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Appl. Environ. Microbiol. doi:10.1128/AEM.00240-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

An in vitro system from Bacillus anthracis supports replication of plasmid pXO2 as well as rolling-circle-replicating plasmids

Department of Molecular Genetics and Biochemistry, and Graduate Program in Molecular Virology and Microbiology, University of Pittsburgh School of Medicine, East 1240 Biomedical Science Tower, Pittsburgh, Pennsylvania 15261

^* To whom correspondence should be addressed. Email: Khan{at}pitt.edu.

	Abstract

The 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 from B. anthracis that can faithfully replicate plasmid DNA in vitro. The newly developed system was shown to support the in vitro replication of the pT181 plasmid that replicates by the rolling-circle mechanism. We also demonstrate that this system supports the replication of the pXO2 plasmid 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.