Nucleic Acid Synthesis in Bacteriophage SPO2c1-Infected Bacillus subtilis

The synthesis of host macromolecules was shut off very slowly and incompletely by bacteriophage SPO2c(1). No change in the rate of incorporation of radioactive precursors into protein and ribonucleic acid (RNA) could be detected after infection, and the rate of incorporation of thymidine was increased only slightly. The relative proportions of phage and host species of nucleic acids at various intervals in the latent period were determined by means of nucleic acid hybridization. Phage-specific RNA populations synthesized early were different from those synthesized late in the latent period. Host deoxyribonucleic acid (DNA) replication continued until 8 to 10 min after SPO2c(1) infection and then decreased markedly as phage-specific DNA synthesis was initiated. Host DNA was not degraded to trichloroacetic acid-soluble fragments, and its nucleotides were not found in either newly synthesized intracellular phage DNA or in progeny phage particles. The average burst size of SPO2c(1) was approximately 200 plaque-forming units per cell.     

Shutoff of Host RNA Synthesis in Bacteriophage T4-Infected Escherichia coli in the Absence of Host DNA Degradation and Nuclear Disruption

In contrast to its effect on host DNA synthesis, nuclear disruption in phage T4-infected Escherichia coli B/5 cells has no effect on the shutoff of host RNA synthesis. Host RNA synthesis is shut off normally after infection with T4 multiple mutants that fail to induce both nuclear disruption and host DNA degradation.     

Morphology and Physiology of the Intracellular Development of Bacillus subtilis Bacteriophage φ25

The morphology of the intracellular development of bacteriophage phi25 in Bacillus subtilis 168M has been correlated with nucleic acid synthesis in infected cells. Host deoxyribonucleic acid (DNA) synthesis was shut off by a phage-induced enzyme within 5 min after infection, and another phage-mediated function extensively degraded host DNA at the time of cell lysis. Synthesis of phage DNA in infected cells began within 5 min and continued until late in the rise period. After phage DNA synthesis and coinciding with lysis, much of the unpackaged, newly synthesized phage DNA was degraded. Studies of thin sections of phi25 infected cells suggested that unfilled capsids may be precursors to filled capsids in the packaging process. To assess dependence of capsid formation on phage DNA replication, cells were either treated with mitomycin C and infected with normal phage or infected with ultraviolet-irradiated (99% killed) phi25. Only empty capsids were found in these cells, indicating that capsid production may be independent of the presence of newly synthesized viral DNA.     

Macromolecular synthesis in mycobacteriophage I3 infected cells

DNA-, RNA- and protein synthesis have been studied inMycobacterium smegmatis cells infected with phage 13. The macromolecular synthesis continued until the end of latent period. Early RNA and protein synthesis were necessary prior to the commencement of DNA replication. The infecting phage DNA sedimented as larger than unit length of genome, after initiation of DNA synthesis. Although the host DNA was not degraded, 90 percent of the RNA synthesized after phage infection hybridized to phage DNA.     

Control of Gene Function in Bacteriophage T4: III. Preventing the Shutoff of Early Enzyme Synthesis

Synthesis of early T4 protein, which is normally shut off at 10 min after infection, continues until lysis when host cells have been preinfected with T3 sam(+). In host cells preinfected with T3 sam(-), synthesis of early enzymes is shut off as normal. Thus, S-adenosylmethionine is required for the turnoff of early T4 functions (at least when host cells have been preinfected with T3).     

Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: shutoff of host DNA and protein synthesis gene dosage experiments, identification of a restrictive host, and possible biological significance.

The shutoff of host DNA synthesis is delayed until about 8 to 10 min after infection when Escherichia coli B/5 cells were infected with bacteriophage T4 mutants deficient in the ability to induce nuclear disruption (ndd mutants). The host DNA synthesized after infection with ndd mutants is stable in the absence of T4 endonucleases II and IV, but is unstable in the presence of these nucleases. Host protein synthesis, as indicated by the inducibility of beta-galactosidase and sodium dodecyl sulfate-polyacrylamide gel patterns of isoptopically labeled proteins synthesize after infection, is shut off normally in ndd-infected cells, even in the absence of host DNA degradation. The Cal Tech wild-type strain of E. coli CT447 was found to restrict growth of the ndd mutants. Since T4D+ also has a very low efficiency of plating on CT447, we have isolated a nitrosoguanidine-induced derivative of CT447 which yields a high T4D+ efficiency of plating while still restricting the ndd mutants. Using this derivative, CT447 T4 plq+ (for T4 plaque+), we have shown that hos DNA degradation and shutoff of host DNA synthesis occur after infection with either ndd98 X 5 (shutoff delayed) or T4D+ (shutoff normal) with approximately the same kinetics as in E. coli strain B/5. Nuclear disruption occurs after infection of CT447 with ndd+ phage, but not after infection with ndd- phage. The rate of DNA synthesis after infection of CT447 T4 plq+ with ndd98 X 5 is about 75% of the rate observed after infection with T4D+ while the burst size of ndd98 X 5 is only 3.5% of that of T4D+. The results of gene dosage experiments using the ndd restrictive host C5447 suggest that the ndd gene product is required in stoichiometric amounts. The observation by thin-section electron microscopy of two distinct pools of DNA, one apparently phage DNA and the other host DNA, in cells infected with nuclear disruption may be a compartmentalization mechanism which separates the pathways of host DNA degradation and phage DNA biosynthesis.     

Isolation and characterization of a bacteriophage T5 mutant deficient in deoxynucleoside 5''-monophosphatase activity.

A bacteriophage T5 mutant has been isolated that is completely deficient in the induction of deoxynucleoside 5'-monophosphatase activity during infection of Escherichia coli F. The mutant bacteriophage has been shown to be deficient in the excretion of the final products of DNA degradation during infection of E. coli F, and about 30% of the host DNA's thymine residues were reinocorporated into phage DNA. During infection with this mutant, host DNA degradation to trichloroacetic acid-soluble products was normal, host DNA synthesis was shut off normally, and second-step transfer was not delayed. However, induction of early phage enzymes and production of DNA and phage were delayed by 5 to 15 min but eventually reached normal levels. The mutant's phenotype strongly suggests that the enzyme's role is to act at the final stage in the T5-induced system of host DNA degradation by hydrolyzing deoxynucleoside 5'-monophosphates to deoxynucleosides and free phosphate; failure to do this may delay expression of the second-step-transfer DNA.     

Effect of the “Ribonucleic Acid Control” Locus in Escherichia coli on T4 Bacteriophage-Specific Ribonucleic Acid Synthesis

Amino acid control of ribonucleic acid (RNA) synthesis in bacteria is known to be governed genetically by the rel locus. We investigated whether the rel gene of the host would also exert its effect on the regulation of phage-specific RNA synthesis in T4 phage-infected Escherichia coli cells. Since T-even phage infection completely shuts off host macromolecular synthesis, phage RNA synthesis could be followed specifically by the cumulative incorporation of radioactivity from labeled precursors into RNA of infected cells. Labeled uracil was shown to accumulate in phage-specific RNA for 30 to 35 min after infection, a phenomenon which probably reflects an expansion of the labile phage-RNA pool. Amino acid starvation was effected by the use of auxotrophic bacterial strains or thienylalanine. The latter substance is an amino acid analogue which induces a chemical auxotrophy by inhibiting the biosynthesis of phenylalanine, tyrosine, and tryptophan. Phage RNA synthesis was strictly dependent on the presence of amino acids, whereas phage deoxyribonucleic acid synthesis was not. By the use of several pairs of bacterial strains which were isogenic except for the rel gene, it was demonstrated that amino acid dependence was related to the allelic state of this gene. If the rel gene was mutated, amino acid starvation did not restrict phage RNA synthesis.     

Control of Bacteriophage-induced Enzyme Synthesis in Cells Infected with a Temperature-sensitive Mutant

The timing of "early" and "late" protein synthesis in Escherichia coli infected with T-even bacteriophage was studied with a temperature-sensitive phage mutant, T4 tsL13. This strain was completely unable to direct the synthesis of phage deoxyribonucleic acid (DNA) at 44 C because it makes a deoxycytidylate hydroxymethylase which cannot act at that temperature. However, the mutant did multiply normally at 30 C. No detectable formation of the late protein, lysozyme, occurred at 44 C, in agreement with the idea, proposed by several workers, that DNA replication is necessary for activation of late genetic functions. However, the formation of an early enzyme, thymidylate synthetase, was shut off at about 10 min, as in normal infection. This implied that separate mechanisms were responsible for cessation of early functions and activation of late ones. That the infected cell at 44 C retained the capacity for synthesis of early enzymes was shown by the fact that DNA synthesis occurred after a culture was transferred from 44 to 30 C as late as 30 min after infection. This synthesis was inhibited by chloramphenicol, indicating that de novo synthesis of an early enzyme can take place at a late period in development. It is suggested that cells infected under normal conditions maintained an appreciable rate of early enzyme synthesis throughout the course of infection.     

Inhibition of Host Protein Synthesis During Infection of Escherichi coli by Bacteriophage T4: III. Inhibition by Ghosts

Deoxyribonucleic acid (DNA)-less T2 "ghosts" were prepared by osmotic shock and purified by KBr density gradient centrifugation. Escherichia coli B was treated with these ghosts in inorganic salts-glycerol medium to see which features of phage infection could be elicited by ghosts. At a multiplicity that was just sufficient to block induction of beta-galactosidase (EC 3.2.1.23), 89% of the bacteria were killed and the rates of ribonucleic acid (RNA) and DNA synthesis were about 10 to 15% of normal. However, protein synthesis was almost completely blocked but resumed after 30 min. During this period, it was possible to induce messenger RNA (mRNA) from the lactose operon, although this mRNA could not be translated into active beta-galactosidase. These results suggest to us that the viable cells surviving ghost infection synthesize nucleic acids at close to a normal rate but are temporarily blocked in protein synthesis. The continued formation of untranslated host mRNA mimics the pattern of bacterial synthesis just after whole-phage infection, and is consistent with the interpretation that the immediate block in the initiation of host translation by these viruses is due to their attachment.     

Potassium Requirement for Synthesis of Macromolecules in Bacillus subtilis Infected with Bacteriophage 2C

A mutant of Bacillus subtilis 168 (strain 168 KW), defective in its ability to concentrate K(+) from low levels in the growth medium, was used to study the role of K(+) in the development of phage 2C. Both the final burst size and the duration of the rise period depended on the K(+) concentration in the medium. During normal infection (in the presence of K(+)), host deoxyribonucleic acid (DNA) synthesis stopped. The synthesis of host messenger ribonucleic acid (RNA) continued throughout infection, albeit at a steadily decreasing rate. The synthesis of ribosomal RNA and its subsequent incorporation into mature ribosomes also proceeded. In contrast to these findings, host DNA and messenger RNA synthesis were not inhibited in cells infected in the absence of K(+). Only "early" phage messenger RNA was synthesized under these conditions of infection. Phage DNA synthesis was dependent on K(+) irrespective of the requirement for this cation in protein synthesis.     

Effect of Bacteriophage Ghost Infection on Protein Synthesis in Escherichia coli

The rate of protein synthesis by Escherichia coli markedly decreased within 1 min after phage T4 infection, whereas a complete cessation of protein synthesis was observed within at least 25 sec after T4 ghost infection. The cellular level of amino acids and aminoacyl-transfer ribonucleic acid (tRNA) did not change drastically upon infection with ghosts, indicating that the inhibition of protein synthesis took place at a step(s) beyond aminoacyl-tRNA formation. The host messenger RNA remained intact and still bound to ribosomes shortly after ghost infection. Kinetic studies of the effect of ghosts on host protein synthesis revealed that nascent peptide chains on ribosomes were not released upon ghost infection.     

Postinfection control in T4 bacteriophage infection: inhibition of the rep function.

We suggest that the general mechanism by which T4 phage turns off host macromolecular synthesis involves specific phage proteins which react with key components in the synthetic pathway. Support for this mechanism exists for the inhibition of host RNA synthesis. Here we note that the host rep function was inhibited after T4 phage infection. Since rep functions are known to be involved in host DNA replication, inhibition of rep might alter the course of host DNA replication.     

Inhibitory proteins in the Newcastle disease virus-induced suppression of cell protein synthesis

Bolognesi, D. P. (Rensselaer Polytechnic Institute, Troy, N.Y.), and D. E. Wilson. Inhibitory proteins in the Newcastle disease virus-induced suppression of cell protein synthesis. J. Bacteriol. 91:1896-1901. 1966.-Infection by Newcastle disease virus brings about a rapid and marked inhibition of cell protein synthesis (CPS) in chick embryo fibroblast monolayers. The block to CPS is initiated about 5 hr after infection, and by 9 hr about 85% of the host protein synthesis is shut off. Azauridine (3 mg/ml), a ribonucleic acid (RNA) synthesis inhibitor, prevents the virus-induced inhibition of CPS when added at the time of infection; but it does not prevent the inhibition when added at 3 hr after infection. When puromycin (60 mug/ml), a protein synthesis inhibitor, was added at 3.5 hr after infection, viral RNA was synthesized in normal amounts, but the virus-induced inhibition of CPS was prevented. Actinomycin D added at the time of infection does not, however, prevent the virus-induced inhibition of CPS. The results of these experiments indicate that proteins synthesized during Newcastle disease virus replication are responsible for the inhibition of host-cell protein synthesis. The synthesis of these inhibitory proteins depends on the prior synthesis of viral RNA.     

Early Synthesis of Membrane Protein After Bacteriophage T4 Infection

Proteins that associate with cellular membrane during the first 5 min after infection with bacteriophage T4 were examined. Several procedures, including electrophoretic separations in three sodium dodecyl sulfate polyacrylamide gel systems and inhibition of host protein synthesis by UV irradiation, were employed to distinguish host-specified proteins from those induced by T4. Residual host protein synthesis was found to account for much of the new protein in preparations of the total membrane and for almost all of the newly synthesized protein in the outer membrane. Preliminary evidence indicates that the synthesis of some host membrane proteins is shut off less rapidly than is host synthesis of soluble protein. One host-directed polypeptide of the outer membrane was unique in that its synthesis or incorporation into the membrane was preferentially inhibited by infection. Also, it was found that the detergent Sarkosyl solubilizes all early T4 membrane proteins; this observation provides the basis for a simple procedure for distinguishing phage proteins from host outer membrane proteins.