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PubMed1974

Bacteriophages of Rhodopseudomonas spheroides: isolation and characterization of a Rhodopseudomonas spheroides bacteriophage.

A DNA-containing bacteriophage, designated RS1, infecting Rhodopseudomonas spheroides 2.4.1, has been isolated from sewage. The buoyant density of RS1 in CsCl equilibrium centrifugation is 1.50 g/cm(3), and the buoyant density of RS1 DNA is 1.706. The phage possesses a polyhedral head, approximately 65 nm in diameter, and a tail 60 nm long. When grown on aerobic cells, RS1 has a latent period of 120 min and an average burst size of 20. When grown on anaerobic cells, RS1 has a latent period of 150 min, and a burst size similar to that observed during aerobic infection. The adsorption rate constant of RS1 to aerobic cells is 1.2 x 10(-9) ml/min, and 0.58 x 10(-9) ml/min to anaerobic cells. Adsorption of RS1 to R. spheroides requires the presence of divalent cations.

Adsorption↗
PubMed1976

Genetic and physiological studies of abortive infections of hydroxymethyluracil-containing bacteriophages in lysogens of temperate Bacillus subtilis bacteriophage SPO2.

Wild-type bacteriophage phie and IS (interference-sensitive) mutants of the related phage SP82G did not productively infect strains of Bacillus subtilis that were lysogenic for temperate phage SPO2. In these abortive infections, the sensitive phages adsorbed to and penetrated the nonpermissive host, phage-directed macromolecular syntheses were initiated, but both viral and bacterial nucleic acid production abruptly stopped about 15 min after addition of the phages. The cessation of RNA and DNA synthesis was preceded or coincident with a reduction in oxygen utilization by the infected cultures. Genetic studies of both phie and SP82G suggest sensitivity to SPO2-mediated abortive infection was controlled by a single gene. A mutant of SPO2, SPO2ehp4-, lysogens of which no longer interfere with the development of SP82GIs, was also isolated. The discovery of this ehp- variant suggests the normal SPO2 prophage synthesized a substance that alters cell physiology in some manner detrimental to SP82GIs development. Since SPO2ehp4- grew on and lysogenized bacteria sensitive to wild-type SPO2, the product of the eph gene was apparently not an essential function of this temperate phage.Overall, these observations exhibit remarkable similarities to the inhibition of T4rII mutants by the product of the rex gene of phage lambda.

Adsorption↗
PubMed1977

Role of the pneumococcal autolysin (murein hydrolase) in the release of progeny bacteriophage and in the bacteriophage-induced lysis of the host cells.

The pneumococcal bacteriophage Dp-1 seems to require the activity of the N-acetylmuramic acid-L-alanine amidase of the host bacterium for the liberation of phage progeny into the medium. This conclusion is based on a series of observations indicating that the exit of progeny phage particles is prevented by conditions that specifically inhibit the activity of the pneumococcal autolysin. These inhibitory conditions are as follows: (i) growth of the bacteria on ethanolamine-containing medium; (ii) growth of the cells at pH values that inhibit penicillin-induced lysis of pneumococcal cultures and lysis in the stationary phase of growth; (iii) addition of trypsin or the autolysin-inhibitory pneumococcal Forssman antigen (lipoteichoric acid) to the growth medium before lysis; (iv) infection of an autolysin-defective pneumococcal mutant at a multiplicity of infection less than 10 (treatment of such infected mutant bacteria with wild-type autolysin from without can liberate the entrapped progeny phage particles); (v) release of phage particles and culture lysis can also be inhibited by the addition of chloramphenicol to infected cultures just before the time at which lysis would normally occur. Bacteria infected with Dp-1 under conditions nonpermissive for culture lysis and phage release secrete into the growth medium a substantial portion of their cellular Forssman antigen in the form of a macromolecular complex that has autolysin-inhibitory activity. We suggest that a phage product may trigger the bacterial autolysin by a mechanism similar to that operating during treatment of pneumococci with penicillin (Tomasz and Waks, 1975).

Amidohydrolases↗
PubMed1978

Restriction endonuclease mapping of bacteriophage phi105 and closely related temperate Bacillus subtilis bacteriophages rho10 and rho14.

Cleavage maps of the three similar Bacillus subtilis temperate bacteriophages, phi105, rho10, and rho14, were constructed by partial digestion analysis utilizing the restriction endonuclease EcoRI. Comparison of the topography of these maps indicates that all phage DNAs posses cohesive ends and a number of EcoRI restriction sites; the fragments are conserved, and the estimated base substitution/nucleotide divergence between these phages is 0.03 to 0.07 based on conserved fragments or between 0.03 and 0.11 based on conserved cleavage sites. These lines of evidence indicate that phi105, rho10, and rho14 are closely related. Double-enzyme digestion analysis reveals that rho14 DNA has unique SalGI and BglII restriction sites and phi105 DNA has a unique SalGI restriction site, making these phages possible cloning vectors for B. subtilis.

Bacillus subtilis↗
PubMed1982

Cloned bacteriophage phi X174 DNA sequence interferes with synthesis of the complementary strand of infecting bacteriophage phi X174.

The insertion of a particular phi X DNA sequence in the plasmid pACYC177 strongly decreased the capacity of Escherichia coli cells containing such a plasmid to propagate bacteriophage phi X174. The smallest DNA sequence tested that showed the effect was the HindII fragment R4. This fragment does not code for a complete protein. It contains the sequence specifying the C-terminal part of the gene H protein and the N-terminal part of the gene A protein, as well as the noncoding region between these genes. Analysis of cells that contain plasmids with the "reduction sequence" showed that (i) the adsorption of the phages to the host cells is normal, (ii) in a single infection cycle much less phage is formed, (iii) only 10% of the infecting viral single-stranded DNA is converted to double-stranded replicative-form DNA, and (iv) less progeny replicative form DNA is synthesized. The reduction process is phi X174 specific, since the growth of the related G4 and St-1 phages was not affected in these cells. The effect of the recombinant plasmids on infecting phage DNA shows similarity to the process of superinfection exclusion.

Bacteriophage phi X 174↗
PubMed1983

Formation and possible functions of alpha-putrescinylthymine in bacteriophage phi W-14 DNA: analysis of bacteriophage mutants with decreased levels of alpha-putrescinylthymine in their DNAs.

The DNA synthesized in the nonpermissive host by the noncomplementing mutants am36 and am42 of bacteriophage phi W-14 contains about half the wild-type level of alpha-putrescinylthymine (putThy) and a correspondingly greater level of thymine. The mechanisms whereby thymine nucleotides are excluded from replicating DNA are functional in both mutants because neither of them incorporates exogenous thymidine into DNA. It is proposed that (i) in wild-type phi W-14, the conversion of hydroxymethyluracil to putThy at the polynucleotide level is sequence specific, but that to thymine is nonspecific; and (ii) in the mutants, the sequence-specific recognition is impaired so that more thymine and less putThy are formed. The thymine-rich DNA can be packaged into phage particles. In the case of am42, the phage particles are morphologically indistinguishable from and have essentially the same polypeptide composition as wild-type particles. However, the DNA molecules they contain are about 11% shorter than those in wild-type phage, am42rev4, a revertant of am42, contains DNA with about 70% of the normal level of putThy; these molecules are about 3% shorter than wild-type DNA. The properties of am42 and am42rev4 are consistent with the suggestion that putThy facilitates the very tight packing of phi W-14 DNA (Scraba et al., Virology 124:152-160, 1983). It also appears that the putThy content of phi W-14 DNA can be reduced by no more than 30% without adversely affecting the production of viable progeny; for example, the burst size of am42rev4 is about 25% of that of the wild type.

Bacteriophages↗
PubMed1984

Vibrio cholerae bacteriophage CP-T1: characterization of bacteriophage DNA and restriction analysis.

Temperature bacteriophage CP-T1 of Vibrio cholerae has a capsid that is 45 nm in diameter, a contractile tail 65 nm long and 9.5 nm wide, and a baseplate with several spikes or short tail fibers. The linear double-stranded DNA is 43.5 +/- 1.4 kilobases long, and the phage genome is both terminally redundant and partially circularly permuted. The extent of terminal redundancy is ca. 4%, and circular permutation is up to ca. 44%. Circular restriction maps have been constructed for the enzymes HindIII, EcoRI, BamHI, and PstI. By restriction endonuclease and heteroduplex analyses of phage DNA, the presence and location of a site (pac) at which packaging of phage DNA is initiated was established.

Bacteriophages↗
PubMed1980

[Pseudomonas aeruginosa bacteriophages with DNA structure similar to the DNA structure of Mu1 phage. II. Evidence for similarity between D3112, B3, and B39 bacteriophages: analysis of DNA splits by restriction endonucleases, isolation of D3112 and B3 recombinant phages].

It is found that bacteriophages B3 and B39 specific for Pseudomonas aeruginosa have the same genome structure as previously described phage D3112. On the right (S) end of their genomes a variable non-phage DNA is located (approximately 0.9-2.5 kilobases for different phages). It is probable that this variable DNa has its origin from different regions of bacterial chromosome. In genome of one of the phages, B3 phage, such variable DNA (not more than 150 base pairs) was found on the left end of DNA molecule. Isolation of a viable B3XD3112 recombinant phage and analysis of its genome with restriction technique and with studies of homo- and heteroduplex molecules had confirmed genetical relationship of B3 and D3112. Some essential non-homology of B3 and D3112 DNAs have been found on the right ends of genomes of the phages.

Bacteriophages↗
PubMed1997

The crystal structure of bacteriophage GA and a comparison of bacteriophages belonging to the major groups of Escherichia coli leviviruses.

The three-dimensional structure of the small T=3 RNA bacteriophage GA has been determined at 3.4 A resolution. The structure was solved by molecular replacement, using the phage MS2 as an initial model. A comparison of the protein shells of the four related phages GA, MS2, fr and Qbeta was carried out in order to define structural features of particular importance for their assembly and specific RNA interaction. A high degree of similarity was found in the RNA binding sites, whereas larger structural differences are located in the loop regions of the coat proteins, especially in the FG loops forming 5-fold and quasi-6-fold contacts. The overall arrangement of the protein subunits in the shells of these phages is very similar, although the details of the interactions differ. The few conserved interactions are suggested to govern the subunit packing during assembly.

Amino Acid Sequence↗
PubMed1982

On the role of the single-stranded DNA binding protein of bacteriophage T4 in DNA metabolism. I. Isolation and genetic characterization of new mutations in gene 32 of bacteriophage T4.

The product of gene 32 of bacteriophage T4 is a single-stranded DNA binding protein involved in T4 DNA replication, recombination and repair. Functionally differentiated regions of the gene 32 protein have been described by protein chemistry. As a preliminary step in a genetic dissection of these functional domains, we have isolated a large number of missense mutants of gene 32. Mutant isolation was facilitated by directed mutagenesis and a mutant bacterial host which is unusually restrictive for missense mutations in gene 32. We have isolated over 100 mutants and identified 22 mutational sites. A physical map of these sites has been constructed and has shown that mutations are clustered within gene 32. The possible functional significance of this clustering is considered.

Base Sequence↗
PubMed1986

Mechanism of inhibition of bacteriophage T7 DNA synthesis in Escherichia coli B cells infected by alkylated bacteriophage T7.

Quantitative analysis of DNA replication, in E. coli B cells infected by methyl methanesulfonate-treated bacteriophage T7, showed that production of phage DNA was delayed and decreased. The cause of the delay appeared to be a delay in host-DNA breakdown, the process which provides nucleotides for phage-DNA synthesis. In addition, reutilisation of host-derived nucleotides was impaired. These observations can be accounted for by a model in which methyl groups on phage DNA slow down DNA injection and also reduce the replicational template activity of the DNA once it has entered the cell. Repair of alkylated phage DNA may be required not only for replication but also for normal injection of DNA.

DNA Repair↗
PubMed1981

Assembly of bacteriophage T7. Dimensions of the bacteriophage and its capsids.

The dimensions of bacteriophage T7 and T7 capsids have been investigated by small-angle x-ray scattering. Phage T7 behaves like a sphere of uniform density with an outer radius of 301 +/- 2 A (excluding the phage tail) and a calculated volume for protein plus nucleic acid of 1.14 +/- 0.05 x 10(-16) ml. The outer radius determined for T7 phage in solution is approximately 30% greater than the radius measured from electron micrographs, which indicates that considerable shrinkage occurs during preparation for electron microscopy. Capsids that have a phagelike envelope and do not contain DNA were obtained from lysates of T7-infected Escherichia coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA by means of temperature shock (capsid IV). In both cases the peak protein density is at a radius of 275 A; the outer radius is 286 +/- 4 A, approximately 5% smaller than the envelope of T7 phage. The thickness of the envelope of capsid II is 22 +/- 4 A, consistent with the thickness of protein estimated to be 23 +/- 5 A in whole T7 phage, as seen on electron micrographs in which the internal DNA is positively stained. The volume in T7 phage available to package DNA is estimated to be 9.2 +/- 0.4 x 10(-17) ml. The packaged DNA adopts a regular packing with 23.6 A interplanar spacing between, DNA strands. The angular width of the 23.6 A reflection shows that the mean DNA-DNA spacing throughout the phage head is 27.5 +/- less than 2.2 A. A T7 precursor capsid (capsid I) expands when pelleted for x-ray scattering in the ultracentrifuge to essentially the same outer dimensions as for capsids II and IV. This expansion of capsid I can be prevented by fixing with glutaraldehyde; fixed capsid I has peak density at a radius of 247 A, 10% less than capsid II or IV.

Capsid↗
PubMed1983

Gene 20 product of bacteriophage T4. II. Its structural organization in prehead and bacteriophage.

The location of gene 20 product of bacteriophage T4 in phage and phage percursors has been determined by immunochemical analysis of polyacrylamide gels, immunoturbidimetry and immunoelectron microscopy. The protein is present at the membrane attachment site of the prehead, a head precursor, and is accessible to the antibodies in the solution. It is present at the tail attachment site of the capsid, partially buried in the structure. In complete phage particles it is totally buried in the structure. It is in contact with the major shell proteins, gp23 and gp23*, respectively, in preheads and capsids, as revealed by partial crosslinking experiments. It forms the upper collar of the neck in necked tails. The lower collar is constructed from other gene products. On the basis of these data a structural model of the neck region of the phage has been derived. This model is consistent with a number of events in phage assembly, such as the role of gp20 in head assembly and DNA packaging, prehead detachment from the bacterial membrane and head-tail attachment. The symmetry mismatch known to occur between head and tail has been localized at the gp20-gp23* contact area.

Antibodies, Viral↗
PubMed1984

Defining a bacteriophage T4 late promoter: bacteriophage T4 gene 55 protein suffices for directing late promoter recognition.

The RNA polymerase from bacteriophage T4-infected Escherichia coli, which specifically initiates transcription at phage T4 late promoters, is extensively modified by ADP-ribosylation of core subunits and by binding several virus-encoded subunits. We show here that one of these subunits, the phage T4 gene 55 protein, designated gp55, alone endows unmodified RNA polymerase core enzyme from uninfected E. coli with the ability to selectively initiate transcription at the phage T4 late promoters, without participation by E. coli RNA polymerase o- subunit.

DNA-Directed RNA Polymerases↗
PubMed1976

On the nature of cis-acting regulatory proteins and genetic organization in bacteriophage: the example of gene Q of bacteriophage lambda.

We note the existence of a "partially cis-acting" regulatory protein of bacteriophage lambda: the product of the phage Q gene. We suggest that there may be a complete spectrum from "all cis" to "all trans" for such regulatory proteins. This behavior might arise because a DNA-binding protein either acts at a nearby (cis) site soon after synthesis or becomes "lost" for its trans activity on another genome through nonspecific interactions with DNA. Our proposed explanation provides one evolutionary basis for the linkage of genes for regulatory proteins and the sites at which such proteins act; it also suggests a possible rationale for the "metabolic instability" of certain regulatory proteins.

Coliphages↗
PubMed1981

Partial exclusion of bacteriophage T2 by bacteriophage T4: induction of early enzymes by excluded T2.

In crosses between bacteriophages T2 and T4 most early genes of T2 are partially excluded from the progeny. Six genes of T4 affect the exclusion of six corresponding exclusion-sensitive sites in T2, each gene being specific for the exclusion of one site. Mutants of T4 in these genes have been isolated (ex mutants). The induction of the gene product (deoxycytidine triphosphate nucleotidohydrolase, dCTPase) of the strongly excluded T2 gene 56 was determined. The dCTPase was induced in the presence of the replication inhibitor oxolinic acid to prevent possible artefacts from unequal replicaton rates of T2 and T4. The rate of T2 dCTPase induction was 30% of the control in T2 X T4 infections in which all six exclusion-sensitive sites were excluded and was 57% in infections in which the sites except 56 were excluded. The dCTPase induction was 67% of the control with exclusion of the 56 site only and equalled the control when no T2 site was excluded. Inhibition of dCTPase induction under conditions of exclusion is partly due to exclusion of neighboring sites and partly due to exclusion of the 56 site.

Deoxycytosine Nucleotides↗
PubMed1987

Bacteriophage Resistance Plasmid pTR2030 Inhibits Lytic Infection of r(1)t Temperate Bacteriophage but Not Induction of r(1)t Prophage in Streptococcus cremoris R1.

The effects of pTR2030 on the replication of four small isometric bacteriophages were examined in Streptococcus cremoris R1. Three lytic phages (652, 720, and 751), which were isolated independently over a 29-year period, were unable to form plaques on a pTR2030 transconjugant of S. cremoris R1. The fourth phage evaluated, phage r(1)t, was a temperate phage induced from S. cremoris R1 by treatment with mitomycin C. A prophage-cured derivative of S. cremoris R1, designated R1Cs, was isolated and served as a lytic indicator for phage r(1)t. Strain R1Cs and a derivative of this strain that was relysogenized with r(1)t, designated R1Cs(r(1)t), were used as conjugal recipients for transfer of the phage resistance plasmid pTR2030. pTR2030 transconjugants of strains R1Cs and R1Cs(r(1)t) were evaluated for sensitivity to r(1)t phage and induction of r(1)t prophage, respectively. The temperate phage r(1)t adsorbed eficiently but did not form plaques on the prophage-cured, pTR2030 transconjugant strain T-R1Cs. However, in the r(1)t lysogen [T-R1Cs(r(1)t)], pTR2030 did not inhibit prophage induction with mitomycin C, cell lysis, or production of infective r(1)t phage particles. The data demonstrated that pTR2030-induced resistance inhibited lytic infection by r(1)t phage from without but did not retard lytic development after prophage induction within the cell. It was suggested that pTR2030-encoded phage resistance to small isometric phages may, therefore, act at the cell surface or membrane to prevent phage DNA passage into the host cell or inhibit early events required for lytic replication of externally infecting phage.

Journal Article↗