The Helicobacter pylori genome is modified at CATG by the product of hpyIM

Abstract

To understand mechanisms of DNA methylation in Helicobacter pylori, a human pathogen associated with peptic ulcer disease and gastric adenocarcinoma, we cloned a putative DNA methyltransferase gene, hpyIM. This gene contains a 990-bp open reading frame encoding a 329-amino-acid protein, M.HpyI. Sequence analysis revealed that M.HpyI was closely related to CATG-recognizing adenine DNA methyltransferases, including M.NlaIII in N. lactamica. hpyIM was present in all H. pylori strains tested. DNA from wild-type H. pylori strains was resistant to digestion by SphI and NlaIII, which recognize DNA at sites containing CATG, whereas their isogenic hpyIM mutants were susceptible, indicating lack of modification. Overexpression of hpyIM in Escherichia coli rendered DNA from these cells resistant to NlaIII digestion, confirming the role of hpyIM in modifying CATG sites. We conclude that hpyIM encodes a DNA methyltransferase, M.HpyI, that is well conserved among diverse H. pylori strains and that modifies H. pylori genomes at CATG sites.Helicobacter pylori is a gram-negative, spiral-shaped bacterium that is an important human pathogen (6,16). This bacterium colonizes the gastric mucus layer, leading to gastric inflammation and, in some persons, to peptic ulceration and adenocarcinoma (7). H. pylori is highly diverse at the genetic level (1,4,15,21,22,49,51), and strain differences are related to variations in clinical outcomes of infection (4,9,10,13,51). This high degree of genetic diversity may arise from spontaneous mutation, intragenomic recombination, and horizontal gene transfer. Differences in virulence may be related to the presence or absence of particular genes or to variations in gene expression. DNA methylation has been demonstrated to be involved in several important cellular processes, such as hostspecific defense mechanisms (3, 32), DNA mismatch repair (17,28,34), regulation of initiation of chromosomal replication (33, 44), regulation of gene transcription (5, 8), and DNA transposition (39). As such, the study of DNA methylation in H. pylori may improve our understanding of the pathogenic mechanisms of this organism. A previous study suggests that chromosomal modification in H. pylori is different from that in Escherichia coli (38), and restriction digestion results (54) also suggest that DNA in H. pylori is highly methylated at both adenine and cytosine residues; however, the mechanisms of DNA methylation in H. pylori have not been identified. In this paper, we report the cloning and identification of a conserved DNA methyltransferase gene, hpyIM, in H. pylori and the characterization of the specific modification sites of its product. Our previous data (37) show that iceA, a gene inducible by contact with epithelial cells, has two genotypes (iceA1 and iceA2) and that infection with iceA1 strains is closely associated with peptic ulcer disease. Since hpyIM lies downstream of iceA (37), we further sought to detect hpyIM among various H. pylori strains to investigate whether the genotypes of hpyIM are correlate...

Promoters of the CATG-Specific Methyltransferase Gene hpyIM Differ between iceA1 and iceA2 Helicobacter pylori Strains

“…The strains and plasmids used in this study are listed in Table 1. Growth conditions for Escherichia coli and H. pylori strains were similar to those described previously (27). Restriction enzymes and digestion buffers were obtained from New England Biolabs (Beverly, Mass.)…”

“…All DNA techniques, including chromosomal and plasmid DNA preparation, PCR, Southern blotting, DNA sequencing, and DNA transformation, have been described (27). Computer analyses of DNA sequences and database similarity searches were performed with the Genetics Computer Group programs (2).…”

“…An insert was constructed by ligation of three DNA fragments: 5Ј and 3Ј flanking fragments from the cysEiceA-hpyIM locus and a central fragment (xylE/kan cassette). The 2.4-kb xylE/kan cassette containing a reporter gene and a kanamycin resistance gene was generated as described previously (27). The 5Ј flanking fragment was created by PCR using 60190 chromosomal DNA as a template and CysE-1 (5ЈTCATGCT AGATCTGTTTTATAGCCT3Ј) and IceA-R as primers, followed by digestion with KpnI.…”

“…The hpyIM open reading frame (ORF) is highly conserved among various strains (27). However, the sequences upstream of hpyIM, where its promoter presumably is located, are substantially different, as shown in studies of iceA, the gene immediately upstream (19).…”

“…However, despite their potential importance, mechanisms of DNA methylation in H. pylori are not well studied. hpyIM, a CATG-specific methylase gene (27) in H. pylori, has been cloned and identified. Study of hpyIM may help us understand DNA methylation in H. pylori.…”