Role of plasmid multimers in mutation to tetracycline resistance

As an additional system for analysing mutations that appear to be specifically induced or directed, we have used a plasmid that contains the mnt repressor gene inserted as an operon fusion with the tet gene of the plasmid pBR322. Thus, the mnt gene product acts as a negative transcriptional regulator of tet gene expression. Mutations inactivating the Mnt repressor are recessive while those destroying operator recognition (Oc) are dominant in conferring tetracycline resistance on the host. When resistance mutations were isolated on plates with high levels of tetracycline they were preferentially mnt- and the plasmids were monomers. Pre-exposure to low concentrations increased the frequency of resistant mutants by 100- to 1000-fold, and the mutations were now mostly Oc, located on one unit of a plasmid multimer. Recessive repressor mutations on one unit would not have been selected. We suggest that the high frequency of mutation in tandem multimeric plasmids may be caused by the formation of single-stranded and hence highly mutable regions by homologous pairing out of register. The role of tetracycline in promoting mutations is discussed.