(A) Growth of WT M. smegmatis (MGM8007), pyc::tn (MGM8008) bioA::tn (MGM8009) and bioB::tn (MGM8010), and pyc::tn complemented with MSMEG_2412 (MGM8011), MSMEG_2413 (MGM8012), or MSMEG_2414 (MGM8013) on biotin containing (left) or biotin free (right) media. Rows are ten-fold serial dilutions. EV=empty vector. (B-D) Growth of WT (B), bioA::tn (C), and pyc::tn (D) in biotin-replete medium (500 ng/mL, dark blue), medium with decreasing concentrations of biotin (50 ng/mL: light blue; 5 ng/mL: green; 0.5 ng/mL: yellow), and biotin-deficient medium (orange). Each line is the mean of three biological replicates. (E) Survival during biotin depletion. WT (circles), bioA::tn (squares), and pyc::tn (triangles) were grown in medium containing (solid lines) or lacking biotin (dashed lines), and viable CFU was assessed by culturing serial dilutions on agar media. Error bars are standard deviations of three biological replicates.