Transcription of the P1 plasmid addiction operon, a prototypical toxin-antitoxin system, is negatively autoregulated by the products of the operon. The Phd repressor-antitoxin protein binds to 8-bp palindromic Phd-binding sites in the promoter region and thereby represses transcription. The toxin, Doc, mediates cooperative interactions between adjacent Phd-binding sites and thereby enhances repression. Here, we describe a homologous operon from Salmonella enterica serovar Typhimurium which has the same pattern of regulation but an altered repressor-operator specificity. This difference in specificity maps to the seventh amino acid of the repressor and to the symmetric first and eighth positions of the corresponding palindromic repressor-binding sites. Thus, the repressor-operator interface has coevolved so as to retain the interaction while altering the specificity. Within an alignment of homologous repressors, the seventh amino acid of the repressor is highly variable, indicating that evolutionary changes in repressor specificity may be common in this protein family. We suggest that the robust properties of the negative feedback loop, the fuzzy recognition in the operator-repressor interface, and the duplication and divergence of the repressor-binding sites have facilitated the speciation of this repressor-operator interface. These three features may allow the repressoroperator system to percolate within a nearly neutral network of single-step mutations without the necessity of invoking simultaneous mutations, low-fitness intermediates, or other improbable or rate-limiting mechanisms.Bacteriophage P1 can be transmitted horizontally, as a viral particle, or vertically, as the P1 plasmid prophage (36). The low-copy-number P1 plasmid (61) is remarkably stable (67) due to faithful replication (6), partition (5), resolution (7), and addiction (43) functions. Replication and resolution system functions act to maintain a copy number of approximately one to two separate plasmid molecules per chromosome, while the partition function ensures that each daughter cell gets at least one plasmid copy. Should these systems fail, the P1 plasmid addiction operon, a classic toxin-antitoxin system, acts to arrest the plasmid-free cell. Elimination or arrest of plasmid-free cells may leave more resources for plasmid containing cells (17,74).The P1 plasmid addiction operon encodes a 73-amino-acid repressor-antitoxin, Phd, which prevents host death, and a 126-amino-acid toxin, Doc, which is responsible for death on curing (plasmid loss) (43). The Phd antitoxin is both synthesized (43) and degraded at a higher rate than the toxin, Doc. Degradation of Phd is dependent upon the host-encoded, ATP-dependent ClpXP protease (44). While the plasmid is maintained, there is enough antitoxin present to neutralize the toxin. When the plasmid is lost or if expression is halted due to some other cause, the continuing degradation of the antitoxin, in the absence of new synthesis, unveils the toxin and thus arrests the cell.Transcription of the P1 p...