An indigenous posttranscriptional modification in the ribosomal peptidyl transferase center confers resistance to an array of protein synthesis inhibitors

Abstract

A number of nucleotide residues in ribosomal RNA (rRNA) undergo specific posttranscriptional modifications. The roles of most modifications are unclear, but their clustering in functionally important regions of rRNA suggests that they might either directly affect the activity of the ribosome or modulate its interactions with ligands. Of the 25 modified nucleotides in Escherichia coli 23S rRNA, 14 are located in the peptidyl transferase center, the main antibiotic target in the large ribosomal subunit. Since nucleotide modifications have been closely associated with both antibiotic sensitivity and antibiotic resistance, loss of some of these posttranscriptional modifications may affect the susceptibility of bacteria to antibiotics. We investigated the antibiotic sensitivity of E. coli cells in which the genes of 8 rRNA-modifying enzymes targeting the peptidyl transferase center were individually inactivated. The lack of pseudouridine at position 2504 of 23S rRNA was found to significantly increase the susceptibility of bacteria to peptidyl transferase inhibitors. Therefore, this indigenous posttranscriptional modification may have evolved as an intrinsic resistance mechanism protecting bacteria against natural antibiotics.

Figure 1

Secondary structure of central loop segment of domain V of the E. coli 23S rRNA . Posttranscriptionally modified residues shown in bold and indicated by arrows (when the enzyme responsible for the modification is known) or arrowheads (when the corresponding enzyme is yet unknown). Enzymes responsible for individual modifications were described in the following references: ^{; ; ; -} . Two out of three RluC targets are located in domain V (positions 2504 and 2580). Ψ 2504, whose presence renders cells resistant to several peptidyl transferase targeting antibiotics, is circled.

Figure 2

Status of the U2504 modification in the strains expressing 23S rRNA with the U955C and U2580C mutations. The strain SQ171 lacking chromosomal rrn alleles was transformed with either pLK35 plasmid expressing wild type rRNA or plasmid pUCUC carrying rrnB operon with the mutations U955C and U2580C in 23S rRNA gene. After plasmid exchange and verification that the strain expresses exclusively mutant ribosome, the rluC::kan allele was transduced from the strain JW1072 from the Keio collection . The status of U2504 modification was verified using the procedure of Ofengand et al. based on selective modification of pseudouridine residues by N³-1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC). Samples that are modified (+) or not modified (−) with CMC are indicated. G,A,U,C – sequencing lanes. Note the lack of the CMC-modified band at position 2504 in the RNA sample prepared from the rluC⁻ derivative of SQ171 cells expressing U955C/U2580C double-mutant 23S rRNA. Ψ residue at position 2457, which is introduced by RluE, remains unaffected.