Involvement of Escherichia coli YbeX/CorC in ribosomal metabolism

YbeX of Escherichia coli, a member of the CorC protein family, is encoded in the same operon with ribosome‐associated proteins YbeY and YbeZ. Here, we report the involvement of YbeX in ribosomal metabolism. The ΔybeX cells accumulate distinct 16S rRNA degradation intermediates in the 30S particles and the 70S ribosomes. E. coli lacking ybeX has a lengthened lag phase upon outgrowth from the stationary phase. This growth phenotype is heterogeneous at the individual cell level and especially prominent under low extracellular magnesium levels. The ΔybeX strain is sensitive to elevated growth temperatures and to several ribosome‐targeting antibiotics that have in common the ability to induce the cold shock response in E. coli. Although generally milder, the phenotypes of the ΔybeX mutant overlap with those caused by ybeY deletion. A genetic screen revealed partial compensation of the ΔybeX growth phenotype by the overexpression of YbeY. These findings indicate an interconnectedness among the ybeZYX operon genes, highlighting their roles in ribosomal assembly and/or degradation.


| INTRODUC TI ON
Ribosome biogenesis is a highly regulated process encompassing concomitant transcription, processing, degradation, modification, and folding of ribosomal RNAs, equimolar synthesis, and incorporation into ribosomes of more than 50 different ribosomal proteins (Davis & Williamson, 2017).In bacteria, this is catalyzed, chaperoned, and generally facilitated by dozens of dedicated proteins working in several partially overlapping and redundant pathways (Shajani et al., 2011).
However, due to its sheer complexity, our understanding of this process relies on isolated fragments of processing/folding pathways, with minimal knowledge of many individual factors' precise mechanisms of action.
It has long been known that Mg 2+ is necessary for ribosomal assembly and translation (McCarthy et al., 1962).More recently, it was discovered that intracellular free Mg 2+ concentrations and rRNA transcription rates are actively co-regulated for achieving optimal ribosomal assembly and translation (Pontes et al., 2016).Also, Mg 2+ influx can alleviate ribosomal stress phenotypes, probably by stabilizing the ribosomal structure (Lee et al., 2019).ybeX encodes a putative Co 2+ /Mg 2+ efflux protein, which, while highly conserved in bacteria, is poorly characterized (Anantharaman & Aravind, 2003;Kazakov et al., 2003).The ybeX/corC gene was initially discovered in Salmonella enterica serovar Typhimurium in a screen for cobalt resistance and was proposed to contribute, possibly as a co-effector of the metal transport protein CorA, to the efflux of divalent cations (Gibson et al., 1991).Notwithstanding over 97% sequence similarity between Escherichia coli and Salmonella ybeX/corC, a corresponding study in E. coli is absent.
The levels of YbeX (but not YbeY and YbeZ) mRNA and protein are about two-fold reduced under low-magnesium conditions (Caglar et al., 2017).Intriguingly, as a serendipitous finding, E. coli cells that rely for growth on an artificial ribosome variant, where fused rRNAs covalently tether the subunits, have increased growth rate caused by a nonsense mutation in the ybeX gene together with a missense mutation in the ribosomal protein gene rpsA (Orelle et al., 2015).
In the E. coli genome, ybeX is located in the ybeZYX-lnt operon (Figure 1a), transcripts of which have not been fully mapped.The lnt gene, which encodes an essential inner membrane protein, is predicted to be under the control of the heat shock sigma factor σ 24 (RpoE), which responds to various stresses including osmotic stress, metal exposure, while interacting with the inner membrane and promoting the degradation of misfolded proteins (Keseler et al., 2013).The transcription of ybeZ, ybeY, ybeX, and lnt is regulated by the primary heat shock sigma factor σ 32 (RpoH) (Nonaka et al., 2006).
The most-studied member of the ybeZYX-lnt operon is the ybeY, whose importance in ribosomal metabolism is beyond dispute, while its precise mode of action remains unclear (Davies et al., 2010).The YbeY is, by sequence homology and structural studies, a zinc-dependent RNA endonuclease.It is extremely highly conserved, has strong, albeit heterogeneous phenotypes in every organism that has been looked into, and is required to correctly process the 3′ end of 16S rRNA (Liao et al., 2021).Moreover, ybeY mutants have been shown to be defective in translation and to accumulate defective ribosomes in several bacterial species, mitochondria, and chloroplasts (D 'Souza et al., 2021;Liao et al., 2021;Liu et al., 2015).And yet, in the purified form, its RNase activity seems to be limited to short RNA oligonucleotides (Babu et al., 2020;Jacob et al., 2013), while in vitro processing of the 16S rRNA 3′-end can be achieved without it (Smith et al., 2018).Longer and more structured RNA substrates were found to be cleaved by human YBEY in vitro (Ghosal et al., 2017;Summer et al., 2020), while RNases required for in vivo processing of the mitochondrial 12S rRNA are all accounted for.
The ybeZ gene is located upstream of ybeY, having four nucleotides overlap.ybeZ encodes a phosphate starvation-regulated PhoH subfamily protein with the NTP hydrolase domain (Kim et al., 1993).
YbeZ has phosphatase activity and is a putative RNA helicase through sequence homology (Andrews & Patrick, 2022;Kazakov et al., 2003).A physical interaction between YbeY and YbeZ was suggested based on bacterial two-hybrid system experiments in E. coli (Vercruysse et al., 2016).Their interaction has been biochemically verified in Pseudomonas aeruginosa (Xia et al., 2020).In an E. coli interactome study, it has been observed that YbeZ not only interacts with YbeY but also exhibits interactions with numerous ribosomal proteins (Butland et al., 2005).
In this work, we investigate the effects of ybeX deletion on E. coli growth and ribosomal metabolism.

| RE SULTS
2.1 | Deletion of ybeX leads to heat sensitivity and longer outgrowth from the stationary phase ybeX belongs to the RpoH heat response regulon (Nonaka et al., 2006).We tested by a spot assay the effect of elevated growth temperature on the ΔybeX strain from the Keio collection (Baba et al., 2006), compared to the isogenic wild-type (WT) strain.After overnight growth in the LB liquid medium, serial dilutions of the cultures were spotted on LB agar plates and incubated at 20°C, 37°C, or 42°C overnight.Disruption of the ybeX gene hindered growth at 42°C but not at 20°C (Figure 1b; Figure S1a).For verification, ybeX deletion was reintroduced in two strain backgrounds, MG1655 and BW25113 (see Figure S1b,c for strain construction).Heat sensitivity was retained in newly constructed ΔybeX strains (Figure S1d), demonstrating that the observed phenotype is ybeX-inflicted.We used the ybeX deletion strain of the Keio collection in further experiments.
Next, we assessed whether the lack of the YbeX protein caused the heat sensitivity, as secondary effects of the chromosomal deletion could be responsible for the phenotype.We reintroduced ybeX on a single-copy TransBac library plasmid (Otsuka et al., 2015) and found that the leaky expression of YbeX in the absence of the inducer (isopropylβ-D-1-thiogalactopyranoside; IPTG) is sufficient to rescue the heat sensitivity phenotype of the ΔybeX mutant.The empty vector and TransBac plasmids carrying ybeY or ybeZ had no effect on growth in the presence or absence of the inducer (Figure 1b; Figure S1e).Thus, the heat sensitivity of the ΔybeX strain is caused by the absence of the YbeX protein rather than through polar effects on neighboring genes.
To find which growth phase is affected by the ybeX deletion, we monitored bacterial growth in liquid LB medium at 37°C in 96well plates.We did not notice differential growth of WT and ΔybeX strains when cultures were started from freshly grown single colonies (data not shown).In contrast, when cultures were inoculated with bacteria from the stationary phase overnight cultures, the ΔybeX mutant had a much longer lag phase (300-350 min) than the WT (100-150 min; Figure 1c; Figure S2a).Both strains reached similar optical densities in the stationary phase with similar growth rates (Figure 1c; Table S1).A similar number of colonies of WT and ΔybeX strains (Figure 1b) indicates that the delay of the visible growth of the ΔybeX mutant is not caused by decreased survival in the stationary phase.The expression of ybeX from a single-copy TransBac library plasmid completely complemented the prolonged lag phase.In contrast, complementation with plasmids carrying ybeY, ybeZ, or lnt had no effect, confirming that lack of the YbeX protein is causing the delay of regrowth while again excluding polar effect as the cause of the ΔybeX phenotype (Figure 1c; Figure S2b).
To investigate whether the longer lag phase of the ΔybeX strain is due to lower metabolic activity in the mutant cells, we used the Alamar Blue reagent, a quantitative indicator of the oxidation-reduction potential of cell membranes, as a proxy for metabolic activity (Rampersad, 2012).In a control experiment conducted in PBS buffer lacking the nutrients necessary for the resumption of growth, both strains show similarly low Alamar Blue signal, indicating similar levels of metabolic activity (the superimposed black lines in Figure S2c).

F I G U R E 1
Growth phenotypes of ΔybeX strain and compensation with single-copy plasmid.(a) The Escherichia coli ybeZYX-lnt operon chromosomal organization with sigma factors (σ 32 and σ 24 ) and promoters (ybeZp and the predicted lntp4).(b) Dot spot assay of wild-type (WT) and ΔybeX strains, along with the strains harboring the TransBac library plasmid backbone (WT/pEmpty or ΔybeX/pEmpty), and the ΔybeX strain with the YbeZ, YbeY, or YbeX-expressing single-copy TransBac library plasmid.LB agar plates were incubated at 37°C or 42°C.(c) Regrowth assay of cultures grown overnight in LB medium.The experiment was done in a 96-well plate reader, and lag times were calculated using Gen5 software (BioTek).The growth rates were calculated using R::growthcurver package (Sprouffske & Wagner, 2016)  When cells were diluted into fresh LB medium, the Alamar Blue fluorescence immediately started to increase for both strains, indicating similar levels of cellular metabolism (Figure S2d,e).While the initial rate of increase in the Alamar Blue fluorescence is indistinguishable in WT and ΔybeX cells, the WT acquires a faster rate of metabolism after about 100 min, while the ΔybeX cells continue as before for about 200 more minutes (Figure S2d).As shown by the OD 600 measurements (Figure S2a), for both the WT and the ΔybeX cells, the increase in fluorescence is accompanied by the start of regrowth (Figure 1e; Figure S2e).These results indicate that the extended lag phase of the ΔybeX strain is not caused by lower levels of metabolic activity upon transfer from the stationary phase culture into the regrowth medium.

| The delayed outgrowth of the ΔybeX mutant is heterogeneous at the individual cell level
When streaking out mutant strains from glycerol stocks and overnight grown stationary phase cultures, we noticed that the ΔybeX strain produces colonies of different sizes.Re-streaking of small and large ΔybeX colonies resulted in similar levels of heterogeneity with WT in second-generation colonies, indicating that the heterogeneous phenotype is not caused by a genetic mutation (data not shown).We hypothesized that the colony size heterogeneity of ΔybeX is a result of the delayed outgrowth of individual bacteria and indicates physiological heterogeneity of the stationary phase inoculum.
Inspection of colony sizes plated from overnight grown bacterial cultures showed, in agreement with our previous observations, that ΔybeX cells tend to form smaller colonies than WT cells when grown overnight in LB or MOPS minimal medium supplemented with 0.3% glucose (Figure 2a; Figure S3a).To quantify this phenotype at the individual cell level, colony radii of ΔybeX and WT were quantified from four independent stationary phase outgrowth experiments using AutocellSeg (Khan et al., 2018).Overnight-grown cells were plated from LB or MOPS minimal media, and plates were incubated at 37°C or 42°C overnight.When plated from the LB medium, ΔybeX cells formed smaller and more heterogeneous colonies than WT cells (Figure 2a,b).In contrast, when plated from the MOPS minimal medium, the ΔybeX colonies appear to be consistently smaller and more homogeneous in size (Figure 2c).We found that a fraction of ΔybeX  cells lost the ability of colony formation when plates were incubated at 42°C (Figure 2d).When plated from LB, this drop was about 10fold (p < 0.0001) and from MOPS medium about two-fold (p = 0.055, Figure 2d).Nonetheless, the quantified colony radii on plates incubated at 42°C resembled those at 37°C (Figure 2b,c).
While the ΔybeX colony sizes were increased after 48 h of incubation at 37°C, they consistently remained heterogeneous in size in the absence or the presence of the kanamycin resistance cassette (Figure S3b,c).In conclusion, the ΔybeX cells grow in at least two distinct regimes, one similar to WT growth, while the other results in up to two-fold smaller colonies.

|
The ΔybeX strain is sensitive to ribosome-targeting antibiotics ybeX disruption has been reported to cause decreased survival in the presence of chloramphenicol (CAM; Smith et al., 2007).Therefore, we explored the effects of various antibiotics on the ΔybeX cells.
We further inspected the effects of these antibiotics using the dot spot assay described above, except that the LB agar plates were supplemented with sub-MIC concentrations of indicated antibiotics (see Section 4).The ΔybeX strain exhibited severe sensitivity to sub-MIC concentrations of CAM, tetracycline, erythromycin, clindamycin, and fusidic acid (Figure 3a).The presence of ybeX single-copy plasmid in the absence of inducer fully rescued the described antibiotic sensitivities (Figure 3b).In contrast, protein synthesis-targeting antibiotics that do not induce the cold shock response (amikacin, streptomycin, kanamycin, tobramycin, and mupirocin) exhibit a similar inhibitory effect on ΔybeX and WT strains.The RNA synthesis inhibitor rifampicin also revealed no differential effect on the ΔybeX strain (Figure 3b; Figure S4b).
To exclude strain-specific effects, we tested two isogenic WT strains, MG1655 and BW25113 and the corresponding deletion strains ΔybeX::kan MG and ΔybeX::kan BW under sub-inhibitory antibiotic concentrations (Figure S4b).Both genetic backgrounds exhibited similar antibiotic sensitivities, and removal of the kanamycin resistance cassette (in strains ΔybeX/−kan MG and ΔybeX/-kan BW ) had no effect.metabolic activity to the WT cells during this lag phase, as well as similar exponential growth rate (Figure 1d).This led us to hypothesize that any cellular defects conferred by the lack of YbeX could accumulate stochastically during late growth, preceding entry into the stationary phase and/or in the stationary phase itself, which in turn could lead to the observed single-cell heterogeneity during outgrowth (Figure 2).Accordingly, we tested whether the phenotypes of ΔybeZ, ΔybeY, and ΔybeX depend on the growth phase of the culture from where the cells originate.We surmised that if a gradual accumulation of harm causes the ΔybeX phenotype, then cells that have had more time to accumulate such harm should exhibit stronger phenotypes.

| The delayed outgrowth and antibiotic sensitivity of ΔybeX depend on the growth history of bacteria
Overnight-grown cultures of ΔybeZ, ΔybeY, ΔybeX, and WT strains were serially diluted and spotted on LB agar plates to assay the heat and antibiotic sensitivity.The same stationary phase cultures were diluted a hundred-fold into fresh LB medium and then grown at 37°C for four to five cell divisions until OD 600 reached 0.2-0.4,after which the cells were diluted and spotted on LB agar plates to assay the heat and antibiotic sensitivity of exponentially growing cells.
The ΔybeX stationary phase inoculum exhibited sensitivity to sub-MIC concentrations of CAM and erythromycin but no sensitivity to rifampicin in comparison to WT (Figure 4a).In contrast, the exponentially growing ΔybeX had WT-like sensitivity to all tested antibiotics.
In comparison, ΔybeZ cultures had intermediate levels of sensitivity to CAM, regardless of the growth history of cells, while they are not more sensitive to erythromycin, rifampicin, and tetracycline as compared to WT (Figure 4a).Exponentially growing ΔybeZ cells in MOPS minimal medium, supplemented with 0.3% glucose, also exhibited sensitivity to CAM (Figure S4a).ΔybeY cells were very sensitive to all tested antibiotics, notwithstanding the growth phase of the spotted culture.
In liquid media, cultures started directly from stationary phase inocula again showed a lengthened lag phase for ΔybeX but not for  were very similar to WT (Figure 4b).The ΔybeY strain behaves similarly, regardless of the growth phase of inoculums, exhibiting a reduced exponential growth rate and reaching a lower maximal cell density.In contrast, when the cells were outgrown from exponential phase cultures, the WT, ΔybeZ and ΔybeX strains grew equally well, with no visible lag phase, while the ΔybeY strain had a reduced growth rate and a lower growth end-point, as expected (Figure 4c).

| The ΔybeX cells accumulate rRNA fragments in stationary phase
As ybeX is located in the same operon with ybeY, which is implicated in ribosome assembly, we assessed the rRNA profiles of total cellular RNA of the WT and the ΔybeZ, ΔybeY, and ΔybeX strains by Northern blotting.For membrane hybridization, we used fluorescence probes specific for the 17S precursor rRNA, the mature 16S rRNA and the 23S rRNA (Figure 5a).
In exponentially growing ΔybeY cells, we saw a substantial accumulation of immature 17S rRNA, while ΔybeX and ΔybeZ cells had comparable levels of 17S rRNA to wild type (Figure 5b,d,e).ΔybeY cells also accumulate a faster migrating 16S rRNA species, labeled as 16S* (Figure 5b; see also (Davies et al., 2010)).The ΔybeX lysates do not contain the 16S* rRNA species.
When we assessed the total RNA extracted from stationary phase ΔybeX cell cultures, we observed an accumulation of 16S and 23S rRNA fragments (Figure 5b,c).These fragments were not present in material obtained from exponentially grown ΔybeX cells.WT,  In addition, the stationary phase ΔybeX cells accumulate a wide spectrum of 17S pre-rRNA degradation intermediates ranging from a couple of hundred nucleotides to almost full-length 16S rRNA, as detected by the 17S 5′-end-specific oligonucleotide (Figure 5d).
Figure 5e, where we use a 3′-end-specific oligonucleotide, indicates that these decay intermediates lack the 17S 3′-end.WT, ΔybeZ, and ΔybeY lysates lack such degradation intermediates.Thus, the ΔybeX cells have a unique and disparate mixture of 17S rRNA and 16S rRNA degradation intermediates.

| The ΔybeX strain accumulates distinct rRNA species already during the late exponential growth
As there is neither substantial assembly nor degradation of mature ribosomes in the stationary phase (Piir et al., 2011), we conjectured that the accumulated fragments observed in ΔybeX cells were likely getting there by the late exponential phase.Accordingly, we purified ribosomal subunits from late exponential cells by sucrose gradient fractionation and assayed the rRNA composition of the 70S ribosomes, 50S and 30S ribosomal subunits by Northern blotting.
The sucrose gradient profiles for WT and ΔybeX lysates are very similar, with the vast majority of ribosomal particles being in the 70S ribosome fraction and the relatively minor free subunit fractions exhibiting no apparent abnormalities (Figure 6a).Northern blots revealed full-length 17S pre-rRNA in the 30S fractions of both the WT and the ΔybeX strains, as detected via 16S and 17S rRNA-specific oligonucleotides (Figure 6b-e).In the ΔybeX strain, the mature 16S rRNA is substantially cleaved at certain positions in the 30S fraction (Figure 6b,c).Thus, in the ΔybeX cells, the 30S (SSU) fraction was unlikely to contain many functionally active small ribosomal subunits.
In  We tested the effect of CAM, a well-studied protein synthesis inhibitor (Wilson, 2014), on the ribosomes by sucrose gradient fractionation and Northern blotting (Figure S5a).The sucrose gradient profiles of the WT and ΔybeX strain lysates were similar, while CAMtreated ribosomal particles sedimented notably differently from those of mature subunits (Figure S5b; see also Siibak et al., 2009).
However, in the ΔybeX strain, in Northern blots, we observed distinct aberrant 16S rRNA fragments, which were absent in the WT (Figure S5c).In addition, the CAM treatment, which leads to bacterial growth arrest, stabilizes the ΔybeX 30S particles.While CAM 30S particles contain a good measure of 16S rRNA and 17S pre-rRNA in the ΔybeX strain, the ΔybeX 30S particles from the control experiment without CAM treatment contain fragments of SSU rRNA and reduced amounts of mature SSU rRNA (Figure S5c).These findings indicate that the accumulation of decay intermediates in ΔybeX is not an artifact that occurs during the purification of ribosomal samples but occurs in vivo.This interpretation is further supported by the fact that hot phenol-extracted total RNA samples harbor similar fragments of rRNA (Figure 5).
These results indicate that in the late exponential phase, RNA in most of the free 30S subunits of the ΔybeX strain is being fragmented.Moreover, the degradation fragments captured by the 16S rRNA and 17S rRNA-specific probes strongly suggest that in the ΔybeX strain, pre-ribosomes (in the 30S fraction) and mature ribosomes (in the 70S fraction) are susceptible to degradation.

| The ΔybeX mutant phenotypes can be suppressed by MgCl 2 supplementation
As YbeX has been implicated in Mg 2+ efflux (Gibson et 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 (Figure 7a; Figure S6a).When bacteria were grown in LB medium that was supplemented with 10 mM MgCl 2 , the antibiotic and heat sensitivity of ΔybeX mutant upon plating disappeared (Figure 7a).To test whether the effect of Mg 2+ is media dependent, we used the SOB medium, which contains 10 mM MgCl 2 .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 (Figure 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 (Li et 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 (505 min [95% CI = 491; 518]) than in LB (319 min [95% CI = 307; 331], Figure S6c), while WT cells grown in LB or PBM did not differ in outgrowth (see Table S1).The heat sensitivity upon plating was more severe in PBM than in LB medium (Figure 7b; Figure S6d,e).Supplementation of LB medium with 50 μM MgCl 2 fully suppressed the outgrowth delay of ΔybeX at 37 and 42°C (Figure S6d).In contrast, PBM with 50 and 100 μM MgCl 2 partially suppressed and 200 μM MgCl 2 completely suppressed the outgrowth delay of ΔybeX at 37 and 42°C (Figure 7b; Figure S6e).
Furthermore, contrary to YbeX Mg 2+ efflux annotation, supplementation of LB with 100 mM MgCl 2 was not detrimental to the ΔybeX, and had no impact on growth compared to WT (Figure S7a).
We further tested the Mg 2+ sensitivity of the ΔybeX mutant in the MOPS minimal medium supplemented with 0.5% glucose.Unlike in rich complex growth media, we can precisely control the magnesium levels in the MOPS medium (Neidhardt et al., 1974).The ΔybeX cultures achieved similar optical density plateaus to the wild type.
This holds for a wide range of Mg 2+ concentrations in the MOPS minimal medium (Figure S7b).When WT cells were grown into stationary phase in the MOPS minimal medium, the Mg 2+ concentration had no effect on the time-to-outgrowth (Figure 7c, the left panel).
Neither was there any effect on the growth rates (Table S1).Under matching conditions, the ΔybeX strain grown in ≤50 μM Mg 2+ exhibited extended lag phases of 350-400 min (see Table S1 for details).
The presence of ≥75 μM MgCl 2 decreases the lag time to about 200 min, after which additional magnesium has little effect on the duration of the lag phase (Figure 7c, the right panel).These results indicate that the effects of ybeX deletion can be compensated by elevated Mg 2+ concentration of the growth medium.
To examine the potential impact of other divalent metals on suppression of ΔybeX phenoytpes, we assessed the ability of Mn 2+ , Cu 2+ , Ni 2+ , Fe 2+ , Ca 2+ , Co 2+ , and Zn 2+ to compensate the prolonged lag phase.Only Mn 2+ exhibited a suppressive effect.LB medium with 200 μM MnCl 2 partially suppressed and with 500 μM MnCl 2 completely suppressed the outgrowth delay of ΔybeX at 37°C (Figure 7d).

| ΔybeX phenotypes are prominent under low extracellular magnesium and during the transition into the stationary phase
Our ability to control the ΔybeX phenotype by Mg 2+ allowed us to pinpoint the growth phase dependence of the ΔybeX phenotype more precisely.Therefore, in the next experiment, we first grew the cultures overnight into the stationary phase in MOPS minimal medium supplemented with 10 mM MgCl 2 and 0.5% glucose, where ΔybeX phenotype does not occur.Then, the cells were washed thrice with MOPS minimal medium lacking Mg 2+ , after which the regrowth assay was set up by suspending the cells in MOPS containing 10 μM MgCl 2 (Figure 8a).
As expected, there is no difference in the duration of the outgrowth lag phase between the WT and ΔybeX, and the exponential growth rates were the same (Figure 8b).To look for the emerging ΔybeX phenotype, we plated spots of samples from the outgrowth agar plates (R2A stimulates the growth of stressed bacteria).The plates were incubated overnight at 37°C or 42°C.While until the 4-h time point there was no difference between the colony formation of WT and ΔybeX (Figure 8c), upon transition into the stationary phase, in the 5.5-h time point, there is a growth delay of the ΔybeX strain on both LB or R2A agar plates, which is more pronounced at 42°C.
Similarly, the sensitivity of the ΔybeX strain to tetracycline, erythromycin, and CAM antibiotics appears only at the 5.5-h time point (Figure 8d).We conclude that the delay of regrowth and antibiotic sensitivity of the ΔybeX strain appears under low extracellular magnesium at the transition from exponential to stationary growth phase.

| Overexpression of YbeY partially rescues the ΔybeX phenotype
We  et al., 2010), to search for genes that compensate for the deletion of ybeX.The selection was carried out in two independent experiments for three 12-h rounds, resulting in enriched plasmid pools (Figure 9a).
We sequenced about 150 clones from the enriched plasmid pools.The ybeY-coding plasmid was predominant (>90%) among sequenced clones (Table S3).Surprisingly, we did not recover a single ybeX coding plasmid, suggesting the harmfulness of ybeX overexpression.Accordingly, the multi-copy ybeX-coding plasmid led to equally strong growth inhibition in WT and ΔybeX cells (Figure S8a).In contrast, the ybeY-coding plasmid had no visible detrimental effect on the growth of the ΔybeX mutant.
We also measured growth in liquid LB medium.In this experiment, the multi-copy ybeX plasmid in the ΔybeX background further increased the duration of the lag phase while also leading to a lower plateau of the growth curve (Figure S8b).In contrast, the multi-copy ybeY plasmid in the ΔybeX background partially rescues the lag phase phenotype (Figure S8b).In the light of this partial rescue of the ΔybeX phenotype by the multi-copy ybeY coding plasmid, we next tested whether overexpression of YbeY from an inducible pET-based multicopy plasmid under the control of the tac promoter can further rescue the ΔybeX lag phenotype.We found that overexpression of YbeY in ΔybeX strain in the presence of 1 mM IPTG did not result in complete rescue of the ΔybeX lag phase phenotype (Figure 9b).
We further tested whether overexpression of YbeX and YbeY to a strong growth rate reduction and a lower final culture density.
Furthermore, we conjugated the TransBac library plasmid overexpressing YbeX into three additional Keio deletion strains of genes whose products are associated with 30S ribosomal assembly.Deletion of rimM and yjeQ is known to impede growth at 37°C, while ksgA deletion does not affect bacterial growth at 37°C (Shajani et al., 2011).We found that the growth of ΔksgA was not significantly affected by YbeX overexpression, while ΔrimM and ΔyjeQ strains exhibited growth inhibition (Figure 9d,e).Therefore, sensitivity to ybeX overexpression is not a YbeY-specific phenomenon but seems to be associated with defective ribosomal assembly in general.

| DISCUSS ION
This work shows that the putative Co 2+ /Mg 2+ 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 (Figure 6b-f), which necessitates a longer lag phase upon outgrowth in a fresh medium.During this prolonged lag phase (Figures 1c and 4b), the ΔybeX cells are metabolically active (Figure 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 (Figure 5b-e).
During the transition from exponential growth to stationary phase, lack of the YbeX leads to sensitivity to erythromycin, CAM, tetracycline, clindamycin, and fusidic acid, which are known to induce cold-shock proteins or block the induction of heat-shock proteins (Figures 3a and 8).Although the late-exponential phase ΔybeX cells accumulate rRNA degradation products, to some extent, even in the 70S fraction (Figure 6b,d), they have WT-like sucrose gradient profiles (Figure 6a), indicating no accumulation of significant defective ribosome-like particles.In addition, the exponential growth rate of the ΔybeX cells is indistinguishable from WT, as are the growth endpoints (Figures 1c, 7c, and 8b).
The involvement of the YbeX in ribosomal metabolism is further supported by the partial rescue of its deletion phenotype by overexpression of YbeY from multi-copy plasmid (Figure 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 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).
Nevertheless, in Francisella tularensis which does not have a corA homolog, still has YbeX contributing to pathogenesis and innate immunity evasion.As yet, there is no mechanistic function ascribed to YbeX, and while Mg 2+ 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 Mg 2+ 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 MgCl 2 (Figure 7a; Figure S7a).Thus, our findings are inconsistent with a magnesium protective role for YbeX in E. coli.Intriguingly, the rescue of growth of the ΔybeX strain by MgCl 2 occurs through a threshold effect, whereby something happens between 50 and 75 μM MgCl 2 that essentially abolishes the phenotype (Figure 7c 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 hetero-  S4 and S5.
E. coli DH5α strain was used for plasmid cloning and propagation.
The TransBac library, an unpublished new E. coli overexpression library based on a single-copy vector, was obtained from Dr Hirotada Mori (Nara Institute of Science and Technology, Japan) as a stab stock (Otsuka et al., 2015).
Hfr + donor strains require DAP to grow because of the deletion of the dapA.Well-grown donor and acceptor cells were mixed (1:1) in a 1.5-mL polypropylene tube and incubated at 37°C for 1 h without shaking.

| Construction of the TransBac empty (pTB-empty) plasmid
The single-copy TransBac library plasmid coding ybeX was purified using an in-house alkaline lysis method followed by purifica-  S5).The plasmid was electroporated into WT and ΔybeX strains.

| Preparation of the PBM
Growth in a PBM is magnesium limited (Christensen et al., 2017).

| Colony size characterization and quantification
Keio WT and ΔybeX strains were grown overnight in LB or defined MOPS minimal medium (Neidhardt et al., 1974).Well-grown bacterial cell cultures were serially diluted and plated on LB agar plates using glass beads (Hecht Assistent, #41401004).We aimed to have approximately 100-125 colonies per plate.The plates were incubated overnight at 37°C or 42°C and scanned using EPSON Expression 1680pro scanner with a pre-defined scan setting.The images were subjected to AutoCellSeg software (Khan et al., 2018), a MATLAB-based supervised automatic and robust image segmentation method.The colonies were first picked automatically using program default settings, and then, as a second step, manual picking was applied (picking small colonies, deselecting adherent colonies, etc.).The data were analyzed in the R packages tidyverse and visualized with ggplot2 (R Core Team, 2022; Wickham et al., 2019).

| Growth monitoring in the 96-well plate reader
Cells were diluted in the appropriate growth media to OD 600 = 0.55, and 10 μL of the diluted cells were transferred into 100 μL of growth medium in a 96-well plate (Anicrin, Flat bottom, #18EMP.I.).The 96-well plate edges were filled with distilled water or 1 × PBS.The remaining 60 wells were used to monitor the growth.At least one column was always set as a sterility control.Alamar Blue reagent (BioRad, #BUF012B) was used per the manufacturer's protocol (excitation 545 nm, emission 590 nm).BioTek Synergy MX or H1 microplate readers were used.

| Purification of the pooled E. coli K-12 ORFeome plasmid library
A previously described E. coli ORFeome plasmid library (Rajagopala et al., 2010) was received as a stab stock in a 96-well plate format (National BioResource Project, Japan).The clones were grown in LB medium supplemented with 50 μg/mL Zeocin for 24 h at 37°C (Velp Scientifica FTC 90I).The cell cultures were pooled, and the plasmids were purified using a NucleoBond Xtra Midi kit (Macherey-Nagel).

| Library selection experiment
The purified plasmid library was electroporated into ΔybeX::kan.The cells were recovered in LB medium for 1 h at 37°C 850 rpm shaking thermostat (Eppendorf Thermomixer compact).The recovery culture was diluted in LB medium supplemented with 50 μg/mL Zeocin and grown for 12 h at 37°C 200 rpm shaking (Infors HT Minitron shaker).After 12 h, the cell cultures were diluted 100× in fresh LB containing 50 μg/mL Zeocin and grown for an additional 12 h at 37°C.The previous step was repeated, and the plasmids were purified at every stage (12, 24, and 36 h).The purified plasmid pools were cleaved via FastDigest SfiI (Thermo Scientific) restriction enzyme at 50°C.Excision of the ORF inserts of the plasmid pools revealed in agarose electrophoresis several bands, of which the most prominent was slightly over 500 kb (Figure S8c).The enriched plasmid pools were introduced into DH5α Inoue chemical competent cells (Green & Sambrook, 2020) and plated on LB agar plates containing 25 μg/ mL Zeocin (Invivogen).Colony PCR was performed using ISM1 (GGC TTG GCC CTG AGG GCC) and ISM2 (GTG GCG GCC GCA TAG GCC) primers and employing the experimental conditions for TransBac library plasmids.

| Construction of pET-based YbeY overexpression plasmid
The pET-ybeY overexpression plasmid was constructed using the CPEC (Circular Polymerase Extension Cloning) method (Quan & Tian, 2011).The primers were designed to amplify the pET-Orange plasmid backbone and the coding region of the ybeY gene from the E. coli genome (sequences are detailed in Table S5).Phusion™ HF DNA Polymerase (Thermo Scientific, #F530L) was used following the manufacturer's protocol.

| Sucrose gradient fractionation
E. coli strains from the Keio collection were streaked onto LB agar plates and grown overnight at 37°C.A single colony of each strain was inoculated into LB and aerated at 37°C overnight.The following morning, the culture densities were determined via spectrophotometer (Biochrom Ultrospec 7000); the cells were diluted to a final OD 600 of 0.05-0.06 in LB medium (150-250 mL) and grown until OD 600 = 0.3-0.35.The cultures were then split into two flasks, in which the CAM treatment was carried out, while the other was grown as a control for 2 h.
The cells were transferred into centrifugation bottles, cooled on ice and pelleted at 4000×g, at +4°C for 10 min.The supernatant was removed, and the cell pellet was snap-frozen in liquid nitrogen and stored at −80°C.For cell lysis, the frozen cell pellets were thawed on ice and then taken up in 1 mL of lysis buffer consisting of 25 mM Tris-HCl pH 7.9, 60 mM KCl, 60 mM NH 4 Cl, 6 mM MgCl 2 , 5% glycerol supplemented with 1 mM PMSF, protease inhibitor (Roche, #04693159001) and 5 mM βME added freshly to the buffer.The cells were lysed using FastPrep homogenizer (MP Biomedicals) by three 40-s pulses at 4.0 m/s, chilling on ice for 5 min between the cycles.The beads were purchased from BioSpec Products, and 0.4 g . The empty circles represent biological replicates, and error bars denote the 95% CI-s.(d, e) The stationary phase outgrowth of the wild-type and the ΔybeX strains in liquid LB medium.Panel (d) shows the OD 600 signal, and panel (e) shows the Alamar Blue fluorescence reading.Both signals are normalized to one.Individual measurements from independent experiments, each presented as the mean of three technical replicates, are shown as dots.Curves are modeled with splines, and 95% credible intervals are shown as shaded areas (see Section 4 for details).pybeX, pybeZ, and pybeY denote the YbeX, YbeZ, and YbeY expressing plasmid.
antibiotics have been shown to induce cold-shock proteins or block the induction of heat-shock proteins(Cruz-Loya et al., 2019; While the ΔybeX cells have a lengthened lag phase during outgrowth from the stationary phase, they appear to retain similar levels of F I G U R E 3 ΔybeX cells exhibit severe sensitivity to sub-minimal inhibitory concentration (MIC) concentrations of cold-shock-inducing, ribosome-binding antibiotics.(a) The wild-type (WT) and the ΔybeX cells were grown overnight in LB liquid medium, serially diluted, and spotted on LB agar plates supplemented with sub-MIC concentrations of indicated antibiotics.For controls, plates without antibiotics (No AB) were used.The plates were incubated at 37°C overnight.(b) Representative images are given from a dot spot assay with strains described in Figure 1.pYbeX denotes the YbeXexpressing TransBac library plasmid.Images are representative of at least three independent experiments.
ΔybeZ, while the exponential growth rates of both ΔybeX and ΔybeZ F I G U R E 4 The ΔybeX-dependent antibiotic sensitivity and outgrowth delay depend on the growth phase of the inoculum.(a) Drops of the stationary phase and exponential phase cultures of the Keio wild-type (WT), ΔybeX, ΔybeZ, and ΔybeY strains were spotted on LB-agar plates.The cells were cultured in LB medium, and the LB agar plates were incubated at 37°C, except for the 42°C plates.(b-c) Stationary and exponential phase cells of wild-type, ΔybeY, ΔybeZ, and ΔybeX strains were inoculated into liquid LB medium, and the growth was monitored by measuring OD 600 at 37°C using a 96-well plate reader.The growth curves, summarizing three independent experiments, are shown as fitted logistic curves.Shaded areas represent the 95% CIs.

5
Deletion of ybeX leads to the accumulation of rRNA fragments during the stationary phase.(a) rRNA operon scheme with locations of the Cy5-labeled oligonucleotides indicated as red stars.(b-e) Hot-phenol extracted total RNA samples from the exponential and stationary phase cultures of wild-type, ΔybeY, ΔybeZ, and ΔybeX strains were separated on 1.5% denaturing agarose gel for Northern blotting.The cells were grown in LB medium at 37°C with aeration.The membrane was hybridized with 16S-EUB (b), 23S h25 (c), 17S 5′-end (d), and 17S 3′-end (e) rRNA targeting fluorescent oligos.
ΔybeZ, and ΔybeY cells exhibited no such fragments in stationary or exponentially growing cells.Both ΔybeX and ΔybeY stationary cells accumulate 17S rRNA (Figure5d,e).
17S rRNA fragments were present only in the 30S fraction of ΔybeX, as detected via 17S 5′ and 3′ ends rRNAspecific probes (Figure6d,e).Truncated 17S pre-rRNA fragments, presumably arising from the precursor SSU particles, are absent in the 70S and 50S fractions (Figure6d,e).Decay intermediates in the 30S fraction indicate that most pre-SSU particles are inactive and degradation-bound in late-exponential phase ΔybeX cultures.In contrast, the 23S rRNA-specific probe reveals only few specific prominent fragments in degradation patterns between WT and ΔybeX strains (Figure6f).

F I G U R E 7
Magnesium supplementation rescues the ΔybeX phenotypes in various growth media.(a) The WT and ΔybeX cells were grown overnight in LB, SOB growth media, or LB supplemented with 10 mM MgCl 2 .The cells were serially diluted and spotted on LB agar plates.The plates with antibiotics were incubated at 37°C.There were also controls without antibiotics, denoted "No AB", at 37°C or 42°C, as indicated in the first two sub-panels.(b) A single colony of WT or ΔybeX was grown overnight in the magnesium-limited peptide-based medium (PBM).0 μM denotes no MgCl 2 supplementation; otherwise, PBM is supplemented with 50, 100, and 200 μM MgCl 2 .The cells were plated as in panel (a).(c) WT and ΔybeX cells were grown overnight at 37°C in MOPS minimal medium supplemented with indicated concentrations of MgCl 2 and 0.5% glucose.The outgrowth from these stationary phase cultures was tested in 1 × MOPS minimal medium (0.5% glucose, 525 μM MgCl 2 ) in 96-well plates at 37°C with aeration.The growth curves summarize three independent experiments shown as fitted logistic curves.Shaded areas represent the 95% CI-s.(d) Regrowth assay of cultures grown overnight in LB medium supplemented with divalent metal salts at 100, 200, or 500 μM.The experiment was done in a 96-well plate reader and lag times were calculated using Gen5 software version 3.10 (BioTek).The empty circles represent biological replicates, and error bars denote the 95% CI-s.time points (Figure8b) onto both LB and R2A used a constitutively expressed E. coli open reading frame (ORF) plasmid library representing 3974 ORFs, cloned in a F I G U R E 8 The growth transition into the stationary phase leads to the ΔybeX phenotype.(a) A scheme of the experimental setup is given.A single colony was inoculated into MOPS minimal medium supplemented with 10 mM MgCl 2 and grown overnight at 37°C.The next day, saturated cultures were washed three times to remove residual magnesium and regrown in 10 μM MgCl 2 -containing MOPS.Aliquots for plating on LB agar were taken at 2, 3, 4 and 5.5 h.The LB agar plates either contained or did not contain antibiotics, as shown on panels (c, d), and they were incubated overnight at 37°C or 42°C.(b) The growth of the wild-type and the ΔybeX strains was monitored at an optical density of 600 nm in MOPS minimal medium supplemented with 10 μM MgCl 2 and 0.5% glucose.The mean optical densities of three biological replicates are shown with 95% CI-s.(c) Dot spot experiments were conducted as in Figure 7a, and the cells were collected from indicated time points, as in panel (b).The ΔybeX cells differed from wild-type in growth phenotype only when collected for the outgrowth spot assay at the 5.5 h time point.(d) When the outgrowth spot assay plates contained tetracycline, erythromycin, or chloramphenicol (at sub-minimal inhibitory concentration concentrations listed in Section 4), severe growth inhibition was observed at the 5.5 h time point.zeocin-resistant Gateway™ pENTR/Zeo plasmid (Rajagopala from a single-copy TransBac library plasmid influences the growth of ΔybeY phenotype (Figure 9c).Overexpression of YbeX and YbeY conferred no growth effect on WT cells.When induced in ΔybeY cells, the single-copy ybeY plasmid compensated for the lack of chromosomal ybeY.In contrast, overexpression of YbeX in ΔybeY cells led F I G U R E 9 YbeY overexpression partially rescues the delayed outgrowth in ΔybeX mutant.(a) A scheme of E. coli open reading frame plasmid library selection experiment for the search of compensatory plasmids rescuing the ΔybeX extended lag phase phenotype.The selection involved sequential regrowth in 25 mL LB medium containing 50 μg/mL Zeocin.Every 12 h, a 1:100 culture sample was transferred to a fresh LB and incubated for another 12 h at 37°C with aeration.(b) Overexpression of YbeY from a pET-based plasmid under the control of tac promoter in the presence of 1 mM IPTG leads to shortened growth lag in the ΔybeX background.(c) YbeX overexpression from a single copy TransBac library plasmid in the presence of 1 mM IPTG has a profound negative effect on the growth of the ΔybeY.The wild-type and ΔybeY strains were conjugated with the backbone TransBac library plasmid (empty), ybeX or ybeY overexpressing single-copy plasmids.The cells were grown in LB liquid medium with tetracycline and 1 mM IPTG.(d, e) ΔrimM and ΔyjeQ Keio mutant strains were conjugated with the indicated TransBac library plasmids as in panel (c).The outgrowth from stationary phase cultures was monitored with aeration in 96well plates in LB medium at 37°C.The logistic curve fits summarize three to four biological replicates, each comprising a minimum of three technical replicates.Shaded areas represent 95% CIs.
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 show opposite effects on YbeX overexpression in the ΔybeY background and YbeY overexpression in the ΔybeX background (Figure9b,c).A similar phenomenon has been noted for many ribosome assembly factors where overexpression of one factor may improve the phenotype of the other, whereas the opposite may even exacerbate the phenotype(Naganathan & Culver, 2022).

;
Figure S6d,e).A previous study examined the effects of E. coli corA, mgtA, yhiD, and corC/ybeX gene deletion with every combination of single, double, and quadruple mutants on Mg 2+ transport in E. coli.(Ishijima et al., 2015) No difference in growth was observed with corC single and double deletion mutants in LB with 0-100 mM MgCl 2 , inconsistent with YbeX involvement in Mg 2+ efflux.Understanding the role of YbeX in Mg 2+ metabolism requires more experimental work.

|
geneity 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 (Figures2b and 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 degradation under magnesium-limited conditions.The specific molecular mechanisms underlying the function of YbeX are yet to be elucidated.Bacterial strains, plasmids, and growth media Genotypes of the bacterial strains, plasmid descriptions, and sequences of primers used in this study are listed in Tables tion via FavorPrep™ plasmid DNA extraction mini kit (Favorgen, #FAPDE300).The cloning site was sequenced by Sanger sequencing (University of Tartu).The ybeX coding region was removed via restriction enzyme cleavage of FastDigest XmaJI and Sfi I (Thermo Scientific).The sticky ends were filled using Klenow fragment (Thermo Scientific), and the linear plasmid was ligated using T4 DNA ligase (Thermo Scientific™) following manufacturer protocols.The ligation reaction was introduced into Inoue E. coli DH5α chemical competent cells(Green & Sambrook, 2020), and the TransBac empty backbone plasmid was purified as mentioned above.The size of the plasmid DNA was determined via agarose gel electrophoresis, and the cloning site was sequenced using SO10 primer (see Table Characterization and quantification of ΔybeX and Keio wild-type (WT) colony sizes at 37 and 42°C.(a) Representative LB agar plates with colonies of WT and ΔybeX strains.As indicated on the panel's top, the cells were plated from LB or MOPS minimal media and the agar plates were incubated at 37°C overnight.(b-c) Density plots presenting the distribution of quantified colony radii of ΔybeX and isogenic WT strains in pixels at 37 and 42°C.The colonies were quantified from four independent experiments as of the plates in panel (a).
(d) Colony counts for WT and ΔybeX strains grown in LB or MOPS minimal media as in panel (a).Values of four biological replicates with three technical replicates each are shown.The plot includes p-values derived from a two-sided Student's t-test with unequal variances.