In vitro analysis of the two-component system MtrB-MtrA from Corynebacterium glutamicum

Abstract

The two-component system MtrBA is involved in the osmostress response of Corynebacterium glutamicum. MtrB was reconstituted in a functionally active form in liposomes and showed autophosphorylation and phosphatase activity. In proteoliposomes, MtrB activity was stimulated by monovalent cations used by many osmosensors for the detection of hypertonicity. Although MtrB was activated by monovalent cations, they lead in vitro to a general stabilization of histidine kinases and do not represent the stimulus for MtrB to sense hyperosmotic stress.

FIG. 1.

Phosphoryl transfer from MtrB to MtrA. Proteoliposomes containing MtrB were mixed with [γ-³³P]ATP for 10 min before MtrA was added to the mixture in a 4:1 ratio of MtrA and MtrB. Samples were taken at the indicated time points after the addition of MtrA. (A) Autoradiogram of the phosphotransfer reaction. (B) Quantification of the phosphorelay. The degree of phosphorylation of MtrB before the addition of MtrA was set at 100%. Since in the presence of MtrA, the shorter degradation fragments of MtrB also undergo the phosphotransfer reaction, all bands of MtrB were used for the quantification of the phosphoryl signal. Circles, phosphorylation of MtrB; triangles, phosphorylation of MtrA. The data are a typical example of results from more than four independent experiments performed with reconstituted MtrB.

FIG. 2.

Dephosphorylation of MtrA. To measure the phosphatase activity of MtrB, phosphorylated MtrA was prepared as described in the legend to Fig. 1. After the separation of MtrA from the proteoliposomes and residual ATP and ADP, phosphorylated MtrA was incubated for 60 min in the presence and absence of MtrB. (A) Influence of the reaction buffer on the dephosphorylation of phosphorylated MtrA. (B) Influence of MtrB-containing IMV on the dephosphorylation of phosphorylated MtrA. (C) Dephosphorylation of phosphorylated MtrA in the presence of MtrB-containing IMV and 1.6 mM ADP. (D) Dephosphorylation of phosphorylated MtrA in the presence of MtrB-free IMV and 1.6 mM ADP. (E) Quantification of the radioactive signals of phosphorylated MtrA shown in panels A to D. Circles, panel A; squares, panel B; closed triangles, panel C; open, inverted triangles, panel D. The data set is an example of findings from two independent experiments with almost identical results.

FIG. 3.

Influence of different monovalent cations on the phosphorylation activities of MtrB and DcuS in liposomes. The reaction was performed for 10 min after the addition of [γ-³³P]ATP. (A) Influence of different monovalent cations on the autokinase activity of MtrB (upper panel, autoradiogram; lower panel, quantification of the phosphorylation activity indicated above). (B) Influence of different monovalent cations on the autokinase activity of DcuS (upper panel, autoradiogram; lower panel, quantification of the phosphorylation activity indicated above). The data sets are examples of findings from at least two independent experiments with almost identical results. −, no cations; mosm, milliosmoles.