From Mimivirus to Mirusvirus: The Quest for Hidden Giants.

Our perception of viruses has been drastically evolving since the inception of the field of virology over a century ago. In particular, the discovery of giant viruses from the Nucleocytoviricota phylum marked a pivotal moment. Their previously concealed diversity and abundance unearthed an unprecedented complexity in the virus world, a complexity that called for new definitions and concepts. These giant viruses underscore the intricate interactions that unfold over time between viruses and their hosts, and are themselves suspected to have played a significant role as a driving force in the evolution of eukaryotes since the dawn of this cellular domain. Whether they possess exceptional relationships with their hosts or whether they unveil the actual depths of evolutionary connections between viruses and cells otherwise hidden in smaller viruses, the attraction giant viruses exert on the scientific community and beyond continues to grow. Yet, they still hold surprises. Indeed, the recent identification of mirusviruses connects giant viruses to herpesviruses, each belonging to distinct viral realms. This discovery substantially broadens the evolutionary landscape of Nucleocytoviricota. Undoubtedly, the years to come will reveal their share of surprises.

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.

A self-transmissible plasmid from a hyperthermophile that facilitates genetic modification of diverse Archaea.

Conjugative plasmids are self-transmissible mobile genetic elements that transfer DNA between host cells via type IV secretion systems (T4SS). While T4SS-mediated conjugation has been well-studied in bacteria, information is sparse in Archaea and known representatives exist only in the Sulfolobales order of Crenarchaeota. Here we present the first self-transmissible plasmid identified in a Euryarchaeon, Thermococcus sp. 33-3. The 103 kbp plasmid, pT33-3, is seen in CRISPR spacers throughout the Thermococcales order. We demonstrate that pT33-3 is a bona fide conjugative plasmid that requires cell-to-cell contact and is dependent on canonical, plasmid-encoded T4SS-like genes. Under laboratory conditions, pT33-3 transfers to various Thermococcales and transconjugants propagate at 100 °C. Using pT33-3, we developed a genetic toolkit that allows modification of phylogenetically diverse Archaeal genomes. We demonstrate pT33-3-mediated plasmid mobilization and subsequent targeted genome modification in previously untransformable Thermococcales species, and extend this process to interphylum transfer to a Crenarchaeon.

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.

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.

Viral origin of eukaryotic type IIA DNA topoisomerases.

Type II DNA topoisomerases of the family A (Topo IIAs) are present in all Bacteria (DNA gyrase) and eukaryotes. In eukaryotes, they play a major role in transcription, DNA replication, chromosome segregation, and modulation of chromosome architecture. The origin of eukaryotic Topo IIA remains mysterious since they are very divergent from their bacterial homologs and have no orthologs in Archaea. Interestingly, eukaryotic Topo IIAs have close homologs in viruses of the phylum Nucleocytoviricota, an expansive assemblage of large and giant viruses formerly known as the nucleocytoplasmic large DNA viruses. Topo IIAs are also encoded by some bacterioviruses of the class Caudoviricetes (tailed bacteriophages). To elucidate the origin of the eukaryotic Topo IIA, we performed in-depth phylogenetic analyses on a dataset combining viral and cellular Topo IIA homologs. Topo IIAs encoded by Bacteria and eukaryotes form two monophyletic groups nested within Topo IIA encoded by Caudoviricetes and Nucleocytoviricota, respectively. Importantly, Nucleocytoviricota remained well separated from eukaryotes after removing both Bacteria and Caudoviricetes from the data set, indicating that the separation of Nucleocytoviricota and eukaryotes is probably not due to long-branch attraction artifact. The topologies of our trees suggest that the eukaryotic Topo IIA was probably acquired from an ancestral member of the Nucleocytoviricota of the class Megaviricetes, before the emergence of the last eukaryotic common ancestor (LECA). This result further highlights a key role of these viruses in eukaryogenesis and suggests that early proto-eukaryotes used a Topo IIB instead of a Topo IIA for solving their DNA topological problems.

Life at high temperature observed in vitro upon laser heating of gold nanoparticles.

Thermophiles are microorganisms that thrive at high temperature. Studying them can provide valuable information on how life has adapted to extreme conditions. However, high temperature conditions are difficult to achieve on conventional optical microscopes. Some home-made solutions have been proposed, all based on local resistive electric heating, but no simple commercial solution exists. In this article, we introduce the concept of microscale laser heating over the field of view of a microscope to achieve high temperature for the study of thermophiles, while maintaining the user environment in soft conditions. Microscale heating with moderate laser intensities is achieved using a substrate covered with gold nanoparticles, as biocompatible, efficient light absorbers. The influences of possible microscale fluid convection, cell confinement and centrifugal thermophoretic motion are discussed. The method is demonstrated with two species: (i) Geobacillus stearothermophilus, a motile thermophilic bacterium thriving around 65 °C, which we observed to germinate, grow and swim upon microscale heating and (ii) Sulfolobus shibatae, a hyperthermophilic archaeon living at the optimal temperature of 80 °C. This work opens the path toward simple and safe observation of thermophilic microorganisms using current and accessible microscopy tools.

Archaea: A Goldmine for Molecular Biologists and Evolutionists.

The rebuttal of the prokaryote-eukaryote dichotomy and the elaboration of the three domains concept by Carl Woese and colleagues has been a breakthrough in biology. With the methodologies available at this time, they have shown that a single molecule, the 16S ribosomal RNA, could reveal the global organization of the living world. Later on, mining archaeal genomes led to major discoveries in archaeal molecular biology, providing a third model for comparative molecular biology. These analyses revealed the strong eukaryal flavor of the basic molecular fabric of Archaea and support rooting the universal tree between Bacteria and Arcarya (the clade grouping Archaea and Eukarya). However, in contradiction with this conclusion, it remains to understand why the archaeal and bacterial mobilomes are so similar and so different from the eukaryal one. These last years, the number of recognized archaea lineages (phyla?) has exploded. The archaeal nomenclature is now in turmoil and debates about the nature of the last universal common ancestor, the last archaeal common ancestor, and the topology of the tree of life are still going on. Interestingly, the expansion of the archaeal eukaryome, especially in the Asgard archaea, has provided new opportunities to study eukaryogenesis. In recent years, the application to Archaea of the new methodologies described in the various chapters of this book have opened exciting avenues to study the molecular biology and the physiology of these fascinating microorganisms.

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.

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.

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.

Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states.

Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.

[Viruses and the evolution of modern eukaryotic cells]. / Les virus et l'émergence des cellules eucaryotes modernes.

It is now well accepted that viruses have played an important role in the evolution of modern eukaryotes. In this review, we suggest that interactions between ancient eukaryoviruses and proto-eukaryotes also played a major role in eukaryogenesis. We discuss phylogenetic analyses that highlight the viral origin of several key proteins in the molecular biology of eukaryotes. We also discuss recent observations that, by analogy, could suggest a viral origin of the cellular nucleus. Finally, we hypothesize that mechanisms of cell differentiation in multicellular organisms might have originated from mechanisms implemented by viruses to transform infected cells into virocells.

Title: Les virus et l'émergence des cellules eucaryotes modernes. Abstract: Il est maintenant bien établi que les virus ont joué un rôle important dans l'évolution des eucaryotes modernes. Dans cette revue, nous commentons le rôle qu'ils ont pu jouer dans l'eucaryogenèse. Nous discutons les analyses phylogénétiques qui mettent en évidence l'origine virale de plusieurs protéines clés de la biologie moléculaire des eucaryotes et des observations récentes qui, par analogie, pourraient suggérer une origine virale du noyau cellulaire. Nous mettons en parallèle la complexité des eucaryotes avec l'unicité de leur virosphère et avançons l'hypothèse selon laquelle des mécanismes de la différenciation cellulaire auraient leur source dans ceux mis en œuvre par les virus pour transformer les cellules infectées en cellules virales.

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.

Phylogeny of the Varidnaviria Morphogenesis Module: Congruence and Incongruence With the Tree of Life and Viral Taxonomy.

Double-stranded DNA viruses of the realm Varidnaviria (formerly PRD1-adenovirus lineage) are characterized by homologous major capsid proteins (MCPs) containing one (kingdom: Helvetiavirae) or two ß-barrel domains (kingdom: Bamfordvirae) known as the jelly roll folds. Most of them also share homologous packaging ATPases (pATPases). Remarkably, Varidnaviria infect hosts from the three domains of life, suggesting that these viruses could be very ancient and share a common ancestor. Here, we analyzed the evolutionary history of Varidnaviria based on single and concatenated phylogenies of their MCPs and pATPases. We excluded Adenoviridae from our analysis as their MCPs and pATPases are too divergent. Sphaerolipoviridae, the only family in the kingdom Helvetiavirae, exhibit a complex history: their MCPs are very divergent from those of other Varidnaviria, as expected, but their pATPases groups them with Bamfordvirae. In single and concatenated trees, Bamfordvirae infecting archaea were grouped with those infecting bacteria, in contradiction with the cellular tree of life, whereas those infecting eukaryotes were organized into three monophyletic groups: the Nucleocytoviricota phylum, formerly known as the Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs), Lavidaviridae (virophages) and Polintoviruses. Although our analysis mostly supports the recent classification proposed by the International Committee on Taxonomy of Viruses (ICTV), it also raises questions, such as the validity of the Adenoviridae and Helvetiavirae ranking. Based on our phylogeny, we discuss current hypotheses on the origin and evolution of Varidnaviria and suggest new ones to reconcile the viral and cellular trees.

Archaeal extracellular vesicles are produced in an ESCRT-dependent manner and promote gene transfer and nutrient cycling in extreme environments.

Membrane-bound extracellular vesicles (EVs), secreted by cells from all three domains of life, transport various molecules and act as agents of intercellular communication in diverse environments. Here we demonstrate that EVs produced by a hyperthermophilic and acidophilic archaeon Sulfolobus islandicus carry not only a diverse proteome, enriched in membrane proteins, but also chromosomal and plasmid DNA, and can transfer this DNA to recipient cells. Furthermore, we show that EVs can support the heterotrophic growth of Sulfolobus in minimal medium, implicating EVs in carbon and nitrogen fluxes in extreme environments. Finally, our results indicate that, similar to eukaryotes, production of EVs in S. islandicus depends on the archaeal ESCRT machinery. We find that all components of the ESCRT apparatus are encapsidated into EVs. Using synchronized S. islandicus cultures, we show that EV production is linked to cell division and appears to be triggered by increased expression of ESCRT proteins during this cell cycle phase. Using a CRISPR-based knockdown system, we show that archaeal ESCRT-III and AAA+ ATPase Vps4 are required for EV production, whereas archaea-specific component CdvA appears to be dispensable. In particular, the active EV production appears to coincide with the expression patterns of ESCRT-III-1 and ESCRT-III-2, rather than ESCRT-III, suggesting a prime role of these proteins in EV budding. Collectively, our results suggest that ESCRT-mediated EV biogenesis has deep evolutionary roots, likely predating the divergence of eukaryotes and archaea, and that EVs play an important role in horizontal gene transfer and nutrient cycling in extreme environments.

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.

Eukaryotic virus composition can predict the efficiency of carbon export in the global ocean.

The biological carbon pump, in which carbon fixed by photosynthesis is exported to the deep ocean through sinking, is a major process in Earth's carbon cycle. The proportion of primary production that is exported is termed the carbon export efficiency (CEE). Based on in-lab or regional scale observations, viruses were previously suggested to affect the CEE (i.e., viral "shunt" and "shuttle"). In this study, we tested associations between viral community composition and CEE measured at a global scale. A regression model based on relative abundance of viral marker genes explained 67% of the variation in CEE. Viruses with high importance in the model were predicted to infect ecologically important hosts. These results are consistent with the view that the viral shunt and shuttle functions at a large scale and further imply that viruses likely act in this process in a way dependent on their hosts and ecosystem dynamics.