IDENTIFICATION AND CHARACTERIZATION OF A NEW DISULFIDE ISOMERASE-LIKEPROTEIN (DSBD) IN ESCHERICHIA-COLI

Previous studies have established that DsbA and DsbC, periplasmic prot eins of Escherichia coli, are two key players involved in disulfide bo nd formation, A search for extragenic mutations able to compensate for the lack of dsbA function in vivo led us to the identification of a n ew gene, designated dsbD, Lack of DsbD protein leads to some, but not all, of the phenotypic defects observed with other dsb mutations, such as hypersensitivity to dithiothreitol and to benzylpenicillin. In add ition, unlike the rest of the dsb genes, dsbD is essential for bacteri al growth at temperatures above 42 degrees C, Cloning of the wild-type gene and sequencing and overexpression of the protein show that dsbD is part of an operon and encodes an inner membrane protein, A 138 amin o acid subdomain of the protein was purified and shown to possess an o xido-reductase activity in vitro, Expressing this subdomain in the per iplasmic space helped restore the phenotypic defects associated with a dsbD null mutation, Interestingly, this domain shares 45% identity wi th the portion of the eukaryotic protein disulfide isomerase carrying the active site. We further show that in dsbD mutant bacteria the dith iol active sites of DsbA and DsbC proteins are mostly oxidized, as com pared with wild-type bacteria, Our results argue that DsbD generates a reducing source in the periplasm, which is required for maintaining p roper redox conditions, The finding that overexpression of DsbD leads to a Dsb(-) phenotype, very similar to that exhibited by dsbA null mut ants, is in good agreement with such a model.