Mutational Analysis of the Nor Gene Cluster which Encodes Nitric‐Oxide Reductase from Paracoccus denitrificans

Abstract

The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower NOR activity compared with the wild-type strain.

Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3

“…The nnrU product, like that of nnrT, is hydrophobic, and hydropathy analysis suggests that it has seven transmembranespanning regions (data not shown). The nnrU product has no significant similarity with the ORF2 product from P. aeruginosa (4) or with the norE product of P. denitrificans (8); both of these are hydrophobic proteins similar to the subunit III of cytochrome c oxidases and have been suggested to be important for Nor function. Sequence comparison with the entire database also failed to yield any proteins with significant identity to the nnrU product.…”

“…Sequence analysis of other denitrifiers has identified ORFs in close proximity to the nor region that encode small hydrophobic proteins. For example, ORF3 in P. aeruginosa encodes a product of 85 residues (4), and norF from P. denitrificans encodes a product of 77 residues (8). Neither of these gene products has any significant similarity to the nnrT product.…”

“…The nonstructural gene in the P. aeruginosa operon is also found immediately downstream of the nor operon in P. stutzeri, but in the latter denitrifier it is independently transcribed. In Paracoccus denitrificans, the nor operon apparently consists of six genes, which include homologs of the three genes in the P. aeruginosa operon (8). Regulation of nor expression appears to be under the control of the nnrR product in P. denitrificans and Rhodobacter sphaeroides 2.4.3 and an apparent functional homolog of NnrR in P. aeruginosa (2,35,37).…”

Formation of a Distinctive Complex between the Inducible Bacterial Lysine Decarboxylase and a Novel AAA+ ATPase

“…Both NorD and CbbO were shown to be important in the activation of functional nitric-oxide reductase and Rubisco, respectively (11)(12)(13). The VWA domain is a metal-binding domain often mediating proteinprotein interactions (19).…”

Iron as a possible mediator of the oxic‐to‐anoxic transition in Paracoccus denitrificans

“…The concerted [ 16] regulation of nitrite reductase (nir) [17] and nitric oxide reduotase (nor) [18] operons of Paracoccus denitrificans involves the nnr gene product [13] which displays significant deletions at the N-terminus, resulting in a loss of the iron-sulfur cluster. This puzzling feature of the Nnr protein may indicate that, in contrast to the Fnr-dependent genes [19][20][21][22][23], the control of nir and nor does not require iron.…”