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Type IV CRISPR-Cas are a distinct variety of highly derived CRISPR-Cas systems that appear to have evolved from type III systems through the loss of the target-cleaving nuclease and partial deterioration of the large subunit of the effector complex. All known type IV CRISPR-Cas systems are encoded on plasmids, integrative and conjugative elements (ICEs), or prophages, and are thought to contribute to competition between these elements, although the mechanistic details of their function remain unknown. There is a clear parallel between the compositions and likely origin of type IV and type I systems recruited by Tn7-like transposons and mediating RNA-guided transposition. We investigated the diversity and evolutionary relationships of type IV systems, with a focus on those in Acidithiobacillia, where this variety of CRISPR is particularly abundant and always found on ICEs. Our analysis revealed remarkable evolutionary plasticity of type IV CRISPR-Cas systems, with adaptation and ancillary genes originating from different ancestral CRISPR-Cas varieties, and extensive gene shuffling within the type IV loci. The adaptation module and the CRISPR array apparently were lost in the type IV ancestor but were subsequently recaptured by type IV systems on several independent occasions. We demonstrate a high level of heterogeneity among the repeats with type IV CRISPR arrays, which far exceed the heterogeneity of any other known CRISPR repeats and suggest a unique adaptation mechanism. The spacers in the type IV arrays, for which protospacers could be identified, match plasmid genes, in particular those encoding the conjugation apparatus components. Both the biochemical mechanism of type IV CRISPR-Cas function and their role in the competition among mobile genetic elements remain to be investigated.

Detection and classification of CRISPR-Cas systems in metagenomic data have become increasingly prevalent in recent years due to their potential for diverse applications in genome editing. Traditionally, CRISPR-Cas systems are classified through reference-based identification of proximate cas genes. Here, we present a machine learning approach for the detection and classification of CRISPR loci using repeat sequences in a cas-independent context, enabling identification of unclassified loci missed by traditional cas-based approaches. Using biological attributes of the CRISPR repeat, the core element in CRISPR arrays, and leveraging methods from natural language processing, we developed a machine learning model capable of accurate classification of CRISPR loci in an extensive set of metagenomes, resulting in an F1 measure of 0.82 across all predictions and an F1 measure of 0.97 when limiting to classifications with probabilities >0.85. Furthermore, assessing performance on novel repeats yielded an F1 measure of 0.96. Although the performance of cas-based identification will exceed that of a repeat-based approach in many cases, CRISPRclassify provides an efficient approach to classification of CRISPR loci for cases in which cas gene information is unavailable, such as metagenomes and fragmented genome assemblies.

In 1977, the world witnessed both the eradication of smallpox and the beginning of the modern age of genomics. Over the following half-century, 7 epidemic viruses of international concern galvanized virologists across the globe and led to increasingly extensive virus genome sequencing. These sequencing efforts exerted over periods of rapid adaptation of viruses to new hosts, in particular, humans provide insight into the molecular mechanisms underpinning virus evolution. Investment in virus genome sequencing was dramatically increased by the unprecedented support for phylogenomic analyses during the COVID-19 pandemic. In this review, we attempt to piece together comprehensive molecular histories of the adaptation of variola virus, HIV-1 M, SARS, H1N1-SIV, MERS, Ebola, Zika, and SARS-CoV-2 to the human host. Disruption of genes involved in virus-host interaction in animal hosts, recombination including genome segment reassortment, and adaptive mutations leading to amino acid replacements in virus proteins involved in host receptor binding and membrane fusion are identified as the key factors in the evolution of epidemic viruses.

CRISPR-associated Rossmann Fold (CARF) and SMODS-associated and fused to various effector domains (SAVED) are key components of cyclic oligonucleotide-based antiphage signaling systems (CBASS) that sense cyclic oligonucleotides and transmit the signal to an effector inducing cell dormancy or death. Most of the CARFs are components of a CBASS built into type III CRISPR–Cas systems, where the CARF domain binds cyclic oligoA (cOA) synthesized by Cas10 polymerase-cyclase and allosterically activates the effector, typically a promiscuous ribonuclease. Additionally, this signaling pathway includes a ring nuclease, often also a CARF domain (either the sensor itself or a specialized enzyme) that cleaves cOA and mitigates dormancy or death induction. We present a comprehensive census of CARF and SAVED domains in bacteria and archaea, and their sequence- and structure-based classification. There are 10 major families of CARF domains and multiple smaller groups that differ in structural features, association with distinct effectors, and presence or absence of the ring nuclease activity. By comparative genome analysis, we predict specific functions of CARF and SAVED domains and partition the CARF domains into those with both sensor and ring nuclease functions, and sensor-only ones. Several families of ring nucleases functionally associated with sensor-only CARF domains are also predicted.

Significance The ongoing COVID-19 pandemic is an urgent, global threat. Here we analyze the genome of the virus that causes COVID-19, SARS-CoV-2, along with other members of the coronavirus family. Our analysis identifies crucial genomic features that are unique to SARS-CoV-2 and two other deadly coronaviruses, SARS-CoV and MERS-CoV. These features correlate with the high fatality rate of these coronaviruses as well as their ability to switch hosts from animals to humans. The identified features could represent crucial elements of coronavirus virulence, and allow for detecting animal coronaviruses that have the potential to make the jump to humans in the future., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an immediate, major threat to public health across the globe. Here we report an in-depth molecular analysis to reconstruct the evolutionary origins of the enhanced pathogenicity of SARS-CoV-2 and other coronaviruses that are severe human pathogens. Using integrated comparative genomics and machine learning techniques, we identify key genomic features that differentiate SARS-CoV-2 and the viruses behind the two previous deadly coronavirus outbreaks, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), from less pathogenic coronaviruses. These features include enhancement of the nuclear localization signals in the nucleocapsid protein and distinct inserts in the spike glycoprotein that appear to be associated with high case fatality rate of these coronaviruses as well as the host switch from animals to humans. The identified features could be crucial contributors to coronavirus pathogenicity and possible targets for diagnostics, prognostication, and interventions.

Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNAPyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNAPyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNAPyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNAPyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNAPyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.

The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria., Archaea and bacteria often have gene pairs with overlapping stop and start codons, suggesting translational coupling. Here, Huber et al. analyse overlapping gene pairs from 720 genomes, and validate translational coupling via termination-reinitiation for 14 gene pairs in Haloferax volcanii and Escherichia coli.

Significance Cancer is caused by the effects of somatic mutations known as drivers. Although a number of major cancer drivers have been identified, it is suspected that many more comparatively rare and conditional drivers exist, and the interactions between different cancer-associated mutations that might be relevant for tumor progression are not well understood. We applied an advanced neural network approach to learn the sequence of mutations and the mutational burden in colon and lung cancers and to identify mutations that are associated with individual drivers. A significant ordering of driver mutations is demonstrated, and numerous, previously undetected conditional drivers are identified. These findings broaden the existing understanding of the mechanisms of tumor progression and have implications for therapeutic strategies., Cancer arises through the accumulation of somatic mutations over time. Understanding the sequence of mutation occurrence during cancer progression can assist early and accurate diagnosis and improve clinical decision-making. Here we employ long short-term memory (LSTM) networks, a class of recurrent neural network, to learn the evolution of a tumor through an ordered sequence of mutations. We demonstrate the capacity of LSTMs to learn complex dynamics of the mutational time series governing tumor progression, allowing accurate prediction of the mutational burden and the occurrence of mutations in the sequence. Using the probabilities learned by the LSTM, we simulate mutational data and show that the simulation results are statistically indistinguishable from the empirical data. We identify passenger mutations that are significantly associated with established cancer drivers in the sequence and demonstrate that the genes carrying these mutations are substantially enriched in interactions with the corresponding driver genes. Breaking the network into modules consisting of driver genes and their interactors, we show that these interactions are associated with poor patient prognosis, thus likely conferring growth advantage for tumor progression. Thus, application of LSTM provides for prediction of numerous additional conditional drivers and reveals hitherto unknown aspects of cancer evolution.

The survival of viruses depends on their ability to resist host defenses and, of all animal virus families, the poxviruses have the most antidefense genes. Orthopoxviruses (ORPV), a genus within the subfamily Chordopoxvirinae, infect diverse mammals and include one of the most devastating human pathogens, the now eradicated smallpox virus. ORPV encode ∼200 genes, of which roughly half are directly involved in virus genome replication and expression as well as virion morphogenesis. The remaining ∼100 “accessory” genes are responsible for virus-host interactions, particularly counter-defense of innate immunity. Complete sequences are currently available for several hundred ORPV genomes isolated from a variety of mammalian hosts, providing a rich resource for comparative genomics and reconstruction of ORPV evolution. To identify the provenance and evolutionary trends of the ORPV accessory genes, we constructed clusters including the orthologs of these genes from all chordopoxviruses. Most of the accessory genes were captured in three major waves early in chordopoxvirus evolution, prior to the divergence of ORPV and the sister genus Centapoxvirus from their common ancestor. The capture of these genes from the host was followed by extensive gene duplication, yielding several paralogous gene families. In addition, nine genes were gained during the evolution of ORPV themselves. In contrast, nearly every accessory gene was lost, some on multiple, independent occasions in numerous lineages of ORPV, so that no ORPV retains them all. A variety of functional interactions could be inferred from examination of pairs of ORPV accessory genes that were either often or rarely lost concurrently. IMPORTANCE Orthopoxviruses (ORPV) include smallpox (variola) virus, one of the most devastating human pathogens, and vaccinia virus, comprising the vaccine used for smallpox eradication. Among roughly 200 ORPV genes, about half are essential for genome replication and expression as well as virion morphogenesis, whereas the remaining half consists of accessory genes counteracting the host immune response. We reannotated the accessory genes of ORPV, predicting the functions of uncharacterized genes, and reconstructed the history of their gain and loss during the evolution of ORPV. Most of the accessory genes were acquired in three major waves antedating the origin of ORPV from chordopoxviruses. The evolution of ORPV themselves was dominated by gene loss, with numerous genes lost at the base of each major group of ORPV. Examination of pairs of ORPV accessory genes that were either often or rarely lost concurrently during ORPV evolution allows prediction of different types of functional interactions.

At the time of this writing, December 2021, potential emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlap with the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Nonetheless, variants with multiple mutations in the RBD have rose to dominance. Non-additive, epistatic relationships among RBD mutations are apparent, and assessing the impact of such epistasis on the mutational landscape is crucial. Epistasis can substantially increase the risk of vaccine escape and cannot be completely characterized through the study of the wild type (WT) alone. We employed protein structure modeling using Rosetta to compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for the WT, Delta, Gamma, and Omicron variants. Overall, epistasis at the RBD interface appears to be limited and the effects of most multiple mutations are additive. Epistasis at the Delta variant interface weakly stabilizes NAb interaction relative to ACE2 interaction, whereas in the Gamma variant, epistasis more substantially destabilizes NAb interaction. Although a small, systematic trend towards NAb destabilization not observed for Delta or Gamma was detected for Omicron, and despite bearing significantly more RBD mutations, the epistatic landscape of the Omicron variant closely resembles that of Gamma. These results suggest that, although Omicron poses new risks not observed with Delta, structural constraints on the RBD hamper continued evolution towards more complete vaccine escape. The modest ensemble of mutations relative to the WT that are currently known to reduce vaccine efficacy is likely to comprise the majority of all possible escape mutations for future variants, predicting continued efficacy of the existing vaccines.SignificanceEmergence of vaccine escape variants of SARS-CoV-2 is arguably the most pressing problem during the COVID-19 pandemic as vaccines are distributed worldwide. We employed a computational approach to assess the risk of antibody escape resulting from mutations in the receptor-binding domain of the spike protein of the wild type SARS-CoV-2 virus as well as the Delta, Gamma, and Omicron variants. At the time of writing, December, 2021, Omicron is poised to replace Delta as the dominant variant worldwide. The efficacy of the existing vaccines against Omicron could be substantially reduced relative to the WT and the potential for vaccine escape is of grave concern. Our results suggest that although Omicron poses new evolutionary risks not observed for the Delta variant, structural constraints on the RBD make continued evolution towards more complete vaccine escape unlikely. The modest set of escape-enhancing mutations already identified for the wild type likely include the majority of all possible mutations with this effect.

Molecular mechanisms involved in biological conflicts and self vs nonself recognition in archaea remain poorly characterized. We apply phylogenomic analysis to identify a hypervariable gene module that is widespread among Thermococcales. These loci consist of an upstream gene coding for a large protein containing several immunoglobulin (Ig) domains and unique combinations of downstream genes, some of which also contain Ig domains. In the large Ig domain containing protein, the C-terminal Ig domain sequence is hypervariable, apparently, as a result of recombination between genes from different Thermococcales. To reflect the hypervariability, we denote this gene module VARTIG (VARiable Thermococcales IG). The overall organization of the VARTIG modules is similar to the organization of Polymorphic Toxin Systems (PTS). Archaeal genomes outside Thermococcales encode a variety of Ig domain proteins, but no counterparts to VARTIG and no Ig domains with comparable levels of variability. The specific functions of VARTIG remain unknown but the identified features of this system imply three testable hypotheses: (i) involvement in inter-microbial conflicts analogous to PTS, (ii) role in innate immunity analogous to the vertebrate complement system, and (iii) function in self vs nonself discrimination analogous to the vertebrate Major Histocompatibility Complex. The latter two hypotheses seem to be of particular interest given the apparent analogy to the vertebrate immunity.

Background: It is often assumed that pathogens evolve towards reduced virulence, but counterexamples abound. Faced with a new pathogen, such as SARS-CoV-2, it is highly desirable to be able to forecast the case fatality rate (CFR) and overall disease burden into the future. Considerable effort has been invested towards the development of a mathematical framework for predicting virulence evolution. Although many approaches accurately recapitulate complex outcomes, most rely on an assumed trade-off between CFR and infection rate. It is often impractical to empirically validate this constraint for human pathogens. Methods: A compartment model with parameters tuning the degree to which symptomatic individuals are isolated and the duration of immunity is constructed and evaluated at both short timescales and at equilibrium (when it exists). Results: We reveal kinetic constraints where the variation of multiple parameters in concert leads to decreased CFR and increased pathogen fitness, whereas independent variation of the parameters decreases pathogen fitness. Smallpox, SARS-CoV-2, and influenza are analyzed as diverse representatives of human respiratory viruses. We show that highly virulent viruses, such as smallpox, are likely often constrained by host behavior, whereas moderately virulent viruses, such as SARS-CoV-2, appear to be typically constrained by the relationship between the duration of immunity and CFR. Conclusions: Evolution of human respiratory epidemics appears to be often kinetically constrained and a reduction in CFR should not be assumed. Our findings imply that, without continued public health intervention, SARS-CoV-2 is likely to continue presenting a substantial disease burden. The existence of a parameter regime admitting endemic equilibrium suggests that herd immunity is unachievable. However, we demonstrate that even partial isolation of symptomatic individuals can have a major effect not only by reducing the number of fatalities in the short term but also by potentially changing the evolutionary trajectory of the virus towards reduced CFR.

Ribosomal proteins (RPs) are highly conserved across the bacterial and archaeal domains. Although many RPs are essential for survival, genome analysis demonstrates the absence of some RP genes in many bacterial and archaeal genomes. Furthermore, global transposon mutagenesis and/or targeted deletion studies showed that elimination of some RP genes had only a moderate effect on the bacterial growth rate. Here, we systematically analyzed the evolutionary conservation of RPs in prokaryotes by compiling a list of the ribosomal genes that are missing from one or more genomes in the recently updated version of the Clusters of Orthologous Genes (COG) database. Some of these absences occurred because the respective genes carried frameshifts, presumably resulting from sequencing errors, while others were overlooked and not translated during genome annotation. Apart from these annotation errors, we identified multiple genuine losses of RP genes in a variety of bacteria and archaea. Some of these losses are clade specific, whereas others occur in symbionts and parasites with dramatically reduced genomes. The lists of computationally and experimentally defined nonessential ribosomal genes show a substantial overlap, revealing a common trend in prokaryote ribosome evolution that could be linked to the architecture and assembly of the ribosomes. Thus, RPs that are located at the surface of the ribosome and/or are incorporated at a late stage of ribosome assembly are more likely to be nonessential and to be lost during microbial evolution, particularly in the course of genome compaction. IMPORTANCE In many prokaryote genomes, one or more ribosomal protein (RP) genes are missing. Analysis of 1,309 prokaryote genomes included in the Clusters of Orthologous Genes (COG) database shows that only about half of the RPs are universally conserved in bacteria and archaea. In contrast, up to 16 other RPs are missing in some genomes, primarily tiny (

CrAssphage is the most abundant human-associated virus and the founding member of a large group of bacteriophages, discovered in animal-associated and environmental metagenomes, that infect bacteria of the phylum Bacteroidetes. We analyze 4907 Circular Metagenome Assembled Genomes (cMAGs) of putative viruses from human gut microbiomes and identify nearly 600 genomes of crAss-like phages that account for nearly 87% of the DNA reads mapped to these cMAGs. Phylogenetic analysis of conserved genes demonstrates the monophyly of crAss-like phages, a putative virus order, and of 5 branches, potential families within that order, two of which have not been identified previously. The phage genomes in one of these families are almost twofold larger than the crAssphage genome (145-192 kilobases), with high density of self-splicing introns and inteins. Many crAss-like phages encode suppressor tRNAs that enable read-through of UGA or UAG stop-codons, mostly, in late phage genes. A distinct feature of the crAss-like phages is the recurrent switch of the phage DNA polymerase type between A and B families. Thus, comparative genomic analysis of the expanded assemblage of crAss-like phages reveals aspects of genome architecture and expression as well as phage biology that were not apparent from the previous work on phage genomics., Here, the authors analyze 4907 Circular Metagenome Assembled Genomes from human microbiomes and identify and characterize nearly 600 diverse genomes of crAss-like phages, finding two putative families with unusual genomic features, including high density of self-splicing introns and inteins.

Ribosomal proteins (RPs) are highly conserved across the bacterial and archaeal domains. Although many RPs are essential for survival, genome analysis demonstrates the absence of some RP genes in many bacterial and archaeal genomes. Furthermore, global transposon mutagenesis and/or targeted deletion showed that elimination of some RP genes had only a moderate effect on the bacterial growth rate. Here, we systematically analyze the evolutionary conservation of RPs in prokaryotes by compiling the list of the ribosomal genes that are missing from the one or more genomes in the recently updated version of the Clusters of Orthologous Genes (COG) database. Some of these absences occurred because the respective genes carried frameshifts, presumably, resulting from sequencing errors, while others were overlooked and not translated during genome annotation. Apart from these annotation errors, we identified multiple genuine losses of RP genes in a variety of bacteria and archaea. Some of these losses are clade-specific, whereas others occur in symbionts and parasites with dramatically reduced genomes. The lists of computationally and experimentally defined non-essential ribosomal genes show a substantial overlap, revealing a common trend in prokaryote ribosome evolution that could be linked to the architecture and assembly of the ribosomes. Thus, RPs that are located at the surface of the ribosome and/or are incorporated at a late stage of ribosome assembly are more likely to be non-essential and to be lost during microbial evolution, particularly, in the course of genome compaction.IMPORTANCEIn many prokaryote genomes, one or more ribosomal protein (RP) genes are missing. Analysis of 1,309 prokaryote genomes included in the COG database shows that only about half of the RPs are universally conserved in bacteria and archaea. In contrast, up to 21 other RPs are missing in some genomes, primarily, tiny (Escherichia coli and Bacillus subtilis. These results reveal general trends and common constraints in the architecture and evolution of ribosomes in prokaryotes.

Intratumour heterogeneity and phenotypic plasticity, sustained by a range of somatic aberrations, as well as epigenetic and metabolic adaptations, are the principal mechanisms that enable cancers to resist treatment and survive under environmental stress. A comprehensive picture of the interplay between different somatic aberrations, from point mutations to whole-genome duplications, in tumour initiation and progression is lacking. We posit that different genomic aberrations generally exhibit a temporal order, shaped by a balance between the levels of mutations and selective pressures. Repeat instability emerges first, followed by larger aberrations, with compensatory effects leading to robust tumour fitness maintained throughout the tumour progression. A better understanding of the interplay between genetic aberrations, the microenvironment, and epigenetic and metabolic cellular states is essential for early detection and prevention of cancer as well as development of efficient therapeutic strategies.

Gene transfer agents (GTAs) are virus-like particles encoded and produced by many bacteria and archaea. Unlike viruses, GTAs package fragments of the host genome instead of the genes that encode the components of the GTA itself. As a result of this non-specific DNA packaging, GTAs can transfer genes within bacterial and archaeal communities. GTAs clearly evolved from viruses and are thought to have been maintained in prokaryotic genomes due to the advantages associated with their DNA transfer capacity. The most-studied GTA is produced by the alphaproteobacterium Rhodobacter capsulatus (RcGTA), which packages random portions of the host genome at a lower DNA density than usually observed in tailed bacterial viruses. How the DNA packaging properties of RcGTA evolved from those of the ancestral virus remains unknown. To address this question, we reconstructed the evolutionary history of the large subunit of the terminase (TerL), a highly conserved enzyme used by viruses and GTAs to package DNA. We found that RcGTA-like TerLs grouped within viruses that employ the headful packaging strategy. Because distinct mechanisms of viral DNA packaging correspond to differences in the TerL amino acid sequence, our finding suggests that RcGTA evolved from a headful packaging virus. Headful packaging is the least sequence-specific mode of DNA packaging, which would facilitate the switch from packaging of the viral genome to packaging random pieces of the host genome during GTA evolution.

CRISPR-Cas systems typically consist of a CRISPR array and cas genes that are organized in one or more operons. However, a substantial fraction of CRISPR arrays are not adjacent to cas genes. Definitive identification of such isolated CRISPR arrays runs into the problem of false-positives, with unrelated types of repetitive sequences mimicking CRISPR. We developed a computational pipeline to eliminate false CRISPR predictions and found that up to 25% of the CRISPR arrays in complete bacterial and archaeal genomes are located away from cas genes. Most of the repeats in these isolated arrays are identical to repeats in cas-adjacent CRISPR arrays in the same or closely related genomes, indicating an evolutionary relationship between isolated arrays and arrays in typical CRISPR-cas loci. The spacers in isolated CRISPR arrays show nearly as many matches to viral genomes as spacers from complete CRISPR-cas loci, suggesting that the isolated arrays were either functionally active recently or continue to function. Reconstruction of evolutionary events in closely related bacterial genomes suggests three routes of evolution of isolated CRISPR arrays: (1) loss of cas genes in a CRISPR-cas locus, (2) de novo generation of arrays from off-target spacer integration into sequences resembling the corresponding repeats, and (3) transfer by mobile genetic elements. Both combination of de novo emerging arrays with cas genes and regain of cas genes by isolated arrays via recombination likely contribute to functional diversification in CRISPR-Cas evolution.

The Clusters of Orthologous Genes (COG) database, also referred to as the Clusters of Orthologous Groups of proteins, was created in 1997 and went through several rounds of updates, most recently, in 2014. The current update, available at https://www.ncbi.nlm.nih.gov/research/COG, substantially expands the scope of the database to include complete genomes of 1187 bacteria and 122 archaea, typically, with a single genome per genus. In addition, the current version of the COGs includes the following new features: (i) the recently deprecated NCBI’s gene index (gi) numbers for the encoded proteins are replaced with stable RefSeq or GenBank\ENA\DDBJ coding sequence (CDS) accession numbers; (ii) COG annotations are updated for >200 newly characterized protein families with corresponding references and PDB links, where available; (iii) lists of COGs grouped by pathways and functional systems are added; (iv) 266 new COGs for proteins involved in CRISPR-Cas immunity, sporulation in Firmicutes and photosynthesis in cyanobacteria are included; and (v) the database is made available as a web page, in addition to FTP. The current release includes 4877 COGs. Future plans include further expansion of the COG collection by adding archaeal COGs (arCOGs), splitting the COGs containing multiple paralogs, and continued refinement of COG annotations.

SummaryAdvances in metagenomics enable massive discovery of diverse, distinct microbes and viruses. Bacteriophages, the most abundant biological entity on Earth, evolve rapidly, and therefore, detection of unknown bacteriophages in sequence datasets is a challenge. The existing methods rely on sequence similarity to known bacteriophage sequences, impeding the identification and characterization of distinct bacteriophage families. We present Seeker, a deep-learning tool for reference-free identification of phage sequences. Seeker allows rapid detection of phages in sequence datasets and clean differentiation of phage sequences from bacterial ones, even for phages with little sequence similarity to established phage families. We comprehensively validate Seeker’s ability to identify unknown phages and employ Seeker to detect unknown phages, some of which are highly divergent from known phage families. We provide a web portal (seeker.pythonanywhere.com) and a user-friendly python package (https://github.com/gussow/seeker) allowing researchers to easily apply Seeker in metagenomic studies, for the detection of diverse unknown bacteriophages.

Gene transfer agents (GTAs) are virus-like particles encoded and produced by many bacteria and archaea. Unlike viruses, GTAs package fragments of the host genome instead of the genes that encode the components of the GTA itself. As a result of this non-specific DNA packaging, GTAs can transfer genes within bacterial and archaeal communities. GTAs clearly evolved from viruses and are thought to have been maintained in prokaryotic genomes due to the advantages associated with their DNA transfer capacity. The most-studied GTA is produced by the alphaproteobacterium Rhodobacter capsulatus (RcGTA), which packages random portions of the host genome at a lower DNA density than usually observed in tailed bacterial viruses. How the DNA packaging properties of RcGTA evolved from those of the ancestral virus remains unknown. To address this question, we reconstructed the evolutionary history of the large subunit of the terminase (TerL), a highly conserved enzyme used by viruses and GTAs to package DNA. We found that RcGTA-like TerLs grouped within viruses that employ the headful packaging strategy. Because distinct mechanisms of viral DNA packaging correspond to differences in the TerL amino acid sequence, our finding suggests that RcGTA evolved from a headful packaging virus. Headful packaging is the least sequence-specific mode of DNA packaging, which would facilitate the switch from packaging of the viral genome to packaging random pieces of the host genome during GTA evolution.

Driver mutations (DM) are the genetic impetus for most cancers. The DM are assumed to be deleterious in species evolution, being eliminated by purifying selection unless compensated by other mutations. We present deep phylogenies for 84 cancer driver genes and investigate the prevalence of 434 DM across gene-species trees. The DM are rare in species evolution, and 181 are completely absent, validating their negative fitness effect. The DM are more common in unicellular than in multicellular eukaryotes, suggesting a link between these mutations and cell proliferation control. 18 DM appear as the ancestral state in one or more major clades, including 3 among mammals. We identify within-gene, compensatory mutations for 98 DM and infer likely interactions between the DM and compensatory sites in protein structures. These findings elucidate the evolutionary status of DM and are expected to advance the understanding of the functions and evolution of oncogenes and tumor suppressors., Rochman et al. present deep phylogenies for 84 cancer driver genes and examine the prevalence of driver mutations across gene-species trees. Their results show that driver mutations are rare in species evolution and give insight into the evolution of driver mutations and oncogenes.

RNA viruses in aquatic environments remain poorly studied. Here, we analysed the RNA virome from approximately 10 l water from Yangshan Deep-Water Harbour near the Yangtze River estuary in China and identified more than 4,500 distinct RNA viruses, doubling the previously known set of viruses. Phylogenomic analysis identified several major lineages, roughly, at the taxonomic ranks of class, order and family. The 719-member-strong Yangshan virus assemblage is the sister clade to the expansive class Alsuviricetes and consists of viruses with simple genomes that typically encode only RNA-dependent RNA polymerase (RdRP), capping enzyme and capsid protein. Several clades within the Yangshan assemblage independently evolved domain permutation in the RdRP. Another previously unknown clade shares ancestry with Potyviridae, the largest known plant virus family. The ‘Aquatic picorna-like viruses/Marnaviridae’ clade was greatly expanded, with more than 800 added viruses. Several RdRP-linked protein domains not previously detected in any RNA viruses were identified, such as the small ubiquitin-like modifier (SUMO) domain, phospholipase A2 and PrsW-family protease domain. Multiple viruses utilize alternative genetic codes implying protist (especially ciliate) hosts. The results reveal a vast RNA virome that includes many previously unknown groups. However, phylogenetic analysis of the RdRPs supports the previously established five-branch structure of the RNA virus evolutionary tree, with no additional phyla., Metagenomic analysis of a single RNA virome from the Yangshan Deep-Water Harbour in China enabled the recovery of more than 4,500 distinct RNA viruses, doubling the known set of RNA viruses to date, and provided insights into their biology.

BackgroundDNA and RNA of most cellular life forms and many viruses contain an expansive repertoire of modified bases. The modified bases play diverse biological roles that include both regulation of transcription and translation, and protection against restriction endonucleases and antibiotics. Modified bases are often recognized by dedicated protein domains. However, the elaborate networks of interactions and processes mediated by modified bases are far from being completely understood.ResultsWe present a comprehensive census and classification of EVE domains that belong to the PUA/ASCH domain superfamily and bind various modified bases in DNA and RNA. Prokaryotes encode two classes of EVE domain proteins, slow-evolving and fast-evolving. The slow-evolving EVE domains in α-proteobacteria are embedded in a conserved operonic context that implies involvement in coupling between translation and respiration, in particular, cytochrome c biogenesis, potentially, via binding 5-methylcytosine in tRNAs. In β and γ-proteobacteria, the conserved associations implicate the EVE domains in the coordination of cell division, biofilm formation, and global transcriptional regulation by non-coding 6S small RNAs, which are potentially modified and bound by the EVE domains. Down-regulation of the EVE-encoding operons might cause dormancy or programmed cell death (PCD). In eukaryotes, the EVE-domain-containing THYN1-like proteins appear to inhibit PCD and regulate the cell cycle, likely, via binding 5-methylcytosine and its derivatives in DNA and/or RNA. Thus, the link between PCD and cytochrome c that appears to be universal in eukaryotes might have been inherited from the α-proteobacterial, proto-mitochondrial endosymbiont and, unexpectedly, could involve modified base recognition by EVE domains. In numerous prokaryotic genomes, fast-evolving EVE domains are embedded in defense contexts, including toxin-antitoxin modules and Type IV restriction systems, all of which can also induce PCD. These EVE domains likely recognize modified bases in invading DNA molecules and target them for restriction. We additionally identified EVE-like prokaryotic Development and Cell Death (DCD) domains that are also implicated in defense functions including PCD. This function was inherited by eukaryotes but, in animals, the DCD proteins apparently were displaced by the extended Tudor family, whose partnership with Piwi-related Argonautes became the centerpiece of the piRNA system.ConclusionsRecognition of modified bases in DNA and RNA by EVE-like domains appears to be an important, but until now, under-appreciated, common denominator in a variety of processes including PCD, cell cycle control, antivirus immunity, stress response and germline development in animals.

CrAssphage is the most abundant virus identified in the human gut virome and the founding member of a large group of bacteriophages that infect bacteria of the phylum Bacteroidetes and have been discovered by metagenomics of both animal-associated and environmental habitats. By analysis of circular contigs from human gut microbiomes, we identified nearly 600 genomes of crAss-like phages. Phylogenetic analysis of conserved genes demonstrates the monophyly of crAss-like phages, which can be expected to become a new order of viruses, and of 5 distinct branches, likely, families within that order. Two of these putative families have not been identified previously. The phages in one of these groups have large genomes (145-192 kilobases) and contain an unprecedented high density of self-splicing introns and inteins. Many crAss-like phages encode suppressor tRNAs that enable readthrough of UGA or UAG stop-codons, mostly, in late phage genes, which could represent a distinct anti-defense strategy. Another putative anti-defense mechanism that might target an unknown defense system in Bacteroidetes inhibiting phage DNA replication involves multiple switches of the phage DNA polymerase type between A and B families. Thus, comparative genomic analysis of the expanded assemblage of crAss-like phages reveals several unusual features of genome architecture and expression as well as phage biology that were not apparent from the previous crAssphage analyses.

The principal function of archaeal and bacterial CRISPR-Cas systems is antivirus adaptive immunity. However, recent genome analyses identified a variety of derived CRISPR-Cas variants at least some of which appear to perform different functions. Here, we describe a unique repertoire of CRISPR-Cas-related systems that we discovered by searching archaeal metagenome-assemble genomes of the Asgard superphylum. Several of these variants contain extremely diverged homologs of Cas1, the integrase involved in CRISPR adaptation as well as casposon transposition. Strikingly, the diversity of Cas1 in Asgard archaea alone is greater than that detected so far among the rest of archaea and bacteria. The Asgard CRISPR-Cas derivatives also encode distinct forms of Cas4, Cas5, and Cas7 proteins, and/or additional nucleases. Some of these systems are predicted to perform defense functions, but possibly not programmable ones, whereas others are likely to represent previously unknown mobile genetic elements.

International audience; Letter to the Editor

Kin selection and group selection remain controversial topics in evolutionary biology. We argue that these types of selection are likely to operate in bacterial populations by showing that bacterial gene transfer agents (GTAs), but not related viruses, evolve under conditions of positive selection for the reduction of the energy cost of GTA particle production. We hypothesize that GTAs are dedicated devices mediating the survival of bacteria under conditions of nutrient limitation. The benefits conferred by GTAs under nutritional stress conditions appear to include horizontal dissemination of genes that could provide bacteria with enhanced capabilities for nutrient utilization and increases of nutrient availability occurring through the lysis of GTA-producing bacteria., Gene transfer agents (GTAs) are virus-like elements integrated into bacterial genomes, particularly, those of Alphaproteobacteria. The GTAs can be induced under conditions of nutritional stress, incorporate random fragments of bacterial DNA into miniphage particles, lyse the host cells, and infect neighboring bacteria, thus enhancing horizontal gene transfer. We show that GTA genes evolve under conditions of pronounced positive selection for the reduction of the energy cost of protein production as shown by comparison of the amino acid compositions with those of both homologous viral genes and host genes. The energy saving in GTA genes is comparable to or even more pronounced than that in the genes encoding the most abundant, essential bacterial proteins. In cases in which viruses acquire genes from GTAs, the bias in amino acid composition disappears in the course of evolution, showing that reduction of the energy cost of protein production is an important factor of evolution of GTAs but not bacterial viruses. These findings strongly suggest that GTAs represent bacterial adaptations rather than selfish, virus-like elements. Because GTA production kills the host cell and does not propagate the GTA genome, it appears likely that the GTAs are retained in the course of evolution via kin or group selection. Therefore, we hypothesize that GTAs facilitate the survival of bacterial populations under energy-limiting conditions through the spread of metabolic and transport capabilities via horizontal gene transfer and increases in nutrient availability resulting from the altruistic suicide of GTA-producing cells.

Few aquatic animal negative-sense RNA viruses have been characterized, and their role in disease is poorly understood. Here, we describe a virus isolated from diseased freshwater turtles from a Florida farm in 2007 and from an ongoing epizootic among free-ranging populations of Florida softshell turtles (Apalone ferox), Florida red-bellied cooters (Pseudemys nelsoni), and peninsula cooters (Pseudemys peninsularis). Affected turtles presented with similar neurological signs, oral and genital ulceration, and secondary microbial infections. Microscopic lesions were most severe in the softshell turtles and included heterophilic/histiocytic meningoencephalitis, multi-organ vasculitis, and cytologic observation of leukocytic intracytoplasmic inclusions. The virus was isolated using Terrapene heart (TH-1) cells. Ultrastructurally, viral particles were round to pleomorphic and acquired an envelope with prominent surface projections by budding from the cell membrane. Viral genomes were sequenced from cDNA libraries of two nearly identical isolates and determined to be bi-segmented, with an ambisense coding arrangement. The larger segment encodes a predicted RNA-directed RNA polymerase (RdRP) and a putative zinc-binding matrix protein. The smaller segment encodes a putative nucleoprotein and an envelope glycoprotein precursor (GPC). Thus, the genome organization of this turtle virus resembles that of arenaviruses. Phylogenetic analysis shows that the RdRP of the turtle virus is highly diverged from the RdRPs of all known negative-sense RNA viruses and forms a deep branch within the phylum Negarnaviricota, that is not affiliated with any known group of viruses, even at the class level. In contrast, the GPC protein of the turtle virus is confidently affiliated with homologs from a distinct group of fish hantaviruses. Thus, the turtle virus is expected to become the founder of a new taxon of negative-sense RNA viruses, at least with a family rank, but likely, an order or even a class. These viruses probably evolved either by reassortment or by intrasegment recombination between a virus from a distinct branch of negarnaviruses distant from all known groups and a hanta-like aquatic virus. We suggest the provisional name Tosoviridae for the putative new family, with Turtle fraservirus 1 (TFV1) as the type species within the genus Fraservirus. A conventional RT-PCR assay, targeting the TFV1 RdRP, confirmed the presence of viral RNA in multiple tissues and exudates from diseased turtles. The systemic nature of the TFV1 infection was further supported by labeling of cells within lesions using in situ hybridization targeting the RNA of the TFV1 RdRP.

Transcriptome-based exon capture methods provide an approach to recover several hundred markers from genomic DNA, allowing for robust phylogenetic estimation at deep timescales. We applied this method to a highly diverse group of venomous marine snails, Conoidea, for which published phylogenetic trees remain mostly unresolved for the deeper nodes. We targeted 850 protein coding genes (678,322 bp) in ca. 120 samples, spanning all (except one) known families of Conoidea and a broad selection of non-Conoidea neogastropods. The capture was successful for most samples, although capture efficiency decreased when DNA libraries were of insufficient quality and/or quantity (dried samples or low starting DNA concentration) and when targeting the most divergent lineages. An average of 75.4% of proteins was recovered, and the resulting tree, reconstructed using both supermatrix (IQ-tree) and supertree (Astral-II, combined with the Weighted Statistical Binning method) approaches, are almost fully supported. A reconstructed fossil-calibrated tree dates the origin of Conoidea to the Lower Cretaceous. We provide descriptions for two new families. The phylogeny revealed in this study provides a robust framework to reinterpret changes in Conoidea anatomy through time. Finally, we used the phylogeny to test the impact of the venom gland and radular type on diversification rates. Our analyses revealed that repeated losses of the venom gland had no effect on diversification rates, while families with a breadth of radula types showed increases in diversification rates, thus suggesting that trophic ecology may have an impact on the evolution of Conoidea.

Amino acid frequencies in proteins may not be at equilibrium. We consider two possible explanations for the nonzero net residue fluxes in drosophilid proteins. First, protein interiors may have a suboptimal residue composition and be under a selective pressure favoring stability, that is, leading to the loss of polar (and the gain of large) amino acids. One would then expect stronger net fluxes on the protein interior than at the exposed sites. Alternatively, if most of the polarity loss occurs at the exposed sites and the selective constraint on amino acid composition at such sites decreases over time, net loss of polarity may be neutral and caused by disproportionally high occurrence of polar residues at exposed, least constrained sites. We estimated net evolutionary fluxes of residue polarity and volume at sites with different solvent accessibility in conserved protein families from 12 species of Drosophila. Net loss of polarity, miniscule in magnitude, but consistent across all lineages, occurred at all sites except the most exposed ones, where net flux of polarity was close to zero or, in membrane proteins, even positive. At the intermediate solvent accessibility the net fluxes of polarity and volume were similar to neutral predictions, whereas much of the polarity loss not attributable to neutral expectations occurred at the buried sites. These observations are consistent with the hypothesis that residue composition in many proteins is structurally suboptimal and continues to evolve toward lower polarity in the protein interior, in particular in proteins with intracellular localization. The magnitude of polarity and volume changes was independent from the protein's evolutionary age, indicating that the approach to equilibrium has been slow or that no such single equilibrium exists.

Evolution of bacteria and archaea involves an incessant arms race against an enormous diversity of genetic parasites. Accordingly, a substantial fraction of the genes in most bacteria and archaea are dedicated to antiparasite defense. The functions of these defense systems follow several distinct strategies, including innate immunity; adaptive immunity; and dormancy induction, or programmed cell death. Recent comparative genomic studies taking advantage of the expanding database of microbial genomes and metagenomes, combined with direct experiments, resulted in the discovery of several previously unknown defense systems, including innate immunity centered on Argonaute proteins, bacteriophage exclusion, and new types of CRISPR-Cas systems of adaptive immunity. Some general principles of function and evolution of defense systems are starting to crystallize, in particular, extensive gain and loss of defense genes during the evolution of prokaryotes; formation of genomic defense islands; evolutionary connections between mobile genetic elements and defense, whereby genes of mobile elements are repeatedly recruited for defense functions; the partially selfish and addictive behavior of the defense systems; and coupling between immunity and dormancy induction/programmed cell death.

All cellular life forms are afflicted by diverse genetic parasites, including viruses and other types of mobile genetic elements (MGEs), and have evolved multiple, diverse defence systems that protect them from MGE assault via different mechanisms. Here, we provide our perspectives on how recent evidence points to tight evolutionary connections between MGEs and defence systems that reach far beyond the proverbial arms race. Defence systems incur a fitness cost for the hosts; therefore, at least in prokaryotes, horizontal mobility of defence systems, mediated primarily by MGEs, is essential for their persistence. Moreover, defence systems themselves possess certain features of selfish elements. Common components of MGEs, such as site-specific nucleases, are ‘guns for hire’ that can also function as parts of defence mechanisms and are often shuttled between MGEs and defence systems. Thus, evolutionary and molecular factors converge to mould the multifaceted, inextricable connection between MGEs and anti-MGE defence systems. Incessant encounters of all cellular life forms with mobile genetic elements (MGEs) have driven the evolution of diverse defence mechanisms, including CRISPR–Cas and restriction–modification systems. In this Perspective, Koonin, Makarova, Wolf and Krupovic describe the surprisingly intricate interplay between MGEs and host defence systems. Not only do defence systems commonly show high horizontal mobility but many molecular components are ‘guns for hire’ that have been co-opted by defence systems from MGEs and vice versa.

Class 2 CRISPR-Cas systems are characterized by effector modules that consist of a single multidomain protein, such as Cas9 or Cpf1. We designed a computational pipeline for the discovery of novel class 2 variants and used it to identify six new CRISPR-Cas subtypes. The diverse properties of these new systems provide potential for the development of versatile tools for genome editing and regulation. In this Analysis article, we present a comprehensive census of class 2 types and class 2 subtypes in complete and draft bacterial and archaeal genomes, outline evolutionary scenarios for the independent origin of different class 2 CRISPR-Cas systems from mobile genetic elements, and propose an amended classification and nomenclature of CRISPR-Cas. Keywords: Bacterial evolution; Bacterial genetics; CRISPR-Cas systems, US National Institute of Mental Health (Grant 5DP1-MH100706), US National Institute of Mental Health (Grant 1R01-MH110049)

Bacteria and archaea typically possess small genomes that are tightly packed with protein-coding genes. The compactness of prokaryotic genomes is commonly perceived as evidence of adaptive genome streamlining caused by strong purifying selection in large microbial populations. In such populations, even the small cost incurred by nonfunctional DNA because of extra energy and time expenditure is thought to be sufficient for this extra genetic material to be eliminated by selection. However, contrary to the predictions of this model, there exists a consistent, positive correlation between the strength of selection at the protein sequence level, measured as the ratio of nonsynonymous to synonymous substitution rates, and microbial genome size. Here, by fitting the genome size distributions in multiple groups of prokaryotes to predictions of mathematical models of population evolution, we show that only models in which acquisition of additional genes is, on average, slightly beneficial yield a good fit to genomic data. These results suggest that the number of genes in prokaryotic genomes reflects the equilibrium between the benefit of additional genes that diminishes as the genome grows and deletion bias (i.e., the rate of deletion of genetic material being slightly greater than the rate of acquisition). Thus, new genes acquired by microbial genomes, on average, appear to be adaptive. The tight spacing of protein-coding genes likely results from a combination of the deletion bias and purifying selection that efficiently eliminates nonfunctional, noncoding sequences.

Almost all cellular life forms are hosts to diverse genetic parasites with various levels of autonomy including plasmids, transposons and viruses. Theoretical modeling of the evolution of primordial replicators indicates that parasites (cheaters) necessarily evolve in such systems and can be kept at bay primarily via compartmentalization. Given the (near) ubiquity, abundance and diversity of genetic parasites, the question becomes pertinent: are such parasites intrinsic to life? At least in prokaryotes, the persistence of parasites is linked to the rate of horizontal gene transfer (HGT). We mathematically derive the threshold value of the minimal transfer rate required for selfish element persistence, depending on the element duplication and loss rates as well as the cost to the host. Estimation of the characteristic gene duplication, loss and transfer rates for transposons, plasmids and virus-related elements in multiple groups of diverse bacteria and archaea indicates that most of these rates are compatible with the long term persistence of parasites. Notably, a small but non-zero rate of HGT is also required for the persistence of non-parasitic genes. We hypothesize that cells cannot tune their horizontal transfer rates to be below the threshold required for parasite persistence without experiencing highly detrimental side-effects. As a lower boundary to the minimum DNA transfer rate that a cell can withstand, we consider the process of genome degradation and mutational meltdown of populations through Muller’s ratchet. A numerical assessment of this hypothesis suggests that microbial populations cannot purge parasites while escaping Muller’s ratchet. Thus, genetic parasites appear to be virtually inevitable in cellular organisms.

The Antarctic Conoidean fauna is critically reviewed based on published data and specimens in the collections of the USNM, IORAS and MNHN. Forty-two species and subspecies of the superfamily Conoidea are recorded as occurring within the Antarctic Convergence (excluding the fauna of the Kerguelen Islands) and are attributed to 14 genera and seven families. These include the new taxa: Antarctospira n. gen. (type species—Leucosyrinx badenpowelli Dell, 1990); Drilliola antarctica n. sp.; Pleurotomella (Pleutoromella) tippetti n. sp.; Pleurotomella (Anomalotomella) petiti n. sp.; Xanthodaphne pastorinoi n. sp. Aforia watsoni is introduced as a new name for Pleurotoma (Surcula) lepta Watson, 1881, non Pleurotoma lepta Edwards, 1861. A lectotype is designated for Conorbella antarctica (Strebel, 1908). New combinations are also proposed. Antarctospira badenpowelli (Dell, 1990), n. comb. (previously assigned to Leucosyrinx); Antarctospira principalis (Thiele, 1912), n. comb. (previously assigned to Typhlom...

International audience; To explore the diversity of mobile genetic elements (MGE) associated with archaea of the phylum Thaumarchaeota, we exploited the property of most MGE to integrate into the genomes of their hosts. Integrated MGE (iMGE) were identified in 20 thaumarchaeal genomes amounting to 2 Mbp of mobile thaumarchaeal DNA. These iMGE group into five major classes: (i) proviruses, (ii) casposons, (iii) insertion sequence‐like transposons, (iv) integrative‐conjugative elements and (v) cryptic integrated elements. The majority of the iMGE belong to the latter category and might represent novel families of viruses or plasmids. The identified proviruses are related to tailed viruses of the order Caudovirales and to tailless icosahedral viruses with the double jelly‐roll capsid proteins. The thaumarchaeal iMGE are all connected within a gene sharing network, highlighting pervasive gene exchange between MGE occupying the same ecological niche. The thaumarchaeal mobilome carries multiple auxiliary metabolic genes, including multicopper oxidases and ammonia monooxygenase subunit C (AmoC), and stress response genes, such as those for universal stress response proteins (UspA). Thus, iMGE might make important contributions to the fitness and adaptation of their hosts. We identified several iMGE carrying type I‐B CRISPR‐Cas systems and spacers matching other thaumarchaeal iMGE, suggesting antagonistic interactions between coexisting MGE and symbiotic relationships with the ir archaeal hosts.

We appreciate the interest of Aouad and colleagues1 in our work on the archaeal class Methanonatronarchaeia2,3 and their effort to clarify the phylogenetic position of this unique group of extremely halophilic, methyl-reducing methanogens. In our analysis, Methanonatronarchaeia formed a clade with the class Halobacteria — the non-methanogenic euryarchaeal extreme halophiles. Notably, this phylogenetic placement is 100% bootstrap-supported in maximum-likelihood (ML) phylogenetic trees for both 16S ribosomal (r)RNA and concatenated alignments of ribosomal proteins3. Given the congruence of the two trees, the strong support for the Methanonatronarchaeia–Halobacteria clade, the biological plausibility of this affinity and the fact that these trees conformed with the currently favoured solutions for difficult problems in archaeal phylogeny (such as the monophyly of the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaeota (DPANN) superphylum and the euryarchaeal assemblage including Class I methanogens and Thermococci), we did not perform a more thorough phylogenetic analysis. Such an in-depth analysis was undertaken by Aouad and colleagues1. Their results suggest a different position for Methanonatronarchaeia — much deeper in the archaeal tree, sitting outside the branch that consists of Methanomicrobia (formerly, Class II Methanogens), including Halobacteria (denoted ‘Stenosarchaea’ by Aouad et al.) and the class Archaeoglobi, and at the base of the group (which Aouad et al. denote the ‘superclass Methanotecta’)1. This difference between the results of the two phylogenetic analyses stems primarily from the increasingly stringent removal of fast-evolving sites from the alignment prior to the phylogenetic tree construction that was applied by Aouad and colleagues. After a certain fraction of the fastest sites was removed, the tree topology abruptly transitioned to the deep placement of Methanonatronarchaeia. This procedure is supposed to eliminate the false signal produced by sites with multiple substitutions, and therefore Aouad et al. conclude that the affinity of Methanonatronarchaeia with Halobacteria was an artefact caused by such sites. Aouad et al. also obtained the ‘deeper’ placement of Methanonatronarchaeia with extended sets of conserved protein families and expanded taxon sampling in these cases, even without removing the fast-evolving sites.

Prokaryotic toxin–antitoxin (TA) modules are highly abundant and are involved in stress response and drug tolerance. The most common type II TA modules consist of two interacting proteins. The type II toxins are diverse enzymes targeting various essential intracellular targets. The antitoxin binds to cognate toxin and inhibits its function. Recently, TA modules whose toxins are GNAT-family acetyltransferases were described. For two such systems, the target of acetylation was shown to be aminoacyl-tRNA: the TacT toxin targets aminoacylated elongator tRNAs, while AtaT targets the amino acid moiety of initiating tRNAMet. We show that the itaRT gene pair from Escherichia coli encodes a TA module with acetyltransferase toxin ItaT that specifically and exclusively acetylates Ile-tRNAIle thereby blocking translation and inhibiting cell growth. ItaT forms a tight complex with the ItaR antitoxin, which represses the transcription of itaRT operon. A comprehensive bioinformatics survey of GNAT acetyltransferases reveals that enzymes encoded by validated or putative TA modules are common and form a distinct branch of the GNAT family tree. We speculate that further functional analysis of such TA modules will result in identification of enzymes capable of specifically targeting many, perhaps all, aminoacyl tRNAs.

Significance How mutation and selection co-determine the course of cancer evolution remains an open, fundamental question. We construct a mutation-selection phase diagram, using tumor mutation load (ML) and selection strength (dN/dS) as key variables, and assess their association with clinical outcome. The results reveal a biphasic evolutionary regime whereby beyond a critical ML, tumor fitness decreases with the number of mutations, although the proteome evolves near neutrality—that is, without strong selection. Deviations from neutrality at extreme ML show how positive selection (at low ML) and purifying selection (at high ML) may act to maintain tumor fitness. These results corroborate the existence of a critical state in cancer evolution predicted by theory and have fundamental and likely clinical implications., How mutation and selection determine the fitness landscape of tumors and hence clinical outcome is an open fundamental question in cancer biology, crucial for the assessment of therapeutic strategies and resistance to treatment. Here we explore the mutation-selection phase diagram of 6,721 tumors representing 23 cancer types by quantifying the overall somatic point mutation load (ML) and selection (dN/dS) in the entire proteome of each tumor. We show that ML strongly correlates with patient survival, revealing two opposing regimes around a critical point. In low-ML cancers, a high number of mutations indicates poor prognosis, whereas high-ML cancers show the opposite trend, presumably due to mutational meltdown. Although the majority of cancers evolve near neutrality, deviations are observed at extreme MLs. Melanoma, with the highest ML, evolves under purifying selection, whereas in low-ML cancers, signatures of positive selection are observed, demonstrating how selection affects tumor fitness. Moreover, different cancers occupy specific positions on the ML–dN/dS plane, revealing a diversity of evolutionary trajectories. These results support and expand the theory of tumor evolution and its nonlinear effects on survival.

We have recently shown in 2 large-scale surveys (1, 2) that human leukocyte antigen (HLA) haplotypes frequency distributions are better characterized by purifying selection than by balancing selection. In their Letter, Hedrick and Klitz claim that the HLA locus is a classical example of balancing selection and that our recent results contradict this traditional view (3). They criticize 3 main aspects of our analyses: [↵][1]1To whom correspondence may be addressed. Email: koonin{at}ncbi.nlm.nih.gov or louzouy{at}math.biu.ac.il. [1]: #xref-corresp-1-1

Tonnoideans are marine carnivorous caenogastropods that prey on different invertebrates, namely polychaetes, sipunculids, bivalve and gastropod molluscs, and echinoderms. The morphology of the digestive system of 20 species from five families of the Tonnoidea was examined (for most of these species for the first time), and the salivary glands of six of them were studied using serial histological sections. Most of the studied families are rather similar anatomically, except Personidae (Distorsio), which differs both in proboscis morphology and the structure of the salivary glands. In most tonnoideans the salivary glands are split morphologically and functionally into anterior and posterior lobes, the latter synthesising strong sulfuric acid. The ducts of the posterior lobes are lined with non-ciliated epithelium and receive usually paired ciliated ducts from the anterior lobes to form a non-ciliated common duct, opening into the buccal cavity. In Personidae, the salivary glands are not separated in...

Evolution and maintenance of genetic recombination and its relation to the mutational process is a long-standing, fundamental problem in evolutionary biology that is linked to the general problem of evolution of evolvability. We explored a stochastic model of the evolution of recombination using additive fitness and infinite allele assumptions but no assumptions on the sign or magnitude of the epistasis and the distribution of mutation effects. In this model, fluctuating negative epistasis and predominantly deleterious mutations arise naturally as a consequence of the additive fitness and a reservoir from which new alleles arrive with a fixed distribution of fitness effects. Analysis of the model revealed a nonmonotonic effect of recombination intensity on fitness, with an optimal recombination rate value which maximized fitness in steady state. The optimal recombination rate depended on the mutation rate and was evolvable, that is, subject to selection. The predictions of the model were compatible with the observations on the dependence between genome rearrangement rate and gene flux in microbial genomes.