Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.

Restriction-modification (R-M) systems are rudimentary bacterial immune systems. The main components include restriction enzyme (R), which cuts specific unmethylated DNA sequences, and the methyltransferase (M), which protects the same DNA sequences. The expression of R-M system components is considered to be tightly regulated, to ensure successful establishment in a naïve bacterial host. R-M systems are organized in different architectures (convergent or divergent) and are characterized by different features, i.e. binding cooperativities, dissociation constants of dimerization, translation rates, which ensure this tight regulation. It has been proposed that R-M systems should exhibit certain dynamical properties during the system establishment, such as: i) a delayed expression of R with respect to M, ii) fast transition of R from “OFF” to “ON” state, iii) increased stability of the toxic molecule (R) steady-state levels. It is however unclear how different R-M system features and architectures ensure these dynamical properties, particularly since it is hard to address this question experimentally. To understand design of different R-M systems, we computationally analyze two R-M systems, representative of the subset controlled by small regulators called ‘C proteins’, and differing in having convergent or divergent promoter architecture. We show that, in the convergent system, abolishing any of the characteristic system features adversely affects the dynamical properties outlined above. Moreover, an extreme binding cooperativity, accompanied by a very high dissociation constant of dimerization, observed in the convergent system, but absent from other R-M systems, can be explained in terms of the same properties. Furthermore, we develop the first theoretical model for dynamics of a divergent R-M system, which does not share any of the convergent system features, but has overlapping promoters. We show that i) the system dynamics exhibits the same three dynamical properties, ii) introducing any of the convergent system features to the divergent system actually diminishes these properties. Our results suggest that different R-M architectures and features may be understood in terms of constraints imposed by few simple dynamical properties of the system, providing a unifying framework for understanding these seemingly diverse systems. We also provided predictions for the perturbed R-M systems dynamics, which may in future be tested through increasingly available experimental techniques, such as re-engineering R-M systems and single-cell experiments.

C-proteins control restriction–modification (R–M) systems’ genes transcription to ensure sufficient levels of restriction endonuclease to allow protection from foreign DNA while avoiding its modification by excess methyltransferase. Here, we characterize transcription regulation in C-protein dependent R–M system Kpn2I. The Kpn2I restriction endonuclease gene is transcribed from a constitutive, weak promoter, which, atypically, is C-protein independent. Kpn2I C-protein (C.Kpn2I) binds upstream of the strong methyltransferase gene promoter and inhibits it, likely by preventing the interaction of the RNA polymerase sigma subunit with the -35 consensus element. Diminished transcription from the methyltransferase promoter increases transcription from overlapping divergent C-protein gene promoters. All known C-proteins affect transcription initiation from R–M genes promoters. Uniquely, the C.Kpn2I binding site is located within the coding region of its gene. C.Kpn2I acts as a roadblock stalling elongating RNA polymerase and decreasing production of full-length C.Kpn2I mRNA. Mathematical modeling shows that this unusual mode of regulation leads to the same dynamics of accumulation of R–M gene transcripts as observed in systems where C-proteins act at transcription initiation stage only. Bioinformatics analyses suggest that transcription regulation through binding of C.Kpn2I-like proteins within the coding regions of their genes may be widespread.