Amino Acid Control over Deoxyribonucleic Acid Synthesis in Escherichia coli Infected With T-Even Bacteriophage

Starvation for a required amino acid of normal or RC(str)Escherichia coli infected with T-even phages arrests further synthesis of phage deoxyribonucleic acid (DNA). This amino acid control over phage DNA synthesis does not occur in RC(rel)E. coli mutants. Heat inactivation of a temperature-sensitive aminoacyl-transfer ribonucleic acid (RNA) synthetase similarly causes an arrest of phage DNA synthesis in infected cells of RC(str) phenotype but not in cells of RC(rel) phenotype. Inhibition of phage DNA synthesis in amino acid-starved RC(str) host cells can be reversed by addition of chloramphenicol to the culture. Thus, the general features of amino acid control over T-even phage DNA synthesis are entirely analogous to those known for amino acid control over net RNA synthesis of uninfected bacteria. This analogy shows that the bacterial rel locus controls a wider range of macromolecular syntheses than had been previously thought.     

Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-Infected Bacillus subtilis I. Bacteriophage Deoxyribonucleic Acid Synthesis and Fate of Host Deoxyribonucleic Acid in Normal and Polymerase-Deficient Strains

The effect of bacteriophage SPO1 infection of Bacillus subtilis and a deoxyribonucleic acid (DNA) polymerase-deficient (pol⁻) mutant of this microorganism on the synthesis of DNA has been examined. Soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. This inhibition of host DNA synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate nucleotidohydrolase. Examination of the host chromosome for genetic linkage throughout the lytic cycle indicated that no extensive degradation occurred. After the inhibition of host DNA synthesis, a new polymerase activity arose which directed the synthesis of phage DNA. This new activity required deoxyribonucleoside triphosphates as substrates, Mg²⁺ ions, and a sulfhydryl reducing agent, and it was stimulated in the presence of adenosine triphosphate. The phage DNA polymerase, like that of its host, was associated with a fast-sedimenting cell membrane complex. The pol⁻ mutation had no effect on the synthesis of phage DNA or production of mature phage particles.     

Effect of Bacteriophage R17 Infection on Hostdirected Synthesis of Ribosomal Ribonucleates

Studies were performed on the synthesis of ribosomal ribonucleates in cells of Escherichia coli K-12 infected by the ribonucleic acid (RNA) bacteriophage R17. Host-specific RNA was measured in the presence of phage RNA by in vitro hybridization of the purified ribonucleates with E. coli deoxyribonucleic acid. The results showed that, although the overall rate of RNA synthesis was only slightly affected by phage infection, the level of host RNA synthesis was decreased by 70 to 80%. Fractionation of the purified ribonucleates by sucrose gradient sedimentation, followed by hybridization of fractions sedimenting in the 23S and 16S regions, revealed that the level of ribosomal RNA synthesis was also decreased by 70 to 80%, and that this inhibition occurred during the first 15 to 20 min after infection. These findings are discussed in light of what is known about the inhibition of host RNA synthesis by other virus systems.     

Ribonucleic Acid Bacteriophage Release: Requirement for Host-Controlled Protein Synthesis

The release of the ribonucleic acid (RNA)-containing phage MS2 from Escherichia coli is accompanied by cellular lysis at 37 C, whereas at 30 C phage are released from intact cells. Chloramphenicol or rifampin prevents the release of progeny phage particles at both temperatures. Neither drug causes an immediate cessation of phage release and after inhibition of protein synthesis by chloramphenicol phage release proceeds for about 17 min at 37 C and about 35 min at 30 C. Rifampin does not inhibit phage release from mutant cells possessing a rifampin-resistant deoxyribonucleic acid-dependent RNA polymerase. The results indicate that a short-lived host-controlled protein(s) is essential for the release of RNA phage particles at both temperatures.     

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.     

Mating Reaction in Saccharomyces cerevisiae. IV. Retardation of Deoxyribonucleic Acid Synthesis

When a and a type haploid cells of Saccharomyces cere-visiae were mixed and cultured, deoxyribonucleic acid synthesis was retarded but ribonucleic acid and protein syntheses were not. It was found that culture filtrate of a type cells inhibited deoxyribonucleic acid synthesis of a type cells and that of a type cells inhibited that of a type cells. Thus, sex-specific diffusible substances secreted by opposite mating type cells are thought, at least partly, to be responsible for the retardation of deoxyribonucleic acid synthesis.     

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.     

Effects of penicillin on macromolecular synthesis and surface growth of a tolerant streptococcus as studied by computer reconstruction methods.

Strains of Streptococcus mutans are very susceptible to growth inhibition by benzylpenicillin, but are tolerant to lysis when exposed to even high concentrations of this drug. These properties enabled this study of S. mutans GS-5 surface growth and peptidoglycan, ribonucleic acid, protein, and deoxyribonucleic acid syntheses in the absence of osmotic stabilization. Inhibition of syntheses of peptidoglycan, ribonucleic acid, and protein was dose dependent. Synthesis of peptidoglycan was most susceptible. Substantial but less severe inhibitions of ribonucleic acid and protein syntheses rapidly followed decreased peptidoglycan synthesis, whereas inhibition of deoxyribonucleic acid synthesis was delayed and minimal. Computer-assisted reconstructions of surface growth zones and poles observed in electron micrographs of replicas were performed and indicated that at low concentrations of benzylpenicillin (0.03 micrograms/ml), growth sites reached abnormally large sizes and surface/volume ratios. The observed shifts in surface/volume ratio were attributed to an inhibition of the normal constrictive division mechanism. The poles of these cells also increased in size over those of the controls, but the relatively smaller change in surface/volume ratio confirmed the visual impression that the shape of the poles was much less altered than the shape of the growth sites. As the concentration of benzylpenicillin used was raised from 0.03 to 2 micrograms/ml, the ability of growth sites and poles to enlarge was restricted in a manner that most closely agreed with the extent of inhibition of peptidoglycan (rather than deoxyribonucleic acid, ribonucleic acid, or protein) synthesis. This correlation suggested that increases in cell size may be regulated by the supply of peptidoglycan precursors.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

Synthesis of Replicative Form Deoxyribonucleic Acid and Messenger Ribonucleic Acid by Gene IV Mutants of Bacteriophage S13

Gene IV mutants of bacteriophage S13 are known to be blocked in infectious replicative form (RF) DNA synthesis, producing only a small fraction of the RF formed by wild-type phage. This investigation shows that gene IV mutants form only parental RF and are blocked in the synthesis of any progeny RF, either infectious or noninfectious. This was determined by density labeling of RF in cells treated with mitomycin C to suppress host deoxyribonucleic acid (DNA) synthesis. RF synthesis was also studied in untreated cells, using methylated albumin columns to separate RF from host DNA. In this case it was also found that synthesis of progeny RF by gene IV mutants is negligible. It has been found by DNA-ribonucleic acid (RNA) hybridization experiments that gene IV mutants form at least as much or more messenger RNA than wild-type phage. Therefore, parental RF alone can form messenger RNA in appreciable amounts.     

Host-Bacteriophage Interaction in Agrobacterium tumefaciens I. Characterization of Bacteriophage R4 and Physiological Changes in the Infected Host Cell

Infection of Agrobacterium tumefaciens B6, a tumor-producing plant pathogen, by bacteriophage R4, does not immediately shut off host deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis. Viral DNA synthesis begins soon after infection, but the host DNA is not shut off until after 35 min; net RNA and protein synthesis are not inhibited until 30 min after infection. The pattern of synthesis of phage particles was confirmed by electron microscopy of thin sections during the infection cycle. The phage particle consists of a polyhedral head, 65 nm in diameter, and a long flexible tail 210 nm long and 10 nm wide with helically arranged subunits. By gel electrophoresis, four major protein components with the following molecular weights were found in the capsid: 72,000, 45,000, 28,000, and 14,500. The phage DNA has a molecular weight of 30 million and a guanine-cytosine content of 59%.     

Genetic and Physiological Studies of Bacteriophage T5 III. Patterns of Deoxyribonucleic Acid Synthesis Induced by Mutants of T5 and the Identification of Genes Influencing the Appearance of Phage-Induced Dihydrofolate Reductase and Deoxyribonuclease

Patterns of deoxyribonucleic acid (DNA) metabolism in nonpermissive cells infected with amber mutants representing 29 genes of T5 are reported. A group of 7 contiguous genes are essential for the synthesis of phage DNA, whereas 20 other genes, when defective, permit varying degrees of phage DNA synthesis. Two further genes are essential for complete transfer of phage DNA to host cells, and therefore indirectly do not permit the synthesis of phage DNA. The structural genes for an early T5 deoxyribonuclease and for T5 DNA polymerase, as well as a gene that affects the synthesis of dihydrofolate reductase, have been identified in the genetic map of T5.     

A Bacteriophage of Bacillus subtilis Which Forms Plaques Only at Temperatures Above 50 C III. Inhibition of TSP-1-Specific Deoxyribonucleic Acid Synthesis at 37 C and 45 C

The effect of temperature on phage-specific deoxyribonucleic acid (DNA) synthesis was studied in TSP-1-infected Bacillus subtilis. This was facilitated by selectively inhibiting host DNA synthesis with 6-(p-hydroxyphenylazo)-uracil. The results indicated that TSP-1 DNA synthesis did not continue at 37 C and was immediately shut down after transfer to this temperature. Incubation at 45 C greatly reduced TSP-1 DNA synthesis. Phage-specific DNA synthesis could resume at 53 C, however, when the infected culture was returned to 53 C after a 2-min incubation period at 37 C. The results suggest that the inhibition of phage DNA synthesis at 37 C is reversible. Since infected cultures returned to 53 C after 2 min at 37 C could not complete the replicative cycle, the irreversible inhibition of yet another intermediate step was suggested.     

Host Cell Participation in Small Virus Replication I. Replication of M-13 in a Strain of Escherichia coli with a Temperature-sensitive Lesion in Deoxyribonucleic Acid Synthesis

The replication of M-13 in a strain of Escherichia coli with a thermosensitive lesion in deoxyribonucleic acid synthesis was studied. M-13 failed to replicate at the restrictive temperature, even when the parental replicative form was allowed to form at the permissive temperature. When cells which were actively producing phage at the permissive temperature were shifted to the restrictive temperature, phage production continued. The incorporation of radioactive label into phage particles at 42 C indicated that continued single-strand synthesis was unaffected by the lesion in the host cell.     

Effect of cessation of phospholipid synthesis on the synthesis of a specific membrane-associated bacteriophage protein in Escherichia coli.

The major coat protein of the bacteriophage f1 is synthesized during infection of Escherichia coli and becomes tightly associated with the host membrane. This synthesis was studied in conjunction with the strain BB26-36, a mutant defective in phospholipid synthesis, to investigate basic questions concerning membrane protein and phospholipid synthesis. Coat protein synthesis is decreased in the absence of net phospholipid synthesis. The coat protein produced under these conditions is still found tightly associated with the membrane. Resumption of phospholipid synthesis leads to an increase in the synthesis and accumulation of the coat protein. Therefore, a correlation between coat protein and phospholipid synthesis seems to exist. However, the packaging of phage deoxyribonucleic acid into phage particles proceeds in the absence of phospholipid synthesis, and the number of phage particles produced appears to depend only on the amount of coat protein in the membrane.     

Inhibitory Effect of Bacteriophage P22 Infection on Host Cell Deoxyribonuclease Activity

Infection of Salmonella typhimurium with phage P22 causes a decrease in the activity of host deoxyribonuclease which degrades single-stranded deoxyribonucleic acid (DNA). This decrease is reversed when the infecting phage is P22c(+); it is not reversed if the infecting phage kills the cell. The decrease does not occur in infections with P22ts25.1 (which only adsorbs and injects DNA) or in infections of a lysogen by a nonvirulent phage. It does occur, however, after infections with other phages which are blocked in phage DNA synthesis. Inhibiting protein synthesis with chloramphenicol does not in itself cause the decrease in uninfected cells, but it does prevent infected cells from showing this effect.     

Regulation of Bacteriophage T5 Development by ColI Factors

The I-type colicinogenic factor ColIb transforms Escherichia coli from a permissive to a nonpermissive host for bacteriophage T5 reproduction by preventing complete expression of the phage genome. T5-infected ColIb(+) cells synthesize only class I (early) phage protein and ribonucleic acid (RNA). Neither phage-specific class II proteins [associated with viral deoxyribonucleic acid (DNA) replication] nor class III proteins (phage structural components) are formed due to the failure of the infected ColIb(+) cells to synthesize class II or class III phage-specific messenger RNA. Comparable studies with T5-infected cells colicinogenic for the related ColIa factor revealed no decrease in the yield of progeny phage although the presence of the ColIa factor leads to a significant reduction in the amount of phage-directed class III protein synthesis.     

Mode of Action of Colicins of Types E1, E2, E3, and K

The effect of colicins on deoxyribonucleic acid and protein synthesis, and also their effect on the ability of T4 phage to replicate in Escherichia coli K-12, were studied. Colicins of type K inhibited deoxyribonucleic acid synthesis, protein synthesis, and phage growth. Among colicins of type E, there was an absolute correlation between mode of action and subdivision into types E(1), E(2), and E(3).