A spontaneous kanamycin resistance and capreomycin resistance mutation, A1408G, in the decoding center of 16S rRNA, was identified in the extreme thermophile Thermus thermophilus. Unexpectedly, this mutation also confers resistance to streptomycin. We propose a novel mechanism of streptomycin resistance by which A1408G influences conformational changes in 16S rRNA during tRNA selection.Ribosomes from the extreme thermophile Thermus thermophilus have proven to be a rich source of structural information regarding the mechanism of tRNA selection during protein synthesis and have provided insights into the mechanism by which antibiotics can disrupt this process (3,17). This species is also amenable to genetic manipulation (11,12), and antibiotic resistance mutations in ribosomal protein and rRNA genes are readily isolated (2,7,8). The ability to apply both genetics and X-ray crystallography to ribosomes from a single organism creates the exciting possibility of elucidating antibiotic resistance mechanisms at atomic level resolution.The aminoglycoside antibiotics, including streptomycin and kanamycin, have long been known to cause misreadings of the genetic code, and it is now confirmed from crystallographic analysis of the T. thermophilus 30S subunit that these antibiotics bind at or near the decoding center and in distinct, nonoverlapping sites (3). Indeed, aminoglycosides, including the kanamycins and neomycins, make contact exclusively with 16S rRNA helix 44 independently of the remainder of the 30S subunit (5, 24). In a striking contrast, streptomycin contacts multiple structural elements of the T. thermophilus 30S subunit, including ribosomal protein S12 and 16S rRNA helices 1, 18, 27, and 44 (3). It has been proposed that streptomycin stabilizes a series of intermolecular and intramolecular contacts within the 30S subunit during decoding (16).What is not yet clear from high-resolution structural studies is the mechanism by which some ribosomal mutations confer resistance to streptomycin. A number of mutations occur at positions of the ribosome which do not make direct contact with streptomycin, and in these instances, simple models of binding site distortion will not suffice. Here we describe the isolation of a spontaneous kanamycin-resistant and capreomycin-resistant mutant expressing a novel streptomycin resistance phenotype and propose one possible mechanism of resistance.Isolation of kanamycin-resistant and capreomycin-resistant mutants of T. thermophilus. Spontaneous mutants of T. thermophilus IB-21 (ATCC 43615) (13) were obtained by plating 10 9 cells onto plates of Thermus enhanced medium (ATCC medium 1598) containing kanamycin sulfate (30 g/ml) or capreomycin sulfate (200 or 400 g/ml). Capreomycin is a member of the tuberactinomycin group of antibiotics, which includes the structurally related compound viomycin, and is known primarily as an inhibitor of translocation (6). Mutants were purified by being restreaked several times and were stored as frozen glycerol stocks at Ϫ80°C. Sequencing of PCR produc...