MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5′-TCCRAC-3′. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5′-GTYGGA-3′. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities.     

Characterization of a Novel Type II Restriction-Modification System, Sth368I, Encoded by the Integrative Element ICESt1 of Streptococcus thermophilus CNRZ368

A novel type II restriction and modification (R-M) system, Sth368I, which confers resistance to ST84, was found in Streptococcus thermophilus CNRZ368 but not in the very closely related strain A054. Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNRZ368 but not by A054, revealed a divergent cluster of two genes, sth368IR and sth368IM. The protein sequence encoded by sth368IR is related to the type II endonucleases R.LlaKR2I and R.Sau3AI, which recognize and cleave the sequence 5′-GATC-3′. The protein sequence encoded by sth368IM is very similar to numerous type II 5-methylcytosine methyltransferases, including M.LlaKR2I and M.Sau3AI. Cell extracts of CNRZ368 but not A054 were found to cleave at the GATC site. Furthermore, the C residue of the sequence 5′-GATC-3′ was found to be methylated in CNRZ368 but not in A054. Cloning and integration of a copy of sth368IR and sth368IM in the A054 chromosome confers on this strain phenotypes similar to those of CNRZ368, i.e., phage resistance, endonuclease activity of cell extracts, and methylation of the sequence 5′-GATC-3′. Disruption of sth368IR removes resistance and restriction activity. We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes the sequence 5′-GATC-3′ and is related to the Sau3AI and LlaKR2I restriction systems.     

Haemophilus influenzae phasevarions have evolved from type III DNA restriction systems into epigenetic regulators of gene expression

Phase variably expressed (randomly switching) methyltransferases associated with type III restriction-modification (R-M) systems have been identified in a variety of pathogenic bacteria. We have previously shown that a phase variable methyltransferase (Mod) associated with a type III R-M system in Haemophilus influenzae strain Rd coordinates the random switching of expression of multiple genes, and constitutes a phase variable regulon—‘phasevarion’. We have now identified the recognition site for the Mod methyltransferase in H. influenzae strain Rd as 5′-CGAAT-3′. This is the same recognition site as the previously described HinfIII system. A survey of 59 H. influenzae strains indicated significant sequence heterogeneity in the central, variable region of the mod gene associated with target site recognition. Intra- and inter-strain transformation experiments using Mod methylated or non-methylated plasmids, and a methylation site assay demonstrated that the sequence heterogeneity seen in the region encoding target site specificity does correlate to distinct target sites. Mutations were identified within the res gene in several strains surveyed indicating that Res is not functional. These data suggest that evolution of this type III R-M system into an epigenetic mechanism for controlling gene expression has, in some strains, resulted in loss of the DNA restriction function.     

Evidence for horizontal transfer of SsuDAT1I restriction-modification genes to the Streptococcus suis genome

Different strains of Streptococcus suis serotypes 1 and 2 isolated from pigs either contained a restriction-modification (R-M) system or lacked it. The R-M system was an isoschizomer of Streptococcus pneumoniae DpnII, which recognizes nucleotide sequence 5′-GATC-3′. The nucleotide sequencing of the genes encoding the R-M system in S. suis DAT1, designated SsuDAT1I, showed that the SsuDAT1I gene region contained two methyltransferase genes, designated ssuMA and ssuMB, as does the DpnII system. The deduced amino acid sequences of M.SsuMA and M.SsuMB showed 70 and 90% identity to M.DpnII and M.DpnA, respectively. However, the SsuDAT1I system contained two isoschizomeric restriction endonuclease genes, designated ssuRA and ssuRB. The deduced amino acid sequence of R.SsuRA was 49% identical to that of R.DpnII, and R.SsuRB was 72% identical to R.LlaDCHI of Lactococcus lactis subsp. cremoris DCH-4. The four SsuDAT1I genes overlapped and were bounded by purine biosynthetic gene clusters in the following gene order: purF-purM-purN-purH-ssuMA-ssuMB-ssuRA-ssuRB-purD-purE. The G+C content of the SsuDAT1I gene region (34.1%) was lower than that of the pur region (48.9%), suggesting horizontal transfer of the SsuDAT1I system. No transposable element or long-repeat sequence was found in the flanking regions. The SsuDAT1I genes were functional by themselves, as they were individually expressed in Escherichia coli. Comparison of the sequences between strains with and without the R-M system showed that only the region from 53 bp upstream of ssuMA to 5 bp downstream of ssuRB was inserted in the intergenic sequence between purH and purD and that the insertion target site was not the recognition site of SsuDAT1I. No notable substitutions or insertions could be found, and the structures were conserved among all the strains. These results suggest that the SsuDAT1I system could have been integrated into the S. suis chromosome by an illegitimate recombination mechanism.     

Purification and characterisation of a novel DNA methyltransferase,M.AhdI

We have cloned the M and S genes of the restriction-modification (R-M) system AhdI and have purified the resulting methyltransferase to homogeneity. M.AhdI is found to form a 170 kDa tetrameric enzyme having a subunit stoichiometry M₂S₂ (where the M and S subunits are responsible for methylation and DNA sequence specificity, respectively). Sedimentation equilibrium experiments show that the tetrameric enzyme dissociates to form a heterodimer at low concentration, with K_d ≈ 2 µM. The intact (tetrameric) enzyme binds specifically to a 30 bp DNA duplex containing the AhdI recognition sequence GACN₅GTC with high affinity (K_d ≈ 50 nM), but at low enzyme concentration the DNA binding activity is governed by the dissociation of the tetramer into dimers, leading to a sigmoidal DNA binding curve. In contrast, only non-specific binding is observed if the duplex lacks the recognition sequence. Methylation activity of the purified enzyme was assessed by its ability to prevent restriction by the cognate endonuclease. The subunit structure of the M.AhdI methyltransferase resembles that of type I MTases, in contrast to the R.AhdI endonuclease which is typical of type II systems. AhdI appears to be a novel R-M system with properties intermediate between simple type II systems and more complex type I systems, and may represent an intermediate in the evolution of R-M systems.     

The Neisseria gonorrhoeae S.NgoVIII restriction/modification system: a type IIs system homologous to the Haemophilus parahaemolyticus HphI restriction/modification system.

Strains of Neisseria gonorrhoeae possess numerous restriction-modification (R-M) systems. One of these systems, which has been found in all strains tested, encodes the S. NgoVIII specificity (5'TCACC 3') R-M system. We cloned two adjacent methyltransferase genes (dcmH and damH), each encoding proteins whose actions protect DNA from digestion by R.HphI or R.Ngo BI (5'TCACC 3'). The damH gene product is a N 6-methyladenine methyltransferase that recognizes this sequence. We constructed a plasmid containing multiple copies of the S.NgoVIII sequence, grew it in the presence of damH and used the HPLC to demonstrate the presence of N 6-methyladenine in the DNA. A second plasmid, containing overlapping damH and Escherichia coli dam recognition sequences in combination with various restriction digests, was used to identify which adenine in the recognition sequence was modified by damH. The predicted dcmH gene product is homologous to 5-methylcytosine methyltransferases. The products of both the dcmH and damH genes, as well as an open reading frame downstream of the damH gene are highly similar to the Haemophilus parahaemolyticus hphIMC , hphIMA and hphIR gene products, encoding the Hph I Type IIs R-M system. The S.NgoVIII R-M genes are flanked by a 97 bp direct repeat that may be involved in the mobility of this R-M system.     

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.     

Molecular Characterization of the Lactococcus lactis LlaKR2I Restriction-Modification System and Effect of an IS982 Element Positioned between the Restriction and Modification Genes

The nucleotide sequence of the plasmid-encoded LlaKR2I restriction-modification (R-M) system of Lactococcus lactis subsp. lactis biovar diacetylactis KR2 was determined. This R-M system comprises divergently transcribed endonuclease (llaKR2IR) and methyltransferase (llaKR2IM) genes; located in the intergenic region is a copy of the insertion element IS982, whose putative transposase gene is codirectionally transcribed with llaKR2IM. The deduced sequence of the LlaKR2I endonuclease shared homology with the type II endonuclease Sau3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5′ GATC 3′. In addition, M·LlaKR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M·Sau3AI. Both of these proteins shared notable homology throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M·LlaKR2I, all remained undigested after treatment with Sau3AI despite the presence of multiple 5′ GATC 3′ sites. The combination of these data suggested that the specificity of the LlaKR2I R-M system was likely to be 5′ GATC 3′, with the cytosine residue being modified to 5-methylcytosine. The IS982 element located within the LlaKR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked by two 7-bp direct repeats. A perfect extended promoter consensus, which represented the likely original promoter of the llaKR2IR gene, was shown to overlap the direct repeat sequence on the other side of IS982. Specific deletion of IS982 and one of these direct repeats via a PCR strategy indicated that the LlaKR2I R-M determinants do not rely on elements within IS982 for expression and that the efficiency of bacteriophage restriction was not impaired.     

Esp1396I restriction-modification system: structural organization and mode of regulation

Esp1396I restriction–modification (RM) system recognizes an interrupted palindromic DNA sequ ence 5′-CCA(N)₅TGG-3′. The Esp1396I RM system was found to reside on pEsp1396, a 5.6 kb plasmid naturally occurring in Enterobacter sp. strain RFL1396. The nucleotide sequence of the entire 5622 bp pEsp1396 plasmid was determined on both strands. Identified genes for DNA methyltransferase (esp1396IM) and restriction endonuclease (esp1396IR) are transcribed convergently. The restriction endonuclease gene is preceded by the small ORF (esp1396IC) that possesses a strong helix-turn-helix motif and resembles regulatory proteins found in PvuII, BamHI and few other RM systems. Gene regulation studies revealed that C.Esp1396I acts as both a repressor of methylase expression and an activator of regulatory protein and restriction endonuclease expression. Our data indicate that C protein from Esp1396I RM system activates the expression of the Enase gene, which is co-transcribed from the promoter of regulatory gene, by the mechanism of coupled translation.     

Families of restriction enzymes: an analysis prompted by molecular and genetic data for type ID restriction and modification systems

Current genetic and molecular evidence places all the known type I restriction and modification systems of Escherichia coli and Salmonella enterica into one of four discrete families: type IA, IB, IC or ID. StySBLI is the founder member of the ID family. Similarities of coding sequences have identified restriction systems in E.coli and Klebsiella pneumoniae as probable members of the type ID family. We present complementation tests that confirm the allocation of EcoR9I and KpnAI to the ID family. An alignment of the amino acid sequences of the HsdS subunits of StySBLI and EcoR9I identify two variable regions, each predicted to be a target recognition domain (TRD). Consistent with two TRDs, StySBLI was shown to recognise a bipartite target sequence, but one in which the adenine residues that are the substrates for methylation are separated by only 6 bp. Implications of family relationships are discussed and evidence is presented that extends the family affiliations identified in enteric bacteria to a wide range of other genera.     

Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition.

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R-M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5' half site of the wild type site is repeated in inverse orientation. The additional base pair in the non-specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl-terminal DNA binding domain which recognizes the 3' half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino-terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R-M systems will be discussed.     

Characterization of thePac25I Restriction-Modification Genes Isolated from the Endogenous pRA2 Plasmid ofPseudomonas alcaligenesNCIB 9867

Genes for the class IIPseudomonas alcaligenesNCIB 9867 restriction-modification (R-M) system,Pac25I, have been cloned from its 33-kb endogenous plasmid, pRA2. ThePac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp. The deduced amino acid sequence of thePac25I methylase revealed significant similarity with theXcyI,XmaI,Cfr9I, andSmaI methylases. High sequence similarity was displayed between thePac25I endonuclease and theXcyI,XmaI, andCfr9I endonucleases which cleave between the external cytosines of the recognition sequence (i.e., 5′-C↓CCGGG-3′) and are thus perfect isoschizomers. However, no sequence similarity was detected between thePac25I endonuclease and theSmaI endonuclease which cleaves between the internal CpG of the recognition sequence (i.e., 5′-CCC↓GGG-3′). Both thePac25I methylase and endonuclease were expressed inEscherichia coli.An open reading frame encoding a protein which shows significant similarity to invertases and resolvases was located immediately upstream of thePac25I R-M operon. In addition, a transposon designated Tn5563was located 1531 bp downstream of the R-M genes. The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests horizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria.     

A Type IC Restriction-Modification System in Lactococcus lactis

Three genes coding for the endonuclease, methylase, and specificity subunits of a type I restriction-modification (R-M) system in the Lactococcus lactis plasmid pIL2614 have been characterized. Plasmid location, sequence homologies, and inactivation studies indicated that this R-M system is most probably of type IC.     

Inactivation of the SauI Type I Restriction-Modification System Is Not Sufficient To Generate Staphylococcus aureus Strains Capable of Efficiently Accepting Foreign DNA

Genetic manipulation of Staphylococcus aureus is limited by the availability of only a single strain, RN4220, that is capable of easily accepting foreign DNA. Inactivation of the hsdR gene of the SauI type I restriction-modification system was shown previously to be responsible for the high transformation efficiency of RN4220 (D. E. Waldron and J. A. Lindsay, J Bacteriol. 188:5578-5585, 2006). However, deletion of this gene in three different S. aureus strains was not sufficient to make them readily transformable, which would be remarkably useful for genetic studies of this pathogenic organism. These results indicate that another unknown factor(s) is required for the transformable phenotype in S. aureus.Staphylococcus aureus is a major pathogen that causes both nosocomial and community-acquired infections, which range from superficial skin infections to severe systemic diseases. It is an extremely versatile pathogen which has developed resistance to virtually all known classes of antibiotics and which expresses various virulence factors that allow it to cause infection in different environments.Molecular genetic studies of S. aureus have resulted in a better understanding of both the virulence and antibiotic resistance mechanisms of this organism, which is crucial for discovery of new approaches to treat staphylococcal infections. One of the limitations in genetically manipulating S. aureus is the fact that there is only a single strain available which can easily accept plasmid DNA isolated from Escherichia coli. This strain, RN4220, is a chemical mutant obtained in the early 1980s by highly mutagenizing NCTC8325-4 (= RN450) with nitrosoguanidine and selecting for a mutant that was able to accept and maintain S. aureus plasmids (10; B. Kreiswirth, personal communication).One of the most important bacterial defenses against uptake of foreign DNA is restriction-modification (R-M) systems. These systems, comprising restriction endonucleases and methyltransferases, recognize and modify specific DNA sequences, protecting “self” DNA from restriction while eliminating potentially harmful foreign DNA which lacks appropriate modification (12). There are three distinct well-characterized types of classical R-M systems, including type II restriction enzymes, which cut DNA within specific recognition sequences and are therefore widely used as molecular biology tools (3). S. aureus strains may contain different R-M systems, including the Sau3AI and Sau96I type II systems (present in isolates of particular lytic groups) (19, 20) or the Sau42I BcgI-like R-M system expressed by S. aureus φ42 lysogens (5). However, the only chromosomal R-M system widely distributed in the sequenced S. aureus isolates is the SauI type I system (21). Type I R-M systems require the products of three genes, hsdR (restriction), hsdM (modification), and hsdS (sequence specificity), and cut DNA at sites remote from the recognition sequence (12). The staphylococcal SauI system includes a single hsdR gene and two copies of the hsdM and hsdS genes, and there is substantial variation between the hsdS genes from different isolates (21). It was recently shown that transformable RN4220 carries a stop mutation in the sauI hsdR gene and that complementation with a functional copy of this gene restores a nontransformable phenotype (21). An hsdR mutant does not cleave foreign unmodified DNA, but it does modify incoming DNA, which therefore is not cleaved when it is transferred to other S. aureus strains that contain an identical R-M system. For this reason, RN4220 has become an essential intermediate for laboratory manipulation of S. aureus, despite its limited clinical relevance. DNA first introduced into RN4220 by electroporation can then be transferred into other laboratory strains (for example, by phage transduction).The relevance of the SauI type I R-M system for horizontal transfer of foreign DNA in different S. aureus isolates in nature has been confirmed with bovine isolates that are hypersusceptible to gene transfer from enterococci and have stop mutations in each of the two SauI hsdS gene copies (18). These “hyperrecipient” strains may be ideal backgrounds for the acquisition of new antibiotic resistance markers, such as the vanA gene complex present in vancomycin-resistant S. aureus strains (18). However, the existence of a second pathway in S. aureus that blocks horizontal transfer of foreign DNA or of an unknown but necessary factor essential for SauI activity has also been proposed based on the fact that some strains that are hypersusceptible to gene transfer (such as B111 used by Noble et al. [13]) do not have a mutation in any of the five hsd genes (18).The availability of laboratory and clinical strains other than RN4220 that are capable of accepting foreign DNA would be remarkably useful for genetic studies of S. aureus, including studies of virulence and antibiotic resistance mechanisms present only in relevant clinical isolates. Therefore, we have tried to reproduce the hsdR mutation in RN4220 in different backgrounds of widely used laboratory strains in order to generate useful, easily transformable strains.     

Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N6-adenine DNA methyltransferases of Neisseria meningitidis

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N⁶-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N⁶-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5′-CGY^m6AG-3′), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5′-AC^m6ACC-3′) and ModD1 (exemplified by M.Nme579I) (5′-CC^m6AGC-3′). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5′-CGY^m6AG-3′. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5′-GCGC^m6AGG-3′ sites, to 100% at 5′-ACGT^m6AGG-3′ sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.     

Identification of restriction endonucleases sensitive to 5-cytosine methylation at non-CpG sites,including expanded (CAG)n/(CTG)n repeats

Most epigenetic studies assess methylation of 5′-CpG-3′ sites but recent evidence indicates that non-CpG cytosine methylation occurs at high levels in humans and other species. This is most prevalent at 5′-CHG-3′, where H = A, C or T, and it preferentially occurs at 5′-CpA-3′ and 5′-CpT-3′ sites. With the goal of facilitating the detection of non-CpG methylation, the restriction endonucleases ApeKI, BbvI, EcoP15I, Fnu4HI, MwoI and TseI were assessed for their sensitivity to 5-methylcytosine at GpCpA, GpCpT, GpCpC or GpCpG sites, where methylation is catalyzed by the DNA 5-cytosine 5′-GpC-3′ methyltransferase M.CviPI. We tested a variety of sequences including various plasmid-based sites, a cloned disease-associated (CAG)83•(CTG)83 repeat and in vitro synthesized tracts of only (CAG)500•(CTG)500 or (CAG)800•(CTG)800. The repeat tracts are enriched for the preferred CpA and CpT motifs. We found that none of the tested enzymes can cleave their recognition sequences when they are 5′-GpC-3′ methylated. A genomic site known to convert its non-CpG methylation levels upon C2C12 differentiation was confirmed through the use of these enzymes. These enzymes can be useful in rapidly and easily determining the most common non-CpG methylation status in various sequence contexts, as well as at expansions of (CAG)n•(CTG)n repeat tracts associated with diseases like myotonic dystrophy and Huntington disease.Key words: non-CpG methylation, CpG methylation, 5-methylcytosine, trinucleotide repeats, ApeKI, BbvI, EcoP151, Fnu4HI, MwoI and TseI     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

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.