Assembly of bacteriophage T7. Dimensions of the bacteriophage and its capsids.

The dimensions of bacteriophage T7 and T7 capsids have been investigated by small-angle x-ray scattering. Phage T7 behaves like a sphere of uniform density with an outer radius of 301 +/- 2 A (excluding the phage tail) and a calculated volume for protein plus nucleic acid of 1.14 +/- 0.05 x 10(-16) ml. The outer radius determined for T7 phage in solution is approximately 30% greater than the radius measured from electron micrographs, which indicates that considerable shrinkage occurs during preparation for electron microscopy. Capsids that have a phagelike envelope and do not contain DNA were obtained from lysates of T7-infected Escherichia coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA by means of temperature shock (capsid IV). In both cases the peak protein density is at a radius of 275 A; the outer radius is 286 +/- 4 A, approximately 5% smaller than the envelope of T7 phage. The thickness of the envelope of capsid II is 22 +/- 4 A, consistent with the thickness of protein estimated to be 23 +/- 5 A in whole T7 phage, as seen on electron micrographs in which the internal DNA is positively stained. The volume in T7 phage available to package DNA is estimated to be 9.2 +/- 0.4 x 10(-17) ml. The packaged DNA adopts a regular packing with 23.6 A interplanar spacing between, DNA strands. The angular width of the 23.6 A reflection shows that the mean DNA-DNA spacing throughout the phage head is 27.5 +/- less than 2.2 A. A T7 precursor capsid (capsid I) expands when pelleted for x-ray scattering in the ultracentrifuge to essentially the same outer dimensions as for capsids II and IV. This expansion of capsid I can be prevented by fixing with glutaraldehyde; fixed capsid I has peak density at a radius of 247 A, 10% less than capsid II or IV.     

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.     

In Vitro Packaging of UV Radiation-Damaged DNA from Bacteriophage T7

When DNA from bacteriophage T7 is irradiated with UV light, the efficiency with which this DNA can be packaged in vitro to form viable phage particles is reduced. A comparison between irradiated DNA packaged in vitro and irradiated intact phage particles shows almost identical survival as a function of UV dose when Escherichia coli wild type or polA or uvrA mutants are used as the host. Although uvrA mutants perform less host cell reactivation, the polA strains are identical with wild type in their ability to support the growth of irradiated T7 phage or irradiated T7 DNA packaged in vitro into complete phage. An examination of in vitro repair performed by extracts of T7-infected E.coli suggests that T7 DNA polymerase may substitute for E. coli DNA polymerase I in the resynthesis step of excision repair. Also tested was the ability of a similar in vitro repair system that used extracts from uninfected cells to restore biological activity of irradiated DNA. When T7 DNA damaged by UV irradiation was treated with an endonuclease from Micrococcus luteus that is specific for pyrimidine dimers and then was incubated with an extract of uninfected E. coli capable of removing pyrimidine dimers and restoring the DNA of its original (whole genome size) molecular weight, this DNA showed a higher packaging efficiency than untreated DNA, thus demonstrating that the in vitro repair system partially restored the biological activity of UV-damaged DNA.     

In vitro packaging of bacteriophate T7 DNA synthesized in vitro.

An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome.     

Simultaneous banding of rat embryo DNA, RNA, and protein in cesium trifluoroacetate gradients

A procedure for the simultaneous banding of cellular DNA, RNA, and protein by centrifugation in cesium trifluoroacetate (CsTFA) gradients is described. Starting with homogenates of Day 11 rat embryos, this procedure was used to separate total DNA, RNA, and protein. Under the conditions used DNA banded at a peak density of 1.63 g/ml, RNA at a peak density of 1.83 g/ml, and protein at a peak density of 1.40 g/ml. Nucleic acids isolated from CsTFA gradients were judged to be protein free. RNA isolated by this method is apparently free of DNA contamination; however, DNA isolated by this method does contain some RNA (less than 5% contamination).     

Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo

The tightly packaged double-stranded DNA (dsDNA) genome in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when reconstructed from cryo-electron micrographs. However, recent single-particle DNA packaging force measurements have suggested that incompletely packaged DNA (ipDNA) is less ordered when it is shorter than ∼ 25% of the full genome length. The study presented here initially achieves both the isolation and the ipDNA length-based fractionation of ipDNA-containing T3 phage capsids (ipDNA-capsids) produced by DNA packaging in vivo; some ipDNA has quantized lengths, as judged by high-resolution gel electrophoresis of expelled DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ipDNA-capsids are a minor component (containing ∼ 10^− 4 of packaged DNA in all particles) and are initially detected by nondenaturing gel electrophoresis after partial purification by buoyant density centrifugation. The primary contaminants are aggregates of phage particles and empty capsids. This study then investigates ipDNA conformations by the first cryo-electron microscopy of ipDNA-capsids produced in vivo. The 3-D structures of DNA-free capsids, ipDNA-capsids with various lengths of ipDNA, and mature bacteriophage are reconstructed, which reveals the typical T = 7l icosahedral shell of many tailed dsDNA bacteriophages. Though the icosahedral shell structures of these capsids are indistinguishable at the current resolution for the protein shell (∼ 15 Å), the conformations of the DNA inside the shell are drastically different. T3 ipDNA-capsids with 10.6 kb or shorter dsDNA (< 28% of total genome) have an ipDNA conformation indistinguishable from random. However, T3 ipDNA-capsids with 22 kb DNA (58% of total genome) form a single DNA ring next to the inner surface of the capsid shell. In contrast, dsDNA fully packaged (38.2 kb) in mature T3 phage particles forms multiple concentric rings such as those seen in other tailed dsDNA bacteriophages. The distance between the icosahedral shell and the outermost DNA ring decreases in the mature, fully packaged phage structure. These results suggest that, in the early stage of DNA packaging, the dsDNA genome is randomly distributed inside the capsid, not preferentially packaged against the inner surface of the capsid shell, and that the multiple concentric dsDNA rings seen later are the results of pressure-driven close-packing.     

Behavior of bacteriophage Mu DNA upon infecton of Escherichia coli cells.

The question whether the ends of bacteriophage Mu DNA are fused to form a ring in host cells is critical to the understanding of the mechanism of integrative recombination between Mu DNA and host DNA. We have examined the fate of ³²P-labeled Mu DNA, after infection of sensitive and immune (lysogenic) cells, by sedimentation in sucrose gradients, ethidium bromide/CsCl density centrifugation and by electrophoresis of parental Mu DNA and its fragments in agarose gels. We find that the parental Mu DNA cannot be detected as covalently closed circles at any stage during the Mu life cycle. An interesting form of Mu DNA can be seen after superinfection of immune cells. This form sediments about twice as fast as the mature phage DNA marker in neutral sucrose gradients but yields linear molecules upon phenol extraction. Upon infection of sensitive cells, most of the parental DNA associates with a large complex, presumably containing the host chromosome. When Mu-sensitive cells are infected with unlabeled Mu particles and Mu DNA examined at different times after infection by fractionation in 0.3% agarose gels and hybridization with ³²P-labeled Mu DNA, Mu sequences are found to appear with the bulk host DNA as the phage lytic cycle progresses. However, no distinct replicative or integrative intermediate of Mu, that behaves differently from linear Mu DNA and is separate from the host DNA, can be detected.     

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.     

Viral DNA packaging studied by fluorescence correlation spectroscopy

The DNA packaging machinery of bacteriophage T4 was studied in vitro using fluorescence correlation spectroscopy. The ATP-dependent translocation kinetics of labeled DNA from the bulk solution, to the phage interior, was measured by monitoring the accompanied decrease in DNA diffusibility. It was found that multiple short DNA fragments (100 basepairs) can be sequentially packaged by an individual phage prohead. Fluorescence resonance energy transfer between green fluorescent protein donors within the phage interior and acceptor-labeled DNA was used to confirm DNA packaging. Without ATP, no packaging was observed, and there was no evidence of substrate association with the prohead.     

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.     

Packaging of genomes in bacteriophages: a comparison of ssRNA bacteriophages and dsDNA bacteriophages.

In complex DNA bacteriophages like lambda, T4, T7, P22, P2, the DNA is packaged into a preformed precursor particle which sometimes has a smaller size and often a shape different from that of the phage head. This packaging mechanism is different from the one suggested for the RNA phages, according to which RNA nucleates the shell formation. The different mechanisms could be understood by comparing the genomes to be packaged: single stranded fII RNA has a very compact structure with high helix content. It might easily form quasispherical structures in solution (as seen in the electron microscope by Thach & Thach (1973)) around which the capsid could assemble. Double stranded phage DNA, on the other hand, is a rigid molecule which occupies a large volume in solution and has to be concentrated 15-fold during packaging into the preformed capsid, and the change in the capsid structure observed hereby might provide the necessary DNA condensation energy.     

Intermediates in genetic recombination of bacteriophage T7 DNA. Biological activity and the roles of gene 3 and gene 5

When Escherichia coli cells were infected with ³²P- and 5-bromodeoxyuridine-labeled T7 bacteriophage defective in genes 1.3, 2.3, 4 and 5, doubly branched T7 DNA molecules with “H” or “X”-like configurations were found in the half-heavy density fractions. Physical study showed that they are dimeric molecules composed of two parental DNA molecules (Tsujimoto & Ogawa, 1977a). The transfection assay of these molecules revealed that they were infective. Genetic analysis of progeny in infective centers obtained by transfection of dimeric molecules formed by infection of genetically marked T7 phage showed that these dimeric molecules were genetically biparental.To elucidate the roles of the products of gene 3 (endonuclease I) and gene 5 (DNA polymerase) of phage T7 in the recombination process, the ³²P/BrdUrd hybrid DNA molecules which were formed in the infected cells in the presence of these gene products were isolated, and their structures were analyzed. The presence of T7 DNA polymerase seems to stimulate and/or stabilize the interaction of parental DNAs. At an early stage of infection few dimeric molecules were formed in the absence of T7 DNA polymerase, whereas a significant number of doubly branched molecules were formed in its presence. With increasing incubation time, the multiply branched DNA molecules with a high sedimentation velocity accumulated.In contrast to the accumulation of multiply branched molecules in phage with mutations in genes 2, 3 and 4, almost all of the ³²P/BrdUrd hybrid DNA formed in phage with mutations in genes 2 and 4 were monomeric linear molecules. Shear fragmentation of monomeric linear ³²P/BrdUrd-labeled DNA shifted the density of [³²P]DNA to almost fully light density. It was also found that approximately 50% of [³²P]DNA was linked covalently to BrdUrd-labeled DNA. These linear monomer DNA molecules had infectivity and some of those formed by infection of genetically marked parents yielded recombinant phages. Therefore the gene 3 product seems to process the branched intermediates to linear recombinant molecules by trimming the branches.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems.

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

Transduction of bacteriophage lambda by bacteriophage T1.

When bacteriophage T1 was grown on bacteriophage lambda-lysogenic cells, phenotypically mixed particles were formed which had the serum sensitivity, host range, and density of T1 but which gave rise to lambda phage. T1 packaged lambda genomes more efficiently both when the length of the prophage was less than that of wild-type lambda and when the host cell was polylysogenic. Expression of the red genes of lambda or the recE system of Escherichia coli during T1 growth enhanced pickup of lambda by T1, whereas packaging was reduced in recB cells. If donors were singly lysogenic, the expression of transduced lambda genomes as a PFU required lambda-specified excisive recombination, whereas lambda genomes transduced from polylysogens required only lambda- or E. coli-specified general recombination to give a productive infection.     

Genetic deletions between directly repeated sequences in bacteriophage T7

Summary DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences. To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase. This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase. The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats. Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage. It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends. In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10⁻¹⁰, while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10⁻⁶. The deletion events were independent of a hostrecA mutation.     

Langevin dynamics simulations of genome packing in bacteriophage

We use Langevin dynamics simulations to study the process by which a coarse-grained DNA chain is packaged within an icosahedral container. We focus our inquiry on three areas of interest in viral packing: the evolving structure of the packaged DNA condensate; the packing velocity; and the internal buildup of energy and resultant forces. Each of these areas has been studied experimentally, and we find that we can qualitatively reproduce experimental results. However, our findings also suggest that the phage genome packing process is fundamentally different than that suggested by the inverse spool model. We suggest that packing in general does not proceed in the deterministic fashion of the inverse-spool model, but rather is stochastic in character. As the chain configuration becomes compressed within the capsid, the structure, energy, and packing velocity all become dependent upon polymer dynamics. That many observed features of the packing process are rooted in condensed-phase polymer dynamics suggests that statistical mechanics, rather than mechanics, should serve as the proper theoretical basis for genome packing. Finally we suggest that, as a result of an internal protein unique to bacteriophage T7, the T7 genome may be significantly more ordered than is true for bacteriophage in general.     

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.     

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.     

Renaturation of denatured DNA in NaI gradients

We show that denatured DNA from Tetrahymena mitochondria or phage T₇, partially renatures during centrifugation in NaI equilibrium density gradients. This makes these gradients unsuitable for the analysis of single-stranded complementary nucleic acids, especially if their complexity and mole percent G+C are low.