Biology subjects

Gophna, U.

Publications and source records attributed to Gophna, U..

At least 19 recordsLinked to original sources

bioRxiv2026

Temporal dynamics of RNA phage infection reveal bacterial recovery despite ongoing viral replication

Bacterial defenses against DNA phages are well documented, yet responses to RNA phages remain largely unexplored. Here, we characterize the population dynamics and host responses of Escherichia coli infected by the lytic RNA phage MS2 across multiple timescales, using bulk and single-cell transcriptomics, fluorescence microscopy, whole-genome sequencing, and genetic perturbations. Despite rapid viral takeover and extensive cell death, the bacterial population subsequently rebounds, revealing an unusual state in which host proliferation persists under intense phage pressure. Early in infection, heterogeneous subpopulations are evident, including one with low or undetectable viral transcript levels, low expression of the F-plasmid genes encoding the F-pilus (the MS2 receptor), and a distinct metabolic state. Strikingly, the rebounding population carries MS2 RNA in the great majority of cells, with evidence for active viral replication and continued cell division despite high viral loads. This rebound coincides with strong induction of biofilm-associated genes, including the pga operon, which synthesizes the biofilm-matrix exopolysaccharide PNAG. Deletion of pgaA causes earlier population decline and reduced population survival, indicating that pga-dependent matrix production delays viral lysis. Over longer timescales, phage-exposed populations show heterogeneous F-plasmid prevalence and plasmid-wide expression, consistent with dynamic cycling between plasmid-bearing and plasmid-free states: MS2 selects against F-plasmid-bearing cells, whereas conjugation may restore plasmid carriage. We propose that the biofilm-associated matrix delays viral lysis relative to continued cell division, long enough for rare F-plasmid-free daughter cells to arise and expand. Our results suggest that bacterial survival is determined by a dynamic race between viral lysis and host-cell division.

microbiology↗

bioRxiv2026

Screening metatranscriptomes for ultrastable RNA secondary structures reveals hidden bacteriophages and novel capsid nanomaterials

Metatranscriptomics has transformed our view of RNA bacteriophage diversity, revealing vast numbers of single-stranded RNA (ssRNA) phages whose protein capsids can be engineered for biotechnology applications. However, many ssRNA phages remain hidden from current detection methods, which require protein-level similarity to known phages. Here we show that RNA structure provides an additional signal for the detection of ssRNA phages in metatranscriptomes, including hidden phages missed by prior protein-based methods. By computationally folding each contig and screening for exceptionally stable RNA secondary structures, we find evidence of thousands of previously unrecognized phages encoding novel coat proteins. We express a library of 12,000 such coat proteins in E. coli and find that most assemble into nuclease-resistant capsids. We determine the 3D structure of one such capsid by cryo-electron microscopy and demonstrate that it can be disassembled and reassembled in vitro to package heterologous RNA--a key step toward repurposing these particles as RNA delivery vehicles. We compile the newly discovered ssRNA phages with previously known ones into a database that contains sequence and structural information for over 460,000 unique RNA molecules and over 100,000 distinct coat proteins, providing a comprehensive resource for microbiology and nanomaterials research.

biochemistry↗

bioRxiv2025

CRISPR-Cas targeting in Haloferax volcanii promotes within-species gene exchange by triggering homologous recombination

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea against mobile genetic elements, but the role they play in gene exchange and speciation remains unclear. Here, we investigated how CRISPR-Cas targeting affects mating and gene exchange in the halophilic archaeon Haloferax volcanii. Surprisingly, we found that CRISPR-Cas targeting significantly increased mating efficiency between members of the same species, in contrast to its previously documented role in reducing inter-species mating. This enhanced mating efficiency was dependent on the Cas3 nuclease/helicase and extended beyond the targeted genomic regions. Further analysis revealed that CRISPR-Cas targeting promoted biased recombination in favour of the targeting strain during mating, resulting in an increased proportion of recombinant progeny that are positive for CRISPR-Cas. To test whether an increase in recombination is sufficient to increase mating efficiency, we tested whether strains lacking the MRE11-RAD50 complex, which are known to have elevated recombination activity, also exhibited higher mating success. Indeed, these strains showed higher mating, as did cells that were exposed to DNA damage using methyl methanesulfonate. These findings suggest that CRISPR-Cas systems may contribute to speciation by facilitating within-species gene exchange while limiting between-species genetic transfer, thereby maintaining species boundaries.

genetics↗

bioRxiv2025

A TIR-SAVED effector mediates antiviral immunity via a conserved host signal

Cyclic oligonucleotide-based anti-phage signalling systems (CBASS) are widespread prokaryotic antiviral defense mechanisms that function through coordinated cyclase-effector interactions. Upon sensing viral infection, the cyclase produces a signal molecule that activates effector function and causes cell dormancy or death. However, the evolutionary origins and functional independence of CBASS components remain unclear. Type II CBASS systems commonly employ TIR-SAVED domain effector proteins that deplete cellular NAD+ during viral infection. Here, we demonstrate that a TIR-SAVED effector protein can operate as a standalone antiviral defense, causing significant growth inhibition and approximately 50% viral clearance during infection in the complete absence of its cognate cyclase. Remarkably, we show that the TIR-SAVED effector can sense cyclic di-AMP, a conserved second messenger produced by the host diadenylate cyclase DacZ, when the canonical CBASS signal is absent. This antiviral activity was associated with depletion of cellular NAD+ and required intact conserved functional residues within both the TIR and SAVED domains. These findings reveal a novel mechanism of antiviral signalling that expands the functional repertoire of CBASS. They also provide insights into the modular evolution of complex prokaryotic immune systems, suggesting that what are now CBASS effectors might have evolved as independent defense components before being integrated into multi-protein systems.

microbiology↗

bioRxiv2025

Evolutionary insights into provirus-encoded CRISPR-Cas systems in halophilic archaea

Prokaryotic microorganisms coexist with mobile genetic elements (MGEs), which can be both genetic threats and evolutionary catalysts. In Haloferax lucentense, a halophilic archaeon, we have recently identified an unusual genomic arrangement: a complete type I-B CRISPR-Cas system encoded on a mega-plasmid coexists with a partial counterpart within an integrated provirus in the main chromosome. The provirus-encoded system lacks the adaptation genes (cas1, cas2, and cas4), suggesting its potential reliance on the plasmid-encoded CRISPR-Cas module for the acquisition of new spacers. This arrangement suggests a potential instance of "adaptive outsourcing," where a provirus might leverage a co-resident MGE for a key function. Through comparative genomics, we show that similar proviral CRISPR-Cas systems are found in distantly related haloarchaea (e.g., Natrinema and Halobacterium), indicating probable virus-mediated horizontal transfer and suggesting they may function as mobile defense modules. Phylogenetic analysis highlights distinct evolutionary origins of the two systems: the plasmid system clusters with other Haloferax CRISPR-Cas systems, while the proviral system clusters with those from other genera, consistent with horizontal acquisition. Interestingly, spacer analysis reveals that the proviral systems predominantly target viral sequences, while the plasmid system appears to target both plasmids and viral sequences, a distribution mirroring broader trends observed in other plasmid- and chromosome-encoded CRISPR systems. This observed targeting preference suggests a potential for complementarity that could support a model of cooperative immunity, where each system may protect its genetic "owner" from competition and, indirectly, the host.

evolutionary biology↗

bioRxiv2025

Planetary-scale metagenomic search reveals new patterns of CRISPR targeting

Interactions between microbes and their mobile genetic elements (MGEs), including viruses and plasmids, are critical drivers of microbiome structures and processes. CRISPR-Cas systems are important regulators of these host-MGE interactions, but a global understanding of CRISPR loci diversity, activity, and roles across Earths biomes is still lacking. Here, we use an optimized computational approach to search short-read data and collect [~]800 million CRISPR spacers across [~]450,000 public metagenomes. From this extensive CRISPR spacer dataset, we identified 1.18 billion hits between 41 million spacers and 2.5 million viruses and plasmids. Focusing on the role of CRISPR as anti-phage defense, we observed an over-representation of prevalent and conserved spacers consistent with a positive selection pressure associated with phage targeting. Identification of sustained CRISPR targeting by microbes not expected to be viable hosts suggested that some phage genomes may frequently enter non-host microbial cells. Taken together, these results outline the extensive diversity of CRISPR loci across microbiomes, and highlight key parameters driving CRISPR targeting patterns likely to influence strain-level diversification and selection processes within microbial populations.

microbiology↗

bioRxiv2024

Optimized Plaque Assay for Detecting Chronically Infecting Viruses of Haloarchaea

Archaeal viruses predominantly exhibit a chronic lifestyle, where viral particles are released without causing host cell death. Therefore, conventional plaque assays, which are well-suited for identifying lytic viruses, usually fail to detect chronically infecting viruses due to their non-lytic nature. To address this limitation, we developed an optimized plaque assay protocol for detecting chronically infecting viruses of haloarchaea, particularly species within the Haloferax genus. By enhancing viral diffusibility and infectivity through adjustments in agar concentration and lowering incubation temperature, this modified protocol improved plaque formation, enabling the detection of viruses that cause mild growth delays. We demonstrate successful plaque formation for two chronically infecting viruses, Haloferax volcanii pleomorphic virus 1 (HFPV-1) and lemon-shaped virus 48N (LSV-48N), on representative Haloferax strains and highlight the potential for isolating novel viruses from environmental samples. This improved plaque assay is an effective method for the detection and quantification of chronically infecting archaeal viruses, providing a new tool for discovering new viral families in extreme environments.

microbiology↗

bioRxiv2024

Internal in-frame translation generates Cas11b, which is important for effective interference in an archaeal CRISPR-Cas system

CRISPR-Cas is a sophisticated defence system used by bacteria and archaea to fend off invaders. CRISPR-Cas systems vary in their Cas protein composition and have therefore been divided into different classes and types. Type I systems of bacteria have been shown to contain the small Cas11 protein as part of the interference complex. Here we show for the first time that an archaeal CRISPR-Cas type I system also contains a Cas11 protein. In addition, we show for the first time an internal in-frame translation of an archaeal protein. The Cas11b protein from the Haloferax volcanii type I-B system is encoded in the cas8b gene. Translation initiation at an internal methionine of the cas8b open reading frame results in synthesis of Cas11b. Cas11b is required for an effective interference reaction and without Cas11b fewer Cascade complexes form. Comparison of transcriptomes from wild type and a Cas11b less strain show that the depletion of Cas11b results in differential regulation of many genes. Taken together Cas11b is important for the defence reaction of the type I-B CRISPR-Cas system and seems to play an additional cellular role.

molecular biology↗

bioRxiv2024

Navigating a fine balance: point-mutant cheater viruses disrupt the viral replication cycle

Cheater viruses, alternatively denoted as defective interfering viruses, cannot replicate on their own yet replicate faster than the wild type (WT) when the two viruses coinfect the same cell. Cheaters must possess dual genetic features: a defect, which leads to their inability to infect cells on their own, and a selective advantage over WT during co-infection. Previously, we have discovered two point-mutant cheaters of the MS2 bacteriophage. Here, we set out to discover the possible repertoire of cheater MS2 viruses by performing experimental evolution at a very high multiplicity of infection (MOI). Our results revealed a third point-mutant cheater that arose in eight biological replicas. Each of the three cheaters disrupts the fine balance necessary for phage replication, in different ways that create a defect + advantage. We found that over time, the point mutant cheaters accumulate additional "helper" mutations, which alter other stages of the viral replication cycle, complementing the disruptions created by the original cheater. Intriguingly, cheater and helper mutations almost always reside in very close proximity on the genome. This region encodes for multiple functions: overlapping reading frames as well as overlapping RNA structures critical for transitioning from one stage to another in the viral replication cycle. This region of overlap explains the dual functions of cheaters, as one mutation can have pleiotropic effects. Overall, these findings underscore how viruses, whose dense genomes often have overlapping functions, can easily evolve point-mutant cheaters, and how cheaters can evolve to alter the intricate balance of the viral replication cycle.

evolutionary biology↗

bioRxiv2024

An archaeal CBASS system eliminates viruses without killing the host cells

Many cyclic-oligonucleotide-based anti-phage signalling systems (CBASS) defend against viral infections by depleting cellular NAD+ levels, eventually leading to dormancy or death. This abortive infection strategy is beneficial in stopping fast lytic infections, as cells die before spreading the virus to neighbouring cells. However, in chronic viral infections, which often occur in archaea, abortive infection could be detrimental, as the cost of immunity may outweigh that of infection. Here we study an archaeal CBASS system (H-CBASS2) that was expressed in the model species Haloferax volcanii DS2 and Haloferax gibbonsii LR2-5. We show that the system provides protection to H. gibbonsii against a lytic tailed haloarchaeal virus HFTV1 by depleting NAD+, similar to what has been observed for lytic phages in bacteria. H-CBASS2 is also triggered, though with much slower activity, during infection with the chronic, non-lytic virus HFPV-1, and promotes virus clearance after several passages without killing host cells. Moreover, cells that clear the HFPV-1 infection become substantially more resistant to subsequent infections, due to mutations in envelope-associated proteins. Cell death by NAD+ depletion only occurs after a very long infection with HFPV-1 on solid medium. These findings suggest that the magnitude of H-CBASS2 response is somehow tuned to the infection type can benefit the host during non-lytic infections, potentially explaining why such systems are relatively common in archaea.

microbiology↗

bioRxiv2024

A new archaeal virus that suppresses the transcription of host immunity genes

In some extreme environments, archaeal cells have been shown to have chronic viral infections, and such infections are well-tolerated by the hosts and may potentially protect against more lethal infections by lytic viruses. We have discovered that a natural Haloferax strain (48N), which is closely related to the model organism Haloferax volcanii, is chronically-infected by a lemon-shaped virus, which we could purify from the medium. The chronic infection by this virus, which we named LSV-48N, is never cleared, despite the multiple defense systems of the host that include CRISPR-Cas, and two CBASS systems. Curing 48N of its virus by genetic engineering, led to radical changes in the gene expression profile of 48N and a dramatic improvement in its growth rate. Remarkably, the cured 48N is the fastest-growing haloarchaeon reported to date, with a generation time of approximately 1 hour at 45{degrees}C compared to the typical 2.5 hours of H. volcanii or its infected isogen, and faster than any known haloarchaeon. The virus subverts host defenses by reducing their transcription and interfering with the CRISPR spacer acquisition machinery. Our results suggest that the slow growth of many halophilic archaea could be due to the effects of proviruses within their genomes that consume resources and alter the gene expression of their hosts.

microbiology↗

bioRxiv2023

Using the pan-genomic framework for the discovery of genomic islands in the haloarchaeon Halorubrum ezzemoulense

In this study, we use pan-genomics to characterize the organized variability from the widely dispersed halophilic archaeal species Halorubrum ezzemoulense. We include a multi-regional sampling of newly sequenced, high-quality draft genomes. Using the pan-genome graph of the species, we discover 50 genomic islands which represent rare accessory genetic capabilities available to members of the species. 19 of these islands are likely the remnant of mobile genetic elements and are enriched for genomic dark matter. 10 islands encode for niche adapting solute transporters, with a cosmopolitan but limited distribution throughout the strains. We also observe rearrangements which have led to the insertion/recombination/replacement of mutually exclusive genomic islands in equivalent genome positions ("homeocassettes"). These conflicting islands encode for similar functions, but homologs from islands located between the same core genes exhibit extreme divergence on the amino acid level. Homeocasettes provide variations for a homologous function, which may confer a greater range of adaptability to the species group. We observe some islands that appear geographically restricted; however, we also observe the coexistence of genomes, in a single geographic region, with and without certain genomic islands, demonstrating the retention and spread of rare genes in the pan-genome. ImportanceUnderstanding the evolution of genome content is a key puzzle in evolutionary biology. Despite its importance, this area hasnt received thorough investigation. This is especially true of Archaeal organisms, which constitute a large fraction of Earths diversity, but are often referred to as the "forgotten" or "third" domain of life. This study dives into those questions by finding rare genes amongst a group of closely related Archaeal species, and describes how their transfer, utilization and persistence may contribute to the speciation and specialization of the group.

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

Differences in homologous recombination and maintenance of heteropolyploidy between Haloferax volcanii and Haloferax mediterranei

Polyploidy, the phenomenon of having more than one copy of the genome in an organism, is common among haloarchaea. While providing short-term benefits for DNA repair, polyploidy is generally regarded as an evolutionary trap that by the notion of the Mullers ratchet will inevitably conclude in the species decline or even extinction due to a gradual reduction in fitness. In most reported cases of polyploidy in archaea, the genetic state of the organism is considered as homoploidy i.e. all copies of the genome are identical. Here we demonstrate that while this is indeed the prevalent genetic status in the halophilic archaeon H. volcanii, its close relative H. mediterranei maintains a prolonged heteroploidy state in a non-selective environment once a second allele is introduced. Moreover, a strong genetic linkage was observed between two distant loci in H. mediterranei indicating a low rate of homologous recombination while almost no such linkage was shown in H. volcanii indicating a high rate of recombination in the latter species. We suggest that H. volcanii escapes Mullers ratchet by means of an effective chromosome-equalizing gene-conversion mechanism facilitated by highly active homologous recombination, whereas H. mediterranei must elude the ratchet via a different, yet to be elucidated mechanism.

genetics↗

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

Escherichia coli strains from patients with inflammatory bowel diseases have disease-specific genomic adaptations

ObjectiveEscherichia coli is over-abundant in the gut microbiome of patients with IBD, yet most studies have focused on the adherent-invasive E. coli pathotype. Here, we aimed to identify IBD-specific or phenotype-specific genomic functions of diverse E. coli lineages. DesignWe investigated E. coli from patients with UC, CD and a pouch and healthy subjects. The majority of E. coli genomes were reconstructed directly from metagenomic samples, including publicly available and newly sequenced fecal metagenomes. Clinical metadata and biomarkers were collected. Functional analysis at the gene and mutation level and genome replication rates of E. coli strains were performed, and correlated with IBD phenotypes and biomarkers. ResultsOverall, 530 E. coli genomes were analysed. A specific E. coli lineage (B2) was more prevalent in UC compared to other IBD phenotypes. Genomic metabolic capacities varied across E. coli lineages and IBD phenotypes. Specifically, sialidases involved in host mucin utilization, were exclusively present in a single lineage and were depleted in patients with a pouch. In contrast, enzymes that hydrolyze inulin were enriched in patients with a pouch. E. coli from patients with UC were twice as likely to encode the genotoxic molecule colibactin than strains from patients with CD or pouch. Strikingly, patients with a pouch showed the highest E. coli growth rates, even in the presence of antibiotics. Fecal calprotectin did not correlate with the relative abundance of E. coli. Finally, we identified multiple IBD-specific loss-of function mutations in E. coli genes encoding for bacterial cell envelope and secretion components. ConclusionThis study presents E. coli as a commensal species better adapted to the overly-active mucosal immune milieu in IBD, that may benefit from intestinal inflammation, rather than causing it. The evidence given here suggests adaptive evolution toward attenuated virulence in some E. coli strains, coupled with a rapid growth despite the presence of antibiotics.

genomics↗

bioRxiv2020

Microbiome-related aspects of locust density-dependent phase transition

Locust plagues are an ancient phenomenon, with references going back to the Old Testament. These swarming pests are notorious for their tendency to aggregate and perform long migrations, consuming vast amounts of vegetation and decimating the cultivated fields on their path. However, when population density is low, locusts will express a solitary, cryptic, non-aggregating phenotype that is not considered as an agricultural pest. Although transition of locusts from the solitary to the gregarious phase has been well studied, the shifts in the locust microbiome composition associated with this phase-transition have yet to be addressed. Here, using 16S rRNA amplicon sequencing, we compared the bacterial composition of solitary desert locusts before and after a crowding-induced phase-transition. Our findings reveal that the microbiome is altered during the phase transition. We also show that this significant change in bacterial composition includes the acquisition of a specific bacterial species - Weissella cibaria (Firmicutes), which has been previously shown to induce aggregation in cockroaches. Our findings led us to hypothesize that the locust microbiome may play a role in inducing aggregation behavior, contributing to the formation and maintenance of a swarm. Employing a mathematical model, we demonstrate the potential evolutionary advantage of inducing aggregation under various environmental conditions; and specifically, when the aggregation-inducing microbe exhibits a relatively high horizontal transmission rate. This is a first description of a previously unknown and important aspect of locust phase transition, demonstrating that the phase shift includes a shift in the gut and integument bacterial composition.

microbiology↗

bioRxiv2020

The maternal foam plug constitutes a reservoir for the desert locust's bacterial symbionts

A hallmark of the desert locusts ancient and deserved reputation as a devastating agricultural pest is that of the long-distance, multi-generational migration of locust swarms to new habitats. The bacterial symbionts that reside within the locust gut comprise a key aspect of its biology, augmenting its immunity and having also been reported to be involved in the swarming phenomenon through the emission of attractant volatiles. However, it is still unclear whether and how these beneficial symbionts are transmitted vertically from parent to offspring. Using comparative 16S rRNA amplicon sequencing and direct experiments with engineered bacteria, we provide here evidence of the vertical transmission of locust gut bacteria. The females perform this activity by way of inoculation of the egg-pods foam plug, through which the larvae pass upon hatching. Furthermore, analysis of the biochemical structure of the foam revealed chitin to be its major component, along with immunity-related proteins such as lysozyme, which could be responsible for the inhibition of some bacteria in the foam while allowing other, more beneficial, strains to proliferate. Our findings reveal a potential vector for the transgenerational transmission of symbionts in locusts, which contributes to the locust swarms ability to invade and survive in new territories.

ecology↗