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The substrates of the Brazilian campos rupestres, a grassland ecosystem, have extremely low concentrations of phosphorus and nitrogen, imposing restrictions to plant growth. Despite that, this ecosystem harbors almost 15% of the Brazilian plant diversity, raising the question of how plants acquire nutrients in such a harsh environment. Here, we set out to uncover the taxonomic profile, the compositional and functional differences and similarities, and the nutrient turnover potential of microbial communities associated with two plant species of the campos rupestres-dominant family Velloziaceae that grow over distinct substrates (soil and rock). Using amplicon sequencing data, we show that, despite the pronounced composition differentiation, the plant-associated soil and rock communities share a core of highly efficient colonizers that tend to be highly abundant and is enriched in 21 bacterial families. Functional investigation of metagenomes and 522 metagenome-assembled genomes revealed that the microorganisms found associated to plant roots are enriched in genes involved in organic compound intake, and phosphorus and nitrogen turnover. We show that potential for phosphorus transport, mineralization, and solubilization are mostly found within bacterial families of the shared microbiome, such as Xanthobacteraceae and Bryobacteraceae. We also detected the full repertoire of nitrogen cycle-related genes and discovered a lineage of Isosphaeraceae that acquired nitrogen-fixing potential via horizontal gene transfer and might be also involved in nitrification via a metabolic handoff association with Binataceae. We highlight that plant-associated microbial populations in the campos rupestres harbor a genetic repertoire with potential to increase nutrient availability and that the microbiomes of biodiversity hotspots can reveal novel mechanisms of nutrient turnover.

We present 49 metagenome assemblies of the microbiome associated with Sphagnum (peat moss) collected from ambient, artificially warmed, and geothermally warmed conditions across Europe. These data will enable further research regarding the impact of climate change on plant-microbe symbiosis, ecology, and ecosystem functioning of northern peatland ecosystems.

Identifying and characterizing mobile genetic elements (MGEs) in sequencing data is essential for understanding their diversity, ecology, biotechnological applications, and impact on public health. Here, we introduce geNomad, a classification and annotation framework that combines information from gene content and a deep neural network to identify sequences of plasmids and viruses. geNomad uses a large dataset of marker proteins to provide functional gene annotation and taxonomic assignment of viral genomes. Using a conditional random field model, geNomad also detects proviruses integrated into host genomes with high precision. In benchmarks that included diverse MGE and chromosome sequences, geNomad significantly outperformed other tools in all evaluated clades of plasmids and viruses. Leveraging geNomad's speed and scalability, we were able to process public metagenomes and metatranscriptomes, leading to the discovery of millions of new viruses and plasmids that are available through the IMG/VR and IMG/PR databases. We anticipate that geNomad will enable further advancements in MGE research, and it is available at https://portal.nersc.gov/genomad.

Metagenomics has enabled accessing the genetic repertoire of natural microbial communities. Metagenome shotgun sequencing has become the method of choice for studying and classifying microorganisms from various environments. To this end, several methods have been developed to process and analyze the sequence data from raw reads to end-products such as predicted protein sequences or families. In this article, we provide a thorough review to simplify such processes and discuss the alternative methodologies that can be followed in order to explore biodiversity at the protein family level. We provide details for analysis tools and we comment on their scalability as well as their advantages and disadvantages. Finally, we report the available data repositories and recommend various approaches for protein family annotation related to phylogenetic distribution, structure prediction and metadata enrichment.

The power of next-generation sequencing has resulted in an explosive growth in the number of projects aiming to understand the metagenomic diversity of complex microbial environments. The interdisciplinary nature of this microbiome research community, along with the absence of reporting standards for microbiome data and samples, poses a significant challenge for follow-up studies. Commonly used names of metagenomes and metatranscriptomes in public databases currently lack the essential information necessary to accurately describe and classify the underlying samples, which makes a comparative analysis difficult to conduct and often results in misclassified sequences in data repositories. The Genomes OnLine Database (GOLD) (https:// gold.jgi.doe.gov/) at the Department of Energy Joint Genome Institute has been at the forefront of addressing this challenge by developing a standardized nomenclature system for naming microbiome samples. GOLD, currently in its twenty-fifth anniversary, continues to enrich the research community with hundreds of thousands of metagenomes and metatranscriptomes with well-curated and easy-to-understand names. Through this manuscript, we describe the overall naming process that can be easily adopted by researchers worldwide. Additionally, we propose the use of this naming system as a best practice for the scientific community to facilitate better interoperability and reusability of microbiome data.

Bacterial strain A52C2T was isolated from the endophytic microbial community of a Pinus pinaster tree trunk and characterized. Strain A52C2T stained Gram-negative and formed rod-shaped cells that grew optimally at 30 °C and at pH 6.0–7.0. The G+C content of the DNA was 65.1 mol %. The respiratory quinone was ubiquinone 10, and the major fatty acids were cyclo-C19:0 ω8c and C18:0, representing 70.1 % of the total fatty acids. Phylogenetic analyses based on the 16S rRNA gene sequences placed strain A52C2T in a distinct lineage within the order Hyphomicrobiales , family Pleomorphomonadaceae . The 16S rRNA gene sequence similarities of A52C2T to that of Mongoliimonas terrestris and Oharaeibacter diazotrophicus were 93.15 and 93.2 %, respectively. The draft genome sequence of strain A52C2T comprises 4 196 045 bases with a 195-fold mapped coverage of the genome. The assembled genome consists of 43 contigs of more than 1 000 bp (N50 contig size was 209 720 bp). The genome encodes 4033 putative coding sequences. The phylogenetic, phenotypic and chemotaxonomic data showed that strain A52C2T (=UCCCB 130T=CECT 8949T=LMG 29042T) represents the type of a novel species and genus, for which we propose the name Faunimonas pinastri gen. nov., sp. nov.

We report here the genome sequences of three Aquimarina megaterium strains isolated from the octocoral Eunicella labiata . We reveal a coding potential for versatile carbon metabolism and biosynthesis of natural products belonging to the polyketide, nonribosomal peptide, and terpene compound classes.

The Integrated Microbial Genomes & Microbiomes system (IMG/M: https://img.jgi.doe.gov/m/) at the Department of Energy (DOE) Joint Genome Institute (JGI) continues to provide support for users to perform comparative analysis of isolate and single cell genomes, metagenomes, and metatranscriptomes. In addition to datasets produced by the JGI, IMG v.7 also includes datasets imported from public sources such as NCBI Genbank, SRA, and the DOE National Microbiome Data Collaborative (NMDC), or submitted by external users. In the past couple years, we have continued our effort to help the user community by improving the annotation pipeline, upgrading the contents with new reference database versions, and adding new analysis functionalities such as advanced scaffold search, Average Nucleotide Identity (ANI) for high-quality metagenome bins, new cassette search, improved gene neighborhood display, and improvements to metatranscriptome data display and analysis. We also extended the collaboration and integration efforts with other DOE-funded projects such as NMDC and DOE Biology Knowledgebase (KBase).

Viruses are widely recognized as critical members of all microbiomes. Metagenomics enables large-scale exploration of the global virosphere, progressively revealing the extensive genomic diversity of viruses on Earth and highlighting the myriad of ways by which viruses impact biological processes. IMG/VR provides access to the largest collection of viral sequences obtained from (meta)genomes, along with functional annotation and rich metadata. A web interface enables users to efficiently browse and search viruses based on genome features and/or sequence similarity. Here, we present the fourth version of IMG/VR, composed of >15 million virus genomes and genome fragments, a ≈6-fold increase in size compared to the previous version. These clustered into 8.7 million viral operational taxonomic units, including 231 408 with at least one high-quality representative. Viral sequences in IMG/VR are now systematically identified from genomes, metagenomes, and metatranscriptomes using a new detection approach (geNomad), and IMG standard annotation are complemented with genome quality estimation using CheckV, taxonomic classification reflecting the latest taxonomic standards, and microbial host taxonomy prediction. IMG/VR v4 is available at https://img.jgi.doe.gov/vr, and the underlying data are available to download at https://genome.jgi.doe.gov/portal/IMG_VR.

The Genomes OnLine Database (GOLD) (https://gold.jgi.doe.gov/) at the Department of Energy Joint Genome Institute (DOE-JGI) continues to maintain its role as one of the flagship genomic metadata repositories of the world. The ever-increasing number of projects and metadata are freely available to the user community world-wide. GOLD’s metadata is consumed by scientists and remains an important source for large-scale comparative genomics analysis initiatives. Encouraged by this active user engagement and growth, GOLD has continued to add new components and capabilities. The new features such as a public Application Programming Interface (API) and Ecosystem landing page as well as the growth of different entities in this current GOLD v.9 edition are described in detail in this manuscript.

Background The role of bacterial symbionts that populate octocorals (Cnidaria, Octocorallia) is still poorly understood. To shed light on their metabolic capacities, we examined 66 high-quality metagenome-assembled genomes (MAGs) spanning 30 prokaryotic species, retrieved from microbial metagenomes of three octocoral species and seawater. Results Symbionts of healthy octocorals were affiliated with the taxa Endozoicomonadaceae, Candidatus Thioglobaceae, Metamycoplasmataceae, unclassified Pseudomonadales, Rhodobacteraceae, unclassified Alphaproteobacteria and Ca. Rhabdochlamydiaceae. Phylogenomics inference revealed that the Endozoicomonadaceae symbionts uncovered here represent two species of a novel genus unique to temperate octocorals, here denoted Ca. Gorgonimonaseunicellae and Ca. Gorgonimonasleptogorgiae. Their genomes revealed metabolic capacities to thrive under suboxic conditions and high gene copy numbers of serine-threonine protein kinases, type 3-secretion system, type-4 pili, and ankyrin-repeat proteins, suggesting excellent capabilities to colonize, aggregate, and persist inside their host. Contrarily, MAGs obtained from seawater frequently lacked symbiosis-related genes. All Endozoicomonadaceae symbionts harbored endo-chitinase and chitin-binging protein-encoding genes, indicating that they can hydrolyze the most abundant polysaccharide in the oceans. Other symbionts, including Metamycoplasmataceae and Ca. Thioglobaceae, may assimilate the smaller chitin oligosaccharides resulting from chitin breakdown and engage in chitin deacetylation, respectively, suggesting possibilities for substrate cross-feeding and a role for the coral microbiome in overall chitin turnover. We also observed sharp differences in secondary metabolite production potential between symbiotic lineages. Specific Proteobacteria taxa may specialize in chemical defense and guard other symbionts, including Endozoicomonadaceae, which lack such capacity. Conclusion This is the first study to recover MAGs from dominant symbionts of octocorals, including those of so-far unculturable Endozoicomonadaceae, Ca. Thioglobaceae and Metamycoplasmataceae symbionts. We identify a thus-far unanticipated, global role for Endozoicomonadaceae symbionts of corals in the processing of chitin, the most abundant natural polysaccharide in the oceans and major component of the natural zoo- and phytoplankton feed of octocorals. We conclude that niche partitioning, metabolic specialization, and adaptation to low oxygen conditions among prokaryotic symbionts likely contribute to the plasticity and adaptability of the octocoral holobiont in changing marine environments. These findings bear implications not only for our understanding of symbiotic relationships in the marine realm but also for the functioning of benthic ecosystems at large.

Novel bacterial isolates with the capabilities of lignin depolymerization, catabolism, or both, could be pertinent to lignocellulosic biofuel applications. In this study, we aimed to identify anaerobic bacteria that could address the economic challenges faced with microbial-mediated biotechnologies, such as the need for aeration and mixing. Using a consortium seeded from temperate forest soil and enriched under anoxic conditions with organosolv lignin as the sole carbon source, we successfully isolated a novel bacterium, designated 159R. Based on the 16S rRNA gene, the isolate belongs to the genus Sodalis in the family Bruguierivoracaceae. Whole-genome sequencing revealed a genome size of 6.38 Mbp and a GC content of 55 mol%. To resolve the phylogenetic position of 159R, its phylogeny was reconstructed using (i) 16S rRNA genes of its closest relatives, (ii) multilocus sequence analysis (MLSA) of 100 genes, (iii) 49 clusters of orthologous groups (COG) domains, and (iv) 400 conserved proteins. Isolate 159R was closely related to the deadwood associated Sodalis guild rather than the tsetse fly and other insect endosymbiont guilds. Estimated genome-sequence-based digital DNA-DNA hybridization (dDDH), genome percentage of conserved proteins (POCP), and an alignment analysis between 159R and the Sodalis clade species further supported that isolate 159R was part of the Sodalis genus and a strain of Sodalis ligni. We proposed the name Sodalis ligni str. 159R (=DSM 110549 = ATCC TSD-177). IMPORTANCE Currently, in the paper industry, paper mill pulping relies on unsustainable and costly processes to remove lignin from lignocellulosic material. A greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. Anaerobic bacteria are a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable by-products. We presented Sodalis ligni str. 159R and its characteristics as another example of potential mechanisms that can be developed for lignocellulosic applications.

BackgroundThe role of bacterial symbionts that populate octocorals (Cnidaria, Octocorallia) is still poorly understood. To shed light on their metabolic capacities, we examined 66 high-quality metagenome-assembled genomes (MAGs) spanning 30 prokaryotic species, retrieved from microbial metagenomes of three octocoral species and seawater. ResultsSymbionts of healthy octocorals were affiliated with the taxa Endozoicomonadaceae, Candidatus Thioglobaceae, Metamycoplasmataceae, unclassified Pseudomonadales, Rhodobacteraceae, unclassified Alphaproteobacteria and Ca. Rhabdochlamydiaceae. Phylogenomics inference revealed that the Endozoicomonadaceae symbionts uncovered here represent two species of a novel genus unique to temperate octocorals, here denoted Ca. Gorgonimonas eunicellae and Ca. Gorgonimonas leptogorgiae. Their genomes revealed metabolic capacities to thrive under suboxic conditions and high gene copy numbers of serine-threonine protein kinases, type III-secretion system, type IV-pili, and ankyrin-repeat proteins, suggesting excellent capabilities to colonize, aggregate, and persist inside their host. All Endozoicomonadaceae MAGs harbored endo-chitinase and chitin-binging protein-encoding genes, enabling these symbionts to hydrolyze the most abundant polysaccharide in the oceans. Other symbionts, including Metamycoplasmataceae and Ca. Thioglobaceae, may assimilate the smaller chitin-oligosaccharides resulting from chitin breakdown and engage in chitin deacetylation, respectively, providing evidence for substrate cross-feeding and a role for the coral microbiome in overall chitin turnover. We also observed sharp differences in secondary metabolite production capacities between symbiotic lineages. Specific Proteobacteria taxa may specialize in chemical defense and guard other symbionts, including Endozoicomonadaceae, which lack such capacity. ConclusionThis is the first study to recover MAGs from dominant symbionts of octocorals, including those of so-far unculturable Endozoicomonadaceae, Ca. Thioglobaceae and Mycoplasmoidales symbionts. We identify a thus-far unanticipated, global role for Endozoicomonadaceae symbionts of corals in the processing of chitin, the most abundant natural polysaccharide in the oceans and major component of the natural zoo- and phytoplankton feed of octocorals. We conclude that niche partitioning, metabolic specialization, and adaptation to low oxygen conditions among prokaryotic symbionts likely contribute to the plasticity and adaptability of the octocoral holobiont in changing marine environments. These findings bear implications not only for our understanding of symbiotic relationships in the marine realm abut also for the functioning of benthic ecosystems at large.

With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek’s Hydrogenobacter sp. population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure.

Metagenomics is unearthing the previously hidden world of soil viruses. Many soil viral sequences in metagenomes contain putative auxiliary metabolic genes (AMGs) that are not associated with viral replication. Here, we establish that AMGs on soil viruses actually produce functional, active proteins. We focus on AMGs that potentially encode chitosanase enzymes that metabolize chitin – a common carbon polymer. We express and functionally screen several chitosanase genes identified from environmental metagenomes. One expressed protein showing endo-chitosanase activity (V-Csn) is crystalized and structurally characterized at ultra-high resolution, thus representing the structure of a soil viral AMG product. This structure provides details about the active site, and together with structure models determined using AlphaFold, facilitates understanding of substrate specificity and enzyme mechanism. Our findings support the hypothesis that soil viruses contribute auxiliary functions to their hosts.

Metagenomics is unearthing the previously hidden world of soil viruses1,2. Many soil viral sequences in metagenomes contain putative auxiliary metabolic genes (AMGs) that are not associated with viral replication. To date only one soil viral AMG has been expressed3 and none has a solved structure. Here, we aimed to establish that AMGs on soil viruses actually produce functional, active proteins. We focused on AMGs that potentially encode chitosanase enzymes that metabolize chitin – a common carbon polymer. Glycoside hydrolase family 75 viral chitosanase genes were identified from environmental metagenomes. Several of these were expressed and functionally screened. One expressed protein showing endo-chitosanase activity (V-Csn) was crystalized and structurally characterized at ultra-high resolution, thus representing the first structure of a soil viral AMG product. A structure for an inactive mutant with a bound substrate (chitosan oligomer) was also determined. Conserved active site residues in V-Csn resided in a cleft between two domains. These structures provided details about the active site, and together with structure models determined using AlphaFold2, facilitated understanding of substrate specificity and enzyme mechanism. These findings support the hypothesis that soil viruses contribute auxiliary functions to their hosts. Soil viral chitosanase AMG products that assist chitin decomposition may therefore play a previously unrecognized essential role in soil carbon cycling.

Small genes (40,000 small-gene families in ~2.3 million phage genome contigs. We find that small genes in phage genomes are approximately 3-fold more prevalent than in host prokaryotic genomes. Our approach enriches for small genes that are translated in microbiomes, suggesting the small genes identified are coding. More than 9,000 families encode potentially secreted or transmembrane proteins, more than 5,000 families encode predicted anti-CRISPR proteins, and more than 500 families encode predicted antimicrobial proteins. By combining homology and genomic-neighborhood analyses, we reveal substantial novelty and diversity within phage biology, including small phage genes found in multiple host phyla, small genes encoding proteins that play essential roles in host infection, and small genes that share genomic neighborhoods and whose encoded proteins may share related functions.

The phylum Actinobacteria includes important human pathogens like Mycobacterium tuberculosis and Corynebacterium diphtheriae and renowned producers of secondary metabolites of commercial interest, yet only a small part of its diversity is represented by sequenced genomes. Here, we present 824 actinobacterial isolate genomes in the context of a phylum-wide analysis of 6,700 genomes including public isolates and metagenome-assembled genomes (MAGs). We estimate that only 30%–50% of projected actinobacterial phylogenetic diversity possesses genomic representation via isolates and MAGs. A comparison of gene functions reveals novel determinants of host-microbe interaction as well as environment-specific adaptations such as potential antimicrobial peptides. We identify plasmids and prophages across isolates and uncover extensive prophage diversity structured mainly by host taxonomy. Analysis of >80,000 biosynthetic gene clusters reveals that horizontal gene transfer and gene loss shape secondary metabolite repertoire across taxa. Our observations illustrate the essential role of and need for high-quality isolate genome sequences. The work (proposal DOI[s]: https://doi.org/10.46936/10.25585/60001024; https://doi.org/10.46936/10.25585/60000886; https://doi.org/10.46936/10. 25585/60001401; https://doi.org/10.46936/10.25585/60001087; https:// doi.org/10.46936/10.25585/60001079; https://doi.org/10.46936/10.25585/ 60001044) conducted by the US Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy operated under contract no. DE-AC02-05CH11231. Design and synthesis of peptide constructs were supported by the Biosystems Design program (DOE Office of Science contract DE-SC0018260) and the US Department of Energy Joint Genome Institute (DOE Office of Science contract DEAC02-05CH1123), respectively. Characterization of the peptide was supported by the Secured Biosystem Design project entitled ‘‘Rapid Design and Engineering of Smart and Secure Microbiological Systems’’ (DOE Office of Science contract DE-AC02-05CH1123). We thank the following researchers for their support of this study by providing free use of their public genome data: Kristen De-Angelis, Grace Pold, Mallory Choudoir, Camila Carlos-Shanley, Paul Carini, H. Corby C. Kistler, James Elkins, Javier A. Izquierdo, Dimitris Hatzinikolaou, Daniel Schachtman, Paul R. Jensen, Aindrila Mukhopadhyay, John Vogel, Carolin Frank, Paul M. D’Agostino, Ann M. Hirsch, Satoshi Yuzawa, Regina Lamendella, Bernhard Fuchs, Dale Pelletier, Laila P. Partida-Martinez, Cameron Currie, Seth De-Bolt, Jeff Dangl, David Mead, Susannah Tringe, David A. Baltrus, Seung Bum Kim, Linda Kinkel, Kelly Wrighton, William Mohn, Ludmila Christoserdova, Sarah Lebeis, Janet Janssen, Sandra Baena Garzon, and Nicholas Coleman. Special thanks to Marie Louise Ballon at the JGI Communications and Outreach office for all her help with the graphical abstract and many other details.