ΔybeX phenotype appears during the transition into the stationary growth phase
Our ability to control the ∆ybeX phenotype by Mg2+ allows us to pinpoint the growth-phase dependence of the ∆ybeX phenotype more precisely. Therefore, in the next experiment, we start by growing the cells under high magnesium, where∆ybeX phenotype does not occur, and then do the outgrowth under low magnesium to look for the gradual appearance of the phenotype. We first grew the cultures overnight into the stationary phase, starting from a single colony in MOPS minimal medium supplemented with 10 mM MgCl2. This protocol was used to suppress the∆ybeX phenotype. These cells were then pelleted and washed thrice in MOPS minimal media lacking Mg2+. Then after the regrowth assay was set up by suspending the cells in the MOPS minimal medium containing 10 µM MgCl2 so that the starting optical density of the regrowth culture would be 0.05 OD600 units (Fig. 9A ). Here, we expected to see the gradual onset of the ∆ybeX phenotype.
As expected, there is now no difference in the duration of the pre-outgrowth lag phase between the strains. During outgrowth, the exponential growth rates were the same for wild-type and ∆ybeX(Fig. 9B ). There is also no difference in growth during the gradual onset of the stationary phase, as the ∆ybeX cells end up in a very similar end-of-growth plateau, which holds for a wide range of Mg2+ concentrations in the medium (Fig. 9C ). To look for the emerging ∆ybeX phenotype, we dot spotted samples from the outgrowth cultures at designated time points (as shown inFig. 9B ) onto both LB agar and the R2A agar plates at 37°C and onto LB agar at 42°C. This experiment shows that during the exponential growth phase (up until 4 hrs time point), there is no growth phenotype for the ∆ybeX strain (Fig. 9D) . In contrast, during the transition into the stationary phase, as measured in the 5.5h time point, there is an apparent growth effect of the ∆ybeX strain at both growth media, which is more pronounced at 42°C heat shock conditions. Similarly, the relative sensitivity of the ∆ybeXstrain vs. the WT strain to the antibiotics Tetracyclin, Erythromycin and Chloramphenicol appears only at the 5.5h time point, the effect being much stronger than for the previous outgrowth assay done without the antibiotics (Fig. 9E ). From these experiments we conclude that the growth phenotype of the ∆ybeX strain, and its antibiotic sensitivity, appear only at the transition between the exponential and stationary growth phases.