Fig. 4
From: Two-step chromosome segregation in the stalked budding bacterium Hyphomonas neptunium
Effect of ParA depletion on chromosome partitioning. a Immunoblot showing the levels of ParA-Venus in strain AJ46 (ΔparA PZn::PZn-parA-venus) under inducing and non-inducing conditions. Cells were grown in MB medium containing inducer (0.3 mM ZnSO4), washed, shifted to ZnSO4-free medium, and cultivated for another 45 h to deplete ParA-Venus. Subsequently, cells were transferred to MB medium containing 0.5 mM ZnSO4 and cultivated for 24 h to re-induce the synthesis of the fusion protein. Samples were subjected to immunoblot analysis with anti-GFP antibodies. A full scan of the immunoblot is shown in Supplementary Fig. 9A. b Microscopic analysis of cells depleted of ParA. Cells of strain AJ46 (ΔparA PZn::PZn-parA-venus) were grown in MB medium with or without 0.3 mM ZnSO4 and incubated for 20 min with DAPI to stain the chromosomal DNA prior to imaging. Shown are overlays of DIC and fluorescence micrographs. The asterisk denotes an anucleate cell. Bars: 5 µm. c Box plots showing the length distributions of budding and amorphous cells from the cultures described in (b). The horizontal line indicates the median, the box the interquartile range, and the whiskers the 5th and 95th percentiles. The number of cells analyzed is given underneath the boxes. Wild-type cells grown in MB medium were analyzed as a control. Source data are provided as a Source Data file. d Demographic analysis of the distribution of chromosomal DNA in budding cells from the cultures described in (b). The fluorescence intensity profiles of random subpopulations of cells were stacked on top of each other according to cell length, with the mother cell body positioned to the left and the bud positioned to the right. e Flow cytometric analysis of the chromosome content of ParA-depleted cells. Strain AJ46 (ΔparA PZn::PZn-parA-venus) was grown in MB medium without or with 0.3 mM ZnSO4. After staining of the chromosomal DNA with Vybrant DyeCycle Orange for 25 min, cells were subjected to flow cytometry. A wild-type culture grown in MB medium was analyzed as a control
