The gut microbiota operates at the interface of host-environment interactions to influence human homoeostasis and metabolic networks. Environmental factors that unbalance gut microbial ecosystems can therefore shape physiological and disease-associated responses across somatic tissues. However, the systemic impact of the gut microbiome on the germline-and consequently on the F offspring it gives rise to-is unexplored. Here we show that the gut microbiota act as a key interface between paternal preconception environment and intergenerational health in mice. Perturbations to the gut microbiota of prospective fathers increase the probability of their offspring presenting with low birth weight, severe growth restriction and premature mortality. Transmission of disease risk occurs via the germline and is provoked by pervasive gut microbiome perturbations, including non-absorbable antibiotics or osmotic laxatives, but is rescued by restoring the paternal microbiota before conception. This effect is linked with a dynamic response to induced dysbiosis in the male reproductive system, including impaired leptin signalling, altered testicular metabolite profiles and remapped small RNA payloads in sperm. As a result, dysbiotic fathers trigger an elevated risk of in utero placental insufficiency, revealing a placental origin of mammalian intergenerational effects. Our study defines a regulatory 'gut-germline axis' in males, which is sensitive to environmental exposures and programmes offspring fitness through impacting placenta function.

Current metagenome assembled human gut phage catalogs contained mostly fragmented genomes. Here, comprehensive gut virome detection procedure is developed involving virus-like particle (VLP) enrichment from ≈500 g feces and combined sequencing of short- and long-read. Applied to 135 samples, a Chinese Gut Virome Catalog (CHGV) is assembled consisting of 21,499 non-redundant viral operational taxonomic units (vOTUs) that are significantly longer than those obtained by short-read sequencing and contained ≈35% (7675) complete genomes, which is ≈nine times more than those in the Gut Virome Database (GVD, ≈4%, 1,443). Interestingly, the majority (≈60%, 13,356) of the CHGV vOTUs are obtained by either long-read or hybrid assemblies, with little overlap with those assembled from only the short-read data. With this dataset, vast diversity of the gut virome is elucidated, including the identification of 32% (6,962) novel vOTUs compare to public gut virome databases, dozens of phages that are more prevalent than the crAssphages and/or Gubaphages, and several viral clades that are more diverse than the two. Finally, the functional capacities are also characterized of the CHGV encoded proteins and constructed a viral-host interaction network to facilitate future research and applications.

Small open reading frames (smORFs) shorter than 100 codons are widespread and perform essential roles in microorganisms, where they encode proteins active in several cell functions, including signal pathways, stress response, and antibacterial activities. However, the ecology, distribution and role of small proteins in the global microbiome remain unknown. Here, we construct a global microbial smORFs catalog (GMSC) derived from 63,410 publicly available metagenomes across 75 distinct habitats and 87,920 high-quality isolate genomes. GMSC contains 965 million non-redundant smORFs with comprehensive annotations. We find that archaea harbor more smORFs proportionally than bacteria. We moreover provide a tool called GMSC-mapper to identify and annotate small proteins from microbial (meta)genomes. Overall, this publicly-available resource demonstrates the immense and underexplored diversity of small proteins.

The gut microbiota play a critical role in maintaining a healthy human body and their dysregulation is associated with various diseases. In this study, we investigated the influence of the gut microbiome diversity on chronic lymphocytic leukemia (CLL) development. Stool sample analysis of 59 CLL patients revealed individual and heterogeneous microbiome compositions, but allowed for grouping of patients according to their microbiome diversity. Interestingly, CLL patients with a lower microbiome diversity and an enrichment of bacteria linked to poor health suffered from a more advanced or aggressive form of CLL. In the Eμ-TCL1 mouse model of CLL, we observed a faster course of disease when mice were housed in high hygiene conditions. Shotgun DNA sequencing of fecal samples showed that this was associated with a lower microbiome diversity which was dominated by Mucispirillum and Parabacteroides genera in comparison to mice kept under lower hygiene conditions. In conclusion, we applied taxonomic microbiome analyses to demonstrate a link between the gut microbiome diversity and the clinical course of CLL in humans, as well as the development of CLL in mice. Our novel data serve as a basis for further investigations to decipher the pathological and mechanistic role of intestinal microbiota in CLL development.

Pharmaceuticals can directly inhibit the growth of gut bacteria, but the degree to which such interactions manifest in complex community settings is an open question. Here, we compared the effects of 30 drugs on a 32-species synthetic community with their effects on each community member in isolation. While most individual drug-species interactions remained the same in the community context, communal behaviors emerged in 26% of all tested cases. Cross-protection during which drug-sensitive species were protected in community was 6 times more frequent than cross-sensitization, the converse phenomenon. Cross-protection decreased and cross-sensitization increased at higher drug concentrations, suggesting that the resilience of microbial communities can collapse when perturbations get stronger. By metabolically profiling drug-treated communities, we showed that both drug biotransformation and bioaccumulation contribute mechanistically to communal protection. As a proof of principle, we molecularly dissected a prominent case: species expressing specific nitroreductases degraded niclosamide, thereby protecting both themselves and sensitive community members.

Genetic variations are instrumental for unraveling phage evolution and deciphering their functional implications. Here, we explore the underlying fine-scale genetic variations in the gut phageome, especially structural variations (SVs). By using virome-enriched long-read metagenomic sequencing across 91 individuals, we identified a total of 14,438 nonredundant phage SVs and revealed their prevalence within the human gut phageome. These SVs are mainly enriched in genes involved in recombination, DNA methylation, and antibiotic resistance. Notably, a substantial fraction of phage SV sequences share close homology with bacterial fragments, with most SVs enriched for horizontal gene transfer (HGT) mechanism. Further investigations showed that these SV sequences were genetic exchanged between specific phage-bacteria pairs, particularly between phages and their respective bacterial hosts. Temperate phages exhibit a higher frequency of genetic exchange with bacterial chromosomes and then virulent phages. Collectively, our findings provide insights into the genetic landscape of the human gut phageome.

The Interactive Tree Of Life (https://itol.embl.de) is an online tool for the management, display, annotation and manipulation of phylogenetic and other trees. It is freely available and open to everyone. iTOL version 6 introduces a modernized and completely rewritten user interface, together with numerous new features. A new dataset type has been introduced (colored/labeled ranges), greatly upgrading the functionality of the previous simple colored range annotation function. Additional annotation options have been implemented for several existing dataset types. Dataset template files now support simple assignment of annotations to multiple tree nodes through substring matching, including full regular expression support. Node metadata handling has been greatly extended with novel display and exporting options, and it can now be edited interactively or bulk updated through annotation files. Tree labels can be displayed using multiple simultaneous font styles, with precise positioning, sizing and styling of each individual label part. Various bulk label editing functions have been implemented, simplifying large scale changes of all tree node labels. iTOL's automatic taxonomy assignment functions now support trees based on the Genome Taxonomy Database (GTDB), in addition to the NCBI taxonomy. The functionality of the optional user account pages has been expanded, simplifying the management, navigation and sharing of projects and trees. iTOL currently handles more than one and a half million trees from >130 000 individual user accounts.

Patients with cirrhosis are susceptible to develop bacterial infections that trigger acute decompensation (AD) and acute-on-chronic liver failure (ACLF). Infections with multidrug-resistant organisms (MDRO) are associated with deleterious outcome. MDRO colonisation frequently proceeds MDRO infections and antibiotic therapy has been associated with MDRO colonisation.

The human gut microbiome is a key contributor to health, and its perturbations are linked to many diseases. Small-molecule xenobiotics such as drugs, chemical pollutants and food additives can alter the microbiota composition and are now recognized as one of the main factors underlying microbiome diversity. Mapping the effects of such compounds on the gut microbiome is challenging because of the complexity of the community, anaerobic growth requirements of individual species and the large number of interactions that need to be quantitatively assessed. High-throughput screening setups offer a promising solution for probing the direct inhibitory effects of hundreds of xenobiotics on tens of anaerobic gut bacteria. When automated, such assays enable the cost-effective investigation of a wide range of compound-microbe combinations. We have developed an experimental setup and protocol that enables testing of up to 5,000 compounds on a target gut species under strict anaerobic conditions within 5 d. In addition, with minor modifications to the protocol, drug effects can be tested on microbial communities either assembled from isolates or obtained from stool samples. Experience in working in an anaerobic chamber, especially in performing delicate work with thick chamber gloves, is required for implementing this protocol. We anticipate that this protocol will accelerate the study of interactions between small molecules and the gut microbiome and provide a deeper understanding of this microbial ecosystem, which is intimately intertwined with human health.

Many of Earth's microbes remain uncultured and under-studied, limiting our understanding of the functional and evolutionary aspects of their genetic material, which remain largely overlooked in most metagenomic studies. Here, we analyzed 149,842 environmental genomes from multiple habitats and compiled a curated catalog 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 catalog is enriched in clade-specific traits, including 1,034 novel families that can distinguish entire uncultivated phyla, classes, and orders, likely 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, which we employed to experimentally validate 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.

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.

Meta'omic data on microbial diversity and function accrue exponentially in public repositories, but derived information is often siloed according to data type, study or sampled microbial environment. Here we present SPIRE, a Searchable Planetary-scale mIcrobiome REsource that integrates various consistently processed metagenome-derived microbial data modalities across habitats, geography and phylogeny. SPIRE encompasses 99 146 metagenomic samples from 739 studies covering a wide array of microbial environments and augmented with manually-curated contextual data. Across a total metagenomic assembly of 16 Tbp, SPIRE comprises 35 billion predicted protein sequences and 1.16 million newly constructed metagenome-assembled genomes (MAGs) of medium or high quality. Beyond mapping to the high-quality genome reference provided by proGenomes3 (http://progenomes.embl.de), these novel MAGs form 92 134 novel species-level clusters, the majority of which are unclassified at species level using current tools. SPIRE enables taxonomic profiling of these species clusters via an updated, custom mOTUs database (https://motu-tool.org/) and includes several layers of functional annotation, as well as crosslinks to several (micro-)biological databases. The resource is accessible, searchable and browsable via http://spire.embl.de.

The rising prevalence of liver diseases related to obesity and excessive use of alcohol is fuelling an increasing demand for accurate biomarkers aimed at community screening, diagnosis of steatohepatitis and significant fibrosis, monitoring, prognosis and prediction of treatment efficacy. Breakthroughs in omics methodologies and the power of bioinformatics have created an excellent opportunity to combine clinical needs with technological advancements. Omics technologies allow for advanced investigations into biological processes from the genes to transcription and regulation, to circulating protein, metabolite and lipid levels, as well as the microbiome including bacteria, viruses and fungi. We consequently find ourselves in a period of rapid progress in technology and bioinformatics that may allow for development of precision biomarkers for personalised medicine. However, there are important barriers to consider in omics biomarker discovery and validation, including the use of semi-quantitative measurements from untargeted platforms, which may exhibit high analytical, inter- and intra-individual variance. Standardising methods and the need to validate across diverse populations, presents a challenge, partly due to disease complexity and the dynamic nature of biomarker expression in different disease stages. Lack of validity causes lost opportunities when studies fail to provide the knowledge needed for regulatory approvals, all of which contributes to a delayed translation of these discoveries into clinical practice. While no omics-based biomarkers have matured to clinical implementation, the extent of data generated through omics-technologies holds the power of hypothesis-free discovery of a plethora of candidate biomarkers to be further validated. To explore the many opportunities of omics technologies, hepatologists need detailed knowledge of commonalities and differences between the various omics layers, and both the barriers to and advantages of these approaches.

The effects of probiotic interventions on colonization with resistant bacteria and early microbiome development in preterm infants remain to be clarified.

The host-microbiota co-metabolite trimethylamine N-oxide (TMAO) is linked to increased cardiovascular risk but how its circulating levels are regulated remains unclear. We applied "explainable" machine learning, univariate, multivariate and mediation analyses of fasting plasma TMAO concentration and a multitude of phenotypes in 1,741 adult Europeans of the MetaCardis study. Here we show that next to age, kidney function is the primary variable predicting circulating TMAO, with microbiota composition and diet playing minor, albeit significant, roles. Mediation analysis suggests a causal relationship between TMAO and kidney function that we corroborate in preclinical models where TMAO exposure increases kidney scarring. Consistent with our findings, patients receiving glucose-lowering drugs with reno-protective properties have significantly lower circulating TMAO when compared to propensity-score matched control individuals. Our analyses uncover a bidirectional relationship between kidney function and TMAO that can potentially be modified by reno-protective anti-diabetic drugs and suggest a clinically actionable intervention for decreasing TMAO-associated excess cardiovascular risk.

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/.

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.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

The fungal component of the human gut microbiome, also known as the mycobiome, plays a vital role in intestinal ecology and human health. However, the overall structure of the gut mycobiome as well as the inter-individual variations in fungal composition remains largely unknown. In this study, we collected a total of 3363 fungal sequencing samples from 16 cohorts across three continents, including 572 newly profiled samples from China.

The InterPro database (https://www.ebi.ac.uk/interpro/) provides an integrative classification of protein sequences into families, and identifies functionally important domains and conserved sites. Here, we report recent developments with InterPro (version 90.0) and its associated software, including updates to data content and to the website. These developments extend and enrich the information provided by InterPro, and provide a more user friendly access to the data. Additionally, we have worked on adding Pfam website features to the InterPro website, as the Pfam website will be retired in late 2022. We also show that InterPro's sequence coverage has kept pace with the growth of UniProtKB. Moreover, we report the development of a card game as a method of engaging the non-scientific community. Finally, we discuss the benefits and challenges brought by the use of artificial intelligence for protein structure prediction.

For decades, marine plankton have been investigated for their capacity to modulate biogeochemical cycles and provide fishery resources. Between the sunlit (epipelagic) layer and the deep dark waters, lies a vast and heterogeneous part of the ocean: the mesopelagic zone. How plankton composition is shaped by environment has been well-explored in the epipelagic but much less in the mesopelagic ocean. Here, we conducted comparative analyses of trans-kingdom community assemblages thriving in the mesopelagic oxygen minimum zone (OMZ), mesopelagic oxic, and their epipelagic counterparts. We identified nine distinct types of intermediate water masses that correlate with variation in mesopelagic community composition. Furthermore, oxygen, NO and particle flux together appeared as the main drivers governing these communities. Novel taxonomic signatures emerged from OMZ while a global co-occurrence network analysis showed that about 70% of the abundance of mesopelagic plankton groups is organized into three community modules. One module gathers prokaryotes, pico-eukaryotes and Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) from oxic regions, and the two other modules are enriched in OMZ prokaryotes and OMZ pico-eukaryotes, respectively. We hypothesize that OMZ conditions led to a diversification of ecological niches, and thus communities, due to selective pressure from limited resources. Our study further clarifies the interplay between environmental factors in the mesopelagic oxic and OMZ, and the compositional features of communities.

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.

DNA methylation plays a crucial role in the survival of bacteriophages (phages), yet the understanding of their genome methylation remains limited. In this study, DNA methylation patterns are analyzed in 8848 metagenome-assembled high-quality phages from 104 fecal samples using single-molecule real-time sequencing. The results demonstrate that 97.60% of gut phages exhibit methylation, with certain factors correlating with methylation densities. Phages with higher methylation densities appear to have potential viability advantages. Strikingly, more than one-third of the phages possess their own DNA methyltransferases (MTases). Increased MTase copies are associated with higher genome methylation densities, specific methylation motifs, and elevated prevalence of certain phage groups. Notably, the majority of these MTases share close homology with those encoded by gut bacteria, suggesting their exchange during phage-bacterium interactions. Furthermore, these MTases can be employed to accurately predict phage-host relationships. Overall, the findings indicate the widespread utilization of DNA methylation by gut DNA phages as an evasion mechanism against host defense systems, with a substantial contribution from phage-encoded MTases.

Much of the complexity within cells arises from functional and regulatory interactions among proteins. The core of these interactions is increasingly known, but novel interactions continue to be discovered, and the information remains scattered across different database resources, experimental modalities and levels of mechanistic detail. The STRING database (https://string-db.org/) systematically collects and integrates protein-protein interactions-both physical interactions as well as functional associations. The data originate from a number of sources: automated text mining of the scientific literature, computational interaction predictions from co-expression, conserved genomic context, databases of interaction experiments and known complexes/pathways from curated sources. All of these interactions are critically assessed, scored, and subsequently automatically transferred to less well-studied organisms using hierarchical orthology information. The data can be accessed via the website, but also programmatically and via bulk downloads. The most recent developments in STRING (version 12.0) are: (i) it is now possible to create, browse and analyze a full interaction network for any novel genome of interest, by submitting its complement of encoded proteins, (ii) the co-expression channel now uses variational auto-encoders to predict interactions, and it covers two new sources, single-cell RNA-seq and experimental proteomics data and (iii) the confidence in each experimentally derived interaction is now estimated based on the detection method used, and communicated to the user in the web-interface. Furthermore, STRING continues to enhance its facilities for functional enrichment analysis, which are now fully available also for user-submitted genomes.

Alcohol disturbs hepatic lipid synthesis and transport, but the role of lipid dysfunction in alcohol-related liver disease (ALD) is unclear. In this biopsy-controlled, prospective, observational study, we characterized the liver and plasma lipidomes in patients with early ALD.

Multi-omics analyses are used in microbiome studies to understand molecular changes in microbial communities exposed to different conditions. However, it is not always clear how much each omics data type contributes to our understanding and whether they are concordant with each other. Here, we map the molecular response of a synthetic community of 32 human gut bacteria to three non-antibiotic drugs by using five omics layers (16S rRNA gene profiling, metagenomics, metatranscriptomics, metaproteomics and metabolomics). We find that all the omics methods with species resolution are highly consistent in estimating relative species abundances. Furthermore, different omics methods complement each other for capturing functional changes. For example, while nearly all the omics data types captured that the antipsychotic drug chlorpromazine selectively inhibits Bacteroidota representatives in the community, the metatranscriptome and metaproteome suggested that the drug induces stress responses related to protein quality control. Metabolomics revealed a decrease in oligosaccharide uptake, likely caused by Bacteroidota depletion. Our study highlights how multi-omics datasets can be utilized to reveal complex molecular responses to external perturbations in microbial communities.

Transposons are mobile genetic elements that drive evolution and adaptation throughout the tree of life. In bacteria, they often transfer antibiotic resistance genes and contribute to the emergence of multidrug-resistant pathogens. In this talk, I will present our recent discoveries on a group of transposons that efficiently propagate antibiotic resistance genes across diverse microbial communities. Using a dedicated computational pipeline, we mapped the most wide-spread transposons in bacterial genomes and characterized their diversity, genetic cargo, and transfer dynamics. Selected elements were experimentally reconstituted to describe their molecular mechanisms and biochemical pathways. We determined numerous high-resolution crystal and cryo-EM structures of the protein-DNA assemblies involved in transposon movement. The structures capture various stages of transposition and reveal an intricate interplay between the core transposition machinery and host-encoded proteins. Protein interactions shape transposon and genomic DNA in unique ways to cut, exchange and re-join strands in a tightly regulated manner. Importantly, our data also explain how specific DNA distortion and cleavage mechanisms enable the transposons to move between diverse genomic sites, expanding gene transfer across many species. These results shed new light onto the molecular strategies of antibiotic resistance carrying transposons and show how they evolved to effectively spread key genetic traits. Our insights also open new opportunities to develop strategies for blocking transposition and thereby help control resistance spreading.

Gut microbiota is a key component in obesity and type 2 diabetes, yet mechanisms and metabolites central to this interaction remain unclear. We examined the human gut microbiome's functional composition in healthy metabolic state and the most severe states of obesity and type 2 diabetes within the MetaCardis cohort. We focused on the role of B vitamins and B7/B8 biotin for regulation of host metabolic state, as these vitamins influence both microbial function and host metabolism and inflammation.

Microbial genes encode the majority of the functional repertoire of life on earth. However, despite increasing efforts in metagenomic sequencing of various habitats, 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.

Previous microbiome and metabolome analyses exploring non-communicable diseases have paid scant attention to major confounders of study outcomes, such as common, pre-morbid and co-morbid conditions, or polypharmacy. Here, in the context of ischemic heart disease (IHD), we used a study design that recapitulates disease initiation, escalation and response to treatment over time, mirroring a longitudinal study that would otherwise be difficult to perform given the protracted nature of IHD pathogenesis. We recruited 1,241 middle-aged Europeans, including healthy individuals, individuals with dysmetabolic morbidities (obesity and type 2 diabetes) but lacking overt IHD diagnosis and individuals with IHD at three distinct clinical stages-acute coronary syndrome, chronic IHD and IHD with heart failure-and characterized their phenome, gut metagenome and serum and urine metabolome. We found that about 75% of microbiome and metabolome features that distinguish individuals with IHD from healthy individuals after adjustment for effects of medication and lifestyle are present in individuals exhibiting dysmetabolism, suggesting that major alterations of the gut microbiome and metabolome might begin long before clinical onset of IHD. We further categorized microbiome and metabolome signatures related to prodromal dysmetabolism, specific to IHD in general or to each of its three subtypes or related to escalation or de-escalation of IHD. Discriminant analysis based on specific IHD microbiome and metabolome features could better differentiate individuals with IHD from healthy individuals or metabolically matched individuals as compared to the conventional risk markers, pointing to a pathophysiological relevance of these features.

Recent evidence suggests a role for the microbiome in pancreatic ductal adenocarcinoma (PDAC) aetiology and progression.

Prokaryotic Mobile Genetic Elements (MGEs) such as transposons, integrons, phages and plasmids, play important roles in prokaryotic evolution and in the dispersal of cargo functions like antibiotic resistance. However, each of these MGE types is usually annotated and analysed individually, hampering a global understanding of phylogenetic and environmental patterns of MGE dispersal. We thus developed a computational framework that captures diverse MGE types, their cargos and MGE-mediated horizontal transfer events, using recombinases as ubiquitous MGE marker genes and pangenome information for MGE boundary estimation. Applied to ∼84k genomes with habitat annotation, we mapped 2.8 million MGE-specific recombinases to six operational MGE types, which together contain on average 13% of all the genes in a genome. Transposable elements (TEs) dominated across all taxa (∼1.7 million occurrences), outnumbering phages and phage-like elements (<0.4 million). We recorded numerous MGE-mediated horizontal transfer events across diverse phyla and habitats involving all MGE types, disentangled and quantified the extent of hitchhiking of TEs (17%) and integrons (63%) with other MGE categories, and established TEs as dominant carriers of antibiotic resistance genes. We integrated all these findings into a resource (proMGE.embl.de), which should facilitate future studies on the large mobile part of genomes and its horizontal dispersal.

Meconium constitutes infants' first bowel movements postnatally. The consistency and microbial load of meconium are different from infant and adult stool. While recent evidence suggests that meconium is sterile , rapid colonization occurs after birth. The meconium microbiome has been associated with negative health outcomes, but its composition is not well described, especially in preterm infants. Here, we characterized the meconium microbiomes from 330 very preterm infants (gestational ages 28 to 32 weeks) from 15 hospitals in Germany and in fecal samples from a subset of their mothers (N = 217). Microbiome profiles were compiled using 16S rRNA gene sequencing with negative and positive controls. The meconium microbiome was dominated by , Staphylococcus, and spp. and was associated with gestational age at birth and age at sample collection. Bifidobacterial abundance was negatively correlated with potentially pathogenic genera. The amount of bacterial DNA in meconium samples varied greatly across samples and was associated with the time since birth but not with gestational age or hospital site. In samples with low bacterial load, human mitochondrial sequences were highly amplified using commonly used, bacterial-targeted 16S rRNA primers. Only half of the meconium samples contained sufficient bacterial material to study the microbiome using a standard approach. To facilitate future meconium studies, we present a five-level scoring system ("MecBac") that predicts the success of 16S rRNA bacterial sequencing for meconium samples. These findings provide a foundational characterization of an understudied portion of the human microbiome and will aid the design of future meconium microbiome studies. Meconium is present in the intestines of infants before and after birth and constitutes their first bowel movements postnatally. The consistency, composition and microbial load of meconium is largely different from infant and adult stool. While recent evidence suggests that meconium is sterile , rapid colonization occurs after birth. The meconium microbiome has been associated with short-term and long-term negative health outcomes, but its composition is not yet well described, especially in preterm infants. We provide a characterization of the microbiome structure and composition of infant meconium and maternal feces from a large study cohort and propose a method to evaluate meconium samples for bacterial sequencing suitability. These findings provide a foundational characterization of an understudied portion of the human microbiome and will aid the design of future meconium microbiome studies.

Alterations to the gut microbiome have been linked to multiple chronic diseases. However, the drivers of such changes remain largely unknown. The oral cavity acts as a major route of exposure to exogenous factors including pathogens, and processes therein may affect the communities in the subsequent compartments of the gastrointestinal tract. Here, we perform strain-resolved, integrated meta-genomic, transcriptomic, and proteomic analyses of paired saliva and stool samples collected from 35 individuals from eight families with multiple cases of type 1 diabetes mellitus (T1DM).

To identify gut and oral metagenomic signatures that accurately predict pancreatic ductal carcinoma (PDAC) and to validate these signatures in independent cohorts.

Natural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups, this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds. However, studying this diversity to identify genomic pathways for the synthesis of such compounds and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters ('Candidatus Eudoremicrobiaceae') that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environments.

We report a droplet microfluidic method to target and sort individual cells directly from complex microbiome samples and to prepare these cells for bulk whole-genome sequencing without cultivation. We characterize this approach by recovering bacteria spiked into human stool samples at a ratio as low as 1:250 and by successfully enriching endogenous to the level required for de novo assembly of high-quality genomes. Although microbiome strains are increasingly demanded for biomedical applications, a vast majority of species and strains are uncultivated and without reference genomes. We address this shortcoming by encapsulating complex microbiome samples directly into microfluidic droplets and amplifying a target-specific genomic fragment using a custom molecular TaqMan probe. We separate those positive droplets by droplet sorting, selectively enriching single target strain cells. Finally, we present a protocol to purify the genomic DNA while specifically removing amplicons and cell debris for high-quality genome sequencing.

Biogeographical studies have traditionally focused on readily visible organisms, but recent technological advances are enabling analyses of the large-scale distribution of microscopic organisms, whose biogeographical patterns have long been debated. Here we assessed the global structure of plankton geography and its relation to the biological, chemical, and physical context of the ocean (the 'seascape') by analyzing metagenomes of plankton communities sampled across oceans during the Oceans expedition, in light of environmental data and ocean current transport. Using a consistent approach across organismal sizes that provides unprecedented resolution to measure changes in genomic composition between communities, we report a pan-ocean, size-dependent plankton biogeography overlying regional heterogeneity. We found robust evidence for a basin-scale impact of transport by ocean currents on plankton biogeography, and on a characteristic timescale of community dynamics going beyond simple seasonality or life history transitions of plankton.

Taxonomic profiling is a fundamental task in microbiome research that aims to detect and quantify the relative abundance of microorganisms in biological samples. Available methods using shotgun metagenomic data generally depend on the deposition of sequenced and taxonomically annotated genomes, usually from cultures of isolated strains, in reference databases (reference genomes). However, the majority of microorganisms have not been cultured yet. Thus, a substantial fraction of microbial community members remains unaccounted for during taxonomic profiling, particularly in samples from underexplored environments. To address this issue, we developed the mOTU profiler, a tool that enables reference genome-independent species-level profiling of metagenomes. As such, it supports the identification and quantification of both "known" and "unknown" species based on a set of select marker genes.

Microorganisms play a role in the progression of various cancers. In this issue of Cancer Cell, Ghaddar et al. traced bacteria in pancreatic tumors at single-cell resolution and associated their intracellular presence with cell-type-specific transcriptional shifts, with links to clinical prognosis.

Fecal microbiota transplantation (FMT) is a therapeutic intervention for inflammatory diseases of the gastrointestinal tract, but its clinical mode of action and subsequent microbiome dynamics remain poorly understood. Here we analyzed metagenomes from 316 FMTs, sampled pre and post intervention, for the treatment of ten different disease indications. We quantified strain-level dynamics of 1,089 microbial species, complemented by 47,548 newly constructed metagenome-assembled genomes. Donor strain colonization and recipient strain resilience were mostly independent of clinical outcomes, but accurately predictable using LASSO-regularized regression models that accounted for host, microbiome and procedural variables. Recipient factors and donor-recipient complementarity, encompassing entire microbial communities to individual strains, were the main determinants of strain population dynamics, providing insights into the underlying processes that shape the post-FMT gut microbiome. Applying an ecology-based framework to our findings indicated parameters that may inform the development of more effective, targeted microbiome therapies in the future, and suggested how patient stratification can be used to enhance donor microbiota colonization or the displacement of recipient microbes in clinical practice.

Taxonomic analysis of microbial communities is well supported at the level of species and strains. However, species can contain significant phenotypic diversity and strains are rarely widely shared across global populations. Stratifying the diversity between species and strains can identify "subspecies", which are a useful intermediary. High-throughput identification and profiling of subspecies is not yet supported in the microbiome field. Here, we use an operational definition of subspecies based on SNV patterns within species to identify and profile subspecies in metagenomes, along with their distinctive SNVs and genes. We incorporate this method into metaSNV v2, which extends existing SNV-calling software to support further SNV interpretation for population genetics. These new features support microbiome analyses to link SNV profiles with host phenotype or environment and niche-specificity. We demonstrate subspecies identification in marine and faecal metagenomes. In the latter, we analyse 70 species in 7,524 adult and infant subjects, supporting a common subspecies population structure in the human gut microbiome and illustrating some limits in subspecies calling.

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here we use advances in cryo-electron tomography and sub-tomogram analysis to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.

Whereas DNA viruses are known to be abundant, diverse, and commonly key ecosystem players, RNA viruses are insufficiently studied outside disease settings. In this study, we analyzed ≈28 terabases of Global Ocean RNA sequences to expand Earth's RNA virus catalogs and their taxonomy, investigate their evolutionary origins, and assess their marine biogeography from pole to pole. Using new approaches to optimize discovery and classification, we identified RNA viruses that necessitate substantive revisions of taxonomy (doubling phyla and adding >50% new classes) and evolutionary understanding. "Species"-rank abundance determination revealed that viruses of the new phyla "," a missing link in early RNA virus evolution, and "" are widespread and dominant in the oceans. These efforts provide foundational knowledge critical to integrating RNA viruses into ecological and epidemiological models.

The deep sea, the largest ocean's compartment, drives planetary-scale biogeochemical cycling. Yet, the functional exploration of its microbial communities lags far behind other environments. Here we analyze 58 metagenomes from tropical and subtropical deep oceans to generate the Malaspina Gene Database. Free-living or particle-attached lifestyles drive functional differences in bathypelagic prokaryotic communities, regardless of their biogeography. Ammonia and CO oxidation pathways are enriched in the free-living microbial communities and dissimilatory nitrate reduction to ammonium and H oxidation pathways in the particle-attached, while the Calvin Benson-Bassham cycle is the most prevalent inorganic carbon fixation pathway in both size fractions. Reconstruction of the Malaspina Deep Metagenome-Assembled Genomes reveals unique non-cyanobacterial diazotrophic bacteria and chemolithoautotrophic prokaryotes. The widespread potential to grow both autotrophically and heterotrophically suggests that mixotrophy is an ecologically relevant trait in the deep ocean. These results expand our understanding of the functional microbial structure and metabolic capabilities of the largest Earth aquatic ecosystem.

The SARS-CoV-2 pandemic is currently leading to increasing numbers of COVID-19 patients all over the world. Clinical presentations range from asymptomatic, mild respiratory tract infection, to severe cases with acute respiratory distress syndrome, respiratory failure, and death. Reports on a dysregulated immune system in the severe cases call for a better characterization and understanding of the changes in the immune system.

Bacteria and fungi are of uttermost importance in determining environmental and host functioning. Despite close interactions between animals, plants, their associated microbiomes, and the environment they inhabit, the distribution and role of bacteria and especially fungi across host and environments as well as the cross-habitat determinants of their community compositions remain little investigated. Using a uniquely broad global dataset of 13 483 metagenomes, we analysed the microbiome structure and function of 25 host-associated and environmental habitats, focusing on potential interactions between bacteria and fungi. We found that the metagenomic relative abundance ratio of bacteria-to-fungi is a distinctive microbial feature of habitats. Compared with fungi, the cross-habitat distribution pattern of bacteria was more strongly driven by habitat type. Fungal diversity was depleted in host-associated communities compared with those in the environment, particularly terrestrial habitats, whereas this diversity pattern was less pronounced for bacteria. The relative gene functional potential of bacteria or fungi reflected their diversity patterns and appeared to depend on a balance between substrate availability and biotic interactions. Alongside helping to identify hotspots and sources of microbial diversity, our study provides support for differences in assembly patterns and processes between bacterial and fungal communities across different habitats.

Protein intake has been associated with the development of pre-diabetes (pre-T2D) and type 2 diabetes (T2D). The gut microbiota has the capacity to produce harmful metabolites derived from dietary protein. Furthermore, both the gut microbiota composition and metabolic status (e.g., insulin resistance) can be modulated by diet and ethnicity. However, to date most studies have predominantly focused on carbohydrate and fiber intake with regards to metabolic status and gut microbiota composition.

The InterPro database (https://www.ebi.ac.uk/interpro/) provides an integrative classification of protein sequences into families, and identifies functionally important domains and conserved sites. InterProScan is the underlying software that allows protein and nucleic acid sequences to be searched against InterPro's signatures. Signatures are predictive models which describe protein families, domains or sites, and are provided by multiple databases. InterPro combines signatures representing equivalent families, domains or sites, and provides additional information such as descriptions, literature references and Gene Ontology (GO) terms, to produce a comprehensive resource for protein classification. Founded in 1999, InterPro has become one of the most widely used resources for protein family annotation. Here, we report the status of InterPro (version 81.0) in its 20th year of operation, and its associated software, including updates to database content, the release of a new website and REST API, and performance improvements in InterProScan.

Ocean plankton comprise organisms from viruses to fish larvae that are fundamental to ecosystem functioning and the provision of marine services such as fisheries and CO sequestration. The latter services are partly governed by variations in plankton community composition and the expression of traits such as body size at community-level. While community assembly has been thoroughly studied for the smaller end of the plankton size spectrum, the larger end comprises ectotherms that are often studied at the species, or group-level, rather than as communities. The body size of marine ectotherms decreases with temperature, but controls on community-level traits remain elusive, hindering the predictability of marine services provision. Here, we leverage Tara Oceans datasets to determine how zooplankton community composition and size structure varies with latitude, temperature and productivity-related covariates in the global surface ocean. Zooplankton abundance and median size decreased towards warmer and less productive environments, as a result of changes in copepod composition. However, some clades displayed the opposite relationships, which may be ascribed to alternative feeding strategies. Given that climate models predict increasingly warmed and stratified oceans, our findings suggest that zooplankton communities will shift towards smaller organisms which might weaken their contribution to the biological carbon pump.

Gut microbial products are involved in regulation of host metabolism. In human and experimental studies, we explored the potential role of hippurate, a hepatic phase 2 conjugation product of microbial benzoate, as a marker and mediator of metabolic health.

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: (i) de novo gene prediction from raw contigs, (ii) built-in pairwise orthology prediction, (iii) fast protein domain discovery, and (iv) automated GFF decoration. eggNOG-mapper v2 is available as a standalone tool or as an online service at http://eggnog-mapper.embl.de.

During the transition from a healthy state to cardiometabolic disease, patients become heavily medicated, which leads to an increasingly aberrant gut microbiome and serum metabolome, and complicates biomarker discovery. Here, through integrated multi-omics analyses of 2,173 European residents from the MetaCardis cohort, we show that the explanatory power of drugs for the variability in both host and gut microbiome features exceeds that of disease. We quantify inferred effects of single medications, their combinations as well as additive effects, and show that the latter shift the metabolome and microbiome towards a healthier state, exemplified in synergistic reduction in serum atherogenic lipoproteins by statins combined with aspirin, or enrichment of intestinal Roseburia by diuretic agents combined with beta-blockers. Several antibiotics exhibit a quantitative relationship between the number of courses prescribed and progression towards a microbiome state that is associated with the severity of cardiometabolic disease. We also report a relationship between cardiometabolic drug dosage, improvement in clinical markers and microbiome composition, supporting direct drug effects. Taken together, our computational framework and resulting resources enable the disentanglement of the effects of drugs and disease on host and microbiome features in multimedicated individuals. Furthermore, the robust signatures identified using our framework provide new hypotheses for drug-host-microbiome interactions in cardiometabolic disease.

DNA mismatch repair deficiency drives microsatellite instability (MSI). Cells with MSI accumulate numerous frameshift mutations. Frameshift mutations affecting cancer-related genes may promote tumorigenesis and, therefore, are shared among independently arising MSI tumors. Consequently, such recurrent frameshift mutations can give rise to shared immunogenic frameshift peptides (FSPs) that represent ideal candidates for a vaccine against MSI cancer. Pathogenic germline variants of mismatch repair genes cause Lynch syndrome (LS), a hereditary cancer syndrome affecting approximately 20-25 million individuals worldwide. Individuals with LS are at high risk of developing MSI cancer. Previously, we demonstrated safety and immunogenicity of an FSP-based vaccine in a phase I/IIa clinical trial in patients with a history of MSI colorectal cancer. However, the cancer-preventive effect of FSP vaccination in the scenario of LS has not yet been demonstrated.

OGEE is an Online GEne Essentiality database. Gene essentiality is not a static and binary property, rather a context-dependent and evolvable property in all forms of life. In OGEE we collect not only experimentally tested essential and non-essential genes, but also associated gene properties that contributes to gene essentiality. We tagged conditionally essential genes that show variable essentiality statuses across datasets to highlight complex interplays between gene functions and environmental/experimental perturbations. OGEE v3 contains gene essentiality datasets for 91 species; almost doubled from 48 species in previous version. To accommodate recent advances on human cancer essential genes (as known as tumor dependency genes) that could serve as targets for cancer treatment and/or drug development, we expanded the collection of human essential genes from 16 cell lines in previous to 581. These human cancer cell lines were tested with high-throughput experiments such as CRISPR-Cas9 and RNAi; in total, 150 of which were tested by both techniques. We also included factors known to contribute to gene essentiality for these cell lines, such as genomic mutation, methylation and gene expression, along with extensive graphical visualizations for ease of understanding of these factors. OGEE v3 can be accessible freely at https://v3.ogee.info.

Human gut bacterial strains can co-exist with their hosts for decades, but little is known about how these microbes persist and disperse, and evolve thereby. Here, we examined these processes in 5,278 adult and infant fecal metagenomes, longitudinally sampled in individuals and families. Our analyses revealed that a subset of gut species is extremely persistent in individuals, families, and geographic regions, represented often by locally successful strains of the phylum Bacteroidota. These "tenacious" bacteria show high levels of genetic adaptation to the human host but a high probability of loss upon antibiotic interventions. By contrast, heredipersistent bacteria, notably Firmicutes, often rely on dispersal strategies with weak phylogeographic patterns but strong family transmissions, likely related to sporulation. These analyses describe how different dispersal strategies can lead to the long-term persistence of human gut microbes with implications for gut flora modulations.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.

The Interactive Tree Of Life (https://itol.embl.de) is an online tool for the display, manipulation and annotation of phylogenetic and other trees. It is freely available and open to everyone. iTOL version 5 introduces a completely new tree display engine, together with numerous new features. For example, a new dataset type has been added (MEME motifs), while annotation options have been expanded for several existing ones. Node metadata display options have been extended and now also support non-numerical categorical values, as well as multiple values per node. Direct manual annotation is now available, providing a set of basic drawing and labeling tools, allowing users to draw shapes, labels and other features by hand directly onto the trees. Support for tree and dataset scales has been extended, providing fine control over line and label styles. Unrooted tree displays can now use the equal-daylight algorithm, proving a much greater display clarity. The user account system has been streamlined and expanded with new navigation options and currently handles >1 million trees from >70 000 individual users.

SMART (Simple Modular Architecture Research Tool) is a web resource (https://smart.embl.de) for the identification and annotation of protein domains and the analysis of protein domain architectures. SMART version 9 contains manually curated models for more than 1300 protein domains, with a topical set of 68 new models added since our last update article (1). All the new models are for diverse recombinase families and subfamilies and as a set they provide a comprehensive overview of mobile element recombinases namely transposase, integrase, relaxase, resolvase, cas1 casposase and Xer like cellular recombinase. Further updates include the synchronization of the underlying protein databases with UniProt (2), Ensembl (3) and STRING (4), greatly increasing the total number of annotated domains and other protein features available in architecture analysis mode. Furthermore, SMART's vector-based protein display engine has been extended and updated to use the latest web technologies and the domain architecture analysis components have been optimized to handle the increased number of protein features available.

Protein-metabolite interactions play an important role in the cell's metabolism and many methods have been developed to screen them in vitro. However, few methods can be applied at a large scale and not alter biological state. Here we describe a proteometabolomic approach, using chromatography to generate cell fractions which are then analyzed with mass spectrometry for both protein and metabolite identification. Integrating the proteomic and metabolomic analyses makes it possible to identify protein-bound metabolites. Applying the concept to the thermophilic fungus Chaetomium thermophilum, we predict 461 likely protein-metabolite interactions, most of them novel. As a proof of principle, we experimentally validate a predicted interaction between the ribosome and isopentenyl adenine.

Resource competition and metabolic cross-feeding are among the main drivers of microbial community assembly. Yet the degree to which these two conflicting forces are reflected in the composition of natural communities has not been systematically investigated. Here, we use genome-scale metabolic modelling to assess the potential for resource competition and metabolic cooperation in large co-occurring groups (up to 40 members) across thousands of habitats. Our analysis reveals two distinct community types, which are clustered at opposite ends of a spectrum in a trade-off between competition and cooperation. At one end are highly cooperative communities, characterized by smaller genomes and multiple auxotrophies. At the other end are highly competitive communities, which feature larger genomes and overlapping nutritional requirements, and harbour more genes related to antimicrobial activity. The latter are mainly present in soils, whereas the former are found in both free-living and host-associated habitats. Community-scale flux simulations show that, whereas competitive communities can better resist species invasion but not nutrient shift, cooperative communities are susceptible to species invasion but resilient to nutrient change. We also show, by analysing an additional data set, that colonization by probiotic species is positively associated with the presence of cooperative species in the recipient microbiome. Together, our results highlight the bifurcation between competitive and cooperative metabolism in the assembly of natural communities and its implications for community modulation.

Antibiotics are used to fight pathogens but also target commensal bacteria, disturbing the composition of gut microbiota and causing dysbiosis and disease. Despite this well-known collateral damage, the activity spectrum of different antibiotic classes on gut bacteria remains poorly characterized. Here we characterize further 144 antibiotics from a previous screen of more than 1,000 drugs on 38 representative human gut microbiome species. Antibiotic classes exhibited distinct inhibition spectra, including generation dependence for quinolones and phylogeny independence for β-lactams. Macrolides and tetracyclines, both prototypic bacteriostatic protein synthesis inhibitors, inhibited nearly all commensals tested but also killed several species. Killed bacteria were more readily eliminated from in vitro communities than those inhibited. This species-specific killing activity challenges the long-standing distinction between bactericidal and bacteriostatic antibiotic classes and provides a possible explanation for the strong effect of macrolides on animal and human gut microbiomes. To mitigate this collateral damage of macrolides and tetracyclines, we screened for drugs that specifically antagonized the antibiotic activity against abundant Bacteroides species but not against relevant pathogens. Such antidotes selectively protected Bacteroides species from erythromycin treatment in human-stool-derived communities and gnotobiotic mice. These findings illluminate the activity spectra of antibiotics in commensal bacteria and suggest strategies to circumvent their adverse effects on the gut microbiota.

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 oxide–sensitive 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.

Genomes are critical units in microbiology, yet ascertaining quality in prokaryotic genome assemblies remains a formidable challenge. We present GUNC (the Genome UNClutterer), a tool that accurately detects and quantifies genome chimerism based on the lineage homogeneity of individual contigs using a genome's full complement of genes. GUNC complements existing approaches by targeting previously underdetected types of contamination: we conservatively estimate that 5.7% of genomes in GenBank, 5.2% in RefSeq, and 15-30% of pre-filtered "high-quality" metagenome-assembled genomes in recent studies are undetected chimeras. GUNC provides a fast and robust tool to substantially improve prokaryotic genome quality.

Alcohol is the most common cause of liver-related mortality and morbidity. We therefore aimed to assess and compare the prognostic performance of elastography and blood-based markers to predict time to the first liver-related event, severe infection, and all-cause mortality in patients with a history of excess drinking.

The role of the Arctic Ocean ecosystem in climate regulation may depend on the responses of marine microorganisms to environmental change. We applied genome-resolved metagenomics to 41 Arctic seawater samples, collected at various depths in different seasons during the Tara Oceans Polar Circle expedition, to evaluate the ecology, metabolic potential and activity of resident bacteria and archaea. We assembled 530 metagenome-assembled genomes (MAGs) to form the Arctic MAGs catalogue comprising 526 species. A total of 441 MAGs belonged to species that have not previously been reported and 299 genomes showed an exclusively polar distribution. Most Arctic MAGs have large genomes and the potential for fast generation times, both of which may enable adaptation to a copiotrophic lifestyle in nutrient-rich waters. We identified 38 habitat generalists and 111 specialists in the Arctic Ocean. We also found a general prevalence of 14 mixotrophs, while chemolithoautotrophs were mostly present in the mesopelagic layer during spring and autumn. We revealed 62 MAGs classified as key Arctic species, found only in the Arctic Ocean, showing the highest gene expression values and predicted to have habitat-specific traits. The Artic MAGs catalogue will inform our understanding of polar microorganisms that drive global biogeochemical cycles.

The SARS-CoV-2 virus is the causative agent of the global COVID-19 infectious disease outbreak, which can lead to acute respiratory distress syndrome (ARDS). However, it is still unclear how the virus interferes with immune cell and metabolic functions in the human body. In this study, we investigated the immune response in acute or convalescent COVID-19 patients. We characterized the peripheral blood mononuclear cells (PBMCs) using flow cytometry and found that CD8 T cells were significantly subsided in moderate COVID-19 and convalescent patients. Furthermore, characterization of CD8 T cells suggested that convalescent patients have significantly diminished expression of both perforin and granzyme A. Using H-NMR spectroscopy, we characterized the metabolic status of their autologous PBMCs. We found that fructose, lactate and taurine levels were elevated in infected (mild and moderate) patients compared with control and convalescent patients. Glucose, glutamate, formate and acetate levels were attenuated in COVID-19 (mild and moderate) patients. In summary, our report suggests that SARS-CoV-2 infection leads to disrupted CD8 T cytotoxic functions and changes the overall metabolic functions of immune cells.

Cellular life depends on a complex web of functional associations between biomolecules. Among these associations, protein-protein interactions are particularly important due to their versatility, specificity and adaptability. The STRING database aims to integrate all known and predicted associations between proteins, including both physical interactions as well as functional associations. To achieve this, STRING collects and scores evidence from a number of sources: (i) automated text mining of the scientific literature, (ii) databases of interaction experiments and annotated complexes/pathways, (iii) computational interaction predictions from co-expression and from conserved genomic context and (iv) systematic transfers of interaction evidence from one organism to another. STRING aims for wide coverage; the upcoming version 11.5 of the resource will contain more than 14 000 organisms. In this update paper, we describe changes to the text-mining system, a new scoring-mode for physical interactions, as well as extensive user interface features for customizing, extending and sharing protein networks. In addition, we describe how to query STRING with genome-wide, experimental data, including the automated detection of enriched functionalities and potential biases in the user's query data. The STRING resource is available online, at https://string-db.org/.

Liver fibrosis accumulation is considered a turnover disease, with formation exceeding degradation, although this hypothesis has never been tested in humans.

The human gut microbiome has emerged as a major player in human health and disease. The liver, as the first organ to encounter microbial products that cross the gut epithelial barrier, is affected by the gut microbiome in many ways. Thus, the gut microbiome might play a major part in the development of liver diseases. The common end stage of liver disease is decompensated cirrhosis and the further development towards acute-on-chronic liver failure (ACLF). These conditions have high short-term mortality. There is evidence that translocation of components of the gut microbiota, facilitated by different pathogenic mechanisms such as increased gut epithelial permeability and portal hypertension, is an important driver of decompensation by induction of systemic inflammation, and thereby also ACLF. Elucidating the role of the gut microbiome in the aetiology of decompensated cirrhosis and ACLF deserves further investigation and improvement; and might be the basis for development of diagnostic and therapeutic strategies. In this Review, we focus on the possible pathogenic, diagnostic and therapeutic role of the gut microbiome in decompensation of cirrhosis and progression to ACLF.

Gut viruses are important, yet often neglected, players in the complex human gut microbial ecosystem. Recently, the number of human gut virome studies has been increasing; however, we are still only scratching the surface of the immense viral diversity. In this study, 254 virus-enriched fecal metagenomes from 204 Danish subjects were used to generate the anish nteric irme atalog (DEVoC) containing 12,986 nonredundant viral scaffolds, of which the majority was previously undescribed, encoding 190,029 viral genes. The DEVoC was used to compare 91 healthy DEVoC gut viromes from children, adolescents, and adults that were used to create the DEVoC. Gut viromes of healthy Danish subjects were dominated by phages. While most phage genomes (PGs) only occurred in a single subject, indicating large virome individuality, 39 PGs were present in more than 10 healthy subjects. Among these 39 PGs, the prevalences of three PGs were associated with age. To further study the prevalence of these 39 prevalent PGs, 1,880 gut virome data sets of 27 studies from across the world were screened, revealing several age-, geography-, and disease-related prevalence patterns. Two PGs also showed a remarkably high prevalence worldwide-a crAss-like phage (20.6% prevalence), belonging to the tentative subfamily, and a previously undescribed circular temperate phage infecting Bacteroides dorei (14.4% prevalence), called LoVEphage because it encodes ots f iral lements. Due to the LoVEphage's high prevalence and novelty, public data sets in which the LoVEphage was detected were assembled, resulting in an additional 18 circular LoVEphage-like genomes (67.9 to 72.4 kb). Through generation of the DEVoC, we added numerous previously uncharacterized viral genomes and genes to the ever-increasing worldwide pool of human gut viromes. The DEVoC, the largest human gut virome catalog generated from consistently processed fecal samples, facilitated the analysis of the 91 healthy Danish gut viromes. Characterizing the biggest cohort of healthy gut viromes from children, adolescents, and adults to date confirmed the previously established high interindividual variation in human gut viromes and demonstrated that the effect of age on the gut virome composition was limited to the prevalence of specific phage (groups). The identification of a previously undescribed prevalent phage illustrates the usefulness of developing virome catalogs, and we foresee that the DEVoC will benefit future analysis of the roles of gut viruses in human health and disease.

Tracing of SARS-CoV-2 transmission chains is still a major challenge for public health authorities, when incidental contacts are not recalled or are not perceived as potential risk contacts. Viral sequencing can address key questions about SARS-CoV-2 evolution and may support reconstruction of viral transmission networks by integration of molecular epidemiology into classical contact tracing.

Fast and reliable detection of patients with severe and heterogeneous illnesses is a major goal of precision medicine. Patients with leukaemia can be identified using machine learning on the basis of their blood transcriptomes. However, there is an increasing divide between what is technically possible and what is allowed, because of privacy legislation. Here, to facilitate the integration of any medical data from any data owner worldwide without violating privacy laws, we introduce Swarm Learning-a decentralized machine-learning approach that unites edge computing, blockchain-based peer-to-peer networking and coordination while maintaining confidentiality without the need for a central coordinator, thereby going beyond federated learning. To illustrate the feasibility of using Swarm Learning to develop disease classifiers using distributed data, we chose four use cases of heterogeneous diseases (COVID-19, tuberculosis, leukaemia and lung pathologies). With more than 16,400 blood transcriptomes derived from 127 clinical studies with non-uniform distributions of cases and controls and substantial study biases, as well as more than 95,000 chest X-ray images, we show that Swarm Learning classifiers outperform those developed at individual sites. In addition, Swarm Learning completely fulfils local confidentiality regulations by design. We believe that this approach will notably accelerate the introduction of precision medicine.

The human microbiome is increasingly mined for diagnostic and therapeutic biomarkers using machine learning (ML). However, metagenomics-specific software is scarce, and overoptimistic evaluation and limited cross-study generalization are prevailing issues. To address these, we developed SIAMCAT, a versatile R toolbox for ML-based comparative metagenomics. We demonstrate its capabilities in a meta-analysis of fecal metagenomic studies (10,803 samples). When naively transferred across studies, ML models lost accuracy and disease specificity, which could however be resolved by a novel training set augmentation strategy. This reveals some biomarkers to be disease-specific, with others shared across multiple conditions. SIAMCAT is freely available from siamcat.embl.de .

Plant nutrient acquisition strategies drive soil processes and vegetation performance, but their effect on the soil microbiome remains poorly understood. This knowledge is important to predict the shifts in microbial diversity and functions to increasing changes in vegetation traits under global change. Here we documented the topsoil microbiomes of 145 boreal and temperate terrestrial sites in the Baltic region that broadly differed in vegetation type and nutritional traits, such as mycorrhizal types and symbiotic nitrogen-fixation. We found that sites dominated by arbuscular mycorrhizal (AM) vegetation harbor relatively more AM fungi, bacteria, fungal saprotrophs, and pathogens in the topsoil compared with sites dominated by ectomycorrhizal (EM) plants. These differences in microbiome composition reflect the rapid nutrient cycling and negative plant-soil feedback in AM soils. Lower fungal diversity and bacteria-to-fungi ratios in EM-dominated habitats are driven by monodominance of woody vegetation as well as soil acidification by EM fungi, which are associated with greater diversity and relative abundance of carbohydrate-active enzymes. Our study suggests that shifts in vegetation related to global change and land use may strongly alter the topsoil microbiome structure and function.

Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is needed for understanding skewed immune responses and defining predictors of outcome. Here, we performed a longitudinal multi-omics study using a two-center cohort of 14 patients. We analyzed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients. Severe COVID-19 was characterized by an increase of proliferating, metabolically hyperactive plasmablasts. Coinciding with critical illness, we also identified an expansion of interferon-activated circulating megakaryocytes and increased erythropoiesis with features of hypoxic signaling. Megakaryocyte- and erythroid-cell-derived co-expression modules were predictive of fatal disease outcome. The study demonstrates broad cellular effects of SARS-CoV-2 infection beyond adaptive immune cells and provides an entry point toward developing biomarkers and targeted treatments of patients with COVID-19.

Untargeted mass spectrometry is a powerful method for detecting metabolites in biological samples. However, fast and accurate identification of the metabolites' structures from MS/MS spectra is still a great challenge.

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.

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/.

DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells.

Microbiota-host-diet interactions contribute to the development of metabolic diseases. Imidazole propionate is a novel microbially produced metabolite from histidine, which impairs glucose metabolism. Here, we show that subjects with prediabetes and diabetes in the MetaCardis cohort from three European countries have elevated serum imidazole propionate levels. Furthermore, imidazole propionate levels were increased in subjects with low bacterial gene richness and Bacteroides 2 enterotype, which have previously been associated with obesity. The Bacteroides 2 enterotype was also associated with increased abundance of the genes involved in imidazole propionate biosynthesis from dietary histidine. Since patients and controls did not differ in their histidine dietary intake, the elevated levels of imidazole propionate in type 2 diabetes likely reflects altered microbial metabolism of histidine, rather than histidine intake per se. Thus the microbiota may contribute to type 2 diabetes by generating imidazole propionate that can modulate host inflammation and metabolism.

Inspired by recent proteomic data demonstrating the upregulation of carbon and glycogen metabolism in aging human hematopoietic stem and progenitor cells (HPCs, CD34+ cells), this report addresses whether this is caused by elevated glycolysis of the HPCs on a per cell basis, or by a subpopulation that has become more glycolytic. The average glycogen content in individual CD34+ cells from older subjects (> 50 years) was 3.5 times higher and more heterogeneous compared to younger subjects (< 35 years). Representative glycolytic enzyme activities in HPCs confirmed a significant increase in glycolysis in older subjects. The HPCs from older subjects can be fractionated into three distinct subsets with high, intermediate, and low glucose uptake (GU) capacity, while the subset with a high GU capacity could scarcely be detected in younger subjects. Thus, we conclude that upregulated glycolysis in aging HPCs is caused by the expansion of a more glycolytic HPC subset. Since single-cell RNA analysis has also demonstrated that this subpopulation is linked to myeloid differentiation and increased proliferation, isolation and mechanistic characterization of this subpopulation can be utilized to elucidate specific targets for therapeutic interventions to restore the lineage balance of aging HPCs.

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRCD11c inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.

A planetary-scale understanding of the ocean ecosystem, particularly in light of climate change, is crucial. Here, we review the work of Tara Oceans, an international, multidisciplinary project to assess the complexity of ocean life across comprehensive taxonomic and spatial scales. Using a modified sailing boat, the team sampled plankton at 210 globally distributed sites at depths down to 1,000 m. We describe publicly available resources of molecular, morphological and environmental data, and discuss how an ecosystems biology approach has expanded our understanding of plankton diversity and ecology in the ocean as a planetary, interconnected ecosystem. These efforts illustrate how global-scale concepts and data can help to integrate biological complexity into models and serve as a baseline for assessing ecosystem changes and the future habitability of our planet in the Anthropocene epoch.

Studying within-species variation has traditionally been limited to culturable bacterial isolates and low-resolution microbial community fingerprinting. Metagenomic sequencing and technical advances have enabled culture-free, high-resolution strain and subspecies analyses at high throughput and in complex environments. This holds great scientific promise but has also led to an overwhelming number of methods and terms to describe infraspecific variation. This Review aims to clarify these advances by focusing on the diversity within bacterial and archaeal species in the context of microbiomics. We cover foundational microevolutionary concepts relevant to population genetics and summarize how within-species variation can be studied and stratified directly within microbial communities with a focus on metagenomics. Finally, we describe how common applications of within-species variation can be achieved using metagenomic data. We aim to guide the selection of appropriate terms and analytical approaches to facilitate researchers in benefiting from the increasing availability of large, high-resolution microbiome genetic sequencing data.

Microbiome community typing analyses have recently identified the Bacteroides2 (Bact2) enterotype, an intestinal microbiota configuration that is associated with systemic inflammation and has a high prevalence in loose stools in humans. Bact2 is characterized by a high proportion of Bacteroides, a low proportion of Faecalibacterium and low microbial cell densities, and its prevalence varies from 13% in a general population cohort to as high as 78% in patients with inflammatory bowel disease. Reported changes in stool consistency and inflammation status during the progression towards obesity and metabolic comorbidities led us to propose that these developments might similarly correlate with an increased prevalence of the potentially dysbiotic Bact2 enterotype. Here, by exploring obesity-associated microbiota alterations in the quantitative faecal metagenomes of the cross-sectional MetaCardis Body Mass Index Spectrum cohort (n = 888), we identify statin therapy as a key covariate of microbiome diversification. By focusing on a subcohort of participants that are not medicated with statins, we find that the prevalence of Bact2 correlates with body mass index, increasing from 3.90% in lean or overweight participants to 17.73% in obese participants. Systemic inflammation levels in Bact2-enterotyped individuals are higher than predicted on the basis of their obesity status, indicative of Bact2 as a dysbiotic microbiome constellation. We also observe that obesity-associated microbiota dysbiosis is negatively associated with statin treatment, resulting in a lower Bact2 prevalence of 5.88% in statin-medicated obese participants. This finding is validated in both the accompanying MetaCardis cardiovascular disease dataset (n = 282) and the independent Flemish Gut Flora Project population cohort (n = 2,345). The potential benefits of statins in this context will require further evaluation in a prospective clinical trial to ascertain whether the effect is reproducible in a randomized population and before considering their application as microbiota-modulating therapeutics.

Alcohol-related liver disease is often undetected until irreversible late-stage decompensated disease manifests. Consequently, there is an unmet need for effective and economically reasonable pathways to screen for advanced alcohol-related fibrosis. We used real-world data from a large biopsy-controlled study of excessive drinkers recruited from primary and secondary care, to evaluate the cost-effectiveness of four primary care initiated strategies: 1) routine liver function tests with follow-up ultrasonography for test-positives, 2) the enhanced liver fibrosis (ELF) test, with hospital liver stiffness measurement (LSM) for positives, 3) three-tier strategy using Forns Index to control before strategy two, 4) direct referral of all to LSM. We used linked decision trees and Markov models to evaluate outcomes short-term (cost-per-accurate-diagnosis) and long-term (quality-adjusted life-years, QALYs). For low-prevalence populations, ELF with LSM follow-up was most cost-effective, both short-term (accuracy 96%, 196$ per patient), and long-term incremental cost-effectiveness ratio (ICER) $5,387-8,430/QALY, depending on whether diagnostic testing had lasting or temporary effects on abstinence rates. Adding Forns Index decreased costs to $72 per patient and accuracy to 95%. The strategy resulted in less QALY's due to more false negatives, but an ICER of $3,012, making this strategy suited for areas with restricted access to ELF and TE, or lower willingness-to-pay. For high-prevalence populations, direct referral to LSM was highly cost-effective (accuracy 93%, $297 per patient), with ICERs between $490 and $1,037/QALY CONCLUSION: Non-invasive screening for advanced, alcohol-related fibrosis is a cost-effective intervention when different referral pathways are used according to the prevalence of advanced fibrosis. Patients in the primary healthcare sector should be tested with the ELF test followed by LSM if test-positive, whereas direct referral to LSM is highly cost-effective in high-prevalence cohorts.

High-throughput studies of microbial communities suggest that Archaea are a widespread component of microbial diversity in various ecosystems. However, proper quantification of archaeal diversity and community ecology remains limited, as sequence coverage of Archaea is usually low due to the inability of available prokaryotic primers to efficiently amplify archaeal compared to bacterial rRNA genes. To improve identification and quantification of Archaea, we designed and validated the utility of several primer pairs to efficiently amplify archaeal 16S rRNA genes based on up-to-date reference genes. We demonstrate that several of these primer pairs amplify phylogenetically diverse Archaea with high sequencing coverage, outperforming commonly used primers. Based on comparing the resulting long 16S rRNA gene fragments with public databases from all habitats, we found several novel family- to phylum-level archaeal taxa from topsoil and surface water. Our results suggest that archaeal diversity has been largely overlooked due to the limitations of available primers, and that improved primer pairs enable to estimate archaeal diversity more accurately. This article is protected by copyright. All rights reserved.

The vast majority of Parkinson's disease (PD) cases are of sporadic origin and despite extensive research in recent years, the etiology still remains unclear. Several current case control studies are aiming to characterize a putative PD-specific composition of the gut microbiome, reflecting the potential relevance of microbiota in the pathogenesis of PD. Although methodologies and cohort sizes differed, the currently available studies showed reproducible or consistent results in terms of PD-specific alterations to the intestinal bacteria. By applying metagenomic sequencing procedures, it is even possible to distinguish PD cases from healthy individuals at a very early disease stage by means of individually modified microbiota. Among others, microbiota that are associated with an altered intestinal barrier or immune function, such as Akkermansia, Lactobacillus, Faecalibacterium and Prevotella were significantly over-represented or under-represented. There may even be a prodromal microbiome, as a comparable microbial shift is also found in patients with rapid eye movement (REM) sleep behavior disorder (RBD), a risk factor for the later development of synucleinopathies, such as PD.

Shotgun metagenomes contain a sample of all the genomic material in an environment, allowing for the characterization of a microbial community. In order to understand these communities, bioinformatics methods are crucial. A common first step in processing metagenomes is to compute abundance estimates of different taxonomic or functional groups from the raw sequencing data. Given the breadth of the field, computational solutions need to be flexible and extensible, enabling the combination of different tools into a larger pipeline.

Some species responded successfully to prehistoric changes in climate [1, 2], while others failed to adapt and became extinct [3]. The factors that determine successful climate adaptation remain poorly understood. We constructed a reference genome and studied physiological adaptations in the Alpine marmot (Marmota marmota), a large ground-dwelling squirrel exquisitely adapted to the "ice-age" climate of the Pleistocene steppe [4, 5]. Since the disappearance of this habitat, the rodent persists in large numbers in the high-altitude Alpine meadow [6, 7]. Genome and metabolome showed evidence of adaptation consistent with cold climate, affecting white adipose tissue. Conversely, however, we found that the Alpine marmot has levels of genetic variation that are among the lowest for mammals, such that deleterious mutations are less effectively purged. Our data rule out typical explanations for low diversity, such as high levels of consanguineous mating, or a very recent bottleneck. Instead, ancient demographic reconstruction revealed that genetic diversity was lost during the climate shifts of the Pleistocene and has not recovered, despite the current high population size. We attribute this slow recovery to the marmot's adaptive life history. The case of the Alpine marmot reveals a complicated relationship between climatic changes, genetic diversity, and conservation status. It shows that species of extremely low genetic diversity can be very successful and persist over thousands of years, but also that climate-adapted life history can trap a species in a persistent state of low genetic diversity.

Microbes drive most ecosystems and are modulated by viruses that impact their lifespan, gene flow, and metabolic outputs. However, ecosystem-level impacts of viral community diversity remain difficult to assess due to classification issues and few reference genomes. Here, we establish an ∼12-fold expanded global ocean DNA virome dataset of 195,728 viral populations, now including the Arctic Ocean, and validate that these populations form discrete genotypic clusters. Meta-community analyses revealed five ecological zones throughout the global ocean, including two distinct Arctic regions. Across the zones, local and global patterns and drivers in viral community diversity were established for both macrodiversity (inter-population diversity) and microdiversity (intra-population genetic variation). These patterns sometimes, but not always, paralleled those from macro-organisms and revealed temperate and tropical surface waters and the Arctic as biodiversity hotspots and mechanistic hypotheses to explain them. Such further understanding of ocean viruses is critical for broader inclusion in ecosystem models.

Here we describe a proteomic data resource for the NCI-60 cell lines generated by pressure cycling technology and SWATH mass spectrometry. We developed the DIA-expert software to curate and visualize the SWATH data, leading to reproducible detection of over 3,100 SwissProt proteotypic proteins and systematic quantification of pathway activities. Stoichiometric relationships of interacting proteins for DNA replication, repair, the chromatin remodeling NuRD complex, β-catenin, RNA metabolism, and prefoldins are more evident than that at the mRNA level. The data are available in CellMiner (discover.nci.nih.gov/cellminercdb and discover.nci.nih.gov/cellminer), allowing casual users to test hypotheses and perform integrative, cross-database analyses of multi-omic drug response correlations for over 20,000 drugs. We demonstrate the value of proteome data in predicting drug response for over 240 clinically relevant chemotherapeutic and targeted therapies. In summary, we present a novel proteome resource for the NCI-60, together with relevant software tools, and demonstrate the benefit of proteome analyses.

With the technological advances of the last decade, it is now feasible to analyze microbiome samples, such as human stool specimens, using multi-omic techniques. Given the inherent sample complexity, there exists a need for sample methods which preserve as much information as possible about the biological system at the time of sampling. Here, we analyzed human stool samples preserved and stored using different methods, applying metagenomics as well as metaproteomics. Our results demonstrate that sample preservation and storage have a significant effect on the taxonomic composition of identified proteins. The overall identification rates, as well as the proportion of proteins from were much higher when samples were flash frozen. Preservation in RNAlater overall led to fewer protein identifications and a considerable increase in the share of , as well as . Additionally, a decrease in the share of metabolism-related proteins and an increase of the relative amount of proteins involved in the processing of genetic information was observed for RNAlater-stored samples. This suggests that great care should be taken in choosing methods for the preservation and storage of microbiome samples, as well as in comparing the results of analyses using different sampling and storage methods. Flash freezing and subsequent storage at -80 °C should be chosen wherever possible.

The composition of the healthy human adult gut microbiome is relatively stable over prolonged periods, and representatives of the most highly abundant and prevalent species have been cultured and described. However, microbial abundances can change on perturbations, such as antibiotics intake, enabling the identification and characterisation of otherwise low abundant species.

eggNOG is a public database of orthology relationships, gene evolutionary histories and functional annotations. Here, we present version 5.0, featuring a major update of the underlying genome sets, which have been expanded to 4445 representative bacteria and 168 archaea derived from 25 038 genomes, as well as 477 eukaryotic organisms and 2502 viral proteomes that were selected for diversity and filtered by genome quality. In total, 4.4M orthologous groups (OGs) distributed across 379 taxonomic levels were computed together with their associated sequence alignments, phylogenies, HMM models and functional descriptors. Precomputed evolutionary analysis provides fine-grained resolution of duplication/speciation events within each OG. Our benchmarks show that, despite doubling the amount of genomes, the quality of orthology assignments and functional annotations (80% coverage) has persisted without significant changes across this update. Finally, we improved eggNOG online services for fast functional annotation and orthology prediction of custom genomics or metagenomics datasets. All precomputed data are publicly available for downloading or via API queries at http://eggnog.embl.de.

The ocean is home to myriad small planktonic organisms that underpin the functioning of marine ecosystems. However, their spatial patterns of diversity and the underlying drivers remain poorly known, precluding projections of their responses to global changes. Here we investigate the latitudinal gradients and global predictors of plankton diversity across archaea, bacteria, eukaryotes, and major virus clades using both molecular and imaging data from Tara Oceans. We show a decline of diversity for most planktonic groups toward the poles, mainly driven by decreasing ocean temperatures. Projections into the future suggest that severe warming of the surface ocean by the end of the 21 century could lead to tropicalization of the diversity of most planktonic groups in temperate and polar regions. These changes may have multiple consequences for marine ecosystem functioning and services and are expected to be particularly significant in key areas for carbon sequestration, fisheries, and marine conservation. VIDEO ABSTRACT.

The Interactive Tree Of Life (https://itol.embl.de) is an online tool for the display, manipulation and annotation of phylogenetic and other trees. It is freely available and open to everyone. The current version introduces four new dataset types, together with numerous new features. Annotation options have been expanded and new control options added for many display elements. An interactive spreadsheet-like editor has been implemented, providing dataset creation and editing directly in the web interface. Font support has been rewritten with full support for UTF-8 character encoding throughout the user interface. Google Web Fonts are now fully supported in the tree text labels. iTOL v4 is the first tool which supports direct visualization of Qiime 2 trees and associated annotations. The user account system has been streamlined and expanded with new navigation options, and currently handles >700 000 trees from more than 40 000 individual users. Full batch access has been implemented allowing programmatic upload and export of trees and annotations.

The healthy 'eubiosis' microbiome in infancy is regarded as the microbiome derived from term, vaginally delivered, antibiotic free, breastfed infants at 4-6 months. Dysbiosis is regarded as a deviation from a healthy state with reduced microbial diversity and deficient capacity to control drug-resistant organisms. Preterm infants are highly sensitive to early gut dysbiosis. Latter has been associated with sepsis and necrotising enterocolitis, but may also contribute to long-term health problems. Probiotics hold promise to reduce the risk for adverse short-term outcomes but the evidence from clinical trials remains inconclusive and none has directly assessed the effects of probiotics on the microbiome at high resolution.

Metagenomic sequencing has greatly improved our ability to profile the composition of environmental and host-associated microbial communities. However, the dependency of most methods on reference genomes, which are currently unavailable for a substantial fraction of microbial species, introduces estimation biases. We present an updated and functionally extended tool based on universal (i.e., reference-independent), phylogenetic marker gene (MG)-based operational taxonomic units (mOTUs) enabling the profiling of >7700 microbial species. As more than 30% of them could not previously be quantified at this taxonomic resolution, relative abundance estimates based on mOTUs are more accurate compared to other methods. As a new feature, we show that mOTUs, which are based on essential housekeeping genes, are demonstrably well-suited for quantification of basal transcriptional activity of community members. Furthermore, single nucleotide variation profiles estimated using mOTUs reflect those from whole genomes, which allows for comparing microbial strain populations (e.g., across different human body sites).

The InterPro database (http://www.ebi.ac.uk/interpro/) classifies protein sequences into families and predicts the presence of functionally important domains and sites. Here, we report recent developments with InterPro (version 70.0) and its associated software, including an 18% growth in the size of the database in terms on new InterPro entries, updates to content, the inclusion of an additional entry type, refined modelling of discontinuous domains, and the development of a new programmatic interface and website. These developments extend and enrich the information provided by InterPro, and provide greater flexibility in terms of data access. We also show that InterPro's sequence coverage has kept pace with the growth of UniProtKB, and discuss how our evaluation of residue coverage may help guide future curation activities.

Proteotypes, like genotypes, have been found to vary between individuals in several studies, but consistent molecular functional traits across studies remain to be quantified. In a meta-analysis of 11 proteomics datasets from humans and mice, we use co-variation of proteins in known functional modules across datasets and individuals to obtain a consensus landscape of proteotype variation. We find that individuals differ considerably in both protein complex abundances and stoichiometry. We disentangle genetic and environmental factors impacting these metrics, with genetic sex and specific diets together explaining 13.5% and 11.6% of the observed variation of complex abundance and stoichiometry, respectively. Sex-specific differences, for example, include various proteins and complexes, where the respective genes are not located on sex-specific chromosomes. Diet-specific differences, added to the individual genetic backgrounds, might become a starting point for personalized proteotype modulation toward desired features.

We present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere.

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.

The gastrointestinal tract is abundantly colonized by microbes, yet the translocation of oral species to the intestine is considered a rare aberrant event, and a hallmark of disease. By studying salivary and fecal microbial strain populations of 310 species in 470 individuals from five countries, we found that transmission to, and subsequent colonization of, the large intestine by oral microbes is common and extensive among healthy individuals. We found evidence for a vast majority of oral species to be transferable, with increased levels of transmission in colorectal cancer and rheumatoid arthritis patients and, more generally, for species described as opportunistic pathogens. This establishes the oral cavity as an endogenous reservoir for gut microbial strains, and oral-fecal transmission as an important process that shapes the gastrointestinal microbiome in health and disease.

Proteins and their functional interactions form the backbone of the cellular machinery. Their connectivity network needs to be considered for the full understanding of biological phenomena, but the available information on protein-protein associations is incomplete and exhibits varying levels of annotation granularity and reliability. The STRING database aims to collect, score and integrate all publicly available sources of protein-protein interaction information, and to complement these with computational predictions. Its goal is to achieve a comprehensive and objective global network, including direct (physical) as well as indirect (functional) interactions. The latest version of STRING (11.0) more than doubles the number of organisms it covers, to 5090. The most important new feature is an option to upload entire, genome-wide datasets as input, allowing users to visualize subsets as interaction networks and to perform gene-set enrichment analysis on the entire input. For the enrichment analysis, STRING implements well-known classification systems such as Gene Ontology and KEGG, but also offers additional, new classification systems based on high-throughput text-mining as well as on a hierarchical clustering of the association network itself. The STRING resource is available online at https://string-db.org/.

Several studies have investigated links between the gut microbiome and colorectal cancer (CRC), but questions remain about the replicability of biomarkers across cohorts and populations. We performed a meta-analysis of five publicly available datasets and two new cohorts and validated the findings on two additional cohorts, considering in total 969 fecal metagenomes. Unlike microbiome shifts associated with gastrointestinal syndromes, the gut microbiome in CRC showed reproducibly higher richness than controls (P < 0.01), partially due to expansions of species typically derived from the oral cavity. Meta-analysis of the microbiome functional potential identified gluconeogenesis and the putrefaction and fermentation pathways as being associated with CRC, whereas the stachyose and starch degradation pathways were associated with controls. Predictive microbiome signatures for CRC trained on multiple datasets showed consistently high accuracy in datasets not considered for model training and independent validation cohorts (average area under the curve, 0.84). Pooled analysis of raw metagenomes showed that the choline trimethylamine-lyase gene was overabundant in CRC (P = 0.001), identifying a relationship between microbiome choline metabolism and CRC. The combined analysis of heterogeneous CRC cohorts thus identified reproducible microbiome biomarkers and accurate disease-predictive models that can form the basis for clinical prognostic tests and hypothesis-driven mechanistic studies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human gut microbiome studies are mainly bacteria- and archaea-oriented, overlooking the presence of single-cell eukaryotes such as an enteric stramenopiles with worldwide distribution. Here, we surveyed the prevalence and subtype variation of in faecal samples collected as part of the Flemish Gut Flora Project (FGFP), a Western population cohort. We assessed potential links between subtypes and identified microbiota-host covariates and quantified microbiota differentiation relative to subtype abundances.

Association studies have linked microbiome alterations with many human diseases. However, they have not always reported consistent results, thereby necessitating cross-study comparisons. Here, a meta-analysis of eight geographically and technically diverse fecal shotgun metagenomic studies of colorectal cancer (CRC, n = 768), which was controlled for several confounders, identified a core set of 29 species significantly enriched in CRC metagenomes (false discovery rate (FDR) < 1 × 10). CRC signatures derived from single studies maintained their accuracy in other studies. By training on multiple studies, we improved detection accuracy and disease specificity for CRC. Functional analysis of CRC metagenomes revealed enriched protein and mucin catabolism genes and depleted carbohydrate degradation genes. Moreover, we inferred elevated production of secondary bile acids from CRC metagenomes, suggesting a metabolic link between cancer-associated gut microbes and a fat- and meat-rich diet. Through extensive validations, this meta-analysis firmly establishes globally generalizable, predictive taxonomic and functional microbiome CRC signatures as a basis for future diagnostics.

Soils harbour some of the most diverse microbiomes on Earth and are essential for both nutrient cycling and carbon storage. To understand soil functioning, it is necessary to model the global distribution patterns and functional gene repertoires of soil microorganisms, as well as the biotic and environmental associations between the diversity and structure of both bacterial and fungal soil communities. Here we show, by leveraging metagenomics and metabarcoding of global topsoil samples (189 sites, 7,560 subsamples), that bacterial, but not fungal, genetic diversity is highest in temperate habitats and that microbial gene composition varies more strongly with environmental variables than with geographic distance. We demonstrate that fungi and bacteria show global niche differentiation that is associated with contrasting diversity responses to precipitation and soil pH. Furthermore, we provide evidence for strong bacterial-fungal antagonism, inferred from antibiotic-resistance genes, in topsoil and ocean habitats, indicating the substantial role of biotic interactions in shaping microbial communities. Our results suggest that both competition and environmental filtering affect the abundance, composition and encoded gene functions of bacterial and fungal communities, indicating that the relative contributions of these microorganisms to global nutrient cycling varies spatially.

Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.

The spread of antimicrobial resistance has become a serious public health concern, making once-treatable diseases deadly again and undermining the achievements of modern medicine. Drug combinations can help to fight multi-drug-resistant bacterial infections, yet they are largely unexplored and rarely used in clinics. Here we profile almost 3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in six strains from three Gram-negative pathogens-Escherichia coli, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa-to identify general principles for antibacterial drug combinations and understand their potential. Despite the phylogenetic relatedness of the three species, more than 70% of the drug-drug interactions that we detected are species-specific and 20% display strain specificity, revealing a large potential for narrow-spectrum therapies. Overall, antagonisms are more common than synergies and occur almost exclusively between drugs that target different cellular processes, whereas synergies are more conserved and are enriched in drugs that target the same process. We provide mechanistic insights into this dichotomy and further dissect the interactions of the food additive vanillin. Finally, we demonstrate that several synergies are effective against multi-drug-resistant clinical isolates in vitro and during infections of the larvae of the greater wax moth Galleria mellonella, with one reverting resistance to the last-resort antibiotic colistin.

While our knowledge about the roles of microbes and viruses in the ocean has increased tremendously due to recent advances in genomics and metagenomics, research on marine microbial eukaryotes and zooplankton has benefited much less from these new technologies because of their larger genomes, their enormous diversity, and largely unexplored physiologies. Here, we use a metatranscriptomics approach to capture expressed genes in open ocean Tara Oceans stations across four organismal size fractions. The individual sequence reads cluster into 116 million unigenes representing the largest reference collection of eukaryotic transcripts from any single biome. The catalog is used to unveil functions expressed by eukaryotic marine plankton, and to assess their functional biogeography. Almost half of the sequences have no similarity with known proteins, and a great number belong to new gene families with a restricted distribution in the ocean. Overall, the resource provides the foundations for exploring the roles of marine eukaryotes in ocean ecology and biogeochemistry.

Gut microbes influence their hosts in many ways, in particular by modulating the impact of diet. These effects have been studied most extensively in humans and mice. In this work, we used whole genome metagenomics to investigate the relationship between the gut metagenomes of dogs, humans, mice, and pigs.

Population stratification is a useful approach for a better understanding of complex biological problems in human health and wellbeing. The proposal that such stratification applies to the human gut microbiome, in the form of distinct community composition types termed enterotypes, has been met with both excitement and controversy. In view of accumulated data and re-analyses since the original work, we revisit the concept of enterotypes, discuss different methods of dividing up the landscape of possible microbiome configurations, and put these concepts into functional, ecological and medical contexts. As enterotypes are of use in describing the gut microbial community landscape and may become relevant in clinical practice, we aim to reconcile differing views and encourage a balanced application of the concept.

In the version of this Perspective originally published, the first and last name of co-author Manimozhiyan Arumugam were switched. This has now been corrected in all versions of the Perspective.

iPath3.0 (http://pathways.embl.de) is a web-application for the visualization and analysis of cellular pathways. It is freely available and open to everyone. Currently it is based on four KEGG global maps, which summarize up to 158 traditional KEGG pathway maps, 192 KEGG modules and other metabolic elements into one connected and manually curated metabolic network. Users can fully customize these networks and interactively explore them through its redesigned, fast and lightweight interface, which highlights general metabolic trends in multi-omics data. It also offers navigation at various levels of details to help users further investigate those trends and ultimately uncover novel biological insights. Support for multiple experimental conditions and time-series datasets, tools for generation of customization data, programmatic access, and a free user accounts system were introduced in this version to further streamline its workflow.

The acquisition and development of the infant microbiome are key to establishing a healthy host-microbiome symbiosis. The maternal microbial reservoir is thought to play a crucial role in this process. However, the source and transmission routes of the infant pioneering microbes are poorly understood. To address this, we longitudinally sampled the microbiome of 25 mother-infant pairs across multiple body sites from birth up to 4 months postpartum. Strain-level metagenomic profiling showed a rapid influx of microbes at birth followed by strong selection during the first few days of life. Maternal skin and vaginal strains colonize only transiently, and the infant continues to acquire microbes from distinct maternal sources after birth. Maternal gut strains proved more persistent in the infant gut and ecologically better adapted than those acquired from other sources. Together, these data describe the mother-to-infant microbiome transmission routes that are integral in the development of the infant microbiome.

Phages invade microbes, accomplish host lysis and are of vital importance in shaping the community structure of environmental microbiota. More importantly, most phages have very specific hosts; they are thus ideal tools to manipulate environmental microbiota at species-resolution. The main purpose of MVP (Microbe Versus Phage) is to provide a comprehensive catalog of phage-microbe interactions and assist users to select phage(s) that can target (and potentially to manipulate) specific microbes of interest. We first collected 50 782 viral sequences from various sources and clustered them into 33 097 unique viral clusters based on sequence similarity. We then identified 26 572 interactions between 18 608 viral clusters and 9245 prokaryotes (i.e. bacteria and archaea); we established these interactions based on 30 321 evidence entries that we collected from published datasets, public databases and re-analysis of genomic and metagenomic sequences. Based on these interactions, we calculated the host range for each of the phage clusters and accordingly grouped them into subgroups such as 'species-', 'genus-' and 'family-' specific phage clusters. MVP is equipped with a modern, responsive and intuitive interface, and is freely available at: http://mvp.medgenius.info.

Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms remain elusive. Here, we present proteome-wide atlases of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) and five other cell populations that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified is assessed in 59 human subjects from different ages. As the HPCs become older, pathways in central carbon metabolism exhibit features reminiscent of the Warburg effect, where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation. Ageing causes alterations in the bone marrow niche too, and diminishes the functionality of the pathways involved in HPC homing. The data represent a valuable resource for further analyses, and for validation of knowledge gained from animal models.

There is growing recognition that the gut microbial community regulates a wide variety of important functions in its animal hosts, including host health. However, the complex interactions between gut microbes and environment are still unclear. Honey bees are ecologically and economically important pollinators that host a core gut microbial community that is thought to be constant across populations. Here, we examined whether the composition of the gut microbial community of honey bees is affected by the environmental landscape the bees are exposed to. We placed honey bee colonies reared under identical conditions in two main landscape types for 6 weeks: either oilseed rape farmland or agricultural farmland distant to fields of flowering oilseed rape. The gut bacterial communities of adult bees from the colonies were then characterized and compared based on amplicon sequencing of the 16S rRNA gene. While previous studies have delineated a characteristic core set of bacteria inhabiting the honey bee gut, our results suggest that the broad environment that bees are exposed to has some influence on the relative abundance of some members of that microbial community. This includes known dominant taxa thought to have functions in nutrition and health. Our results provide evidence for an influence of landscape exposure on honey bee microbial community and highlight the potential effect of exposure to different environmental parameters, such as forage type and neonicotinoid pesticides, on key honey bee gut bacteria. This work emphasizes the complexity of the relationship between the host, its gut bacteria, and the environment and identifies target microbial taxa for functional analyses.

The gut microbiome is recognised as playing an integral role in the health and ecology of a wide variety of animal taxa. However, the relationship between social behavioural traits and the microbial community has received little attention. Honey bees are highly social and the workers perform different behavioural tasks in the colony that cause them to be exposed to different local environments. Here we examined whether the gut microbial community composition of worker honey bees is associated with the behavioural tasks they perform, and therefore also the local environment they are exposed to. We set up five observation hives, in which all workers were matched in age and observed the behaviour of marked bees in each colony over 4 days. The gut bacterial communities of bees seen performing predominantly foraging or predominantly in nest tasks were then characterised and compared based on amplicon sequencing of the 16S rRNA gene. Our results show that some core members of the unique honey bee gut bacterial community are represented in different relative abundances in bees performing different behavioural tasks. The differentially represented bacterial taxa include some thought to be important in carbohydrate metabolism and transport, and also linked to bee health. The results suggest an influence of task-related local environment exposure and diet on the honey bee gut microbial community and identify focal core taxa for further functional analyses.

In many families with suspected Lynch syndrome (LS), no germline mutation in the causative mismatch repair (MMR) genes is detected during routine diagnostics. To identify novel causative genes for LS, the present study investigated 77 unrelated, mutation-negative patients with clinically suspected LS and a loss of MSH2 in tumor tissue. An analysis for genomic copy number variants (CNV) was performed, with subsequent next generation sequencing (NGS) of selected candidate genes in a subgroup of the cohort. Genomic DNA was genotyped using Illumina's HumanOmniExpress Bead Array. After quality control and filtering, 25 deletions and 16 duplications encompassing 73 genes were identified in 28 patients. No recurrent CNV was detected, and none of the CNVs affected the regulatory regions of MSH2. A total of 49 candidate genes from genomic regions implicated by the present CNV analysis and 30 known or assumed risk genes for colorectal cancer (CRC) were then sequenced in a subset of 38 patients using a customized NGS gene panel and Sanger sequencing. Single nucleotide variants were identified in 14 candidate genes from the CNV analysis. The most promising of these candidate genes were: (i) PRKCA, PRKDC, and MCM4, since a functional relation to MSH2 is predicted by network analysis; and (ii) CSMD1, as this is commonly mutated in CRC. Furthermore, six patients harbored POLE variants outside the exonuclease domain, suggesting that these might be implicated in hereditary CRC. Analyses in larger cohorts of suspected LS patients recruited via international collaborations are warranted to verify the present findings. This article is protected by copyright. All rights reserved.

Vertical transmission of bacteria from mother to infant at birth is postulated to initiate a life-long host-microbe symbiosis, playing an important role in early infant development. However, only the tracking of strictly defined unique microbial strains can clarify where the intestinal bacteria come from, how long the initial colonisers persist and whether colonisation by other strains from the environment can replace existing ones. Using rare single nucleotide variants in fecal metagenomes of infants and their family members, we show strong evidence of selective and persistent transmission of maternal strain populations to the vaginally born infant, and their occasional replacement by strains from the environment, including those from family members, in later childhood. Only strains from the classes Actinobacteria and Bacteroidia, which are essential components of the infant microbiome, are transmitted from the mother and persist for at least one year. In contrast, maternal strains of Clostridia, a dominant class in the mother's gut microbiome, are not observed in the infant. Caesarean-born infants show a striking lack of maternal transmission at birth. After the first year, strain influx from the family environment occurs, and continues even in adulthood. Fathers appear to be more frequently donors of novel strains to other family members than receivers. Thus, the infant gut is seeded by selected maternal bacteria, which expand to form a stable community, with a rare but stable continuing strain influx over time.

Changes in the gut microbiota are increasingly recognised to be involved in many diseases. This ecosystem is known to be shaped by many factors, including climate, geography, host nutrition, lifestyle and medication. Thus, knowledge of varying populations with different habits is important for a better understanding of the microbiome.

SMART (Simple Modular Architecture Research Tool) is a web resource (http://smart.embl.de) for the identification and annotation of protein domains and the analysis of protein domain architectures. SMART version 8 contains manually curated models for more than 1300 protein domains, with approximately 100 new models added since our last update article (1). The underlying protein databases were synchronized with UniProt (2), Ensembl (3) and STRING (4), doubling the total number of annotated domains and other protein features to more than 200 million. In its 20th year, the SMART analysis results pages have been streamlined again and its information sources have been updated. SMART's vector based display engine has been extended to all protein schematics in SMART and rewritten to use the latest web technologies. The internal full text search engine has been redesigned and updated, resulting in greatly increased search speed.

A few commonly used non-antibiotic drugs have recently been associated with changes in gut microbiome composition, but the extent of this phenomenon is unknown. Here, we screened more than 1,000 marketed drugs against 40 representative gut bacterial strains, and found that 24% of the drugs with human targets, including members of all therapeutic classes, inhibited the growth of at least one strain in vitro. Particular classes, such as the chemically diverse antipsychotics, were overrepresented in this group. The effects of human-targeted drugs on gut bacteria are reflected on their antibiotic-like side effects in humans and are concordant with existing human cohort studies. Susceptibility to antibiotics and human-targeted drugs correlates across bacterial species, suggesting common resistance mechanisms, which we verified for some drugs. The potential risk of non-antibiotics promoting antibiotic resistance warrants further exploration. Our results provide a resource for future research on drug-microbiome interactions, opening new paths for side effect control and drug repurposing, and broadening our view of antibiotic resistance.

To minimize the impact of antibiotics, gut microorganisms harbour and exchange antibiotics resistance genes, collectively called their resistome. Using shotgun sequencing-based metagenomics, we analysed the partial eradication and subsequent regrowth of the gut microbiota in 12 healthy men over a 6-month period following a 4-day intervention with a cocktail of 3 last-resort antibiotics: meropenem, gentamicin and vancomycin. Initial changes included blooms of enterobacteria and other pathobionts, such as Enterococcus faecalis and Fusobacterium nucleatum, and the depletion of Bifidobacterium species and butyrate producers. The gut microbiota of the subjects recovered to near-baseline composition within 1.5 months, although 9 common species, which were present in all subjects before the treatment, remained undetectable in most of the subjects after 180 days. Species that harbour β-lactam resistance genes were positively selected for during and after the intervention. Harbouring glycopeptide or aminoglycoside resistance genes increased the odds of de novo colonization, however, the former also decreased the odds of survival. Compositional changes under antibiotic intervention in vivo matched results from in vitro susceptibility tests. Despite a mild yet long-lasting imprint following antibiotics exposure, the gut microbiota of healthy young adults are resilient to a short-term broad-spectrum antibiotics intervention and their antibiotics resistance gene carriage modulates their recovery processes.

Quantitative mass spectrometry enables to monitor the abundance of thousands of proteins across biological conditions. Currently, most data analysis approaches rely on the assumption that the majority of the observed proteins remain unchanged across compared samples. Thus, gross morphological differences between cell states, deriving from, e.g., differences in size or number of organelles, are often not taken into account. Here, we analyzed multiple published datasets and frequently observed that proteins associated with a particular cellular compartment collectively increase or decrease in their abundance between conditions tested. We show that such effects, arising from underlying morphological differences, can skew the outcome of differential expression analysis. We propose a method to detect and normalize morphological effects underlying proteomics data. We demonstrate the applicability of our method to different datasets and biological questions including the analysis of sub-cellular proteomes in the context of aging. Our method provides a complementary perspective to classical differential expression analysis and enables to uncouple overall abundance changes from stoichiometric variations within defined group of proteins.

We recently presented a three-pronged association study that integrated human intestinal microbiome data derived from shotgun-based sequencing with untargeted serum metabolome data and measures of host physiology. Metabolome and microbiome data are high dimensional, posing a major challenge for data integration. Here, we present a step-by-step computational protocol that details and discusses the dimensionality-reduction techniques used and methods for subsequent integration and interpretation of such heterogeneous types of data. Dimensionality reduction was achieved through a combination of data normalization approaches, binning of co-abundant genes and metabolites, and integration of prior biological knowledge. The use of prior knowledge to overcome functional redundancy across microbiome species is one central advance of our method over available alternative approaches. Applying this framework, other investigators can integrate various '-omics' readouts with variables of host physiology or any other phenotype of interest (e.g., connecting host and microbiome readouts to disease severity or treatment outcome in a clinical cohort) in a three-pronged association analysis to identify potential mechanistic links to be tested in experimental settings. Although we originally developed the framework for a human metabolome-microbiome study, it is generalizable to other organisms and environmental metagenomes, as well as to studies including other -omics domains such as transcriptomics and proteomics. The provided R code runs in ~1 h on a standard PC.

Conjugative transposition drives the emergence of multidrug resistance in diverse bacterial pathogens, yet the mechanisms are poorly characterized. The Tn1549 conjugative transposon propagates resistance to the antibiotic vancomycin used for severe drug-resistant infections. Here, we present four high-resolution structures of the conserved Y-transposase of Tn1549 complexed with circular transposon DNA intermediates. The structures reveal individual transposition steps and explain how specific DNA distortion and cleavage mechanisms enable DNA strand exchange with an absolute minimum homology requirement. This appears to uniquely allow Tn916-like conjugative transposons to bypass DNA homology and insert into diverse genomic sites, expanding gene transfer. We further uncover a structural regulatory mechanism that prevents premature cleavage of the transposon DNA before a suitable target DNA is found and generate a peptide antagonist that interferes with the transposase-DNA structure to block transposition. Our results reveal mechanistic principles of conjugative transposition that could help control the spread of antibiotic resistance genes.

Our understanding of the human gut microbiome continues to evolve at a rapid pace, but practical application of thisknowledge is still in its infancy. This review discusses the type of studies that will be essential for translating microbiome research into targeted modulations with dedicated benefits for the human host.

Single-celled eukaryotes (protists) are critical players in global biogeochemical cycling of nutrients and energy in the oceans. While their roles as primary producers and grazers are well appreciated, other aspects of their life histories remain obscure due to challenges in culturing and sequencing their natural diversity. Here, we exploit single-cell genomics and metagenomics data from the circumglobal Tara Oceans expedition to analyze the genome content and apparent oceanic distribution of seven prevalent lineages of uncultured heterotrophic stramenopiles. Based on the available data, each sequenced genome or genotype appears to have a specific oceanic distribution, principally correlated with water temperature and depth. The genome content provides hypotheses for specialization in terms of cell motility, food spectra, and trophic stages, including the potential impact on their lifestyles of horizontal gene transfer from prokaryotes. Our results support the idea that prominent heterotrophic marine protists perform diverse functions in ocean ecology.

Bacterial metabolism plays a fundamental role in gut microbiota ecology and host-microbiome interactions. Yet the metabolic capabilities of most gut bacteria have remained unknown. Here we report growth characteristics of 96 phylogenetically diverse gut bacterial strains across 4 rich and 15 defined media. The vast majority of strains (76) grow in at least one defined medium, enabling accurate assessment of their biosynthetic capabilities. These do not necessarily match phylogenetic similarity, thus indicating a complex evolution of nutritional preferences. We identify mucin utilizers and species inhibited by amino acids and short-chain fatty acids. Our analysis also uncovers media for in vitro studies wherein growth capacity correlates well with in vivo abundance. Further value of the underlying resource is demonstrated by correcting pathway gaps in available genome-scale metabolic models of gut microorganisms. Together, the media resource and the extracted knowledge on growth abilities widen experimental and computational access to the gut microbiota.

A unique collection of oceanic samples was gathered by the Tara Oceans expeditions (2009-2013), targeting plankton organisms ranging from viruses to metazoans, and providing rich environmental context measurements. Thanks to recent advances in the field of genomics, extensive sequencing has been performed for a deep genomic analysis of this huge collection of samples. A strategy based on different approaches, such as metabarcoding, metagenomics, single-cell genomics and metatranscriptomics, has been chosen for analysis of size-fractionated plankton communities. Here, we provide detailed procedures applied for genomic data generation, from nucleic acids extraction to sequence production, and we describe registries of genomics datasets available at the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena). The association of these metadata to the experimental procedures applied for their generation will help the scientific community to access these data and facilitate their analysis. This paper complements other efforts to provide a full description of experiments and open science resources generated from the Tara Oceans project, further extending their value for the study of the world's planktonic ecosystems.

Parkinson's disease (PD) presently is conceptualized as a protein aggregation disease in which pathology involves both the enteric and the central nervous system, possibly spreading from one to another via the vagus nerves. As gastrointestinal dysfunction often precedes or parallels motor symptoms, the enteric system with its vast diversity of microorganisms may be involved in PD pathogenesis. Alterations in the enteric microbial taxonomic level of L-DOPA-naïve PD patients might also serve as a biomarker.

Proteomics techniques can identify thousands of phosphorylation sites in a single experiment, the majority of which are new and lack precise information about function or molecular mechanism. Here we present a fast method to predict potential phosphorylation switches by mapping phosphorylation sites to protein-protein interactions of known structure and analysing the properties of the protein interface. We predict 1024 sites that could potentially enable or disable particular interactions. We tested a selection of these switches and showed that phosphomimetic mutations indeed affect interactions. We estimate that there are likely thousands of phosphorylation mediated switches yet to be uncovered, even among existing phosphorylation datasets. The results suggest that phosphorylation sites on globular, as distinct from disordered, parts of the proteome frequently function as switches, which might be one of the ancient roles for kinase phosphorylation.

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

OGEE is an Online GEne Essentiality database. To enhance our understanding of the essentiality of genes, in OGEE we collected experimentally tested essential and non-essential genes, as well as associated gene properties known to contribute to gene essentiality. We focus on large-scale experiments, and complement our data with text-mining results. We organized tested genes into data sets according to their sources, and tagged those with variable essentiality statuses across data sets as conditionally essential genes, intending to highlight the complex interplay between gene functions and environments/experimental perturbations. Developments since the last public release include increased numbers of species and gene essentiality data sets, inclusion of non-coding essential sequences and genes with intermediate essentiality statuses. In addition, we included 16 essentiality data sets from cancer cell lines, corresponding to 9 human cancers; with OGEE, users can easily explore the shared and differentially essential genes within and between cancer types. These genes, especially those derived from cell lines that are similar to tumor samples, could reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer types, and can be further screened to identify targets for cancer therapy and/or new drug development. OGEE is freely available at http://ogee.medgenius.info.

We present a 3D-fluorescence imaging and classification tool for high throughput analysis of microbial eukaryotes in environmental samples. It entails high-content feature extraction that permits accurate automated taxonomic classification and quantitative data about organism ultrastructures and interactions. Using plankton samples from the Tara Oceans expeditions, we validate its applicability to taxonomic profiling and ecosystem analyses, and discuss its potential for future integration of eukaryotic cell biology into evolutionary and ecological studies.

Population genomics of prokaryotes has been studied in depth in only a small number of primarily pathogenic bacteria, as genome sequences of isolates of diverse origin are lacking for most species. Here, we conducted a large-scale survey of population structure in prevalent human gut microbial species, sampled from their natural environment, with a culture-independent metagenomic approach. We examined the variation landscape of 71 species in 2,144 human fecal metagenomes and found that in 44 of these, accounting for 72% of the total assigned microbial abundance, single-nucleotide variation clearly indicates the existence of sub-populations (here termed subspecies). A single subspecies (per species) usually dominates within each host, as expected from ecological theory. At the global scale, geographic distributions of subspecies differ between phyla, with Firmicutes subspecies being significantly more geographically restricted. To investigate the functional significance of the delineated subspecies, we identified genes that consistently distinguish them in a manner that is independent of reference genomes. We further associated these subspecies-specific genes with properties of the microbial community and the host. For example, two of the three Eubacterium rectale subspecies consistently harbor an accessory pro-inflammatory flagellum operon that is associated with lower gut community diversity, higher host BMI, and higher blood fasting insulin levels. Using an additional 676 human oral samples, we further demonstrate the existence of niche specialized subspecies in the different parts of the oral cavity. Taken together, we provide evidence for subspecies in the majority of abundant gut prokaryotes, leading to a better functional and ecological understanding of the human gut microbiome in conjunction with its host.

We present metaSNV, a tool for single nucleotide variant (SNV) analysis in metagenomic samples, capable of comparing populations of thousands of bacterial and archaeal species. The tool uses as input nucleotide sequence alignments to reference genomes in standard SAM/BAM format, performs SNV calling for individual samples and across the whole data set, and generates various statistics for individual species including allele frequencies and nucleotide diversity per sample as well as distances and fixation indices across samples. Using published data from 676 metagenomic samples of different sites in the oral cavity, we show that the results of metaSNV are comparable to those of MIDAS, an alternative implementation for metagenomic SNV analysis, while data processing is faster and has a smaller storage footprint. Moreover, we implement a set of distance measures that allow the comparison of genomic variation across metagenomic samples and delineate sample-specific variants to enable the tracking of specific strain populations over time. The implementation of metaSNV is available at: http://metasnv.embl.de/.

Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

InterPro (http://www.ebi.ac.uk/interpro/) is a freely available database used to classify protein sequences into families and to predict the presence of important domains and sites. InterProScan is the underlying software that allows both protein and nucleic acid sequences to be searched against InterPro's predictive models, which are provided by its member databases. Here, we report recent developments with InterPro and its associated software, including the addition of two new databases (SFLD and CDD), and the functionality to include residue-level annotation and prediction of intrinsic disorder. These developments enrich the annotations provided by InterPro, increase the overall number of residues annotated and allow more specific functional inferences.

Orthology assignment is ideally suited for functional inference. However, because predicting orthology is computationally intensive at large scale, and most pipelines are relatively inaccessible (e.g. new assignments only available through database updates), less precise homology-based functional transfer is still the default for (meta-)genome annotation. We therefore developed eggNOG-mapper, a tool for functional annotation of large sets of sequences based on fast orthology assignments using precomputed clusters and phylogenies from the eggNOG database. To validate our method, we benchmarked Gene Ontology predictions against two widely used homology-based approaches: BLAST and InterProScan. Orthology filters applied to BLAST results reduced the rate of false positive assignments by 11%, and increased the ratio of experimentally validated terms recovered over all terms assigned per protein by 15%. Compared to InterProScan, eggNOG-mapper achieved similar proteome coverage and precision while predicting, on average, 41 more terms per protein and increasing the rate of experimentally validated terms recovered over total term assignments per protein by 35%. EggNOG-mapper predictions scored within the top-5 methods in the three Gene Ontology categories using the CAFA2 NK-partial benchmark. Finally, we evaluated eggNOG-mapper for functional annotation of metagenomics data, yielding better performance than interProScan. eggNOG-mapper runs ∼15x faster than BLAST and at least 2.5x faster than InterProScan. The tool is available standalone and as an online service at http://eggnog-mapper.embl.de.

Microbial cell factories based on renewable carbon sources are fundamental to a sustainable bio-economy. The economic feasibility of producer cells requires robust performance balancing growth and production. However, the inherent competition between these two objectives often leads to instability and reduces productivity. While algorithms exist to design metabolic network reduction strategies for aligning these objectives, the biochemical basis of the growth-product coupling has remained unresolved. Here, we reveal key reactions in the cellular biochemical repertoire as universal anchor reactions for aligning cell growth and production. A necessary condition for a reaction to be an anchor is that it splits a substrate into two or more molecules. By searching the currently known biochemical reaction space, we identify 62 C-C cleaving anchor reactions, such as isocitrate lyase (EC 4.1.3.1) and L-tryptophan indole-lyase (EC 4.1.99.1), which are relevant for biorefining. The here identified anchor reactions mark network nodes for basing growth-coupled metabolic engineering and novel pathway designs.

Blowflies and houseflies are mechanical vectors inhabiting synanthropic environments around the world. They feed and breed in fecal and decaying organic matter, but the microbiome they harbour and transport is largely uncharacterized. We sampled 116 individual houseflies and blowflies from varying habitats on three continents and subjected them to high-coverage, whole-genome shotgun sequencing. This allowed for genomic and metagenomic analyses of the host-associated microbiome at the species level. Both fly host species segregate based on principal coordinate analysis of their microbial communities, but they also show an overlapping core microbiome. Legs and wings displayed the largest microbial diversity and were shown to be an important route for microbial dispersion. The environmental sequencing approach presented here detected a stochastic distribution of human pathogens, such as Helicobacter pylori, thereby demonstrating the potential of flies as proxies for environmental and public health surveillance.

The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known-at both the protein and complex levels-this study constitutes another step forward toward a molecular understanding of subcellular organization.

The global biogeography of microorganisms remains poorly resolved, which limits the current understanding of microbial resilience toward environmental changes. Using high-throughput 16S rRNA gene amplicon sequencing, we characterized the microbial diversity of terrestrial and lacustrine biofilms from the Arctic, Antarctic and temperate regions. Our analyses suggest that bacterial community compositions at the poles are more similar to each other than they are to geographically closer temperate habitats, with 32% of all operational taxonomic units (OTUs) co-occurring in both polar regions. While specific microbial taxa were confined to distinct regions, representing potentially endemic populations, the percentage of cosmopolitan taxa was higher in Arctic (43%) than in Antarctic samples (36%). The overlap in polar microbial OTUs may be explained by natural or anthropogenically-mediated dispersal in combination with environmental filtering. Current and future changing environmental conditions may enhance microbial invasion, establishment of cosmopolitan genotypes and loss of endemic taxa.

Current treatment for pediatric IBD patients is often ineffective, with serious side effects. Manipulating the gut microbiota via fecal microbiota transplantation (FMT) is an emerging treatment approach but remains controversial. We aimed to assess the composition of the fecal microbiome through a comparison of pediatric IBD patients to their healthy siblings, evaluating risks and prospects for FMT in this setting. A Case-Control (Sibling) Study was conducted analyzing fecal samples of six children with Crohn's Disease (CD), six children with Ulcerative Colitis (UC) and 12 healthy siblings by metagenomic sequencing. In addition, lifetime antibiotic intake was retrospectively determined. Species richness and diversity were significantly reduced in UC patients compared to control (MWU FDR = 0.011). In UC, bacteria positively influencing gut homeostasis e.g. Eubacterium rectale and Faecalibacterium prausnitzii were significantly reduced in abundance (MWU FDR = 0.05). Known pathobionts like Escherichia coli were enriched in UC patients (MWU FDR = 0.084). Moreover, E. coli abundance correlated positively with that of several virulence genes (SCC > 0.65, FDR < 0.1). A shift towards antibiotic resistant taxa in both IBD groups distinguished them from controls (MWU BY FDR = 0.062 in UC, MWU BY FDR = 0.019 in CD). The collected results confirm a microbial dysbiosis in pediatric UC, and to a lesser extent in CD patients, replicating associations found previously using different methods. Taken together, these observations suggest microbiotal remodeling therapy from family donors, at least for children with UC, as a viable option.

The availability of microbial genomes has opened many new avenues of research within microbiology. This has been driven primarily by comparative genomics approaches, which rely on accurate and consistent characterization of genomic sequences. It is nevertheless difficult to obtain consistent taxonomic and integrated functional annotations for defined prokaryotic clades. Thus, we developed proGenomes, a resource that provides user-friendly access to currently 25 038 high-quality genomes whose sequences and consistent annotations can be retrieved individually or by taxonomic clade. These genomes are assigned to 5306 consistent and accurate taxonomic species clusters based on previously established methodology. proGenomes also contains functional information for almost 80 million protein-coding genes, including a comprehensive set of general annotations and more focused annotations for carbohydrate-active enzymes and antibiotic resistance genes. Additionally, broad habitat information is provided for many genomes. All genomes and associated information can be downloaded by user-selected clade or multiple habitat-specific sets of representative genomes. We expect that the availability of high-quality genomes with comprehensive functional annotations will promote advances in clinical microbial genomics, functional evolution and other subfields of microbiology. proGenomes is available at http://progenomes.embl.de.

Post-translational modifications (PTMs) are an important source of protein regulation; they fine-tune the function, localization, and interaction with other molecules of the majority of proteins and are partially responsible for their multifunctionality. Usually, proteins have several potential modification sites, and their patterns of occupancy are associated with certain functional states. These patterns imply cross talk among PTMs within and between proteins, the majority of which are still to be discovered. Several methods detect associations between PTMs; these have recently combined into a global resource, the PTMcode database, which contains already known and predicted functional associations between pairs of PTMs from more than 45,000 proteins in 19 eukaryotic species.

The rapidly expanding microbiomics field is generating increasingly larger datasets, characterizing the microbiota in diverse environments. Although classical numerical ecology methods provide a robust statistical framework for their analysis, software currently available is inadequate for large datasets and some computationally intensive tasks, like rarefaction and associated analysis.

A system-wide understanding of cellular function requires knowledge of all functional interactions between the expressed proteins. The STRING database aims to collect and integrate this information, by consolidating known and predicted protein-protein association data for a large number of organisms. The associations in STRING include direct (physical) interactions, as well as indirect (functional) interactions, as long as both are specific and biologically meaningful. Apart from collecting and reassessing available experimental data on protein-protein interactions, and importing known pathways and protein complexes from curated databases, interaction predictions are derived from the following sources: (i) systematic co-expression analysis, (ii) detection of shared selective signals across genomes, (iii) automated text-mining of the scientific literature and (iv) computational transfer of interaction knowledge between organisms based on gene orthology. In the latest version 10.5 of STRING, the biggest changes are concerned with data dissemination: the web frontend has been completely redesigned to reduce dependency on outdated browser technologies, and the database can now also be queried from inside the popular Cytoscape software framework. Further improvements include automated background analysis of user inputs for functional enrichments, and streamlined download options. The STRING resource is available online, at http://string-db.org/.

The human microbiome - the vast amount of microbes that colonize our body - play an important role in maintaining our health. Changes in microbiome composition have been linked to multiple diseases including cancer. Although mechanisms and causalities of these associations still have to be uncovered, microbiome alterations across various stages of disease can be utilized for novel diagnostic and prognostic tests. Research on biomarkers extracted from the gut microbiome has in particular focused on colorectal cancer, where clinical use is already on the horizon. For example, multiple microbial taxonomic markers such as Fusobacterium nucleatum and other oral pathogens have been identified in human feces with potential for non-invasive diagnostics and prognostics. The article summarizes the recent developments, but also limitations and challenges for the development of microbiome-based biomarkers for cancer early detection.

Drug resistance is becoming a serious problem that leads to the failure of standard treatments, which is generally developed because of genetic mutations of certain molecules. Here, we present GEAR (A database of Genomic Elements Associated with drug Resistance) that aims to provide comprehensive information about genomic elements (including genes, single-nucleotide polymorphisms and microRNAs) that are responsible for drug resistance. Right now, GEAR contains 1631 associations between 201 human drugs and 758 genes, 106 associations between 29 human drugs and 66 miRNAs, and 44 associations between 17 human drugs and 22 SNPs. These relationships are firstly extracted from primary literature with text mining and then manually curated. The drug resistome deposited in GEAR provides insights into the genetic factors underlying drug resistance. In addition, new indications and potential drug combinations can be identified based on the resistome. The GEAR database can be freely accessed through http://gear.comp-sysbio.org.

A Western lifestyle with high salt consumption can lead to hypertension and cardiovascular disease. High salt may additionally drive autoimmunity by inducing T helper 17 (TH17) cells, which can also contribute to hypertension. Induction of TH17 cells depends on gut microbiota; however, the effect of salt on the gut microbiome is unknown. Here we show that high salt intake affects the gut microbiome in mice, particularly by depleting Lactobacillus murinus. Consequently, treatment of mice with L. murinus prevented salt-induced aggravation of actively induced experimental autoimmune encephalomyelitis and salt-sensitive hypertension by modulating TH17 cells. In line with these findings, a moderate high-salt challenge in a pilot study in humans reduced intestinal survival of Lactobacillus spp., increased TH17 cells and increased blood pressure. Our results connect high salt intake to the gut-immune axis and highlight the gut microbiome as a potential therapeutic target to counteract salt-sensitive conditions.

Achieving high accuracy in orthology inference is essential for many comparative, evolutionary and functional genomic analyses, yet the true evolutionary history of genes is generally unknown and orthologs are used for very different applications across phyla, requiring different precision-recall trade-offs. As a result, it is difficult to assess the performance of orthology inference methods. Here, we present a community effort to establish standards and an automated web-based service to facilitate orthology benchmarking. Using this service, we characterize 15 well-established inference methods and resources on a battery of 20 different benchmarks. Standardized benchmarking provides a way for users to identify the most effective methods for the problem at hand, sets a minimum requirement for new tools and resources, and guides the development of more accurate orthology inference methods.

Efficient nutrient usage is a trait under universal selection. A substantial part of cellular resources is spent on making nucleotides. We thus expect preferential use of cheaper nucleotides especially in transcribed sequences, which are often amplified thousand-fold compared with genomic sequences. To test this hypothesis, we derive a mutation-selection-drift equilibrium model for nucleotide skews (strand-specific usage of 'A' versus 'T' and 'G' versus 'C'), which explains nucleotide skews across 1,550 prokaryotic genomes as a consequence of selection on efficient resource usage. Transcription-related selection generally favours the cheaper nucleotides 'U' and 'C' at synonymous sites. However, the information encoded in mRNA is further amplified through translation. Due to unexpected trade-offs in the codon table, cheaper nucleotides encode on average energetically more expensive amino acids. These trade-offs apply to both strand-specific nucleotide usage and GC content, causing a universal bias towards the more expensive nucleotides 'A' and 'G' at non-synonymous coding sites.

We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated '-omics' data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes.

The unicellular cyanobacterium UCYN-A, one of the major contributors to nitrogen fixation in the open ocean, lives in symbiosis with single-celled phytoplankton. UCYN-A includes several closely related lineages whose partner fidelity, genome-wide expression and time of evolutionary divergence remain to be resolved. Here we detect and distinguish UCYN-A1 and UCYN-A2 lineages in symbiosis with two distinct prymnesiophyte partners in the South Atlantic Ocean. Both symbiotic systems are lineage specific and differ in the number of UCYN-A cells involved. Our analyses infer a streamlined genome expression towards nitrogen fixation in both UCYN-A lineages. Comparative genomics reveal a strong purifying selection in UCYN-A1 and UCYN-A2 with a diversification process ∼91 Myr ago, in the late Cretaceous, after the low-nutrient regime period occurred during the Jurassic. These findings suggest that UCYN-A diversified in a co-evolutionary process, wherein their prymnesiophyte partners acted as a barrier driving an allopatric speciation of extant UCYN-A lineages.

Data on bioactivities of drug-like chemicals is rapidly accumulating in public repositories, creating new opportunities for research in computational systems pharmacology. However, integrative analysis of these data sets is difficult due to prevailing ambiguity between chemical names and identifiers and a lack of cross-references between databases.

The biological carbon pump is the process by which CO2 is transformed to organic carbon via photosynthesis, exported through sinking particles, and finally sequestered in the deep ocean. While the intensity of the pump correlates with plankton community composition, the underlying ecosystem structure driving the process remains largely uncharacterized. Here we use environmental and metagenomic data gathered during the Tara Oceans expedition to improve our understanding of carbon export in the oligotrophic ocean. We show that specific plankton communities, from the surface and deep chlorophyll maximum, correlate with carbon export at 150 m and highlight unexpected taxa such as Radiolaria and alveolate parasites, as well as Synechococcus and their phages, as lineages most strongly associated with carbon export in the subtropical, nutrient-depleted, oligotrophic ocean. Additionally, we show that the relative abundance of a few bacterial and viral genes can predict a significant fraction of the variability in carbon export in these regions.

The Environment for Tree Exploration (ETE) is a computational framework that simplifies the reconstruction, analysis and visualization of phylogenetic trees and multiple sequence alignments. Here we present ETE v3, featuring numerous improvements in the underlying library of methods, and providing a novel set of standalone tools to perform common tasks in comparative genomics and phylogenetics. The new features include i) building gene-based and supermatrix-based phylogenies using a single command, ii) testing and visualizing evolutionary models, iii) calculating distances between trees of different size or including duplications, and iv) providing seamless integration with the NCBI taxonomy database. ETE is freely available at http://etetoolkit.org.

eggNOG is a public resource that provides Orthologous Groups (OGs) of proteins at different taxonomic levels, each with integrated and summarized functional annotations. Developments since the latest public release include changes to the algorithm for creating OGs across taxonomic levels, making nested groups hierarchically consistent. This allows for a better propagation of functional terms across nested OGs and led to the novel annotation of 95 890 previously uncharacterized OGs, increasing overall annotation coverage from 67% to 72%. The functional annotations of OGs have been expanded to also provide Gene Ontology terms, KEGG pathways and SMART/Pfam domains for each group. Moreover, eggNOG now provides pairwise orthology relationships within OGs based on analysis of phylogenetic trees. We have also incorporated a framework for quickly mapping novel sequences to OGs based on precomputed HMM profiles. Finally, eggNOG version 4.5 incorporates a novel data set spanning 2605 viral OGs, covering 5228 proteins from 352 viral proteomes. All data are accessible for bulk downloading, as a web-service, and through a completely redesigned web interface. The new access points provide faster searches and a number of new browsing and visualization capabilities, facilitating the needs of both experts and less experienced users. eggNOG v4.5 is available at http://eggnog.embl.de.

Early-life antibiotic use is associated with increased risk for metabolic and immunological diseases, and mouse studies indicate a causal role of the disrupted microbiome. However, little is known about the impacts of antibiotics on the developing microbiome of children. Here we use phylogenetics, metagenomics and individual antibiotic purchase records to show that macrolide use in 2-7 year-old Finnish children (N=142; sampled at two time points) is associated with a long-lasting shift in microbiota composition and metabolism. The shift includes depletion of Actinobacteria, increase in Bacteroidetes and Proteobacteria, decrease in bile-salt hydrolase and increase in macrolide resistance. Furthermore, macrolide use in early life is associated with increased risk of asthma and predisposes to antibiotic-associated weight gain. Overweight and asthmatic children have distinct microbiota compositions. Penicillins leave a weaker mark on the microbiota than macrolides. Our results support the idea that, without compromising clinical practice, the impact on the intestinal microbiota should be considered when prescribing antibiotics.

Unwanted side effects of drugs are a burden on patients and a severe impediment in the development of new drugs. At the same time, adverse drug reactions (ADRs) recorded during clinical trials are an important source of human phenotypic data. It is therefore essential to combine data on drugs, targets and side effects into a more complete picture of the therapeutic mechanism of actions of drugs and the ways in which they cause adverse reactions. To this end, we have created the SIDER ('Side Effect Resource', http://sideeffects.embl.de) database of drugs and ADRs. The current release, SIDER 4, contains data on 1430 drugs, 5880 ADRs and 140 064 drug-ADR pairs, which is an increase of 40% compared to the previous version. For more fine-grained analyses, we extracted the frequency with which side effects occur from the package inserts. This information is available for 39% of drug-ADR pairs, 19% of which can be compared to the frequency under placebo treatment. SIDER furthermore contains a data set of drug indications, extracted from the package inserts using Natural Language Processing. These drug indications are used to reduce the rate of false positives by identifying medical terms that do not correspond to ADRs.

MOCAT2 is a software pipeline for metagenomic sequence assembly and gene prediction with novel features for taxonomic and functional abundance profiling. The automated generation and efficient annotation of non-redundant reference catalogs by propagating pre-computed assignments from 18 databases covering various functional categories allows for fast and comprehensive functional characterization of metagenomes.

Interactive Tree Of Life (http://itol.embl.de) is a web-based tool for the display, manipulation and annotation of phylogenetic trees. It is freely available and open to everyone. The current version was completely redesigned and rewritten, utilizing current web technologies for speedy and streamlined processing. Numerous new features were introduced and several new data types are now supported. Trees with up to 100,000 leaves can now be efficiently displayed. Full interactive control over precise positioning of various annotation features and an unlimited number of datasets allow the easy creation of complex tree visualizations. iTOL 3 is the first tool which supports direct visualization of the recently proposed phylogenetic placements format. Finally, iTOL's account system has been redesigned to simplify the management of trees in user-defined workspaces and projects, as it is heavily used and currently handles already more than 500,000 trees from more than 10,000 individual users.

Fecal microbiota transplantation (FMT) has shown efficacy in treating recurrent Clostridium difficile infection and is increasingly being applied to other gastrointestinal disorders, yet the fate of native and introduced microbial strains remains largely unknown. To quantify the extent of donor microbiota colonization, we monitored strain populations in fecal samples from a recent FMT study on metabolic syndrome patients using single-nucleotide variants in metagenomes. We found extensive coexistence of donor and recipient strains, persisting 3 months after treatment. Colonization success was greater for conspecific strains than for new species, the latter falling within fluctuation levels observed in healthy individuals over a similar time frame. Furthermore, same-donor recipients displayed varying degrees of microbiota transfer, indicating individual patterns of microbiome resistance and donor-recipient compatibilities.

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.

The precise effects of HIV-1 on the gut microbiome are unclear. Initial cross-sectional studies provided contradictory associations between microbial richness and HIV serostatus and suggested shifts from Bacteroides to Prevotella predominance following HIV-1 infection, which have not been found in animal models or in studies matched for HIV-1 transmission groups. In two independent cohorts of HIV-1-infected subjects and HIV-1-negative controls in Barcelona (n = 156) and Stockholm (n = 84), men who have sex with men (MSM) predominantly belonged to the Prevotella-rich enterotype whereas most non-MSM subjects were enriched in Bacteroides, independently of HIV-1 status, and with only a limited contribution of diet effects. Moreover, MSM had a significantly richer and more diverse fecal microbiota than non-MSM individuals. After stratifying for sexual orientation, there was no solid evidence of an HIV-specific dysbiosis. However, HIV-1 infection remained consistently associated with reduced bacterial richness, the lowest bacterial richness being observed in subjects with a virological-immune discordant response to antiretroviral therapy. Our findings indicate that HIV gut microbiome studies must control for HIV risk factors and suggest interventions on gut bacterial richness as possible novel avenues to improve HIV-1-associated immune dysfunction.

Recent large-scale studies revealed cell-type specific proteomes. However, protein complexes, the basic functional modules of a cell, have been so far mostly considered as static entities with well-defined structures. The co-expression of their members has not been systematically charted at the protein level.

Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin-resistant individuals is characterized by increased levels of branched-chain amino acids (BCAAs), which correlate with a gut microbiome that has an enriched biosynthetic potential for BCAAs and is deprived of genes encoding bacterial inward transporters for these amino acids. Prevotella copri and Bacteroides vulgatus are identified as the main species driving the association between biosynthesis of BCAAs and insulin resistance, and in mice we demonstrate that P. copri can induce insulin resistance, aggravate glucose intolerance and augment circulating levels of BCAAs. Our findings suggest that microbial targets may have the potential to diminish insulin resistance and reduce the incidence of common metabolic and cardiovascular disorders.

The human gastrointestinal (GI) tract is the habitat for hundreds of microbial species, of which many cannot be cultivated readily, presumably because of the dependencies between species(1). Studies of microbial co-occurrence in the gut have indicated community substructures that may reflect functional and metabolic interactions between cohabiting species(2,3). To move beyond species co-occurrence networks, we systematically identified transcriptional interactions between pairs of coexisting gut microbes using metagenomics and microarray-based metatranscriptomics data from 233 stool samples from Europeans. In 102 significantly interacting species pairs, the transcriptional changes led to a reduced expression of orthologous functions between the coexisting species. Specific species-species transcriptional interactions were enriched for functions important for H2 and CO2 homeostasis, butyrate biosynthesis, ATP-binding cassette (ABC) transporters, flagella assembly and bacterial chemotaxis, as well as for the metabolism of carbohydrates, amino acids and cofactors. The analysis gives the first insight into the microbial community-wide transcriptional interactions, and suggests that the regulation of gene expression plays an important role in species adaptation to coexistence and that niche segregation takes place at the transcriptional level.

Ocean microbes drive biogeochemical cycling on a global scale. However, this cycling is constrained by viruses that affect community composition, metabolic activity, and evolutionary trajectories. Owing to challenges with the sampling and cultivation of viruses, genome-level viral diversity remains poorly described and grossly understudied, with less than 1% of observed surface-ocean viruses known. Here we assemble complete genomes and large genomic fragments from both surface- and deep-ocean viruses sampled during the Tara Oceans and Malaspina research expeditions, and analyse the resulting 'global ocean virome' dataset to present a global map of abundant, double-stranded DNA viruses complete with genomic and ecological contexts. A total of 15,222 epipelagic and mesopelagic viral populations were identified, comprising 867 viral clusters (defined as approximately genus-level groups). This roughly triples the number of known ocean viral populations and doubles the number of candidate bacterial and archaeal virus genera, providing a near-complete sampling of epipelagic communities at both the population and viral-cluster level. We found that 38 of the 867 viral clusters were locally or globally abundant, together accounting for nearly half of the viral populations in any global ocean virome sample. While two-thirds of these clusters represent newly described viruses lacking any cultivated representative, most could be computationally linked to dominant, ecologically relevant microbial hosts. Moreover, we identified 243 viral-encoded auxiliary metabolic genes, of which only 95 were previously known. Deeper analyses of four of these auxiliary metabolic genes (dsrC, soxYZ, P-II (also known as glnB) and amoC) revealed that abundant viruses may directly manipulate sulfur and nitrogen cycling throughout the epipelagic ocean. This viral catalog and functional analyses provide a necessary foundation for the meaningful integration of viruses into ecosystem models where they act as key players in nutrient cycling and trophic networks.

Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales.

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane.

The Rvb1/Rvb2 complex is an essential component of many cellular pathways. The Rvb1/Rvb2 complex forms a dodecameric assembly where six copies of each subunit form two heterohexameric rings. However, due to conformational variability, the way the two rings pack together is still not fully understood. Here, we present the crystal structure and two cryo-electron microscopy reconstructions of the dodecameric, full-length Rvb1/Rvb2 complex, all showing that the interaction between the two heterohexameric rings is mediated through the Rvb1/Rvb2-specific domain II. Two conformations of the Rvb1/Rvb2 dodecamer are present in solution: a stretched conformation also present in the crystal, and a compact conformation. Novel asymmetric features observed in the reconstruction of the compact conformation provide additional insight into the plasticity of the Rvb1/Rvb2 complex.

Interactions between proteins and small molecules are an integral part of biological processes in living organisms. Information on these interactions is dispersed over many databases, texts and prediction methods, which makes it difficult to get a comprehensive overview of the available evidence. To address this, we have developed STITCH ('Search Tool for Interacting Chemicals') that integrates these disparate data sources for 430 000 chemicals into a single, easy-to-use resource. In addition to the increased scope of the database, we have implemented a new network view that gives the user the ability to view binding affinities of chemicals in the interaction network. This enables the user to get a quick overview of the potential effects of the chemical on its interaction partners. For each organism, STITCH provides a global network; however, not all proteins have the same pattern of spatial expression. Therefore, only a certain subset of interactions can occur simultaneously. In the new, fifth release of STITCH, we have implemented functionality to filter out the proteins and chemicals not associated with a given tissue. The STITCH database can be downloaded in full, accessed programmatically via an extensive API, or searched via a redesigned web interface at http://stitch.embl.de.

Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient power to detect associations that are reproducible and significant after correction for multiple testing.

The assimilation of organic nutrients by autotrophs, a form of mixotrophy, has been demonstrated in the globally abundant marine picocyanobacterial genera Prochlorococcus and Synechococcus. However, the range of compounds used and the distribution of organic compound uptake genes within picocyanobacteria are unknown. Here we analyze genomic and metagenomic data from around the world to determine the extent and distribution of mixotrophy in these phototrophs. Analysis of 49 Prochlorococcus and 18 Synechococcus isolate genomes reveals that all have the transporters necessary to take up amino acids, peptides and sugars. However, the number and type of transporters and associated catabolic genes differ between different phylogenetic groups, with low-light IV Prochlorococcus, and 5.1B, 5.2 and 5.3 Synechococcus strains having the largest number. Metagenomic data from 68 stations from the Tara Oceans expedition indicate that the genetic potential for mixotrophy in picocyanobacteria is globally distributed and differs between clades. Phylogenetic analyses indicate gradual organic nutrient transporter gene loss from the low-light IV to the high-light II Prochlorococcus. The phylogenetic differences in genetic capacity for mixotrophy, combined with the ubiquity of picocyanobacterial organic compound uptake genes suggests that mixotrophy has a more central role in picocyanobacterial ecology than was previously thought.The ISME Journal advance online publication, 3 May 2016; doi:10.1038/ismej.2016.64.

Phosphorylation and proteolysis are among the most common post-translational modifications (PTMs), and play critical roles in various biological processes. More recent discoveries imply that the crosstalks between these two PTMs are involved in many diseases. In this work, we construct a post-translational regulatory network (PTRN) consists of phosphorylation and proteolysis processes, which enables us to investigate the regulatory interplays between these two PTMs. With the PTRN, we identify some functional network motifs that are significantly enriched with drug targets, some of which are further found to contain multiple proteins targeted by combinatorial drugs. These findings imply that the network motifs may be used to predict targets when designing new drugs. Inspired by this, we propose a novel computational approach called NetTar for predicting drug targets using the identified network motifs. Benchmarking results on real data indicate that our approach can be used for accurate prediction of novel proteins targeted by known drugs.

Systematic interrogation of mutation or protein modification data is important to identify sites with functional consequences and to deduce global consequences from large data sets. Mechismo (mechismo.russellab.org) enables simultaneous consideration of thousands of 3D structures and biomolecular interactions to predict rapidly mechanistic consequences for mutations and modifications. As useful functional information often only comes from homologous proteins, we benchmarked the accuracy of predictions as a function of protein/structure sequence similarity, which permits the use of relatively weak sequence similarities with an appropriate confidence measure. For protein-protein, protein-nucleic acid and a subset of protein-chemical interactions, we also developed and benchmarked a measure of whether modifications are likely to enhance or diminish the interactions, which can assist the detection of modifications with specific effects. Analysis of high-throughput sequencing data shows that the approach can identify interesting differences between cancers, and application to proteomics data finds potential mechanistic insights for how post-translational modifications can alter biomolecular interactions.

Quest for Orthologs (QfO) is a community effort with the goal to improve and benchmark orthology predictions. As quality assessment assumes prior knowledge on species phylogenies, we investigated the congruency between existing species trees by comparing the relationships of 147 QfO reference organisms from six Tree of Life (ToL) / species tree projects: the NCBI taxonomy, Opentree of Life, the sequenced species/species Tree of Life (sToL), the 16S rRNA database, and trees published by Ciccarelli et al in 2006, and by Huerta-Cepas et al in 2014. Our study reveals that each species tree suggests a different phylogeny: 87 out of 146 (60%) possible splits of a dichotomous and rooted tree are congruent, while all other splits are incongruent in at least one of the species trees. Topological differences are observed not only at deep speciation events, but also within younger clades, such as Hominidae, Rodentia, Laurasiatheria, or rosids. The evolutionary relationships of 27 archaea and bacteria are highly inconsistent. By assessing 458,108 gene trees from 65 genomes we show that consistent species topologies are more often supported by gene phylogenies than contradicting ones. The largest concordant species tree includes 77 of the QfO reference organisms at the most. Results are summarized in the form of a consensus ToL (http://swisstree.vital-it.ch/species_tree) that can serve different benchmarking purposes.

The ocean is the largest ecosystem on Earth, and yet we know very little about it. This is particularly true for the plankton that inhabit the ocean. Although these organisms are at least as important for the Earth system as the rainforests and form the base of marine food webs, most plankton are invisible to the naked eye and thus are largely uncharacterized. To study this invisible world, the multinational Tara Oceans consortium, with use of the 110-foot research schooner Tara, sampled microscopic plankton at 210 sites and depths up to 2000 m in all the major oceanic regions during expeditions from 2009 through 2013.

Viruses influence ecosystems by modulating microbial population size, diversity, metabolic outputs, and gene flow. Here, we use quantitative double-stranded DNA (dsDNA) viral-fraction metagenomes (viromes) and whole viral community morphological data sets from 43 Tara Oceans expedition samples to assess viral community patterns and structure in the upper ocean. Protein cluster cataloging defined pelagic upper-ocean viral community pan and core gene sets and suggested that this sequence space is well-sampled. Analyses of viral protein clusters, populations, and morphology revealed biogeographic patterns whereby viral communities were passively transported on oceanic currents and locally structured by environmental conditions that affect host community structure. Together, these investigations establish a global ocean dsDNA viromic data set with analyses supporting the seed-bank hypothesis to explain how oceanic viral communities maintain high local diversity.

Marine plankton support global biological and geochemical processes. Surveys of their biodiversity have hitherto been geographically restricted and have not accounted for the full range of plankton size. We assessed eukaryotic diversity from 334 size-fractionated photic-zone plankton communities collected across tropical and temperate oceans during the circumglobal Tara Oceans expedition. We analyzed 18S ribosomal DNA sequences across the intermediate plankton-size spectrum from the smallest unicellular eukaryotes (protists, >0.8 micrometers) to small animals of a few millimeters. Eukaryotic ribosomal diversity saturated at ~150,000 operational taxonomic units, about one-third of which could not be assigned to known eukaryotic groups. Diversity emerged at all taxonomic levels, both within the groups comprising the ~11,200 cataloged morphospecies of eukaryotic plankton and among twice as many other deep-branching lineages of unappreciated importance in plankton ecology studies. Most eukaryotic plankton biodiversity belonged to heterotrophic protistan groups, particularly those known to be parasites or symbiotic hosts.

Horizontal gene transfer has emerged as a crucial driving force for the evolution of eukaryotes. This also includes Plasmodium falciparum and related economically and clinically relevant apicomplexan parasites, whose rather small genomes have been shaped not only by natural selection in different host populations but also by horizontal gene transfer following endosymbiosis. However, there is rather little reliable data on horizontal gene transfer between animal hosts or bacteria and apicomplexan parasites. Here we show that apicomplexan homologues of peroxiredoxin 5 (Prx5) have a prokaryotic ancestry and therefore represent a special subclass of Prx5 isoforms in eukaryotes. Using two different immunobiochemical approaches, we found that the P. falciparum Prx5 homologue is dually localized to the parasite plastid and cytosol. This dual localization is reflected by a modular Plasmodium-specific gene architecture consisting of two exons. Despite the plastid localization, our phylogenetic analyses contradict an acquisition by secondary endosymbiosis and support a gene fusion event following a horizontal prokaryote-to-eukaryote gene transfer in early apicomplexans. The results provide unexpected insights into the evolution of apicomplexan parasites as well as the molecular evolution of peroxiredoxins, an important family of ubiquitous, usually highly concentrated thiol-dependent hydroperoxidases that exert functions as detoxifying enzymes, redox sensors and chaperones.

In recent years, several associations between common chronic human disorders and altered gut microbiome composition and function have been reported. In most of these reports, treatment regimens were not controlled for and conclusions could thus be confounded by the effects of various drugs on the microbiota, which may obscure microbial causes, protective factors or diagnostically relevant signals. Our study addresses disease and drug signatures in the human gut microbiome of type 2 diabetes mellitus (T2D). Two previous quantitative gut metagenomics studies of T2D patients that were unstratified for treatment yielded divergent conclusions regarding its associated gut microbial dysbiosis. Here we show, using 784 available human gut metagenomes, how antidiabetic medication confounds these results, and analyse in detail the effects of the most widely used antidiabetic drug metformin. We provide support for microbial mediation of the therapeutic effects of metformin through short-chain fatty acid production, as well as for potential microbiota-mediated mechanisms behind known intestinal adverse effects in the form of a relative increase in abundance of Escherichia species. Controlling for metformin treatment, we report a unified signature of gut microbiome shifts in T2D with a depletion of butyrate-producing taxa. These in turn cause functional microbiome shifts, in part alleviated by metformin-induced changes. Overall, the present study emphasizes the need to disentangle gut microbiota signatures of specific human diseases from those of medication.

Inverse gene expression profiling was recently shown to help drug repositioning. We showed that this approach works best for cancer and predicted novel drug candidates that may reduce metastasis in colorectal cancer. Antimetastatic activity of our predicted candidate, citalopram, was validated in an orthotopic mouse model of metastatic colorectal cancer.

SMART (Simple Modular Architecture Research Tool) is a web resource (http://smart.embl.de/) providing simple identification and extensive annotation of protein domains and the exploration of protein domain architectures. In the current version, SMART contains manually curated models for more than 1200 protein domains, with ∼200 new models since our last update article. The underlying protein databases were synchronized with UniProt, Ensembl and STRING, bringing the total number of annotated domains and other protein features above 100 million. SMART's 'Genomic' mode, which annotates proteins from completely sequenced genomes was greatly expanded and now includes 2031 species, compared to 1133 in the previous release. SMART analysis results pages have been completely redesigned and include links to several new information sources. A new, vector-based display engine has been developed for protein schematics in SMART, which can also be exported as high-resolution bitmap images for easy inclusion into other documents. Taxonomic tree displays in SMART have been significantly improved, and can be easily navigated using the integrated search engine.

Species interaction networks are shaped by abiotic and biotic factors. Here, as part of the Tara Oceans project, we studied the photic zone interactome using environmental factors and organismal abundance profiles and found that environmental factors are incomplete predictors of community structure. We found associations across plankton functional types and phylogenetic groups to be nonrandomly distributed on the network and driven by both local and global patterns. We identified interactions among grazers, primary producers, viruses, and (mainly parasitic) symbionts and validated network-generated hypotheses using microscopy to confirm symbiotic relationships. We have thus provided a resource to support further research on ocean food webs and integrating biological components into ocean models.

Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome-reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non-coding RNAs (ncRNAs) non-overlapping with essential genes is 5% higher than of non-transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non-coding regions, thus changing the focus when aiming to define the minimal essential genome.

Histone deacetylases (HDACs) and acetyltransferases control the epigenetic regulation of gene expression through modification of histone marks. Histone deacetylase inhibitors (HDACi) are small molecules that interfere with histone tail modification thus altering chromatin structure and epigenetically controlled pathways. They promote apoptosis in proliferating cells and are promising anti-cancer drugs. While some HDACis have already been approved for therapy and others are in different phases of clinical trials, the exact mechanism of action of this drug class remains elusive. Previous studies have shown that HDACis cause massive changes in chromatin structure but only moderate changes in gene expression. To which extent these changes manifest at the protein level has never been investigated on a proteome-wide scale. Here, we have studied HDACi-treated cells by large-scale mass spectrometry based proteomics. We show that HDACi treatment affects primarily the nuclear proteome and induces a selective decrease of bromodomain containing proteins (BCPs), the main readers of acetylated histone marks. By combining time-resolved proteome and transcriptome profiling, we show that BCPs are affected at the protein level as early as 12 hours after HDACi treatment and that their abundance is regulated by a combination of transcriptional and post-transcriptional mechanisms. Using gene silencing, we demonstrate that the decreased abundance of BCPs is sufficient to mediate important transcriptional changes induced by HDACi. Our data reveals a new aspect of the mechanism of action of HDACi that is mediated by an interplay between histone acetylation and the abundance of BCPs. Data are available via ProteomeXchange with identifier PXD001660 and NCBI Gene Expression Omnibus with identifier GSE64689.

The post-translational regulation of proteins is mainly driven by two molecular events, their modification by several types of moieties and their interaction with other proteins. These two processes are interdependent and together are responsible for the function of the protein in a particular cell state. Several databases focus on the prediction and compilation of protein-protein interactions (PPIs) and no less on the collection and analysis of protein post-translational modifications (PTMs), however, there are no resources that concentrate on describing the regulatory role of PTMs in PPIs. We developed several methods based on residue co-evolution and proximity to predict the functional associations of pairs of PTMs that we apply to modifications in the same protein and between two interacting proteins. In order to make data available for understudied organisms, PTMcode v2 (http://ptmcode.embl.de) includes a new strategy to propagate PTMs from validated modified sites through orthologous proteins. The second release of PTMcode covers 19 eukaryotic species from which we collected more than 300 000 experimentally verified PTMs (>1 300 000 propagated) of 69 types extracting the post-translational regulation of >100 000 proteins and >100 000 interactions. In total, we report 8 million associations of PTMs regulating single proteins and over 9.4 million interplays tuning PPIs.

The InterPro database (http://www.ebi.ac.uk/interpro/) is a freely available resource that can be used to classify sequences into protein families and to predict the presence of important domains and sites. Central to the InterPro database are predictive models, known as signatures, from a range of different protein family databases that have different biological focuses and use different methodological approaches to classify protein families and domains. InterPro integrates these signatures, capitalizing on the respective strengths of the individual databases, to produce a powerful protein classification resource. Here, we report on the status of InterPro as it enters its 15th year of operation, and give an overview of new developments with the database and its associated Web interfaces and software. In particular, the new domain architecture search tool is described and the process of mapping of Gene Ontology terms to InterPro is outlined. We also discuss the challenges faced by the resource given the explosive growth in sequence data in recent years. InterPro (version 48.0) contains 36 766 member database signatures integrated into 26 238 InterPro entries, an increase of over 3993 entries (5081 signatures), since 2012.

Aging is associated with the decline of protein, cell, and organ function. Here, we use an integrated approach to characterize gene expression, bulk translation, and cell biology in the brains and livers of young and old rats. We identify 468 differences in protein abundance between young and old animals. The majority are a consequence of altered translation output, that is, the combined effect of changes in transcript abundance and translation efficiency. In addition, we identify 130 proteins whose overall abundance remains unchanged but whose sub-cellular localization, phosphorylation state, or splice-form varies. While some protein-level differences appear to be a generic property of the rats' chronological age, the majority are specific to one organ. These may be a consequence of the organ's physiology or the chronological age of the cells within the tissue. Taken together, our study provides an initial view of the proteome at the molecular, sub-cellular, and organ level in young and old rats.

The Tara Oceans expedition (2009–2013) sampled contrasting ecosystems of the world oceans, collecting environmental data and plankton, from viruses to metazoans, for later analysis using modern sequencing and state-of-the-art imaging technologies. It surveyed 210 ecosystems in 20 biogeographic provinces, collecting over 35,000 samples of seawater and plankton. The interpretation of such an extensive collection of samples in their ecological context requires means to explore, assess and access raw and validated data sets. To address this challenge, the Tara Oceans Consortium offers open science resources, including the use of open access archives for nucleotides (ENA) and for environmental, biogeochemical, taxonomic and morphological data (PANGAEA), and the development of on line discovery tools and collaborative annotation tools for sequences and images. Here, we present an overview of Tara Oceans Data, and we provide detailed registries (data sets) of all campaigns (from port-to-port), stations and sampling events.

The Tara Oceans expedition (2009-2013) sampled contrasting ecosystems of the world oceans, collecting environmental data and plankton, from viruses to metazoans, for later analysis using modern sequencing and state-of-the-art imaging technologies. It surveyed 210 ecosystems in 20 biogeographic provinces, collecting over 35,000 samples of seawater and plankton. The interpretation of such an extensive collection of samples in their ecological context requires means to explore, assess and access raw and validated data sets. To address this challenge, the Tara Oceans Consortium offers open science resources, including the use of open access archives for nucleotides (ENA) and for environmental, biogeochemical, taxonomic and morphological data (PANGAEA), and the development of on line discovery tools and collaborative annotation tools for sequences and images. Here, we present an overview of Tara Oceans Data, and we provide detailed registries (data sets) of all campaigns (from port-to-port), stations and sampling events.

Assembly of protein complexes is considered a post-translational process involving random collision of subunits. We show that within the Escherichia coli cytosol, bacterial luciferase subunits LuxA and LuxB assemble into complexes close to the site of subunit synthesis. Assembly efficiency decreases markedly if subunits are synthesized on separate mRNAs from genes integrated at distant chromosomal sites. Subunit assembly initiates cotranslationally on nascent LuxB in vivo. The ribosome-associated chaperone Trigger Factor delays the onset of cotranslational interactions until the LuxB dimer interface is fully exposed. Protein assembly thus is directly coupled to the translation process, and involves spatially confined, actively chaperoned cotranslational subunit interactions. Bacterial gene organization into operons therefore reflects a fundamental mechanism for spatiotemporal regulation vital to effective cotranslational protein complex assembly.

Microbes are dominant drivers of biogeochemical processes, yet drawing a global picture of functional diversity, microbial community structure, and their ecological determinants remains a grand challenge. We analyzed 7.2 terabases of metagenomic data from 243 Tara Oceans samples from 68 locations in epipelagic and mesopelagic waters across the globe to generate an ocean microbial reference gene catalog with >40 million nonredundant, mostly novel sequences from viruses, prokaryotes, and picoeukaryotes. Using 139 prokaryote-enriched samples, containing >35,000 species, we show vertical stratification with epipelagic community composition mostly driven by temperature rather than other environmental factors or geography. We identify ocean microbial core functionality and reveal that >73% of its abundance is shared with the human gut microbiome despite the physicochemical differences between these two ecosystems.

In molecular systems biology, data are flooding us at an ever�?increasing pace, from genomic to transcriptomic and proteomic information, complemented by spatially and time�?resolved data obtained at multiple scales. Computational biology usually integrates these layers of information at the cellular and biochemical levels. But how does the interplay between experimental and computational biology work if this information is coming not only from a cellular system, but from an entire ecosystem and if this ecosystem spans the entire Earth? Here, we illustrate some of the computational challenges and promises of large�?scale eco�?systems biology studies (Raes & Bork, 2008) in the context of the Tara Oceans project (Fig 1), which has arguably been one of the wettest wet laboratory experiments ever.

The many functional partnerships and interactions that occur between proteins are at the core of cellular processing and their systematic characterization helps to provide context in molecular systems biology. However, known and predicted interactions are scattered over multiple resources, and the available data exhibit notable differences in terms of quality and completeness. The STRING database (http://string-db.org) aims to provide a critical assessment and integration of protein-protein interactions, including direct (physical) as well as indirect (functional) associations. The new version 10.0 of STRING covers more than 2000 organisms, which has necessitated novel, scalable algorithms for transferring interaction information between organisms. For this purpose, we have introduced hierarchical and self-consistent orthology annotations for all interacting proteins, grouping the proteins into families at various levels of phylogenetic resolution. Further improvements in version 10.0 include a completely redesigned prediction pipeline for inferring protein-protein associations from co-expression data, an API interface for the R computing environment and improved statistical analysis for enrichment tests in user-provided networks.

Rapid development of high-throughput (HTS) molecular identification methods has revolutionized our knowledge about taxonomic diversity and ecology of fungi. However, PCR-based methods exhibit multiple technical shortcomings that may bias our understanding of the fungal kingdom. This study was initiated to quantify potential biases in fungal community ecology by comparing the relative performance of amplicon-free shotgun metagenomics and amplicons of nine primer pairs over seven nuclear ribosomal DNA (rDNA) regions often used in metabarcoding analyses. The internal transcribed spacer (ITS) barcodes ITS1 and ITS2 provided greater taxonomic and functional resolution and richness of operational taxonomic units (OTUs) at the 97% similarity threshold compared to barcodes located within the ribosomal small subunit (SSU) and large subunit (LSU) genes. All barcode-primer pair combinations provided consistent results in ranking taxonomic richness and recovering the importance of floristic variables in driving fungal community composition in soils of Papua New Guinea. The choice of forward primer explained up to 2.0% of the variation in OTU-level analysis of the ITS1 and ITS2 barcode data sets. Across the whole data set, barcode-primer pair combination explained 37.6–38.1% of the variation, which surpassed any environmental signal. Overall, the metagenomics data set recovered a similar taxonomic overview, but resulted in much lower fungal rDNA sequencing depth, inability to infer OTUs, and high uncertainty in identification. We recommend the use of ITS2 or the whole ITS region for metabarcoding and we advocate careful choice of primer pairs in consideration of the relative proportion of fungal DNA and expected dominant groups.

Agulhas rings provide the principal route for ocean waters to circulate from the Indo-Pacific to the Atlantic basin. Their influence on global ocean circulation is well known, but their role in plankton transport is largely unexplored. We show that, although the coarse taxonomic structure of plankton communities is continuous across the Agulhas choke point, South Atlantic plankton diversity is altered compared with Indian Ocean source populations. Modeling and in situ sampling of a young Agulhas ring indicate that strong vertical mixing drives complex nitrogen cycling, shaping community metabolism and biogeochemical signatures as the ring and associated plankton transit westward. The peculiar local environment inside Agulhas rings may provide a selective mechanism contributing to the limited dispersal of Indian Ocean plankton populations into the Atlantic.

Metagenomics has become a prominent approach for exploring the role of the gut microbiota in human health. However, the temporal variability of the healthy gut microbiome has not yet been studied in depth using metagenomics and little is known about the effects of different sampling and preservation approaches. We performed metagenomic analysis on fecal samples from seven subjects collected over a period of up to two years to investigate temporal variability and assess preservation-induced variation, specifically, fresh frozen compared to RNALater. We also monitored short-term disturbances caused by antibiotic treatment and bowel cleansing in one subject.

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block-although compositionally identical-engage in different local sets of interactions and conformations.

Many cellular processes involve the recruitment of proteins to specific membranes, which are decorated with distinctive lipids that act as docking sites. The phosphoinositides form signaling hubs, and we examine mechanisms underlying recruitment. We applied a physiological, quantitative, liposome microarray-based assay to measure the membrane-binding properties of 91 pleckstrin homology (PH) domains, the most common phosphoinositide-binding target. 10,514 experiments quantified the role of phosphoinositides in membrane recruitment. For most domains examined, the observed binding specificity implied cooperativity with additional signaling lipids. Analyses of PH domains with similar lipid-binding profiles identified a conserved motif, mutations in which-including some found in human cancers-induced discrete changes in binding affinities in vitro and protein mislocalization in vivo. The data set reveals cooperativity as a key mechanism for membrane recruitment and, by enabling the interpretation of disease-associated mutations, suggests avenues for the design of small molecules targeting PH domains.

Different DNA mismatch repair (MMR)-deficient mouse strains have been developed as models for the inherited cancer predisposing Lynch syndrome. It is completely unresolved, whether coding mononucleotide repeat (cMNR) gene mutations in these mice can contribute to intestinal tumorigenesis and whether MMR-deficient mice are a suitable molecular model of human microsatellite instability (MSI)-associated intestinal tumorigenesis. A proof-of-principle study was performed to identify mouse cMNR-harboring genes affected by insertion/deletion mutations in MSI murine intestinal tumors. Bioinformatic algorithms were developed to establish a database of mouse cMNR-harboring genes. A panel of five mouse noncoding mononucleotide markers was used for MSI classification of intestinal matched normal/tumor tissues from MMR-deficient (Mlh1(-/-) , Msh2(-/-) , Msh2(LoxP/LoxP) ) mice. cMNR frameshift mutations of candidate genes were determined by DNA fragment analysis. Murine MSI intestinal tumors but not normal tissues from MMR-deficient mice showed cMNR frameshift mutations in six candidate genes (Elavl3, Tmem107, Glis2, Sdccag1, Senp6, Rfc3). cMNRs of mouse Rfc3 and Elavl3 are conserved in type and length in their human orthologs that are known to be mutated in human MSI colorectal, endometrial and gastric cancer. We provide evidence for the utility of a mononucleotide marker panel for detection of MSI in murine tumors, the existence of cMNR instability in MSI murine tumors, the utility of mouse subspecies DNA for identification of polymorphic repeats, and repeat conservation among some orthologous human/mouse genes, two of them showing instability in human and mouse MSI intestinal tumors. MMR-deficient mice hence are a useful molecular model system for analyzing MSI intestinal carcinogenesis. © 2014 Wiley Periodicals, Inc.

Microbial communities populate most environments on earth and play a critical role in ecology and human health. Their composition is thought to be largely shaped by interspecies competition for the available resources, but cooperative interactions, such as metabolite exchanges, have also been implicated in community assembly. The prevalence of metabolic interactions in microbial communities, however, has remained largely unknown. Here, we systematically survey, by using a genome-scale metabolic modeling approach, the extent of resource competition and metabolic exchanges in over 800 communities. We find that, despite marked resource competition at the level of whole assemblies, microbial communities harbor metabolically interdependent groups that recur across diverse habitats. By enumerating flux-balanced metabolic exchanges in these co-occurring subcommunities we also predict the likely exchanged metabolites, such as amino acids and sugars, that can promote group survival under nutritionally challenging conditions. Our results highlight metabolic dependencies as a major driver of species co-occurrence and hint at cooperative groups as recurring modules of microbial community architecture.

Gut microbiota has been implicated as a pivotal contributing factor in diet-related obesity; however, its role in development of disease phenotypes in human genetic obesity such as Prader-Willi syndrome (PWS) remains elusive. In this hospitalized intervention trial with PWS (n = 17) and simple obesity (n = 21) children, a diet rich in non-digestible carbohydrates induced significant weight loss and concomitant structural changes of the gut microbiota together with reduction of serum antigen load and alleviation of inflammation. Co-abundance network analysis of 161 prevalent bacterial draft genomes assembled directly from metagenomic datasets showed relative increase of functional genome groups for acetate production from carbohydrates fermentation. NMR-based metabolomic profiling of urine showed diet-induced overall changes of host metabotypes and identified significantly reduced trimethylamine N-oxide and indoxyl sulfate, host-bacteria co-metabolites known to induce metabolic deteriorations. Specific bacterial genomes that were correlated with urine levels of these detrimental co-metabolites were found to encode enzyme genes for production of their precursors by fermentation of choline or tryptophan in the gut. When transplanted into germ-free mice, the pre-intervention gut microbiota induced higher inflammation and larger adipocytes compared with the post-intervention microbiota from the same volunteer. Our multi-omics-based systems analysis indicates a significant etiological contribution of dysbiotic gut microbiota to both genetic and simple obesity in children, implicating a potentially effective target for alleviation.

Gene content differences in human gut microbes can lead to inter-individual phenotypic variations such as digestive capacity. It is unclear whether gene content variation is caused by differences in microbial species composition or by the presence of different strains of the same species; the extent of gene content variation in the latter is unknown. Unlike pan-genome studies of cultivable strains, the use of metagenomic data can provide an unbiased view of structural variation of gut bacterial strains by measuring them in their natural habitats, the gut of each individual in this case, representing native boundaries between gut bacterial populations. We analyzed publicly available metagenomics data from fecal samples to characterize inter-individual variation in gut bacterial species.

It has been drawn to our attention that the methods described in the main text and the Supplementary Information of this Article have been considered by some researchers to be insufficient to enable them to identify enterotypes in their own data sets. Enterotypes were originally defined in this Article (page 177) as “densely populated areas in a multi-dimensional space of community composition�? and should not be seen as discrete clusters, but as a way of stratifying samples to reduce complexity. Additionally, the Fig. 2 legend should not imply that between-class analysis is simply a method of visualizing principal component analysis (PCA); rather, it is a supervised rather than an unsupervised analysis of data because it incorporates the outcome of clustering of data. To simplify enterotype identification in the original and other data sets, we have developed a comprehensive tutorial at http://enterotype.embl.de—which is a website on enterotypes that will be updated as methods improve. We thank Ivica Letunic and Paul Costea from EMBL for setting up the tutorial.

The thermophilic fungus Chaetomium thermophilum holds great promise for structural biology. To increase the efficiency of its biochemical and structural characterization and to explore its thermophilic properties beyond those of individual proteins, we obtained transcriptomics and proteomics data, and integrated them with computational annotation methods and a multitude of biochemical experiments conducted by the structural biology community. We considerably improved the genome annotation of Chaetomium thermophilum and characterized the transcripts and expression of thousands of genes. We furthermore show that the composition and structure of the expressed proteome of Chaetomium thermophilum is similar to its mesophilic relatives. Data were deposited in a publicly available repository and provide a rich source to the structural biology community.

Vor 20 Jahren wurden Bioinformatiker belächelt oder als Datenpiraten verunglimpft. Heute kommt die Biologie nicht mehr ohne sie aus. Die Bioinformatik ist teilweise nur noch ein Sammelbegriff unter dem sich verschiedenste Teildiziplinen gruppieren. Eigentlich ist Bioinformatik heute viel spannender, vielfältiger - auch erfolgsträchtiger - als vor 20 Jahren und kann disziplinübergreifend eingesetzt werden.

Morphogenesis is the process whereby cell collectives are shaped into differentiated tissues and organs. The self-organizing nature of morphogenesis has been recently demonstrated by studies showing that stem cells in three-dimensional culture can generate complex organoids, such as mini-guts, optic-cups and even mini-brains. To achieve this, cell collectives must regulate the activity of secreted signalling molecules that control cell differentiation, presumably through the self-assembly of microenvironments or niches. However, mechanisms that allow changes in tissue architecture to feedback directly on the activity of extracellular signals have not been described. Here we investigate how the process of tissue assembly controls signalling activity during organogenesis in vivo, using the migrating zebrafish lateral line primordium. We show that fibroblast growth factor (FGF) activity within the tissue controls the frequency at which it deposits rosette-like mechanosensory organs. Live imaging reveals that FGF becomes specifically concentrated in microluminal structures that assemble at the centre of these organs and spatially constrain its signalling activity. Genetic inhibition of microlumen assembly and laser micropuncture experiments demonstrate that microlumina increase signalling responses in participating cells, thus allowing FGF to coordinate the migratory behaviour of cell groups at the tissue rear. As the formation of a central lumen is a self-organizing property of many cell types, such as epithelia and embryonic stem cells, luminal signalling provides a potentially general mechanism to locally restrict, coordinate and enhance cell communication within tissues.

We show how metagenomic analysis of the human gut antibiotic resistome, compared across large populations and against environmental or agricultural resistomes, suggests a strong anthropogenic cause behind increasing antibiotic resistance in bacteria. This area has been the subject of intense and polarized debate driven by economic and political concerns; therefore such recently available insights address an important need. We derive and compare antibiotic resistomes of human gut microbes from 832 individuals from ten different countries. We observe and describe significant differences between samples from these countries in the gut resistance potential, in line with expectations from antibiotic usage and exposure in medical and food production contexts. Our results imply roles for both of these sources in increased resistance among pathogens in recent history. In contrast, other available metadata such as age, body mass index, sex, or health status have little effect on the antibiotic resistance potential of human gut microbes. Also watch the Video Abstract.

The large number of macromolecular structures deposited with the Protein Data Bank (PDB) describing complexes between proteins and either physiological compounds or synthetic drugs made it possible a systematic analysis of the interactions occurring between proteins and their ligands. In this work, the binding pockets of about 4000 PDB protein-ligand complexes were investigated and amino acid and interaction types were analyzed. The residues observed with lowest frequency in protein sequences, Trp, His, Met, Tyr, and Phe, turned out to be the most abundant in binding pockets. Significant differences between drug-like and physiological compounds were found. On average, physiological compounds establish with respect to drugs about twice as many hydrogen bonds with protein atoms, whereas drugs rely more on hydrophobic interactions to establish target selectivity. The large number of PDB structures describing homologous proteins in complex with the same ligand made it possible to analyze the conservation of binding pocket residues among homologous protein structures bound to the same ligand, showing that Gly, Glu, Arg, Asp, His, and Thr are more conserved than other amino acids. Also in the cases in which the same ligand is bound to unrelated proteins, the binding pockets showed significant conservation in the residue types. In this case, the probability of co-occurrence of the same amino acid type in the binding pockets could be up to thirteen times higher than that expected on a random basis. The trends identified in this study may provide an useful guideline in the process of drug design and lead optimization. Copyright © 2014 John Wiley & Sons, Ltd.

16S ribosomal DNA (rDNA) amplicon sequencing is frequently used to analyse the structure of bacterial communities from oceans to the human microbiota. However, computational power is still a major bottleneck in the analysis of continuously enlarging metagenomic data sets. Analysis is further complicated by the technical complexity of current bioinformatics tools.

Background: 16S ribosomal DNA (rDNA) amplicon sequencing is frequently used to analyse the structure of bacterial communities from oceans to the human microbiota. However, computational power is still a major bottleneck in the analysis of continuously enlarging metagenomic data sets. Analysis is further complicated by the technical complexity of current bioinformatics tools. Results: Here we present the less operational taxonomic units scripts (LotuS), a fast and user-friendly open-source tool to calculate denoised, chimera-checked, operationa l taxonomic units (OTUs). These are the basis to generate taxonomic abundance tables and phylogenetic trees from multiplexed, next-generation sequencing data (454, illumina MiSeq and HiSeq). LotuS is outstanding in its execution speed, as it can process 16S rDNA data up to two orders of magnitude faster than other existing pipelines. This is partly due to an included stand-alone fast simultaneous demultiplexer and quality filter C++ program, simple demultiplexer (sdm), which comes packaged with LotuS. Additionally, we sequenced two MiSeq runs with the intent to validate future pipelines by sequencing 40 technical replicates; these are made available in this work. Conclusion: We show that LotuS analyses microbial 16S data with comparable or even better results than existing pipelines, requiring a fraction of the execution time and providing state-of-the-art denoising and phylogenetic reconstruction. LotuS is available through the following URL: http://psbweb05.psb.ugent.be/lotus. Keywords: OTU, 16S rDNA gene, Pipeline, Metagenomics, Demultiplexing [This corrects the article on p. 30 in vol. 2.].

STITCH is a database of protein-chemical interactions that integrates many sources of experimental and manually curated evidence with text-mining information and interaction predictions. Available at http://stitch.embl.de, the resulting interaction network includes 390 000 chemicals and 3.6 million proteins from 1133 organisms. Compared with the previous version, the number of high-confidence protein-chemical interactions in human has increased by 45%, to 367 000. In this version, we added features for users to upload their own data to STITCH in the form of internal identifiers, chemical structures or quantitative data. For example, a user can now upload a spreadsheet with screening hits to easily check which interactions are already known. To increase the coverage of STITCH, we expanded the text mining to include full-text articles and added a prediction method based on chemical structures. We further changed our scheme for transferring interactions between species to rely on orthology rather than protein similarity. This improves the performance within protein families, where scores are now transferred only to orthologous proteins, but not to paralogous proteins. STITCH can be accessed with a web-interface, an API and downloadable files.

Many analyses of the human gut microbiome depend on a catalog of reference genes. Existing catalogs for the human gut microbiome are based on samples from single cohorts or on reference genomes or protein sequences, which limits coverage of global microbiome diversity. Here we combined 249 newly sequenced samples of the Metagenomics of the Human Intestinal Tract (MetaHit) project with 1,018 previously sequenced samples to create a cohort from three continents that is at least threefold larger than cohorts used for previous gene catalogs. From this we established the integrated gene catalog (IGC) comprising 9,879,896 genes. The catalog includes close-to-complete sets of genes for most gut microbes, which are also of considerably higher quality than in previous catalogs. Analyses of a group of samples from Chinese and Danish individuals using the catalog revealed country-specific gut microbial signatures. This expanded catalog should facilitate quantitative characterization of metagenomic, metatranscriptomic and metaproteomic data from the gut microbiome to understand its variation across populations in human health and disease.

Sequencing of 16S rDNA polymerase chain reaction (PCR) amplicons is the most common approach for investigating environmental prokaryotic diversity, despite the known biases introduced during PCR. Here we show that 16S rDNA fragments derived from Illumina-sequenced environmental metagenomes (mi tags) are a powerful alternative to 16S rDNA amplicons for investigating the taxonomic diversity and structure of prokaryotic communities. As part of the Tara Oceans global expedition, marine plankton was sampled in three locations, resulting in 29 subsamples for which metagenomes were produced by shotgun Illumina sequencing (ca. 700 Gb). For comparative analyses, a subset of samples was also selected for Roche-454 sequencing using both shotgun (m454 tags; 13 metagenomes, ca. 2.4 Gb) and 16S rDNA amplicon (454 tags; ca. 0.075 Gb) approaches. Our results indicate that by overcoming PCR biases related to amplification and primer mismatch, mi tags may provide more realistic estimates of community richness and evenness than amplicon 454 tags. In addition, mi tags can capture expected beta diversity patterns. Using mi tags is now economically feasible given the dramatic reduction in high-throughput sequencing costs, having the advantage of retrieving simultaneously both taxonomic (Bacteria, Archaea and Eukarya) and functional information from the same microbial community.

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

With the increasing availability of various 'omics data, high-quality orthology assignment is crucial for evolutionary and functional genomics studies. We here present the fourth version of the eggNOG database (available at http://eggnog.embl.de) that derives nonsupervised orthologous groups (NOGs) from complete genomes, and then applies a comprehensive characterization and analysis pipeline to the resulting gene families. Compared with the previous version, we have more than tripled the underlying species set to cover 3686 organisms, keeping track with genome project completions while prioritizing the inclusion of high-quality genomes to minimize error propagation from incomplete proteome sets. Major technological advances include (i) a robust and scalable procedure for the identification and inclusion of high-quality genomes, (ii) provision of orthologous groups for 107 different taxonomic levels compared with 41 in eggNOGv3, (iii) identification and annotation of particularly closely related orthologous groups, facilitating analysis of related gene families, (iv) improvements of the clustering and functional annotation approach, (v) adoption of a revised tree building procedure based on the multiple alignments generated during the process and (vi) implementation of quality control procedures throughout the entire pipeline. As in previous versions, eggNOGv4 provides multiple sequence alignments and maximum-likelihood trees, as well as broad functional annotation. Users can access the complete database of orthologous groups via a web interface, as well as through bulk download.

Lipids have a role in virtually all biological processes, acting as structural elements, scaffolds and signaling molecules, but they are still largely under-represented in known biological networks. Here we describe a liposome microarray-based assay (LiMA), a method that measures protein recruitment to membranes in a quantitative, automated, multiplexed and high-throughput manner.

Accurate orthology prediction is crucial for many applications in the post-genomic era. The lack of broadly accepted benchmark tests precludes a comprehensive analysis of orthology inference. So far, functional annotation between orthologs serves as a performance proxy. However, this violates the fundamental principle of orthology as an evolutionary definition, while it is often not applicable due to limited experimental evidence for most species. Therefore, we constructed high quality "gold standard" orthologous groups that can serve as a benchmark set for orthology inference in bacterial species. Herein, we used this dataset to demonstrate 1) why a manually curated, phylogeny-based dataset is more appropriate for benchmarking orthology than other popular practices and 2) how it guides database design and parameterization through careful error quantification. More specifically, we illustrate how function-based tests often fail to identify false assignments, misjudging the true performance of orthology inference methods. We also examined how our dataset can instruct the selection of a "core" species repertoire to improve detection accuracy. We conclude that including more genomes at the proper evolutionary distances can influence the overall quality of orthology detection. The curated gene families, called Reference Orthologous Groups, are publicly available at http://eggnog.embl.de/orthobench2.

Drug-induced gene expression profiles that invert disease profiles have recently been illustrated to be a starting point for drug-repositioning. In this study, we validate this approach and focus on prediction of novel drugs for colorectal cancer (CRC), for which there is a pressing need to find novel anti-metastatic compounds. We computationally predicted three novel and still unknown compounds against CRC; citalopram (an anti-depressant), troglitazone (an anti-diabetic) and enilconazole (a fungicide). We verified the compounds by in vitro assays of clonogenic survival, proliferation and migration and in a subcutaneous mouse model. We found evidence that the mode-of-action of these compounds may be through inhibition of TGF-β signaling. Furthermore, one compound, citalopram, reduced tumor size as well as the number of circulating tumor cells and metastases in an orthotopic mouse model of CRC. This study proposes citalopram as a potential therapeutic option for CRC patients illustrating the potential of systems pharmacology.

Bacteriophages have key roles in microbial communities, to a large extent shaping the taxonomic and functional composition of the microbiome, but data on the connections between phage diversity and the composition of communities are scarce. Using taxon-specific marker genes, we identified and monitored 20 viral taxa in 252 human gut metagenomic samples, mostly at the level of genera. On average, five phage taxa were identified in each sample, with up to three of these being highly abundant. The abundances of most phage taxa vary by up to four orders of magnitude between the samples, and several taxa that are highly abundant in some samples are absent in others. Significant correlations exist between the abundances of some phage taxa and human host metadata: for example, 'Group 936 lactococcal phages' are more prevalent and abundant in Danish samples than in samples from Spain or the United States of America. Quantification of phages that exist as integrated prophages revealed that the abundance profiles of prophages are highly individual-specific and remain unique to an individual over a 1-year time period, and prediction of prophage lysis across the samples identified hundreds of prophages that are apparently active in the gut and vary across the samples, in terms of presence and lytic state. Finally, a prophage-host network of the human gut was established and includes numerous novel host-phage associations.

Several bacterial species have been implicated in the development of colorectal carcinoma (CRC), but CRC-associated changes of fecal microbiota and their potential for cancer screening remain to be explored. Here, we used metagenomic sequencing of fecal samples to identify taxonomic markers that distinguished CRC patients from tumor-free controls in a study population of 156 participants. Accuracy of metagenomic CRC detection was similar to the standard fecal occult blood test (FOBT) and when both approaches were combined, sensitivity improved > 45% relative to the FOBT, while maintaining its specificity. Accuracy of metagenomic CRC detection did not differ significantly between early- and late-stage cancer and could be validated in independent patient and control populations (N = 335) from different countries. CRC-associated changes in the fecal microbiome at least partially reflected microbial community composition at the tumor itself, indicating that observed gene pool differences may reveal tumor-related host-microbe interactions. Indeed, we deduced a metabolic shift from fiber degradation in controls to utilization of host carbohydrates and amino acids in CRC patients, accompanied by an increase of lipopolysaccharide metabolism.

Protein post-translational modifications (PTMs) allow the cell to regulate protein activity and play a crucial role in the response to changes in external conditions or internal states. Advances in mass spectrometry now enable proteome wide characterization of PTMs and have revealed a broad functional role for a range of different types of modifications. Here we review advances in the study of the evolution and function of PTMs that were spurred by these technological improvements. We provide an overview of studies focusing on the origin and evolution of regulatory enzymes as well as the evolutionary dynamics of modification sites. Finally, we discuss different mechanisms of altering protein activity via post-translational regulation and progress made in the large-scale functional characterization of PTM function.

One of the most exciting scientific advances in recent years has been the realization that commensal microorganisms are not simple 'passengers' in our bodies, but instead have key roles in our physiology, including our immune responses and metabolism, as well as in disease. These insights have been obtained, in part, through the work of large-scale, consortium-driven metagenomic projects. Here, five experts in the field of microbiome research discuss the most surprising and exciting new findings, and outline the future steps that will be necessary to elucidate the numerous roles of the microbiota in human health and disease and to develop viable therapeutic strategies.

Mendelian disorders are often caused by mutations in genes that are not lethal but induce functional distortions leading to diseases. Here we study the extent of gene duplicates that might compensate genes causing monogenic diseases. We provide evidence for pervasive functional redundancy of human monogenic disease genes (MDs) by duplicates by manifesting 1) genes involved in human genetic disorders are enriched in duplicates and 2) duplicated disease genes tend to have higher functional similarities with their closest paralogs in contrast to duplicated non-disease genes of similar age. We propose that functional compensation by duplication of genes masks the phenotypic effects of deleterious mutations and reduces the probability of purging the defective genes from the human population; this functional compensation could be further enhanced by higher purification selection between disease genes and their duplicates as well as their orthologous counterpart compared to non-disease genes. However, due to the intrinsic expression stochasticity among individuals, the deleterious mutations could still be present as genetic diseases in some subpopulations where the duplicate copies are expressed at low abundances. Consequently the defective genes are linked to genetic disorders while they continue propagating within the population. Our results provide insight into the molecular basis underlying the spreading of duplicated disease genes.

The interactions between proteins and nucleic acids have a fundamental function in many biological processes, including gene transcription, RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins that bind individual mRNAs in mammalian cells has been greatly augmented by recent surveys, no systematic study on the non-sequence-specific engagement of native human proteins with various types of nucleic acids has been reported.

Despite increasing concerns over inappropriate use of antibiotics in medicine and food production, population-level resistance transfer into the human gut microbiota has not been demonstrated beyond individual case studies. To determine the "antibiotic resistance potential" for entire microbial communities, we employ metagenomic data and quantify the totality of known resistance genes in each community (its resistome) for 68 classes and subclasses of antibiotics. In 252 fecal metagenomes from three countries, we show that the most abundant resistance determinants are those for antibiotics also used in animals, and for antibiotics that have been available longer. Resistance genes are also more abundant in samples from Spain, Italy and France than from Denmark, the US, or Japan. Where comparable country-level data on antibiotic use in both humans and animals are available, differences in these statistics match the observed resistance potential differences. The results are robust over time as the antibiotic resistance determinants of individuals persist in the human gut flora for at least a year.

Complete knowledge of all direct and indirect interactions between proteins in a given cell would represent an important milestone towards a comprehensive description of cellular mechanisms and functions. Although this goal is still elusive, considerable progress has been made-particularly for certain model organisms and functional systems. Currently, protein interactions and associations are annotated at various levels of detail in online resources, ranging from raw data repositories to highly formalized pathway databases. For many applications, a global view of all the available interaction data is desirable, including lower-quality data and/or computational predictions. The STRING database (http://string-db.org/) aims to provide such a global perspective for as many organisms as feasible. Known and predicted associations are scored and integrated, resulting in comprehensive protein networks covering >1100 organisms. Here, we describe the update to version 9.1 of STRING, introducing several improvements: (i) we extend the automated mining of scientific texts for interaction information, to now also include full-text articles; (ii) we entirely re-designed the algorithm for transferring interactions from one model organism to the other; and (iii) we provide users with statistical information on any functional enrichment observed in their networks.

Nucleo-cytoplasmic large DNA viruses (NCLDVs) constitute a group of eukaryotic viruses that can have crucial ecological roles in the sea by accelerating the turnover of their unicellular hosts or by causing diseases in animals. To better characterize the diversity, abundance and biogeography of marine NCLDVs, we analyzed 17 metagenomes derived from microbial samples (0.2-1.6 μm size range) collected during the Tara Oceans Expedition. The sample set includes ecosystems under-represented in previous studies, such as the Arabian Sea oxygen minimum zone (OMZ) and Indian Ocean lagoons. By combining computationally derived relative abundance and direct prokaryote cell counts, the abundance of NCLDVs was found to be in the order of 10(4)-10(5) genomes ml(-1) for the samples from the photic zone and 10(2)-10(3) genomes ml(-1) for the OMZ. The Megaviridae and Phycodnaviridae dominated the NCLDV populations in the metagenomes, although most of the reads classified in these families showed large divergence from known viral genomes. Our taxon co-occurrence analysis revealed a potential association between viruses of the Megaviridae family and eukaryotes related to oomycetes. In support of this predicted association, we identified six cases of lateral gene transfer between Megaviridae and oomycetes. Our results suggest that marine NCLDVs probably outnumber eukaryotic organisms in the photic layer (per given water mass) and that metagenomic sequence analyses promise to shed new light on the biodiversity of marine viruses and their interactions with potential hosts.The ISME Journal advance online publication, 11 April 2013; doi:10.1038/ismej.2013.59.

In pharmacology, it is crucial to understand the complex biological responses that drugs elicit in the human organism and how well they can be inferred from model organisms. We therefore identified a large set of drug-induced transcriptional modules from genome-wide microarray data of drug-treated human cell lines and rat liver, and first characterized their conservation. Over 70% of these modules were common for multiple cell lines and 15% were conserved between the human in vitro and the rat in vivo system. We then illustrate the utility of conserved and cell-type-specific drug-induced modules by predicting and experimentally validating (i) gene functions, e.g., 10 novel regulators of cellular cholesterol homeostasis and (ii) new mechanisms of action for existing drugs, thereby providing a starting point for drug repositioning, e.g., novel cell cycle inhibitors and new modulators of α-adrenergic receptor, peroxisome proliferator-activated receptor and estrogen receptor. Taken together, the identified modules reveal the conservation of transcriptional responses towards drugs across cell types and organisms, and improve our understanding of both the molecular basis of drug action and human biology.

Viruses are the most abundant biological entities on earth and encompass a vast amount of genetic diversity. The recent rapid increase in the number of sequenced viral genomes has created unprecedented opportunities for gaining new insight into the structure and evolution of the virosphere. Here we present an update of the Phage Orthologous Groups (POGs), a collection of 4,542 clusters of orthologous genes from bacteriophages that now also includes viruses infecting archaea and encompasses more than 1,000 distinct virus genomes. Analysis of this expanded dataset shows that the number of POGs keeps growing without saturation and that a substantial majority of the POGs remain specific to viruses, lacking homologues in prokaryotic cells, outside of known proviruses. Thus, the great majority of virus genes apparently remains to be discovered. A complementary observation is that numerous viral genomes remain poorly if at all covered by POGs. The genome coverage by POGs is expected to increase as more genomes are sequenced. Taxon-specific, single-copy signature genes, that are not observed in prokaryotic genomes outside of detected proviruses, were identified for two-thirds of the 57 taxa (those with genomes available from at least 3 distinct viruses, with half of these present in all members of the respective taxon. These signatures can be used to specifically identify the presence and quantify the abundance of viruses from particular taxa in metagenomic samples and thus gain new insights into the ecology and evolution of viruses in relation to their hosts.

Side effect similarities of drugs have recently been employed to predict new drug targets, and networks of side effects and targets have been used to better understand the mechanism of action of drugs. Here, we report a large-scale analysis to systematically predict and characterize proteins that cause drug side effects. We integrated phenotypic data obtained during clinical trials with known drug-target relations to identify overrepresented protein-side effect combinations. Using independent data, we confirm that most of these overrepresentations point to proteins which, when perturbed, cause side effects. Of 1428 side effects studied, 732 were predicted to be predominantly caused by individual proteins, at least 137 of them backed by existing pharmacological or phenotypic data. We prove this concept in vivo by confirming our prediction that activation of the serotonin 7 receptor (HTR7) is responsible for hyperesthesia in mice, which, in turn, can be prevented by a drug that selectively inhibits HTR7. Taken together, we show that a large fraction of complex drug side effects are mediated by individual proteins and create a reference for such relations.

We are facing a global metabolic health crisis provoked by an obesity epidemic. Here we report the human gut microbial composition in a population sample of 123 non-obese and 169 obese Danish individuals. We find two groups of individuals that differ by the number of gut microbial genes and thus gut bacterial richness. They contain known and previously unknown bacterial species at different proportions; individuals with a low bacterial richness (23% of the population) are characterized by more marked overall adiposity, insulin resistance and dyslipidaemia and a more pronounced inflammatory phenotype when compared with high bacterial richness individuals. The obese individuals among the lower bacterial richness group also gain more weight over time. Only a few bacterial species are sufficient to distinguish between individuals with high and low bacterial richness, and even between lean and obese participants. Our classifications based on variation in the gut microbiome identify subsets of individuals in the general white adult population who may be at increased risk of progressing to adiposity-associated co-morbidities.

The exponentially increasing number of sequenced genomes necessitates fast, accurate, universally applicable and automated approaches for the delineation of prokaryotic species. We developed specI (species identification tool; http://www.bork.embl.de/software/specI/), a method to group organisms into species clusters based on 40 universal, single-copy phylogenetic marker genes. Applied to 3,496 prokaryotic genomes, specI identified 1,753 species clusters. Of 314 discrepancies with a widely used taxonomic classification, >62% were resolved by literature support.

Post-translational modifications (PTMs) are involved in the regulation and structural stabilization of eukaryotic proteins. The combination of individual PTM states is a key to modulate cellular functions as became evident in a few well-studied proteins. This combinatorial setting, dubbed the PTM code, has been proposed to be extended to whole proteomes in eukaryotes. Although we are still far from deciphering such a complex language, thousands of protein PTM sites are being mapped by high-throughput technologies, thus providing sufficient data for comparative analysis. PTMcode (http://ptmcode.embl.de) aims to compile known and predicted PTM associations to provide a framework that would enable hypothesis-driven experimental or computational analysis of various scales. In its first release, PTMcode provides PTM functional associations of 13 different PTM types within proteins in 8 eukaryotes. They are based on five evidence channels: a literature survey, residue co-evolution, structural proximity, PTMs at the same residue and location within PTM highly enriched protein regions (hotspots). PTMcode is presented as a protein-based searchable database with an interactive web interface providing the context of the co-regulation of nearly 75 000 residues in >10 000 proteins.

To understand the structure and function of large molecular machines, accurate knowledge of their stoichiometry is essential. In this study, we developed an integrated targeted proteomics and super-resolution microscopy approach to determine the absolute stoichiometry of the human nuclear pore complex (NPC), possibly the largest eukaryotic protein complex. We show that the human NPC has a previously unanticipated stoichiometry that varies across cancer cell types, tissues and in disease. Using large-scale proteomics, we provide evidence that more than one third of the known, well-defined nuclear protein complexes display a similar cell type-specific variation of their subunit stoichiometry. Our data point to compositional rearrangement as a widespread mechanism for adapting the functions of molecular machines toward cell type-specific constraints and context-dependent needs, and highlight the need of deeper investigation of such structural variants.

SUMMARY: Drug versus Disease (DvD) provides a pipeline, available through R or Cytoscape, for the comparison of drug and disease gene expression profiles from public microarray repositories. Negatively correlated profiles can be used to generate hypotheses of drug-repurposing whilst positively correlated profiles may be used to infer side-effects of drugs. DvD allows users to compare drug and disease signatures with dynamic access to databases Array Express, GEO and data from the Connectivity Map.Availability and Implementation: R package (submitted to Bioconductor) under GPL 3 and Cytoscape plug-in freely available for download at www.ebi.ac.uk/saezrodriguez/DVD/ CONTACT: saezrodriguez@ebi.ac.uk SUPPLEMENTARY INFORMATION: Accompanies this paper.

Whereas large-scale efforts have rapidly advanced the understanding and practical impact of human genomic variation, the practical impact of variation is largely unexplored in the human microbiome. We therefore developed a framework for metagenomic variation analysis and applied it to 252 faecal metagenomes of 207 individuals from Europe and North America. Using 7.4 billion reads aligned to 101 reference species, we detected 10.3 million single nucleotide polymorphisms (SNPs), 107,991 short insertions/deletions, and 1,051 structural variants. The average ratio of non-synonymous to synonymous polymorphism rates of 0.11 was more variable between gut microbial species than across human hosts. Subjects sampled at varying time intervals exhibited individuality and temporal stability of SNP variation patterns, despite considerable composition changes of their gut microbiota. This indicates that individual-specific strains are not easily replaced and that an individual might have a unique metagenomic genotype, which may be exploitable for personalized diet or drug intake.

To quantify known and unknown microorganisms at species-level resolution using shotgun sequencing data, we developed a method that establishes metagenomic operational taxonomic units (mOTUs) based on single-copy phylogenetic marker genes. Applied to 252 human fecal samples, the method revealed that on average 43% of the species abundance and 58% of the richness cannot be captured by current reference genome-based methods. An implementation of the method is available at http://www.bork.embl.de/software/mOTU/.

The breakthrough of next generation sequencing-technologies has enabled large-scale studies of natural microbial communities and the 16S rRNA genes have been widely used as a phylogenetic marker to study community structure. However, major limitations of this approach are that neither strain-level resolution nor genomic context of microorganisms can be provided. This information, however, is crucial to answer fundamental questions about the temporal stability and distinctiveness of natural microbial communities.

ABSTRACT: BACKGROUND: Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. RESULTS: Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. Thermophilum (Arx1), we could also characterise the molecular consequences of some of these mutations. CONCLUSIONS: The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.

Despite the high abundance of Archaea in the global ocean, their metabolism and biogeochemical roles remain largely unresolved. We investigated the population dynamics and metabolic activity of Thaumarchaeota in polar environments, where these microorganisms are particularly abundant and exhibit seasonal growth. Thaumarchaeota were more abundant in deep Arctic and Antarctic waters and grew throughout the winter at surface and deeper Arctic halocline waters. However, in situ single-cell activity measurements revealed a low activity of this group in the uptake of both leucine and bicarbonate (<5% Thaumarchaeota cells active), which is inconsistent with known heterotrophic and autotrophic thaumarchaeal lifestyles. These results suggested the existence of alternative sources of carbon and energy. Our analysis of an environmental metagenome from the Arctic winter revealed that Thaumarchaeota had pathways for ammonia oxidation and, unexpectedly, an abundance of genes involved in urea transport and degradation. Quantitative PCR analysis confirmed that most polar Thaumarchaeota had the potential to oxidize ammonia, and a large fraction of them had urease genes, enabling the use of urea to fuel nitrification. Thaumarchaeota from Arctic deep waters had a higher abundance of urease genes than those near the surface suggesting genetic differences between closely related archaeal populations. In situ measurements of urea uptake and concentration in Arctic waters showed that small-sized prokaryotes incorporated the carbon from urea, and the availability of urea was often higher than that of ammonium. Therefore, the degradation of urea may be a relevant pathway for Thaumarchaeota and other microorganisms exposed to the low-energy conditions of dark polar waters.

OGEE is an Online GEne Essentiality database. Its main purpose is to enhance our understanding of the essentiality of genes. This is achieved by collecting not only experimentally tested essential and non-essential genes, but also associated gene features such as expression profiles, duplication status, conservation across species, evolutionary origins and involvement in embryonic development. We focus on large-scale experiments and complement our data with text-mining results. Genes are organized into data sets according to their sources. Genes with variable essentiality status across data sets are tagged as conditionally essential, highlighting the complex interplay between gene functions and environments. Linked tools allow the user to compare gene essentiality among different gene groups, or compare features of essential genes to non-essential genes, and visualize the results. OGEE is freely available at http://ogeedb.embl.de.

Recently duplicated genes are believed to often overlap in function and expression. A priori, they are thus less likely to be essential. While this was indeed observed in yeast, mouse singletons and duplicates were reported to be equally often essential. This contradiction can only partly be explained by experimental biases. We herein show that older genes (i.e., genes with earlier phyletic origin) are more likely to be essential, regardless of their duplication status. At a given phyletic gene age, duplicates are always less likely to be essential compared to singletons. The 'paradoxical' high essentiality among mouse gene duplicates is then caused by different age profiles of singletons and duplicates, with the latter tending to be derived from older genes.

The genome of Mycobacterium tuberculosis (H37Rv) contains 4,019 protein coding genes, of which more than thousand have been categorized as 'hypothetical' implying that for these not even weak functional associations could be identified so far. We here predict reliable functional indications for half of this large hypothetical orfeome: 497 genes can be annotated based on orthology, and another 125 can be linked to interacting proteins via integrated genomic context analysis and literature mining. The assignments include newly identified clusters of interacting proteins, hypothetical genes that are associated to well known pathways and putative disease-relevant targets. All together, we have raised the fraction of the proteome with at least some functional annotation to 88% which should considerably enhance the interpretation of large-scale experiments targeting this medically important organism.

Transient myeloproliferative disorder (TMD) of the newborn and acute megakaryoblastic leukaemia (AMKL) in children with Down syndrome (DS) represent paradigmatic models of leukaemogenesis. Chromosome 21 gene dosage effects and truncating mutations of the X-chromosomal transcription factor GATA1 synergize to trigger TMD and AMKL in most patients. Here, we report the occurrence of TMD, which spontaneously remitted and later progressed to AMKL in a patient without DS but with a distinct dysmorphic syndrome. Genetic analysis of the leukaemic clone revealed somatic trisomy 21 and a truncating GATA1 mutation. The analysis of the patient's normal blood cell DNA on a genomic single nucleotide polymorphism (SNP) array revealed a de novo germ line 2·58 Mb 15q24 microdeletion including 41 known genes encompassing the tumour suppressor PML. Genomic context analysis of proteins encoded by genes that are included in the microdeletion, chromosome 21-encoded proteins and GATA1 suggests that the microdeletion may trigger leukaemogenesis by disturbing the balance of a hypothetical regulatory network of normal megakaryopoiesis involving PML, SUMO3 and GATA1. The 15q24 microdeletion may thus represent the first genetic hit to initiate leukaemogenesis and implicates PML and SUMO3 as novel components of the leukaemogenic network in TMD/AMKL.

InterPro (http://www.ebi.ac.uk/interpro/) is a database that integrates diverse information about protein families, domains and functional sites, and makes it freely available to the public via Web-based interfaces and services. Central to the database are diagnostic models, known as signatures, against which protein sequences can be searched to determine their potential function. InterPro has utility in the large-scale analysis of whole genomes and meta-genomes, as well as in characterizing individual protein sequences. Herein we give an overview of new developments in the database and its associated software since 2009, including updates to database content, curation processes and Web and programmatic interfaces.

The increased availability of large-scale open-access resources on bioactivities of small molecules has a significant impact on pharmacology facilitated mainly by computational approaches that digest the vast amounts of data. We discuss here how computational data integration enables systemic views on a drug's action and allows to tackle complex problems such as the large-scale prediction of drug targets, drug repurposing, the molecular mechanisms, cellular responses or side effects. We particularly focus on computational methods that leverage various cell-based transcriptional, proteomic and phenotypic profiles of drug response in order to gain a systemic view of drug action at the molecular, cellular and whole-organism scale.

To facilitate the study of interactions between proteins and chemicals, we have created STITCH, an aggregated database of interactions connecting over 300 000 chemicals and 2.6 million proteins from 1133 organisms. Compared to the previous version, the number of chemicals with interactions and the number of high-confidence interactions both increase 4-fold. The database can be accessed interactively through a web interface, displaying interactions in an integrated network view. It is also available for computational studies through downloadable files and an API. As an extension in the current version, we offer the option to switch between two levels of detail, namely whether stereoisomers of a given compound are shown as a merged entity or as separate entities. Separate display of stereoisomers is necessary, for example, for carbohydrates and chiral drugs. Combining the isomers increases the coverage, as interaction databases and publications found through text mining will often refer to compounds without specifying the stereoisomer. The database is accessible at http://stitch.embl.de/.

MOCAT is a highly configurable, modular pipeline for fast, standardized processing of single or paired-end sequencing data generated by the Illumina platform. The pipeline uses state-of-the-art programs to quality control, map, and assemble reads from metagenomic samples sequenced at a depth of several billion base pairs, and predict protein-coding genes on assembled metagenomes. Mapping against reference databases allows for read extraction or removal, as well as abundance calculations. Relevant statistics for each processing step can be summarized into multi-sheet Excel documents and queryable SQL databases. MOCAT runs on UNIX machines and integrates seamlessly with the SGE and PBS queuing systems, commonly used to process large datasets. The open source code and modular architecture allow users to modify or exchange the programs that are utilized in the various processing steps. Individual processing steps and parameters were benchmarked and tested on artificial, real, and simulated metagenomes resulting in an improvement of selected quality metrics. MOCAT can be freely downloaded at http://www.bork.embl.de/mocat/.

SMART (Simple Modular Architecture Research Tool) is an online resource (http://smart.embl.de/) for the identification and annotation of protein domains and the analysis of protein domain architectures. SMART version 7 contains manually curated models for 1009 protein domains, 200 more than in the previous version. The current release introduces several novel features and a streamlined user interface resulting in a faster and more comfortable workflow. The underlying protein databases were greatly expanded, resulting in a 2-fold increase in number of annotated domains and features. The database of completely sequenced genomes now includes 1133 species, compared to 630 in the previous release. Domain architecture analysis results can now be exported and visualized through the iTOL phylogenetic tree viewer. 'metaSMART' was introduced as a novel subresource dedicated to the exploration and analysis of domain architectures in various metagenomics data sets. An advanced full text search engine was implemented, covering the complete annotations for SMART and Pfam domains, as well as the complete set of protein descriptions, allowing users to quickly find relevant information.

Due to the complexity of the protocols and a limited knowledge of the nature of microbial communities, simulating metagenomic sequences plays an important role in testing the performance of existing tools and data analysis methods with metagenomic data. We developed metagenomic read simulators with platform-specific (Sanger, pyrosequencing, Illumina) base-error models, and simulated metagenomes of differing community complexities. We first evaluated the effect of rigorous quality control on Illumina data. Although quality filtering removed a large proportion of the data, it greatly improved the accuracy and contig lengths of resulting assemblies. We then compared the quality-trimmed Illumina assemblies to those from Sanger and pyrosequencing. For the simple community (10 genomes) all sequencing technologies assembled a similar amount and accurately represented the expected functional composition. For the more complex community (100 genomes) Illumina produced the best assemblies and more correctly resembled the expected functional composition. For the most complex community (400 genomes) there was very little assembly of reads from any sequencing technology. However, due to the longer read length the Sanger reads still represented the overall functional composition reasonably well. We further examined the effect of scaffolding of contigs using paired-end Illumina reads. It dramatically increased contig lengths of the simple community and yielded minor improvements to the more complex communities. Although the increase in contig length was accompanied by increased chimericity, it resulted in more complete genes and a better characterization of the functional repertoire. The metagenomic simulators developed for this research are freely available.

Various post-translational modifications (PTMs) fine-tune the functions of almost all eukaryotic proteins, and co-regulation of different types of PTMs has been shown within and between a number of proteins. Aiming at a more global view of the interplay between PTM types, we collected modifications for 13 frequent PTM types in 8 eukaryotes, compared their speed of evolution and developed a method for measuring PTM co-evolution within proteins based on the co-occurrence of sites across eukaryotes. As many sites are still to be discovered, this is a considerable underestimate, yet, assuming that most co-evolving PTMs are functionally associated, we found that PTM types are vastly interconnected, forming a global network that comprise in human alone >50 000 residues in about 6000 proteins. We predict substantial PTM type interplay in secreted and membrane-associated proteins and in the context of particular protein domains and short-linear motifs. The global network of co-evolving PTM types implies a complex and intertwined post-translational regulation landscape that is likely to regulate multiple functional states of many if not all eukaryotic proteins.

Orthologous relationships form the basis of most comparative genomic and metagenomic studies and are essential for proper phylogenetic and functional analyses. The third version of the eggNOG database (http://eggnog.embl.de) contains non-supervised orthologous groups constructed from 1133 organisms, doubling the number of genes with orthology assignment compared to eggNOG v2. The new release is the result of a number of improvements and expansions: (i) the underlying homology searches are now based on the SIMAP database; (ii) the orthologous groups have been extended to 41 levels of selected taxonomic ranges enabling much more fine-grained orthology assignments; and (iii) the newly designed web page is considerably faster with more functionality. In total, eggNOG v3 contains 721 801 orthologous groups, encompassing a total of 4 396 591 genes. Additionally, we updated 4873 and 4850 original COGs and KOGs, respectively, to include all 1133 organisms. At the universal level, covering all three domains of life, 101 208 orthologous groups are available, while the others are applicable at 40 more limited taxonomic ranges. Each group is amended by multiple sequence alignments and maximum-likelihood trees and broad functional descriptions are provided for 450 904 orthologous groups (62.5%).

Protein post-translational modifications (PTMs) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. We systematically investigate the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae. Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein-specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N-acetyltransferases affects protein phosphorylation, confirming cross-talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co-occur within the same protein and that they are frequently observed at interaction interfaces and in multifunctional proteins. The results imply previously unreported hidden layers of post-transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell.

Despite the current wealth of sequencing data, one-third of all biochemically characterized metabolic enzymes lack a corresponding gene or protein sequence, and as such can be considered orphan enzymes. They represent a major gap between our molecular and biochemical knowledge, and consequently are not amenable to modern systemic analyses. As 555 of these orphan enzymes have metabolic pathway neighbours, we developed a global framework that utilizes the pathway and (meta)genomic neighbour information to assign candidate sequences to orphan enzymes. For 131 orphan enzymes (37% of those for which (meta)genomic neighbours are available), we associate sequences to them using scoring parameters with an estimated accuracy of 70%, implying functional annotation of 16 345 gene sequences in numerous (meta)genomes. As a case in point, two of these candidate sequences were experimentally validated to encode the predicted activity. In addition, we augmented the currently available genome-scale metabolic models with these new sequence-function associations and were able to expand the models by on average 8%, with a considerable change in the flux connectivity patterns and improved essentiality prediction.

Here, we report the genome-wide identification of small RNAs associated with transcription start sites (TSSs), termed tssRNAs, in Mycoplasma pneumoniae. tssRNAs were also found to be present in a different bacterial phyla, Escherichia coli. Similar to the recently identified promoter-associated tiny RNAs (tiRNAs) in eukaryotes, tssRNAs are associated with active promoters. Evidence suggests that these tssRNAs are distinct from previously described abortive transcription RNAs. ssRNAs have an average size of 45 bases and map exactly to the beginning of cognate full-length transcripts and to cryptic TSSs. Expression of bacterial tssRNAs requires factors other than the standard RNA polymerase holoenzyme. We have found that the RNA polymerase is halted at tssRNA positions in vivo, which may indicate that a pausing mechanism exists to prevent transcription in the absence of genes. These results suggest that small RNAs associated with TSSs could be a universal feature of bacterial transcription.

The human gastrointestinal tract (GI tract) harbors a complex community of microbes. The microbiota composition varies between different locations in the GI tract, but most studies focus on the fecal microbiota, and that inhabiting the colonic mucosa. Consequently, little is known about the microbiota at other parts of the GI tract, which is especially true for the small intestine because of its limited accessibility. Here we deduce an ecological model of the microbiota composition and function in the small intestine, using complementing culture-independent approaches. Phylogenetic microarray analyses demonstrated that microbiota compositions that are typically found in effluent samples from ileostomists (subjects without a colon) can also be encountered in the small intestine of healthy individuals. Phylogenetic mapping of small intestinal metagenome of three different ileostomy effluent samples from a single individual indicated that Streptococcus sp., Escherichia coli, Clostridium sp. and high G+C organisms are most abundant in the small intestine. The compositions of these populations fluctuated in time and correlated to the short-chain fatty acids profiles that were determined in parallel. Comparative functional analysis with fecal metagenomes identified functions that are overrepresented in the small intestine, including simple carbohydrate transport phosphotransferase systems (PTS), central metabolism and biotin production. Moreover, metatranscriptome analysis supported high level in-situ expression of PTS and carbohydrate metabolic genes, especially those belonging to Streptococcus sp. Overall, our findings suggest that rapid uptake and fermentation of available carbohydrates contribute to maintaining the microbiota in the human small intestine.

Despite decades of research, the structure and assembly of the nuclear pore complex (NPC), which is composed of ∼30 nucleoporins (Nups), remain elusive. Here, we report the genome of the thermophilic fungus Chaetomium thermophilum (ct) and identify the complete repertoire of Nups therein. The thermophilic proteins show improved properties for structural and biochemical studies compared to their mesophilic counterparts, and purified ctNups enabled the reconstitution of the inner pore ring module that spans the width of the NPC from the anchoring membrane to the central transport channel. This module is composed of two large Nups, Nup192 and Nup170, which are flexibly bridged by short linear motifs made up of linker Nups, Nic96 and Nup53. This assembly illustrates how Nup interactions can generate structural plasticity within the NPC scaffold. Our findings therefore demonstrate the utility of the genome of a thermophilic eukaryote for studying complex molecular machines.

Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.

In pharmacology, it is essential to identify the molecular mechanisms of drug action in order to understand adverse side effects. These adverse side effects have been used to infer whether two drugs share a target protein. However, side-effect similarity of drugs could also be caused by their target proteins being close in a molecular network, which as such could cause similar downstream effects. In this study, we investigated the proportion of side-effect similarities that is due to targets that are close in the network compared to shared drug targets. We found that only a minor fraction of side-effect similarities (5.8 %) are caused by drugs targeting proteins close in the network, compared to side-effect similarities caused by overlapping drug targets (64%). Moreover, these targets that cause similar side effects are more often in a linear part of the network, having two or less interactions, than drug targets in general. Based on the examples, we gained novel insight into the molecular mechanisms of side effects associated with several drug targets. Looking forward, such analyses will be extremely useful in the process of drug development to better understand adverse side effects.

We describe the draft genome of the microcrustacean Daphnia pulex, which is only 200 megabases and contains at least 30,907 genes. The high gene count is a consequence of an elevated rate of gene duplication resulting in tandem gene clusters. More than a third of Daphnia's genes have no detectable homologs in any other available proteome, and the most amplified gene families are specific to the Daphnia lineage. The coexpansion of gene families interacting within metabolic pathways suggests that the maintenance of duplicated genes is not random, and the analysis of gene expression under different environmental conditions reveals that numerous paralogs acquire divergent expression patterns soon after duplication. Daphnia-specific genes, including many additional loci within sequenced regions that are otherwise devoid of annotations, are the most responsive genes to ecological challenges.

Single copy genes, universally distributed across the three domains of life and encoding mostly ancient parts of the translation machinery, are thought to be only rarely subjected to horizontal gene transfer (HGT). Indeed it has been proposed to have occurred in only a few genes and implies a rare, probably not advantageous event in which an ortholog displaces the original gene and has to function in a foreign context (orthologous gene displacement, OGD). Here, we have utilised an automatic method to identify HGT based on a conservative statistical approach capable of robustly assigning both donors and acceptors. Applied to 40 universally single copy genes we found that as many as 68 HGTs (implying OGDs) have occurred in these genes with a rate of 1.7 per family since the last universal common ancestor (LUCA). We examined a number of factors that have been claimed to be fundamental to HGT in general and tested their validity in the subset of universally distributed single copy genes. We found that differing functional constraints impact rates of OGD and the more evolutionarily distant the donor and acceptor, the less likely an OGD is to occur. Furthermore, species with larger genomes are more likely to be subjected to OGD. Most importantly, regardless of the trends above, the number of OGDs increases linearly with time, indicating a neutral, constant rate. This suggests that levels of HGT above this rate may be indicative of positively selected transfers that may allow niche adaptation or bestow other benefits to the recipient organism.

The identification of single copy (1-to-1) orthologs in any group of organisms is important for functional classification and phylogenetic studies. The Metazoa are no exception, but only recently has there been a wide-enough distribution of taxa with sufficiently high quality sequenced genomes to gain confidence in the wide-spread single copy status of a gene.Here, we present a phylogenetic approach for identifying overlooked single copy orthologs from multigene families and apply it to the Metazoa. Using 18 sequenced metazoan genomes of high quality we identified a robust set of 1,126 orthologous groups that have been retained in single copy since the last common ancestor of Metazoa. We found that the use of the phylogenetic procedure increased the number of single copy orthologs found by over a third more than standard taxon-count approaches. The orthologs represented a wide range of functional categories, expression profiles and levels of divergence.To demonstrate the value of our set of single copy orthologs, we used them to assess the completeness of 24 currently published metazoan genomes and 62 EST datasets. We found that the annotated genes in published genomes vary in coverage from 79% (Ciona intestinalis) to 99.8% (human) with an average of 92%, suggesting a value for the underlying error rate in genome annotation, and a strategy for identifying single copy orthologs in larger datasets. In contrast, the vast majority of EST datasets with no corresponding genome sequence available are largely under-sampled and probably do not accurately represent the actual genomic complement of the organisms from which they are derived.

The structure, robustness, and dynamics of ocean plankton ecosystems remain poorly understood due to sampling, analysis, and computational limitations. The Tara Oceans consortium organizes expeditions to help fill this gap at the global level.

Interactive Tree Of Life (http://itol.embl.de) is a web-based tool for the display, manipulation and annotation of phylogenetic trees. It is freely available and open to everyone. In addition to classical tree viewer functions, iTOL offers many novel ways of annotating trees with various additional data. Current version introduces numerous new features and greatly expands the number of supported data set types. Trees can be interactively manipulated and edited. A free personal account system is available, providing management and sharing of trees in user defined workspaces and projects. Export to various bitmap and vector graphics formats is supported. Batch access interface is available for programmatic access or inclusion of interactive trees into other web services.

Using metagenomic 'parts lists' to infer global patterns on microbial ecology remains a significant challenge. To deduce important ecological indicators such as environmental adaptation, molecular trait dispersal, diversity variation and primary production from the gene pool of an ecosystem, we integrated 25 ocean metagenomes with geographical, meteorological and geophysicochemical data. We find that climatic factors (temperature, sunlight) are the major determinants of the biomolecular repertoire of each sample and the main limiting factor on functional trait dispersal (absence of biogeographic provincialism). Molecular functional richness and diversity show a distinct latitudinal gradient peaking at 20°N and correlate with primary production. The latter can also be predicted from the molecular functional composition of an environmental sample. Together, our results show that the functional community composition derived from metagenomes is an important quantitative readout for molecular trait-based biogeography and ecology.

An essential prerequisite for any systems-level understanding of cellular functions is to correctly uncover and annotate all functional interactions among proteins in the cell. Toward this goal, remarkable progress has been made in recent years, both in terms of experimental measurements and computational prediction techniques. However, public efforts to collect and present protein interaction information have struggled to keep up with the pace of interaction discovery, partly because protein-protein interaction information can be error-prone and require considerable effort to annotate. Here, we present an update on the online database resource Search Tool for the Retrieval of Interacting Genes (STRING); it provides uniquely comprehensive coverage and ease of access to both experimental as well as predicted interaction information. Interactions in STRING are provided with a confidence score, and accessory information such as protein domains and 3D structures is made available, all within a stable and consistent identifier space. New features in STRING include an interactive network viewer that can cluster networks on demand, updated on-screen previews of structural information including homology models, extensive data updates and strongly improved connectivity and integration with third-party resources. Version 9.0 of STRING covers more than 1100 completely sequenced organisms; the resource can be reached at http://string-db.org.

The increasing number of sequenced genomes has prompted the development of several automated orthology prediction methods. Tests to evaluate the accuracy of predictions and to explore biases caused by biological and technical factors are therefore required. We used 70 manually curated families to analyze the performance of five public methods in Metazoa. We analyzed the strengths and weaknesses of the methods and quantified the impact of biological and technical challenges. From the latter part of the analysis, genome annotation emerged as the largest single influencer, affecting up to 30% of the performance. Generally, most methods did well in assigning orthologous group but they failed to assign the exact number of genes for half of the groups. The publicly available benchmark set (http://eggnog.embl.de/orthobench/) should facilitate the improvement of current orthology assignment protocols, which is of utmost importance for many fields of biology and should be tackled by a broad scientific community.

iPath2.0 is a web-based tool (http://pathways.embl.de) for the visualization and analysis of cellular pathways. Its primary map summarizes the metabolism in biological systems as annotated to date. Nodes in the map correspond to various chemical compounds and edges represent series of enzymatic reactions. In two other maps, iPath2.0 provides an overview of secondary metabolite biosynthesis and a hand-picked selection of important regulatory pathways and other functional modules, allowing a more general overview of protein functions in a genome or metagenome. iPath2.0's main interface is an interactive Flash-based viewer, which allows users to easily navigate and explore the complex pathway maps. In addition to the default pre-computed overview maps, iPath offers several data mapping tools. Users can upload various types of data and completely customize all nodes and edges of iPath2.0's maps. These customized maps give users an intuitive overview of their own data, guiding the analysis of various genomics and metagenomics projects.

Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences-the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The 'environmental packages' apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.

Combinatorial therapy is a promising strategy for combating complex disorders due to improved efficacy and reduced side effects. However, screening new drug combinations exhaustively is impractical considering all possible combinations between drugs. Here, we present a novel computational approach to predict drug combinations by integrating molecular and pharmacological data. Specifically, drugs are represented by a set of their properties, such as their targets or indications. By integrating several of these features, we show that feature patterns enriched in approved drug combinations are not only predictive for new drug combinations but also provide insights into mechanisms underlying combinatorial therapy. Further analysis confirmed that among our top ranked predictions of effective combinations, 69% are supported by literature, while the others represent novel potential drug combinations. We believe that our proposed approach can help to limit the search space of drug combinations and provide a new way to effectively utilize existing drugs for new purposes.

SUMMARY: SmashCommunity is a stand-alone metagenomic annotation and analysis pipeline suitable for data from Sanger and 454 sequencing technologies. It supports state-of-the-art software for essential metagenomic tasks such as assembly and gene prediction. It provides tools to estimate the quantitative phylogenetic and functional compositions of metagenomes, to compare compositions of multiple metagenomes and to produce intuitive visual representations of such analyses. AVAILABILITY: SmashCommunity is freely available at http://www.bork.embl.de/software/smash CONTACT: bork@embl.de.

The spectacular escalation in complexity in early bilaterian evolution correlates with a strong increase in the number of microRNAs. To explore the link between the birth of ancient microRNAs and body plan evolution, we set out to determine the ancient sites of activity of conserved bilaterian microRNA families in a comparative approach. We reason that any specific localization shared between protostomes and deuterostomes (the two major superphyla of bilaterian animals) should probably reflect an ancient specificity of that microRNA in their last common ancestor. Here, we investigate the expression of conserved bilaterian microRNAs in Platynereis dumerilii, a protostome retaining ancestral bilaterian features, in Capitella, another marine annelid, in the sea urchin Strongylocentrotus, a deuterostome, and in sea anemone Nematostella, representing an outgroup to the bilaterians. Our comparative data indicate that the oldest known animal microRNA, miR-100, and the related miR-125 and let-7 were initially active in neurosecretory cells located around the mouth. Other sets of ancient microRNAs were first present in locomotor ciliated cells, specific brain centres, or, more broadly, one of four major organ systems: central nervous system, sensory tissue, musculature and gut. These findings reveal that microRNA evolution and the establishment of tissue identities were closely coupled in bilaterian evolution. Also, they outline a minimum set of cell types and tissues that existed in the protostome-deuterostome ancestor.

A major challenge in the human metagenomics field is to identify associations of the bacterial genes and human phenotypes and act to modulate microbial populations in order to improve human health and wellbeing. MetaHIT project addresses this ambitious challenge by developing and integrating a number of necessary approaches within the context of the gut microbiome. Among the first results is the establishment of a broad catalog of the human gut microbial genes, which was achieved by an original application of the new generation sequencing technology. The catalog contains 3.3 million non-redundant genes, 150-fold more than the human genome equivalent and includes a large majority of the gut metagenomic sequences determined across three continents, Europe, America and Asia. Its content corresponds to some 1000 bacterial species, which likely represent a large fraction of species associated with humankind intestinal tract. The catalog enables development of the gene profiling approaches aiming to detect associations of bacterial genes and phenotypes. These should lead to the speedy development of diagnostic and prognostic tools and open avenues to reasoned approaches to the modulation of the individual's microbiota in order to optimize health and well-being.

Protein-metabolite networks are central to biological systems, but are incompletely understood. Here, we report a screen to catalog protein-lipid interactions in yeast. We used arrays of 56 metabolites to measure lipid-binding fingerprints of 172 proteins, including 91 with predicted lipid-binding domains. We identified 530 protein-lipid associations, the majority of which are novel. To show the data set's biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live-cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. Validated interactions with Slm1, a regulator of actin polarization, show that PH domains can have unexpected lipid-binding specificities and can act as coincidence sensors for both phosphatidylinositol phosphates and phosphorylated sphingolipids.

ABSTRACT: BACKGROUND: Extensive transcription of non-coding RNAs has been detected in eukaryotic genomes and is thought to constitute an additional layer in the regulation of gene expression. Despite this role, their transcription through the cell cycle has not been studied; genome-wide approaches have only focused on protein-coding genes. To explore the complex transcriptome architecture underlying the budding yeast cell cycle, we used 8 bp tiling arrays to generate a 5 minute-resolution, strand-specific expression atlas of the whole genome. RESULTS: We discovered 523 antisense transcripts, of which 80 cycle or are located opposite periodically expressed mRNAs, 135 unannotated intergenic non-coding RNAs, of which 11 cycle, and 109 cell-cycle-regulated protein-coding genes that had not previously been shown to cycle. We detected periodic expression coupling of sense and antisense transcript pairs, including antisense transcripts opposite of key cell-cycle regulators, like FAR1 and TAF2. CONCLUSIONS: Our dataset presents the most comprehensive resource to date on gene expression during the budding yeast cell cycle, revealing both protein-coding and non-coding RNA periodicity of expression and the first that profiles non-annotated RNAs. It enables hypothesis-driven mechanistic studies concerning the functions of non-coding RNAs.

SUMMARY: Recent advances in single-cell manipulation technology, whole genome amplification and high-throughput sequencing have now made it possible to sequence the genome of an individual cell. The bioinformatic analysis of these genomes however is far more complicated than the analysis of those generated using traditional, culture-based methods. In order to simplify this analysis we have developed SmashCell (Simple Metagenomics Analysis SHell-for sequences from single Cells). It is designed to automate the main steps in microbial genome analysis - assembly, gene prediction, functional annotation - in a way that allows parameter and algorithm exploration at each step in the process. It alsomanages the data created by these analyses and provides visualisation methods to allow rapid analysis of the results. AVAILABILITY: The SmashCell source code and a comprehensive manual are available at http://asiago.stanford.edu/SmashCell CONTACT: eoghanh@stanford.edu.

Drug perturbations of human cells lead to complex responses upon target binding. One of the known mechanisms is a (positive or negative) feedback loop that adjusts the expression level of the respective target protein. To quantify this mechanism systems-wide in an unbiased way, drug-induced differential expression of drug target mRNA was examined in three cell lines using the Connectivity Map. To overcome various biases in this valuable resource, we have developed a computational normalization and scoring procedure that is applicable to gene expression recording upon heterogeneous drug treatments. In 1290 drug-target relations, corresponding to 466 drugs acting on 167 drug targets studied, 8% of the targets are subject to regulation at the mRNA level. We confirmed systematically that in particular G-protein coupled receptors, when serving as known targets, are regulated upon drug treatment. We further newly identified drug-induced differential regulation of Lanosterol 14-alpha demethylase, Endoplasmin, DNA topoisomerase 2-alpha and Calmodulin 1. The feedback regulation in these and other targets is likely to be relevant for the success or failure of the molecular intervention.

As biology is becoming a data-driven discipline, ontologies become increasingly important for systematically capturing the existing knowledge. This essay discusses current trends and how ontologies can also be used for discovery.

The molecular understanding of phenotypes caused by drugs in humans is essential for elucidating mechanisms of action and for developing personalized medicines. Side effects of drugs (also known as adverse drug reactions) are an important source of human phenotypic information, but so far research on this topic has been hampered by insufficient accessibility of data. Consequently, we have developed a public, computer-readable side effect resource (SIDER) that connects 888 drugs to 1450 side effect terms. It contains information on frequency in patients for one-third of the drug-side effect pairs. For 199 drugs, the side effect frequency of placebo administration could also be extracted. We illustrate the potential of SIDER with a number of analyses. The resource is freely available for academic research at http://sideeffects.embl.de.

Over the last years, the publicly available knowledge on interactions between small molecules and proteins has been steadily increasing. To create a network of interactions, STITCH aims to integrate the data dispersed over the literature and various databases of biological pathways, drug-target relationships and binding affinities. In STITCH 2, the number of relevant interactions is increased by incorporation of BindingDB, PharmGKB and the Comparative Toxicogenomics Database. The resulting network can be explored interactively or used as the basis for large-scale analyses. To facilitate links to other chemical databases, we adopt InChIKeys that allow identification of chemicals with a short, checksum-like string. STITCH 2.0 connects proteins from 630 organisms to over 74 000 different chemicals, including 2200 drugs. STITCH can be accessed at http://stitch.embl.de/.

SUMMARY: Multiple Sequence Alignment (MSA) is a central tool in most modern biology studies. However, despite generations of valuable tools, human experts are still able to improve automatically generated MSAs. In an effort to automatically identify the most reliable MSA for a given protein family, we propose a very simple protocol, named AQUA for "Automated quality improvement for multiple sequence alignments". Our current implementation relies on 2 alignment programs (MUSCLE and MAFFT), one refinement program (RASCAL) and one assessment program (NORMD) but other programs could be incorporated at any of the 3 steps. AVAILABILITY: AQUA is implemented in Tcl/Tk and runs in command line on all platforms. The source code is available under the GNU GPL license. Source code, README and Supplementary data are available at http://www.bork.embl.de/Docu/AQUA. CONTACT: muller@embl.de, bork@embl.de.

The identification of orthologous relationships forms the basis for most comparative genomics studies. Here, we present the second version of the eggNOG database, which contains orthologous groups (OGs) constructed through identification of reciprocal best BLAST matches and triangular linkage clustering. We applied this procedure to 630 complete genomes (529 bacteria, 46 archaea and 55 eukaryotes), which is a 2-fold increase relative to the previous version. The pipeline yielded 224 847 OGs, including 9724 extended versions of the original COG and KOG. We computed OGs for different levels of the tree of life; in addition to the species groups included in our first release (i.e. fungi, metazoa, insects, vertebrates and mammals), we have now constructed OGs for archaea, fishes, rodents and primates. We automatically annotate the non-supervised orthologous groups (NOGs) with functional descriptions, protein domains, and functional categories as defined initially for the COG/KOG database. In-depth analysis is facilitated by precomputed high-quality multiple sequence alignments and maximum-likelihood trees for each of the available OGs. Altogether, eggNOG covers 2 242 035 proteins (built from 2 590 259 proteins) and provides a broad functional description for at least 1 966 709 (88%) of them. Users can access the complete set of orthologous groups via a web interface at: http://eggnog.embl.de.

We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.

Cis-acting short sequence motifs play important roles in alternative splicing. It is now possible to identify such sequence motifs as conserved sequence patterns in genome sequence alignments. Here, we report the systematic search for motifs in the neighboring introns of alternatively spliced exons by using comparative analysis of mammalian genome alignments. We identified 11 conserved sequence motifs that might be involved in the regulation of alternative splicing. These motifs are not only significantly overrepresented near alternatively spliced exons, but they also co-occur with each other, thus, forming a network of cis-elements, likely to be the basis for context-dependent regulation. Based on this finding, we applied the motif co-occurrence to predict alternatively skipped exons. We verified exon skipping in 29 cases out of 118 predictions (25%) by EST and mRNA sequences in the databases. For the predictions not verified by the database sequences, we confirmed exon skipping in 10 additional cases by using both RT-PCR experiments and the publicly available RNA-Seq data. These results indicate that even more alternative splicing events will be found with the progress of large-scale and high-throughput analyses for various tissue samples and developmental stages.

Several cyclic processes take place within a single organism. For example, the cell cycle is coordinated with the 24 h diurnal rhythm in animals and plants, and with the 40 min ultradian rhythm in budding yeast. To examine the evolution of periodic gene expression during these processes, we performed the first systematic comparison in three organisms (Homo sapiens, Arabidopsis thaliana and Saccharomyces cerevisiae) by using public microarray data. We observed that although diurnal-regulated and ultradian-regulated genes are not generally cell-cycle-regulated, they tend to have cell-cycle-regulated paralogues. Thus, diverged temporal expression of paralogues seems to facilitate cellular orchestration under different periodic stimuli. Lineage-specific functional repertoires of periodic-associated paralogues imply that this mode of regulation might have evolved independently in several organisms.

About 15% of human colorectal cancers and, at varying degrees, other tumor entities as well as nearly all tumors related to Lynch syndrome are hallmarked by microsatellite instability (MSI) as a result of a defective mismatch repair system. The functional impact of resulting mutations depends on their genomic localization. Alterations within coding mononucleotide repeat tracts (MNRs) can lead to protein truncation and formation of neopeptides, whereas alterations within untranslated MNRs can alter transcription level or transcript stability. These mutations may provide selective advantage or disadvantage to affected cells. They may further concern the biology of microsatellite unstable cells, e.g. by generating immunogenic peptides induced by frameshifts mutations. The Selective Targets database (http://www.seltarbase.org) is a curated database of a growing number of public MNR mutation data in microsatellite unstable human tumors. Regression calculations for various MSI-H tumor entities indicating statistically deviant mutation frequencies predict TGFBR2, BAX, ACVR2A and others that are shown or highly suspected to be involved in MSI tumorigenesis. Many useful tools for further analyzing genomic DNA, derived wild-type and mutated cDNAs and peptides are integrated. A comprehensive database of all human coding, untranslated, non-coding RNA- and intronic MNRs (MNR_ensembl) is also included. Herewith, SelTarbase presents as a plenty instrument for MSI-carcinogenesis-related research, diagnostics and therapy.

Recently, approaches have been developed to sample the genetic content of heterogeneous environments (metagenomics). However, by what means these sequences link distinct environmental conditions with specific biological processes is not well understood. Thus, a major challenge is how the usage of particular pathways and subnetworks reflects the adaptation of microbial communities across environments and habitats-i.e., how network dynamics relates to environmental features. Previous research has treated environments as discrete, somewhat simplified classes (e.g., terrestrial vs. marine), and searched for obvious metabolic differences among them (i.e., treating the analysis as a typical classification problem). However, environmental differences result from combinations of many factors, which often vary only slightly. Therefore, we introduce an approach that employs correlation and regression to relate multiple, continuously varying factors defining an environment to the extent of particular microbial pathways present in a geographic site. Moreover, rather than looking only at individual correlations (one-to-one), we adapted canonical correlation analysis and related techniques to define an ensemble of weighted pathways that maximally covaries with a combination of environmental variables (many-to-many), which we term a metabolic footprint. Applied to available aquatic datasets, we identified footprints predictive of their environment that can potentially be used as biosensors. For example, we show a strong multivariate correlation between the energy-conversion strategies of a community and multiple environmental gradients (e.g., temperature). Moreover, we identified covariation in amino acid transport and cofactor synthesis, suggesting that limiting amounts of cofactor can (partially) explain increased import of amino acids in nutrient-limited conditions.

To study basic principles of transcriptome organization in bacteria, we analyzed one of the smallest self-replicating organisms, Mycoplasma pneumoniae. We combined strand-specific tiling arrays, complemented by transcriptome sequencing, with more than 252 spotted arrays. We detected 117 previously undescribed, mostly noncoding transcripts, 89 of them in antisense configuration to known genes. We identified 341 operons, of which 139 are polycistronic; almost half of the latter show decaying expression in a staircase-like manner. Under various conditions, operons could be divided into 447 smaller transcriptional units, resulting in many alternative transcripts. Frequent antisense transcripts, alternative transcripts, and multiple regulators per gene imply a highly dynamic transcriptome, more similar to that of eukaryotes than previously thought.

The InterPro database (http://www.ebi.ac.uk/interpro/) integrates together predictive models or 'signatures' representing protein domains, families and functional sites from multiple, diverse source databases: Gene3D, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY and TIGRFAMs. Integration is performed manually and approximately half of the total approximately 58 000 signatures available in the source databases belong to an InterPro entry. Recently, we have started to also display the remaining un-integrated signatures via our web interface. Other developments include the provision of non-signature data, such as structural data, in new XML files on our FTP site, as well as the inclusion of matchless UniProtKB proteins in the existing match XML files. The web interface has been extended and now links out to the ADAN predicted protein-protein interaction database and the SPICE and Dasty viewers. The latest public release (v18.0) covers 79.8% of UniProtKB (v14.1) and consists of 16 549 entries. InterPro data may be accessed either via the web address above, via web services, by downloading files by anonymous FTP or by using the InterProScan search software (http://www.ebi.ac.uk/Tools/InterProScan/).

Functional partnerships between proteins are at the core of complex cellular phenotypes, and the networks formed by interacting proteins provide researchers with crucial scaffolds for modeling, data reduction and annotation. STRING is a database and web resource dedicated to protein-protein interactions, including both physical and functional interactions. It weights and integrates information from numerous sources, including experimental repositories, computational prediction methods and public text collections, thus acting as a meta-database that maps all interaction evidence onto a common set of genomes and proteins. The most important new developments in STRING 8 over previous releases include a URL-based programming interface, which can be used to query STRING from other resources, improved interaction prediction via genomic neighborhood in prokaryotes, and the inclusion of protein structures. Version 8.0 of STRING covers about 2.5 million proteins from 630 organisms, providing the most comprehensive view on protein-protein interactions currently available. STRING can be reached at http://string-db.org/.

ABSTRACT: A recent trend in computational methods for annotation of protein function is that many prediction tools are combined in complex workflows and pipelines to facilitate the analysis of feature combinations, for example, the entire repertoire of kinase-binding motifs in the human proteome.

The Alternative Splicing and Transcript Diversity database (ASTD) gives access to a vast collection of alternative transcripts that integrate transcription initiation, polyadenylation and splicing variant data. Alternative transcripts are derived from the mapping of transcribed sequences to the complete human, mouse and rat genomes using an extension of the computational pipeline developed for the ASD (Alternative Splicing Database) and ATD (Alternative Transcript Diversity) databases, which are now superseded by ASTD. For the human genome, ASTD identifies splicing variants, transcription initiation variants and polyadenylation variants in 68%, 68% and 62% of the gene set, respectively, consistent with current estimates for transcription variation. Users can access ASTD through a variety of browsing and query tools, including expression state-based queries for the identification of tissue-specific isoforms. Participating labs have experimentally validated a subset of ASTD-predicted alternative splice forms and alternative polyadenylation forms that were not previously reported. The ASTD database is accessed at http://www.ebi.ac.uk/astd.

The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.

Simple modular architecture research tool (SMART) is an online tool (http://smart.embl.de/) for the identification and annotation of protein domains. It provides a user-friendly platform for the exploration and comparative study of domain architectures in both proteins and genes. The current release of SMART contains manually curated models for 784 protein domains. Recent developments were focused on further data integration and improving user friendliness. The underlying protein database based on completely sequenced genomes was greatly expanded and now includes 630 species, compared to 191 in the previous release. As an initial step towards integrating information on biological pathways into SMART, our domain annotations were extended with data on metabolic pathways and links to several pathways resources. The interaction network view was completely redesigned and is now available for more than 2 million proteins. In addition to the standard web access to the database, users can now query SMART using distributed annotation system (DAS) or through a simple object access protocol (SOAP) based web service.

The emerging coverage of diverse habitats by metagenomic shotgun data opens new avenues of discovering functional novelty using computational tools. Here, we apply three different concepts for predicting novel functions within light-mediated microbial pathways in five diverse environments. Using phylogenetic approaches, we discovered two novel deep-branching subfamilies of photolyases (involved in light-mediated repair) distributed abundantly in high-UV environments. Using neighborhood approaches, we were able to assign to BLUF domain-containing proteins (involved in light-sensing) seven novel functional partners in luciferase synthesis, nitrogen metabolism, and quorum sensing. Finally, by domain analysis, we predict for RcaE proteins (involved in chromatic adaptation) sixteen novel domain architectures that indicate novel functionalities in habitats with little or no light. Quantification of protein abundance in the various environments supports our findings that bacteria utilize light for sensing, repair, and adaptation far more widely than previously thought. While the discoveries illustrate the opportunities in function discovery, we also discuss the immense conceptual and practical challenges that come along with this new type of data.

Despite only becoming popular at the beginning of this decade, biomolecular networks are now frameworks that facilitate many discoveries in molecular biology. The nodes of these networks are usually proteins (specifically enzymes in metabolic networks), whereas the links (or edges) are their interactions with other molecules. These networks are made up of protein-protein interactions or enzyme-enzyme interactions through shared metabolites in the case of metabolic networks. Evolutionary analysis has revealed that changes in the nodes and links in protein-protein interaction and metabolic networks are subject to different selection pressures owing to distinct topological features. However, many evolutionary constraints can be uncovered only if temporal and spatial aspects are included in the network analysis.

To understand basic principles of bacterial metabolism organization and regulation, but also the impact of genome size, we systematically studied one of the smallest bacteria, Mycoplasma pneumoniae. A manually curated metabolic network of 189 reactions catalyzed by 129 enzymes allowed the design of a defined, minimal medium with 19 essential nutrients. More than 1300 growth curves were recorded in the presence of various nutrient concentrations. Measurements of biomass indicators, metabolites, and (13)C-glucose experiments provided information on directionality, fluxes, and energetics; integration with transcription profiling enabled the global analysis of metabolic regulation. Compared with more complex bacteria, the M. pneumoniae metabolic network has a more linear topology and contains a higher fraction of multifunctional enzymes; general features such as metabolite concentrations, cellular energetics, adaptability, and global gene expression responses are similar, however.

Targets for drugs have so far been predicted on the basis of molecular or cellular features, for example, by exploiting similarity in chemical structure or in activity across cell lines. We used phenotypic side-effect similarities to infer whether two drugs share a target. Applied to 746 marketed drugs, a network of 1018 side effect-driven drug-drug relations became apparent, 261 of which are formed by chemically dissimilar drugs from different therapeutic indications. We experimentally tested 20 of these unexpected drug-drug relations and validated 13 implied drug-target relations by in vitro binding assays, of which 11 reveal inhibition constants equal to less than 10 micromolar. Nine of these were tested and confirmed in cell assays, documenting the feasibility of using phenotypic information to infer molecular interactions and hinting at new uses of marketed drugs.

BACKGROUND: Polyketides are a diverse group of biotechnologically important secondary metabolites that are produced by multi domain enzymes called polyketide synthases (PKS). METHODOLOGY/PRINCIPAL FINDINGS: We have estimated frequencies of type I PKS (PKS I) - a PKS subgroup - in natural environments by using Hidden-Markov-Models of eight domains to screen predicted proteins from six metagenomic shotgun data sets. As the complex PKS I have similarities to other multi-domain enzymes (like those for the fatty acid biosynthesis) we increased the reliability and resolution of the dataset by maximum-likelihood trees. The combined information of these trees was then used to discriminate true PKS I domains from evolutionary related but functionally different ones. We were able to identify numerous novel PKS I proteins, the highest density of which was found in Minnesota farm soil with 136 proteins out of 183,536 predicted genes. We also applied the protocol to UniRef database to improve the annotation of proteins with so far unknown function and identified some new instances of horizontal gene transfer. CONCLUSIONS/SIGNIFICANCE: The screening approach proved powerful in identifying PKS I sequences in large sequence data sets and is applicable to many other protein families.

Bacterial nitrile hydratase (NHases) are important industrial catalysts and waste water remediation tools. In a global computational screening of conventional and metagenomic sequence data for NHases, we detected the two usually separated NHase subunits fused in one protein of the choanoflagellate Monosiga brevicollis, a recently sequenced unicellular model organism from the closest sister group of Metazoa. This is the first time that an NHase is found in eukaryotes and the first time it is observed as a fusion protein. The presence of an intron, subunit fusion and expressed sequence tags covering parts of the gene exclude contamination and suggest a functional gene. Phylogenetic analyses and genomic context imply a probable ancient horizontal gene transfer (HGT) from proteobacteria. The newly discovered NHase might open biotechnological routes due to its unconventional structure, its new type of host and its apparent integration into eukaryotic protein networks.

The molecular basis of drug action is often not well understood. This is partly because the very abundant and diverse information generated in the past decades on drugs is hidden in millions of medical articles or textbooks. Therefore, we developed a one-stop data warehouse, SuperTarget that integrates drug-related information about medical indication areas, adverse drug effects, drug metabolization, pathways and Gene Ontology terms of the target proteins. An easy-to-use query interface enables the user to pose complex queries, for example to find drugs that target a certain pathway, interacting drugs that are metabolized by the same cytochrome P450 or drugs that target the same protein but are metabolized by different enzymes. Furthermore, we provide tools for 2D drug screening and sequence comparison of the targets. The database contains more than 2500 target proteins, which are annotated with about 7300 relations to 1500 drugs; the vast majority of entries have pointers to the respective literature source. A subset of these drugs has been annotated with additional binding information and indirect interactions and is available as a separate resource called Matador. SuperTarget and Matador are available at http://insilico.charite.de/supertarget and http://matador.embl.de.

SUMMARY: Sircah is a flexible tool for the detection, analysis and visualisation of alternative transcripts. It takes as input gene models or spliced alignments and creates a database of alternative transcription events: alternative transcription initiation and polyadenylation, alternative 3' and 5' splice-site usage, skipped exons and retained introns. The results can be visualised in a variety of ways, allowing the creation of publication quality images. RESULTS: AVAILABILITY: The Sircah is available for download under a creative commons license along with additional documentation and a tutorial from http://www.bork.embl.de/Sircah. CONTACT: bork@embl.de.

Continuing improvements in DNA sequencing technologies are providing us with vast amounts of genomic data from an ever-widening range of organisms. The resulting challenge for bioinformatics is to interpret this deluge of data and place it back into its biological context. Biological networks provide a conceptual framework with which we can describe part of this context, namely the different interactions that occur between the molecular components of a cell. Here, we review the computational methods available to predict biological networks from genomic sequence data and discuss how they relate to high-throughput experimental methods.

In the major animal model species like mouse, fish or fly, detailed spatial information on gene expression over time can be acquired through whole mount in situ hybridization experiments. In these species, expression patterns of many genes have been studied and data has been integrated into dedicated model organism databases like ZFIN for zebrafish, MEPD for medaka, BDGP for Drosophila or GXD for mouse. However, a central repository that allows users to query and compare gene expression patterns across different species has not yet been established. Therefore, we have integrated expression patterns for zebrafish, Drosophila, medaka and mouse into a central public repository called 4DXpress (expression database in four dimensions). Users can query anatomy ontology-based expression annotations across species and quickly jump from one gene to the orthologues in other species. Genes are linked to public microarray data in ArrayExpress. We have mapped developmental stages between the species to be able to compare developmental time phases. We store the largest collection of gene expression patterns available to date in an individual resource, reflecting 16 505 annotated genes. 4DXpress will be an invaluable tool for developmental as well as for computational biologists interested in gene regulation and evolution. 4DXpress is available at http://ani.embl.de/4DXpress.

BACKGROUND: Environments and their organic content are generally not static and isolated, but in a constant state of exchange and interaction with each other. Through physical or biological processes, organisms, especially microbes, may be transferred between environments whose characteristics may be quite different. The transferred microbes may not survive in their new environment, but their DNA will be deposited. In this study, we compare two environmental sequencing projects to find molecular evidence of transfer of microbes over vast geographical distances. METHODOLOGY: By studying synonymous nucleotide composition, oligomer frequency and orthology between predicted genes in metagenomics data from two environments, terrestrial and aquatic, and by correlating with phylogenetic mappings, we find that both environments are likely to contain trace amounts of microbes which have been far removed from their original habitat. We also suggest a bias in direction from soil to sea, which is consistent with the cycles of planetary wind and water. CONCLUSIONS: Our findings support the Baas-Becking hypothesis formulated in 1934, which states that due to dispersion and population sizes, microbes are likely to be found in widely disparate environments. Furthermore, the availability of genetic material from distant environments is a possible font of novel gene functions for lateral gene transfer.

: A response to Combined analysis reveals a core set of cycling genes by Y Lu, S Mahony, PV Benos, R Rosenfeld, I Simon, LL Breeden and Z Bar-Joseph. Genome Biol 2007, 8:R146.

The identification of orthologous genes forms the basis for most comparative genomics studies. Existing approaches either lack functional annotation of the identified orthologous groups, hampering the interpretation of subsequent results, or are manually annotated and thus lag behind the rapid sequencing of new genomes. Here we present the eggNOG database ('evolutionary genealogy of genes: Non-supervised Orthologous Groups'), which contains orthologous groups constructed from Smith-Waterman alignments through identification of reciprocal best matches and triangular linkage clustering. Applying this procedure to 312 bacterial, 26 archaeal and 35 eukaryotic genomes yielded 43 582 course-grained orthologous groups of which 9724 are extended versions of those from the original COG/KOG database. We also constructed more fine-grained groups for selected subsets of organisms, such as the 19 914 mammalian orthologous groups. We automatically annotated our non-supervised orthologous groups with functional descriptions, which were derived by identifying common denominators for the genes based on their individual textual descriptions, annotated functional categories, and predicted protein domains. The orthologous groups in eggNOG contain 1 241 751 genes and provide at least a broad functional description for 77% of them. Users can query the resource for individual genes via a web interface or download the complete set of orthologous groups at http://eggnog.embl.de.

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.