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.     

Nuclear Disruption After Infection of Escherichia coli with a Bacteriophage T4 Mutant Unable to Induce Endonuclease II

Nuclear disruption after infection of Escherichia coli with a bacteriophage T4 mutant deficient in the ability to induce endonuclease II indicates that either (i) the endonuclease II-catalyzed reaction is not the first step in host deoxyribonucleic acid (DNA) breakdown or (ii) nuclear disruption is independent of nucleolytic cleavage of the host chromosome. M-band analysis demonstrates that the host DNA remains membrane-bound after infection with either an endonuclease II-deficient mutant or T4 phage ghosts.     

Unfolding of the Host Genome After Infection of Escherichia coli with Bacteriophage T4

The folded genome of Escherichia coli is converted to a slower-sedimenting form within 5 min after infection with bacteriophage T4 or T4nd28(den A)-amN82(44). Chloramphenicol sensitivity and response to UV-irradiation of the phage suggest participation of viral-induced functions.     

Analysis of Nuclear Disruption and Binding of Intermediates in Host DNA Breakdown to Membranes After Infection of Escherichia coli with Bacteriophages T4 and T7

Escherichia coli DNA polymerase I is implicated in the binding of intermediates in host DNA breakdown to membrane in T4-infected, but not T7-infected, cells. Nuclear disruption is observed in T4-infected polA1 mutant cells.     

Degradation of Escherichia coli Chromosome After Infection by Bacteriophage T4: Role of Bacteriophage Gene D2a

Mutations in the D2a gene of bacteriophage T4 have recently been shown to result in the stabilization of cytosine-containing phage deoxyribonucleic acid (DNA) made after infection by phage gene 56 (deoxycytidine triphosphatase) mutants. In the experiments reported here, we investigate the role of the D2a gene in the degradation of the host chromosome. We find that if T4 endonuclease II, a product of the phage gene denA, is active, host chromosome degradation appears normal, regardless of the presence of the D2a gene product. However, if T4 endonuclease II is absent, a small amount of host chromosome degradation occurs, but only if the D2a product is present. These results are interpreted in terms of the hypothesis that D2a controls a nuclease which degrades cytosine-containing DNA. Neither D2a nor denA mutations affect the shut-off of host DNA synthesis.     

Partial Suppression of Bacteriophage T4 Ligase Mutations by T4 Endonuclease II Deficiency: Role of Host Ligase

Endonuclease II-deficient, ligase-deficient double mutants of phage T4 induce considerably more deoxyribonucleic acid (DNA) synthesis after infection of Escherichia coli B than does the ligase-deficient single mutant. Furthermore, the double mutant can replicate 10 to 15% as well as wild-type T4, whereas the single mutant fails to replicate. When the E. coli host is also deficient in ligase, the double mutant resembles the single mutant. The results indicate that host ligase can substitute for phage ligase when the host DNA is not attacked by the phage-induced endonuclease II.     

Mutants in a Nonessential Gene of Bacteriophage T4 Which Are Defective in the Degradation of Escherichia coli Deoxyribonucleic Acid

Wild-type bacteriophage T4 was enriched for mutants which fail to degrade Escherichia coli deoxyribonucleic acid (DNA) by the following method. E. coli B was labeled in DNA at high specific activity with tritiated thymidine ((3)H-dT) and infected at low multiplicity with unmutagenized T4D. At 25 min after infection, the culture was lysed and stored. Wild-type T4 degrades the host DNA and incorporates the (3)H-dT into the DNA of progeny phage; mutants which fail to degrade the host DNA make unlabeled progeny phage. Wild-type progeny are eventually inactivated by tritium decay; mutants survive. Such mutants were found at a frequency of about 1% in the survivors. Eight mutants are in a single complementation group called denA located near gene 63. Four of these mutants which were examined in detail leave the bulk of the host DNA in large fragments. All eight mutants exhibit much less than normal T4 endonuclease II activity. The mutants produce somewhat fewer phage and less DNA than does wild-type T4.     

Bacteriophage T4 Inhibits Colicin E2-Induced Degradation of Escherichia coli Deoxyribonucleic Acid II. Inhibition by T4 Ghosts and by T4 in the Absence of Protein Synthesis

The deoxyribonucleic acid (DNA) of Escherichia coli B is converted by colicin E2 to products soluble in cold trichloroacetic acid; we showed previously that this DNA degradation (hereafter termed solubilization) is subject to inhibition by infection with phage T4 and that at least two modes of inhibition can be differentiated on the basis of their sensitivity to chloramphenicol (CM). This report deals exclusively with the inhibition of E2 produced by T4, or T4 ghosts, in the absence of protein synthesis. The following observations are described. (i) The stage of T4 infection that inhibits E2 occurs after reversible adsorption of the phage to the bacterial surface, but probably prior to injection of T4 DNA into the cell's interior. (ii) The extent of inhibition increases as the T4 multiplicity is increased; however, the fraction of bacterial DNA that eventually is solubilized is virtually independent of the phage multiplicity. (iii) Phage ghosts (DNA-less phage particles) possess an approximately 15-fold greater inhibitory capacity toward E2 than do intact phage; however, because highly purified T4 (completely freed of ghost contamination) still inhibit E2, we discount the possibility that preparations of "intact phage" inhibit exclusively by virtue of contaminating ghosts. (iv) T4 infection does not liberate an extracellular inactivator of E2. In fact, infection with sufficiently high multiplicities of T4 produces a supernatant factor that protects E2 from nonspecific inactivation at 37 C. This protective factor does not interfere with the colicin's ability to induce DNA solubilization. (v) Inhibition of E2 occurs even when phage are added well after initiation of DNA solubilization by E2, suggesting that a late stage of E2 action is the target of inhibition by T4 infection. (vi) Increasing the CM concentration from 50 mug/ml to 200 mug/ml appears to reduce the inhibition appreciably; however, this can be attributed to an enhancement by CM of the rate of E2-induced DNA solubilization. (vii) The same degree of inhibition of E2 by T4 seen in CM is observed when CM is replaced by puromycin or rifampin. (viii) Others have shown that raising the multiplicity of E2 increases the rate of DNA solubilization. We find that the fractional inhibition (i), [i = (1 - y(i)/y(o)), where y(i) and y(o) represent the inhibited and uninhibited rates of solubilization of DNA, respectively], produced by a given T4 multiplicity is independent of the multiplicity of E2 and hence is independent of the rate of DNA solubilization induced by E2.     

Bacteriophage-induced Inhibition of Host Functions : I. Degradation of Escherichia coli Deoxyribonucleic Acid After T4 Infection

The kinetics of degradation of bacterial deoxyribonucleic acid (DNA) after infection of Escherichia coli with T4D, ultraviolet-irradiated T4D, and two amber mutants, N122 and N94, was studied by zone sedimentation through linear glycerol gradients. Within 5 min after infection with any of the bacteriophages, breakdown of host genome was evident. The first product was a high-molecular-weight material (50S to 70S) and further degradation appeared to occur in discrete steps. Rapid and extensive breakdown of bacterial DNA was seen after infection with am N122 and T4D. Infection with ultraviolet-irradiated phage or with am N94 resulted in an accumulation of high-molecular-weight material. These results suggest that the observed degradation of host DNA begins early and requires sequential action of several phage-induced endo- as well as exodeoxyribonucleases.     

Inhibition of Host Protein Synthesis During Infection of Escherichia coli by Bacteriophage T4: II. Induction of Host Messenger Ribonucleic Acid and Its Exclusion from Polysomes

Two gene clusters on the Escherichia coli chromosome were induced at early times after T4 infection when >99% of the cells were infected: the lactose (lac) operon and prophage lambda. Their messenger ribonucleic acid (mRNA) was detected by hybridization to phi80 dlac deoxyribonucleic acid (DNA) and lambdaDNA, respectively. Synthesis of host mRNA could be initiated during the first few minutes after T4 infection, although no beta-galactosidase activity could be detected. Hybridization analyses of selected fractions from sucrose gradients revealed that most of this lac mRNA induced at very early times of T4 infection was not associated with ribosomes. In contrast, virtually all lac mRNA in uninfected bacteria was associated with polysomes. This exclusion affected all host mRNA; about 70% of E. coli(3)H-mRNA, labeled from 2 to 3 min after T4 infection, was excluded from polysomes. Infection even reduced the yield of beta-galactosidase from lac mRNA induced before infection. Gradients from rifampicin-inhibited cells showed the normal growth of lac mRNA polysomes; in contrast, T4 infection prevented growth of the preinduced lac polysomes. It is concluded that T4 infection interferes within seconds with the reassociation of ribosomes to host mRNA.     

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.     

Degradation of Escherichia coli B Deoxyribonucleic Acid After Infection with Deoxyribonucleic Acid-Defective Amber Mutants of Bacteriophage T7

The degradation of bacterial deoxyribonucleic acid (DNA) was studied after infection of Escherichia coli B with DNA-negative amber mutants of bacteriophage T7. Degradation occurred in three stages. (i) Release of the DNA from a rapidly sedimenting cellular structure occurred between 5 and 6 min after infection. (ii) The DNA was cleaved endonucleolytically to fragments having a molecular weight of about 2 x 10(6) between 6 and 10 min after infection. (iii) These fragments of DNA were reduced to acid-soluble products between 7.5 and 15 min after infection. Stage 1 did not occur in the absence of the gene 1 product (ribonucleic acid polymerase sigma factor), stage 2 did not occur in the absence of the gene 3 product (phage T7-induced endonuclease), and stage 3 did not occur in the absence of the gene 6 product.     

Synthesis of Bacteriophage and Host DNA in Toluene-Treated Cells Prepared from T4-Infected Escherichia coli: Role of Bacteriophage Gene D2a

We investigated the synthesis of DNA in toluene-treated cells prepared from Escherichia coli infected with bacteriophage T4. If the phage carry certain rII deletion mutations, those which extend into the nearby D2a region, the following results are obtained: (i) phage DNA synthesis occurs unless the phage carries certain DNA-negative mutations; and (ii) host DNA synthesis occurs even though the phage infection has already resulted in the cessation of host DNA synthesis in vivo. The latter result indicates that the phage-induced cessation of host DNA synthesis is not due to an irreversible inactivation of an essential component of the replication apparatus. If the phage are D2a(+), host DNA synthesis in toluene-treated infected cells is markedly reduced; phage DNA synthesis is probably also reduced somewhat. These D2a effects, considered along with our earlier work, suggest that a D2a-controlled nuclease, specific for cytosine-containing DNA, is active in toluene-treated cells.     

Point Mutants in the D2a Region of Bacteriophage T4 Fail to Induce T4 Endonuclease IV

We have studied the properties of presumptive point mutants in the D2a region of bacteriophage T4. Dominance tests showed that the D2a mutation was recessive to the wild-type allele. The mutations were shown to map in the D2a region by complementation against rII deletions. The D2a mutations were also located between gene 52 and rIIB by two- and three-factor crosses. The mutants are located at at least two distinct loci in the D2a region. The point mutants grow normally on all hosts tested and none of the mutants makes T4 endonuclease IV. We propose the name "denB" for the D2a locus.     

Site-specific recognition of bacteriophage T4 DNA by T4 type II DNA topoisomerase and Escherichia coli DNA gyrase

The site specificity of bacteriophage T4-induced type II DNA topoisomerase action on double-stranded DNA has been explored by studying the sites where DNA cleavages are induced by the enzyme. Oxolinic acid addition increases the frequency at which phi X174 duplex DNA is cut by the enzyme by about 100-fold, to the point where nearly every topoisomerase molecule causes a double-stranded DNA cleavage event. The effect of oxolinic acid on the enzyme is very similar to its effect on another type II DNA topoisomerase, the Escherichia coli DNA gyrase. A filter-binding method was developed that allows efficient purification of topoisomerase-cleaved DNA fragments by selecting for the covalent attachment of this DNA to the enzyme. Using this method, T4 topoisomerase recognition of mutant cytosine-containing T4 DNA was found to be relatively nonspecific, whereas quite specific recognition sites were observed on native T4 DNA, which contains glucosylated hydroxymethylcytosine residues. The increased specificity of native T4 DNA recognition seems to be due entirely to the glucose modification. In contrast, E. coli DNA gyrase shows a high level of specificity for both the mutant cytosine-containing DNA and native T4 DNA, recognizing about five strong cleavage sites on both substrates. An unexpected feature of DNA recognition by the T4 topoisomerase is that the addition of the cofactor ATP strongly stimulates the topoisomerase-induced cleavage of native T4 DNA, but has only a slight effect on cleavage of cytosine-containing T4 DNA.     

Inhibition of Host Deoxyribonucleic Acid Synthesis by T4 Bacteriophage in the Absence of Protein Synthesis

The requirement for phage protein synthesis for the inhibition of host deoxyribonucleic acid synthesis has been investigated by using a phage mutant unable to catalyze the production of any phage deoxyribonucleic acid. It has been concluded that the major pathway whereby phage inhibit host syntheses requires protein synthesis. The inhibition of host syntheses by phage ghosts is not affected by inhibitors of protein synthesis.     

Rescue of DNA Replication and Bacteriophage Production After Infection with T4 DNA Ligase Mutants

By preventing phage DNA synthesis during a critical period, conditions have been found under which DNA replication and phage production are rescued after infection with T4 DNA ligase mutants.     

Genetic Location of a Mutant of Bacteriophage T4 Deficient in the Ability to Induce Endonuclease II

Reciprocal three-factor crosses and the use of a partial revertant of a putative ribonucleotide reductase mutant of Escherichia coli B/5 as indicator have made it possible to map denA (deficient in endonuclease II) between nrd-11 (ribonucleotide reductase gene B) and amM69 (gene 63) on the bacteriophage T4 chromosome.     

Loss of Lysis Inhibition in Filamentous Escherichia coli Infected with Wild-Type Bacteriophage T4

The plaque enlargement of wild-type T4 bacteriophage observed when assayed in the presence of low concentrations of mitomycin C or after exposure to very low doses of ultraviolet light was studied by using solid as well as liquid culture media. It was found that the filamentous cell formed by the treatment with the agents is responsible for the phenomenon. The filamentous cell was also shown to be characterized not only by the loss of capacity of lysis inhibition but also by a shortening of the latent period. No difference in cellular rigidity could be seen between the filamentous cell and normal cell as far as the analysis from the outside of the cell was concerned, whereas the former cell was shown to be more readily susceptible to phage-induced lysozyme from the inside of the cell. A possible change in the membrane of the filamentous cell and a possible mechanism for lysis inhibition are discussed.