Fig 4. Acetyl and uridyltransferase activities are independently essential.

(A) and (B) Schematic and cartoon representation of GlmU_Mtb depicting different domains, active site residues and the deletion mutants. (C) GlmU_Mtb and GlmU_Mtb-mutants were purified as described earlier [38] and the 1 μg of the purified proteins were resolved on 10% SDS-PAGE and stained with coomassie. (D) Uridyltransferase (left panel) and acetyltransferase (right panel) activities were carried out as describe in Methods using 0.5 to 20 pmoles of wild type or mutant GlmU_Mtb proteins. Activity was defined as μM product formed / min / pmole of enzyme. Relative activities of the mutants were calculated with respect to the activity of GlmU_Mtb, which was normalized to 100%. The experiment was repeated three times and the error bars indicate s.e.m. (E) Wild type and mutated GlmU_Mtb genes were cloned into pNit vector without any N- or C- terminal tag. pNit-glmU _wt or pNit-glmU _mutant constructs were electroporated into Rv∆glmU, and the WCLs prepared from Rv∆glmU and Rv∆glmU::glmU _mutant cultures were resolved and probed with anti-GlmU and anti-GroEL1 antibodies. Bands corresponding to FLAG-GlmU_Mtb, complemented GlmU_wt/mutant and the deletion fragments of GlmU are indicated. (F) Rv∆glmU and Rv∆glmU::glmU _mutant cultures were seeded at an initial A₆₀₀ of 0.1 and grown for five days in the absence or presence of ATc. The experiment was performed in triplicates and the error bars represent s.e.m.