Passage through stationary phase and High Mg²⁺ cause OFF cells to prime to ON state.

<p>Priming is defined as the deterministic conversion of OFF cells to the ON state. (A and B) Passage through stationary phase primes OFF cells in minimal medium. Starting from OFF cells, two lineages (STA and EXP) were established in minimal medium with various magnesium concentrations as shown schematically in panel A and in detail in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003706#pgen.1003706.s002" target="_blank">Figure S2</a>. Cells from these lineages were imaged under a microscope as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003706#s4" target="_blank">Methods</a> and the percentage of ON cells in the mid-exponential culture obtained at the end was plotted (panel B). (C and D) Overnight culture at slow growth rates and high Mg²⁺ results in priming. Overnight cultures inoculated with OFF colonies were set up in indicated conditions, diluted 1000-fold into Minimal A medium with 100 µM Mg²⁺, grown to mid-exponential phase at 37°C (even for 30°C overnight cultures) and imaged under a microscope (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003706#s4" target="_blank">Methods</a>). The percentage of ON cells present in the images of mid-exponential cultures is shown. Panel C shows the effect of increasing [Mg²⁺] in LB, while panel D documents the effect of changing growth rate in low [Mg²⁺] media. In panels B, C and D, error bars indicate half the range of two independent experiments. The range is less than 0.5% in the instances where error bars are not visible.