Interactions of CcdB with DNA gyrase - Inactivation of GyrA, poisoning of the gyrase-DNA complex, and the antidote action of CcdA

The F plasmid-carried bacterial toxin, the CcdB protein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blo cking the passage of polymerases and leading to double-strand breakage of t he DNA. Alternatively, in cells that overexpress CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB, We have rec onstituted the inactive GyrA-CcdB complex by denaturation and renaturation of the purified GyrA dimer in the presence of CcdB, This inactivating inter action involves the N-terminal domain of GyrA, because similar inactive com plexes were formed by denaturing and renaturing N-terminal fragments of the GyrA protein in the presence of CcdB, Single amino acid mutations, both in GyrA and in CcdB, that prevent CcdB-induced DNA cleavage also prevent form ation of the inactive complexes, indicating that some essential interaction sites of GyrA and of CcdB are common to both the poisoning and the inactiv ation processes. Whereas the lethal effect of CcdB is most probably due to poisoning of the gyrase-DNA complex, the inactivation pathway may prevent c ell death through formation of a toxin antitoxin-like complex between CcdB and newly translated GyrA subunits, Both poisoning and inactivation can be prevented and reversed in the presence of the F plasmid-encoded antidote, t he CcdA protein. The products of treating the inactive GyrA-CcdB complex wi th CcdA are free GyrA and a CcdB-CcdA complex of approximately 44 kDa, whic h may correspond to a (CcdB),(CcdA), heterotetramer.