Ancestral and homoplastic mutations in lldD2 augment Mtb fitness in lactate.

(A) Growth curves of Mtb clinical strains from L1, L2, and L4. Strains are grouped for comparison of the lldD2 homoplastic mutation strains to closely related lineage ancestral strains. All cultures started at OD₆₀₀ 0.005 at day 0. Triplicate replicates shown, error bars represent the standard deviation. (B) Quantification of the area under the curves (AUCs) from A. The AUC in 7H12–0.2% L-lactate is normalized to that of 7H9 for a given strain. The AUC is averaged for the V3I and V253M L4 strains and for the two L1 ancestral strains that were paired to -18 G>T. P-value indicates results of an unpaired t-test. (C) and (E) Schematic depicting the construction of recombinant lldD2 strains. (D) and (F) Growth curves of the strains indicated in (C) and (E). Hyg^R, Kan^R, and Zeo^R refer to hygromycin, kanamycin, and zeocin resistance respectively. All cultures started at OD₆₀₀ 0.005 at day 0. Triplicate replicates shown, error bars represent the standard deviation. Representative of two independent experiments.