DISCUSSION
This work shows that the putative Co2+/Mg2+ efflux protein YbeX is functionally involved in ribosome metabolism in E. coli . For a possible mechanism that is consistent with experimental results, we propose that growth without YbeX leads to the accumulation of 17S pre-rRNA and 16S rRNA partial degradation intermediates in the late-exponential growth phase (Fig. 6B-F ), which necessitates a longer lag phase upon outgrowth in a fresh medium. During this prolonged lag phase (Fig. 1C, Fig. 4B ), the ΔybeX 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 (Fig. 5B-E ). During the transition from exponential growth to stationary phase, lack of the YbeX leads to sensitivity to erythromycin, chloramphenicol, tetracycline, clindamycin and fusidic acid, which are known to induce cold-shock proteins or block the induction of heat-shock proteins (Fig. 3A , Fig. 8 ). Although the late-exponential phase ΔybeX cells accumulate rRNA degradation products, to some extent, even in the 70S fraction (Fig. 6B, D ), they have WT-like sucrose gradient profiles (Fig. 6A ), indicating no accumulation of significant defective ribosome-like particles. In addition, the exponential growth rate of the ΔybeXcells is indistinguishable from WT, as are the growth end-points (Fig. 1C, Fig. 7C, Fig. 8B ).
The involvement of the YbeX in ribosomal metabolism is further supported by the partial rescue of its deletion phenotype by overexpression of YbeY (Fig. 9B ), which is part of the ybeZYX operon and involved in ribosomal small subunit assembly and degradation, possibly through its enzymatic activity (Liao et al. , 2021). Although generally milder, ybeX deletion in E. coli leads to overlapping phenotypes with ybeY deletion. This raises the question of whether the products of these genes might work in the same pathway of ribosomal metabolism. Both YbeY and the YbeX exert an influence on cell growth through their effects on the ribosomal assembly and/or degradation of rRNA. One could posit a possible auxiliary role of YbeX in support of YbeY function. However, the function of the metalloprotein YbeY, with its zinc cofactor, remains enigmatic, primarily due to the lack of in vivo experimental data. Our results showing opposite effects on YbeX overexpression in ΔybeYbackground and YbeY overexpression in ΔybeX background (Fig. 9B, C ) are logically inconsistent with causal schemes where YbeX and YbeY work in a single pathway, one after another, to influence cell growth.
The ybeX /corC gene was discovered in a Salmonella enterica serovar Typhimurium screen for resistance to cobalt. It was proposed that CorC contributes to the efflux of divalent cations, possibly by sensing cations or as a co-effector of CorA (Gibson et 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 (Armitano et al. , 2016). Essentially, YbeX is a cytoplasmic protein (Sueki et al. , 2020), for which we have no direct evidence that it might be involved in Mg2+-efflux. The anticipated phenotype for a magnesium efflux mutant would be sensitivity to increased magnesium levels. In contrast, ybeX null mutant phenotypes were rescued in our experiments when cells were exposed to high magnesium, reaching as high as 100 mM MgCl2 (Fig. 7A , Fig. S7a ). Thus, our findings are inconsistent with a magnesium protective role for YbeX in E. coli . Intriguingly, the rescue of growth of theΔybeX strain by MgCl2 occurs through a threshold effect, whereby something happens between 50 μM and 75 μM MgCl2 that essentially abolishes the phenotype (Fig. 7C, Fig. S6d, e ). Understanding the role of YbeX in Mg2+ metabolism requires more experimental work.
What could be the mechanistic role of YbeX in the E. coli cell? Unlike its neighboring gene products, YbeZ and YbeY, there is no evidence that YbeX binds to the ribosome or any ribosome-associated proteins. The transition from the exponential phase to the stationary phase, where ybeX mutant phenotypes are prominent, results in the degradation of the ribosome (Piir et al. , 2011). A recent study using rRNA-FISH in E. coli and Salmonella shows a heterogeneous 16S rRNA decrease occurs during this growth transition, stabilizing into low-uniform levels in the stationary phase (Ciolli Mattioli et al. , 2023). This observation could account for the colony heterogeneity in ΔybeX strain; while a subpopulation of cells regrows faster because ribosomes are efficiently degraded, the remaining cells tend to form relatively smaller colonies because of ineffective clearance of the toxic rRNA fragments from the cell (Fig. 2B , Fig. 5B-E ). It is plausible that YbeX is needed for efficient processing and removal of rRNA decay intermediates. According to this hypothesis, during the lag phase preceding exit from the stationary phase, exponential growth starts after the degradation of the decay intermediates is completed.
Overall, our results indicate a role for YbeX in ribosome assembly and/or rRNA degradation under magnesium-limited conditions. The specific molecular mechanisms underlying the function of YbeX are yet to be elucidated.