Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria

RNA. 2009 Feb;15(2):327-36. doi: 10.1261/rna.1371409.

Abstract

The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfr- strains with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m(2)A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position and is independent of methylation at the 2 position of A2503. This investigation is, to our knowledge, the first instance where the 8-methyladenosine modification has been described in natural RNA molecules.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives*
  • Adenosine / chemistry
  • Adenosine / metabolism
  • Anti-Bacterial Agents / pharmacology
  • Catalysis
  • Chromatography, High Pressure Liquid
  • Chromatography, Liquid
  • Drug Resistance, Bacterial / genetics*
  • Escherichia coli / drug effects
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / classification
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Methylation
  • Methyltransferases / classification
  • Methyltransferases / genetics
  • Methyltransferases / metabolism*
  • Nucleic Acid Conformation
  • RNA, Ribosomal, 23S / chemistry
  • RNA, Ribosomal, 23S / metabolism*
  • Spectrometry, Mass, Electrospray Ionization

Substances

  • Anti-Bacterial Agents
  • Escherichia coli Proteins
  • RNA, Ribosomal, 23S
  • 8-methyladenosine
  • Cfr protein, E coli
  • Methyltransferases
  • Adenosine