ΔybeX phenotypes are prominent under low extracellular magnesium and during the transition into the stationary phase
Our ability to control the ΔybeX phenotype by Mg2+ allowed us to pinpoint the growth phase dependence of the ΔybeX phenotype more precisely. Therefore, in the next experiment, we first grew the cultures overnight into the stationary phase in MOPS minimal medium supplemented with 10 mM MgCl2 and 0.5% glucose, where ΔybeX phenotype does not occur. Then, the cells were washed thrice with MOPS minimal medium lacking Mg2+, after which the regrowth assay was set up by suspending the cells in MOPS containing 10 µM MgCl2 (Fig. 8A ).
As expected, there is no difference in the duration of the outgrowth lag phase between the wild-type and ΔybeX , and the exponential growth rates were the same (Fig. 8B ). To look for the emergingΔybeX phenotype, we plated spots of samples from the outgrowth cultures at designated time points (Fig. 8B ) onto both LB and R2A agar plates (R2A stimulates the growth of stressed bacteria). The plates were incubated overnight at 37°C or 42°C. While until the 4h time point there was no difference between the colony formation of WT andΔybeX (Fig. 8C) , upon transition into the stationary phase, in the 5.5h time point, there is a growth delay of theΔybeX strain on both LB or R2A agar plates, which is more pronounced at 42°C. Similarly, the sensitivity of the ΔybeXstrain to tetracycline, erythromycin and chloramphenicol antibiotics appears only at the 5.5h time point (Fig. 8D ). We conclude that the delay of regrowth and antibiotic sensitivity of the ΔybeXstrain appears under low extracellular magnesium at the transition from exponential to stationary growth phase.