Low-level expression of the Type II restriction–modification system confers potent bacteriophage resistance in Escherichia coli

Abstract

Restriction–modification systems (R–M) are one of the antiviral defense tools used by bacteria, and those of the Type II family are composed of a restriction endonuclease (REase) and a DNA methyltransferase (MTase). Most entering DNA molecules are usually cleaved by the REase before they can be methylated by MTase, although the observed level of fragmented DNA may vary significantly. Using a model EcoRI R–M system, we report that the balance between DNA methylation and cleavage may be severely affected by transcriptional signals coming from outside the R–M operon. By modulating the activity of the promoter, we obtained a broad range of restriction phenotypes for the EcoRI R–M system that differed by up to 4 orders of magnitude in our biological assays. Surprisingly, we found that high expression levels of the R–M proteins were associated with reduced restriction of invading bacteriophage DNA. Our results suggested that the regulatory balance of cleavage and methylation was highly sensitive to fluctuations in transcriptional signals both up- and downstream of the R–M operon. Our data provided further insights into Type II R–M system maintenance and the potential conflict within the host bacterium.

Cells with stochastically increased methyltransferase to restriction endonuclease ratio provide an entry for bacteriophage into protected cell population

“…Earlier, we used mathematical modeling to show how the ability of a phage to overcome protection afforded by an RM system can be determined by the relative ratio of MTase and REase activities in the cell ( 34 ). This result seems to be collaborated by recent experimental analysis, where it was shown that the level of protection afforded by a plasmid-borne EcoRI system is not directly related to the absolute amounts of RM enzymes and, in fact, can be even negatively correlated with RM enzymes abundance ( 20 ). To further verify the model predictions we here experimentally perturbed the intracellular amounts of MTase and REase of C protein-dependent RM system Esp1396I ( 30 , 35 ).…”

“…The intracellular amounts/activity levels of the MTase and REase enzymes shall be tightly controlled because an imbalance can either lead to the death of uninfected (excess REase activity) ( 19 , 20 ) or infected (excess MTase activity) cells ( 20 ). Indeed, diverse regulatory mechanisms that ensure the amounts of RM enzymes are tightly controlled have been described (reviewed in ( 21 )).…”

MamA essentiality in Mycobacterium smegmatis is explained by the presence of an apparent cognate restriction endonuclease

“…Considering the essentiality of mamA in M. smegmatis, we suspected that this methylase could be part of an R-M system, and that lack of MamA is toxic because an active restriction endonuclease recognizing the same sequence cleaves the genome when unmethylated. In addition, it has been observed that disturbance of R-M systems increases cell filamentation in bacteria [16][17][18], supporting the idea that the unbalance in the R-M system could be the reason for the mamA KD phenotype. We previously found that mamA is co-transcribed with the downstream gene, MSMEG_3214 [14].…”

Regulator-dependent temporal dynamics of a restriction-modification system's gene expression upon entering new host cells: single-cell and population studies

“…Second, the overexpression might explain why the Esp1396I R-M system acceptor cells in that study showed a DNA damage phenotype, manifested by abnormal, filamentous cell morphology, possibly due to excess REase production. Under conditions with an active SOS response, cells cannot grow with normal generation times and have substantially-disturbed patterns of gene expression ( 77 , 78 ), perhaps contributing to this timing.…”