Functional analysis of the M.HpyAIV DNA methyltransferase of Helicobacter pylori
| Autor: | Margareta Krabbe, Britta Björkholm, Cecilia Jernberg, Anna Skoglund, Lars Engstrand, Cristofer Enroth, Christina Nilsson, Katja Schirwitz, Anders F. Andersson |
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| Rok vydání: | 2007 |
| Předmět: |
DNA
Bacterial Candidate gene Mutant Mutagenesis (molecular biology technique) Gene Expression Biology Gene Mutant Microbiology Cell Line Escherichia coli Humans Cloning Molecular Molecular Biology Gene DNA Modification Methylases Regulation of gene expression Genetics Microbial Viability Helicobacter pylori Interleukin-8 Wild type Epithelial Cells DNA Restriction Enzymes Gene Expression Regulation Bacterial Molecular biology Enzymes and Proteins Recombinant Proteins Restriction enzyme Mutagenesis Insertional Gene Deletion Protein Binding |
| Zdroj: | Journal of bacteriology. 189(24) |
| ISSN: | 1098-5530 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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