Characterization of a single-strand origin, ssoU, required for broad host range replication of rolling-circle plasmids

Single-stranded DNA (ssDNA) promoters are the key components of the single- strand origins (ssos) of replication of rolling-circle (RC) replicating pla smids. The recognition of this origin by the host RNA polymerase and the sy nthesis of a short primer RNA are critical for initiation of lagging-strand synthesis. This step is thought to be a limiting factor for the establishm ent of RC plasmids in a broad range of bacteria, because most of the ssos d escribed are fully active only in their natural hosts. A special type of ss o, the ssoU, is unique in the sense that it can be efficiently recognized i n a number of different Gram-positive hosts. We have experimentally deduced the folded structure and characterized the ssDNA promoter present within t he ssoU using P1 nuclease digestion and DNase I protection assays with the Bacillus subtilis and Staphylococcus aureus RNA polymerases. We have also i dentified the RNA products synthesized from this ssDNA promoter and mapped the initiation points of lagging-strand synthesis in vivo from ssoU-contain ing plasmids. Through gel mobility shift experiments, we have found that ss DNA containing the ssoU sequence can efficiently interact with the RNA poly merase from two different Gram-positive bacteria, S. aureus and B. subtilis . We have also realigned the narrow and broad host range sso sequences of R C plasmids, and found that they contain significant homology. Our data supp ort the notion that the strength of the RNA polymerase-ssoU interaction may be the critical factor that confers the ability on the ssoU to be fully fu nctional in a broad range of bacteria.