Intracellular Inactivation of Bacteriophage T4 Early Deoxyribonucleic Acid

As previously shown, a small amount of polynucleotide material is added to parental T4 deoxyribonucleic acid (DNA) molecules within the first 5 min of infection. I have asked whether this process is essential for phage replication. Two approaches-one involving decay of (32)P incorporated into this "early DNA" and the other involving photoinactivation of bromodeoxyuridine-containing early DNA-indicate that it is.     

Inhibition of Replication of Ribonucleic Acid Bacteriophage f2 by Superinfection with Bacteriophage T4

Superinfection by phage T4 of cells infected by the ribonucleic acid (RNA) phage f2 results in inhibition of further f2 production. Experiments using rifampin show that the exclusion of f2 requires T4 gene function soon after T4 infection. By using a sensitive new peptide-mapping procedure to identify f2 coat protein in infected cells, we show that synthesis of the f2 coat occurs at a reduced level until 4 min after T4 superinfection and then ceases abruptly. Within 4 min after T4 superinfection, there are also several changes in f2 RNA metabolism, all of which require T4 gene function: preexisting f2 replicative intermediate RNA and f2 single-stranded RNA are degraded to small but still acid-precipitable fragments, and most f2-specific RNA is released from polyribosomes. We favor the hypothesis that T4 induces the synthesis of a specific endoribonuclease which degrades f2 RNA and that the inhibition of f2 protein synthesis may be a consequence of this degradation, rather than a direct effect of T4 upon translation.     

Ribonucleotides Covalently Linked to Deoxyribonucleic Acid in T4 Bacteriophage

Bacteriophage T4 was grown in the presence of labeled uridine. The deoxyribonucleic acid (DNA) of the phage was shown to contain covalently attached ribonucleotides. The label appears not to be internal in the DNA strands. Presumably, it is at the ends of the DNA strands and this may be related to DNA initiation.     

Role of Gene 46 in Bacteriophage T4 Deoxyribonucleic Acid Synthesis

In an attempt to establish whether Escherichia coli B infected with N130 (an amber mutant defective in gene 46) is recombination-deficient, the postinfection fate of (14)C-labeled N130 parental deoxyribonucleic acid (DNA) was followed, its amount in complex with the host cell membrane being determined in sucrose gradients after mild lysis of the infected cells. The parental DNA was found to undergo gradual detachment from the membrane during infection. Pulse-chase experiments similarly showed that newly synthesized DNA is normally attached to the host cell membrane and is detached by endonucleolytic breakage at a late stage of infection. The conclusion is that only attached DNA molecules are replicated by membrane-bound replicase, whereas those detached by endonucleolytic breakage are not. It thus seems that the gene 46 product controls the activity of a nuclease whose main function is recombination of DNA nicked by endonuclease, thereby attaching it to the host cell membrane. The rate of T4 DNA synthesis is apparently governed by the efficiency of recombination. Supporting evidence was found in experiments with the double mutant N130 x N134 (genes 46, 33).     

Capsid Size and Deoxyribonucleic Acid Length: the Petite Variant of Bacteriophage T4

A mutant which produces a small-headed ("petite") variant of bacteriophage T4 is described. The mutation (E920g) maps in a new gene (66) between genes 23 and 24. Petite phage particles composed up to 70% of the phage yield. The petite phage was nonviable upon single infection but produced progeny when two or more infected a cell. Its genome was shortened by a random deletion of about 30%, and deoxyribonucleic acid (DNA) extracted from the particles was 0.68 the length of normal T4 DNA. The reduction in DNA length was accompanied by a proportional reduction in head volume. Double mutants between E920g and head-defective mutants in gene 21 produced unusually high frequencies of spherical capsidlike structures (tau-particles).     

Replicative Intermediates of Bacteriophage T7 Deoxyribonucleic Acid

After infection with bacteriophage T7, parental and newly synthesized deoxyribonucleic acid (DNA) exhibit an extremely fast sedimentation rate in neutral sucrose gradients. This fast-sedimenting component (intermediate I) has a sedimentation constant of about 1,500S and contains T7 DNA as determined by DNA-DNA hybridization experiments. Pulse-chase experiments indicate that the fast-sedimenting material is metabolically active and serves as a precursor to the formation of T7 DNA. Intermediate I contains about 2.5 to 7% of the total ³H-labeled protein formed between 3 and 9.5 min after T7 infection. Treatment of intermediate I with Pronase results in the release of the DNA from the complex. At early times after infection, a second intermediate (intermediate II) can be detected which contains both parental and newly synthesized DNA sedimenting slower than intermediate I but 2 to 3 times as fast as mature T7 DNA. Intermediates I and II containing parental DNA are formed after infection of the nonpermissive host with an amber mutant in gene 1, a gene whose expression is necessary for the synthesis of most T7 proteins. The two intermediates are also observed when infection with T7 wild type is carried out in the presence of chloramphenicol.     

Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid I. Tertiary Structure of Early Replicative and Recombining Deoxyribonucleic Acid

A replicative hybrid resulting from the infection of heavy (substituted with 5-bromodeoxyuridine) bacteria with light (not substituted with 5-bromodeoxyuridine) radioactive bacteriophage was isolated from a CsCl density gradient. Sedimentation studies indicate that 60% of the deoxyribonucleic acid (DNA) behaves as if it were in units more than four times as large as an intact reference molecule. Under the electron microscope, hybrid molecules appeared tangled, showed puffs and loops, occupied a small area, and often had a total length twice that of mature phage. This indicates that sucrose gradient sedimentation is not applicable as a method for estimating the relative molecular size of replicative forms of DNA. After denaturation, the separated strands of hybrid were of the same size as those of reference DNA. CsCl density gradient analysis revealed no terminal covalent addition of new material to the old parental strand. The possibility of a continuous growth of the DNA molecule, either on a single-stranded level or as a double helical structure, is disproved. When chloramphenicol (CM) was added at critical times after infection, DNA synthesis continued at a constant rate. The parental label soon assumed and retained a hybrid density, despite concomitant synthesis of DNA, throughout the rest of the period of incubation in CM. The hybrid moiety, however, actively participated in replication and exchanged its partner strand for a new one; this was demonstrated by changing the density label during incubation in CM. A new enzyme synthesized shortly after infection introduced single-stranded "nicks" into the parental DNA. Since nicking can be inhibited by chloramphenicol, the responsible enzyme is not of host origin. The time of the appearance of this enzyme coincided with the onset of molecular recombination. Another enzyme, which mediates the repair of the continuity of the polynucleotide chain after recombination, appeared after recombination. If selectively inhibited by chloramphenicol, recombinant molecules remained unrepaired, and, upon denaturation, the parental fragment was liberated in pure form.     

Association of Replicative T4 Deoxyribonucleic Acid and Bacterial Membranes

Experiments utilizing CsCl density gradient analysis and radioactive labels specific for bacteriophage T4 deoxyribonucleic acid (DNA) and membranes have shown that replicative T4 DNA is associated with host membranes. The association is inhibited by chloramphenicol and takes place just prior to semi-conservative replication of the phage DNA.     

Effect of Myxin on Deoxyribonucleic Acid Synthesis in Escherichia coli Infected with T4 Bacteriophage

Exposure of Escherichia coli cells to myxin results in the almost complete inhibition of new deoxyribonucleic acid (DNA) synthesis, extensive degradation of pre-existing intracellular DNA, and a rapid loss of viability in these cells (9). After exposure to myxin for 30 min (<1% survivors and >25% degradation of DNA), infection of these cells by T4 bacteriophage results in the renewal of DNA synthesis at a rate essentially equal to that found in T4-infected cells in the absence of myxin. This DNA was characterized as T4 DNA by hybridization and by hydroxyapatite chromatography. These results suggest that the primary site of action of myxin does not involve the biochemical pathways involved in either the energy metabolism or the biosynthesis of DNA precursors in the uninfected host cell. The yield of infectious T4 particles was reduced when myxin was present during multiplication. This effect may be partly accounted for by the finding that a significant fraction of the T4 DNA synthesized in the presence of myxin is apparently not properly enclosed by the bacteriophage protein coat since it is shown to be degraded by exogenous nuclease.     

Defective Deoxyribonucleic Acid Replication of T4rII Bacteriophage in Lambda-Lysogenic Host Cells

Sedimentation of the replicative deoxyribonucleic acid through alkaline sucrose gradients showed that rII single chains reached the half-mature size at a time when wild-type molecules formed long chains (dimers and trimers of genome size). Long rII single chains could be observed on substitution of tris(hydroxymethyl)aminomethane buffer for Na⁺K⁺ phosphate in the growth medium.     

Host Cell Deoxyribonucleic Acid Polymerase I and the Support of T4 Bacteriophage Growth

Ultraviolet irradiation of Escherichia coli polA(-) cells reduces their capacity to support the growth of T4 phage. There is no additional loss of capacity observed in pol tsA(-)recA(-) double mutants at the nonpermissive temperature. The reversion frequency of a T4 rII mutant after ultraviolet irradiation is not changed by the absence of host deoxyribonucleic acid polymerase I.     

Localization of Parental Deoxyribonucleic Acid from Superinfecting T4 Bacteriophage in Escherichia coli

High-resolution autoradiography has been employed to localize the nonsolubilized but genetically excluded deoxyribonucleic acid (DNA) of T4 bacteriophage superinfecting endonuclease I-deficient Escherichia coli. This DNA was found to be associated with the cell envelope (this term is used here to include all cellular components peripheral to and including the cytoplasmic membrane); in contrast, T4 DNA in primary infected cells, like host DNA in uninfected E. coli, was found to be near the cell center. The envelope-associated DNA from super-infecting phage was not located on the outermost surface of the cell since it was insensitive to deoxyribonuclease added to the medium. These results suggest that DNA from superinfecting T-even phage is trapped within the cell envelope.     

Polyamines and the Delay in Deoxyribonucleic Acid Synthesis in Some Bacteriophage T4 Infections

Escherichia coli B infected by the DD mutant of T4, am N116, is stimulated to initiate deoxyribonucleic acid accumulation by 1 to 10 mm spermidine but not by 10 mm putrescine. The syntheses of putrescine and spermidine in cells infected by T4D and the mutant are similar, although slight differences are observed in the intracellular concentration of free spermidine. Unlike r-K12 (lambda) systems, am N116-infected cells do not leak polyamine.     

Equal Incorporation of Both Parental Bacteriophage T7 Deoxyribonucleic Acid Strands into Intracellular Concatemeric Deoxyribonucleic Acid

After infection of Escherichia coli B with radiolabeled T7 bacteriophage, the parental deoxyribonucleic acid label was found in both polynucleotide chains of the intracellular T7 concatemer.     

Repair of Alkylated Bacteriophage T4 Deoxyribonucleic Acid by a Mechanism Involving Polynucleotide Ligase

Methyl methanesulfate-induced lesions in bacteriophage T4 are repaired primarily by a mechanism involving polynucleotide ligase. Apparently, other recombinational and ultraviolet repair functions aren't involved.     

Transfection of Staphylococcus aureus with Bacteriophage Deoxyribonucleic Acid

Staphylococcus aureus cells of strain 8325 (N) are competent for phage deoxyribonucleic acid (DNA) when harvested in the early exponential growth phase. Phenotypic expression of the competence requires divalent cations, and calcium ions are most effective. Treatment of phage DNA with deoxyribonuclease completely destroys infectivity and heat-denaturated DNA is not infectious. The highest frequency of transfection is around 10(4) plaque-forming units per mug of DNA.     

Correlation Between Degradation of Bacteriophage T2 Deoxyribonucleic Acid and the Resistance of Escherichia coli to Infection

The ability of certain strains of Escherichia coli to degrade T2 deoxyribonucleic acid to acid-soluble fragments is correlated with their high capacity to survive T2 infection.     

Infection of Bacillus stearothermophilus with Bacteriophage Deoxyribonucleic Acid

Early stationary-phase cells are most susceptible to infection with deoxyribonucleic acid from bacteriophage TP-1C. Transfection is destroyed by deoxyribonuclease and unaffected by phage antiserum.