COREPRESSION OF THE P1 ADDICTION OPERON BY PHD AND DOC

The P1 plasmid addiction operon encodes Doc, a toxin that kills plasmi d-free segregants, and Phd, an unstable antidote that neutralizes the toxin, Additionally, these products repress transcription of the opero n. The antidote binds to two adjacent sites in the promoter. Here we p resent evidence concerning the regulatory role of the toxin, which we studied with the aid of a mutation, docH66Y. The DocH66Y protein retai ned the regulatory properties of the wild-type protein, but not its to xicity, In vivo, DocH66Y enhanced repression by Phd but failed to affe ct repression in the absence of Phd, suggesting that DocH66Y contacts Phd. In vitro, a MalE-DocH66Y fusion protein was found to bind Phd. Bi nding of toxin to antidote may be the physical basis for the neutraliz ation of toxin. DocH66Y failed to hind DNA in vitro yet enhanced the a ffinity, cooperativity, and specificity with which Phd hound the opera tor, Although DocH66Y enhanced the binding of Phd to two adjacent Phd- binding sites, DocH66Y had relatively little effect on the binding of Phd to a single Phd-binding site, indicating that DocH66Y mediates coo perative interactions between adjacent Phd-binding sites. Several elec trophoretically distinct protein-DNA complexes were observed with diff erent amounts of DocH66Y relative to Phd. Maximal repression and speci ficity of DNA binding were observed with subsaturating amounts of DocH 66Y relative to Phd. Analogous antidote-toxin pairs appear to have sim ilar autoregulatory circuits. Autoregulation, by dampening fluctuation s in the levels of toxin and antidote, may prevent the inappropriate a ctivation of the toxin.