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

The Escherichia coli protein YbeX/CorC is encoded in the same operon as the ribosome biogenesis factor YbeY, and its deletion leads to accumulation of 17S pre-rRNA and degradation intermediates of 16S rRNA under magnesium limitation. To further investigate the ybeX deletion phenotype, we used rRNA fluorescence in situ hybridization coupled with flow cytometry (rRNA-FISH-flow) to quantify 16S rRNA, 23S rRNA, and 17S pre-rRNA levels at single-cell resolution in{Delta} ybeX and{Delta} ybeY strains.{Delta} ybeX cells grown under limiting Mg2+ develop striking cell-to-cell heterogeneity in 17S pre-rRNA content during the transition to stationary phase, with up to 25-fold differences between individual cells. Upon regrowth from the stationary phase,{Delta} ybeX cultures display a bimodal distribution of 17S pre-rRNA, revealing two distinct subpopulations -- one retaining high levels of unprocessed pre-rRNA and the other with low levels -- whose relative proportions shift over time, until visible growth resumes. The stoichiometry between mature 16S and 23S rRNAs remains tight in both strains, indicating that the heterogeneity is specific to pre-rRNA processing, rather than a general disruption of ribosome homeostasis. The{Delta} ybeY mutant accumulates 17S pre-rRNA more uniformly across cells and primarily during exponential growth in rich medium, consistent with its direct role in 16S rRNA maturation. These single-cell data suggest that YbeX and YbeY affect ribosomal RNA metabolism through distinct mechanisms and that the extended lag phase of{Delta} ybeX is caused by a heterogeneous clearing of pre-ribosomal intermediates in individual cells.