Biology subjects

Krupovic, M.

Publications and source records attributed to Krupovic, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Jumping DNA polymerases in bacteriophages

Viruses with double-stranded (ds) DNA genomes in the realm Duplodnaviria share a conserved structural gene module but show a broad range of variation in their repertoires of DNA replication proteins. Some of the duplodnaviruses encode (nearly) complete replication systems whereas others lack (almost) all genes required for replication, relying on the host replication machinery. DNA polymerases (DNAPs) comprise the centerpiece of the DNA replication apparatus. The replicative DNAPs are classified into 4 unrelated or distantly related families (A-D), with the protein structures and sequences within each family being, generally, highly conserved. More than half of the duplodnaviruses encode a DNAP of family A, B or C. We showed previously that multiple pairs of closely related viruses in the order Crassvirales encode DNAPs of different families. Here we identify four additional groups of tailed phages in the class Caudoviricetes in which the DNAPs apparently were swapped on multiple occasions, with replacements occurring both between families A and B, or A and C, or between distinct subfamilies within the same family. The DNAP swapping always occurs "in situ", without changes in the organization of the surrounding genes. In several cases, the DNAP gene is the only region of substantial divergence between closely related phage genomes, whereas in others, the swap apparently involved neighboring genes encoding other proteins involved in phage replication. We hypothesize that DNAP swapping is driven by selection for avoidance of host antiphage mechanisms targeting the phage DNAP that remain to be identified, and/or by selection against replicon incompatibility. In addition, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication.

microbiology↗

bioRxiv2024

Egoviruses: distant relatives of poxviruses abundant in the gut microbiome of humans and animals worldwide

Large and giant double-stranded DNA viruses are diverse and highly prevalent in the environment where they substantially affect the ecology and evolution of eukaryotes1-4. Until now, they were only sporadically found in the digestive system of vertebrates5-7. Here, we present the discovery of a diversified group of DNA viruses with large linear genomes dubbed egoviruses that comprise a putative viral order most closely related to animal-infecting poxviruses. Egoviruses are found worldwide, almost exclusively in the digestive system of humans, livestock and wild animals. One egovirus is human-specific, evolutionarily constrained, and spread across continents, demonstrating a long-term association between egoviruses and the human population. Analysis of eukaryotic homologs of egovirus core genes suggests that these viruses primarily infect Trichostomatia (Ciliates), which play a central role in ruminant digestive processes8, and Parabasalia (Metamonada), which directly influence animal immunity and gut microbiome ecology9. Egoviruses also contributed to the genetic makeup of polintons, endogenous viruses that proliferated in genomes of Trichomonas vaginalis10 and its close relatives among the Parabasalia. Unlike poxviruses that form brick-shaped virions, egoviruses are predicted to form multilayered icosahedral capsids resembling those of asfuviruses and likely representing the ancestral virion structure. Egoviruses currently are the only known clade of eukaryotic DNA viruses abundant in the animal digestive system. Phylogenomic analysis suggests an evolutionary scenario whereby egovirus-like ancestors of poxviruses infected anaerobic unicellular eukaryotes inside the gut of ancestral animals, before transitioning to animal cell infection which was accompanied by the virion metamorphosis.

microbiology↗

bioRxiv2024

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We employed protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a supefamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains, and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution. SignificanceStructural modeling of protein domains using advanced artificial intelligence-based methods such as AlphaFold2 may lead to insights into evolutionary relationships among proteins that are unreachable by sequence analysis. We applied this approach to elucidate the evolutionary relationships of four major groups of eukaryotic viruses: polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We identified previously uncharacterized protein domains predicted to be essential for virus genome replication. Analysis of the presence/absence and inactivation of these domains suggests a complete scenario for the origin and evolution of this major part of the eukaryotic virosphere.

microbiology↗

bioRxiv2024

Culture- and genome-based characterization of a tripartite interaction between patescibacterial epibionts, methylotrophic proteobacteria, and a jumbo phage in freshwater ecosystems

Patescibacteria form a very diverse and widely distributed phylum of small bacteria inferred to have an episymbiotic lifestyle. However, the prevalence of this lifestyle within the phylum and their host specificity remain poorly known due to the scarcity of cultured representatives. Here, we describe a complex system consisting of a patescibacterium, its gammaproteobacterial hosts, and their respective phages based on enrichment cultures and metagenomic data from two shallow, geographically close, freshwater ecosystems. The patescibacterium, Strigamonas methylophilicida sp. nov., defines a new genus within the family Absconditicoccaceae. It grows as epibiont on cells of methanotrophic species of the gammaproteobacterial family Methylophilaceae. Strigamonas cells grow tightly attached to the host, sometimes forming stacks that connect two host cells. Despite a surprisingly large genome (1.9 Mb) compared to many other Patescibacteria, S. methylophilicida lacks many essential biosynthetic pathways, including the complete biosynthesis of phospholipids, amino acids, and nucleic acids, implying a dependence on the host to obtain these molecules. We also identified and assembled the complete genomes of one patescibacterial phage that might represent a new virus family within the class Caudoviricetes, and two Methylophilaceae phages predicted to have head-tailed and filamentous virions, respectively. The patesciphage uses a modified genetic code similar to that of its host and encodes four tRNA genes, including the suppressor tRNA gene for the UGA stop codon, which is reassigned to glycine in many Patescibacteria. Our results confirm a prevalent episymbiotic lifestyle in Absconditicoccaceae and further suggest a clade-specific adaptation of this patescibacterial family for gammaproteobacterial hosts. IMPORTANCEPatescibacteria are ultra-small bacteria with reduced genomes that rely on symbiotic interactions with other prokaryotes, yet their host specificity and associated viral parasites remain poorly characterized due to limited cultured representatives. Here, we describe Strigamonas methylophilicida, a new patescibacterial species of the family Absconditicoccaceae that grows as an epibiont on various methylotrophic Gammaproteobacteria. This expands the host range for this family beyond previously documented photosynthetic partners. Using enrichment cultures and metagenomics, we retrieved complete genomes of novel phages infecting S. methylophilicida and its hosts, including one phage that uses a modified genetic code matching that of the patescibacterium. These findings reveal a previously unrecognized patescibacteria- methylotroph-phage tripartite interaction in freshwater environments, highlight the adaptations of patescibacterial phages, and shed light on the complex ecology and evolution of host-parasite-phage dynamics in understudied bacterial lineages.

microbiology↗

bioRxiv2024

Mriyaviruses: Small Relatives of Giant Viruses

The phylum Nucleocytoviricota consists of large and giant viruses that range in genome size from about 100 kilobases (kb) to more than 2.5 megabases. Here, using metagenome mining followed by extensive phylogenomic analysis and protein structure comparison, we delineate a distinct group of viruses with double-stranded (ds) DNA genomes in the range of 35-45 kb that appear to be related to the Nucleocytoviricota. In phylogenetic trees of the conserved double jelly-roll major capsid proteins (MCP) and DNA packaging ATPases, these viruses do not show affinity to any particular branch of the Nucleocytoviricota and accordingly would comprise a class which we propose to name "Mriyaviricetes" (after Ukrainian Mriya, dream). Structural comparison of the MCP suggests that, among the extant virus lineages, mriyaviruses are the closest one to the ancestor of the Nucleocytoviricota. In the phylogenetic trees, mriyaviruses split into two well-separated branches, the family Yaraviridae and proposed new family "Gamadviridae". The previously characterized members of these families, Yaravirus and Pleurochrysis sp. endemic viruses, infect amoeba and haptophytes, respectively. The genomes of the rest of the mriyaviruses were assembled from metagenomes from diverse environments, suggesting that mriyaviruses infect various unicellular eukaryotes. Mriyaviruses lack DNA polymerase, which is encoded by all other members of the Nucleocytoviricota, and RNA polymerase subunits encoded by all cytoplasmic viruses among the Nucleocytoviricota, suggesting that they replicate in the host cell nuclei. All mriyaviruses encode a HUH superfamily endonuclease that is likely to be essential for the initiation of virus DNA replication via the rolling circle mechanism. ImportanceThe origin of giant viruses of eukaryotes that belong to the phylum Nucleocytoviricota is not thoroughly understood and remains a matter of major interest and debate. Here we combine metagenome database searches with extensive protein sequence and structure analysis to describe a distinct group of viruses with comparatively small genomes of 35-45 kilobases that appears to comprise a distinct class within the phylum Nucleocytoviricota that we provisionally named "Mriyaviricetes". Mriyaviruses appear to be the closest identified relatives of the ancestors of the Nucleocytoviricota. Analysis of proteins encoded in mriyavirus genomes suggest that they replicate their genome via the rolling circle mechanism that is unusual among viruses with double-stranded DNA genomes and so far not described for members of Nucleocytoviricota.

microbiology↗

bioRxiv2024

Diversity, Origin and Evolution of the ESCRT Systems

Endosomal Sorting Complexes Required for Transport (ESCRT) play key roles in protein sorting between membrane-bounded compartments of eukaryotic cells. Homologs of many ESCRT components are identifiable in various groups of archaea, especially in Asgardarchaeota, the archaeal phylum that is currently considered to include the closest relatives of eukaryotes, but not in bacteria. We performed a comprehensive search for ESCRT protein homologs in archaea and reconstructed ESCRT evolution using the phylogenetic tree of Vps4 ATPase (ESCRT IV) as a scaffold, using sensitive protein sequence analysis and comparison of structural models to identify previously unknown ESCRT proteins. Several distinct groups of ESCRT systems in archaea outside of Asgard were identified, including proteins structurally similar to ESCRT-I and ESCRT-II, and several other domains involved in protein sorting in eukaryotes, suggesting an early origin of these components. Additionally, distant homologs of CdvA proteins were identified in Thermoproteales which are likely components of the uncharacterized cell division system in these archaea. We propose an evolutionary scenario for the origin of eukaryotic and Asgard ESCRT complexes from ancestral building blocks, namely, the Vps4 ATPase, ESCRT-III components, wH (winged helix-turn-helix fold) and possibly also coiled-coil, and Vps28-like domains. The Last Archaeal Common Ancestor likely encompassed a complex ESCRT system that was involved in protein sorting. Subsequent evolution involved either simplification, as in the TACK superphylum, where ESCRT was co-opted for cell division, or complexification as in Asgardarchaeota. In Asgardarchaeota, the connection between ESCRT and the ubiquitin system that was previously considered a eukaryotic signature was already established. ImportanceAll eukaryotic cells possess complex intracellular membrane organization. ESCRT (Endosomal Sorting Complexes Required for Transport) plays a central role in membrane remodeling which is essential for cellular functionality in eukaryotes. Recently, it has been shown that Asgard archaea, the archaeal phylum that includes the closest known relatives of eukaryotes, encode homologs of many components of the ESCRT systems. We employed protein sequence and structure comparisons to reconstruct the evolution of ESCRT systems in archaea and identified several previously unknown homologs of ESCRT subunits, some of which can be predicted to participate in cell division. The results of this reconstruction indicate that the Last Archaeal Common ancestor already encoded a complex ESCRT system that was involved in protein sorting. In Asgard archaea, ESCRT systems evolved towards greater complexity, and in particular, the connection between ESCRT and the ubiquitin system that was previously considered a eukaryotic signature was established.

evolutionary biology↗

bioRxiv2023

Diversity of Bathyarchaeia viruses in metagenomes and virus-encoded CRISPR system components

Bathyarchaeia represent a class of archaea common and abundant in sedimentary ecosystems. The virome of Bathyarchaeia so far has not been characterized. Here we report 56 metagenome-assembled genomes of Bathyarchaeia viruses identified in metagenomes from different environments. Gene sharing network and phylogenomic analyses led to the proposal of four virus families, including viruses of the realms Duplodnaviria and Adnaviria, and archaea-specific spindle-shaped viruses. Genomic analyses uncovered diverse CRISPR elements in these viruses. Viruses of the proposed family Fuxiviridae harbor an atypical type IV-B CRISPR-Cas system and a Cas4 protein that might interfere with host immunity. Viruses of the family Chiyouviridae encode a Cas2-like endonuclease and two mini-CRISPR arrays, one with a repeat identical to that in the host CRISPR array, potentially allowing the virus to recruit the host CRISPR adaptation machinery to acquire spacers that could contribute to competition with other mobile genetic elements or to inhibition of host defenses. These findings present an outline of the Bathyarchaeia virome and offer a glimpse into their counter-defense mechanisms.

microbiology↗

bioRxiv2023

DNA polymerase diversity reveals multiple incursions of Polintons during nematode evolution

Polintons are dsDNA, virus-like self-synthesizing transposons widely found in eukaryotic genomes. Recent metagenomic discoveries of Polinton-like viruses are consistent with the hypothesis that Polintons invade eukaryotic host genomes through infectious viral particles. Nematode genomes contain multiple copies of Polintons and provide an opportunity to explore the natural distribution and evolution of Polintons during this process. We performed an extensive search of Polintons across nematode genomes, identifying multiple full-length Polinton copies in several species. We provide evidence of both ancient Polinton integrations and recent mobility in strains of the same nematode species. In addition to the major nematode Polinton family, we identified a group of Polintons that are overall closely related to the major family, but encode a distinct protein-primed B family DNA polymerase (pPolB) that is related to homologs from a different group of Polintons present outside of the Nematoda. Phylogenetic analyses on the pPolBs support the evolutionary scenarios in which these extrinsic pPolBs that seem to derive from Polinton families present in oomycetes and molluscs replaced the canonical pPolB in subsets of Polintons found in terrestrial and marine nematodes, respectively, suggesting inter-phylum horizontal gene transfers. The pPolBs of the terrestrial nematode and oomycete Polintons share a unique feature, an insertion of a HNH nuclease domain, whereas the pPolBs in the marine nematode Polintons share an insertion of a VSR nuclease domain with marine mollusc pPolBs. We hypothesize that horizontal gene transfer occurs among Polintons from widely different but cohabiting hosts.

evolutionary biology↗

bioRxiv2023

Two dramatically distinct archaeal type IV pili structures formed by the same pilin

Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from relatively small pilin proteins, can be many microns long and serve diverse functions, including adhesion, biofilm formation, motility, and intercellular communication. Using cryo-electron microscopy (cryo-EM), we determined atomic structures of two dramatically different T4P from Saccharolobus islandicus REY15A. Unexpectedly, both pili were assembled from the same pilin protein but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal T4P structures where all subunits are equivalent, whereas in the other filament, the tri-pilus, the same protein exists in three different conformations. The three conformations involve different orientations of the outer immunoglobulin (Ig)-like domains, mediated by a very flexible linker, and all three of these conformations are very different from the single conformation found in the mono-pilus. Remarkably, the outer domains rotate nearly 180{degrees} between the mono- and tri-pilus conformations, formally similar to what has been shown for outer domains in bacterial flagellar filaments, despite lack of homology between bacterial flagella and archaeal T4P. Interestingly, both forms of pili require the same ATPase and TadC-like membrane pore for assembly, indicating that the same secretion system can produce structurally very different filaments. However, the expression of the ATPase and TadC genes was significantly different under the conditions yielding mono- and tri-pili. While archaeal T4P are homologs of archaeal flagellar filaments, our results show that in contrast to the rigid supercoil that the flagellar filaments must adopt to serve as helical propellers, archaeal T4P are likely to have fewer constraints on their structure and enjoy more internal degrees of freedom.

microbiology↗

bioRxiv2023

Distinct groups of RNA viruses associated with thermoacidophilic bacteria

Recent massive metatranscriptome mining substantially expanded the diversity of the bacterial RNA virome, suggesting that additional groups of riboviruses infecting bacterial hosts remain to be discovered. We employed full length double-stranded (ds) RNA sequencing for identification of riboviruses associated with microbial consortia dominated by bacteria and archaea in acidic hot springs in Japan. Whole sequences of two groups of multisegmented riboviruses genomes were obtained. One group, which we denoted hot spring riboviruses (HsRV), consists of unusual viruses with distinct RNA-dependent RNA polymerases (RdRPs) that seem to be intermediates between typical ribovirus RdRPs and viral reverse transcriptases. We also identified viruses encoding HsRV-like RdRPs in moderate aquatic environments, including marine water, river sediments and salt marsh, indicating that this previously overlooked ribovirus group is not restricted to the extreme ecosystem. The HsRV-like viruses are candidates for a distinct phylum or even kingdom within the viral realm Riboviria. The second group, denoted hot spring partiti-like viruses (HsPV), is a distinct branch within the family Partitiviridae. All genome segments in both these groups of viruses display the organization typical of bacterial riboviruses, where multiple open reading frames encoding individual proteins are preceded by ribosome-binding sites. Together with the identification in bacteria-dominated habitats, this genome architecture indicates that riboviruses of these distinct groups infect thermoacidophilic bacterial hosts.

microbiology↗

bioRxiv2023

A 2-million-year-old microbial and viral communities from the Kap Kobenhavn Formation in North Greenland

Environmental DNA (eDNA) from the 2-million-year-old Kap Kobenhavn Formation of northern Greenland has revealed an ecosystem of plants and animals with no contemporary analogue1. Here, we reconstruct the microbial (bacterial, archaeal, and viral) communities that thrived at the site during this time. By leveraging a novel analytical framework that integrates taxonomic profiling, DNA damage estimates, and functional reconstructions, we identify and distinguish pioneer microbial communities from later permafrost microbial assemblages. We show that at the time of sediment deposition, the terrestrial input at the Kap Kobenhavn site originated from a palustrine wetland, suggesting warmer, non-permafrost conditions. During this period, the detection of methanogenic archaea and signals of their carbon metabolism is consistent with Kap Kobenhavn and similar northern ecosystems contributing moderate methane emissions. Intriguingly, we discover a remarkable nucleotide sequence similarity--exceeding 98%--between pioneer methanogens and present-day analogues in thawing permafrost. This aligns with the concept of "time-traveling" microbes2 surviving across geological time and waiting for conditions to turn favourable rather than evolving to adapt to changing conditions. Importantly, in contrast to the plant and animal communities of the Kap Kobenhavn, a striking similarity in microbial composition to that of a contemporary thawing Arctic suggests that microbial communities may serve as the first indication of broader climate-driven ecosystem disruptions.

microbiology↗

bioRxiv2023

Widespread PRC barrel proteins play critical roles in archaeal cell division

Cell division is fundamental to all cellular life. Most of the archaea employ one of two alternative division machineries, one centered around the prokaryotic tubulin homolog FtsZ and the other around the endosomal sorting complex required for transport (ESCRT). However, neither of these mechanisms has been thoroughly characterized in archaea. Here, we show that three of the four PRC (Photosynthetic Reaction Center) barrel domain proteins of Haloferax volcanii (renamed Cell division proteins B1/2/3 (CdpB1/2/3)), play important roles in division. CdpB1 interacts directly with the FtsZ membrane anchor SepF and is essential for division, whereas deletion of cdpB2 and cdpB3 causes a major and a minor division defect, respectively. Orthologs of CdpB proteins are also involved in cell division in other haloarchaea. Phylogenetic analysis shows that PRC barrel proteins are widely distributed among archaea, including the highly conserved CdvA protein of the crenarchaeal ESCRT-based division system. Thus, diverse PRC barrel proteins appear to be central to cell division in most if not all archaea. Further study of these proteins is expected to elucidate the division mechanisms in archaea and their evolution.

microbiology↗

bioRxiv2023

Cbp1-Cren7 chromatinization of CRISPR arrays favours transcription from leader- over cryptic promoters

CRISPR arrays form the physical memory of CRISPR adaptive immune systems by incorporating foreign DNA as spacers that are often AT-rich and derived from viruses. As promoter elements such as the TATA-box are AT-rich, CRISPR arrays are prone to harbouring cryptic promoters. Sulfolobales harbor extremely long CRISPR arrays spanning several kilobases, a feature that is accompanied by the CRISPR-specific transcription factor Cbp1. Aberrant Cbp1 expression modulates CRISPR array transcription, but the molecular mechanisms underlying this regulation are unknown. Here, we characterise the genome-wide Cbp1 binding at nucleotide resolution and characterise the binding motifs on distinct CRISPR arrays, as well as on unexpected non-canonical binding sites associated with transposasons. Cbp1 recruits Cren7 forming together chimeric chromatin-like structures at CRISPR arrays. We dissect Cbp1 function in vitro and in vivo and show that the third HTH domain is responsible for Cren7 recruitment, and that Cbp1-Cren7 chromatinization plays a dual role in the transcription of CRISPR arrays. It suppresses spurious transcription from cryptic promoters within CRISPR arrays but enhances CRISPR RNA transcription directed from their cognate promoters in their leader region. Our results show that Cbp1-Cren7 chromatinization drives the productive expression of long CRISPR arrays.

microbiology↗

bioRxiv2022

Viral DNA SIP: the isotopic composition of virions is correlated with that of the substrate used for host growth

DNA Stable Isotope Probing is emerging as a powerful tool to study host-virus interactions. Indeed, since all viruses depend on a host for virion production, a link between the isotopic compositions of hosts and the virions they produce is expected. However, stable isotope probing applied to viral DNA has never been evaluated on simple biological models. Here, this method was tested on the bacteriophage T4 and its host Escherichia coli. To validate that E. coli cells cultivated using a substrate enriched in 13C isotope were resulting on the production of 13C-labeled T4 DNA. T4 DNA buoyant density in CsCl gradient was overall higher than the values predicted by a previously established empirical model, highlighting the need to adapt this type of models when analysing modified viral DNA. Moreover, our results show a strong correlation between the proportion of 13C6-D-glucose in the substrate used for host growth and the buoyant density of T4 DNA, validating the use of DNA SIP in viral ecology, to identify viruses infecting hosts with a specific metabolism.

microbiology↗

bioRxiv2022

A vast world of viroid-like circular RNAs revealed by mining metatranscriptomes

Viroids and viroid-like agents are unique, minimal RNA replicators that typically encode no proteins and hijack cellular enzymes for their genome replication. As the extent and diversity of viroid-like agents are poorly understood, we developed a computational pipeline to identify viroid-like covalently closed circular (ccc) RNAs and applied it to 5,131 global metatranscriptomes and 1,344 plant transcriptomes. The search resulted in 11,420 viroid-like, ribozyme-containing cccRNAs spanning 4,409 species-level clusters, which is a five-fold increase compared to the previously known set of viroids and viroid-like RNA agents. Within this diverse collection, we identified numerous putative novel viroids, satellite RNAs, retrozymes, and ribozylike viruses. We also found previously unknown ribozyme combinations and unusual ribozymes within the cccRNAs. Self-cleaving ribozymes were identified in both RNA strands of ambiviruses and some mito-like viruses as well as in capsid-encoding satellite virus-like cccRNAs. The broad presence of viroid-like cccRNAs in diverse transcriptomes and ecosystems implies that their host range is not limited to plants, and matches between viroid-like cccRNAs and CRISPR spacers suggest that some of them might replicate in prokaryotes.

microbiology↗

bioRxiv2022

Varidnaviruses in the human gut: a major expansion of the order Vinavirales

BackgroundBacteriophages play key roles in the dynamics of the human microbiome. By far the most abundant components of the human gut virome are tailed bacteriophages of the realm Duplodnaviria, in particular, crAss-like phages. However, apart from duplodnaviruses, the gut virome has not been dissected in detail. ResultsHere we report a comprehensive census of a minor component of the gut virome, the tailless bacteriophages of the realm Varidnaviria. Tailless phages are primarily represented in the gut by prophages of the families Corticoviridae and Autolykiviridae that jointly comprise the order Vinavirales and are mostly integrated as prophages in genomes of Alphaproteobacteria and Verrucomicrobia. Phylogenetic analysis of the major capsid proteins (MCP) and packaging ATPases suggests that at least three new families within Vinavirales should be established to accommodate the diversity of prophages from the human gut virome. Previously, only the MCP and ATPase genes were reported to be conserved in all members of Vinavirales. Here we identify a core set of 12 proteins that are shared by most of these viruses including previously undetected lysis enzymes. We further demonstrate that replication system components are frequently replaced in the genomes of Vinavirales, suggestive of selective pressure for escape from yet unknown host defenses or avoidance of incompatibility with coinfecting related viruses. ConclusionsThe results of this analysis show that, in a sharp contrast to marine viromes, varidnaviruses are a minor component of the human gut virome. Moreover, they are primarily represented by proviruses, suggesting that there are few if any active varidnavirus infections in the gut at any given time. These findings complement the existing knowledge of the human gut virome by exploring a group of viruses that was virtually overlooked in previous work.

microbiology↗

bioRxiv2022

A novel RHH family transcription factor aCcr1 and its viral homologs dictate cell cycle progression in archaea

Cell cycle regulation is of paramount importance for all forms of life. Here we report that a conserved and essential cell cycle-specific transcription factor (designated as aCcr1) and its viral homologs control cell division in Sulfolobales. We show that the transcription level of accr1 reaches peak during active cell division (D-phase) subsequent to the expression of CdvA, an archaea-specific cell division protein. Cells over-expressing the 58-aa-long RHH (ribbon-helix-helix) family cellular transcription factor as well as the homologs encoded by large spindle-shaped viruses Acidianus two-tailed virus (ATV) and Sulfolobus monocaudavirus 3 (SMV3) display significant growth retardation and cell division failure, manifested as enlarged cells with multiple chromosomes. aCcr1 over-expression results in downregulation of 17 genes (>4-folds) including cdvA. A conserved motif, aCcr1-box, located between the TATA-binding box and the translation initiation site in the promoters of 13 out of the 17 highly repressed genes, is critical for aCcr1 binding. The aCcr1-box is present in the promoters of cdvA genes across Sulfolobales, suggesting that aCcr1-mediated cdvA repression is an evolutionarily conserved mechanism by which archaeal cells dictate cytokinesis progression, whereas their viruses take advantage of this mechanism to manipulate the host cell cycle.

microbiology↗

bioRxiv2022

Evolutionary Diversification of Methanotrophic Ca. Methanophagales (ANME-1) and Their Expansive Virome

Candidatus Methanophagales (ANME-1) is a major order-level clade of archaea responsible for methane removal in deep-sea sediments through the anaerobic oxidation of methane. Yet the extent of their diversity and factors which drive their dynamics and evolution remain poorly understood. Here, by sampling hydrothermal rocks and sediments, we expand their phylogenetic diversity and characterize a new deep-branching, thermophilic ANME-1 family, Candidatus Methanospirareceae (ANME-1c). They are phylogenetically closest to the short-chain-alkane oxidizers Candidatus Syntrophoarchaeales and Candidatus Alkanophagales, and encode ancestral features including a methyl coenzyme M reductase chaperone McrD and a hydrogenase complex. Global phylogeny and near-complete genomes clarified that the debated hydrogen metabolism within ANME-1 is an ancient trait that was vertically inherited but differentially lost during lineage diversification. Our expanded genomic and metagenomic sampling allowed the discovery of viruses constituting 3 new orders and 16 new families that so far are exclusive to ANME-1 hosts. These viruses represent 4 major archaeal virus assemblages, characterized by tailless icosahedral, head-tailed, rod-shaped, and spindle-shaped virions, but display unique structural and replicative signatures. Exemplified by the analyses of thymidylate synthases that unveiled a virus-mediated ancestral process of host gene displacement, this expansive ANME-1 virome carries a large gene repertoire that can influence their hosts across different timescales. Our study thus puts forth an emerging evolutionary continuum between anaerobic methane and short-chain-alkane oxidizers and opens doors for exploring the impacts of viruses on the dynamics and evolution of the anaerobic methane-driven ecosystems.

microbiology↗