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Konstantin Severinov - One of the best experts on this subject based on the ideXlab platform.

  • effects of population dynamics on establishment of a Restriction Modification System in a bacterial host
    Molecules, 2019
    Co-Authors: Stefan Graovac, Konstantin Severinov, Andjela Rodic, M Djordjevic, Marko Djordjevic
    Abstract:

    In vivo dynamics of protein levels in bacterial cells depend on both intracellular regulation and relevant population dynamics. Such population dynamics effects, e.g., interplay between cell and plasmid division rates, are, however, often neglected in modeling gene expression regulation. Including them in a model introduces additional parameters shared by the dynamical equations, which can significantly increase dimensionality of the parameter inference. We here analyse the importance of these effects, on a case of bacterial Restriction-Modification (R-M) System. We redevelop our earlier minimal model of this System gene expression regulation, based on a thermodynamic and dynamic System modeling framework, to include the population dynamics effects. To resolve the problem of effective coupling of the dynamical equations, we propose a “mean-field-like” procedure, which allows determining only part of the parameters at a time, by separately fitting them to expression dynamics data of individual molecular species. We show that including the interplay between kinetics of cell division and plasmid replication is necessary to explain the experimental measurements. Moreover, neglecting population dynamics effects can lead to falsely identifying non-existent regulatory mechanisms. Our results call for advanced methods to reverse-engineer intracellular regulation from dynamical data, which would also take into account the population dynamics effects.

  • regulation of gene expression in Restriction Modification System eco29ki
    Nucleic Acids Research, 2011
    Co-Authors: Maxim Nagornykh, A S Protsenko, M V Zakharova, A S Solonin, E A Bogdanova, Konstantin Severinov
    Abstract:

    The Eco29kI Restriction-Modification (R-M) System consists of two partially overlapping genes, eco29kIR, encoding a Restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M System in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

  • transcription regulation of Restriction Modification System esp1396i
    Nucleic Acids Research, 2009
    Co-Authors: E A Bogdanova, M V Zakharova, Tomasz Heyduk, Geoff Kneale, Simon Streeter, James E Taylor, Konstantin Severinov
    Abstract:

    Restriction-Modification (R-M) System Ecl18kI is representative of R-M Systems whose coordinated transcription is achieved through a separate DNA-binding domain of the methyltransferase. M.Ecl18kI recognizes an operator sequence located in the noncoding region that separates the divergently transcribed R and M genes. Here we show that, contrary to previous predictions, the two ecl18kI promoters are not divergent, but actually face one another. The binding of M.Ecl18kI to its operator prevents RNA polymerase (RNAP) binding to the M promoter by steric exclusion, but has no direct effect on RNAP interaction with the R promoter. The start point for R transcription is located outside of the intergenic region, opposite the initiation codon of the M gene. Regulated transcription of the potentially toxic ecl18kI R gene is accomplished (i) at the stage of promoter complex formation, through direct competition from complexes formed at the M promoter, and (ii) at the stage of promoter clearance, since R promoter-bound RNAP escapes the promoter more slowly than RNAP bound to the M promoter.

  • regulation of gene expression in type ii Restriction Modification System
    Russian Journal of Genetics, 2008
    Co-Authors: Maxim Nagornykh, E S Bogdanova, A S Protsenko, M V Zakharova, A S Solonin, Konstantin Severinov
    Abstract:

    Type II Restriction-Modification Systems are comprised of a Restriction endonuclease and methyltransferase. The enzymes are coded by individual genes and recognize the same DNA sequence. Endonuclease makes a double-stranded break in the recognition site, and methyltransferase covalently modifies the DNA bases within the recognition site, thereby down-regulating endonuclease activity. Coordinated action of these enzymes plays a role of primitive immune System and protects bacterial host cell from the invasion of foreign (for example, viral) DNA. However, uncontrolled expression of the Restriction-Modification System genes can result in the death of bacterial host cell because of the endonuclease cleavage of host DNA. In the present review, the data on the expression regulation of the type II Restriction-Modification enzymes are discussed.

  • transcription regulation of the type ii Restriction Modification System ahdi
    Nucleic Acids Research, 2008
    Co-Authors: E A Bogdanova, Marko Djordjevic, Ioanna Papapanagiotou, Tomasz Heyduk, Geoff Kneale, Konstantin Severinov
    Abstract:

    The Restriction-Modification System AhdI contains two convergent transcription units, one with genes encoding methyltransferase subunits M and S and another with genes encoding the controller (C) protein and the Restriction endonuclease (R). We show that AhdI transcription is controlled by two independent regulatory loops that are well-optimized to ensure successful establishment in a naive bacterial host. Transcription from the strong MS promoter is attenuated by methylation of an AhdI site overlapping the -10 element of the promoter. Transcription from the weak CR promoter is regulated by the C protein interaction with two DNA-binding sites. The interaction with the promoter-distal high-affinity site activates transcription, while interaction with the weaker promoter-proximal site represses it. Because of high levels of cooperativity, both C protein-binding sites are always occupied in the absence of RNA polymerase, raising a question how activated transcription is achieved. We develop a mathematical model that is in quantitative agreement with the experiment and indicates that RNA polymerase outcompetes C protein from the promoter-proximal-binding site. Such an unusual mechanism leads to a very inefficient activation of the R gene transcription, which presumably helps control the level of the endonuclease in the cell.

A M Gibbins - One of the best experts on this subject based on the ideXlab platform.

  • type ii dna Restriction Modification System and an endonuclease from the ruminal bacterium fibrobacter succinogenes s85
    Journal of Bacteriology, 1992
    Co-Authors: S F Lee, Cecil W. Forsberg, A M Gibbins
    Abstract:

    Fibrobacter succinogenes is an important cellulolytic bacterium found in the rumen and cecum of herbivores. Numerous attempts to introduce foreign DNA into F. succinogenes S85 have failed, suggesting the presence of genetic barriers in this organism. Results from this study clearly demonstrate that F. succinogenes S85 possesses a type II Restriction endonuclease, FsuI, which recognizes the sequence 59-GG(A/T)CC-39. Analysis of the Restriction products on sequencing gels showed that FsuI cleaves between the two deoxyguanosine residues, yielding a 3-base 59 protruding end. These data demonstrate that FsuI is an isoschizomer of AvaII. A methyltransferase activity has been identified in the cell extract of F. succinogenes S85. This activity modified DNA in vitro and protected the DNA from the Restriction by FsuI and AvaII. DNA modified in vivo by a cloned methylase gene, which codes for M.Eco47II, also protected the DNA from Restriction by FsuI, suggesting that FsuI is inhibited by methylation at one or both deoxycytosine residues of the recognition sequence. The methyltransferase activity in F. succinogenes S85 is likely modifying the same deoxycytosine residues, but the exact site(s) is unknown. A highly active DNase (DNase A) was also isolated from the cell extract of this organism. DNase A is an endonuclease which showed high activity on all forms of DNA (single stranded, double-stranded, linear, and circular) but no activity on RNA. In vitro, the DNase A hydrolyzed F. succinogenes S85 DNA extensively, indicating the lack of protection against hydrolysis by this enzyme. In the presence of Mg2+, DNA was hydrolyzed to fragments of 8 to 10 nucleotides in length. The presence of DNase A and the type II Restriction-Modification System of F. succinogenes S85 may be the barriers preventing the introduction of foreign DNA into this bacterium. Images

M V Zakharova - One of the best experts on this subject based on the ideXlab platform.

  • an alternative approach to study the enzymatic specificities of the cfrbi Restriction Modification System
    Heliyon, 2019
    Co-Authors: M V Zakharova, Irina V Beletskaya, Elena M Ibryashkina, A S Solonin
    Abstract:

    Abstract Restriction–Modification Systems (RMS) are the main gene-engineering tools and a suitable model to study the molecular mechanisms of catalysis and DNA–protein interactions. Research into the catalytic properties of these enzymes, determination of hydrolysis and DNA-methylation sites remain topical. In our previous work we have cloned and sequenced the CfrBI Restriction–Modification System (strain Citrobacter freundii), which recognizes the nucleotide sequence 5′-CCWWGG-3′. In this article we describe the cloning of the methyltransferase and Restriction endonuclease genes (gene encoding CfrBI DNA methyltransferase (cfrBIM) and gene encoding CfrBI Restriction endonuclease (cfrBIR)) separately to obtain strains overproducing the enzymes of this System. His6-CfrBI, which had been purified to homogeneity, was used to establish the DNA-hydrolysis point in its recognition site. CfrBI was shown to cleave DNA after just the first 5′C within the recognition site and then to generate 4-nt 3′ cohesive ends (5′-C/CWWGG-3′). To map the site of methylation by M.CfrBI, we exploited the fact that the CfrBI site partially overlaps with the recognition sites of the well-documented enzymes KpnI and ApaI. The M.CfrBI- induced hemimethylation of the internal C residue of the ApaI recognition sequence (GGGCN4mCC) was observed to block cleavage by ApaI. In contrast, KpnI was able to digest its M.CfrBI-hemimethylated site (GGTAN4mCC). KpnI was used to restrict a fragment of DNA harbouring the CfrBI and KpnI sites, in which the CfrBI site was methylated in vitro by His6-M.CfrBI using [3H]-SAM. The subsequent separation of hydrolysis products by electrophoresis and the enumeration of incorporated [H3]-methyl groups in each of the fragments made it possible to determine that external cytosine undergoes Modification in the recognition site.

  • peculiarities of the regulation of gene expression in the ecl18ki Restriction Modification System
    Acta Naturae, 2013
    Co-Authors: Yu O Burenina, A S Protsenko, M V Zakharova, A S Solonin, E A Fedotova, Yu A Ryazanova, A S Karyagina, T S Oretskaya, E A Kubareva
    Abstract:

    ABSTRACT Transcription regulation in bacterial Restriction–Modification (R–M) Systems is an important proc-ess, which provides coordinated expression levels of tandem enzymes, DNA methyltransferase (MTase) and Restriction endonuclease (RE) protecting cells against penetration of alien DNA. The present study focuses on (cytosine-5)-DNA methyltransferase Ecl18kI (M.Ecl18kI), which is almost identical to DNA methyltrans-ferase SsoII (M.SsoII) in terms of its structure and properties. Each of these enzymes inhibits expression of the intrinsic gene and activates expression of the corresponding RE gene via binding to the regulatory site in the promoter region of these genes. In the present work, complex formation of M.Ecl18kI and RNA polymerase from Escherichia сoli with the promoter regions of the MTase and RE genes is studied. The mechanism of regulation of gene expression in the Ecl18kI R–M System is thoroughly investigated. M.Ecl18kI and RNA polymerase are shown to compete for binding to the promoter region. However, no direct contacts between M.Ecl18kI and RNA polymerase are detected. The properties of M.Ecl18kI and M.SsoII mutants are studied. Amino acid substitu-tions in the N-terminal region of M.Ecl18kI, which performs the regulatory function, are shown to influence not only M.Ecl18kI capability to interact with the regulatory site and to act as a transcription factor, but also its ability to bind and methylate the substrate DNA. The loss of methylation activity does not prevent MTase from performing its regulatory function and even increases its affinity to the regulatory site. However, the presence of the domain responsible for methylation in the M.Ecl18kI molecule is necessary for M.Ecl18kI to perform its regulatory function.KEYWORDS Restriction–Modification Systems; (cytosine-5)-DNA methyltransferase; DNA–protein interactions; transcriptional regulation.ABBREVIATIONS MTase – DNA methyltransferase; PAGE – polyacrylamide gel electrophoresis; RNAP – RNA polymerase; R–М System – Restriction–Modification System; RE – Restriction endonuclease; AdoMet – S-ad-enosyl-L-methionine; М.Ecl18kI – DNA methyltransferase Ecl18kI; М.SsoII – DNA methyltransferase SsoII; R.Ecl18kI – Restriction endonuclease Ecl18kI. Prefix “d” for designating deoxyribonucleosides, oligodeoxyribo-nucleotides, and DNA duplexes is omitted.INTRODUCTIONRestriction–Modification (r–M) Systems are abundant in bacterial cells; they contain genes that encode re-striction endonucleases (re) and DnA methyltrans-ferases (Mtases). re hydrolyzes a certain sequence in a double-stranded DnA (dsDnA), while Mtase meth-ylates the same sequence at a strictly determined po-sition, thus preventing its cleavage by re. the r–M functions as a primitive immune System that protects a host bacterium from penetration by alien DnA: re hydrolyses the intruding DnA that is not methylated by the corresponding Mtase [1]. the activity levels of

  • regulation of gene expression in Restriction Modification System eco29ki
    Nucleic Acids Research, 2011
    Co-Authors: Maxim Nagornykh, A S Protsenko, M V Zakharova, A S Solonin, E A Bogdanova, Konstantin Severinov
    Abstract:

    The Eco29kI Restriction-Modification (R-M) System consists of two partially overlapping genes, eco29kIR, encoding a Restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M System in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

  • transcription regulation of Restriction Modification System esp1396i
    Nucleic Acids Research, 2009
    Co-Authors: E A Bogdanova, M V Zakharova, Tomasz Heyduk, Geoff Kneale, Simon Streeter, James E Taylor, Konstantin Severinov
    Abstract:

    Restriction-Modification (R-M) System Ecl18kI is representative of R-M Systems whose coordinated transcription is achieved through a separate DNA-binding domain of the methyltransferase. M.Ecl18kI recognizes an operator sequence located in the noncoding region that separates the divergently transcribed R and M genes. Here we show that, contrary to previous predictions, the two ecl18kI promoters are not divergent, but actually face one another. The binding of M.Ecl18kI to its operator prevents RNA polymerase (RNAP) binding to the M promoter by steric exclusion, but has no direct effect on RNAP interaction with the R promoter. The start point for R transcription is located outside of the intergenic region, opposite the initiation codon of the M gene. Regulated transcription of the potentially toxic ecl18kI R gene is accomplished (i) at the stage of promoter complex formation, through direct competition from complexes formed at the M promoter, and (ii) at the stage of promoter clearance, since R promoter-bound RNAP escapes the promoter more slowly than RNAP bound to the M promoter.

  • regulation of gene expression in type ii Restriction Modification System
    Russian Journal of Genetics, 2008
    Co-Authors: Maxim Nagornykh, E S Bogdanova, A S Protsenko, M V Zakharova, A S Solonin, Konstantin Severinov
    Abstract:

    Type II Restriction-Modification Systems are comprised of a Restriction endonuclease and methyltransferase. The enzymes are coded by individual genes and recognize the same DNA sequence. Endonuclease makes a double-stranded break in the recognition site, and methyltransferase covalently modifies the DNA bases within the recognition site, thereby down-regulating endonuclease activity. Coordinated action of these enzymes plays a role of primitive immune System and protects bacterial host cell from the invasion of foreign (for example, viral) DNA. However, uncontrolled expression of the Restriction-Modification System genes can result in the death of bacterial host cell because of the endonuclease cleavage of host DNA. In the present review, the data on the expression regulation of the type II Restriction-Modification enzymes are discussed.

Ichizo Kobayashi - One of the best experts on this subject based on the ideXlab platform.

  • Restriction Modification System with methyl inhibited base excision and abasic site cleavage activities
    Nucleic Acids Research, 2015
    Co-Authors: Masaki Fukuyo, Yoshikazu Furuta, Ken Ishikawa, Miki Watanabematsui, Toshiaki Nakano, Yingbiao Zhang, Hirokazu Yano, Takeshi Hamakawa, Hiroshi Ide, Ichizo Kobayashi
    Abstract:

    The Restriction-Modification Systems use epigenetic Modification to distinguish between self and nonself DNA. A Modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate Restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the Restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a Restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5′-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5′-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA–R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes Restriction because the Restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

  • A sequence-specific DNA glycosylase mediates Restriction-Modification in Pyrococcus abyssi
    Nature Communications, 2014
    Co-Authors: Ken-ichi Miyazono, Ichizo Kobayashi, Yoshikazu Furuta, Miki Watanabe-matsui, Takuya Miyakawa, Tomoko Ito, Masaru Tanokura
    Abstract:

    Restriction enzymes are generally thought to act as sequence-specific endonucleases. Here Miyazono et al. demonstrate that one of these enzymes, R.PabI, is instead an adenine DNA glycosylase, expanding the known mechanisms by which a Restriction-Modification System can operate.

  • evolutionary genome engineering using a Restriction Modification System
    Nucleic Acids Research, 2011
    Co-Authors: Yoko Asakura, Hiroyuki Kojima, Ichizo Kobayashi
    Abstract:

    Modification of complex microbial cellular processes is often necessary to obtain organisms with particularly favorable characteristics, but such experiments can take many generations to achieve. In the present article, we accelerated the experimental evolution of Escherichia coli populations under selection for improved growth using one of the Restriction–Modification Systems, which have shaped bacterial genomes. This resulted in faster evolutionary changes in both the genome and bacterial growth. Transcriptome/genome analysis at various stages enabled prompt identification of sequential genome rearrangements and dynamic gene-expression changes associated with growth improvement. The changes were related to cell-to-cell communication, the cell death program, as well as mass production and energy consumption. These observed changes imply that improvements in microorganism population growth can be achieved by inactivating the cellular mechanisms regulating fraction of active cells in a population. Some of the mutations were shown to have additive effects on growth. These results open the way for the application of evolutionary genome engineering to generate organisms with desirable properties.

  • antisense rna associated with biological regulation of a Restriction Modification System
    Nucleic Acids Research, 2011
    Co-Authors: Iwona Mruk, Yaoping Liu, Ichizo Kobayashi
    Abstract:

    Restriction–Modification Systems consist of a Modification enzyme that methylates a specific DNA sequence and a Restriction endonuclease that cleaves DNA lacking this epigenetic signature. Their gene expression should be finely regulated because their potential to attack the host bacterial genome needs to be controlled. In the EcoRI System, where the Restriction gene is located upstream of the Modification gene in the same orientation, we previously identified intragenic reverse promoters affecting gene expression. In the present work, we identified a small (88nt) antisense RNA (Rna0) transcribed from a reverse promoter (PREV0) at the 3 0 end of the Restriction gene. Its antisense transcription, as measured by transcriptional gene fusion, appeared to be terminated by the PM1,M2 promoter. PM1,M2 promoter-initiated transcription, in turn, appeared to be inhibited by PREV0. Mutational inactivation of PREV0 increased expression of the Restriction gene. The biological significance of this antisense transcription is 2-fold. First, a mutation in PREV0 increased Restriction of incoming DNA. Second, the presence of the antisense RNA gene (ecoRIA) in trans alleviated cell killing after loss of the EcoRI plasmid (post-segregational killing). Taken together, these results strongly suggested the involvement of an antisense RNA in the biological regulation of this Restriction–Modification System.

  • is linked movement of a Restriction Modification System
    PLOS ONE, 2011
    Co-Authors: Noriko Takahashi, Seishi Ohashi, Marat R Sadykov, Yoko Mizutaniui, Ichizo Kobayashi
    Abstract:

    Potential mobility of Restriction-Modification Systems has been suggested by evolutionary/bioinformatic analysis of prokaryotic genomes. Here we demonstrate in vivo movement of a Restriction-Modification System within a genome under a laboratory condition. After blocking replication of a temperature-sensitive plasmid carrying a PaeR7I Restriction-Modification System in Escherichia coli cells, the plasmid was found integrated into the chromosome of the surviving cells. Sequence analysis revealed that, in the majority of products, the Restriction-Modification System was linked to chromosomal insertion sequences (ISs). Three types of products were: (I) apparent co-integration of the plasmid and the chromosome at a chromosomal IS1 or IS5 copy (24/28 analyzed); (II) de novo insertion of IS1 with the entire plasmid except for a 1–3 bp terminal deletion (2/28); and (III) reciprocal crossing-over between the plasmid and the chromosome involving 1–3 bp of sequence identity (2/28). An R-negative mutation apparently decreased the efficiency of successful integration by two orders of magnitude. Reconstruction experiments demonstrated that the Restriction-dependence was mainly due to selection against cells without proper integration: their growth was inhibited by the Restriction enzyme action. These results demonstrate collaboration of a mobile element and a Restriction-Modification System for successful joint migration. This collaboration may have promoted the spread and, therefore, the long-term persistence of these complexes and Restriction-Modification Systems in a wide range of prokaryotes.

Kelli L Palmer - One of the best experts on this subject based on the ideXlab platform.

  • a type i Restriction Modification System associated with enterococcus faecium subspecies separation
    Applied and Environmental Microbiology, 2019
    Co-Authors: Wenwen Huo, Hannah M Adams, Cristian Trejo, Rohit Badia, Kelli L Palmer
    Abstract:

    Enterococcus faecium is a leading cause of hospital-acquired infections around the world. Rising antibiotic resistance in certain E. faecium lineages leaves fewer treatment options. The overarching aim of this work was to determine whether Restriction-Modification (R-M) Systems contribute to the structure of the E. faecium species, wherein hospital-epidemic and non-hospital-epidemic isolates have distinct evolutionary histories and highly resolved clade structures. R-M provides bacteria with a type of innate immunity to horizontal gene transfer (HGT). We identified a type I R-M System that is enriched in the hospital-epidemic clade and determined that it is active for DNA Modification activity and significantly impacts HGT. Overall, this work is important because it provides a mechanism for the observed clade structure of E. faecium as well as a mechanism for facilitated gene exchange among hospital-epidemic E. faecium isolates.

  • a type i Restriction Modification System associated with enterococcus faecium subspecies separation
    bioRxiv, 2018
    Co-Authors: Wenwen Huo, Hannah M Adams, Cristian Trejo, Rohit Badia, Kelli L Palmer
    Abstract:

    The gastrointestinal colonizer Enterococcus faecium is a leading cause of hospital-acquired infections. Multidrug-resistant (MDR) E. faecium are particularly concerning for infection treatment. Previous comparative genomic studies revealed that subspecies referred to as Clade A and Clade B exist within E. faecium. MDR E. faecium belong to Clade A, while Clade B consists of drug-susceptible fecal commensal E. faecium. Isolates from Clade A are further grouped into two sub-clades, A1 and A2. In general, Clade A1 isolates are hospital epidemic isolates whereas Clade A2 isolates are isolates from animals and sporadic human infections. Such phylogenetic separation indicates that reduced gene exchange occurs between the clades. We hypothesize that endogenous barriers to gene exchange exist between E. faecium clades. Restriction-Modification (R-M) Systems are such barriers in other microbes. We utilized bioinformatics analysis coupled with second generation and third generation deep sequencing platforms to characterize the methylome of two representative E. faecium strains, one from Clade A1 and one from Clade B. We identified a Type I R-M System that is Clade A1-specific, is active for DNA methylation, and significantly reduces transformability of Clade A1 E. faecium. Based on our results, we conclude that R-M Systems act as barriers to horizontal gene exchange in E. faecium and propose that R-M Systems contribute to E. faecium subspecies separation.