The Mechanism of Inactivation of Bacteriophage ϕX174 by Autoxidizable Synthetic Polysaccharides

Autoxidizable synthetic polysaccharides prepared by polycondensation of reducing aldose or ketose in dimethyl sulfoxide containing pohsphorus pentaoxide [Polymer, 13, 190 (1972)] inactivated phage ?X174. Another autoxidizable polysaccharides obtained by oxidation of natural glucans with the same oxidant also inactivated ?X174. The ?X174 inactivation was due to strand scission of viral DNA in the virion. The inactivation reaction was stimulated by Cu²⁺ and inhibited by EDTA, Superoxide dismutase, catalase and several radical scavengers. These results suggest that oxygen radicals produced during autoxidation of polysaccharides are responsible for ?X174 inactivation.     

Inhibition of Bacteriophage ϕX174 Replicaiive-form DNA Replication by Rifampicin

?X174 DNA synthesis as well as phage production was inhibited by rifampicin when added in early phase of infection. Rifampicin did not inhibit the formation of parental duplex replicative-form, RF, and it inhibited the synthesis of progeny RF under conditions where protein synthesis was not necessary to be synthesized continuously. In addition, replication of parental RF into progeny RF was inhibited by rifampicin under conditions where a high concentration of chloramphenicol did not affect the replication. Consequently, it could be concluded that RNA synthesis other than that required for protein synthesis was necessary for both the initiation and continuation of RF replication.     

Effect of Chloramphenicol on the DNA Synthesis of Bacteriophage ϕX174

In bacteriophage ?X174 infection, the net synthesis of replicative form DNA ceased between 15 and 20 min after infection. When 30 μg of chloramphenicol/ml was added, net RF synthesis, however, continued beyond the normal time and level of turn-off. Experiments with ?X174 mutants unable to synthesize single-stranded DNA showed that a protein synthesis was required for the cessation of net RF synthesis and the protein was synthesized between 10 and 15 min after infection.     

Photodynamic Inactivation of Antigenic Determinants of Single-Stranded DNA Bacteriophage φX174

Bacteriophage phiX174 when photodynamically inactivated (i.e., when rendered unable to produce plaques as a result of exposure to visible light in air in the presence of proflavine) progressively lost their capacity to bind efficiently with homologous antiserum. Such loss of serum-blocking power was evident with heat-inactivated but not with UV-irradiated phage. The ability of the phages to adsorb to host cells, however, remained practically unaltered even after photodynamic inactivation. It thus appears that photodynamic damages in the so-called "jacket" component of the phiX174 coat proteins are partly responsible for the loss of plaque-forming ability, whereas the "spikes" are either poor antigens or insensitive to photodynamic treatment.     

Mode of Host Cell Penetration by Bacteriophage φX174

Bacteriophage phiX174 is an icosahedral phage which attaches to host cells without the aid of a complex tail assembly. When phiX174 was mixed with cell walls isolated from the bacterial host, the virions attached to the wall fragments and the phage deoxyribonucleic acid (DNA) was released. Attachment was prevented if the cell walls were treated with chloroform. Release of phage DNA, but not viral attachment, was prevented if the cell walls were incubated with lysozyme or if the virions were inactivated with formaldehyde. Treatment of the cell walls with lysozyme released structures which were of uniform size (6.5 by 25 nm). These structures attached phiX174 at the tip of one of its 12 vertices, but the viral DNA was not released. The virions attached to these structures were oriented with their fivefold axis of symmetry normal to the long axis of the structure. No virions were attached to these structures by more than one vertex. Freeze-etch preparations of phiX174 adsorbed to intact bacteria showed that the virions were submerged to one half their diameter into the host cell wall, and the fivefold axis of symmetry was normal to the cell surface. A second cell could not be attached to the outwardly facing vertex of the adsorbed phage and thus the phage could not cross-link two cells. When the virions were labeled with (3)H-leucine, purified, and adsorbed to Escherichia coli cells, about 15% of the radioactivity was recovered as low-molecular-weight material from spheroplasts formed by lysozyme-ethylenediaminetetraacetic acid. Other experiments revealed that about 7% of the total parental virus protein label could be recovered in newly formed progeny virus.     

Effect of Rifampicin and Chloramphenicol on Progeny Single-stranded DNA Synthesis of Bacteriophage ϕX174

Neither bacteriophage ?X174 single-stranded DNA synthesis nor phage growth was affected by rifampicin (200 μg/ml) once it started, whereas a low concentration of chloramphenicol (30 μg/ml) inhibited the phage growth when added in a late phase of infection. When rifampicin was added at a stage where double-stranded duplex (RF) DNA replication proceeded preferentially in the presence of chloramphenicol, or even after chloramphenicol was removed before the addition of rifampicin, both single-stranded DNA synthesis and phage growth were inhibited. These results suggest that RNA synthesis sensitive to rifampicin was necessary to initiate single-stranded DNA synthesis, but no longer needed once ?X174 DNA synthesis started.     

The Effect of Colicin E2 on Bacteriophage ϕX 174-Infected Cells of Escherichia coli

The structure of SF-1836 substance, which is produced by Streptomyces zaomyceticus SF-1836 and has an antimicrobial activity against Xanthomonas species, was determined to be 2-azabicyclo[2.1.0]pentane-3-(S)-carboxilic acid by chemical and spectroscopic studies.     

DNA Products in In Vitro DNA Synthesis Using Bacteriophage ϕX174 Single-stranded DNA

We described product analysis of DNA synthesized in chloroplast lysate from liverwort Marchantia polymorpha L. cell suspension cultures. Characteristics of in vitro DNA synthesis by chloroplast lysate using bacteriophage ?X174 single-stranded DNA were very similar to those in the case of double-stranded calf thymus DNA reported previously. Autoradiographic analysis clearly showed the incorporation of radioactive [α-³²P]-dCTP into DNA molecules associated with bacteriophage ?X174 single-stranded template DNA, indicating conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III, double-stranded linear molecule). Experiments on the fate of [³²P]-labeled single-stranded DNA also showed a clear conversion of the single-stranded DNA to double-stranded DNA. Furthermore, patterns of sucrose density gradient centrifugations (neutral and alkaline) showed the production of two major components in in vitro DNA synthesis by chloroplast lysate. This also indicated conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III form). Our results suggest that the mechanism of chloroplast DNA replication could be the mode of strand-displacement DNA synthesis as seen in animal mitochondrial DNA synthesis.     

Reactivation of Photoinactivated Single-Stranded DNA Bacteriophage φX174 by UV-Irradiated Escherichia coli Cells

Reactivation of single-stranded DNA phage, photodynamically inactivated in the presence of proflavine sulfate, by three isogenic Escherichia coli strains having different DNA repair capabilities has been studied. It was found that reactivation of photoinactivated phiX174 was possible only if the host cells were recombination proficient (recA(+)) and had been lightly irradiated with UV light prior to infection; the presence of the uvrA(+) gene was not essential. Only a small part of the proflavine-mediated photodynamic damage in phiX174 could be repaired in this fashion. Burst sizes of reactivated phages were, however, comparable to those of normal unirradiated phages.     

Involvement of a Cell Envelope Component of Escherichia coli in the Early Stages of Infection with Bacteriophage ϕX174

Envelope fraction I prepared from a ?X174 sensitive host, KD4301, showed a strong eclipsing activity, while the lipopolysaccharide (LPS) fraction showed a weak activity. The eclipsing activity in envelope fraction I was sensitive to heat treatment, while that in the LPS fraction was insensitive. When the complete phage particles (114S) were treated with envelope fraction I, the eclipsed particles (70S) and a rapidly sedimenting component were obtained, but when they were treated with LPS, only 70S eclipsed particles were obtained. Electron microscopic observation showed that there were two types of eclipsed particles formed on treatment with fraction I; in one of them phage DNA was extruded from the phage particles as a thick bundle, and in the other more than 95% of the phage DNA was extruded from the phage particles. The rapidly sedimenting component was the membrane-eclipsed particle complex. LPS gave only one type of eclipsed particles in which DNA was extruded as a thick bundle. These results indicate that a heat labile component in the cell envelopes other than LPS is involved in the extrusion of ?X174 DNA.     

Genetic Recombination in Bacteriophage φX174

Genetic recombination in bacteriophage X174 usually takes place early in the infection process and involves two parental replicative form (double-stranded) DNA molecules. The host recA protein is required; none of the nine known X174 cistron products is essential. The products of a single recombination event are nonreciprocal and asymmetric. Typically, only one of the parental genotypes and one recombinant genotype are recovered from a single cell. An alternative, less efficient recombination mechanism which requires an active X174 cistron A protein is observed in the absence of the host recA gene product.     

Requirement of Protein Synthesis for Bacteriophage φX174 Superinfection Exclusion

Within 5 to 10 min at 37 C, bacteria infected by bacteriophage phiX174 acquire the ability to exclude superinfecting phiX particles from reproducing themselves. The superinfecting phage are blocked at a stage prior to synthesis of the parental replicative form molecule; the superinfecting deoxyribonucleic acid remains as intact (infective) single strands. Establishment of superinfection exclusion and its maintenance require protein synthesis.     

Effect of Rifampicin and Uracil Depletion on Bacteriophage ϕX174 DNA Synthesis in an E. coli dnaHts Mutant

The fluorescent antibody technique was used to trace an inoculated Nocardia erythropolis strain which was capable of rapidly degrading phthalate esters in soil column and activated sludge systems. The reaction of antibody to Nocardia erythropolis S-1 was highly strain specific, i. e., only one of twelve other strain of N. erythropolis was stained with this fluorescent antibody. All other species of Nocardia and other genera of bacteria and a strain of Candida were not stained. Using this technique it was demonstrated that N. erythropolis S-1 inoculated into activated sludge and soil column systems was successfully distinguished from many other microorganisms in mixed culture systems, and the distribution of this strain was appreciated.     

Specific Endonuclease R Fragments of Bacteriophage φX174 Deoxyribonucleic Acid

The restriction enzyme from Hemophilus influenzae, endonuclease R, cleaves phiX174 replicative-form deoxyribonucleic acid (DNA) into at least 13 specific limit fragments. The molecular weights of 12 of the fragments have been estimated by gel electrophoresis and electron microscopy. Using the genetic assay for small fragments of phiX DNA, we have shown that we can salvage markers from the endonuclease R phiX-RF fragments.     

Second-Site Suppressors of a Cold-Sensitive Prohead Accessory Protein of Bacteriophage φX174

This study describes the isolation of second-site suppressors which correct for the defects associated with cold-sensitive (cs) prohead accessory proteins of bacteriophage phi X174. Five phenotypically different suppressors were isolated. Three of these suppressors confer novel temperature-sensitive (ts) phenotypes. They were unable to complement a ts mutation in gene F which encodes the major coat protein of the phage. All five suppressor mutations confer nucleotide changes in the gene F DNA sequence. These changes define four amino acid sites in the gene F protein. Three suppressor mutations placed into an otherwise wild-type background display a cold resistant phenotype in liquid culture infections when compared to a wild-type phi X174 control.     

Genetic Assay for Small Fragments of Bacteriophage φX174 Deoxyribonucleic Acid

The double-stranded replicative form deoxyribonucleic acid (RF-DNA) of bacteriophage phiX174 was fragmented by pancreatic deoxyribonuclease, and the complementary strand fragments were then annealed to intact viral single strands. When such complexes infected Escherichia coli spheroplasts, some of the progeny virus bore genetic markers derived from the RF-DNA fragments. In this way, genetic markers have been salvaged from DNA fragments less than 50 nucleotides in length. This method is potentially useful as a specific assay to aid in the purification of genetically defined DNA fragments and also as a mechanism for the incorporation of small chemically synthesized DNA sequences into viral genomes.