Transfer of mobile genetic elements between prokaryotes is limited by restriction-modification systems. Restriction-modification systems consist of a modification enzyme that epigenetically methylates a specific DNA sequence, and a restriction endonuclease (restriction enzyme) that cuts DNA lacking this epigenetic mark. These elements were discovered because they attack mobile genetic elements. However, recent studies have revealed that they are themselves mobile. In some cases, the mobility of restriction-modification systems is through symbiosis with other forms of mobile elements. In other cases, movement is unlinked to other mobile elements. The systems may insert into the genome with long and variable target duplication, or into the intergenic region of an operon. Insertion of restriction-modification systems induces other genome rearrangements such as amplification and inversion. Even a domain within a protein can be the unit of mobility: some restriction-modification system subunits that recognize a target DNA sequence contain mobile amino acid sequences that can apparently move between different domains of a protein through recombination of DNA sequences encoding them. This mobility extends the biological significance of restriction-modification systems beyond defense: the systems define, and sometimes even force, epigenetic order on a genome. The multilevel conflicts involving these mobile epigenetic elements may drive prokaryotic evolution.

Introduction: Restriction-Modification Systems in Epigenetic Conflicts

DNA methyltransferases methylate specific bases in recognition sequences and generate three types of modified base: 5-methylcytosine (m5C), N4-methylcytosine (m4C), and N6-methyladenine (m6A). This is a type of epigenetic modification because it is passed on through maintenance methylation after DNA replication.¹ Epigenetic DNA methylation can be involved in gene regulation, and variation in DNA methyltransferases can potentially provide diversity in the gene expression status of the prokaryotic cell.

Many prokaryotic DNA methyltransferases are paired with restriction enzymes, which were first discovered through their ability to restrict phage infection.² Restriction enzymes are DNA endonucleases that recognize specific DNA sequences and introduce a break (Fig. 1A). This activity restricts the establishment of invading DNAs that lack proper DNA methylation, such as bacteriophage DNA genomes, plasmids, and DNA fragments delivered through natural transformation machinery (Fig. 1B). The potentially lethal cleavage of cellular DNA in cells that harbor a restriction enzyme is prevented by epigenetic DNA methylation by the cognate DNA methyltransferase (Fig. 1A,B). Genes encoding the restriction enzyme and the methyltransferase are often located next to each other and form a unit called a restrictionmodification (RM) system. RM systems are classified into four types, Type I, II, III, and IV, based on their genetic and biochemical characteristics.

Figure 1.

Type II restriction-modification system. A) A modification enzyme and a restriction endonuclease of an RM system. B) Attack on incoming DNA. C) A hypothetical life cycle. D) Epigenetic conflicts and postsegregational killing involving RM systems. DNA (more...)

The primary biological significance of RM systems is often assumed to be their activity as a defense system for host cells against invading DNA such as bacteriophages. However, an RM system will not defend a bacterial cell from invasion of DNA from a bacterial cell carrying the same RM system. An RM system will attack invading DNA only when it lacks its epigenetic mark. The essence of the RM phenomena is conflict involving an epigenetic system rather than defense against invading DNA. In response to violation of epigenetic status by invasion of foreign DNA or loss of an RM system, RM systems induce DNA breakage and, consequently, reactions such as DNA damage repair, cell death and genome rearrangements (Fig. 1C,D).

RM systems are themselves mobile and can therefore be designated epigenetic mobile elements. This review introduces the mobility of RM systems first and then the conflicts between RM systems and their host cells and between RM systems themselves. These conflicts, which can involve host cell death, affect the conservation or change of the host epigenetic status.

Abundance of RM systems

RM gene homologs that have been identified in completely sequenced bacterial genomes are in the REBASE database.³ Some genomes—for example, those from Hemophilus influenzae, Methanococcus jannaschii, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, and Xylella fastidiosa—have large numbers of RM gene homologs, although many RM gene homologs are specific to a single strain within a given species. No RM system is native to eukaryotes, although a family of viruses that use the unicellular eukaryotic alga Chlorella as a host produce RM systems.⁴

Types of Restriction Systems

RM systems are currently classified into four types (I-IV), each with a unique mechanism for target recognition (Fig. 2).² Type I systems consist of R, M and specificity (S) subunit genes. Formation of a multisubunit structure is necessary for modification (SM) and restriction (SMR) activities.⁵ Sequence recognition is determined by the target recognition domains (TRDs) in the S subunit. Each of the two TRDs, TRD1 and TRD2, recognizes one half of a bipartite target sequence.⁶ DNA methylation takes place within the recognition sequence, whereas the cleavage site is at a variable distance from the recognition sequence. After binding to an unmodified recognition sequence, restriction enzyme complexes are thought to translocate DNA toward themselves from both directions, in a reaction coupled to ATP hydrolysis. DNA is cleaved where two restriction enzyme complexes meet.⁷

Figure 2.

Gene organization of various types of restriction-modification systems. See text for explanation. TRD: target recognition domain.

Type II systems consist of separate R and M enzymes, which independently recognize a target sequence and catalyze reactions.^2,8 M proteins have amino acid sequence motifs that are common to DNA methyltransferases and are well conserved, and their target recognition domain can be easily identified.⁹ R proteins share much less similarity.¹⁰

RM systems of the Type IIG subgroup are defined by the presence of a single polypeptide that apparently results from a fusion of R and M proteins. This Type IIG subgroup can be further classified into two subgroups by sequence similarity to either Type II or Type I systems.¹¹ The class similar to Type II (Fig. 2C) has the same TRD structure as typical Type II M enzymes within an RM polypeptide.^2,11-13 The other class similar to Type I, such as AloI,¹⁴ has a fused S subunit in addition to the RM fusion polypeptide (Fig. 2D). As in Type I systems, the S subunit counterpart has two TRDs. Although R and M are always fused in Type IIG systems, the S subunit is sometimes separated from the RM fused gene (Fig. 2E).¹⁵

Type III systems consist of res and mod genes (Fig. 2F). The mod gene product has modification activity by itself, while the complex of the two gene products has restriction enzyme activity.¹⁶ The Mod subunit is responsible for target recognition and its TRD can be easily identified, similar to M enzymes of Type II systems.^17-19

Type IV systems contain a class of enzymes that cleave DNA only when the recognition site is methylated (Fig. 2F).² McrA, McrBC, and Mrr are prototype Escherichia coli enzymes in this class that show different restriction spectra.^1,20-23

Gene Organization

There are many variations to the simplified schemes above.³ A Type II system may have two different modification enzymes for the top and bottom strands of the recognition sequence. It may carry two different nucleases for the different strands. Some RM systems have a gene for a regulatory protein (C protein, Fig. 3A, also see below).

Figure 3.

Gene order in restriction–modification systems. A) PvuII RM system, which has a control (C) gene in addition to R and M genes. B) Type I RM system in Staphylococcus aureus. A single R gene may interact with two sets of M and S genes. C) Type I (more...)

The relative direction of the constituent genes can vary. In the simplest case of a Type II system with one R gene and one M gene, the two genes may lie in the same orientation, or in opposite orientations.

In most cases, genes in an RM system are clustered into a single locus, but in some cases they are unlinked or in a more complex context.²⁴ In a Type I system of Staphylococcus aureus, one pair of M and S genes lie on one genomic island, another M and S pair are on another genomic island, and an R gene is outside of both islands (Fig. 3B). The hypothesis is that the R gene product can associate with either MS pair to form a two-faced restriction enzyme that recognizes two different sequences.²⁵ Lactococcus lactis has plasmids that carry an S gene but not an R or an M gene (Fig. 3C). The plasmid-encoded S gene products form active Type I RM systems with R and M products from genes on the chromosome.²⁶ In Mycoplasma pulmonis, two S genes in opposing orientation flank an R gene and an M gene in the inverted orientation (Fig. 3D). Recombination between the two S genes is discussed below.²⁷

Mobility of RM Systems Revealed from Molecular Evolutionary Analyses

Evolutionary analyses suggest that RM genes have undergone extensive horizontal transfer between different groups of microorganisms.^28,29 Early studies found that close homologs occur in distantly related organisms such as Eubacteria and Archaea (archaebacteria).³⁰ Extensive sequence alignment and phylogenetic tree construction now provide strong support for this point.^31-34 Additional evidence for extensive horizontal transfer of RM genes comes from incongruencies between methyltransferase phylogenetic trees and rRNA gene trees from the same species.³¹ Moreover, the GC content and/or codon usage of RM genes often differs from the majority of other genes in the genome.^31,35-37 This indicates that some RM genes may have joined the genome relatively recently through horizontal transfer from distantly related bacteria.

RM Systems on Mobile Genetic Elements

Genome comparisons revealed that the RM systems are mobile and involved in genome rearrangements. In some cases, mobility is acquired by carriage on other mobile elements (Table 1) such as plasmids,^38-45 phages,^45-52 conjugative elements/genomic islands,^25,53-57 transposons,^58-60 and integrons.^61,62 Indeed, some RM systems are located immediately adjacent to mobility-related genes such as those for transposases, integrases, or resolvases.^63-66 RM systems are also found that form a composite transposon by insertion of insertion sequence (IS) to both sides.⁶⁷

Table 1.

RM systems on mobile genetic elements.

The mobile elements themselves can be lost from the genome. Carrying a Type II RM system may allow their stable maintenance through postsegregational killing, as demonstrated for plasmids.^68,69 This may represent the symbiosis of two genetic elements that provides mutual benefits of maintenance and mobility.

RM Systems as Mobile Genetic Elements

In other cases, RM systems are found inserted in the genome, but not linked to a mobile element (Table 2). They sometimes insert into an operon-like gene cluster (Fig. 4A (i))^70-72 by a recombination process that could be related to the DNA cleavage activity of the restriction enzyme.⁷³ Insertion might give a competitive advantage to the operon as to the mobile elements. Some RM systems are substituted by another RM system at the same locus.^70,74-76

Table 2.

Genome rearrangements associated with RM systems.

Figure 4.

Restriction-modification systems and genome rearrangements. A) Various patterns of genome rearrangements linked to an RM system (or a DNA methyltransferase gene (vi)). B) A transposon-like RM system. A Type II RM gene pair flanked by long (65 bp), imperfect (more...)

In Helicobacter pylori, insertion of several RM systems has occurred with the duplication of a long target sequence (Fig. 4A (iii)).⁷³ The generality of this mode was confirmed by systematic comparison analysis of RM systems on the completely sequenced genomes.⁷⁴ This survey also led to the discovery of RM systems with a transposon-like structure where RM genes replaced the transposase gene (Fig. 4B).⁷⁴

Attack on the Host Bacterial Genome: Type II RM Systems

Works have demonstrated that RM systems also act as a watchdog maintaining epigenetic order in cells (Fig. 5). Alteration in the epigenetic status might lead to double-strand breakage of the self genome by the restriction enzyme,^34,68,77,78 which can result in cell death or genome rearrangements. This may eliminate unstable cells and maintain the epigenome status.

Figure 5.

Host attack by restriction systems in conflict with epigenetic systems. A) Postsegregational killing by Type II systems. When a resident RM gene complex is replaced by a competitor genetic element, a decrease in the modification enzyme level exposes newly (more...)

Some Type II RM systems cause chromosomal cleavage of their host cells when their genes are eliminated, for example, by a competitor genetic element (Figs. 1D and 5A).⁶⁸ When an RM system is stably maintained in a cell, the restriction enzyme does not cleave the genomic DNA because of protection through epigenetic methylation by the cognate methyltransferases. However, when the RM system is lost, the concentration of the restriction and modification enzymes is decreased through cell division,⁷⁹ resulting in undermethylated sites on newly replicated chromosomes.⁸⁰ The remaining restriction enzyme molecules cleave the unmethylated recognition sequence and cause cell death. The net result is survival of cells that were not invaded by the competitor. This process is called "postsegregational killing" or "genetic addiction".²⁴ The capability of an RM system in forcing maintenance on its host can become stronger by a mutation in its methyltransferase.⁸¹

Figure 1E visualizes the effect of postsegregational killing during the formation of bacterial colony. An unstable plasmid in the bacterial cell is lost during colony formation and leads to formation of papillae (Fig. 1E, left). However, when the EcoRI RM system is present on the plasmid, no papillae are formed because the plasmid-free cells are killed (Fig. 1E, right).

Postsegregational killing occurs because of a conflict between the RM system (or the plasmid) and the host bacteria and is an example of intragenomic conflicts. A theoretical work demonstrated that starting from very few copies, a postsegregational killing gene can increase in a population in the presence of a spatial structure (Fig. 1F (ii)). In the absence of the spatial structure, the gene is quickly lost unless it is abundant at the beginning (Fig. 1F (i)).⁸²

Post-segregational cell killing by one RM system is inhibited by the presence of another RM system recognizing the same DNA sequence, because the M protein of the latter system protects the genome from cleavage by the R protein of the former system.⁸³ This indicates the presence of competition for recognition sequences between RM systems. Thus, a recognition sequence of RM systems defines an incompatibility group. This competition explains the individual specificity and collective diversity in RM systems' sequence recognition. The competition may be one-sided when the recognition sequence of one RM is included in the recognition sequence of the other RM.⁸⁴ Another incompatibility relationship between RM systems is found in a regulatory protein that delays expression of the R protein upon entry of an RM system into a new host bacterial cell.⁸⁵

Recent studies revealed a common pathway of stress-induced cell death in bacteria.^86,87 Transcriptome analysis during postsegregational killing by a Type II RM system revealed its similarity to killing by several bacteriocidal antibiotics.⁷⁸ Thus, RM systems switch on the death pathway intrinsic to the host bacterial cells. Gene products that program bacterial cell death, such as the restriction enzymes discussed here, are likely to work upstream of the common cell death pathway.

Attack on the Host Bacterial Genome: Type IV Restriction Systems

Several studies demonstrated that phages or plasmids carrying a DNA methyltransferase gene cannot be propagated in an strain of E. coli which harbors mcrBC, a methylated DNA specific restriction enzyme.⁸⁸ Whether the block to propagation is caused by repeated methylation and subsequent cleavage of the introduced DNA,⁸⁸ or to host genome methylation and its cleavage was not known. Fukuda et al. demonstrated that McrBC inhibits establishment of the gene for the DNA methyltransferase PvuII (M.PvuII, 5′CAGm4CTG) in E. coli,³⁴ even when the gene is on a plasmid lacking its recognition sequence. This result suggests that the transferred DNA does not need to have methylated sites for McrBC-dependent inhibition,³⁴ suggesting that host genome cleavage accompanied by cell death inhibits the establishment of the methyltransferase gene (Fig. 5B). The underlying mechanism of cell death was revealed by observing E. coli chromosomal DNA infected with lambda phage carrying the M.PvuII gene.³⁴ Accumulation of large linear DNAs corresponding to broken chromosomes, and smaller DNAs of variable size was observed, which likely reflected chromosomal degradation. The mcrBC-dependence strongly suggests that M.PvuII-mediated chromosomal methylation triggers chromosomal cleavage by McrBC, followed by chromosomal degradation. This, in turn, indicates that inhibition of phage multiplication (restriction) is caused by host death.³⁴ This type of conflict between DNA methyltransferase genes carried by bacteriophages and methyl-specific restriction enzymes is biologically relevant because DNA methyltransferase genes are often found in bacteriophage genomes.^89-92

In addition to M.PvuII, the M.SinI (GGWm⁵CC) and M.MspI (m⁵CCGG) cause McrBC-dependent cell death, whereas M.SsoII (Cm⁵CNGG) does not. These results are consistent with the RmC sequence specificity of McrBC observed in vitro.⁹³ McrBC has the potential to act as a defense system against many DNA methyltransferases with an appropriate specificity. Such conflicts between McrBC and invading epigenetic DNA methylation systems may have driven diversification of sequence recognition by the methyltransferases and by the McrBC family.⁹⁴

Type II systems cause cell death when a particular mode of epigenetic DNA methylation decreases, while this Type IV system causes cell death when an epigenetic DNA methylation mode increases. Nonetheless, the result of both systems is the maintenance of an epigenetic order defined by DNA methylation (Fig. 5).

The DNA replication fork may be the site of action of McrBC, which can cleave a model DNA replication fork in vitro.⁷⁷ Cleavage of a fork requires methylation on both arms and results in removal of one or both arms. Most cleavage events remove the methylated sites from the fork. This suggests that acquisition of even rare modification patterns will be recognized and rejected efficiently by modification-dependent restriction systems that recognize two sites.

Attack on the Host Bacterial Genome: Type I RM Systems

Restriction alleviation is a phenotypic decrease in restriction activity against invading DNA that can be induced by DNA-damaging agents; this also occurs constitutively in some bacterial mutants. The underlying mechanisms vary among the restriction system types.^95-98 Restriction alleviation is proposed to be a mechanism for protecting chromosomes from restriction at newly generated replication forks that produce unmethylated restriction sites.⁵

A recent work demonstrated that a Type I restriction enzyme cleaves model replication forks at their branch point in vitro.⁷⁷ Cleavage was dependent on a recognition sequence on one of the arms and was inhibited when the site was hemi-methylated. The results suggested that the enzyme binds to DNA at the recognition sequence and tracks along the DNA, cleaving when it encounters a branch point.

Fork cleavage may take place on chromosomal DNA under conditions of extra replication initiation. From an unmethylated recognition sequence, the restriction enzyme tracks on the DNA. If the fork is moving forward during replication, DNA cleavage might not occur. However, when the enzyme meets an arrested replication fork, one arm is cleaved, possibly leading to cell death or a round of repair through recombination and replication. This mechanism might lead to elimination of cells with an unstable genome and to maintenance of an intact genome.²⁶ This hypothesized function is similar to that of programmed cell death in multicellular organisms.

Anti-Restriction Systems

The anti-restriction modification systems evolved by bacteriophages represent examples of co-evolution with the host bacterium.¹⁶ Host bacteria cells have evolved anti-restriction features, such as solitary methyltransferases that protect restriction sites from lethal attack by an RM system.^81,99 Another sign of adaptation, restriction avoidance, is discussed below.

RM Systems and Genome Rearrangements

The attack of RM systems on the host genome induces repair by recombination and replication and may induce genome rearrangements (Fig. 4A). Systematic genome comparison shows the involvement of RM systems in genome rearrangements (Table 2). RM systems can be inserted into the genomes with other mobile elements (see above) or inserted by themselves with long target duplication (Fig. 4A (iii)).^73,74 A large genome inversion event is seen in the neighborhood of RM insertion in Helicobacter pylori (Fig. 4A (v)).¹⁰⁰ Activity of an RM system likely induced unequal homologous recombination at IS3 sequences in E. coli, causing genome-wide rearrangements.⁸⁰ A methyltransferase gene in Helicobacter pylori is linked to an event of DNA duplication associated with inversion (Fig. 4A (vi)), a mechanism formally similar to replicative inversion in several DNA transposons.¹⁰¹ A Type II restriction-modification system accelerates genome and phenotype changes in bacterial experimental evolution.¹⁰²

We found that the bamHI gene complex, flanked by long direct repeats, amplifies in the Bacillus subtilis chromosome in its restriction activity dependent manner.¹⁰³ These results led us to propose that RM gene complexes increase in frequency in the cell population in a life cycle similar to that of a DNA virus (Fig. 1C). Insertion with long target duplication (Fig. 4A (iii), discussed above) results in formation of direct repeats flanking an RM system, giving it the potential for amplification.

Impact on Genome Evolution

The presence of an RM gene complex¹⁰⁴ and the action of a restriction enzyme¹⁰⁵ induce the SOS response, as does the action of a methylated DNA-specific endonuclease.¹⁰⁶ Global mutagenesis generates heterogeneity in the cell population, similar to other stressful conditions,¹⁰⁷ and may help the survival of the genome. These mechanisms could contribute to the evolution of restriction avoidance (discussed below) and to the inactivation of a RM gene complex. Genomes show signs of strong selection against restriction sites. Systematic avoidance of potential restriction sites (palindromes) in bacterial genomes is called restriction avoidance and has been characterized by informatics.^108-110 This is likely the result of selection after host attack by RM systems. (However, evaluating the contribution, if any, of selection by restriction attacks on groups of transferred genomic DNAs is difficult.) In many genomes, restriction avoidance is more pronounced in the genome proper than in prophages.^109,110 This is probably because the genome has experienced long-term selection by RM systems, while the prophages, as newcomers, have not yet undergone selection.^109,110 This restriction avoidance would lead to a decrease in the virulence of the particular RM system against the genome, which is almost always beneficial for the host and can be beneficial to the RM system itself under some conditions.

DNA methylation may locally increase mutation. 5-methylcytosine shows a higher rate of deamination than cytosine. Its deamination at the C/G pair in duplex DNA results in the formation of thymine and hence the generation of a T/G mispair.^111,112 Very short patch mismatch correction by a vsr gene linked to a solitary cytosine methyltransferase dcm in E. coli, can repair the mispair generated by methylation by dcm product and restore the mC/G pair.¹¹² A homolog of the vsr gene is linked to several RM genes that produce 5-methylcytosine,³ some of which are active. Local mutagenesis induced by the action of the RM systems leads to loss of the restriction sites in the genome, which can be beneficial to the host. The anti-mutagenesis activity of Vsr homologs linked to RM systems will prevent this loss and may be of immediate benefit to the RM system.

Domain Sequence Movement in the Specificity Subunit

The above results revealed that RM systems have many features as mobile genetic elements. Recent work demonstrates that parts of RM system genes are variable and even mobile.

DNA sequence recognition of the Type I RM system specificity subunit is mediated by TRD1 and TRD2. These TRDs show diversity among strains, consistent with the diversification of recognition sequences.^113-117 In some cases, diversification occurred by a specific genome rearrangement mechanism. In Mycoplasma pulmonis, two S genes flank the R and M genes, which are in an inverted orientation and prone to recombine with each other, resulting in TRD shuffling.²⁷ In Lactococcus lactis, two copies of the S genes are on different plasmids, interacting through homologous recombination to create two chimeric S genes for one RM system with shuffled recognition sequences.¹¹⁸ S genes tolerates exchange of the sequences between TRD1 and TRD2 by circular permutation.¹¹⁹ Weak sequence similarity (36% identity in amino acid sequence) was detected between TRD1 and TRD2 of from different species, that is TRD1 of StySKI from Salmonella enterica and TRD2 of EcoR124I from Escherichia coli.¹²⁰

Surprisingly, several sequences are shared by TRD1 and TRD2 genes at the same locus in several bacteria: these domain sequences appear to have moved between two positions within a single protein (Fig. 6).¹²¹ The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y are repeated. Movement probably occurs by recombination at these flanking DNA repeats. Lateral domain movements within a protein, which we have designated DOMO (domain movement), represent novel routes for the diversification of proteins.

Figure 6.

Domain movement. Movement of an amino-acid sequence from one protein domain to another domain of the same protein. The underlying mechanism is movement of the corresponding DNA sequence through recombination at sequences flanking the domain sequences. (more...)

Gene Regulation

RM systems possess mechanisms that tightly regulate their gene expression to suppress the potential of a lethal attack on the host bacteria. When RM systems enter a new host bacterial cell with a genome that lacks proper methylation, they avoid killing the cell by expressing the modification enzyme first (Fig. 1C).¹²² The restriction endonuclease and modification enzyme activities must be carefully regulated not only during RM system establishment in a new host, but also during maintenance. When RM genes are lost from a cell or the epigenetic status is disturbed, the restriction enzyme will attack the chromosome through postsegregational killing, leading to cell death or genome repair by recombination and replication. DNA fragments encoding RM systems may be released into the environment after cell death, invading other cells and establishing in the genome of a new host. This life cycle is similar to other mobile elements such as lysogenic phages and DNA transposons.

The regulatory machinery of mobile RM systems is expected to be host-independent, to bypass differences in the host factors affecting their establishment, maintenance and host attack. Thus far, regulatory mechanisms have been studied mainly at the transcriptional level,

as three main modes of regulation. One employs C-proteins,¹²³ which specifically bind a DNA operator sequence through a helix-turn-helix (HTH) motif to temporally control expression of the restriction enzyme, the modification enzyme or both.^124,125 This tight, finely tuned regulation operates via transcriptional feedback circuits.¹²⁶ Moreover, C proteins can efficiently delay expression of the restriction enzyme during establishment in a new host cell.^85,122 In the second type of regulation, the modification enzyme represses transcription of its own gene and occasionally stimulates restriction gene expression by DNA binding via its HTH domain.^127-129 In the third mode, the coordinated expression of R-M systems depends on the methylation status of a cognate recognition site(s) in their promoter region.^130,131

Recent analyses revealed regulation by intragenic reverse promoters from which antisense RNAs are transcribed.^132-135

Conclusion and Perspective

RM systems, originally found to be a barrier to gene mobility, turn out to be mobile elements themselves. They are mobile epigenetic elements because they define, and sometimes even force an epigenetic status on a genome. Multilevel conflicts involving these epigenetic systems may drive prokaryotic evolution.

This model is based on laboratory experiments and genome comparisons and needs to be examined through additional experimental and theoretical studies.¹³⁶ The expanding accumulation of bacterial genome sequences, especially within a species, may allow more detailed analysis. The concept of conflicts between epigenetic systems may provide information for understanding eukaryotic evolution and the origin of life.

Acknowledgments

We thank Noriko Takahashi for providing Figure 1E.

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