Papers In Press, published online ahead of print December 7, 2001
J. Biol. Chem, 10.1074/jbc.M105505200
Submitted on June 14, 2001
Revised on November 28, 2001
Accepted on November 28, 2001
Intricate interactions within the ccd plasmid addiction system
Minh-Hoa Dao-Thi, Daniel Charlier, Remy Loris, Dominique Maes, Joris Messens, Lode Wyns, and Jan Backmann
Ultrastructure, Institute of Molecular Biology, Sint-Genesius-Rode B-1640
Corresponding Author: dommaes{at}vub.ac.be
The ccd addiction system plays a crucial role in the stable maintenance of the E. coli F-plasmid. It codes for a stable toxin (CcdB) and a less stable antidote (CcdA). Both are expressed at low levels during normal cell growth. Upon plasmid loss, CcdB outlives CcdA and kills the cell by poisoning gyrase. The interactions between CcdB, CcdA and its promoter DNA were analyzed. In solution, the CcdA-CcdB interaction is complex, leading to various complexes with different stoichiometry. CcdA has two binding sites for CcdB and vice versa, permitting soluble hexamer formation but also causing precipitation, especially at CcdA:CcdB ratios close to one. CcdA alone, but not CcdB binds to promoter DNA with high on- and off-rates. The presence of CcdB enhances the affinity and the specificity of CcdA-DNA binding and results in a stable CcdA-CcdB-DNA-complex with a CcdA:CcdB ratio of one. This (CcdA2CcdB2)n complex has multiple DNA-binding sites and spirals around the 120 bp promoter region.