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.     

Pulse-chase analysis of the in vivo assembly of the bacteriophage T4 tail

The in vivo assembly pathway of the complex tail of bacteriophage T4 virus was determined using pulse-chase analysis as a non-invasive alternative to the in vitro experiments previously used to map assembly. Bacteriophage T4 mutants defective in head assembly were used to infect cultures of Escherichia coli in order to study tail assembly in isolation. Beginning with the onset of late protein synthesis, the cultures were labeled continuously with [(3)H]leucine to normalize against subsequent sample losses. After completed tails had begun to accumulate at a constant rate, the cultures were pulsed with [(35)S]methionine, and then chased. Completed tails were purified at one minute intervals for the next 30 minutes and their proteins separated electrophoretically and counted by liquid scintillation. Total (35)S incorporation into each protein rose and then leveled off as the chase of unlabeled methionine flushed the label through the pools of soluble proteins and assembly intermediates and into completed tails. The inflection point in the sigmoidal (35)S-incorporation curve of each protein marks the maximal uptake of (35)S within that pool just before the effect of the chase becomes apparent and the curve begins to level off. The length of the delay in the apparent chase time reflects the position of that protein in the pathway. The closer the assembly point to the end of the pathway, the sooner the chase appears, revealing the relative order of assembly. As predicted, tail completion proteins such as gp18 (tail sheath) and 19 (tail tube) show the earliest inflection, while those earlier in the pathway take longer to chase. Of the 17 tail proteins analyzed, 14 are in agreement with the established in vitro pathway. The other three, gp15, gp10 and gp53, have helped us to develop a model that offers a plausible explanation for their altered chase times.     

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.     

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.     

Primary structure of the tail sheath protein of bacteriophage T4 and its gene

Complete sequence determination of gene 18 encoding the tail sheath protein was carried out mainly by the Maxam-Gilbert method. Approximately 40 peptides contained in a tryptic digest and a lysyl endopeptidase digest of gp 18 were isolated by reversed-phase high-performance liquid chromatography. All the peptides were identified along the nucleotide sequence of gene 18 based on the amino acid compositions. These peptides cover 88% of the total primary structure. Furthermore, the amino acid sequences of 9 of the 40 peptides were determined by a gas-phase protein sequencer; one of them turned to be the N-terminal one. The C-terminal peptide in the tryptic digest was isolated from the unadsorbed fraction of affinity chromatography on immobilized anhydrotrypsin and the amino acid sequence was also determined. Thus, the complete primary structure of gp 18 was determined; it has 658 amino acid residues and a molecular weight of 71,160.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Morphogenesis of the tail fiber of bacteriophage T4

Summary A component of T₄ phage tail fiber was purified from the lysate of E. coli strain Bb infected with gene 35 defective mutant of T₄D (amB252). The purified component which occupies a part of the distal half fiber is formed under the control of genes 36, 37 and 38. The purified component was characterized and compared with the genes 35-36-37-38 directed half fiber. Although the components resembled each other, differences were observed in length, stability and chemical compositions. The results of a further decomposition of this component and the correlating characters of the gene 35 and 36 directed products were discussed.     

Morphogenesis of the tail fiber of bacteriophage T4

Summary The formation of the tail fiber of bacteriophage T4 is controlled by genes 34, 35, 36, 37, 38 and 57. The gene 35 product was partially purified by IRC-50 column chromatography and by ammonium sulfate precipitation. The genes 36-37-38 directing component was purified 570 fold using the method of salting in and out and a sucrose density gradient centrifugation.Some characters of the purified components and the complementation reaction between these two components were investigated.     

Morphogenesis of the tail fiber of bacteriophage T4

Summary The tail fiber component ofcoli phage T4 was purified and partially characterized. The material was purified approximately 1 200 fold over the original lysate obtained fromE. coli B/1 cells infected with a mutant in gene 34 (am A455). The purified material was ultracentrifugally, electrophoretically and electron microscopically homogeneous. Its chemical composition were also analyzed.The purified component was characterized to be a half fiber controlled by at least four genes, 35, 36, 37, and 38.This work was supported by a grant from the Ministry of Education, Japan, and a grant (No. 5 ROI GM-10982) from the National Institute of Health, U.S.A.     

Attachment of tail fibers in bacteriophage T4 assembly: role of the phage whiskers.

The collar and whiskers of bacteriophage T4 extend outward from the top of the tail and play a role in regulating retraction of the tail fibers (Conley &; Wood, 1975). The collar and whiskers also are required for efficient tail fiber attachment during phage assembly. The structural gene for the collar/whisker protein is called wac. In vitro, infected-cell extracts that contain tail fibers activate whiskerless (wac) tail fiberless particles and ordinary (wac⁺) tail fiberless particles at equal rates if the extracts contain the wac⁺ gene product. However, extracts that contain tail fibers but no wac⁺ gene product activate wac particles about ten times more slowly. In vivo, whiskers are not essential for plaque formation, but a wac mutation causes a delay in the appearance of intracellular phage and a fivefold decrease in the burst size of infectious particles.The effect of the whiskers on tail fiber attachment is due to an interaction between the whisker and the distal half of the tail fiber, similar if not identical to the interaction that controls tail fiber retraction in complete phage. The following observations support this view: a slow rate of in vitro tail fiber attachment similar to that described above is seen with wac⁺ particles when they are pretreated with anti-whisker serum, or when the tail fibers carry a mutational alteration in gp36, a structural protein in the distal half fiber near the central kink. Lack of whiskers does not affect the slow rate of attachment of proximal half fibers to the baseplate of fiberless particles, but lack of whiskers greatly decreases the rate at which particles with attached proximal half fibers are activated by addition of distal half fibers. Since whiskers normally are attached to the phage only after head—tail union (Coombs &; Eiserling, 1977; Terzaghi et al., 1978), these findings explain why tail fibers do not attach efficiently to the baseplates of free tails.     

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.     

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.     

Stability of bacteriophage T4 short tail fiber

Adsorption of T-even bacteriophages to the E. coli host cell is mediated by long and short tail fibers. Bacteriophage T4 short tail fiber protein p12 was used to investigate the stability against thermal and chemical denaturation. Purified p12 is thermostable with a melting point of 78 degrees C. Guanidinium chloride-induced denaturation displayed strong hysteresis and an intermediate between 2 and 3 M denaturant. The transitions occur at 1.5 and 3.2 M denaturant as revealed by fluorescence spectroscopy and circular dichroism. The data suggest an equilibrium unfolding intermediate with a separate unfolding of the C-terminal knob domain and the shaft region.     

Engineering of bacteriophage T4 tail sheath protein

Gene product 18 (gp18, 659 amino acids) forms bacteriophage T4 contractile tail sheath. Recombinant protein assembles into different length polysheaths during expression in the cell, which complicates the preparation of protein crystals for its spatial structure determination. To design soluble monomeric gp18 mutants unable to form polysheaths and useful for crystallization, we have used Bal31 nuclease for generation deletions inside gene 18 encoding the Ile507-Gly530 region. Small deletions in the region of Ile507-Ile522 do not affect the protein assembly into polysheaths. Protein synthesis termination occurs because of reading frame failure in the location of deletions. Some fragments of gp18 containing short pseudoaccidental sequence in the C-terminal, while being soluble, have lost the ability for polysheath assembly. For the first time we succeeded in obtaining crystals of a soluble gp18 fragment containing 510 amino acids which, according to trypsin resistance, is similar to native protein monomer.     

Molecular architecture of bacteriophage T4

In studying bacteriophage T4—one of the basic models of molecular biology for several decades—there has come a Renaissance, and this virus is now actively used as object of structural biology. The structures of six proteins of the phage particle have recently been determined at atomic resolution by X-ray crystallography. Three-dimensional reconstruction of the infection device—one of the most complex multiprotein components—has been developed on the basis of cryo-electron microscopy images. The further study of bacteriophage T4 structure will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1463–1476.Original Russian Text Copyright © 2004 by Mesyanzhinov, Leiman, Kostyuchenko, Kurochkina, Miroshnikov, Sykilinda, Shneider.