Association of Replicating Bacteriophage T7 DNA with Bacterial Membranes

Infection of Escherichia coli with bacteriophage T7 leads to the formation of an association between host membranes and newly synthesized T7 DNA. Evidence for this conclusion is suggested by the findings that replicating T7 DNA cosediments with host membranes in sucrose and cesium chloride density gradients. Furthermore, the sedimentation rate of T7 DNA is dependent on the integrity of membrane structure. Finally, an association between DNA and membranes can be demonstrated by electron microscope studies.     

Role of gene 2 in bacteriophage T7 DNA synthesis.

Studies have been carried out to elucidate the in vivo function of gene 2 in T7 DNA synthesis. In gene 2-infected cells the rate of incorporation of (3-H)thymidine into acid-insoluble material is about 60% that of cells infected with T7 wild type. Gene 2 mutants do not however produce viable phage after infection of the nonpermissive host. In T7 wild type-infected cells, a major portion of the newly alkaline sucrose gradients. The concatemers serve as precursors for the formation of mature T7 DNA as demonstrated in pulse-chase experiments. In similar studies carried out with gene 2-infected cells, concatemers are not detected when the intracellular DNA is analyzed at several different times during the infection process. The DNA made during a gene 2 infection is present as duplex structures with a sedimentation rate close to mature T7 DNA.     

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.     

Studies on bacteriophage T7 DNA synthesis in vitro. II. Reconstitution of the T7 replication system using purified proteins.

Summary DNA synthesis in vitro using intact duplex T7 DNA as template is dependent on a novel group of three phage T7-induced proteins: DNA-priming protein (activity which complements a cell extract lacking the T7 gene 4-protein), T7 DNA polymerase (gene 5-protein plus host factor), and T7 DNA-binding protein. The reaction requires, in addition to the four deoxyribonucleoside triphosphates, all four ribonucleoside triphosphates and is inhibited by low concentrations of actinomycin D. Evidence is presented that the priming protein serves as a novel RNA polymerase to form a priming segment which is subsequently extended by T7 DNA polymerase. T7 RNA polymerase (gene 1-protein) can only partially substitute for the DNA-priming protein. At 30°C, deoxyribonucleotide incorporation proceeds for more than 2 hours and the amount of newly synthesized DNA can exceed the amount of template DNA by 10-fold. The products of synthesis are not covalently attached to the template and sediment as short (12S) DNA chains in alkaline sucrose gradients. Sealing of these fragments into DNA of higher molecular weight requires the presence of E. coli DNA polymerase I and T7 ligase. Examination of the products in the electron microscope reveals many large, forked molecules and a few eye-shaped structures resembling the early replicative intermediates normally observed in vivo.     

Physical and genetical analysis of bacteriophage T4 generalized transduction

This report describes a comparison of the efficiency of transduction of genes in E. coli by the generalized transducing bacteriophages T4GT7 and P1CM. Both phages are capable of transducing many genetic markers in E. coli although the frequency of transduction for particular genes varies over a wide range. The frequency of transduction for most genes depends on which transducing phage is used as well as on the donor and recipient bacterial strains. Analysis of T4GT7 phage lysates by cesium chloride density gradient centrifugation shows that transducing phage particles contain primarily bacterial DNA and carry little, if any, phage DNA. In this regard transducing phages P1CM and T4GT7 are similar; both phages package either bacterial or phage DNA but not both DNAs into the same particle.     

DNA packaging in vitro by an isolated bacteriophage T7 procapsid.

The results of previous in vivo studies indicate that a DNA-free procapsid (capsid I) packages bacteriophage T7 DNA during infection of Escherichia coli. It was shown here that capsid I, isolated by electrophoresis in metrizamide density gradients, packaged DNA and formed infectious phage particles when incubated in vitro with extracts deficient in capsid proteins.     

Purification and structures of recombining and replicating bacteriophage T7 DNA.

During the infection of Escherichia coli by bacteriophage T7, there is a gradual conversion of host DNA to T7 DNA. Recombination and replication occur during this time. We have devised a new way of examining the physical structures of the intermediates of these processes. It is based on the observation that there are no sites in T7 DNA susceptible to cleavage by the restriction endonuclease EcoRI. E. coli DNA, on the other hand, is susceptible to degradation by EcoRI. Thus, phage and host DNA can be separated by sucrose gradient centrifugation after treatment with EcoRI. Concatemeric T7 DNA contains a high proportion of branched, gapped, and whiskered structures. These appear to be intermediates of replication and recombination. This approach also monitors the conversion process from host to T7 DNA.     

Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.

Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy. T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl. In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl. Thioredoxin does not bind to gene 2.5 protein. Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1. Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin. The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E. coli single-stranded DNA-binding protein, T4 gene 32 protein, and E. coli recA protein. All except E. coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E. coli recA protein inhibits these activities.     

Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates

The DNA polymerase activity induced after bacteriophage T7 infection of Escherichia coli is found in a complex of two proteins, the T7 gene 5 protein and a host protein, thioredoxin. Gene 5 protein is a DNA polymerase and a 3' to 5' exonuclease. Thioredoxin binds tightly to the gene 5 protein and increases the processivity of polymerization some 1000-fold. Gene 5 protein forms a short-lived complex with the primer-template, poly(dA).oligo(dT), in the absence of Mg2+ and nucleotides. Thioredoxin increases the half-life of the preformed primer-template-polymerase complex from less than a second to approximately 5 min. The dissociation is accelerated by excess single-stranded DNA in an apparent second order reaction, indicating direct transfer of polymerase between DNA fragments. Thioredoxin also reduces the equilibrium dissociation constant, Kd, of the gene 5 protein -poly(dA).oligo(dT) complex 20- to 80-fold. The salt dependence of Kd indicates that thioredoxin stabilizes the primer-template-polymerase complex mainly through additional charge-charge interactions, increasing the estimated number of interactions from 2 to 7. The affinity of gene 5 protein for single-stranded DNA is at least 1000-fold higher than for double-stranded DNA and is little affected by thioredoxin. Under conditions of steady state synthesis the effect of thioredoxin on the polymerization rate is determined by two competing factors, an increase in processivity and a decrease of the dissociation rate of polymerase and replicated template.     

Gene 19 of bacteriophage T7. Overexpression, purification, and characterization of its product

Gene 18 and 19 proteins of bacteriophage T7 are essential for DNA maturation and packaging. The phage capsid is the site of both maturation and packaging of T7 DNA. Both gene 18 and 19 proteins bind to capsid intermediates during DNA packaging but are not found in mature virions, suggesting that they play a direct role in the enzymatic mechanisms of DNA maturation and packaging. As part of an effort to reconstitute T7 DNA maturation and packaging with purified components, we have cloned and overexpressed T7 gene 19 in Escherichia coli. Gene 19 has been inserted downstream from the bacteriophage PL promoter controlled by the temperature-sensitive lambda repressor encoded by c1857. Upon thermal induction, most of the overproduced gene 19 protein is insoluble and inactive. However, by attenuation of the expression of gene 19 from the PL promoter, significant levels of soluble and active gene 19 protein are produced. Soluble gene 19 protein can be monitored by its ability to complement extracts of T7-infected cells for packaging of exogenous DNA. We have used this assay to monitor the activity of gene 19 protein during purification. The native protein is a monomer of molecular weight 66,000. We have also tested for the formation of a stable complex between gene 18 and 19 proteins. Coproduction of gene 18 and 19 proteins has no effect on either the solubility or activity of gene 19 protein, despite the fact that gene 18 protein is produced at at least 10-fold greater rates. Furthermore, we find no evidence for any interaction between soluble gene 18 and 19 proteins in extracts or between the purified proteins.     

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.     

Properties of condensed bacteriophage T4 DNA isolated from Escherichia coli infected with bacteriophage T4.

Methods developed for isolating bacterial nucleoids were applied to bacteria infected with phage T4. The replicating pool of T4 DNA was isolated as a particle composed of condensed T4 DNA and certain RNA and protein components of the cell. The particles have a narrow sedimentation profile (weight-average s=2,500S) and have, on average, a T4 DNA content similar to that of the infected cell. Their dimensions observed via electron and fluorescence microscopy are similar to the dimensions of the intracellular DNA pool. The DNA packaging density is less than that of the isolated bacterial nucleoid but appears to be roughly similar to its state in vivo. Host-cell proteins and T4-specific proteins bound to the DNA were characterized by electrophoresis on polyacrylamide gels. The major host proteins are the RNA polymerase subunits and two envelope proteins (molecular weights, 36,000 and 31,000). Other major proteins of the host cell were absent or barely detectable. Single-strand breaks can be introduced into the DNA with gamma radiation or DNase without affecting its sedimentation rate. This and other studies of the effects of intercalated ethidium molecules have suggested that the average superhelical density of the condensed DNA is small. However, these studies also indicated that there may be a few domains in the DNA that become positively supercoiled in the presence of high concentrations of ethidium bromide. In contrast to the Escherichia coli nucleoid, the T4 DNA structure remains condensed after the RNA and protein components have been removed (although there may be slight relaxation in the state of condensation under these conditions).     

Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis

Three proteins catalyze RNA-primed DNA synthesis on the lagging strand side of the replication fork of bacteriophage T7. Oligoribonucleotides are synthesized by T7 gene 4 protein, which also provides helicase activity. DNA synthesis is catalyzed by gene 5 protein of the phage, and processivity of DNA synthesis is conferred by Escherichia coli thioredoxin, a protein that is tightly associated with gene 5 protein. T7 DNA polymerase and gene 4 protein associate to form a complex that can be isolated by filtration through a molecular sieve. The complex is stable in 50 mM NaCl but is dissociated by 100 mM NaCl, a salt concentration that does not inhibit RNA-primed DNA synthesis. T7 DNA polymerase forms a stable complex with single-stranded M13 DNA at 50 mM NaCl as measured by gel filtration, and this complex requires 200 mM NaCl for dissociation, a salt concentration that inhibits RNA-primed DNA synthesis. Gene 4 protein alone does not bind to single-stranded DNA. In the presence of MgCl2 and dTTP or beta, gamma-methylene dTTP, a gene 4 protein-M13 DNA complex that is stable at 200 mM NaCl is formed. The affinity of DNA polymerase for both gene 4 protein and single-stranded DNA leads to the formation of a gene 4 protein-DNA polymerase-M13 DNA complex even in the absence of nucleoside triphosphates. However, the binding of each protein to DNA plays an important role in mediating the interaction of the proteins with each other. High concentrations of single-stranded DNA inhibit RNA-primed DNA synthesis by diluting the amount of proteins bound to each template and reducing the frequency of protein-protein interactions. Preincubation of gene 4 protein, DNA polymerase, and M13 DNA in the presence of dTTP forms protein-DNA complexes that most efficiently catalyze RNA-primed DNA synthesis in the presence of excess single-stranded competitor DNA.     

Bacteriophage proteins associated with the bacterial membrane after bacteriophage T7 infection.

The majority of the phage-induced proteins made after T7 infection of Escherichia coli are tightly associated with the bacterial membrane. Many of these have been identified. Selective extraction of proteins from these membranes by the detergent Sarkosyl or the chaotropic agent guanidine-hydrochloride indicated that most of these proteins are an integral part of the cytoplasmic membrane and the cell wall. No major changes in the distribution of bacterial proteins in the membrane were observed as a consequence of phage T7 infection.     

Folded, concatenated genomes as replication intermediates of bacteriophage T7 DNA.

A complex form of bacteriophage T7 DNA, containing up to several hundred phage equivalents of DNA, arises during replication of T7. The complex was stable to treatment with ionic detergent, Pronase, and phenol. The complex form normally exists for only a short time, corresponding to the phase of rapid T7 DNA synthesis. It is then converted to shorter molecules, both concatemers and unit-size DNA. The complex was stable up to the temperature of denaturation of the bihelix. It consisted of a series of loops amanating from a dense central core, as shownby electron microscopy. The complex form is similar to the relaxed Escherichia coli folded chromosome ('nucleoid'). The loops contained an average of 0.7 to 0.8 phage equivalent of DNA. During infection by phage with an amber mutation in gene 3 (endonuclease), formation of the complex occurred normally, but its maturation to unit-size DNA blocked. Before treatment with phenol, the complex contained short fragments of newly replicated DNA. These were released as single-stranded pieces during phenol treatment. A pathway for T7 DNA replication is indicated in which the flow of material is from unit-size DNA to linear concatemers to the complex form, and then back to unit-size DNA by way of linear concatemers.     

Inducible reactivation of bacteriophage T7 damaged by methyl methanesulfonate or UV light.

We examined the effects of host mutations affecting "SOS"-mediated UV light reactivation on the survival of bacteriophage T7 damaged by UV light or methyl methanesulfonate (MMS). Survival of T7 alkylated with MMS was not affected by the presence of plasmid pKM101 or by a umuC mutation in the host. The survival of UV light-irradiated T7 was similar in umuC+ and umuC strains but was slightly enhanced by the presence of pKM101. When phage survival was determined on host cells preirradiated with a single inducing dose of UV light, these same strains permitted higher survival than that seen with noninduced cells for both UV light- and MMS-damaged phage. The extent of T7 reactivation was approximately proportional to the UV light inducing dose inflicted upon each bacterial strain and was dependent upon phage DNA damage. Enhanced survival of T7 after exposure to UV light or MMS was also observed after thermal induction of a dnaB mutant. Thus, lethal lesions introduced by UV light or MMS are apparently repaired more efficiently when host cells are induced for the SOS cascade, and this inducible reactivation of T7 is umuC+ independent.     

Ribosomes after infection with bacteriophage T4 and T7

Summary The synthesis of E. coli ribosomal proteins ceases after infection with bacteriophages T4 or T7 as does the synthesis of most other host proteins. The shut-off does not affect all ribosomal proteins to the same extent. After T7 infection no new proteins were detected in NH₄Cl-washed ribosomal particles. Bacteriophage T4, however, induces 3–4 new protein bands demonstrated by one-dimensional gel electrophoresis. The appearance of these bands is prevented by the addition of rifampicin at the time of infection but not when rifampicin is added one minute after infection. The NH₄Cl-washed ribosomal particles present at the time of T7 or T4 infection do not show any structural changes by sedimentation, subunit dissociation, or protein analysis on two-dimensional polyacrylamide gels. However, by labeling the T7 infected cells with ³²P-phosphate, it is seen that the ribosomes become phosphorylated. The ³²P-label comigrates with ribosomal proteins. This phosphorylating activity depends on a T7 gene. The T7 protein phosphokinase utilizes ribosomes as phosphate acceptor in vitro. The T7 ribosomes (NH₄Cl-washed) still function in vitro as do ribosomal particles from uninfected cells.Paper No. 83 on Ribosomal Proteins. Preceding paper is by Isono et al., Mol. gen. Genet. 127, 191–195 (1973).