Epitranscriptomic control of stress adaptations in Escherichia coli

Bacterial stress responses have been studied at the phenotypic, transcriptional, and translational levels, demonstrating the presence of an “alarm” phase immediately after stress exposure. However, the contributions of RNA modifications during stress adaptation remain largely unexplored. Here, we map the epitranscriptomic changes of Escherichia coli after exposure to oxidative and acid stress using direct RNA sequencing of mRNA, rRNA, and tRNA, combined with mass spectrometry, deletion mutant phenotyping, and single-nucleotide PCR. We identified widespread, dynamic RNA modifications that include central metabolism transcripts and increased levels of rRNA methylations (m4Cm and m5C) under both stresses, with potential consequences for translation. In uncharged tRNAs, stress-specific modifications via the Mnm and Q pathways accumulated at the wobble position; these modifications proved crucial for survival. Together, these findings reveal a multifaceted layer of post-transcriptional regulation, establishing the first comprehensive view of the bacterial epitranscriptome during the alarm phase of stress adaptation. Overall design: Total RNA was extracted from two biological replicates each of wild-type E. coli cultured under standard growth conditions (no stress), medium with HCl added (acid stress), or medium with H2O2 added (oxidative stress) to measure RNA modifications throughout the transcriptome (mRNAe samples). Two replicates of in vitro transcribed RNA were synthesized from no-stress RNA samples as unmodified controls. Putative modifications were detected after samples were processed with direct RNA sequencing via Nanopore. A tRNA-specific preparation and sequencing protocol (Nano-tRNAseq) was also employed for the no-stress, acid-stress, and oxidative-stress samples.