THE BIOGENESIS OF C-TYPE CYTOCHROMES IN ESCHERICHIA-COLI REQUIRES A MEMBRANE-BOUND PROTEIN, DIPZ, WITH A PROTEIN DISULFIDE ISOMERASE-LIKE DOMAIN

A mutant of Escherichia cell K-12, JCB606, which lacks all five c-type cytochromes synthesized during anaerobic growth in the presence of ni trite or trimethylamine-N-oxide (TMAO), was totally defective in Nrf a ctivity and also partially defective in TMAO reductase activity. The m utation in strain JCB606 was shown to affect expression of the tor ope ron, which contributes almost equally with the products of the dms ope ron to the rate of TMAO reduction by bacteria during anaerobic growth in the presence of TMAO. The mutation in strain JCB606, dipZ, was mapp ed by P1 transduction close to the mel operon at co-ordinate 4425 on t he E. coli chromosome, the gene order being nrf-fdhF-mel-dipZ-ampC. Re combinant plasmids that restored Nrf activity to test-tube cultures of the mutant were isolated from a cosmid library. A 2.7 kb EcoRV-Smal f ragment (co-ordinates 4443 to 4446 kb on the physical map of the E. ce ll chromosome) was found potentially to encode three genes arranged in at least two operons. The second gene, dipZ, was sufficient to comple ment the JCB606 mutation. The translated DNA sequence predicts that Di pZ is a 53 kDa integral membrane protein with a 37 kDa N-terminal doma in including at least six membrane-spanning helices and a 16 kDa carbo xy-terminal hydrophilic domain which includes a protein disulphide iso merase-like motif, It is suggested that DipZ is essential for maintain ing cytochrome c apoproteins in the correct conformations for the cova lent attachment of haem groups to the appropriate pairs of cysteine re sidues.