Fig. 3: Allosteric variants dysregulate L-histidine production in M. tuberculosis.
A Western blotting with anti-ATP-PRT serum showing a lack of changes in protein levels. Lane 1 molecular weight marker, lane 2 H37Rv, lane 3 D216V, lane 4 T248’V and lane 5 A249K. Anti-antigen 85 serum was used as a loading control. The black dot shows the position of the monomeric ATP-PRT protein (MW = 30.5 kDa). B Growth kinetics of wild-type M. tuberculosis H37Rv and the D216V, T248’V and A249K strains in Middlebrook 7H9 complete medium at 37 °C. The data show is from two biological replicates and is representative of three independent experiments. C Growth kinetics of H37Rv and the A249K strains in Middlebrook 7H9 medium with increasing amounts of glycerol (0.2, 0.4 and 0.6 %). Final biomass for each strain was lower than for parent strain, however, due to clumping, the data obtained was not plotted. D Overproduction of l-histidine in the mutant strains D216V, T248’V and A249K, compared with H37Rv. E Relative changes (mutant/wild-type strain) in the pool size of proteinogenic amino acids and other key metabolites in the D216V, T248’V and A249K strains. No Asn was detected in metabolomics experiments consistent with the knowledge that M. tuberculosis does not synthesize this amino acid in free form. The abundance of Ser is not reported as the peak for this metabolite overlapped with a contaminant. Metabolomic data show is the average ± standard error of three biological replicates and is representative of two independent experiments. F Scheme for the biosynthesis of ergothioneine from l-histidine and the pool size of key intermediates in H37Rv and in the D216V, T248’V and A249K strains. Data shown are representative of the mean, whereas error bars represent standard error SEM, n = 3, representative of at least two independent experiments. P-values are indicated as: * P ≤ 0.05, ** P ≤ 0.001, *** P ≤ 0.0001, **** P ≤ 0.00001.
