Pseudomonas aeruginosa bacteriophage phi PLS27-lipopolysaccharide interactions

We investigated the phi PLS27 receptor in Pseudomonas aeruginosa strain PAO lipopolysaccharide (LPS) by analyzing a resistant mutant. This mutant, which was designated AK1282, had the most defective LPS yet reported for a P. aeruginosa rough mutant; this LPS contained only lipid A, 2-keto-3-deoxyoctonate, heptose, and alanine as major components. In addition, this LPS lacked galactosamine, which is present in the inner core of the LPS of other rough mutants. The loss of galactosamine but only a small decrease in the alanine content indicated that the core of strain PAO LPS differed from the core structure which has been suggested for the LPS of other well-characterized P. aeruginosa strains. Our analysis also indicated that galactosamine residues may be crucial for phi PLS27 receptor activity of the LPS. Electrodialysis of LPS and conversion to salt forms (sodium or triethylamine) influenced the phage-inactivating capacity of the LPS, as did the medium in which the inactivation occurred; experiments performed in 1/10-strength broth resulted in much lower PhI50 (concentration of LPS causing a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) values than experiments performed in regular-strength broth. Sonication of the LPS also increased the phage-inactivating capacities of the LPS preparations.     

Localization of canonical single-strand breaks in DNA of the Pseudomonas aeruginosa bacteriophage phi kF77

Bacteriophages phi k of P. aeruginosa were characterized by the presence of T4 DNA-ligase-repaired, single-chain breaks in their genome. A restriction map was constructed for one of these phages (phi kF77) with restriction endonucleases SalI, HindIII, EcoRI, MluI, XbaI and ClaI. phi kF77 DNA was resistant to the cleavage by BamHI, BglII, HpaI, PstI, PvuII and XhoI endonucleases. Single-chain breaks were mapped by means of electron microscopy of partially denatured DNA molecules, electrophoretic studies of denatured DNA and S1-analysis. Four major nicks were thus located which were revealed in 33 to 83% of DNA molecules. On the basis of mutual hybridization of single-strand DNA fragments it was shown that all nicks are located in one of the phi kF77 DNA chains. S1-treated hybrids of 32P-labeled single-strand fragments with intact DNA chain were used for DNA orientation. The physical map of phi kF77 DNA was constructed.     

Nucleotide sequence at the site of single-strand DNA breaks in Pseudomonas aeruginosa bacteriophage phi kF77

It is known that DNA molecules from the phage group phi k specific to Pseudomonas aeruginosa possess single-strand breaks (nicks). The sequences around the nicks in the bacteriophage phi kF77 DNA have been determined by various methods. In addition, an EcoRV-HindIII fragment, containing a nick, was cloned into the plasmid pUC9 and sequenced by Maxam-Gilbert technique. The sequence common for all nicks was CCTAohpCTCCGG.     

Characterization of the origins of replication of bacteriophage phi 29 DNA.

The origins of replication of phi 29 DNA have been studied by analyzing the activity as templates in the phi 29 in vitro replication system of E. coli recombinant plasmids and M13 derivatives containing phi 29 DNA terminal sequences. Plasmid pITR, containing the 6 bp long inverted terminal repeat of phi 29 DNA, was shown to be essentially inactive. The analysis of a series of deletion derivatives of plasmid pID13, that contains the 73 and 269 bp from the left and right phi 29 DNA ends, respectively, indicated that the minimal origins of replication are comprised within the mutagenesis at these sequences was carried out. Changes of the second or third A into a C completely abolished the template activity. In the case of changes at position from 4 to 12, only 3 out of 14 mutations reduced the template activity; these 3 mutations were double changes and 2 of them affected the inverted terminal repeat. The results suggest that the sequence requirement at the end-proximal region of the origin of replication is more strict than that at the distal region.     

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.     

Electron microscope study of the intracellular development of the Pseudomonas aeruginosa bacteriophage phi KZ

The study of the ultrathin sections of cells infected with virulent phage phi KZ has confirmed the presence of a specific cylindrical formation, an inner body, in the head of this phage and revealed the spiral structure of this inner body. The formation of DNA condensates whose structure resembles a spring wound around the core (the inner body) has been shown to occur in the cells in the process of the ultracellular development of phage phi KZ. This development leads to characteristic changes in the cellular structure, and in particular in the cell walls and the nucleoid.     

Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule.

The structure of DNA from the temperate Bacillus subtilis phage phi105 was examined by using the restriction endonuclease EcoRI and by sedimentation analysis. The DNA contains six EcoRI cleavage sites. Although eight DNA fragments were identified in the EcoRI digests, the largest of these was shown to consist of the two fragments that carry the cohesive ends of the phage DNA. In neutral gradients, the majority of whole phi105 DNA sedimented as nicked circles and the remainder as oligomers. No unit-length linear structures were detected. The associated cohesive ends could be sealed by DNA ligase from Escherichia coli and could be cleaved by S1 nuclease. On the basis of these results and previously reported studies, it appears that, as isolated from phage particles, phi105 DNA is a circular molecule that is formed from the linear structure by the association of complementary single-stranded DNA.     

Nucleotide sequence analysis of DNA. X. Complete nucleotide sequence of the cohesive ends of bacteriophage phi80 DNA

The base sequence of the cohesive ends of bacteriophage φ80 DNA has been shown to be identical to the base sequence of the cohesive ends of bacteriophage lambda DNA.     

Characterization of the small RNA of the bacteriophage phi 29 DNA packaging machine.

The prohead connector of the bacteriophage luminal diameter 29 DNA packaging machine was reconstructed with the small RNA that regulates DNA packaging in vitro. The complete sequence of the 120 nucleotide RNA proved its origination from the promoter PE1(A1) of the left early region of phi 29 DNA, the end packaged first during assembly. The prohead RNA was clearly distinct from eubacterial 5S rRNA in sequence and composition.     

Physical map of Pseudomonas aeruginosa phage phi kF77 genome. Localization of sites sensitive to restriction endonucleases

A physical map of the P. aeruginosa bacteriophage phi kF77 has been constructed using the restriction endonucleases SalI, HindIII, EcoRI, EcoRV, MuI, XbaI, ClaI. The phi kF77 DNA is resistant to cleavage by the restriction endonucleases BamHI, BglII, HpaI, PstI, PvuII, SmaI, XhoI.     

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.     

Effects of genome size on bacteriophage phi X174 DNA packaging in vitro

Effects of the size of template DNA on the DNA packaging reaction of bacteriophage phi X174 were studied using plasmids of various sizes which contain the phi X174 origin of DNA replication and the in vitro phage synthesizing system (Aoyama, A., Hamatake, R. K., and Hayashi, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4195-4199). DNA between 78.5% and 101% of the length of phi X174 DNA produced infectious particles efficiently. Packaging of DNA smaller or larger than this range produced uninfectious defective particles. Although these particles contained circular single-stranded DNA, they suffered structural changes which altered the sedimentation properties or the ability to adsorb to the cells. Mutant phage were found from the packaging reaction of DNA larger than 101% of phi X174 DNA. These mutants deleted the termination region of DNA, suggesting that they were produced by early termination of the phage synthesizing reaction.     

New temperate Pseudomonas aeruginosa phage, phi297: Specific features of genome structure

The genome structure and some specific features of temperate Pseudomonas aeruginosa phage phi297 are considered. Analysis of sequencing data and genome annotation suggest that the phi297 genome displays a mosaic structure, which has formed through combining gene blocks from bacteria of taxonomically remote groups and/or their phages. The results of a comparison of the phi297 DNA homology level and pattern with the genome sequences of the currently known related P. aeruginosa bacteriophages are interpreted from the perspective of assumed active migration of these phages between different bacterial species.     

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.     

Functional domains in the bacteriophage phi 29 terminal protein for interaction with the phi 29 DNA polymerase and with DNA.

Deletion mutants at the amino- and carboxyl-ends of the phi 29 terminal protein, as well as internal deletion and substitution mutants, whose ability to prime the initiation of phi 29 DNA replication was affected to different extent, have been assayed for their capacity to interact with DNA or with the phi 29 DNA polymerase. One DNA binding domain at the amino end of the terminal protein has been mapped. Two regions involved in the binding to the DNA polymerase, an internal region near the amino-terminus and a carboxyl-terminal one, have been also identified. Interaction with both DNA and phi 29 DNA polymerase are required to led to the formation of terminal protein-dAMP initiation complex to start phi 29 DNA replication.