BT3563 degrades nucleic acids in the extracellular matrix of bile-dependent biofilms. (A) Incubation of B. thetaiotaomicron genomic DNA (23 ng/µL final concentration) with the supernatant of 48-h bile-free cultures, as indicated, and loaded immediately on a 1% agarose gel (t = 0 h) and after an overnight incubation at 37 °C (t = O/N). A control using DNase I rather than supernatant is shown in the last lane. pE, empty vector; p3563, vector constitutively expressing BT3563. (B) A 96-well plate crystal violet biofilm assay after 48-h growth in BHIS + 0.5% bile extract, with or without DNase I. Mean of WT in BHIS is adjusted to 100% (not represented). Min-max boxplot of 9 to 21 biological replicates for each strain, each replicate is the mean of two technical replicates. **P value <0.005, Wilcoxon matched-pairs signed rank test. (C) Extracellular DNA concentration (ng/µL) in the purified extracellular matrix of bile-dependent biofilms grown in BHIS + 0.5% bile extract for 48 h. Min-max boxplot of 6 biological replicates for each strain. **P value <0.005, Mann–Whitney U test. pE, empty vector; p3563, vector constitutively expressing BT3563.