title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Rasmus N
authors.initials: RN
authors.lastname: Klitgaard
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Eleni
authors.initials: E
authors.lastname: Ntokou
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Katrine
authors.initials: K
authors.lastname: Nørgaard
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Daniel
authors.initials: D
authors.lastname: Biltoft
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Lykke H
authors.initials: LH
authors.lastname: Hansen
authors.affiliation: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark.
authors.forename: Nicolai M
authors.initials: NM
authors.lastname: Trædholm
authors.affiliation: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark.
authors.forename: Jacob
authors.initials: J
authors.lastname: Kongsted
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark b.vester@bmb.sdu.dk.
authors.forename: Birte
authors.initials: B
authors.lastname: Vester
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D000069349:Linezolid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; D000069349:Linezolid
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