FIGURE 4.
EgtD is a substrate of multiple M. tuberculosis STPKs. A, in vitro phosphorylation of EgtD by multiple kinases. M. tuberculosis STPKs purified as GST or His fusions were incubated with His-tagged EgtD and [γ-32P]ATP. Samples were separated by SDS-PAGE and stained with Coomassie Blue followed by visualization by autoradiography. Upper bands represent autophosphorylation activity of each kinase (Pkn); lower bands reflect phosphorylated EgtD. B, interaction between EgtD and M. tuberculosis STPKs facilitates the reassembly of complementary fragments F1 and F2 and fragment F3 of murine dihydrofolate reductase and thus confers M. smegmatis resistance to trimethoprim (TMP). Growth was monitored over 4 days on kanamycin/hygromycin plates supplemented with 0 and 10 μg/ml trimethoprim. Control plates without trimethoprim revealed growth of all strains. Positive Control, M. tuberculosis ESAT-6 (F1 and F2) and CFP-10 (F3); Negative Control, EgtD (F1 and F2) with F3 alone. Experiments are shown in duplicates.