Figure 3. Mutations in specific PA-PhoQ residues lead to loss of Mg²⁺-mediated repression.

(A) Mutations in PA-PhoQ result in loss of repression of the PhoN reporter. Strain MB147, which carries a PhoQ-null allele and the plasmid pBAD24-FL-PA-PhoQ, was used as the wild type strain. The indicated mutations were made in pBAD24-FL-PA-PhoQ by Quickchange mutagenesis. Cultures were grown in N minimal medium supplemented with 10 mM MgCl₂.

(B) Several mutants still show Mg²⁺-mediated repression of the PhoN reporter. A subset of the mutant strains from Fig 3A were grown in N minimal medium supplemented with 10 mM (black bars) or 10 μM (gray bars) MgCl₂.

(C) Several mutations result in complete loss of Mg²⁺-mediated repression of the PhoN reporter. The remaining mutant strains from Fig 3A were grown in N minimal medium supplemented with 10 mM (black bars) or 10 μM (gray bars) MgCl₂. All graphed values are mean ± standard deviation of cultures grown in duplicate and are representative of at least three independent trials.

(D) Mutations in PA-PhoQ alter Mg²⁺-binding saturation. MgCl₂ was added at indicated concentrations to 1 μM purified wild type or mutant PA-PhoQ-PD. Peak fluorescence values were fit to a simple equilibrium model.