Figure 6
Klebsiella PmrD interacts with PmrA to prevent dephosphorylation. (A) Bacterial two-hybrid analysis of PmrD/PmrA interaction in vivo. The E. coli XL1-Blue cells co-transformed with various combinations of pTRG and pBT-derived plasmids were diluted serially and spotted onto the indicator plate. The bacterial growth after 36 h was investigated and photographed. Combinations of plasmids carried by each strain were indicated above the figure. (B) Klebsiella PmrD prevents the dephosphorylation of PmrA by its cognate sensor protein. The phosphorylation state of the recombinant His-PmrA protein was monitored upon the addition of the sensor protein His-PmrBC276 in the presence (PmrD) or absence (-) of purified His-PmrD protein at specific time points as indicated. The arrows indicate phospho-PmrA (P-PmrA) and phospho-PmrBC276 (P-PmrBC276). (C) Kinase/phosphatase assay was carried out using the recombinant His-PmrA (final concentration 5 μM) and His-PmrBC276 (final concentration 2.5 μM) in the presence (PmrD) or absence (-) of the recombintant His-PmrD protein (final concentration 5 μM). The small cationic proteins RNase A and cytochrome C were introduced individually as a negative control at a final concentration of 5 μM. (D) Autokinase assay of the recombinant His-PmrBC276 (final concentration 2.5 μM) was performed in the presence (PmrD) or absence (-) of the recombintant His-PmrD protein (final concentration 5 μM).