InTechOpen uses cookies to offer you the best online experience. By continuing to use our site, you agree to our Privacy Policy.

Biochemistry, Genetics and Molecular Biology » "Methylation - From DNA, RNA and Histones to Diseases and Treatment", book edited by Anica Dricu, ISBN 978-953-51-0881-8, Published: November 28, 2012 under CC BY 3.0 license. © The Author(s).

Chapter 9

Host-Mimicking Strategies in DNA Methylation for Improved Bacterial Transformation

By Hirokazu Suzuki
DOI: 10.5772/51691

Article top

Host-Mimicking Strategies in DNA Methylation for Improved Bacterial Transformation

Hirokazu Suzuki1

1. Introduction

In 1928, Griffith [1] reported that soluble substances from virulent pneumococcal cells transformed non-virulent pneumococcus to virulent forms. This substance has now been demonstrated to be DNA [2-4]. This is considered to be the first report on genetic transformation of bacteria by exogenous DNA. Subsequently, natural competence of Bacillus subtilis was reported in 1958 by Young and Spizizen [5]. They also demonstrated genetic transformation of natural competent B. subtilis cells using exogenous DNA. It was in 1970 that genetic transformation of Escherichia coli using chemically competent cells was reported [6]. Thus, genetic transformation of common bacterial models was established at an early stage in the development of bacteriology. The alternative view is that bacterial models such as B. subtilis and E. coli have become the mainstay of this field because of high transformation ability. Genetic transformation techniques remain important for studying numerous bacteria and for the advancement of bacteriology, biochemistry, applied microbiology, and microbial biotechnology. Moreover, recent developments in the search for new bacteria and genome sequencing have provided numerous effective bacteria that are useful for biological studies and industrial applications. With these developments, there is a greater demand for establishing genetic transformation methods for more bacteria.

DNA introduction is an essential process for transforming target bacterium by exogenous DNA. Various methods for introducing DNA into bacteria have been developed to date, including chemotransformation, electroporation, sonopolation, tribos, and conjugational transfer [7]. In spite of these developments, it is often difficult to establish transformation methods for target bacterium. A possible reason is the difficulty faced while exploring suitable conditions for introducing DNA, which requires not only theoretical understanding but also a trial and error approach. Circumventing bacterial RM systems is a major challenge. These systems defend bacteria against transformation by exogenous DNA, such as bacteriophages, and effectively hamper genetic transformation by exogenous plasmids. RM systems selectively digest exogenous DNA by differentiating them from host-endogenous DNA on the basis of host-specific DNA methylation [8]. Therefore, DNA that imitates the methylation patterns of the host bacterium (host-mimicking DNA) is incorporated into the bacterium without restriction. Thus, RM systems can be overcome by theoretical host-mimicking strategies (Figure 1), rather than exploring conditions for introducing DNA. This chapter explains host-mimicking strategies and provides tips for establishing transformation methods for new bacteria.


Figure 1.

Host-mimicking strategies for circumventing restriction–modification (RM) systems in bacteria.

RM systems serve to defend bacteria against invasion by exogenous DNA, and thereby hamper genetic transformation by exogenous plasmids. In typical RM systems, restriction endonuclease (RE) digests exogenous DNA but not endogenous DNA that has been methylated by cognate DNA methyltransferase (MT). Host-mimicking DNA that imitates the methylation patterns of the bacterial host is recognized as endogenous DNA by the host because RM systems depend on host-specific DNA methylation to distinguish between exogenous and endogenous DNA.

2. RM systems

In 1962, Arber and Dussoix [9] found that bacteriophage λ carried specificity for E. coli strains in which they were produced. For examples, bacteriophage λ from E. coli K-12 was efficiently transfected into K-12 and C strains, but not strains B and K(P1) (efficiencies were <10−4 fold). Meanwhile, bacteriophage λ produced by E. coli C efficiently transformed strain C, but not strains K-12, B, and K(P1) (efficiencies were <4×10−4 fold). Thus, bacteriophage λ vectors readily infected E. coli strains that produced them but the infection to other strains was “restricted”. This occurrence, termed restriction, is explained by E. coli RM systems that act as host defense against exogenous DNA.

RM systems selectively digest exogenous DNA and greatly influence the efficiency of genetic transformation by exogenous DNA. Numerous RM systems have been found in bacteria and archaea, and are classified into four main types (type I–IV) [10]. Their general properties are summarized in Table 1. The REBASE database ( [11] has accumulated large amounts of information about RM systems, including types, gene sequences, recognition sites, and origin organisms. Among the four types of RM systems, type II consists of RE and MT. In this type, RE cuts exogenous DNA at specific sites but not endogenous DNA that has been methylated by MT (Figure 1). Type I and III systems also cut exogenous DNA by similar mechanisms, but comprise protein complexes of some subunits. Type IV is known as a modification-specific restriction system to cut DNA with heterologous modifications. Intriguingly, RM systems behave as selfish elements like viruses and transposons [12], implying that RM systems have been irreversibly distributed in bacteria. The following sections describe more details of RM systems.

2.1. DNA modifications involved in DNA restriction

Several DNA modifications have been elucidated and in almost all cases the modifications that are involved in restriction are nucleobase methylations. The main forms of methylated nucleobases are N 6-methyladenine (6mA; Figure 2), 5-methylcytosine (5mC), and N 4-methylcytosine (4mC). These modifications are performed for double-stranded DNA using methyltransferases or other methyltransfer machinery. In addition, some bacteriophage DNA contain 5-hydroxymethylcytosine (5hmC) instead of cytosine. This modification is incorporated during phage DNA replication using 5-hydroxymethyldeoxycytidine triphosphate as the substrate [13]. The hydroxyl group is further glucosylated to produce β-glucosyl-5-hydroxymethylcytosine (ghmC) in a phage-specific pattern by glucosyltransferase [14]. This modification has various biological functions, including circumvention of restriction barriers of RM systems in phage hosts [13, 14]. The modified cytosine 5hmC is also found in mammalians [15-17]. Unlike phages, it is produced by oxidation of 5mC in double-stranded DNA [18]. In bacteria, there are no known type I–III RM systems involving 5hmC or ghmC. However, several type IV systems that restrict DNA containing 5hmC and/or ghmC have been reported [19-21].

RM systemRestrictionMethylation
MachineryCleavage siteMachineryNucleobase
Type IR2M2SVariableM2S 6mA
Type IIREFixedMT 6mA, 5mC, 4mC
Type IIIR2M2 VariableM2 6mA
Type IVREVariable

Table 1.

General properties of four types of RM systems.

Type I comprises R, M, and S subunits. Methylation is performed by subunits M and S. Type III comprises R and M subunits. The M subunit alone catalyzes methylation. Type II comprises two independent proteins, RE and MT. Type IV comprises only RE and restricts DNA with heterologous modifications. Methylation produces 6mA, 5mC, or 4mC.


Figure 2.

Chemical structures of modified nucleobases (modification moieties are indicated in red).

In addition to the methyl-based modifications described above, sulfur modification (phosphorothioation) of DNA backbones has been observed [22, 23]. Notably, it is suggested that this modification is involved in DNA restriction in Salmonella enterica [24]. The gene cluster for this RM system consists of eight genes, of which four are involved in phosphorothioation, while seven genes are essential for restricting unphosphorothioated DNA. Similar gene clusters, along with DNA containing phosphorothiol bonds, are found in many bacteria, implying that phosphorothioation is a widespread DNA modification [24, 25]. A type IV system that restricts phosphorothioated DNA has been also reported [26].

2.2. Type I RM systems

The E. coli strain K-12 harbors one type I RM system (EcoKI) encoded by the genes hsdR (R subunit), hsdM (M subunit), and hsdS (S subunit). This system constructs a multi-subunit complex that comprises two R subunits, two M subunits, and one S subunit (R2M2S), and scans double-stranded DNA after replication [27, 28]. When the complex recognizes DNA that is unmethylated at recognition sites, it acts as an ATP dependent endonuclease to digest DNA. The sequences of recognition sites are asymmetric but not palindromic. Examples include 5′-AACN6GTGC-3′ and 5′-GCACN6GTT-3′ for EcoKI, 5′-TGAN8TGCT-3′ and 5′-AGCAN8TCA-3′ for EcoBI, and 5′-TTAN7GTCY-3′ and 5′-RGACN7TAA-3′ for EcoDI (methylated adenine is underlined; R: A/G, Y: C/T, N: A/C/G/T) [29, 30].

The cleavage positions are distal from recognition sites and are variable. It is believed that the complex of type I RM system, while binding to recognition sites, translocates (or pulls) the DNA along in an ATP dependent fashion, and cleaves DNA when the translocation is impended by collision and/or by stalling with another translocating complex [27, 31, 32]. Electron microscopy analysis has been used to detect ATP-dependent formation of loop DNA during DNA cleavage by a type I RM complex [32]. The R subunit is responsible for ATP hydrolysis, translocation, and endonuclease activity, but not DNA binding. The binding depends on subunits M and S, which are therefore essential for both endonuclease and methyltransferase activities.

The complex of type I RM system acts similar to methyltransferase when it recognizes DNA that is hemimethylated (methylated on one strand) at recognition sites [27]. The methylation is performed by M and S subunits using S-adenosyl-L-methionine as the methyl donor [8]. The M subunit has the binding site for S-adenosyl-L-methionine, while the S subunit is essential for determining recognition sites. The R subunit is unnecessary for methyltransferase activity. In all cases reported so far, methylation by type I RM systems occurs in adenine to produce 6mA.

2.3. Type II RM systems

Type II RM systems are extremely diverse and are currently classified into 11 subfamilies [8]. Generally, these comprise two enzymes, RE and MT. Cleavages of exogenous DNA at unmethylated recognition sites is carried out by RE, which spares endogenous DNA that has been methylated by the cognate MT. Most REs require Mg2+ ions as a cofactor for cleavage. Although RE may form monomers, dimers, or tetramers, it functions without forming complexes with the cognate MT. The recognition of cleavage sites is highly precise and recognition sequences are often palindromic. Such sites include 5′-GAATTC-3′ for EcoRI and 5′-GGATCC-3′ for BamHI, which are cleaved symmetrically within the sites. Because of these useful properties, more than 3,500 REs have been characterized, and many are widely utilized in recombinant DNA technology [8]. The enzyme MT catalyzes methylation at recognition sites using S-adenosyl-L-methionine as the substrate and generally acts as a monomer. The nucleobases produced are 6mA, 5mC, or 4mC. For example, EcoRI and BamHI methyltransferases produce 5′-GA6mATTC-3′ and 5′-GGAT4mCC-3′, respectively.

2.4. Type III RM systems

Type III RM systems operate with multi-subunit machinery comprising two R subunits and two M subunits (R2M2) [33]. Subunit M contains recognition domain for binding to specific sites and also a methyltransferase domain. It can thereby bind at recognition sites independently, and methylate DNA using S-adenosyl-L-methionine as a substrate. The nucleobase produced is 6mA in all cases reported so far. Unlike type I and II RM systems, full modification is actually hemimethylation (methylation on one strand). The recognition sequences are asymmetric, such as 5′-CAGCAG-3′ for EcoP15I (methylated adenine is underlined).

Subunit R has an ATP dependent DNA helicase and endonuclease domains that are responsible for DNA cleavage. This subunit is unable to bind to DNA and therefore requires subunit M to cleave DNA. Two unmethylated sites that are inversely oriented (head-to-head orientation) serve as the target for DNA cleavage. Cleavage occurs at 25–27 bp downstream of one of the recognition sites, which is chosen randomly from the two sites. Even DNA with 3.5 kb between the two sites is cleaved. The cleavage requires ATP similar to type I RM systems. However, the amount of ATP consumed is only ~1% of that required for cleavage by type I RM systems. This fact makes it difficult to transpose the translocation model that is proposed in type I RM systems to type III RM systems. Thus, some alternative models have now been proposed [33].

2.5. Type IV restriction systems

Several enzymes specifically restrict modified DNA [10, 19-21, 26, 34-36]. These systems offer very efficient to defense from bacteriophages with highly-modified DNA. Among these, the enzymes for which cleavage sites are very specific and precise are classified into the M subfamily of type II RM systems [21]. Examples include DpnI (recognition sequence: 5′-G6mATC-3′), GlaI (5′-G5mCG5mC-3′), BisI (5′-G5mCNGC-3′; N: A/G/C/T), and MspJI (5′-5mCNNR-3′; R: G/A). The enzymes with non-specific and variable cleavage sites are classified as type IV restriction systems. The E. coli strain K-12 harbors three type IV systems encoded by mcrA, mcrB-mcrC, and mrr. The enzyme McrA recognizes 5′-Y5mCGR-3′ site (Y: C/T; R: G/A) [37], whereas McrBC recognizes pairs of 5′-RmC-3′ (mC: 5mC or 4mC) separated by 40–3000 bp, and cleaves DNA ~30 bp distal from one of the sites [36]. The Mrr system recognizes DNA containing 6mA, 5mC, or 4mC, but its recognition sites have not been well defined [36]. The enzyme SauUSI of Staphylococcus aureus recognizes 5′-S5mCNGS-3′ and 5′-S5hmCNGS-3′ (S: C/G; N: A/G/C/T) and cleaves at position 2–18 bp downstream of the recognition site [21]. The enzyme GmrSD restriction system of E. coli CT596 cuts DNA containing ghmC [20]. Thus, all species of modified nucleobases are potentially restricted by type IV restriction systems.

MicrobeHost-mimicking DNAReference
Bacillus anthracis In vivo (MF)Electroporation [50]
Bacillus cereus In vitro (EX)Electroporation[51]
Bacillus weihenstephanensis In vitro (EX)Electroporation[51]
Bifidobacterium adolescentis In vivo (GM)Electroporation[52]
Bifidobacterium longum In vitro (MT/SD)Electroporation[53]
Borrelia burgdorferi In vitro (MT)Electroporation[54]
Clostridium acetobutylicum In vivo (HG)Electroporation[55]
Clostridium difficile In vivo (HG/SD)Conjugation[56]
Clostridium thermocellum In vivo (IG)Electroporation[57]
Geobacillus kaustophilus In vivo (GM/IG)Conjugation[41]
Helicobacter pylori In vitro (EX)Competency [58]
Salmonella typhimurium In vivo (DS)Competency [59]
Staphylococcus aureus In vivo (DS)Electroporation [60]
Streptomyces avermitilis In vivo (MF)Protoplast [61]
Streptomyces bambergiensis In vivo (MF)Conjugation [62]
Streptomyces coelicolor In vivo (MF)Protoplast[35]
Streptomyces griseus IFO 13350 In vivo (GM/HG)Protoplast [40]
In vivo (DS)Protoplast[63]
Streptomyces griseus NRRL B-2682 In vitro (MT)Protoplast[64]
Streptomyces natalensis In vivo (MF)Conjugation [65]
Sulfolobus acidocaldarius In vivo (HG)Electroporation[38]
Thermoanaerobacter sp. X514 In vivo (IG)Sonoporation [66]

Table 2.

Microbial transformation using host-mimicking DNA.

DS: in vivo methylation in a strain that is related to the target bacterium and is deficient in restriction and proficient in methylation; EX: in vitro methylation using a crude extract of the target bacterium; GM: in vivo methylation using methyltransferase genes in the target bacterial genome; HG: in vivo methylation using heterologous genes; IG: in vivo methylation using E. coli intrinsic genes; MF: methyl-free DNA; MT: in vitro methylation using commercially available methyltransferases; and SD: transformation using DNA with specifically abolished recognition sites.

3. Host-mimicking strategies

Circumvention of RM systems is critical for establishing transformation methods for target bacteria (Table 2). This is true not only for bacteria but also archaea [38]. This section describes some strategies for circumventing RM systems and focuses on host-mimicking. Because all types of RM systems digest exogenous DNA after distinguishing it from endogenous DNA on the basis of host-specific methylation patterns, DNA modification that mimic these patterns evade digestion. A general flowchart for producing host-mimicking DNA is shown in Figure 3. The details are described in the following sections.


Figure 3.

General flowchart for producing host-mimicking DNA to target bacteria.

The chromatogram is the data from HPLC analysis of deoxynucleosides prepared from G. kaustophilus chromosomes [41]. It includes 2′-deoxyadenosine (dA), 2′-deoxycytidine (dC), 2′-deoxyguanosine (dG), 2′-deoxythymidine (dT), and N 6-methyl-2′-deoxyadenosine (6mdA) but not 5-methyl-2′-deoxycytidine (5mdC) or N 4-methyl-2′-deoxycytidine (4mdC).

3.1. A brief survey of functional RM systems

As mentioned earlier, type I–III RM systems involve DNA methylation in their functions. Therefore, the presence of methylated DNA in bacterial chromosomes indicates a type I–III RM system in the bacterium. Hence analysis of methylated DNA is an effective survey method to identify functional RM systems in target bacterium. Methylated DNA in chromosomes can be analyzed using high-performance liquid chromatography (HPLC) [15, 18, 39-45]. This method determines the presence of deoxynucleosides and methylated deoxynucleosides. Deoxynucleosides are prepared by hydrolyzing chromosomal DNA with nuclease P1 and alkaline phosphatase [39-41], are separated using reverse mode C18-based silica columns, and are detected by ultraviolet absorption at 260 and/or 280 nm. Authentic 6mdA, 5mdC, and 4mdC are commercially available or can be prepared from methylated 2′-deoxynucleoside-5′-triphosphate by dephosphorylation using E. coli alkaline phosphatase [40]. Note that this analysis requires large amounts of DNA (>10 µg). Contamination with RNA, proteins, and chemicals that absorb ultraviolet radiation may also complicate accurate analysis. In some reports [18, 42, 43], deoxynucleosides are more accurately identified by combining HPLC and mass spectrometry analysis. In addition, immunochemical methods are available for analyzing methylated DNA [46-49]. Several anti-5mC antibodies have been developed and are commercially available. Although commercially available antibodies against other methylated DNA are limited, Kong et al. [49] have reported successful production of rabbit polyclonal antibodies against 6mA and 4mC.

DeoxynucleosideRelative coefficient
Detection at 260 nmDetection at 280 nm

Table 3.

Relative coefficients for determining deoxynucleoside molar ratios by HPLC analysis; [Relative amount of deoxynucleoside] = [Coefficient] × [Peak area on HPLC chromatogram].

Analysis of DNA using HPLC allows determination of deoxynucleoside composition and methylation frequency in chromosomes. The coefficients in Table 3 may be used for estimation. For example, HPLC analysis of S. griseus chromosomes revealed 5mdC but not 6mdA or 4mdC [40]. The composition ratio of 5mdC to dC was 0.7 mol%, suggesting that S. griseus possesses approximately one 5mC per 0.5 kb of chromosomal DNA with 67% GC content. The chromosome of G. kaustophilus contains 6mdA and the composition ratio to dA is 2.0 mol% [41]. This suggests that G. kaustophilus possesses approximately one 6mA per 0.1 kb of chromosomal DNA with 52% GC content. Although DNA methylation unrelated to RM systems have been observed, such as E. coli Dam and Dcm methylation [67, 68], high frequency methylations imply that the bacteria may harbor a considerable type I–III RM system. Meanwhile, the chromosomes of S. avermitilis, S. coelicolor, and S. lividans contain no methylated DNA (my unpublished data). This observation suggests that these bacteria harbor no type I–III RM systems. However, the possibility remains that a functional type IV system exists in these species. Potent methyl-specific restrictions have been observed in S. avermitilis [61], S. coelicolor [35], S. bambergiensis [62], and S. natalensis [65].

3.2. Methylation site analysis

When significant DNA methylation is observed in the target bacterium, preliminary determination of DNA methylation sites is generally required to produce host-mimicking DNA. Recent epigenetic studies have developed many methods to analyze DNA methylation [29, 30, 69-76]. Although most of these studies aimed to analyze 5-methylation of cytosine at specific sites, or differential DNA methylation in chromosomes, there are a few methods that exhaustively determine methylated consensus sites in chromosomes as follows.

In S. griseus, bisulfite-based analysis of a plasmid library isolated from this bacterium (Figure 4A) was used to determine the two consensus sites 5′-GAG5mCTC-3′ and 5′-GC5mCGGC-3′ [40]. Note that this method is not employed for methylation analysis in bacteria that cannot be transformed with exogenous plasmids, and in bacteria that have methylated nucleobases other than 5mC. For determining 6mA consensus sites in G. kaustophilus, chromosome digestion using methyl-sensitive restriction enzymes (Figure 4B) was used to reveal 5′-GG6mATC-3′ and 5′-G6mATCC-3′ site [41]. Methods using methyl-sensitive restriction enzymes help identify several methylation species, including 6mA, 5mC, and 4mC. However, methylation sites that can be identified by these methods are limited due to the lack of commercially available restriction enzymes. Recently, direct detection using real-time DNA sequencing has been reported [74, 75]. This method potentially enables the exhaustive determination of all methylation sites in chromosomal DNA. Although this method requires special equipments, which has limited availability, it may become one of the most promising methods for methylation analysis in the future. Also, the author has now developed a versatile immunological method for determining consensus sequences with methylated nucleobases.

3.3. Production of host-mimicking DNA

If the target bacterium contains no or negligible methylated DNA, methyl-free DNA should be used as host-mimicking DNA because the bacterium may harbor type IV restriction systems, as exemplified by transformation of S. avermitilis [61], S. coelicolor [35], and S. natalensis [65]. Methyl-free DNA can be readily produced using E. coli strains deficient in DNA methyltransferase genes (dam dcm hsd ), such as E. coli IR27, ET12567, IBEC58, and HST04 (Table 4). Methyl-restrained DNA from E. coli GM2929 and SCS110 (dam dcm hsd +) were also used for efficient transformation of S. bambergiensis [62] and B. anthracis [50], respectively. Because Hsd mediated methylation is of low frequency in E. coli, deficiency of this methyltion may not be essential for circumventing type IV systems.

If the target bacterium contains considerable methylated DNA, host-mimicking DNA that reconstitutes the methylation pattern of the target bacterium should be used for transformation. There are two main approaches to produce heterologous methylation of DNA. One is in vitro methylation using methyltransferases [53, 54, 64], such as Dam (catalyzing 5′-G6mATC-3′ methylation), M.TaqI (5′-TCG6mA-3′), M.AluI (5′-AG5mCT-3′), M.BamHI (5′-GGATC4mC-3′), M.SssI (5′-5mCG-3′), M.EcoRI (5′-GA6mATTC-3′), M.CviPI (5′-GC5m-3′), M.HaeIII (5′-GGC5mC-3′), M.HhaI (5′-G5mCGC-3′), M.HpaII (5′-C5mCGG-3′), and M.MspI (5′-5mCCGG-3′). This approach is very simple and effective but has low cost-performance and low versatility due to the limited number of commercially available methyltransferases. In transformations of B. cereus [51], B. weihenstephanensis [51], and H. pylori [58], crude extracts prepared from the respective bacterium were used for DNA methylation. This method has high cost-performance and high versatility but may not be efficient because of low methyltransferase activity in crude extracts and DNA degradation by nucleases. Moreover, type I and III methylation pattern cannot be achieved by this method.


Figure 4.

Methylation site analysis in target bacterium.

(A) Bisulfite-based analysis to determine 5mC consensus sites. Bisulfite treatment converts methyl-free cytosine to uracil without affecting 5mC. Therefore, 5mC positions can be determined by comparing bisulfite-treated and -untreated DNA sequences. (B) Chromosomal digestion by methyl-sensitive restriction enzymes is used to analyze 5′-G6mATC-3′ methylation. The restriction enzyme DpnI cuts 5′-G6mATC-3′ but not 5′-GATC-3′, DpnII cuts 5′-GATC-3′ but not 5′-G6mATC-3′, and Sau3AI cuts 5′-GATC-3′ and 5′-G6mATC-3′.

Another approach is in vivo methylation by expressing methyltransferase genes in E. coli cells [38, 40, 41, 52, 55, 56]. Type II and III methylation can be reconstituted by expressing MT and M subunit genes, respectively, and type I methylation can be reconstituted by simultaneous expression of M and S subunit genes. Numerous gene sequences of methyltransferases are accumulated in the REBASE database along with their methylation sites [11]. Either plasmids or chromosomal integration can be used as expression vectors for methyltransferase genes. When the genome sequence of the target bacterium has been determined, methyltransferase genes in the genome may be used for in vivo methylation [40, 41, 52]. Methylation site analysis is not essential for this approach; however, functional expression of methyltransferase genes requires confirmation by HPLC analysis of recombinant E. coli chromosomes. A methyltransferase gene of S. griseus, responsible for 5′-GAG5mCTC-3′ methylation, was found to be nonfunctional in E. coli. Hence, an alternative methyltransferase gene from S. achromogenes (M.SacI) was used for DNA methylation [40]. The E. coli host used to produce host-mimicking DNA must be a methylation-deficient strain (Table 4) because the target bacterium may have type IV systems in addition to type I–III RM systems, as exemplified by G. kaustophilus transformation [41]. In addition, it is desirable that the E. coli host is deficient in type IV system genes (mcrA, mcrBC, and mrr) because these may restrict heterologous methylation in E. coli cells. In this regard, E. coli strains IR27 and HST04 are appropriate for producing host-mimicking DNA through in vivo methylation. Although in vivo methylation may be more complicated than in vitro methylation, this approach often has excellent cost-performance, versatility, and efficiency.

A derivative of the target bacterium with methylation activity and reduced restriction activity can also be used for the production of host-mimicking DNA. In transformations of Salmonella typhimurium and Staphylococcus aureus, plasmids isolated from E. coli strains were initially introduced and propagated in the restriction-deficient strains LB5000 [59] and RN4220 [60], respectively, and were then used for transformation of other strains. In S. griseus transformation, mutant HH1 that reduces restriction activity compared to the wild-type has been used [63]. This approach enables production of perfect host-mimicking DNA, although it is not easy to find a strain that is both deficient in restriction and proficient in methylation.

StrainRelevant genotypeReference
IR21 e14(mcrA ) Δdam::metB Δ(mrr-hsdRMS-mcrBC)114::IS10 rpsL104 (StrR)[41]
IR24 e14(mcrA ) Δdcm::lacZ Δ(mrr-hsdRMS-mcrBC)114::IS10 rpsL104 (StrR)[41]
IR27 e14(mcrA ) Δdam::metB Δdcm::lacZ Δ(mrr-hsdRMS-mcrBC)114::IS10 rpsL104 (StrR) [40]
ET12567 dam-13::Tn9 (CmR) dcm-6 hsdRM zjj-202::Tn10 (TetR) rpsL136 (StrR) [77]
IBEC58Δdam Δdcm ΔhsdRMS [35]
HST04Δ(mrr-hsdRMS-mcrBC) ΔmcrA dam dcm rpsL (StrR)TB
JM110 dam dcm rpsL (StrR)AT
SCS110 dam dcm rpsL (StrR) endA AT
INV110 dam dcm Δ(mrr-hsdRMS-mcrBC)102::Tn10 (TetR)
rpsL (StrR) endA
GM48 dam-3 dcm-6CGSC
GM272 dam-3 dcm-6 hsdS21CGSC
GM2929 dam-13::Tn9 (CmR) dcm-6 hsdR2 mcrA mcrB rpsL136 (StrR)CGSC

Table 4.

E. coli strains deficient in genes involved in DNA methylation and/or DNA restriction.

CmR: chloramphenicol resistance; TetR: tetracycline resistance; StrR: streptomycin resistance; TB: Takara Bio Inc. (; AT: Agilent Technologies Inc. (; LT: Life Technologies Corporation (; and CGSC: The Coli Genetic Stock Center (

3.4. Alternative methods for circumventing RM systems

In addition to host-mimicking strategies, there are some simple approaches for circumventing RM systems. One is to abolish sites recognized by RM systems. In Clostridium difficile transformation, five CdiI sites in plasmids were abolished and used to demonstrate improved efficiency [56]. Similarly, a plasmid with three abolished SacII sites was used for efficient transformation of Bifidobacterium longum [53]. When methyltransferase enzymes or genes are unavailable, this approach can be an effective alternative. One other approach is to reduce the restriction activity in the target bacterium temporarily by heat treatment. In B. amyloliquefaciens transformation, heat treatment at 46°C for 6 min increased transformation efficiency [78]. More forcible heat treatment (higher temperature and longer time) inactivates RM systems more efficiently but concurrently reduces viability of cells. Although this approach is very simple and is available for most bacteria, the heat conditions required for inactivating RM systems are not predictable and thereby may have to be determined by repeated trials.

4. Conclusion

In this chapter, RM systems are reviewed and some approaches to produce host-mimicking DNA are described. Analysis of chromosomal DNA using HPLC is a simple method to elucidate functional RM systems in target bacterium, and is therefore highly recommended for establishing bacterial transformation methods. When negligible DNA methylation is observed, methyl-free DNA is suitable for transformation of the bacterium. On the other hand, when significant DNA methylation is observed, a host-mimicking strategy involving methylation needs to be utilized. One weak point of this strategy is that there are no methods to exhaustively, readily, and rapidly determine methylation sites in target bacterium. When this analytical method becomes more widespread, this strategy will become a crucial technique for establishing efficient bacterial transformation methods.


This work was supported by Grant-in-Aid for Young Scientists (B) of Japan Society for the Promotion of Science (20780080 and in part 23750083). The author would like to thank Enago ( for the English language review.


1 - F. Griffith, 1928 The Significance of Pneumococcal Types. J. hyg. (Lond) 27 113 159
2 - O. T. Avery, C. M. MacLeod, M. McCarty, 1944 Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III. J. exp. med. 79 137 158
3 - M. McCarty, O. T. Avery, 1946 Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: II. Effect of Desoxyribonuclease on the Biological Activity of the Transforming Substance. J. exp. med. 83 89 96
4 - M. McCarty, O. T. Avery, 1946 Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal types: III. An Improved Method for the Isolation of the Transforming Substance and its Application to Pneumo coccus Types II, III, and VI. J. exp. med. 83 97 104
5 - J. Spizizen, 1958 Transformation of Biochemically Deficient Strains of Bacillus subtilis by Deoxyribonucleate Proc. natl. acad. sci. USA. 44 1072 1078
6 - M. Mandel, A. Higa, 1970 Calcium-dependent Bacteriophage DNA Infection. J. mol. biol. 53 159 162
7 - T. E. Aune, F. L. Aachmann, 2010 Methodologies to Increase the Transformation Efficiencies and the Range of Bacteria that can be Transformed. Appl. microbiol. biotechnol. 85 1301 1313
8 - R. J. Roberts, M. Belfort, T. Bestor, A. S. Bhagwat, T. A. Bickle, J. Bitinaite, R. M. Blumenthal, S. K. Degtyarev, D. T. F. Dryden, K. Dybvig, K. Firman, E. S. Gromova, R. I. Gumport, S. E. Halford, S. Hattman, J. Heitman, D. P. Hornby, A. Janulaitis, A. Jeltsch, J. Josephsen, A. Kiss, T. R. Klaenhammer, I. Kobayashi, H. Kong, D. H. Krüger, S. Lacks, M. G. Marinus, M. Miyahara, R. D. Morgan, N. E. Murray, V. Nagaraja, A. Piekarowicz, A. Pingoud, E. Raleigh, D. N. Rao, N. Reich, V. E. Repin, E. U. Selker, P. C. Shaw, D. C. Stein, B. L. Stoddard, W. Szybalski, T. A. Trautner, J. L. Van Etten, J. M. B. Vitor, G. G. Wilson, S. Y. Xu, 2003 A Nomenclature for Restriction Enzymes, DNA Methyltransferases, Homing Endonucleases and their Genes. Nucleic acids res. 31 1805 1812
9 - W. Arber, D. Dussoix, 1962 Host Specificity of DNA Produced by Escherichia coli: I. Host Controlled Modification of Bacteriophage. J. mol. biol. 5 18 36
10 - R. J. Roberts, T. Vincze, J. Posfai, D. Macelis, 2003 REBASE: Restriction Enzymes and Methyltransferases Nucleic acids res. 31 418 420
11 - R. J. Roberts, T. Vincze, J. Posfai, D. Macelis, 2010 REBASE-a Database for DNA Restriction and Modification: Enzymes, Genes and Genomes. Nucleic acids res. 38 D 234 D236
12 - I. Kobayashi, 2001 Behavior of Restriction-Modification Systems as Selfish Mobile Elements and their Impact on Genome Evolution. Nucleic acids res. 29 3742 3756
13 - L. Snyder, L. Gold, E. Kutter, 1976 A Gene of Bacteriophage T4 whose Product Prevents True Late Transcription on Cytosine-containing T4 DNA Proc. natl. acad. sci. USA. 73 3098 3102
14 - S. Moréra, A. Imberty, U. Aschke-Sonnenborn, W. Rüger, P. S. Freemont, 1999 T4 Phage β-Glucosyltransferase: Substrate Binding and Proposed Catalytic Mechanism. J. mol. biol. 292 717 730
15 - S. Kriaucionis, N. Heintz, 2009 The Nuclear DNA Base 5-Hydroxymethylcytosine is Present in Purkinje Neurons and the Brain Science 324 929 930
16 - M. Münzel, D. Globisch, T. Carell, 2011 Hydroxymethylcytosine, the Sixth Base of the Genome. Angew. chem. int. ed. 50 6460 6468
17 - M. Tahiliani, K. P. Koh, Y. Shen, W. A. Pastor, H. Bandukwala, Y. Brudno, S. Agarwal, L. M. Iyer, D. R. Liu, L. Aravind, A. Rao, 2009 Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1. Science 324 930 935
18 - H. Wu, Y. Zhang, 2011 Mechanisms and Functions of Tet Protein-mediated 5-Methylcytosine Oxidation. Genes dev. 25 2436 2452
19 - Y. Zheng, D. Cohen-Karni, D. Xu, H. G. Chin, G. Wilson, S. Pradhan, R. J. Roberts, 2010 A Unique Family of Mrr-like Modification-dependent Restriction Endonucleases. Nucleic acids res. 38 5527 5534
20 - C. L. Bair, L. W. Black, 2007 A Type IV Modification Dependent Restriction Nuclease that Targets Glucosylated Hydroxymethyl Cytosine Modified DNAs J. mol. biol. 366 768 778
21 - S. Y. Xu, A. R. Corvaglia, S. H. Chan, Y. Zheng, P. Linder, 2011 A Type IV Modification-dependent Restriction Enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300 Nucleic acids res. 39 5597 5610
22 - X. Zhou, X. He, J. Liang, A. Li, T. Xu, T. Kieser, JD J. D. Helmann, Z. Deng, 2005 A Novel DNA Modification by sulphur. Mol. microbiol. 57 1428 1438
23 - L. R. Wang, S. Chen, T. Xu, K. Taghizadeh, J. S. Wishnok, X. Zhou, D. You, Z. Deng, P. C. Dedon, 2007 Phosphorothioation of DNA in Bacteria by dnd Genes. Nat. chem. biol. 3 709 710
24 - T. Xu, F. Yao, X. Zhou, Z. Deng, D. You, 2010 A Novel Host-specific Restriction System Associated with DNA Backbone S-Modification in SalmonellaNucleic acids res.38 7133 7141
25 - L. Wang, S. Chen, K. L. Vergin, S. J. Giovannoni, S. W. Chan, M. S. De Mott, K. Taghizadeh, O. X. Cordero, M. Cutler, S. Timberlake, E. J. Alm, M. F. Polz, J. Pinhassi, Z. Deng, P. C. Dedon, 2011 DNA Phosphorothioation is Widespread and Quantized in Bacterial Genomes. Proc. natl. acad. sci. USA. 108 2963 2968
26 - G. Liu, H. Y. Ou, T. Wang, L. Li, H. Tan, X. Zhou, K. Rajakumar, Z. Deng, X. He, 2010 Cleavage of Phosphorothioated DNA and Methylated DNA by the Type IV Restriction Endonuclease ScoMcrA PLoS genet. 6 e1001253
27 - N. E. Murray, 2000 Type I Restriction Systems: Sophisticated Molecular Machines (A Legacy of Bertani and Weigle) Microbiol. mol. biol. rev. 64 412 434
28 - C. K. Kennaway, A. Obarska-Kosinska, J. H. White, I. Tuszynska, L. P. Cooper, J. M. Bujnicki, J. Trinick, D. T. F. Dryden, 2009 The Structure of M.EcoKI Type I DNA Methyltransferase with a DNA Mimic Antirestriction Protein Nucleic acids res. 37 762 770
29 - J. Ryu, E. Rowsell, 2008 Quick Identification of Type I Restriction Enzyme Isoschizomers Using Newly Developed pTypeI and Reference Plasmids Nucleic acids res. 36 e81
30 - V. Nagaraja, M. Stieger, C. Nager, S. M. Hadi, T. A. Bickle, 1985 The Nucleotide Sequence Recognized by the Escherichia coli D Type-I Restriction and Modification Enzyme. Nucleic acids res. 13 389 399
31 - L. R. García, I. J. Molineux, 1999 Translocation and Specific Cleavage of Bacteriophage T7 DNA in vivo by EcoKI Proc. natl. acad. sci. USA. 96 12430 12435
32 - R. Yuan, D. L. Hamilton, J. Burckhardt, 1980 DNA Translocation by the Restriction Enzyme from E. coli K. Cell 20 237 244
33 - D. T. F. Dryden, J. M. Edwardson, R. M. Henderson, 2011 DNA Translocation by Type III Restriction Enzymes: A Comparison of Current Models of their Operation Derived from Ensemble and Single-molecule Measurements Nucleic acids res. 39 4525 4531
34 - E. Sutherland, L. Coe, E. A. Raleigh, 1992 McrBC: A Multisubunit GTP-dependent Restriction Endonuclease. J. mol. biol. 225 327 348
35 - G. González-Cerón, O. J. Miranda-Olivares, L. Servín-González, 2009 Characterization of the Methyl-specific Restriction System of Streptomyces coelicolor A3(2) and of the Role Played by Laterally Acquired Nucleases. FEMS microbiol. lett. 301 35 43
36 - P. A. Waite-Rees, C. J. Keating, L. S. Moran, B. E. Slatko, L. J. Hornstra, J. S. Benner, 1991 Characterization and Expression of the Escherichia coli Mrr Restriction System. J. bacteriol. 173 5207 5219
37 - E. A. Mulligan, E. Hatchwell, S. R. McCorkle, J. J. Dunn, 2010 Differential Binding of Escherichia coli McrA Protein to DNA Sequences that Contain the Dinucleotide m5CpG. Nucleic acids res. 38 1997 2005
38 - N. Kurosawa, D. W. Grogan, 2005 Homologous Recombination of Exogenous DNA with the Sulfolobus acidocaldarius Genome: Properties and Uses. FEMS microbiol. lett. 253 141 149
39 - C. W. Gehrke, R. A. McCune, M. A. Gama-Sosa, M. Ehrlich, K. C. Kuo, 1984 Quantitative Reversed-phase High-performance Liquid Chromatography of Major and Modified Nucleosides in DNA. J. chromatogr. 301 199 219
40 - H. Suzuki, S. Takahashi, H. Osada, K. Yoshida, 2011 Improvement of Transformation Efficiency by Strategic Circumvention of Restriction Barriers in Streptomyces griseus J. microbiol. biotechnol. 21 675 678
41 - H. Suzuki, K. Yoshida, 2012 Genetic Transformation of Geobacillus kaustophilus HTA426 by Conjugative Transfer of Host-mimicking Plasmids J. microbiol. biotechnol. 22 1279 1287
42 - R. S. Annan, G. M. Kresbach, R. W. Giese, P. Vouros, 1989 Trace Detection of Modified DNA Bases via Moving-belt Liquid Chromatography-Mass Spectrometry Using Electrophoretic Derivatization and Negative Chemical Ionization. J. chromatogr. 465 285 296
43 - R. del Gaudio, R. Di Giaimo, G. Geraci, 1997 Genome Methylation of the Marine Annelid Worm Chaetopterus variopedatus: Methylation of a CpG in an Expressed H1 Histone Gene. FEBS lett. 417 48 52
44 - M. Ehrlich, G. G. Wilson, K. C. Kuo, C. W. Gehrke, 1987 N4-Methylcytosine as a Minor Base in Bacterial DNA. J. bacteriol. 169 939 943
45 - M. Ehrlich, M. A. Gama-Sosa, L. H. Carreira, L. G. Ljungdahl, K. C. Kuo, C. W. Gehrke, 1985 DNA Methylation in Thermophilic Bacteria: N4-Methylcytosine, 5-Methylcytosine, and N6-Methyladenine. Nucleic acids res. 13 1399 1412
46 - S. Banerjee, R. Chowdhury, 2006 An Orphan DNA (Cytosine-5-)-Methyltransferase in Vibrio cholerae. Microbiology 152 1055 1062
47 - H. J. Störl, H. Simon, H. Barthelmes, 1979 Immunochemical Detection of N6-Methyladenine in DNA. Biochim. biophys. acta. 564 23 30
48 - L. F. Lin, J. Posfai, R. J. Roberts, H. Kong, 2001 Comparative Genomics of the Restriction-Modification Systems in Helicobacter pyloriProc. natl. acad. sci. USA.98 2740 2745
49 - H. Kong, L. F. Lin, N. Porter, S. Stickel, D. Byrd, J. Posfai, R. J. Roberts, 2000 Functional Analysis of Putative Restriction-Modification System Genes in the Helicobacter pylori J99 Genome. Nucleic acids res. 28 3216 3223
50 - R. Sitaraman, S. H. Leppla, 2012 Methylation-dependent DNA Restriction in Bacillus anthracis Gene 494 44 50
51 - M. N. Groot, F. Nieboer, T. Abee, 2008 Enhanced Transformation Efficiency of Recalcitrant Bacillus cereus and Bacillus weihenstephanensis Isolates Upon in vitro Methylation of Plasmid DNA Appl. environ. microbiol. 74 7817 7820
52 - K. Yasui, Y. Kano, K. Tanaka, K. Watanabe, M. Shimizu-Kadota, H. Yoshikawa, T. Suzuki, 2009 Improvement of Bacterial Transformation Efficiency Using Plasmid Artificial Modification Nucleic acids res. 37 e3
53 - J. Y. Kim, Y. Wang, M. S. Park, G. E. Ji, 2010 Improvement of Transformation Efficiency Through in vitro Methylation and SacII Site Mutation of Plasmid Vector in Bifidobacterium longum MG1 J. microbiol. biotechnol. 20 1022 1026
54 - Q. Chen, J. R. Fischer, V. M. Benoit, N. P. Dufour, P. Youderian, J. M. Leong, 2008 In vitro CpG Methylation Increases the Transformation Efficiency of Borrelia burgdorferi Strains Harboring the Endogenous Linear Plasmid lp56 J. bacteriol. 190 7885 7891
55 - L. D. Mermelstein, E. T. Papoutsakis, 1993 In vivo Methylation in Escherichia coli by the Bacillus subtilis Phage ϕ 3T I Methyltransferase to Protect Plasmids from Restriction Upon Transformation of Clostridium acetobutylicum ATCC 824. Appl. environ. microbiol. 59 1077 1081
56 - D. Purdy, T. A. O’Keeffe, M. Elmore, M. Herbert, A. McLeod, M. Bokori-Brown, A. Ostrowski, N. P. Minton, 2002 Conjugative Transfer of Clostridial Shuttle Vectors from Escherichia coli to Clostridium difficile Through Circumvention of the Restriction Barrier Mol. microbiol. 46 439 452
57 - M. V. Tyurin, S. G. Desai, L. R. Lynd, 2004 Electrotransformation of Clostridium thermocellumAppl. environ. microbiol.70 883 890
58 - J. P. Donahue, D. A. Israel, R. M. Peek, M. J. Blaser, G. G. Miller, 2000 Overcoming the Restriction Barrier to Plasmid Transformation of Helicobacter pylori Mol. microbiol. 37 1066 1074
59 - L. R. Bullas, J. I. Ryu, 1983 Salmonella typhimurium LT2 Strains which are r m+ for All Three Chromosomally Located Systems of DNA Restriction and Modification. J. bacteriol. 156 471 474
60 - S. Schenk, R. A. Laddaga, 1992 Improved Method for Electroporation of Staphylococcus aureus. FEMS microbiol. lett. 94 133 138
61 - D. J. MacNeil, 1988 Characterization of a Unique Methyl-specific Restriction System in Streptomyces avermitilis. J. bacteriol. 170 5607 5612
62 - S. B. Zotchev, H. Schrempf, C. R. Hutchinson, 1995 Identification of a Methyl-specific Restriction System Mediated by a Conjugative Element from Streptomyces bambergiensis. J. bacteriol. 177 4809 4812
63 - H. Yamazaki, Y. Ohnishi, S. Horinouchi, 2003 Transcriptional Switch on of ssgA by A-factor, which is Essential for Spore Septum Formation in Streptomyces griseus J. bacteriol. 185 1273 1283
64 - J. Kwak, H. Jiang, K. E. Kendrick, 2002 Transformation Using in vivo and in vitro Methylation in Streptomyces griseus. FEMS microbiol. lett. 209 243 248
65 - L. L. Enríquez, M. V. Mendes, N. Antón, S. Tunca, S. M. Guerra, J. F. Martín, J. F. Aparicio, 2006 An Efficient Gene Transfer System for the Pimaricin Producer Streptomyces natalensis FEMS microbiol. lett. 257 312 318
66 - L. Lin, H. Song, Y. Ji, Z. He, Y. Pu, J. Zhou, J. Xu, 2010 Ultrasound-mediated DNA Transformation in Thermophilic Gram-positive Anaerobes. PLoS one 5 e12582
67 - K. T. Militello, R. D. Simon, M. Qureshi, R. Maines, M. L. Van Horne, S. M. Hennick, S. K. Jayakar, S. Pounder, 2012 Conservation of Dcm-mediated Cytosine DNA Methylation in Escherichia coli. FEMS microbiol. lett. 328 78 85
68 - M. G. Marinus, J. Casadesus, 2009 Roles of DNA Adenine Methylation in Host-pathogen Interactions: Mismatch Repair, Transcriptional Regulation, and More. FEMS microbiol. rev. 33 488 503
69 - R. M. Brena, T. H. M. Huang, C. Plass, 2006 Quantitative Assessment of DNA Methylation: Potential Applications for Disease Diagnosis, Classification, and Prognosis in Clinical Settings J. mol. med. 84 365 377
70 - E. J. Oakeley, 1999 DNA Methylation Analysis: A Review of Current Methodologies. Pharmacol. ther. 84 389 400
71 - A. Harrison, A. Parle-McDermott, 2011 DNA Methylation: A Timeline of Methods and Applications. Front genet. 2 74
72 - S. D. Fouse, R. P. Nagarajan, J. F. Costello, 2010 Genome-scale DNA Methylation Analysis Epigenomics 2 105 117
73 - A. Sulewska, W. Niklińska, M. Kozlowski, L. Minarowski, W. Naumnik, J. Nikliński, K. Dąbrowska, L. Chyczewski, 2007 Detection of DNA Methylation in Eucaryotic Cells. Folia histochem. cytobiol. 45 315 324
74 - J. Eid, A. Fehr, J. Gray, K. Luong, J. Lyle, G. Otto, P. Peluso, D. Rank, P. Baybayan, B. Bettman, A. Bibillo, K. Bjornson, B. Chaudhuri, F. Christians, R. Cicero, S. Clark, R. Dalal, A. de Winter, J. Dixon, M. Foquet, A. Gaertner, P. Hardenbol, C. Heiner, K. Hester, D. Holden, G. Kearns, X. Kong, R. Kuse, Y. Lacroix, S. Lin, P. Lundquist, C. Ma, P. Marks, M. Maxham, D. Murphy, I. Park, T. Pham, M. Phillips, J. Roy, R. Sebra, G. Shen, J. Sorenson, A. Tomaney, K. Travers, M. Trulson, J. Vieceli, J. Wegener, D. Wu, A. Yang, D. Zaccarin, P Zhao, F. Zhong, J. Korlach, S. S. Turner, 2009 Real-time DNA Sequencing from Single Polymerase Molecules. Science 323 133 138
75 - B. A. Flusberg, D. Webster, R.J. H. Lee, K. J. Travers, E. C. Olivares, T. A. Clark, J. Korlach, S. W. Turner, 2010 Direct Detection of DNA Methylation During Single-molecule, Real-time Sequencing. Nat. methods 7 461 465
76 - A. Bart, M. W. J. van Passel, K. van Amsterdam, A. van der Ende, 2005 Direct Detection of Methylation in Genomic DNA Nucleic acids res. 33 e124
77 - D. J. MacNeil, K. M. Gewain, C. L. Ruby, G. Dezeny, P. H. Gibbons, T. MacNeil, 1992 Analysis of Streptomyces avermitilis Genes Required for Avermectin Biosynthesis Utilizing a Novel Integration Vector. Gene 111 61 68
78 - G. Q. Zhang, P. Bao, Y. Zhang, A. H. Deng, N. Chen, T. Y. Wen, 2011 Enhancing Electro-transformation Competency of Recalcitrant Bacillus amyloliquefaciens by Combining Cell-wall Weakening and Cell-membrane Fluidity Disturbing Anal. biochem. 409 130 137