The ccd toxin-antitoxin system of the F plasmid encodes CcdB, a protein that poisons the essential Escherichia coli DNA gyrase, unique type IIA topoisomerase able to introduce negative supercoils into DNA. Based on CcdB structure, a series of linear peptide analogues were obtained by the solid-phase methodology. One of these peptides (CcdBET2) displayed inhibition of the supercoiling activity of bacterial DNA gyrase with a concentration required for complete inhibition (IC(100)=10 microM) lower than the wild type CcdB. For Topo IV, a second type IIA bacterial topoisomerase, CcdBET2 was better inhibited the relaxation activity with an IC(100) of 5 microM (wt CcdB>10 microM). The replacement of Gly, present in the three C-terminal amino acid residues, by Glu, abolished the capacity to inhibit the gyrase but not the Topo IV activities. These findings demonstrate that the mechanism by which CcdBET2 inhibits DNA gyrase is different of the mechanism by which inhibits Topo IV. Therefore, CcdBET2 is a new type II topoisomerase inhibitor with specificity for Topo IV.