Physiological and Genetic Aspects of Abortive Infection of a Shigella sonnei Strain by Coliphage T7

Phage T7 adsorbed to and lysed cells of Shigella sonnei D(2) 371-48, although the average burst size was only 0.1 phage per cell (abortive infection). No mechanism of host-controlled modification was involved. Upon infection, T7 rapidly degraded host deoxyribonucleic acid (DNA) to acid-soluble material. Phage-directed DNA synthesis was initiated normally, but after a few minutes the pool of phage DNA, including the parental DNA, was degraded. Addition of chloramphenicol, at the time of phage infection, prevented both the initiation of phage-directed DNA synthesis and the degradation of parental phage DNA. Addition of chloramphenicol 4.5 min after phage was added permitted the onset of phage-directed DNA synthesis but prevented breakdown of phage DNA. Mutants of T7 (ss(-) mutants) have been isolated which show normal growth in strain D(2) 371-48. Upon mixed infection of this strain with T7 wild type and an ss(-) mutant, infection was abortive; no complementation occurred. The DNA of the ss(-) mutants was degraded in mixed infection like that of the wild type. Revertant mutants which have lost their ability to grow on D(2) 371-48 were isolated from ss(-) mutants; they are, in essence, phenotypically like T7 wild type. Independently isolated revertants of ss(-) mutants did not produce ss(-) recombinants when they were crossed among themselves. When independently isolated ss(-) mutants were crossed with each other, wild-type recombinants were found; ss(-) mutants could then be mapped in a cluster compatible with the length of one cistron. We concluded that T7 codes for an active, chloramphenicol-sensitive function [ss(+) function (for suicide in Shigella)] which leads to the breakdown of phage DNA in the Shigella host.     

Defect in synthesis of deoxyribonucleotides by a bacteriophage T4 nrdB mutant is suppressed on mutation of T4 DNA topoisomerase gene

Bacteriophage T4 infection is known to induce the formation of a complex of enzymes effecting the de novo synthesis of deoxyribonucleoside triphosphates, which in turn are channeled into T4 DNA replication. The first step in this pathway is catalyzed by a ribonucleoside diphosphate reductase, comprised of subunits coded by T4 genes nrdA and nrdB. Maximum rates of synthesis of the pyrimidine deoxyribonucleotides and of DNA replication in vivo also require a type II DNA topoisomerase encoded by T4 genes 39, 52, and 60. We report the identification of a unique mutant, nrdB93, and the suppression of its defective deoxyribonucleotide synthesis by a gene 39 mutation, 39-01. After infection by 39-01, DNA synthesis and plaque formation were temperature-sensitive, but nearly wild type rates of deoxyribonucleotide synthesis were retained at all temperatures. The nrdB93 mutation had a profound effect on deoxyribonucleotide synthesis at 41 degrees C; even at the permissive temperature of 30 degrees C, synthesis was reduced to 30% of that of wild type or 39-01. However, on infection at 30 degrees C by the double mutant, 39-01 nrdB93, the level of deoxyribonucleotide synthesis again reached that of wild type phage infections; involvement of the comparable host enzyme in the suppression process has been excluded. Suppression of the effect of nrdB93 by 39-01 implicates the gene 39 product in the regulation of nrdB expression. The accompanying paper (Cook, K. S., Wirak, D. O., Seasholtz, A. F., and Greenberg, G. R. (1988) J. Biol. Chem. 263, 6202-6208) examines the nature of the suppression process at the molecular level.     

In vitro host cell reactivation of alkylated bacteriophage T7 deoxyribonucleic acid by repair-deficient strains of Escherichia coli.

An in vitro system capable of packaging bacteriophage T7 deoxyribonucleic acid (DNA) into phage heads to form viable phage particles has been used to monitor the biological consequences of DNA dam aged by alkylating agents, and an in vitro DNA replication system has been used to examine the ability of alkylated T7 DNA to serve as template for DNA synthesis. The survival of phage resulting from in vitro packaging of DNA preexposed to various concentrations of methyl methane sulfonate or ethyl methane sulfonate closely paralleled the in vivo situation, in which intact phage were exposed to the alkylating agents. Host factors responsible for survival of alkylated T7 have been examined by using wild-type strains of EScherichia coli and mutants deficient in DNA polymerase I (polA) or 3-methyladenine-DNA glycosylase (tag). For both in vivo and in vitro situations, a deficiency in 3-methyladenine-DNA glycosylase dramatically reduced phage survival relative to that in the wild type, whereas a deficiency in DNA polymerase I had an intermediate effect. Furthermore, when the tag mutant was used as an indicator strain, phage survival was enhanced when alkylated DNA was packaged with extracts prepared from a wild-type strain in place of the tag mutant or by complementing a tag extract with an uninfected tag+ extract, indicating in vitro repair during packaging.     

Replication and Recombination of Gene 59 Mutant of Bacteriophage T4D

After infection of Escherichia coli B with phage T4D carrying an amber mutation in gene 59, recombination between two rII markers is reduced two- to three-fold. This level of recombination deficiency persists even when burst size similar to wild type is induced by the suppression of the mutant DNA-arrest phenotype. In the background of two other DNA-arrest mutants in genes 46 and 47, a 10- to 11-fold reduction in recombination is observed. The cumulative effect of gene 59 mutation on gene 46-47 mutant suggests that complicated interactions must occur in the production of genetic recombinants. The DNA-arrest phenotype of gene 59 mutant can be suppressed by inhibiting the synthesis of late phage proteins. Under these conditions, DNA replicative intermediates similar to those associated with wild-type infection are induced. Synthesis of late phage proteins, however, results in the degradation of mutant 200S replicative intermediate into 63S DNA molecules even in the absence of capsid assembly. Although these 63S molecules are associated with membrane, they do not replicate. These results suggest a role for gene 59 product, in addition to a possible requirement of concatemeric DNA in late replication of phage T4 DNA.     

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.     

Studies related to the head-maturation pathway of bacteriophages T4 and T2: II. Nuclear disruption,protein synthesis and particle formation with the mutant 43−·30−·46−

We describe the aberrant phage multiplication of the triple conditional lethal mutant 43^?(polymerase)· 30^?(ligase)·46^?(exonuclease) of bacteriophage T4D in which phage DNA replication is arrested but some late protein synthesis occurs (33). The nuclear disruption is indistinguishable from wild type. Forty-five empty small and empty large particles are assembled per cell when the multiplicity of infection (m.o.i.) is 100. This number corresponds closely to the 38 phage equivalents of cleaved major head protein determined biochemically. By reducing the m.o.i. the number of observable particles decreases, reaching 1–5 per cell at an m.o.i. of 5(+5). The total synthesis of phage related proteins is not significantly dependant on the m.o.i. The synthesis of late proteins is about 10% of that of wild type at high m.o.i. and decreases with the m.o.i. The different early and late proteins do not show the same relative proportions as in wild type and respond differently to an increased m.o.i. These and other results are discussed with respect to the role of phage DNA in prehead assembly and head maturation.     

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.     

Bypass of a primase requirement for bacteriophage T4 DNA replication in vivo by a recombination enzyme, endonuclease VII.

A primase, the product of phage T4 gene 61, is required to initiate synthesis of Okazaki pieces and to allow bidirectional replication from several T4 origins. However, primase-defective T4 gene 61 mutants are viable. In these mutants, leading-strand DNA synthesis starts at the same time as in wild type infections, but, in contrast to wild type, initiation is unidirectional and the first replicative intermediates are large displacement loops. Rapid double-strand DNA replication occurs later after infection, generating multiple branched concatemers, which are cut and packaged into viable progeny particles, as in wild-type T4. Evidence is presented that this late double-strand DNA replication requires functional endonuclease VII (endo VII), the product of the T4 gene 49. We propose that endo VII can provide a backup mechanism when primase is defective, because it cuts recombinational junctions, generating 3' ends. These ends can prime DNA synthesis to copy the DNA strands that had been displaced during the initial origin-dependent replication. We explain the DNA-delay phenotype and the commonly observed temperature dependence of DNA replication in primase-deficient gene 61 mutants as a consequence of temperature-dependent translational control of gene 49 expression. In the presence or absence of functional primase endo VII is essential for correct packaging of DNA. The powerful selection that keeps the function of endo VII and expression of its gene at levels that are optimal for T4 development determines both the efficiency and the limitations of the bypass mechanism.     

DNA helicase requirements for DNA replication during bacteriophage T4 infection.

The lytic bacteriophage T4 uses multiple mechanisms to initiate the replication of its DNA. Initiation occurs predominantly at replication origins at early times of infection, but there is a switch to genetic recombination-dependent initiation at late times of infection. The T4 insertion-substitution system was used to create a deletion in the T4 dda gene, which encodes a 5'-3' DNA helicase that stimulates both DNA replication and recombination reactions in vitro. The deletion caused a delay in T4 DNA synthesis at early times of infection, suggesting that the Dda protein is involved in the initiation of origin-dependent DNA synthesis. However, DNA synthesis eventually reached nearly wild-type levels, and the final number of phages produced per bacterium was similar to that of the wild type. When the dda mutant phage also contained a mutation in T4 gene 59 (a gene normally required only for recombination-dependent DNA replication), essentially no DNA was synthesized. Recent in vitro studies have shown that the gene 59 protein loads a component of the primosome, the T4 gene 41 DNA helicase, onto DNA. A molecular model for replication initiation is presented that is based on our genetic data.     

Recombination of bacteriophage T7 in vivo

Summary Most recombination following infection with T7 was found to coincide with the time of most rapid DNA synthesis, at about 20 min after infection at 30° in minimal medium. Recombining DNA was investigated electron microscopically. Multiply branched DNA structures were observed after infection with T7 wild type, gene 3 ^–, gene 6 ^– and genes 3 ^–, 6 ^– phage, but not after infection with T7 gene 5 ^– phage. Evidence is presented indicating that these structures are T7 DNA molecules in the process of recombining. The detailed structures of these recombinational intermediates suggest mechanisms by which T7 DNA initiates recombination.     

Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA.

The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4 DNA synthesis-negative mutatants (Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and 5-hydroxylxytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathways from [6-3H]uracil including those in DNA and any degradation products excreted into the medium. The ratio of the de novo synthesis of Thy/HmCyt derivatives remained constant at 2.1 +/- 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/HmCyt ratio in T4 DNA. On infection by phage mutated in the Dna-genes 32, 41, 44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about 100-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dna- mutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivates were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of enzymes synthesizing and channeling deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.     

Replication of bacteriophages in Escherichia coli mutants thermosensitive in DNA synthesis

Summary In E. coli mutants thermosensitive in DNA synthesis the capacity for replication of bacteriophages , P1 and T4 was studied in order to obtain more information about the biochemical lesions in such strains. Two mutant types were used. In one of them DNA synthesis stops immediately at the restrictive temperature (mutant 165/70). In the other type DNA synthesis continues at the elevated temperature for a residual time period before it comes to a halt (mutant 252). The thermolabile synthetic steps involved in both mutant types are presently still unknown.The temperate phages and P1 differ in their ability to replicate in the mutant types at temperatures non-permissive for host cell DNA synthesis. Replication of phage is blocked in 165/70 but can still take place in 252 after host DNA synthesis has come to a halt. Phage P1 shows the opposite behaviour. It grows in the mutant 165/70 but its ability to replicate in 252 at 42° C is restricted to the period of residual host cell DNA synthesis observed in uninfected cells. Replication of phage T4 on the other hand is unimpeded in both mutants at restrictive temperatures.     

Characterization of a camptothecin-resistant human DNA topoisomerase I in an in vitro system for Simian virus 40 DNA replication.

DNA topoisomerase I was required for bidirectional DNA replication in an in vitro system for Simian virus 40 (SV40) DNA replication with purified proteins in which the replication fork moved at the rate of 260 nucleotides/min on average. DNA topoisomerase I purified from camptothecin-resistant human lymphoblastoid cells, which confers high resistance of cellular DNA replication to camptothecin [Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, Y. & Okada, K. (1987) Proc. Natl Acad. Sci. USA 84, 5565-5569], was characterized using this system. The activity of stimulating bidirectional DNA replication was comparable between two topoisomerase I from parental and resistant cells, i.e. in its dose-response relationship and in its time course for DNA synthesis. Camptothecin severely inhibited the leading as well as the lagging strand synthesis in the reaction containing the wild type topoisomerase I but not the mutant type topoisomerase I. The mutant type topoisomerase I was over 125-fold as resistant to camptothecin as the wild type topoisomerase I. These results are in good agreement with those on the sensitivity of cellular DNA synthesis to camptothecin in the resistant cells. These findings suggest that topoisomerase I is involved in cellular DNA replication as a swivelase and the mutation conferring camptothecin-resistance on the enzyme does not affect its functional efficiency in this system.     

Bacillus subtilis DNA Polymerase III Is Required for the Replication of the Virulent Bacteriophage φe

The virulent phage phie of Bacillus subtilis which contains hydroxymethyluracil in its DNA requires host DNA polymerase III for its DNA replication. DNA polymerase III(ts) mutant cells infected with phie at restrictive temperatures do not support phage DNA synthesis. However, phie grows normally both at low and high temperatures in the mutant's parent strain and in spontaneous DNA polymerase III(+) revertants isolated from the mutant strain. Temperature-shift-down experiments with phie-infected cells having thermosensitive DNA polymerase III (pol III(ts)) indicate that at 48 C the thermolabile DNA polymerase III is irreversibly inactivated and has to be synthesized de novo after the shift to 37 C, before phage DNA synthesis can begin. Temperature-shift-up experiments with phie-infected mutant cells show that phage replication is arrested immediately after the temperature shift and indicate that phie requires DNA polymerase III throughout its replication stage.     

New late gene, dar, involved in the replication of bacteriophage T4 DNA. III. DNA replicative intermediates of T4 dar and a gene 59 mutant suppressed by dar.

A mutation in the dar gene of phage T4 restored the arrested DNA synthesis caused by the gene 59 mutation. We have studied the DNA replicative intermediates in cells infected with a dar mutant and a dar-amC5 (gene 59) mutant by velocity sedimentation in neutral and alkaline sucrose gradients. In T4 dar-infected cells, compared to the wild type, three kinds of abnormalities were observed in DNA replication (i) There were unusually rapidly sedimenting intermediates (800S). (ii) When centrifuged in alkaline gradients, there was less single-stranded DNA exceeding 1 phage unit. (iii) The rate of repair of DNA intermediates was slower. It has been proposed by others that the 200S DNA replicative intermediates are required for DNA packaging, but our results showed that the 800S DNA of dar does not have to be converted into the 200S form to undergo conversion to mature viral DNA. Therefore, 200S DNA may not be an obligatory intermediate for mature viral DNA formation. In amC5 (gene 59)-infected cells, the DNA was completely converted 2 to 3 min after intiation of replication to the biologically inactive 63S DNA, and DNA synthesis was concomitantly arrested. However, in dar-am-C5 (gene 59)-infected cells, the formation of abnormal 63S DNA did not occur and 200S DNA appeared instead. An endonucleolytic activity, normally associated with the cell membrane and capable of making double-stranded cuts, was found in the cytoplasm of T4 dar-infected cells. Because the total activity of this endonuclease is the same for both wild-type T4D and the dar mutant, it seems unlikely that the dar protein has endonucleolytic activity itself. However, the finding does explain the abnormal sedimentation of dar DNA intermediates (800S) as well as the proposed suppression mechanism of the gene 59 mutation.     

Selective Allele Loss in Mixed Infections with T4 Bacteriophage

Evidence is presented that when E. coli B is mixedly infected with T4D wild type and rII deletion mutants, the excess DNA of the wild type allele is lost. No loss is seen in mixed infections with rII point mutants and wild type. In similar experiments with lysozyme addition mutants, the mutant allele is lost. We believe these results demonstrate a repair system which removes "loops" in heteroduplex DNA molecules. A number of phage and host functions have been tested for involvement in the repair of the excess DNA, and T4 genes x and v have been implicated in this process.     

Specific Suppression of Mutations in Genes 46 and 47 by das, a New Class of Mutations in Bacteriophage T4D

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.