(A) Visualisation of condensation of a single DNA molecule induced by 250 nM ParBAF binding. The volume of the bead causes the DNA to be slightly tilted with respect to the surface such that emission of fluorescence is limited by the penetration depth of the excitation field. The DNA does not condense while held at non-permissive force of 1 pN. However, as in conventional magnetic tweezers experiments, at a permissive force of 0.2 pN a progressive reduction of the DNA length is observed. (B) Condensation experiment as in Figure 5A but in the presence of 2.5 μM non-fluorescent CTD showing that at a constant force of 0.2 pN, ParB is not able to condense to the inhibition of the CTD. Co-incubation with the CTD does not inhibit ParB binding to DNA, as indicated by clearly visible fluorescence filaments. (C) Condensation experiment as in Figure 5B. The CTD inhibits ParB-dependent DNA condensation, even at forces as low as 0.07 pN. Note that some residual condensation was observed at very low forces, visible at the anchoring point (arrow) as shown by the bright dot. (D) Percentage of DNA molecules that were condensed (more than 90% extension reduction), partially condensed or not condensed by ParBAF (less than 10% extension reduction), in the presence (n = 47) or absence of an excess of the CTD (n = 36). (E) Integrated fluorescence intensity in a laminar flow experiment. Intensity remains constant throughout the cycles of ParBAF and ParBAF + CTD (shadowed area), indicating that the excess of CTD is not competing for the binding sites on the DNA, but rather ‘capping’ the DNA-bound ParBAF. Intensity changes when alternating ParBAF with a combination of ParBAF and unlabelled ParB (shadowed areas). (F) Integrated intensity of ParBAF plotted versus the integrated intensity of ParBAF + CTD or ParBAF + ParB. ParBAF + CTD shows a slope of 1.00 ± 0.02 corresponding to unchanged intensity, while ParBAF + ParB shows a slope of 1.23 ± 0.02. (n ~ 10–16 molecules). All experiments were conducted in ParB reaction buffer supplemented with 4 mM Mg2+.