Partial exclusion of genes of bacteriophage T2 with T4-glucosylated DNA in crosses with bacteriophage T4

A recombinant strain (D41) between phage T2 and T4 was isolated which possessed the T2 region of the genome between genes 32 and 39 and both the T4 genesgt ⁺ andgt ⁺ for glucosyltransferase. D41 was crossed with T4amber mutants in the genes for early functions and in some genes for late funcitions. The progeny of the crosses was examined for the frequency of theam ⁺ markers from D41. Genes 32, 60 and 39 in the T2 region of the recombinant strain were as sensitive to exclusion as those in standard-type T2. The T4 glucosylation of the DNA of these T2 genes did not protect them against partial exclusion by T4. However, genes in the region from gene 56 to 55 in the recombinant were resistent to exclusion. In standard-type T2 this region of the genome is sensitive to partial exclusion by T4. There are at least four exclusion sensitive sites in T2: one near gene 32, one near gene 60, one linked to gene 56 and one between genes 42 and 55.This investigation was carried out partially within the frame of the Association between Euratom and the University of Leiden, contract nr. 052-64-1-BIAN.     

Crystalline T4 bacteriophage

Concentrated suspensions of T4 phage crystallize spontaneously in the absence of tryptophan. The crystals, in one plane, contain repeated bilayers of phage in head to head and tail to tail contact, probably with tail fibers retracted. In the plane of each layer there is hexagonal packing of the phage.     

Properties of condensed bacteriophage T4 DNA isolated from Escherichia coli infected with bacteriophage T4.

Methods developed for isolating bacterial nucleoids were applied to bacteria infected with phage T4. The replicating pool of T4 DNA was isolated as a particle composed of condensed T4 DNA and certain RNA and protein components of the cell. The particles have a narrow sedimentation profile (weight-average s=2,500S) and have, on average, a T4 DNA content similar to that of the infected cell. Their dimensions observed via electron and fluorescence microscopy are similar to the dimensions of the intracellular DNA pool. The DNA packaging density is less than that of the isolated bacterial nucleoid but appears to be roughly similar to its state in vivo. Host-cell proteins and T4-specific proteins bound to the DNA were characterized by electrophoresis on polyacrylamide gels. The major host proteins are the RNA polymerase subunits and two envelope proteins (molecular weights, 36,000 and 31,000). Other major proteins of the host cell were absent or barely detectable. Single-strand breaks can be introduced into the DNA with gamma radiation or DNase without affecting its sedimentation rate. This and other studies of the effects of intercalated ethidium molecules have suggested that the average superhelical density of the condensed DNA is small. However, these studies also indicated that there may be a few domains in the DNA that become positively supercoiled in the presence of high concentrations of ethidium bromide. In contrast to the Escherichia coli nucleoid, the T4 DNA structure remains condensed after the RNA and protein components have been removed (although there may be slight relaxation in the state of condensation under these conditions).     

Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.     

Ribosomes after infection with bacteriophage T4 and T7

Summary The synthesis of E. coli ribosomal proteins ceases after infection with bacteriophages T4 or T7 as does the synthesis of most other host proteins. The shut-off does not affect all ribosomal proteins to the same extent. After T7 infection no new proteins were detected in NH₄Cl-washed ribosomal particles. Bacteriophage T4, however, induces 3–4 new protein bands demonstrated by one-dimensional gel electrophoresis. The appearance of these bands is prevented by the addition of rifampicin at the time of infection but not when rifampicin is added one minute after infection. The NH₄Cl-washed ribosomal particles present at the time of T7 or T4 infection do not show any structural changes by sedimentation, subunit dissociation, or protein analysis on two-dimensional polyacrylamide gels. However, by labeling the T7 infected cells with ³²P-phosphate, it is seen that the ribosomes become phosphorylated. The ³²P-label comigrates with ribosomal proteins. This phosphorylating activity depends on a T7 gene. The T7 protein phosphokinase utilizes ribosomes as phosphate acceptor in vitro. The T7 ribosomes (NH₄Cl-washed) still function in vitro as do ribosomal particles from uninfected cells.Paper No. 83 on Ribosomal Proteins. Preceding paper is by Isono et al., Mol. gen. Genet. 127, 191–195 (1973).     

Physical mapping of bacteriophage T4

Summary The 134 positions of the cleavage sites of the restriction endonucleases XbaI, HaeII and EcoRI were determined for a cytosine-containing DNA of bacteriophage T4. This physical map was aligned with the genetic map. The T4 early regions were further identified by hybridization of RNA synthesized in vitro to the restriction fragments and two promoter regions were localized by filter binding tests and R-loop analysis.     

Properties of bacteriophage T4 modified with cetyltrimethylammonium bromide

Treating of the bacteriophage T4B with cetyltrimethylammonium bromide results in extension of long fibers and contraction of tail sheaths. The unreorganized hexagonal baseplate attachment to the distal end of the tail core remains intact. Such aberrantly contracted phages are shown to retain the ability to absorb on the bacterial surface. The absorption is inhibited by sucrose and does not require the presence of 1-tryptophan. The aberrantly contracted phages lose the infection ability.     

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.     

Marker rescue of the adsorption properties of bacteriophage T 4 by bacteriophage T 2

Rescue of adsorption properties from UV-irradiated T4 by T2 as a helper phage, revealed progeny phage with intermediate properties. Fourteen independent progeny phages, plating onE. coli B/2, were plated on several indicator strains and their adsorption properties were also studied with specific T4 antibodies. Two of these, plating onE. coli KS/4, were not inactivated by the T4 antiserum, and were T2h without apparent T4 properties. The other 12 progeny phages did not plate on KS/4, and were inactivated, but at a slower rate than the parental T4. Their mean efficiency of plating onE. coli B/2 (0.83) was significantly lower than that of the parental T4. The efficiency of plating was positively correlated with the velocity of inactivation by T4 antiserum. The observations were explained by assuming that the progeny phages were recombinants of T4 and T2 loci for adsorption sites. Plating of these 12 progeny phages on several indicator strains showed that they were allrII mutants and all, except one, wererI mutants too. In addition, two weretu andh ⁴, respectively. The condition for the appearance of multiple mutants might be a complementation by T2 of UV-damaged functions, which otherwise fail to induce the completion of the lytic cycle in monocomplexes of extracellularly irradiated T4.     

Deletion formation in bacteriophage T4

We have manipulated the dispensable region of the rIIB gene of bacteriophage T4 in order to study the generation of deletions involving direct repeats. We show that recombination between different parental chromosomes is one source of the deletions we have studied. We have also investigated the effects of structure, base composition and distance on deletion formation. We demonstrate that the potential to form structure in single-stranded DNA has variable effects on the frequency of deletion formation and conclude that, in some cases, slipped mispairing during DNA synthesis can make a substantial contribution to deletion frequencies. The G + C richness of the direct repeats involved in deletion formation is an important parameter of the frequency of deletion formation. We have confirmed that increasing the distance between direct repeats decreases deletion frequency.