Pf16 and phiPMW: Expanding the realm of Pseudomonas putida bacteriophages.

We present the analysis of two novel Pseudomonas putida phages, pf16 and phiPMW. Pf16 represents a peripherally related T4-like phage, and is the first of its kind infecting a Pseudomonad, with evidence suggesting cyanophage origins. Extensive divergence has resulted in pf16 occupying a newly defined clade designated as the pf16-related phages, lying at the interface of the Schizo T-Evens and Exo T-Evens. Recombination with an ancestor of the P. putida phage AF is likely responsible for the tropism of this phage. phiPMW represents a completely novel Pseudomonas phage with a genome containing substantial genetic novelty through its many hypothetical proteins. Evidence suggests that this phage has been extensively shaped through gene transfer events and vertical evolution. Phylogenetics shows that this phage has an evolutionary history involving FelixO1-related viruses but is in itself highly distinct from this group.

Complete nucleotide sequence of phiCHU: a Luz24likevirus infecting Pseudomonas aeruginosa and displaying a unique host range.

A complete nucleotide sequence of the new Pseudomonas aeruginosa Luz24likevirus phiCHU was obtained. This virus was shown to have a unique host range whereby it grew poorly on the standard laboratory strain PAO1, but infected 26 of 46 clinical isolates screened, and strains harbouring IncP2 plasmid pMG53. It was demonstrated that phiCHU has single-strand interruptions in its genome. Analysis of the phiCHU genome also suggested that recombination event(s) participated in the evolution of the leftmost portion of the genome, presumably encoding early genes.

Genomic analysis of Pseudomonas putida phage tf with localized single-strand DNA interruptions.

The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure--a blunt right end and a 4-nucleotide 3'-protruding left end--was observed. Secondly, 14 single-chain interruptions (nicks) were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5'-TACT/RTGMC-3'. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages.

Complete genome sequence of the giant virus OBP and comparative genome analysis of the diverse ΦKZ-related phages.

The 283,757-bp double-stranded DNA genome of Pseudomonas fluorescens phage OBP shares a general genomic organization with Pseudomonas aeruginosa phage EL. Comparison of this genomic organization, assembled in syntenic genomic blocks interspersed with hyperplastic regions of the ΦKZ-related phages, supports the proposed division in the "EL-like viruses," and the "phiKZ-like viruses" within a larger subfamily. Identification of putative early transcription promoters scattered throughout the hyperplastic regions explains several features of the ΦKZ-related genome organization (existence of genomic islands) and evolution (multi-inversion in hyperplastic regions). When hidden Markov modeling was used, typical conserved core genes could be identified, including the portal protein, the injection needle, and two polypeptides with respective similarity to the 3'-5' exonuclease domain and the polymerase domain of the T4 DNA polymerase. While the N-terminal domains of the tail fiber module and peptidoglycan-degrading proteins are conserved, the observation of C-terminal catalytic domains typical for the different genera supports the further subdivision of the ΦKZ-related phages.

The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties.

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a 'T7-like virus' with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10(4) and 10(6) pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.