Genetic determinants of R391 stability.

<p>(A) Quantification of R391 integration/excision and replication. The DNA targets (<i>attP</i>, <i>attB</i>, <i>int</i> and <i>prfC</i>) measured by real-time quantitative PCR are shown by black bars. (B) Schematic representation of R391 and R391K structures. The position and orientation of open reading frames (ORFs) are indicated by arrowed boxes. The colors depict the function deduced from functional analyses and BLAST comparisons: dark blue, toxin/antitoxin system; black, site-specific recombination; light blue, active partitioning system; yellow, DNA processing; orange; mating pair formation; light grey, mercury resistance (Hg^R); green, transcriptional activators; red, transcriptional repressor; white, other or unknown functions. The putative centromere-like region (<i>srpC</i>) and the origin of transfer (<i>oriT</i>) are depicted by a light blue rectangle and a yellow circle, respectively. <i>hipAB</i> and the mercury resistance genes are not conserved features of SXT/R391 ICEs. To construct R391K, the reporter gene <i>galK</i> (in magenta) under the control of the IPTG-inducible <i>P</i>_<i>lac</i> promoter was inserted between <i>traG</i> and <i>merR</i>, the first gene of the mercury resistance operon. (C) Colony color-based assay for the detection of R391K-containing colonies on MacConkey D-galactose indicator agar. Colonies bearing <i>galK</i>-tagged R391 (R391K) appeared red on the indicator medium (R391K⁺) while white colonies lacked the ICE (R391K^-). Sectored colonies forming during growth on agar plates are indicative of high instability of R391K mutants.