Phage P22 Research Articles

Accurate model annotation of a near-atomic resolution cryo-EM map

Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo-EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.

Genetic and functional characterisation of the lactococcal P335 phage-host interactions

BackgroundDespite continuous research efforts, bacterio(phages) infecting Lactococcus lactis starter strains persist as a major threat to dairy fermentations. The lactococcal P335 phages, which are currently classified into four sub-groups (I-IV), are the second most frequently isolated phage group in an industrial dairy context.ResultsThe current work describes the isolation and comparative genomic analysis of 17 novel P335 group phages. Detailed analysis of the genomic region of P335 phages encoding the so-called “baseplate”, which includes the receptor binding protein (RBP) was combined with a functional characterization of the RBP of sub-group III and IV phages. Additionally, calcium-dependence assays revealed a specific requirement for calcium by sub-group IV phages while host range analysis highlighted a higher number of strains with CWPS type A (11 of 39 strains) are infected by the P335 phages assessed in this study than those with a C (five strains), B (three of 39 strains) or unknown (one of 39 strains) CWPS type.ConclusionsThese analyses revealed significant divergence among RBP sequences, apparently reflecting their unique interactions with the host and particularly for strains with a type A CWPS. The implications of the genomic architecture of lactococcal P335 phages on serving as a general model for Siphoviridae phages are discussed.

Visualizing Infection Initiation of Bacteriophage P22 by Cryo-Electron Tomography

For successful infection, bacteriophages must overcome multiple barriers in the bacterial cell envelope to translocate viral DNA and proteins into the host cell. However, the molecular mechanisms underlying the phage infection initiation remain poorly understood. Here, we use cryo-electron tomography of Salmonella cells infected by phage P22 to capture intermediates during infection. P22 particle initially attaches to the cell surface obliquely through interactions between phage tail spikes and bacterial lipopolysaccharides. Subsequently the phage needle penetrates the host membrane. Three ejection proteins (gp7, gp16, gp20), which are originally stored in capsid, are ejected and assembled to create a 40 nm long trans-envelope channel between the phage tail and the cytoplasmic membrane. The novel channel serves as the conduit for genome translocation. Using mutant particles lacking one or more of the ejection proteins, our in situ structural analysis demonstrates that gp7 forms an extra-cellular channel, extending from the phage tail to the outer membrane; gp20 forms a mushroom-like structure spanning the periplasm, while gp16 is involved in channel formation in the cytoplasmic membrane. Together with atomic resolution information from mature particles and their proteins, our studies reveal a series of key intermediate infection structures at unprecedented detail. They also unveil a massive remodeling of both the infecting phage and the bacterial cell envelope. The structures we obtain provide the first direct evidence illuminating the functions of the three ejection proteins and their distinct role in phage genome delivery.

Optimization of a Cryo-EM Structural Model Representation

Electron cryo-microscopy (cryoEM) has been used to resolve atomic resolution biological structures. However, the resulting density maps and models still lack assessment of accuracy and reproducibility. The densities of some residues particularly, the negative charged residues have been found to be consistently absent or have low values in near-atomic resolution cryoEM maps. We thus introduce a procedure to derive an atomic model that faithfully replicates the experimental map and then use this map to ensure data accuracy and reproducibility. We have applied this protocol to describe the 3.3 A structure of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. This structure identifies previously unknown molecular interactions between capsid subunits, crucial to maintaining particle stability.Acknowledgement: This research has been supported by NIH grants (R01GM079429, P01GM063210, T15LM007093) and R Welch Foundation (Q1242).

Optimization of a Cryo-EM Structural Model Representation

Electron cryo-microscopy (cryoEM) has been used to resolve atomic resolution biological structures. However, the resulting density maps and models still lack assessment of accuracy and reproducibility. The densities of some residues particularly, the negative charged residues have been found to be consistently absent or have low values in near-atomic resolution cryoEM maps. We thus introduce a procedure to derive an atomic model that faithfully replicates the experimental map and then use this map to ensure data accuracy and reproducibility. We have applied this protocol to describe the 3.3 Å structure of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. This structure identifies previously unknown molecular interactions between capsid subunits, crucial to maintaining particle stability. Acknowledgement: This research has been supported by NIH grants (R01GM079429, P01GM063210, T15LM007093) and R Welch Foundation (Q1242)

Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid’) built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging’ is a DNA-dependent symmetrization of portal protein.

Marker Removal in Transgenic Plants Using Cre Recombinase Delivered with Potato Virus X.

In this chapter we present an alternative method to develop marker-free transgenic plants. It makes use of the Cre/loxP recombination system from bacteriophage P1 and consists of two essential components. The first component is the transgenic plant containing a loxP-flanked marker gene. The second component is a cre transient expression vector based on potato virus X. The great benefit of this transient delivery method consists in the avoidance of stable integration of the cre recombinase gene into the plant genome. Upon infection of the loxP-target plant with PVX-Cre, the virus spreads systemically through the plant and causes the recombinase-mediated excision of the marker gene. Marker-free transgenic loci can be transmitted to the progeny by plant regeneration from PVX-Cre systemically infected leaves or self-pollination of virus-infected plants. The protocol covers generation of loxP-target transgenic plants, PVX-mediated delivery of Cre recombinase protein, phenotypic and molecular analysis of recombination events, and transmission of marker-free transgenic loci to the next generation. The transient expression system described in this chapter can be adapted for marker gene removal in other plant species that are amenable for virus infection.

Modular interior loading and exterior decoration of a virus-like particle.

Virus-like particles (VLPs) derived from the bacteriophage P22 offer an interesting and malleable platform for encapsulation and multivalent presentation of cargo molecules. The packaging of cargo in P22 VLP is typically achieved through genetically enabled directed in vivo encapsulation. However, this approach does not allow control over the packing density and composition of the encapsulated cargos. Here, we have adopted an in vitro assembly approach to gain control over cargo packaging in P22. The packaging was controlled by closely regulating the stoichiometric ratio of cargo-fused-scaffold protein and wild-type scaffold protein during the in vitro assembly. In a "one-pot assembly reaction" coat protein subunits were incubated with varied ratios of wild-type scaffold protein and cargo-fused-scaffold protein, which resulted in the encapsulation of both components in a co-assembled capsid. These experiments demonstrate that an input stoichiometry can be used to achieve controlled packaging of multiple cargos within the VLP. The porous nature of P22 allows the escape and re-entry of wild-type scaffold protein from the assembled capsid but scaffold protein fused to a protein-cargo cannot traverse the capsid shell due to the size of the cargo. This has allowed us to control and alter the packing density by selectively releasing wild-type scaffold protein from the co-assembled capsids. We have demonstrated these concepts in the P22 system using an encapsulated streptavidin protein and have shown its highly selective interaction with biotin or biotin derivatives. Additionally, this system can be used to encapsulate small molecules coupled to biotin, or display large proteins, that cannot enter the capsid and thus remain available for the multivalent display on the exterior of the capsid when attached to a flexible biotinylated linker. Thus, we have developed a P22 system with controlled protein cargo composition and packing density, to which both small and large molecules can be attached at high copy number on the interior or exterior of the capsid.

Rapid Identification of E. coli Bacteriophages using Mass Spectrometry

Objective: The current increasing interest in the application of mass spectrometry, in particular MALDI-TOF MS, to identification of bacteria and fungi calls for a need to utilise this technology for identification of other infectious agents such as viruses. The aim of the present study was to develop a rapid and reliable mass spectrometry-based proteomic method for identification of Escherichia coli phages. Methods: The approach was based on rapid in-solution tryptic digestion of suspensions of plaque-purified bacteriophage followed by mass spectral analysis. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography – tandem mass spectrometry (LC-MS) were used to analyse the tryptic digests. Processing of tandem mass spectrometry data and interpretation of results were achieved using Mascot software and the Swiss-Prot database. Results: Five bacteriophage species (Enterobacteria phages P2, T4, T5, T7 and Lambda) isolated in E. coli cultures were identified. The viral proteins were identified from a complex mixture of host bacterial proteins. In addition, using a single ion monitoring method, a Lambda prophage derived protein was also identified. Conclusion: The data obtained demonstrate that LC-MS/MS can be used for accurate identification of E.coli- specific bacteriophages in both lytic and lysogenic cycles

Bacteriophage P1vir-induced cell-to-cell plasmid transformation in Escherichia coli.

Bacteria undergo horizontal gene transfer via various mechanisms. We recently reported that cell-to-cell transfer of nonconjugative plasmids occurs between strains of Escherichia coli in co-cultures, and that a specific strain (CAG18439) causes frequent plasmid transfer involving a DNase-sensitive mechanism, which we termed “cell-to-cell transformation”. Here we found that CAG18439 is a type of P1 bacteriophage lysogen that continuously releases phages. We tested the ability of P1vir bacteriophage to induce horizontal plasmid transfer and demonstrated that such a horizontal plasmid transfer was caused by adding culture supernatants of P1vir-infected cells harboring plasmids to other plasmid-free cells. This plasmid transfer system also reproduced the major features of plasmid transfer involving CAG18439, suggesting that P1vir-induced plasmid transfer is equivalent or very similar to plasmid transfer involving CAG18439. We further revealed that approximately two-thirds of the P1vir-induced plasmid transfer was DNase-sensitive, but that complete abolition of plasmid transfer was observed when proteins were denatured or removed, despite the presence or absence of DNase. Therefore, we concluded that P1vir-induced plasmid transfer is largely due to the occurrence of cell-to-cell transformation, which involves the assistance of some proteinaceous factor, and partly due to the occurrence of plasmid transduction, which is mediated by phage virions. This is the first demonstration of the P1-phage-induced cell-to-cell transformation.

Self-assembly of P22 protein cages with polyamidoamine dendrimer and inorganic nanoparticles

Abstract

NMR assignments for the insertion domain of bacteriophage Sf6 coat protein.

The P22 bacteriophage group is a subgroup of the λ phagesupercluster, comprised of the three major sequence types Sf6, P22, and CUS-3, based on their capsid proteins. Our goal is to investigate the extent to which structure-function relationships are conserved for the viral coat proteins and I-domains in this subgroup. Sf6 is a phage that infects the human pathogen Shigella flexneri. The coat protein of Sf6 assembles into a procapsid, which further undergoes maturation during DNA packaging into an infectious virion. The Sf6 coat protein contains a genetically inserted domain, termed the I-domain, similar to the ones present in the P22 and CUS-3 coat proteins. Based on the P22 example, I-domains play important functional roles in capsid assembly, stability, viability, and size-determination. Here we report the 1H, 15N, and 13C chemical shift assignments for the I-domain of the Sf6 phage coat protein. Chemical shift-based secondary structure prediction and hydrogen-bond patterns from a long-range HNCO experiment indicate that the Sf6 I-domain adopts a 6-stranded β-barrel fold like those of P22 and CUS-3 but with important differences, including the absence of the D-loop that is critical for capsid assembly and the addition of a novel disordered loop region.

The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage

Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7–49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29–98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4–10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

Virus Matryoshka: A Bacteriophage Particle-Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus.

Immature human immunodeficiency virus type 1 (HIV-1) is approximately spherical, but is constructed from a hexagonal lattice of the Gag protein. As a hexagonal lattice is necessarily flat, the local symmetry cannot be maintained throughout the structure. This geometrical frustration presumably results in bending stress. In natural particles, the stress is relieved by incorporation of packing defects, but the magnitude of this stress and its significance for the particles is not known. In order to control this stress, we have now assembled the Gag protein on a quasi-spherical template derived from bacteriophage P22. This template is monodisperse in size and electron-transparent, enabling the use of cryo-electron microscopy in structural studies. These templated assemblies are far less polydisperse than any previously described virus-like particles (and, while constructed according to the same lattice as natural particles, contain almost no packing defects). This system gives us the ability to study the relationship between packing defects, curvature and elastic energy, and thermodynamic stability. As Gag is bound to the P22 template by single-stranded DNA, treatment of the particles with DNase enabled us to determine the intrinsic radius of curvature of a Gag lattice, unconstrained by DNA or a template. We found that this intrinsic radius is far larger than that of a virion or P22-templated particle. We conclude that Gag is under elastic strain in a particle; this has important implications for the kinetics of shell growth, the stability of the shell, and the type of defects it will assume as it grows.

Construction of chromosomally encoded lacZ and gfp reporter strains of Escherichia coli for the study of global regulation of metabolism

We developed plasmid systems pJLIC and pJLRCS that allow the easy cloning of promoter regions upstream of reporter genes (lacZ and gfp) and the subsequent transfer of the resulting reporter cassettes into the chromosome of Escherichia coli by recombineering. Thus, a promoter region can be inserted upstream of lacZ gene in the context of the lactose operon of recipient E. coli strains. Moreover, a promoter–GFP reporter is inserted at a nonessential site of the chromosome. These systems allow for selection and promoter strength analysis at the single copy level. We also provide a collection of E. coli knock‐out mutants in genes for global regulation of C, N, and O metabolism, and combinations of these mutants with the newly constructed chromosomal lacZ and gfp reporter system. This allows assaying transcriptional activities of promoters before the background of different mutants. Both lacZ and gfp reporter cassettes (including appropriate antibiotic resistance markers) and the regulator knock‐outs may be easily transferred by phage P1 mediated transduction to other E. coli strains. As a proof of principle, ß‐galactosidase activity and GFP fluorescence were compared for lacZ and gfp reporter systems placed under the control of the PrssA promoter both in a wild‐type and an isogenic rpoS‐mutant strain.

Construction of Marker-Free Transgenic Strains of Chlamydomonas reinhardtii Using a Cre/loxP-Mediated Recombinase System.

The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct marker-free transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble-(linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after introduction of the ble-(linker)-CrCRE expression cassette. The ble-(linker)-CrCRE expression cassette remained in the genome after excision of the aphVIII expression cassette, and it was subsequently removed by crossing with the wild-type strain. This precise Cre-mediated deletion method applicable to transgenic C. reinhardtii could further increase the potential of this organism for use in basic and applied research.

Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging.

ABSTRACTThe P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three “ejection proteins” (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins’ locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection.

Measurement of the accurate mass of a 50MDa infectious virus.

Bacteriophage P22 is believed to contain a total of 521 copies of 9 different proteins and a 41,724 base pair genome. Despite its enormous size and complexity, phage P22 can be electrosprayed, and it remains intact in ultra-high vacuum where its molar mass distribution has been measured. Phage P22 virions were generated by complementation in Salmonella enterica and purified. They were transferred into 100mM ammonium acetate and then electrosprayed. The masses of individual virions were determined using charge detection mass spectrometry. The stoichiometry of the protein components of phage P22 is sufficiently well known that the theoretical molar mass can be determined to within a narrow range. The measured average molar mass of phage P22, 52,180±59kDa, is consistent with the theoretical molar mass and supports the proposed stoichiometry of the components. The intrinsic width of the phage P22 mass distribution can be accounted for by the distribution of DNA packaged by the headful mechanism. At over 50MDa, phage P22 is the largest object with a well-defined molar mass to be analyzed by mass spectrometry. The narrow measured mass distribution indicates that the virions survive the transition into the gas phase intact. Copyright © 2016 John Wiley & Sons, Ltd.

Computational determination of the effects of virulent Escherichia coli and salmonella bacteriophages on human gut

Background and objectiveSalmonella and Escherichia coli are different types of bacteria that cause food poisoning in humans. In the elderly, infants and people with chronic conditions, it is very dangerous if Salmonella or E. coli gets into the bloodstream and then they must be treated by phage therapy. Treating Salmonella and E. coli by phage therapy affects the gut flora. This research paper presents a system for detecting the effects of virulent E. coli and Salmonella bacteriophages on human gut. MethodsA method based on Domain–Domain Interactions (DDIs) model is implemented in the proposed system to determine the interactions between the proteins of human gut bacteria and the proteins of bacteriophages that infect virulent E. coli and Salmonella. The system helps gastroenterologists to realize the effect of injecting bacteriophages that infect virulent E. coli and Salmonella on the human gut. ResultsBy testing the system over Enterobacteria phage 933W, Enterobacteria phage VT2-Sa and Enterobacteria phage P22, it resulted in four interactions between the proteins of the bacteriophages that infect E. coli O157:H7, E. coli O104:H4 and Salmonella typhimurium and the proteins of human gut bacterium strains. ConclusionSeveral effects were detected such as: antibacterial activity against a number of bacterial species in human gut, regulation of cellular differentiation and organogenesis during gut, lung, and heart development, ammonia assimilation in bacteria, yeasts, and plants, energizing defense system and its function in the detoxification of lipopolysaccharide, and in the prevention of bacterial translocation in human gut.

Characterization and adsorption of Lactobacillus virulent phage P1

Bacteriophage infection of lactic acid bacteria is considered an important problem worldwide in the food fermentation industry, as it may produce low quality or unsafe foods, cause fermentation failure, and result in economic losses. To increase current knowledge on the properties of Lactobacillus virulent phages, we evaluated the effect of divalent cations, temperature, pH, and chloramphenicol on the adsorption ability of Lactobacillus virulent phage P1. Phage P1 was isolated from the abnormal fermentation liquid of Lactobacillus plantarum IMAU10120. The results showed that this phage belonged to the Siphoviridae family. The latent period of this phage was 45min, and the burst time was 90min. Burst size was 132.88±2.37 phage counts expressed per milliliter per infective center. This phage showed good tolerance at different temperatures, but incubation at 50°C only affected its adsorption. Adsorption rate reached a maximum value between 30 and 42°C. A high adsorption value of phage infectivity was obtained from pH 6 to 8. Moreover, calcium ions promoted and increased the adsorption capacity of phage P1, but magnesium ions had negative effects. Chloramphenicol had no effect on phage adsorption. This study increased current knowledge on the characterization and biological aspects of Lactobacillus virulent phages, and may provide some basic information that can be used to design successful antiphage strategies in the food industry.