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.     

Structure and inherent properties of the bacteriophage lambda head shell. IV. Small-head mutants

Missense mutants of bacteriophage lambda that produce small proheads were found among prophage mutants defective in the major head protein gpE. Measurements of the sedimentation coefficient and molecular weight of the small proheads showed that they have the T = 4 structure composed of 240 molecules of gpE instead of the wild-type T = 7 structure composed of 420 molecules of gpE. When the phage mutants were grown in groE mutants of Escherichia coli, they produced small unprocessed proheads, which contained a smaller number (about 60) of the core protein (gpNu3) molecules than normal unprocessed proheads, which contain about 180 molecules of gpNu3. This shows that the major head protein determines the size of not only the shell but also the core of unprocessed proheads. These mutants by themselves produce very few mature small-headed phage particles, partly because the lambda DNA molecule, whose cos sites are separated at a distance of 48,500 bases, is too long to be packaged into the small proheads. However, the small proheads can package shorter DNA in vivo and in vitro at somewhat reduced efficiency, if the length or a multiple of the length between the cos sites of the DNA is 13,000 to 19,000 bases.     

Genetic Control of Capsid Length in Bacteriophage T4 I. Isolation and Preliminary Description of Four New Mutants

Four new mutants are described whose phenotypic expression affects the length of the head of bacteriophage T4D. All mutants produce some phenotypically normal phage particles. Mutant pt21-34 also produces at least two size classes of phage particle which have heads that are shorter than normal. The other three mutants, ptg19-2, ptg19-80, and ptg191, produce, in addition to phages with normal and with shorter-than-normal heads, giant phages with heads from 1.5 to at least 10 times the normal length. All mutations are clustered near gene 23. Giant phage particles have the following properties: they are infectious and contain and inject multiple genomes as a single continuous bihelical DNA molecule of greater-than-unit length. Their frequency, relative to the total plaque-former population, increases late in the infectious cycle. They have a normal diameter, variable length, and a buoyant density range in CsCl from equal to slightly greater than that of normal phage. The arrangement of capsomers is visible in the capsids, which are composed of cleaved gene 23 protein.     

Characterization of two Salmonella newport bacteriophages

Salmonella newport phages 16--19 and 7--11 have very long heads and are members of two rare and so far little-known phage groups. Both produce various morphological aberrations. Preparations of phage 7--11 contain numerous polyheads and about 0.4% short heads belonging to nine size classes. In addition, one giant phage particle was observed. The head of phage 7--11 seems to be an icosahedron which became elongated by adding successive rows of subunits. Phages 16--19 and 7--11 have buoyant densities in CsCl of 1.43 and 1.48 g/mL and particle weights of 103 and 204 x 10(6) respectively. Both viruses contain double-stranded DNA, internal proteins, and sugars. Phage 16--19 contains 46.5% DNA of 35 x 10(6) molecular weight, and glucose. Phage 7--11 contains 47.5% DNA of 108 x 10(6) molecular weight, and mannose. Base compositions of phage and S. newport DNAs were determined from buoyant densities, melting point, and acid hydrolysis. Phage 16--19 contains 5.4% 5-methylcytosine.     

The two-disulphide intermediates and the folding pathway of reduced pancreatic trypsin inhibitor.

We have studied bacteriophage λ head assembly under conditions in which the normal pathways for late phage DNA (concatemer) synthesis are blocked, and early (monomeric circular) DNA replication products accumulate. Our results show that under such conditions, the amount of late protein per amount of DNA is normal, but the amount of phage produced is not. Electron microscopic examination of thin sections of these bacteria shows that large numbers of “empty” head-shaped particles are produced. We conclude that the packaging of λ DNA depends on some structure (or property) possessed by DNA concatemers and absent in monomeric circular molecules and that the empty head-shaped particles which accumulate when concatemer production is blocked are head precursors which would normally accept concatemer DNA.These empty particles are the same size (approximately 550 Å vertex-to-vertex diameter) as the electron-dense, DNA-filled particles observed in similar sections of wild-type infected bacteria. In lysates the empty particles are approximately the same size as they are within the bacteria. However, filled heads observed in thin sections (or in negatively stained preparations) of lysates are larger than they are within the bacteria. This observation is contrary to what was previously suspected, since there seems to be little or no change in the size of intracellular λ capsids as a direct consequence of DNA packaging. Instead, an increase in the size of completed phage heads seems to take place as a consequence of cell lysis.     

Morphogenesis of bacteriophage lambda deletion mutants. I. Abnormal head-related structures produced in normal Escherichia coli.

Late in the morphogenesis of bacteriophage lambda, DNA condenses into the nascent head and is cut from a concatemeric replicative intermediate by a nucleolytic function, Ter, acting at specific sites, called cos. As a result of this process, heads of lambda deletion mutants contain less DNA than those of the wild-type phage. It has been reported that phage with very large deletions (22% of the genome or more) grow poorly but that normal growth can be restored by the non-specific addition of DNA to the genome. This finding implies that DNA content may exert a physical effect on some stage of head assembly.We have investigated the effects of two long deletions, b221 and tdel33, on head assembly. Bacteria infected with the mutants were lysed with non-ionic detergent under conditions favoring stabilization of labile structures containing condensed DNA. It has proved possible to isolate two aberrant head-related structures produced by the deletion mutants. One of these (“overfilled heads”) contains DNA which is longer than the deletion mutant genome and is about the same size as that found in wild-type heads. These structures appear to be unable to attach tails. The second type of structure (“incompletely filled heads”) contains a short piece of DNA, 40% of the length of the mutant genome. The incompletely filled heads are found both with and without attached tails. Both of these abnormal structures are initially attached to the replicating DNA but are released by treatment with DNAase. The nature of these abnormal structures indicates that very small genomes affect a late stage of head morphogenesis, after the DNA is complexed with a capsid of normal size. The results presented suggest that underfilling of the capsid interferes with the ability of the Ter function to properly cleave cos.     

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.     

Defective Bacteriophage PBSH in Bacillus subtilis: I. Induction, Purification, and Physical Properties of the Bacteriophage and Its Deoxyribonucleic Acid

PBSH, a defective phage of Bacillus subtilis strain 168, is described. Conditions are given for optimal induction of the prophage with mitomycin C. After a latent period of 90 min, cells were lysed and phage-like particles were released with a burst size of approximately 100 to 400 phage per bacterium. Since no known host supports phage replication after infection, burst size was determined by electron microscope count. Purification procedures and criteria for purity are described. The molecular weight of deoxyribonucleic acid (DNA) extracted from PBSH was estimated by length measurement and sedimentation. No circular DNA molecules were found by either technique. PBSH DNA molecules are linear, double-stranded, and of homogeneous molecular weight, about 12 x 10(6) daltons. There is no evidence for single-strand breaks. The majority of PBSH DNA molecules show a sedimentation behavior dependent on ionic strength. It is inferred that most of the DNA molecules are less hydrodynamically rigid than native DNA having a similar average base composition and molecular weight. Possible reasons for the sedimentation behavior are discussed.     

Structure of Erwinia carotovora temperate bacteriophage 59 and its DNA.

The temperate phage 59 from Erwinia carotovora and its DNA were studied. The phage particles have an icosahedral head and a long noncontractile tail with a base plate. The virus DNA makes up 50% of the total virus and exists as a linear molecule (molecular weight, 2.6 X 10(7)). A model of virus structural organization is presented.     

Bacteriophage T4 head morphogenesis: host DNA enzymes affect frequency of petite forms

Petite T4 phage particles have a shorter head than normal T4 phage and contain less DNA. They are not viable in single infections but are able to complement each other in multiply infected cells. Such particles normally make up 1 to 3% of T4 lysates. We show here that lysates of T4 grown on Escherichia coli H560 (end-A^?, pol-A^?) contain 33% of such petite particles. These particles are identical in physical and biological properties to those described previously, only their high frequency is abnormal. The frequency of petite particles in lysates grown on H560 is controlled by the presence or absence of the gene for DNA polymerase I (pol-A1) and apparently also a gene for endonuclease I (end-A). The involvement of these host DNA enzymes with T4 head morphology and DNA content indicates that DNA is directly involved in head morphogenesis. Such an involvement is incompatible with models of T4 head morphogenesis in which dimensionally stable, preformed empty heads are precursors of filled heads. The processing or repair of DNA apparently helps decide whether the assembly of T4 head subunits produces normal or petite heads.     

Packaging of ColE1 DNA having a lambda phage cohesive end site.

The mechanism of lambda phage-mediated transduction of hybrid colicin E1 DNAs of various lengths was studied, and factors influencing the formation of these transducing particles were investigated. The results were as follows: 1. The presence of a cohesive end site of lambda phage (coslambda) on colicin E1 DNA was essential for packaging of the DNA. 2. Packaging of colicin E1 DNAs, which carry coslambda with molecular sizes corresponding to 68% of that of lambda phage DNA, was observed in the absence of all known recombination functions of E. coli K-12 and of lambda phage. 3. Hybrid colicin E1 DNAs having coslambda with molecular sizes corresponding to 28% of that of lambda phage DNA were packaged within lambda phage particles as trimers; hybrid DNAs with coslambda of 40 and 47% of the length of lambda phage DNA were packaged as dimers; and those with molecular sizes of 68% of that of lambda phage DNA were packaged mostly as monomers. These results demonstrated that two factors are essential for the packaging of DNAs within lambda phage particles; the presence of coslambda on the DNA molecule and an appropriate size of DNA.     

Bacteriophage T4 Head Morphogenesis II. Studies on the Maturation of Gene 49-Defective Head Intermediates

An investigation into the metabolic requirements for maturation of gene 49-defective heads indicated that adenosine triphosphate energy and continued deoxyribonucleic acid (DNA) but not ribonucleic acid synthesis were needed. The fate of DNA present at restrictive temperatures (41.5 C) in tsC9 (gene 49)-infected cells was also examined. After lysis of infected cells, the 12 to 32% deoxyribonuclease-resistant DNA associated with isolated gene 49-defective heads was found to be attached to a deoxyribonuclease-sensitive complex associated with the debris. Pulsechase experiments where (3)H-thymidine was used to label the DNA at 41.5 C suggested that more DNA from this pool was present in phage recovered after rescue of the gene 49 function than could be accounted for by the deoxyribonuclease-resistant portion. Further, when these experiments were repeated with an additional density shift ((15)N(13)C-glucose to (14)N(12)C-glucose), the DNA extracted from phage rescued at 10 min after the temperature shift-down was found to be 90% conserved. These results suggest a model whereby DNA packaging into capsid precursors is separated from DNA replication and the energy from DNA synthesis provides the driving force for packaging. Pulse-chase, temperature-shift experiments with E920g (gene 66) or E920g;tsC9 mutant-infected cells showed that gene (49, 66)-defective heads, which were isolated as small, isometric-shaped unfilled heads, were a precursor to "petite" phage. This suggests that the maturation process is independent of the size and shape of the head membrane. Similar experiments with the double mutant tsC9;amN120 indicate that gene 49-defective heads can also be filled in the absence of tails.     

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.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA protein gp3.

The assembly of phage phi 29 occurs by a single pathway, and the DNA protein (DNA-gp3) of "packaging intermediates" can be obtained after DNase I interruption of in vitro complementation. A broad spectrum of DNA molecules of variable length was isolated from DNase I-treated proheads. Restriction endonuclease EcoRI digestion and electrophoretic analysis of these DNA molecules suggested that DNA-gp3 packaging was oriented with respect to the physical map and was a complex process. Proteinase K-treated exogenous DNA was not packaged. When exogenous DNA-gp3 was predigested with the restriction endonucleases BstEII. EcoRI, HpaI, and HpaII, the left-end fragments, ranging in size from 8 to 0.9 megadaltons, were selectively and efficiently packaged. During in vivo and in vitro assembly, DNA-gp3 is packaged into proheads, the "core-scaffolding" protein gp7 exits from the particles, and the DNA-filled heads assume the angular morphology of phage phi 29. The packaging of a 4.1-megadalton DNA-gp3 left-end fragment (one third of the genome) resulted in the exit of gp7 and the transition to angularity.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway

We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis.In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8. Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3, which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26.Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3^? infected cells are convertible to mature phage in vivo, with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads.Detailed examination of 8^? lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well.All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3, the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.     

Real-time imaging of single DNA molecules with fluorescence microscopy.

A fluorescence microscopy technique was used to image the dynamics of individual DNA molecules. Lambda, calf thymus, cosmid (circular), and T4 DNA were studied with the fluorescent dye acridine orange. Experiments with DNAase I were conducted, and the results indicate that these observations correspond to DNA molecules. The results of experiments with circular DNA provide strong evidence that these were single DNA molecules. Molecules were observed free in solution or attached to a glass or copper surface at one or several points. The Brownian motion of these molecules was observed, indicating that DNA in solution exists in a partially supercoiled state. Some molecules appeared stretched and were attached to the surface by their termini; the lengths of these molecules were measured. Such molecules also exhibited elastic behavior upon breaking. The power of this technique is demonstrated in images of cosmid DNA molecules, catenanes, and DNA extending from T4 phage particles. These results suggest immediate applications to molecular biology, such as examining the dynamics of protein-DNA interactions. Areas of ongoing research are discussed.     

Coordinate Variation in Lengths of Deoxyribonucleic Acid Molecules and Head Lengths in Morphological Variants of Bacteriophage T4

We have investigated three classes of small bacteriophage T4 particles which differ from normal T4 particles in length of their deoxyribonucleic acid (DNA), in head length, in protein content, and in density. The different particles contain DNA molecules measuring 0.90, 0.77, or 0.67, respectively, of the normal T4 length. An additional class of viable particles contains DNA molecules of 1.1 unit length. These discrete differences in DNA length correspond to discrete differences in length (but not width) of the respective heads and are roughly proportional to the resulting differences in head volumes. The measured relative dimensions of the different heads fit best the relative dimensions predicted by a quasi-icosahedral model in which the smallest T4 head corresponds to an icosahedron with a triangulation number T = 21. The mid-portion of this structure is thought to be elongated by adding successive rows of gene 23 protein hexamers, the normal T4 head having three added rows. Different mutants produce small particles of the three classes in varying proportions, but no mutant produces exclusively particles of a single class. Particles of each class, with indistinguishable DNA content, show additional minor differences in protein content, as measured by differences in buoyant density and in the relative ratio of (32)P to (35)S.     

Electrogenic cyclic redox chain in bacterial photosynthesis: Two regimes of operation in intact cells of Rhodospirillum rubrum

Bacteriophage K7 is specific for Escherichia coli strains harbouring R factors of incompatability group W, including hybrid coliphage P1-Myxococcus virescens plasmids. The phage has an unusual morphology with an isometric head and long tail of variable length. The tail lengths appear to fall into classes corresponsing to simple multimers of a unit length. Partially purified lysates of the phage include material that may represent phage particles in the process of biogenesis and other material demonstrating attachment of phage to cell envelope. Newly released phage DNA contains single standed ends. In the course of work, E. coli strains that harbour R factor Sa were found to be apparently restrictive.     

Structural aberrations in T-even bacteriophage. IX. Effect of mixed infection on the production of giant bacteriophage.

To date, the production of T-even bacteriophage with giant heads has been achieved in two ways: (i) by use of canavanine-arginine treatment of Escherichia coli B cultures infected by wild-type bacteriophage (Cummings and Bolin, Bacteriol. Rev. 40:314-359, 1976; Cummings et al., Virology 54:245-261, 1973), which give a size distribution of giants that is phage specific (Cummings et al., Virology 54:245-261, 1973); and (ii) by infection with certain missense mutants of T4D gene 23 (Doermann et al., J. Virol. 12:374-385, 1973; ICN-UCLA Symposium on Molecular Biology, p. 243-285, 1973) or temperature-sensitive mutants of gene 24 (Aebi et al., J. Supramol. Struct. 2:253-275, 1974; Biljenga et al., J. Mol. Biol. 103:469-498, 1976). We now report the effect of mixed infection with several mutants of T4D on both the production and the size of giant bacteriophage. We found that gene 24 mutant is a critical partner for the production of giants. Infection using T4.24 mutants together with either T4.23 mutants, T4B+ or T6+ led to the formation of giants with heads 10- to 14-fold longer than normal-length heads. Infection with amber 24-bypass 24 double mutants of T4D led to the production of giants when gene 23 mutant was used to co-infect. Addition of canavanine to the co-infected cultures could alter the size distribution of giants, depending on which phage were used to coinfect. Gene 22 mutants had a modifying effect on these results. In the absence of canavanine co-infection with gene 22 mutants prevented the production of giants, and in the presence of canavanine giants of 1.5 to 5 head lengths were found. We have interpreted these results to mean that critical concentrations of gene products 22, 23, and 24 interact to control head length in T-even bacteriophage.     

Replication and packaging of choleraphage phi 149 DNA.

The intercellular replication of the circularly permuted DNA of choleraphage phi 149 involves a concatemeric DNA structure with a size equivalent to six genome lengths. The synthesis of both monomeric and concatemeric DNAs during replication of phi 149 occurred in the cytoplasm. The concatemers served as the substrate for the synthesis of mature phage DNA, which was eventually packaged by a headful mechanism starting from a unique pac site in the concatemeric DNA. Packaging of DNA into phage heads involved binding of concatemeric DNA to the cell membrane. A scheme involving sequential packaging of five headfuls proceeding in the counterclockwise direction from the pac site is proposed. After infection under high-phosphate conditions, the concatemeric DNA intermediates were not formed, although synthesis of monomeric molecules was unaffected.