FIGURE AND TABLE LEGENDS
Fig. 1. Growth phenotypes of ∆ybeX strain and compensation with single-copy plasmid. (A ) The E. coli ybeZYX-lnt operon chromosomal organization with designed sigma factors (σ32 and σ24). (B ) Dot spot assay with wild type (WT) and ∆ybeX strain, along with the wild type strain harbouring empty plasmid (WT/pEmpty), ∆ybeX strain transformed with empty plasmid, and ∆ybeX strain conjugated with YbeZ, YbeY, or YbeX-expressing single copy TransBac library plasmid. LB agar plates without any antibiotics were incubated at 37°C or 42°C. (C-D ) The stationary phase outgrowth of wild type and∆ybeX strains harbouring empty plasmid in liquid LB medium. Panel C shows the OD600 signal, and panel D shows the alamarBlue fluorescence reading normalized to one. Individual measurements from independent experiments, each presented as the mean value of three technical replicates, are shown as dots. Curves are presented as modelled splines, and 95% credible intervals are shown as shaded areas (see Materials and methods for details). pybeX, pybeZ and pybeY denote the YbeX, YbeZ and YbeY expressing plasmid.
Fig. 2. Characterization and quantification of ∆ybeX and Keio wild-type (WT) strain colony sizes at 37°C and 42°C. (A ) Visual inspection of colony appearance of WT and ∆ybeX strains on LB agar plates. The cells were grown in LB or MOPS minimal media, serially diluted, and plated on LB agar plates. The plates were incubated at 37°C overnight. (B-C ) Density plots of the distribution of quantified colony radiuses of ∆ybeX and isogenic WT strains at 37°C and 42°C. (D ) Colony counts for WT and ∆ybeXstrains grown in LB or MOPS MM are presented. Diluted cells were plated on LB agar plates and incubated overnight at 37°C or 42°C.
Fig. 3. ∆ybeX cells exhibit severe sensitivity to sub-lethal concentrations of ribosome-binding antibiotics. (A ) BW25113 (WT) and ∆ybeX cells were grown overnight in LB liquid medium, serially diluted and spotted on LB agar plates supplemented with sub-inhibitory concentrations of indicated antibiotics or without antibiotics (No AB). The plates were incubated at 37°C overnight. (B ) Representative plates from a dot spot assay with strains described in Fig. 1 are presented. pybeX denotes the YbeX expressing plasmids.
Fig. 4. Growth phenotypes of ∆ybeX are growth-phase dependent. (A ) Experimental scheme for bacterial growth in LB medium. Overnight cultures were directly used for the Stationary phase experiments, while the cells were diluted into fresh LB and regrown for Exponential phase experiments. (B ) Dot spot experiments of Keio wild-type (WT) and ybeX , ybeZ , and ybeY deletion strains are given. The plates were incubated at 37°C, except for the 42°C plate. (C and D ) Growth curves of indicatedE. coli strains grown on 96-well plates. The monitored growth of stationary (C ) and exponential (D ) phase cells of wild type and ybeY , ybeZ , and ybeX deletion strains in liquid LB medium at 37°C. The growth curves are presented as curves for four biological replicates from two independent experiments, where error bars represent the 95% CI-s.
Fig. 5. Accumulation of ribosomal RNA fragments in stationary phase ∆ybeX cells. (A ) Denaturing agarose gel electrophoresis of hot phenol extracted total RNA samples from wild type and ybeY , ybeZ , and ybeX deletion strains. The empty triangle marks the accumulated shortened rRNA species. (B ) Northern blot analysis of hot phenol extracted total RNA samples from wild type and ybeY , ybeZ , and ybeXdeletion strains. The membrane was hybridized with 16S rRNA targeting oligonucleotide.
Fig. 6. Deletion of ybeX leads to the accumulation of distinct rRNA species. (A ) rRNA operon illustration with locations of the Cyanine 5 (red star) labelled oligonucleotides. (B ) Sucrose gradient profiles of WT and ∆ybeX strains grown at 37°C for 2 hours after the OD600 reached 0.3 (see Fig. 7A). 10-30% sucrose gradients were used for sedimentation. The profiles are representative of four independent experiments. (C -G ) Northern blot hybridization of the same membrane using different Cyanine 5 (Cy5) labelled oligonucleotides (see Table S3). Truncated ribosomal RNA species are annotated as ”trunc.”.
Fig. 7. Accumulated distinct ribosomal RNA species are formed in vivo. (A ) An experimental scheme where stationary phase cells (denoted as STAT) were grown to exponential phase (marked as EXP) followed by chloramphenicol (CAM) treatment (7µg/mL) for 2 hours. (B ) 10-30% sucrose gradient fractionation of clarified WT and∆ybeX strains lysates. (C -D ) Northern blot analysis of purified rRNA of sucrose gradient fractions separated on denaturing 1.5% agarose gel. The Northern blot was performed using 16S rRNA-specific oligo. The lower panels present the more prolonged exposure of the distinct accumulated rRNA species for more precise visualization.
Fig. 8. Magnesium supplementation rescues the ∆ybeXphenotypes in various growth media. (A ) The WT and∆ybeX cells were grown overnight in LB, SOB growth media, or LB supplemented with 10 mM MgCl2. The cells were serially diluted and spotted on LB agar plates. The plates with antibiotics were incubated at 37°C. There are also controls without antibiotics, denoted ”No AB”, at 37°C and at 42°C, as indicated in the first two sub-panels. (B ) A single colony of WT or ∆ybeX was grown overnight in the magnesium-limited peptide-based medium (PBM). 0 µM denotes no MgCl2 supplementation; otherwise, PBM is supplemented with 50, 100 and 200 µM MgCl2. (C ) WT or∆ybeX cells were grown overnight in a defined MOPS minimal medium supplemented with indicated concentrations of MgCl2 and 0.3% glucose as a carbon source. The outgrowth from these stationary phase cultures was done in MOPS minimal medium supplemented with 525 µM of MgCl2 (this is the prescribed optimal magnesium concentration of the 1x MOPS minimal medium). The regrowth of the cells was monitored at 37°C using a 96-well plate reader. The growth curves are presented as LOESS curves for six biological replicates from three independent experiments, where shaded areas represent the 95% CI-s for the fitted LOESS curves.
Fig. 9. The growth transition into the stationary phase leads to the ∆ybeX phenotype. (A) A scheme of the experimental setup. A single colony was inoculated into MOPS minimal medium supplemented with 10 mM MgCl2 and grown overnight. The next day saturated cultures were washed three times to remove residual magnesium and regrown in 10 µM MgCl2-containing MOPS. Aliquots for plating on LB agar were taken at 2, 3, 4 and 5.5 hours. The LB agar plates either contained or did not contain antibiotics as shown on panels D and E, and they were incubated overnight at 37 °C or 42 °C. (B ) The growth of the wild-type and the ∆ybeX strains in liquid MOPS medium supplemented with 10 µM MgCl2 as monitored at 600 nm. (C ) The wild-tpe and ∆ybeX cells were grown to saturation overnight in MOPS minimal medium containing the indicated amount of MgCl2. The mean optical densities of four biological replicates are shown with 95% CI-s. (D ) The∆ybeX cells had a growth phenotype only when collected for the outgrowth spot assay at the 5.5h time point. (E) When the outgrowth spot assay plates contained tetracycline, erythromycin or chloramphenicol (at subinhibitory concentrations listed in Materials and Methods), the growth phenotype seen at the 5.5h time point was more severe.