The Dictyostelium discoideum genome lacks significant DNA methylation and uncovers palindromic sequences as a source of false positives in bisulfite sequencing

DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, is an evolutionarily conserved epigenetic mark involved in a number of different biological functions in eukaryotes, including transcriptional regulation, chromatin structural organization, cellular differentiation and development. In the social amoeba Dictyostelium, previous studies have shown the existence of a DNA methyltransferase (DNMA) belonging to the DNMT2 family, but the extent and function of 5-methylcytosine in the genome are unclear. Here, we present the whole genome DNA methylation profile of Dictyostelium discoideum using deep coverage replicate sequencing of bisulfite-converted gDNA extracted from post-starvation cells. We find an overall very low number of sites with any detectable level of DNA methylation, occurring at significant levels in only 303-3432 cytosines out of the ∼7.5 million total cytosines in the genome depending on the replicate. Furthermore, a knockout of the DNMA enzyme leads to no overall decrease in DNA methylation. Of the identified sites, significant methylation is only detected at 11 sites in all four of the methylomes analyzed. Targeted bisulfite PCR sequencing and computational analysis demonstrate that the methylation profile does not change during development and that these 11 cytosines are most likely false positives generated by protection from bisulfite conversion due to their location in hairpin-forming palindromic DNA sequences. Our data therefore provide evidence that there is no significant DNA methylation in Dictyostelium before fruiting body formation and identify a reproducible experimental artifact from bisulfite sequencing. © 2023 The Author(s). Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics

Examining Signatures of Natural Selection in Antifungal Resistance Genes Across Aspergillus Fungi

Certain Aspergillus fungi cause aspergillosis, a set of diseases that typically affect immunocompromised individuals. Most cases of aspergillosis are caused by Aspergillus fumigatus, which infects millions of people annually. Some closely related so-called cryptic species, such as Aspergillus lentulus, can also cause aspergillosis, albeit at lower frequencies, and they are also clinically relevant. Few antifungal drugs are currently available for treating aspergillosis and there is increasing worldwide concern about the presence of antifungal drug resistance in Aspergillus species. Furthermore, isolates from both A. fumigatus and other Aspergillus pathogens exhibit substantial heterogeneity in their antifungal drug resistance profiles. To gain insights into the evolution of antifungal drug resistance genes in Aspergillus, we investigated signatures of positive selection in 41 genes known to be involved in drug resistance across 42 susceptible and resistant isolates from 12 Aspergillus section Fumigati species. Using codon-based site models of sequence evolution, we identified ten genes that contain 43 sites with signatures of ancient positive selection across our set of species. None of the sites that have experienced positive selection overlap with sites previously reported to be involved in drug resistance. These results identify sites that likely experienced ancient positive selection in Aspergillus genes involved in resistance to antifungal drugs and suggest that historical selective pressures on these genes likely differ from any current selective pressures imposed by antifungal drugs.RS was supported by the Brazilian São Paulo Research Foundation (FAPESP) grant numbers 2017/21983-3 and 2019/07526-4. JS and AR are supported by the Howard Hughes Medical Institute through the James H. Gilliam Fellowships for Advanced Study Program. AR’s laboratory received additional support from a Discovery grant from Vanderbilt University, the Burroughs Wellcome Fund, the National Science Foundation (DEB-1442113), and the National Institutes of Health/National Institute of Allergy and Infectious Diseases (R56AI146096). GHG was supported by FAPESP (2016/07870-9) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq).S

Examination of Genome-Wide Ortholog variation in clinical and environmental isolates of the fungal pathogen Aspergillus fumigatus

Aspergillus fumigatus is a cosmopolitan species of fungus responsible for thousands of cases of invasive disease annually. Clinical and environmental isolates of A. fumigatus exhibit extensive phenotypic differences, including differences related to virulence and antifungal drug resistance. Aspergillus fumigatus is both an environmental saprobe and an opportunistic human fungal pathogen. Knowledge of genomic variation across A. fumigatus isolates is essential for understanding the evolution of pathogenicity, virulence, and resistance to antifungal drugs. Here, we investigated 206 A. fumigatus isolates (133 clinical and 73 environmental isolates), aiming to identify genes with variable presence across isolates and test whether this variation was related to the clinical or environmental origin of isolates. The PanOrtho genome of A. fumigatus consists of 13,085 ortholog groups, of which 7,773 (59.4\%) are shared by all isolates (core groups) and 5,312 (40.6\%) vary in their gene presence across isolates (accessory groups plus singletons). Despite differences in the distribution of orthologs across all isolates, no significant differences were observed among clinical versus environmental isolates when phylogeny was accounted for. Orthologs that differ in their distribution across isolates tend to occur at low frequency and/or be restricted to specific isolates; thus, the degree of genomic conservation between orthologs of A. fumigatus is high. These results suggest that differences in the distribution of orthologs within A. fumigatus cannot be associated with the clinical or environmental origin of isolates. IMPORTANCE Aspergillus fumigatus is a cosmopolitan species of fungus responsible for thousands of cases of invasive disease annually. Clinical and environmental isolates of A. fumigatus exhibit extensive phenotypic differences, including differences related to virulence and antifungal drug resistance. A comprehensive survey of the genomic diversity present in A. fumigatus and its relationship to the clinical or environmental origin of isolates can contribute to the prediction of the mechanisms of evolution and infection of the species. Our results suggest that there is no significant variation in ortholog distribution between clinical and environmental isolates when accounting for evolutionary history. The work supports the hypothesis that environmental and clinical isolates of A. fumigatus do not differ in their gene contents.We thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 2020/10536-9 (M.A.C.H.) and 2016/07870-9 (G.H.G.) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 301058/2019-9 and 404735/2018-5 (G.H.G.), both in Brazil, and National Institutes of Health/National Institute of Allergy and Infectious Diseases (R01AI153356) (A.R. and G.H.G.), in the United States.Peer ReviewedPostprint (published version

Gene Essentiality Analyzed by In Vivo Transposon Mutagenesis and Machine Learning in a Stable Haploid Isolate of Candida albicans

This work was supported by European Research Council Advanced Award 340087 (RAPLODAPT) to J.B., the Dahlem Centre of Plant Sciences (DCPS) of the Freie Universität Berlin (R.K.), Israel Science Foundation grant no. 715/18 (R.S.), the Wellcome Trust (grants 086827, 075470, 101873, and 200208) and the MRC Centre for Medical Mycology (N006364/1) (N.A.R.G.). Data availability.All of the code and required dependencies for analysis of the TnSeq data are available at https://github.com/berman-lab/transposon-pipeline. Library insertion sequences are available at NCBI under project PRJNA490565 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA490565). Datasets S1 through S9 are available at https://doi.org/10.6084/m9.figshare.c.4251182.Peer reviewedPublisher PD

Extensive loss of cell-cycle and DNA repair genes in an ancient lineage of bipolar budding yeasts

Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine–Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 (WHI5), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [MAD1], Mitotic Arrest-Deficient 2 [MAD2]) and DNA-damage–checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [MEC3], RADiation sensitive 9 [RAD9]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 (MAG1), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 (PHR1), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [POL4] and POL32) and telomere-associated genes (e.g., Repressor/ activator site binding protein-Interacting Factor 1 [RIF1], Replication Factor A 3 [RFA3], Cell Division Cycle 13 [CDC13], Pbp1p Binding Protein [PBP2]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them.Fil: Steenwyk, Jacob L.. Vanderbilt University; Estados UnidosFil: Opulente, Dana A.. University of Wisconsin; Estados UnidosFil: Kominek, Jacek. University of Wisconsin; Estados UnidosFil: Shen, Xing-Xing. Vanderbilt University; Estados UnidosFil: Zhou, Xiaofan. South China Agricultural University; ChinaFil: Labella, Abigail L.. Vanderbilt University; Estados UnidosFil: Bradley, Noah P.. Vanderbilt University; Estados UnidosFil: Eichman, Brandt F.. Vanderbilt University; Estados UnidosFil: Cadez, Neza. University of Ljubljana; EsloveniaFil: Libkind Frati, Diego. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; ArgentinaFil: DeVirgilio, Jeremy. United States Department of Agriculture. Agricultural Research Service; ArgentinaFil: Hulfachor, Amanda Beth. University of Wisconsin; Estados UnidosFil: Kurtzman, Cletus P.. United States Department of Agriculture. Agricultural Research Service; ArgentinaFil: Hittinger, Chris Todd. University of Wisconsin; Estados UnidosFil: Rokas, Antonis. Vanderbilt University; Estados Unido

Mapping the Fungal Battlefield: Using in situ Chemistry and Deletion Mutants to Monitor Interspecific Chemical Interactions Between Fungi

Fungi grow in competitive environments, and to cope, they have evolved strategies, such as the ability to produce a wide range of secondary metabolites. This begs two related questions. First, how do secondary metabolites influence fungal ecology and interspecific interactions? Second, can these interspecific interactions provide a way to “see” how fungi respond, chemically, within a competitive environment? To evaluate these, and to gain insight into the secondary metabolic arsenal fungi possess, we co-cultured Aspergillus fischeri, a genetically tractable fungus that produces a suite of mycotoxins, with Xylaria cubensis, a fungus that produces the fungistatic compound and FDA-approved drug, griseofulvin. To monitor and characterize fungal chemistry in situ, we used the droplet-liquid microjunction-surface sampling probe (droplet probe). The droplet probe makes a microextraction at defined locations on the surface of the co-culture, followed by analysis of the secondary metabolite profile via liquid chromatography-mass spectrometry. Using this, we mapped and compared the spatial profiles of secondary metabolites from both fungi in monoculture versus co-culture. X. cubensis predominantly biosynthesized griseofulvin and dechlorogriseofulvin in monoculture. In contrast, under co-culture conditions a deadlock was formed between the two fungi, and X. cubensis biosynthesized the same two secondary metabolites, along with dechloro-5′-hydroxygriseofulvin and 5′-hydroxygriseofulvin, all of which have fungistatic properties, as well as mycotoxins like cytochalasin D and cytochalasin C. In contrast, in co-culture, A. fischeri increased the production of the mycotoxins fumitremorgin B and verruculogen, but otherwise remained unchanged relative to its monoculture. To evaluate that secondary metabolites play an important role in defense and territory establishment, we co-cultured A. fischeri lacking the master regulator of secondary metabolism laeA with X. cubensis. We found that the reduced secondary metabolite biosynthesis of the ΔlaeA strain of A. fischeri eliminated the organism’s ability to compete in co-culture and led to its displacement by X. cubensis. These results demonstrate the potential of in situ chemical analysis and deletion mutant approaches for shedding light on the ecological roles of secondary metabolites and how they influence fungal ecological strategies; co-culturing may also stimulate the biosynthesis of secondary metabolites that are not produced in monoculture in the laboratory

Nutritional Heterogeneity Among Aspergillus fumigatus Strains Has Consequences for Virulence in a Strain- and Host-Dependent Manner

Acquisition and subsequent metabolism of different carbon and nitrogen sources have been shown to play an important role in virulence attributes of the fungal pathogen Aspergillus fumigatus, such as the secretion of host tissue-damaging proteases and fungal cell wall integrity. We examined the relationship between the metabolic processes of carbon catabolite repression (CCR), nitrogen catabolite repression (NCR) and virulence in a variety of A. fumigatus clinical isolates. A considerable amount of heterogeneity with respect to the degree of CCR and NCR was observed and a positive correlation between NCR and virulence in a neutropenic mouse model of pulmonary aspergillosis (PA) was found. Isolate Afs35 was selected for further analysis and compared to the reference strain A1163, with both strains presenting the same degree of virulence in a neutropenic mouse model of PA. Afs35 metabolome analysis in physiological-relevant carbon sources indicated an accumulation of intracellular sugars that also serve as cell wall polysaccharide precursors. Genome analysis showed an accumulation of missense substitutions in the regulator of protease secretion and in genes encoding enzymes required for cell wall sugar metabolism. Based on these results, the virulence of strains Afs35 and A1163 was assessed in a triamcinolone murine model of PA and found to be significantly different, confirming the known importance of using different mouse models to assess strain-specific pathogenicity. These results highlight the importance of nitrogen metabolism for virulence and provide a detailed example of the heterogeneity that exists between A. fumigatus isolates with consequences for virulence in a strain-specific and host-dependent manner

The dawn of relaxed phylogenetics.

Tracing the history of evolution across time is a primary goal of evolutionary biology. The 2006 publication of a landmark study on relaxed phylogenetics in PLOS Biology enabled biologists to shed light on evolution's tempo and shaped the future of evolutionary studies

Copy Number Variation in Fungi and Its Implications for Wine Yeast Genetic Diversity and Adaptation

In recent years, copy number (CN) variation has emerged as a new and significant source of genetic polymorphisms contributing to the phenotypic diversity of populations. CN variants are defined as genetic loci that, due to duplication and deletion, vary in their number of copies across individuals in a population. CN variants range in size from 50 base pairs to whole chromosomes, can influence gene activity, and are associated with a wide range of phenotypes in diverse organisms, including the budding yeast Saccharomyces cerevisiae. In this review, we introduce CN variation, discuss the genetic and molecular mechanisms implicated in its generation, how they can contribute to genetic and phenotypic diversity in fungal populations, and consider how CN variants may influence wine yeast adaptation in fermentation-related processes. In particular, we focus on reviewing recent work investigating the contribution of changes in CN of fermentation-related genes in yeast wine strains and offer notable illustrations of such changes, including the high levels of CN variation among the CUP genes, which confer resistance to copper, a metal with fungicidal properties, and the preferential deletion and duplication of the MAL1 and MAL3 loci, respectively, which are responsible for metabolizing maltose and sucrose. Based on the available data, we propose that CN variation is a substantial dimension of yeast genetic diversity that occurs largely independent of single nucleotide polymorphisms. As such, CN variation harbors considerable potential for understanding and manipulating yeast strains in the wine fermentation environment and beyond