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An approach to correlate functions with cistrons in bacteriophage PM2 is presented. Two of the temperature-sensitive mutants obtained differed from the wild type in heat sensitivity and four differed in host range. Comparison of the electrophoretic patterns of DNA from cells infected at the restrictive temperature with those obtained in wild-type infection revealed that viral DNA bands were absent with three additional mutants. Complementation analysis assigned the four host range mutants to cistron I and the three mutants defective in DNA synthesis to cistron IV. Recombination frequencies were used to locate cistrons III and IV on the partial genetic map of PM2.

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.

Four hundred strains of enterobacteria, isolated before the introduction of trimethoprim as a single agent in Chile, were studied. One hundred and thirty six strains (34%) were resistant to greater than or equal to 8 mg/l of trimethoprim and 118 of these (87%) were also resistant to sulphamethoxazole (156 mg/l). Plasmids were detected in 79% of the trimethoprim-resistant strains; 56% of the strains carried two or more plasmids. Trimethoprim resistance could be transferred by conjugation in 66% of the plasmid-carrying strains; this resistance was co-transferred with a variety of other antibiotic resistance determinants.

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.