PknG senses amino acid availability to control metabolism and virulence of Mycobacterium tuberculosis
Fig 5
GarA phosphorylation in M. smegmatis was regulated by nutrient availability.
The reporter strain of M. smegmatis was cultured in different media and cell lysates analysed by Western blot and densitometry. (A) Glutamate and related amino acids triggered phosphorylation of GarA: the nitrogen source is indicated and the carbon source was glucose. (B) The supplied carbon source affected phosphorylation of GarA: the carbon source is indicated and the nitrogen source was NH4Cl. (C) Phosphorylation of GarA occurred rapidly when cells were cultured in poor medium and then given supplementary nutrients (initially 1% glucose with 10 mM NH4Cl and 0.05% tyloxapol, then 1% v/v glycerol and 30 mM asparagine were added at time zero). (D) Dephosphorylation of GarA occurred slowly when cells switched from rich to poor medium (initially 1% glycerol with 30 mM asparagine and 0.05% Tween 80 then switched to 1% glucose with 10 mM NH4Cl and 0.05% tyloxapol). (E) GarA was predominantly unphosphorylated when M. smegmatis were in stationary phase or starved in PBS. The reporter strain of M. smegmatis was grown in Sauton’s medium with shaking for 5 days. For the starvation experiment exponentially growing M. smegmatis were washed with PBS and incubated in PBS with 0.05% tyloxapol for 5 days. Values represent mean and standard deviation of at least three independent replicates.