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Volume 171, Issue 11

Eliminating the type I restriction endonuclease from PAO1 for optimized phage isolation Open Access

Graphical Abstract

Graphical Abstract

The type I R-M system is comprised of a methyltransferase (one HsdS and two HsdM subunits) and a restriction endonuclease (one HsdS, two HsdM and two HsdR subunits). In wildtype PAO1, the restriction endonuclease destroys phage DNA with unmethylated restriction sites, while the methyltransferase methylates host recognition sites to protect self-DNA from restriction. ∆hsdR is optimised for phage isolation and propagation as it lacks the restriction endonuclease, preventing phage DNA cleavage and facilitating efficient phage proliferation. The methyltransferase remains active, so both host and phage DNA are methylated, protecting phage progeny from R-M systems in future hosts. (Created in BioRender.com)

Abstract

Phage therapy is a promising treatment for multidrug-resistant bacterial infections. Due to their high host specificity, phages must be matched to the target clinical strains. Efficiently identifying appropriate phages and producing sufficient titres for clinical use requires comprehensive phage libraries and multiple propagation hosts. An idealized system would use a highly promiscuous bacterial host to isolate a broader range of phages and streamline optimized phage production. Anti-phage defences constrain bacterial host promiscuity, such as restriction-modification systems that recognize and cleave foreign DNA. Here, the type I restriction endonuclease, HsdR, was deleted from PAO1 to make a more promiscuous phage isolation and propagation host. Removal of this endonuclease more than doubled the efficiency of phage propagation on solid media, improved yields from hard-to-propagate phages in liquid bulk-ups and yielded seven times more phages from freshwater samples than wild-type PAO1 – an important step in producing an optimized strain for isolating and propagating phages for clinical phage therapy.

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Keyword(s): anti-phage defence , hsdR , phage isolation , phage therapy , Pseudomonas aeruginosa and restriction-modification
Funding
This study was supported by the:
  • InnovateUK (Award 10070793)
    • Principal Award Recipient: BenTemperton
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2025-11-21
2025-12-16

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/content/journal/micro/10.1099/mic.0.001634
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Eliminating the type I restriction endonuclease from Pseudomonas aeruginosa PAO1 for optimized phage isolation
Microbiology 171, 001634 (2025); https://doi.org/10.1099/mic.0.001634
/content/journal/micro/10.1099/mic.0.001634
/content/journal/micro/10.1099/mic.0.001634
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