Intricate Interactions within the ccd Plasmid Addiction System

Abstract

The ccd addiction system plays a crucial role in the stable maintenance of the Escherichia coli F plasmid. It codes for a stable toxin (CcdB) and a less stable antidote (CcdA). Both are expressed at low levels during normal cell growth. Upon plasmid loss, CcdB outlives CcdA and kills the cell by poisoning gyrase. The interactions between CcdB, CcdA, and its promoter DNA were analyzed. In solution, the CcdA-CcdB interaction is complex, leading to various complexes with different stoichiometry. CcdA has two binding sites for CcdB and vice versa, permitting soluble hexamer formation but also causing precipitation, especially at CcdA:CcdB ratios close to one. CcdA alone, but not CcdB, binds to promoter DNA with high on and off rates. The presence of CcdB enhances the affinity and the specificity of CcdA-DNA binding and results in a stable CcdA⅐ CcdB⅐DNA complex with a CcdA:CcdB ratio of one. This (CcdA 2 CcdB 2 ) n complex has multiple DNA-binding sites and spirals around the 120-bp promoter region.

Energetics of Structural Transitions of the Addiction Antitoxin MazE

“…10 shows that, according to the criterion introduced by Uversky (51,52,54) and Uversky et al (53), the addiction antitoxins in the monomeric form have a high tendency to be partially unstructured, whereas the corresponding toxins tend to be structured even as monomers. The prediction of structuring based on the mean hydrophobicity and mean net charge of the protein polypeptide chain is in good agreement with our observations and with recently reported results on addiction modules (11,47,89,90,102). Moreover, the large amount of the random-coil form found in free antitoxins in solutions (11,47,89,90,102) represents high vulnerability for their proteolytic cleavage (25,101,103).…”

“…9b shows that, in this range, the most populated antitoxin-toxin complexes contain more T 2 than A 2 molecules (A 2 T 4 and A 4 T 6 ). The A 2 T 4 form was also observed experimentally (48,90,92). By contrast, DNA is in the 10 Ϫ7 to 10 Ϫ6 M range occupied mainly by complexes in which A 2 and T 2 molecules are at a 1:1 ratio (Fig.…”

“…In the case of addiction antitoxins, such conformational alterations have been observed upon both toxin and DNA binding (11,47,48,90,92). Moreover, we have shown that the antitoxin molecules may spend a significant amount of time as monomers with a higher potential for structural adaptability than the dimeric form.…”

“…. ϭ A 2i T 2j (46,48,90,92). The expression of the antitoxin and toxin can be inhibited either by low affinity binding of the antitoxin to the promoter/operator DNA (10,11,47,89,90) or by binding of the antitoxin-toxin complexes to the same part of the DNA with higher affinity (10,48,90,92).…”

The ParE2–PaaA2 toxin–antitoxin complex fromEscherichia coliO157 forms a heterodocecamer in solution and in the crystal

“…A number of other toxinantitoxin pairs (e.g. CcdAB, Kis-Kid and Phd-Doc) have also been shown to form larger aggregates under certain conditions, but in these cases nonglobular chains of variable length consisting of alternating toxin and antitoxin molecules were identified (Dao-Thi et al, 2002;Kamphuis et al, 2007;Garcia-Pino et al, 2010). These extended structures have recently been interpreted in terms of a specific mechanism of transcription regulation called 'conditional cooperativity' (De Jonge et al, 2009;Garcia-Pino et al, 2010).…”