Genomic and Proteomic Analyses of the Terminally Redundant Genome of the Pseudomonas aeruginosa Phage PaP1: Establishment of Genus PaP1-Like Phages

Lu, Shuguang; Le, Shuai; Tan, Yinling; Zhu, Jing; Li, Ming; Rao, Xiancai; Zou, Lingyun; Li, Shu; Wang, Jing; Jin, Xiaolin; Huang, Guangtao; Zhang, Lin; Zhao, Xia; Hu, Fuquan

doi:10.1371/journal.pone.0062933

Cited by 73 publications

(88 citation statements)

References 61 publications

(71 reference statements)

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“…The smaller range also corresponds to the genome sizes for the nine phages characterized by DNA sequencing (i.e., Psa374). JG004, vB_PaeM_c2-10_Ab1, PaP1, and PAK_P1 are highly similar phages, with ϳ93-kb genomes, which infect P. aeruginosa (58,65,76,77). Genome comparison of Psa374 to JG004 revealed a low level of similarity in several genomic regions, suggesting that Psa374 and the other eight sequenced phages are more distantly related to JG004 than other members of this related group of Myoviridae that infect P. aeruginosa.…”

Section: Figmentioning

confidence: 99%

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Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

Frampton

Taylor

Moreno

et al. 2014

Appl Environ Microbiol

113

105

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c Pseudomonas syringae pv. actinidiae is a reemerging pathogen which causes bacterial canker of kiwifruit (Actinidia sp.). Since 2008, a global outbreak of P. syringae pv. actinidiae has occurred, and in 2010 this pathogen was detected in New Zealand. The economic impact and the development of resistance in P. syringae pv. actinidiae and other pathovars against antibiotics and copper sprays have led to a search for alternative management strategies. We isolated 275 phages, 258 of which were active against P. syringae pv. actinidiae. Extensive host range testing on P. syringae pv. actinidiae, other pseudomonads, and bacteria isolated from kiwifruit orchards showed that most phages have a narrow host range. Twenty-four were analyzed by electron microscopy, pulse-field gel electrophoresis, and restriction digestion. Their suitability for biocontrol was tested by assessing stability and the absence of lysogeny and transduction. A detailed host range was performed, phage-resistant bacteria were isolated, and resistance to other phages was examined. The phages belonged to the Caudovirales and were analyzed based on morphology and genome size, which showed them to be representatives of Myoviridae, Podoviridae, and Siphoviridae. Twenty-one Myoviridae members have similar morphologies and genome sizes yet differ in restriction patterns, host range, and resistance, indicating a closely related group. Nine of these Myoviridae members were sequenced, and each was unique. The most closely related sequenced phages were a group infecting Pseudomonas aeruginosa and characterized by phages JG004 and PAK_P1. In summary, this study reports the isolation and characterization of P. syringae pv. actinidiae phages and provides a framework for the intelligent formulation of phage biocontrol agents against kiwifruit bacterial canker.

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Section: Figmentioning

confidence: 99%

“…This spike in coverage is likely due to terminal redundancy at the genome ends (64), suggesting a linear genome. Indeed, the genome of the related Pseudomonas phage, PaP1, contains 1,190 bp of terminal redundancy (65).…”

Section: Figmentioning

confidence: 99%

Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

Frampton

Taylor

Moreno

et al. 2014

Appl Environ Microbiol

113

105

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“…By contrast, most dsDNA phage genome contains numerous genes with unknown functions1723; some potentially encode virulent genes. Potential virulence is a safety concern for dsDNA phage therapy.…”

Section: Resultsmentioning

confidence: 99%

“…Proteomic analysis was performed as previously described23. Briefly, purified particles were denatured with heat and loaded onto a 15% (w/v) polyacrylamide gel.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the first double-stranded RNA bacteriophage infecting Pseudomonas aeruginosa

Yang

Shen

et al. 2016

Sci Rep

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Bacteriophages (phages) are widely distributed in the biosphere and play a key role in modulating microbial ecology in the soil, ocean, and humans. Although the role of DNA bacteriophages is well described, the biology of RNA bacteriophages is poorly understood. More than 1900 phage genomes are currently deposited in NCBI, but only 6 dsRNA bacteriophages and 12 ssRNA bacteriophages genome sequences are reported. The 6 dsRNA bacteriophages were isolated from legume samples or lakes with Pseudomonas syringae as the host. Here, we report the first Pseudomonas aeruginosa phage phiYY with a three-segmented dsRNA genome. phiYY was isolated from hospital sewage in China with the clinical P. aeruginosa strain, PAO38, as a host. Moreover, the dsRNA phage phiYY has a broad host range, which infects 99 out of 233 clinical P. aeruginosa strains isolated from four provinces in China. This work presented a detailed characterization of the dsRNA bacteriophage infecting P. aeruginosa.

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“…PaP1 and PaP3 were propagated and purified in accordance with previously described protocols [9, 18, 19] with slight modifications. In brief, the liquid culture of the host bacteria during the log growth phase was inoculated with phages (multiplicity of infection of 1/100) and incubated at 37°C with shaking at 200 rpm.…”

Section: Methodsmentioning

confidence: 99%

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity

Tan

et al. 2014

BMC Genomics

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BackgroundWhole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome.ResultsAfter persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51 N-6-methyladenines and 152 N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi- mutant of Escherichia coli DH5α as a host bacterium to construct a shotgun library.ConclusionsThis work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-803) contains supplementary material, which is available to authorized users.

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Genomic and Proteomic Analyses of the Terminally Redundant Genome of the Pseudomonas aeruginosa Phage PaP1: Establishment of Genus PaP1-Like Phages

Cited by 73 publications

References 61 publications

Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

Characterization of the first double-stranded RNA bacteriophage infecting Pseudomonas aeruginosa

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity

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