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Regulation and sequence of the structural gene for cytochrome c552 from Escherichia coli: not a hexahaem but a 50 kDa tetrahaem nitrite reductase

Darwin A; Hussain H; Griffiths L; Grove J; Sambongi Y; Busby S; Cole J
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
PMID: 7934939
ISSN: 0950-382x
CID: 23552

Identification of the formate dehydrogenases and genetic determinants of formate-dependent nitrite reduction by Escherichia coli K12

Darwin A; Tormay P; Page L; Griffiths L; Cole J
The formate dehydrogenases of Escherichia coli involved in electron transfer from formate to nitrite (Nrf activity: nitrite reduction by formate) have been identified. No previously undescribed selenoprotein was detected in bacteria grown under conditions optimal for the expression of Nrf activity. The Nrf activities of single mutants defective in either FdhN or FdhH were between 50 and 60% that of the parental strain. A double mutant defective in both FdhN and FdhH retained less than 10% of the activity of the FdhN+ FdhH+ strain. No Nrf activity was detected in a triple mutant defective in FdhN, FdhH and FdhO or in the selC strain. It is concluded that all three of the known formate dehydrogenases of E. coli can contribute to the transfer of electrons from formate to the Nrf pathway. Mutants defective in Nrf activity and cytochrome c552 synthesis were isolated by insertion mutagenesis or identified amongst strains received from the E. coli Genetic Stock Center. The mutations were located in at least three regions of the chromosome, including the 92 to 94 minute region which includes fdhF, the gene encoding FdhH required for formate hydrogenlyase activity. Fine structure mapping by P1 transduction established that the nrf mutations in the fdhF region were due to defects in three separable loci, all of which were independent of but close to fdhF. Clones were isolated from a cosmid library that complemented a deletion extending from fdhF into a region essential for Nrf activity. From these clones, plasmids were isolated that complemented only some of the Nrf- mutations in the 92 to 94 minute region, confirming the presence of different operons essential for Nrf activity and cytochrome c552 synthesis in this region. Suggested reasons for this genetic complexity include the need for proteins involved in electron transfer from the various formate dehydrogenases to cytochrome c552, for the attachment of the haem group to the apocytochrome and for cytochrome c552 export into the periplasm
PMID: 8409924
ISSN: 0022-1287
CID: 23553