The ∆ybeX phenotype can be suppressed by MgCl2
As YbeX has been implicated in Mg+2 efflux (Gibsonet al. , 1991), we tested whether supplementing growth media with magnesium chloride affects the ∆ybeX phenotype. First, we compared the growth of WT and ∆ybeX in LB medium with and without magnesium supplementation (Fig. 8A, Fig. S5a ). When the LB medium was supplemented with 10 mM MgCl2, the antibiotic sensitivity and heat shock phenotypes of ∆ybeX disappeared (Fig. 8A ). To test whether the effect is media-dependent, we used the SOB medium, which contains a high 10 mM concentration of MgCl2. Again, the phenotypes of ∆ybeX disappeared (Fig. S5a,b ). Thus, excess magnesium in the growth media, either LB or SOB, fully rescues the outgrowth growth phenotypes of the∆ybeX cells.
To test whether magnesium-deficient-rich media could increase the severity of the growth phenotype, we used the peptide-based medium (PBM), a rich, magnesium-limited, buffered, complex growth medium (Christensen et al. , 2017). PBM is advantageous because it is free of any cell extract, which is the primary source of magnesium in almost all complex media (Li et al. , 2020). To avoid diauxic inhibition, we modified it to contain casamino acids instead of glucose as the carbon source (see Materials and Methods). ∆ybeX cells had longer lag times on outgrowth compared to when grown in PBM (Fig. S5c ), while wild-type cells grown in LB or PBM didn’t differ. When PBM is not supplemented with MgCl2, the heat shock phenotype is more substantial than in LB medium (Fig. 8B , compare Fig. S5d with Fig. S5e ). Supplementing PBM with 50 µM and 100 µM MgCl2 partially suppresses the phenotype, first at 37°C and then at 42°C, and supplementation with 200 µM MgCl2 completely suppresses it under both temperature conditions (Fig. 8B ). Therefore, we have a potentially sensitive regulatable system for driving the growth phenotype of the∆ybeX strain.
To test the sensitivity and robustness of such an experimental system, we did a liquid medium growth experiment in defined MOPS minimal medium, with glucose as the carbon source. Unlike with the PBM, in the MOPS medium, we can precisely control the magnesium levels by adding MgCl2 from 10 µM to 525 µM (the ”normal” optimal level for this medium; (Neidhardt et al. , 1974)). When the WT cells grew into an overnight stationary phase in different Mg2+-depleted MOPS media, there was no Mg2+ supplementation effect for the outgrowth lag phase duration (Fig. 8C , the left panel). As expected, there was no effect on the actual growth rate after the lag phase. Under the same conditions, the Mg-supplementation effect on ∆ybeX lag phase was very different (Fig. 8C , the right panel). There appears a threshold effect, whereby Mg2+-supplementations by 50 µM and less produce a slight gradual shortening of the lag phase from around 400 minutes to 350 minutes, while supplementation with 75 µM MgCl2 suddenly shifts the lag time to about 200 minutes, after which additional magnesium has little effect on the duration of the lag phase.