Restriction modification (R-M) systems are one of the most widespread and due their often plasmid-based nature easily transmittable systems bacteria can utilize to protect themselves against infections by bacteriophages (1). The here studied CfrBI R-M system is a type II R-M system and consists of a methyltransferase (MT) and a restriction endonuclease (RE) that are divergently expressed and share a promoter region (2,3). The MT and RE recognize the same DNA sequence, with the MT conferring protection against the RE that can only act on unmethylated DNA. Interestingly, in the promoter region of the CfrBI R-M system a single CfrBI recognition site is present that has been shown to regulate the expression of the system (4,5). Here, we show that the expression dynamics of the Crib R-M system, as well as its protective properties, depend on the copy number of the plasmid harboring it. The higher the plasmid copy number, the higher the overall expression, but the expression dynamics and MT to RE expression ratios in the background of a medium copy number plasmid interestingly conferred the highest phage resistance. After transformation in naïve cells however, expression of the RE that only occurs after methylation of the CfrBI site in the promoter was fastest in the high copy plasmid background. To conclude, the results indicate that while for protection against phages the overall expression strength might, in case of the CfrBI R-M system, not be the predominant factor, while for the swiftness of establishment of the system the initial expression rate of the MT seems to be the determining factor.

(1) Loenen, W.A.M., et al. (2014) Nucleic Acids Res. 42: 3–19. doi: 10.1093/nar/gkt990.

(2) Kravets, A.N., et al. (1992) Mol Gen Mikrobiol Virusol. 7-8: 4–7.

(3) Zakharova, M.V., et al. (1993) Gene. 129: 77–81. doi: 10.1016/0378-1119(93)90698-3.

(4) Beletskaya, I.V., et al. (2000) Nucleic Acids Res. 28: 3817–3822. doi: 10.1093/nar/28.19.3817.

(5) Zakharova M.V., et al. (2004) J Mol Biol. 335: 103–111. doi: 10.1016/j.jmb.2003.09.081.