Prokaryotic RNA N1-Methyladenosine Erasers Maintain tRNA m1A Modification Levels in

tRNA modifications help maintain tRNA structure and facilitate translation and stress response. Found in all three kingdoms of life, mA tRNA modification occurs in the T loop of many tRNAs, stabilizes tertiary tRNA structure, and impacts translation. MA in the T loop is reversible by three mammalian demethylase enzymes, which bypasses the need of turning over the tRNA molecule to adjust its mA levels in cells. However, no prokaryotic tRNA demethylase enzyme has been identified that acts on endogenous RNA modifications. Using as a model organism, we confirmed the presence and quantitative mA tRNA signatures using mass spectrometry and high-throughput tRNA sequencing. We identified two RNA demethylases that can remove mA in tRNA and validated the activity of a previously annotated tRNA mA writer. Using single-gene knockouts of these erasers and the mA writer, we found dynamic changes of mA levels in many tRNAs under stress conditions. Phenotypic characterization highlighted changes in their growth and altered antibiotic production. Our identification of the first prokaryotic tRNA demethylase enzyme paves the way for investigating new mechanisms of translational regulation in bacteria.