Control of pT181 replication II. Mutational analysis.

We describe the isolation and analysis of mutations affecting the regulation of Staphylococcus aureus plasmid pT181 replication. Previous results suggested that regulation is achieved by control of the synthesis of RepC, a plasmid-coded replication protein and that the primary negative control element is CopA RNA, which consists of two transcripts that are complementary to the 5' region of the repC mRNA leader. CopA inhibition probably involves a base pairing interaction with the complementary region of the RepC mRNA leader which would facilitate the formation of a downstream stem-loop in the leader that occludes the repC ribosome binding site. RepC is freely diffusible so that regulation of pT181 replication is indirect. Both CopA RNA-sensitive (recessive) and -insensitive (dominant) mutants were isolated. The recessives have defects in CopA RNA structure or activity, the dominants have defects in the site of action (target) of the inhibitor. Some dominants were located within the copA coding sequence. These therefore affect the structure of CopA RNA as well as that of its target. Other dominant mutations mapped outside of the copA gene and therefore produced wild-type CopA RNA. In contrast to directly regulated plasmids, pT181 copy mutants producing wild-type inhibitor could be co-maintained with the wild-type plasmid and mutational changes in inhibitor-target specificity did not change incompatibility specificity.