Ribosome State Distributions Define Escherichia coli Persister Physiology: Links to Formation, Stress Responses, and Resuscitation Dynamics

Persister cells survive antibiotic exposure and contribute to infection relapse, yet the molecular features that distinguish them from actively growing cells remain incompletely defined. Here, we used sucrose gradient-based ribosome sedimentation profiling to characterise ribosome complex distributions in Escherichia coli persister cells and monitored their dynamics during resuscitation. Rifampicin-induced persister cells were characterised by pronounced enrichment of translationally inactive 90-100S ribosome complexes and a concomitant reduction in 70S ribosomes relative to exponentially growing cells. Upon nutrient replenishment, ribosome distributions progressively shifted toward higher 70S and polysome (complexes of multiple ribosomes simultaneously translating a single mRNA) levels, coinciding with growth recovery, indicating that resuscitation involves gradual remodelling of ribosome states rather than abrupt restoration of active translation. Functional analysis of ribosome-associated factors demonstrated that RMF, HPF and RaiA promote 100S ribosome accumulation and enhance persister formation, whereas deletion of rmf severely impaired both 100S formation and persistence. In contrast, loss of HflX did not measurably affect persister formation, consistent with a role downstream of persister establishment. In multiple stress-induced persister models including rifampicin, tetracycline, CCCP and starvation, as well as in a clinically relevant E. coli O157: H7 (EHEC) strain, ribosome distributions consistently exhibited a quantitative reversal of the AUC₇0S/AUC₁00S ratio (Ratio 1. 0). Collectively, these findings demonstrate that this shift in the 70S-to-100S balance is a consistent and shared feature of E. coli persister physiology and that ribosome state distributions link persister formation to resuscitation dynamics. These findings provide a quantitative ribosome-state framework that may inform the development of anti-persistence strategies targeting ribosome hibernation factors.