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J Biol Chem, Vol. 275, Issue 11, 8213-8219, March 17, 2000

Stoichiometry of P1 Plasmid Partition Complexes^*

Jean-Yves Bouet, Jennifer A. Surtees, and Barbara E. Funnell^§

From the Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site. This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS. We have investigated the assembly of ParB at parS and its stoichiometry of binding. Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I + B1. We observed that as ParB concentration was increased, a second, larger complex (I + B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex. Shift Western blotting experiments indicated that the I + B2 complex contained twice as much ParB as the I + B1 complex. Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I + B1 complex contains one dimer of ParB. Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend in parS. Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.

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^* This work was supported by a University of Toronto Open Fellowship (to J. A. S.) and a grant from the Medical Research Council of Canada (to B. E. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Laboratoire de Microbiologie et de Génétique Moléculaire du CNRS, 118 route de Narbonne, 31062 Toulouse cedex, France.

^§ To whom correspondence should be addressed: Dept. of Molecular and Medical Genetics, Medical Sciences Bldg., University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-1665; Fax: 416-978-6885; E-mail: b.funnell@utoronto.ca.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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