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

<p>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 <i>lacO</i> regions at the <i>lac</i> promoter before <i>phoBR</i>. The presence or absence of <i>lacI</i> is indicated by “+” or “−” symbols. Sequences of the <i>lacO</i>1 site from adaptive mutants are shown with dots representing identical sequences. (E) Repression of PhoB expression by additional copies of <i>lacI</i>. Indicated colonies were transformed with the <i>lacI_q</i>-containing plasmid pRG177 and probed for PhoB expression in the absence of IPTG.