An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins

Abstract

A fusion between tsr (encoding the inner membrane protein Tsr) and phoA (encoding the periplasmic protein alkaline phosphatase, AP) generates a membrane-bound hybrid protein (Tsr-AP 2) with AP enzymatic activity. The hybrid protein is proteolytically unstable and is broken down to yield a smaller, soluble species with AP activity. We devised a genetic screen to distinguish between cells containing only membrane-bound AP and those containing soluble AP. The screen depends on diffusion of soluble AP away from cells with a leaky outer membrane to produce a halo of AP activity around colonies on solid growth medium. Several mutants lacking this halo show reduced degradation of Tsr-AP 2. One mutant is also defective in breakdown of five other abnormal periplasmic proteins but not of two cytoplasmic proteins. The mutation in this strain, degP4::Tn5, defines a locus distinct from previously identified loci that affect protein stability or protease activities. This strain may be useful for preventing the breakdown of unstable foreign proteins in Escherichia coli.