Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose, and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune. Comparative genomics and transcriptomics on two wild isolates revealed a Zn2Cys6-type transcription factor gene (roc1) that was highly upregulated during growth on cellulose, compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase gene expression and enzyme activity were reduced in the Δroc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A conserved motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota. IMPORTANCE Wood-degrading fungi in the phylum Basidiomycota play a crucial role in nutrient recycling by breaking down all components of wood. Fungi have evolved transcriptional networks that regulate expression of wood-degrading enzymes, allowing them to prioritize one nutrient source over another. However, to date all these transcription factors have been identified in the phylum Ascomycota, which is only distantly related to the phylum Basidiomycota. Here, we identified the transcription factor Roc1 as a key regulator of cellulose degradation in the mushroom-forming and wood-degrading fungus Schizophyllum commune. Roc1 is highly conserved in the phylum Basidiomycota. Using comparative genomics, transcriptomics, ChIP-Seq and promoter analysis we have identified direct targets of Roc1, as well as other aspects of the transcriptional response to cellulose.

Anaerobic gut fungi (Neocallimastigomycetes) live in the digestive tract of large herbivores, where they are vastly outnumbered by bacteria. It has been suggested that anaerobic fungi challenge growth of bacteria owing to the wealth of biosynthetic genes in fungal genomes, although this relationship has not been experimentally tested. Here, we cocultivated the rumen bacteria Fibrobacter succinogenes strain UWB7 with the anaerobic gut fungi Anaeromyces robustus or Caecomyces churrovis on a range of carbon substrates and quantified the bacterial and fungal transcriptomic response. Synthetic cocultures were established for at least 24 h, as verified by active fungal and bacterial transcription. A. robustus upregulated components of its secondary metabolism in the presence of Fibrobacter succinogenes strain UWB7, including six nonribosomal peptide synthetases, one polyketide synthase-like enzyme, and five polyketide synthesis O-type methyltransferases. Both A. robustus and C. churrovis cocultures upregulated S-adenosyl-l-methionine (SAM)-dependent methyltransferases, histone methyltransferases, and an acetyltransferase. Fungal histone 3 lysine 27 trimethylation marks were more abundant in coculture, and heterochromatin protein-1 was downregulated. Together, these findings suggest that fungal chromatin remodeling occurs when bacteria are present. F. succinogenes strain UWB7 upregulated four genes in coculture encoding drug efflux pumps, which likely protect the cell against toxins. Furthermore, untargeted nonpolar metabolomics data revealed at least one novel fungal metabolite enriched in coculture, which may be a defense compound. Taken together, these data suggest that A. robustus and C. churrovis produce antimicrobials when exposed to rumen bacteria and, more broadly, that anaerobic gut fungi are a source of novel antibiotics. IMPORTANCE Anaerobic fungi are outnumbered by bacteria by 4 orders of magnitude in the herbivore rumen. Despite their numerical disadvantage, they are resilient members of the rumen microbiome. Previous studies mining the genomes of anaerobic fungi identified genes encoding enzymes to produce natural products, which are small molecules that are often antimicrobials. In this work, we cocultured the anaerobic fungus Anaeromyces robustus or Caecomyes churrovis with rumen bacteria Fibrobacter succinogenes strain UWB7 and sequenced fungal and bacterial active genes via transcriptome sequencing (RNA-seq). Consistent with production of a fungal defense compound, bacteria upregulated genes encoding drug efflux pumps, which often export toxic molecules, and fungi upregulated genes encoding biosynthetic enzymes of natural products. Furthermore, tandem mass spectrometry detected an unknown fungal metabolite enriched in the coculture. Together, these findings point to an antagonistic relationship between anaerobic fungi and rumen bacteria resulting in the production of a fungal compound with potential antimicrobial activity.

The genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. To demonstrate the utility of the individual mutants, we profiled gene expression in mutants of six regulatory genes and used these data, together with 1,313 high-confidence transcription start sites identified by tiling microarrays and transcriptome sequencing (5' RNA-Seq), to update the regulons of Fur and Rex and to confirm the predicted regulons of LysX, PhnF, PerR, and Dde_3000, a histidine kinase. In addition to enabling single mutant investigations, the D. alaskensis G20 transposon mutants also contain DNA bar codes, which enables the pooling and analysis of mutant fitness for thousands of strains simultaneously. Using two pools of mutants that represent insertions in 2,369 unique protein-coding genes, we demonstrate that the hypothetical gene Dde_3007 is required for methionine biosynthesis. Using comparative genomics, we propose that Dde_3007 performs a missing step in methionine biosynthesis by transferring a sulfur group to O-phosphohomoserine to form homocysteine. Additionally, we show that the entire choline utilization cluster is important for fitness in choline sulfate medium, which confirms that a functional microcompartment is required for choline oxidation. Finally, we demonstrate that Dde_3291, a MerR-like transcription factor, is a choline-dependent activator of the choline utilization cluster. Taken together, our data set and genetic resources provide a foundation for systems-level investigation of a poorly studied group of bacteria of environmental and industrial importance.

Microbial fermentation of agro-industrial waste holds great potential for reducing the environmental impact associated with the production of lipids for industrial purposes from plant biomass. However, the chemical complexity of many residues currently prevents efficient conversion into lipids, creating a high demand for strains with the ability to utilize all energy-rich components of agricultural residues. Here, we present results of genome and transcriptome analyses of Trichosporon oleaginosus. This oil-accumulating yeast is able to grow on a wide variety of substrates, including pentoses and N-acetylglucosamine, making it an interesting candidate for biotechnological applications. Transcriptomics shows specific changes in gene expression patterns under lipid-accumulating conditions. Furthermore, gene content and expression analyses indicate that T. oleaginosus is well-adapted for the utilization of chitin-rich biomass. We also focused on the T. oleaginosus mating type, because this species is a member of the Tremellomycetes, a group that has been intensively analyzed as a model for the evolution of sexual development, the best-studied member being Cryptococcus neoformans. The structure of the T. oleaginosus mating-type regions differs significantly from that of other Tremellomycetes and reveals a new evolutionary trajectory paradigm. Comparative analysis shows that recruitment of developmental genes to the ancestral tetrapolar mating-type loci occurred independently in the Trichosporon and Cryptococcus lineages, supporting the hypothesis of a trend toward larger mating-type regions in fungi.

In 1970, the Southern Corn Leaf Blight epidemic ravaged U.S. fields to great economic loss. The outbreak was caused by never-before-seen, supervirulent, Race T of the fungus Cochliobolus heterostrophus. The functional difference between Race T and O, the previously known, far less aggressive strain, is production of T-toxin, a host-selective polyketide. Supervirulence is associated with ~1 Mb of Race T-specific DNA; only a fraction encodes T-toxin biosynthetic genes (Tox1). Tox1 is genetically and physically complex, with unlinked loci (Tox1A, Tox1B) genetically inseparable from breakpoints of a Race O reciprocal translocation that generated hybrid Race T chromosomes. Previously, we identified 10 genes for T-toxin biosynthesis. Unfortunately, high-depth, short-read sequencing placed these genes on four small, unconnected scaffolds surrounded by repeated A+T rich sequence, concealing context. To sort out Tox1 topology and pinpoint the hypothetical Race O translocation breakpoints corresponding to Race T-specific insertions, we undertook PacBio long-read sequencing which revealed Tox1 gene arrangement and the breakpoints. Six Tox1A genes are arranged as three small islands in a Race T-specific sea (~634 kb) of repeats. Four Tox1B genes are linked, on a large loop of Race T-specific DNA (~210 kb). The race O breakpoints are short sequences of race O-specific DNA; corresponding positions in race T are large insertions of race T-specific, A+T rich DNA, often with similarity to transposable (predominantly Gypsy) elements. Nearby, are 'Voyager Starship' elements and DUF proteins. These elements may have facilitated Tox1 integration into progenitor Race O and promoted large scale recombination resulting in race T. IMPORTANCE In 1970 a corn disease epidemic ravaged fields in the United States to great economic loss. The outbreak was caused by a never-before seen, supervirulent strain of the fungal pathogen Cochliobolus heterostrophus. This was a plant disease epidemic, however, the current COVID-19 pandemic of humans is a stark reminder that novel, highly virulent, pathogens evolve with devastating consequences, no matter what the host-animal, plant, or other organism. Long read DNA sequencing technology allowed in depth structural comparisons between the sole, previously known, much less aggressive, version of the pathogen and the supervirulent version and revealed, in meticulous detail, the structure of the unique virulence-causing DNA. These data are foundational for future analysis of mechanisms of DNA acquisition from a foreign source.