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.