lldD2 homoplastic mutations modulate gene and enzyme activity.

(A) lldD2 expression as measured by qPCR. Gene expression is normalized to that of -18 T>G. Each dot represents average of technical replicates from one of three independent experiments. Error bars indicate standard deviation. P-value indicates results of an ordinary one-way ANOVA test with Dunnett’s multiple comparison correction. (B) LldD2 production as measured by a Renilla luciferase assay. Luminescence normalized to that of the Msm strain expressing the L4 ancestral version of lldD2. Each dot represents average of technical replicates from an independent experiment, error bars represent the standard deviation. P-value indicates results of an ordinary one-way ANOVA test with Dunnett’s multiple comparison correction. Nat^R refers to nourseothricin resistance. (C) Alamar blue assay of clofazimine (CFZ). P-value indicates the results of two-way ANOVA with Dunnett’s multiple test correction. Triplicate replicates shown, error bars represent the standard deviation. (D) Growth curves of the indicated strains. All cultures started at OD₆₀₀ 0.005 at day 0. Triplicate replicates shown, error bars represent the standard deviation. Representative of two independent experiments. (E) Schematic of ¹³C-Lactate metabolic flux assay. Grey box indicates a proposed pathway. Dotted arrow represents the spontaneous conversion of DHAP to methylglyoxal. Dashed arrows represent abbreviated steps. (F) ¹³C-Lactate metabolic flux assay results. Error bars represent the standard deviation of three technical replicates. Dark grey indicates labeled carbon ions, light grey represents total carbon ions.