The Staphylococcus aureus plasmid pT181 contains a cis-acting sequence element, cmp (for competition), whose deletion impairs the plasmid's ability to coexist with an incompatible plasmid. The element, which is external to the minimal plasmid replicon, affects the ability of the plasmid's origin of replication to utilize the plasmid-encoded initiator protein. The goal of this study is to understand the molecular mechanism by which cmp affects the site-specific interaction between ori and RepC over a distance. Structural requirements for cmp will be elucidated in terms of minimal sequence, DNA bending and distance from ori. Comparative studies with cmp-like elements in other plasmids will define common structural and functional features. These studies will be carried out by using an in vivo assay for the Cmp phenotype using plasmids having progressive deletions of the cmp region, having cmp located at various distances from the origin, or having the pT181 cmp replaced by cmp-like elements from other plasmids. Cmp plasmids have a lower titratable negative supercoiling than Cmp+ plasmids. The nature of the linking difference associated with cmp will be studied by assaying levels of superhelical tension in vivo in cmp+ and cmp plasmids by means of a supercoiling-sensitive promoter. The contribution of other plasmid elements to cmp-associated supercoiling will be examined by determining the linking difference between non-pT181 plasmids carrying pT181 cmp and/or ori in the presence/absence of RepC. The possibility of interactions between cmp and other plasmid elements and between cmp and host factor(s) will be investigated by isolation and characterization of plasmid-and host-linked mutations that suppress the Cmp phenotype. A knowledge of cmp structure and function will provide the first evidence for the participation of enchancer-like elements in DNA replication. This study will contribute to our understanding of the mechanisms by which auxiliary factors and DNA elements govern site-specific interactions between proteins and DNA.
