Biology subjects

Pogliano, J.

Publications and source records attributed to Pogliano, J..

At least 19 recordsLinked to original sources

bioRxiv2026

A bacteriophage split DNA polymerase incorporates a sliding clamp-derived processivity subunit

Viruses that infect bacteria, termed bacteriophages or phages, typically encode their own DNA replication machinery that rapidly generates many copies of the phage genomic DNA in an infected cell. Nucleus-forming phages of the chimallivirus family possess a distinctive repertoire of predicted DNA replication proteins, with two proteins annotated as DNA polymerases plus a DnaB-like replicative helicase, but lacking an identified primase or sliding clamp protein. Here, we demonstrate that in the Pseudomonas aeruginosa-infecting chimallivirus PhiKZ, the core DNA polymerase comprises a stable 1:1 complex of two DNA polymerase subunits, termed DNAPN and DNAPC. A 2.1 A resolution cryoelectron microscopy structure of this core complex reveals homology to other phage and bacterial B-family DNA polymerases. Next, we identify a direct but transient interaction between the core DNA polymerase complex and a third protein termed Rhc, which possesses an N-terminal RNase H domain and a C-terminal domain related to DNA sliding clamp proteins. Cryoelectron microscopy structures of a stabilized DNA polymerase-Rhc complex, and of this complex bound to a primer-template DNA, reveal a single Rhc subunit that cooperates with the DNA polymerase thumb domain to bind double-stranded DNA emerging from the DNA polymerase active site. While the Rhc RNase H domain is not resolved in our structures, this domain is situated such that it could degrade RNA associated with an incoming template DNA strand. In vitro primer-extension assays show that Rhc strongly boosts DNA polymerase activity, and that this boost relies on the Rhc-DNA polymerase interface we observe in our structures. Overall, our findings reveal a distinctive chimallivirus-specific DNA replication machine with an integrated DNA-binding subunit related to sliding clamp proteins, and a flexibly-linked RNase H domain.

biochemistry↗

bioRxiv2026

A bifunctional RNase H protein supports DNA replication and transcription in nucleus-forming Chimalliviridae phages

Jumbo bacteriophages of the Chimalliviridae family replicate their genomes within a proteinaceous phage nucleus that spatially segregates viral DNA replication and transcription from translation. While the phage nucleus protects viral DNA from host defenses, it also imposes unique constraints on genome replication and gene expression. Here we identify the phage-encoded protein Rhc (RNase H-clamp) as a central mediator of genome transactions within the phage nucleus. Using the Escherichia coli infecting bacteriophage Goslar, we show that Rhc functions as an essential component of a non-canonical three-subunit DNA polymerase complex composed of DNAPN, DNAPC and Rhc. Rhc physically interacts with the DNAPC subunit, and both the catalytic activity of Rhc's N-terminal RNase H domain and its C-terminal sliding clamp-derived DNA binding domain are required for genome replication, nuclear maturation, and productive infection. Further, Goslar Rhc also supports transcription within the phage nucleus through a function that is separable from its DNA replication function. Rhc depletion results in reduced transcription of a subset of late genes, in a pattern that suggests a role for Rhc in resolving R-loops. Imaging and cryo-electron tomography reveal that loss of Rhc arrests phage development at a nascent nuclear stage, uncoupling nuclear assembly from maturation. Together, these findings identify Rhc as a multifunctional nuclear factor that mediates both DNA replication and transcription in nucleus-forming jumbo phages, revealing how viruses repurpose a conserved enzymatic activity to coordinate gene expression within a compartmentalized replication environment.

cell biology↗

bioRxiv2026

Structure of the Chimalliviridae bacteriophage Goslar reveals host recognition and infection machinery

Large-genome bacteriophages (phages) of the Chimalliviridae family possess a distinctive life cycle, building compartments inside infected bacterial cells that broadly protect their genomes from host defenses. Here, we use cryoelectron microscopy to determine a high-resolution structure of the E. coli-infecting Chimalliviridae phage Goslar, generating a composite model with 2,888 protein chains from 28 different structural proteins, totaling over 1.4 million amino acid residues. Our structure reveals the architecture of the Goslar capsid, portal, tail, and baseplate, highlighting structural similarities and differences with other tailed phages. Combining our structural data with quantitative mass spectrometry of Goslar virions, we identify several high-copy virion-associated proteins that likely play key roles when injected into host cells upon infection. We also identify a Chimalliviridae-specific family of proteins that form long, flexible filaments anchored at the phage baseplate and which incorporate carbohydrate-binding domains, suggesting a key role in host recognition.

microbiology↗

bioRxiv2025

Chlorotonils exhibit potent activity against Mycobacterium tuberculosis, while resistance is mediated by MmpR5-MmpL5

Treatment of Mycobacterium tuberculosis (Mtb) is challenging and requires administration of at least four different antibiotics. Unfortunately, multi-drug resistant Mtb strains continue to emerge, undermining the effectiveness of current treatment regimens and highlighting the urgent need for new therapeutics. In this study, we evaluated the potential of natural product-derived chlorotonils as anti-Mtb agents. We demonstrate that chlorotonils exhibit nanomolar potency against a range of attenuated and virulent Mtb strains. Mechanistic studies and resistance profiling in Mtb revealed that chlorotonils affect both lipid and energy metabolism. Through systems biology approaches, including the construction of an Mtb CRISPRi library specifically designed for chemical-genomic profiling, we identified MmpR5/MmpL5 as major driver of chlorotonil-resistance in Mtb leading also to cross-resistance with bedaquiline. Our findings highlight chlorotonils as valuable chemical tools to further dissect the role and function of the MmpS5-MmpL5 efflux pump in drug-resistant Mtb.

microbiology↗

bioRxiv2025

Thienopyrimidine amide analogs target MmpL3 in Mycobacterium tuberculosis

ObjectivesThe identification of novel agents with mechanisms of action distinct from those currently utilized in tuberculosis treatment remains a significant challenge. The mycobacterial protein MmpL3 has emerged as a promising drug target due to its essential role in the synthesis of the cell wall of Mycobacterium tuberculosis. We previously identified novel thienopyrimidine amides with good anti-tubercular activity. MethodsWe profiled a subset of thienopyrimidine amides determining activity against intracellular bacteria and bactericidal activity against replicating bacteria. We ran assays to determine mode of action by measuring cell wall stress, ATP production, and bacterial cytological profiling. We determined activity against a strain of M. tuberculosis with mutations in MmpL3. We isolated and sequenced resistant mutants. ResultsWe tested five analogs against a strain of M. tuberculosis with mutations in MmpL3 and determined that they lost potency. Analogs induced PiniBAC, a reporter for cell wall stress, and led to an ATP boost characteristic of cell wall inhibitors. Bacterial cytological profiling of a representative compound revealed a morphological profile consistent with other MmpL3 inhibitors. ConclusionsTogether, our data support MmpL3 as the most probable drug target for the TPA analogs and add to the growing list of scaffolds that can inhibit this vulnerable transporter.

microbiology↗

bioRxiv2025

A miniature CRISPR-Cas10 enzyme confers immunity by an inverse signaling pathway

Microbial and viral co-evolution has created immunity mechanisms involving oligonucleotide signaling that share mechanistic features with human anti-viral systems1. In these pathways, including CBASS and type III CRISPR systems in bacteria and cGAS-STING in humans, oligonucleotide synthesis occurs upon detection of virus or foreign genetic material in the cell, triggering the antiviral response2-4. In a surprising inversion of this process, we show here that the CRISPR-related enzyme mCpol synthesizes cyclic oligonucleotides constitutively as part of an active mechanism that maintains cell health. Cell-based experiments demonstrated that the absence or loss of mCpol-produced cyclic oligonucleotides triggers cell death, preventing spread of viruses that attempt immune evasion by depleting host cyclic nucleotides. Structural and mechanistic investigation revealed mCpol to be a di-adenylate cyclase whose product, c-di-AMP, prevents toxic oligomerization of the effector protein 2TM{beta}. Analysis of cells by fluorescence microscopy showed that lack of mCpol allows 2TM{beta}-mediated cell death due to inner membrane collapse. These findings unveil a powerful new defense strategy against virus-mediated immune suppression, expanding our understanding of oligonucleotides in cell health and disease. These results raise the possibility of similar protective roles for cyclic oligonucleotides in other organisms including humans.

biochemistry↗

bioRxiv2024

Identification of a large cohort of Enterobacter jumbo phages with broad host ranges across pathogenic Gammaproteobacteria

ESKAPE pathogens cause most hospital-acquired infections globally and often carry antibiotic resistance. Many of them have been the target of bacteriophage therapies. Enterobacter is an ESKAPE pathogen but is less frequently a target for phage therapy due to a relative lack of available phages. We isolated eight jumbo phages with genomes ranging from 223 to 366 kbp targeting Enterobacter spp. and found that they belonged to separate phage clades. Six of them formed nucleus-like structures confirmed by DAPI-staining, and were phylogenetically related to Chimalliviridae. Two jumbo phages did not form nucleus-like structures and did not cluster with Chimalliviridae. Although these jumbo phages were found on Enterobacter, many were closely related to phages with non-Enterobacter hosts. To test whether these phages may have had expanded host ranges, we examined 14 pathogenic Gammaproteobacteria and found that these phages were capable of creating plaques on 8 of them. These species included Escherichia coli, Klebsiella aerogenes, Serratia marcescens, Salmonella spp., Shigella spp., Providencia spp., Citrobacter spp., and Cronobacter sakazakii. We verified that there was phage amplification in these microbes rather than lysis from without by performing qPCR to confirm DNA replication in each species. Phages typically have narrow host ranges, a benefit for microbiome-sparing compared to antibiotics. However, the broad host ranges of these Gammaproteobacteria jumbo phages suggests that not all phages have the same risk/benefit ratios. While this broad range could aid their development as antibiotic alternatives, further study is needed to assess potential microbiome disruption. SignificanceWith the growing threat of antibiotic resistant bacteria, alternative treatments like bacteriophages have emerged. Bacteriophages typically have narrow host ranges, a disadvantage compared to antibiotics. We discovered eight jumbo phages that kill antibiotic resistant Enterobacter, but were diverse phylogenetically. Six of them formed nucleus-like structures and were members of the Chimalliviridae family and the other two did not form nucleus-like structures. We confirmed that each phage had broad host ranges capable of lysing at least eight different human Gammaproteobacteria including pathogens such as E. coli, K. aerogenes, and S. marcescens. By identifying broad host range jumbo phages that attack pathogens, we may have identified phages with spectrums of activity more similar to antibiotics than have been traditionally attributed to phages.

microbiology↗

bioRxiv2024

An arylsulphonamide that targets cell wall biosynthesis in Mycobacterium tuberculosis

We investigated the mechanism of action of an arylsulphonamide with whole cell activity against Mycobacterium tuberculosis. We newly synthesized the molecule and confirmed it had activity against both extracellular and intracellular bacilli. The molecule had some activity against HepG2 cells but maintained some selectivity. Bacterial cytological profiling suggested that mechanism of action was via disruption of cell wall synthesis, with similarities to an inhibitor of the mycolic acid exporter MmpL3. The compound induced expression from the IniB promoter and caused a boost in ATP production but did not induce reactive oxygen species. A mutation in MmpL3 (S591I) led to low-level resistance. Taken together these data confirm the molecule targets cell wall biosynthesis with MmpL3 as the most probable target.

microbiology↗

bioRxiv2024

A phage nucleus-associated protein from the jumbophage Churi inhibits bacterial growth through protein translation interference

Antibacterial proteins inhibiting Pseudomonas aeruginosa have been identified in various phages and explored as antibiotic alternatives. Here, we isolated a phiKZ-like phage, Churi, which encodes 364 open reading frames. We examined 15 early-expressed phage proteins for their ability to inhibit bacterial growth, and found that gp335, closely related to phiKZ-gp14, exhibits antibacterial activity. Similar to phiKZ-gp14, recently shown to form a complex with the P. aeruginosa ribosome, we predict experimentally that gp335 interacts with ribosomal proteins, suggesting its involvement in protein translation. GFP-tagged gp335 clusters around the phage nucleus as early as 15 minutes post-infection and remains associated with it throughout the infection, suggesting its role in protein expression in the cell cytoplasm. CRISPR-Cas13-mediated deletion of gp355 reveals that the mutant phage has a prolonged latent period. Altogether, we demonstrate that gp335 is an antibacterial protein of nucleus-forming phages that associates with the ribosomes at the phage nucleus.

molecular biology↗

bioRxiv2024

Asesino: a nucleus-forming phage that lacks PhuZ

As nucleus-forming phages become better characterized, understanding their unifying similarities and unique differences will help us understand how they occupy varied niches and infect diverse hosts. All identified nucleus-forming phages fall within the proposed Chimalliviridae family and share a core genome of 68 unique genes including chimallin, the major nuclear shell protein. A well-studied but non-essential protein encoded by many nucleus-forming phages is PhuZ, a tubulin homolog which aids in capsid migration, nucleus rotation, and nucleus positioning. One clade that represents 24% of all currently known chimalliviruses lacks a PhuZ homolog. Here we show that Erwinia phage Asesino, one member of this PhuZ-less clade, shares a common overall replication mechanism with other characterized nucleus-forming phages despite lacking PhuZ. We show that Asesino replicates via a phage nucleus that encloses phage DNA and partitions proteins in the nuclear compartment and cytoplasm in a manner similar to previously characterized nucleus-forming phages. Consistent with a lack of PhuZ, however, we did not observe active positioning or rotation of the phage nucleus within infected cells. These data show that some nucleus-forming phages have evolved to replicate efficiently without PhuZ, providing an example of a unique variation in the nucleus-based replication pathway.

microbiology↗

bioRxiv2024

An essential and highly selective protein import pathway encoded by nucleus-forming phage

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts. Significance StatementThe phage nucleus is an enclosed replication compartment built by Chimalliviridae phages that, similar to the eukaryotic nucleus, separates transcription from translation and selectively imports certain proteins. This allows the phage to concentrate proteins required for DNA replication and transcription while excluding DNA-targeting host defense proteins. However, the mechanism of selective trafficking into the phage nucleus is currently unknown. Here we determine the region of a phage nuclear protein that targets it for nuclear import and identify a conserved, essential nuclear shell-associated protein that plays a key role in this process. This work provides the first mechanistic model of selective import into the phage nucleus.

microbiology↗

bioRxiv2024

Hachiman is a genome integrity sensor

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman comprises a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact dsDNA. When the HamAB complex detects DNA damage, HamB helicase activity liberates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating phantom cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and eukaryotic enzymes suggest this bacterial immune system has been repurposed for diverse functions across all domains of life.

biochemistry↗

bioRxiv2024

Architecture and infection-sensing mechanism of the bacterial PARIS defense system

Bacteria and the viruses that infect them (bacteriophages or phages) are engaged in an evolutionary arms race that has resulted in the development of hundreds of bacterial defense systems and myriad phage-encoded counterdefenses1-5. While the mechanisms of many bacterial defense systems are known1, how these systems avoid toxicity outside infection yet activate quickly upon sensing phage infection is less well understood. Here, we show that the bacterial Phage Anti-Restriction-Induced System (PARIS) operates as a toxin-antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefense protein Ocr. Using cryoelectron microscopy (cryoEM), we show that AriA is structurally similar to dimeric SMC-family ATPases but assembles into a distinctive homohexameric complex through two distinct oligomerization interfaces. In the absence of infection, the AriA hexamer binds up to three monomers of AriB, maintaining them in an inactive state. Ocr binding to the AriA-AriB complex triggers rearrangement of the AriA hexamer, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease whose activation arrests cell growth and inhibits phage propagation by globally inhibiting protein translation. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defense system that evolved in response to a phage counterdefense protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor.

biochemistry↗

bioRxiv2023

A mobile intron facilitates interference competition between co-infecting viruses

Mobile introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. Here we studied a mobile intron in bacteriophage {Phi}PA3 and found its homing endonuclease gp210 contributes to viral competition by interfering with the virogenesis of co-infecting phage {Phi}KZ. We show that gp210 targets a specific sequence in its competitor {Phi}KZ, preventing the assembly of progeny viruses. This work reports the first demonstration of how a mobile intron can be deployed to engage in interference competition and provide a reproductive advantage. Given the ubiquity of introns, this selective advantage likely has widespread evolutionary implications in nature.

microbiology↗

bioRxiv2023

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection

Large-genome bacteriophages (jumbo phages) of the Chimalliviridae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.

microbiology↗

bioRxiv2023

Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses1-3. Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA4-10. Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae, but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART11 in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

cell biology↗

bioRxiv2023

Identification of the bacteriophage nucleus protein interaction network

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against DNA-targeting immune factors. By segregating the genome from the host cytoplasm, however, the "phage nucleus" introduces the need to specifically transport mRNA and proteins through the nuclear shell, and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggests that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging, and may also participate in mRNA and/or protein transport.

biochemistry↗

bioRxiv2023

Jumbo phages are active against extensively-drug-resistant eyedrop-associated Pseudomonas aeruginosa infections.

Antibiotic resistant bacteria present an emerging challenge to human health as the pressure instituted on the microbial world through the liberal use of antibiotics has resulted in their emergence across the globe. Those bacteria that acquire mobile genetic elements such as plasmids are especially concerning because those plasmids may be shared readily with other microbes that then can also become antibiotic resistant. Serious infections have recently been related to contamination of preservative-free eyedrops with extensively drug resistant (XDR) isolates of Pseudomonas aeruginosa, already resulting in three deaths. These drug-resistant isolates cannot be managed with most conventional antibiotics. We sought to identify alternatives to conventional antibiotics for lysis of these XDR isolates, and identified multiple bacteriophages (viruses that attack bacteria) that killed them efficiently. We found both jumbo phages (>200kb in genome size) and non-jumbo phages that were active against these isolates, the former killing more efficiently. Jumbo phages effectively killed the 3 separate XDR P. aeruginosa isolates both on solid and liquid medium. Given the ongoing nature of the XDR P. aeruginosa eyedrop outbreak, the identification of phages active against them provides physicians with several novel potential alternatives for treatment.

microbiology↗