This application proposes using exiting and new in vivo selection procedures to isolate new antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli. In addition, an existing chloramphenicol-erythromycin resistance mutation will be further characterized. The isolation of these mutations in rRNA genes is possible due to selection for mutations on a multicopy plasmid that carries a ribosomal RNA operon. This plasmid contributes to a substantial fraction of the cellular ribosomes, thereby allowing antibiotic resistance phenotypes to be expressed. Ribosomes with mutations in rRNA will be characterized both in vivo and in vitro. The mutations will also be characterized by DNA sequencing after mapping the mutations to small regions of DNA. Genetic mapping will employ versatile genetic procedures developed in this laboratory. These mapping methods are called plasmid-plasmid marker rescue and shotgun recombination mapping. Existing antiboiotic resistance mutations in rRNA genes will be used to help isolate and characterize an inducible lambda PL-rrn fusion operon that will result in conditional synthesis of rRNA. This conditionally expressed fusion operon will assist in isolation and characterization of potentially lethal or detrimental mutations constructed by site-directed mutagenesis. Oligonucleotide-directed mutagenesis will be used to construct mutations in rRNA genes in the lambda PL-rrn fusion operon and in a wild-type rrn operon. The rRNA genes used as substrates for this site-directed mutagenesis will incorporate existing spectinomycin and erythromycin resistance mutations in 16S and 23S rRNA genes, respectively, to enable characterization of the funtional defects caused by the site-directed mutations. Mutagenesis will be site-directed to specific sequences with proposed involvement in intermolecular base-pairing interactions important to mRNA binding and ribosomal subunit association. The mutations described in this proposal will help elucidate the role of rRNA in antibiotic action and ribosome function. These mutations have clinical significance because an existing mutation in an E.coli rRNA gene closely mimics the mechanism responsible for MLS antibiotic resistance in clinical bacterial isolates. Mutations in Escherichia coli rRNA genes may predict other clinical resistance mechanisms.
