REVIEWS

The bacterial epigenome

María A. Sánchez-​Romero and Josep Casadesús * Abstract | In all domains of life, genomes contain epigenetic information superimposed over the nucleotide sequence. Epigenetic signals control DNA–protein interactions and can cause phenotypic change in the absence of mutation. A nearly universal mechanism of epigenetic signalling is DNA methylation. In bacteria, DNA methylation has roles in genome defence, chromosome replication and segregation, nucleoid organization, cell cycle control, DNA repair and regulation of transcription. In many bacterial species, DNA methylation controls reversible switching (phase variation) of gene expression, a phenomenon that generates phenotypic cell variants. The formation of epigenetic lineages enables the adaptation of bacterial populations to harsh or changing environments and modulates the interaction of pathogens with their eukaryotic hosts.

Methylome In the second half of the 20th century, eukaryotic genomes cell lineages. At the turn of the century, DNA adenine Overall DNA methylation were found to contain information superimposed over methylation was found to be essential for the patho- pattern in a genome. the nucleotide sequence1. In multicellular eukaryotes, genesis of Salmonella enterica subsp. enterica serovar this additional epigenetic information controls differen- Typhimurium11,12, and later of other pathogens13,14. Adaptive value In population genetics, the tiation and development, and its perturbation can cause Together, these studies extended to bacteria the notion 2 contribution of a phenotypic pathological conditions . The main epigenetic signals in that DNA methylation can exert epigenetic control over trait to the fitness of an eukaryotic genomes are DNA methylation3 and post-​ the phenotype6,7,15–17. The possibility that phosphoro- individual or a population. translational modification of histones4, and both mech- thioation of the DNA backbone may provide additional anisms contribute to establish the transcription patterns epigenetic marks, perhaps in a manner complementary Restriction–modification 18,19 (R-M)​ systems that govern the formation of specialized cell types. DNA to DNA methylation, remains open . Machine systems that methylation controls the binding of proteins to DNA and A hurdle in the investigation of the roles of DNA eliminate foreign DNA provides a primary layer of information to shape the methylation in bacteria was that most studies focused molecules by endonucleolytic eukaryotic epigenome3. DNA methylation is also found on 6mA, which is a base modification that was difficult cleavage and protect the genome by modification of the in bacterial genomes, where it controls DNA–protein to detect. This technical limitation has been overcome 5–8 cognate endonuclease target. interactions as in eukaryotes (Box 1). through the development of DNA-sequencing​ techno­l­ A frequent type of protection The methylated bases found in bacteria are N6-methyl-​ ogies that enable the high-​throughput analysis of DNA is DNA methylation. adenine (6mA) and C5-methyl-​cytosine (5mC), which methylation across the genome (the methylome). Two also exist in eukaryotes, and N4-methyl-cytosine​ (4mC), such technologies, single-​molecule real-​time sequenc- which is found only in bacteria and archaea (Table 1). ing (SMRT)20,21 and nanopore sequencing22, are currently Base methylation is post-​replicative, and the bacte- ‘gold standards’ in the study of bacterial methylomes23. rial genome is methylated at both DNA strands in the SMRT technology permits researchers to infer the pres- absence of DNA replication. Upon DNA replication, ence of methylated bases on the template DNA strand base methyl­ation is present in one strand only (hem- from kinetic signatures that are specific for each type imethylation)5. Early studies in Escherichia coli and of methylated base20. Nanopore sequencing identifies other gammaproteobacteria unveiled roles for DNA methylated nucleotides by detecting changes in electric adenine methylation in chromosome replication, mis- current density upon electrophoretic passage of the sam- match repair, control of transposon activity and regu- ple through an orifice22. Methylome analysis has shown lation of transcription5,8. Proteins that can discriminate that base methylation is widespread in the genomes the methyl­ation state (methylated or hemimethyl­ated) of bacteria and archaea24, including the small genomes of of cognate binding sites were also identified5. In alpha­ certain (but not all) obligate parasites25. The wealth Departamento de Genética, proteobacteria, DNA adenine methylation was found to of information provided by methylome analysis has Facultad de Biología, 9 Universidad de Sevilla, govern the cell cycle . Seminal studies in uropathogenic also updated (and sometimes changed) classic notions Sevilla, Spain. E. coli unveiled a role for DNA adenine methylation in about the physiological relevance and adaptive value of 10 *e-mail:​ [email protected] the formation of phenotypic variants of bacterial cells , DNA methylation in bacteria. For instance, the DNA https://doi.org/10.1038/ in a manner reminiscent of the involvement of DNA methyl­transferases of restriction–modification (R-​M) s41579-019-0286-2 cytosine methylation in the formation of eukaryotic systems were thought to function in genome defence

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37 Box 1 | The epigenomes of eukaryotes and bacteria DNA methyltransferases are active as monomers . For the methylation reaction, the target base is rotated out 3 • In eukaryotes, epigenetic modification of the genome involves DNA methylation (‘flipped out’) of the DNA helix and inserted into the 4 and histone modification . Bacteria lack histones, and epigenetic control relies on catalytic pocket of the enzyme35,36. Depending on DNA methylation only6. the position of the target base in the double helix, DNA • In eukaryotes, de novo and maintenance forms of DNA methylation are performed methyltransferases are classified into two groups: exo- by separate enzymes2. Bacterial DNA methyltransferases have both de novo and cyclic (for 6mA and 4mC) and endocyclic (for 5mC)37. maintenance activities37. Aside from this difference, all DNA methyltransferases • In eukaryotes, two main mechanisms exist to erase DNA methylation marks: active show structural similarity, and the SAM-binding​ domain demethylation by dedicated proteins (Tet enzymes), and passive demethylation by the hindrance of DNA methylase activity upon DNA replication35. In bacteria, DNA (or catalytic domain) is especially well conserved across demethylation is usually passive66, and the relevance of active demethylation by kingdoms. By contrast, the target recognition domain DNA repair remains to be evaluated82. (TRD) shows high variability in sequence and structure, 38 • In both bacteria and eukaryotes, transcriptional repression by DNA methylation is which correlates with the manifold target specificities . common3,6. Transcriptional activation of bacterial genes under DNA methylation Structural similarity and conservation of motifs between control often involves demethylation (partial or complete, single- or double-stranded)​ different groups of DNA methyltransferases may be of promoters or regulatory regions57,72,89,90,94,158. indicative of a monophyletic origin, and diversification • The methylated base typically involved in the control of eukaryotic transcription is across kingdoms may have occurred upon the insertion C5-methyl-cytosine​ 3, whereas in bacteria it is often N6-methyl-adenine​ 7,14. However, and permutation of domains39. direct control of bacterial transcription by C5-methyl-cytosine​ has been demonstrated DNA methyltransferases associated with R-​M sys- recently126. Transcriptional control by N4-methyl-cytosine​ may also exist130. tems are abundant, with an average of more than two • In multicellular eukaryotes, the DNA methylation pattern of the genome is R-​M systems per genome40,41. Species with higher num- reprogrammed during gametogenesis and during early embryonic development2. bers of R-​M systems exist, and extreme examples are In bacteria, reprogramming does not occur, and the DNA methylation pattern can be found in Helicobacter pylori strains that encode more transmitted unaltered across generations. However, the acquisition and loss of DNA than 50 R-​M systems42. In addition, many bacterial and 41 methyltransferase genes and recombinational shuffling of DNA methyltransferase archaeal genomes harbour at least one orphan DNA domains27,33,143 can produce novel methylation patterns in bacterial genomes. methyltransferase24. Orphan DNA methyltransferase • In both bacteria and eukaryotes, DNA methylation controls the formation of phenotypic genes are also found in about one fifth of phage genomes43. variants of genetically identical cells. However, DNA methylation-dependent​ formation Most orphan DNA methyltransferases are conserved at of bacterial cell lineages can show programmed reversion (phase variation)15,27,93,111. the genus level; the most highly represented are the Dam family in gammaproteobacteria and the CcrM family in only, and housekeeping functions were ascribed to alphaproteobacteria24. Bioinformatic detection of a gene solitary (‘orphan’) DNA methyltransferases not asso- encoding a DNA methyltransferase does not imply that ciated with R-​M systems. This distinction is not valid the enzyme is active, or even expressed: for example, anymore, as a number of R-​M methyltransferases have transcription of the gene encoding the YhdJ methyltrans- been shown to have roles in addition to genome defence, ferase of gammaproteobacteria is low or absent under including transcriptional regulation and the formation laboratory conditions44. of phenotypic cell variants26–28. The initial notion that Most DNA methyltransferases recognize specific solitary DNA methyltransferases were rare also did not DNA targets: for instance, Dam and CcrM have a single hold up, as methylome analysis has shown that they are target site (GATC and GANTC, respectively), whereas abundant24. Finally, the semantics of the bacterial epige- Dcm has two target sites, CCAGG and CCTGG5. Orphan nome, which traditionally had centred on 6mA, has been DNA methyltransferases that have several TRDs (and extended to 5mC and 4mC. thus are multispecific) have been described in Bacillus In this Review, we summarize historic and recent subtilis phages43. Nonspecific DNA methyltransferases developments in epigenetic signalling by DNA methyl­ also exist, but they will not be discussed here45. ation, with an emphasis on its contribution to pheno- The DNA methyltransferases of R-M systems methyl­ typic heterogeneity in bacterial populations. This bias ate DNA in a distributive manner; that is, they methylate may be justified by the growing evidence that DNA only one target upon DNA binding37. Distributive methylation-​mediated control of lineage formation is DNA methylation can be seen as a crucial adaptation a widespread phenomenon in bacteria14,15,27,28, whereas of these enzymes to their biological function, as the need the control of physiological processes such as chromo- to rebind DNA after each methylation event can slow some replication and mismatch repair may be restricted down the methylation of phage DNA, thus facilitating to certain bacterial clades8. An emphasis on phenotypic restriction by the cognate R-M endo­nuclease37. Orphan heterogeneity may be further warranted by theoretical DNA methyltransferases are generally processive, and analysis that has outlined its adaptive value29,30. We also so methylate multiple targets without dissociating discuss, often in a speculative manner, evolutionary from DNA37,46. However, the CcrM methyl­transferase aspects of bacterial DNA methylation. Complementary of alphaproteobacteria seems to be distributive47. Pro­ information can be found in other reviews27,31–34. cessivity can vary depending on the genome context; for instance, certain DNA sequences that flank the DNA methyltransferases GATC site can decrease the processivity of the Dam The formation of 6mA, 5mC and 4mC is catalysed by methyltransferase48. DNA methyltransferases, which transfer a methyl group Bacterial DNA methyltransferases can be essential or from S-​adenosyl-methionine (SAM) to DNA35,36. Most dispensable7,13,49. In the genus Yersinia, Dam-​dependent

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SOS regulon methylation is essential in Yersinia enterocolitica, and Hemimethylation and demethylation Regulatory network that dispensable in two related species, Yersinia pseudo­ Bacterial DNA methyltransferases are active on both responds to DNA damage tuberculosis and Yersinia pestis50–52. Growth conditions nonmethylated and hemimethylated DNA46,63, thus in gammaproteobacteria. can also determine whether a DNA methyltransferase is acting as both writers of the methylome and copyists essential or dispensable: for instance, the CcrM methyl­ that transmit the methylation pattern of the genome to ase of Caulobacter crescentus is only essential in rich daughter cells. Because every round of DNA replica- medium53. The phenotypes of strains that lack or tion produces hemimethylated DNA from methylated overproduce a DNA methyltransferase can be indic- DNA, methylome inheritance requires methylation of ative of the physiological roles that DNA methyla- the newly synthesized DNA strand (Fig. 1). The duration tion might have in the species under study14,49,54,55. of the hemimethylated state after DNA replication varies Moreover, altered patterns of gene expression can among bacterial clades, and a crucial factor is the avail- provide evidence that the methylation state of promot- ability of the DNA methyltransferase. In gammaproteo- ers or regulatory regions controls the activity of the bacteria, Dam methylase is synthesized at every stage of transcriptional machinery56,57. However, altered gene growth64, and the hemimethylated state of GATC sites is expression does not necessarily imply direct transcrip- short-​lived because the Dam methylase trails the DNA tional control, because indirect effects are possible. For replication fork at a short distance, estimated to be less instance, increased levels of SAM in DNA methylase than 10 kbp65. By contrast, the genome of alphaproteo- mutants may induce changes in gene expression due bacteria remains hemimethylated during most of the cell to alterations in one-​carbon metabolism58. Similarly, cycle, because the CcrM methylase is only synthesized transcriptome differences can reflect indirect control. at the final stage of chromosome replication9,32,34. For instance, upregulation of the SOS regulon in dam As a rule, hemimethylation after DNA replication is mutants of E. coli and S. enterica is not caused by Dam-​ transient if the cognate DNA methyltransferase is avail- dependent transcriptional control, but by DNA strand able. However, at specific sites methylation of the daugh- breakage59,60. Enigmatic cases of post-​transcriptional ter strand can be hindered by the binding of proteins that regulation by Dam-​dependent methylation have also prevent DNA methyltransferase activity, causing methyl­ been described61,62, and a tentative explanation is that ation loss in a passive manner (Fig. 1). This phenome- Dam methylation may regulate transcription of genes non is well documented in E. coli and S. Typhimurium: involved in control of mRNA stability, mRNA translation stable hemimethylated GATC sites are formed when a or other post-transcriptional​ events. DNA-​binding protein protects hemimethylated DNA

Table 1 | Examples of writers and readers of the bacterial epigenome Methylated base Writer Target readers Physiological process or sequence phenotype under control (5′–3′) N6-methyl-adenine​ Dam GATC DnaA102, SeqA86, Chromosome replication and MutHLS105, RNA segregation86,102,104, nucleoid H C 3 NH polymerase91, organization87,164, mismatch repair105, transposase91,134, transposition91,134, conjugal transfer88, N N Lrp69,88,89,163, OxyR70–72,94, motility56, synthesis and secretion Fur73 and HdfR74,75 of virulence determinants11,12,56,62,165, N N 166 H envelope structure , bacteriophage resistance107 and antibiotic resistance155 CcrM GANTC GcrA90, MucR76,167 and Cell cycle control32,34 RNA polymerase53,57 ModA13 AGAAA Unknown Antibiotic resistance, epithelial cell invasion and biofilm formation113 SpnD39III Allele-​specific Unknown Opacity (capsule synthesis) and (six variants) virulence123 C5-methyl-cytosine​ Dcm CCAGG and Unknown General stress response128 and CCTGG drug transport129 NH2

H3C JHP1050 GCGC Unknown Cell morphology , competence, N outer membrane composition and copper resistance126 N O H

N4-methyl-cytosine​ M2.Hpy.AII TCTTC Unknown Virulence (adhesion to host cells)130 H C 3 NH

N

N O H

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from Dam methylase activity6,14,66. If Dam methyla- OxyR70–72, Fur73 and HdfR74,75 are examples of DNA-​ tion hindrance persists during two consecutive rounds binding proteins that are involved in ‘passive’ erasure of of genome replication, a nonmethylated GATC site is methyl groups (see below). Undermethylated GANTC formed14,15. Undermethylation requires that the pro- sites have also been detected in alphaproteobacterial cessivity of Dam methylase is reduced, which occurs at genomes, and an example of protection from CcrM GATC sites that are flanked by specific, A–T-rich​ DNA methylation has been shown to involve a transcription sequences48,67. The effect of sequences outside the GATC factor, MucR76. Passive demethylation may be a common site is not surprising, as DNA-bound​ Dam methyltrans- phenomenon in the bacterial world: methylatable sites ferase spans 10 bp68. The transcription factors Lrp69, that lack methylation in one or both DNA strands are

a b 5′ 3′

Methylated 5′ 3′ MBF Dam G C CH A G G 3 T CH A C A CH T A CH3 3 3 C G T T C T C A T C Methylation G A hindrance G 3′ 5′

3′ 5′ 5′ 3′ DNA replication

5′ 3′ 5′ 3′ Hemimethylated (stable) G C G C CH A T A T 3 CH 5′ 3′ T A T A 3 C G C G Hemimethylated Dam 3′ 5′ 3′ 5′

G G DNA replication CH A A CH 3 C C 3 T T T C T C 5′ 3′ A A G G

3′ 5′ 5′ 3′ G G Methylation A C A hindrance T T C T C A T C G A G

3′ 5′ 5′ 3′

Nonmethylated Methylated G C G C G C G C CH3 A T CH3 A T A T A T T A CH3 T A CH3 T A T A C G C G C G C G

Most GATC sites Special GATC sites

Fig. 1 | Methylation, hemimethylation and passive demethylation of gATc sites in gammaproteobacteria. a | The bacterial genome is methylated at both DNA strands in the absence of DNA replication. Upon DNA replication, methylation is present in the template strand only (hemimethylation). Hemimethylation after DNA replication is transient as Dam, an orphan DNA methyltransferase that trails the DNA replication fork , targets GATC sites for methylation to restore the methylated state. This cycle occurs once per round of DNA replication and affects most GATC sites5. b | Binding of a protein that functions as a methylation-blocking​ factor (MBF) can prevent methylation of a hemimethylated GATC site by the Dam methyltransferase. If hindrance of methylation persists during two consecutive rounds of DNA replication, a nonmethylated GATC site is formed66. DNA methylation hindrance by blocking factors occurs only at GATC sites flanked by DNA sequences that decrease the processivity of the Dam methylase48 (not shown). Undermethylated (hemimethylated or nonmethylated) GATC site states can be transmitted to daughter cells in certain cases.

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common in the genomes of gamma- and alphaproteo- nonmethylated sites can be heritable, thus transmitting bacteria24,77–80, and some of those sites are located within epigenetic states to daughter cells6,15. In gammaproteo­ protein-binding​ sites24,80. In E. coli, the methylation state bacteria, Dam-​dependent methylation patterns are of certain GATC sites depends on growth conditions, found at the transcriptional control regions of bistable which suggests that environmental effects can contribute loci, and the elements involved in the formation of OFF to shape the methylome81. and ON states are more complex than those in Fig. 2a. ‘Active’ DNA demethylation has been described in For instance, the E. coli pap operon contains six bind- E. coli, and its physiological relevance remains to be fully ing sites for Lrp and two GATC sites93; the S. enterica understood. AlkB, a DNA repair protein that removes opvAB and gtr operons contain four OxyR binding sites alkyl groups from nucleic acids, also targets 6mA and and four GATC sites72,94; the E. coli sci1 locus contains removes its methyl group82. Because AlkB expression three Fur binding sites and three GATCs73; and the is strongly induced upon DNA damage, widespread S. Typhimurium std operon contains one binding site demethylation of 6mA may occur under such con- for HdfR and three GATC sites75 (Fig. 2b). In all cases, the ditions. It is thus conceivable that the demethylation ON and OFF states are dictated by the binding pattern of physiologically relevant, critical GATC sites might of the cognate protein. Binding causes passive demethyl­ contribute to survival, perhaps by delaying genome ation, which can increase the affinity of the regulator replication or cell division82. towards its cognate site (or sites), thus generating a posi­ tive feedback loop that stabilizes the epigenetic state, Readout of methylation marks which can sometimes be heritable. The methyl group of 6mA, 5mC and 4mC protrudes Certain bistable loci that are controlled by Dam-​ from the major groove of the double helix, which pro- dependent methylation switch between alternative vides a platform for DNA-​binding proteins to bind to states, and the system is thus reversible (‘phase-variable’;​ cognate nucleotide sequences. As a consequence, the see below)15,66. Switching between states requires DNA methylation state of critical sites can either favour or replication with concomitant formation of hemimethyl- hinder the interaction between a DNA-binding​ domain ated DNA, and a crucial factor that determines the fre- of the protein and a cognate DNA site7. In addition, base quency of switching is the affinity of the DNA-binding​ methylation can induce or enhance DNA curvature83–85, protein for its binding sites72,95 (Fig. 2a). Ancillary factors which can further modulate DNA–protein interactions. may affect switching: for instance, in the pap operon a Inhibition of the activity of DNA restriction endo­ protein named PapI is necessary for switching from OFF nuclease by methylation of the target is a classic example to ON and for maintenance of the ON state95. of a methylation-​sensitive DNA–protein interaction. Frequencies of ON–OFF switching are locus specific. However, discrimination of DNA methylation states For instance, under laboratory conditions the GtrOFF and can be far more complex. Certain methylation readers GtrON subpopulations of S. enterica have roughly iden- (for example, the GATC sequestration factor SeqA) tical sizes96, whereas the OpvAB and Std switches are can bind both methylated and hemimethylated DNA lopsided towards the OFF state72,97. However, switching targets, albeit with different affinities86,87. Other pro- can be under environmental influence: for instance, the teins (for example, the transcription factor Lrp) bind StdOFF and StdON subpopulations have different sizes in both hemi­methylated and nonmethylated targets, but the laboratory and in the animal intestine97,98 (see below). the binding patterns and the affinities are different88,89. Additional evidence for environmental control has come Another remarkable bias in protein binding to hemi- from the observation that housekeeping proteins respon- methylated sites is strand specificity, which can favour sive to environmental cues can alter switching frequen- binding to only one of the hemimethylated DNA spe- cies. Examples include the global regulator CRP99, the cies produced by DNA replication89–91. An unproven signal transduction proteins CpxAR100 and the nucleoid but reasonable speculation is that the overall methyl­ proteins H-​NS and HU72,101. ation state of the genome may also influence the bind- ing of proteins to specific regions, by moulding nucleoid Cell cycle signalling by DNA methylation topology34,92. In gammaproteobacteria, the initiation of chromo- Especially complex DNA–protein interactions occur some replication and segregation of newly replicated at regions that harbour several protein-​binding sites, nucleoids is controlled by readers that discriminate which in turn contain methylatable motifs6,15,66. In such methylation from hemimethylation. One such reader is cases the protein-binding​ pattern determines the methyl­ DnaA, which initiates chromosome replication if GATC ation state, because methylation hindrance causes passive sites within the origin of replication (oriC) are methyl- demethylation of specific sites. In turn, the methyl­ ated102. Another reader of the methylation state is SeqA, ation pattern determines the protein-​binding pattern. which binds hemimethylated GATC sites at the oriC The same protein is thus the eraser of methyl groups and delays Dam methyltransferase activity86. Transient and the reader of the resulting DNA methylation pattern. hemimethylation of­ the oriC by SeqA also represses In the imaginary example presented in Fig. 2a, the exist- transcription of the nearby dnaA gene, which contains ence of two methylatable binding sites permits alternative, GATC sites in the promoter region103. In addition, the mutually exclusive binding patterns, and each binding binding of SeqA to hemimethylated GATC sites gov- pattern determines a distinct DNA methyl­ation pattern, erns the condensation and segregation of newly repli- because methylation is hindered at the sites bound by cated nucleoids104. Transient hemimethylation upon the regulator6,66. Such combinations of methylated and replication fork passage is also a signal for repair of

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mismatched nucleotide pairs, which relies on the ability transcription upon passage of the replication fork. of the MutHLS complex to cleave the daughter strand at Classic examples in gammaproteobacteria are the trans- hemimethylated GATCs105. posase (tnp) gene of insertion element IS10 (ref.91) and In C. crescentus, the initiation of chromosome repli­ the plasmid-​borne traJ gene, which encodes an activa- cation by DnaA requires GANTC methylation at the tor of conjugal transfer88,89. Because the hemimethyla- replication origin (Cori)32,34. However, Cori remains tion state is transient, the expression of tnp and traJ is spontaneously hemimethylated, without sequestration, short-​lived, and synthesis of the corresponding gene because the CcrM methylase becomes available only at products is restrained. An additional sophistication of the end of every round of chromosome replication32. tnp and traJ hemimethylation readout is asymmetry: Hence, the DnaA proteins of gamma- and alphaproteo- the activation of tnp and traJ transcription takes place bacteria read analogous signals at oriC and Cori, respec- in only one of the hemimethylated DNA species pro- tively, but the mechanisms that control methylation of duced by DNA replication89,91. The adaptive value of the replication origin are different7,32. strand-​specific transcription of tnp and traJ may be Reading of DNA hemimethylation signals by RNA tentatively understood: asymmetric reading restrains polymerase and transcription factors can activate synthesis of dangerous (transposase) or energetically a b OFF state ON state Lrp Lrp Papl

G A T C CH3 CH3 pap State A State B CH3 CH3 GATC 1 GATC 2 GATC 1 GATC 2

CH3 CH3 Fur CH3 CH3 CH3

CH3 CH3 sci1

DNA replication CH3 CH3 CH3

OxyR OxyR

CH3 CH3 CH3 CH3 CH3 CH3 gtr

CH3 CH3 CH3 CH3

CH3 CH3 OxyR OxyR

CH3 CH3 CH3 CH3 opvAB

CH3 CH3 CH3 CH3

CH CH 3 3 HdfR CH3 CH3 CH3 CH3 std

CH3 CH3 CH3 CH3 CH3 CH3

Fig. 2 | control of bistable loci by the formation of alternative Dam six binding sites for Lrp in pap93, four for OxyR in gtr94 and opvAB106, three for methylation patterns. a | Shown is an imaginary locus that harbours two Fur in sci1 (ref.73), and one binding site for HdfR in std75. The transcription protein-​binding sites, each containing a GATC (GATC 1 and GATC 2). factor binding sites contain GATC sites: two in pap93, three in sci1 (ref.73) and Two binding patterns are possible, and each binding pattern causes passive std75, and four in opvAB107 and gtr94. The methylation state of such sites demethylation of a specific GATC site because methylation is hindered at is dictated by the binding pattern of the transcription factor: GATCs the sites bound by the regulator. Nonmethylation can increase the affinity located within sites bound by the transcription factor undergo passive of the protein for its cognate binding site, thus creating a positive feedback demethylation, while GATCs within unbound sites are methylated. This loop that stabilizes the DNA methylation pattern and makes it heritable. mutual exclusion between Dam methylation and transcription factor However, the system can be reset when hemimethylated DNA substrates binding produces distinct patterns of DNA methylation in the regulatory are formed upon DNA replication. Switching occurs if the protein moves region of OFF and ON cells. In PapOFF cells, the upstream GATC site is from one cognate site to the other, and a major factor that determines the methylated and the downstream GATC is nonmethylated, and the opposite frequency of switching in each direction is the affinity of the protein GATC methylation pattern is found in the PapON lineage93. In the regulatory towards each binding site72,95. Ancillary factors (not shown) can skew regions of gtr and opvAB, cells in OFF and ON states display opposite Dam switching, either in a stochastic manner or in response to environmental methylation patterns involving two methylated and two nonmethylated signals100,101. b | Epigenetic states of bistable loci of Escherichia coli (pap and GATC sites72,94. The formation of nonmethylated GATC sites is likewise sci1) and Salmonella enterica (gtr, opvAB and std) under the control of Dam observed in Sci1OFF cells as the consequence of Fur binding73, and in StdON methylation. The diagrams show regulatory regions upstream of the cells as the consequence of HdfR binding75. Under laboratory conditions, promoters, with the promoter-​proximal side on the right. Transcription is the formation of ON and OFF subpopulations is reversible (phase variable) repressed in the OFF state and activated in the ON state. In all cases, the in pap10, gtr94, opvAB106 and std97, but not in sci1 (ref.73). In pap, switching to regulatory region contains binding sites for a transcription factor: ON also requires an ancillary factor, PapI95.

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expensive (conjugation) cell products. Asymmetric state quickly reproduces a population in which >99% transcription from a hemimethylated promoter has also of cells are OpvABOFF (Fig. 3a). The phase variation of been observed in the creS gene of C. crescentus76, which the opvAB operon may thus be viewed as a trade-​off suggests that the transcription factor involved (GrcA) between virulence and bacteriophage resistance107. may be a hemimethylation reader with DNA strand Another phase-variable​ locus, std, provides a unique specificity. An attractive speculation is that asymmetric example of the power of Dam-​dependent phase varia- reading of GANTC hemimethylation may contribute to tion to produce phenotypic diversity. The std operon is differentiation of the two cell types34. found in the genome of S. Typhimurium and encodes fimbriae that facilitate adhesion to the mucus layer Bistability and phase variation of the large intestine98,108. In addition, the std operon Heritable DNA adenine methylation patterns. The encodes two transcription factors, StdE and StdF, that formation of alternative Dam-​dependent methylation are activators or repressors of more than one hundred patterns, a phenomenon discussed above, controls the genes97. Pleiotropic control thus splits the bacterial pop- expression of bistable loci and produces phenotypic ulation into two subpopulations that differ in multiple variants of genetically identical cells in gammaproteo­ phenotypic traits, a phenomenon that may be viewed bacteria66. Dam-​dependent bistability can be phase-​ as a rudimentary differentiation program97 (Fig. 3b). The variable15. Paradigms of Dam-dependent​ phase variation molecular mechanisms that control std phase variation in E. coli are the pap operon of uropathogenic strains, are more complex than those in other Dam methylation-​ which encodes fimbriae for adhesion to the urinary dependent loci: std transcription is activated by HdfR, epithelium93, and the agn43 gene, which encodes a transcription factor of the LysR family, and the tran- a non-​fimbrial adhesin involved in bacterial auto­ scription of hdfR is activated by an std product, StdE75. aggregation70,71. Descriptions of Dam-dependent​ control The binding of HdfR upstream of the std promoter is of pap and agn43 phase variation can be found in other hindered by Dam-​dependent methylation, and HdfR reviews6,7,66,93 and will not be discussed here. binding prevents methylation of two GATC sites in the Natural isolates of non-​typhoidal S. enterica carry regulatory region75 (Fig. 2b). In addition, the formation of one to four operons that encode glucosyltransferases, StdON cells requires autogenous activation of transcrip- which add sugars to the lipopolysaccharide O-antigen​ 96. tion by StdF, which binds the std promoter in a Dam Such operons, named gtr, are associated with prophages methylation-independent​ fashion75. or prophage remnants present in the genome, and each operon may produce a distinct modification of the Phase-variable​ DNA methyltransferases of R-M​ systems. O-​antigen. The resulting variants of the bacterial sur- Two decades ago, a Haemophilus influenzae gene (mod) face may adapt Salmonella to interact with different host homologous to genes encoding the DNA adenine receptors, and they may also alter phage susceptibility96. methyltransferases of type III R-​M systems was found The expression of gtr is phase-​variable, and the GtrOFF to undergo phase variation due to contraction and/or and GtrON subpopulations harbour alternative patterns expansion of nucleotide repeats109. Another phase-​ of methylated and nonmethylated GATC sites within a variable mod gene belonging to a type III R-​M system regulatory region upstream of the gtr promoter94. The was thereafter described in H. pylori, and phase vari- transcription factor OxyR is both an eraser of methyl ation was found to be caused by length variation of a groups and a reader of the resulting gtr methylation homopolymeric nucleotide tract in the 5′-coding region patterns: OxyR binding to upstream cognate sites pre- of a gene located upstream of mod110. The type III

vents methylation of GATC1 and GATC2 and activates R-M​ Mod enzyme of H. influenzae turned out to be more transcription, whereas binding to downstream sites pre- than just a modification enzyme involved in genome pro-

vents methylation of GATC3 and GATC4 and represses tection: transcriptome analysis identified genes that were transcription94,96 (Fig. 2b). either up- or downregulated in a mod mutant, which Another phase-​variable operon under the control indicates that adenine methylation of specific genome of Dam-​dependent methylation and OxyR is opvAB, sequences controlled the expression of specific genes. whose products are cytoplasmic membrane proteins Switching in the availability of active DNA methyltrans- that reduce the length of the S. enterica lipopolysaccha- ferase thus produced a phase-​variable regulon (‘phase­ ride O-​antigen106. As in gtr, the control of phase varia- varion’)111. An additional, remarkable observation was tion is transcriptional. A regulatory region upstream of that the restriction enzyme of certain type III R-M​ sys- the opvAB promoter contains four OxyR binding sites tems was inactive in a number of Haemophilus strains, and four methylatable GATC motifs; OpvABOFF and which suggests that such R-M​ systems had evolved into OpvABON cell lineages display opposite patterns of OxyR epigenetic regulators of gene expression112. Further stud- binding, which in turn generate opposite patterns of ies described 6mA phasevarions in Neisseria meningitidis GATC methylation72 (Fig. 2b). Transcriptional switching and Neisseria gonorrhoeae113. Phase variation involved is skewed towards the OpvABOFF state, and the OpvABON variations in the length of simple sequence repeats lineage comprises only 0.3% of cells106. Shortening of the (Fig. 4), and the loci under the control of Mod-​mediated O-​antigen renders OpvABON cells resistant to phages methylation included genes with roles in envelope that use the O-​antigen as a receptor. OpvABON cells structure, virulence and stress responses114. The power Homopolymeric nucleotide 107 tract are unable to infect animal hosts ; however, as soon of phasevarions as sources of epigenetic variation is DNA region that contains only as phage challenge ceases, phase variation produces remarkable: in contrast with the phase variation systems AT or GC nucleotide pairs. OpvABOFF cells, and lopsided switching towards the OFF that generate heterogeneity of a single phenotypic trait,

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a OpvABOFF b Fimbriae Long O-antigen Flagella T3SS Dam-dependent StdON switching StdOFF

ON OpvAB ILEUM Short O-antigen

LPS Dam-dependent switching

OpvABOFF OpvABON Selection of M cell Epithelial cell the OpvABON subpopulation CAECUM

Bacteriophage Reconstruction of the Selection of Mucus layer orignal population, the OpvABON predominantly virulent subpopulation

Resuscitation of Goblet cell the OpvABOFF subpopulation

Fig. 3 | Formation of subpopulations controlled by Dam-dependent​ methylation. a | Phase-variable​ transcription of the Salmonella enterica opvAB operon produces a lineage of OpvABOFF cells with long O-​antigen chains in the lipopolysaccharide­ (LPS) (OpvABOFF) and a lineage with shorter O-​antigen chains (OpvABON)106. Shortening of the O-​antigen renders the OpvABON lineage avirulent but resistant to bacteriophages (many Salmonella-targeting​ phages use the O-​antigen as a receptor). In the presence of a phage, the OpvABOFF subpopulation will be eliminated, while the OpvABON subpopulation will survive. When the phage challenge ceases, OpvABOFF cells produced by phase variation will survive, and virulence will be regained. However, a small subpopulation of avirulent OpvABON cells will sustain preadaptation to future phage challenge107. b | Phase-variable​ transcription of the std operon produces subpopulations of StdOFF and StdON cells97. The StdOFF lineage is motile (producing flagella) and synthesizes the type 3 secretion system (T3SS), promoting invasion of epithelial cells in the ileum of infected animals, which is the initial stage of systemic Salmonella spp. infection97,162. In turn, the StdON lineage produces fimbriae that permit adhesion to the mucus layer of the caecum in the large intestine98,108 but harbours neither flagella nor a T3SS97. Adhesion of the Std fimbriae to the caecal mucosa increases inflammation98, which promotes the survival of Salmonella. StdOFF cells can thus cause acute infection, whereas StdON cells cause chronic infection, and std phase variation can be viewed either as a division of labour or as a bet-hedging​ strategy.

bacterial lineages under phasevarion control can differ the presence of 6mA at promoters or transcription in multiple phenotypic traits. control regions120. Additional examples of phase-variable​ type III R-M​ Phase variation of DNA adenine methyltransferases systems have been described in the past decade33,115–117, belonging to type I R-​M systems was first identified in and bioinformatic analysis predicts that nearly one fifth Mycoplasma pulmonis121, and later in other human patho­ of type III mod genes may be phase variable, as judged gens27,122. A frequent mechanism of switching is recom- from the presence of sequence repeats117. In certain bac- bination between inverted repeats of type I R-​M (hsd) terial species (for example, Neisseria spp.), mod alleles genes, which can be present in multiple copies, complete show high homology except in the target recognition or incomplete, in a genome27 (Fig. 4). DNA inversion by domain, thus providing evidence for rapid evolution site-​specific recombination and the expansion and/or by recombinational shuffling113. Target diversifica- contraction of single sequence repeats can also mediate tion, together with frequent horizontal transfer of mod the switching of hsd genes27,33 (Fig. 4). An example of the 118 119 Transparent–opaque genes , may further promote phenotypic diversity . contribution of type I R-​M systems to phase variation transition A DNA adenine methyltransferase of a phase-​ has been described in Streptococcus pneumoniae, in Formation of phenotypic variable type II R-M​ system has been shown to regulate which genetic rearrangements generate six 6mA vari­ variants (phase variation) of gene expression and virulence in Campylobacter jejunii, ants of a type I DNA methyltransferase that controls the Streptococcus pneumoniae transparent–opaque transition123–125 that is involved in where phase variation is controlled by the insertion or . Each DNA methyl­ pneumococcal carriage deletion of repeat units. However, it remains to be estab- transferase variant generates a distinct methylation and invasive infection. lished whether or not gene expression control involves pattern in the genome, which results in different gene

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a ON–OFF switching b Methylome diversification IR IR IR IR Genome methylated ON (full length) Recombination between inverted repeats SSR

Contraction and/or expansion of repeats

Stop Variation of genome methylation pattern SSR

OFF (truncated)

Genome not methylated Shuffling of target recognition domains

Fig. 4 | Mechanisms of switching and methylome diversification in phasevarions. a | Phase variation (ON–OFF switching) by the expansion and/or contraction of single sequence repeats (SSRs). Frameshifting in the coding sequence of a DNA methyltransferase gene causes premature termination, as a stop codon is introduced, generating a cell lineage whose genome lacks DNA methylation27,33,114. However, further frameshifting events can either revert or suppress the frameshift, and the restoration of a functional reading frame can permit DNA methyltransferase synthesis and genome methylation. If base methylation affects the DNA–protein interactions that are crucial for gene expression, each bacterial lineage will show a distinct expression pattern of methylation-sensitive​ loci114. b | Formation of DNA methyltransferase variants by recombination between inverted repeats (IRs) or between target recognition domains. DNA methyltransferase variants with distinct specificities generate allele-specific​ patterns of genome methylation, which can in turn have phenotypic consequences27.

expression profiles and produces pneumococcal lineages DNA methylation and bacterial evolution with different virulence capacities123–125. DNA methylation is both a conservative and an inno- vative force in bacterial evolution. R-​M systems func- Transcriptional control by 5mC and 4mC tion as barriers for the acquisition of genetic elements JHP1050 is a 5mC DNA methyltransferase derived from by horizontal transfer26,40. Furthermore, R-​M systems an ancestral R-M​ system that is highly conserved among can limit recombination between bacterial clades that H. pylori strains126. Mutants that lack JHP1050 show harbour distinct R-​M repertoires131,132. A likely mecha­ alterations in adherence to host cells, natural competence nism is cleavage of incoming DNA molecules, with a for DNA uptake, bacterial cell shape and susceptibility to concomitant shortening of recombination tracts. These copper126. These phenotypes correlate with altered pat- conservative roles are strengthened by the behaviour of terns of gene expression, and site-directed​ mutagenesis of R-​M systems as toxin–antitoxin addiction modules: if GCGC targets at promoter regions has provided evidence an R-​M system is lost, the decrease in the number of for direct transcriptional control by 5mC126. The forma- DNA methyltransferase molecules may permit restric- tion of 5mC may further influence gene expression in an tion, and a single chromosome break can be sufficient to indirect manner, perhaps by changes in DNA topology, cause cell death40,133. Among orphan methyltransferases, as was previously proposed for 6mA92. Dam contributes to genome maintenance by the repres- In Vibrio cholerae, the VhcM orphan 5mC methyl- sion of transposable elements91,134 and prophages135,136, transferase is necessary for optimal growth, both in vitro as well as by reduction of the mutation rate via mis- and during infection, and modulates stress responses127. match repair105. Interestingly, Dam methyltransferase Transcriptome changes in vhcM deletion mutants may can also be viewed as an addictive component of the be a consequence of direct transcriptional control, but genome, as its loss causes genome instability upon DNA indirect effects are also conceivable127. A similar situation strand breakage59,137. may apply to upregulation of the general stress response A major role of both Dam and phase-​variable DNA in E. coli mutants lacking the Dcm 5mC methyltrans- methyltransferases is the formation of phenotypic line- ferase128, as well as to RpoS-​dependent control of a drug ages, which enables division of labour in a community or transporter129. prepares the community for future changes in the envi- The literature also contains one example of epigenetic ronment (bet-hedging)​ 138. An organism can only have a signalling by 4mC: M2.Hpy.AII, a DNA methyltrans- limited set of traits, and improvement of a given pheno­ ferase that introduces a methyl group at the N4 position type comes at the expense of another phenotype139. in the first C of TCTTC motifs, controls gene expression Lineage formation solves this problem by allotting spe- and virulence-​related traits in H. pylori130. As in other cific phenotypes to each cell type in a manner that can studies discussed above, transcriptional control may be benefit the whole population. Formation of epigenetic either direct or indirect130. cell variants may also provide a strategy for mutation

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avoidance: mutations are often deleterious, and their costs. An example is provided by the Dam methylation-​ accumulation in a population can decrease fitness140,141. dependent OpvAB bet-​hedging system of S. enterica, In fact, bacterial populations with high mutation rates which confers bacteriophage resistance by modification adapt quickly to a given environment but are outcom- of the O-​antigen107 (see above). In an opvAB deletion peted by non-​mutators in the long run142. By contrast, mutant strain, bacteriophage-​resistant mutants can be non-​mutational variation can adapt bacterial popu- isolated; however, such mutants often harbour mutations lations to environmental challenge with lower fitness that impair fitness107.

Box 2 | Applications of DNA methylation in biotechnology and synthetic biology Lack of DNA methylation attenuates virulence, sometimes severely, in a large number of bacterial pathogens13. In species in which DNA methylation is essential and mutants are not viable, the attenuation of virulence has been observed following the overproduction of DNA methyltransferases13. This relationship between DNA methylation and virulence has fostered novel preventive and therapeutic approaches to combat bacterial infections. For instance, live vaccines against Salmonella spp. have been developed using strains that carry dam mutations, alone or combined with other mutations that attenuate virulence159,160. Another example is an oral Yersinia pseudotuberculosis vaccine, which has been shown to be protective in mice52. A different approach involves the use of DNA methyltransferase inhibitors as antibacterial agents152. In gammabacterial pathogens in which Dam-dependent​ methylation is dispensable, the inhibition of Dam methylase activity can produce phenocopies of dam mutants, thus reducing virulence without affecting viability. As a consequence, the selection of inhibitor-resistant​ variants may not be strong. An additional advantage is that inhibitors of DNA adenine methylation may be efficient against various bacterial pathogens, as DNA adenine methyltransferases show structural similarity in different bacterial clades38. Inhibitors of the DNA adenine methyltransferases of alphaproteobacteria and of Gram-​positive bacteria have been also described153,154. The inhibition of DNA adenine methylation was initially considered harmless for the mammalian host. However, the recent discovery of DNA adenine methylation in the human genome161 raises the concern of possible side effects. A recent study has suggested the possibility of using Dam methylase inhibitors to enhance the therapeutic activity of antibiotics155. Lack of Dam methylation increases the bactericidal activity of β-lactams,​ because exposure to the antibiotic causes oxidative damage, which in turn induces SOS-dependent​ error-​prone repair. In wild-​type Escherichia coli, Dam-dependent mismatch repair corrects the errors. However, in the absence of Dam methylation, the MutHLS mismatch repair system generates double-strand​ breaks in the DNA. Such breaks further activate the SOS response, thus generating a toxic feedback loop that potentiates the lethal action of the antibiotic155. DNA methylation may also have application in phage therapy: the use of phages that encode DNA methyltransferases can be expected to boost the elimination of infectious bacteria, by delaying the ability of the host R-M​ systems to kill the incoming phage43. An appealing but largely unexplored application of bacterial DNA methylation is the development of DNA methylation-​ based biosensors (see the figure). An example is provided by a recent study that describes synthetic memory systems that are able to store information in E. coli in the form of CcrM-​dependent methylation patterns156. In the negative control system (see the figure, part a), signal detection dislodges a repressor from the control region of a DNA methylase gene (mtase), and methylation of the control region prevents repressor binding. The resulting feedback loop will keep the sensor active even if the signal disappears, and the DNA methylation pattern will be transmitted to daughter cells, thus providing memory of a past event156. In the positive control system (see the figure, part b), signal detection enables a transcription factor to activate the expression of a DNA methylase gene. In both systems, genome methylation will occur in response to the signal. The addition of a fluorescent protein gene downstream of the DNA methylase gene (not shown in the figure) can help detect sensor activation. Variants of these systems have been engineered to detect various environmental stimuli including heat, DNA-​damaging agents and nutrients. A device for signal reset can be added to memory sensors (see the figure, part c): induced synthesis of a protease (Lon) permits the degradation of an engineered DNA methylase variant that contains a protease-​sensitive tag156.

a Negative control b Positive control c Signal reset OFF state OFF state

mtase mtase Signal lon Repressor Inactive activator

ON state ON state Protease

mtase Signal mtase

DNA methyltransferase Activator Protease-sensitive tag Signal Inactive mtase repressor

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Although Dam methylation-dependent​ switches and may represent a burden in others. However, asymmet- phasevarions may have analogous roles in the forma- ric mutation towards AT seems to be a universal phe- tion of bacterial lineages, they must be viewed as the nomenon in bacterial genomes146, and the bias may be products of distinct evolutionary strategies, each with explained by a high rate of 5mC deamination147. advantages and drawbacks. Dam-​dependent switches are the products of long-term​ co-evolution​ with the core Conclusions and future perspectives genome, and the formation of Dam-​dependent methyl- Conservation of the DNA methyltransferase structure ation patterns may be an especially robust mechanism suggests its early evolutionary origin38, and the wide- for transmission of epigenetic memory to daughter cells. spread methylation of bacterial and archaeal genomes Furthermore, crosstalk with the housekeeping machin- may be indicative of positive selection during evolu- ery may permit adjustment of the switching frequency tion24. A conceivable scenario is that DNA methylation in response to environmental cues72,100,101. However, arose as a genome defence mechanism associated with integration into host regulatory networks may heav- restriction40. An alternative possibility is that restric- ily constrain further evolution. By contrast, stochastic tion enzymes did not initially evolve to destroy foreign switching of phasevarions may be prone to perturbation DNA molecules, but to guarantee that DNA methyl­ but at the same time be more evolvable, and independ- transferases remained active24. High rates of hori- ence from host genome control may further increase zontal transfer of DNA methyltransferase genes have evolvability. Recombination between DNA methyl- contributed to the spread of DNA methylation in the transferase alleles may be a powerful mechanism for the bacterial world41, and exchange and shuffling of DNA generation of DNA methyltransferase alleles with novel methyltransferase genes have promoted methylome TRDs, especially in species that harbour multiple DNA diversification24,27,118,143. methyltransferase genes113,118. During evolution, and perhaps very early, the addi- High rates of horizontal transfer of DNA methylase tion of epigenetic information to the nucleotide sequence genes, either R-​M or orphan, can further promote turned out to confer adaptive traits to bacterial cells. One bacterial diversification40,41. Indeed, DNA-​recognition such trait is the ability to form phenotypic cell variants, domains seem to be highly mobile143. In R-​M systems, often in a reversible manner15,27,111. An asset of reversibil- a change of DNA methylation specificity can permit ity is the ability to restore phenotypic heterogeneity after the acquisition of novel foreign elements, including a bottleneck, a circumstance that is especially frequent prophages, transposons, plasmids and other mobile ele- inside animal hosts27. The abundance of phasevarions in ments, perhaps accompanied by the loss of resident, old bacterial pathogens may support this view27,33,114. components of the peripheral genome41. Furthermore, In certain bacterial clades, long-​term co-​evolution the acquisition of a novel methylation pattern may mod- with the host genome has permitted DNA methyltrans- ify the transcriptome if the transcriptional machinery ferases to participate in the control of housekeeping of the recipient cell contains appropriate readers. The processes, including the cell cycle32,34, chromosome fate of the new-​born pattern of epigenetic regulation replication and segregation6–8, DNA repair105 and the will be determined by natural selection, and it might secretion of virulence determinants13. Functional analy­ instantly produce a novel bacterial clade, in the case sis of bacterial methylomes may reveal novel roles in of high adaptive value. Shuffling and exchange of TRD years to come. modules may thus be viewed as a trial-​and-error game Modification of the host epigenome by bacterial path- for bacterial diversification. ogens is a well-established​ phenomenon, the significance The use of 5mC as an epigenetic mark has an enig- of which remains to be understood148–150. Epigenetic matic side because 5mC is unstable in all cells, including memory of a bacterial infection might benefit either bacteria, and 5mC deamination produces thymine144. the host (for example, by boosting immune responses), In the resulting T:G mismatches, discrimination of the the pathogen (for example, by weakening immunity) or incorrect base is not feasible, as both T and G are nor- both organisms (for example, by facilitating commensal mal bases. If left unrepaired, a GC to AT transition will or symbiotic co-​existence). An intriguing possibility is occur upon DNA replication, and one of the daughter that DNA methyltransferases of intracellular pathogens DNA molecules will contain a base pair substitution. may directly methylate the eukaryotic genome151. Gammaproteobacteria possess a repair system (very Future applications of bacterial DNA methylation in short patch repair) that corrects T:G mismatches by biotechnology may include the use of inhibitors of DNA replacing T with C145. However, a system of this kind adenine methylation as antibacterial drugs152–155 and is absent in many bacterial clades: for instance, 5mC the design of synthetic switches, sensors and memory deamination contributes to the high mutation frequency devices156,157 (Box 2). of H. pylori126. One can speculate that a high mutation rate may have adaptive value in certain species and Published online xx xx xxxx

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