An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains

Area of Science:

• Microbiology
• Bacteriology
• Virology

Background:

• Prophages, or integrated bacteriophages, are prevalent in *Pseudomonas aeruginosa*, an opportunistic pathogen.
• Prophages contribute to bacterial genetic diversity and can enhance pathogenicity.
• Studying prophages in *P. aeruginosa* is challenging due to the lack of effective curing methods.

Purpose of the Study:

• To develop and validate a novel, targeted prophage-curing strategy for *Pseudomonas aeruginosa*.
• To facilitate research into prophage biology and its impact on bacterial traits.

Main Methods:

• A targeted-curing approach involving ampicillin resistance (ampR) cassette tagging and sacB counter-selection.
• Temporal insertion of the sacB counter-selection marker into the prophage region.
• Validation using local PCR amplification and Whole Genome Sequencing.

Main Results:

• The developed method successfully cured Pf4 prophage from the laboratory strain PAO1.
• The strategy was also effective in curing the PR2 prophage from the clinically relevant and difficult-to-manipulate strain 39016.
• High efficiency was observed in both laboratory and clinical strains.

Conclusions:

• The novel targeted-curing method provides an efficient way to remove prophages from *P. aeruginosa*.
• This technique can advance the study of prophage biology in *P. aeruginosa* and other Gram-negative bacteria.