Fig. 4.

In vivo comparison of PhoB^WT and PhoB^F20D. (A) Phosphorylation of PhoB^WT and PhoB^F20D at Pi-depleted conditions. Strain RU1621 carrying pRG298 (P_lac-phoB^F20DphoR) or pRG226 (P_lac-phoB^WTphoR), and the wild-type strain BW25113 (WT) were analyzed with Phos-tag gels (Upper) and normal SDS gels (Lower). (B and C) Comparison of phosphorylation percentages (B) and levels (C) of PhoB^WT and PhoB^F20D. Phosphorylation percentages were quantified for plasmid-expressed PhoB^WT (open squares) and PhoB^F20D (solid circles), respectively. Phosphorylation levels were calculated as the product of phosphorylation percentages and total PhoB amount. The solid line indicates the best fit of the PhoB^F20D data with a C_p of 10 ± 1.8 pmol and a C_t of 2.3 ± 0.8 pmol. The dotted line is a simulated curve for PhoB^WT with a C_p of 1.2 pmol and a C_t of 0.1 pmol. Error bars are SDs from three independent experiments, and unseen error bars are smaller than symbols.