ABSTRACT Yeasts are model organisms and protein production hosts of choice, in part due to their ease of genetic manipulation. Yeast transformation with DNA on plasmids or integrated into the genome has been developed since the 1970s and is a routine method in many laboratories. However, transformation frequencies are surprisingly low, with common methods yielding around 1% of transformed cells in budding yeast Saccharomyces cerevisiae . In alternative species, there is lower transformation frequency and unreasonably large quantities of input DNA may be required. Surprisingly, the mechanisms by which yeast cells take up DNA are not known. Here we argue that three approaches could deliver a step‐change in yeast transformation frequency and efficiency. First, investigating the mechanisms of DNA uptake. The requirement for salts and extraneous polymers—carrier DNA and PEG—in standard protocols suggests that phase separation of DNA may enhance DNA uptake into cells or subsequent nuclear delivery. Second, accounting for the physiological or growth state of the yeast including cell cycle phase and nutrient or stress conditions. Third, systematically determining the factors that affect transformation efficiency using statistical Design of Experiments. These approaches could allow superefficient transformation of S. cerevisiae , and accelerate genetic manipulation of non‐model yeasts.
ABSTRACT Insects are essential partners for yeasts, playing a key role in their dispersal to new environments. Stingless bees (Apidae: Corbiculata: Meliponini) are social corbiculate bees which lack a functional sting. They are important pollinators in tropical and subtropical regions, foraging for nectar, pollen, and resins, thereby enriching their nests with plant‐associated microbes, including yeasts. Here we assessed the yeast diversity associated with stingless bees and their provisions, by thoroughly sampling guts, brood cells, pollen, and honey pots, across seven different Meliponini species: Frieseomelitta varia , Melipona quadrifasciata , Scaptotrigona depilis , Scaptotrigona postica , Tetragona clavipes , Tetragonisca angustula , and Trigona cilipes . Yeast diversity was analyzed for the most extensively sampled bees, S. depilis and T. clavipes . Among the 20 yeast and yeast‐like species recovered, we found a core dominant yeast community, primarily composed of Starmerella and Wickerhamiella yeasts, which are notorious to be associated with flowers and floricolous insects. Although the communities are dominated by specific yeast taxa with similar metabolic profiles, it differs among stingless bee species that occur in the same geographic area, as observed for S. depilis and T. clavipes . We suggest that yeast communities associated with stingless bees and their provisions contain overrepresented taxa and are shaped by host foraging choices.
Endosomal sorting complex required for transport (ESCRT-III) is a membrane remodeling complex involved in a large number of cellular processes. It appears to perform an essential function in eukaryotes, since to date no eukaryotic organism completely devoid of ESCRT-III has been found. Yet, yeast cells with a deletion of all eight known ESCRT-III genes are viable. We therefore searched for new, previously undiscovered ESCRT-III like proteins in yeast. HHPred uncovered several proteins with similarity to Snf7. The similarity was mostly restricted to the α1-α2 hairpin region of Snf7. A conserved pattern of amino acids was detected in this region. One of the proteins with an ESCRT-III like sequence pattern, which strikingly resembled Snf7 in its secondary structure, was studied more closely. We named the protein encoded by ORF YPL199c Etl1 (ESCRT-three-like 1). Etl1 is palmitoylated and localizes to the plasma membrane. In contrast to other palmitoylated proteins, Etl1 does not appear to be associated with lipid rafts, since it could be easily extracted from the membrane by Triton X-100 treatment. When ETL1 was deleted in the octuple ESCRT-III deletion background, the yeast cells were still viable. So far, despite a number of experiments, a bona fide ESCRT-III function could not be demonstrated for Etl1.
All living organisms can enter a non-dividing state known as quiescence, which often functions as a survival strategy. In many cases, quiescent cells retain the ability to re-enter the cell cycle when conditions become favorable. Therefore, preserving the integrity of non-dividing cells is essential not only to prevent their deterioration, but also to ensure proper population re-establishment once growth resumes. This also applies to the genome, whose stability must be maintained during both proliferation and quiescence. For several years, we have investigated this aspect of quiescence in fission yeast, which enters a G0 state in response to nitrogen starvation. We have shown that wild-type G0 cells accumulate mutations over time, with a mutational spectrum distinct from that observed during proliferation. We are now extending this work to mutants defective in various DNA repair pathways. In doing so, we developed a robust protocol ensuring highly reproducible results when assessing mutagenesis in quiescent cells. Here, we describe this protocol in detail.
Together with other fungi, yeasts make up a significant component of the plant microbiome. As the planet warms, cacti expand their range. Cactus-associated yeasts are known to exhibit signatures of adaptation to the cactus host. Our previous isolation of a wild Saccharomyces paradoxus yeast from a cactus in a forest of oaks populated by S. cerevisiae prompted us to further explore cactus-associated fungi and look for genomic and phenotypic signatures of adaptation. Here we characterize seven yeast isolates, five from cacti and two from adjacent non-cactus plants, among which was a novel species of Coniochaeta isolated from wild grape that we name C. udwismasis. Closely-related isolates from distinct plant hosts exhibited distinct features, including differences in thermotolerance, freeze-thaw tolerance, pigmentation, and predicted septin protein complex assembly, providing new insights into possible mechanisms of cactus adaptation by the yeast microbiome.
