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Antimicrobial Agents and Chemotherapy, October 2001, p. 2877-2884, Vol. 45, No. 10
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.10.2877-2884.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Mechanisms of Streptomycin Resistance: Selection of Mutations in the 16S rRNA Gene Conferring Resistance

Burkhard Springer, Yishak G. Kidan, Therdsak Prammananan, Kerstin Ellrott, Erik C. Böttger, and Peter Sander^*

Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany

Received 23 March 2001/Returned for modification 8 May 2001/Accepted 25 July 2001

Chromosomally acquired streptomycin resistance is frequently due to mutations in the gene encoding the ribosomal protein S12, rpsL. The presence of several rRNA operons (rrn) and a single rpsL gene in most bacterial genomes prohibits the isolation of streptomycin-resistant mutants in which resistance is mediated by mutations in the 16S rRNA gene (rrs). Three strains were constructed in this investigation: Mycobacterium smegmatis rrnB, M. smegmatis rpsL³⁺, and M. smegmatis rrnB rpsL³⁺. M. smegmatis rrnB carries a single functional rrn operon, i.e., rrnA (comprised of 16S, 23S, and 5S rRNA genes) and a single rpsL⁺ gene; M. smegmatis rpsL³⁺ is characterized by the presence of two rrn operons (rrnA and rrnB) and three rpsL⁺ genes; and M. smegmatis rrnB rpsL³⁺ carries a single functional rrn operon (rrnA) and three rpsL⁺ genes. By genetically altering the number of rpsL and rrs alleles in the bacterial genome, mutations in rrs conferring streptomycin resistance could be selected, as revealed by analysis of streptomycin-resistant derivatives of M. smegmatis rrnB rpsL³⁺. Besides mutations well known to confer streptomycin resistance, novel streptomycin resistance conferring mutations were isolated. Most of the mutations were found to map to a functional pseudoknot structure within the 530 loop region of the 16S rRNA. One of the mutations observed, i.e., 524GC, severely distorts the interaction between nucleotides 524G and 507C, a Watson-Crick interaction which has been thought to be essential for ribosome function. The use of the single rRNA allelic M. smegmatis strain should help to elucidate the principles of ribosome-drug interactions.

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^* Corresponding author. Present address: Institut für Medizinische Mikrobiologie, Universität Zürich, Gloriastr. 30/32, CH-8028 Zurich, Switzerland. Phone: 41-1-634-2684. Fax: 41-1-634-4906. E-mail: sander.peter{at}gmx.de.

Present address: Division of Mycology and Mycobacteriology, Faculty of Medicine, Mahidol University, Bangkok 10700, Thailand.

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Antimicrobial Agents and Chemotherapy, October 2001, p. 2877-2884, Vol. 45, No. 10
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.10.2877-2884.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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