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Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. We showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization.

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Here we analyse biospecimens from a randomized, controlled trial of a microbiome-directed complementary food (MDCF-2) that produced superior rates of weight gain compared with a calorically more dense conventional ready-to-use supplementary food in 12-18-month-old Bangladeshi children with moderate acute malnutrition4. We reconstructed 1,000 bacterial genomes (metagenome-assembled genomes (MAGs)) from the faecal microbiomes of trial participants, identified 75 MAGs of which the abundances were positively associated with ponderal growth (change in weight-for-length Z score (WLZ)), characterized changes in MAG gene expression as a function of treatment type and WLZ response, and quantified carbohydrate structures in MDCF-2 and faeces. The results reveal that two Prevotella copri MAGs that are positively associated with WLZ are the principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilizing the component glycans of MDCF-2. The predicted specificities of carbohydrate-active enzymes expressed by their polysaccharide-utilization loci are correlated with (1) the in vitro growth of Bangladeshi P. copri strains, possessing varying degrees of polysaccharide-utilization loci and genomic conservation with these MAGs, in defined medium containing different purified glycans representative of those in MDCF-2, and (2) the levels of faecal carbohydrate structures in the trial participants. These associations suggest that identifying bioactive glycan structures in MDCFs metabolized by growth-associated bacterial taxa will help to guide recommendations about their use in children with acute malnutrition and enable the development of additional formulations.

Microbial communities in the world ocean are affected strongly by oceanic circulation, creating characteristic marine biomes. The high connectivity of most of the ocean makes it difficult to disentangle selective retention of colonizing genotypes (with traits suited to biome specific conditions) from evolutionary selection, which would act on founder genotypes over time. The Arctic Ocean is exceptional with limited exchange with other oceans and ice covered since the last ice age. To test whether Arctic microalgal lineages evolved apart from algae in the global ocean, we sequenced four lineages of microalgae isolated from Arctic waters and sea ice. Here we show convergent evolution and highlight geographically limited HGT as an ecological adaptive force in the form of PFAM complements and horizontal acquisition of key adaptive genes. Notably, ice-binding proteins were acquired and horizontally transferred among Arctic strains. A comparison with Tara Oceans metagenomes and metatranscriptomes confirmed mostly Arctic distributions of these IBPs. The phylogeny of Arctic-specific genes indicated that these events were independent of bacterial-sourced HGTs in Antarctic Southern Ocean microalgae.

Increases in snack consumption associated with Westernized lifestyles provide an opportunity to introduce nutritious foods into poor diets. We describe two 10-wk-long open label, single group assignment human studies that measured the effects of two snack prototypes containing fiber preparations from two sustainable and scalable sources; the byproducts remaining after isolation of protein from the endosperm of peas and the vesicular pulp remaining after processing oranges for the manufacture of juices. The normal diets of study participants were supplemented with either a pea- or orange fiber-containing snack. We focused our analysis on quantifying the abundances of genes encoding carbohydrate-active enzymes (CAZymes) (glycoside hydrolases and polysaccharide lyases) in the fecal microbiome, mass spectrometric measurements of glycan structures (glycosidic linkages) in feces, plus aptamer-based assessment of levels of 1,300 plasma proteins reflecting a broad range of physiological functions. Computational methods for feature selection identified treatment-discriminatory changes in CAZyme genes that correlated with alterations in levels of fiber-associated glycosidic linkages; these changes in turn correlated with levels of plasma proteins representing diverse biological functions, including transforming growth factor type β/bone morphogenetic protein-mediated fibrosis, vascular endothelial growth factor-related angiogenesis, P38/MAPK-associated immune cell signaling, and obesity-associated hormonal regulators. The approach used represents a way to connect changes in consumer microbiomes produced by specific fiber types with host responses in the context of varying background diets.

Processed foods often include food additives such as xanthan gum, a complex polysaccharide with unique rheological properties, that has established widespread use as a stabilizer and thickening agent. Xanthan gums chemical structure is distinct from those of host and dietary polysaccharides that are more commonly expected to transit the gastrointestinal tract, and little is known about its direct interaction with the gut microbiota, which plays a central role in digestion of other dietary fibre polysaccharides. Here we show that the ability to digest xanthan gum is common in human gut microbiomes from industrialized countries and appears contingent on a single uncultured bacterium in the family Ruminococcaceae. Our data reveal that this primary degrader cleaves the xanthan gum backbone before processing the released oligosaccharides using additional enzymes. Some individuals harbour Bacteroides intestinalis that is incapable of consuming polymeric xanthan gum but grows on oligosaccharide products generated by the Ruminococcaceae. Feeding xanthan gum to germfree mice colonized with a human microbiota containing the uncultured Ruminococcaceae supports the idea that the additive xanthan gum can drive expansion of the primary degrader Ruminococcaceae, along with exogenously introduced B. intestinalis. Our work demonstrates the existence of a potential xanthan gum food chain involving at least two members of different phyla of gut bacteria and provides an initial framework for understanding how widespread consumption of a recently introduced food additive influences human microbiomes.

Changing food preferences brought about by westernization that have deleterious health effects1,2-combined with myriad forces that are contributing to increased food insecurity-are catalysing efforts to identify more nutritious and affordable foods3. Consumption of dietary fibre can help to prevent cardiovascular disease, type 2 diabetes and obesity4-6. A substantial number of reports have explored the effects of dietary fibre on the gut microbial community7-9. However, the microbiome is complex, dynamic and exhibits considerable intra- and interpersonal variation in its composition and functions. The large number of potential interactions between the components of the microbiome makes it challenging to define the mechanisms by which food ingredients affect community properties. Here we address the question of how foods containing different fibre preparations can be designed to alter functions associated with specific components of the microbiome. Because a marked increase in snack consumption is associated with westernization, we formulated snack prototypes using plant fibres from different sustainable sources that targeted distinct features of the gut microbiomes of individuals with obesity when transplanted into gnotobiotic mice. We used these snacks to supplement controlled diets that were consumed by adult individuals with obesity or who were overweight. Fibre-specific changes in their microbiomes were linked to changes in their plasma proteomes indicative of an altered physiological state.

Greater understanding of the spatial relationships between members of the human gut microbiota and available nutrients is needed to gain deeper insights about community dynamics and expressed functions. Therefore, we generated a panel of artificial food particles with each type composed of microscopic paramagnetic beads coated with a fluorescent barcode and one of 60 different dietary or host glycan preparations. Analysis of 160 Bacteroides and Parabacteroides strains disclosed diverse strain-specific and glycan-specific binding phenotypes. We identified carbohydrate structures that correlated with binding by specific bacterial strains in vitro and noted strain-specific differences in the catabolism of glycans that mediate adhesion. Mixed in vitro cultures revealed that these adhesion phenotypes are maintained in more complex communities. Additionally, orally administering glycan beads to gnotobiotic mice confirmed specificity in glycan binding. This approach should facilitate analyses of how strains occupying the same physical niche interact, and it should advance the development of synbiotics, more nutritious foods, and microbiota-based diagnostics.

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 h in nylon bags within the rumen of cannulated dairy cows. Across a gene catalog covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of hemicelluloses, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8, and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggest that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B12, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents ∼75% of the genus-level bacterial and archaeal taxa present in the rumen.

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

To study how microbes establish themselves in a mammalian gut environment, we colonized germ-free mice with microbial communities from human, zebrafish, and termite guts, human skin and tongue, soil, and estuarine microbial mats. Bacteria from these foreign environments colonized and persisted in the mouse gut; their capacity to metabolize dietary and host carbohydrates and bile acids correlated with colonization success. Cohousing mice harboring these xenomicrobiota or a mouse cecal microbiota, along with germ-free "bystanders," revealed the success of particular bacterial taxa in invading guts with established communities and empty gut habitats. Unanticipated patterns of ecological succession were observed; for example, a soil-derived bacterium dominated even in the presence of bacteria from other gut communities (zebrafish and termite), and human-derived bacteria colonized germ-free bystander mice before mouse-derived organisms. This approach can be generalized to address a variety of mechanistic questions about succession, including succession in the context of microbiota-directed therapeutics.

Basidiomycota (basidiomycetes) make up 32percent of the described fungi and include most wood decaying species, as well as pathogens and mutualistic symbionts. Wood-decaying basidiomycetes have typically been classified as either white rot or brown rot, based on the ability (in white rot only) to degrade lignin along with cellulose and hemicellulose. Prior genomic comparisons suggested that the two decay modes can be distinguished based on the presence or absence of ligninolytic class II peroxidases (PODs), as well as the abundance of enzymes acting directly on crystalline cellulose (reduced in brown rot). To assess the generality of the white rot/brown rot classification paradigm we compared the genomes of 33 basidiomycetes, including four newly sequenced wood decayers, and performed phylogenetically-informed Principal Components Analysis (PCA) of a broad range of gene families encoding plant biomass-degrading enzymes. The newly sequenced Botryobasidium botryosum and Jaapia argillacea genomes lack PODs, but possess diverse enzymes acting on crystalline cellulose, and they group close to the model white rot species Phanerochaete chrysosporium in the PCA. Furthermore, laboratory assays showed that both B. botryosum and J. argillacea can degrade all polymeric components of woody plant cell walls, a characteristic of white rot. We also found expansions in reducing polyketide synthase genes specific to the brown rot fungi. Our results suggest a continuum rather than a dichotomy between the white rot and brown rot modes of wooddecay. A more nuanced categorization of rot types is needed, based on an improved understanding of the genomics and biochemistry of wood decay.

The role of specific gut microbes in shaping body composition remains unclear. We transplanted fecal microbiota from adult female twin pairs discordant for obesity into germ-free mice fed low-fat mouse chow, as well as diets representing different levels of saturated fat and fruit and vegetable consumption typical of the U.S. diet. Increased total body and fat mass, as well as obesity-associated metabolic phenotypes, were transmissible with uncultured fecal communities and with their corresponding fecal bacterial culture collections. Cohousing mice harboring an obese twin's microbiota (Ob) with mice containing the lean co-twin's microbiota (Ln) prevented the development of increased body mass and obesity-associated metabolic phenotypes in Ob cage mates. Rescue correlated with invasion of specific members of Bacteroidetes from the Ln microbiota into Ob microbiota and was diet-dependent. These findings reveal transmissible, rapid, and modifiable effects of diet-by-microbiota interactions.

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the button mushroom forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non?lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period.

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.

The wood degrading fungus Schizophyllum commune is a model system for mushroom development. Here, we describe the 38.5 Mb assembled genome of this basidiomycete and application of whole genome expression analysis to study the 13,210 predicted genes. Comparative analyses of the S. commune genome revealed unique wood degrading machinery and mating type loci with the highest number of reported genes. Gene expression analyses revealed that one third of the 471 identified transcription factor genes were differentially expressed during sexual development. Two of these transcription factor genes were deleted. Inactivation of fst4 resulted in the inability to form mushrooms, whereas inactivation of fst3 resulted in more but smaller mushrooms than wild-type. These data illustrate that mechanisms underlying mushroom formation can be dissected using S. commune as a model. This will impact commercial production of mushrooms and the industrial use of these fruiting bodies to produce enzymes and pharmaceuticals