FIG 3.
Two lysines in the MgrB cytosolic region regulate PhoQ activity through their charged side chain. (A) The activities of MgrB mutants with single alanine substitutions in the cytosolic region were tested using the PhoQ/PhoP-regulated transcriptional reporter PmgtLA-gfp in a ΔmgrB strain. MgrB mutants were constructed in a pBAD33 vector, and their expression was induced with 0.008% arabinose. (B) The activities of MgrB K2 K3 double mutants were tested as for panel A. The reporter activity change was significant (P = 0.0045) when comparing MgrB K2D K3D to the no-MgrB control. (C) FRET efficiencies between PhoQ-mNeonGreen and mCherry (−), mCherry-tagged wild-type MgrB (WT), or MgrB mutants (K2A, K3A, K2D K3D, and K2E K3E) were measured. The MG1655 ΔphoQ ΔmgrB strain was transformed with pBAD33 phoQ-mneongreen and pTrc99a mcherry-mgrB variants. Protein expression was induced with 0.008% arabinose and 10 μM IPTG. FRET was measured for each protein pair, and the efficiencies were calculated as described in Materials and Methods. All the data points represent averages of the results of at least three independent experiments, and the error bars show SD. The statistical significances of changes in reporter activity or FRET efficiency were calculated with t tests by comparison to the wild type or as specified and are indicated with asterisks (***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant).