title: Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein.
abstract: The nirIX gene cluster of Paracoccus denitrificans is located between the nir and nor gene clusters encoding nitrite and nitric oxide reductases respectively. The NirI sequence corresponds to that of a membrane-bound protein with six transmembrane helices, a large periplasmic domain and cysteine-rich cytoplasmic domains that resemble the binding sites of [4Fe-4S] clusters in many ferredoxin-like proteins. NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence. NirI and NirX are homologues of NosR and NosX, proteins involved in regulation of the expression of the nos gene cluster encoding nitrous oxide reductase in Pseudomonas stutzeri and Sinorhizobium meliloti. Analysis of a NirI-deficient mutant strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N-oxides. The NirX-deficient mutant transiently accumulated nitrite in the growth medium, but it had a final growth yield similar to that of the wild type. Transcription of the nirIX gene cluster itself was controlled by NNR, a member of the family of FNR-like transcriptional activators. An NNR binding sequence is located in the middle of the intergenic region between the nirI and nirS genes with its centre located at position -41.5 relative to the transcription start sites of both genes. Attempts to complement the NirI mutation via cloning of the nirIX gene cluster on a broad-host-range vector were unsuccessful, the ability to express nitrite reductase being restored only when the nirIX gene cluster was reintegrated into the chromosome of the NirI-deficient mutant via homologous recombination in such a way that the wild-type nirI gene was present directly upstream of the nir operon.
authors.affiliation: Department of Molecular Cell Physiology, Faculty of Biology, BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands.
authors.forename: N F
authors.initials: NF
authors.lastname: Saunders
authors.forename: E N
authors.initials: EN
authors.lastname: Houben
authors.forename: S
authors.initials: S
authors.lastname: Koefoed
authors.forename: S
authors.initials: S
authors.lastname: de Weert
authors.forename: W N
authors.initials: WN
authors.lastname: Reijnders
authors.forename: H V
authors.initials: HV
authors.lastname: Westerhoff
authors.forename: A P
authors.initials: AP
authors.lastname: De Boer
authors.forename: R J
authors.initials: RJ
authors.lastname: Van Spanning
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D004268:DNA-Binding Proteins; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D009572:Nitrite Reductases / Q000235:genetics* / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C120062:NNR protein, Paracoccus denitrificans; C401644:NirI protein, Paracoccus denitrificans; C401643:NirX protein, Paracoccus denitrificans; D014157:Transcription Factors; D009572:Nitrite Reductases