REGULATION AND SEQUENCE OF THE STRUCTURAL GENE FOR CYTOCHROME-C552 FROM ESCHERICHIA-COLI - NOT A HEXAHAEM BUT A 50 KDA TETRAHAEM NITRITE REDUCTASE

The structural gene, nrfA, for cytochrome c552, which is the terminal reductase of the formate-dependent pathway for nitrite reduction to am monia, has been located at co-ordinate 4366 on the physical map of the Escherichia coli chromosome. The DNA sequence of nrfA encodes a tetra haem c-type cytochrome with a predicted M(r) for the unprocessed produ ct of 53788. Cleavage of the putative signal peptide at Ala-26 would r esult in a mature, periplasmic cytochrome of M(r) 50580 rather than a larger hexahaem cytochrome, as has been widely reported previously. A cytochrome of this size was detected by staining SDS-polyacrylamide ge ls for covalently bound haem. This cytochrome was partially purified b y anion exchange chromatography and confirmed to be cytochrome c552 by difference spectroscopy. Similar cytochromes were detected in five ot her E. coli strains including strain ST 249, which was used previously to purify and characterize the protein. A plasmid with an in-phase de letion within nrfA directed the synthesis of a truncated haemoprotein of the predicted mass. In-phase translational fusions to lacZ were use d to locate the nrfA translation start, and the transcription start si te was found by S1 mapping. Expression from the FNR-dependent nrfA pro moter was almost totally repressed during aerobic growth, partially in duced during anaerobic growth in the absence of nitrite or in the pres ence of nitrate, but fully induced only during anaerobic growth in the presence of nitrite. No nitrate repression was detected in a narL mut ant, but nitrite induction was unaffected, indicating that the nitrite -sensing mechanism is independent of the NarL protein. Expression from the nrfA promoter was subject to glucose repression but regulation wa s independent of the CRP-cAMP complex.