Mutations in ribosomal protein L16 conferring reduced susceptibility to evernimicin (SCH27899): Implications for mechanism of action

A clinical isolate of Streptococcus pneumoniae (SP#5) that showed decreased susceptibility to evernimicin (MIC, 1.5 mu g/ml) was investigated. A 4,255 -bp EcoRI fragment cloned from SP#5 was identified by its ability to transf orm evernimicin-susceptible S. pneumoniae R6 (MIC, 0.03 mu g/ml) such that the evernimicin MIC was 1.5 mu g/ml. Nucleotide sequence analysis of this f ragment revealed that it contained portions of the S10-spc ribosomal protei n operons, The nucleotide sequences of resistant and susceptible isolates w ere compared, and a point mutation (thymine to guanine) that causes an Ile5 2-Ser substitution in ribosomal protein L16 was identified. The role of thi s mutation in decreasing susceptibility to evernimicin was confirmed by dir ect transformation of the altered L16 gene. The presence of the L16 mutatio n in the resistant strain suggests that evernimicin is an inhibitor of prot ein synthesis. This was confirmed by inhibition studies using radiolabeled substrates, which showed that the addition of evernimicin at sub MIC levels resulted in a rapid decrease in the incorporation of radiolabeled isoleuci ne in a susceptible isolate (SP#3) but was much less effective against SP#5 , The incorporation of isoleucine showed a linear response to the dose leve l of evernimicin. The incorporation of other classes of labeled substrates was unaffected or much delayed, indicating that these were secondary effect s.