Fig 2. Acidic pH/high salt concentration-dependent phosphorylation of PhoP requires the presence of PhoR.

(A) Schematic diagram showing construction of PhoR-deleted M. tuberculosis H37Rv (ΔphoR-H37Rv) by ‘mycobacterial recombineering’. (B) We utilized RT-qPCR experiments to compare mRNA level of phoR in the mutant and WT-H37Rv, as described in the Methods. Importantly, expression of phoR remains undetectable in the mutant relative to WT-H37Rv, but is significantly restored in the complemented mutant. As a control, gapdh levels remain comparable in WT-H37Rv, ΔphoR-H37Rv mutant and the complemented mutant strain. The results are derived from average of biological duplicates, each with one technical repeat (**P≤0.01). (C) To probe in vivo phosphorylation, Phos-tag Western blot analysis were performed using cell lysates prepared from ΔphoR-H37Rv, and WT-H37Rv harbouring His-tagged PhoP protein, respectively. The relevant strains were grown under normal or indicated stress conditions, as described in the Methods. Sample detection and data analysis were carried out as mentioned in the legend to Fig 1.