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

• 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.

Ämnesord

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

Nyckelord

Cell Line

Cloning

Molecular

DNA Modification Methylases/*genetics/*metabolism

DNA Restriction Enzymes/metabolism

DNA

Bacterial/metabolism

Epithelial Cells/microbiology

Escherichia coli/genetics

Gene Deletion

Gene Expression

Gene Expression Regulation

Bacterial

Helicobacter pylori/*enzymology/*genetics

Humans

Interleukin-8/biosynthesis

Microbial Viability

Mutagenesis

Insertional

Protein Binding

Recombinant Proteins/genetics/isolation & purifica

Biology

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)