Interactions of CcdB with DNA Gyrase

Abstract

The F plasmid-carried bacterial toxin, the CcdB pro- tein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breakage of the DNA. Alternatively, in cells that overex- press CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB. We have reconstituted the inactive GyrA-CcdB complex by dena- turation and renaturation of the purified GyrA dimer in the presence of CcdB. This inactivating interaction in- volves the N-terminal domain of GyrA, because similar inactive complexes were formed by denaturing and re- naturing 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 formation of the inactive com- plexes, indicating that some essential interaction sites of GyrA and of CcdB are common to both the poisoning and the inactivation processes. Whereas the lethal effect of CcdB is most probably due to poisoning of the gyrase- DNA complex, the inactivation pathway may prevent cell death through formation of a toxin-antitoxin-like complex between CcdB and newly translated GyrA subunits. Both poisoning and inactivation can be pre- vented and reversed in the presence of the F plasmid- encoded antidote, the CcdA protein. The products of treating the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA complex of approximately 44 kDa, which may correspond to a (CcdB)2(CcdA)2 heterotetramer.