Bacteriophage T4 Head Morphogenesis IV. Comparison of Gene 16-, 17-, and 49-Defective Head Structures2

Defective heads present in extracts of bacteriophage T4 gene 16, 17, or 49 mutant-infected cells have been characterized. All appeared as empty shells when examined by negative-stain electron microscopy and showed essentially the same polypeptide pattern on sodium dodecyl sulfate-acrylamide gels. However, when analyzed by several other methods, gene 16- and 17-defective heads were shown to differ markedly from phage heads present in gene 49-defective extracts. First, the gene 16- and 17-defective structures were found to possess a large number of attached tails (50%, rather than about 5%). Second, they contained less nuclease-resistant deoxyribonucleic acid (DNA) (3 versus 18% of a phage equivalent), had a smaller sedimentation coefficient (240 versus 315S), and a lighter density (1.31 vs. 1.34 g/ml) than gene 49-defective heads. Third, they were not attached to the intracellular DNA pool through a deoxyribonuclease-sensitive linkage. Finally, 8-nm diameter capsomers were clearly revealed on the surface of many gene 16- and 17-defective structures. There was a total of 305 ± 25 capsomers per particle, which yielded an approximate molecular weight of 84 × 10⁶ for these heads. The capsomers were presumably not seen on gene 49-defective heads because of the large amount (18%) of associated DNA.     

Bacteriophage T4 head morphogenesis : On the nature of gene 49-defective heads and their role as intermediates

Following infection under non-permissive conditions, T4 mutants defective in gene 49 accumulate structures which appear in the electron microscope to be empty phage heads. These structures are seen in extracts prepared under a variety of conditions, as well as in sections of the mutant-infected cells. The 49-defective heads (300 s) can be separated from phage particles (1000 s) by sedimentation through a sucrose gradient. A temperature-sensitive gene 49 mutant, tsC9, accumulates 300 s heads following infection at 41.5 °C, but can be “rescued” by a shift-down to 25 °C during the latter half of the latent period. Evidence from pulse-chase isotopic labeling experiments suggests that the 49-defective heads are intermediates in head formation. ¹⁴C-Labeled lysine, incorporated into the 300 s fraction at 41.5 °C, is rapidly and almost quantitatively transferred into the 1000 s phage particle fraction following a chase with an excess of unlabeled lysine and a shift to low temperature. The same result is observed when puromycin (200 μg/ml.) or chloramphenicol (200 μg/ml.) is added to the culture before temperature shift, suggesting that the inactive gene 49 product produced at high temperature becomes active at low temperature. In pulse-chase experiments carried out with wild-type T4-infected cells during the latter half of the latent period, the labeling kinetics of the 300 s and phage particle fractions support a precursor-product relationship. Conservation of the 300 s head structures during conversion to phage is demonstrated by ¹³C-¹⁵N density labeling of tsC9-infected cells at 41.5 °C followed by transfer to ¹²C-¹⁴N medium, shift to low temperature, isolation and lysis of the phage particles formed and centrifugation of the phage ghosts to equilibrium in CsCl solution.     

Bacteriophage T4 head morphogenesis. VII. Terminal stages of head maturation.

Several aspects of the terminal stages of T4 head maturation were investigated using ts and am mutants blocked at single steps of the assembly pathway. We had previously found that cells infected with mutants of gene 13, e.g., tsN38 and amE609, accumulated both stable (10 to 20%)- and fragile (80%)-filled head precursors (Hamilton and Luftig, 1972). Here we showed the following for such gene 13-defective, mutant-infected cells. (i) Using thin-section analysis the pool of phage precursor structures observed under nonpermissive conditions was one-third of that observed when the cells were cultured under permissive conditions. (ii) In order for complete conversion of the precursors into viable phage to occur, there were apparent requirements of metabolic energy, protein, and DNA synthesis. (iii) The intracellular DNA pool under nonpermissive conditions exhibited a 50% distribution between 63S (mature size) and 200 S (concatenate size) DNA, with the latter DNA serving as a precursor pool. Further, this DNA pool when spread onto a protein monolayer exhibited a dispersed array of DNA, strands around a core, which was less dense than that found for the greater than 1,000S DNA concatenate isolated from gene 49-defective infected cells. (iv) When precuations were taken to stabilize the head precursors, such as lysis of the cells into glutaraldehyde, there was a 30% increase in the yield of 1,200S filled heads. Correlating these results and previous results concerning gene 49-defective unfilled heads, we propose that there are several forms of gene 13 fragile head precursors which serve as intermediates between gene 49 unfilled heads and gene 13 stable filled heads. We cannot, however, rule out the possibility that all gene 13-defective heads represent a single class of unstable particles, which decay slowly. In either case, we have shown that gene 13-defective particles are unstable to some degree inside the cell and are highly unstable outside the cell; yet all particles can still be efficiently converted to phage in vivo.     

Maturation of the head of bacteriophage T4. III. DNA packaging into preformed heads

An estimate was made of the amount of DNA packaged into gene 49-defective heads when P49 is activated by a temperature shift. The uptake of DNA into preformed heads following activation of P49 was studied using bromo-deoxyuridine as a label. The rate of inactivation by visible light of the phage matured in the presence of BrdU as well as their buoyant density in CsCl, indicate that over half of the particles package, on the average, at least 25% of the DNA complement following P49 activation. This is a minimum estimate, since the BrdU-labeled DNA has to compete with unlabeled DNA. Analysis on alkaline sucrose gradients of the size of the DNA extracted from phage matured in the presence of BrdU following irradiation reveals that extended irradiation at 313 nm breaks the DNA close to half of its original size. These experiments clearly show that up to half of the DNA can be packaged into the preformed heads made at high temperature following activation of the product of gene 49 (P49), strongly supporting the pathway for phage head maturation described by Laemmli &; Favre (1973).The so-called τ-particles, which accumulate in 24-defective cells, can serve as precursors of the mature phage (Bijlenga et al., 1973). We have measured the uptake of BrdU-labeled DNA into τ-particles during their maturation. We find that a very large proportion of DNA made after activation of P24 is apparently incorporated into preformed τ-particles as these particles are converted into mature heads. This indicates that the τ-particles contain very little or no DNA prior to P24 activation and supports the pathway described by Laemmli &; Favre (1973).     

Bacteriophage T4 Head Morphogenesis III. Some Novel Properties of Gene 13-Defective Heads

The nature of phage precursors in gene 13-defective infected cells was studied by electron microscopy and pulse-chase isotopic labeling experiments. Our results suggest that both stable (20%) and fragile (70%) filled-head precursors accumulated in the absence of gene 13 product. Upon extraction, the fragile heads were found to lose most of their deoxyribonucleic acid and appeared unfilled with an average density of 1.34 g/cm(3) and a sedimentation coefficient of 300S. These unfilled heads differed from empty gene 13-defective heads which did not have any associated deoxyribonucleic acid and banded at an average density of 1.31 g/cm(3). Furthermore, it was found that a tsN38 (temperature-sensitive mutant in gene 13)- infected culture maintained at 41.5 C for increasing times led to a decrease in specific infectivity of 1,000S phagelike particles. Electron microscopy of these particles revealed that the decreased infectivity was due to an improper union of head and tails.     

Function of gene 49 of bacteriophage T4. II. Analysis of intracellular development and the structure of very fast-sedimenting DNA.

With the exception of mutants in gene 49, all mutants in phage T4 defective in the process of head filling accumulate a normal replicative DNA intermediate of 200S. Mutants in gene 49 produce a very fast-sedimenting (VFS) DNA with s values of greater than 1,000S. The intracellular development of the VFS-DNA generated in gene 49-defective phage-infected cells was followed by sedimentation analysis of crude lysates on neutral sucrose gradients. It was observed that the production of a 200S replicative intermediate is one step in the development of VFS-DNA. After restoring permissive conditions the development of the VFS-DNA can be reversed, but the 200S form is not regenerated under these conditions. The process of head filling can take place from the VFS-DNA under permissive conditions. From the absence of other components in the VFS-DNA complexes, its high resistance to shearing, its resistance against the attack of the single-strand-specific nuclease S1, and from its appearance in the electron microscope, a complex structure of tightly packed DNA is inferred. The demonstration by the electron microscope of branched DNA structures sometimes closely related to partially filled heads is taken in support of the idea that the process of head filling in gene 49-defective phage-infected cells is blocked by some steric hindrance in the DNA. In light of these results, the role of gene 49 is discussed as a control function for the clearance of these structures. A fixation procedure for cross-linking of gene 49-defective heads to the VFS-DNA allowed us to study progressive stages in the process of head filling. Electron microscopic evidence is presented which suggests that during the initial events the DNA accumulates in the vertexes of the head.     

Involvement of Gene 49 in Recombination of Bacteriophage T4

The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants. When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype. However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor. Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvsX, but recombination between rII markers was not.     

Bacteriophage T4 Head Maturation: Release of Progeny DNA from the Host Cell Membrane

We have presented a new approach to studying bacteriophage T4 head maturation. Using a modified M-band technique, we have shown that progeny deoxyribonucleic acid (DNA) was synthesized on the host cell membrane throughout infection. This DNA was released from the membrane later in infection as the result of formation of the phage head; detachment of the DNA required the action of gene products 20, 21, 22, 23, 24, 31, 16, 17 and 49, known to be necessary for normal head formation. Gene products 2, 4, 50, 64, 65, 13 and 14, also involved in head morphogenesis were not required to detach progeny DNA from the membrane; the presence of the phage tail and tail fibers also was not required. DNA was released in the form of immature heads and initially was sensitive to deoxyribonuclease (DNase). Conversion to DNase resistance followed rapidly. The amount of phage precursors present at the time of DNA synthesis determined the time of onset and detachment rate of DNA from the M band as well as the kinetics by which the detached DNA become DNase resistant.     

Capsid Size and Deoxyribonucleic Acid Length: the Petite Variant of Bacteriophage T4

A mutant which produces a small-headed ("petite") variant of bacteriophage T4 is described. The mutation (E920g) maps in a new gene (66) between genes 23 and 24. Petite phage particles composed up to 70% of the phage yield. The petite phage was nonviable upon single infection but produced progeny when two or more infected a cell. Its genome was shortened by a random deletion of about 30%, and deoxyribonucleic acid (DNA) extracted from the particles was 0.68 the length of normal T4 DNA. The reduction in DNA length was accompanied by a proportional reduction in head volume. Double mutants between E920g and head-defective mutants in gene 21 produced unusually high frequencies of spherical capsidlike structures (tau-particles).     

A promiscuous DNA packaging machine from bacteriophage T4

Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.     

Model for DNA packaging into bacteriophage T4 heads.

The mechanism of DNA packaging into bacteriophage T4 heads in vivo was investigated by glucosylation of hydroxymethylcytosine residues in a conditionally glucose-deficient host. Cytoplasmic DNA associated with partially packaged ts49 heads can be fully glucosylated, whereas DNA already packaged into these heads is shown to be resistant to glucosylation. After temperature shift and completion of arrested packaging into the reversible temperature-sensitive ts49 head, the structure of the DNA in the mature ts49 phage was investigated by restriction enzyme digestion, autoradiography, and other techniques. Such mature DNA appears to be fully glucosylated along part of its length and nonglucosylated on the remainder. Its structure suggests that the DNA is run into the head linearly and unidirectionally from one mature end and that there is little sequence specificity in that portion of the T4 DNA which first enters the capsid. This technique should be useful in investigation of the three-dimensional structure of first- and last-packaged DNA within the head; preliminary studies including autoradiography of osmotically shocked phage suggest that the DNA which first enters the head is deposited toward the center of the capsid and that the end of the DNA which first enters the head exits first upon injection. In conjunction with studies of the structure of condensed DNA, the positions and functions of T4 capsid proteins in DNA packaging, and the order of T4 packaging functions [Earnshaw and Harrison, Nature (London) 268:598-602, 1977; Hsiao and Black, Proc. Natl. Acad. Sci. U.S.A. 74:3652-3656, 1977; Müller-Salamin et al., J. Virol. 24:121-134, 1977; Richards et al., J. Mol. Biol. 78:255-259, 1973], the features described above suggest the following model: the first DNA end is fixed to the proximal apex of the head at p20 and the DNA is then pumped into the head enzymatically by proteins (p20 + p17) which induce torsion in the DNA molecule.     

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.     

Studies related to the head-maturation pathway of bacteriophages T4 and T2: I. Morphology and kinetics of intracellular particles produced by mutants in the maturation genes

Mutants in the genes governing the maturation of the head of bacteriophage T4 and in gene 24 were studied by electron microscopy of thin sections. We define morphologically: black particles, comprising mature, stable heads and immature, fragile heads, which break down upon lysis; grizzled particles, which apparently are partially filled or partially emptied; empty large particles without DNA or core Which are all the same size as normal heads; empty small particles without DNA and without core which are of the size of the τ particle, which is the prehead of phage T4. The study of single and double mutants of the maturation genes demonstrates that the phenotypes are only different by the proportions of the different particles made except for 17^? where only empty small and empty large particles accumulate. The mutants in gene 24 are epistatic on all other mutants. Mutants in gene 17 are epistatic on the remaining ones. The results are consistent with the hypothesis that the products of several of the maturation genes act on DNA to render it competent for packaging while the others act directly on the particle. By this uncoupling, bypasses and abortive pathways can result.     

Synthesis of a trans-Acting Inhibitor of DNA Maturation by Prohead Mutants of Phage λ

Bacteriophage lambda with mutations in genes that control prohead assembly and other head precursors cannot mature their DNA. In this paper we present evidence that the failure of these phage mutants to mature DNA is a reflection of a mechanism that modulates terminase nicking activity during normal phage development. We have constructed plasmids that contain the lambda-cohesive end site (cos) and the genes that code for DNA terminase, the enzyme that matures DNA by cutting at cos. The DNA terminase genes are under control of a thermosensitive cI repressor. These plasmids lack most of the genes involved in prohead morphogenesis and other head precursors. However, when repression is lifted by destruction of the thermosensitive repressor, the terminase synthesized is able to cut almost 100% of the plasmids. Therefore, these plasmids can mature in the absence of proheads and other head gene products. The plasmids are also able to complement mutants of lambda deficient in terminase and DNA maturation. However, in these complementation experiments, if the phage carry mutations in prohead genes E or B, not only is phage DNA maturation blocked, but the plasmid also fails to mature. These experiments show that, in the absence of proheads, phage lambda produces a trans-acting inhibitor of maturation. The genetic determinant of this inhibitor maps in a region extending from the middle of gene B to the end of gene C. A model is proposed in which the nicking activity of DNA-bound terminase is inhibited by the trans-acting inhibitor. Prohead (and other factors) binding to this complex would release the block to allow DNA cleavage and packaging.     

Identification of sequences necessary for packaging DNA into lambda phage heads

Several species of DNA molecules are packaged into lambda phage heads if they carry the region around the cohesive end site of lambda phage (cos lambda). The minimal functional sequence around cos lambda needed for packaging was examined by cloning in pBR322. The results showed that the minimal region contained 85 bp around cos lambda; 45 bp of the left arm of lambda phage and 40 bp of the right arm. A 75-bp region located to the right of the minimal region seems to enhance packaging. A 223-bp fragment containing these regions can be used as a portable element for plasmid DNA packaging into lambda phage heads. Plasmid ppBest 322, a derivative of pBR322 carrying this portable packager and both amp and tet genes, was constructed. This plasmid is useful for cloning of large DNA fragments.     

Structure and inherent properties of the bacteriophage lambda head shell. VI. DNA-packaging-defective mutants in the major capsid protein

Some amino acid substitutions in the major capsid protein (gene E product) of lambda phage are found to cause a defect in DNA packaging. These substitutions permit initiation of DNA packaging and expansion of the prohead. However, cleavage of the concatemer DNA at the cos site takes place only to a very small extent, and the capsid eventually becomes empty. Interestingly, the mutations are suppressed by a decrease of the DNA length between the cos sites by 8000 to 10,000 bases. These properties are similar to those of amber mutants in gene D, which codes for the capsid outer-surface protein. Studies on the E missense.D amber double mutant show that the E protein and the D protein contribute additively to the stabilization of the condensed form of the DNA molecule in phage heads.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: DNA-gp3 intermediate in in vivo and in vitro assembly

The assembly of phage phi 29 occurs by a single pathway, and DNA-protein (DNA-gp3) has been shown to be an intermediate on the assembly pathway by a highly efficient in vitro complementation. At 30 degrees C, about one-half of the viral DNA synthesized was assembled into mature phage, and the absolute plating efficiency of phi 29 approached unity. DNA packaging at 45 degrees C was comparable to that at 30 degrees C, but the burst size was reduced by one-third. When cells infected with mutant ts3(132) at 30 degrees C to permit DNA synthesis were shifted to 45 degrees C before phage assembly, DNA synthesis ceased and no phage were produced. However, a variable amount of DNA packaging occurred. Superinfection by wild-type phage reinitiated ts3(132) DNA synthesis at 45 degrees C, and if native gp3 was covalently linked to this DNA during superinfection replication, it was effectively packaged and assembled. Treatment of the DNA-gp3 complex with trypsin prevented in vitro maturation of phi 29, although substantial DNA packaging occurred. A functional gp3 linked to the 5' termini of phi 29 DNA is a requirement for effective phage assembly in vivo and in vitro.     

Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations

SaPI1 and SaPIbov1 are chromosomal pathogenicity islands in Staphylococcus aureus that carry tst and other superantigen genes. They are induced to excise and replicate by certain phages, are efficiently encapsidated in SaPI-specific small particles composed of phage virion proteins and are transferred at very high frequencies. In this study, we have analysed three SaPI genes that are important for the phage–SaPI interaction, int (integrase) terS (phage terminase small subunit homologue) and pif (phage interference function). SaPI1 int is required for SaPI excision, replication and packaging in a donor strain, and is required for integration in a recipient. A SaPI1 int mutant, following phage induction, produces small SaPI-specific capsids which are filled with partial phage genomes. SaPIbov1 DNA is efficiently packaged into full-sized phage heads as well as into SaPI-specific small ones, whereas SaPI1 DNA is found almost exclusively in the small capsids. TerS, however, determines DNA packaging specificity but not the choice of large versus small capsids. This choice is influenced by SaPIbov1 gene 12, which prevents phage DNA packaging into small capsids, and which is also primarily responsible for interference by SaPIbov1 with phage reproduction.     

New late gene, dar, involved in the replication of bacteriophage T4 DNA. II. Overproduction of DNA binding protein (gene 32 protein) and further characterization.

We have previously shown that the arrested DNA synthesis of mutant defective in T4 phage gene 59 can be reversed by a mutation in dar. In this paper, we have examined the effect of the dar mutation on the kinetics of gene 32 protein (DNA binding protein) synthesis, DNA packaging, progeny formation, and several other porcesses. Several lines of evidence are presented showing that the regulation of synthesis of gene 32 protein is abnormal in dar 1-infected cells. In these cells, gene 32 protein, an early protein, is also expressed late in the infectious cycle. Our data also indicate that the packaging og DNA into T4 phage heads is delayed in dar mutant-infected cells, and this in turn results in a 6- to 8-min delay in intracellular progeny formation, although the synthesis of late proteins appears to be normal, as shown by gel electrophoresis. We have also studied the phenotypes of the double mutant dar-amC5 (gene 59). The increased sensitivity to hydroxyurea caused by a mutation in the dar gene can be alleviated by a second mutation in gene 59, but an increased sensitivity to UV irradiation caused by a mutation in gene 59 cannot be alleviated by a second mutation in the dar gene. Therefore, the double mutant still exhibits abnormalities in the repair of UV lesions.     

Bacteriophage T4D Head Morphogenesis. VIII. DNA-Protein Associations in Intermediate Head Structures That Accumulate in Gene 49? Mutant-Infected Cells

We have utilized the gene 49⁻ mutant-infected cells of bacteriophage T4D to accumulate large numbers of nucleic acid-protein intermediate head structures. These heads were used as substrates for experiments in the investigations of the mechanism of DNA packaging. Specifically, we have examined: (i) the susceptibility of the DNA in these structures to digestion by a variety of nucleases after a series of increasing temperature pulses from 25 to 100°C, (ii) the physicochemical characteristics of the DNA inside these heads, and (iii) the mechanism by which proteins are displaced from the interior of the head after treatment with basic proteins. We isolated DNA from these gene 49⁻ heads by use of gradient centrifugation procedures. The DNA had a molecular weight of 8 × 10⁶ and a density of 1.697 ± 0.005 g/cm³, and it contained a short resistant fraction (SRF) which, when associated with the gene 49⁻ heads, exhibited AT-protected regions that were not susceptible to micrococcal nuclease digestion. Such a fraction may contain pieces which are important in the initial association of the DNA with the prohead. Exposure of the gene 49⁻ intermediate capsid structures to basic proteins, such as bovine trypsin inhibitor, lysozyme, and l-polylysine-70, caused a displacement of an amorphous-appearing structure which may be a complex of the gene 49⁻ DNA and interior components of the capsid (e.g., internal proteins, polyamines). Our general conclusion is that in the gene 49⁻ intermediate head structures which are only partly filled with DNA, this DNA is held inside the head by strong electrostatic linkages with interior polypeptides and polyamines.