Yeast

Environmental surveys of wild yeasts typically rely on ribosomal barcodes, which cannot resolve cryptic species, interspecific gene flow, mixed cultures, or population structure. To determine what genome-scale characterization adds, we sequenced a representative panel of wild yeasts spanning the genera Saccharomyces, Schizosaccharomyces, and Lachancea using Oxford Nanopore long-read whole-genome sequencing and placed each isolate within published reference datasets. Whole-genome analyses revealed biologically important features that barcoding alone could not detect. A shagbark-hickory isolate resolved as a genuine two-species co-culture. An oak-bark isolate proved to be Schizosaccharomyces versatilis, a recently reinstated species represented by very few known strains, and its analysis demonstrated that standard assembly-quality benchmarks can be misleading for deep-branching taxa. Three Lachancea thermotolerans isolates formed a distinct, previously unsampled population within the wild tree-associated lineage, extending its known geographic range. In contrast, an apparent signal of Saccharomyces eubayanus introgression in two beer-associated S. cerevisiae isolates disappeared after analysis with matched negative controls and de novo assemblies, showing that it reflected mapping artifacts rather than genuine ancestry. Together, these results demonstrate that inexpensive long-read whole-genome sequencing transforms wild-yeast bioprospecting from species identification into a genome-scale framework for resolving cryptic diversity, population structure, and mixed cultures while providing stronger support - and stronger limits - for evolutionary inference. SIGNIFICANCEMost surveys of wild yeasts identify isolates using short DNA barcodes, which are well suited for naming species but often miss the evolutionary relationships and hidden diversity within them. By applying inexpensive whole-genome sequencing to a diverse collection of environmental yeasts, we uncovered previously undetected mixed cultures, a rare recently recognized species, and a distinct wild population, while also showing that an apparent case of interspecies gene exchange was instead a technical artifact. These results demonstrate that genome-scale analysis can both reveal biological diversity that simpler methods overlook and provide the evidence needed to avoid misleading evolutionary conclusions, making it a powerful new approach for studying natural microbial populations.

The budding yeast Saccharomyces cerevisiae is a well-established model organism to study cellular stress response and underlying mechanistic regulation. Although glucose starvation fundamentally alters gene regulation and cell behaviour, inconsistent deprivation protocols often trigger gross morphological artefacts. These non-specific changes confound findings by activating pathways independently of true glucose-signalling mechanisms. Furthermore, a thorough transcriptomic profile of glucose starvation using non-confounding conditions remains lacking. Consequently, the precise transcriptional impact of losing key metabolic regulators that mediate adaptation to glucose starvation remains undefined. Here we have employed a refined glucose starvation protocol, utilising raffinose exchange, which shows induction of vast transcriptional stress response with minimal impact on cellular morphology confirmed by label-free imaging. Transcriptomic profiling revealed shifts in metabolic regulation, ATP turnover, and cell-to-cell communication as acute glucose deprivation driving cells towards oxidation-driven metabolism. Additionally, we characterise transcriptional alterations seen in deletion mutants of SNF12 and SPT20, known regulators of cellular metabolism, showing previously unappreciated transcriptional conservation, in part mimicking glucose starvation response. Finally, we identified cargo and stress-specific expression related to both eisosome components and surface transporters that are critical for metabolic adaptation. Overall, this dataset provides a comprehensive transcriptomic resource for dissecting stress signalling and driving novel hypothesis generation.

Three-dimensional live-cell fluorescence imaging of yeast cells is crucial for studying cell-cycle mechanics and regulation. However, extracting multi-channel phenotypes within dense cell clusters remains an image-processing bottleneck. Standard deep-learning models segment cells but fail to track mother-bud boundaries, mitotic spindle shapes and spindle-localizing proteins. Investigators rely on labour-intensive manual coordinate plotting, introducing observer bias and often exclude clustered cell data due to visual complexity. Here, we present an open-source Fiji pipeline for automated yeast cell image processing and deterministic classification of cell-cycle, spindle and protein dynamics. The workflow utilizes a dual-segmentation architecture via custom Cellpose models to capture the mother-bud cell boundaries. Extracted masks are integrated with multi-channel fluorescence data using a Difference-of-Gaussians framework to resolve SPB coordinates and localized protein kinetics, which a rule-based decision-tree maps to precise mitotic phenotypes. Validation demonstrates a 50-fold acceleration with ~6% deviation from manual analysis. Availability: Zenodo at https://doi.org/10.5281/zenodo.22083016.

Brettanomyces bruxellensis is a yeast species associated with diverse fermentation environments and characterized by extensive genetic diversity, including diploid, autotriploid, and allotriploid lineages resulting from independent hybridization events. These lineages are associated with distinct ecological niches and provide a framework for studying metabolic trait evolution in complex genomes. Nitrate assimilation is a relatively uncommon trait among yeasts and has been reported in B. bruxellensis, but its distribution and evolutionary history within the species remain poorly understood. Here, we combined phenotypic characterization of 151 strains with genomic analyses of 946 whole-genome sequences to investigate nitrate assimilation. Growth assays revealed that nitrate assimilation is widespread but unevenly distributed across genetic lineages, with some populations largely retaining the trait whereas others have frequently lost it. Genomic analyses identified extensive variation affecting the nitrate assimilation gene cluster composed of YNR1, YNI1, and YNT1. Nitrate assimilation was strongly associated with both gene copy number and predicted gene functionality, with nitrate-assimilating strains generally carrying more functional copies of the cluster. Leveraging the complex genomic architecture of the species, we independently analyzed primary and acquired genomes in allotriploid lineages and uncovered contrasting evolutionary trajectories following hybridization. While nitrate assimilation genes were generally maintained in primary genomes, acquired genomes showed a higher prevalence of gene loss and predicted loss-of-function variants, revealing asymmetric dynamics between subgenomes. Altogether, our results suggest that nitrate assimilation represents an ancestral trait that has been differentially maintained across B. bruxellensis lineages through a combination of copy number variation, gene degeneration, and genome-specific evolutionary dynamics. These findings provide new insights into how genome architecture and polyploid evolution shape the maintenance and loss of metabolic traits in an industrially relevant yeast species.

The coprophilous agaricomycete Coprinopsis cinerea is a model organism for antagonistic fungal-bacterial interactions. Previous studies showed that C. cinerea responds to antagonistic bacteria with strong induction of a set of genes encoding secreted antibacterial molecules. However, little is known about the elicitors of this response. Key open questions in this respect include whether individual antibacterial defence genes are induced by different bacteria and/or by specific bacterial soluble molecules. Here, we present a new C. cinerea reporter system to monitor antibacterial defence induction and address related outstanding issues with minimal hands-on time. In this system, the promoter of the endogenous bacterial-induced gene cclys1 drives the expression of cnluc, which encodes a secreted variant of the deep-sea shrimp luciferase Nluc. We show that cNluc allows to detect and quantify cclys1 induction by measuring luminescence directly in the culture medium of reporter strain colonies. Building on these features, we successfully leveraged the inducible cNluc reporter strain for the development of a novel 96-well plate assay that allows the high-throughput screening of antibacterial defence elicitors. As cNluc can be subject to degradation by secreted proteases of fungal or bacterial origin in the culture medium, we coupled this assay to confirmatory qRT-PCR. Testing this set-up by confronting the reporter strain with several different bacteria revealed that cclys1 induction occurs independently of the bacterial ecological niche. Based on these results, we also recommend qRT-PCR exclusively for validation of negative results. We conclude that cNluc offers significant advantages over cytoplasmic reporter proteins, especially for preliminary rapid screening of multiple conditions.

Recombinase polymerase amplification (RPA) enables rapid nucleic acid testing in low-resource environments, but poorly characterized byproducts can compromise assay specificity and cause false-positive results. Here, we amplified the thirteen original CODIS core loci and Amelogenin to characterize recurrent RPA artefacts and establish conditions that reduce their formation. First, RPA products were analyzed for two reference samples by Oxford Nanopore Technologies sequencing. This revealed two distinct classes of multimeric products: primer multimers and amplicon multimers, consisting of repeated primer or amplicon sequences, respectively. Individual artefacts contained up to 281 primer copies or 22 amplicon copies, demonstrating the extensive range of these products. Next, we performed an optimization study to evaluate the effects of reaction temperature and reagent concentrations at two representative loci, D3S1358 and D5S818. Among the conditions tested, temperature had the most pronounced effect. Reducing the temperature from 42{degrees}C to 34{degrees}C increased the relative target amplicon fraction from 15% to 83% for D3S1358 and from 84% to 98% for D5S818, while maintaining or increasing absolute target concentration. Lower primer concentrations and higher T4 UvsX concentrations also reduced multimer formation, although lower primer concentrations reduced target yield and caused allelic dropout. Finally, amplification at 34{degrees}C was evaluated across all fourteen loci by sequencing. Relative to 42{degrees}C, the target read fraction increased by more than 5 percentage points for 7/14 loci in one reference sample and 9/14 loci in the other, with the largest improvements at multimer-prone loci. These findings identify multimers as an important class of RPA artefacts and establish reaction temperature and T4 UvsX concentration as promising conditions to improve RPA specificity.

Forest-associated Saccharomycotina occur at low relative abundance, limiting inference about their diversity and dynamics. We sampled leaf litter weekly for 47 weeks across a subtropical forest in northern Taiwan. Enrichment, isolation and ITS sequencing recovered 687 isolates, including 613 Saccharomycotina representing 56 described species and 77 putatively novel operational taxonomic units. Rarefaction indicated unsampled culturable diversity. Among litter traps, community dissimilarity was high and dominated by taxon replacement, but neither topography nor geographic distance was associated with composition, and turnover matched randomised expectations. Richness peaked during warm, wet periods and declined in winter, and minimum temperature showed the strongest statistical association. Composition was associated with maximum temperature, minimum relative humidity, precipitation and solar radiation. Selected isolates' thermal optima covaried with collection-week temperatures, and two October Magnusiomyces magnusii isolates had higher optima than four winter isolates. Together, these findings reveal substantial culturable diversity and seasonal community restructuring consistent with temperature-related filtering.

Sporulation is an integral process in the lifecycle of many fungal pathogens, including Histoplasma, a primary human pathogen that causes respiratory infections. Histoplasma conidia, or asexual spores, are the primary infectious particle but very little is known about them, in part due to the need for Biosafety Level 3 containment and inconsistency in generating viable conidia under laboratory conditions. Here, we identify media that consistently promote Histoplasma conidiation, yielding both micro- and macroconidia, and conditions that promote high levels of germination. We show that conidiation media and duration affect the proportion of macroconidia produced, and we demonstrate that Histoplasma strains vary in their response to these conidiation parameters. Finally, imaging studies of chitin, exposed chitin, and cell wall mannoproteins show that while micro- and macroconidia have similar cell wall compositions, strain type and conidiation media variation result in qualitative differences in staining. These optimized methods for Histoplasma conidial preparations will enable more detailed investigations into this understudied aspect of the biology of an important human fungal pathogen.

SummaryPangenome graphs capture extensive genetic diversity but introduce analytical challenges due to the redundant representation of structural variations (SVs). While existing tools effectively address cross-sample redundancy or cross-locus redundancy, none specifically target the intra-locus allelic redundancy inherent to pangenome graphs. Here, we present PanSVmerger, an open-source tool designed to consolidate redundant multiallelic SVs within individual loci using three complementary clustering strategies: adaptive k-mer-based Jaccard distance, global alignment distance via VSEARCH, and length distribution. Validation on HPRC pangenome data demonstrates that PanSVmerger effectively reduces multiallelic complexity (e.g., AC [≥] 3 loci from 62.4% to 4.7% using Strategy A) with a modest trade-off: Recall decreased from 97.13% to 93.58%, while precision improved from 94.95% to 96.56%, yielding an overall F1-score of 95.05%. These results demonstrate that PanSVmerger effectively consolidates redundant allele representations with only a minimal loss of sensitivity, making it well-suited for downstream applications that require clean, non-redundant variants. Availability and implementationPanSVmerger is implemented in Python 3.8+ and freely available under the MIT license at GitHub: https://github.com/tingting100/PanSVmerger. The software requires vcflib, bcftools, and optionally VSEARCH. Comprehensive documentation and tutorials are provided.

Quantification of microbial growth inhibition is central to assays ranging from antibiotic susceptibility of bacteria to sensitivity of yeasts to antifungal therapeutics. Classical analysis approaches derive from zone-of-inhibition (termed halo) formats using filter paper discs, spanning methods from laser detection to machine learning. However, these tools struggle with non-uniform halos, fail to account for lawn density variability despite its experimental influence, and lack accessible, reproducible code. Here, we present Halo Unbiased Measurement of growth Inhibition (HaloUMI); an open-source Python graphical user interface for automated, high-throughput analysis of lawn-based microbial assays. HaloUMI integrates robust image processing with physics-informed models to quantify inhibition zones irrespective of shape, enabling accurate segmentation of uniform and irregular halo phenotypes. This analysis pipeline incorporates the critical correction for spatial heterogeneity in lawn density, improving reproducibility across experimental conditions. The software enhances usability without sacrificing precision, allowing rapid batch processing and intuitive parameter control. HaloUMI can be applied to multiple assay types, including yeast toxin halo, microbial mating, and conventional filter paper disc assays. It yields high-precision measurement of halo size and morphology, with improved consistency compared to standard thresholding and circular fitting. By combining accessibility, flexibility, and biophysical modelling, HaloUMI provides a quantitative framework for irregularly shaped halos of lawns of varying growth potential, enabling generalisable analysis of broad microbial interactions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/743694v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1bd7c88org.highwire.dtl.DTLVardef@13ac9b6org.highwire.dtl.DTLVardef@9108e1org.highwire.dtl.DTLVardef@1de06bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microbial growth screens generate thousands of curves, but cross-experiment comparison and mapping growth phenotypes to genotypes or environments routinely require custom code. GUIbiont is a no-code browser application for quality control, curve fitting, clustering and metadata-linked analysis with machine learning techniques. Interactive sessions export as Julia scripts, allowing users to reproduce or extend browser analyses. Validated across 3,885 E. coli deletion strains and 13,608 defined-media curves, GUIbiont recovered known auxotrophic and nutrient-dependent phenotypes.

Brook trout (Salvelinus fontinalis) exhibit strain-level variation in growth performance, environmental tolerance, and survival, yet the biochemical mechanisms underlying these differences remain poorly understood. We developed and applied a ratiometric biochemical framework integrating the pentose-phosphate pathway (PPP) and glutathione metabolism to characterize strain-specific hepatic metabolic organization in brook trout. Five strains reared under standardized conditions differed significantly in hepatic soluble protein density, glutathione pool size, total NADP(H) concentration, and activities of glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), and transketolase (TKT). These differences were not uniformly coordinated across pathways, demonstrating that metabolic phenotype cannot be inferred from individual biomarkers alone. Derived ratios describing oxidative-to-non-oxidative PPP capacity (G6PDH/TKT) and glutathione buffering relative to recycling capacity ((GSH+GSSG)/GR) resolved distinct patterns of metabolic allocation among strains. Despite shared ancestry, the Temiscamie (TEM) strain and its domestic x TEM hybrid (TXD) exhibited markedly divergent metabolic phenotypes, demonstrating that closely related strains can differ substantially in hepatic metabolic organization. Together, these findings identify relative allocation among interconnected metabolic pathways as an axis of physiologic diversity and establish a ratiometric approach for comparing metabolic organization across populations and species. Graphical abstractHepatic metabolic phenotypes of brook trout strains were characterized by integrating pentose phosphate pathway enzyme capacities, glutathione metabolism, NADP(H) availability, and soluble protein into a ratiometric framework. Ratios distinguish investment in oxidative versus non-oxidative PPP capacity (G6PDH/TKT), antioxidant buffering versus glutathione recycling capacity (total glutathione/GR), and hepatic protein density (soluble protein/liver mass), revealing distinct metabolic organization among strains. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/743818v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1694676org.highwire.dtl.DTLVardef@90f2d4org.highwire.dtl.DTLVardef@365327org.highwire.dtl.DTLVardef@8d56ca_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA ratiometric framework was developed to characterize hepatic metabolic organization in brook trout C_LIO_LIGlutathione buffering and recycling capacity distinguish alternative redox phenotypes C_LIO_LIInvestment in oxidative and non-oxidative PPP capacity varies independently among strains C_LIO_LIG6PDH/TKT and total glutathione (GSH+GSSG)/GR reveal distinct metabolic phenotypes C_LIO_LIRatiometric indices provide a framework for interpreting redox metabolism and carbon allocation C_LI

Plant cell walls are polysaccharide-rich extracellular matrices composed of multiple complex carbohydrate polymer networks, including cellulose, hemicelluloses, pectins, and glycosylated proteins. Polysaccharide deposition critically impacts cell wall structure, and structural microheterogeneity within cell wall glycans also influences polymer rigidity and polymer-polymer interactions. Collections of monoclonal antibodies (mAbs) have been developed to target unique carbohydrate epitopes within cell wall polysaccharides and to investigate how these structural changes impact cellular and plant development. Here, we implement generalizable methods to attach unique DNA barcodes to mAbs that recognize major cell wall polysaccharide classes. By applying these mAbs individually to polysaccharide standards, we demonstrate that bound DNA barcoded antibody abundance can be measured via quantitative PCR. Additionally, we demonstrate that DNA conjugated antibodies can be pooled to quantitatively analyze polysaccharide epitope composition of polysaccharide standards and fractionated cell wall material by amplifying their unique barcodes via qPCR. These results demonstrate that barcoded polysaccharide-directed mAbs offer sensitive, quantitative insights into cell wall polysaccharide composition and facilitate multiplexed profiling of cell wall polysaccharide abundance. This approach will also enable multiple future high-throughput applications, such as glycome profiling, spatial glycomics, and glycan interaction measurements, that will further our understanding of cell wall compositional impacts on plant physiology.

Sexual coral propagation is an emerging technique capable of producing large numbers of corals for coral transplantation and reef rehabilitation. In contrast to asexual coral propagation, sexual propagation increases genotypic diversity and can be used for selective breeding to enhance coral heat tolerance or other desirable traits. However, implementation at meaningful ecological scales is hindered by high mortality during early life stages, high costs associated with nursery rearing facilities, and labour-intensive outplanting methods. To overcome these issues, we developed the CoralAssist Plug (CAP), a ceramic device designed for the rapid and cost-effective outplanting of sexually propagated corals in large numbers that maximises post-outplant survivorship. CAPs combine three important functional features: 1) built-in microrefugia to protect juvenile corals from grazing, 2) a relatively small size, 3 by 1 cm, that is easy to handle and stack efficiently without compromising the survivorship of corals, and 3) a hole in the middle that facilitates handling and attachment. CAPs were settled with Acropora aff. digitifera and outplanted to a reef crest after 1 to 6 months of ex situ nursery rearing. A 3-person dive team was able to outplant ~120 CAPs in one 90-minute shallow dive (just over 2 minutes per CAP per person). With longer nursery durations of 6 months, it was possible to achieve 36 % yield (i.e., the proportion of devices with a surviving coral) 4-years post-outplant. With nursery durations shortened to 1 month, we were able to attain 24 % yield 3-years post-outplant. Microrefugia significantly enhanced post-outplant survivorship leading to an 11 % increase in yield 4 years post outplant compared to devices without microrefugia. Outplanted corals that had reached adult size, were self-attached and were reproductively mature after 4 years. Our results suggest that CAPs can play a meaningful role in reef rehabilitation by efficiently introducing sexually propagated corals into natural populations with clear applications to assisted evolution techniques, such as selective breeding.

Over the past decade there has been increasing interest in the use of microhaplotype markers in autopolyploid taxa. This has been driven by theoretical and observed improvements in signals of allelic dosage, linkage, and heritability. Yet, to date there has been little investigation into the suitability of microhaplotype markers for estimating kinship. Here, we develop the theory of kinship estimation from microhaplotypes, introduce the MCHap microhaplotype caller for autopolyploid populations, and apply these methods to a highly diverse germplasm population of mixed-ploidy Actinidia (kiwifruit and relatives). We find that microhaplotype-based kinship estimates are generally superior to equivalent single nucleotide variant based estimates. This is because microhaplotypes minimize the coalescent signal among alleles which may bias estimates within the context of a recent reference population. Hence, kinship estimates from microhaplotypes more accurately capture the recent demographic history of a population. These findings are supported by both coalescent simulations and the analysis of real data. Our findings are relevant to organisms of any ploidy, but most actionable in highly heterozygous taxa such as Actinidia.

Prolonged low-temperature exposure may extend the preservation window of mammalian cells but can also disrupt cellular homeostasis and ultimately compromise cell viability. This study investigated the time-dependent phenotypic and transcriptomic responses of primary canine dermal fibroblasts to sustained hypothermic stress. Passage-three fibroblasts were continuously maintained at 15 for up to 15 days, with samples collected on Days 0, 3, 6, 9, 12, and 15. Cellular morphology, metabolic activity and viability, and apoptosis were evaluated using bright-field microscopy, Cell Counting Kit-8 assays, and Annexin V-FITC/propidium iodide flow cytometry, respectively. RNA sequencing was performed to characterize dynamic transcriptional changes throughout the exposure period. Early low-temperature exposure was associated with relatively preserved cellular morphology and viability, suggesting a transient adaptive response. With increasing exposure duration, fibroblasts exhibited progressive morphological deterioration, reduced metabolic activity, loss of adhesion, and increased apoptosis. Time-series transcriptomic analysis further revealed temporally coordinated and stage-dependent gene-expression programs associated with metabolic regulation, cellular stress responses, structural homeostasis, and cell survival. Integration of phenotypic and transcriptomic data demonstrated that the response of primary canine dermal fibroblasts to 15 was dynamic rather than linear, progressing from early adaptation to cumulative dysfunction during prolonged exposure. These findings provide a framework for defining the low-temperature tolerance of primary canine dermal fibroblasts and may inform the optimization of protocols for their short- to medium-term preservation and transportation.

The morphology of cyanobacterial colonies plays a key role in harmful cyanobacterial blooms, with implications for their vertical migration, resistance against grazing, and light availability. In this study, we introduce the use of Optical Coherence Tomography (OCT) to investigate the three-dimensional morphology of cyanobacterial colonies. The technique enables non-invasive 3D imaging of colonies up to several millimeters in size, providing access to detailed mesoscale morphological features. Gas vesicles inside cells were shown to strongly improve image quality. We describe the sample preparation and image acquisition protocol, as well as an image processing pipeline that extracts mesoscale morphological features and provides a volumetric visualization of colonies. The method was tested for representative colonies of different cyanobacterial species while a dataset of volumetric images and measured mesoscale features was acquired for natural colonies of Microcystis. We demonstrate the utility of 3D imaging by quantifying the effects of irregular colony morphologies on their flotation velocity and the light availability within colonies. We anticipate OCT to become a key imaging technique to monitor populations of cyanobacterial colonies and investigate colony formation, with potential extensions to other colonial and aggregated organisms in freshwater and marine environments.

Snow algae are major biological drivers of snow darkening in polar and high-alpine environments. However, the direct contribution of algal pigmentation to snow reflectance has remained difficult to quantify because field observations cannot disentangle the effects of pigmentation from variation in biomass, species composition, and snow physical properties. Here, we characterized the optical effects of pigmentation using hyperspectral spectroradiometry to compare green, orange, and red cyst-like cells of a snow-derived Haematococcus isolate while controlling for developmental stage and cell abundance. Cysts became more red with increasing astaxanthin concentrations while chlorophyll-a concentrations remained relatively constant. Relative to green cysts, mean reflectance decreased by approximately 30% in orange cysts and 40% in red cysts. Integrated reflectance across the visible spectrum (350-800 nm) was negatively correlated with astaxanthin concentration. These results provide direct experimental evidence that algal pigmentation alone substantially reduces reflectance after controlling for cell abundance and developmental stage, and indicate that differences in snow physical properties may partly obscure this effect under natural field conditions. Our findings identify astaxanthin accumulation as an intrinsic driver of biological snow darkening and suggest that algal pigmentation, which may vary with species identity and physiological state, should be considered alongside biomass when predicting the radiative effects of snow algal blooms.

Blue light using Flavin (BLUF) proteins are microbial photoreceptors that are involved in various physiological responses. Their occurrence and biochemical properties in fungi remain poorly understood. Here, we investigated a putative BLUF photoreceptor from the corn-smut fungus Mycosarcoma maydis (MmBLUF). Domain analysis, multiple sequence alignment of BLUF core regions, and structural modelling indicated conserved canonical BLUF fold and flavin-pocket residues. However, when heterologously expressed, UV-visible and fluorescence spectroscopy revealed different spectral behaviour than canonical BLUF protein. Further, we tested the role of extended N-terminus in modulation of chromophore binding by expressing N-terminus truncated protein variants. Our results suggest that the unusual spectral behaviour is not linked to the truncation construct (extended N-terminus), which also showed similar spectral features, indicating that the extended N-terminus is unlikely to account for an unusual photodynamics characteristics. Our findings support MmBLUF as a structurally conserved putative fungal BLUF-like photoreceptor with different photochemical properties. Further studies are required to establish its chromophore identity, photocycle and function of this unusual BLUF-like domain from fungal system.

NicA2 is a flavin-bound amine dehydrogenase from Pseudomonas putida S16 that converts nicotine to the pharmacologically inactive N-methylmyosmine. In animal models of nicotine addiction, injection of NicA2 can decrease nicotine-seeking behavior 10-fold. Accordingly, NicA2-related enzymes have been investigated as smoking-cessation therapeutics. However, efficient catalysis by NicA2 in Pseudomonas putida relies on electron transfer to CycN, a cytochrome c, and not directly to O2. Impractically high amounts of NicA2 are thus necessary to achieve a pharmacological effect in the absence of CycN. Directed evolution has improved the ambient-O2 value of kcat from 0.007 s-1 to 1 s-1 for NicA2, but further improvements have been challenging. Here, we identify a strain of Peribacillus frigoritolerans NIC8 which encodes two flavin amine oxidoreductases, Ncox and Pnox. In the presence of oxygen, Ncox and Pnox act on nicotine and pseudooxynicotine respectively with apparent kcat values of 7.7 s-1 and 3.9 s-1. Transient kinetics establishes bimolecular rate constants of 51100 M-1s-1 and 81000 M-1s-1 for the half-reactions between Ncox and O2 and between Pnox and O2 respectively, consistent with Ncox and Pnox being bona-fide oxidases. Transcriptomics shows enhanced expression of Ncox and Pnox under nicotine-dependent growth as well as supporting the identification of downstream enzymes. Phylogenetic analysis suggests that Ncox and Pnox arose out of repurposing of homologous enzymes found in Bacillus species. The enzymes we describe may be useful for the development of nicotine addiction therapeutics and for bioconversion of nicotine in waste streams.