Intracellular morphogenesis of bacteriophage T4. II. Head morphogenesis

The relative phage yields of cells of Escherichia coli infected with both wild-type and amber mutant phages deficient in head morphogenesis were determined. The decrease in burst size as a function of the ratio of mutant:wild-type-infecting phage was linear and proportional for mutants in genes 20, 22, and 23, while for mutants in genes 21, 31, and 24 the results suggest an excess of intracellular gene product. The initiation of assembly of phage particles was not delayed at reduced gene product levels; only a reduction in the rate of phage assembly was observed. The effects on burst size of pairs of mutations in genes 20 and 23, 22 and 23, and 22 and 24, in both cis and trans arrangements, were identical. Experiments using the mutant E920g in gene 23 show that varying the kind and intracellular amounts of the major capsid protein (gp23) with respect to the major core or scaffold protein (gp22) had a profound effect on the length of the T4 head. Head length determination must therefore depend on the proper intracellular balance between these two proteins.     

Requirement of E. coli DNA synthesis functions for the lytic replication of bacteriophage P1

P1 lytic growth was examined in a number of different temperature sensitive mutants of E. coli that affect chromosomal replication. Growth was analyzed by measurements of phage burst sizes and specific DNA synthesis. Efficient P1 growth required each of the bacterial elongation functions dnaE (polC), dnaZ (sub units of E. coli polymerase III holoenzyme), and dnaG (primase) but was not dependent on the elongation function dnaB (mobile promoter). Of two initiation functions tested the dnaA function was found to be dispensable for normal growth whereas the dnaC function was essential. Temperature shift experiments with different dnaC mutants showed that the initiation component of the dnaC function was needed continuously throughout at least the first half of the lytic cycle, while the dnaC elongation activity was probably required during the entire cycle for normal phage yields. In two respects the dependence of P1 lytic growth on E. coli DNA synthesis functions was significantly different from that reported for P1 plasmid replication (Scott and Vapnek, 1980). Thus, lytic replication was far more dependent on a functional polC gene product than was plasmid replication and did not require the bacterial dnaB product.     

A pilot protein participates in the initiation of P22 procapsid assembly

The gene 16 protein of the bacteriophage P22 is required as a pilot protein aiding the transfer of DNA from the phage into the Salmonella typhimurium host cell. During assembly 10-20 copies of the 63,000-Da gp 16 protein are incorporated into the procapsid shell prior to DNA packaging. The protein has been purified from isolated procapsids and behaved as a monomer in solution. Upon incubation with purified coat and scaffolding subunits in vitro, it assembled into procapsids with the correct stoichiometry. The addition of physiological quantities of gp 16 resulted in an increased rate of procapsid assembly. Sedimentation of mixtures of coat and gp 16 protein subunits revealed association/dissociation behavior. It is likely that the added gp 16 is acting to stabilize a transient oligomeric coat protein species that functions as the in vitro initiation complex for procapsid assembly.     

Functions of two new genes in Salmonella phage P22 assembly.

The capsid of bacteriophage P22 is composed of at least eight protein species, six of which had been identified as the products of known P22 genes. The two smallest proteins, pX of 18,000 daltons, and pa of 15,000 daltons, were not the products of any of the known P22 genes. We have isolated and characterized phage carrying amber mutations in the two genes which code for pX and pα (designated gene 7 and gene 4, respectively). The products of these genes are essential for the formation of viable phage. The gene 7 protein is not needed for particle assembly, but rather for particle infectivity. Restrictive cells infected with a mutant in gene 7 accumulate structures with the sedimentation properties and electron microscopic appearance of wild-type phage. Although the defective 7^? particles adsorb to cells, they are unable to recombine with a defective prophage inside the cell or express phage-specific protein synthesis. The 7^? phenotype is similar to the phenotypes of amber mutants in genes 16 and 20 [Botstein et al., J. Mol. Biol.80, 669–695 (1973)]. Genes 7, 16, and 20 map contiguously. The gene 4 product seems to be necessary for the stabilization of newly DNA-filled phage heads. Restrictive cells infected with a mutant of gene 4 accumulate structures with the sedimentation properties and electron microscopic appearance of the “empty heads” that occur in smaller relative amounts in wild-type lysates. These 4^? empty heads are derived from unstably packaged heads inside the cell. The product of gene 4 is not required for cutting phage DNA into mature length pieces. The 4^? phenotype is similar to the 10^? and 26^? phenotypes [Lenk et al., Virology68, 182–199 (1975)]. Genes, 4, 10, and 26 map contiguously. All the proteins found in P22 phage and phage precursor particles have been matched with their genes.     

A late gene product of phage P22 affecting virus infectivity

Gene 14 is a recently discovered late gene of phage P22, mapping between the DNA injection and head completion genes (P. Youderian and M. Susskind (1980), Virology 107, 258-269). The gene 14 product has not been detected in phage particles. We have studied the defective phenotype of amber mutants in gene 14 to determine the role of gp14. The yield of physical particles from 14- infections is normal, but the infectivity of those particles is reduced by 60-80%. The noninfectious particles adsorb to but do not kill the host cell, as if they were defective in DNA injection. No differences in morphology, DNA composition, DNA permutation, or protein composition have been detected between 14- and wild-type particles. Procapsids, the capsid precursor to DNA packaging, exhibit a similar reduction in viability when isolated from 14- infected cells, assayed by in vitro DNA packaging. This is consistent with the gene 14 product functioning in the assembly or maturation of the procapsid. The three DNA injection proteins, encoded by genes 7, 16, and 20, are assembled into the particle at the procapsid stage. The defect in 14- particles may arise from improper organization or modification of one or more of the three proteins needed for DNA injection.     

Nucleotide sequence of the bacteriophage P22 gene 19 to 3 region: identification of a new gene required for lysis

The nucleotide sequence of a 2558-bp region of bacteriophage P22 at the right end of the genetic map between genes 19 and 3 was determined. A new gene that is partially required for lytic growth, named gene 15, was noted. P22 mutants were constructed which lack gene 15 function, and the gene 15 product was found to be required for lysis in the presence of some divalent cations. It has extensive amino acid sequence similarity with the phage lambda Rz gene, which has a similar function, and weak similarity to the phage T7 18.5 gene which previously had no known function. A hybrid P22 phage, in which the T7 18.5 gene replaces the P22 gene 15, exhibits the plating properties of wild-type P22, strongly suggesting that the two genes have similar functions. In addition, deletions were constructed which show that phage P22 has no additional genes required for lytic growth of lysogeny between genes 19 and 3.     

Genetic analysis of the erf region of the bacteriophage P22 chromosome

Derivatives of P22 with deletions of DNA sequences around and including the erf gene were obtained by crossing phages with plasmids containing fragments of the P22 chromosome. In some cases, the parent phages carried a large insertion in sequences not borne by the plasmids. In these cases, deletion of DNA from the phage chromosome to restore terminal repetition (a selectable trait) could be accomplished by recombination between phage and plasmid DNA in chosen sequences flanking the insertion on both sides and borne by the plasmid. In other cases, the parent phages had deletions of a selectable gene, which could be acquired from the plasmid parents only by acquisition of an overlapping deletion. Deletion-bearing P22 strains were tested for growth and homologous genetic recombination in wild-type, recA-, and rec(B or C)- hosts. This analysis indicated the existence of a gene, mapping to the left of erf, that is helpful (but not completely essential) for growth of P22 in a wild-type host. Because P22 lacking this gene grows as well as wild-type P22 on a recBC- host, it has been designated abc (anti-recBC). The abc gene does not appear to be essential for homologous genetic recombination in any host. A plasmid bearing a 1900 base pair fragment of P22 DNA, that expresses erf and abc under the control of the E. coli lac promoter, was constructed. It supports growth and recombination in a recA- host by a phage that lacks all of the genes known to lie between 24 and 9.     

Studies on the morphopoiesis of the head of T-even phage. IX. Gamma-particles: their morphology, kinetics of appearance and possible precursor function

The τ-particles produced by bacteriophage T4 mutants in gene 21 or 24 are morphologically similar. Functionally, however, they are different. Particle counts in electron micrographs obtained from in situ lysed cells show that the number of τ-particles produced by a temperature-sensitive mutant in gene 24 decreases after temperature shift, whereas the number produced by a mutant in gene 21 remains constant after shift. Also, sucrose gradient centrifugation of lysates from pulse-labeled cells infected with a mutant in gene 24 shows that after shift to the permissive temperature, 50% of the radioactivity, when compared to wild-type, or 33% of the total label on the sucrose gradient migrates into a position corresponding to that of intact phage particles. Such a transfer of label is not observed in the corresponding experiment with a mutant in gene 21. Finally, gel electrophoretic analysis in the presence of sodium dodecyl sulfate (SDS) shows for mutants in gene 24 that about 50% of the major head protein, P23, and the phage specific proteins P22 and IP III are modified after temperature shift in a similar way as during infection with wild-type phage. Such modifications do not occur to a comparable extent with mutants in gene 21. Based on these results it is postulated that τ-particles produced by 24^? mutants can serve as precursors to mature phage heads if the functional gene 24 product is supplied. τ-Particles produced by 21^? mutants, on the other hand, are abortive.     

Cloning and expression in Escherichia coli of the streptolysin O determinant from Streptococcus pyogenes: characterization of the cloned streptolysin O determinant and demonstration of the absence of substantial homology with determinants of other thiol-activated toxins.

A gene bank of Streptococcus pyogenes Richards was constructed in Escherichia coli by using the bacteriophage replacement vector lambda L47.1, and hybrid phage expressing streptolysin O (SLO) were identified among the recombinants. DNA sequences encoding SLO were subcloned from an slo+ hybrid phage into a low-copy-number vector plasmid to yield an slo+ hybrid plasmid, pMK157. This plasmid contains 5.6 kilobase pairs of cloned streptococcal DNA sequences, is stable, and expresses SLO at easily detectable levels in E. coli. Transposon gamma delta insertion mutants and in vitro-generated deletion mutants of pMK157 were isolated and analyzed. This analysis showed that a single gene is sufficient for production of SLO in E. coli and allowed this slo gene to be mapped to within +/- 100 base pairs. Two forms of the slo gene product, with molecular weights of 68,000 and 61,000, were detected in E. coli minicells harboring slo+ plasmids and by immunoblotting of E. coli whole cells harboring slo+ plasmids. Southern blotting hybridization experiments with the cloned SLO DNA sequences as probes failed to demonstrate homology between the cloned SLO determinant and DNA isolated from bacteria expressing thiol-activated cytolysins related to SLO.     

Role of gene 8 product in morphogenesis of bacteriophage T3

The product of gene 8 (gp8) of T3 phage is one of the minor head proteins located at the phage head-tail junction. To determine the role of gp8, an amber (8-) and four temperature-sensitive mutants (ts8) were characterized by sedimentation analysis, polyacrylamide gel electrophoresis, and extract complementation. Neither DNA-containing particles nor empty particles were formed in cells infected with 8-. In addition, prohead assembly was greatly reduced. Prohead assembly was also blocked in cells infected with all ts8 mutants at 42 degrees and with some ts8 even at 37 degrees. Proheads containing gpts8 were converted to empty heads when cell lysates were treated with chloroform. The protein compositions of proheads showed that the minor head proteins, gp8, gp15, and gp16, were lost from proheads formed in cells infected with ts8, but these minor proteins were present in proheads formed in cells infected with double mutants of ts8 and 5- or 19-, which are defective in DNA synthesis or DNA maturation, respectively. In vitro complementation experiments suggested that a ts mutation in gene 8 affected not only DNA packaging but also subsequent assembly steps. From these results, it is concluded that gp8 plays multiple roles in T3 phage morphogenesis, including prohead assembly, prohead stabilization, DNA packaging, and subsequent events.     

Encapsulation of phage P22 DNA in vitro.

The major steps in phage P22 morphogenesis include formation of proheads, DNA encapsulation, head stabilization, and tail addition. P22 DNA encapsulation and later assembly steps can proceed in vitro in a “DNA donor extract.” This extract is made from an induced lysogen carrying mutations in the coat protein gene. Assembly of phage in the extract is dependent on addition of P22 proheads and ATP. It is not dependent on protein or DNA synthesis, or on genetic recombination in vitro. Active proheads can be purified from cells infected with wild-type phage or phage carrying mutations in genes required for post-prohead formation assembly steps. Proheads lacking any of the normal structural proteins are defective and are not repaired in this in vitro system.     

Biological activity of purified bacteriophage T3 prohead and prohead-like structure as precursors for in vitro head assembly

Proheads were purified by differential centrifugation followed by sucrose density gradient centrifugation from Escherichia coli cells infected with a bacteriophage T3 mutant defective in either DNA synthesis (gene 5) or DNA maturation (gene 19). The prohead could be converted to infectious phage very efficiently by incubation in vitro with an extract prepared from cells infected with mutants defective in head genes 8, 9, 10, 13, 14, 15, or 16 (8^? extract and so on) but not with 19^? extract. Under the most favorable condition, 40% of added proheads were converted to infectious phage. The efficiency of the conversion did not depend upon the prohead concentration.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that prohead contained the proteins coded by genes 8, 9, 10, 13, 14, 15, and 16 but did not contain the product of gene 19. Prohead-like structures were isolated from cells infected with double mutants of gene 5 and one of the head protein genes 8, 13, 14, 15, and 16. These structures were not converted to infectious phage when incubated with 10^? extract (lacking the major head protein and had protein compositions differing from biologically active proheads. From these results, we discuss the possible role of head gene products in head assembly of phage T3.     

Gene-III protein of filamentous phages: evidence for a carboxyl-terminal domain with a role in morphogenesis

A filamentous phage derivative, fCA55, bearing a nonpolar deletion in gene III, has been constructed and characterized to study the functions of that gene. The deletion eliminates most of gene III without disturbing its reading frame or the putative promoter for the downstream gene, VI. Therefore it is assumed that any abnormalities exhibited by fCA55 are a direct effect of the gene-III lesion itself, and not polar effects on other genes. fCA55 Is abnormal in two respects. First, it is noninfective; in this it resembles another nonpolar gene-III deletion mutant, fKN16, which is missing 507 bp encompassing roughly the first half of the gene. Second, it is secreted as polyphage--very long particles containing many unit-length DNA molecules; in this respect, fCA55 differs from fKN16. When the viral proteins of these two mutants were analyzed with antibody directed against gene-III protein, it was found that fKN16 contains an altered gene-III protein, while fCA55 is unreactive. It was concluded that the gene-III protein has two functional domains: the N-terminal domain, missing in both mutants, is required for viral infectivity; while the C-terminal domain, partly missing in fCA55 but retained in fKN16, is incorporated into the virion, and is responsible for the protein's role in generating normal, unit-length particles.     

Filamentous phage DNA cloning vectors: a noninfective mutant with a nonpolar deletion in gene III

A filamentous phage derivative, fKN16, has been constructed from the tetracyclineresistance transducing phage fd-tet by deleting a 507-base-pair (bp) segment of phage gene III. In accord with the importance of the gene III protein in infection, the infectivity of fKN16 phage is less than 10^?8 that of fd-tet phage. In contrast to most gene III amber mutants, which are polar on the downstream phage genes VI and I, fKN16 should be a nonpolar mutant since its 507-bp deletion spans an integral number of coding triplets. And indeed, two phage traits that may depend on gene VI and I function—the level of phage production and packaging into unit-length virions—appear to be normal in fKN16. High phage production coupled with very low infectivity make fKN16 suitable as a vector for DNA cloning experiments requiring a high level of biological containment. The characteristics of fKN16 as a vector were investigated in detail, using HindIII fragments of phage λ DNA as model foreign DNA. fKN16 may also be useful in studying the role of the gene III protein in the filamentous phage life cycle.     

Intracellular visualization of precursor capsids in phage P22 mutant infected cells

The morphogenesis of the double-stranded DNA Salmonella phage P22 has been studied by electron microscopy of sections of wild type and mutant-infected cells. Previous work had shown that the precursor capsid structure that encapsidates DNA is a complex of two major protein species, the gene 5 coat protein and the gene 8 scaffolding protein. Scaffolding protein exits from the precursor capsid in coupling with DNA encapsidation and then recycles.No organized structures were seen in cells infected with amber mutants of the coat protein gene. Cells infected with an amber mutant of the scaffolding gene contained small numbers of aberrant particles, including empty petit capsids and giant nested or spiral shell structures.In cells infected with mutants blocked in DNA encapsidation (genes 1, 2, and 3) precursor capsids (proheads) accumulate amid the vegetative DNA and not along the membrane, as in phage T4. The proheads appear as double-shell structures about the size of mature phage, with the inner cell diameter about two-thirds the dimension of the outer shell.Mature phage and defective particles containing DNA form paracrystalline arrays within infected cells. Empty capsids, lacking both DNA and the inner shell of proheads, appear within the paracrystalline arrays of filled heads in cells infected with mutants blocked in head completion (genes 10 and 26). These empty capsids are presumably derived from filled but incomplete heads that have lost their DNA intracellularly.Use of temperature-sensitive mutants blocked in the encapsidation steps allowed visualization of the first filled heads upon shift to permissive temperature. These particles tended to appear at the edge of the DNA pool. Partially filled particles with dense central cores often were seen associated with the growing paracrystalline arrays, and they probably represented intermediates in encapsidation.These experiments, in conjunction with others, suggest that the scaffolding protein, which functions in prohead assembly and perhaps in DNA encapsidation, is organized into an inner shell within the precursor capsid.     

Structure of products of protein reassembly and reconstruction of potato virus X.

The gene H protein of bacteriophage ?X174 is a coat component located in the spikes at the vertices of the polyhedron. In extracts of ?X174-infected cells, the gene H protein binds to exogenous single-stranded phage DNA enhancing its infectivity in transfection of spheroplasts. This stimulation was not observed with extracts deficient in gene H protein. Transfection by the gene H protein-DNA complex was specifically inhibited by antibody to gene H protein. Lipopolysaccharide derived from ?X174-sensitive cells blocked transfection by the DNA complex with gene H protein but not by the DNA alone, suggesting that the gene H protein functions as the phage adsorption protein by recognizing the phage lipopolysaccharide receptor. Gene H protein from ?X174 stimulated the transfection by single-stranded DNA from the closely related phage S13; this stimulation was blocked by antibody to gene H protein and by the ?X174 lipopolysaccharide receptor. These actions in phage-receptor recognition and in transfection suggest that the gene H protein has multiple functions and behaves as a “pilot protein” in guiding the phage and its DNA through several stages of the infection cycle.     

Bacteriophage T7 morphogenesis: phage-related particles in cells infected with wild-type and mutant T7 phage.

Extracts from cells infected with wild-type T7 phage were found to contain three classes of particles: proheads, empty heads, and mature phage. These three particles differed with respect to their sedimentation properties, protein composition, and appearance in the electron microscope. All three structures contained the proteins specified by genes 8, 10, 13, 14, 15, and 16. Proheads and empty heads also contained the proteins coded for by genes 9 and 19. However, proheads contained more of the gene 9 product and less of the gene 19 product than empty heads. Phage did not contain the products of genes 9 or 19 but did contain three tail proteins, those specified by genes 11, 12, and 17. The product of gene 18 was found only in empty heads. In the electron microscope, proheads appeared to exclude the negative stain to a considerable degree, were round in outline, had thick shells, and contained eccentrically placed cores. Empty heads were easily penetrated by stain, had thinner shells than proheads, and often appeared to be collapsed or broken. Phage had polyhedral heads and small conical tails. Lysates of Su° cells infected with phage bearing amber mutations were examined for the presence of head-related structures. In the absence of the products of gene 9 or 10, no head-like structures were produced. After infection with a mutant in gene 8, some proheads but no empty heads or full heads were synthesized. Mutants in genes 14, 15, and 16 produced proheads, empty heads, and phage, although in reduced amounts relative to wild-type. The phage produced were noninfectious. The proheads produced after infection with mutants in genes 8, 14, 15, and 16 had aberrant protein compositions and appeared to lack cores when examined in the electron microscope. In the absence of the products of genes 18 or 19, or in the absence of T7 DNA, proheads accumulated but no other head structures were formed. After infection with mutants in genes 11, 12, and 17, head assembly and DNA packaging proceeded normally but tail assembly was defective. In the absence of the proteins specified by genes 7 and 13, phage assembly proceeded efficiently but the phage-like particles which were formed were noninfectious. On the basis of these data, we have proposed a model for phage T7 assembly in which the prohead, in the presence of the products of genes 18 and 19, packages DNA and loses P9 to give a full head to which are added the tail proteins. According to this model, the empty head is a breakdown product of a prohead which has initiated but not yet completed DNA packaging.