Fig. 5: Spatial organization and triggered stress responses in biofilms.

a Schematic illustration of the flow channel used for the cultivation and real-time imaging of a living RGB-S E. coli biofilm grown under continuous flow of 10 µl/min LB+kan. The “centre” and “edge” positions are indicated. b A mature biofilm grown for 96 h mainly shows a low heterogenic response caused by random occurrence of individual stressed cells (upper row). Near the edge of the flow channel (lower row), GFP-expressing cells dominate the top of the biofilm whereas RFP-expressing cells accumulate at the bottom. Scale bar is 100 µm for top and bottom views, and 25 µm for side views. c After growing the biofilm for 96 h, only a basal background signal can be observed as detected at centre positions (no stress, I) indicating the normal state of the biofilm without particular stress. After treatment with nalidixic acid for another 24 h (NA, II), the cells in the biofilm displayed an elevated GFP signal (genotoxicity). Subsequent administration of glyphosate (Gly, III) leads to decreased GFP response with concomitant occurrence of an elevated RFP (physiological stress) response. Further treatment with methanol (MeOH, IV) leads to induction of strong BFP signals (cytotoxicity). Scale bar is 100 µm. Quantitative red, green and blue bars (on the right) represent the intensity average of RFP, GFP and BFP, respectively, acquired from nine 100 µm² regions. (*) indicate statistical significance of response means (p ≤ 0.05), evaluated by One-way ANOVA for parametric groups, and Kruskal-Wallis Test for nonparametric groups. Error bars represent the standard deviation (SD). Additional images of different centre positions are shown in Supplementary Fig. 13. d Internal organization of the three stress responses inside the final MeOH treated biofilm, imaged at a centre position. The biofilm displays a blue fluorescence with a second internal red fluorescent layer and a final inner thin SOS layer. 3D scale bar is 25 µm.