An efficient, counter-selection-based method for prophage curing in pseudomonas aeruginosa strains

Esther Shmidov, Itzhak Zander, Ilana Lebenthal-Loinger, Sarit Karako-Lampert, Ehud Banin, Sivan Shoshani

Research output: Contribution to journalArticlepeer-review


Prophages are bacteriophages in the lysogenic state, where the viral genome is inserted within the bacterial chromosome. They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were shown to confer specific traits that can promote strain pathogenicity. The main difficulty of studying those regions is the lack of a simple prophage-curing method for P. aeruginosa strains. In this study, we developed a novel, targeted-curing approach for prophages in P. aeruginosa. In the first step, we tagged the prophage for curing with an ampicillin resistance cassette (ampR) and further used this strain for the sacB counter-selection marker’s temporal insertion into the prophage region. The sucrose counter-selection resulted in different variants when the prophage-cured mutant is the sole variant that lost the ampR cassette. Next, we validated the targeted-curing with local PCR amplification and Whole Genome Sequencing. The application of the strategy resulted in high efficiency both for curing the Pf4 prophage of the laboratory wild-type (WT) strain PAO1 and for PR2 prophage from the clinical, hard to genetically manipulate, 39016 strain. We believe this method can support the research and growing interest in prophage biology in P. aeruginosa as well as additional Gram-negative bacteria.

Original languageEnglish
Article number336
Issue number2
StatePublished - 21 Feb 2021


  • Bacteriophages
  • Counter-selection
  • Curing
  • Lysogen
  • Prophages
  • Pseudomonas aeruginosa

All Science Journal Classification (ASJC) codes

  • Infectious Diseases
  • Virology


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