Characterization and Genomic Analyses of Pseudomonas aeruginosa Podovirus TC6: Establishment of Genus Pa11virus

Abstract

Phages have attracted a renewed interest as alternative to chemical antibiotics. Although the number of phages is 10-fold higher than that of bacteria, the number of genomically characterized phages is far less than that of bacteria. In this study, phage TC6, a novel lytic virus of Pseudomonas aeruginosa, was isolated and characterized. TC6 consists of an icosahedral head with a diameter of approximately 54 nm and a short tail with a length of about 17 nm, which are characteristics of the family Podoviridae. TC6 can lyse 86 out of 233 clinically isolated P. aeruginosa strains, thus showing application potentials for phage therapy. The linear double-stranded genomic DNA of TC6 consisted of 49796 base pairs and was predicted to contain 71 protein-coding genes. A total of 11 TC6 structural proteins were identified by mass spectrometry. Comparative analysis revealed that the P. aeruginosa phages TC6, O4, PA11, and IME180 shared high similarity at DNA sequence and proteome levels, among which PA11 was the first phage discovered and published. Meanwhile, these phages contain 54 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore proposed that these four phages can be classified as Pa11virus, comprising a new phage genus of Podoviridae that infects Pseudomonas spp. The results of this work promoted our understanding of phage biology, classification, and diversity.

Keywords: Pa11virus; Pseudomonas aeruginosa; comparative genomic analysis; phage TC6; podovirus; structural protein.

FIGURE 1

Electron micrograph of TC6 phage particles. Sample was stained with phosphotungstate. Scale bar represents 100 nm. White arrows indicate short tails.

FIGURE 2

Plaques and one-step growth curve of TC6. (A) TC6 plaques on double agar plate. Phages were incubated at 37°C for 12 h. (B) One-step growth curve of phage TC6. Experiment was repeated thrice with duplicate samples at a MOI of 10:1.

FIGURE 3

Circular presentation of the TC6 genome. The products of predicted genes were indicated in the outermost region. The outermost ring denotes genes on the plus strand, followed by rings that depict genes on the minus strand, GC content (black), GC skew (purple/green), functional modules, and position scales.

FIGURE 4

Identification of TC6 structural proteins. Proteins were visualized by SDS-PAGE analysis in a 10% (w/v) gel. The detailed results of HPLC-MS analysis were indicated on the right side of the figure. ^aMW value was theoretically calculated. ^bMW value was experimentally estimated. ^cDistinct summed MS/MS search score.

FIGURE 5

Circos diagram of nucleotide sequence alignment. Ribbons represent the similarity levels among the linked phage genome sequences. Different ranges of BLAST alignment bitscores are shown with distinct colors.

FIGURE 6

Visualized tBlastX comparison of phage genomes. Arrowheads denote genes, and the identity cut-off is set at 43%. Other function indicates putative pectate lyase, putative DNA cytosine methyltransferase, thioredoxin-like protein, PhoH-like protein, DUF3310 domain-containing protein, putative 3′–5′ exonuclease, putative 5′–3′ exonuclease, putative endonuclease, putative ATP-grasp protein, putative amidoligase, putative COOH-NH2 ligase, putative amidotransferase, putative glutamine amidotransferase, homing endonuclease, or putative serine/threonine protein phosphatase.

FIGURE 7

Phylogenetic relationships of Podoviridae phages infecting Pseudomonas spp. Major capsid proteins and neighbor-joining method were used to construct the phylogenetic tree. Different clades are marked with different colors. The name of each clade is identical with the GenBank database, except that the Pa11virus (marked in red) was proposed in this work. The scale length of relative evolution distance is 0.2.

FIGURE 8

Venn diagram of genomes of Pa11virus. The results of core genes analysis are shown. The number of proteins were indicated in parenthesis.