The levels of ParB_Bb and ParA_Bb vary differently during the cell cycle.

(A) ParBBb protein levels are cell-cycle dependent. Western blots of whole-cell protein extracts from WT B. bacteriovorus were probed with an anti-ParBBb antibody, as represented in the schematics. WT protein samples were isolated at time points throughout the predatory cell cycle: 1h, 2h, 3h, 4h, 5h after mixing with prey (GP), and AP. All samples were run on the same gel; for enhanced figure clarity, sample positions have been rearranged, as indicated by the dashed line. Graphical illustration of the cell cycle stages corresponding to the indicated time points. Arrowhead points to ParBBb (detected only during GP). Ponceau staining served as a loading control and quantification from full lanes (displayed in S6A Fig) was used to normalize band intensities. Quantifications are shown as the means of independent triplicates. Error bars represent standard deviations. All replicates are shown in S6A Fig. (B) ParBBb-msfGFP fluorescence intensity level drops during cell division while ParABb-msfGFP fluorescence intensity levels remain high. B. bacteriovorus parABb::parABb-msfgfp parBBb::parBBb-msfgfp (GL2154) was mixed with prey and imaged in time-lapse after 75 min with 8-min intervals. Top: overlay of phase contrast and fluorescence images for ParABb-msfGFP and ParBBb-mCherry for selected late time points; Bottom: mean fluorescence intensities of msfGFP (in green) and mCherry (in magenta) plotted over time for the same cell. The time window corresponding to the images displayed on top starts at the dashed line. ParBBb-mCherry fluorescence intensity decreases during this interval, unlike ParABb-msfGFP. (C) ParABb protein levels do not decrease in the attack phase. Western blots of whole-cell protein extracts from B. bacteriovorus parABb::parABb-msfgfp (GL2134) probed with an anti-msfGFP antibody, as represented in the schematics. GL2134 protein samples are isolated at time points throughout the predatory cell cycle: 1h, 2h, 3h, 4h, and 5h after mixing with prey (GP), and AP. All samples were run on the same gel; for enhanced figure clarity, sample positions have been rearranged, as indicated by the dashed line. Graphical illustration of the cell cycle stages corresponding to the indicated time points. Arrowhead points to ParABb-msfGFP (detected also in the AP). Ponceau staining served as a loading control and quantification from full lanes (displayed in S6B Fig) was used to normalize band intensities. Quantifications are shown as the means of independent triplicates. Error bars represent standard deviations. All replicates are shown in S6B Fig. (D) Top: Western blots of whole-cell protein extracts from AP cells of B. bacteriovorus WT, parBBb::parBBb-msfgfp (ParBBb, GL1654) and parABb::parABb-msfgfp (ParABb, GL2134) strains were probed with an anti-msfGFP antibody. Only ParABb-msfGFP protein is detected in AP. Bottom: quantifications are shown as the means of independent triplicates, normalized as in A and C. Error bars represent standard deviations. All replicates and Ponceau staining are shown in S6C Fig. (E) Overproduction of ParBBb-msfGFP does not lead to the parS-bound focus in AP. Left: representative phase contrast and fluorescence images of AP cells of a WT B. bacteriovorus strain constitutively producing ParBBb-msfGFP (GL1003). Right: histogram representing the percentage of cells with zero, one, or two ParBBb-msfGFP foci in the same strain; n indicates the number of cells analyzed in a representative experiment. Scale bars are 1 μm. All experiments were performed at least twice. See also S3 Fig.