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.