The levels of ParB_Bb and ParA_Bb vary differently during the cell cycle, and ParB_Bb is unable to form foci when overproduced.

Related to Fig 3. (A) The anti-ParBBb antibody detects overproduced (right) but not native levels (right) of untagged ParBBb in AP; control experiment for Fig 3A. Western blots of whole-cell protein extracts from AP cells of WT B. bacteriovorus and a strain constitutively producing the untagged ParBBb (GL1261) were probed with an anti-ParBBb antibody represented in the schematics on the left. (B) Same as in Fig 3B for the strain natively producing ParBBb-msfGFP (GL1654). (C) The protein levels of natively produced ParBBb-msfGFP or ParBBb-mCherry detected with the same anti-ParBBb antibody compare with the endogenous untagged ParBBb profile during a synchronized B. bacteriovorus cell cycle. Western blots of whole-cell protein extracts from B. bacteriovorus strains parBBb::parBBb-msfgfp (GL1654) and parBBb::parBBb-mcherry (GL906) were probed with an anti-ParBBb antibody, as represented in the schematics. Protein samples are isolated at time points throughout the predatory cell cycle: AP, 1h, 2h, 3h, and 4h after mixing with prey. Arrowhead indicates detected ParBBb protein during the growth phase. Ponceau staining of the same membranes (where bands were most visible, ~30-50 kDa) is illustrated below each blot as a loading control. Molecular weight markers (kDa) are shown on the side. (D) ParABb protein is detected in the AP. Western blots of whole-cell protein extracts from Caulobacter crescentus (positive control), E. coli MG1655 (negative control), and WT B. bacteriovorus (in triplicates), were probed with an anti-ParACc antibody, as represented in the schematics on the left. Untagged ParABb (~29 kDa) is detected in the AP in all three samples. Ponceau staining of the same membranes (where bands were most visible, ~30-50 kDa) is illustrated below each blot as a loading control. Molecular weight markers (kDa) are shown on the side. (E) Same as in Fig 3B for the strain natively producing ParABb-msfGFP (GL2134). (F) Mean msfGFP fluorescence measured for AP cells of WT, parBBb::parBBb-msfgfp (parBBb-msfgfp, GL1654) and WT / pTNV215-parBBb-msfgfp (parBBb-msfgfp++, GL1003). n indicate the number of cells analyzed in a representative experiment; mean values are represented. Error bars indicate standard deviations. (G) Western blots of whole-cell protein extracts from AP cells of WT B. bacteriovorus parBBb::parBBb-msfgfp (parBBb-msfgfp, GL1654, left) and WT / pTNV215-parBBb-msfgfp (parBBb-msfgfp++, GL1003, right) strains were probed with an anti-msfGFP antibody, represented in the schematics on the left. Arrowhead points to the ParBBb-msfGFP protein detected in AP when overproduced. (H) Overproduction of ParBBb-msfGFP leads to morphological and chromosome segregation defects. Violin plots of cell length and nucleoid area distributions for the cells in Fig 3E 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. (TIF)