Isolation of Deoxyribonucleic Acid Methylase Mutants of Escherichia coli K-12

Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N(6)-methyladenine (N(6)-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict lambda. None of the mutations had the effect of being lethal.     

Bacterial DNA methylation and gene transfer efficiency

The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide sequence of plasmid DNA used in gene transfer. These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/T)GG sequences. These modifications which introduce 6-methyladenine and 5-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of 5-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, but the possible consequences of the presence of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown. Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam(-), dcm(-)) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam(+), dcm(+)) or JM110 (dam(-), dcm(-)) bacteria. Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer. On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam(+), dcm(+)) bacteria gives greater amounts of luciferase than the same transfection performed with a plasmid amplified in the mutated JM110 (dam(-), dcm(-)) counterpart. Therefore, these data do not suggest that the use of (dam(-), dcm(-)) bacteria to amplify plasmid DNA may increase gene transfer efficiency. However, the persistence of the use of (dam(+), dcm(+)) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in eukaryotic cells or organisms.     

Deoxyribonucleic acid methylation in mycobacteria.

Deoxyribonucleic acid modification in six strains of mycobacteria was investigated. The presence of 5-methylcytosine in the virulent strain Mycobacterium tuberculosis H37Rv and its absence in the avirulent strain M. tuberculosis H37Ra and other saprophytic, fast-growing mycobacteria appear to be the salient features. However, deoxyribonucleic acid from M. smegmatis SN2 lysogenized with the temperature phage I3 showed the presence of 5-methylcytosine. All of the strains had N6-methyladenine.     

Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli

In Escherichia coli, cytosine DNA methylation is catalyzed by the DNA cytosine methyltransferase (Dcm) protein and occurs at the second cytosine in the sequence 5'CCWGG3'. Although the presence of cytosine DNA methylation was reported over 35?years ago, the biological role of 5-methylcytosine in E.?coli remains unclear. To gain insight into the role of cytosine DNA methylation in E.?coli, we (1) screened the 72 strains of the ECOR collection and 90 recently isolated environmental samples for the presence of the full-length dcm gene using the polymerase chain reaction; (2) examined the same strains for the presence of 5-methylcytosine at 5'CCWGG3' sites using a restriction enzyme isoschizomer digestion assay; and (3) quantified the levels of 5-methyl-2'-deoxycytidine in selected strains using liquid chromatography tandem mass spectrometry. Dcm-mediated cytosine DNA methylation is conserved in all 162 strains examined, and the level of 5-methylcytosine ranges from 0.86% to 1.30% of the cytosines. We also demonstrate that Dcm reduces the expression of ribosomal protein genes during stationary phase, and this may explain the highly conserved nature of this DNA modification pathway.     

Analysis of genomic DNA from Chlamydia trachomatis for Dam and Dcm methylation

Chlamydia trachomatis is a Gram-negative eubacterium with a dimorphic developmental cycle and obligate intracellular growth in the eucaryotic host. The Dam transmethylase of Escherichia coli methylates at the N6 position of adenine in the sequence 5'-GATC-3' and the Dcm transmethylase adds methyl groups to the C5 position of the internal cytosines in the sequences 5'-CCWGG-3'. In contrast to E. coli, C. trachomatis DNA appears to have unmethylated Dam sites and only low level Dcm methylation.     

Salmonella typhimurium SA host specificity system is based on deoxyribonucleic acid-adenine methylation.

We have determined the nature of the deoxyribonucleic acid (DNA) modification governed by the SA host specificity system of Salmonella typhimurium. Two lines of evidence indicate that SA modification is based on methylation of DNA-adenine residues. (i) The SA+ locus of Salmonella was transferred into Escherichia coli B, a strain that does not contain 5-methylcytosine in its DNA; although the hybrid strain was able to confer SA modification, its DNA still did not contain 5-methylcytosine. (ii) the N6-methyladenine content of phage L DNA was measured after growth in various host strains; phage lacking SA modification contained fewer N6-methyladenine residues per DNA. We also investigated the possibility, suggested by others (32), that SA modification protects phage DNA against restriction by the RII host specificity system. Phages lambda, P3, and L were grown in various SA+ and SA- hosts and tested for their relative plating ability on strains containing or lacking RII restriction; the presence or absence of SA modification had no effect on RII restriation. In vitro studies revealed, however, that Salmonella DNA is protected against cleavage by purified RII restriction endonuclease (R-EcoRII). This protection is not dependent on SA modification; rather, it appears to be due to methylation by a DNA-cytosine methylase which has overlapping specificity with the RII modification enzyme, but which is not involved in any other known host specificity system.     

Direct detection of methylation in genomic DNA

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N⁶-methyladenine, 5-methylcytosine and N⁴-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N⁶-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N⁴-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote.     

Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing

DNA methylation is the most common form of DNA modification in prokaryotic and eukaryotic genomes. We have applied the method of single-molecule, real-time (SMRT®) DNA sequencing that is capable of direct detection of modified bases at single-nucleotide resolution to characterize the specificity of several bacterial DNA methyltransferases (MTases). In addition to previously described SMRT sequencing of N6-methyladenine and 5-methylcytosine, we show that N4-methylcytosine also has a specific kinetic signature and is therefore identifiable using this approach. We demonstrate for all three prokaryotic methylation types that SMRT sequencing confirms the identity and position of the methylated base in cases where the MTase specificity was previously established by other methods. We then applied the method to determine the sequence context and methylated base identity for three MTases with unknown specificities. In addition, we also find evidence of unanticipated MTase promiscuity with some enzymes apparently also modifying sequences that are related, but not identical, to the cognate site.     

The mealybug chromosome system I: Unusual methylated bases and dinucleotides in DNA of aPlanococcus species

The methylation status of the nuclear DNA from a mealybug, aPlanococcus species, has been studied. Analysis of this DNA by High Performance Liquid Chromatography and Thin Layer Chromatography revealed the presence of significant amounts of 5-—methylcytosine. Since analysis of DNA methylation using the Msp I/Hpa II system showed only minor differences in susceptibility of the DNA to the two enzymes, it seemed possible that 5-methylcytosine (5mC) occurred adjacent to other nucleotides in addition to its usual position, next to guanosine. This was verified by dinucleotide analysis of DNA labelledin vitro by nick translation. These data show that the total amount of 5-methylcytosine in this DNA is slightly over 2.3 mol %, of which 0.61% occurs as the dinucleotide 5mCpG, 0.68% as 5mCpA, 0.59% as 5mCpT and 0.45% as 5mCpC. 5mCpG represents approximately 3.3% of all CpG dinucleotides. The experimental procedure would not have permitted the detection of 5mCp5mC, if it occurs in this system. Unusually high amounts of 6-methyladenine (approximately 4 mol %) and 7-methylguanine (approximately 2 mol %) were also detected, 6-methyladenine and 7-methylguanine occurred adjacent to all four nucleotides. The total G+C content was 33.7% as calculated from dinucleotide data and 32.9% as determined from melting profiles.     

Comparative study of DNA methylation in three unicellular eucaryotes.

We have analyzed the nature/content of methylated bases in the nuclear DNA of three unicellular eucaryotes. The pattern of methylation was different for each of the three organisms studied: Saccharomyces cerevisiae contained only 5-methylcytosine; Tetrahymena pyriformis contained only N6-methyladenine; and Chlamydomonas reinhardi contained both modified bases.     

Sites of methylation of DNA bases by the action of organophosphorus insecticides in vitro

Methylation in vitro of DNA by three methyl-14C-labelled organophosphorus insecticides has been studied. The ability of methylbromphenvinphos, methylparathion and malathion to methylate N-7 of guanine in DNA can be expressed as 100:40:15. Among the methylation products, no O6-methylguanine, a known mutagen, was found. Both in the reaction with dsDNA and with ssDNA 7-methyl-guanine was the main methylation product. However, all methyl derivatives of adenine (3-methyladenine, 1-methyladenine and 7-methyladenine) constituted about 40% and 50% of all methylation products in the case of dsDNA and ssDNA, respectively. The only methyl derivative of pyrimidine we have identified was 3-methylcytosine. In the case of dsDNA 3-methylcytosine appeared in small amounts but in the alkylated ssDNA 3-methylcytosine C constituted about 20% of all alkylation products.     

Deoxyribonucleic acid methylation and development in Caulobacter bacteroides

The stalked bacterium Caulobacter bacteroides, which displays a series of developmental changes during its life cycle, was shown to contain the methylated deoxyribonucleic acid (DNA) nucleotide bases N(6)-methyladenine and 5-methylcytosine as well as the enzymes required for the synthesis of these bases. A difference in the specific activity of these enzymes was observed between swarmer cells, which are low in activity, and stalked cells, which are high in activity. This difference was not reflected in the methylation patterns in the DNA of the two cell types since C. bacteroides DNA is essentially completely methylated with respect to C. bacteroides methylases.     

Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

ABSTRACT: BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.     

DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine.

While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C). Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy. Eight of the bacterial DNAs contained m4C. Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile. The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs. Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns. Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes. Instead, some of them have deamination-resistant m4C residues.     

Plasmid-controlled variation in the content of methylated bases in single-stranded DNA bacteriophages M13 and fd

The N⁶-methyladenine and 5-methylcytosine contents in the DNA of bacteriophages M13 and fd have been analyzed. The results are summarized as follows. (1) After growth in bacteria harboring the N-3ft^? drug resistance-factor, fd and M13 are observed to contain approximately 1 to 2 more 5-methylcytosine residues per DNA molecule than after growth in the parental drug-sensitive host; no effect on the N⁶-methyladenine content is produced by the plasmid. (2) After growth in bacteria harboring P1 prophage, fd and M13 are observed to contain approximately 2 to 3 more N⁶-methyladenine residues per DNA molecule than after growth in the parental P1-sensitive host; no apparent effect on the 5-methylcytosine content was produced by the P1 plasmid. (3) In agreement with others, fd carrying B-host specificity (fd·B) is observed to contain 2 more N⁶-methyladenine residues/DNA molecule than fd·K.     

The Neisseria gonorrhoeae S.NgoVIII restriction/modification system: a type IIs system homologous to the Haemophilus parahaemolyticus HphI restriction/modification system.

Strains of Neisseria gonorrhoeae possess numerous restriction-modification (R-M) systems. One of these systems, which has been found in all strains tested, encodes the S. NgoVIII specificity (5'TCACC 3') R-M system. We cloned two adjacent methyltransferase genes (dcmH and damH), each encoding proteins whose actions protect DNA from digestion by R.HphI or R.Ngo BI (5'TCACC 3'). The damH gene product is a N 6-methyladenine methyltransferase that recognizes this sequence. We constructed a plasmid containing multiple copies of the S.NgoVIII sequence, grew it in the presence of damH and used the HPLC to demonstrate the presence of N 6-methyladenine in the DNA. A second plasmid, containing overlapping damH and Escherichia coli dam recognition sequences in combination with various restriction digests, was used to identify which adenine in the recognition sequence was modified by damH. The predicted dcmH gene product is homologous to 5-methylcytosine methyltransferases. The products of both the dcmH and damH genes, as well as an open reading frame downstream of the damH gene are highly similar to the Haemophilus parahaemolyticus hphIMC , hphIMA and hphIR gene products, encoding the Hph I Type IIs R-M system. The S.NgoVIII R-M genes are flanked by a 97 bp direct repeat that may be involved in the mobility of this R-M system.     

Undetectable levels of N6-methyl adenine in mouse DNA: Cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase

Three methylated bases, 5-methylcytosine, N4-methylcytosine and N6-methyladenine (m6A), can be found in DNA. However, to date, only 5-methylcytosine has been detected in mammalian genomes. To reinvestigate the presence of m6A in mammalian DNA, we used a highly sensitive method capable of detecting one N6-methyldeoxyadenosine per million nucleosides. Our results suggest that the total mouse genome contains, if any, less than 10(3) m6A. Experiments were next performed on PRED28, a putative mammalian N6-DNA methyltransferase. The murine PRED28 encodes two alternatively spliced RNA. However, although recombinant PRED28 proteins are found in the nucleus, no evidence for an adenine-methyltransferase activity was detected.     

The M.AluI DNA-(cytosine C5)-methyltransferase has an unusually large, partially dispensable, variable region.

The DNA methyltransferase of the AluI restriction-modification system, from Arthrobacter luteus, converts cytosine to 5-methylcytosine in the sequence AGCT. The gene for this methyltransferase, aluIM, was cloned into Escherichia coli and sequenced. A 525-codon open reading frame was found, consistent with deletion evidence, and the deduced amino acid sequence revealed all ten conserved regions common to 5-methylcytosine methyltransferases. The aluIM sequence predicts a protein of M(r) 59.0k, in agreement with the observed M(r), making M.AluI the largest known methyltransferase from a type II restriction-modification system. M.AluI also contains the largest known variable region of any monospecific DNA methyltransferase, larger than that of most multispecific methyltransferases. In other DNA methyltransferases the variable region has been implicated as the sequence-specific target recognition domain. An in-frame deletion that removes a third of this putative target-recognition region leaves the Alu I methyltransferase still fully active.