Targeted hypermutation of putative antigen sensors in multicellular bacteria.

Diversity-generating retroelements (DGRs) are used by bacteria, archaea, and viruses as a targeted mutagenesis tool. Through error-prone reverse transcription, DGRs introduce random mutations at specific genomic loci, enabling rapid evolution of these targeted genes. However, the function and benefits of DGR-diversified proteins in cellular hosts remain elusive. We find that 82% of DGRs from one of the major monophyletic lineages of DGR reverse transcriptases are encoded by multicellular bacteria, which often have two or more DGR loci in their genomes. Using the multicellular purple sulfur bacterium Thiohalocapsa sp. PB-PSB1 as an example, we characterized nine distinct DGR loci capable of generating 10282 different combinations of target proteins. With environmental metagenomes from individual Thiohalocapsa aggregates, we show that most of PB-PSB1's DGR target genes are diversified across its biogeographic range, with spatial heterogeneity in the diversity of each locus. In Thiohalocapsa PB-PSB1 and other bacteria hosting this lineage of cellular DGRs, the diversified target genes are associated with NACHT-domain anti-phage defenses and putative ternary conflict systems previously shown to be enriched in multicellular bacteria. We propose that these DGR-diversified targets act as antigen sensors that confer a form of adaptive immunity to their multicellular consortia, though this remains to be experimentally tested. These findings could have implications for understanding the evolution of multicellularity, as the NACHT-domain anti-phage systems and ternary systems share both domain homology and conceptual similarities with the innate immune and programmed cell death pathways of plants and metazoans.

Pontiella agarivorans sp. nov., a novel marine anaerobic bacterium capable of degrading macroalgal polysaccharides and fixing nitrogen.

Marine macroalgae produce abundant and diverse polysaccharides, which contribute substantially to the organic matter exported to the deep ocean. Microbial degradation of these polysaccharides plays an important role in the turnover of macroalgal biomass. Various members of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum are degraders of polysaccharides in widespread anoxic environments. In this study, we isolated a novel anaerobic bacterial strain NLcol2T from microbial mats on the surface of marine sediments offshore Santa Barbara, CA, USA. Based on 16S ribosomal RNA (rRNA) gene and phylogenomic analyses, strain NLcol2T represents a novel species within the Pontiella genus in the Kiritimatiellota phylum (within the PVC superphylum). Strain NLcol2T is able to utilize various monosaccharides, disaccharides, and macroalgal polysaccharides such as agar and É©-carrageenan. A near-complete genome also revealed an extensive metabolic capacity for anaerobic degradation of sulfated polysaccharides, as evidenced by 202 carbohydrate-active enzymes (CAZymes) and 165 sulfatases. Additionally, its ability of nitrogen fixation was confirmed by nitrogenase activity detected during growth on nitrogen-free medium, and the presence of nitrogenases (nifDKH) encoded in the genome. Based on the physiological and genomic analyses, this strain represents a new species of bacteria that may play an important role in the degradation of macroalgal polysaccharides and with relevance to the biogeochemical cycling of carbon, sulfur, and nitrogen in marine environments. Strain NLcol2T (= DSM 113125T = MCCC 1K08672T) is proposed to be the type strain of a novel species in the Pontiella genus, and the name Pontiella agarivorans sp. nov. is proposed.IMPORTANCEGrowth and intentional burial of marine macroalgae is being considered as a carbon dioxide reduction strategy but elicits concerns as to the fate and impacts of this macroalgal carbon in the ocean. Diverse heterotrophic microbial communities in the ocean specialize in these complex polymers such as carrageenan and fucoidan, for example, members of the Kiritimatiellota phylum. However, only four type strains within the phylum have been cultivated and characterized to date, and there is limited knowledge about the metabolic capabilities and functional roles of related organisms in the environment. The new isolate strain NLcol2T expands the known substrate range of this phylum and further reveals the ability to fix nitrogen during anaerobic growth on macroalgal polysaccharides, thereby informing the issue of macroalgal carbon disposal.

Targeted rRNA depletion enables efficient mRNA sequencing in diverse bacterial species and complex co-cultures.

IMPORTANCE: Microbes present one of the most diverse sources of biochemistry in nature, and mRNA sequencing provides a comprehensive view of this biological activity by quantitatively measuring microbial transcriptomes. However, efficient mRNA capture for sequencing presents significant challenges in prokaryotes as mRNAs are not poly-adenylated and typically make up less than 5% of total RNA compared with rRNAs that exceed 80%. Recently developed methods for sequencing bacterial mRNA typically rely on depleting rRNA by tiling large probe sets against rRNAs; however, such approaches are expensive, time-consuming, and challenging to scale to varied bacterial species and complex microbial communities. Therefore, we developed EMBR-seq+, a method that requires fewer than 10 short oligonucleotides per rRNA to achieve up to 99% rRNA depletion in diverse bacterial species. Finally, EMBR-seq+ resulted in a deeper view of the transcriptome, enabling systematic quantification of how microbial interactions result in altering the transcriptional state of bacteria within co-cultures.

High-quality RNA extraction and the regulation of genes encoding cellulosomes are correlated with growth stage in anaerobic fungi.

Anaerobic fungi produce biomass-degrading enzymes and natural products that are important to harness for several biotechnology applications. Although progress has been made in the development of methods for extracting nucleic acids for genomic and transcriptomic sequencing of these fungi, most studies are limited in that they do not sample multiple fungal growth phases in batch culture. In this study, we establish a method to harvest RNA from fungal monocultures and fungal-methanogen co-cultures, and also determine an optimal time frame for high-quality RNA extraction from anaerobic fungi. Based on RNA quality and quantity targets, the optimal time frame in which to harvest anaerobic fungal monocultures and fungal-methanogen co-cultures for RNA extraction was 2-5 days of growth post-inoculation. When grown on cellulose, the fungal strain Anaeromyces robustus cocultivated with the methanogen Methanobacterium bryantii upregulated genes encoding fungal carbohydrate-active enzymes and other cellulosome components relative to fungal monocultures during this time frame, but expression patterns changed at 24-hour intervals throughout the fungal growth phase. These results demonstrate the importance of establishing methods to extract high-quality RNA from anaerobic fungi at multiple time points during batch cultivation.

Structure and enzymatic characterization of CelD endoglucanase from the anaerobic fungus Piromyces finnis.

Anaerobic fungi found in the guts of large herbivores are prolific biomass degraders whose genomes harbor a wealth of carbohydrate-active enzymes (CAZymes), of which only a handful are structurally or biochemically characterized. Here, we report the structure and kinetic rate parameters for a glycoside hydrolase (GH) family 5 subfamily 4 enzyme (CelD) from Piromyces finnis, a modular, cellulosome-incorporated endoglucanase that possesses three GH5 domains followed by two C-terminal fungal dockerin domains (double dockerin). We present the crystal structures of an apo wild-type CelD GH5 catalytic domain and its inactive E154A mutant in complex with cellotriose at 2.5 and 1.8 Å resolution, respectively, finding the CelD GH5 catalytic domain adopts the (ß/α)8-barrel fold common to many GH5 enzymes. Structural superimposition of the apo wild-type structure with the E154A mutant-cellotriose complex supports a catalytic mechanism in which the E154 carboxylate side chain acts as an acid/base and E278 acts as a complementary nucleophile. Further analysis of the cellotriose binding pocket highlights a binding groove lined with conserved aromatic amino acids that when docked with larger cellulose oligomers is capable of binding seven glucose units and accommodating branched glucan substrates. Activity analyses confirm P. finnis CelD can hydrolyze mixed linkage glucan and xyloglucan, as well as carboxymethylcellulose (CMC). Measured kinetic parameters show the P. finnis CelD GH5 catalytic domain has CMC endoglucanase activity comparable to other fungal endoglucanases with kcat = 6.0 ± 0.6 s-1 and Km = 7.6 ± 2.1 g/L CMC. Enzyme kinetics were unperturbed by the addition or removal of the native C-terminal dockerin domains as well as the addition of a non-native N-terminal dockerin, suggesting strict modularity among the domains of CelD. KEY POINTS: • Anaerobic fungi host a wealth of industrially useful enzymes but are understudied. • P. finnis CelD has endoglucanase activity and structure common to GH5_4 enzymes. • CelD's kinetics do not change with domain fusion, exhibiting high modularity.

Identification and expression of small multidrug resistance transporters in early-branching anaerobic fungi.

Membrane-embedded transporters impart essential functions to cells as they mediate sensing and the uptake and extrusion of nutrients, waste products, and effector molecules. Promiscuous multidrug exporters are implicated in resistance to drugs and antibiotics and are highly relevant for microbial engineers who seek to enhance the tolerance of cell factory strains to hydrophobic bioproducts. Here, we report on the identification of small multidrug resistance (SMR) transporters in early-branching anaerobic fungi (Neocallimastigomycetes). The SMR class of transporters is commonly found in bacteria but has not previously been reported in eukaryotes. In this study, we show that SMR transporters from anaerobic fungi can be produced heterologously in the model yeast Saccharomyces cerevisiae, demonstrating the potential of these proteins as targets for further characterization. The discovery of these novel anaerobic fungal SMR transporters offers a promising path forward to enhance bioproduction from engineered microbial strains.

Expression and characterization of spore coat CotH kinases from the cellulosomes of anaerobic fungi (Neocallimastigomycetes).

Anaerobic fungi (Neocallimastigomycetes) found in the guts of herbivores are biomass deconstruction specialists with a remarkable ability to extract sugars from recalcitrant plant material. Anaerobic fungi, as well as many species of anaerobic bacteria, deploy multi-enzyme complexes called cellulosomes, which modularly tether together hydrolytic enzymes, to accelerate biomass hydrolysis. While the majority of genomically encoded cellulosomal genes in Neocallimastigomycetes are biomass degrading enzymes, the second largest family of cellulosomal genes encode spore coat CotH domains, whose contribution to fungal cellulosome and/or cellular function is unknown. Structural bioinformatics of CotH proteins from the anaerobic fungus Piromyces finnis shows anaerobic fungal CotH domains conserve key ATP and Mg2+ binding motifs from bacterial Bacillus CotH proteins known to act as protein kinases. Experimental characterization further demonstrates ATP hydrolysis activity in the presence and absence of substrate from two cellulosomal P. finnis CotH proteins when recombinantly produced in E. coli. These results present foundational evidence for CotH activity in anaerobic fungi and provide a path towards elucidating the functional contribution of this protein family to fungal cellulosome assembly and activity.

Lignin deconstruction by anaerobic fungi.

Lignocellulose forms plant cell walls, and its three constituent polymers, cellulose, hemicellulose and lignin, represent the largest renewable organic carbon pool in the terrestrial biosphere. Insights into biological lignocellulose deconstruction inform understandings of global carbon sequestration dynamics and provide inspiration for biotechnologies seeking to address the current climate crisis by producing renewable chemicals from plant biomass. Organisms in diverse environments disassemble lignocellulose, and carbohydrate degradation processes are well defined, but biological lignin deconstruction is described only in aerobic systems. It is currently unclear whether anaerobic lignin deconstruction is impossible because of biochemical constraints or, alternatively, has not yet been measured. We applied whole cell-wall nuclear magnetic resonance, gel-permeation chromatography and transcriptome sequencing to interrogate the apparent paradox that anaerobic fungi (Neocallimastigomycetes), well-documented lignocellulose degradation specialists, are unable to modify lignin. We find that Neocallimastigomycetes anaerobically break chemical bonds in grass and hardwood lignins, and we further associate upregulated gene products with the observed lignocellulose deconstruction. These findings alter perceptions of lignin deconstruction by anaerobes and provide opportunities to advance decarbonization biotechnologies that depend on depolymerizing lignocellulose.

Lipid membrane mimetics and oligomerization tune functional properties of proteorhodopsin.

The functional properties of proteorhodopsin (PR) have been found to be strongly modulated by oligomeric distributions and lipid membrane mimetics. This study aims to distinguish and explain their effects by investigating how oligomer formation impacts PR's function of proton transport in lipid-based membrane mimetic environments. We find that PR forms stable hexamers and pentamers in both E. coli membranes and synthetic liposomes. Compared with the monomers, the photocycle kinetics of PR oligomers is ∼2 and ∼4.5 times slower for transitions between the K and M and the M and N photointermediates, respectively, indicating that oligomerization significantly slows PR's rate of proton transport in liposomes. In contrast, the apparent pKa of the key proton acceptor residue D97 (pKaD97) of liposome-embedded PR persists at 6.2-6.6, regardless of cross-protomer modulation of D97, suggesting that the liposome environment helps maintain PR's functional activity at neutral pH. By comparison, when extracted directly from E. coli membranes into styrene-maleic acid lipid particles, the pKaD97 of monomer-enriched E50Q PR drastically increases to 8.9, implying that there is a very low active PR population at neutral pH to engage in PR's photocycle. These findings demonstrate that oligomerization impacts PR's photocycle kinetics, while lipid-based membrane mimetics strongly affect PR's active population via different mechanisms.

Co­cultivation of anaerobic fungi with Clostridium acetobutylicum bolsters butyrate and butanol production from cellulose and lignocellulose.

A system for co-cultivation of anaerobic fungi with anaerobic bacteria was established based on lactate cross-feeding to produce butyrate and butanol from plant biomass. Several co-culture formulations were assembled that consisted of anaerobic fungi (Anaeromyces robustus, Neocallimastix californiae, or Caecomyces churrovis) with the bacterium Clostridium acetobutylicum. Co-cultures were grown simultaneously (e.g., 'one pot'), and compared to cultures where bacteria were cultured in fungal hydrolysate sequentially. Fungal hydrolysis of lignocellulose resulted in 7-11 mM amounts of glucose and xylose, as well as acetate, formate, ethanol, and lactate to support clostridial growth. Under these conditions, one-stage simultaneous co-culture of anaerobic fungi with C. acetobutylicum promoted the production of butyrate up to 30 mM. Alternatively, two-stage growth slightly promoted solventogenesis and elevated butanol levels (∼4-9 mM). Transcriptional regulation in the two-stage growth condition indicated that this cultivation method may decrease the time required to reach solventogenesis and induce the expression of cellulose-degrading genes in C. acetobutylicum due to relieved carbon-catabolite repression. Overall, this study demonstrates a proof of concept for biobutanol and bio-butyrate production from lignocellulose using an anaerobic fungal-bacterial co-culture system.

Enzyme Discovery in Anaerobic Fungi (Neocallimastigomycetes) Enables Lignocellulosic Biorefinery Innovation.

Lignocellulosic biorefineries require innovative solutions to realize their full potential, and the discovery of novel lignocellulose-active enzymes could improve biorefinery deconstruction processes. Enzymatic deconstruction of plant cell walls is challenging, as noncarbohydrate linkages in hemicellulosic sidechains and lignin protect labile carbohydrates from hydrolysis. Highly specialized microbes that degrade plant biomass are attractive sources of enzymes for improving lignocellulose deconstruction, and the anaerobic gut fungi (Neocallimastigomycetes) stand out as having great potential for harboring novel lignocellulose-active enzymes. We discuss the known aspects of Neocallimastigomycetes lignocellulose deconstruction, including their extensive carbohydrate-active enzyme content, proficiency at deconstructing complex lignocellulose, unique physiology, synergistic enzyme complexes, and sizeable uncharacterized gene content. Progress describing Neocallimastigomycetes and their enzymes has been rapid in recent years, and it will only continue to expand. In particular, direct manipulation of anaerobic fungal genomes, effective heterologous expression of anaerobic fungal enzymes, and the ability to directly relate chemical changes in lignocellulose to fungal gene regulation will accelerate the discovery and subsequent deployment of Neocallimastigomycetes lignocellulose-active enzymes.

Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota).

Establishing a solid taxonomic framework is crucial for enabling discovery and documentation efforts. This ensures effective communication between scientists as well as reproducibility of results between laboratories, and facilitates the exchange and preservation of biological material. Such framework can only be achieved by establishing clear criteria for taxa characterization and rank assignment. Within the anaerobic fungi (phylum Neocallimastigomycota), the need for such criteria is especially vital. Difficulties associated with their isolation, maintenance and long-term storage often result in limited availability and loss of previously described taxa. To this end, we provide here a list of morphological, microscopic, phylogenetic and phenotypic criteria for assessment and documentation when characterizing newly obtained Neocallimastigomycota isolates. We also recommend a polyphasic rank-assignment scheme for novel genus-, species- and strain-level designations for newly obtained Neocallimastigomycota isolates.

Biofilm disruption enhances growth rate and carbohydrate-active enzyme production in anaerobic fungi.

Anaerobic gut fungi (AGF) are lignocellulose degraders that naturally form biofilms in the rumen of large herbivores and in standard culture techniques. While biofilm formation enhances biomass degradation and carbohydrate-active enzyme (CAZyme) production in some bacteria and aerobic fungi, gene expression and metabolism in AGF biofilms have not been compared to non-biofilm cultures. Here, using the tunable morphology of the non-rhizoidal AGF, Caecomyces churrovis, the impacts of biofilm formation on AGF gene expression, metabolic flux, growth rate, and xylan degradation rate are quantified to inform future industrial scale-up efforts. Contrary to previous findings, C. churrovis upregulated catabolic CAZymes in stirred culture relative to biofilm culture. Using a de novo transcriptome, 197 new transcripts with predicted CAZyme function were identified. Stirred cultures grew and degraded xylan significantly faster than biofilm-forming cultures with negligible differences in primary metabolic flux, offering a way to accelerate AGF biomass valorization without altering the fermentation product profile. The rhizoidal AGF, Neocallimastix lanati, also grew faster with stirring on a solid plant substrate, suggesting that the advantages of stirred C. churrovis cultures may apply broadly to other AGF.

GPCR-FEX: A Fluoride-Based Selection System for Rapid GPCR Screening and Engineering.

The directed evolution of proteins comprises a search of sequence space for variants that improve a target phenotype, yet identification of desirable variants is inherently limited by library size and screening ability. Selections that couple protein phenotype to cell viability accelerate identification of promising variants by depleting libraries of undesirable variants en masse. Here, we introduce GPCR-FEX, a stringent selection platform that couples G-protein coupled receptor (GPCR) signaling to expression of a fluoride ion exporter (FEX)-GFP fusion gene and concomitant cellular fluoride tolerance in yeast. The GPCR-FEX platform works to deplete inactive GPCR variants from the library prior to high-throughput fluorescence-based cell sorting for rapid, inexpensive screening of receptor libraries that sample an expanded sequence space. Using this system, FEX1 was placed under the control of either PFUS1 or PFIG1, promoters activated upon agonist binding by the native yeast GPCRs, Ste2p or Ste3p. Addition of a C-terminal degron to FEX1p enhanced the dynamic range of cell growth between agonist-treated and untreated cells. Using deep sequencing to enumerate population members, we show rapid selection of a previously engineered Ste2p receptor mutant strain over wild-type Ste2p in a model library enrichment experiment. Overall, the GPCR-FEX platform provides a mechanism to rapidly engineer GPCRs, which are important cellular sensors for synthetic biology.

Identification of novel membrane proteins for improved lignocellulose conversion.

Lignocellulose processing yields a heterogeneous mixture of substances, which are poorly utilized by current industrial strains. For efficient valorization of recalcitrant biomass, it is critical to identify and engineer new membrane proteins that enable the broad uptake of hydrolyzed substrates. Whereas glucose consumption rarely presents a bottleneck for cell factories, there is also a lack of transporters that allow co-consumption of glucose with other abundant biomass sugars such as xylose. This review discusses recent efforts to bioinformatically identify membrane proteins of high biotech potential for lignocellulose conversion and metabolic engineering in both model and nonconventional organisms. Of particular interest are transporters sourced from anaerobic gut fungi resident to large herbivores, which produce Sugars Will Eventually be Exported Transporters (SWEETs) that enhance xylose transport in the yeast Saccharomyces cerevisiae and enable glucose and xylose co-utilization. Additionally, recently identified fungal cellodextrin transporters are valuable alternatives to mitigate glucose repression and transporter inhibition.

Co­cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules, dockerins, and pyruvate formate lyases on specific substrates.

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal-methanogen physical associations and fungal cell wall development and remodeling.

Microbial communities and their enzymes facilitate degradation of recalcitrant polymers in anaerobic digestion.

Microbial consortia efficiently degrade complex biopolymers found in the organic fraction of municipal solid waste (OFMSW). Through enzyme production and division of labor during anaerobic digestion, microbial communities break down recalcitrant polymers and make fermentation products, including methane. However, microbial communities remain underutilized for waste degradation as it remains difficult to characterize and predict microbial interactions during waste breakdown, especially as cultivation conditions change drastically throughout anaerobic digestion. This review discusses recent progress and opportunities in cultivating natural and engineered consortia for OFMSW hydrolysis, including how recalcitrant substrates are degraded by enzymes as well as the critical factors that govern microbial interactions and culture stability. Methods to measure substrate degradation are also reviewed, and we demonstrate the need for increased standardization to enable comparisons across different environments.

Non-destructive quantification of anaerobic gut fungi and methanogens in co-culture reveals increased fungal growth rate and changes in metabolic flux relative to mono-culture.

BACKGROUND: Quantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design. RESULTS: We present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail. CONCLUSIONS: The method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

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.

Cellulosome Localization Patterns Vary across Life Stages of Anaerobic Fungi.

Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores are powerful biomass-degrading organisms that enhance their degradative ability through the formation of cellulosomes, multienzyme complexes that synergistically colocalize enzymes to extract sugars from recalcitrant plant matter. However, a functional understanding of how fungal cellulosomes are deployed in vivo to orchestrate plant matter degradation is lacking, as is knowledge of how cellulosome production and function vary throughout the morphologically diverse life cycle of anaerobic fungi. In this work, we generated antibodies against three major fungal cellulosome protein domains, a dockerin, scaffoldin, and glycoside hydrolase (GH) 48 protein, and used them in conjunction with helium ion and immunofluorescence microscopy to characterize cellulosome localization patterns throughout the life cycle of Piromyces finnis when grown on simple sugars and complex cellulosic carbon sources. Our analyses reveal that fungal cellulosomes are cell-localized entities specifically targeted to the rhizoids of mature fungal cells and bodies of zoospores. Examination of cellulosome localization patterns across life stages also revealed that cellulosome production is independent of growth substrate in zoospores but repressed by simple sugars in mature cells. This suggests that further exploration of gene regulation patterns in zoospores is needed and can inform potential strategies for derepressing cellulosome expression and boosting hydrolytic enzyme yields from fungal cultures. Collectively, these findings underscore how life cycle-dependent cell morphology and regulation of cellulosome production impact biomass degradation by anaerobic fungi, insights that will benefit ongoing efforts to develop these organisms and their cellulosomes into platforms for converting waste biomass into valuable bioproducts. IMPORTANCE Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses. We developed and used imaging tools to observe cellulosome spatial localization patterns across life stages of the anaerobic fungus Piromyces finnis under different environmental conditions. The resulting spatial details of how anaerobic fungi orchestrate biomass degradation and uncovered relationships between life cycle progression and regulation of cellulosome production will benefit ongoing efforts to develop anaerobic fungi and their cellulosomes into useful biomass-upgrading platforms.