FIGURE 2: Expression of colibactin is modulated by L- and D-amino acids. Relative clbB expression was measured by RT-qPCR. CFT073 was grown in M9 minimal media supplemented with amino acids to a final concentration of 1 mM for 5 h. (A) Shows relative expression in the presence of L-amino acids and (B) indicates expression in the presence of D-amino acids. The orange dashed line indicates baseline expression with bars above and below this line representing up and downregulation, respectively. Statistical significance was determined from three biological replicates using an unpaired Student's t-test with, * and ** indicating significance, P < 0.05 and 0.01, respectively. (C) Amino acids significantly affecting clbB expression in CFT073 were tested in Nissle 1917 under the same growth conditions as discussed above. (D) clbBp:gfp reporter activity in MEM-HEPES in the presence and absence of 1 mM D-serine. Bacteria were sampled at 4 h post-inoculation, clbB expression was measured as GFP/OD₆₀₀. Columns represent mean +/- standard error of the means (SEM) with individual experimental observations indicated by data points. Statistical significance was assessed using a paired Student's t-test with *, ***, denoting P < 0.05 and 0.0001, respectively.