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Journal of Bacteriology, May 2004, p. 2717-2723, Vol. 186, No. 9
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.9.2717-2723.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Isolation of a Minireplicon of the Virulence Plasmid pXO2 of Bacillus anthracis and Characterization of the Plasmid-Encoded RepS Replication Protein

Department of Molecular Genetics and Biochemistry and Graduate Program in Molecular Virology and Microbiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261,¹ Department of Microbiology and Molecular Genetics, The University of Texas—Houston Health Science Center, Houston, Texas 77030²

Received 11 December 2003/ Accepted 21 January 2004

A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5' and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.

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