EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain als o exhibited high-level resistance to evernimicin, a drug undergoing evaluat ion as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibit ed a dramatic reduction in evernimicin binding, shown by both cell-free tra nslation assays and direct-binding assays. The resistance determinant was c loned from strain 9631355; sequence alignments suggested it was a methyltra nsferase and therefore it was designated emtA for evernimicin methyltransfe rase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was u nique to the 23S rRNA extracted from resistant ribosomes. The pause corresp onded to methylation of residue G2470 (Escherichia coli numbering). RNA foo tprinting revealed that G2470 is located within the evernimicin-binding sit e on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.