Genomic and proteomic analyses of the terminally redundant genome of the Pseudomonas aeruginosa phage PaP1: establishment of genus PaP1-like phages

Abstract

We isolated and characterized a new Pseudomonas aeruginosa myovirus named PaP1. The morphology of this phage was visualized by electron microscopy and its genome sequence and ends were determined. Finally, genomic and proteomic analyses were performed. PaP1 has an icosahedral head with an apex diameter of 68-70 nm and a contractile tail with a length of 138-140 nm. The PaP1 genome is a linear dsDNA molecule containing 91,715 base pairs (bp) with a G+C content of 49.36% and 12 tRNA genes. A strategy to identify the genome ends of PaP1 was designed. The genome has a 1190 bp terminal redundancy. PaP1 has 157 open reading frames (ORFs). Of these, 143 proteins are homologs of known proteins, but only 38 could be functionally identified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-mass spectrometry allowed identification of 12 ORFs as structural protein coding genes within the PaP1 genome. Comparative genomic analysis indicated that the Pseudomonas aeruginosa phage PaP1, JG004, PAK_P1 and vB_PaeM_C2-10_Ab1 share great similarity. Besides their similar biological characteristics, the phages contain 123 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore propose that these four phages be classified as PaP1-like phages, a new phage genus of Myoviridae that infects Pseudomonas aeruginosa.

Figure 1. Electron micrographs of purified PaP1 phage particles.

A shows two PaP1 particles with uncontracted tails and an empty head (uranyl acetate). B shows the contracted tail with straight tail fibers (phosphotungstate). C shows a pentagonal head (uranyl acetate). The scale bar represents 100 nm. These micrographs were taken by Hans-Wolfgang Ackermann, School of Medicine, Laval University, Quebec, Canada.

Figure 2. One-step growth curve of phage PaP1.

Experiments were repeated three times with duplicate samples. The Y-axis shows the log of plaque forming units per milliliter (PFU/mL).

Figure 3. Circular conformation of GC skew and GC plot of the PaP1 genome.

The GC skew is calculated as (G-C)/(G+C) and the GC plot shows GC% content plot. The outer circle represents the GC skew (red for positive and blue for negative); the inner circle represents the GC plot (pink for above-average and powder blue for below-average). The red pane indicates the putative replication origin of the PaP1 genome.

Figure 4. Identification of PaP1 genome ends.

(A) Digestion of the PaP1 genome DNA by NarI and NotI. The red arrow indicates the 3′ end fragment of the PaP1 genome. (B) NarI and NotI restriction map of the PaP1 genome. (C) PaP1 DNA was digested by FspI and the recovered 5′ end fragment (5′ Frag., indicated by the red arrow) was digested by S1 nuclease. (D) Strategy designed to identify PaP1 genome ends. Primers P1 and P2 are annealed to 3′ and 5′ end fragments of the PaP1 genome DNA, respectively. Terminal run-off sequencing of the two ends (also shown in Figure 5C) is then performed. Case 1 (blunt end): The two sequences obtained by P1 and P2 do not have repeated regions and they can be assembled to the PaP1 genome sequence with no gap between them. Case 2 (3′-protruded end): The two sequences obtained by P1 and P2 also do not have repeated regions; however, a gap is observed between the sequences once assembled to the PaP1 genome sequence. The sequence within the gap is the 3′-protruded cohesive sequence. Case 3 (5′-protruded end or terminal redundancy): A repeat between the two obtained sequences is observed. If the repeated sequence is less than 100 bp, it is regarded as the 5′-protruded cohesive sequence , , ; however, if the repeated sequence is over 100 bp, it is regarded as a terminally redundant sequence , , .

Figure 5. Structure of PaP1 terminal redundancy.

(A) PaP1 genome DNA with terminal redundancy at both ends. The sequence data of the PaP1 genome are calculated from 1 bp to 91,715 bp. (B) Putative genes within the PaP1 terminal redundancy. (C) Terminal run-off sequencing chromatograms of both ends of the PaP1 genome. The sequencing direction of primers P1 and P2 is indicated by the black arrows.

Figure 6. Diagram of the PaP1 genome with functional annotations.

Blue arrowheads show genes on the plus strand; brownish red arrowheads show genes on the minus strand; green words refer to the amino acid transferred by the corresponding tRNA. NPR: nicotinamide phosphoribosyl, PRP: phosphoribosylpyrophosphate, RDR: ribonucleotide diphosphate reductase.

Figure 7. SDS-PAGE analysis of the structural proteins of phage PaP1.

Proteins were visualized in a 15% (w/v) gel and identified by HPLC-MS analysis (Table 4).

Figure 8. Pairwise nucleotide sequence comparison of phages closely related to PaP1.

Comparisons were conducted using BLAST 2.25 and displayed using ACT . Highly related sequences are shown by the blue shadings. The intensity of the blue coloration indicates the level of sequence similarity. The minimum score cutoff is 100 and the minimum identity cutoff is 50%. ^#The full name is vB_PaeM_C2-10_Ab1.

Figure 9. Dot plot of genome sequences of six phages using the program Gepard.

The word length used is 9 bp; other parameters are set by default. The black dots indicate that the corresponding genome regions of the abscissa and the ordinate show similarity to each other. ^#The full name is vB_PaeM_C2-10_Ab1.

Figure 10. Phylogenetic analysis of major capsid protein amino acid sequences.

The diagram was constructed using the MEGA5 program . The relative distances of each main branch are shown in the figure. Both PaP2 and LUZ24-like phages belong to the family of Podoviridae; all other phage groups belong to the Myoviridae phage family. The phages in the same group are marked with the same color. The group of “PaP1-like phages” is first presented in this work. ^#The full name is vB_PaeM_C2-10_Ab1.