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PubMed1977

Intracellular organization of bacteriophage T7 DNA: analysis of parenteral bacteriophage T7 DNA-membrane and DNA-protein complexes.

After infection of Escherichia coli with bacteriophage T7, the parenteral DNA forms a stable association with host cell membranes. The DNA-membrane complex isolated in cesium chloride gradients is free of host DNA and the bulk of T7 RNA. The complex purified through two cesium chloride gradients contains a reproducible set of proteins which are enriched in polypeptides having molecular weights of 54,000, 34,000, and 32,000. All proteins present in the complex are derived from host membranes. Treatment of the complex with Bruij-58 removes 95% of the membrane lipid and selectively releases certain protein components. The Brij-treated complex has an S value of about 1,000 and the sedimentation rate of this material is not altered by treatment with Pronase or RNase.

Bacterial Proteins↗
PubMed1977

Assembly of bacteriophage phi X174: identification of a virion capsid precursor and proposal of a model for the functions of bacteriophage gene products during morphogenesis.

A capsomeric structure sedimenting with an S value of 108 in sucrose gradients was isolated from Escherichia coli infected with bacteriophage phi X174. The 108S material contained viral proteins F, G, H, and D, and the relative amounts of these proteins in the 108S material were similar to those in the infectious 132S particle, which has previously been described as a possible intermediate in the assembly of 114S phage particles. Electron micrographs indicated that the size and shape of the 108S material resemble those of the 132S particle. The 108S material contained no DNA, and its formation occurred independently of DNA synthesis. The 108S material accumulated in infected cells when viral DNA replication was prevented either by mutation in phage genes A or C or by removal of thymidine from a culture infected with wild-type phage or with a lysis gene E mutant. Upon restoration of thymidine to cells infected with the lysis gene E mutant and then starved of thymidine, the accumulated 108S material was converted to 132S particles and to 114S phage particles, implying that the 108S material is a precursor of phage particles. A model that proposes possible functions for the products of phi X174 genes A, B, C, D, F, and G during viral replication and phage maturation is described.

Capsid↗
PubMed1977

Slow switchover from host RNA synthesis to bacteriophage RNA synthesis after infection of Escherichia coli with a T4 mutant defective in the bacteriophage T4-induced unfolding of the host nucleoid.

Most, if not all, host RNA synthesis was shut off after infection of Escherichia coli strain B/5 with a bacteriophage T4 multiple mutant defective in the abilities to induce (i) unfolding of the host nucleoid (unf-), (ii) nuclear disruption (ndd-), and (iii) host DNA degradation (denA-, denB-). The shutoff of host RNA synthesis and turn-on of phage RNA synthesis were slower after infection of E. coli with unf- phage than after infection with unf+ phage. This delay in the switchover from host RNA synthesis to phage RNA synthesis in unf- infections did not result in a measurable delay in the onset of nuclear disruption, deoxyribonucleoside monophosphate kinase synthesis, or DNA synthesis. unf39 did not complement alc (allows late transcription on cytosine-containing DNA) mutants, supporting the proposal of Sirotkin et al. [Nature (London) 265:28-32, 1977] that alc and unf are possibly the same gene.

Cell Nucleus↗
PubMed1979

Recombination of bacteriophage phi X174 by the red function of bacteriophage lambda.

Recombination of bacteriophage phi X174 was effectively promoted when the Red function of lambda was supplied by either co-infection with lambda or induction of lambda lysogens. Mutations in red alpha and red beta genes of lambda abolished recombination nearly completely, whereas a mutation in gam gene reduced it only slightly. The Red-promoted recombination of phi X174 occurred in recA, recB, and polA mutants as well as in wild-type strains of Escherichia coli. It was further stimulated when phi X174 mutants were irradiated with UV light before infection.

Coliphages↗
PubMed1969

Bacteriophage-induced inhibition of host functions. II. Evidence for multiple, sequential bacteriophage-induced deoxyribonucleases responsible for degradation of cellular deoxyribonucleic acid.

Degradation of bacterial deoxyribonucleic acid (DNA) after infection with T4 bacteriophage was studied in an endonuclease I-deficient host. The kinetics of degradation were similar to those seen in other hosts with a normal level of this enzyme. Irradiation of extracellular phage with ultraviolet (UV) destroyed the capacity of the infecting virus to induce extensive breakdown of host DNA, which was, however, converted to high-molecular-weight material. Addition of chloramphenicol to T4-infected cells provided data which can be interpreted to indicate the involvement of at least two endodeoxyribonucleases and one exodeoxyribonuclease having a high degree of specificity. A model is proposed showing the sequential action of two endodeoxyribonucleases followed by an exodeoxyribonuclease in the degradation of host DNA. The appearance of these hydrolytic enzymes requires protein synthesis. Infections leading to partial degradation only (UV-irradiated phages, gene 46 mutants) effectively inhibited the synthesis of bacterial messenger ribonucleic acid and of beta-galactosidase.

Chloramphenicol↗
PubMed1980

High-molecular-weight DNA and the sedimentation coefficient: a new perspective based on DNA from T7 bacteriophage and two novel forms of T4 bacteriophage.

The DNA molecules from T7 bacteriophage and a recently obtained mutant form of T4D were studied. The DNA of this T4 mutant contains cytosine in place of all of the glucosylated hydroxymethylcytosines normally present in T4. Molecular weights were measured with an electron microscope technique, and sedimentation coefficients were determined in isokinetic sucrose gradients. T7 DNA was found to have an Mr of 26.5 x 10(6). The T4 mutant, which we have termed T4c, produces two distinct phage head and DNA size clases. DNA from the standard heads (T4c DNA) has an Mr of 114.9 x 10(6), and DNA from the petite heads (T4cp DNA) has an Mr of 82.9 x 10(6). This enabled the derivation of an equation of sedimentation coefficient at zero concentration corrected to water at 20 degrees C versus Mr for the molecular weight range of 25 x 10(6) to 115 x 10(6) that is based solely on cytosine-containing DNA standards, thereby avoiding possible anomalies introduced by the glucosylation and hydroxymethylation of cytosine. The theory of Gray et al. provided the best description of the sedimentation coefficient versus Mr relationship, based on the sedimentation coefficients and the molecular weights of the three DNA standards and other evidence.

Base Composition↗
PubMed1980

Head maturation pathway of bacteriophages T4 and T2. V. Maturable epsilon-particle accumulating an acridine-treated bacteriophage T4-infected cells.

A maturable head-related particle of bacteriophage T4 has been identified and characterized. This epsilon-particle has the same size as the prehead, but its shell is made of the cleaved product of gene 23 (gp23*). It contains internal matter, most likely the processed core proteins, which is lost or modified by experimental manipulations. It accumulates, together with partially filled ("grizzled") heads, in T4 infected cells that are treated with 9-aminoacridine. On sections of "well-preserved" cells the epsilon-particles are not identifiable with certainty; a more or less empty breakdown product of them becomes visible when cytoplasmic leakage is induced. The number of particles per cell is then in agreement with the biochemically and with the number of particles counted in lysates. Morphologically and biochemically, the isolated epsilon-particles closely resemble the empty small particles of 17- -infected cells described in previous papers of this series. Both are composed of gp23* and are still unexpanded, so that they are not yet able to bind the minor head proteins soc and hoc. We discuss the possibility of the epsilon-particle being an intermediate on the normal T4 wild-type head maturation pathway.

Aminacrine↗
PubMed1981

Salmonella bacteriophage glycanases: endorhamnosidase activity of bacteriophages P27, 9NA, and KB1.

Four bacteriophages, P22, P27, 9NA, and KB1, active on smooth Salmonella strains belonging to serogroups A, B, and D1 were investigated for endoglycosidase activity and specificity in enzyme hydrolysis assays. Purified phage was incubated with phenol-water-extracted lipopolysaccharide preparations which had been partially delipidated. Dialyzable oligosaccharides, released by phage glycosidase activity, were analyzed by sugar and methylation analyses. Phages P27, 9NA, and KB1, as well as P22 assayed earlier (U. Eriksson et al., J. Gen. Virol. 43: 503-511, 1979; S. Iwashita and S. Kanegasaki, Biochem. Biophys. Res. Commun. 55:403-409, 1973), were all found to have phage-associated endorhamnosidase activity hydrolyzing the O-polysaccharide chain common to bacteria of serogroups A, B, and D1 [Formula: see text] between the l-rhamnose and d-galactose residues. The nature of the R monosaccharide, abequose, tyvelose, or paratose, had no effect on the activity or specificity of the endorhamnosidase, whereas a change of the d-galactose --> d-mannose linkage from alpha1,2 to alpha1,6 made the O-polysaccharide chain resistant to the endorhamnosidases. Modification of the O chain by glucosylation of the d-galactose residue at O-4 or O-6 revealed two glycosidase specificities: the phage P22 and P27 enzymes hydrolyzed O chains glucosylated at O-4 but not O-6, whereas the phage 9NA and KB1 enzymes hydrolyzed chains glucosylated at O-6 but not O-4. Phage KB1, like P22 and P27, had a short, noncontractile tail containing a base plate with tail spikes (morphologically Bradley group C), whereas 9NA had a long, flexible tail ending with a base plate-like appendage (Bradley group B), which suggests that the endorhamnosidase activity can be associated with different tail structures.

Glycoside Hydrolases↗
PubMed1982

Genetic analysis of bacteriophage T4 transducing bacteriophages.

Mutations in the genes for nuclear disruption (ndd), endonuclease IV (denB), and the D1 region of the T4 genome are essential for converting bacteriophage T4 into a generalized transducing phage. These mutations gave rise to a very low frequency of transduction, about 10(-8) per infected bacterium. The addition of an rII mutation raised the transduction frequency about 20-fold. An additional 100-fold increase in the transduction frequency was observed with mutations in genes 42, 56, and alc. High-frequency generalized transduction by T4 results from the cumulative effect of these mutations.

Chromosome Deletion↗
PubMed1970

Bacteriophage tail components. I. Pteroyl polyglutamates in T-even bacteriophages.

A pteroylpolyglutamate has been found to be a constituent of all Escherichia coli T-even bacteriophages and has been characterized with regard to its oxidation state, molecular weight, origin, and location on the phage particle. The phage compound has been shown to be a dihydropteroyl penta- or hexaglutamate on the basis of its chemical and physical properties. Analyses of extracts of uninfected and T2L-infected E. coli have indicated that the phage dihydropteroyl polyglutamate was present only in infected cells. Its synthesis was sensitive to the addition of chloramphenicol before infection, and the compound appeared to be specifically induced by phage infection. Analyses of isolated phage ghosts and tail substructures have shown that each phage particle contains between two and six phage-specific pteroyl derivatives and that the juncture of the phage tail plate with the tail tube is the most likely site of binding of the phage-induced pteroyl compound.

Aminobenzoates↗
PubMed1970

Bacteriophage tail components. II. Dihydrofolate reductase in T4D bacteriophage.

The protein component of the T-even bacteriophage coat which binds the phage-specific dihydropteroyl polyglutamate has been identified as the phage-induced dihydrofolate reductase. Dihydrofolate reductase activity has been found in highly purified preparations of T-even phage ghosts and phage substructures after partial denaturation. The highest specific enzymatic activity was found in purified tail plate preparations, and it was concluded that this enzyme was a structural component of the phage tail plate. Phage viability was directly correlated with the enzymological properties of the phage tail plate dihydrofolate reductase. All reactions catalyzed by this enzyme which changed the oxidation state of the phage dihydrofolate also inactivated the phage. Properties of two T4D dihydrofolate reductase-negative mutants, wh1 and wh11, have been examined. Various lines of evidence support the view that the product of the wh locus of the phage genome is normally incorporated into the phage tail structure. The effects of various dihydrofolate reductase inhibitors on phage assembly in in vitro complementation experiments with various extracts of conditional lethal T4D mutants have been examined. These inhibitors were found to specifically block complementation when added to extracts which did not contain preformed tail plates. If tail plates were present, inhibitors such as aminopterin, did not affect further phage assembly. This specific inhibition of tail plate formation in vitro confirms the analytical and genetic evidence that this phage-induced "early" enzyme is a component of the phage coat.

Aminopterin↗
PubMed1971

Superinfection in bacteriophage S13 and determination of the number of bacteriophage particles which can function in an infected cell.

Bacteriophage S13 shows exclusion of superinfecting homologous phage, but the exclusion is only partial. The superinfecting phage can form infectious replicative form deoxyribonucleic acid (RF), can direct protein synthesis, and can form progeny particles even at a superinfection time as late as 60 min after the first infection. Exclusion is also only partial for the closely related phage phiX174. Seven min after the first infection, the exclusion mechanism begins to operate, requiring continuous phage-specified protein synthesis. The gene A protein (required for synthesis of progeny RF) appears to be involved in the exclusion mechanism. In superinfection experiments, it was found that at least 40 phage particles per cell can replicate and can carry out protein synthesis, though the number of sites for binding of RF to the membrane is only about 15 per cell. The results suggest that attachment of RF to a binding site is not required for protein synthesis. Evidence is presented that non-attached parental RF can serve as a template for single-stranded deoxyribonucleic acid synthesis.

Binding Sites↗
PubMed1972

Deoxyribonucleic acid synthesis in bacteriophage SPO1-infected Bacillus subtilis. I. Bacteriophage deoxyribonucleic acid synthesis and fate of host deoxyribonucleic acid in normal and polymerase-deficient strains.

The effect of bacteriophage SPO1 infection of Bacillus subtilis and a deoxyribonucleic acid (DNA) polymerase-deficient (pol(-)) mutant of this microorganism on the synthesis of DNA has been examined. Soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. This inhibition of host DNA synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate nucleotidohydrolase. Examination of the host chromosome for genetic linkage throughout the lytic cycle indicated that no extensive degradation occurred. After the inhibition of host DNA synthesis, a new polymerase activity arose which directed the synthesis of phage DNA. This new activity required deoxyribonucleoside triphosphates as substrates, Mg(2+) ions, and a sulfhydryl reducing agent, and it was stimulated in the presence of adenosine triphosphate. The phage DNA polymerase, like that of its host, was associated with a fast-sedimenting cell membrane complex. The pol(-) mutation had no effect on the synthesis of phage DNA or production of mature phage particles.

Adenosine Triphosphate↗
PubMed1984

[Effect of the polymerase activity of DNA polymerase I on the development of moderate bacteriophages Mu, lambda red- and lambda red-gam-. II. The effect of the polymerase activity of DNA polymerase I on the development of bacteriophage Mu].

The paper reports on the influence of polymerizing activity of DNA-polymerase I on different developmental stages of temperate bacteriophage Mu in Escherichia coli K-12 cells. This activity is shown to be necessary for optimization of phage Mu primary integration into cell chromosomes. The relative frequency of Mu integration into bacterial chromosomes is 5-6 times lower in polA cells than in isogenic polA+ control strains, the phage yield from cells being delayed during the phage infectious development, but not in the course of induction from the prophage state. Data have been obtained that show the process of phage Mu DNA integration into the plasmid pRP1 .2 and the process of Mu transposition from the cell chromosome into the plasmid to be independent of the polymerizing activity of DNA-polymerase I.

Adsorption↗
PubMedDate not supplied

[Antigenic structure of bacteriophage lambda. Identification of the chief antigens of bacteriophage lambda].

The antigenic composition of the bacteriophages lambdaC1857 and lambdagt-lambdaC was investigated by modified immunoelectrophoresis in a 1,2% agarose gel involving 1% Triton X-100 and 0,25% sodium desoxycholate. The phages lambdaC1857 and lambdagt-lambdaC were shown to have identical antigenic compositions and to comprise three basic antigens, such as a1, a2, a3. The main structural proteins of the phage such as pE, pV and pD were isolated by preparative electrophoresis in 13% polyacrilamide gel. The immunophoresis of individual proteins indicated the antigens a1, a2 and a3 to be proteins pV, pD and pE, respectively.

Antigens, Viral↗
PubMed1976

Effect of ts mutation in gene 43 of bacteriophage T4 on recombination of the bacteriophage.

Certain ts mutations in gene 43 of bacteriophage T4 are known to exhibit mutator or antimutator activities with respect to different rII mutations of this phage. The effect of these mutations on recombination frequencies of double mutant strains of phage T4 with genotype rII ts--homoallelic with respect to the ts trait--was examined. The results implicate the essential role of the genetic background in the investigated process.

Coliphages↗