Targeted discovery of tetrapeptides and cyclic polyketide-peptide hybrids from a fungal antagonist of farming termites

Herein, we report the targeted isolation and characterization of four linear nonribosomally synthesized tetrapeptides (pseudoxylaramide A–D) and two cyclic nonribosomal peptide synthetase- polyketide synthase-derived natural products (xylacremolide A and B) from the termite-associated stowaway fungus Pseudoxylaria sp. X187. The fungal strain was prioritized for further metabolic analysis based on its taxonomical position and morphological and bioassay data. Metabolic data were dereplicated based on high-resolution tandem mass spectrometry data and global molecular networking analysis. The structure of all six new natural products was elucidated based on a combination of 1D and 2D NMR analysis, Marfey’s analysis and X-ray crystallography.The Deutsche Forschungsgemeinschaft (DFG)https://chemistry-europe.onlinelibrary.wiley.com/journal/14397633am2020BiochemistryGeneticsMicrobiology and Plant Patholog

Adaptations of Pseudoxylaria towards a comb-associated lifestyle in fungus-farming termite colonies

Characterizing ancient clades of fungal symbionts is necessary for understanding the evolutionary process underlying symbiosis development. In this study, we investigated a distinct subgeneric taxon of Xylaria (Xylariaceae), named Pseudoxylaria, whose members have solely been isolated from the fungus garden of farming termites. Pseudoxylaria are inconspicuously present in active fungus gardens of termite colonies and only emerge in the form of vegetative stromata, when the fungus comb is no longer attended ("sit and wait" strategy). Insights into the genomic and metabolic consequences of their association, however, have remained sparse. Capitalizing on viable Pseudoxylaria cultures from different termite colonies, we obtained genomes of seven and transcriptomes of two Pseudoxylaria isolates. Using a whole-genome-based comparison with free-living members of the genus Xylaria, we document that the association has been accompanied by significant reductions in genome size, protein-coding gene content, and reduced functional capacities related to oxidative lignin degradation, oxidative stress responses and secondary metabolite production. Functional studies based on growth assays and fungus-fungus co-cultivations, coupled with isotope fractionation analysis, showed that Pseudoxylaria only moderately antagonizes growth of the termite food fungus Termitomyces, and instead extracts nutrients from the food fungus biomass for its own growth. We also uncovered that Pseudoxylaria is still capable of producing structurally unique metabolites, which was exemplified by the isolation of two novel metabolites, and that the natural product repertoire correlated with antimicrobial and insect antifeedant activity

Disease-free monoculture farming by fungus-growing termites

Fungus-growing termites engage in an obligate mutualistic relationship with Termitomyces fungi, which they maintain in monocultures on specialised fungus comb structures, without apparent problems with infectious diseases. While other fungi have been reported in the symbiosis, detailed comb fungal community analyses have been lacking. Here we use culture-dependent and -independent methods to characterise fungus comb mycobiotas from three fungus-growing termite species (two genera). Internal Transcribed Spacer (ITS) gene analyses using 454 pyrosequencing and Illumina MiSeq showed that non-Termitomyces fungi were essentially absent in fungus combs, and that Termitomyces fungal crops are maintained in monocultures as heterokaryons with two or three abundant ITS variants in a single fungal strain. To explore whether the essential absence of other fungi within fungus combs is potentially due to the production of antifungal metabolites by Termitomyces or comb bacteria, we performed in vitro assays and found that both Termitomyces and chemical extracts of fungus comb material can inhibit potential fungal antagonists. Chemical analyses of fungus comb material point to a highly complex metabolome, including compounds with the potential to play roles in mediating these contaminant-free farming conditions in the termite symbiosis

Adaptations of Pseudoxylaria towards a comb-associated lifestyle in fungus-farming termite colonies

DATA AVAILABILITY: Supporting Information of this article is free of charge and contains list of accession numbers of sequences used for analysis, phylogenetic trees, cultivation studies including co-cultivation, analyses of genomic and metabolomic data, NMR and MS-data of isolated metabolites and data of insect feeding studies including statistical analyses.Characterizing ancient clades of fungal symbionts is necessary for understanding the evolutionary process underlying symbiosis development. In this study, we investigated a distinct subgeneric taxon of Xylaria (Xylariaceae), named Pseudoxylaria, whose members have solely been isolated from the fungus garden of farming termites. Pseudoxylaria are inconspicuously present in active fungus gardens of termite colonies and only emerge in the form of vegetative stromata, when the fungus comb is no longer attended (“sit and wait” strategy). Insights into the genomic and metabolic consequences of their association, however, have remained sparse. Capitalizing on viable Pseudoxylaria cultures from different termite colonies, we obtained genomes of seven and transcriptomes of two Pseudoxylaria isolates. Using a whole-genome-based comparison with free-living members of the genus Xylaria, we document that the association has been accompanied by significant reductions in genome size, protein-coding gene content, and reduced functional capacities related to oxidative lignin degradation, oxidative stress responses and secondary metabolite production. Functional studies based on growth assays and fungus-fungus co-cultivations, coupled with isotope fractionation analysis, showed that Pseudoxylaria only moderately antagonizes growth of the termite food fungus Termitomyces, and instead extracts nutrients from the food fungus biomass for its own growth. We also uncovered that Pseudoxylaria is still capable of producing structurally unique metabolites, which was exemplified by the isolation of two novel metabolites, and that the natural product repertoire correlated with antimicrobial and insect antifeedant activity.The German Research Foundation (DFG, Deutsche Forschungsgemeinschaft), the Germany´s Excellence Strategy, the European Research Council and The Danish Council for Independent Research. Open Access funding enabled and organized by Projekt DEAL.https://www.nature.com/ismejBiochemistryGeneticsMicrobiology and Plant Patholog

Genome reduction and relaxed selection is associated with the transition to symbiosis in the basidiomycete genus Podaxis

Insights into the genomic consequences of symbiosis for basidiomycete fungi associated with social insects remain sparse. Capitalizing on viability of spores from centuries-old herbarium specimens of free-living, facultative, and specialist termite-associated Podaxis fungi, we obtained genomes of 10 specimens, including two type species described by Linnaeus >240 years ago. We document that the transition to termite association was accompanied by significant reductions in genome size and gene content, accelerated evolution in protein-coding genes, and reduced functional capacities for oxidative stress responses and lignin degradation. Functional testing confirmed that termite specialists perform worse under oxidative stress, while all lineages retained some capacity to cleave lignin. Mitochondrial genomes of termite associates were significantly larger; possibly driven by smaller population sizes or reduced competition, supported by apparent loss of certain biosynthetic gene clusters. Our findings point to relaxed selection that mirrors genome traits observed among obligate endosymbiotic bacteria of many insects.SUPPLEMENTARY MATERIAL: DOCUMENT S1. Figures S1–S10 and Tables S1, S2, S3, and S8TABLE S4. Orthogroups which were present in at least two genomes with the lifestyles in which they were found, related to Figure 2BTABLE S5. Predicted CAZymes, EC numbers and substrates from HotPep analysis, related to Figures 3A–3ETABLE S6. Functionally-enriched GO terms based on dcGO with an adjusted p-value cutoff of padj = 0.05, related to Figure 2BTABLE S7. Growth measurements for Podaxis strains grown on YMEA enriched with 0, 5 and 20 mM H2O2, related to Figure 3FThe Danish Council for Independent Research, the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) and the Slovenian Research Agency.https://www.cell.com/iscience/homehj2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Pseudoxylallemycins A-F, cyclic tetrapeptides with rare allenyl modifications isolated from <i>Pseudoxylaria</i> sp. X802:a competitor of fungus-growing termite cultivars

Based on fungus-fungus pairing assays and HRMS-based dereplication strategy, six new cyclic tetrapeptides, pseudoxylallemycins A-F (1-6), were isolated from the termite-associated fungus Pseudoxylaria sp. X802. Structures were characterized using NMR spectroscopy, HRMS, and Marfey's reaction. Pseudoxylallemycins B-D (2-4) possess a rare and chemically accessible allene moiety amenable for synthetic modifications, and derivatives A-D showed antimicrobial activity against Gram-negative human-pathogenic Pseudomonas aeruginosa and antiproliferative activity against human umbilical vein endothelial cells and K-562 cell lines.</p

LC-ESIMS/MS data for fractionated comb extracts

LC-ESIMS/MS data for fractionated comb extracts, acquired in negative ionisation mod

Data from: Disease-free monoculture farming by fungus-growing termites

Fungus-growing termites engage in an obligate mutualistic relationship with Termitomyces fungi, which they maintain in monocultures on specialised fungus comb structures, without apparent problems with infectious diseases. While other fungi have been reported in the symbiosis, detailed comb fungal community analyses have been lacking. Here we use culture-dependent and -independent methods to characterise fungus comb mycobiotas from three fungus-growing termite species (two genera). Internal Transcribed Spacer (ITS) gene analyses using 454 pyrosequencing and Illumina MiSeq showed that non-Termitomyces fungi were essentially absent in fungus combs, and that Termitomyces fungal crops are maintained in monocultures as heterokaryons with two or three abundant ITS variants in a single fungal strain. To explore whether the essential absence of other fungi within fungus combs is potentially due to the production of antifungal metabolites by Termitomyces or comb bacteria, we performed in vitro assays and found that both Termitomyces and chemical extracts of fungus comb material can inhibit potential fungal antagonists. Chemical analyses of fungus comb material point to a highly complex metabolome, including compounds with the potential to play roles in mediating these contaminant-free farming conditions in the termite symbiosis

The termite fungal cultivar Termitomyces combines diverse enzymes and oxidative reactions for plant biomass conversion

Please read abstract in the article.The Deutsche Forschungsgemeinschaft (DFG; German Research Foundation), the Danish Council for Independent Research, a European Research Council consolidator grant and the state budget of the Slovenian Research Agency.https://journals.asm.org/journal/mbiohj2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

The Termite Fungal Cultivar <i>Termitomyces</i> Combines Diverse Enzymes and Oxidative Reactions for Plant Biomass Conversion

Macrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using predigested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive omics and activity-based evidence that Termitomyces employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that Termitomyces induces hydroquinone-mediated Fenton chemistry (Fe(2+) + H(2)O(2) + H(+) → Fe(3+) + (•)OH + H(2)O) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MH(2)Q, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insect’s agricultural symbiosis is accomplished