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The cycHJKL genes of Rhizobium meliloti involved in cytochrome c biogenesis are required for “respiratory” nitrate reduction ex planta and for nitrogen fixation during symbiosis

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Abstract

We report the genetic and biochemical analysis of Rhizobium meliloti mutants defective in symbiotic nitrogen fixation (Fix−) and “respiratory” nitrate reduction (Rnr−). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus. By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr− and Fix− phenotypes. Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins. Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes. In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively. The predicted protein product of cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multi-subunit cytochrome c haem lyase. Moreover, our results indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules.

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Author notes

  1. Krystyna Slaska-Kiss

    Present address: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, PO Box 521, H-6701, Szeged, Hungary

  2. Zsofia Banfalvi

    Present address: Agricultural Biotechnology Centre, PO Box 170, H-2101, Gödöllô, Hungary

Authors and Affiliations

  1. Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, H-6701, PO Box 521, Szeged, Hungary

    Attila Kereszt, Krystyna Slaska-Kiss & Peter Putnoky

  2. Institute des Sciences Végétales, CNRS, Avenue de la Terrasse, F-91198, Gif-sur-Yvette Cedex, France

    Zsofia Banfalvi & Adam Kondorosi

Authors
  1. Attila Kereszt
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  2. Krystyna Slaska-Kiss
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  3. Peter Putnoky
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  4. Zsofia Banfalvi
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  5. Adam Kondorosi
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Communicated by J. Lengeler

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Kereszt, A., Slaska-Kiss, K., Putnoky, P. et al. The cycHJKL genes of Rhizobium meliloti involved in cytochrome c biogenesis are required for “respiratory” nitrate reduction ex planta and for nitrogen fixation during symbiosis. Molec. Gen. Genet. 247, 39–47 (1995). https://doi.org/10.1007/BF00425819

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