Experimental evolution of Streptococcus pneumoniae under combined antibiotic and immune pressure reveals that convergent mutations in rny encoding RNase Y confer broad-spectrum antibiotic tolerance by preventing transcriptional collapse through a bet-hedging strategy where a resilient minority maintains baseline transcription while a quiescent majority undergoes selective RNA degradation and prioritized ribosomal recovery, identifying RNA turnover as a tunable master regulator of bacterial survival that enables tolerance rather than classical resistance as the primary adaptive strategy in vivo.

The mechanisms underlying adaptation to antibiotic pressure within complex host environments remain incompletely understood. By experimentally evolving Streptococcus pneumoniae subjected to various antibiotics and immune states, we demonstrate populations adopting distinct adaptive strategies depending on specific selective context. General antibiotic stress drives convergent mutations in rny, encoding the RNA degradosome scaffold RNase Y, that exhibit broad-spectrum antibiotic tolerance and accelerated recovery. Single-cell transcriptomics revealed antibiotic-induced death is driven by transcriptional collapse, a catastrophic loss of RNA quantity and integrity. In contrast, rny mutants avert this via a bet-hedging strategy: a resilient minority maintains a baseline transcriptional profile, while a quiescent majority undergoes selective RNA degradation to preserve transcript fidelity. Upon stress removal, these populations execute a prioritized transcriptional ribosomal reboot, facilitating accelerated recovery. These findings identify RNA turnover as a tunable master regulator to survive combined pressures of antibiotics and immunity.