The Plasmid Addiction Operon
Upon infecting the bacterium Escherichia coli, bacteriophage P1 may express its lytic functions, produce 100-200 new bacteriophage and lyse the infected bacterium. Alternatively, the infecting bacteriophage may repress its lytic functions and transmit its genome vertically to the descendants of the infected bacterium.
In the latter case, the bacteriophage genome is maintained as a large, stable, low-copy plasmid. This plasmid prophage carries several genetic elements that contribute to its stability. One of these, the plasmid addiction operon, increases plasmid stability by eliminating plasmid-free segregants. The operon, and thus the plasmid, is addictive in that its withdrawal is lethal.
The plasmid addiction operon encodes two small proteins: a stable, 126 amino acid toxin and an unstable 73 amino acid antitoxin. While the plasmid is present, the antitoxin is expressed in sufficient quantities to bind and neutralize the toxin. Upon plasmid loss, the continuing degradation of the antitoxin by the host-encoded ClpXP protease unveils the toxin. The toxin then kills the plasmid-free segregant.
In addition to their activities as toxin and antitoxin, Phd and Doc cooperate to regulate transcription of the operon. Phd binds to DNA (as a dimer) and represses transcription. Doc binds to Phd, mediates cooperative interactions between two adjacent Phd-binding sites, and thus enhances repression.
The Phd protein is highly modular. Point mutations affecting repressor and antitoxin activity map to different residues and are separated rather than interspersed in the linear sequence. Deletion mutations show that the N-terminal 3/4 of the protein is essential for repressor but not antitoxin and activity and the C-terminal 1/3 of the protein is essential for antitoxin but not repressor activity. Thus, the Phd protein appears to be a concatenation of a repressor moiety and an antitoxin moiety. Analysis of homologs and hemihomologs of Phd indicate that modular exchange has contributed to the diversification of this protein family.
Modular Exchange:
Promoter Operator Repressor-Antitoxin Toxin
Promoter Operator Repressor-Antitoxin Toxin
Promoter Operator Repressor-Antitoxin Toxin
The analysis of Phd and certain close homologs indicates that protein-ligand covariation has also contributed to the diversification of this protein family.
Repressor-Operator Covariation:
Promoter Operator Repressor-Antitoxin Toxin
Promoter Operator Repressor-Antitoxin Toxin
Current research questions include:
1) How does the toxin kill the cell?
2) What is the function of the transcriptional autoregulation?
3) What structural elements are required for the interactions between:a) operator and repressor4) What is the three dimensional structure of the various components and complexes?
b) repressor and repressor (dimerization)
c) antitoxin and protease
d) antitoxin and toxin
e) toxin and target
5) How do the various protein-ligand interactions coevolve?
6) How does modular exchange affect the structure of the operon?
7) Under what conditions will a self-selecting genetic parasite persist or spread?
Selected Theses (with some minor typological problems relating to file conversions)
Roy David Magnuson, Ph.D. Thesis, 1995. Characterization of a Competence Pheromone in Bacillus subtilis. [PDF, 18.1 MB, Colorized]
Jeremy Allen Smith, Master's Thesis, 2002. Mapping the Antidote and Autoregulatory Domains of Phd. [PDF, 316 KB, Some conversion typos]
Xueyan "Snow" Zhao, Master's Thesis, 2003. Repressor-Operator Specificity Determinants of the P1 Plasmid Addiction Operon. [PDF, 2 MB, pretty clean]
James Estle McKinley, Master's Thesis, 2004. Genetic Analysis of the Phd Antitoxin from Bacteriophage P1. [PDF, 1.5 MB, with minor corrections]