Investigation of the cfr and rlmN genes in linezolid resistance

Linezolid is an effective antibiotic against pathogens like Staphylococcus aureus. Recently, the Cfr methyltransferase was found to confer resistance to linezolid by methylating C8 of adenine 2503 of the large ribosomal subunit. A2503 is also methylated at C2 by the housekeeping enzyme RlmN, which is an evolutionary relative of Cfr. We first investigated the fitness cost of cfr expression. Acquisition of the cfr gene does not produce much reduction in the cell growth rate, indicating that it may have the ability to disseminate and persist in the clinical setting. We also examined the genetic environment of cfr in the clinical S. epidermidis 426-3147L strain. Cfr is associated with a transposon that increases cfr transcription to a higher level. In addition, we show that a mutation in RlmN in a clinical strain abolishes enzyme activity and increases resistance to linezolid. This is the first report linking RlmN activity and linezoid resistance in the clinical setting. We further characterize the distinctive enzymatic activity of Cfr and RlmN, focusing on the rRNA substrate requirements and specificity.