Fig. 5.

Tryptophan biosynthesis enzymes are significantly increased in RM33 biofilms. a The enzymatic pathway for synthesis of tryptophan was adapted from KEGG. The concentration of amino acids within the biofilms of 86-028NP (circle) and RM33 (square) and secreted indole were quantitated from three independent biological replicates as described in the “Methods” section. The TnaB importer of tryptophan from the periplasm (pp) to the cytoplasm (cyto) was not significantly different between the two strains. Statistical significance was determined using a two-tailed unpaired Student’s t test (*p = 0.03; **p = 0.007; ***p < 0.0001). b Representative image of a biofilm of the parent (86-028NP) grown for 48 h and stained with LIVE/DEAD to visualize DNA stain with orthogonal views of the three-dimensional rendering of a 26.4 µm biofilm. c Representative image of a biofilm of 86-028NP trpBA mutant grown for 48 h and stained with LIVE/DEAD to visualize bacteria with orthogonal views of the three-dimensional rendering of a 6 µm biofilm. d Representative image of a biofilm of 86-028NP trpBA tnaAB mutant grown for 48 h and stained with LIVE/DEAD to visualize bacteria with orthogonal views of the three-dimensional rendering of a 6 µm biofilm. e Quantitative assessment of the height of biofilms formed by the parental strain (86-028 NP), 86-028NP trpBA, or 86-028NP trpBA tnaAB. The height of the biofilm was measured at ten random locations on three independent occasions (n = 30 per strain). Each point represents an individual measurement. Statistical significance was determined by a two-tailed unpaired Student’s t test. f The culture supernatant from three biofilms grown for 48 h on three independent occasions were used to quantify the level of indole as described in the “Methods” section. The dashed horizontal line indicates the limit of detection. The mean and s.e.m. are reported. Statistical significance was determined using a two-tailed unpaired Student’s t test. Scale bar indicates 25 μm