Long-term culturing demonstrates the metastability of the ON state in LB.

<p>Seven independent lineages of <i>phoQ (T281R)</i> OFF and <i>phoQ (T281R)</i> ON were established and maintained as depicted in detail in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003706#pgen.1003706.s004" target="_blank">Figure S4</a>. OFF and ON colonies were inoculated in LB and grown for 24 hours to generate a Day 1 culture. For each lineage, the Day 1 culture was diluted 10⁶-fold to generate a corresponding Day 2 culture, and this procedure was repeated for a total of 5 days. Day 1, 3, and 5 cultures were also diluted and plated in duplicate on minimal medium plates. The mean percentage of YFP-bright colonies on these plates was plotted in maroon (<i>phoQ (T281R)</i> OFF) or blue (<i>phoQ (T281R)</i> ON) with different symbols representing independent lineages. Representative merged images of Day 3 plates are shown on the right. These were constructed by merging the background-subtracted CFP image with its corresponding background-subtracted YFP image as the red and green channels respectively.