DNA Methylation in Prokaryotes: Regulation and Function

Reference work entry
Part of the Handbook of Hydrocarbon and Lipid Microbiology book series (HHLM)


Methylation of DNA in prokaryotes is known since the 1950s, but its role is still elusive and therefore under intense investigation. Differently from eukaryotes, the most important methylation in bacteria takes place on adenines (in position N6). The enzymes responsible for DNA methylation are often associated with restriction enzymes acting as a defense mechanism against foreign DNA (Restriction-Modification or R-M system). Other methyltransferases are solitary that function independently of the presence of a cognate restriction enzyme and are mostly involved in controlling replication of chromosome, DNA mismatch repair systems, or modulating gene expression. This is the case of the methylase Dam in gamma-proteobacteria or CcrM in alpha-proteobacteria. In this chapter, we will discuss the role of the R-M system and the activity of Dam and CcrM.

1 Introduction

All living organisms store the life information in a coded form in their DNA that is transmitted to the progeny. However, this inherited information can be modified by methylation of DNA, leading to an epigenetic reversible control of the genetic program. The nucleotides that can be modified by transferring a methyl group are adenosines (prevalent in prokaryotes) and cytosines (prevalent in eukaryotes). In bacteria, this DNA modification controls many important processes, including, for example, the restriction systems, regulation of gene expression, or the control of DNA replication. In this chapter we’ll focus on adenosine methylation, while cytosine methylation, of which its role is less understood, is described elsewhere (Casadesús 2016; Adhikari and Curtis 2016).

The first evidence of DNA methylation in the bacteria was found studying bacterial infection by phages (Bertani and Weigle 1953). The DNA of phages and the bacterial host cell can have different methylation patterns. This mechanism encoded in many bacterial genomes is composed by a DNA methyltransferase that “marks” the DNA with specific methylation signatures, therefore protecting the DNA from the activity of the cognate restriction enzyme. This R-M (restriction-modification) system functions as a form of bacterial immune system that is able to protect the host bacterium from foreign DNA invasion.

Several DNA methyltransferases are reported which do not belong to the R-M systems. In fact, there are orphan (or solitary) DNA methyltransferases without a cognate restriction enzyme that are involved in important cellular mechanisms. Deoxyadenosine methylase or Dam is one of the most studied orphan adenine methyltransferase in the gamma-proteobacteria, being first discovered in Escherichia coli (Boye and Løbner-Olesen 1990). In the model alpha-proteobacterium Caulobacter crescentus another methyltransferase, CcrM (Cell Cycle Regulated Methylase), plays a crucial role in the regulation of cell cycle (Zweiger et al. 1994). The origin of these solitary methyltransferases could be related to a loss of the corresponding restriction endonucleases as suggested by the discovery of R-M systems in the genome of Helicobacter pylori in which the restriction enzyme has lost its activity while the methyltransferase is still functional (Fox et al. 2007). However, the high number of solitary methyltransferases in bacterial genomes may suggest the exact opposite hypothesis with an early evolution of methyltransferring enzymes later associated with endonucleases (Blow et al. 2016).

2 Bacterial Restriction-Modification Systems

DNA methylation in bacterial systems was initially discovered as a primitive defense mechanism employed by the bacteria to restrict the influx of extraneous DNA from bacteriophages (Luria and Human 1952; Bertani and Weigle 1953). This defense mechanism, known as restriction-modification (R-M) systems, consists of two enzyme components, a DNA methyltransferase (MTase) and a restriction endonuclease (REase). While the MTase methylates a specific site in the DNA sequence, the REase cleaves the DNA in a sequence specific manner. Because of the presence of both restriction and modification components of the R-M system, the bacteria are inherently capable of discriminating between the “self” and the “non-self” genetic material. As the DNA molecules originating from the bacteriophages have different methylation pattern than the host cell, the restriction enzymes produced by the host can cleave the incoming DNA and restrict the bacteriophage infection. These properties of the bacterial R-M systems are akin to an innate immune response mechanism, in contrast to the CRISPR-Cas systems that provide an adaptive form of defense against bacteriophage invasions (Vasu and Nagaraja 2013).

At the biochemical level, REases produce 5′ or 3′ overhangs or blunt ends at the site of their action by cleaving a phosphodiester bond. MTases modify the target nucleotides by adding a methyl group to the N6 amino group of the adenine or the C5 carbon or the N4 amino group of the cytosine residues. Both adenine and cytosine residues are modified in the bacterial and archeal genomes by methylation. At the functional level, the R-M systems exhibit considerable diversity and, therefore, can be categorized into four distinct types (Type I–IV) according to their recognition sequence, subunit composition, cofactor requirements, and site of cleavage (Roberts et al. 2003).

2.1 Four Types of Restriction-Modification Systems

The Type I enzymes consist of subunits for the restriction and methylation activities. The enzymes function as hetero-oligomers and cleave the DNA sequence from 100 bp to several Kbp away from their recognition sites. These enzymes are encoded by three different genes, namely hsdR, hsdM, and hsdS (Ershova et al. 2015). The enzyme recognition site consists of two parts separated by an intervening sequence of nucleotides, with this sequence AAC(N)6GTGC being a representative (Ershova et al. 2015). Type I MTase is a complex of two methyltransferase subunits and a specificity determining subunit (M2S1). The S subunit possesses two target recognition domains that interact with the two recognition sites, whereas the MTase dimer methylates both the DNA strands simultaneously. The restriction enzyme binds to the unmethylated target site and translocates along the DNA till it finds any other DNA binding protein, where it cleaves the DNA in an ATP-dependent manner. Because of this activity the cleavage occurs far from the recognition site of the enzyme (Roberts et al. 2003; Ershova et al. 2015).

The Type II R-M systems consist of distinct restriction and methyltransferase enzyme components that are encoded by two different genes. The restriction enzyme functions either in homodimeric or homotetrameric form and specifically cleaves the DNA within or very close to their recognition site (Vasu and Nagaraja 2013). The recognition sequence is often a short palindrome consisting of 4–8 bp sequences (for example GAATTC). The type II MTases act as monomer and methylate both DNA strands after binding. Because of their precise site specificity and their diversity, they are very useful in genetic manipulation experiments and, therefore, most extensively studied. The type II R-M systems are further subdivided into 11 groups on the basis of their biochemical properties, though the subtyping is not mutually exclusive (Roberts et al. 2003; Ershova et al. 2015).

The type III R-M systems are encoded by the two closely located genes, mod and res, that produce the methyltransferase and the restriction components, respectively (Janscak et al. 2001; Dryden et al. 2001; Mücke et al. 2001). The Type III enzymes function as heterotrimers or heterotetramers consisting of restriction, methylation, and DNA-dependent NTPase activities. The restriction and methylation enzymes compete for the same catalytic reaction leading to incomplete reactions on most occasions. The restriction enzyme recognizes a 5–6 bp long nonpalindromic sequence and cleaves at the 3′ site at a distance of ~25–27 bp (Dryden et al. 2001; Vasu and Nagaraja 2013).

In contrast to the already described R-M systems, the enzymes belonging to the type IV systems are not R-M systems in true sense, as they do not have a methylation component. Also, the restriction enzyme only cleaves the DNA substrate that has been modified previously such as methylated, hydroxyl-methylated, and glucosyl-hydroxy-methylated, although the sequence specificity is not very well defined among these enzymes. Because of the lack or low- sequence specificity they protect the host from a broad range of extraneous DNA having different methylation patterns (Ershova et al. 2015; Loenen and Raleigh 2014). Interestingly, enzymes belonging to the type II M are also methylation directed and, therefore, have been proposed to be included in the type IV (Loenen and Raleigh 2014).

2.2 Occurrence and Prevalence of R-M Systems in the Bacterial Genome

With the advancement of the genome sequencing techniques and the availability of a great number of prokaryotic genomes, it is now apparent that very diverse sets of R-M systems exist in nature. The R-M systems are ubiquitous among the prokaryotes. So far approximately 4000 enzymes are known that show 300 different specificities (Roberts et al. 2010). According to the REBASE (restriction enzyme database), out of the 8500 sequenced genomes of prokaryotes only 385 have no recognizable R-M systems (Roberts et al. 2015; Ershova et al. 2015). A more recent study using SMRT (Small Molecule Real Time) sequencing methods surveying 217 bacterial and 13 archaeal species, from 19 different phyla and 37 different classes, reported an extensive dataset of novel methylated motifs in these genomes (Blow et al. 2016). This study reported a total of 858 distinct methylation motifs present in 93% of the genomes tested. Interestingly, the methyltransferases show considerable sequence conservation, while restriction enzymes are quite diverse. It could be reasoned that while sequence conservation of the MTases limits the number of recognizable extraneous sequences, at the same time broad REase specificity would provide the scope to cleave a large number of nonself DNA that the bacterial cell encounters. The genome sequences have provided information that more than 80% of the sequenced genome contains multiple R-M systems. Also, the number of R-M systems present in a genome is related to its size. For example, three R-M systems are present in organisms with a genome size between 2 and 3 Mbp, and four R-M systems in organisms with genome size between 3 and 4 Mbp (Vasu and Nagaraja 2013). However, there are notable exceptions such as Helicobacter and Campylobacter species of genome sizes between 1.5 and 2 Mbp that possess around 30 R-M systems (Vasu and Nagaraja 2013). The significance of such a large number of R-M systems in these species is not fully understood. In contrast, some obligate intracellular pathogens such as Chlamydia, Coxiella, and Rickettsia of genome size between 1 and 2.5 Mbp have no R-M systems. Considering the environmental niche where these organisms live, they may not be encountering any bacteriophages, therefore, the absence of the R-M systems (Vasu and Nagaraja 2013). Another interesting feature with respect to the genome size and the recognition motif of the R-M systems is that the larger proportion of the R-M systems present in organisms with a larger genome (Bacillus, Pseudomonas, Streptomyces, etc.) can recognize longer palindromic sequences (Vasu and Nagaraja 2013). Considering that a larger genome has a higher number of 4 bp or 6 bp recognition motifs than the 8 bp ones, it is reasonable to assume that having more number of R-M systems that prefer the longer recognition motifs would limit nonspecific cleavage of the host genome leading to random double strand breaks.

The widespread presence of the R-M systems in the prokaryotic genomes and the diversity of their recognition sequences have been attributed to their importance in their life cycle and also to their “selfish” nature by which they maintain in the genome. Previous studies have demonstrated that type II R-M systems, carried by plasmids, ensure their retention during postreplication segregation in the bacterial host, indicating the selfish nature of this R-M systems (Naito et al. 1995; Kobayashi 2001). Because of horizontal gene transfer events bacteria can also acquire new R-M systems, which is evident from the fact that different strains of the same bacterial species possess different R-M systems (Oliveira et al. 2014; Croucher et al. 2014). Specific regions in the bacterial genomes have been detected that encode several R-M systems and named as Immigration Control Regions (ICR) involved in the defense mechanisms. Investigations with the ICR flanking regions suggest that these regions are acquired from other genomes by horizontal gene transfer (Kobayashi 2001). In addition, R-M genes are colocalized with mobile genetic elements in several bacterial species such as Staphylococcus aureus and Helicobacter pylori (Alm et al. 1999; Kobayashi 2001; Lindsay 2010; Xu et al. 2011). The presence of a number of REase pseudogenes in the vicinity of the MTase genes, orphan REases that no longer function as endonuclease and nonfunctional MTases in the bacterial genomes, indicates a loss of R-M systems (Seshasayee et al. 2012; Furuta et al. 2014). Apart from the mechanisms mentioned above, prokaryotic R-M systems can alter their sequence specificity leading to the emergence of new R-M systems in the genome (Furuta et al. 2014; Sánchez-Romero et al. 2015).

2.3 Functions of the R-M Systems

The discovery of R-M systems was made in the context of the bacterial defense mechanisms against invading bacteriophages (Luria and Human 1952; Bertani and Weigle 1953). However, bacteriophages also employ several counterstrategies to avoid the host restriction, such as the modification of nucleotides (methylation, glucosylation, etc.). Bacterial species also possess restriction enzymes (type II M and type IV) that target such modified nucleotides from the bacteriophages leading to a “coevolutionary arms race” that has produced several diverse R-M systems. Even though it is suggested that the arms race between the host and the bacteriophages has produced so much diversity with respect to the R-M systems, however, it is not clear how a very specific restriction enzyme would provide protection against a broad range of bacteriophages. Also, it is not properly understood why certain bacterial species for example naturally competent ones like Helicobacter pylori, Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria gonorrohoeae possess multiple R-M systems in their genome (Vasu and Nagaraja 2013; Ershova et al. 2015).

Discrimination between the self and nonself genetic material is the primary role of the R-M systems in the bacterial genome. However, the existence of the different subpopulations of a bacterial species is always beneficial for the species survival. In this context, R-M systems help to maintain the genetic diversity by limiting the gene transfer from other bacterial cells (Raleigh 1992; Sibley and Raleigh 2004). Horizontal gene transfer events are limited because of the presence of R-M systems in several bacterial species. The most notable examples are Burkholderia pseudomallei, Streptococcus pneumoniae, and Neisseria meningitidis. In the case of B. pseudomallei, the R-M systems have been shown to function as a barrier to genetic exchange. Three phylogenetic groups are identified among the analyzed B. pseudomallei genomes, showing almost no genetic exchanges between them. Representatives from the groups showed the presence of different R-M systems and further studies indicated that they are primarily responsible for restricting gene transfer among the different phylogenetic groups. In the case of S. pneumoniae 15 monophyletic groups has been recognized that possess distinct sets of R-M systems. In the case of N. meningitidis, analysis using 20 sequenced genomes indicated the presence of eight different phylogenetic groups possessing 22 different R-M systems (Ershova et al. 2015). To understand the impact of the presence of R-M systems on horizontal gene transfer events, Budroni et al. (2011) showed that the size of the fragments that were transferred among the different phylogenetic groups in N. meningitidis were significantly shorter than the fragments exchanged within the group (680 bp vs. 3890 bp).

Even though the orphan methyltransferases are primarily involved in the epigenetic regulation of gene expression (see next sections), MTases that are part of the R-M systems sometimes influence the expression of genes in several bacterial species. In the case of H. pylori, the transcriptome analysis indicated differences in the expression of genes in the strains carrying different methylation patterns (Furuta et al. 2014). Another consequence of alternate gene expression is phase variation among the bacterial strains, which is a heritable yet on-or-off switching of transcription. This is adopted by several pathogens to increase their survival and fitness in different environmental niches (Hallet 2001; van der Woude and Bäumler 2004). Several pathogens use phase variation to generate diversity in their surface antigenic structure, such as pili, flagella, capsules, lipopolysaccharides, etc. (Weiser et al. 1990; van der Woude and Bäumler 2004). Based upon nucleotide sequence analyses and other genetic studies, it has been shown among several pathogens such as H. influenzae, H. pylori, and N. meningitidis that type I and III enzymes are involved in phase variation mechanisms (Dybvig et al. 1998; De Bolle et al. 2000; de Vries et al. 2002; Fox et al. 2007). Though the R-M systems are implicated in phase variation mechanisms in several bacterial species, their significance is not completely comprehended as yet.

The restriction enzymes cleave the nonself DNA into smaller fragments that sometimes act as substrates for homologous recombination with the host genome. Though the primary role of the R-M systems is to restrict the entry of foreign DNA, the homologous recombination that follows after an initial cleavage could be a byproduct of the process (Price and Bickle 1986; Vasu and Nagaraja 2013). In several bacterial species, nonhomologous recombination events are also a consequence of restriction mechanisms, in which a small homologous DNA sequence is used to recombine and integrate a larger nonhomologous region into the host genome. Such illegitimate recombination events, leading to genome rearrangements, have been observed with the type I R-M enzyme EcoKI (Kusano et al. 1997).

3 Dam Methyltransferase

In gammaproteobacteria, methylation by orphan (or solitary) methyltransferases plays several important roles, such as DNA mismatch repair, control of chromosome replication, and regulation of gene expression. Loss of function dam mutants in E. coli and Salmonella enterica showed an increased mutation rate (Marinus and Morris 1974; Torreblanca and Casadesús 1996) and an abnormal rate of the initiation of chromosome replication (Boye et al. 1988). The Dam methylase (Deoxyadenosine methyltransferase) is a 32 KDa monomer that uses S-adenosyl-L-methionine (AdoMet) as methyl group donor to the adenine N6 atom (Herman and Modrich 1982). Dam transfers a methyl group to 5′-GATC-3′ sequences that are usually fully methylated (the adenosines on both strands are modified), as Dam is constitutively expressed, except for a short period following DNA replication (Marinus and Casadesus 2009). Accordingly the optimal substrate of Dam is hemimethylated DNA although this enzyme can also methylate de novo unmethylated target sites (Herman and Modrich 1982). In E. coli, Dam methylates the ca. 20000 sites in a highly processive manner (Urig et al. 2002) ensuring a rapid and robust remethylation of the entire chromosome. But what is the function(s) of Dam methylation?

3.1 Dam-Dependent DNA Mismatch Repair

In E. coli, mismatches, due to erroneous incorporation of bases, is corrected by a mechanism called methyl-directed mismatch repair (MMR) (Fig. 1). Whereas in the base excision repair and nucleotide excision repair, the wrong nucleotide is directly detected by the repair system, in the case of a mismatch the two replicated strands are composed of unmethylated nucleotides. The presence of a methylated strand (nonmutated) dictates the correct repair (Lu et al. 1983; Pukkila et al. 1983). Mismatch regions are detected by MutS, the first DNA binding protein that then recruits MutL and MutH (Caillet-Fauquet et al. 1984; Au et al. 1992; Iyer et al. 2006). In this ternary complex, MutH has endonuclease activity on nonmethylated DNA strand near the GATC methylation site (Welsh et al. 1987), while MutL connects MutS with MutH. The exonuclease UvrD then digests the single strand mutated DNA and DNA polymerase III in complex with single strand binding proteins and DNA ligase completely repairs the mutated region (Iyer et al. 2006).
Fig. 1

Schematic view of the mismatch repair system in E. coli. Mismatch regions in the E. coli genome (asterisk) are detected by MutS, the DNA-binding protein that then recruits MutL and MutH. In this ternary complex, MutH has endonuclease activity on nonmethylated DNA strand near the GATC methylation site, while MutL connects MutS with MutH. The exonuclease UvrD then digests the single-strand mutated DNA and DNA polymerase III in complex with single-strand binding proteins (dotted grey line) and DNA ligase completely repair the mutated region. Finally Dam remethylates the repaired strand

Concentration of Dam methylase in the cells is tightly regulated (Løbner-Olesen et al. 1992). As mentioned earlier, the absence of Dam in E. coli and Salmonella results in an increasing rate of mutations. However, overexpression of Dam also causes an augmentation of mutations (Pukkila et al. 1983; Torreblanca and Casadesús 1996). This apparent contradiction depends on the specificity of MutH for hemimethylated DNA sequences, while the endonuclease activity of MutH is basically absent on fully methylated templates, when Dam is overexpressed (Herman and Modrich 1981). Therefore, too much or no methylation cause the same MutH inactivity and then accumulation of mutations.

3.2 Chromosome Replication Control

The origin of replication (oriC) in E. coli is controlled at multiple levels, including Dam methylation. As initiation of replication depends on the activity of DnaA, regulation of dnaA transcription and modulation of its activity are two important mechanisms at the onset of DNA replication (Mott and Berger 2007). A third mechanism directly controls the accessibility of the origin of replication (Waldminghaus and Skarstad 2009). Dam methylation is responsible for two of those mechanisms: regulation of expression of dnaA and oriC binding, both phenomena depending on SeqA (Fig. 2). Most of the 20000 GATC sites in the E. coli genome are remethylated in few seconds (Stancheva et al. 1999). However, oriC and the promoter of dnaA, containing respectively 11 and 8 GATC sites, remain hemimethylated for at least 10 min, although Dam methylase is present in the cells. This “protection” is performed by the dimeric protein SeqA (Campbell and Kleckner 1990), which has affinity for hemimethylated GATC sites that are also closely spaced (Kang et al. 1999, 2005). Binding of SeqA to the oriC and the dnaA promoter in turn blocks further access to those sites, stalling both the access to the origin of replication by the replisome and the transcription of dnaA. This time gap allows the conversion of the active DnaA-ATP to inactive DnaA-ADP and the blocking of DnaA production by SeqA repression of the promoter region (Katayama et al. 1998; Kato and Katayama 2001). In conclusion, Dam methylation and SeqA are intimately linked with respect to the initiation of DNA replication. Accordingly, seqA loss of function mutants show high similarity to Dam-overproducing cells (Løbner-Olesen et al. 2003).
Fig. 2

Mechanism of action of methylation dependent chromosome replication initiation in the E. coli. In G1 cells of E. coli, the origin of replication and the promoter of dnaA are completely methylated. At the onset of DNA replication, the protein DnaA binds the oriC sites initiating replication. The two newly replicated oriCs will be then hemimethylated. In this form the protein SeqA replaces DnaA from oriC and blocks transcription of dnaA. Shortly Dam methylase remethylates all sites blocking early reinitiation of DNA replication until a new cycle is possible

3.3 Regulation of Gene Expression

Methylation sites, such as E. coli Dam GATC, are widespread along the genome and occur approximately every 200–300 bp; however, their presence in promoter regions, similarly to SeqA and the promoter of dnaA, may affect binding and activity of transcription factors and RNA polymerase, especially when the number of methylation sites is above average distribution. Expression profiles of dam-deficient cells have been recorded revealing genes whose transcription change without methylation (Torreblanca and Casadesús 1996; Oshima et al. 2002). However, not all affected genes are directly controlled by Dam methylation, such as the SOS regulon that is on the contrary activated by the abnormal activity of the MutSHL system in the absence of methylation (Marinus and Casadesus 2009). In other words, although methylation affects the expression of many genes, only a few subsets of promoters, associated with specific DNA-binding proteins, may be directly linked to a methylation control.

In Salmonella, among genes in the conjugative plasmid containing virulence factors, traJ encodes a transcription factor, whose expression is under the control of the global regulator Lrp (Camacho and Casadesús 2002, 2005). The promoter of traJ has two Lrp binding sites required for the activation of transcription. A fully methylated GATC site, present in one of those Lrp binding sites, is able to repress traJ transcription, while only hemimethylation on the adenosine of the noncoding strand is able to trigger the transcription of the gene (Camacho and Casadesús 2005). Therefore, when the plasmid is replicated, only one daughter DNA sequence is expressed although plasmids have the same genetic information. As activation of traJ requires DNA replication, a physiological state associated with nutrient abundance and absence of stress, this epigenetic activation ensures that energy-consuming conjugation will take place in a suitable environment. Similar mechanisms of epigenetic regulation are described in the regulation of the IS10 transposase, whose activity is kept at a low level by this kind of methylation control (Roberts et al. 1985).

Some bacterial species show the phenomenon called “phase variation ” where genetically identical populations of cells express certain factors at different levels. This is a bet-hedging strategy used by the bacterial species, to prepare them to face changing environmental conditions. Although several mechanisms of phase variations require the variation of the DNA sequence, some mechanisms are epigenetically controlled by DNA methylation. For example, in the uropathogenic E. coli, the synthesis of Pap pili is under the control of the methylase Dam (Fig. 3). During infection, the population contains bacteria with (ON) and without Pap pili (OFF). This dual expression pattern derives from two opposite characteristics of Pap pili: on one hand those pili are highly immunogenic triggering immune responses; on the other hand Pap pili are indispensable for the colonization of the upper urinary tract. Therefore, this dual nature of the population keeps the potential ability to colonize new infection niches without triggering too much the immune response (Roberts et al. 1994; Hernday et al. 2002). This epigenetic regulation is based on the presence of two Dam methylation sites: GATC-I or distal (upstream the papI gene) and GATC-II or proximal (upstream the pap operon). In the OFF state, GATC-II is nonmethylated while GATC-I is methylated; in the ON state, the methylation pattern is the opposite. Differently from the rest of the genome, GATC sites in the pap operon can show a nonmethylated state because of the DNA binding of the Lrp protein that prevents Dam methylation. In the OFF state, Lrp is bound to GATC-II, keeping its state nonmethylated and preventing Lrp to bind GATC-I, which is indeed methylated. In the OFF state, Lrp binding represses the pap operon transcription. This situation is stable unless a second factor (PapI), whose fluctuations of expression are noisy, reaches a threshold able to dislocate Lrp from GATC-II in favor of GATC-I. Once GATC-II is free from Lrp, Dam is able to methylate the GATC-II site therefore stabilizing the ON state. Several cycles of DNA replication will free the GATC-I site from methylation as Lrp and PapI stably protect this region (Casadesús and Low 2006, 2013).
Fig. 3

Role of DNA methylation in phase variation . Pap pili expression is under the control of the methylase Dam. During infection, the population contains bacteria with Pap pili (ON) and cells with no pili (OFF). GATC-I and GATC-II are present (black box). In the OFF state, GATC-II is nonmethylated while GATC-I is methylated; in the ON state, the methylation pattern is opposite. In the OFF state, Lrp is bound to GATC-II, keeping its state non methylated and preventing Lrp to bind GATC-I, which is indeed methylated. In the OFF state, Lrp binding represses the pap operon transcription. In the ON-state, accumulation of PapI moves Lrp to GATC-I, freeing GATC-II allowing Dam to methylate the GATC-II site therefore stabilizing the ON state

Dam methylation and Lrp also control other fimbrial operons such as foo, clp, and pef (Casadesús and Low 2006). However, Dam can also be associated with other factors regulating phase variation. For example, the locus agn43, encoding a membrane protein of the outer membrane involved in biofilm formation and interaction with the infected host, is regulated by Dam methylation and the protein OxyR (Henderson and Owen 1999; Danese et al. 2000; Waldron et al. 2002; Wallecha et al. 2002, 2003; Kaminska and van der Woude 2010). Finally, OxyR and Dam are also involved in the epigenetic control of the Salmonella opvAB operon encoding a protein modifying the length of the O-antigen in lipopolysaccharide (Cota et al. 2012).

4 CcrM Methyltransferase

Among the cell cycle regulators in Caulobacter crescentus, the methyltransferase CcrM plays a crucial role in coordinating important developmental processes by transcriptional regulation (Table 1). This methylase and its functions are possibly conserved in other alpha-proteobacteria although its role has been less studied (Wright et al. 1997; Robertson et al. 2000; Kahng and Shapiro 2001). In contrast to gamma-proteobacteria in which the hemimethylation state of DNA is not stable and limited in time, in C. crescentus all CcrM loci remain hemimethylated during the S-phase until the replication of DNA is complete. This cell cycle-dependent methylation pattern has an important consequence on the regulation of gene expression over time in coordination with DNA replication progression (Mohapatra et al. 2014).
Table 1

CcrM-dependent genes involved in cell cycle and polarity in C. crescentus



Regulation (hemi/full)




Cell cycle regulator



(Reisenauer and Shapiro 2002; Holtzendorff et al. 2004; Fioravanti et al. 2013; Murray et al. 2013; Gonzalez et al. 2014)


Polarity determining protein



(Holtzendorff et al. 2004; Fioravanti et al. 2013; Murray et al. 2013)


Cell division plane positioning ATPase



(Holtzendorff et al. 2004; Fioravanti et al. 2013; Murray et al. 2013)


Cell division protein



(Gonzalez and Collier 2013; Murray et al. 2013)


Cell division protein


(Fioravanti et al. 2013; Murray et al. 2013; Gonzalez et al. 2014)


Polarity determining protein


(Fioravanti et al. 2013)


Polarity determining protein


(Fioravanti et al. 2013; Kozdon et al. 2013)




(Fioravanti et al. 2013)


Cell shape

(Gonzalez et al. 2014)

ftsW, ftsE

Cell division protein

(Gonzalez et al. 2014)

gyrA, gyrB

DNA replication

(Kozdon et al. 2013; Gonzalez et al. 2014)


Chromosome segregation

(Gonzalez et al. 2014)


Stalk biogenesis

(Kozdon et al. 2013; Gonzalez et al. 2014)


Pole organizing protein

(Gonzalez et al. 2014)

F+, activated when fully methylated; F−, repressed when fully methylated; H+, activated when hemimethylated, “–” no information available.

C. crescentus produces two different cell types at every division that are morphologically and functionally different, a sessile replication competent stalked cell and a vegetative and replication incompetent swarmer cell (Fig. 4a). The stalked cell, capable of replicating the circular chromosome and producing new cells (Brown et al. 2009), possesses a polar tubular appendix, the stalk, having the same composition of the cell envelope (Jenal 2000).
Fig. 4

DNA Methylation and progression of cell cycle in Caulobacter crescentus. (a) C. crescentus cell cycle progression. Every cell division C. crescentus produce two different cell types: a vegetative swarmer cells, possessing a flagellum and pili, that is able to swim; a stalked cell capable of DNA replication and attached to substrate by stalk in continuity with the cell envelope. The swarmer cell must differentiate in a stalk cell in order to initiate a division cycle. After cell division, the stalked cell is able to immediately initiate the DNA replication. C. crescentus has a single circular chromosome, whose origin has a polar localization and it is replicated only once per cell division. (b) The C. crescentus chromosome in G1 is fully methylated by CcrM (four sites are schematically represented here in dark grey). As the replication initiates the sites proximal to the origin of replication (oriC) become hemimethylated (grey and white). As the replication fork proceeds, all sites become hemimethylated until the methylase CcrM (black bar) accumulates at the end of S-phase

In C. crescentus, CcrM methylates adenosines of the palindromic 5′-GAnTC-3′ sites in the DNA double helix (Zweiger et al. 1994). CcrM is present and active only for a short window of time at the end of the S-phase in the late predivisional cells. In fact, the gene encoding CcrM is under the control of the cell cycle master regulator CtrA that has its highest activity in late S-phase (Collier et al. 2007). At the same time, CcrM is degraded by the Lon protease before cell division so that swarmer cells have no CcrM (Wright et al. 1996). Although there are other methylases in Caulobacter, CcrM is the only one showing cell cycle-regulated activity and affecting cell cycle progression (Nierman et al. 2001; Kozdon et al. 2013). The chromosome remains fully methylated in both strands in G2 and G1 phases. The time of conversion during the S-phase into two hemimethylated copies of each GAnTC sequence depends on the chromosomal location of the individual methylation sites (Zweiger et al. 1994; Kozdon et al. 2013) (Fig. 4b).

CcrM methylates the 4542 potential GAnTC sites in C. crescentus (Nierman et al. 2001) using S-adenosyl-L-methionine as substrate (Zweiger et al. 1994) in a distributive manner that is similar to E. coli Dam (Albu et al. 2012), and that is characterized by a preferential binding for a hemimethylated template (Berdis et al. 1998; Albu et al. 2012). Approximately a quarter of these CcrM motifs are located in the intergenic regions suggesting a potential regulation of transcription. Using single-molecule real-time (SMRT) DNA sequencing, all sites methylated by CcrM were identified at base-pair resolution (Kozdon et al. 2013). Accordingly to the previous knowledge, almost all GAnTC sites progressed from full- to hemimethylation and back again to a full methylation stage with the progression of the replication fork during the S-phase. However, 27 GAnTC sites remained permanently unmethylated (Kozdon et al. 2013) during the S-phase. Some of these hypomethylated sequences have been recently shown to be associated with the regulator MucR (Ardissone et al. 2016), although their functional role is still unclear.

CcrM methylation controls many important genes in C. crescentus (Mohapatra et al. 2014). This control is mostly positive (activation on hemi or, less frequently, on fully methylated DNAs) or negative (repression by the full methylation) (Table 1).

Under the control of CcrM-methylation, ctrA (Quon et al. 1996) probably is the most significant gene, as it encodes the master regulator of cell cycle in C. crescentus. Two promoters are responsible for ctrA transcription: ctrAP1 contains a GAnTC site at its −35 region and is activated only in the hemimethylated state, when the replication fork progresses through it, while the promoter ctrAP2 is auto-induced by phosphorylated CtrA (Reisenauer and Shapiro 2002).

The full methylation state keeps the promoter ctrAP1 in a repressed mode and only the conversion to a hemimethylated form allows transcription (Reisenauer and Shapiro 2002). The importance of temporal control of CcrM expression is evident as a strain constitutively expressing CcrM throughout the cell cycle results in aberrant cell types with multiple chromosome duplication events because of a fully methylated chromosome (Zweiger et al. 1994). The transcription from ctrAP1 clearly depends on the amount of time it remains fully methylated during the replication, as ctrAP1 when placed proximal to the origin of replication, showed highest activity, whereas a terminus proximal location of ctrAP1 had basically no expression (Reisenauer and Shapiro 2002). These observations prompted the question concerning the molecular mechanism connecting the methylation state to RNA polymerase activity. Studies have suggested that CcrM is associated with GcrA (Holtzendorff et al. 2004) among the members of the Alphaproteobacteria group (Brilli et al. 2010; Murray et al. 2013). GcrA is a DNA binding protein with sites spread all over the Caulobacter genome (Fioravanti et al. 2013; Haakonsen et al. 2015). Although GcrA target regions are generally associated with GAnTC sites, it also binds to regions in the chromosome having no methylation (Fioravanti et al. 2013). Coherent to previous results (Holtzendorff et al. 2004), GcrA binds the promoter P1 of ctrA; interestingly the CcrM full methylation, which corresponds in vivo to inhibition (Reisenauer and Shapiro 2002), shows the highest binding efficiency in comparison with the two hemimethylation arrangements, which were still more efficient than the nonmethylated sequence (Fioravanti et al. 2013). GcrA affects RNA polymerase by interacting with Sigma70 favoring the open complex formation (Haakonsen et al. 2015).

CcrM orthologs have been investigated in other alpha-proteobacteria, such as Sinorhizobium meliloti, Brucella abortus, and Agrobacterium tumefaciens (Wright et al. 1997; Robertson et al. 2000; Kahng and Shapiro 2001). In contrast to Caulobacter, in which the GAnTC methylation is dispensable (Gonzalez and Collier 2013; Fioravanti et al. 2013; Murray et al. 2013), CcrM in other alpha-proteobacteria is essential, although its exact role has not been elucidated yet (Brilli et al. 2010). Interestingly, CcrM and GcrA orthologs can complement, at least partially, functions in other alpha-proteobacteria, suggesting a similar role (Wright et al. 1997; Robertson et al. 2000; Kahng and Shapiro 2001; Fioravanti et al. 2013). Furthermore, the cell cycle-regulated activity of CcrM, peaking at the end of S-phase, has been demonstrated in A. tumefaciens (Kahng and Shapiro 2001) and S. meliloti (De Nisco et al. 2014).

5 Research Needs

DNA methylation in bacteria has been described since many years, but its real importance is still not fully understood. For example, the role of the solitary methylase CcrM controlling cell cycle in C. crescentus appears still at the beginning of its elucidation. Multiple DNA-binding proteins controlling transcription have been associated with DNA methylation, such as SeqA, Lrp in E. coli or GcrA in C. crescentus, however, there could be many more transcription factors that could detect the methylation state and modulate global transcriptional programs in bacterial cells. It is worth noticing that many CcrM methylation sites are independent of GcrA.

New techniques, such SMRT sequencing (see previous sections), have been developed in order to understand DNA methylation at the genomic scale, revealing that multiple methylases are indeed responsible for DNA methylation in bacterial cells. Next few years will definitely open new surprising frontiers in the epigenetic regulation in bacteria. What is clear for now is that although simple, bacteria possess a complexity in regulatory mechanisms, among which DNA methylation plays a crucial role.


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Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Genetic Engineering, School of BioengineeringSRM UniversityKattankulathurIndia
  2. 2.Aix Marseille University, CNRS, IMM, LCBMarseilleFrance

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