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 ).