Recombineering reveals a diverse collection of ribosomal proteins L4 and L22 that confer resistance to macrolide antibiotics

Abstract

Mutations in ribosomal proteins L4 and L22 confer resistance to erythromycin and other macrolide antibiotics in a variety of bacteria. L4 and L22 have elongated loops whose tips converge in the peptide exit tunnel near the macrolide-binding site, and resistance mutations typically affect residues within these loops. Here, we used bacteriophage lambda Red-mediated recombination, or "recombineering," to uncover new L4 and L22 alleles that confer macrolide resistance in Escherichia coli. We randomized residues at the tips of the L4 and L22 loops using recombineered oligonucleotide libraries and selected the mutagenized cells for erythromycin-resistant mutants. These experiments led to the identification of 341 resistance mutations encoding 278 unique L4 and L22 proteins-the overwhelming majority of which are novel. Many resistance mutations were complex, involving multiple missense mutations, in-frame deletions, and insertions. Transfer of L4 and L22 mutations into wild-type cells by phage P1-mediated transduction demonstrated that each allele was sufficient to confer macrolide resistance. Although L4 and L22 mutants are typically resistant to most macrolides, selections carried out on different antibiotics revealed macrolide-specific resistance mutations. L22 Lys90Trp is one such allele that confers resistance to erythromycin but not to tylosin and spiramycin. Purified L22 Lys90Trp ribosomes show reduced erythromycin binding but have the same affinity for tylosin as wild-type ribosomes. Moreover, dimethyl sulfate methylation protection assays demonstrated that L22 Lys90Trp ribosomes bind tylosin more readily than erythromycin in vivo. This work underscores the exceptional functional plasticity of the L4 and L22 proteins and highlights the utility of Red-mediated recombination in targeted genetic selections.

Figure 1

L4 and L22 loops and the ribosomal macrolide-binding site. (a) The E. coli large ribosome subunit viewed from the cytoplasmic opening of the exit tunnel. All ribosomal proteins except L4 and L22 have been omitted from the rendering. (b) View of the macrolide-binding site. Residues A2058 and A2059 of 23S rRNA form a hydrophobic crevice into which macrolide antibiotics bind. Residue A2062 has been shown to form a reversible covalent bond with the aldehyde moiety of tylosin and spiramycin . The tips of the L4 and L22 loops are shown in orange and yellow, respectively. Structures were derived from PDB file 2AW4 and rendered using PyMol. (c) L4 and L22 nucleotide and protein sequences. L4 residues Gln62 to Arg67 (in orange) and L22 residues Arg88 to Ala93 (in yellow) were mutagenized by oligonucleotide library recombineering as described in the text.

Figure 2

Isolation frequencies of L4 Gln62, L4 Lys63, L4 Gly66, and L22 Lys90 missense alleles. Frequencies are expressed as the percent of total mutants isolated from each selection. The total number of mutants identified for each position is indicated. L4 Gln62, L4 Lys63, and L4 Gly66 mutants isolated by spiramycin selection are shown in grey. Complex mutations isolated from these libraries are not shown in the histograms, but were included in the calculation of allele frequencies. See Supplemental Tables II & III for a complete enumeration of all isolated mutations.

Figure 3

In vivo methylation protection assays. Wild-type and mutant cells were treated with dimethyl sulfate (DMS) in the presence and absence of macrolide antibiotics (150 μg/mL erythromycin and 1 mg/mL tylosin). Binding of macrolides to the ribosome inhibits DMS-mediated methylation of 23S rRNA residue A2058. Methylation was monitored by primer extension analysis as described in Materials and Methods. Primer extension products corresponding to DMS-mediated N¹-methylation of A2058 and A2062 are indicated. Erythromycin had a greater inhibitory effect on DMS-mediated A2058 methylation in wild-type cells compared with the two L22 mutants. Tylosin significantly inhibited DMS-mediated A2058 methylation in wild-type and L22 Lys90Trp cells, but had less of an effect in L22 Ala89Glu-Lys90Pro mutant. A control reaction from cells overproducing the ErmC methyltransferase was included to serve as a marker for A2058 methylation.