Methylated Bases of Bacillus megaterium KM Nucleic Acids: Comparison with Escherichia coli

Parallel studies were performed with methionineless derivatives of Escherichia coli 15 T(-) and Bacillus megaterium KM: T(-). Methylated bases are present in the total cell ribonucleic acid (RNA) of B. megaterium. The level of RNA methylation in E. coli is about 60% greater than that in B. megaterium. Although E. coli deoxyribonucleic acid (DNA) was found to contain 0.12% 5-methylcytosine (5-MC) and 0.24% 6-methylaminopurine (6-MA), methylated bases were not detected in the DNA of B. megaterium. Assuming a molecular weight of 7 x 10(9) daltons for B. megaterium DNA, it was calculated that this organism could not contain more than one molecule of 5-MC or 6-MA per genome, and that possibly no methylated bases were present. Methylated bases were also not detected in the DNA of thymine-starved B. megaterium. Crude extracts of this organism possess RNA methylase activity but no detectable DNA methylase activity.     

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.     

Plasmid-Controlled Variation in the Content of Methylated Bases in Bacteriophage Lambda Deoxyribonucleic Acid

The N(6)-methyladenine (MeAde) and 5-methylcytosine (MeC) contents in deoxyribonucleic acid (DNA) of bacteriophage lambda has been analyzed as a function of host specificity. The following facts have emerged: (i) lambda grown on strains harboring the P1 prophage contain ca. 70 more MeAde residues/DNA molecule than lambda grown either in the P1-sensitive parent, or in a P1 immune-defective lysogen which does not confer P1 modification; (ii) lambda grown on strains harboring the N-3 drug-resistance factor contain ca. 60 more MeC residues/DNA molecule than lambda grown on the parental strain lacking the factor; (iii) lambda grown in Escherichia coli B strains is devoid of MeC, whereas lambda grown in a B (N-3) host contains a high level of MeC; (iv) the MeAde content in lambda DNA is not affected by the N-3 factor. These results suggest that P1 controls an adenine-specific DNA methylase, and that the N-3 plasmid controls a cytosine-specific DNA methylase. The N-3 factor has been observed previously to direct cytosine-specific methylation of phage P22 DNA and E. coli B DNA in vivo; in vitro studies presented here demonstrate this activity.     

Genetic Control of the Secondary Modification of Deoxyribonucleic Acid in Escherichia coli

The wild-type restriction and modification alleles of Escherichia coli K-12 and B were found to have no measurable effect on the patterns of methylated bases in the deoxyribonucleic acid (DNA) of these strains. The genetic region controlling the methylation of cytosine in E. coli K-12 was mapped close to his, and the presence or absence of this gene in E. coli B or E. coli K had no effect on the restriction and modification properties of these strains. Thus, only a few of the methylated bases in the DNA of these strains are involved in host modification, and the biological role of the remainder remains obscure.     

Analysis of bacteriophage deoxyribonucleic acid sequences methylated by host- and R-factor-controlled enzymes.

Phages lambda and fd were propagated in Escherichia coli strains that have either host K-12 or the N-3 R-factor deoxyribonucleic acid-cytosine methylase activity. Pyrimidine tracts containing 3H-labeled 5-methylcytosine (MeC) were analyzed; in all cases, the major methylated sequence was 5' ... C-MeC-T ... 3'.     

Specificity and functions of guanine methylase of Shigella sonnei DDVI phage.

DNA methylase methylating adenine with formation of 6-methylaminopurine has been identified in Shigella sonnei 1188 cells which are the natural host of DDVI phage. At the same time, in DNA of DDVI phage replicating both in Sh. sonnei 1188 cells and in Escherichia coli B cells 7-methylguanine was found as the only minor base in amounts of 0.25 and 0.27 mol per 100 mol of nucleotides, respectively. The extract of the infected cells was found to contain both kinds of DNA methylases: virus-specific guanine methylase and cellular adenine methylase. The lack of 6-methylaminopurine in DNA of this phage is explained by reversible inhibition of the cell enzyme in the infected cells. The amount of methyl groups transferred by DDVI-specific methylase on DNA does not depend on the species of the infected cells and is similar in the case of unmodified SD phage DNA and DNA of T2 phage methylated by E. coli B enzyme. Guanine methylase has been shown to be a DDVI-induced modification enzyme and to protect against restriction of B-type. It methylates double-stranded DNAs only and is inhibited by S-adenosylhomocysteine.     

Change in Methylation of Salmonella Bacteriophage P3 Deoxyribonucleic Acid with Host-Controlled Modification by Escherichia coli

Modification of bacteriophage P3 by passage through Escherichia coli K was correlated with a 54% decrease in the content of 6-methylaminopurine in the phage deoxyribonucleic acid.     

Study of the methylation process and DNA methylase specificity in Shigella]

The nature and content of minor bases in DNA of 3 Shigella strains are investigated. DNAs from Shigella stutzeri 2, Sh. sonnei 1188 and Sh. sonnei 311 are found to contain 0.43, 0.56 and 0.45 mol.% of N6-methyladenine respectively. 5-methylcytosine (0.16 mol.%) is discovered in Sh. sonnei 311. Substrate specificity of adenine methylase from Sh. sonnei 1188 with respect to phage DNAs of different host modification is investigated. Recognition sites for guanine methylase of DDVI phage and for adenine methylase of Sh. sonnei 1188 turned to be different. DNA of DDII phage grown in Sh. stutzeri 2 cells does not accept methyl groups under the treatment with Sh. sonnei 1188 extracts, but it is methylated by Escherichia coli extract. Adenine methylases of Sh. sonnei 1188 and Sh. stutzeri 2 are suggested to be either the same enzyme, or enzymes, which recognition sites are partially overlapped.     

Absence in Bacillus subtilis and Staphylococcus aureus of the sequence-specific deoxyribonucleic acid methylation that is conferred in Escherichia coli K-12 by the dam and dcm enzymes

Restriction analysis of plasmid pHV14 deoxyribonucleic acid isolated from Escherichia coli K-12, Bacillus subtilis, and staphylococcus aureus with restriction endonucleases MboI, Sau3AI, and EcoRII was used to study the methylation of those nucleotide sequences which in E. coli contain the major portions of N6-methyladenine and 5-methylcytosine. The results showed that neither B. subtilis nor S. aureus methylates deoxyribonucleic acid at the same sites and nucleotides which are recognized and methylated by dam and dcm enzymes in E. coli K-12.     

Variation of 6-Methylaminopurine Content in Bacteriophage P22 Deoxyribonucleic Acid as a Function of Host Specificity

The 6-methylaminopurine (MAP) content of P22 deoxyribonucleic acid has been analyzed as a function of the host specificity it carries. A 40 to 50% reduction in MAP level occurs as a result of growth in host cells defective in the ability to confer LT specificity.     

Methylation Pattern of Lambda Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from phage lambda grown on Escherichia coli K-12 strain W4032 had 113 +/- 10 5-methylcytosine residues and 215 +/- 20 6-methyl adenine residues per genome, as determined by three independent methods. These methylated nucleotides were distributed equally among the two strands of lambda DNA. Shearing of double-stranded DNA to half-length fragments revealed a slight deficiency of 5-methyl cytosine in the 55% guanine plus cytosine half. Shearing the DNA to fragments of smaller length showed that the distribution of methylated nucleotides along the double helix was uniform with the exception of an undermethylated fragment arising from the center of the lambda DNA molecule. The implication of these results for the function of methylated nucleotides in the lambda DNA molecule is discussed.     

Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes.

Deoxyribonucleic acid (DNA)-cytosine methylation specified by the wild-type Escherichia coli K 12 mec+ gene and by the N-3 drug resistance (R) factor was studied in vivo and in vitro. Phage lambda and fd were propagated in the presence of L-[methyl-3H]methionine in various host bacteria. The in vivo labeled DNA was isolated from purified phage and depurinated by formic acid-diphenylamine treatment. The resulting pyrimidine oligonucleotide tracts were separated according to size and base composition by chromatography on diethylaminoethyl-cellulose in 7 M urea at pH 5.5 and 3.5, respectively. The distribution of labeled 5-methylcytosine in DNA pyrimidine tracts was identical for phage grown in mec+ and mec minus (N-3) cells. For phage lambda the major 5-methylcytosine containing tract was the tripyrimidine, C2T; for both fd-mec minus (N-3) DNA and fd-mec+DNA, C2T was the sole 5-methylcytosine-containing tract. When various lambda DNAs were methylated to saturation in vitro by crude extracts from mec+ and mec minus (N-3) cells, the extent of cytosine methylation was the same. This is in contrast to in vivo methylation where lambda-mec minus (N-3) DNA contains twice as many 5-methylcytosines per genome as lambda-mec+ DNA. Therefore, we suggest that the K12 met+ cytosine methylase and the N-3 plasmid modification methylase are capable of recognizing the same nucleotide sequences, but that the in vivo methylation rate is lower in mec+ cells.     

Methylation of satellite deoxyribonucleic acid in mouse neoplastic and non-neoplastic cell cultures

Summary Comparisons of nucleic acid methylation between paired neoplastic and non-neoplastic mouse cell lines have shown a striking difference in the deoxyribonucleic acid (DNA) peak eluted from methylated albumin-kieselguhr columns (R. Gantt and V. J. Evans, 1969, Cancer Res. 29: 536–541). Since mouse satellite DNA is relatively highly methylated, its 5-methylcytosine content was compared with mainband DNA in these two paired cell lines to determine whether this might account for the observed differences. The cell DNA was labeled with methyl-labeled methionine and isolated from the cells by repeated neutral cesium chloride isopycnic centrifugation. The satellite DNA strands were then separated in an alkaline cesium chloride gradient. Both the 5-methylcytosine content and the relative amounts of satellite DNA were indistinguishable in the paired cell lines. Further, the results showed that both strands of satellite DNA had virtually equal amounts of 5-methylcytosine, although the heavy strand contains 1.5 times more cytosine than the light strand.     

The construction of Streptomyces cyaneus genomic libraries in Escherichia coli is dependent upon the use of Mcr-deficient strains.

Streptomyces cyaneus genomic DNA ligated into either lambda phage or plasmid vectors was very inefficiently cloned into standard Escherichia coli host strains. However, the same material could be efficiently cloned using Mcr-deficient E. coli strains. These results suggest that the S. cyaneus genome contains 5-methylcytosine residues, some of which occur within the recognition sequences of the E. coli Mcr restriction system.     

Deoxyribonucleic acid modifications and restriction endonuclease production in Neisseria gonorrhoeae.

Modification of gonococcal deoxyribonucleic acid (DNA) was investigated, and the relationship with endonuclease production was explored. Both chromosomal and plasmid DNA from different gonococcal strains, irrespective of their plasmid content, was poorly cleaved by the restriction endonucleases HaeII, HaeIII, SacII, and BamHI. The fragment pattern of the Tn3 segment present on the 7.2-kilobase gonococcal resistance plasmid, when compared to its known DNA sequence, allowed us to conclude that the HaeIII and BamHI resistance was due to modification of these sites. A comparison of the fragment pattern of the resistance plasmid, when isolated from Escherichia coli or Neisseria gonorrhoeae, revealed that the resistance of HaeII must also be due to modification of its recognition sequence. Isoschizomers of HaeII and HaeIII can be found in isolates of N. gonorrhoeae (NgoI and NgoII, respectively). A new restriction endonuclease in gonococci, NgoIII, with a specificity similar to SacII, is reported here. High-pressure liquid chromatography of gonococcal DNA showed the presence of 5-methylcytosine. It is suggested that the methylation of cytosine residues in the HaeII (NgoI), HaeIII (NgoII), and SacII (NgoIII) recognition sites is the basis for the resistance of gonococcal DNA to cleavage by these enzymes. This methylation may be part of a host restriction modification system. In two out of five gonococcal strains the sequence -GATC- was modified. One strain unable to modify this sequence was a spontaneous mutant of a strain carrying such a modifying function.     

Separation of T-even Bacteriophage Deoxyribonucleic Acid from Host Deoxyribonucleic Acid by Hydroxyapatite Chromatography

A simple and rapid method is described for separation of T-even bacteriophage deoxyribonucleic acid (DNA) from host (Escherichia coli) DNA by hydroxyapatite column chromatography with a shallow gradient of phosphate buffer at neutral pH. By this method, bacteriophage T2, T4, and T6 DNA (but not T5, T7, or lambda DNA) could be separated from host E. coli DNA. It was found that glucosylation of the T-even phage DNA is an important factor in separation.     

Partial purification of the Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase: in vitro methylation completely protects bacteriophage lambda deoxyribonucleic acid against cleavage by R-EcoRII.

A procedure is described for the partial purification of the deoxyribonucleic acid (DNA)-cytosine methylases controlled by the RII plasmid and by the Escherichia coli mec+ gene. The two enzymes exhibit similar but distinct chromatographic behavior on diethylaminoethyl-cellulose and phosphocellulose. Preliminary studies on the two methylases indicate that they are indistinguishable with respect to their Km for S-adenosylmethionine and their pH (in tris (hydroxymethyl)aminomethane buffer) and NaCl concentration optima. In vitro methylation of various phage lambda DNA substrates by the mec'r RII enzyme modifies the DNA to a form that is completely resistant to double-stranded cleavage by the RII restriction endonuclease (R-EcoRII). These results are consistent with our earlier proposal that the mec8ethylase recognizes RII host specificity sites.     

Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine.

Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JA1 and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JA1 cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JA1. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JA1 restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In contrast, JA1 cells (which contain no methylated DNA bases) have a newly discovered type of restriction-modification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JA1 cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JA1, which retains the JA1 modification phenotype, was isolated, indicating that JA1 cells have a gene product with restriction specificity for DNA containing 5-methylcytosine.     

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.     

Mutants of the N-3 R-Factor Conditionally Defective in hspII Modification and Deoxyribonucleic Acid-Cytosine Methylase Activity

The N-3 drug resistance (R) factor specifies a deoxyribonucleic acid (DNA)-cytosine methylase and a DNA restriction-modification (hspII) system. We have isolated three independent mutants that are conditionally defective in their ability to modify bacteriophage lambda and to methylate DNA-cytosine residues. The ratio of 5-methylcytosine to N(6)-methyladenine in bacterial DNA and in the DNA of phages lambda and fd was determined after labeling with [methyl-(3)H]methionine at various growth temperatures. Although the ability of the wild-type N-3 factor to modify phage lambda and to methylate DNA-cytosine residues was unaffected with increasing temperature, two of the mutants exhibited a parallel loss in modification and cytosine methylation ability. The ability of the third mutant to carry out these functions was dependent on the presence or absence of an amber suppressor mutation in the host genome. These results offer further support for the notion that hspII modification is mediated by a DNA-cytosine methylase. Evidence is also presented that the modification methylase is responsible for the in vivo methylation of phage fd DNA (which is not subject to hspII restriction in vivo).