Identification of recombinants that replace the ICR only.

<p>(A) Schematic of the mating configuration designed to allow rapid genetic screening of the proximal ICR border status. In the recipient, A KnR cassette replaced a gene inside the proximal border of the ICR (<i>yjiT</i>::<i>npt</i>) and a CmR cassette replaced a nearby gene outside the proximal border (<i>yjiP</i>::<i>cat</i>). We call these ICR proximal border recipients. With this arrangement, a recombinant with donor DNA confined to the ICR will lose KnR but keep CmR and thus can be quickly identified by antibiotic screening. The embedded table displays the distribution of recombinant phenotypes in matings with different recipient backgrounds. The Δ<i>recA</i> Δ<i>repE</i>* donor was crossed with recA⁺, Δ<i>recA</i> or Δ<i>recA yjiPc-rhaBp</i> proximal border recipients (Crosses 7, 8, and 10 respectively); at least 100 recombinants from at least three independent matings of each cross were tested. The proximal crossover yielding complete donor replacement (top configuration) dominated Rec+, with some recombinants showing proximal crossover within the ICR (bottom configuration). In <i>recA</i>, a larger fraction showed proximal crossover within the ICR, and a minority showed proximal crossover within the ICR border region (middle configuration). When YjiPc was overexpressed this distribution did not change greatly. (B) Detecting distal crossover events with PCR and Sanger sequencing. The schematic shows SNP configurations at the distal ICR border. Sanger sequencing of the closest two SNPs distal to the ICR in the ten KnS, CmR recombinants from Cross 8 showed that seven shared the SNPs of the donor, while three (ER3482, ER3483, and ER3510) shared the SNPs of the recipient. For these three, the distal-side limit of the replacement event lies within a ~13 Kb region that includes the downstream edge of the ICR.