FIGURE 6: Overexpression of the wildtype allele of cycA provides resistance to DMB during growth with D-alanine. (A) Location of the gain-of-function mutation in strain JE26530. This strain was originally isolated from D-alanine minimal medium supplemented with DMB (0.7 mM). The cycA transcription start site is denoted by + 1 [31]. (B) RT-qPCR of cycA expression in wildtype S. Typhimurium (JE9426) or its DMB resistant derivative (JE26530) grown with D-alanine plus DMB (0.2 mM). Fold change values were calculated by normalizing relative expression values to the lowest cycA⁺ expression. Error bars represent standard deviation of three biological replicates. (C) Growth analysis to study DMB resistance of cells growing with D-alanine. Cells were grown in NCE minimal medium supplemented with D-alanine (20 mM), glucose (0.5 mM) and DMB (0.7 mM). (D) The DMB resistant strain has a higher sensitivity for D-Cycloserine (D-Cyc). Cells were grown in NCE minimal medium supplemented with glycerol (20 mM), with or without D-Cyc (20 μM) as indicated. l-(+)-Arabinose (0.5 mM) was used to induce of plasmid-borne cycA⁺ in all experiments. Strain JE26530 was the DMB resistant strain used in these studies; str. LT2 is an abbreviation of the wildtype strain of S. Typhimurium. Error bars represent standard deviation of three technical replicates and each experiment was completed with three biological replicates, with a representative graph shown.