The ΔybeX mutant phenotypes can be suppressed by MgCl2 supplementation
As YbeX has been implicated in Mg+2 efflux (Gibsonet al. , 1991), we tested whether supplementing growth media with magnesium chloride affects the ΔybeX -caused phenotypes. First, we grew the WT and ΔybeX strains in LB medium with and without magnesium supplementation (Fig. 7A, Fig. S6a ). When bacteria were grown in LB medium that was supplemented with 10 mM MgCl2, the antibiotic and heat sensitivity ofΔybeX mutant upon plating disappeared (Fig. 7A ). To test whether the effect of Mg+2 is media-dependent, we used the SOB medium, which contains 10 mM MgCl2. Again, the phenotypes of ΔybeX disappeared. Thus, excess magnesium in the growth media, either LB or SOB, fully rescued the growth and ribosomal phenotypes of the ΔybeX cells (Fig. S6a, b ).
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) that is free of any cell extract, which is the primary source of magnesium in almost all complex media (Liet al. , 2020). We modified it to contain casamino acids instead of glucose to avoid diauxic inhibition. ΔybeX cells had longer lag times during outgrowth in PBM than in LB (Fig. S6c ), while wild-type cells grown in LB or PBM did not differ in outgrowth. The heat sensitivity upon plating was more severe in PBM than in LB medium (Fig. 7B , Fig. S6d, e ). Supplementation of PBM with 50 µM and 100 µM MgCl2 partially suppressed and 200 µM MgCl2 completely suppressed the outgrowth delay ofΔybeX at 37°C and 42°C (Fig. 7B ). Supplementation of LB with 100 mM MgCl2 completely suppressed the outgrowth delay of ΔybeX, with no impact on the growth rates of wild-type and ΔybeX strains (Fig. S7a ).
We further tested the Mg2+-sensitivity of theΔybeX mutant in the MOPS minimal medium supplemented with 0.5% glucose. Unlike in PBM, we can precisely control the magnesium levels in the MOPS medium (Neidhardt et al. , 1974). The ΔybeXcultures achieved similar optical density plateaus to the wild-type. This holds for a wide range of Mg2+ concentrations in the MOPS minimal medium (Fig. S7b ). When WT cells were grown into stationary phase in the MOPS minimal medium, the Mg2+ concentration had no effect on the time-to-outgrowth (Fig. 7C , the left panel). Neither was there any effect on the growth rates. Under matching conditions, theΔybeX strain grown in ≤50 µM Mg2+ exhibited extended lag phases of 350 to 400 minutes. The presence of ≥75 µM MgCl2 decreases the lag time to about 200 minutes, after which additional magnesium has little effect on the duration of the lag phase (Fig. 7C , the right panel). These results indicate that the effects of ybeX deletion can be compensated by elevated Mg2+ concentration of the growth medium.