Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus

We have isolated spontaneous streptomycin-resistant, streptomycin-dependent and streptomycin-pseudo-dependent mutants of the thermophilic bacterium Th ermus thermophilus IB-21. All mutant phenotypes were found to result from s ingle amino acid substitutions located in the rpsL gene encoding ribosomal protein S12. Spontaneous suppressors of streptomycin dependence were also r eadily isolated. Thermus rpsL, mutations were found to be very similar to r psL mutations identified in mesophilic organisms. This similarity affords g reater confidence in the utility of the crystal structures of Thermus ribos omes to interpret biochemical and genetic data obtained with Escherichia co li ribosomes. In the X-ray crystal structure of the T. thermophilus HB8 30 S subunit, the mutated residues are located in close proximity to one anoth er and to helices 18, 27 and 44 of 16 S rRNA. X-ray crystallographic analys is of ribosomes from streptomycin-resistant, streptomycin-pseudo-dependent and streptomycin-dependent mutants described here is expected to reveal fun damental insights into the mechanism of tRNA selection, translocation, and conformational dynamics of the ribosome. (C) 2001 Academic Press.