Discovery of antimicrobial peptides in the global microbiome with machine learning.

Novel antibiotics are urgently needed to combat the antibiotic-resistance crisis. We present a machine-learning-based approach to predict antimicrobial peptides (AMPs) within the global microbiome and leverage a vast dataset of 63,410 metagenomes and 87,920 prokaryotic genomes from environmental and host-associated habitats to create the AMPSphere, a comprehensive catalog comprising 863,498 non-redundant peptides, few of which match existing databases. AMPSphere provides insights into the evolutionary origins of peptides, including by duplication or gene truncation of longer sequences, and we observed that AMP production varies by habitat. To validate our predictions, we synthesized and tested 100 AMPs against clinically relevant drug-resistant pathogens and human gut commensals both in vitro and in vivo. A total of 79 peptides were active, with 63 targeting pathogens. These active AMPs exhibited antibacterial activity by disrupting bacterial membranes. In conclusion, our approach identified nearly one million prokaryotic AMP sequences, an open-access resource for antibiotic discovery.

A global survey of prokaryotic genomes reveals the eco-evolutionary pressures driving horizontal gene transfer.

Horizontal gene transfer, the exchange of genetic material through means other than reproduction, is a fundamental force in prokaryotic genome evolution. Genomic persistence of horizontally transferred genes has been shown to be influenced by both ecological and evolutionary factors. However, there is limited availability of ecological information about species other than the habitats from which they were isolated, which has prevented a deeper exploration of ecological contributions to horizontal gene transfer. Here we focus on transfers detected through comparison of individual gene trees to the species tree, assessing the distribution of gene-exchanging prokaryotes across over a million environmental sequencing samples. By analysing detected horizontal gene transfer events, we show distinct functional profiles for recent versus old events. Although most genes transferred are part of the accessory genome, genes transferred earlier in evolution tend to be more ubiquitous within present-day species. We find that co-occurring, interacting and high-abundance species tend to exchange more genes. Finally, we show that host-associated specialist species are most likely to exchange genes with other host-associated specialist species, whereas species found across different habitats have similar gene exchange rates irrespective of their preferred habitat. Our study covers an unprecedented scale of integrated horizontal gene transfer and environmental information, highlighting broad eco-evolutionary trends.

Expanded phylogeny of extremely halophilic archaea shows multiple independent adaptations to hypersaline environments.

Extremely halophilic archaea (Haloarchaea, Nanohaloarchaeota, Methanonatronarchaeia and Halarchaeoplasmatales) thrive in saturating salt concentrations where they must maintain osmotic equilibrium with their environment. The evolutionary history of adaptations enabling salt tolerance remains poorly understood, in particular because the phylogeny of several lineages is conflicting. Here we present a resolved phylogeny of extremely halophilic archaea obtained using improved taxon sampling and state-of-the-art phylogenetic approaches designed to cope with the strong compositional biases of their proteomes. We describe two uncultured lineages, Afararchaeaceae and Asbonarchaeaceae, which break the long branches at the base of Haloarchaea and Nanohaloarchaeota, respectively. We obtained 13 metagenome-assembled genomes (MAGs) of these archaea from metagenomes of hypersaline aquatic systems of the Danakil Depression (Ethiopia). Our phylogenomic analyses including these taxa show that at least four independent adaptations to extreme halophily occurred during archaeal evolution. Gene-tree/species-tree reconciliation suggests that gene duplication and horizontal gene transfer played an important role in this process, for example, by spreading key genes (such as those encoding potassium transporters) across extremely halophilic lineages.

Functional and evolutionary significance of unknown genes from uncultivated taxa.

Many of the Earth's microbes remain uncultured and understudied, limiting our understanding of the functional and evolutionary aspects of their genetic material, which remain largely overlooked in most metagenomic studies1. Here we analysed 149,842 environmental genomes from multiple habitats2-6 and compiled a curated catalogue of 404,085 functionally and evolutionarily significant novel (FESNov) gene families exclusive to uncultivated prokaryotic taxa. All FESNov families span multiple species, exhibit strong signals of purifying selection and qualify as new orthologous groups, thus nearly tripling the number of bacterial and archaeal gene families described to date. The FESNov catalogue is enriched in clade-specific traits, including 1,034 novel families that can distinguish entire uncultivated phyla, classes and orders, probably representing synapomorphies that facilitated their evolutionary divergence. Using genomic context analysis and structural alignments we predicted functional associations for 32.4% of FESNov families, including 4,349 high-confidence associations with important biological processes. These predictions provide a valuable hypothesis-driven framework that we used for experimental validatation of a new gene family involved in cell motility and a novel set of antimicrobial peptides. We also demonstrate that the relative abundance profiles of novel families can discriminate between environments and clinical conditions, leading to the discovery of potentially new biomarkers associated with colorectal cancer. We expect this work to enhance future metagenomics studies and expand our knowledge of the genetic repertory of uncultivated organisms.

Comparison of gene clustering criteria reveals intrinsic uncertainty in pangenome analyses.

BACKGROUND: A key step for comparative genomics is to group open reading frames into functionally and evolutionarily meaningful gene clusters. Gene clustering is complicated by intraspecific duplications and horizontal gene transfers that are frequent in prokaryotes. In consequence, gene clustering methods must deal with a trade-off between identifying vertically transmitted representatives of multicopy gene families, which are recognizable by synteny conservation, and retrieving complete sets of species-level orthologs. We studied the implications of adopting homology, orthology, or synteny conservation as formal criteria for gene clustering by performing comparative analyses of 125 prokaryotic pangenomes. RESULTS: Clustering criteria affect pangenome functional characterization, core genome inference, and reconstruction of ancestral gene content to different extents. Species-wise estimates of pangenome and core genome sizes change by the same factor when using different clustering criteria, allowing robust cross-species comparisons regardless of the clustering criterion. However, cross-species comparisons of genome plasticity and functional profiles are substantially affected by inconsistencies among clustering criteria. Such inconsistencies are driven not only by mobile genetic elements, but also by genes involved in defense, secondary metabolism, and other accessory functions. In some pangenome features, the variability attributed to methodological inconsistencies can even exceed the effect sizes of ecological and phylogenetic variables. CONCLUSIONS: Choosing an appropriate criterion for gene clustering is critical to conduct unbiased pangenome analyses. We provide practical guidelines to choose the right method depending on the research goals and the quality of genome assemblies, and a benchmarking dataset to assess the robustness and reproducibility of future comparative studies.

Computational exploration of the global microbiome for antibiotic discovery.

Novel antibiotics are urgently needed to combat the antibiotic-resistance crisis. We present a machine learning-based approach to predict prokaryotic antimicrobial peptides (AMPs) by leveraging a vast dataset of 63,410 metagenomes and 87,920 microbial genomes. This led to the creation of AMPSphere, a comprehensive catalog comprising 863,498 non-redundant peptides, the majority of which were previously unknown. We observed that AMP production varies by habitat, with animal-associated samples displaying the highest proportion of AMPs compared to other habitats. Furthermore, within different human-associated microbiota, strain-level differences were evident. To validate our predictions, we synthesized and experimentally tested 50 AMPs, demonstrating their efficacy against clinically relevant drug-resistant pathogens both in vitro and in vivo. These AMPs exhibited antibacterial activity by targeting the bacterial membrane. Additionally, AMPSphere provides valuable insights into the evolutionary origins of peptides. In conclusion, our approach identified AMP sequences within prokaryotic microbiomes, opening up new avenues for the discovery of antibiotics.

Atlas of mRNA translation and decay for bacteria.

Regulation of messenger RNA stability is pivotal for programmed gene expression in bacteria and is achieved by a myriad of molecular mechanisms. By bulk sequencing of 5' monophosphorylated mRNA decay intermediates (5'P), we show that cotranslational mRNA degradation is conserved among both Gram-positive and -negative bacteria. We demonstrate that, in species with 5'-3' exonucleases, the exoribonuclease RNase J tracks the trailing ribosome to produce an in vivo single-nucleotide toeprint of the 5' position of the ribosome. In other species lacking 5'-3' exonucleases, ribosome positioning alters endonucleolytic cleavage sites. Using our metadegradome (5'P degradome) sequencing approach, we characterize 5'P mRNA decay intermediates in 96 species including Bacillus subtilis, Escherichia coli, Synechocystis spp. and Prevotella copri and identify codon- and gene-level ribosome stalling responses to stress and drug treatment. We also apply 5'P sequencing to complex clinical and environmental microbiomes and demonstrate that metadegradome sequencing provides fast, species-specific posttranscriptional characterization of responses to drug or environmental perturbations. Finally we produce a degradome atlas for 96 species to enable analysis of mechanisms of RNA degradation in bacteria. Our work paves the way for the application of metadegradome sequencing to investigation of posttranscriptional regulation in unculturable species and complex microbial communities.

proGenomes3: approaching one million accurately and consistently annotated high-quality prokaryotic genomes.

The interpretation of genomic, transcriptomic and other microbial 'omics data is highly dependent on the availability of well-annotated genomes. As the number of publicly available microbial genomes continues to increase exponentially, the need for quality control and consistent annotation is becoming critical. We present proGenomes3, a database of 907 388 high-quality genomes containing 4 billion genes that passed stringent criteria and have been consistently annotated using multiple functional and taxonomic databases including mobile genetic elements and biosynthetic gene clusters. proGenomes3 encompasses 41 171 species-level clusters, defined based on universal single copy marker genes, for which pan-genomes and contextual habitat annotations are provided. The database is available at http://progenomes.embl.de/.

eggNOG 6.0: enabling comparative genomics across 12 535 organisms.

The eggNOG (evolutionary gene genealogy Non-supervised Orthologous Groups) database is a bioinformatics resource providing orthology data and comprehensive functional information for organisms from all domains of life. Here, we present a major update of the database and website (version 6.0), which increases the number of covered organisms to 12 535 reference species, expands functional annotations, and implements new functionality. In total, eggNOG 6.0 provides a hierarchy of over 17M orthologous groups (OGs) computed at 1601 taxonomic levels, spanning 10 756 bacterial, 457 archaeal and 1322 eukaryotic organisms. OGs have been thoroughly annotated using recent knowledge from functional databases, including KEGG, Gene Ontology, UniProtKB, BiGG, CAZy, CARD, PFAM and SMART. eggNOG also offers phylogenetic trees for all OGs, maximising utility and versatility for end users while allowing researchers to investigate the evolutionary history of speciation and duplication events as well as the phylogenetic distribution of functional terms within each OG. Furthermore, the eggNOG 6.0 website contains new functionality to mine orthology and functional data with ease, including the possibility of generating phylogenetic profiles for multiple OGs across species or identifying single-copy OGs at custom taxonomic levels. eggNOG 6.0 is available at http://eggnog6.embl.de.

New globally distributed bacterial phyla within the FCB superphylum.

Microbes in marine sediments play crucial roles in global carbon and nutrient cycling. However, our understanding of microbial diversity and physiology on the ocean floor is limited. Here, we use phylogenomic analyses of thousands of metagenome-assembled genomes (MAGs) from coastal and deep-sea sediments to identify 55 MAGs that are phylogenetically distinct from previously described bacterial phyla. We propose that these MAGs belong to 4 novel bacterial phyla (Blakebacterota, Orphanbacterota, Arandabacterota, and Joyebacterota) and a previously proposed phylum (AABM5-125-24), all of them within the FCB superphylum. Comparison of their rRNA genes with public databases reveals that these phyla are globally distributed in different habitats, including marine, freshwater, and terrestrial environments. Genomic analyses suggest these organisms are capable of mediating key steps in sedimentary biogeochemistry, including anaerobic degradation of polysaccharides and proteins, and respiration of sulfur and nitrogen. Interestingly, these genomes code for an unusually high proportion (~9% on average, up to 20% per genome) of protein families lacking representatives in public databases. Genes encoding hundreds of these protein families colocalize with genes predicted to be involved in sulfur reduction, nitrogen cycling, energy conservation, and degradation of organic compounds. Our findings advance our understanding of bacterial diversity, the ecological roles of these bacteria, and potential links between novel gene families and metabolic processes in the oceans.

PhyloCloud: an online platform for making sense of phylogenomic data.

Phylogenomics data have grown exponentially over the last decades. It is currently common for genome-wide projects to generate hundreds or even thousands of phylogenetic trees and multiple sequence alignments, which may also be very large in size. However, the analysis and interpretation of such data still depends on custom bioinformatic and visualisation workflows that are largely unattainable for non-expert users. Here, we present PhyloCloud, an online platform aimed at hosting, indexing and exploring large phylogenetic tree collections, providing also seamless access to common analyses and operations, such as node annotation, searching, topology editing, automatic tree rooting, orthology detection and more. In addition, PhyloCloud provides quick access to tools that allow users to build their own phylogenies using fast predefined workflows, graphically compare tree topologies, or query taxonomic databases such as NBCI or GTDB. Finally, PhyloCloud offers a novel tree visualisation system based on ETE Toolkit v4.0, which can be used to explore very large trees and enhance them with custom annotations and multiple sequence alignments. The platform allows for sharing tree collections and specific tree views via private links, or make them fully public, serving also as a repository of phylogenomic data. PhyloCloud is available at https://phylocloud.cgmlab.org.

GeCoViz: genomic context visualisation of prokaryotic genes from a functional and evolutionary perspective.

Synteny conservation analysis is a well-established methodology to investigate the potential functional role of unknown prokaryotic genes. However, bioinformatic tools to reconstruct and visualise genomic contexts usually depend on slow computations, are restricted to narrow taxonomic ranges, and/or do not allow for the functional and interactive exploration of neighbouring genes across different species. Here, we present GeCoViz, an online resource built upon 12 221 reference prokaryotic genomes that provides fast and interactive visualisation of custom genomic regions anchored by any target gene, which can be sought by either name, orthologous group (KEGGs, eggNOGs), protein domain (PFAM) or sequence. To facilitate functional and evolutionary interpretation, GeCoViz allows to customise the taxonomic scope of each analysis and provides comprehensive annotations of the neighbouring genes. Interactive visualisation options include, among others, the scaled representations of gene lengths and genomic distances, and on the fly calculation of synteny conservation of neighbouring genes, which can be highlighted based on custom thresholds. The resulting plots can be downloaded as high-quality images for publishing purposes. Overall, GeCoViz offers an easy-to-use, comprehensive, fast and interactive web-based tool for investigating the genomic context of prokaryotic genes, and is freely available at https://gecoviz.cgmlab.org.

Towards the biogeography of prokaryotic genes.

Microbial genes encode the majority of the functional repertoire of life on earth. However, despite increasing efforts in metagenomic sequencing of various habitats1-3, little is known about the distribution of genes across the global biosphere, with implications for human and planetary health. Here we constructed a non-redundant gene catalogue of 303 million species-level genes (clustered at 95% nucleotide identity) from 13,174 publicly available metagenomes across 14 major habitats and use it to show that most genes are specific to a single habitat. The small fraction of genes found in multiple habitats is enriched in antibiotic-resistance genes and markers for mobile genetic elements. By further clustering these species-level genes into 32 million protein families, we observed that a small fraction of these families contain the majority of the genes (0.6% of families account for 50% of the genes). The majority of species-level genes and protein families are rare. Furthermore, species-level genes, and in particular the rare ones, show low rates of positive (adaptive) selection, supporting a model in which most genetic variability observed within each protein family is neutral or nearly neutral.

Profiling cellular diversity in sponges informs animal cell type and nervous system evolution.

The evolutionary origin of metazoan cell types such as neurons and muscles is not known. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identified 18 distinct cell types. These include nitric oxide­sensitive contractile pinacocytes, amoeboid phagocytes, and secretory neuroid cells that reside in close contact with digestive choanocytes that express scaffolding and receptor proteins. Visualizing neuroid cells by correlative x-ray and electron microscopy revealed secretory vesicles and cellular projections enwrapping choanocyte microvilli and cilia. Our data show a communication system that is organized around sponge digestive chambers, using conserved modules that became incorporated into the pre- and postsynapse in the nervous systems of other animals.

eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale.

Even though automated functional annotation of genes represents a fundamental step in most genomic and metagenomic workflows, it remains challenging at large scales. Here, we describe a major upgrade to eggNOG-mapper, a tool for functional annotation based on precomputed orthology assignments, now optimized for vast (meta)genomic data sets. Improvements in version 2 include a full update of both the genomes and functional databases to those from eggNOG v5, as well as several efficiency enhancements and new features. Most notably, eggNOG-mapper v2 now allows for: 1) de novo gene prediction from raw contigs, 2) built-in pairwise orthology prediction, 3) fast protein domain discovery, and 4) automated GFF decoration. eggNOG-mapper v2 is available as a standalone tool or as an online service at http://eggnog-mapper.embl.de.

Prevalence and Specificity of Chemoreceptor Profiles in Plant-Associated Bacteria.

Chemosensory pathways are among the most abundant prokaryotic signal transduction systems, allowing bacteria to sense and respond to environmental stimuli. Signaling is typically initiated by the binding of specific molecules to the ligand binding domain (LBD) of chemoreceptor proteins (CRs). Although CRs play a central role in plant-microbiome interactions such as colonization and infection, little is known about their phylogenetic and ecological specificity. Here, we analyzed 82,277 CR sequences from 11,806 representative microbial species covering the whole prokaryotic phylogeny, and we classified them according to their LBD type using a de novo homology clustering method. Through phylogenomic analysis, we identified hundreds of LBDs that are found predominantly in plant-associated bacteria, including several LBDs specific to phytopathogens and plant symbionts. Functional annotation of our catalogue showed that many of the LBD clusters identified might constitute unknown types of LBDs. Moreover, we found that the taxonomic distribution of most LBD types that are specific to plant-associated bacteria is only partially explained by phylogeny, suggesting that lifestyle and niche adaptation are important factors in their selection. Finally, our results show that the profile of LBD types in a given genome is related to the lifestyle specialization, with plant symbionts and phytopathogens showing the highest number of niche-specific LBDs. The LBD catalogue and information on how to profile novel genomes are available at https://github.com/compgenomicslab/CRs. IMPORTANCE Considering the enormous variety of LBDs at sensor proteins, an important question resides in establishing the forces that have driven their evolution and selection. We present here the first clear demonstration that environmental factors play an important role in the selection and evolution of LBDs. We were able to demonstrate the existence of LBD families that are highly enriched in plant-associated bacteria but show a wide phylogenetic spread. These findings offer a number of research opportunities in the field of single transduction, such as the exploration of similar relationships in chemoreceptors of bacteria with a different lifestyle, like those inhabiting or infecting the human intestine. Similarly, our results raise the question whether similar LBD types might be shared by members of different sensor protein families. Lastly, we provide a comprehensive catalogue of CRs classified by their LBD region that includes a large number of putative new LBD types.

The Quest for Orthologs benchmark service and consensus calls in 2020.

The identification of orthologs-genes in different species which descended from the same gene in their last common ancestor-is a prerequisite for many analyses in comparative genomics and molecular evolution. Numerous algorithms and resources have been conceived to address this problem, but benchmarking and interpreting them is fraught with difficulties (need to compare them on a common input dataset, absence of ground truth, computational cost of calling orthologs). To address this, the Quest for Orthologs consortium maintains a reference set of proteomes and provides a web server for continuous orthology benchmarking (http://orthology.benchmarkservice.org). Furthermore, consensus ortholog calls derived from public benchmark submissions are provided on the Alliance of Genome Resources website, the joint portal of NIH-funded model organism databases.

Disentangling the impact of environmental and phylogenetic constraints on prokaryotic within-species diversity.

Microbial organisms inhabit virtually all environments and encompass a vast biological diversity. The pangenome concept aims to facilitate an understanding of diversity within defined phylogenetic groups. Hence, pangenomes are increasingly used to characterize the strain diversity of prokaryotic species. To understand the interdependence of pangenome features (such as the number of core and accessory genes) and to study the impact of environmental and phylogenetic constraints on the evolution of conspecific strains, we computed pangenomes for 155 phylogenetically diverse species (from ten phyla) using 7,000 high-quality genomes to each of which the respective habitats were assigned. Species habitat ubiquity was associated with several pangenome features. In particular, core-genome size was more important for ubiquity than accessory genome size. In general, environmental preferences had a stronger impact on pangenome evolution than phylogenetic inertia. Environmental preferences explained up to 49% of the variance for pangenome features, compared with 18% by phylogenetic inertia. This observation was robust when the dataset was extended to 10,100 species (59 phyla). The importance of environmental preferences was further accentuated by convergent evolution of pangenome features in a given habitat type across different phylogenetic clades. For example, the soil environment promotes expansion of pangenome size, while host-associated habitats lead to its reduction. Taken together, we explored the global principles of pangenome evolution, quantified the influence of habitat, and phylogenetic inertia on the evolution of pangenomes and identified criteria governing species ubiquity and habitat specificity.

proGenomes2: an improved database for accurate and consistent habitat, taxonomic and functional annotations of prokaryotic genomes.

Microbiology depends on the availability of annotated microbial genomes for many applications. Comparative genomics approaches have been a major advance, but consistent and accurate annotations of genomes can be hard to obtain. In addition, newer concepts such as the pan-genome concept are still being implemented to help answer biological questions. Hence, we present proGenomes2, which provides 87 920 high-quality genomes in a user-friendly and interactive manner. Genome sequences and annotations can be retrieved individually or by taxonomic clade. Every genome in the database has been assigned to a species cluster and most genomes could be accurately assigned to one or multiple habitats. In addition, general functional annotations and specific annotations of antibiotic resistance genes and single nucleotide variants are provided. In short, proGenomes2 provides threefold more genomes, enhanced habitat annotations, updated taxonomic and functional annotation and improved linkage to the NCBI BioSample database. The database is available at http://progenomes.embl.de/.

Gene Expression Changes and Community Turnover Differentially Shape the Global Ocean Metatranscriptome.

Ocean microbial communities strongly influence the biogeochemistry, food webs, and climate of our planet. Despite recent advances in understanding their taxonomic and genomic compositions, little is known about how their transcriptomes vary globally. Here, we present a dataset of 187 metatranscriptomes and 370 metagenomes from 126 globally distributed sampling stations and establish a resource of 47 million genes to study community-level transcriptomes across depth layers from pole-to-pole. We examine gene expression changes and community turnover as the underlying mechanisms shaping community transcriptomes along these axes of environmental variation and show how their individual contributions differ for multiple biogeochemically relevant processes. Furthermore, we find the relative contribution of gene expression changes to be significantly lower in polar than in non-polar waters and hypothesize that in polar regions, alterations in community activity in response to ocean warming will be driven more strongly by changes in organismal composition than by gene regulatory mechanisms. VIDEO ABSTRACT.