Fig. 6

Arginine 179 of DppA is essential for heme binding and utilization by M. tuberculosis. a Purification of DppA_R179A from E. coli. Lanes: (1) E. coli lysate containing mbp_6his-dppA_R179A expression vector, combined fractions after Ni(II)-affinity (2) and amylose affinity (3), (4) purified DppA_R179A post TEV cleavage after Ni(II) recapture of MBP_6His. b Difference absorption spectroscopy of DppA_wt and DppA_R179A. The free heme spectra were subtracted from the heme-incubated protein spectra at protein concentrations of 10 µM. Growth of avirulent Mtb mc²6206 (circles), the Δdpp mutant ML2436 (triangles), ML2436 complemented with wt dpp operon genes (squares) and ML2436 complemented with dpp operon genes but expressing dppA_R179A (diamonds). Strains were grown in HdB minimal medium with 2.5 µM human hemoglobin (c) or 10 µM hemin (d). Medium with hemin and hemoglobin contained 20 µM of 2,2′-dipyridyl to prevent utilization of trace ferric iron. Error bars represent standard errors of mean values of biological triplicates. Please note that the data in Fig. 5c, d for wt (circles), ML2436 (triangles), and ML2436 complemented with wt dpp operon genes (squares) are the same as in Fig. 1b. Growth experiments for all strains were performed at the same time. Source data are provided in the Source Data file