Simple Matching Using QIIME 2 and RDP Reveals Misidentified Sequences and an Underrepresentation of Fungi in Reference Datasets

Simple nucleotide matching identification methods are not as accurate as once thought at identifying environmental fungal sequences. This is largely because of incorrect naming and the underrepresentation of various fungal groups in reference datasets. Here, we explore these issues by examining an environmental metabarcoding dataset of partial large subunit rRNA sequences of Basidiomycota and basal fungi. We employed the simple matching method using the QIIME 2 classifier and the RDP Classifier in conjunction with the latest releases of the SILVA (138.1, 2020) and RDP (11, 2014) reference datasets and then compared the results with a manual phylogenetic binning approach. Of the 71 query sequences tested, 21 and 42% were misidentified using QIIME 2 and the RDP Classifier, respectively. Of these simple matching misidentifications, more than half resulted from the underrepresentation of various groups of fungi in the SILVA and RDP reference datasets. More comprehensive reference datasets with fewer misidentified sequences will increase the accuracy of simple matching identifications. However, we argue that the phylogenetic binning approach is a better alternative to simple matching since, in addition to better accuracy, it provides evolutionary information about query sequences.

Early-diverging fungal phyla: taxonomy, species concept, ecology, distribution, anthropogenic impact, and novel phylogenetic proposals

The increasing number of new fungal species described from all over the world along with the use of genetics to define taxa, has dramatically changed the classification system of early-diverging fungi over the past several decades. The number of phyla established for non-Dikarya fungi has increased from 2 to 17. However, to date, both the classification and phylogeny of the basal fungi are still unresolved. In this article, we review the recent taxonomy of the basal fungi and re-evaluate the relationships among early-diverging lineages of fungal phyla. We also provide information on the ecology and distribution in Mucoromycota and highlight the impact of chytrids on amphibian populations. Species concepts in Chytridiomycota, Aphelidiomycota, Rozellomycota, Neocallimastigomycota are discussed in this paper. To preserve the current application of the genus Nephridiophaga (Chytridiomycota: Nephridiophagales), a new type species, Nephridiophaga blattellae, is proposed.

Bacterial-like nonribosomal peptide synthetases produce cyclopeptides in the zygomycetous fungus Mortierella alpina

Fungi are traditionally considered as reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early diverging fungi such as Mortierella spec. Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC32222. The molecular structures of malpicyclins were elucidated by HR-MS/MS, Marfey's method, and 1D and 2D NMR spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative qRT-PCR expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPS), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered l-amino acids and the subsequent incorporation of d-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicate a bacterial origin and a horizontal gene transfer into the fungal genome. We report on the as yet unexplored nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as novel and underrated resource of natural products. IMPORTANCE Fungal natural compounds are industrially produced with application in antibiotic treatment, cancer medications and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but re-identification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, denoted malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much closer related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella. Both enzymes were biochemically characterized and are involved in as yet unknown biosynthetic pathways of natural products in basal fungi. Hence, this report establishes early diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

Bacterial-like nonribosomal peptide synthetases produce cyclopeptides in the zygomycetous fungus Mortierella alpina

Fungi are traditionally considered as reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early diverging fungi such as Mortierella spec. Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC32222. The molecular structures of malpicyclins were elucidated by HR-MS/MS, Marfey’s method, and 1D and 2D NMR spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative qRT-PCR expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPS), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered l-amino acids and the subsequent incorporation of d-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicate a bacterial origin and a horizontal gene transfer into the fungal genome. This is the first report of nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as novel and underrated resource of natural products.IMPORTANCEFungal natural compounds are industrially produced with application in antibiotic treatment, cancer medications and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but re-identification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, denoted malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much closer related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella. Both enzymes are the first biochemically characterized natural product biosynthesis enzymes of basal fungi. Hence, this report establishes early diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription

Rediscovered as a potential eukaryotic epigenetic mark, DNA N6-adenine methylation (6mA) varies across species in abundance and its relationships with transcription. Here we characterize AMT1—representing a distinct MT-A70 family methyltransferase—in the ciliate Tetrahymena thermophila. AMT1 loss-of-function leads to severe defects in growth and development. Single Molecule, Real-Time (SMRT) sequencing reveals that AMT1 is required for the bulk of 6mA and all symmetric methylation at the ApT dinucleotides. The detection of hemi-methylated ApT sites suggests a semi-conservative mechanism for maintaining symmetric methylation. AMT1 affects expression of many genes; in particular, RAB46, encoding a Rab family GTPase involved in contractile vacuole function, is likely a direct target. The distribution of 6mA resembles H3K4 methylation and H2A.Z, two conserved epigenetic marks associated with RNA polymerase II transcription. Furthermore, strong 6mA and nucleosome positioning in wild-type cells is attenuated in ΔAMT1 cells. Our results support that AMT1-catalyzed 6mA is an integral part of the transcription-associated epigenetic landscape. AMT1 homologues are generally found in protists and basal fungi featuring ApT hyper-methylation associated with transcription, which are missing in animals, plants, and true fungi. This dichotomy of 6mA functions and the underlying molecular mechanisms may have implications in eukaryotic diversification.

Mitovirus and Mitochondrial Coding Sequences from Basal Fungus Entomophthora muscae

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina, phylum Zoopagamycota) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina, phylum Mucoromycota, phylum Basidiomycota and phylum Ascomycota) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses.

Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi

Although cell cycle control is an ancient, conserved, and essential process, some core animal and fungal cell cycle regulators share no more sequence identity than non-homologous proteins. Here, we show that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor through horizontal gene transfer. Cell cycle evolution in the fungal ancestor then proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in many basal fungi. We hypothesize that a virally-derived SBF may have initially hijacked cell cycle control by activating transcription via the cis-regulatory elements targeted by the ancestral cell cycle regulator E2F, much like extant viral oncogenes. Consistent with this hypothesis, we show that SBF can regulate promoters with E2F binding sites in budding yeast.