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Journal of Bacteriology, October 2000, p. 5325-5331, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Oxazolidinone Resistance Mutations in 23S rRNA of Escherichia coli Reveal the Central Region of Domain V as the Primary Site of Drug Action

Liqun Xiong,1 Patricia Kloss,1 Stephen Douthwaite,2 Niels Møller Andersen,2 Steven Swaney,3 Dean L. Shinabarger,3 and Alexander S. Mankin1,*

Center for Pharmaceutical Biotechnology, University of Illinois, Chicago, Illinois 606071; Department of Biochemistry and Molecular Biology, Odense University, DK-5230 Odense M, Denmark2; and Infectious Diseases Research, Pharmacia Corporation, Kalamazoo, Michigan 490013

Received 1 May 2000/Accepted 14 July 2000

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant 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 mutation, also conferred resistance to oxazolidinone. All the characterized resistance mutations were clustered in the vicinity of the central loop of domain 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 domain V is an essential integral component of the ribosomal peptidyl transferase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptidyl transferase center.

* Corresponding author. Mailing address: Center for Pharmaceutical Biotechnology-m/c 870, University of Illinois, 900 S. Ashland Ave., Chicago, IL 60607-7173. Phone: (312) 413-1406. Fax: (312) 413-9303. E-mail: shura{at}uic.edu.

Journal of Bacteriology, October 2000, p. 5325-5331, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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