Protease-dependent streptomycin sensitivity in E. coli--a system for protease inhibitor selection

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Streptomycin resistance in E. coli is conferred by mutations in ribosomal protein S12 and is typically recessive.
• Developing sensitive screening systems for protease inhibitors is crucial for drug discovery.

Purpose of the Study:

• To engineer a bacterial cell system where heterologous protease activity confers a dominant streptomycin-sensitive phenotype.
• To establish a sensitive method for identifying protease inhibitors using this engineered system.

Main Methods:

• Constructed a bacterial system expressing a heterologous protease that induces streptomycin sensitivity.
• Utilized the recessive nature of streptomycin resistance and dominant sensitivity conferred by protease activity.
• Co-expressed a protease with an inactive fusion protein (S12-protease substrate) to demonstrate protease-dependent phenotype induction.

Main Results:

• Protease activity conferred a dominant streptomycin-sensitive (strs) phenotype.
• Streptomycin-resistant (strr) E. coli strains showed a 100-1000 fold increase in streptomycin sensitivity upon co-expression of wild-type S12.
• Co-expression of protease and S12-substrate fusion increased streptomycin sensitivity ~100-fold, dependent on substrate cleavage.

Conclusions:

• The developed bacterial system effectively uses protease-dependent dominant phenotypes for sensitive streptomycin sensitivity screening.
• This system is well-suited for identifying protease inhibitors from various libraries.
• Protease-dependent dominant phenotypes offer advantages over recessive phenotypes for certain protease applications.