title: Antibiotic resistance evolved via inactivation of a ribosomal RNA methylating enzyme.
abstract: Modifications of the bacterial ribosome regulate the function of the ribosome and modulate its susceptibility to antibiotics. By modifying a highly conserved adenosine A2503 in 23S rRNA, methylating enzyme Cfr confers resistance to a range of ribosome-targeting antibiotics. The same adenosine is also methylated by RlmN, an enzyme widely distributed among bacteria. While RlmN modifies C2, Cfr modifies the C8 position of A2503. Shared nucleotide substrate and phylogenetic relationship between RlmN and Cfr prompted us to investigate evolutionary origin of antibiotic resistance in this enzyme family. Using directed evolution of RlmN under antibiotic selection, we obtained RlmN variants that mediate low-level resistance. Surprisingly, these variants confer resistance not through the Cfr-like C8 methylation, but via inhibition of the endogenous RlmN C2 methylation of A2503. Detection of RlmN inactivating mutations in clinical resistance isolates suggests that the mechanism used by the in vitro evolved variants is also relevant in a clinical setting. Additionally, as indicated by a phylogenetic analysis, it appears that Cfr did not diverge from the RlmN family but from another distinct family of predicted radical SAM methylating enzymes whose function remains unknown.
authors: Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA.
authors: Vanja
authors: V
authors: Stojković
authors: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel.
authors: Lianet
authors: L
authors: Noda-Garcia
authors: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel dan.tawfik@weizmann.ac.il.
authors: Dan S
authors: DS
authors: Tawfik
authors: Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA Department of Pharmaceutical Chemistry, University of California San Francisco, 600 16th St, MC2280 San Francisco, CA 94158, USA danica.fujimori@ucsf.edu.
authors: Danica Galonić
authors: DG
authors: Fujimori
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D000068536:Bacillota / Q000145:classification / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D010802:Phylogeny; D011487:Protein Conformation; D012335:RNA, Ribosomal / Q000235:genetics* / Q000378:metabolism*; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000900:Anti-Bacterial Agents; D012335:RNA, Ribosomal; D012343:RNA, Transfer; D008780:Methyltransferases