An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis

Bacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogen Enterococcus faecalis. We analyze the tRNA epitranscriptome of E. faecalis in response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identify large decreases in N2-methyladenosine (m2A) in both 23 S ribosomal RNA and transfer RNA. This we determine to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gives rise to a proteome that mimics the oxidative stress response, with an increase in levels of superoxide dismutase and decrease in virulence proteins. While tRNA modifications were established to be dynamic for fine-tuning translation, here we report the discovery of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to a model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, adding a different paradigm in which RNA modifications can directly regulate the proteome.Ministry of Education (MOE)National Research Foundation (NRF)Published versionThis research was supported by the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program, through the Singapore–MIT Alliance for Research and Technology Antimicrobial Resistance Interdisciplinary Research Group. We acknowledge funding support from NMRC OFIRG20nov-0079. Contributions of LNL were supported by the National Research Foundation and Ministry of Education Singapore under its Research Center of Excellence Program at SCELSE. A.S. and J.L. acknowledge support from the Singapore‐MIT Alliance (SMA) Graduate Fellowship and MOE Tier 2 Grant MOE2018-T2-2-13. Proteomics work was performed in part in the MIT Center for Environmental Health Sciences Bioanalytical Core, which is supported by Center grant P30‐ES002109 from the National Institute of Environmental Health Sciences with the aid of Dr. Michael Demott