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Characterization of the Phd repressor-antitoxin boundary.
James Estle McKinley1, Roy David Magnuson
1Department of Biological Sciences, University of Alabama in Huntsville, Wilson Hall, Room 258, 301 Sparkman Dr., Huntsville, AL 35758, USA.
Journal of Bacteriology
|January 5, 2005
Summary
The P1 plasmid addiction operon
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The P1 plasmid addiction operon features Phd (repressor-antitoxin) and Doc (toxin).
- Previous studies indicated distinct N-terminal repressor and C-terminal antitoxin functions for Phd.
- A modular organization of Phd was hypothesized based on minimal functional domains.
Purpose of the Study:
- To investigate the modular organization of the Phd protein.
- To identify specific amino acid residues critical for Phd's repressor and antitoxin activities.
Main Methods:
- Construction and characterization of 30 point mutations in the Phd protein.
- Deletion analysis to determine the minimal antitoxin domain.
- Assessing repressor and antitoxin activities of mutated and deleted Phd variants.
Main Results:
- Four point mutations (PhdA36H, V37A, I38A, F44A) impaired repressor activity.
- Five point mutations (PhdD53A, D53R, E55A, F56A, F60A) impaired antitoxin activity.
- Mutations affecting repressor and antitoxin functions were distinct and spatially separated.
- The C-terminal 24 amino acids of Phd retained full antitoxin activity.
Conclusions:
- The Phd protein exhibits a modular organization with distinct functional domains.
- Repressor activity is localized to specific residues in the third and fourth quarters of Phd.
- Antitoxin activity is primarily conferred by the C-terminal 24 amino acids.