Bacteriophage HK97 Research Articles

Structural basis for DNA recognition by a viral genome-packaging machine

DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.

Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny.

A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some β-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.IMPORTANCEThe lysis-lysogeny decision is made by most bacterial viruses (bacteriophages, phages), determining whether to kill their host or go dormant within it. With over half of the bacteria containing phages waiting to wake, this is one of the most important behaviors in all of biology. These phages are also considered unusable for therapy because of this behavior. In this paper, we show that many antibiotics bias this behavior to "wake" the dormant phages, forcing them to kill their host, but some also prevent dormancy in the first place. These will be important tools to study this critical decision point and may enable the therapeutic use of these phages.

The Parasporal Body of Bacillus thuringiensis subsp. israelensis: A Unique Phage Capsid-Associated Prokaryotic Insecticidal Organelle.

The three most important commercial bacterial insecticides are all derived from subspecies of Bacillus thuringiensis (Bt). Specifically, Bt subsp. kurstaki (Btk) and Bt subsp. aizawai (Bta) are used to control larval lepidopteran pests. The third, Bt subsp. israelensis (Bti), is primarily used to control mosquito and blackfly larvae. All three subspecies produce a parasporal body (PB) during sporulation. The PB is composed of insecticidal proteins that damage the midgut epithelium, initiating a complex process that results in the death of the insect. Among these three subspecies of Bt, Bti is unique as it produces the most complex PB consisting of three compartments. Each compartment is bound by a multilaminar fibrous matrix (MFM). Two compartments contain one protein each, Cry11Aa1 and Cyt1Aa1, while the third contains two, Cry4Aa1/Cry4Ba1. Each compartment is packaged independently before coalescing into the mature spherical PB held together by additional layers of the MFM. This distinctive packaging process is unparalleled among known bacterial organelles, although the underlying molecular biology is yet to be determined. Here, we present structural and molecular evidence that the MFM has a hexagonal pattern to which Bti proteins Bt152 and Bt075 bind. Bt152 binds to a defined spot on the MFM during the development of each compartment, yet its function remains unknown. Bt075 appears to be derived from a bacteriophage major capsid protein (MCP), and though its sequence has markedly diverged, it shares striking 3-D structural similarity to the Escherichia coli phage HK97 Head 1 capsid protein. Both proteins are encoded on Bti's pBtoxis plasmid. Additionally, we have also identified a six-amino acid motif that appears to be part of a novel molecular process responsible for targeting the Cry and Cyt proteins to their cytoplasmic compartments. This paper describes several previously unknown features of the Bti organelle, representing a first step to understanding the biology of a unique process of sorting and packaging of proteins into PBs. The insights from this research suggest a potential for future applications in nanotechnology.

A symmetry mismatch unraveled: How phage HK97 scaffold flexibly accommodates a 12-fold pore at a 5-fold viral capsid vertex.

Tailed bacteriophages and herpesviruses use a transient scaffold to assemble icosahedral capsids with hexameric capsomers on the faces and pentameric capsomers at all but one vertex where a 12-fold portal is thought to nucleate the assembly. How does the scaffold orchestrate this step? We have determined the portal vertex structure of the bacteriophage HK97 procapsid, where the scaffold is a domain of the major capsid protein. The scaffold forms rigid helix-turn-strand structures on the interior surfaces of all capsomers and is further stabilized around the portal, forming trimeric coiled-coil towers, two per surrounding capsomer. These 10 towers bind identically to 10 of 12 portal subunits, adopting a pseudo-12-fold organization that explains how the symmetry mismatch is managed at this early step.

Insights into a viral motor: the structure of the HK97 packaging termination assembly.

Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of individual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.

Isolation and Characterization of Phages That Bypass the Requirement for RNA-Mediated Antitermination.

The rpoCY75N mutation in the zinc-binding domain of the β' subunit of Escherichia coli RNA polymerase blocks the RNA-based mechanism of transcription antitermination utilized by bacteriophage HK022. Mutant phages that overcome the block imposed by the rpoCY75N mutation are described. These phages, designated "orc" (overcomes rpoC), carry mutations that create new promoters. Promoter activity was assessed by cloning the respective regions from the wild-type and orc phages into a promoterless lacZ reporter vector. Reporter assays showed that the sequence originating from orc phages had significant promoter activity when compared with the equivalent sequence cloned from the parental phage. The newly created promoters facilitate the expression of phage genes that are essential for growth on the rpoCY75N strain by bypassing transcription terminators. The small plaque phenotype of orc phages, when grown on the mutant host, suggests that suppression of the rpoCY75N mutation is incomplete.

Engineering the HK97 virus-like particle as a nanoplatform for biotechnology applications.

The research described here looks at the development of virus-like particles (VLPs) derived from bacteriophage HK97 as versatile scaffolds for bionanomaterials construction. Based on molecular models, the Prohead I HK97 VLP was engineered to allow attachment of small molecules to the interior by introducing a reactive cysteine into the genetic sequence of the HK97 GP5 protein that self assembles to form the VLP structure. In addition, methods for entrapping large protein macromolecules were evaluated and found to produce high encapsulation numbers of green fluorescent proteins (GFP) in the internal space of the HK97 VLP. A method for modular modification of the external surface was engineered by constructing a plasmid allowing the addition of peptide sequences to the C-terminus of the GP5 protein, which was validated by appending the sortase recognition peptide sequence, LPETG, to the C-terminus of GP5 and showing the attachment of a polyglycine-GFP to the HK97 VLP through sortase mediated ligation. To demonstrate the potential for advanced applications, an HK97 VLP covalently labeled on the interior surface with fluorescein and containing an externally displayed integrin binding peptide sequence (RGD) was evaluated and found to be preferentially localized at C2C12 cells relative to the HK97 VLP lacking the RGD peptide. Together, these results support the potential of the HK97 VLP as a versatile nanoparticle platform that can be modified internally and externally in a modular fashion for the purpose of programming the VLP for desired applications.

Structural basis of transcriptional regulation by a nascent RNA element, HK022 putRNA

Transcription, in which RNA polymerases (RNAPs) produce RNA from DNA, is the first step of gene expression. As such, it is highly regulated either by trans-elements like protein factors and/or by cis-elements like specific sequences on the DNA. Lambdoid phage HK022 contains a cis-element, put, which suppresses pausing and termination during transcription of the early phage genes. The putRNA transcript solely performs the anti-pausing/termination activities by interacting directly with the E.coli RNAP elongation complex (EC) by an unknown structural mechanism. In this study, we reconstituted putRNA-associated ECs and determined the structures using cryo-electron microscopy. The determined structures of putRNA-associated EC, putRNA-absent EC, and σ70-bound EC suggest that the putRNA interaction with the EC counteracts swiveling, a conformational change previously identified to promote pausing and σ70 might modulate putRNA folding via σ70-dependent pausing during elongation.

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system.

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

Complete Genome Sequence of Stenotrophomonas maltophilia Siphophage Silvanus.

ABSTRACTStenotrophomonas maltophilia is an opportunistic Gram-negative bacterium capable of causing respiratory infections. S. maltophilia siphophage Silvanus was isolated, and its 45,678-bp genome is not closely related to known phages based on whole-genome comparative genomics analysis. It is predicted to use cos-type packaging due to the similarity of its large terminase subunit to that of phage HK97.

High Resolution Structure of the Mature Capsid of Ralstonia solanacearum Bacteriophage ϕRSA1 by Cryo-Electron Microscopy.

The ϕRSA1 bacteriophage has been isolated from Ralstonia solanacearum, a gram negative bacteria having a significant economic impact on many important crops. We solved the three-dimensional structure of the ϕRSA1 mature capsid to 3.9 Å resolution by cryo-electron microscopy. The capsid shell, that contains the 39 kbp of dsDNA genome, has an icosahedral symmetry characterized by an unusual triangulation number of T = 7, dextro. The ϕRSA1 capsid is composed solely of the polymerization of the major capsid protein, gp8, which exhibits the typical “Johnson” fold first characterized in E. coli bacteriophage HK97. As opposed to the latter, the ϕRSA1 mature capsid is not stabilized by covalent crosslinking between its subunits, nor by the addition of a decoration protein. We further describe the molecular interactions occurring between the subunits of the ϕRSA1 capsid and their relationships with the other known bacteriophages.

Temperate phage-antibiotic synergy eradicates bacteria through depletion of lysogens.

There is renewed interest in bacterial viruses (phages) as alternatives to antibiotics. All phage treatments to date have used virulent phages rather than temperate ones, as these can integrate into the genome of the bacterial host and lie dormant. However, temperate phages are abundant and easier to isolate. To make use of these entities, we leverage stressors known to awaken these dormant, integrated phages. Co-administration of the temperate phage HK97 with sub-inhibitory concentrations of the antibiotic ciprofloxacin results in bacterial eradication (≥8 log reduction) invitro. This synergy is mechanistically distinct from phage-antibiotic-synergy described for virulent phages. Instead, the antibiotic specifically selects against bacteria in which the phage has integrated. As the interaction between temperate phages and stressors such as ciprofloxacin are known to be widespread, this approach may be broadly applicable and enable the use of temperate phages to combat bacterial infections.

Temperate Phage-Antibiotic Synergy Eradicates Bacteria Through Depletion of Lysogens

Mounting stress from multidrug-resistant bacteria has renewed interest in bacteriophage (bacterial virus, phage) therapy as an alternative to antibiotics. While virulent phages quickly infect and kill their host through a lytic lifecycle, temperate phages can stably integrate into the host’s genome until exposed to stressors that can cause a switch to a lytic cycle. Dormant phages can protect their host, known as lysogen, from further phage infections. In vitro this results in rapid host regrowth, which would likely complicate treatment. All case studies and clinical trials to date use virulent phages. However, temperate phages are far easier to find and more abundant in (and on) humans, playing a vital role in vivo. To make use of these abundant entities, we propose a synergy using stressors such as antibiotics, which are known to awaken dormant phages. Here we demonstrate that co-administration of the temperate phage HK97 with sub-inhibitory concentrations of a fluoroquinolone antibiotic, ciprofloxacin, results in bacterial eradication (≥8 log reduction) in vitro. This synergy is up to 4.5 x105 fold greater than the multiplicative effects of the phage and antibiotic alone. Furthermore, there is no large increase in final phage titre, latency period or burst size – as such, this synergy is distinct from traditional phage-antibiotic-synergy described with virulent phages. We demonstrate the presence of the antibiotic prevents lysogen colony formation over time, the biggest hurdle in the current use of temperate phages in therapy. As the interaction between temperate phages and stressors such as ciprofloxacin are known to be widespread, the findings from this work may allow for the use of these phages in combination with antibiotics for the treatment of antimicrobial-resistant infections.

HK97 gp74 Possesses an α-Helical Insertion in the ββα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities.

The last gene in the genome of the bacteriophage HK97 encodes gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and they adopt a ββα structure. gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage cos site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here, we present nuclear magnetic resonance (NMR) studies demonstrating that gp74 contains an α-helical insertion in the Ω-loop, which connects the two β-strands of the ββα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ββα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the cos site and contribute to the phage replication cycle.IMPORTANCE This study demonstrates that residues outside the canonical ββα fold, namely, the Ω-loop α-helical insert and a disordered C-terminal tail, regulate the activity of the HNH endonuclease gp74. The increased divalent metal ion binding when the Ω-loop insert is removed compared to reduced cos site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.

Phylogenetic Grouping of Human Ocular Escherichia coli Based on Whole-Genome Sequence Analysis.

Escherichia coli is a predominant bacterium in the intestinal tracts of animals. Phylogenetically, strains have been classified into seven phylogroups, A, B1, B2, C, D, E, and F. Pathogenic strains have been categorized into several pathotypes such as Enteropathogenic (EPEC), Enterotoxigenic (ETEC), Enteroinvasive (EIEC), Enteroaggregative (EAEC), Diffusely adherent (DAEC), Uropathogenic (UPEC), Shiga-toxin producing (STEC) or Enterohemorrhagic (EHEC) and Extra-intestinal pathogenic E. coli (ExPEC). E. coli also survives as a commensal on the ocular surface. However, under conditions of trauma and immune-compromised states, E. coli causes conjunctivitis, keratitis, endopthalmitis, dacyrocystitis, etc. The phylogenetic affiliation and the pathotype status of these ocular E. coli strains is not known. For this purpose, the whole-genome sequencing of the 10 ocular E. coli strains was accomplished. Based on whole-genome SNP variation, the ocular E. coli strains were assigned to phylogenetic groups A (two isolates), B2 (seven isolates), and C (one isolate). Furthermore, results indicated that ocular E. coli originated either from feces (enteropathogenic and enterotoxigenic), urine (uropathogenic), or from extra-intestinal sources (extra-intestinal pathogenic). A high concordance was observed between the presence of AMR (Antimicrobial Resistance) genes and antibiotic resistance in the ocular E. coli strains. Furthermore, several virulent genes (fimB to fimI, papB to papX, etc.) and prophages (Enterobacteria phage HK97, Enterobacteria phage P1, Escherichia phage D108 etc.) were unique to ocular E. coli. This is the first report on a whole-genome analysis of ocular E. coli strains.

Capsids and Portals Influence Each Other’s Conformation During Assembly and Maturation

The portal proteins of tailed bacteriophage and Herpesvirus capsids form dodecameric rings that occupy one capsid vertex and are incorporated during the assembly of capsid precursors called procapsids or proheads. Portals are essential and serve as the pore for DNA transit and the site of tail attachment; however, bacteriophage HK97 capsid proteins assemble efficiently without a portal when expressed from plasmids. Following portal co-expression, portals were incorporated into about half of the proheads that were made. In the absence of active capsid maturation protease, uncleaved proheads formed dimers, trimers, and tetramers of proheads during purification, but only if they had portals. These appeared bound to membrane-like fragments by their portals and could be disaggregated by detergents, supporting a role for membranes in their formation and in capsid assembly. The precursors to prohead oligomers were detected in cell extracts. These were able to bind to Octyl-Sepharose and could be released by detergent, while uncleaved proheads without portal or cleaved proheads with portal did not bind. Our results document a discrete change in the HK97 portal’s hydrophobicity induced by cleavage of the procapsid shell in which it is embedded. Additionally, we detected an increase in the rate of expansion induced by the presence of a portal complex in cleaved HK97 proheads. These results suggest that portals and capsids influence each other’s conformation during assembly. The formation of prohead oligomers also provides a rapid and sensitive assay for identification and analysis of portal incorporation mutants.

Reconstruction of Stochastic 3D Signals With Symmetric Statistics From 2D Projection Images Motivated by Cryo-Electron Microscopy.

Cryo-electron microscopy provides 2D projection images of the 3D electron scattering intensity of many instances of the particle under study (e.g., a virus). Both symmetry (rotational point groups) and heterogeneity are important aspects of biological particles and both aspects can be combined by describing the electron scattering intensity of the particle as a stochastic process with a symmetric probability law and, therefore, symmetric moments. A maximum likelihood estimator implemented by an expectation-maximization algorithm is described, which estimates the unknown statistics of the electron scattering intensity stochastic process from the images of instances of the particle. The algorithm is demonstrated on the bacteriophage HK97 and the virus [Formula: see text]. The results are contrasted with the existing algorithms, which assume that each instance of the particle has the symmetry rather than the less restrictive assumption that the probability law has the symmetry.

Strategies for purification of the bacteriophage HK97 small and large terminase subunits that yield pure and homogeneous samples that are functional

Packaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease gp74 in the bacteriophage HK97. Gp74 enhances the activity of terminase-mediated digestion of the cohesive (cos) site that connects individual genomes in phage concatemeric DNA, a pre-requisite to DNA packaging, and this enhancement requires an intact HNH motif in gp74. Testing of whether gp74 alters the terminase DNA binding or enzymatic activities requires obtaining isolated samples of pure TerS and TerL, which has been challenging owing to the poor solubility of these proteins. To this end, we developed methods to obtain purified TerS and TerL proteins that are active. TerS is expressed solubly in E. coli as a fusion with SUMO, which can be removed during purification to yield a TerS nonamer (TerS9). Homogenous samples of a TerL monomer are also obtained, but the homogeneity of the sample depends on the solution conditions, as seen for other terminases. DNA binding, ATPase, and nuclease assays demonstrate that our preparations of TerS9 and TerL are functional, and that they also function with gp74. Purified TerS9 and TerL enable studies into the molecular basis by which gp74 regulates terminase activity in phage maturation.

PH-induced morphological changes of proteinaceous viral shells

Changes in environmental pH can induce morphological changes in empty proteinaceous shells of bacteriophages in vitro that are very similar to changes occurring in viral capsids in vivo after encapsidation of DNA. These changes in capsid shape and size cannot be explained with a simple elastic model alone. We propose a new theoretical framework that combines the elasticity of thin icosahedral shells with the pH dependence of capsid charge distribution. Minimization of the sum of elastic and electrostatic free energies leads to equilibrium shapes of viral shells that depend on a single elastic parameter and the detailed configuration of the imbedded protein charges. Based on the in vitro shell reconstructions of bacteriophage HK97 we elucidate the details of how the reversible transition between Prohead II and Expansion Intermediate II states of the HK97 procapsid is induced by pH changes, as well as some other features of the bacteriophage maturation.

Genome packaging within icosahedral capsids and large-scale segmentation in viral genomic sequences

The assembly and maturation of viruses with icosahedral capsids must be coordinated with icosahedral symmetry. The icosahedral symmetry imposes also the restrictions on the cooperative specific interactions between genomic RNA/DNA and coat proteins that should be reflected in quasi-regular segmentation of viral genomic sequences. Combining discrete direct and double Fourier transforms, we studied the quasi-regular large-scale segmentation in genomic sequences of different ssRNA, ssDNA, and dsDNA viruses. The particular representatives included satellite tobacco mosaic virus (STMV) and the strains of satellite tobacco necrosis virus (STNV), STNV-C, STNV-1, STNV-2, Escherichia phages MS2, ϕX174, α3, and HK97, and Simian virus 40. In all their genomes, we found the significant quasi-regular segmentation of genomic sequences related to the virion assembly and the genome packaging within icosahedral capsid. We also found good correspondence between our results and available cryo-electron microscopy data on capsid structures and genome packaging in these viruses. Fourier analysis of genomic sequences provides the additional insight into mechanisms of hierarchical genome packaging and may be used for verification of the concepts of 3-fold or 5-fold intermediates in virion assembly. The results of sequence analysis should be taken into account at the choice of models and data interpretation. They also may be helpful for the development of antiviral drugs.