THE ROLE OF RIBOSOMAL-RNAS IN MACROLIDE RESISTANCE

Macrolides are bacteriostatic antibiotics which interfere with the pep tidyltransfer function of the ribosome. We have investigated the molec ular mechanisms underlying macrolide resistance in Mycobacterium smegm atis, an eubacterium carrying two rRNA operons. Surprisingly, drug res istance was associated not with alterations in ribosomal proteins, but with a single point mutation in the peptidyltransferase region of one of the two 23S RNA genes, i.e. A2058-->G or A2059-->G. This mutation resulted in a heterozygous organism with a mutated and a wild-type rRN A operon respectively. Reverse transcriptase sequencing indicated the expression of both wild-type and mutated rRNAs. The mutated operon was introduced into genetically engineered rrn(-) strains of M. smegmatis carrying a single functional rRNA operon and into parental M. smegmat is with two chromosomal rRNA operons, using gene transfer as well as g ene replacement techniques. The results obtained demonstrate the domin ant nature of resistance. As exemplified in our results on macrolide r esistance, a complete set of genetic tools is now available, which all ows questions of dominance vs. recessivity and gene dosage effects in eubacterial ribosomal nucleic acids to be addressed experimentally in vivo.