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The DNA sequence and derived amino-acid sequence of a 5618-base region in the 74-min area of the Escherichia coli chromosome has been determined in order to locate the structural gene, nirB, for the NADH-dependent nitrite reductase and a gene, cysG, required for the synthesis of the sirohaem prosthetic group. Three additional open reading frames, nirD, nirE and nirC, were found between nirB and cysG. Potential binding sites on the NirB protein for NADH and FAD, as well as conserved central core and interface domains, were deduced by comparing the derived amino-acid sequence with those of database proteins. A directly repeated sequence, which includes the motif -Cys-Xaa-Xaa-Cys-, is suggested as the binding site for either one [4Fe-4S] or two [2Fe-2S] clusters. The nirD gene potentially encodes a soluble, cytoplasmic protein of unknown function. No significant similarities were found between the derived amino-acid sequence of NirD and either NirB or any other protein in the database. If the nirE open reading frame is translated, it would encode a 33-amino-acid peptide of unknown function which includes 8 phenylalanyl residues. The product of the nirC gene is a highly hydrophobic protein with regions of amino-acid sequence similar to cytochrome oxidase polypeptide 1.

The 74-min region of the Escherichia coli chromosome includes five open reading frames of known sequence. The first and last of these genes, nirB and cysG, are transcribed in the same direction and both are essential for NADH-dependent nitrite reductase activity. The functions of the other genes, nirD, nirE and nirC, which are located between nirB and cysG, are unknown. The nirB gene is transcribed from a promoter which is anaerobically induced, expression being dependent on the transcription activator protein, Fnr. Here we show that the nirD, nirE, nirC and cysG genes are also expressed from the nirB promoter. After subcloning cysG, a second promoter was located less than 100 bases upstream of cysG. Two groups of transcription start points separated by 40 bases were detected in this region by S1 mapping. Rates of transcription from the isolated cysG promoter were the same during aerobic growth and anaerobic growth in the presence or absence of nitrite. However, when the nirB gene and its promoter were cloned back upstream from the cysG promoter, the rate of transcription was higher during anaerobic growth than during aerobic growth and was further induced by nitrite. These increases were totally dependent on a functional fnr gene and were shown by S1 mapping experiments to be due to transcriptional read-through from the Fnr-dependent nirB promoter. No promoter activity was associated with DNA fragments between the BamHI site located within the N-terminal coding region of the nirB gene and the cysG promoter located at the C-terminus of nirC.

The DNA sequence containing the start of the Escherichia coli nirB gene is reported. The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point. Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon. By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208. Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth. Optimal activity was found only after anaerobic growth in the presence of nitrite. The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.