Methylomic and phenotypic analysis of the ModH5 phasevarion of Helicobacter pylori

Abstract

The Helicobacter pylori phase variable gene modH, typified by gene HP1522 in strain 26695, encodes a N6-adenosine type III DNA methyltransferase. Our previous studies identified multiple strain-specific modH variants (modH1 – modH19) and showed that phase variation of modH5 in H. pylori P12 influenced expression of motility-associated genes and outer membrane protein gene hopG. However, the ModH5 DNA recognition motif and the mechanism by which ModH5 controls gene expression were unknown. Here, using comparative single molecule real-time sequencing, we identify the DNA site methylated by ModH5 as 5′-Gm6ACC-3′. This motif is vastly underrepresented in H. pylori genomes, but overrepresented in a number of virulence genes, including motility-associated genes, and outer membrane protein genes. Motility and the number of flagella of H. pylori P12 wild-type were significantly higher than that of isogenic modH5 OFF or ΔmodH5 mutants, indicating that phase variable switching of modH5 expression plays a role in regulating H. pylori motility phenotypes. Using the flagellin A (flaA) gene as a model, we show that ModH5 modulates flaA promoter activity in a GACC methylation-dependent manner. These findings provide novel insights into the role of ModH5 in gene regulation and how it mediates epigenetic regulation of H. pylori motility.

The core genome m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in orchestrating gene expression in Helicobacter pylori

“…The flaA promoter, whose expression is governed mainly by the transcription factor σ 28 , was shown by extensive mutagenesis t be strongly modulated in a topology-dependent manner during the growth phase (65). This also fits to the previously described methylation-dependent indirect regulation of the flaA promoter (39). Finally, several direct and indirect means of methylation-mediated regulatory mechanisms might not exclude each other, generating an intricate network fine-tuning gene expression, which depends on genome-wide methylation.…”

“…This provides strong and direct evidence that methylation of the GCGC motif within a promoter sequence affects gene transcription. Similar findings were previously reported for G m6 ACC motifs methylated by the H. pylori ModH5 MTase, which are involved in the control of the activity of the flaA promoter in strain P12 (39). The exact mechanism(s) how methylated sequence motifs within promoters and most likely also within coding sequences influence gene expression in H. pylori is still unknown.…”

“…Despite the diversity of methylation patterns, a small number of target motifs were shown to be methylated in all (one motif, GCGC) or almost all (3 motifs protected in >99% of strains) H. pylori strains in a study by Vale et al ., who tested genomic DNAs purified from 221 H. pylori strains for susceptibility to cleavage by 29 methylation-sensitive restriction enzymes, and in those studies investigating the methylomes of multiple H. pylori strains (33,34,36,37). R-M systems have also previously been shown to contribute to gene regulation in H. pylori; the phase-variable MTase ModH5 is involved in the control of the expression of virulence-associated genes like hopG or flaA in strain P12 (38,39).…”

Kinetic and catalytic properties of M.HpyAXVII, a phase-variable DNA methyltransferase from Helicobacter pylori

“…The multiple-sequence alignment obtained from the above analysis provided more information about the phase-variable nature of the mod 4a/4b loci across H. pylori strains. The role of homopolymeric base repeats in phase-variable gene regulation is well-known at the transcriptional level (20) as well as the translational level (21). Here, we have analyzed the status of Figure 1.…”

Pathogenesis of Helicobacter pylori infection

“…Two studies deepened our understanding of the roles of specific DNA methylases in regulating gene expression by leveraging SMRT PacBio sequencing to identify the target specificity of the N4‐cysteine DNA methylase M2.HpyAII and the N6‐adenosine methylase Mod5 . The target sequences for both of these methylases are underrepresented in the H. pylori genome.…”