Resistance mutations in 23 S rRNA identify the site of action of the protein synthesis inhibitor linezolid in the ribosomal peptidyl transferase center

Oxazolidinones represent a novel class of antibiotics that inhibit protein synthesis in sensitive bacteria. The mechanism of action and location of th e binding site of these drugs is not clear. A new representative of oxazoli dinone antibiotics, linezolid, was found to be active against bacteria and against the halophilic archaeon Halobacterium halobium. The use of H. halob ium, which possess only one chromosomal copy of rRNA operon, allowed isolat ion of a number of linezolid-resistance mutations in rRNA. Four types of li nezolid-resistant mutants were isolated by direct plating of H. halobium ce l:Ls on agar medium containing antibiotic. In addition, three more linezoli d-resistant mutants were identified among the previously isolated mutants o f H. halobium containing mutations in either 16 S or 23 S rRNA genes. All t he isolated mutants were found to contain single-point mutations in 23 S rR NA. Seven mutations affecting six different positions in the central loop o f domain V of 23 S rRNA were found to confer resistance to linezolid. Domai n V of 23 S rRNA is known to be a component of the ribosomal peptidyl trans ferase center. Clustering of linezolid-resistance mutations within this reg ion strongly suggests that the binding site of the drug is located in the i mmediate vicinity of the peptidyl transferase center. However, the antibiot ic failed to inhibit peptidyl transferase activity of the H. halobium ribos ome, supporting the previous conclusion that linezolid inhibits translation at a step different from the catalysis of the peptide bond formation. (C) 1999 Academic Press.