Fig. 3: SOS response and RecA responsible for integrating extracellular R-loops into viscoelastic eDNA network of P. aeruginosa biofilm.
From: Stress-induced toxic genomic R-loops support biofilm extracellular matrix formation
a Box plot representation (n = 3) showing normalised relative abundance (%) of upregulated SOS gene recA in RNA extracted from 16 h P. aeruginosa planktonic cells, 1, 2, 3 and 5 d P. aeruginosa biofilm samples, both intracellular and extracellular. b Three-dimensional (3D) confocal micrographs (n = 5) of mature (5 d) P. aeruginosa wildtype and ΔrecA transposon mutant biofilms showing eDNA fibres in top panel stained with TOTO-1(green), and R-loop specific s9.6 antibody in bottom panel (red). All 3D biofilms were imaged with a Z-stack thickness of 10 µm. c Quantification of biofilm biomass of P. aeruginosa wildtype and ΔrecA transposon mutants across days 2–5 using crystal violet assay (n = 3). d Quantification of biofilm biomass of mature P. aeruginosa (n = 3) PAO1 wildtype and ΔrecA transposon mutants after treatment with different concentrations of SOS response inducing chemical methyl methane sulfonate (MMS). e Two-dimensional (2D) confocal micrographs (n = 4) of mature P. aeruginosa wildtype and ΔrecA biofilms showing RecA specific antibody (1 mg/ml; red) across eDNA fibre (green). All scale bars represent 10 µm. n represents the number of biological replicates, with the exception of microscopy images, where it is the number of representative microscopy images collected. For (a), error bars represent mean ± standard deviation (SD) and box plots display the 25th and 75th percentiles (lower and upper edges of the box). Whiskers extend to the minimum and maximum values within 1.5 times the interquartile range. The centre point indicates the mean of the data. For (a), significance was determined using DeSeq2 to identify differentially expressed genes which incorporates two-tailed Wald test with a P  <  0.01 and for (c, d), significance between and within different treatment groups was determined using both one and two factor ANOVA with replication with P  <  0.05. Source data are provided as a source data file.
