Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup

Abstract

Lytic Pseudomonas aeruginosa phages LKD16 and LKA1 were locally isolated and morphologically classified as Podoviridae. While LKD16 adsorbs weakly to its host, LKA1 shows efficient adsorption (ka = 3.9 x 10(-9) ml min(-1)). LKA1, however, displays a narrow host range on clinical P. aeruginosa strains compared to LKD16. Genome analysis of LKD16 (43,200 bp) and LKA1 (41,593 bp) revealed that both phages have linear double-stranded DNA genomes with direct terminal repeats of 428 and 298 bp and encode 54 and 56 genes, respectively. The majority of the predicted structural proteins were experimentally confirmed as part of the phage particle using mass spectrometry. Phage LKD16 is closely related to bacteriophage phiKMV (83% overall DNA homology), allowing a more thoughtful gene annotation of both genomes. In contrast, LKA1 is more distantly related, lacking significant DNA homology and showing protein similarity to phiKMV in 48% of its gene products. The early region of the LKA1 genome has diverged strongly from phiKMV and LKD16, and intriguing differences in tail fiber genes of LKD16 and LKA1 likely reflect the observed discrepancy in infection-related properties. Nonetheless, general genome organization is clearly conserved among phiKMV, LKD16, and LKA1. The three phages carry a single-subunit RNA polymerase gene adjacent to the structural genome region, a feature which distinguishes them from other members of the T7 supergroup. Therefore, we propose that phiKMV represents an independent and widespread group of lytic P. aeruginosa phages within the T7 supergroup.

FIG. 1.

Electron micrographs of negatively stained LKA1 and LKD16 particles. Bar, 50 nm.

FIG. 2.

Summary of the bioinformatic analysis of the φKMV group. (A) Pair-wise nucleotide identity of LKD16 and φKMV, compared ORF by ORF while using a sliding window of 50 bp. Insertions/deletions are indicated by gaps in the coding sequence. (B, D, and E) Genome comparison of LKD16, φKMV, and LKA1, respectively. Open reading frames and their amino acid identities with the corresponding φKMV proteins are indicated. Newly defined and extended φKMV genes are designated in hatched boxes. Putative protein functions encoded by the φKMV-like phages are shown in the right column (F), while experimentally confirmed structural proteins are marked by an asterisk. Putative host promoters (A), phage promoters (P), and terminators (T) are indicated. (C) Circular mutation analysis of the φKMV genome in order to find breakpoints where changes in strand-specific degenerate-octomeric skew are the strongest (19). The predicted origin and termination of replication are indicated by arrows.