Figure 5. Long-term culturing demonstrates the metastability of the ON state in LB.

Seven independent lineages of phoQ (T281R) OFF and phoQ (T281R) ON were established and maintained as depicted in detail in Figure S4. 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 (phoQ (T281R) OFF) or blue (phoQ (T281R) 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.