Biology subjects

Severinov, K.

Publications and source records attributed to Severinov, K..

At least 19 recordsLinked to original sources

bioRxiv2026

H-NS silences antiviral immunity through 3D chromatin compaction

Bacterial antiviral immunity systems can be toxic to their hosts and must be tightly regulated. Immunity genes are often encoded by AT-rich mobile genetic elements subjected to silencing by nucleoid binding protein H-NS. Using RNA-seq, ChIP-seq, and chromosome 3D reconstruction with Micro-C, we find that H-NS binds and compacts bacterial immunity loci. H-NS deletion reveals anti-phage activity in model Escherichia coli strains generally considered phage-sensitive. Extending this approach to environmental isolates, we show that removal of H-NS silencing enhances defense, allowing bacteria to restrict phages with anti-defense proteins. We also discover Madara, a novel immunity system that complements the co-regulated BREX defense. Our results establish H-NS as a master regulator of bacterial immunity and highlight the importance of expression levels for anti-phage activity in native hosts.

molecular biology↗

bioRxiv2026

OLD sentinel: an abortive tRNase surveys phage replication and DNA defects in RecBCD-compromised cells

OLD, an abortive immunity protein from prophage P2, consists of an ABC ATPase sensor and a TOPRIM nuclease effector -- a core architecture shared by a large protein family, including components of antiphage systems Gabija, PARIS, Septu, and Lamassu. OLD was originally identified for its lethality in recB-deficient cells and inhibition of bacteriophage {lambda} infection, but the mechanisms governing its activation have remained elusive. Here, we present the cryo-EM structure of an inactive OLD tetramer and show that destabilization into dimeric form opens the TOPRIM catalytic site, stimulating tRNA cleavage. This activity arrests translation, a phenotype rescued by phage-encoded tRNAs. We demonstrate that OLD activation is not strictly RecBCD-dependent: OLD binds aberrant DNA structures in recB-deficient cells, but activation during infection requires recognition of single-stranded DNA hairpins at the phage replication origin. Collectively, our findings reveal how host and phage DNA processing factors create a complex landscape controlling OLD-mediated immunity.

molecular biology↗

bioRxiv2026

Phages infecting the common gut commensal Escherichia coli HS reveal tropism for its Klebsiella-like capsule

Tailed dsDNA phages are ubiquitous and thought to infect almost any bacterial species. Escherichia coli HS - a commensal strain used as a model in gut colonization studies - has not been reported as a host for dsDNA phages and is resistant to more than 100 coliphages from the BASEL collection. Here, we report the first phages infecting E. coli HS, characterize their interaction with an endogenous BREX system, and provide a detailed genomic and phylogenetic description. Phages {phi}HS1 (Queuovirinae) and {phi}HS2 (Ackermannviridae) possess dPreQ and 5-NeOmdU modifications that confer resistance to restriction digestion. {phi}HS3 is a temperate phage, related to the native HS prophage {phi}HS4, and represents a founding member of a novel genus (Hueyvirus) of P22-like phages capable of lateral transduction. We reveal exchange of tailspike genes between {phi}HS2, {phi}HS3, and Klebsiella-specific phages. Furthermore, we demonstrate that E. coli HS encodes a Klebsiella-like K47 capsule required for phage infection. These results suggest that the broad phage resistance of E. coli HS may be linked to its capsular type.

microbiology↗

bioRxiv2026

A virion protein of an archaeal virus inhibits a Type V BREX defense system in Haloarcula hispanica

Bacteriophage Exclusion (BREX) systems are a diverse family of bacterial and archaeal defense mechanisms defined by two conserved genes encoding a putative alkaline phosphatase BrxZ (PglZ) and an ATPase BrxC (PglY). Here, we characterize a Type V BREX system from the archaeon Haloarcula hispanica. Similar to bacterial Type I BREX systems, host-virus discrimination relies on methylation of specific non-palindromic DNA motifs. Notably, the H. hispanica BREX locus encodes two methyltransferases, BrxX1 and BrxX2, which independently target distinct motifs (GTAYCCG and GACCCC). The system protects H. hispanica against three of seven tested archaeal viruses. However, two related viruses, SH1 and HHIV-2, despite encoding multiple BREX target sites, escape BREX-mediated defense. We demonstrate that a large virion protein, VP1, encoded by these viruses inhibits the BREX system, as mutant viruses carrying partial deletions of VP1 lose resistance to the host defense. Together, these findings provide the first characterization of a Type V BREX system and demonstrate that archaeal viruses can counteract BREX through virion-associated anti-defense proteins.

molecular biology↗

bioRxiv2025

Plasmid copy number affects the DNA methylation-driven expression dynamics of the CfrBI restriction-modification system and impacts phage restriction

Restriction-modification (R-M) systems are one of the most widespread and, due to their often plasmid-based nature, transmittable anti-phage systems bacteria have. The CfrBI R-M system studied here consists of a methyltransferase (MT) and a restriction endonuclease (RE) that are divergently expressed and share a promoter region that harbors a single CfrBI recognition site. Previously, the methylation of this site has been shown to regulate the expression of the R-M system. Here, we show that the expression dynamics of the CfrBI R-M system and its protective properties depend on the copy number of the plasmid harboring it. A higher copy number results in higher expression, but the expression on a medium-copy number plasmid interestingly conferred the highest phage resistance. After transformation of naive cells however, expression of the RE was fastest in the high-copy plasmid background. In vivo we show that the expression strength of the MT inhibits its own expression, while enhancing RE expression. To conclude, the results indicate that for phage resistance the overall expression strength might not be the predominant factor in case of the CfrBI R-M system, while for the establishment of the system the initial expression rate of the MT seems to be the determining factor.

microbiology↗

bioRxiv2025

A census of anti-CRISPR proteins reveals AcrIE9 as an inhibitor of Escherichia coli K12 Type IE CRISPR-Cas system

CRISPR-Cas adaptive immunity systems provide defense against mobile genetic elements and are often countered by diverse anti-CRISPR (Acr) proteins. The Type IE CRISPR-Cas of Escherichia coli K12 has been a model for structural and functional studies and is a part of the species core genome. However, this system is transcriptionally silent, which has fueled questions about its true biological function. To clarify the role of this system in defense, we carried out a census of Acr proteins found in Enterobacterales and identified AcrIE9 as a potent inhibitor of the E. coli K12 Type IE CRISPR-Cas system. While sharing little sequence identity, AcrIE9 proteins from Pseudomonas and Escherichia both interact with the Cas7 subunit of the Cascade complex, thus preventing its binding to DNA. We further show that AcrIE9 is genetically linked to AcrIE10, forming the most widespread anti-CRISPR cluster in Enterobacterales, and this module often co-occurs with a novel HTH-like protein with unusual architecture.

microbiology↗

bioRxiv2025

The number of immune defense and counter-defense systems sustained in the arms race between procaryotes and viruses

Prokaryotes have evolved various mechanisms to counter viruses, which in their turn developed numerous strategies to avoid defenses of the hosts. Dozens of such defense and counter-defense mechanisms have recently been discovered, yet the number of such systems held by a given virus or its host is limited. Here, we present numerical and theoretical arguments for the existence of the maximal number of ecologically and evolutionary sustainable defense and counter-defense systems maintained by both sides at any time of the never-ending evolutionary arms race. We find that the number of such systems is of the order of 10 for a broad range of assumptions about the costs and benefits of defense and counter-defense mechanisms and their specificity. This optimum in the number of defense and counter-defense systems appears as a result of a compromise between the metabolic and autoimmune costs of adding a new layer of defense and the benefits it conveys. 1 Significance statementAlmost all living creatures are either predators or prey (and many are both). At all scales of life, from single cells to largest mammals, prey evolve defenses against predators and predators in their turn evolve counter-defense mechanisms. As a result of this ever-lasting arms race, each organism usually possesses multilayer defense and counter-defense arsenals. Is there a limit to the size of these arsenals? In this work we present a proof-of-principle argument that such a limit indeed exists. Using for definity an example of prokaryotes as the prey and their viruses as predators, we show that the number of distinct defense and counter-defense system that the prey and predators possess is limited by a number of the order of 10 and is only weakly dependent on the ecology of predator-prey interactions. Similar principles and limits likely emerge universally in various antagonistic conflicts such as crime and law enforcement or real arms race.

evolutionary biology↗

bioRxiv2024

New viruses infecting hyperthermophilic bacterium Thermus thermophilus

Highly diverse phages infecting thermophilic bacteria of the Thermus genus have been isolated over the years from hot springs around the world. Many of these phages are unique, rely on highly unusual developmental strategies, and encode novel enzymes. The variety of Thermus phages is clearly undersampled, as evidenced, for example, by a paucity of phage-matching spacers in Thermus CRISPR arrays. Using water samples collected from hot springs in the Kunashir Island from the Kuril archipelago and from the Tsaishi and Nokalakevi districts in the Republic of Georgia we isolated several distinct phages infecting laboratory strains of Thermus thermophilus. Genomic sequence analysis of 11 phages revealed both close relatives of previously described Thermus phages isolated from geographically distant sites, as well as phages with very limited similarity to earlier isolates. Comparative analysis allowed us to predict several accessory phage genes whose products may be involved in host defense/interviral warfare, including a putative Type V CRISPR-cas system.

microbiology↗

bioRxiv2024

Molecular basis of foreign DNA recognition by BREX anti-phage immunity system

Anti-phage systems of the BREX (BacteRiophage EXclusion) superfamily rely on epigenetic DNA methylation to discriminate between the host and invading DNA, but their mechanism of protection remains enigmatic. We demonstrate that in Type I BREX systems, both defense and methylation are based on site-specific DNA recognition by the BrxX (PglX) methyltransferase and require the S-adenosyl methionine cofactor. We present a 2.2-[A] cryoEM structure of Escherichia coli BrxX bound to target dsDNA, which reveals the molecular details of DNA recognition by BREX and paves the way for rational engineering of BREX specificity. We show that BrxX alone does not support methylation, and BREX activity requires an assembly of a supramolecular BrxBCXZ immune complex. Finally, we present a cryoEM structure of BrxX bound to a phage-encoded inhibitor Ocr that sequesters an inactive dimeric form of BrxX. Together, these results allow us to propose a model of BREX-mediated DNA sensing and anti-phage defense.

molecular biology↗

bioRxiv2024

Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

biochemistry↗

bioRxiv2023

Characterization of Streptococcus uberis Cas9 (SuCas9) - a Type II-A Ortholog Functional in Human Cells

Type II CRISPR-Cas9 RNA-guided nucleases are commonly used for genome engineering. To date, all characterized Cas9-based genome editors, including the widely used SpCas9, have limitations such as their relatively large size and restriction of targets flanked by a specific PAM sequence. Here, we biochemically characterized more compact SpCas9 ortholog, SuCas9, from Streptococcus uberis, a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5'-NNAAA-3' PAM, efficiently cleaves DNA in vitro, and is active in human cells. The study of SuCas9 has the potential to expand the range of applications of CRISPR-Cas9 enzymes in medicine and biotechnology.

molecular biology↗

bioRxiv2023

The Effect of Pseudoknot Base Pairing on Cotranscriptional Structural Switching of the Fluoride Riboswitch

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of E. coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37 {degrees}C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65 {degrees}C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

molecular biology↗

bioRxiv2023

The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early genes products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection was studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

molecular biology↗

bioRxiv2023

Interference Requirements of Type III CRISPR-Cas Systems from Thermus thermophilus

Among the diverse prokaryotic adaptive immunity mechanisms, the Type III CRISPR-Cas systems are the most complex. The multisubunit Type III effectors recognize RNA targets complementary to CRISPR RNAs (crRNAs). Target recognition causes synthesis of cyclic oligoadenylates that activate downstream auxiliary effectors, which affect cell physiology in complex and poorly understood ways. Here, we studied the ability of III-A and III-B CRISPR-Cas subtypes from Thermus thermophilus to interfere with plasmid transformation. We find that for both systems, requirements for crRNA-target complementarity sufficient for interference depend on the target transcript abundance, with more abundant targets requiring shorter complementarity segments. This result and thermodynamic calculations indicate that Type III effectors bind their targets in a simple bimolecular reaction with more extensive crRNA-target base pairing compensating for lower target abundance. Since the targeted RNA used in our work is non-essential for either the host or the plasmid, the results also establish that a certain number of target-bound effector complexes must be present in the cell to interfere with plasmid establishment. For the more active III-A system, we determine the minimal length of RNA-duplex sufficient for interference and show that the position of this minimal duplex can vary within the effector. Finally, we show that the III-A immunity is dependent on the HD nuclease domain of the Cas10 subunit. Since this domain is absent from the III-B system the result implies that the T. thermophilus III-B system must elicit a more efficient cyclic oligoadenylate dependent response to provide the immunity.

microbiology↗

bioRxiv2023

Single-molecule studies reveal the off-pathway elemental pause state as a target of streptolydigin inhibition of RNA polymerase and its dramatic enhancement by Gre factors

Antibiotic streptolydigin (Stl) inhibits bacterial transcription by blocking the trigger loop folding in the active center of RNA polymerase (RNAP), which is essential for catalysis. We use acoustic force spectroscopy to characterize the dynamics of transcription elongation in ternary elongation complexes of RNAP (ECs) in the presence of Stl at a single-molecule level. We found that Stl induces long-lived stochastic pauses while the instantaneous velocity of transcription between the pauses is unaffected. Stl enhances the short-lived pauses associated with an off-pathway elemental paused state of the RNAP nucleotide addition cycle. Unexpectedly, we found that transcript cleavage factors GreA and GreB, which were thought to be Stl competitors, do not alleviate the streptolydigin-induced pausing; instead, they synergistically increase transcription inhibition by Stl. This is the first known instance of a transcriptional factor enhancing antibiotic activity. We propose a structural model of the EC-Gre-Stl complex that explains the observed Stl activities and provides insight into possible cooperative action of secondary channel factors and other antibiotics binding at the Stl-pocket. These results offer a new strategy for high-throughput screening for prospective antibacterial agents.

biophysics↗

bioRxiv2023

Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in Clostridioides difficile

CRISPR-Cas systems provide their prokaryotic hosts with adaptive immunity against mobile genetic elements. Many bacteriophages encode anti-CRISPR (Acr) proteins that inhibit host defense. The identification of Acr proteins is challenging due to their small size and high sequence diversity, and only a limited number has been characterized to date. In this study, we report the discovery of a novel Acr protein, AcrIB2, encoded by the {varphi}CD38-2 Clostridioides difficile phage that efficiently inhibits interference by the type I-B CRISPR-Cas system of the host and likely acts as a DNA mimic. Most C. difficile strains contain two cas operons, one encoding a full set of interference and adaptation proteins and another encoding interference proteins only. Unexpectedly, we show that only the partial operon is required for interference and is subject to inhibition by AcrIB2.

microbiology↗

bioRxiv2023

Phage T3 overcomes the BREX defence through SAM cleavage and inhibition of SAM synthesis

Bacteriophage T3 encodes a SAMase that through cleavage of S-adenosyl-methionine (SAM) circumvents the SAM-dependent Type I Restriction-Modification defence of the host bacterium Escherichia coli. Here, we show that the SAMase also allows T3 to evade BREX defence. SAM degradation weakly affects BREX methylation of host DNA, but completely inhibits the defensive function of BREX, suggesting that SAM is required as a co-factor for BREX-mediated exclusion of phage DNA. The anti-BREX activity of the T3 SAMase is mediated by two independent mechanisms: enzymatic degradation of SAM and downregulation of SAM synthesis through direct inhibition of the host SAM synthase MetK. We determined a 2.8 [A] cryo-EM structure of the eight-subunit T3 SAMase-MetK complex. Structure guided mutagenesis of the SAMase-MetK interface revealed that the interaction with MetK stabilizes the T3 SAMase in vivo, thus further stimulating its anti-BREX activity. This work provides insights in the versatility and intricacy of bacteriophage counter-defence mechanisms and highlights the role of SAM as an important co-factor of diverse phage-defence systems.

microbiology↗

bioRxiv2022

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP)1 that defines the length of its extraordinarily long 800 nm tail2,3. We found that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular and viral two-barrel RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We determined the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab claw-like architecture similar to previously reported two-barrel RNAPs. The virion RNAP adopts a unique flat structure without a clamp, which likely reflects the requirement for its extrusion through the narrow channel in the phage tail for delivery into the cell. Structure and sequence comparisons of the P23-45 RNAPs with other phage and cellular RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate remarkable adaptability of two-barrel RNAPs that can be attained within a single virus species.

molecular biology↗