In this study, we introduce a novel approach for the high-throughput quantitative analysis of mitotic defects in the fission yeast Schizosaccharomyces pombe using Imaging Flow Cytometry (IFC). Fission yeast is a valuable model organism for cell cycle research, and the ‘cut’ phenotype, indicative of mitotic catastrophe, has been instrumental in discovering cell cycle regulators. Traditional fluorescence microscopy methods for quantifying ‘cut’ events suffer from subjectivity and limited throughput. Our IFC pipeline overcomes these limitations by automating the detection of ‘cut’ cells based on the unique characteristic of daughter cell nuclei becoming trapped in the cell wall during aberrant mitosis. We demonstrate the pipeline’s effectiveness using wild-type and mutant strains, with results validated against manual scoring. Our study establishes IFC as a powerful tool for investigating mitotic fidelity in fission yeast, with implications for advancing cell biology research.

Transcription presents challenges to genome stability both directly, by altering genome topology and exposing single-stranded DNA to chemical insults and nucleases, and indirectly by introducing potential obstacles to the DNA replication machinery. Such obstacles include the RNA polymerase holoenzyme itself, DNA bound regulatory factors, G-quadruplexes and RNA::DNA hybrid structures known as R-loops. Here we review the detrimental impacts of transcription on genome stability in budding yeast, as well as the mitigating effects of transcription-coupled DNA repair and of systems that maintain DNA replication fork processivity and integrity. We conclude that the impacts of transcription on DNA replication by the replisome must be very mild with little direct mutagenic outcome under normal conditions. However, transcription can significantly impair the fidelity of replication fork rescue mechanisms, particularly Break Induced Replication (BIR), which is used to restart collapsed replication forks when other means fail. This leads to de novo mutations, structural variation and extrachromosomal circular DNA formation that contribute to genetic heterogeneity. By re-analysing published datasets, we show that different classes of genes have different interactions with the replisome, and that highly transcribed environment dependant genes in S. cerevisiae tend to be located close to replication origins. We have previously implicated interactions between replication origins and BIR forks in adaptive transcription-induced copy number variation events, which indicates that environment dependent genes are preferentially located in genomic environments more prone to instability, particularly under replication stress.

Atacama is the most hyper-arid Desert in the world. In this study, we describe a novel species, Nakazawaea atacamensis f. a., sp. nov., isolated from plant samples in the Atacama Desert of Chile. In total, three isolates of N. atacamensis were obtained from independent Neltuma chilensis bark samples (synonym Prosopis chilensis, Algarrobo). The novel species was delineated based on morphological, physiological, biochemical, and molecular characteristics. A neighbour-joining analysis using the sequences of the D1/D2 domains of LSU rRNA revealed that N. atacamensis sp. nov. clustered with Nakazawaea pomicola. The sequence of N. atacamensis differed from closely related species by 1.3% to 5.2% in the D1/D2 domains. A phylogenomic analysis based on single nucleotide polymorphism’s data confirms that the novel species belongs to the genus Nakazawaea, and placed N. atacamensis closer to N. peltata. Phenotypic comparisons demonstrated that N. atacamensis sp. nov. exhibited distinct carbon assimilation patterns compared to its related species. Genome sequencing of the ATA-11A-B T strain revealed a genome size of approximately 12.4 Mbp, similar to other Nakazawaea species, with 5,116 protein-coding genes annotated using InterProScan. In addition, N. atacamensis exhibited the capacity to ferment synthetic wine must, representing a potential new yeast for mono or co-culture wine fermentations. This comprehensive study expands our understanding of the genus Nakazawaea and highlights the ecological and industrial potential of these yeasts in fermentation processes. The holotype of N. atacamensis sp. nov. is CBS 18375 T. The Mycobank number is MB 849680.

The yeast Saccharomyces cerevisiae and most eukaryotes carry two 5’→3’ exoribonuclease paralogues that are very similar. In yeast, they are called Xrn1, which shuttles between the nucleus and cytoplasm and executes major cytoplasmic mRNA decay, and Rat1, which carries a strong nuclear localization sequence (NLS) and localizes in the nucleus. Xrn1 is 40% homologous to Rat1 but has an extra ~500 amino acids C-terminal extension. In the cytoplasm, Xrn1 can degrade decapped mRNAs during the last round of translation by ribosomes “co-translational mRNA decay”. The division of labor between the two enzymes is still enigmatic and can serve as a paradigm for division of labor of many other paralogues. Here we show that Rat1 is capable of functioning in cytoplasmic mRNA decay, provided that Rat1 remains cytoplasmic due to its NLS disruption (cRat1). This indicates that the actual segregation of the two paralogues plays roles in their specific functions. However, segregation is not sufficient for fully complementing Xrn1 function. Specifically, cRat1 can only partially recover cell volume, mRNA stability, proliferation rate, 5’→3’ decay alterations that characterize xrn1Δ cells. In particular, co-translational decay is only little complemented by cRat1. Adding the Xrn1 C-terminal domain to Rat1 does not improve the phenotypes indicating that lack of C-terminal is not the reason for the partial complementation. Collectively, it seems that during evolution the two paralogues acquire unique features that make the division of work beneficial.

While flocculation has demonstrated its efficacy in enhancing yeast robustness and ethanol production, its potential application for lactic acid fermentation remains largely unexplored. Our study delved into the impact of flocculating behavior on the metabolic dynamics of Saccharomyces cerevisiae strains incorporating an exogenous lactic acid pathway in different cell densities and in the presence of lignocellulose-derived byproducts. Comparative gene expression profiles revealed that cultivating a nonflocculant strain at higher cell density yielded a substantial upregulation of genes associated with glycolysis, energy metabolism, and other key pathways, resulting in elevated levels of fermentation products. Meanwhile, the flocculating strain displayed an inherent ability to sustain high glycolytic activity regardless of the cell density. Moreover, our investigation revealed a significant reduction in glycolytic activity under chemical stress, potentially attributable to diminished ATP supply during the energy investment phase. Conversely, the formation of flocs in the flocculating strain conferred protection against toxic chemicals present in the medium, fostering more stable lactic acid production levels. We postulated that the cell flocculation phenomenon induced oxygen-limited conditions, resulting in increased lactate dehydrogenase activity, and helped protect the cells against acid stress, contributing to the overall robustness of the flocculant strain. Additionally, the distinct flocculation traits observed between the two examined strains may be attributed to differences in the amino acid sequences within the tandem repeat regions of the flocculin protein. This study uncovers the potential of flocculation for enhanced lactic acid production in yeast, offering insights into metabolic mechanisms and potential gene targets for strain improvement.

Ntšhe, a Setswana name for sweet reeds, a sweet sorghum variety ( Sorghum bicolor (L)) is a commercial crop consumed as a delicacy in enormous quantities in Botswana and in southern Africa at large. Pre and post-harvest losses due to infestation by larval stages of stem borer moths, Chilo partellus often lead to severe financial losses as consumers condemn the worm-infested sweet reeds as unpalatable, “ Mo gase ntšhe tota tota” (this is not a sweet reed). Valorisation of condemned sweet reeds is one attractive route to reduce economic losses. Here, we took advantage of our understanding of yeast-insect interactions to isolate yeasts associated with larval stages of the stem borer moths and investigated their potential for production of an alcoholic sweet reed beverage. We isolated 33 yeast strains representing from the galleries and frass as well as from the guts of the larval moths. Assessment of their ability to ferment the simplest sugar, glucose, resulted in identification of 14 strains belonging to Hanseniaspora and Candida genera. These strains were further assessed for their capacity to ferment by calculating the rate of accumulation of carbon dioxide and ethanol when grown in the principal sugars found in sweet sorghum juice as sole carbon sources as well in sweet sorghum juice. In addition, as an industrially relevant trait, we tested the potential of the strains to tolerate brewing/fermentation-associated stresses. Furthermore, we assessed the aromatic complexity of the produced beverage. Our results suggest that non-conventional yeasts associated with the larval moths have potential for valorization of condemned sweet sorghum stalks to produce a sweet sorghum beverage.