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 (MLSB) 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 MLSB 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.
Key WordsrRNA methylation ribosomal antibiotic resistance RNA mass spectrometry
We thank Birte Vester and Lykke Haastrup Hansen for discussions. Support from the Danish Research Agency (FNU Grants #21-04-0505 and #21-04-0520), the Nucleic Acid Center of the Danish Grundforskningsfond, and CDC funds are gratefully acknowledged.
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