PknG senses amino acid availability to control metabolism and virulence of Mycobacterium tuberculosis
Fig 4
Investigation of GarA phosphorylation in M. smegmatis and M. tuberculosis.
(A) Addition of Phos-tag reagent to SDS-PAGE allowed separation of phosphorylated GarA (GarA-P) from GarA. Cell extracts were prepared from M. smegmatis and M. tuberculosis wild type and pknG deletion strains and complemented strains. (B) The same cell extracts of M. tuberculosis wild type and pknG deletion strain were analysed by LC-MS/MS to detect the tryptic peptide of GarA carrying the ETTS phosphorylation motif. The abundance of the peptide with no phosphorylation is shown in black and the T21-phosphorylated peptide is shown in red. A peptide from another part of the protein was also measured as a control (blue). (C) A reporter strain of ΔgarAMs carrying plasmids encoding hexahistidine-tagged garA, or variants of garA, confirmed that most phosphorylation occurred at the first threonine in the ETTS motif. GarA from cell lysates was visualised by Western blotting. Images shown are representative of three or more independent replicates.