Regulation and sequence of the structural gene for cytochrome C552 from Escherichia coli: not a hexahaem but a 50kDa tetrahaem nitrite reductase

Abstract

The structural gene, nrfA, for cytochrome c552, which is the terminal reductase of the formate-dependent pathway for nitrite reduction to ammonia, has been located at co-ordinate 4366 on the physical map of the Escherichia coli chromosome. The DNA sequence of nrfA encodes a tetrahaem c-type cytochrome with a predicted M(r) for the unprocessed product of 53,788. Cleavage of the putative signal peptide at Ala-26 would result in a mature, periplasmic cytochrome of M(r) 50,580 rather than a larger hexahaem cytochrome, as has been widely reported previously. A cytochrome of this size was detected by staining SDS-polyacrylamide gels for covalently bound haem. This cytochrome was partially purified by anion exchange chromatography and confirmed to be cytochrome c552 by difference spectroscopy. Similar cytochromes were detected in five other E. coli strains including strain ST 249, which was used previously to purify and characterize the protein. A plasmid with an in-phase deletion within nrfA directed the synthesis of a truncated haemoprotein of the predicted mass. In-phase translational fusions to lacZ were used to locate the nrfA translation start, and the transcription start site was found by S1 mapping. Expression from the FNR-dependent nrfA promoter was almost totally repressed during aerobic growth, partially induced during anaerobic growth in the absence of nitrite or in the presence of nitrate, but fully induced only during anaerobic growth in the presence of nitrite. No nitrate repression was detected in a narL mutant, 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 was independent of the CRP-cAMP complex.