Fig. 1

ROS accumulation triggers the DNA damage response (DDR) in a subpopulation of cells in a B. subtilis biofilm. a Older biofilms accumulate ROS. B. subtilis NCIB 3610 cells collected from 0, 24, and 48 h biofilm pellicles were treated with a superoxide-specific dye and observed under fluorescent microscopy. Cells with strong superoxide accumulation show a red color. Scale bar represents 10 μm. b There is an increase in ROS accumulation in cells from older biofilms. Quantification of cells from a was performed using ImageJ.⁴⁴ Each dot represents one cell and the y-axis shows fluorescent intensity. The dotted horizontal line indicates the threshold used to define ROS^ON cells (described in Methods). c Older biofilms accumulate DNA damage. B. subtilis YCN036 cells containing the P _yneA -gfp DDR reporter were collected from 24, 48, and 72 h pellicle biofilms and observed under fluorescent microscopy (green cells). Scale bar represents 10 μm. d ROS^ON cells and DDR^ON cells overlap. Same cells applied in c were collected from 48 h pellicle biofilms and stained with superoxide-specific dye (red cells). An overlay image of ROS^ON cells (in red) and DDR^ON cells (in green) is shown. Scale bar represents 10 μm. e ROS negatively impacts B. subtilis biofilm development. In the top panels, the B. subtilis 3610 biofilm pellicles treated with catalase (1 mg ml⁻¹) was more robust than the one without treatment. Images were taken after 24 h of incubation. Bottom panels show that the pellicle biofilms treated with H₂O₂ (0.001%, v/v) or pyocyanin (PCN) (2.5 μg ml⁻¹) are much weaker than without treatment. Images were taken after 48 h of incubation. Scale bar represents 2.5 mm