The delayed outgrowth and antibiotic sensitivity of ΔybeX
depend on the growth history of bacteria
While the ΔybeX cells have a lengthened lag phase during
outgrowth from the stationary phase, they appear to retain similar
levels of metabolic activity to the WT cells during this lag phase, as
well as similar exponential growth rate (Fig. 1D ). This led us
to hypothesize that any cellular defects conferred by the lack of YbeX
could accumulate stochastically during late growth, preceding entry into
the stationary phase and/or in the stationary phase itself, which in
turn could lead to the observed single-cell heterogeneity during
outgrowth (Fig. 2 ). Accordingly, we tested whether the
phenotypes of ΔybeZ , ΔybeY, and ΔybeX depend on the
growth phase of the culture from where the cells originate. We surmised
that if a gradual accumulation of harm causes the ΔybeXphenotype, then cells that have had more time to accumulate such harm
should exhibit stronger phenotypes.
Overnight-grown cultures of ΔybeZ , ΔybeY , ΔybeX and
wild-type (WT) strains were serially diluted and spotted on LB agar
plates to assay the heat and antibiotic sensitivity. The same stationary
phase cultures were diluted a hundred-fold into fresh LB medium and then
grown at 37°C for four to five cell divisions until
OD600 reached 0.2-0.4, after which the cells were
diluted and spotted on LB agar plates to assay the heat and antibiotic
sensitivity of exponentially growing cells.
The ΔybeX stationary phase inoculum exhibited sensitivity to
sub-MIC concentrations of chloramphenicol and erythromycin but no
sensitivity to rifampicin in comparison to WT (Fig. 4A ). In
contrast, the exponentially growing ΔybeX had WT-like sensitivity
to all tested antibiotics. In comparison, ΔybeZ cultures had
intermediate levels of sensitivity to chloramphenicol, regardless of the
growth history of cells, while they are not more sensitive to
erythromycin, rifampicin, and tetracycline as compared to WT
(Fig. 4A ). Exponentially growing ΔybeZ cells in MOPS
minimal medium, supplemented with 0.3% glucose, also exhibited
sensitivity to chloramphenicol (Fig. S4a ). ΔybeY cells
were very sensitive to all tested antibiotics, notwithstanding the
growth phase of the spotted culture.
In liquid media, cultures started directly from stationary phase inocula
again showed a lengthened lag phase for ΔybeX but not forΔybeZ , while the exponential growth rates of both ΔybeXand ΔybeZ were very similar to WT (Fig. 4B ). TheΔybeY strain behaves similarly, regardless of the growth phase of
inoculums, exhibiting a reduced exponential growth rate and reaching a
lower maximal cell density. In contrast, when the cells were outgrown
from exponential phase cultures, the WT, ΔybeZ and ΔybeXstrains grew equally well, with no visible lag phase, while theΔybeY strain had a reduced growth rate and a lower growth
end-point, as expected (Fig. 4C ).