A Bacillus anthracis-based in vitro system supports replication of plasmid pXO2 as well as rolling-circle-replicating plasmids.

Capsule-encoding virulence plasmid pXO2 of Bacillus anthracis is predicted to replicate by a unidirectional theta-type mechanism. To gain a better understanding of the mechanism of replication of pXO2 and other plasmids in B. anthracis and related organisms, we have developed a cell-free system based on B. anthracis that can faithfully replicate plasmid DNA in vitro. The newly developed system was shown to support the in vitro replication of plasmid pT181, which replicates by the rolling-circle mechanism. We also demonstrate that this system supports the replication of plasmid pXO2 of B. anthracis. Replication of pXO2 required directional transcription through the plasmid origin of replication, and increased transcription through the origin resulted in an increase in plasmid replication.