Yeast

Yeasts ability to invade surfaces has important implications for infections and food contamination. Invasive growth in yeast is influenced by genetic and environmental factors. In this exploratory study, we investigated the effects of sodium sulfide, gene deletions, and environmental conditions on the invasive behaviour of the wine yeast strain AWRI 796. Sodium sulfide enhanced invasion in the (parent) AWRI 796 strain under nitrogen-limiting conditions, although its effect was obscured by experimental variability and pre-culture conditions. Genetic factors had a major effect on the overall invasive phenotype, with deletion of key genes suppressing invasion. Most gene-deletion mutants did not significantly affect how the colony responded to sulfide. In addition to sulfide and genotype, environmental conditions also influenced invasive behaviour. The pre-2xSLAD pre-culture condition was best for detecting sulfide-induced growth, and later plate washing time and decreased nutrient levels enhanced invasiveness. Our experimental design and findings provide a framework for understanding the determinants of yeast invasiveness, which may inform future studies on filamentous yeast behaviour.

Purine moieties are essential for many functions within the eukaryotic cell, including energy, signaling and nucleic acid synthesis. While purine starvation is known to induce stress resistance in eukaryotic model organism budding yeast Saccharomyces cerevisiae, it remains unclear whether the physiological response is related to disruption of synthesis pathway in particular position or it is uniform across all genetic deficiencies within the de novo adenine biosynthesis pathway. It is also not known how purine starved cells perceive purine shortage - weather they share the same signaling elements with nitrogen starvation or not. MethodsWe characterised physiology of strains with deletions in adenine biosynthesis pathway when cultivated in full or purine deficient and compared to cell physiological parameters when cultivated in nitrogen deficient media. We tested stress tolerance, carbon flux, cell cycle arrest and did transcription profiling (RNA-seq). ResultsOur findings demonstrate that purine starvation-induced stress resistance is significantly modulated by the specific step at which the pathway is interrupted. Transcriptional analysis revealed that purine starvation in many aspects phenocopies nitrogen starvation, particularly - in both starvations strong downregulation of ribosome related genes occurs. In the same time several metabolic features which differ from N- and ade- starvations: pentose phosphate pathway is specifically upregulated within ade4{Delta}-ade2{Delta} and downregulated in N-cells. Notably, the expression of stress-responsive genes such as HSP12, HSP26, and GRE1 varied between mutants, suggesting that the accumulation of pathway intermediates (e.g., AIR in ade2{Delta}) or the absence of downstream precursors (AICAR) alters the perception of starvation especially in the case of ade16{Delta}ade17{Delta} strain. ConclusionsMetabolic and stress-tolerance phenotypes of purine auxotrophs are not merely a result of purine depletion but seems that the response is signalled via the same pathways, like TOR1. The results suggest that strains having mutations within various positions of the purine pathway "perceive" purine limitation a bit differently - especially when we compare the end of the pathway with the other mutants. Different phenotypic outcomes of the occasional purine depletion might give preferences for organisms which have mutations in the beginning rather at the end of the pathway. Besides, our findings might have implications in the design of synthetic pathways and the use of auxotrophic markers in yeast research.

We present a comparative analysis of 13 yeasts available for mead (honey wine) fermentation, a source of Saccharomyces cerevisiae diversity that has not yet been analyzed in detail. Using genomic, phenotyping, and analytic methods, we show that currently available mead yeasts belong to various clades of the species, most commonly to the Commercial Wine clade (5 of 13 samples). Mead yeasts in this group displayed genome structure variations and occasional loss of killer activity, despite being closely related. Historic European and traditional African mead isolates with sequenced genomes were found not to be closely related to any contemporary mead yeast product. The 13 yeasts tested here displayed high variability in oenological characteristics and in aroma production. Maximum ethanol tolerance ranged from 15 to 22% v/v, however, the most tolerant strain produced lower ethanol levels and retained high fructose content in experimental meads. The most abundant aroma components produced in meads were ethyl acetate, ethyl caprylate, isoamyl alcohol, and ethyl caprate, with similar aroma profiles in members of the Commercial Wine clade, and pronounced differences among other yeasts. Our results contribute to the knowledge of Saccharomyces yeasts in various fermentation environments, adding mead to the list of alcoholic beverages with a known diversity of starter cultures. Our results may aid strain selection for honey wine fermentations and inspire strain improvement.

Agrobacterium-mediated transformation (AMT) is a critical method for genetic manipulation of non-conventional fungi, yet it remains a laborious and inefficient technique. Here we demonstrate that eliminating the time-consuming filtration step frequently incorporated in AMT fungal transformation protocols not only reduces hands-on time from over four hours to 15 minutes, but also increases transformation efficiency by 69% in the lipogenic yeast, Rhodotorula toruloides. We further optimized the Agrobacterium:yeast cell ratio and culture resuspension volume to achieve an efficiency > 210,000 CFU per transformation representing a 2-3 fold improvement over previously implemented protocols. This simplified "unfiltered" spot-plating method was successfully applied to seven yeast species, including one for which genetic transformation has not previously been reported, Botryozyma nematodophila. This approach enables high-throughput transformation workflows that are critical for genome-scale functional studies across diverse yeast systems.

Sugar-induced cell death (SICD) remains an intriguing but poorly studied phenomenon in the physiology of Saccharomyces cerevisiae. Recently, it was shown that SICD development largely depends on the redirection of glucose fluxes between glycolysis and the pentose phosphate pathway (PPP). In particular, inhibition of glycolysis by iodoacetamide (IAA) was observed to reduce SICD levels. This study is devoted to further investigation of the relationship between SICD and the functionality of the two PPP branches. It was shown that deletion of the ZWF1 gene does not affect the decrease in SICD levels in IAA-treated cells. This allows us to conclude that the oxidative branch of the PPP is not involved in the suppression of SICD/ROS. Deletion of the GLR1 gene and attenuation of the TRR1 gene also did not restore SICD levels in cells after IAA treatment. The obtained results indicate that the level of reduced glutathione or thioredoxin does not affect SICD genesis. The addition of 5 mM ribose-5-phosphate (R5P) to the incubation medium led to suppression of SICD by 79%. At the same time, the addition of 5 mM ribose + 5 mM Pi suppressed SICD by only 20%. Suppression of SICD by 5 mM R5P in the{Delta} pho3 strain (83%) excludes the mechanism of extracellular dephosphorylation of R5P to ribose, its subsequent transport into the cell, and re-phosphorylation inside the cell. Furthermore, more than 70% suppression of SICD in the{Delta} end3 strain with 5 mM R5P excludes endocytosis as a mechanism of R5P import into the cell. The observed effect of R5P can be explained by the moonlighting function of some unknown protein. Thus, SICD development in S. cerevisiae cells depends on the final product of the non-oxidative PPP--R5P.

Sourdough starters contain simple microbial communities typically consisting of a few bacterial species and one or two yeast species. The yeast Maudiozyma humilis and the lactic acid bacterium Fructilactobacillus sanfranciscensis often co-occur in sourdough starters, and have been presumed to exist in a trophic relationship supported by glucose cross-feeding. However, previous research has highlighted a lack of evidence showing that yeast strains consume the glucose that F. sanfranciscensis produces. We have investigated the interaction between sourdough isolates of M. humilis and F. sanfranciscensis in a synthetic wheat sourdough medium, allowing us to control substrate composition and use flow cytometry to enumerate living and dead cells. M. humilis fitness was found to be lower in co-culture with F. sanfranciscensis than when grown alone. Analysis of spent medium composition highlighted the reliance of M. humilis on glucose rather than maltose for growth. Comparisons of predicted and measured co-culture metabolite content also revealed that F. sanfranciscensis consumed less maltose in co-culture than when grown alone. For the first time, we examined potential amino acid cross-feeding between M. humilis and F. sanfranciscensis, and found that within the pairing, F. sanfranciscensis was the main producer of amino acids. Our findings suggest that the M. humilis-F. sanfranciscensis interaction is likely to be neutral, or even competitive, with the strain identity of F. sanfranciscensis playing a defining role in the observed dominance of the bacteria and spent medium metabolite composition. ImportanceThe association of the yeast Maudiozyma humilis and the bacterium Fructilactobacillus sanfranciscensis in sourdough starters is well-documented, and together this pairing makes key functional and organoleptic contributions to the final bread product. Their relationship has historically been thought to be stabilised by cross-feeding of glucose to M. humilis. However, this theory has been drawn into question by recent research which found no evidence that M. humilis consumes the glucose produced by F. sanfranciscensis. Our understanding of cooperation, coexistence, and competition in microbial consortia affects approaches to ecosystem management in a broad variety of applied fields. The significance of our research is in demonstrating that this pairing does not interact mutualistically within a specified setting, providing support for neutral or competitive interactions as drivers of ecological stability. Research areas:

Yarrowia lipolytica is a yeast of industrial interest exhibiting remarkable lipolytic and proteolytic capacities, with a high potential for white biotechnology applications. This yeast can be isolated from a wide range of natural, polluted or anthropogenic environments, including various food products. The present study aims to increase the data on Y. lipolytica phenotypic diversity by evaluating the growth parameters and secreted enzymatic activities of 28 wild-type Y lipolytica (and Yarrowia sp.) strains isolated from various environments across 10 countries. These data could facilitate the selection of appropriate strains for specific research purposes, particularly when wild-type strains are prioritized over genetically engineered ones, like for food-related applications. Notably, strain SWJ-1b exhibited an outstanding combination of favourable characteristics, with optimum (or near) performances for both growth and enzymatic parameters.

The transcription factor Pho4 is crucial for the response to phosphate starvation in many fungi, and it has been linked to tolerance to alkalinization of the medium and to pathogenicity. It is widely accepted that it is encoded by a single gene. However, the industrially relevant yeast Komagataella phaffii might contain two Pho4-encoding genes (PAS_chr1-1_0265 and PAS_chr2-1_0177, designated here PHO4(A) and PHO4(B), respectively), which have never been functionally characterized. The phenotypic analysis of single and double mutants suggests that Pho4(B) plays a major role in the adaptation to Pi scarcity. While single mutants exhibited limited and non-overlapping phenotypic defects, the pho4(A) pho4(B) strain was sensitive to multiple types of stress, including phosphate starvation and alkaline pH. Transcriptomic analysis confirms that Pho4(B) is crucial for the transcriptional response to phosphate starvation, including induction of typical gene markers (PHO5, PHO89, VTC1, etc.). However, by using a GFP reporter we found that PHO4(A) also participates in the induction of PHO89 under high pH stress. Expression of both PHO4(A) and PHO4(B) in S. cerevisiae complemented the pho4 mutation under phosphate limitation by restoring growth, expression of the Pho84 transporter and secreted phosphatase activity. These results indicate that both transcription factors display partially overlapping functions, responding differently to diverse stimuli, and that together they constitute a key component in the adaptation to a variety of stresses. Therefore, K. phaffii is an exceptional example among fungi that encodes two Pho4 functional transcription factors.

Yeasts have long served as key experimental systems in genetics, cell biology, and fermentation research; however, these studies have largely focused on a limited number of model species. In contrast, yeasts are a fungal group with remarkable physiological and ecological diversity. To provide a global overview of yeast diversity, we compiled and visualized phenotypic information for approximately 1,300 yeast species documented in The Yeasts (5th edition), including carbon utilization profiles, fermentation capacity, growth temperature ranges, and reported isolation sources. Taxonomic reconciliation revealed extensive reannotation, with approximately 44% of the species undergoing name changes and recognized genera increasing from 143 to 233. Integrative analyses revealed pronounced phylogenetic structuring of metabolic breadth. Many basidiomycetous yeasts, particularly Agaricomycotina, exhibited broader, generalist-like carbon utilization, whereas ascomycetous yeasts, especially Saccharomycotina, more frequently displayed sugar-centered, specialist-like narrower profiles; model yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe fell at the narrow end of this spectrum. Fewer than half of all species fermented glucose, a trait largely confined to Saccharomycotina. In addition, nearly one-fifth of species failed to grow at 30 {degrees}C, the standard laboratory temperature. By reconstructing and visualizing decades of dispersed taxonomic knowledge accumulated in The Yeasts, this study reframes yeasts not merely as laboratory model organisms but as metabolically diverse fungi whose phenotypic diversity reflects diverse ecological contexts. The analytical framework presented here provides a foundation for integrating standardized quantitative phenotypes and newly described species and offers a starting point for exploring the latent ecological and metabolic potential of yeast diversity.

Rock-inhabiting fungi thrive in subaerial oligotrophic environments such as desert rocks, solar panels and marble monuments where organic carbon and nitrogen are scarce. We tested whether the rock-inhabiting fungus Knufia petricola showed a preference regarding nitrogen ([Formula] or [Formula]) and carbon (glucose or sucrose) sources and whether it was sensitive towards carbon and nitrogen limitation. As this fungus produces the carbon-rich, nitrogen-free 1,8-dihydroxynaphthalene (DHN) melanin, we tested whether a melanin-deficient mutant would be less sensitive to carbon limitation. The carbon and nitrogen concentrations were the primary predictors of growth, with a broad optimum partially explained by an optimal fungal C:N ratio. Limiting carbon or nitrogen supply decreased biomass formation, CO2 production and biofilm thickness but promoted substratum penetration through filamentous growth. The nitrogen content of the biomass was flexible within limits, increasing upon increasing nitrogen supply or decreasing carbon supply. The carbon use efficiency was fairly constant, whereas melanization correlated with a higher nitrogen content of the biomass despite melanin being nitrogen-free. In conclusion, in vitro, K. petricola switches to explorative growth under nutrient limitations, like fast-growing fungi, revealing universal fungal resource-acquisition patterns. Graphical abstract text and imageCarbon and nitrogen availability affect biofilm growth and morphology of the extremotolerant fungus Knufia petricola Abolfazl Dehkohneh, Julia Schumacher, Bastiaan J. R. Cockx, Karin Keil, Tessa Camenzind, Jan-Ulrich Kreft, Anna A. Gorbushina, Ruben Gerrits Growth of the rock-inhabiting fungus Knufia petricola was studied by varying carbon and nitrogen sources and concentrations. Overall, growth was best predicted by the carbon and nitrogen concentrations. Carbon and nitrogen limitation promoted substratum penetration through filamentous growth. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/712823v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@6d98bdorg.highwire.dtl.DTLVardef@146aac5org.highwire.dtl.DTLVardef@757fa8org.highwire.dtl.DTLVardef@ff709_HPS_FORMAT_FIGEXP M_FIG C_FIG

Measuring the growth rate of filamentous fungi is an essential phenotype assay in fungal biology, enabling the comparison of nutrient-related fitness metrics across various isolates, species and genera. Conventional methods are time consuming and labor intensive, which prohibits the adaptation and implementation of high-throughput phenotyping. Here, we suggest a high-throughput methodological pipeline to study fungal growth on solid media combining the use of 24-well plates, an automated image acquisition system, and human assisted deep learning analysis of acquired images. Training a deep learning model through an iterative process - with continuous feedback and corrective annotations - enabled the development of a satisfying model that automatically segments pixels belonging to either fungus or background within a few hours. We evaluated this deep learning model by applying it to two test sets: First, a set of 336 images was used to validate the results by comparison with manual measurements. We demonstrate that the automated segmentation approach provides robust estimation of fungal growth not significantly different to manually segmented data. Second, a larger test set consisting of 2,016 images was used to illustrate the scalability of the model. After training the model for less than two hours, the deep learning model segmented the entire image data set automatically within minutes. The presented method is easily scalable and adjustable to other fungi and growth morphologies, due to the interactive training. Moreover, by combining 24-well plates and automatic image acquisition, measurements can be sped up as growth is detected across a smaller surface area than a standard six or nine cm diameter petri dish. The proposed methodological pipeline thus offers a new tool for estimating fungal growth rates, which can accelerate measurements, reduce bias, and increase throughput.

IntroductionThe use of Saccharomyces cerevisiae to ferment alcoholic beverages is an ancient tradition, with genetic evidence indicating origins in Neolithic Asia, although the domestication process of the species is not fully understood. Kveik is a group of traditional yeasts used in farmhouse brewing in western Norway preserved through generations of rural brewing practice. While recent studies have highlighted the distinctiveness of kveik, its precise phylogenetic position, genetic diversity, and domestication history remain unclear. ResultsWe performed whole-genome sequencing on 62 samples representing 25 unique Norwegian strains selected using cultural heritage criteria, and generated telomere-to-telomere (T2T) assemblies for representative isolates. Phylogenomic and population genetic analyses reveal that kveik forms a paraphyletic and early diverging group with respect to other domesticated S. cerevisiae strains. Most strains exhibit low within-strain diversity, strong geographic clustering, and little evidence of gene-flow or admixture. Mitochondrial genomes and Ty1 retrotransposon profiles corroborate this distinct lineage history. We further show that previously reported signals of gene flow between kveik and Asian fermentation strains are likely artifacts caused by population structure and selection. Divergence time estimates suggest that the common ancestor of beer, kveik, and other liquid-phase fermenting strains originated from ancestral populations 4,000 to 8,000 years ago. ConclusionKveik yeasts represent a relic of early S. cerevisiae domestication, shaped by ancient human practices, migrations, and the spread of agriculture. Our genomic resource sheds light on yeast evolution and domestication. They likely comprise some of the oldest domesticated lineages in continuous use until today, connecting endangered intangible cultural heritage to an early genetic origin.

Maudiozyma humilis is the second most frequently encountered yeast species in sourdough bread. Despite its ecological and food relevance, little is known about its evolutionary trajectories and phenotype traits of interest. Here we investigated the genomic and phenotypic diversity of a world-wide collection of 55 M. humilis strains, including 52 from sourdough, by combining genomic analysis, flow cytometry and high-throughput phenotyping of fermentation kinetics and fitness. Population genomic analysis revealed six genetically distinct clades, three diploid and three triploid, with no geographical or substrate-specific structuring. Phylogenetic, loss of heterozygosity (LOH) distribution and allele specific analysis indicated that triploid strains originated from both recent and more ancient hybridization events involving multiple diploid lineages. The absence of the HO gene, and mating-type silent cassettes, revealed that M. humilis is not able to carry mating-type switching. In addition, high linkage disequilibrium (LD) and variable LOH accumulation were observed consistent with a predominantly clonal reproduction. Last, an exceptionally high level of heterozygosity was detected, suggesting that occasional hybridization is the major driver of genetic diversity. Phenotypic characterization in a synthetic sourdough medium revealed variation in fermentation kinetics and fitness statistically associated with the genetic clades. Interestingly, genetic distance between clades and strains better explain the phenotypic variation than difference in ploidy. Altogether, our findings highlight the complex evolutionary history of M. humilis, shaped by hybridization and ploidy variation and reveal that historical contingency, more than ploidy, shapes the phenotypic landscape of this species. Beyond providing the first analysis of M. humilis evolution, our result challenges the hypothesis that increases in ploidy are necessarily beneficial in domesticated species.

Chromosome copy number variation (CNV) is a major contributor to genome plasticity and adaptation in Candida albicans, a leading fungal pathogen of humans. Aneuploidy, defined as deviations from the normal diploid chromosome set, rapidly alters gene dosage, enabling tolerance to host-imposed and antifungal stress. Accurate detection and quantification of chromosomal copy number changes are thus essential to dissect the mechanisms by which C. albicans adapts and evolves. Here, we describe the development, optimization, and validation of a six-color, 16-plex droplet digital PCR assay for simultaneous quantification of all C. albicans chromosome arms in a single reaction. Each target is detected by a unique dual-color or single-color combination of probes, enabling high-order multiplexing through binary fluorescence encoding. Following optimization of probe concentrations, PCR cycling parameters, genomic DNA extraction and pre-treatment with restriction enzymes, the assay provides accurate, reproducible chromosome-level copy number estimates that correlate closely with WGS results across euploid and aneuploid isolates. Compared to whole-genome sequencing, the assay is rapid, cost-effective, and scalable, requiring minimal DNA input and allowing high-throughput analysis of large isolate collections. The 16-plex assay thus provides a platform for dissecting genome instability and adaptive evolution in C. albicans. Article SummaryWe developed and validated a 16-plex droplet digital PCR assay that estimates chromosome dosage across the entire genome of the human fungal pathogen C. albicans in a single reaction. The assay uses six fluorescent colors and unique color combinations to track one marker on each chromosome arm, enabling rapid detection of aneuploidy (extra or missing chromosomes). Results closely matched whole-genome sequencing for isolates with simple aneuploid forms and detected low-frequency trisomic clones in mixed populations. With optimized DNA preparation, this method provides a practical tool for screening genome instability in research and clinical settings.

Using its 9 sequentially-placed cohesin (Coh) domains as bait, each CipA scaffoldin chain in a Clostridium thermocellum cellulosome binds to (and displays) some combination of 9 out of over [~]70 available dockerin (Doc) domain-bearing lignocellulose-degrading enzymes, at any time. Domains numbered Coh3 through Coh8 show over 94 % pairwise sequence identity with each other, but only [~] 61-76 % identity with Coh1, Coh2, and Coh9. Such identities are much lower ([~] 40-60 %) amongst Doc domains; however, amongst both Coh and Doc domains, polypeptide backbone folds are highly conserved, suggesting that loading preferences of enzyme-bearing Doc domains upon Coh domains must depend upon their relative abundances, and pairwise affinities. To explore this further, we used microscale thermophoresis (MST), size exclusion chromatography (SEC), native polyacrylamide gel electrophoresis (NPGE), mass spectrometry (MS) and bioinformatics-based approaches (BIBA), to examine 28 Coh-Doc pairwise interactions involving recombinant Coh [Coh1, Coh2, Coh3, Coh9] and enzyme-bearing Doc [Cel8A, Cel9F, Man26/5H, Cel9R, Xyn10C, Xyn11D, Xyn10Z] domains. Interactions were found to occur with varying affinities, suggesting that Coh1 prefers Xyn11D; Coh2 prefers Xyn10C; Coh3 prefers Cel9R; Coh9 prefers Cel9R; Coh1/Coh2 prefer Xyn partners; Coh3/Coh9 prefer Cel partners. Dual modes of binding are shown by Coh1 with Xyn10C and Xyn11D; Coh2 with Xyn10Z, Cel8A, and Cel9R; Coh3 with Cel8A, and Cel9R; and Coh9 with Xyn10C, suggesting that Doc domains use either of their two homologous helices (1 and 3) to bind to Coh domains, as earlier proposed. ImportanceBacteria such as Clostridium thermocellum use extracellular enzyme complexes called cellulosomes to degrade and use cellulose. Each complex uses a linear chain of nine cohesin (Coh) domains called a scaffoldin to bind to (and display) any nine of over seventy available xylan or cellulose-degrading enzymes that bear dockerin (Doc) domains. Understanding interactions between Coh and Doc domains facilitates an appreciation of how cellulosomes are assembled and supports the building of protein-engineered constructs that utilize such interactions for many conceivable enzymatic and other applications. The significance of the presented research lies in its demonstration of the differential modes and pairwise affinities of different Coh-Doc interactions, using recombinant protein constructs and a combination of quantitative, semi-quantitative and qualitative analytical methods.

Coordination of metabolism, cell growth and cell division is essential to life. Recent single-cell measurements in S. cerevisiae have shown that metabolic processes and the cellular redox state are dynamic along the cell cycle. However, it is unknown whether similar metabolic oscillations also occur in other organisms. Until now, the dynamics of metabolism in other eukaryotes have predominantly been studied in cell cycle synchronised populations. Since cell cycle synchronisation methods can perturb metabolism, they may also introduce artefacts in the recorded dynamics. Here, we performed time-lapse microscopy analyses of exponentially growing single cells of the budding yeast S. cerevisiae, the fission yeast S. pombe and murine leukaemia L1210 cells. Measuring the NAD(P)H autofluorescence and the cell surface area growth rate in unsynchronised cells, we discovered oscillations along the cell cycle of the cellular redox state and lipid metabolism, respectively. Thus, our work shows that metabolism is dynamic along the cell cycle of these three evolutionarily distant eukaryotic organisms. This finding suggests that such metabolic oscillations could be a conserved characteristic among eukaryotes.

Drug interaction outcomes-synergism, additivity, or antagonism-represent complex phenotypes. While drug repurposing aims to identify compounds that potentiate conventional antifungals, when given in combination, the modulators of these interactions in fungi remain largely unexplored. We hypothesize that the response to repurposed-conventional antifungal pairs is a complex trait modulated by genetic and environmental factors. To study the impact of genotype on the outcome, we screened six diverse Saccharomyces cerevisiae isolates, including clinical, wild, and fermentation strains, for their responses to combinations of ibuprofen with either clotrimazole or caspofungin. We evaluated the role of the environment using rich and minimal media and assessed the influence of assay type by comparing solid- and liquid-rich media assays. Our results reveal that ibuprofen-clotrimazole interactions are highly dynamic, predominantly antagonistic, with limited synergy observed. These outcomes are significantly modulated by genetic background, media composition, assay type, and, in specific genotypes, even by the drug dosage, reflecting a complex, multi-parametric phenotype. However, the ibuprofen-caspofungin combination is more predictable, exhibiting only synergy or additivity. Interaction outcomes correlate with baseline sensitivity to caspofungin: caspofungin-resistant isolates consistently demonstrate synergy, while sensitive strains exhibit additivity. These findings shift the paradigm of drug discovery by demonstrating that synergism and antagonism are not static properties of drug pairs but are dynamic, context-dependent outcomes. This study highlights the need to use clinically relevant models and patient-specific isolates before clinical application, as drug interactions cannot be generalized from a single dosage, strain, or environmental condition.

Bacteria increase in biomass and divide, but determining precisely when cell division completes is technically challenging. To aid time-lapse imaging and cell-cycle tracking, we set out to identify a protein in Bacillus subtilis, which when fused with a fluorophore would cause the membrane to fluoresce in a manner that was constitutive, uniform, and bright. A forward genetic transposon-based approach combined with fluorescence-activated cell sorting was used to identify a fluorescent fusion to the glucose PTS transport transmembrane protein PtsG with all desired properties. Moreover, PtsG-GFP was constitutive and neutral to growth under all conditions tested and also labeled membranes during sporulation. We used PtsG-GFP to track cell growth in microfluidic channels and determine when cytokinesis occurred, defined as when fluorescence reached a local maximum at the division plane. Simultaneous imaging with a compatible fluorescent fusion to the cell division protein FtsZ indicated that FtsZ peak intensity occurred midway through septum constriction and that Z-ring recycling coincided with cytokinesis. We conclude that PtsG-GFP is a powerful tool for membrane imaging and cell cycle tracking. As such, we provide constructs with fluorophores that emit across the visible spectrum and antibiotic resistance cassettes to facilitate deployment in B. subtilis. IMPORTANCEBacterial cells are fully divided when new membrane separates the cytoplasm of each daughter. Reproducibly staining of bacterial membranes with exogenous labels for fluorescence microscopy can be challenging, particularly during chemostatic growth in microfluidic devices. Here, we report that fusion of a fluorescent protein to the glucose transport protein PtsG causes the membrane of Bacillus subtilis to give off bright and even fluorescence under a variety of conditions. We use PtsG-GFP to operationally define when cytokinesis occurs during growth, and we note that a fluorescent PtsG fusion would likely make fluorescent staining of the membrane more facile theoretically in any organism.

The efficient production of food and biochemicals using microorganisms that utilize single-carbon feedstocks presents a promising approach for advancing a circular bioeconomy. Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast already widely used in industry, making it an attractive host for such applications. Recently, K. phaffii was converted into an autotrophic strain capable of assimilating CO2 into both biomass and secreted organic acids, using energy derived from dissimilation of methanol to CO2. In these strains, methanol oxidation is catalysed by an alcohol oxidase (Aox2), which transfers electrons to oxygen without conserving reducing equivalents. To address this limitation, in this study we explored redirecting methanol dissimilation through the native alcohol dehydrogenase (Adh2), coupling methanol oxidation with NADH generation to improve carbon efficiency. By deleting AOX2 and overexpressing ADH2, we generated Adh2-based autotrophic strains that exhibited growth rates comparable to the parental strain (0.007 h-{superscript 1}), while reducing specific CO2 production by 53% and increasing biomass yield (YX/MeOH) by 59%. We further applied this strategy to convert previously developed autotrophic strains producing itaconic acid and lactic acid into Adh2-dependent strains. Optimizing ADH2 expression through multicopy integration resulted in strains with approximately two-fold higher molar carbon efficiency (Y(X+P)/CO2) while achieving elevated product titers--2.2-fold for itaconic acid and 3.8-fold for lactic acid--relative to the parental strains. Our findings demonstrate that alcohol dehydrogenase-mediated methanol dissimilation can significantly improve yield and productivity of autotrophic K. phaffii strains, with broad implications for sustainable bioproduction from one-carbon substrates.

The growth of cultures and formation of mucilage blooms in reaction to salt stress of cyanobacterial cultures are investigated with a focus on the influence of pH. In non-buffered medium, cultures show their pH increasing from 6.5 just after inoculation, up to 11 during the exponential phase. We record the time-evolution of concentration and pH, with different initial OD0. In a second set of experiments, we extract the doubling time of the unbuffered cultures in comparison with those inoculated in pH-buffered BG11 media at four different pH from 6.3 to 10.5 : in the most acid media, all cultures die or grow very slowly. At pH = 10.5, we obtain the fastest growth for all four strains, allowing to qualify these cyanobacteria as being alkaliphiles, though for all strains with comparable initial OD0, the doubling time is shorter for unbuffered cultures. Following a previous study [31]), we finally investigate the influence of pH on mucilage formation and biomass uplift induced by salt stress, involving EPS floculation by cations. Our results show that operating in buffered media significantly influences the mucilage formation, though the observed regimes cannot be simply correlated to the pH value.