The antibiotic sensitivity of ∆ybeX depends on the growth
history of cells
Our finding that while the ∆ybeX cells have a lengthened lag
phase during outgrowth from the stationary phase, they appear to retain
similar levels of metabolic activity during this lag phase to the WT
cells, as well as similar exponential growth rate (Fig. 1D ),
led us to hypothesize that any cellular defects conferred by the lack of
YbeX may accumulate during the late growth, preceding entry into the
stationary phase and/or in the stationary phase itself. Such a
stochastic process could lead to the observed single-cell level growth
heterogeneity (Fig. 2 ). Accordingly, we assayed whether the
phenotypes of ∆ybeZ , ∆ybeY, and ∆ybeX depend on the
growth phase where the cells originate. We surmised that if the∆ybeX phenotype is caused by a gradual accumulation of harm, then
cells that have been given ample time to accumulate such harm, should
exhibit a stronger phenotype than cells with only a few divisions.
First, we tested the antibiotic sensitivity phenotype. In this
experimental setup, we start by growing a single bacterial colony for 12
hours into the early stationary phase (Fig. 4A ). Then, the
experiment is divided into two. In the first arm, to assay the
antibiotic sensitivity during outgrowth, stationary liquid cultures are
directly dot spotted into agar plates containing sub-inhibitory
concentrations of antibiotics. In the second arm, to assay the
antibiotic sensitivity of exponentially growing cells, the same
stationary cultures are first diluted a hundred-fold into fresh liquid
media and grown at 37°C for four to five cell divisions until
OD600 reaches 0.2-0.4, after which they are dot spotted.
The ∆ybeX strain exhibited very strong chloramphenicol and
erythromycin sensitivity in cells originating from the early stationary
phase but no sensitivity to Rifampin (Fig. 4B ). In contrast,
the ∆ybeX cells plated on the antibiotic after only a few rounds
of the division had WT-like sensitivity to all tested antibiotics. In
comparison, ∆ybeZ cultures had similar intermediate levels of
sensitivity to chloramphenicol, regardless of the growth history of
cells, while they are not sensitive to erythromycin, rifampicin, and
tetracycline (Fig. 4B ). Exponentially growing ∆ybeZcells in MOPS minimal medium, supplemented with 0.3% glucose as the
carbon source, also exhibited sensitivity to chloramphenicol
(Figure S4a ). ∆ybeY cells had a very strong sensitivity
to all tested antibiotics under both growth conditions. This is not
surprising, considering its strong growth phenotype.
Testing the culture growth in liquid media, after diluting the culture
directly from the early stationary phase, again showed a lengthened lag
phase for ∆ybeX but not for ∆ybeZ , while the exponential
growth rates of both ∆ybeX and ∆ybeZ were very similar to
WT (Fig. 4C ). The ∆ybeY strain behaves similarly in both
experiments, exhibiting a reduced exponential growth rate and reaching a
lower maximal cell density. In contrast, when the cells are outgrown
from exponential phase cultures, the WT, ∆ybeZ and ∆ybeXstrains grow equally well, with no visible lag phase, while the∆ybeY strain has a reduced growth rate and a lower growth
end-point, as expected (Fig. 4D ).