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.