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, blocking the passage of polymerases and leading to double-strand breakage of the 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 reconstituted the inactive GyrA-CcdB complex by denaturation and renaturation of the purified GyrA dimer in the presence of CcdB. This inactivating interaction involves the N-terminal domain of GyrA, because similar inactive complexes 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 formation of the inactive complexes, 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 prevented 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.
Bahassi
E M
EM
Laboratoire de Génétique des Procaryotes, Département de Biologie Moléculaire, Université Libre de Bruxelles, rue des Chevaux 67, B-1640 Rhode-Saint-Genèse, Belgium.
O'Dea
M H
MH
Allali
N
N
Messens
J
J
Gellert
M
M
Couturier
M
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D003603:Cytotoxins / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D009154:Mutation; D011485:Protein Binding; D059005:Topoisomerase II Inhibitors
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D003603:Cytotoxins; D059005:Topoisomerase II Inhibitors; D004247:DNA; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II