Fig. 2.

DDR^ON cells and matrix producers are mutually exclusive and spatially separated in the B. subtilis biofilm. a DDR^ON cells and matrix producers are mutually exclusive cell types. Pellicle biofilms by the dual reporter strain (YCN040: P_yneA-gfp, green, DDR reporter; P_tapA-mKate2, red, matrix reporter) were collected after 24, 48, and 72 h of incubation and examined under fluorescent microscopy for DDR (P_yneA-gfp) and matrix gene expression (P_tapA-mKate2). An overlay image of cells expressing the two reporters is shown. Scale bar represents 10 μm. b–d Quantification of DDR^ON and matrix producer cells. Statistic analyses of cells collected at 24, 48, and 72 h from a using MicrobeJ plugin for ImageJ.⁴⁴ Each dot represents a cell, y-axis represents red fluorescent intensity (matrix) while x-axis represents green fluorescent intensity (DDR). The horizontal and vertical dotted lines represent the thresholds used to define DDR^ON and matrix producing cells. Provided data set represents one independent replicate. The values in the upright corner of each panel are the average percentage ratio with standard devation of cells that fell into the corresponding area in the plot, determined from three biological replicates. e DDR^ON cells and matrix producers have distinct spatial localization in the biofilm. Thin-section fluorescent microscopic analysis of a 48-h colony biofilm formed by the dual reporter strain showing spatial localization of DDR^ON cells (P_yneA-gfp) and matrix producers (P_tapA-mKate2). Scale bar represents 1 mm