Figure 5. Adapted LAC₀ cells carry promoter regulation mutations for optimal expression of PhoB.

AP activities (A) and PhoB levels (B) of individual colonies isolated from LAC₀ and LAC₁₅₀ cultures. Indicated strains were grown in batch cultures with 50 µM (Lo) or 2 mM Pi (Hi) for 3 h. PhoB levels are shown for cells grown in Pi-replete medium (2 mM) except for WT. Error bars are SDs of at least four independent experiments. (C) IPTG-induced PhoB expression of indicated strains under Pi-deplete conditions. (D) Schematic representation of adaptive mutations. In the LAC strain, LacI binds to lacO regions at the lac promoter before phoBR. The presence or absence of lacI is indicated by “+” or “−” symbols. Sequences of the lacO1 site from adaptive mutants are shown with dots representing identical sequences. (E) Repression of PhoB expression by additional copies of lacI. Indicated colonies were transformed with the lacI_q-containing plasmid pRG177 and probed for PhoB expression in the absence of IPTG.