The role of ATP and membrane potential in the penetration of phage T17 DNA into the cell during infection

The role of ATP and membrane potential in phage T7 DNA injection into E. coli during infection has been studied. Entrance of phage T7 genes of class II and III was shown to be prevented by arsenate, indicating the requirement for phosphorylated macroergs in the phage DNA injection. The injection process was also inhibited by exposition of the cells to the uncoupler of oxidative phosphorylation. Dependence of the injection efficiency on the membrane-potential value has been shown to be sigmoidal, which suggests a regulatory role of the membrane potential in phage T7 DNA injection from the virion into the host cell.     

DNA injection during bacteriophage T4 infection of Escherichia coli.

The process of phage T4 DNA injection into the host cell was studied under a fluorescent microscope, using 4',6-diamidino-2-phenylindole as a DNA-specific fluorochrome. The phage DNA injection was observed when spheroplasts were infected with the artificially contracted phage particles having a protruding core. The DNA injection was mediated by the interaction of the core tip with the cytoplasmic membrane of the spheroplast. A membrane potential was not required for the process of DNA injection. On the other hand, DNA injection upon infection by intact noncontracted phage of the intact host cell was inhibited by an energy poison. Based on these observations, together with results from previous work, a model for the T4 infection process is presented, and the role of the membrane potential in the infection process is discussed.     

The possible involvement of protein synthesis in the injection of PL-1 phage genome into its host, Lactobacillus casei.

The process of genome DNA injection, after adsorption, by phage PL-1 into host cells of Lactobacillus casei was monitored by using the electron microscope. Injection of DNA was inhibited by the protein-synthesis inhibitors chloramphenicol and erythromycin at concentrations where the colony-forming ability of cells not infected by phage was unaffected. The results suggest that protein synthesis may be involved in some way in the process of genome injection.     

Biological Activity and Mode of Action of a Specific Antiphage Substance,AFS

Purified AFS (anti-filamentous phage substance) produced by Streptomyces lavendulae AM–7a showed specific antiphage activity against the male specific, deoxyribonucleic acid-containing filamentous phages of Escherichia coli without any activity against other DNA-phages nor the male-specific ribonucleic acid-containing phages of E. coli. AFS brought about no inactivation of free particles of filamentous phage, fl, nor the receptor of the host cells for the phage, while it showed strong killing effect against the fl-infected host cells at the concentration below 0.01 μg/ml. Antiphage activity of AFS might be due to its highly specific killing effect only on the E. coli cells infected with the filamentous DNA phages, while it exerted no effect on the growth of the unifected E. coli nor other microorganisms. Killing by AFS seemed to require the energy metabolism of the phage-infected host cells. Macro-molecular synthesis and respiration of the infected host cells were inhibited soon after the addition of small amounts of AFS without any cell lysis.     

Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction?

Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases.     

Studies on energy supply for genetic processes. Requirement for membrane potential in Escherichia coli infection by phage T4

In this study the hypothesis considering the requirement for an electrochemical proton gradient in the injection of phage T4 DNA into Escherichia coli cell has been verified experimentally. The phage caused a reversible depolarization of cell membrane, while phage 'ghosts' induced an irreversible depolarization. The phage infection was strictly dependent on E. coli membrane potential value when phage/cell ratio was 5 and higher. When the ratio was close to 1, the decrease in the membrane potential up to -100 mV caused practically no effect on the phage infection. The infection inhibition was observed when the membrane potential was lowered below this 'threshold' value. On the other hand, the decrease in the membrane potential caused no effect on the phage infection under conditions promoting a concomitant increase in the value of the transmembranous pH gradient. The phage DNA transfer through the membrane of ATPase-deficient cells was reversibly inhibited by switching off the respiratory chain - the sole generator of a protonmotive force in these mutant cells. The membrane should be kept in the energized state during the phage DNA entrance into the cell. Adsorption of the phage on E. coli was followed by the reversible release of the respiratory control. Thus the results presented here indicate the requirement of the electrochemical proton gradient across the plasma membrane for injection of phage T4 DNA into E. coli. They support the concept postulating an expenditure of host cell metabolic energy for phage T4 DNA transfer through the membrane.     

Is the injection of DNA enough to cause bacteriophage P22-induced changes in the cellular transport process of Salmonella typhimurium?

It was demonstrated earlier in this laboratory that phage P22 induces a transient depression in the cellular transport processes of the host Salmonella typhimurium immediately after infection and that an effective injection process is enough to cause the depression. By using defective phage particles that contain host DNA instead of phage DNA for infection, it has been demonstrated that the injection of phage-specific DNA is essential for this. The defective particles adsorbed to the host and injected their DNA, but the cellular transport processes of the host were not altered. Thus, the injection of host DNA by the phage fails to affect the transport process. Insensitivity of the phage DNA-induced depression in transport to chloramphenicol rules out the involvement of newly synthesized protein in this change and indirectly suggests the possible role of phage DNA-associated internal proteins of P22.     

Possible involvement of a calcium-stimulated ATP-hydrolyzing activity associated with mycobacteriophage I3 in the DNA injection process

Ca2+ ions are necessary for the successful propagation of mycobacteriophage I3. An assay for the phage DNA release in the presence of an isolated cell wall preparation from the host was established, and in this system Ca2+ ions also stimulated the release of DNA. The inhibition of phage DNA injection caused by Tween 80 (polyoxyethylene sorbitan monooleate), a nonionic detergent routinely used in mycobacterial cultures, was reversed by Ca2+. The presence of a phage-associated ATP-hydrolyzing activity was demonstrated. This enzyme was stimulated by Ca2+ ions and inhibited by Tween 80. From this and the behavior of the two agents at the level of DNA injection, as well as the fact that phage I3 has a contractile tail structure, we conclude that the phage-associated ATPase is involved in the DNA injection process.     

Increased permeability and subsequent resealing of the host cell membrane early after infection of Escherichia coli with bacteriophage T1.

The addition of T1 to cells growing at 37 degrees C in a minimal medium at 0.4 mM Mg2+ rapidly induced an irreversible loss of K+ and Mg2+ and uptake of Na+ by the cells. Both the ATP pool of the cells and the transmembrane proton motive force were reduced. These cells did not lyse from within, since viral DNA replication and the maturation of the 36,000-molecular-weight phage head protein were inhibited. By contrast, cells lysed when infected at 5.4 mM Mg2+. In these cells, T1 initially induced K+ efflux and Na+ influx and lowered the cytoplasmic ATP concentration. After a few minutes, the cation gradients and ATP pool were restored to levels close to that of control cells. At 5.4 mM Mg2+, the shutoff of host protein synthesis was delayed and coincided with the restoration of the ATP pool. In an ATP synthase-negative mutant, infection with T1 did not affect the cytoplasmic ATP concentration but inhibited host protein synthesis with the same rate as it did in wild-type cells.     

Effect of spermidine on the development of bacteriophage SP6

Bacteriophage SP6 is a virulent phage of Salmonella typhimurium which behaves differently than other phages of the same host. The effect of spermidine on SP6 infection of S. typhimurium has been found to depend on the time of addition of spermidine with respect to the time of addition of the phage and also on the composition of the growth medium. If spermidine was added prior to or within a short time after infection, the cells survived. Under this condition the invading DNA appeared to remain trapped in the cell membrane, and there was no expression of the phage genome. If spermidine was added after the initiation of the infection process, the replication of the phage was inhibited but the cells did not survive. Furthermore, if spermidine was added after DNA synthesis was over, there was no effect of spermidine on phage multiplication. Spermidine was found to affect phage DNA synthesis but not host DNA synthesis.     

Inhibition of Phage Growth by an Antibiotic Rugulosin Isolated from Myrothecium verucaria

It was found that (?)rugulosin, an antibiotic isolated from Myrothecium verucaria, had a potent anti-phage effect on RNA phages (MS2, GA and Qβ) and DNA phages (δA and T4). The effect was almost independent of the host bacterial strains used. By a detailed investigation using MS2, it was revealed that the antibiotic did not affect the free phage or host bacterium alone but inhibited phage multiplication, and the degree of the inhibition depended on the multiplicity of infection. The inhibition was not mainly due to a drop in the burst size but rather was due to a decrease of the phage-producing cells during the early stages of phage infection and replication.     

Maturation of bacteriophage 9NA DNA is influenced by the fatty acid composition of the host cell membrane

The fatty acid composition of the membrane of the conditional auxotroph fabB2 can be altered by allowing the cells to grow at non-permissive temperature (37°C) in the presence of a cis-unsaturated fatty acid. The phage 9NA, a virulent phage ofSalmonella typhimurium, can not multiply in fabB2. Synthesis and maturation of the phage DNA are differentially affected by variation in the fatty acid composition of the cell membrane. The replicating DNA associates with the membrane complex, the site of DNA synthesis. The association is comparatively weak in oleic, claidic, palmitoleic, palmitelaidic and linolelaidic acid enriched cells. When the cells are grown in the presence of palmitoleic acid, a large pool of concatemeric phage DNA accumulates in the cytoplasm within 10 min of infection. The conversion of concatemeric DNA to monomeric one i.e., mature phage length DNA, is inhibited in such cells. The presence of concatemeric DNA can be visualized by electron microscope. Such a situation is not observed when the cells are grown in media supplemented with other types of unsaturated fatty acids. The mechanism by which the host cell membrane lipid controls phage development is yet to be worked out.     

Putrescine and certain polyamines can inhibit DNA injection from bacteriophage lambda

The injection of λDNA from attached phage into a host bacterium can be reversibly inhibited by putrescine. The concentration of di- or polyamine required to inhibit injection varies with the Mg²⁺ concentration and the amount of DNA in the phage head. In a series of n-alkyl diamines, those with more than five or fewer than three CH₂ groups between amino groups were ineffective.     

Effect of spermidine on bacteriophage P22 infection.

The effect of spermidine on phage P22 infection of Salmonella typhimurium has been found to depend on the time of addition of spermidine with respect to the time of addition of the phage and also on the composition of the growth medium. If spermidine was added prior to or within a short time after infection, the cells survived. Under this condition the invading DNA appeared to remain trapped in the cell membrane, and there was no expression of the phage genome. If spermidine was added after the initiation of the infective process, the replication of the phage was inhibited but the cells did not survive. If spermidine was added after DNA synthesis was over, there was no effect of spermidine on phage multiplication. Spermidine was found to affect phage DNA synthesis but not host DNA synthesis.     

Mutagenesis of ultraviolet-irradiated lambda phage by host cell irradiation: induction of Weigle mutagenesis is not an all-or-none process

Summary Ultraviolet mutagenesis of lambda phage to clear plaque formers is the same in the total phage population and in subpopulations of phage which have also mutated to gam ^- or at an amber codon. This is true for phage assayed in host cells in which Weigle mutagenesis has been either partially induced by low levels of ultraviolet irradiation, or fully induced by higher levels. If induction of Weigle mutagenesis were all-or-none, clear plaque formers in phage subpopulations selected for another mutation elsewhere would come mainly from induced cells; then the clear plaque mutation rate would always be that for fully induced host cells. Therefore, induction requires more than one lesion in host cell DNA.Although thymine starvation of cells induces synthesis of recA protein, it does not induce Weigle mutagenesis; in fact starvation inhibits induction of this process on subsequent ultraviolet irradiation of the cells.     

Inducible reactivation of UV-irradiated cholera phage e5 in Vibrio cholerae MAK757

Summary The survival of UV-irradiated cholera phage e5 was found to increase when the host cells, Vibrio cholerae MAK757, were exposed to a low dose of UV irradiation before phage infection (Weigle reactivation), indicating the existence of a UV-inducible DNA repair pathway (SOS repair) in V. cholerae MAK757. The induction signal generated by UV irradiation was transient in nature and lasted about 20–30 min at 37°C. Maximal weigle reactivation of the phage was obtained when the host cells were irradiated with a UV dose of 16 J/m². V. cholerae MAK757 was also found to possess efficient photoreactivation and host cell reactivation of UV-damaged DNA in phage e5.     

W-reactivation of phage lambda in X-irradiated mutants ofEscherichia coli K-12

Summary The survival of UV irradiated phage lambda was increased on X-irradiatedE. coli K-12 host cells over that on unirradiated cells. The frequency of c mutants among the surviving phages was to a similar extent increased by the X-ray exposure of the host cells as by UV light. This W-reactivation of phage lambda occurred inuvrA, polA, andrecB mutants besides the wild type at about equal X-ray doses, however, at a reduced reactivation efficiency compared with the wild type. W-reactivation was undetectable inrecA mutants. While maximal UV induced W-reactivation occurred 30 min after irradiation, the maximal X-ray induced reactivation was found immediately after irradiation. Chloramphenicol (100 µg/ml) and nitrofurantoin (50 µg/ml) inhibited W-reactivation of phage lambda if added before irradiation of the host cells, indicating the necessity of protein synthesis for W-reactivation.     

Presence of mycobacteriophage I3-like DNA sequences in the genome of its host Mycobacterium smegmatis

The genomes of phage I3 and its host Mycobacterium smegmatis have been compared. From thermal melting studies the GC contents of DNA from mycobacteriophage I3 and its host M. smegmatis were found to be 66%. A new method, based only on the initial rates of reassociation, has been developed for calculating the DNA homology. Analysis of DNA reassociation kinetics suggested the presence of one equivalent of the phage I3 genome within the M. smegmatis genome. Southern analysis revealed the presence of almost all of the phage I3 specific sequences within the host genome.     

Biochemical Studies on Polyamine and its Analogues

Preincubation with spermine, of λ T 7 and P 465 phages which were sensitive to oxidized spermine, resulted in a decrease of their susceptibility to the action of oxidized spermine. Phages resistant to oxidized spermine such as T 4 and ?X 174 became susceptible to this agent after dialysis.

The mechanism of phagocidal action of oxidized spermine was examined with ³²P-labelled λ phage. Oxidized spermine interfered neither with the absorption of λ phage, nor with the injection of its DNA into the host cells. The injected DNA, however, did not lead to the formation of mature phage.

The interaction of oxidized spermine with the DNA of phages T 4 and T 7 was investigated by thermal denaturation studies. DNA treated with oxidized spermine showed the same Tm as untreated DNA. However, the treated DNA was decreased in its hyperchromicity and was renatured to a great extent, even after rapid cooling. These facts are explained by the formation of cross-links which prevents the separation of complementary DNA strands.     

Infection of transformable cells ofHaemophilus influenzae by bacteriophage and bacteriophage DNA

Summary A phage HP1, infecting transformable cells ofHaemophilus influenzae Rd, has been isolated. The general properties of the wild type and of a clear plaquemutantc1 employed for most of the experiments are described. Phage DNA is infective for transformableHaemophilus cells with an efficiency (plaqueforming units of the original phage recovered as DNA-infected cells) of up to 6×10^–3. The competence ofHaemophilus cells for infection with phage DNA parallels the competence for transformation with bacterial DNA.Both HP1 and thec1 mutant are able to lysogenize their host, and the lysogenic cells are readily induced by UV. Competent non-lysogenicHaemophilus cells can be infected by DNA of lysogenic cells, thereby giving rise to phage progeny. Thus, the phage genetic material can be introduced into competentHaemophilus cells in three different ways: injection from intact phage, and infection with either phage DNA or with bacterial DNA carrying the prophage.The UV inactivation curves for infectious phage DNA and for complete phages are similar, both indicating the occurrance of host-cell reactivation. Photoreactivationin vitro of infectious phage DNA takes place to about the same high extent as observed with bacterial transforming DNA.The usefulness of this system for investigating bacterial transformation and biological effects ofin vitro treatment of DNA is discussed.with the technical assistance ofSandra J. Antoine With 4 Figures in the TextPreliminary report presented at the 7th Annual Bacterial Transformation Meeting, Aspen, Colorado, June 17–19, 1963.Supported by a travel grant from the Deutsche Forschungsgemeinschaft.Supported by Research Carreer Development Award GM-K3-7500 and Research Grant RH 00221 from the U.S. Public Health Service.