Fig. 3: mRNA is detected in eDNA fibres upon mild DNase I pretreatment.

Extracellular lasB mRNA visualisation using the smiFISH method. smiFISH confocal micrographs of five-day P. aeruginosa biofilms showing lasB mRNA smiFISH probes (red), eDNA specific TOTO-1 (green), merged image i.e (red + green) and colocalised region (yellow): a without DNase I treatment showing no affinity of lasB probe for lasB mRNA in the eDNA fibre, b treated with 0.02 mg ml⁻¹ DNase I indicating weak dispersed lasB RNA signal on the eDNA fibre, and c treated with 0.05 mg ml⁻¹ and d 0.1 mg ml⁻¹ of DNase I respectively showing increased lasB RNA signal intensity (yellow; c and d colocalised region panel). Scale bars represent 10 µm. e Three-dimensional (3-D) confocal micrograph of five-day P. aeruginosa biofilm (Z-stack = 5 µm) showing overall spatial distribution of lasB mRNA (red) and their colocalisation (yellow) with TOTO-1 stained eDNA fibre (green) (n = 4). Scale bars represent 10 µm. f Fluorescence intensity quantification (n = 4) of eDNA (green bars) and lasB mRNA (red bars) expression in five-day P. aeruginosa biofilms treated with 0, 0.02, 0.05 and 0.1 mg ml⁻¹ DNase I. g Colocalisation analysis (n = 4) showing Mander’s coefficients for eDNA and lasB mRNA colocalisation in five-day P. aeruginosa PAO1 wildtype biofilms at 0, 0.02, 0.05 and 0.1 mg ml⁻¹ DNase I. h Confocal micrograph of DNase I pre-treated (0.1 mg ml⁻¹) five-day P. aeruginosa biofilms performed using smiFISH probes specific for IpdV (27 primary probes), antB (13 primary probes), and bkDA-2 (8 primary probes) mRNA (red), which colocalise with TOTO-1 (green) stained eDNA fibres (reddish yellow streaks in top panel of merged images). Mander’s coefficients for Ipdv, antB, and bkDA-2 of 16 ± 5%, 11 ± 3%, and 18 ± 6%, respectively (n = 5 images each), were determined based on the overlap of the mRNA signal with TOTO−1 stained (green) eDNA fibres (colocalised region panel at bottom; yellow). Scale bars represent 10 µm. i smiFISH confocal micrograph fluorescence intensity quantification (n = 5) of eDNA (green bars) and highly enriched extracellular mRNA bkDA-2, IpDV and antB (red bars) in five-day P. aeruginosa biofilms. The standard deviation bars indicated in Fig. 3f, g, i are generated based on the mean values calculated from biological triplicates. Relevant source data for Fig. 3f, g and i are provided as a source data file.