Identification of P48 and P54 as components of bacteriophage T4 baseplates.

The involvement of two bacteriophage T4 gene products in the initiation of T4 tail tube and sheath polymerization on mature baseplates has been studied by radioautography of acrylamide gels of various partially completed tail structures. The products of genes 48 and 54 (P48[the nomenclature P48 refers to the protein product of bacteriophage T4 gene 48] and P54), which are known to be required for the synthesis of mature baseplates, have been shown to be structural components of the baseplate. These gene products have molecular weights of 42,000 and 33,000, respectively. The addition of P54 to the baseplate not only permits the polymerization of the core protein, P19, onto the baseplate, but also caused the disappearance of a polypeptide of molecular weight about 15,000 from the supernatant fraction of infected cells. Another gene product, P27, has been identified in the crude extracts of infected cells. This gene product, which is required for the synthesis of baseplate structures, has the same mobility as one of the unidentified structural polypeptides of the baseplate and is therefore probably also a baseplate component.     

Bacteriophage T4 tail length is controlled by its baseplate

Purified T4 baseplate, when treated with high concentrations of pancreatic RNase, are inactive in $\text{in}\text{vitro}$ complementation assays. Their ability to initiate tail tube assembly is not altered; but the most probably length of the tube-baseplate formed is only 800A, compared to 1000A, the normal tube length, when untreated baseplates are used. Thus, baseplates help to regulate tube length, possibly by a template mechanism. Several minor baseplate proteins which may be involved in determining the length, including gp54, are missing from RNase - treated base-plates. These effects may be due to an unidentified protease contaminant of the RNase, since they are inhibited by phenylmethane sulfonyl fluoride.     

Structure and location of gene product 8 in the bacteriophage T4 baseplate

Many bacteriophages, such as T4, T7, RB49, and phi29, have complex, sometimes multilayered, tails that facilitate an almost 100% success rate for the viral particles to infect host cells. In bacteriophage T4, there is a baseplate, which is a multiprotein assembly, at the distal end of the contractile tail. The baseplate communicates to the tail that the phage fibers have attached to the host cell, thereby initiating the infection process. Gene product 8 (gp8), whose amino acid sequence consists of 334 residues, is one of at least 16 different structural proteins that constitute the T4 baseplate and is the sixth baseplate protein whose structure has been determined. A 2.0A resolution X-ray structure of gp8 shows that the two-domain protein forms a dimer, in which each monomer consists of a three-layered beta-sandwich with two loops, each containing an alpha-helix at the opposite sides of the sandwich. The crystals of gp8 were produced in the presence of concentrated chloride and bromide ions, resulting in at least 11 halide-binding sites per monomer. Five halide sites, situated at the N termini of alpha-helices, have a protein environment observed in other halide-containing protein crystal structures. The computer programs EMfit and SITUS were used to determine the positions of six gp8 dimers within the 12A resolution cryo-electron microscopy image reconstruction of the baseplate-tail tube complex. The gp8 dimers were found to be located in the upper part of the baseplate outer rim. About 20% of the gp8 surface is involved in contacts with other baseplate proteins, presumed to be gp6, gp7, and gp10. With the structure determination of gp8, a total of 53% of the volume of the baseplate has now been interpreted in terms of its atomic structure.     

Bacteriophage T4 baseplate components. I. Binding and location of the folic acid.

Two different proteins with high affinities for the pteridine ring of folic acid have been used to determine the location of this portion of the folate molecule in the tail plate of T4D and other T-even bacteriophage particles. The two proteins used were (i) antibody specific for folic acid and (ii) the folate-binding protein from bovine milk. Both proteins were examined for their effect on various intact and incomplete phage particles. Intact T2H was weakly inactivated by the antiserum but not by the milk protein. No other intact T-even phage, including T4D, was affected by these two proteins. When incomplete T4D particles were exposed in an in vitro morphogenesis system, it was found that neither of the two proteins affected either the addition of the long tail fibers to fiberless particles or the addition of tail cores to tail plates. On the other hand, these two proteins specifically blocked the addition of T4D gene 11 product to the bottom of T4D baseplates. After the addition of the gene 11 protein, these two reagents did not inhibit the further addition of the gene 12 protein to the baseplate. It can be concluded that the phage folic acid is a tightly bound baseplate constituent and that the pteridine portion of the folic acid is largely covered by the gene 11 protein.     

Structural and Functional Studies of gpX of Escherichia coli Phage P2 Reveal a Widespread Role for LysM Domains in the Baseplates of Contractile-Tailed Phages

A variety of bacterial pathogenicity determinants, including the type VI secretion system and the virulence cassettes from Photorhabdus and Serratia, share an evolutionary origin with contractile-tailed myophages. The well-characterized Escherichia coli phage P2 provides an excellent system for studies related to these systems, as its protein composition appears to represent the “minimal” myophage tail. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of gpX, a 68-residue tail baseplate protein. Although the sequence and structure of gpX are similar to those of LysM domains, which are a large family associated with peptidoglycan binding, we did not detect a peptidoglycan-binding activity for gpX. However, bioinformatic analysis revealed that half of all myophages, including all that possess phage T4-like baseplates, encode a tail protein with a LysM-like domain, emphasizing a widespread role for this domain in baseplate function. While phage P2 gpX comprises only a single LysM domain, many myophages display LysM domain fusions with other tail proteins, such as the DNA circulation protein found in Mu-like phages and gp53 of T4-like phages. Electron microscopy of P2 phage particles with an incorporated gpX-maltose binding protein fusion revealed that gpX is located at the top of the baseplate, near the junction of the baseplate and tail tube. gpW, the orthologue of phage T4 gp25, was also found to localize to this region. A general colocalization of LysM-like domains and gpW homologues in diverse phages is supported by our bioinformatic analysis.     

Attachment of tail fibers in bacteriophage T4 assembly. Identification of the baseplate protein to which tail fibers attach

A phage-neutralizing rabbit antiserum collected after immunization with tail-fiberless bacteriophage T4 particles was adsorbed with complete T4 phage. The resulting adsorbed serum inhibited tail fiber attachment in vitro. To identify the antigens against which this inhibitory activity was directed, blocking experiments were carried out with the adsorbed serum. Isolated complete baseplates and mutant-infected-cell extracts lacking known baseplate gene products but containing gene 9 product showed similar high levels of blocking activity. By contrast, both tail-fiberless particles lacking gene 9 product and infected-cell extracts made with gene 9 mutants showed 30-fold to 100-fold lower blocking activity. These results strongly support the conclusion that gene 9 product is the baseplate protein to which tail fibers attach.     

Evolution of bacteriophage tails: Structure of T4 gene product 10

The success of tailed bacteriophages to infect cells far exceeds that of most other viruses on account of their specialized tail and associated baseplate structures. The baseplate protein gene product (gp) 10 of bacteriophage T4, whose structure was determined to 1.2 A resolution, was fitted into the cryo-electron microscopy structures of the pre and post-infection conformations of the virus. gp10 functions as a molecular lever that rotates and extends the hinged short tail fibers to facilitate cell attachment. The central folding motif of the gp10 trimer is similar to that of the baseplate protein gp11 and to the receptor-binding domain of the short tail fiber, gp12. The three proteins comprise the periphery of the baseplate and interact with each other. The structural and functional similarities of gp10, gp11, and gp12 and their sequential order in the T4 genome suggest that they evolved separately, subsequent to gene triplication from a common ancestor. Such events are usual in the evolution of complex organelles from a common primordial molecule.     

Structure of the bacteriophage T4 baseplate as determined by chemical cross-linking.

We have carried out a series of reversible chemical cross-linking experiments using the reagent ethylene glycol-bis(succinimidylsuccinate) with the goal of determining the three-dimensional structure of the bacteriophage T4 baseplate. In a previous report, we investigated the near-neighbor contacts in baseplate precursors and substructures (N.R.M. Watts and D.H. Coombs, J. Virol. 63:2427-2436, 1989). Here we report completion of the analysis by examining finished baseplates and tails. Most of the previous contacts were confirmed, and we report several new contacts, including those within the central hub (gp5-gptd2, gp26-gptd), between the hub and the outer wedges (gp6-gp27(2], between baseplate and sheath (gp54-gp18), and between sheath and core (gp19-gp18). On the basis of this and other available information, a partial three-dimensional model of the baseplate is proposed.     

Localization of the proteins gp7, gp8 and gp10 in the bacteriophage T4 baseplate with colloidal gold: F(ab)2 and undecagold: Fab' conjugates

We report the localization of the proteins gp7, gp8 and gp10 in the bacteriophage T4 baseplate. Proceeding on the assumption that these proteins occupy discrete locations, we have decorated baseplates and tails with immunological probes. Using 5 nm diameter colloidal gold: F(ab')2 conjugates, we show that proteins gp7 and gp10 are located directly at the vertex, with gp10 positioned in the pin directly below gp7. gp8 is located beside gp7 towards the centre of the baseplate. Using a novel undecagold: Fab' conjugate we have also determined the radial positions of gp7 and gp8 in baseplates that have transformed to stars. A mechanism for the nature of the hexagon-to-star transformation is proposed.     

Structural changes in proteins of the bacteriophage T4 basal plate resulting from the attachment of long fibrils

The effect of the attachment of long tail fibers on the structure of proteins of the bacteriophage T4 baseplate was studied by digital processing of electron microscopic images. The attachment of the long fibers was found to result in dramatical changes of the proteins of the baseplate plag, while the wedges, to which the long fibers are attached, undergo only slight changes. We studied the baseplates with one to six attached fibers and found that the attachment of one fiber resulted in the change of the entire baseplate, although the wedge located in the vicinity of the fiber attachment changed to a greater extent. Only after the attachment of three and more fibers the changes of the same kind occurred through the entire baseplate.     

The structure of bacteriophage T4 gene product 9: the trigger for tail contraction.

BACKGROUND: The T4 bacteriophage consists of a head, filled with double-stranded DNA, and a complex contractile tail required for the ejection of the viral genome into the Escherichia coli host. The tail has a baseplate to wh?ch are attached six long and six short tail fibers. These fibers are the sensing devices for recognizing the host. When activated by attachment to cell receptors, the fibers cause a conformational transition in the baseplate and subsequently in the tail sheath, which initiates DNA ejection. The baseplate is a multisubunit complex of proteins encoded by 15 genes. Gene product 9 (gp9) is the protein that connects the long tail fibers to the baseplate and triggers the tail contraction after virus attachment to a host cell. RESULTS: The crystal structure of recombinant gp9, determined to 2.3 A resolution, shows that the protein of 288 amino acid residues assembles as a homotrimer. The monomer consists of three domains: the N-terminal domain generates a triple coiled coil; the middle domain is a mixed, seven-stranded beta sandwich with a topology not previously observed; and the C-terminal domain is an eight-stranded, antiparallel beta sandwich having some resemblance to 'jelly-roll' viral capsid protein structures. CONCLUSIONS: The biologically active form of gp9 is a trimer. The protein contains flexible interdomain hinges, which are presumably required to facilitate signal transmission between the long tail fibers and the baseplate. Structural and genetic analyses show that the C-terminal domain is bound to the baseplate, and the N-terminal coiled-coil domain is associated with the long tail fibers.     

The function of tail fibers in triggering baseplate expansion of bacteriophage T4

Normal particles of bacteriophage T4 have six long tail fibers attached to a hexagonal baseplate. T4 particles having various complements of tail fibers were prepared by in vitro addition of fibers to fiberless particles, and the infectivity of the particles was determined. Particles having fewer than six fibers (partially fibered) were found to have a decreased probability of infection. Partially fibered particles having T4 fibers were completed by addition of T6 fibers, and the infectivity was determined on a host that lacked the T6 tail fiber receptor. Attachment of the additional fibers increased the infectivity even though the T6 fibers could not bind to the host cell. The infectivity of particles having mixtures of T4 and T6 fibers was determined on cells having only one type of receptor. The results indicated that particles bound by only three fibers have a low probability of infection. The effect of thermolabile baseplate mutations was also examined. Studies of partially fibered particles and particles with mixtures of fibers indicated that particles with altered baseplates have a less stringent requirement for binding of the tail fibers for infection.     

The tail structure of bacteriophage T4 and its mechanism of contraction

Bacteriophage T4 and related viruses have a contractile tail that serves as an efficient mechanical device for infecting bacteria. A three-dimensional cryo-EM reconstruction of the mature T4 tail assembly at 15-A resolution shows the hexagonal dome-shaped baseplate, the extended contractile sheath, the long tail fibers attached to the baseplate and the collar formed by six whiskers that interact with the long tail fibers. Comparison with the structure of the contracted tail shows that tail contraction is associated with a substantial rearrangement of the domains within the sheath protein and results in shortening of the sheath to about one-third of its original length. During contraction, the tail tube extends beneath the baseplate by about one-half of its total length and rotates by 345 degrees , allowing it to cross the host's periplasmic space.     

The tail lysozyme complex of bacteriophage T4

The tail baseplate of bacteriophage T4 contains a structurally essential, three-domain protein encoded by gene 5 in which the middle domain possesses lysozyme activity. The gene 5 product (gp5) undergoes post-translational cleavage, allowing the resultant N-terminal domain (gp5*) to assemble into the baseplate as a trimer. The lysozyme activity of the undissociated cleaved gp5 is inhibited until infection has been initiated, when the C-terminal portion of the molecule is detached and the rest of the molecule dissociates into monomers. The 3D structure of the undissociated cleaved gp5, complexed with gp27 (another component of the baseplate), shows that it is a cell-puncturing device that functions to penetrate the outer cell membrane and to locally dissolve the periplasmic cell wall.     

Functional relationships and structural determinants of two bacteriophage T4 lysozymes: a soluble (gene e) and a baseplate-associated (gene 5) protein

Lysozymes have proved useful for analyzing the relation between protein structure and function and evolution. In bacteriophage T4, the major soluble lysozyme is the product of the e gene, gpe (gene product = gp). This lysozyme destroys the wall of its host, Escherichia coli, at the end of infection to release progeny particles. Phage T4 contains two additional lysozymes that facilitate penetration of the baseplates into host cell walls during adsorption. At least one of these, a 44-kD protein, is encoded by gene 5. We show here that a segment of the gp5 lysozyme amino acid sequence, deduced from the DNA sequence of gene 5, is remarkably similar to that of the T4 gene e lysozyme. Both T4 lysozymes are somewhat similar to the lysozyme of the Salmonella phage P22, but there is little significant DNA sequence homology among the two T4 lysozyme genes and the P22 lysozyme gene. We speculate that these lysozymes are adapted to differences in the composition of the cell walls of E. coli and S. typhimurium. The cloned gene 5 of the phage T4 directs synthesis of a 63-kD precursor protein that is approximately 19 kD larger than the gene 5 protein isolated from baseplates. Gp5 first associates with gp26 to form the central hub of this structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the central hub of bacteriophage Mu baseplate determined by X-ray crystallography of gp44

Bacteriophage Mu is a double-stranded DNA phage that consists of an icosahedral head, a contractile tail with baseplate and six tail fibers, similar to the well-studied T-even phages. The baseplate of bacteriophage Mu, which recognizes and attaches to a host cell during infection, consists of at least eight different proteins. The baseplate protein, gp44, is essential for bacteriophage Mu assembly and the generation of viable phages. To investigate the role of gp44 in baseplate assembly and infection, the crystal structure of gp44 was determined at 2.1A resolution by the multiple isomorphous replacement method. The overall structure of the gp44 trimer is similar to that of the T4 phage gp27 trimer, which forms the central hub of the T4 baseplate, although these proteins share very little primary sequence homology. Based on these data, we confirm that gp44 exists as a trimer exhibiting a hub-like structure with an inner diameter of 25A through which DNA can presumably pass during infection. The molecular surface of the gp44 trimer that abuts the host cell membrane is positively charged, and it is likely that Mu phage interacts with the membrane through electrostatic interactions mediated by gp44.     

A simple purification method and morphology and component analyses for carotovoricin Er, a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er.

Carotovoricin Er has been isolated as a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er [Kamimiya, S. et al., (1977), Agric. Biol. Chem. 41, 911-912]. However, the fine morphology and structural composition of carotovoricin Er remained to be studied because a large amount of contracted carotovoricin Er were present in the bacteriocin preparations so far obtained. To obtain intact carotovoricin Er and its major parts, we developed simple and efficient purification methods including the use of sucrose density gradient centrifugation in the presence of 10-20% (v/v) ethanol. Electron microscopy for the purified carotovoricin Er showed the presence of a novel antenna-like structure at the proximal end of the phage-tail-like particle, which consisted of a sheath-and-core part, a baseplate, and tail fibers. Contracted sheath and inner core were purified as hollow cylindrical structures with longitudinal lengths of 69 and 174 nm, respectively, and tail fibers were purified as a fibrous structure with length of 63 nm. SDS-polyacrylamide gel electrophoresis showed the presence of single major proteins of 50, 20, and 68 kDa in the isolated sheath, core, and tail fiber, respectively. Three other minor proteins of 46, 44, and 35 kDa were also identified as the structural proteins of carotovoricin Er, which may be the candidate proteins for the antenna-like and the base plate structures. Thus carotovoricin Er consists of at least 6 protein components.     

Protein interactions in the assembly of the tail of bacteriophage T4

Protein interactions in the assembly of the baseplate have been investigated. The baseplate of the phage T4 tail consists of a hub and six wedges which surround the former. Both reversible and irreversible interactions were found. Reversible association includes gp5 and gp27 (gp: gene product) which form a complex in a pH-dependent manner and gp18 polymerization, i.e. the tail sheath formation depends on the ionic strength. These reversible interactions were followed by irreversible or tight binding which pulls the whole association reaction to complete the assembly. The wedge assembly is strictly ordered which means that if one of the seven wedge proteins is missing, the assembly proceeds to that point and the remaining molecules stay non-associated. The strictly sequential assembly pathway is suggested to be materialized by successive conformational change upon binding, which can be shown by proteolytic probe.     

Extra-long bacteriophage T4 tails produced under in vitro conditions

Extra-long bacteriophage T4 tails have been produced under in vitro conditions from purified tails of normal length. These tails show a range of lengths suggesting that the basic increment of increased length is the 41 Å (Moody, 1971) axial repeating unit rather than the length of a normal tail. Some extra-long tails and tubes attached to baseplates show stain penetration down the central tunnel of the tube to approximately the normal tail length. The stain-penetrated tunnel, as visualised by three-dimensional reconstruction from the electron micrographs, has a diameter between 30 and 40 Å, sufficient to allow the passage of DNA. The exclusion of stain from the tunnel in the baseplate-near segment of the tube is interpreted as being due to the presence of additional material in the tunnel. The relevance of these observations to the assembly and length-regulation of the tail is discussed.     

Isolation of a Gallic Acid-producing Microorganism with Sake Cake Medium and Production of Gallic Acid

Carotovoricin Er has been isolated as a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er [Kamimiya, S. et al., (1977), Agric. Biol. Chem. 41, 911-912]. However, the fine morphology and structural composition of carotovoricin Er remained to be studied because a large amount of contracted carotovoricin Er were present in the bacteriocin preparations so far obtained. To obtain intact carotovoricin Er and its major parts, we developed simple and efficient purification methods including the use of sucrose density gradient centrifugation in the presence of 10-20% (v/v) ethanol. Electron microscopy for the purified carotovoricin Er showed the presence of a novel antenna-like structure at the proximal end of the phage-tail-like particle, which consisted of a sheath-and-core part, a baseplate, and tail fibers. Contracted sheath and inner core were purified as hollow cylindrical structures with longitudinal lengths of 69 and 174 nm, respectively, and tail fibers were purified as a fibrous structure with length of 63 nm. SDS-polyacrylamide gel electrophoresis showed the presence of single major proteins of 50, 20, and 68 kDa in the isolated sheath, core, and tail fiber, respectively. Three other minor proteins of 46, 44, and 35 kDa were also identified as the structural proteins of carotovoricin Er, which may be the candidate proteins for the antenna-like and the base plate structures. Thus carotovoricin Er consists of at least 6 protein components.