PROTEASE-DEPENDENT STREPTOMYCIN SENSITIVITY IN ESCHERICHIA-COLI - A SYSTEM FOR PROTEASE INHIBITOR SELECTION

We have developed a bacterial cell system in which the activity of an expressed heterologous protease confers a dominant streptomycin-sensitive (str(s)) phenotype on the cells. This phenotype owes its high selectivity to the fact that streptomycin (strep) resistance, which is conferred on E. coli by mutants of ribosomal protein S12, is highly recessive to strep sensitivity, Thus, when strep-resistant (str(r)) strains of E. coli are transformed to co-express the wild-type allele of S12 in addition to the mutant allele, their sensitivity to strep increases by a factor of 100-1000. Similarly, we found that when the same str(r) strains were transformed to co-express a heterologous protease and an inactive fusion of S12 with a substrate of the protease, the strep sensitivity of the cells increased similar to 100-fold, This effect was strictly dependent on correct cleavage of the S12 precursor, required only modest levels of expression of protease and substrate, and could be competitively inhibited by co-expression of an alternative substrate gene, This system thus appears to be well-suited to the identification of protease inhibitors, either by selection from libraries of endogenously expressed random peptide-encoding genes, or by screening synthetic or natural products libraries, Protease-dependent dominant phenotypes may be more sensitive and appropriate than the more commonly used recessive phenotypes for proteases which are activating enzymes.