XerH/<i>difH</i> site-specific recombination assay in <i>Helicobacter pylori.</i>

<p>(A) Schematic depiction of XerH excision assay. The <i>difH</i> repeat cassette, consisting of streptomycin susceptibility-chloramphenicol resistance genes (<i>rpsL</i>-<i>cat</i>) flanked by <i>difH</i> sequence, was introduced into the <i>H. pylori</i> 26695^Str (WT) genome by natural transformation and homologous recombination between genes HP0203 and HP0204 or in place of genes <i>ureA</i> and <i>ureB</i>. Recombination at <i>difH</i> sites leads to excision of <i>rpsL</i>-<i>cat</i> and one <i>difH</i> sequence, detectable by a PCR fragment 1.5 kb smaller than that from parental <i>difH</i> repeat cassette containing DNA. A cassette with <i>nondif</i> sequence flanking <i>rpsL-cat</i> served as a negative control. (B) Results of <i>difH</i> recombination assay in <i>H. pylori</i> 26695^Str (WT) with 40 bp <i>difH</i> direct repeats or 40 bp <i>nondif</i> DNA direct repeats at the HP0203-HP0204 locus. With each of these cassettes, two independent clones were tested by diagnostic PCR. The 2.1 kb and 3.6 kb PCR products come <i>difH</i> recombinant and parental (<i>difH</i> repeat containing) DNAs, respectively. Lane C, control from wild-type <i>H. pylori</i> without <i>difH</i> repeat cassette. (C) <i>difH</i> recombination frequencies for <i>difH</i> repeat cassette located at HP0203-HP0204 or <i>ureAB</i> loci. Cells were grown on non-selective media for two or four days, re-streaked for single colonies, and ∼100–200 colonies were tested for retention or loss of <i>rpsL</i> and <i>cat</i> genes by replica plating to streptomycin and to chloramphenicol containing media. Experiments were performed in triplicates; horizontal bars indicate means and standard deviation.