Fig. 1.

The M. tuberculosis Pup-proteasome system (PPS) is required for growth in nitrate. (A) The PPS does not promote survival of M. tuberculosis during complete nitrogen starvation. Survival of M. tuberculosis wild-type (WT), mpa (MHD149), and prcBA strains was measured by number of colony-forming units (CFU) per milliliter of culture at the indicated time points. At week 3, the fold change in CFU from input was determined to be statistically insignificant (one-way ANOVA, P > 0.05) for mpa and prcBA strains compared with the WT strain. Experiment represents data from six replicate cultures. (B) The PPS is not essential for growth of M. tuberculosis in ideal nitrogen sources. Growth of M. tuberculosis strains in Proskauer–Beck (PB) minimal media supplemented with single nitrogen sources asparagine (PB-Asn) or glutamate (PB-Glu) was measured by optical density at 580 nm (OD₅₈₀). (C) An intact PPS is essential for M. tuberculosis growth when provided nitrate as the sole nitrogen source. M. tuberculosis strains were grown in PB supplemented with arginine (PB-Arg), nitrate (PB-nitrate), or ammonium (PB-ammonium). For each condition in B and C, the growth defect of the mpa mutant compared with the WT strain was statistically significant (one-way ANOVA, P < 0.01). (D) Complementation of the mpa mutant growth defect in PB-nitrate. (E) Pupylation and proteasomal degradation are required for M. tuberculosis nitrate utilization, as assessed by the growth of pafA (MHD2), mpa (MHD5), and prcBA strains in PB-nitrate. (F) Schematic of the M. tuberculosis enzymes that catalyze reduction of nitrate to ammonium (33, 34). (G) PPS mutants (as in E) secrete excess nitrite into culture supernatants during growth in PB-nitrate. Statistical significance was determined using one-way ANOVA; ***P < 0.001. Experiments in B–E and G each contain data from three replicate cultures.