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Nucleotide sequence and genetic analysis of the Rhodobacter capsulatus ORF6-nifU I SVW gene region: possible role of Nif W in homocitrate processing

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Abstract

DNA sequence analysis of a 3494-bp HindIII-Bc1I fragment of the Rhodobacter capsulatus nif region A revealed genes that are homologous to ORF6, nifU, nifS, nifV and nifW from Azotobacter vinelandii and Klebsiella pneumoniae. R. capsulatus nifU, which is present in two copies, encodes a novel type of NifU protein. The deduced amino acid sequences of NifUI and NifUII share homology only with the C-terminal domain of NifU from A. vinelandii and K. pneurnoniae. In contrast to nifA andnifB which are almost perfectly duplicated, the predicted amino acid sequences of the two NifU proteins showed only 39% sequence identity. Expression of the ORF6-nifU ISVW operon, which is preceded by a putative σ54-dependent promoter, required the function of NifA and the nif-specific rpoN gene product encoded by nifR4. Analysis of defined insertion and deletion mutants demonstrated that only nifS was absolutely essential for nitrogen fixation in R. capsulatus. Strains carrying mutations in nifV were capable of very slow diazotrophic growth, whereas ORF6, nifU I and nifW mutants as well as a nifU I/nifUII, double mutant exhibited a Nif+ phenotype. Interestingly, R. capsulatus nifV mutants were able to reduce acetylene not only to ethylene but also to ethane under conditions preventing the expression of the alternative nitrogenase system. Homocitrate added to the growth medium repressed ethane formation and cured the NifV phenotype in R. capsulatus. Higher concentrations of homocitrate were necessary to complement the NifV phenotype of a polar nifV mutant (NifV−NifW−), indicating a possible role of NifW either in homocitrate transport or in the incorporation of this compound into the iron-molybdenum cofactor of nitrogenase.

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Authors and Affiliations

  1. Lehrstuhl für Genetik, Fakultät für Biologic, Universität Bielefeld, Postfach 10 0131, W-4800, Bielefeld 1, Germany

    Bernd Masepohl, Sieglinde Angermüller, Silke Hennecke & Werner Klipp

  2. Department of Microbiology, Biozentrum der Universität Basel, Klingelbergstrasse 70, CH-4056, Basel, Switzerland

    Philipp Hübner

  3. Departamento de Bioquimica, Biologia Molecular y Fisiologia, Facultad de Ciencias, Avda. S. Alberto Magno, Universidad de Cordoba, 14071, Cordoba, Spain

    Conrado Moreno-Vivian

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  1. Bernd Masepohl
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  2. Sieglinde Angermüller
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  3. Silke Hennecke
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  4. Philipp Hübner
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  5. Conrado Moreno-Vivian
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  6. Werner Klipp
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Communicated by A. Kondorosi

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Masepohl, B., Angermüller, S., Hennecke, S. et al. Nucleotide sequence and genetic analysis of the Rhodobacter capsulatus ORF6-nifU I SVW gene region: possible role of Nif W in homocitrate processing. Molec. Gen. Genet. 238, 369–382 (1993). https://doi.org/10.1007/BF00291996

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