The bacterium Helicobacter pylori is one of the most common infectious agents found in the human stomach. H. pylori has an unusually large number of DNA methyltransferases (MTases), prompting speculation that they may be involved in the cancerization of epithelial cells. The mod-4a/4b locus, consisting of the hp1369 and hp1370 ORFs, encodes for a truncated and inactive MTase in H. pylori strain 26695. However, slipped-strand synthesis within the phase-variable polyguanine tract in hp1369 results in expression of an active HP1369 -1370 fusion N 6 -adenine methyltransferase, designated M.HpyAXVII. Sequence analysis of the mod-4a/4b locus across 74 H. pylori strain genomes has provided insights into the regulation of M.HpyAX-VII expression. To better understand the role of M.HpyAXVII in the H. pylori biology, here we cloned and overexpressed the hp1369-70 fusion construct in Escherichia coli BL21(DE3) cells. Results from size-exclusion chromatography and multi-angle light scattering (MALS) analyses suggested that M.HpyAXVII exists as a dimer in solution. Kinetic studies, including product and substrate inhibition analyses, initial velocity dependence between substrates, and isotope partitioning, suggested that M.HpyAXVII catalyzes DNA methylation in an ordered Bi Bi mechanism in which the AdoMet binding precedes DNA binding and AdoMet's methyl group is then transferred to an adenine within the DNA recognition sequence. Altering the highly conserved catalytic motif (DPP(Y/F)) as well as the AdoMetbinding motif (FXGXG) by site-directed mutagenesis abolished the catalytic activity of M.HpyAXVII. These results provide insights into the enzyme kinetic mechanism of M.HpyAXVII. We propose that AdoMet binding conformationally "primes" the enzyme for DNA binding.Restriction endonucleases cleave DNA at a particular recognition sequence, and the cognate MTases 4 methylate a base(s) in this sequence, thereby making it refractory to cleavage. R-M systems have evolved to protect bacteria from bacteriophages. The MTase of an R-M system ensures that the host DNA is methylated, thereby identified as "self," whereas the nuclease cleaves the phage DNA, which is unmethylated (1). DNA MTases can be broadly classified into exocyclic or aminomethyltransferases and endocyclic or ring methyltransferases. Exocyclic methylation gives rise to N 6 -adenine as well as N 4 -cytosine methylation. Ring methylation gives rise to C 5 -cytosine methylation (2). MTases could also be involved in epigenetic regulation of gene expression by differential methylation of specific sites within a genome (3). N 6 -Adenine methylation has been identified as the predominant epigenetic signal in prokaryotic cells (4), whereas C 5 -cytosine methylation is thought to be mainly associated with R-M system-based protection of bacteria against bacteriophages. However, we have previously reported that M.HpyAVIB, a phase-variable C 5 -cytosine MTase, is involved in the modulation of gene expression in Helicobacter pylori, which results in a hypermutator phenotype (5). We ...

There was an error in Fig. S3A. While compiling data from a doctoral thesis, the wrong image and quantification from a similar experiment was incorporated into the article. This error has now been corrected and does not affect the results or conclusions of the work. The authors apologize for the error.