Fig. 5: G-quadruplex DNA observed in P. aeruginosa biofilms and its loss coincides with the disappearance of matrix fibres.

Confocal micrographs of 3-day P. aeruginosa rugose small colony variant (RSCV) pellicle biofilm showing the presence of eDNA fibres, visualised by binding to DNA-specific dye propidium iodide (PI; red) (A), the binding of anti-DNA G-quadruplex structure antibody to the eDNA fibres, visualised with GFP-labelled goat anti-mouse IgG (green) (B) and overlapping bindings of both PI and anti-DNA G-quadruplex antibody (C). The zoomed-in insets from the three images show that the anti-DNA G-quadruplex antibody binds to eDNA fibres. The horizontal line denotes the region used to describe the colocalisation of PI and G-quadruplex antibodies in terms of channel pixel intensity as a function of location, where PI and G-quadruplex colocalise at the sixth pixel of the line (Supplementary Figure 9). Confocal micrograph of the RSCV pellicle biofilm with pre-heating to 60 °C (D) and pre-heating to 70 °C (E), with PI and anti-DNA G-quadruplex antibody with GFP-labelled goat anti-mouse IgG (green) overlapping, showing almost complete disappearance of G-quadruplex structures by 60 °C, and partial and complete disappearance of eDNA fibres at 60 and 70 °C, respectively. Confocal micrograph of DNase-treated 3-day P. aeruginosa rugose small colony variant pellicle biofilm overlapping (PI) and anti-DNA G-quadruplex structures’ antibody with GFP-labelled goat anti-mouse IgG (green), showing that the eDNA fibres disappear coincident with the loss of anti-DNA G-quadruplex antibody binding (F). Scale bars represent 10 µm.