The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones.

Microbial competition within plant tissues affects invading pathogens' fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce's disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography-mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

Cynipid wasps systematically reprogram host metabolism and restructure cell walls in developing galls.

Many insects have evolved the ability to manipulate plant growth to generate extraordinary structures called galls, in which insect larva can develop while being sheltered and feeding on the plant. In particular, cynipid (Hymenoptera: Cynipidae) wasps have evolved to form morphologically complex galls and generate an astonishing array of gall shapes, colors, and sizes. However, the biochemical basis underlying these remarkable cellular and developmental transformations remains poorly understood. A key determinant in plant cellular development is cell wall deposition that dictates the physical form and physiological function of newly developing cells, tissues, and organs. However, it is unclear to what degree cell walls are restructured to initiate and support the formation of new gall tissue. Here, we characterize the molecular alterations underlying gall development using a combination of metabolomic, histological, and biochemical techniques to elucidate how valley oak (Quercus lobata) leaf cells are reprogrammed to form galls. Strikingly, gall development involves an exceptionally coordinated spatial deposition of lignin and xylan to form de novo gall vasculature. Our results highlight how cynipid wasps can radically change the metabolite profile and restructure the cell wall to enable the formation of galls, providing insights into the mechanism of gall induction and the extent to which plants can be entirely reprogrammed to form unique structures and organs.

Reproducible growth of Brachypodium in EcoFAB 2.0 reveals that nitrogen form and starvation modulate root exudation.

Understanding plant-microbe interactions requires examination of root exudation under nutrient stress using standardized and reproducible experimental systems. We grew Brachypodium distachyon hydroponically in fabricated ecosystem devices (EcoFAB 2.0) under three inorganic nitrogen forms (nitrate, ammonium, and ammonium nitrate), followed by nitrogen starvation. Analyses of exudates with liquid chromatography-tandem mass spectrometry, biomass, medium pH, and nitrogen uptake showed EcoFAB 2.0's low intratreatment data variability. Furthermore, the three inorganic nitrogen forms caused differential exudation, generalized by abundant amino acids-peptides and alkaloids. Comparatively, nitrogen deficiency decreased nitrogen-containing compounds but increased shikimates-phenylpropanoids. Subsequent bioassays with two shikimates-phenylpropanoids (shikimic and p-coumaric acids) on soil bacteria or Brachypodium seedlings revealed their distinct capacity to regulate both bacterial and plant growth. Our results suggest that (i) Brachypodium alters exudation in response to nitrogen status, which can affect rhizobacterial growth, and (ii) EcoFAB 2.0 is a valuable standardized plant research tool.

Conservation of beneficial microbes between the rhizosphere and the cyanosphere.

Biocrusts are phototroph-driven communities inhabiting arid soil surfaces. Like plants, most photoautotrophs (largely cyanobacteria) in biocrusts are thought to exchange fixed carbon for essential nutrients like nitrogen with cyanosphere bacteria. Here, we aim to compare beneficial interactions in rhizosphere and cyanosphere environments, including finding growth-promoting strains for hosts from both environments. To examine this, we performed a retrospective analysis of 16S rRNA gene sequencing datasets, host-microbe co-culture experiments between biocrust communities/biocrust isolates and a model grass (Brachypodium distachyon) or a dominant biocrust cyanobacterium (Microcoleus vaginatus), and metabolomic analysis. All 18 microbial phyla in the cyanosphere were also present in the rhizosphere, with additional 17 phyla uniquely found in the rhizosphere. The biocrust microbes promoted the growth of the model grass, and three biocrust isolates (Bosea sp._L1B56, Pseudarthrobacter sp._L1D14 and Pseudarthrobacter picheli_L1D33) significantly promoted the growth of both hosts. Moreover, pantothenic acid was produced by Pseudarthrobacter sp._L1D14 when grown on B. distachyon exudates, and supplementation of plant growth medium with this metabolite increased B. distachyon biomass by over 60%. These findings suggest that cyanobacteria and other diverse photoautotrophic hosts can be a source for new plant growth-promoting microbes and metabolites.

BLINK enables ultrafast tandem mass spectrometry cosine similarity scoring.

Metabolomics has a long history of using cosine similarity to match experimental tandem mass spectra to databases for compound identification. Here we introduce the Blur-and-Link (BLINK) approach for scoring cosine similarity. By bypassing fragment alignment and simultaneously scoring all pairs of spectra using sparse matrix operations, BLINK is over 3000 times faster than MatchMS, a widely used loop-based alignment and scoring implementation. Using a similarity cutoff of 0.7, BLINK and MatchMS had practically equivalent identification agreement, and greater than 99% of their scores and matching ion counts were identical. This performance improvement can enable calculations to be performed that would typically be limited by time and available computational resources.

A combinatorial droplet microfluidic device integrated with mass spectrometry for enzyme screening.

Mass spectrometry (MS) enables detection of different chemical species with a very high specificity; however, it can be limited by its throughput. Integrating MS with microfluidics has a tremendous potential to improve throughput and accelerate biochemical research. In this work, we introduce Drop-NIMS, a combination of a passive droplet loading microfluidic device and a matrix-free MS laser desorption ionization technique called nanostructure-initiator mass spectrometry (NIMS). This platform combines different droplets at random to generate a combinatorial library of enzymatic reactions that are deposited directly on the NIMS surface without requiring additional sample handling. The enzyme reaction products are then detected with MS. Drop-NIMS was used to rapidly screen enzymatic reactions containing low (on the order of nL) volumes of glycoside reactants and glycoside hydrolase enzymes per reaction. MS "barcodes" (small compounds with unique masses) were added to the droplets to identify different combinations of substrates and enzymes created by the device. We assigned xylanase activities to several putative glycoside hydrolases, making them relevant to food and biofuel industrial applications. Overall, Drop-NIMS is simple to fabricate, assemble, and operate and it has potential to be used with many other small molecule metabolites.

Decomposition decreases molecular diversity and ecosystem similarity of soil organic matter.

Soil organic matter (SOM) is comprised of a diverse array of reactive carbon molecules, including hydrophilic and hydrophobic compounds, that impact rates of SOM formation and persistence. Despite clear importance to ecosystem science, little is known about broad-scale controls on SOM diversity and variability in soil. Here, we show that microbial decomposition drives significant variability in the molecular richness and diversity of SOM between soil horizons and across a continental-scale gradient in climate and ecosystem type (arid shrubs, coniferous, deciduous, and mixed forests, grasslands, and tundra sedges). The molecular dissimilarity of SOM was strongly influenced by ecosystem type (hydrophilic compounds: 17%, P < 0.001; hydrophobic compounds: 10% P < 0.001) and soil horizon (hydrophilic compounds: 17%, P < 0.001; hydrophobic compounds: 21%, P < 0.001), as assessed using metabolomic analysis of hydrophilic and hydrophobic metabolites. While the proportion of shared molecular features was significantly higher in the litter layer than subsoil C horizons across ecosystems (12 times and 4 times higher for hydrophilic and hydrophobic compounds, respectively), the proportion of site-specific molecular features nearly doubled from the litter layer to the subsoil horizon, suggesting greater differentiation of compounds after microbial decomposition within each ecosystem. Together, these results suggest that microbial decomposition of plant litter leads to a decrease in SOM α-molecular diversity, yet an increase in ß-molecular diversity across ecosystems. The degree of microbial degradation, determined by the position in the soil profile, exerts a greater control on SOM molecular diversity than environmental factors, such as soil texture, moisture, and ecosystem type.

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past.

Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

Information theory and machine learning illuminate large-scale metabolomic responses of Brachypodium distachyon to environmental change.

Plant responses to environmental change are mediated via changes in cellular metabolomes. However, <5% of signals obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) can be identified, limiting our understanding of how metabolomes change under biotic/abiotic stress. To address this challenge, we performed untargeted LC-MS/MS of leaves, roots, and other organs of Brachypodium distachyon (Poaceae) under 17 organ-condition combinations, including copper deficiency, heat stress, low phosphate, and arbuscular mycorrhizal symbiosis. We found that both leaf and root metabolomes were significantly affected by the growth medium. Leaf metabolomes were more diverse than root metabolomes, but the latter were more specialized and more responsive to environmental change. We found that 1 week of copper deficiency shielded the root, but not the leaf metabolome, from perturbation due to heat stress. Machine learning (ML)-based analysis annotated approximately 81% of the fragmented peaks versus approximately 6% using spectral matches alone. We performed one of the most extensive validations of ML-based peak annotations in plants using thousands of authentic standards, and analyzed approximately 37% of the annotated peaks based on these assessments. Analyzing responsiveness of each predicted metabolite class to environmental change revealed significant perturbations of glycerophospholipids, sphingolipids, and flavonoids. Co-accumulation analysis further identified condition-specific biomarkers. To make these results accessible, we developed a visualization platform on the Bio-Analytic Resource for Plant Biology website (https://bar.utoronto.ca/efp_brachypodium_metabolites/cgi-bin/efpWeb.cgi), where perturbed metabolite classes can be readily visualized. Overall, our study illustrates how emerging chemoinformatic methods can be applied to reveal novel insights into the dynamic plant metabolome and stress adaptation.

Mass spectrometry imaging-based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme.

Aminotransferases (ATs) catalyze pyridoxal 5'-phosphate-dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging-based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI-mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime-mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.

Tracing Carbon Metabolism with Stable Isotope Metabolomics Reveals the Legacy of Diverse Carbon Sources in Soil.

Tracking the metabolic activity of whole soil communities can improve our understanding of the transformation and fate of carbon in soils. We used stable isotope metabolomics to trace 13C from nine labeled carbon sources into the water-soluble metabolite pool of an agricultural soil over time. Soil was amended with a mixture of all nine sources, with one source isotopically labeled in each treatment. We compared changes in the 13C enrichment of metabolites with respect to carbon source and time over a 48-day incubation and contrasted differences between soluble sources (glucose, xylose, amino acids, etc.) and insoluble sources (cellulose and palmitic acid). Whole soil metabolite profiles varied singularly by time, while the composition of 13C-labeled metabolites differed primarily by carbon source (R2 = 0.68) rather than time (R2 = 0.07), with source-specific differences persisting throughout incubations. The 13C labeling of metabolites from insoluble carbon sources occurred slower than that from soluble sources but yielded a higher average atom percent (atom%) 13C in metabolite markers of biomass (amino acids and nucleic acids). The 13C enrichment of metabolite markers of biomass stabilized between 5 and 15 atom% 13C by the end of incubations. Temporal patterns in the 13C enrichment of tricarboxylic acid cycle intermediates, nucleobases (uracil and thymine), and by-products of DNA salvage (allantoin) closely tracked microbial activity. Our results demonstrate that metabolite production in soils is driven by the carbon source supplied to the community and that the fate of carbon in metabolites do not generally converge over time as a result of ongoing microbial processing and recycling. IMPORTANCE Carbon metabolism in soil remains poorly described due to the inherent difficulty of obtaining information on the microbial metabolites produced by complex soil communities. Our study demonstrates the use of stable isotope probing (SIP) to study carbon metabolism in soil by tracking 13C from supplied carbon sources into metabolite pools and biomass. We show that differences in the metabolism of sources influence the fate of carbon in soils. Heterogeneity in 13C-labeled metabolite profiles corresponded with compositional differences in the metabolically active populations, providing a basis for how microbial community composition correlates with the quality of soil carbon. Our study demonstrates the application of SIP-metabolomics in studying soils and identifies several metabolite markers of growth, activity, and other aspects of microbial function.

Substrate Utilization and Competitive Interactions Among Soil Bacteria Vary With Life-History Strategies.

Microorganisms have evolved various life-history strategies to survive fluctuating resource conditions in soils. However, it remains elusive how the life-history strategies of microorganisms influence their processing of organic carbon, which may affect microbial interactions and carbon cycling in soils. Here, we characterized the genomic traits, exometabolite profiles, and interactions of soil bacteria representing copiotrophic and oligotrophic strategists. Isolates were selected based on differences in ribosomal RNA operon (rrn) copy number, as a proxy for life-history strategies, with pairs of "high" and "low" rrn copy number isolates represented within the Micrococcales, Corynebacteriales, and Bacillales. We found that high rrn isolates consumed a greater diversity and amount of substrates than low rrn isolates in a defined growth medium containing common soil metabolites. We estimated overlap in substrate utilization profiles to predict the potential for resource competition and found that high rrn isolates tended to have a greater potential for competitive interactions. The predicted interactions positively correlated with the measured interactions that were dominated by negative interactions as determined through sequential growth experiments. This suggests that resource competition was a major force governing interactions among isolates, while cross-feeding of metabolic secretion likely contributed to the relatively rare positive interactions observed. By connecting bacterial life-history strategies, genomic features, and metabolism, our study advances the understanding of the links between bacterial community composition and the transformation of carbon in soils.

A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria.

Exometabolomics is an approach to assess how microorganisms alter, or react to their environments through the depletion and production of metabolites. It allows the examination of how soil microbes transform the small molecule metabolites within their environment, which can be used to study resource competition and cross-feeding. This approach is most powerful when used with defined media that enable tracking of all metabolites. However, microbial growth media have traditionally been developed for the isolation and growth of microorganisms but not metabolite utilization profiling through Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Here, we describe the construction of a defined medium, the Northen Lab Defined Medium (NLDM), that not only supports the growth of diverse soil bacteria but also is defined and therefore suited for exometabolomic experiments. Metabolites included in NLDM were selected based on their presence in R2A medium and soil, elemental stoichiometry requirements, as well as knowledge of metabolite usage by different bacteria. We found that NLDM supported the growth of 108 of the 110 phylogenetically diverse (spanning 36 different families) soil bacterial isolates tested and all of its metabolites were trackable through LC-MS/MS analysis. These results demonstrate the viability and utility of the constructed NLDM medium for growing and characterizing diverse microbial isolates and communities.

SIMILE enables alignment of tandem mass spectra with statistical significance.

Interrelating small molecules according to their aligned fragmentation spectra is central to tandem mass spectrometry-based untargeted metabolomics. Current alignment algorithms do not provide statistical significance and compounds that have multiple delocalized structural differences and therefore often fail to have their fragment ions aligned. Here we align fragmentation spectra with both statistical significance and allowance for multiple chemical differences using Significant Interrelation of MS/MS Ions via Laplacian Embedding (SIMILE). SIMILE yields spectral alignment inferred structural connections in molecular networks that are not found with cosine-based scoring algorithms. In addition, it is now possible to rank spectral alignments based on p-values in the exploration of structural relationships between compounds and enhance the chemical connectivity that can be obtained with molecular networking.

Protist impacts on marine cyanovirocell metabolism.

The fate of oceanic carbon and nutrients depends on interactions between viruses, prokaryotes, and unicellular eukaryotes (protists) in a highly interconnected planktonic food web. To date, few controlled mechanistic studies of these interactions exist, and where they do, they are largely pairwise, focusing either on viral infection (i.e., virocells) or protist predation. Here we studied population-level responses of Synechococcus cyanobacterial virocells (i.e., cyanovirocells) to the protist Oxyrrhis marina using transcriptomics, endo- and exo-metabolomics, photosynthetic efficiency measurements, and microscopy. Protist presence had no measurable impact on Synechococcus transcripts or endometabolites. The cyanovirocells alone had a smaller intracellular transcriptional and metabolic response than cyanovirocells co-cultured with protists, displaying known patterns of virus-mediated metabolic reprogramming while releasing diverse exometabolites during infection. When protists were added, several exometabolites disappeared, suggesting microbial consumption. In addition, the intracellular cyanovirocell impact was largest, with 4.5- and 10-fold more host transcripts and endometabolites, respectively, responding to protists, especially those involved in resource and energy production. Physiologically, photosynthetic efficiency also increased, and together with the transcriptomics and metabolomics findings suggest that cyanovirocell metabolic demand is highest when protists are present. These data illustrate cyanovirocell responses to protist presence that are not yet considered when linking microbial physiology to global-scale biogeochemical processes.

Novel metabolic interactions and environmental conditions mediate the boreal peatmoss-cyanobacteria mutualism.

Interactions between Sphagnum (peat moss) and cyanobacteria play critical roles in terrestrial carbon and nitrogen cycling processes. Knowledge of the metabolites exchanged, the physiological processes involved, and the environmental conditions allowing the formation of symbiosis is important for a better understanding of the mechanisms underlying these interactions. In this study, we used a cross-feeding approach with spatially resolved metabolite profiling and metatranscriptomics to characterize the symbiosis between Sphagnum and Nostoc cyanobacteria. A pH gradient study revealed that the Sphagnum-Nostoc symbiosis was driven by pH, with mutualism occurring only at low pH. Metabolic cross-feeding studies along with spatially resolved matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) identified trehalose as the main carbohydrate source released by Sphagnum, which were depleted by Nostoc along with sulfur-containing choline-O-sulfate, taurine and sulfoacetate. In exchange, Nostoc increased exudation of purines and amino acids. Metatranscriptome analysis indicated that Sphagnum host defense was downregulated when in direct contact with the Nostoc symbiont, but not as a result of chemical contact alone. The observations in this study elucidated environmental, metabolic, and physiological underpinnings of the widespread plant-cyanobacterial symbioses with important implications for predicting carbon and nitrogen cycling in peatland ecosystems as well as the basis of general host-microbe interactions.

Biofilm Interaction Mapping and Analysis (BIMA) of Interspecific Interactions in Pseudomonas Co-culture Biofilms.

Pseudomonas species are ubiquitous in nature and include numerous medically, agriculturally and technologically beneficial strains of which the interspecific interactions are of great interest for biotechnologies. Specifically, co-cultures containing Pseudomonas stutzeri have been used for bioremediation, biocontrol, aquaculture management and wastewater denitrification. Furthermore, the use of P. stutzeri biofilms, in combination with consortia-based approaches, may offer advantages for these processes. Understanding the interspecific interaction within biofilm co-cultures or consortia provides a means for improvement of current technologies. However, the investigation of biofilm-based consortia has been limited. We present an adaptable and scalable method for the analysis of macroscopic interactions (colony morphology, inhibition, and invasion) between colony-forming bacterial strains using an automated printing method followed by analysis of the genes and metabolites involved in the interactions. Using Biofilm Interaction Mapping and Analysis (BIMA), these interactions were investigated between P. stutzeri strain RCH2, a denitrifier isolated from chromium (VI) contaminated soil, and 13 other species of pseudomonas isolated from non-contaminated soil. One interaction partner, Pseudomonas fluorescens N1B4 was selected for mutant fitness profiling of a DNA-barcoded mutant library; with this approach four genes of importance were identified and the effects on interactions were evaluated with deletion mutants and mass spectrometry based metabolomics.

Long-read metagenomics of soil communities reveals phylum-specific secondary metabolite dynamics.

Microbial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 712 full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night primarily in cyanobacteria, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts.

Cocultivation of Anaerobic Fungi with Rumen Bacteria Establishes an Antagonistic Relationship.

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.