Single-cell analysis of pre-rRNA in Escherichia coli indicates distinct pathways of action for YbeX and YbeY proteins in ribosome biogenesis

Technical Summary: Single-cell analysis of pre-rRNA in Escherichia coli indicates distinct pathways of action for YbeX and YbeY proteins in ribosome biogenesis

Problem Statement
The Escherichia coli proteins YbeX (also known as CorC) and YbeY are encoded in the same operon (ybeZYX-lnt) and are both implicated in ribosome biogenesis. While YbeY is established as a factor in 16S rRNA maturation, the specific function of YbeX remains unclear. Previous bulk-level studies indicated that deletion of ybeX ($\Delta$ybeX) leads to the accumulation of 17S pre-rRNA and 16S rRNA degradation intermediates, particularly under magnesium (Mg$^{2+}$) limitation. This phenotype is accompanied by a prolonged lag phase upon regrowth from stationary phase and heterogeneous colony sizes, suggesting cell-to-cell variability. However, bulk measurements cannot distinguish whether these defects represent a uniform per-cell impairment or a severe defect confined to a specific subpopulation. Furthermore, it is unknown whether YbeX and YbeY perturb ribosome metabolism through the same or distinct mechanisms.

Methodology
To resolve these questions at single-cell resolution, the authors employed rRNA fluorescence in situ hybridization coupled with flow cytometry (rRNA-FISH-flow).

• Strains: Isogenic E. coli BW25113 (Wild Type, WT), $\Delta$ybeX, and $\Delta$ybeY strains were utilized.
• Growth Conditions:
  • $\Delta$ybeX and WT were grown in MOPS minimal medium under Mg$^{2+}$ limitation (50 $\mu$M MgCl$_2$) to induce the specific phenotype. The experiment involved two rounds: (1) growth into late exponential phase to accumulate pre-rRNA, and (2) regrowth from stationary phase to observe clearance.
  • $\Delta$ybeY and WT were grown in rich LB medium, as $\Delta$ybeY exhibits a constitutive growth defect independent of Mg$^{2+}$ limitation.
• FISH-Flow Protocol: Cells were fixed, permeabilized with ethanol, and hybridized with Cy3- and Cy5-labeled DNA oligonucleotide probes targeting 16S rRNA, 23S rRNA, and 17S pre-rRNA. DAPI staining was used to exclude non-cellular particles.
• Analysis: Flow cytometry (BD LSR Fortessa) quantified fluorescence signals for individual cells. Data analysis focused on median signal intensities, interquartile ranges (cell-to-cell variation), and Pearson correlation coefficients between rRNA species and DNA content. The authors specifically analyzed the top 10% of cells with the highest 17S pre-rRNA signals to assess extreme phenotypes.

Key Contributions and Results

1. Distinct Single-Cell Signatures of $\Delta$ybeX vs. $\Delta$ybeY:

   • $\Delta$ybeX (Mg$^{2+}$-dependent, Heterogeneous): Under Mg$^{2+}$ limitation, $\Delta$ybeX cells exhibited a striking, time-dependent broadening of the 17S pre-rRNA distribution. During the transition to stationary phase, cell-to-cell variation in 17S levels increased from ~5-fold to ~25-fold. Upon regrowth from stationary phase, the population displayed a bimodal distribution of 17S pre-rRNA. Two distinct subpopulations emerged: one retaining high levels of unprocessed pre-rRNA and another with low levels. Visible growth resumed only when these subpopulations reached approximately equal proportions, suggesting the lag phase is driven by the time required for the "high-17S" subpopulation to clear intermediates.
   • $\Delta$ybeY (Constitutive, Uniform): In contrast, $\Delta$ybeY cells accumulated 17S pre-rRNA uniformly across the population during early exponential growth in rich medium. The distribution remained narrow and unimodal (~8-fold higher than WT), indicating a direct, constitutive defect affecting all cells similarly, consistent with YbeY's known role in 16S 3'-end maturation.

2. Stoichiometry and Homeostasis:

   • In both mutants, the stoichiometry between mature 16S and 23S rRNAs remained tightly correlated and balanced, similar to the wild type. This indicates that the transcription of rRNA operons and the assembly of mature subunits are not generally disrupted.
   • The defect in $\Delta$ybeX is specific to pre-rRNA processing. The correlation between 17S pre-rRNA and mature 16S rRNA was weaker in $\Delta$ybeX and varied with growth phase, particularly breaking down during the clearance phase in the regrowth experiment.

3. Temporal Decoupling:

   • In $\Delta$ybeX, the accumulation of 17S pre-rRNA began in the mid-exponential phase, yet growth rates remained indistinguishable from WT for several hours. Growth arrest only occurred at the late-exponential/stationary transition. This suggests that 17S accumulation is not immediately translationally limiting but that a threshold (e.g., in the ratio of mature to immature subunits or Mg$^{2+}$ cofactor depletion) must be crossed to trigger the lag phase.

Significance and Claims
The paper claims that single-cell analysis reveals that YbeX and YbeY affect ribosomal RNA metabolism through qualitatively distinct mechanisms:

• YbeY acts via a direct biochemical mechanism (likely enzymatic processing), resulting in a uniform, population-wide defect.
• YbeX acts via an indirect mechanism, likely related to intracellular Mg$^{2+}$ homeostasis (consistent with its CorC domain). The defect manifests only under Mg$^{2+}$ limitation and only in a subset of cells, leading to phenotypic heterogeneity and bistability.

The authors conclude that the extended lag phase of $\Delta$ybeX is caused by a heterogeneous clearing of pre-ribosomal intermediates in individual cells, rather than a uniform slowdown. This work provides a framework for distinguishing between direct enzymatic lesions and indirect physiological defects in bacterial stress responses, suggesting that cell-to-cell heterogeneity can serve as a diagnostic flag for the underlying mechanism of action. The study does not definitively resolve whether YbeX is a direct Mg$^{2+}$ sensor regulating transcription or a factor driving assembly under low Mg$^{2+}$, noting that kinetic experiments and single-cell Mg$^{2+}$ measurements are required for further resolution.