Mechanism of inhibition of bacteriophage T7 DNA synthesis in Escherichia coli B cells infected by alkylated bacteriophage T7

Quantitative analysis of DNA replication, in E. coli B cells infected by methyl methanesulfonate-treated bacteriophage T7, showed that production of phage DNA was delayed and decreased. The cause of the delay appeared to be a delay in host-DNA breakdown, the process which provides nucleotides for phage-DNA synthesis. In addition, reutilisation of host-derived nucleotides was impaired. These observations can be accounted for by a model in which methyl groups on phage DNA slow down DNA injection and also reduce the replicational template activity of the DNA once it has entered the cell. Repair of alkylated phage DNA may be required not only for replication but also for normal injection of DNA.     

Binding of ethidium to bacteriophage T7 and T7 deletion mutants

Equilibrium binding of ethidium, quantitated by fluorescence enhancement, to DNA packaged in bacteriophage T7 and T7 deletion mutants has been compared with the binding of this dye to DNA released from its capsid (free DNA). During achievement of apparent equilibrium binding, no change in bacteriophage T7 structure occurred, by the criterion of agarose gel electrophoresis. However, excessive incubation with ethidium bromide caused detectable changes in bacteriophage structure, a possible explanation of disagreements in similar studies previously performed with T-even bacteriophages. Scatchard plots for packaged DNA had a curvature greater than the previously demonstrated [Bresloff, J. L. & Crothers, D. M. (1981) Biochemistry 20 , 3547–3553] curvature for free DNA. By treating plots for packaged DNA as though they were biphasic, it was found that binding to most sites occurred with an apparent association constant (K_ap) 3.3–4.3 times lower than the K_ap of free DNA. The number of these sites increased significantly as the density of packaged DNA was decreased by use of the deletion mutants. Values of ΔH° for these sites were negative and equal to the ΔH° for free DNA; values of ΔS° were positive and about half the ΔS° for free DNA. A second class of sites, roughly 1.2% of the total, had a significantly higher K_ap and more negative ΔH° than those of the majority of sites.     

Biological consequences of infection of Escherichia coli B by alkylated T7 bacteriophage.

Alkylation of T7 bacteriophage considerably delayed phage development and reduced the phage's killing action on host cells. Only a small fraction of infected cells produced phage. For these phages, the latent period was markedly prolonged but the burst was equivalent to or only slightly lower than that of untreated phage. In the progeny of alkylated phage, there was an increase in the fraction of defective particles as well as a change in their morphology. These data show that infection with alkylated T7 bacteriophage is to a large degree abortive; hence, biological consequences of this infection are very different from those characteristic of a normal virus infection.     

Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant.

We have analyzed the structure of the late cytoplasmic RNAs made after infection with wild-type simian virus 40 and a set of viable mutants, four of which have deletions and one an insertion within the nucleotide sequence specifying the leader segment of the 16S and 19S mRNA's. The principal findings are: (i) simian virus 40 16S and 19S mRNA's made during infections with wild-type virnds and possibly in the nucleotide sequence comprising the "leader" segments. (II) "Spliced" 16S and 19S mRNA's are made during infections with each of the mutants although, in some cases, the ratio of 19S to 16S mRNA species is reduced. (iii) The deletion or insertion of nucleotides within the DNA segment defined by map position 0.70 to 0.75 causes striking alterations in the types of leader structures in the late mRNAs. (iv) Many of the late RNA leader segments produced after infection with the mutants appear to be multiply spliced, i.e., instead of the major 200- to 205-nucleotide-long leader segment present in wild-type 16S mRNA, the RNAs produced by several of the deletion mutants have leaders with whort discontiguous segments.     

Phosphorylation of elongation factor G and ribosomal protein S6 in bacteriophage T7-infected Escherichia coli

Bacteriophage T7 expresses a serine/threonine-specific protein kinase activity during Infection of Its host, Escherichia coli. The protein kinase (gpO.7 PK), encoded by the T7 early gene 0.7, enhances phage reproduction under sub-optimal growth conditions. It was previously shown that ribosomal protein S1 and translation initiation factors IF1, IF2, and IF3 are phosphoryiated in T7-infected cells, and it was suggested that phosphorylation of these proteins may serve to stimulate translation of the phage late mRNAs. Using high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation, we show that elongation factor G and ribosomal protein S6 are phosphorylated following T7 infection. The gel electro-phoretic data moreover indicate that elongation factor P is phosphorylated in T7-infected cells. T7 early and late mRNAs are processed by ribonuclease III, whose activity is stimulated through phosphorylation by gp0.7 PK. Specific overexpression and phosphorylation was used to locate the RNase III polypeptide in the standard two-dimensional gel pattern, and to confirm that serine is the phosphate-accepting amino acid. The two-dimensional gels show that the in vivo expression of gp0.7 PK results in the phosphorylation of over 90 proteins, which Is a significantly higher number than previous estimates. The protein kinase activities of the T7-related phages T3 and BA14 produce essentially the same pattern of phosphorylated proteins as that of T7. Finally, several experimental variables are analysed which influence the production and pattern of phosphorylated proteins in both uninfected and T7-rnfected cells.     

Growth of bacteriophage Mu in Escherichia coli dnaA mutants.

In one-step growth experiments we found that bacteriophage Mu grew less efficiently in nonreplicating dnaA mutants than in dnaA+ strains of Escherichia coli. Phage development in dnaA hosts was characterized by latent periods that were 15 to 30 min longer and an average burst size that was reduced by 1.5- to 4-fold. The differences in phage Mu development in dnaA and dnaA+ strains were most pronounced in cells infected at a low multiplicity and became less pronounced in cells infected at a high multiplicity. Many of these differences could be eliminated by allowing the arrested dnaA cells to restart chromosome replication just before infection. In continuous labeling experiments we found that infected dnaA strains incorporated 5 to 40 times more [methyl-3H]thymidine than did uninfected cells, depending on the multiplicity of infection. DNA-DNA hybridization assays showed that greater than 90% of this label was contained in phage Mu DNA sequences and that only small amounts of the label appeared in E. coli sequences. In contrast, substantial amounts of label were incorporated into both host and viral DNA sequences in infected dnaA+ cells. Although our results indicated that phage Mu development is not absolutely dependent on concurrent host chromosomal DNA replication, they did strongly suggest that host replication is necessary for optimal growth of this phage.     

Frameshift mutagenesis in bacteriophage T7.

We have undertaken an initial characterization of frameshift mutagenesis in bacteriophage T7 and have identified a subset of very low reversion frameshift mutations in the T7 ligase gene (gene 1.3). We used this information to construct bacteriophage T7 strains that contain one extra or one less base pair in gene 1.3 such that a frameshift event restores the reading frame of that gene. These events can be quantified and the frameshift mutation isolated within a localized region of the ligase gene. We have also identified a portion of the T7 ligase protein that will accept tracts of nonsense amino acids yet still give a ligase positive phenotype. This allows flexibility in the design of the target DNA sequence with which to study frameshift mutagenesis. These assays for frameshift mutagenesis performed in E. coli cells infected with the appropriate T7 strain, were used to measure the frequency of both plus and minus frameshifts in vivo.     

Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels.

Agarose gel electrophoresis of the following was performed in 0.05 M sodium phosphate-0.001 M MgCl2 (pH 7.4): (i) bacteriophage T7; (ii) a T7 precursor capsid (capsid I), isolated from T7-infected Escherichia coli, which has a thicker and less angular envelope than bacteriophage T7; (iii) a second capsid (capsid II), isolated from T7-infected E. coli, which has a bacteriophage-like envelope; and (iv) capsids (capsid IV) produced by temperature shock of bacteriophage T7. Bacteriophage T7 and all of the above capsids migrated towards the anode. In a 0.9% agarose gel, capsid I had an electrophoretic mobility of 9.1 +/- 0.4 X 10(-5) cm2/V.s; bacteriophage T7 migrated 0.31 +/- 0.02 times as fast as capsid I. The mobilities of different preparations of capsid II varied in such gels: the fastest-migrating capsid II preparation was 0.51 +/- 0.03 times as fast as capsid I and the slowest was 0.37 +/- 0.02 times as fast as capsid I. Capsid IV with and without the phage tail migrated 0.29 +/- 0.02 and 0.42 +/- 0.02 times as fast as capsid I. The results of the extrapolation of bacteriophage and capsid mobilities to 0% agarose concentration indicated that the above differences in mobility are caused by differences in average surface charge density. To increase the accuracy of mobility comparisons and to increase the number of samples that could be simultaneously analyzed, multisample horizontal slab gels were used. Treatment with the ionic detergent sodium dodecyl sulfate converted capsid I to a capsid that migated in the capsid II region during electrophoresis through agarose gels. In the electron microscope, most of the envelopes of these latter capsids resembled the capsid II envelope, but some envelope regions were thicker than the capsid II envelope.     

Incomplete entry of bacteriophage T7 DNA into F plasmid-containing Escherichia coli.

The penetration of bacteriophage T7 DNA into F plasmid-containing Escherichia coli cells was determined by measuring Dam methylation of the entering genome. T7 strains that cannot productively infect F-containing cells fail to completely translocate their DNA into the cell before the infection aborts. The entry of the first 44% of the genome occurs normally in an F-containing cell, but the entry of the remainder is aberrant. Bypassing the normal mode of entry of the T7 genome by transfecting naked DNA into competent cells fails to suppress F exclusion of phage development. However, overexpression of various nontoxic T7 1.2 alleles from a high-copy-number plasmid or expression of T3 1.2 from a T7 genome allows phage growth in the presence of F.     

Interruption-deficient mutants of bacteriophage T5: analysis of single-site mutants.

The properties of viable mutants of bacteriophage T5 that lack, singly, each of the four major sites at which single-chain interruptions normally occur in T5 DNA are described. The mutations responsible for loss of each interruption were mapped by analysis with HhaI, a restriction endonuclease with a cleavage site (pGCGC) that occurs at the 5' termini of the major interruptions (B. P. Nichols and J. E. Donelson, J. Virol. 22:520-526, 1977). For each mutant tested, loss of a specific interruption resulted in loss of a specific HhaI cleavage site. Multiple single-site mutants were constructed to determine the effect of loss of more than one interruption on phage viability. These recombinants, including a phage that lacks the four major interruptible sites, were fully viable and did not exhibit a compensating increase in the frequency of minor interruptions. The effect of loss of a specific interruption on genetic recombination was tested in two-factor crosses with markers that occur close to, but on opposite sites of, the interruption. Loss of the interruptible site did not affect recombination frequency.