Figure 6.

Overexpression of FtsK_c abolishes the requirement for homologous recombination and for the location of dif in DAZ. (A) Plasmid resolution assay in WT (AB1157), RecA⁻ (GR19), FtsK_c⁻ (MA3), RecA⁻ FtsK_c⁻ (GR59), and XerC⁻ (DS8029) cells under conditions of repression (glucose) or induction (0.4% arabinose) of 818 expression. The bands corresponding to the supercoiled substrate and supercoiled replicative product are indicated by the top and bottom black triangle, respectively. Numbers indicate the proportion of supercoiled product to total supercoiled substrate in each lane. (B) dif-Km-dif cassette resolution assay. RecA⁺ (FC235) and RecA⁻ (FC217) cells were transformed with a control expression vector (neg) or with the vector expressing the 818 FtsK derivative. They then were transformed with pFC225, which carries a functional xerC gene. The proportion of kanamycin resistant cells in the cultures (Km^R) was quantitated after overnight growth in the presence of 0.2% arabinose. Results from a typical experiment are shown. (C) Plasmid integration assay. FtsK_c⁺ and FtsK_c⁻ cells carrying a single chromosomal dif site in DAZ or in lacZ (DS941, NL250, DS9041, MA2) were transformed with a control expression vector (neg), with the 818 expression vector or with the fl expression vector. They then were transformed with pFH127, which carries a single dif site. The proportion of cells having integrated this plasmid was quantitated after overnight growth at 30°C in the presence of glucose (white bars) or 0.2% arabinose (gray bars). Shown is the result of at least two independent experiments.