Functional analysis of the M.HpyAIV DNA methyltransferase of Helicobacter pylori

Abstract

A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen Helicobacter pylori. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the H. pylori strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase. H. pylori strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from H. pylori strain 26695 was overexpressed in Escherichia coli, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of H. pylori strains 26695 and J99 resulted in a number of candidate genes. katA, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.

FIG. 1.

Schematic representation of the genetic organization of the R.HpyAIV-M.HpyAIV genes in strains 26695, J99, and HPAG1. The M.HpyAIV genes are closely homologous in the three strains. The R.HpyAIV gene in HPAG1, HPAG_1299, is truncated, and the adjacent gene, HPAG_1298, is homologous to the 3′ end of the genes in strains 26695 and J99. The lined area shows the nucleotide position where the adenine repeat was found to be variable in active and inactive strains (start at 601 bp).

FIG. 2.

Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S-adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

FIG. 3.

Intergenic regions containing one or more GANTC sites in H. pylori strain 26695. Gray bars indicate region lengths. Black bars indicate that the J99 ortholog has upstream GANTC sites, while dark gray bars indicate the absence of upstream GANTC sites. White bars indicate that an ortholog in J99 is missing. Arrows indicate the direction of ORFs.

FIG. 4.

GANTC site distribution along intergenic regions of strain 26695 for true and randomized data. Intergenic regions containing one GANTC site were divided into 10-nt nonoverlapping windows, and the occurrences of GANTC sites (start positions) in each window were summarized over all intergenic regions (dark gray bars). Randomized data were obtained by randomly positioning the GANTC sites within each intergenic region and counting occurrences of GANTCs as before. The procedure was repeated 10,000 times, and average counts were calculated (light gray bars). *, P < 0.005; 38 of 10,000 randomizations displayed at least as many occurrences as observed. Intergenic regions extending 260 nt are not shown.