A Staphylococcus phage recombination cassette that enables recombineering across distant bacteria
- 1University of Rochester
Abstract
Modifying bacterial genomes with phage recombinases, a process termed recombineering, has enabled precise genome editing, but its reach is limited by host specificity: each new organism generally requires its own recombinase to be identified and optimized. Using known phage recombination genes as synteny anchors, we mined Staphylococcus genomes and recovered a diverse collection of phage recombination cassettes, spanning the RAD52-like annealase superfamily (Sak, Erf) and the RAD51/RecA-like Sak4 family. Functional testing of representatives in S. aureus identified several highly efficient systems that support recombineering. We engineered the most efficient of these into a single-plasmid tool that yields markerless editing in S. aureus in four days. Notably, where other recombineering systems rely on host-specific factors for nuclease evasion and ssDNA loading, this cassette encodes both activities itself: a predicted broad-spectrum nuclease inhibitor and a dedicated single-stranded DNA-binding protein. We therefore tested the cassette across phylogenetically distant hosts, and observed recombination in Streptococcus pyogenes, Enterococcus faecium, Francisella novicida, and Escherichia coli. These results identify a self-sufficient class of phage recombination systems and provide a portable tool for
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BibTeX
@misc{maguin2026staphylococcus,
title = {{A Staphylococcus phage recombination cassette that enables recombineering across distant bacteria}},
author = {P. Maguin and M. Aghahosseini and M. Brodmann and V. Chen and J. Zhu and K. Szenkiel and L. A. Marraffini and A. Varble},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.30.761037},
url = {https://www.biorxiv.org/content/10.64898/2026.10.30.761037v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - A Staphylococcus phage recombination cassette that enables recombineering across distant bacteria
AU - P. Maguin
AU - M. Aghahosseini
AU - M. Brodmann
AU - V. Chen
AU - J. Zhu
AU - K. Szenkiel
AU - L. A. Marraffini
AU - A. Varble
PY - 2026
DA - 2026/10/30
PB - bioRxiv
DO - 10.64898/2026.10.30.761037
UR - https://www.biorxiv.org/content/10.64898/2026.10.30.761037v1
AB - Modifying bacterial genomes with phage recombinases, a process termed recombineering, has enabled precise genome editing, but its reach is limited by host specificity: each new organism generally requires its own recombinase to be identified and optimized. Using known phage recombination genes as synteny anchors, we mined Staphylococcus genomes and recovered a diverse collection of phage recombination cassettes, spanning the RAD52-like annealase superfamily (Sak, Erf) and the RAD51/RecA-like Sak4 family. Functional testing of representatives in S. aureus identified several highly efficient systems that support recombineering. We engineered the most efficient of these into a single-plasmid tool that yields markerless editing in S. aureus in four days. Notably, where other recombineering systems rely on host-specific factors for nuclease evasion and ssDNA loading, this cassette encodes both activities itself: a predicted broad-spectrum nuclease inhibitor and a dedicated single-stranded DNA-binding protein. We therefore tested the cassette across phylogenetically distant hosts, and observed recombination in Streptococcus pyogenes, Enterococcus faecium, Francisella novicida, and Escherichia coli. These results identify a self-sufficient class of phage recombination systems and provide a portable tool for
ER -