A high-frequency single nucleotide polymorphism in the MtrB sensor kinase in clinical strains of Mycobacterium tuberculosis alters its biochemical and physiological properties
Fig 3
Effect of the mutation on the phosphatase activity of the SK on RR~P and its DNA binding ability.
A. Dephosphorylation analysis of MtrA~P through wild type or mutant MrB. MtrA~P was generated as described in the Methods section and incubated with the wild type or mutant MtrB for the indicated time points. M, marker. B. Quantitative measurement of the amount of MtrA~P remaining at the indicated time points is shown in Fig 3A. The signal recorded with MtrA~P in the absence of SK was taken as 100, and the other time points were normalized to it to calculate the percentage of phosphorylated RR (n = 3). C. Titration of MtrA RR protein concentration to determine the minimal concentration of unphosphorylated MtrA which can bind to the oriC. Labeled DNA were incubated with increasing concentration of MtrA protein (as indicated) and tested for mobility shift of the probe by EMSA. The RR KdpE was used as a negative control to evaluate nonspecific binding (lane 2). D. A comparative analysis of MtrA binding to oriC DNA as a function of phosphorylation through wild-type or mutant MtrB’ SK proteins used at different concentrations. For all experiments, n = 3 biological replicates and a representative image is shown.