Gene Transfer Agents Research Articles

New evidence supports the prophage origin of RcGTA.

Gene transfer agents (GTAs) are phage-like entities that package and transfer random host genome fragments between prokaryotes. RcGTA, produced by Rhodobacter capsulatus, is hypothesized to originate from a prophage ancestor. Most of the evidence supporting this hypothesis came from the finding of RcGTA-like genes in phages. More than 75% of the RcGTA genes have a phage homolog. However, only a few RcGTA homologs have been identified in a (pro)phage genome, leaving the hypothesis that GTAs evolved from prophages through gene loss with only weak evidence. We herein report the discovery of an inducible prophage (vB_MseS-P1) from a Mesorhizobium sediminum strain that contains the largest number (12) of RcGTA homologs found in a phage genome to date. We also identified three putative prophages and two prophage remnants harboring 12-14 RcGTA homologs in a Methylobacterium nodulans strain. The protein remote homology detection also revealed more RcGTA homologs from other phages than we previously thought. Moreover, the head-tail gene architecture of these newly discovered prophage-related elements closely resembles that of RcGTA. Furthermore, vB_MseS-P1 virions have structural proteins similar to RcGTA particles. Close phylogenetic relationships between certain prophage genes and RcGTA-like genes in Alphaproteobacteria further support the shared ancestry between RcGTA and prophages. Our findings provide new relatively direct evidence of the origin of RcGTA from a prophage progenitor.IMPORTANCEGTAs are important genetic elements in certain groups of bacteria and contribute to the genetic diversification, evolution, and ecological adaptation of bacteria. RcGTA, a common type of GTA, is known to package and transfer random fragments of the bacterial genome to recipient cells. However, the origin of RcGTA is still elusive. It has been hypothesized that RcGTA evolved from a prophage ancestor through gene loss. However, the few RcGTA homologs identified in a (pro)phage genome leave the hypothesis lacking direct evidence. This study uncovers the presence of a large number of RcGTA homologs in an inducible prophage and several putative prophages. The similar head-tail gene architecture and structural protein compositions of these newly discovered prophage-related elements and RcGTA further demonstrate an unprecedentedly observed close evolutionary relationship between prophages and RcGTA. Together, our findings provide more direct evidence supporting the origin of RcGTA from prophage.

The extent and characteristics of DNA transfer between plasmids and chromosomes

Plasmids are extrachromosomal genetic elements that reside in prokaryotes. The acquisition of plasmids encoding beneficial traits can facilitate short-term survival in harsh environmental conditions or long-term adaptation of new ecological niches. Due to their ability to transfer between cells, plasmids are considered agents of gene transfer. Nonetheless, the frequency of DNA transfer between plasmids and chromosomes remains understudied. Using a novel approach for detection of homologous loci between genome pairs, we uncover gene sharing with the chromosome in 1,974 (66%) plasmids residing in 1,016 (78%) taxonomically diverse isolates. The majority of homologous loci correspond to mobile elements, which may be duplicated in the host chromosomes in tens of copies. Neighboring shared genes often encode similar functional categories, indicating the transfer of multigene functional units. Rare transfer events of antibiotics resistance genes are observed mainly with mobile elements. The frequent erosion of sequence similarity in homologous regions indicates that the transferred DNA is often devoid of function. DNA transfer between plasmids andchromosomes thus generates genetic variation that is akin to workings of endosymbiotic gene transfer in eukaryotic evolution. Our findings imply that plasmid contribution to gene transfer most often corresponds to transfer of the plasmid entity rather than transfer of protein-coding genes between plasmids and chromosomes.

Control of a gene transfer agent cluster in Caulobacter crescentus by transcriptional activation and anti-termination

Gene Transfer Agents (GTAs) are phage-like particles that cannot self-multiply and be infectious. Caulobacter crescentus, a bacterium best known as a model organism to study bacterial cell biology and cell cycle regulation, has recently been demonstrated to produce bona fide GTA particles (CcGTA). Since C. crescentus ultimately die to release GTA particles, the production of GTA particles must be tightly regulated and integrated with the host physiology to prevent a collapse in cell population. Two direct activators of the CcGTA biosynthetic gene cluster, GafY and GafZ, have been identified, however, it is unknown how GafYZ controls transcription or how they coordinate gene expression of the CcGTA gene cluster with other accessory genes elsewhere on the genome for complete CcGTA production. Here, we show that the CcGTA gene cluster is transcriptionally co-activated by GafY, integration host factor (IHF), and by GafZ-mediated transcription anti-termination. We present evidence that GafZ is a transcription anti-terminator that likely forms an anti-termination complex with RNA polymerase, NusA, NusG, and NusE to bypass transcription terminators within the 14 kb CcGTA cluster. Overall, we reveal a two-tier regulation that coordinates the synthesis of GTA particles in C. crescentus.

Characterisation of PVL-Positive Staphylococcus argenteus from the United Arab Emirates.

Staphylococcus argenteus is a recently described staphylococcal species that is related to Staphylococcus aureus but lacks the staphyloxanthin operon. It is able to acquire both resistance markers such as the SCCmec elements and mobile genetic elements carrying virulence-associated genes from S. aureus. This includes those encoding the Panton-Valentine leukocidin (PVL), which is associated mainly with severe and/or recurrent staphylococcal skin and soft tissue infections. Here, we describe the genome sequences of two PVL-positive, mecA-negative S. argenteus sequence type (ST) 2250 isolates from the United Arab Emirates in detail. The isolates were found in a dental clinic in the United Arab Emirates (UAE). Both were sequenced using Oxford Nanopore Technology (ONT). This demonstrated the presence of temperate bacteriophages in the staphylococcal genomes, including a PVL prophage. It was essentially identical to the published sequence of phiSa2wa_st78 (GenBank NC_055048), a PVL phage from an Australian S. aureus clonal complex (CC) 88 isolate. Besides the PVL prophage, one isolate carried another prophage and the second isolate carried two additional prophages, whereby the region between these two prophages was inverted. This "flipped" region comprised about 1,083,000 bp, or more than a third of the strain's genome, and it included the PVL prophage. Prophages were induced by Mitomycin C treatment and subjected to transmission electron microscopy (TEM). This yielded, in accordance to the sequencing results, one or, respectively, two distinct populations of icosahedral phages. It also showed prolate phages which presumptively might be identified as the PVL phage. This observation highlights the significance bacteriophages have as agents of horizontal gene transfer as well as the need for monitoring emerging staphylococcal strains, especially in cosmopolitan settings such as the UAE.

An autonomous plasmid as an inovirus phage satellite.

Bacterial viruses (phages) are potent agents of lateral gene transfer and thus are important drivers of evolution. A group of mobile genetic elements, referred to as phage satellites, exploits phages to disseminate their own genetic material. Here, we isolated a novel member of the family Inoviridae, Shewanella phage Dolos, along with an autonomously replicating plasmid, pDolos. Dolos causes a chronic infection in its host Shewanella oneidensis by phage production with only minor effects on the host cell proliferation. When present, plasmid pDolos hijacks Dolos functions to be predominantly packaged into phage virions and released into the environment and, thus, acts as a phage satellite. pDolos can disseminate further genetic material encoding, e.g., resistances or fluorophores to host cells sensitive to Dolos infection. Given the rather simple requirements of a plasmid for takeover of an inovirus and the wide distribution of phages of this group, we speculate that similar phage-satellite systems are common among bacteria.IMPORTANCEPhage satellites are mobile genetic elements, which hijack phages to be transferred to other host cells. The vast majority of these phage satellites integrate within the host's chromosome, and they all carry remaining phage genes. Here, we identified a novel phage satellite, pDolos, which uses an inovirus for dissemination. pDolos (i) remains as an autonomously replicating plasmid within its host, (ii) does not carry recognizable phage genes, and (iii) is smaller than any other phage satellites identified so far. Thus, pDolos is the first member of a new class of phage satellites, which resemble natural versions of phagemids.

Gene transfer agents: The ambiguous role of selfless viruses in genetic exchange and bacterial evolution.

Gene transfer agents (GTAs) are genetic elements derived from ancestral bacteriophages that have become domesticated by the host. GTAs are present in diverse prokaryotic organisms, where they can facilitate horizontal gene transfer under certain conditions. Unlike typical bacteriophages, GTAs do not exhibit any preference for the replication or transfer of the genes encoding them; instead, they exhibit a remarkable capacity to package chromosomal, and sometimes extrachromosomal, DNA into virus-like capsids and disseminate it to neighboring cells. Because GTAs resemble defective prophages, identification of novel GTAs is not trivial. The detection of candidates relies on the genetic similarity to known GTAs, which has been fruitful in α-proteobacterial lineages but challenging in more distant bacteria. Here we consider several fundamental questions: What is the true prevalence of GTAs in prokaryote genomes? Given there are high costs for GTA production, what advantage do GTAs provide to the bacterial host to justify their maintenance? How is the bacterial chromosome recognized and processed for inclusion in GTA particles? This article highlights the challenges in comprehensively understanding GTAs' prevalence, function and DNA packaging method. Going forward, broad study of atypical GTAs and use of ecologically relevant conditions are required to uncover their true impact on bacterial chromosome evolution.

Co-evolution of gene transfer agents and their alphaproteobacterial hosts.

Gene transfer agents (GTAs) are enigmatic elements that resemble small viruses and are known to be produced during nutritional stress by some bacteria and archaea. The production of GTAs is regulated by quorum sensing, under which a small fraction of the population acts as GTA producers, while the rest becomes GTA recipients. In contrast to canonical viruses, GTAs cannot propagate themselves because they package pieces of the producing cell's genome. In alphaproteobacteria, GTAs are mostly vertically inherited and reside in their hosts' genomes for hundreds of millions of years. While GTAs' ability to transfer genetic material within a population and their long-term preservation suggest an increased fitness of GTA-producing microbes, the associated benefits and type of selection that maintains GTAs are poorly understood. By comparing rates of evolutionary change in GTA genes to the rates in gene families abundantly present across 293 alphaproteobacterial genomes, we detected 59 gene families that likely co-evolve with GTA genes. These gene families are predominantly involved in stress response, DNA repair, and biofilm formation. We hypothesize that biofilm formation enables the physical proximity of GTA-producing cells, limiting GTA-derived benefits only to a group of closely related cells. We further conjecture that the population structure of biofilm-forming sub-populations ensures that the trait of GTA production is maintained despite the inevitable rise of "cheating" genotypes. Because release of GTA particles kills the producing cell, maintenance of GTAs is an exciting example of social evolution in a microbial population.IMPORTANCEGene transfer agents (GTAs) are viruses domesticated by some archaea and bacteria as vehicles for carrying pieces of the host genome. Produced under certain environmental conditions, GTA particles can deliver DNA to neighboring, closely related cells. The function of GTAs remains uncertain. While making GTAs is suicidal for a cell, GTA-encoding genes are widespread in genomes of alphaproteobacteria. Such GTA persistence implies functional benefits but raises questions about how selection maintains this lethal trait. By showing that GTA genes co-evolve with genes involved in stress response, DNA repair, and biofilm formation, we provide support for the hypothesis that GTAs facilitate DNA exchange during the stress conditions and present a model for how GTAs persist in biofilm-forming bacterial populations despite being lethal.

Vibrio cyclitrophicus phage encoding gene transfer agent fragment, representing a novel viral family

Vibrio is a prevalent bacterial genus in aquatic environments and exhibits diverse metabolic capabilities, playing a vital role in marine biogeochemical cycles. This study isolated a novel virus infecting Vibrio cyclitrophicus, vB_VviC_ZQ26, from coastal waters near Qingdao, China. The vB_VviC_ZQ26 comprises a linear double-stranded DNA genome with a length of 42,982 bp and a G + C content of 43.21 %, encoding 72 putative open reading frames (ORFs). Transmission electron microscope characterization indicates a siphoviral-morphology of vB_VviC_ZQ26. Nucleic-acids-wide analysis indicates a tetranucleotide frequency deviation for genomic segments encoding putative gene transfer agent protein (GTA) and coil-containing protein, implying divergent origins occurred in different parts of viral genomes. Phylogenetic and genome-content-based analysis suggest that vB_VviC_ZQ26 represents a novel vibriophage-specific family designated as Coheviridae. From the result of biogeographic analysis, Coheviridae is mainly colonized in the temperate and tropical epipelagic zones. This study describes a novel vibriophage infecting V. cyclitrophicus, shedding light on the evolutionary divergence of different parts of the viral genome and its ecological footprint in marine environments.

XRE transcription factors conserved in Caulobacter and φCbK modulate adhesin development and phage production.

The xenobiotic response element (XRE) family of transcription factors (TFs), which are commonly encoded by bacteria and bacteriophage, regulate diverse features of bacterial cell physiology and impact phage infection dynamics. Through a pangenome analysis of Caulobacter species isolated from soil and aquatic ecosystems, we uncovered an apparent radiation of a paralogous XRE TF gene cluster, several of which have established functions in the regulation of holdfast adhesin development and biofilm formation in C. crescentus. We further discovered related XRE TFs throughout the class Alphaproteobacteria and its phages, including the φCbK Caulophage, suggesting that members of this cluster impact host-phage interactions. Here we show that a closely related group of XRE transcription factors encoded by both C. crescentus and φCbK can physically interact and function to control the transcription of a common gene set, influencing processes including holdfast development and the production of φCbK virions. The φCbK-encoded XRE paralog, tgrL, is highly expressed at the earliest stages of infection and can directly inhibit transcription of host genes including hfiA, a potent holdfast inhibitor, and gafYZ, an activator of prophage-like gene transfer agents (GTAs). XRE proteins encoded from the C. crescentus chromosome also directly repress gafYZ transcription, revealing a functionally redundant set of host regulators that may protect against spurious production of GTA particles and inadvertent cell lysis. Deleting the C. crescentus XRE transcription factors reduced φCbK burst size, while overexpressing these host genes or φCbK tgrL rescued this burst defect. We conclude that this XRE TF gene cluster, shared by C. crescentus and φCbK, plays an important role in adhesion regulation under phage-free conditions, and influences host-phage dynamics during infection.

Orientia tsutsugamushi: comprehensive analysis of the mobilome of a highly fragmented and repetitive genome reveals the capacity for ongoing lateral gene transfer in an obligate intracellular bacterium.

Obligate intracellular bacteria, or those only capable of growth inside other living cells, have limited opportunities for horizontal gene transfer with other microbes due to their isolated replicative niche. The human pathogen Ot, an obligate intracellular bacterium causing scrub typhus, encodes an unusually high copy number of a ~40 gene mobile genetic element that typically facilitates genetic transfer across microbes. This proliferated element is heavily degraded in Ot and previously assumed to be inactive. Here, we conducted a detailed analysis of this element in eight Ot strains and discovered two strains with at least one intact copy. This implies that the element is still capable of moving across Ot populations and suggests that the genome of this bacterium may be even more dynamic than previously appreciated. Our work raises questions about intracellular microbial evolution and sounds an alarm for gene-based efforts focused on diagnosing and combatting scrub typhus.

Extracellular vesicles are the main contributor to the non-viral protected extracellular sequence space

Environmental virus metagenomes, commonly referred to as “viromes”, are typically generated by physically separating virus-like particles (VLPs) from the microbial fraction based on their size and mass. However, most methods used to purify VLPs, enrich extracellular vesicles (EVs) and gene transfer agents (GTAs) simultaneously. Consequently, the sequence space traditionally referred to as a “virome” contains host-associated sequences, transported via EVs or GTAs. We therefore propose to call the genetic material isolated from size-fractionated (0.22 µm) and DNase-treated samples protected environmental DNA (peDNA). This sequence space contains viral genomes, DNA transduced by viruses and DNA transported in EVs and GTAs. Since there is no genetic signature for peDNA transported in EVs, GTAs and virus particles, we rely on the successful removal of contaminating remaining cellular and free DNA when analyzing peDNA. Using marine samples collected from the North Sea, we generated a thoroughly purified peDNA dataset and developed a bioinformatic pipeline to determine the potential origin of the purified DNA. This pipeline was applied to our dataset as well as existing global marine “viromes”. Through this pipeline, we identified known GTA and EV producers, as well as organisms with actively transducing proviruses as the source of the peDNA, thus confirming the reliability of our approach. Additionally, we identified novel and widespread EV producers, and found quantitative evidence suggesting that EV-mediated gene transfer plays a significant role in driving horizontal gene transfer (HGT) in the world’s oceans.

Viruses, Vesicles, and other Biological Nanoparticles: The Sub-cellular Biosphere of a Deeply Buried 2km-Deep, 20-Million-Year-Old Coalbed Community

Horizontal gene transfer is an important driver of adaptation and evolution in microorganisms. Transducing biological nanoparticles such as viral particles are believed to be key facilitators of horizontal gene transfer. In deep subseafloor sediments, energy can be highly limiting, supporting only extremely slow metabolisms. In such low-energy, isolated environments where communities may subsist for millions of years, the mechanisms of subsurface microbial adaptation and evolution remain a mystery. Virus particles have been found everywhere that life has been found, including deep subsurface environments. Although microorganisms are abundant and active in the Earth's subsurface, the role of viruses in shaping and influencing these slow-growing communities is only recently starting to be explored. Here, we analyzed the deeply buried microbial community from a lignite coalbed layer 2km below the seafloor offshore Shimokita, Japan (IODP Expedition 337) that had been buried for 20 million years. We harvested cells (>0.2µm) and biological nanoparticles (<0.2µm) from a bioreactor enrichment seeded by lignite core samples. We sequenced DNA from the cells and nanoparticles and subsequently analyzed the metagenomes. Within the nanoparticle metagenome, numerous complete novel virus genomes were reconstructed. Comparison of the virus genomes to the prokaryotic MAGs (metagenome assembled genomes) revealed that many of the virus genomes had been integrated prophage within bacterial genomes, suggesting the potential for virus-host interactions to occur in the deep subseafloor. Additionally, lysogeny may be an important survival mechanism for viruses in deeply buried, low-energy environments. Host genes were found to be packaged by viral particles, demonstrating the potential for specialized and general transduction by viruses. Not only viral particles, but there was also evidence that membrane vesicles and gene transfer agents may participate in transduction in this deep subsurface community. Horizontal gene transfer mediated by biological nanoparticles may be an important mechanism of adaptation for deep subsurface microbial communities and may provide insight into possible evolutionary processes shaping microbial communities in the deep subsurface. These results may also shed some light onto the nature of viral infection in the subsurface, potentially revealing insights about the long-term persistence of life under extreme energy limitation and how viruses may survive this over geological timescales.

In Silico Analysis of Antibiotics Resistance Genes in Lactobacillus plantarum Kita-3 isolated from Halloumi Cheese

Previous studies found that antibiotics resistance genes on plasmids in Lactobacillus make them unsafe for food purposes due to the capability of genes to transfer to pathogenic microorganisms. In contrast, Lactobacillus is widely used as a probiotic. This study assessed the antibiotics susceptibility of Lactobacillus plantarum Kita-3 isolated from Halloumi cheese using eight antibiotics. Genome sequencing was performed using the NovaSeq 6000 sequencing platform to detect the presence of antibiotics resistance genes on chromosomes and plasmids. L. plantarum Kita-3 was resistant to clindamycin, streptomycin, chloramphenicol and susceptible to tetracycline, ampicillin, kanamycin, erythromycin, ciprofloxacin. Genome sequencing of the L. plantarum Kita-3 verified the presence of the tetracycline, fluoroquinolones, β-lactamase resistance genes, and multidrug resistance efflux. Kita-3 did not have transposable elements, gene transfer agents, and plasmid-related functions. Overall, this study resulted in the antibiotics resistance profile of L. plantarumKita-3 to assess the risk of antibiotics resistance genes transfer to other bacteria. This study can provide essential data on the safe use of Lactobacillus plantarum Kita-3 as probiotics.

Changes to virus taxonomy and the ICTV Statutes ratified by the International Committee on Taxonomy of Viruses (2023).

This article reports changes to virus taxonomy and taxon nomenclature that were approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in April 2023. The entire ICTV membership was invited to vote on 174 taxonomic proposals that had been approved by the ICTV Executive Committee in July 2022, as well as a proposed revision of the ICTV Statutes. All proposals and the revised ICTV Statutes were approved by a majority of the voting membership. Of note, the ICTV continued the process of renaming existing species in accordance with the recently mandated binomial format and included gene transfer agents (GTAs) in the classification framework by classifying them as viriforms. In total, one class, seven orders, 31 families, 214 genera, and 858 species were created.

Virus-like Particles from Wolbachia-Infected Cells May Include a Gene Transfer Agent.

Wolbachia are obligate intracellular bacteria that occur in insects and filarial worms. Strains that infect insects have genomes that encode mobile genetic elements, including diverse lambda-like prophages called Phage WO. Phage WO packages an approximately 65 kb viral genome that includes a unique eukaryotic association module, or EAM, that encodes unusually large proteins thought to mediate interactions between the bacterium, its virus, and the eukaryotic host cell. The Wolbachia supergroup B strain, wStri from the planthopper Laodelphax striatellus, produces phage-like particles that can be recovered from persistently infected mosquito cells by ultracentrifugation. Illumina sequencing, assembly, and manual curation of DNA from two independent preparations converged on an identical 15,638 bp sequence that encoded packaging, assembly, and structural proteins. The absence of an EAM and regulatory genes defined for Phage WO from the wasp, Nasonia vitripennis, was consistent with the possibility that the 15,638 bp sequence represents an element related to a gene transfer agent (GTA), characterized by a signature head-tail region encoding structural proteins that package host chromosomal DNA. Future investigation of GTA function will be supported by the improved recovery of physical particles, electron microscopic examination of potential diversity among particles, and rigorous examination of DNA content by methods independent of sequence assembly.

Extracellular Polysaccharide Receptor and Receptor-Binding Proteins of the Rhodobacter capsulatus Bacteriophage-like Gene Transfer Agent RcGTA.

A variety of prokaryotes produce a bacteriophage-like gene transfer agent (GTA), and the alphaproteobacterial Rhodobacter capsulatus RcGTA is a model GTA. Some environmental isolates of R. capsulatus lack the ability to acquire genes transferred by the RcGTA (recipient capability). In this work, we investigated the reason why R. capsulatus strain 37b4 lacks recipient capability. The RcGTA head spike fiber and tail fiber proteins have been proposed to bind extracellular oligosaccharide receptors, and strain 37b4 lacks a capsular polysaccharide (CPS). The reason why strain 37b4 lacks a CPS was unknown, as was whether the provision of a CPS to 37b4 would result in recipient capability. To address these questions, we sequenced and annotated the strain 37b4 genome and used BLAST interrogations of this genome sequence to search for homologs of genes known to be needed for R. capsulatus recipient capability. We also created a cosmid-borne genome library from a wild-type strain, mobilized the library into 37b4, and used the cosmid-complemented strain 37b4 to identify genes needed for a gain of function, allowing for the acquisition of RcGTA-borne genes. The relative presence of CPS around a wild-type strain, 37b4, and cosmid-complemented 37b4 cells was visualized using light microscopy of stained cells. Fluorescently tagged head spike fiber and tail fiber proteins of the RcGTA particle were created and used to measure the relative binding to wild-type and 37b4 cells. We found that strain 37b4 lacks recipient capability because of an inability to bind RcGTA; the reason it is incapable of binding is that it lacks CPS, and the absence of CPS is due to the absence of genes previously shown to be needed for CPS production in another strain. In addition to the head spike fiber, we found that the tail fiber protein also binds to the CPS.

Metagenome-derived virus-microbe ratios across ecosystems.

It is generally assumed that viruses outnumber cells on Earth by at least tenfold. Virus-to-microbe ratios (VMR) are largely based on counts of fluorescently labelled virus-like particles. However, these exclude intracellular viruses and potentially include false positives (DNA-containing vesicles, gene-transfer agents, unspecifically stained inert particles). Here, we develop a metagenome-based VMR estimate (mVRM) that accounts for DNA viruses across all stages of their replication cycles (virion, intracellular lytic and lysogenic) by using normalised RPKM (reads per kilobase of gene sequence per million of mapped metagenome reads) counts of the major capsid protein (MCP) genes and cellular universal single-copy genes (USCGs) as proxies for virus and cell counts, respectively. After benchmarking this strategy using mock metagenomes with increasing VMR, we inferred mVMR across different biomes. To properly estimate mVMR in aquatic ecosystems, we generated metagenomes from co-occurring cellular and viral fractions (>50 kDa-200 µm size-range) in freshwater, seawater and solar saltern ponds (10 metagenomes, 2 control metaviromes). Viruses outnumbered cells in freshwater by ~13 fold and in plankton from marine and saline waters by ~2-4 fold. However, across an additional set of 121 diverse non-aquatic metagenomes including microbial mats, microbialites, soils, freshwater and marine sediments and metazoan-associated microbiomes, viruses, on average, outnumbered cells by barely two-fold. Although viruses likely are the most diverse biological entities on Earth, their global numbers might be closer to those of cells than previously estimated.

Structural Study of the Cobetia marina Bacteriophage 1 (Carin-1) by Cryo-EM.

Most of studied bacteriophages (phages) are terrestrial viruses. However, marine phages are shown to be highly involved in all levels of oceanic regulation. They are, however, still largely overlooked by the scientific community. By inducing cell lysis on half of the bacterial population daily, their role and influence on the bacterial biomass and evolution, as well as their impact in the global biogeochemical cycles, is undeniable. Cobetia marina virus 1 (Carin-1) is a member of the Podoviridae family infecting the γ-protoabacteria C. marina. Here, we present the almost complete, nearly-atomic resolution structure of Carin-1 comprising capsid, portal, and tail machineries at 3.5 Å, 3.8 Å and 3.9 Å, respectively, determined by cryo-electron microscopy (cryo-EM). Our experimental results, combined with AlphaFold2 (AF), allowed us to obtain the nearly-atomic structure of Carin-1 by fitting and refining the AF atomic models in the high resolution cryo-EM map, skipping the bottleneck of de-novo manual building and speeding up the structure determination process. Our structural results highlighted the T7-like nature of Carin1, as well as several novel structural features like the presence of short spikes on the capsid, reminiscent those described for Rhodobacter capsulatus gene transfer agent (RcGTA). This is, to our knowledge, the first time such assembly is described for a bacteriophage, shedding light into the common evolution and shared mechanisms between gene transfer agents and phages. This first full structure determined for a marine podophage allowed to propose an infection mechanism different than the one proposed for the archetypal podophage T7. IMPORTANCE Oceans play a central role in the carbon cycle on Earth and on the climate regulation (half of the planet's CO2 is absorbed by phytoplankton photosynthesis in the oceans and just as much O2 is liberated). The understanding of the biochemical equilibriums of marine biology represents a major goal for our future. By lysing half of the bacterial population every day, marine bacteriophages are key actors of these equilibriums. Despite their importance, these marine phages have, so far, only been studied a little and, in particular, structural insights are currently lacking, even though they are fundamental for the understanding of the molecular mechanisms of their mode of infection. The structures described in our manuscript allow us to propose an infection mechanism that differs from the one proposed for the terrestrial T7 virus, and might also allow us to, in the future, better understand the way bacteriophages shape the global ecosystem.

Viriforms-A New Category of Classifiable Virus-Derived Genetic Elements.

The International Committee on Taxonomy of Viruses (ICTV) recently accepted viriforms as a new polyphyletic category of classifiable virus-derived genetic elements, juxtaposed to the polyphyletic virus, viroid, and satellite nucleic acid categories. Viriforms are endogenized former viruses that have been exapted by their cellular hosts to fulfill functions important for the host's life cycle. While morphologically resembling virions, particles made by viriforms do not package the viriform genomes but instead transport host genetic material. Known viriforms are highly diverse: members of family Polydnaviriformidae (former Polydnaviridae) have thus far been found exclusively in the genomes of braconid and ichneumonid parasitoid wasps, whereas the completely unrelated gene transfer agents (GTAs) are widely distributed among prokaryotes. In addition, recent discoveries likely extend viriforms to mammalian genomes. Here, we briefly outline the properties of these viriform groups and the first accepted and proposed ICTV frameworks for viriform classification.

Archaeal Host Cell Recognition and Viral Binding of HFTV1 to Its Haloferax Host.

Viruses are highly abundant and the main predator of microorganisms. Microorganisms of each domain of life are infected by dedicated viruses. Viruses infecting archaea are genomically and structurally highly diverse. Archaea are undersampled for viruses in comparison with bacteria and eukaryotes. Consequently, the infection mechanisms of archaeal viruses are largely unknown, and most available knowledge stems from viruses infecting a select group of archaea, such as crenarchaea. We employed Haloferax tailed virus 1 (HFTV1) and its host, Haloferax gibbonsii LR2-5, to study viral infection in euryarchaea. We found that HFTV1, which has a siphovirus morphology, is virulent, and interestingly, viral particles adsorb to their host several orders of magnitude faster than most studied haloarchaeal viruses. As the binding site for infection, HFTV1 uses the cell wall component surface (S)-layer protein. Electron microscopy of infected cells revealed that viral particles often made direct contact with their heads to the cell surface, whereby the virion tails were perpendicular to the surface. This seemingly unfavorable orientation for genome delivery might represent a first reversible contact between virus and cell and could enhance viral adsorption rates. In a next irreversible step, the virion tail is orientated toward the cell surface for genome delivery. With these findings, we uncover parallels between entry mechanisms of archaeal viruses and those of bacterial jumbo phages and bacterial gene transfer agents. IMPORTANCE Archaeal viruses are the most enigmatic members of the virosphere. These viruses infect ubiquitous archaea and display an unusually high structural and genetic diversity. Unraveling their mechanisms of infection will shed light on the question if entry and egress mechanisms are highly conserved between viruses infecting a single domain of life or if these mechanisms are dependent on the morphology of the virus and the growth conditions of the host. We studied the entry mechanism of the tailed archaeal virus HFTV1. This showed that despite "typical" siphovirus morphology, the infection mechanism is different from standard laboratory models of tailed phages. We observed that particles bound first with their head to the host cell envelope, and, as such, we discovered parallels between archaeal viruses and nonmodel bacteriophages. This work contributes to a better understanding of entry mechanisms of archaeal viruses and a more complete view of microbial viruses in general.