Figure 2. TDE1909 synthesizes c-di-AMP.

(A) The genes for TDE1909 and TDE1908 were cloned into pET-45b(+) and transformed into E. coli BL21(DE3) cells. Cells with empty pET-45b(+) served as a negative control. Protein expression was induced with IPTG prior to determining the intracellular levels of c-di-AMP by ELISA (Cayman Chemicals). Data are the average and standard deviation from replicate cultures (n=3) analyzed by ANOVA with Dunnett’s post-hoc test. (B) Recombinant proteins for TDE1909 and TDE1908 were incubated with 150 µM ATP for 1 hr and reactions products were analyzed by RP-HPLC. Purified ATP and c-di-AMP were fractionated as controls to determine elution profiles for each nucleotide. Data shown is representative of triplicate experiments.