Biology subjects

Krupovic, M.

Publications and source records attributed to Krupovic, M..

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bioRxiv2022

DeepTracer ID: De Novo Protein Identification from Cryo-EM Maps

The recent revolution in cryo-electron microscopy has made it possible to determine macromolecular structures directly from cell extracts. However, identifying the correct protein from the cryo-EM map is still challenging and often needs additional sequence information from other techniques, such as tandem mass spectrometry and/or bioinformatics. Here, we present DeepTracer-ID, a server-based approach to identify the candidate protein in a user-provided organism de novo from a cryo-EM map, without the need for additional information. Our method first uses DeepTracer to generate a protein backbone model that best represents the cryo-EM map, and this model is then searched against the library of AlphaFold2 predictions for all proteins in the given organism. This method is highly accurate and robust: in all 13 experimental maps tested blindly, DeepTracer-ID identified the correct proteins as the top candidates. Eight of the maps were of known structures, while the other five unpublished maps were validated by prior protein annotation and careful inspection of the model refined into the map. The program also showed promising results for both homomeric and heteromeric protein complexes. This platform is possible because of the recent breakthroughs in large-scale protein 3D structure prediction. Statement of SignificanceWhile it has now become routine for cryo-EM maps of proteins to reach a near-atomic resolution, potentially allowing for reliable atomic models to be built, there are a growing number of instances where the protein identity may not be known. Without knowing the protein sequence, it is impossible to build an atomic model. DeepTracer-ID is a server-based approach to surmount this problem by identifying the proteins in a given organism that are found in the cryo-EM map. A free web service for global academic access is provided.

biophysics↗

bioRxiv2022

Archaeal bundling pili of Pyrobaculum calidifontis reveal similarities between archaeal and bacterial biofilms

While biofilms formed by bacteria have received great attention due to their importance in pathogenesis, much less research has been focused on the biofilms formed by archaea. It has been known that extracellular filaments in archaea, such as Type IV pili, hami and cannulae, play a part in the formation of archaeal biofilms. We have used cryo-electron microscopy to determine the atomic structure of a previously uncharacterized class of archaeal surface filaments from hyperthermophilic Pyrobaculum calidifontis. These filaments, which we call archaeal bundling pili (ABP), assemble into highly ordered bipolar bundles. The bipolar nature of these bundles most likely arises from the association of filaments from at least two different cells. The component protein shows homology, both at the sequence and structural level, to the bacterial protein TasA, a major component of the extracellular matrix in bacterial biofilms, contributing to biofilm stability. We show that ABP forms very stable filaments in a manner similar to the donor-strand exchange of bacterial TasA fibers and chaperone-usher pathway pili where a {beta}-strand from one subunit is incorporated into a {beta}-sheet of the next subunit. Our results reveal mechanistic similarities and evolutionary connection between bacterial and archaeal biofilms, and suggest that there could be many other archaeal surface filaments that are as yet uncharacterized. SignificanceBiofilms are communities of microbes where cells attach to each other as well as to surfaces, and bacterial biofilms have been intensively studied due to their importance in many infections. Much less has been known about archaeal biofilms, where archaea are a third domain of life. Using cryo-electron microscopy, we have determined the atomic structure of a surface filament in archaea that forms bi-polar bundles connecting cells. We show that this protein has common ancestry with a protein known to be an important component of bacterial biofilms. This adds to our understanding of the evolutionary relationship between bacteria and archaea and may provide new insights into bacterial biofilms.

biophysics↗

bioRxiv2022

Viral origin of eukaryotic type IIA DNA topoisomerases

Type II DNA topoisomerases of the family A (Topo IIA) 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 homologues and have no orthologues in Archaea. Interestingly, eukaryotic Topo IIA have close homologues in viruses of the phylum Nucleocytoviricota, an expansive assemblage of large and giant viruses formerly known as the nucleocytoplasmic large DNA viruses (NCLDV). Topo IIA 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 combining viral and cellular Topo IIA homologs. Topo IIA 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 dataset, indicating that the separation of Nucleocytoviricota and eukaryotes is probably not due to long branch attraction artefact. The topology of our tree suggests 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.

evolutionary biology↗

bioRxiv2022

Identification of a new Inovirus in the absence of bacterial infection in the human virome

Viruses infecting bacteria, known as bacteriophages, represent the most abundant viral particles in the human body. They participate in the control of the human associated bacterial communities and play an important role in the dissemination of virulence genes. Here we present the identification of a new genetic element, named RIP1, in the human virome. RIP1 shares conserved structural genes with the single-stranded DNA viruses of the Inoviridae family. Furthermore structure-function studies identified nuclear subcellular localization of the RIP1 DNA replication/recombination machinery. Additional metagenomics analysis and polymerase chain reaction detected RIP1 in multiple body sites including blood, cerebrospinal pericardial and amniotic fluids, nasal swabs and feces in the absence of concomitant bacterial infection, uncovering inovirus phage persistence in the human virome.

microbiology↗

bioRxiv2022

A five-fold expansion of the global RNA virome reveals multiple new clades of RNA bacteriophages

High-throughput RNA sequencing offers unprecedented opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA viral contigs. Via analysis of the 330k novel RNA-dependent RNA polymerases (RdRP), this expansion corresponds to a five-fold increase of RNA virus diversity. Extended RdRP phylogeny supports monophyly of the five established phyla, reveals two putative new bacteriophage phyla and numerous putative novel classes and orders. The dramatically expanded Lenarviricota phylum, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome diversity. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts. Gene content analysis revealed multiple domains previously not found in RNA viruses and implicated in virus-host interactions. This vast collection of new RNA virus genomes provides insights into RNA virus evolution and should become a major resource for RNA virology.

genomics↗

bioRxiv2021

A closed Candidatus Odinarchaeum genome exposes Asgard archaeal viruses

Asgard archaea have recently been identified as the closest archaeal relatives of eukaryotes. Their ecology remains enigmatic, and their virome, completely unknown. Here, we describe the closed genome of Ca. Odinarchaeum yellowstonii LCB_4, and, from this, obtain novel CRISPR arrays with spacer targets to several viral contigs. We find related viruses in sequence data from thermophilic environments and in the genomes of diverse prokaryotes, including other Asgard archaea. These novel viruses open research avenues into the ecology and evolution of Asgard archaea.

microbiology↗

bioRxiv2021

Viruses of Asgard archaea

Asgardarchaeota encode many eukaryotic signature proteins and are widely considered to represent the closest archaeal relatives of eukaryotes. Whether similarities between Asgard archaea and eukaryotes extend to their viromes remains unknown. Here we present 20 metagenome-assembled genomes of Asgardarchaeota from deep-sea sediments of the basin off the Shimokita Peninsula, Japan. By combining CRISPR spacer search of metagenomic sequences with phylogenomic analysis, we identify three family-level groups of viruses associated with Asgard archaea. The first group, Verdandiviruses, includes tailed viruses of the realm Duplodnaviria, the second one, Skuldviruses, consists of viruses with predicted icosahedral capsids that belong to the realm Varidnaviria, and the third group, Wyrdviruses, is related to spindle-shaped viruses previously identified in other archaea. More than 90% of the proteins encoded by these putative viruses of Asgard archaea show no sequence similarity to proteins encoded by other known viruses. Nevertheless, all three proposed families consist of viruses typical of prokaryotes, providing no indication of a specific evolutionary relationship between viruses infecting Asgard archaea and eukaryotes. Verdandiviruses and skuldviruses are likely to be lytic, whereas wyrdviruses, similar to all other known spindle-shaped viruses, probably establish chronic infection and are released without host cell lysis. All three groups of viruses were identified in sediment samples from distinct geographical locations and are expected to play important roles in controlling the Asgard archaea populations in deep-sea ecosystems.

microbiology↗

bioRxiv2021

Diversity, taxonomy and evolution of archaeal viruses of the class Caudoviricetes

The archaeal tailed viruses (arTV), evolutionarily related to tailed double-stranded DNA bacteriophages of the class Caudoviricetes, represent the most common isolates infecting halophilic archaea. Only a handful of these viruses have been genomically characterized, limiting our appreciation of their ecological impacts and evolution. Here, we present 37 new genomes of haloarchaeal tailed virus isolates, more than doubling the current number of sequenced arTVs. Analysis of all 63 available complete genomes of arTVs, which we propose to classify into 14 new families, suggests ancient divergence of archaeal and bacterial tailed viruses and points to an extensive sharing of genes involved in DNA metabolism and counter defense mechanisms, illuminating common strategies of virus-host interactions with tailed bacteriophages. Coupling of the comparative genomics with the host range analysis on a broad panel of haloarchaeal species uncovered four distinct groups of viral tail fiber adhesins controlling the host range expansion. The survey of metagenomes using viral hallmark genes suggests that the global architecture of the arTV community is shaped through recurrent transfers between different biomes, including hypersaline, marine and anoxic environments.

microbiology↗

bioRxiv2021

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 diverse 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 suggest that, similar to eukaryotes, production of EVs in S. islandicus depends on the archaeal ESCRT machinery. 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. 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.

microbiology↗

bioRxiv2020

New virus isolates from Italian hydrothermal environments underscore the biogeographic pattern in archaeal virus communities

Viruses of hyperthermophilic archaea represent one of the least understood parts of the virosphere, showing little genomic and morphological similarity to viruses of bacteria or eukaryotes. Here, we investigated virus diversity in the active sulfurous fields of the Campi Flegrei volcano in Pozzuoli, Italy. Virus-like particles displaying eight different morphotypes, including lemon-shaped, droplet-shaped and bottle-shaped virions, were observed and five new archaeal viruses proposed to belong to families Rudiviridae, Globuloviridae and Tristromaviridae were isolated and characterized. Two of these viruses infect neutrophilic hyperthermophiles of the genus Pyrobaculum, whereas the remaining three have rod-shaped virions typical of the family Rudiviridae and infect acidophilic hyperthermophiles belonging to three different genera of the order Sulfolobales, namely, Saccharolobus, Acidianus and Metallosphaera. Notably, Metallosphaera rod-shaped virus 1 is the first rudivirus isolated on Metallosphaera species. Phylogenomic analysis of the newly isolated and previously sequenced rudiviruses revealed a clear biogeographic pattern, with all Italian rudiviruses forming a monophyletic clade, suggesting geographical structuring of virus communities in extreme geothermal environments. Furthermore, we propose a revised classification of the Rudiviridae family, with establishment of five new genera. Collectively, our results further show that high-temperature continental hydrothermal systems harbor a highly diverse virome and shed light on the evolution of archaeal viruses.

microbiology↗

bioRxiv2019

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger1-3, activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction4,5. Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions6,7. Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

biochemistry↗

bioRxiv2019

Cryptic inoviruses are pervasive in bacteria and archaea across Earth's biomes

Bacteriophages from the Inoviridae family (inoviruses) are characterized by their unique morphology, genome content, and infection cycle. To date, a relatively small number of inovirus isolates have been extensively studied, either for biotechnological applications such as phage display, or because of their impact on the toxicity of known bacterial pathogens including Vibrio cholerae and Neisseria meningitidis. Here we show that the current 56 members of the Inoviridae family represent a minute fraction of a highly diverse group of inoviruses. Using a new machine learning approach leveraging a combination of marker gene and genome features, we identified 10,295 inovirus-like genomes from microbial genomes and metagenomes. Collectively, these represent six distinct proposed inovirus families infecting nearly all bacterial phyla across virtually every ecosystem. Putative inoviruses were also detected in several archaeal genomes, suggesting that these viruses may have occasionally transferred from bacterial to archaeal hosts. Finally, we identified an expansive diversity of inovirus-encoded toxin-antitoxin and gene expression modulation systems, alongside evidence of both synergistic (CRISPR evasion) and antagonistic (superinfection exclusion) interactions with co-infecting viruses which we experimentally validated in a Pseudomonas model. Capturing this previously obscured component of the global virosphere sparks new avenues for microbial manipulation approaches and innovative biotechnological applications.

microbiology↗

bioRxiv2019

Gene sharing networks to automate genome-based prokaryotic viral taxonomy

Viruses of bacteria and archaea are likely to be critical to all natural, engineered and human ecosystems, and yet their study is hampered by the lack of a universal or scalable taxonomic framework. Here, we introduce vConTACT 2.0, a network-based application to establish prokaryotic virus taxonomy that scales to thousands of uncultivated virus genomes, and integrates confidence scores for all taxonomic predictions. Performance tests using vConTACT 2.0 demonstrate near-identical correspondence to the current official viral taxonomy (>85% genus-rank assignments at 96% accuracy) through an integrated distance-based hierarchical clustering approach. Beyond "known viruses", we used vConTACT 2.0 to automatically assign 1,364 previously unclassified reference viruses to tentative taxa, and scaled it to modern metagenomic datasets for which the reference network was robust to adding 16,000 viral contigs. Together these efforts provide a systematic reference network and an accurate, scalable taxonomic analysis tool that is critically needed for the research community.

bioinformatics↗

bioRxiv2018

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 a subject of intense debate. Since the characterization of the mimivirus in 2003, many big and giant viruses have been discovered at a steady pace, offering a vast material for evolutionary investigations. In parallel, phylogenetic tools are constantly being improved, offering more rigorous approaches for reconstruction of deep evolutionary history of viruses and their hosts. 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 two 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 seemingly transferred from two clades of NCLDVs to proto-eukaryotes, giving rise to two of the three 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.

evolutionary biology↗

bioRxiv2018

Origins and Evolution of the Global RNA Virome

Viruses with RNA genomes dominate the eukaryotic virome, reaching enormous diversity in animals and plants. The recent advances of metaviromics prompted us to perform a detailed phylogenomic reconstruction of the evolution of the dramatically expanded global RNA virome. The only universal gene among RNA viruses is the RNA-dependent RNA polymerase (RdRp). We developed an iterative computational procedure that alternates the RdRp phylogenetic tree construction with refinement of the underlying multiple sequence alignments. The resulting tree encompasses 4,617 RNA virus RdRps and consists of 5 major branches, 2 of which include positive-sense RNA viruses, 1 is a mix of positive-sense (+) RNA and double-stranded (ds) RNA viruses, and 2 consist of dsRNA and negative-sense (-) RNA viruses, respectively. This tree topology implies that dsRNA viruses evolved from +RNA viruses on at least two independent occasions, whereas -RNA viruses evolved from dsRNA viruses. Reconstruction of RNA virus evolution using the RdRp tree as the scaffold suggests that the last common ancestors of the major branches of +RNA viruses encoded only the RdRp and a single jelly-roll capsid protein. Subsequent evolution involved independent capture of additional genes, particularly, those encoding distinct RNA helicases, enabling replication of larger RNA genomes and facilitating virus genome expression and virus-host interactions. Phylogenomic analysis reveals extensive gene module exchange among diverse viruses and horizontal virus transfer between distantly related hosts. Although the network of evolutionary relationships within the RNA virome is bound to further expand, the present results call for a thorough reevaluation of the RNA virus taxonomy.\n\nIMPORTANCEThe majority of the diverse viruses infecting eukaryotes have RNA genomes, including numerous human, animal, and plant pathogens. Recent advances of metagenomics have led to the discovery of many new groups of RNA viruses in a wide range of hosts. These findings enable a far more complete reconstruction of the evolution of RNA viruses than what was attainable previously. This reconstruction reveals the relationships between different Baltimore Classes of viruses and indicates extensive transfer of viruses between distantly related hosts, such as plants and animals. These results call for a major revision of the existing taxonomy of RNA viruses.

microbiology↗

bioRxiv2017

Candidatus Nitrosocaldus cavascurensis, an ammonia oxidizing, extremely thermophilic archaeon with a highly mobile genome

Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first cultivated thermophilic representative with a sequenced genome, which allows to search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota. Candidatus Nitrosocaldus cavascurensis has been cultivated from a hot spring in Ischia, Italy. It grows optimally at 68{degrees}C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 25h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome of Ca. N. cavascurensis harbors an amoAXCB gene cluster encoding ammonia monooxygenase, genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although a bona fide gene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the importance of this compound for AOA metabolism. Ca. N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.

microbiology↗

bioRxiv2017

Genomic, proteomic, and phylogenetic analysis of spounaviruses indicates paraphyly of the order Caudovirales

It is almost a cliche that tailed bacteriophages of the order Caudovirales are the most abundant and diverse viruses in the world. Yet, their taxonomy still consists of a single order with just three families: Myoviridae, Siphoviridae, and Podoviridae. Thousands of newly discovered phage genomes have recently challenged this morphology-based classification, revealing that tailed bacteriophages are genomically even more diverse than once thought. Here, we evaluate a range of methods for bacteriophage taxonomy by using a particularly challenging group as an example, the Bacillus phage SPO1-related viruses of the myovirid subfamily Spounavirinae. Exhaustive phylogenetic and phylogenomic analyses indicate that the spounavirins are consistent with the taxonomic rank of family and should be divided into at least five subfamilies. This work is a case study for virus genomic taxonomy and the first step in an impending massive reorganization of the tailed bacteriophage taxonomy.

microbiology↗

bioRxiv2017

Numerous cultivated and uncultivated viruses encode ribosomal proteins

Text Text METHODS References Viruses modulate ecosystems by directly altering host metabolisms through auxiliary metabolic genes, which are obtained through random sampling of the host genome and rise to fixation, presumably through improved viral fitness by alleviating key metabolic bottlenecks during infection. Conspicuously, however, viral genomes are not known to encode the core components of translation machinery, such as ribosomal proteins (RPs), though genes for RPs S1 and S21 have been detected in viral metagenomes1,2. Here we augment this little-noticed observation using available reference genomes, global-scale viral metagenomic datasets, and functional assays for select proteins. We identify 15 different RPs across diverse viral genomes arising from cultivated viral isolates (5 RPs in 16 genomes) and metag ...

microbiology↗