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.     

The effect of biocides on proteins of Pseudomonas aeruginosa PAO bacteriophage F116

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the effect of several biocides on the protein content of Pseudomonas aeruginosa PAO phage F116. Eight major protein bands were identified and used as controls. The test biocides altered the control band pattern in different ways. Similarities were noticed between closely related biocides. Moreover, the alteration in the protein band pattern did not always correlate with phage inactivation.     

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.     

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.     

Studies on the Pseudomonas aeruginosa PAO1 bacteriophage receptors

Receptor for phage PIK specific for Pseudomonas aeruginosa strain PAO1 was studied. Phage PIK was strongly inactivated by lipopolysaccharide (LPS) in vitro, exhibiting a PhI50 of 4.8 micrograms/ml. Further it was noted that this inactivation by LPS was reduced to 50% by several mono- and disaccharides when tested in vitro. D-glucosamine, D-mannose and L-rhamnose were found to be most effective at the concentration of 0.045 M, 0.25 M and 0.35 M respectively. This suggests the possibility that phage PIK receptor in LPS contains D-mannose, L-rhamnose and D-glucosamine. Either one of the former two could be located at a terminal position alpha-linked to the adjacent residue or located internally in the polysaccharide chain linked through its C-4 position. A theoretical approach to the interpretation of phage cell interaction was also investigated.     

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.     

Basic characterization of a Pseudomonas aeruginosa PAO1 bacteriophage

A bacteriophage of Pseudomonas aeruginosa PAO1 was characterized. Bacteriophage PIK was found to adsorb on the cell wall of the host organism. Electron microscopy of the phage PIK revealed that it had a bipyramidal hexagonal prismatic head of 110 nm in diameter, a tail which was 158 nm long and a tail plate of 47 nm width. This paper describes its basic characters, and a quantitative study was made of its adsorption to exponential phase cells of two different strains of P. aeruginosa. PIK was found to contain double stranded DNA and it appears to be virulent towards its host, P. aeruginosa PAO1. It was classified into the group of phages possessing a contractile tail.     

Influence of pH value on viability and transduction frequency of Pseudomonas aeruginosa phage F116

The effect of pH value on viability and transduction frequency of the Pseudomonas aeruginosa phage F116 was studied. Plaques were formed at pH 5·0–10·0 and transduction occurred at pH 5·0–8·0. Outside the range pH 4·0–11·0 phages rapidly lost viability, but retained the capacity to transduce.     

The nature of Pseudomonas aeruginosa strain PAO bacteriophage receptors.

Receptors for phages specific to Pseudomonas aeruginosa strain PAO were studied. Phages 16, 44, 109, F8, and PBI are lipopolysaccharide (LPS) specific as shown by neutralization tests. The PhI50's of the LPS, adsorption rate constants with strain PAO and the plaque morphologies of these five phages were quite similar. Phages 1214 and 7 also appear to be LPS-specific on the basis of host-range studies. Phage 73 is pilus-specific, while phages 21 and 68 fall into a group which does not attach to pili, flagella, or LPS. A theoretical approach to the interpretation of phage-cell interactions is presented.     

Frequency of F116-mediated transduction of Pseudomonas aeruginosa in a freshwater environment.

Transduction of Pseudomonas aeruginosa streptomycin resistance by a generalized transducing phage, F116, was shown to occur during a 10-day incubation in a flow-through environmental test chamber suspended in a freshwater reservoir. Mean F116 transduction frequencies ranged from 1.4 X 10(-5) to 8.3 X 10(-2) transductants per recipient during the in situ incubation. These transduction frequencies were comparable to transduction frequencies determined in preliminary laboratory transduction experiments. The results demonstrate the potential for naturally occurring transduction in aquatic environments and concurrent environmental and ecological ramifications.     

O-antigen conversion in Pseudomonas aeruginosa PAO1 by bacteriophage D3.

The lysogenization of Pseudomonas aeruginosa PAO by phage D3 results in derivatives which are resistant to superinfection by phage D3c by virtue of the fact that homologous phage cannot adsorb to these cells. The serologically and morphologically unrelated phage E79 showed a markedly decreased adsorption rate to the lysogen PAO(D3). Since both of these phages are lipopolysaccharide specific, these results suggested lysogenic conversion of the phage receptor. The lipopolysaccharide was extracted from strain PAO by the hot phenol-water technique, but this procedure was ineffective with PAO(D3). We developed a technique involving cold trichloroacetic acid extraction, followed by ultracentrifugation, digestion of the high-speed pellet with proteinase K, and ultimate purification on CsCl step gradients. The lipopolysaccharide from the wild type had inactivating activity against D3 and E79, whereas that from PAO(D3) inactivated neither. Chromatographic analysis indicated that the convertant lipopolysaccharide was smooth, and quantitative chemical analyses of the two preparations showed no differences in the level of the major fatty acids, amino compounds, or neutral sugars. On the other hand, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the side chains had a decreased migration rate through the gel matrix. The application of 1H and 13C nuclear magnetic resonance spectroscopic analysis revealed that the PAO side chain is chemically identical to that of serotype O:2a,d, containing 2,3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, and 2-acetamido-2,6-dideoxy-D-galactose (D-fucosamine). The molecular basis of the conversion event was (i) the introduction of an acetyl group into position 4 of the fucosamine residue and a change in the bonding between trisaccharide repeating units from alpha 1 leads to 4 to beta 1 leads to 4.     

Transformation of Pseudomonas aeruginosa strain PAO with bacteriophage and plasmid DNA.

A procedure has been developed which allows transformation of P. aeruginosa strain PAO with plasmid and bacteriophage DNA at a frequency of 10(-6) per recipient cell. The method is similar in outline to that developed for Escherichia coli. It involves growing the recipient cells to 3-5 x 10(8) per ml in nutrient broth, washing the cells with 0.1 M MgCl2, resuspending in 0.175 M CaCl2 for 20 min, exposing to DNA for 1 h and then heat pulsing at 42 degrees C for 1 min. Some plasmid markers are expressed immediately, whereas others require time for phenotypic expression.     

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.     

Transducing phages of Pseudomonas aeruginosa related to phage F116L

New phages K104 and B26, which are relative to F116L by a number of biological characters, appeared to show general transducing activity. Phage K104 transduces all tested markers with higher frequency than the phage B26. Linkage of the bacterial markers pair ilv202--met28 durspectively. When recipient bacteria lysogenic for phages K104 and B26 are used, frequencies of transduction by phage F116L are decreased. In the presence of F116L prophage the frequency of transduction by phage B26 is 10-fold increased. Phages B26 and F116L do not grow on bacteria lysogenic for these phages. Phage F116L does not grow on the lawn of bacteria, lysogenic for phage K104, while phage B26 grows on the same lawn with the efficiency of plating about 10(-2).     

Alginate synthesis by mucoid strains of Pseudomonas aeruginosa PAO

Summary Alginic acid production by Pseudomonas aeruginosa PAO strains was studied in yeast extract/2% (w/v) gluconate medium. In all of the five strains studied, synthesis of the alginic acid was shown to occur in the stationary phase of growth. Each strain produced similar amounts of alginic acid at both 30° C and 37° C. However the amount of alginic acid varied from 7.5–11.5 gl^–1 depending upon the strain. The alginic acid was isolated, purified and its chemical composition determined. All strains produced a polysaccharide rich in polymannuronic acid which contained only polymannuronic acid blocks, polyguluronic acid blocks appeared to be absent. The amount of O-acetylation varied considerably from 2.3–14.7%. Analysis of the chain length distribution by poly-acrylamide gel electropheresis indicated that a homogeneous size of polymer was synthesised when compared to a high viscosity algal sample.     

Size of the Chromosome of Pseudomonas aeruginosa PAO

Electron microscope examination and velocity sedimentation analysis of the deoxyribonucleic acid released from Pseudomonas aeruginosa spheroplasts indicate that this organism carries the bulk of its genetic determinants in a single duplex deoxyribonucleic acid molecule having a molecular mass of 2.1 x 10(9) daltons.     

Identification of the cell wall receptor for bacteriophage E79 in Pseudomonas aeruginosa strain PAO.

Bacteriophage E79 was shown to interact with the lipopolysaccharide (LPS) of Pseudomonas aeruginosa strain PAO. LPS isolated from an E79-sensitive, smooth strain inactivated the phage, exhibiting a Phl50 value (concentration of LPS that caused a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) of 0.04 microgram/ml, whereas the LPS isolated from a rough mutant derived from the wild type showed no neutralizing activity towards E79. EDTA and sodium deoxycholate were demonstrated to abolish the neutralizing capacity of the smooth LPS. One E79 receptor site was shown to be equivalent to 10(-16) g of LPS.     

The chromosome map of Pseudomonas aeruginosa PAO

A revised chromosomal map of Pseudomonas aeruginosa is presented and the role of a variety of mapping procedures is discussed.     

Phenotypic mixing of pyocin R2 and bacteriophage PS17 in Pseudomonas aeruginosa PAO.

Previous results indicate that a group of bacteriocins in Pseudomonas aeruginosa, named R-type pyocins, have a structure resembling bacteriophage tails and share some serological homology with certain bacteriophages. This paper presents genetic evidence which strongly suggests that components of pyocin R2, an R-type pyocin of P. aeruginosa PAO, and tail components of bacteriophage PS17 are interchangeable. Complementation tests with pyocin R2-deficient mutants of PAO and ts mutants of PS17 revealed that various phenotypic interactions occur between the pyocin and bacteriophage in PAO cells lysogenized or infected with PS17. (i) Certain pyocin R2-deficient mutations were phenotypically suppressed in cells carrying PS17 prophage. (ii) A temperature-sensitive mutant of PS17, tsQ31, was phenotypically suppressed in PAO cells treated with mitomycin C. (iii) Phenotypically mixed phages with receptor and serological specificities of pyocin R2 were formed in PS17 lysogens of certain pyocin R2-deficient mutants.     

Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning

In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (egl-9) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.