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Epitranscriptomic regulation of the oxidative stress response by the radical S-adenosylmethionine methyltransferase RlmN

ABSTRACT: 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. Following exposure to menadione or sublethal doses of ROS-inducing erythromycin and chloramphenicol, analysis of 25 modified ribonucleosides of the epitranscriptome revealed large decreases in N2-methyladenosine (m2A) in both 23S ribosomal RNA (rRNA) and transfer RNA (tRNA) caused by ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Loss of RlmN altered protein expression in a way that mimicked menadione exposure, such as increased superoxide dismutase and decreased virulence proteins. These studies suggest that RlmN acts as a redox-sensitive molecular switch that links environmental and antibiotic-induced ROS exposure to epitranscriptome dynamics in ribosomal and transfer RNA to effect translation of stress response proteins.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Enterococcus Faecalis (streptococcus Faecalis)

SUBMITTER: AMEYA SINHA

LAB HEAD: Peter Dedon

PROVIDER: PXD038178 | Pride | 2023-05-09

REPOSITORIES: Pride

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Project description:Legionella pneumophila (Lp) is an opportunistic pathogen and its survival in water is critical for human infection. Therefore, identifying the genes of Lp that are required for survival in water may help devise strategies to prevent Legionella outbreaks. In this study, we exposed Lp in rich medium and in an artificial freshwater medium (Fraquil) for 2, 6 and 24 hours to uncover the global transcriptomic changes of Lp in water. The repression of major metabolic pathways, such as division, transcription and translation, suggests that Lp enters a dormant state in water. The induction of the flagellar associated genes (flg, fli and mot), enhance entry genes (enh) and some Icm/Dot effectors suggests that Lp may be waiting to establish intracellular replication in suitable host. Moreover, many genes involved in resistance to antibiotic and oxidative stress were induced, suggesting that Lp may be more tolerant to environmental stresses in water. Indeed, Lp exposed to water is more resistant to erythromycin, gentamycin and kanamycin than those cultured in rich medium. Apart from this, the gene bdhA involved in the degradation of the intracellular energy storage compound poly-hydroxybutyrate is highly expressed in water. Further characterization shows that bdhA is positively regulated by RpoS during short-term exposure to water. The deletion mutant of bdhA had a survival defect in water at 37°C, demonstrating that this gene is important for maintaining the long-term survivorship of Lp in water. Other identified genes highly induced upon exposure to water could also be necessary for Lp to survive in water. Legionella pneumophila Philadelphia-1 strain JR32 was grown in AYE broth at 25°C shaking to OD600 of 1 in triplicate. Samples were taken for analysis; this is the control. Then the cultures were washed three times in Fraquil and resuspended in Fraquil to an OD600 of 1 and transfered to vessels of bioreactior (Biostat Q-plus). Samples were taken after 2h, 6h and 24h.

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Epitranscriptomic regulation of the oxidative stress response by the radical S-adenosylmethionine methyltransferase RlmN

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