DEFECTIVE EXPORT IN ESCHERICHIA-COLI CAUSED BY DSBA'-PHOA HYBRID PROTEINS WHOSE DSBA' DOMAIN CANNOT FOLD INTO A CONFORMATION RESISTANT TO PERIPLASMIC PROTEASES

The disulfide bond-forming factor DsbA and the alkaline phosphatase ar e stable in the Escherichia coli periplasmic space and can be overprod uced without significant perturbation of the cell's physiology. By con trast, DsbA'-PhoA hybrid proteins resulting from TnphoA insertions int o different regions of a plasmid-borne dsbA gene could become toxic (l ethal) to bacteria. Toxicity was concomitant with an impairment of som e step of the export mechanism and depended on at least three paramete rs, i.e., (i) the rate of expression of the hybird protein, (ii) the a bility of the amino-terminal DsbA' domain of the hybrid protein to fol d into a protease-resistant conformation in the periplasmic space, and (iii) the activity of the DegP periplasmic protease. Even under viabl e conditions of low expression, DsbA' folding-deficient hybrid protein s accumulated more than the folding-proficient ones in the insoluble m aterial and this was aggravated in a strain lacking the DegP protease. When production was more elevated, the folding-deficient hybrid prote ins became lethal, but only in strains lacking the DegP activity, whil e the folding-proficient ones were not. Under conditions of very high production by degP(+) or degP strains, both types of hybrid proteins a ccumulated as insoluble preproteins. Meanwhile, the export machinery w as dramatically handicapped and the cells lost viability. However, the folding-deficient hybrid proteins had a higher killing efficiency tha n the folding-proficient ones. Free DshA'-truncated polypeptides, alth ough not toxic, were processed more slowly when they could not fold in to a protease-resistant form in the periplasmic space. This provides i ndications in E. coli for a direct or indirect influence of the foldin g of a protein in the periplasmic environment on export efficiency.