Fig 4.

Genetic requirements for excision and transfer of MGIVflInd1. Mobilization assays of MGIVflInd1::aph or its Δcds4, Δcds8, or Δcds9 mutants by ICEVflInd1 were carried out using E. coli CAG18439 containing ICEVflInd1 and MGIVflInd1 mutants as donors. When indicated, the donor expressed cds9, cds8, or cds9 and cds8 from p9, p8, or p9-8, respectively. E. coli VB112 (Rf^r) was used as the recipient strain. The frequency of exconjugant formation was calculated by dividing the number of exconjugants (Rf^r Kn^r CFU) by the number of donors (Tc^r CFU). Real-time quantitative PCR was used to determine the percentage of unoccupied attB sites resulting from the circularization of MGIVflInd1 or of its Δcds9 mutant in E. coli CAG18439 harboring ICEVflInd1, p9, p8, or p9-8. The bars indicate the mean values and standard deviations obtained from three independent experiments. ND, not determined. Asterisks indicate that the frequency of exconjugant formation or the percentage of attB sites was below the limit of detection of the assays (<1 × 10⁻⁸ or 0.0004%, respectively).