Complete Genome Sequence of Pseudomonas aeruginosa Lytic Bacteriophage PA1Ø Which Resembles Temperate Bacteriophage D3112

Abstract

A novel Pseudomonas aeruginosa lytic bacteriophage (phage), PA1Ø, was isolated, and its genome was sequenced completely. This phage is able to lyse not only P. aeruginosa but also Staphylococcus aureus. Genome analysis of PA1Ø showed that it is similar to a P. aeruginosa temperate phage, D3112, with the exception of the absence of a c repressor-encoding gene, which is known to play a critical role in the maintenance of the lysogenic state of D3112 in P. aeruginosa.

GENOME ANNOUNCEMENT

Lytic bacteriophages (phages) have been aimed at treating infectious bacterial diseases (7). Pseudomonas aeruginosa is one of the major life-threatening opportunistic pathogens that infect cystic fibrosis and immunocompromised patients. This Gram-negative bacterium can colonize various organs, including the lungs, kidneys, skin, and urinary tract, and cause inflammation and sepsis (4, 6, 15). Since P. aeruginosa is resistant to most antimicrobial agents and forms biofilms in organs, it is difficult to eliminate using traditional antimicrobial drugs. Recently, several clinical trials of phage therapy have been performed against P. aeruginosa-induced human and animal infections (8, 10). For therapeutic purposes, the phages should be obligatorily lytic and not harmful to humans or animals.

We report here a novel lytic P. aeruginosa phage, PA1Ø, which was isolated from sewage samples from a livestock farmhouse in South Korea. PA1Ø was amplified and purified by the agar overlay method using P. aeruginosa strain PAO1 as a propagating host. Further tests for determining the host range revealed that PA1Ø showed strong lytic activities against Gram-negative bacteria such as P. aeruginosa and Shigella sonnei and even against Gram-positive bacteria such as Staphylococcus aureus, Streptococcus salivarius, and Listeria monocytogenes (data not shown). This extraordinary ability of PA1Ø led us to determine the complete genome sequence of this phage.

Genomic DNA of PA1Ø was purified using a Qiagen Lambda midikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Purified genomic DNA was fragmented by physical shearing, and fragmented DNAs were blunt end repaired and dephosphorylated for ligation with the pCB31 vector (Macrogen, Seoul, South Korea) to create a shotgun library. Ligated product was introduced into transformation-competent Escherichia coli DH10B cells; recovered plasmids were subjected to sequencing (ABI Prism 3730xl instrument; Applied Biosystems, Foster City, CA) to 8.52-fold coverage of the phage genome. Open reading frames (ORFs) were predicted by Glimmer version 3.0 (3), and genes were annotated by homology searches of the GenBank (1), COG (13), Swiss-Prot (5), and PIR protein databases (9).

The PA1Ø genome was found to be 34,553 bp in length, with 51 putative protein-coding genes with a G+C content of 64.8%. Among the ORFs identified, 40 were annotated as hypothetical proteins; homology searches identified transposase A, DNA transposition protein, host nuclease inhibitor protein, putative integral membrane protein, putative structural protein, portal protein, virion morphogenesis proteins, protease and scaffold proteins, putative major head subunit protein, and putative tail component protein. The PA1Ø genome is highly homologous to the genomes of phages MP29 (GenBank accession number EU272036.1) and 3112 (GenBank accession number AY394005.1), with identities of 93% and 90%, respectively. Both MP29 and D3112 are Pseudomonas phages belonging to the Siphoviridae family, indicating that PA1Ø is one of the Pseudomonas Siphoviridae phages. There have been no reports yet about MP29, but phage D3112 is a well-known lysogenic phage and has been used for molecular-biology studies of P. aeruginosa (2, 14). D3112 infects via the type IV pilus of P. aeruginosa (11). Consistent with this, PA1Ø also requires the type IV pilus for P. aeruginosa infection (data not shown). However, PA1Ø differs from D3112 by its lack of a c repressor-encoding gene. Since this gene is known to play a critical role in the maintenance of the lysogenic state of D3112 in P. aeruginosa (12), we are currently investigating whether the absence of a c repressor-encoding gene in PA1Ø is related to its strong lytic activity.

To our knowledge, PA1Ø is the first phage that can lyse both Gram-positive and Gram-negative bacteria. Characterization of PA1Ø may facilitate its development as a phage therapy and alternative antimicrobial agent applicable to the treatment of mixed bacterial infections with P. aeruginosa and S. aureus.

Nucleotide sequence accession number.

The whole-genome sequence of phage PA1Ø has been deposited in GenBank under accession no. HM624080.