SummaryThe ccd operon of the F plasmid encodes CcdB, a toxin targeting the essential gyrase of Escherichia coli, and CcdA, the unstable antidote that interacts with CcdB to neutralize its toxicity. Although work from our group and others has established that CcdA and CcdB are required for transcriptional repression of the operon, the underlying mechanism remains unclear. The results presented here indicate that, although CcdA is the DNA-binding element of the CcdA -CcdB complex, the stoichiometry of the two proteins determines whether or not the complex binds to the ccd operator -promoter region. Using electrophoretic mobility shift assays, we show that a (CcdA)2 -(CcdB)2 complex binds DNA. The addition of extra CcdB to that protein -DNA complex completely abolishes DNA retardation. Based on these results, we propose a model in which the ratio between CcdA and CcdB regulates the repression state of the ccd operon. When the level of CcdA is superior or equal to that of CcdB, repression results. In contrast, derepression occurs when CcdB is in excess of CcdA. By ensuring an antidote -toxin ratio greater than one, this mechanism could prevent the harmful effect of CcdB in plasmid-containing bacteria.

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal tldE (pmbA) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently, tldE as well as tldD mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the ccd poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the ccd system, we found that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.

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 plasmidencoded 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.The ability to modulate the topological state of DNA is essential to bacteria. One of the enzymes responsible for this critical function is DNA gyrase (a bacterial type II topoisomerase). In Escherichia coli, DNA gyrase consists of two subunits, GyrA 1 and GyrB, of molecular masses 97 and 90 kDa, respectively, encoded by the gyrA and gyrB genes; the active enzyme is an A 2 B 2 complex. The A subunits mediate the ability of the enzyme to introduce and rejoin double-strand breaks in DNA. The B subunits mediate energy transduction and ATP hydrolysis (for recent reviews, see Refs. 1-3).Gyrase was first identified by its ability to convert relaxed circular DNA into a negatively supercoiled form at the expense of ATP hydrolysis (4). Given its role in the maintenance of proper levels of negative supercoiling, and the overall role of supercoiling in transcription, replication, and recombination (1, 2, 5), gyrase influences nearly all DNA transactions in prokaryotic cells.DNA gyrase is the target of several antibacterial agents, including the coumarin and quinolone families of antibiotic drugs (6 -9). The coumarin antibiotics inhibit supercoiling activity by interfering with ATP binding and hydrolysis. The quinolones block all activities of gyrase that involve DNA strand passage. However, quinolone drugs tend to exert their bactericidal action through complex formation with gyrase and DNA rather than through simple elimination of gyrase activity. The quinolones are thought to trap a reaction inter...

The types of topoisomerase alterations in genomically diverse epidemic and sporadic strains of methicillin- and fluoroquinolone-resistant Staphylococcus aureus isolated from European hospitals between 1984 and 1994 were characterized. Convergent dual mutations in gyrA (codon 83, 84, or 88) and grlA (codon 79 and/or 80) were found in all strains exhibiting high-level resistance to ciprofloxacin (MIC, 16 to > or = 128 microg/ml). In some epidemic strains, the resistant phenotype and genotype appeared in the 1990s and persisted thereafter.