The binding of ParB_Bb on plasmidic <i>parS</i>_<i>Bb</i> is specific; titration of the free ParB_Bb by <i>parS</i>_<i>Bb</i> rescues the <i>parB</i> overexpression phenotype.

Related to Fig 5. (A) From left to right: representative phase contrast and fluorescence images of AP cells of WT B. bacteriovorus constitutively producing ParBBb-msfGFP from a plasmid carrying a mutated –parSBb (parSBb*, GL1541). Right: histogram representing the percentage of cells with zero, one, or two ParBBb-msfGFP foci in the same strain. (B) Only the cytosolic excess ParBBb is toxic for B. bacteriovorus AP cells. Violin plots of cell length for the cells in Fig 5A–5C and WT B. bacteriovorus. The lines indicate the 25, 50, and 75 percent quantiles from bottom to top. Mean, standard deviation, and coefficient of variation (CV) values are shown on top of the corresponding plot. n indicate the number of cells analyzed in a representative experiment; all experiments were performed at least twice. (C) Diffuse ParBBb-msfGFP signal in AP cells carrying plasmidic parSextended sequences is not due to protein instability. Western blots of whole-cell protein extracts from AP cells of B. bacteriovorus WT / pTNV215-parBBb-msfgfp-parS (GL1750, AP, fluorescent focus), WT / pTNV215-parBBb-msfgfp-parS1extended (GL1749, AP, diffuse signal), WT / pTNV215-parBBb-msfgfp-parS1extended (GL1925, AP, diffuse signal), WT / pTNV215-parBBb-msfgfp (GL1003, AP, diffuse signal) and WT / pTNV215-msfgfp (GL1208, control) strains were probed with an anti-msfGFP antibody, as represented in the schematics on the left. (D-E) ParBBb-msfGFP is able to form foci in the growth phase in strains carrying plasmidic parSextended sequences. Time-course experiment with strains (D) GL1749 (WT / pTNV215-parBBb-msfgfp-parS1extended) and (E) GL1825 (WT / pTNV215-parBBb-msfgfp-parS2extended). Cells were mixed with prey and imaged every hour. Phase contrast and fluorescence images of selected timepoints are shown; the white arrowheads indicate the appearance of ParBBb-msfGFP foci. (TIF)