Biphasic expression of the <i>parA</i>_<i>Bb</i> and <i>parB</i>_<i>Bb</i> genes and proper balancing of <i>parAB</i>_<i>Bb</i> expression contributes to progressive <i>ori</i> segregation.

Related to Fig 2. (A) Expression of the parABb and parBBb genes, individually, follow the same biphasic pattern as the fragment covering both genes. RT-PCR experiment as in Fig 2A using primer pairs hybridizing within the parABb (left) or the parBBb gene (right). (B) Overexpression of parABb leads to chromosome segregation defects. B. bacteriovorus strain parBBb::parBBb-mcherry / pTNV215-parABb (GL2129) was mixed with prey and imaged in time-lapse after 120 min with 8-min intervals. Left: phase contrast and fluorescence images of selected time points; arrowhead points to an altered ParBBb-mCherry behavior (patches instead of well-separated foci) during the cell cycle. (C) Overexpression of parBBb or parABb leads to pronounced phenotypes, largely rescued by overexpression of both. Violin plots of cell length, cell area, and nucleoid area distributions in WT / pTNV215-parBBb (parBBb ++, GL1261); WT / pTNV215-parABb (parABb ++, GL1460); WT / pTNV215-parBBb-parABb (parABBb ++, GL1004) and WT B. bacteriovorus from cells in Fig 2B. 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. (D) Overexpression of both parABb and parBBb has no obvious effect on ori segregation. B. bacteriovorus strain WT / pTNV215-parBBb-mcherrystop-RBS-parABb-msfgfp (GL1004) was mixed with prey and imaged in time-lapse after 75 min with 8-min intervals. Left: phase contrast and fluorescence images of selected time points; arrowheads point to well-distributed ParBBb-mCherry foci (which mark chromosomal ori) during the cell cycle; ParABb-msfGFP not shown for simplicity. All experiments were performed at least twice. Scale bars are 1 μm. (TIF)