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.     

DNA injection proteins are targets of acridine-sensitized photoinactivation of bacteriophage P22

Viruses and other nucleoprotein complexes are inactivated on exposure to white light in the presence of acridine and related dyes. The mechanism is thought to involve generation of singlet oxygen or related species, but the actual molecular targets of the inactivating event have not been well defined. We have re-examined the mechanism of dye-sensitized photoinactivation taking advantage of the well characterized bacteriophage P22. Though the inactivated phage absorb to their host cells, the cells are not killed and genetic markers cannot be rescued from the inactivated phage. These observations indicate that the chromosome is not injected into the host cell. However, the DNA of the damaged particles shows no evidence of double-stranded breaks or crosslinking.The DNA injection process of P22 requires three particle-associated proteins, the products of genes 7, 16 and 20. Gp16, which can act in trans during injection, is inactivated in the killed particles. Sodium dodecyl sulfate/polyacrylamide gel analysis reveals that gp16, gp7 and gp20 are progressively covalently damaged during photoinactivation. However, this damage does not occur in particles lacking DNA, indicating that it is DNA-mediated. Similar findings were obtained with acridine orange, acridine yellow, proflavin and acriflavin.These results indicate that the actual targets for inactivation are the DNA injection proteins, and that the lethal events represent absorption of photons by acridine molecules stacked in a region of DNA closely associated with the injection proteins.     

Functional analysis of the bacteriophage T4 DNA-packaging ATPase motor

Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp255) preceded by four hydrophobic residues (251MIYI254), which are predicted to form a beta-strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the beta-strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp255 mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging.     

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.     

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.     

Assembly of Bacillus subtilis phage phe29. 2. Mutants in the cistrons coding for the non-structural proteins.

The effect on phage morphogenesis of sus mutations in the cistrons coding for nonstructural proteins has been studied. Mutants in three cistrons analyzed that are involved in phage DNA synthesis, as well as in cistron 16 which codes for a late nonstructural protein, produce prolate capsids which are more rounded at the corners than complete phage heads and have an internal core; they contain the head proteins, the upper collar protein and protein p7, not present in mature phage particles. Mutants in cistron 7 do not produce capsids nor other phage-related structures; this result and the presence of p7 in phage capsids suggest an essential role in capsid assembly for this protein. The protein product of cistron 13 is probably needed for a stable DNA encapsulation since mutants in this cistron produce mainly DNA-free complete phage particles and only about 10% of uninfective DNA-containing complete phage. Cistron 15 codes for a late, partially dispensable, nonstructural protein which is present in the DNA-free capsids produced after infection with the delayed-lysis mutant sus14(1242), used as the wild-type control, or with mutants in cistrons 9, 11,12 and 13. Proteins p15 and p16 are probably involved in the encapsulation of viral DNA in a prohead.     

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.     

Peptidoglycan hydrolytic activities associated with bacteriophage virions

Murein hydrolases appear to be widespread in the virions of bacteriophages infecting Gram‐positive or Gram‐negative bacteria. Muralytic activity has been found in virions of the majority of a diverse collection of phages. Where known, the enzyme is either part of a large protein or found associated with other structural components of the virion that limit enzyme activity. In most cases, the lack of genetic and structural characterization of the phage precludes making a definitive identification of the enzymatic protein species. However, three proteins with muralytic activity have been unequivocally identified. T7gp16 is a 144 kDa internal head protein that is ejected into the cell at the initiation of infection; its enzyme activity is required only when the cell wall is more highly cross‐linked. P22gp4 is part of the neck of the particle and is essential for infectivity. The activity associated with virions of Bacillus subtilis phage ø29 and its relatives lies in the terminal protein gp3. These studies lead to a general mechanism describing how phage genomes are transported across the bacterial cell wall.     

Assembly of Bacillus subtilis phage phi29. 1. Mutants in the cistrons coding for the structural proteins.

The effect of mutations in the cistrons coding for the phage structural proteins has been studied by analyzing the phage-related structures accumulated after restrictive infection. Infection with susmutants in cistron 8, lacking both the major head and the fiber protein, does not produce any phage-related structure, suggesting a single route for the assembly of phage phi29; infection with ts mutants in this cistron produces isometric particles. Mutants is cistron 9, coding for the tail protein, TP1, produce DNA-free prolate heads with an internal core; these particles are abortive and contain the head proteins HPO, HP1 and HP3, the upper collar protein NP2 and the nonstructural proteins p7, p15 and p16. Mutants in cistron 10, coding for the upper collar protein, NP2, produce DNA-free isometric heads also with an internal core; they contain the head proteins and the nonstructural protein p7, suggesting that this protein forms the internal core. Mutants in cistrons 11 and 12, coding for the lower collar protein, NP3, and the neck appendages, NP1, respectively, give rise to the formation of DNA-containing normal capsids and DNA-free prolate particles, more rounded at the corners than the normal capsids and with an internal core; the DNA-containing 11-particles are formed by the head proteins and the upper collar protein; the DNA-free 11-particles contain, besides these proteins, the nonstructural protein p7 and a small amount of proteins p15 and 16. The DNA-containing 12-particles have all the normal phage structural proteins except the neck appendages, formed by protein NP1; the DNA-free particles are similar to the DNA-free 11-particles. After restricitive infection mutant sus14(1241) has a delayed lysis phenotype and produces a phage burst higher than normal, after artificial lysis. It produces DNA-containing particles, identical to wild-type phage, which have all the normal phage structural proteins, and DNA-free prolate particles, more rounded at the corners than the final phage particles and with an internal core; the last particles contain the same proteins as the DNA-free 11 or 12-particles. These particles could represent a prohead state, ready for DNA encapsulation. None of the DNA-containing particles have the nonstructural proteins p7, p15 or p16, suggesting that these proteins are released from the proheads upon DNA encapsulation.     

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.     

Single molecule force spectroscopy of salt-dependent bacteriophage T7 gene 2.5 protein binding to single-stranded DNA

The gene 2.5 protein (gp2.5) encoded by bacteriophage T7 binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication and recombination in the phage-infected cell. gp2.5 lowers the phage lambda DNA melting force as measured by single molecule force spectroscopy. T7 gp2.5-Delta26C, lacking 26 acidic C-terminal residues, also reduces the melting force but at considerably lower concentrations. The equilibrium binding constants of these proteins to single-stranded DNA (ssDNA) as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 +/- 0.6) x 10(5) m(-1) in a 50 mm Na(+) solution, whereas the truncated protein binds to ssDNA with a much higher affinity of (7.8 +/- 0.9) x 10(7) m(-1) under the same solution conditions. T7 gp2.5-Delta26C binding to single-stranded DNA also exhibits a stronger salt dependence than the full-length protein. The data are consistent with a model in which a dimeric gp2.5 must dissociate prior to binding to ssDNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component.     

Sequential interactions of structural proteins in phage phi 29 procapsid assembly.

The mechanism of viral capsid assembly is an intriguing problem because of its fundamental importance to research on synthetic viral particle vaccines, gene delivery systems, antiviral drugs, chimeric viruses displaying antigens or ligands, and the study of macromolecular interactions. The genes coding for the scaffolding (gp7), capsid (gp8), and portal vertex (gp10) proteins of the procapsid of bacteriophage phi 29 of Bacillus subtilis were expressed in Escherichia coli individually or in combination to study the mechanism of phi 29 procapsid assembly. When expressed alone, gp7 existed as a soluble monomer, gp8 aggregated into inclusion bodies, and gp10 formed the portal vertex. Circular dichroisin spectrum analysis indicated that gp7 is mainly composed of alpha helices. When two of the proteins were coexpressed, gp7 and gp8 assembled into procapsid-like particles with variable sizes and shapes, gp7 and gp10 formed unstable complexes, and gp8 and gp10 did not interact. These results suggested that gp7 served as a bridge for gp8 and gp10. When gp7, gp8, and gp10 were coexpressed, active procapsids were produced. Complementation of extracts containing one or two structural components could not produce active procapsids, indicating that no stable intermediates were formed. A dimeric gp7 concatemer promoted the solubility of gp8 but was inactive in the assembly of procapsid or procapsid-like particles. Mutation at the C terminus of gp7 prevented it from interacting with gp8, indicating that this part of gp7 may be important for interaction with gp8. Coexpression of the portal protein (gp20) of phage T4 with phi 29 gp7 and gp8 revealed the lack of interaction between T4 gp20 and phi 29 gp7 and/or gp8. Perturbing the ratio of the three structural proteins by duplicating one or another gene did not reduce the yield of potentially infectious particles. Changing of the order of gene arrangement in plasmids did not affect the formation of active procapsids significantly. These results indicate that phi 29 procapsid assembly deviated from the single-assembly pathway and that coexistence of all three components with a threshold concentration was required for procapsid assembly. The trimolecular interaction was so rapid that no true intermediates could be isolated. This finding is in accord with the result of capsid assembly obtained by the equilibrium model proposed by A. Zlotnick (J. Mol. Biol. 241:59-67, 1994).     

Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA

Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.     

Role of the Gp16 lytic transglycosylase motif in bacteriophage T7 virions at the initiation of infection

The predicted catalytic glutamate residue for transglycosylase activity of bacteriophage T7 gp16 is not essential for phage growth, but is shown to be beneficial during infection of Escherichia coli cells grown to high cell density, conditions in which murein is more highly cross-linked. In the absence of the putative transglycosylase, internalization of the phage genome is significantly delayed during infection. The lytic transglycosylase motif of gp16 is essential for phage growth at temperatures below 20 degrees C, indicating that these growth conditions also lead to increased cross-linking of peptidoglycan. Overexpression of sltY, E. coli soluble lytic transglycosylase, partially complements the defect in infection of mutant phage particles, allowing them to infect at higher efficiencies. Conversely, an sltY deletion increases the latent period of wild-type phage.     

Genomic structure of the Salmonella enterica serovar Typhimurium DT 64 bacteriophage ST64T: evidence for modular genetic architecture

The complete sequence of the double-stranded DNA genome of a serotype-converting temperate bacteriophage, ST64T, was determined. The 40,679-bp genomic sequence of ST64T has an overall GC content of 47.5% and was reminiscent of a number of lambdoid phages, in particular, P22. Inferred proteins of ST64T which exhibited a high degree of sequence similarity to P22 proteins (>90%) included the functional serotype conversion cassette, integrase, excisionase, Abc1, Abc2, early antitermination (gp24), NinD, NinH, NinZ, packaging (gp3 and gp2), head (with the exception of gp26, gp7, gp20, and gp16), and tail proteins. The putative immunity genes were highly related to those of Salmonella enterica serotype Typhimurium phage L, whereas the lysis genes were almost identical to those of S. enterica serovar Typhimurium PS3.     

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.     

Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29

The ATPase activity of the DNA packaging protein gp16 (gene product 16) of bacteriophage phi 29 was studied in the completely defined in-vitro assembly system. ATP was hydrolyzed to ADP and Pi in the packaging reaction that included purified proheads, DNA-gp3 and gp16. Approximately one molecule of ATP was used in the packaging of 2 base-pairs of phi 29 DNA, or 9 X 10(3) ATP molecules per virion. The hydrolysis of ATP by gp16 was both prohead and DNA-gp3 dependent. gp16 contained both the "A-type" and the "B-type" ATP-binding consensus sequences (Walker et al., 1982) and the predicted secondary structure for ATP binding. The A-type sequence of gp16 was "basic-hydrophobic region-G-X2-G-X-G-K-S-X7-hydrophobic", and similar sequences were found in the phage DNA packaging proteins gpA of lambda, gp19 of T7 and gp17 of T4. Having both the ATP-binding and potential magnesium-binding domains, all of these proteins probably function as ATPases and may have common prohead-binding capabilities. The phi 29 protein gp3, covalently bound to the DNA, may be analogous in function to proteins gpNul of lambda and gpl of phi 21 that bind the DNA.