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Communication of ClpXP protease hypersensitivity to bacteriophage Mu repressor isoforms
D J Welty1, J M Jones, H Nakai
1Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd NW, Washington, DC 20007, USA.
Abstract:
The immunity repressor (Rep) of bacteriophage Mu establishes and maintains lysogeny by shutting down transposition functions needed for phage DNA replication. Although Rep is stable in vivo, an altered immunity repressor (Vir) encoded by virulent, trans-dominant Mu mutants is rapidly degraded by Escherichia coli ClpXP protease. Rep and Vir are degraded at approximately the same maximal velocity (Vmax) by ClpXP, but the Km for Rep (3.6 microM) is over 20-fold higher than the Km for Vir (0.15 microM). Rep is also highly resistant to degradation in the presence of DNA whereas Vir is not. Vir increases the rate of Rep degradation by reducing its Km and imparts to Rep ClpXP sensitivity in the presence of DNA. Vir can drive at an accelerated rate the complete degradation of Rep molecules that outnumber Vir by eightfold or more. So long as Vir is present at a concentration of 0.1 microM or higher, Rep is degraded with a Km that is indistinguishable from that of Vir. These characteristics of repressor may be an important means of transducing physiological signals that induce Mu transposition in response to growth conditions or environmental stress, ClpXP hypersensitivity being disseminated among Rep molecules for the induction of Mu transposition.
Insights
Bacteriophage Mu
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- The immunity repressor (Rep) of bacteriophage Mu is crucial for establishing and maintaining lysogeny by inhibiting phage DNA replication.
- Virulent mutants express an altered repressor (Vir) that is rapidly degraded by Escherichia coli ClpXP protease, unlike the stable Rep.
Purpose of the Study:
- To investigate the degradation kinetics of bacteriophage Mu's Rep and Vir repressors by E. coli ClpXP protease.
- To elucidate the role of DNA in the degradation of Rep and Vir by ClpXP.
- To understand how Vir influences Rep degradation and ClpXP sensitivity.
Main Methods:
- Enzyme kinetics assays to determine degradation rates (Vmax) and substrate affinities (Km) of Rep and Vir by ClpXP.
- Comparative analysis of Rep and Vir degradation in the presence and absence of DNA.
- Investigating the effect of varying Vir concentrations on Rep degradation.
Main Results:
- Rep and Vir share similar Vmax, but Vir has a 20-fold lower Km (0.15 microM) than Rep (3.6 microM) for ClpXP.
- Rep is resistant to ClpXP degradation in the presence of DNA, while Vir is not.
- Vir accelerates Rep degradation by reducing its Km and conferring DNA sensitivity, even at substoichiometric concentrations.
Conclusions:
- The Vir repressor enhances the degradation of Rep by ClpXP, particularly in the presence of DNA.
- This mechanism allows for rapid induction of Mu transposition in response to environmental cues.
- The modulation of repressor stability by ClpXP is a key regulatory switch for bacteriophage Mu life cycle.