During our attempts to rescue the high-pressure induced SOS response in S. typhimurium LT2 by introducing MrrMG1655 of E. coli MG1655, we observed that the presence of this Type IV REase conferred constitutive (i.e. without the need for activation by high pressure) genotoxicity in LT2, while a similar toxicity was totally absent in MG1655. Since our knowledge about Mrr functionality is still limited and fragmentary, we decided to dissect the molecular basis of this apparent discrepancy between its impact on MG1655 and LT2. As such, we were able to show that the observed genotoxicity was elicited by the activity of a Type III MTase present in LT2 (i.e. ModLT2 of the StyLTI system), which most likely makes the chromosome susceptible to degradation by MrrMG1655. This feature, however, was not limited to ModLT2 as another Type III MTase encoded by E. coli ED1A (i.e. ModED1A) could similarly elicit MrrMG1655 activity. Furthermore, because of this functional antagonism, loci encoding MrrMG1655 and ModLT2/ED1A were shown to readily counter-select each other. In the same context, we revealed a strong and more general anti-correlation between close homologues of mrrMG1655 and modLT2 in the genome database, suggesting that their incompatibility might well-extend beyond the genus of Escherichia and Salmonella. As such, this study is the first to provide evidence for Type III MTases as natural triggers of Mrr activity.
The observed interference between MrrMG1655
has a number of interesting repercussions. As such, Type III MTases only methylate one specific strand of its 5–6
bp asymmetric target sequence, indicating that MrrMG1655
is, in fact, able to restrict hemi-methylated DNA. Moreover, during the writing of this article, in vitro
evidence was published that revealed a similar activity for an Mrr-like REase (i.e. MspJI) from Mycobacterium sp JLS
). In addition to hemi-methylation, all currently characterized Type III MTases seem to modify adenine (45
), indicating that their activity remains undetected by other characterized Type IV REases (i.e. McrBC and McrA) which specifically target modified cytosines (46
). Finally, our results also indicate that inactivation or deletion of the resLT2
gene greatly attenuates ModLT2
toxicity in LT2. In turn, this might suggest that the expression and/or activity of ModLT2
could be affected by ResLT2
, which challenges the current assumption that Type III MTases function independently of their corresponding REases (48
Interestingly, their specificity for methylated or modified DNA has allowed the solitary Type IV REases to fulfill a number of different activities or roles in the cell. As such, it was demonstrated that McrA, McrBC and GmrSD are able to restrict invading phages that chemically disguise their chromosome with increasingly complex base modifications (8
). Indeed, while McrA and McrBC are able to restrict DNA containing hydroxymethyscytosine (HMC), GmrSD has developed specificity for glucosylated HMC, and this differentiation is thought to reflect the evolutionary arms race between phage modification systems and the host REases. Most recently, however, Fukuda et al
) demonstrated that the deliberate introduction of Type II MTases sharing the Rm
C sequence specificity of McrBC could trigger cell death in E. coli
by eliciting McrBC-dependent chromosome degradation, thereby counter-selecting the establishment or maintenance of the MTase. As a result, these authors postulated a novel function for Type IV REases in warding off epigenetic regulation by incoming MTases (50
). The functional and evolutionary antagonism between Type IV Mrr and Type III Mod systems observed in this report seems to corroborate this hypothesis. Moreover, aside from being subject to horizontal gene transfer, some Type III MTases have actually been identified as dedicated epigenetic regulators, and have recently been shown to direct the expression of a number of genes (including virulence genes) in species of Neisseria
Interestingly, and fuelling their importance as epigenetic regulators, the coding sequence of many Type III MTases harbors homopolymeric tracts or tandem repeats, allowing frequent frame-shifts to occur and making their expression prone to phase variation at the translational level (48
). In turn, this results in a phase variable ON/OFF switching of Mod-dependent regulons (i.e. phasevarions), which is believed to be a strategy that increases the adaptive potential of populations facing fluctuating environmental conditions. Although these phenomena have not been thoroughly investigated in S. enterica
or E. coli
, it should be noted that the corresponding genes contain a number of 6–7
bp homopolymeric tracts. Moreover, we have shown such tracts to support phasevariation in the modLT2
gene upon functional counter-selection of its gene product by MrrMG1655
. Furthermore, it is clear from the genome database that a number of S. enterica
strains naturally contain out-of-frame mod
alleles. The foil pathogen S.
Gallinarum str. 287/91, for example, harbors an inactive mod
allele because of a +1 frame shift in a poly-G tract. Whether Mod also contributes to epigenetic regulation in strains of E. coli
and S. enterica
still remains to be established.
Aside from the control of phasevarions, however, frequent phase variation of Mod activity would have a number of interesting consequences with respect to its antagonism with Mrr. As such, lateral acquisition of Mrr could readily select for a population phenotypically remaining in the OFF state by counteracting those cells in which the mod
allele switches ON. On the other hand, transient OFF states might actually have evolved in order to allow safe horizontal passage or dissemination of mod
alleles through strains exhibiting Mrr activity, thus mitigating restriction or cell death. Finally, one could even imagine that switching Mod activity in the presence of a cognate and functional Mrr would temporarily allow a small subpopulation to experience bursts of DNA damage and increased mutation rates, and to behave as transient mutators that can generate genotypic variability. The transient nature of increased mutation rates is essential, as constitutive mutators eventually succumb to the accumulation of deleterious random mutations (54
). Obviously, the validity and relevance of the possible phenomena stated above require further investigation.
In summary, we present data supporting the functional interference and corresponding genomic incompatibility between Type IV Mrr and Type III Mod activity within E. coli and S. enterica, and extrapolate that these observations might extend to other species. Furthermore, this apparent antagonism fits within the recently emerging view of Type III MTases as phase variable epigenetic regulators and the hypothesis that Type IV REases might have evolved to counteract the establishment of specific genome methylation systems.