A PLEIOTROPIC ACID PHOSPHATASE-DEFICIENT MUTANT OF ESCHERICHIA-COLI SHOWS PREMATURE TERMINATION IN THE DSBA GENE - USE OF DSBA--PHOA FUSIONS TO LOCALIZE A STRUCTURALLY IMPORTANT DOMAIN IN DSBA

A one-step mutant of Escherichia coli K-12 lacking both glucose-1-phos phatase (Agp) and pH 2.5 acid phosphatase (AppA) activities in the per iplasmic space was isolated. The mutation which mapped close to chlB, at 87 min on the E. coli linkage map, also caused the loss of alkaline phosphatase (PhoA) activity, even when this activity was expressed fr om TnphoA fusions to genes encoding periplasmic or membrane proteins. A DNA fragment that complements the mutation was cloned and shown to c arry the dsbA gene, which encodes a periplasmic disulphide bond-formin g factor. The mutant had an ochre triplet in dsbA, truncating the prot ein at amino acid 70. Introduction of TnphoA fusions into a plasmid-bo rne dsbA gene resulted in DsbA-PhoA hybrid proteins that were all expo rted to the periplasmic space in both dsbA(+) and dsbA strains. They b elong to three different classes, depending on the length of the DsbA fragment fused to PhoA. When PhoA was fused to an amino-terminal DsbA heptapeptide, the protein was only seen in the periplasm of a dsbA(+) strain, as in the case of wildtype PhoA. Hybrid proteins missing up to 29 amino acids at the carboxy-terminus of DsbA were stable and retain ed both the DsbA and PhoA activities. Those with shorter DsbA fragment s that still carried the -Cys-Pro-His-Cys- motif were rapidly degraded (no DsbA activity). The presence is discussed of a structural domain lying around amino acid 170 of DsbA and which is probably essential fo r its folding into a proteolytic-resistant and enzymatically active fo rm.