Assessment of the autophosphorylation and autokinase activity of the sensor kinase MtrB carrying M517L mutation.

A. Autophosphorylation time-course analysis for the mutated and wild-type MtrB SK protein. M, Marker. B. Quantitative measurement of phosphorylation of the WT and mutant MtrB proteins at various time points (as shown in Fig 2A). The signal recorded for both proteins at the first time point was taken as 1 and the later time points were normalized to it (n = 3). C. Phosphotransfer time course to analyze the effect of the mutation on the phosphotransfer efficiency to the RR MtrA. The mutated or the wild-type MtrB protein were incubated with the RR MtrA (after autophosphorylation) in the phosphotransfer assay. M, marker. D and E. Quantitation of phosphorylated SK MtrB~P and RR MtrA~P protein at various time points (generated through wild type or and mutant MtrB as shown in Fig 2C). The signal recorded by the phosphotransfer of the SK at the first time point was taken as 100%, and the subsequent time points were normalized to it (n = 3) (p values; *≤ 0.05, **≤ 0.01).