Isolation and characterization of a bacteriophage T5 mutant deficient in deoxynucleoside 5''-monophosphatase activity.

A bacteriophage T5 mutant has been isolated that is completely deficient in the induction of deoxynucleoside 5'-monophosphatase activity during infection of Escherichia coli F. The mutant bacteriophage has been shown to be deficient in the excretion of the final products of DNA degradation during infection of E. coli F, and about 30% of the host DNA's thymine residues were reinocorporated into phage DNA. During infection with this mutant, host DNA degradation to trichloroacetic acid-soluble products was normal, host DNA synthesis was shut off normally, and second-step transfer was not delayed. However, induction of early phage enzymes and production of DNA and phage were delayed by 5 to 15 min but eventually reached normal levels. The mutant's phenotype strongly suggests that the enzyme's role is to act at the final stage in the T5-induced system of host DNA degradation by hydrolyzing deoxynucleoside 5'-monophosphates to deoxynucleosides and free phosphate; failure to do this may delay expression of the second-step-transfer DNA.     

Effect of biocides on Pseudomonas aeruginosa phage F116

Pseudomonas aeruginosa phage F116 is of interest in understanding the virucidal mechanisms of disinfectant action. Phage F116 has been used to test several disinfectants. The bacteriophage was relatively resistant to several biocides commonly used in disinfection processes. Only 0.05% cetylpyridinium chloride, 0.1% peracetic acid and 2% phenol were highly active against the phage.     

Bacteriophage influence Campylobacter jejuni types populating broiler chickens

The characteristics that allow one Campylobacter jejuni genotype to succeed over another under the influence of bacteriophage predation have been examined in experimental broiler chickens following the observation that this succession appeared to occur in naturally colonized broiler chicken flocks. Examination of three C. jejuni strains from a single flock indicated that horizontal transfer of at least 112 kb of genomic DNA from strain F2C10 (bacteriophage sensitive) to strain F2E1 (bacteriophage insensitive) had created strain F2E3. Transfer of this DNA was associated with acquisition of sensitivity to 6 of 25 lytic bacteriophage isolated from the same flock. All strains tested were capable of colonizing broiler chickens but cocolonization revealed that the bacteriophage sensitive strains F2E3 and F2C10 had a competitive advantage over the bacteriophage insensitive strain F2E1. With the addition of lytic bacteriophage the situation was completely reversed, with F2E1 dominating. The inability to replicate bacteriophage is associated with a significant fitness cost that renders the insensitive strain competitive only in the presence of bacteriophage. We demonstrate that interstrain recombination in vivo can generate genome diversity in C. jejuni and that bacteriophage predation is a strong selective pressure that influences the relative success of emergent strains in broiler chickens.     

Effects of biocides on the transduction of Pseudomonas aeruginosa PAO by F116 bacteriophage

The transduction of Pseudomonas aeruginosa PAO by the F116 bacteriophage was monitored after phage treatment with seven biocides commonly used in disinfection processes. Ethanol (70% and 100%), isopropanol (100%) and phenol (2%) did not affect the ability of the phage to transduce. However, chlorhexidine diacetate (1%), cetylpyridinium chloride (0·05%), glutaraldehyde (2%), peracetic acid (1%) and sodium hypochlorite (0·1%) significantly inhibited F116 transduction. The transduction process was more sensitive to biocides than the phage itself. Inactivation of F116 and inhibition of the transduction process appeared to involve two distinct mechanisms implying that the process depends on cellular target sites not affected by some biocides.     

Plasmid pR386 renders Escherichia coli cells restrictive to the growth of bacteriophage T4 unf mutants.

The introduction of the F1 incompatibility group plasmid pR386 Tc into several common laboratory strains of Escherichia coli rendered them restrictive to the growth of bacteriophage T4 unf mutants, which are defective in unfolding the host genome. The growth inhibition was temperature dependent. The single mutant unf39 x 5 exhibited an efficiency of plating of less than 10(-8) at 27 degrees C. However, at 37 degrees C, complete growth inhibition occurred only when host DNA degradation was also absent.     

Energy dispersive analysis of X-rays study of the distribution of chlorhexidine diacetate and cetylpyridinium chloride on the Pseudomonas aeruginosa bacteriophage F116

Using an energy dispersive analyser of X-rays fitted to a scanning electron microscope, chlorhexidine was shown not to bind onto F116 bacteriophage, unlike cetylpyridinium chloride, which possibly penetrated the phage. This could explain the difference in viricidal activity between the two compounds.     

Evidence for phage-mediated gene transfer among Pseudomonas aeruginosa strains on the phylloplane.

As the use of genetically engineered microorganisms for agricultural tasks becomes more frequent, the ability of bacteria to exchange genetic material in the agricultural setting must be assessed. Transduction (bacterial virus-mediated horizontal gene transfer) is a potentially important mechanism of gene transfer in natural environments. This study investigated the potential of plant leaves to act as surfaces on which transduction can take place among microorganisms. Pseudomonas aeruginosa and its generalized transducing bacteriophage F116 were used as a model system. The application of P. aeruginosa lysogens of F116 to plant leaves resulted in genetic exchange among donor and recipient organisms resident on the same plant. Transduction was also observed when these bacterial strains were inoculated onto adjacent plants and contact was made possible through high-density planting.     

Development of T7 phage and T7 phage containing apurinic sites in an exonuclease III, endonuclease IV double mutant of Escherichia coli.

The development of bacteriophage T7 was examined in an Escherichia coli double mutant defective for the two major apurinic, apyrimidinic endonucleases (exonuclease III and endonuclease IV, xth nfo). In cells infected with phages containing apurinic sites, the defect in repair enzymes led to a decrease of phage survival and a total absence of bacterial DNA degradation and of phage DNA synthesis. These results directly demonstrate the toxic action of apurinic sites on bacteriophage T7 at the intracellular level and its alleviation by DNA repair. In addition, untreated T7 phage unexpectedly displayed reduced plating efficiency and decreased DNA synthesis in the xth nfo double mutant.     

Mapping of a gene controlling the production of phospholipase C and alkaline phosphatase in Pseudomonas aeruginosa

Summary Using plasmid R68.45 mediated conjugation and transduction with bacteriophage F116L, a genetic locus controlling at least two orthophosphate repressible proteins in Pseudomonas aeruginosa has been mapped at about 22–23 min on the PAO chromosome.     

Effect of Myxin on Deoxyribonucleic Acid Synthesis in Escherichia coli Infected with T4 Bacteriophage

Exposure of Escherichia coli cells to myxin results in the almost complete inhibition of new deoxyribonucleic acid (DNA) synthesis, extensive degradation of pre-existing intracellular DNA, and a rapid loss of viability in these cells (9). After exposure to myxin for 30 min (<1% survivors and >25% degradation of DNA), infection of these cells by T4 bacteriophage results in the renewal of DNA synthesis at a rate essentially equal to that found in T4-infected cells in the absence of myxin. This DNA was characterized as T4 DNA by hybridization and by hydroxyapatite chromatography. These results suggest that the primary site of action of myxin does not involve the biochemical pathways involved in either the energy metabolism or the biosynthesis of DNA precursors in the uninfected host cell. The yield of infectious T4 particles was reduced when myxin was present during multiplication. This effect may be partly accounted for by the finding that a significant fraction of the T4 DNA synthesized in the presence of myxin is apparently not properly enclosed by the bacteriophage protein coat since it is shown to be degraded by exogenous nuclease.     

Inactivation of bacteriophage PM2 during storage

The kinetics of bacteriophage inactivation in the medium that is optimal for its storage has been studied at temperatures from 4 to 55 degrees C. The plot of Arrhenius dependence of the constant of inactivation rate consists of the two linear parts with the energies of activation Ea = 25 kcal/mol for 4-37 degrees C and Ea = 91 kcal/mol for 37-55 degrees C. The DNA of inactivated bacteriophage remained mostly in superspiralized form and completely preserved its biological activity as tested by transfection in spheroplasts. The analysis of inactivation kinetics suggests ageing of virions cultivated at 4 degrees C. The addition of watersoluble antioxidant amoxipin did not change the inactivation kinetics. The addition of antioxidant ionol with twin-80 increased the inactivation that was paralleled by the bacteriophage DNA degradation.     

Damage to Pseudomonas aeruginosa PAO1 bacteriophage F116 DNA by biocides

The mechanism of action of biocides against viruses has not been widely studied, although two main targets are viral proteins (capsids, enzymes) and the viral genome. This study was undertaken in order to investigate the efficacy of several disinfectants against the nucleic acid of the Pseudomonas aeruginosa PAO bacteriophage F116. Of all the biocides tested, only peracetic acid affected significantly the phage genome. However, it is not clear whether the nucleic acid was damaged inside the phage capsid or when released into the surrounding medium.     

Role of F Pili in the Penetration of Bacteriophage fl

Early stages of infection of Escherichia coli with the filamentous bacteriophage f1 were examined in the electron microscope. Purified phage-bacteria complexes were prepared at various time intervals after the initiation of synchronous infection. Cells were scored for the total number of F pili, the number of F pili with f1 attached, the number of intact phage particles which occurred at the surface of the cell, and F pilus length. Electron microscope autoradiographs were also prepared at each time interval. The results showed that the average number of F pili with f1 attached decreased with time as phage deoxyribonucleic acid (DNA) entered the cell. Concomitant with this loss, the remaining F pili became shorter. The rate of entry of phage DNA into the cell followed, with a short lag, the rate of loss of F pili with f1 attached. During the lag period, intact phage particles accumulated at the surface of the cell. The results from radioautographs showed that no phage DNA could be located within the F pilus. These results suggest that F pili are resorbed by the cell during infection with the bacteriophage f1. Parallel experiments with noninfected cultures further suggest that pilus resorption may be a normal cellular phenomenon.     

Infection by choleraphage phi 138: bacteriophage DNA and replicative intermediates.

Choleraphage phi 138 contains a linear, double-stranded, circularly permuted DNA molecule of 30 X 10(6) daltons or 45 kilobase pairs. Upon infection, the host DNA is degraded, and synthesis of phage-specific DNA is detectable 20 min after infection. The phage utilizes primarily the host DNA degradation products for its own DNA synthesis. A physical map of phi 138 DNA was constructed with the restriction endonucleases Bg/II, HindIII, and PstI. A concatemeric replicative DNA intermediate equivalent to eight mature genome lengths was identified. The concatemer was shown to be the precursor for the synthesis of mature bacteriophage DNA which is subsequently packaged by a headful mechanism.     

178-Nucleotide sequence surrounding the cos site of bacteriophage lambda DNA.

A nucleotide sequence of 61 nucleotides at the left end and 117 nucleotides at the right end of DNA from bacteriophage lambdacI857Sam7 was determined by the Maxam and Gilbert method. A perfect inverted repeat sequence of 10 nucleotides is near the left end, and one of 15 nucleotides is near the right end. DNA from another closely related lambda strain, lambdacI857prm116Sam7, has about 10% divergence in the sequence of the first 110 nucleotides at the right end and has a 17-member perfect inverted repeat sequence.     

Formation of small circular DNA molecules via an in vitro site-specific recombination system

The Cre-lox site-specific recombination system of bacteriophage P1 has been used to investigate the role of DNA flexibility in recombination. We have determined that a minimal distance of 82 bp must separate two loxP sites located on the same DNA molecule to allow these sites to undergo intramolecular recombination with one another. As a result of recombination, DNA circles as small as 116bp have been produced. In addition, we have demonstrated that the nuclease BAL 31 recognizes distortions in the DNA helix resulting from the formation of small DNA circles whose length is not a multiple of the helical repeat.     

Specialized transduction of Pseudomonas aeruginosa PAO by bacteriophage D3.

D3, a temperate bacteriophage of Pseudomonas aeruginosa PAO, was found to specifically transduce the alleles met-49 and met-117. Induction of established lysogens with UV light was necessary for the production of transducing lysates. Transduced cells were immune to superinfection by phage D3 and could give rise to high-frequency transducing lysates. Cotransduction of these two alleles could not be demonstrated. met-117 was mapped to 26 min on the PAO genetic map. Complementation studies using the generalized transducing phage F116L indicated that met-49 is an allele of met-9011 which maps at 55 min. The integrated D3 prophage was shown to be coinherited with met-117 and with met-49.     

Prophage F116: Evidence for Extrachromosomal Location in Pseudomonas aeruginosa Strain PAO

F116 is a temperate-generalized transducing phage of Pseudomonas aeruginosa. Genetic evidence leads to the conclusion that F116 prophage DNA is maintained extrachromosomally as a plasmid. Preliminary physical evidence is presented to support this hypothesis.     

Interactions of Tn7 and temperate phage F116L of Pseudomonas aeruginosa

Summary Tn7 insertions into the genome of F116L, a Pseudomonas aeruginosa generalized transducing phage, were isolated by repeated cycles of transducing phage, were of strains lysogenic for F116cts mutants with selection for trimethoprim resistance (Tp¹). Two non-defective F116Lcts:Tn7 phage were characterized. They have reduced plaquing ability, produced non-lysogenic Tp^r transductants, and have yielded a deletion mutant of the phage genome upon selection for plaque formation in single infection. F116L DNA is circularly permuted and terminally redundant. A circular restriction map of 61.7 kb has been defined, and a cleavage site common to many enzymes has been identified at coordinate 23.3 kb on the map. It is presumed that this site represents the sequence for the initiation of DNA encapsidation by a headful packaging mode. The Tn7 insertion targets and a 13.4 kb deletion define regions of the F116L genome non-essential for either vegetative growth or lysogenization. The restriction map of Tn7 has been determined for five enzymes. Non-lysogenic Tp^r transuctants reveal a Tn7 insertion hot-spot in the P. aeruginosa genome.