The activity of rRNA resistance methyltransferases assessed by MALDI mass spectrometry.
Resistance to antibiotics that target the bacterial ribosome is often conferred by methylation at specific nucleotides in the rRNA. The nucleotides that become methylated are invariably key sites of antibiotic interaction. The addition of methyl groups to each of these nucleotides is catalyzed by a specific methyltransferase enzyme. The Erm methyltransferases are a clinically prevalent group of enzymes that confer resistance to the therapeutically important macrolide, lincosamide, and streptogramin B (MLS B) antibiotics. The target for Erm methyltransferases is at nucleotide A2058 in 23S rRNA, and methylation occurs before the rRNA has been assembled into 50S ribosomal particles. Erm methyltransferases occur in a phylogenetically wide range of bacteria and differ in whether they add one or two methyl groups to the A2058 target. The dimethylated rRNA confers a more extensive MLS B resistance phenotype. We describe here a method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to determine the location and number of methyl groups added at any site in the rRNA. The method is particularly suited to studying in vitro methylation of RNA transcripts by resistance methyltransferases such as Erm.
Douthwaite
Stephen
S
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej, Denmark.
Jensen
Rikke Lind
RL
Kirpekar
Finn
F
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase