ParA_Bb subcellular localization and interaction with ParB_Bb.

Related to Fig 1. (A) The endogenous ParABb-msfGFP fusion is functional. Violin plots of cell length, cell area, and nucleoid area distributions in parABb::parABb-msfgfp (GL2134) and WT B. bacteriovorus strains, measured from cells in Fig 1E. 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. (B) ParABb is not oriented towards any cell pole in AP cells. Left to right: representative phase contrast and fluorescence images of AP cells of parABb::parABb-msfgfp romR::romR-mcherry strain (GL2155); demographs of the corresponding fluorescent signals in the same cells sorted by length and oriented based on RomR-mCherry signal intensity; heatmaps represent relative fluorescence intensities; mean pole-to-pole profiles of relative fluorescence intensity of the corresponding fusions in the same cells. Scale bar is 1 μm. (C) ParABb and ParBBb interact in a bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins fused to the T18 or T25 adenylate cyclase domain were spotted on X-gal agar plates supplemented with IPTG. Interaction between two proteins results in blue colony color. The Zip-Zip interaction serves as a positive control. (D) Schematic representation of the POLAR assay. POLAR takes advantage of PopZ from C. crescentus (grey), which spontaneously forms clusters at the cell poles or septa when produced in E. coli. The unlabeled PopZ is produced along with an msfGFP-H3H4 fusion to the bait protein of choice (here ParBBb), depicted in magenta; H3H4 is a PopZ self-interacting domain (shown in blue) that is sufficient to draw the bait protein to PopZ clusters. The prey protein (ParABb) is depicted in green. The prey protein is recruited to the PopZ-bait cluster if prey and bait proteins interact (upper half of the cell), whereas a lack of interaction leaves the localization of the prey protein unmodified by the bait protein (lower part of the cell). (E) ParABb does not interact with msfGFP-H3H4 (control). Representative fluorescence images of E. coli cells expressing ParABb-mScarlet in the absence (left) or presence (right) of msfGFP-H3H4 without bait fusion. ParABb-mScarlet (expressed from GL2295) displays a nucleoid-bound pattern and is not recruited to the polar PopZ cluster via msfGFP-H3H4; pole-to-pole profiles of relative fluorescence intensity of the corresponding fusions in the one cell. (F) ParABb and ParBBb interact in the POLAR assay. Same as in (E) for cells producing ParABb-mScarlet as prey (green) in the absence (left) or presence (right) of ParBBb-msfGFP as bait fused to msfGFP-H3H4 (magenta). ParABb partially colocalizes with the ParBBb-msfGFP-H3H4 cluster (expressed from GL2294) visible as fluorescent foci (arrowheads); pole-to-pole profiles of relative fluorescence intensity of the corresponding fusions in the same cell. Scale bar is 2 μm. (G) A strain in which both native ParABb and ParBBb are labeled displays morphology and chromosome aspect ratio similar to wild-type. Violin plots of cell length, cell area, and nucleoid area distributions in parABb::parABb-msfgfp parBBb::parBBb-mcherry (GL2154) and WT B. bacteriovorus strains, measured from cells in Fig 1G. The lines indicate the 25, 50, and 75 percent quantiles from bottom to top. Mean and standard deviation 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)