Deletion of E. coli ybeX leads to heat sensitivity and longer outgrowth from the stationary phase
ybeX is a part of the RpoH (heat response) regulon (Nonakaet al. , 2006). We tested by a spot assay the effect of elevated growth temperature on the ∆ybeX strain from the Keio collection (Baba et al. , 2006), compared to the isogenic BW25113. After overnight growth in the LB liquid medium, serial dilutions of the culture were spotted on LB agar plates and incubated at 20°C, 37°C or 42°C overnight. Disruption of ybeX 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. We verified the deletion of ybeX and the presence of kanamycin resistance cassette by PCR analysis (Fig. S1b, c ). Heat sensitivity occurred in both newly constructed ∆ybeX strains (Fig. S1d). This demonstrates that the observed phenotype isybeX -inflicted. We used the ybeX deletion strain of the Keio collection in further studies.
Next, we assessed whether the lack of the YbeX protein caused heat sensitivity. Alternatively, secondary effects of the chromosomal deletion might be responsible for this phenotype. We reintroducedybeX on a single-copy TranBac library plasmid (Otsuka et al. , 2015) and found the leaky YbeX expression in the absence of the inducer (isopropyl-β-D-1-thiogalactopyranoside; IPTG) was sufficient to rescue the heat sensitivity of the ∆ybeX mutant. The empty vector (pEmpty) and TranBac plasmids carrying ybeY or ybeZ had no effect on the growth (Fig. 1B ). Thus, the heat sensitivity of the ∆ybeX strain was 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 cultures in liquid LB medium at 37°C on a 96-well plate reader. We did not notice differences between the growth of WT and∆ybeX strains when cultures were started from freshly grown single colonies (data not shown). When cultures were inoculated with bacteria from the stationary phase overnight cultures, the ∆ybeXmutant had a much longer lag phase (300-350 min.) compared to the WT (100-150 min.; Fig. 1C , Fig. S2a ). Both strains reached the same optical density in the stationary phase. A similar number of colonies after dilution and plating WT and ∆ybeX(Fig. 1B ) indicates that the delay of the visible growth of theΔybeX mutant is not caused by decreased survival in the stationary phase but reflects later regrowth of the same number of live bacteria. Expression of ybeX from a single-copy plasmid abolished the prolonged lag phase completely. In contrast, complementation with the plasmids carrying either ybeY , ybeZ or lnt had no effect confirming that lack of the YbeX protein is causing the delay of regrowth, while further excluding the polar effect as a cause of theΔybeX phenotype (Fig. 1C; Fig. S2b ).
To investigate whether the longer lag phase of ΔybeX strain is due to lower metabolic activity in the mutant cells, we used the alamarBlue reagent, a quantitative indicator of the oxidation-reduction potential of cell membranes, as a proxy for metabolic activity (Rampersad, 2012). In a negative control experiment conducted in PBS buffer lacking the nutrients necessary for the resumption of growth, both strains show similarly low alamarBlue signal, indicating similar levels of metabolic activity (the superimposed black lines inFig. S2c ). When diluted into fresh LB medium, the alamarBlue signal immediately starts to increase for both strains, indicating activation of similar levels of cellular metabolism (Fig. S2d, e ). While the initial rate of increase in the alamarBlue signal, and by implication the cellular metabolism levels, are equal for WT andΔybeX cells, after about 100 minutes, the WT acquires a still faster rate of signal increase, while the ΔybeX cells continue as before for about 200 more minutes, before the rate of their signal growth increases to WT levels (Fig. S2d ). As shown by the OD600 measurements (Fig. S2a ), for both the WT and the ΔybeX cells, this phase shift in redox power is accompanied by the start of cell divisions (Fig. S2e ). These results indicate that the longer lag phase of the ∆ybeX strain is not caused by lower levels of metabolic activity in the ΔybeXcells whilst they are preparing for the resumption of cell divisions. Nor is it caused by a later onset of said metabolic activity.