Abstract
Phage O4 of Pseudomonas aeruginosa was previously visualized as a short-tailed virus using a transmission electron microscope. In this work, the O4 genome was characterized to be a linear dsDNA molecule comprising 50509 bp with 76 predicted genes located in five clusters. Mass spectrometry showed that the O4 virion contains 6 putative structural proteins, 2 putative enzymes, and 7 hypothetical proteins. By analyzing a Tn5G transposon mutation library, eight genes, wbpR, wbpV, wbpO, wbpT, wbpS, wbpL, galU, and wzy, were identified and confirmed responsible for the phage-resistant phenotype; all of them are related to the synthesis of O-specific antigen (OSA) of lipopolysaccharide (LPS), indicating that OSA is the receptor for the adsorption of phage O4. Comparative genomic analysis revealed that the phage O4 genome shares little similarity to any known podovirus, indicating that phage O4 is classifiable as a novel member of the Podoviridae family.
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This work is supported by The National Natural Science Foundation of China (Grant No. 31370205 and 30970114).
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Figure S1
Characteristics of phage O4. a: One-step growth curve of PAK infected by phage O4 with an MOI of 0.001. L: latent phase. R: rise phase. P: plateau phase. b: Inhibition effects on the growth of PAK by phage O4 (JPEG 541 kb)
Figure S2
Identification of the O4 termini. a: Schematic diagram of the assembled genome of phage O4. The fragments are the StuI digested fragments projected from the draft genomic sequence. b: Electrophoresis profiles. Lane 1: The genomic DNA of phage O4. Lane 2: The BglII digested DNA. Lane 3: The SnaBI digested DNA. Lane 4: The StuI digested DNA. The predicted 7.5 kb and 3.2 kb were replaced with 6.8 kb and 4.2 kb, respectively. Lane 5-9: the purified StuI fragments of 20.6 kb, 12.6 kb, 6.8 kb, 5.9 kb, and 4.2 kb, respectively. Lane 10: Negative amplification of the 4.2 kb StuI fragment using the primers F2 and R2. Lane 11: Negative amplification of the 6.8 kb StuI fragment using the primers F2 and R2. Lane 12-14: Positive amplifications of the StuI fragments of 20.6 kb, 12.6 kb, and 5.9 kb using the primers F1 and R1, F3 and R3, and F4 and R4, respectively. Lane 15-16: Positive amplification of the 4.2 kb StuI fragment using the primers F5 and R5. Lane 17-18: Positive amplification of the 6.8 kb StuI fragment using the primers F6 and R6. c: Schematic diagram of the curated genome. The blue arrows stand for primers used in inverse PCR for analysis of the StuI fragments ends (JPEG 1459 kb)
Figure S3
Characterization of the phage-resistant mutants. a: Relative adsorption rates of the phage-resistant mutants. The parent strain PAK was used as a control. b: Complementation of the phage-resistant mutants with the target genes. The upper panel showed the spotting results of the strains carrying the vector pUCP18. The lower panel showed the spotting results of the strains carrying the corresponding target genes, respectively (JPEG 1839 kb)
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Zhang, F., Huang, K., Yang, X. et al. Characterization of a novel lytic podovirus O4 of Pseudomonas aeruginosa. Arch Virol 163, 2377–2383 (2018). https://doi.org/10.1007/s00705-018-3866-y
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DOI: https://doi.org/10.1007/s00705-018-3866-y