ROD21 is lost in bacteria undergoing type 2 excision.

<p>(<b>A</b>) Schematic representation of <i>tetRA</i> insertion in the ROD21::<i>tetRA</i> strain. This strain was used to isolate bacteria that have lost ROD21. The ROD21::<i>tetRA</i> strain was grown in the contraselection BM medium and the arising colonies were tested for the presence of ROD21 by PCR. The lines denominated 1 and 2 represent the expected PCR products that would be generated if ROD21 was inserted in the chromosome. (<b>B</b>) Detection of ROD21 by PCR analysis in WT <i>S.</i> Enteritidis and in one of the ΔROD21 strains isolated in a contraselection assay. This figure shows a representative agarose gel (1%) resolving the PCR products 1 and 2 (which denote each boundary of the integrated form of ROD21) obtained for WT and ΔROD21 <i>S.</i> Enteritidis strains. In addition, the <i>attB</i> sequences generated after type 1 (<i>attB-1</i>) and type 2 (<i>attB-2</i>) excisions were also detected by PCR. As a positive control, the <i>rpoD</i> gene was amplified by PCR. Data shown derive from one representative <i>S.</i> Enteritidis ΔROD21 strain selected out of 6 strains recovered in two independent experiments in which the <i>attB-2</i> sequence was detected. (<b>C</b>) C57BL/6 mice were orally infected with 1×10⁶ CFUs of either WT or ΔROD21 <i>S.</i> Enteritidis strains and the survival rate was measured daily. Uninfected mice were included as controls. Data shown are averages of two independent experiments, each including at least 4 mice per group. (<b>D and E</b>) Competitive infection assays, consisting of C57BL/6 mice infected either orally or intravenously with a mixture of the WT (Kn^R) and ΔROD21 (Cm^R) <i>S.</i> Enteritidis strains (input ratio equal to 1∶1). After 72 h, bacteria were recovered from spleens and livers of infected mice and the ratio of WT (Kn^R) over ΔROD21 (Cm^R) <i>S.</i> Enteritidis was calculated and compared to the input. Data shown in graphs are average values from two independent experiments for bacteria recovered from spleens and livers after intragastric gavage (<b>D</b>) or intravenous (<b>E</b>) infections of 3 mice per group.