Identification of the 9-aminoacridine/DNA complex responsible for photodynamic inactivation of P22

Acridine dyes bound to the condensed DNA within phage particles sensitize them to inactivation by visible light. The mechanism involves absorption of photons by an acridine/DNA complex, generating singlet oxygen, which covalently damages nearby proteins needed for DNA injection [Bryant, J., & King, J. (1985) J. Mol. Biol. 180, 837-863]. Acridines and related dyes interact with double-stranded DNA through a number of binding modes. To determine in condensed phage DNA the binding mode responsible for this inactivation, we have studied the formation of the DNA/acridine target complexes for photoinactivation. Analysis of the kinetics of 9-aminoacridine binding to Salmonella phage P22 particles revealed the formation of two binding species, one of which appeared more rapidly and was apparently an intermediate in the formation of the second. The rapidly forming species represented DNA sites with intercalated acridines, while the more slowly forming species represented the subsequent binding of additional acridine molecules to the DNA backbone of sites already containing intercalated dye. The rates of photoinactivation correlated with the rate of binding of 9-aminoacridine to the DNA backbone. This suggests that the most effective species for sensitizing phage to light-induced damage has acridine molecules stacked alongside the backbone of a region with intercalated molecules.     

E proteins of bacteriophage P22. I. Identification and ejection from wild-type and defective particles.

Of the nine proteins found in the virion of phage P22, four are ejected into the cell after adsorption. The four ejected proteins, termed E proteins, are gp16, gp20, gp26, and gp7. This was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled phage that had been adsorbed to cells and then eluted off the surface with distilled water. Phage particles that lack gp7 (7- particles) or gp20 (20- particles) successfully eject all their E proteins. The 16- particles do not eject gp7. Analysis of phage ghosts showed that they lack gp16, gp20, and gp7, but they have gp26 in close to normal quantities. Our results suggest roles for gp16 and gp26 in DNA and E protein ejection. All four E proteins are possible candidates for roles in helping the phage DNA cross the plasma membrane.     

Is the injection of DNA enough to cause bacteriophage P22-induced changes in the cellular transport process of Salmonella typhimurium?

It was demonstrated earlier in this laboratory that phage P22 induces a transient depression in the cellular transport processes of the host Salmonella typhimurium immediately after infection and that an effective injection process is enough to cause the depression. By using defective phage particles that contain host DNA instead of phage DNA for infection, it has been demonstrated that the injection of phage-specific DNA is essential for this. The defective particles adsorbed to the host and injected their DNA, but the cellular transport processes of the host were not altered. Thus, the injection of host DNA by the phage fails to affect the transport process. Insensitivity of the phage DNA-induced depression in transport to chloramphenicol rules out the involvement of newly synthesized protein in this change and indirectly suggests the possible role of phage DNA-associated internal proteins of P22.     

Oligopeptidase A is required for normal phage P22 development.

The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing.     

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.     

Transport of phage P22 DNA across the cytoplasmic membrane

Although a great deal is known about the life cycle of bacteriophage P22, the mechanism of phage DNA transport into Salmonella is poorly understood. P22 DNA is initially ejected into the periplasmic space and subsequently transported into the host cytoplasm. Three phage-encoded proteins (gp16, gp20, and gp7) are coejected with the DNA. To test the hypothesis that one or more of these proteins mediate transport of the DNA across the cytoplasmic membrane, we purified gp16, gp20, and gp7 and analyzed their ability to associate with membranes and to facilitate DNA uptake into membrane vesicles in vitro. Membrane association experiments revealed that gp16 partitioned into the membrane fraction, while gp20 and gp7 remained in the soluble fraction. Moreover, the addition of gp16, but not gp7 or gp20, to liposomes preloaded with a fluorescent dye promoted release of the dye. Transport of ³²P-labeled DNA into liposomes occurred only in the presence of gp16 and an artificially created membrane potential. Taken together, these results suggest that gp16 partitions into the cytoplasmic membrane and mediates the active transport of P22 DNA across the cytoplasmic membrane of Salmonella.     

Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel

The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. The gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.     

Role of gene 10 protein in the hierarchical assembly of the bacteriophage P22 portal vertex structure

The portal vertex structure of the phage P22 is a 2.8 MDa molecular machine that mediates attachment and injection of the viral genome into the host Salmonella enterica serovar Typhimurium. Five proteins form this molecular machine: the portal protein, gp1; the tail-spike, gp9; the tail-needle, gp26, and the tail accessory factors, gp4 and gp10. In order to understand the assembly of the portal vertex structure, we have isolated the gene encoding tail accessory factor gp10 and defined its structural composition and assembly within the portal vertex structure. In solution, monomeric gp10 is a beta-sheet-rich protein with a stable conformational structure, which spontaneously assembles into hexamers, likely via a dimeric intermediate. This oligomerization enhances the structural stability of the protein, which then becomes competent for assembly to a preformed portal protein:gp4 complex, and acts as a structural adaptor bridging the nascent phage tail to gp26 and gp9. Notably, in vitro purified tail accessory factors gp4, gp10, and gp26 do not significantly interact with each other in solution, but their assembly takes place efficiently when these factors are added sequentially onto an immobilized portal protein. This suggests that the assembly of the P22 tail is a highly sequential and cooperative process, likely mediated by structural rearrangements in the assembly components. The assembled portal vertex structure represents both a membrane-binding and penetrating device as well as a plug that retains the pressurized phage DNA inside the capsid.     

The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32(-) particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.     

Bacteriophage P22 virion protein which performs an essential early function. II. Characterization of the gene 16 function.

P16 is a virion protein and, as such, is incorporated into the phage head as a step in morphogenesis. The role of P16 in assembly is not essential since particles are formed without this protein which appear normal by electron microscopy. P16 is essential when the particle infects a cell in the following cycle of infection. In the absence of functional P16, the infection does not appear to proceed beyond release of phage DNA from the capsid. No known genes are expressed, no DNA is transcribed, and the host cell survives the infection, continuing to grow and divide normally. The P16 function is required only during infection for the expression of phage functions. Induction in the absence of P16 proceeds with the expression of early and late genes and results in particle formation. P16 must be incorporated during morphogenesis into progeny particles after both infection and induction for the progeny to be infectious. The P16 function is necessary for transduction as well as for infection. Its activity is independent of new protein synthesis and it is not under immunity control. P16 can act in trans, but appears to act preferentially on the phage or phage DNA with which it is packaged. The data from complementation studies are compatible with P16 release from the capsid with the phage DNA. In the absence of P16 the infection is blocked, but the phage genome is not degraded. The various roles which have been ruled out for P16 are: (i) an early regulatory function, (ii) an enzymatic activity necessary for phage production, (iii) protection of phage DNA from host degradation enzymes, (iv) any generalized alteration of the host cell, (v) binding parental DNA to the replication complex, and (vi) any direct involvement in the replication of P22 DNA. P16 can be responsible for: (i) complete release of the DNA and disengagement from the capsid, (ii) bringing the released DNA to some necessary cell site or compartment such as the cytoplasm, (iii) removal of other virion proteins from the injected DNA, and (iv) alterations of the structure of the injected DNA.     

Participation of the host protein(s) in the morphogenesis of bacteriophage P22

Summary Spontaneous mutants of S. typhimurium resistant to thiolutin are conditionally non-permissive for phage P22 development (Joshi and Chakravorty 1979). At 40° C non-infective phage particles are produced. Phage development in two nonpermissive hosts (18/MC4 and 153/MC4) has been studied in detail. The steps at which the phage morphogenesis is interfered with differ in the two mutants. The electron micrograph of the particles produced in the mutant 18/MC4 reveals the presence of normal-looking particles; these particles contain phage DNA, adsorb to the permissive host but fail to inject their DNA. The particles produced in the mutant 153/MC4 which fail to adsorb to the host are found to be tail fibre-less. These observations indicate the involvement of host protein(s) in phage P22 morphogenesis.     

Sites and gene products involved in lambdoid phage DNA packaging.

21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage. The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence. The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific. DNA packaging by 21 is dependent on a host protein, integration host factor. A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1. gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase. orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI. orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth. 21 was also not able to complement a lambda FI mutant.     

Cloning, overexpression and purification of the terminase proteins gp16 and gp17 of bacteriophage T4. Construction of a defined in-vitro DNA packaging system using purified terminase proteins

Terminases of double-stranded DNA bacteriophages are required for packaging and generation of terminii in replicated concatemeric DNA molecules. Genetic evidence suggests that these functions in phage T4 are carried out by the products of genes 16 and 17. We cloned these T4 genes into a heat-inducible cI repressor-lambda PL promoter vector system, and overexpressed them in Escherichia coli. We developed an in-vitro DNA packaging system, which, consistent with the genetic data, shows an absolute requirement for the terminase proteins. The overexpressed terminase proteins gp16 and gp17 appear to form a specific complex and an ATP binding site is present in the gp17 molecule. We purified the terminase proteins either as individual gp16 or gp17 proteins, or as a gp16-gp17 complex. The gp16 function of the terminase complex is dispensable for packaging mature DNA, whereas gp17 is essential for packaging DNA under any condition tested. We constructed a defined in-vitro DNA packaging system with the purified terminase proteins, purified proheads and a DNA-free phage completion gene products extract. All the components of this system can be stored at -90 degrees C without loss of packaging activity. The terminase proteins, therefore, may serve as useful reagents for mechanistic studies on DNA packaging, as well as to develop T4 as a packaging-cloning vector.     

Steps in the stabilization of newly packaged DNA during phage P22 morphogenesis

The protein products of three adjacent P22 genes, 4, 10 and 26, are required for the stabilization of DNA newly packaged into P22 phage capsids. We have isolated unstable DNA containing capsids from cells infected with mutants defective in these genes. All three classes could be converted into mature phage in vitro, confirming that they represent intermediates in particle maturation. The first of the three proteins to add to the newly filled capsids is gp4, followed by gp10 and gp26. The active form of gp4 sediments at 3 S, while the active forms of both gp10 and gp26 sediment at 5 S. These soluble subunits appear to polymerize onto the newly filled capsids to form the neck of the mature phage, the channel for DNA injection. Since gp4 is the first protein to act after DNA packaging, the unstable DNA containing capsids from 4- -infected cells must represent the direct product of the packaging of DNA into procapsids. The major fraction of these capsids lost activity with a half-life of 1.1 minutes at 23 degrees C, though they were much more stable at 0 degree C. Electron microscopic observations indicated that the loss of activity was due to the DNA exiting from the incomplete capsids. The marginal stability of the condensed DNA molecules within capsids is consistent with models of ATP-driven condensation and spontaneous DNA ejection. The basis of the stability of these highly condensed molecules remains to be determined.     

The Bacteriophage HK97 gp15 Moron Element Encodes a Novel Superinfection Exclusion Protein

A phage moron is a DNA element inserted between a pair of genes in one phage genome that are adjacent in other related phage genomes. Phage morons are commonly found within phage genomes, and in a number of cases, they have been shown to mediate phenotypic changes in the bacterial host. The temperate phage HK97 encodes a moron element, gp15, within its tail morphogenesis region that is absent in most closely related phages. We show that gp15 is actively expressed from the HK97 prophage and is responsible for providing the host cell with resistance to infection by phages HK97 and HK75, independent of repressor immunity. To identify the target(s) of this gp15-mediated resistance, we created a hybrid of HK97 and the related phage HK022. This hybrid phage revealed that the tail tube or tape measure proteins likely mediate the susceptibility of HK97 to inhibition by gp15. The N terminus of gp15 is predicted with high probability to contain a single membrane-spanning helix by several transmembrane prediction programs. Consistent with this putative membrane localization, gp15 acts to prevent the entry of phage DNA into the cytoplasm, acting in a manner reminiscent of those of several previously characterized superinfection exclusion proteins. The N terminus of gp15 and its phage homologues bear sequence similarity to YebO proteins, a family of proteins of unknown function found ubiquitously in enterobacteria. The divergence of their C termini suggests that phages have co-opted this bacterial protein and subverted its activity to their advantage.     

The minor capsid protein gp7 of bacteriophage SPP1 is required for efficient infection of Bacillus subtilis

Gp7 is a minor capsid protein of the Bacillus subtilis bacteriophage SPP1. Homologous proteins are found in numerous phages but their function remained unknown. Deletion of gene 7 from the SPP1 genome yielded a mutant phage (SPP1del7) with reduced burst-size. SPP1del7 infections led to normal assembly of virus particles whose morphology, DNA and protein composition was undistinguishable from wild-type virions. However, only approximately 25% of the viral particles that lack gp7 were infectious. SPP1del7 particles caused a reduced depolarization of the B. subtilis membrane in infection assays suggesting a defect in virus genome traffic to the host cell. A higher number of SPP1del7 DNA ejection events led to abortive release of DNA to the culture medium when compared with wild-type infections. DNA ejection in vitro showed that no detectable gp7 is co-ejected with the SPP1 genome and that its presence in the virion correlated with anchoring of released DNA to the phage particle. The release of DNA from wild-type phages was slower than that from SPP1del7 suggesting that gp7 controls DNA exit from the virion. This feature is proposed to play a central role in supporting correct routing of the phage genome from the virion to the cell cytoplasm.     

Bacteriophage P22 tail accessory factor GP26 is a long triple-stranded coiled-coil

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.     

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 Å crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.     

Identification of the Block in the Intracellular Replication of Single-Stranded DNA of Photodynamically Inactivated Bacteriophage φX174

(32)P-labeled single-stranded DNA phage phiX174 was photodynamically inactivated by irradiation in air with visible light in the presence of the acridine dye, proflavine sulfate. The inactivated phages could adsorb to the host cells but failed to lyse them. Formation of intracellular mature phages was almost completely inhibited. Photodynamic lesions in phiX174 DNA caused intracellular formation of defective double-stranded replicative form molecules which ultimately reverted to the single-stranded configuration.