Mirusviruses link herpesviruses to giant viruses.

DNA viruses have a major influence on the ecology and evolution of cellular organisms1-4, but their overall diversity and evolutionary trajectories remain elusive5. Here we carried out a phylogeny-guided genome-resolved metagenomic survey of the sunlit oceans and discovered plankton-infecting relatives of herpesviruses that form a putative new phylum dubbed Mirusviricota. The virion morphogenesis module of this large monophyletic clade is typical of viruses from the realm Duplodnaviria6, with multiple components strongly indicating a common ancestry with animal-infecting Herpesvirales. Yet, a substantial fraction of mirusvirus genes, including hallmark transcription machinery genes missing in herpesviruses, are closely related homologues of giant eukaryotic DNA viruses from another viral realm, Varidnaviria. These remarkable chimaeric attributes connecting Mirusviricota to herpesviruses and giant eukaryotic viruses are supported by more than 100 environmental mirusvirus genomes, including a near-complete contiguous genome of 432 kilobases. Moreover, mirusviruses are among the most abundant and active eukaryotic viruses characterized in the sunlit oceans, encoding a diverse array of functions used during the infection of microbial eukaryotes from pole to pole. The prevalence, functional activity, diversification and atypical chimaeric attributes of mirusviruses point to a lasting role of Mirusviricota in the ecology of marine ecosystems and in the evolution of eukaryotic DNA viruses.

Dry mass photometry of single bacteria using quantitative wavefront microscopy.

Quantitative phase microscopy (QPM) represents a noninvasive alternative to fluorescence microscopy for cell observation with high contrast and for the quantitative measurement of dry mass (DM) and growth rate at the single-cell level. While DM measurements using QPM have been widely conducted on mammalian cells, bacteria have been less investigated, presumably due to the high resolution and high sensitivity required by their smaller size. This article demonstrates the use of cross-grating wavefront microscopy, a high-resolution and high-sensitivity QPM, for accurate DM measurement and monitoring of single microorganisms (bacteria and archaea). The article covers strategies for overcoming light diffraction and sample focusing, and introduces the concepts of normalized optical volume and optical polarizability (OP) to gain additional information beyond DM. The algorithms for DM, optical volume, and OP measurements are illustrated through two case studies: monitoring DM evolution in a microscale colony-forming unit as a function of temperature, and using OP as a potential species-specific signature.

Expanded Dataset Reveals the Emergence and Evolution of DNA Gyrase in Archaea.

DNA gyrase is a type II topoisomerase with the unique capacity to introduce negative supercoiling in DNA. In bacteria, DNA gyrase has an essential role in the homeostatic regulation of supercoiling. While ubiquitous in bacteria, DNA gyrase was previously reported to have a patchy distribution in Archaea but its emergent function and evolutionary history in this domain of life remains elusive. In this study, we used phylogenomic approaches and an up-to date sequence dataset to establish global and archaea-specific phylogenies of DNA gyrases. The most parsimonious evolutionary scenario infers that DNA gyrase was introduced into the lineage leading to Euryarchaeal group II via a single horizontal gene transfer from a bacterial donor which we identified as an ancestor of Gracilicutes and/or Terrabacteria. The archaea-focused trees indicate that DNA gyrase spread from Euryarchaeal group II to some DPANN and Asgard lineages via rare horizontal gene transfers. The analysis of successful recent transfers suggests a requirement for syntropic or symbiotic/parasitic relationship between donor and recipient organisms. We further show that the ubiquitous archaeal Topoisomerase VI may have co-evolved with DNA gyrase to allow the division of labor in the management of topological constraints. Collectively, our study reveals the evolutionary history of DNA gyrase in Archaea and provides testable hypotheses to understand the prerequisites for successful establishment of DNA gyrase in a naive archaeon and the associated adaptations in the management of topological constraints.

Giant Viruses Encode Actin-Related Proteins.

The emergence of the eukaryotic cytoskeleton is a critical yet puzzling step of eukaryogenesis. Actin and actin-related proteins (ARPs) are ubiquitous components of this cytoskeleton. The gene repertoire of the Last Eukaryotic Common Ancestor (LECA) would have therefore harbored both actin and various ARPs. Here, we report the presence and expression of actin-related genes in viral genomes (viractins) of some Imitervirales, a viral order encompassing the giant Mimiviridae. Phylogenetic analyses suggest an early recruitment of an actin-related gene by viruses from ancient protoeukaryotic hosts before the emergence of modern eukaryotes, possibly followed by a back transfer that gave rise to eukaryotic actins. This supports a coevolutionary scenario between pre-LECA lineages and their viruses, which could have contributed to the emergence of the modern eukaryotic cytoskeleton.

The emergence, spread and vanishing of a French SARS-CoV-2 variant exemplifies the fate of RNA virus epidemics and obeys the Mistigri rule.

The nature and dynamics of mutations associated with the emergence, spread, and vanishing of SARS-CoV-2 variants causing successive waves are complex. We determined the kinetics of the most common French variant ("Marseille-4") for 10 months since its onset in July 2020. Here, we analyzed and classified into subvariants and lineages 7453 genomes obtained by next-generation sequencing. We identified two subvariants, Marseille-4A, which contains 22 different lineages of at least 50 genomes, and Marseille-4B. Their average lifetime was 4.1 ± 1.4 months, during which 4.1 ± 2.6 mutations accumulated. Growth rate was 0.079 ± 0.045, varying from 0.010 to 0.173. Most of the lineages exhibited a bell-shaped distribution. Several beneficial mutations at unpredicted sites initiated a new outbreak, while the accumulation of other mutations resulted in more viral heterogenicity, increased diversity and vanishing of the lineages. Marseille-4B emerged when the other Marseille-4 lineages vanished. Its ORF8 gene was knocked out by a stop codon, as reported in SARS-CoV-2 of mink and in the Alpha variant. This subvariant was associated with increased hospitalization and death rates, suggesting that ORF8 is a nonvirulence gene. We speculate that the observed heterogenicity of a lineage may predict the end of the outbreak.

The hyperthermophilic archaeon Thermococcus kodakarensis is resistant to pervasive negative supercoiling activity of DNA gyrase.

In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Archaea, but its emergent function remains a mystery. To better understand the challenges associated with the establishment of pervasive negative supercoiling activity, we expressed the gyrase of the bacterium Thermotoga maritima in a naïve archaeon Thermococcus kodakarensis which naturally has positively supercoiled DNA. We found that the gyrase was catalytically active in T. kodakarensis leading to strong negative supercoiling of plasmid DNA which was stably maintained over at least eighty generations. An increased sensitivity of gyrase-expressing T. kodakarensis to ciprofloxacin suggested that gyrase also modulated chromosomal topology. Accordingly, global transcriptome analyses revealed large scale gene expression deregulation and identified a subset of genes responding to the negative supercoiling activity of gyrase. Surprisingly, the artificially introduced dominant negative supercoiling activity did not have a measurable effect on T. kodakarensis growth rate. Our data suggest that gyrase can become established in Thermococcales archaea without critically interfering with DNA transaction processes.

Structural basis for peptide recognition by archaeal oligopeptide permease A.

Oligopeptide permease A (OppA) plays an important role in the nutrition of cells and various signaling processes. In archaea, OppA is a major protein present in membrane vesicles of Thermococcales. Because there being no crystal structures of archaeal OppAs determined to date, we report the crystal structure of archaeal OppA from Thermococcus kodakaraensis (TkOppA) at 2.3 Å resolution by the single-wavelength anomalous dispersion method. TkOppA consists of three domains similarly to bacterial OppAs, and the inserted regions not present in bacterial OppAs are at the periphery of the core region. An endogenous pentapeptide was bound in the pocket of domains I and III of TkOppA by hydrogen bonds of main-chain atoms of the peptide and hydrophobic interactions. No hydrogen bonds of side-chain atoms of the peptide were observed; thus, TkOppA may have low peptide selectivity but some preference for residues 2 and 3. TkOppA has a relatively large pocket and can bind a nonapeptide; therefore, it is suitable for the binding of large peptides similarly to OppAs of Gram-positive bacteria.

Diversity and evolution of B-family DNA polymerases.

B-family DNA polymerases (PolBs) represent the most common replicases. PolB enzymes that require RNA (or DNA) primed templates for DNA synthesis are found in all domains of life and many DNA viruses. Despite extensive research on PolBs, their origins and evolution remain enigmatic. Massive accumulation of new genomic and metagenomic data from diverse habitats as well as availability of new structural information prompted us to conduct a comprehensive analysis of the PolB sequences, structures, domain organizations, taxonomic distribution and co-occurrence in genomes. Based on phylogenetic analysis, we identified a new, widespread group of bacterial PolBs that are more closely related to the catalytically active N-terminal half of the eukaryotic PolEpsilon (PolEpsilonN) than to Escherichia coli Pol II. In Archaea, we characterized six new groups of PolBs. Two of them show close relationships with eukaryotic PolBs, the first one with PolEpsilonN, and the second one with PolAlpha, PolDelta and PolZeta. In addition, structure comparisons suggested common origin of the catalytically inactive C-terminal half of PolEpsilon (PolEpsilonC) and PolAlpha. Finally, in certain archaeal PolBs we discovered C-terminal Zn-binding domains closely related to those of PolAlpha and PolEpsilonC. Collectively, the obtained results allowed us to propose a scenario for the evolution of eukaryotic PolBs.

Diversification of giant and large eukaryotic dsDNA viruses predated the origin of modern eukaryotes.

Giant and large eukaryotic double-stranded DNA viruses from the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) assemblage represent a remarkably diverse and potentially ancient component of the eukaryotic virome. However, their origin(s), evolution, and potential roles in the emergence of modern eukaryotes remain subjects of intense debate. Here we present robust phylogenetic trees of NCLDVs, based on the 8 most conserved proteins responsible for virion morphogenesis and informational processes. Our results uncover the evolutionary relationships between different NCLDV families and support the existence of 2 superclades of NCLDVs, each encompassing several families. We present evidence strongly suggesting that the NCLDV core genes, which are involved in both informational processes and virion formation, were acquired vertically from a common ancestor. Among them, the largest subunits of the DNA-dependent RNA polymerase were transferred between 2 clades of NCLDVs and proto-eukaryotes, giving rise to 2 of the 3 eukaryotic DNA-dependent RNA polymerases. Our results strongly suggest that these transfers and the diversification of NCLDVs predated the emergence of modern eukaryotes, emphasizing the major role of viruses in the evolution of cellular domains.

Exploring short k-mer profiles in cells and mobile elements from Archaea highlights the major influence of both the ecological niche and evolutionary history.

BACKGROUND: K-mer-based methods have greatly advanced in recent years, largely driven by the realization of their biological significance and by the advent of next-generation sequencing. Their speed and their independence from the annotation process are major advantages. Their utility in the study of the mobilome has recently emerged and they seem a priori adapted to the patchy gene distribution and the lack of universal marker genes of viruses and plasmids. To provide a framework for the interpretation of results from k-mer based methods applied to archaea or their mobilome, we analyzed the 5-mer DNA profiles of close to 600 archaeal cells, viruses and plasmids. Archaea is one of the three domains of life. Archaea seem enriched in extremophiles and are associated with a high diversity of viral and plasmid families, many of which are specific to this domain. We explored the dataset structure by multivariate and statistical analyses, seeking to identify the underlying factors. RESULTS: For cells, the 5-mer profiles were inconsistent with the phylogeny of archaea. At a finer taxonomic level, the influence of the taxonomy and the environmental constraints on 5-mer profiles was very strong. These two factors were interdependent to a significant extent, and the respective weights of their contributions varied according to the clade. A convergent adaptation was observed for the class Halobacteria, for which a strong 5-mer signature was identified. For mobile elements, coevolution with the host had a clear influence on their 5-mer profile. This enabled us to identify one previously known and one new case of recent host transfer based on the atypical composition of the mobile elements involved. Beyond the effect of coevolution, extrachromosomal elements strikingly retain the specific imprint of their own viral or plasmid taxonomic family in their 5-mer profile. CONCLUSION: This specific imprint confirms that the evolution of extrachromosomal elements is driven by multiple parameters and is not restricted to host adaptation. In addition, we detected only recent host transfer events, suggesting the fast evolution of short k-mer profiles. This calls for caution when using k-mers for host prediction, metagenomic binning or phylogenetic reconstruction.

Pervasive Suicidal Integrases in Deep-Sea Archaea.

Mobile genetic elements (MGEs) often encode integrases which catalyze the site-specific insertion of their genetic information into the host genome and the reverse reaction of excision. Hyperthermophilic archaea harbor integrases belonging to the SSV-family which carry the MGE recombination site within their open reading frame. Upon integration into the host genome, SSV integrases disrupt their own gene into two inactive pseudogenes and are termed suicidal for this reason. The evolutionary maintenance of suicidal integrases, concurring with the high prevalence and multiples recruitments of these recombinases by archaeal MGEs, is highly paradoxical. To elucidate this phenomenon, we analyzed the wide phylogenomic distribution of a prominent class of suicidal integrases which revealed a highly variable integration site specificity. Our results highlighted the remarkable hybrid nature of these enzymes encoded from the assembly of inactive pseudogenes of different origins. The characterization of the biological properties of one of these integrases, IntpT26-2 showed that this enzyme was active over a wide range of temperatures up to 99 °C and displayed a less-stringent site specificity requirement than comparable integrases. These observations concurred in explaining the pervasiveness of these suicidal integrases in the most hyperthermophilic organisms. The biochemical and phylogenomic data presented here revealed a target site switching system operating on highly thermostable integrases and suggested a new model for split gene reconstitution. By generating fast-evolving pseudogenes at high frequency, suicidal integrases constitute a powerful model to approach the molecular mechanisms involved in the generation of active genes variants by the recombination of proto-genes.

The reverse gyrase TopR1 is responsible for the homeostatic control of DNA supercoiling in the hyperthermophilic archaeon Sulfolobus solfataricus.

Maintaining an appropriate DNA topology with DNA-based processes (DNA replication, transcription and recombination) is crucial for all three domains of life. In bacteria, the homeostatic regulation for controlling DNA supercoiling relies on antagonistic activities of two DNA topoisomerases, TopoI and gyrase. In hyperthermophilic crenarchaea, the presence of such a regulatory system is suggested as two DNA topoisomerases, TopoVI and reverse gyrase, catalyze antagonistic activities. To test this hypothesis, we estimated and compared the number of the TopoVI with that of the two reverse gyrases, TopR1 and TopR2, in Sulfolobus solfataricus cells maintained either at 80 or at 88°C, or reciprocally shifted from one temperature to the other. From the three DNA topoisomerases, TopR1 is the only one exhibiting significant quantitative variations in response to the up- and down-shifts. In addition, the corresponding intrinsic activities of these three DNA topoisomerases were tested in vitro at both temperatures. Although temperature modulates the three DNA topoisomerases activities, TopR1 is the sole topoisomerase able to function at high temperature. Altogether, results presented in this study demonstrate, for the first time, that the DNA topological state of a crenarchaeon is regulated via a homeostatic control, which is mainly mediated by the fine-tuning of TopR1.

The first head-tailed virus, MFTV1, infecting hyperthermophilic methanogenic deep-sea archaea.

Deep-sea hydrothermal vents are inhabited by complex communities of microbes and their viruses. Despite the importance of viruses in controlling the diversity, adaptation and evolution of their microbial hosts, to date, only eight bacterial and two archaeal viruses isolated from abyssal ecosystems have been described. Thus, our efforts focused on gaining new insights into viruses associated with deep-sea autotrophic archaea. Here, we provide the first evidence of an infection of hyperthermophilic methanogenic archaea by a head-tailed virus, Methanocaldococcus fervens tailed virus 1 (MFTV1). MFTV1 has an isometric head of 50 nm in diameter and a 150 nm-long non-contractile tail. Virions are released continuously without causing a sudden drop in host growth. MFTV1 infects Methanocaldococcus species and is the first hyperthermophilic head-tailed virus described thus far. The viral genome is a double-stranded linear DNA of 31 kb. Interestingly, our results suggest potential strategies adopted by the plasmid pMEFER01, carried by M. fervens, to spread horizontally in hyperthermophilic methanogens. The data presented here open a new window of understanding on how the abyssal mobilome interacts with hyperthermophilic marine archaea.

WASPS: web-assisted symbolic plasmid synteny server.

MOTIVATION: Comparative plasmid genome analyses require complex tools, the manipulation of large numbers of sequences and constitute a daunting task for the wet bench experimentalist. Dedicated plasmid databases are sparse, only comprise bacterial plasmids and provide exclusively access to sequence similarity searches. RESULTS: We have developed Web-Assisted Symbolic Plasmid Synteny (WASPS), a web service granting protein and DNA sequence similarity searches against a database comprising all completely sequenced natural plasmids from bacterial, archaeal and eukaryal origin. This database pre-calculates orthologous protein clustering and enables WASPS to generate fully resolved plasmid synteny maps in real time using internal and user-provided DNA sequences. AVAILABILITY AND IMPLEMENTATION: WASPS queries befit all current browsers such as Firefox, Edge or Safari while the best functionality is achieved with Chrome. Internet Explorer is not supported. WASPS is freely accessible at https://archaea.i2bc.paris-saclay.fr/wasps/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei.

Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1-Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3' end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR-Cas systems.

The Evolution of Reverse Gyrase Suggests a Nonhyperthermophilic Last Universal Common Ancestor.

Reverse gyrase (RG) is the only protein found ubiquitously in hyperthermophilic organisms, but absent from mesophiles. As such, its simple presence or absence allows us to deduce information about the optimal growth temperature of long-extinct organisms, even as far as the last universal common ancestor of extant life (LUCA). The growth environment and gene content of the LUCA has long been a source of debate in which RG often features. In an attempt to settle this debate, we carried out an exhaustive search for RG proteins, generating the largest RG data set to date. Comprising 376 sequences, our data set allows for phylogenetic reconstructions of RG with unprecedented size and detail. These RG phylogenies are strikingly different from those of universal proteins inferred to be present in the LUCA, even when using the same set of species. Unlike such proteins, RG does not form monophyletic archaeal and bacterial clades, suggesting RG emergence after the formation of these domains, and/or significant horizontal gene transfer. Additionally, the branch lengths separating archaeal and bacterial groups are very short, inconsistent with the tempo of evolution from the time of the LUCA. Despite this, phylogenies limited to archaeal RG resolve most archaeal phyla, suggesting predominantly vertical evolution since the time of the last archaeal ancestor. In contrast, bacterial RG indicates emergence after the last bacterial ancestor followed by significant horizontal transfer. Taken together, these results suggest a nonhyperthermophilic LUCA and bacterial ancestor, with hyperthermophily emerging early in the evolution of the archaeal and bacterial domains.

Vesiduction: the fourth way of HGT.

Besides the canonical gene transfer mechanisms transformation, transduction and conjugation, DNA transfer involving extracellular vesicles is still under appreciated. However, this widespread phenomenon has been observed in the three domains of life. Here, we propose the term 'Vesiduction' as a fourth mode of intercellular DNA transfer.

Structure-function analysis of Sua5 protein reveals novel functional motifs required for the biosynthesis of the universal t6A tRNA modification.

N6-threonyl-carbamoyl adenosine (t6A) is a universal tRNA modification found at position 37, next to the anticodon, in almost all tRNAs decoding ANN codons (where N = A, U, G, or C). t6A stabilizes the codon-anticodon interaction and hence promotes translation fidelity. The first step of the biosynthesis of t6A, the production of threonyl-carbamoyl adenylate (TC-AMP), is catalyzed by the Sua5/TsaC family of enzymes. While TsaC is a single domain protein, Sua5 enzymes are composed of the TsaC-like domain, a linker and an extra domain called SUA5 of unknown function. In the present study, we report structure-function analysis of Pyrococcus abyssi Sua5 (Pa-Sua5). Crystallographic data revealed binding sites for bicarbonate substrate and pyrophosphate product. The linker of Pa-Sua5 forms a loop structure that folds into the active site gorge and closes it. Using structure-guided mutational analysis, we established that the conserved sequence motifs in the linker and the domain-domain interface are essential for the function of Pa-Sua5. We propose that the linker participates actively in the biosynthesis of TC-AMP by binding to ATP/PPi and by stabilizing the N-carboxy-l-threonine intermediate. Hence, TsaC orthologs which lack such a linker and SUA5 domain use a different mechanism for TC-AMP synthesis.

Lokiarchaea are close relatives of Euryarchaeota, not bridging the gap between prokaryotes and eukaryotes.

The eocyte hypothesis, in which Eukarya emerged from within Archaea, has been boosted by the description of a new candidate archaeal phylum, "Lokiarchaeota", from metagenomic data. Eukarya branch within Lokiarchaeota in a tree reconstructed from the concatenation of 36 universal proteins. However, individual phylogenies revealed that lokiarchaeal proteins sequences have different evolutionary histories. The individual markers phylogenies revealed at least two subsets of proteins, either supporting the Woese or the Eocyte tree of life. Strikingly, removal of a single protein, the elongation factor EF2, is sufficient to break the Eukaryotes-Lokiarchaea affiliation. Our analysis suggests that the three lokiarchaeal EF2 proteins have a chimeric organization that could be due to contamination and/or homologous recombination with patches of eukaryotic sequences. A robust phylogenetic analysis of RNA polymerases with a new dataset indicates that Lokiarchaeota and related phyla of the Asgard superphylum are sister group to Euryarchaeota, not to Eukarya, and supports the monophyly of Archaea with their rooting in the branch leading to Thaumarchaeota.