INTRODUCING MUTATIONS INTO A CHROMOSOMAL RIBOSOMAL-RNA GENE USING A GENETICALLY-MODIFIED EUBACTERIAL HOST WITH A SINGLE RIBOSOMAL-RNA OPERON

Gene-inactivation techniques were employed to construct a eubacterial organism harbouring a single functional rRNA operon. This mutant of My cobacterium smegmatis permits replacement of the single remaining rRNA operon with a homologous fragment from a vector-borne gene. By homolo gous recombination with the chromosome a plasmid-borne rDNA segment wi th resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, resulting in a homogeneous population of muta ted ribosomes in the cell. As a first result we demonstrate that the s ingle allelic knock-out strain allows for isolation of rRNA mutants wi th a drug-resistant phenotype, circumventing the problem of recessivit y which prohibits the isolation of such mutants in organisms with mult iple rRNA operons. Subsequently, by allelic exchange experiments, it w as demonstrated that the rRNA mutation found indeed confers drug resis tance in vivo. This system provides intriguing potential for the study of the structure and function of ribosomal RNAs.