Major Tail Protein Research Articles

Structural proteins and DNA characteristics of 14 Listeria typing bacteriophages

The major structural proteins of 13 temperate and one virulent Listeria typing bacteriophages were analysed and compared using isoelectric focusing in immobilized pH gradients (IPG), ultrathin-layer two-dimensional electrophoresis, amino acid analysis and N-terminal amino acid sequences of selected proteins. Isoelectric points for major capsid and tail proteins of the 12 members of the siphoviridae family included in this study ranged from 4.70 to 5.92, whereas one of the two myoviridae investigated (B054) showed structural proteins in the 6.1 to 6.3 range. In comparison to protein profiles from one-dimensional SDS gels, the IPG technique gave better resolution and improved discrimination of phage proteins. Combination of this technique and SDS gel electrophoresis made it possible to correlate M(r) and isoelectric points of major structural proteins. Tail polypeptides of all siphoviridae are smaller and, with one exception, more acidic than their corresponding capsid counterparts. We also determined the amino acid composition of capsid and tail proteins. When compared with an average protein, they were found to be fairly rich in acidic and short-chain hydrophobic amino acids, as well as in lysine. In addition, the N-terminal amino acid sequences of major capsid and tail proteins of four representative listeriaphages were compared. The base composition of listeriaphage DNAs was between 37% and 39% G + C, reflecting that of their bacterial hosts. Each phage had a distinct restriction endonuclease pattern, and genome sizes ranged from 35 to 116 kb. DNA-DNA hybridization permitted the identification of five DNA homology groups. The two myoviruses studied (A511 and B054) showed no DNA homology to other phages, confirming their unique nature. The 12 siphoviruses were classified into three DNA homology groups with little cross-homology. Furthermore, phage A006 was found to share little DNA homology with the other investigated members of species 2671. Therefore, a new species (A006) is proposed. With respect to phage classification and taxonomy, a good correlation between the various approaches was observed, mostly corresponding to particle morphology.

Sequence of a Leishmania major gene encoding an ubiquitin fusion protein.

A gene encoding an ubiquitin-tail protein fusion was isolated from the parasitic protozoan, Leishmania major, and sequenced. The L. major tail protein shares 97, 96, 67, 62, 62 and 61% sequence identity with the tail proteins of Trypanosoma brucei, Trypanosoma cruzi, yeast, Dictyostelium discoideum, human, and Arabidopsis thaliana, respectively. The putative 'zinc finger' nucleic acid-binding domain found in all ubiquitin 'tail' or 'extension' proteins described is also conserved in the L. major sequence. The upstream sequence indicated that this gene is not located at the end of a polyubiquitin sequence.

Further biological and molecular characterization of actinophage VWB

The development cycle of the temperate actinophage VWB was investigated. Adsorption of most phage particles occurred within 30 min and the adsorption constant was 0.6 x 10(-8) ml min-1. The latent and rise periods were 140 and 100 min, respectively, and the burst size was estimated to be 130-250 p.f.u. Although phage VWB could infect only Streptomyces venezuelae ETH 14630 (ATCC 40755), of six different S. venezuelae strains tested, phage DNA could be introduced by transfection into most non-infectible strains. Upon transfection, phage DNA was propagated in these non-infectible strains and phage particles were released. In addition, the transfected strains could be lysogenized. By comparison of restriction fragments of VWB DNA, either free or integrated in the chromosomal DNA of the S. venezuelae ETH 14630 lysogen, the attachment site was localized. PAGE of the phage proteins revealed at least 17 different proteins with three major bands estimated as 16.5, 27.2 and 43 kDa in size. The N-terminal amino acid sequence of these supposed major head and tail proteins was determined. The corresponding DNA sequences on the phage genome were localized using oligonucleotides synthesized on the basis of the N-terminal amino acid sequences. The genes coding for the major structural proteins were shown to be clustered, as has been observed for other bacteriophages.

Mechanism of length determination in bacteriophage lambda tails

The mechanism of length determination in bacteriophage lambda tails is discussed as a model for regulation in protein assembly systems. The lambda tail is a long flexible tube ending in a conical part and a single tail fiber. Its length is exactly determined in the sense that the number of major tail protein (gpV) molecules, which comprise more than 80% of the mass of the tail, is exactly the same in all tails. Assembly of gpV is regulated by the initiator complex, which contains the tail fiber and the conical part, and by the terminator protein gpU. There are two key points in the assembly of gpV with respect to length determination. (1) Assembly of gpV on the initiator pauses at the correct tail length. Binding of gpU to the tail only fixes the pause firmly. (2) When the tail length is too short, binding of gpU to tails is inhibited. Deletions and a duplication (both in frame) in gene H, which codes for one of the proteins in the initiator, result in production of phage particles with altered tail length. Moreover, the tail length is roughly proportional to the length of the mutated versions of gene H. This shows that the tail length is measured by the length of gene H protein (gpH), which seems to be approximately as long as the tail tube, if extended like a thread, according to secondary structure prediction (alpha-helices connected by other structures). Various pieces of evidence show that about six molecules of gpH are attached to the remaining portion of the initiator by the C-terminal part and folded into a somewhat compact form, while they are elongated as they are enclosed in the tail tube during assembly of gpV. Unlike interaction between the length-measuring genome RNA and the coat protein of tobacco mosaic virus, the major tail protein gpV does not bind specifically to the ruler protein gpH. Rather, gpH determines the tail length by inhibiting the binding of gpU to short tails and by signalling the pause when the correct tail length is attained.

Characterization of the phage P1 dam gene

The genome of enterobacterial phage T1 has been sequenced, revealing that its 50.7-kb terminally redundant, circularly permuted sequence contains 48,836 bp of nonredundant nucleotides. Seventy-seven open reading frames (ORFs) were identified, with a high percentage of small genes located at the termini of the genomes displaying no homology to existing phage or prophage proteins. Of the genes showing homologs (47%), we identified those involved in host DNA degradation (three endonucleases) and T1 replication (DNA helicase, primase, and single-stranded DNA-binding proteins) and recombination (RecE and Erf homologs). While the tail genes showed homology to those from temperate coliphage N15, the capsid biosynthetic genes were unique. Phage proteins were resolved by 2D gel electrophoresis, and mass spectrometry was used to identify several of the spots including the major head, portal, and tail proteins, thus verifying the annotation.

Phage head assembly in bacteriophage T1

Phage-related structures found in wild-type and mutant Tl infections were examined by sedimentation analysis, electron microscopy, and polyacrylamide gel electrophoresis. Phage-particle polypeptides P7 (molecular weight 33,000) and P11 (molecular weight 16,000) were identified as major head proteins and P10 (molecular weight 26,000) was shown to be the major tail protein. A DNA-free head-like structure containing P7 but not P11 was present in wild-type and a number of mutant infections. In view of its possible role as a precursor to mature heads, this structure was termed the prohead. Mutants in two genes, am10 (gene 13) and am45 (gene 14), synthesised only tails in nonpermissive infections. Mutants in eight of the head genes, am23 (gene 4), am283 (gene 13.3), am216 (gene 13.7), ts257 (gene 14.5), am11 (gene 15), am4 (gene 16), am7 (gene 17), and am30 (gene 18), synthesised proheads and tails but not other structures. am37 (gene 12) synthesised proheads, tails, and empty heads. Studies with a tail-defective mutant suggested that empty heads were produced from the breakdown of DNA-filled heads. P11, the T1 gene 13.3 product, is analogous to the phage λ gene D protein: both are major capsid proteins which appear to stabilise the later stages of head filling. These data are reviewed according to the general principles established for phage-head assembly and a tentative pathway for T1-head assembly is proposed.

Structure and function of the major tail protein of bacteriophage lambda

Abstract Viable mutants of bacteriophage lambda having small major tail protein molecules in their virion have been isolated as pseudo-revertants of a defective prophage mutant ( defK244 ) in gene V , which codes for the major tail protein. According to deletion mapping, the defK244 mutation is located near the translation terminal of gene V , whereas some mappable reversion mutations leading to small major tail protein molecules map upstream to defK244 but still downstream to all the amber mutations tested. This suggests (if not proves) that the removable part is located at or near the carboxyl terminal of the major tail protein. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and buoyant density measurements of the mutant phage particles show that as much as one-third of the major tail protein molecule can be removed without losing its capacity to maintain the total shape and infectivity of the phage particles. In the three-dimensional structure of the tail the removable part of the molecule exists as a protrusion at the outer part of the tail tube according to electron microscopy and hydrodynamic calculations based on sedimentation velocity experiments.

Purification and characterization of the major protein and the terminator protein of the bacteriophage λ tail

Two proteins of the tail of bacteriophage λ were purified in an active form and characterized for the study of phage assembly. The terminator protein (product of gene U), which stops the polymerization of the major tail protein at the correct tail length, was purified from crude lysates. It exists as a globular monomer of 16,000 daltons in the absence of magnesium ions and as a ring-like hexamer in the presence of 20 m M MgSO 4. It is a very acidic protein poor in lysine and lacking cysteine residues. Its secondary structure is rich in β-sheet and relatively poor in α-helix. Experiments using its antibody show that it is located at the proximal end of the tail. The major tail protein (product of gene V) was purified from dissociated tails or phage ghosts. This preparation was indistinguishable from the unassembled major tail protein in tail-defective lysates with respect to various properties except that the in vitro complementation activity was significantly lower. The gene V product consists of a polypeptide chain of about 25,000 daltons, which is rich in valine and threonine and which has no cysteine and no or an extremely small amount of histidine. Its secondary structure contains a large amount of random-coil, a relatively small amount of β-sheet, and very small amounts of α-helix. In solution it exists in a monomer-dimer equilibrium, and polymerizes only in the presence of the initiator for its assembly. The antibody against the major tail protein attaches all over the tubular part but not to the basal part of the tail.

Morphogenesis of bacteriophage lambda tail. Polymorphism in the assembly of the major tail protein

The major tail protein of bacteriophage lambda can polymerize into three different structures: (1) the tail, (2) the polytail, and (3) the polytube. They are characterized, respectively, as (1) a structure of exactly determined length having the tail tip, (2) a structure of undetermined length having the tail tip, and (3) a structure of undetermined length lacking the tail tip. This report shows that the polytube is made by aberrant initiation, whereas the polytail is made by incorrect termination of assembly. Genetic evidence suggests that the polymerization of the major tail protein (gene V product) into the polytube takes place on a pseudo-initiator, the formation of which is controlled by genes M and L . The assembly of both the tail and the polytail starts on the normal initiator which requires at least seven gene products for its formation. In this case, the elongation reaction pauses at the correct tail length even in the absence of the terminator protein (gene U product), and yields a tail precursor which we call “ U − -tail”. If the terminator protein attaches to the top of the U − -tail, it completes the termination of polymerization, and further action of the gene Z product makes it the tail. However, if more major tail protein molecules attach to the top of the U − -tail before the terminator protein attaches, the elongation reaction resumes and finally yields the polytail. Two abnormal reactions occur in the polytail pathway. (1) The gene Z product can act on the polytail without the action of the gene U product, and (2) the gene H product is released during the formation of the polytail.

Morphogenesis of the tail of bacteriophage lambda: III. Morphogenetic pathway

Precursors of the tail of bacteriophage λ have been detected by measurements of in vitro complementation activities and serum blocking activity in sucrose gradients of lysates defective in tail genes. On the basis of these measurements, a pathway for the assembly of the λ tail is proposed: The morphogenesis of the λ tail starts from the tail fiber (product of gene J) located at the distal end of the tail, and proceeds to the proximal end. Gene J by itself produces a 15 S structure with serum blocking activity but without any detectable in vitro complementation activity, which may be the least advanced precursor of the λ tail or an abortive product. Functions of genes J, I, K, L are required for the formation of a 15 S precursor that has in vitro complementation activities with J −, I −, K − and L − lysates and serum blocking activity. If the products of genes G and H act on the latter 15 S precursor, a 25 S precursor is made, but this precursor seems either to be in equilibrium with the 15 S precursor or to degrade easily into the 15 S precursor. Gene M has a function of stabilizing the 25 S precursor. After the action of gene M product, the 25 S precursor is ready to serve as a nucleus on which the product of gene V (the major tail protein) assembles. However, gene U product is also necessary at this step for the correct assembly of the major tail protein on the 25 S precursor. Without gene U product the assembly of the major tail protein does not stop at the correct length and a polytail is formed instead of a morphologically normal tail. Finally, gene Z product acts on the morphologically normal tail and makes it a biologically active tail. Without the action of gene Z product, the defective tail binds to a head and forms a phage-like particle which is only very weakly infectious. (The position of gene T in the pathway is not determined, because no sus mutant is available in gene T.) Two abnormal, less efficient pathways are also present in vitro. (1) If gene U product acts on a polytail in an U − lysate, the polytail finally binds to a head and forms a phage particle with an extra long tail which is infectious to a small extent. (2) The function of gene K seems to be bypassed to some extent: K − lysates accumulate particles which sediment as fast as normal phage and which are complemented by other tail − lysates.

Morphogenesis of the tail of bacteriophage λ: II. In vitro formation and properties of phage particles with extra long tails

λ phage particles with extra long tails are formed in vitro in U−-lysate after the addition of V−-lysate or of dilute H−-lysate, whereas mainly normal λ particles are made after the addition of concentrated H−-lysate. These long-tailed phage particles have a broad distribution of tail lengths, a smaller sedimentation coefficient and smaller density than normal phage particles. Relative to uv absorbance at 260 nm, the number of plaque forming units of long-tailed particles is about 0.04% of that of normal phage.We discovered precursors for long and normal tails by testing in vitro complementation activity in sucrose gradient fractions of defective lysates. We propose the following pathways. (1) After being acted on by 2 S material, probably pU, from V−- or H−-lysates, 60 S material, free polytails, from a U−-lysate binds heads and forms long-tailed particles. (2) Unpolymerized major tail protein (gene V product) in an H−-lysate assembles on a 25 S precursor originating from a U−-lysate. In the presence of pU this reaction yields normal tails that bind heads and finally form normal phage.

Structure of the bacteriophage P2 tail

Analysis of bacteriophage P2 particle proteins by SDS gel electrophoresis indicates that at least six proteins are present in the tail. A tail protein of 71,000 daltons has been identified as the product of the H gene, and a tail protein of 94,000 daltons has been tentatively identified as the T gene product. The T protein appears to be stabilized by the E gene product. The major tail proteins, of 46,000 and 19,600 daltons, are missing from cells infected with an F amber mutant, but not from cells infected with amber mutants in any of the genes downstream from F. These two proteins are designated F I and F II, respectively. We propose that one of these proteins is coded for a gene adjacent to F which has not yet been defined by mutation. On the basis of their relative proportions in the phage and their molecular weights, we propose that F I and F II are the tail sheath and tube proteins, respectively, and that the T protein may be a tail-fiber component. High-resolution (minimal beam exposure) electron microscopy has revealed the structure of the contractile P2 tail in greater detail. At the head-proximal end of free tails are two disclike structures of different thickness and diameter. In preparations of whole phage, only the thinner of these discs is seen to lie outside the phage head (at the head-tail junction). We, therefore, presume that the thicker disc lies inside the head. At the distal end of the tail is a small, slender spike, and six tail fibers which are composed of two subsections. Electron microscopic examination of cells infected with different tail amber mutants indicates that the tail genes R and S are involved in determining tail length, since these mutants produce giant tails and giant tail sheaths, respectively.

A regulator protein for the length determination of bacteriophage lambda tail

AbstractMutants in gene U of phage λ produce polytails. Those polytails have a tail fiber and a basal part like normal tails, but their major tubular part is longer than that of normal tails and shows a wide length distribution.We established the morphogenetic pathway of the λ tail and found that U gene product (pU) acts throughout the assembly of the major tail protein (pV). Polytails in U− lysate are activated by pU in vitro and form long‐tailed phage which are infectious to a small extent.If the formation of the basal part of the tail is blocked, pV (the major tail protein) remains unassembled as long as pU is present in the cell. However, we found that part of pV assembles into giant polytubes of several microns in length in lysates of a double mutant U− · H− in which both the basal part and pU are absent.pV in purified tails can be dissociated into disks (about 10S) or smaller units (about 2.5S) in vitro under extreme conditions. The disks form polytubes efficiently under physiological conditions, but the smaller units do not form polytubes efficiently. The smaller units have in vitro complementation activity with V− lysate. In vitro complementation activities with V−, U−, and Z− lysates are detected in the dialyzed extracts of SDS gel electrophoresis of purified tails. The molecular weights of the polypeptide chains containing those activities are estimated to be 31,000, 14,000 and 20,000 daltons, respectively.

Caulobacter crescentus bacteriophage φCbK: Structure and in vitro self-assembly of the tail

Electron micrographs of negatively stained and platinum-shadowed bacteriophage φCbK have been analyzed by optical diffraction and computer Fourier transformation. The results show that the phage tail is a helical “stacked disc” structure with an annular repeat of about 38 Å and with 3-fold rotational symmetry about the helix axis. Phage tails exhibited lateral and rotational flexibility and were found to possess variable helical parameters. The smaller angle of rotation about the helix axis between equivalent asymmetric units on adjacent discs measured from a number of tail images was found to have an average value of 41.5±0.9 °. Cross-sectional views of short tail fragments were obtained after sonication at 0 °C. These views confirmed the 3-fold symmetry of the 38 Å annular unit, which most probably consists of three identical subunits of the major tail protein. Formation of extended tail polymers, both linear and circular, was found to take place spontaneously in vitro after sonication. On the basis of these results, a low-resolution model for the tail helix is presented. The questions of head-tail symmetry mismatch in the phage and of tail length regulation are discussed.

Bacteriophage T4 tail assembly: Structural proteins and their genetic identification

We have identified the structural proteins of phage T4 precursor tails. Complete tails, labeled with 14C-labeled amino acids, were isolated from cells infected with mutants blocked in head assembly. The proteins were characterized by sodium dodecyl sulfate-acrylamide gel electrophoresis and subsequent autoradiography. The complete tails are made up of at least fifteen different species of phage proteins. To identify the genes specifying these proteins we prepared 14C-labeled amino acid lysates made with amber mutants defective in each of the twenty-one genes involved in tail assembly. Comparison of the gel pattern of the amber mutant lysates with wild type lysates enabled us to identify the following gene products, with molecular weights in parentheses: P6 (85,000); P7 (140,000); P8 (46,000); P9 (34,000); P10 (88,000); P11 (26,000); P12 (55,000); P15 (35,000); P18 (80,000); P19 (21,000); P29 (77,000). These eleven species are all structural proteins of the tail. The genetically unidentified tail proteins have molecular weights of 42,000, 41,000, 40,000 and 35,000. They are likely to be the products of known phage genes which were not resolved in the crowded middle region of the whole lysate gel patterns. The major tail proteins are all synthesized during the late part of the phage growth cycle. The mobilities of the proteins derived from tails did not differ from the mobilities of the proteins when derived from the unassembled pools of subunits accumulating in mutant infected cells, or when derived from complete phage particles. The genes for at least seven of the structural proteins are contiguous on the genetic map. Genes for proteins needed in many copies seem to be clustered separ- ately from genes whose products are needed in only a few copies. Consideration of protein sizes and published mapping data on phage T4 also suggest that the phage structural proteins are, on the average, much larger than the non-structural proteins. The requirement that at least fifteen different species of proteins must come together in forming a phage tail emphasizes the complexity of this morphogenetic process.

LPP-1 infection of the blue-green alga Plectonema boryanum. 3. Protein synthesis.

The structural proteins of mature LPP-1 particles and the patterns of protein synthesis after LPP-1 infection have been examined by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. Structural proteins account for 35% of the LPP-1 genome, and proteins that would require about 65% of the total coding capacity have been detected after infection. The major head proteins have molecular weights of 39,000 and 13,000, whereas the major tail protein is an 80,000-molecular-weight species. Host protein synthesis is depressed soon after infection and appears to be entirely shut off by 5 hr. Three classes of viral proteins are distinguished in infected cells, based on their time course of synthesis and their presence in mature virions.