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.