Giant Phage Research Articles

Genetic control of capsid length in bacteriophage T4: DNA sequence analysis of petite and petite/giant mutants.

The T4 gene 23 product (gp23) encodes the major structural protein of the mature capsid. Mutations in this gene have been described which disrupt the normal length-determining mechanism (A.H. Doermann, F.A. Eiserling, and L. Boehner, J. Virol. 12:374-385, 1973). Mutants which produce high levels of petite and giant phage (ptg) are restricted to three tight clusters in gene 23 (A.H. Doermann, A. Pao, and P. Jackson, J. Virol. 61:2823-2827, 1987). Twenty-six of these ptg mutations were cloned, and their DNA sequence alterations were determined. Each member of this set of ptg mutants arose from a single mutation, and the set defined 10 different sites at which ptg mutations can occur in gene 23. Two petite (pt) mutations in gene 23 (pt21-34 and ptE920g), which produce high frequencies of petite particles but no giants, were also sequenced. Both pt21-34 and ptE920g were shown to include multiple mutations. The phenotypes attributed to both pt and ptg mutations are discussed relative to the mechanism of capsid morphogenesis. A site-directed mutation (SD-1E) was created at the ptgNg191 site, and its phenotypic consequences were examined. Plaque morphology revertants arising from a gene 23 mutant derivative of pt21-34 and from SD-1E were isolated. A preliminary mapping of the mutation(s) responsible for their revertant phenotypes suggested that both intra- and extragenic suppressors of the petite phenotype can be isolated by this method.

Genetic control of capsid length in bacteriophage T4: phenotypes displayed by ptg mutants.

The phenotypic characteristics of 26 ptg mutations in T4 gene 23 are described. All were located in three tight clusters in that gene and, by definition of ptg mutations, all produced giant phage. Intermediate petite phage, which invariably made up a substantial fraction of the progeny of these mutants, appeared to be a unique product of gene 23 mutations. Isometric petite phage were produced in significant numbers by strains with mutations at only 4 of the 10 sites identified with the PTG phenotype. The data presented indicate that there was little if any variation in the lengths of the normal, the intermediate petite, and the isometric petite classes. The frequencies of those capsid types were fairly specific for the individual mutations. The giant capsids that resulted from ptg mutations also had characteristic length distributions, of which three types were distinguished. These highly specific effects of gene 23 ptg mutations on capsid length regulation of T4 imply that the product of gene 23, gp23, plays a significant role in controlling the length of its capsid. The restrictions these observations place on a model for T4 capsid length regulation are discussed briefly.

Proteolytic cleavage and structural transformation: Their relationship in bacteriophage T4 capsid maturation

Giant T4 phage capsoids formed in canavanine-treated cultures infected by phage mutants in genes 21 and 17, respectively, differ with regard to cleavage of the major capsid protein, gp23, and in the fine structure of their hexagonal surface lattices. Quantitative computer processing of electron micrographs shows that the significant differences in capsomer morphology amount to six symmetrically placed features present in the uncleaved hexamer but absent after cleavage. These features may be related with the N-terminal portions of gp23 monomers excised by phage-specific proteolysis. Cleaved 17- giants can be induced to undergo a further structural transformation (expansion). Structural characteristics of partially transformed giant particles give clues about the dynamics of the cleavage and expansion transformations. Both processes appear to be polar, initiating in one cap and propagating along the particle. The transition zone of partial cleavage is diffuse, whereas the transition between unexpanded and expanded areas is confined to a narrow band of some 20 nm width.

New method for localizing proteins in periodic structures: Fab fragment labeling combined with image processing of electron micrographs.

Fab fragments prepared from antisera directed against purified bacteriophage T4 structural proteins and head-related structures were used to label proteins on the surface of T-even giant phage capsids. Optically filtered electron micrographs of the Fab-labeled capsids reveal both the location of specific proteins within the capsomeres and differing conformational states of the protein subunits. We describe parameters affecting the utility of this technique for the study of molecular organization and protein conformation in periodic biological structures.

Capsid fine structure of T-even bacteriophages binding and localization of two dispensable capsid proteins into the P23 * surface lattice

Two methods have been used to identify the stain-excluding regions in the P23 * matrix of the T-even phage capsid, and to investigate the bonding of the dispensable proteins hoc and soc into this matrix. (1) T2L giant phage capsids, which lack hoc and soc, have been titrated with these two proteins. (2) hoc, soc and other capsid proteins have been differentially dissociated from T4D and from hoc and soc complemented T2L capsids, by reversible acylation with citraconic anhydride. Changes in the capsomeres of giant capsids have been monitored by electron microscopy followed by optical filtration of the electron micrographs. The results indicate that the stain-excluding region in the centre of the capsomere is associated with binding of one molecule of hoc protein, and the six stain-excluding regions “bridging” adjacent capsomeres are associated with the binding of six molecules of soc protein. Further, the binding of soc in the absence of hoc is highly co-operative, whereas the presence of hoc in the P23 * lattice induces a marked heterogeneity in the soc binding sites.

Structural aberrations in T-even bacteriophage VIII. Surface morphology of T4 Lollipops

The surface of canavanine-induced T4B and T4D giant phages was investigated with the electron microscope using freeze-etching and shadow casting methods. On the relatively smooth surface of the elongated phage heads, a left-handed helical arrangement of subunits of about 4.0 to 4.5 nm diameter was found with interhelical distances ∼6.7 nm and an angle of 15°, measured normal to the long axis of the particle. These helices were the most obvious; they were intercepted at about 60° by two other lattice lines, one of 75° relative to the equator, the other one at an angle of 45°. Both of these lattice lines were about equally spaced but of lesser definition than the first helix. This “smooth” surface pattern changed drastically after exposure of the phage to temperatures between 42 and 48° for 10 to 60 min; the interhelical distance then measured 11.1 ± 0.4 nm, and “capsomeres” of 7.0 to 7.4 nm diameter became visible, producing a rather coarse hexameric lattice. No significant change in the angles between the lattice lines was observed. During storage of suspensions of the “smooth” phage at 2 to 4°, most virus surfaces had assumed the “coarse,” 11.1 nm arrangement. By employing acrylamide electrophoresis, we found that the heat treatment releases a protein with molecular weight of about 11,000. Lollipops of T2L, a phage lacking a capsid protein of similar size, revealed an ill-defined but “coarse” lattice which did not change after heat treatment.

Molecular organization of the shell of T-even bacteriophage head: II. Arrangement of subunits in the head shells of giant phages

Optically filtered images of the giant heads of T-even phage revealed the surface lattice at the level of subunit arrangements. Phage strains studied were wild type T4D, T2H and T2L, and three defective T4D mutants, namely T4soc, T4hoc and T4soc hoc ( Ishii and Yanagida, 1975 , Ishii and Yanagida, 1977 ). All these have a head lattice of near-hexagonal symmetry when flattened, and the same unit cell length (12·5 nm) and pitch angle (about −14°). Their protein distributions are strikingly different, however, and four different types of protein arrangement are found. The head shell of wild type T4D consists of repeating units of “hexamer+a centre unit+six bridges” as illustrated in Figure 13. The hexamer and centre unit are located at the 6-fold position, while the bridges are at the 3-fold or 2-fold positions in the unit cell. The filtered image of the T4hoc giant head showed a much weaker centre unit, otherwise indistinguishable from that of the wild type. T4soc lacks the bridging units and is composed of repeating units of “hexamer+a centre unit”. T4soc hoc is built simply of hexamers. T2L is identical to T4soc hoc, while T2H is similar to T4hoc. Comparison and interpretation of the filtered images using biochemical and genetical evidence lead to the conclusion that the hexamer, six bridges and a central unit correspond to six P23* molecules, six soc polypeptides and one hoc polypeptide, respectively.

Structure of T4 polyheads: II. A pathway of polyhead transformations as a model for T4 capsid maturation

We have studied the aberrant tubular polyheads of bacteriophages T4D and T2L as a model system for capsid maturation. Six different types of polyhead surface lattice morphology, and the corresponding protein compositions are reported and discussed. Using in vitro systems to induce transformations between particular polyhead types, we have deduced that the structural classes represent successive points in a transitional pathway. In the first step, coarse polyheads (analogous to the prohead τ-particle) are proteolytically cleaved by a phagecoded protease, a fragment of the gene 21 product. This cleavage of P23 to P23 ∗ induces a co-operative lattice transformation in the protein of the surface shell, to a conformation equivalent to that of T2L giant phage capsids. These polyheads (derived either from T4 or T2L lysates) can accept further T4-coded proteins. In doing so, they pass through intermediate structural states, eventually reaching an end point whose unit cell morphology is indistinguishable from that of the giant T4 capsids. At least one protein (called soc (Ishii & Yanagida, 1975)) is bound stoichiometrically to P23 ∗ in the end-state conformation. The simulation of several aspects of capsid maturation (cleavage of P23 to P23 ∗, stabilization, and lattice expansion) in the polyhead pathway suggest that it parallels the major events of phage T-even capsid maturation, decoupled from any involvement of DNA packaging.

Properties and structure of a gene 24-controlled T4 giant phage

Giant T4 bacteriophage were found by Doermann et al. (1973 a) with point mutants in gene 23 and by Cummings et al. (1973) after l-canavanine induction followed by an arginine chase. We now find T4 giant phage with 14 out of 15 tested temperature-sensitive mutants in gene 24 grown at intermediate temperatures between 33 °C and 37 °C. For one of these mutants, T4,24( tsB86), we found that (a) the optimum temperature for giant phage production is 34.8 °C, (b) the head-length distribution peaks sharply between 10 and 12 normal T4 phage head lengths, (c) about 75% of our giant phage have two tails, (d) the buoyant density in CsCl is greater than that of normal phage, (e) they are infectious and show an increased u.v. resistance, (f) their sodium dodecyl sulphate gel electrophoresis pattern is qualitatively similar to that of normal T4 phage, although the relative intensities of some of the bands are different, showing for example, a decreased P24 ∗ P23 ∗ † † P23, P24, product of gone 23, gene 24 etc. ratio, (g) optical diffraction and filtering of the flattened cylindrical part of the giant heads show a p6 surface net with a lattice constant of approximately 130 Å, a unique u v ratio of 15 5 and a capsomer morphology of the type 1 + 6 + 6. Mixed infections with T4 wild type and T4.24( amN65) also yield giant phage. These are produced in highest amounts with a multiplicity of infection ratio of 5:5; no giants are observed at ratios of 1:9 or 9:1, suggesting that their formation may be caused by a dosage effect of P24.

Folding and capsomere morphology of the P23 surface shell of bacteriophage T4 polyheads from mutants in five different head genes

Mutants in five different “head formation” genes ( 20, 22, 24, 40, IPIII) † † Abbreviations used: Gene products indicated by P— (e.g. P23); cleaved proteins by asterisk (e.g. P23∗). Exception: the “internal proteins” IPI, IPII, IPIII. of bacteriophage T4 produce polyheads. “Coarse” polyheads, which contain uncleaved P23, constitute over 90% of these tubular particles in fresh lysates. Using optical diffraction and filtration, we show that the pseudo-hexagonal net and the capsomere morphology are common to all coarse polyheads, regardless of genetic origin or polyhead diameter. Micropolymorphism is exhibited in each genetic class with respect to the cylindrical folding of the hexagonal net. We find that the frequency distribution of the diameters and pitch angles is significantly different for polyheads made by mutants affecting either of the major prohead core proteins (IPIII and P22). In every case, the foldings differ from the unique folding characteristic of giant phage capsid, suggesting that the assembly error responsible for producing polyheads instead of proheads involves a misdirection in arranging the P23 shell. By analysing the properties common to the various structures which may be formed out of this net (single-layered polyheads, multi-layered polyheads, proheads), we find that the P23 molecules possess form-determining specificity in terms of an intrinsic curvature of the capsomere bonding. These observations are discussed within the context of form determination of the phage prohead (τ-particle) and of its subsequent conservative maturation to the head of the infective wild-type phage.

Comparison of the structural and chemical composition of giant T‐even phage heads

A study has been made of the structure of the capsids of T4D giant phage produced from mutants in gene 23 and temperature-sensitive mutants in gene 24, and T4D and T2L giant phage formed by the addition of L-canavanine followed by an Larginine chase in the growth medium. All the giant phage capsids have been shown to be built according to the same geometrical architecture. This consists of a near-hexagonal surface net, lattice constant 129.5 A, folded into a left-handed T = 13 prolate icosahedron elongated along one of its fivefold symmetry axes. Their only apparent difference from wild-type T-even phage capsids is their abnormally elongated tubular part. A comparison of the capsomere morphologies and protein compositions of the giant phage capsids showed that all T4D giants are identical but differ from T2L: The T4D capsomere has a complex (6 + 6 + 1)-type morphology, whereas the T2L has a simple 6-type. T2L phage, however, lack two capsid proteins, "soc" and "hoc", present in T4D. The difference in capsomere morphology can therefore be related to the difference in the protein compositions of these two phage. Possible differences between the initiation and means of length regulation of giant phage heads and the aberrant polyheads are discussed.

Structural aberrations in T-even bacteriophage. VII. In vitro analysis of the canavanine-mediated inhibition of proteolytic cleavage.

Canavanine arrests a critical function in head morphogenesis and the potential for forming giant T-even phage particles termed lollipops is induced. Formation of the particles requires the addition of arginine and the restoration of normal functions. We now report on an investigation into the effects of canavanine on both the T4-induced proteolytic activity and on the substrate proteins. Using an in vitro cleavage assay we have shown that the gene 21-dependent proteolytic activity from canavanine-treated extracts is markedly inhibited, whereas the substrate proteins retain a high susceptibility for cleavage. The proteolytic activity in extracts treated with canavanine followed by arginine is readily detectable, and proteins previously synthesized in the presence of canavanine can be cleaved. Protein synthesis is apparently required for the appearance of the proteolytic activity after the canavanine-arginine treatment. Mixing experiments suggest the requirement for a component of the gene 21-dependent proteolytic activity that is not coded for by gene 21.

Molecular organization of the shell of the Teven bacteriophage head

Bacteriophage T 4 head shells contain two species of non-essential structural proteins ( M r 10,000 and 40,000); viable mutants missing either or both of them were isolated. Evidence is presented that these as well as P23* constitute basic components of the head surface lattice. The purified proteins were found to associate in vitro with the phage particles lacking them. Optically filtered images of the giant phage heads reveal the surface lattice at the level of subunit arrangement: a unit cell (125 Å in length) of near hexagonal symmetry contains six P23*, six 10,000 molecular weight and possibly one 40,000 molecular weight polypeptides, forming a “(6+1)+bridges” morphological unit.

The transformation of tau particles into T4 heads. II. Transformations of the surface lattice and related observations on form determination.

AbstractIn part I of this paper (1) we give evidence that the P23‐capsoid of τ‐particles is transformed in situ into the P23*‐capsid of normal phage. Using the polymorphism of phage T4, we have chosen polyheads as representative of P23 assemblies and giant phages as representative of P23* assemblies in order to study their surface crystals by optical filtration of micrographs. We found for polyheads a lattice constant of 112 Å with the typical hexameric, ringlike capsomer and for the giants a lattice constant of 124 Å with quite a different capsomer morphology, of the type (6+1). From the stoichiometry of the proteins composing the normal capsid we conclude that the protomer is a single P23* molecule and that the minor capsid‐proteins must be in singular positions on the surface lattice or on the polyhedral head (center of capsomers, vertices, or basal part).We extrapolate the findings on the giant head to the normal head and give a geometric model which is consistent with 1,100 molecules of P23* per capsid.We discuss the part of form inheritance contributed by P23 and the other formgiving gene products and give evidence that morphologic characters are the result of pairs of a reaction chain of interacting gene products. The example we give is the giant head produced by a ts mutant in gene 24 at 36°C.

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.