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Article

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

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The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel
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The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 5290002, Israel
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Scientific Equipment Center, The Mina & Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat Gan 5290002, Israel
*
Author to whom correspondence should be addressed.
Academic Editor: Dann Turner
Viruses 2021, 13(2), 336; https://doi.org/10.3390/v13020336
Received: 2 February 2021 / Revised: 11 February 2021 / Accepted: 18 February 2021 / Published: 21 February 2021
(This article belongs to the Special Issue Prophage Biology)
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. View Full-Text
Keywords: Pseudomonas aeruginosa; prophages; bacteriophages; curing; counter-selection; lysogen Pseudomonas aeruginosa; prophages; bacteriophages; curing; counter-selection; lysogen
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MDPI and ACS Style

Shmidov, E.; Zander, I.; Lebenthal-Loinger, I.; Karako-Lampert, S.; Shoshani, S.; Banin, E. An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains. Viruses 2021, 13, 336. https://doi.org/10.3390/v13020336

AMA Style

Shmidov E, Zander I, Lebenthal-Loinger I, Karako-Lampert S, Shoshani S, Banin E. An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains. Viruses. 2021; 13(2):336. https://doi.org/10.3390/v13020336

Chicago/Turabian Style

Shmidov, Esther, Itzhak Zander, Ilana Lebenthal-Loinger, Sarit Karako-Lampert, Sivan Shoshani, and Ehud Banin. 2021. "An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains" Viruses 13, no. 2: 336. https://doi.org/10.3390/v13020336

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