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A Mutagenesis Method for the Addition and Deletion of Highly Repetitive DNA Regions: The Paradigm of EPIYA Motifs in the cagA Gene of H elicobacter pylori
Author(s) -
Papadakos Konstantinos S.,
Sougleri Ioanna S.,
Mentis Andreas F.,
Sgouras Dionyssios N.
Publication year - 2013
Publication title -
helicobacter
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.206
H-Index - 79
eISSN - 1523-5378
pISSN - 1083-4389
DOI - 10.1111/hel.12029
Subject(s) - mutagenesis , gene , dna , genetics , site directed mutagenesis , chemistry , microbiology and biotechnology , biology , mutation , mutant
Background CagA protein of W estern origin H elicobacter pylori isolates contains at its carboxyl‐terminal end repeating types of EPIYA motifs, depending on the surrounding sequence, which dictate hierarchic tyrosine phosphorylation. To produce, in an isogenic background, mutant strains expressing CagA protein with variable numbers of EPIYA ‐C terminal motifs, we have adopted a mutagenesis assay using a megaprimer approach. Materials and Methods The H . pylori P12 reference strain containing two terminal EPIYA ‐C motifs was utilized. Initially, we cloned, full‐length cagA gene, next to the C ampylobacter jejuni kanamycin‐resistance cassette, followed by the 1200‐bp region located immediately after cagA gene (meta cagA region). Then, we generated a megaprimer consisting of three consecutive copies of the EPIYA ‐C coding sequence of cagA gene, followed by the 140‐bp region of the cagA genomic sequence present immediately after the second EPIYA ‐C repeat. We utilized these two products to perform a QuikChange mutagenesis assay and were able to obtain all desired combinations of EPIYA ‐C motifs, followed by Kan r cassette and meta cagA region. These constructions were used to perform natural transformation of the P12 parental strain, by directional homologous recombination. Results We produced isogenic H . pylori strains that express CagA with variable number of EPIYA ‐C motifs ( AB , ABC , ABCCC ) and their phosphorylation‐deficient counterparts. They exhibited similar growth characteristics to the parental strain, adhered equally well to gastric cells and successfully translocated CagA, following pilus induction. Conclusions Our method can be used in other cases where highly repetitive sequences need to be reproduced.