DISCUSSION
This work shows that the putative
Co2+/Mg2+ efflux protein YbeX is
functionally involved in ribosome metabolism in Escherichia coli .
For a possible mechanism that is consistent with experimental results,
we propose that during growth without ybeX, there is an
accumulation of harm in the late-exponential growth phase involving
pre-17S rRNA and 16S rRNA partial degradation products (Fig.
6C-G ), which necessitates a longer lag phase upon outgrowth in a fresh
medium. During this prolonged lag phase, the ∆ybeX cells are
metabolically active (Fig. 1D ) and would be busy cleaning up
the inactive and/or partially degraded ribosomal particles before new
ribosome synthesis and subsequent cell division can commence. An
inability to do so properly in the absence of the YbeX protein then
leads to the growth phenotype and antibiotic sensitivity during the
shift from exponential growth to stationary phase (Fig. 9 ).
Intriguingly, although the late-exponential phase ∆ybeX cells
accumulate rRNA degradation products, to some extent, even in the 70S
fraction (Fig. 6C, E ), they have WT-like sucrose gradient
profiles (Fig. 6B ), indicating no accumulation of significant
ribosome-like particles. In addition, although the ∆ybeX cells
have a clear growth phenotype, manifested in a lengthened outgrowth lag
phase and in sensitivity to antibiotics, the exponential growth rate of
the ∆ybeX cells is indistinguishable from WT, as are the growth
end-points (Fig. 1C, Fig. 8C, Fig. 9B ).
We find that the growth phenotype of ∆ybeX is
Mg2+-dependent, being present in
Mg2+-limiting growth conditions (Fig. 8 ).
This result is consistent with its proposed role in
Mg2+ efflux in Salmonella typhimurium (Gibson
et al. 1991). However, to mechanistically tie the YbeX protein with
Mg2+ metabolism, requires considerably more
experimental work. Currently, the totality of evidence is highly
suggestive of the role of YbeX in regulating Magnesium homeostasis, but
the exact mechanism should still be considered as open.
What could be the mechanism of action of the YbeX protein on the
ribosome? Unlike its neighbouring gene products, the YbeY and the YbeZ,
there is no evidence that YbeX binds to the ribosome or to any
ribosome-associated protein. Nonetheless, at this stage, we cannot
exclude the possibility of a direct action of the YbeX on the ribosome.
The ybeX /corC gene was initially recovered in S.
typhimurium in a screen for resistance to cobalt and proposed to
contribute, possibly as a co-effector of the trans-membrane metal
transport protein CorA, to the efflux of divalent cations (Gibsonet al. , 1991). As yet, there is no mechanistic function ascribed
to YbeX, and while Mg2+ influx is generally
well-studied, its efflux is poorly understood in bacteria (Armitanoet al. , 2016). Essentially, YbeX is a cytoplasmic protein (Suekiet al. , 2020), for which we have indirect evidence that it might
be somehow involved in Mg2+-efflux. Our finding that
the growth phenotype of the ∆ybeX strain needs low extracellular
Mg2+ is consistent with the role of YbeX in
Mg2+ efflux, as Mg2+ efflux is
inhibited at low extracellular magnesium (Nelson and Kennedy, 1971) and
can be activated by adding 1mM MgCl2 to the growth
medium for S. typhimurium (Gibson et al. , 1991). Our
results suggest that this activation, which occurs by an unknown
mechanism, may involve a discontinuous switch, occurring somewhere
between 50 µM and 75 µM MgCl2 concentration
(Fig. 8C ). When thinking about the activation of
Mg2+ efflux by increasing extracellular
Mg2+ concentrations, we also need to consider the
effect of low extracellular Mg2+ on cellular
physiology. The extracellular Mg2+ acts as a
counterion to neutralize the phosphate groups of outer-membrane
lipopolysaccharides, and it binds to many membrane proteins, stabilizing
their structures (Groisman and Chan, 2021). Accordingly, a lack of
extracellular Mg2+ leads to permeabilization of the
outer membrane, including for hydrophobic antibiotics like Erythromycin
and Rifampin (Vaara, 1992).
YbeX has been genetically connected to translation, as E. colicells that rely for growth on an artificial ribosome variant, where the
subunits are covalently tethered by fused rRNAs, need for faster growth
a nonsense mutation in the ybeX gene, together with a missense
mutation in the rpsA (Orelle et al. , 2015). A mechanism of
action could be that the suppressed Mg2+ efflux in the
absence of ybeX leads to increased cytoplasmic
Mg2+ concentration, stabilizing the artificial
ribosomes and thus activating them for protein synthesis.
On the other hand, we have found that a very high, sublethal,
Mg2+ concentration (200 mM) in LB liquid media
gradually leads to cell death and the emergence of aggregates, giving
the ∆ybeX strain a survival advantage over the WT, which
increases to an order of magnitude during 6 hours of incubation
(Fig. S6 ). While this positive effect can only be seen under
very high Mg2+ concentrations (between 200 mM and 100
mM, data not shown), it is the opposite of what one expects to see when
growing an Mg2+-efflux deficient strain in extremely
high extracellular MgCl2. Clearly, the mechanistic role
of the YbeX in MgCl2 homeostasis awaits further
clarification.
We currently favor the provisional model whereby the effect ofybeX deletion on ribosomal metabolism is indirect, happening
through an increased concentration of intracellular
Mg2+. According to this model, the YbeX-promoted
Mg2+ efflux is needed in the late exponential phase,
when cell growth rates begin to fall, and ribosomes are degraded (Piiret al. , 2011), releasing some of the ribosome-bound, as well as
NTP-bound, Mg2+ into the free Mg2+pool. As both very low and very high Mg2+concentrations are detrimental to cells, mainly through translation, the
intracellular free Mg2+ is tightly controlled between
1 mM and 5 mM (Akanuma, 2021) and actively regulated
Mg2+ efflux can be an integral part of metal
homeostasis management in bacteria (Wendel et al. , 2022).In vitro translation is very sensitive to increased
Mg2+ concentration, already being >95%
inhibited at 6 mM MgCl2 (Borg and Ehrenberg, 2015).
Intriguingly, and in accordance with the role of YbeX in maintaining
Mg2+-homeostasis, we found that expression of theybeX from a high-copy plasmid is toxic to both WT and∆ybeX cells, even in the absence of an inducer.
In conclusion, our work emphasizes that the role of magnesium
homeostasis in ribosomal metabolism should become an increasingly
fertile field of study.