Architecture and activation mechanism of the bacterial PARIS defence system.

Bacteria and their viruses (bacteriophages or phages) are engaged in an intense evolutionary arms race1-5. While the mechanisms of many bacterial antiphage defence systems are known1, how these systems avoid toxicity outside infection yet activate quickly after 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 counterdefence protein Ocr. Using cryo-electron microscopy, we show that AriA is related to SMC-family ATPases but assembles into a distinctive homohexameric complex through two oligomerization interfaces. In uninfected cells, the AriA hexamer binds to up to three monomers of AriB, maintaining them in an inactive state. After Ocr binding, the AriA hexamer undergoes a structural rearrangement, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease, the activation of which arrests cell growth and inhibits phage propagation by globally inhibiting protein translation through specific cleavage of a lysine tRNA. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defence system triggered by a phage counterdefence protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor.

Prokaryotic viperins produce diverse antiviral molecules.

Viperin is an interferon-induced cellular protein that is conserved in animals1. It has previously been shown to inhibit the replication of multiple viruses by producing the ribonucleotide 3'-deoxy-3',4'-didehydro (ddh)-cytidine triphosphate (ddhCTP), which acts as a chain terminator for viral RNA polymerase2. Here we show that eukaryotic viperin originated from a clade of bacterial and archaeal proteins that protect against phage infection. Prokaryotic viperins produce a set of modified ribonucleotides that include ddhCTP, ddh-guanosine triphosphate (ddhGTP) and ddh-uridine triphosphate (ddhUTP). We further show that prokaryotic viperins protect against T7 phage infection by inhibiting viral polymerase-dependent transcription, suggesting that it has an antiviral mechanism of action similar to that of animal viperin. Our results reveal a class of potential natural antiviral compounds produced by bacterial immune systems.

Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase.

Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2'-deoxyguanosine-5'-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host "arms race." We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt-Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt-Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.

Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope.

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15-gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

Novel Host Recognition Mechanism of the K1 Capsule-Specific Phage of Escherichia coli: Capsular Polysaccharide as the First Receptor and Lipopolysaccharide as the Secondary Receptor.

K1 capsule-specific phages of Escherichia coli have been reported in recent years, but the molecular mechanism involved in host recognition of these phages remains unknown. In this study, the interactions between PNJ1809-36, a new K1-specific phage, and its host bacterium, E. coli DE058, were investigated. A transposon mutation library was used to screen for receptor-related genes. Gene deletion, lysis curve determination, plaque formation test, adsorption assay, and inhibition assay of phage by lipopolysaccharide (LPS) showed that capsular polysaccharide (CPS) was the first receptor for the initial adsorption of PNJ1809-36 to E. coli DE058 and that LPS was a secondary receptor for the irreversible binding of the phage. The penultimate galactose in the outer core was identified as the specific binding region on LPS. Through antibody blocking assay, fluorescence labeling and high-performance gel permeation chromatography, the tail protein ORF261 of phage PNJ1809-36 was identified as the receptor-binding protein on CPS. Given these findings, we propose a model for the recognition process of phage PNJ1809-36 on E. coli DE058: the phage PNJ1809-36 tail protein ORF261 recognizes and adsorbs to the K1 capsule, and then the K1 capsule is partially degraded, exposing the active site of LPS which is recognized by phage PNJ1809-36. This model provides insight into the molecular mechanisms between K1-specific phages and their host bacteria. IMPORTANCE It has been speculated that CPS is the main receptor of K1-specific phages belonging to Siphoviridae. In recent years, a new type of K1-specific phage belonging to Myoviridae has been reported, but its host recognition mechanisms remain unknown. Here, we studied the interactions between PNJ1809-36, a new type of K1 phage, and its host bacterium, E. coli DE058. Our research showed that the phage initially adsorbed to the K1 capsule mediated by ORF261 and then bound to the penultimate galactose of LPS to begin the infection process.

Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence.

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction-modification systems, is also active against BREX. In contrast to the wild-type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R-M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.

Evaluation of an E. coli Cell Extract Prepared by Lysozyme-Assisted Sonication via Gene Expression, Phage Assembly and Proteomics.

Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols and optimized reaction conditions have been established for the production of cell-free gene expression systems. One of the crucial steps during the preparation of cell extract-based expression systems is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here we evaluate the utility of an E. coli cell extract, which was prepared using a combination of lysozyme incubation and a gentle sonication step. As quality measure, we demonstrate the cell-free expression of YFP at concentrations up to 0.6 mg/mL. In addition, we produced and assembled T7 bacteriophages up to a titer of 108  PFU/mL. State-of-the-art quantitative proteomics was used to compare the produced extracts with each other and with a commercial extract. The differences in protein composition were surprisingly small between lysozyme-assisted sonication (LAS) extracts, but we observed an increase in the release of DNA-binding proteins for increasing numbers of sonication cycles. Proteins taking part in carbohydrate metabolism, glycolysis, amino acid and nucleotide related pathways were found to be more abundant in the LAS extract, while proteins related to RNA modification and processing, DNA modification and replication, transcription regulation, initiation, termination and the TCA cycle were found enriched in the commercial extract.

Combined Solution and Crystal Methods Reveal the Electrostatic Tethers That Provide a Flexible Platform for Replication Activities in the Bacteriophage T7 Replisome.

Recent structural studies of the bacteriophage T7 DNA replication system have shed light on how multiple proteins assemble to copy two antiparallel DNA strands. In T7, acidic C-terminal tails of both the primase-helicase and single-stranded DNA binding protein bind to two basic patches on the DNA polymerase to aid in replisome assembly, processivity, and coordinated DNA synthesis. Although these electrostatic interactions are essential for DNA replication, the molecular details for how these tails bind the polymerase are unknown. We have determined an X-ray crystal structure of the T7 DNA polymerase bound to both a primer/template DNA and a peptide that mimics the C-terminal tail of the primase-helicase. The structure reveals that the essential C-terminal phenylalanine of the tail binds to a hydrophobic pocket that is surrounded by positive charge on the surface of the polymerase. We show that alterations of polymerase residues that engage the tail lead to defects in viral replication. In the structure, we also observe dTTP bound in the exonuclease active site and stacked against tryptophan 160. Using both primer/extension assays and high-throughput sequencing, we show how mutations in the exonuclease active site lead to defects in mismatch repair and an increase in the level of mutagenesis of the T7 genome. Finally, using small-angle X-ray scattering, we provide the first solution structures of a complex between the single-stranded DNA binding protein and the DNA polymerase and show how a single-stranded DNA binding protein dimer engages both one and two copies of DNA polymerase.

Colorimetric detection of Escherichia coli using engineered bacteriophage and an affinity reporter system.

Reporter phage systems have emerged as a promising technology for the detection of bacteria in foods and water. However, the sensitivity of these assays is often limited by the concentration of the expressed reporter as well as matrix interferences associated with the sample. In this study, bacteriophage T7 was engineered to overexpress mutated alkaline phosphatase fused to a carbohydrate-binding module (ALP*-CBM) following infection of E. coli to enable colorimetric detection in a model system. Magnetic cellulose particles were employed to separate and concentrate the overexpressed ALP*-CBM in bacterial lysate. Infection of E. coli with the engineered phage resulted in a limit of quantitation of 1.2 × 105 CFU, equating to 1.2 × 103 CFU/mL in 3.5 h when using a colorimetric assay and 100 mL sample volume. When employing an enrichment step, < 101 CFU/mL could be visually detected from a 100 mL sample volume within 8 h. These results suggest that affinity tag modified enzymes coupled with a material support can provide a simple and effective means to improve signal sensitivity of phage-based assays. Graphical abstract.

Phenotypic consequences of RNA polymerase dysregulation in Escherichia coli.

Many bacterial adaptive responses to changes in growth conditions due to biotic and abiotic factors involve reprogramming of gene expression at the transcription level. The bacterial RNA polymerase (RNAP), which catalyzes transcription, can thus be considered as the major mediator of cellular adaptive strategies. But how do bacteria respond if a stress factor directly compromises the activity of the RNAP? We used a phage-derived small protein to specifically perturb bacterial RNAP activity in exponentially growing Escherichia coli. Using cytological profiling, tracking RNAP behavior at single-molecule level and transcriptome analysis, we reveal that adaptation to conditions that directly perturb bacterial RNAP performance can result in a biphasic growth behavior and thereby confer the 'adapted' bacterial cells an enhanced ability to tolerate diverse antibacterial stresses. The results imply that while synthetic transcriptional rewiring may confer bacteria with the intended desirable properties, such approaches may also collaterally allow them to acquire undesirable traits.

Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome.

DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

Direct visualization of single virus restoration after damage in real time.

We use the nano-dissection capabilities of atomic force microscopy to induce structural alterations on individual virus capsids in liquid milieu. We fracture the protein shells either with single nanoindentations or by increasing the tip-sample interaction force in amplitude modulation dynamic mode. The normal behavior is that these cracks persist in time. However, in very rare occasions they self-recuperate to retrieve apparently unaltered virus particles. In this work, we show the topographical evolution of three of these exceptional events occurring in T7 bacteriophage capsids. Our data show that single nanoindentation produces a local recoverable fracture that corresponds to the deepening of a capsomer. In contrast, imaging in dynamic mode induced cracks that separate the virus morphological subunits. In both cases, the breakage patterns follow intratrimeric loci.

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery.

Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading- and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.

Synthetic poliovirus and other designer viruses: what have we learned from them?

Owing to known genome sequences, modern strategies of DNA synthesis have made it possible to recreate in principle all known viruses independent of natural templates. We describe the first synthesis of a virus (poliovirus) in 2002 that was accomplished outside living cells. We comment on the reaction of laypeople and scientists to the work, which shaped the response to de novo syntheses of other viruses. We discuss those viruses that have been synthesized since 2002, among them viruses whose precise genome sequence had to be established by painstakingly stitching together pieces of sequence information, and viruses involved in zoonosis. Synthesizing viral genomes provides a powerful tool for studying gene function and the pathogenic potential of these organisms. It also allows modification of viral genomes to an extent hitherto unthinkable. Recoding of poliovirus and influenza virus to develop new vaccine candidates and refactoring the phage T7 DNA genome are discussed as examples.

DNA buckling in bacteriophage cavities as a mechanism to aid virus assembly.

While relatively simple biologically, bacteriophages are sophisticated biochemical machines that execute a precise sequence of events during virus assembly, DNA packaging, and ejection. These stages of the viral life cycle require intricate coordination of viral components whose structures are being revealed by single molecule experiments and high resolution (cryo-electron microscopy) reconstructions. For example, during packaging, bacteriophages employ some of the strongest known molecular motors to package DNA against increasing pressure within the viral capsid shell. Located upstream of the motor is an elaborate portal system through which DNA is threaded. A high resolution reconstruction of the portal system for bacteriophage ϕ29 reveals that DNA buckles inside a small cavity under large compressive forces. In this study, we demonstrate that DNA can also buckle in other bacteriophages including T7 and P22. Using a computational rod model for DNA, we demonstrate that a DNA buckle can initiate and grow within the small confines of a cavity under biologically-attainable force levels. The forces of DNA-cavity contact and DNA-DNA electrostatic repulsion ultimately limit cavity filling. Despite conforming to very different cavity geometries, the buckled DNA within T7 and P22 exhibits near equal volumetric energy density (∼1kT/nm(3)) and energetic cost of packaging (∼22kT). We hypothesize that a DNA buckle creates large forces on the cavity interior to signal the conformational changes to end packaging. In addition, a DNA buckle may help retain the genome prior to tail assembly through significantly increased contact area with the portal.

Lysis delay and burst shrinkage of coliphage T7 by deletion of terminator Tφ reversed by deletion of early genes.

Bacteriophage T7 terminator Tϕ is a class I intrinsic terminator coding for an RNA hairpin structure immediately followed by oligo(U), which has been extensively studied in terms of its transcription termination mechanism, but little is known about its physiological or regulatory functions. In this study, using a T7 mutant phage, where a 31-bp segment of Tϕ was deleted from the genome, we discovered that deletion of Tϕ from T7 reduces the phage burst size but delays lysis timing, both of which are disadvantageous for the phage. The burst downsizing could directly result from Tϕ deletion-caused upregulation of gene 17.5, coding for holin, among other Tϕ downstream genes, because infection of gp17.5-overproducing Escherichia coli by wild-type T7 phage showed similar burst downsizing. However, the lysis delay was not associated with cellular levels of holin or lysozyme or with rates of phage adsorption. Instead, when allowed to evolve spontaneously in five independent adaptation experiments, the Tϕ-lacking mutant phage, after 27 or 29 passages, recovered both burst size and lysis time reproducibly by deleting early genes 0.5, 0.6, and 0.7 of class I, among other mutations. Deletion of genes 0.5 to 0.7 from the Tϕ-lacking mutant phage decreased expression of several Tϕ downstream genes to levels similar to that of the wild-type phage. Accordingly, phage T7 lysis timing is associated with cellular levels of Tϕ downstream gene products. This suggests the involvement of unknown factor(s) besides the known lysis proteins, lysozyme and holin, and that Tϕ plays a role of optimizing burst size and lysis time during T7 infection. IMPORTANCE Bacteriophages are bacterium-infecting viruses. After producing numerous progenies inside bacteria, phages lyse bacteria using their lysis protein(s) to get out and start a new infection cycle. Normally, lysis is tightly controlled to ensure phage progenies are maximally produced and released at an optimal time. Here, we have discovered that phage T7, besides employing its known lysis proteins, additionally uses its transcription terminator Tϕ to guarantee the optimal lysis of the E. coli host. Tϕ, positioned in the middle of the T7 genome, must be inactivated at least partially to allow for transcription-driven translocation of T7 DNA into hosts and expression of Tϕ downstream but promoter-lacking genes. What role is played by Tϕ before inactivation? Without Tϕ, not only was lysis time delayed but also the number of progenies was reduced in this study. Furthermore, T7 can overcome Tϕ deletion by further deleting some genes, highlighting that a phage has multiple strategies for optimizing lysis.

Front propagation speeds of T7 virus mutants.

We propose a new reaction-diffusion model with an eclipse time to study the spread of viruses on bacterial populations. This new model is both biologically and physically sound, unlike previous ones. We determine important parameter values from experimental data, such as the one-step growth. We verify the proposed model by comparing theoretical and experimental data of the front propagation speed for several T7 virus strains.

Coordinating DNA replication by means of priming loop and differential synthesis rate.

Genomic DNA is replicated by two DNA polymerase molecules, one of which works in close association with the helicase to copy the leading-strand template in a continuous manner while the second copies the already unwound lagging-strand template in a discontinuous manner through the synthesis of Okazaki fragments. Considering that the lagging-strand polymerase has to recycle after the completion of every Okazaki fragment through the slow steps of primer synthesis and hand-off to the polymerase, it is not understood how the two strands are synthesized with the same net rate. Here we show, using the T7 replication proteins, that RNA primers are made 'on the fly' during ongoing DNA synthesis and that the leading-strand T7 replisome does not pause during primer synthesis, contrary to previous reports. Instead, the leading-strand polymerase remains limited by the speed of the helicase; it therefore synthesizes DNA more slowly than the lagging-strand polymerase. We show that the primase-helicase T7 gp4 maintains contact with the priming sequence during ongoing DNA synthesis; the nascent lagging-strand template therefore organizes into a priming loop that keeps the primer in physical proximity to the replication complex. Our findings provide three synergistic mechanisms of coordination: first, primers are made concomitantly with DNA synthesis; second, the priming loop ensures efficient primer use and hand-off to the polymerase; and third, the lagging-strand polymerase copies DNA faster, which allows it to keep up with leading-strand DNA synthesis overall.

Large terminase conformational change induced by connector binding in bacteriophage T7.

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.

Electrospun water-soluble polymer nanofibers for the dehydration and storage of sensitive reagents.

The ability to preserve and deliver reagents remains an obstacle for the successful deployment of self-contained diagnostic microdevices. In this study we investigated the ability of bacteriophage T7 to be encapsulated and preserved in water soluble nanofibers. The bacteriophage T7 was added to mixtures of polyvinylpyrrolidone and water and electrospun onto a grounded plate. Trehalose and magnesium salts were added to the mixtures to determine their effect on the infectivity of the bacteriophage following electrospinning and during storage. The loss of T7 infectivity was determined immediately following electrospinning and during storage using agar overlay plating and plaque counting. The results indicate that the addition of magnesium salts protects the bacteriophage during the relatively violent and high voltage electrospinning process, but is not as effective as a protectant during storage of the dried T7. Conversely, the addition of trehalose into the electrospinning mix has little effect on the electrospinning, but a more significant role as a protectant during storage.