Moderately thermostable phage Phi11 Cro repressor has novel DNA-binding capacity and physicochemical properties

Area of Science:

• Bacteriophage biology
• Molecular genetics
• Protein biochemistry

Background:

• Temperate bacteriophages, such as Staphylococcus aureus phage Phi11, possess regulatory genes like cI and cro, analogous to lambdoid phages.
• The Cro repressor protein plays a crucial role in phage genetic regulation.

Purpose of the Study:

• To characterize the biophysical and DNA-binding properties of the Phi11 Cro repressor.
• To compare Phi11 Cro with other well-characterized Cro repressors from different phage families.

Main Methods:

• Purification of extremely pure Phi11 Cro protein.
• Analysis of protein structure and dimerization using biophysical techniques.
• Thermostability assays using thermolysin and aggregation studies.
• DNA binding assays with homologous operator sequences (O1, O2, O3).

Main Results:

• Phi11 Cro exhibits a single-domain, largely alpha-helical structure and forms dimers in solution.
• The protein demonstrates moderate thermostability, with sensitivity to thermolysin between 55-75°C and aggregation around 63°C.
• Phi11 Cro specifically binds to the O3 operator, located upstream of the cI gene, with high efficiency.
• Comparative analysis reveals distinct DNA binding, structural, and dimerization properties compared to P22 Cro and lambda Cro.

Conclusions:

• Phi11 Cro is a moderately thermostable protein with unique DNA-binding specificity for the O3 operator.
• The distinct characteristics of Phi11 Cro highlight the diversity among Cro repressor proteins from different phage families.