Bernard Henrissat

Sirenians, such as manatees, are the only extant mammals fully adapted to aquatic herbivory. Through specialized hindgut fermentation, they play a role in modulating nutrient fluxes and carbon cycling across tropical coastal and riverine ecosystems. However, our understanding of their gut microbiome and its metabolic potential remains elusive. Here we present an ultradeep genomic atlas of the gut microbiome from 47 freshwater and marine manatees spanning distinct developmental stages, comprising 1,215 non-redundant prokaryotic metagenome-assembled genomes and their associated virome. The manatee microbiome encodes a multilayered enzymatic repertoire for plant cell wall deconstruction, including free enzymes, cellulosomes and CAZyme-enriched outer membrane vesicles, alongside diverse pathways for aromatic catabolism. Combining biochemical and structural approaches, we characterize a previously unrecognized enzymatic pathway involving epilactase, which enables a specific Bacillota species to exploit a carbohydrate inaccessible to most microbial life, defining a specialized biochemical strategy for nutrient niche partitioning. Together, these findings uncover the microbial and metabolic basis of aquatic mammalian herbivory.

Summary: Dark septate endophytes (DSE) are widespread root‐colonising fungi with poorly understood lifestyle and ecological functions. We investigated their genomic traits, sexual reproduction signatures, carbohydrate‐active enzyme (CAZyme) and secondary metabolites profiles, and tested whether genomic features distinguish DSEs and other endophytes from fungi with other lifestyles. We also reconstructed ancestral states to clarify the evolutionary origins of DSEs. We analysed 107 Ascomycota genomes representing diverse lifestyles, including nine newly sequenced DSE genomes. The dataset allowed us to test whether secreted enzyme profiles differ between fungi colonising roots vs shoots and those associated with grass vs nongrass hosts. DSEs evolved through multiple independent trajectories and diverse lifestyles and show remarkable metabolic diversity. They possess extensive degradative capacities and some of the largest biosynthetic gene cluster (BGC) richness among fungi. Despite sharing BGC richness with other endophytes, DSEs form a distinct subgroup with fewer cellulose‐ and more hemicellulose‐targeting enzymes. Genomes also revealed genomic signatures associated with fungi colonising grass vs nongrass hosts and root‐ vs shoot‐associated niches. Our comparative genomics approach highlights how genome‐level data can deepen our understanding of endophytic lifestyle and the biology of the enigmatic DSE fungi.

Deciphering the functional potential of the unusually large and repetitive genomes of trichomonads has remained challenging since the first Trichomonas vaginalis genome assembly in 2007. These genomes are characterised by extensive gene duplication, widespread lateral gene transfer from prokaryotes, and numerous hypothetical proteins. Here, we systematically re-annotated five parasitic trichomonad genomes using the CAZy annotation pipeline, identifying 1,406 CAZyme-encoding genes (which encompass all four core CAZyme classes and carbohydrate-binding modules (CBMs)) - including 516 genes previously annotated as encoding hypothetical proteins - and generating predictions of CAZy genes originating from potential lateral gene transfer, encoding candidate secreted proteins, and their candidate metabolic pathway involvement. Comparative analyses revealed a conserved, highly duplicated repertoire of candidate mucin O-glycan-targeting CAZymes across all five species, alongside broad conservation of a candidate antibacterial CAZyme toolkit. Together, these findings substantially enrich functional annotation of these genomes and highlight in particular CAZymes and CBMs as promising targets for the future characterisation of the molecular and cellular basis of parasite-host and parasite-microbiota interactions underlying mucosal disease and dysbiosis associated with trichomonads. Significance Statement: Parasitic trichomonads possess unusually large and complex genomes that are challenging to annotate, hindering the identification of virulence factors and other genes of interest. Glycans are central to interactions between these parasites and their hosts and host microbiota, influencing processes such as attachment, nutrient acquisition and microbial competition. This study enhances the annotation of five parasitic trichomonad genomes by incorporating CAZy-classified protein and module information (e.g. glycoside hydrolases and carbohydrate binding modules) and comparative analyses, revealing novel candidate virulence factors within this functionally important gene set. The resulting dataset provides a rich and robust resource for the research community to further investigate the biology and the molecular and cellular basis of the pathogenic potential of these understudied parasites that infect various mucosal sites across a broad range of hosts across birds and mammals, including humans.

Degradation of mucins at the host-microbial mucus interphase involves glycosidases that release monosaccharides from O-glycans and mucinases that cleave the mucin protein backbone. Mucinases recognize and cleave peptide bonds at specific sequence motifs with varying O-glycan structures required and/or permissible. Mucinases that digest mucins with intact O-glycans can potentially destroy the protective mucus, while mucinases that only digest mucins with partially degraded O-glycans may serve at a later stage of nutrient sourcing from mucins. Here, we discovered nine CBM-bearing M60-like mucinases across gut commensals and opportunists, including a conserved Bacteroides fragilis mucinase denoted HC11. We also investigated the previously described Bacteroides thetaiotaomicron mucinase BT4244, which together delineates two functional classes with distinct preferences: BT4244 for bis-Tn (GalNAcα1-O-Ser/Thr) and HC11 for bis-T (Galβ1-3GalNAcα1-O-Ser/Thr) O-glycans. Both mucinases harbor carbohydrate-binding modules (CBM32) that bind their cognate O-glycan motifs and are required - together with the catalytic domains - for efficient cleavage of extended mucin domains, which is consistent with cooperative engagement, but are not required for the cleavage of short glycopeptides. We show B. fragilis strains secrete HC11 and degrade mucins only after the removal of sialic acids. Together, these findings expand the mucinase repertoire by nine enzymes spanning commensals and opportunists, demonstrate that CBM32 domains are essential for efficient cleavage of extended mucin substrates likely by promoting multivalent engagement and substrate positioning, and nominateidentify CBM-catalytic cooperation as a mechanism and intervention point for controlling mucus turnover and barrier integrity.

Trichoderma fungi support sustainable agriculture by suppressing plant diseases and improving crop performance. However, emerging pathogenicity of Trichoderma warrants further ecological and genetic characterization. Here we used machine learning to correlate genomic data from 37 Trichoderma strains with over 140 phenotypic traits, spanning metabolic versatility, biotic interactions, stress tolerance and reproductive strategies. We determined Trichoderma to be an ancient, genetically cohesive and physiologically diverse genus with spores capable of germination in water and dispersal via air and water droplets. Metabolic preferences indicate universal adaptation to mycoparasitism and to niches like arboreal microbial mats, alongside broader saprotrophic versatility. Our analyses are consistent with character displacement among close relatives and convergent evolution in distant lineages, with both processes shaping ecological plasticity and traits including dispersal modes, terrestrialization or endophytism. Our findings reveal that while some Trichoderma species show traits of biosafety concern, its vast ecophysiological diversity enables the development of safe, targeted bioeffectors.

The mucus lining wet body surfaces forms the interphase and barrier for the microbiota and resident microbiomes. Large mucin proteins densely decorated with O-glycans make up the mucus lining to entrap, feed and shape the microbiota, and repress biofilm formation and virulence. How mucins exert these effects is poorly understood and critical is how the microbiota recognize, sense, and break down mucins. Here, we provide structural molecular evidence that a small mucin-binding module designated X409 recognizes clustered saccharide patches comprised of rows of inner monosaccharides in adjacent O-glycans. These patches are unique to mucins and binding to these provides an elegant mechanism to retain adherence to mucins despite trimming of O-glycans during microbial scavenging of monosaccharides from mucins. Realization of clustered saccharide patch-binding motifs provides a hitherto overlooked scenario of contextual glycan epitopes and impetus for discovery of new classes of glycan-binding proteins.

Basidiomycete fungi are the main decomposers of dead wood with an impact on the global carbon cycle. Their degradative mechanisms have been well-studied under aerobic conditions. Here, we study their activity in oxygen-depleted environments. We use metaproteomics in a field study to identify active wood-decomposing fungi and their enzymes at different depths from the wood surface, including in oxygen-depleted conditions. In vitro, we observe that the brown-rot fungus Fomitopsis pinicola can grow on wood in complete anoxia. Using ¹³C solid-state NMR, we demonstrate the degradation of plant cell-wall polysaccharides and fungal growth in the absence of oxygen. Proteomic analyses reveal that F. pinicola switches from a Fenton chemistry-based process under aerobic conditions to the secretion of plant cell wall-active enzymes in anoxia. Our finding that wood decay fungi can thrive in complete anoxia provides a deeper understanding of lignocellulose degradation mechanisms in nature and raises opportunities for the development of bio-inspired anaerobic processes.

The breakdown of cellulose is one of the most important reactions in nature^1,2 and is central to biomass conversion to fuels and chemicals³. However, the microfibrillar organization of cellulose and its complex interactions with other components of the plant cell wall poses a major challenge for enzymatic conversion⁴. Here, by mining the metagenomic ‘dark matter’ (unclassified DNA with unknown function) of a microbial community specialized in lignocellulose degradation, we discovered a metalloenzyme that oxidatively cleaves cellulose. This metalloenzyme acts on cellulose through an exo-type mechanism with C1 regioselectivity, resulting exclusively in cellobionic acid as a product. The crystal structure reveals a catalytic copper buried in a compact jelly-roll scaffold that features a flattened cellulose binding site. This metalloenzyme exhibits a homodimeric configuration that enables in situ hydrogen peroxide generation by one subunit while the other is productively interacting with cellulose. The secretome of an engineered strain of the fungus Trichoderma reesei expressing this metalloenzyme boosted the glucose release from pretreated lignocellulosic biomass under industrially relevant conditions, demonstrating its biotechnological potential. This discovery modifies the current understanding of bacterial redox enzymatic systems devoted to overcoming biomass recalcitrance^5,6,7. Furthermore, it enables the conversion of agro-industrial residues into value-added bioproducts, thereby contributing to the transition to a sustainable and bio-based economy.

Synthetic glycans (SGs) containing glycosidic linkages and structures not identified in nature offer a means for deliberately altering microbial community properties. Here pools of SG oligosaccharides were generated via polymerization of monosaccharides and screened for their ability to increase saccharolytic Bacteroides in ex vivo cultures of human fecal samples. A lead SG preparation was orally administered to gnotobiotic mice harboring a consortium of 56 cultured, phylogenetically diverse human gut bacteria and fed a Western diet. The abundances of 3 of 15 Bacteroides strains increased, most prominently B. intestinalis. Underlying mechanisms were characterized by analyzing in vivo expression of the carbohydrate utilization machinery, using retrievable microscopic paramagnetic particles with bound SG oligosaccharides and assaying SG degradation by individual purified B. intestinalis glycoside hydrolases. The results reveal that SGs can selectively co-opt carbohydrate utilization machinery in different human gut Bacteroides and demonstrate a means for identifying artificial carbohydrate structures for targeted bacterial manipulation.