The genomic context of <i>parS</i>_<i>Bb</i> plays a role in ParB_Bb localization.

(A) Changing the native context of the parSBb enables ParBBb binding. Left: representative phase contrast and fluorescence images of AP cells of WT B. bacteriovorus strain constitutively producing ParBBb-msfGFP from a plasmid carrying a copy of the parSBb sequence (GL1751). Right: histogram representing the percentage of cells with zero, one, or two ParBBb-msfGFP foci in the same strain. (B-C) Short chromosomal regions around each parSBb site are enough to prevent ParBBb from clustering on the centromere. Top: schematic representation of the parAB operon in B. bacteriovorus and the upstream parS sequences; chromosomal regions around parSBb sequences (“extended parS”) cloned in the plasmids are indicated; plasmid schematics are shown on the right. Bottom: representative phase contrast and fluorescence images of AP cells of WT strain constitutively producing ParBBb-msfGFP from a plasmid carrying parS1extended (B, GL1749) or parS2extended (C, GL1925); histogram representing the percentage of cells with zero, one, or two ParBBb-msfGFP foci in the same strain. Scale bars are 1 μm. (D) The genomic context of the parSBb prevents ParBBb focus formation only in B. bacteriovorus. Top: representative phase contrast and fluorescence images of E. coli strains carrying plasmids as in Figs 3D, 5A–5C, constitutively producing ParBBb-msfGFP in the absence of parS (GL1661), or in the presence of parSBb (GL1669), parS1extended (GL1737) or parS2extended (GL1899), respectively. Schematics illustrate the parBBb–msfgfp expression plasmid with or without (extended) parS sequences. Bottom: histogram representing the percentage of cells with zero, one, or two ParBBb-msfGFP foci in the same strains. The scale bar is 1 μm. n indicates the number of cells analyzed in a representative experiment; all experiments were performed at least twice. See also S5 Fig.