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.     

In vitro polymerization of bacteriophage T4D tail core subunits

The in vitro polymerization of bacteriophage T4 core protein subunits (P19) onto baseplates is examined by electron microscopy. Procedures for purifying P19 and baseplates, both of which have in vitro complementation activity, are described. It is found that when P19 and baseplates are allowed to react over a wide range of initial concentration ratios, the most probable core length is about 1000 Å, the length found in vivo. Very few structures longer than this are observed if the polymerization is not allowed to proceed for much longer than one hour. Some physical properties of the baseplates were determined. These are s_20,W= 72 S, D_20,W = 8.56 × 10^?8 cm²/s, andM_r = 7.4 ± 0.3 × 10⁶.     

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.     

The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage

Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in beta-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.     

Maturation of the head of bacteriophage T4. II. Head-related, aberrant tau-particles

Three somewhat different types of particle accumulate in cells infected with a phage carrying a mutation in gene 21 (in addition to the tubular variant (polyhead) of the head). The major type is the so-called τ-particle. These particles are very fragile, associated with the cell membrane, and have a sedimentation coefficient of about 420 S. They possess no DNA if isolated, and contain predominantly the precursor proteins P23, P24, P22 and the internal protein IP III, in addition to protein P20 and several proteins of unknown genetic origin.The remainder of the particles are partially or completely filled with DNA. The ratio of τ-particles to these partially or completely filled particles depends upon the particular mutant (in gene 21) phage used. In cells infected with a phage carrying the amber mutation (N90) in gene 21, about 10% of the precursor head protein P23 is cleaved to P231, and correspondingly about 10% of the particles are partially or completely filled with DNA. In cells infected with the temperature-sensitive mutant (N8) in gene 21, about 1% of the particles are fully or partially filled, and correspondingly about 1% of the P23 is cleaved to P231. In either case, the DNA-associated particles contain predominantly the cleaved proteins P231 and IP III1, and have none of the P22 and IP III found in τ-particles. This observation, and the correlation of the amount of partially or completely filled particles with the extent of the cleavage of P23 in the lysates, strongly suggest that cleavage of the head proteins is required for DNA packaging to occur.The τ-particles have properties similar to the so-called prohead I particles which we have isolated as intermediates in wild-type head assembly (preceding paper). However, temperature shift-down experiments, using several different phage carrying temperature-sensitive mutations in gene 21, indicate that the bulk of the τ-particles cannot be used for normal phage production.     

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 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 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.     

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.     

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.     

Evidence for an internal component of the bacteriophage T4D tail core: a possible length-determining template.

The length of the T4 tail is precisely regulated in vivo at the time of polymerization of the tail core protein onto the baseplate. Since no mutations which alter tail length have been identified, a study of in vivo-assembled tail cores was begun to determine whether the structural properties of assembled cores would reveal the mechanism of length regulation. An assembly intermediate consisting of a core attached to a baseplate (core-baseplate) was purified from cells infected with a T4 mutant in gene 15. When core-base plates were treated with guanidine hydrochloride, cores were released from baseplates. The released cores had the same mean length as cores attached to baseplates. Electron micrographs of these cores showed partial penetration of negative stain into one end, and, at the opposite end, a modified tip which often appeared as a short fiber projecting from the core. When cores were purified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two minor proteins and the major core protein were detected. One minor protein, the product of gene 48 (gp48), was present in at least 72% of the amount found in core-baseplates, relative to the amount of the major core protein. These findings suggest that cores contain a fibrous structure, possibly composed of gp48, which may form a "ruler" that specifies the length of the T4 tail.     

The role of structural changes in T4 bacteriophage tail proteins

Conformational changes in bacteriophage tail proteins after heating and ionic strength alteration leading to dissociation of tail sheath have been studied using protein fluorescence, differential scanning microcalorimetry and electron microscopy methods. Autonomous structural changes in tube-baseplate proteins have been revealed. They take place under the same conditions as those which release the bonds holding the sheath protein subunits to those of the tube in isolated sheathed tails. The conformational changes in the tube-baseplates are reversible similarly to the process of assembly and disassembly of the extended sheath. Morphological changes in the tube have been found at the temperature above the transition registered by protein fluorescence but not by calorimetry. This suggests that revealed spectral alterations reflect changes in quaternary structure of tail tube in particular.     

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.