Kinetic, Catalytic and Epigenetic Properties of HpyAXVII DNA Methyltransferase – a Phasevarion in the Human Pathogen Helicobacter pylori

Abstract

Helicobacter pylori is the leading cause of gastric cancer in human beings. It is a helix-shaped, Gram-negative, motile bacteria that colonizes the gastric mucosa and eventually the gastric epithelium. It has an overabundance of Restriction-Modification (R-M) systems, a large majority of them being strain-specific. Several Type III DNA MTases have been identified as phasevarions (phase variable regulons) that can reversibly switch ON or OFF depending on the repeat length at a phase variable locus within the gene. This study is a comprehensive approach to understand the biochemical and enzyme kinetic nature of M.HpyAXVII, a phase variable N6-adenine DNA MTase from H. pylori 26695. Initial velocity, product inhibition and substrate inhibition analyses revealed that M.HpyAXVII binds to AdoMet first followed by DNA to form a ternary complex in an ordered Bi Bi reaction. Comparison of M.HpyAXVII amino acid sequence with its homologues from H. pylori strains, for which full sequence data is available, showed that the protein is highly conserved except for the TRD domain. The high homology at the region flanking the TRD means that this region is amenable for domain swapping. Combined with phase variable switching between ON and OFF states, H. pylori has evolved a powerful way to switch between genome methylation status, or methylomes, thereby increasing fitness. Comparison of hpyaxvii DNA sequences of homologs across strains revealed that the poly-G repeat length is variable. In almost half the strains the poly-G is maintained in the OFF position, i.e., a full-length fusion protein is not expressed. However, addition or deletion of a single guanosine can result in the expression of a potentially functional M.HpyAXVII in these strains. In a small subset of strains, the status of the phase variable locus is in the phase variably ON position. Interestingly, there are a few strains where the locus is always ON, i.e., there is no poly-G tract at all. Thus, the ontogeny of hpyaxvii phase variation in different strains provides a snapshot into the phylogeny of hpyaxvii phase evolution across different H. pylori strains. To investigate the effects of the total absence of M.HpyAXVII from H. pylori 26695, as in, constitutively OFF genotype, a deletion mutant was generated, designated Δhpyaxvii. Comparison of total mRNA levels between H. pylori 26695 Δhpyaxvii and WT revealed several genes that were differentially expressed. Several proteins that have been associated with acid-stress and iron-starvation stress were downregulated in Δhpyaxvii cells. This study provides a holistic understanding of M.HpyAXVII, a phasevarion from H. pylori 26695. The phase variable switching from a truncated, inactive DNA MTase to a functional, full-length DNA MTase has been shown before in Haemophilus influenzae strain Rd (ModA) and Helicobacter pylori strain P12 (ModH5). However, this is the first study where the biochemical and enzyme kinetic properties of such a phase variable DNA MTase have been elucidated.