Sequence analysis of an internal 9.72-kb segment from the 30-kb denitrification gene cluster of Pseudomonas stutzeri.

The DNA segment was sequenced that links the nir-nor and nos gene clusters for denitrification of Pseudomonas stutzeri ATCC 14405. Of 10 predicted gene products, four are putative membrane proteins. Sequence similarity was detected with the subunit III of cytochrome-c oxidase (ORF175), PQQ3 of the biosynthetic pathway for pyrrolo-quinoline quinone (ORF393), S-adenosylmethionine-dependent uroporphyrinogen-III C-methyltransferase (ORF278), the cytochrome cd1 nitrite reductase and the NirF protein involved in the biosynthesis of heme d1 (ORF507), LysR type transcriptional regulators (ORF286), short-chain alcohol dehydrogenases (ORF247), and a hypothetical protein, YBEC, of Escherichia coli (ORF57). The current data together with previous work establish a contiguous DNA sequence of 29.2 kb comprising the supercluster of nos-nir-nor genes for denitrification in this bacterium.

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd1-free background (NirS-) of Pseudomonas stutzeri.

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of "Achromobacter cycloclastes" nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd1 nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d1, indicating that both heterologous hosts synthesized a cytochrome cd1 without the d1-group.