Mutational Analysis of 16S and 23S rRNA Genes of Thermus thermophilus

Abstract

Structural studies of the ribosome have benefited greatly from the use of organisms adapted to extreme environments. However, little is known about the mechanisms by which ribosomes or other ribonucleoprotein complexes have adapted to functioning under extreme conditions, and it is unclear to what degree mutant phenotypes of extremophiles will resemble those of their counterparts adapted to more moderate environments. It is conceivable that phenotypes of mutations affecting thermophilic ribosomes, for instance, will be influenced by structural adaptations specific to a thermophilic existence. This consideration is particularly important when using crystal structures of thermophilic ribosomes to interpret genetic results from nonextremophilic species. To address this issue, we have conducted a survey of spontaneously arising antibioticresistant mutants of the extremely thermophilic bacterium Thermus thermophilus, a species which has featured prominently in ribosome structural studies. We have accumulated over 20 single-base substitutions in T. thermophilus 16S and 23S rRNA, in the decoding site and in the peptidyltransferase active site of the ribosome. These mutations produce phenotypes that are largely identical to those of corresponding mutants of mesophilic organisms encompassing a broad phylogenetic range, suggesting that T. thermophilus may be an ideal model system for the study of ribosome structure and function.Members of the bacterial genus Thermus are extreme thermophiles first described by Brock and Freeze in 1969 (2) and have since been found in terrestrial and marine thermal environments throughout the world (60). Together with Deinococcus, Meiothermus, Marinithermus, Oceanithermus, and Vulcanithermus, they form a deeply branching phylum now known to be monophyletic (23,59). The close affiliation between Thermus and Deinococcus has been confirmed by complete genome sequences of Thermus thermophilus (30) and Deinococcus radiodurans (59). The finding of slightly thermophilic species related to Deinococcus, together with the thermophilic nature of the other genera of this phylum, suggests that thermophily is a primitive character of this clade (59).Recent advances in structural biology have produced a vast reservoir of high-resolution structural information regarding components of the protein synthetic machinery from members of the Deinococcus-Thermus phylum, including high-resolution crystal structures of the T. thermophilus 30S subunit (47, 61), medium-resolution structures of the entire T. thermophilus 70S ribosome (65), and high-resolution structures of the D. radiodurans 50S subunit (26). The value of these structures is magnified by their interpretation in light of several decades of genetics and biochemistry using ribosomes from mesophiles such as Escherichia coli. However, the ability to make such interpretations is potentially compromised by the absence of genetic and biochemical data obtained for ribosomes from members of the Deinococcus-Thermus phylum. It is assumed that nucleotide sequence c...

Genome sequencing of linezolid-resistant Streptococcus pneumoniae mutants reveals novel mechanisms of resistance

“…The mutations A2503G and A1743T in 1974M2 are associated with linezolid resistance for the first time. A recent crystal structure of linezolid bound to the 50S ribosomal subunit has shown interaction between the antibiotic and A2503 (Leach et al 2007;Ippolito et al 2008;Wilson et al 2008), and mutation of this residue was observed in some chloramphenicol-resistant mutants (Gregory et al 2005). Interestingly, methylation of the A2503 residue by the acquired cfr gene also led to linezolid resistance (Toh et al 2007).…”

Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity

“…Given that a guanine lies at the corresponding position in eukaryotes, A1408 was concluded to be the main determinant of the prokaryotic specificity of aminoglycosides (Hobbie et al 2006a). However, the A1408G mutation in E. coli 16S rRNA conferred only an intermediate level of resistance to aminoglycosides with a hydroxyl group at the 69 position of ring I, such as paromomycin and geneticin (G418) (Recht et al 1999b;Gregory et al 2005). In contrast, mutations at G1491 conferred much greater resistance to these compounds (Pfister et al 2005b).…”

“…This has led to several studies of the decoding site of bacterial ribosomes (Pfister et al 2003a,b;2005a,b). The A1408G mutation, among all decoding site mutations, conferred the most significant resistance to aminoglycosides (Sander et al 1996;Recht et al 1999a,b;Pfister et al 2003a;Gregory et al 2005;Hobbie et al 2005). Given that a guanine lies at the corresponding position in eukaryotes, A1408 was concluded to be the main determinant of the prokaryotic specificity of aminoglycosides (Hobbie et al 2006a).…”

Genetic and structural analysis of base substitutions in the central pseudoknot of Thermus thermophilus 16S ribosomal RNA

“…These arose at a frequency of around 10 À7 , comparable to the frequencies of other spontaneous antibiotic-resistant mutants of T. thermophilus that we have described previously (Gregory et al 2001a, b;Cameron et al 2004;Gregory et al 2005b). We tentatively identified the mutations by PCR amplification and sequencing of rrsB and rpsL.…”

“…We obtained such markers from our collection of spontaneous mutants resistant to either of the antibiotics capreomycin or hygromycin B. We had previously characterized such mutants derived from T. thermophilus strain IB-21 (Gregory et al 2005b) and readily isolated similar mutants of T. thermophilus HB8 for the purpose of this study. These included the hygromycin B-resistance mutation U1406A, and the capreomycin-resistance mutation G1491A.…”