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. 2022 Jul 14:13:946251.
doi: 10.3389/fmicb.2022.946251. eCollection 2022.

Isolation and characterization of two homolog phages infecting Pseudomonas aeruginosa

Niu Yuanyuan  1   2 , Yang Xiaobo  2 , Wang Shang  2 , Yang Yutong  2 , Zhou Hongrui  2 , Li Chenyu  2 , Xue Bin  2 , Zhang Xi  2 , Zhao Chen  2 , Shen Zhiqiang  2 , Wang Jingfeng  2 , Ling Yun  1 , Yu Pingfeng  3 , Qiu Zhigang  1   2
Affiliations

Isolation and characterization of two homolog phages infecting Pseudomonas aeruginosa

Niu Yuanyuan et al. Front Microbiol. .

Abstract

Bacteriophages (phages) are capable of infecting specific bacteria, and therefore can be used as a biological control agent to control bacteria-induced animal, plant, and human diseases. In this study, two homolog phages (named PPAY and PPAT) that infect Pseudomonas aeruginosa PAO1 were isolated and characterized. The results of the phage plaque assay showed that PPAT plaques were transparent dots, while the PPAY plaques were translucent dots with a halo. Transmission electron microscopy results showed that PPAT (65 nm) and PPAY (60 nm) strains are similar in size and have an icosahedral head and a short tail. Therefore, these belong to the short-tailed phage family Podoviridae. One-step growth curves revealed the latent period of 20 min and burst time of 30 min for PPAT and PPAY. The burst size of PPAT (953 PFUs/infected cell) was higher than that of PPAY (457 PFUs/infected cell). Also, the adsorption rate constant of PPAT (5.97 × 10-7 ml/min) was higher than that of PPAY (1.32 × 10-7 ml/min) at 5 min. Whole-genome sequencing of phages was carried out using the Illumina HiSeq platform. The genomes of PPAT and PPAY have 54,888 and 50,154 bp, respectively. Only 17 of the 352 predicted ORFs of PPAT could be matched to homologous genes of known function. Likewise, among the 351 predicted ORFs of PPAY, only 18 ORFs could be matched to genes of established functions. Homology and evolutionary analysis indicated that PPAT and PPAY are closely related to PA11. The presence of tail fiber proteins in PPAY but not in PPAT may have contributed to the halo effect of its plaque spots. In all, PPAT and PPAY, newly discovered P. aeruginosa phages, showed growth inhibitory effects on bacteria and can be used for research and clinical purposes.

Keywords: Pseudomonas aeruginosa; bacteriophages; genome; phage therapy; tail fiber protein.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Plaque morphology. (A) PPAT and (B) PPAY.
Figure 2
Figure 2
Transmission electron micrographs showing the virion particle morphology. (A) PPAT and (B) PPAY. Scale bar: 100 nm.
Figure 3
Figure 3
The phase adsorption curves. (A) PPAT and (B) PPAY. The adsorption curve was drawn with time as abscissa and the percentage of free phage as ordinate. Error bars indicate standard deviations of biological triplicates.
Figure 4
Figure 4
One-step growth curves of phages. (A) PPAT and (B) PPAY. Error bars indicate standard deviations of biological triplicates.
Figure 5
Figure 5
SDS-PAGE analysis of phage proteins.
Figure 6
Figure 6
Cluster of phage genes. Genes with the same color denote homologs.
Figure 7
Figure 7
Genomic collinearity between PPAT and PPAY.
Figure 8
Figure 8
The phylogenetic tree of PPAT and PPAY. (A) Phylogenetic tree of PPAT and PPAY genomes, related sequences of PPAT and PPAY were obtained from GenBank and the neighbor-joining phylogenetic tree was constructed by Mega X with 3,000 bootstrap replicates. (B) A phylogenetic tree was generated using the sequences of the large terminase subunit. The proteins showing relatedness with large terminase subunit in PPAT and PPAY were obtained from GenBank and the neighbor-joining phylogenetic tree was constructed by Mega X with 3,000 bootstrap replicates.
Figure 9
Figure 9
Phage infection inhibits bacterial growth.
See this image and copyright information in PMC

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