The Helicobacter pylori Methylome is Acid-Responsive due to Regulation by the Two-Component System ArsRS and the Type I DNA Methyltransferase HsdM1 (HP0463)
Helicobacter pylori colonizes the gastric epithelium and is the leading cause of gastric adenocarcinoma. There is increasing evidence that DNA methylation regulates gene expression in addition to its role in genome protection. In this study, we characterize the impact of acidity, the Two-Component System (TCS) ArsRS, Autoinducer-2, and the Type I m6A DNA methyltransferase HsdM1 (HP0463) on the Helicobacter pylori methylome. Transcription of hsdM1 increases at least 4-fold in the absence of the sensory histidine kinase ArsS, the major acid sensing protein of H. pylori. HsdM1 exists in the phase-variable operon hsdR1-hsdM1. Phase-locking hsdR1 (HP0464), the restriction endonuclease gene, has significant impacts on the transcription of hsdM1. To determine the impacts of methyltransferase transcription patterns on the methylome, we conducted methylome sequencing on samples that had undergone prolonged acid-exposure experiment. We found differentially methylated motifs between pH 7 and pH 5 growth conditions, and that deletions of arsS and hsdM1 interfere with the epigenetic acid response. Deletion of arsS leads to altered hsdM1 expression and alters the activity of multiple methyltransferases in both conditions indicating that the ArsRS TCS, in addition to its role in acid acclimation via direct effects on regulon member transcription, may also indirectly impact gene expression via regulation of the methylome. We characterized the target motif of HsdM1 (HP0463) to be (H)HTCAVN6TGY. HsdM1 may hierarchically regulate the Type II m5C DNA methyltransferase HP1121. This complex regulation of DNA methyltransferases, and thus differential methylation patterns, may have implications for the decades-long persistent infection by H. pylori. Overall design: Three Helicobacter pylori 26695 mutant strains were used for our experiments: a metranidazole-resistant control strain (?rdxA, referenced as 1' or control mutant), a strain with a deletion of the hsdM1 gene (?rdxA-?hsdM1, referenced as ?hsdM1), and a strain with a deletion of the arsS gene (?rdxA-?arsS, referenced as ?arsS). Each strain underwent a 72 hour growth in either pH 5 or pH 7 SFBB-cholesterol-vancomycin broth culture conditions, following a subculture procedure every 24 hours. Genomic DNA was extracted using the Bio-Rad gDNA extraction kit. Sequencing was performed via the PacBio Sequel Machine using Single Molecule, Real-Time (SMRT) sequencing. Azenta Life Sciences performed SMRT-seq and data analysis with the SMRT Analysis software through PacBio.