Fig. 3: DmxA is recruited to the division site during cytokinesis by the divisome and its function depends on DGC activity.

a Localization of DmxA-mVenus in the presence and absence of cephalexin. The mean ± SD percentage of cells with a DmxA-mVenus cluster (white) or a constriction (pink) is indicated; n = 600 from three biological replicates with each 200 cells. Scale bars, 5 µm. b DmxA-mVenus localization during the cell cycle. Left panel, epifluorescence and phase-contrast images from time-lapse microscopy of a representative cell expressing DmxA-mVenus. Images recorded every 5 min; arrowhead indicates completion of cytokinesis (defined as the first frame at which daughters were clearly separated); right panels, histograms of the timing of cluster appearance relative to the completion of cytokinesis and lifetime of DmxA-mVenus clusters (n = 100 division events from three biological replicates, number of events per replicate in brackets). The first time point after completion of cytokinesis is defined as t = 0 and indicated by a gray vertical bar. Error bar, mean ± SD. c Localization of DmxA-mVenus during FtsZ depletion. Cells were grown in the presence of 10 µM vanillate before the depletion. The percentage of cells with a cluster (white) or a constriction (pink) are indicated (n = 200 from one biological replicate). White arrowheads indicate DmxA-mVenus clusters. Scale bar, 10 µm. d, e Proximity labeling using DmxA-miniTurbo-FLAG or FtsK-miniTurbo-FLAG as baits compared to sfGFP-miniTurbo-FLAG. Volcano plots show proteins enriched by DmxA-miniTurbo-FLAG (d) and FtsK-miniTurbo-FLAG (e). DmxA-miniTurbo-FLAG and FtsK-miniTurbo-FLAG were expressed from the pilA promoter, and sfGFP-miniTurbo-FLAG from the P_van in the presence of 100 µM vanillate added 18 h before the addition of 100 µM biotin and cephalexin for 4 h. Samples from three biological replicates were analysed. X-axis, log₂-fold enrichment in experimental samples compared to sfGFP-miniTurbo-FLAG (negative control) calculated based on normalized intensities. Y-axis, -log₁₀ of p value. Significantly enriched proteins in the experimental samples (log₂ ratio ≥3; p value ≤ 0.01 (−log₁₀ ≥ 2.0) are indicated by the stippled lines. DmxA and FtsK are shown in blue and red, respectively, and other enriched proteins are in yellow. Enriched proteins other than FtsK and DmxA are listed in Tables S1 and S2. p values were calculated using eBayes moderated t-statistics¹⁰⁹ and are listed in the Tables S1 and S2. f, g Analysis of DmxA-mVenus variants. Domain architecture of variants (f) and population-based motility assay for T4P-dependent motility ((g), upper panels) and localization ((g), lower panels). Motility was analysed as in Fig. 2a. In lower panels, the mean ± SD percentage of cells with cluster at mid-cell is indicated. White arrowheads indicate clusters. In (g), the data for WT are the same as in (a), and were included for presentation purposes. For all strains, n = 600 from three biological replicates with each 200 cells. Scale bars, 1 mm (upper panels), 5 μm (lower panels). Source data are provided as a Source Data file.