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bioRxiv · 10.64898/2025.12.04.692298

Plasmid copy number affects the DNA methylation-driven expression dynamics of the CfrBI restriction-modification system and impacts phage restriction

Gibhardt, J.·Hoang, T. D.·Severinov, K.·Khodorkovskii, M. A.·Morozova, N. E.
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Abstract

Restriction-modification (R-M) systems are one of the most widespread and, due to their often plasmid-based nature, transmittable anti-phage systems bacteria have. The CfrBI R-M system studied here consists of a methyltransferase (MT) and a restriction endonuclease (RE) that are divergently expressed and share a promoter region that harbors a single CfrBI recognition site. Previously, the methylation of this site has been shown to regulate the expression of the R-M system. Here, we show that the expression dynamics of the CfrBI R-M system and its protective properties depend on the copy number of the plasmid harboring it. A higher copy number results in higher expression, but the expression on a medium-copy number plasmid interestingly conferred the highest phage resistance. After transformation of naive cells however, expression of the RE was fastest in the high-copy plasmid background. In vivo we show that the expression strength of the MT inhibits its own expression, while enhancing RE expression. To conclude, the results indicate that for phage resistance the overall expression strength might not be the predominant factor in case of the CfrBI R-M system, while for the establishment of the system the initial expression rate of the MT seems to be the determining factor.

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Gibhardt, J., Hoang, T. D., Severinov, K., Khodorkovskii, M. A., Morozova, N. E.. 2025-12-06. Plasmid copy number affects the DNA methylation-driven expression dynamics of the CfrBI restriction-modification system and impacts phage restriction. https://doi.org/10.64898/2025.12.04.692298

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