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

2004; American Society for Microbiology; Volume: 186; Issue: 9 Linguagem: Inglês

10.1128/jb.186.9.2717-2723.2004

1098-5530

Eowyn Tinsley, Asma Naqvi, Agathe Bourgogne, Theresa M. Koehler, Saleem A. Khan,

Bacteriophages and microbial interactions

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.