Schematic representation of the modified genetic regions of the strains used in the <i>S.</i> Enteritidis-to-<i>S.</i> Enteritidis mating assays.

<p>(<b>A</b>) Donor strain, showing excision type 1 and 2; (<b>B</b>) recipient strain and (<b>C</b>) transconjugant strain. Numbers above of each scheme represent the coordinates in the chromosome of <i>S.</i> Enteritidis. Asparagine tRNA genes are designated as <i>asnT-1</i>, <i>-2</i> and <i>-3</i>. DRS stands for <u>D</u>irect <u>R</u>epeated <u>S</u>equence. Black arrows represent the inserted antibiotic gene (<i>aph</i> or <i>cat</i>). Thin arrows represent primers used for PCR verification of the strains: (1) left-end of ROD21 (1,011 bp), (2) DRS (914 bp), (3) right-end (903 bp), (4) insertion of <i>cat</i> gene (1,030 bp without <i>cat</i> insert, 1,943 bp with <i>cat</i> insert), and (5) insertion of <i>aph</i> gene (2,469 bp). Below each scheme, agarose gels show PCR products obtained for the recipient and transconjugant <i>S.</i> Enteritidis strain.</p

Transfer of ROD21 to <i>S.</i> Typhimurium.

<p>(<b>A</b>) Scheme of the genome of the <i>S.</i> Typhimurium recipient strain with <i>cat</i> gene inserted in the putAP region (black arrow) and double-resistant strain with <i>aph</i> gene inserted in ROD21 and <i>cat</i> gene inserted in the putAP region (black arrows). Numbers above each scheme represent the coordinates in the chromosome of <i>S.</i> Typhimurium. Asparagine tRNA genes are designated as <i>asnT-1</i>, <i>-2</i> and <i>-3</i>. DRS stands for <u>D</u>irect <u>R</u>epeated <u>S</u>equence. Thin arrows represent primers used for PCR verification of the strains: (1) left-end of ROD21 (1,011 bp), (2) DRS (914 bp), (3) right-end of ROD21 (903 bp), (4) insertion of <i>cat</i> gene (1,838 bp), (5) insertion of <i>aph</i> gene (2,469 bp), and (6) left-end of Gifsy-1 (1,110 bp). Below the scheme, agarose gels show PCR products obtained for the recipient and transconjugant <i>S.</i> Typhimurium strains. (<b>B</b>) Schematic representation showing the location of a 50 bp region present in <i>S.</i> Enteritidis, downstream DRS, but absent in <i>S.</i> Typhimurium. The figure below shows the alignments of the theoretical sequence of the above mentioned region for <i>S.</i> Enteritidis PT4 and <i>S.</i> Enteritidis 14028 and the experimental sequences obtained for <i>S.</i> Enteritidis PT1, <i>S.</i> Typhimurium 14028 and 7 transconjugants of <i>S.</i> Typhimurium-ROD21 obtained in a representative transfer assay. (<b>C</b>) Survival of C57BL/6 mice intragastrically infected with 1×10⁵ CFU of <i>S.</i> Typhimurium wild type and <i>S.</i> Typhimurium-ROD21 strain. Data shown are the averages of three independent experiments, which included 4 mice per group. ***, P<0.05 Kaplan-Meier and Mantel-Cox post test.</p

Excision of ROD21 in different conditions.

<p>Donor <i>S.</i> Enteritidis strain was grown in LB medium and (<b>A</b>) LB medium supplemented with hydrogen peroxide, (<b>B</b>) lower pH (pH 5), and (<b>C</b>) lower and higher temperatures (23°C and 43°C respectively) in order to evaluate excision of ROD21. The frequency of <i>attB1</i> excision was quantified by qPCR using genomic DNA and was expressed as a relative value equal to the ratio between the copy number of <i>attB1</i> over the copy number of <i>rpoD</i> gene. Error bars represent standard deviation from the mean (N = 3). ***, P<0.001, **, P = 0.016 (one-way ANOVA and Tukey's post-test).</p

ROD21 transfer requires excision.

<p>Transfer frequency of ROD21 from donor <i>S.</i> Enteritidis, from <i>S.</i> Enteritidis ΔDRSΔSEN1970, from <i>S.</i> Enteritidis ΔSEN1980 and from <i>S.</i> Enteritidis/pBAD-SEN1998 supplemented with arabinose to (<b>A</b>) recipient <i>S.</i> Enteritidis and (<b>B</b>) recipient <i>S.</i> Typhimurium. Graph shows the frequency of ROD21 transfer, expressed as the ratio between transconjugant bacteria and the total amount of donors calculated in each assay. Results are the average of four independent experiments (Y axis are expressed as reverse of log values). ***, P<0.001, Student's <i>t</i> test. ND = Non-detected.</p

Transfer of ROD21 to <i>S.</i> Enteritidis and <i>S.</i> Typhimurium in different environmental conditions.

<p>Strains were subjected to transfer assays in LB medium and (<b>A</b>) LB medium supplemented with hydrogen peroxide, (<b>B</b>) pH 7 and pH 5, and (<b>C</b>) 23°C and 43°C. Error bars represent standard deviation from the mean (N = 3). NS = non-significant, Student t-Test (Two tailed). ***, p<0.001 one-way ANOVA and Tukey post-test.</p

Primers and probes used in this study.

<p>The location of primers pairs from 1 to 6 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090626#pone-0090626-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090626#pone-0090626-g002" target="_blank">2</a>. Lowercase indicate the region that hybridizes to the 5′ or 3′ end of the pKD3 or pKD4 plasmids.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

ROD21 excision frequency increases when <i>S.</i> Enteritidis infects phagocytic cells.

<p>Bone marrow-derived DCs (<b>A</b>) and J774.3 macrophages (<b>B</b>) were infected with <i>S.</i> Enteritidis (MOI equal to 25). After 2, 18 and 24 h post infection (hpi) intracellular bacteria were recovered and the copy number of the <i>attB</i> sequence generated by type 1 excision was detected by quantitative PCR, using as template the genomic DNA obtained from intracellular bacteria. Frequency of excision is expressed as the ratio between the copy number of the <i>attB-1</i> sequence determined for intracellular and extracellular bacteria. The DNA amount was normalized by calculating the copy number of the <i>rpoD</i> gene. Data shown in graphs are average values of at least three independent experiments. The amount of intracellular bacteria recovered after 2, 18 and 24 hpi from DCs (<b>C</b>) and J774.3 (<b>D</b>) was determined by lysing either 1,000 DCs or 100 J774.3 cells with PBS-triton X100 (0.1%) and seeding the lysates in LB plates. Data shown are the average of at least 3 independent experiments.</p

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.</p

ROD21 excision can be generated by means of two different recombination events.

<p>Amplification of <i>attB</i> and <i>attP</i> sequences generated after type 1 and type 2 excisions were detected by nested PCR in LK5, PT4, PT1 and PT21 strains of <i>S.</i> Enteritidis, using primer pairs described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026031#pone-0026031-t002" target="_blank">Table 2</a> and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026031#pone-0026031-g002" target="_blank">Figure 2</a>. PCR products for <i>attB</i> (<b>A</b>) and <i>attP</i> (<b>B</b>) sequences for each type of excision were resolved in 1% agarose gels. The sequence of each PCR product was obtained (chromatograms in each Figure) and compared with the <i>attB</i> and <i>attP</i> sequences deduced for type 1 and type 2 excisions (labeled as theoretical). <i>attB</i> and <i>attP</i> sequences are highlighted in red in both alignments and chromatograms. Expected size for each PCR product: 591 bp for type 1 excision <i>attB</i>, 657 bp for type 2 excision <i>attB</i>, 995 bp for type 1 excision <i>attP</i> and 1050 bp for type 2 excision <i>attP</i>.</p