Mutations in the bacterial ribosomal protein l3 and their association with antibiotic resistance

Abstract

Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations in E. coli was used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted in E. coli, although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin.

FIG 1

The ribosomal protein L3 mutations in the 50S ribosomal subunit. (A) An X-ray structure of the E. coli 50S ribosomal subunit (PDB file 3R8T, now superseded by 4V9D), looking down the peptide exit tunnel (open circle in the middle) with 5S RNA in light green on the top. 23S RNA is shown as gray tubing and r-proteins in light blue, except L3, which is shown as violet. At the right is a closeup of part of the PTC with linezolid (red) bound. Nucleotides G2056, U2504, G2576, and C2610, shown in green, have been calculated to exhibit structural changes upon L3 mutations. (B) Amino acid sequence alignment of a partial fragment of L3 ribosomal proteins of E. coli, S. aureus, Brachyspira hyodysenteriae, and M. tuberculosis with the mutations from this study shown above the alignment. (C) Representation of the E. coli L3 backbone in tubing marked with the positions of the mutated amino acids.

FIG 2

A schematic illustration of the exchange procedure replacing plasmid-carried wild-type L3 with plasmid-carried mutated L3. Filled circles illustrate pBR322AmpL3 (Amp^r) and open circles pBR322mutatedL3 (Tet^r). In the case shown, the selection was performed with tetracycline.

FIG 3

Illustration of the computational modulations. (A) A selection of structural changes upon mutations measured in terms of RMSD with respect to the wild type. The selected nucleotides are shown on the x axis with the RMSD (Å) from all L3 mutations (color code beneath the graph) on the y axis. (B) A closeup cutaway image of the 60-Å docking sphere from the X-ray structure of the E. coli 50S (PDB file 3OFC) with the L3 mutation R149 modeled into the structure. The nucleotides most affected (see panel A above) by the mutation are shown as green sticks together with the calculated position of linezolid (ball and stick). The wild-type N149 is superimposed in blue. The distances from the mutated R149 amino acid to nucleotides G2056 and U2504 are shown. (C) Superposition of U2504, G2056, and linezolid from the N149R L3 and the wild-type L3 docking structures. The curved line points to the calculated structural shift of linezolid resulting from the presence of the mutated R149 amino acid.