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Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction inParacoccus denitrificans

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Abstract

By using the gene encoding the C-terminal part of thecd 1-type nitrite reductase ofPseudomonas stutzeri JM300 as a heterologous probe, the corresponding gene fromParacoccus denitrificans was isolated. This gene,nirS, codes for a mature protein of 63144 Da having high homology withcd 1-type nitrite reductases from other bacteria. Directly downstream fromnirS, three othernir genes were found in the ordernirECF. The organization of thenir gene cluster inPa. denitrificans is different from the organization ofnir clusters in some Pseudomonads.nirE has high homology with a S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (uro'gen III methylase). This methylase is most likely involved in the hemed 1 biosynthesis inPa. denitrificans. The third gene,nirC, codes for a small cytochromec of 9.3 kDa having high homology with cytochromec 55X ofPs. stutzeri ZoBell. The 4th gene,nirF, has no homology with other genes in the sequence databases and has no relevant motifs. Inactivation of either of these 4 genes resulted in the loss of nitrite and nitric oxide reductase activities but not of nitrous oxide reductase activity.nirS mutants lack thecd 1-type nitrite reductase whilenirE, nirC andnirF mutants produce a small amount ofcd 1-type nitrite reductase, inactive due to the absence of hemed 1. Upstream from thenirS gene the start of a gene was identified which has limited homology withnosR, a putative regulatory gene involved in nitrous oxide reduction. A potential FNR box was identified between this gene andnirS.

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Abbreviations

SDS:

sodium dodecyl sulfate

NBT:

nitroblue tetrazolium

PAGE:

polyacrylamide gel electrophoresis

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, 1081 HV, Amsterdam, The Netherlands

    Anthonius P. N. de Boer, Willem N. M. Reijnders, Adriaan H. Stouthamer & Rob J. M. van Spanning

  2. Laboratory of Microbiology and Enzymology, Delft University of Technology, Julianalaan 67A, 2628 BC, Delft, The Netherlands

    J. Gijs Kuenen

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  1. Anthonius P. N. de Boer
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  2. Willem N. M. Reijnders
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  4. Adriaan H. Stouthamer
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  5. Rob J. M. van Spanning
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de Boer, A.P.N., Reijnders, W.N.M., Kuenen, J.G. et al. Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction inParacoccus denitrificans . Antonie van Leeuwenhoek 66, 111–127 (1994). https://doi.org/10.1007/BF00871635

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