Spontaneous Erythromycin Resistance Mutation in a 23S rRNA Gene, rrlA , of the Extreme Thermophile Thermus thermophilus IB-21

Abstract

Spontaneous, erythromycin-resistant mutants of Thermus thermophilus IB-21 were isolated and found to carry the mutation A2058G in one of two 23S rRNA operons. The heterozygosity of these mutants indicates that A2058G confers a dominant or codominant phenotype in this organism. This mutation provides a valuable tool for the genetic manipulation of the 23S rRNA genes of Thermus.Genetic analysis of rRNAs and ribosomal proteins has been an essential tool in elucidating the mechanism of protein synthesis. While numerous ribosomal protein and rRNA mutants have been isolated, none has been examined structurally in atomic detail. This is primarily due to the inadequacy of ribosomes from genetically tractable organisms such as Escherichia coli for forming crystals that diffract X-rays at sufficient resolution. Ribosomes suitable for X-ray crystallographic analysis have thus far been obtained exclusively from thermophiles (4, 23, 33) or halophiles (1). Regrettably, only limited effort has been devoted to studying the ribosomes of these organisms using genetic approaches (see, however, reference 26). Determining the structures of mutant ribosomes of such an organism (an organism producing ribosomes suitable for X-ray crystallographic analysis) should define conformational perturbations that result in specific functional consequences. We have, therefore, initiated a genetic dissection of ribosomes of the extreme thermophile Thermus thermophilus. Despite the promise of Thermus as a genetic system (16, 21), the lack of selectable genetic markers such as transposons or antibiotic resistance mutations makes the mapping of mutations to ribosomal protein and RNA genes problematic. Here we report the isolation of a 23S rRNA mutation conferring resistance to the antibiotics erythromycin and lincomycin. This selectable genetic marker will facilitate the identification, by transformation, of 23S rRNA mutations affecting 50S subunit structure and function.Thermus spp. have an unusual rRNA gene composition of two 16S rRNA genes unlinked from two 23S rRNA-5S rRNAtRNA Gly operons (3, 12, 13). Thus, rRNA mutations are potentially obtainable by direct selection if they exhibit a dominant or codominant phenotype or if gene conversion occurs at a sufficiently high frequency. For these experiments we chose the halotolerant strain T. thermophilus IB-21 (ATCC 43615). Although the strain was originally described as Thermus sp. strain IB-21 (17), the designation T. thermophilus is indicated by DNA-DNA hybridization experiments (32) and our own DNA sequence analysis (S. Gregory, J. H. D. Cate, and A. E. Dahlberg, unpublished results).Isolation of spontaneous erythromycin-resistant mutants. We conducted a search for erythromycin resistance mutations in 23S rRNA which have been isolated for a number of organisms with multiple rRNA operons (reviewed in reference 30). In several instances, including in the cases of Streptomyces ambofaciens (22), Helicobacter pylori (29), and Streptococcus pneumoniae (27), heterozygous mutants indicate that this mutati...

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

“…2; Table 1). While the T. thermophilus A2058G erythromycin resistance mutation was found only in the heterogeneous populations (21), in the present study, the A2059G and A2062G mutations were each found in both pure and mixed populations of ribosomes.…”

“…Our ability to obtain a large number of rRNA mutants of T. thermophilus is made possible in part by the presence of only two rRNA gene copies. In our earlier search for rRNA mutants, we had obtained the A2058G mutation in 23S rRNA (21). All three isolates of this mutant that we examined proved to contain heterogeneous ribosome populations.…”

“…The peptidyltransferase active site is the target of a number of structurally unrelated classes of antibiotics. We previously described the isolation of mutants resistant to the 14-atom macrolide erythromycin by virtue of the A2058G mutation (21). Selections on the 16-atom macrolide tylosin (100 g/ml) led to the identification of two other mutations in this same region, A2059G and A2062G ( Fig.…”

“…Foremost is the ease of genetic manipulation of this organism (29,35). The presence of only two copies of rRNA genes (30,56) facilitates the isolation of rRNA mutants (21), including mutants with homogeneous ribosome populations, most likely resulting from gene conversion between homologous rRNA genes (4). For conducting mutational analysis of ribosomes, antibiotic resistance mutations are advantageous, as T. thermophilus is susceptible to a wide range of antibiotics, providing the opportunity for multiple genetic selections which can target individual functional sites.…”

A Mutation in the Decoding Center of Thermus thermophilus 16S rRNA Suggests a Novel Mechanism of Streptomycin Resistance

“…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.…”