A spontaneous point mutation in the single 23S rRNA gene of the thermophilic arachaeon Sulfolobus acidocaldarius confers multiple drug resistance

Abstract

Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes. Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius. Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees. Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol. Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA). The single-site mutation also conferred resistance to carbomycin. The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archaeal vector.The abundance of a special class of introns in the rRNA genes of thermophilic archaea, which sometimes encode "homing" endonucleases (9,19) chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton strongly inhibited growth of wild-type cells, while anisomycin, clindamycin, erythromycin, kanamycin, lincomycin, and spectinomycin had little or no effect on growth, even at the highest drug concentration tested (250 jig/ml). To ensure that the antibiotics were active at the high growth temperature (70'C), they were tested at the same temperature, and under similar conditions, on the thermophilic bacterium Thermus aquaticus (4) (

Erythromycin resistance by ribosome modification

“…Moreover, L22 in ribosomes from both resistant mutants and wild-type cells have been reported to form covalent complexes with erythromycin-based photoaffinity probes (1). In interpreting the results of affinity labeling studies it should be noted that covalent complex formation reflects only the proximity between probe and protein and implies nothing regarding the mechanism.…”

“…Purified protein L15, in solution, has been reported to bind to erythromycin (121) and, while associated with and part of a functional ribosome, to bind to erythromycin-based photoaffinity probes (1). An affinity constant of 2 ϫ 10 Ϫ5 liters/mol for the association of L15 with erythromycin was determined by Teraoka and Nierhaus (121).…”

Interference probing of rRNA with snoRNPs: A novel approach for functional mapping of RNA in vivo

“…Less information is available about the importance of the C2817 (C2452) site. In archaeal rRNAs, point mutations at the equivalent position have been shown to create resistance to drugs that inhibit PTase activity, such as chloramphenicol (Aagaard et al 1994) and sparsomycin (Tan et al 1996). This situation suggests that C2817 (C2452) is required for the binding or action of the antibiotics.…”

The Genome ofSulfolobus acidocaldarius, a Model Organism of theCrenarchaeota

“…Thus, S. acidocaldarius was employed to demonstrate the similarity of the archaeal and eukaryal transcription apparatuses (6,36,46). Moreover, its sensitivity to a wide range of ribosomal antibiotics (1) and ease of transformation (3) have rendered S. acidocaldarius a focus for in vivo genetic studies. Proteins responsible for chromatin folding (Sac7c) and the highly abundant Sac10b (Alba) protein, implicated in the regulation of chromatin and/or cellular RNAs in Sulfolobus (7,30), were first characterized for this organism (29).…”