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  • 1
    ISSN: 1432-072X
    Keywords: Nucleotide sequence ; Apocytochrome cd 1 ; Heme d 1 incorporation ; Denitrification ; Copper coordination ; Signal peptide ; Escherichia coli
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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 cd 1 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 d 1, indicating that both heterologous hosts synthesized a cytochrome cd 1 without the d 1-group.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-072X
    Keywords: Nitrous oxide reduction ; Copper proteins ; Cytochrome patterns ; Denitrification ; Pseudomonas
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Copper is the essential transition element for nitrous oxide respiration in Pseudomonas perfectomarinus. Two novel kinds of copper proteins were detected in this organism. Their distribution was studied under different growth conditions and in other pseudomonads, as well as their association with N2O reduction of intact cells. A low molecular mass copper protein (M r 38,000) with a single absorption band at 340 nm (oxidized form), was found only in P. perfectomarinus and was not required for N2O reduction. N2O respiration was consistently associated with a high molecular mass copper protein (M r 120,000) in P. perfectomarinus, Pseudomonas stutzeri, and in strains of Pseudomonas fluorescens that were capable of this type of respiration. The oxidized protein was violet to pink with absorption bands at 350, 480, 530, 620, and 780 nm. Pseudomonas chlororaphis and Pseudomonas aureofaciens which did not respire with N2O as electron acceptor, did not contain the novel type of copper protein. Cytochrome patterns were compared in these denitrifying pseudomonads to search for the physiological electron carrier to N2O reductase. The content and nature of the soluble c-type cytochromes depended strongly on the species and the particular growth condition.
    Type of Medium: Electronic Resource
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