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Phosphatidylethanolamine is the only phosphatide present in purified bacteriophage PM2 when obtained from bacteria grown and infected in minimal medium. The growth cycle of bacteriophage PM2 shows the basic general features described for virulent bacteriophages. PM2 infection of Pseudomonas BAL-31 causes a pronounced increase in deoxyribonucleic acid synthesis, but no detectable effect on the incorporation of phosphorus into lipid. When 32 P-prelabeled bacteria are infected with PM2, the specific activity of the phosphatide phosphorus in the virus is almost the same as that in the host bacteria labeled before infection. It is concluded that the viral phosphatide is not synthesized de novo after infection, but it probably is derived from preexisting cellular phosphatidylethanolamine.

Shortly after infection with bacteriophage PM2, host DNA synthesis is inhibited while synthesis of the supercoiled viral DNA begins at a low rate. This rate increases at about ten minutes after infection to reach a value that is thereafter maintained until lysis of the cells. Pulse-chase experiments suggest the existence of several precursors of supercoiled PM2 DNA. Two of them, one which sediments as nicked circular PM2 DNA (21 s) and another, appearing earlier in time, sedimenting 1.15 times faster (24 s), have been partially characterized. The 24 s component seems to contain linear single strands both longer and shorter than the strands of PM2 DNA. It has been purified, examined in the electron microscope and shown to consist of double-stranded rings of the circumference of PM2 DNA, with one double-stranded DNA branch of distinct length, but always shorter than PM2 DNA. From these characteristics the 24 s precursor seems to be a PM2 DNA molecule with a growing chain in accordance with the hypothesis that chain growth involves addition of nucleotides to the end of one parental strand while a second strand is initiated de novo. The pulse-labeled precursor which sediments like nicked circular PM2 DNA does not contain label in single-strand rings but only in open strands. Experiments based on transfer of the infected cell from heavy to light medium suggest a mechanism of replication which involves random selection of the viral DNA molecule to be duplicated from among these present in the cell.

1. 1. After γ-irradiation (0.9–18.0·10 4 R) of ribomononucleotides in aqueous solution a decrease in absorbancy at 260 mμ of at least two different components is observed. This decrease is an exponential function of the dose. 2. 2. Alkaline and enzymic hydrolysis with RNAase or phosphodiesterase of the irradiated samples produces an hyperchromic effect. 3. 3. Paper chromatography shows that the irradiated product has a lower R F than the mononucleotides. After alkaline or phosphodiesterase hydrolysis the R F approaches again that of the controls. 4. 4. The irradiated samples were chromatographed on a DEAE-cellulose column. Stronger anions (polymers) with a nucleic acid-like absorption spectrum were separated from the original mononucleotides by elution with increasing concentrations of NH 4 HCO 3 . 5. 5. The polymers formed by radiation decrease or disappear if the samples are incubated with alkali or enzymes before adsorption on the column. 6. 6. There is a correlation between the size of the polymers formed and the dose of γ-rays used. 7. 7. Irradiated samples excited at 310 mμ have a fluorescence at 400 mμ, not shown by the mononucleotides, directly related to the dose and decreasing after alkaline hydrolysis. 8. 8. Irradiated deoxyribomononucleotides (14.4·10 4 R) show a decrease in absorbancy at 260 mμ, which is partially reversed after incubation with deoxyribonuclease. 9. 9. The polymerization through phosphodiester bonds after γ-radiation of ribomononucleotides is discussed.

The cytological pattern of infection of a host pseudomonad with PM2, a lipid-containing bacterial virus, was investigated by electron microscopy. Normal and infected cells frequently contain a myelin figure, which is found in the nucleoid region or at the periphery of the cell. The most striking finding in this investigation was that completed virions are found in the cell adjacent to or in association with the cytoplasmic membrane. This localization is precise; virions are not found elsewhere in infected cells. The completed virions occasionally appear to be attached to the cytoplasmic membrane. The virus contains a darkly staining core surrounded by a tripartite envelope of a thickness of approximately 70 A, which is identical to the thickness of the cytoplasmic membrane. Lysing cells appear to undergo extensive damage of the cytoplasmic membrane prior to rupture of the L layer of the cell wall.

Procedures are described for the isolation in pure form of the bacterial virus PM2. This virus isolated from seawater is shown to contain lipids by its sensitivity to ethyl ether and by chemical analysis of the purified virus. The virus has a buoyant density in CsCl of 1.28 g cm −3 and a sedimentation constant in 1 M NaCl of 230 S ; it contains 14% by weight of DNA and a minimum of 10% by weight of lipids. Phosphatidylethanolamine is the main lipid present in weight percent. The virus is disrupted in 0.05% Sarkosyl or sodium dodecyl sulfate. Analysis in acrylamide gel electrophoresis indicates the presence in the virus of at least two proteins, probably lipoproteins since they can be stained with Black Sudan. The virus appears to be a polyhedral object 60 mμ in diameter; the coat seems to have an unusual structure, in that an apparent double-layered wall can be observed in the electron microscope.

The DNA molecules isolated from mature bacteriophage PM2 are closed double-stranded rings of a molecular weight of six million. Direct evidence for the circularity and supercoiling of PM2 DNA was obtained by electron microscopy. Other properties, such as sedimentation behavior at neutral and alkaline pH, unusually high buoyant density in alkaline CsCl, and stability upon heating, are consistent with this structure. Only the four common nucleotides are found after hydrolysis and chromatographic analysis of PM2 DNA.

A marine bacterium, which acts as host for bacteriophage PM2 isolated from the same environment, has been characterized as a Pseudomonas species. This Pseudomonas cannot be assigned to any of the subgeneric categories or “groups” described by Stanier, Palleroni, and Doudoroff. The psychrophilic and halophilic nature of the organism and its requirement for NaCl suggest an indigenous marine origin.

Vegetative supercoiled DNA of bacteriophage PM2 has a lower superhelix density than does the viral supercoiled PM2 DNA for which it is a precursor.