Effects of mutations in ribosomal protein L16 on susceptibility and accumulation of evernimicin

Chemical mutagenesis of Staphylococcus aureus RN450 generated two strains t hat displayed a stable reduction (30- to 60-fold) in susceptibility to ever nimicin. Cell-free translation reactions demonstrated that the resistance d eterminant was located in the ribosomal fraction. Compared to ribosomes iso lated from a wild-type strain, ribosomes from the mutant strains displayed an 8- to 10-fold reduction in affinity for [C-14] evernimicin. In contrast, the mutants displayed no alteration in either binding affinity or in vitro susceptibility to erythromycin. Exponential cultures of the mutant strains accumulated significantly less [C-14]evernimicin than the wild-type strain , suggesting that accumulation is dependent on the high affinity that evern imicin displays for its binding site. Sequencing rplP (encodes ribosomal pr otein L16) in the mutant strains revealed a single base change in each stra in, which resulted in a substitution of either cysteine or histidine for ar ginine at residue 51. Introduction of a multicopy plasmid carrying wild-typ e rplP into the mutant strains restored sensitivity to evernimicin, confirm ing that the alterations in rplP were responsible for the change in suscept ibility. Overexpression of the mutant alleles in S. aureus RN450 had no eff ect on susceptibility to evernimicin, demonstrating that susceptibility is dominant over resistance.