Construction and characterization of hybrids containing genes coding for proteins of the central part of bacteriophage T4 baseplate.

The central part (hub or plug) of bacteriophage T4 baseplate consist of several proteins which are present in only few copies per phage particle. The presence of these minor baseplate components was inferred from the genetic data but only some of them were identified as distinct proteins species by biochemical analysis. We have constructed a number of plasmids containing segments of bacteriophage T4 genome coding for baseplate proteins. The following genes were cloned into expression vectors: 54, 48, 29, 28, 27, 51, 26 and 25. The presence of a particular gene product was confirmed by in vivo complementation test. On the basis of these results we could more precisely localize the position of a particular gene on T4 phage genetic map. The hybrids contain sets of genes which make aggregation impossible, so bacteria harbouring these plasmids are convenient starting point for the purification of baseplate proteins.     

Three-dimensional structure of bacteriophage T4 baseplate

The baseplate of bacteriophage T4 is a multiprotein molecular machine that controls host cell recognition, attachment, tail sheath contraction and viral DNA ejection. We report here the three-dimensional structure of the baseplate-tail tube complex determined to a resolution of 12 A by cryoelectron microscopy. The baseplate has a six-fold symmetric, dome-like structure approximately 520 A in diameter and approximately 270 A long, assembled around a central hub. A 940 A-long and 96 A-diameter tail tube, coaxial with the hub, is connected to the top of the baseplate. At the center of the dome is a needle-like structure that was previously identified as a cell puncturing device. We have identified the locations of six proteins with known atomic structures, and established the position and shape of several other baseplate proteins. The baseplate structure suggests a mechanism of baseplate triggering and structural transition during the initial stages of T4 infection.     

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.     

Identification of bacteriophage T4D gene products 26 and 51 as baseplate hub structural components

Products of two bacteriophage T4D genes, 26 and 51, both known to be essential for the formation of the central hub of the phage tail baseplate, have been partially characterized chemically, and their biological role has been examined. The gene 26 product was found to be a protein with a molecular size of 41,000 daltons and the gene 51 product a protein of 16,500 daltons. The earlier proposal (L. M. Kozloff and J. Zorzopulos, J. Virol. 40:635-644), from observations of a 40,000-dalton protein in labeled hubs, that the gene 26 product is a structural component of the baseplate, has been confirmed. The gene 51 product, not yet detected in phage particles, appears from indirect evidence also to be a structural component of the baseplate hub. These current conclusions about the gene 26 and 51 products are based on properties of T4 mutant particles containing altered gene 26 or 51 products and include (i) changes in heat lability, (ii) changes in adsorption rates, and (iii) changes in plating efficiencies on different hosts, and with the results of previous isotope incorporation experiments indicate that T4 particles contain three copies of the gene 26 product and possibly one or at most two copies of the gene 51 product. Properties of these mutant particles indicate that the gene 26 product, together with the other hub components such as the gene 28 product, plays a critical role in phage DNA injection into the host cell, whereas the 51 product seems essential in initiating baseplate hub assembly.     

Assembly of the tail of bacteriophage T4

The protein products of at least 21 phage genes are needed for the formation of the tail of bacteriophage T4. Cells infected with amber mutants defective in these genes are blocked in the assembly process. By characterizing the intermediate structures and unassembled proteins accumulating in mutant-infected cells, we have been able to delineate most of the gene-controlled steps in tail assembly. Both the organized structures and unassembled proteins serve as precursors for in vitro tail assembly. We review here studies on the initiation, polymerization, and termination of the tail tube and contractile sheath and the genetic control of these processes. These studies make clear the importance of the baseplate; if baseplate formation is blocked (by mutation) the tube and sheath subunits remain essentially unaggregated, in the form of soluble subunits. Seventeen of the 21 tail genes specify proteins involved in baseplate assembly. The genes map contiguously in two separate clusters, one of nine genes and the other of eight genes. Recent studies show that the hexagonal baseplate is the end-product of two independent subassembly pathways. The proteins of the first gene cluster interact to form a structure which probably represents one-sixth of the outer radius. The products of the other gene cluster interact to form the central part of the baseplate. Most of the phage tail precursor proteins appear to be synthesized in a non-aggregating form; they are converted to a reactive form upon incorporation into preformed substrate complexes, without proteolytic cleavage. Thus reactive sites are limited to growing structures.     

Evidence of interactions between Gp27 and Gp28 constituents of the central part of bacteriophage T4 baseplate

The central part of the bacteriophage T4 baseplate consists of several proteins. However, for a number of the constituents the manner of incorporation are not convincingly established. Recently, we have presented evidence that gp28 is the structural component of the central part of the baseplate, which possesses a hydrophobic region and is membrane bound [Nieradko et al., 1998]. By utilizing extracts prepared from Escherichia coli cells that overexpressed genes 27 and 28 of phage T4, we proved that gp28 forms a complex with an another baseplate structural components: gp27. This complex was located in the membrane fraction. Its affinity to the inner membrane indicates that the identified complex may function as an initiator of the central hub assembly. It was subsequently established that these products interact in the ratio 1:1. We have also demonstrated that the particular components of the complex can be separated by action of SDS and to a lesser extent by Triton X-100.     

Effect of thermoinduced changes in T4 bacteriophage structure on the process of molecular recognition of 'host' cells.

By means of high-precision acoustic measurements and by methods of fluorescent and electron microscopy, investigations have been performed of thermoinduced conformational changes in T4 bacteriophage and its thermolabile mutants altered in baseplate proteins (gene products 7, 8, 10). A relationship was found between the conformational changes in T4 bacteriophage structure in the temperature range of 33-45 degrees C and the efficiency of bacteriophage adsorption and the changes in the orientation of long tail fibers. The possibility of heat regulation of 'recognition' of 'host' cells by bacterial viruses is suggested.     

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.     

Molecular assembly and structure of the bacteriophage T4 tail

The tail of bacteriophage T4 undergoes large structural changes upon infection while delivering the phage genome into the host cell. The baseplate is located at the distal end of the contractile tail and plays a central role in transmitting the signal to the tail sheath that the tailfibers have been adsorbed by a host bacterium. This then triggers the sheath contraction. In order to understand the mechanism of assembly and conformational changes of the baseplate upon infection, we have determined the structure of an in vitro assembled baseplate through the three-dimensional reconstruction of cryo-electron microscopy images to a resolution of 3.8 Å from electron micrographs. The atomic structure was fitted to the baseplate structure before and after sheath contraction in order to elucidate the conformational changes that occur after bacteriophage T4 has attached itself to a cell surface. The structure was also used to investigate the protease digestion of the assembly intermediates and the mutation sites of the tail genes, resulting in a number of phenotypes.     

Structural role of the polyglutamate portion of the folate found in T4D bacteriophage baseplate.

Three types of reagents were used to determine the structural role and location of the polyglutamate portion of the Escherichia coli T4D bacteriophage baseplate dihydropteroyl hexaglutamate. These reagents were examined for their effect in vitro on some of the final steps in phage baseplate morphogenesis. The reagents were (i) a series of oligopeptides composed solely of glutamic acid residues but with various chemical linkages and chain lengths; (ii) a homogeneous preparation of carboxypeptidase G1, an exopeptidase that hydrolyzes carboxyl-terminal glutamates (or aspartates) from simple oligopeptides, including the gamma-glutamyl bonds on folyl polyglutamates as well as the bond between the carboxyl group of the p-aminobenzoyl moiety and the amino group of the first glutamic acid residue of folic acid; and (iii) antisera prepared against a polyglutamate hapten. All three types of reagent markedly inhibited the attachment of the phage long tail fibers to the baseplate. Other steps in baseplate assembly such as the addition of T4D gene 11 or gene 12 products were not affected by any of these reagents. These results indicate that the polyglutamate portion of the folate is located near the attachment site on the bacteriophage baseplate for the long tail fibers.     

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.     

The bacteriophage T4 DNA injection machine

The tail of bacteriophage T4 consists of a contractile sheath surrounding a rigid tube and terminating in a multiprotein baseplate, to which the long and short tail fibers of the phage are attached. Upon binding of the fibers to their cell receptors, the baseplate undergoes a large conformational switch, which initiates sheath contraction and culminates in transfer of the phage DNA from the capsid into the host cell through the tail tube. The baseplate has a dome-shaped sixfold-symmetric structure, which is stabilized by a garland of six short tail fibers, running around the periphery of the dome. In the center of the dome, there is a membrane-puncturing device, containing three lysozyme domains, which disrupts the intermembrane peptidoglycan layer during infection.     

Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host

The contractile tail of bacteriophage T4 undergoes major structural transitions when the virus attaches to the host cell surface. The baseplate at the distal end of the tail changes from a hexagonal to a star shape. This causes the sheath around the tail tube to contract and the tail tube to protrude from the baseplate and pierce the outer cell membrane and the cell wall before reaching the inner cell membrane for subsequent viral DNA injection. Analogously, the T4 tail can be contracted by treatment with 3 M urea. The structure of the T4 contracted tail, including the head-tail joining region, has been determined by cryo-electron microscopy to 17 A resolution. This 1200 A-long, 20 MDa structure has been interpreted in terms of multiple copies of its approximately 20 component proteins. A comparison with the metastable hexagonal baseplate of the mature virus shows that the baseplate proteins move as rigid bodies relative to each other during the structural change.     

Identification of a second gene-27 product (27bis) of bacteriophage T4, and its involvement in regulatation of expression of gene 51

The 27 gene of bacteriophage T4 has been shown of encode two proteins of 44 and 39 kilodaltons (designated 27-44 and 27-39 bis, respectively) as a result of independent translational initiation at two different start codons within the same reading frame. The first product is the structural component of the viral baseplate. The latter with molecular weight 39 kDa probably plays significant role in regulation of expression of gene 51.     

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.     

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.     

Molecular reorganization in the hexagon to star transition of the baseplate of bacteriophage T4

The baseplate of bacteriophage T4 is a complex structure containing at least 14 different structural proteins. It undergoes a transition from a hexagonal to a star-shaped configuration during infection of the host bacterial cell. We have used a combination of genetics and image processing of electron micrographs to analyse both the wild-type structure and a series of mutant structures lacking specific gene products. Besides describing the basic anatomy of the hexagon and star configurations, we have been able to locate the products of genes 9, 11 and 12.Gene 9 product occupies a peripheral position in hexagons and stars consistent with its providing a binding site for the long tail fibres. Gene 11 product in the hexagon forms the distal part of the tail pin, which folds out to form the point of the hexagram in the star configuration. Gene 12 product is visualized as an extended 350 Å fibre in stars and broken baseplates but appears to have a more compact configuration in hexagons and intact phage.We demonstrate the structural relationship between the hexagonal and starshaped configurations and show how the positions of the specific gene products alter as a result of the structural transition. We suggest a speculative model for the role of gene 9 and gene 12 products in triggering the rearrangement of the baseplate and tail contraction.     

Isolation and reassembly of bacteriophage T4 core proteins

The products of genes 22, 67 and 68, and the internal proteins IPI, IPII and IPIII, as components of the scaffolding core of the bacteriophage T4 prohead, have been isolated and purified by hydroxylapatite column chromatography. Under conditions promoting reassembly in vitro, the proteins associated into elongated particles of practically constant width but variable length that we have called polycores. Preliminary optical diffraction experiments indicate that polycores may have an ordered structure, possibly helical, as has been suggested for the polyhead core. The coassembly of core proteins and the purified shell protein gp23 results in the formation of core-containing polyheads. Occasionally, prolate core-like particles have been observed but their reproducible formation has not been attained. Attempts to investigate the role of the minor prohead component gp20 in core assembly have been made through the cloning of the corresponding gene in an expression vector and subsequent purification of the protein.     

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.     

Cryo-EM study of the Pseudomonas bacteriophage phiKZ

The phiKZ virus is one of the largest known bacteriophages. It infects Pseudomonas aeruginosa, which is frequently pathogenic in humans, and, therefore, has potential for phage therapy. The phiKZ virion consists of an approximately 1450 A diameter icosahedral head and an approximately 2000 A long contractile tail. The structure of the phiKZ tail has been determined using cryo-electron microscopy. The phiKZ tail is much longer than that of bacteriophage T4. However, the helical parameters of their contractile sheaths, surrounding their tail tubes, are comparable. Although there is no recognizable sequence similarity between the phiKZ and T4 tail sheath proteins, they are similar in size and shape, suggesting that they evolved from a common ancestor. The phiKZ baseplate is significantly larger than that of T4 and has a flatter shape. Nevertheless, phiKZ, similar to T4, has a cell-puncturing device in the middle of its baseplate.