Oxazolidinone resistance mutations in 23S rRNA of Escherichia coli reveal the central region of domain V as the primary site of drug action

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interactin g with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which the se drugs inhibit translation. To investigate the drug-ribosome interaction, we selected Escherichia coli oxazolidinone-resistant mutants, which contai ned a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2 032 to A, was identified in the 23S rRNA genes of several independent resis tant isolates. Engineering of this mutation by site-directed mutagenesis in the wild-type rRNA operon produced an oxazolidinone resistance phenotype, establishing that the G2032A substitution was the determinant of resistance . Engineered U and C substitutions at G2032, as well as a G2447-to-U mutati on, also conferred resistance to oxazolidinone. All the characterized resis tance mutations were clustered in the vicinity of the central loop of domai n V of 23S rRNA, suggesting that this rRNA region plays a major role in the interaction of the drug with the ribosome. Although the central loop of do main V is an essential integral component of the ribosomal peptidyl transfe rase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptid yl transferase center.