Novel mutations in ribosomal proteins L4 and L22 that confer erythromycin resistance in Escherichia coli

Abstract

L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated 'tentacles' that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. Mutations in the tentacles of L4 and L22 confer macrolide resistance in a variety of pathogenic and non-pathogenic bacteria. In Escherichia coli, a Lys-to-Glu mutation in L4 and a three-amino-acid deletion in the L22 had been reported. To learn more about the roles of the tentacles in ribosome assembly and function, we isolated additional erythromycin-resistant E. coli mutants. Eight new mutations mapped in L4, all within the tentacle. Two new mutations were identified in L22; one mapped outside the tentacle. Insertion mutations were found in both genes. All of the mutants grew slower than the parent, and they all showed reduced in vivo rates of peptide-chain elongation and increased levels of precursor 23S rRNA. Large insertions in L4 and L22 resulted in very slow growth and accumulation of abnormal ribosomal subunits. Our results highlight the important role of L4 and L22 in ribosome function and assembly, and indicate that a variety of changes in these proteins can mediate macrolide resistance.

Fig. 1

Mutations in L4 and L22 conferring erythromycin resistance in E. coli. A. Structure model of L4 (derived using CHIMERA from PDB file A2WB) showing locations of missense and insertion mutations in the tentacle of L4. Alpha helices are shown in green, and beta sheets are shown in purple. B. Positions in the L4 gene of mutations conferring erythromycin resistance. Changes are indicated in red. C. Structure model of L22 (derived using CHIMERA from PDB file A2WB) showing locations of deletion and insertion mutations in L22. D. Positions in the L22 gene of mutations conferring erythromycin resistance. Changes are indicated in red.

Fig. 2

β-Galactosidase induction lag times. Cells growing in minimal medium were induced with IPTG. Aliquots were removed and assayed at various times after induction (see Experimental procedures) to detect the time at which the enzyme activity increased above the basal level. Results for the wild-type parent and several of the mutants are shown. The y-axis represents arbitrary β-galactosidase activities, but the x-axis is the same for all the strains.

Fig. 3

Sucrose gradient sedimentation profiles of AB301 and ery-R mutants. Crude ribosomes isolated from cells grown in LB with (+Ery) or without (–Ery) erythromycin were centrifuged through a 10–50% (w/v) sucrose gradient prepared in buffer containing 1 mM MgCl₂ and 200 mM NH₄Cl. Absorption profiles were recorded at 260 nm. The absorption peaks of free 30S and 50S subunits are identified; aberrant ribosomal particles are indicated by vertical arrows. Fractions from the L22-99/15 –Ery gradient used later for primer extension (Fig. 4B) are indicated. The results for all of the mutants are summarized in Table 1, column 7.

Fig. 4

Primer extension analysis of 23S rRNA in ery-R mutants. A. Total RNA was purified from a mid-log phase culture and used as template. A ³²P-labelled oligonucleotide hybridizing internal to the 5′ end of 23S rRNA was used as the primer for the extension reaction. The products were analysed on a 8% acrylamide gel together with a sequencing ladder. Precursor and mature 23S rRNA are indicated. B. RNA was extracted from L22-99/15 high-salt sucrose gradient fractions (see Fig. 3) and analysed by primer extension as described above. The AB301 lane contained RNA from a total-cell extract. C. RNA was extracted from cells grown in LB containing the indicated concentration of fusidic acid or erythromycin, and analysed as described above.

Fig. 5

Model of the L4 and L22 tentacles and key nucleotides in the PTC and PTC-proximal region of the tunnel. The backbones of the L4 (red) and L22 (green) tentacles are shown. Positions of point mutations, insertions and deletions are indicated in yellow for L4 and black for L22. Nucleotides are shown in blue. A2058 and A2059 are located the beginning of the tunnel and form part of the binding site for erythromycin and other macrolides. A2450 and A2451 are key nucleotides in the PTC (Nissen et al., 2000; Hansen et al., 2002b). The model was drafted using RasMol and PDB structure 2I2T (Berk et al., 2006).