Restoration by Chloramphenicol of Bacteriophage Production in Escherichia coli B Infected with a Ligase-Deficient Amber Mutant

The addition of chloramphenicol (CM) 5 min after infection of the nonpermissive host Escherichia coli B with the ligase-negative T4 amber, T4 AmH39X, allowed replication of parental deoxyribonucleic acid (DNA) and the production of high-molecular-weight progeny DNA, composed mostly of subunits with a D₂/D₁ of 0.6. When CM was removed after the accumulation of a large pool of this DNA, most of the infected bacteria were able to produce viable progeny phage, with an average yield of approximately 15 bacteriophage per bacterium. This phenomenon is called CM rescue of the ligase-negative T4 Am. CsCl and sucrose gradient analyses showed both the resulting phage and DNA extracted from them to be similar to the phage and DNA produced on the permissive host. The total transfer of the parental label to progeny phages was as high as 20%. In contrast, in bacteria not treated with CM or in bacteria to which CM was added after phage-coded nucleases had already been synthesized, both parental and progeny (newly synthesized) DNA was composed of very short fragments. Phage which are produced under conditions other than those of CM rescue are dead, light in CsCl, and contain only very short fragments of DNA. Parent-to-progeny transfer in this case is below 1%. When light radio-active parental DNA was used to infect heavy bacteria, DNA replicating in the CM rescue conditions assumed only a hybrid density. After removal of CM and maturation, the parental DNA was incorporated into progeny molecules in fragments constituting approximately 7 to 10% of its mass. This pattern of distribution is essentially what is observed in similar experiments in the permissive host. The role of ligase as an enzyme which compensates for the lethal action of phage-coded nuclease and which is stringently required for the repair of single-stranded nicks is emphasized. The possibility of specific sites for a unique cutting enzyme is discussed in connection with the hypothesis of a circularly permuted assembly of sets.     

On the mechanism of production of tandem genetic duplications in phage lambda

We have asked whether the mechanism by which tandem genetic duplications arise in the chromosome of phage lambda is inter- or intramolecular. Two parental phages carrying genetic markers at opposite ends of the phage chromosome have been grown in mixed infection, and progeny phages carrying newly-arising tandem duplications have been analysed to determine whether they carry the markers in parental or recombinant configuration. Ordinary genetic recombination of the markers has been prevented by mutations in the phage and host. Phages carrying tandem duplications are isolated by use of CsCl density gradients and an Escherichia coli strain that does not plate deletion phages. Of the duplication mutants isolated under these conditions, 13% carry the input markers in recombinant configuration. This suggests that tandem duplications can be produced via an intermolecular route which joins sequences originally present on different DNA molecules.     

Inactivation of bacteriophages by decay of incorporated radioactive phosphorus

The inactivation of the phages T1, T2, T3, T5, T7, and lambda by decay of incorporated P(32) has been studied. It was found that these phages fall into two classes of sensitivity to P(32) decay: at the same specific activity of P(32) in their deoxyribonucleic acid (DNA), T2 and T5 are inactivated three times as rapidly as T1, T3, T7, and lambda. Since the strains of the first class were found to contain about three times as much total phosphorus per phage particle as those of the second) it appears that the fraction of all P(32) disintegrations which are lethal is very nearly the same in all the strains. This fraction alpha depends on the temperature at which decay is allowed to proceed, being 0.05 at -196 degrees C., 0.1 at +4 degrees C., and 0.3 at 65 degrees C. Decay of P(32) taking place only after the penetration of the DNA of a radioactive phage particle into the interior of the bacterial cell can still prevent the reproduction of the parental phage, albeit inactivation now proceeds at a slightly reduced rate. T2 phages inactivated by decay of P(32) can be cross-reactivated; i.e., donate some of their genetic characters to the progeny of a mixed infection with a non-radioactive phage. They do not, however, exhibit any multiplicity reactivation or photoreactivation. The fact that at low temperatures less than one-tenth of the P(32) disintegrations are lethal to the phage particle and the dependence of the fraction of lethal disintegrations on temperature can be accounted for by the double stranded structure of the DNA macromolecule.     

Characteristics of PR5, a lipid-containing plasmid-dependent phage.

An extensive characterization of plasmid-dependent phage PR5 isolated from sewage has been carried out. The phage has a head diameter of 65--68 nm, is isometric with a double-layered capsid, and a minority possess tails. It adsorbs to many but not all types of bacteria possessing P, N, or W plasmids. The phage contains 20% lipid, 15.1% DNA, and 64.9% protein by weight and has a buoyant density of 1.265 g/ml in CsCl. The DNA is double-stranded with a G + C content of 49% and a molecular weight of 7.4 +/- 0.6 x 10 (6) as shown by electron microscopy. Phospholipid content is 66% of lipid and consists of cardiolipin (13%), phosphatidylethanolamine (43%), and phosphatidylglycerol (44%) and differ quantitatively from that of host bacteria. Anti-PR5 serum inactivates other similar phages, PR3 and PR4. Phage adsorption is impaired in deep rough mutants of Salmonella minnesota.     

Intermediates in genetic recombination of bacteriophage T7 DNA. Biological activity and the roles of gene 3 and gene 5

When Escherichia coli cells were infected with ³²P- and 5-bromodeoxyuridine-labeled T7 bacteriophage defective in genes 1.3, 2.3, 4 and 5, doubly branched T7 DNA molecules with “H” or “X”-like configurations were found in the half-heavy density fractions. Physical study showed that they are dimeric molecules composed of two parental DNA molecules (Tsujimoto & Ogawa, 1977a). The transfection assay of these molecules revealed that they were infective. Genetic analysis of progeny in infective centers obtained by transfection of dimeric molecules formed by infection of genetically marked T7 phage showed that these dimeric molecules were genetically biparental.To elucidate the roles of the products of gene 3 (endonuclease I) and gene 5 (DNA polymerase) of phage T7 in the recombination process, the ³²P/BrdUrd hybrid DNA molecules which were formed in the infected cells in the presence of these gene products were isolated, and their structures were analyzed. The presence of T7 DNA polymerase seems to stimulate and/or stabilize the interaction of parental DNAs. At an early stage of infection few dimeric molecules were formed in the absence of T7 DNA polymerase, whereas a significant number of doubly branched molecules were formed in its presence. With increasing incubation time, the multiply branched DNA molecules with a high sedimentation velocity accumulated.In contrast to the accumulation of multiply branched molecules in phage with mutations in genes 2, 3 and 4, almost all of the ³²P/BrdUrd hybrid DNA formed in phage with mutations in genes 2 and 4 were monomeric linear molecules. Shear fragmentation of monomeric linear ³²P/BrdUrd-labeled DNA shifted the density of [³²P]DNA to almost fully light density. It was also found that approximately 50% of [³²P]DNA was linked covalently to BrdUrd-labeled DNA. These linear monomer DNA molecules had infectivity and some of those formed by infection of genetically marked parents yielded recombinant phages. Therefore the gene 3 product seems to process the branched intermediates to linear recombinant molecules by trimming the branches.     

Control of the Replication Complex of Bacteriophage P22

A replication complex for the vegetative synthesis of the deoxyribonucleic acid (DNA) of the temperate phage P22 previously has been described. This complex is an association of parental phage DNA, most of the newly synthesized phage DNA made during pulses with (3)H-thymidine, and other cell constituents, and has a sedimentation rate in neutral sucrose gradients of at least 1,000S. The complex is one of the intermediates, intermediate I, in the synthesis and maturation of phage P22 DNA after infection or induction. Evidence supporting the replicative nature of intermediate I is presented. Phage replication is repressed in lysogenic bacteria. On superinfection of P22 lysogens with nonvirulent phage, little association of the input phage DNA with a rapidly sedimenting fraction is demonstrable. However, after induction with ultraviolet light, the superinfecting parental phage DNA quickly acquires the rapid sedimentation rate characteristic of intermediate I; phage DNA synthesis follows; and progeny phages are produced. Infection with a virulent mutant of P22 produces progeny phages in lysogens. Its DNA associates with intermediate I. In mixed infection with the virulent phage, replication of nonvirulent phage P22 is still repressed, even though the virulent replicates normally. The nonvirulent input DNA does not associate with intermediate I. The repressor of the lysogenic cell prevents replication by interfering with the physical association of template material with intermediate I. A phage function is required for association of phage template with the replication machinery.     

Effects of UV irradiation on the fate of 5-bromodeoxyuridine-substituted bacteriophage T4 DNA.

We have carried out a series of experiments designed to characterize the impact of UV irradiation (260 nm) on 5-bromodeoxyuridine-labeled (heavy) T4 bacteriophage, both before and after infection of Escherichia coli. In many respects, these effects differ greatly from those previously described for non-density-labeled (light) phage. Moreover, our results have led us to propose a model for a novel mechanism of host-mediated repair synthesis, in which excision of UV-damaged areas is followed by initiation of replication, strand displacement, and a considerable amount of DNA replication. UV irradiation of 5-bromodeoxyuridine-labeled phage results in single-stranded breaks in a linear, dose-dependent manner (1.3 to 1.5 breaks per genomic strand per lethal hit). This damage does not interfere with injection of the phage genome, but some of the UV-irradiated heavy phage DNA undergoes additional intracellular breakdown (also dose dependent). However, a minority (25%) of the injected parental DNA is protected, maintaining its preinjection size. This protected moiety is associated with a replicative complex of DNA and proteins, and is more efficiently replicated than is the parental DNA not so associated. Most of the progeny DNA is also found with the replicative complex. The 5-bromodeoxyuridine of heavy phage DNA is debrominated by UV irradiation, resulting in uracil which is removed by host uracil glycosylase. Unlike the simple gap-filling repair synthesis after infection with UV-irradiated light phage, the repair replication of UV-irradiated heavy phage is extensive as determined by density shift of the parental label in CsC1 gradients. The newly synthesized segments are covalently attached to the parental fragments. The repair replication takes place even in the presence of chloramphenicol, a protein synthesis inhibitor, suggesting it is host mediated. Furthermore, the extent of the repair replication is greater at higher doses of UV irradiation applied to the heavy phage. This abundant synthesis results ultimately in dispersion of the parental sequences as short stretches in the midst of long segments of newly synthesized progeny DNA. Together, the extensive replication and the resulting distribution pattern of parental sequences, without significant solubilization of parental label, are most consistent with a model of repair synthesis in which the leading strand displaces, rather than ligates to, the encountered 5' end.     

Absence of interparental recombination in multiplicity reconstitution from incomplete bacteriophage T4 genomes.

Interparental recombination between injected T4 DNA molecules is indetectable for incomplete petite phages (carrying a terminally deficient genome and therefore unable to circularize) as well as for genetically complete phages. The nonvialbe petite phages can individually replicate their DNA repeatedly, and they aso undergo multiplicity reconstitution, producing complete phages, provided that a host bacterium is infected by several petite particles that carry genetically complementary segments of DNA. The formation of complete phages in multiplicity reconstitution must be due to recombination among incomplete progeny fragments, i.e., partial replicas of the T4 genomes. It evidently does not result from interparental recombination. To test for interparental recombination, light bacteria (containing no bromouracil) were simultaneously infected in light medium with light radioactive phage in minority (usually less than one per cell) and heavy (bromouracil-labeled) phage in majority (usually about nine per cell). Any interparental recombination should, under these circumstances of infection, head to movement of the radioactive label of the minority light phage DNA to a position of higher density. That possibility was not observed.     

Intracellular Development of Bacteriophage φR: II. Fractionation of Replicative Form Deoxyribonucleic Acid Associated with Rapidly Sedimenting Host Cell Components

When Escherichia coli is infected with bacteriophage phiR, parental deoxyribonucleic acid (the single- or double-stranded DNA containing the isotopic label of the infecting phage) becomes firmly attached to a cellular structure and can be isolated as a rapidly sedimenting component as described earlier for phiX174. If this component is centrifuged to equilibrium, two peaks of infective DNA are observed at densities of 1.30 and 1.15 g/ml. At low multiplicities of infection, (32)P-labeled parental DNA is found associated with only the cellular components in the dense band; as the multiplicities of infection are increased, the dense band becomes saturated and parental DNA molecules are then found at the light density as well. Actively replicating host DNA is found only in the dense band, whereas progeny DNA, which does not replicate semiconservatively, can become associated with cellular components in the light band. This fractionation of cellular components on the basis of their buoyant density separates primary sites of DNA replication associated with the dense band from nonfunctional binding sites in the light band.     

The rolling circle . capsid complex as an intermediate in phi X DNA replication and viral assembly

Late in the life cycle of the single-stranded DNA phage phi X, the synthesis of positive strand DNA is coupled to the maturation of progeny virions. DNA synthesis and packaging take place in a replication-assembly complex, which we have purified to homogeneity and characterized. The following conclusions can be drawn: 1. The DNA component of the replication-assembly complex is a rolling circle with a single-stranded DNA tail which is less than or equal to genome length. 2. The major protein component of the replication-assembly complex is an intact viral capsid, as shown by gel analysis of 35S-labeled complexes. As replication proceeds at the DNA growing point, the positive strand tail of the rolling circle is displaced directly into the capsid. In addition to the capsid, the complex contains at least 1 molecule of the phi X gene A nicking protein, which appears to be covalently linked to the DNA. 3. The rolling circle . capsid complex can be purified to homogeneity by taking advantage of its uniform sedimentation velocity (35 S) and its uniform density upon equilibrium centrifugation in CsCl (1.50 g/cc). 4. The replication-assembly complex can be visualized in the electron microscope. An electron-dense particle, which has the dimensions of a viral capsid, is observed attached to a rolling circle at the DNA growing point. 5. Hybridization of specific phi X restriction fragments to the deproteinized, single-stranded tails of intact rolling circles has allowed the use of these replicating intermediates to determine both the origin/terminus and the direction of phi X positive strand DNA synthesis. The ends of the rolling circle tails map in the Hae III restriction Fragment Z6b, at the position on the phi X genome at which the gene A endonuclease is known to cut. This result indicates that this endonuclease participates in the "termination" of each round of synthesis by cutting off unit-length viral genomes. 6. Rolling circle . capsid complexes were also isolated from two other icosahedral, single-stranded DNA phages: G4 and St-1. The rolling circle . capsid complex seen in the case of the single-stranded DNA phages represents the first example of a structure in which DNA synthesis and viral assembly occur in a coupled manner. This tight coordination explains why double-stranded DNA circles are the net product of synthesis early in the viral life cycle while only single-stranded DNA circles are produced later. The single-stranded tails of the rolling circle intermediates are available for conversion to the duplex state at early times, whereas the concentration of preformed capsids later is high enough to bind to all of the replicating molecules and package the emerging positive strand DNA.     

Synthesis of the Unusual DNA of Bacillus subtilis Bacteriophage SP-15

Cultures of Bacillus subtilis infected with phage SP-15 were examined to investigate the metabolic origin of two of the unique components of the phage DNA: the component responsible for the unusually high buoyant density in CsCl and the unusual pyrimidine, 5-(4', 5'-dihydroxypentyl) uracil (DHPU). Newly synthesized pulse-labeled DNA was light in buoyant density and shifted to the high density of mature phage DNA upon further incubation. Parental DNA was converted to a light-density intermediate form prior to replication. When labeled uracil, thymidine, or DHPU were added to infected cells, it was found that only uracil served as the precursor to DHPU and thymine in phage DNA. Analysis of the bases from hydrolyzed DNA of labeled phage or infected cells indicated that the uracil was incorporated into the DNA as such (presumably via deoxyuridine triphosphate) and later converted to DHPU and thymine at the macromolecular level. The sequence of events after phage infection appeared to be: (i) injection of parental DNA; (ii) conversion of parental DNA to a light form; (iii) DNA replication, yielding light DNA containing uracil; (iv) conversion of uracil to DHPU and thymine; and (v) addition of the heavy component.     

The molecular structure of the transducing particles of Salmonella phage P22. II. Density gradient analysis of DNA

Summary CsCl density gradient analysis showed that the DNA of plaque forming particles ofSalmonella phageP22 is lighter than the host DNA. The DNA of transducing phages exhibits an intermediate density, but close to host DNA. BU labelling of DNA synthesized in the cells after phage infection resulted in a density increase of transducing DNA of about 0.004 gxcm^-3, whereas infectious DNA increased by about 0.045 gxcm^-3. Shearing of isolated DNA molecules from unlabelledP22 lysates demonstrated that transducing DNA consists of two pieces of DNA of different density: 90% stem from the bacterial host whereas 10% are phage DNA and therefore responsible for the BU lable in transducing phages.     

The effect of 5-Iodouracil on the growth and biosynthetic processes of bacteriophage T4td8 in the absence of light

5-Iodouracil (IUra)-substituted progeny bacteriophage T4td8 were grown under conditions such that, upon CsCl equilibrium isopycnic gradient centrifugation, progeny with density distributions about the median similar to that of unsubstituted phage are obtained. In the absence of light a monotonie relationship exists between decreasing progeny viability and increasing percent IUra substitution. IUra is equivalent to thymine as a growth factor on a molar basis, and at concentrations of IUra plus thymine above that required for maximum particle production, the percent IUra substitution in phage DNA is determined by the mole fraction of IUra in the medium. The lethal effects of 5-iodo-2'-deoxyuridine (IdUrd) and IUra are equivalent, and are not produced by a direct effect on the phage particles. At equivalent percent substitution in phage DNA the order of lethality is IUra > 5-bromouracil (BrUra) > 5-chlorouracil (ClUra). There is no interference with the transfer of thymine from host cell to progeny phage by the presence of IUra in the medium, and IUra affects neither the time of lysis nor the content of phage DNA in the infected cells.     

Recombinational-type transfer of viral DNA during bacteriophage 2C replication in Bacillus subtilis.

The Bacillus subtilis phage 2C contains one molecule of double-stranded DNA of about 100 x 10(6) daltons in which thymine is replaced by hydroxymethyluracil; the two strands have different buoyant densities. Parental DNA, labeled with either [3H]uracil of [32P]phosphate, was quite effectively transferred to offspring phage, and the efficiency of transfer was the same for the two strands. Labeled nucleotide compositions of the H and L strands from parental and progeny virions were very close. These data exclude a degradation of the infecting DNA and reutilization of nucleotides. Upon infection of light unlabeled cells with heavy radioactive viruses, no DNA with either heavy or hybrid density was extracted from offspring phage. Instead, an heterogeneous population of DNA molecules of densities ranging from that of almost hybrid to that of fully light species was obtained. Shear degradation of such progeny DNA to fragments of decreasing molecular weight produced a progressive shift to the density of hybrid molecules. Denaturation of sheared DNA segments caused the appearance of labeled and heavy single-stranded segments. These findings indicate that 2C DNA replicates semiconservatively and then undergoes extensive genetic recombination with newly formed viral DNA molecules within the vegatative pool, thus mimicking a dispersive transfer of the infecting viral genome. The pieces of transferred parental DNA have an average size of 10 x 10(6) daltons.     

Mechanism of the Inactivation of the Bacteriophage T1 in Aerosols

The mechanism of inactivation of bacteriophage T(1) in aerosols was studied by using (32)P-labeled phage. During inactivation, viability decreased in parallel with the adsorption of (32)P to the host, showing either that inactivated phage does not adsorb or that the deoxyribonucleic acid (DNA) has left the coat. A (32)P band at the density of free DNA was found when inactivated phage was analyzed in a CsCl gradient.     

Characterization of Inducible Bacteriophages in Bacillus licheniformis

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.     

Monoclonal Antibodies Raised against Native Major Capsid Proteins of Lactococcal c2-Like Bacteriophages

Phage Q38, a representative member of the c2 species, was purified by CsCl gradient and used to immunize BALB/c mice. Monoclonal antibodies (MAbs) were raised and then characterized by enzyme-linked immunosorbent assay. Two MAbs of isotype immunoglobulin G2a, designated 2A5 and 6G7, reacted only with phages belonging to the c2 species and not with phages of the 936 and P335 species, with a Lactococcus lactis cell extract, or with phage DNA. Immunoelectron microscopy showed that both MAbs recognized only phage head proteins. They did not react with any denatured phage proteins in Western blot assays. However, when the nitrocellulose membranes were treated with a Triton-based buffer to assist in protein renaturation, MAbs 2A5 and 6G7 recognized the two major capsid proteins with molecular masses of 80 and 170 kDa. Competitive inhibition tests showed that the two MAbs bind to overlapping epitopes. These MAbs may be a useful tool for monitoring c2 bacteriophages during dairy fermentation and in genetic studies.     

Characterization of the bacteriophage B2 of Lactobacillus plantarum ATCC 8014

Bacteriophage B2 of Lactobacillus plantarum ATCC 8014, isolated in 1971, belonged to Bradley's group B. Electron microscopy revealed an isometric head (110 nm) and a long non-contractile and flexible tail (500 nm) containing about 75 regularly aligned lateral striations. Burst size was 12-14 phages per infectious centre. The latent period for phage development was 75 min and the rise period approximately 90 min. The phage particle contained 5 major proteins. The buoyant density of the phage in CsCl was measured as 1.575 g/cm3. B2 genome was a linear double-stranded DNA molecule of 37 +/- 1% guanosine-cytosine. Its size was 73 kilobase pairs (kbp). Restriction analysis of the genome showed that 4 restriction enzymes (Xba I, Sac I, Bgl II and Sma I) gave single site cuts in the DNA, while Ava I and Sal I formed 2 and 5 cuts, respectively.     

Study of the diversity in a group of phages of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> species PB1 (Myoviridae) and their behavior in adsorbtion-resistant bacterial mutants

A group of 12 Pseudomonas aeruginosa virulent bacteriophages of different origin scored with regard to the plaque phenotype are assigned to PB1-like species based on the similarity in respect to morphology of particles and high DNA homology. Phages differ in restriction profile and the set of capsid major proteins. For the purpose of studying adsorption properties of these phages, 20 random spontaneous mutants of P. aeruginosa PAO1 with the disturbed adsorption placed in two groups were isolated. Mutants of the first group completely lost the ability to adsorb all phages of this species. It is assumed that their adsorption receptors are functionally inactive or lost at all, because the attempt to isolate phage mutants or detect natural phages of PB1 species capable of overcoming resistance of these bacteria failed. The second group includes five bacterial mutants resistant to the majority of phages belonging to species PB1. These mutants maintain the vigorous growth of phage SN and poor growth of phage 9/3, which forms turbid plaques with low efficiency of plating. In the background of weak growth, phage 9/3 yields plaques that grew well. The examination of the progeny of phage 9/3, which can grow on these bacteria, showed that its DNA differed from DNA of the original phage 9/3 by restriction profile and is identical to DNA of phage PB1 with regard to this trait. Data supported a suggestion that this phage variant resulted from recombination of phage 9/3 DNA with the locus of P. aeruginosa PAO1 genome encoding the bacteriocinogenic factor R. However, this variant of phage 9/3 did not manifest the ability to grow on phage-resistant mutants of the first group. Possible reasons for the difference between phages 9/3 or SN and the remaining phages of PB1 species are discussed. A preliminary formal scheme of the modular structure for adsorption receptors on the surface of P. aeruginosa PAO1 bacteria was constructed based on the analysis of growth of some other phage species on adsorption mutants of the first type.     

Independent functions of viral protein and nucleic acid in growth of bacteriophage

1. Osmotic shock disrupts particles of phage T2 into material containing nearly all the phage sulfur in a form precipitable by antiphage serum, and capable of specific adsorption to bacteria. It releases into solution nearly all the phage DNA in a form not precipitable by antiserum and not adsorbable to bacteria. The sulfur-containing protein of the phage particle evidently makes up a membrane that protects the phage DNA from DNase, comprises the sole or principal antigenic material, and is responsible for attachment of the virus to bacteria. 2. Adsorption of T2 to heat-killed bacteria, and heating or alternate freezing and thawing of infected cells, sensitize the DNA of the adsorbed phage to DNase. These treatments have little or no sensitizing effect on unadsorbed phage. Neither heating nor freezing and thawing releases the phage DNA from infected cells, although other cell constituents can be extracted by these methods. These facts suggest that the phage DNA forms part of an organized intracellular structure throughout the period of phage growth. 3. Adsorption of phage T2 to bacterial debris causes part of the phage DNA to appear in solution, leaving the phage sulfur attached to the debris. Another part of the phage DNA, corresponding roughly to the remaining half of the DNA of the inactivated phage, remains attached to the debris but can be separated from it by DNase. Phage T4 behaves similarly, although the two phages can be shown to attach to different combining sites. The inactivation of phage by bacterial debris is evidently accompanied by the rupture of the viral membrane. 4. Suspensions of infected cells agitated in a Waring blendor release 75 per cent of the phage sulfur and only 15 per cent of the phage phosphorus to the solution as a result of the applied shearing force. The cells remain capable of yielding phage progeny. 5. The facts stated show that most of the phage sulfur remains at the cell surface and most of the phage DNA enters the cell on infection. Whether sulfur-free material other than DNA enters the cell has not been determined. The properties of the sulfur-containing residue identify it as essentially unchanged membranes of the phage particles. All types of evidence show that the passage of phage DNA into the cell occurs in non-nutrient medium under conditions in which other known steps in viral growth do not occur. 6. The phage progeny yielded by bacteria infected with phage labeled with radioactive sulfur contain less than 1 per cent of the parental radioactivity. The progeny of phage particles labeled with radioactive phosphorus contain 30 per cent or more of the parental phosphorus. 7. Phage inactivated by dilute formaldehyde is capable of adsorbing to bacteria, but does not release its DNA to the cell. This shows that the interaction between phage and bacterium resulting in release of the phage DNA from its protective membrane depends on labile components of the phage particle. By contrast, the components of the bacterium essential to this interaction are remarkably stable. The nature of the interaction is otherwise unknown. 8. The sulfur-containing protein of resting phage particles is confined to a protective coat that is responsible for the adsorption to bacteria, and functions as an instrument for the injection of the phage DNA into the cell. This protein probably has no function in the growth of intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from the experiments presented.