Deletion of ybeX leads to heat sensitivity and longer outgrowth from the stationary phase
ybeX belongs to the RpoH heat response regulon (Nonaka et al. , 2006). We tested by a spot assay the effect of elevated growth temperature on the ΔybeX strain from the Keio collection (Babaet al. , 2006), compared to the isogenic wild-type strain. After overnight growth in the LB liquid medium, serial dilutions of the cultures were spotted on LB agar plates and incubated at 20°C, 37°C or 42°C overnight. Disruption of the ybeX gene hindered growth at 42°C but not at 20°C (Fig. 1B , Fig. S1a ). For verification, ybeX deletion was reintroduced in two strain backgrounds, MG1655 and BW25113 (see Fig. S1b, c for strain construction). Heat sensitivity was retained in newly constructedΔybeX strains (Fig. S1d) , demonstrating that the observed phenotype is ybeX -inflicted. We used the ybeXdeletion strain of the Keio collection in further experiments.
Next, we assessed whether the lack of the YbeX protein caused the heat sensitivity, as secondary effects of the chromosomal deletion could be responsible for the phenotype. We reintroduced ybeX on a single-copy TransBac library plasmid (Otsuka et al. , 2015) and found that the leaky expression of YbeX in the absence of the inducer (isopropyl-β-D-1-thiogalactopyranoside; IPTG) is sufficient to rescue the heat sensitivity phenotype of the ΔybeX mutant. The empty vector and TransBac plasmids carrying ybeY or ybeZ had no effect on growth (Fig. 1B ). Thus, the heat sensitivity of theΔybeX strain is caused by the absence of the YbeX protein rather than through polar effects on neighbouring genes.
To find which growth phase is affected by the ybeX deletion, we monitored bacterial growth in liquid LB medium at 37°C in 96-well plates. We did not notice differential growth of WT and ΔybeXstrains when cultures were started from freshly grown single colonies (data not shown). In contrast, when cultures were inoculated with bacteria from the stationary phase overnight cultures, the ΔybeXmutant had a much longer lag phase (300-350 min.) than the WT (100-150 min.; Fig. 1C , Fig. S2a ). Both strains reached similar optical densities in the stationary phase. A similar number of colonies of WT and ΔybeX strains (Fig. 1B ) indicates that the delay of the visible growth of the ΔybeX mutant is not caused by decreased survival in the stationary phase. The expression of ybeX from a single-copy TransBac library plasmid completely complemented the prolonged lag phase. In contrast, complementation with plasmids carryingybeY , ybeZ or lnt had no effect, confirming that lack of the YbeX protein is causing the delay of regrowth while again excluding polar effect as the cause of the ΔybeX phenotype (Fig. 1C; Fig. S2b ).
To investigate whether the longer lag phase of the ΔybeX strain is due to lower metabolic activity in the mutant cells, we used the Alamar Blue reagent, a quantitative indicator of the oxidation-reduction potential of cell membranes, as a proxy for metabolic activity (Rampersad, 2012). In a control experiment conducted in PBS buffer lacking the nutrients necessary for the resumption of growth, both strains show similarly low Alamar Blue signal, indicating similar levels of metabolic activity (the superimposed black lines in Fig. S2c ). When cells were diluted into fresh LB medium, the Alamar Blue fluorescence immediately started to increase for both strains, indicating similar levels of cellular metabolism (Fig. S2d, e ). While the initial rate of increase in the Alamar Blue fluorescence is indistinguishable in WT and ΔybeX cells, the WT acquires a faster rate of metabolism after about 100 minutes, while the ΔybeX cells continue as before for about 200 more minutes (Fig. S2d ). As shown by the OD600 measurements (Fig. S2a ), for both the WT and the ΔybeX cells, the increase in fluorescence is accompanied by the start of regrowth (Fig. S2e ). These results indicate that the extended lag phase of the ΔybeX strain is not caused by lower levels of metabolic activity upon transfer from the stationary phase culture into the regrowth medium.
The delayed outgrowth of the ΔybeX mutant is heterogeneous at the individual cell level
When streaking out mutant strains from glycerol stocks and overnight grown stationary phase cultures, we noticed that the ΔybeX strain produces colonies of different sizes. Re-streaking of small and largeΔybeX colonies resulted in similarly-sized second-generation colonies, indicating that the heterogeneous phenotype is not caused by a genetic mutation (data not shown). We hypothesized that the colony size heterogeneity of ΔybeX is a result of the delayed outgrowth of individual bacteria and indicates physiological heterogeneity of the stationary phase inoculum.
Inspection of colony sizes plated from overnight grown bacterial cultures showed, in agreement with our previous observations, thatΔybeX cells tend to form smaller colonies than wild-type (WT) cells when grown overnight in LB or MOPS minimal medium supplemented with 0.3% glucose (Fig. 2A , Fig. S3a ). To better understand the nature of the ΔybeX phenotype at the individual cell level, colony radiuses of ΔybeX and WT were quantified from four independent stationary phase outgrowth experiments using AutocellSeg (Khan et al. , 2018). Overnight-grown cells were plated from LB or MOPS minimal media, and plates were incubated at 37°C or 42°C overnight. When plated from the LB medium, ΔybeX cells formed smaller and more heterogeneous colonies than wild-type cells (Fig. 2A , B ). In contrast, when plated from the MOPS minimal medium, the ΔybeX colonies appear to be consistently smaller and more homogeneous in size (Fig. 2C ). We found that a fraction of ΔybeX cells lost the ability of colony formation when plates were incubated at 42°C (Fig. 2D ). When plated from LB, this drop was about 10-fold (p < 0.0001) and from MOPS medium about two-fold (p=0.055, Fig. 2D ). Nonetheless, the quantified colony radiuses on plates incubated at 42°C resembled those at 37°C (Fig. 2B , C ).
While the ΔybeX colony sizes were increased after 48 hours of incubation at 37°C, they consistently remained heterogeneous in size in the absence or presence of the kanamycin resistance cassette (Fig. S3b , c ). In conclusion, the ΔybeX cells grow in at least two distinct regimes, one similar to WT growth, while the other results in up to two-fold smaller colonies.