Methyltransferases (MTases) of procaryotes affect general cellular processes such as mismatch repair, regulation of transcription, replication, and transposition, and in some cases may be essential for viability. As components of restriction-modification systems, they contribute to bacterial genetic diversity. The genome of Helicobacter pylori strain 26695 contains 25 open reading frames encoding putative DNA MTases. To assess which MTase genes are active, strain 26695 genomic DNA was tested for cleavage by 147 restriction endonucleases; 24 were found that did not cleave this DNA. The specificities of 11 expressed MTases and the genes encoding them were identified from this restriction data, combined with the known sensitivities of restriction endonucleases to specific DNA modification, homology searches, gene cloning and genomic mapping of the methylated bases m 4 C, m 5 C, and m 6 A.The bacterium Helicobacter pylori chronically infects the upper gastrointestinal tract of the majority of people worldwide and is major cause of chronic gastritis and peptic ulcer disease and an early risk factor for gastric cancer (39). H. pylori strains are diverse genetically (1,2,4,16,21,44), and certain strain differences may contribute to the diversity of clinical outcome of infection (13,15,47). Analyses of genome sequences of H. pylori strains 26695 (46) and J99 (3) revealed that each strain contained more than two dozen genes likely to encode DNA methyltransferases (MTases), far more than have been detected in other bacterial genomes to date (20). These MTase genes comprise a large fraction of all genes that were specific to one or the other strain. Bacterial MTases exist either as components of restriction-modification (R-M) systems or as separate enzymes. The former are strain specific, and their main function is to protect host DNA from damage by a cognate restriction enzyme (RE). Separate MTases, which tend to be present in all strains of a species (species-specific MTases), are involved in general cellular processes such as regulation of transcription of specific genes (5, 48), DNA replication (32), mismatch repair (33), or DNA transposition (40). Certain of them can be essential for viability (43) or contribute to specificity in bacterium-host interactions or virulence in pathogens (8,17).Most MTase genes found in the genomes of H. pylori strains 26695 and J99 probably belong to R-M systems. That certain R-M systems might also affect bacterium-host interactions was suggested by induction of transcription of iceA1, a homolog of RE NlaIII, by H. pylori-host cell contact (36). Although iceA1 does not encode a functional protein in most H. pylori strains (14), it is adjacent to a generally active MTase gene (hpyIM) specific for sites recognized by NlaIII. Thus, the hpyI-iceA1 pair may represent a vestige of an R-M system that has evolved some special function relevant to host-pathogen interactions (14). By extrapolation, the same might be true for the MTases of other putative R-M systems in H. pylori. The identification ...