrations ranging from 365 to 752 g∙L-1 identified the osmotolerant Zygosaccharomyces rouxii, Candida (Starmerella) lactis-condensi and Candida zemplinina (Starmerella bacillaris) as the dominating species. Ten additional species, mostly known as osmotolerant spoilage yeasts or biofilm-producing yeasts, were detected as minor components of the populations. The high phenotypical and molecular (karyotype, mtDNA restriction fragment length polymorphism (RFLP) and microsatellite-primed PCR (MSP-PCR)) diversity of the conspecific strains indicated that diverse clones of the species coexisted in the wines. Genetic segregation of certain clones and interactions (antagonism and crossfeeding) of the species also appeared to shape the fermenting yeast biota.

A new, rapid multiplex PCR method identifies frequent probiotic origin among clinical Saccharomyces isolates.

An increasing number of infections originating from probiotic use are reported worldwide, with the majority of such cases caused by the yeast Saccharomyces 'boulardii', a subtype of S. cerevisiae. Reliably linking infectious cases to probiotic products requires unequivocal genotyping data, however, these techniques are often time-consuming and difficult to implement in routine diagnostics. This leads to a widespread lack of genetic data regarding the origin of Saccharomyces infections. We propose a quick and reliable PCR-based protocol for the identification of S. 'boulardii' based on a combined analysis of interdelta fingerprinting and microsatellite typing. By applying various typing methods and our proposed method to the clinical yeast collection of a Hungarian hospital we show that probiotic origin is common among clinical Saccharomyces, and that the new multiplex method enables rapid and unequivocal identification of probiotic yeast infections. This method can be applied for the identification of yeast infection sources, helping decisions on probiotic use.

Assembly of Schizosaccharomyces cryophilus chromosomes and their comparative genomic analyses revealed principles of genome evolution of the haploid fission yeasts.

The fission yeast clade, which has a distinct life history from other yeasts, can provide important clues about evolutionary changes. To reveal these changes the large S. cryophilus supercontigs were assembled into chromosomes using synteny relationships and the conserved pericentromeric, subtelomeric genes. Togetherness of the supercontigs was confirmed by PCR. Investigation of the gene order revealed localisation of the rDNA arrays, more than 300 new conserved orthologues and proved that S. cryophilus supercontigs were mosaics of collinear blocks. PFGE analysis showed that size of the S. cryophilus chromosomes differ from the S. pombe chromosomes. Comparative genomic analyses of the newly assembled chromosomes confirmed that the closest relative of S. cryophilus was S. octosporus not just in sequence similarity but also in a structural way, and revealed that preservation of the conserved regions did not arise from the lower number of chromosomal rearrangements. Translocations were more typical in the closely related species, while the number of inversions increased with the phylogenetic distances. Our data suggested that sites of the chromosomal rearrangements were not random and often associated with repetitive sequences, structural- and nucleotide evolution might correlate. Chromosomal rearrangements of the fission yeasts compared to other lineages were also discussed.

Commercial strain-derived clinical Saccharomyces cerevisiae can evolve new phenotypes without higher pathogenicity.

SCOPE: Saccharomyces cerevisiae is one of the most important microbes in food industry, but there is growing evidence on its potential pathogenicity as well. Its status as a member of human mycobiome is still not fully understood. METHODS AND RESULTS: In this study, we characterize clinical S. cerevisiae isolates from Hungarian hospitals along with commercial baking and probiotic strains, and determine their phenotypic parameters, virulence factors, interactions with human macrophages, and pathogenicity. Four of the clinical isolates could be traced back to commercial strains based on genetic fingerprinting. Our observations indicate that the commercial-derived clinical isolates have evolved new phenotypes and show similar, or in two cases, significantly decreased pathogenicity. Furthermore, immunological experiments revealed that the variability in human primary macrophage activation after coincubation with yeasts is largely donor and not isolate dependent. CONCLUSION: Isolates in this study offer an interesting insight into the potential microevolution of probiotic and food strains in human hosts. These commensal yeasts display various changes in their phenotypes, indicating that the colonization of the host does not necessarily impose a selective pressure toward higher virulence/pathogenicity.

Genomic expression patterns in cell separation mutants of Schizosaccharomyces pombe defective in the genes sep10 ( + ) and sep15 ( + ) coding for the Mediator subunits Med31 and Med8.

Cell division is controlled by a complex network involving regulated transcription of genes and postranslational modification of proteins. The aim of this study is to demonstrate that the Mediator complex, a general regulator of transcription, is involved in the regulation of the second phase (cell separation) of cell division of the fission yeast Schizosaccharomyces pombe. In previous studies we have found that the fission yeast cell separation genes sep10 ( + ) and sep15 ( + ) code for proteins (Med31 and Med8) associated with the Mediator complex. Here, we show by genome-wide gene expression profiling of mutants defective in these genes that both Med8 and Med31 control large, partially overlapping sets of genes scattered over the entire genome and involved in diverse biological functions. Six cell separation genes controlled by the transcription factors Sep1 and Ace2 are among the target genes. Since neither sep1 ( + ) nor ace2 ( + ) is affected in the mutant cells, we propose that the Med8 and Med31 proteins act as coactivators of the Sep1-Ace2-dependent cell separation genes. The results also indicate that the subunits of Mediator may contribute to the coordination of cellular processes by fine-tuning of the expression of larger sets of genes.

Gradual genome stabilisation by progressive reduction of the Saccharomyces uvarum genome in an interspecific hybrid with Saccharomyces cerevisiae.

Considerable amounts of molecular and genetic data indicate that interspecific hybridisation may not be rare among natural strains of Saccharomyces sensu stricto. Although a post-zygotic barrier operating during meiosis usually prevents the production of viable spores, stable hybrids can arise which can even evolve into distinct species. This study was aimed to analyse the genome of a fertile Saccharomyces cerevisiae x S. uvarum hybrid and monitor its changes over four filial generations of viable spores. The molecular genetic analysis demonstrated that the two species did not contribute equally to the formation and stabilisation of the hybrid genome. S. cerevisiae provided the mitochondrial DNA and the more stable part of the nuclear genome. The S. uvarum part of the hybrid nuclear genome became progressively smaller by loosing complete chromosomes and genetic markers in the course of successive meiotic divisions. Certain S. uvarum chromosomes were eliminated and/or underwent rearrangements in interactions with S. cerevisiae chromosomes. Numerous S. uvarum chromosomes acquired S. cerevisiae telomere sequences. The gradual elimination of large parts of the S. uvarum genome was associated with a progressive increase of sporulation efficiency. We hypothesise that this sort of genomic alterations may contribute to speciation in Saccharomyces sensu stricto.