Structure and synthesis of a lipid-containing bacteriophage. XIX. Reconstitution of bacteriophage PM2 in vitro.

In order to construct a physical map of the bacteriophage fd genome, the doubly closed replicative form (RFI) DNA of phage fd was cleaved into unique fragments by four different restriction endonucleases (Hap, Hga, HinH and Hind) prepared from Haemophilus strains H. aphirophilus, H. gallinarum, H. influenzae H-I and H. influenzae Rd, respectively. As Hind cleaved RFI DNA at a single site, this site was used as a reference point for mapping. HinH cleaved RFI DNA at three sites, Hga at six sites and Hap at 13 sites, respectively. The 5′-termini of the fragments produced by either HinH or Hga were labelled with ³²P in the polynucleotide kinase reaction. The labelled fragments were separated and further cleaved by other enzymes. The re-digestion products of partially digested fragments were also analysed. On the basis of these data and estimates of the size of each fragment, a cleavage map of the phage fd genome was constructed.     

Structure and synthesis of a lipid-containing bacteriophage. Chemical modifications of bacteriophage PM2 and the resulting alterations in acyl-chain motion in the PM2 membrane.

The nucleocapsid proteins of bacteriophage PM2 and the inner lamella of the lipid bilayer, containing most of the phosphatidlethanolamine residues, were selectively cross-linked in the presence of 0.1-0.5% glutaraldehyde, 5 mM dimethylsuberimidate, or 0.05% toluene 2,4-diisocyanate. The biological activity (p.f.u.) of PM2 modified by these reagents decreased 10(6)-fold in all cases. The spike and coat proteins were selectively cross-linked in the presence of 7.5 mM N,N'-p-phenylenedimaleimide. The biological activity of virus modified by this reagen was unaffected. The electron paramagnetic resonance spectra of fatty acid spin labels incorporated into native and chemically modified viral membranes were qualitatively similar but show quantitative differences. Fixation with glutaraldehyde increased the rigidity of the membrane while Triton X-100 induced a more flexible structure. There was no change in the electron paramagnetic resonance spectrum of virus treated with N,N'-p-phenylenedimaleimide, however.     

Structure and synthesis of a lipid-containing bacteriophage. A polynucleotide-dependent polynucleotide-pyrophosphorylase activity in bacteriophage PM2.

A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 degrees C. The activity is a polynucleotide-pyrophosphorylating reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nuclei- acids or polynucleotides as effectors. The product is not covalently linked to the effector.     

Control of membrane morphogenesis in bacteriophage PM2

The regulation of membrane formation in bacteriophage PM2 serves as a simple model for changes in membrane structure in eukaryotic cells. Prior to Pseudomonas host lysis, wild-type virions mature to an icosahedral morphology at the inner face of the cytoplasmic membrane. The proliminary charcterization of two temperature-sensitive mutants of PM2 is described. In cells infected at the restrictive temperature with ts 1, an abundance of “empty” virus-size membrane vesicles are seen. Synthesis of DNA is also reduced in ts 1 infected cells. The preponderance of vesicles is not sen in cells infected with wil-type virus or with ts 1 at the permissive temperature. The “empty” appearance of the viral membranes suggests that viral DNA is not encapsulated. The major viral capsid protein (MW 26,000) is located just out side the viral membrane and normallyl sediments with host and virus membranes; insted, large amounts of capsid protein can be precipitated from the supernatant with TCA. Compared to cells infected with wild type virus, cells infected with is 5 at th restrictive temperature produce inside the cell an aboundance of virus-soze membrane vesicles. Taken Together, These results with viral mutants suggest that formation of a viral membrane of the proper size does not require a DNA core around which to form, or an outer scaffolding of coat protein against which to form a spherical bilayer.     

Cloning of bacteriophage PM2 DNA in Escherichia coli K12.

Summary DNA fragments of phage PM2 restricted with Hin dIII endonuclease was cloned in the vector pBR 322 in an Escherichia coli K 12 host. The attempt to clone full length PM2 DNA restricted with PstI endonuclease has been unsuccesful. From six randomly chosen recombinant clones DNA was purified and analysed with EcoRI, PstI and HindIII endonucleases. The physical map of three chimeric plasmids was unequivocally established. It was shown, that the whole PM2 genome was cloned, although in separate fragments. However, most of the recombinant clones were instable in the absence of selective pressure.     

Structure and synthesis of a lipid-containing bacteriophage. Amphiphilic properties of protein IV of bacteriophage PM2

Interactions between lipids and the DNA-binding protein (protein IV) purified from bacteriophage PM2 were studied in vitro. The efficiency of incorporation of protein IV into single-walled liposomes was more than 90%. Protein IV embedded in liposomes interacted more strongly with PM2 DNA than protein IV alone. The DNA--protein-IV--liposome complex was relatively stable as observed by sedimentation behavior on a sucrose gradient. The interaction between DNA and the protein-IV--liposome was abolished by tryptic digestion, even though 40% of the protein remained in the vesicle. More than 70% of the amino acids of this embedded peptide segment were hydrophobic. Carboxypeptidase digestion of the protein-IV--liposome caused a release of 20% of the radioactivity of the vesicle without changing the DNA-binding ability of the complexes. Modification of the protein-IV--liposome with the chemical probe, 2,4-dinitrofluorobenzene, and analysis of the tryptic peptides released from the protein-IV--liposome demonstrated that the N-terminal basic amino acid cluster segment responsible for the DNA binding was located on the outer surface of the bilayer. These results support an earlier model in which protein IV anchors itself in the inner leaflet of the PM2 bilayer membrane, interacting with the DNA in the virion.     

Molecular cloning and physical map of bacteriophage PM2 DNA

The entire genome and the DNA fragments of the lipid-containing bacteriophage pM2 were cloned in the pBR322 plasmid vector. A physical map including the sites for the following restriction enzymes was obtained: HpaII, HaeIII, TthI, Sau96I, AvaII, PstI, BstNI, AccI, HincII, HpaI and HindIII. No restriction sites on PM2 DNA were found for BalI, BamHI, BclI, BglI, BglII, BstEII, KpnI, PvuII, SacI, SalI, Sau3A, XbaI and XhoI.     

Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila

PM2 is an Aeromonas-specific bacteriophage isolated on A. hydrophila strain AH-3. The bacteriophage receptor for this phage was found to be the lipopolysaccharide (LPS), specifically a low-molecular weight LPS fraction (LPS-core oligosaccharides). Mutants resistant to this phage were isolated and found to be devoid of LPS O-antigen and altered in the LPS-core. No other outer-membrane (OM) molecules appeared to be involved in phage binding.     

Photoinactivation of bacteriophage PM2 by cyanine dyes]

Photoinactivation of the lipid-containing bacteriophage PM2 by visible light and cyanine dyes (carbo- and dicarbocyanines), aluminum phtalocyanine tetrasulfonate and methylene blue was studied. It was concluded that cyanine dye aggregates adsorbed on phage particles and oxygen are essential for phage photoinactivation.     

Mutant ts1 of bacteriophage PM2 is defective in the major capsid protein and fails to package its DNA.

Infection of Alteromonas espejiana at restrictive temperature with mutant ts1 of bacteriophage PM2 resulted in the intracellular accumulation of virus-sized empty-appearing membrane vesicles. The DNA associated with purified vesicles was fully susceptible to digestion with DNase. Sedimentation analysis and electron microscopy suggested a full-length linear form of the normally circular viral genome. A pulse-chase-shift experiment suggested that [3H]thymidine-labeled DNA made under restrictive conditions is assembled into virions after shift to permissive temperature. A defective structural protein in the ts1 virion appears to be the cause of a rapid rate of thermal inactivation of infectivity. Analysis of the proteins of ts1 by isoelectric focusing indicated a more alkaline isoelectric mobility of the major capsid protein, sp27. Six spontaneous revertants of ts1 showed reversion to the wild-type isoelectric form of sp27. These results identify sp27 as the defective gene product of ts1. Taken together, these results suggest that the membrane of PM2 is formed without the aid of an inner core or an outer scaffolding.     

Calcium requirement for assembly of the lipid-containing bacteriophage PM2

The bacteriophage PM2 requires extracellular Ca²⁺ at concentrations greater than 3 · 10⁻⁴ M for the production of viable virus, whereas the host cell Pseudomonas BAL-31 grows normally in medium containing 3 · 10⁻⁵ M Ca²⁺ (low calcium). Virus attachment occurs normally in low calcium, the infected cultures partially lyse, but no infectious virus particles are released. Sucrose gradient analysis shows that lysates made in low calcium contain no PM2-like particles. The addition of calcium very late in the infectious cycle completely restores virus production to cultures infected in low calcium, whereas removal of calcium after infection prevents virus production. Our experiments indicate that Ca²⁺ is essential for some process late in the lytic cycle, such as the final assembly of stable, infectious PM2 particles.     

Sedimentation and intrinsic viscosity behavior of PM2 bacteriophage DNA in alkaline solution

The sedimentation coefficient and intrinsic viscosity of nicked and closed circular PM2 bacteriophage DNA have been measured as a function of pH in the alkaline region. A gradual increase in the sidimentation coefficient, and a corresponding decrease in the intrinsic viscosity, are observed for the superhelical (closed) circle in the pH region from 10.5 to about 10.9. This has been tentatively interpreted in terms of the known dependence of sedimentation coefficient upon the number of superhelical turns. At slightly higher pH values, the curve passes through the minimum (sedimentation coefficient) and maximum (intrinsic viscosity) expected when the superhelical turns present at neutral pH are unwound by partial alkaline denaturation. Sedimentation studies of the relaxed (nicked) circular species have revealed the existence of DNA forms in the pH region from 11.27 to 11.37 which sediment considerably faster than the closed circle in the same pH region. These have been identified as partially denatured nicked circles, in which varying fractions of the duplex structure have undergone alkaline denaturation, but strand separation has not yet occurred. Varying fractions of a slower species, either undenatured or completely denatured nicked circles, are also observed in some of these experiments. A corresponding result is observed in the intrinsic viscosity vs. pH curve. When nicked circular PM2 DNA is exposed to various alkaline pH's, rapidly neutralized, and sedimented at neutral pH, the expected sharp transition from native to denatured (strand-separated) molecules is seen. However, a very narrow pH range is noted in which native and denatured forms coexist in a single experiment. The above experiments carried out upon the closed form also reveal a narrow pH range in which the bulk of the transition from native closed circles to the collapsed cyclic coil takes place, in acccord with an earlier study on a different DNA. This transition is shown never to be completely effected, however, as there is a fraction (7–8%)of the closed circles which renature to the native form, regardless of the alkaline pH employed. This same phenomenon was not observed in the case of artificially closed λb₂b₅c DNA circles. Possible explanations for some of the above results are discussed.     

Penetration of membrane-containing double-stranded-DNA bacteriophage PM2 into Pseudoalteromonas hosts

The icosahedral bacteriophage PM2 has a circular double-stranded DNA (dsDNA) genome and an internal lipid membrane. It is the only representative of the Corticoviridae family. How the circular supercoiled genome residing inside the viral membrane is translocated into the gram-negative marine Pseudoalteromonas host has been an intriguing question. Here we demonstrate that after binding of the virus to an abundant cell surface receptor, the protein coat is most probably dissociated. During the infection process, the host cell outer membrane becomes transiently permeable to lipophilic gramicidin D molecules proposing fusion with the viral membrane. One of the components of the internal viral lipid core particle is the integral membrane protein P7, with muralytic activity that apparently aids the process of peptidoglycan penetration. Entry of the virion also causes a limited depolarization of the cytoplasmic membrane. These phenomena differ considerably from those observed in the entry process of bacteriophage PRD1, a dsDNA virus, which uses its internal membrane to make a cell envelope-penetrating tubular structure.