N4-methylcytosine as a minor base in bacterial DNA

Abstract

The DNA base composition, including the minor base content, of 26 strains of bacteria was determined. The studied bacteria are sources of widely used restriction endonucleases. Approximately 35% of the bacterial DNAs contained N4-methylcytosine, about 60% contained 5-methylcytosine, and about 90% had N6-methyladenine.Both N6-methyladenine (m6A) and 5-methylcytosine (m5C) have been long known to be minor bases in bacterial DNA (7,9,16). One or both of these methylated bases are present in most bacterial DNAs examined (4,11,33). Recently, a third minor base, N4-methylcytosine (m4C), has been found in DNA from eight types of thermophilic bacteria (11) and in that from one type of mesophilic bacteria (19,20). Previously, m4C residues had been detected only in the RNA of the small ribosomal subunit of bacteria and of mammalian and insect mitochondria (8,13,40). This modified base is not present in a variety of eucaryotic DNAs (13; Gehrke et al., unpublished results).The minor base composition of bacterial DNA is partially determined by restriction-modification systems (30). The level of minor bases should be consistent with the known specificities of the host restriction and modification enzymes. Also, it can reveal the presence of new DNA methyltransferases. For example, if the known restriction enzymes in a bacterium have recognition sites containing only G. C base pairs and if m6A as well as m5C is found in this cellular DNA, then one or more other unrelated modification pathways must be present. These could be involved in previously unidentified restriction-modification systems in the bacterium or in the control of various DNA functions (3,15,29,35).The present study demonstrates that genomic m4C is not mostly limited to the DNA of thermophilic bacteria. Rather, it is present as a minor base in the DNA of many bacterial mesophiles. Of the 26 bacterial species examined in this study, 9 contained m4C in their genomes. Most of the examined bacteria are mesophiles, and all of them are sources of commercially available restriction endonucleases. The prevalence of m4C as a minor base in these bacterial DNAs indicates that many restriction endonucleases may be inhibited by N4-methylation of cytosine residues in their recognition sites in vivo and that bacterial DNA cytosine methyltransferases must be carefully checked to determine whether they catalyze the formation of m4C. MATERIALS AND METHODSStrains. All bacteria came from the New England BioLabs strain collection except Xanthomonas campestris pv. oryzae, which was from the collection of M. Ehrlich. The * Corresponding author. strains are listed in Table 1 with the sources from which they were originally obtained and the temperature at which the cultures were grown.Purification of bacterial DNA. DNA was prepared from 10 g of wet packed cells suspended in 20 ml of 25% sucrose-50 mM Tris hydrochloride (pH 8.0), and then 10 ml of 0.25 M disodium EDTA (pH 8.0) and 6 ml of 10-mg/ml lysozyme in 0.25 M Tris (pH 8.0) were added. After 2 h at 0°C, 24 ml of 1% Triton X-100 in ...

Characterization of a unique methyl-specific restriction system in Streptomyces avermitilis

“…are common (1,6,8,17,26,35), but this report is the first description of a methyl-specific restriction system in Streptomyces spp. Some bacteria methylate DNA to produce N4-methylcytosine (5,9). The S. avermitilis restriction system has not been tested with this form of modification.…”

Nucleic Acid Modifications in Regulation of Gene Expression

“…It should be noted that besides 6mA, bacterial genomes also contain N 4 -methylcytosine (4mC or m 4 dC) and 5mC (Figure 2A) (Ehrlich et al, 1987; Vanyushin et al, 1968). These cytosine modifications are also used by bacterial restriction–modification (R-M) systems as defense mechanisms.…”

Biochemical and structural characterization of a DNA N6-adenine methyltransferase fromHelicobacter pylori

“…By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown. N 4 -cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC) [4]. N 4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages [5].…”