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1. Discovery, diversity and distribution of functional dark matter through global metagenomics.

   Pavlopoulos, G.A. (October 2023, Nature)

   Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities1,2. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database3. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matter. 

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2. Deep learning of a bacterial and archaeal universal language of life enables transfer learning and illuminates microbial dark matter

   https://doi.org/10.1038/s41467-022-30070-8  

   Hoarfrost, A; Aptekmann, A; Farfañuk, G; Bromberg, Y (December 2022, Nature Communications)

   Abstract The majority of microbial genomes have yet to be cultured, and most proteins identified in microbial genomes or environmental sequences cannot be functionally annotated. As a result, current computational approaches to describe microbial systems rely on incomplete reference databases that cannot adequately capture the functional diversity of the microbial tree of life, limiting our ability to model high-level features of biological sequences. Here we present LookingGlass, a deep learning model encoding contextually-aware, functionally and evolutionarily relevant representations of short DNA reads, that distinguishes reads of disparate function, homology, and environmental origin. We demonstrate the ability of LookingGlass to be fine-tuned via transfer learning to perform a range of diverse tasks: to identify novel oxidoreductases, to predict enzyme optimal temperature, and to recognize the reading frames of DNA sequence fragments. LookingGlass enables functionally relevant representations of otherwise unknown and unannotated sequences, shedding light on the microbial dark matter that dominates life on Earth. 

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3. Prevalence and diversity of TAL effector-like proteins in fungal endosymbiotic Mycetohabitans spp.

   https://doi.org/10.1099/mgen.0.001261  

   Carpenter, Sara_C D; Bogdanove, Adam J; Abbot, Bhuwan; Stajich, Jason E; Uehling, Jessie K; Lovett, Brian; Kasson, Matt T; Carter, Morgan E (June 2024, Microbial genomics)

   EndofungalMycetohabitans(formerlyBurkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus,Rhizopus microsporus. The one known secreted effector family fromMycetohabitansconsists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenicXanthomonasandRalstoniaspp. to activate host genes that promote disease. These ‘BurkholderiaTAL-like (Btl)’ proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of twoMycetohabitansstrains revealed that the single Btl in oneMycetohabitans endofungorumstrain enhances fungal membrane stress tolerance, while others in aMycetohabitans rhizoxinicastrain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nineMycetohabitansspp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal–bacterial metagenomes. We show thatbtlgenes are present across diverseMycetohabitansstrains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested thatMycetohabitansmight contain more species than previously recognized. Within our data set, Btl proteins were more conserved acrossM. rhizoxinicastrains than acrossM. endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial–fungal symbioses in myriad ways. 

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4. Global deep-sea hydrothermal deposit metagenomes and metagenome-assembled genomes over time and space

   https://doi.org/10.1038/s41597-026-06612-w  

   St_John, Emily; Reysenbach, Anna-Louise (January 2026, Scientific Data)

   Not AvailableActively venting high temperature deep-sea hydrothermal vent deposits along tectonic spreading centers and in backarc basins harbor a rich diversity of thermophilic Bacteria and Archaea, many of which have no representatives in cultivation nor any genomic representation in databases. Here, in order to produce a global-scale time series metagenomic resource for studying the microbial functional and genomic diversity in these high temperature ecosystems, we obtained 70 metagenomes from collections across spatial and temporal gradients from 21 different vent fields spanning 16 years (1993–2009). The dataset (Deep-Sea Hydrothermal Vent dataset (DSV70)) includes 3.56 Tbp of raw DNA sequence reads, that have been assembled to produce 7,422 medium- to high-quality (based on CheckM2) metagenome-assembled genomes (MAGs) of Bacteria (6,063 MAGs) and Archaea (1,359 MAGs). Collectively, this DSV70 dataset and the published 40 metagenomes from more recent deep-sea collections (2004 to 2018), represent a valuable resource for exploring the functional and phylogenomic diversity of the deep-sea hydrothermal microbiomes, and provide many reference genomes for studies in the taxonomy and systematics of poorly studied microbial lineages. Further, with the interest in mining the mineral resources at deep-sea vents, the DSV70 provides a genomic legacy for monitoring impacts on the microbial communities in these systems. 

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5. Complementary Metagenomic Approaches Improve Reconstruction of Microbial Diversity in a Forest Soil

   https://doi.org/10.1128/mSystems.00768-19  

   Alteio, L. V.; Schulz, F.; Seshadri, R.; Varghese, N.; Rodriguez-Reillo, W.; Ryan, E.; Goudeau, D.; Eichorst, S. A.; Malmstrom, R. R.; Bowers, R. M.; et al (April 2020, mSystems)

   Jansson, Janet K. (Ed.)

   ABSTRACT Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this “uncultivated majority” remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes . Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages. IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism. 

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