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.