Mutations in conserved helix 69 of 23S rRNA of Thermus thermophilus that affect capreomycin resistance but not posttranscriptional modifications

Abstract

Translocation during the elongation phase of protein synthesis involves the relative movement of the 30S and 50S ribosomal subunits. This movement is the target of tuberactinomycin antibiotics. Here, we describe the isolation and characterization of mutants of Thermus thermophilus selected for resistance to the tuberactinomycin antibiotic capreomycin. Two base substitutions, A1913U and mU1915G, and a single base deletion, DeltamU1915, were identified in helix 69 of 23S rRNA, a structural element that forms part of an interribosomal subunit bridge with the decoding center of 16S rRNA, the site of previously reported capreomycin resistance base substitutions. Capreomycin resistance in other bacteria has been shown to result from inactivation of the TlyA methyltransferase which 2'-O methylates C1920 of 23S rRNA. Inactivation of the tlyA gene in T. thermophilus does not affect its sensitivity to capreomycin. Finally, none of the mutations in helix 69 interferes with methylation at C1920 or with pseudouridylation at positions 1911 and 1917. We conclude that the resistance phenotype is a consequence of structural changes introduced by the mutations.

FIG. 1.

Deletion of genes for 23S RNA and TlyA. Wild-type HB8 was transformed with plasmid DNA containing the appropriate deletion allele, selecting kanamycin resistance. (A) Deletion of rrlB-rrfB-glyT. Lanes: 1, wild-type HB8; 2, TM200; P, PCR using pUC18 plasmid DNA template; bla, PCR using primers specific for the bla gene carried by pUC18. Primers A, B, C, D, E, and F are primers 23S-A, 23S-B, 23S-C, 23S-D, HTK1, and HTK2, respectively. (B) Deletion of tlyA. Lanes: 1, wild-type HB8; 2, TM469; P, PCR using pUC18 template; bla, PCR for the bla gene. Primers G and H are primers tlyA5 and tlyA6, respectively. Lane L and lane values, molecular weight markers (in thousands).

FIG. 2.

Capreomycin resistance mutations. (A) Locations of capreomycin resistance mutations in the secondary structure of T. thermophilus 23S rRNA (31). (B) Locations of sites of capreomycin resistance mutations in 23S rRNA identified in this study, as well as sites of mutations in 16S rRNA identified previously (11) in the crystal structure of the T. thermophilus 70S ribosome (39). Also shown are sites of 2′-O methylation by TlyA (16, 27).

FIG. 3.

Effects of mutations in helix 69 of 23S rRNA on posttranscriptional modifications. (A) Primer extension analysis of Cm1920 in capreomycin-resistant mutants A1913U, mU1915G, and ΔmU1915 and in the ΔtlyA::htk1 deletion mutant (ΔtlyA). rRNA from the wild-type (WT) strain was used for dideoxynucleotide sequencing (lanes C, U, A, and G). Decreasing concentrations of dGTP in the extension reaction mixture (1 μM, 0.5 μM, and 0.1 μM) are indicated by wedges. Extension reactions contained dATP, dCTP, and ddTTP at 40 μM. ddTTP caused termination at A1919 of all products extending past Cm1920. (B) Primer extension analysis to detect the presence of pseudouridine (Ψ) at positions 1911 and 1917 and of methylation at U1915, using CMCT. The presence or absence of CMCT modification is indicated by + and −, respectively.

FIG. 4.

Subunit association defect of the 23S rRNA (ΔmU1915) mutation. Ribosomes and ribosomal subunits were separated on a 10 to 40% sucrose gradient containing 10 mM MgCl₂ as described in Materials and Methods.